Study on Enterococci and Bacillus Cereus Isolated from Minced Meat

Study On Enterococci And Bacillus Cereus isolated from minced meat

Amr Babiker Awadelkareem

B.V.Sc., (university of Khartoum, 2003)

Supervisor: Dr. Khalid Mohamed Suleman Athesis submitted for the fulfillment of the requirement of the master degree in microbiology

Department of microbiology Faculty of veterinary midicine University of Khartoum December, 2006

Dedication

I dedicate this work to my parents who paved the way for me to attain my ultimate aim. My gratitude also goes to my brother, sisters and friends

With love Amr

Acknowledgment

I would like to express my sincere thanks and gratitude to my supervisor Dr. Khalid Mohammed Suleiman, for his advice, interest, encouragement, guidance, criticism and patience through out the course of the study . Thanks are also extended to the staff of the Department of Microbiology, Faculty of Veterinary Medicine, University of Khartoum, including the head department, technicians for their co- operation assistance, with special regard to Abdalagis, Fauzia and one great appreciation to my colleagues and friends for their co- operation and assistance My deepest gratitude's are extended to Ustaza Nuha , Rasha , Department of Surgery for their help I would like to give due to one who is special , My father for his moral , support and encouragement

Amr Babiker

II Abstract This is a descriptive study to determine the prevalence of multiple- drug resistant Enterococci, and the possible occurrence of vancomycin resistant Enterococci (VRE) in raw minced beef samples, human stool and in chicken droppings. Moreover, the study aimed to explore the possible contamination of minced meat with enterotoxigenic Bacillus cereus. One hundred and eighty raw minced beef samples, thirty human stool specimens and thirty chicken droppings specimens were bacteriologically investigated for the presence enterococci. The isolation rate of enterococci in beef samples was 61%, 73% for stool specimens and (53%) for chicken droppings. The isolates were identified by conventional cultural and biochemical methods. The most frequent enterococci isolated from beef samples was Enterococci faecalis, while E. faecium was the predominant isolates from stool and chicken droppings.

All isolated enterococci were tested for antibiotic sensitivity by disk diffusion method. Almost, all isolates had shown resistance to both cloxacillin and Penicillin, in addition all isolates from chicken droppings and human stools were found to be resistant or intermediately resistant to Meat isolates were found to be susceptible to Tetracycline gentamycin. and ampicillin while stool isolates were found to be susceptible to ampicillin and chloramphenicol. The resistance to vancomycin in all isolates was intermediate.

Fifty raw beef samples were collected from different butcher shops in Omdurman area. Ten Bacillus cereus (20%) were isolated. Using the rabbit ileal loop test, 80% of B. cereus isolates were found to be interotoxigenic.

In conclusion, our study documented that Enterococci commonly contaminate meat, and their antibiotic sensitivity profile demonstrated high resistance level to numerous antibiotics. Also this work reported high

III contamination rate of meat with B. cereus, and many isolates of which proved to be enterotoxigenic.

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هﺬﻩ دراﺳﺔ وﺻﻔﻴﺔ ﺣﺪدت ﻣﺪى إﻧﺘﺸﺎر اﻟﻤﻘﺎوﻣﺔ اﻟﻤﺘﻌﺪدة ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ ﻓىﺎﻟﻤﻜﻮرات اﻟﻤﻌﻮﻳﺔ، وإﺣﺘﻤﺎﻟﻴﺔ وﺟﻮد ﻋﺰﻻت اﻟﻤﻜﻮرات اﻟﻤﻌﻮﻳﺔ اﻟﻤﻘﺎوﻣﺔ ﻟﻌﻘﺎر اﻟﻔﺎﻧﻜﻮﻣﺎﻳﺴﻴﻦ ﻓﻰ ﻋﻴﻨﺎت اﻟﻠﺤﻢ اﻟﻤﻔﺮوم وﻋﻴﻨﺎت اﻟﺒﺮاز ﻣﻦ أﻓﺮاد ﻣﻌﺎﻓﻴﻦ وﻋﻴﻨﺎت ﺑﺮاز ﻣﻦ اﻟﺪواﺟﻦ .آﺬﻟﻚ ﺳﻠﻄﺖ هﺬﻩ اﻟﺪر اﺳﺔ اﻟﻀﻮء ﻋﻠﻰ ﺗﻠﻮث اﻟﻠﺤﻢ اﻟﻤﻔﺮوم ﺑﺒﻜﺘﺮﻳﺎ اﻟﺒﺎﺳﻠﺲ ﺳﺮس اﻟﻤﺤﺮرة ﻟﻠﺰﻳﻔﺎن اﻟﻤﻌﻮي. ﻟﻌﺰل اﻟﻤﻜﻮرات اﻟﻤﻌﻮﻳﺔ ﺟﻤﻌﺖ 180 ﻋﻴﻨﺔ ﻟﺤﻢ ﻣﻔﺮوم و30 ﻋﻴﻨﺔ ﺑﺮاز ﻣﻦ أﻓﺮاد ﻣﻌﺎﻓﻴﻦ و30 ﻋﻴﻨﺔ ﺑﺮاز ﻣﻦ اﻟﺪواﺟﻦ، ﻋﺰﻟﺖ اﻟﻤﻜﻮرات اﻟﻤﻌﻮﻳﺔ ﻓﻲ 110 ﻣﻦ ﻋﻴﻨﺎت اﻟﻠﺤﻢ ﺑﻨﺴﺒﺔ %61 وﻓﻰ 22 ﻣﻦ ﻋﻴﻨﺎت ﺑﺮاز اﻻﺷﺨﺎص اﻟﻤﻌﺎﻓﻴﻦ ﺑﻨﺴﺒﺔ %73 وﻓﻰ 16 ﻋﻴﻨﺔ ﻣﻦ ﺑﺮاز اﻟﺪواﺟﻦ ﺑﻨﺴﺒﺔ %53. ﺗﻢ ﺗﻌﺮﻳﻒ اﻟﻤﻜﻮرات اﻟﻤﻌﻮﻳﺔ ﺑﺎﺳﺘﺨﺪام اﻷوﺳﺎط اﻟﺘﻘﻠﻴﺪﻳﺔ واﻻﺧﺘﺒﺎرات اﻟﻜﻴﻤﻴﺎﺋﻴﺔ. اﻟﻤﻜﻮرات اﻟﻤﻌﻮﻳﺔ اﻟﺒﺮازﻳﺔ آﺎﻧﺖ اآﺜﺮ ﺗﻜﺮارا ﻓﻰ ﻋﺰﻻت اﻟﻠﺤﻢ اﻟﻤﻔﺮوم ﺑﻴﻨﻤﺎ اﻟﻤﻜﻮرات اﻟﻤﻌﻮﻳﺔ ﺗﻔﻞ اﻟﺒﺮازﻳﺔ هﻰ اﻻآﺜﺮ ﺗﻜﺮارا ﻓﻰ ﻋﺰﻻت اﻟﺒﺮاز. ﺗﻢ إﺟﺮاء إﺧﺘﺒﺎر اﻟﺤﺴﺎﺳﻴﺔ ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ ﻟﻌﺰﻻت اﻟﻤﻜﻮرات اﻟﻤﻌﻮﻳﺔ. اﻇﻬﺮت اﻟﺪراﺳﺔ إن اﻟﻤﻜﻮرات اﻟﻤﻌﻮﻳﺔ اﻟﻤﻌﺰوﻟﺔ ﻣﻦ اﻟﺤﻢ اﻟﻤﻔﺮوم أﺑﺪت ﻣﻘﺎوﻣﺔ ﻟﻠﻜﻠﻮآﺴﺎﺳﻠﻴﻦ واﻟﺒﻨﺴﻠﻴﻦ ، ﺑﻴﻨﻤﺎ وﺟﺪ إن ﻋﺰﻻت ﺑﺮاز اﻟﺪواﺟﻦ واﻷﻓﺮاد اﻟﻤﺘﻄﻮﻋﻴﻦ أﻇﻬﺮت ﻣﻘﺎوﻣﺔ آﺎﻣﻠﺔ وﻣﺘﻮﺳﻄﺔ ﻟﻌﻘﺎر اﻟﺠﻨﺘﻤﺎﻳﺴﻴﻦ. آﺎﻧﺖ هﻨﺎك ﺣﺴﺎﺳﻴﺔ واﺿﺤﺔ ﻟﻌﻘﺎرى اﻟﺘﺘﺮاﺳﺎﻳﻜﻠﻴﻦ واﻷﻣﺒﺴﻠﻴﻦ ﻣﻦ ﻋﺰﻻت اﻟﻠﺤﻤﺔ اﻟﻤﻔﺮوﻣﺔ ﺑﻴﻨﻤﺎ ﻋﺰﻻت اﻟﺒﺮاز ﻣﻦ اﻷﻓﺮاد اﻟﻤﺘﻄﻮﻋﻴﻦ أﻇﻬﺮت ﺣﺴﺎﺳﻴﺔ ﻟﻌﻘﺎرى اﻷﻣﺒﺴﻠﻴﻦ واﻟﻜﻠﻮرﻣﻔﻴﻨﻜﻮل، اﻟﻤﻘﺎوﻣﺔ ﻟﻌﻘﺎر اﻟﻔﺎﻧﻜﻮﻣﺎﻳﺴﻴﻦ آﺎﻧﺖ ﻣﺘﻮﺳﻄﺔ. ﺗﻢ ﺟﻤﻊ 50 ﻋﻴﻨﺔ ﻟﺤﻤﺔ ﻣﻔﺮوﻣﺔ ﻣﻦ ﺟﺰارات ﻣﺨﺘﻠﻔﺔ ﻓﻰ ﻣﻨﻄﻘﺔ أﻣﺪرﻣﺎن ﻟﻌﺰل ﺑﻜﺘﺮﻳﺎ اﻟﺒﺎﺳﻠﺲ ﺳﺮس. ﺗﻢ ﻋﺰل اﻟﺒﻜﺘﺮﻳﺎ ﻣﻦ10 ﻋﻴﻨﺎت ﻓﻘﻂ وأﻇﻬﺮت 8 ﻣﻨﻬﺎ اﺳﺘﺠﺎﺑﺔ ﻣﻮﺟﺒﺔ ﻓﻲ ﻋﺮوة اﻟﻠﻔﺎﻳﻔﻰ اﻟﻤﺮﺑﻮﻃﺔ ﻓﻲ اﻷرﻧﺐ. ﺧﻠﺼﺖ هﺬﻩ اﻟﺪراﺳﺔ إن اﻟﻤﻜﻮرات اﻟﻤﻌﻮﻳﺔ ﻣﻠﻮﺛﺎت أﺳﺎﺳﻴﺔ ﻟﻠﺤﻢ, وهﻲ ﺗﻤﺘﺎز ﺑﻤﻘﺎوﻣﺔ ﻋﺎﻟﻴﺔ ﻷﻧﻮاع ﻣﺨﺘﻠﻔﺔ ﻣﻦ اﻟﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ ، أﻳﻀﺎ أوﺿﺤﺖ اﻟﺪراﺳﺔ أن هﻨﺎك ﻧﺴﺒﺔ ﺗﻠﻮث آﺒﻴﺮة ﺑﺒﻜﺘﺮﻳﺎ اﻟﺒﺎﺳﻠﺲ ﺳﺮس ﻓﻲ اﻟﻠﺤﻢ، و أن ﻟﻠﻌﺪﻳﺪ ﻣﻨﻬﺎ اﻟﻤﻘﺪرة ﻋﻠﻰ إﻧﺘﺎج زﻳﻔﺎﻧﺎت ﻣﻌﻮﻳﺔ.

