A Study of Bacterial Agents Associated with Diarrhoeal Cases in the Federal Capital Territory, Abuja

A Study of Bacterial Agents Associated with Diarrhoeal Cases in the Federal Capital Territory, Abuja.

ABSTRACT

One hundred and six faecal samples were analyzed to detect the sporadic involvement of E. coli O157:H7, other Enterohaemorrhagic E. coli and common bacterial pathogens in enteric infections within the Federal Capital Territory, Abuja.

Enrichment culture in modified peptone water followed by immunomagnetic separation (IMS) with magnetic beads coated with an antibody against Escherichia coli O157 was used in addition to Direct culture on Sorbitol MacConkey agar and Cefixime-Tellurite Sorbitol MacConkey agar for the isolation of E. coli O157: H7.

For the identification of non-O157 STEC among the Non-sorbitol fermenting E. coli isolated, PCR for virulence markers was used. Verocytotoxicity Assay was used to detect free faecal toxin in the stool samples for the identification of other Enterohaemorrhagic E. coli (EHEC) infections.

Routine methods were used for the isolation of common bacterial pathogens and for identification of isolates. Antimicrobial susceptibility tests were carried out on the isolates using the disc diffusion technique while interpretation of zone sizes was done using the NCCLS interpretative chart.

Stool Microscopy for ova or cyst of parasites was also done using the formol ether sedimentation technique. No E. coli O157:H7 was isolated.

However, free faecal toxin was demonstrated in 16 (15.09%) of the 106 faecal samples analyzed indicating the involvement of non-O157 VTEC in diarrhoeal diseases in the Federal Capital Territory, Abuja. None of the non-sorbitol fermenting E. coli was found to be STEC.

Enteric pathogens isolated from the study include V. cholerae (1.9% of the faecal sample), S. typhi (1.9%), S. paratyphi B (0.95%), S. paratyphi C (0.95%), unidentified Shigella species (0.95%), Shigella dysenteriae A1 (1.9%), Shigella flexneri (4.7%) Entamoeba histolytica (0.95%), Hookworm (0.95%), Schistosoma mansoni (0.95%) and Candida spp (6.6%).

Sorbitol MacConkey agar was found to be more sensitive than the Deoxycholate Citrate agar and Selenite F enrichment broth routinely used for the isolation of Shigella flexneri.

Thus, for optimal isolation of Shigella flexneri, the inclusion of MacConkey agar among the media used for routine cultures is recommended. V. cholerae isolates were 100% sensitive to tetracycline and ciprofloxacin, Shigella species were 100% resistant to tetracycline and 100% sensitive to ciprofloxacin.

The Salmonella isolates were 100% sensitive to ciprofloxacin while showing varied degrees of resistance to tetracycline, chloramphenicol and trimethoprim-sulphamethoxazole.

These results show the need to promote rational use of antibiotics in FCT. The results, also, suggest that the incidence of non-O157 VTEC involvement in diarrhoeal diseases may be quite common and should be considered as an agent in diarrhoeal illnesses especially among children.

TABLE OF CONTENTS

Certification–ii
Declaration —iii
Dedication—-iv
Acknowledgements—–v
Table of contents—-viii
List of tables—-xiv
List of figures—xvi
List of plates—xvii
List of appendices——xviii
List of abbreviations–xix
Abstract——-xxi

CHAPTER ONE: INTRODUCTION

1.1 BACKGROUND OF THE STUDY———1
1.1.1 Diarrhoeal Disease and its prevalence —–1
1.1.2 Principal Mechanisms of Diarrhoea ——-2
1.1.3 Agents of Gastrointestinal Infection—–4
1.2 Limitations to the study——11
1.3 PREAMBLE ——11
1.4 AIMS AND OBJECTIVES—12

CHAPTER TWO: LITERATURE REVIEW

2.1 SALMONELLA——-13
2.1.1 Description of the Genus—13
2.1.2 Taxonomy of Salmonella–13
2.1.3 Nomenclature for Salmonella and distribution of serotypes—-14
2.1.4 Pathogenicity——15
2.1.5 Epidemiology of Salmonellosis—-16
2.1.6 Isolation procedures—-18
2.1.7 Identification——-20
2.1.8 Antimicrobial susceptibilities—–20
2.2 SHIGELLA —-21
2.2.1 Description of the genus—-21
2.2.2 Taxonomy of Shigella ——21
2.2.3 Nomenclature of Shigella —22
2.2.4 Pathogenicity——–22
2.2.5 Epidemiology of Shigellosis——23
2.2.6 Isolation procedures—24
2.2.7 Identification ——–25
2.2.8 Antimicrobial Susceptibilities—-26
2.3 VIBRIO —————27
2.3.1 Description of the Genus —–27
2.3.2 Taxonomy —–28
2.3.3 Pathogenicity ——29
2.3.4 Epidemiology of Vibrio Species—–33
2.3.5 Isolation Procedures ———36
2.3.6 Identification ——–38
2.3.7 Antimicrobial Susceptibilities —38
2.4 ESCHERICHIA —–39
2.4.1 Description of the Genus —-39
2.4.2 Pathogenesis—–39
2.4.3 Diarrhoeagenic E.coli —–40
2.4.4 Isolation Methods for ETEC, EPEC, EIEC and the Putative Diarrhoeagenic E.coli –43
2.4.5 Screening procedures for ETEC, EPEC and EIEC strains —–
2.4.6 Antimicrobial Susceptibilities ——–45
2.5 ESCHERICHIA COLI O157:H7 AND OTHER ENTEROHAEMORRHAGIC ESCHERICHIA COLI —45
2.5.1 History of E.coli O157:H7 —-46
2.5.2 Nomenclature of Enterohaemorrhagic Escherichia coli –48
2.5.3 Epidemiology —–51
2.5.4 Transmission —61
2.5.5 Epidemiological Investigation of Verocytotoxin-producing Escherichia coli by Molecular Epidemiology-69
2.5.6 Pathogenesis —–77
2.5.7 Pathophysiology -83
2.5.8 Immunity —-84
2.5.9 Clinical Manifestations —86
2.5.10 Complications of Infections with Enterohaemorrahagic E.coli —87
2.5.11 Risk factors that determine the outcome of EHEC infections–90
2.5.12 Diagnosis ——-93
2.5.13 Therapy/Treatments —-104
2.5.14 Convalescent faecal Excretion of E.coli O157:H7 —–111
2.5.15 Recognition of and Response to outbreaks ——112

