MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH HANOI MEDICAL UNIVERSITY LE KHANH TRAM DETERMINATION OF CARRIER STATUS AND TOXIN GENES OF Staphylococcus aureus IN FOOD HANDLERS Specialization: MEDICAL MICROBIOLOGY Code: 62720115 PHD MEDICAL THESIS SUMMARY HANOI-2013 THE WORKS WERE COMPLETED AT THE HANOI MEDICAL UNIVERSITY Supervisors: 1. Assoc. Professor Dinh Huu Dung, MD, PhD 2. Assoc. Professor Nguyen Vu Trung, MD, PhD Referee 1: Assoc. Professor, PhD. Le Van Phung Referee 2: Assoc. Professor, PhD. Nguyen Thi Khanh Tram Referee 3: PhD. Tran Huy Thinh Examination committee: Institutional Dissertation Council, Hanoi Medical University Examination date: The thesis can be found at: 1. National library. 2. Central library of medicine information 3. Library of Hanoi Medical University, SCIENTIFIC PUBLICATIONS RELEVANT TO THE THESIS 1. Le Khanh Tram, Dinh Huu Dung, Nguyen Vu Trung (2011), Determination of carriage prevalence and carrier status of Staphylococcus aureus, Journal of medical research, 77 (6), pp. 29-33. 2. Le Khanh Tram, Dinh Huu Dung, Pham Thi Thuy Duong, Nguyen Vu Trung (2012), Distribution of coagulase gene in genotypes of Staphylococcus aureus, Journal of medical research, 78 (1), pp. 1-5. 3. Le Khanh Tram, Dinh Huu Dung, Nguyen Vu Trung (2012), Detection of enterotoxin genes in Staphylococcus aureus strains, Journal of medical research, 80 (3), pp. 11-16. 4. Le Khanh Tram, Pham Thi Thuy Duong, Dinh Huu Dung, Nguyen Vu Trung (2010), Determination of carriage prevalence and classification of Staphylococcus aureus based in coagulase encoding gene of staffs working in restaurants in Hanoi, at 16 th Scientific Conference for PhDs, Hanoi Medical University, Won the third prize. 5. Le Khanh Tram, Nguyen Vu Trung, Dinh Huu Dung (2011), Detection of enterotoxin genes in Staphylococcus aureus strains, at 17 th Scientific Conference for PhDs, Hanoi Medical University, Won the third prize. 1 INTRODUCTION Staphylococcus aureus is a member of the bacterial flora in humans. About 30% of healthy people carry S. aureus. These bacteria can be found in many areas of human body such as on skin of the necks, chest, abdominal, hands…and many natural cavities of the body, especially the anterior nares. S. aureus can be persistent, intermittent or non- carry on the human body. When there were skin lesions, destroyed mucous membranes, the immunity defence of body would decrease, giving the opportunities for the infection by S. aureus. The presence and number of S. aureus in the body depends on the immune status, sanitation, and many other factors of the hosts as well as the bacteria. S. aureus is one of the leading causes of food poisoning throughout the world and it is still an unresolved problem. Food poisoning caused by S. aureus accounted for about 25% of cases and 10% of patients hospitalized for food poisoning treatment. There have been severe cases of danger to life in Vietnam. S. aureus is the main cause of contamination in food. Due to the hot and humid climate, the lack of appropriate storage conditions, the presence of S. aureus with high numbers in foods would potentially lead to the break out of food poisoning, and consequently affect the economy. The individuals working in the close contacts with food such as food processing, food saling would be highly potential sources of contaminating S. aureus for processing, manufacturing, packaging, transportation and storage of food. In Vietnam there have been many studies on the pathogenicity and antibiotic resistance of S. aureus but, no research on carrier status as well as very low number of studies on toxin genes carried of these pathogen. Thus, our study with the title: “Determination of carrier status and toxin genes of Staphylococcus aureus in food handlers” with the following two main purposes: 1. To determine the carriage prevalence and the carrier status of Staphylococcus aureus in the nose and hands of staffs working in restaurants in Hanoi. 2. To determine the distribution of coagulase gene polymorphism 2 and enterotoxins genes of the isolated S. aureus strains. NEW CONTRIBUTIONS OF THE THESIS 1. This is the first study on the carrier status of S. aureus in healthy people in Viet Nam. 2. Providing scientific information contributing to the carriage prevalence of Staphylococcus aureus of food handlers working in restaurants in Hanoi. 