Emetic Strains Research Articles

Phylogenetic and toxinogenic characteristics of Bacillus cereus group members isolated from spices and herbs

The foodborne pathogen Bacillus (B.) cereus is a common contaminant in spices and herbs. To further characterise B. cereus and its closely related group members present in spices and herbs, we analysed presumptive B. cereus strains isolated from six different condiments with view to B. cereus group species, phylogenetic affiliation and toxinogenic potential.Of a total of 59 isolates 44 were identified as B. cereus sensu stricto (s.s.), four as B. toyonensis-like, five as B. thuringiensis, one as B. weihenstephanensis, two as B. pseudomycoides/B. mycoides and three as undefined B. cereus group species. A maximum of three different species occurred simultaneously in the same spice sample. The isolates comprised 33 multilocus (ML) sequence types (STs), which can be assigned to three different phylogenetic groups. Except two B. pseudomycoides/B. mycoides strains, all isolates were able to produce enterotoxins and one strain the emetic toxin cereulide as detected by an immunoassay and LC-MS, respectively. The prevalence of toxin genes was 96.6% for nheA, 94.9% for hblD, 50.8% for cytK-2 and 1.7% for ces. The emetic strain was characterised by ST 869, which for the first time was assigned to an emetic B. cereus (s.s.) strain and is not part of the previously known two emetic MLST clusters.Our results demonstrate that not only B. cereus (s.s.) but also toxin producing B. thuringiensis, B. weihenstephanensis and B. toyonensis-like strains could be detected in condiments. For some isolates MLST revealed disagreements between phylogenetic relationship and the classification as B. weihenstephanensis and B. mycoides based on previously described species markers.

Bacillus cereus-induced food-borne outbreaks in France, 2007 to 2014: epidemiology and genetic characterisation.

The aim of this study was to identify and characterise Bacillus cereus from a unique national collection of 564 strains associated with 140 strong-evidence food-borne outbreaks (FBOs) occurring in France during 2007 to 2014. Starchy food and vegetables were the most frequent food vehicles identified; 747 of 911 human cases occurred in institutional catering contexts. Incubation period was significantly shorter for emetic strains compared with diarrhoeal strains A sub-panel of 149 strains strictly associated to 74 FBOs and selected on Coliphage M13-PCR pattern, was studied for detection of the genes encoding cereulide, diarrhoeic toxins (Nhe, Hbl, CytK1 and CytK2) and haemolysin (HlyII), as well as panC phylogenetic classification. This clustered the strains into 12 genetic signatures (GSs) highlighting the virulence potential of each strain. GS1 (nhe genes only) and GS2 (nhe, hbl and cytK2), were the most prevalent GS and may have a large impact on human health as they were present in 28% and 31% of FBOs, respectively. Our study provides a convenient molecular scheme for characterisation of B. cereus strains responsible for FBOs in order to improve the monitoring and investigation of B. cereus-induced FBOs, assess emerging clusters and diversity of strains.

Characterization of three autolysins with activity against cereulide-producing Bacillus isolates in food matrices

Bacillus cereus is a pathogen related with diarrhoeal or emetic food poisoning cases, of which the latter caused by the cereulide-producing isolates are more severe with several reported lethal cases. It is therefore necessary to develop an effective strategy to prevent the propagation of B. cereus in the food supply. In this study, three autolysins from the cereulide-producing B. cereus group isolates, LysIS075, LysF8819.1 and LysCER057, were identified and characterized. The results showed that the three autolysins were highly lytic and bactericidal to the tested cereulide-producing B. cereus group strains and cross-lytic against other tested B. cereus group strains, and they could inhibit the spore germination and propagation of their tested derived emetic strains. Physical and chemical characterization showed that all the three autolysins were alkalophilic with the optimal activity at pH9.0 or 9.5 with one exception of LysF8819.1 also having significant lytic activity at pH5.0, and they all had relative strong lytic activity at 37–50°C during the 30minute assay. However, LysCER057 showed relative susceptibility to thermo-condition. Remarkably, the separate or cock-tail addition of the three autolysins in food matrices (milk and rice porridge) showed effective bactericidal activity within the tested 2h. All the results revealed that the three autolysins might be potential candidates to control emetic B. cereus strains in different applications.

