食源性产志贺毒素大肠杆菌的分离及菌株特征分析

了解不同食品中产志贺毒素大肠杆菌的流行情况、菌株特征及潜在致病性。方法 对我国不同地区采集的355份食品样品进行产志贺毒素大肠杆菌分离鉴定,对菌株进行stx1/stx2基因分型、eae等毒力基因检测,并对菌株进行多位点序列分型(MLST)分析。结果 355份样品中44份stx2基因阳性,共分离出11株非O157 产志贺毒素大肠杆菌,其中3株携带stx2a亚型,3株携带stx2e亚型,1株携带stx2b亚型,4株不能分型;5株携带ehxA、saa毒力基因,2株携带subA基因,1株携带katP基因;MLST将11株菌分为7个不同的ST型,存在与溶血性尿毒综合症患者肠出血性大肠杆菌分离株(HUS-associated enterohemorrhagic E.coli,HUSEC)及主要流行血清群产志贺毒素大肠杆菌亲缘关系较近的ST型别。结论 我国食品中存在一定程度的非O157产志贺毒素大肠杆菌污染,部分菌株具有潜在致病性,应加强对食品中STEC的监测。     

多位点序列分析方法在克罗诺杆菌属菌株溯源分析上的应用

克罗诺杆菌(Cronobacter)是一种急性细菌病原体,最初因与新生儿重症监护室爆发的几起致死性疾病(脑膜炎,坏死性小肠结肠炎)相关而受到人们广泛关注。目前Cronobacter PubMLST基因组和序列定义数据库(http://pubmlst.org/cronobacter/)中已包含超过2400多株克罗诺杆菌分离菌株的序列信息,阪崎克罗诺杆菌1 774株,丙二酸盐阳性克罗诺杆菌295株是其中的优势菌种,这些菌株共分离自40多个国家和地区。通过将7个位点的多位点序列分型(7-loci multilocus sequence typing,7-loci MLST)方案应用于2438株克诺罗菌株,共揭示了591种可定义的序列型(sequence type,ST),其中ST4(334株)、ST1(280株)、ST7(78株)和ST13(67株)为主要序列型。7-loci MLST对于克罗诺杆菌的致病型分型鉴定有很大帮助,但由于MLST所针对的七个基因位点在基因组总量中占比较小,导致同一ST型中包含地理来源,分离时间,宿主等不相关的菌株,而对菌株溯源结果适得其反。为了解决这一问题,本研究进一步采用基于直系同源基因簇的多位点序列分析方法(Clusters of Orthologous Genes MLST, cogMLST(1865-loci)),对PubMLST中240株已完成全基因组测序的菌株,包括阪崎克罗诺杆菌ST1(67株),ST4(95株),ST13(43株)和丙二酸盐阳性克罗诺杆菌ST7(35株)进行了全基因组序列分析。结果显示,cogMLST可对ST型相同,但无相关性的菌株进行进一步分型,且不同聚类与菌株分离国家及来源之间存在一定相关性。这或许对于相同ST克罗诺杆菌属菌株的全球溯源分析及食源性疾病监测有较大帮助。     

出入境动物源性食品中单增李斯特菌的多位点序列分型分析

对出入境动物源性食品中分离的单增李斯特菌进行多位点序列分型（mutilocus sequence typing,MLST）分析,了解其序列型分布特点及不同菌株之间的亲缘关系。提取单增李斯特菌基因组DNA,选择其7 个管家基因进行聚合酶链式反应扩增并测序。将测序结果截成标准序列的长度后上传到MLST数据库进行比对分析,获得7 个管家基因的等位基因谱和序列分型编码,并将结果采用不加权算术平均组对（unweighted pair group method usingarithmetic averages,UPGMA）法进行聚类分析。89 株单增李斯特菌共获得51 个STs,其中26 个为新获得的STs（STnew1～STnew26）;数量最多的5 个STs为ST8（9.0%）,ST121（9.0%）、ST7（5.6%）、ST87（5.6%）及新发现的STnew3（7.8%）;其中ST456、ST34、ST343、ST19、ST517、ST201、ST98、ST330和ST73为在国内首次获得。采用UPGMA算法得到的进化树可将89 株菌株分为3 大类群,分类的结果与单增李斯特菌血清学家系分类结果一致。MLST结果对了解出入境动物源性食品中分离的单增李斯特菌的亲缘关系及流行病学溯源有重要意义。     