Table of Contents 1. Introduction 1 2. Literature Review 4 2.1 Enterococci 4 2.1.1 Definition 4 2.1.2 Classification 4 2.1.3 Natural habitats 5 2.1.4 Pathogenicity 5 2.1.5 Cultural characteristics 5 2.1.6 Intrinsic resistance of enterococci 6 2.1.7 Acquired resistance to B_Lactams and aminoglycosides 6 2.1.7.1 B-lactam antibiotic 6 2.1.7.2 Aminoglycoside resistance 7 2.2 Vancoymcin 7 2.2.1 Mode of action 7 2.2.2 Resistance to vancomycin 8 2.2.2.1 History 8 2.2.2.2 Mechanism of vancomycin resistance 8 2.2.2.3 Genes of vancomycin resistance 9 2.2.2.4 Phenotypic description 9

VI 2.2.2.5 Genotypic classification of VRE 10 2.2.2.5.1 Van A glycopeptide resistance 10 2.2.2.5.2 Van B glycopeptide resistance 11 2.2.2.5.3Van C glycopeptide 11 2.2.2.5.4 Van E glycopeptide resistance 11 2.2.2.5.5 Van D glycopeptide resistance 12 2.3 Antimicrobial resistance of enterococcus species isolated from retail meat 12 2.3.1 The percentage of enterococcus spp in retail meat 12 2.3.2 Antimicrobial susceptibility testing for enterococci 12

2.3.3 Prevalence of VRE isolated from retail meat 13 2.3.4 VRE in the community 14 2.3.5 Colonization and Infection 15 2.3.6 Reservoirs 15 2.3.7 Mode of transmission 16 2.3.8 Prevention and control of VRE 17

2.3.8.1 Prevention and control of VRE in minced beef meat 17 2.3.8.2 Prevention and control of the nosocomial transmission of VRE 17 2.3.8.2.1 Education programs 18 VII 2.3.8.2.2 Role of the microbiology laboratory in the detection of VRE 18 2.3.8.2.3 Treatment 19 2.4 Bacillus cereus 21 2.4.1 Definition 21 2.4.2 Natural habitats 21 2.4.3 Bacillus cereus food poisoning 21 2.4.3.1 Contamination of diary products with B. cereus 22 2.4.3.2 Contamination of fried rice with B. cereus 22 2.4.4 Factors affecting the growth of Bacillus cereus in food 22 2.4.5 Bacillus cereus toxins 23 2.4.5.1 Diarrhoeal toxins 23 2.4.5.2 Emetic toxins 24 2.4.5.3 Other toxins 24 2.4.6 The illness 25 2.4.7 Rabbit ileal loop assays (RIL) 26

2.4.8 prevention and control of Bacillus cereus food’s contamination sss 26

3. Material and Method

3.1 Study area 29

3.2 Sampling of isolation of enterococci 29 VIII 3.2.1 Minced meat samples 29 3.2.2 Chicken dropping specimens 29

2.2.3 Stool specimen 29 3.3 Sterilization 29 3.3.1 Hot air oven 29

3.3.2 Autoclaving 30

3.3.3 Disinfection of benches 30

3.4 Culture media 30 3.4.1 Blood Agar (Oxiod) 30 3.4.2 Nutrient agar 31 3.4.3 MacConkey’s agar (Oxiod) 31 3.4.4 Peptone water sugar (Oxiod) 32

3.4.5 Bile Aesculin Agar (Oxiod) 33 3.4.6 Diagnostic Sensitivity Test Agar D.S.T Agar (Oxiod) 33

3.4.7 Craigie tube medium 34

3.4.8 Peptone water medium (CM9) 34

3.4.9 Blood – Tellurite Agar 34 3.4.10 Ammonium Salt Sugar (ASS) 34 3.4.11 Citrate media 35 3.4.12 Starch Agar 35

IX 3.5 Cultivation of samples to isolate enterococcci 36

3.5.1 Minced meat samples 36 3.5.2 Chicken droppings specimens 36

3.5.3 Stool specimens 36 3.6 Sub culturing and purification 36

3.7 Preservation of isolates 36

3.8 Biochemical methods 36

3.9 Identification of Enterococci 37 3.9.1 Cultured characteristic 37 3.9.2 Primary tests 37 3.9.2.1 Gram’ stain 37

3.9.2.2 Catalase test 37

3.9.2.3 Motility test 37 3.9.3 Secondary tests 37

3.9.3.1 Bile Aesculin hydrolysis test 38 3.9.3.2 Salt tolerance test 38

3.9.3.3 Tellurite tolerance test 38

3.9.3.4 Sugars tests 38

3.9.3.4 Growth at 45oC 38

X 3.9.4 Antibiotic Sensitivity of enterococci 39 3.10 Sampling for isolation B. cereus 40 3.11 Cultivation of samples to isolate B. cereus 40 3.12 Identification of B. cereus 40 3.12.1 Primary tests 40

3.12.2 Secondary tests 40

3.12.2.1 Sugars tests 41 3.12.2.2 Starch hydrolysis 41 3.13 Enterotoxigenicity of B. cereus 41

4. RESULTS 4.1 Samples investigated for enterococci 43 4.2 Samples investigated for B. cereus 43 4.3 Cultural characteristics 43 4.5 Identification of enterococcus species 44 4.5 Identification of B. cereus 44 4.6 Antibiotic Sensitivity test for enterococcus species 50 4.7 Sensitivity of enterococci to Vancomycin 50 4.8 Vancomycin susceptibility in different species of Enterococci 50 XI 4.9 Enterotoxigenicity of B. cereus 55 5. Discussion 59 6. Recommendations 66

7. References 67

Appendix 84

XII list of Tables

Table Subject Page No No Tab. 1: Biochemical reaction for enterococcus spp isolates 45

Tab. 2: Biochemical reaction for B. cereus isolates 46 52 Tab. 3: Result of antibiotic sensitivity tests of enterococci isolated from meat samples 52 Tab. 4: Result of antibiotic sensitivity tests of enterococci isolated from stool specimens 53 Tab. 5: Result of antibiotic sensitivity tests of enterococci isolated from chicken droppings specimens 58 Tab. 6: RIL activity of various B. cereus strains

XIII

list of figures

Fig. 1: Relative distribution of 110 isolates of enterococcal species 47 from raw minced beef samples.

Fig. 2: Relative distribution of 22 isolates of enterococcal species 48 from stool specimens.

Fig. 3: Relative distribution of 16 isolates of enterococcal species 49 in chicken droppings specimens.

Fig. 4: Enterococci on blood agar. 51

Fig. 5: Antibiotic sensitivity of enterococus isolates. 51

Fig. 6: Sensitivity of enterococci isolated from different specimens 54 to vancomycin.

Fig. 7: Heamolytic activity of Bacillus cereus. 56

Frequency of bacillus cereus in raw minced beef samples. 56 Fig. 8:

Fig. 9: Rabbit ileal loop test of bacillus cereus isolates. 57

XIV Chapter One Introduction Protection of the food supply includes considerations of the microbiological quality and safety of commodities available for public consumption. While these concerns often address specific pathogenic micro organisms that present on commensal components of flora associated with food-producing animals that may also impact consumer, species of the genus Enterococcus and B. cereus comprise a large proportion of the microflora associated with meat contamination. In the last decade, enterococci become the second most frequently reported cause of surgical wound infections and nosocomial urinary tract infections (E liopoulos, 1993; Murray, 1990; Willey et al., et al 1994) and the third most frequently reported cause of bacteremia (lemmen and Daschner, 1996; Schaberg and Gaynes, 1991). In spite of their low level of virulence the increasing importance of enterococci is largely due to their resistance to many antimicrobial agents, including B-lactam antibiotics, aminoglycoside and recently, glycopeptides (Eliopoulos, 1993; Hall, 1993; Moellering, 1992). The glycopeptide antibiotics vancomycin and teicoplanin are important substances for treatment of several hospital infections. Disease caused by enterococci which are resistant to B. lactam ampicillin and aminoglycoside antibiotics can be treated only with glycopeptides (Calia, 1996; lerner, 1996). Unfortunately, resistance to vancomycin and teicoplanin has also been reported. In the United States, there was 20-fold increase in the occurrence of vancomycin – resistant enterococci (VRE) associated with nosocomial infections from 1989 to 1990 (Center for Disease Control and Prevention, 1993).

Therefore, the therapeutic use of glycopeptides should be restricted. Otherwise, infections caused by such multi drugs resistant enterococci will become untreatable (Bodnar et al., 1996; Uttley et al., 1988). Moreover, glycopeptide resistant E. faecium strains have recently been isolated from farm animals in Europe (Klare et al., 1995). The high level of resistance to glycopeptides is inducible and is coded by the Van A gene cluster, which is carried on transposons. These characteristics make it very easy for bacteria to transfer antibiotic resistance to other cell of the same (or different) species (walsh and Christopher, 2001). The consumption of meat carrying antibiotic resistant enterococci population has been reported as possible route of transfer and could result in either colonization or transfer of resistance determinants to host adapted strains (Hayes et al., 2003). This study was performed in order to measure the prevalence of multi- antimicrobial resistance of enterococcal spp from raw beef samples, chicken droppings specimens and stool specimens from healthy volunteers, and it determined the rate of VRE among the isolates. B. cereus has been recognized as a causative agent of food poisoning for 20 years (Spira and Goepfert, 1972), and as common inhabitant of soil, plant and wide variety of foods. It causes two different form of food poisoning; an emetic illness and a diarrheal illness, the emetic illness is mediated by a highly stable toxin that survives at high temperatures and exposure to trypsin, pepsin and pH extremes. The diarrheal illness is mediated by a heat- and acid- labile enterotoxin (Notermans and Batt,. 1998). On the basis of the ability of culture filtrates of B. cereus to induce fluid accumulation in ligated rabbit ileal loops (RIL), it was concluded that B. cereus secreted a diarrheal enterotoxin. This enterotoxin posses hemolytic, cytotoxic, dermonecrotic, and vascular permeability activities (Beecher et al., 1995; Thompson et al., 1984). This study was performed to determine the contamination rate of meat associated with B. cereus, and the use of ligated rabbit ileal loop assay as an experimental model of B. cereus food poisoning. The objectives of this study to investigate the occurrence and resistance of enterococci isolated from meat to abroad range of antimicrobial substances that are used either as therapeutic agents or as feed additives. Also investigation of the possible occurrence of VRE is detected. Moreover, to study contamination rates of meat with B. cereus and to demonstrate the enterotoxigenic of B. cereus by using rabbit ileal loop assay.

Chapter Tow Literature Review 2.1 Enterococci : 2.1.1 Definition: Enterococci are Gram positive cocci, occurring singly, in pairs or in short chains, non-spore forming, facultatively anaerobic. Motility is observed in some species. Glucose fermentation results in production of lactic acid (FackLam et al., 1998). Sherman(1937) further recommended that the term "Enterococci" should be used specifically for streptococci that grow at both 10 and 45˚C, at pH 9.6 and in 6.5% NaCl and survive at 60˚C for 30 min. These organisms also hydrolyze asculin in the presence of bile, catalase and oxidase negative (Sherman, 1937), and are also characterizes by production of pyrrolidonyl arylamidase (Gordts et al., 1995).

2.1.2 Classification Enterococci were originally classified as enteric gram- positive cocci and they were later included in genus streptococcus (Murray, 1990). In 1930, with the establishment of the lancefield serological typing system, enterococci were classified as group D streptococci and were differentiated from the non-enterococcal group D streptococci such as streptococcus bovis by distinctive biochemical characteristics (Lancefield, 1933). In 1980, based on genetic differences, enterococci were removed from the genus streptococcus and placed in a sparate genus, enterococcus (Schleifer et al., 1984). Enterococcus was designated to nineteen species until recently, E. faecalis and E. faecuim had been the predominant enterococcal species than other enterococcus species such as E. gallinarum, E. durans E. casseliflavus, E. avium, E. hirae and E. raffinosus (Hayes et al., 2003). 2.1.3 Natural habitats Enterococci occur almost everywhere, including soil, plant, food, animals, birds, and insects (Hayes et al., 2003).

