Properties of Bacillus cereus and other bacilli contaminating biomaterial-based industrial processes

This paper is an overview on bacilli in industrial processes, with focus on food grade paper and paperboard production. Paperboards mainly contain sporeforming bacteria belonging to the genera Bacillus, Paenibacillus and Brevibacillus, usually found in quantities from <50 to 250 cfu g⁻¹ homogenized paperboard. Of those frequently found, Bacillus cereus group, B. licheniformis, B. subtilis and Brevibacillus brevis are important for food hygiene because of their hydrolytic activities on food components and the ability of some strains to produce food poisoning toxins or to grow at refrigerated temperatures. We found that the phenotypic properties (lecithinase activity, nitrate reduction) used in standard methods (e.g., ISO, FDA, IDF) to recognize B. cereus, were unreliable for industrial isolates. Whole cell fatty acid composition of a group of the industrial isolates deviated so much from those in a widely used commercial database that the strains were not or only poorly recognized as B. cereus. Industrial isolates, including toxigenic ones, often missed one or more of these characters, even in cases where 100% 16S rDNA identity was found with B. cereus or with B. thuringiensis. 11-Methyldodecanoic acid and trans-9-hexadecenoic acid were found without exception in over 200 industrial B. cereus group isolates and in over 30 culture collection strains. The detection of these fatty acids is a secure method for the identification of B. cereus. Negative reaction for starch hydrolysis and for BCET-RPLA test and a specific ribotype were found in all B. cereus strains producing the emetic toxin.     

生食蔬菜模拟污染米饭样本中蜡样芽胞杆菌和呕吐毒素基因分布状况研究

目的 分析被生食蔬菜模拟污染的米饭样本中蜡样芽胞杆菌（Bc）和呕吐毒素基因（ces）的分布状况,为Bc食物中毒科学防控提供基础数据。方法 采集生食蔬菜样本50件,每件样本用0.85%生理盐水盥洗后污染“新煮熟米饭”,置于30 ℃、70% RH培养箱中放置24 h。对生食蔬菜和“污染米饭”进行Bc的定量计数、荧光PCR检测和数字PCR检测。对基于不同采集地点、不同蔬菜类型分组的生食蔬菜样本和及被其污染的“污染米饭”的各项检出率指标进行统计学分析。结果 生食蔬菜样本中Bc检出率为80.00%（40/50）,ces基因和Bac16s RNA基因检出率分别为0（0/50）和10.00%（5/50）;“污染米饭”样本Bc检出率为94.00%（47/50）,ces基因和Bac16s RNA基因检出率分别为14.00%（7/50）和90.00%（45/50）。采集自农贸市场和农户土地的2组生食蔬菜中Bc检出率差异有统计学意义（χ²=11.063,P=0.000 88校正）,被上述2组生食蔬菜类污染的“污染米饭”Bac16s RNA基因检出率和ces基因检出率差异均有统计学意义（χ²=3.926,P=0.047 5校正;χ²=5.444,P=0.019 6校正）。7件“污染米饭”基于荧光PCR检测ces基因阳性,Ct值介于24.12~37.73,数字PCR结果介于6.8 copes/μL~6.2×10⁶ copes/μL。结论 被生食蔬菜模拟污染的米饭样本可具有导致Bc食物中毒风险的病原学特征。     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

Changes in membrane fatty acids and murein composition of Bacillus cereus and Salmonella Typhi induced by gamma irradiation treatment

This present study was carried out to evaluate the effects of gamma irradiation on the fatty acids (FA) and mureins composition of two different radiotolerant bacteria. Bacillus cereus LSPQ 2872 and Salmonella Typhi ATCC 19430 were used for this study. The bacterial strains were treated with a sublethal radiation dose of 1 kGy to cause a cellular damage. Gas chromatography (GC) and high performance liquid chromatography (HPLC) analysis were performed to demonstrate respectively the modification of the FA composition and the changes in muropeptide profile. Results obtained show, for both bacteria, that this treatment had a significant effect (P ≤ 0.05) on the FA content with an increase of unsaturated FA percentage. Substantial changes were also noticed for the relative percentage and the number of the muropeptides. This study represents one of the few to demonstrate the modifications on bacterial membrane as a cellular response to survive the ionising radiation stress.     

