Studies on Extended-Spectrum β-Lactamase Producing

Escherichia coli and Klebsiella pneumoniae

By

Masroor Hussain

Department of Microbiology

Quaid-i-Azam University

Islamabad, Pakistan

2013

Studies on Extended-Spectrum β-Lactamase Producing

Escherichia coli and Klebsiella pneumoniae

A thesis

Submitted in the Partial Fulfillment of the

Requirements for the Degree of

DOCTOR OF PHILOSOPHY

IN

MICROBIOLOGY

By

Masroor Hussain

Department of Microbiology

Quaid-i-Azam University

Islamabad, Pakistan

2013

DECLARATION

The material contained in this thesis is my original work and I have not presented any part

of this thesis/work elsewhere for any other degree.

Masroor Hussain

TO

MY PARENTS

CERTIFICATE

This thesis, submitted by Mr. Masroor Hussain is accepted in its present form by the

Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University,

Islamabad as satisfying the thesis requirement for the degree of Doctor of Philosophy

(PhD) in Microbiology.

Internal Examiner: _______________________________

(Dr. Fariha Hasan)

External Examiner: _____________________________

(Dr. Arshad Pervez)

External Examiner: ______________________________

(Dr. Ghazala Kaukab)

Chairperson: ______________________________

(Prof. Dr. Safia Ahmed)

Dated: August 15, 2013

CONTENTS

S. No Title Page. No

1. List of Abbreviations i

2. List of Tables ii

3. List of Figures iii

4. Acknowledgements v

5. Abstract vii

6. Chapter 1: Introduction 1

7. Chapter 2: Literature Review 15

8. Chapter 3: Materials and Methods 36

9. Chapter 4: Results 50

10. Chapter 5: Discussion 105

11. Conclusions 113

12. Future prospects 114

13. References 115

14. Appendix

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i

LIST OF ABBREVIATIONS

× g Times Gravity

µg/ml Microgram per milliliter

API Analytical profile index

CI Confidence Interval

CLSI Clinical and Laboratory Standard Institute

dNTPs Deoxyribonucleotides

EMB Eosin methylene blue

ESBL Extended-spectrum β-lactamase

GI Gastrointestinal

ICU Intensive care unit

Kb Kilo base pair Kilo basepairs

kDA Kilo Daltons Kilo Dalton

mg/l Milligram per liter Milligram/liter

MH Muller-Hinton agar Mueller-Hinton

MIC minimum inhibitory concentration Minimum Inhibitory Concentration

MRL Microbiology Research Laboratory

ng Nanogram

OMP outer membrane protein

OPD Out-patient department

PBP Penicillin Binding Proteins

PCR Polymerase chain reaction

PIMS Pakistan Institute of Medical Sciences

RNase Ribonuclease

rpm Revolution per minute

SAP Shrimp alkaline phosphatase

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

U/ml Unit per Millilitre

UTI urinary tract infections

VRE Vancomycin-resistant enterococci

w/v Weight by volume

μm Micrometer

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ii

List of Tables

S. No Title

Page. No

1 Oligonucleotide sequence of primers used in this study 47

2 Interpretation of conventional biochemical tests for E. coli and

K. pneumoniae

53

3 Prevalence and association of bacterial isolates with variable 60

4 ESBL status in E. coli and its association with different risk

factors.

65

5 ESBL status in K. pneumonia and its association with different

risk factors

69

6 AmpC status of E. coli isolates and its association with different

risk factors.

74

7 AmpC status of K. pneumonia and its association with different

risk factors

78

8 CLSI breakpoints for MIC of antibiotics used in present study 81

9 Distribution of ESBL and AmpC β-lactamase enzymes in E. coli

based on sample sources and hospitalization

88

10 Distribution of ESBL and AmpC β-lactamase enzymes in E. coli

based on hospitalization

89

11 Distribution of ESBL and AmpC β-lactamase enzymes in E. coli

based on age groups and gender

90

12 Distribution of ESBL and AmpC β-lactamases in K. pneumoniae

based on sample sources and hospitalization

91

13 Distribution of ESBL and AmpC β-lactamase enzymes in K.

pneumoniae based on hospitalization

92

14 Distribution of ESBL and AmpC β-lactamase enzymes in K.

pneumoniae based on age groups and gender

93

15 Different gene combinations in E. coli 95

16 Different gene combinations in K. pneumoniae 96

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iii

List of Figures

S. No Title

Page. No

1 API 20E 39

2 Pink colonies indicating lactose fermentation by E. coli and K.

pneumoniae on MacConkey agar

51

3 E. coli colonies on EMB agar with green metallic sheen 51

4 Klebsiella pneumoniae colonies on EMB agar 52

5 Double Disc Synergy Test for detecting ESBLs 54

6 Overall distribution of E. coli and Klebsiella pneumoniae in the

study group

55

7 Overall distribution of E. coli and Klebsiella pneumoniae in

different age categories

57

8 Gender distribution of E. coli and Klebsiella pneumoniae 58

9 Percentage distribution of E. coli and Klebsiella pneumoniae on

the basis of sample origin

58

10 Percentage distribution of E. coli and Klebsiella pneumoniae on

the basis of sample source

59

11 Overall percentage distribution of ESBLs producer strains of E.

coli and Klebsiella pneumoniae in the study group

61

12 Overall distribution of ESBL producing E. coli among different

age groups

63

13 Gender distribution of ESBL producing E. coli 63

14 Percentage distribution of ESBL producing E. coli strains on the

basis of sample origin

64

15 Percentage distribution of ESBL producing E. coli strains on the

basis of sample source

64

16 Overall distribution of ESBL producing K. pneumoniae among

different age groups

67

17 Gender distribution of ESBL producing K. pneumoniae 67

18 Percentage distribution of ESBL producing K. pneumoniae

strains on the basis of sample origin

68

19 Percentage distribution of ESBL producing K. pneumoniae

strains on the basis of sample source

68

20 Overall percentage distribution of AmpC producing E. coli and

K. pneumoniae in the study group

70

21 Overall distribution of AmpC producing E. coli among different

age groups

72

22 Gender distribution of AmpC producing E. coli 72

23 Percentage distribution of AmpC producing E. coli strains on the

basis of sample origin

73

24 Percentage distribution of AmpC producing E. coli strains on the

basis of sample source

73

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iv

25 Overall distribution of AmpC producing K. pneumoniae among

different age groups

76

26 Gender distribution of ESBL producing K. pneumoniae 76

27 Percentage distribution of AmpC producing K. pneumoniae

strains on the basis of sample origin

77

28 Percentage distribution of AmpC producing K. pneumoniae

strains on the basis of sample source

77

29 Antimicrobial resistance patterns of E. coli isolates 80

30 Antimicrobial resistance patterns of K. pneumoniae isolates 80

31 MIC of ESBL producing E. coli 82

32 MIC of ESBL producing K. pneumoniae 82

33 Agarose gel with PCR fragments for TEM gene of E. coli 85

34 Agarose gel with PCR fragments for CTX-M1 gene of E. coli 85

35 Agarose gel with PCR fragments for CMY gene of E. coli 86

36 Agarose gel with PCR fragments for SHV gene of E. coli 86

37 Agarose gel with PCR fragments for SHV gene of K.

pneumoniae

87

38 Phylogenetic Tree of the TEM showing genetic relationships

with the reported genes

99

39 Phylogenetic Tree of the SHV showing genetic relationships

with the reported genes

100

40 Phylogenetic Tree of the CTX-M1 showing genetic relationships

with the reported genes

101

41 Phylogenetic Tree of the CTX-M9 showing genetic relationships

with the reported genes

102

42 Phylogenetic Tree of the CIT showing genetic relationships with

the reported genes

103

43 Variation in translated product of CIT the CIT-172 (E. coli MS

172), CMY-2 and CMY-32 (Already reported sequences)

104

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v

ACKNOWLEDGEMENTS

All praises be to Allah, the most beneficent, the most merciful. His prophet Muhammad

(P.B.U.H), the most perfect of human beings ever born, is the source of guidance and

knowledge for humanity, forever.

I wish to initiate this acknowledgement with the deep indebtedness to Dr. Fariha Hasan,

Associate Professor, Department of Microbiology, Quaid-i-Azam University, Islamabad,

Pakistan, who extended full support in planning and execution of this work, and thesis

writing. I appreciate her vast knowledge of microbiology, understanding of life,

unprecedented laboratory and writing skills, and uncompromising quest for excellence

that enabled me to successfully complete this huge task. She was always there to help me

and suggest appropriate remedies, whenever I needed. I would like to thank her for all the

support and motivation she provided during my Ph.D. studies.

The affectionate guidance and whole-hearted cooperation of Prof. Dr. Safia Ahmed, the

Chairperson, Department of Microbiology, Quaid-i-Azam University, Islamabad, and

sustained academic support of Dr. Aamer Ali Shah, did work wonders in producing and

reforming my research. I must appreciate and MRL staff who were always forth-coming

and helpful in rendering any help sought after.

Thanks are also due to Prof. Dr. Abdul Hameed, Ex-Chairman and Dean, Faculty of

Biological Sciences, for his interest in my work and providing the necessary research

facilities. Many thanks are due to members of Microbiology Department, Pakistan

Institute of Medical Sciences, Islamabad, especially Dr. Shagufta Hussain, who provided

samples and trained me in laborious yet very interesting task of data collection and

processing. Mohammad Shafiq, a microbiologist and friend, has always been there in

Microbiology Laboratory, PIMS, Islamabad, to help me in collection of isolates.

I would also like to thank Prof. Han Sang Yoo, Department of Infectious Diseases,

College of Veterinary Medicine, Seoul National University, Seoul, Korea, for inviting me

on a six month research visit in his lab. This work would not have seen the light of the

day without her guidance, help and mentoring. My lab fellows at Yoo Lab; Nabin, Seung-

Bin and Myunghwan made my stay in Seoul, a memorable experience of my life.

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vi

I am grateful to Dr. Khalid Mehmood for his encouragement, valuable suggestions and

advices at each and every stage of research work and write up of this thesis.

The co-workers and M. Phil student Sadia Mumtaz at MRL, need to be appreciated for

her whole-hearted cooperation in planning of many laborious experiments, and data-

collection and processing. Their cheerful presence made my working very interesting and

their intriguing questions kept me thoughtful during our discussions. My lab fellows and

friends at MRL, Fazal, Zia, Sami, Saadia Andleeb, Lubna, Khurram, Bashir, Naima,

Maryam, Pir Bux, Zulfiqar, Nida, Farah, Iffat, Sadaf (without prejudice to those whose

names are not annotated) were my real strength in giving me work support, sustained

environment, unbiased positive criticism and all available help. Their moral and material

support eased my resolve to work with dedication and tirelessness.

I would be failing my duty if I do not acknowledge the moral, material and spiritual

support of my loving parents, my wife, my brothers, my sisters, my parents-in-law, and

all the other family members who bore with me during testing times.

Last but not the least, my loving daughter Amal Masroor, a relaxation and harmony, was

source of inspiration for me. She filled my life with thrill, happiness and joy, and is the

most beautiful gift of my life.

Masroor Hussain

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vii

Abstract

This study was conducted to assess the prevalence of resistance genotypes of

extended-spectrum and AmpC β-lactamase producing Escherichia coli and Klebsiella

pneumoniae. Out of 632 samples, suspected for E. coli and K. pneumoniae, collected from

different units of Pakistan Institute of Medical Sciences, Islamabad, Pakistan, the number of

positive samples for E. coli and K. pneumoniae were 593 (93.8 %). Out of these 593 isolates,

61.6% (n=365) were identified as E. coli and 38.4% (n=228) were K. pneumoniae. Common

age group for sample isolation was 13-25 years for both E. coli and K. pneumoniae, from

which 29.9% of E. coli (n=109) and 27.6% of K. pneumoniae (n=63) were isolated. However,

none of the age groups achieved statistical significance. Higher percentage of E. coli was

isolated from females as compared to males, while the ratio of K. pneumoniae was higher in

male patients (p=0.012). Most of the isolates were recovered from specimens collected from

outdoor patients and were mainly from urine samples. ESBL production was detected in

46.20% (n = 274) of the 593 isolates by phenotypic method. Out of total 365 E. coli strains,

49.3% (n = 180) were found to be ESBL producers and 41.2% (n =94) of total 228 K.

pneumoniae isolates, were ESBL producers. Statistical analysis indicated that age groups

have significant association with the presence of ESBLs (p= 0.007) in E. coli isolates. No

significant association was observed in ESBL producing K. pneumoniae with age, gender,

sample source or origin. AmpC β-lactamase production was detected in 25.8% (n = 94) of the

total 365 E. coli and 20.6% (n =47) of total 228 K. pneumoniae isolates. There was significant

association between males (p=0.018) and samples collected from surgical ward (p=0.01) with

AmpC positive status in E. coli isolates. No significant association (p=0.88) was found in

AmpC producing K. pneumoniae and gender. However, like AmpC producing E. coli,

isolation from surgical wards had a statistically significant association with AmpC positive K.

pneumoniae (p=0.001). Out of these 593 isolates, 200 samples of the phenotypically

confirmed ESBLs or AmpC producers, E. coli and K. pneumoniae, were processed for

antibiotic susceptibility analysis and detection of the selected genes. Out of 200, 131 were E.

coli and 69 were K. pneumoniae. The highest resistance (90.1%) was observed against

sulphamethoxazole, followed by tetracycline (88.5%) and ciprofloxacin (80%) among E. coli

isolates. In case of β-lactam antibiotics, high resistance (87.8%) was observed against

cefotaxime and amoxicillin/clavulanic acid, followed by cefepime (81.7%) and aztreonam

(79.4%). Out of the total 131 E. coli isolates, 100 (76.3%) were found resistant to ceftazidime

having an MIC >32μg/ml. Highest resistance was observed in case of amoxicillin/clavulanic

acid, in which 117 isolates (89.3%) were resistant, followed by cefotaxime (116, 89.3%).

About 45 (34.3%) isolates of E. coli showed resistance to cefoxitin with a maximum range of

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viii

256 μg/ml. PCR amplification revealed that CTX-M-1 was the most frequently (77 isolates,

58.7%) detected ESBL gene group, followed by TEM (25 isolates, 19%) and SHV (19

isolates, 14.5%). CTX-M-9 group was observed in only 4 bacterial isolates. Among AmpC β-

lactamases, MOX gene was detected in 19 (14.5%) E. coli isolates, CIT in 17 (13%), CMY

gene in 7 (5%), EBC gene in 5 (4%), and 2 isolates showed FOX AmpC β-lactamases. A total

of 26 different patterns of genes were detected in 112 E. coli isolates, while no candidate gene

was found in 19 E. coli isolates. Among K. pneumoniae, higher resistance was observed in

case of tetracycline (98.6%), amoxicillin/clavulanic acid (97.1%) and sulphamethoxazole

(95.7%). In case of β-lactam antibiotics, cefoxitin was the most successful antibiotic showing

resistance to 20 (29%) isolates, followed by ceftazidime and cefepime (69.6%) and aztreonam

(75.4%). MIC results revealed that fifty isolates (72.5%) were found resistant to ceftazidime

with a maximum range of 512 μg/ml, while 19 (27.5%) were found susceptible. Fifty six

(81%) isolates were resistant to cefotaxime and 61 (88.4%) to amoxicillin/clavulanic acid.

Cefoxitin was the most successful antibiotic, effective against 47 (68.1%) of the total 69 K.

pneumoniae isolates tested CTX-M-1 type ESBLs were detected in 43 (62.3%) isolates, SHV

in 9 (13%), TEM in 8 (11.6%) and CTX-M-9 in 2 isolates (3%). Six (9%) isolates showed

CIT type AmpC genes while 4 (6%) had CMY, 3 (4%) each FOX and MOX and 2 (3%) had

EBC type genes. Eighty genes showed amplification in 69 K. pneumoniae isolates. A total of

18 different patterns of genes were detected in K. pneumoniae in a total of 58 isolates, while

in 11 isolates, no gene was detected. Our study showed that both class A and class C β-

lactamases contributed to cephalosporin resistance in E. coli and K. pneumoniae, thereby

limiting therapeutic options. Co-expression of these enzymes may further hinder the

identification of ESBLs, which is a critical step for designing a successful treatment for

multidrug-resistant E. coli and K. pneumoniae. Sequence analyses revealed 99-100%

homology with already reported ESBL genes from around the world. However, mutations in

CIT gene were found which indicates possible amino acid substitutions in more than one

position

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1

Introduction

Bacteria live with other inhabitants of the earth in harmony and in some instances are

the causes of some infections in humans mostly entering the body by chance (Levy,

1997). Infectious diseases have remained a major cause of death and disability in the

history of mankind. Infection related mortality and morbidity were greatly reduced in

the industrialized nations after the start of antibiotic use (Yoshikawa, 2002). In the

past 60 years, antibiotics have been used clinically in the treatment of bacterial

infectious diseases, which are commonly considered as the most important discovery

in the drugs’ history. Now we are seeing the change in bacteria themselves (Levy,

1997). There was an increase in antibiotic resistance among several bacteria in the last

two decades of the 20th

century. The major pathogens in nosocomial infections are the

threat to resistance (Adam, 2002). Increase in the number of multi-drug resistant

pathogenic and opportunistic bacteria is associated with the intense use and misuse of

the antibiotics (Barbosa and Levy, 2000).

In the evolutionary process, bacteria have acquired such well-developed, complex and

adaptable system of resistance that existed antibiotics are of no advantage (Rice,

2001). The overuse of antibiotics in both humans and animals, have increased the

resistance (Andersson et al., 1998). Multidrug resistant bacterial strains, resistant to

several antibiotic classes have appeared with new mechanisms of resistance, also

called the “superbugs” (Alanis, 2005). The emergence and spread of antibiotic

resistance is considered the product of the development of natural selection, related to

the use of antibiotics. Only the resistant strains survived under the selective pressure

of antibiotics resulting in the spread of resistance. The main way of resistance

acquisition seems to be the horizontal transfer of resistance genes (Blazquez et al.,

2002). These mechanisms are diverse and complex due to which bacteria developed

resistance to various classes of antibiotics (Alanis, 2005).

In the recent years, there has been less improvement in the field of research and

development for the search of new antibiotics. The number of new antimicrobial

agents approved by drug authorities is lower than the past, in last two decades and far

behind the speedy evolution of resistance genes spreading among both Gram-positive

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2

and Gram-negative bacteria (Gootz, 2010). The research and development sectors of

pharmaceutical industries, institutions and governments are not interested in new, safe

and effective antibiotics because of the high expenditures. In most cases, large

companies ended their research programs due to financial reasons and lengthy

procedures for the approval (Alanis, 2005). The recent development in genomics may

provide new targets resulting in the flow of new antimicrobial agents as genomes of

more than 200 bacteria are now available (Black and Hodgson, 2005).

β-lactamases

Penicillin was introduced into clinical use as the first β-lactam antibiotic. All β-

lactams have a reactive four member ring and inhibit cell wall synthesis. The class

now includes penicillin, cephalosporins, monobactams and carbapenems.

Chromosome encoded β-lactamase occur in many Gram negative bacteria in nature.

The evolution of β-lactamases is traced back to transpeptidases (Ghuysen, 1994).

Bacterial cell wall is composed mainly of peptidoglycan giving them shape and

protecting them from osmotic lysis. Peptidoglycan is a polymer containing two sugar

derivatives, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), and

several different amino acids. The backbone of the polymer consists of alternating

NAG and NAM subunits. NAM has short peptide stems connected to the carboxyl

group. Chains of linked peptidoglycan strands are joined by cross-linking of these

peptides giving it the characteristic net structure. This cross-linking is catalyzed by

Penicillin Binding Proteins (PBP) or bacterial transpeptidases. β-lactams have a

structure similar to the D-alanyl-D-alanine attached to NAM. PBPs (transpeptidases)

use β-lactams mistakenly as a substrate in the synthesis of the cell wall and are

acylated. The acylated transpeptidase is unable to hydrolyze the β-lactam and lead the

cell to lysis by the activity of autolysins (Ghuysen et al., 1996; Goffin and Ghuysen,

1998).

Bacteria can avoid the effect of β-lactams by any of the three strategies; a) Production

of β-lactam hydrolyzing β-lactamases: the enzymes able to hydrolyze the active β-

lactam ring and antibiotic is inactivated before reaching its target (Massova and

Mobashery, 1998), b) Alteration in the structure of PBP: utilization of transpeptidases

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3

insensitive to β-lactams have reduced affinity for β-lactams and are resistant to

penicillin (Chambers, 1997), c) Loss of outer membrane protein (OMPs): active

expulsion of β-lactam molecules by efflux pumps and loss of OMPs in Gram negative

bacteria reduces the entry of β-lactam antibiotics into the periplasm (Livermore, 2001;

Gootz, 2004; Jacoby et al., 2004; Wilke et al., 2005).

Staphylococcus aureus among Gram positive bacteria and the members of the

Enterobacteriaceae family among Gram negative bacteria are the most important β-

lactamase producing pathogens (Dolapci, 2005).

Classification of β-lactamases

β-lactamases are divided into 4 classes on the bases of similarity in amino acid

sequence (Ambler Classification) and on substrate and inhibitor profile (Bush-Jacoby-

Medeiros classification). Ambler classes are designated as Class A, B, C and D while

Bush-Jacoby-Medeiros classified them into Group 1, 2, 3 and 4.

Ambler Class A or Bush Group 2 are penicillinases susceptible to β-lactamase

inhibitors. The PC1 β-lactamase of Staphylococcus aureus and TEM-1 and SHV-1 β-

lactamase in E. coli and K. pneumoniae represents Group 2a and Group 2b,

respectively.

Ambler Class B or Bush Group 3 enzymes are Metallo-β-lactamases (MBL)

containing zinc. They use one of the Zinc (Zn+2

) atoms to inactivate penicillins and

cephalosporins. Bacteria having MBL are the most resistant phenotypes conferring

resistance to carbapenems, penicillins and cephalosporins.

Ambler Class C or Bush Group 1 are chromosomally encoded AmpC type β-

lactamases found in members of Enterobacteriaceae and P. aeruginosa. AmpC type β-

lactamases confer resistance to penicillins, β-lactamase inhibitors, cefoxitin, cefotetan,

ceftazidime, ceftriaxone and cefotaxime. They are usually susceptible to aztreonam

and cefepime.

Ambler Class D or Bush Group 2f includes serine β-lactamases able to hydrolyze

oxacillin. The OXA (oxacillinase or OXA β-lactamase) enzymes of Acinetobacter

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4

baumannii and P. aeruginosa are structurally more diverse and rapidly growing β-

lactamases.

ESBLs

TEM-1 was the first plasmid encoded β-lactamase found in early 1960s in E. coli

strain isolated from blood culture in Greece. The enzyme was named after the person

Temoniera, and was designated as TEM (Medeiros, 1984). The later generations of

cephalosporins were introduced to overcome the resistance of earlier β-lactamases.

Within two years, the already existing TEM and SHV plasmid-mediated β-lactamases

were able to inactivate the new drugs by simple amino acid substitutions around the

active site of the enzyme (Du Bois et al., 1995).

First described in the early 1980s in Europe, there has been an increase in the

incidence and prevalence of ESBLs all over the world. Bacteria producing these

broad-spectrum plasmid mediated enzymes can hydrolyze and inactivate penicillins,

oxyimino-cephalosporins and aztreonam. ESBLs have been isolated from different

species worldwide mostly from Enterobacteriaceae (Bradford, 2001; Colodner, 2005).

ESBLs have been most commonly reported in E. coli and Klebsiella spp.

Enterobacter, Proteus, Citrobacter, Salmonella, Serratia marcescens, Morganella

morganii, Pseudomonas aeruginosa, Acinetobacter baumannii, Shigella dysenteriae

and Burkhilderia cepacia have also been reported for ESBL production (Komatsu et

al., 2000; Pagani et al., 2002; Muller et al., 2011).

Although the effects of ESBLs could be restricted by β-lactamase inhibitors like

clavulanic acid, sulbactam and tazobactam, ESBLs are still considered as a threat

because the enzymes are plasmid encoded and can transfer between species easily

(Dolapci, 2005).

ESBLs leave carbapenems as the only option for β-lactam treatment. The number of

ESBL producing pathogens is increasing in the community acquired infections. It is

evident that carbapenemases accompany many bacteria having ESBL (Babic et al.,

2006).

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5

ESBL Classification

New β-lactamases evolved after the introduction of 3rd

and 4th

generation

cephalosporins able to hydrolyze these antibiotics. ESBLs are the most important

group of these enzymes. Majority of the ESBLs are generally the mutants of

penicillinases (TEM-1, TEM-2 and SHV-1) (Hrabak, 2007). TEM-1 is most

frequently found in plasmid encoded β-lactamases of Gram negative bacilli resistant

to ampicillin, while SHV-1 is commonly produced by K. pneumoniae. TEM-2 is not

as much common having biochemical properties similar to TEM-1. They are unable

to hydrolyze 3rd

and 4th

generation cephalosporins and monobactams (aztreonam)

(Livermore, 1995).

Their classification in Group 2be by Bush-Jacoby-Medeiros shows that these β-

lactamases derived from penicillinases of the group 2b (inactivates penicillin and

ampicillin and to some extent carbenicillin by hydrolysis) (Bush et al., 1995). On the

basis of similarity in their peptide sequence all ESBLs are grouped in molecular Class

A with the exemption of OXA-type β-lactamases included in class D in the Ambler

classification of β-lactamases (Harada et al., 2008).

These enzymes are many, and constant mutations have increased the spectrum of their

activity to a greater number of β-lactams (Bissett, 2007). There are 200 TEM and 150

SHV (sulphydryl variable) type β-lactamases commonly found in enterobacteriaceae

and other Gram negative bacilli (http://www.lahey.org/Studies/). CTX-M type is able

to hydrolyze a greater variety of antibiotics. They rapidly spread in the members of

Enterobacteriaceae in the last 10 years. Increasing prevalence of CTX-M type β-

lactamases has been reported in some epidemiological studies in certain areas of the

world (Harada et al., 2008).

