一起空肠弯曲菌引起食源性疾病事件的病原学研究及溯源分析

目的 对一起食源性疾病事件进行病原菌检测，了解病原菌毒力基因携带情况并进行溯源分析。方法 对事件采集的样本经FilmArray多重PCR系统进行快速初筛，同时进行细菌分离培养鉴定。使用PCR检测技术对分离菌株进行毒力基因检测，采用16S rRNA基因序列分析与PFGE分型方法对分离菌株进行同源性分析。结果 2份患者肛拭子样本和4份食堂厨工肛拭子样本检出空肠弯曲菌，检出菌株均携带flaA、cadF、imaA、cdtA、cdtB、cdtC等毒力基因。16S rRNA基因序列分析表明6株分离菌株均为空肠弯曲菌，1株菌株与其他5株菌株分子发育距离稍远。6株菌株经PFGE分型可分为3种带型，3株菌和2株菌分别呈现同一带型，2种带型相似性为52.2%；另1株菌为另一带型，与其他菌株带型相似性仅为26.7%。结论 实验室结果表明这是一起由不同克隆株的空肠弯曲菌感染引起的食源性疾病事件。     

实时荧光PCR检测弯曲菌感染病例的粪便和肛拭子样本

目的 通过对3起弯曲菌暴发事件中粪便和肛拭子样本采用实时荧光PCR和培养法检测结果的比较,为实验室快速有效地应对此类事件奠定基础。方法 收集3起弯曲菌感染暴发事件中病例的生物样本;使用过滤培养法进行弯曲菌培养检测,分别使用原始样本、增菌24 h和增菌48 h样本提取DNA进行弯曲菌实时荧光PCR检测;使用Kappa检验对实时荧光PCR检测结果和培养法结果进行一致性分析。结果 原始样本、增菌24 h和增菌48 h实时荧光PCR检测灵敏度分别为90.91%、97.22%和100%,特异度分别为75.00%、84.00%和78.95%,与培养法结果一致性分析的Kappa值分别为0.643、0.813和0.785。结论 实时荧光PCR检测与培养法结合使用是弯曲菌暴发事件处置的有效实验室检测方法。     

北京市通州区食源性疾病监测病例中副溶血弧菌的病原特征分析

目的 了解通州区2015—2021年食源性疾病监测病例中副溶血弧菌的脉冲场凝胶电泳（PFGE）优势带型、耐药情况和毒力基因携带情况,为副溶血弧菌感染防控和治疗提供参考数据。方法 对2015—2021年通州区定点监测医院门诊腹泻病例的粪便样本进行副溶血弧菌分离培养,对其进行血清分型、毒力基因检测、PFGE聚类分析及药敏试验。结果 2 828份粪便标本中检出副溶血弧菌100株,检出率为3.54%,每年7~9月是检出高峰月份。不同年份副溶血弧菌检出率差异具有统计学意义（χ²=53.94,P<0.001）。100株副溶血弧菌中有一株未携带tlh基因,89.00%的菌株携带致病性毒力基因tdh。副溶血弧菌优势血清型为O3∶K6（66/100）,其次是O4∶K8（9/100）。98株菌副溶血弧菌（2株降解）PFGE分型结果显示副溶血性弧菌有39个PFGE带型,命名为V1-V39,条带相似度在79.6%~100%之间,基因分布呈高度多态性,V22和V25是通州区副溶血弧菌的优势带型。菌株对头孢唑林的耐药率最高（32.00%）,其次是氨苄西林（14.00%）和多黏菌素E（13.00%）,对四环素类、氯霉素类、氨基糖苷类、碳青霉烯类四类药物100%敏感。结论 通州区2015—2021年食源性疾病监测病例中检出的副溶血弧菌主要是O3∶K6型tdh⁺- trh^-菌株,对头孢唑林,氨苄西林和多黏菌素E耐药。PFGE图谱主要流行菌株带型相似度在93.1%以上,存在暴发风险,食品安全相关部门需做好副溶血弧菌监测及暴发预警工作,预防食源性疾病暴发。     