1株食源性蜡样芽胞杆菌分离株的分子特征毒性及耐药性研究

目的 研究株蜡样芽胞杆菌SCY分离株的毒力和耐药性。方法 使用选择性培养基从银川市某餐厅鱼肉菜肴中培养分离获得蜡样芽胞杆菌SCY分离株,经23S rRNA基因PCR测序鉴定后,通过二代全基因组测序技术分析SCY分离株的基因组特征,进而分别采用免疫组化技术和药敏纸片实验研究SCY分离株的毒性和耐药性。结果 SCY分离株的基因组大小为5.82 Mb,编码基因个数为5 767,编码区总长度占全基因组的85.50%。SCY分离株基因组中共含有11个基因岛、16个CRISPR和5个前噬菌体;其中,SCY编码基因分别在PHI和VFDB毒力因子数据库中注释到了14个和62个毒力基因（Identiy>80,Evalue<0.05）,在CARD耐药数据库中注释到了215个耐药基因（Best Hit evalue<0.05）。小鼠肠道组织切片免疫组化结果表明,炎性因子IL-1β、CASP1和Nlrp3的表达水平发生差异显著性上调。22种细菌抗生素药敏纸片试验结果显示,SCY分离株对氯霉素、克林霉素等高度敏感,对四环素、头孢唑啉、头孢哌酮、氨苄青霉素表现为中介,对青霉素、苯唑西林、哌拉西林、杆菌肽、头孢他啶等10种抗生素表现为耐药,SCY分离株多重耐药情况较为严重。结论 本研究发现的SCY分离株属于肠毒株,携带多种肠毒素毒力基因,且具有典型的多重耐药特征。研究结果为揭示蜡样芽胞杆菌分离株的致病机制提供参考依据。     

蜡样芽孢杆菌高密度发酵条件与过程的优化

蜡样芽孢杆菌的生物量对其生物功能具有重要影响。为了提高蜡样芽孢杆菌的产量与芽孢率,通过单因素实验筛选出最适宜其生长的碳源与氮源,分别为质量比1∶1复配的葡萄糖与水溶性淀粉和质量比1∶1复配的大豆蛋白胨与酵母提取物。在此基础上,于7 L发酵罐中探究流加方式、p H、搅拌转速和通气量等因素对其生物量及芽孢率的影响。最优发酵条件为：在0～9 h之间,转速250 r/min,通气量3 L/min,pH恒定6.5;9～18 h时,转速升高至350 r/min,通气量升高至4.5 L/min,并以0.45 mL/min流量补加培养基浓缩液;18 h时,转速降低至150 r/min,通气量降低至2.25 L/min,pH调高至7.5,停止补料至发酵结束。基于以上发酵策略,在18 h时,蜡样芽孢杆菌活菌数达2.2×10¹⁰CFU/mL,是未优化前的4.88倍;发酵结束时,芽孢率超过90%。本研究结果为蜡样芽孢杆菌的工业化应用提供了一定基础。     

一起疑似由蜡样芽胞杆菌引起的食源性疾病事件的分子溯源研究

目的 对一起食源性疾病事件中食品分离蜡样芽胞杆菌（B. cereus）进行溯源分析,为确定污染源、切断传播途径提供技术支撑。方法 本研究建立脉冲场凝胶电泳（PFGE）方法,对12株B. cereus进行分子分型,同时对10株B. cereus进行全基因组测序（WGS）,利用BioNumerics软件对测序数据进行拼接组装、多位点序列分型（MLST）、毒力基因、核苷酸多态性（SNP）分析。结果 本起食源性疾病事件中分离自不同食品的12株B. cereus的PFGE分型显示为8种带型,其中3株ST1435型 B. cereus带型相同,且SNP分析显示这3株B. cereus只有3个碱基差异;2株ST24型B. cereus 的PFGE带型完全相同,且SNP分析显示只有1个碱基差异,提示3株ST1435和2株ST24菌株分别为克隆株。2株ST24型B. cereus携带溶血性肠毒素hlbA、hlbC和hlbD,另有4株B. cereus携带腹泻毒力基因（hlbA、hlbC和hlbD）。结论 B. cereus引起的食源性疾病事件比较复杂,污染源也比较复杂,因此加强原辅料监测、从业人员的卫生监测、环境和设备及环节的清洗消毒,对预防控制由其引起的食源性疾病事件非常重要。     

