View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by KhartoumSpace Detection of Staphylococcus aureus and other Coagulase Positive Staphylococci in Bovine Raw Milk in Khartoum State By Ikhtyar Ahmed Hassan Ali B.C.Sc (2003) Supervisor Prof. Mohammed Taha Shigiddi Department of Microbiology Faculty of Veterinary medicine A dissertation submitted to University of Khartoum in partial fulfillment for the requirement of the Degree of Master of Science in Microbiology Department of Microbiology Faculty of veterinary Medicine University of Khartoum 2010 Dedication to my father, mother, brothers and sisters with love I Table of Contents Subject Page Dedication………………………………………………………. I Table of Contents………………………………………………. II List of Figures…………………………………………………… VII List of Table…………………………………………………….. VIII Acknowledgments………………………………………………. IX Abstract…………………………………………………………. X Abstract (Arabic)……………………………………………… XI Introduction…………………………………………………… 1 Chapter One: Literature Review…………………………….. 3 1.1. Health Hazards of Raw Milk…………………………………… 4 1.2. Pathogenic bacteria in milk........................................................ 5 1.3. Microbial quality of raw milk.................................................... 6 1.4. Staphylococci........................................................................... 7 1.4.1. Coagulase positive staphylococci (CPS)……………………… 8 1.4.2. Coagulase negative staphylococci (CNS)……………………… 10 1.5. Staphylococcus aureus………………………………………… 10 1.5.1. Virulence characteristics of S. aureus…………………………... 12 1.5.2. Toxins………………………………………………………….. 13 II 1.5.2.1. Exfoliative toxins (EF)………………………………………… 13 1.5.2.2. Other toxins……………………………………………………. 14 1.5.3. Protein A……………………………………………………….. 14 1.6. Mastitis.................................................................................... 14 1.7. Microbial Contamination from the Exterior of the Udder……. 15 1.8. Staphylococci and food poisoning……………………………. 16 Chapter Two: Materials and Methods……………………… 2.1 Study area……………………………………………………….. 18 2.2 Raw milk Samples ……………………………………………… 18 2.3 Sterilization Procedures…………………………………………. 19 2.3.1 Hot air oven……………………………………………………... 19 2.3.2 Autoclaving……………………………………………............... 19 2.3.3 Steaming………………………………………………………… 19 2.3.4 U .V. light……………………………………………………….. 19 2.3.5 Red heat…………………………………………………………. 19 2.4 Disinfectants……………………………………………………. 19 2.5 Solutions, chemicals and reagent……………………………….. 20 2.5.1 Normal saline solutions…………………………………………. 