ESBL producing organisms are considered resistant to penicillins and extended-

spectrum cephalosporins including the monobactam and aztreonam, as a rule, while

the same plasmid mostly have genes for the resistance of other types of antibiotics

like trimethoprim–sulphamethaxazole, gentamicin, amikacin and other

aminoglycosides. AmpC β-lactamases are also reported to exist on the same plasmid

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6

with ESBLs which are poorly inhibited by β-lactamase inhibiters like clavulanic acid

(Bissett, 2007; Gupta et al., 2007).

TEM

The TEM-type ESBL originated from TEM-1 and TEM-2. TEM-1 was first described

in 1965, capable of hydrolyzing ampicillin with great efficiency as compared to

carbenicillin, oxacillin, or cephalothin and has no activity against extended-spectrum

β-lactams. TEM-1, TEM-2 and TEM-13 have a similar hydrolytic profile and are not

ESBLs. However, in 1984, a novel plasmid encoded β-lactamase was found in France

to have increased activity against cefotaxime and was named CTX-1. The enzyme is

now listed as TEM-3, different from TEM-2 by only two amino acid substitutions.

Some of the TEM-type β-lactamases are reported with reduced affinity for inhibitors,

but maintaining the same hydrolytic activity for 3rd

generation cephalosporins. They

are designated as are complex mutants (CMT-1 to 4) (Sirot et al., 1997).

TEM-type ESBLs have been isolated from many countries of the world from different

species of enterobacteriaceae and other Gram negative bacteria. Although they are

most commonly found in Klebsiella pneumoniae and Escherichia coli, TEM-type

ESBLs have further spread into the members of Gram negative bacteria, as there are

reports of TEM-type ESBLs in other members of the Enterobacteriaceae such as

Proteus mirabilis, Enterobacter aerogenes, Morganella morganii, Enterobacter

cloacae and Salmonella spp. Whereas, Pseudomonas aeruginosa is a non-

enterobacteriaceae having TEM-type ESBL (Susic, 2004).

SHV

The SHV-type of ESBL can be found more commonly than any other type of ESBLs

in clinical isolates. In 1983, a mutant of SHV-1 called SHV-2, capable of hydrolyzing

oxyimino-cephalosporins, was first isolated from a Klebsiella ozaenae strain from

Germany (Knothe et al., 1983). The enzyme was actively hydrolyzing cefotaxime and

has lesser activity against ceftazidime. Sequence studies of the amino acids of the β-

lactamase showed that it is different from SHV-1 only by single amino acid. At

position 238, glycine was substituted by serine. There are few derivatives of SHV-1

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7

as compared to TEM-type ESBLs. In some of the variants of SHV, glutamate is

substituted by lysine at position 240. The serine residue at 238 is important for

hydrolysis of cefotaxime, whereas both serine at 238 and lysine at 240 are needed for

hydrolysis of ceftazidime (Huletsky et al., 1993).

About 130 SHV varieties have been reported worldwide. Most of these types have

ESBL phenotype and in few of the SHV enzymes, reduced affinity to inhibitors have

been reported (Prinarakis et al., 1997). SHV-type of ESBLs has been observed in

many members of Enterobacteriaceae. Acinetobacter spp. and Pseudomonas

aeruginosa have been also reported to produce SHV.

CTX-M

CTX-M, a novel family of class A ESBLs with extended-spectrum properties was

described in 2000 and is now the most prevailing type worldwide. CTX-type ESBLs

have enhanced activity against cefotaxime as the name represents, while MICs of

ceftazidime and aztreonam have been found susceptible and variable, respectively.

However some types confer resistance to ceftazidime by actively hydrolyzing this

cephalosporin. CTX-M-type β-lactamases have an efficient catalytic activity against

cefepime. Tazobactam is a good inhibitor of CTX-M as compared to sulbactam and

clavulanic acid (Tzouvelekis et al., 2000).

CTX-M type β-lactamases are determined by transportable plasmids and found in a

variety of enterobacteria, mostly E. coli, K. pneumoniae, S. typhimurium and Proteus

mirabilis. CTX-M β-lactamases are not very similar to TEM or SHV-type ESBLs but

are more similar (70 to 75%) to chromosomal β-lactamases of Klebsiella oxytoca

(Tzouvelekis et al., 2000).

Some reports state that CTX-M β-lactamases are derivatives of the chromosomal β-

lactamases of the Kluyvera species, from where they were acquired by the plasmids.

The chromosomal β-lactamase was reported to be 95 to 100% similar to some of the

plasmid-mediated CTX-M-type β-lactamases. In past decade, CTX-M-type ESBLs

have been frequently reported from all parts of the world and the prevalence of CTX-

M-type β-lactamases is expanding (Humeniuk et al., 2002).

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8

AmpC β-lactamases

AmpC β-lactamases were initially reported as chromosome-encoded inducible

enzymes responsible for resistance to ampicillin and first generation cephalosporins in

the Gram negative bacteria including; Citrobacter, Enterobacter, Morganella,

Providencia, Serratia and Pseudomonas spp. (Bush et al., 1995; Jones, 1998). The

chromosomal AmpC gene of E. coli was reported to be weakly expressed (Mulvey et

al., 2005; Tracz et al., 2007), while in Salmonella and Klebsiella sp. the chromosomal

AmpC gene was not present. Chromosomal AmpC β-lactamases are generally

inducible as compared to plasmid-mediated AmpC enzymes, except for DHA

enzymes (Coudron et al., 2000; Fortineau et al., 2001).

AmpC β-lactamases are cephalosporinases belonging to Group 1 enzymes or

molecular class C which are present on the chromosomes of most of the

Enterobacteriaceae and a small number of other Gram negative bacteria (Jacoby,

2009). They actively hydrolyze cephalosporins in comparison to benzylpenicillin and

usually are not susceptible to inhibition by clavulanic acid. They are active on

cephamycins (cefoxitin). In contrast to the class A cephalosporinases, they have a

greater affinity for the monobactams (Bush et al., 1982; Bush, 1988). Like ESBLs,

plasmid-mediated cephalosporinases have emerged in the recent times. AmpC β-

lactamases are class C cephalosporinases, linked to chromosomally located AmpC

cephalosporinases of Citrobacter freundii.

Later, plasmid-encoded AmpC β-lactamases have been discovered throughout the

world. There have been variations in the nomenclature of AmpC β-lactamases. CMY,

FOX, MOX and LAT have been named due to its resistance to cephamycins,

cefoxitin, moxalactam and latamoxef, respectively. ACC and ACT are referred to as

Ambler class C and AmpC type, MIR and DHA due to its sites of discovery in the

Miriam and Dhahran hospital, and BIL named after Bilal, the patient from whom the

bacteria was isolated (Philippon et al., 2002).

AmpC β-lactamases are chromosomal or plasmid-mediated. In most of the Gram

negative bacteria, including; C. freundii, E. cloacae, S. marcescens and P.

aeruginosa, AmpC β-lactamases are expressed in very small quantity, but the

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9

expression may be induced when exposed to β-lactams like penicillins, carbapenems

and β-lactamase inhibitors (Bush et al., 1985; Livermore, 1987; Weber and Sanders,

1990; Jacoby, 2009). In E. coli, a very low level of enzyme have been produced by

the chromosomal AmpC because of a weak promoter and transcriptional attenuator

(Nelson and Elisha, 1999). AmpC β-lactamases are expressed constitutively as a

result of deregulation of the chromosomal AmpC gene or by acquisition of mobile

plasmid-mediated ampC gene. Production in larger quantities can provide resistance

to carbapenems, particularly ertapenem, mainly in a host with reduced β-lactam

accumulation (Bradford et al., 1997; Jacoby et al., 2004; Quale et al., 2006).

Inducible chromosomal ampC genes were identified on plasmids in 1980s and were

introduced into organisms, which lack or didn’t express chromosomal AmpC type β-

lactamase, like Klebsiella spp., E. coli or Salmonella spp. (Philippon et al., 2002;

Liebana et al., 2004; Hopkins et al., 2006; Woodford et al., 2007).

Plasmid-mediated class C or group 1 β-lactamases in the MIR, CMY, DHA, ACT,

FOX, and other families have been identified since 1989 but at present are not

common like plasmid-mediated class A or subgroup 2be ESBLs (Jacoby, 2009). The

new subgroup 1e β-lactamases are variants of group 1, with higher affinity for

ceftazidime and other oxyimino-β-lactams resulted from substitutions, insertions, or

deletions of amino acid (Nordmann and Mammeri, 2007). Considerable resistance

have been most often observed in case of porin mutation (Mammeri et al., 2008b).

Similarly, most of the clinical microbiologists have not been successful in identifying

plasmid-mediated AmpC β-lactamases due to difficulty in phenotypic detection

(Hanson, 2003).

Escherichia coli

Escherichia coli is member of the Gamma Proteobacteria class of bacteria. It is a

gram negative rod which belongs to a large bacterial family, Enterobacteriaceae.

Most E. coli strains are not harmful. They are the part of microflora of the

gastrointestinal tract and beneficial to the host, as they produce vitamin K2 (Bentley

and Meganathan, 1982) and keep away pathogenic bacteria from colonizing the gut

(Hudault et al., 2001). Over 700 serotypes (on the basis of O, H, and K antigens) of E.

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10

coli are recognized. Enteric E. coli are classified on the basis of antigenic

characteristics and their virulence properties (Nataro and Kaper, 1998). Transmission

of pathogenic E. coli mostly occurs through oral-fecal transmission (Gehlbach et al.,

1973).

The diseases caused by a particular strain of E. coli depend on distribution and

expression of an array of virulence factors, including adhesins, toxins, invasins and

abilities to withstand defenses of the host (Johnson, 1991). An E. coli infection may

also arise due to environmental exposure. Pathogenic strains of E. coli are responsible

for various types of infections in humans: urinary tract infections (UTI), intestinal

diseases (gastroenteritis), neonatal meningitis, abdominal cavity (peritonitis) and

biliary tract infections (Wirth et al., 2006).

Klebsiella pneumoniae

Klebsiella spp. are found everywhere in environment. K. pneumoniae probably have

two common habitats, in the nature, they are found in sewage, on plants, surface water

and soil, and the other habitat is mucosal surfaces of mammals such as humans,

horses, or swine, which they colonize (Seidler et al., 1975; Edberg et al., 1986).

The urinary tract infection is the most common type of infection. Klebsiella is

responsible for 6 to 17% of the total hospital-acquired urinary tract infections (UTI)

and shows an even higher prevalence in particular groups of patients at risk (Bennett

et al., 1995; Schoevaerdts et al., 2011). Klebsiella is only second to E. coli as a reason

of nosocomial Gram negative bacteremia (Pittet et al., 1993).

Epidemiology

Although statistical data from most parts of the world is unavailable, the available

data shows that resistance to extended- spectrum cephalosporins in E. coli and, in

particular, K. pneumoniae is an emerging global concern (Paterson and Bonomo,

2005), with only some particular areas in the USs and northern Europe have a

relatively low resistance and are relatively spared. Also, the recent reports on the

prevalence of community acquired ESBL-producing Enterobacteriaceae are

becoming a new threat, since this will turn into a powerful reservoir providing

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11

continuous supply of the resistant isolates into hospitals (Pitout et al., 2005; Ben-Ami

et al., 2009).

Taken together, these reports constantly show that extended spectrum β-lactamase

producing Enterobacteriaceae are related to delay in proper antibiotic treatment

initiation, resulting in prolonged hospital stay, increased costs and greater risk of

death (Engel, 2010).

Because ESBL-producing isolates often happen in focal outbreaks, their frequencies

vary to a great extent from one place to another and even over time. As a result, local

and regional estimates are more useful to clinical decision-making than global

assessments. An additional limitation about the data is the difference in criteria used

to determine an ESBL-produces organism (Pfaller and Segreti, 2006).

Resistance to non- β-lactam antibiotics

ESBL producers are frequently observed resistant to fluoroquinolones, co-

trimoxazole, and trimethoprim (Schwaber et al., 2005; Colodner et al., 2007). Thus,

ESBL production may be a good indicator to the MDR phenotype. The carbapenems

are the drugs of choice in treatment of infections caused by extended-spectrum

cephalosporin- resistant E. coli and K. pneumoniae; however, resistance to

carbapenem is increasing in certain areas (Giakkoupi et al., 2003; Lee et al., 2006;

Navon-Venezia et al., 2006).

Multiple-drug resistance is common in E. coli-expressing ESBLs. ESBL producing

organisms are not only resistant to β-lactam antimicrobial agents but also show

resistance to other antibiotics like; ciprofloxacin, amikacin and gentamicin (Sharma et

al., 2010). Earlier studies of plasmids harbouring ESBLs also demonstrated multiple

resistance genes, including aminoglycoside, sulfonamide and tetracycline resistance

genes (Jacoby and Sutton, 1991).

E. coli confers resistance to aminoglycosides by enzymatic modifications. Three

classes of modifying enzymes are responsible for aminoglycoside resistance. They

include the acetyltranferase, phosphotransferase, and adenyltransferase. As a result of

these modifications, the binding affinity of the drug to ribosomes is altered, resulting

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12

in resistance. In addition intrinsic and adaptive resistance that results in decreased

uptake have also been found in aminoglycoside-resistant Gram negative bacteria (Siu,

2002). The major mechanism of resistance to chloramphenicol is through enzymatic

modification by chloramphenicol acetyltranferase, using acetyl-coenzyme A as the

acyl donor to eventually convert chloramphenicol to 1,3-diacetoxychloramphinicol.

The product then loses its ability to bind to the peptidyltransferase component of the

50S ribosomal subunit rendering the drug inactive (Shaw et al., 1985). Another

mechanism is chromosomal mutation, which causes a lack of the entry porin, ompF

leading to membrane impermeability to the drug (Toro et al., 1990).

Significance of the study

As resistance resulted due to the extensive use of antibiotics, it is important that the

use of drugs is selective by exercising careful judgment and not unnecessary. The

inappropriate use of new drugs with extended- spectrum further complex the problem.

The actual prevalence of resistance should be constantly observed each year. The

impact of resistance to an antibiotic and its specific mechanism, as well as

transmission, must be carefully studied (Siu, 2002). Information regarding patterns of

resistance of bacteria in an area will help to direct towards proper antibiotic use.

Restricted use of antibiotics can lead to the reversal of antibiotic resistance by

reduction in selective pressure, which will finally result in reduction of resistant

bacteria (Mathur et al., 2002).

Antibiotics are sold as over the counter drugs and anyone can purchase antibiotics

without prescription. There is also no control on the use of antibiotics in farm animals

and poultry. Most of the general physicians prescribe broad-spectrum antibiotics

without antibiotic susceptibility tests. In most of the health outlets in the country, the

facilities to perform antibiotics susceptibility tests are inadequate. There is no

systematic national surveillance of antibiotic resistance, and insufficient data is

available to quantify the problem. Storage conditions at pharmacy level are also

inadequate and the climatic conditions are at extreme in most parts of the country,

especially in remote rural areas. As self-medication is also a common practice, it

ultimately results in inappropriate dosage and inadequate length of treatment.

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13

Moreover, the hygienic conditions in most of the hospitals and health facilities in

Pakistan are also the causative factors in increase of antibiotic resistance.

In Pakistan, a number of studies have reported a high prevalence of ESBL producing

Gram negative bacteria (Shah et al., 2004; Ullah et al., 2009). This, therefore,

necessitates the need for research on prevalence of ESBL producing bacteria and

ofcourse collaborative studies are required on epidemiology of ESBLs to establish the

role of plasmids in transfer of different antibiotics resistance. As β-lactamase genes

are mostly plasmid-mediated, carrying resistance genes to sulfonamides, tetracyclines,

aminoglycosides and other antimicrobial agents, therefore the results of unusual

resistance to these antibiotics should urge the need for further studies.

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Aims and objectives

The main theme of the study is to find out prevalence of ESBLs in Pakistan focusing

on the selected genotypes of ESBLs in E. coli and K. pneumoniae isolates.

Isolation and identification of E. coli and K. pneumoniae from different

clinical specimens.

Primary screening of extended-spectrum β-lactamase producing E. coli and K.

pneumoniae and those resistant to cefoxitin or clavulanic acid or both.

Detection of AmpC β- lactamase producing E. coli and K. pneumoniae.

Determination of antibiotic susceptibility pattern of extended-spectrum and

AmpC producing E. coli and K. pneumoniae.

Determination of MIC of extended-spectrum and AmpC producing E. coli and

K. pneumoniae.

Frequency distribution and relationship of extended-spectrum and AmpC β-

lactamase producing E. coli and K. pneumoniae with age, gender, sample

origin and sample source.

Molecular characterization of genes responsible for extended-spectrum and

AmpC β-lactamase production in E. coli and K. pneumoniae.

Determination of gene combinations in ESBL and AmpC producers.

Homology studies of the selected genes by sequencing and computational

analysis.

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Literature Review

Beta-lactam drugs inhibit the synthesis of bacterial cell wall at the last stage. These

drugs constitute one of the largest families of antimicrobial agents widely used in

contemporary clinical practice. β-lactam drugs are preferred due to their bactericidal

action, broader coverage and high safety profile. Modifications in the original

structure gave new β-lactam drugs having better antimicrobial activity. Development

of resistance against β-lactam drugs has however limited their use in some clinical

situations. In spite of this issue, β-lactams remain the treatment of choice in wide

range of indications i.e. hospital-acquired infections and infection caused by multi-

drug resistant strains (Suarez and Gudiol, 2009).

Cephalosporins are broad-spectrum antibiotics used empirically for treatment of both

suspected and culture-proven Gram-negative and Gram-positive bacterial infections.

Cephalosporins vary to a considerable extent in their spectra of bactericidal activity,

pharmacokinetics profile and susceptibility to β-lactamases. The first and second

generation cephalosporins are effective against Gram-positive bacteria like

streptococci and staphylococci. The third-generation agents are most active against

Gram-negative bacteria like Pseudomonas and members of Enterobacteriaceae.

Cefepime is member of fourth-generation cephalosporins having outstanding activity

against both Gram-negative and Gram-positive pathogens, including multidrug-

resistant Enterobacteriaceae. The selection of most suitable member of cephalosporin

group for specific ailment still remains a challenge in clinical practice (Gustaferro and

Steckelberg, 1991; Klein and Cunha, 1995).

Carbapenems are β-lactam agents with a remarkably broad spectrum of activity.

Similar to other β-lactam antibiotics, carbapenems also inhibit the synthesis of

bacterial cell wall. Unlike to cephalosporins, carbapenems are stable against β-

lactamases including class A and class C β-lactamases. Carbapenems have similar

spectra, although there are significant differences in their antimicrobial activity, on

the basis of which a clinician determines their specific clinical indications. Agents

like meropenem, imipenem and doripenem have broad-spectrum activity against

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16

many Gram-positive, Gram-negative anaerobic bacteria but have no or little activity

against methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium

and Stenotrophomonas maltophilia (Zhanel et al., 2007; Martinez et al., 2010).

Mechanism of action of β-lactam antibiotics

β-lactams and glycopeptides are important classes of antibiotics that interfere with

bacterial cell wall synthesis by blocking the machinery responsible for assembly. The

machinery is specific to bacteria and is easy to access as compared to the other

organelles like ribosome which are considered the next important target. One of the

main groups represents some of the proteins collectively called penicillin-binding

proteins (PBPs). These are the transpeptidases, which catalyze the cross-linking of the

D-alanyl-D-alanine chains of adjoining peptidoglycan strands. These transpeptidases

are difficult to study as the enzymes are membrane-bound, and have not been isolated

until the past decade. The solubilized analogues were studied by X-ray

crystallography for structural analysis (Gordon et al., 2000; Lim and Strynadka,

2002). Transpeptidases polymerize and modify peptidoglycan of the bacterial cell

wall. They assist in creating the morphology of the peptidoglycan skeleton with

skeleton proteins that control formation of the septum and shape of the cell.

Microscopic and genetic analysis reveal clear involvement of class A and class B

PBPs and suggest that shape determination mechanism involves localization of

differential protein and interacts with specific cell components (Popham and Young,

2003).

Analysis of the amino acid sequences of number of low- and high-molecular-weight

PBPs and serine active-site β-lactamases have supported the view that the two types

of proteins have a common, but different on the basis of evolution. Findings of

resemblance in the 3D arrangement of a low-molecular-weight transpeptidases and

class A β-lactamases provided strong evidence for this view. In some Gram-negative

bacteria, β-lactam resistance mediated by modification of PBPs has been described

(Spratt and Cromie, 1988).

More than 40 structurally different β-lactam antibiotics are available in about 73

formulations and most of them are in clinical use at present. β-lactams are

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17

antimicrobial agents of choice with minimum side effects. β-lactamases produced by

bacteria represent severe risk for the clinical use of β-lactams. β-lactamase inhibitors

were considered to solve the issue of resistance to β-lactams, but bacteria have

evolved new resistance mechanism to inactivate β-lactamase inhibitors (Therrien and

Levesque, 2000; Drawz and Bonomo, 2010).

Currently, four β-lactam/β-lactam-inhibitor combinations are available for medical

use: amoxicillin/clavulanic acid, piperacillin/tazobactam, ticarcillin/clavulanic acid,

and ampicillin/sulbactam. Piperacillin/tazobactam and ticarcillin/clavulanic acid

possess broadest spectrum of microbiologic activity. Pharmacodynamics and activity

of these two combinations is influenced by many factors (Lister, 2000).

Resistance Mechanisms in Pathogenic Bacteria

Scarcity in new antibiotic research and emerging resistance to the available antibiotics

has created an important issue in public health. Gram negative multidrug-resistance

has received little attention as compared to MRSA and other Gram-positive threats.

Gram negative bacteria like Acinetobacter baumannii and Pseudomonas aeruginosa

are cause of dangerous hospital outbreaks and possess a variety of mechanisms for

resistance. In several cases, they have been reported to be resistant to all the available

antimicrobial agents. The increasing prevalence of community acquired infections by

extended-spectrum β-lactamase-producing organisms is alarming. Furthermore, the

carbapenems are now susceptible to various organisms, once considered as the most

successful class of antibiotics (Siegel, 2008).

Increase in antibiotic resistance in pathogenic bacteria is a result of overuse and

misuse of antibiotics. In spite of constant warnings, poor infection-control practice

and negligent antibiotic use and have led to the development of wide-ranging

resistance problems worldwide. Multidrug-resistant bacteria with greater virulence

and increasing resistance are commonly reported from community and nosocomial

outbreaks. For example MRSA, Vancomycin-resistant enterococci (VRE), ESBLs and

carbapenemase production in Gram negative bacteria, and toxin-hyperproducing

Clostridium difficile (French, 2010).

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Several aspects of the irrepressible and rapid increase in antibiotic resistance of

microorganisms are of special concern. Number of resistance mechanisms appear and

spread within bacterial populations having broad spectra of activity, making them

resistant to one antibiotic or the whole group. These mechanisms restrict the use of

last-choice antimicrobial agents in the treatment of a variety of infections. Some

mechanisms require specific detection procedures and are of great clinical

importance, as they may not show clear resistance in vitro using standard

susceptibility testing. Numerous mechanisms affecting the same and/or different

groups of antibiotics exist and are even selected together in new and more pathogenic

bacterial strains. Multiple β-lactamases with activity against broad spectra of

antibiotics exemplify all the incidents stated above. They comprise of key antibiotic

resistance mechanism of Gram negative rods. Three important groups of β-lactamases

are usually notable, Class A extended-spectrum β-lactamases (ESBLs), class

C cephalosporinases (AmpC), and different types of β-lactamases with

carbapenemase activity, of which the so-called class B metallo-β-lactamases (MBLs)

are of the greatest concern (Gniadkowski, 2001).

Numerous surveillance reports have confirmed that resistance is increasing among

prevalent pathogens at an alarming rate, resulting in an increase in morbidity and

mortality from hospital-acquired infections. The most important causes among Gram

positive organisms include methicillin-(oxacillin-) resistant Staphylococcus aureus

(MRSA), multidrug-resistant and β-lactam-resistant pneumococci, and vancomycin-

resistant enterococci (VRE). Important resistant pathogens of Gram negative

resistance are extended-spectrum β-lactamases (ESBLs) in K. pneumoniae, E. coli,

and P. mirabilis, high-level third-generation cephalosporin (Amp C) β-

lactamase resistance among Citrobacter freundii and Enterobacter species, and

multidrug-resistance genes observed in Acinetobacter, P. aeruginosa and

Stenotrophomonas maltophilia. Current data imply that because of increase in ESBLs

and high-level amp C β-lactamase producing bacteria, use of cephalosporins may be

unproductive in most of the patients with nosocomially-acquired infections. Beside

this, the use of these antibiotics may allow the overgrowth of naturally resistant

enterococci. The use of fluoroquinolones in the empiric treatment of hospital-acquired

infections is also being restricted by increasing resistance levels and new resistance

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19

patterns. Carbapenems, piperacillin/tazobactam, and cefepime have good activity

against many resistant pathogens among the available antimicrobials. Appropriate

antimicrobial selection, surveillance systems, and effective infection-control

procedures are important factors in limiting spread and occurrence of antimicrobial-

resistant pathogen (Jones, 2001).

There have been an increase in multidrug resistance among Gram negative rods and is

connected with the expression of plasmid-mediated as well as chromosomal β-

lactamase enzymes, whose number is now more than 890. The novel β-lactamases

have broad-spectrum catalytic activity and hydrolyze most of the β-lactams. The most

important plasmid-mediated β-lactamases consist of (a) AmpC β-lactamases are

expressed in higher quantities, (b) ESBLs like CTX-M β-lactamases having activity

against 3rd

generation cephalosporins and monobactams, and (c) carbapenemases from

several molecular classes that are capable of hydrolyzing almost all β-lactam

antibiotics, including the carbapenems. Important plasmid-mediated carbapenemases

include (a) the KPC β-lactamases basically evolved in K. pneumoniae isolates and

now moving to other pan-resistant Gram negative bacteria worldwide and (b) metallo-

β-lactamases that have evolved in bacteria by deletions in other β-lactamases, having

resistance to all β-lactam antibiotics except aztreonam. Beta-lactamase genes

encoding these enzymes are mostly present on plasmids carrying resistance genes for

other non-β-lactam antibiotic. As a result, some Gram negative infections have a

limited choice of antibiotic therapy. Gram negative bacteria having multidrug

resistance have been reported in both hospital- and community-acquired infections,

eradication of these resistant strains is complicated (Bush, 2010b).

