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.     

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.     

基于环介导等温扩增技术的产呕吐毒素 蜡样芽胞杆菌特异性检测

本研究旨在建立食品中产呕吐毒素蜡样芽胞杆菌的快速检测方法.基于产呕吐毒素蜡样芽胞杆菌合成酶基因cesB靶基因,设计4条特异性引物(2条内引物、2条外引物),建立环介导等温扩增技术(LAMP).然后采用2株产呕吐毒素蜡样芽胞杆菌、19株蜡样芽胞杆菌和41株非蜡样芽胞杆菌验证了该LAMP具有很好的特异性.LAMP检测的灵敏...     

芥末等8种食物过敏原的多重PCR检测技术

依据芥末Sin a I基因、羽扇豆核糖体内转录间隔区(ITS)基因、胡桃Jug r 2基因、榛果Oleosin基因、芹菜Mtd基因、杏仁Pru du 1基因、燕麦Avenin基因和芝麻2S albumin mRNA基因设计的特异性引物序列,在普通PCR方法的基础上,通过2组4重PCR扩增,建立了同时检测芹菜、杏仁、燕麦、芝麻、芥末、羽扇豆、胡桃和榛果等8种食物过敏原的方法。该方法特异性强,灵敏性高,可应用于食品中多种食物过敏原的快速检测和监控。     

多重PCR方法对大豆转基因食品的定性检测

为了同时检测转基因食品中所含的多个目标基因序列,本文采用多重PCR分析技术对转基因大豆食品进行了检测。为了排除扩增结果的假阴性,大豆外源凝集素基因和肌动蛋白基因被作为内部对照以评价所有PCR反应的效率。当转基因大豆含量仅为0.15%时,仍然可以对转基因食品进行可靠的鉴定,从而表明该方法的高度敏感性。该文所述的多重PCR系统是一种简单、准确并且敏感的检测方法,只需要进行一个反应就可以检测多个目标基因序列,因而可以用来对转基因原材料及加工成品进行高精度、高灵敏的检测。     

大肠埃希O157:H7等致病菌多重PCR法快速检测试剂盒的研制

目的:研制一种能够同时快速检测大肠埃希O157:H7、志贺氏菌和致病性蜡样芽孢杆菌的三重PCR试剂盒.方法:以大肠埃希O157:H7的hlyAB基因、痢疾志贺氏菌的IpaH基因和致病性蜡样芽孢杆菌的hblA基因作为目的基因片段O157:H7、痢疾志贺氏菌和致病性蜡样芽孢杆菌的特异性抗原基因序列,分别设计1对引物,并对其反应条件进行优化,建立1种多重PCR检测试剂盒.结果:本试剂盒可准确检测出生肉和即食肉制品中的上述3种致病菌,O157:H7检出极限为19.8 cfu/mL,志贺氏菌检出极限为17 cfu/mL,蜡样芽孢杆菌检出极限为17.7 cfu/mL.可在5 h内完成全部反应过程,得出检测结果.结论:本试剂盒在理论和实际应用方面均具有优越性,能够同时检测上述3种病原菌,可用于食品及其原料的生物安全检测,也可用于兽医临床诊断.     

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.     

2012～2016年云南省婴幼儿食品和即食食品中蜡样芽胞杆菌污染状况调查评估

目的 调查分析和评估云南省2012~2016年婴幼儿食品和即食食品中蜡样芽胞杆菌污染状况。方法 本研究的样品包括2012年到2016年采集自云南省16个地州的1014份婴幼儿食品和3361份即食食品。按照GB 4789.14-2014《食品安全国家标准 食品微生物学检验 蜡样芽胞杆菌检验》第一法平板计数法进行检验, 并用国际上现有的限量标准进行评估分析。结果 婴幼儿食品中蜡样芽胞杆菌的平均检出率分别为18.1%(2012)、5.5%(2013)、18.2%(2014)、2.5%(2016), 超标产品的比例分别为1.1%(2012)、1.9%(2013)、1.7%(2014)。即食食品的平均检出率分别为7.6%(2012)、1.3%(2013)、13.9%(2014)、6.5%(2015)、10.1%(2016)。结论 建议对婴幼儿食品中的蜡样芽胞杆菌以102 CFU/g为临界值进行污染评估、对普通即食食品针对儿童与成人分别以103~105 CFU/g和大于105 CFU/g 2个值作为临界值进行评估。     