In human, potentially pathogenic and non pathogenic species may be present on the skin and on the mucous membranes of the female genital tract and upper respiratory tract (Carter, 1986) E. faecalis and E. faecium inhibit the gastrointestinal tract of human, poultry and other animals. E. avium and E. gallinarum were isolated from chicken faeces E. casseliflavus was recovered from plant, soil and rarely from the faeces of chicken. E. hirae isolated from chicken crops, faeces and gastrointestinal tract of other animals. (Elmer et al., 1997).

2.1.4 Pathogenicity Enterococci was recognized as common cause of hospital acquired infection in the middle to late 1970 (Murray, 1990) Infections caused by the genus enterococcus (most notably E. faecalis which account for 80% - 90% of all infections, and E. faecium had account for 5 to 15% (other enterococcus species had account less than 5% of all infection) include urinary tract infection, bacteremia, intra-abdominal infection and endocarditis (Moellering et al., 1992). Enterococci are also usually found in wound and soft tissue infection with other facultative and anaerobic bacteria (Murray, 1990). Respiratory tract infections caused by enterococci are rare (Berk et al., 1983).

2.1.5 Cultural characteristics: Enterococci show small lactose fermenting colonies on MacConkey's agar plates. E. faecalis and E. durans are beta- hemolytic. They produce clear zone of complete hemolysis on blood gar plates containing erythrocytes from rabbit, horses and human. All other species are usually Alpha hemolytic, or non hemolytic (Facklam et al., 1998).

2.1.6 Intrinsic resistance of enterococci

Soon after the introduction of penicillin in the early 1940. There were reports that penicillin treatment for enterococcal endocarditis produced worse outcomes than penicillin treatment for streptococcal endocarditis. This fact was consistant with the observation that enterococci were considerably less susceptible to penicillin than streptococci For example, E. faecalis is typically 10 to 100 less susceptible to penicillin than are most streptococci, while E. faecium is at least 4 to16 times less susceptible than E. faecalis. (Murray, 1990). While most isolates of E. faecalis are inhibited by concentrations of penicillin or ampicillin (1 to8µg/ml) easily achievable in humans, isolates of E. faecium usually require an average of 16 to 64µg/ml to inhibit growth, although some isolates are even more resistant (Murray, 1997). Even those enterococci that are susceptible to killing by pencillin can develop tolerance to this bactericidal effect (Hodges et al., 1992). 2.1.7 Acquired resistance to B-Lactams and aminoglycoside 2.1.7.1 B- lactam antibiotic: Many strains of enterococci, particulary E. faecium; are intrinsically resistant to B- Lactam antibiotics because they posses proteins with low binding affinity for these drugs (Leclercq et al., 1989). Resistance to penicillin is directly proportional to the amount of penicillin binding Protein5 (specific PBP) (Fontana et al., 1983). Unlike most staphylococci, where B- lactamase production is inducible, B- lactamase production in enterococci is constitutive, low leveled, and is inoculum dependent (Murray, 1992).

2.1.7.2 Aminoglycoside resistance There are mechanisms for enterococcal resistant to aminoglycosides. Aminoglyconside- modifying enzymes, which include phosphotransferase, nucleotidyl transferases, and acetyl transferases, are produced constitutively on plasmids (Leclercq et al., 1992). They resemble the enzymes found in staphylococci rather than those produced in gram- negative bacilli. Early studies demonstrated that two types of streptomycin resistance occur in enterococci: (i) moderate-level resistance (MIC, 62 to 500µg/ml), because of low permeability, which can be overcomed with a penicillin (which increase the cellular up take of the aminoglycoside); and (ii) high level resistance (MIC > 2,000 µg/ml), which is either ribosomally mediated or due to the production of aminoglycosid- inactivating enzymes (Cetinkaya et al., 2000) Detection of high level resistance of enterococci to aminoglycosides is important because these strains are not inhibited synergistically by a combination therapy with B- lactam antibiotics (Eliopoulos et al., 1988; Fuller et al, 1990; Spiegel, 1988).

2.2 Vancomycin: 2.2.1 Mode of action of vancomycin: Vancomycin is a bactericidal glycopeptide antibiotic, that acts principally against gram- positive organisms, vancomycin interrupts cell wall synthesis by forming a complex with the C- terminal D- alanine residues of peptidoglycan precursors. Complex formation at the outer surface of the cytoplasmic membrane prevents the transfer of the precursors from a lipid carrier to the growing peptidoglycan wall by transglycosidases (Wu, el at., 1995; Zaman et al,. 1996)

2.2.2 Resistance to vancomycin 2.2.2.1 History Scientists introduced vancomycin into hospitals more than 40 years ago in response to new strains of staphylococci developing resistance to pencillin. And it has been in clinical use for more than 30 years without the emergence of market resistance (Ingerman and Santora,1989). The introduction of methicillin decreased the use of vancomycin for a few years; however, when methicillin- resistant S. aureus trains appeared in the past two decades, the glycopeptide antibiotic was reinstated as a therapeutic agent. Vancomycin is now seen as the last-resort drug because it offers the last opportunity that a physician may have to eliminate infections caused by multidrug resistant bacteria (Cetinkaya,Yesim et al., 2000)

In 1988, Utelly et al, was the first to report the isolates of vancomysin- resistant E. faecalis and E. faecium in England shortly after the first isolates of VRE were reported by investigators in the United Kindom and France (Uttley et al., 1988). Certain transposable genetic elements encode special cell wall synthesizing enzymes which change the structure of the normal D- alanyl-d-alanine side chain in the peptidoglycan assembly pathway. The altered side chain (D- Ala- D- lac) does not bind vancomycin and allows normal peptidoglycan polymerization to occur in the presence of the drug (Wu, el at., 1995; Zaman et al,. 1996). .2.2.2.3 Genes of vancomycin resistance: Five types of vancomycin resistant enterococcus (VRE) genes have been repoted (Van A, Van B, Van C, Van D and Van E) (Leclercq and Courvalin, 1997; Fines et al., 1999). The mechanism of resistance have been best characterized for Van A. It consists of cluster of seven genes found on the transposable (mobile) genetic element Tn/546. In the presence of an inducer like vancomycin, transcription of the genes necessary for resistance to vancomycin is activated as a result of the interactions of a sensory kinase and a response regulator, the transcribed genes are translated into enzymes, some of which make cell-wall precursors ending in d-alanyl, d-lactate (d-Ala- d-lac), which vancomycin binds with very low affinity. Others prevent synthesis or modify endogenous cell- wall precursors ending in D. alanyl- D- alanine (D- Ala, D- Ala), to which vancomycin binds with high affinity (Evers et al; 1996; Arthur et al; 1998). By binding to these residues with high affinity, the antibiotic prevents them from being accessible to the active side of the transpeptidases. Peptide cross- linking therefore can not occur, and the structural integrity of the peptidoglycan is compressed, causing the cell to lyse (Walsh and Christopher., 2001)

2.2.2.4 Phenotypic description There are five recognized phenotypic of vancomycin resistance. Van A, Van B, Van C, Van D, and Van E. Two of these (Van A and Van B) are mediated by newly acquired gene clusters not previously found in enterococci (Arthur and Courvalin, 1993; Arthur et al., 1993). Van A and Van B types of resistance are primarily found among enterococci isolated from clinical, veterinary, and food specimens (Klare et al., 1995), but not commonly found in intestinal or environmental enterococci. In the laboratory, however, these genes can be transferred from enterococci to other bacteria. Van A and Van B resistance phenotypes were described primarily in E.faecalis and E. faecium. Van A resistant strains possess inducible, high level resistance to vancomycin and teicoplanin (Arthur and Courvalin; 1993). The details of vancomycin resistance have been best documented with the van A gene cluster on the transposon, Tn 1546 (Arthur and Courvalin 1993; Arthur et al., 1993). Van B isolates were initially believed to be inducible resistant to more moderest levels of vancomycin but are susceplible to teicoplanin. Van B resistance determinants also reside on large mobile elements that can be transferred from one strain of enterococcus to another (Quintiliani and Courvalin, 1994) The Van C resistance phenotype was described in E. gallinarum which demonstrate low- level resistance to vancomycin and susceptible to teicoplanin. In the United States, Van A and Van B account for approximately 60% and 40% of VRE isolates respectively (Clark et al., 1993). Certain limitations of this classification method have become evident over time. For example, the genetic determinates of the Van A phenotype have now appeared in E. gallinarum and other enterococcal species (Dutka Malen et al., 1994). Nevertheless, this phenotypic classification is still useful, because, it usually corresponds well to the genetic classification and utilized information that can be derived simply and inexpensively in laboratory (Elipoulos, 1997).

2.2.2.5 Genotypic classification of VRE: 2.2.2.5.1 Van A glycopeptide resistance: The Van A gene and other genes involved in the regulation and expression of vancomycin resistance (Van R, Van S, Van H, Van X, and Van Z) are located on a

10'581'bp transposon (Tn1546) of E. faecium, which often resides on plasmid (Arthur et al; 1993). The advantage of accumulating genes in plasmid is that, these regions of DNA can replicate independently of the bacterial genome, and can also be readily transferred from one cell to another. Resistance can therefore easily speed between species. In addition, these Vancomycin- resistance genes are located on transposon element, which can cut themselves out of one segment of DNA and move to another segment. These characteristics make it very easy for bacteria to transfer antibiotic resistance to other cell of the same (or different) species (Walsh and christopher., 2001).

2.2.2.5.2 Van B glycoptide resistance: The Van B cluster genes is often located on the short chromosome and initially was thought not to be transferable to other bacteria, however, it can also occur on plasmids, and, even when it is chromosomal, this gene cluster has been transferable as part of large mobile element, perhaps related to large conjugative transposon (Quintiliani et al., 1993).

2.2.2.5.3 Van C glycopepetide resistance Van C phenotype constitutes a low level resistance to vancomycin (Clark et al ., 1998). The genes encoding the Van C type of vancomycin resistance are endogenous, species- specific components of E. gallinarum (Van C-1) and E. casseliflavus / E. flavescens (Van C-2/ Van C-3) (Navarro and Courvalin., 1994).