All Enterobacteriaceae produce inherent chromosomal encoded β-lactamases, except

for Salmonella spp. which are responsible for intrinsic resistance of individual species

among Enterobacteriaceae. E. coli and Shigella spp. produce a very small quantity of

AmpC β-lactamases and are not resistant to ampicillin and other β-lactam antibiotic

agents. Serratia spp., C. freundii, Enterobacter spp, P. stuartii, P. rettgeri and M.

morganii produce very minute quantities of inducible AmpC β-lactamases which are

not susceptible to inhibition by β-lactamases inhibitor, a reason of natural resistance

to ampicillin, amoxicillin/clavulanic acid and 1st generation cephalosporins. Small

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20

amounts of SHV-1 β-lactamases are produced by K. pneumoniae, making it resistant

to ampicillin, carbenicillin, ticarcillin and attenuated zone of inhibition to piperacillin,

as compared to piperacillin with tazobactam. They are not resistant to inhibition by β-

lactamase inhibitors. While Proteus mirabilis showed a very little expression of

chromosomal β-lactamases, Proteus vulgaris produce chromosomally encoded β-

lactamases of class A (cefuroximases), making it resistant to ticarcillin, ampicillin,

and 1st and 2

nd generation cephalosporins. In the course of evolution antibiotics are

the reason of appearance of acquired or secondary β-lactamases, with the only

function to protect bacteria from antibiotics. A high level of β-lactamases leads to

resistance to their inhibitors. Most of them are derivatives of classic TEM- and SHV-

β-lactamase types. Unlike these parent enzymes, ESBLs hydrolyze oxyimino-

cephalosporins such as cefuroxime, ceftriaxone, cefotaxime, ceftazidime, ceftizoxime,

cefpirome and cefepime, aztreonam, as well as penicillins and other generations of

cephalosporins, with the exception of cephamycin (cefoxitin and cefotetan). β-

lactamase inhibitors inhibit theses β-lactams. AmpC β-lactamases are chromosomal

and inducible in most Enterobacter spp., M. morganii, C. freundii, Serratia and

Providentia spp. They show resistance to almost all penicillins and cephalosporins, to

β-lactamase inhibitors and aztreonam as well, and are susceptible to cefepime and

carbapenems. Plasmid-encoded AmpC β-lactamases have arisen through the

transmission of chromosomal genes for the inducible AmpC β-lactamase onto

plasmids. All plasmid-encoded AmpC β-lactamases have substrate profiles similar to

the parental enzymes, from which they appear to be derived. The only difference is

that plasmid-encoded AmpCs are uninducible unlike chromosomal AmpCs. The

Clinical and Laboratory Standards Institute (CLSI), previously known as National

Committee for Clinical Laboratory Standards (NCCLS) has issued guidelines for

ESBL screening and confirmation for isolates of E. coli, K. pneumoniae and K.

oxytoca. But no CLSI guidelines are available for detecting plasmid-mediated AmpC

β-lactamases or ESBLs detection and reporting of other organisms. Elevated

expression of AmpC β-lactamase may mask ESBL production in species with

production of chromosomally mediated inducible AmpC β-lactamase. AmpC-

inducible species (for example C. freundii and Enterobacter spp.) can be detected by

cefoxitin/cefotaxime disc antagonism tests. Since bacterial pathogens with novel types

of antibiotic resistance are first encountered by clinical laboratories, they need proper

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21

equipment and training to detect these bacteria, including appropriate staff with

sufficient time and tools to follow up important observations. Because bacteria

constantly evolve different strategies, training must be carried out continuously

(Susic, 2004).

In Gram negative bacteria, outer membrane work as a barrier for penetration of

hydrophilic compounds. Loss of porins (water-filled protein channels) is also a reason

to antimicrobial resistance, mainly when the organism is expressing other resistance

mechanisms. There is little information about outer membrane proteins (OMPs) in

Gram negative clinical isolates. In K. pneumoniae, two major OMPs, OmpK35 and

OmpK36, are expressed, but OmpK35 is not expressed in many ESBL-producing K.

pneumoniae. Loss of both OMPs in ESBL-producing K. pneumoniae account for

resistance to cefoxitin, increased resistance to expanded-spectrum cephalosporins, and

decreased susceptibility to carbapenems, particularly ertapenem. OMPs loss also

reduces susceptibility of ESBL-producing organisms to other non-β-lactam

compounds, such as fluoroquinolones (Martinez-Martinez, 2008).

Extended-Spectrum β-Lactamases

Extended-spectrum β-lactamases hydrolyze the third-generation cephalosporins. Such

resistance derives from genes for TEM-1, TEM-2, or SHV-1 which alters the

configuration of amino acid. A large number of ESBLs other than TEM or SHV

lineage have been reported. The presence of often plasmid encoded ESBLs carries

remarkable clinical significance. Plasmids responsible for ESBL production often

carry genes that encode resistance to other classes of drugs like aminoglycosides. This

makes use of antibiotic for treating ESBL-producing bacteria extremely limited.

Although carbapenems are the drugs of choice for infections caused by ESBL-

producing organisms, isolates resistant to carbapenems have been reported. Treatment

of ESBL-producing organisms with some extended-spectrum cephalosporins still

remains an option but failure rates are higher. The alteration in cephalosporin

breakpoints for the Enterobacteriaceae has been proposed so that the need for

detecting ESBLs could be obviated. Clinical and Laboratory Standards Institute

(formerly the National Committee for Clinical Laboratory Standards) provides the

guidelines for such detection in Klebsiella spp. and Escherichia coli. The

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22

enhancement of extended-spectrum cephalosporins activity against ESBL-producing

organisms in the presence of clavulanic acid is a common phenomenon in all

detection methods. Presence of ESBLs in Gram negative bacteria is major cause for

development of new antibiotic resistance mechanisms which in turn necessitates

development of new antimicrobial compounds (Paterson and Bonomo, 2005).

Extended spectrum β-lactamases, resistant TEM- or SHV-derived β-lactamases,

plasmid mediated cephalosporinases and carbapenem hydrolyzing β-lactamases are

among the most problematic β-lactamases (Bush, 1999).

ESBLs which hydrolyze extended-spectrum cephalosporins are being frequently

reported among members of Enterobacteriaceae. In community acquired infections by

Escherichia coli, SHV and TEM enzymes are often reported replaced with CTX-M

enzymes that are becoming prevalent type of ESBLs. Usually infection of the urinary

tract, bloodstream and abdomen are included among such serious infections which

often warrant hospitalization. Diverse underlying risk factors have been reported in

affected patients. The expression of ESBL in Enterobacteriaceae strains isolated from

nosocomial patients has substantially increased in many countries. The

epidemiological findings of these infections are frequently complex as multiple strains

were reported for outbreaks that may co-exist sporadically. Relevant infection-control

measures should focus on reducing patient-to-patient transmission via the inanimate

environment and hospital personnel. Rational use of antibiotics, sterilization of

medical equipment and ensuring proper cleaning can reduce the onset of new

infections. The data regarding effectiveness of different treatment regimens for

ESBLs associated infections are scarce. Beta-lactam/β-lactamase inhibitor

combinations may be useful, but the supporting evidence is not significant.

Carbapenems, however, are the agents of choice, and may prove more effective for

serious infections than fluoroquinolones. Tigecycline and polymyxins have

considerable activity against ESBL-producing Enterobacteriaceae. Also the addition

of fosfomycin to this regime has been effective but warrant more evidence (Falagas

and Karageorgopoulos, 2009).

Due to the problems associated with ESBLs, such organisms pose exceptional

challenges for clinicians, microbiologists, infection control professionals and research

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23

scientists. ESBLs are the enzymes that can hydrolyse penicillins, cephalosporins and

monobactams. ESBLs are generally derived from TEM and SHV-type enzymes and

are often located on transferable plasmids. Although the prevalence of ESBLs is not

exactly known, it is believed that these are expressed in 10-40% of Escherichia coli

and Klebsiella pneumoniae strains throughout the world. Enterobacteriaceae with

ESBLs expression have been associated with infection outbreaks. Control of such

infections has been a great challenge due to number of factors. Some of these factors

are; complex nature of laboratory detection of ESBLs and multi-resistance to

antibacterial agents. Various ESBL-producers also express AmpC β-lactamases that

can be co-transferred with plasmid mediating resistance to aminoglycoside. Also

association between fluoroquinolone resistance and ESBL production is being

observed quite often (Rupp and Fey, 2003).

Patterson (2006) reported that 20% of infections caused by Klebsiella pneumoniae

and 31% by Enterobacter spp., in intensive care units of the United States hospitals,

involved strains that were resistant to third-generation cephalosporins. He concluded

that Escherichia coli, K. pneumoniae and other members of Enterobacteriaceae as

ESBL-producers with multidrug resistance are being frequently encountered in

healthcare settings. Salmonella and other Enterobacteriaceae responsible for

gastroenteritis may also express ESBLs. Resistance to third-generation cephalosporins

by Enterobacter spp. is mostly caused by overproduction of AmpC β-lactamases.

Some strains of Enterobacter cloacae are both ESBL and AmpC producers,

conferring resistance to both third- and fourth-generation cephalosporins. Among

Enterobacteriaceae, quinolone resistance is usually due to chromosomal mutations

that lead to alterations in target enzymes or drug accumulation. Plasmid-mediated

quinolone resistance associated with acquisition of the qnr gene has also been

reported in E. coli and K. pneumonia.

Diversity of ESBL Types

TEM

Datta and Kontomichalou (1965), first characterized TEM-1 penicillinase from E.

coli isolated from blood of an infected patient. Later plasmid containing TEM-1-

were found in various members of the Enterobacteriaceae family, Haemophilus

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24

influenzae, Pseudomonas aeruginosa and Neisseria gonorrhoeae. Du Bois et al.,

(1995) isolated TEM-2 enzyme with the same substrate specificity but single amino

acid mutation Gln39Lys. TEM-3 was characterized by another mutation of

Glu104Lys in addition to the one at position 39, thus increasing its substrate spectrum

to cephalosporins. Perilli et al., (1997) reported that amino acid substitutions within

the TEM enzyme occurred at limited positions resulting in various alterations in the

ESBL phenotypes. More than 190 members of TEM have already been described and

this number is still growing (http://www.lahey.org/studies/).

There are 288 amino acid residues in the primary structure of TEM-1. The derivatives

of TEM are variants of TEM-1 β-lactamase that usually differ from it by a single

amino acid substitution. Mutations are found at 60 positions; however, mutation

frequency is different at each position. Most frequent mutations were seen at positions

21, 39, 69, 104, 164, 182, 238, 240, 244, 265 and 275. Mutations at positions 104,

164, 238, and 240 are related with increasing the substrate specificity. Bradford,

(2001) observed that mutation Gly238Ser resulted in ability to destroy cefotaxime and

ceftazidime with equal efficiency, whereas mutation Arg164Ser was highly active

against ceftazidime but less against cefotaxime. Chaibi et al., (1999) reported that

some mutations resulted in inhibitor-resistant enzymes (IRT, inhibitor resistant

phenotype, subgroup 2br). Mutations at positions 69, 244, 275, and 276 define

resistance to inhibitors (Strynadka et al., 1992). Combination of these two mutations

in a single TEM have been found associated with both ESBL and IRT types of

resistance. Petrosino and Palzkill, (1996) used random and directed mutagenesis and

Hayes et al. (1997) used insertional mutagenesis to synthesize mutants having

enhanced activity spectrum and even new subtypes of TEM were forecasted.

Resistant infections of Escherichia coli, Klebsiella pneumoniae and Citrobacter

freundii were observed recently in France and Germany linked to new plasmid-

mediated TEM-3 to TEM-7 β-lactamases. Sougakoff et al., (1988) cloned a BamHI

plasmid encoding TEM-3 in E. coli and determined the nucleotide sequence of

corresponding gene blaTEM-3. They observed that the amino acid sequence of TEM-

3 was different from that of the TEM-2 enzyme in two positions: serine (TEM-3) for

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25

glycine (TEM-2) at residue 238 and lysine (TEM-3) was substituted for glutamic acid

(TEM-2) at residue 104 (Sougakoff et al., 1988).

SHV

These enzymes were discovered after TEM type β-lactamases. SHV-1 β-lactamase

was the first type these enzymes, encoded by Klebsiella pneumoniae chromosomes

(Knothe et al., 1983; Livermore, 1995). SHV-2 mutant was the first described ESBL

which differed by a single Gly238Ser mutation (Kliebe et al., 1985; Bradford, 1999).

SHV β-lactamases are derivatives of SHV-1 enzyme and differ by the presence of

point mutations as well as by deletions (SHV-9 and SHV-10) or inserts (SHV-16).

Substrate specificity in mutant SHV depends on the substitution type. The common

positions considered as the key ones for changes in the substrate specificity were

found to be as 35, 238, and 240. SHV-10 with IRT resistance phenotype was not

having an ESBL phenotype, although it differs only by Ser130Gly from SHV-9

(Rubtsova et al., 2010).

The plasmid-mediated SHV β-lactamase SHV-1 has 23 variants, mostly having

extended spectrum activity against the broad spectrum cephalosporins. The ancestor is

thought to be a chromosomal penicillinase of K. pneumoniae. SHV enzymes belong to

class A serine β-lactamases and share functional and structural similarities with TEM

β-lactamases. SHV-1 β-lactamase behaves like a characteristic penicillinase capable

of hydrolyzing penicillins and first generation cephalosporins. SHV-1 β-lactamase is

mostly encountered in K. pneumoniae. ES SHV β-lactamases; the most prevalent ES

β-lactamases are found in increasing rates in K. pneumoniae and other enterobacterial

isolates. These ES SHV β-lactamases conferring resistance to β-lactams and

monobactams are usually encoded by mobile multi-resistant plasmids (Tzouvelekis

and Bonomo, 1999).

CTX-M

CTX-M Extended-spectrum β-lactamase were observed for the first time in the later

half of the 1980s and were reported in many countries and spread very quickly in the

last 10 years to become the commonly found β-lactamase type in most parts of the

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26

world. CTX-M does not belong to TEM or SHV families’ highlights, that these

enzymes are more efficient in hydrolyzing cefotaxime as compared to ceftazidime

(Bonnet, 2004).

First CTX-M β-lactamase was reported from an E. coli strain isolated in 1989

(Bauernfeind et al., 1990). Later on this enzyme was named as CTX-M-1. Genes of

this β-lactamase type are localized on plasmid. More than 120 β-lactamases of this

type have been reported (http://www.lahey.org/studies/). Among class A extended-

spectrum β-lactamase, the CTX-M type of enzymes are one of the most diverse

groups concerning the genes coding then and sequence of the amino acids,

respectively. The CTX-M group of enzymes are now classified into five sub-classes

(Bonnet, 2004). Each sub-class includes the main CTX-M β-lactamase (CTX-M-1,

CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25) and its variants which differ only

by single or few mutations. CTX-M group of enzymes and other class A types of

enzymes are marginally homologous to each other (below 40%) (Tzouvelekis et al.,

2000). They are more homologous (over 70%) to the chromosomally encoded β-

lactamases from K. oxytoca, C. diversus, P. vulgaris, and S. fonticola (Bonnet et al.,

1999). Which shows that the plasmid mediated CTX-M β-lactamases are originally

derived from β-lactamases whose genes were present on the chromosomes. The

variation of CTX-M group of enzymes on one hand from the TEM and SHV group of

β-lactamases on the other hand concerning to specificity of their substrate profile is

that about all of CTX-M group of enzymes have hydrolytic activity against

cephalosporins, which make them extended-spectrum β-lactamases. Differences

between CTX-M group of β-lactamases confer changes in its substrate specificity

towards various cephalosporins like cefotaxime, ceftazidime and cefepime. It was also

observed that main mutations for this type of β-lactamases lead to the variation in its

catalytic activity against specific substrates, for example the mutations at positions

167 and 240. CTX-M group of enzymes are mostly reported in outdoor patients of the

hospitals (Harada et al., 2008).

The CTX-M-encoding genes have jumped from the chromosome of Kluyvera spp.

onto transferable plasmid that mediated their dissemination among pathogenic

enterobacteria. CTX-M-type ESBLs shows good activity against cefotaxime and

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27

ceftriaxone but have a poor activity against ceftazidime, which is important for

detection in laboratory. However, numerous CTX-M derivatives with improved

activity against ceftazidime have been reported. These ESBLs are now predominantly

reported enzymes in Enterobacteriaceae in some areas (Rossolini et al., 2008).

Among the extended-spectrum β-lactamases, the cefotaximases represent a rapidly

increasing group of β-lactamases that have spread globally. The cefotaximases, which

efficiently hydrolyze cefotaxime, are commonly carried by transferable plasmids, and

they are predominantly found in Enterobacteriaceae, mostly in E. coli, K.

pneumoniae, P. mirabilis and Salmonella typhimurium. Isolates of Aeromonas

hydrophila, Acinetobacter baumannii and Vibrio cholerae having cefotaximases have

also been reported. The cefotaximases are molecular class A β-lactamase enzymes,

and are functionally classified as ESBLs conferring a good activity against penicillins,

extended-spectrum cephalosporins and aztreonam, and are susceptible to inhibition by

β-lactamase inhibitors. Generally, the CTX-M enzymes are more efficient in

hydrolyzing cefotaxime as compared to ceftazidime, which is revealed by

considerably higher minimum inhibitory concentrations (MICs) to cefotaxime as

compared to ceftazidime. On evolutionary point of view, the cefotaximases are

classified into four sub-classes that are probably derived from β-lactamases genes of

the chromosome of Kluyvera spp. Insertion sequences, mainly the ISEcp1, have been

observed close to the genes coding for β-lactamases of all four sub-classes (Walther-

Rasmussen and Hoiby, 2004).

Since 2000, cefotaximase enzymes producing E. coli (mainly CTX-M-15) have been

an important cause of the community acquired bloodstream and urinary tract

infections (UTIs) due to bacteria producing extended-spectrum β-lactamase (ESBL)

all over the world. Molecular characterization studies showed that the sudden increase

of CTX-M-15-producing E. coli all over the world is mainly because of a single strain

known as ST131 and that foreign travel to high-risk areas such as the Indian

subcontinent might playing its part in the spread of this strain across different

continents (Pitout, 2010).

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Epidemiology of ESBL-Producing Organisms

The increasing prevalence of multidrug-resistance in the members of

Enterobacteriaceae is causing difficulties in the treatment of hospital-acquired

infections. In most parts of the world, resistance to the second and third- generations

cephalosporins is more than 10% in total nosocomial infections and about 30% of

strains isolated from the intensive care unit. β-lactamase-mediated resistance is related

with plasmid-mediated extended-spectrum β-lactamases (ESBLs) and

carbapenemases, specifically the CTX-M family of ESBLs, the KPC family of serine

carbapenemases, and the VIM, IMP, and NDM-1 metallo-β-lactamases. Although

clonal dispersion of resistant isolates was seen initially, more diverse genetic

platforms are being observed as variations of mobile elements are transferred

worldwide. These enzymes are now appearing in multiple combinations of ESBLs

and carbapenemases, thereby conferring resistance to virtually all β-lactam antibiotics

In addition, these mobile segments of DNA frequently carry genes for other β-lactam

and non-β-lactam antibiotics. (Endimiani and Paterson, 2007; Bush, 2010a).

Kiratisin et al., (2008) investigated the trends in occurrence and antibiotic resistance

patterns among ESBL-producing E. coli and K. pneumoniae over the two year period

in Thailand. The results indicated a very high prevalence (up to 65.9% among sputum

isolates) of ESBL positive strains. These isolates verified a considerable increase in

rates of resistance to various non-β-lactam antibiotics and also expressed a multidrug

resistance phenotype at a high rate.

Mulvey et al., (2004) reported prevalence of ESBL-producing E. coli and Klebsiella

spp. from Canada. They screened a total of 29,323 E. coli and 5,156 Klebsiella spp.

isolated from 12 participating sites. Of these, 505 non-duplicate isolates showing

reduced susceptibility to the CLSI-recommended β-lactams were investigated in a

central laboratory over a 12 months period. Phenotypic confirmation results indicated

that a total of 116 isolates were ESBL producers. Molecular characterization revealed

the occurrence of TEM-11 (n = 1), TEM-12 (n = 1), TEM-29 (n = 1), TEM-52 (n =

4), CTX-M-13 (n = 1), CTX-M-14 (n = 15), CTX-M-15 (n = 11), SHV-2 (n = 2),

SHV-2a (n = 12), SHV-5 (n = 6), SHV-12 (n = 45) and SHV-30 (n = 2). Sequence

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29

analysis showed five novel β-lactamases, which were designated as TEM-115 (n = 2),

TEM-120 (n = 1), SHV-40 (n = 2), SHV-41 (n = 4), and SHV-42 (n = 1). Besides, no

gene was identified for five isolates confirmed as ESBL producers by phenotypic

assays.

Ode et al., (2009) reported the connection of aminoglycoside resistance and plasmid-

mediated quinolone resistance among cephalosporin-resistant E. coli (n=46) and K.

oxytoca (n=28) strains isolated in Japan. A total of 73 isolates were extended-

spectrum β-lactamase (ESBL) producers and one K. oxytoca strain was producing

IMP-1 metallo-β-lactamase (MBL). PCR and sequence analysis confirmed that 8

CTX-M-9/SHV-12-producing isolates, one IMP-1-producing K. oxytoca isolate, and 6

ESBL-positive E. coli isolates, respectively had plasmid-mediated quinolone

resistance genes qnrA1, qnrB6, and aac(6')-Ib-cr. All qnr-positive isolates also have

aminoglycoside acetyltransferase genes, either aac(6')-Ib or aac(6')-IIc. Resistance

genes to β-lactams, quinolones and aminoglycosides were present on a plasmid of ca.

140 kb.

In Brooklyn, Enterobacteriaceae samples were collected from 15 hospitals. ESBLs

were reported in 44% of the total 409 K. pneumoniae. Six of these isolates had a low

susceptibility to carbapenems, while two of these that were resistant to all antibiotics

tested. There was similarity in the resistant strains as revealed by Pulsed field gel

electrophoresis. The incidence of ESBL producing isolates were associated with the

use of cephalosporin (P = 0.055). ESBLs were also present in 4.7% of E. coli and

9.5% of P. mirabilis isolates (Saurina et al., 2000).

A total of 252 GNB isolates, 155 (113 Klebsiella species, 21 Escherichia coli and 21

other) were subjected to drug susceptibility testing, ESBL phenotyping and testing for

clonal relatedness of ESBL strains by PFGE. The results demonstrated that Klebsiella

species and E. coli are the most common GNB causes of neonatal sepsis in India, and

over one-third are ESBL producers in both community and hospital settings. ESBL-

producing strains exhibited frequent co-resistance to aminoglycosides and

ciprofloxacin, but remained susceptible to imipenem. PFGE analysis revealed

extensive genetic diversity within the ESBL-producing isolates (Chandel et al., 2011).

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Since 2000's, community-acquired ESBLs producing bacteria have been reported

worldwide. Previous use of cephalosporin and fluoroquinolone are the two main

common risk factors known in patients that have ESBL-producing bacteria. Data

reports have revealed an alarming association of resistance to additional classes of

antibiotics among isolates. The emergence of ESBL-producing isolates limits the

choice of treatment considerably. Carbapenems are drugs of choice for serious

infections caused by ESBL-producing bacteria. Prevention on the spread and proper

management of the infections caused by community-acquired ESBLs producing

bacteria is necessary (Zahar et al., 2009).

Detection of ESBL in the Clinical Microbiology Laboratory

Extended-spectrum β-lactamases (ESBLs) are plasmid-mediated β-lactamases that

confer resistance to a wide range of β-lactams. They are derivatives of native β-

lactamases found in Gram-negative rods by genetic mutation, especially the

pathogenic isolates of E. coli and Klebsiella species. Genetic modifications have

expanded the substrate specificity of β-lactamases to inactivate third-generation

cephalosporins like ceftazidime. Resistance to the commonly available antibiotics has

complicated the treatment strategies and poses a serious global health concern.

Overproduction of chromosomal or plasmid-encoded AmpC β-lactamases is another

common resistance mechanism in members of Enterobacteriaceae. Unlike most

ESBLs, AmpC enzymes are able to escape clavulanate and similar β-

lactamase inhibitors. Technological improvements in testing and in the development

of uniform standards for both ESBL detection and confirmatory testing, helps in

accurate identification of ESBL-producing organisms in clinical laboratories (Pfaller

and Segreti, 2006).

Methods for detecting ESBL-producing Enterobacteriaceae begin by a correct

interpretation of the susceptibility profiles, applying the usual criteria for

interpretative reading of the antibiogram. Appropriate confirmatory methods will be

consequently chosen, based on the inhibition of the enzyme by β-lactamases

inhibitors, generally clavulanic acid. In case of non-AmpC-producing

Enterobacteriaceae, at least two substrates should be used -cefotaxime or ceftriaxone

and ceftazidime- to detect enzymes with a low hydrolytic activity against both

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31

substrates. Cefepime or AmpC-inhibitors should be recommended for AmpC-

producing microorganisms. The identification of the enzymes responsible for the

confirmed ESBL phenotype can be performed, either in the clinical laboratory or in

reference centres, following a protocol of biochemical and molecular reactions able to

detect and characterize, at least, those genes more frequently related to the

predominant phenotypic profiles in our region. It is important to know which are the

most prevalent combinations enzyme-microorganism, the vehicles for the genetic

transmission involved in their dissemination, and the main epidemiological

characteristics of the infections that they produce, in order to establish the dimensions

of the problem and conduct surveillance studies, with the aim of achieving measures

to control the wide spread (Garcia et al., 2010).

ESBL producing microorganisms resistant to non-β-lactam antibiotics

There is an increase in antibiotic Resistance throughout the world. Pathogens and

opportunistic microorganisms are evolving mechanisms of resistance to all known

antibiotics. Prevalence of plasmid-mediated extended-spectrum β-lactamases and

other β-lactamases is on the rise. Carbapenemases present on mobile genes encoded

resistance to other classes of antibiotics. Numerous plasmid-mediated resistance

mechanisms against aminoglycosides and fluoroquinolones have been described.

