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CHAPTER 6
ANTIBIOTIC RESISTANCE OF THE ENTEROCOCCAL ISOLATES
6.1. INTRODUCTION
Antibiotics are extensively used to prevent or to treat microbial
infections in human and veterinary medicine. Steadily increasing antibiotic
resistance and decreasing numbers of newer antibiotics has become a global
problem. Over the last two decades, nosocomial infections caused by
Enterococcus particularly VRE have emerged and have subsequently been
isolated from all over the world. (Rice, 2000; Oteo et al., 2007; Top et al.,
2008; Suchitra and Laksmidevi, 2009). The emergence of E. faecalis and E.
faecium was paralleled by the increase in glycopeptide and high-level
aminoglycoside resistance which are important drugs for the treatment of
gram positive bacterial infections (Shepard and Gilmore, 2002).
To date six gene clusters like vanA, vanB, vanC, vanD, vanE and
vanG (Boyd et al., 2006) have been found to be associated with vancomycin
resistance in Enterococcus species. It is very likely that this resistance could
be horizontally transferred to other organisms (Arthur and Courvalin, 1993).
The epidemiology of vancomycin resistant E.faecium is yet to be well
understood. There is difference in opinion about the emergence of
vancomycin resistance. Nosocomial spread of VREF has been reported by
Bonten et al., (2001). Others revealed that commensal microbiota of some
animals and humans act as reservoirs of VREF (Jensen et al., 1998; Klare et
al., 1995). VREF of food borne origin is also known to cause human
colonization (Bogaard et al., 1996). However the role played by non human
124 Chapter 6
sources and reservoirs other than hospitalized patients in the spread of
Enterococcus is ambiguous (Turnidge, 2004). Many molecular techniques
are used for studying the genetic relatedness of Enterococcus. The
effectiveness of RAPD in enterococcus typing was described by Barbier et
al., (1996).
Concern over increasing resistance to antimicrobial agents has
highlighted the need for surveillance of antimicrobial resistance at a local,
national and international level to tackle this problem. In this study, a
population of Enterococcus species from various sources was examined for
the occurrence of resistance against antimicrobials employed in veterinary
and in human medicine and also to compare antimicrobial sensitivity against
Enterococcus isolated from different sources. In the study, RAPD analysis
was conducted to compare the genetic similarities of VREF isolates from
clinical and poultry sources.
6.2. MATERIALS AND METHODS
Different enterococcal strains obtained from clinical and nonclinical
isolates especially from water, chicken faeces and human faeces were
screened for antibiotic resistance.
6.2.1. Antimicrobial susceptibility of Enterococcus
Antimicrobial susceptibility of the isolate was tested by the disc
diffusion method according to Bauer et al., (1966). Bacterial cultures were
prepared from overnight growth on a blood agar plate by suspending seven
to eight morphologically similar colonies in peptone water. Each inoculum
was adjusted to 0.5 Mc Farland standards. The inoculum was swabbed on to
Mueller -Hinton Agar. Commercially prepared antibiotic discs obtained from
Hi-Media (Appendix-2) with a diameter of 6 mm were used to determine the
Antibiotic Resistance of the Enterococcal Isolates 125
susceptibility pattern of Enterococcus species. The plates were incubated at
37o C over night. Zone of inhibition was measured and the results were
interpreted according to the National Committee for Clinical Laboratory
Standards, (2003).
6.2.2. MIC determination
E-test antibiotic strips (HiMedia) were used to determine the
susceptibility of the strains to ampicillin, benzyl penicillin, streptomycin,
gentamicin, vancomycin and teicoplanin. Mueller–Hinton agar plates were
lawn-cultured with enterococci, and antibiotic strips were placed onto the
plates according to the manufacturer's instructions. Plates were incubated at
37°C, and results were read after 24 and 48 h of incubation. The MIC values
were determined from the concentration of antibiotic at which the zone
intersects the test strip (Huang et al., 1992)
6.2.3. Beta lactamase test:
Production of beta lactamase was determined by using cefinase discs
(BD Diagnostic Systems) which were impregnated with chromogenic
cephalosporin, nitrocefin. Colonies were smeared onto the surface of
moistened disc and observed for colour change (Montgomery et al., 1979).
6.2.4. PCR amplification of vancomycin resistance gene
Three vancomycin resistant isolates (1 vanA & 2vanB phenotypes)
were selected for the detection of resistance gene determinants by
Polymerase Chain Reaction (PCR). The selected isolates were subjected to
both genomic DNA isolation and plasmid purification.
126 Chapter 6
6.2.4.1. Genomic DNA Isolation
Genomic DNA isolation was carried out as per the methods
explained by Kitao et al., (2010). For this, 1.2×104 CFU of the pure cultured
bacteria were mixed with 0.5mL IM NaCl supplemented 10 mM Tris HCl
buffer (pH 8.0) in a microtube. This was followed by the addition of 5µl
(1,000 U/mL) lysostaphin (Sigma – Aldrich) and incubated at 37°C for 1 h.
The sample was further kept for 15min on a boiling water bath and was then
centrifuged at 12,000 rpm for 10 min. The concentration of DNA in the
supernatant was measured at 260nm, and was further diluted to 100ng/µl
concentration for the PCR.
