DETECTION OF EXTENDED SPECTRUM BETA-LACTAMASE AND AmpC BETA-LACTAMASE-PRODUCING

ENTEROBACTERIACEAE CLINICAL ISOLATES AND TESTING THEIR SUSCEPTIBILITY TO NOVEL

ANTIBIOTICS

Thesis

Submitted for partial Fulfillment of the Master Degree in Medical Microbiology and Immunology

By

Noha Yousry Elsayed (M.B.B.CH.)

Supervised by

Prof. Dr. Magda Ibrahim Ayoub

Professor of Medical Microbiology and Immunology Faculty of Medicine, Cairo University

Prof. Dr. Maisa Mohamed Omar

Professor of Medical Microbiology and Immunology Theodor Bilharz Research Institute

Dr. Alaa Mohamed Reda

Lecturer of Medical Microbiology and Immunology, Faculty of Medicine, Cairo University

Faculty of Medicine Cairo University

2015

To My Family

ABSTRACT

Extended spectrum β-lactamases (ESBLs) and AmpC β-lactamase are enzymes produced by a variety of Gram-negative bacteria which confer an increased

resistance to commonly used antibiotics and represent a substantial clinical threat. There are currently no standard diagnostic tests available for detection of AmpC. A

total of 100 Enterobacteriaceae isolates were collected. ESBL production was screened by using the disc diffusion test and was confirmed by the combination disc diffusion test, screening of AmpC production was done by cefoxitin disc test, disc

approximation test and confirmation was done by AmpC disc test. Isolates screened positive for ESBL were investigated for their susceptibility to novel antibiotics

(Temocillin, Tigecycline, Colistin and Doripenem) by E-test. Results showed that among 100 Enterobacteriaceae isolates, 45 were screened positive for ESBL-

production using the disc diffusion test and 36 were confirmed by the combination disc test. Nine isolates were screened for AmpC-production using the cefoxitin disc

test and 5 isolates were confirmed as AmpC producers by AmpC disc test Using MAST D68C set, 35 isolates were ESBL producers, 2 were AmpC producers, one isolate was both ESBL and AmpC producers. All isolates were found sensitive to

Tigecycline and Doripenem. Forty-three isolates were sensitive to Colistin, while for Temocillin, thirty-seven isolates were found sensitive. MAST D68C test appears to be a promising way to detect isolates producing ESBL and/or AmpC. Tigecycline, Doripenem, Temocillin and Colistin revealed excellent activity against ESBL an

KEY WORDS DETECTION OF EXTENDED SPECTRUM BETA-LACTAMASE AND AMPC BETA-LACTAMASE-PRODUCING ENTEROBACTERIACEAE CLINICAL ISOLATES AND TESTING THEIR SUSCEPTIBILITY TO NOVEL ANTIBIOTICS

To My Family

Acknowledgement First and foremost, I wish to express my sincerest thanks to

ALLAH for his care and generosity throughout my life

I wish to express my sincere appreciation and deepest gratitude to

Prof. Dr. Magda Ibrahim Ayoub, Professor of Medical Microbiology

and Immunology, Faculty of Medicine, Cairo University, for her

supervision, illuminating guidance, valuable instructions as well as her

support throughout this work.

I am deeply grateful to Prof. Dr. Maisa Mohamed Omar, Professor

of Medical Microbiology and Immunology, Theodor Bilharz Research

Institute, for her continuous help, support and direct supervision of the

work and for her fruitful thinking which was behind the progress of the

work.

My sincere thanks are due to Dr. Alaa Mohamed Reda, Lecturer of

Medical Microbiology and Immunology, Faculty of Medicine, Cairo

University, for her supervision, help and cooperation throughout this

work.

I am deeply indebted to Dr. Doaa Gamal Desouky, Lecturer of

Medical Microbiology and Immunology, Theodor Bilharz Research

Institute, for her kind help, encouragement, precious advice and great

support throughout the whole work.

Special thanks and gratitude to Dr. Inas El Defrawy, current head of

Microbiology Department, Theodor Bilharz Research Institute, for her

inspiring enthusiasm and sincere advice that guided me through my research.