2.2.2.5.4 Van D resistance The strain carrying this resistance is E. faecium that inhibited by vancomycin at 64 µg/ml. Van D appears to be located on the chromosome and is not transferable to other enterococci (Perichon et al,. 1997)

2.2.2.5.5 Van E glycopeptide resistance The Van E vancomycin resistance gene has recently been described in E. faecalis, which is resistant to low levels of vancomycin (MICs 16µg/ml). (Fines, et al., 1999). 2.3 Antimicrobial Resistance of enetrococcus species Isolated from retail meat 2.3.1 The percentage of enterococcus in retail meat Enterococci are common components of the microflora community of mammals, birds, insects, and reptiles. Retail meat of chicken, pork and beef were investigated bacteriologically for Enterococci. E. faecium was the predominant species recovered (61%), followed by E. faecalis (29%) and E. hirae (5.7%), E. casseliflavus (2.1)E. durans (1.2%), E. gallinarum (0.7%) and E. avium (0.1%) ( Hayes et al, 2003). Klein et al (1998) also studied the prevalence of enterococci in minced beef in Germany and they showed that, E. faecalis was the predominant species recovered (87%) and (4%) were identified as E. faecium isolates, (3%) E. casseliflavus, (2%) E. durans, (2%) E. gallinarum, (1%) E. hirae, (<1%) E. avium. 2.3.2 Antimicrobial susceptibility testing for Enterococci Hayes et al, (2003) examined a population of E. faecium (n = 825) and E. faecalis (n = 388) for their antimicrobial resistance profiles. Resistance to chloramphenicol gave a very low level (<1%) a cross the population of E. faecalis recovered, while a resistant subpopulation of E. faecalis was observed only among population isolated from pork. Resistance to ciprofloxacin was observed at a higher frequency among E. faecium isolates than among E. faecalis isolates. E. faecium was also more often resistance to penicillin and Tetracycline, whereas resistance to vancomycin or linezolid was not observed among E. faecium or E. faecalis isolates and resistance to tetracycline was frequent, with over 70% of all isolates displaying resistance, resistance to high level aminoglycosides was prevalent a cross all species recovered, the patterns of susceptibility to these groups were interesting in that resistance to kanamycin was the most prevalent, followed by resistance to streptomycin and resistance to gentamicin. The high- level aminoglycosides resistance among E. faecalis and E. faecium isolates was observed with rates 5% and 47%, respectively. Klein et al (1998) also studied susceptibility of enterococci to several antibiotic and they observed that, a total of 144out of 209 isolates exhibited intermediate resistance or were resistance to erythromycin and all of the isolates except two enterococci were susceptible to streptomycin and 42- 6 strains exhibited intermediate resistance or were resistance to tetracycline and chloramphenicol, respectively.

2.3.3 Prevalence of VRE Isolated from Retail meat The food chain, especially raw minced meat, is thought to be responsible for an increase in the incidence of VRE in human nosocomial infections, 555 samples of minced beef and pork from a European Union- licensed meat- processing plant were screened for the occurrence of VRE. Enterococci were isolate directly from enterococcosal selective agar plates and also from enterococcal selective agar plates supplemented with 32mg of vancomycin per liter. In addition, buffer peptone water (BPW) was used in a preenrichment procedure. When the direct method was used, VRE were found in 3 of 555 samples (0.5%) (No enrichment procedure was performed), whereas in each 46 of 555 samples (8.3%) VRE were found after overnight enrichment in BPW (Klein et al, 1998). How ever, in previous studies to detect VRE from meat, five vancomycin resistant E. faecium strains with Van A resistant from meat samples obtained from 13 different butchers shops( Klare et al., 1995) Wegener et al (1997), found that food contaminated with vancomycin- resistant E. faecium strains was an important possible source of infections in the community, based on their study in Denmark and a study performed in Belgium. The persistance of VRE on farms that have discontinued the use of avoparcin as a growth promoter illustrated the impact posed by antimicrobial usage in food animal production environment (Bager et al., 1997)

2.3.4 VRE in the community In the United states, attention has focused on the epidemiology of VRE mainly in hospitals, and there is little evidence to suggest that transmission of VRE to healthy adults occurs to any significant extent in the community. In Texas, investigators failed to find any VRE in the feces or carcasses of chicken. In addition VRE could not be isolated from healthy volunteers in two studies (Murray, B. E.,1995). The situation in Europe is quite different from that in the United States. In Europe, VRE have been isolated from sewage and various animals. It has been suggested that the use of glycopeptide- containing animal feeds in some regions of Europe may have contributed to such differences. In one study, Van A resistant E. faecium was isolated from frozen poultry and pork and feces of 12 of 100 non hospitalized inhabitants in a rural area (Klare et al., 1995) Van A VRE have also be found in the feces or in intestines of other farm animals (Devriese et al., 1996). These observations suggest a potentiality for VRE or the resistance genes of VRE to reach humans through the food chain or through contact with domesticated animals (Gordst et al., 1995).

2.3.5 Colonization and Infection The emergence of vancomycin resistance in enterococci in addition to the increasing incidence of high- level enterococcal resistance to pencillin and aminoglycosides presents a serious challenge for physicians treating patients with infections due to these microrganisms (Cetinkaya et al.,2000). In many affected institutions, most patients, from whom VRE are recovered, are colonized rather than infected with the organism (Boyce et al., 1994; Jordens et al., 1994; Leclercq and Courvalin, 1997). The ratio of colonized to infected patients may reach as high as 10: 1 at hospitals in which pre-rectal or rectal swab specimens from high- risk patients are screened for VRE (Jordens et al., 1994 and Monteclvo et al., 1994). The extend to which VRE causes morbidity and mortality is often not known because most affected patients have serious underlying diseases that cause substantial morbidity and death and because VRE are often recovered in mixed cultures with other potential pathogens (Boyce, 1997).

2.3.6 Reservoirs Although much has been learnt about the epidemiology of VRE in recent years, the most important reservoirs of VRE has not been identified. Subsequent investigation revealed that several of these patients resided in farms and that chickens and swine present on the farms were colonized with vancomycin resistant E. faecium. VRE has subsequently been recovered from various animals sources in different European countries. The occurrence of VRE in such animals could be related to the fact that avoparcin (a glycopeptide) has been available as a feed additive for more than 15 years in the United Kingdom and other European countries (Aarestrup 1995; Witte, W., and klare, I. 1995). It has been fed to chickens, swine and cattle. In United States, avoparcin has not been used as food additive for animals, and surveys of a limited number of chickens in several cities have failed to detect VRE (Hayes et al.,2004). Further studies on animals based food products are needed to determine if food items represent a community reservoir for VRE in that country (Boyce., 1997).

2.3.7 Mode of transmission: Transmission of VRE by health care workers whose hands become contaminated with the organism while caring for patients is probably the most common mode of nonsocial transmission (Tornieporth et al., 1996; zervos et al; 1987). Transmission of VRE may also occur by way of contaminated medical equipment, although this is probably much less important than transmission by the hands of personnel. Electronic thermometers contaminated with the outbreak strain were implicated in some outbreaks (Cetinkaya et al., 2000). Since enterococci may remain viable for several days to weeks on dry surfaces , it seems possible that contaminated surfaces may act as a source from which personnel may contaminate their hands or clothing (Boyce et al., 1994)

Recovery of VRE from animals' resources in part of Europe suggests that food-borne transmission may occur in certain geographic areas (Bates and Griffithiths, 1995). VRE of humans are linked to food- animals production through the use of avoparcin as growth promoter in swine and poultry (Klare et al., 1995) Epidemiological studies in animals showed that avoparcin usage selected VRE (Bager et al., 1997). The genetic similarity of the strains isolated from animals and workers indicates that VRE can be transmitted from animals to humans (Jensen et al., 1998).

2.3.8 Prevention and control of VRE 2.3.8.1 Prevention and control of VRE in minced beef meat The epidemiology of VRE has not been completely elucidated; Minced meat created a risk that microbiological contamination would occur, so that the use of plastic gloves by workers was very important to avoid contamination of meat with enterococci and VRE from human (Wegener, et al 1997). On the other hand, epidemiologic studies are documented on association between avoparcin–containing feed and VRE recovery from animals (Mc Donald, et al 1997), these observation have led to an European ban on the use of avoparcin (Aarestrup, et al 1995).

2.3.8.2 Prevention and control of nosocomial transmission of VRE. Certain patient populations are at increased risk for VRE infection or colonization. These include critically ill patients or those with severe underling disease or immunosuppression, such as intensive care unit (ICU) patients or patients in oncology or transplantation wards. Those who have had on intra-abdominal or cardiothoracic surgical procedure, in addition to that those who have had prolonged hospital stay or received multiple antimicrobial agents (Friden et al., 1993 and Montecalve et al., 1994). However, recent reports have demonstrated that enterococci, including VRE, can be spread by direct patient – to patient contact or

indirectly via transient carriage on the hands of personnel contaminated environmental surfaces or patient care equipment (Boyce et al., 1994). In an effort to control the nosocomial Infection of VRE, Hospital infection Control Practices Advisory Committee (HICPAC) in USA published recommendations in February 1995 (Center for Disease Control and Prevention 1995). These recommendation mainly focused on; prudent use of vancomycin, education of hospital staff, effective use of the microbiology laboratory and implementation of infection control measures (including the use of gloves and gowns, and isolation of patients, as appropriate to specific conditions) (Boyle, 1993) The current isolation precautions recommended by HICPAC to prevent patient - to patient transmission of VRE are as follows: (i) Place VRE infected or – colonized patients in single room or in the same room as other patients with VRE. (Boyce, et al., 1994). (ii) Wear clean non-sterile gloves and gown when entering the room of VRE infected or – colonized patient (Boyce, et al., 1994). (iii) Remove gloves and gowns before leaving the patients room and wash hand immediately with an antiseptic agent (Doebbeling, et al., 1992; Jones, et al., 1981).

2.3.8.2.1 Education programs Educational programs for hospital staff (including student, pharmacy personnel, nurses and laboratory personnel) should include information about the epidemiology of VRE. Because detection and containment of VRE require high performance standards for hospital personnel, special awareness and education sessions may be indicated (Boyce, 1997).

2.3.8.2.2 Role of the microbiology laboratory in the detection of VRE The microbiology laboratory is the first line of defense against the spread of VRE in the hospital. The ability of the laboratory to identify enterococci and to detect vancomycin resistance accurately is essential in recognizing VRE colonization and infection and avoiding complex, costly containment efforts that are required when recognition of the problem is delayed (Cetinkaya et al., 2000). Entercocci may also be tested for vancomycin resistance by using polymerase Chain Reaction (PCR) assay designed to detect the genes responsible for glycopeptide resistance in these organisms (Clark, et al., 1993). Such tests may be particularly helpful in detecting Van B or Van C containing strains with low-level resistance to Vancomycin (swensen et al., 1992). Testing VRE isolates for susceptibility to teicoplanin by using simple disk diffusing test will differentiate between Van A (teicoplanin-resistant) and Van B (teicoplanin–susceptible ) strains in most instances (Satake et al., 1997).

2.3.8.2.3 Treatment Treatment of infections, especially those caused by E. faecium, is extremely problematic, because these organisms are resistant to multiple antibiotics. Penicillin or ampicillin with or without synergizing aminoglycoside would be a reasonable choice in the non-allergic patient infected with vancomycin resistant Entercocci. Chloramphenical is one of the few agents that retains in vitro activity against many strains of multiple drug–resistant E. faecium (Cetinkaya et al., 2000). Teicoplanin is another glycopeptide that is active in vitro against most Van B- type enterococci. It shown some efficacy in animal molecule of endocardities when combined with an aminoglycoside to which the infecting strain is not highly resistant (Hayden et al.,1994). In vitro data suggest that Novobiocin is very active against Van A or Van B E. faecium strains, even these strains are concomitantly resistant to penicillin and ampicillin (French and Frainow, 1993). Novobiocin plus ciprofloxacin was found to be effective in a rabbit model of endocardities (Gordst et al., 1995). One of the most active agents against VRE is a semisynthetic glycopeptide designated LY333328, which showed bactericiodal as well as bacteristatic activity against enterococci (Nawas et al., 2000). Ramoplanin, a lipoglycodepsiptide, is even more active in all investigations, all strains of all species, including vancomycin – resistant E. faecalis and E. faecium, were inhabited by 8 µg/ml and the MIC90s (in all but one study) ranged from ≤ 0.25 to 1.6 µg/ml (Bartoloni et al., 1990; Billot – Klein., 1994). The Oxazolidinones are new class of synthetic antibiotics with a good anti- enterococcal activity and different from any other class (Eliopoulos et al., 1996). The mechanism of activity demonstrated has been the inhibition of protein synthesis leading to bacteriostatic effect against most species (Daly et al., 1988). Mechanisms of resistance that effect the antibiotics in current clinical use do not affect the activities of oxazolidinones. Eperzolid and linezolid are two investigational oxazolidione agents which showed excellent activity against multi antibiotic – resistant enterococci characterized by low MIC (Bostic et al., 1998).