鸡源大肠埃希氏菌mcr-1基因携带及分析

目的 了解在农业农村部禁止使用多黏菌素作为动物促生长使用后四川部分地区鸡源大肠埃希氏菌（E.coli）mcr-1基因的携带情况,为制定进一步防控措施提供依据。方法 采集四川部分地区市场售卖点肉鸡直肠拭子,用含有多黏菌素（终浓度4 μg/mL）的EC肉汤增菌接种含多黏菌素（终浓度4 μg/mL）的麦康凯平板,挑取可疑菌落,采用PCR方法鉴定菌株并检测mcr-1基因;微量肉汤稀释法测定mcr-1基因阳性菌株对临床常见抗菌药物耐药情况。脉冲场凝胶电泳（PFGE）对mcr-1基因阳性菌株进行同源分析。耐药基因质粒结合实验验证mcr-1基因传播途径。结果 从70份肉鸡样本中的13份检出mcr-1基因阳性大肠埃希氏菌,检出率18.57%（13/70）,对实验的13种抗生素,除13株mcr-1阳性菌株对头孢西丁有12株敏感以外,对其他抗生素都表现出不同程度的耐药,其中四环素和甲氧苄啶/磺胺甲恶唑耐药率最高,达到了100%（13/13）;其次是氨苄西林和氯霉素,耐药率为84.62%（11/13）。PFGE显示13株mcr-1阳性大肠埃希氏菌分属13个不同的型别;质粒结合实验显示mcr-1基因能够通过质粒传播。结论 mcr-1基因在鸡大肠内大肠杆菌中检测率比较高,且鸡大肠中mcr-1阳性大肠埃希氏菌的耐药情况比较严重。     

一起肠炎沙门菌引起的校园食源性疾病暴发事件调查溯源分析

目的 调查一起肠炎沙门菌引起的学校食源性疾病暴发事件,并对危险因素开展分析溯源,为预防类似事件的发生提供科学依据。方法 运用描述性流行病学方法分析事件的流行病学特征;运用病例对照研究调查可疑食物;通过环境卫生学方法调查追溯食品污染的过程;对病例、食物、环境样品进行病原菌分离、血清分型和脉冲场凝胶电泳（PFGE）检测。结果 本起暴发事件共报告病例71例,流行曲线符合持续同源暴发模式的特点。病例对照、环境卫生调查和病因溯源研究提示学校超市售卖的三明治是引起本次事件的危险食品（OR=302.09,95%CI=75.18~1 213.97）。采集的病例、食品、环境样品中有8份（12.70%,8/63）检出了肠炎沙门菌,分离菌株的PFGE图谱条带完全一致,证实了学校超市出售的三明治使用的沙拉酱在制作过程中被肠炎沙门菌污染,是导致这次食源性疾病暴发的原因。结论 本次事件是由肠炎沙门菌污染食品引起的食源性疾病暴发,可疑食品为被自制沙拉酱污染的三明治。建议禁止各类未经充分热处理的含生鲜鸡蛋成分的食品进入学校,相关行政部门应加强对该类食品的卫生监督。     

一起疑似产气荚膜梭菌食物中毒事件的病原学分析

目的 分析一起疑似产气荚膜梭菌食物中毒事件的病原学。方法 利用荧光定量PCR方法对一起食物中毒患者的粪便进行产气荚膜梭菌基因的初筛, 根据荧光定量PCR初筛的提示结果进行细菌的分离培养, 对分离到的菌株进行质谱和生化鉴定、毒力基因检测和PFGE分析。结果 从5份食物中毒患者粪便中分离到5株携带肠毒素基因cpe的A型产气荚膜梭菌, 并且这5株菌的PFGE带型完全一致。结论 此次食物中毒致病因子为携带肠毒素基因cpe的A型产气荚膜梭菌, PFGE结果提示5株菌可能有同一来源。     

