Detection of Escherichia Coli Serotype

O25b-ST131clone among Patients with

Urinary Tract Infection

Submitted for Fulfillment of the Master Degree in

Clinical and Chemical Pathology

By

Dr. Mai Farouk Shalan M.B.B.CH, Faculty of Medicine

Cairo University

Supervised by

Prof. Dr. Soheir Fathy Helal Professor of Clinical and chemical pathology

Faculty of medicine Cairo University

Prof. Dr. Nada Nabil Nawar Professor of Clinical and chemical Pathology

Faculty of Medicine Cairo University

Prof. Dr. Mervat Gaber El Anany

Professor of Clinical and chemical Pathology Faculty of Medicine

Cairo University

Faculty of Medicine Cairo University

2012

� ر�ما��ورة ط� )114(

Thanks first and last to AllahAllahAllahAllah for His great care, support, and mercy that

illuminate every step of our life.

No words of gratitude would be enough to express my deep indebtedness and

sincere appreciation to Prof. Dr. Prof. Dr. Prof. Dr. Prof. Dr. Soheir Fathy Soheir Fathy Soheir Fathy Soheir Fathy HelalHelalHelalHelal, Professor of Clinical and

chemical Pathology, Faculty of Medicine, Kasr El Aini University for her continuous encouragement, valuable guidance, help, and overall her moral support that gave me and this work a lift to the scope of light.

My words stand short of my supreme gratitude and thanks to Prof. Prof. Prof. Prof. Dr. Dr. Dr. Dr.

Nada Nabil NawarNada Nabil NawarNada Nabil NawarNada Nabil Nawar, Professor of Clinical and chemical Pathology, Faculty of

Medicine, Kasr El Aini University who freely gave her precious time, effort, and experience along with continuous guidance in completing this work.

I am heartily thankful to the generous help offered by Prof. Dr.Prof. Dr.Prof. Dr.Prof. Dr. Mervat Mervat Mervat Mervat

Gaber El AnanyGaber El AnanyGaber El AnanyGaber El Anany, Professor of Clinical and chemical Pathology, Faculty of

Medicine, Kasr El Aini University, who devoted her time, effort, and experience to accomplish this work.

I would like to express my deepest gratitude and sincere appreciation to Prof.Prof.Prof.Prof.

Dr.Dr.Dr.Dr. AdelAdelAdelAdel Khalil Gohar Khalil Gohar Khalil Gohar Khalil Gohar , Professor of Clinical Pathology, Faculty of veterinary

medicine, cairo University for his continuous encouragement, his generous help, expert advice, and assistance throughout this work.

Words of thanks are so little for the great help offered by my colleagues in the Clinical Microbiology unit in Kasr El Aini Hospitals labs , Faculty of Medicine, Cairo University.

My grateful thanks to all my colleagues in the sixth of October Hospital, Health insurance for their continuous support and encouragement.

Finally, but not last, a very special and deep appreciation to my mother, my husband and my family for supporting and encouraging me in every step of my life.

Acknowledgement

Abstract

We aimed in our work to screen ESBL producing E.coli in UTI and

detection of O25-ST131clone in ESBL producing E.coli with

fluoroquinolone resistance. This study was carried out in the Clinical

Microbiology Laboratory at Kasr El Aini Hospital, Faculty of Medicine,

Cairo University, Egypt, during 10 months from November 2010 to August

2011, where three thousand, nine hundred and twenty two urine samples

were collected from patients with cystitis and the patients data were

collected.

All isolates were subjected to routine culture on blood agar and

MacConky agar media and incubated for 24 to 48 hrs at 35°C. Identification

of the isolates was done using Gram stain morphology and conventional

biochemical tests. Antimicrobial susceptibility testing and phenotypic

detection of ESBL production were carried on Muller-Hinton agar by using

the combination disk as recommended by CLSI, (2010) guidelines.

Identification revealed 492 E.coli isolates.

Key Words :

Amikacin – Aztreonam – Ciprofloxacin .

