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Occurrence of Yersinia Enterocolitica and Aeromonas Hydrophila in Clinical, Food and Environmental Samples in Gaza Strip

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Occurrence of Yersinia Enterocolitica and Aeromonas Hydrophila in Clinical, Food and Environmental Samples in Gaza Strip The Islamic University-Gaza Deanship of Graduate Studies Faculty of Science Biological Sciences Master Program Microbiology Occurrence of Yersinia enterocolitica and Aeromonas hydrophila in Clinical, Food and Environmental Samples in Gaza Strip A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Biological Sciences/ Microbiology By Naima I. Ferwana Supervisor Dr. Abdelraouf A. Elmanama Ph. D Microbiology November 2007 DEDICATION I dedicate this thesis to my supervisor, Dr. Abdelraouf A. Elmanama. I would like to thank him so much for always helping me and being by my side. Also, this thesis is dedicated to my family and to my friend Lila Basher who supported me all the way since the beginning of this study. Finally, this thesis is dedicated to all those believe in the richness of learning ii Declaration "I herby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substational extent has been accepted for the award of any other degree of the university or other institute, except where due acknowledgment has been made in the text" Author Naima Ferwana Copyright © by Naima Ferwana, 2007 All Right Reserved: No part of this work can be copied, translated or stored in retrieval system, without prior permission of the authors. iii Occurrence of Yersinia enterocolitica and Aeromonas hydrophila in Clinical, Food and Environmental Samples in Gaza Strip Abstract The interest on the occurrence of Yersinia enterocolitica and Aeromonas hydrophila, their pathogenicity and antimicrobial resistance is increasing worldwide because both were linked to acute and chronic gastroenteritis, septicemia and wound infections. Though reports on the occurrence of both pathogens among human that are available from certain areas, no published data are available from Gaza strip. Moreover, there are no routine methods for the detection of Yersinia and Aeromonas in clinical or environmental samples. Hence this study investigated the occurrence of both Y. enterocolitica and A. hydrophila in clinical and environmental samples. Of the 473 clinical and environmental samples, 28 (5.9%) were positive for Y. enterocolitica and 179 (38.1%) for