I am also grateful to my colleagues in the Microbiology Department at

Theodor Bilharz Research Institute, for their kind help and support.

I would like to thank my beloved mother, father and my sister for their

support, love and faith in me throughout my life.

My warmest appreciation goes to my dear husband for his continuous

encouragement, sacrifices and for being there for me.

Special thanks to my little baby Candy.

Noha Yousry

2015

I

LIST OF CONTENTS

Pages

LIST OF ABBREVIATIONS I

LIST OF TABLES II

LIST OF FIGURES III

INTRODUCTION AND AIM OF THE WORK 1

REVIEW OF LITERATURE

CHAPTER I: BETA-LACTAM ANTIBIOTICS

CHAPTER II: BETA-LACTAMASES

CHAPTER III: EXTENDED SPECTRUM BETA-LACTAMASE

CHAPTER IV: AmpC BETA-LACTAMASES

CHAPTER V:ESBL AND AmpC CO-PRODUCTION

Chapter VI: INFECTION CONTROL MEASURES TO OVERCOME ESBL AND/OR AmpC-PRODUCING ORGANISMS

Chapter VII: NOVEL ANTIBIOTICS IN TREATING ESBL AND AmpC PRODUCING ENTEROBACTERIACEAE

MATERIALS AND METHODS

RESULTS

DISCUSSION

SUMMARY

CONCLUSIONS

RECOMMENDATIONS

REFERENCES

ARABIC SUMMARY

I

LIST OF ABBREVIATIONS

AK Amikacin

AmpC-R AmpC-mediated resistance

AmpR AmpC regulator

ASC Advanced spectrum cephalosporins

ATCC American type culture collection

ATM Aztreonam

BA Boronic acid

BES Brazilian extended-spectrum cephalosporinase

BD Becton Dickinson

BIL Bilal

BLI(s) β-lactamase inhibitor(s)

CA Clavulanic acid

CAD Cloxacillin agar dilution

CAM Cefoxitin-agar medium

CAMHB Cation-adjusted Mueller-Hinton broth

CAZ Ceftazidime

CIP Ciprofloxacin

CLSI Clinical Laboratory Standards Institute

Clox-DDST Cloxacillin double disk synergy test

CMY Cephamycin

CN E test with cefotetan

CNI E test with cefotetan +cloxacillin

CSB Cell Suspension buffer

CTX Cefotaxime

DDST Double-disk synergy test

DHA Dhahran hospital

DPT Disk-potentiation test

ELISA Enzyme-linked immunosorbent assay

ESBL(s) Extended-spectrum β-lactamase(s)

II

ESC Extended spectrum cephalosporins

FEP Cefepime

FDA Food and Drug Administration

FOX Cefoxitin

ICU Intensive care unit

IPM Imipenem

IRT Inhibitor resistant TEM

KB Kirby-Bauer

MBL(s) Metallo-β-lactamase(s)

MDDST Modified double disk synergy test

MHA Muller-Hinton agar

MIC(s) Minimum inhibitory concentration(s)

MIR Miriam Hospital

MOX Moxalactam

NaCl Sodium chloride

PABL(s) Plasmid-mediated AmpC β-lactamases enzyme(s)

pAmpC Plasmid-mediated AmpC

PBP(s) Penicillin-binding protein(s)

TBRI Theodor Bilharz Research Institute

TDT Three-dimensional test

TE Tris EDTA

TZP Piperacillin-tazobactam

UTI Urinary tract infection

VEB Vietnam extended-spectrum ß-lactamase

β-lactam Beta-lactam

β-lactamases Beta-lactamases

III

LIST OF TABLES

Table No. Table Title Page

Table 1: Groups and examples of β-lactam antimicrobial agents 6

Table 2: Classification schemes for bacterial β-lactamases 12

Table 3: Screening and confirmatory tests for ESBLs in Klebsiella pneumonia, Escherichia coli and Proteus mirabilis

20-

21

Table 4: Distribution of Enterobacteriaceae isolates recovered from

different clinical specimens.

60

Table 5: Results of screening and confirmatory tests used for

detection of ESBL-producing Enterobacteriaceae isolates in

relation to type of isolate

62

Table 6: Results of different methods used for detection of AmpC-

producing Enterobacteriaceae isolates in relation to type of

isolate.

