ABSTRACT
The aim of the study was to detect extended spectrum B-lactamase (ESBL) in Klebsiella pneumoniae isolated from patients’ sputum attending public hospitals in Abia State. A total of 64 sputum specimens were collected from public hospitals in Abia State, majorly; Federal Medical Centre, Umuahia, Abia State Specialist Hospital, Umuahia and Seventh Day Adventists Hospital, Aba between the period of June and September 2017. Bacteriological tests of sputum specimens were performed for Klebsiella pneumoniae including inoculation on MacConkey agar and blood agar. The identity of the isolates was confirmed by biochemical tests. Out of 64 sputum specimens, 21 (32%) Klebsiella pneumoniae were recovered from patients’ sputum. Susceptibility test were carried out for each Isolate and samples 14, 55, 58 and 63 had highest susceptibility and Ceftazidime (CAZ 30µg), gentamicin (GEN 10µg), Ofloxacin (OFL 5µg) Nitrofurantoin (NIT 300µg) and Aztreonam (ATM 30µg) shows the highest susceptibility pattern. While Ciprofloxacin (CPR 5µg), Augumentin (AUG 30µg) and Ceftriaxone (30µg) showed the lowest susceptibility pattern. Screening tests for ESBLs were performed against the 21 Isolates and 15 were sensitive while 6 were resistant. Double-disc synergy test (DDST) was done for this 15 isolates and 9 were positive and 6 were negative. This study showed that Klesiella pneumoniae is considered to be one of ESBLs producers.
TABLE OF CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgements iv
Table of Contents v
List of Tables vi
List of Figures viii
Abstract ix
Chapter One
1.0 Introduction 1
1.1 Statement of the Problem 4
1.2 Aim of the Study 5
1.3 Objective of the Study 5
Chapter Two
Literature Review
2.0 History 6
2.1 Clinical Significance 6
2.2
Antimicrobial Resistance Mechanism in Klebsiella Species 7
2.3 Beta-Lactam (Β-Lactam)
Antibiotics 7
2.3.1 Structure 7
2.3.2 Mechanism of Action 10
2.4 Overview of Beta-Lactamases 10
2.4.1 Synthesis, Location and Mode
of Transfer of Beta-Lactamases 12
2.4.2 Classification of Beta-Lactamases 12
2.4.2.1 Functional Classification of
Beta-Lactamases 12
2.4.2.2 Molecular Classification 14
2.5
Βeta-Lactamase Active Site and Hydrolytic Mechanisms 17
2.5.1 Serine Β-Lactamases
(Ser-Β-Lactamase) 17
2.5.1.1 Mechanism of Hydrolysis of Ser-Β-Lactamases 19
2.5.1.2 Β-Lactamase Inhibitors and
Pathway of Inhibition by Clavulanic Acid 21
2.6 Definition of Extended-Spectrum
B–Lactamases (Esbls) 23
2.7 Diversity of ESBLS Types 24
2.7.1 SHV 24
2.7.2 TEM 25
2.7.3 CTX-M 26
2.7.4 OXA 26
2.8 Epidemiology of ESBLS 27
Chapter Three
3.0 Materials and Methods 28
3.1 Study Area 28 3.2 Collection of Specimen 28
3.3 Sterilization Method 28
3.4 Isolation and Identification of
Bacteria from Patients 28
3.4.1 Colony Purification 29
3.4.2 Gram Staining 29
3.4.3 Motility Test 29
3.4.4 Wet Mount Preparation 30
3.5 Biochemical Test 30
3.5.1 Catalase Reaction 30
3.5.2 Citrate Test 30
3.5.3 Oxidase Test 31
3.5.4 Methyl red test 31
3.5.5 Indole test 31
3.5.6 Urease test 32
3.6 In-Vitro Antimicrobial
Susceptibility Test 32
3.7 ESBL Detection 32
Chapter Four
Result
4.0 Result 34
Chapter Five
Discussion,
Conclusion and Recommendation
5.1 Discussion 40
5.2 Conclusion 41
5.3 Recommendation 41
References 42
Appendix I 47
Appendix II 48
Appendix III 49
Appendix IV 50
LIST OF TABLES
|
Table
|
Title
|
Page
|
|
2.1
|
Classification scheme for
beta-lactam antibiotics, based on chemical structure
|
9
|
|
2.2
|
Evolution of functional
classification of beta-lactamases
|
15
|
|
2.3
|
Tabular Representative of
Functional And molecular Characteristics of the Major Groups of β-Lactamases
|
16
|
|
2.4
|
Conserved motifs (elements) in the
active site of Ser-β-lactamases
|
18
|
|
4.1
|
Incidence and percentage
occurrence of the K. pneumoniae in sputum samples
|
35
|
|
4.2
|
Identification and
Characterization of Klebsiella
pneumoniae Isolate
|
36
|
|
4.3
|
Antibiotics susceptibility pattern
of the isolated Klebsiella pneumoniae
Isolates from the sputum samples
|
37
|
|
4.4
|
Screening for ESBLs on CLSI
breakpoint
|
38
|
|
4.5
|
Confirmation of ESBLs among the K. pneumoniae Isolates based on the
Double disc synergy text (DDST)
|
39
|
LIST OF FIGURES
|
Figure
|
Title
|
Page
|
|
2.1
|
Action of Ser- β-lactamases
(Matagne et al., 1998)
|
20
|
|
2.2
|
Mechanism of action of clavulanate
and amoxicillin
|
22
|
|
2.3
|
Representation of the general
mechanism of action of irreversible inhibitors.