20 2.5.2 Oxidase test reagent…………………………………………….. 20 2.5.3 Potassium hydroxide solution, 40% w/v……………………….. 20 III 2.5.4 Andrade’s indicator……………………………………………... 20 2.5.5 Hydrochloric acid, 1 mol/l (1N)………………………………… 20 2.5.6 Alpha naphthol solution, 5% w/v……………………………….. 21 2.5.7 Hydrogen peroxide 3% aqueous solution………………………. 21 2.5.8. EDTA…………………………………………………………... 21 2.6 Culture media for bacteriological examinations………………… 21 2.6.1 Solid media……………………………………………………… 21 2.6.1.1 Nutrient agar (Oxoid Code: CM0003 (Powder)………………… 21 .2.6.1.2 Sheep Blood Agar base: Code: (Oxoid CM0854) BASE……… 22 2.6.1.3. Deoxyribonuclease (DNase) medium Code (OxoidCM0321)….. 23 2.6.2 Semi-Solid media……………………………………………….. 24 2.6.2.1. Hugh and Leifson's (O/F) medium (Barrow and Feltham, 1993). 25 2.6.3 Liquid media…………………………………………………….. 26 2.6.3.1. Nutrient broth (Oxoid Code: CM0001)……………………….. 26 2.6.3.2 Methyl Red -Voges Proskauer medium (MR- VP)……………... 26 2.6.3.3. Carbohydrate fermentation media (Barrow and Feltham, 1993)... 27 2.6.4 Selective & differential media…………………………………... 27 2.6.4.1. Mannitol Salt Agar (Code: Oxoid CM0085 )…………………… 27 2.7 Cultural method………………………………………………… 28 2.7.1 Media used for primary isolation……………………………….. 28 IV 2.7.2 Incubation of culture…………………………………………….. 28 2.7.3 Examination of cultures…………………………………………. 28 2.7.4 Purification and storage of maintenance medium……………..... 28 2.8 Identification of isolates………………………………………… 29 2.8.1 Microscopic examination……………………………………….. 29 2.8.1.1 Gram’s stain…………………………………………………… 29 2.8.1.1.2 Staining procedure………………………………………………. 29 2.8.1.1.3 Motility test……………………………………………………... 30 2.8.2 Biochemical tests………………………………………………... 30 2.8.2.1 Oxidase test……………………………………………………... 30 2.8.2.2 Catalase test…………………………………………………….. 30 2.8.2.3 Sugar fermentation test…………………………………………. 31 2.8.2.4. Oxidation fermentation test (O.F)………………………………. 31 2.8.3 Secondary tests…………………………………………………. 31 2.8.3.1 Tube coagulase test……………………………………………… 31 2.8.3.1.1 Collection of Plasma……………………………………………. 31 2.8.3.1.1.2 Test procedure…………………………………………………... 32 2.8.3.1.1.2 Test procedure…………………………………………………. 32 2.8.3.2 Deoxyribonuclease (DNase) test………………………………... 32 V 2.8.3.3 Acetylmethylcarbinol (acetoin) production (Voges-proskauer reaction)………………………………………………………… 32 2.8.3.4. Maltose fermentation test……………………………………….. 33 2.8.3.5. Haemolysis on blood agar………………………………………. 