CHAPTER THREE: MATERIALS AND METHODS

3.1 ETHICAL CONSIDERATIONS ——-115
3.2 STUDY CENTRES ———115
3.3 INCLUSION CRITERIA —–116
3.4 SAMPLING TECHNIQUE —–117
3.5 DETERMINATION OF SAMPLE SIZE —-117
3.6 COLLECTION OF SAMPLES ——118
3.7 ISOLATION OF ESCHERICHIA COLIO157: H7—–119
3.7.1 Direct culture of E.coli O157:H7 -119
3.7.2 Enrichment culture followed by Immunomagnetic separation —–120
3.8 POLYMERASE CHAIN REACTION (PCR) FOR THE DIAGNOSIS OF DIARRHOEAGENIC ESCHERICHIA COLI —-122
3.8.1 Introduction ——-122
3.8.2 Primers and Probes—– 123
3.8.3 Extraction of DNA Template——124
3.8.4 Mastermixes for one sample—–125
3.8.5 Controls———127
3.8.6 Procedures ——–127
3.9 DETECTION OF OTHER SEROTYPES OF ENTEROHAEMORRHAGIC E.COLI (EHEC) 128
3.9.1 Preparation of faecal extracts —–129
3.9.2 Verocytotoxicity Assay ——-129
3.10 ISOLATION OF SALMONELLA/SHIGELLA SPECIES —–132
3.11 CHARACTERIZATION OF SHIGELLA FLEXNERI ISOLATES ——133
3.12 ISOLATION OF VIBRIO CHOLERAE ——–134
3.13 ANTIMICROBIAL SUSCEPTIBILITY TESTING —–135
3.14 EXAMINATION OF FAECAL SPECIMENS FOR PARASITES/YEASTS (FUNGI) —-135
3.15 PROCEDURE FOR EXAMINATION OF ALL THE FAECAL SAMPLES —137

CHAPTER FOUR: RESULTS 

CHAPTER FIVE: DISCUSSIONS

SUMMARY OF RESULTS —-185
CONTRIBUTION TO KNOWLEDGE -187
REFERENCES ——188
APPENDICES ———215

INTRODUCTION

1.1. Background of the Study

1.1.1 Diarrhoeal Disease and its Prevalence Diarrhoeal diseases of the bowel make up a veritable augean stable of entities. Microbiologic agents cause many; others arise in the setting of malabsorptic disorders and idiopathic inflammatory bowel disease (Crawford, 1999).

A precise definition of diarrhoea is elusive, given the considerable variation in normal bowel habits.

An increase in stool mass, stool frequency or stool fluidity is perceived as diarrhoea by most patients (Crawford, 1999).

For many individuals, this consists of daily stool production in excess of 250 gm, containing 70 – 95% water. More than 14 litres of fluid may be lost per day in severe cases of diarrhoea (i.e the equivalent of the circulating blood volumes).

Diarrhoea is often accompanied by pain, urgency, perianal discomfort, and incontinence. Low-volume, painful, bloody diarrhoea is known as dysentery (Crawford, 1999).

Diarrhoeal disease is a common cause of morbidity and mortality, with worldwide distribution and is of significant public health concern (Snyder & Merson, 1982; Ling & Chen, 1993).

It is one of the commonest illnesses of children and one of the major causes of infant and childhood mortality in developing countries (Bern, Martins, de Zoysa, and Glass, 1992) of which Nigeria is one.

The magnitude of the problem cannot be overemphasized with an estimated 1,000 million episodes and 3.3 million deaths (range 1.5 – 5.1 million) occurring each year among under 5 year 2 olds (Bern et. al., 1992). In the general population, there are an estimated 4 billion episodes of diarrhoeal diseases largely foodborne and waterborne resulting in over 2.2 million deaths (Murray & Lopez, 1996; WHO, 2000).

Diarrhoeal disease caused by microbiologic agents is principally a foodborne and waterborne illness. Foodborne and waterborne illnesses are leading global health problems, accounting for more morbidity and mortality than tuberculosis and Malaria (Besser, Beebe, and Swaminathan, 2003).

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