Genetic diversity, antimicrobial resistance and toxigenic profiles of Bacillus cereus isolated from food in Brazil over three decades

Bacillus cereus is an ever-present problem. It is widely distributed in several environments such as soil and plants and is commonly isolated from food and additives. In this study we analyzed 97 foodborne B. cereus sensu stricto strains isolated in Brazil in the 1980's, 1990's and 2000's in order to investigate the genetic diversity (assessed by Rep-PCR), antimicrobial resistance and toxigenic profiles (presence of hblA, hblC and hblD; nheA, nheB and nheC as well as cytK, ces and entFM genes) of such strains. The majority of the strains (79, 81.4%) were β-hemolytic. The NHE complex was found in 82 strains (84.5%) and HBL complex was found in 61 (62.9%) strains. All strains were negative to ces. The cytK-2 gene was found in 44 (45.4%) strains. The predominant toxigenic pattern was type I (32, 33%) which included strains positive for all toxin genes but ces. Computer assisted cluster analysis of Rep-PCR profiles showed a high genetic diversity. Seven major clusters comprising two or more strains were found and cluster 1 was predominant (ten strains, nine of them showing 100% similarity). This cluster included strains isolated in the 1980's and the 1990's. Cluster analysis of Rep-PCR profiles based on decade of isolation, source, hemolytic pattern, toxigenic and antibiotic resistance patterns revealed a similar clustering pattern as found in the analysis including all strains. The inability to observe a predominant band pattern when Rep-PCR cluster analysis was based on decade of isolation suggests that this diversity has been maintained over time. All strains were susceptible to gentamicin. We detected resistance to tetracycline (11 strains showing intermediate resistance and nine completely resistant strains), clindamycin (ten intermediate strains) and vancomycin (one strain). Clindamycin resistance showed statistical association with strains isolated in 2000's. The predominant resistance pattern was type A (72, 72.2%) which included strains susceptible to all drugs tested. Our results suggest that the majority of the strains present in several types of food in Brazil pose a potential risk to cause food poisoning due to the high prevalence of toxin genes found in these strains. However, additional studies involving cytotoxicity tests and affiliation of these strains to phylogenetic groups based on molecular data would be useful to better evaluate this potential and could provide a more accurate indication of the risk.     

Prevalence of Bacillus cereus in dried milk products used by Chilean School Feeding Program

The prevalence of Bacillus cereus, in a total of 381 samples of dried milk products (milk with rice, milk substitute, milk powder, milk-cereal-rice, pudding milk, flan, and mousse) used by the Chilean School Feeding Program, was investigated. The potential of 94 selected isolates of B. cereus to produce diarrhoeal enterotoxin (by the BCET-RPLA test) in BHI culture, as well as the ability of enterotoxigenic-strains to grow at psychrotrophic temperatures were also verified. B. cereus was found in 175 of 381 of the samples analysed (45.9%), reaching levels from 3.0 to 10(4) spores g(-1). As expected, the higher prevalence and counts were observed in those products that contained whole rice, cereals and pulses extruded, and food additives. Of the 94 isolates of B. cereus tested for diarrhoeal enterotoxin production, 28 (29.8%) were positive, and none of these was able to grow at < or = 7 degrees C. The prevalence of B. cereus in dried milk products analysed was fairly high, although it was present in low number. However, as they were composed to a large extent of enterotoxigenic mesophilic strains, the potential risk for the safety of reconstituted products held at improper temperature should not be neglected.     

Toxin profile, antibiotic resistance, and phenotypic and molecular characterization of Bacillus cereus in Sunsik

Sunsik, a ready-to-eat food in Korea, is comprised of various agricultural and marine products, and has been an important concern in Bacillus cereus food poisoning. The aim of this study was to investigate the toxin profiles, genotypic and phenotypic patterns as well as antibiotic resistance of B. cereus strains isolated from Sunsik. A subtyping method known as automated repetitive sequence-based PCR system (DiversiLab™) was used to assess the intraspecific biodiversity of these isolates. Thirty-five B. cereus strains were isolated from 100 commercial Sunsik samples, all of which harbored at least 1 enterotoxin gene. The detection rates of nheABC, hblCDA, cytK, and entFM enterotoxin gene among all isolates were 97%, 86%, 77%, and 100%, respectively. Most strains also produced corresponding enterotoxins such as HBL (83%) and NHE (94%). One strain (2.9%) carried the emetic toxin genes, including ces and EM1, and was positive for the HEp-2 cell emetic toxin assay. Most strains were positive for various biochemical tests such as salicin hydrolysis (86%), starch fermentation (89%), hemolysis (89%), motility test (100%) and lecithinase hydrolysis (89%). All isolates were susceptible to most antibiotics although they were highly resistant to β-lactam antibiotics. By using the automated rep-PCR system, all isolates were successfully differentiated, indicating the diversity of B. cereus strains present in Sunsik.     