Production of high degree polymerized chitooligosaccharides in a membrane reactor using purified chitosanase from Bacillus cereus

Crude chitosanase from Bacillus cereus NTU-FC-4 was separated by a cation exchanger to three fractions named CBCI, CBCII, and CBCIII. The CBCI hydrolyzed chitosan to yield dimers. The primary hydrolytic products of CBCII were low degree polymerized (DP) chitooligosaccharides. The CBCIII had the fastest reaction rate and yielded high DP chitooligosaccharides (heptamer and higher DP oligomers). When CBCIII was used in the ultrafiltration membrane reactor with enzyme/substrate ratio 0.06 unit/mg and 100 min of residence time (RT), the concentration of high DP oligomers was 9.78 mg/mL which occupied ca. 48% of total oligomers in the final product as compared to ca. 29% resulted from the crude enzyme. Decrease of RT to 50 min and 33 min, the high DP oligomers in the products were ca. 61% and 69%, respectively. This system could be operated for at least 24 h and kept a constant permeate flux and product output rate.     

2014—2019年岳阳市食源性蜡样芽胞杆菌溶血素hblA与磷脂酶C plc基因特征分析

目的 了解湖南省岳阳市2014－2019年食源性蜡样芽胞杆菌菌株病原学特征,并为蜡样芽胞杆菌引起的食物中毒事件的科学防控提供依据。方法 对2014—2019年分离自湖南省岳阳市城区餐饮门店的26株蜡样芽胞杆菌菌株的致病毒力因子溶血素BL的hblA基因和磷脂酶C的plc基因进行序列扩增和测序,并使用Seqman和MEGA X软件对蜡样芽胞杆菌的hblA和plc基因进行遗传进化分析。结果 从岳阳市分离到的蜡样芽胞杆菌hblA、plc毒力基因的同源性与GenBank中的蜡样芽胞杆菌群相比均大于93.0%。结论 岳阳市分离的蜡样芽胞杆菌的hblA和plc基因与GenBank中的蜡样芽胞杆菌群的同源性高,具有一定的亲缘关系。本研究为进一步了解和科学防控蜡样芽胞杆菌引起的食物中毒事件奠定了研究基础。     

16S与gyrB基因联合建树快速鉴定一株蜡样芽孢杆菌

开发一种可用于保守序列相似度较高的菌群快速鉴定的方法,并用其鉴定一株从土壤采集的芽孢杆菌。从厦门市同安国家农业科技园区施用动物肥的种植区采集土壤,稀释涂布平板,分离纯化菌株后,扩增16S与gyrB基因序列并测序,在Genbank选择序列相似的ATCC菌株进行比对,将16S序列与gyrB序列线性拼接后,利用Paup* 4.0构建进化树,通过生理生化实验对鉴定结果进行验证。在16S序列与gyrB基因序列单独建树均无法鉴定到种的情况下,通过16S与gyrB碱基线性拼接序列联合建树,将土壤中分离得到的芽孢杆菌HX2016002鉴定为蜡样芽孢杆菌,该方法所构建的进化树自展值高,鉴定结果与生理生化实验一致。对Genbank中已知的蜡样芽孢杆菌序列建树分析表明,该方法在蜡样芽孢杆菌中具有普适性。利用16S与gyrB基因拼接序列联合建树,在保守序列相似度高的属内菌种鉴定中具有进一步研究的价值。     

Synergistic effect of Pulsed Electric Fields and CocoanOX 12% on the inactivation kinetics of Bacillus cereus in a mixed beverage of liquid whole egg and skim milk

