广州市市售食品食源性致病菌污染状况调查

目的了解广州市市售食品中食源性致病菌的污染状况。方法采用随机抽样方法分别对我市10个区以及两个地级市的集贸市场、超市、宾馆饭店及个体熟食销售点的7类食品共363份样品中沙门氏菌、金黄色葡萄球菌、副溶血性弧菌、单增李斯特菌、肠出血性大肠杆菌O157∶H7、空肠弯曲菌等6种致病菌依据国标的方法进行了监测。结果363件样品中检出致病菌72株。其中副溶血性弧菌检出率为33.93%,主要污染水产品;金黄色葡萄球菌为3.00%,主要污染熟食品;沙门氏菌和单增李斯特菌检出率分别为1.93%和0.66%;未检出大肠杆菌O157：H7和空肠弯曲菌。在各类食品中,致病菌带菌数有显著性差异（以χ^2检验,P〈0.005）。水产品致病菌带菌率最高,以副溶血性弧菌为主;其次为生肉类,以检出沙门氏菌为多。结论我市市售食品存在食源性致病菌污染,水产品和生肉类是主要污染食品。应加强市售食品监督管理,以减少可能引起食源性疾病的因素。     

广州市市售食品沙门菌污染状况及耐药性分析

目的按国家食源性致病菌监测项目要求,对广州市市售食品的沙门菌污染状况以及对抗生素的药物敏感性进行调查。方法采集广州肉菜市场、大型超市、饭店提供的肉、水产品、凉拌菜及沙拉冷饮食品,按国标方法进行沙门菌检测,并用纸片法进行抗生素敏感性测定。结果 413份食品中有41份检出沙门菌,检出率为9.9%,其中生肉类食品的污染状况最为严重,检出率达18.8%,其次是生吃水产品为14.0%,熟肉和凉拌菜较低分别为4.5%和3.9%。在检出的17种血清型中德尔卑沙门菌比例最高为29.3%。对16株抗生素的药敏试验结果显示,对四环素耐药率最高,达61.0%;并且发现有6株多重耐药菌株以及对头孢类耐药的菌株。结论广州市市售食品中存在沙门菌污染,并存在有多重耐药和对头孢类抗生素耐药的现象。     

吉兰-巴雷综合征相关空肠弯曲菌WLAX基因序列分析

目的了解吉兰-巴雷综合征(GBS)相关空肠弯曲菌的WLAX基因序列特征,为进一步研究GBS相关空肠弯曲菌的分子致病机制打下基础。方法通过PCR方法对该基因进行扩增,将PCR产物克隆到质粒载体上,然后进行测序,将测序结果通过DNAstar软件进行比较和聚类分析。结果与非GBS相关空肠弯曲菌比较,GBS相关空肠弯曲菌的WLAX核苷酸序列发生变异的频率增大;菌株的核苷酸序列与全基因测序空肠弯曲菌NCTC11168比较存在差异;聚类关系反映了空肠弯曲菌具有一定的区域特征。结论GBS相关空肠弯曲菌中WLAX基因发生突变的概率明显增大,这些突变与GBS致病性的关系有待于进一步确定。     

2010年广州某医疗中心儿童夏季细菌性腹泻的病原学调查

目的研究广州地区小儿夏季细菌性腹泻的病原菌分布。方法采集2010年5～7月广州市妇女儿童医疗中心珠江新城院区腹泻患儿的大便标本进行常规病原菌的分离培养,通过生化反应和血清凝集试验进行鉴定和分型,并使用金标法对空肠弯曲菌抗原进行检测。结果从110份标本中检出44株病原菌,检出率为40.0%。其中致病性大肠埃希菌17株,2岁以下患儿检出15株;空肠弯曲菌12株,2岁以下患儿检出10株;沙门菌6株;念珠菌纯生长6株;产气荚膜杆菌3株。结论广州地区夏季儿童细菌性腹泻的主要病原菌是致病性大肠埃希菌及空肠弯曲菌,两者的易感人群以2岁以下婴幼儿为主。     

