空肠和结肠弯曲菌鞭毛蛋白基因fla A的检测

目的 运用一种快速、敏感、特异的检测空肠和结肠弯曲菌的方法.方法 以空肠和结肠弯曲菌所共有特异的鞭毛蛋白基因 fla A的一段高度保守序列为引物,用PCR法扩增fla A基因上的一段约1 700 bp的片断.用该引物对空肠和结肠弯曲菌的标准株、福建省的食品分离株进行PER扩增检测,并同时检测该PCR方法的敏感性.结果 扩增片断表现出极好的特异性,2株空肠和结肠弯曲菌标准菌株、8株分离自不同食品样品的空肠穹曲菌和结肠弯曲菌菌株均为阳性,且敏感性实验显示该PCR方法的反应体系最低检出菌量为6 CFU.结论 该方法快速、敏感、特异,可用于突发性食物中毒和暴发感染的调查.     

2007年江苏省食源性致病菌监测分析

目的监测江苏省主要食源性致病菌污染状况,确定本地区高危食品,为预防控制食源性疾病提供科学依据。方法依据GB/T 4789—2003食品卫生微生物学检验方法,对样品分别进行沙门菌、单核细胞增生性李斯特菌、大肠杆菌O157∶H7、空肠弯曲菌和副溶血性弧菌进行分离,生化及血清学鉴定。结果共监测生畜禽肉、熟肉制品、生食蔬菜、水产品8类样品共804件,检出致病菌113株,总检出率14.1%(113/804)。其中沙门菌检出33株,检出率为4.1%(33/804),单核细胞增生性李斯特菌检出41株,检出率5.6%(41/738);副溶血性弧菌检出13株,检出率为11.2%(13/116);金黄色葡萄球菌检出率15.9%(24/151);大肠杆菌O157∶H7检出2株,检出率0.41%(2/493);空肠弯曲菌和阪崎肠杆菌未检出。33株沙门菌经血清型鉴定,分离出9种血清型,主要是德尔卑沙门菌,其次为罗米他沙门菌和鸭沙门菌。结论江苏省地区食品存在食源性致病菌污染,其中水产品和生肉是主要污染食品品种,而熟肉和生食水产品导致食源性疾病的风险较高。     

2006-2008年北京市大兴区食品中食源性致病菌的污染状况

目的:了解北京市大兴区市售食品中食源性致病菌污染状况.方法:2006-2008年,采集大兴辖区內的10类食品,对沙门菌、大肠埃希菌0157:H7、金黄色葡萄球菌、单核细胞增生李斯特菌、空肠弯曲茼和副溶血性弧菌等6种食源性致病菌进行监测分析.结果:食源性致病菌检出率为11.96%(67/560).金黄色葡萄球菌的检出率最高(6.79%,38/560),其次为单核细胞增生李斯特菌(3.57%,20/560)和沙门菌(11.61%,9/560).560份食品中均未检出大肠埃希菌0157:H7、空肠弯曲菌和副溶血性弧菌.生牛奶中金黄色葡萄球菌检出率最高(30.67%,23/75),其次为生肉(15.29%,39/255)、鲜冻水产品(5.45%,3/55)和熟肉制品(2.67%,2/75).结论:大兴区市售食品中食源性致病菌污染较为普遍,生牛奶、生肉类、鲜冻水产品及散装熟肉制品可能是导致食源性疾病的高危食品.     

2006年东阳市食品致病菌污染状况监测分析

目的对东阳市食品中6种致病菌的分布状况进行监测,确定污染致病菌的高危食品,为食物中毒监测提供科学依据。方法依据国标方法和2006年中国疾病预防控制中心食品污染菌调查工作手册,采用进口显色培养基、生物梅里埃-ATB鉴定系统,对采集的食品样本分别进行沙门菌、单核细胞增生性李斯特菌、EHEC O157：H7、弯曲菌、金黄色葡萄球菌和副溶血性弧菌的分离和生化鉴定。结果检测生肉（牛、羊、鸡、猪）、熟肉、非生食水产品、生食水产品、生食蔬菜、蔬菜色拉、鲜榨果汁共7类样品143份,检出致病菌38株（其中有2份生猪肉同时检出2株沙门菌）,总检出率25.2％。其中分离到副溶血性弧菌6株,金黄色葡萄球菌12株,沙门菌17株,单核细胞增生性李斯特菌3株。EHEC O157：H7和弯曲菌未栓出。7类食品中致病菌阳性率最高的为非生食水产品（70．0％）,其次为蔬菜色拉（50.0%）、生肉（39.5％）、熟肉制品（15．2％）,生食蔬菜和生食水产品中均未检出致病菌。结论东阳市居民主要消费食品存在食源性致病菌污染,其中非生食水产品和生肉是主要被污染食品。主要污染致病菌是副溶血性孤菌、金黄色葡萄球菌和沙门菌。     

