DETECTION OF EXTENDED SPECTRUM BETA-LACTAMSE IN READY- TO -EAT FOOD (AGIDI AND MOI-MOI)

ABTRACT

The presence of extended spectrum beta lactamase producing bacteria from ready to eat foods (agidi and moi moi) sold in Michael Okpara University of Agriculture Umudike was accessed. Using standard isolation and identification techniques, the isolated organisms were identified as Escherichia coli sp, Salmonella sp, Klebsiella sp, Shigella sp. The antimicrobial susceptibility Testing of all identified isolates was done using the Kirby Bauer disk diffusion method. All the isolates were screened according to CLSI break point and confirmed using the Double disk, synergy test(DDST) The percentage occurrence of the isolates indicated (37%) for E.coli, (23%) were Salmonella spp, (21%) were Klebsiella spp, and (19%) were Shigella sp. The antibiogram done on the isolates showed that Escherichia coli sp isolates showed high resistance to Cefotaxime, Cefixime, Oflaxcin, Amoxicillin and Ceftriazone, Salmonella spp isolates exhibited high resistance to Cefotaxime, Gentamycin and Oflaxcin, Klebsiella spp isolates showed greatest resistance to cefotaxime and gentamycin and that of Shigella sp isolate exhibited highest resistance to Cefotaxime and Cetrafiazone. The ESBL screening of the isolates showed that E: coli sp had the highest occurrence of 43.8%, and Klebsiella sp had the lowest occurrence with 0%. The DDST confirmatory test for the isolates showed that E. coli and Salmonella sp had the highest occurrence with 37.5% and 30.0% respectively. This research work has confirmed the presence of extended spectrum beta lactamase producing enteric bacteria in ready to eat food (Agidi and Moi-Moi) sold within Michael Okpara University of Agriculture, Umudike. There is a need for the vendors to strictly observe food safety principles and personal hygiene during preparation, packaging and selling of food.

TABLE OF CONTENTS

Cover page                                                                                                             i

Certification                                                                                               ii

Dedication                                                                                                 iii

Acknowledgement                                                                                    iv

Table of tables                                                                                                           v

List of tables                                                                                                         ix

Abstracts                                                                                                     x

CHAPTER ONE

1.0 INTRODUCTION                                                                                      1

1.2    Aim and Objective                          4

CHAPTER TWO

2.0 LTERATURE REVIEW                                                                                                    5

2.1    Agidi                                                                                                   5

2.3     Preparation of Agidi                                                    6

2.4     Moi-Moi  6

2.5     Micro-organisms associated with spoilage of Moi-Moi  7

2.6     Handling and preparation of street Foods                                                                                                          8

2.7     Extended spectrum beta-lactamase                                                                                                    9

2.8     Resistance to beta-lactams                                                                                                      11

2.9     ESBL Detection                                                                                                  12

2.9.1     Screening for ESBL producers                                                                                                  12

2.9.1.1     Disc-diffusion method  12

2.9.1.2     Screening dilution antimicrobial susceptibility tests       13

2.9.2     Confirmatory Test for ESBLs Production

2.9.2.1    Double disc synergy test                                                             13

2.9.2.2     Cephalosporin/Clavulanate Combination disk                           14

2.9.2.3     Broth micro-dilution                                                                   15

CHAPTER THREE

3.0 MATERIALS AND METHODS                                                       16

3.1    Collection of samples                                                                        16

3,2Materials and media used                                                                    16

3.3              Sterilization                                                                                16

3.4     Isolation and enumeration of organisms                                          17

3.5     Morphological and biochemical characteristics of the isolates                                                                                                      17

3.5.1    Gram staining                                                                                 17

3.5.2     Citrate utilization test                                                                    18

3.5.3     Motility test                                                                                   18

3.5.4     Indole test                                                                                      19

3.5.5     Urease test                                                                                     19

3.5.6     Triple sugar iron test                                                                     19

3.7 Detection of ESBL producing Gram-negative isolates                                               20

3.7.1    Screening test for ESBL production                                          20

3.7.2     Double disc synergy test (DDST) for ESBL confirmation                                        20

CHAPTER FOUR

4.0 RESULTS                                                                                        21

CHAPTER FIVE

5.0 DISCUSSION                                                                                  31

5 1 Conclusion                                                                                       32

5.2 Recommendation                                                                             32

Reference                                                                                               33

Appendix                                                                                                36

LIST OF TABLES

Tables                                           Titles                                  Pages

Table 4.1 Colonial morphological and identification of Bacterial Isolates                                                                                                      23

Table 4.2 Total viable bacteria count and the total coliform count of bacteria isolates                                   24

Table 4.3 Percentage occurrence of the bacterial isolates from the sample                                          25

Table 4.4 Antibiogram of the Escherichia coli isolates.                             26

Table 4.5 Antibiogram of Salmonella sp isolates.                                27

Table 4.6 Antibiogram of Klebsiella sp isolates.                             28

Table 4.7 Antibiogram of Shigella sp isolates.                                 29

Table 4.8 ESBL screening on the bacteria isolates.                                            30

Table 4.9 DDST confirmatory test of the bacteria isolates.                                 30

CHAPTER ONE

1.0 INTRODUCTION

Ready-To-Eat (RTE) are food substance ingested or consumed by human for the continuance of life. However, foods consumed by human serves as favorable media for rapid growth of microorganisms because of the abundant organic matter, moisture and suitable pH contained and because of the fact that the microorganisms are equally living organisms. Presence of these in food warrants their rapid multiplication which renders the food unfit and dangerous for consumption Yusha’u et al., (2010).

