1
Genotyping of Methicillin Resistant Staphylococcus aureus
(MRSA) from Local Hospital of Rawalpindi/Islamabad,
Pakistan
By
YASRAB ARFAT
Department of Biochemistry
Faculty of Biological Sciences
Quaid-i-Azam University, Islamabad
2013
2
Genotyping of Methicillin Resistant Staphylococcus aureus
(MRSA) from Local Hospital of Rawalpindi/Islamabad,
Pakistan
A Thesis Submitted in the Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biochemistry
By
YASRAB ARFAT
Department of Biochemistry
Faculty of Biological Sciences
Quaid-i-Azam University, Islamabad
2013
3
CERTIFICATE
This thesis by Mr. Yasrab Arfat is accepted in its present form by the
Department of Biochemistry, Quaid-i-Azam University, Islamabad as
satisfying the thesis requirements for the degree of Doctor of Philosophy
(PhD) in Biochemistry/Molecular Biology.
External Examiner: (Dr. ____________)
External Examiner: (Dr. ____________)
Supervisor & Chairman: (Prof. Dr. S.A. Malik) Chairperson: _____________ (Prof. Dr. Bushra Mirza)
Dated: _______________
4
IN THE NAME OF ALLAH,
MOST GRACIOUS, MOST MERCIFUL
Allah Almighty initiates the creation of everything
according to His Law and then, while passing it through
various stages of development, takes it to its destined
point. And all this happens very easily. All the plans, both
in the heavens and on earth, are cast in the patterns set
by the Almighty and are therefore most sublime.
Almighty’s Law, in fact, contains extremely beautiful
combinations in itself; it has force and power as well as
rationale and wisdom.
(Al-Quran: Surrah ArRûm, Verse: 27)
5
DEDICATION
Dedicated to my beloved parents and family members
i
CONTENTS
ACKNOWLEDGEMENTS V
LIST OF ABBREVIATIONS VII
LIST OF FIGURES IX
LIST OF TABLES XIV
ABSTRACT XVI
CHAPTER 1 1
INTRODUCTION 1
Staphylococcus aureus 1
Taxonomy of Staphylococcus aureus 2
Diseases caused by Staphylococcus aureus 2
Staphylococcus aureus Pathogenicity 4
Surface proteins 4
Invasins 6
Surface factors 6
Immunological disguises 7
Membrane-damaging toxins 7
Exotoxins 8
Antibiotic Resistance in Staphylococcus aureus 8
Mechanism of Antimicrobial resistance in Staphylococcus aureus 10
Production of beta lactamase (Penicillinase) 11
Production of an altered penicillin binding protein (PBP 2’ or
PBP2a)
11
Hyperproduction of beta-lactamase or methicillinase 11
Production of modified penicillin binding protein other than PBP2a 12
The unknown mechanism exhibited by small colony variants 12
Methicillin Resistant Staphylococcus aureus 12
Factors affecting Methicillin Resistance 16
Effect of Beta lactamases genes on methicillin resistance 16
ii
Effect of Plasmids encoding Staphylococci beta-lactamase on
methicillin Resistance 16
Effects of fem and aux factors on methicillin resistance 17
Other Chromosomal loci affecting the expression of methicillin
resistance 17
External factors affecting the expression of methicillin resistance 17
Heterogeneous and homogenous resistance to methicillin 18
Treatment against Staphylococcal Infections 18
Vaccines 19
Epidemiology of MRSA 20
Phenotypic methods of strain differentiation 20
Antibiogram 20
Phage Typing 21
Immunobloting 21
Multilocus Enzyme Electrophoresis (MLEE) 21
Serotyping 22
Genome Based Methods of Typing MRSA 22
Plasmid Analysis 22
Restriction enzyme analysis of whole genomic DNA 22
Ribotyping 23
Pulse Field Gel Electrophoresis of Chromosomal DNA 23
Ribosome spacer PCR 24
SCCmec region 24
PCR Restriction Modification (RM) Typing 25
Multilocus Variable Number of Tandem Repeat analysis (MLVA) 25
SPA Sequence typing 27
Multilocus sequence typing (MLST) 29
STAR Analysis 30
Aim and Objectives of Study 30
CHAPTER 2 32
MATERIALS AND METHODS 32
iii
Isolation and identification of Staphylococcus aureus 32
Gram staining and Biochemical Tests 33
Bacterial culture and DNA Isolation 34
16S-rDNA Identification of Staphylococcus aureus 35
Mec A- gene Identification and SCCmec typing of S. aureus 36
Detection of PVL Gene 37
Determination of MRSA Lineages Using Restriction Modification
system (RM-Test) 37
Typing of Staphylococcus aureus for STAR Element 39
Multiplex PCR Based MLVA Assay 40
Calculation of VNTRs and MLVA Data analysis 42
Phylogenetic analysis 42
SPA typing 42
Multilocus sequence typing (MLST) 44
DNA Sequencing Procedure 46
DNA Ladder 47
Solutions and Buffers 47
Statistical and Sequence Analysis 49
CHAPTER 3
Results 50
Staphylococcus aureus identification by 16S-rRNA Gene 50
MRSA Identification by mecA Gene 55
Detection of Panton Valentine Leukocidin Gene in MRSA isolates
from Pakistan
61
Multilocus variable Number of Tandem Repeats Analysis (MLVA)
of MRSA Isolates from Pakistan
64
Lineage Determination by Using RM Test 81
Spa Sequence Typing of Pakistani MRSA Isolates 90
Multilocus Sequence Typing of Staphylococcus aureus 94
iv
Analysis of STAR Element of Staphylococcus aureus isolates from
Pakistan 104
CHAPTER 4 124
Discussion 124
Recommendations 132
Future Directions 133
CHAPTER 5 134
REFERENCES 134
APPENDIX-I 162
APPENDIX-II 182
v
ACKNOWLEDGEMENTS
Praise be to Thee! We do not know anything except what Thou hast made
known to us: indeed Thou art the best Knower, the Wisest (Al-Quran).
I am greatly thankful to Almighty Allah who has enabled me to undertake this task
and complete it successfully. The writing of a dissertation can be a lonely and
isolating experience, yet it is obviously not possible without the personal and
practical support of my Supervisor, parents, my friends, colleagues, lab fellows, other
associates and staff.
I am most indebted to my Supervisor Dr. Salman Akbar Malik, Professor and former
Chairman Department of Biochemistry, Faculty of Biological Sciences (FBS) Quaid-
i-Azam University (QAU), for his affectionate guidance, encouraging behavior,
constant support and advice.
I am also thankful to Dr. M. Fayaz Chaudry, former Dean FBS, Professor Dr.
Waseem Ahmed and Professor and Chairperson Dr. Bushra Mirza for their co-
operation during my research work.
My research work for this dissertation was made more efficient because of Dr.
Christopher D. Bayliss and Dr. Julie Morrissey, Department of Genetics, University
of Leicester, UK. Thus I gladly express my gratitude to Dr. Christopher D. Bayliss
and Dr. Julie Morrissey for their guidance and provision of laboratory and sequencing
facility while I was in University of Leicester, UK. I am also thankful to my lab
fellows Dr. Miranda Johnson, Dr. Juanna Purvees. Dr. Randeep Sandhu, Dr. Alex
Woodcare and Mr. Isfahan Touseef for their kind support and cooperation during my
stay in University of Leicester, UK. I am also thankful to Deleo FR, for providing me
the picture showing the position of genes associated with methicillin.
I am also thankful to my Lab fellows Dr. Waseem Shehzad Abasi, Dr. Elahi Bukhsh,
Dr. Lubna Khatoon, Dr. Imran Qadir, Ammad Farooqi, Zahid Majeed and all others
who bestowed me with their sincere and valuable suggestions.
vi
I owe to my warmest gratitude to all of my family members. I am immensely thankful
to my elder sister, brothers, Nazar Hussain, Khan Badshah and their wives for their
all around unconditional support throughout my life.
I have no words to express my feelings of gratitude to my wife, Uzma Noureen for all
of her sacrifices and untiring support. She has helped me to undertake this work and
continuously supported me in high and lows. I cannot forget to mention my son
Muhammad Ahmad Shahweiz, Muhammad Abdullah Arfat and my cute and loving
daughter Aleena Fatima for their love and freshening smiles that kept me alive. I am
also thankful to my in laws for their prayers and continued support.
I extend my sincere gratitude to my friends and colleagues Prof. Ghulab Khan, Prof.
Mehboob Hussain, Prof. Abdul Ghazafar, Prof. Najm-ul-Hassan, Dr. Ghazanfar Ali,
Dr. Salman Chishti and Dr. Peter John.
I appreciate Farooq Ahmed, Noor Habib, Amjad Ali and Tariq Mahmood at
Biochemistry Department, QAU for their support and cooperation.
I am indebted to Higher Education Commission (HEC), Government of Pakistan for
awarding me scholarship under the indigenous Ph.D. fellowship program and also
financing my foreign research training International Research Support Initiative
Program.
Finally, I extend my deepest gratitude to my respected Late Father for his inspiration
and struggle for us and my loving mother who has always prayed for my success.
Yasrab Arfat
vii
Abbreviations aroC Carbamate Kiase
aroE Shikimate Kinase
BHI Agar Brain Heart Infusion Agar
BHI Broth Brain Heart infusion Broth
Bla Beta-lactamase
Bp Base Pairs
BURST Based Upon Related Sequence Type
CA-MRSA Community Acquired Methicillin Resistant Staphylococcus aureus
CC Clonal Complex
Chp Conserved Hypothetical Region
CTAB Cetyl Trimethyl Ammonium Bromide
EDTA Ehylene Diamine Tetra Acetate
EtBr Ethidium Bromide
IgG Immunoglobulin G
GC Guanine Cytosine
glpF Glycerol Kinase
g Gram
agr Accessory Gene Regulator
gmk Guanylate Kinase
gapR Glycolytic Operon Regulator
geh Glycerol Ester Hydrolase
HA-MRSA Hospital Acquird Methicillin Resistant Staphylococcus aureus
IAA Isoamylalcohol
Ica N-glycosyl transferase
IPA Isopropanol
ml Milliliter
MLEE Multilocus Enzyme Electrophoresis
MLVA Multilocus variable number of tandem repeats analysis
MRSA Methicillin Resistant Staphylococcus aures
viii
Abbreviations MLST Multilocus Sequence Typing
MT MLVA Type
PCR Polymerase Chain Reaction
pta Phosphate Acetyl Transferase
RAPD Randomly Amplified Polymorphic DNA
REA Restriction Enzyme Analysis
RM Restriction Modification
SAR Staphyloccal Accessory Regulator
SDS Sodium Dodecyl Sulphate
S.aureus Staphylococcus aureus
S.epidermidis Staphylococcus epidermidis
SPA Staphylococcus aureus Protein A
ST Sequence Type
STAR Staphylococcus aureus repeats
UPGMA Unweighted Pair Group Method with arithmetic Mean
uvrA Exonuclease ABC subunit A
VNTR Variable Number of Tandem Repeats
TAE Tris Acetic Acid EDTA
TBE Tris Borate EDTA
TE Tris EDTA
Tpi Triose Phosphate Isomerase
TSST Toxic Shock Syndrome Toxin
ET Exofolian Toxin
VNTR Variable number of tandem repeats
Yqil Acetyl Coenzyme A Acetyl Transferase
ix
LIST OF FIGURES
Figure No. Title Page No.
1 Gram stain of Staphylococcus aureus in pustular exudates. 3
2 Sites of Infection and Diseases caused by S. aureus in Humans.
5
3 Virulence determinant of Staphylococcus aureus and their target cell in host.
9
4 Chromosome of MRSA showing position of genes associated with methicillin.
14
5 Primer binding positions and predicted product sizes. 26
6 Indicating spa gene repeats sequence and repeat IDs of spa type t253
28
7 Norgen Full Ranger 100bp DNA Ladder and Ø×174 RF DNA Hae III Ladder is shown.
48
8 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of 16S-rRNA Gene for identification of Staphylococcus aureus.
51
9 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of 16S rRNA Gene for identification of Staphylococcus aureus.
53
10 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of 16S rRNA Gene for identification of Staphylococcus aureus.
53
11 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of 16S rRNA Gene for identification of Staphylococcus aureus.
54
12 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of 16S rRNA Gene for identification of Staphylococcus aureus.
54
13 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of 16S rRNA Gene for identification of Staphylococcus aureus.
56
x
LIST OF FIGURES
Figure No. Title Page No.
14 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of mecA gene for the identification of methicillin resistant Staphylococcus aureus.
57
15 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of mecA gene for the identification of methicillin resistant Staphylococcus aureus.
59
16 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of mecA gene for the identification of methicillin resistant Staphylococcus aureus..
60
17 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of mecA gene for the identification of methicillin resistant Staphylococcus aureus..
60
18 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of mecA gene for the identification of methicillin resistant Staphylococcus aureus.
62
19 Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing the PVL PCR Results with Pakistani MRSA isolates.
63
20 Showing MLVA results of ClfB, sdrD, spa sspa with thirty Pakistani MRSA strain.
65
21 Showing MLVA results of ClfA, sdrC, sav1078 with Pakistani MRSA strain.
67
22 Showing MLVA results of ClfB, sdrD, spa and sspa with a representative Pakistani MRSA strain P.843.
69
23 Showing MLVA results of ClfB, sdrd, spa and sspa with a representative Pakistani MRSA strain P.2285.
70
24 Showing MLVA results of ClfB, sdrD, spa and sspa with a representative Pakistani MRSA strain A. 9313.
71
25 Showing MLVA results of ClfB, sdrd, spa and sspa with a representative Pakistani MRSA strain P.1.
72
26 Minimum spanning tree of 123 Pakistani MRSA isolates derived from VNTRs for clfA, clfB, sdrD, sdrC, spa and sspa
75
xi
LIST OF FIGURES
Figure No. Title Page No.
loci using Bionumerics software.
27 Dendrogram base on clustering with respect to MLVA types of Pakistani MRSA Isolates with similarity calculated by Dice coefficient and represented by UPGMA.
79-80
28 RM tests with Pakistani Staphylococcus aureus (MRSA) isolates.
82
29 RM3 test Results with Pakistani Staphylococcus aureus (MRSA) isolates.
85
30 RM3 test Results with Pakistani Staphylococcus aureus (MRSA) isolates.
86
31 RM3 test Results with Pakistani Staphylococcus aureus (MRSA) isolates.
87
32 RM tests with Pakistani MRSA P.1286 and P.16809. 88
33 RM3 test Results with Pakistani Staphylococcus aureus MRSA isolates.
89
34 Showing spa X-region sequences of a representative MRSA isolate A.14256.
95
35 Showing spa X-region sequences of a representative MRSA isolate P.2113.
96
36 Showing spa X-region sequences of a representative MRSA isolate A.9313.
97
37 spa X-region sequences of a representative MRSA isolate A.13282.
98
38 Showing spa X-region sequences of a representative MRSA isolate P.1286.
99
39 Showing spa X-region sequences of a representative MRSA isolate A.26163.
100
40 Showing spa X-region sequences of a representative MRSA isolate P.1287.
101
41 Electropherogram of an ethidium bromide stained 1.5% agarose gel showing size of GapR Upstream non coding
109
xii
LIST OF FIGURES
Figure No. Title Page No.
Region having STAR Repeat element.
42 Electropherogram of an ethidium bromide stained 1.5% agarose gel showing size of GapR Upstream non coding Region having STAR REPEAT element.
111
43 Electropherogram of an ethidium bromide stained 1.5% agarose gel showing size of uvrA-hprK intergenic Region having STAR REPEAT element in the Representative Pakistani MRSA isolates.
118
44 Electropherogram of an ethidium bromide stained 1.5% agarose gel showing size of uvrA-hprK intergenic Region having STAR REPEAT element in the Representative Pakistani MRSA isolates.
119
45 A representative Electropherogram of an ethidium bromide stained 1.5% agarose gel showing size of ica-geh intergenic region of five strains having STAR element.
120
46 A representative Electropherogram of an ethidium bromide stained 1.5% agarose gel showing size of ica-geh intergenic region of sixteen representative strains having STAR element.
122
47 Median Joining Tree drawn by using Network 4.5 for the STAR present in the intergenic region of three loci Chp-gapR, Uvr-hprk and Ica-geh.
123
48 Showing Sequence data of arcC gene locus of S. aureus strain P.2113 in fasta format.
182
49 Showing Sequence data of aroE gene locus of S. aureus strain P.2113 in fasta format.
182
50 Showing Sequence data of glp gene locus of S. aureus strain P.2113 in fasta format.
183
51 Showing Sequence data of gmk gene locus of S. aureus strain P.2113 in fasta format.
183
52 Showing Sequence data of pta gene locus of S. aureus strain P.2113 in fasta format.
184
53 Showing Sequence data of tpi gene locus of S. aureus strain 184
xiii
LIST OF FIGURES
Figure No. Title Page No.
P.2113 in fasta format.
54 Showing Sequence data of yqil gene locus of S. aureus strain P.2113 in fasta format.
185
55 Showing Sequence data of arcC gene locus of S. aureus strain A.13282 in fasta format.
186
56 Showing Sequence data of aroE gene locus of S. aureus strain A.13282 in fasta format.
186
57 Showing Sequence data of glp gene locus of S. aureus strain A.13282 in fasta format.
187
58 Showing Sequence data of gmk gene locus of S. aureus strain A.13282 in fasta format.
187
59 Showing Sequence data of pta gene locus of S. aureus strain A.13282 in fasta format.
188
60 Showing Sequence data of pta gene locus of S. aureus strain A.13282 in fasta format.
188
61 Showing Sequence data of yqil gene locus of S. aureus strain A.13282 in fasta format.
189
62 Showing Sequence data of arcC gene locus of S. aureus strain P.1286 in fasta format.
190
63 Showing Sequence data of aroE gene locus of S. aureus strain P.1286 in fasta format.
190
64 Showing Sequence data of glp gene locus of S. aureus strain P.1286 in fasta format.
191
65 Showing Sequence data of gmk gene locus of S. aureus strain P.1286 in fasta format.
191
66 Showing Sequence data of pta gene locus of S. aureus strain P.1286 in fasta format.
192
67 Showing Sequence data of tpi gene locus of S. aureus strain P.1286 in fasta format.
192
68 Showing Sequence data of yqil gene locus of S. aureus strain P.1286 in fasta format.
193
xiv
LIST OF TABLES
Table No. Title Page No.
1 Oligonucleotide Primers used in RM typing 38
2 RM typing Product sizes for different Clonal Complexes 38
3 Oligonucleotide Primers used in Typing of Staphylococcus aureus for STAR Element.
41
4 Oligonucleotide Primers used in MLVA Typing of Staphylococcus aureus
43
5 Oligonucleotide primers used in MLST typing. 45
6 Showing the Spa sequence repeat profile obtained from Ridom spa server after sequencing of selected 26 MRSA isolates from Pakistan.
92
7 Multi locus Sequence Typing profiles and Clonal Complexes of MRSA isolates from Pakistan.
103
8 Abundance of individual STAR element repeat motifs and STAR element loci in different Staphylococcal genomes
105
9 Amplicon sizes and abundance of STAR motifs present in intergenic region of CHP-gapR, uv-hprk and ica-geh of ten sequenced S. aureus strains.
107
10 STAR element repeats of the representative sequenced Pakistani MRSA Isolates.
108
11 Amplicon sizes of CHP-gapR, Uvr-hprk and Ica-geh intergenic region containing STAR element of MRSA isolates from Pakistan.
112
12 Amplicon sizes of Chp-gapR, Uvr-hprk and Ica-geh intergenic region containing STAR element of MRSA isolates form Pakistan.’
115
13 Pakistani MRSA Isolates obtained from Hospitals of Rawalpindi/Islamabad.
162
14 Showing the amplicon sizes for the 123 Pakistani MRSA Isolates and NCTC 8325 and Mu50 (Positive control strains) obtained after Gene Scan for the clfA, clfB, sdrD,sdrC,spa sspa and sav1078 loci.
164
xv
LIST OF TABLES
Table No. Title Page No.
15 Showing the IDs of isolates, amplicon sizes, length of VNTR region present in these amplicon, total number of repeats for the 123 Pakistani MRSA isolates and NCTC 8325 and Mu50 (Positive control strains) obtained after Gene Scan for the clfA, clfB, sdrD, sdrC, spa and sspa.
168
16 Showing the Spa sequencing results with selected 26 MRSA isolates from Pakistan.
175
xvi
ABSTRACT
Methicillin resistant S. aureus (MRSA) is a versatile and dangerous human’s
pathogen and is a common cause of nosocomial and community acquired infections.
S. aureus causes several types of infections such as bacteremia, folliculitis, sepsis,
mastitis, meningitis and toxic shock syndrome and staphylococcal pneumonia. S.
aureus has developed resistance to the antibiotic ‘methicillin’ and continued spread of
methicillin-resistant S. aureus (MRSA) strains poses a significant risk to patients and
contributes to a substantial financial burden on healthcare resources. HA-MRSA
isolates usually belong to six lineages (CC1, CC5, CC8, CC22, CC30 and CC45) out
of ten dominant clonal complexes (CCs) or lineages. Various methods have been
employed to identify and characterize S. aureus strains. Phenotypic techniques have
been replaced by more robust and accurate molecular techniques. The commonly
used molecular techniques are Pulse Field Gel Electrophoresis (PFGE), Multilocus
variable number of Tandem repeat analysis (MLVA), Restriction modification tests
(RM), multilocus sequence typing (MLST) and spa typing.
Present work focuses on genotyping of clinical MRSA isolates from three tertiary
care hospital located in “Rawalpindi/Islamabad”, in order to examine the types and
phylogenetic relationship of the isolates. In order to gain the understanding of the
distribution of MRSA clones in Pakistan, where unregulated antibiotic use is
widespread and distributions of MRSA is supposed to be high, an epidemiological
relationships between 123 methicillin resistant Staphylococcus aureus strains,
isolated between 2006 and 2008 from three tertiary care hospitals of Rawalpinidi and
Islamabad, were examined using MLVA scheme, combined with RM typing, PVL
sceening, STAR element analysis, spa typing and MLST to investigate the
phylogenetic relationships of the Pakistani MRSA isolates. Six loci (clfA, clfB, sdrC,
sdrD, spa and sspa) were used in a multilocus variable number tandem repeat
analysis (MLVA). A total of 63 MTs/haplotypes were obtained by MLVA. Analysis
of restriction modification (RM) genes detected, an RM3 type, associated with CC8,
in most strains and an RM1 type, associated with CC30, in only two strains. On
xvii
further typing of selected strains by Spa typing and MLST, it was found that the
RM3/CC8 isolates were ST113-t064, ST113-t451 or ST239, with one of four spa
types, whilst the RM1/CC30 isolates were ST30-t021. Analysis of STAR element of
these strains for three loci showed their close resemblance; only the strains belonging
to CC30 showed no STAR motif in gapR upstream region, confirming their genetic
homology to other CC30 strains. Furthermore, the ST30 strains were also found
positive for PVL gene.
The present genotypic study showed that in Pakistan, the isolates belonging to clonal
complex eight (CC8) are dominant in clinical settings. They belong to ST239, ST113
or ST8. The other clonal complex found was CC30 with presence of PVL gene and
isolates belong to ST30. The use of MLVA in resource poor laboratories as a rapid
and robust method for grouping noscomial MRSA isolates into clusters for
identification of localized outbreaks is quite fruitful and MLVA may also provide an
understanding of the evolutionary processes as changes in the number of repeats at
different loci, may be indicative of which loci are prone to natural selection resulting
in higher levels of variation, thus VNTRs serve as evolutionary clock for
investigating an outbreaks and transmission events. In this study, we observed more
variation in clfA and clfB than in sdrC, sdrD, spa and sspa. We also found that a
change in repeat number was not necessarily gradual but may have occurred as a
result of large jumps. Some isolates with significant differences in repeat numbers at
single locus but being identical numbers in all other loci. Further evidence is provided
by the spa typing results, i.e. with a loss of four repeats resulting in a shift from t987
to t030 and a two repeat difference changed t021 to t275. These large jumps might be
due to deletions or insertions mediated by recombination or as result of deletions due
to slip-strand impairing during DNA replication.
Thus MRSA infections have become a challenge across the globe. The MRSA
isolates which were once endemic to Europe and America, Africa has now been
reported from Asia and thus suggesting that the MRSA isolates which once was
endemic to a certain geographical area are no more confined to those boundaries.
More over the pandemic spread of one type of MRSA clone across the globe is the
result of antibiotic resistance. Therefore a joint global effort will be effective for the
xviii
control of MRSA infection. Although this study was carried out on limited number of
isolated but it is quite useful to strengthen the MRSA data in Pakistan and to develop
the genetic profile of MRSA in Pakistan and then to link it globally. This study also
helped to under stand that, although there are only two lineages in these hospitals as
in most of other Asian countries but there is a diversity at subspecies level as some of
the isolates assumed a specific genetic profile as they evolved locally after they were
imported to this region.
The work presented in the thesis has been published in the following articles:
1- 2- Arfat Y1*, Johnson M2, Malik SA1, Morrissey J2 and Bayliss CD2 (2013).
Epidemiology of Methicillin Resistant Staphylococcus aureus (MRSA) Isolates
from Pakistan. African Journal of Microbiology Research; Vol.7 (7): 568-576.
H5 Index: 15.
2- Joanne Purvees1*, Mathew Blades2, Yasrab Arfat3, Salman A Malik1,
Christopher D Bayliss1 and Julie Morrissey1 (2012). Variation in the genomic
locations and sequence conservation of STAR elements among Staphylococcus
aureus species provides insight into DNA repeat evolution. Genomics; 13: 515.
Impact factor 4.07.
Chapter 1 Introduction
1
INTRODUCTION
Staphylococcus aureus
Staphylococci (staph) are Gram-positive spherical bacteria (1 micrometer in diameter)
that occur in microscopic clusters resembling grapes. The term Staphylococci was first
introduced by Ogston in 1881. Later in 1884, Rosenbach described two types of
Staphylococci, Staphylococcus aureus (Yellow Colony) and Staphylococcus albus
(White Colony) which is now named as Staphylococcus epidermidis. Although there are
more than twenty species have been identified but only Staphylococcus aureus and
Staphylococcus epidermidis are important in interaction with humans (Bergey’s Manual,
2001). Staphylococcus aureus colonizes mainly the nasal passages, but it may also
colonelize the skin, oral cavity and gastrointestinal tract while Staphylococcus
epidermidis is only the inhabitant of the skin (Dancer & Noble, 1991; Bannerman, 2003;
Garrity et al., 2004; Graham et al, 2006).
The cell wall of S. aureus is composed of numerous peptidoglycan layers which are
interspersed with teichoic acid and lipoteichoic acid (Giesbrecht et al., 1998). Inside the
cell wall is gelatinous periplasm prior to plasma membrane. Some strains also have a
shiny exopolysaccharide layer (Wilkinson, 1983). The genome size of different S. aureus
strains ranges from 2.820MB to 2.903MB (Baba et al., 2001; Holden et al., 2004; Gill et
al., 2005). The genome of S. aureus is co-linear and 95% of the genome consists of
coding sequences. Sequences of S. aureus can be classified into two types, the stable
sequences that are meant for long term selection and evolution, variable sequences that
are meant for acquisition virulence and resistance. The GC contents of S. aureus genome
range from 30 to 33% (Kuroda et al., 2001). Antibiotic resistance genes are present on
plasmids and other satellite structures. Virulence factors are encoded by the
staphylococcal cassette chromosome, Pathogenicity Island, plasmids and phages (Holden
et al., 2006; Baba et al., 2008). Apart from virulence factors antibiotic resistance is also
conferred by transposons (Foster, 1983; Lyon and Skurray, 1987).
Chapter 1 Introduction
2
Taxonomy of Staphylococcus aureus
The genus Staphylococcus is in the bacterial family Staphylococcaceae, which also
contains three lesser known genera, Gamella, Macrococcus and Salinicoccus. Taxonomic
classification of Staphylococcus aureus is as under as described by Kwok & Chow
(Kwok & Chow, 2003) and Garrity (Garrity et al., 2004).
Domain Bacteria
Kingdom Eubacteria
Phylum Firmicutes
Class Cocci
Order Bacillales
Family Staphylococcaceae
Genus Staphylococcus
Species aureus
The genus Staphylococcus consist of 32 species and seventeen out of the 32 are
frequently detected in clinical samples taken from human (Ryan et al., 2004).
Staphylococcus aureus forms a fairly large yellow colony on rich medium and is often
hemolytic on blood agar, however in case of toxic shock syndrome it act as non
hemolytic (Barbour, 1981; Chow et al., 1982). Staphylococci grow by aerobic respiration
or by fermentation that yields principally lactic acid, thus they are facultative anaerobes.
These bacteria are catalase-positive and oxidase-negative (Koneman et al., 1997).
Staphylococcus aureus can grow at a temperature range of 15 oC to 45 oC and tolerate as
high NaCl concentrations as 15 percent. The coagulase enzyme is produced by almost all
S. aureus strains but in the recent years coagulase negative S. aureus are also frequently
reported (Henderson and Brodie, 1963; Yoshida, 1970; Kloos & Jorgensen, 1985;
Landolo, 1990).
Diseases caused by Staphylococcus aureus
Staphylococcus aureus causes a variety of pus-forming infections and produces toxins in
Chapter 1 Introduction
3
Figure 1. Gram stain of Staphylococcus aureus in pustular exudates. (Text book of
Microbiology, Tudor, 2008)
Chapter 1 Introduction
4
humans [Fig.2] (Archer, 1998; Lowy, 1998). It also causes a variety of superficial skin
lesions (boils, styes, furuncules); serious infections (pneumonia, mastitis, phlebitis,
meningitis, urinary tract infections); and deep-seated infections (osteomyelitis and
endocarditis) (Klein et al., 2007). The major cause of hospital acquired infections of
surgical wounds and infections associated with indwelling medical devices is
Staphylococcus aureus. It also releases enterotoxins into food, thus causing food
poisoning and toxic shock syndrome by releasing super antigens into blood (Todd, 1978;
Shands et al., 1980; Dinges et al., 2000). In human, staphylococcal infections are
frequent, but mostly they remain localized at the portal of entry like hair follicle or
minute break in the skin or a surgical wound, due to normal host defense system. Some
times respiratory tract also serves as portal of entry which may lead to Staphylococcal
pneumonia. Inflammation, an elevated temperature at the site of infection, swelling,
accumulation of pus, and necrosis of tissue are host’s response to staphylococcal
infection. A fibrin clot may also form around the inflamed area, walling off the bacteria
and leukocytes, as a result a characteristic pus-filled boil, abscess or even serious skin
infections may occur, such as furuncles or impetigo and some time bone infection
osteomyelitis. Some times Staphylococcus aureus also invade the blood stream resulting
septicemia. Bacteremia may result serious consequences causing internal abscesses, other
skin lesions, or infections in the lung, kidney, heart, skeletal muscle or meningitis (del
Poso & Patel, 2007; Ashby et al., 2009).
Staphylococcus aureus Pathogenicity
Staphylococcus aureus pathogenicity is caused by many potential virulence factors.
Although the determination of role of a particular virulence factor in a disease is difficult
to identify but a correlation has been found between a particular disease and presence of a
specific virulence determinants, suggesting its role in that particular disease (Foster &
Hook, 1998). The Potential virulence factors can be classified as under:
Surface proteins
These proteins promote Staphylococcus aureus attachment to host proteins laminin,
fibronectin and form the extracellular matrix of epithelial and endothelial surfaces.
Chapter 1 Introduction
5
Figure 2. Sites of Infection and Diseases caused by S. aureus in Humans. (Text book of
Microbiology, Tudor, 2008)
Chapter 1 Introduction
6
Most strains also express a fibrin/fibrinogen binding protein (clumping factor) which
promotes attachment to blood clots and traumatized tissue. Majority of the
Staphylococcus aureus strains express both fibronectin and fibrinogen-binding proteins.
In strains that cause osteomyelitis and septic arthritis, an adhesin is present that promotes
attachment to collagen. Interaction with collagen is important in promoting bacterial
attachment to damaged tissue where the underlying layers have been exposed (Patti et al,
1994; Foster & Hook, 1998; Tung et al., 2000; Menzies, 2003).
Invasins
These extra cellular proteins promote bacterial spread in host tissues. Leukocidin, kinases
and hyaluronidase are the examples of invasions (Foster, 2005; Wang et al., 2007).
