Objective To investigate Cronobacter spp.and Enterobacteriaceae contamination in nutritious noodles manufacture process and study the molecular typing and traceability analysis of Cronobacter spp.Methods Totally 101 samples were collected during the process of production,including raw materials,intermediate products,environment swabs,manufacturing facilities,personnel swabs and final products.Enterobacteriaceae and Cronobacter spp.were detected.And pulsed-field gel electrophoresis (PFGE) assay was utilized for the molecular typing of Cronobacter spp.Results The isolation rate of Enterobacteriaceae and Cronobacter spp.were 53.5％ (54/101) and 29.7％ (30/101),respectively.The detection rate of Enterobacteriaceae was the highest in the environment swabs (72.7％,16/22),and the prevalence of Cronobacter spp.was 50.0％ (5/10) in the final products.A high degree of genetic diversity was observed among the 31 isolates with 20 pulse-types dispersed throughout the dendrogram.And the isolates could be grouped into 6 clusters.Conclusion It should be paid more attention that Cronobacter spp.contamination existed in nutritious noodles manufacturing process,which might be due to the contaminated materials.In addition,effective control strategies were supposed to be developed during the manufacturing process.

目的 分析5株阪崎克罗诺杆菌(Cronobacter sakazakii,C sakazakii)是否能够侵染人肠上皮细胞及其侵染能力.方法 将阪崎克罗诺杆菌与Caco-2细胞以100∶1的接种率接种后共培养4h,裂解细胞将进入细胞的菌落释放出来稀释涂布于LB琼脂平板,计数,计算侵染率.结果 从食源性疾病儿童病例中分离出的两种菌株C sakazakii13SJFB217、C sakazakii 14SJFB412对Caco-2细胞的侵染率分别达到0.0 219％和0.0 165％,从婴幼儿奶粉中分离的两株阪崎克罗诺杆菌C sakazakii 14SJ430、C sakazakii 14SJ431侵染率分别为0.0 071％和0.0 083％;来源于人的阪崎克罗诺菌株比来源于食品菌株对肠上皮细胞有更高的侵染能力.结论 石家庄市腹泻儿童粪便中分离到的2株阪崎克罗诺杆菌属于高侵染能力的菌株.

目的 收集室内外空气中的细菌进行鉴定,研究环境中可培养细菌的分布种类.方法 使用空气撞击法采集室外环境和公共场所内的细菌,分离目的细菌并纯化培养后提取基因组,以16S rDNA的27F和1492R为引物,进行菌株的16S rDNA序列分析鉴定,并利用16S rDNA的高变区域V2-V9对分离的环境菌株进行聚类分析.结果 从环境空气中共分离到可培养细菌237株,分别属于35个菌属,以放线菌门、厚皮菌门和变形菌门细菌为主,以芽孢杆菌属、葡萄球菌属、考克菌属和链霉菌属细菌居多.分离的空气菌株聚类成两大类群,A类群细菌来自外环境空气和公共场所空气,B类群细菌均分离自公共场所.结论 环境空气中的细菌以芽孢杆菌属为主,其他革兰阳性杆菌和球菌分布广泛,公共场所内的空气细菌与外环境空气中的细菌存在差异.

Objective To investigate the distribution and drug resistance of Campylobacter spp.from slaughters and markets in Shijiazhuang City.Methods Chicken samples including anal swab before slaughter,ketones smear,chicken and liver were collected.Campylobacter spp.were isolated and identified according to GB/T 4789.9-2014 and multiplex polymerase chain reaction (PCR) method.The antibiotics susceptibility testing was conducted by the method of KirbyBauer with 12 kinds of antibiotics of 6 categories.Results Totally 17 strains of Campylobacter spp.were detected from 439 samples and the detection rate was 3.9％ (17/439).The resistance rates of Campylobacter jejuni to nalidixic acid,levofloxacin,ciprofloxacin and tetracycline were 85.7％ (6/7).All Campylobacter jejuni isolates were susceptible to gentamicin,streptomycin,amikacin,azithromycin and florfenicol.Furthermore,the resistance rate of Campylobacter coli to azithromycin,doxycycline and florfenicol was less than 50.0％,while was up to 80.0％ to ciprofloxacin,levofloxacin,nalidixic acid,gentamicin,amikacin,tetracycline,streptomycin and clindamycin.The multi-drug resistance rate of Campylobacter was 70.6％ (12/17),which showed 11 resistance patterns.The drug resistance patterns from market isolates were more than those from the slaughterhouse,and both had different dominant drug resistance profiles.Conclusion There was a certain degree of Campylobacter contamination in the slaughter and market in Shijiazhuang.The multi-drug resistance of the isolates was serious.