Food safety doesn’t begin at the grocery store or in the kitchen, it begins on the farm. In the developed world of Europe, concerns about food safety, welfare of plants and animal as well as traceability are more preferable than the food products being supplied in plenitude suggested EUROSTAT, (2008). The reverse is the case in developing nations especially in Africa where famine is the order of the day in many countries or parts of a certain country, Nigeria inclusive. This therefore hampers the need for food safety as the need for food at any condition is at its peak. Foods commercialized in markets have likelihood of being exposed to pathogenic microorganisms because of poor handling by the sellers. Waterborne and other related pathogens including bacteria are spread in water either through human ingestion of contaminated water or Yusha'u et al,. (2010).

Meanwhile, fecal present in contaminated water used in the production of some food are due to ineffective sanitary measures. The pathogens as a result, get into the locally made food which require water as medium and ingredient. Water contaminated with these pathogenic organisms may be a source of some diseases comprising of typhoid and paratyphoid fevers, dysentery and allied diarrheal infections including cholera

They have been described as Ready to Eat Food (RTE), because of their status as food being ready for immediate consumption at the point of sale, Lovet et al,. (2012). The consumption of ready to eat food is due to the convenience of modem lifestyle, economic downturn, industrialization, materialism, the quest for wealth, lack of time to proper meal and the low purchasing power Nielsen (2006). The major concern with ready-to-eat food is their microbiological safety mainly because vending prepares such food in places that may have poor sanitation. In some African countries, such food have tested positive for various microorganisms of public health concern, including fecal coliforms like Escherichia coli, Salmonella spp. And Bacillus cereus. A study conducted in 2011 by Oranusi and friends observed that the presence of Escherichia coli, Enterococcus, Klebsiella spp. and Staphylococcus aureus are of concern and further support the possibility of fecal contamination of product due to poor sanitation Oranusi et zzZ,.(2011) and Hazariwala, (2002 ).

In 2007, World Health Organization estimated that a significant proportion of the approximately 1.5 billion episodes of diarrhea and more than three million deaths globally recorded annually results from the consumption of food with microbial pathogens and toxins Oluwafemi and Simisaye (2005).These bacteria produce an enzyme, extended specrum beta-lactamase (ESBL) a typical example of which bacteria cause disease of public health concern.

BETA LACTAMASE

Beta-lactamase are enzymes produced by some bacteria that provide resistance to 0-lactam antibiotics like penicillins, cephalosporins, and carbepenems (ertapenem), although carbapenams are relatively resistant to beta-lactamase provides antibiotic resistance by breaking the antibiotic structure. These antibiotics have a common element in their molecular structure of a six-atom ring known as 0-lactam. However, through hydrolysis the lactamase enzyme breaks the 0-lactam

ring open, deactivating the molecule’s antibacterial properties. Beta-lactam antibiotics are typically used to treat a broad spectrum of Gram-positive and Gram-negative bacteria. Beta­lactamase produced by Gram-negative organisms are usually secreted especially when antibiotics are present in the environment Neu, (1969).

Beta-lactamase are enzymes produced by some bacteria that provide resistance to |3-lactam antibiotics like penicillins, cephalosporins, and carbepenems (ertapenem), although carbapenams are relatively resistant to beta-lactamase provides antibiotic resistance by breaking the antibiotic structure. These antibiotics have a common element in their molecular structure of a six-atom ring known as 0-lactam. However, through hydrolysis the lactamase enzyme breaks the 0-lactam ring open, deactivating the molecule’s antibacterial properties. Beta-lactam antibiotics are typically used to treat a broad spectrum of Gram-positive and Gram-negative bacteria. Beta­lactamase produced by Gram-negative organisms are usually secreted especially when antibiotics are present in the environment Neu, (1969).

Extended Spectrum Beta Lactamase were first described in 1980s and have been detected in Klebsiella spp and later in Escherichia coli, Pseudomonas aeroginisa, Serratia marcescens and other Gram-negative bacilli. Extended spectrum0-lactamase are enzymes conferring broad resistance to Penicillins, Aztreonamand, Cephalosporins. These enzymes are able to hydrolyze 3^rd and 4rd generations of Cephalosporins and Monobactams. They are an increasing important cause of transferable multidrug resistance in Gram-negative bacteria throughout the world Bali et al., (2010). One of the important features of Extended Spectrum Beta Lactamase producing strains is resistant to multiple clinically important antibiotics Shacheraghi et al., (2010).

1.2. AIM AND OBJECTIVES 1. The aim of this study is to screen ready to eat foods (Moimoi and Agidi) for the presence of extended spectrum beta-lactamase producers and identify organisms of public health

My objectives are

1. To determine the antibiotic susceptibility pattern of the isolates

2.. To identify beta-lactamase producing bacteria from these foods