实时荧光PCR方法快速检测空肠弯曲菌方法的建立

目的建立实时荧光PCR快速检测空肠弯曲菌的方法。方法以空肠弯曲杆菌HipO基因的保守序列为模板设计特异性引物探针,建立一种能快速检测样本中空肠弯曲杆菌的实时荧光PCR方法;对方法的特异性和敏感性进行评价,并以正常人粪便为空白样本,添加一定量空肠弯曲菌标准株菌液进行检测,以对方法的检测效果进行初步评价。结果该实时荧光PCR方法只对空肠弯曲杆菌进行特异扩增,同种属的结肠弯曲菌及其他常见食源性病原菌均不能扩增;整个检测过程只需要80min,对空肠弯曲菌菌悬液可检测至5个细菌,对加标粪便样本可检测至10-100个细菌。结论本研究建立的实时荧光PCR检测空肠弯曲菌方法不仅能实现对空弯菌的快速检测,而且还为空弯菌的快速诊断及其引起的食源性疾病的监控溯源提供有意义的参考。     

格林-巴利综合征相关空肠弯曲菌flaA基因的克隆及序列特征

近年来研究发现，空肠弯曲菌感染与部分格林一巴利综合征（Guillain-Barre syndrome，GBS）发病密切相关。空肠弯曲菌引起GBS的发病机制非常复杂，由于GBS常发生于少数几种特殊血清型的空肠弯曲菌感染，提示这些空肠弯曲菌可能具有某种独特的毒力特征，而这种毒力特征与引起GBS有关。空肠弯曲菌的鞭毛是其一个重要的毒力因子，在空肠弯曲菌感染的致病机制中起重要作用。鞭毛微丝由两个高度同源的基因flaA和flaB编码，     

格林—巴利综合征相关空肠弯曲菌脂多糖抗原初步分析

用格林－巴利综合征相关性空肠弯曲菌复制动物模型成功后的动物免疫血清对河北省格林－巴利综合征病人分离株空肠弯曲菌、动物来源株空肠弯曲菌及北京对照用人源株空肠弯曲菌及其脂多糖抗原性进行分析。结果显示，发生了典型的格林－巴利综合征样疾病的动物已产生了明显的抗格林－巴利综合征相关性空肠弯曲菌的免疫反应，其抗脂多糖抗体与当地其它来源株空肠弯曲菌有部分交叉反应，与北京分离株空肠弯曲菌无明显交叉反应。说明格林－巴利综合征相关性空肠弯曲菌脂多糖具有其独持的抗原性。支持某些特殊血清型的空肠弯曲菌参与格林－巴利综合征的致病过程。     

空肠弯曲菌67KD 外膜蛋白的提取及其对淋巴细胞的多克隆激活作用

本室曾建立空肠弯曲菌诱导自身免疫综合征小鼠模型.本文用盐析法及离子交换层析法从空肠弯曲菌(CJ-S131)中提取了67KD 的外膜蛋白.免疫印迹法证实该蛋白为空肠弯曲菌的主要抗原成份之一,具有较强的免疫原性;体外对小鼠脾淋巴细胞具有明显的多克隆激活作用.本实验为从分子水平研究感染与自身免疫的关系提供了重要的物质基础和新的实验依据.     

The significance of Campylobacter jejuni infection in poultry: a review

Campylobacter is a significant cause of enterocolitis in consumers of undercooked poultry meat. Campylobacter jejuni is the most significant of the three thermophilic Campylobacter species, and is responsible for intestinal colonization in poultry and food-borne enteritis in humans. Generally, C. jejuni is apathogenic in poultry, although newly hatched chicks and turkeys may develop a transient diarrhoea following infection. Modern intensive poultry production favours the introduction of infection into commercial growing units, resulting in intestinal colonization during the second to fourth weeks inclusive. Routes of infection include contaminated fomites, infected water supply, rodents, insects, and free-living birds. Vertical transmission is considered unlikely. Contamination of poultry meat is enhanced by deficiencies in transport and processing of broilers and turkeys. Scalding, defeathering and evisceration represent the significant points of cross-contamination during processing. Epidemiological correlation has been established between consumption of contaminated chicken and outbreaks of human campylobacteriosis. Amelioration of infection by application of improved standards of hygiene and decontamination is possible in the context of commercial poultry production. Improvement in washing of carcasses, and the application of chemical disinfectants and gamma irradiation have the potential to reduce the prevalence of C. jejuni contamination in poultry meat. These innovations, together with improved storage and handling of meat products, will reduce the risk of campylobacteriosis to consumers.     

Isolation of Campylobacter fetus subsp. jejuni from migratory waterfowl.