In order to study the distribution of virulence genes of Listeria monocytogenes (Lm) in Hebei Province,29 virulence genes of Lm,including Listeria monocytogenes pathogenicity islands Ⅰ (LIPI-Ⅰ:prfA,plcA,plcB,hlyA,mpl and actA),10 internalins genes (inlA,inlB,inlC,inlD,inlE,inlF,inlG,inlH /C2,inlI and inlJ) and the other 13 virulence-associated genes (bsh,srtA,iap,sigB,virR,mprF,dltA,dltB,dltC,dltD,srtB,fbpA and hpt) were detected by PCR.Results showed that in the 91 Lm strains,the detection rate of 23 virulence genes were 100％.The 29 virulence genes of 26 Lm strains were all detected,and 65 Lm strains had different deletion of 6 virulence genes inlD,inlF,inlG,inlH /C2,inlJ and mpl.The deletion rate of inlG and inlF were 60.44％ and 54.95％,respectively,following by mpl gene,with a deletion rate of 19.78％.According to the absence of virulence genes,91 strains could be divided into 10 subtypes,and the dominant virulence subtypes was type Ⅰ with all 23 virulence genes.The deletion rate of virulent genes in Shijiazhuang was higher than that in northern Hebei.It is suggested that the rate of virulence gene of food-borne Lm in Hebei Province is high,and the virulence gene deletion patterns has diversity and regional differences.

目的 通过比较蜡样芽胞杆菌(以下简称蜡样杆菌)脉冲场凝胶电泳(PFGE)技术方法的影响因素,优化蜡样杆菌PFGE分型方法.方法 在细菌收集、细胞裂解、胶块制备和电泳参数等环节设立对照,选取3株蜡样杆菌比较不同实验方案的电泳图谱结果.细菌收集设立LB固体培养基和液体培养基集菌法;细胞裂解设立灌制胶块前溶菌酶孵育组与未孵育组,设立单独溶菌酶裂解组和溶菌酶与溶葡萄球菌酶联合裂解组;细菌的胶块内裂解设立溶菌酶不同浓度和不同作用时间(2、4和16h)实验组;选取22株蜡样杆菌对3组电泳参数进行分析评价.结果 LB固体培养基集菌法与液体集菌法相比,获得了同样清晰或更清晰的条带.灌制胶块前溶菌酶与菌悬液进行一定时间的孵育可明显改善条带的清晰度.溶菌酶对胶块内细菌的裂解,4h为最佳裂解时间.溶葡萄球菌酶与溶菌酶联合使用未见对细菌裂解有改善作用.3组电泳参数(EP)的图谱差异有统计学意义(P＜0.05),EP-a的相似性系数最小,为最佳电泳参数.结论 试验确定了最佳蜡样杆菌PFGE实验方案,简化和优化了目前已有的蜡样杆菌PFGE实验方法,可应用于蜡样杆菌引起的暴发疫情分析及食源性病原菌的实验室分子分型研究.

目的 了解河北省食源性单核细胞增生李斯特氏菌(Lm)脉冲场凝胶电泳(Pulsed-field gel electrophoresis,PFGE)基因分型分布特征,建立分子分型数据库,为Lm分子流行病学研究提供资料.方法 分别以Apa Ⅰ和Asc Ⅰ两种内切酶对食品中分离的57株Lm进行PFGE基因分型检测,并以BioNumerics Version 6.6软件绘制进化树.结果 57株食源性Lm经ApaⅠ和Asc Ⅰ双酶切后,可产生32个基因型别(PFGE types,PT),分辨率为0.9 516.优势型别为PT11、PT12、PT5和PT10型.结论 河北省食品中污染的Ln菌型分散,来源于不同的克隆株.

目的 对多种分子分型方法在单核细胞增生李斯特氏菌(Listeria monocytogenes,Lm)分型中的应用进行分析和比较,确定适合于不同实验室的分型检测方法,并进一步掌握食源性Lm的病原特征.方法 采用分子血清分型、二元基因分型与多位点序列分型方法对分离自食品的69株Lm进行分型检测,并对结果进行分析.结果 69株Lm食品分离株中,1/2a型菌株有29株,1/2b型有19株,1/2c型有7株,4b型有14株.二元基因分型法将69株Lm食品分离株分为19个二元基因型(Binary type,BT型),DI值为91.60％;主要的BT型为BT146、BT254、BT222、BT82、BT159和BT190.多位点序列分型法将69株Lm食品分离株分为16个序列型(Sequencing type,ST型),DI值为89.98％;主要的ST型为ST8、ST87、ST2、ST1、ST3和ST9.结论 分子血清学方法和二元基因分型方法具有操作简便、检测结果易于分析、较快提供检测结果的优点,可作为实验室的一线技术用以对李斯特氏菌病暴发的发现和确认.

Objective To evaluate the fungi contamination of wheat harvested in 2015 in order to provide the basis for fungi and mycotoxin control and study in predictive microbiology.Methods Organoleptic examinations and thousand-grain weight check were conducted for wheat kernel samples collected without storage from five regions of Anhui Prov-ince harvested in 2015.Wheat kernels were sterilized and inoculated onto potato dextrose agar medium plates which contained 0.1 g /L chloramphenicol.The colony-forming units of fungi germinated were enumerated,classified and identified after incubation for 5 d at (28 ±1)℃.Results Varied amounts of gibberella damaged wheat kernels were observed in each sample,the frequencies ranging from 8.0% to 20.0%.The average value of thousand-grain weight ranged from 39.8 to 48.5 g.The frequency of scabby wheat kernels had negative correlation with thousand-grain weight (r =-0.98).The frequency of wheat samples contaminated by fungi was up to 100.0%,and the predominant was Alternaria species.Conclusion The wheat kernels collected from five regions of Anhui Province harvested in 2015 are seriously contaminated by fungi and the frequency of scabby wheat kernels is pretty high.There is a need to focus on the detection of mycotoxins combined with a risk assessment of different mycotoxins in wheat and its products to Chinese population.