Complete Genome Sequence of Bacteriophage Deep-Blue Infecting Emetic Bacillus cereus.

The Bacillus cereus emetic pathotype is responsible for important food-borne intoxications. Here, we describe the complete genome sequence of bacteriophage Deep-Blue, which is able to infect emetic strains of B. cereus. Deep-Blue is a 159-kb myophage of the Bastille-like group within the Spounavirinae.

MALDI-TOF MS portrait of emetic and non-emetic Bacillus cereus group members.

The number of foodborne intoxications caused by emetic Bacillus cereus isolates has increased significantly. As such, rapid and reliable methods to identify emetic strains appear to be clinically relevant. In this study, intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to differentiate emetic and non-emetic bacilli. The phyloproteomic clustering of 34 B. cereus emetic and 88 non-emetic isolates classified as B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus mycoides, showed (i) a clear separation of both groups at a similarity level of 43%, and (ii) a high relatedness among the emetic isolates (similarity of 78%). Specifically, 83 mass peak classes were recognized in the spectral window range between m/z 4000 and 12000 that were tentatively assigned to 41 protein variants based on a bioinformatic approach. Mass variation between the emetic and the non-emetic subsets was recorded for 27 of them, including ten ribosomal subunit proteins, for which inter-strain polymorphism was confirmed by gene sequencing. Additional peaks were assigned to other proteins such as small acid soluble proteins, cold shock proteins and hypothetical proteins, e.g., carbohydrate kinase. Moreover, the results were supported by in silico analysis of the biomarkers in 259 members of B. cereus group, including Bacillus anthracis, based on their whole-genome sequences. In conclusion, the proteomic profiling by MALDI-TOF MS is a promising and rapid method for pre-screening B. cereus to identify medically relevant isolates and for epidemiologic purposes.

Characterization of Bacillus cereus isolates from local dairy farms in China.

Bacillus cereus is an important opportunistic foodborne pathogen. In the present work, a total of 306 milk and environmental samples were collected from 10 local dairy farms in Beijing, China. Of the 92 B. cereus-like isolates, 88 and 4 belonged to B. cereus and B. thuringiensis, respectively. The prevalence of B. cereus isolates in bedding, feces, feed, liquid manure and raw milk was 93.3%, 78.9%, 41.2%, 100.0% and 9.8%, respectively. Three main toxin genes nhe, hbl and ces were detected with rates of 100.0%, 78.3% and 1.1%, but no strain harbored cytK1 The production of Nhe, Hbl and cereulide could be confirmed by specific monoclonal antibodies-based enzyme immunoassays in 94.6%, 70.7% and 1.1% of all isolates, respectively. Cytotoxicity tests were used to further corroborate the results of genetic and protein-based assays; 91.3% of the isolates showed cytotoxicity to Vero cells. All isolates were tested for antimicrobial resistance against 17 antibiotics. All isolates were resistant to lincomycin, retapamulin, tiamulin and valnemulin, while two strains were susceptible to ampicillin and ceftiofur. A total of 16 isolated strains were resistant to tetracycline. Since spores of B. cereus are not inactivated during manufacturing of most milk products, contamination of milk with B. cereus on the farm level may represent a potential hazard, particularly with respect to emetic toxin-producing strains.

Identification of Cereulide-Producing Bacillus cereus by Nucleic Acid Chromatography and Reverse Transcription Real-Time PCR.

RNA extracts were analyzed with a nucleic acid sequence-based amplification (NASBA) - nucleic acid chromatography and a reverse transcription-quantitative PCR assay (RT-qPCR) based on the TaqMan probe for identification of cereulide-producing Bacillus cereus. All 100 emetic B. cereus strains were found to give positive results, but 50 diarrheal B. cereus strains and other bacterial species showed negative results in the NASBA-chromatography. That is, the assay could selectively identify the emetic strains among B. cereus strains. Also, the B. cereus contents of more than 10(7) cfu/ml were required for the identification of the cereulide-producing strains in this assay. In qRT-PCR assays, all 100 emetic type strains of B. cereus produced 10(2) - 10(4) copy numbers per ng of the RNA preparation, and the strains produced 10(4) copies including ones which had the high vacuolation activities of HEp-2 cells.