Staphylococcus aureus strains also express a plasminogen activator called staphylokinase
which lyses fibrin. Staphylokinase and plasminogen form a complex which activates
plasmin which causes dissolution of fibrin clots. Although there is no strong evidence
that staphylokinase is a virulence factor, but it seems reasonable to imagine that localized
fibrinolysis might aid in bacterial spreading (Chen et al., 2002).
Surface factors
Surface factors include capsule and protein A, which inhibit phagocytic engulfment by
the host immune cells. Most of the clinical isolates of S aureus express a surface
polysaccharide of either serotype 5 or 8 which is known as a microcapsule because it can
be visualized only by electron microscopy unlike the true capsules of some bacteria
which are readily visualized by light microscopy. Staphylococcus aureus strains isolated
from infections express high levels of the polysaccharide but loses when cultured in the
laboratory. The function of the capsule in virulence is not entirely clear but it hinders
phagocytosis in the absence of complement. Protein A is a surface protein of
Staphylococcus aureus which binds IgG molecules by their Fc region. In serum, the
bacteria will bind IgG molecules in the wrong orientation on their surface, which disrupts
opsonization and phagocytosis (Projan and Novick, 1997).
Chapter 1 Introduction
7
Immunological Disguises
These include Protein A, coagulase and clotting factor. Coagulase which is an
extracellular protein binds to prothrombin in the host to form a complex called
staphylothrombin. The protease activity of thrombin is activated in the complex, resulting
in the conversion of fibrinogen to fibrin. Coagulase test is used for identifying of S.
aureus in the clinical microbiology laboratory. But it is not clear that whether that it is a
virulence factor or not, but it is reasonable to speculate that the bacteria could protect
themselves from phagocytic and immune defenses by causing localized clotting
(Friedrich et al., 2006; Janwithayanuchit et al., 2006). Clumping factor is the fibrinogen-
binding determinant on the S. aureus cell surface. Genetic studies have shown that
coagulase and clumping factor are different entities. It has been observed that specific
mutants lacking coagulase retain clumping factor activity, while clumping factor mutants
express coagulase normally (Moreillon et al., 1995).
Membrane-Damaging Toxins
These are the proteins that lyse eukaryotic cell membranes include hemolysins,
leukotoxin, leukocidin (Patti et al., 1994). Alpha toxin is the best characterized and most
potent membrane-damaging toxin of Staphylococcus aureus. Alpha toxin is expressed as
a monomer and binds to the membrane of susceptible cells and then oligomerize to form
heptameric rings with a central pore through which cellular contents leak. Monocytes and
platelets are more vulnerable to α toxin (Bhakdi and Tranum-Jensen, 1991). ß-toxin
which damages membranes rich in lipid such as sphingomyelinase but the majority of
human isolates of Staphylococcus aureus do not express ß-toxin. Delta toxin produced by
most strains of S. aureus is a very small peptide toxin and it possesses detergent like
activity resulting in disruption of host membrane (Bladon et al., 1992). Leukocidin which
is a multicomponent protein toxin forms a hetero-oligomeric transmembrane pore
composed of four LukF and four LukS subunits, thus forming an octameric pore in the
affected membrane. Although only 2% of all of Staphylococcus aureus isolates express
leukocidin, but nearly 90% of the strains isolated from severe dermonecrotic lesions
express this toxin, which indicates that it is an important factor in necrotizing skin
infections (Gladstone and Van Heyningan, 1957, Foster, 1995).
Chapter 1 Introduction
8
Exotoxins
These proteins damage host tissues or otherwise provoke symptoms of disease (SE A-K,
TSST, and ET). There are eleven superantigen (SE A-K) are known to date for
Staphylococcal enterotoxins. New SE and SE like toxin which are heat stable are
reported. These are mostly produced in foods which are rich in proteins and
carbohydrates causing Staphylococcal food poisonings (Su and Wong, 1995; Jorrand et
al., 2001; Orwin et al., 2001). Toxic shock syndrome toxins (TSST) are released
systematically and cause toxic shock syndrome (TSS). Menstrual toxic shock syndrome
(75% cases) is caused by (TSST-1) (Murray et al., 1996). Exofoliation toxins are the
cause of scalded skin syndrome in new born babies and they are present in 5-10% of
clinical S. aureus isolates.
Antibiotic Resistance in Staphylococcus aureus
Hospital strains of S. aureus are usually resistant to a variety of different antibiotics. A
few strains are resistant to all clinically useful antibiotics except vancomycin (Chamber
and Deleo, 2009). Methicillin resistance is widespread and most methicillin-resistant
strains are also multiple drug-resistant. In addition, S. aureus exhibits resistance to
antiseptics and disinfectants, such as quaternary ammonium compounds, which may aid
its survival in the hospital environment. In the case of HA-MRSA, patients who already
have an MRSA infection or who carry the bacteria on their bodies but do not have
symptoms (are colonized) are the most common sources of transmission. MRSA
infections that occur in otherwise healthy people who have not been recently (within the
past year) hospitalized or had a medical procedure (such as dialysis, surgery, catheters)
are categorized as community-associated (CA-MRSA) infections. These infections are
usually skin infections, such as abscesses, boils, and other pus-filled lesions. About 75
percent of CA-MRSA infections are localized to skin and soft tissue and usually can be
treated effectively. However, CA-MRSA strains display enhanced virulence, spread more
rapidly and cause more severe illness than traditional HA-MRSA infections, and can
affect vital organs leading to widespread infection (sepsis), toxic shock syndrome and
pneumonia. It is not known why some healthy people develop CA-MRSA skin infections
Chapter 1 Introduction
9
Figure 3. Virulence determinant of Staphylococcus aureus and their target cell in host.
(Text book of Microbiology, Tudor, 2008)
Chapter 1 Introduction
10
that are treatable whereas others infected with the same strain develop severe, fatal
infections. Drug resistance has developed in the staphylococci within a very short time
after introduction of antibiotic penicillin in the 1940's. Penicillinase-producing
Staphylococcus aureus was first described by Kirby in 1944. Penicillin became widely
available after the end of World War II, and the prevalence of Penicillin-resistant
Staphylococcus aureus began to rise, such that by 1948, most hospital isolates were
resistant to penicillin (Barber and Dowzenko, 1948). In contrast to hospital situation in
the 1950s community strains of Staphylococcus aureus were largely susceptible to
penicillin, and penicillin remained the drug of first choice for the strains until the early
1970s (Weinstein, 1975)
Methicillin and related compounds were developed in response to the emergence of
penicillin resistance Staphylococcus aureus. Methicillin resistance in Staphylococcus
aureus was reported almost immediately after its use. Methicillin resistance in
Staphylococcus aureus was first reported in 1961 from the UK, by Jevons. MRSA was
also reported in Poland by Borowski et al ( Borowski et al., 1964), and MRSA for the
first time reported in the United States in 1968 by Barrett (Barrett et al., 1968) and in
Turkey in 1962 by Cetin & Ang (Cetin & Ang, 1962) and in India by Pal and Ghosh in
1964 (Pal and Ghosh in 1964), while the data with respect to Pakistan that when the first
case of MRSA was found, is not available but in one study by Hafiz et al has found that
the frequency of Methicillin resistant Staphylococcus aureus (MRSA) in different
hospitals was varying between 2-61% (Hafiz et al., 2002) and in another study Butt et al
has found that the frequency of MRSA among nosocomial isolates of S. aureus increased
from 39% in 1996 to 51% in 2003 (Butt et al., 2004).
Mechanism of Antimicrobial resistance in Staphylococcus
aureus
Staphylococcus aureus can be resistant to beta lactams via several mechanisms, a few of
them are described here.
Chapter 1 Introduction
11
Production of beta lactamase (Penicillinase)
The production of Beta lactamase results in resistance to penicillin (Penicillin
Resistance). Beta lactams are known to bind to cell wall proteins called penicillin binding
proteins (PBPs) which polymerize the catalization and cross linking of peptidoglycan in
the bacterial cell wall (Berger-Bachi, 1997). Beta lactams are substrate analogue that
covalently bound to PBP active site serine, inactivating the enzyme (Chambers, 1997a).
Penicillin resistance in Staphylococcus aureus is mediated by the production of beta
lactamase (Richmond, 1965). A system of genes concerned with beta-lactamase
production in S. aureus has been identified. Regulatory genes include blal which code for
a repressor protein (Richmond, 1967) and blaR2 which code for a protein which act as
intracellular inducer (Cohen and Sweeney, 1968). blaZ code for beta lactamases itself.
BlaZ, blal and blaRl are found on large plasmids, but blaR2 is always chromosomal.
Beta-lactams bind to the C terminus of blaRl, resulting in signal being passed to blal,
causing it not to bind to the operator region and as a result transcription of blaZ and
blaR1 and blal increases, resulting in an increased synthesis of beta lactamase and
regulatory proteins (Dyke and Gregory, 1997).
Production of an altered penicillin binding protein (PBP 2’ or PBP2a)
The production of an altered penicillin binding protein results in resistance to all beta
lactams including methicillin in Staphylococcus aureus (Ubukata et al., 1989; Hackbarth
and chambers, 1993). The regulatory genes bla Rl and bla1 affect synthesis of PBP2a.
The altered Penicillin binding protein mecA gene coding for PBP2a, could be derived
from a fusion product of upstream region of blaZ with a normal penicillin binding protein
gene (Song et al., 1987).
Hyperproduction of beta-lactamase or methicillinase
The mechanism of resistance in border line Staphylococcus aureus is either by the
production of beta lactamase (methicillinase) with a higher affinity for methicillin
(Massida et al., 1992) or by hyper production of beta lactamase (Mc Dougal and
Thornsberry, 1986). In the case of border line Staphylococcus aureus, production of this
enzyme is inducible and the enzyme has molecular weight slightly lower than the plasmid
Chapter 1 Introduction
12
encoded penicillinase (Barg et al., 1991; Varaldo, 1993b; Massida et al., 1994). Detection
of mecA with PCR distinguishes MRSA strain from border line Staphylococcus aureus
(Bignandri et al., 1996) as does the disc diffusion if 5ug methicillin disc on agar with
0.5% NaCl and inoculated with 5x 105 organisms and incubated at 30 oC (Petersson et al.,
1999).
Production of modified penicillin binding protein other than PBP2a
S. aureus has raised resistance to methicillin by point mutations in the penicillin binding
domains of PBPs 1, 2 and 4, which results in reduction in affinity for beta-lactams
(Berger Bachi et al., 1986). These altered penicillin binding PBPs binds penicillin more
slowly and release penicillin more rapidly (Chambers et al., 1994). These binding
alterations are due to point mutations (Hachbarth et al., 1995). These strains are referred
as modified S. aureus (Berger-Bachi, 1997).
The unknown mechanism exhibited by small colony variants
Small colony variants appear as minute, colorless and slow growing colonies on agar
plates and often mistaken as coagulase negative Staphylococci (Proctor et al., 1995). It
has been observed that their production is inducible by exposure to antibiotics such as
beta lactams, gentamycin, quiniolones and cotrimoxazole (Anear and Grubb, 1973;
Pelleiter et al., 1979; Mitsuyama et al., 1997; Proctor and Peter, 1998). Most of the small
colony variants have defective electron transport chain and can not take cationic
antibiotics and show more resistance to methicillin. Their frequent isolation from patient
with persistent relapsing infection indicates that invitro resistance is clinically significant
(Proctor et al., 1994).
Methicillin Resistant Staphylococcus aureus
Methicillin Resistant Staphylococcus aureus are resistant to the action of methicillin and
related beta-lactam antibiotics (e.g. penicillin, oxacillin, amoxacillin). MRSA have
evolved resistance not only to beta-lactam antibiotics, but to several classes of antibiotics.
Although methicillin-resistant Staphylococcus aureus (MRSA) is causing infections in
hospital settings for several decades, MRSA strains have recently emerged outside the
hospital and known as community associated-MRSA (CA-MRSA) or superbug strains of
Chapter 1 Introduction
13
the organism, which now account for the majority of staphylococcal infections HA-
MRSA occurs most frequently among patients who undergo invasive medical procedures
or who have weakened immune systems and are being treated in hospitals and healthcare
facilities such as nursing homes and dialysis centers.
MRSA strains possess a 30-50kb segment of chromosomal DNA not found in MSSA
strains (Ito and Hiramatsu, 1998, Ito and Hiramatsu et al., 1999; Ito et al., 1999;
Katayama et al., 2000). The segment of DNA has various synonyms: mec determinant,
mec, staphylococcal cassette chromosome mec (SCCmec), and mec associated DNA.
SCCmec, is always located near to pur-nov-his gene cluster on Staphylococcus aureus
chromosome (Kuhl et al., 1978), and between spa (encoding protein A) and purA
(encoding for adenine requirement) (Berger-Bachi, 1997). SCC mec contains mecA, and
usually the regulatory genes mecl and mecRl. Only the presence of mecA is required for
methicillin resistance as demonstrated by cloning experiments in which cloned mecA was
introduced into a methicillin susceptible strain of S. aureus, resulting in expression of
PBP2a in the recipient strains and being resistant to methicillin (Tech et al., 1988; Inglis
et al., 1988).
MRSA strains are resistant to all beta lactam antibiotics (Penicillins, cephalosporin,
monobactams and carbapenems) due to the presence of altered PBP in cell wall known as
PBP2a which is the product of the mecA gene (Ubukata et al., 1989a). PBP2a is a 76KDa
protein with the characteristics of a membrane bound PBP, having a putative
transglycosylaze domain and the characteristic conserved motifs of a transpeptidase. It
probably acts as trans-peptidase taking over the function of other PBPs when they are
inhibited by beta lactams (Reynolds and Brown, 1985; Wu et al., 1994). PBP 2a can
substitute for the essential fuctions of the high affinity PBPs 1, 2 and in concentration of
beta-lactams that would otherwise be lethal (Chambers, 1997a)
SCCmec contains up to 100 open reading frames (Beck et al., 1986; Hiramatsu et al.,
1966; Mathew et al., 1987). MecA is always present, but mecl, and mecRl, which are
mecA regulatory genes, may or may not be present. Mecl and mecRl are located
upstream of mecA [Fig.4] and are separated from mecA by its promoter and operator
(Hiramatsu et al., 1992a; Tech et al., 1990).
Chapter 1 Introduction
14
Figure 4 Chromosome of MRSA showing position of genes associated with methicillin.
(Natalia Malanchowa & Deleo FR, 2010).
Chapter 1 Introduction
15
Transposons and insertion sequences are present, such as Tn554 located 5’ of mecA, 1-4
copies of IS431, at least one of which IS431 is located 3’ of mecA (Archer and
Niemeyer, 1994; Hiramatsu et al., 1996). Deletion rearrangement and recombination
commonly occur between mecA and IS431. IS431 is a common staphylococcal
chromosomal and plasmid insertion sequence, and it is associated with various resistance
determinants and probably explains the propensity for MRSA strains to become
multidrug resistant (Chambers, 1997).
MecA is highly conserved amongst S. aureus and co-agulase negative staphylococci (
Bech et al., 1986; Chambers, 1987; Ubukata et al., 1990; Wu et al., 1992; Arsher et al.,
1994). The following genes involved in beta-lactamase production and methicillin
resistance respectively and have sequence similarities; BlaZ and the first 300 nucleotides
of mecA, blaRl and mecRl and blal and mecl (Matsuhashi et al., 1980; Song et al., 1987).
Mecl and mecRl, regulatory genes located upstream of mecA promoter (Tech et al.,
1990; Hiramatsu et al., 1992) and possess similar molecular organization, function and
regulation to the beta lactamase regulatory genes blal and blaRl respectively (Berger-
Bachi, 1997).
It was found that strains possessing functional mecl and mecRl genes exhibit strong
suppression of mecA transcription, and require induction by methicillin to express mecA,
produce PBP2a and exhibit methicillin resistance (Ryffel et al., 1992). MRSA strains can
possess mecI and mecRI sequences yet express PBP 2a constitutively and in high
amounts. These strains usually have point mutations or deletions inactivating mecl
(Suzuki et al., 1993). Strains isolated since 1980 generally do not have deletion of
regulatory genes but do exhibit mecl polymorphism and mecA promoter mutations
(Hiramatsu et al., 1996). Intact regulatory genes strongly suppress expression of mecA.
Such strains can appear methicillin-susceptible on the bench. Insertional inactivation of
mecl and mecRl results in homogenous methicillin resistance (Ryffel et al., 1992;
Kuwahara-Arai et al., 1996; Niemeyer et al., 1996).
Genes other than mecl and mecRl affect transcription of mecA. The same mecl and
mecRl elements appear to be associated with different methicillin resistance phenotypes
in different strains of MRSA (Niemeyer et al., 1996). There is also no clear relationship
Chapter 1 Introduction
16
between the amount of PBP2a produced and whether resistance is constitutive or
inducible or homogeneous or heterogeneous. It seems likely that the mecl polymorphism
and mecA promoter mutation reflect the selective pressure of betalactams for mutants
lacking strong repressor activity, so that the amount of PBP2a produced will confer a
survival advantage. Both SCCmec and beta lactamases regulator genes control PBP 2a
production in these strains (Hackbarth and Chambers, 1993).
Factors affecting Methicillin Resistance
There are many factors that appear to affect the methicillin resistance phenotype. These
factors include the beta lactamase gene, the plasmids encoding staphylococcal beta
lactamase, fem factors, llm, agr, and other chromosomal loci.
Effect of Beta lactamases genes on methicillin resistance
Blal and blaR1, having great structural and functional similarities to mec1 and mecR1
respectively, can also regulate the expression of PBP2a (Hackbarth and Chambers, 1993;
Ubukata et al., 1989), i.e. the production of PBP 2a will be inducible. Repression by bla1
is weaker than mec1, some PBP2a is produced by the uninduced strain, and induction by
methicillin is as rapid as beta lactamase induction (Ryffel et al., 1992). It has been
postulated that as blaR2 interferes with the bla regulation, it may also interfere with the
regulation of mecA (Cohen and Sweny, 1968). If the strain lacks bla1 and blaR1, then
PBP 2a production is constitutive. The interaction between mec1, mecR1, bla1 and blaR1
are complex and it effects the expression of methicillin resistance (Berger-Bachi, 1997).
Effect of Plasmids encoding Staphylococci beta-lactamase on methicillin
Resistance
There are several ways in which the presence of the plasmid encoding staphylococcal
betalactamases may effect the expression of methicillin resistance. This plasmid may
prevent spontaneous deletion of SCCmec, thereby increasing its stability and preventing
reversion to susceptibility (Hiramatsu et al., 1990). Introduction of a beta–lactamase
plasmid into homogeneously resistant recipient can result in heterogenous resistance
(Boyce et al 1990). Mutations altering the inducibility of beta-lactamases are associated
with reduced methicillin resistance (Cohen et al., 1972). Inactivation of blaR1 reduces
Chapter 1 Introduction
17
the expression of methicillin resistance (Hackbarth et al., 1994), presumably by the
unopposed action of bla1 on mecA. Strain with functional SCCmec regulatory genes
produce little PBP2a and are poorly inducible with many beta-lactams (Kuwahara-Arai et
al., 1996).
Effects of fem and aux factors on methicillin resistance
Fem (factors essential for methicillin resistance) and aux factors are chromosomal genes
and are essential for the full expression of methicillin resistance. Fem and aux genes are
also present in MSSA strains and represent house keeping genes. Although they are
required for the full expression of methicillin resistance but they are not the part of the
complex of genes conferring methicillin resistance (Berger-Bachi, 1983; Berger-Bachi,
1997; Murakami and Tomasz, 1989).
Other Chromosomal loci affecting the expression of methicillin
resistance
Other chromosomal loci affecting the expression of methicillin resistance include llm,
autolysis genes, agr (accessory gene regulator) and sar (staphyloccal accessory regulator).
llm inactivation results in conversion of homogenious resistance to heterogenous and an
increase in autolysis (Maki et al., 1994). Increased autolysis is seen in strains
demonstrating heterogeneous methicillin resistance (Qoronflesh and Wilkinson, 1986;
Chambers and Hackbarth, 1987; Gustafson and Wilkison, 1989). The agr and sar loci
control the expression of exoprotein including virulence factors. Inactivation of either
locus in heterogenous strains resulted in a minor decrease in the number of highly
resistant cells (Pirtz et al., 1996).
External factors affecting the expression of methicillin resistance
Temperature, osmolality, light, divalent cations concentration, presence of chelating
agents and an aerobiosis all affect all affect the expression of methicillin resistance
(Matthew and Stewart, 1984; Madiraju et al., 1987). Lowering the temperature and
increasing the concentration of sodium chloride enhance the expression of methicillin
Chapter 1 Introduction
18
resistance and are recommended in routine testing (National Committee for Clinical
Standards, 1999).
Heterogeneous and homogenous resistance to methicillin
It is significant as strains of S. aureus with heterogenous resistance may be falsely
classified as methicillin susceptible in routine laboratory tests (National Committee for
clinical laboratory Standards, 2000). Heterogeneous strains typically have >99.9% of the
cells susceptible to low concentration of methicillin (the basal resistance level) with a
small minority resistant to concentration >50mg/L, as determined by population analysis
profile (Chambers, 1997). Growth in hypertonic NaCl or incubation at 30°C can result in
strain appear in homogenous (>1% of cells growing at 50mg/L) (Sabat and Wallace
1974). Addition of EDTA or incubation at 37-43°C may suppress resistance entirely. The
population analysis profile is growth phase dependent. This suggests that the changes that
confer the high level resistance are cell cycle dependent (Seligman, 1969).
MSSA strains have slow inducibility of transcription of mecA if mecR1/mec1 is intact
and fully functional (Berger-Bachi, 1997). Repression of mecA by the bla1 can convert a
highly homogenously resistant MRSA strain into heterogeneously resistant strains if
blaR1 (the beta lactamase signal transducer for staphylococcal beta lactamase) is
inactivated (Hackbarth et al., 1994). This suggests that a certain threshold for PBP2a
production is needed for the expression of methicillin resistance.
Treatment against Staphylococcal Infections
Phagocytosis is the major mechanism for combating staphylococcal infection. Antibodies
are produced which neutralize toxins and promote opsonization. However, the bacterial
capsule and protein A may interfere with phagocytosis. Biofilm growth on implants is
also impervious to phagocytes. Staphylococci may be difficult to kill after phagocytic
engulfment because they produce carotenoids and catalase which neutralize single
oxygen and superoxide, which are primary phagocytic killing mechanisms within the
phagolysosome.
Hospital acquired infection is often caused by antibiotic resistant strains (e.g. MRSA) and
can successfully treated with vancomycin or an alternative. The community associated
Chapter 1 Introduction
19
staphylococcal infections are resistant to penicillins and cephalosporins. Therefore
infections have been treated with combination therapy using sulfa drugs and minocycline
or rifampin (Bermingham et al., 2003). A relatively newer drug, Linezolid belonging to
oxazolidinons class is effective against both CA-MRSA and HA-MRSA
(Mongkolrattanothai et al., 2003). Some glycopeptides and teichoplanin and
vancomycins are also effectively used to control MRSA infections (Schentag et al.,
1998). Linezolid, quinpristin/daflopristine, daptomycin and tigecyclin are used when
therapy fails with glycopeptides such as vancomycin (Mongkolrattanothai et al., 2003).
Phage therapy is also being tested that involves infecting and destroying MRSA with
phage particles. This approach has been proven to be effective against 95% of MRSA
isolates tested (Matsuzaki et al., 2003)
Vaccines
There is no such vaccine is generally available that stimulates active immunity against
staphylococcal infections in humans however a vaccine based on fibronectin binding
protein induces protective immunity against mastitis in cattles. Vaccine therapies are
relatively new approach in combating with S. aureus bacterial infections. Monoclonal
antibodies directed towards surface components (e.g. capsular polysaccharide or surface
protein adhesions) can be used to prevent bacterial adherence and promote phagocytosis
by opsonization of bacterial cells. These monoclonal antibodies can also be given to
hospital patients to create passive immunity during surgery (Cegelski et al., 2008; Ragle
and Wardenburg, 2009).
The vaccine called StaphVAX, composed of S. aureus type 5 and 8 capsular
polysaccharides conjugated to nontoxic recombinant Pseudomonas aeruginosa exotoxin
was introduced in 2002. The vaccine was administered to 892 hemodialysis patients and
reported to be safe and immunogenic for approximately 40 weeks in patients with end-
stage renal disease. Approximately 90% of patients who received the vaccine developed
antibodies to the two capsular polysaccharides than the control group who did not receive
the vaccine. A decrease in vaccine efficiency after week 40 correlated with a decrease in
S. aureus antibodies. Although the vaccine has limited time frame in protection against S.
aureus infections but it can be useful in cases where healthy individuals come into the
Chapter 1 Introduction
20
hospital for elective surgery, such as a joint replacement, organ transplant or coronary
bypass surgery. Such patients do not require protection for the rest of their lives; what
they need is protection for a short period while they are in the hospital (Cegelski et al.,
2008, Fattom et al., 2004).
Epidemiology of MRSA
Genotyping is the process of determining the genotype of an individual by the use of
different biological assays. Genotyping helps in controlling the spread of pathogens, by
tracing the origin of outbreaks. Genotyping is also known as molecular epidemiology or
forensic microbiology. Many outbreaks of infectious diseases are usually due to exposure
to organism from a common source. It is considered that organism in the affected
individual are highly related to each other. Bacterial typing system has been developed to
confirm this hypothesis and demonstrate that isolates from epidemiologically identical
cases are identical (Tenover et al., 1992).
Bacterial typing techniques can be classified into two main groups: phenotypic methods
and molecular methods (Arbeit, 1995). Phenotypic methods involve the detection of
features expressed by the organism. They are thus limited with capacity of organism to
change the expression of underlying genes. Genotyping involve the direct study of
genomic material and consequently all bacterial strains are typeable. The major
disadvantage is the cost (Tenover et al., 1994) and difficulties with interpreting results
(Tenover et al., 1995).
Phenotypic methods of strain differentiation
Antibiogram
Antibiogram is widely used typing technique which involves the testing of isolates
against antibiotics susceptibility following the Kirby Bauer Disk Diffusion Method. The
reagents and equipment are easily available and relatively cheaper. Normally the medium
used is Muller Hinton agar containing antibiotic erythromycin, Clindamycin,
cotrimoxazole, gentamycin and ciprofloxacin disk (Blanc et al., 1994). This method is
limited by phenotypic variation and antibiotic selection pressure. The same strain may
reflect a different antibiogram due to loss or gain of antibiotic resistance plasmid or
Chapter 1 Introduction
21
transposable genetic element. Thus results are not reproducible even for the same
isolates, making the method unreliable (Costas et al., 1989; Tenover et al., 1994).
Phage Typing
Phage typing was first developed by Fisk in 1942 (Fisk, 1942) and later on this method
was described in 1961 by Blair and William (Blair and William, 1961). As phages are
host specific and there is a narrow range of viruses which can be used to produce a
typical lyses pattern for a specific strain. There is a set of 23 phages recommended for
typing Staphylococcus. MRSA strains are not type able with these and supplementary
phages are used, consisting of international set of experimental phages for MRSA
(Blumberg et al., 1992; Richardson et al., 1999). Plaques i.e. lyses region by each phage
are recorded either as a weak or strong reaction. Strong reaction is interpreted as more
than fifty plaques and used as distinguishing character of isolates while the weaker
reaction is recorded but not counted toward final typing (Parker, 1972).
In certain outbreaks many strains were untypeable. Some strains of even diverse origin
grouped into the same cluster reflecting its low discriminatory power. (Harstein et al.,
1989; Dominguez et al., 1994; Vickery et al., 1997b). The technique is technically
demanding. (Hartstein et al., 1989; Tenover et al 1994; Arbeit, 1995) and results are
often non reproducible (Rhinehart et al., 1987).
Immunobloting
Immunobloting consist of electrophoresis of proteins on polysaccharide gel, blotting the
protein on nitrocellulose, reacting this with human serum and detecting antibody with
anti human IgG (Tsang et al., 1983). The method is held to be discriminatory (Couto et
al., 1995) but the resulting bands are very difficult to interpret (Couto et al., 1995;
Maslow et al., 1993a).
Multilocus Enzyme Electrophoresis (MLEE)
Multilocus enzyme electrophoresis (MLEE) involves the extraction of S. aureus
enzymes, separated elecrophoretically and then selectively stained and detecting various
isoenzymes by mean of their mobility (Selander et al., 1986). All isolates are typeable
because multi enzymes are used but single amino acid substitution results in 80% shift of
Chapter 1 Introduction
22
electrophoresis pattern. Variation in enzyme reflects the difference in the origin of gene
loci. Thus the proportion of loci which are different can give the measure of relatedness
between two isolates and these can be categorized into electrophoresis type (Musser and
Kapur, 1992). It is moderately discriminatory for clinical isolates and most useful for
studying sets of MRSA separated both in time and place (Mulligan and Arbeit, 1991;
Blumberg et al., 1992; Goering and Winter, 1992; Maslow et al., 1993a). MLEE pattern
are easy to read but difficult to compare (Tenover et al., 1994). This technique is highly
technically demanding and labor intensive (Maslow et al., 1993a).
Serotyping
There are eleven capsular polysaccharides found in S. aureus but 85% of clinical MRSA
isolates express only two of eight capsular polysaccharides types i.e. type 5 and type 8.
So this method is of little epidemiological value due to limited diversity of capsular
polysaccharide found among MRSA (Branger et al., 1990).
Genome Based Methods of Typing MRSA
Plasmid Analysis
Plasmid analysis was the first ever molecular typing method used to type MRSA. It
involves extraction of plasmid DNA, running the digest on an agarose gel which is
stained with ethedium bromide and photographed with UV light. The resulting banding
pattern can be analyzed (McGowan et al., 1979; Pfalleret et al., 1992). It is an easy
technique to perform and explain but the major draw back is that plasmids are easily
gained and lost thus making the analysis doubtful (Tenover, 1996; Yoshida, 1997;
Weller, 2000). Most strains of MRSA are also not type able due to absence of plasmids
(Harstein et al., 1989; Zuccarelli et al., 1990; Couto et al., 1995).
Restriction enzyme analysis of whole genomic DNA
This involves the extraction of chromosomal DNA, digesting it with restriction enzyme,
subjecting the product to electrophoresis on an agarose gel and detecting the resulting
band patterns with ethidium bromide photographed with UV light. The resulting pattern
is called restriction fragment length polymorphism (RFLPs). This technique has been
Chapter 1 Introduction
23
useful in confirming MRSA outbreaks (Veneza et al., 1992). However standard
restriction enzymes result in numerous bands and interpretation is difficult. This has led
to the development of various probing techniques that yield patterns that are easier to
interpret (Goering and Winters, 1992).
Ribotyping
Ribosomal genes are present in the MRSA genome in multiple copies at several positions
and are conserved with in a species (Bush and Nitschko, 1999; Arbeit, 1999, Weller,
2000). Ribotyping is a form of RFLP analysis in which transcriptional sequence of rRNA
is amplified and PCR product is subjected to restriction enzyme analysis (Popvic et al.,
1993). Restriction enzyme EcoRI has shown more bands than other restriction enzymes
which include ClaI and HindIII (Preheim et al 1991; Blumberg et al., 1992; Prevost et
al., 1992). Multiple enzymes have been tried to achieve greater resolution but EcoRI
seemed to produce equivalent results (Richardson et al., 1994). Ribotyping of MRSA has
successfully been used in several epidemiological studies with slight variation in the use
of restriction enzymes or probes and is discriminatory and reproducible (Haley et al.,
1982; Hadorn et al., 1990; Tenover et al., 1997).
Pulse Field Gel Electrophoresis of Chromosomal DNA
Pulse Field Gel Electrophoresis (PFGE) was developed by Schwartz and Cantor require
intact chromosomal DNA. It involves isolating micro-organism, embedding them in low
melting point agarose, lysing the organism in situ, digesting the DNA with rare cutting
restriction enzyme and then using special alternating electric fields to separate the large
DNA fragments produced (Schwarz and Cantor, 1984; Haley, 1991; Maslow et al.,
1993b). It takes two to four days to prepare the DNA for PFGE (Arbeit, 1995). SmaI is
commonly used restriction enzyme as it has few restriction sites and produces relatively
large fragments of DNA (10-800kb). The interpretation of banding patterns from pulse
field gel electrophoresis is done according to the criteria set by Tenover (Tenover et al.,
1995). One genetic difference results in a 2-3 band change and isolates are regarded as
being closely related and probably part of an outbreak. Two genetic changes results in a
4-6 band difference and isolates are regarded as possibly related and possibly part of an
outbreak. If there are three or more genetic differences, a difference of seven or more
Chapter 1 Introduction
24
band occurs. Such isolates are regarded as different and not part of an out break. Tenover
et al emphasized that these criteria were applicable only to PFGE performed on a set
epidemiological related organisms. Suggested restriction enzymes for typing MRSA
includes SmaI which gives 15-20 fragments, EagI gives 15-20 bands, SstII which gives
8-22 fragments, and ApaI, which gives 30-40 fragments. NotI, AscII, RsrII, and CspI,
give too few fragments. Kpul, EcoRI, Xhol and BamHI give multiple small fragments
that run of gel (Ichiyama et al., 1991; Carles-Nurit et al., 1992; Hollis et al., 1995;
Maslow et al., 1993b; Richardson and Reith, 1993; Winter et al 1993; Dominguez et al.,
1994; Struelens et al 1995; Tenover et al., 1995).
