红河州食源性蜡样芽胞杆菌毒力基因研究

目的了解红河州食源性蜡样芽胞杆菌毒力基因的携带情况,为食源性食物中毒的防治提供参考依据。方法采用PCR方法对2011年1月至2014年9月红河州收集到的31株食源性蜡样芽胞杆菌10种毒力基因进行检测。结果分离菌株普遍携带肠毒素毒力基因并且所有菌株均至少携带一种肠毒素基因,而呕吐型毒力基因只有一株菌携带。结论红河州食源性蜡样芽胞杆菌基因携带以腹泻型为主,且携带率较高,对食品安全和公共健康存在潜在威胁。     

我国食源性蜡样芽孢杆菌毒力基因和药物敏感性研究

目的 了解我国不同地区食源性蜡样芽孢杆菌的毒力基因携带特点及其对抗生素的敏感性,为食源性食物中毒的防治提供参考依据.方法 采用PCR方法对2011年我国不同地区收集的238株食源性蜡样芽孢杆菌10种毒力基因进行检测;采用微量肉汤稀释法测定其抗生素敏感性.结果 溶血素BL基因、肠毒素T基因和细孢毒素K基因是我国食源性蜡样芽孢杆菌的主要毒力基因,至少携带一个毒力基因的菌株达到检出菌总数的87.4％;蜡样芽孢杆菌对庆大霉素、万古霉素、环丙沙星、复方新诺明的敏感率为100％,对红霉素、四环素、氯霉素、克林霉素的敏感率分别为88.8％、90.2％、99.6％、87.1％,对氨苄西林和头孢噻肟的敏感率仅为0.4％和5.4％.结论 我国食源性蜡样芽孢杆菌毒力基因携带率较高,对食品安全和公共健康构成潜在的威胁;蜡样芽孢杆菌对氨苄西林、头孢噻肟的敏感性差,不应作为经验用药和预防用药.     

食源性蜡样芽孢杆菌的危害及其检测方法研究进展

蜡样芽孢杆菌是一种常见的食源性致病菌,可通过污染乳制品、米饭、散装熟肉制品和豆制品等食品引起婴幼儿及成人食物中毒。采用准确、高效的蜡样芽孢杆菌检测方法,是预防食源性蜡样芽孢杆菌病及食品安全质量控制的关键。蜡样芽孢杆菌检测方法主要包括细菌培养分离鉴定法、免疫学检测方法和核酸检测方法等。本文总结了各类检测方法的核心技术特征和应用实例,为食源性蜡样芽孢杆菌的快速检测方法的研发和使用提供思路。     

RE-PCR variability and toxigenic profile of food poisoning, foodborne and soil-associated Bacillus cereus isolates from Brazil

Twenty-three Bacillus cereus isolates from food poisoning outbreaks associated with a diarrheal-type syndrome, fourteen foodborne isolates not associated with food poisoning and fifteen isolates from Brazilian soil samples were analyzed for the presence and genetic diversity (by RE-PCR) of the virulence genes ces (emetic toxin, cereulide), plcR-papR (pleiotropic regulator PlcR and peptide PapR), nheA (a component of the NHE complex), bceT (diarrheal enterotoxin bc-D-ENT), gyrB (B subunit of DNA gyrase), cytK-2 (necrotic enterotoxin cytotoxin K-2), and plcA (phosphatidylcholine-specific phospholipase C). Additionally, these isolates were phenotypically characterized for motility, hemolytic and lecithinase activities, as well as HBL enterotoxin production. The group of isolates associated with food poisoning had the highest occurrence of the phenotypically analyzed factors and the most frequent occurrence and highest genetic diversity of the plcR-papR, nheA, bceT, cytK-2, plcA, and gyrB genes. An analysis of molecular variance (AMOVA), in which all loci were analyzed, demonstrated that the genetic variation intragroup of isolates (92%) was significantly higher than that intergroup (8%) (P < 0.05). These results were corroborated by an analysis of the genetic differentiation between the groups, which was low/moderate, the result of a high degree of allele sharing. Our results suggest that B. cereus isolates with the potential to cause food poisoning outbreaks do not have a specific genetic profile characterized by the presence of a particular gene or allele among the genes assessed. On the contrary, different combinations of genes encoding virulence factors may be present in different isolates of B. cereus that potentially cause food poisoning outbreaks.     