3. This is the first study in Viet Nam on coagulase and enterotoxin coding genes of S. aureus isolated from noses and hands of food handlers working in restaurants in Hanoi. 4. Providing a better understanding in molecular epidemiology of coagulase and enterotoxin coding genes of S. aureus. THE LAYOUT OF THE DISSERTATION THESIS The thesis comprises of 117 pages. Introduction 2 page; the conclusions 1 page; recommendations 1 page. The main content is divided into four chapters: Chapter 1: overview (35 pages); Chapter 2: the object and methodology (20 pages); Chapter 3: study results (27 pages); Chapter 4: discussion (30 pages); There are 40 tables, 13 figures and 7 charts, appendices and 131 references of those 28 were in Vietnamese and 103 were in English. CHAPTER 1 OVERVIEW 1.1. MICROFLORA IN HUMAN Normal microflora in human (normal microbial flora) is the complex of microorganisms that live on the healthy human body. Microflora on skin contains mainly Gram-positive bacteria, which are usually Staphylococci (Staphylococcus epidermidis), anaerobic bacteria (Propionibacterium acnes which cause acne). There is also 3 the Staphylococcus aureus, Streptococcus viridians, Bacillus like diphtheria and spore bacteria in the air. It is accounted for about 10 10 bacteria on the skin. Normal microflora in narses are staphylococci and streptococci, Corynebacterium, especially the S. aureus that have the ability to infect and spread. 1.2. CHARACTERISTICS OF S. aureus S. aureus is a Gram-positive cocci, with a diameter of 0.8-1 μm, grouping into the form of grapes, no pilli, no spore and usually no capsula. S. aureus can be easily cultivated, they grow at a temperature of 10 0 C-45 0 C and salt concentrations as high as 10%, appropriate in both anaerobic and aerobic conditions. The genome of S. aureus is completely sequenced in size from 2820 to 2903 bp. G, C contain is around 33% and from 2592 to 2748 bp sequence encodes for proteins. In the genome, 75% of the sequences is highly conserved between the strains of S. aureus. The set of all the sequences of genomic region is called core genome. 1.3. CARIAGE PREVALENCE AND CARRIER STATUS OF S. aureus S. aureus is a member of the bacterial flora in humans. About 20-30% of healthy people carry S. aureus. Long time ago, the relationship between carriers of bacteria and food poisoning caused by S. aureus was mentioned and studied, particularly in those who having direct contact with food during the processes of production, processing, transportation, distribution and storage of food. These bacteria can be persistent, intermittent on the host body. Also, in some individuals the existence of these bacteria has not been found. Carriage prevalence of S. aureus among food handlers is about 20- 40% according to several different studies. The strains of S. aureus 4 isolated from food handlers also possessed a high rate of antibiotic resistance and contained the toxin genes such as the enterotoxin genes se: sea, seb, sec, sed, see (80-90%). 1.4. THE PATHOGENIC FACTORS OF S. aureus There are many factors affecting to the establishment of pathogenicity in S. aureus. These can be divided into three groups: cell surface-associated, extra cellular proteins and others. 1.4.1. Cell surface-associated protein: peptidoglycan, teichoic acid, protein A, capsula, adhesins 1.4.2. Extra cellular proteins: enterotoxins, toxic shock syndrome toxin, hemolyasin, exfoliatin 1.4.3. Other pathogenic factors: coagulase, alpha toxin, leukocidin, staphylokinase enzymes, Panton-Valentine leukocidin, staphyloxanthin, some of others extracellular enzymes 1.5. TOXIN GENES OF S. aureus Pathogenic factors of S. aureus are controlled by a gene or some genes directly or indirectly through enzymes. In this study we just focused on the toxin genes of importance in these bacteria. 1.5.1. Coagulase encoding gene of S. aureus The coagulase gene of S. aureus has 6 basic segments. The signal chain of the sequence starting at the N-end to the D1, D2 regions, central region, the repeated region (segments with 81 bp nucleotid), and the C-end. The classification of the genotype of coagulase gene in S. aureus based on the polymorphism of the DNA fragments treated by the restriction enzyme AluI has a number of advantages in the S. aureus strain identification such as simplicity, 5 fast, and accuracy. 