This method is highly discriminatory and can be applied to any bacterial species (Maslow
et al., 1993b; Tenover et al., 1995). PFGE has been suggested as method of choice for
typing MRSA (Bannerman et al 1995; Goering et al., 1995). This methods is usually
quite discriminating, however another method is often necessary to subdivide some
strains (Carles and Nurit et al., 1992; Tenover et al., 1994; Couto et al., 1995) and often
unrelated strains are found to have identical banding pattern to the outbreak strains
(Fukuda et al., 1998).
Ribosome spacer PCR
Kumari et al compared ribosome spacer PCR (RS-PCR) to PFGE in typing MRSA. The
16S-23S RNA intergenic spacer region is amplified by PCR, and restriction analysis
applied to the amplicons. 180 isolates of MRSA were typed; PFGE generated 17 and RS-
PCR 13 types. PFGE detected minor variants not discriminated by RS-PCR and detected
4 unique strains not identified by PCR. RS-PCR was found useful in typing strains from a
local outbreak. They found RS-PCR was rapid, inexpensive, highly reproducible and
slightly less discriminatory as PFGE in typing MRSA (Kumari et al., 1997).
SCCmec region PCR
The SCC mec region has a hypervariable segment 3’ to mecA, analysis of which can be
used to type MRSA strains. MSSA are non typeable by this method. Nishi et al typed
methicillin-resistant S. aureus and coagulase-negative staphylococci strain by amplifying
the hypervariable region downstream of mecA, and comparing the sizes of the amplicons
Chapter 1 Introduction
25
generated (Nishi et al., 1995). 61 MRSA isolates were grouped into 5 types and 26
coagulase-negative staphylococci were grouped into 5 types with this method.
Restriction Modification (RM) Typing
Staphylococcus aureus isolates belong to one of the ten reported S. aureus lineages. Each
lineage has characteristic external surface proteins that are responsible for interaction
with host. MRSA isolates that are widely distributed in hospitals and community belong
to six predominant S. aureus lineages and they have also acquired SCCmec on mobile
genetic elements (MGEs) (Robinson & Enright, 2003; Lindsay et al., 2006). Hospital
acquired MRSA population of each geographical location have limited clones and evolve
independently. The clones of each geographical location belong to separate lineages and
can be easily discriminated using restriction modification tests. Sequences of S. aureus
hsdS, sau1hsdS1and Sau1hsdS2 are highly conserved with in S. aureus lineages but vary
between the lineages (Waldon and Lindsay, 2006). Each gene has a conserved 5’ end,
central region and occasionally 3’ end. These genes also have a highly variable region in
between and specifically designed primers, one forward and two distinct reverse primers
targeting these variable region. In this way three PCR assays were developed which could
lead to three possibilities, a large amplicon, a small amplicon or no amplification at al.
These three PCR assays were termed as RM1, RM2 and RM3 as explained in figure 5.
Thus each possibility assigns a clonal complex to an isolate. Thus RM typing is rapid
PCR base method for the determination of clonal complex of MRSA isolates (Cockfield
et al., 2007).
Multilocus Variable Number of Tandem Repeat analysis (MLVA)
Genome sequence project of Eukaryotic and prokaryotic organisms has led to the
identification of special DNA sequences that repeat across the genome (Van Belkum,
1998). These repeated sequences were termed as Variable Number of Tandem Repeat
Analysis. These repeats are unique in their length, sequences and have specific copy
number across the genome of an isolate. So the difference in the number of repeat can be
used to measure the degree of relatedness among isolates and also can be used to trace
evolutionary changes in the genome (Jafferys et al., 1992; Tautz and Schlotterer, 1994).
Chapter 1 Introduction
26
Figure 5. Primer binding positions and predicted product sizes. On the left hand
side, the expected PCR product size for each lineage is listed. On the right, the
hybridization positions of the test primers on the variant forms of the
sau1hsdS genes are shown.
Chapter 1 Introduction
27
The locus specific repeats can be amplified through PCR by using the flanking primers
(Keim et al., 2001; Farlow et al., 2001). The PCR base VNTR anaylsis technique has
been reliably used to type clinical MRSA isolates. Sabat et al used five gene loci sdr,
clfA (clumping factor A), clfB (clumping factor B), sspA, spa to type MRSA isolates and
named this VNTR analysis as Mutlilous Variable of Tandem repeat analysis. These genes
code for different surface protein and have an R region present at the 3’ end which
contains the different number of repeats. S. aureus genome has several loci which contain
VNTRs (Francois et al., 2005; Koreen et al., 2005; Malanchowa et al., 2005, Sabat et al.,
2003). It has been observed that this method is robust, cost effective and have greater
discriminatory power (Sabat et al., 2003).
SPA Sequence typing
Direct Sequencing of DNA is considered as the most objective technique. A segment of
genomic DNA is amplified with PCR and the base sequence of amplicon is determined.
A number of major advantages exist (Oliveira et al., 2001; Enright et al., 2000).
Sequencing techniques are highly reliable and all strains are typeable. The method is
faster than PFGE, there is unambiguous data interpretation simplicity of large scale
database interpretation and standardization amongst laboratories is facilitated. The
technique is still very expansive, and only lends itself to the typing of a very small
number of organisms.
SPA typing involves direct sequencing of polymorphic X region of protein A gene. Spa
was used for molecular typing of MRSA (Frenay et al., 1996; Oliveira et al., 2001;
Shopsin et al., l999). The X region of protein A gene contains different number of repeats
which are due to loss and gain of repeats by spontaneous mutation. A numeric code is
assigned to each repeat unit and size of repeat unit is 24bp. Then spa type is determined
by the arrangement of these codes. Spa sequence typing is fast and easy to perform and
easy to interpret and produces unambiguous data. This data can be compared through
ridom spa server (www.ridom.de/spaserver). Spa typing is beneficial to monitor the
transmission of MRSA isolates from one hospital to another (Harmsen et al., 2003).
Chapter 1 Introduction
28
Figure 6. Indicating spa gene repeats sequence and repeat IDs of spa type t253.
Chapter 1 Introduction
29
Multilocus sequence typing (MLST)
Enright et al has developed this method for typing S. aureus which include the
sequencing of seven house keeping genes and then compare these sequences with the
standard available sequence available at MLST data base web (www.mlst.net).Multilocus
sequence typing is considered to be equivalent to Multilocus enzyme electrophoresis.
Instead of studying the enzyme, PCR is used to amplify the regions with in the genes of
these enzymes, and analysis of these sequences used to compare different bacterial
species (Enright and Spratt, 1999; Enright et al., 2000). The gene sequences are good
indicators of evolution with in species (Feil et al., 2003). The allelic profile is obtained by
sequencing internal fragments of seven house keeping genes. These genes include
Carbamate kinase (arcC), Shikimate dehydrogenase (aroE), glycerol kinase (glpF)
guanylate kinase (gmk), phosphate dehydrogenase (pta), triosephosphate dehydrogenase
(tpi) and acetyl coenzyme A acetyltransferase (yqil). Internal fragment of 450bp to 500bp
are sequenced for the seven house keeping genes and each gene loci is assigned a specific
number on the basis of sequence. These numbers are then arranged in the form of allelic
profile which are termed as sequence type. Any new occurring allele has a different DNA
sequence and assigned a distinct new allelic number (Enright et al., 2000).
A specifically designed algorithm BURST (based on related sequence type) is used to
show the relationship with in the clonal complexes (Feil and Chen, 2000). Data is
subdivided into clonal complexes in which members have at least 5 loci in common.
Ancestral genotypes are assigned as the genotype with in the clonal complex which
defines the highest number of single locus variants. Other strains are then assigned
according to their relation with ancestral genotype. Each clonal complex is represented
by series of concentric circles with the ancestral genotype at the centre (Enright et al.,
2002; Feil et al., 2004; Robinson and Enright, 2004).
This technique is highly discriminatory. This data base is available on the internet and
people can submit there sequence data to the database for the comparison, and analysis
(Anonymous, 2000c). Thus MLST produces unambiguous results that are due to high
cost reproducible and portable across different laboratories. MLST typing is most
Chapter 1 Introduction
30
desirable method for typing but high sequencing accuracy makes this method less
adaptable (Enright and Spratt, 1999; Olivera and Bean, 1999; Peacocok et al., 2002).
STAR Analysis
Staphylococcus auerus repeats (STAR) are the repetitive element that is present in
multiple copies in the S. aureus genome. These are present in intergenic region of
Staphylococcus auerus genome. An analysis of PCR product whose size may differ in
few nucleotide or several at several loci would allow the identification and differentiation
of clinically similar isolates at sub species level. The most important feature of the STAR
element is its repeated signature sequence T(/G/A/T)TGTTGG(G/T)GCCC(C/A) which
is present in different copies. There might be some strains which do not have signature
sequence at the particular locus thus lacking STAR element where as other have at that
particular locus either one or in multiple copies. STAR element normally begins with
TGGG[A/C]GTGGGACAGAAATGAT and ends at T[G/A/T]TGTTGGGGCCCCGCC
which is an extended signature sequence. STAR element region is normally followed by
a conserved region. Variation between the strains is in the number of STAR elements and
some time accompanied with the few base pair changes in the conserved region (Cramton
et al., 2000).
Aim and Objectives of Study
The present study is carried out in order to improve evidence based medicine and
valuable health policies in Pakistan against Staphylococcus aureus infections, by looking
into the patterns and distribution MRSA strains in different hospitals of
Islamabad/Rawalpindi. Simply by understanding how and what infection causing the
people sick is important in improving community health. The aim of present work was to
collect MRSA isolates from variety of infections from hospitals of
Islamabad/Rawalpindi, Pakistan and genotype them by using the Multilocus variable
number of tandem repeat analysis as described by Sabat et al., 2003., then with RM
typing, spa typing, MLST and also screened them for the presence of PVL gene and
Staphylococcus aureus repeats (STAR) at three loci. For this purpose a total of 123
MRSA isolates were obtained from three tertiary care hospitals of Islamabad/Rawalpindi.
Chapter 1 Introduction
31
Objectives of present work include:
To investigate the prevalent infectious MRSA strains in Pakistani clinical
environments and to identify the epidemic strains.
To investigate the genetic inter-relationship of the Pakistan MRSA isolates and to
find out whether the strains are restricted with specific hospitals.
To get an insight how these isolates evolved in Pakistan and to understand the
evolutionary mechanism which resulted in the selection of these strains.
To look for the relationship of Pakistani MRSA with global MRSA strains
Finally the objective of this study was the applicability of such molecular techniques
which are less labor intensive and portable across small diagnostic laboratories, at the
same time cheap and easy to perform.
Chapter 2 Materials and Methods
32
MATERIALS AND METHODS
Staphylococcus aureus is the major human pathogen and causes nosocomial as well as
community acquired infections. Since the last decade or so there is an alarming increase
in Staphylococcus aureus infections in Pakistan. Hence an epidemiological study was
designed to investigate the MRSA isolates prevalent in the nosocomial environment of
Rawalpindi/Islamabad, Pakistan and to understand their phylogenetic relationship among
the local isolates and also with globally isolated MRSA strains.
Isolation and Identification of Staphylococcus aureus
All the 123 non duplicate Staphylococcus aureus strains enlisted in table 13 (see
appendix-I) were isolated from patients hospitalized in three tertiary Care hospitals
situated in Islamabad and Rawalpindi (Pakistan Institute of Medical sciences, KRL
Hospital Islamabad and Military Hospital, Rawalpindi). These strains were re-confirmed
through recommended biochemical (Coagulase, Catalase and DNase test) and antibiotic
sensitivity tests initially and later on 16S-rRNA gene primers and mecA gene primers
were employed in order to confirm at molecular level. Staphylococcus aurues resistance
to antibiotics “Methicillin” or “Oxacillin” was determined through Kirby-Bauer Disc
Diffusion Method (Lawrence et al., 1972). The resistant and susceptible MRSA were
recognized on the basis of standard criteria.
Oxacillin
Resistant (MRSA): < 10 mm zone size of inhibition
Intermediate (MRSA) 11-12 mm zone size of inhibition
Susceptible (MSSA) > 13 mm zone size of inhibition
Methicillin
Resistant (MRSA) < 9 mm zone size of inhibition
Intermediate (MRSA) 10-13 mm zone size of inhibition
Susceptible (MSSA) > 14 mm zone size of inhibition
Chapter 2 Materials and Methods
33
Later on these strains were typed by using mecA typing, RM typing and MLVA typing,
STAR typing, spa typing and MLVA typing schemes. The materials and procedure
adapted for this purpose is explained below.
Gram staining and Biochemical Tests
Gram staining and Biochemical tests were performed according to the National Standard
Method developed by Health Protection Agency, UK (http://www.hpa-standardmethods.
org.uk/).
Gram Staining
A single colony of the S. aureus was picked from the fresh culture plate and smeared on
the glass slide, followed by gentle heating to fix the smear. Slide was flooded with 0.5%
methyl crystal violet and left for 60 seconds. Extra stain was washed away by treating the
slide with water for 5 seconds and then flooded the slide with 1% Lugol’s iodine solution
for 60 seconds. Then slide was treated with 95-100% ethanol drop wise in order to
remove the excess iodine from the smear and again rinsed the slide with tap water for 5
seconds. Safranin (0.1%) was used as a counterstain and flooded the slide for 2 minutes.
Again slide was washed with water and dried. Appearance of deep blue or purple cocci
under light microscope was interpreted as gram-positive bacteria.
Catalase Test
The catalase test is important in distinguishing streptococci (catalase-negative) from
staphylococci, which are vigorous catalase-producers. A colony was picked from an 18-
24 hour culture and placed on a clean glass slide. One drop of 3% Hydrogen peroxide
(H2O2) was dropped on slide and observed for bubbling. S. aureus was confirmed if the
bubbles appeared immediately.
Coagulase Test
Rabbit plasma (0.5 ml) was added to a sterile glass tube. A large loop full of a pure
colony of Staphylococci was emulsified into the plasma and incubated at 37°C for 4
hours. Clot formation was checked at different time intervals; 30 minutes, 1 hour and 4
Chapter 2 Materials and Methods
34
hours. The samples which showed absence of clot formation up to 4 hours were
rechecked after 24 hours. Clot formation confirmed the presence of Staphylococcus
aureus. No formation of clot was indicative of the presence of S. epidermidis
Deoxyribonuclease (DNase) Test
Line streak inoculation technique was used to apply a heavy inoculum on the agar and
drawn a line 3-4 cm long from the rim to the centre of the plate. In the other section of
the plate, DNase positive control strain inoculated and incubated at 37oC for 18-24 hours.
DNase activity was detected by flooding 1M Hydrochloric acid on the culture plate after
waiting for a few minutes. Excess hydrochloric acid was decanted and clear zone
formation was examined around the bacterial culture. Zone was compared with the
DNase positive control.
Bacterial culture and DNA Isolation
MRSA strains stored at -80 oC were streaked on BHI agar (Oxoid) and cultured over
night at 37oC. The colonies were visually inspected and then a single colony was picked
and inoculated in BHI Broth (Oxoid) and incubated over night (16-24hrs) in a shaking
incubator. 1.5ml of this fresh culture was taken and centrifuged at 10,000 rpm for 10
mins. The pellet was re-suspended in 250 l P-1buffer (Qiagen) and to this were added
2.5 l (100g/ml) lysostaphin, 2 l (25 mg/ml) proteinase K, mixed and 27 l 10% SDS.
Tubes were inverted 3-4 times and incubated at 37oC for 20-30 mins. Then 98 l 5M
NaCl and 81 l preheated CTAB (incubated CTAB for 20 mins at 65oC) was added.
After mixing, an equal volume of 24:1 chloroform: isoamyl alcohol was added and tubes
were centrifuged at 10,000 rpm for 10 mins. The upper layer was transferred to a fresh
tube and an equal volume of isopropanol was added prior to centrifugation at 10,000 rpm
for 10 min and resuspenion of the DNA pellet in 50 l sterile TE buffer pH8. Extracted
DNA was checked by running on 1.5% agarose gel for 45mins at 100volts.
DNA concentration (µg/µl) was determined by measuring absorbance of the sample at
260 nm wavelength and 280nm wavelength (Hoisington et al., 1994). 10 µl DNA
samples were diluted with 900 µl TE and quantified through UV spectrophotometer
Chapter 2 Materials and Methods
35
(Series 8453, Agilant, USA). The ratio of approximately 1.8-2 was taken as pure DNA.
Following formula was used to calculate the final quantity of the DNA in the sample.
DNA (µg/µl) = [OD260 x 100 (Dilution Factor) × 50 µg/ml]/1000
16S-rDNA Identification of Staphylococcus aureus
The 16SrDNA was used for identification of Staphlococcus aureus, as it is highly
conserved between different species of bacteria. In addition to highly conserved primer
binding sites, 16S rDNA contain hypervariable regions which provide species-specific
signature sequences useful for bacterial identification. As a result, 16S rDNA has become
prevalent in medical microbiology as a rapid, accurate alternative to phenotypic methods
of bacterial identification.
The primers used for the identification of Staphylococcus aureus had been taken from
Johnson et al., 2008. The primer sequences are given as under.
16S-F: 5`-GATCCTGGGTCAGGATG-3`
16S-R: 5`-CTAGAGTTGTCAAAGGATG-3`
The expected amplicon size is 1074bps. Polymerase chain reaction was performed in 0.2
ml tubes (Axygen, USA) containing 10 μl total reaction mixture. The reaction mixture
contained 40 ng (1ul of 1:10 diluted DNA) of genomic DNA,1 μl 10X PCR buffer (MBI
Fermentas, UK), 2.4ul 25mM MgCl2, 1ul 10mM dNTPs, 0.5ul 10uM each primer
(forward and reverse), and one unit of Taq DNA polymerase and 3.6ul PCR grade water.
The reaction mixture was taken through thermocycling conditions consisting: Lid temp
105°C, 2 minutes of 94°C for template DNA denaturation followed by 30 cycles of
amplification each consisting of 3 steps: one minute at 94°C for double stranded DNA
denaturation; one minute at 56°C for primer annealing to their complementary sequences
on either side of the target sequence; and three minute at 72°C for extension of
complementary DNA strands from each primer, final 4 minutes at 74°C for Taq DNA
polymerase to synthesize any unextended strands left. PCR was performed using Triple
Master Mix Thermocycler (Eppendorf).
Chapter 2 Materials and Methods
36
Mec A- gene Identification and SCCmec typing of Staphylococcus
aureus
mecA gene has been used for rapid identification of methicillin-resistant S. aureus
(MRSA). mecA gene was amplified using previously described primers by
Francois et al (Francois et al.,2005).
mecAF-: 5`-CATTGATCGCAACGTTCAATTT -3`
mecAR-: 5`-TGGTCTTTCTGCATTCCTGGA-3`
Polymerase chain reaction was performed in 0.2 ml tubes (Axygen, USA) containing 10
μl total reaction mixture. The reaction mixture contained 40 ng (1ul of 1:10 diluted DNA)
of genomic DNA,1 μl 10X PCR buffer (MBI Fermentas, UK), 2.4μl 25mM MgCl2, 1μl
10mM dNTPs, 1μl 5uM each primer (forward and reverse), and one unit of Taq DNA
polymerase and 2.6μl PCR grade water. The reaction mixture was taken through
thermocycling conditions consisting: Lid temp 105°C, 5 minutes of 94°C for template
DNA denaturation followed by 30 cycles of amplification each consisting of 3 steps:
Thirty seconds at 94°C for double stranded DNA denaturation; Thirty seconds at 55°C
for primer annealing to their complementary sequences on either side of the target
sequence; and thirty seconds at 72°C for extension of complementary DNA strands from
each primer, final five minutes at 72°C for Taq DNA polymerase to synthesize any
unextended strands left. PCR was performed using Triple Master Mix Thermocycler
(Eppendorf).
SCCmec typing was performed in a multiplex PCR as described by Boye et al., 2007.
Multiplex Polymerase chain reaction was performed in 0.2 ml tubes (Axygen, USA)
containing 10 μl total reaction mixture. The reaction mixture contained 2.5μl of genomic
DNA, 5 μl 10X PCR master mix ,2μl 10uM primer mix (forward and reverse), and 0.5μl
of Taq DNA polymerase. The reaction mixture was taken through thermocycling
conditions consisting: Lid temp 105°C, 4 minutes of 94°C for template DNA
denaturation followed by 30 cycles of amplification each consisting of 3 steps: Thirty
seconds at 94°C for double stranded DNA denaturation; Thirty seconds at 55°C for
primer annealing to their complementary sequences on either side of the target sequence;
Chapter 2 Materials and Methods
37
and sixty seconds at 72°C for extension of complementary DNA strands from each
primer, final four minutes at 72°C for Taq DNA polymerase to synthesize any
unextended strands left. PCR was performed using Triple Master Mix Thermocycler
(Eppendorf).
Detection of PVL Gene
The presence of Panton Valetine Leukotoxin gene was also checked by using the primers
Luk-PV-1 5’ATCATTAGGTAAAATGTCTGGACATGATCCA3’
Luk-PV-2 5’GCATCAAGTGTATTGGATAGCAAAAGC3’
The PCR reaction mixture contained 2 l (40 ng) of genomic DNA, 2.5 μl 10X PCR
buffer (MBI Fermentas, UK), 1.5 l 25mM MgCl2, 0.8μl 10mM dNTPs, 1.25 l 5uM
each primer and one unit of Taq DNA polymerase (Fermentas, UK) and 15.5 l PCR
grade water. Thermocycling conditions consisted of one cycle of 5 minutes of 94°C
followed by 30 cycles of amplification each consisting of three steps: forty-five seconds
at 94°C; forty-five seconds at 55°C and sixty-five seconds at 72°C, followed by a final
extension step of 5 minutes at 72°C. PCR was performed using a Triple Master Mix
Thermocycler (Eppendorf, USA).
Determination of MRSA Lineages Using Restriction Modification
system (RM-Test)
Restriction-modification systems (RMS) (restriction endonucleases and modification
methyltransferases) was used to determine lineages of Staphylococcus aureus obtained
from clinical material as described by Lindsay et al. (Lindsay et al., 2007). The primers
AF, AR30, AR22 were used for RM test 1, primers AF, AR45, AR1 for RM test 2 and
BF, BR8, BR5 are used for RM test3. The primer sequences are given in the Table 1 and
the expected product sizes for different RM tests are given in the table 2.
Polymerase chain reaction was performed in 0.2 ml tubes (Axygen, USA) containing 10
μl total reaction mixture. The reaction mixture contained 40 ng (1μl of 1:10 diluted DNA)
Chapter 2 Materials and Methods
38
Table 1: Oligonucleotide Primers used in RM typing
RM Test Primers Sequence (5` 3`)
RM 1 AF
AR30
AR22
AGGGTTTGAAGGCGAATGGG
CAACAGAATAATTTTTTAGTTC
TCAGAGCTCAACAATGATGC
RM-2 AF
AR45
AR1
AGGGTTTGAAGGCGAATGGG
GGAGCATTATCTGGTGTTTTCC
GGGTTGCTCCTTGCATCATA
RM-3 BF
BR8
BR5
CCCAAAGGTGGAAGTGAAAA
CCAGTTGCACCATAGTAAGGGTA
TCGTCCGACTTTTGAAGATTG
Table 2: RM typing Product sizes for different Clonal Complexes.
Clonal Complex
RM-1
Size in bp
RM-2
Size in bp
RM-3
Size in bp
CC30 203 - -
CC22 990 - -
CC45 - 722 -
CC1 - 1037 680
CC8 - - 680
CC5 - - 1071
Chapter 2 Materials and Methods
39
of genomic DNA, 1μl 10X PCR buffer (MBI Fermentas, UK), 2.4μl 25mM MgCl2, 1μl
10mM dNTPs, 1μl 5 μM each primer (forward and reverse), and one unit of Taq DNA -
Polymerase and 1.6μl PCR grade water. The reaction mixture was taken through
thermocycling condition consisting: Lid temp 105°C, 5 minutes of 94°C for template
DNA denaturation followed by 35 cycles of amplification each consisting of 3 steps:
Thirty seconds at 94°C for double stranded DNA denaturation; Thirty seconds at 55°C
for primer annealing to their complementary sequences on either side of the target
sequence; and two minutes at 72°C for extension of complementary DNA strands from
each primer, final five minutes at 72°C for Taq DNA polymerase to synthesize any
unextended strands left. PCR was performed using Triple Master Mix Thermocycler
(Eppendorf).
Typing of Staphylococcus aureus for STAR Element
Staphylococcus aureus repeat (STAR) elements present in the inetrgenic region has been
used for typing of methicillin-resistant Staphylococcus. aureus (MRSA). STAR element
present in intergenic region of CHP-gapR, uvrA-hprk and ica-geh was amplified by using
primers given in the table 2.
Polymerase chain reaction was performed in 0.2 ml tubes (Axygen, USA) containing 10
μl total reaction mixture. The reaction mixture contained 2.5 μl of genomic DNA, 5 μl
2XPCR pre mix D, 1μl 10mM each primer (forward and reverse), and 0.5μl of Taq DNA
polymerase. The reaction mixture was taken through thermocycling conditions
consisting: Lid temp 105°C, 5 minutes of 94°C for template DNA denaturation followed
by 30 cycles of amplification each consisting of 3 steps: Thirty seconds at 94°C for
double stranded DNA denaturation; Thirty seconds at 52°C for primer annealing to their
complementary sequences on either side of the target sequence for gapR-chp, ica-geh and
62°C for uvrA-hprk and thirty seconds for gapR-chp, one minute for ica-geh, and uvrA-
hprk at 72°C for extension of complementary DNA strands from each primer, final five
minutes at 72°C for Taq DNA polymerase to synthesize any unextended strands left.
PCR was performed using Triple Master Mix Thermocycler (Eppendorf, USA).
Chapter 2 Materials and Methods
40
Multiplex PCR Based Multilocus Variable Number of Tandem
Repeats (MLVA) Assay
Seven genes ClfA (Clumping factor A), ClfB (Clumping factor B), sdrC (Ser-Asp-rich
fibrinogen binding proteins), sdrD (Ser-Asp-rich fibrinogen binding proteins), spa, sspa and
sav1078 were selected for the multiplex PCR for MLVA typing. The fluorescent labeled
primers used are given in the table 3, ClfA, sdrC and sav1078 were multiplex separately
from ClfB, sdrD, spa, sspa.
Polymerase chain reaction was performed in 0.2 ml tubes (Axygen, USA) containing 25
μl total reaction mixture. The reaction mixture contained 2μl DNA (40 ng) of genomic
DNA, 2.5 μl 10X PCR buffer (MBI Fermentas, UK), 1.5μl 25mM MgCl2, 0.8μl 10mM
dNTPs, 1.5μl of 5μM each primer ClfA (forward and reverse), sdrC (forward and
reverse), sav1078 (forward and reverse) and one unit of Taq DNA polymerase and 9μl
PCR grade water. In the second PCR ClfB, sdrD, spa and sspa were multiplexed with the
same procedure as mention above.
The reaction mixture was taken through thermocycling conditions consisting: Lid temp
105°C, 5 minutes of 94°C for template DNA denaturation followed by 30 cycles of
amplification each consisting of 3 steps: thirty seconds at 94°C for double stranded DNA
denaturation; thirty seconds at 53°C for primer annealing to their complementary
sequences on either side of the target sequence; and Two minute at 72°C for extension of
complementary DNA strands from each primer, final 5 minutes at 72°C for Taq DNA
polymerase to synthesize any unextended strands left. PCR was performed using Triple
Master Mix Thermocycler (Eppendorf, USA). The amplified products were subjected to
gene scan for size analysis in ABI-sequencer.
Gene Scan Protocol
PCR products (labeled with dyes as given in table 3) were diluted 1:10 with sterile
distilled water. Then 0.5 l of diluted (1:10) sample was added to 0.5 l Liz1200 (ABI)
size standard and 9 l of formamide before heating to 95°C for 3 minutes. Samples were
Chapter 2 Materials and Methods
41
Table 3: Oligonucleotide Primers used in Typing of Staphylococcus aureus for
STAR Element.
Gene Primer Sequence (5` 3`) Tm
gapBF
gapSR
GGGGATCCGCTAATGATAAGTAGTATTTAG
GGGGATCCGTAAATAAGGATATATCACAAC
52
52
uvrA
hprk
GGTCCTGAAGGTGGTAGTGGCGGTGG
GGCTTCGATAATCCTTCTTCACCAGCG
77.7
73.9
icaC
geh
TTATTAAGCTATGTTAAAAACACGCGGTGG
AAAGGGAGGAATTAATAATGACTGCAGACT
69.6
67.8
Chapter 2 Materials and Methods
42
electrophoresed on an ABI DNA Sequencer. Gene scan peaks were analyzed using the
Applied Biosystem Peak Scaner software volume 1.0 to obtain the exact PCR product
size (http://www.appliedbiosystems.com/absite/us/home/supportsoftware-community/
free-ab-software. html).
Calculation of VNTRs and MLVA Data analysis
The number of tandem repeats for all loci were calculated from the standard sequenced
strains 8325 and Mu50 using the Tandem Repeats Finder Program, Version 4.03
(Benson, G. 1999; http://tandem.bu.edu/trf.html ).
PCR products from these strains were used as a reference to calculate the number of
repeats in the test strains. The formula used to calculate the number of repeats was:- size
of the PCR product minus size of the flanking region divided by the size of the repeat
unit.
Phylogenetic analysis
The number of tandem repeats obtained for all the loci for each strain were imported into
bioinformatics software program, Network 4.5 available at a public domain (http//fluxux-
engineering.com/) and also into the MLVA plug-in of Bionumeric (Applied Maths Inc),
in order to calculate a phylogenetic tree. In the trees each circle represents a specific
MLVA type (MT/genotype) and the size and color of the circle represents the number of
strains in this specific type and their source of isolation. Lines between the circles
represent variation in the number of repeats. The isolates varying at one locus are
connected by bold lines and those varying in more than one locus are connected by thin
lines while the standard strains are connected by dotted lines.
SPA typing
DNA sequence analysis of the protein A gene variable repeat region (spa typing) provides
a rapid and accurate method for typing Staphylococcus aureus isolates. The primers used
for the amplification of protein A gene variable repeat region is given in the table 4.
Chapter 2 Materials and Methods
43
Table 4: Oligonucleotide Primers used in MLVA Typing of Staphylococcus aureus.
Gene Name Primer Sequence (5` 3`) Dye Tm (°C)
ClfA
(Clumping factor A)
F-GCATTTAATAACGGATCAGG
R-TGAATTAGGCGGAACTACAT
FAM 58.5
ClfB
(Clumping factor A)
F-ATGGTGATTCAGCAGTAAATCC
R-CATTATTTGGTGGTGTAACTCTT
vic 61.5
sdrC
(Ser-Asp-rich fibrinogen binding proteins)
F-ATGATTTCACACTTGATAATGGC
R- GCTGTTTTATGCTGATCTTTAAC
vic 61.5
sdrD
(Ser-Asp-rich fibrinogen binding proteins)
F-ATGATTTCACACTTGATAATGGC
R- TAACGTTGCGTTATTTGAGCCC
Tet 61.5
Spa
(Protein)
F-AGCACCAAAAGAGGAAGACAA
R-GTTTAACGACATGTACTCCGT
FAM 62
sspA
(Serine protease V8)
F-ATCMATTTYGCMAAYGATGACCA
R-TTGTCTGAATTATTGTTATCGCC
Ned 61.5
Sav1078
(Hypothetical Protein)
F-GTGCATAATGGCTTACGAAT
R-TGGGAGGAATTAATCATGTC
FAM 59
Chapter 2 Materials and Methods
44
Polymerase chain reaction was performed in 0.2 ml tubes (Axygen, USA) containing
25μl total reaction mixture. The reaction mixture contained 2μl DNA (40 ng) of genomic
DNA, 2.5 μl 10X PCR buffer (MBI Fermentas, UK), 1.5μl 25mM MgCl2, 0.2μl 10mM
dNTPs, 1.5μl 5μM each primer spa(forward and reverse), one unit of Taq DNA
polymerase and 15.7μl PCR grade water. The reaction mixture was taken through
thermocycling conditions consisting: Lid temp 105°C, 5 minutes of 94°C for template
DNA denaturation followed by 30 cycles of amplification each consisting of 3 steps:
thirty seconds at 94°C for double stranded DNA denaturation; thirty seconds at 55°C for
primer annealing to their complementary sequences on either side of the target sequence;
and thirty seconds at 72°C for extension of complementary DNA strands from each
primer, final 5 minutes at 72°C for Taq DNA polymerase to synthesize any unextended
strands left. PCR was performed using Triple Master Mix Thermocycler (Eppendorf,
USA).The amplified product was checked by running on 1.5% agrose gel and then
proceeded for sequencing.