6.2.4.2. Plasmid DNA Isolation
Plasmid DNA was isolated by alkali lysis method as described by
Birnboim and Doly, (1979) with slight modification. For this, a test tube
containing 5mL of brain heart infusion broth was inoculated with a single
isolated colony picked from BHIA and this was incubated at 37°C, overnight
with shaking. From this, 1.5mL of culture was taken in a microfuge tube and
was centrifuged for 30 seconds at 12,000g at 4°C. The supernatant was
discarded and the bacterial pellet was resuspended in 100µl of solution I
(Appendix-3) and kept for 5 minutes in ice. This was followed by the
addition of 200µl of solution II (Appendix-3) and was mixed by inverting
the tube gently. The tube was stored on ice for 10 minutes. Then 150µl of
ice cold solution III (Appendix-3) was added and mixed by inverting the
tube gently. The tube was then kept for 15 minutes on ice to form white
precipitate. The tube was again centrifuged for 10 minutes at 14,000 rpm.
The supernatant was transferred to a fresh tube without transferring any of
the white precipitate. This was followed by addition of two volumes of
Antibiotic Resistance of the Enterococcal Isolates 127
ethanol to the supernatant to precipitate the plasmid DNA. This was then
mixed well by inverting the tube several times and the mixture was allowed
to stand for 30 minutes on ice. The precipitated plasmid was collected by
centrifuging at 14,000 rpm for 10min. The supernatant was removed and
discarded and to the pellet 1mL of ice cold 70% ethanol was added and
centrifuged again for 30 seconds. The supernatant was removed and
discarded. The pellet was air dried for 10-30 minutes and then it was
resuspended in 50µl of sterile distilled water and stored at -20°C. The
quality of the plasmid DNA was checked by electrophoresis using 1.2%
agarose gel.
Vancomycin resistant gene was amplified by PCR from genomic
DNA and plasmid DNA using vancomycin specific primers. Three
vancomycin resistant E.faecium genomic DNA were initially screened for
vanA and vanB by PCR. In those cases, where PCR was negative again
subjected to PCR using plasmid DNA as template. The primers used for Van
A gene amplification was VanAF (5’- GGGAAAACGACAATTGC-3’) and
VanAR (5’-GTACAATGCGGCCGTTA-3’). The primers for Van B gene
amplification were VanBF (5’-ACCTACCCTGTCTTTGTGAA-3’) and
VanBR (5’-AATGTCTGCTGGAACGATA-3’) respectively. The primer
sequences were got synthesized from Sigma – Genosys. PCR was carried
out in a final volume of 50ul with 50 ng of genomic DNA/plasmid DNA, 20
pmol of each primer, 200 µM of each dNTPs, 5 µl of 10X PCR buffer and
1.25 U of Taq DNA polymerase in a Mycycler™ (Bio-Rad, USA). The PCR
conditions used were; 5 minutes denaturation at 94°C, followed by 30 cycles
of 1 min denaturation at 94°C, 1 min annealing at 50°C , 1 min extension at
72°C, and a final extension for 7 minutes at 72°C. PCR products were
128 Chapter 6
visualized by 1.5% (w/v) agarose gel. This part of the work was carried out
at Unibiosys lab, Cochin
6.2.4.3. Sequencing and analysis
The PCR product was further gel purified and subjected to DNA
sequencing. This part of the work was carried out at Sci Genome, Cochin.
The sequence data of both vanA and vanB genes were then subjected to
BLAST analysis. The data was also used for multiple sequence alignment
and phylogenetic analysis with other sequences from database.
6.2.5. Resistance gene transfer by conjugation
In order to confirm the transfer of plasmid carrying vancomycin
resistance (VanB) to other enterococci, conjugation studies were conducted.
E.faecium with vanB plasmid was selected as donors based on their
resistance to vancomycin (vanr) and sensitivity to rifampicin (Rif s). This
vanr Rif s strain was mixed with E.faecium which were sensitive to
vancomycin and resistant to rifampicin. Broth mating was done as described
by Ike et al., (1998) with modifications. Here, 0.5 mL of donor culture was
mixed with 0.5 mL of the recipient culture and was added to 5 mL of Luria
Bertani broth (Appendix 3). The mixture was incubated at 37° C for 4 hours
with gentle agitation for the appropriate time. Then the transconjugants were
selected on agar plates containing 16µg/mL vancomycin and 20 µg/mL of
rifampicin. Colonies were counted after 48 h of incubation at 37°C. The
conjugation was confirmed by their antibiotics resistance patterns and
plasmid detection. The conjugation efficiency was determined based on the
number of transconjugants per donor cells.
Antibiotic Resistance of the Enterococcal Isolates 129
6.2.6. Random amplified polymorphic DNA analysis
RAPD analysis was carried out to investigate role of clonal spread of
VREF, in the emergence of van B type of VREF. By detecting and
sequencing the resistance genes, similar plasmid borne vanB type were
detected and to rule out the clonal spread of VREF, RAPD was carried out.
One vanB type of vancomycin resistant enterococci from chicken source and
a vanB type of vancomycin resistant enterococci from blood were randomly
selected for molecular typing by RAPD test. The sequence of the random
primer used for DNA amplification were OPK7: AGCGAGCAAG, OPK11:
AATGCCCCAG, OPK12: TGGCCCTCAC, OPBG 19: GGTCTCGCTC,
OPE6: AAGACCCCTC, OPL7. AGGCGGGAAC, OPAA14:
AACGGGCCAA, OPAA 17: GAGCCCGACT. Each reaction mixture (25
µl) contained template DNA (20-25 ng), 10X Taq buffer , MgCl2 (25mM),
dNTP mix (10mM), Primer (10pmol) Taq DNA polymerase (0.3U). The
thermo cycler was programmed with an initial denaturation at 94° C for
3minutes followed by cyclic denaturation at 94°c for 30cycles for 45
seconds, annealing at 37° C for 1 minute and extension at 72° C for 1
minute. The PCR was conducted for 40 cycles. Finally it was subjected to
extended polymerization at 72° C for 6 minutes. Approximately 10
microliter PCR products were electrophoresed on 2% (w/v) agarose gel
prepared in 1X TBE Buffer at 80 V for 2 h. This was followed by staining
with ethidium bromide (0.5 µg/mL).