Chromosomally encoded resistance mechanisms combining with plasmid-

mediated resistance have resulted in multiple-drug resistant strains that are resistant to

all the major groups of commonly used antibiotics. Carbapenem-resistant strains of

Enterobacteriaceae limits the choice of antimicrobial agents for the treatment of

infections. Tigecycline, temocillin, colistin and fosfomycin are the choices left.

Although reports indicate strong activity by in vitro testing for all these drugs against

carbapenemase producing isolates of Enterobacteriaceae, clinical assessment do not

present strong evidence for any enhanced outcome. ESBL-producing Escherichia coli

in lower urinary tract infections (UTIs) can be effectively treated with oral fosfomycin

tromethamine. In patients having severe infections of carbapenem-resistant Klebsiella

pneumoniae, intravenous fosfomycin may be useful and harmless, when used in

combination with other antibiotic. Tigecycline is approved for community-acquired

pneumonia treatment in the US and is only prescribed for the treatment of skin

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32

structure and complicated skin and intra-abdominal infections in Europe. Clearly,

further research is urgently required on the clinical and safety outcomes of these

existing alternative drugs in the treatment of multidrug-resistant Enterobacteriaceae,

and also on the development of novel and unrelated antibiotics (Schultsz and

Geerlings, 2012).

Epidemiological data suggested that most of the ESBL-producing E. coli isolates were

from patients in the internal medicine wards (72.7 %), while ESBL-producing K.

pneumoniae strains were evenly distributed between ICU (45.8 %) and internal

medicine wards (45.8 %). Majority of the isolates were from urine samples. ESBL-

producing strains exhibited multiple drug resistance, namely to aminoglycosides,

quinolones and trimethoprim-sulfamethoxazole, as compared to ESBL-negative

isolates (Espinar et al., 2011).

Karah et al., (2010) investigated occurrence of the plasmid-encoded

quinolone resistance genes qnr and aac(6')-Ib-cr in clinical isolates of E. coli and

Klebsiella spp. They selected a total of 414 isolates on the basis of reduced

susceptibility to ciprofloxacin and Nalidixic acid. ESBL producers have a higher

frequency of both aac(6')-Ib-cr and qnr (52.3% and 9.1%, respectively). In two

isolates qnrB1 and qnrB7 were detected, whereas 6 isolates were having qnrS1. The

genetic structure surrounding qnrS1 was similar to previously described structures.

Conjugative IncN-type plasmids of about 140 kb were carrying qnrS1 in 2 isolates.

AmpC β-Lactamases

AmpC β-lactamases are important chromosomally-mediated β-lactamases of many of

the Enterobacteriaceae and a few other organisms, hydrolyzing cefoxitin, cefazolin,

cephalothin, most penicillins and are not inhibited by β-lactamase-inhibitors. In many

bacteria, AmpC β-lactamases are inducible and are expressed at high levels after

mutation. Overexpression of AmpC β-lactamase is a quite problematic in

Enterobacter aerogenes and Enterobacter cloacae infections, where the isolate

become resistant after the start of treatment while initially it is susceptible to these

antibiotics. Transferable plasmids have acquired genes for AmpC β-lactamases, which

as a result, appear in bacteria which do not have or weakly expressed chromosomal

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33

blaAmpC gene, such as K. pneumoniae, E. coli and P. mirabilis. Resistance due to

plasmid-encoded AmpC β-lactamase producing bacteria are not common as compared

to ESBL producing bacteria in most parts of the world but these enzymes are difficult

in detection and have a broader spectrum. AmpC β-lactamase-producing isolates can

be identified by available techniques but are in developing stage and are not yet

optimized for the use in clinical laboratory. Carbapenems can generally used in

infections caused by AmpC-producing bacteria, but resistance to carbapenem is

increasing in some bacteria due to OMP loss or efflux pump activation (Jacoby,

2009).

Manchanda and Singh (2003) reported that AmpC β-lactamases could be

distinguished from ESBLs on the basis of their activity against cephamycins as well

as other extended-spectrum cephalosporins. They determined the prevalence of AmpC

β-lactamase producing Gram negative bacilli in a tertiary care facility in India. Using

a modified three dimensional test, 20.7% of the isolates were found harbouring AmpC

β-lactamases among the total 135 clinical isolates tested. AmpC β-lactamase

production was confirmed by inhibition of zone distortion in the presence of

cloxacillin. Majority of the AmpC β-lactamase producers were among Acinetobacter

spp. (42.8%) followed by K. pneumoniae isolates (33.3%). All AmpC β-lactamase

producers were susceptible to cefoxitin.

Pitout et al., (2003) developed a modified double-disc test for successful detection of

ESBLs in Gram negative bacilli producing well-characterized β-lactamases, as

detection of ESBLs in AmpC producing Enterobacteriaceae is problematic. 212

clinical isolates of Enterobacter cloacae, Enterobacter aerogenes, Serratia

marcescens and Citrobacter freundii are also tested. Modified double-disc test

accurately differentiated between ESBL producers and derepressed chromosomal

AmpC mutants.

Nasim et al., (2004) introduced a novel method for detection of AmpC β-lactamase

producing in E. coli and K. pneumoniae. The compared cefoxitin-agar medium

(CAM)-method to the previously reported modified three-dimensional (M3D)

method. Six cefoxitin-sensitive, non-ESBL E. coli isolates, 8 ESBL-producing,

AmpC negative E. coli isolates, 55 cefoxitin-resistant and non-ESBL E. coli isolates,

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34

6 cefoxitin-resistant and non-ESBL K. pneumoniae isolates and 9 E. cloacae isolates

susceptible to extended-spectrum cephalosporins were selected for study. Various

control strains producing AmpC β-lactamase and without β-lactamase production

were used in the study. The M3D assay was negative with all AmpC-negative controls

and positive with all known AmpC-positive controls and the nine E. cloacae clinical

isolates. Fifty four of 55 E. coli strains and 1 of 6 Klebsiella pneumoniae strains were

positive by the M3D method. The CAM method with 4 μg of cefoxitin/ml was

equivalent to the M3D method for detecting AmpC production in E. coli and K.

pneumoniae.

AmpC disc test, based on filter paper discs soaked with EDTA, was developed by

Black et al., (2005) and was found to be a highly specific, sensitive, and suitable for

detecting plasmid-mediated AmpC β-lactamases in bacteria that do not have a

chromosomally mediated AmpC β-lactamase. The test accurately differentiated

AmpC and ESBL production using cefoxitin insusceptibility as a screen, and

distinguished AmpCs from non-β-lactamase mechanisms of cefoxitin resistant, such

as reduced permeability of the outer membrane.

Woodford et al., (2007), tested 173 strains of E. coli and Klebsiella spp. for acquired

AmpC β-lactamases production. Genes encoding 6 phylogenetic groups of acquired

AmpC β-lactamases were detected by PCR. Which were 67 (49%) E. coli and 21

(55%) Klebsiella spp. Sixty isolates produced CIT-type enzymes, 14 had ACC types,

11 had FOX types and 3 had DHA enzymes. The low-level cephalosporin resistance

of the remaining isolates (n = 85; 49%) was inferred to result from reduced

permeability or, in E. coli, from hyper expression of chromosomal ampC. Twenty-

four E. coli isolates from one hospital produced a CIT-type enzyme, with 20 of these

additionally producing a group 1 CTX-M ESBL.

Prevalence of plasmid-mediated AmpC β-lactamases in isolates of Escherichia coli

and Klebsiella spp. in China, was investigated by Li et al., (2008). They collected

1,935 clinical isolates of Escherichia coli, Klebsiella pneumoniae and Klebsiella

oxytoca. About 327 isolates with cefoxitin zone diameters less than 18 mm were

selected for PCR of the blaAmpC genes and sequencing. Fifty-four isolates harbored

plasmid-mediated AmpC β-lactamases, as demonstrated by PCR and isoelectric

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35

focusing. Based on species, plasmid-mediated AmpC β-lactamases were detected in

30 isolates of K. pneumoniae, 23 isolates of E. coli, and 1 isolate of K. oxytoca. DHA-

1 was the most prevalent acquired AmpC β-lactamase and DHA-1 producing K.

pneumoniae was the most prevalent bacterium harboring a plasmid-mediated AmpC

β-lactamases. This was the first report of CMY-2-type AmpC β-lactamases in the

China (Vercauteren, 1997).

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36

Materials and Methods

The present research was conducted from August 2008 to January 2010. Samples

were collected from different medical, surgical wards, ICU and OPDs of Pakistan

Institute of Medical Sciences, Islamabad. These pathological samples were

transported to Microbiology Research Laboratory, Quaid-i-Azam University,

Islamabad, and proceeded for isolation of E. coli and Klebsiella pneumoniae.

Pathogens were identified according to standard identification methods. Antimicrobial

sensitivity, screening and confirmation of Extended-spectrum and AmpC β-lactamase

enzymes, MICs and molecular characterization, performed according to the standard

protocols.

Epidemiology

Patient’s/ clinical data collection

Information regarding age, gender, sample source (blood, urine, pus, cerebrospinal

fluid, sputum, catheters) and nature/status of patient (inpatient/outpatient) was

recorded for each sample. All patients in OPD with no history of hospital admission

for the last one month, were considered as having community-acquired infections,

while in-patients who acquired infection after 2 days of hospital admission were

considered as having hospital-acquired infections.

Transport of samples

Samples were transported in Amies agar gel transport swabs at ambient temperature.

Statistical analysis

To statistically analyze the data, SPSS Statistics 17.0 software (SPSSInc, Chicago) for

Windows was used. For all categories, variables were reported in numbers and

percentages. Association of the variables was analyzed using Chi-Square (X2) test. p

value less than 0.05 was defined as statistically significant.

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37

SAMPLE PROCESSING AND IDENTIFICATION OF CULTURE ISOLATES

Bacterial strains

Quality control was maintained at each step. E. coli ATCC 25922 was used for quality

control of the Gram’s stain, biochemical tests, media preparation, susceptibility

testing, AmpC disc test and MICs determination.

Samples were inoculated on MacConkey and Eosin Methylene Blue agar except

urine, which was inoculated on CLED agar (Oxoid, Hampshire UK). These are

differential and selective media for Gram negative bacterial isolates. These plates

were incubated aerobically at 35oC. After overnight incubation, the identification of

bacterial isolates was made by conventional microbiological tests, which include

colony morphology, Gram’s staining and biochemical characteristics.

Colony Morphology

On the basis of growth on the solid agar media, isolates were identified. Features,

which were observed on the respective media, include; size (large, moderate, small,

pinpoint), pigmentation (color of colony), form (irregular, circular, rhizoid), margins

(entire, undulate, lobate, serrate, filamentatous) elevation (flat, raised, convex).

Gram Staining

Smears of the bacterial isolates were prepared on the glass slides, allowed to air dry,

then heat fixed on flame. Crystal violet was flooded on the smears for one minute.

Washed slides with tap water. Then Gram’s Iodine was poured on the smears for one

minute. Slides were again washed with tap water. These smears were decolorized with

95% ethyl alcohol just for few seconds. Slides were again washed with tap water.

After that, smears were counterstained with safranin for 45 seconds and washed with

tap water. The slides were blot dried with bibulous paper and examined under the

microscope with oil immersion (100X). Color and shape of bacteria were noted.

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38

Biochemical Identification of Isolates

All isolates were identified by using standard biochemical tests which included; triple

sugar iron test, indole production test, methyl red test, Voges-Proskauer test, citrate

utilization test, motility and urease test.

Identification Through BiomérieuxVitek®2 System

Isolates with confusing results were confirmed with Biomérieux Vitek®

2 System

(bioMérieux, Marcy l'Etoile, France). A sterile loop was used to take sufficient

number of pure colonies and suspended in 3 mL of sterile saline (aqueous 0.45% to

0.50% NaCl, pH 4.5 to 7.0) in a 12 x 75 mm clear plastic (polystyrene) test tube. The

turbidity was adjusted 0.50-0.63 using a turbidity meter (DensiChekTM

).

Test tubes having the bacterial culture in suspension form were put into the cassette

and the card with chemicals in wells for identification (GN Card) was put in the slot

in front of tube, while the transfer tube was inserted into the accompanying

suspension tube. The GN card can be used for the identification of 135 different

bacteria of the most important fermenting and non-fermenting Gram-negative rods by

automated biochemical testing. The wells have chemicals for 47 different biochemical

tests with a single well as negative control. The filled cassette was placed manually

into the vacuum chamber station. After the application of vacuum, air was re-

introduced into the station and organism suspension was forced through the transfer

tube into micro-channels that filled all the test wells.

Inoculated GN cards were passed by a mechanism, which cut off the transfer tube and

sealed the card prior to loading into the carousel incubator. The system works by

measuring either turbidity or colored products of substrate metabolism for each test

reaction read after every 15 minutes. Final identification results were printed out from

the system after series of automated analyses in approximately 6 hours.

Test Reactions

In the VITEK 2 Identification system, calculations were performed on raw data and

compared to showtime to determine results for each test reaction. Results of the test

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39

reaction are represented as “+”,”–“, “(–)” or “(+)”. Reaction results in parentheses

indicate weak reactions and they are close to the test threshold.

AmpC β-lactamase producing isolates were identified by API 20E kit (Biomériuex,

USA).

Analytical profile index (API) 20E is a standardized system of 20 miniaturized

biochemical tests used for Enterobacteriaceae. A strip contains dehydrated substrates

in 20 micro tubes (Fig. 1). These tubes were inoculated with bacterial suspension

equal to 0.5 McFarland according to the test requirement. Some tests were incubated

anaerobically by overlaying mineral oil. The strip was incubated for 18-24 hours at

35oC. During incubation, color changes are produced spontaneously by metabolic

products or revealed by the adding reagents. Positive results were noted to determine

seven digits numerical profile, which is looked up in Analytical Profile Index.

Fig. 1. API 20E

ANTIBIOTIC SUSCEPTIBILITY TESTING

Antibiotics

Amoxicillin/clavulanic acid AMC (20/10 g), cefoxitin FOX (30 g), cefepime FEP

(30 g), aztreonam ATM (30 g), ceftazidime CAZ (30 g), cefotaxime CTX (30

g), imipenem IPM (10 g), trimethoprim-sulfamethoxazole SXT (1.25/23.75 g),

tetracycline TE (30 g), ciprofloxacin CIP (5 g), amikacin AK (30 g), gentamicin

CN (10 g), tigecycline TGE (15 g), cefoperazone/sulbactam SCF (95/10 g),

piperacillin/tazobactam TZP (100/10 g) (Oxoid, Hampshire UK) and

cefotaxime/clavulanic acid (30 μg/10 μg) (MAST Diagnostics, UK) were used.

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40

Preparation of 0.5 McFarland Standard

Prepared 1.75% w/v solution of BaCl2, by dissolving 2.35 g of dehydrated barium

chloride in 200 ml of distilled water. Solution of sulphuric acid (1% v/v) was prepared

by adding 1 ml of concentrated sulphuric acid to 99 ml of distilled water. Added 0.5

ml of BaCl2.2H2O solution to 99.5 ml of sulphuric acid solution to make 0.5%

turbidity standard and stirred constantly. The suspension was thoroughly mixed to

make sure that it is homogenous. Matched cuvettes were used to measure absorbance

at a wavelength of 625 nm in a spectrophotometer with a 1 cm light path. Water was

used as a blank standard. The acceptable absorbance for 0.5 McFarland standard was

0.08-0.13.

Disc Diffusion Method

Overnight fresh cultures were used to make lawns on Mueller-Hinton agar (MHA)

(Difco BD, Le Pont-De-Claix, France). The inoculum was suspended in direct saline

by selecting isolated colonies from an 18 to 24 hour old culture. The suspension was

adjusted to match the turbidity of 0.5 McFarland’s standard, adding saline and mixing

by vortex. For streaking on the plate, aseptic cotton swabs were submerged into the

suspension adjusted to the McFarland’s. For removing the excess culture, the swab

was pressed tightly to the walls of the tubes on the inside little above the level of the

fluid and rotated few times. The dried floor of an MH agar plate was streaked by

swabbing over the whole surface of sterile agar. The process was repeated two or

more times, changing the angle of the plate by about 60 every time to ascertain equal

dispersion of culture. As the end, the rim of the MH agar plate was also swabbed.

The plate was allowed to dry for 5 minutes. The antibiotic discs were dispensed onto

the surface of the inoculated MH agar plate. Every disc was pressed down a little to

ascertain full contact with the surface of the agar (Lalitha, 2004). The discs were

having the distance not closer than 20 mm from center to center. Maximum of 12

discs were placed onto a single 150 mm plate or a maximum of 8 discs onto a 90 mm

plate using modified Kirby-Bauer method. The plates were then incubated for 16 to

18 hours in ambient air at 35oC. E. coli ATCC 25922 was used as control. Zone of

inhibitions in millimeters were measured, recorded and the isolates were classified as

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41

“resistant”, “intermediate”, “sensitive” according to clinical laboratory standard

institutes criteria (CLSI, 2006).

ESBL INVESTIGATIONS

Any organism showing reduced susceptibility to cefotaxime, ceftazidime or

cefpodoxime was further investigated for ESBL production. ESBL production of

clinical isolates were investigated by Disc agar Diffusion method using cefotaxime,

ceftazidime, ceftriaxone, aztreonam, and/or cefpodoxime in close proximity of 20 to

30 mm center to center from amoxicillin/clavulanic acid disc. Extended-spectrum β-

lactamase production was confirmed by performing confirmatory test with

ceftazidime and cefotaxime discs alone and in combination with clavulanic acid

(CLSI, 2006). Augmentation of the zone of inhibition by ≥5 mm is considered a

positive test result.

AMPC β-LACTAMASE DETECTION

Strains resistant or intermediate to cefoxitin (zone diameter less than 18 mm) and

resistant to amoxicillin/clavulanic acid were suspected to be AmpC -lactamase

producers. These were further investigated for AmpC enzyme production.

Ceftazidime-Imipenem Antagonism Test (CIAT)

To detect and confirm the presence of inducible AmpC - lactamases among Gram

negative isolates, the ceftazidime-imipenem antagonism test was performed, which

consisted of a imipenem disc (10 µg) placed 20 mm apart (edge-to-edge) from a

ceftazidime disc (30 µg) on a Mueller-Hinton agar plate, previously inoculated with a

0.5 McFarland equivalent bacteria suspended in saline, and incubated for 24 hrs at

35°C±2. For comparison, a cefoxitin disc was also placed 20 mm apart from the

ceftazidime disc. Antagonism, indicated by a visible reduction in the inhibition zone

around the ceftazidime disc adjacent to the imipenem or cefoxitin discs, was regarded

as positive for inducible AmpC -lactamase production (Cantarelli et al, 2007).

AmpC disk test

Tris-EDTA is used to permeabilize a bacterial cell wall, releasing β-lactamases into

the exterior. The test works on this principle. AmpC discs were made in the laboratory

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42

by applying 20 µl of a 1:1 mixture of saline and 100×Tris-EDTA (Merck, Germany).

The discs were allowed to dry and stored at 4°C. Lawns of E. coli ATCC 25922 were

prepared by inoculation on Mueller-Hinton agar plates. AmpC discs were rehydrated

by putting 20 µl of saline prior to use and some colonies of every test organism were

inoculated onto disc. This AmpC disc was dispensed onto the inoculated surface of

the MH agar plate surface with the inoculated disc face in contact with the agar. A 30

µg cefoxitin disc was also placed more or less touching the AmpC disc. The plate was

then incubated for 18 to 24 hours at 35°C. Plates were examined for either flattening

or indentation of the zone of inhibition, indicative of enzymatic inactivation of

cefoxitin (positive result), or the lacking the above characteristics, representing no

inactivation of cefoxitin (negative result) (Black et al., 2005; Singhal et al., 2005;

Bhattacharjee et al., 2008).

Three-dimensional extract test

Three-dimensional extract method was used to test the isolates for AmpC β-lactamase

activity. About 50 μl bacterial suspension of a 0.5 McFarland was prepared from an

overnight culture and inoculated into 12 ml of tryptic soy broth. The culture was

incubated for 4 h at 35°C. The cells were harvested by centrifugation, and crude

enzyme extract was made by sonicating the pellets at 8 μm (in Soniprep, UK) for 15

sec (two cycles) with 10 sec cooling in between sonications; this was repeated four

times. Lawns of E. coli strains (ATCC 25922) were prepared on Mueller-Hinton agar

plate by inoculation according to the standard disc diffusion method; a 30 µg cefoxitin

disc was placed in the centre of inoculated agar. A sharp cut with a sterile scalpel

blade in the agar was made in outward radial direction starting 5 mm from the edge of

the disc. Beginning close to the disk and then moving towards outer direction, about

25 to 30 μl of crude enzyme was put into the slit using a micropipette. Slit overfill

was avoided. Plates were incubated for 18 to 24 hours at 35°C. Enhanced growth of

the E. coli ATCC 25922 at the point of intersection of the slit and the zone of

inhibition was considered as positive three-dimensional test result and was interpreted

as confirmation for the production of AmpC β-lactamase (Coudron et al., 2000; Arora

and Bal, 2005).

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43

Cefoxitin agar medium assay

For the CAM assay, 50 μl bacterial suspension of a 0.5 McFarland was prepared from

one night old culture and inoculated into 12 millilitres of sterile tryptic soy broth. The

inoculum was incubated for 4 h at 35°C. These cells were then harvested by

centrifugation, and crude enzyme extract was made by sonicating the pellets at 8 μm

(in Soniprep, UK) for 15 sec (two cycles) with 10 sec cooling in between sonications;

this was repeated four times. Mueller-Hinton agar plates containing cefoxitin (4

µg/ml) were used. Entire surface of the MH agar plates were inoculated with E. coli

ATCC 25922. Wells of 5 mm diameters were made in the agar with the help of borer

aseptically, and filled with 30 µl of crude enzyme extract from individual strains.

Inoculated agar plates were incubated for 18 to 24 hours at 35°C. Growth of E. coli

ATCC 25922 around the periphery of a well was confirmation for the production of

an AmpC β-lactamase and was reported as positive CAM assay (Nasim et al., 2004).

Inhibitor-Based Method

Discs having boronic acid were made by following procedure: A solution of 120

milligrams of phenylboronic acid was prepared by dissolving it in 3 millilitres of

dimethyl sulfoxide. To this solution, 3 ml of sterile distilled water was added. Twenty

microliters of the stock solution was dispensed onto discs containing 30 µg of

cefoxitin. Discs were allowed to dry for 30 min and used immediately or stored in

airtight vials with desiccant at 4 and at −70°C. The boronic acid disc test was

performed by inoculating Mueller-Hinton agar by the standard disc diffusion method

and placing a disc containing 30 µg of cefoxitin and a disc containing 30 µg of

cefoxitin and 400 µg of boronic acid onto the agar. Inoculated plates were incubated

overnight at 35°C. An organism that demonstrated a zone diameter around the disc

containing cefoxitin and boronic acid that was 5 mm or greater than the zone diameter

around the disc containing cefoxitin was considered an AmpC producer (Coudron,

2005).

Minimum Inhibitory Concentration (MIC)

Agar dilution method was used to determine the MICs of ceftazidime, cefoxitin,

cefotaxime cefepime and imipenem. Standard powders of antibiotics were used to

make stock solutions. Stock solutions were prepared by using the formula:

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44

1000/P x V x C = W

Where P= potency of the antibiotic given by the manufacturer (µg/mg), V= required

volume (ml), C= final concentration of the solution (multiples of 1000) (mg/l), and

W= weight of antimicrobial agent in mg to be dissolved in volume V (ml).

Stock solutions of antibiotic were prepared by adding known quantity of antibiotic

powder in respective sterile diluents. Ceftazidime pentahydrate (GlaxoSmithKline)

was dissolved in saturated NaHCO3 solution; cefotaxime sodium (Aventis Pharma) in

water, cefoxitin sodium (Merck Sharpe & Dohme Ltd.) in water; cefepime

dihydrochloride (Bristol Myers Squibb) in water; and imipenem monohydrate (Merck

Sharpe & Dohme Ltd.) was dissolved in 1 M MOPS. These antibiotic stock solutions

were used to make antibiotic dilution range according to antibiotic. According to the

labeled potencies/concentrations, stock solutions of different concentrations of

antibiotics were prepared. Stock solutions were freshly used (Andrews, 2006).

The volume of stock solution that was added to flasks was calculated by formula:

C1V1=C2V2

C1= Concentration of stock solution

V1= Volume of stock solution

C2= Required concentration of antibiotics

V2= Volume of media to be made

Antibiotic dilution range of 0.25, 0.5, 1.0, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024

μg/ml, was prepared in flasks according to the antibiotic breakpoints for that

particular species. No antibiotic was added to one flask which was antibiotic free

growth control.

Preparation of agar dilution plates

About 20 ml of cooled molten agar (medium was cooled to 50°C before adding to the

antibiotics) was added to each flask, including the antibiotic free control. Mixed well

and poured into the 90 mm Petri dish. Allowed agar to set and used immediately.

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45

Preparation of inoculum

Four colonies of the bacterial isolate were transferred to nutrient broth. Broth was

then placed in incubator shaker at 35-36oC until it was visibly turbid and was same as

or greater than the 0.5 McFarland standard. It was used within 30 minutes of

preparation.

Inoculation

Multipoint inoculator was used to deliver 1-2 μl of suspension on to the surface of the

agar. The plates were then incubated at 35°C in ambient air.

MIC determination

After incubation it was ensured that all of the organisms have grown on the antibiotic-

free control plate. Determined the MIC of each antibiotic as the MIC is the lowest

concentration of antibiotic at which there is no visible growth of organism.

PRESERVATION OF ISOLATES

To prepare an isolate for preservation at -70°C, each isolate was grown overnight (18-

20 hr) in trypticase soy broth. Equal volume of the suspension and 50% v/v glycerol

were mixed vigorously by continuous pipeting. One ml of the mixture was transferred

to storage vials, which were stored at -70°C. Two copies of each ESBL producing E.

coli and Klebsiella pneumoniae were kept for future analyses.