2.4 Bacillus Cereus 2.4.1 Definition The genus Bacillus, to which B. cereus belongs, is the largest of the Bacillaceae, encompassing more than 60 species. The genus Bacillus is described as aerobic or facultative anaerobic Gram- positive, spore- forming rods (Claus and Berkeley,1986)

2.4.2 Natural habitats B. cereus is commonly found in soil, on vegetables and in many raw and processed foods. Cooked meat and vegetables, boiled or fried rice, vanilla sauce, custards, soups and raw vegetable spouts have been incriminated in past outbreaks. Consumption of foods that contain large numbers of B. cereus (105 or more/g) may result in food poisoning especially when foods are prepared and hold several hours without adequate refrigeration before serving. (Notermans and Batt,. 1998).

2.4.3 B. cereus food poisoning B. cereus has been recognized as an agent of food poisoning since 1955. Between 1972 and 1986, 52 outbreaks of food- borne associated with B. cereus were reported (Kenneth Toder, 2005).

In the USA, from 1988 to 1992, twenty-one out breaks caused by B. cereus and 433 persons were affected (Notermans and Batt,. 1998). B. cereus causes two different forms of food poisoning: short- incubation or emetic form of disease, this is mediated by a highly stable toxin that survives high temperatures and exposure to trypsin, pepsin and pH extremes, with molecular weight less than 5000 dalton. And long- incubation or diarrhoeal form of disease, this is mediated by a heat- and acid- labile enterotoxin, with molecular weight 50000 dalton. The emetic form is most often associated with fried rice that has been cooked and then held at worm temperature for several hours, the disease is often associated with Chinese restaurants, in one reported outbreak, macaroni and cheese made from powdered milk turned out to be the source of intoxication. Whereas the diarrhoeal form is associated with meat or vegetable-containing foods after cooking. The bacterium has been isolated from 50% of dried beans and cereals and from 25% of dried foods such as spice, seasoning mixes potatoes (Kenneth Toder, 2005).

2.4.3.1 Contamination of dairy products with B. cereus In China, 293 dairy products were collected from local markets, and they were examined to determine the incidence of B. cereus. The B. cereus was reported in 17% of fermented milk, 52% of ice cream,35% soft ice cream , 2% pasteurized milk and pasteurized fruit-or nut-flavored milk mixes, and 29% of milk powder. The average population of B. cereus in these dairy products was 15 to 280 CFU/ml/g (Wong, et al., 1988). 2.4.3.2 Contamination of fried rice with B. cereus In one study from U.S, an outbreak of B. cereus food contamination was due to consuming fried rice prepared at a local restaurant. Illness occurred in 14(29%) of 48 persons who ate chicken fried rice. B. cereus was isolated from leftover chicken fried rice ( > 105 organism /gram) and from vomitus from one ill child (>106 organisms /gram). Twelve of 14 cases occurred among children aged 2.5-5 years (Khodr et al., 1993).

2.4.4 Factors affecting the growth of Bacillus cereus in food B. cereus has an optimum growth temperature of 28- 35ºC with a minimum of 4- 5ºC and a maximum of 48ºC. The generation time of the organism is 18- 27 min, it grows over a wide pH range 4.9- 9.3 and at salt concentrations of up to 7.5%. Growth is best in the presence of oxygen. Toxin production is lower under anaerobic conditions. The spores are relatively heat resistant and germination of spores is rapid and can occur in some strains within 30 min. (Notermans and Batt, 1998; Kenneth Todar, 2005). There is wide variation in charactestics of B. cereus strains. Psychotropic strains that can grow at temperatures < 7ºC,wheares mesophilic strains grow at temperatures >10ºC. Previous studies indicated that psychotropic strains are physiologically similar to mesosphelic strain (Notermans and Batt, 1998).

2.4.5 Bacillus cereus toxins

2 .4.5.1 Diarrhoeal toxins B. cereus produces one or more toxins that induce diarrhoea. All of these toxins are unstable and are inactivated by low pH as well as digestive enzymes. The most widely used model to evaluate compounds for the diarhoeal response is the rabbit ileal loop assay. In this model, the lower part of the intestine of a rabbit is surgically exposed to allow legation of the ileal region. The sample is then injected into one legated section and the response, in terms of fluid accumulation is measured (Notermans and Batt, 1998.) Beecher and Wong (1994) continuing the work of Thompson, et, al., (1984) isolated a three component toxin (B, L1, L2) that together possess hemolytic, cytotoxic, dermonecrotic and vascular permeability activities. The combination of all three components is required for maximal activity against all of the cells and tissues so far tested. None of the individual components has exhibited any toxic activity. The components are physicochemically very similar, with respective molecular masses of 37.5, 38.2 and 43.5 KDa (Beecher et al., 1994). Previous studies reported that a three component non-hemolytic enterotoxin were isolated from a strain of B. cereus which was involved in a large food poisoning out break in Norway. The components were purified and were found to have molecular mass of 39, 45, and 105M. Optimal cytotoxic activity was observed when the three components were tested together (Notermans and Batt,. 1998).. Recently, the enterotoxin sequences was detected by using PCR-based assay (Agata et al., 1994; Beecher and Wong (1994))

4.2.5.2 Emetic toxins Studies on the emetic toxin have been complicated by the lack of a suitable model system to assay activity. Melling and Capel (1974) reported that a heat- stable, law molecular weight compound produced by B. cereus caused emesis in monkeys. Due to the lack of a suitable animal model, they did not purify the component. Hughes et al., (1988) and Shinagawa et al., (1992) reported a high vacuole response in Hep- 2 cells caused by culture supernates of B. cereus strain which were isolated from outbreaks of vomiting- type food poisoning. Shinagawa, et al., (1992) demonstrated that this activity, which appeared only after spore formation, was stable to heat, photolytic enzymes and exposure to pH 2.0. Agata et al, (1994) isolated and identified a putative emetic toxin that caused Hep-2 cell- vacuole formation. The purified compound was a cyclic dodecadepsiptide (d- O-leu-d-Ala-l-o-Val-l-Val) with a molecular mass of 1.2 Kda. The peptide is stable to photolytic enzymes, low pH and is not inactived by heat.

2.4.5.3 Other toxins A number of genes in B. cereus has been identified for enzymatic activities including haemolysis and sphingomyelinase, activities that would be consistent with virulence factors. One of these virulence factors in B. cereus is cereolysin which again is a multi component cytotoxic complex (Gilmore et al., 1989).

2.4.6 The illness B. cereus associated food borne illness occurs as two distinct syndromes: emetic and diarrhoeal. Emetic syndrome is characterized by rapid onset (1- 5 h) of nausea, vomiting and malaise, in some cases followed by diarrhea of 6- 24 h duration. It is similar to S. aureus food poisoning in its symptoms and incubation period. Diarrhoeal syndrome is characterized by an incubation period of 8- 16 h before the onset of abdominal pain, profuse watery diarrhea, rectal tenesmus, and occasionally nausea that seldom results in vomiting. Symptoms generally resolve within 12- 24h, and it clinically resemble food poisoning caused by Clostridium perfringens (Notermans and Batt, 1998). In small number of cases, patients have exhibited symptoms of both types of illness (Shinagawa et al., 1993). The emetic form of the disease is diagnostic by the isolation of B. cereus from incriminated food. The diarrheal form is diagnostic by isolation of the organism from stool and food. Isolation from stool alone is not sufficient because 14% of healthy adults have been reported to have transient gastrointestinal colonization with B. cereus (Kenneth Todar, 2005)

2.4.7 Rabbit ileal loop assays (RIL) The ligated loop of the rabbit small intestine has been used successfully in the study of various enteropathogenic bacteria. The injection of whole cultures, culture supernatant fluids and cell extracts of vibrio cholerae caused dilation of the loop due to fluid accumulation, this investigative technique has also been used to study the enteropathogenicity of Salmonella, Shigellae and Clostridium perfringens (Spira and Goepfert, 1972) and Escherichia coli (Moon et al., 1970). The usefulness of RIL assay as an experimental model of Bacillus cereus food poisoning was investigated by Beecher et al., (1995), the rabbits were sedated with ketamine and xylazine intramuscularly and the ileum was flushed with 5 ml of sterile saline prior to ligation. Ileal sections (loops) were approximately 2- 4 cm long, and were separated by 2- 4cm enter loops, then the loops were injected with 2 ml of sample. The number of loop tied off in a single animal was usually five and never more than six after preliminary test. Each rabbit received two negative controls, one positive control and three to five samples. Negative control consisted of either sodium phosphate buffer (PH 7.4) or brain heart infusion broth, positive control and samples consisted of culture supernatant. RIL activity is reported as V/L, the ratio of fluid volume (in milliliters) to loop length (in centimeters). Responses were considered positive when V/L was > 0.5. Results were considered usable when both negative controls were negative and positive control was positive. Spira and Goepfert, (1972); Beecher et al., (1995) observed that the loop response in older (larger) rabbits was sporadic and rarely reproducible. In contrast, the loop response in smaller rabbits was consistent. And growth in Brain heart infusion broth engendered the greatest, it was chosen as the standard growth medium for subsequent investigations.

2.4.8 Prevention and control of Bacillus cereus food's contamination

B. cereus spores are extremely heat resistant, so cooking at proper temperatures would destroy most food- borne pathogens including the vegetative cells of B. cereus, but not the spores. While heat resistance is increased by high salt concentrations and gradual heating, the spores loose their heat resistance in acidic environments. Spores can be activated by heat and or improper handling; therefore the 2001 Food Code recommends that hot foods should be maintained at a temperature of 140°F or above. According to the National Institutes of Health (NIH), the National Institute of Allergy and Infectious Diseases (NIAID), and the National Food Processors Association (NFPA), the suggestions below are good examples of how to destroy B. cereus (Schneider et al., 2004):

• Steaming under pressure, roasting, frying and grilling foods can destroy the vegetative cells and spores.

• Foods infested with the diarrheal toxin can be inactivated by heating for 5 minutes at 133°F.

• Foods infested with the emetic toxin need to be heated to 259°F for more than 90 minutes. Reheating foods until they're steaming is not enough to destroy the emetic toxin.

For meat processing facilities, in order to prevent contamination and toxin formation following steps had been recommended:-

• Assure current Good Manufacturing Practices (cGMP) (21CFR 110), are being used in the slaughter houses and processing units.

• Apply approved treatments of carcasses to remove fecal bacteria.

• Use proper cleaning and disinfection of food contact surfaces (FCS) with hypochloride or other approved sanitizers.

• Utilize a heat process to destroy the vegetative cells and a rapid cooling process to prevent the spores from germination.

• Keep hot foods above 60°C and cold foods below 4°C to prevent the formation of spores.

• Wash hands, utensils, (FCSs) with hot soapy water after they touch raw meat or poultry, or before food preparation, and after using the bathroom.

• Cook meat and meat products thoroughly.

• Properly refrigerate leftovers.

Chapter Three Material and Method

3.1 Study Area:

This study was conducted in Khartoum State and included butcher's shops, some healthy volunteers and chicken farms.

3.2 Sampling for isolation of Enterococci 3.2.1 Minced meat samples 180 minced meat samples were collected in clean and sterile bottles from ten different Butcher's shops in Omdurman area. Two grams of minced meat were taken in sterile screw- capped bottles and immediately send to the laboratory. 3.2.2 Chicken droppings specimens: 30 chicken droppings specimens were collected from five farms in Shambat area in Khartoum state. The samples were collected using sterile cotton swabs, moistened by sterile normal saline immediately before collection. Care was taken to avoid contaminating the specimen with organisms from environment. The swabs were labeled and sent immediately to the laboratory. 3.2.3 Stool specimens: Human stool specimens were collected from healthy volunteers in clean, dry, and wide necked containers, the specimen were labeled and transported to the laboratory. 3.3 Sterilization: 3-3-1 Hot air oven: Petri dishes, pipettes, tubes, flasks and glass rods were sterilized in hot air oven at 160°C for one hour.