Bacillus cereus,Bacillus thuringiensis and Bacillus mycoides differentiation using a PCR-RE technique

A method was developed to differentiate between Bacillus cereus, Bacillus mycoides and Bacillus thuringiensis using the polymerase chain reaction combined with a restriction endonuclease (PCR-RE) technique. This fast and simple protocol, applied to pure culture strains, was developed using the gyrB DNA sequence, as previously proposed by other authors. Strains from international collections were used to optimize the method which was then applied to the identification of strains isolated from food samples. Amplifications were specific for the B. cereus group. Only Staphylococcus aureus gave the same size PCR product, but it was easily differentiated from strains in the B. cereus group by using restriction analysis, based on digestion with the RsaI, Sau3AI and EcoRI endonucleases. Specific amplifications and good differentiations were obtained using pure strains, suggesting the possibility of using the method described to identify the B. cereus group directly in food samples.     

Effect of dairy product environment on the growth of Bacillus cereus

pH is one of the most important parameters to manage bacterial replication in foodstuffs. In this study, the ability of 2 Bacillus cereus strains, 1 clinical human isolate (GPe2) and 1 isolate from a dairy product (D43), were investigated for in vitro growth at different pH values (from 3.5 to 7.5) at 2 temperatures (15 and 37°C), showing their ability to grow from 5.5 to 7.5 and from 5.0 to 7.5, respectively. The ability of spores of these 2 microorganisms to germinate in different typologies of dairy products (unflavored yogurt, Taleggio cheese, mascarpone cheese, and raw and pasteurized milk) was also investigated by inoculating the spores and maintaining the products at 15°C. No growth was observed in yogurt, likely due to the combined effect of low pH (<5) and the presence of natural microflora. An inhibitory action of the natural microflora on the growth of B. cereus was also hypothesized for Taleggio cheese and raw milk, as these substrates were characterized by a high natural lactic acid bacteria population and permissive pH values (5.8/6.8 in Taleggio cheese, >7 in raw milk). In pasteurized milk and mascarpone cheese, where pH was not restrictive for B. cereus growth and where no significant natural microflora was present, growth occurred rapidly up to loads close to 7 log cfu/g.     

Application of gaseous ozone to control populations of Escherichia coli, Bacillus cereus and Bacillus cereus spores in dried figs

The effect of ozonation as a method to reduce Escherichia coli, Bacillus cereus and Bacillus cereus spores in dried figs was investigated. Dried figs were sprinkle inoculated with E. coli, B. cereus and B. cereus spores in sterile bags at a level of 10(7)microorganism g(-1), mixed and allowed to dry for 1h at 25 degrees C prior to ozonation. Inoculated samples were exposed to gaseous ozone in a chamber at 20 degrees C and 70% relative humidity. Ozone concentrations of 0.1, 0.5 and 1.0 ppm up to 360 min were used to inactivate E. coli and B. cereus while 1.0, 5.0, 7.0 and 9.0 ppm ozone concentrations for 360 min were used to treat B. cereus spores. E. coli and B. cereus counts were decreased by 3.5 log numbers at 1.0 ppm ozone concentration for 360 min ozone treatment. Up to 2 log reductions in the number of B. cereus spores were observed above 1.0 ppm ozone concentration at the end of 360 min of ozonation. No significant changes in color, pH and moisture content values of dried figs were observed after the ozonation treatments. No significant changes were found between sweetness, rancidity, flavor, appearance and overall palatability of ozonated and non-ozonated dried figs. Ozonation was found to be effective especially in reduction of vegetative cells in dried figs and a promising method for the decontamination of dried figs.     

