【作者】 徐振波；

【导师】 李琳； 石磊；

【作者基本信息】 华南理工大学 ， 制糖工程， 2011， 博士

【摘要】 食品工业是关系国计民生的生命产业,也是一个国家经济发展水平和人民生活质量的重要标志;而其中,食品安全问题越来越引起国家和社会的关注。在食品安全领域的众多问题中,食源性疾病的发病率居各类疾病总发病率的前列,成为最突出的卫生问题。由微生物引起的安全问题,成为多种食源性疾病的主因,其范畴和影响都是一个不断扩大的全球性公共卫生问题。传统的食品微生物安全问题集中于由致病微生物引起的细菌性食物中毒研究,但随着对食品安全认识的加深和范畴的扩展,由于抗生素在动物食品中的滥用而引起食源性微生物的耐药性,以及微生物通过改变生长方式形成生物被膜从而逃逸常规灭菌消毒处理造成食品污染等,均属于重要的食品微生物安全问题。本文基于食品工业领域中的各种微生物安全问题,以最重要的食源致病性微生物葡萄球菌为对象,研究其流行状况包括毒素及耐药基因分布,对菌种进行检测和鉴定并建立开发新型快速鉴定方法,通过研究葡萄球菌的基因组岛和整合子系统分析其耐药分子机制及其与表型的相关性,考察葡萄球菌生物被膜粘附与脱落的基因调控行为,从而前瞻性地提出及科学性地消除各种由葡萄球菌在食品加工及糖加工领域中引起的安全隐患。主要研究内容和结果如下:(1)建立一种能同时对葡萄球菌、金葡菌及甲氧西林耐药因子mecA进行检测与鉴定的多重PCR体系;同时应用该方法于262株葡萄球菌,对其进行金葡菌鉴定及甲氧西林耐药因子的检测。结果表明,262株葡萄球菌包括209株金葡菌和53株凝固酶阴性葡萄球菌,均为甲氧西林耐药,检出率达100%。而对262株葡萄球菌进行常见4种毒素基因的检测结果显示,262株葡萄球菌均不携带4种毒素基因。可见,目前流行的葡萄球菌中以耐药性为最显著的特点,而致毒性则不常见。(2)基于LAMP快速检测技术,建立针对葡萄球菌及其关键耐药因子的快速检测体系,为进一步对葡萄球菌进行全面性监控提供技术方法的支持。针对葡萄球菌属16S rRNA、femA和mecA基因分别设计LAMP引物,建立和优化了环介导恒温核酸扩增体系,成功实现对MRSA、MSSA、MRCNS和MSCNS的快速检测。通过检测41株对照菌株验证LAMP反应的高特异性,同时反应最低检出限能达10 CFU/reaction细菌量和100 fg模板DNA,比PCR反应灵敏度高10-1000倍;LAMP反应快速,耗时少,从模板DNA提取至结果判断,仅需60-80 min;反应过程简便稳定,对模板DNA的要求较低,采用粗提方法即可;不需要精密的温度循环装置,普通水浴锅或其他有稳定热源的装置即可实现,使反应更简便;反应实用性强,应用于118株葡萄球菌的检测,16S rRNA、femA和mecA基因灵敏度分别达100%、98.5%和92.3%。(3)通过对葡萄球菌耐药表型和分子机理的研究,包括传统基因组岛SCCmec和新型整合子系统,为了解及追踪潜在“超级细菌”之一的葡萄球菌耐药性的发展和进化提供科学依据。对262株葡萄球菌的耐药表型以及基因组岛和整合子系统两种耐药分子机制进行研究与分析。262株葡萄球菌中多重耐药性占82.1%(215/262),其中9、27和211株分别携带I、II和III型基因组岛SCCmec,另15株无法分型;262株葡萄球菌中122株携带第一类整合子,包括4种不同的耐药基因盒。在传统基因组岛SCCmec和新型整合子系统等耐药机制作用下,耐药性成为葡萄球菌最显著的特征,多重耐药性进一步增强,严重威胁将来人类的生存。(4)为了解葡萄球菌的传播渠道与侵袭途径、基因来源与克隆起源,以及基因组进化与流行变迁,对葡萄球菌进行指纹图谱分析并且深入地解析其基因组背景。通过对29株MRSA进行指纹图谱及遗传相似性分析,结果显示葡萄球菌可能的侵袭途径包括通过空气和接触进行人与人之间的传播或通过繁殖于各种表面进行人与环境之间的传播;通过对209株MRSA和23株MRCNS进行指纹图谱分析,结果显示整合子在MRCNS中为多克隆来源,以耐药基因的横向水平转移为主,在MRSA中则多为寡克隆来源,但同时存在耐药基因的横向水平转移和纵向垂直传播;对46株MRSA进行多位点序列分型,对22株MRSA进行表面蛋白A和凝固酶基因分型,结果显示目前流行的金葡菌与台湾、香港和除日本和韩国外的大多数亚洲国家,包括新加坡,印尼,泰国等多个地区流行的菌株基因组背景相同,该流行菌株在巴西、葡萄牙和维也纳等地区也是主要的流行类型,其进化与迁移途径有待进一步研究。(5)研究与分析了金葡菌生物被膜中菌体从粘附于生物被膜到脱落成浮游状态过程的分子调控模式。结果显示,2个与生物被膜形成关键的骨架蛋白基因在脱落菌体中转录量显著下降,可能是其从粘附状态脱落成游离菌体的主要原因;在脱落成游离菌体后,多种与生命活动相关的基因显著上调;同时,金葡菌在脱落后,杀白细胞素、肠毒素和外毒素9转录量上升超过1000倍,而具有强免疫原性和过敏原性的蛋白A,转录量也显著上升。更多还原

【Abstract】 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.更多还原