1.5.2. Enterotoxin genes of S. aureus Enterotoxin genes of S. aureus such as sea, seb, sec… encode for more than 20 different types of enterotoxins. Most of the genes coding for enterotoxins have been identified in the mobile genetic factors such as plasmids, bacteriophages. Moreover, the pathogenic genes can be scattered or concentrated into a group called enterotoxin gene cluster. Thus, horizontal transmission of these genes between S. aureus strains is common. 1.6. S. aureus INFECTIOUS DISEASES Accoding to the statistic data of the 16 common disease-causing bacteria in Vietnam, the rate of S. aureus constitutes from 18.5- 21.7%, ranking second only to E. coli. S. aureus can cause several clinical diseases such as infections affecting skin and soft tissues, scalded skin syndrome, infection of bones and joints, endocarditis, bacteraemia, pneumonia, food poisoning and intestinal infections, toxic shock syndrome and nosocomial infections. 1.7. LABORATORY ISOLATION AND IDENTIFICATION OF S. aureus 1.7.1 Common techniques to identify S. aureus: direct microscopic examination; isolation, cultivation, and identification slide; aglutination, ELISA, identification by phages. 1.7.2. Molecular biology techniques: molecular biology techniques have been applied more and more in the diagnosis and study of S. aureus. These methods are faster, less costly in some cases. In particular, these techniques give results with high sensitivity, specificity such as PCR, realtime PCR, nested PCR, RFLP, PFGE, 6 MLST, gene sequencing. CHAPTER 2 OBJECTS, MATERIALS AND METHODS 2.1. OBJECTS 2.1.1. Research object of carriage prevalence and carrier status of S. aureus - Selection criteria: employees working at 14 restaurants in Hanoi have direct contact with food. - Exclusion criteria: employees who do not voluntarily participate in all 4 sampling times, who have acute or chronic or diseases of nose, throat, skin inflammation, infections of the skin and hands, nails, who is taking antibiotics or immune suppressing drugs. 2.1.2. Study objects for the distribution of toxin genes of S. aureus All S. aureus strains isolated from employees. 2.2. RESEARCH METHODS 2.2.1. Study design: This is a cross-sectional study which has been described previously. 2.2.2. Sample size and sample selection - Sample size: n = Z 2 1-α/2 p (1-p)/(εp) 2 n: sample size for the research; Z: coefficient of reliability, with α = 0.05 then Z 1-α/2 = 1.96 p: carriage prevalence of S. aureus in food handlers according to previous studies (33%); ε: accuracy relative to p, ε = 0.2, n = 195. Number of eligible objects are studied was 212. [...]... 1 genotype of the gene encoding coagulase was the most common, 73.58% The prevalence with 2 genotypes constituted 24.53% Only one S aureus- harboring staff showed 3 different genotypes, constituted 1.89% 3.2.2 Study on enterotoxin genes of S aureus 3.2.2.1 Overall carriage prevalence of enterotoxin genes of S aureus Table 3.26: Overall carriage prevalence of enterotoxin genes of S aureus Carrying gene... of 11 800 bp Genotype 14 3 2.97 + 1 0.99 1 0.99 + + + + Total 101 100,00 3.2.1.3 Distribution S aureus- harboring staffs according to the number of the genotype of coagulase gene Table 3.25: Distribution S aureus- harboring staffs by the number of the genotype of coagulase gene Number of genotypes S aureus- harboring staffs (%) 1 39 73.58 2 13 24.53 3 1 1.89 4 0 0.00 15 Total 53 100.00 S aureus carriage... on 13 toxin genes 3.2.1 Coagulase gene polymorphism of S aureus strains 3.2.1.1 Distribution of genotype of coagulase gene of S aureus 20 genotypes were identified based on the length of DNA fragments obtained after digestion of the gene sequence by restriction enzyme AluI The most frequent was genotype 6 (53.47%) which included 3 DNA fragments 465 bp, 250 bp and 165 bp The second was genotype 12 (6.93%)... carriage was statistically significant, p . of coagulase gene in genotypes of Staphylococcus aureus, Journal of medical research, 78 (1), pp. 1-5. 3. Le Khanh Tram, Dinh Huu Dung, Nguyen Vu Trung (2012), Detection of enterotoxin genes in Staphylococcus aureus. the study on p<0,05 13 toxin genes. 3.2.1. Coagulase gene polymorphism of S. aureus strains 3.2.1.1. Distribution of genotype of coagulase gene of S. aureus 20 genotypes were identified based. Distribution S. aureus- harboring staffs according to the number of the genotype of coagulase gene Table 3.25: Distribution S. aureus- harboring staffs by the number of the genotype of coagulase gene Number