Incidence,Antibiotic Susceptibility,and Toxin Profiles of Bacillus cereus sensu lato Isolated from Korean Fermented Soybean Products

Korean fermented soybean products, such as doenjang, kochujang, ssamjang, and cho‐kochujang, can harbor foodborne pathogens such as Bacillus cereus sensu lato (B. cereus sensu lato). The aim of this study was to characterize the toxin gene profiles, biochemical characteristics, and antibiotic resistance patterns of B. cereus sensu lato strains isolated from Korean fermented soybean products. Eighty‐eight samples of Korean fermented soybean products purchased from retails in Seoul were tested. Thirteen of 26 doenjang samples, 13 of 23 kochujang samples, 16 of 30 ssamjang samples, and 5 of 9 cho‐kochujang samples were positive for B. cereus sensu lato strains. The contamination level of all positive samples did not exceed 4 log CFU/g of food (maximum levels of Korea Food Code). Eighty‐seven B. cereus sensu lato strains were isolated from 47 positive samples, and all isolates carried at least one enterotoxin gene. The detection rates of hblCDA, nheABC, cytK, and entFM enterotoxin genes among all isolates were 34.5%, 98.9%, 57.5%, and 100%, respectively. Fifteen strains (17.2%) harbored the emetic toxin gene. Most strains tested positive for salicin fermentation (62.1%), starch hydrolysis (66.7%), hemolysis (98.9%), motility test (100%), and lecithinase production (96.6%). The B. cereus sensu lato strains were highly resistant to β‐lactam antibiotics such as ampicillin, penicillin, cefepime, imipenem, and oxacillin. Although B. cereus sensu lato levels in Korean fermented soybean products did not exceed the maximum levels permitted in South Korea (<10⁴ CFU/g), these results indicate that the bacterial isolates have the potential to cause diarrheal or emetic gastrointestinal diseases.     

Bacillus subtilis HJ18‐4 from Traditional Fermented Soybean Food Inhibits Bacillus cereus Growth and Toxin‐Related Genes

Bacillus subtilis HJ18‐4 isolated from buckwheat sokseongjang, a traditional Korean fermented soybean food, exhibits broad‐spectrum antimicrobial activity against foodborne pathogens, including Bacillus cereus. In this study, we investigated the antibacterial efficacy and regulation of toxin gene expression in B. cereus by B. subtilis HJ18‐4. Expression of B. cereus toxin–related genes (groEL, nheA, nheC, and entFM) was downregulated by B. subtilis HJ18‐4, which also exhibited strong antibacterial activity against B. cereus. We also found that water extracts of soy product fermented with B. subtilis HJ18‐4 significantly inhibited the growth of B. cereus and toxin expression. These results indicate that B. subtilis HJ18‐4 could be used as an antimicrobial agent to control B. cereus in the fermented soybean food industry. Our findings also provide an opportunity to develop an efficient biological control agent against B. cereus.     

食品中蜡样芽孢污染特点及检测方法研究进展

蜡样芽孢杆菌引发的食物污染大都是和人们生活息息相关的食品,在特定条件下对人们的身体健康会带来很大危害.文章综述了粮食制品、含乳制品、豆制品及其他易污染食品中蜡样芽胞杆菌的污染特点,并总结了国内外学者最新研究的检测蜡样芽胞的手段,为全面了解蜡样芽孢杆菌污染状况以供制定相关食品限量标准、完善检测方法提供参考.     

Characterization of Antibiotic Resistance Genes from Lactobacillus Isolated from Traditional Dairy Products