Bacillus cereus, the causative agent of an emetic type of food-borne illness

Bacillus cereus is the causative agent of two distinct forms of gastroenteritic disease connected to food-poisoning. It produces one emesis-causing toxin and three enterotoxins that elicit diarrhea. Due to changing lifestyles and eating habits, B. cereus is responsible for an increasing number of food-borne diseases in the industrial world. In the past, most studies concentrated on the diarrhoeal type of food-borne disease, while less attention has been given to the emetic type of the disease. The toxins involved in the diarrhoeal syndrome are well-known and detection methods are commercially available, whereas diagnostic methods for the emetic type of disease have been limited. Only recently, progress has been made in developing identification methods for emetic B. cereus and its corresponding toxin. We will summarize the data available for the emetic type of the disease and discuss some new insights in emetic strain characteristics, diagnosis, and toxin synthesis.     

Virulence of Bacillus cereus: a multivariate analysis

Biological activity and presence of DNA sequences related to virulence genes were studied in 21 strains of the Bacillus cereus group. The activity of spent culture supernatants and the effect of infection by vegetative bacterial cells were assessed on cultured human enterocytes (Caco-2 cells). The effect of extracellular factors on the detachment, necrosis and mitochondrial dehydrogenase activity of cultured human enterocytes was studied. Hemolytic activity on rabbit red blood cells was also evaluated and the effect of direct procaryotic-eucaryotic interactions was assessed in infection assays with vegetative bacterial cells. Concerning virulence genes, presence of the DNA sequences corresponding to the genes entS, entFM, nhe (A, B and C), sph, hbl (A, B, C and D), piplC and bceT was assessed by PCR. Ribopatterns were determined by an automated riboprinting analysis after digestion of the DNA with EcoRI. Principal component analysis and biplots were used to address the relationship between variables. Results showed a wide range of biological activities: decrease in mitochondrial dehydrogenase activity, necrosis, cell detachment and hemolytic activity. These effects were strain-dependent. Concerning the occurrence of the DNA sequences tested, different patterns were found. In addition, ribotyping showed that strains under study grouped into two main clusters. One of these clusters includes all the strains that were positive for all the DNA sequences tested. Positive and negative correlations between variables under study were evidenced. Interestingly, high detaching strains were positively correlated with the presence of the sequences entS, nheC and sph. Within gene complexes, high correlation was found between sequences of the hbl complex. In contrast, sequences of the nhe complex were not correlated. Some strains clustered together in the biplots. These strains were positive for all the DNA sequences tested and they were able to detach enterocytes upon infection. Our results highlight the multifactorial character of the virulence of the B. cereus group and show the correlation between ribopatterns, occurrence of toxin genes and biological activity of the strains under study.     

一株降解呕吐毒素蜡样芽孢杆菌的筛选与鉴定

目的:分离筛选能够降解呕吐毒素(deoxynivalenol,DON)的芽孢杆菌,以用于该毒素的生物降解。方法:采集霉变秸秆、土壤和粪便样品,先将样品加热80℃后,取上清液接种到以DON为唯一碳源的分离培养基富集DON降解菌。以LB培养基分离纯化富集菌,然后对分离到的菌株进行DON毒素降解能力检测。对降解能力最强的菌株进行形态学观察、生理生化实验和16S r DNA序列鉴定。结果:从16株分离菌中筛选得到一株对DON降解能力最强的菌株B.JG05,降解率最高可达80.61%,且对含DON饲料的降解率为82.68%。该菌株呈短杆状,能形成芽孢;生理生化特性符合蜡样芽孢杆菌的基本特征;16S r DNA序列进化树分析表明该菌株与蜡样芽孢杆菌的亲缘关系最近。结论:筛选获得了一株高效降解DON的蜡样芽孢杆菌B.JG05,为饲料和食品中DON毒素的生物降解提供了可能。     

Antibacterial and anti-adhesive efficiency of Pediococcus acidilactici against foodborne biofilm producer Bacillus cereus attached on different food processing surfaces

Food Science and Biotechnology - This study aimed to assess the biofilm formation by Bacillus cereus on two novel surfaces namely: aluminum and cold steel in comparison study with stainless steel...     