6.3. STATISTICAL ANALYSIS
Significance in percentage difference is calculated by chi square test.
When the Chi square tests were not valid Yates correction were applied and
Fisheres exact test were also done where ever necessary. Statistical analysis
130 Chapter 6
of MIC of antibiotics was carried out by ANOVA. Analysis was done by
using SigmaStat software (Sigma-Aldrich, St. Louis USA). The level of
significance was set up at P <0.05.
6.4. RESULTS
Many of the isolates were resistant to multiple antibiotics. All
E.faecium and E.faecalis isolates tested showed resistance to clindamycin
and all remained susceptible to linezolid. Penicillin resistance was high in the
isolates.
The resistance to different antibiotics shown by the isolates of
different Enterococcus from water is shown in Table 6.1.
Table 6.1.Antibiotic resistance in isolates of Enterococcus from water
Antibiotics
No.&% *of resistant isolates
E.faecium
(n=100)
E.faecalis
(n=56)
E.raffinosus
(n=10)
E.avium
(n=10)
E.durans
(n=10)
E.gallinarum
(n=14)
No. & % No. &% No.&% No.&% No.&% No. & %
Penicillin 100(100) 56(100) 10(100) 5(50) 5(50) 7(50)
Ampicillin 42(42) 46(82.1) 0 0 0 0
Oxacillin 70(70) 45(80.4) 2(20) 4(40) 2(20) 2(14.28)
Cindamycin 100(100) 56(100) 10(100) 5(50) 5(50) 7(50)
Linezolid 0 0 0 0 0 0
Gentamicin 42(42) 36(64.3) 0 0 0 0
Ciprofloxacin 14(14) 36(64.3) 0 0 0 0
Erythromycin 14(14) 36(64.3) 0 0 0 0
Chloramphenicol 20(20) 8(14.3) 0 0 0 0
Tetracycline 14(14) 8(14.3) 10(100) 5(50) 5(50) 7(50)
Amoxyclav 0 8(14.3) 0 0 0 0
Teicoplanin 0 0 0 0 0 0
Gentamicin 120 0 2(3.6) 0 0 0 0
Vancomycin 0 0 0 0 0 0
*: percentage of isolates is given in brackets
Antibiotic Resistance of the Enterococcal Isolates 131
Penicillin resistance was significantly high in water isolates
(p<0.001). None had linezolid, teicoplanin and vancomycin resistance.
Amoxyclav resistance shown by few E.faecalis isolates. Ampicillin
resistance was high in the isolates.
The resistance to different antibiotics shown by the clinical isolates of
enterococci is summarized in Table 6.2.
Table 6. 2. Antibiotic resistance in isolates of Enterococcus from clinical sources
Antibiotic
No and percentage* of resistant isolates
E.faecalis
(n=148)
E.faecium
(n=52)
E.gallinarum
(n=2)
E.raffinosus
(n=2)
E.avium
(n=1)
No.& % No.&% No.&% No.&% No.&%
Penicillin 120(81.1) 38(73.1) 1(50) 1(50) 1(100)
Ampicillin 20(13.5) 14(26.9) 1(50) 1(50) 1(100)
Gentamicin 104(70.3) 34(65.4) 0 0 0
Oxacillin 105(70.9) 30(57.7) 1(50) 1(50) 1(100)
Streptomycin 88(59.5) 18(34.6) 1(50) 0 0
Gentamicin 120 60(40.5) 32(61.5) 0 0 0
Amoxyclav 20(13.5) 14(26.9) 1(50) 0 1(100)
Tetracycline 54(36.5) 14(26.9) 1(50) 0 1(100)
Erythromycin 98(66.2) 28(53.8) 0 0 0
Ciprofloxacin 60(40.5) 18(34.6) 1(50) 0 0
Chloramphenicol 8(5.4) 6(11.5) 0 0 0
Nitrofurantoin 10(6.8) 10(19.2) 0 0 0
Clindamycin 148(100) 52(100) 2(100) 2(100) 1(100)
Vancomycin 0 6(11.5) 0 0 0
Teicoplanin 0 3(5.8) 0 0 0
*: percentage of isolates is given in brackets
132 Chapter 6
Majority of the isolates have shown resistance to most of the tested
antibiotics. Penicillin and oxacillin resistance was found to be high in the
isolates. Six of the E.faecium isolates were vancomycin resistant. Though
ampicillin resistance was less, aminoglycoside resistance was high.
Resistance to gentamicin (30 µg and 120 µg) and amoxyclav was distributed
with high frequency in clinical isolates (P<0.001).
The resistance to different antibiotics shown by different enterococci
isolates from chicken is summarized in Table 6.3.