(I= Inhibitor; E= Enzymes)
|
22
|
CHAPTER ONE
1.0 INTRODUCTION
Klebsiella pneumoniae (K. Pneumoniae) is a member of family Enterobacteriaceae. The organism can produce infection at a
variety of sites with the risk of being increased in patients with impaired
host defences (e.g. Diabetes mellitus, alcoholism, malignancy, chronic
obstruction pulmonary disease and glucorticoid therapy). The introduction of
the third generation cephalosporins (3 GC) was very much helpful in fighting
against the beta- lactamases in clinical practice (Paterson and Bonomo, 2005).
However, resistance to these antibiotics started to emerge rapidly. Because of
their increased spectrum of activity, especially against 3GC, these enzymes
were called extended spectrum beta-lactamases (ESBLs) (Bradford, 2001).
These
enzymes are produced by Enbterobacteriaceae mainly by Escherichia coli, Klebsiella
pneumoniae and K. oxytoca. They
have been detected in other Gram-negative bacilli such as Proteus species, Salmonella
species, Pseudomonas aeruginosa and
other Enteobacteriaceae. The first ESBL-producing organism was isolated in
Germany in 1983. Thereafter, such organisms were reported in the USA following
outbreaks of infections caused by these pathogens. The ESBL enzymes are capable
of hydrolyzing broad spectrum cephalosporins and monobactams but inactive against
cephamycins and imipenem. In addition, ESBL producing organisms exhibit
co-resistance to many other classes of antibiotics resulting in the limitation
of therapeutic option (Astal, et al., 2004).
For
this reason, the significance of such ESBL–mediated infections has been
increasingly reported worldwide. The ESBL have serine at their active site and
attack the amide bond in the lactam ring of antibiotics causing their
hydrolysis. Because of inoculum effect and substrate specificity, their
detection is a major challenge. Two indicators of ESBL are eight- fold
reductionsin MIC and
potentiation of the
inhibitor zone of
third generation cephalosporin in
the presence of clavulanic acid. For this reason, detection of ESBL, using
conventional antimicrobial susceptibility methods and delay in the recognition
and reporting of ESBL production by Gram negative bacilli is associated with
prolonged hospital stay, increase morbidity, mortality and health care
expenses. So, it becomes necessary to know the prevalence of these organisms
and to formulate the treatment policy (Mehrgan and Rahbar,2008).
The
National Committee for Clinical Laboratory Standards (NCCLS) recommended that
microbiology laboratories reported ESBL-producing isolates of E. coli and Klebsiella species are
resistant to all
penicillins, cephalosporins (including
cefepime), and aztreonam, irrespective of their individual in vitro test results. The presence of
ESBL in some K. pneumoniae and E. coli strains poses an important
challenge in clinical practice, since these organisms are common causes of
serious infections (Mehrgan and Rahbar, 2008).
Klebsiella is
ubiquitous in nature,
in human they
colonize the skin,
pharynx or gastro intestinal tract (GIT), they may also
colonize sterile wounds and urine, carriage rate varies with different studies.
Klebsiella may be regarded as normal
flora in many parts of colon, intestinal tract and in the biliary tract;
oropharyngeal carriage has been associated with endothelial incubation,
impaired host defences and antimicrobial use. K. Pneumoniae and K. oxytoca
are the 2 members of the genus responsible for the most human infections. They
are opportunistic pathogens found in the environment and in the mammalian
mucosal surfaces. The principle pathogenic reservoirs of infection are the GIT
of patient and the hands of personnel. Organisms can spread rapidly even leading
to nosocomial outbreaks (Baron, et al.,
1994).