Growth and inhibition by spices of growth from spores of enterotoxigenic Bacillus cereus in cooked rice

Bacillus cereus is a pathogenic spore-forming bacterium implicated in cases of diarrheal and emetic type of foodborne illness. We previously found that enterotoxigenic B. cereus is widely present in retail spices. Here we analyzed the spore heat resistance of nine diarrheal strains isolated from spices. Seven had D95°C values ranging from 0.64 to 3.53 min while two emetic strains had D95°C values of 7.04 min and 6.64 min. The ability of selected strains to grow in cooked rice at temperatures 20 °C, 17 °C and 12 °C was determined as well as their toxin expression capability. After 48 h, B. cereus levels of 1.26 × 107 and 3.8 × 107 CFU/g were obtained in cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 104 CFU/g even after 48 h of incubation. The increase in the aerobic, mesophilic bacterial population (APC) and B. cereus population naturally present in 0.1% crushed pepper added to cooked rice was determined over a period of 48 h at 20 °C and 17 °C. Levels of B. cereus in pepper/rice samples reached a maximum of 1600 MPN/g at 20 °C while the APC was 2.4 × 108/g at this temperature. When thyme, containing the same initial natural level of B. cereus, was added to rice in place of pepper, more than a five-fold greater level of B. cereus was detected at 20 °C. Since thyme contained initial APC of 2.5 log10 less than pepper we conclude that the high APC functions in a competitive-exclusion manner, minimizing the growth of B. cereus and potentially other agents of foodborne illness. This is particularly relevant in instances of temperature abuse of foods and may explain the low incidence of foodborne illness due to B. cereus despite its widespread presence shown in previous surveys of market spices.

Ces locus embedded proteins control the non-ribosomal synthesis of the cereulide toxin in emetic Bacillus cereus on multiple levels.

The emetic toxin cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like megaplasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the non-ribosomal peptide synthetase (NRPS), gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling cereulide synthesis on a transcriptional level, while the ABC transporter CesCD was found to be essential for post-translational control of cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase) embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY.

A novel pentaplex real time (RT)- PCR high resolution melt curve assay for simultaneous detection of emetic and enterotoxin producing Bacillus cereus in food

Bacillus cereus causing emetic and diarrheal type of food poisoning is widely distributed in nature and is therefore considered a major foodborne pathogen. There is a growing demand for fast, accurate, reliable and economic detection of potentially toxigenic B. cereus. To improve differential diagnosis of toxigenic B. cereus, a highly sensitive pentaplex RT- PCR high resolution melt curve assay was developed for simultaneous detection of 4 major enterotoxim genes (cytK, entFM, hblD, nheA) and emetic toxin gene (ces). The average melting temperatures (Tm) of PCR products were 72.2 °C (ces), 74.23 °C (cytK), 76.55 °C (nheA), 78.42 °C (entFM) and 81.90 (hblD). The multiplex assay was evaluated using 71 bacterial strains including 17 emetic B. cereus reference strains, 9 enterotoxic B. cereus reference strains, 4 B. cereus group members, 23 wild B. cereus strains, 18 non-target strains, and was further tested on artificially inoculated foods. The detection limit in food samples was approximately 103 CFU/g without enrichment and 101 CFU/g was observed following 7 h enrichment. The DNA intercalating dye SYTO9 used in this study generated high resolution melt curve peaks for the target strains and genes in which the peaks were sharp and easily distinguishable from each other. Thus, the developed multiplex real-time (RT) PCR approach can be a reliable tool for the identification of emetic and enterotoxic strains of B. cereus present in food and food-related samples.

Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR (PMA-mPCR)

Bacillus cereus can cause emetic and diarrheal food poisoning. It is widespread in nature and therefore, considered a major foodborne pathogen. To develop a sensitive and reliable assay for detecting enterotoxin genes (nheA, entFM, hblD, cytK) and emetic toxin (ces), specific primers each targeting one individual gene were designed. Propidium monoazide (PMA) was coupled with the developed multiplex PCR (mPCR) for the detection of viable B. cereus. The inclusivity and exclusivity of the PMA-mPCR was confirmed using a panel of 44 strains including 17 emetic and 9 enterotoxic B. cereus reference strains and 18 non-target strains. The limit of detection (LOD) without PMA treatment in pure DNA was 2 pg/reaction tube. The LOD of mPCR assay in pure heat-killed dead bacteria was 4.0 × 102 CFU/mL. Also, the LOD on the viable bacteria with or without PMA treatment was similar (3.8 × 102 CFU/mL) showing that the PMA treatment did not significantly decrease sensitivity. Finally, the newly developed PMA-mPCR successfully detected 4.8 × 103 and 3.6 × 103 CFU/g of viable B. cereus F4810/72 (emetic) and B. cereus ATCC 12480 (enterotoxic) reference strains, respectively, in food samples. Hence, this study combines PMA and mPCR to detect viable B. cereus with a wide range of toxin detection (5 toxins). Thus, the novel PMA-mPCR assay developed in this study is a rapid and efficient diagnostic tool for the monitoring of viable B. cereus in food samples and potentially other samples via appropriate DNA extraction.

An emetic Bacillus cereus outbreak in a kindergarten: detection and quantification of critical levels of cereulide toxin.

A Bacillus cereus-related emetic outbreak was reported in a Belgian kindergarten. High levels of emetic B. cereus (>1.5E+07 colony-forming units/g) were detected in the food leftovers, and the presence of an emetic strain was confirmed in feces. Emetic toxin levels ranging up to 4.2 μg/g were also quantified in the leftovers by liquid chromatography coupled to tandem mass spectrometry (LC-MS(2)) analysis. Those levels, although moderate in comparison with earlier published intoxications, provoked profuse-vomiting episodes in 20 toddlers aged between 10 and 18 months. Few studies have focused on the levels of emetic toxin implicated in food intoxications. This publication emphasizes the importance of defining toxic doses of emetic toxin among high-risk population groups.

Analysis of emetic toxin production by Bacillus species using cellular cytotoxicity, molecular, and chromatographic assays

In this study, we analyzed production of the emetic toxin, cereulide, in 30 Bacillus strains by using a cellular cytotoxicity (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; MTT) assay, polymerase chain reaction (PCR), and micellar electrokinetic chromatographycapillary electrophoresis (MEKC-CE) analysis. The Bacillus cereus emetic strains produced 60 ∼ 227 µg/mL of cereulide when analyzed using MEKC-CE. Some other Bacillus species, including B. subtilis, B. pumilus, and B. megaterium, produced putative cereulide at levels similar to those produced by emetic B. cereus, whereas B. mycoides and B. thuringiensis did not produce the putative toxin or produced it at a concentration less than 2 µg/mL. Only B. cereus emetic strains were found to be toxic to bovine fibroblast cells, with the exception of one Bacillus diarrheal strain. The PCR results correlated with the MTT assay results, except in the case of one B. cereus diarrheal strain. This strain may produce either unusually heat-stable enterotoxins or is a co-producer of emetic and diarrheal toxins. Collectively, our results indicate that several Bacillus species produce toxin(s) with structures and properties similar to those of cereulide.

Characterization of three Bacillus cereus strains involved in a major outbreak of food poisoning after consumption of fermented black beans (Douchi) in Yunan, China.