2004-2007年肇庆市食品中食源性致病菌监测与分析

目的了解肇庆市食品中食源性致病菌的污染状况和污染水平,初步确定高危食品的种类,为食源性疾病的监测提供科学的依据。方法按全国食品污染物监测网的工作手册,2004-2007年采集4个监测点的5大类食品(生肉、熟肉、水产品、生吃水产品和生吃蔬菜)共计325份,对沙门菌、单核细胞增生李斯特菌、大肠杆菌O157∶H7和副溶血性弧菌4种食源性致病菌进行监测分析。结果325份食品样品中,检出致病菌27株(8.31%)。其中沙门菌16株(4.92%),单核细胞增生李斯特菌10株(3.08%),大肠杆菌O157∶H7未检出,80份水产品中检出副溶血性弧菌1株(1.25%)。生肉的污染情况最为严重,检出率为11.35%,其次是熟肉,检出率为9.68%;水产品的检出率为5.45%,生食水产品的检出率为4.00%,生食蔬菜的检出率为2.56%。结论沙门菌和单核细胞增生李斯特菌对肇庆市食品的污染普遍存在。生肉、熟肉的污染尤为严重,是重要的高危食品。     

舟山市2006-2008年食源性致病菌污染状况分析

目的了解舟山市市售食品中食源性致病菌污染状况及分布和高危食品种类。方法参照国家食源性疾病监测网工作手册进行。结果检测生畜肉、生禽肉、非定型包装熟肉制品、动物性水产品、蔬菜、海水鱼、淡水鱼、冷菜、鲜榨果汁等共511件,检出沙门氏菌3株,检出单核细胞增生李斯特菌5株,检出空肠弯曲菌3株,副溶血性弧菌68株。结论舟山市居民主要消费食品存在食源性致病菌污染,其中水产品和熟肉制品污染较重,应引起有关部门重视,并加强监督、检查力度,加大对食品卫生安全的宣传教育工作。     

扬州市食品中7种食源性致病菌污染状况及耐药性研究

为确定易受污染的食品,研究食源性病原菌耐药性状,为制定HACCP控制食源性疾病提供科学依据,对扬州市食品中沙门菌、E.coliO157:H7、单核细胞增生李斯特菌、金黄色葡萄球菌、副溶血性弧菌、空肠弯曲菌和小肠结肠炎耶尔森菌污染状况进行分析。在957份食品中共检出沙门菌、单核细胞增生李斯特菌、金黄色葡萄球菌、副溶血性弧菌90株,检出率为9.40%。其中沙门菌检出率为2.09%,单核细胞增生李斯特菌检出率为4.49%,金黄色葡萄球菌检出率为2.72%,副溶血性弧菌检出率为0.10%,未检出E.coliO157:H7。在80件生肉类试样中检出空肠弯曲菌8株,小肠结肠炎耶尔森菌6株。对分离出的沙门菌、单核细胞增生李斯特菌、金黄色葡萄球菌进行药敏试验,结果表明3种致病菌对部分抗生素多重耐药。扬州市食品中主要危害因素为沙门菌、单核细胞增生李斯特菌、金黄色葡萄球菌及其对抗生素的多重耐药。食物链是病原菌耐药性产生的重要环节。加强生肉制品、散装熟食及生牛奶的卫生管理,控制动物饲料抗生素添加剂的使用并严格遵守休药期以防止耐药菌株的产生,对控制食源性疾病、保证食品安全具有很重要的意义。     

2002-2005年北京市顺义区食品致病菌监测分析

目的了解北京市顺义区食品致病菌的污染状况。方法按照GB/T4789─2003食品微生物检验标准方法进行检验。结果2002-2005年共监测了6类食品（590份）中的4种致病菌（沙门菌、金黄色葡萄球菌、肠出血性大肠埃希菌O157：H7、单核细胞增生性李斯特菌）。检出致病菌57株,阳性率为9.7%,其中检出沙门菌2株,金黄色葡萄球菌26株,单核细胞增生性李斯特菌29株,未检出肠出血性大肠埃希菌O157：H7。6类食品中生牛奶、生畜（禽）肉类产品致病菌阳性率较高,分别为15.5%、14.5%;冰淇淋、水产品、散装熟食中也有检出,分别为5.0%、4.4%、1.9%;生食蔬菜中没有检出致病菌。结论生牛奶、生畜（禽）肉类产品是致病菌污染的主要食品,由此造成的二次污染是引起食物中毒的隐患,应该引起足够重视。     