A. hydrophila. With high incidence of Y. enterocolitica and A. hydrophila in sewage (19.1%) and water (46.9%) respectively. The overall incidence of Y. enterocolitica and A. hydrophila in clinical samples was 4.7% and 34.3% respectively, with high frequency of both pathogens in AL-Dorrah and AL-Nasser hospitals. Virulence of isolates was assessed and their antimicrobial resistance to 20 antimicrobial agents was evaluated. Both clinical and environmental isolates possessed virulence factors with higher frequency in clinical samples. Antibiotic susceptibility testing revealed that most of Y. enterocolitica isolates were sensitive to most antibiotics; on the other hand, most of A. hydrophila isolates showed multiple antibiotic resistances. The most effective antimicrobials on A. hydrophila were azetreonam, ciprofloxacin and ofloxacin. KeyWords: Yersinia enterocolitica, Aeromonas hydrophila, clinical samples, Environmental samples, Gaza Strip, Antimicrobial resistance iv ﺗﻮاﺟﺪ اﻟﻴﺮﺳﻴﻨﻴﺎ اﻧﺘﻴﺮوآﻮﻟﺘﻴﻜﺎ و اﻻﻳﺮوﻣﻮﻧﺎس هﻴﺪروﻓﻴﻼ ﻓﻲ ﻋﻴﻨﺎت ﺳﺮﻳﺮﻳﺔ و ﻏﺬاﺋﻴﺔ و ﺑﻴﺌﻴﺔ ﻓﻲ ﻗﻄﺎع ﻏﺰة ﻣﻠﺨﺺ ﻋﺮﺑﻲ اﻹهﺘﻤﺎم ﺑﻮﺟﻮد آﻞ ﻣﻦ اﻟﻴﺮﺳﻴﻨﻴﺎ اﻧﺘﻴﺮوآﻮﻟﺘﻴﻜﺎ و اﻻﻳﺮوﻣﻮﻧﺎس هﻴﺪروﻓﻴﻼ و اﻻﻣﺮاض اﻟﺘﻲ ﺗﺴﺒﺒﻬﺎ و ﻣﻘﺎوﻣﺘﻬﺎ ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ ﻓﻲ ﺗﺰاﻳﺪ ﻣﺴﺘﻤﺮ ﻋﻠﻰ اﻟﻤﺴ ﺘﻮى اﻟﻌﺎﻟﻤﻲ ﻧ ﻈ ﺮ اً ﻻرﺗﺒﺎط آﻼ اﻟﻜﺎﺋﻨﻴﻦ ﺑﻤﺠﻤﻮﻋﺔ ﻣﻦ اﻷﻣﺮاض ﻣﺜﻞ اﻻﺗﻬﺎﺑﺎت اﻟﻤﻌﻮﻳﺔ اﻟﺤﺎدة و اﻟﻤﺰﻣﻨﺔ , ﺧﻤﺞ اﻟﺪم و اﻟﺘﻬﺎﺑﺎت اﻟﺠﺮوح. و ﺑﺎﻟﺮﻏﻢ ﻣﻦ وﺟﻮد ﺗﻘﺎرﻳﺮ ﻋﻦ وﺟﻮد هﺬﻳﻦ اﻟﻜﺎﺋﻨﻴﻦ ﻓﻲ اﻟﺒﺸﺮ ﻓﻲ اﻣﺎآﻦ ﻣﻌﻴﻨﺔ ﻣﻦ اﻟﻌﺎﻟﻢ ﻻﻳﻮﺟﺪ أي ﺑﻴﺎﻧﺎت ﻣﻨﺸﻮرة ﻋﻦ ﻗﻄﺎع ﻏﺰة . ﻓﻀﻼ ﻋﻦ ذﻟﻚ ﻻ ﺗﻮﺟﺪ ﻃﺮق ﻣﺤﺪدة ﻟﻠﻜﺸﻒ ﻋﻦ اﻟﻴﺮﺳﻴﻨﻴﺎ و اﻻﻳﺮوﻣﻮﻧ ﺎس ﻓﻲ اﻟﻌﻴﻨﺎت اﻟﺴﺮﻳﺮﻳﺔ واﻟﺒﻴﺌﻴﺔ و ﻟﻬﺬا اﻟﺴﺒﺐ ﻓﻘﺪ ﺑﺤﺜﺖ هﺬﻩ اﻟﺪراﺳﺔ ﺗﻮاﺟﺪ آ ﻞٍ ﻣﻦ اﻟﻴﺮﺳﻴﻨﻴﺎ اﻧﺘﻴﺮوآﻮﻟﺘﻴﻜﺎ و اﻻﻳﺮوﻣﻮﻧﺎس هﻴﺪروﻓﻴﻼ ﻓﻲ هﺬﻩ اﻟﻌﻴﻨﺎت . اﻟﺒﻜﺘﻴﺮﻳﺎ اﻟﻤﻌﺰوﻟﺔ اﺟﺮي ﻟﻬﺎ اﺧﺘﺒﺎر اﻟﻔﻮﻋﺔ و ﻣﻘﺎوﻣﺘﻬﺎ ﻟﻌﺸﺮﻳﻦ ﻣﻀﺎ د ﺣﻴﻮي . و آﺎﻧﺖ ﻧﺴﺒﺔ اﻟﺒﻜﺘﻴﺮﻳﺎ اﻟﻤﻌﺰوﻟﺔ ﻣﻦ اﻟﻌﻴﻨﺎت اﻟﺴﺮﻳﺮﻳﺔ و اﻟﺒﻴﺌﻴﺔ آﺎﻟﺘﺎﻟﻲ : 28 (5.9%) ﻳﺮﺳﻴﻨﻴﺎ اﻧﺘﻴﺮوآﻮﻟﺘﻴﻜﺎ و 179 (38.1%) اﻳﺮوﻣﻮﻧﺎس هﻴﺪروﻓﻴﻼ و آﺎﻧﺖ أﻋﻠﻰ ﻧﺴﺒﺔ ﺗﻮاﺟﺪ ﻟﻠﻴﺮﺳﻴﻨﻴﺎ اﻧﺘﻴﺮوآﻮﻟﺘﻴﻜﺎ و اﻻﻳﺮوﻣﻮﻧﺎس اﻟﻬﻴﺪروﻓﻴﻼ ﻓﻲ ﻋﻴﻨﺎت اﻟﻤﺠﺎري (19.1%) وﻋﻴﻨﺎت اﻟﻤﺎء ( 46.9%) ﻋﻠﻰ اﻟﺘﻮاﻟﻲ . و آﺎن اﻟﻤﺠﻤﻮع اﻟﻜﻠﻲ ﻟ ﻜ ﻞٍ ﻣﻦ اﻟﻴﺮﺳﻴﻨﻴﺎ اﻧﺘﻴﺮوآﻮﻟﺘﻴﻜﺎ و اﻻﻳﺮوﻣﻮﻧﺎس هﻴﺪروﻓﻴﻼ ﻣﻦ اﻟﻌﻴﻨﺎت اﻟﺴﺮﻳﺮﻳﺔ هﻮ (3.7%) و (%34.3) ﻋﻠﻰ اﻟﺘﻮاﻟﻲ , و آﺎﻧﺖ أﻋﻠﻰ ﻧﺴﺒﺔ ﻟﻜﻞٍ ﻣﻦ اﻟﻜﺎﺋﻨﻴﻦ ﻓﻲ ﻣﺴﺘﺸﻔﻰ اﻟﺪرة وﻣﺴﺘﺸﻔﻰ اﻟﻨﺼﺮ . اﺣﺘﻮت آﻞ ﻣﻦ اﻟﻌﺰﻻت اﻟﺒﻴﺌﻴﺔ و اﻟﺴﺮﻳﺮﻳﺔ ﻋﻠﻰ ﻋﻮاﻣﻞ اﻟﻔﻮﻋﺔ ﻣﻊ زﻳﺎدة واﺿﺤﺔ ﻟﻬﺬﻩ اﻟﻌﻮاﻣﻞ ﻓﻲ اﻟﻌﺰﻻت اﻟﺴﺮﻳﺮﻳﺔ , وﻗﺪ أﻇﻬﺮت ﻧﺘﺎﺋﺞ ﺣﺴﺎﺳﻴﺔ اﻟﻤﻀﺎدات ﺑﺄن اﻟﻴﺮﺳﻴﻨﻴﺎ اﻧﺘﻴﺮوآﻮﻟﺘﻴﻜﺎ ﺣﺴﺎﺳﺔ ﻟﻤﻌﻈﻢ اﻟﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ اﻟﻤﻔﺤﻮﺻﺔ ﺑﻴﻨﻤﺎ أﻇﻬﺮت ﻋﺰﻻت اﻻﻳﺮوﻣﻮﻧﺎس هﻴﺪروﻓﻴﻼ ﻧﺴﺒﺔ ﻋﺎﻟﻴﺔ ﻣﻦ اﻟﻤﻘﺎوﻣﺔ اﻟﻤﺘﻌﺪدة . و آﺎن اﻻزﺗﺮﻳﻮﻧﺎم و ﺳﺒﺮوﻓﻠﻮآﺴﺎﺳﻴﻦ و اﻓﻠﻮآﺴﺎﺳﻴﻦ ﻣﻦ ﺑﻴﻦ اﻟﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ اﻻآﺜﺮ ﻓﻌﺎﻟﻴﺔ ﺿﺪ اﻻﻳﺮوﻣﻮﻧﺎس هﻴﺪروﻓﻴﻼ. • اﻟﻜﻠﻤﺎت اﻟﻤﻔﺘﺎﺣﻴﺔ: ﻳﺮﺳﻴﻨﻴﺎ اﻧﺘﻴﺮوآﻮﻟﺘﻴﻜﺎ, اﻳﺮوﻣﻮﻧﺎس هﻴﺪروﻓﻴﻼ, ﻋﻮاﻣﻞ اﻟﻔﻮﻋﺔ, ﻗﻄﺎع ﻏﺰة v Acknowledgments As with any project, there are a number of people who contributed to complete this research, without their help, this research would not exist. I am deeply grateful to: The Islamic University-Gaza and the Faculty of Science for providing me the opportunity to pursue my post-graduate study. My supervisor Dr. Abdelraouf Elmanama, who introduced me to the world of science, supported and guided me, for his comments, suggestions and excellent proofreading skills, and who encouraged me to complete this work with his incredible optimism. Abboud Y. El Kichaoui, Director of the Biological Sciences Master Program. Special thanks to Lila Basher and Hashem Arafa, for their patience and support. I also wish to acknowledge the efforts of all hospitals directors and staff members who supported this work in a variety of ways. Thanks extended to my family for their sincere encouragement, teaching me perseverance and always encouraging me to pursue my goal. Finally, I thank the countless people who contributed to this research. Anyone who helped me in any form. vi LIST OF ABBREVIATIONS A Ampicillin AA Autoagglutination ADA Ampicillin Dextrin Agar AC Amoxycillin/clavulanate AFLP Amplified Fragment Length Polymorphism Ail Attachment Invasion Locus AK Amikacin Ao Aztreonam API Analytical Profile Index APHA American Public Health Association BHIA Brain Heart Infusion Agar BHIB Brain Heart Infusion Broth BIBG Bile Salts Inositol Brilliant Green Agar BOS Bile-Oxalate-Sorbose Broth C Chloramphenicol Ca Ceftazidim CDC Center for Disease Control and Prevention Cf Ciprofloxacin CFU Colony Forming Unit Ci Ceftriaxone CIN Cefsulodin-Irgasan-Novobiocin agar plate Co Co-Trimoxazole Cp Cephalexin Cu Cefuroxime CV Crystal violet Cz Cefazolin DNA Deoxy Ribonucleic Acid DC Deoxycholate citrate agar Do Doxycycline EPA Environmental Protection Agency EDTA Ethylene Diamine Tetra Acetic Acid FAME Fatty Acid Methyl Ester Analysis FDA Food and Drug Administration FISH Fluorescent In Situ Hybridization G Gentamicin GWWTP Gaza Wastewater Treatment Plant HCl Hydrochloric Acid HE Hektoen Enteric Agar HPI High Pathogenic Islands HUS Hemolytic Uremic Syndrome Inv Invasin ISO