Table 6.3. Antibiotic resistance in isolates of Enterococcus from chicken faeces
Antibiotic
No.and % *of resistant isolates
E.faecium
(n=130)
E.faecalis
(n=4)
E.gallinarum
(n=5)
E.avium
(n=5)
E.raffinosus
(n=10)
E.durans
(n=3)
No.&% No.&% No.&% No.&% No.&% No.&%
Penicillin 120(92.3) 3(75) 4(80) 4(80) 8(80) 2(66.7)
Ampicillin 9(6.9) 2(50) 2(40) 0 0 0
Oxacillin 20(15.4) 0 0 0 0 0
Gentamicin 13(10) 3(75) 1(20) 0 0 0
Gentamicin 120 0 0 0 0 0 0
Amoxyclav 9(6.9) 0 0 0 0 0
Tetracycline 100(76.9) 0 0 0 0 0
Erythromycin 115(88.5) 1(25) 0 0 0 0
Ciprofloxacin 23(17.7) 0 0 0 0 0
Clindamycin 130(100) 4(100) 5(100) 5(100) 10(100) 2(66.7)
Chloramphenicol 7(5.4) 2(50) 0 0 0 0
Linezolid 0 0 0 0 0 0
Nitrofurantoin 0 0 0 0 0 0
Teicoplanin 0 0 0 0 0 0
Vancomycin 20(15.4) 0 0 0 0 0
*: percentage of isolates is given in brackets
Antibiotic Resistance of the Enterococcal Isolates 133
All the Enterococcal isolates from chicken isolates were resistant to
penicillin and had a high clindamycin resistance. A high resistance
percentage was obtained against erythromycin followed by tetracycline.
None were found to be resistant to linezolid and streptomycin (data not
shown). Gentamicin resistance was very low. Vancomycin resistance was
shown by 20 E.faecium isolates. Penicillin, tetracycline and erythromycin
resistance was significantly high in chicken isolates (P<0.05).
The antibiotic resistance pattern shown by human faecal isolates are
presented in table 6.4.
Table 6.4. Antibiotic resistance in isolates of Enterococcus from human faeces
Antibiotic
No.and % *of resistant isolates
E.faecium (n=52)
E.faecalis (n=2)
E.avium (n=12)
No.&% No.&% No.&%
Penicillin 32(61.5) 0(0) 0(0)
Ampicillin 10(19.2) 0(0) 0(0)
Oxacillin 12(23.1) 0(0) 0(0)
Gentamicin 16(30.8) 1(50) 1(8.3)
Gentamicin 120 4(7.7) 0(0) 0(0)
Amoxyclav 0(0) 0(0) 0(0)
Tetracycline 12(23.1) 0(0) 0(0)
Erythromycin 16(30.8) 0(0) 0(0)
Ciprofloxacin 8(15.4) 0(0) 0(0)
Clindamycin 52(100) 2(100) 11(91.7)
Chloramphenicol 8(15.4) 0(0) 0(0)
linezolid 0(0) 0(0) 0(0)
Nitrofurantoin 8(15.4) 0(0) 0(0)
Teicoplanin 0(0) 0(0) 0(0)
Vancomycin 0(0) 0(0) 0(0)
* % of isolates is given in brackets
134
The percentage
antibiotics was
nitrofurantoin 15%.
Resistance to different groups of antibiotics shown by the isolates from
clinical and environmental sources
Figure 6.1(a-m).
Among the beta lactam
more in the environmental isolates compared to the human.
Enterococcus were resistant to penicillin.
0
20
40
60
80
100
120
E.faecium
Pe
rce
nta
ge
The percentage incidence of resistance of E. faecium towards different
antibiotics was ampicillin 19%, gentamycin 31%, HLGR 8%
ntoin 15%.
Resistance to different groups of antibiotics shown by the isolates from
clinical and environmental sources is compared in Figures 6. a-m
Occurrence of antibiotic resistance in isolatesEnterococcus from clinical and environmental
Figure 6.1.a. Resistance to penicillin
Among the beta lactam antibiotics tested, resistance to penicillin was
more in the environmental isolates compared to the human.
were resistant to penicillin.
E.faecium E.faecalis E.avium E.gallinarum
Human Environmental
Chapter 6
towards different
31%, HLGR 8% and
Resistance to different groups of antibiotics shown by the isolates from
m.
of antibiotic resistance in isolates of clinical and environmental sources
antibiotics tested, resistance to penicillin was
All species of
E.raffinosus
Antibiotic Resistance of the Enterococcal Isolates
Figure
Ampicillin resistance in
sources. Ampicillin resistance in
environmental isolates compared to the human isolates.
E.avium, E.gallinarum
environmental isolates.
Figure
Oxacillin resistance was also found in both environmental and human
sources. Oxacillin resistance was seen chiefly in
0
20
40
60
80
100
E.faecium
Pe
rce
nta
ge
0
10
20
30
40
50
60
70
80
E.faecium
Pe
rce
nta
ge
he Enterococcal Isolates
ure 6.1.b. Resistance to ampicillin
Ampicillin resistance in E.faecium was low in isolates from both the
sources. Ampicillin resistance in E.faecalis isolates was higher in
environmental isolates compared to the human isolates. Human isolates of
and E.raffinosus were more resistant than
Figure 6.1.c. Resistance to oxacillin
Oxacillin resistance was also found in both environmental and human
Oxacillin resistance was seen chiefly in E.faecalis.
E.faecalis E.avium E.gallinarum E.raffinosus
Human Environmental
E.faecalis E.avium E.gallinarum E.raffinosus
Human Environmental
135
was low in isolates from both the
isolates was higher in
n isolates of
were more resistant than
Oxacillin resistance was also found in both environmental and human
E.raffinosus
E.raffinosus
136
Amoxyclav
absent in the isolates of
E.raffinosus.
Figure
Gentamicin (low level) resistance was more in human
isolates. A large number of environmental isolates was also found to be
resistant. E.avium
also had this property.