Lactobacilli are widely used as starter cultures or probiotics in yoghurt, cheese, beer, wine, pickles, preserved food, and silage. They are generally recognized as safe (GRAS). However, recent studies have shown that some lactic acid bacteria (LAB) strains carry antibiotic resistance genes and are resistant to antibiotics. Some of them may even transfer their intrinsic antibiotic resistance genes to other LAB or pathogens via horizontal gene transfer, thus threatening human health. A total of 33 Lactobacillus strains was isolated from fermented milk collected from different areas of China. We analyzed (1) their levels of antibiotic resistance using a standardized dilution method, (2) their antibiotic resistance gene profiles by polymerase chain reaction (PCR) using gene‐specific primers, and (3) the transferability of some of the detected resistance markers by a filter mating assay. All Lactobacillus strains were found to be resistant to vancomycin, but susceptible to gentamicin, linezolid, neomycin, erythromycin, and clindamycin. Their susceptibilities to tetracycline, kanamycin, ciprofloxacin, streptomycin, quinupristin/dalfopristin, trimethoprim, ampicillin, rifampicin, and chloramphenicol was different. Results from our PCR analysis revealed 19 vancomycin, 10 ciprofloxacin, and 1 tetracycline‐resistant bacteria that carried the van(X), van(E), gyr(A), and tet(M) genes, respectively. Finally, no transferal of the monitored antibiotic resistance genes was observed in the filter mating assay. Taken together, our study generated the antibiotic resistance profiles of some milk‐originated lactobacilli isolates and preliminarily assessed their risk of transferring antibiotic gene to other bacteria. The study may provide important data concerning the safe use of LAB.     

不同生境中芽孢杆菌的分离鉴定及药敏性检测

为了给农业生产(发酵饲料、堆肥、污水处理)及微生态调节剂的制备提供前期理论依据和优良的微生物资源,本研究以土壤、饲料和肥料为材料,分离筛选出22株优良的芽孢杆菌菌株,按《伯杰氏细菌学鉴定手册》和芽孢杆菌特有的生理生化指标对其进行了初步鉴定,结果显示,其中的T-1为多粘类芽孢杆菌(Bacillu spaenibacillus polymyxa);T-2为桥石短芽孢杆菌(Bacillus choshinensis);T-6为软骨酸类芽孢杆菌(Bacillus paenibacilluschondroitinus);T-8为解淀粉类芽孢杆菌(Bacillus paenibacillus amyloyticus);S-4为饲料类芽孢杆菌(Bacilluspaenibacillus pabuli);S-6为哥本类芽孢杆菌(Bacillus paenibacillus kobensis)。其余的16株芽孢杆菌还有待于进一步鉴定。在此基础上,采用纸片琼脂扩散法测定了已鉴定出的6株芽孢杆菌对常用的12大类35种抗生素的药敏性。结果显示,分离自土壤、饲料及堆肥中的芽孢杆菌均对喹诺酮类、氨基糖甙类、大环内酯类和糖肽类抗菌素表现出敏感性,大部分对多肽类抗菌素表现出耐受性,其他抗菌素则在不同芽孢杆菌菌株间表现出差异性。     

Genome Subtyping of Autochthonous Bacillus Species Isolated from Iru,a Fermented Parkia biglobosa Seed

Phylogenetic relationship and strains sub-typing of Bacillus species isolated from iru, a traditional fermented condiment in Africa were studied using polyphasic genomic approaches and the profiles compared with bacilli isolated from similar Asian condiments. The 16S rRNA gene sequencing identified the strains as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus, and Brevibacillus formosus. The phylogenetic analysis conducted showed five distinct clusters with genetic relatedness among B. subtilis and B. amyloliquefaciens strains from Africa and Asia. Amplified ribosomal DNA restriction analysis (ARDRA) successfully differentiated species of B. subtilis phylogeny from B. cereus. Combined analyses of ARDRA, internal transcribed spacer-polymerase chain reaction (ITS-PCR), ITS-PCR-restriction fragment length polymorphism (ITS-PCR-RFLP) and randomly amplified polymorphic DNA (RAPD-PCR) further confirmed B. subtilis and B. amyloliquefaciens as the dominant Bacillus species associated with fermentation of iru, and revealed high strains genetic diversity, while multilocus sequence analysis (MLSA) data distinguished B. cereus from B. thuringiensis. This information is essential for selection of starter cultures with desirable functional attributes to guarantee product consistency and safety quality of traditional fermented foods.     