After sequencing the trace files were edited by using CLC DNA Work Bench 5.1. The
consensus sequences obtained were checked for the 24bps repeats units as given by
Korean et al., 2004. The 24 base pair repeat units were feed into the spa server and repeat
codes were identified and entered these repeat codes again into spa server and obtained
the spa types of isolated strains
Multilocus sequence typing (MLST) of Staphylococcus aureus
The Staphylococcus aureus MLST scheme uses internal fragments of the following seven
house-keeping genes: arcC (Carbamate kinase), aroE (Shikimate dehydrogenase), glp
(Glycerol kinase), gmk (Guanylate kinase), pta (Phosphate acetyltransferase), tpi
Triosephosphate isomerase), yqil (Acetyle coenzyme A acetyltransferase). The primers
for the amplification and sequencing of these gene fragments were taken from Enright et
al., 2000. The primer sequence is given in the table 5. Polymerase chain reaction was
performed in 0.2 ml tubes (Axygen, USA) containing 20μl total reaction mixture by
using 1μl chromosomal DNA (40ng), 2μl 10X PCR buffer (contains MgCl2), 1.25 μl
25mM MgCl2, 0.4μl 10mM dNTP mix, 2μl of each 2μM primer (forward and reverse),
one unit of Taq polymerase (Kappa-taq 10units/ul), distilled water 11.25μl. The reaction
Chapter 2 Materials and Methods
45
Table 5: Oligonucleotide primers used in MLST typing.
S.NO. Gene Name PCR/Sequencing primers
1 arcC up - 5' TTG ATT CAC CAG CGC GTA TTG TC -3'
dn - 5' AGG TAT CTG CTT CAA TCA GCG -3'
2 aroE up - 5' ATC GGA AAT CCT ATT TCA CAT TC -3'
dn - 5' GGT GTT GTA TTA ATA ACG ATA TC -3'
3 glp up - 5' CTA GGA ACT GCA ATC TTA ATC C -3'
dn - 5' TGG TAA AAT CGC ATG TCC AAT TC -3'
4 gmk up - 5' ATC GTT TTA TCG GGA CCA TC -3'
dn - 5' TCA TTA ACT ACA ACG TAA TCG TA -3'
5 pta up - 5' GTT AAA ATC GTA TTA CCT GAA GG -3'
dn - 5' GAC CCT TTT GTT GAA AAG CTT AA -3'
6 tpi up - 5' TCG TTC ATT CTG AAC GTC GTG AA -3'
dn - 5' TTT GCA CCT TCT AAC AAT TGT AC -3'
7 yqi up- 5' CAG CAT ACA GGA CAC CTA TTG GC -3'
dn- 5' CGT TGA GGA ATC GAT ACT GGA AC -3'
Chapter 2 Materials and Methods
46
mixture was taken through the thermocycling conditions consisting: Lid temp 105°C, 5
minutes of 94°C for template DNA denaturation followed by 30 cycles of amplification
each consisting of 3 steps: one minute at 94°C for double stranded DNA denaturation;
one minute at 55°C for primer annealing to their complementary sequences on either side
of the target sequence except for tpi and gmk used 50°C; and one minute at 72°C for one
minute at 55°C for primer annealing to their complementary sequences on either side of
the target sequence except for tpi and gmk used 50°C; and one minute at 72°C for
extension of complementary DNA strands from each primer, final 5 minutes at 72°C for
Taq DNA polymerase to synthesize any unextended strands left. PCR was performed
using Triple Master Mix Thermocycler (Eppendorf, USA).
The allelic profile of Staphylococcus aureus strain was obtained by sequencing internal
fragments of seven house-keeping genes. The sequence was obtained for both DNA
strands and then consensus sequences were determined by using CLC DNA work bench.
The sequences were trimmed so that they correspond exactly to the region that MLST
server uses to define the alleles. After submitting the seven sequences, got the allelic
profile of isolates with assigned Sequence type (ST).
DNA Sequencing Procedure
Took 0.5μl amplified product to be sequenced in 0.2ml PCR tube and added 1μl forward
and reverse primers in separate reactions. Then added 4μl Big Dye (1:8 diluted), 6.4μl 5x
Sequencing Buffer, 11.6μl distilled water and the reaction mixture was taken through the
thermocycling conditions consisting twenty five cycles each consisting thirty seconds at
96C, fifteen seconds at 50°C and four minutes at 60°C.
Added 2μl 2.2% SDS to 20μl reaction after doing PCR, mixed by flicking tube and spin
briefly. Heated to 98°C for 5 min, Cooled to room temperature and spin briefly. Spin the
cartridges for three minutes at 850xg (3100rpm). Transferred Cartridges to 1.5ml
eppendorf tubes and added sample. Spin the cartridges for 3 min at 850xg (3100rpm).
Retained fluid and used for sequencing. Respinned if obtained less than 16ul of reaction
volume. All the sequencing carried out in the AB sequencer (model and made), PNACLE
facility, University of Leicester, UK and trace files were edited using CLC-DNA work
bench 5.1.
Chapter 2 Materials and Methods
47
100 bp DNA Ladder
The Norgen Full Ranger 100bp DNA Ladder [1mL of premixed DNA ladder
(0.5µg/10µL) in loading buffer (10mM EDTA, 10% glycerol, 0.015% bromophenol blue
and 0.17% SDS)] was used in horizontal gel electrophoresis in order to determine DNA
amplicon sizes. Norgen Full Ranger contains sixteen discrete fragments ranging from
100bp to 5000bp with higher intensity reference bands at 500bp and 1000bp. This Ladder
(Figure 7a) is ideal for accurate visual determination in both large and small cloning
applications.
Ø×174 RF DNA Hae III Ladder
The other marker used was Ø×174 RF DNA Hae III Ladder. Typically 2ul of the ladder
solution was used per well. The fragment sizes of this ladder are given in figure 7b.
Solutions and Buffers
1- 1.5%Agarose
Dissolved 1.5gm of agrose in 98.5ml of distilled water and heated in microwave
for 2mins,cooled down to 60 oC and then added 5ul ETBr per 100ml of agrose and
poured in gel tray and allowed to polymerize at room temperature for 30-45min.
2- 10%SDS
Took 10gm of SDS (Merk) and made the volume upto 100ml.
3- 5M NaCl
Took 292.5gm of Sodium Chloride (Merk) and made volume upto 1000ml.
4- CTAB Solution
Dissolve 4.1g NaCl (Merk) in 80mls H2O, and add 10g CTAB make up to 100mls
and warmed to 65 oC to dissolve.
Chapter 2 Materials and Methods
48
Figure 7 (A&B)
Fig.7A Fig.7B
Norgen Full Ranger 100bp DNA Ladder is shown in Fig.7A and Ø×174 RF DNA Hae III
Ladder is shown in Fig.7B.
Chapter 2 Materials and Methods
49
5- 10XTAE buffer
To make 1L, 48.4g Tris, 20mL of 0.5M EDTA pH 8.0, 11.42mL Glacial Acetic
Acid and added enough H2O to dissolve solids, pH with HCl to 7.6-7.8, then
bring up to final volume of 1000mL.
6- 10XTBE buffer
To make1 L, dissolved 108g Tris base, 55g Boric acid, 40mls 0.5M EDTA (pH
8.0) in enough water and made the final volume upto 1L and autoclaved for 20
min.
7- TE buffer
Took 10ml of 1M Tris-Cl, pH 7.5 and 2ml of 0.5M EDTA pH 8.0, and made the
volume upto 1L.
8- 1M Tris-Cl
Dissolved 60.57g of Tris in 0.5L water and adjusted the pH to 7.5 using HCl.
9- 0.5M EDTA
Dissolved 186.1g of EDTA in IL water by stirring and adjusted the pH to 8.0 by
using NaOH.
Statistical and Sequence Analysis
All statistical (clustering) and sequence analysis was performed using BioNumerics
(Applied Maths, Belgium) and CLC DNA (USA) respectively.
Chapter 3 Results
50
Results
Staphylococcus aureus is the major human pathogen and is the cause of various kinds of
human infection. In order to proceed for epidemiology study initial identification and
confirmation of Staphylococcus aureus is necessary. So all the 123 Staphylococcus
aureus isolates included in this study were identified and confirmed by the Gram
staining, Coagulase, Catalase & DNase tests and also for antibiotic sensitivity for
methicillin and then for 16S-rRNA gene and also for the presence of mecA gene. Then all
of the 123 isolates were subjected to the epidemiological techniques: MLVA typing, RM
typing, PVL typing, Spa, MLST and STAR analysis. The results are presented
accordingly.
Staphylococcus aureus Identification by 16S-rRNA Gene
There are different parameters used for the identification of Staphylococcus aureus. The
most reliable and commonly used to identify Staphylococcus aureus is by 16S rRNA
gene PCR. The most important potential use of 16S-rRNA gene is to provide genus and
species identification for isolates. Specifically designed 16S rRNA gene primers were
used, whose sequence is given in the material and methods section, in order to identify
the Staphylococcus aureus isolates accurately. The expected product size was 1000bps,
as calculated from the standard sequenced strain NCTC-8325 and Mu-50 by using the
software Artimis v. 9.
Pakistani Staphylococcus aureus isolates were screened for 16S-rRNA gene with specific
primers and expected product sizes of 1000bp were obtained with all the 123 isolates
(Fig. 8 to Fig. 13), confirming the isolates as Staphylococcus aureus. The
electropherogram obtained by running the PCR product in 1.5% agarose gel and after
staining with ethedium bromide is shown in the figure 8 (A, B, C). In the figure 8A from
left to right M is 100bp DNA ladder (Norgen Full Ranger), lane one to twenty contains
the standard strain NCTC8325, P.10019, K.3, P.2113, P.2112, P.2054, P.5123, P.13729,
P.18431, P.2896, P.2886, P.1896, P.5, P.10015, P.12006, P. 8431, P.10012, P10021, P.
18119 and P.1966 respectively. Similarly in the figure 8B from left to right M is 100 bps
Chapter 3 Results
51
Figure 8 (A, B, C)
Fig. 8A Fig. 8B
Fig. 8C
Electropherogram of 1.5% ethedium bromide stained gel of Pakistani isolates showing amplification of 16S-rRNA Gene for identification of Staphylococcus aureus. In the figure, M is 100 bp DNA ladder (Norgen Full Ranger); lanes 1 to 20 contain NCTC8325 [Positive control], P.10019, K.3, P.2113, P.2112, P.2054, P.5123, P.13729, P.18431, P.2896, P.2886, P.1896, P.5, P.10015, P.12006, P. 8431, P.10012, P10021, P. 18119 and P.1966 respectively. Figure 8B:- lanes 21 to 40 contain P.1, K.6, P.8, P.8487, P.17120, P.6269, P.11293, P.17121, P.13749, A.9965, P.8668, P.1297, P.16509, P.16849, P.12285, P.1129, P.1286, P.15415, P.18339, P. 8116 respectively. Figure 8C:- lane number forty one to fifty contains P.2285, P.1964, P.5119, P.11291, A.1761, A.35087, A.9313, A.9914, A.9077 and -ive control respectively.
Chapter 3 Results
52
DNA ladder (Norgen Full Ranger), lane twenty to forty contains P.1, K.6, P.8, P.8487,
P.17120, P.6269, P.11293, P.17121, P.13749, A.9965, P.8668, P.1297, P.16509, P.16849,
P.12285, P.1129, P.1286, P.15415, P.18339, P.8116 respectively.
In figure 8C from left to right M is 100bps DNA ladder (Norgen Full Ranger), lane
number forty one to fifty contains P.2285, P.1964, P.5119, P.11291, A.1761, A.35087,
A.9313, A.9914, A.9077 and -ive control respectively. All the amplified DNA fragments
were of approximately 1000bps in size, calculated after comparing with the ladder
confirming that the strains as Staphylococcus aureus.
Similar pattern of amplification was also observed for all the other isolates as indicated in
the Figure 9 to Figure 13. In the figure 9 from left to right M is 100bps DNA ladder
(Norgen Full Ranger), Lane number one to nineteen contain A.13319, A.7145, P.3, P.4,
P.7, K.8, P.8485, P.2, P.2871, P.5020, P.5114, P.1005, P.11296, P.862, P.844, P.843,
P.1843, A.8823 and negative control respectively. Similar results are given in the figure
10. In the figure 10 from left to right M is 100bps DNA ladder (Norgen Full Ranger),
lane number one to sixteen contain A.9781. A.465, A.26163, A.14254, A.15330, A.379,
A.13282, P.10013, P.1003, P.1863, P.3727, P.2887, P.6, P.8486, P.13741 and negative
control respectively. All the amplified DNA fragments were of approximately1000bps in
size, as calculated after comparing with the DNA marker confirming that these strains
were Staphylococcus aureus
Lane number one to eleven from left to right in figure 11 is showing the amplified
product of 16S-rRNA gene of Pakistani Staphylococcus aureus isolates, P.13740, K.10,
K.13, A.5054, A.13730, P.1962, P.2111, P.2255, K.11, P.12007 and negative control
respectively. M is 100bps DNA ladder (Norgen full Ranger). All the amplified DNA
fragments are of 1000bps in size, as calculated after comparing with the ladder
confirming that these strains were Staphylococcus aureus.
Figure 12 showing the amplified product of 16S rRNA gene of Pakistani Staphylococcus
aureus strains. In the figure 12 from left to right M is 100bps DNA ladder (Norgen full
Ranger), lane one to seventeen contain P.1009, P.16809, A.5053, P.3027, P.14947,
P.18176, P.1961, P.1963, P.5019, P.1123, K.12, P.2006, P.14946, P.2885, P.1864,
Chapter 3 Results
53
Figure 9
Electropherogram of 1.5% ethedium bromide stained gel of Pakistani isolates showing amplification of 16S rRNA Gene for identification of Staphylococcus aureus. In the figure from left to right M is 100bps DNA ladder (Norgen Full Ranger), Lane number one to nineteen contain A.13319, A.7145, P.3, P.4, P.7, K.8, P.8485, P.2, P.2871, P.5020, P.5114, P.1005, P.11296, P.862, P.844, P.843, P.1843, A.8823 and negative control respectively.
Figure 10
Electropherogram of 1.5% ethedium bromide stained gel of Pakistani isolates showing amplification of 16S rRNA Gene for identification of Staphylococcus aureus. In the figure from left to right M is 100bps DNA ladder (Norgen Full Ranger), lane no. one to sixteen contain A.9781. A.465, A.26163, A.14254, A.15330, A.379, A.13282, P.10013, P.1003, P.1863, P.3727, P.2887, P.6, P.8486, P.13741 and negative control respectively.
Chapter 3 Results
54
Figure 11
Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of 16S rRNA Gene for identification of Staphylococcus aureus. Lane number one to eleven from left to right in figure is showing the amplified product of 16S rRNA gene of Pakistani Staphylococcus aureus isolates, P.13740, K.10, K.13, A.5054, A.13730, P.1962, P.2111, P.2255, K11, P.12007 and negative control respectively.
Figure 12
Electropherogram of 1.5% ethidium bromide stained gel of Pakistani isolates showing amplification of 16S rRNA Gene for identification of Staphylococcus aureus. In the figure from left to right M is 100bps DNA ladder (Norgen full Ranger), lane no. one to seventeen contain P.1009, P.16809, A.5053, P.3027, P.14947, P.18176, P.1961, P.1963, P.5019, P.1123, K.12, P.2006, P.14946, P.2885, P.1864, P.13947 and negative control.
Chapter 3 Results
55
P.13947 and negative control. All the amplified DNA fragments are of
approximately1000bps in size, as calculated after comparing with the ladder confirming
that these stains were Staphylococcus aureus.
Figure 13 (A, B) showing the amplified product of 16S ribosomal DNA of Pakistani
Staphylococcus aureus strains. In the figure.13A from left to right M is 100bps DNA
ladder (Norgen full ranger), lane no. one to ten P.13714, P.14313, A.9445, A.9703,
A.9954, A.10813, A.11241, A.14075, A.14256, A.25356 and negative control
respectively. All the amplified DNA fragments are of approximately1000bps in size, as
calculated after comparing with the 100bp DNA marker, confirming the isolates as
Staphylococcus aureus.
Same is the case for the remaining isolates as shown in the figure 13B. In the figure 13B
from left to right M is 100bps DNA ladder (Norgen full ranger), lane no. one to seven
contains A.35045, P.1967, and P.1287, P.2119, P.6270, P.13731 and negative control
respectively. All the amplified DNA fragments are of approximately1000bps in size, as
calculated after comparing with the ladder confirming that these stains are
Staphylococcus aureus.
MRSA Identification by mecA Gene
The mecA gene is present in the SCCmec region. Methicillin resistant Staphylococcus
aureus is a carrier of mecA gene which makes this bacterium resistant to methicillin,
penicillin and other penicillin like antibiotics. So it is a good way to identify MRSA by
amplifying the mecA gene fragment by using PCR. It is a rapid and cheaper method for
the identification of MRSA.
The amplified product of mecA gene fragment of 38 Pakistani Staphylococcus aureus
strains are shown in the representative figure 14 (A, B, C). In the figure 14A from left to
right M1 is 100bp DNA ladder (Norgen full ranger) and M2 is the λ-ladder, lane one to
eleven contains the standard strain MRSA252, P.10019, K.3, P.2113, P.2112, P.2054,
P.5123, P.13729, P.18431, P.2896, P.2886, and in the figure 14B M is 100bp DNA ladder
(Norgen full ranger) and M2 is the λ-ladder, lane twelve to twenty four contain P.1896,
Chapter 3 Results
56
Figure 13 (A, B)
Fig. 13A Fig. 13B
Electropherogram of 1.5% ethedium bromide stained gel of Pakistani isolates showing amplification of 16S rRNA Gene for identification of Staphylococcus aureus. In the figure13A from left to right M is 100bps DNA ladder (Norgen full ranger), lane no. one to ten P.13714, P.14313, A.9445, A.9703, A.9954, A.10813, A.11241, A.14075, A.14256, A.25356 and negative control respectively. In the figure 13B from left to right M is 100bps DNA ladder (Norgen full ranger), lane no. one to seven contains A.35045, P.1967, and P.1287, P.2119, P.6270, P.13731 and negative control respectively
Chapter 3 Results
57
Figure 14 (A, B, C)
Fig. 14A Fig. 14B
Fig. 14C
Electropherogram of 1.5% ethedium bromide stained gel of Pakistani isolates showing amplification of mecA gene for the identification of methicillin resistant Staphylococcus aureus. In the figure 14A from left to right M1 is 100bp DNA ladder (Norgen full ranger) and M2 is the λ-ladder, lane one to eleven contains the standard strain MRSA252, P.10019, K.3, P.2113, P.2112, P.2054, P.5123, P.13729, P.18431, P.2896, P.2886, and in the figure 14B M is 100bp DNA ladder (Norgen full ranger) and M2 is the λ-ladder, lane twelve to twenty four contain P.1896, P.5, P.10015, P.12006, P. 8431, P.10012, P10021, P. 18119, P.1966, P.1, K.6, P.8, P.8487 respectively and in Fig 14C from left to right M is the 100bp DNA ladder (Norgen full ranger), lane twenty five to thirty nine contain P.17120, P.6269, P.11293, P.17121, P.13749, A.9965, P.8668, P.1297, P.16509, P.16849, P.12285, P.1129, P.1286, P.15415 and negative control respectively.
Chapter 3 Results
58
P.5, P.10015, P.12006, P. 8431, P.10012, P10021, P. 18119, P.1966, P.1, K.6, P.8,
P.8487 respectively and in Fig 14C from left to right M is the 100bp DNA ladder
(Norgen full ranger), lane twenty five to thirty nine contain P.17120, P.6269, P.11293,
P.17121, P.13749, A.9965, P.8668, P.1297, P.16509, P.16849, P.12285, P.1129, P.1286,
P.15415 and negative control respectively. The amplified products obtained for all the 38
above mentioned isolates are100bps long thus confirming that these stains are methicillin
resistant Staphylococcus aureus.
Figure 15 showing the amplified product of mecA gene fragment of Pakistani
Staphylococcus aureus strains. In the figure 15A from left to right M1 and M2 are 100
base pair DNA ladder (Norgen full ranger), lane one to fourteen contains P.18339,
P.8116, P.2285, P.1964, P.5119, P.11291, A.1761, A.35087, A.9313, A.9914, A.9077,
A.14256, A.9954, and A.14075 respectively, and in the figure.15B from left to right M1 is
100 bps DNA ladder, lane fifteen to twenty eight contains A.11241, A.25356, A.10813,
A.9445, A.35045, A.9703. A.13319, A.7145, P.3, P.4, P.7 K.8, P.8485, P.2 respectively
and in the figure 15C from left to right M1 is 100 bps, lane twenty nine to thirty four
contain P.2871, P.5020, P.5114, P.1005, P.11296 and -ive control respectively. All the
amplified products are of approximately100bps long confirming isolates as methicillin
resistant Staphylococcus aureus.
Figure 16 showing the amplified product of mecA gene fragment. In the figure 16A M1
and M2 is 100 bps DNA ladder (Norgen full ranger), and from left to right, lane one to ten
contain P.862, P.844, P.843, P.1843, P.8823, .A.9781, A.465, A.26163, A.14254,
A.15330, and in the Figure16b from left to right lane eleven to fifteen contain A.379,
A.13282, P.10013, P.1003 and negatve control respectively. All the amplified products
are of approximately 100bps long confirming that these stains are methicillin resistant
Staphylococcus aureus.
Figure .17 showing the amplified product of mecA gene fragment. From left to right M1
and M2 are 100bps DNA ladders (Norgen full ranger). lane one to nineteen contain
P.1863, P.3727, P.2887, P.6, P.8486, P.13741, P.13740, K.10, -ive control, K.13, A.5054,
A.13730, P.1962, P.2111, P.2255, K.11, P.12007, P.1009, P.16809 respectively. All the
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Figure 15 (A, B, C)
Fig. 15A Fig. 15B
Fig. 15C
Electropherogram of 1.5% ethedium bromide stained gel of Pakistani isolates showing amplification of mecA gene for the identification of methicillin resistant Staphylococcus aureus. In the figure 15A, M1 and M2 are 100 base pair DNA ladder (Norgen full ranger), lane one to fourteen contains P.18339, P.8116, P.2285, P.1964, P.5119, P.11291, A.1761, A.35087, A.9313. A.9914, A.9077, A.14256, A.9954, and A.14075 respectively, and in the figure 15B from left to right M1 is 100 bps DNA ladder, lane fifteen to twenty eight contains A.11241, A.25356, A.10813, A.9445, A.35045, A.9703. A.13319, A.7145, P.3, P.4, P.7 K.8, P.8485, P.2 respectively and in the figure 15C from left to right M1 is 100 bps, lane twenty nine to thirty four contain P.2871, P.5020, P.5114, P.1005, P.11296 and -ive control respectively.
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Figure 16 (A, B)
Fig. 16A Fig.16B
Electropherogram of 1.5% ethedium bromide stained gel of Pakistani isolates showing amplification of mecA gene for the identification of methicillin resistant Staphylococcus aureus. In the figure 16A, M1 and M2 are 100 bp DNA ladder (Norgen full ranger), and from left to right, lane one to ten contain P.862, P.844, P.843, P.1843, P.8823, .A.9781, A.465, A.26163, A.14254, A.15330 and in the Figure16B from left to right lane eleven to fifteen contain A.379, A.13282, P.10013, P.1003 and negatve control respectively.
Figure 17
Electropherogram of 1.5% ethedium bromide stained gel of Pakistani isolates showing amplification of mecA gene for the identification of methicillin resistant Staphylococcus aureus. In the figure100bps DNA ladder (Norgen full ranger), lane one to nineteen contain P.1863, P.3727, P.2887, P.6, P.8486, P.13741, P.13740, K.10, -ive control, K.13, A.5054, A.13730, P.1962, P.2111, P.2255, K.11, P.12007, P.1009, P.16809 respectively.
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amplified products are of approximately100bps long confirming that these stains are
methicillin resistant Staphylococcus aureus.
Figure 18 showing the amplified product of mecA gene fragment. In the figure 18A from
left to right M is 100bps DNA ladder (Norgen full Ranger), Lane one to eleven contains
P.5053, P.1963, P.5019, P.1123, K.12, P.2006, P.14946, P.3027, P.14947, P.18176,
P.1961 respectively and in Figure 18B lane twelve to nineteen contain P.2885, P.1864,
P.13947, P.1967, P.1287, P.2119, P.6270, P.13731 and in figure 18C from left to right M
is the 100 bp DNA ladder (Norgen full Ranger) lane twenty to twenty two contains
P.13714, P.14313 and negative control respectively. All the amplified products are of
approximately100bps long confirming that these stains are methicillin resistant
Staphylococcus aureus.
Detection of Panton Valentine Leukocidin Gene in MRSA isolates from Pakistan
Panton Valentine Leukocidin is an additional exoprotein produced by only 2% of
Staphylococcus aureus isolates and belong to a toxin family known as synergy
hymenotropic toxins. Panton Valentine Lecucidin act on cell membrane in combination
with gamma haemolysin to produce a pore in the cell membrane, due this PVL is known
as the most leukocytic toxin in its family.
In the present study we screened all 123 MRSA Isolates from Pakistan for the presence of
PVL gene by the PCR. Only six isolates out of 123 showed positive result with PVL gene
detection PCR giving band size of approximately 420bp. In the figure 19 from left to
right M is the 100bp DNA ladder1 (Bio-line), lane number one to seven contain the F1
(positive control for PVL gene), A.10813, P.1286, P.16809, P.5114, P.13740. P.16849
and negative control respectively. The remaining 117 isolates did not showed
amplification; hence they were confirmed as PVL negative.
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Figure 18 (A, B, C)
Fig. 18A Fig. 18B Fig. 18C
Electropherogram of 1.5% ethedium bromide stained gel of Pakistani isolates showing amplification of mecA gene for the identification of methicillin resistant Staphylococcus aureus. In the figure 18A from left to right M is 100bps DNA ladder (Norgen full Ranger), Lane one to eleven contains P.5053, P.1963, P.5019, P.1123, K.12, P.2006, P.14946, P.3027, P.14947, P.18176, P.1961 respectively and in Figure 18B lane twelve to eighteen contain P.2885, P.1864, P.13947, P.1967, P.1287, P.2119, P.6270 and in figure18C from left to right M is the 100 bp DNA ladder (Norgen full Ranger) lane nineteen to twenty two contains P.13731, P.13714, P.14313 and –ive control respectively.
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Figure 19
Electropherogram of 1.5% ethedium bromide stained gel of Pakistani isolates showing the PVL PCR Results with Pakistani MRSA isolates. In the figure 19 from left to right M is the 100bp DNA ladders (Bioline), lane number one to seven contain the positive control for PVL gene F1, A.10813, P.1286, P.16809, P.5114, P.13740, P.16849 and negative control respectively.
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Multilocus variable Number of Tandem Repeats Analysis
(MLVA) of MRSA Isolates from Pakistan
The identification of specific DNA sequences that repeat across the genome are termed as
variable number of tandem repeats (VNTR) and the analysis of VNTR at different loci is
known as Multilocus Variable Number of Tandem Repeats Analysis (MLVA). VNTR are
unique in their length and copy number. These repeats are present in a specific
organization and copy number with in the genome of an organism. Thus difference in the
number of repeats can be used to measure the degree of relatedness among the
individuals of a population. As these repeats are present universally, so these repeats can
be used to trace evolutionary changes in the genome.
In the present study seven loci, clfA (Clumping factor A), clfB (Clumping factor B), sdrC
(Ser-Asp-rich fibrinogen binding proteins), sdrD (Ser-Asp-rich fibrinogen binding
proteins), spa (staphylococcal protein A), sspa and sav1078 were selected for the purpose
of multiplex PCR in order to analyze Pakistani MRSA isolates on the basis of multilocus
variable number of tandem repeat (MLVA). The MVLA approach was adapted to the
analysis of these strains as a rapid and cost-effective method of establishing their
phylogenetic relationships. The choice of VNTR was based on established MLVA
protocols (Sabat et al., 2003; Francois et al., 2005, Gilbert et al., 2006). In the present
study we used the fluorescent labeled primers in order to perform capillary
electrophoresis assay on ABI gene sequencer as given in material and methods.
These primers were multiplexed in two separate PCR reactions. ClfA, sdrC and sav1078
primers were multiplexed together separately from ClfB, sdrD, spa, sspa in order to
prevent band overlap and to avoid the fake band formation. Before capillary
electrophoresis the amplified product were also checked on polyacrylamide gel stained
with ethedium bromide in order to check the amplification. Some of representative
figures are shown here.
Figure 20 (A, B) is showing multilocus variable number of tandem repeats (MLVA)
results of clfB, sdrD, spa and sspa with thirty Pakistani MRSA strains. In this figure,
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65
Figure 20 (A, B)
Figure 20A
Figure 20B
Showing MLVA results of ClfB, sdrD, spa sspa with thirty Pakistani MRSA strain. In this figure 20A M is the 100bp DNA ladder, lane 1 to 19 are the strains NCTC 8325 (positive control), P.13729, P.15415, P.2255, P.12285, P. 18339, A.5054, P. 1962, P.13749, P.12007, P.1009, P.16809, P.3027, P.1961, P.1963, P.14946, P.1864, P.844, P.843. Similarly in the figure 20B, M is the 100bp DNA ladder (Norgen DNA Full Ranger), lane twenty to thirty one contain P.10013, P.1003, P.13741, P.1843, P.1896, P.8486, A.25356, A.465, P.2886, A.14075,A 13282 and P.1863 respectively. In each lane from top to bottom are the amplified product of ClfB, sdrD, spa and sspa respectively.
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M is the 100bp DNA ladder, lane 1 to 19 contain the strains NCTC 8325 (positive
control), P.13729, P.15415, P.2255, P.12285, P. 18339, A.5054, P. 1962, P.13749,
P.12007, P.1009, P.16809, P.3027, P.1961, P.1963, P.14946, P.1864, P.844, P.843. In
each lane from top to bottom are the amplified product of clfB, sdrD, spa and sspa
respectively. The amplicon sizes of these isolates calculated after gene scan are given in
the table 14 (appendix-I). Similarly in the figure 20B, M is the 100bp DNA ladder
(Norgen DNA Full Ranger), lane twenty to thirty one contain P.10013, P.1003, P.13741,
P.1843, P.1896, P.8486, A.25356, A.465, P.2886, A.14075,A 13282 and P.1863. In each
lane from top to bottom are the amplified product of clfB, sdrD, spa and sspa
respectively. The amplified product sizes of these isolates calculated after gene scan are
given in the table 14 (appendix-I).
Similarly the results obtained with an other thirty seven Pakistani MRSA isolates in
second multiplex PCR for the clfA, sdrC, sav1078 are shown in the figure 21 (A, B). In
this figure 21A, M is the 100bp DNA ladder (Norgen DNA Full Ranger,) lane number
one to seventeen contain the strains NCTC8325, A.465, A.26163, A.14254, A.13282,
A.1964, A.5054, P.1962, P.1123, P.12007, K.12, P.1009, P.16809, P.18176, P.5019,
A.5053, P.14947 respectively. In each lane from top to bottom are the amplified product
of clfA, sdrC and sav1078 respectively. Their amplified product sizes calculated after
gene scan by using ABI Peak scan are given in the table 14 (appendix-1).
