ABSTRACT
Extended spectrum beta-lactamases (ESBL) are enzymes produced by members of the Enterobacteriaceae which can hydrolyze the beta-lactam antibiotics such as penicillins and cephalosporins, and thereby conferring antibiotic resistance on strains producing them. Bacterial isolates producing ESBLs have spread to different parts of the world. The ESBLs are encoded by several different genetic elements borne on the chromosome and plasmids. This study was carried out to investigate the prevalence of ESBL producing bacterial uropathogens among asymptomatic pregnant women in the study area and to characterize the genes associated with ESBL among the isolates. Mid-stream urine sample was collected from a total of 660 pregnant women between July and December 2018. A questionnaire to access the risk factors for acquiring ESBL producing bacteria was filled. The specimens were inoculated on MacConkey agar and incubated at 37oC for 24h. The biochemical characterization of the isolates was done using the Microbact 24E (Oxoid Ltd, UK). Antibiotic susceptibility testing was done by Kirby-Bauer disc agar diffusion method. The isolates were tested for the production of ESBL, using Double Disk Synergy test and CHROMagar ESBL. Genomic and plasmid DNA from ESBL producing strains was extracted and amplified using the Polymerase Chain Reaction (PCR) with primers for blaTEM, blaSHV and blaCTX-M-15genes. A total of 252 uropathogenic bacterial isolates were encountered. Of this number, 231 (92%) were ESBL producers. The distribution of bacterial pathogens were as follows: Enterobacter cloacae (25.7%) followed by Escherichia coli (20.2%), Klebsiella pneumoniae (16.3%), Citrobacter spp. (1.2%), and Hafnia alvei (6.7%). E. cloacae were the most frequently isolated ESBL producer (25%), followed by E. coli (19%) and K. pneumoniae (15%). There was significant association (P<0.005) between age, marital status, previous use of antibiotics, personal hygiene and parity with the occurrence of ESBL producing bacteria. Extended spectrum beta-lactamase producers revealed a higher level of antibiotic resistance (90%) compared to non-ESBLs. Carbapenems were the most effective treatment options for these bacteria. Genotypic characterization of the ESBL producing isolates showed blaCTX-M-15 to be the most prevalent (26%). The carriage of multiple bla genes was low, ranging from 2-6 % of different combinations. This study has shown the existence of multiple bla genes in the Gram-negative bacterial isolates from pregnant women in Ikot Ekpene, Eket and Oron Local Government Areas. This calls for appropriate antibiotic use through creating awareness in order to combat the potential negative effects of the spread of resistant bacteria carrying genes for resistance to extended spectrum beta-lactam antibiotics.
TABLE
OF CONTENTS
TITLE PAGE
Title
Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of Contents vi
List
of Tables x
List
of Figures xi
List
of Plates xii
List
of Appendices xiii
Abstract xiv
CHAPTER 1: INTRODUCTION
1.1 Background
of the Study 1
1.2 Statement
of Research Problem 3
1.3 Justification
of the Study 4
1.4 Aim
and Objectives of the Study 4
1.5 Null
Hypothesis 5
CHAPTER 2: LITERATURE
REVIEW
2.1
Asymptomatic Bacteriuria 6
2.2 Classification
of Urinary Tract Infections 6
2.2.1. Complicated urinary tract infections 6
2.2.2
Uncomplicated urinary tract infections 7
2.2.3 Upper