两种分子分型技术在乳制品克诺罗杆菌污染溯源分析上的比较

对2005-2006年进口婴幼儿配方奶粉等乳制品的原料及产品中分离获得的49株阪崎肠杆菌(Enterobacter sakazakii),通过二代测序技术获得其全基因组序列,并比较两种分子分型技术的优缺点。通过二代测序技术获得49个菌株的全基因组序列,通过fusA确定菌株种属,根据多位点序列分型(Multi-locus Sequence Typing, MLST)确定序列型(ST, Sequence type)。再对32株ST13菌株进行单核苷酸多态性位点分析(single nucleotide polymorphism, SNP),并构建进化树。结果 49株阪崎肠杆菌均确认为阪崎克罗诺杆菌(C.sakazakii)。49株C.sakazakii由8个ST(Sequence type,序列型)组成。ST13的菌株占分离菌株的绝对优势(65.3%, 32/49)。ST13菌株的SNP结果显示32株菌被分成4个组和5个独立的菌株。表明随着高通量测序技术的不断完善,通过全基因组序列的MLST分型方法完成初步分型,而针对遗传相似性很高的菌株,应用SNP分析深入分型,能对被克罗诺杆菌属(Cronobacter spp.)污染的乳粉进行快速溯源,对全球疫情调查有重要意义。     

基于全基因组测序的阪崎克罗诺杆菌婴幼儿食品分离株分子特征研究

目的 研究婴幼儿食品中分离的阪崎克罗诺杆菌(C.sakazakii)分子生物学特征。方法 对婴幼儿食品中C.sakazakii进行分离鉴定、药物敏感性试验和全基因组测序,利用BioNumerics软件对全基因组数据进行拼接组装,对组装基因组开展多位点序列分型(MLST)、核心基因组多位点序列分型(cgMLST)、毒力基因和耐药基因分析,并与PubMLST数据库中ST型进行比较分析。结果 本研究中分离的9株C.sakazakii共分为5个ST型,ST1和ST64型为主要型别,同时也是PubMLST数据库中C.sakazakii的主要型别。3株C.sakazakii携带mcr-9耐药基因,但药敏结果显示所有菌株对包括多粘菌素B和多粘菌素E在内的12种抗生素均敏感。除携带共同毒力基因谱外,ST1、ST64和ST458型携带脂多糖基因gtrB。cgMLST聚类分析显示,9株C.sakazakii呈高度多样性。结论 与临床分离株相关的ST1和ST64型是本研究食品分离株中的主要型别,提示C.sakazakii具有潜在的致病性,有必要对婴幼儿食品中C.sakazakii开展连续监测,为预防控制由其引起的食源性疾病提供依据。     

鸡肉生产中分离金黄色葡萄球菌的基因分型与耐药性分析

为探究鸡肉制品在加工过程中金黄色葡萄球菌的肠毒素基因分布、分子分型和耐药性情况,本研究对某鸡肉制品加工厂的生产加工过程进行取样,通过DNA提取、PCR扩增nuc基因对金黄色葡萄球菌进行分离鉴定,然后分析肠毒素基因分布情况,对获得带有毒素基因的菌株进行多位点序列分型（MLST）和耐药性研究。结果表明,260份样本中分离到38株金黄色葡萄球菌（其中携带肠毒素基因的菌株有24株）,检出率为63.16%;共检测到7种毒素基因型,其中sed携带率最高（60.53%）,依次为seg（26.32%）、see（21.05%）、sei（18.42%）、sec（10.53%）、sea（7.89%）、seh（5.26%）,携带两种及以上肠毒素基因的菌株有14株（36.84%）。24株携带毒素基因的菌株MLST分型都为ST型,分为3种ST分型,16株为ST7序列型（66.67%）;5株为ST5序列型（20.83%）;3株为ST464序列型（12.5%）。耐药性分析实验结果表明,抑菌效果最好的抗生素是万古霉素,38株菌株都对其敏感（100%）;绝大多数菌株都对抗生素有耐药性,依次为青霉素G（86.84%）、环丙沙星（60.53%）、克林霉素（55.26%）、四环素（52.63%）;对3-6种抗生素耐药的金黄色葡萄球菌菌株分别占18.42%、15.79%、23.68%、7.89%。研究结果表明鸡肉产品中存在金黄色葡萄球菌污染的情况,并且其中存在携带多种类型肠毒素基因的菌株,也存在具有多重耐药性的菌株。     