3.3.2 Autoclaving:

Screw capped bottlers, rubber caps, tips of micropipette and culture media were sterilized in the autoclave at 121°C for 15 min. Sugar media were sterilized at 110°C for 10 min.

3.3.3 Disinfection of benches 70% alcohol was used for bench disinfection.

3.4 Culture media: 3.4.1 Blood Agar (Oxiod) Constituents Quantity Proteose 15g Liver digest 2.5g Yeast extract 5g Sodium chloride 5g

Agar No3 12g PH 6.8 -7.3

Twenty eight gram of blood agar base powder were suspended in one liter of distilled water, boiled to dissolve completely in a steamer, mixed and sterilized by autoclaving at 121°C for 15 min, then cooled to 45-50°C in a water bath before the addition of 10% defibrinated ovine blood. Mixed gently and dispensed into sterile petri dishes in 15 ml volume each.

3.4.2 Nutrient agar (Oxiod) Constituents Quantity Proteose peptone 15g Liver digest 2.5g Yeast extract 5g Sodium chloride 12g PH 7.3

Twenty eight grams of powder were added to one liter of distilled water, and dissolved by steaming, mixed and sterilized by autoclaving at 121°C for 15 minutes, then cooled to 45-50°C in a water bath. Then dispended into sterile petri dishes in 15 ml volume each.

3.4.3 MacConkey's agar (Oxiod) Constituents Quantity Peptone 20g Lactose 10g Bile salt 5g Neutral red 12g PH 7.4

The medium was prepared by dissolving 47.0 grams of dehydrated medium in one liter of distilled water. Boiled to dissolve and then autoclaved at 121°C for 15 minutes. After cooling, the mixture was poured aseptically into sterile petri dishes in 15 ml volume.

3.4.4 Peptone water sugar (Oxiod): Constituents Quantity Peptone water 900 ml Andrades indicator 0.10 ml Sugar 10 g

The pH of peptone water was adjusted to 7.1-7.3 before addition of Andrade's indicator. The specific sugar was then added to the mixture, mixed thoroughly, and then distributed into tubes in 5 ml volume each. Which were sterilized by autoclaving at 110°C for 10 minutes.

3.4.5 Bile Aesculin agar Constituents Quantity Peptone 5g Beef extract 3g Ox gut bile 40g Aesculin 1g Ferric citrate 0.5g Agar 15g Distilled water 1 liter PH 7.4

All ingredients except Aesculin were dissolved by boiling. The solution allowed to cool, aesculin added. The medium was then dispensed in screw-capped bottles and sterilized at 115°C for 20 min, and allowed to set as slopes.

3.4.6 Diagnostic Sensitivity Test Agar (D.S.T Agar) (Oxid): Constituents Quantity Proteose Peptone 10g Veal infusion soiled 10g Glucose 2g Sodium chloride 3g Di- sodium phosphate 2g Sodium acetate 1g Adenine sulphate 0.01g Guania hydrochloride 0.01g Uracil 0.01g Xanthine 0.01g Aneurine 0.00002g Agar 12g

Forty grams of D.S.T. agar were suspended in 1 liter of distilled water. The mixture was brought to boiling to dissolve completely and then sterilized by autoclaving at 121°C for 15 minutes.

3.4.7 Craigie tube medium (Barrow and Felthman, 1993)

Constituents Quantity

Dehydrated nutrient broth powder 13g

Agar(Oxoid No. 1) 5g

An amount of 13g nutrient broth were added to 5g agar and dissolved in one liter of distilled water by boiling, the pH was adjusted to 7.2. The medium was distributed in 5ml volumes in the test tubes containing the craigie tubes, and sterilized by autoclaving at 115°C for 15 minutes.

3.4.8 Peptone water medium (CM9)

Constituents Quantity

Peptone 10g

Nacl 5g

Fifteen grams of the medium were dissolved in one liter of distilled water by boiling, mixed well and the pH was adjusted to 7.2, the medium was distributed into 5 ml amounts into miniature bottles, and finally sterilized at 121°C for 15 minutes.

3.4.9 Blood–Tellurite Agar Constituents Quantity

K2 TeO3, 2% aq. sol 16 ml Sterile blood 50 ml Infusion Agar 100 ml

Infusion agar was melted, cooled to 50°C and was aseptically added to the blood and sterile tellurite solution (sterilized by filtration).It was then mixed and distributed. The final concentration of tellurite in this medium is approximately 0.03%. 3.4.10 Ammonium Salt Sugar (ASS) Constituents Quantity

(NH4)2 HPO4 1.0g KCL 0.2g

MgSO4. 7 H2O 0.2g Yeast extract 0.2 g Agar 20 g D.W 1000 g Bromocresol purple, 0.2% aq. sol 4 ml Solids were dissolved in DW by steaming. Then the indicator was added and solution sterilized at 115°C for 20 min. The basal medium was allowed to cool to 60°C. Then appropriate sterile carbohydrate solution added as to give a final concentration of 0.5- 1%. The medium was mixed and aseptically distributed into sterile tubes that were allowed in

3.4.11 Citrate media Constituents Quantity Sodium citrate 3 g Glucose 0.2 g Yeast extract 0.5 g L.cystein hydrochloride 0.1 g Ferric ammonium citrate 0.4 g

KH2PO4 1 g NaCl 5 g

Na2S2O3 0.08 g Agar 20 g Phenol red, 0,2% aq.soln 6 ml

D.W 1000 ml The solids were dissolved in the water by steaming, then the solution was filtered, the indicter was added after PH adjusted to 6.8-6.9, and finally sterilized at 115°C for 20 min.

3.4.12 Starch Agar Constituents Quantity Potato Starch 10 ml D.W 50 ml Nutrient agar 1000 ml

The starch was saturated with the water to smooth cream, and was added to the molten nutrient agar, mixed, and sterilized at 115 for 10min. And then distributed into Petri dishes.

3.5 Cultivation of samples to isolate enterococci 3.5.1 Minced meat sample: Two grams of minced meat were added to five ml nutrient broth and incubated at 37°C for 24 hours, and then culture was used to streak blood agar. Inoculated plates were incubated over night at 37°C aerobically.

3.5.2 Chicken droppings specimens: Fecal chicken swabs were directly streaked on MacConkey’s agar and blood agar and incubated aerobically at 37°C for 24 hours.

3.5.3 Stool specimens: Loop full of each stool specimen was inoculated on MacConkey’s agar blood agar, in the same manner as for the fecal chicken specimens.

3.6 Sub_ culturing and purification: It depended on the characteristics of colonial morphology and gram's reaction. Suspected colonies were picked and sub- cultured on nutrient agar.

3.7 Preservation of isolates: Preservation of the pure culture was done by the inoculation into cooked meat media, incubated at 37°C for 24hrs and then stored at 4°C.

3.8 Biochemical methods: All the biochemical tests were performed according to Barraw and Feltham, (1993).

3.9 Identification of Enterococci 3.9.1 Cultured characteristic Cultures on solid media were examined for growth, colonial morphology and haemolysis on blood agar. Whereas liquid media were examined for turbidity, colour change and/or formation of sediment and pellicle.

3.9.2 Primary tests 3.9.2.1 Gram stain's Smears were prepared from singly isolated colonies, allowed to dry, fixed with heat and stained with Gram’s Method (Barrow and Feltham, 1993). Stained smears were examined under oil immersion lens.

3.9.2.2 Catalase test: A drop of 3% aqueous solution of hydrogen peroxide was placed on a clean microscopic glass slide, a small amount of growth the bacteria under test was placed in the drop using a glass rod. Production of gas bubbles indicated a positive result.

3.9.2.3 Motility test Motility was tested by stabbing the isolated bacteria using straight wire loop into a semi solid medium (Craigie tube method). The medium was then incubated for up to 3 days at 37°C together with uninoculated medium as control. The growth of non motile organism was confined to the stab line, while the motile one radiated away from the stab line.

3.9.3 Secondary tests The suspected colonies for enterococci were inoculated onto MacConkey’s agar plates and examined for small lactose fermenting colonies. Isolates that were lactose fermenting and Gram positive cocci were tested for the production of catalase enzyme. The isolates that were catalase positive were excluded. Lactose fermenting, gram positive cocci and catalase negative isolates were further identified.

3.9.3.1 Bile Aesculin hydrolysis test Bile aesculin agar plates were inoculated with the suspected isolates of enterococci spp and incubated at 37°C for 48 hrs aerobically. The enterococci colonies produced blackening of the medium.

3.9.3.2 Salt tolerance test Two or Three colonies of enterococci were inoculated into nutrient broth containing 6.5% Nacl. The inoculated broth was incubated at 37°C for 3 days aerobically. Growth was indicated by development of turbidity.

3.9.3.3 Tellurite tolerance:

The suspected colonies for enterococci were inoculated in blood tellurite agar plates and incubated at 37°C for 24 hrs. Enterococci are tellurite resistant organisms, and usually show a heavy growth of jet black colonies; tellurite- sensitive organisms fail to grow or show a very light growth.

3.9.3.4 Growth at 45oC:

The suspected colonies for enterococci were inoculated in blood agar plates and incubated at 45oC for 24 hrs Enterococci are heat tolerance organism.

3.9.3.5 Sugar tests Some sugars were used for the biochemical identification of Enterococcus species, the sugars included; Mannitol, Arabinose, Sorbitol, Inulin, Adonitol and Raffinose. Sugar media were inoculated by adding 2-3 drops of test culture grown in peptone water. Inoculated media were incubated at 37°C and examined daily for up to seven days. Acid production was indicated by the development of pinkish colour in the media.

3.9.4 Antibiotic Sensitivity of enterococci Sensitivity of isolated enterococci to a number of antibiotics was demonstrated by modified Kirby Bauer disc diffusion technique (Kirby et al., 1966). Four to five well-isolated colonies were suspended in 4 ml peptone water, incubated over night, then diluted with sterile normal saline until the density of suspension to be inoculated was equal to Mc Farlend 0.5 STD tube. 0.5 ml of diluted culture was poured and spreaded evenly on the surface of D.S.T agar. Excess fluid was aspirated using Pasteur pipette and then the plates were allowed to dry. Using a forceps, antibiotic disc were placed on the agar surface, and pressed gently to ensure full contact with the surface of the culture medium. Plates were left on the bench for 15 minutes, and then incubated overnight at 37°C.

The antibiotic discs used were: (i) Glycopeptide (vancomycin). (ii) Aminoglycoside (Gentamycin, Streptomycin and Erythromycin). (iii) B. lactams (Pencillin and Ampecillin). (iv) Macrolides (Erythromycin) (v) Tetracycline (vi) Amphenicols (choloramphenicol). Inhibition zone diameter was measured after the incubation period and the sensitivity results were interpretated according to the National Committee for Clinical Laboratory Standards (NCCLS) published guidelines (1999)

3.10 Sampling for isolation of B. cereus 30 minced meat samples were collected in clean and sterile bottles from ten Butcher shops. Two grams of minced meat were put in sterile container and promptly send to the laboratory.

3.11 Cultivation of samples to isolate B. cereus Two grams of minced meat were dissolved in five ml normal saline, and then used to streak blood agar. Inoculated plates were incubated for over night at 37°C aerobically

3.12 Identification of B. cereus 3.12.1 Primary tests Tests were performed as described in 3. 9. 2 for identification of enterococci.