弯曲菌及弯曲菌病的流行现状

为阐明弯曲菌在肠内外感染中的重要性 ,综述了弯曲菌病的发病率、流行特点、临床表现及弯曲菌对抗生素的耐药性。弯曲菌感染率在世界范围内呈普遍上升趋势 ,禽肉、水、牛奶、动物是主要传染源。空肠弯曲菌和结肠弯曲菌是弯曲菌感染最常见的两个种。格林巴利综合征是弯曲菌感染后最严重的并发症。近年来 ,弯曲菌对抗生素的耐药株迅速增多 ,最值得注意的是耐氟喹诺酮类弯曲菌。为更好地控制弯曲菌的感染与流行 ,各国 (特别是发展中国家 )有必要建立对弯曲菌的监测系统。     

2010年上海市市售食品中食源性致病菌监测结果分析

目的掌握上海市市售食品中食源性致病菌污染状况。方法在全市监测网点抽检各类食品,开展沙门菌等11种食源性致病菌的监测。结果各类食品中食源性致病菌总体检出率为7.6%,检出食源性致病菌的食品包括剩饭(25.0%)、生禽肉(21.7%)、生畜肉(19.0%)、水产品(14.4%)、餐饮即食食品(4.5%)和速冻熟制米面制品(1.7%)。其中,海产品中的副溶血性弧菌、生禽肉中单增李斯特菌、生猪产品中的沙门菌的检出率分别为21.0%,19.8%和11.6%,是本市食源性致病菌风险来源的主要食品品种和项目。熟食卤味、乳制品、鸡蛋、生食蔬菜、冷饮饮料、糕点、非发酵豆制品等食品均未检出食源性致病菌。结论上海市部分市售食品,特别是部分生食品中食源性致病菌仍有一定的检出率,食品加工过程仍应采取烧熟煮透、防止交叉污染等针对性措施,以提高食品安全性。     

Detection of Campylobacter jejuni and Campylobacter coli in foods by enrichment culture and polymerase chain reaction enzyme-linked immunosorbent assay

A polymerase chain reaction (PCR) assay based on a solution hybridization format with colorimetric end-point detection (PCR ELISA) was investigated for the specific detection of Campylobacter jejuni and Campylobacter coli in food samples following enrichment culture. One hundred fifteen samples of raw meat and offal (poultry, porcine, ovine, and bovine), raw shellfish, and artificially contaminated milk were enriched in blood-free Campylobacter Enrichment Broth for 48 h. Enrichment cultures were subcultured to Campylobacter blood-free selective agar plates, and presumptive isolates were identified by phenotypic methods. DNA was extracted from 1-ml aliquots of the enrichment cultures using a rapid extraction method, and the DNA was used as the template in a PCR ELISA. A comparison of the PCR ELISA with the enrichment culture and subculture to selective agar method showed that the results of 112 of the 115 samples tested were in agreement by both methods. Seventy-one of the various food samples were positive in the PCR ELISA, and 70 samples were positive by culture. The PCR ELISA had a sensitivity of 99% and a specificity of 96%, with a positive predictive value of 97% and a negative predictive value of 98%. The PCR ELISA is a rapid, sensitive, and specific method for the detection of C. jejuni and C. coli in foods following enrichment culture and significantly reduces the time required for their detection.     

Campylobacter and Salmonella in raw red meats in the United Kingdom: prevalence, characterization and antimicrobial resistance pattern, 2003-2005

The prevalence of Campylobacter and Salmonella was assessed in 3959 raw red meats in the UK during 2003-2005. Meats were more frequently contaminated with Campylobacter (7.2%) than with Salmonella (2.4%). Lamb and other meats (e.g. mutton, rabbit) exhibited the highest contamination from Campylobacter (12.6% and 19.8%, respectively), compared with pork (6.3%) and beef (4.9%). Pork however had the highest contamination from Salmonella (3.9%), followed by lamb (2.0%), other meats (2.0%) and beef (1.3%). Offal samples (36.6%) were more frequently contaminated with Campylobacter or Salmonella than muscle tissue (7.0%). C. jejuni predominated in all meat types. C. coli isolates were more likely to exhibit antimicrobial drug resistance, including quinolones, than C. jejuni. Salmonella typhimurium was the most frequent Salmonella serotype isolated from meats; S. typhimurium DT104/104b isolates exhibited higher rates of multiple drug resistance than other serotypes. The findings reinforce the importance of adequate cooking of meat and good hygiene to avoid cross-contamination.     