urinary tract infections 7
2.3 Pathogenesis of Urinary
Tract Infections 8
2.4 Risk
Factors for Asymptomatic Bacteriuria in Pregnancy 9
2.5 Prevalence
of Asymptomatic Bacteriuria in Pregnancy 10
2.6 Virulence
Factors of Uropathogens 11
2.7 Immunology
of the Urinary Tract 12
2.8 Defense
against Uropathogens 13
2.9 Antibiotic
Use in Pregnancy 14
2.10 Commonly Implicated Organisms in
Asymptomatic Bacteriuria 16
2.11 Classification
of Antibiotics 20
2.12 Antibiotic Resistance 33
2.12.1 The advent of antibiotic resistance 34
2.12.2 Causes of antibiotic
resistance 35
2.12.3 Antibiotic resistance transmission 36
2.12.4 Antibiotic resistance and bacterial virulence 37
2.12.5 Mechanism of bacterial resistance 38
2.12.6 Multiple antibiotic resistance index 42
2.12.7 Microbact-24E bacterial identification system 43
2.13 The Biochemical Resistance Mechanism 44
2.14 Beta-lactamases 47
2.15 Beta-lactamase Classifications 48
2.15.1 Mode of action of beta-lactamases 51
2.16 Bacterial Resistance Mechanism to
Beta-lactam drugs 52
2.17 Earliest and Recent β-lactamase
Inhibitor Combinations 54
2.18 Penicillin-binding
Protein (PBP), the Targets of β-lactam
Antibiotics 57
2.19 Extended Spectrum Beta-lactamases (ESBLs) 59
2.19.1 Types of ESBLs 60
2.20 Epidemiology
of ESBL producers 64
2.21 Risk
of Acquiring ESBL Infections with ESBL Producing Bacteria 67
2.22 Detection
of ESBLs in Clinical Laboratory 67
2.22.1 Phenotypic methods 68
2.22.2 Double disk synergy test
(DDST) 68
2.22.3 CLSI phenotypic
confirmatory tests (PCT) 69
2.22.4 ESBLs confirmatory test 71
2.23 Identifying Genes that encode resistance to antibiotics 72
2.23.1
Polymerase chain reactions (PCR) 73
2.23.2 DNA microarrays 74
2.24 PCR-based DNA Finger Printing
Technique 75
2.24.1
Random Amplified Polymorphic DNA (RAPD) 75
2.24.2
Agarose gel electrophoresis 76
2.25 Control of Antimicrobial Resistance 77
CHAPTER 3: MATERIALS AND METHODS
3.1 Study Area 79
3.2 Study Design 79
3.3 Study Population 80
3.4 Inclusion and Exclusion Criteria 80
3.5 Ethical Approval 80
3.6 Sample Size 80
3.7 Sample Collection 81
3.8 Specimen Processing 81
3.8.1 Biochemical identification of
isolates 82
3.8.2 Identification
of bacterial isolates by PCR and DNA sequencing 84
3.8.3 Antibiotic susceptibility test 85
3.8.4 Detection
of Extended-Spectrum Beta-lactamase 85
3.8.5 Double disk synergy test 85
3.9 Molecular Studies 86
3.9.1 Plasmid DNA extraction 86
3.9.2 Genomic DNA
extraction 87
3.9.3 Quantification of plasmid DNA 88
3.9.4 Amplification
of blaCTX-M-15, SHV and TEM resistant gene by PCR 88
3.9.5 Random
amplified polymorphic DNA 91
3.9.6 Statistical analysis 93
CHAPTER 4:
RESULTS AND DISCUSSION
4.1 Results 94
4. 2 Discussion 131
CHAPTER 5: CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion
149
5.2 Recommendations 150
References 151
Appendices 178
LIST
OF TABLES
|
TABLE
|
TITLE
|
PAGE
|
|
2.1
|
Antibiotic use in
pregnancy
|
15
|
|
2.2
|
Beta-lactamases in well
characterized families
|
50
|
|
3.1
|
Oligonucleotide sequences
of the primer pairs
|
90
|
|
3.2
|
Oligonucleotide sequences
for RAPD PCR
|
92
|
|
4.1
|
Socio demographic characteristics of
women investigated for asymptomatic bacteriuria from Ikot Ekpene, Eket and
Oron General Hospital in Akwa Ibom State.
|
95
|
|
4.2
|
Distribution of ESBL producing bacteria in the urine of asymptomatic
Pregnant women in Ikot Ekpene, Eket and Oron General Hospital in Akwa Ibom
State.