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.     

Emetic toxin producing Bacillus cereus Korean isolates contain genes encoding diarrheal-related enterotoxins

Bacillus cereus can cause the diarrheal and emetic type of food poisoning but the symptoms of emetic food poisoning caused by B. cereus occasionally include emesis and diarrhea. The enterotoxin characteristics of emetic toxin (cereulide) producing B. cereus were needed to be determined. Therefore, forty B. cereus strains isolated from various sources in Korea were investigated for the presence of enterotoxin genes. All strains were confirmed to produce the emetic toxin using HPLC-MS methods. The rates of the nheABC, hblCDA, entFM and cytK genes amongst emetic toxin producing B. cereus strains were 82.5, 7.5, 50.0 and 27.5%, respectively. Pattern III harbored nheABC and entFM genes and pattern V processed entFM gene and were shown to be the major patterns, being present in 55.0% (21 of 40) of the emetic toxin producing B. cereus strains. Our findings revealed that 34 (85.0%) of 40 emetic toxin producing B. cereus strains isolated in Korea have the potential to cause diarrheal and emetic type of food poisoning, simultaneously. Thus, emetic toxin and enterotoxin genes should be constantly screened to provide insight into B. cereus food poisoning.     

Minimizing the level of Bacillus cereus spores in farm tank milk

In a year-long survey on 24 Dutch farms, Bacillus cereus spore concentrations were measured in farm tank milk (FTM), feces, bedding material, mixed grass and corn silage, and soil from the pasture. The aim of this study was to determine, in practice, factors affecting the concentration of B. cereus spores in FTM throughout the year. In addition, the results of the survey were used in combination with a previously published modeling study to determine requirements for a strategy to control B. cereus spore concentrations in FTM below the MSL of 3 log₁₀ spores/L. The B. cereus spore concentration in FTM was 1.2 ± 0.05 log₁₀ spores/L and in none of samples was the concentration above the MSL. The spore concentration in soil (4.9 ± 0.04 log₁₀ spores/g) was more than 100-fold higher than the concentration in feces (2.2 ± 0.05 log₁₀ spores/g), bedding material (2.8 ± 0.07 log₁₀ spores/g), and mixed silage (2.4 ± 0.07 log₁₀ spores/g). The spore concentration in FTM increased between July and September compared with the rest of the year (0.5 ± 0.02 log₁₀ spores/L difference). In this period, comparable increases of the concentrations in feces (0.4 ± 0.03 log₁₀ spores/g), bedding material (0.5 ± 0.05 log₁₀ spores/g), and mixed silage (0.4 ± 0.05 log₁₀ spores/g) were found. The increased B. cereus spore concentration in FTM was not related to the grazing of cows. Significant correlations were found between the spore concentrations in FTM and feces (r = 0.51) and in feces and mixed silage (r = 0.43) when the cows grazed. The increased concentrations during summer could be explained by an increased growth of B. cereus due to the higher temperatures. We concluded that year-round B. cereus spores were predominantly transmitted from feeds, via feces, to FTM. Farmers should take measures that minimize the transmission of spores via this route by ensuring low initial contamination levels in the feeds (<3 log₁₀ spores/g) and by preventing growth of B. cereus in the farm environment. In addition, because of the extremely high B. cereus spore concentrations in soil, the contamination of teats with soil needs to be prevented.     

Predictive modeling of Bacillus cereus spores in farm tank milk during grazing and housing periods

The shelf life of pasteurized dairy products depends partly on the concentration of Bacillus cereus spores in raw milk. Based on a translation of contamination pathways into chains of unit-operations, 2 simulation models were developed to quantitatively identify factors that have the greatest effect on the spore concentration in milk. In addition, the models can be used to determine the reduction in concentration that could be achieved via measures at the farm level. One model predicts the concentration when soil is the source of spores, most relevant during grazing of cows. The other model predicts the concentration when feed is the main source of spores, most relevant during housing of cows. It was estimated that when teats are contaminated with soil, 33% of the farm tank milk (FTM) contains more than 3 log₁₀ spores/L of milk. When feed is the main source, this is only 2%. Based on the predicted spore concentrations in FTM, we calculated that the average spore concentration in raw milk stored at the dairy processor during the grazing period is 3.5 log₁₀ spores/L of milk and during the housing period is 2.1 log₁₀ spores/L. It was estimated that during the grazing period a 99% reduction could be achieved if all farms minimize the soil contamination of teats and teat cleaning is optimized. During housing, reduction of the concentration by 60% should be feasible by ensuring spore concentrations in feed below 3 log₁₀ spores/g and a pH of the ration offered to the cows below 5. Implementation of these measures at the farm level ensures that the concentration of B. cereus spores in raw milk never exceeds 3 log₁₀ spores/L.     