Prevalence and characterization of Campylobacter jejuni isolated from pasture flock poultry

The growing interest in organic and natural foods warrants a greater need for information on the food safety of these products. In this study, samples were taken from 2 pasture flock farms (N = 178; feed, water, drag swabs, and insect traps), pasture flock retail carcasses (N = 48) and 1 pasture flock processing facility (N = 16) over a period of 8 mo. A total of 105 Campylobacter isolates were obtained from 53 (30%), 36 (75%), and 16 (100%) samples from the farms, retail carcasses, and processing facility, respectively. Of the 105 isolates collected, 65 were C. jejuni, 31 were C. coli, and 9 were other Campylobacter spp. Using PCR, the C. jejuni isolates were further analyzed for virulence genes involved in colonization and survival (flaA, flaC, cadF, dnaJ, racR, cbrR), invasion (virB11, ciaB, pldA), protection against harsh conditions (sodB, htrA, clpA), toxin production (cdtA, cdtB, cdtC), siderophore transport (ceuE), and ganglioside mimicry (wlaN). In addition, the short variable region of the flaA locus (flaA SVR) was sequenced to determine the genetic diversity of the C. jejuni isolates. The flaA SVR diversity indices increased along the farm to carcass continuum. PCR-based analysis indicated a low prevalence of 5 genes involved in colonization (dnaJ, ciaB, pldA, racR, virB11). The results of this survey indicate that the prevalence of Campylobacter on organic retail carcasses is similar to prevalence reports of Campylobacter on conventional retail carcasses. However, the genetic diversity of the flaA SVR genotypes increased along the farm to carcass continuum that contrasted with conventional poultry studies. PRACTICAL APPLICATION: Campylobacter jejuni is a leading cause of foodborne illness with poultry and poultry products being leading sources of infection. Free-range and pasture flock chickens are becoming more popular; however, there is an inherent biosecurity risk that can increase the prevalence of foodborne pathogens in these flocks. This study aimed to determine sources and characterize C. jejuni isolated from pasture flocks.     

Detection of thermophilic Campylobacter spp. in blood-free enriched samples of inoculated foods by the polymerase chain reaction

The detection of thermophilic Campylobacter spp., as represented by Campylobacter jejuni, by the polymerase chain reaction (PCR) was investigated and compared with the selective agar isolation (SAI) method. Stationary-phase cultures of C. jejuni were inoculated into either blood-free enrichment broth (BFEB) or BFEB that contained 10% broccoli, crabmeat, mushroom, raw milk, and raw oyster rinses. Following a 48-h enrichment period, aliquots of food test portions were removed for simultaneous analysis by PCR and SAI. It was determined that the presence of charcoal and iron in the enrichment broth interfered with the PCR assay. Therefore, three DNA extraction techniques were developed and evaluated using a 16S rRNA primer pair in the PCR assay. The 50% end point (DL50) values (determined upon six initial C. jejuni spiking levels) were used to assess the frequency of isolation utilizing PCR versus SAI for the detection of this organism in the enrichment matrices. There were virtually no differences in detection of C. jejuni among enriched samples analyzed by PCR and SAI. Mean DL50 values (n = 3) for plain BFEB, broccoli, crabmeat, mushroom, raw milk, and raw oyster were, respectively, 0.02 (PCR) versus 0.01 (SAI), 0.01 versus 0.06, 0.07 versus 0.04, 0.03 versus 0.08, 0.01 versus 0.01, and 0.01 versus 0.01 CFU/5 g food. Significant variability in the detection limit of C. jejuni by PCR in the food enrichments was observed among DNA extraction techniques. Using 48-h enrichment cultures followed by PCR analysis could save 1 day of the time required for the presumptive identification of C. jejuni in suspected foods.     

