PREVALENCE OF SHIGELLA FROM STOOL SAMPLES IN DIARRHEA PATIENTS

ABSTRACT

The prevalence of Shigella species in stool samples of diarrhea patient and their antibiogram, their studies. A total of (30) samples comprising 10 samples from each of three hospitals were analyzed for isolation, identification and susceptibility to different antibiotics. Results obtained showed that 3 species of Shigella were isolated; namely Shigella dysenteriae, Shigella sonnei and Shigella flexneriae. In the first hospital (hospital 1, the percentage occurrence of the isolate were 50%, 60% and 60% respectively, while the second hospital give occurrence of 60%, 40% and 50%, and in the third hospital (hospital 3) it was 40%, 50% and 30% respectively. Test on the antibiotic resistance pattern shows that the sensitivity and resistance of the different Shigella species to the different test drugs, vary with significant difference (P<0.05). Shigella flexneriae was resistant to tetracycline and ampycline but susceptible to chloramphenicol (21mm) ciprofloxacin (24mm), gentamicine (21.3mm) and clotrinoxazol (12.7mm). Shigella dysenteriae was resistance to tetracycline but susceptible to ampicillin (9.5mm), chlorophenical (15.0mm), gentamicin (12.50) and ciprofloxacin (18.5mm) while Shigella flexineriae was resistance to ampicillin and tetracycline but sensitive to chloramphenicol (17.3mm). Clotrimazole (17.0mm), gentamicin (23.7mm) and ciprofloxacin (23.6mm). Variations prevalence and antibiotic pattern of different Shigella species isolated from the diarrhea stools, was of significant difference (P<0.05)

TABLE OF CONTENTS

Title page                                                                                                                    i

Declaration                                                                                                                 ii

Certification                                                                                                                iii

Dedication                                                                                                                  iv

Acknowledgement                                                                                                      v

Table of contents                                                                                                        vi

List of tables                                                                                                               viii

Abstract                                                                                                                      ix

CHAPTER ONE

1.0        Introduction                                                                                              1

1.1       Causes of diarrhea                                                                                          5

1.2       Epidemiology of diarrhea                                                                               6

1.3       Aims and objectives                                                                                       6

1.4       Objectives                                                                                                       7

CHAPTER TWO

2.0       Literature review                                                                                            8

2.1       Causes of shigellosis                                                                                      12

CHAPTER THREE

3.0       Materials and methods                                                                                    14

3.1       Source of materials                                                                                         14

3.2       Sample/media preparation                                                                              14

3.3       Media preparation                                                                                           14       

3.4       Sterilization                                                                                                    15

3.5       Enumeration and identification of bacteria                                                    15

3.6       Identification of bacterial isolates                                                                  15

3.7       Gram staining                                                                                                 16

3.8       Biochemical cultural characteristics                                                              16

3.8.1    Catalase test                                                                                                    16

3.8.2    Coagulase test                                                                                                 16

3.8.3    Citrate test                                                                                                       17

3.8.4    Motility, indole, urease test (MIU)                                                                 17

3.8.5    Triple sugar iron test                                                                                       18

3.8.6    Oxidase test                                                                                                    18

3.9       Antibiotic sensitivity testing                                                                           18

3.10     Isolation of Shigella                                                                                        19

CHAPTER FOUR

4.0       Results                                                                                                            20

CHAPTER FIVE                                         

5.0       Discussion, recommendation and conclusion                                                23

5.1       Discussion                                                                                                       23

5.2       Recommendation                                                                                            24

5.3       Conclusion                                                                                                      24

References                                                                                                      25

LIST OF TABLES 

Table      Title                                                                Page

4.1: Occurrence of Shigella Isolate from Stool Samples in Diarrhea Patients           20

4.2: Antibiogram of Shigella species Isolated from Diarrhea Stool Samples                        21

4. 3: Characteristics of Bacteria Isolates from Diarrhea Stoll samples                        29

4.4: Antibiogram of Shigella species Isolated from Diarrhea Stool Samples                      30