Figure 21B is showing MLVA results of clfA, sdrC, sav1078 multiplex PCR with
fourteen Pakistani MRSA strains. In this figure M is the 100 bp DNA ladder, lane
number 18 to 31 contain P1, P.9703, P.12006, P.1286, P.11241, 15330, P.1005, P.2054,
P3, A.9914, P.1967,P.13729, 13730, P.14946 respectively. In each lane from top to
bottom are the amplified product of clfA, sdrC and sav1078 respectively. Their amplified
product sizes calculated by gene scan are given in the table 14 (appendix-I).
Figure 20 and 21 shows that it is difficult to calculate the exact amplicon sizes on the gel
by comparing with the DNA ladder. Because some time the amplicon from two loci over
lap each other or the difference between them is very minute to identify them. Due to this
we used the fluorescently labeled primers and so the PCR products obtained after two
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67
Figure 21 (A, B)
Figure 21A
Figure 21B
Showing MLVA results of ClfA, sdrC, sav1078 with Pakistani MRSA strain. In the figure 15A, M is the 100bp DNA ladder (Norgen DNA Full Ranger,) lane number one to seventeen contain the strains NCTC8325, A.465, A.26163, A.14254, A.13282, A.1964, A.5054, P.1962, P.1123, P.12007, K.12, P.1009, P.16809, P.18176, P.5019, A.5053, P.14947 respectively. Similarly Figure 15B is showing MLVA results of clfA, sdrC, sav1078 multiplex PCR with fourteen Pakistani MRSA strains. In this figure M is the 100 bp DNA ladder, lane number 18 to 31 contain P1, P.9703, P.12006, P.1286, P.11241, 15330, P.1005, P.2054, P3, A.9914, P.1967, P.13729, 13730, P.14946 respectively. In each lane from top to bottom are the amplified product of ClfA, sdrC and sav1078 respectively.
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multiplex PCR were subjected to capillary electrophoresis by using the ABI DNA
sequencer along with fluorescently labeled LIZ 1200 ABI DNA ladder. The data obtained
from capillary electrophoresis in the form of peaks was analyzed by using ABI Peak
scanner V 1.0. and the amplicon sizes obtained for each locus for each isolate are
tabulated in the table 14 (appendix-I).
Figure 22 is showing MLVA results of ClfB, sdrD, spa, sspa with a representative
Pakistani MRSA strain P.843, In the figure orange peaks are of Liz1200 marker, while
from left to right black peak is of sspa, blue is of spa, mixed colored peak is of sdrD and
green peak is of ClfB. On y-axis in the figure is the height of peaks while x-axis is
showing the size of amplicons in bp. The amplicon sizes are 935bp, 756bp, 276bp and
155bp for ClfB, sdrD, spa and sspa respectively.
Figure 23 is showing MLVA results of ClfB, sdrd, spa sspa with with a representative
Pakistani MRSA strain P.2285, In the figure orange peaks are of Liz1200 marker, while
from left to right black peak is of sspa, blue peak is of spa, mixed colored peak is of sdrD
and green peak is of ClfB. On y-axis in the figure is the height of peaks while x-axis is
showing the size of amplicons in bp. The amplicon sizes are 902bp, 756bp, 276bp and
155bp for ClfB, sdrD, spa and sspa respectively.
Figure 24 is showing MLVA results of ClfB, sdrD, spa sspa with with a representative
Pakistani MRSA strain A. 9313, In the figure orange peaks are of Liz1200 marker, while
from left to right black peak is of sspa, blue is of spa, mixed colored peak is of sdrD and
green peak is of ClfB. On y-axis in the figure is the height of peaks while x-axis is
showing the size of amplicons in bp. The amplicon sizes are 992bp, 756bp, 229bp and
155bp for ClfB, sdrD, spa and sspa respectively.
Figure 25 is showing MLVA results of ClfB, sdrd, spa sspa with Pakistani with a
representative MRSA strain P.1. In the figure orange peaks are of Liz1200 marker, while
from left to right black peak is of sspa, blue peak is of spa, mixed colored peak is of sdrD
and green peak is of ClfB. On y-axis in the figure is the height of peaks while x-axis is
showing the size of amplicons in bp. The amplicon sizes are 884bp, 704bp, 229bp and
155bp for ClfB, sdrD, spa and sspa respectively.
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Figure 22
Showing MLVA results of ClfB, sdrD, spa and sspa with a representative Pakistani MRSA strain P.843, In the figure orange peaks are of Liz1200 marker, while from left to right black peak is of sspa, blue is of spa, mixed colored peak is of sdrD and green peak is of ClfB. On y-axis in the figure is the height of peaks while x-axis is showing the size of amplicons in bp.
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Figure 23
Showing MLVA results of ClfB, sdrd, spa and sspa with a representative Pakistani MRSA strain P.2285. In the figure orange peaks are of Liz1200 marker, while from left to right black peak is of sspa, blue peak is of spa, mixed colored peak is of sdrD and green peak is of ClfB. On y-axis in the figure is the height of peaks while x-axis is showing the size of amplicons in bp.
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Figure 24
Showing MLVA results of ClfB, sdrD, spa and sspa with a representative Pakistani MRSA strain A. 9313. In the figure orange peaks are of Liz1200 marker, while from left to right black peak is of sspa, blue is of spa, mixed colored peak is of sdrD and green peak is of ClfB. On y-axis in the figure is the height of peaks while x-axis is showing the size of amplicons in bp
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Figure 25
Showing MLVA results of ClfB, sdrd, spa and sspa with a representative Pakistani MRSA strain P.1. In the figure orange peaks are of Liz1200 marker, while from left to right black peak is of sspa, blue peak is of spa, mixed colored peak is of sdrD and green peak is of ClfB. On y-axis in the figure is the height of peaks while x-axis is showing the size of amplicons in bp.
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For the CLFA loci we got nine different kinds of amplicions in 123 Pakistani isolates
after gene scan analysis which was 1062bp (4 isolates), 1045bp (20 isolate), 1027bp (3
isolates), 953 bp (7 isolates), 900bp (52 isolates), 882bp (15 isolates), 864bp (1 isolate),
846bp (19 isolates) and 540bp (2 isolates). It means that with respect to ClfA loci there
are nine groups of isolates with similar amplicon sizes Table 14 (appendix-I).
In case of ClfB gene loci we got eight different kinds of amplicons with all of 123
Pakistani MRSA isolates. Thèse amplicon sizes were 1102bp (2 isolates), 992bp (2
isolates), 974bp (12 isolates), 935bp (51 isolates), 902bp (28 isolates) 884bp (16 isolates),
864bp (11 isolates) and 822bp (2 isolates). Thus on the basis of amplicon sizes of clfA
gene locus there were eight different groups of MRSA isolates present in Pakistani
MRSA isolates.
In case of sdrD gene locus six different types of amplicons were obtained with Pakistani
MRSA isolates. These amplicon sizes were 756bp (84 isolates), 741bp (6 isolates), 704bp
(29 isolates), 641bp (2 isolates), 595bp (1 isolate) and 501bp (1 isolate). In this only on
the basis of sdrD gene, we got six groups of isolates. Similarly incase of sdrC gene we
got seven different amplicon sizes. These were 658bp (1 isolate) 560bp (29 isolates),
548bp (72 isolates), 524bp (1), 506 (10), 481bp (5 isolates) and 324bp (5 isolates). Thus
with respect to amplicons sizes of sdrC locus, there were seven different groups of
isolates.
Similar results were found in case of spa locus. In case of spa gene, six different
amplicon sizes were obtained. These amplicon were of 276bp (62 isolates), 253bp (9
isolate), 229bp (10 isolates), 205bp (2 isolates), 181bp (32 isolates) and 157bp (8
isolates). Thus if the all Pakistani MRSA isolates were grouped only on the basis of
amplicon of spa locus, the six groups of isolates were present as given in table 14
(appendix-I).
In case of sspa gene locus we got only 155bp amplicon size with all the MRSA isolates
from Pakistan and same was the case sav1078 locus. We got 317bp amplicon with all 123
Pakistani MRSA isolates. Thus when we consider each locus for typing of isolates, only
limited number of groups were present.
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As in MLVA typing a combination of different loci is used to group epidemiologically
similar isolates. So a combination of six isolates as used by Sabat et al., 2003, from these
six loci was used for converting the amplicon sizes into the repeat numbers in order to get
a broader view for phylogentical analysis. Sav1078 was not used in clustering as all the
isolates were similar in PCR product.
The fragment sizes obtained for each locus for all the isolates were converted into the
repeats number. For this purpose the sequence data for the standard strains NCTC8325
and MU50 and sequences of one of Pakistani MRSA isolate P.18431were imported into
the Tandem Repeats Finder, a Program in Bioinformatics written by Gary Benson,
Boston Version 4.0 (Benson G, 1999; http://tandem.bu.edu/trf.html). The software
identified the number of repeats present in the DNA sequences of the seven loci clfA,
clfB, sdrD, sdrC, spa, sspa and sav1078. The size of the flanking regions was assessed by
substracting the repeat containing region from the total size of the amplicon. In this way
we got the length of flaking region, length of VNTR region, and size of repeat unit from
the Tandem repeat finder version 4.0. Then this approach was used to calculate the
number of repeats present in the PCR products obtained from other MRSA isolates. Total
number of repeats was calculated by using the formula:- size of the PCR product minus
size of the flanking region divided by the size of the repeat unit. In this way we got the
number of VNTR for all the loci which are given in the table 15 (appendix-I).
Table 15 (appendix-1) is showing the IDs of isolates, then the amplicon sizes obtained by
gene scan, length of VNTR region present in these amplicons and total number of repeats
as calculated by using the formula mentioned in material and methods. In this way the
number of repeats obtained for six loci was used to generate allelic profile.
In order to draw the minimum spanning tree (Phylogenetic tree), the repeats obtained
with six loci for each isolate were feed into the Bionumeric program. The order of allelic
profile fed into the Bionumeric was clfA-clfB-sdrD-sdrc-spa-sspa. Minimum spanning
tree of 123 Pakistani MRSA isolates derived from VNTRs of clfA, clfB, sdrD, sdrC, spa
and sspa loci using Bionumerics software is shown as figure 26. In the phylogenetic tree
circle sizes are proportional to number of genotypes present in each node while the color
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75
Blood
Catheter tip
Ear infection
Pus
Spinal
SputumStandard Strain
Tissue
Urine
Figure 26
Minimum spanning tree of 123 Pakistani MRSA isolates derived from VNTRs for clfA, clfB, sdrD, sdrC, spa and sspa loci using Bionumerics software. Circle sizes are proportional to number of MTs/haplotype/genotype in each node while the colour represents the sources of each isolate. The isolates varying by repeat at a single locus are connected with bold lines while isolates varying at two or more loci are shown connected with thin lines. Positive control strains are shown connected with dotted lines. The number on the nodes represents the type of MTs/genotype.
121212121212121212
181818181818181818
131313131313131313
151515151515151515
111111111111111111
111111111
666666666272727272727272727
141414141414141414
282828282828282828
292929292929292929
343434343434343434
212121212121212121
252525252525252525
232323232323232323
242424242424242424
191919191919191919
202020202020202020
171717171717171717
555555555
333333333
888888888
303030303030303030
222222222222222222
161616161616161616
222222222777777777
444444444999999999
353535353535353535333333333333333333
414141414141414141
444444444444444444
515151515151515151
262626262626262626
525252525252525252
555555555555555555
505050505050505050
313131313131313131
535353535353535353
474747474747474747
101010101010101010
464646464646464646
545454545454545454
484848484848484848
565656565656565656
494949494949494949
393939393939393939
424242424242424242
404040404040404040
454545454545454545
363636363636363636
383838383838383838
323232323232323232
616161616161616161
575757575757575757
595959595959595959 606060606060606060
585858585858585858
373737373737373737
434343434343434343
656565656565656565
626262626262626262
636363636363636363
646464646464646464
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represents the sources isolation of each isolate. Such as yellow color represents the isolates taken
from pus and red color represents the isolates taken from catheter tip. The isolates varying by
repeat at a single locus are connected with bold lines while isolates varying at two or more loci
are shown connected with thin lines. Positive control strains are shown connected with dotted
lines. The number on the nodes represents the total number of genotypes. All the 123 MRSA
isolates clustered into the 63 MLVA type or genotypes with out applying the cut off i.e.
similarity between repeats at six loci was considered 100%. In figure 26, it is clear that these
isolates are closely linked with each other as majority of the MLVA type are connected with bold
lines and it is also clear in the figure that most of the genotypes are originating from genotype
twelve.
The allelic profile was also used to draw the dendrogram. The dendrogram obtained from the
MLVA types (genotypes) of the 123 Pakistani MRSA isolates and two standard strains is shown
in figure 27. The dendrogram construction method was UPGMA with a cluster cut-off of 80%
and a categorical distance coefficient. Strain names, source of isolate and time of isolation are
also indicated in the figure. All the 123 Pakistani MRSA were clustered into 63 genotypes at
100% similarity (with out cut off) as shown in the dendrogram. Following genotypes were
obtained after clustering. Genotype one contain three isolates P.2113, P.10021and P.13714,
Genotype 2 contains one isolate P.10019 , Genotype 3 contain one isolate P.2111, Genotype 4
contains one isolate P.6270, Genotype 5 contain two isolates P.2119 & P13749, Genotype 6
contain two isolates P.1003& P10013, Genotype 7 has one isolate P.5123, Genotype 8 has one
isolate P.10012, Genotype 9 has one isolate P.11291, Genotype 10 has one isolate P.12007,
Genotype 11 has two isolates K13, 13729 and Genotype 11 has isolates, Genotype 12 has three
isolates P.2112, P.1966 and P.13319, Genotype 13 has ten isolates P.843, P.844, P.1843, P.5020,
P.8485, P.8486, P.8487, P.11296, P.18431 and P.14254. Genotype 14 has three isolates P.6269,
P.3027 and P.17120, Genotype 15 has one isolates A.14075, Genotype 16 has one isolate
P.18119, Genotype 17 has five isolates A.9077, A.9445, A.9781, A.14256, A.25356 isolates.
Genotype 18 has three isolate K.8, A.9914 and A.15330. Genotype 19 has one isolates A.35087,
Genotype 20 has isolates P.8823, Genotype 21 has one isolates A.9954, Genotype 22 has one
isolate P.17121, Genotype 23 has one isolates P.13947, Genotype 24 has isolate P.1967,
Genotype 25 has 6 isolates P.6, P.862, P.1863, P.1864, P.1963 and 1964, Genotype 26 has one
isolates P.1961, Genotype 27 has 2 isolates P.14947 & A.7145, Genotype 28 has 2 isolates
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P.2006 & P.2887, Genotype 29 has three isolates P.1962 K.10 and P.3727, Genotype 30 has
three isolates P.2885, P.2896 and P.8431, Genotype 31 has one isolate P.8, Genotype 32 has two
isolates A.9313 & A.10813, Genotype 33 has one isolates P.15415, Genotype 34 has one isolates
P.7, Genotype 35 has isolates one isolates P.10015, Genotype 36 has one isolates P.5019,
Genotype 37 has one isolates P.2255, Genotype 38 has one isolates , Genotype 39 has six
isolates P.14946, A.5053,A.5054,A.9703, A.11241 and A.13730, Genotype 40 has one isolate
isolates P.2054, Genotype 41 has one isolates P.3, Genotype 42 has one isolates P.18176,
Genotype 43 has one isolates K.12, Genotype 44 has one isolate P.1286 , Genotype 45 has one
isolates P.16809, Genotype 46 has three isolates P.11293, P.13740,P.13741, Genotype 47 has
eleven P.1129, P.1005, P.1009, P.5119, P.12285, P.2285, A.465,P.16509, P.16849, P.18339 and
A.35045 isolates, Genotype 48 has one isolates P.5114, Genotype 49 has one isolates P.1297
Genotype 50 has one isolates P.5, Genotype 51 has one isolate K.6, Genotype 52 has two K.11 &
P.13731 isolate, Genotype 53 has two isolate P.4 & P.1287, Genotype 54 has two isolate P.8668
& K.3, Genotype 55 has two isolates P.2886& P..8116, Genotype 56 has one isolate P.1896,
Genotype 57 has one isolate P.1761, Genotype 58 has one isolate P.1123, Genotype 59 has four
isolate P.1, P.12006, A.9965 and A.26163, Genotype 60 has one isolate P.2871, genotype 61 has
one isolate A.379, Genotype 62 has one isolate P.14313 and Genotype 63 has one isolate
A.13282. Thus there were 63 genotypes present in Pakistani MRSA isolates when we assigned a
different genotype or MLVA type to them if they vary by repeat at one locus from each other.
But when 80% cut off was applied, 63 genotypes clustered into 18 groups as shown in the figure-
27. For example from figure 27 at 80% cut off, the Genotype 1,2, 3 and 4 cluster together to
form a group1. Similarly Genotype 5, 6, 7, 8 clustered together to make another group 2,
Genotype 9,10 clustered together to make group 3, Genotype 11, 12 , 13, 14, 15 clustered into
group 4, Genotype 17, 18, 19, 20 clustered into group 5 , Genotype 21, 22 clustered into group 6
, Genotype 23, 24, 25, 26 cluttered into group 7 genotype , Genotype 27, 28, 29 clustered into
group 8 , Genotype 30, 31 cluster into groups 9, Genotype 33,34 clustered into groups 10,
Genotype 39, 40, 41 clustered into group 11, Genotype 44, 45 clustered into group12, Genotype
46, 47 clustered into group 13, Genotype 50 ,51 clustered into group 14, Genotype 52, 53
clustered into group 15, Genotype 54, 55 clustered into group 16, genotype 59,60 clustered into
group 17 and genotype 62, 63 clustered into group 18 while the Genotype 16, 32, 35,36,37,38,
42,43,48,49, 56, 57,58,61 did not cluster into any group after 80% cut off.
Chapter 3 Results
78
Genotype 13 and Genotype 47 are large clusters of 10 and 11 identical isolates with all these
isolates being derived from pus and from Pakistan Institute of Medical Science Hospital. This
clustering of large number of isolates is suggestive of a common source of infection. Along with
two large clusters of isolates, smaller clusters of isolates with a common source were also
detected. Thus, Genotype 17 consisted of five blood isolates, Genotype 25 of six urine isolates
and Genotype 30 of three spinal cord isolates. These isolates are indicative of clusters of MRSA
infection cases. Thus MLVA typing clustered the isolates with similar genetic make up and
similar kind of infections into same genotype as shown in figure 27.
Chapter 3 Results
79
Figure 27
Dendrogram base on clustering with respect to MLVA types of Pakistani MRSA Isolates with similarity calculated by Dice coefficient and represented by UPGMA. 63 haplotypes are shown at 100% similarity, which clustered into 18 groups when 80%cut off technique was used.
MLVA
100
80
60
40
20
MLVA
ClfA
ClfB
sdrD
sdrC
spa
sspa
37 41 30 24 9 4
37 41 30 24 9 4
37 41 30 24 9 4
40 41 30 24 9 4
36 41 30 24 9 4
35 41 30 24 9 4
36 41 30 24 8 4
36 41 30 24 8 4
37 41 30 24 8 4
37 41 30 24 8 4
40 41 30 24 8 4
34 41 30 24 8 4
37 41 30 25 9 4
37 41 30 25 6 4
40 41 30 24 10 4
40 41 30 24 10 4
37 41 30 24 10 4
37 41 30 24 10 4
37 41 30 24 10 4
34 41 30 24 10 4
34 41 30 24 10 4
34 41 30 24 10 4
34 41 30 24 10 4
34 41 30 24 10 4
34 41 30 24 10 4
34 41 30 24 10 4
34 41 30 24 10 4
34 41 30 24 10 4
34 41 30 24 10 4
46 41 30 24 10 4
46 41 30 24 10 4
46 41 30 24 10 4
36 41 30 24 10 4
34 41 30 25 10 4
45 41 30 12 10 4
45 41 30 12 10 4
45 41 30 12 10 4
45 41 30 12 10 4
45 41 30 12 10 4
45 41 30 24 10 4
45 41 30 24 10 4
45 41 30 24 10 4
45 41 30 23 10 4
45 41 30 30 10 4
37 41 30 20 10 4
36 41 30 20 10 4
45 43 30 24 10 4
45 39 30 24 10 4
45 37 30 24 10 4
45 37 30 24 10 4
45 37 30 24 10 4
45 37 30 24 10 4
45 37 30 24 10 4
45 37 30 24 10 4
35 37 30 24 10 4
37 43 30 24 10 4
37 43 30 24 10 4
37 38 30 24 10 4
37 38 30 24 10 4
37 37 30 24 10 4
37 37 30 24 10 4
37 37 30 24 10 4
n°
42
43
44
72
67
70
52
53
48
49
69
73
68
74
56
57
39
40
41
20
21
22
23
24
25
26
27
28
29
10
11
12
66
71
113
114
115
116
117
7
8
9
13
14
63
64
15
16
1
2
3
4
5
6
75
54
55
50
51
36
37
38
Strains
P.2113
P.10021
P.13714
P.10019
P.2111
P.6270
P.2119
P.13749
P.1003
P.10013
P.5123
P.10012
P.11291
P.12007
K.13
P.13729
P.2112
P.1966
A.13319
P.843
P.844
P.1843
P.5020
P.8485
P.8486
P.8487
P.11296
P.18431
A.14254
P.6269
P.3027
P.17120
A.14075
P.18119
A.9077
A.9445
A.9781
A14256
A.25356
K.8
A.9914
A.15330
A.35087
P.8823
A.9954
P.17121
P.13947
P. 1967
P.6
P.862
P.1863
P.1864
P.1963
P.1964
P.1961
P.14947
A.7145
P.2006
P.2887
P.1962
P.3727
K10
Source
Sputum
Sputum
Pus
Pus
Catheter tip
Catheter tip
Pus
Pus
Tissue fluid
Tissue fluid
Catheter tip
Sputum
Pus
Sputum
Catheter tip
Catheter tip
Sputum
Sputum
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Catheter tip
Catheter tip
Catheter tip
Catheter tip
Catheter tip
Blood
Blood
Blood
Blood
Blood
Catheter tip
Pus
Catheter tip
Pus
Pus
Catheter tip
Catheter tip
Catheter tip
Catheter tip
Urine
Urine
Urine
Urine
Urine
urine
Sputum
Pus
Pus
Tissue fluid
Tissue fluid
Sputum
Sputum
Sputum
Period
Aug,2006
July,2007
Aug,2007
July,2007
Aug,2006
Dec,2006
Aug,2006
Aug,2007
May,2006
July,2007
Oct,2006
July,2007
July,2007
July,2007
Aug,2007
Aug,2007
Aug,2006
July,2006
Jan,2008
May,2006
May,2006
June,2006
Oct,2006
April,2007
April,2007
April,2007
July,2007
Sept,2007
Jan,2008
Dec,2006
Aug,2007
Sept,2007
Jan,2008
Sept,2007
Nov,2007
Nov,2007
Dec,2007
Jan,2008
févr-08
July,2007
Dec,2007
Feb,2008
Feb,2008
May,2007
Dec.2007
Sept,2007
Aug,2007
July,2006
April,2006
May,2006
June,2006
June,2006
June,2006
July,2006
July,2006
Aug,2007
Oct,2007
Aug,2006
Aug,2006
July,2006
Aug,2006
Aug,2007
genotype
1
1
1
2
3
4
5
5
6
6
7
8
9
10
11
11
12
12
12
13
13
13
13
13
13
13
13
13
13
14
14
14
15
16
17
17
17
17
17
18
18
18
19
20
21
22
23
24
25
25
25
25
25
25
26
27
27
28
28
29
29
29
isolates
Arafat042
Arafat043
Arafat044
Arafat072
Arafat067
Arafat070
Arafat052
Arafat053
Arafat048
Arafat049
Arafat069
Arafat073
Arafat068
Arafat074
Arafat056
Arafat057
Arafat039
Arafat040
Arafat041
Arafat020
Arafat021
Arafat022
Arafat023
Arafat024
Arafat025
Arafat026
Arafat027
Arafat028
Arafat029
Arafat010
Arafat011
Arafat012
Arafat066
Arafat071
Arafat113
Arafat114
Arafat115
Arafat116
Arafat117
Arafat007
Arafat008
Arafat009
Arafat013
Arafat014
Arafat063
Arafat064
Arafat015
Arafat016
Arafat001
Arafat002
Arafat003
Arafat004
Arafat005
Arafat006
Arafat075
Arafat054
Arafat055
Arafat050
Arafat051
Arafat036
Arafat037
Arafat038
Chapter 3 Results
80
Figure 27 (Continue)
37 37 30 24 10 4
37 37 30 24 10 4
37 37 30 24 10 4
34 38 30 24 10 4
34 38 30 24 10 4
34 38 30 24 10 4
34 38 30 24 6 4
17 44 30 24 8 4
17 44 30 24 8 4
37 43 27 24 10 4
37 41 27 24 10 4
37 41 27 24 8 4
45 38 27 24 10 4
40 39 27 24 5 4
34 41 27 24 5 4
36 43 30 22 10 4
36 43 30 22 10 4
36 43 30 22 10 4
36 43 30 22 10 4
36 43 30 22 10 4
36 43 30 22 10 4
36 43 30 22 8 4
37 43 30 22 10 4
36 39 30 22 10 4
36 35 30 22 5 4
40 41 24 24 9 4
40 41 24 25 9 4
37 39 29 25 6 4
37 39 29 25 6 4
37 39 29 25 6 4
37 39 27 25 6 4
37 39 27 25 6 4
37 39 27 25 6 4
37 39 27 25 6 4
37 39 27 25 6 4
37 39 27 25 6 4
37 39 27 25 6 4
37 39 27 25 6 4
37 39 27 25 6 4
37 39 27 25 6 4
37 39 27 25 6 4
46 39 27 25 6 4
36 39 29 25 6 4
37 38 30 25 6 4
37 38 30 24 6 4
37 39 30 24 6 4
37 39 30 24 6 4
37 39 30 25 6 4
37 39 30 25 6 4
37 39 27 24 6 4
37 39 27 24 6 4
37 38 27 24 6 4
37 38 27 24 6 4
34 38 27 24 6 4
37 39 21 21 6 4
45 39 29 24 6 4
37 38 27 25 5 4
37 38 27 25 5 4
37 38 27 25 5 4
37 38 27 25 5 4
34 38 27 25 5 4
37 29 30 20 6 4
44 50 27 20 7 4
44 50 16 20 7 4
44 35 25 36 11 4
45 35 31 29 10 5
36
37
38
45
46
47
105
120
121
58
60
62
17
108
111
30
31
32
33
34
35
59
61
65
109
122
123
91
92
93
76
77
78
79
80
81
82
83
84
85
86
19
102
103
104
96
97
94
95
100
101
98
99
107
110
18
87
88
89
90
106
112
118
119
124
125
P.1962
P.3727
K10
P.2885
P.2896
P.8431
P.8
A.9313
A.10813
P.15415
P.7
P.10015
P. 5019
P.2255
P.2
P.14946
A.5053
A.5054
A.9703
A.11241
A.13730
P.2054
P.3
P.18176
K.12
P.1286
P.16809
P.11293
P.13740
P.13741
P.1129
P.1005
P.1009
P.5119
P.12285
P.2285
A.465
P.16509
P. 16849
P.18339
A.35045
P. 5114
P.1297
P.5
K.6
K.11
P.13731
P.4
P.1287
P.8668
K.3
P.2886
P.8116
P.1896
A.1761
P. 1123
P.1
P.12006
A.9965
A.26163
P.2871
A.379
P.14313
A.13282
NCTC 8325
Mu 50
Sputum
Sputum
Sputum
Spinal Card
Spinal Card
Spinal Card
Ear Infection
Pus
Sputum
Catheter tip
Pus
Sputum
Catheter tip
Pus
Pus
Sputum
Catheter tip
Catheter tip
Catheter tip
Pus
Pus
Catheter tip
Pus
Catheter tip
Pus
Sputum
Sputum
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Pus
Catheter tip
Ear Infection
Sputum
Sputum
Pus
Pus
Pus
Pus
Ear infection
Ear infection
Catheter tip
Catheter tip
Ear infection
Ear infection
Catheter tip
Pus
Pus
Pus
Pus
Sputum
Ear infection
Pus
Pus
Standard Strain
Standard Strain
July,2006
Aug,2006
Aug,2007
Aug,2006
Aug,2006
April,2007
April,2006
Nov,2007
Jan,2008
Aug,2007
April,2006
July,2007
Oct,2006
Aug,2006
April,2006
Aug,2007
Sept,2007
Sept,2007
Dec,2007
Jan,2008
Jan,2008
Aug,2006
April,2006
Sept,2007
Aug,2007
May,2006
Sept,2007
July,2007
Aug,2007
Aug,2007
May,2006
May,2006
May,2006
Oct,2006
July,2007
Aug,2006
Aug,2007
Sept,2007
Sept,2007
Sept,2007
Feb,2008
Oct,2006
May,2006
April,2006
July,2007
Aug,2007
Aug,2007
April,2006
May,2006
May,2007
July,2007
Aug,2006
Dec,2006
June,2006
Sept,2007
May,2006
April,2006
July,2007
Dec,2007
Feb,2008
Aug,2006
Aug,2007
Aug,2007
Jan,2008
29
29
29
30
30
30
31
32
32
33
34
35
36
37
38
39
39
39
39
39
39
40
41
42
43
44
45
46
46
46
47
47
47
47
47
47
47
47
47
47
47
48
49
50
51
52
52
53
53
54
54
55
55
56
57
58
59
59
59
59
60
61
62
63
64
65
Arafat036
Arafat037
Arafat038
Arafat045
Arafat046
Arafat047
Arafat105
Arafat120
Arafat121
Arafat058
Arafat060
Arafat062
Arafat017
Arafat108
Arafat111
Arafat030
Arafat031
Arafat032
Arafat033
Arafat034
Arafat035
Arafat059
Arafat061
Arafat065
Arafat109
Arafat122
Arafat123
Arafat091
Arafat092
Arafat093
Arafat076
Arafat077
Arafat078
Arafat079
Arafat080
Arafat081
Arafat082
Arafat083
Arafat084
Arafat085
Arafat086
Arafat019
Arafat102
Arafat103
Arafat104
Arafat096
Arafat097
Arafat094
Arafat095
Arafat100
Arafat101
Arafat098
Arafat099
Arafat107
Arafat110
Arafat018
Arafat087
Arafat088
Arafat089
Arafat090
Arafat106
Arafat112
Arafat118
Arafat119
Arafat124
Arafat125
Chapter 3 Results
81
Lineage Determination by Using RM Test Most Staphylococcus aureus strains belong to one of ten dominant clonal complexes
(CC) Lineages. Each clonal complex has specific external surface proteins and structures
that are responsible for interaction with the host. The restriction modification (RM) test is
a simple and reliable method for typing HA-MRSA. Restriction modification test
identifies six clonal complexes CC1, CC5, CC8, CC22, CC30 and CC45. Thus it
provides the single most important piece of information about the lineage/CC that
correlates with variation in hundreds of genes. The RM test is based on primers that target
the hsd S gene of sau1 type 1 restriction modification system. Three separate PCR
reactions with one forward and two reverse primers are used in RM test which may result
a large amplicon, a small amplicon or no amplification at all. These three PCR assays are
termed as RM1, RM2 and RM3.
Pakistani MRSA isolates (n=123) were typed with restriction modification tests. It was
observed that 121 isolates out of 123 isolates showed the amplification only with RM3
and no amplification with RM2 and RM1 test. Only two isolated showed amplification in
RM-1 test. The results are presented in figure 28 through figure 33.
This figure 28 (A, B, C) shows the amplicons obtained from three RM test with Pakistani
Staphylococcus aureus isolates. The figure 28A is showing RM1, from left to right M is
100 bp DNA ladder (Norgen Full Ranger); lanes 1 to 9 contain MRSA252 [Positive
control for RM1], NCTC8325, P.18431, A.14256, K.3, P.2113, P.2112, A.26163 and a
negative control, respectively. The figure 28B is showing RM2; lanes 1 to 9 contain
CDC-8 [Positive control for RM2], NCTC8325, P.18431, A.14256, K.3, P.2113, P.2112,
A.26163 and a negative control. Similarly figure 28c is showing RM3 results, from left to
right, lanes 1 to 9 contain NCTC8325, Mu50 [Positive controls for RM3], P.18431,
A.14256, K.3, P.2113, P.2112, A.26163 and a negative control respectively. All the
Pakistani MRSA isolates showing positive results with in an RM3 test giving a PCR
product size of 680 bp, indicative of CC8, while Mu50 has a 1071 bp amplicon size
indicative of CC5.