一起阿邦尼沙门菌引起食源性疾病暴发事件流行病学分析及分子溯源调查

目的 查明引起某庙会期间发生的一起食源性疾病暴发事件的原因，确认致病危害因素及其来源，为此类事件预防控制提供参考。方法 开展现场流行病学调查，通过描述分析方法分析病例临床特征、流行病学特征及相关危害因素。开展病例对照研究确定可疑食物，通过脉冲场凝胶电泳（PFGE）法对致病因子进行相似性分析。结果 根据病例定义共收集可疑病例32例，主要临床表现为腹痛（87.50%）、腹泻（78.13%）、发热（75.00%）、头晕恶心（71.88%）。病例对照研究结果显示水煮带壳花生是危险因素（OR=4.000，95%CI：1.409~11.354）。采集28份样品中有7份分离培养出沙门菌，血清型鉴定均为阿邦尼沙门菌，经PFGE图谱分析高度相似。结论 本次事件是由阿邦尼沙门菌引起的食源性疾病暴发，可疑食物为水煮带壳花生，应加强非城区流动摊位散装食品的监管，同时加强食源性疾病监测管理工作。     

2021年新乡市一起山夫登堡沙门菌食物中毒分离株的病原特征分析

目的 分析2021年新乡市一起山夫登堡沙门菌食物中毒分离株的病原特征。方法 对食物中毒事件进行流行病学调查;对采集的11份样本进行致病菌分离鉴定;对分离出的10株沙门菌进行血清学分型、药敏试验、5种毒力岛（SPIs）特征基因片段（mogA、sseL、mgtC、bcfA、araB）检测及脉冲场凝胶电泳（PFGE）分型分析。结果 10株沙门菌血清抗原式均为1,3,19;g,s,t,即山夫登堡沙门菌。10株沙门菌对头孢唑啉、卡那霉素、庆大霉素、阿米卡星的耐药率为100%,其中从患者粪便中分离出的2株对氨苄西林、四环素、多西环素、氯霉素、复方新诺明的耐药率为100%。PFGE图谱聚类分析显示,10株菌（2株病例株,5株食品株,3株环境株）之间条带无差异,高度同源。10株菌中5种毒力岛特征基因片段均检出。结论 本起食物中毒事件致病因子为山夫登堡沙门菌,该菌携带5种毒力岛特征基因片段,2株病例株沙门菌具有多重耐药性,建议相关部门高度关注。     

一起多所幼儿园食源性疾病暴发事件的溯源分析

目的 对一起涉及多所幼儿园的食源性疾病暴发事件的可疑食品和致病因子进行溯源调查,为今后类似事件的防控和处置提供参考依据。方法 采用实时荧光PCR法、质谱技术等快速检测技术,结合分离培养、酶联免疫法（ELISA）等传统鉴定方法对3所幼儿园采集的32份样品开展病原检测。使用描述性流行病学调查方法对事件进行调查,脉冲场凝胶电泳（PFGE）方法对致病因子进行分子溯源分析。结果 3所幼儿园共有幼儿568名,62名病例,患病率为10.92%。临床症状主要为呕吐、腹泻等。从10份生物标本和某配餐公司统一配送的2份留存的拔丝肉松蛋糕检出金黄色葡萄球菌,蛋糕中金黄色葡萄球菌量分别为2.0×10⁷ CFU/g、1.4×10⁷ CFU/g,11株分离自病例和蛋糕的金黄色葡萄球菌同时检出葡萄球菌A型肠毒素基因（sea）和A型肠毒素（SEA）。PFGE指纹图谱为同一带型,提示病例和食品的分离株为同一来源。结论 本起暴发事件是由配餐公司统一配送的拔丝肉松蛋糕污染金黄色葡萄球菌产生肠毒素导致的3所幼儿园的食物中毒,应进一步加强对学校配餐食材的监管。     