3.12.2 Secondary tests: The suspected colonies for B. cereus in inoculated blood agar were examined for large and heamolytic ones, from those colonies, smears were prepared, stained by gram's stain methods and examined microscopically. Isolates that were heamolytic Gram-positive, and spore forming rods were tested for production of catalase enzyme. Gram- positive, spore forming rods and catalase positive isolates, were further identified:

3.12.2.1 Sugars tests: Some sugars were used for the biochemical identification of B. cereus, the sugars included; Mannitol, Arabinose, mannose and salicin. B. cereus cultures were inoculated in ammonium salt sugar (ASS). Inoculated sugars media were incubated at 37°C and examined daily for up to seven days. Acid production was indicated by the development of pinkish color in the media.

3.12.2.2 Starch hydrolysis: The tested organisms were inoculated onto starch agar or nutrient agar containing 0.2% soluble starch and incubated at 30°C for 5 days. The cultured plates were flooded with lugol's iodine solution; if the medium turn blue where starch had not been hydrolysed, clear colorless zones indicated hydrolysis.

3.13 Enterotoxigenicity of B. cereus The tests were performed as described by Beecher et al. (1995): Two rabbits (1 Kg each) were used in this test, they were acclimatized to the experimental house for one week before testing, then the rabbits were fasted for 24 hrs prior to externalizing the ileum. Each of the tow rabbits was sedated with ketamin (30mg/kg) and xylazine (5mg/kg) intramuscularly. After that the rabbit was secured in dorsal recumbancy, and by using scalpel and forceps the middle of abdominal cavity was opened, the small intestines were brought out through the midline incision and the intestinal lumen was flushed with warm saline, then the ileum was segmented and tieded off to form six loops each 5 cm long, separated from one another by 2cm blank loops. Ten strains of B. cereus were inoculated in brain heart infusion broth and after over night incubation at 37°C aerobically, supernatants were taken by sterile syringe, and filtrated through Millipore filter (0.22µm). In each rabbit one loop was injection with 1ml sterile brain heart infusion broth as a negative control, and 1 ml of filtrates were injected intraluminaly in each five test loops. The ileum was returned to the abdominal cavity, the incision was closed with sutures. And after a holding period (7 hours) the rabbits were sedated, and abdominal cavities were opened for examination. The gross appearance of the loops was noted, and fluid inside the loops was measured, RIL activity was reported as the ratio of fluid volume (in milliliters) to loop length (in centimeters) (V/L). Responses were considered positive when V/L was ≥ 0.5 Results were considered usable when negative controls were negative.

Chapter Four RESULT

4.1 Samples investigated for enterococci Out of the 180 raw minced beef samples, 30 human stool specimens and 30 chicken droppings swabs, which were collected and bacteriologically investigated in this study, the bacterial growth was found in all meat samples, 148 (61.6%) Enterococcus species were isolated; 110 isolates were obtained from raw minced beef sample, 22 isolates from stool of healthy volunteers and 16 from the Chicken fecal swabs. The over all isolation rate of Enterococcus species from raw beef samples was 61%, stool Enterococcal carriers was 73% and chicken droppings swabs 53 %.

4.2 Samples investigated for B. cereus Ten B. cereus isolates (20%) were recovered from 50 minced beef meat samples (Fig. 8).

4.3 Cultural characteristics: Enterococcus isolates formed magenta color on the MacConkey's agar. On blood agar medium they formed grey, small, haemolytic or non hemolytic colonies (Fig. 4), these colonies showed a heavy growth of jet, black colonies on blood tellurite agar, growth on 6.5% NaCl nutrient broth and bile was aesculin positive. B. cereus isolates formed large (4-5 mm) and heamolytic colonies on blood agar (Fig. 7), formation pellicle shape in liquid media and hydrolysis starch.

4.5 Identification 4.5.1 Identification of enterococci species

Identification of the genus Enterocccus was achieved according to microscopic examination of stained smear (gram +ve cocci), catalase production, Cultural characteristics and sugars fermentation (Table. 1). The Enterococci isolated from raw minced beef were identified to seven species those are; Enterococus faecalis which was the predominant species recovered (68.2%) followed by E. Faecium (22.8%), E. durans (3.6%), E casseliflavus (1.8%), E. malodoratus (1.8%), E. hirae (0.9%) and E. avium (0.9%), (Fig. 1). Enterococci isolated from stool samples included E. faecium which was the predominant species recovered (63.6%) followed by E. faecalis (27.3%) and E. durans (9.1%) (Fig. 2). The Enterococcus species recovered from chicken droppings samples, were E. faecium (56.25%), E. faecalis (25%), E. durans (6.25%), E. hire (6.25%) and E. gallinarum (6.25%) (Fig. 3).

4.5.2 Identification of B. cereus B. cereus isolates appeared as gram-positive rods that produced oval, sub terminal bulging spores, catalase positive, motile and did not ferment mannitol, mannose and raffinose (Table. 2).

Table. 1: Biochemical reaction for Enterococcus species isolates Species of E. E. E. E. E. E. E. enterococci faecalis faecium durans gallinarum cassaliflavus hirae avium Character Shape Cocci Cocci Cocci Cocci Cocci Cocci Cocci Gram reaction +ve +ve +ve +ve +ve +ve +ve Catalase - + - - - - - Motility - + - + + - -

Haemolycin -/ β - - β - α α Yellow pigment - - - - + - - Growth at 45oC + + + + + + + Bile aesculin + + + + + + + Growth at 6.5 + + + + + + + NaCl Fermentation of sugars: Arabinose - + - + + - + Mannitol + + + + + - + Sorbitol + + - - - - + Raffinose - - - + - - - Inulin ------Adonitol ------+ +ve = positive -ve = negative

Table. 2: Biochemical reaction for B. cereus isolates

Character Result Shape Rodes Chains of cells + Gram reaction + Spore position C/ST Motility + Catalase + Growth in 10% NaCl +/- Galactose - Mannose - Raffinose - Salicin + Xylose - Starch hydrolysis + Utilization of citrate +/- Urease +/-

C = central S/C = sub terminal

68.2 70

Percentage 30 22.8

10 3.6 1.8 1.8 0.9 0.9 0

s s e u m av viu l . hira aecium durans lif E . faecali f e E. a E E. E. . cass E. malodoratus E ٍ:Figure.Species 2

Figure.1:Relative distribution of 16 isolates of enterococcus species from stool specimens .

70 63.6 60

Percentage 30 27.3

9.1 10

0 E. faecium E. faecalis E. durans Species

Figure. 2: Relative distribution of 16 isolates of enterococcus species from stools pecimens.

60 56.25

30 25 Percentage

10 6.25 6.25 6.25

0 E. faecalis E. faecium E. durans E. hirae E. gallinarum Species

Figure. 3: Relative distribution of 22 isolates of enterococcus species from chicken droppings specimens.

Result of this study showed that isolated enterococci were resistant to many antibiotics (Fig. 5). All of the isolates except two were resistance to cloxacillin and penicillin. Resistance to gentamycin was observed among all populations isolated from chicken droppings and most isolates from stool, in addition to that all of the 16 isolates from chicken droppings were found resistance to Erythromycin. All of the 22 isolates from stool except four, exhibited intermediate resistance or resistance to tetracycline and streptomycin (Table 3, 4 and 5). Above all, 89.6% of isolates from meat showed good sensitivity to Ampicilin and 82% of them were sensitive to tetracycline. While most isolates from stool showed good sensitivity to chloramphenicol and Ampicilin (Table 3 and4)

4.7 Sensitivity of enterococci isolated from different specimens to Vancomycin: None of the enterococci isolated in this study exhibited complete resistance to the glycopeptide vancomycin, but some of them showed intermediate resistance to vancomycin. Isolates of intermediate resistance represented 62.5% of chicken dropping, 18.1% of stool isolates and 12.7% of enterococci isolated from minced meat samples (Fig. 6).

4.8 Sensitivity of different species of enterococci to Vancomycin: A total of 14 meat isolates with intermediate resistance to vancomycin were identified up to the species level. 8 of these 14 isolates were identified as E. faecalis, 4 were E. faecium, one was identified as E. hirae and one was identified as E. durans. In contrast, all isolates from chicken droppings and stool samples exhibited intermediate resistance to vancomycin were identified as E. faecium.

Figure. 4: Enterococci on blood agar

4.6 Antibiotic Sensitivity tests for enterococcal species

Table.3: Result of antibiotic sensitivity test to enterococci Isolated from meat samples Antibiotic disc Amp Cxc Chl Ery Gen Pen Str Tet Van No of isolates Susceptible 99 0 54 36 36 0 16 90 96 Intermediate 1 0 26 24 10 0 3 10 14 Resistance 10 110 30 50 64 110 91 10 0 Total 110

Table.4: result of antibiotic sensitivity test to enterococci isolated from stool specimens. Antibiotic disc Amp Cxc Chl Ery Gen Pen Str Tet Van No of isolates Susceptible 9 2 14 2 0 0 2 2 18 Intermediate 5 0 2 2 4 0 2 2 4 Resistance 8 20 6 18 18 22 18 18 0 Total 22

Table. 5: Result of antibiotic sensitivity test to enterococci isolated from chicken droppings specimens. Antibiotic disc Van No of isolates Amp Cxc Chl Ery Gen Pen Str Tet

Susceptible 4 0 3 0 0 0 0 2 6 Intermediate 3 0 0 0 0 0 2 0 10 Resistance 9 16 13 16 16 16 14 14 0

Total 16

Key: Amp: Ampicillin Cxc: Cloxacillin Chl: Chloramphinicol

Ery: Erythromycin Gen: Gentamycin Pen: Penicillin

Str: Streptomycin Tet: Tetracycline Van: Vancomycin

4.9 Entrotoxigenicity of B. cereus Out of fifty minced meat which samples were collected, ten strain of Bacillus cereus were isolated, and they were tested for enterotoxigenicity by fluid accumulation in ligated ileal loops in rabbits (Fig. 9). Eight out of ten (80%) strains exhibited positive response and gave V/L ratios more than 0.5 (>0.5). The greatest response seen in this study was 0.8. The tow negative controls gave V/L ratios <0.5. (Table. 6) The fluid in positive loops ranged from straw colored to red because of the presence of variable amounts of blood.

100 87.3

90 81.8

70 62.5

Percentage 50 37.5

30 18.1 20 12.7

10 000

Intermediate Resistant Susceptible

Figure. 6 Sensitivity of enterococci isolated from different specimens to vancomycin.