Chapter 3 Results
82
Figure 28 (A, B, C)
Fig. 28A Fig. 28B Fig. 28C
RM tests with Pakistani Staphylococcus aureus (MRSA) isolates. This figure shows the amplicons obtained from three RM test with Pakistani Staphylococcus aureus isolates:- RM1 (Fig. A); RM2 (Fig. B); and RM3 (Fig. C). Fig. A:- M, 100 bp DNA ladder (Norgen Full Ranger); lanes 1 to 9 contain MRSA252 [Positive control for RM1], NCTC8325, P.18431, A.14256, K.3, P.2113, P.2112, A.26163 and a negative control, respectively. Fig. B:- lanes 1 to 9 contain CDC-8 [Positive control for RM2], NCTC8325, P.18431, A.14256, K.3, P.2113, P.2112, A.26163 and a negative control. Panel C:- lanes 1 to 9 contain NCTC8325, Mu50 [Positive controls for RM3], P.18431, A.14256, K.3, P.2113, P.2112, A.26163 and a negative control. All the Pakistani MRSA isolates showing positive results with in an RM3 test giving a PCR product size of 680 bp, indicative of CC8, while Mu50 has a 1071 bp amplicon size indicative of CC5.
Chapter 3 Results
83
Similar kinds of results were obtained for all the other 115 MRSA isolates from Pakistan.
Two isolates out of 123 Pakistani isolates showed amplification in RM1 test, so only the
gel pictures showing the RM3 test are included in the results.
Thus Restriction modification three (RM3) results with results with fifty of the Pakistani
Staphylococcus aureus strains are shown in figure number 29 (A, B, C). In Figure 28A
from left to right, M is 100bp DNA ladder (Norgen Full Ranger), Lane one to twenty one
is showing the RM3 results with standard strain 8325-4, Mu50 and Pakistani strains
P.10019, P.2054, P.5123, P.13729, P.2896, P.2886, P.1896, P.5, P.10015, P.12006, P.
8431, P.10012, P10021, P. 18119, P.1966, P.1, K.6, P.8, P.8487, -ive control
respectively. In the figure 29B from left to right M is the 100 DNA ladder (Norgen Full
Ranger), Lane twenty two to forty two contains P.17120, P.6269, P.11293, P.17121,
P.13749, A.9965, P.8668, P.1297, P.16509, P.16849, P.12285, P.1129, P.15415, P.18339,
P.8116, P.2285, P.1964, P.5119, P.11291, A.1761, A.35087 respectively and in the figure
29C, from left to right M is the 100 bp DNA ladder A.9313, A.9914, A.9077, A.9954,
A.14075, A.11241, A.25356 A.10813, A.9445, A.35045 and negative control
respectively. All the fifty isolates from Pakistan showed amplification only with in RM3
thus confirming as CC8
Similarly the amplified product of 19 Pakistani Staphylococcus aureus isolates with RM3
test are shown in Figure. 30A. In the figure from left to right M is the 100bps DNA
ladder, lane number one to eleven contains NCTC8325, Mu50( positive control for
RM3), A.9703, A.13319, A.7145, P.3, P.4, P.7, K.8, P.8485, P.2 respectively and In the
figure 30B from left to right M is 100bps DNA ladder (Norgen Full Ranger), lane number
twelve to twenty two P.2871, P.5020, P.5114, P.1005, P.844, P.843, P.1843, A.8823
A.9781, A465, , and negative -ive control respectively.
Figure 31 (A, B, C) is showing the amplified product of RM3 with thirty three Pakistani
Staphylococcus aureus strains. In the figure 31A from left to right M is the 100bp DNA
ladder (Norgen full Ranger), Lane one to eighteen contains NCTC8325, Mu50, A.14254,
A.15330, P.11296, P.862, A.379, P.1286, A.13282, P.10013, P.16809, P.1003, P.1863,
P.3727, P.2887, P.6, P.8486, P.13741,. In the figure 31B from left to right M is the 100bp
Chapter 3 Results
84
DNA ladder (Norgen full Ranger), Lane 19 to 29 contains P.13740, K.10, K.13, A.5054,
A.13730, P.1962, P.2111, P.2255, K.11, P.12007, P.1009, respectively and In the figure
31C from left to right M is the 100bp DNA ladder, lane thirty to thirty six contain,
A.5053, P.3027, P.14947, P.18176, P.1961, P.1963, and negative control respectively.
All the strains showed amplification for RM3 except two strains, P.1286 and P.16809.
These two strains showed positive results with RM1 and negative with RM2 and RM3
(figure 32), confirming CC30.
Figure No.33A is showing the results of RM3 with Pakistani Staphylococcus aureus
strains. In the figure from left to right M is the 100bp DNA ladder (Norgen full Ranger),
lane one to ten contains NCTC 8325, Mu50, P.5019 P.1123, K.12, P.2006, P.14946,
P.2885, P.1864, P.13947 and in Figure No.19b, from left to right P.1967, P.1287,
P.13714, P.14313, P.2119, P.6270, P.13731 and negative control and negative control.
Chapter 3 Results
85
Figure 29 (A, B, C)
Fig. 29A Fig. 29B
Fig. 29C
RM3 test Results with Pakistani Staphylococcus aureus MRSA isolates. In the figure 29A, M is 100bp DNA ladder (Norgen Full Ranger), Lane one to twenty one is showing the RM3 results with standard strain 8325-4, Mu50 and Pakistani strains P.10019, P.2054, P.5123, P.13729, P.2896, P.2886, P.1896, P.5, P.10015, P.12006, P. 8431, P.10012, P10021, P. 18119, P.1966, P.1, K.6, P.8, P.8487, -ive control respectively. In the figure 29B from left to right M is the 100 DNA ladder (Norgen Full Ranger), Lane twenty two to forty two contains P.17120, P.6269, P.11293, P.17121, P.13749, A.9965, P.8668, P.1297, P.16509, P.16849, P.12285, P.1129, P.15415, P.18339, P.8116, P.2285, P.1964, P.5119, P.11291, A.1761, A.35087 respectively and in the figure 29C, from left to right M is the 100 bp DNA ladder , lane forty three to fifty three contain A.9313, A.9914, A.9077, A.9954, A.14075, A.11241, A.25356 A.10813, A.9445, A.35045 and negative control respectively. All fifty Pakistani MRSA isolates showing positive results with in an RM3 test giving a PCR product size of 680 bp, indicative of CC8, while Mu50 has a 1071 bp amplicon size indicative of CC5.
Chapter 3 Results
86
Figure 30 (A, B)
Fig. 30A Fig. 30B
RM3 test Results with Pakistani Staphylococcus aureus (MRSA) isolates. This figure shows the amplicons obtained from RM3 test with Pakistani Staphylococcus aureus isolates. In the figure 30A from left to right M is the 100bps DNA ladder, lane number one to eleven contains NCTC8325, Mu50( positive control for RM3), A.9703, A.13319, A.7145, P.3, P.4, P.7, K.8, P.8485, P.2 respectively and In the figure 30B from left to right M is 100bps DNA ladder (Norgen Full Ranger), lane number twelve to twenty two P.2871, P.5020, P.5114, P.1005, P.844, P.843, P.1843, A.8823 A.9781, A465, and negative -ive control respectively. All the Pakistani MRSA isolates showing positive results within an RM3 test giving a PCR product size of 680 bp, indicative of CC8, while Mu50 has a 1071 bp amplicon size indicative of CC5.
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Figure 31(A, B, C)
Fig. 31A Fig. 31B
Fig. 31C
RM3 test Results with Pakistani Staphylococcus aureus (MRSA) isolates. In the figure 31A from left to right M is the 100bp DNA ladder (Norgen full Ranger), Lane one to eighteen contains NCTC8325, Mu50 (positive control), A.14254, A.15330, P.11296, P.862, A.379, P.1286, A.13282, P.10013, P.16809, P.1003, P.1863, P.3727, P.2887, P.6, P.8486, P.13741,. In the figure 31B from left to right M is the 100bp DNA ladder (Norgen full Ranger), Lane 19 to 29 contains P.13740, K.10, K.13, A.5054, A.13730, P.1962, P.2111, P.2255, K.11, P.12007, P.1009, respectively and In the figure 31C from left to right M is the 100bp DNA ladder, lane thirty to thirty six contain, A.5053, P.3027, P.14947, P.18176, P.1961, P.1963, and negative control respectively. All the Pakistani MRSA isolates showing positive results with in an RM3 test giving a PCR product size of 680 bp, except P.1286 & P.16809 in lane number 8 &11 respectively, are indicative of CC8, while Mu50 has a 1071 bp amplicon size indicative of CC5.
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88
Figure 32 (A, B, C)
Fig. 32A Fig. 32B Fig. 32C
RM tests with Pakistani MRSA P.1286 and P.16809. This figure shows the amplicons obtained from three RM test with Pakistani Staphylococcus aureus isolates P.1286 and P.16809:- RM1 (Fig. 32A); RM2 (Fig. 32B); and RM3 (Fig. 32C). Fig. 19A:- M, 100 bp DNA ladder (Norgen Full Ranger); lanes 1 to 5 contain NCTC8325, MRSA252 [Positive control for RM1], P.1286, P.16809 and a negative control, respectively. Fig. 19B:- lanes 1 to 5 contain CDC-8 [Positive control for RM2], NCTC8325, P.1286, P.16809 and a negative control. Panel C:- lanes 1 to 5 contain NCTC8325, [Positive controls for RM3], P.18431, MRSA252,P.1286, P.16809 and a negative control. P.1286 and P.16809 showed amplification only in RM1, thus belong to CC30.
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89
Figure 33 (A, B)
Fig. 33A Fig. 33B
M 11 12 13 14 15 16 17 18
RM3 test Results with Pakistani Staphylococcus aureus MRSA isolates. This figure shows the amplicons obtained from RM3 test with Pakistani Staphylococcus aureus isolates. In the figure 33A from left to right M is the 100bps DNA ladder, lane number one to ten contains NCTC 8325, Mu50, P.5019 P.1123, K.12, P.2006, P.14946, P.2885, P.1864, P.13947 and in figure 33B, from left to right P.1967, P.1287, P.13714, P.14313, P.2119, P.6270, P.13731 and negative control and negative control. All the Pakistani MRSA isolates showing positive results within an RM3 test giving a PCR product size of 680 bp, indicative of CC8, while Mu50 has a 1071 bp amplicon size indicative of CC5.
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90
Spa Sequence Typing of Pakistani MRSA Isolates
Spa typing is a single locus sequencing typing technique in which variation in the tandem
repeats of protein A, encoded by the spa gene, and is used to type S. aureus isolates.
These repeats vary both in number and sequence. The spa gene harbors a number of
functionally distinct regions which includes Fc-binding region of immunoglobulin G
(IgG), X-region and C-terminus. The X-region contains a varying number of 24 bp
repeats and this polymorphic X region is used in epidemiological typing of MRSA
isolates. The X-region usually cotains 3 to 15 repeat units. Data from spa typing can be
compared globally through the Ridom Staph Type software and website (Harmsen et al.,
2003). Spa typing is portable and relatively easy and rapid to perform.
In order to understand the phylogenetic relatedness of Pakistani MRSA isolates and to
compare them globally spa sequence typing was performed on selected number of
Pakistani MRSA isolates. Twenty six MRSA isolates were selected for Spa typing after
the multilocus variable number of tandem repeat analysis which clustered 123 Pakistani
MRSA into 63 genotypes. The results obtained with 26 Pakistani MRSA isolates are
given in the table 16 (appendix-I).
Table 16 (appendix-I) is showing the strain IDS, repeat sequences, repeat IDs, Kreiswirth
IDs and spa types for 26 MRSA isolates. The 26 MRSA isolates which were spa
sequenced includes P.2113, P.18431, P.8486, P.5020, P.844, A.14254, P.17120, A.9445,
A.14256, A.15330, A.9914, K.8, P.1863, A.9313, A.10813, A.13282, P.14947, A.5053,
P.1286, P.8, P.13740, P.2285, P.1287, A.26163, P.12006 and P-1. The sequence data
obtained after sequencing on ABI sequencer was used to find the consensus sequences
using CLC DNA work bench V.5.3 and fed into ridom spa server to obtain the repeat
IDs. The repeat IDs were then used in the spa server to query the spa type. The spa types
obtained after querying are given against each of the 26 isolates. Out of the 26 MRSA
isolates twelve isolates P.18431, P.8486, P.5020, P.844, A.14254, P.17120, A.9445,
A.14256, A.15330, A.9914, K.8, P.1863 have the same spa type t064, four isolates P.8,
P.13740, P.2285, P.1287 have spa type t030, three isolates A.26163, P.12006 and P.1
belong to spa type t632, while two isolates A.10813, A.13282 belong to t275 and another
two P.14947, A.5053 belong to t987, one P.2113 belongs to t037, and another one P.1286
Chapter 3 Results
91
belongs to t021. The sequences of repeat of units obtained for each strain and total
number of repeats for each strains are also given in the table 16 (appendix-I).
Spa typing was performed on 26 isolates which also included multiple isolates from the
same MT (genotype). Five isolates P.18431, P.8486, P.5020, P.844, A.14254 were from
MT13, three isolates A.26163, P.12006, P.1 from MT59, three isolates A.15330, A.9914,
K.8 from MT18, two MTs A.9445, A.14256 from MT17 and another two A.9313,
A.10813 from MT32 and other from different MTs as shown in table 6. All isolates from
the same MT had identical spa types indicating that MLVA was grouping highly-related
strains. The separation of isolates having the same spa type into different MTs indicates
the presence of genetic diversity within a spa type as shown in table 6. There is a wide
distribution of spa types within the Pakistani hospitals. Thus, among the 16 isolates from
Hospital P, there were six spa types:- t064 (6 isolates), t030 (4 isolates), t632 (2 isolates),
t987 (2 isolates), t451 (1 isolate) and t021 (1 isolate). In Hospital A, we found four spa
types:- t064 (1 isolate), t275 (2 isolates), t632 (2 isolates) and t037 (1 isolate) and one spa
t064 (1 isolate) type from hospital K. Spa repeat IDs shows (Table 6) that these isolates
are closely related to each other. For example P.2113 belongs to t451 and P.18431 belong
to t064 both of these isolates are similar in spa sequence except that P.2113 has lost one
repeat unit r19 at position two. Similarly the isolates belonging spa t037 and t275 are
identical in spa sequence except that t037 has lost one repeat unit r24 at the end.
Similarly the isolates belonging to t987 and t021 have identical spa sequence except that
t021 has lost one repeat unit r24 at 5th position.
Figure No 34 is showing spa X-region sequences of a representative MRSA isolate
A.14256. The sequences at the top is a consensus sequence obtained after alignment and
the sequences below to consensus sequences are letter representation of forward and
reverse strands of spa X- region containing spa repeats for the isolate A.14256. The
number of spa repeats starts at position 07 and ends at 246. The total number of repeat
present are 10 and spa type found for A.14256 was t064.The repeat IDs obtained for the
strain is shown in Table 6.
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92
Table 6: Showing the Spa sequence repeat profile obtained from Ridom spa server after
sequencing of selected 26 MRSA isolates from Pakistan.
S.No. S. aureus Isolates
Mec Type
MLVA type Spa Repeat Successions/IDs SPA type
1 P.2113 IV MT1 11-12-05-17-34-24-34-22-25 t451 2 P.18431 IV MT13 11-19-12-05-17-34-24-34-22-25 t064 3 P.8486 IV MT13 11-19-12-05-17-34-24-34-22-25 t064 4 P.5020 IV MT13 11-19-12-05-17-34-24-34-22-25 t064 5 P.844 IV MT13 11-19-12-05-17-34-24-34-22-25 t064 6 A.14254 IV MT13 11-19-12-05-17-34-24-34-22-25 t064 7 P.17120 IV MT14 11-19-12-05-17-34-24-34-22-25 t064 8 A.9445 IV MT17 11-19-12-05-17-34-24-34-22-25 t064 9 A.14256 IV MT17 11-19-12-05-17-34-24-34-22-25 t064 10 A.15330 IV MT18 11-19-12-05-17-34-24-34-22-25 t064 11 A.9914 IV MT18 11-19-12-05-17-34-24-34-22-25 t064 12 K.8 IV MT18 11-19-12-05-17-34-24-34-22-25 t064 13 P.1863 IV MT25 11-19-12-05-17-34-24-34-22-25 t064 14 A.9313 III MT32 15-12-16-02-25-17-24-24 t275
15 A.10813 III MT32 15-12-16-02-25-17-24-24 t275
16 A.13282 III MT63 15-12-16-02-25-17-24 t037
17 P.14947 III MT27 15-12-16-02-24-16-02-25-17-24 t987
18 A.5053 III MT39 15-12-16-02-24-16-02-25-17-24 t987
19 P.1286 IV MT44 15-12-16-02-16-02-25-17-24 t021 20 P.8 III MT31 15-12-16-02-24-24 t030 21 P.13740 III MT46 15-12-16-02-24-24 t030 22 P.2285 III MT47 15-12-16-02-24-24 t030 23 P.1287 III MT53 15-12-16-02-24-24 t030 24 A.26163 III MT59 8-16-02-24-24 t632 25 P.12006 III MT59 8-16-02-24-24 t632 26 P-1 III MT59 8-16-02-24-24 t632
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93
Similarly Figure 35 is showing spa X-region sequences of another representative MRSA
isolate P.2113. The sequences at the top is a consensus sequence obtained after alignment
and the sequences below to consensus sequences are letter representation of forward and
reverse strands of spa x region containing spa repeats for the isolate P.2113. The number
of repeats starts at position 07 and ends at 222. The total number of repeat found were 9
and spa found for P.2113 was t451.
Figure 36 is showing spa X-region sequences of a representative MRSA isolate A.9313
The sequences at the top is a consensus sequence obtained after alignment and the
sequences below to consensus sequences are letter representation of forward and reverse
strands of spa X region containing spa repeats for the isolate P.9313. The number of
repeats starts at position 06 and ends at 197. The total number of repeat found were 8 and
spa type for P.9313 was t275
Figure 37 is showing spa X-region sequences of a representative MRSA isolate A.13282
The sequences at the top is a consensus sequence obtained after alignment and the
sequences below to consensus sequences are letter representation of forward and reverse
strands of spa X region containing spa repeats for the isolate A.13282. The number of
repeats starts at position 08 and ends at 175. The total number of repeats found was 7 and
spa type for A.13282 was t037.
Figure 38 is showing spa X-region sequences of a representative MRSA isolate P.1286.
The sequences at the top is a consensus sequence obtained after alignment and the
sequences below to consensus sequences are letter representation of forward and reverse
strands of spa X region containing spa repeats for the isolate P.1286. The number of
repeats starts at position 07 and ends at 222. The total number of repeats found was 9 and
spa type for P.1286 was t021.
Figure 39 is showing spa X-region sequences of a representative MRSA isolate A.26163.
The sequences at the top is a consensus sequence obtained after alignment and the
sequences below to consensus sequences are letter representation of forward and reverse
strands of spa X region containing spa repeats for the isolate P.26163. The number of
repeats starts at position 11 and ends at 130. The total number of repeats found was 5 and
spa type for P.26163 was t632.
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94
Figure 40 is showing spa X-region sequences of a representative MRSA isolate P.1287.
The sequences at the top is a consensus sequence obtained after alignment and the
sequences below to consensus sequences are letter representation of forward and reverse
strands of spa X region containing spa repeats for the isolate P.1287. The number of
repeats starts at position 4and ends at 147. The total number of repeats found was 6 and
spa type for P.1287 was t030.
Multilocus Sequence Typing of Staphylococcus aureus
Multilocus Sequence Typing (MLST) involves the sequencing of internal fragment of
seven house keeping genes which includes arcC (for enzyme carbamate kinase), aroE (for
enzyme shikimate dehydrogenase), glpf (for enzyme glycerol kinase), gmk (for enzyme
guanylate kinase), pta (for enzyme phosphate acetyl transferase), tpi (for triphosphate
isomerase), yqil ( for enzyme acetyl Co enzyme A). The sequences of these internal
fragments of about 500bp are then compared with known allele at each locus to the
MLST database via MLST website (http://www.mlst.net.). In MLST data base every
isolate is described by a seven integer allelic profile that represents a specific sequence
type (ST).
In the present study MLST was carried out on 10 selected MRSA isolates P.2113 (MT1,
spa type t451), P.5020 (MT13, spa type t064), A.15330 (MT18, spa type t064), P.1863
(MT25, t064), A.9313 (MT32, spa type t275), A.13282 (MT63, spa type t037), A.5053
(MT39, spa type t987), P.1286(MT44, spa type t021), P.2285 (MT47, spa typet030) and
A.26163 (MT39, spa type t0632) which belong to different MLVA types
(MTs/genotypes) and spa types. The nucleotide sequences of all seven alleles of these
isolates were used to obtain the allelic profile from the mlst database using the mlst
website (http://www.mlst.net.). In this way the allelic profile obtained is given in the
table 7. The table number is showing the stain IDs, MLVA type, spa type, allic profile in
the order of aro-arc-glp-gmk-pta-tpi-yqil and Sequence type obtained after mlst for the
isolates and their respective clonal complexes. Four out of 10 MRSA isolates P.2113,
P.5020, A.15330, P.1863 were ST113 with allelic profile “3-3-1-1-4-62-3” while five
isolates A.9313, A.13282, A.5053, P.2285 and A.26163 belonged to ST239 having
allelic profile “2-3-1-1-4-4-3” and only one isolate P.1286 was ST30 with allelic profile
Chapter 3 Results
95
Figure No 34
Showing spa X-region sequences of a representative MRSA isolate A.14256. The sequences at the top is a consensus sequence obtained after alignment and the sequences below to consensus sequences are letter representation of forward and reverse strands of spa x region containing spa repeats for the isolate A.14256. The number of repeats starts at position 07 and ends at 246. The total number of repeat present are 10 and spa type found for A.14256 was t064.
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96
Figure 35
Showing spa X-region sequences of a representative MRSA isolate P.2113. The sequences at the top is a consensus sequence obtained after alignment and the sequences below to consensus sequences are letter representation of forward and reverse strands of spa x region containing spa repeats for the isolate P.2113. The number of repeats starts at position 07 and ends at 222. The total number of repeat found were 9 and spa type found for P.2113 was t451.
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97
Figure 36
Showing spa X-region sequences of a representative MRSA isolate A.9313. The sequences at the top is a consensus sequence obtained after alignment and the sequences below to consensus sequences are letter representation of forward and reverse strands of spa X region containing spa repeats for the isolate P.9313. The number of repeats starts at position 06 and ends at 197. The total number of repeat found was 8 and spa type for P.9313 was t275.
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98
Figure No 37
Showing spa X-region sequences of a representative MRSA isolate A.13282. The sequences at the top is a consensus sequence obtained after alignment and the sequences below to consensus sequences are letter representation of forward and reverse strands of spa X region for the isolate A.13282. The number of repeats starts at position 08 and ends at 175. The total number of repeats found was 7 and spa type for A.13282 was t037.
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99
Figure 38
Showing spa X-region sequences of a representative MRSA isolate P.1286. The sequences at the top is a consensus sequence obtained after alignment and the sequences below to consensus sequences are letter representation of forward and reverse strands of spa X region containing spa repeats for the isolate P.1286. The number of repeats starts at position 07 and ends at 222. The total number of repeats found was 9 and spa type for P.1286 was t021.
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100
Figure 39
Showing spa X-region sequences of a representative MRSA isolate A.26163. The sequences at the top is a consensus sequence obtained after alignment and the sequences below to consensus sequences are letter representation of forward and reverse strands of spa X region containing spa repeats for the isolate P.26163. The number of repeats starts at position 11 and ends at 130. The total number of repeats found was 5 and spa type for P.26163 was t632.
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101
Figure 40
Showing spa X-region sequences of a representative MRSA isolate P.1287. The sequences at the top is a consensus sequence obtained after alignment and the sequences below to consensus sequences are letter representation of forward and reverse strands of spa X region containing spa repeats for the isolate P.1287. The number of repeats starts at position 4 and ends at 147. The total number of repeats found was 6 and spa type for P.1287 was t030.
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102
2-2-2-2-6-3-2. The ST113 and ST239 belong to clonal complex eight (CC8), while ST30
belong to clonal complex thirty CC30. Thus majority of Pakistani isolates belong to CC8.
The sequences of seven alleles in fasta format are shown here from figure 41to figure for
the representative strains of ST113, ST239 and ST30 while the allelic profiles of
remaining isolates are given in the table11.
Figure 48 to figure 54 (appendix-II) are showing Sequence data of arc- aro -glp-gmk-pta-
tpi-yqil, gene locus of S. aureus strain P.2113 in fasta format respectively. These
sequences are the consensus sequence obtained after the alignment of forward and
reverse strand. The allelic codes obtained for these sequence from MLST server are 3-3-
1-1-4-62-3 and thus the isolate confirmed as ST113, which belong to clonal complex
eight (CC8)
.Similarly Figure 55 to figure 61 (appendix-II) are showing the sequence data of aro-arc-
glp-gmk-pta-tpi-yqil, gene locus of S. aureus strain A.13282 in fasta format respectively.
These sequences are the consensus sequence obtained after the alignment of forward and
reverse strand. The allelic codes obtained for these sequences from MLST server were 2-
3-1-1-4-4-3 and thus isolate confirmed as ST239, which belong to clonal complex eight
(CC8)
Similarly Figure 62 to figure 68 (appendix-II) is showing Sequence data of aro-arc-glp-
gmk-pta-tpi-yqil, gene locus of S. aureus strain P.1286 in fasta format respectively. These
sequences are the consensus sequence obtained after the alignment of forward and
reverse strand. The allelic code obtained for the sequences from MLST server were 2-2-
2-2-6-3-2 and the isolate confirmed as ST30 which is a part of clonal complex thirty
(CC30). Thus the result obtained after multilocus sequence typing showed that majority
of MRA isolates from Pakistan belong to clonal complex eight with either ST113 or
ST239.
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103
Table 7: Multi locus Sequence Typing profiles and Clonal Complexes of MRSA isolates from Pakistan.
Strain
IDs.
MLVA type
SPA type
aro arc glp gmk pta tpi yqil MLST Profile
arc-aro-glp-gmk-pta-tpi-yqil
MLST
Type
Clonal Complex
P.2113 MT1 t451 3 3 1 1 4 62 3 3-3-1-1-4-62-3 ST113 CC8
P.5020 MT13 t064 3 3 1 1 4 62 3 3-3-1-1-4-62-3 ST113 CC8
A.15330 MT18 t064 3 3 1 1 4 62 3 3-3-1-1-4-62-3 ST113 CC8
P.1863 MT25 t064 3 3 1 1 4 62 3 3-3-1-1-4-62-3 ST113 CC8
A.9313 MT32 t275 2 3 1 1 4 4 3 2-3-1-1-4-4-3 ST239 CC8
A.13282 MT63 t037 2 3 1 1 4 4 3 2-3-1-1-4-4-3 ST239 CC8
A.5053 MT39 t987 2 3 1 1 4 4 3 2-3-1-1-4-4-3 ST239 CC8
P.1286 MT44 t021 2 2 2 2 6 3 2 2-2-2-2-6-3-2 ST30 CC30
P.2285 MT47 t030 2 3 1 1 4 4 3 2-3-1-1-4-4-3 ST239 CC8
A.26163 MT59 t632 2 3 1 1 4 4 3 2-3-1-1-4-4-3 ST239 CC8
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Chapter 4 Discussion
124
DISCUSSION
Methicillin resistant Staphylococcus aureus (MRSA) is the major human pathogen and is
leading cause of nosocomial infections. The distribution of MRSA clones has been
reported world wide. Staphylococcus aureus causes a variety of diseases ranging from
superficial skin infections to deep seated infections like pneumonia and septicemia etc.
Genotyping of bacterial isolates is a tool, which can be used in epidemiological studies
while epidemiology is the study of patterns of disease in populations. Genotyping is the
process of determining the genotype of an individual by the use of different biological
assays. Genotyping of MRSA is necessary in order to study its pattern of spread and
infections. It has been reported that most of outbreaks of infectious diseases are due to
exposure to organism from a common source. It is considered that organism in the
affected individuals are genotypically related to each other. Genotyping is carried out to
investigate that isolates from epidemiologically identical cases are genetically identical
(Tenover, 1992). Thus genotyping helps in controlling the spread of pathogens, by
tracing the origin of outbreaks. Genotyping is also often called as molecular
epidemiology or forensic microbiology. Many molecular methodologies have been
adapted for genotyping of MRSA each having its merits and demerits with respect to
their resolving power, portability, cost effectiveness and ease of use.
In the current epidemiological study we focused on clinical MRSA isolates from three
tertiary care hospitals located in the twin cities of Rawalpindi/Islamabad and examined
the types and phylogenetic relationship of the isolates by using MLVA typing, RM
typing, PVL screening, spa typing, Multilocus sequence typing (MLST), and STAR
analysis. A total of 123 nosocomial MRSA isolates were obtained:- 89 isolates from
hospital PIMS (P) a large tertiary care hospital in Islamabad: 7 isolates from hospital
KRL (K), a medium-size hospital in Islamabad; and 27 isolates from Military hospital
(A), a large unit in Rawalpindi. Isolates were obtained from a range of infection
types/sources such as pus, blood, Catheter tips etc and were from different individuals.
Firstly the 16rRNA gene and mecA gene PCR was used to identify and confirm isolates
as MRSA. 16S rRNA gene has specific sequences which are genus specific, thus
Chapter 4 Discussion
125
specifically designed oligonucleotide primers targeting 16rRNA genes correctly identifies
MRSA (Maes et al., 2002). All the isolates used in the current study showed amplication
with 16rRNA primers and mecA gene primers giving the expected product sizes of
1000bp and 100bp respectively, thus were confirmed as MRSA.
After initial identification of MRSA, an MVLA approach was adapted to the analysis of
these MRSA strains as a rapid and cost-effective method of establishing their
phylogenetic relationships as compared to PFGE. PCR products were analyzed using an
ABI sequencer, as this method of MLVA typing is more accurate and robust as compared
to analyzing fragments on gels due to its ability to separate PCR amplicons with small
differences in size thereby providing accurate sizes for these products. The choice of
VNTR was based on established MLVA protocol (Sabat et al., 2003; Francois et al.,
2005; Gilbert et al., 2006) and available genome sequence data
(http://www.ncbi.nlm.nih.gov/ genomes/prokes.cgi), resulting in selection of six variable
number of tandem repeat (VNTR) loci clumping factor A (clfA), clumping factor (clfB),
serine–aspartate protein (sdrC), serine–aspartate protein D (sdrD), Staphylococcal protein
A (spa) and sspa . All the isolates from Pakistan yielded PCR products with all six
VNTR loci. PCR products of the expected sizes, as predicted from analysis of genome
sequences, were obtained for the control strains NCTC 8325 and Mu50. The PCR
products sizes were then converted into repeat numbers. The length of flanking region
and number of repeats were calculated by using program Tandem Repeat Finder v.4.01
from the sequenced strains, NCTC8325 and Mu50, and from the sequences of six loci of
our isolate P.18431 and also same repeat numbers were found true in 26 isolates
sequenced for spa typing. Some VNTR loci contained large numbers of repeats, e.g. clfA
contained up to 46 repeats and clfB up to 55, while some had limited number of repeats,
sspa had four or five repeats. One VNTR loci sav1078 having repeat unit of 63bp did not
showed variation in amplicon sizes of isolates from Pakistan. All the isolates showed the
PCR product sizes of 317bp. So sav1078 was not included in the VNTR analysis for
clustering and strain typing.
In the majority of diseases caused by S. aureus, pathogenesis is multifactorial, so it is
difficult to determine precisely the role of any given factor in a particular case. So the
selection of several loci for MLVA typing was necessary. The number of repeats
Chapter 4 Discussion
126
calculated for the six loci for all the isolates were used to generate an allelic profile
(Fig.27). Isolates varying in repeats at one locus were assigned a different MT/genotype.
In this way a total of 63 MT (genotypes) were obtained for the Pakistani isolates. Two
large clusters of 10 and 11 identical isolates (MTs 13 and 47) were detected with all these
isolates being derived from pus and Hospital P. This is suggestive of a common source of
infection. The allelic profile of MT13 and MT14 was “34-41-30-24-10-4” and “37-39-27-
25-10-4”. Clusters of isolates with a common source were also detected. Thus, MT17
consisted of five blood isolates (November 2007–February 2008), MT25 of six urine
isolates (April-July 2006), MT29 of three isolates from sputum (July2006-August 2007),
MT30 of three spinal cord isolates (August 2006/April 2007) but few cluster of isolates
with different sources was also present like MT1, MT12 of three isolates each from
sputum and pus. These isolates were indicative of clusters of MRSA infection cases.