Prevalence,Genetic Diversity,and Antibiotic Resistance of Bacillus cereus Isolated from Korean Fermented Soybean Products

Bacillus cereus contamination is a major food safety problem for Korean fermented soybean products, but few studies have assessed its potential to cause foodborne illness. The objectives of this study were to investigate the prevalence and characteristics of B. cereus isolated from Korean fermented soybean products. B. cereus was detected in 110 of 162 (67.9%) samples. The highest B. cereus frequency was observed in deonjang (68 of 93 samples, 73.1%) and cheonggukjang (18 of 25, 72.0%); however, nonhemolytic enterotoxin was detected only in 22 of 162 samples (13.6%). Although the tested B. cereus isolates showed diverse pulsotypes according to repetitive sequence‐PCR banding patterns, they displayed similar antibiotic sensitivity spectra. The low frequency of enterotoxin detection suggests that the potential risk of B. cereus foodborne illness associated with Korean fermented soybean products is lower than generally presumed. However, considering the prevalence of B. cereus and the high content of fermented soybean products in the Korean diet, it is necessary to constantly monitor the level of contamination with B. cereus and its toxins in such Korean food products.     

动物性食品源空肠弯曲杆菌二重PCR检测方法的建立及应用

根据GenBank空肠弯曲杆菌(Campylobacter jejuni,Cj)的16S rDNA及hipO(编码马尿酸酶基因)序列设计两对特异引物,建立检测动物性食品源Cj的二重PCR方法,并应用于样品检测。结果显示只对Cj能特异的扩增出699bp和366bp两个基因片段,而大肠杆菌、沙门氏菌等其他11种细菌均未扩增出条带;Cj标准株ATCC33560的16S rDNA及hipO序列与GenBank其他Cj的相应序列具高度相似性(分别为99.7%~99.9%,98.1%~99.7%);该方法可在27h内完成,其灵敏度为2.4~16 CFU/mL;四川省雅安市鸡肉、猪肉、牛肉和牛奶样品中的Cj阳性率分别为38.0%(19/50)、28.3%(15/53)、17.1%(6/35)和8.6%(4/46)。     

Supplementation of Modified Mannitol‐Yolk‐Polymyxin B Agar with Cefuroxime for Quantitative Detection of Bacillus cereus in Food

The presence of unwanted competing flora has been the most common confounding factor in the enumeration of Bacillus cereus (B. cereus) using selective media such as mannitol‐yolk‐polymyxin B agar (MYPA). The objective of this study was to improve MYPA selectivity for B. cereus by supplementation with a second‐generation cephalosporin, cefuroxime. The performance of cefuroxime‐supplemented MYPA (cefu‐MYPA) was evaluated by comparison with original MYPA in 60 food products with established microbiological standards for B. cereus contamination. Cefu‐MYPA demonstrated superior recoverability and selectivity for B. cereus compared with original MYPA in most tested foods. B. cereus numbers on MYPA and cefu‐MYPA were 363.5 and 462.0 CFU/g, respectively. Competing flora on cefu‐MYPA was detected in significantly less samples (70%) compared to original MYPA (93%). In addition, the detection and isolation of suspected colonies were significantly improved in cefu‐MYPA because of the reduction or elimination of competing flora in all tested foods except fruit juice, indicating superior selectivity of the modified medium. Our findings suggest that cefuroxime supplementation of MYPA would markedly improve the detection rate of B. cereus, particularly in foods with high levels of indigenous flora.     

Multiplex PCR coupled with propidium monoazide for the detection of viable Cronobacter sakazakii,Bacillus cereus,and Salmonella spp. in milk and milk products