List of ContentsList of ContentsList of ContentsList of Contents

Title Title page

List of Figures I

List of Tables II

List of Abbreviations III

Introduction………..……………………...…..……………………………..…… 2

Aim of the work……………………………………….………………………….. 3

Review of Literature: 5

chapterI

:Extended Spectrum Beta Lactamase…………………….. 5

*Definition…………………………………………….. 5

*Beta-lactamases……………………………………… 6

*β-Lactam Antibiotics………………………………… 6

*β-lactam antibiotics structure………………………….………... 6

* Mechanism of action of β-lactam antibiotics………… 7

*ESBL Producer......................................................... 9

* Prevalence of ESBL………………………………… 9

* Genetics of ESBL…………………………………... 11

* Classification of ESBL……………………………… 13

*Laboratory detection of ESBLs……………………… 20

chapter II :E.coli Serotype O25b-ST131clone..…………………….. 28

*Prevalence of O25b-ST131clone…………………….. 28

*Geographical distribution……………………...…….. 29

*Reservoirs of ST131……………………………...…. 32

*Resistance to other antibiotics……………………..... 33

* Human infection…………………………………...… 34

*Detection of O25b-ST131clone.................................. 35

chapter III :Management of ESBL Producers..…………………...…. 36

*Management of O25b-ST131clone............................ 36

Materials and methods…...……………………….………………….. 39

*Study design.............................................................. 39

*Data Collection.......................................................... 39

*Microbiology workup……………............................................. 39

-Routine bacteriological culture and identification….. 39

-ESBL Producers Detection………………………… 42

*Statistical analysis…………....................................... 48

Results ……………………………………………...….…………… 50

Discussion ………………………….………………………………… 67

Conclusions & recommendation…………………….…………………...….. 72

Summary………………………….…………………………………..…………….. 74

References………………………………………………………………………………….…. 77

Arabic summary…………………………………………………………………………...… 91

I

List of FiguresList of FiguresList of FiguresList of Figures

.Figure No Title page

1 Structure of an oxyimino-amino-thiazolyl cephalosporin.... 5

2 Chemical structures of beta-lactam……..………….…..... 7

3

Illustration of the outer membrane, cell wall and plasma membrane of a Gram-positive and Gram-negative bacterium.. ……………

8

4 The β-lactamase enzyme reacting with the substrate…….. 12

5 CLSI ESBL confirmatory disk test…………...……….… 23

6 Detection of ESBL carriage with an E-test ESBL strip…... 26

7 Departments from which samples were collected………... 51

8 Growth of organisms within the collected samples……... 51

9 Antibiotic sensitivity among collected samples…………... 54

10 Disk approximation test………………………………….. 55

11 Combination disk test for ESBL confirmation…………... 56

12 Susceptibility testing of selected isolates……………….… 56

13 ESBL E.coli producers among collected samples……….. 57

14 Resistance to other antibiotic groups……………….…… 59

15 PCR gel electrophoresis, lane(1-28)………………….….. 60

16 PCR gel electrophoresis, lane(29-50)………...………….… 61

17 Relation between recurrence and O25-ST131 Clone gene acquisition…………………………………………………

65

II

List of TablesList of TablesList of TablesList of Tables

.Table No

Title page

1 Classification schemes for β-lactamases……………….... 14

2 Screening for E.coli, Klebsiella spp. and P.mirabilis…… 22

3 ESBL confirmatory tests for E.coli, Klebsiella spp. and P.mirabilis……………………………………………….……

24

4 Zone diameter interpretive standard for the tested antibiotics…................................................................…

41

5 Departments from which samples were collected…….….. 50

6 Colony count of growth among collected samples…….… 52

7 Incidence of organisms detected among collected samples 52

8 Antibiotic sensitivity among collected samples…………… 53

9 Resistance to other antibiotic groups…………………….. 58

10 Multi drug resistance in O25-ST131 isolates……………. 62

11 Relation between chronic disease and O25-ST131clone gene acquisition…………………………………………………

63

12

Relation between antibiotics taken and O25-ST131 clone gene acquisition……………………………………………….

64

13

Relation between recurrence of UTI and O25-ST131 clone gene acquisition………………………………………………..