食源性空肠弯曲菌的分子分型及其遗传多样性分析

为了解在2012~2014年从中国不同城市分离到的食源性空肠弯曲菌的遗传多样性特征,本研究采用多序列位点分型(multilocus sequencing typing,MLST)的方法对分离到的33株食源性空肠弯曲菌进行分子分型。研究中,对33株菌株的管家基因使用7对不同的引物进行PCR扩增,并对扩增的产物使用凝胶电泳鉴定后进行测序。将测序结果同Pub MLST中Campylobacter数据库进行比对,以获得对应菌株ST型,并提交新的ST型数据。利用其MLST数据构建进化树和最小生成树。结果显示33株食源性空肠弯曲菌可以分为27个ST型,其中有14种为新的ST型,可形成11种克隆复合体,优势克隆复合体为CC22和CC45,部分菌株的CC型也曾在临床上发现。共存在91种核苷酸多态性位点,部分等位基因之间存在重组。这表明在2012-2014年间分离得到的菌株具有丰富的遗传多样性,并且有潜在致病风险。     

2014—2018年江西省食源性疾病患者沙门菌分离株MLST分型及流行特征研究

目的 掌握江西省食源性疾病患者沙门菌分离株优势ST（MLST）型及其时空分布特征、流行趋势，分析血清型与ST型的对应关系。方法 以分离自2014—2018年江西省食源性疾病患者的313株沙门菌为研究对象，采用多位点序列分型（MLST）技术对菌株进行基因分型，探讨ST型别的时空分布特征及其与血清型的对应关系。结果 313株沙门菌共分为39种ST型，占比最高的是ST34（32.59%），其次是ST11（15.97%）、ST19（11.50%）。每年的优势ST型基本为ST34、ST11、ST19，地区间ST型分布存在差异：上饶市ST型种类最多（20种），其次是抚州市（15种）、景德镇市（14种）；南昌市优势ST型别为ST11、新余和鹰潭市优势ST型为ST19，而其他地市的优势ST型均为ST34。313株沙门菌共分为30个血清型，优势血清型为4，［5］，12：i：-（30.35%）、肠炎沙门菌（15.97%）、鼠伤寒沙门菌（14.06%）。结论 江西省食源性疾病患者沙门菌分离株的ST型别较多，年度流行ST型以ST34、ST11、ST19为主。这三种优势ST型在江西省不同地区间均有分布，但ST型在时空分布上也存在地区差异。     

即食食品中阪崎克罗诺杆菌的分离鉴定及分子分型

为了解即食食品中污染阪崎克罗诺杆菌的流行情况,分析我国阪崎克罗诺杆菌主要流行菌株,对即食食品中分离的10株阪崎克罗诺杆菌进行基质辅助激光解析/电离飞行时间质谱(MALDI-TOF-MS)、VITEK 2、16s rDNA鉴定,采用多位点序列分型(MLST)方法对分离菌株进行分型,并对PubMLST数据库中322株中国分...     

基于全基因组测序的蜡样芽孢杆菌食品分离株分子特征及耐药性研究

目的 研究上海市食品中分离的蜡样芽孢杆菌(Bacillus cereus)的基因组特征及其耐药性。方法 本研究对上海市食品中分离的蜡样芽孢杆菌进行鉴定、药物敏感性实验和全基因组测序,利用BioNumerics软件对测序数据进行拼接组装,利用拼接数据进行多位点序列分型(MLST)、毒力基因和耐药基因分析,并与PubMLST数据库中的ST型进行比较;对耐药基因和耐药表型进行比较分析。结果 本研究中37株蜡样芽孢杆菌均可分型,其中7株为ST新型。37株蜡样芽孢杆菌分为34个ST型,其中3株菌为ST26型,其余ST型各占1株,呈高度多样性。37株蜡样芽孢杆菌均携带nheA、nheB、nheC基因,hblACD基因簇的携带率为37.8%,而完整ces呕吐基因簇的携带率只有16.2%,其中2株为分离自即食食品的ST26型。药敏实验显示37株蜡样芽孢杆菌对氨苄西林、青霉素和苯唑西林耐药性较高,与其携带耐药基因种类并不完全一致。结论 上海市食品中分离的蜡样芽孢杆菌菌株分子分型呈现高度多样性,应加强食品中蜡样芽孢杆菌分子生物学监测,为预防控制由其引起的食源性疾病提供科学依据。     

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.     

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.     

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.     

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.     

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.