|
97
|
|
4.3
|
Socio
demographic characteristics and recovery of ESBL uropathogens from Urine of
pregnant women in Ikot Ekpene, Eket and Oron LGA
|
100
|
|
4.4
|
Distribution
of ESBLs producing bacteria in
urine of asymptomatic pregnant women in Ikot Ekpene, Eket and Oron LGA
|
104
|
|
4.5
|
Distribution
of ESBL and non-ESBL producing E. cloacae, E. coli and K. pneumoniae in urine of asymptomatic
pregnant women in Ikot Ekpene, Eket and Oron LGA
|
106
|
|
4.6
|
Comparison of Confirmed ESBL
producers detected by Chrom agar ESBL and DDST
|
108
|
|
4.7
|
Antibiotic susceptibility pattern of ESBL producing isolates
|
110
|
|
4.8
|
Antibiotic susceptibility
pattern of non- ESBL producing isolates
|
114
|
|
4.9
|
Multiple antibiotic resistant indices
of ESBL producing E. coli, E. cloacae,
and K. pneumoniae
|
119
|
|
4.10
|
Plasmid DNA Plasmid profile of
ESBL producing E. coli, K. pneumoniae and E. clocae
|
121
|
LIST
OF FIGURES
|
FIGURE
|
TITLE
|
PAGE
|
|
2.1
|
Chemical
structure of beta-lactam structure
|
22
|
|
2.2
|
Structure
of cephalosporins
|
24
|
|
2.3
|
Structure
of monobactam
|
25
|
|
2.4
|
Structure
of carbapenem
|
26
|
|
2.5
|
Structure
of macrolide
|
27
|
|
2.6
|
Structure
of tetracycline
|
28
|
|
2.7
|
Structure
of aminoglycoside
|
29
|
|
2.8
|
General structure of
sulphanomides
|
30
|
|
2.9
|
Relationship between
the use of antibiotic and resistance development
|
36
|
|
2.10
|
Gene transfer mechanism in
bacteria
|
42
|
|
2.11
|
Picture of Microbact 24E strip assay
|
43
|
|
2.12
|
Target sites of antibiotics
|
46
|
|
2.13
|
Structural similarity between beta-lactamase and
PBP
|
51
|
|
2.14
|
Mechanism of hydrolysis of beta-lactam
|
53
|
|
2.15
|
Earliest
β-lactamase inhibitors
|
55
|
|
2.16
|
Positive result for Double disk synergy test
|
69
|
|
2.17
|
Negative result for DDST test
|
69
|
|
2.18
|
Phenotypic confirmatory test for SHV-derivative
extended-spectrum beta-lactamases in several Enterobacteriaceae
|
70
|
|
4.1
|
Plots
showing the antimicrobial resistance pattern of ESBL producing and non-ESBL
producing strains of E. cloacae, E.
coli and K. pneumoniae
recovered from urine of asymptomatic pregnant women in Akwa Ibom State.
|
117
|
|
4.2
|
Distribution of gene encoding blaTEM, blaSHV
and blaCTX-M-15 in ESBL producing E.
cloacae, E. coli and K. pneumoniae
|
123
|
|
4.3
|
Phylogenetic
Tree of isolates showing their affiliation to other known lineages.
|
132
|
LIST OF PLATES
|
PLATE
|
TITLE
|
PAGE
|
|
1
|
Plasmids profile of ESBL producing K. pneumoniae, Enterobacter cloacae and E. coli.
|
124
|
|
2
|
Agarose gel
electrophoresis of amplified blaTEM
PCR product
|
125
|
|
3
|
Gel
electrophoresis of amplified blaCTX-M-15 PCR product
|
126
|
|
4
|
Agarose
gel electrophoresis of amplified blaSHV PCR product
|
127
|
|
5
|
Agarose gel
electrophoresis of RAPD PCR.
|
128
|
|
6
|
ESBL producing
Isolate showing reduced susceptibility to Third generation Cephalosporin
|
129
|
|
7
|
Culture
of ESBL producing isolate with amoxicillin disc.
|
130
|
|
8
|
Growth of Klebsiella, Citrobacter and Enterobacter Spp. on Chrom Agar
|
131
|
LIST OF APPENDICES
|
APPENDIX
|
TITLE
|
PAGE
|
|
I
|
Study location map
|
180
|
|
II
|
Letter
of ethical approval from Akwa Ibom State Ministry of Health
|
181
|
|
III
|
Clinical
Laboratory and Standards Institute (CLSI) Performance Standard for
Antimicrobial Susceptibility Testing (M100, 28th Edition 2018).