Since the sources from which humans acquire Campylobacter enteritis are only partially known, we studied the frequency of carriage of Campylobacter fetus subsp. jejuni in migratory waterfowl. Cecal contents of various species of wild ducks were cultured on selective media that contained antibiotics to inhibit normal flora. Thirty-five percent of the 445 ducks cultured harbored C. fetus subsp. jejuni. Migratory waterfowl are yet another reservoir for this enteric pathogen and may be of public health importance for humans in the contamination of water or when used as food.     

Human serum antibody response inCampylobacter jejuni enteritis as measured by enzyme-linked immunosorbent assay

An ELISA for detection of IgG, IgA, and IgM antibody using an acid-glycine extract from Campylobacter jejuni as antigen was developed. To determine the value of this assay for the diagnosis of acute Campylobacter jejuni infections, the IgG, IgA, and IgM immune response against Campylobacter jejuni was investigated at various timepoints after infection in patients with culture-proven infection. A total of 112 sera from 46 patients and 78 sera from a control group were tested. All but one of the 46 patients with culture-proven Campylobacter jejuni enteritis developed IgG antibodies against Campylobacter jejuni. IgA and IgM ELISA both showed 97% specificity, and sensitivity of 63% and 30% respectively. IgG antibody titers generally remained at a constant level for more than 50 days, whereas IgA and IgM antibody titers declined more rapidly to normal values within 30 to 50 days after onset of clinical symptoms. Detection of Campylobacter jejuni specific IgA antibodies in a single serum sample provided the most useful assay for serological diagnosis of Campylobacter jejuni enteritis. The presence of Campylobacter jejuni specific IgM antibodies was the sole diagnostic criterion in three cases. Serological diagnosis of Campylobacter jejuni enteritis should therefore include both IgA and IgM antibody determination.     

Pet dogs and chicken meat as reservoirs of Campylobacter spp. in Barbados

Campylobacter spp. are the second most common pathogen isolated from stools of patients with gastroenteritis in Barbados. The aim of this study was to identify reservoirs of Campylobacter and the likely source(s) of human infection. Fecal specimens from 596 animals and 311 samples of animal food products were analyzed for the presence of Campylobacter spp. by standard culture techniques. Isolates were characterized by conventional phenotypic tests, confirmed by latex agglutination and PCR with genus-specific primers, and identified by the use of species-specific primers. High isolation rates were obtained for chickens (94.2%), pigs (90.5%), dogs (46.9%), cats (37.3%), and wild birds (39.3%). Campylobacter was also recovered from monkeys (17.1%) and sheep (4.2%) but not from cows. Chicken meat was frequently contaminated with Campylobacter (58.4%), but its recovery from other animal food products was rare. Campylobacter jejuni was the most commonly identified species in humans (63.6%), chickens (86.6%), dogs (51.5%), and chicken meat (79.8%). Porcine isolates were predominantly C. coli (98.4%), while cats harbored mainly C. upsaliensis and C. helveticus. Wild birds alone carried urease-positive thermophilic campylobacters. C. jejuni and C. coli isolates from different sources were compared with isolates from humans by randomly amplified polymorphic DNA typing with the primers OPA 11 and HLWL 85. Genotyping revealed similarities between isolates from chicken meat and those from humans and could not distinguish between two clinical isolates and four canine strains. Our results suggest that dogs are significant reservoirs of Campylobacter and contribute to human enteric infections and that chicken meat is a likely vehicle for the transmission of campylobacters to humans.     

Attempts to isolate Campylobacter jejuni from various body sites.

Clinical material collected from various body sites, but excluding faeces, was cultured on either selective or non-selective media for Campylobacter spp. Campylobacter jejuni was found in only two (0.18%) of 1100 specimens; both positive specimens were urine. From these results it is suggested that C jejuni is an uncommon finding in clinical material other than faeces.     