2016—2021年无锡市副溶血性弧菌毒力基因和耐药性分析及分子分型研究

目的 分析2016—2021年无锡市不同来源副溶血性弧菌的毒力基因、耐药性和分子分型结果。方法 采用多重荧光PCR、微量肉汤稀释法、脉冲场凝胶电泳（PFGE）分别对204株分离自无锡市各类监测样本中的副溶血性弧菌进行tlh/tdh/trh毒力基因检测、耐药试验和分子分型。数据比较采用χ²检验。结果 204株菌tlh基因携带率为100%（204/204），tdh基因携带率为82.35%（168/204），trh基因携带率为2.45%（5/204），食品及环境分离株与病患分离株tdh基因携带率差异具有统计学意义（P<0.001）。菌株对头孢唑啉耐药率最高达96.08%（196/204），对3种及以上抗菌药物的耐药率为2.94%（6/204），食品及环境分离株与病患分离株对氨苄西林、四环素、磺胺甲??唑/甲氧苄啶、环丙沙星耐药率差异具统计学意义（P<0.05）；204株副溶血性弧菌经过聚类分析，分为123个PFGE带型，相似度49.1%~100.0%，按85%的相似度聚类可分为18个带型簇。结论 无锡市副溶血性弧菌病患分离株大部分携带tdh基因；菌株对头孢唑啉耐药率最高；PFGE型别呈多态性，优势带型不明显。     

Arsenic content of some edible mushroom species

The arsenic contents of 162 fruit body samples of 37 common edible mushroom taxa were analyzed. The samples were gathered from different habitats of Hungary (mainly from mountains) between 1984 and 1999. The arsenic content of the samples was measured by the inductively coupled plasma spectrometry method. Very low [lower than 0.05 mg/kg dry matter (DM)] concentrations were found in the samples of 13 taxa, while higher (or very high) contents were quantified in other common taxa (the highest arsenic content was recorded in the fruit body of Laccaria amethysthea at 146.9 mg/kg DM). The species of eight genera (Agaricus, Calvatia, Collybia, Laccaria, Langermannia, Lepista, Lycoperdon, Macrolepiota) belong to the so-called accumulating taxa, and this tendency is evident on all habitats. This arsenic accumulation capability is found in two orders of Basidiomycetes (Agaricales and Gasteromycetales), which is to say this phenomenon occurs in the families Agaricaceae, Tricholomataceae and Gasteromycetaceae. The accumulating taxa found all have a saprotrophic type of nutrition; arsenic accumulation is not detectable in xilophagous or in mycorrhizal species. The consumption of the accumulating species found has only a low toxicological risk for three reasons: the consumed fresh fruit bodies contain about a tenfold lower arsenic level than the dried ones, the majority of arsenic occurs not in poisonous inorganic, but in less dangerous (or not poisonous) organic forms, and the frequency of consumption is low.     

Organization and localization of the dnaA and dnaK gene regions on the multichromosomal genome of Burkholderia multivorans ATCC 17616

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To reveal the distribution and organization of the genes for fundamental cell functions on the genome of this bacterium, the dnaA and dnaK gene regions of ATCC 17616 were cloned and characterized. The gene organization of the dnaA region was rnpA-rmpH-dnaA-dnaN-gyrB with a single consensus DnaA-binding box (TTATCCACA) between the rmpH and dnaA genes. This intergenic region, however, did not work as an autonomously replicating sequence in ATCC 17616. On the other hand, the gene organization of the dnaK region was grpE-orf1 (gene for thioredoxin homologue)-dnaK-dnaJ-pabB (gene for p-aminobenzoate synthetase component homologue). A putative heat-shock promoter that showed good homology to the sigma32-dependent promoter consensus sequence in Escherichia coli was found upstream of the grpE gene, suggesting that these five genes constitute an operon. In M9 succinate minimal medium the dnaJ mutant grew more slowly than the wild-type strain, indicating that this operon is functional. Pulsed-field gel electrophoresis and Southern blot analyses indicated that both the dnaA and dnaK gene regions exist as single copies on the 3.4 Mb chromosome.     