With a view to extending the shelf-life and enhancing the safety of liquid whole egg/skim milk (LWE–SM) mixed beverages, a study was conducted with Bacillus cereus vegetative cells inoculated in skim milk (SM) and LWE–SM beverages, with or without antimicrobial cocoa powder. The beverages were treated with Pulsed Electric Field (PEF) technology and then stored at 5 °C for 15 days. The kinetic results were modeled with the Bigelow model, Weibull distribution function, modified Gompertz equation, and Log-logistic models.Maximum inactivation registered a reduction of around 3 log cycles at 40 kV/cm, 360 µs, 20 °C in both the SM and LWE–SM beverages. By contrast, in the beverages supplemented with the aforementioned antimicrobial compound, higher inactivation levels were obtained under the same treatment conditions, reaching a 3.30 log₁₀ cycle reduction.The model affording the best fit for all four beverages was the four-parameter Log-logistic model.After 15 days of storage, the antimicrobial compound lowered Bacillus cereus survival rates in the samples supplemented with CocoanOX 12% by a 4 log cycle reduction, as compared to the untreated samples without CocoanOX 12%. This could indicate that the PEF-antimicrobial combination has a synergistic effect on the bacterial cells under study, increasing their sensitivity to subsequent refrigerated storage.     

Effect of modified atmosphere and temperature abuse on the growth from spores and cereulide production of Bacillus weihenstephanensis in a cooked chilled meat sausage

The effect of modified atmosphere packaging (MAP) on the germination and growth of toxin producing psychrotolerant Bacillus spp is not well described. A model agar system mimicking a cooked meat product was used in initial experiments. Incubation at refrigeration temperature of 8 °C for 5 weeks of 26 Bacillus weihenstephanensis including two emetic toxin (cereulide) producing strains showed that B. weihenstephanensis is sensitive to MAP containing CO₂. The sensitivity to 20% CO₂ was dependent on strain and oxygen level, being increased when oxygen was excluded from the MAP. Growth from spores was observed at the earliest within 2 weeks when 20% CO₂ was combined with 2% O₂ and in 3 weeks when combined with “0”% O₂ (the remaining atmosphere was made up from N₂). Results were validated in a cooked meat sausage model for two non-emetic and one emetic B. weihenstephanensis strain. The packaging film oxygen transfer rates (OTR) were 1.3 and 40 ml/m²/24 h and the atmospheres were 2% O₂/20% CO₂ and “0”% O₂/20% CO₂. Oxygen availability had a large impact on the growth from spores in the MAP meat sausage, only the most oxygen restricted condition (OTR of 1.3 ml/m²/24 h and “0”% O₂/20 % CO₂) inhibited growth of the three strains during 4 weeks storage at 8 °C. Cereulide production was undetectable during storage at 8 °C irrespective of choice of the MAP (quantified by liquid chromatography mass spectrometry/mass spectrometry). MAP storage at 8 °C for 1 and 3 weeks followed by opening of packages and temperature abuse for 1.5 h daily at 20 °C during 1 week resulted in increased cell counts and variable cereulide production in the meat sausage. A pre-history at 8 °C for 1 week in MAP with OTR of 1.3 or 40 ml/m²/24 h and 2% O₂ resulted in cereulide concentrations of 0.816 – 1.353 µg/g meat sausage, while a pre-history under the most oxygen restricted condition (OTR of 1.3 ml/m²/24 h, “0”% O₂/20 % CO₂) resulted in minimal cereulide production (0.004 µg/g meat sausage) at abuse condition. Extension of MAP storage at 8 °C for 3 weeks followed by abuse resulted in a substantially reduced cereulide production.Data demonstrates that MAP can be used to inhibit growth of a psychrotolerant toxin producing Bacillus spp. during chill storage at 8 °C, and substantially reduce the risk of emetic food poisoning at abuse condition. Results are of relevance for improving safety of ready to eat processed chilled foods of extended durability.     