Campylobacter, from obscurity to celebrity

After its successful isolation from stools in the 1970s, Campylobacter jejuni has rapidly become the most commonly recognised cause of bacterial gastroenteritis in man. Reported cases of human campylobacteriosis represent only a small fraction of the actual number. In industrialised countries, the incidence of C. jejuni/Campylobacter coli infections peaks during infancy, and again in young adults aged 15-44 years. Acute self-limited gastrointestinal illness, characterised by diarrhoea, fever and abdominal cramps, is the most common presentation of C. jejuni/C. coli infection. The introduction of selective media has made the diagnosis of Campylobacter enteritis a simple procedure. In general, Campylobacter enteritis is a self-limiting disease which seldom requires antimicrobial therapy, although one in 1000 infections may lead to the Guillain-Barré syndrome. In industrialised countries, most infections are acquired through the handling and consumption of poultry meat. In developing countries, where the disease is confined to young children, inadequately treated water and contact with farm animals are the most important risk factors. Many infections are acquired during travel. Fluoroquinolone resistance has been reported in C. jejuni since the late 1980s in Europe and Asia, and since 1995 in the USA. The use of fluoroquinolones to treat animals used for food has accelerated this trend of resistance. In Australia, where fluoroquinolones have not been licensed for use in food production animals, C. jejuni remains susceptible to fluoroquinolones. The public health burden of Campylobacter spp. other than C. jejuni/C. coli remains unmeasured. Better diagnostic methods may reveal the true health burden of these organisms.     

Fitness cost of fluoroquinolone resistance in Campylobacter coli and Campylobacter jejuni

In this study, the fitness cost of fluoroquinolone resistance was evaluated in vitro, on food matrices, and in vivo, using Campylobacter coli and Campylobacter jejuni in vitro selected mutants. In vitro, the growth rate of the susceptible (wild type) and resistant (mutant) strains did not differ when cultured separately. However, by conducting sequential passages of mixed cultures, the ratio of the resistant mutant to the susceptible strain decreased for C. coli but not for C. jejuni. When the wild type and the mutant were co-inoculated on food matrices, mutants were no longer detectable 3 to 5 days after artificial contamination, but the wild-type strains remained detectable for over 13 days. In mono-inoculated animals, no difference was observed between wild-type and mutant fecal titers. When co-inoculated into chickens, the susceptible strain outcompeted the resistant mutant for C. coli and for C. jejuni. However, for C. coli, if the resistant strain was already present in animals, it could persist at high titers in the digestive tract even in the presence of the wild-type strain. Together, these findings suggest that, depending on strain and study conditions, fluoroquinolone resistance can impose a fitness cost on Campylobacter.     

Water-borne Campylobacter jejuni infection in a Danish town-a 6-week continuous source outbreak

Objective: To determine the cause and characteristics of illness of a Campylobacter jejuni outbreak in Denmark in 1995-96.
Methods: A retrospective follow-up study was designed for culture-confirmed cases and for residents without a bacteriologic diagnosis. Stored clinical and environmental isolates were analyzed by serotyping and genotyping with restriction endonuclease analysis (REA), pulsed-field gel electrophoresis (PFGE) and ribotyping.
Results: Campylobacter jejuni was isolated from 110 residents and visitors to the area. However, an estimate based on a telephone survey indicated that some 2400 people were affected by the outbreak. Water samples obtained from the community waterworks contained Campylobacter jejuni serotype 02, the same serotype as in all but one of the 30 stored isolates from the outbreak. The water and clinical isolates also showed the same DNA profile, except for the single strain showing the distinct serotype. The contamination of the water supply was traced back to contamination of ground water due to a break in a sewage pipe.
Conclusions: A retrospective and demographic epidemiologic investigation of both culture-confirmed and non-culture-confirmed cases in the town combined with typing of the isolates was crucial in defining the extent and cause of the outbreak.     

Comparative performance of different PCR assays for the identification of Campylobacter jejuni and Campylobacter coli

The sensitivity and specificity of 7 PCR assays described for the identification of Campylobacter jejuni and Campylobacter coli were examined using alkaline cell lysates from a collection of 100 well characterized reference strains of C. jejuni, C. coli, Campylobacter lari and related Campylobacter, Helicobacter and Arcobacter species. Based on a preliminary evaluation, one multiplex test was excluded from further evaluation. The various assays differed considerably in sensitivity and specificity towards their target species. For C. coli, 4 of the 5 assays were 100% specific and sensitive, but for C. jejuni, none of the 5 assays were found to be 100% specific or sensitive. Subsequently, a statistically valid sample (n=263) was taken from a Belgian collection of 1906 human Campylobacter field isolates. This second collection was used to further evaluate two selected multiplex PCR assays. The present study indicates that PCR-based identification using each of the two selected multiplex PCR assays was highly reliable. The R-mPCR assay, followed by species-specific PCR assays or the ceu-oxr mPCR assay if necessary, is our current strategy of choice for the molecular identification of C. jejuni and C. coli. Results presented here should aid researchers in selecting a PCR assay suitable for their specific needs.