A novel cyanobacteriolytic bacterium, Bacillus cereus, isolated from a Eutrophic Lake

Isolation and screening of cyanobacteriolytic bacteria were carried out. Fifteen strains of cyano-bacteriolytic bacteria were isolated by the double layer method using the cyanobacterium, Microcystis, as a sole nutrient. The isolate, N-14, showing the highest cyanobacteriolytic activity was identified as Bacillus cereus based on the 16S rRNA sequence. Components among the extracellular products in the culture supernatant of B. cereus were responsible for the cyanobacteriolytic activity. Lytic assay tests of culture supernatants indicated that the major substances for lytic activity could be non-proteinaceous, and hydrophilic, heat stable, and with a molecular weight of less than 2 kDa. The highest lytic activity was obtained under alkaline conditions, indicating an advantage for the practical application of water bloom control in eutrophic lakes where the pH is usually in the alkaline region. The lytic substance of B. cereus N-14 were compared with enterotoxins and an emetic toxin produced by a pathogenic strain of B. cereus, and also with a known algicide produced by Bacillus brevis, gramicidin. From these results, the lytic substance seemed to be a novel algicide.     

Germination of Bacillus cereus spores adhered to stainless steel

Adhered spores of Bacillus cereus represent a significant part of the surface-derived contamination in processing equipment used in the dairy industry. As germinated spores lose their resistance capacities instantaneously, efficient germination prior to a cleaning in place treatment could aid to the disinfecting effect of such a treatment. Therefore, spores of B. cereus ATCC 14579 and that of the environmental isolate B. cereus CMCC 3328 were assessed for their germination behaviour when adhered to a stainless steel surface. A mixture of l-alanine and inosine initiated germination of adhered spores efficiently, resulting in 3.2 decimal logarithms of germination. Notably, implementation of a germination-inducing step prior to a representative cleaning in place procedure reduced the number of survivors with over 3 decimal log units, while an alkali treatment alone, as part of the cleaning in place procedure, did not show any effect on B. cereus spore viability. These results show that implementation of a germination step enhances the disinfection effect of currently used cleaning in place procedures.     

Isolation and characterization of an antimicrobial substance from Bacillus subtilis BY08 antagonistic to Bacillus cereus and Listeria monocytogenes

A wild-type bacteria producing an antimicrobial substance was isolated from Korean traditional fermented soybean paste, and identified as Bacillus subtilis. The antimicrobial substance purified by TLC, tentatively named UV254-B, displayed a specific antimicrobial activity against pathogenic Bacillus cereus and Listeria monocytogenes, without inhibiting the growth of soybean-fermenting Bacillus species. The antimicrobial substance was susceptible to proteinase K and lipase but resistant to esterase. Antimicrobial activity was observed over a wide range of pH from 3 to 11, with the maximum activity at pH 9, and thermal stability up to 80°C. The minimum inhibitory concentration of the antimicrobial substance was found to be 64 μg/mL for B. cereus and 128 μg/mL for L. monocytogenes. This antimicrobial substance has a putative molecular weight either at 1,133.6 or 1,700.5, which differs from that of other antimicrobial substances described for B. subtilis such as iturin, surfactin, fengycin, and subtilisin.     

The ability of Schisandra chinensis fruit to inhibit the growth of foodborne pathogenic bacteria and the viability and heat resistance of Bacillus cereus spores

The objective of this study was to investigate the influence of Schisandra chinensis fruit on the growth of spoilage and pathogenic bacteria and on the viability and heat resistance of Bacillus cereus spores. Schisandra chinensis fruit was extracted with one of three different solvents (50% ethanol, 100% ethanol and distilled water), and the extracts exhibited antimicrobial activity against all the bacteria tested. Particularly, the ethanol extracts of S. chinensis fruit had the strongest activity, in a concentration‐dependent manner. Fractionation of extracts by ion chromatography revealed that the antimicrobial activity of S. chinensis fruit is mainly due to organic acids such as citric acid and malic acid. Meanwhile, S. chinensis fruit extract (10%) significantly reduced the viability and heat resistance of B. cereus spores. Therefore, this study suggests that S. chinensis fruit extract has potential as a natural food preservative and/or sanitising agent for the reduction of spoilage and pathogenic contamination.     