MOLECULAR CHARACTERIZATION

DNA Extraction

Genomic DNA was extracted by simple boiling method with some modification.

Bacterial strains were grown overnight in trypticase soy broth. Samples were washed

twice at 13,000 rpm for 5 min (Eppendorf, Centrifuge 5424, Germany) with normal

saline. The pellet was then suspended in distilled water and boiled at 95°C for 10 min

(Labnet, D 1200, US), followed by centrifugation at 13,000 rpm for 5 min. The

supernatant was transferred to another eppendorf tube containing equal amount of

70% ethanol and mixed by inversion. The mixture was centrifuged at high speed for 2

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46

minutes. The supernatant was decanted carefully and remaining ethanol was

evaporated by placing the tube at 80°C water bath for 2 minutes. Concentrated DNA

pellet was rehydrated with 100 µl of distilled water.

Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) was performed to detect ESBL genes (blaTEM,

blaSHV, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8 and blaCTX-M-9) and AmpC β-

lactamase genes with primers shown in Table 1.

PCR mixes were 50 μL: 5 μL 10 × ExTaq buffer (Mg2+

free), 1 μL extracted template

genomic DNA, 0.1 mM MgCl2, 0. 015 mM each dNTP, 50 pmol each primer, and 2.5

units of Taq polymerase (iNtRON Biotechnology, Korea).

PCR conditions

After initial denaturation at 94°C for 5 min, 35 cycles of 94°C for 30 S, 60°C

(annealing temperature of each primer) for 30 s, and initial extension at 72°C for 3

min were set with a final extension for 10 min at 72°C. Reactions were performed on

a Veriti 96-well thermal cycler (Applied Biosciences, USA).

Gel electrophoresis

PCR products and DNA samples were analyzed by gel electrophoresis using 1.5%

and 0.8% gel (Seakem, USA), respectively in TAE buffer (50 x TAE buffer: 242 g/L

Tris, 18.61 g/L NaEDTA. 2H2O, 57 ml glacial acetic acid). For all samples, 1 µl

loading dye (30% v/v glycerol and 0.25 % w/v each of bromophenol blue and xylene

cyanol FF) was used in 10% final concentration in the sample. Low mass DNA ladder

(iNtRON Biotechnology, Korea) was loaded as size marker in the first well and gel

was run at 60 V for one hour (Whatman Biometra, Germany). After 20 minutes

staining with ethidium bromide (0.5 µg/ml in TAE buffer as stock solution), gel was

de-stained with TAE buffer or dH2O for 5-10 minutes and observed for bands under

UV using UVP Biospectrum 300 imaging system (Bio-Rad, CA, US).

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47

Table 1. Oligonucleotide sequence of primers used in this study.

Target

Genes

Primer Primer sequence (5`-3`) Annealing

Temp. (°C)

Amplicon

Size

Acces.

No.

Reference

TEM Forward TCGGGGAAATGTGCG 60 971 J01749.1 Vercauteren et

al., 1997 Reverse TGCTTAATCAGTGAGGCACC

SHV Forward GCCGGGTTATTCTTATTTGTCGC 60 1007 X98100 De Gheldre et

al., 2003 Reverse ATGCCGCCGCCAGTCA

CTX-M-

1

Forward CGTCACGCTGTTGTTAGGAA 55 780 AJ63211

9.1

(Kim et al.,

2005) Reverse ACGGCTTTCTGCCTTAGGTT

CTX-M-

2

Forward TTAATGATGACTCAGAGCATTC 58 901 X92507.

1

(Kim et al.,

2005) Reverse GATACCTCGCTCCATTTATTG

CTX-M-

8

Forward CGCTTTGCCATGTGCAGCACC 58 307 AF1897

21

(Pitout et al.,

2004) Reverse GCTCAGTACGATCGAGCC

CTX-M-

9

Forward TATTGGGAGTTTGAGATGGT 52 932 AF4546

633.2

(Kim et al.,

2005) Reverse TCCTTCAACTCAGCAAAAGT

ACC Forward AACAGCCTCAGCAGCCGGTTA 57 346 AJ13312

1

(Perez-Perez

and Hanson,

2002) Reverse TTCGCCGCAATCATCCCTAGC

LAT-1 to

LAT-4,

CMY-2

to CMY-

7, BIL-1

Forward TGGCCAGAACTGACAGGCAAA 58 462 X78117 (Perez-Perez

and Hanson,

2002) Reverse TTTCTCCTGAACGTGGCTGGC

DHA-1,

DHA-2

Forward AAC TTT CAC AGG TGT GCT

GGG T

54 405 Y16410 (Perez-Perez

and Hanson,

2002) Reverse CCG TAC GCA TAC TGG CTT

TGC

MIR-1T

ACT-1

Forward TCGGTAAAGCCGATGTTGCGG 58 302 M37839 (Perez-Perez

and Hanson,

2002) Reverse CTTCCACTGCGGCTGCCAGTT

FOX-1 to

FOX-5b

Forward AACATGGGGTATCAGGGAGATG 58 190 X77455 (Perez-Perez

and Hanson,

2002) Reverse CAAAGCGCGTAACCGGATTGG

MOX-1,

MOX-2,

CMY-1,

CMY-8

to CMY-

11

Forward GCTGCTCAAGGAGCACAGGAT 60 520 D13304 (Perez-Perez

and Hanson,

2002) Reverse CACATTGACATAGGTGTGGTGC

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48

Gel purification

Bands were excised with sharp blade under UV light and PCR product was extracted

using gel purification kit (GE Healthcare, UK) as per manufacturer's instructions.

DNA was eluted with 50 µl of DNAase free water for sequencing. The nucleic acid

content thus obtained was stored at 4ºC until sequenced.

Determination of DNA concentration

DNA concentration of samples was determined using Nanodrop1000 (Thermo

Scientific, Rockford, USA) as per standard procedure. Briefly, the probe was washed

with sufficient amount of dH2O. The reading was set as blank with 2 µl of distilled

water and then wiped with Kimwipe. Two µl of each sample was used to determine

DNA concentration, wiped the probes each time for new sample.

Sequencing

For nucleotide sequencing, ESBLs and AmpC gene allele-positive isolates were

randomly selected. PCR was performed in 50- μl volumes using DNA polymerase,

LA Taq (Takara, Otsu, Japan). The reaction conditions were same as for the detection

of ESBLs and AmpC genes. The amplicons were purified using a DNA extraction kit

(Qiagen, Hilden, Germany).

DNA extraction from the Agarose Gel

DNA was extracted from the agarose gel using QIAquick Gel Extraction Kit.

DNA fragment was excised from the agarose gel with a clean, sharp scalpel. Gel slice

was weighed in a colorless tube and 3 volumes of Buffer QG were added to 1 volume

of gel (100 mg ~ 100 μl). The tubes were incubated at 50°C until the gel slice has

been completely dissolved. Tubes were vortexed every 2–3 min during the incubation

to dissolve the gel. A QIAquick spin column was placed in a 2 ml collection tube. To

bind DNA, 800 μl of the sample was added to reservoir of the QIAquick column, and

centrifuged for 1 min. For sample volumes of more than 800 μl, the samples were

loaded to the same QIAquick column and centrifuged again. Flow-through was

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49

discarded and QIAquick columns were placed back in the same collection tube. To

wash, add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1 min. Flow-

through was discarded and QIAquick column was centrifuged for an additional 1 min

at 17,900 x g (13,000 rpm) to completely remove residual ethanol. QIAquick column

was placed in a clean 1.5 ml microcentrifuge tube and 50 μl of water (pH 7.0–8.5)

was added to the center of the QIAquick membrane and centrifuged the column for 1

min. The purified DNA was analyzed using Nanodrop1000 (Thermo Scientific,

Rockford, USA).

Homology

Sequences obtained in .abi format were viewed in Finch TV program and FASTA

formats was blast in NCBI BlastN program to check the homology with NCBI

database. Results were presented in the form of table.

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50

Results

The present study was conducted on Escherichia coli and Klebsiella pneumoniae

strains isolated from Pakistan Institute of Medical Sciences, a tertiary care hospital in

Islamabad, Pakistan from August 2008 to January 2010. The frequency of ESBL and

ampC producers and the genes involved in their production were determined in a

varied sample set. Total 632 isolates were collected from clinical specimens from

different units of Pakistan Institute of Medical Sciences Islamabad. Some strains were

recovered from mix cultures. Out of 632 samples collected, the number of positive

samples for E. coli and Klebsiella pneumoniae was 593 (93.8 %). Out of these 593

isolates, 200 samples of the phenotypically confirmed ESBLs or AmpC producers, E.

coli and K. pneumoniae, were processed to detect the frequency of selected genes.

IDENTIFICATION OF SAMPLES

All the 593 samples were identified by culturing on MacConkey agar, EMB agar and

CLED agar and identified using Gram staining and conventional biochemical tests. E.

coli formed characteristic pink colonies, indicating lactose fermentation on

MacConkey agar while Klebsiella pneumoniae appeared as lactose fermenting,

mucoid and string type colonies, respectively (Fig 4.1-4.3). For confirmation, six

standard biochemical tests were performed for every isolate of E. coli and K.

pneumoniae (Table 2). API E20 identification system was used for some of the

isolates which were not clearly identified by conventional biochemical method. For

few isolates VITEK 2 Systems was used.

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51

Fig. 4.1: Pink colonies indicating lactose fermentation by E. coli and K. pneumoniae

on MacConkey agar

Fig. 4.2: E. coli colonies on EMB agar with green metallic sheen

K. pneumoniae E. coli

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52

Fig. 4.3: Klebsiella pneumoniae colonies on EMB agar

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53

Table 2: Interpretation of conventional biochemical tests for E. coli and K. pneumoniae

Isolates

MacCkonkey

agar

Indole Motility

TSI

Citrate Urease Oxidase Slope Butt Gas H2S

Escherichia

Coli

Lactose

fermenting

colonies.

+ ve + ve Y Y -ve -ve -ve - ve -ve

Klebsiella

pneumoniae

Lactose

fermenting,

mucoid string

type growth

produced

-ve -ve Y Y +

ve -ve + ve + ve -ve

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54

Detection of ESBL and AmpC

ESBL producing organisms were detected by double disc synergy test. Extension of

the zone of inhibition towards any of the cephalosporin used was confirmed as ESBL

producer (Fig 4.4).

For AmpC production, any organism resistant to cefoxitin or amoxicillin/clavulanic

acid or both was considered as suspected AmpC producer. For confirmation of the

AmpC β-lactamase, any of the ceftazidime-imipenem antagonism test, AmpC disc

test, three-dimensional extract test, cefoxitin agar medium assay or inhibitor-based

method was used. Any organism showing positive result with the AmpC test was

considered as confirmed AmpC producer. In case of negative result the other tests

were used for the confirmation.

Fig. 4.4: Double Disc Synergy Test for detecting ESBLs.

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55

Sample Distribution and Statistics

Total samples

Of the total 593 isolates identified as E. coli or K. pneumonia, 61.6% (n=365) were E.

coli and 38.4% (n=228) were K. pneumoniae (Fig. 4.5).

Fig. 4.5: Overall distribution of E. coli and K. pneumoniae in the study group.

_____________________________________________________________________

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56

Age distribution

The age of patients was categorized into six groups; up to 12 years (n=13, 2.2%), 13-

25 years (n=172, 29%), 26-35 years (n=90, 15.2%), 36-50 (n=161, 27.2%), 51-60

(n=79, 13.3%) and above 60 years (n=78, 13.2%). Mean age of the patients was 39.1

years. Seven out of 365 samples (1.9%) were confirmed to be E. coli while 2.6%

(n=6) of the total 228 K. pneumoniae in patients of age group up to 12 years. K.

pneumoniae were found to be more prevalent in patients of age group up to 12 years.

In case of age category 13-25 years, 109 samples (29.9%) were E. coli while 27.6%

(n=63) were K. pneumoniae. Among age category 26-35 years, 51 bacterial isolates

(14%) were E. coli while 17.1% (n=39) were K. pneumoniae. One hundred and six

samples (29%) in age group of 36-50 years were E. coli while 24.1% (n=55) were

found to be K. pneumoniae. In case of age category 51-60 years, 45 samples (12.3%)

were E. coli while 14.9% (n=34) were K. pneumonia, whereas, in patients aged above

60 years, 47 strains (12.9%) were E. coli while 13.6 % (n=31) were K. pneumoniae.

Common age group for sample isolation was 13-25 years for E. coli and K.

pneumoniae from which 29.9% of E. coli (n=109) and 27.6% of K. pneumoniae

(n=63) were isolated (Fig 4.6). However, none of the age group achieved statistical

significance (Table 3).

Gender distribution

A higher percentage of E. coli isolates was reported from females as compared to

males, while the ratio of K. pneumoniae was higher in male patients (p=0.012) (Table

3). Of 365 E. coli isolates, 160 (43.84%) were obtained from male patients and 205

(56.16%) from female patients. Out of 228 K. pneumoniae isolates, 124 (54.4%) were

from male patients and 104 (45.6%) from female patients (Fig 4.7).

Ward distribution

Most of the isolates were recovered from outdoor patients’ specimens followed by

medical ward and intensive care unit. The frequency of E. coli isolates recovered from

the total 365 specimens were as medical ward 20% (n=73), surgical ward, 11% (n =

40); OPD 53.7% (n = 196) and ICU 15.3% (n = 56). While K. pneumoniae recovered

_____________________________________________________________________

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57

from the total 228 specimens were as medical ward 19.7% (n=45), surgical ward,

11.4% (n = 26); OPD 53.5% (n = 122) and ICU 15.4% (n = 35) (Fig 4.8).

Sample source

Isolates were mainly recovered from pus and urine specimens followed by fluid and

blood. The frequency of E. coli isolates recovered from the total 365 specimens were;

urine 41.4% (n=151), blood, 9.9% (n = 36); pus 27.1% (n = 99); fluid, 11% (n = 40);

devices, 7.4% (n = 27) and sputum 3.3% (n=12). While K. pneumoniae recovered

from the total 228 specimens were as urine 39% (n=89), blood, 11.4% (n = 26); pus

27.6% (n = 63); fluid, 8.8% (n = 20); devices, 8.8% (n = 20) and sputum 4.4% (n=10)

(Fig 4.9).

Fig. 4.6: Overall distribution of E. coli and K. pneumoniae in different age

categories.

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58

Fig. 4.7: Gender distribution of E. coli and K. pneumoniae.

Fig. 4.8: Percentage distribution of E. coli and K. pneumoniae on the basis of

sample origin

_____________________________________________________________________

_____________________________________________________________________

59

Fig. 4.9: Percentage distribution of E. coli and K. pneumoniae on the basis of

sample source

_____________________________________________________________________

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60

Table 3: Prevalence and association of bacterial isolates with variable

Variable Value Bacterial Strains Total

(n=593)

Pearson’s Chi-square test

E. coli K.

pneumoniae

(n = 365) (n = 228) Value df

Asymp. Sig.

(2-sided)

Age distribution

(years)

Upto 12

7

53.8%

6

46.2%

13 3.483 5 0.626

13-25 109

63.4%

63

36.6%

172

26-35 51

56.7%

39

43.3%

90

36-50 106

65.8%

55

34.2%

161

51-60 45

57.0%

34

43.0%

79

Above 60 47

60.3%

31

39.7%

78

Gender distribution

Male 160

56.3%

124

43.7%

284 6.259 1 0.012

Female 205

66.3%

104

33.7%

309

Ward distribution Medical ward 73

61.9%

45

38.1%

118 0.031 3 .999

Surgical ward 40

60.6%

26

39.4%

66

OPD 196

61.6%

122

38.4%

318

ICU 56

61.5%

35

38.5%

91

Sample Source Urine 151

62.9%

89

37.1%

240 1.975 5 0.853

Blood 36

58.1%

26

41.9%

62

Pus 99

61.1%

63

38.9%

162

Fluids 40

66.7%

20

33.3%

60

Devices 27

57.4%

20

42.6%

47

Sputum 12

54.5%

10

45.5%

22

AmpC Negative 271

60.0%

181

40.0%

452

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61

ESBL Producing strains of E. coli and K. pneumoniae

ESBL production was detected in 46.20% (n = 274) isolates by phenotypic method.

Out of total 365 E. coli strains, 49.3% (n = 180) were found to be ESBL producers

against 50.7% (n = 185) non-ESBL producers. ESBL producing K. pneumoniae were

41.2% (n =94) out of total 228, while 58.8% (n=134) were non-ESBL producing K.

pneumoniae (Fig 4.2).

Fig. 4.2: Overall percentage distribution of ESBLs producer strains of E. coli and K.

pneumoniae in the study group.

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62

ESBL producing strains of E. coli

ESBL status of E. coli isolates and its association with different risk factors is given in

Table 4.

Age distribution

From age group of up to 12 years of age, 1.7% (n=3) were ESBL producers while

2.2% (n=4) were found as ESBL negative. Among age groups 13-25 and 26-35 years,

23.9% (n=43) and 13.9% (n=25) were reported to be ESBL positive, respectively.

While a higher trend was observed among the age group 36-50 years having 35.6%

(n=64) ESBL positive E. coli, and 22.7% (n=42) were detected as ESBL negative.

Among the age group 51-60 years 15.6% (n=28) and above 60 years 9.4% (n=17)

were found to be ESBL producers (Fig. 4.2.1). Statistical analysis indicated that age

groups have significant association with the presence of ESBLs (p= 0.007).

Gender distribution

Among 180 ESBL producing E. coli, 72 (40%) were found to be males and 108

(60%) were females (4.2.2). Higher proportion of ESBL producing E. coli was found

in females as compared to males. However, it was not observed to be statistically

significant (p=0.145). Details of gender distribution of patients having ESBL

producers and non- producer E. coli are shown in Table 4.

Ward distribution

Out of total 196 E. coli isolated from OPD patients, 51.1% (n = 92) were found to be

ESBL producers and 56.2 % (n=104) as non- ESBL producers. The ESBL producing

E. coli isolated from other wards were; Medical ward (23.9%, n=43), surgical ward

(10%, n=18) and ICU (15%, n=27) (p=0.327) (Fig. 4.2.3).

Sample source

The highest number of ESBL producing E. coli isolates was recovered from urine

samples. Out of total ESBL producing E. coli, the number of ESBL producers in urine

was 37.8% (n=68). The ESBL producing E. coli in other specimens were; blood

10.6% (n=19), pus 30.6% (n=55), fluids 10.6% (n=19), devices 8.3% (n=15) and

sputum 2.2% (n=4), of the total ESBL producing E. coli (p=0.477) (Fig. 4.2.4).

_____________________________________________________________________

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63

Fig. 4.2.1: Overall distribution of ESBL producing E. coli among different age

groups

Fig. 4.2.2: Gender distribution of ESBL producing E. coli.

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64

Fig. 4.2.3: Percentage distribution of ESBL producing E. coli strains on the basis

of sample origin.

Fig. 4.2.4: Percentage distribution of ESBL producing E. coli strains on the basis

of sample source.

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65

Table 4: ESBL status in E. coli and its association with different risk factors.

Variable Value ESBL Total

(n=365)

Pearson’s Chi-square test

ESBL

Positive

ESBL

Negative

(n = 180) (n =185 ) Value df Asymp. Sig.

(2-sided)

Age distribution

Up to

12 years 3

42.9%

4

57.1%

7 15.801 5 0.007

13-25 years 43

39.4%

66

60.6%

109

26-35 years 25

49.0%

26

51.0%

51

36-50 years 64

60.4%

42

39.6%

106

51-60 years 28

62.2%

17

37.8%

45

Above 60

years

17

36.2%

30

63.8%

47

Gender

distribution

Male 72

45.0%

88

55.0%

160 2.122 1 0.145

Female 108

52.7%

97

47.3%

205

Ward distribution Medical ward 43

58.9%

30

41.1%

73 3.453 3 0.327

Surgical ward 18

45.0%

22

55.0%

40

OPD 92

46.9%

104

53.1%

196

ICU 27

48.2%

29

51.8%

56

Sample Source Urine 68

45.0%

83

55.0%

151 4.522 5 0.477

Blood 19

52.8%

17

47.2%

36

Pus 55

55.6%

44

44.4%

99

Fluids 19

47.5%

21

52.5%

40

Devices 15

55.6%

12

44.4%

27

Sputum 4

33.3%

8

66.7%

12

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66

ESBL producing strains of K. pneumoniae

ESBL status of K. pneumonia isolates and its association with different risk factors is

detailed in Table 5.

Age distribution

Among 94 ESBL producing K. pneumoniae, 1.1% (n=1) ESBLs positive and 3.7%

(n=5) non-ESBL producers were from the age group upto 12 years of age. Among age

group 13-25 years, 27.7% (n=26), and age group 26-35 years, 16% (n=15) isolates

were found to be ESBL producers. Among age group 36-50 years and 51-60 years,

ESBL producing K. pneumoniae were 21.3% (n=20) and 19.1% (n=18), respectively.

Among the age group above 60 years, 14.9% (n=14) out of 94 ESBL producing K.

pneumoniae isolates were detected (4.2.5). However no significant association was

observed (p=0.5).

Gender distribution

Out of the total 94 ESBL producing K. pneumoniae, 56.4% (n=53) ESBL producers

were from the males, while number of positive isolates observed among the females

were 43.6% (n=41) (Fig. 4.2.6). Among 134 non-ESBL producing K. pneumoniae,

53% (n=73) and 47% (n=63) samples were from male and female patients,

respectively (p=0.612).

Ward distribution

Out of 94 ESBL producing K. pneumoniae, 53.2% (n=50) were isolated from OPD. In

wards, 20.2% (n = 19) ESBL producing K. pneumoniae were isolated from medical

ward, 7.4% (n=7) from surgical ward and 19.1% (n=18) were isolated from ICU (Fig.

4.2.7). In 134 non-ESBLs producing K. pneumoniae, the distribution of samples in the

wards was as: Medical ward 19.4% (n=26), Surgical ward 14.2% (19), OPD 53.7%

(72) and ICU 12.7% (17) (p=0.293).

Sample source

Among 94 total ESBL producing K. pneumoniae isolates, 29.8% (n=28) were

obtained from urine samples, 17% (n=16) from blood samples, 28.7% (n=27) from

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67

pus, 10.6% (n=10) from different body fluids, 8.5% (n=8) from medical devices and

5.3% (n=5) from the sputum (p=0.114) (Fig. 4.2.8).

Fig. 4.2.5: Overall distribution of ESBL producing K. pneumoniae among

different age groups

Fig. 4.2.6: Gender distribution of ESBL producing K. pneumoniae.

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68

Fig. 4.2.7: Percentage distribution of ESBL producing K. pneumoniae strains on

the basis of sample origin.

Fig. 4.2.8: Percentage distribution of ESBL producing K. pneumoniae strains on

the basis of sample source.

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69

Table 5: ESBL status in K. pneumonia and its association with different risk factors

Variable Value ESBL Total

(n=228)

Pearson’s Chi-square test

ESBL

Positive

ESBL

Negative

(n =94 ) (n =134 ) Value df Asymp. Sig.

(2-sided)

Age distribution

(years)

Up to

12 1

16.7%

5

83.3%

6 4.277 5 0.510

13-25 26

41.3%

37

58.7%

63

26-35 15

38.5%

24

61.5%

39

36-50 years 20

36.4%

35

63.6%

55

51-60 18

52.9%

16

47.1%

34

Above 60 14

45.2%

17

54.8%

31

Gender distribution

Male 53

42.7%

71

57.3%

124 0.257 1 0.612

Female 41

39.4%

63

60.6%

104

Ward distribution Medical

ward

19

42.2%

26

57.8%

45 3.720 3 0.293

Surgical

ward

7

26.9%

19

73.1%

26

OPD 50

41.0%

72

59.0%

122

ICU 18

51.4%

17

48.6%

35

Sample Source Urine 28

31.5%

61

68.5%

89 8.965 5 0.110

Blood 16

61.5%

10

38.5%

26

Pus 27

42.9%

36

57.1%

63

Fluids 10

50.0%

10

50.0%

20

Devices 8

40.0%

12

60.0%

20

Sputum 5

50.0%

5

50.0%

10

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70

AmpC Producing strains of E. coli and K. pneumoniae

AmpC β-lactamases were detected in 23.8% (n = 141) of total isolates when

phenotypic tests were employed for AmpC detection. AmpC β-lactamase production

was detected in 25.8 % (n = 94), among total 365 E. coli strains. AmpC β-lactamase

producing K. pneumoniae were found to be 20.6% (n =47) of total 228 K. pneumoniae

isolates, indicating a higher trend in the number of AmpC producing E. coli than that

of K. pneumoniae (Fig. 4.3).

Fig. 4.3: Overall percentage distribution of AmpC producing E. coli and K.

pneumoniae in the study group.

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71

AmpC Producing strains of E. coli

Age distribution

Out of total 94 AmpC producing E. coli, 3.2% (n=3) were isolated from the patients

of 12 years of age and 21.3% (n=20) from the age group 13-25 years. Twelve out of

94 AmpC producing E. coli (12.8%) were from the age group 26-35 years, while

27.7% (n=26) were from the age group 36-50 years. Among the age groups 51-60

years, and above 60 years AmpC producers were 14.9% (n=14) and 20.2% (n=19),

respectively (p =0.065) (Fig. 4.3.1).

Gender distribution

Among 94 AmpC positive E. coli isolates, 54.3% (n=51) were from male patients

while 45.7% (n=43) were from the female patients. In 271 AmpC negative 40.2%

(n=109) were from male patients and 59.8% (n=162) were from female patients (Fig.

4.3.2). Males were found to have a statistically significant association with AmpC

positive status in E. coli isolates (p=0.018).

Ward distribution

Isolation from surgical ward had a statically significant association with AmpC

positive status of E. coli (p=0.01) (Table 6). About 17% (n=16) AmpC producing E.

coli were recovered from medical ward, 19.1% (n=18) from surgical ward, 44.7%

(n=42) from OPD and 19.1% (n=18) were isolated from ICU. In case of AmpC

negative E. coli, 21% (n=57) were from the medical ward, 8.1% (n=22) from surgical

ward, 56.8% (n=154) from OPD and 14% (n=38) of the total 271 AmpC negative E.

coli were isolated from ICU (Fig. 4.3.3).