0
10
20
30
40
50
60
Pe
rce
nta
ge
0
10
20
30
40
50
60
70
80
E.faecium
Pe
rce
nta
ge
Figure 6.1.d. Resistance to amoxyclav
moxyclav resistance was very less in all isolates and was totally
solates of E.avium, environmental isolates of E.gallinarum
Figure 6.1.e. Resistance to gentamicin(30µg)
Gentamicin (low level) resistance was more in human
isolates. A large number of environmental isolates was also found to be
E.avium human isolates and E.gallinarum environmental isolates
also had this property. Resistance was absent in E.raffinosus.
E.faecium E.faecalis E.avium E.gallinarum E.raffinosus
Human Environmental
E.faecium E.faecalis E.avium E.gallinarum E.raffinosus
Human Environmental
Chapter 6
and was totally
E.gallinarum and
Gentamicin (low level) resistance was more in human E.faecalis
isolates. A large number of environmental isolates was also found to be
environmental isolates
E.raffinosus
E.raffinosus
Antibiotic Resistance of the Enterococcal Isolates
Figure 6.1
HLGR was predominantly seen
was not present in the isolates
Figure
Tetracycline resistance was more prevalent among environmental
isolates of E.faecium. E.faecalis
be more resistant than environmental isolates.
0
5
10
15
20
25
30
35
40
45
E.faecium
Pe
rce
nta
ge
0
10
20
30
40
50
60
E.faecium
Pe
rce
nta
ge
he Enterococcal Isolates
6.1.f. Resistance to gentamicin(120µg)
HLGR was predominantly seen E.faecalis of human origin.
isolates of E.avium, E.gallinarum and E.raffinosus
6.1.g. Resistance to tetracycline
resistance was more prevalent among environmental
E.faecalis isolates from human sources were found to
be more resistant than environmental isolates. Resistance was found in the
E.faecalis E.avium E.gallinarum E.raffinosus
Human Environmental
E.faecalis E.avium E.gallinarum E.raffinosus
Human Environmental
137
HLGR
E.raffinosus.
resistance was more prevalent among environmental
solates from human sources were found to
Resistance was found in the
E.raffinosus
E.raffinosus
138
human isolates of
the environmental isolates of
Figure
Human isolates of
Environmental E.faecium
isolates of E.faecium
Figure
Ciprofloxacin resistance was high in environmental isolates of
E.faecalis. E.faecium
0
10
20
30
40
50
60
70
E.faecium
Pe
rce
nta
ge
0
10
20
30
40
50
60
70
E.faecium
Pe
rce
nta
ge
isolates of E.raffinosus ,E.avium and E.gallinarum. It was absent in
the environmental isolates of E.raffinosus and E.gallinarum.
Figure 6.1.h.Resistance to erythromycin
Human isolates of E.faecalis had a high resistance to erythromycin.
E.faecium isolates exhibited more resistance than human
E.faecium. Other enterococci were found to be sensitive.
Figure 6.1.i. Resistance to ciprofloxacin
Ciprofloxacin resistance was high in environmental isolates of
.faecium isolates and E.avium from human sources
E.faecium E.faecalis E.avium E.gallinarum E.raffinosus
Human Environmental
E.faecium E.faecalis E.avium E.gallinarum E.raffinosus
Human Environmental
Chapter 6
It was absent in
had a high resistance to erythromycin.
more resistance than human
Other enterococci were found to be sensitive.
Ciprofloxacin resistance was high in environmental isolates of
from human sources were more
E.raffinosus
E.raffinosus
Antibiotic Resistance of the Enterococcal Isolates
resistant than environmental isolates.
and all isolates of E.avium
Figure 6.1
Chloramphenicol
isolates of E.faecalis. E.faecium
chloramphenicol. All other enterococci species were sensitive.
Figure 6.1
Nitrofurantoin resistance was
E.faecium and E.faecalis.
0
5
10
15
20
E.faecium
Pe
rce
nta
ge
0
5
10
15
20
E.faecium
Pe
rce
nta
ge
he Enterococcal Isolates
resistant than environmental isolates. Environmental isolates of E.gallinarum
E.avium and E.raffinosus were sensitive to the drug.
6.1.j. Resistance to chloramphenicol
resistance was more frequently in environmental
E.faecium from human sources was more resistant
All other enterococci species were sensitive.
6.1.k. Resistance to nitrofurantoin
resistance was found in human isolates in the case of
E.faecalis. All other isolates tested were sensitive.
E.faecalis E.avium E.gallinarum E.raffinosus
Human Environmental
E.faecalis E.avium E.gallinarum E.raffinosus
Human Environmental
139
E.gallinarum
were sensitive to the drug.
in environmental
resistant to
in the case of
E.raffinosus
E.raffinosus
140
Teicoplanin resistance was found only in the human isolates of
E.faecium. All other
Figure
The vancomycin resistance was more in isolates fr
sources compared to human isolates.
E.gallinarum isolates
found only in the isolates of
0
0.5
1
1.5
2
2.5
3
3.5
E.faecium
0
2
4
6
8
10
E.faecium
Pe
rce
nta
ge
Figure 6.1.l. Resistance to teicoplanin
Teicoplanin resistance was found only in the human isolates of
other isolates tested were sensitive to the drug.
Figure 6.1.m. Resistance to vancomycin
The vancomycin resistance was more in isolates from environmental
sources compared to human isolates. E.faecalis, E.avium, E.raffinosus
isolates tested were sensitive to vancomycin. Resistance was
found only in the isolates of E.faecium.