64

III

List of AbbreviationsList of AbbreviationsList of AbbreviationsList of Abbreviations

AK Amikacin

AMC Amoxicillin/ clavulanic acid

AmpC Ambler class C enzymes

ATCC American Type Culture Collection

ATM Aztreonam

BSAC The British Society for Antimicrobial Chemotherapy

CAMHB Cation-adjusted Mueller-Hinton broth

CAUTI community-acquired urinary tract infections

CAZ Ceftazidime

CAZ/CLA Ceftazidime in combination with clavulanate

CES Cefoperazone/ sulbactam

CIP Ciprofloxacin

CLED Cysteine Lactose Electrolyte Deficient

CLSI Clinical Laboratory Standards Institute

CN Gentamicin

CRO Ceftriaxone

CT Cefotaxime in E-test

CTL Cefotaxime with clavulanate in E-test

CTX Cefotaxime

CTX/CLA Cefotaxime in combination with clavulanate

DDW double distilled water

DEPC Diethylpyrocarbonate

DNA Deoxyribonucleic acid

IV

E.coli Escherichia coli

E-test Epsilometer test

EDTA Ethylene diamine tetra acetic acid

ESBL Extended spectrum beta lactamase

F Nitrofurantoin

FEP Cefepime

ICUs Intensive care units

IPM Imipenem

IRTs Inhibitor Resistant TEMs

K.oxytoca Klebsiella oxytoca

K. pneumoniae Klebsiella pneumoniae

LIA Lysine iron agar

MBLs Metallo-β-Lactamase

MDR Multi drug resistance

MEM Meropenem

MgCl2 Magnesium chloride

MIC Minimal inhibitory concentration

MIO Motility indole ornithine

MLST Multi locus sequence typing

NAG N-acetylglucosamine

NAM N-acetylmuramic acid

NNIS National Nosocomial Infections Surveillance System

O25 O antigen type 25

OXA oxacillinases

V

P.B.S phosphate Buffer saline

PCR Polymerase chain reaction

PM Cefepime in E-test

P. mirabilis Proteus mirabilis

PML Cefepime with clavulanate in E-test

QC Quality Control

SAM Ampicillin/ sulbactam

SCF Cefoperazone

SD Standard Deviation

SDS Sodium dodecyl sulphat

SFM The Societe Française de Microbiologie

SMART The Study for Monitoring Antimicrobial Resistance Trends

Spp Species

ST131 Sequence Type 131

SXT Trimethoprim/sulfamethoxazole

TAE Tris Acetate EDTA

TE Tris EDTA

TSI Triple sugar iron agar

TZ Ceftazidime in E-test

TZL Ceftazidime with clavulanate in E-test

UK The United Kingdom

USA The United state of America

UTI urinary tract infection

Introduction & Aim of the work

2

INTRODUCTION

More than 90% of all uncomplicated urinary tract infection (UTI) is

caused by Escherichia coli infection. The recurrence rate after a first

E.coli infection is 44% (Madappa and Chi Hiong, 2010). In recent

years, the epidemiology of extended spectrum β-lactamase (ESBL) has

radically changed, with the emergence of E.coli producing CTX-M

enzymes, both in hospitals and in the community (Clermont et al., 2009).

Recently, an E.coli clone producing ESBL CTX-M-15 with a high

virulence potential has been reported all over the world, representing a

major public health problem, the new serotype of CTX-M15, ST131, is a

major cause of serious antimicrobial resistant E.coli infections in the

United States since 2007. In one study, researchers analyzed resistant

E.coli isolates collected during 2007 from hospitalized patients and they

identified 54 ST131 isolates, which accounted for 67 percent to 69

percent of E.coli isolates exhibiting fluoroquinolone and extended

spectrum cephalosporin resistance (Johnson et al., 2010 a).

The E.coli ST131 findings add to the growing concerns about drug

resistance in common infections such as UTI. For nearly two decades,

doctors and scientists have watched and worried as resistance mounted

(Johnson et al., 2010 b).

So it is important to identify the clone rapidly to choice the best

antibiotic for UTI treatment which lead to prevention of spread and

eradication of the infection.

Introduction & Aim of the work

3

Aim of The Work

The aim of our work was screening of ESBL producing E.coli in

patients suffering from UTI during a period of 10 months in Kasr El

Aini hospitals and detection of O25b-ST131 clone in ESBL producing

E.coli isolates associated with fluoroquinolone resistance.