Three Bacillus cereus strains isolated from an outbreak of food poisoning caused by the consumption of fermented black beans (douchi) containing B. cereus is described. The outbreak involved 139 persons who had nausea, vomiting, and diarrhea. The strains were isolated from vomit and the unprepared douchi. Two of the strains produced the emetic toxin cereulide, as evidenced by polymerase chain reaction analysis for the presence of the nonribosomal synthetase cluster responsible for the synthesis of cereulide and by chemical analysis by high-performance liquid chromatography-mass spectrometry. These two strains belong to genetic group III of B. cereus, and multiple locus sequence typing revealed that the type was ST26, as a major part of B. cereus emetic strains. One of these strains produced significantly more cereulide at 37°C than the type cereulide producer (F4810/72), and it was also able to produce the toxin at 40°C and 42°C. The third strain belongs to genetic group IV, and it is a new multiple locus sequence type closely related to strains that are cytotoxic and enterotoxigenic. It possesses genes for hemolysin BL, nonhemolytic enterotoxin, and cytotoxin K2; however, it varies from the majority of strains possessing genes for hemolysin BL by not being hemolytic. Thus, two B. cereus strains producing the emetic toxin cereulide and a strain producing enterotoxins might have been involved in this food-poisoning incident caused by the consumption of a natural fermented food. The ability of one of the strains to produce cereulide at ≥37°C makes it possible that it is produced in the human gut in addition to occurring in the food.

A case of intoxication due to a highly cytotoxic Bacillus cereus strain isolated from cooked chicken

Outbreaks of Bacillus cereus infection/intoxication are not commonly reported because symptoms are often mild, and the disease is self-limiting. However, hypervirulent strains increase health risks. We report a case, which occurred in Argentina, of severe food poisoning illness on a healthy adult woman associated to B. cereus strain MVL2011. The studied strain was highly cytotoxic, showed high ability to detach Caco-2 cells and was positive for the hblA, hblB, and hblC genes of the hbl complex, bceT, entS and ces. As it is considered that B. cereus emetic cluster evolved from a panmictic population of diarrheal strains, B. cereus MVL2011 could constitute an intermediate strain between diarrheal and emetic strains.

Diversity of pulsed-field gel electrophoresis patterns of cereulide-producing isolates ofBacillus cereusandBacillus weihenstephanensis

Bacillus cereus is an important foodborne pathogen causing diarrhoea, emesis and in, rare cases, lethal poisonings. The emetic syndrome is caused by cereulide, a heat-stable toxin. Originally considered as a rather homogenous group, the emetic strains have since been shown to display some diversity, including the existence of two clusters of mesophilic B. cereus and psychrotolerant B. weihenstephanensis. Using pulsed-field gel electrophoresis (PFGE) analysis, this research aimed to better understand the diversity and spatio-temporal occurrence of emetic strains originating from environmental or food niches vs. those isolated from foodborne cases. The diversity was evaluated using a set of 52 B. cereus and B. weihenstephanensis strains isolated between 2000 and 2011 in ten countries. PFGE analysis could discriminate 17 distinct profiles (pulsotypes). The most striking observations were as follows: (1) more than one emetic pulsotype can be observed in a single outbreak; (2) the number of distinct isolates involved in emetic intoxications is limited, and these potentially clonal strains frequently occurred in successive and independent food poisoning cases; (3) isolates from different countries displayed identical profiles; and (4) the cereulide-producing psychrotolerant B. weihenstephanensis were, so far, only isolated from environmental niches.

Emetic Bacillus cereus are more volatile than thought: recent foodborne outbreaks and prevalence studies in Bavaria (2007-2013).

Several Bacillus cereus strains possess the genetic fittings to produce two different types of toxins, the heat-stable cereulide or different heat-labile proteins with enterotoxigenic potential. Unlike the diarrheal toxins, cereulide is (pre-)formed in food and can cause foodborne intoxications shortly after ingestion of contaminated food. Based on the widely self-limiting character of cereulide intoxications and rarely performed differential diagnostic in routine laboratories, the real incidence is largely unknown. Therefore, during a 7-year period about 4.300 food samples linked to foodborne illness with a preliminary report of vomiting as well as food analysed in the context of monitoring programs were investigated to determine the prevalence of emetic B. cereus in food environments. In addition, a lux-based real-time monitoring system was employed to assess the significance of the detection of emetic strains in different food matrices and to determine the actual risk of cereulide toxin production in different types of food. This comprehensive study showed that emetic strains are much more volatile than previously thought. Our survey highlights the importance and need of novel strategies to move from the currently taxonomic-driven diagnostic to more risk orientated diagnostics to improve food and consumer safety.