E.faecalis E.faecalis E.gallinarum E.raffinosus
Human Environmental
E.faecium E.faecalis E.faecalis E.gallinarum
Human Environmental
Chapter 6
Teicoplanin resistance was found only in the human isolates of
m environmental
E.raffinosus and
Resistance was
E.raffinosus
E.raffinosus
Antibiotic Resistance of the Enterococcal Isolates 141
The minimum inhibitory concentrations of the selected antibiotics
towards different Enterococcus species are shown in Table 6.5
Table 6. 5. Minimum Inhibitory Concentration of drugs against enterococci
Antibiotic
Range of MIC
E.faecalis (n=30)
E.faecium (n=30)
E.gallinarum (n=5)
E.raffinosus (n=5)
E.avium (n=5)
Penicillin 8 to 16 8 to 16 8 8 8to16
Streptomycin 0.1 to >240 0.1 to >240 0.1 0.5 16--32
Teicoplanin .01 to 0.05 0.05 to 64 0.01-4 0.01 0.01
Vancomycin 0.5 to 2 0.5 to 128 0.5-1 1 0.5
Gentamicin 0.128 - >1024 0.512 - >1024 0.128 0.512 0.128
Ampicillin 0.5 – 32 0.5 – 32 0.5 0.5 0.5
*No. of isolates tested is given in brackets
Though penicillin resistance was expressed by most of the strains, the
MIC was below 16µg/mL. Ampicillin MIC observed in this study was also
between 0.5 to 32µg/mL. HLGR and HLSR were distributed among the
isolates. Vancomycin MIC was significantly more in E.faecium than other
species (p<0.001). High-level resistance to vancomycin and teicoplanin
(MIC >128& 64 µg/mL) observed in this study corresponds to vanA
phenotype. Low level resistance to vancomycin (MIC >16-32 µg/mL) and
sensitivity to teicoplanin matches to vanB phenotype.
Antibiotic sensitivity pattern shown by Enterococcus is displayed in
Plates 6.1. Plate 6.2 shows the pattern of MIC towards different antibiotics
obtained by the E test.
142 Chapter 6
Plate.6.1: Antibiotic sensitivity test by disc diffusion method
Plate 6.2: E test for detection of MIC
Antibiotic Resistance of the Enterococcal Isolates 143
Genomic DNA and plasmid DNA was isolated from selected
isolates. Plate 6.3(A) shows the gel pictures of DNA isolated from sample
VREF1 (clinical), VREF2 (clinical) & VREF4 (chicken) and plate 6.3. (B)
shows the gel picture of plasmids from VREF1&VREF4
Plate 6.3. Agarose gel electrophoresis of genomic DNA and Plasmid
DNA of VREF
(A) . Genomic DNA (B). Plasmid DNA
Agarose gel electrophoresis analysis of genomic and plasmid DNA: Plate
6.3(A) shows the gel pictures of isolated DNA; Lane 1, 2 & 3 are
samplesVREF1, VREF2 & VREF4 respectively. Plate 6.3 (B) shows the
gel picture of plasmids isolated from samples VREF 1 & VREF 4.
144 Chapter 6
Results of the phenotypic analysis for vancomycin resistance was,
confirmed by PCR analysis using primers specific to vancomycin resistance.
The isolate with vanA phenotype formed expected PCR product of 900bp
corresponding to the size of vanA gene. Similarly the PCR using primers
specific to vanB gene formed product of 700 bp size (Plates 6.4(A) & (B) .
Plate 6.4 .PCR detection of vanA and vanB genes of VREF
(A) (B)
Plate 6.4.(A): Agarose gel electrophoresis analysis & PCR amplification of
vanB genes from various strains of VREF are shown in plate 6.4.(A).Lane
1:Molecular size markers(100bp),Lane 2:VREF1,Lane 3:VREF 4.
Plate 6.4 (B): Agarose gel electrophoresis analysis & PCR amplification of
vanA gene from VREF2 is shown in Plate 6.4 (B). Lane 1: Molecular size
markers (100bp), Lane 2:VREF2.
Antibiotic Resistance of the Enterococcal Isolates 145
To confirm the accuracy of PCR product formed, the product was gel
purified and subjected to DNA sequencing. This part of the work was
carried out at Sci Genome, Cochin. The sequence data of both vanA and
vanB genes were then subjected to BLAST analysis in which vanA gene
sequence showed its maximum identity to 922bp and that of vanB showed its
maximum identity to 706bp.
To confirm the plasmid borne nature and transferability of vanB
gene, the isolated plasmid was also used for mating experiments which
resulted in the formation of vancomycin resistant transconjugants. This was
confirmed by the growth of tranconjugants in BEA agar- media containing
16 µg/mL concentrations of vancomycin and 20 µg/mL concentrations of
rifampicin. Transconjugants showed same MIC as that of the donor E.
faecium and the presence of plasmid in the transconjugants was also
confirmed by plasmids isolation. The efficiency of conjugation ranged from
10-6 to 10-7 per donor cell.
Plate.4. illustrates the gel pictures of RAPD analysis of VRREF
samples.
146 Chapter 6
Plate 6.5: RAPD-PCR profile of VREF 6.5(A) 6.5 (B)
6.5(C) 6.5(D)
Plate 6.5. RAPD-PCR profiles among VREF isolates of clinical and chicken sources generated with each oligonucleotide. OPK7, OPK11, OPA14 and OPAA 17 in plate 6.5(A) & 6.5(B). RAPD-PCR profiles among VREF isolates of clinical and chicken sources generated with each oligonucleotide OPE6, OPL7, OPK12, and OPBG19 in plate 6.5(C) & 6.5(D). (M: 100 bp DNA ladder. Lanes 2, 4, 6 & 8: VREF1isolates from clinical sources; lanes 3, 5, 7 & 9: VREF4 isolate from chicken sources.