Cronobacter sakazakii, Bacillus cereus, and Salmonella spp. are common food-borne pathogens. The aim of this study was to develop a sensitive, specific, and rapid method for the simultaneous detection of these 3 pathogens in milk and milk products. Three specific primers were designed based on ompA, invA, and cesB of C. sakazakii, Salmonella spp. and B. cereus, respectively, for use in a multiplex PCR (mPCR). To eliminate false-positive results, cells were pretreated with propidium monoazide (PMA) for the selective elimination of the genomic DNA of dead cells. An internal amplification control was applied as an indicator of false-negative results from the interference of inhibitors in the food matrix. Results showed that, in pure culture, the limits of detection of the assay for C. sakazakii, Salmonella Enteritidis, and B. cereus were 9.5 × 10⁴, 7.4 × 10², and 7.5 × 10² cfu/mL, respectively. Moreover, 8 cfu/mL of viable B. cereus cells were detected after 5 h of enrichment, and 9 cfu/mL of viable C. sakazakii and 7 cfu/mL of Salmonella Enteritidis were detected after 7 h of enrichment in spiked pure milk, walnut peanut milk, and whole-wheat milk. To validate the PMA-mPCR assay, the PMA-mPCR assay and the traditional culture method were performed to detect the 3 bacterial strains in 1,165 milk product samples. The PMA-mPCR assay obtained the same results as the culture-based method. Results demonstrated that the PMA-mPCR assay has excellent sensitivity and specificity for the simultaneous detection of viable C. sakazakii, Salmonella Enteritidis, and B. cereus in milk and milk products.     

Characterization of a novel protease from Bacillus cereus and evaluation of an eco‐friendly hydrolysis of a brewery byproduct

Proteases and proteolytic enzymes constitute one of the most important groups of enzymes and are attracting worldwide attention in attempts to exploit their physiological and biotechnological applications. In this study, partial purifications and biochemical and antimicrobial characterizations of a protease from Bacillus cereus spp., originally isolated from fermented cabbage, were carried out. The crude extract obtained after purification, involving ammonium sulphate precipitation and dialysis, was designated as a partially purified protease (PPP). The obtained PPP had a specific activity of 0.395–2.539 U/g at 32 °C, with maximum activities for the fractions precipitated at 60 and 80% ammonium sulphate. The PPP activity ranged between 20 and 55 °C, with an optimum temperature at 40 °C. At 60 °C, the PPP retained more than 30% of its activity. The optimum pH for the PPP was achieved at pH 9, indicating the alkaline source of the enzyme. Protease production was specifically dependent on the calcium concentration in the culture medium. Also the robustness of the protease on brewer's spent grain hydrolysis was demonstrated. This suggests a potential eco‐friendly application of the enzyme. Finally, it was found that the PPP inhibited the growth of Escherichia coli O157:H7. This novel property of the PPP liberated by the B. cereus spp. could provide important future benefits to industry. Copyright © 2015 The Institute of Brewing & Distilling     

Multiplex PCR identification of Listeria monocytogenes isolates from milk and milk‐processing environments

A multiplex PCR procedure based on genes iap (coding for the invasion‐associated 60 kDa protein or p60) and hly (coding for listeriolysin O) of Listeria monocytogenes was to used to investigate the status of its contamination along the major milk‐processing environments. Duplex PCR amplified fragments of the iap gene at about 1.45 kb from all strains of major Listeria spp. tested and a 420 bp fragment of hly from L. monocytogenes reference strains. With triplex PCR, all L. monocytogenes strains exhibited a 420 bp fragment of hly as well as a 700 bp fragment of iap instead of its 1.45kb PCR product. The tentative L. monocytogenes isolates (n = 27) out of 566 samples of milk and milk‐processing environments in the PALCAM agar from cultures of buffered listeria enrichment broth were further subjected to both API and triplex PCR identification. Both methods identified the same 20 isolates as L. monocytogenes. The triplex PCR procedure detected as low as 3.2 × 10¹ cfu mL^?1 of listerial cells even in the presence of L. innocua (10⁸ cfu mL^?1). It could detect 1.4 × 10² listerial cells mL^?1 directly from milk artificially contaminated with the bacteria. Lower levels of L. monocytogenes cells in milk (1.45 × 10¹ to 1.45 × 10⁰ cfu mL^?1) could be detectable if the inoculated milk samples were cultured for 3–6 h. The bacterium was found not only in raw milk, sewage water and vessel surfaces but also in pasteurized milk. Copyright © 2005 Society of Chemical Industry     