酸奶中保加利亚乳杆菌药物敏感性分析

从酸奶中分离保加利亚乳杆菌,对其遗传多样性和药物敏感性进行分析,并进一步对耐药菌株的抗性基因进行检测。利用改良MRS培养基,从国内不同品牌酸奶中分离获得18株保加利亚乳杆菌。18株分离菌先经RAPD分型后采用琼脂稀释法测定其对11种抗生素的药敏性,并通过PCR对耐药菌株中可能存在的抗性基因进行检测。结果显示,18株受试菌具有明显的遗传多样性和耐药表型多样性。18株菌全部对罗红霉素敏感,而全部对卡那霉素耐药;对氨苄青霉素、青霉素G、金霉素、氯霉素、四环素、林克霉素、链霉素、新霉素及庆大霉素等9种抗生素均表现出不同程度的耐药性。通过检测耐药菌株的抗性基因,从1株菌(B-8)中检出四环素抗性基因tet(M),从2株菌(B-8和B-41)中检出链霉素抗性基因ant(6),从4株菌(B-43、B-47、B-49和B-51)中检出卡那霉素抗性基因aph(3’)-Ⅲa。结果表明,供试18株保加利亚乳杆菌多重耐药现象比较严重。     

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.     

中国传统发酵豆制品γ-氨基丁酸研究

选择全国不同地区生产的61种传统发酵豆制品(包括豆豉、腐乳、豆酱和酱油),使用高效液相色谱法(HPLC)柱前衍生测定其γ-氨基丁酸(GABA)含量。研究表明,中国传统发酵豆制品富含GABA,从所试样品来看,腐乳样品GABA平均含量最高,为277.26mg/100g干重,含量最高的样品达1 159.46 mg/100g干重。所试豆豉、豆酱和酱油中平均GABA含量分别为116.81 mg/100g干重、68.81mg/100g干重和141.51mg/100mL。同一种类不同品牌发酵豆制品中GABA含量存在较大差异,这与发酵豆制品的不同生产工艺相关。通过工艺改进,可望富集GABA,生产富含GABA的功能性发酵豆制品。     

郫县豆瓣酱中芽孢杆菌进化关系及酶活性和抗生素特性分析

结合宏基因组和纯培养方法,对郫县豆瓣酱中已分离到的66株芽孢杆菌进行进化关系研究,并对代表性菌株进行酶活性和抗生素特性分析,以期加深对郫县豆瓣酱中芽孢杆菌多样性的认识,同时对筛选到的菌株后续利用提供一定依据.结果表明:采用宏基因组方法共鉴定出24种芽孢杆菌,全长16S rDNA序列系统分类鉴定,郫县豆瓣酱中芽孢杆菌可分...     

Genetic Diversity and Antibiotic Resistance Patterns of Staphylococcus Aureus Isolated from Leaf Vegetables in Korea

Staphylococcus aureus is an important foodborne pathogen on global basis. The current study investigated the genetic patterns in S. aureus isolates from leaf vegetables (n = 53). Additional isolates from livestock (n = 31) and humans (n = 27) were compared with the leaf vegetable isolates. Genes associated with toxins, antibiotic resistance, and pulsed‐field gel electrophoresis (PFGE) patterns were analyzed. At least 1 enterotoxin‐encoding gene (sea, seb, sec, sed, and see) was detected in 11 of 53 (20.75%) leaf vegetable isolates. When the agr (accessory gene regulator) grouping was analyzed, agr II was the major group, whereas agr IV was not present in leaf vegetable isolates. All S. aureus isolates from leaf vegetables were resistant to more than one of the antibiotics tested. Nineteen of 53 (35.85%) isolates from leaf vegetables exhibited multidrug‐resistance, and 11 of these were MRSA (methicillin‐resistant S. aureus). A dendrogram displaying the composite types of S. aureus isolates from 3 origins was generated based on the combination of the toxin genes, agr genes, antibiotic resistance, and PFGE patterns. The isolates could be clustered into 8 major composite types. The genetic patterns of S. aureus isolates from leaf vegetables and humans were similar, whereas those from livestock had unique patterns. This suggests some S. aureus isolates from leaf vegetables to be of human origin.     