Microbial profiles of frozen trimmings and silver sides prepared at Indian buffalo meat packing plants

To assess microbiological quality of buffalo meat trimmings (TT = 114) and silver sides (SS = 41), samples were collected from four different Indian meat packing plants. The aim of this study was: (i) to evaluate standard plate count (SPC), psychrotrophic count (PTC), Enterococcus feacalis count (EFC), Staphylococcus aureus count (SAC) and Escherichia coli count (ECC) and the presence of Salmonella spp. and Listeria monocytogenes; and (ii) also to determine vero toxic E. coli (VTEC) by polymerase chain reaction (PCR). TT samples had significantly higher (P < 0.001) SPC, PTC, EFC, and SAC than SS, while across the meat types there was no difference (P > 0.05) in ECC. E. coli was recovered from 32.4% TT and 19.5% SS samples. The prevalence rate of Salmonella spp. and L. monocytogenes in TT was 1.75% and 0.87%, respectively. But no SS sample was found to be positive for any of these two pathogens. VTEC was found in 2.58% of all the tested samples. This finding suggests that TT contain higher microbes but only small numbers of pathogens of latent zoonotic importance. The present study confirmed the importance of maintaining good process hygiene at meat plants for microbiological status of buffalo meat.     

Susceptibility of stored product insects to high concentrations of ozone at different exposure intervals

Ozone is a highly reactive gas with insecticidal activity. Past studies have indicated that ozone technology has potential as a management tool to control insect pests in bulk grain storage facilities. The objective of this study was to determine the efficacy of short periods of exposure to high ozone concentrations to kill all life stages of red flour beetle (Tribolium castaneum (Herbst)) (Coleoptera: Tenebrionidae), and Indianmeal moth (Plodia interpunctella (Hübner)) (Lepidoptera: Pyralidae), adult maize weevil (Sitophilus zeamais (Motsch.)) (Coleoptera: Curculionidae) and adult rice weevil (S. oryzae (L)) (Coleoptera: Curculionidae). Insects were treated with six ozone concentrations between 50 and 1800 ppm. The specific objective was to determine minimal time needed to attain 100% mortality. The most ozone-tolerant stages of T. castaneum were pupae and eggs, which required a treatment of 180 min at 1800 ppm ozone to reach 100% mortality. Eggs of P. interpunctella also required 180 min at 1800 ppm ozone to reach 100% mortality. Ozone treatments of 1800 ppm for 120 min and 1800 ppm for 60 min were required to kill all adult S. zeamais and adult S. oryzae, respectively. The results indicate that high ozone concentrations reduce the treatment times significantly over previously described results. Our results also provide new baseline information about insect tolerance to ozone treatment.     

Effect of Zataria multiflora Boiss. essential oil and nisin on Salmonella typhimurium and Staphylococcus aureus in a food model system and on the bacterial cell membranes

The effects of different concentrations of Zataria multiflora Boiss. essential oil (EO: 0, 5, 15 and 30 μl 100 ml⁻¹) and nisin (N: 0, 0.25 and 0.5 μg ml⁻¹), temperatures (T: 25 and 8 °C), and storage times (up to 21 days) on growth of Salmonella typhimurium and Staphylococcus aureus in a commercial barley soup were evaluated in a factorial design study. The growth of S. typhimurium was significantly (P < 0.05) decreased by EO concentrations and their combinations with N concentrations at 8 °C. For S. aureus, the viable count was significantly (P < 0.05) inhibited by EO and N concentrations and their combinations, incubated at both storage temperatures. The mechanism of the antimicrobial action of EO, N, and their combinations against cell membranes of the tested organisms were also studied by measurement of the release of cell constituents and by the electronic microscopy observations of the cells. The significant increase of the cell constituents’ release of both organisms was observed as a result of treatments with EO and EO in combination with N. Electronic microscopy observations revealed that the cell membranes of S. typhimurium treated by EO and EO in combination with N were significantly damaged, while cells treated with only N looked similar to untreated cells. The electron micrographs of treated cells of S. aureus with EO, N, and their combination also showed important morphological damages and disrupted membranes.     