65

Table 7: Results of different methods used for detection of ESBL-

producing Enterobacteriaceae isolates in relation to type of

isolate.

66

Table 8: Result of Mast D68C ESBL and AmpC detection set in

relation to type of isolate.

67

Table 9: Performance of Mast D68C set in detection of ESBL

production in relation to combined disc diffusion method in

the 45 isolates screened positive for ESBL

68

Table 10: Performance of Mast D68C set in detection of AmpC

production in relation to AmpC disc test in 9 isolates

screened positive for AmpC

69

Table 11: Antibiotic Susceptibility Patterns of bacterial isolates 71

Table 12: E-test result of novel antibiotics 74

IV

LIST OF FIGURES

Fig. No. Figure title Page

Fig. 1: Penicillin and most other β-lactam antibiotics act by

inhibiting penicillin-binding proteins, which normally catalyze

cross-linking of bacterial cell walls

7

Fig. 2: Mechanism of action of β-lactamases 13

Fig. 3: Metallo beta lactamase mechanism 13

Fig. 4: Scheme of AmpC induction process 26

Fig. 5: Three-dimensional extract test. Positive results in the form

of distortion in the cefoxitin inhibition zone near the agar

slits that contain extracts of the AmpC positive test isolate

36

Fig. 6: AmpC disk test results 37

Fig. 7: CAM assay: AmpC-positive extracts produce a zone of

growth around wells

38

Fig. 8: APB-DPT showing an increase in zone diameter in CAZ+APB

compared to CAZ only

40

Fig. 9: APB-broth microdilution test: an eightfold or greater

decrease in the MIC of CAZ with the addition of APB is

indicative of the production of class C β-Lactamases

41

Fig. 10: DDST showing synergy between cloxacillin containing disk

and both CAZ and CTX disks

42

Fig. 11: An AmpC positive isolate with cefotetan (CN) MIC 4,

cefotetan + cloxacillin CNI) MIC 0.5 (i.e. 8 folds reduction

in MIC)

39

Fig. 12: Screening for ESBL production by the disc diffusion test: 61

Fig. 13: Confirmation of ESBL production by the combined disc

method

62

Fig. 14: Screening for AmpC production by the cefoxitin disc test. 63

Fig. 15: Screening for AmpC production by disc approximation test. 64

Fig. 16: Positive AmpC disc test confirming AmpC production 64

Fig. 17: Modified double disc synergy test showing positive result

for ESBL production

65

V

Fig. No. Figure title Page

Fig. 18: Result of Mast D68C ESBL and AmpC detection set 67

Fig. 19: Detection of ESBL production by Mast D68C set showing

ESBL positive result

68

Fig. 20: Detection of AmpC production by the Mast D68C set

showing AmpC positive result

69

Fig. 21: Detection of ESBL and AmpC Co- production by the Mast

D68C set

70

Fig. 22: Tigecycline E-test showing a sensitive isolate 72

Fig. 23: Doripenem E-test showing a sensitive isolate 72

Fig. 24: Colistin E-test showing a sensitive isolate 73

Fig. 25: Temocillin E-test showing a resistant isolate 73

Introduction

1

INTRODUCTION

Enterobacteriaceae are major pathogens responsible for various

nosocomial and community acquired infections (Fraser et al., 2013).

Beta-lactam (β-Lactam) antibiotics are considered the cornerstone in

treating Enterobacteriaceae infections, this is due to their broad spectrum of

activity and safety profile (Jacoby and Munoz-Price, 2005).

The spread of extended spectrum beta-lactamases (ESBLs) and AmpC

among isolated Enterobacteriaceae from both community and health-care

settings reduces the reliability and effectiveness of β-lactam antibiotics as

options in therapy (Falagas and Karageorgopoulos, 2009).