糙米中蜡样芽孢杆菌的分离鉴定及其毒力基因与药敏性检测

本文从一份糙米样品中分离纯化出5株细菌,分别命名为BR1、BR2、BR3、BR4和BR5,对其进行形态学观察、生理生化鉴定、16S rRNA及gyrB基因测序鉴定,并对其毒力基因及药物敏感性等生物学特征进行研究。结果表明,5株分离菌株生化特性符合蜡样芽孢杆菌（Bacillus cereus）的特性,结合16S rRNA及gyrB基因测序结果,菌株BR1和BR4鉴定为蜡样芽孢杆菌。毒力基因的PCR检测结果表明,菌株BR1和BR4携带肠毒素基因（hbl、nhe、entFM和bceT）和细胞毒素K基因（cytK）,未检测到呕吐型毒力基因（cer和ces）。菌株BR1和BR4对青霉素、氨苄西林和头孢噻肟等药物耐药,对阿莫西林中度敏感,对诺氟沙星、环丙沙星和庆大霉素等药物敏感。本研究通过分离鉴定糙米源蜡样芽孢杆菌,发现分离菌株携带多种毒力基因,并且对青霉素类、头孢菌素类及磺胺类等药物耐药,表明糙米中的蜡样芽孢杆菌有引起食源性疾病的风险。     

酱香型酒醅产香芽孢杆菌的分离鉴定及其代谢产物分析

本文研究了酱香型酒醅中的产香芽孢杆菌及其优势代谢产物。通过平板分离法从酱香型酒醅中筛选出5株芽孢杆菌,采用PLFA技术对其鉴定,分别为:蜡样芽孢杆菌(Bacillus ereus)、泛酸枝芽孢杆菌(Virgibacillus pantothenticus)、巨大芽孢杆菌(Bacillus megaterium)、枯草芽孢杆菌(Bacillus subtilis)和地衣芽孢杆菌(Bacillus licheniformis);革兰氏染色试验结果说明:这五类菌株均为革兰氏阳性菌;生化分析发现:枯草芽孢杆菌产淀粉酶能力最强,巨大芽孢杆菌蛋白质水解能力最强;以高粱粉为底物进行固态发酵,采用GC-MS分析技术对发酵代谢产物进行分析表明:菌株的优势产物主要为3-羟基-2-丁酮,另外还有少量的2,3-丁二醇和酯类等芳香物质。蜡样芽孢杆菌、泛酸枝芽孢杆菌、巨大芽孢杆菌、枯草芽孢杆菌和地衣芽孢杆菌这五类菌株是酱香型白酒主要的产香功能菌。     

豆瓣中腐败菌蜡样芽孢杆菌性能及对抗生素敏感性研究

该实验以豆瓣中分离的蜡样芽孢杆菌为实验菌株,对蜡样芽孢杆菌最适生长条件及对抗生素敏感性进行了检测。结果显示：该菌的最适生长温度是35 ℃,5 ℃以下和50 ℃以上不生长;最适生长盐含量为2%,在含量5%时生长缓慢,在盐含量10%以上时不生长;最适生长pH值为7.0,pH值3.0以下和pH值10.0以上该菌不生长;另外,在检测的25种抗生素中,有21种抗生素对该菌的生长具有抗性。     

响应面法优化以豆渣为基质发酵纳豆菌

为提高豆渣的附加值,以豆渣为基质液体发酵纳豆菌;首先用Plackett-Burman方法对影响纳豆菌发酵因素的效应进行评价并筛选出了具有显著效应的三个因素:初始pH值、发酵温度和麸皮含量,其他因素对纳豆菌发酵无显著影响;然后采用最陡爬坡实验逼近最大响应区域,最后由响应面实验确定了主要影响因素的最佳条件:初始pH6．02、发酵温度36．4℃、麸皮0．31%;在优化培养条件下,纳豆菌发酵液活菌数达到4．35×10~9CFU/ml。     

5株发酵食品源乳酸菌的抗药性分析

以实验室前期分离自混合果蔬酵素的植物乳杆菌(Lactobacillus plantarum S)、分离自树莓酵素的植物乳杆菌(L. plantarum WD)和分离自不同奶制品的保加利亚乳杆菌(L. bulgaricus LB-DR)、鼠李糖乳杆菌(L. rhamnosus Lr-05-281)与干酪乳杆菌(L. casei D-400)5株乳酸菌为研究对象,采用平板药敏纸片扩散法、PCR及RT-PCR技术从表型、基因型以及基因表达几个方面分析菌株的抗药性。结果表明5株菌对氨基糖苷类、喹诺酮类、糖肽类抗生素、多粘菌素B均有抗性;对大环内酯类、四环素类、磺胺类抗生素、呋喃妥因均敏感,并有较为相似的耐药谱;而对不同的β-内酰胺类抗生素敏感性不同;5株乳酸菌均含有质粒,供试的12个抗性基因中,在质粒上检测到erm、aph、vanⅠ、aacⅠ4种抗性基因,基因组DNA上检测到aph、erm、vanⅠ、blaⅡ、aacⅠ、aacⅡ6种抗性基因。部分抗性基因在MRS和加抗生素的MRS培养下会表达,不表达的抗性基因在相应抗生素诱导的条件下其抗性基因不表达。L. plantarum WD菌株质粒上因只含一种vanⅠ抗性基因,其应用安全性较好。     