Sample source

A higher percentage (36.2%, n=34) of AmpC producing E. coli were recovered from

the urine specimens having positive isolates. In case of the other specimen 7.4%

(n=7) were from blood, 28.7% (n=27) from pus, 13.8% (n=13) from fluids, 9.6%

(n=9) from the devices and 4.3% (n=4) were isolated from blood (Fig. 4.3.4). Out of

271, AmpC negative E. coli were; urine 43.2% (n=117), blood 10.7% (n=29), pus

26.6% (n=72), fluids 10% (n=27), medical devices 6.6% (n=18), and 3% (n=8) were

isolated from sputum (p=0.57).

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72

Fig. 4.3.1: Overall distribution of AmpC producing E. coli among different age

groups

Fig. 4.3.2: Gender distribution of AmpC producing E. coli.

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73

Fig. 4.3.3: Percentage distribution of AmpC producing E. coli strains on the basis

of sample origin.

Fig. 4.3.4: Percentage distribution of AmpC producing E. coli strains on the basis

of sample source.

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74

Table 6: AmpC status of E. coli isolates and its association with different risk factors.

Variable Value AmpC Status Total

(n=365)

Pearson Chi-square

AmpC

Positive

AmpC

Negative

(n =94) (n =271 ) Value df Asymp. Sig.

(2-sided)

Age distribution

(years)

Up to

12 3

42.9%

4

57.1%

7 10.379 5 0.065

13-25 20

18.3%

89

81.7%

109

26-35 12

23.5%

39

76.5%

51

36-50 26

24.5%

80

75.5%

106

51-60 14

31.1%

31

68.9%

45

Above 60 19

40.4%

28

59.6%

47

Gender distribution

Male 51

31.9%

109

68.1%

160 5.583 1 0.018

Female 43

21.0%

162

79.0%

205

Ward distribution Medical

ward

16

21.9%

57

78.1%

73 11.424 3 0.010

Surgical

ward

18

45.0%

22

55.0%

40

OPD 42

21.4%

154

78.6%

196

ICU 18

32.1%

38

67.9%

56

Sample Source Urine 34

22.5%

117

77.5%

151 3.820 5 0.576

Blood 7

19.4%

29

80.6%

36

Pus 27

27.3%

72

72.7%

99

Fluids 13

32.5%

27

67.5%

40

Devices 9

33.3%

18

66.7%

27

Sputum 4

33.3%

8

66.7%

12

_____________________________________________________________________

_____________________________________________________________________

75

AmpC Producing strains of K. pneumoniae

Age distribution

Among the total 47 AmpC producing and 181 non-AmpC producing K. pneumoniae,

no AmpC positive isolate was detected in the age group up to 12 years of age and

3.3% (n=6) were AmpC negative. Among age group 13-25 years of age, 29.8%

(n=14) were AmpC positive, while 27.1% (n=49) were AmpC negative. Nine isolates

(19.1%) were found as AmpC producers in the age group 26-35 years, 23.4% (n=11)

in the age group 36-50 years, 12.8% (n=6) and 14.9% (n=7) were detected in age

group above 60 years of age (Fig. 4.3.5).

Gender distribution

Prevalence of AmpC production was relatively higher in males as compared to

females with no significance (p=0.88). Out of total 47 AmpC positive K. pneumoniae

55.3% (n=26) were from male patients while 44.7% (n=21) were from the female

patients (Fig. 4.3.6).

Ward distribution

Like AmpC producing E. coli, isolation from surgical ward had a statistically

significant association with AmpC positive K. pneumoniae (p=0.001) (Table 7).

AmpC producing K. pneumoniae from the medical ward were 19.9% (n=9), 27.7%

(n=13) were from the surgical ward, 36.2% (n=17) were from OPD and 17% (n=8)

were from the ICU (Fig. 4.3.7).

Sample source

Among the total 47 K. pneumoniae showing positive AmpC test, 44.7% (n=21) were

from the urine specimen, 4.3% (n=2) from the blood, 25.5% (n=12) from pus, 12.8%

(n=6) from fluids, 6.4% (n=3) from devices and 6.4% (n=3) were from sputum

(p=0.388) (Fig. 4.3.8).

_____________________________________________________________________

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76

Fig. 4.3.5: Overall distribution of AmpC producing K. pneumoniae among

different age groups

Fig. 4.3.6: Gender distribution of ESBL producing K. pneumoniae.

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77

Fig. 4.3.7: Percentage distribution of AmpC producing K. pneumoniae strains on

the basis of sample origin.

Fig. 4.3.8: Percentage distribution of AmpC producing K. pneumoniae strains on

the basis of sample source.

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78

Table 7: AmpC status of K. pneumonia and its association with different risk factors.

Variable Value AmpC Status Total

(n=228)

Pearson’s Chi-square test

AmpC

Positive

AmpC

Negative

(n = 47) (n =181 ) Value df Asymp. Sig.

(2-sided)

Age distribution

Up to

12 years 0

0%

6

100.0%

6 2.071 5 0.839

13-25 years 14

22.2%

49

77.8%

63

26-35 years 9

23.1%

30

76.9%

39

36-50 years 11

20.0%

44

80.0%

55

51-60 years 6

17.6%

28

82.4%

34

Above 60

years

7

22.6%

24

77.4%

31

Gender

distribution

Male 26

21.0%

98

79.0%

124 .021 1 0.885

Female 21

20.2%

83

79.8%

104

Ward distribution Medical

ward

9

20.0%

36

80.0%

45 17.164 3 0.001

Surgical

ward

13

50.0%

13

50.0%

26

OPD 17

13.9%

105

86.1%

122

ICU 8

22.9%

27

77.1%

35

Sample Source Urine 21

23.6%

68

76.4%

89 5.231 5 0.388

Blood 2

7.7%

24

92.3%

26

Pus 12

19.0%

51

81.0%

63

Fluids 6

30.0%

14

70.0%

20

Devices 3

15.0%

17

85.0%

20

Sputum 3

30.0%

7

70.0%

10

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79

Antibiotic Susceptibility

Disc diffusion test

E. coli

Out of 131 E. coli isolates randomly selected for further study, 75 E. coli (57.3%)

were isolated from outpatients, and 23 (17.5%) from medical ward, 11 (8.4%) from

surgical ward and 22 (16.8%) were from ICU patients. About 62 (47.3%) of the

isolates were from urine, 38 (29%) were from pus, 18 (13.7%) from fluids, and 6

(4.6%) from blood; 4 isolates (3.1%) were from medical devices and 3 (2.3%) from

sputum. All 131 (100%) isolates were sensitive to imipenem and tigecycline, making

it the most effective antibiotic in this study; cefoxitin was the second most effective

antibiotic (63.4%). The maximum resistance (90.1%) was observed against

sulphamethaxazole, followed by tetracycline (88.5%). In case of β-lactam antibiotics,

high resistance (87.8%) was observed to cefotaxime and amoxicillin/clavulanic acid,

followed by cefepime (81.7%) and aztreonam (79.4%). Forty four (33.6%) were

found resistant to amikacin, while 106 isolates (80.9%) showed resistance to

ciprofloxacin (Fig. 4.5.1, Fig. 4.5.3).

K. pneumoniae

All 69 (100%) K. pneumoniae isolates were found susceptible to tigecycline making it

the most effective antibiotic, followed by imipenem (98.6%). Higher resistance was

observed in case of tetracycline (98.6%), amoxicillin/clavulanic acid (97.1%) and

sulphamethaxazole (95.7%). In case of β-lactam antibiotics, cefoxitin was the most

successful antibiotic showing resistance to 20 (29%) isolates, followed by ceftazidime

and cefepime (69.6%) and aztreonam (75.4%). Fifty six isolates (81.2%) were found

resistant to cefotaxime and 50 (72.5%) resistant to ciprofloxacin (Fig. 4.5.2).

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80

Fig. 4.5.1:Antimicrobial resistance patterns of E. coli isolates

Fig 4.5.2: Antimicrobial resistance patterns of K. pneumoniae isolates

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81

MIC test

MIC was determined by broth microdilution according to the break points mentioned

in CLSI guidelines (Table 8).

Table 8: CLSI breakpoints for MIC of antibiotics used in present study

Antibiotics MIC (μg/ml)

Susceptible Intermediate Resistant

Cefoxitin <8 16 >32

Ceftazidime <8 16 >32

Cefotaxime <8 16-32 >64

AMC <8/4 16/8 >32/16

E. coli

Out of the total 131 E. coli isolates, 100 (76.3%) were found resistant to ceftazidime

having an MIC >32μg/ml. Highest resistance was observed in case of

amoxicillin/clavulanic acid, in which 117 isolates (89.3%) were resistant, followed by

cefotaxime (116, 89.3%). About 45 (34.3%) isolates of E. coli showed resistance to

cefoxitin with a maximum range of 256 μg/ml (Fig. 4.5.4).

K. pneumoniae

Fifty isolates (72.5%) were found resistant to ceftazidime with a maximum range of

512 μg/ml, while 19 isolates (27.5%) were susceptible. Fifty six (81%) isolates were

resistant to cefotaxime and 61 (88.4%) were resistant to amoxicillin/clavulanic acid.

Cefoxitin was the most successful antibiotic, effective against 47 (68.1%) of the total

69 K. pneumoniae isolates tested (4.5.5).

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82

Fig 4.5.4 MIC of ESBL producing E. coli

Fig. 4.5.5MIC of ESBL producing K. pneumoniae

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83

Molecular Characterization

E. coli

All 131 E. coli ESBL producers were examined for the presence of specific ESBL

gene groups; TEM, SHV and CTX-M. AmpC detection was done in the same samples

with primers specific for CIT, MOX, FOX, CMY, DHA and EBC genes. A total of

175 gene amplifications were obtained. PCR amplification revealed that CTX-M-1

was the most frequently (77 isolates, 58.7%) detected ESBL gene group, followed by

TEM (25 isolates, 19%) and SHV (19 isolates, 14.5%). CTX-M-9 group was observed

in only 4 bacterial isolates (Fig. 4.6.1-5). Among AmpC β-lactamases, MOX gene

was detected in 19 (14.5%) E. coli isolates, CIT in 17 (13%), CMY gene in 7 (5%),

EBC gene in 5 (4%), and 2 isolates showed FOX AmpC β-lactamases (Table 9).

Among AmpC producing E. coli strains, MOX and CIT were the most prevalent gene

types. Among ESBL specific gene groups, CTX-M-1 group was detected most

frequently in E. coli isolated from urine, followed by those isolated from pus (Table

10).

Of the 77 isolates in which CTX-M-1 group were detected, included, 36 (47%) from

male patients and 41 (53%) from female patients. Forty three (56%) were obtained

from OPD and 34 (44%) were from hospitalized patients. Out of total 34 isolates from

hospitalized patients, 15 were recovered from medical ward, 13 from ICU and 6 from

surgical ward (Table 10). Five blood isolates showed amplification of CTX-M-1

group; 4 alone, and one in combination with MOX AmpC β-lactamase. No DHA

AmpC β-lactamase was detected in our study. Nineteen E. coli isolates did not show

amplification with any of the primers.

K. pneumoniae

Sixty nine K. pneumoniae isolates were analyzed by PCR amplification for the

presence of selected genes responsible for ESBL and AmpC β-lactamase production.

CTX-M-1 type ESBLs were detected in 43 (62.3%) isolates, SHV in 9 (13%), TEM

in 8 (11.6%) and CTX-M-9 in 2 isolates (3%). Six (9%) isolates showed CIT type

AmpC genes while 4 (6%) had CMY, 3 (4%) each FOX and MOX, and 2 isolates

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84

(3%) had EBC type genes. Eighty genes showed amplification in 69 K. pneumoniae

isolates.

Out of 43 CTX-M-1 isolates, 16 (37%) were from the urine samples, 9 (21%) from

blood, 8 (19%) from pus, 4 (9%) from fluids and 3 (7%) each from medical devices

and sputum (Table 12). Twenty one (49%) of the 43 CTX-M-1 type genes were

detected in the samples from outpatients (Table 13). Details of the different genes on

the basis of age groups and gender are given in Table 13. No genes were detected in

11 K. pneumoniae isolates.

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85

Fig. 4.6.1. Agarose gel with showing PCR fragments for TEM gene of E. coli Lane

M: 1kb DNA ladder; Lanes 1-10: Strains positive for TEM, Lane 11: Negative control

Fig. 4.6.2. Agarose gel with PCR fragments for CTX-M1 gene of E. coli, Lane M:

1kb DNA ladder; Lanes 1-10: strains positive for CTX-M1, Lane 11: Negative

control.

M 1 2 3 4 5 6 7 8 9 10 11

M 1 2 3 4 5 6 7 8 9 10 11

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86

Fig 4.6.3 Agarose gel with PCR fragments for CMY gene of E. coli Lane M: 1kb

DNA ladder; Lanes 2, 4, 6-8 and 10: Strains positive for CMY, Lane 11: Negative

control

Fig. 4.6.4. Agarose gel with PCR fragments for SHV gene of E. coli, Lane M: 1 kb

DNA ladder; Lanes 1-10: strains positive for SHV, Lane 11: Negative control

M 1 2 3 4 5 6 7 8 9 10 11

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87

Fig. 4.6.5. Agarose gel with PCR fragments for SHV gene of K. pneumoniae, Lane

M: 1 kb DNA ladder; Lanes 1-6 and 8-10,: strains positive for SHV, Lane 11:

Negative control

M 1 2 3 4 5 6 7 8 9 10 11

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88

Table 9: Distribution of ESBL and AmpC β-lactamases in E. coli based on sample sources and hospitalization

ESBL and

AmpC

β-lactamase

group

Source of sample

Urine Pus Fluids Blood Devices Sputum Total

TEM 9 7 4 - 4 1 25

SHV 8 4 5 - 1 1 19

CTX-M-1 33 23 13 5 2 1 77

CTX-M-9 3 - - - 1 - 4

CMY 4 3 - - - - 7

CIT 8 4 3 1 1 17

DHA - - - - - - -

EBC 3 2 - - - - 5

FOX 1 1 - - - - 2

MOX 9 3 4 1 - 2 19

TOTAL 78 47 29 6 9 6 175

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89

Table 10: Distribution of ESBL and AmpC β-lactamases in E. coli based on hospitalization

ESBL and

AmpC

β-lactamase

group

Sample Origin

Medical

Ward

Surgical

Ward

OPD ICU Total

TEM 4 2 14 5 25

SHV 3 2 13 1 19

CTX-M-1 15 6 43 13 77

CTX-M-9 - 1 3 - 4

CMY - 1 4 2 7

CIT 5 3 5 4 17

DHA - - - - -

EBC 2 1 2 - 5

FOX 1 1 - - 2

MOX 6 3 7 3 19

TOTAL 36 20 91 28 175

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90

Table 11: Distribution of ESBL and AmpC β-lactamases in E. coli based on age groups and gender

ESBL and

AmpC

β-lactamase

group

Age groups Gender

Up to

12 years

13-25

years

26-35

years

36-50

years

51-60

years

Above

60 years

Total Male Female Total

TEM 0 8 2 7 3 5 25 11 14 25

SHV 0 9 3 5 0 2 19 9 10 19

CTX-M-1 1 32 10 20 6 8 77 36 41 77

CTX-M-9 0 0 1 0 2 1 4 2 2 4

CMY 1 2 0 2 2 0 7 4 3 7

CIT 1 3 4 6 2 1 17 10 7 17

DHA - - - - - - - - - -

EBC 0 1 2 1 1 0 5 1 4 5

FOX 0 0 1 1 0 0 2 - 2 2

MOX 1 6 3 4 3 2 19 9 10 19

TOTAL 4 61 26 46 19 19 175 82 93 175

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91

Table 12: Distribution of ESBL and AmpC β-lactamases in K. pneumoniae based on sample sources and hospitalization

ESBL and

AmpC

β-lactamase

group

Source of sample

Urine Pus Fluids Blood Devices Sputum Total

TEM 4 1 2 1 - - 8

SHV 2 4 - 1 2 - 9

CTX-M-1 16 8 4 9 3 3 43

CTX-M-9 - - 1 1 - - 2

CMY - 1 1 - 1 1 4

CIT 3 - - - 1 2 6

DHA - - - - - - -

EBC - - - - - 2 2

FOX 2 - - - 1 - 3

MOX 3 - - - - - 3

TOTAL 30 14 8 12 8 8 80

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92

Table 13: Distribution of ESBL and AmpC β-lactamases in K. pneumoniae based on hospitalization

ESBL and

AmpC

β-lactamase

group

Sample Origin

Medical

Ward

Surgical

Ward

OPD ICU Total

TEM - 1 4 3 8

SHV 2 1 5 1 9

CTX-M-1 8 5 21 9 43

CTX-M-9 - - - 2 2

CMY 1 2 1 - 4

CIT 1 1 4 - 6

DHA - - - - -

EBC 1 1 - - 2

FOX 2 - - 1 3

MOX - - 3 - 3

TOTAL 15 11 38 16 80

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93

Table 14: Distribution of ESBL and AmpC β-lactamases in K. pneumoniae based on age groups and gender

ESBL and

AmpC

β-lactamase

group

Age groups Gender

Up to

12 years

13-25

years

26-35

years

36-50

years

51-60

years

Above 60

years

Total Male Female Total

TEM - 1 - - 1 6 8 4 4 8

SHV - 1 3 - 1 4 9 5 4 9

CTX-M-1 - 14 7 12 4 6 43 23 20 43

CTX-M-9 - 1 - 1 - - 2 2 - 2

CMY - 2 - 1 - 1 4 3 1 4

CIT - 3 - 2 - 1 6 1 5 6

DHA - - - - - - - - - -

EBC - 1 - - - 1 2 - 2 2

FOX - 1 - 2 - - 3 2 1 3

MOX - - 1 1 - 1 3 1 2 3

TOTAL - 24 11 19 6 20 80 41 39 80

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94

Gene Combinations

E. coli

Both Extended- spectrum and AmpC β-lactamase genes were observed to be present

alone and also in combinations. A total of 26 different patterns of genes were detected

in 112 E. coli isolates, while no candidate gene was found in 19 E. coli isolates. Most

of the genes were found in combination with CTX-M1. TEM was detected in 10 E.

coli isolates in combination with CTX-M1, while SHV was found in seven E. coli

isolates along with CTX-M1. Details of the gene combination patterns are given in the

Table 15.

K. pneumoniae

A total of 18 different patterns of genes were detected in K. pneumoniae in a total of

58 isolates, while in 11 isolates no gene was detected. CTX-M1 was the most

prevalent gene in combination as well as alone. TEM was detected in 6 isolates

having CTX-M1 gene. Three K. pneumoniae isolates were found to have 3 genes

each, while in single K. pneumoniae isolate, 4 genes were detected (Table 16).

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95

Table 15: Different gene combinations in E. coli

Gene combinations Number of Isolates

CIT 03

CIT, MOX 01

CIT, CMY, MOX 01

CIT, TEM 01

CMY, MOX 02

CTX-M-1 42

CTX-M-1, CIT, EBC 01

CTX-M-1, CIT, MOX 05

CTX-M-1, CMY, MOX 01

CTX-M-1, FOX, EBC 01

CTX-M-1, MOX 04

CTX-M-1,CIT 02

CTX-M-1, CIT, TEM 01

CTX-M-1, CMY 01

CTX-M-1, SHV 07

CTX-M-1, TEM 10

CTX-M-1, TEM, CIT, MOX 01

CTX-M-1, TEM, CMY, MOX 01

CTX-M-9 03

CTX-M-9, TEM 01

EBC, CIT, MOX 01

MOX 02

SHV 10

SHV, TEM 02

TEM 07

TEM, FOX, CMY, EBC 01

No Detection 19

Total 131

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96

Table 16: Different gene combinations in K. pneumoniae

Gene combinations Number of Isolates

CIT 02

CMY 01

CMY,SHV 01

CTX-M-1 26

CTX-M-1, CIT 01

CTX-M-1, CIT, CMY, EBC 01

CTX-M-1, CIT, EBC 01

CTX-M-1, CIT, MOX 01

CTX-M-1, CMY 01

CTX-M-1, CTX-M-9 01

CTX-M-1, FOX, SHV 01

CTX-M-1, MOX 02

CTX-M-1, SHV 02

CTX-M-1, TEM 06

CTX-M-9 01

FOX 02

SHV 06

TEM 02

No Detection 11

Total 69

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97

Sequence Analysis

Genes from different isolates having TEM, SHV, CTX-M-1, CTX-M-9 and CIT were

sequenced. Sequences thus obtained were subjected to BLAST using NCBI BLASTn

program. The sequences were aligned using mafft v6.903b alignment software

(http://mafft.cbrc.jp/alignment/software). On the basis of homologous sequences,

phylogenetic tree was constructed using MEGA 4.0.2 program.

TEM

TEM from three isolates, one from E. coli (E. coli MS 50) and two from K.

pneumoniae (K. pneumoniae MS 88 and K. pneumoniae MS 130) were subjected to

BLASTn. The published sequences of TEM were aligned using mafft v6.903b

alignment software. On the basis of homologous sequences, phylogenetic tree was

constructed using MEGA 4.0.2 program. All the amplified sequences were found to

be homologous with TEM-1 ESBL gene reported from different parts of the world

(Fig. 4.7.1).

SHV

Among SHV isolates, the sequence from a single K. pneumoniae (K. pneumoniae MS

81) was subjected to BLASTn and aligned with the already published SHV genes. We

observed 100% homology with a novel gene from SHV family of ESBL genes; SHV-

49, an inhibitor resistant ESBL (Fig. 4.7.2).

CTX-M-1

Five isolates showing positive PCR results for CTX-M-1 were sequenced. BLASTn

data and phylogenetic tree from the MEGA4 showed that the CTX-M-1 sequences

from one E. coli and 4 K. pneumoniae (E. coli MS 26, K. pneumoniae MS 81, K.

pneumoniae MS 83, K. pneumoniae MS 10, and K. pneumoniae MS 41) were 100%

homologous to already published CTX-M-15 (Fig. 4.7.3).

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98

CTX-M-9

Sequencing results from a single E. coli isolate (E. coli MS 37) for CTX-M-9 were

analyzed. The BLASTn data showed that the sequence was homologous to CTX-M-

27 and CTX-M-14 ESBLs (Fig. 4.7.4).

CIT

CIT genes were similar to CMY-2 and CMY-32 genes on the basis of BLASTn

results with 2 nucleotide difference with Adenine replacing Guanine at position 609

and Guanine replacing Adenine at position 609 (Appendix). The nucleotide sequences

were translated to amino acid sequences by using an online program Just-Bio

translator (http://www.justbio.com/index.php?page=translator). The amino acid

sequences thus obtained were subjected to BLASTp

(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). The sequences were then

aligned with the nearest hits using ClustalW program

(http://www.genome.jp/tools/clustalw/). These two nucleotides confer change in the

two amino acids (Fig. 4.7.5-6).

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99

K. pneumoniae MS88-TEM-PAK

K. pneumoniae MS130-TEM-PAK

E. coli MS50-TEM-PAK

E. coli-73B-Ecuador(EU352898)

E. coli-pCY1-Ch(JQ941741)

Leclercia adecarboxylata-S86b-Ch(JF91...

Mixed culture-USA(GQ343049)

E.coli-15B-Ecuador(EU352902)

Enterobacter cloacae-pEC2-France(HQ66...

Salmonella enteric-Mala(HQ625489)

Enterobacter sp-O050230-Ind(EU979562)

K. pneumoniae-O050110-Ind(EU979561)

E. coli-O050222-Ind(EU979560)

E. coli-0157-Jpn(AB201242)

E. coli-Jpn(AB194682)

E. coli-13-5-Ch(HQ174461)

E. coli-8-5-Ch(HQ174460)

Salmonella enteric-pST3553-Jpn(AB571794)

Proteus mirabilis-H. Kong(AY874538)

S. marcescens-ES-71-Jpn(AY538702)

H.parainfluenzae-Aus(AM849806)

Mixed culture-USA(GQ343053)

Mixed culture-USA(GQ343159)

Mixed culture-USA(GQ343004)

Mixed culture-USA(GQ343054)

Mixed culture-USA(GQ343047)

Mixed culture-USA(GQ343006)

Mixed culture-USA(GQ343050)

Fig. 4.7.1. Phylogenetic Tree of the TEM showing genetic relationships with the

reported genes

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100

K. pneumoniae P050111 SHV-5-IND(EU441...

K. pneumoniae KPLA-1-SHV-5a-SWT(X98105)

E. coli SP060035 SHV-5-IND(EU441171)

K. pneumoniae KPGE-2-SHV-5-SWT(X98103)

A. baumannii SHV-5-USA(EF653399)

K. pneumoniae P060388 SHV-12-IND(FJ44...

C. freundii SHV-12-SWT(AY940490)

P. aeruginosa SHV-2a-France(AM988779)

P. aeruginosa plasmid shv-2a-France(A...

K.pneumoniae KPAA-1 SHV-1-SWT(X98098)

K. pneumoniae KPZU-12-SHV-1a-SWT(X98101)

K. pneumoniae SHV-26-Taiwan(AF227204)

K. pneumoniae KPZU-13-SHV-1-SWT(X98099)

S. dysenteriae bla gene-IND(Y18299)

K. pneumoniae SHV-1-USA (AF124984)

K. pneumoniae blaSHV-49-France(AY528718)

K. ozaenae SHV-2 (X53433)

K. pneumoniae-SHV-USA(X62115)

K. pneumoniae-SHV-33-Spain(AY037779)

K. pneumoniae U070018 SHV-28-IND(EU44...

K. pneumoniae MS81-SHV-PAK

K pneumoniae SHV-11-France (AY528717)

K. pneumoniae ARS9 SHV-121-GER(GQ428198)

K. pneumoniae SHV-32-Spain(AY037778)

K. pneumoniae SHV-105-USA(FJ194944)

K. pneumoniae P050111-SHV-38-IND(EU97...

K. pneumoniae SHV-38-France(AY079099)

K. pneumoniae SHV-63-Rus(EU342351)

K. pneumoniae SHV-USA (FJ494816)

Fig. 4.7.2. Phylogenetic Tree of the SHV showing genetic relationships with the

reported genes

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101

K. pneumoniae L17-CTXM-15-Uganda (JQ6...

Escherichia coli 996--CTXM-15-Uganda ...