蜡样芽孢杆菌烯醇还原酶基因的克隆、表达和酶学性质研究

从蜡样芽孢杆菌WZY004基因组DNA中克隆获得了烯醇还原酶基因,并在大肠杆菌BL21(DE3)中以可溶形式过量表达。重组烯醇还原酶经Ni-NTA亲和层析分离纯化后,对其酶学性质进行了详细表征。该重组酶经SDS-PAGE检测为单一条带,其分子量为38.2 k D。该酶最适p H和温度分别为7.5与60°C。对氧代异佛尔酮的Km和Vmax值分别为1.83 mmol/L与1.13 U/mg。底物特异性分析表明该酶对柠檬醛、2-甲基-2-戊烯醛和反式-2-癸烯醛均有较高活力,其中对柠檬醛的比酶活高达1.33 U/mg。     

蜡样芽胞杆菌生长特性及大蒜精油对其抑菌活性的研究

主要研究了37℃条件下3种蜡样芽胞杆菌在营养肉汤中的生长状况,建立了37℃下蜡样芽胞杆菌在营养肉汤中的Boltzmann牛长模犁,3条牛长曲线相关系数鼯均大于0．97;检测了不同培养时间蜡样芽胞杆菌的产芽胞情况,结果表明1号菌株和14号菌株较早产芽胞,培养相同时间,产芽胞数：1号菌株,4号菌株〉标准菌株;采用牛沣杯法和平板计数法研究了大蒜精油对蜡样芽胞杆菌的抑制效果,结果表明浓度为lO。的人蒜精油对3种蜡样芽胞杆菌都有很好的抑制效果,3种菌株中l号菌株最难抑制。     

食源性致病菌免疫学检测方法研究进展

食源性致病菌广泛存在,因其能引起人的疾病甚至死亡,而越来越受世界各国的关注。食源性致病菌传统检测方法为培养法,其费时耗力,不适用于快速检测。本文介绍的免疫学方法具有方便、快速等优点,因而越来越被广泛应用。免疫学检测方法主要有酶联免疫吸附法、免疫层析法、免疫磁珠分离技术、化学发光免疫分析法和乳胶凝集法等。本文着重对这些方法的原理及其应用进行综述,阐述其优缺点,同时对免疫学检测方法未来的发展趋势进行了展望。     

Bacillus cereus endospores exhibit a heterogeneous response to heat treatment and low-temperature storage

Bacillus cereus endospores were challenged by heat treatments simulating typical domestic/industrial cooking regimes and the resulting effects on germination, viability and sub-lethal heat damage determined using differential plate counting on a rich versus selective medium, flow cytometry (FCM), beta-D-glucuronidase (GUD) activity and OD(600) measurement. Additionally, these techniques were used to investigate the effect on endospores of storage in a non-nutrient medium at 4 degrees C for 1 month. Plate counting revealed that heating generated sub-populations of sub-lethally damaged endospores, with the more severe heat treatments generating larger proportions of sub-lethally damaged endospores. These findings were also reflected in FCM analyses, which detected large amounts of heterogeneity among the populations of heat-treated endospores and uncovered differences in the proportions of membrane-damaged endospores and those displaying esterase activity pre- and post-treatment. Plate count data suggested that both the control and heat-treated endospores lost viability during storage, with FCM data indicating that the proportion of membrane-damaged endospores increased and those displaying the esterase activity decreased. The FCM, GUD and OD(600) data suggested that germination rates decreased with the increasing severity of heat treatment. This study demonstrates that a combination of plate counting and FCM can be used to detect heterogeneity in the response of endospores to insults.