【作者】 马立才；

【导师】 吴聪明； 汪洋；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2014， 博士

【摘要】 弯曲菌是一类重要的食源性肠道病原菌,世界范围内每年弯曲菌感染病例数约4-5亿。随着抗菌药物在食品动物养殖中的大量使用,弯曲菌的耐药现状越来越严重。调查研究表明,加工和消费受到弯曲菌污染的鸡肉是人类弯曲菌感染的主要途径之一。在我国,对于弯曲菌流行和耐药性的监测还不完善,肉鸡中弯曲菌的流行和耐药性的报道也相对较少。本研究旨在了解弯曲菌在我国肉鸡生产链中的污染、传播及其抗菌药物耐药性情况,进而在已有数据的基础之上开展肉鸡源弯曲菌耐药性风险评估研究。在2012至2013年,本研究从上海和青岛的两个典型肉鸡生产链中分别采集了肉鸡盲肠样本142和118份,屠宰胴体样本135和110份,以及市售鸡肉样本128和128份,在青岛肉鸡生产链中还采集了分割鸡肉样品104份,各分离得到弯曲菌259(空肠弯曲菌：106；结肠弯曲菌：153)株和282(空肠弯曲菌：142；结肠弯曲菌：140)株。值得注意的是,肉鸡中结肠弯曲菌的分离率已经达到甚至超过了空肠弯曲菌。药敏结果显示,分离自上海和青岛肉鸡生产链的541株弯曲菌均至少耐1种抗菌药物。两地区所分离的弯曲菌对环丙沙星耐药率最高(>99.0%)；其次是对四环素的耐药率(86.0%)。另外,克林霉素、庆大霉素和红霉素的耐药率也比较高,均在62%以上；上海和青岛弯曲菌的氟苯尼考耐药率分别为20.1%和4.6%。结肠弯曲菌对四环素、克林霉素、庆大霉素和红霉素的耐药率明显高于空肠弯曲菌,氟苯尼考耐药率则相反。多重耐药情况非常严重,两地弯曲菌的多重耐药率分别达到了87.9%和73.4%,远高于其它国家等。分子分型结果显示,不同来源的弯曲菌可能具有相同PFGE谱型,这说明弯曲菌可以随着肉鸡生产链传播。基于OIE风险分析框架和FDA152号抗菌药物耐药性风险评估指南,本研究结合我国细菌耐药风险评估的现状建立了肉鸡源细菌耐药性定性风险评估模型。该模型包括：危害特征描述、释放评估、暴露评估、后果评估和风险估计五个部分,在评估过程中使用“矩阵法”和“非矩阵法”两种方法对风险参数进行整合。之后,运用所建立的评估模型对我国肉鸡源耐氟喹诺酮类药物(FQs)弯曲菌进行了评估。结果表明,我国肉鸡源耐FQs弯曲菌的释放风险“极高”；消费者因消费鸡肉而暴露于耐FQs弯曲菌的风险“较高”；弯曲菌的FQs耐药性对弯曲菌病治疗造成的影响“非常严重”。利用“矩阵法”综合上述结果得到如下结论：在我国肉鸡养殖中使用FQs所导致的耐FQs弯曲菌对我国消费者的健康构成了“高水平”的风险。本研究基于OIE风险分析框架和FDA风险评估指南,并结合我国目前开展动物源细菌耐药性风险评估所具备的基本条件建立了一个“农场-餐桌”的肉鸡源细菌耐药性随机定量风险评估模型。该模型利用统计分析软件(如@riskTM等)进行蒙特卡罗模拟获得一个包含95%置信区间的风险评估结果平均值,然后通过敏感性分析确定各模型参数对风险评估结果不确定度的影响大小。最后,运用所建立的模型评估了我国肉鸡源耐FQs弯曲菌对我国消费者健康的影响。评估结果表明,我国2010年和2011年受到肉鸡源耐FQs弯曲菌感染影响的病例数分别为：N2010=0.2630×106(95%CI:0.038-0.728)和N2011=0.2637×106(95%CI:0.039-0.728)敏感性分析结果表明,服用FQs治疗耐FQs弯曲菌感染的概率(PFQ)对模型结果Nm的不确定度的影响最大,其次为PF和PA,而参数PT的影响最小。综上所述,我国肉鸡源弯曲菌的耐药情况非常严重,多重耐药的比例远高于其它国家。结肠弯曲菌正逐渐替代空肠弯曲菌成为肉鸡中的优势弯曲菌群,且其对四环素、克林霉素、庆大霉素和红霉素的耐药率显著地高于空肠弯曲菌。PFGE分型结果表明,肉鸡源弯曲菌可以随着肉鸡的生产链传播。风险评估结果显示,我国消费者受到肉鸡源耐FQs弯曲菌影响的风险较高。总之,我国肉鸡源弯曲菌耐药性的严峻形势,以及耐药弯曲菌随着肉鸡生产链传播给消费者并对其健康造成影响的事实理应受到更多的关注。更多还原