|
182
|
|
IV
|
Synthesis
report by InqabaBiotec, West Africa
|
183
|
|
V
|
Reagents
|
188
|
|
|
|
|
|
VI
|
Equipment
|
192
|
|
VII
|
Information
letter/consent form
|
193
|
|
VIII
|
List of
plates
|
194
|
|
IX
|
List of abbreviations
|
195
|
|
X
|
Result of biochemical tests
|
197
|
|
XI
|
Identification of isolates by Sanger
sequencing
|
219
|
CHAPTER
1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Urinary tract infections (UTIs) are recurrent infections with
potentially fatal symptoms. The rise in antibiotics resistance, together with our heightened
awareness of the adverse effects of broad-spectrum antibiotics on beneficial
host microbiota, has generated difficulties in the existing treatment trend for
UTIs (Roger and Scott, 2020). The most prevalent form
of infection among women is urinary tract infection (UTI) most notably pregnant
women, due to hormonal and morphological changes that occur during pregnancy. In various studies, this prevalence
ranges between 5 and 20% and differs depending on the health status in the
respective countries (Tadesse et
al., 2014; Szweda and Jóźwik,
2016)
There are three distinct clinical types of UTI linked to
pregnancy; asymptomatic bacteriuria, cystitis, and pyelonephritis. Obstetric complications can lead to death and small
birth weights (Oladeinde et
al., 2015; Szweda and Jóźwik,
2016). The most common reason for antibiotic use in primary care is
urinary tract infections (UTIs) (Plate et al., 2020). Abuse of antibiotics used
in treating UTIs contributes to high incidence of resistance (Rodriguez et al., 2019).
Studies showed that asymptomatic infection alone affects
2–12 % of women (Steele et al., 2017).
Urinary tract infection was found to be prevalent in 12.5 % of
pregnant women (Azami et al., 2019). A prevalence rate of 31% and 6.4%
has been reported in Ogun State and Akwa Ibom State respectively (Ochei et
al., 2018; Nseobong
et al., 2019). Other researchers reported a prevalence rate of
21.2%, 29.1% and 6.3% respectively in Ethopia, Kenya, and Iraq (Tadesse et
al., 2018a; Taye et
al., 2018; Goruntla et al.,
2019).
Beta-lactam antibiotics are one of the commonly used drug classes
with a wide range of therapeutic applications. Since their introduction in the 30s
of the twentieth century they, have dramatically
altered the scenario of the war against bacterial infectious diseases. However,
their use conflicts with the disturbing occurrence of antimicrobial resistance,
which is now a global health problem (Thakuria et al.,
2013). Resistance to β-lactams is growing increasingly as new
β-lactamases (enzymes which destroy β-lactams) are discovered daily; example is
the discovery of β-lactamases (ESBL) with the ability to inactivate certain
cephalosporins by hydrolyzing the beta-lactam ring. Extended spectrum betalactamases diversity is
rapidly growing, so far
more than 170 variants have been identified for a single genotype, encoding the
bla CTX-M ESBL (Sadeeq et al., 2018 and Warawan et al., 2018).
Extended spectrum beta-lactamases are frequently found in the
Enterobacteriaceae family of Gram negative organisms, especially the Klebsiella species, Escherichia coli and E.
cloacae but also in other species such as Pseudomonas aeroginosa, Citrobacter
species and Enterobacter species (Giwa
et al., 2018). Several studies
have identified the prevalence of ESBLs producing uropathogens in Nigeria
(Onanuga et al., 2019). In
North Western Nigeria, Giwa et al.
(2018) recorded a prevalence rate of 34.3 %. Kalaivani et al. (2018) have also documented a prevalence rate of 73.3 %. In
addition, Oloso et al. (2018)
recorded a nationwide geographic distribution trend of antimicrobial resistance
based on geographical zones which showed that the highest number of reports
were from South West Nigeria and, in descending order, from South-South,
North-West, North-Central, North-East and South-East as lowest.