K. pneumoniae 639-CTXM-15-Uganda (JQ6...

E. coli E4-CTX-M-15-Portugal (JQ397655)

E. coli E11-CTX-M-15-Portugal (JQ397662)

E. coli E5-CTX-M-15-Portugal (JQ397656)

E. coli 10/136- CTX-M-22-Austria (JN6...

Aeromonas hydrophila E2-CTX-M-3-Portu...

K. pneumoniae 51-CTXM-15-Uganda (JQ68...

K. pneumoniae MS81-CTXM1-PAK

E. coli E12-CTX-M-15-Portugal (JQ397663)

E. coli 10/148-CTX-M-15-Austria (JN67...

K. pneumoniae L4-CTXM-15-Uganda(JQ686...

K. pneumoniae MS83-CTXM1-PAK

E. coli E7 -CTX-M-15-Portugal (JQ397658)

E. coli E9-CTX-M-15-Portugal (JQ397660)

Shigella sonnei CTX-M-15-Jpn (AB698974)

Shigella sonnei CTX-M-15-Jpn (AB698975)

E. coli E16-CTX-M-15-Portugal (JQ397667)

Pseudomonas sp. E18-CTX-M-15-Portugal...

E. coli 09/116-CTX-M-15-Austria (JN67...

E. coli 09/137-CTX-M-15-Austria (JN67...

E. coli 09/133-CTX-M-15-Austria (JN67...

E. coli 10/133-CTX-M-15-Austria (JN67...

E. coli 10/152-CTX-M-15-Austria (JN67...

E. coli L11-CTXM-15-Uganda (JQ686200)

E. coli 1030-CTXM-15-Uganda (JQ686201(2)

E. coli 1030-CTXM-15-Uganda (JQ686201)

K. pneumoniae MS10-CTXM1-PAK

K. pneumoniae MS41-CTXM1-PAK

E. coli MS26-CTXM1-PAK

Fig. 4.7.3. Phylogenetic Tree of the CTX-M1 showing genetic relationships with the

reported genes

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102

E. coli-S-514-CTX-M-14-RUS(GQ385325)

K. pneumoniae Kpc1-CTX-M-14-CH(HQ650134)

E. coli PY-5-plasmid-CTX-CH(FJ424731)

E. coli PY-6-plasmid-CTX-CH(FJ424732)

E. coli PY-7 plasmid CTX-CH(FJ424733)

E coli FJW10-CTX-M-14-CH(GQ896551)

E. coli FJ8-CTX-M-14-CH(GQ896554)

K. pneumoniae 1-CTX-M-14-CH(GU211011)

E. coli K-26-CTX-M-14-RUS(GQ385321)

E. coli strain 09/136-CTX-M-14-Austri...

E. coli 10/144 CTX-M-14-Austria(JN676...

E. coli GD-3 CTX-M-27-CH(FJ405220)

E. coli-MS37-CTX-M9-Pak

E. coli GD-1-CTX-M-27-CH(FJ405222)

Synthetic construct CTX-M-27-SPN(HQ73...

E. coli GD-4-CTX-M-27-CH(FJ405219)

E. coli CTX-M-121-CH(JN790862)

E. coli g25-ESBL-CH(GU226841)

E. coli A2-CTX-m-14-like-CH(EU350506)

E. coli G200-CTX-M-98-CH(HQ637575)

E coli FJG11-CTX-M-27-CH(GQ896552)

E. coli CTX-M-27-FR(AY156923)

E coli CTX-M-93 (HQ166709)

E. coli FJ9-CTX-M-27-CH(GQ896550)

E. coli CTX-M-27-SKR(EU916273)

E. coli ECSB23-CTX-M-102-CH(HQ398215)

Fig. 4.7.4. Phylogenetic Tree of the CTX-M9 showing genetic relationships with the

reported genes

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103

S. enterica 15C3 CMY-2-Canada(GU393325)

S. enterica 8Db CMY-2-Canada(GU393323)

S. enterica 22C1 CMY-2-Canada(GU393326)

S. enterica 19C1 CMY-2-Canada(GU393328)

S. enterica 21C1 CMY-2-Canada(GU393329)

E. coli CMY-2-USA(JF300163)

S. enterica 22D2 CMY-2-Canada(GU393327)

E. coli N8091181 CMY-61-Belgium(JF460...

E. coli CMY-2-(EF648208)

S. enterica 12Ca CMY-2-Canada(GU393324)

Salmonella sp. S138 CMY-2-CH(EU113220)

C. freundii W701 CMY-2-Spain (JQ733574)

E. coli YDC107 CMY-44-USA(FJ437066)

E. coli 1285 CMY-33-USA(EU496816)

E. coli 71047 CMY-55-Spain(HM544040)

E. coli CMY-2-CH(AM779746)

E. coli CMY-2-CH(AM779745)

E. coli CMY-2-CH(AM779747)

E. coli CMY-2-CH(AM779748)

S. typhimurium CMY-2-CH(EU113221)

K. pneumoniae CMY-56-Spain(HQ322613)

S. enterica yuhs04-31 CMY-2-Mexico(FJ...

E. coli cmy-28-Ireland(EF561644)

E. coli CMY-7-UK(AJ011291)

S. typhimurium CMY-7-USA(AY324388)

E. coli ARL05-909-CMY-29-N.Z(EF685371)

E. coli CMY-7-UK(DQ173300)

E. coli MS172-CIT-PAK

Fig 4.7.5 Phylogenetic Tree of the CIT showing genetic relationships with the

reported genes

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104

CMY-2 ---------------------------TFNGVLGGDAIARGEIKLSDPVTKYWPELTGKQ

CMY-32 FTWGKADIANNHPVTQQTLFELGSVSKTFNGVLGGDAIARGEIKLSDPVTKYWPELTGKQ

CIT-172 ----------------------------------------------------WPELTGKQ

********

CMY-2 WQGIRLLHLATYTAGGLPLQIPDDVRDKAALLHFYQNWQPQWTPGAKRLYANSSIGLFGA

CMY-32 WQGIRLLHLATYTAGGLPLQIPDDVRDKAALLHFYQNWQPQWTPGAKRLYANSSIGLFGA

CIT-172 WQGIRLLHLATYTAGGLPLQIPDDVRDKAALLHFYQNWQPQWTPGAKRLYANSSIGLFGE

***********************************************************

CMY-2 LAVKPSGMSYEEAMTRRVLQPLKLAHTWITVPQNEQKDYAWGYREGKPVHVSPGQLDAEA

CMY-32 LAVKPSGMSYEEAMTRRVLQPLKLAHTWITVPQNEQKDYAWGYREGKPVHVSPEQLDAEA

CIT-172 LAVKPSGMSYEEAMTRRVLQPLKLAHTWITVPQNEQKDYAWGYREGKPVHVSPRQLGAEA

***************************************************** **.***

CMY-2 YGVKSSVIDMARWVQANMDASHVQEKTLQQGIALA-------------------------

CMY-32 YGVKSSVIDMARWVQANMDASHVQEKTLQQGIALAQSRYWRIGDMYQGLGWEMLNWPLKA

CIT-172 YGVKSSVIDMARWVQANMDASHVQEK----------------------------------

**************************

Fig. 4.7.6. Variation in translated product of CIT the CIT-172 (E. coli MS 172),

CMY-2 and CMY-32 (Already reported sequences)

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105

Discussion

With the advancement of modern antimicrobial chemotherapy, cephalosporins are

extensively prescribed for treatment of several of community- and hospital-acquired

against Gram positive and Gram negative pathogens infections (Andes and Craig,

2005). However, increased use of broad-spectrum cephalosporins resulted in the

emergence of β-lactamase enzymes, most importantly the extended-spectrum β-

lactamases (ESBLs) (Paterson et al., 2004; Paterson and Bonomo, 2005; Yang and

Guglielmo, 2007; Pitout and Laupland, 2008).

The production of one or more than one β-lactamases is the most important

mechanism of β-lactam antibiotics resistance in the members of Enterobacteriaceae,

although additional mechanisms such as porin loss can also contribute to resistance

(Nikaido and Normark, 1987; Livermore, 1992). The introduction of oxyimino-

cephalosporins in the chemotherapy of enterobacterial infections resistant to the

widespread TEM-1 and SHV-1 enzymes was a major breakthrough, but the

emergence of ESBLs, which are now throughout the world, is reducing the

effectiveness of those agents and is one of the frequent causes of therapeutic failure

(Bradford, 2001).

Enterobacteriaceae, including ESBL-producing strains, are one of the most important

causes of threatening hospital acquired bacterial infections and community onset

infections in humans. These organisms resistant to β-lactam antibiotics are becoming

an emerging problem in the field of public health for providers of health care (Pitout

et al., 2005; Pitout, 2008).

E. coli is one of the most important pathogens causing urinary tract infections (Piatti

et al., 2008). E. coli infections have been widely treated with β-lactam antibiotics;

however, treatment of UTIs with β-lactams has become progressively more

problematic due to the prevalence of ESBLs, including the derivatives of TEM, SHV,

and CTX-M. The genes encoding CTX-M β-lactamases have spread rapidly in the

past decades and are now the predominant type of ESBLs in E. coli in many parts of

the world (Bonnet, 2004; Kim et al., 2005).

Detection of ESBLs with rapidity and accuracy and other plasmid-mediated β-

lactamases in Enterobacteriaceae is important for appropriate treatment and proper

infection control measures. Many tests for ESBLs and PABLs detection have been

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106

reported, but some methods are very difficult to take place in general practice, hard to

interpret, and time consuming (Drieux et al., 2008; Jacoby, 2009). The detection is

made more difficult by extension of the class A carbapenemases, with involving of

KPC β-lactamases, (Bratu et al., 2005; Queenan and Bush, 2007).

This study was carried out for the purpose of assessing the prevalence of different

pathogens, including the resistance genotypes of ESBL and AmpC-producing E. coli

and Klebsiella pneumoniae, causing infections in patients in Islamabad, Pakistan, as

well as their antimicrobial resistance profiles and patterns. The present study is the

first report in Pakistan on assessment of antimicrobial resistance related to

surveillance of genotypes of different ESBLs and AmpC β-lactamases. Total 632

suspected E. coli and Klebsiella pneumoniae were collected from urine, blood,

sputum, pus, fluids and medical devices. Out of these, 593 isolates were confirmed as

E. coli and Klebsiella pneumoniae. Out of 593 isolates, 61.6% were E. coli and 38.4%

were Klebsiella pneumoniae. Various reports on the prevalence and susceptibility of

different members of Enterobacteriaceae have been reported. Increasing incidence of

multidrug resistant E. coli has been described in various reports (Livermore et al.,

2008). Most of our isolates, 41.4% of E. coli and 39% of Klebsiella pneumoniae were

from urine samples. A study in Iran also supports our results, where most of the E.

coli and Klebsiella pneumoniae (56.7%) were from urine (Mohammadi-Mehr and

Feizabadi, 2011).

The effect of antibiotic exposure on ESBL resistance has also been studied by several

individual patient-level risk factor analyses, yielding diverse results. The use of

ceftazidime (Lin et al., 2003), broad-spectrum cephalosporins and aminoglycosides

(Asensio et al., 2000), ciprofloxacin and/or trimethoprim-sulfamethoxazole (Wiener

et al., 1999), and cephalosporins, fluoroquinolones, and penicillins (Colodner et al.,

2004) was identified as a risk factors in isolating ESBL-producing Klebsiella.

pneumoniae in different studies.

ESBL producing E. coli and Klebsiella pneumoniae were detected by double disc

synergy test and combination disc synergy test according to the CLSI guidelines. The

Clinical and Laboratory Standards Institute (CLSI) have approved standard broth

microdilution (BMD) and disc diffusion susceptibility test methods for screening and

confirmation of ESBL production in E. coli, Klebsiella pneumoniae, K. oxytoca and

P. mirabilis. ESBLs were detected in 46.2% of the total isolates. In E. coli strains,

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107

49.3% were found to be ESBL producers, while 41.2% of the K. pneumoniae strains

were ESBL producers. High prevalence of ESBLs among the isolates of E. coli and

Klebsiella pneumoniae (46.6%) was reported in another study (Mohammadi-Mehr

and Feizabadi, 2011). Reports from 1994 to 1999 placed the incidence of ESBL

producing Klebsiella spp. in Europe between 23 and 25%, and 5.4% for E. coli, with

wide variance among geographical locations (Babini and Livermore, 2000; Winokur

et al., 2001). ESBL-positive Enterobacteriaceae have been reported to be isolated

frequently in health care centres in Algeria, and the overall prevalence of ESBL

producers from January to June 2005 was 20.4% (Ramdani-Bouguessa et al., 2006).

The reported prevalence of ESBL-producing Gram negative isolates in various

hospitals in India was in the range of 19-60% (Bhattacharya, 2011). The high rate of

ESBL producing bacteria in the developing nations is the point to worry about;

mortality and morbidity due to these infections are increasing for the reason of

deficiency in health related funds for effectively controlling these infections and their

restricted accession to the most effective antibiotics. Shigella sonnei WA7593 was

reported from a fecal sample in August 2004, and was positive for the production of

an ESBL by confirmatory disc diffusion test. The patient with this infection had

visited Pakistan and likely has contracted the infection there and became ill. He was

still sick when he returned to the US (Kim et al., 2007).

High resistance was observed in both ESBL and AmpC β-lactamase producing E. coli

and Klebsiella pneumoniae to cephalosporins and other non- β-lactam antibiotics

tested during the present study. However, imipenem and tigecycline were the most

successful drugs. Similar results were reported by Segatore et al., (2004), where

meropenem was the most effective drug. Another study of the Asia Pacific, Europe,

Latin and North America also reported the effectiveness of imipenem and tigecycline

(Reinert et al., 2007).

Rapid evolution of bacterial resistance may be due to a complex interaction of several

factors such as higher burden of infectious diseases, treatment uncertainty, lack of

treatment guidelines, inadequate access to standard laboratory facilities, self-

medication, prescription based on availability, government support to pharmaceutical

industries, market forces, antibiotics prescribed by unqualified health professionals,

less strict law enforcement, fragmented public health system, poor population-wide

insurance coverage, inadequate adherence to universal hygiene and infection control

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108

measures and to low population-wide education level (Kamat and Nichter, 1998;

Sahoo et al., 2010).

The CTX-M–type β-lactamases are non-TEM and non-SHV plasmid-encoded, class

A, ESBLs. The CTX-M–type β-lactamase enzymes have recently evolved and as the

most important and common group of ESBLs, with distribution worldwide (Vanhove

et al., 1995). The CTX-M enzymes are grouped into five major phylogenetic trees

(CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25) that are based on

sequences of amino acid similarities (Bonnet, 2004). About 100 CTX-M β-lactamases

are included in the Lahey Clinic database for ESBL (www.lahey.org/studies).

E. coli expressing CTX-M β-lactamase enzymes are the most common type of

organisms widespread globally (Canton et al., 2008). The epidemiology of ESBL

genes is complex because there is both clonal spread of resistant strains and spread of

specific plasmids and/or other mobile genetic elements. The distribution of ESBLs is

far from uniform and there are considerable geographical differences in the

prevalence of ESBL (al Naiemi et al., 2006).

CTX-M-1 was the most commonly found gene in the present study. It was detected in

58.8% and 62.3% of the 131 and 69 ESBLs producing E. coli and Klebsilla

pneumoniae, respectively. Sequencing results showed that these genes were similar to

CTX-M-15 reported from Austria, Uganda and Portugal. This is the first report of

CTX-M-15 from Pakistan. CTX-M-15 has been reported by several researchers from

India (Ensor et al., 2006; Muzaheed et al., 2008). CTX-M-15 have also been reported

from different parts of the world (Baraniak et al., 2002; Leflon-Guibout et al., 2004;

Markovska et al., 2004).

Invasive infections due to ESBL producing E. coli are a major problem in neonates,

because of restricting the drugs of choice. It is assumed that the emergence of CTX-M

enzymes has been favored the extensive use of cefotaxime and ceftriaxone (Soge et

al., 2006).

The production of a CTX-M type, by ESBL-producing isolates is not reported very

commonly in the United States. The only other reference was from a multistate study

in 2001-2002 that confirmed CTX-M type from E. coli isolated from urine, blood and

sputum (Moland et al., 2003). The epidemiology of CTX-M enzyme-producing

organisms is different from those of TEM or SHV ESBL-producing organisms (Pitout

et al., 2005). Community-acquired infections caused by organisms producing CTX-M

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109

enzymes, have been described frequently, typically as urinary tract infections by E.

coli (Song et al., 2009).

In our study, TEM genes were detected in 19.1% of the 131 ESBL producing E. coli.

Whereas, in 11.5% of the 69 ESBL K. pneumoniae, positive results for the TEM

genes were observed on the basis of polymerase chain reaction. Sequence analysis

showed that the sequence is 99% similar to TEM-1 already reported from Ecuador,

France, Japan and China. Previously, TEM-1 have been reported from Venezuela

(Araque and Rivera, 2004), Taiwan (Yan et al., 2001) and China (Zhang et al., 2008).

Out of the 131 ESBL producing E. coli, 14.5% and out of 69 ESBL producing K.

pneumoniae, 13% were found having SHV gene. NCBI BLAST and gene tree

analysis revealed that it was 99% similar to SHV-49 reported from France and SHV-

38 from India (Dubois et al., 2004). SHV have been previously reported from various

parts of the world. SHV have been detected in 18 out of 20 K. pneumoniae from

Thailand (Udomsantisuk et al., 2011). Kaftandzieva et al., (2011) reported that 19.5%

E. coli and K. pneumoniae were having SHV gene in Macedonia. Reports of other

places also suggest the presence of SHV in different parts (Colom et al., 2003;

Bedenic et al., 2005).

In our study, CTX-M-9 was found in 3% of the ESBL producing E. coli and 2.8% of

the ESBL producing K. pneumoniae. Homology results showed that the gene is

similar to CTX-M-27 reported from China, Spain and France, and to CTX-M-14

reported from Austria. CTX-M-9 have been previously reported in India but the

frequency was low (Roy et al., 2011). CTX-M-27 have also been reported from

United Kingdom (Doumith et al., 2012).

AmpC enzymes are serine cephalosporinases that can be inhibited by cloxacillin and

boronic acid (Pitout et al., 2010), AmpC-producing E. coli resistant to 3rd

generation

different from those producing ESBL by their ability to inhibit cefoxitin and

amoxicillin/clavulanic acid and are resistant to them, susceptible to cefepime, and

inhibit the activity of the cephalosporin even in inherence of clavulanate. This type of

AmpC β-lactamase may have been resulted from the excessive expression of the

chromosome-mediated AmpC (c-AmpC) β-lactamase enzyme (Potz et al., 2006;

Corvec et al., 2007) or by acquiring a transmissible plasmid encoded ampC gene (p-

AmpC) (Perez-Perez and Hanson, 2002; Mata et al., 2010; Naseer et al., 2010).

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110

No standard method is available for the detection of AmpC β-lactamase in CLSI

guidelines. However, different methods described for AmpC β-lactamase detection

are in use in research laboratories. Three of these methods were used to detect AmpC

β-lactamase enzymes in the present study. Our study showed that in 365 E. coli

isolates, AmpC β-lactamase production was detected in 25.8% of the isolates while

20.6% of the 228 K. pneumoniae isolates produced AmpC β-lactamase. Studies

performed in India have shown that plasmid-borne and chromosomally mediated

AmpC and cephalosporinase-producing pathogens are common in resistant E. coli and

Klebsiella pneumoniae isolates (Bhattacharya, 2011; Castanheira et al., 2011). One

surveillance study conducted in the United States reported detection of plasmid-

encoded AmpC β-lactamase and ESBL in 4 and 40% of E. coli isolates with reduced

susceptibility to broad-spectrum cephalosporins collected between 1992 and 2000,

respectively (Jacoby et al., 2006).

Unlike ESBLs, the detection method of plasmid-encoded AmpC β-lactamase enzyme

including CMY-type β-lactamase has not been standardized by the CLSI or any other

authorities, which is a major barrier in defining its epidemiology. As of now, the

isolates producing this group of β-lactamases are typically labeled as ESBL-negative

and would not be tested further. However, there has been a growing interest in using

boronic acid compounds as specific AmpC inhibitors for detection and confirmation

of plasmid-encoded AmpC β-lactamase enzyme production in E. coli and Klebsiella

spp. (Yagi et al., 2005; Jacoby et al., 2006). Routine use of boronic acid-based

method on isolates which are positive for the initial screen test for ESBL production

(i.e. reduced susceptibility to broad-spectrum cephalosporins) would greatly enhance

detection of E. coli (Sidjabat et al., 2009).

Although not frequent yet, pAmpC producing Enterobacteriaceae are causing

nosocomial, healthcare-associated and community infections in Spain (Oteo et al.,

2010). Most of the infections were caused by E. coli, but also Proteus mirabilis and

Klebsiella pneumoniae accounted for a significant proportion of cases. They reported

that more than 50% of the cases were community-onset. Many of these were

healthcare-associated. Similar results were reported by Sidjabat et al. (2009) in

Pittsburgh, studying only CMY-producing E. coli.

Among the other AmpC β-lactamases during the present study were MOX, EBC and

FOX. MOX gene was detected most frequently in E. coli isolates and CIT was

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111

frequently detected in Klebsiella pneumoniae. Present study showed CIT as the

second most prevalent AmpC β-lactamase gene detected by polymerase chain reaction

in E. coli. These results are in concordance with other reports from Europe (Haldorsen

et al., 2008; Naseer et al., 2010) and the reports from other parts of the world

(Laupland et al., 2005; Mammeri et al., 2008a; Iabadene et al., 2009; Mata et al.,

2010).

Gene sequencing results of the CIT in E. coli MS 172 showed similarity to already

reported CMY-2 and CMY-7. However, there were three nucleotide differences from

the reported genes. Protein translation of the sequence revealed that the sequence is

different at three positions from CMY-2 and CMY-32. These could be novel

mutations in CMY from this region.

As we found in this study, the CIT type β-lactamases, are the most frequently isolated

AmpC β-lactamase enzymes observed in E. coli and K. pneumoniae, while the other

AmpC β-lactamase enzymes types are not prevalent and are found more in other

Enterobacteriaceae generally (Mata et al., 2009; Naseer et al., 2009; Haldorsen et al.,

2008; Sidjabat et al., 2009; Mammeri et al., 2008a; Woodford et al., 2007; Li et al.,

2008). For example, in the plasmid encoded AmpC–producing Klebsiella pneumoniae

isolates, DHA-type β-lactamase enzymes were the more frequently observed β-

lactamases in China (Li et al., 2008), and ACC β-lactamase and FOX β-lactamase

enzymes were the most commonly reported β-lactamases in Ireland and UK (Roche et

al., 2008; Woodford et al., 2007). CMY2 producing organisms have also been

reported as common β-lactamases in the United States and Canada in the last few

years (Baudry et al., 2009; Hanson et al., 2008; Mataseje et al., 2009; Pitout et al.,

2007). In another report, 96.3% of the 27 E. coli isolates from Canadian intensive care

units were producing CMY-2 (Baudry et al., 2009). Further, out of the 142 cefoxitin-

resistant E. coli, isolated from water samples from the sources across Canada, 77.5%

of the bacteria were producing CMY-2 β-lactamase (Mataseje et al., 2009).

One of the most important mechanisms of β-lactam resistance is the production of

multiple β-lactamase enzymes. In these cases, the combined activity of various

enzymes can broaden the range of hydrolysable substrates and eventually cover the

spectrum of all molecules in clinical use (Essack et al., 2001). Many genes were in the

combination of two or more than two genes in a single isolate. The combination of

TEM-1, SHV-12, and a variant of IMP-2 metallo-β-lactamase, designated IMP-8 were

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112

reported to occur on a single multidrug resistance plasmid from a clinical isolate of K.

pneumoniae (Yan et al., 2001). armA, blaCTX-M-15 and blaTEM-1 genes were detected

in K. oxytoca 157 in China (Zhang et al., 2008). Two or more types for ESBL were

detected in 61% of ESBL isolates; bla(TEM) + bla(SHV) being the most commonly

encountered combination (Kaftandzieva et al., 2011).

Other mutations in further non-information positions of the gene may exist but remain

undiscovered, as in molecular diagnostics only the expected size of the PCR amplicon

is considered and no nucleotide sequencing is performed routinely.

Our study has some limitations. We did not include a control group to try and identify

specific risk factors or to compare the prognostic implications. Even though our

screening protocol was designed to detect pAmpC producing organisms with high

sensitivity and specificity, in some cases might not have been detected. Finally, the

low number of cases in some subgroups preclude from obtaining robust conclusions.

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113

Conclusions

ESBL and AmpC producers pose serious problems in managing patients with

hospital and community acquired infections. E. coli accounted for more infections as

compared to K. pneumoniae in the positive samples.

Phenotypic screening methods were successful for determining ESBL and AmpC

production. E. coli samples gave a slightly higher prevalence of ESBL and AmpC

production than in K. pneumoniae.

Out of these 593 isolates, 200 samples of the phenotypically confirmed ESBLs or

AmpC producers, E. coli and Klebsiella pneumoniae, were processed. Imipenem and

tigecycline were the most effective antibiotics for ESBL producing E. coli and

Klebsiella pneumoniae. Maximum resistance was shown against sulphamethoxazole

and tetracycline in ESBL producing E. coli and Klebsiella pneumoniae. Higher

resistance was observed for all six β-lactam antibiotics tested.

Age was significant risk factor for ESBL, whereas gender and sample origin were

significant risk factors for AmpC production in E. coli. No variable was having

significance association for ESBL production in K. pneumoniae, whereas sample

origin was found to be the only significant risk factor for AmpC production in K.

pneumoniae.

CTX-M1 was the most prevalent (60%) out of six ESBL genes tested (TEM, SHV,

CTX-M1, CTX-M2, CTX-M8 and CTX-M9) in both E. coli and K. pneumoniae.