MLVA profiles were clustered into minimum spanning trees using Bionumerics in order
to show the genetic relationships among the various MLVA types (Fig.26). In the tree,
repeat numbers were treated as categories. Thus two MT differing in repeats at one locus
are clustered together and connected by bold line and two MT varying in more than one
loci are represented by light line while standard strains are shown by dotted lines. This
distribution of strains from different hospitals into the same MT suggests that there is no
association between specific strains and hospitals. This might be due to the fact that these
hospitals are not geographically isolated from each other and visits of patients between
hospitals are common resulting in frequent exchange of strains between these closely-
linked hospitals.
In order to establish the clonal relationship of these isolates to other global collections of
MRSA isolates, further analyses were carried out. Firstly we investigated the clonal
relationships of these isolates by analyzing the RM systems as described by Cockfield et
al., 2007. Staphylococcus aureus isolates belong to one of the ten reported S. aureus
lineages. MRSA isolates that are widely distributed in hospitals belong to six (CC1, CC5,
CC8, CC22, CC30 and CC45) predominant S. aureus lineages (Robinson & Enright,
2003; Lindsay et al., 2006). Clonal complex of an isolate can be confirmed by RM test.
The RM test in the present study showed that 121 isolates were positive for an RM3 type
and only two strains, P.1286 and P.16809, for an RM1 type. This indicated that the
Chapter 4 Discussion
127
majority of isolates belonged to CC8, which is associated with an RM3 type and only
small number of isolates belong to CC30.
Thus CC8 is the most dominant clonal complex in Pakistan and its presence has been
reported from many other countries including UK, USA. Uganda, Germany, Sweden,
Denmark, Australia, Malaysia, India, Kuwait (Moore & Lindsay, 2002; Aires de Sousa &
de Lencastre, 2003; Perez-Roth et al, 2004 Ghebremendhin et al., 2005; Deurenberg et
al., 2007; Ghaznavi et al., 2010). The presence of this clonal complex is also reported
earlier in Pakistani hospitals (Shabir et al., 2010) and it appears that CC8 is the most
successful MRSA clonal complex in this country. The presence of CC8 MRSA has also
been reported from other Asian countries. One study in Asia detected two major lineages
with CC5 strains being dominant in Korea and Japan and CC8 in other countries (Ko et
al., 2005).
CC30 (1.6%) was the second clonal complex found in Pakistani isolates. Only 2 isolates
out of 123 belonged to CC30. This clonal complex first reported in UK and has now
spread to Australia, Belgium, Canada, Greece, Finland, Denmark, Ireland, Spain, Swede,
USA (Deurenberg et al., 2007; Donnio et al., 2007). Thus RM test correctly assigned
clonal complex to isolates which were later on confirmed by doing spa typing and MLST
on selected number of isolates.
Spa typing is a single locus sequencing typing technique in which variation in the tandem
repeats of protein A, encoded by the spa gene was used to type S. aureus isolates. These
repeats vary both in number and sequence. The spa gene harbors a number of
functionally distinct regions which includes Fc-binding region of immunoglobulin G
(IgG), X-region and C-terminus. The X-region contains a varying number of 24 bp
repeats and this polymorphic X region is used in epidemiological typing of MRSA
isolates. The X-region usually contains 3 to 15 repeat units. Data from spa typing can be
compared globally through the Ridom Staph Type software and website (Harmsen et al.,
2003). Spa typing was performed on 26 isolates which also included multiple isolates
from the same MT such as five isolates from MT13, two from MT17, three from MT18,
two isolates from MT32 and three isolates from MT59 and remaining from different MT
(table 6). All isolates from the same MT had identical spa types indicating that MLVA
Chapter 4 Discussion
128
grouped highly-related strains while the separation of isolates having the same spa type
into different MTs such as MT13, MT14, MT17, MT18 and MT25 indicates the presence
of genetic diversity within a spa type.
There was a wide distribution of spa types within these three Pakistani hospitals. Thus,
among the 16 isolates from hospital P which were spa typed, we found six spa types:-
t064 (6 isolates), t030 (4 isolates), t632 (2 isolates), t987 (2 isolates), t451 (1 isolate) and
t021 (1 isolate) and among the 9 isolates from Hospital A, we found four spa types:- t064
(1 isolate), t275 (2 isolates), t632 (2 isolates) and t037 (1 isolate) and one isolates from
KRL hospital belong to t064.
Spa-t064, spa-t030, spa-t451, spa-t021 has been reported from many countries around the
world which include Australia, Canada, China, Germany, India, Netherlands, Sweden,
Norway, UK , USA and UAE while t632 has been reported from Germany and Austria
and t987 has been reported from Norway (spa.ridom.de/).
MLST was carried out for 10 isolates representing different spa types. Out of 5 isolates
from hospital A, we found one ST113 and four ST239 while out of 5 from Hospital
PIMS, three were ST113, one was ST239 and one was ST30 (see Table 7). Thus nine
out-of-ten isolates had MLST types associated with CC8 and one with CC30. CC8
consist of 182 STs in total. The CC8 isolates from Pakistan were resolved into two
sequence types (ST), ST239 and ST113. The ST239 is a single locus variant of ST 8 and
has MLST profile “2-3-1-1-4-4-3”. The ST239 has global distribution and predominant
clone in Asia and Africa. It has been reported from many Asian countries i.e. Saudi
Arabia, Egypt, Tunisia, Singapore, Vietnam, Indonesia, Philippines, Japan, China, Sri
Lanka, India (Chongtrakool et al., 2006; Nickerson et al., 2006; Smyth et al., 2009;
Kechrid et al., 2010, Shabir et al.,2010). ST239 originated from Sweden in 1970s and
then spread around the globe in a very short time due its hybrid nature (Smyth et al.,
2009). Its presence in Pakistan is indicative of its spread in South Asian countries. The
other ST found among Pakistani isolates was ST113 which also belong to CC8 having
MLST profile of 3-3-1-1-4-62-3 and which is a single locus variant of ST8. ST 8 is a
pioneer of group 1 of STs of MLST scheme and reported from many European, Asian
and Middle East countries (Olivera et al., 2001; Enright et al., 2002; Udo et al., 2010)
Chapter 4 Discussion
129
ST30 was also found among isolates from Pakistan which belong to CC30 and has MLST
profile 2-2-2-2-6-3-2. The frequency of ST30 (1.6%) is less as compared to ST239,
ST113 and ST8 in Pakistan. ST 30 was first reported in UK and it has spread now to
Europe, Asia, America and Australia (Vandenesch et al., 2003). ST30 has 101 single
locus variants and 34 double locus variants and thus ST30 belong to CC30 which has 135
ST in total (www.mlst.net).
Thus the genotyping of MRSA isolates from three hospitals located in
Islamabad/Rawalpindi indicates the predominance of CC8 clones, particularly ST239 and
ST113. This is an extension of the observation that ST239 may account for more than
90% of hospital acquired MRSA infections in South East Asia (Feil et al., 2008) to
include Pakistan. The presence of CC8 especially ST239 has also been also been reported
from India (Arakere et al., 2005; Ko et al., 2006).
These two CC8 and CC30 have also been reported in previous studies from Pakistan.
Thus our results complement previous studies by Shabir et al., 2010 on 49 isolates from
Pakistan and Zafar et al., 2011 on 126 isolates. In our study, the majority of isolates were
found to be from CC8 with the following types being represented: - ST239, ST113 and a
few isolates belong to CC30 having sequence type 30 (ST30). Our study also
complements with two larger studies (Ko et al., 2005; Song et al., 2011) which did not
examine isolates from Pakistan but found that, with the exception of Korea and Japan,
Asian countries are dominated by CC8 strains particularly ST239. ST239 is the
Brazalian/Hnngarian clone and evolved from the highly successful and unrelated lineages
CC8 (ST8) and CC30 (ST30). The exact mechanism of DNA transfer between these
lineages is unknown but it has been considered that fusion of two isolates may have
occurred (Robinson et al., 2004; Lindsay et al., 2006). Thus ST239 with an increased
virulence is the most widely distributed clone of MRSA (Edgeworth et al., 2007).
Comparing typing efficiency of MLVA and its use in epidemiology it has been shown in
previous study that it is comparable to PFGE and even more resolution some time (Sabat
et al., 2003).
Although there is significant diversity at the micro level, as shown by variations in the
VNTRs, the majority of MRSA strains present in the hospitals of Pakistan are from CC8
Chapter 4 Discussion
130
and a limited number from CC30. The dominant STs present are ST239 with mec type-
III, and ST113 with mec type-IV with only a limited number of strains with an ST30 mec
type-IV which may be due to local clonal evolution. These results are the indicative of
clonal polymorphism which resulted to evolve ST239 and ST113 from ST8. Unlike CC8
which is predominant in Asian countries, other isolates belonged to CC30 which is
predominant in Europe and the Pacific Islands (Smith et al, Udo et al., 2008). ST30 is a
community acquired MRSA in origin. The identification of ST30 isolates with SCCmec
IV and positive for the Panton Valentine Leukocidin in Pakistan indicates its
dissemination in South East Asia as seen previously in China and Singapore (Ho et al.,
2004; Hsu et al., 2006).
After the confirmation and identification of the isolates lineages by RM typing, Spa
typing and MLST these isolated were analyzed for Staphylococcus aureus repeat (STAR)
elements present in the intergenic region of three loci i.e. between conserved hypothetical
region-gapR, uvrA-hprK and icaC-geh. STAR element contain 14bp GC rich direct
repeats of consensus sequences T(G/A/T)TGTTG(G/T)GGCCC(C/A) interspersed with
40bp of recurring sequences and the number of repeats at particular locus varies between
strains (Cramton et al., 2000).
On the basis of STAR element containing repeats present in the upstream region of gapR
the MRSA isolates from Pakistan are grouped into 4 clusters, 0 repeats (2 isolates), 1
repeat (60 isolates), 2 repeats (56), 4 repeats (5 isolates). The isolates P.1286 and P.16809
with no repeats in the gapR upstream region are similar to MRSA252 which do not have
the STAR repeat in gapR upstream region and thus there is a sequence similarity between
them. This is the confirmation that there is as similarity between the isolates in STAR
element belonging to same lineages as P.1286, P.16809 and MRSA252 belong to CC30.
The remaining isolates having 1 repeat, 2 repeat or 4 repeats in gapR upstream region
belong to clonal complex eight having variable STAR element length. While in Case of
STAR element present in the intergenic regions of uvrA-hprK, there were either two 3
repeats (100 isolates) or 2 repeats (23 isolates) were found in Pakistani MRSA isolates
and incase of ica-geh intergenic region, isolates with 1 repeat (2 isolates), 2 repeats (109
isolates), 3 repeats (12 isolates) were found. The phylogenetic tree drawn by using STAR
present in these intergenic region , resulted into seven groups that were group one contain
Chapter 4 Discussion
131
two isolates with STAR profile 0-2-1. This group included two CC30 isolates only.
Group two contain seven isolates with allelic profile 1-2-2, groups three contain thirteen
isolates with profile 2-2-2, group four contain 47 isolates with profile 1-3-2, group five
contain 42 isolates with profile 2-3-2, group six contain 7 isolates with profile 1-3-3 and
group seven contain five isolates with profile 4-3-3. Thus there is a genetic diversity at
subspecies level in the isolates belonging to CC8 as compared to CC30 as shown by the
Multilocus Variable number of tandem repeat analysis because repetitive elements can
evolve rapidly due to mismatch repair during DNA replication and can result a change in
repeat number after a single generation This is the basis of phase variation gene
regulation, resulting in subpopulations that are better adapted to environmental changes.
Mutation in tandem repeats occur 100-10,000 more frequently than point mutation (Van
Ende et al., 2000; Wis derniewski et al., 2008; Vinces et al., 2009).
Conclusion
The present genotypic study showed that in Pakistan the isolates belonging to clonal
complex eight (CC8) are dominant in clinical settings. They belong to ST239, ST113 or
ST8. The other clonal complex found was CC30 with presence of PVL gene and isolates
belong to ST30. The use of MLVA in resource poor laboratories as a rapid and robust
method for grouping noscomial MRSA isolates into clusters for identification of
localized outbreaks is quite fruitful and MLVA may also provide an understanding of the
evolutionary processes as changes in the number of repeats at different loci, may be
indicative of which loci are prone to natural selection resulting in higher levels of
variation, thus VNTRs serve as evolutionary clock for investigating an outbreaks and
transmission events. In this study, we observed more variation in clfA and clfB than in
sdrC, sdrD, spa and sspa. We also found that a change in repeat number was not
necessarily gradual but may have occurred as a result of large jumps. Some isolates with
significant differences in repeat numbers at single locus but being identical numbers in all
other loci. Further evidence is provided by the spa typing results, i.e. with a loss of four
repeats resulting in a shift from t987 to t030 and a two repeat difference changed t021 to
t275 (see Table 6). These large jumps might be due to deletions or insertions mediated by
Chapter 4 Discussion
132
recombination or as result of deletions due to slip-strand impairing during DNA
replication.
Thus MRSA infections have become a challenge across the globe. The MRSA isolates
which were once endemic to Europe and America, Africa has now been reported from
Asia and thus suggesting that the MRSA isolates which once was endemic to a certain
geographical area are no more confined to those boundaries. More over the pandemic
spread of one type of MRSA clone across the globe is the result of antibiotic resistance.
Therefore a joint global effort will be effective for the control of MRSA infection.
Although this study was carried out on limited number of isolated but it is quite useful to
strengthen the MRSA data in Pakistan and to develop the genetic profile of MRSA in
Pakistan and then to link it globally. This study also helped to under stand that although
there are only two lineages in these hospitals as in most of other Asian countries but there
is a diversity at subspecies level as some of the isolates assumed a specific genetic profile
as they evolved locally after they were imported to this region.
Recommendations
There is an urgent need of continuation of epidemiological studies of MRSA in Pakistan
in order to develop a local data base which will be helpful to determine the impact of
novel strains. We need to be vigilant to recognize MRSA strains. Surveillance need to be
coordinated at regional level and also at national level. If we do not control MRSA at
local and national level then these strains will increase exponentially. We need to update
our health care workers, especially physicians, microbiologists, infection control
practioners and bureaucrats managing health care about MRSA emergence and clonal
spread. These people also need to be aware of changing epidemiology of existing MRSA
strains and also about the emergence of novel strains of MRSA in Pakistan. It is essential
for the doctors to check the Staphylococcus aureus properly for antibiotic resistance
before doing treatment. It is also necessary to educate the public about MRSA infections
and also about the importance of hygienic measures which will help to reduce the MRSA
infections.
Chapter 4 Discussion
133
Future Directions
There is a need to perform multicentre studies all around the Pakistan with same
genotyping tools such as MLVA, spa and MLST in order to delineate the prevalence and
epidemiology of MRSA. Thus a data bank regarding MRSA infection, prevalence, mode
of transmissions, genotypes, clonal complexes and novelty can be prepared and can be
used for updating Health Care Policies.
Chapter 5 References
134
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162
Appendix- I
Table 13: Pakistani MRSA Isolates obtained from Hospitals of Rawalpindi/Islamabad.
S. No. S. aureus
Isolates
Clinical Source S. No. S. aureus
Isolates
Clinical Source
1 P.1 Pus 30 P.2006 Tissue Fluid
2 P.2 Pus 31 P.2054 Catheter tip
3 P.3 Pus 32 P.2111 Catheter tip
4 P.4 Pus 33 P.2112 Sputum
5 P.5 Sputum 34 P.2113 Sputum
6 P.6 Urine 35 P.2119 Pus
7 P.7 Pus 36 P.2255 Pus
8 P.8 Ear Infection 37 P.2285 Pus
9 P.843 Pus 38 P.2871 Sputum
10 P.844 Pus 39 P.2885 Spinal Card
11 P.862 Urine 40 P.2886 Catheter tip
12 P.1003 Tissue fluid 41 P.2887 Tissue Fluid
13 P.1005 Pus 42 P.2896 Spinal card
14 P.1009 Pus 43 P.3027 Catheter tips
15 P.1123 Catheter tip 44 P.3727 Sputum
16 P.1129 Pus 45 P.5019 Catheter tip
17 P.1286 Sputum 46 P.5020 Pus
18 P.1287 Pus 47 P.5114 Catheter tip
19 P.1297 Ear Infection 48 P.5119 Pus
20 P.1843 Pus 49 P.5123 Catheter tip
21 P.1863 Urine 50 P.6269 Catheter tip
22 P.1864 Urine 51 P.6270 Catheter tip
23 P.1896 Ear Infection 52 P.8116 Catheter tip
24 P.1961 Sputum 53 P.8431 Spinal Card
25 P.1962 Sputum 54 P.8485 Pus
26 P.1963 Urine 55 P.8486 Pus
27 P.1964 Urine 56 P.8487 Pus
28 P.1966 Sputum 57 P.8668 Ear Infection
29 P.1967 Catheter tip 58 P.8823 Pus
Chapter 5 References
163
Table 13 (Continue)
S. No. S. aureus
Isolates
Clinical Source S. No. S. aureus
Isolates
Clinical Source
59 P.10012 Sputum 89 P.18431 Pus
60 P.10013 Tissue Fluid 90 K.3 Ear Infection
61 P.10015 Sputum 91 K.6 Sputum
62 P.10019 Pus 92 K.8 Catheter tip
63 P.10021 Sputum 93 K.10 Sputum
64 P.11291 Pus 92 K.8 Catheter tip
65 P.11293 Pus 93 K.10 Sputum
66 P.11296 Pus 94 K.11 Pus
67 P.12006 Pus 95 K.12 Pus
68 P.12007 Sputum 96 K.13 Catheter tip
69 P.12285 Pus 97 A.379 Ear Infection
70 P.13714 Pus 98 A.465 Pus
71 P.13729 Catheter Tip 99 A.1761 Ear Infection
72 P.13731 Pus 100 A.5053 Catheter tips
73 P.13740 Pus 101 A.5054 Catheter tip
74 P.13741 Pus 102 A.7145 Pus
75 P.13749 Pus 103 A.9077 Blood
76 P.13947 Catheter tip 104 A.9313 Pus
77 P.14313 Pus 105 A.9445 Blood
78 P.14946 Sputum 106 A.9703 Catheter tip
79 P.14947 Pus 107 A.9781 Blood
80 P.15415 Catheter tip 108 A.9914 Pus
81 P.16509 Pus 109 A.9954 Catheter tip
82 P.16809 Sputum 110 A.9965 Pus
83 P.16849 Pus 111 A.10813 Pus
84 P.17120 Catheter tip 112 A.11241 Pus
85 P.17121 Catheter tip 113 A.13282 Pus
86 P.18119 Catheter tip 114 A.13319 Pus
87 P.18176 Catheter tip 115 A.13730 Pus
88 P.18339 Pus 116 A.14075 Catheter tip
Chapter 5 References
164
Table 14: Showing the amplicon sizes for the 123 Pakistani MRSA Isolates and NCTC 8325 and Mu50 (Positive control strains) obtained after Gene Scan for the clfA, clfB, sdrD,sdrC,spa sspa and sav1078 loci.
S.No. Strains IDs
ClfA band size
ClfB band size
sdrD band size
sdrC band size
spa band size
Sspa band size
Sav1078 band size
1 NCTC 8325
1027 822 659 762 300 155 317
2 Mu-50 1050 822 768 634 276 174 317
3 P.1 900 884 704 560 157 155 317
4 P.2 846 935 704 548 157 155 317
5 P.3 900 974 756 506 276 155 317
6 P.4 900 902 756 560 181 155 317
7 P.5 900 884 756 560 181 155 317
8 P.6 1045 864 756 548 276 155 317
9 P.7 900 935 704 548 276 155 317
10 P.8 846 884 756 548 181 155 317
11 P.843 846 935 756 548 276 155 317
12 P.844 846 935 756 548 276 155 317
13 P.862 1045 864 756 548 276 155 317
14 P.1003 900 935 756 548 229 155 317
15 P.1005 900 902 704 560 181 155 317
16 P.1009 900 902 704 560 181 155 317
17 P.1123 1045 902 741 548 181 155 317
18 P.1129 900 902 704 560 181 155 317
19 P.1286 953 935 641 548 253 155 317
20 P.1287 900 902 741 560 181 155 317
21 P.1297 882 902 741 560 181 155 317
22 P.1843 846 935 756 548 276 155 317
23 P.1863 1045 864 756 548 276 155 317
24 P.1864 1045 864 756 548 276 155 317
25 P.1896 846 884 704 548 181 155 317
26 P.1961 864 864 756 548 276 155 317
27 P.1962 900 864 756 548 276 155 317bp
28 P.1963 1045 864 756 548 276 155 317
29 P.1964 1045 864 756 548 276 155 317
30 P.1966 900 935 756 548 276 155 317
31 P.1967 1045 902 756 548 276 155 317
Chapter 5 References
165
Table 14 (Continue)
S.No. Strains IDs
ClfA band size
ClfB band size
sdrD band size
sdrC band size
spa band size
Sspa band size
Sav1078 band size
32 P.2006 900 864 756 548 276 155 317
33 P.2054 882 974 756 506 229 155 317
34 P.2111 882 935 756 548 253 155 317
35 P.2112 900 935 756 548 276 155 317
36 P.2113 900 935 756 548 253 155 317
37 P.2119 882 935 756 548 229 155 317
38 P.2255 953 902 704 548 157 155 317
39 P.2285 900 902 704 560 181 155 317
40 P.2871 846 884 704 560 157 155 317
41 P.2885 846 884 756 548 276 155 317
42 P.2886 900 884 704 548 181 155 317
43 P.2887 900 884 756 548 276 155 317
44 P.2896 846 884 756 548 276 155 317
45 P.3027 1062 935 756 548 276 155 317
46 P.3727 900 864 756 548 276 155 317
47 P.5019 1045 884 704 548 276 155 317
48 P.5020 846 935 756 548 276 155 317
49 P.5114 1062 902 704 560 181 155 317
50 P.5119 900 902 704 560 181 155 317
51 P.5123 953 935 756 548 229 155 317
52 P.6269 1062 935 756 548 276 155 317
53 P.6270 864 935 756 548 253 155 317
54 P.8116 900 884 704 548 181 155 317
55 P.8431 846 884 756 548 276 155 317
56 P.8485 846 935 756 548 276 155 317
57 P.8486 846 935 756 548 276 155 317
58 P.8487 846 935 756 548 276 155 317
59 P.8668 900 902 704 548 181 155 317
60 P.8823 1045 935 756 658 276 155 317
61 P.10012 846 935 756 548 229 155 317
62 P.10013 900 935 756 548 229 155 317
Chapter 5 References
166
Table 14 (Continue)
S.No. Strains ID
ClfA band size
ClfB band size
sdrD band size
sdrC band size
spa band size
Sspa band size
Sav1078 band size
63 P.10015 900 935 704 548 229 155 317
64 P.10019 953 935 756 548 253 155 317
65 P.10021 900 935 756 548 253 155 317
66 P.11291 900 935 756 560 253 155 317
67 P.11293 900 902 741 560 181 155 317
68 P.11296 846 935 756 548 276 155 317
69 P.12006 900 884 704 560 157 155 317
70 P.12007 900 935 756 560 181 155 317
71 P.12285 900 902 704 560 181 155 317
72 P.13714 900 935 756 548 253 155 317
73 P.13729 953 935 756 548 276 155 317
74 P.13731 900 902 756 548 181 155 317
75 P.13740 900 902 741 560 181 155 317
76 P.13741 900 902 741 560 181 155 317
77 P.13749 882 935 756 548 229 155 317
78 P.13947 1045 974 756 548 276 155 317
79 P.14946 882 974 756 506 276 155 317
80 P.14313 1027 1102 704 481 205 155 317
81 P.14947 900 974 756 548 276 155 317
82 P.15415 900 974 704 548 276 155 317
83 P.16509 900 902 704 560 181 155 317
84 P.16809 953 935 641 560 253 155 317
85 P.16849 900 902 704 560 181 155 317
86 P.17120 1062 935 756 548 276 155 317
87 P.17121 882 935 756 481 276 155 317
88 P.18119 846 935 756 560 276 155 317
89 P.18176 882 902 756 506 276 155 317
90 P.18339 900 902 704 560 181 155 317
91 P.18431 846 935 756 548 276 155 317
92 K.3 900 902 704 548 181 155 317
93 K.6 900 884 756 548 181 155 317
Chapter 5 References
167
Table 14 (Continue)
S.No. Strains ID
ClfA band size
ClfB band size
sdrD band size
sdrC band size
spa band size
Sspa band size
Sav1078 band size
94 K.8 1045 935 756 548 276 155 317
95 K.10 900 864 756 548 276 155 317
96 K.11 900 902 756 548 181 155 317
97 K.12 882 822 756 506 157 155 317
98 K.13 953 935 756 548 276 155 317
99 A.379 900 902 756 481 181 155 317
100 A.465 900 902 704 560 181 155 317
101 A.1761 900 902 595 560 181 155 317 102 A.5053 882 974 756 506 276 155 317
103 A.5054 882 974 756 506 276 155 317
104 A.7145 900 974 756 548 276 155 317 105 A.9077 1045 935 756 324 276 155 317 106 A.9313 540 992 756 548 229 155 317 107 A.9445 1045 935 756 324 276 155 317 108 A.9703 882 974 756 506 276 155 317 109 A.9781 1045 935 756 324 276 155 317
110 A.9914 1045 935 756 548 276 155 317 111 A.9954 900 935 756 481 276 155 317 112 A.9965 900 884 704 560 157 155 317
113 A.10813 540 992 756 548 229 155 317 114 A.11241 882 974 756 506 276 155 317 115 A.13282 1027 1102 501 481 205 155 317
116 A.13319 900 935 756 548 276 155 317 117 A.13730 882 974 756 506 276 155 317
118 A.14075 882 935 756 548 276 155 317 119 A.14254 846 935 756 548 276 155 317
120 A.14256 1045 935 756 324 276 155 317 121 A.15330 1045 935 756 548 276 155 317
122 A.25356 1045 935 756 324 276 155 317 123 A.26163 900 884 704 560 157 155 317
124 A.35045 900 902 704 560 181 155 317 125 A.35087 1045 935 756 524 276 155 317
Chapter 5 References
168
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169
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170
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171
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172
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173
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174
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175
Table 16: Showing the Spa sequencing results with selected 26 MRSA isolates from Pakistan.
S. No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
1 P.2113 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACAACAAAAAGCCTGGC
4- AAAGAAGACGGCAACAAGCCTGGT
5- AAAGAAGACAACAAAAAACCTGGT
6- AAAGAAGATGGCAACAAGCCTGGT
7- AAAGAAGACAACAAAAAACCTGGT
8- AAAGAAGACGGCAACAAGCCTGGC
9- AAAGAAGATGGCAACAAACCTGGT
r11
r12
r05
r17
r34
r24
r34
r22
r25
YGCMBQBLO t451
2. P.18431 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
3. P.8486 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
4 P.5020 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
r11
r19
r12
r05
YHGCMBQBLO t064
Chapter 5 References
176
Table 16 (Continue)
S.No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r17
r34
r24
r34
r22
r25
5 P.844 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
6 A.14254 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
7 P.17120 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
r11
r19
r12
r05
r17
r34
YHGCMBQBLO t064
Chapter 5 References
177
Table 16 (Continue)
S.No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r24
r34
r22
r25
8 A.9445 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
9 A.14256 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
10 A.15330 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
YHGCMBQBLO t064
Chapter 5 References
178
Table 16 (Continue)
S.No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT r22
r25
11 A.9914 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
12 K.8 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
13 P.1863 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
Chapter 5 References
179
Table 16 (Continue)
S. No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
14 A.9313
1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAACCTGGT
6- AAAGAAGACGGCAACAAGCCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r25
r17
r24
r24
WGKAOMQQ t275
15 P.10813
1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAACCTGGT
6- AAAGAAGACGGCAACAAGCCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r25
r17
r24
r24
WGKAOMQQ t275
16 A.13282
1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAACCTGGT
6- AAAGAAGACGGCAACAAGCCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r25
r17
r24
WGKAOMQ t037
17 P.14947 1-GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6- AAAGAAGACGGCAACAAACCTGGT
7- AAAGAAGACAACAAAAAACCTGGC
8- AAAGAAGATGGCAACAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGT
10- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r16
r02
r25
r17
r24
WGKAQKAOMQ t987
Chapter 5 References
180
Table 16 (Continue)
S. No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
18 A.5053 1-GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6- AAAGAAGACGGCAACAAACCTGGT
7- AAAGAAGACAACAAAAAACCTGGC
8- AAAGAAGATGGCAACAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGT
10- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r16
r02
r25
r17
r24
WGKAQKAOMQ t987
19 P.1286
1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGACGGCAACAAACCTGGT
6- AAAGAAGACAACAAAAAACCTGGC
7- AAAGAAGATGGCAACAAACCTGGT
8- AAAGAAGACGGCAACAAGCCTGGT
9- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r16
r02
r25
r17
r24
WGKAKAOMQ t021
20 P.8 1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r24
WGKAQQ t030
21 P.13740 1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r24
WGKAQQ t030
Chapter 5 References
181
Table 16 (Continue)
S.No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
22 P.2285 1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3-AAAGAAGACGGCAACAAACCTGGT
4-AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6-AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r24
WGKAQQ t030
23 P.1287 1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r24
WGKAQQ t030
24 A.26163
1- GAGGAAGACAACAACAAGCCTGGT
2- AAAGAAGACGGCAACAAACCTGGT
3- AAAGAAGACAACAAAAAACCTGGC
4- AAAGAAGATGGCAACAAGCCTGGT
5- AAAGAAGATGGCAACAAGCCTGGT
r08
r16
r02
r24
r24
XKAQQ t632
25 P.12006
1- GAGGAAGACAACAACAAGCCTGGT
2- AAAGAAGACGGCAACAAACCTGGT
3- AAAGAAGACAACAAAAAACCTGGC
4- AAAGAAGATGGCAACAAGCCTGGT
5- AAAGAAGATGGCAACAAGCCTGGT
r08
r16
r02
r24
r24
XKAQQ t632
26 P.1
1- GAGGAAGACAACAACAAGCCTGGT
2- AAAGAAGACGGCAACAAACCTGGT
3- AAAGAAGACAACAAAAAACCTGGC
4- AAAGAAGATGGCAACAAGCCTGGT
5- AAAGAAGATGGCAACAAGCCTGGT
r08
r16
r02
r24
r24
XKAQQ t632
Chapter 5 References
182
Figure 48
Showing Sequence data of arcC gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Figure 49
Showing Sequence data of aroE gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Chapter 5 References
183
Figure 50
Showing Sequence data of glp gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 1.
Figure 51
Showing Sequence data of gmk gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 1.
Chapter 5 References
184
Figure 52
Showing Sequence data of pta gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 4.
Figure 53
Showing Sequence data of tpi gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 62.
Chapter 5 References
185
Figure 54
Showing Sequence data of yqil gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Chapter 5 References
186
Figure 55
Showing Sequence data of arcC gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.
Figure 56
Showing Sequence data of aroE gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Chapter 5 References
187
Figure 57
Showing Sequence data of glp gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 1.
Figure 58
Showing Sequence data of gmk gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 1.
Chapter 5 References
188
Figure 59
Showing Sequence data of pta gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 4.
Figure 60
Showing Sequence data of tpi gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 4.
Chapter 5 References
189
Figure 61
Showing Sequence data of yqil gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Chapter 5 References
190
Figure 62
Showing Sequence data of arcC gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.
Figure 63
Showing Sequence data of aroE gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.
Chapter 5 References
191
Figure 64
Showing Sequence data of glp gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.
Figure 65
Showing Sequence data of gmk gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.
Chapter 5 References
192
Figure 66
Showing Sequence data of pta gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 6.
Figure 67
Showing Sequence data of tpi gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Chapter 5 References
193
Figure 68
Showing Sequence data of yqil gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.
Appendix-I
162
Appendix- I
Table 13: Pakistani MRSA Isolates obtained from Hospitals of Rawalpindi/Islamabad.
