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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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14
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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15
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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18
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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28
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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30
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
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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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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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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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(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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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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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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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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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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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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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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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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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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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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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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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
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___________________________________________________________________________
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
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___________________________________________________________________________
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
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___________________________________________________________________________
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