Extended spectrum β-lactamase producing organisms have enzymes that

hydrolyze the β-lactam ring of β-lactam antibiotics like penicillins,

cephalosporins and monobactams but not cephamycins (cefoxitin and

cefotetan) and carbapenems rendering them ineffective. However, they are

inhibited by β-lactamase inhibitors (clavulanic acid, sulbactam and

tazobactam). The ESBLs are frequently plasmid encoded. Plasmids responsible

for ESBL production frequently carry genes encoding resistance to other

classes of antimicrobial drugs such as fluroquinolones, co-trimoxazole,

tetracyclines and aminoglycosides. Therefore, antibiotic options in the

treatment of ESBL-producing organisms are extremely limited (Paterson and

Bonomo, 2005).

Class C β-lactamases (AmpC) are cephalosporinases whose encoding

genes were presumed to be chromosomally-mediated. They have been

described in many bacterial species e.g. Citrobacter freundii, Enterobacter

spp., Hafnia alvei, Morganella morganii, Pseudomonas aeruginosa and

Serratia marcescens. In these genera, AmpC is inducible via a system

Introduction

2

involving 3 cellular proteins (AmpD, AmpG and AmpR). Escherichia coli also

possess AmpC gene which is normally expressed at a low level and is non-

inducible since AmpR is missing (Moland, 2008).

Since the late 1980s AmpC has disseminated on plasmids. Plasmid-

mediated AmpC represent a substantial clinical threat as their presence renders

the bacteria resistant to most β-lactams including cephamycins and β-lactam/β-

lactamase inhibitors combinations (Decré et al., 2002).

Transmissible plasmids have acquired genes for AmpC enzymes, which

consequently can now appear in bacteria poorly expressing a chromosomal

blaAmpC gene, such as E. coli, or lacking AmpC gene as K. pneumoniae, P.

mirabilis and Salmonella spp..

Techniques to identify AmpC β-lactamase production are still evolving

and are not yet optimized for the clinical laboratory, which probably now

underestimates the prevalence of this resistance mechanism (Jacoby, 2009).

Detection of AmpC in a strain with a coexisting ESBL is even more

challenging (Philippon et al., 2002). Also the presence of an ESBL can be

masked by the expression of an AmpC. So the co-existence of AmpCs and

ESBLs in the same strain may result in false negative tests for the detection of

ESBLs by the current CLSI criteria (Thomson, 2001).

Infections caused by such resistant organisms can increase the length of

hospital stay and result in intensive care unit (ICU) admission. Also

inappropriate treatment of these complex infections can increase mortality and

morbidity. Whereas, rapid detection of these enzymes allows for de-escalation

to more targeted therapy and it is also an important infection control issue;

therefore there is a requirement for a simple and reliable diagnostic test for

confirmation of AmpC and ESBL production (Naveen et al., 2013).

Introduction

3

Carbapenems are a class of antimicrobials that are structurally related to

penicillin They can usually be used to treat infections due to AmpC-producing

bacteria, but carbapenem resistance can arise in some organisms by mutations

that reduce influx (outer membrane porin loss) or enhance efflux (efflux pump

activation) (Jacoby, 2009).

Doripenem, the newest addition to the carbapenem class of antibiotics,

was approved by the Food and Drug Administration (FDA) to treat intra-

abdominal infections and urinary tract infections caused by ESBL and AmpC-

producing Enterobacteriaceae (Shanthi and Sekar, 2011).

Tigecycline had good activity against most ESBL-producing and AmpC-

hyperproducing Enterobacteriaceae, and may be a therapeutic alternative to

carbapenems in some infections caused by ESBL and AmpC-producing

isolates, many of which are also multiresistant to quinolones, aminoglycosides

and classical tetracyclines (Meagher et al., 2005).

Temocillin (the 6-α-methoxy derivative of ticarcillin) has been re-

launched in the UK. This modification increases stability to β-lactamases,

including AmpC and extended-spectrum types. It is a potential alternative to

carbapenems against the growing number of infections, particularly those of the

urinary tract, that are caused by ESBL producers and other cephalosporin-

resistant strains (Livermore et al., 2006).

Colistin, an intravenous formulation of a polymyxin, has fairly reliable

in vitro activity against the ESBL and AmpC β-lactamase–producing

Enterobacteriaceae, and it might be useful in the treatment of co-infection with

these organisms (Zohreh et al. 2013).