S. No. S. aureus
Isolates
Clinical Source S. No. S. aureus
Isolates
Clinical Source
1 P.1 Pus 30 P.2006 Tissue Fluid
2 P.2 Pus 31 P.2054 Catheter tip
3 P.3 Pus 32 P.2111 Catheter tip
4 P.4 Pus 33 P.2112 Sputum
5 P.5 Sputum 34 P.2113 Sputum
6 P.6 Urine 35 P.2119 Pus
7 P.7 Pus 36 P.2255 Pus
8 P.8 Ear Infection 37 P.2285 Pus
9 P.843 Pus 38 P.2871 Sputum
10 P.844 Pus 39 P.2885 Spinal Card
11 P.862 Urine 40 P.2886 Catheter tip
12 P.1003 Tissue fluid 41 P.2887 Tissue Fluid
13 P.1005 Pus 42 P.2896 Spinal card
14 P.1009 Pus 43 P.3027 Catheter tips
15 P.1123 Catheter tip 44 P.3727 Sputum
16 P.1129 Pus 45 P.5019 Catheter tip
17 P.1286 Sputum 46 P.5020 Pus
18 P.1287 Pus 47 P.5114 Catheter tip
19 P.1297 Ear Infection 48 P.5119 Pus
20 P.1843 Pus 49 P.5123 Catheter tip
21 P.1863 Urine 50 P.6269 Catheter tip
22 P.1864 Urine 51 P.6270 Catheter tip
23 P.1896 Ear Infection 52 P.8116 Catheter tip
24 P.1961 Sputum 53 P.8431 Spinal Card
25 P.1962 Sputum 54 P.8485 Pus
26 P.1963 Urine 55 P.8486 Pus
27 P.1964 Urine 56 P.8487 Pus
28 P.1966 Sputum 57 P.8668 Ear Infection
29 P.1967 Catheter tip 58 P.8823 Pus
Appendix-I
163
Table 13 (Continue)
S. No. S. aureus
Isolates
Clinical Source S. No. S. aureus
Isolates
Clinical Source
59 P.10012 Sputum 89 P.18431 Pus
60 P.10013 Tissue Fluid 90 K.3 Ear Infection
61 P.10015 Sputum 91 K.6 Sputum
62 P.10019 Pus 92 K.8 Catheter tip
63 P.10021 Sputum 93 K.10 Sputum
64 P.11291 Pus 92 K.8 Catheter tip
65 P.11293 Pus 93 K.10 Sputum
66 P.11296 Pus 94 K.11 Pus
67 P.12006 Pus 95 K.12 Pus
68 P.12007 Sputum 96 K.13 Catheter tip
69 P.12285 Pus 97 A.379 Ear Infection
70 P.13714 Pus 98 A.465 Pus
71 P.13729 Catheter Tip 99 A.1761 Ear Infection
72 P.13731 Pus 100 A.5053 Catheter tips
73 P.13740 Pus 101 A.5054 Catheter tip
74 P.13741 Pus 102 A.7145 Pus
75 P.13749 Pus 103 A.9077 Blood
76 P.13947 Catheter tip 104 A.9313 Pus
77 P.14313 Pus 105 A.9445 Blood
78 P.14946 Sputum 106 A.9703 Catheter tip
79 P.14947 Pus 107 A.9781 Blood
80 P.15415 Catheter tip 108 A.9914 Pus
81 P.16509 Pus 109 A.9954 Catheter tip
82 P.16809 Sputum 110 A.9965 Pus
83 P.16849 Pus 111 A.10813 Pus
84 P.17120 Catheter tip 112 A.11241 Pus
85 P.17121 Catheter tip 113 A.13282 Pus
86 P.18119 Catheter tip 114 A.13319 Pus
87 P.18176 Catheter tip 115 A.13730 Pus
88 P.18339 Pus 116 A.14075 Catheter tip
Appendix-I
164
Table 14: Showing the amplicon sizes for the 123 Pakistani MRSA Isolates and NCTC 8325 and Mu50 (Positive control strains) obtained after Gene Scan for the clfA, clfB, sdrD,sdrC,spa sspa and sav1078 loci.
S.No. Strains IDs
ClfA band size
ClfB band size
sdrD band size
sdrC band size
spa band size
Sspa band size
Sav1078 band size
1 NCTC 8325
1027 822 659 762 300 155 317
2 Mu-50 1050 822 768 634 276 174 317
3 P.1 900 884 704 560 157 155 317
4 P.2 846 935 704 548 157 155 317
5 P.3 900 974 756 506 276 155 317
6 P.4 900 902 756 560 181 155 317
7 P.5 900 884 756 560 181 155 317
8 P.6 1045 864 756 548 276 155 317
9 P.7 900 935 704 548 276 155 317
10 P.8 846 884 756 548 181 155 317
11 P.843 846 935 756 548 276 155 317
12 P.844 846 935 756 548 276 155 317
13 P.862 1045 864 756 548 276 155 317
14 P.1003 900 935 756 548 229 155 317
15 P.1005 900 902 704 560 181 155 317
16 P.1009 900 902 704 560 181 155 317
17 P.1123 1045 902 741 548 181 155 317
18 P.1129 900 902 704 560 181 155 317
19 P.1286 953 935 641 548 253 155 317
20 P.1287 900 902 741 560 181 155 317
21 P.1297 882 902 741 560 181 155 317
22 P.1843 846 935 756 548 276 155 317
23 P.1863 1045 864 756 548 276 155 317
24 P.1864 1045 864 756 548 276 155 317
25 P.1896 846 884 704 548 181 155 317
26 P.1961 864 864 756 548 276 155 317
27 P.1962 900 864 756 548 276 155 317
28 P.1963 1045 864 756 548 276 155 317
29 P.1964 1045 864 756 548 276 155 317
30 P.1966 900 935 756 548 276 155 317
31 P.1967 1045 902 756 548 276 155 317
Appendix-I
165
Table 14 (Continue)
S.No. Strains IDs
ClfA band size
ClfB band size
sdrD band size
sdrC band size
spa band size
Sspa band size
Sav1078 band size
32 P.2006 900 864 756 548 276 155 317
33 P.2054 882 974 756 506 229 155 317
34 P.2111 882 935 756 548 253 155 317
35 P.2112 900 935 756 548 276 155 317
36 P.2113 900 935 756 548 253 155 317
37 P.2119 882 935 756 548 229 155 317
38 P.2255 953 902 704 548 157 155 317
39 P.2285 900 902 704 560 181 155 317
40 P.2871 846 884 704 560 157 155 317
41 P.2885 846 884 756 548 276 155 317
42 P.2886 900 884 704 548 181 155 317
43 P.2887 900 884 756 548 276 155 317
44 P.2896 846 884 756 548 276 155 317
45 P.3027 1062 935 756 548 276 155 317
46 P.3727 900 864 756 548 276 155 317
47 P.5019 1045 884 704 548 276 155 317
48 P.5020 846 935 756 548 276 155 317
49 P.5114 1062 902 704 560 181 155 317
50 P.5119 900 902 704 560 181 155 317
51 P.5123 953 935 756 548 229 155 317
52 P.6269 1062 935 756 548 276 155 317
53 P.6270 864 935 756 548 253 155 317
54 P.8116 900 884 704 548 181 155 317
55 P.8431 846 884 756 548 276 155 317
56 P.8485 846 935 756 548 276 155 317
57 P.8486 846 935 756 548 276 155 317
58 P.8487 846 935 756 548 276 155 317
59 P.8668 900 902 704 548 181 155 317
60 P.8823 1045 935 756 658 276 155 317
61 P.10012 846 935 756 548 229 155 317
62 P.10013 900 935 756 548 229 155 317
Appendix-I
166
Table 14 (Continue)
S.No. Strains ID
ClfA band size
ClfB band size
sdrD band size
sdrC band size
spa band size
Sspa band size
Sav1078 band size
63 P.10015 900 935 704 548 229 155 317
64 P.10019 953 935 756 548 253 155 317
65 P.10021 900 935 756 548 253 155 317
66 P.11291 900 935 756 560 253 155 317
67 P.11293 900 902 741 560 181 155 317
68 P.11296 846 935 756 548 276 155 317
69 P.12006 900 884 704 560 157 155 317
70 P.12007 900 935 756 560 181 155 317
71 P.12285 900 902 704 560 181 155 317
72 P.13714 900 935 756 548 253 155 317
73 P.13729 953 935 756 548 276 155 317
74 P.13731 900 902 756 548 181 155 317
75 P.13740 900 902 741 560 181 155 317
76 P.13741 900 902 741 560 181 155 317
77 P.13749 882 935 756 548 229 155 317
78 P.13947 1045 974 756 548 276 155 317
79 P.14946 882 974 756 506 276 155 317
80 P.14313 1027 1102 704 481 205 155 317
81 P.14947 900 974 756 548 276 155 317
82 P.15415 900 974 704 548 276 155 317
83 P.16509 900 902 704 560 181 155 317
84 P.16809 953 935 641 560 253 155 317
85 P.16849 900 902 704 560 181 155 317
86 P.17120 1062 935 756 548 276 155 317
87 P.17121 882 935 756 481 276 155 317
88 P.18119 846 935 756 560 276 155 317
89 P.18176 882 902 756 506 276 155 317
90 P.18339 900 902 704 560 181 155 317
91 P.18431 846 935 756 548 276 155 317
92 K.3 900 902 704 548 181 155 317
93 K.6 900 884 756 548 181 155 317
Appendix-I
167
Table 14 (Continue)
S.No. Strains ID
ClfA band size
ClfB band size
sdrD band size
sdrC band size
spa band size
Sspa band size
Sav1078 band size
94 K.8 1045 935 756 548 276 155 317
95 K.10 900 864 756 548 276 155 317
96 K.11 900 902 756 548 181 155 317
97 K.12 882 822 756 506 157 155 317
98 K.13 953 935 756 548 276 155 317
99 A.379 900 902 756 481 181 155 317
100 A.465 900 902 704 560 181 155 317
101 A.1761 900 902 595 560 181 155 317 102 A.5053 882 974 756 506 276 155 317
103 A.5054 882 974 756 506 276 155 317
104 A.7145 900 974 756 548 276 155 317 105 A.9077 1045 935 756 324 276 155 317 106 A.9313 540 992 756 548 229 155 317 107 A.9445 1045 935 756 324 276 155 317 108 A.9703 882 974 756 506 276 155 317 109 A.9781 1045 935 756 324 276 155 317
110 A.9914 1045 935 756 548 276 155 317 111 A.9954 900 935 756 481 276 155 317 112 A.9965 900 884 704 560 157 155 317
113 A.10813 540 992 756 548 229 155 317 114 A.11241 882 974 756 506 276 155 317 115 A.13282 1027 1102 501 481 205 155 317
116 A.13319 900 935 756 548 276 155 317 117 A.13730 882 974 756 506 276 155 317
118 A.14075 882 935 756 548 276 155 317 119 A.14254 846 935 756 548 276 155 317
120 A.14256 1045 935 756 324 276 155 317 121 A.15330 1045 935 756 548 276 155 317
122 A.25356 1045 935 756 324 276 155 317 123 A.26163 900 884 704 560 157 155 317
124 A.35045 900 902 704 560 181 155 317 125 A.35087 1045 935 756 524 276 155 317
Appendix-I
168
Table 15: Showing the IDs of isolates, amplicon sizes, length of VNTR region present in these amplicon, total number of repeats for the 123 Pakistani
MRSA isolates and NCTC 8325 and Mu50 (Positive control strains) obtained after Gene Scan for the clfA, clfB, sdrD, sdrC, spa and sspa.
S.
No.
Strains ID
ClfA band size
Rep. size
Rep. Nos.
ClfB band size
Rep. size
Rep. Nos.
sdrD band size
Rep. size
Rep. Nos.
sdrC band size
Rep. size
Rep. Nos.
spa band size
Rep. size
Rep. Nos.
sspa band size
Rep. size
Rep.Nos.
1 NCTC 8325
1027 792 44 822 630 35 659 450 25 762 648 36 300 264 11 155 72 4
2 Mu-50 1050 815 45 822 630 35 768 558 31 634 522 29 276 240 10 174 91 5
3 P.1 900 665 37 884 688 38 704 486 27 560 450 25 157 121 5 155 72 4
4 P.2 846 611 34 935 739 41 704 486 27 548 432 24 157 121 5 155 72 4
5 P.3 900 665 37 974 778 43 756 540 30 506 396 22 276 240 10 155 72 4
6 P.4 900 665 37 902 706 39 756 540 30 560 450 25 181 145 6 155 72 4
7 P.5 900 665 37 884 688 38 756 540 30 560 450 25 181 145 6 155 72 4
8 P.6 1045 810 45 864 668 37 756 540 30 548 432 24 276 240 10 155 72 4
9 P.7 900 665 37 935 739 41 704 486 27 548 432 24 276 240 10 155 72 4
10 P.8 846 611 34 884 688 38 756 540 30 548 432 24 181 145 6 155 72 4
11 P.843 846 611 34 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
12 P.844 846 611 34 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
13 P.862 1045 810 45 864 668 37 756 540 30 548 432 24 276 240 10 155 72 4
14 P.1003 900 665 37 935 739 41 756 540 30 548 432 24 229 193 8 155 72 4
15 P.1005 900 665 37 902 706 39 704 486 27 560 450 25 181 145 6 155 72 4
16 P.1009 900 665 37 902 706 39 704 486 27 560 450 25 181 145 6 155 72 4
17 P.1123 1045 810 45 902 706 39 741 540 30 548 432 24 181 145 6 155 72 4
Appendix-I
169
Table 15 (Continue)
S.
No.
Strains ID
ClfA band size
Rep. size
Rep. Nos.
ClfB band size
Rep. size
Rep. Nos.
sdrD band size
Rep. size
Rep. Nos.
sdrC band size
Rep. size
Rep. Nos.
spa band size
Rep. size
Rep. Nos.
sspa band size
Rep. size
Rep.Nos.
18 P.1129 900 665 37 902 706 39 704 486 27 560 450 25 181 145 6 155 72 4
19 P.1286 953 718 40 935 742 41 641 432 24 548 432 24 253 217 9 155 72 4
20 P.1287 900 665 37 902 706 39 741 540 30 560 450 25 181 145 6 155 72 4
21 P.1297 882 647 36 902 706 39 741 522 29 560 450 25 181 145 6 155 72 4
22 P.1843 846 611 34 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
23 P.1863 1045 810 45 866 670 37 756 540 30 548 432 24 276 240 10 155 72 4
24 P.1864 1045 810 45 864 668 37 756 540 30 548 432 24 276 240 10 155 72 4
25 P.1896 846 611 34 884 688 38 704 486 27 548 432 24 181 145 6 155 72 4
26 P.1961 864 629 35 864 668 37 756 540 30 548 432 24 276 240 10 155 72 4
27 P.1962 900 665 37 864 668 37 756 540 30 548 432 24 276 240 10 155 72 4
28 P.1963 1045 810 45 864 668 37 756 540 30 548 432 24 276 240 10 155 72 4
29 P.1964 1045 810 45 864 668 37 756 540 30 548 432 24 276 240 10 155 72 4
30 P.1966 900 665 37 935 739 41 756 540 30 548 432 24 277 241 10 155 72 4
31 P.1967 1045 810 45 904 708 39 756 540 30 548 432 24 276 240 10 155 72 4
32 P.2006 900 665 37 864 668 38 756 540 30 548 432 24 276 240 10 155 72 4
33 P.2054 882 647 36 974 779 43 756 540 30 506 396 22 229 193 8 155 72 4
34 P.2111 882 647 36 935 739 41 756 540 30 548 432 24 253 217 9 155 72 4
35 P.2112 900 665 37 935 739 41 756 540 30 548 432 24 277 241 10 155 72 4
36 P.2113 900 665 37 935 739 41 756 540 30 548 432 24 253 217 9 155 72 4
Appendix-I
170
Table 15 (Continue)
S. No.
Strains ID
ClfA band size
Rep. size
Rep. Nos.
ClfB band size
Rep. size
Rep. Nos.
sdrD band size
Rep. size
Rep. Nos.
sdrC band size
Rep. size
Rep. Nos.
spa band size
Rep. size
Rep. Nos.
sspa band size
Rep. size
Rep.Nos.
37 P.2119 882 647 36 935 739 41 756 540 30 548 432 24 229 193 8 155 72 4
38 P.2255 953 718 40 902 706 39 704 486 27 548 432 24 157 121 5 155 72 4
39 P.2285 900 665 37 902 706 39 705 486 27 560 448 25 181 145 6 155 72 4
40 P.2871 846 611 34 884 688 38 704 486 27 560 450 25 157 121 5 155 72 4
41 P.2885 846 611 34 884 688 38 756 540 30 548 432 24 276 240 10 155 72 4
42 P.2886 900 665 37 884 688 38 704 486 27 548 432 24 181 145 6 155 72 4
43 P.2887 900 665 37 884 688 38 756 540 30 548 432 24 276 240 10 155 72 4
44 P.2896 846 611 34 884 688 38 756 540 30 548 432 24 277 241 10 155 72 4
45 P.3027 1062 827 46 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
46 P.3727 900 665 37 864 668 37 756 540 30 548 432 24 276 240 10 155 72 4
47 P.5019 1045 810 45 884 688 38 704 486 27 548 432 24 276 240 10 155 72 4
48 P.5020 846 611 34 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
49 P.5114 1062 827 46 902 706 39 704 486 27 560 450 25 181 145 6 155 72 4
50 P.5119 900 665 37 902 706 39 705 486 27 561 450 25 181 145 6 155 72 4
51 P.5123 953 718 40 935 739 41 756 540 30 548 432 24 229 193 8 155 72 4
52 P.6269 1062 827 46 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
53 P.6270 864 629 35 935 739 41 756 540 30 548 432 24 253 217 9 155 72 4
54 P.8116 902 667 37 882 686 38 705 486 27 541 432 24 181 145 6 155 72 4
Appendix-I
171
Table 15 (Continue)
S.
No.
Strains ID
ClfA band size
Rep. size
Rep. Nos.
ClfB band size
Rep. size
Rep. Nos.
sdrD band size
Rep. size
Rep. Nos.
sdrC band size
Rep. size
Rep. Nos.
spa band size
Rep. size
Rep. Nos.
sspa band size
Rep. size
Rep.Nos.
55 P.8431 846 611 34 884 688 38 756 540 30 548 432 24 276 240 10 155 72 4
56 P.8485 846 611 34 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
57 P.8486 846 611 34 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
58 P.8487 846 611 34 935 739 41 756 540 30 548 432 24 277 241 10 155 72 4
59 P.8668 900 665 37 902 706 39 704 486 27 548 432 24 181 145 6 155 72 4
60 P.8823 1045 810 45 935 739 41 756 540 30 658 540 30 276 240 10 155 72 4
61 P.10012 846 611 34 935 739 41 756 540 30 548 432 24 229 193 8 155 72 4
62 P.10013 900 665 37 935 739 41 756 540 30 548 432 24 229 193 8 155 72 4
63 P.10015 900 665 37 935 739 41 704 540 30 548 432 24 229 193 8 155 72 4
64 P.10019 953 719 40 935 739 41 756 547 30 548 432 24 253 217 9 155 72 4
65 P.10021 900 665 37 935 739 41 756 540 30 548 432 24 253 217 9 155 72 4
66 P.11291 900 665 37 935 739 41 756 540 30 560 450 25 253 217 9 155 72 4
67 P.11293 900 665 37 902 706 39 741 522 29 560 450 25 181 145 6 155 72 4
68 P.11296 846 611 34 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
69 P.12006 900 665 37 884 688 38 704 540 30 560 450 25 157 121 5 155 72 4
70 P.12007 900 665 37 935 739 41 756 540 30 560 450 25 181 145 6 155 72 4
71 P.12285 900 665 37 902 706 39 704 486 27 560 450 25 181 145 6 155 72 4
72 P.13714 900 665 37 935 739 41 756 540 30 548 432 24 253 217 9 155 72 4
Appendix-I
172
Table 15 (Continue)
S.
No.
Strains ID
ClfA band size
Rep. size
Rep. Nos.
ClfB band size
Rep. size
Rep. Nos.
sdrD band size
Rep. size
Rep. Nos.
sdrC band size
Rep. size
Rep. Nos.
spa band size
Rep. size
Rep. Nos.
sspa band size
Rep. size
Rep.Nos.
73 P.13729 953 719 40 935 739 41 756 540 30 548 432 24 277 241 10 155 72 4
74 P.13731 900 665 37 902 706 39 756 540 30 548 432 24 181 145 6 155 72 4
75 P.13749 882 642 36 935 739 41 756 540 30 548 432 24 229 193 8 155 72 4
76 P.13947 1045 810 45 974 778 43 756 540 30 548 432 24 276 240 10 155 72 4
77 P.14313 1027 792 44 1102 906 50 704 486 27 481 360 20 205 169 7 155 72 4
78 P.13740 900 665 37 902 706 39 741 540 30 560 450 25 181 145 6 155 72 4
79 P.13741 900 665 37 902 706 39 741 540 30 560 450 25 181 145 6 155 72 4
80 P.14946 882 647 36 974 778 43 756 540 30 505 396 22 276 240 10 155 72 4
81 P.14947 900 665 37 974 778 43 756 540 30 548 432 24 276 240 10 155 72 4
82 P.15415 900 665 37 974 778 43 704 486 27 548 432 24 276 240 10 155 72 4
83 P.16509 900 665 37 902 706 39 704 486 27 560 450 25 181 145 6 155 72 4
84 P.16809 953 718 40 935 742 41 641 432 24 560 450 25 253 217 9 155 72 4
85 P.16849 900 665 37 902 706 39 704 486 27 560 450 25 181 145 6 155 72 4
86 P.17120 1062 827 46 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
87 P.17121 882 641 36 935 739 41 756 540 30 480 360 20 276 240 10 155 72 4
88 P.18119 846 611 34 935 739 41 756 540 30 560 450 25 277 241 10 155 72 4
89 P.18176 882 647 36 902 706 39 756 540 30 505 396 22 276 240 10 155 72 4
90 P.18339 900 665 37 902 706 39 704 486 27 560 450 25 181 145 6 155 72 4
91 P.18431 846 611 34 935 739 41 756 540 30 548 432 24 277 241 10 155 72 4
Appendix-I
173
Table 15 (Continue)
S.
No.
Strains ID
ClfA band size
Rep. size
Rep. Nos.
ClfB band size
Rep. size
Rep. Nos.
sdrD band size
Rep. size
Rep. Nos.
sdrC band size
Rep. size
Rep. Nos.
spa band size
Rep. size
Rep. Nos.
sspa band size
Rep. size
Rep.Nos.
92 K.3 900 665 37 902 706 39 704 495 27 548 432 24 181 145 6 155 72 4
93 K.6 900 665 37 884 688 38 756 540 30 548 432 24 181 145 6 155 72 4
94 K.8 1045 810 45 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
95 K.10 900 665 37 864 668 37 756 540 30 548 432 24 276 240 10 155 72 4
96 K11 900 665 37 902 706 39 756 540 30 548 432 24 181 145 6 155 72 4
97 K.12 882 647 36 822 626 35 756 540 30 506 396 22 157 121 5 155 72 4
98 K.13 953 718 40 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
99 A.379 900 665 37 902 706 39 756 540 30 481 360 20 181 145 6 155 72 4
100 A.465 900 665 37 902 706 39 698 486 27 560 450 25 181 145 6 155 72 4
101 A.1761 900 665 37 902 706 39 595 378 21 560 450 25 181 145 6 155 72 4
102 A.5053 882 647 36 974 779 43 756 540 30 505 396 22 276 240 10 155 72 4
103 A.5054 882 647 36 974 778 43 756 540 30 505 396 22 276 240 10 155 72 4
104 A.7145 900 665 37 974 778 43 756 540 30 548 432 24 276 240 10 155 72 4
105 A.9077 1045 810 45 935 739 41 756 540 30 324 216 12 276 240 10 155 72 4
106 A.9313 540 305 17 992 796 44 756 540 30 542 432 24 229 193 8 155 72 4
107 A.9445 1045 810 45 935 739 41 756 540 30 324 216 12 276 240 10 155 72 4
108 A.9703 882 647 36 974 778 43 756 540 30 506 396 22 276 240 10 155 72 4
109 A.9781 1045 810 45 935 739 41 756 540 30 324 216 12 276 240 10 155 72 4
110 A.9914 1045 810 45 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
Appendix-I
174
Table 15 (Continue)
S.
No.
Strains ID
ClfA band size
Rep. size
Rep. Nos.
ClfB band size
Rep. size
Rep. Nos.
sdrD band size
Rep. size
Rep. Nos.
sdrC band size
Rep. size
Rep. Nos.
spa band size
Rep. size
Rep. Nos.
sspa band size
Rep. size
Rep.Nos.
111 A.9954 900 665 37 935 739 41 756 540 30 481 360 20 276 240 10 155 72 4
112 A.9965 900 665 37 884 688 38 704 486 27 560 450 25 157 121 5 155 72 4
113 A.10813 540 305 17 992 796 44 756 540 30 542 432 24 229 193 8 155 72 4
114 A.11241 882 647 36 974 778 43 756 540 30 506 396 22 276 240 10 155 72 4
115 A.13282 1026 791 44 1102 905 50 501 288 16 481 360 20 205 169 7 155 72 4
116 A.13319 900 665 37 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
117 A.13730 882 647 36 974 778 43 756 540 30 505 396 22 276 240 10 155 72 4
118 A.14075 882 647 36 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
119 A.14254 846 611 34 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
120 A.14256 1045 810 45 935 739 41 756 540 30 324 216 12 276 240 10 155 72 4
121 A.15330 1045 810 45 935 739 41 756 540 30 548 432 24 276 240 10 155 72 4
122 A.25356 1045 810 45 935 739 41 756 540 30 324 216 12 276 240 10 155 72 4
123 A.26163 900 665 37 884 688 38 704 486 27 560 450 25 157 121 5 155 72 4
124 A.35045 900 665 37 902 706 39 704 486 27 560 450 25 181 145 6 155 72 4
125 A.35087 1045 810 45 935 741 41 756 540 30 523 414 23 276 240 10 155 72 4
Appendix-I
175
Table 16: Showing the Spa sequencing results with selected 26 MRSA isolates from Pakistan.
S. No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
1 P.2113 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACAACAAAAAGCCTGGC
4- AAAGAAGACGGCAACAAGCCTGGT
5- AAAGAAGACAACAAAAAACCTGGT
6- AAAGAAGATGGCAACAAGCCTGGT
7- AAAGAAGACAACAAAAAACCTGGT
8- AAAGAAGACGGCAACAAGCCTGGC
9- AAAGAAGATGGCAACAAACCTGGT
r11
r12
r05
r17
r34
r24
r34
r22
r25
YGCMBQBLO t451
2. P.18431 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
3. P.8486 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
4 P.5020 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
r11
r19
r12
r05
YHGCMBQBLO t064
Appendix-I
176
Table 16 (Continue)
S.No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r17
r34
r24
r34
r22
r25
5 P.844 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
6 A.14254 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
7 P.17120 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
r11
r19
r12
r05
r17
r34
YHGCMBQBLO t064
Appendix-I
177
Table 16 (Continue)
S.No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r24
r34
r22
r25
8 A.9445 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
9 A.14256 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
10 A.15330 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
YHGCMBQBLO t064
Appendix-I
178
Table 16 (Continue)
S.No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT r22
r25
11 A.9914 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
12 K.8 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
13 P.1863 1- GAGGAAGACAATAACAAGCCTGGC
2- AAAGAAGACAATAACAAGCCTGGC
3- AAAGAAGACAACAACAAGCCTGGT
4- AAAGAAGACAACAAAAAGCCTGGC
5- AAAGAAGACGGCAACAAGCCTGGT
6- AAAGAAGACAACAAAAAACCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGACAACAAAAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGC
10- AAAGAAGATGGCAACAAACCTGGT
r11
r19
r12
r05
r17
r34
r24
r34
r22
r25
YHGCMBQBLO t064
Appendix-I
179
Table 16 (Continue)
S. No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
14 A.9313
1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAACCTGGT
6- AAAGAAGACGGCAACAAGCCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r25
r17
r24
r24
WGKAOMQQ t275
15 P.10813
1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAACCTGGT
6- AAAGAAGACGGCAACAAGCCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
8- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r25
r17
r24
r24
WGKAOMQQ t275
16 A.13282
1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAACCTGGT
6- AAAGAAGACGGCAACAAGCCTGGT
7- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r25
r17
r24
WGKAOMQ t037
17 P.14947 1-GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6- AAAGAAGACGGCAACAAACCTGGT
7- AAAGAAGACAACAAAAAACCTGGC
8- AAAGAAGATGGCAACAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGT
10- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r16
r02
r25
r17
r24
WGKAQKAOMQ t987
Appendix-I
180
Table 16 (Continue)
S. No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
18 A.5053 1-GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6- AAAGAAGACGGCAACAAACCTGGT
7- AAAGAAGACAACAAAAAACCTGGC
8- AAAGAAGATGGCAACAAACCTGGT
9- AAAGAAGACGGCAACAAGCCTGGT
10- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r16
r02
r25
r17
r24
WGKAQKAOMQ t987
19 P.1286
1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGACGGCAACAAACCTGGT
6- AAAGAAGACAACAAAAAACCTGGC
7- AAAGAAGATGGCAACAAACCTGGT
8- AAAGAAGACGGCAACAAGCCTGGT
9- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r16
r02
r25
r17
r24
WGKAKAOMQ t021
20 P.8 1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r24
WGKAQQ t030
21 P.13740 1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r24
WGKAQQ t030
Appendix-I
181
Table 16 (Continue)
S.No. Strain
ID
Repeat Sequences Repeat IDs
Kreiswirth IDs SPA type
22 P.2285 1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3-AAAGAAGACGGCAACAAACCTGGT
4-AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6-AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r24
WGKAQQ t030
23 P.1287 1- GAGGAAGACAACAACAAGCCTGGC
2- AAAGAAGACAACAACAAGCCTGGT
3- AAAGAAGACGGCAACAAACCTGGT
4- AAAGAAGACAACAAAAAACCTGGC
5- AAAGAAGATGGCAACAAGCCTGGT
6- AAAGAAGATGGCAACAAGCCTGGT
r15
r12
r16
r02
r24
r24
WGKAQQ t030
24 A.26163
1- GAGGAAGACAACAACAAGCCTGGT
2- AAAGAAGACGGCAACAAACCTGGT
3- AAAGAAGACAACAAAAAACCTGGC
4- AAAGAAGATGGCAACAAGCCTGGT
5- AAAGAAGATGGCAACAAGCCTGGT
r08
r16
r02
r24
r24
XKAQQ t632
25 P.12006
1- GAGGAAGACAACAACAAGCCTGGT
2- AAAGAAGACGGCAACAAACCTGGT
3- AAAGAAGACAACAAAAAACCTGGC
4- AAAGAAGATGGCAACAAGCCTGGT
5- AAAGAAGATGGCAACAAGCCTGGT
r08
r16
r02
r24
r24
XKAQQ t632
26 P.1
1- GAGGAAGACAACAACAAGCCTGGT
2- AAAGAAGACGGCAACAAACCTGGT
3- AAAGAAGACAACAAAAAACCTGGC
4- AAAGAAGATGGCAACAAGCCTGGT
5- AAAGAAGATGGCAACAAGCCTGGT
r08
r16
r02
r24
r24
XKAQQ t632
Appendix-II
182
Figure 48
Showing Sequence data of arcC gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Figure 49
Showing Sequence data of aroE gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Appendix-II
183
Figure 50
Showing Sequence data of glp gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 1.
Figure 51
Showing Sequence data of gmk gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 1.
Appendix-II
184
Figure 52
Showing Sequence data of pta gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 4.
Figure 53
Showing Sequence data of tpi gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 62.
Appendix-II
185
Figure 54
Showing Sequence data of yqil gene locus of S. aureus strain P.2113 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Appendix-II
186
Figure 55
Showing Sequence data of arcC gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.
Figure 56
Showing Sequence data of aroE gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Appendix-II
187
Figure 57
Showing Sequence data of glp gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 1.
Figure 58
Showing Sequence data of gmk gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 1.
Appendix-II
188
Figure 59
Showing Sequence data of pta gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 4.
Figure 60
Showing Sequence data of tpi gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 4.
Appendix-II
189
Figure 61
Showing Sequence data of yqil gene locus of S. aureus strain A.13282 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Appendix-II
190
Figure 62
Showing Sequence data of arcC gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.
Figure 63
Showing Sequence data of aroE gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.
Appendix-II
191
Figure 64
Showing Sequence data of glp gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.
Figure 65
Showing Sequence data of gmk gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.
Appendix-II
192
Figure 66
Showing Sequence data of pta gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 6.
Figure 67
Showing Sequence data of tpi gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 3.
Appendix-II
193
Figure 68
Showing Sequence data of yqil gene locus of S. aureus strain P.1286 in fasta format. This sequence is consensus sequence obtained after the alignment of forward and reverse strand. The allelic code obtained for this sequence from MLST server is 2.