Review of literature

5

Review of Literature

Chapter I :(Extended Spectrum Beta Lactamase)ESBL

Definition

Extended spectrum beta lactamases (ESBL) are a large rapidly

evolving group of plasmid-mediated enzymes. That confers resistance to

oxyimino-cephalosporins (e.g., ceftazidime, ceftriaxone, cefotaxime) as well

as oxyimino-monobactam (aztreonam) (not carbapenems and cephamycins)

or any beta-lactamase mutant, within a family, that has an enhanced ability to

do so. They are inhibited by clavulanate (CA), sulbactam, or tazobactam.

Originally observed in E.coli and Klebsiella spp. ESBL production has

now been documented in other gram-negative bacilli, including

Enterobacter spp., Proteus mirabilis, and Providencia stuartii (Figure 1)

(Bradford, 2001 and Neelam and Meera, 2008).

Fig. (1): Structure of an oxyimino-amino-thiazolyl cephalosporin. The C=N OR group, shaded, is held rigid and shields the ß-lactam ring from attack by classic ß-lactamase, but not by extended-spectrum ß-lactamase. Cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefepime and cefpirome are all designed on this scaffold (Livermore, 2008).

Review of literature

6

Beta-lactamases

Beta lactamases are enzymes special bacteria produce them , these

enzymes make one of important causes for resistance to beta-lactam

antibiotics like penicillin, cephamycins, and carbapenems (ertapenem).

(Cephalosporin are relatively resistant to beta-lactamase.) These

antibiotics have a common element in their molecular structure: a four-

atom ring known as a beta-lactam. The lactamase enzyme breaks that ring

open, deactivating the molecule's antibacterial properties (Livermore,

2008).

ββββ-Lactam Antibiotics

β-lactam antibiotics are widely used all over the world due to high

effectiveness, low cost, easiness of delivery, as well as their minimal side

effects. Also their activity can be modulated according to the side groups

attached to the β-lactam ring (Mark et al., 2005 and Maja et al., 2006).

ββββ-lactam antibiotics structure

The unique structural feature of a β-lactam is the highly reactive four-

membered β-lactam ring. It can be attached to saturated or unsaturated five-

or six-membered heterocyclic rings with double bond between positions 3

and 4. The heteroatom in position 1 may be sulfur (penams, cephems, and

penems), carbon (carbapenems and carbacephems), or oxygen (clavams,

oxapenems, and oxacephems). Monobactam, a structurally distinct class of

agents, consist of unfused β-lactam rings (Figure 2).

Review of literature

7

Fig. (2):Chemical structures of beta-lactam (1-4), site of action of beta lactamase (5),and chemical structures of beta-lactamase inhibitors used in clinical practice (6-8) (Maja et al., 2006).

Mechanism of action of ββββ-lactam antibiotics

Bacterial cell wall synthesis is inhibited by the bactericidal effect of β-

lactam antibiotics. The bacterial cell wall is a complex structure composed

of a tightly cross-linked peptidoglycan net which “corsets” maintaining cell

shape despite a high internal osmotic pressure. The glycan component of

this rigid structure consists of alternating units of N-acetylmuramic acid

(NAM) and N-acetylglucosamine (NAG). The former having short peptide

stems attached to it which are cross-linked; producing the characteristic net

structure of the peptidoglycan. This varies among the Gram-negative and

Gram-positive species, but always terminates in two D-alanine residues

(Figure3) (Melckebeke et al., 2006).

Review of literature

8

Fig. (3): Illustration of the outer membrane, cell wall and plasma membrane of a Gram-positive & Gram-negative bacterium. Note: in Gram-positive, the wall is relatively thick and consists of many layers of peptidoglycan interspersed with teichoic acids. While in Gram-negative bacterium, the wall is relatively thin and contains much less peptidoglycan also, teichoic acids are absent. However, the Gram negative cell wall consists of an outer membrane that is outside of the peptidoglycan layer. The outer membrane is attached to the peptidoglycan sheet by a unique group of lipoprotein molecules (Nikaido, 2003).