CHAPTER ONE

1.0   INTRODUCTION

Diarrhea is the passage of unusually loose or watery stools, usually at least three times within 24 hour period. Prolonged diarrhea may lead to excessive loss of fluid, salt and nutrient in the faeces. The main cause of death from acute diarrhea is dehydration, which result from loss of fluid and electrolyte in stool. Another important cause of death is dysentery and under nutrition (Sinclair et al., 2003). Diarrhea is an important cause of under nutrition because patients eat less during diarrhea and their ability to absorb nutrients is reduced. Moreover, nutrient requirement is increased as a result of infection (Sinclair et al., 2003). Risk factors that predispose children to diarrhea include poor sanitation, poor social and economic status and malnutrition (Andu et al., 2002).Diarrhoeal diseases are major attendance at health facilities, a common cause of admission to many of the hospitals in the country, and a significant and often preventable cause of death. The clinical symptoms of diarrhea include the passage of frequent loose or watery stool without visible blood (Bahal et al., 2001), vomiting may occur and fever may be present. The most important cause of acute watery diarrhea in young children in Nigeria include rotavirus, enterotoxigenic Escherichia coli, Shigella, Campylobacter jejuni, and Cryptosporidia, Vibrio cholerae, Salmonella and enteropathogenic Escherichia coli (Bahal et al., 2001). Another clinical symptom of diarrhea is dysentery, which refers to diarrhea with visible blood in faeces, the symptoms of which include anorexia, rapid weight loss and damage to the intestinal mucosa by invasive bacteria. The organisms implicated in this type of diarrhea include Shigella, Campylobacter jejuni, Salmonella and very rarely Entamoeba histolytica (Bahal et al., 2001). Transmission of agents that cause diarrhea are usually by the faecal oral route, which include the ingestion of faecal contaminated water or food, person to person contact and direct contact with infected faeces. Host factors that increase susceptibility to diarrhea include under nutrition, current or recent measles and immune deficiency or immuno supression (Andu et al., 2002). Diarrhea disease is part of the social problems in Nigeria and in other developing countries in the tropics. Diarrhea disease is a leading cause of morbidity and mortality among young children in low income countries. The diarrhea specific mortality in children less than 5 years of age in Africa has been estimated at about 106 per 1000. Therefore, the prevalence is not fully understood and the isolating technique is rather too expensive (Olowe et al., 2003). Diarrhoea is a leading cause of morbidity and mortality in developing countries, particularly in children. Diarrhoea are caused by bacterial, viral and parasitic infections, as well as food intolerance, reaction to medicines, and other physiological disorders (Abrami et al., 1998). Escherichia coli and Shigella species have now been established asaetiological agents of diarrhoea diseases of humans in developing countries (Asghar, 2002; Ekwenye and Kazi, 2007), but remains as an occasional cause of diarrhoea among children in industrialized countries, particularly in settings such as day care. The risk of diarrhoea in developing countries is attributed to deficiencies in environmental sanitation and personal hygiene (Prado et al., 1998). Antimicrobial resistance among enteric pathogens is a serious problem in developing countries. Increased antimicrobial usage is the main driving force leading to evolution of drug-resistant bacteria (WHO, 2001). Tetracycline is not commonly used to treat diarrhoea in children between the ages of 0-5 yrs in Nigeria (Blake et al., 2003). In spite of this, tetracycline resistance rate is high. Studies showed that, once evolved, resistance genes could spread through the world’s bacterial populations, irrespective of the pattern of antimicrobial use in an area (O’Brien, 2002). Therefore, mechanisms other than selective pressure might exist for maintaining a resistant bacteria pool.