Infection
with Klebsiella organisms occur in
the lung, where they cause destruction changes, necrosis, inflammation and
hemorrhage occur within lung tissue, sometimes producing thick, bloody, mucoid
sputum described as currant jelly sputum. Pneumonia caused by Klebsiella is a necrotizing process with
predilection for debilitated people; it has a high mortality rate with
approximately 50% even with antimicrobial therapy. The mortality rate
approaches 100% for person with alcoholism and bactremia. Klebsiella pneumoniae infection it causing inflammation of lung
characterized by fever, chills, muscle stiffness, chest pain, cough and short
of breath. Pneumonia may be caused by bacteria, virus or fungus and sometimes
by physical and chemical irritants. It occurs in patients with allergic but
young children and the elderly as well as immune compromised and immune defect
patients are especially at risk. Septic patients may be at increased risk for
acquiring antimicrobial resistant
pneumonia because of the prior exposure to various types of antibiotics,
factors that is known to play an important
role in the generating of
antimicrobial resistance. The bacterial spectrum and antimicrobial
resistance may vary temporarily and geographically; each institution must
undertake its own local evaluation. Such an evaluation may also be useful to
detect emerging trends of antimicrobial resistance (Wagenlehner, et al., 2008).
K. Pneumoniae are resistant to multiple antibiotics
which is plasmid mediated propriety of K.
Pneumoniae due to production of beta lactamase enzyme. Carbapenemase
producing K. Pneumoniae is the recent
addition to the pool of multi drug resistant nosocomial pathogen (Desphande, et al., 2006). Extensive use of broad
spectrum antibiotic in hospitalized patients has led to
both increased carriage
of Klebsiella and subsequently
the development of multidrug resistant strains that produce
extended spectrum beta lactamase (ESBL). These strains are highly virulent show
capsular type K55, and have an extraordinary ability to spread. Most outbreaks
are due to single clone or single gene. The bowel is the major site of
colonization with infection of the urinary tract, respiratory tract and wounds.
Bactremia and significant increased mortality have resulted from infection with
these species. In addition to prior antibiotic use, risk factors for infection
include the presence of an indwelling catheter, feeding tube or central venous
catheter, poor health status and treatment in intensive care units or nursing
home. Acquisition of these species has major problem in most hospital because
of resistance of multiple antibiotic and potential transfer of plasmid to other
organisms (Tortora, et al., 2004).
Morbidity and mortality rate are comparable to those for other Gram-ve
organisms that cause sepsis and septic shock. In neonatal units, outbreaks
caused by ESPL producing strains present a more serious problem and may be
associated with increased mortality (Tortora, et al., 2004).
ESBLs
are beta-lactamases that hydrolyse expanded spectrum cephalosporin with an
oxyimino side chain these include cefotaxime, ceftriaxone and ceftazidime as
well as the oxyimino-monobactam (aztreonam).
The ESBLs confer resistant to these antibiotics and related
oxyimino-beta lactams. Typically, they derived from genes for TEM-1, TEM-2 or
SHV-1 by mutations that alter the amino acid configuration around the active
site of these beta lactamases. This extends the spectrum of β.lactam antibiotics
susceptible to hydrolysis by these enzymes. An increasing number of ESBLs not
of TEM or SHV lineage has recently been described. The ESBLs are frequently
plasmid encoded (Emery and Weymouth, 1997). Carbapenems are the treatment of
choice for serious infections due to ESBL producing organisms, yet carbapenem
resistant isolates have recently been reported. ESBL producing organisms may
appear susceptible to some extended spectrum cephalosporins. However, treatment
with such antibiotic has been associated with high failure rates (Paterson, et al., 2003).
1.1 STATEMENT
OF THE PROBLEM
Carbapenem
resistant Enterobacteriaceae have become a serious health concern all over the
world. This is due to the indiscriminate use of antibiotics, poor sanitation in
hospitals and unhealthy life styles. Evidences of outbreaks and spread are well
documented. Unfortunately, no information or data about the incidence or
otherwise is available in Michael Okpara University of Agriculture, Umudike
despite the fact that it is a significant health problem even in the developed
world.
1.2 AIM
OF THE STUDY
The
aim of this study is to investigate the incidence of beta-lactamase including
extended spectrum beta-lactamase in Klebsiella
pneumoniae screened from sputum samples from patients in public hospitals,
Abia State.
1.3 OBJECTIVE
OF THE STUDY
•
To screen Klebsiella
pneumoniae from patients’ sputum samples attending public hospitals in Abia
State.
•
To determine the pattern of antibiotics susceptibility of the
isolates.
•
To determine the prevalence of extended spectrum beta-lactamase
(ESBLs) in sputum samples of patients attending public hospitals in Abia State.
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