33 Chapter Three: Results 3.1. Isolation of bacteria…………………………………………… 34 Chapter Four: Discussion……………………………………... 4.1 Discussion…………………………… ……………………….. 41 4.2 Conclusions ……………………………………………. 45 4.3 Recommendations……………………………………… 45 References……………………………………………………… 47 VI LIST OF FIGURES Figures Title Page 1 Colonies of Staphylococcus aureus showing fermentation and yellow discoloration on mannitol salt agar ……………………………………………………. 38 2 Staph. aureus on Manitol salt agar showing Positive manitol fermentation (left ) , other coagulase positive Staph .hyicus showing negative manitol fermentation 39 (right)………………………………………………… 3 Staphylococcus intermedius on blood agar showing 40 clear hemolysis………………………………………... VII LIST OF TABLES Table No. Title Page 1 Frequency of coagulase positive Staphylococcus spp in bovine raw milk in Khartoum State 36 ………………………….. 2 Characteristics of coagulase positive Staphylococcus spp from bovine raw milk………………. 37 VIII ACKNOWLEDGMENTS First of all, thanks and praise be to Allah, the compassionate and the most merciful for availability of which I need. I wish to express my deepest gratitude and appreciation to my supervisor Prof. M. T. Shigiddi, Department of Microbiology, Faculty of veterinary medicine , university of Khartoum , for his unfailing guidance , constructive criticism and encouragement throughout this work. I wish to express my deep appreciation to Prof. S. M.Elsanousi, Department of Microbiology, Faculty of veterinary medicine, university of Khartoum and my husband for his assistance in the collection of samples used in this study , Thanks are also be extended to Dr. Moaaz , Department of Virology ,Faculty of veterinary medicine, University of Nyala , Tech. Mustafa, Department of Microbiology, university of Khartoum Dr. Mohammed Sayed, Department of parasitology ,Faculty of veterinary medicine, University of Nyala and Dr. Altegany ,Department of Microbiology ,Faculty of veterinary medicine, University of Nyala. I would also like to express my thanks to the members of Department of Microbiology, Faculty of Veterinary medicine University of Khartoum, Tech. Mona and Naglaa, Miss Huda Yousif. My gratitude to my family, they were greatly helpful and encouraging especially my mother and my sister Seham. IX Abstract The present study was undertaken to isolate and characterize coagulase positive staphylococci with emphasis on Staphylococcus aureus, an important food‐borne pathogen, from bulk raw bovine milk in Khartoum State. Milk samples