食品中蜡样芽孢杆菌污染的预测模型及风险评估进展

蜡样芽孢杆菌（Bacillus cereus）是一种食品中常见的食源性条件致病菌,其引发的食源性疾病不仅对食用者健康造成严重损伤,也对社会经济造成严重损失。本文综述了近年来国内外对食品中Bacillus cereus污染预测模型的研究及其在风险评估中的应用进展。将影响因素、食品基质、生产链环节、初级模型和次级模型常用拟合模型等进行总结,发现当前预测模型研究最多的是温度、食品成分、水分活度、pH值等因素对Bacillus cereus增殖影响,而温度是主要影响因素;食品主要集中于动物性食品和大米及其制品。Gompertz模型、Logistic模型、Baranyi模型、Weibull等模型常用于拟合生长或失活曲线,建立初级模型;二次多项式模型和平方根模型常用来拟合生长速率或延滞期的变化,建立次级模型;在此基础上,用软件系统建立三级模型。当前的风险评估研究仅涉及到生产环节或从销售到消费环节,尚未见到某类食品从原料环节到消费环节Bacillus cereus预测模型研究的报道。本文展望了食品中Bacillus cereus污染的风险评估研究,以期为今后的研究提供参考。     

北京市某区2021年5—9月生鲜蔬菜中蜡样芽胞杆菌检出情况和耐药特征分析

目的 对生鲜蔬菜中蜡样芽胞杆菌的分布和耐药特征进行分析。方法 采集北京市顺义区2021年5—9月生鲜蔬菜样本120份,对样本进行蜡样芽胞杆菌平板计数检测、增菌培养及增菌液呕吐毒素基因(ces)、蜡样芽胞杆菌16S r DNA基因实时荧光PCR检测,对分离株进行14种抗生素敏感性检测。结果 生鲜蔬菜中蜡样芽胞杆菌检出率为84.17%(101/120)。根茎类和叶菜类生鲜蔬菜蜡样芽胞杆菌检出率差异有统计学意义(χ²=14.181,P_校正=0.000);超市、农贸市场和农户菜地3类采集地点蜡样芽胞杆菌检出率差异有统计学意义(χ²=11.050,P=0.004),基于样本增菌液的ces检出率差异有统计学意义(P_Fisher=0.001)。12件ces⁺的生鲜蔬菜增菌液检测蜡样芽胞杆菌16S rDNA基因和ces基因Ct值均值分别为19.96和31.80,但均未能分离到ces⁺蜡样芽胞杆菌菌落。蜡样芽胞杆菌分离株对氨苄西林、青霉素、复方磺胺耐药率分别为100%、99...     

Immunization of the industrial fermentation starter culture strain of Saccharomyces cerevisiae to a contaminating killer toxin-producing Candida tropicalis

K3 killer trait was introduced into the fermentation starter strain of Saccharomyces cerevisiae BSP 1 in order to construct immune industrial strain that produces K3 type killer toxin and was resistant to Candida tropicalis (K⁺) contamination. Protoplasts of respiration-deficient Rho^o strain of S. cerevisiae NCYC 761 (K3) and S. cerevisiae BSP 1 were fused. The resulting respiration-competent hybrid with K3 type killer activity was selected on media containing a non-fermentable carbon source and by a killer zone assay in a plate test, respectively. The fusant was similar to the parent strain in its fermentation and sugar utilization patterns, growth rate, dough-raising properties and osmotolerance. The newly constructed S. cerevisiae BSP 1 (K3) inhibited the growth of C. tropicalis in a pH range from 3.5 to 5.0 and over a temperature range of 20–30°C.     

Kinetic studies on high-pressure inactivation of Bacillus stearothermophilus spores suspended in food matrices

Bacillus stearothermophilus spores ATCC 7953 can effectively be inactivated by high-pressure treatment, but only if it is applied at elevated temperatures; however, these temperatures are much lower compared to the temperature level used in heat inactivation under atmospheric pressure. Temperature and pressure in a range between 60 and 120°C and 50–600 MPa were applied to inactivate spores suspended in mashed broccoli and in cocoa mass. Utilizing an empirical mathematical model, derived from nth order kinetics, the survival curves of the spore strain investigated could be described accurately. The model can predict the impact of combined action of pressure and temperature on spore reduction. It was demonstrated that the inactivation of B. stearothermophilus spores ATCC 7953 improved with increasing treatment intensity. Beside intrinsic microbial inactivation mechanisms, the role of the pressure-induced shift in crystallization temperature of fat on spore inactivation in cocoa mass is discussed.     