The genetic diversity analysis shown by RAPD had difference in
bands when oligonucleotides OPK11, OPK12 and OPBG19 were used for
the PCR.
Antibiotic Resistance of the Enterococcal Isolates 147
6.5. DISCUSSION
Analysis of antibiotic resistance pattern shown by the isolates was
included in this study. The results of this study indicate that multi-drug
resistance is common among isolates of enterococci from all sources.
Penicillin resistance was high, nevertheless it was less in isolates from
human faeces compared to the other sources (P<0.05). According to some
earlier studies, a high percentage of enterococci from poultry and water were
resistant to penicillin (Garcia-Migura et al., 2005; Grammenou et al., 2006).
The percentage incidence of penicillin resistance observed in clinical isolates
was similar with the earlier findings reported across India and in some other
countries (Mendiratta et al., 2008). In this study the oxacillin resistance was
very common. Glew et al., (1975) have reported that the penicillinase-
resistant penicillins were less active than penicillin against enterococci.
Though ampicillin is the drug of choice for treating enterococcal
infections, most studies conducted across India and other parts of the world
have reported high ampicillin resistance (Randhawa et al., 2004). However
in this study the incidence of ampicillin resistance among the clinical isolates
of E.faecalis and E.faecium were much lower than these studies. Until
recently, ARE were recovered sporadically from animals and humans outside
the nosocomial environment (Biavasco et al., 2007). But in this study
enterococci from water had a higher rate of ampicillin resistance. While
considering the MIC of ampicillin in the present study, it was in the range of
0.5 to32 µg/mL. But E. faecium strains expressing very high levels of
ampicillin resistance (MIC >128 µg/mL) have been reported by Grayson et
al., (1991). Nevertheless in this study no such high level resistance was
observed.
148 Chapter 6
Though a high percentage of the isolates showed antibiotic resistance
to penicillin, none expressed detectable beta-lactamase activity when tested
with cefinase disks. It is quite possible that the penicillin resistance observed
could be due to the altered penicillin binding protein. Many studies
conducted in northern part of India have reported beta lactamase activity
(Mohanty et al., 2005).
In the present study a high percentage of E.faecium and E.faecalis
isolates from clinical sources were resistant to low and high levels of
gentamicin. Earlier studies showed both low and high level of gentamicin
resistance among clinical isolates of Enterococi (Udo et al., 2003).
Aminoglycosides are used in clinical practice because of its synergistic effect
with cell wall synthesis inhibitors like penicillin or vancomycin. Rosvoll,
(2012) also found the high incidence of HLGR among E.faecium isolates
from clinical sources. Hence the aminoglycoside resistance is of great
concern in the treatment. High-level aminoglycoside resistance may be
acquired by chromosomal mutation or by acquisition of plasmids encoding
aminoglycoside-modifying enzymes (Krogstad et al., 1978; Eliopoulos et al.,
1984). Therefore, HLGR deserve further attention.
In this study only a few enterococcal isolates from chicken sources
exhibited resistance to gentamicin. The degree of high level resistance was
nil in these isolates. Similar results were seen in another study (Hayes et al.,
2004). Gentamicin is seldom used in veterinary medicine and the low
incidence of gentamicin resistance observed in enterococci from chicken
sources can due to this.
In this study the enterococcal strains from all sources except human
faecal isolates showed resistance to amoxyclav. Amoxyclav resistance was
Antibiotic Resistance of the Enterococcal Isolates 149
distributed in 14% of E. faecalis and 27% of E. faecium of clinical origin.
These observations strengthen the reports of other studies from India
(Miskeen and Deodhar, 2002), in which amoxyclav resistance was found to
be present, though studies from West Indies and Iran have reported 100%
sensitivity for amoxyclav (Orrett and Connors, 2000).
In this study a high percentage of enterococci from chickens were
resistant to tetracycline, compared to clinical sources. There are similar
reports from U.S. and abroad (Wiggins, 1996); (Yoshimura et al., 2000).
Their resistance level has been attributed to the extensive use of broad-
spectrum antibiotics for disease prevention in poultry production.
Chloramphenicol resistance in this study was found to be
comparatively more in environmental isolates of E.faecalis than from other
sources. This can be correlated with the less frequent use of chloramphenicol
for human treatment, because of its side effects. Despite of this low use in
humans, chloramphenicol resistance occurred in human enterococci isolates
of this and other studies in Denmark (Aarestrup et al., 2000). The use of
other antibiotics (e.g., penicillins, macrolides, and cephalosporins) appears to
drive chloramphenicol resistance, which is often a part of gene clusters that
encode for multidrug resistance. Chloramphenicol-resistant strains spread to
people from cattle through food have been discussed by Davis et al., (1999).
Clindamycin resistance in all the isolates was very high in this study.
Similar high resistance against clindamycin has been reported in some other
studies (Singh et al., 2002). Graham et al., (2009) also had found that all
enterococcal isolates had resistance to clindamycin.
Erythromycin resistance was more in enterococcal isolates from
chicken than in clinical isolates. The rapid development of erythromycin
150 Chapter 6
resistance in the poultry production environment as the result of medicated
feed use has been documented (Aarestrup et al., 2000). Resistance displayed
by clinical isolates may be due to the over use of erythromycin and other
macrolides.
A high incidence of ciprofloxacin resistance was shown by the water
and clinical isolates. The prevalence of resistance to fluoroquinolones in
human infections acquired from animals through the food chain is increasing.