Pathogenic organisms have developed a number of elaborate mechanisms for acquiring and disseminating antibiotic resistance.  Diarrheal diseases constitute a major burden of disease in the world, especially in low and middle-income countries. Dehydration resulting from diarrhea causes approximately 1.8 million deaths every year. These illnesses are particularly dangerous for young children, who are more susceptible to dehydration and nutritional losses during the episode of acute diarrhea (Ahs et al., 2010). Diarrheal diseases can be caused by many etiological agents, but mainly by Entero bacteriaceae (Paniagua et al., 2007). Among the different pathogens responsible for diarrheal diseases, Shigella species play an important role in causing inflammatory diarrhea and dysentery (Hui et al., 2001). Shigellosis, an acute diarrheal disease, is caused Shigella species. Supply of untreated water, poor sanitation, and overcrowding contribute to the spread of shigellosis both by human contacts and supplies of contaminated water (Schroeder and Hilbi, 2008).   Diarrhoea is a significant health problem worldwide, especially in the developing world where adequate sanitation facilities are lacking (Okeke et al., 2000). Globally diarrhoeal diseases account for almost a fifth of all deaths of children below five years of age, with an estimated 2.2 million deaths annually (Black et al., 2003). Epidemiological studies of diarrhoea have been reported from several African countries including South Africa, Gabon, Egypt and Kenya (Sang et al., 1996).  In the year 2001, diarrhoea was the most common illness reported by the United States military service members deployed to Africa for strategic training and contingency operations. Out of 15,000 US military personnel who participated, more than 500 service members were affected by acute diarrhoea (Sanders et al., 2005). The service members represent an immunologically naïve group to the various enteric pathogens and are likely to be at higher risks for contracting acute infectious diarrhoea.  The causes of diarrhoea include a wide array of viruses, parasites and bacteria. Shigella, Salmonella, Cryptosporidium species and Giardia lamblia are found throughout the world while Campylobacter jejuni and cytotoxigenic Clostridium difficile are seen with increasing frequency in developed countries (Guerrant 1990). The bacterial pathogen most commonly associated with childhood diarrhoea is Escherichia coli and at least six categories have been described: entero pathogenic E. coli (EPEC); entero toxigenic E. coli (ETEC); entero invasive E. coli (EIEC); entero hemorrhagic E. coli (EHEC), also known as shigato xigenic E. coli (STEC); diffusely adherent E. coli (DAEC); and entero aggregative E. coli (EAEC). The associated clinical pictures comprise childhoodand traveller’s diarrhoea (ETEC), bloody diarrhoea and hemolytic uremic syndrome (EHEC), infantile diarrhoea (EPEC), and bacillary dysentery-like diarrhoea (EIEC). Entero aggregative E. coli have been associated with acute and persistent diarrhoea in children and adults in industrial and developing countries in Europe, America, Asia and Africa. New virulent enteric pathogens are emerging throughout the world, Africa included. A multi-drug resistant entero aggregative E. coli, O44, which is associated with acute and persistent diarrhoea, has been reported in Kenyan children (Sang et al., 1997). Very recently, E. coli O157 was reported for the first time as the etiologic cause of a large dysentery outbreak in Swaziland (Effler et al., 2001). Also, during a study on bacterial diarrhoeal diseases involving children below five years of age in Kenya, the KEMRI/JICA Research and Control of Infectious Diarrhea Project (between 1990 and 1995) reported the first confirmed case of hemorrhagic colitis due to E. coli serotype O157:H7 in Kenya (Sang et al., 1996). This particular isolate produced only vero toxin II (VT2). In the same study, entero toxigenic E. coli (ETEC) strains that elaborated at least one member of two defined groups of entero toxins, heat-stable (ST) and heat-labile (LT) toxins, were isolated (Sang et al., 1996).  Antimicrobial resistance surveillance has been conducted only at the institutional levels (e.g., referral and private hospitals), with limited sharing of information and analysis of data. As a result, the actual scale of regional or national antimicrobial drug resistance is not well defined.

1.1       CAUSES OF DIARRHEA

The causes of acute diarrhoea include a wide range of viruses, bacteria, and parasites. However, enteric bacteria and parasites are more prevalent than viruses. Bacterial entero pathogens that cause diarrhoea include Diarrhoea genic E. coli, Shigella , Salmonella species, and Campylobacter species (Guarino et al., 2008; Viswanathan  et al., 2009). Shigellosis is a highly infectious disease of world significance. Its prevalence is highest in tropical and subtropical parts of the world where living standards are very low and access to safe and adequate drinking water and proper excreta disposal systems are often limited (Abera, 2004). Salmonella infections also remain as an important public health problem particularly in developing countries (Huang and Dupont, 2005). Like other developing nations, Shigellosis and Salmonellosis are among the common causes of morbidity and mortality in Ethiopia (Viswanathan et al., 2009; Abera, 2004). Moreover, emergence and spread of antibiotic resistance is posing serious problems in antimicrobial treatment worldwide (Murray et al., 2002). In view of increasing antibiotic resistance rate in the country, stimulates us to assess the prevalence and resistance patterns of Shigella and Salmonella species in patients who were presenting with diarrhoea at selected health facilities.

1.2       EPIDEMIOLOGY OF DIARRHEA

Severe presentations of acute diarrhea constitute one of the commonest challenges faced by the medical team in pediatric ambulatories and emergency rooms in the developing world. Supportive anti-dehydration treatment is the cornerstone of therapy and must be promptly started, but specific antimicrobial treatment may be required, depending on the severity of the disease and on the risk of complications. As stool cultures take several days to provide adequate information about pathogens and their susceptibility patterns, empirical treatment must be immediately adopted in such cases. To guide the empirical choice of antibiotics, it is crucial to know both which pathogens are most likely to be infecting the patient in a particular geographic area and the most effective antibiotics for treating them. All over the world, severe acute bacterial gastroenteritis is caused mainly by Shigella, whereas Salmonella, E. coli (chiefly entero pathogenic E. coli, or EPEC, but also entero hemorrhagic E. coli or EHEC, entero invasive E. coli or EIEC and other types), Campylobacter and Vibrio spp. have also been shown to play a role in the epidemiology of diarrhea, especially in certain areas of the globe WHO (1998); Wasfy et al., 2000).

1.3       AIMS AND OBJECTIVES

1.              The aim of the study is to assess the association of bacteria isolated from diarrhea.

2.              This study identified the bacterial causes of diarrhoea, the virulence properties associated with pathogens.

1.4       OBJECTIVES

1.              The objective of this study was to determine the prevalence of Shigella species and their antimicrobial resistance patterns among diarrheal patients.

2.              This study was carried out to identify and to establish the antimicrobial susceptibility pattern of the most important pathogens.

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