Contamination with storage fungi of human food from Cameroon

In a mycological study, a total of 95 human food samples were investigated to evaluate the incidence of fungal contamination in Cameroon by conventional identification method and partly confirmed by DNA sequencing. The isolated fungal spp. were further studied to determine their toxigenic potentials. The investigation revealed the predominance of Aspergillus and Penicillium with 96% of samples contaminated with at least one species of these fungi, whereas the incidence of co-contamination of samples was 85%. Aspergillus flavus and Aspergillus parasiticus (Flavi section) were the most predominant species contaminating mainly maize and peanuts. In addition, P. crustosum and P. polonicum were the most common contaminants belonging to the genus Penicillium. On the other hand, A. ochraceus (Circumdati section) registered a low incidence rate of 5%, including other members of the Aspergillus group. Other members of the genera Rhizopus and Alternaria spp. were also registered in the study. A majority of fungal strains of A. ochraceus, A. parasiticus, P. crustosum and P. polonicum isolated were toxigenic, producing the mycotoxins tested for, while none was detected in cultures of A. fumigatus. The high incidence rate of fungi contamination coupled with their potentials in producing mycotoxins gives a strong indication that the samples tested may likely be contaminated with various mycotoxins. There is need for further study to assess the incidence of mycotoxins contamination in similar food samples.     

Cloning and expression analysis of two catalase genes from Aspergillus oryzae

Fungi contain distinct genes encoding the same class of enzyme that are differentially regulated according to conditions. We cloned two catalase genes, catA and catB, from Aspergillus oryzae. The catA gene predicts a 747-amino-acid polypeptide sharing 81% identity with Aspergillus fumigatus catalase (catA) and 77% with Aspergillus nidulans catalase (catA). The catB gene predicts a 725-amino-acid polypeptide sharing 82% identity with A. fumigatus catalase (catB) and 75% with A. nidulans catalase (catB). However, the catA and catB genes share little homology (41%) with one another, suggesting that each gene belongs to a distinct gene family. Overexpression studies demonstrated that both genes encode a functional catalase. Promoter assays indicated that the catA gene is developmentally regulated as it was preferentially expressed in solid-state cultures undergoing sporulation. However, its expression was not affected by hydrogen peroxide treatment. Conversely, the catB gene was highly expressed under all culture conditions tested, and it was induced by hydrogen peroxide treatment. These results suggest that the catB gene may be mainly used for detoxification of oxidative stress while the catA gene may have another role such as chaperoning proteins in the spore.     

Formation of cereulide and enterotoxins by Bacillus cereus in fermented African locust beans

Afitin, iru and sonru are three spontaneously fermented African locust bean Benin condiments. The fermentation processes are exothermic, with temperatures mostly being above 40 °C. A total of 19 predominant Bacillus cereus isolates from afitin, iru and sonru, were investigated. The enterotoxin genes nhe (A, B, C) were present in all 19 isolates, the hbl (A, C, D) in one (afitin), and the cytK gene in three isolates (afitin). Levels of cytotoxicity to Vero cells and NheA production in BHI-broth was within the range of known diarrheal outbreak strains. Autoclaved cooked African locust beans inoculated with emetic (cereulide producing) B. cereus Ba18H2/RIF supported growth at 25, 30 and 40 °C with highly different maximum cereulide productions of 6 ± 5, 97 ± 3 and 0.04 ± 0.02 μg/g beans, respectively (48 h). For non-autoclaved cooked beans inoculated with 2, 4 and 6 log₁₀B. cereus Ba18H2/RIF spores/g beans, cereulide production was 5 ± 4, 64 ± 8 and 69 ± 34 μg/g beans, respectively at 24 h, while it was 70 ± 43, 92 ± 53 and 99 ± 31 μg/g at 48 h of fermentation at 30 °C. Even though high toxin levels were observed, to date there are no known reports on diarrhea or vomiting due to the consumption or afitin, iru and sonru in Benin, which also according to the present study is likely to be expected from the low levels of cereulide produced at 40 °C.