弯曲菌快速检测技术研究及应用进展

弯曲菌是引起人类食源性腹泻的主要病原菌之一,快速及特异地检测该致病菌对控制弯曲菌病传播至关重要。本文综述了近年来基于蛋白、核酸和其他水平的技术在弯曲菌快速检测中的应用研究,并对未来弯曲菌检测技术的发展趋势和研究方向进行展望,以期为快速、准确、高效、低成本、高通量的弯曲菌检测方法的建立提供参考。     

动物性食品源空肠弯曲杆菌二重PCR检测方法的建立及应用

根据GenBank空肠弯曲杆菌(Campylobacter jejuni,Cj)的16S rDNA及hipO(编码马尿酸酶基因)序列设计两对特异引物,建立检测动物性食品源Cj的二重PCR方法,并应用于样品检测。结果显示只对Cj能特异的扩增出699bp和366bp两个基因片段,而大肠杆菌、沙门氏菌等其他11种细菌均未扩增出条带;Cj标准株ATCC33560的16S rDNA及hipO序列与GenBank其他Cj的相应序列具高度相似性(分别为99.7%~99.9%,98.1%~99.7%);该方法可在27h内完成,其灵敏度为2.4~16 CFU/mL;四川省雅安市鸡肉、猪肉、牛肉和牛奶样品中的Cj阳性率分别为38.0%(19/50)、28.3%(15/53)、17.1%(6/35)和8.6%(4/46)。     

基质辅助激光解析电离-飞行时间质谱法快速鉴定餐饮食品中的空肠弯曲菌

目的建立基质辅助激光解析电离-飞行质谱法(matrix assisted laser desorption ionization-time of flight mass spectrometry,MALDI-TOF-MS)快速鉴定餐饮食品中空肠弯曲菌的方法。方法从餐饮样品中分离的疑似空肠弯曲菌经不同配比的基质液处理后进行质谱鉴定,通过分析鉴定概率值及其特征峰的信号强度来评价不同配比基质液对空肠弯曲菌鉴定的影响,并与16S rRNA鉴定结果进行比较。结果通过16S rRNA与MALDI-TOF-MS 2种鉴定结果一致,获得MALDI-TOF-MS鉴定空肠弯曲菌的最佳质谱基质液配比V_(乙醇):V_(乙腈):V_水=1:1.5:1,2.5%α-氰-4-羟基肉桂酸,5.0%三氟乙酸,鉴定概率值达99.9%。结论致病菌的前处理与优化基质液配比可以提高基质辅助激光解析电离-飞行时间质谱鉴定的概率值。     

Addition of Rifampicin to Bolton Broth to Inhibit Extended‐Spectrum β‐Lactamase‐Producing Escherichia coli for the Detection of Campylobacter

Exponential growth of extended‐spectrum β‐lactamase (ESBL)‐producing Escherichia coli in Campylobacter media has become a common problem for the detection of Campylobacter in chicken meats. We investigated the minimum inhibitory concentration of 40 ESBL‐producing E. coli isolates from meats obtained from various countries against antibacterial agents in Bolton broth (cefoperazone, vancomycin, and trimethoprim). All ESBL‐producing E. coli strains were resistant to cefoperazone and vancomycin, whereas 50% of them were resistant to trimethoprim and grew in Bolton broth. We found that 20 μg/mL of rifampicin inhibited the growth of trimethoprim‐resistant E. coli strains. Hence, we added 20 μg/mL of rifampicin to Bolton broth to improve the isolation of Campylobacter from chicken carcass rinses. The isolation rate of Campylobacter was significantly higher in the modified broth (44 out of 58, 75.9%, P < 0.05) than in the normal broth (0 out of 58, 0%). Furthermore, the number of agar plates with non‐Campylobacter spp. was much lower after enrichment in the modified broth (4 out of 58, 6.9%, P < 0.05) than in the normal broth (58 out of 58, 100%).     

扬州市市售鸡肉中弯曲菌定性定量流行病学分析

目的调查分析超市和农贸市场零售鸡肉中弯曲菌定量污染水平,为弯曲菌污染的定量风险评估、危害分析和防控提供数据。方法选择扬州市内各大型超市和农贸市场作为采样点,采取未分割的鸡胴体,在3 h内送到实验室开展检测。运用选择性CCDA平板直接计数法对弯曲菌进行定量检测,涂布平板法计数,用PCR进行鉴定。结果采集的40只整鸡,检测出阳性样品19份,平均阳性率为47.5%,平均带菌量为229.2 CFU/g。其中超市的8份阳性整鸡中,平均带菌量为28.3 CFU/g;农贸市场的11份阳性整鸡中,平均带菌量为313.6CFU/g。共分离出56株弯曲菌,其中空肠弯曲菌29株,结肠弯曲菌23株,其他弯曲菌4株。结论定性及定量结果显示,零售鸡中存在弯曲菌污染,农贸市场的零售鸡肉比超市的零售鸡肉污染严重,需要加强防控措施。     