The individual peptidoglycan units are produced inside the cell, but

their final cross-linking is catalyzed outside the cytoplasmic membrane

by a group of membrane anchored bacterial enzymes known as the cell-

wall transpeptidase. Transpeptidases perform their catalytic cycle by way of

an acylation/deacylation pathway (Andersson et al., 2001).

These transpeptidases are the target of β-lactam agents; therefore these

enzymes are often termed penicillin binding proteins (PBPs). However, the

mechanism of action of β-lactam agents is more complex than initially

thought and likely involves several interrelated cellular processes

(Andersson et al., 2001, Hall and Barlow, 2004).

The mechanism of action of β-lactam agents begins by inhibition

transpeptidation. Beta-lactam are similar to the penultimate D-Ala-D-Ala of

the pentapeptide that is attached to NAM; hence PBPs mistakenly use

penicillin as a substrate for cell wall synthesis and the transpeptidase (or

carboxypeptidase) is acylated. The acylated PBP cannot hydrolyze the β-

Review of literature

9

lactam and subsequent steps in cell wall synthesis are hindered while

autolysis by cell wall degrading (autolytic) enzymes continues. Bacterial

cells become permeable to water, rapidly take up fluid, and eventually lyses

(Melckebeke et al., 2006).

ESBL Producers

Extended spectrum beta-lactamase has been found in a wide range of

Gram-negative rods e.g. family Enterobacteriaceae. As Klebsiella

pneumoniae consider the major ESBL producer , it survives longer than

other enteric bacteria on hands and environmental surfaces, facilitating

occurrence of Nosocomial infections (Al-Jasser, 2006).

E.coli is considered the second important ESBL producer organism.

ESBL production is relatively rare with Pseudomonas aeruginosa,

Acinetobacter spp, Burkholderia cepacia, and Alcaligenes fecalis. It is

important to note the growing incidence of ESBLs in Salmonella spp.

(non-Enterobacteriaceae) (Levison, 2002).

ESBL organisms can be detected on routine culture of blood, sputum,

urine, or stool specimens and can be detected in rectal or wound swabs.

Infections caused by ESBL organisms are treated with antibiotics, but

colonization of the bowel is not treated as it does not cause illness

(Livermore, 2008).

Prevalence of ESBL

Nowadays, Extended spectrum β-lactamase producing Gram-negative

bacilli with resistance to broad-spectrum oxyimino ß-lactam have been

threating as described worldwide resulting in a growing public health crisis

(Pitout and Laupland, 2008).

Review of literature

10

Extended spectrum β-lactamase were expressed as a serious problem

to clinicians and epidemiologists for many reasons; first, ESBLs have

wide substrate specificity that restricts therapeutic options. Second, the

dynamic evolution and epidemiology of these infections is often

complex; multiple clonal strains causing focal outbreaks may co-exist

with sporadic ones. Third, prevalence of ESBL-producing organisms is

under estimated worldwide, because of the significant diagnostic

challenges in the clinical microbiology laboratory. Finally, the biggest

challenge lies in overcoming widespread unawareness among clinicians

regarding their prevention and infection control issues (Rodriguez and

Pascual, 2008).

In Germany in 1983, a new group of enzymes (ESBLs) were detected,

Which hydrolyzed extended-spectrum cephalosporin with an oxyimino

side chain. Various types of ESBLs constitute one of the major

mechanisms of resistance of gram-negative bacteria (Livermore,1995,

Rahal,2000, Gniadkowski, 2001 and Coque et al., 2008).

During the 1990 ESBLs were mostly found in Klebsiella species,

often in intensive care units threatening the most vulnerable patients.

However, a new class of ESBLs (called CTX-M enzymes) had emerged

,detected among E.coli bacteria. This ESBL producing E.coli was able to

resist penicillin and cephalosporin and was found most often in urinary

tract infections e.g. cystitis. They had been found in the community as

well as in hospitals (Boyd et al.,2004).

By 1994 the Center for Disease Control and Prevention National

Nosocomial Infections Surveillance System (NNIS) reported that 8% of

Klebsiella spp. had ESBLs producers predominately in few large centers

(Burwen et al.,1994).