| Staphylococcus aureus (S.aureus) is an important pathogenic bacteria which can cause clinical infections and food poisoning. S.aureus enterotoxin is the main reason to cause bacterial food poisoning in S.aureus. The infection of S.aureus in livestock animals can cause animal diseases, when antibiotics used to prevent and treat the animal diseases, it would make S.aureus produce drug-resistance or increase drug-resistance, thus it lead to the result that the research about drug-resistant S.aureus in food contamination has become a hot research of the world. With the widely use of antibiotics in livestock breeding in China, enterotoxigenic and drug-resistant S.aureus were widely present in foods, especially in milk and its products, and it can cause harm to human health through direct effects or indirect effects of the environment and the food chain. To Isolation and identification of S.aureus strains, analyse the enterotoxin distribution (sea, seb, sec, sed), detect the drug resistance and resistance genes(mecA, blaZ) of S.aureus in the dairy production chain(raw milk-semi-finished milk-milk products), samples of dairy production chain were collected and tested in Sichuan Province. The followings are the results.1. The analysis of contamination of S.aureus in the dairy production chainA total of893samples in the dairy production chain(raw milk-semi-finished milk-milk products) were collected from2010to2011in Sichuan Province. According to the standard of GB4789.10-2010and using chromogenic medium CHROMagar,196S.aureus were isolated and identified,the total detectable rate of S.aureus was21.9%(196/893), in which, the positive rate in raw milk, semi-finished milk and milk products were37.5%,7.1%and5.7%respectively.2. The analysis of enterotoxin of S.aureus in the dairy production chainTo analysis the presence of staphylococcal enterotoxins(SEs) genes in126S. aureus, SEs genes sea to see was studied by Multiplex Polymerase Chain Reaction(M-PCR) method. The results indicated that23.0%(29) strains produced enterotoxin genes(sea-sed), in which, the positive rate in raw milk, semi-finished milk and milk products were23.5%(27),0.0%(0) and40.0%(2), respectively.From126isolates, the positive rate of sea,seb,sec and sed were14.3%(18),10.3%(13),5.6%(7),3.2%(4), respectively. From milk products S.aureus,40%(2) isolates were found positive for sea gene.There were difference between Enzyme-Linked Immunosorbent Assay (ELISA) method and M-PCR method when SEs genes were detected.3isolates produced SE gene by M-PCR method, while those strains were negative to determine the SE production by ELISA method.3. The analysis of drug resistance of S.aureus in the dairy production chain126S.aureus were tested against12antibiotics (combination) using broth micro-dilution method recommended by Clinical and Laboratory Standards Institute (CLSI). The susceptibility results were determinated by CLSI standards (2010). The results show that all the isolates were most resistance to ampicillin (97.6%), followed by resistance to penicillin (93.7%), amoxicillin/clavulanic acid (56.3%), trimethoprim/sulfamethoxazole (50.0%), tetracycline (37.3%), erythromycin (29.4%) and clindamycin (29.4%). All strains were susceptible to oxacillin, ceftiofur, florfenicol. The drug resistance of S.aureus from3production processes had some difference.57.9%of the isolates were multi-drug resistants and a total of26kinds of drug resistance patterns were presented. The whole strains demonstrated were multi-drug resistance mainly to penicillins, sulfonamides, erythromycin, tetracycline and lincomycin. M-PCR method was used to analyze the heat nuclease gene (nuc) and the beta-within lactams resistance gene (mecA, blaZ) of the126isolates. The results demonstrated that the positive rates for detection of nuc, blaZ and mecA genes were100.0%,60.5%and0.0%respectively. The highest detected rate of blaZ was100.0%(5/5) from raw milk, followed by70.8%(80/113) from semi-finished milk and50%(4/8) from fresh milk. There were a lot of correlations between the drug restence to beta-lactams and the detected rate of the resistance gene (blaZ) of S.aureus.This study analysed the contamination of S.aureus in the dairy production chain, and conducted a survey about its enterotoxigenic and drug resistance, it provided the basic data for risk assessment of food-borne S.aureus,at the same time,it provided the theoretical basis for the establishment of HACCP in dairy production and the future analysisis of the change of drug resistance of S.aureus. The thesis results have certain theoretical significance and practical value. |
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