were collected from sale points in Khartoum, Omdurman and Khartoum North during January and February 2010. A total of 75 samples were collected; 25 samples from Khartoum (15 from Alshagara and 10 from Yathrib), 25samples from Omdurman (all from Waddrow sale point) and 25 samples from Khartoum North (15 from Helat Kuku and 10 from a sale point near the Faculty of Veterinary Medicine, University of Khartoum). All samples were transported to the laboratory in an ice box and were cultured on mannitol salt agar on the same day. A total of 163 coagulase positive staphylococcus isolates was obtained from all samples. Of these S. aureus constituted 32.6 % of the isolates: 39.6% from Khartoum, 47.2% from Omdurman and 13.2% from Khartoum North. Other coagulase positive staphylococcus constituted 67.4% of the isolates. It appears that raw bovine milk contains a high percentage of pathogenic S. aureus which makes it inherently dangerous and should not be consumed before pasteurization or boiling. X ﺍﻟﻤﺴﺘﺨﻠﺹ ﺼﻤﻤﺕ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺒﻐﺭﺽ ﻋﺯل ﻭﺍﻟﺘﻌﺭﻑ ﻋﻠﻲ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻌﻨﻘﻭﺩﻴﺔ ﺍﻟﻤﺠﻠﻁﺔ ﻟﻠﺒﻼﺯﻤﺎ (coagulase positive) ﺒﺎﻟﺘﺭﻜﻴﺯ ﻋﻠﻰ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻌﻨﻘﻭﺩﻴﺔ ﺍﻟﺫﻫﺒﻴﺔ ﻭﻫﻲ ﻤﻥ ﺃﻫﻡ ﺍﻷﻨﻭﺍﻉ ﺍﻟﻤﺴﺒﺒﺔ ﻟﻠﺘﺴﻤﻡ ﺍﻟﻐﺫﺍﺌﻲ. ﺠﻤﻌﺕ 75 ﻋﻴﻨﺔ ﻤﻥ ﺍﻟﻠﺒﻥ ﺍﻟﺒﻘﺭﻱ ﺍﻟﻁﺎﺯﺝ ﺍﻟﻤﺄﺨﻭﺫ ﻤﻥ ﻤﺭﺍﻜﺯ ﺒﻴﻊ ﺍﻟﺒﻥ ﻓﻲ ﻭﻻﻴﺔ ﺍﻟﺨﺭﻁﻭﻡ ﺨﻼل ﺸﻬﺭﻱ ﻴﻨﺎﻴﺭ ﻭ ﻓﺒﺭﺍﻴﺭ ﻤﻥ ﻋﺎﻡ 2010 ﻭﺍﻟﺘﻲ ﺘﻡ ﺠﻤﻌﻬﺎ ﻜﺎﻵﺘﻲ 25 ﻋﻴﻨﺔ ﻤﻥ ﻤﺤﺎﻓﻅﺔ ﺍﻟﺨﺭﻁﻭﻡ ، (15 ﻋﻴﻨﺔ ﻤﻥ ﺍﻟﺸﺠﺭﺓ ﻭ 10 ﻋﻴﻨﺎﺕ ﻤﻥ ﻴﺜﺭﺏ)، ﻤﺤﺎﻓﻅﺔ ﺃﻡ ﺩﺭﻤﺎﻥ 25ﻋﻴﻨﺔ (ﻜل ﺍﻟﻌﻴﻨﺎﺕ ﻤﻥ ﻨﻘﻁ ﺒﻴﻊ ﺍﻟﻠﺒﻥ ﻓﻲ ﻭﺩ ﺩﺭﻭ) ﻭﻤﻥ ﺍﻟﺨﺭﻁﻭﻡ ﺒﺤﺭﻱ25 ﻋﻴﻨﺔ (15 ﻋﻴﻨﺔ ﻤﻥ ﺤﻠﺔ ﻜﻭﻜﻭ ﻭ10 ﻋﻴﻨﺎﺕ ﻤﻥ ﻤﺭﻜﺯ ﺘﺠﻤﻊ ﺒﻴﻊ ﺍﻟﻠﺒﻥ ﺒﺎﻟﻘﺭﺏ ﻤﻥ ﺠﺎﻤﻌﺔ ﺍﻟﺨﺭﻁﻭﻡ ﻜﻠﻴﺔ ﺍﻟﺒﻴﻁﺭﺓ) ﺘﻡ ﻨﻘل ﺠﻤﻴﻊ ﺍﻟﻌﻴﻨﺎﺕ ﻓﻲ ﺒﺭﺍﺩ ﺍﻟﻰ ﺍﻟﻤﻌﻤل ﻟﻀﻤﺎﻥ ﺍﻟﺤﻔﻅ ﻭﺘﻡ ﺍﻟﺘﺯﺭﻴﻊ ﻓﻲ ﻨﻔﺱ ﺍﻟﻴﻭﻡ ﻓﻲ ﺍﻟﻭﺴﻁ ﺍﻟﻤﻌﺭﻭﻑ ﺒﺎﺴﻡ ﻤﺎﻨﺘﻭل ﻭ ﺍﻟﻤﻠﺢ (Manitol – salt agar ) . XI ﺘﻡ ﻋﺯل 163 ﻋﻴﻨﺔ ﻤﻥ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻤﺠﻠﻁﺔ ﻟﻠﺒﻼﺯﻤﺎ (coagulase positive) ﻭ ﻜﺎﻨﺕ ﻨﺴﺒﺔ ﻋﺯل ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻌﻨﻘﻭﺩﻴﺔ ﺍﻟﺫﻫﺒﻴﺔ ﻟﻤﺠﻤﻭﻉ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻤﺠﻠﻁﺔ ﻟﻠﺒﻼﺯﻤﺎ ﺍﻷﺨﺭﻯ (32.6%) , ﻭﻋﻠﻰ ﻤﺴﺘﻭﻯ ﺍﻟﻤﺤﺎﻓﻅﺎﺕ ﻜﺎﻨﺕ ﺍﻟﻨﺴﺏ ﻜﺎﻻ~ﺘﻲ (%39.6) ﻓﻲ ﺍﻟﺨﺭﻁﻭﻡ , (%47.2) ﻓﻲ ﺍﻡ ﺩﺭﻤﺎﻥ ﻭ(%13.2) ﻓﻲ ﺍﻟﺨﺭﻁﻭﻡ ﺒﺤﺭﻱ. ﻭﺸﻜﻠﺕ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻤﺠﻠﻁﺔ ﻟﻠﺒﻼﺯﻤﺎ ﺍﻻﺨﺭﻯ (coagulase Positive) ﻨﺴﺒﺔ (%4 .67) ﻤﻥ ﻤﺠﻤﻭﻉ ﺍﻟﻌﺯل . ﺘﺒﻴﻥ ﺃﻥ ﺍﻟﻠﺒﻥ ﺍﻟﺒﻘﺭﻱ ﺍﻟﻁﺎﺯﺝ ﻴﺤﻭﻱ ﻨﺴﺒﺔ ﻋﺎﻟﻴﺔ ﻤﻥ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻌﻨﻘﻭﺩﻴﺔ ﺍﻟﺫﻫﺒﻴﺔ ﺍﻟﻤﻤﺭﻀﺔ