International Organization for Standardization ITC Irgasan-Ticarcillin-Potassium Chlorate broth KOH Potassium hydroxide LPS Lipopolysaccharide MCA MacConkey agar vii MEM Meropenem MF Membrane Filtration method MRB Modified Rappaport Broth Na Nalidixic Acid Nacl Sodium chloride NCCLS National Committee for Clinical Laboratory Standard Ofx Ofloxacin OMP Outer Membrane Proteins PAB Precipitation After Boiling PBS Phosphate-Buffered Saline Broth PBSSB Phosphate-Buffered Saline Broth with Sorbitol and Bile Salts PCR Polymerase Chain Reaction PFGE Pulsed-Field Gel Electrophoresis PMO Pasteurized Milk Ordinance PYZ Pyrazinamidase pYV Plasmid for Yersinia Virulence SEL Selenite Broth SAA Starch Ampicillin Agar SAP Surface Array Protein SGAP-10 Starch Glutamate Ampicillin Penicillin SP Self pelleting SS Salmonella-Shigella agar SSDC Salmonella-Shigella-Sodium Deoxycholate-Calcium Chloride agar Te Tetracycline TSB Trypticase Soya Broth TSB-30 Tryptose Broth containing Ampicillin (ampicillin 30 mg/L) Ure Urease gene USA United State of America USFDA United States Food and Drug Administration VYE Virulent Yersinia enterocolitica agar XLD Xylose Lysine Deoxycholate agar YadA Yersinia Adhesin A YER Yeast Extract-Rose bengal Broth Yop Yersinia Outer Membrane Protein Ybt Yersiniabactin locus Ysc Yop Secretion Yst Yersinia Heat Stable Enterotoxin WHO World Health Organization viii List of contents Dedication…………………………………………………………………………. ii Abstract……………………………………………………………………………. iv Acknowledgements………………………………………………………………. vi List of abbreviations……………………………………………………………… vii List of content…………………………………………………………………….. x List of tables………………………………………………………………………. xiii List of figure……………………………………………………………………….. xv Chapter 1: Introduction 1 1.1 Overview………………………………………………………………….….. 1 1.2 Objectives……………………………………………………………….….. 4 1.3 Significance …………………………………………………………….….. 5 Chapter 2: Literature Review ………………………………………………… 6 2.1 Y. enterocolitica …………………………………………….……………… 6 2.1.1 Historical background and taxonomy………….…………………… 6 2.1.2 Ecology and host range …………………………………………….. 8 2.1.3 Pathogenicity…………………………………………………………. 8 2.1.3.1 Plasmid-encoded virulence factors …………………...……. 9 2.1.3.2 Chromosome-encoded virulence factors …………………… 10 2.1.4 Factors affecting growth ……………..……………………………… 13 2.1.5 Isolation and identification of pathogenic Yersinia enterocolitica from clinical, foods and environmental sample………………………….
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