Toxin producing Bacillus cereus persist in ready-to-reheat spaghetti Bolognese mainly in vegetative state

The potential of Bacillus cereus to cause a diarrheal toxico-infection is related to its ability to perform de novo enterotoxin production in the small intestine. A prerequisite for this is presence of sufficient numbers of B. cereus that have survived gastro-intestinal passage. It is known that the percentage of survival is much smaller for vegetative cells in comparison to spores and it is therefore important to know the state in which B. cereus is ingested. The results of the current study performed on twelve B. cereus strains, comprising both diarrheal and emetic type, indicate that exposure via contaminated foods mainly concerns vegetative cells. Inoculated vegetative cells grew to high counts, with the growth dynamic depending on the storage temperature. At 28°C growth to high counts resulted in spore formation, in general, after 1day of storage. One strain was an exception, producing spores only after 16days. At 12°C obtained high counts did not result in spore formation for 11 of 12 tested strains after two weeks of storage. The highest counts and time to sporulation were different between strains, but no difference was observed on the group level of diarrheal and emetic strains. The spore counts were always lower than vegetative cell counts and occurred only when food was obviously sensory spoiled (visual and odor evaluation). Similar observations were made with food inoculated with B. cereus spores instead of vegetative cells. Although the prospect of consuming spores was found very weak, the numbers of vegetative B. cereus cells were high enough, without obvious sensory deviation, to survive in sufficient level to cause diarrheal toxico-infection.

PCR detection of cytK gene in Bacillus cereus group strains isolated from food samples

A method for detection of the cytotoxin K cytK structural gene and its active promoter preceded by the PlcR-binding box, controlling the expression level of this enterotoxin, was developed. The method was applied for the purpose of the analysis of 47 bacterial strains belonging to the Bacillus cereus group isolated from different food products. It was found that the majority of the analyzed strains carried the fully functional cytK gene with its PlcR regulated promoter. The cytK gene was not detected in four emetic strains of Bacillus cereus carrying the cesB gene and potentially producing an emetic toxin — cereulide. The cytotoxin K gene was detected in 4 isolates classified as Bacillus mycoides and one reference strain B. mycoides PCM 2024. The promoter region and the N-terminal part of the cytK gene from two strains of B. mycoides (5D and 19E) showed similarities to the corresponding sequences of Bacillus cereus W23 and Bacillus thuringiensis HD-789, respectively. It was shown for the first time that the cytK gene promoter region from strains 5D and 19E of Bacillus mycoides had a similar arrangement to the corresponding sequence of Bacillus cereus ATCC 14579. The presence of the cytK gene in Bacillus mycoides shows that this species, widely recognized as nonpathogenic, may pose potential biohazard to human beings.

Germination and proliferation of emetic Bacillus cereus sensu lato strains in milk

The Bacillus cereus sensu lato group includes potentially pathogenic bacteria that are ubiquitous in the environment and, importantly, could also be present in food products. This study focuses on emetic isolates which presumably could cause acute food poisoning and emetic syndrome. Here, we evaluate the ability of psychrotolerant Bacillus weihenstephanensis MC118 (isolated from soil) and mesophilic B. cereus BOD3/9 isolated from milk to germinate and multiply at 7 and 30°C. Whereas the rates of germination at 30°C in milk and nutrient broth of MC118 and BOD3/9 were similar, MC118, but not BOD3/9, proliferated to achieve relatively high numbers (∼10(6) colony-forming units/g) within 7days of incubation at 7°C. Mesophilic BOD3/9 showed a slight decrease of cell concentration in similar studies at 7°C. Genotyping with repetitive extragenic palindromic sequence-based PCR and pulsed field gel electrophoresis revealed significant similarities between BOD3/9 and emetic reference B. cereus F4810/72 strain, while the B. weihenstephanensis MC118 isolate was more similar to the B. weihenstephanensis non-emetic reference DSMZ11821 strain. Our data suggest that emetic isolates that are also psychrotolerant, such as MC118, could constitute a hazard in the dairy industry, where milk could be a suitable medium for germination and growth.