Species identification by genotyping and determination of antibiotic resistance in Campylobacter jejuni and Campylobacter coli from humans and chickens in Sweden

Campylobacter is today the most common cause of human bacterial enteritis in Sweden, as well as in most other industrialized countries. Common sources of infection are undercooked chicken meat, unpasteurized milk and contaminated drinking water. One aim with our present study was to identify the species Campylobacter jejuni and Campylobacter coli strains from humans and chickens using a polymerase chain reaction/restriction enzyme analysis (PCR/REA) method, as well as traditional hippurate hydrolysis test. Another aim was to investigate the antibiotic resistance pattern of the human domestic C. jejuni/C. coli isolates from infected patients and isolates from healthy Swedish chicken, as well as isolates from humans infected abroad. If discrimination between C. jejuni and C. coli was based on testing for hippurate hydrolysis, 95% of the human domestic strains and 88% of the chicken strains were identified as C. jejuni. Based on genotyping by PCR/REA, 100% of the human domestic strains and 98% of the chicken strains were attributed to C. jejuni. The E-test and disc diffusion methods were used for phenotypic antibiotic resistance studies. The two methods gave similar results. Most Swedish C. jejuni/C. coli isolates both from humans and chickens were sensitive to doxycycline and erythromycin, which are antibiotics used to treat human infection. Only 7% of the human domestic strains and 2% of the chicken strains were resistant to the quinolones tested. As a comparison, more than 94% of strains isolated from travelers to Asia and southern Europe showed antibiotic resistance to one or more drugs.     

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%).     

Performance comparison of the BioSys optical assay and the violet red bile agar method for detecting coliforms in food products

Coliform counts in a variety of foods, including dairy products (raw milk, pasteurized milk, yogurt, butter, and ice cream), meats (pork sausage, ground beef, and raw chicken), raw eggs, and chocolate, were performed by the rapid automated BioSys optical assay and the conventional method with violet red bile agar (VRBA). The standard deviation (SD) among five replicate counts for the optical assay was similar to or better than that obtained with VRBA plates for all foods tested. The average SD for all foods tested was 0.21 for the optical assay and 0.30 for the VRBA plates. At very low concentrations of coliforms (1 to 10 CFU/ml for liquid products and 10 to 100 CFU/g for solid samples), the average SDs were 0.26 and 0.47, respectively. The optical assay was less susceptible to interference by noncoliform organisms. In naturally contaminated samples, bacteria such as Serratia liquefaciens, Pantoea spp., Vibrio fluvialis, Aeromonas hydrophilia, and Pseudomonas spp. formed typical colonies in VRBA, resulting in false-positive results or a need to verify colonies in brilliant green lactose broth. The optical assay appeared to be more selective than the VRBA conventional method, detecting fewer noncoliforms. There was close agreement in test results between the two methods, as indicated by correlation coefficients of 0.92 to 0.99 obtained for the regression analysis of the two methods. In most cases both methods distinguished accurately between positive samples containing coliforms and negative controls. All products tested using the automated BioSys Optical Assay for coliforms yielded results more quickly (typically 10 to 12 h) than did those tested with the conventional VRBA method (24 to 72 h with confirmation).     

Automated simultaneous detection of low levels of listeriae and salmonellae in foods

Salmonella spp. and Listeria monocytogenes continue to be major pathogens of concern to food processors. However, routine screening of food samples to detect these pathogens is generally labor intensive and costly. Automated optical procedures for the detection of salmonellae and listeriae in foods were developed in our laboratory. In the present study we report their adaptation to a simultaneous recovery and detection procedure. Milk, shell eggs, fresh and ready-to-eat (RTE) meats or raw chicken contaminated with a combination of sub-lethally injured salmonellae and listeriae (10-50 cells each) were incubated for 6 h at 35 degrees C in modified universal pre-enrichment broth (MUPB). Volumes (4 ml) were then transferred to vials containing selective liquid media for these pathogens (4 ml), and incubated overnight at 35 degrees C in a BioSys instrument. The presence of the pathogens was identified by a black coloration of the media and a sharp drop in light transmittance caused by hydrogen sulfide production (Salmonella organisms), or esculin hydrolysis (Listeria organisms). There was no difference in the detection time of salmonellae when incubated alone or with listeriae, but listeriae grew at a slower rate in the presence of salmonellae, resulting in a delay of < or = 1 h in their detection. Overall, the detection of 10-50 salmonellae and 10-50 listeriae in 25 g of the tested foods required a total of 24 h. Confirmation of the pathogens by PCR-based assay (6 h) was completed the following day directly from positive vials, requiring a total of < or = 30 h for detection and confirmation. Negative samples required no confirmation. The testing system was confirmed in 70 naturally contaminated foods.