【Abstract】 Campylobacter spp. are the most frequently identified foodborne bacteria causing gastroenteritis throughout the world. Campylobacter cause approximately400to500million human infection cases worldwide each year. Along with the antimicrobial use in food-producing animal, Campylobacter are increasingly resistant to some important antibiotics, which compromises clinical therapy and presents a major threat to public health. Epidemiological studies have demonstrated that handling and consumption of contaminated broiler meat, is a major source of human Campylobacter infections. In China, an active surveillance system to monitor the prevalence and antimicrobial resistance of Campylobacter from farm to retail is not yet available, and little information has been reported on Campylobacter in broiler. The prevalence and distribution of Campylobacter along the broiler production chain is unknown in China, which hampers the implementation of interventions. This study was to investigate the prevalence and antimicrobial resistance of Campylobacter in broiler production chain, under which two antimicrobial resistance risk assessment models on Campylobacter in broiler were developed.During2012-2013,142&118broiler ceca,135&110broiler carcasses, and128&128broiler meat samples were randomly collected from broiler farm, slaughterhouse and supermarket of the boiler production chains in Shanghai&Qingdao, respectively. Additional104boiler meat product samples were collected from slaughterhouse in Qingdao. A total of259(C.jejuni:106; C. coli:153) and282(C. jejuni:142; C. coli:140) Campylobacter were isolated from Shanghai and Qingdao, respectively. It worth noting that C. coli is becoming the predominant species in broiler in China. All541Campylobacter isolates were resistant to at least one of the tested antimicrobial agents. Notably, nearly100%of the isolates were resistant to ciprofloxacin. Tetracycline resistance of Campylobacter was also very severe. Besides, the resistance rates of these isolates to clindamycin, gentamicin, and erythromycin all exceeded62%. The lowest resistance observed in these Campylobacter isolates was to florfenicol, and florfenicol resistance rate of Campylobacter isolated from Shanghai and Qingdao were20.1%and4.6%, respectively. Contrary to florfenicol resistance, the resistance rates of C.coli to tetracycline, clindamycin, gentamicin, and erythromycin were higher than that of C. jejuni. The rate of MDR for Campylobacter from Shanghai and Qingdao were respectively87.9%and73.4%, which were much higher than that reported in other countries. Furthermore, isolates from different production stages might have the same PFGE patterns, indicating their transmission along the broiler production chain.Referring to risk analysis framework recommended by OIE and FDA Guidance Document152, we developed a qualitative risk assessment model on antimicrobial resistance from food-producing animals. The model contains five parts:hazard characterization, release assessment, exposure assessment, consequence assessment and risk eatimate. Throughout the model, both "metrix method" and "non-metrix method" were used to combined the qualitative probabilities/parameters. Then, the qualitative model was used to evaluate the risk from FQs-resistant Campylobacter attributed to the use of FQs in the broiler rearing. The results revealed that the risk of developing FQs resistance, contributed to the use of FQs in broiler production, for Campylobacter was extremely high; the probability of consumer exposure to FQs-resistant Campylobacter in broiler meat was "high", and the influence of FQs-resistant Campylobacter cases attributable to chicken and affected by the FQs resistance was "highly mmportant" in China.Based on risk analysis framework recommended by OIE and the anmicrobial resistance risk assessmenr theory of FDA, we established a "farm-consumption"quantitative risk assessment model on antimicrobial resistance in food-producing animals. The software program@riskTM was used to simulate the risk assessment model. A total of10,000simulations were run and medians and the output distributions (95%CI) were reported. By sensitivity analysis, we can lern about the uncertainty in the value foroutput contributed to each input of the assessment model. The quantitative model was used to evaluate the risk from FQs-resistant Campylobacter attributed to the use of FQs in the broiler production. The results of the risk assessment revealed that the theoretical numbers of FQs-resistant Campylobacter cases attributable to chicken and affected by the FQs resistance in2010and2011were0.2630×106and0.2637×106, respectively, and the probability of taking FQs for Campylobacter-associated diarrhea (PFQ) contributed the most toward uncertainty of quantitative risk assessment model.In summary, the antimicrobial resistance level of Campylobacter in China was very high, and higher than that in other countries. The resistance of C.coli to tetracycline, clindamycin, gentamicin, and erythromycin were severer than that of C. jejuni, and C. coli is becoming the predominant Campylobacter species in chickens in China. The PFGE results indicated the transmission of Campylobacter along the broiler production chain. Antimicrobial resistance risk assessment showed that human health risks due to FQs-selected resistance of Campylobacter in broiler were high. Taken together, the hazard for Campylobacter resistance in broiler deserves further investigation.更多还原