Resistance to ciprofloxacin has emerged in enterococci over the last few
years and is now spreading clonally (Schaberg et al., 1992). High-level
resistance to fluoroquinolones is due to mutations in regions encoding
subunits of DNA gyrase and topoisomerase IV.
Nitrofurantoin is a successful antibiotic used to treat urinary tract
infections. Resistance to nitrofurantoin was observed to be more in clinical
isolates compared to other sources. Among the enterococcal isolates tested in
this study 7% of E.faecalis and 19% of E.faecium were resistant to
nitrofurantoin. Nitrofurantoin resistance was very less in nonclinical isolates.
Resistance was found only in low percentages or is absent (Rudy et al.,
2004).
None of the isolates in the present study was resistant to linezolid.
However there were few reports of linezolid resistance from other parts of
world. Many incidences of linezolid-resistant enterococci (LRE) have been
reported (Seedat et al., 2006; Auckland et al., 2002). There are even reports
of linezolid-resistant and vancomycin-resistant E. faecium (LRVRE)
(Gonzales et al., 2001).
Resistance towards glycopeptides, like vancomycin and teicoplanin
was significantly low and was shown only by the isolates of E.faecium.
Antibiotic Resistance of the Enterococcal Isolates 151
Those phenotypic expressions of vanA gene is high level resistance to
vancomycin and resistance to teicoplanin and that of vanB gene is with low
levels of resistance to these antibiotics. Only 6 isolates of VRE (3vanA 3
vanB phenotypes) could be isolated from clinical sources in this study.
Although in many studies conducted in other countries high percentage of
glycopeptides resistance was reported (Pfaller et al., 1998 & Perlada et al.,
1997), the result of the present study is in accordance with the situation in
northern India where the incidence was low (Mathur et al., 2003& Ghoshal
et al., 2006). Vancomycin resistant enterococci have the ability to spread and
cause hospital outbreaks (Patel, 2003).
In this study from chicken sources, 20 vancomycin resistant
E.faecium isolates were obtained and all exhibited vanB type of vancomycin
resistance. In discrepancy, vanA was the most frequently recovered
vancomycin resistant phenotype observed in isolates from animal sources in
earlier studies (Bates et al., 1994). The low prevalence of vancomycin-
resistant E. faecium in normal intestinal flora of humans is probably due to
low glycopeptide consumption in humans.
Genotypic analysis of vancomycin resistance has shown that the same
genotype was observed in accordance with the resistant phenotype. Results
of PCR amplification on 3 strains verified that 1 clinical strain had vanA & 1
had vanB gene and the chicken isolate also had vanB gene. The occurrence
of a phenotype not in constant with the genotype has been reported (Bell et
al., 1998). In this study the phenotypic characters and genotypes were
conforming in the two selected isolates. Also the results of the study confirm
vanB genes as plasmid coded and vanA as chromosomally encoded. A
previous report also indicates that the vanB resistance determinants may be
plasmid carried (Showsh, 2001).
152 Chapter 6
The results of the study also confirmed the ability of vanB coding
plasmids of chicken VREF (sample no.210) to transfer conjugatively to other
enterococci by broth mating. The recovery of transconjugants with the same
MIC as that of the donor E. faecium proved this. Similar to the current
study, vanB transfer has also been noticed in vivo and in vitro in previous
reports (Dahl et al., 2007). The results of some other studies are also in
agreement with this study where transferable vanB plasmids from strains of
E. faecium, transferred at rates of 10−7 to 2 × 10−7/ recipient (Rice et al.,
1998). Also there are studies demonstrating gene transfer by conjugation
from E.faecium to E. faecalis (Leclercq et al., 1989). In addition to this,
Noble et al., (1992) reported transfer of genes from E. faecalis to S. aureus.
In this study, VREF strains harboring vanB genes from chicken and
clinical sources were found to be polymorphic by RAPD typing. Earlier
studies proved that the RAPD methods as a powerful tool to investigate VRE
isolates in cases of nosocomial infection (Barbier et al.,1996) Results of
recent studies also support RAPD as effective tool for epidemiological
investigations of enterococci (Werner et al., 2003). The RAPD analysis
clearly indicated that the strains from chicken samples and clinical sources
were not identical.
The answer to the question on the origin of human VRE remains
obscure because of the lack of evidence for the spread of strains from
animals to humans (Donnelly et al., 1996). However there are studies also
explaining with strong evidence to support infection potential of animal
enterococci (Iversen et al., 2002). Even though the present study excludes
the possibility of such a spread from chicken sources, there is the possibility
of horizontal transfer of vanB vancomycin resistance plasmids by
Antibiotic Resistance of the Enterococcal Isolates 153
conjugation to other enterococcal strains and this was in agreement with the
findings of previous studies (Chow et al., 1993; Carias et al., 1998,).
The low prevalence of vancomycin resistance among the isolates in
this study indicates that vancomycin is still retaining its therapeutic efficacy
against the majority of enterococci. Since linezolid resistance is absent in
these isolates, it can also be used in treatment.
This work also establishes a baseline of resistance among
Enterococcus species in this part of the country which will be useful in
monitoring the dynamics of resistance. Drug resistant enterococci from
different sources can not only cause infections in immune compromised
hosts, but also may serve as important reservoirs of resistance genes. The
present study points to the necessity to control the spread of the antibiotic
resistance, by judicious use of antibiotics. Use of antibiotics in veterinary
therapy and bacterial infection prevention in animals should be minimized by
improving methods of animal husbandry and disease eradication.
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