This trend is similar to that observed in other countries of Europe and Asia. No CTX-

M2 and CTX-M8 could be detected indicating that these genes do not contribute

towards ESBL production in Pakistan.

Presence of multiple genes in single ESBL producer indicates stubborn nature of

resistant isolates. This trend of gene combinations was commonly found in the

present study. CTX-M1 and TEM were mostly found 2-gene combination followed

by CTX-M1 and SHV in both ESBL producing E. coli and Klebsiella pneumoniae.

We could also observe 3- and 4-gene combinations also.

Sequence analyses revealed 99-100% homology with already reported ESBL genes

from around the world. Computational analyses revealed amino acid substitutions in

more than one positions in CIT isolates. SHV was found to be 100% homologous to

an already reported novel inhibitor resistant SHV-49 gene from France.

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114

Future prospects

Antibiotic resistance is an under- studied prospective of medical research in

Pakistan. There is a dire need to establish databases for antibiotic resistance

and its association with various risk factors in Pakistan.

Extensive research in antibiotic resistance is required in Pakistan as most of

the antibiotics are not working properly now.

Further investigations are required to evaluate the impact of antibiotic

selection, dynamic flow of organisms and genes between hospital and

community, community colonization, and the multiple origins of the isolates.

Plasmid profiling can be done to determine the number and types of plasmids

and to detect other types of resistance.

PFGE and RAPD analysis could be performed to detect the source of the

infection.

Further investigations are required to detect novel genes and novel mutations.

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115

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i

Appendix

A-1. Overall distribution of E. coli and Klebsiella pneumoniae in the study group

Bacterial Isolate Number (n) Percentage (%)

E. coli 365 61.6

K. pneumoniae 228 38.4

Total 593 100.0

A-2. Gender distribution of E. coli and K. pneumoniae

A-3.Overall distribution of E. coli and Klebsiella pneumoniae in different age categories

Bacterial Isolate Age Groups (Years) Total

Upto

12 13-25 26-35 36-50 51-60

Above

60

E. coli Number (n) 7 109 51 106 45 47 365

Percentage

(%)

1.9 29.9 14.0 29.0 12.3 12.9 100.0

K.

pneumoniae

Number (n) 6 63 39 55 34 31 228

Percentage

(%)

2.6 27.6 17.1 24.1 14.9 13.6 100.0

Total Number (n) 13 172 90 161 79 78 593

Percentage

(%)

2.2 29.0 15.2 27.2 13.3 13.2 100.0

Bacterial Isolate Gender Total

Male Female

E. coli Number (n) 160 205 365

Percentage (%) 43.8 56.2 100.0

K. pneumoniae Number (n) 124 104 228

Percentage (%) 54.4 45.6 100.0

Total Number (n) 284 309 593

Percentage (%) 47.9 52.1 100.0

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___________________________________________________________________________

ii

A-4. Percentage distribution of E. coli and Klebsiella pneumoniae on the basis of sample

origin

Bacterial Isolate Sample origin Total

Medical

ward

Surgical

ward OPD ICU

E. coli Number (n) 73 40 196 56 365

Percentage

(%)

20.0 11.0 53.7 15.3 100.0

K. pneumoniae Number (n) 45 26 122 35 228

Percentage

(%)

19.7 11.4 53.5 15.4 100.0

Total Number (n) 118 66 318 91 593

Percentage

(%)

19.9 11.1 53.6 15.3 100.0

A-5. Percentage distribution of E. coli and Klebsiella pneumoniae on the basis of sample

source

Bacterial Isolate Sample source Total

Urine Blood Pus Fluids Devices Sputum

E. coli Number (n) 151 36 99 40 27 12 365

Percentage

(%)

41.4 9.9 27.1 11.0 7.4 3.3 100.0

K.

pneumoniae

Number (n) 89 26 63 20 20 10 228

Percentage

(%)

39.0 11.4 27.6 8.8 8.8 4.4 100.0

Total Number (n) 240 62 162 60 47 22 593

Percentage

(%)

40.5 10.5 27.3 10.1 7.9 3.7 100.0

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iii

A-6. Overall percentage distribution of ESBLs producer strains of E. coli and Klebsiella

pneumoniae in the study group

Bacterial Isolate ESBL status Total

Yes No

E. coli Number (n) 180 185 365

Percentage (%) 49.3 50.7 100.0

K. pneumoniae Number (n) 94 134 228

Percentage (%) 41.2 58.8 100.0

Total Number (n) 274 319 593

Percentage (%) 46.2 53.8 100.0

A-7. Overall distribution of ESBL producing E. coli among different age groups

ESBL status Age Groups (Years) Total

Upto

12 13-25 26-35 36-50 51-60

Above

60

Yes Number (n) 3 43 25 64 28 17 180

Percentage (%) 1.7 23.9 13.9 35.6 15.6 9.4 100.0

No Number (n) 4 66 26 42 17 30 185

Percentage (%) 2.2 35.7 14.1 22.7 9.2 16.2 100.0

Total Number (n) 7 109 51 106 45 47 365

Percentage (%) 1.9 29.9 14.0 29.0 12.3 12.9 100.0

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___________________________________________________________________________

iv

A-8. Gender distribution of ESBL producing E. coli

ESBL status Gender Total

Male Female

Yes Number (n) 72 108 180

Percentage (%) 40.0 60.0 100.0

No Number (n) 88 97 185

Percentage (%) 47.6 52.4 100.0

Total Number (n) 160 205 365

Percentage (%) 43.8 56.2 100.0

A-9. Percentage distribution of ESBL producing E. coli strains on the basis of sample origin

ESBL status Sample origin Total

Medical

ward

Surgical

ward OPD ICU

Yes Number (n) 43 18 92 27 180

Percentage (%) 23.9 10.0 51.1 15.0 100.0

No Number (n) 30 22 104 29 185

Percentage (%) 16.2 11.9 56.2 15.7 100.0

Total Number (n) 73 40 196 56 365

Percentage (%) 20.0 11.0 53.7 15.3 100.0

___________________________________________________________________________

___________________________________________________________________________

v

A-10. Percentage distribution of ESBL producing E. coli strains on the basis of sample

source

ESBL status Sample source Total

Urine Blood Pus Fluids Devices Sputum

Yes Number (n) 68 19 55 19 15 4 180

Percentage (%) 37.8 10.6 30.6 10.6 8.3 2.2 100.0

No Number (n) 83 17 44 21 12 8 185

Percentage (%) 44.9 9.2 23.8 11.4 6.5 4.3 100.0

Total Number (n) 151 36 99 40 27 12 365

Percentage (%) 41.4 9.9 27.1 11.0 7.4 3.3 100.0

A-11. Overall distribution of ESBL producing K. pneumoniae among different age groups

ESBL status Age Groups (Years) Total

Upto

12 13-25 26-35 36-50 51-60

Above

60

Yes Number (n) 1 26 15 20 18 14 94

Percentage (%) 1.1 27.7 16.0 21.3 19.1 14.9 100.0

No Number (n) 5 37 24 35 16 17 134

Percentage (%) 3.7 27.6 17.9 26.1 11.9 12.7 100.0

Total Number (n) 6 63 39 55 34 31 228

Percentage (%) 2.6 27.6 17.1 24.1 14.9 13.6 100.0

___________________________________________________________________________

___________________________________________________________________________

vi

A-12. Gender distribution of ESBL producing K. pneumoniae

ESBL status Gender Total

Male Female

Yes Number (n) 53 41 94

Percentage (%) 56.4 43.6 100.0

No Number (n) 71 63 134

Percentage (%) 53.0 47.0 100.0

Total Number (n) 124 104 228

Percentage (%) 54.4 45.6 100.0

A-13. Percentage distribution of ESBL producing K. pneumoniae strains on the basis of

sample origin

ESBL status Sample origin Total

Medical

ward

Surgical

ward OPD ICU

Yes Number (n) 19 7 50 18 94

Percentage (%) 20.2 7.4 53.2 19.1 100.0

No Number (n) 26 19 72 17 134

Percentage (%) 19.4 14.2 53.7 12.7 100.0

Total Number (n) 45 26 122 35 228

Percentage (%) 19.7 11.4 53.5 15.4 100.0

___________________________________________________________________________

___________________________________________________________________________

vii

A-14. Percentage distribution of ESBL producing K. pneumoniae strains on the basis of

sample source

ESBL status Sample source Total

Urine Blood Pus Fluids Devices Sputum

Yes Number (n) 28 16 27 10 8 5 94

Percentage (%) 29.8 17.0 28.7 10.6 8.5 5.3 100.0

No Number (n) 61 10 36 10 12 5 134

Percentage (%) 45.5 7.5 26.9 7.5 9.0 3.7 100.0

Total Number (n) 89 26 63 20 20 10 228

Percentage (%) 39.0 11.4 27.6 8.8 8.8 4.4 100.0

A-15. Overall percentage distribution of AmpC producing E. coli and Klebsiella

pneumoniae in the study group

Bacterial Isolate AmpC status Total

Yes No

E. coli Number (n) 94 271 365

Percentage (%) 25.8 74.2 100.0

K. pneumoniae Number (n) 47 181 228

Percentage (%) 20.6 79.4 100.0

Total Number (n) 141 452 593

Percentage (%) 23.8 76.2 100.0

___________________________________________________________________________

___________________________________________________________________________

viii

A-16. Overall distribution of AmpC producing E. coli among different age groups

AmpC status Age Groups (Years) Total

Upto

12 13-25 26-35 36-50 51-60

Above

60

Yes Number (n) 3 20 12 26 14 19 94

Percentage (%) 3.2 21.3 12.8 27.7 14.9 20.2 100.0

No Number (n) 4 89 39 80 31 28 271

Percentage (%) 1.5 32.8 14.4 29.5 11.4 10.3 100.0

Total Number (n) 7 109 51 106 45 47 365

Percentage (%) 1.9 29.9 14.0 29.0 12.3 12.9 100.0

A-17. Gender distribution of AmpC producing E. coli

AmpC status Gender Total

Male Female

Yes Number (n) 51 43 94

Percentage (%) 54.3 45.7 100.0

No Number (n) 109 162 271

Percentage (%) 40.2 59.8 100.0

Total Number (n) 160 205 365

Percentage (%) 43.8 56.2 100.0

___________________________________________________________________________

___________________________________________________________________________

ix

A-18. Percentage distribution of AmpC producing E. coli strains on the basis of sample

origin

AmpC status Sample origin Total

Medical

ward

Surgical

ward OPD ICU

Yes Number (n) 16 18 42 18 94

Percentage (%) 17.0 19.1 44.7 19.1 100.0

No Number (n) 57 22 154 38 271

Percentage (%) 21.0 8.1 56.8 14.0 100.0

Total Number (n) 73 40 196 56 365

Percentage (%) 20.0 11.0 53.7 15.3 100.0

A-19. Percentage distribution of AmpC producing E. coli strains on the basis of sample

source

AmpC status Sample source Total

Urine Blood Pus Fluids Devices Sputum

Yes Number (n) 34 7 27 13 9 4 94

Percentage (%) 36.2 7.4 28.7 13.8 9.6 4.3 100.0

No Number (n) 117 29 72 27 18 8 271

Percentage (%) 43.2 10.7 26.6 10.0 6.6 3.0 100.0

Total Number (n) 151 36 99 40 27 12 365

Percentage (%) 41.4 9.9 27.1 11.0 7.4 3.3 100.0

___________________________________________________________________________

___________________________________________________________________________

x

A-20. Overall distribution of AmpC producing K. pneumoniae among different age groups

AmpC status Age Groups (Years) Total

Upto

12 13-25 26-35 36-50 51-60

Above

60

Yes Number (n) 0 14 9 11 6 7 47

Percentage (%) .0 29.8 19.1 23.4 12.8 14.9 100.0

No Number (n) 6 49 30 44 28 24 181

Percentage (%) 3.3 27.1 16.6 24.3 15.5 13.3 100.0

Total Number (n) 6 63 39 55 34 31 228

Percentage (%) 2.6 27.6 17.1 24.1 14.9 13.6 100.0

A-21. Gender distribution of ESBL producing K. pneumoniae

AmpC status Gender Total

Male Female

Yes Number (n) 26 21 47

Percentage (%) 55.3 44.7 100.0

No Number (n) 98 83 181

Percentage (%) 54.1 45.9 100.0

Total Number (n) 124 104 228

Percentage (%) 54.4 45.6 100.0

___________________________________________________________________________

___________________________________________________________________________

xi

A-22. Percentage distribution of AmpC producing K. pneumoniae strains on the basis of

sample origin

AmpC status Sample origin Total

Medical

ward

Surgical

ward OPD ICU

Yes Number (n) 9 13 17 8 47

Percentage (%) 19.1 27.7 36.2 17.0 100.0

No Number (n) 36 13 105 27 181

Percentage (%) 19.9 7.2 58.0 14.9 100.0

Total Number (n) 45 26 122 35 228

Percentage (%) 19.7 11.4 53.5 15.4 100.0

A-23. Percentage distribution of AmpC producing K. pneumoniae strains on the basis of

sample source

AmpC status Sample source Total

Urine Blood Pus Fluids Devices Sputum

Yes Number (n) 21 2 12 6 3 3 47

Percentage (%) 44.7 4.3 25.5 12.8 6.4 6.4 100.0

No Number (n) 68 24 51 14 17 7 181

Percentage (%) 37.6 13.3 28.2 7.7 9.4 3.9 100.0

Total Number (n) 89 26 63 20 20 10 228

Percentage (%) 39.0 11.4 27.6 8.8 8.8 4.4 100.0

___________________________________________________________________________

_____________________________________________________________________

xii

Sequences >K. pneumoniae MS88-TEM-PAK

TCTAATACATTCAATATGTATCCGCTCATGAGACAATAACCCTGGTAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATT

TTCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCA

GTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGC

ACTTTTAAAGTTCTGCTATGTGGTGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT

TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC

TGCTGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGT

TGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTG

GCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGC

TGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATC

GTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT

>K. pneumoniae MS130-TEM-PAK

TCTAATACATTCAATATGTATCCGCTCATGAGACAATAACCCTGGTAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATT

TTCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCA

GTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGC

ACTTTTAAAGTTCTGCTATGTGGTGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT

TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC

TGCTGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGT

TGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTG

GCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGC

TGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATC

GTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATTGACAGATCGC

>E. coli MS50-TEM-PAK

AATACATTCAATATGTATCCGCTCATGAGACAATAACCCTGGTAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTTC

GTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTT

GGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACT

TTTAAAGTTCTGCTATGTGGTGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGG

TTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGC

TGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGG

GAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCG

AACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGG

CTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTA

GTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGAC

> K. pneumoniae MS81-SHV-PAK

TTATCGGCCCTCACTCAAGGATGTATTGTGGTTATGCGTTATATTCGCCTGTGTATTATCTCCCTGTTAGCCACCCTGCCGCTGGCGGTACACG

CCAGCCCGCAGCCGCTTGAGCAAATTAAACAAAGCGAAAGCCAGCTGTCGGGCCGCGTAGGCATGATAGAAATGGATCTGGCCAGCGGCCGCAC

GCTGACCGCCTGGCGCGCCGATGAACGCTTTCCCATGATGAGCACCTTTAAAGTAGTGCTCTGCGGCGCAGTGCTGGCGCGGGTGGATGCCGGT

GACGAACAGCTGGAGCGAAAGATCCACTATCGCCAGCAGGATCTGGTGGACTACTCGCCGGTCAGCGAAAAACACCTTGCCGACGGCATGACGG

TCGGCGAACTCTGCGCCGCCGCCATTACCATGAGCGATAACAGCGCCGCCAATCTGCTGCTGGCCACCGTCGGCGGCCCCGCAGGATTGACTGC

CTTTTTGCGCCAGATCGGCGACAACGTCACCCGCCTTGACCGCTGGGAAACGGAACTGAATGAGGCGCTTCCCGGCGATGCCCGCGACACCACT

ACCCCGGCCAGCATGGCCGCGACCCTGCGCAAGCTGCTGACCAGCCAGCGTCTGAGCGCCCGTTCGCAACGGCAGCTGCTGCAGTGGATGGTGG

ACGATCGGGTCGCCGGACCGTTGATCCGCTCCGTGCTGCCGGCGGGCTGGTTTATCGCCGATAAGACCGGAGCTGGCGAACGGGGTGCGCGCGG

GATTGTCGCCCTGCTTGGCCCGAATAACAAAGCAGAGCGCATTGTGGTGATTTATCTGCGGGATACCCCGGCGAGCATGGCCGAGCGAAATCAG

CAAATCGCCGGGATCGGCGCGGCGCTGATCGAGCACTGGCAACGCTAACCCGGCGGTGGCCGCGCGCGTTATCCGGCTCGTAGCACTTCGCAGG

C >E. coli MS26-CTXM1-PAK

GGACGTACAGCAAAAACTTGCCGAATTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGGCATTGATTAACACAGCAGATAATTCGCAAATACTT

TATCGTGCTGATGAGCGCTTTGCGATGTGCAGCACCAGTAAAGTGATGGCCGCGGCCGCGGTGCTGAAGAAAAGTGAAAGCGAACCGAATCTGT

TAAATCAGCGAGTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAATGGGACGATGTCACTGGCTGAGCT

TAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGCGATGAATAAGCTGATTGCTCACGTTGGCGGCCCGGCTAGCGTCACCGCGTTCGCCCGA

CAGCTGGGAGACGAAACGTTCCGTCTCGACCGTACCGAGCCGACGTTAAACACCGCCATTCCGGGCGATCCGCGTGATACCACTTCACCTCGGG

CAATGGCGCAAACTCTGCGGAATCTGACGCTGGGTAAAGCATTGGGCGACAGCCAACGGGCGCAGCTGGTGACATGGATGAAAGGCAATACCAC

CGGTGCAGCGAGCATTCAGGCTGGACTGCCTGCTTCCTGGGTTGTGGGGGATAAAACCGGCAGCGGTGGCTATGGCACCACCAACGATATCGCG

GTGATCTGGCCAAAAGATCGTGCGCCGCTGATTCTGGTCAC

>K. pneumoniae MS41-CTXM1-PAK

GTTAGGAAGTGTGCCGCTGTATGCGCAAACGGCGGACGTACAGCAAAAACTTGCCGAATTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGGCA

TTGATTAACACAGCAGATAATTCGCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTGCAGCACCAGTAAAGTGATGGCCGCGGCCGCGG

TGCTGAAGAAAAGTGAAAGCGAACCGAATCTGTTAAATCAGCGAGTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAA

GCACGTCAATGGGACGATGTCACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGCGATGAATAAGCTGATTGCTCACGTT

GGCGGCCCGGCTAGCGTCACCGCGTTCGCCCGACAGCTGGGAGACGAAACGTTCCGTCTCGACCGTACCGAGCCGACGTTAAACACCGCCATTC

CGGGCGATCCGCGTGATACCACTTCACCTCGGGCAATGGCGCAAACTCTGCGGAATCTGACGCTGGGTAAAGCATTGGGCGACAGCCAACGGGC

GCAGCTGGTGACATGGATGAAAGGCAATACCACCGGTGCAGCGAGCATTCAGGCTGGACTGCCTGCTTCCTGGGTTGTGGGGGATAAAACCGGC

AGCGGTGGCTATGGCACCACCAACGATATCGCGGTGATCTGGCCAAAAGATCGTGCGCCGCTGATTCTGGTCACTTACTTCACCCAGCCTCAAC

CTAAGG

>K. pneumoniae MS83-CTXM1-PAK

GCGCAAACGGCGGACGTACAGCAAAAACTTGCCGAATTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGGCATTGATTAACACAGCAGATAATT

CGCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTGCAGCACCAGTAAAGTGATGGCCGCGGCCGCGGTGCTGAAGAAAAGTGAAAGCGA

ACCGAATCTGTTAAATCAGCGAGTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAATGGGACGATGTCA

CTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGCGATGAATAAGCTGATTGCTCACGTTGGCGGCCCGGCTAGCGTCACCG

CGTTCGCCCGACAGCTGGGAGACGAAACGTTCCGTCTCGACCGTACCGAGCCGACGTTAAACACCGCCATTCCGGGCGATCCGCGTGATACCAC

TTCACCTCGGGCAATGGCGCAAACTCTGCGGAATCTGACGCTGGGTAAAGCATTGGGCGACAGCCAACGGGCGCAGCTGGTGACATGGATGAAA

GGCAATACCACCGGTGCAGCGAGCATTCAGGCTGGACTGCCTGCTTCCTGGGTTGTGGGGGATAAAACCGGCAGCGGTGGCTATGGCACCACCA

ACGATATCGCGGTGATCTGGCCAAAAGATCGTGCGCCGC

___________________________________________________________________________

_____________________________________________________________________

xiii

>K. pneumoniae MS10-CTXM1-PAK

TGCGCAAACGGCGGACGTACAGCAAAAACTTGCCGAATTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGGCATTGATTAACACAGCAGATAAT

TCGCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTGCAGCACCAGTAAAGTGATGGCCGCGGCCGCGGTGCTGAAGAAAAGTGAAAGCG

AACCGAATCTGTTAAATCAGCGAGTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAATGGGACGATGTC

ACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGCGATGAATAAGCTGATTGCTCACGTTGGCGGCCCGGCTAGCGTCACC

GCGTTCGCCCGACAGCTGGGAGACGAAACGTTCCGTCTCGACCGTACCGAGCCGACGTTAAACACCGCCATTCCGGGCGATCCGCGTGATACCA

CTTCACCTCGGGCAATGGCGCAAACTCTGCGGAATCTGACGCTGGGTAAAGCATTGGGCGACAGCCAACGGGCGCAGCTGGTGACATGGATGAA

AGGCAATACCACCGGTGCAGCGAGCATTCAGGCTGGACTGCCTGCTTCCTGGGTTGTGGGGGATAAAACCGGCAGCGGTGGCTATGGCACCACC

AACGATATCGCGGTGATCTGGCCAAAAGATCGTGCGCCGCTGATTCTGGTCACTT

>K. pneumoniae MS81-CTXM1-PAK

TTGTTAGGAAGTGTGCCGCTGTATGCGCAAACGGCGGACGTACAGCAAAAACTTGCCGAATTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGG

CATTGATTAACACAGCAGATAATTCGCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTGCAGCACCAGTAAAGTGATGGCCGCGGCCGC

GGTGCTGAAGAAAAGTGAAAGCGAACCGAATCTGTTAAATCAGCGAGTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAA

AAGCACGTCAATGGGACGATGTCACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGCGATGAATAAGCTGATTGCTCACG

TTGGCGGCCCGGCTAGCGTCACCGCGTTCGCCCGACAGCTGGGAGACGAAACGTTCCGTCTCGACCGTACCGAGCCGACGTTAAACACCGCCAT

TCCGGGCGATCCGCGTGATACCACTTCACCTCGGGCAATGGCGCAAACTCTGCGGAATCTGACGCTGGGTAAAGCATTGGGCGACAGCCAACGG

GCGCAGCTGGTGACATGGATGAAAGGCAATACCACCGGTGCAGCGAGCATTCAGGCTGGACTGCCTGCTTCCTGGGTTGTGGGGGATAAAACCG

G

>E. coli-MS37-CTX-M9-Pak

ACGGATGATGTTCGCGGCGGCGGCGTGCATTCCGCTGCTGCTGGGCAGCGCGCCGCTTTATGCGCAGACGAGTGCGGTGCAGCAAAAGCTGGCG

GCGCTGGAGAAAAGCAGCGGAGGGCGGCTGGGCGTCGCGCTCATCGATACCGCAGATAATACGCAGGTGCTTTATCGCGGTGATGAACGCTTTC

CAATGTGCAGTACCAGTAAAGTTATGGCGGCCGCGGCGGTGCTTAAGCAGAGTGAAACGCAAAAGCAGCTGCTTAATCAGCCTGTCGAGATCAA

GCCTGCCGATCTGGTTAACTACAATCCGATTGCCGAAAAACACGTCAACGGCACAATGACGCTGGCAGAACTGAGCGCGGCCGCGTTGCAGTAC

AGCGACAATACCGCCATGAACAAATTGATTGCCCAGCTCGGTGGCCCGGGAGGCGTGACGGCTTTTGCCCGCGCGATCGGCGATGAGACGTTTC

GTCTGGATCGCACTGAACCTACGCTGAATACCGCCATTCCCGGCGACCCGAGAGACACCACCACGCCGCGGGCGATGGCGCAGACGTTGCGTCA

GCTTACGCTGGGTCATGCGCTGGGCGAAACCCAGCGGGCGCAGTTGGTGACGTGGCTCAAAGGCAATACGACCGGCGCAGCCAGCATTCGGGCC

GGCTTACCGACGTCGTGGACTGTGGGTGATAAGACCGGCAGCGGCGGCTACGGCACCACCAATGATATTGCGGTGATCTGGCCGCAGGGTCGTG

CGCCGCTGGTTCTGGTGACCTATTTTACCCAGCCGCAACAGAACGCAGAGAGCCGCCGCGATGTGCTGGCTTCAGCGGCGAGAATCATCGCCGA

AGGGCTG > E. coli MS172-CIT-PAK

TGGCCAGAACTGACAGGCAAACAGTGGCAGGGTATCCGCCTGCTGCACTTAGCCACCTATACGGCAGGCGGCCTACCGCTGCAGATCCCCGATG

ACGTTAGGGATAAAGCCGCATTACTGCATTTTTATCAAAACTGGCAGCCGCAATGGACTCCGGGCGCTAAGCGACTTTACGCTAACTCCAGCAT

TGGTCTGTTTGGCGAGCTGGCGGTGAAACCCTCAGGAATGAGTTACGAAGAGGCAATGACCAGACGCGTCCTGCAACCATTAAAACTGGCGCAT

ACCTGGATTACGGTTCCGCAGAACGAACAAAAAGATTATGCCTGGGGCTATCGCGAAGGGAAGCCCGTACACGTTTCTCCGAGACAACTTGGCG

CCGAAGCCTATGGCGTGAAATCCAGCGTTATTGATATGGCCCGCTGGGTTCAGGCCAACATGGATGCCAGCCACGTTCAGGAGAAAA