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Antibiotic Sensitivity Profile of Enteric Bacteria Isolated from Soil

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Antibiotic Sensitivity Profile of Enteric Bacteria Isolated from Soil antibiotic sensitivity pr o file of en ter ic bacteria ISOLATED FROM SOIL SAMPLES AROUND KEMRI (CMR) AND ITS ENVIRONS IN NAIROBI KENYA By: Peter Shigoli Mashedi (BSc Medical Microbiology, JKUAT) \ thesis submitted in partial fulfillment of the requirements for the award of the degree of Master of Science in Microbiology of the University of Nairobi. November, 2012 i pec I a ration flie work described herein is my original work as part of ongoing project. Articles and texts hed have been acknowledged. The contents in this thesis have not been submitted previously to ly other Univeristy in whole or part, for the award of any degree or academic titles >tter Shigoli Mashedi lture.. te.... 3o his thesis has been submitted with the approval as the University supervisor. 'Jr Miriam M. Jumba Jchool of Biological Sciences Jniversity of Nairobi Signature ...^ )ate................/ .10 Is3L Q J 5- 'his thesis has been submitted with the approval as the External supervisor. )r. Christine Bii -enter for Microbiology Research (CMR) Kenya Medical Research Institute, Nairobi * ) Box 54840-00200 NAIROBI. Signature....... ~ ................................... p a te ....................... ................................................................... II Dedication I dedicate this study to my family members. Acknowledgment I would like to thank the Almighty God lor giving me the strength and guidance to have completed this project. I also thank my supervisors Dr. Miriam Jumba and Dr. Christine Bii for guiding me with the project and also the entire stall at Opportunistic Infections Laboratory in KEMRI Nairobi, for the assistance they gave me. IV Abstract Soil is able to contain enteric bacteria and other pathogens in great concentrations, as it is normally a recipient of solid and liquid waste materials frequently. Recent studies elicit that soil may have a greater role in the transmission of enteric diseases than previously expected, even though its role as a reservoir of certain bacterial pathogens is not in doubt. Enteric bacteria are responsible for causing most gastrointestinal infections, for example salmonellosis, dysentery, typhoid fever and other infections caused by Yersinia sp. and Escherichia coli 0157:H7 and many other strains. The study was aimed at determining the prevalence of enteric bacteria from various soil samples collected around Nairobi, and to compare their drug susceptibility profile with those from clinical samples. The soil samples were collected from various locations in Nairobi within a radius of 30km from Kenya Medical Research Institute, Centre for Microbiology Research in Nairobi, with their Global Position System (GPS) location recorded down, then transported to the laboratory. Ten grams of each of the soil samples were serially diluted then plated on Mueller-I Iinton agar and incubated at 30°C overnight, the colonies were Gram stained and the Gram-negative colonies inoculated on Analytic Profile Index kit (API 20E) for further identification. Antibiotic sensitivity testing was done using Disc Diffusion method and then compared with clinical isolates. Out of the soil samples (n=236) inoculated onto Mueller- Hinton agar, 17 were positive for Proteus salmonicida, which represents a prevalence of 7.2% of enteric bacteria in the soil. The other isolated Gram negative bacteria were Myroides spp, Pseudomonas putida. Pseudomonas aeruginosa, Stenotrophomonas maltophila and Alcaligenes spp. Proteus salmonicida showed a higher sensitivity to the antibiotics compared to the clinical Proteus except for Cefotaxime antibiotic which was resistant to it. In conclusion, soil may be a significant a reservoir for the enteric bacteria contributing to antibiotic resistance as indicated by Proteus salmonicida with resistance to Cefotaxime antibiotic, compared to Proteus species from the clinical sources which was sensitive to the same antibiotic. v TABLE OF CONTENTS Declaration..........................................................................................................................................ii Dedication...........................................................................................................................................iii Acknowledgement..............................................................................................................................iv Abstract.............................................................................................................................................. v 1.1 Introduction................................................................................................................................1 2.0 CHAPTER TWO.........................................................................................................................5 2.1 Literature review............................... !...................................................................................... 5 2.1.1 Enteric bacteria.....................................................................................................................5 2.1.2 Isolation, identification and drug susceptibility testing of enteric bacteria.......................6 2.1.3 Sources of soil contamination by enteric pathogens..........................................................7 2.1.4 Fate of enteric bacteria in the soil....................................................................................... 9 2.1.5 The role of soil as a reservoir for bacteria in contributing to antibtiobitc resistance ... 10 2.2 Justification...............................................................................................................................12 2.3 Hypothesis.................................................................................................................................13 2.4 Objectives................................................................................................................................13 2.4.1 Overall objective.................................................................................................................13 2.4.2 Specific objectives..................................;.........................................................................13 3.0 CHAPTER THREE..................................................................................................................14 3.1 Materials and methods..............................................................................................................14 3.1.1 Study site................................................................................................ 14 3.1.2 Sampling.............................................................................................................................15 3.1.3 Sample size and its justification........................................................................................15 3.1.4 Sample collection................................................................................................................15 3.2 Laboratory methodology..........................................................................................................16 3.2.1 Sample processing and identification of enteric bacteria................................................16 3.2.2 Antibiotic susceptibility testing for the enteric bacteria...................................................16 3.2.4 Variables................................................. ;.........................................................................17 3.3 Experimental design..................................................................................................................17 VI 4.0 CHAPTER FOUR........................................................................................................................18 4.1 Results........................................................................................................................................18 4.1.3 Colour reactions on API 20E kit after incubation and addition of reagents...................22 4.1.4 Distribution of the enteric soil isolates...............................................................................24 4.1.5 Antibiotic sensitivity profile of the soil and clinical isolates........................................ 25 4.2 Data analysis.............................................................................................................................29 5.0 CHAPTER FIVE...................................................................................................................... 30 5.1 Discussion.....................................................................................................i......................... 30 5.2 Conclusion.................................................................................................................................33 5.3 Recommendations.................................................................................................................... 33 6.0 REFERENCES.......................................................................................................................34 APPENDIX....................................................................... 40 vii LIST OF TABLES 1: Gram stain reactions of isolates 19 2: Antibiotic sensitivity profile of Proteus salmonicida from soil 25 3: Antibiotic sensitivity profile of Proteus species from clinical isolate 26 4: Antibiotic amounts impregnated on disc and breakpoints for enteric bacteria 26 viii . * LIST OF FIGURES
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