Role of rodents in transmission of Salmonella and Campylobacter

Salmonella and Campylobacter are generally regarded as the most important food‐borne pathogens in the world. Reduction or elimination of these pathogens in the first part of the food chain (on the farm) is important to prevent disease among consumers of animal products. In organic farming, elimination becomes more difficult, as food animals are allowed outdoors and have easy access to potential sources of hazardous pathogens. Whilst rodents are often associated by organic farmers with infrastructural damage and eating or spoiling of stored feed and products, their zoonotic risks are frequently underestimated. They can amplify the number of pathogens in the environment and transfer them to food animals. Thus organic farmers should be aware of the need for rodent control from a food safety perspective. Preferably, rodent control should form an integral part of a total package of hygiene measures to prevent transfer of food‐borne pathogens. These should also include e.g. control of wild birds and flies and obligatory disinfection of boots/clothes and equipment for farm workers and visitors. Copyright © 2007 Society of Chemical Industry     

Prevalence and risk factors associated with campylobacter infections in broiler flocks in Shiraz, southern Iran

Campylobacter species are among the most common bacterial causes of human gastroenteritis in many countries, and poultry meat is considered as a major source of human campylobacteriosis. The present study was conducted to determine the prevalence of infection by Campylobacter jejuni and Campylobacter coli in broiler flocks in Shiraz and to investigate the possible risk factors for the campylobacter infections in this area. For detection of campylobacter, multiplex polymerase chain reaction (mPCR) was used. Between August and September 2009, a total of 100 broiler flocks from 100 commercial broiler farms were selected at slaughter and campylobacter status was determined by mPCR on caecal samples. Data about farms and flocks were collected by questionnaires. Approximately 76% (95% CI: 67-84%) of the flocks were positive for C. jejuni or C. coli. Twenty two percent were positive for C. jejuni, 32% for C. coli and 22% for both species. Results of the statistical analysis using multivariable logistic regression showed that the odds of flock infection decreased when level of owner's education (years) increased (OR = 0.86, P = 0.04), also odds of infection was nearly five times higher when age at slaughter was ≥ 45 days compared with < 45 days (OR = 5.3, P = 0.003) and use of antibiotic medications at early stage of production period was negatively associated with the infection status of the flock (OR = 0.33, OR = 0.059). We found no evidence of the effects of any other factors such as time interval between successive flocks, hygiene measures and number of broiler houses on the farm on the prevalence of campylobacter infection. Getting more attention to the health education issues and planning qualitative studies to reveal the behavioral aspects of the management policy, may be subjects of interest for future researches.     

江苏地区肉鸡屠宰链弯曲菌分离菌株毒力基因分布及分子分型研究

目的了解江苏地区肉鸡屠宰生产链中弯曲菌分离菌株毒力基因分布及不同环节分离菌株遗传相关性。方法利用特异性引物对肉鸡屠宰过程不同环节分离所得的96株弯曲菌分离菌株的10种致病相关毒力基因进行聚合酶链式反应(polymera sechainre action,PCR)检测,应用多位点序列分型(multilocus sequence typing,MLST)方法对分离菌株进行分子分型研究,通过与弯曲菌MLST数据库比对获得各株菌等位基因值、序列型(sequence types,STs)及克隆复合体。结果96株分离菌株中,flaA、cadF和cdtB毒力基因携带率均为100%,其次为cdtC、iam和cdtA毒力基因,携带率较高,分别为96.9%、86.5%和84.4%,另外3个毒力基因flhA、virB11、ciaB携带率均较低,分别为25.0%、15.6%、11.5%;格林-巴利综合症相关毒力基因wlaN在所有菌株中均无检出。MLST分型结果显示,96株弯曲菌可分为10个STs,其中2个为新STs,形成1种优势克隆复合体CC828(56株)和4种未定义的克隆复合体,表现为较低的遗传多样性。结论肉鸡屠宰生产链中弯曲菌毒力基因分布广泛,不同环节弯曲菌分离菌株遗传多样性较低,表明弯曲菌在肉鸡屠宰生产链中存在交叉污染。     

Contaminants and microorganisms in Dutch organic food products: a comparison with conventional products

Organic products were analysed for the presence of contaminants, microorganisms and antibiotic resistance and compared with those from conventional products. No differences were observed in the Fusarium toxins deoxynivalenol and zearalenone in organic and conventional wheat, during both a dry period and a very wet period which promoted the production of these toxins. Nitrate levels in head lettuce produced organically in the open field were much lower than those in conventional products. In iceberg lettuce and head lettuce from the greenhouse, no differences were detected. Organically produced carrots contained higher nitrate levels than conventional products. Both organic and conventional products contained no residues of non-polar pesticides above the legal limits, although some were detected in conventional lettuce. Organic products contained no elevated levels of heavy metals. Salmonella was detected in 30% of pig faeces samples obtained from 30 organic farms, similar to the incidence at conventional farms. At farms that switched to organic production more then 6 years ago no Salmonella was detected, with the exception of one stable with young pigs recently purchased from another farm. No Salmonella was detected in faeces at the nine farms with organic broilers, and at one out of ten farms with laying hens. This is comparable with conventional farms where the incidence for Salmonella lies around 10%. Campylobacter was detected in faeces at all organic broiler farms, being much higher than at conventional farms. One of the most remarkable results was the fact that faeces from organic pigs and broilers showed a much lower incidence of antibiotic resistant bacteria, except for Campylobacter in broilers. It is concluded that the organic products investigated scored as equally well as conventional products with regard to food safety and at the same time show some promising features with respect to antibiotic resistance.