Negative Negative Control r e s p o n c e Positive

Figure.9: Rabbit ileal loops test of bacillus cereus isolates

Figure. 7: Haemolytic activity of Bacillus cereus

20% positive meat sample to B- cereus

Negative meat samples to B- 80% cereus

Figure. 8: Frequency of B. cereus in raw minced beef samples

Tabsle. 6: RIL activity of various B. cereus strains

Fluid volume to length ratio 1 0.8 2 0.75 3 0.65 4 0.65 5 0.6 6 0.6 7 0.55 8 0.55 9 0.3 10 0.25 Negative control (1) 0.2 Negative control (2) 0.2

Chapter five Discussion In the last decade, enterococci have been recognized as the leading cause of nosocomial bacteremia, surgical wound infections and urinary tract infections. The increasing importance of enterococci is largely due to their resistance to many antimicrobial agents mainly vancomycin. This study documents the distribution of enterococci among raw minced beef, stool from healthy volunteers and chicken droppings, and establishes a base line for antimicrobial resistance among isolated enterococci of human and veterinary importance. Isolation and identification of enterococci to the genus level was readily achieved by using a set of biochemical methods. In this study the frequency of enterococci isolates from minced meat samples was 61%, comparable results obtained by Klein et al (1998), who reported isolation rates of 75% from minced meat samples. Enterococci were recovered from 73% of the stool samples, and this agrees with the fact that enterococci are part of the flora in the gastrointestinal tract of healthy individuals (Silverman et al., 1998). Enterococci were recovered from 53% from chicken’s droppings samples, and this is inconsistence with the previous studies which reported that, enterococci are common components of the micro floral community of animals (Hayes et al., 2003). In the present study the majority of isolates from beef samples were identified as E. faecalis (68.2%), followed by E. faecium (22.8%), and the percentages of other species represented (9.0%). A Similar result was reported in Germany, where E. faecalis isolated from raw beef accounted (87%) and E. faecium (4%) (Klein et al., 1998). On the contrary, in the United States, E. faecium was more frequently isolated from beef (67%), followed by E. faecalis (29%), and other species of enterococci accounted (10%) of the isolates. This agrees with our result (9.0%). In this study, E. faecium was the predominant isolates from stool (63.7%) and chicken droppings (56.3%). Similar results were reported by Cetinkaya,. et al (2000) who found that E. faecium was identified in 24 out of the 36 (66.6%) stool samples. Studies from Japan also concluded that E. faecium was the dominant enterococcal species of poultry fecal flora (Yoshimuro et al., 2000). While in other part of the United States (Welton, et al., 1998), as well as in United Kingdom (Kaukas and linter, 1986) and Denimark (Aarestrup, et al., 2000) E. faecalis was the predominant enterococci in poultry production environments. On the other hand, Silverman et al., (1998) observed that, E. faecalis represented (68%) and E. faecium (24%) of isolates from stool. This study documented the presence of multiple-drug resistant enterococci in isolates from minced beef, human stool samples and chickens droppings. (87%) of enterococci isolates from chickens droppings were resistant to six antibiotics (penicillin, cloxacillin, erythromycin, streptomycin, gentamycin and tetracycline, while (91%) of isolates from stool were resistant to three antibiotics, namely; penicillin, gentemycin and streptomycin. In contrast, previous studies showed that, of the 73 E. faecalis isolated from stool, only 8(11%) demonstrated high-level gentamicin plus high-level streptomycin resistance (Silverman et al., 1998). The observation that all enterococci isolates in this study were resistant to penicillin is consistent with and that, enterococci are typically tolerant to B-lactams. The major mechanism underlying this resistance is the production of low-affinity PBP5 (Cetinkaya et al., 2000) 58.2%, 82.8% and 45.5% of enterococci isolates from beef sample exhibited highly resistance to gentamycin, streptomycin and erythromycin, respectively. Previous studies reported that, none of enterococcal isolates from retail meat exhibited resistance to the gentamycin, and all isolates except two enterococci (0.9%) were susceptible to streptomycin, and 69% of isolates exhibited intermediate resistance or were resistant to erythromycin (Klein et al., 1998). (89.6%) and (82%) of enterococci isolated from meat were susceptible to ampicillin and tetracycline respectively. This finding is in agreement with the results of Klein et al., (1998) studies, who found that, all enterococci isolated, including VRE, from raw minced meat were susceptible to ampicillin and (80%) of them to tetracycline, and they suggested that, in case of infections caused by enterococci from

meat, patients can be treated with ampicillin as the antibiotic of choice and usage of glycopeptides is not necessary. Resistance to tetracycline among Enterococcus spp is very common, especially among those of poultry origin. Tetracycline resistance has also been previously demonstrated to be linked closely to poultry production environments (Molitoris et al., 1986). While these observations are similar to the results presented here, which reported 87% of enterococcus spp from chicken droppings exhibited resistance to tetracycline, this percentage is high compared to the values reported from United States (68%) (Hayes et al., 2004), this high result may be due to misusage of this antibiotic in poultry environments in Sudan. Abdalgalil, (2004) reported that the enterococci isolated from stool showed high sensitivity to chloramphenicol, this agrees with the (72%) of the isolates from stool which were susceptible to chloraphenicol in this study. There were no vancomycin-resistant enterococci (VRE) among all isolates in this study. But 12.7%, 18.2% and 62.5% of the isolates from beef, stool and chicken droppings respectively exhibited intermediate resistance to vancomycin. The absence of VRE from domestic retail meat in this study is consistent with previous observations (Hayes et al., 2003; Coque et al., 1996; Knudtson and Hartman., 1993., and Thal et al., 1995). Similar studies reported the absence of VRE from poultry production environment (Hayes et al., 2004) and from stool sample of healthy volunteers (Murray, 1997). In the United State VRE appear to have spread in hospitals, but community acquisition of strains has not been reported (coque et al., 1996). In Europe, however, vancomycin resistant strains have been isolated from a symptomatic outpatients and from animals, and sewage, showing various community reservoir (Aarestrup et al., 1995; Bates et al., 1995; Klare, et al., 1993; Thal, et al., 1995), these findings indicated important differences in the epidemiology of VRE in Europe and the United States. These differences may be related to the use of avoparcin in animal feed in

Europe and the over use of vancomycin in hospitalized patients in the United States (Silverman et al., 1998). Abdalgalil, (2004) reported that, 20% of the isolates from faeces of hospitalized patients were VRE. Detection of VRE from hospitalized patients strongly suggested that VRE isolated were acquired from the hospital environment, this hypothesis was supported by other studies which showed that environmental surfaces and medical equipment items in the patient's room frequently become contaminated with VRE and may also serve as reservoir for the organism in the hospital (Boyce et al., 1994). Other studies showed that VRE colonization rates among the community vary greatly from one study to another; low rates were found in the Netherlands (2%) (Endtz et al., 1997), the United Kindom (2%) (Jordens and Griffiths, 1994). On the other hand, other studies had reported high prevalence rates; 12% in Germany (Klare et al., 1995). In contrast, Silverman et al, (1998), had not reported VRE in 200 fecal samples from patients admitted to a community hospital in the United States. Our study demonstrated that all vancomycin intermediate resistance entercocci from stool and feces of chicken were identified as E. faecium, but the intermediate resistance to vancomycin from beef was higher among E. faecalis (58.5%) than E. faecium (25.5%). Low susceptibility to vancomycin among E. faecium is in agreement with the previous studies from human, animal and environmental sources (Gambarotto, et al., 2000; Schouten, et al., 2000). These findings were suggesting that, food contaminated with E. faecium plays a major role in the epidemiology of VRE. Other studies on poultry showed that, among 82 VRE isolates E. faecalis was 89% and E. faecium was 11% of the isolates (Manson et al., 2000), VRE was not reported in this study and this might be attributed to many causes; vancomycin is not commonly used as a drug of choice in clinical fields, and it is not included as a feed additive to animals in Sudan. Although studies in Germany detected VRE from meat after banning of avoparcin and this suggested that the use of avoparcin may not have been responsible for the development of vancomycin resistance in animals (Klein et al., 1998).

Highly multi resistance to penicillin, streptomycin, gentamycin and tetracycline among enterococcus spp isolated from human led necessitate the evaluation of usage of antimicrobial for treatment of human infection. An analysis of reported food borne disease reveals that B. cereus is frequently diagnosed as the cause of gastrointestinal disorders. . in USA, from 1988 to 1992, 2423 food borne disease out breaks affecting 77375 persons were reported, twenty-one outbreaks were caused by B. cereus and 433 persons were affected (Notermans and Batt, 1998 ). Result of this study showed that, the percentage of B. cereus in raw beef samples was 20%

In this study, 8 of 10 isolates of B. cereus elicited fluid accumulation in rabbit ileal loops, these high positive responses are in agreement with Spira Goepefert, (1972) who reported that 19 of 22 strains of B. cereus produced positive response in rabbit ileal loop test. On the contrary, Turn bull, (1976) reported that, only 2 of 11 isolates of B. cereus from food poisoning investigations, exhibited positive response.

The observation of vascular permeability and hemolytic activities of B. cereus was seen in his study, and was supported by Beecher et al., (1995) results which showed that, the fluid positive loops had variable amounts of blood and exhibited necrosis and sub mucosal oedema. The growth medium and rabbit’s weight in which the test is performed markedly affected the ability of a given strain of B. cereus to provoke a response. In the RIL test, we used Brain Heart Infusion (BHI) medium to grow B. cereus, also the rabbits used were weighting 1000g each. (Turnbull, 1976; Beecher et al., 1995) observed that, BHI medium and animals weighting less than 1200g proved to give the best response. It appears that these experimental animals are able to develop resistance, as they grow older. In a number of studies, it was concluded that the hemolytic activity of B. cereus is separable from enterotoxic activity (Glatz et al., 1974). Spira and Goepfert, (1972) results offer presumptive evidence that such illness in humans is the result of an enterotoxication rather than infection.

The presence of some strains which of B. cereus which didn’t exhibit positive response in RIL tests might explain that not all B. cereus strains cause enterotoxaemia. Psychrotrophic strains grow very poorly at 37Co suggesting that they would be unable to grow in the ileum and probably not cause diarrhea, these observations need, however, to be confirmed, in addition to that frequent exposure to B. cereus in food may build up a resistance in human, B. cereus may need specific food components to produce the enterotoxin (Notermans and Batt, 1998). Over all, it’s still not known if diarrhea is caused by ingestion of performed enterotoxins or if enterotoxins are produced in the gut. Clarification of the role of potential virulence factors and their interaction will allow further exploration of the nature of the public health risk posed by this organism (Beecher et al,. 1995). In conclusion, our study documented that Enterococcus spp commonly contaminate meat, and the consumption of such meat which might carry antibiotic resistant enterococci could be a possible route of transfer of resistance determinant to bacteria adapted in human. VRE was not reported and this is might be attributed to the fact that, vancomycin is not commonly used as a drug of choice and it is not included as a feed additive in Sudan. The presence of intermediate resistance Enterococci to vancomycin in this study represents of development of VRE. This work also reported high contamination rate of meat with B. cereus, which was proved to be enterotoxigenic by the RIl assay.

Recommendation: Based on this study, we recommend the following: 1/ Implementation of effective control strategies aiming at reducing enterococcal contamination of minced meat may become more significant in the future. 2/ More research should be carried out to examine genotypic relationships between human and animals enterococcal isolates. 3/ Further studies, to illustrate if antibiotic resistant enterococci isolates from meat pose a risk to consumers and the efficiency of therapeutic antimicrobials to treat disease. 4/ Drugs of choice should be given in full therapeutic doses for adequate period. 5/ Application of proper hygienic methods during food processing by the use cleaning and disinfection methods for surfaces, equipments, tools….etc with to prevent contamination and toxin formation by B. cereus. 6/ further studies, to illustrate mode of action of the enterotoxin of B. cereus to induce diarrheal response. 7/ further studies should be carried out to illustrate whether diarrhea caused by B. cereus induced is by ingestion of preformed enterotoxin or if enterotoxin is produced in the gut.

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Appendix

NCCL Zone Diameter Interpretive Standards And Equivalent Minimum Inhibitory Concentration (MIC) for enterococci Antimicrobial Disk Resistant Intermediate Susceptible Agent content Amp 10 mcg 22 or less 23 – 30 31 or more Clx 30 mcg 12 or less 13 – 17 18 or more Tet 5 mcg 9 or less 10 – 13 14 or more Str 15 mcg 13 or less 14 – 22 23 more Gen 10 mcg 12 or less 13 – 14 15 or more Ery 10 mcg 11 or less 12 – 14 15 or more Chl 30 mcg 14 or less 15 – 18 19 or more Pen 1 IU 20 or less 21 – 28 29 or more Van 30 mcg 9 or less 10 – 11 12 or more

Amp: Ampicillin Cxc : Cloxacillin Tet: Tetracyclin Str : Sterptomycine Gen : Gentamycin Ery: Erythromycin Chl : Chlormphenicol Pen : Penicillin Van: Vancomycin