Clinically, ESBLs limit the efficacy of
β-lactams including extended-spectrum cephalosporins, and are associated with
high morbidity and mortality (Rawat
and Nair, 2010). More than 2.8 million antimicrobial-resistant infections occur
in the U.S. each year, and more than 35,000 people die as a result. In 2017,
there were an estimated 197,400 cases of ESBL-producing Enterobacterales among
hospitalized patients and 9,100 estimated deaths in the United States (CDC,
2019).
Colonization of pregnant women with extended spectrum β-lactamase
(ESBL)-producing micro-organisms might lead to the transmission of these bacteria to
neonates resulting in severe and extreme morbidity due to these pathogens (Jalilian et al., 2019). Detection and case management of an ESBL infection is critical now
as ESBL infections tend to be ascribable to resistant organisms
(Sahni et al., 2018). The
aim of this study is to investigate the prevalence of ESBL producing
uropathogens among pregnant women in the study area and to characterize the
genes associated with ESBL among the bacterial isolates.
1.2 STATEMENT
OF THE PROBLEM
Antibiotic
resistance has become a global issue due to the exponential increase of
multidrug-resistant infections. In particular, there is a high rate of urinary
tract infection and treatment failure in pregnant women, which has resulted in
increased mortality and morbidity, as well as longer hospital stays. Antibiotics
are available for purchase without prescription and even when prescribed, there
is poor patients’ compliance in underdeveloped nations. There is inadequate information on the prevalence of bacteria
that produce ESBLs in pregnant women from Ikot Ekpene, Oron, and Eket Local
Government Areas of Akwa Ibom. This study will bridge the knowledge gap by
determining the prevalence of ESBLs producing organism in these three study
areas and also characterise the ESBLs genes harboured by these bacteria using
molecular biology. The knowledge of antibiotic susceptibility pattern of ESBLs
producing isolates from the pregnant women will guide the clinicians in
prescriptions and will reduce maternal mortality and treatment failure hence
help in more efficient health care of pregnant women.
1.3 JUSTIFICATION
OF THE STUDY
Owing
to the spread of multidrug-resistant bacteria, their encoding genes on easily
transferrable plasmids, and shortage of successful eradication methods for
virulent strains that exist in humans, ESBL-producing bacteria pose a difficult
challenge to address (De Kraker et al., 2013; Tacconelli et al.,
2014). The consequences of morbidity and death are numerous (O'Neill, 2016; Musicha
et al., 2017). Therefore, more information about the precise prevalence
of ESBL-producing bacteria and the characterization of antibiotic resistant
genes in pregnant women is necessary to help health policymakers and providers
deliver adequate prevention and treatment measures to pregnant women.
1.4 AIMS
AND OBJECTIVES
The aim of this study is to
investigate the prevalence of ESBL producing uropathogens among pregnant women
in the study area and to characterize the genes associated with ESBL among the
bacterial isolates.
Specific objectives
1.
To access the risk factors
for acquiring ESBL producing bacteria using questionnaire.
2.
To isolate and identify bacterial uropathogens.
3.
To assess the prevalence of bacterial uropathogens among
pregnant women attending antenatal at General Hospital, Ikot Ekpene, Eket and
Oron.
4.
To determine the antibiotic susceptibility pattern of
bacterial isolates from urine specimens of pregnant women attending antenatal
at General Hospital, Ikot Ekpene, Eket, and Oron.
5.
To screen resistant isolates for Extended Spectrum Beta
Lactamases (ESBL) production using two different methods (Double disk synergy
test and Chrom Agar ESBL).
6.
To characterize the ESBL genes in ESBLs producing isolates
using Polymerase Chain Reaction (PCR).
7.
To conduct a molecular epidemiology of some of the isolates
using RAPD.
1.5 NULL
HYPOTHESIS
1. At
least 90% of the bacterial isolates from the urine specimen of pregnant women
in the study area are not susceptible to all commonly used antimicrobial agents
available in the study area.
2. Antimicrobial
resistance that may be detected among the bacterial isolates from the pregnant
women in the study area is not due to ESBL production.
3. Antimicrobial
resistant bacterial isolates in the study area carry unrelated resitance genes.
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