云南边贸进口即食食品中蜡样芽胞杆菌污染状况及其呕吐毒素基因型检测

为探究云南省边贸进口即食食品中蜡样芽胞杆菌的污染状况及其呕吐毒素基因型携带情况,从云南边境各口岸进口食品贸易集散点(边民互市点、农贸市场以及进出口产品店等地)共采集市售包装样品224份。通过VITEK 2 compact全自动微生物鉴定系统和双重实时荧光聚合酶链式反应(Double real-time fluorescent polymerase chain reaction,Dual real-time PCR)技术对分离得到的蜡样芽胞杆菌进行鉴定,并对阳性菌株中的呕吐毒素基因进行检测。结果表明:蜡样芽胞杆菌在所采集样品中的检出率为20.09%(45/224),天保、金水河、瑞丽以及畹町各口岸检出率均高于10%。检出率较高的食品类别为:酱腌菜60%(3/5)、调味品50%(15/30)、水产及其制品46.67%(7/15);从45份阳性样品中共分离出113株蜡样芽胞杆菌,其中呕吐型毒素基因cesB的携带率为0.88%(1/113),呕吐型毒素基因携带率较低,但是蜡样芽胞杆菌的污染风险仍不可忽视。该研究结果对边贸进口食品安全、食源性疾病的监测及预防提供了理论依据。     

应用通用引物PCR法对葡萄球菌B、C1、C2、C3型肠毒素基因的鉴定与分型研究

本实验采用通用引物P1、P2聚合酶链反应(PCR)法对产肠毒素葡萄球菌的entB、entC1、entC2、entC3基因进行了扩增,结果表明,含有这四种肠毒素基因的葡萄球菌都可以产生595bp的特异性扩增片段,其余菌扩增均为阴性;扩增产物分别用在产物内部只有单一识别位点的限制酶HinfⅠ、BclⅠ、MboⅡ和MboⅡ酶切后产生大小不同的片段,因而可以达到酶切分型的目的。经敏感性试验表明,该方法可以检出10~0个细菌。     

A Multiplex PCR Assay for the Rapid and Sensitive Detection of Methicillin‐Resistant Staphylococcus aureus and Simultaneous Discrimination of Staphylococcus aureus from Coagulase‐Negative Staphylococci

Abstract: Methicillin‐resistant Staphylococcus aureus (MRSA) is a global health concern, which had been detected in food and food production animals. Conventional testing for detection of MRSA takes 3 to 5 d to yield complete information of the organism and its antibiotic sensitivity pattern. So, a rapid method is needed to diagnose and treat the MRSA infections. The present study focused on the development of a multiplex PCR assay for the rapid and sensitive detection of MRSA. The assay simultaneously detected 4 genes, namely, 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, and one internal control. It was rapid and yielded results within 4 h. The analytical sensitivity and specificity of the multiplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the multiplex PCR assay at the DNA level was 10 ng DNA. The analytical specificity was evaluated with 10 reference staphylococci strains and was 100%. The diagnostic evaluation of MRSA was carried out using 360 foodborne staphylococci isolates, and showed 99.1% of specificity, 96.4% of sensitivity, 97.5% of positive predictive value, and 97.3% of negative predictive value compared to the conventional method. The inclusion of an internal control in the multiplex PCR assay is important to exclude false‐negative cases. This test can be used as an effective diagnostic and surveillance tool to investigate the spread and emergence of MRSA.     

Original article: Modelling the effects of food ingredients and pH on high‐pressure processing inactivation of Bacillus cereus spores: a laboratorial study

The combination of high pressure and heat on Bacillus cereus spores in food matrix was investigated with the purpose of achieving a predictive model of microbial inactivation. The high‐pressure processing (HPP) conditions were fixed at 540 MPa and 71 °C for 16.8 min, which were determined as the optimum conditions considering six‐log‐cycle reductions of B. cereus spores. The effects of soybean protein, sucrose, soybean oil and pH on the inactivation of B. cereus spores by HPP were evaluated, and a quadratic predictive model for the effects of food ingredients and pH on the reductions of B. cereus spores by HPP was built using response surface methodology. The experimental results showed that soybean protein, sucrose and pH significantly affected the reductions of B. cereus spores. The predictive model is significant, because the level of significance was P < 0.0001 and the calculated F‐value is much greater than the tabulated F‐value. The adequacy of the predictive model equation was verified effectively using the experimental test data that were not used in the development of the model.     

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R² values of 0.9969 and 0.9958 respectively. Linear correlations between the log₁₀ input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10¹ CFU/mL to 1.65 × 10⁶ CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.