| As a major industry in public health, food industry is very important and is a symbol of the development level of a country, as well as the quality of the living level of human being, of which, food safety has been drawing more and more attraction globally. Of many issues associated with food safety, food-borne diseases have a high rate of incidence in all diseases and become the leading problem in public health. In food-borne diseases, diseases mediated by microbes, have become the cause of many food-borne diseases and considered to be an expanding issue for public health globally. Conventionally, food pathogen safety had been limited to food poisoning cases caused by pathogens. However, as investigation on food safety develops, issues including indiscriminate use of antibiotics in veterinary industry which leads to antibiotic resistance in microbes, as well as the formation of biofilm which leads to survival of microbes under regular anti-bacterial process and causes food contamination, have now been regarded as significant problems in food pathogen safety.This study focused on one of the most important food-borne pathogen- Staphylococcus, and aimed to investigate the occurrence of toxins and antibiotic resistant genes, develop and apply rapid diagnosis and detection methods and demonstrate the genetic modulation of biofilm attachment and detachment, in order to pre-propose and eliminate various potential food-borne safety issues during food production and sugar production. The main content of this study were as follows:(1) The prevalence of staphylococci was investigated and analyzed systematically via detection and identification of bacteria and the toxins. This study established a multiplex-PCR assay which was able to detect staphylococci, Staphylococcus aureus and mecA resistance, and this assay had been further applied to detect 262 staphylococci strains. According to the results, 209 and 53 strains were found to be S. aureus and coagulase-negative staphylococci, and all of them carried mecA gene, which showed resistance to methicillin and the detection rate reached 100%. Prevalence of 4 common toxins had also been identified, and none of the tested strains was positive for each individual toxin. In conclusion, for the prevalent staphylococci isolates, the most obvious characteristics was antibiotic resistance rather than toxicity.(2) For systematic surveillance of food-borne staphylococci, this study established rapid assays for detection of staphylococci, S. aureus and mecA, based on loop-mediated isothermal amplification (LAMP). Targeted at staphylococci 16S rRNA, femA and mecA and design specific primers, this study developed and evaluated rapid LAMP detection methods, for differentiation of MRSA, MSSA, MRCNS, MSCNS and non-staphylococci strains. It was showed thatthe LAMP assays had high specificity with confirmation of 41 reference strains, as well as high sensitivity with the detection limit of 10 CFU/reaction for bacteria and 100 fg for DNA template, which was 10-1000 times higher than PCR. Lamp assays were rapid and reproducible, even with rapid and rough DNA template process, the whole procedure could be done within 60-80 min, and only simple equipments as water baths and heat block were involved. For the application of LAMP assays, detection sensitivities for 16S rRNA?femA and mecA reached 100%, 98.5% and 92.3%, respectively.(3) To track the potential“Super Bugs”- Staphylococcus, this study investigated the antibiotic resistance phenotypes and molecular mechanisms, including conventional genomic island SCCmec and novel integron system. 262 staphylococci strains were performed to test their antibiotic resistance phenotype and molecular mechanisms. 82.1% of 262 staphylococci strains (215 strains) exhibited the multiple antibiotic resistance with 9, 27 and 211 carried type I, II and III SCCmec, 15 strains remained untypable. 122 strains were positive for class 1 integron, with 4 different types of gene cassettes. With double antibiotic resistance mechanisms, resistance had become the most obvious characteristics of staphylococci strains. As the last solution for therapy of MRSA and MRCNS, the ineffectiveness of vancomycin may make multiple resistant staphylococci develop as“Super Bugs”, which will definitely be a great threaten to human being in the future.(4) To understand the contamination and infection mediation, genetic and clonal origin, as well as the evolution of genomes and epidemiologic change, this study analyzed and implemented staphylococci strains by DNA fingerprinting and genome analysis techniques. According to the results of DNA fingerprinting and genetic relatedness analysis, there existed possible staphylococci-mediated infection between air or direct contact through people and people, or between environment and people via clones on surfaces. Investigation on DNA fingerprinting analysis of 209 MRSA and 23 MRCNS showed that, the origin of integrons in MRCNS was found to be multi-clones, with horizontal transfer of antibiotic resistant genes. While in MRSA, the origin of a large part of integrons was oligo-clones, indicating the existence of both horizontal and vertical transfer of antibiotic resistant genes. At last, according to investigation of MLST on 46 MRSA and spa and coa typing on 22 MRSA, the prevalent strains in this study were genetically identical to those reported in Taiwan, Hong Kong and most Asian countries except Japan and Korea, such as Singapore, Indonesia and Thailand, as well as in Brazil, Vienna and Portugal. However, the specific evolution and migration requires further investigation. (5) This study investigated the molecular modulation mechanism of staphylococci biofilm attachment to detachment, and according to the results, 2 genes in respect with the formation and structure of biofilm had been significantly down-regulated in detachment, which might probably be the main cause for the detachment. In the meanwhile, during detachment, many of genes related with bacteria growth had been significantly up-regulated, and the expression level of leukocidin, enterotoxin and exotoxin-9 had been increased more than 1000 times, and strong antigens protein A had also been dramatically up-regulated. |
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