Short communication: Presence of neutral metallopeptidase (npr) gene and proteolytic activity of Bacillus cereus isolated from dairy products

The control of proteolytic microorganisms is one of the main challenges of the dairy industry, due to their spoilage activity that jeopardizes the quality of their products. Seventy-four Bacillus cereus strains isolated from powdered, UHT, and pasteurized milks were tested for the presence of the neutral metallopeptidase (npr) gene and proteolytic activity at 7, 10, 25, 30, and 37°C. All strains had the npr gene, and proteolytic activity increased with the incubation temperature. The obtained results highlight the relevance of B. cereus as a spoiling agent in the dairy industry in terms of its genetic predisposition for proteolytic capacity, especially at room temperature.     

Formation of pyrazines and 2-acetyl-1-pyrroline by Bacillus cereus

The production of alkylpyrazines and 2-acetyl-1-pyrroline by different Bacillus cereus strains, which has been previously reported, was studied in detail. B. cereus ATCC 27522 produced the highest amounts of flavour compounds when grown as surface cultures on plate count agar. Pyrazine, methylpyrazine, 2,5-dimethylpyrazine, trimethylpyrazine and 3-ethyl-2,5-dimethylpyrazine were produced in low amounts. Since they were also detected in control flasks, an enzymatic formation was concluded to be unlikely. Only the production of 3-ethyl-2,5-dimethylpyrazine was in all cases significantly different from the control. Detailed precursor studies revealed that the production of 2-acetyl-1-pyrroline by B. cereus ATCC 27522 proceeds via acetylation of 1-pyrroline, a metabolic degradation product of proline and ornithine. Comparison of results obtained from dynamic headspace and simultaneous steam distillation – solvent extraction showed that the use of a non-thermal extraction method is essential to obtain reliable results on the biological formation of these Maillard flavour compounds.     

Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus

In this study, an effective substance was isolated from Bacillus subtilis SC-8, which was obtained from traditionally fermented soybean paste, cheonggukjang. The substance was purified by HPLC, and its properties were analyzed. It had an adequate antagonistic effect on Bacilluscereus, and its spectrum of activity was narrow. When tested on several gram-negative and gram-positive foodborne pathogenic bacteria such as Salmonella enterica, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes, no antagonistic effect was observed. Applying the derivative from B. subtilis SC-8 within the same genus did not inhibit the growth of major soybean-fermenting bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens. The range of pH stability of the purified antagonistic substance was wide (from 4.0 to >10.0), and the substance was thermally stable up to 60 °C. In the various enzyme treatments, the antagonistic activity of the purified substance was reduced with proteinase K, protease, and lipase; its activity was partially destroyed with esterase. Spores of B. cereus did not grow at all in the presence of 5 μg/mL of the purified antagonistic substance. The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254 because it absorbed to UV radiation at 254 nm.     

Low temperatures and fermentative metabolism limit peptidoglycan digestion of Bacillus cereus. Impact on colony forming unit counts

The impact of fermentative metabolism at low temperature on cell division of Bacillus cereus was studied. Fermentation at 37 °C had no influence on the division of bacteria. Aerobic cultures at 15 °C produced larger cells than at 37 °C, but cell division was normal. In fermentative cultures at 15 °C, no increase in CFU ml⁻¹ was observed. However, A₆₀₀ increased, due to formation of long filaments. Transmission electronic microscopy and light microscopy with fluorescent staining showed several nucleic acid entities separated by a hydrophobic membrane, indicating that each filament contained several individual cells attached by peptidoglycan. When left in air at room temperature, one filament gave several daughter cells, this means that one CFU formed by one filament may represent a greater contamination potential than one CFU formed by a single cell. Division was observed in cultures at 15 °C with anaerobic respiration in the presence of nitrates. Possible filamentous growth must thus be taken into account to avoid underestimating B. cereus growth in vacuum or modified atmosphere packaged foods stored at low temperature.