发酵豆制品酿造过程中组分和营养功能因子的变化及调控

发酵豆制品是发酵食品中的一大类,在亚洲国家人民饮食中占重要地位,并逐渐受到西方国家青睐。近年来,发酵豆制品酿造过程中产生的功能因子不断被报道,其抗氧化、降血压、降血糖、溶血栓、抗突变等功能性不断被揭示,引起世界广泛关注。发酵豆制品组分变化的调控对增强营养和富集功能因子意义重大,有利于进一步提升其食用价值,促进人类健康。本文综述发酵豆制品生产过程中与营养和功能相关的部分组分的变化及调控,并对该领域的科学问题进行展望。     

黑龙江省种植大豆品种遗传多样性分析及与性状关联SSR标记筛选

为从分子水平鉴定黑龙江省种植大豆品种的遗传多样性,并筛选与蛋白质、脂肪、百粒重和水分等大豆主要性状显著关联的SSR分子标记,利用18对SSR引物,采用荧光毛细管电泳法分析黑龙江省75个种植大豆品种遗传多样性,并与性状进行关联分析.结果 表明:18个SSR标记中的10个标记可以实现单对引物准确鉴别42个大豆品种,18个S...     

Enterotoxigenic Profiling of Emetic Toxin‐ and Enterotoxin‐Producing Bacillus cereus,Isolated from Food,Environmental, and Clinical Samples by Multiplex PCR

Bacillus cereus comprises the largest group of endospore‐forming bacteria and can cause emetic and diarrheal food poisoning. A total of 496 B. cereus strains isolated from various sources (food, environmental, clinical) were assessed by a multiplex PCR for the presence of enterotoxin genes. The detection rate of nheA, entFM, hblC, and cytK enterotoxin genes among all B. cereus strains was 92.33%, 77.21%, 59.47%, and 47.58%, respectively. Enterotoxigenic profiles were determined in emetic toxin‐ (8 patterns) and enterotoxin‐producing strains (12 patterns). The results provide important information on toxin prevalence and toxigenic profiles of B. cereus from various sources. Our findings revealed that B. cereus must be considered a serious health hazard and Bacillus thuringiensis should be considered of a greater potential concern to food safety among all B. cereus group members. Also, there is need for intensive and continuous monitoring of products embracing both emetic toxin and enterotoxin genes.     

Prevalence and characterisation of Bacillus cereus in vacuum packed potato puree

Refrigerated processed foods of extended durability (REPFED) potato puree was analysed for Bacillus cereus contamination along the production line and during the product shelf‐life. Isolated B. cereus strains were tested for their psychrotrophic character and the ability to produce enterotoxins. Bacillus cereus contamination during four subsequent productions was in the range of 2.3–4.0 log cfu g^?1. Productions five and six were significantly less contaminated with B. cereus (≤1 log cfu g ^?1). All B. cereus isolates from the first four productions were able to grow at 7 ° and 10 °C, whereas the majority of the isolates from productions five and six did not. No B. cereus isolates grew at 4 °C. randomly amplified polymorphic DNA (RAPD) fingerprinting showed that the most of B. cereus contamination originated from one source. In total, 30.4% of isolates expressed enterotoxic character. The present study points out the necessity to prevent an ‘in house’ colonisation and contamination during food processing in order to accomplish the safety of REPFED throughout the shelf‐life. It also indicates the most critical steps in the production line of ready‐to‐eat potato puree and impact of failures regarding the food safety. The data provided can be used for risk assessment studies regarding B. cereus in REPFED.