Objective To establish a standard pulsed-field gel electrophoresis(PFGE) assay for the detection of Bacillus cereus.Methods Fifty-one B.cereus strains isolated from different source were selected to evaluate the discriminatory power of the established PFGE assay.Results The established PFGE assay for the detection of B.cereus successfully identified the genotypes of 51 strains of B.cereus.Conclusion The PFGE assay was established for the genetic classification of B.cereus which can provide technical support for the control and prevention of food poisoning and nosocomial infection caused by B.cereus.

目的 研究石家庄市食源性蜡样芽胞杆菌毒力基因的分布及毒力活性,了解蜡样芽胞杆菌的潜在威胁.方法 采用PCR方法,对食品风险监测中分离到的131株蜡样芽胞杆菌进行肠毒素、呕吐毒素9种毒力基因扩增检测,用血平板检测的方法分析蜡样芽胞杆菌的毒力.结果 毒力基因携带率较高,至少携带一个毒力基因的菌株达到检出菌总数的99.2％(130/131),溶血素BL基因(hblACD)和肠毒素FM基因(entFM)是石家庄市食源性蜡样芽胞杆菌的主要毒力基因;检出的蜡样芽胞杆菌均产生溶血素BL,检出率为100％.结论 腹泻型肠毒素在食品中的分布比较广泛,检出的蜡样芽胞杆菌均具有溶血素,对进食者存在潜在的危险性,今后应加强监控蜡样芽胞杆菌的污染,预防和控制蜡样芽胞杆菌食源性疾病的发生.

目的 建立针对食品来源的单核细胞增生李斯特氏菌(Listeria monocytogenes,Lm)分离株的多位点串联重复序列分型(Multiple-Locus Variable number tandem repeat Analysis,MLVA)方法,为暴发确认和溯源检测提供实验室支持.方法 对2005-2014年间分离自食品的91株Lm进行14个可变数目串联重复序列(Variable Number of Tandem Repeats,VNTR)位点的检测,评估最优检测位点组合并分析检测结果.结果 通过采用软件分析,由LMV1、LMV2、LMV7、Lm10、Lm11、Lm23、LM-TR6、TR3和Lm15等9个VNTR位点组成的位点组合为最优MLVA检测位点,可以将91株Lm分离株分为70个型别,分型能力达到0.987 1.结论 本研究建立的基于全自动毛细管电泳的由9个检测位点组成的Lm的MLVA分型方法,具有操作简便、快速、结果客观、操作标准化、易于在不同实验室间比较的优势,可作为一线检测方法用于李斯特菌病的暴发确认和溯源检测.

目的 了解目前市售婴幼儿配方奶粉、谷基辅助食品样品中蜡样芽胞杆菌的污染及其毒素基因的携带情况.方法 采集石家庄市23个区县市售婴幼儿配方粉、谷基辅助食品共399份,依照国家标准GB/T 4789.14-2003《食品卫生微生物学检验蜡样芽胞杆菌检验》和《食源性致病菌监测工作手册》进行蜡样芽胞杆菌检测并计数,应用荧光PCR方法检测蜡样分离株的溶血素基因和非溶血素基因.结果 399份样品中蜡样芽胞杆菌检出85份,其中婴幼儿配方奶粉检出36份,检出率22.8％ (36/158);谷基辅助食品检出49份,检出率20.3％(49/241).85份阳性样品中有48份呈溶血素基因阳性,检出率56.5％,非溶血素基因均为阴性.结论 婴幼儿食品中蜡样芽胞杆菌的污染较严重,存在潜在的食品风险.分析结果可为婴幼儿食品卫生学检验标准及监督管理等方面提供参考.

Objective To study the gene characteristics and serotype of adenovirus strains in an outbreak through a municipal swimming pool.Methods The serotype of sixteen adenovirus positive samples detected by using respiratory virus multiple PCR joint inspection kit were tested with type specific primers of adenovirus type 3,7 and 21.And the completely hexon genes and fiber genes of three adenovirus positive specimens were amplified by polymerase chain reaction(PCR) and completely sequence determination was conducted.Results All the sixteen adenovirus positive samples were human adenovirus type 7.The three adenovirus serotype 7 isolates had the same hexon gene and fiber gene sequence,the nucleic acid homology was 100%.Compared with the adenovirus serotype 7 outbreak in Shaanxi province,China,which associated with a severe lower respiratory tracted disease in infants,the three adenovirus positive samples and Shaanxi adenovirus strain shared the same sequence,and no significant genetic variations were found.Conclusion The viral pathogen related to the adenovirus in the outbreak through municipal swimming pool is the type 7.Sequences of the hexon gene and fiber gene are similar to the adenovirus serotype 7 strain in the outbreak in Shaanxi province,none variation is found.