Bacillus cereus in free-stall bedding

To increase the understanding of how different factors affect the bacterial growth in deep sawdust beds for dairy cattle, the microbiological status of Bacillus cereus and coliforms in deep sawdust-bedded free stalls was investigated over two 14-d periods on one farm. High counts of B. cereus and coliforms were found in the entire beds. On average, 4.1 log₁₀B. cereus spores, 5.5 log₁₀B. cereus, and 6.7 log₁₀ coliforms per gram of bedding could be found in the upper layers of the sawdust likely to be in contact with the cows’ udders. The highest counts of B. cereus spores, B. cereus, and coliforms were found in the bedding before fresh bedding was added, and the lowest immediately afterwards. Different factors of importance for the growth of B. cereus in the bedding material were explored in laboratory tests. These were found to be the type of bedding, pH, and the type and availability of nutrients. Alternative bedding material such as peat and mixtures of peat and sawdust inhibited the bacterial growth of B. cereus. The extent of growth of B. cereus in the sawdust was increased in a dose-dependent manner by the availability of feces. Urine added to different bedding material raised the pH and also led to bacterial growth of B. cereus in the peat. In sawdust, a dry matter content greater than 70% was needed to lower the water activity to 0.95, which is needed to inhibit the growth of B. cereus. In an attempt to reduce the bacterial growth of B. cereus and coliforms in deep sawdust beds on the farm, the effect of giving bedding daily or a full replacement of the beds was studied. The spore count of B. cereus in the back part of the free stalls before fresh bedding was added was 0.9 log units lower in stalls given daily bedding than in stalls given bedding twice weekly. No effect on coliform counts was found. Replacement of the entire sawdust bedding had an effect for a short period, but by 1 to 2 mo after replacement, the counts of B. cereus spores in the beds had increased about 2 log units and were as high as they were before bed replacement. Therefore, free-stall management could, to a limited extent, reduce the content of B. cereus spores in the beds by daily bedding and entire bed replacement.     

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.     

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.     

Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme

The objective of this work was to study high hydrostatic pressure (HHP) inactivation of spores of Bacillus cereus ATCC 9139 inoculated in model cheeses made of raw milk, together with the effects of the addition of nisin or lysozyme. The concentration of spores in model cheeses was approximately 6-log10 cfu/g of cheese. Cheeses were vacuum packed and stored at 8 degrees C. All samples except controls were submitted to a germination cycle of 60 MPa at 30 degrees C for 210 min, to a vegetative cells destruction cycle of 300 or 400 MPa at 30 degrees C for 15 min, or to both treatments. Bacillus cereus counts were measured 24 h and 15 d after HHP treatment. The combination of both cycles improved the efficiency of the whole treatment. When the second pressure-cycle was of 400 MPa, the highest inactivation (2.4 +/- 0.1 log10 cfu/g) was obtained with the presence of nisin (1.56 mg/L of milk), whereas lysozyme (22.4 mg/L of milk) did not increase sensitivity of the spores to HHP. For nisin (0.05 and 1.56 mg/L of milk), no significant differences were found between counts at 24 h and 15 d after treatment. Considering that mesophilic spore counts usually range from 2.6 to 3.0 log10 cfu/ml in raw milk, HHP at mild temperatures with the addition of nisin may be useful for improving safety and preservation of soft curd cheeses made from raw milk.     

Inhibition of Bacillus cereus spore outgrowth and multiplication by chitosan

Bacillus cereus is an endospore-forming bacterium able to cause food-associated illness. Different treatment processes are used in the food industry to reduce the number of spores and thereby the potential of foodborne disease. Chitosan is a polysaccharide with well-documented antibacterial activity towards vegetative cells. The activity against bacterial spores, spore germination and subsequent outgrowth and growth (the latter two events hereafter denoted (out)growth), however, is poorly documented. By using six different chitosans with defined macromolecular properties, we evaluated the effect of chitosan on Bacillus cereus spore germination and (out)growth using optical density assays and a dipicolinic acid release assay. (Out)growth was inhibited by chitosan, but germination was not. The action of chitosan was found to be concentration-dependent and also closely related to weight average molecular weight (M_w) and fraction of acetylation (F_A) of the biopolymer. Chitosans of low acetylation (F_A = 0.01 or 0.16) inhibited (out)growth more effectively than higher acetylated chitosans (F_A = 0.48). For the F_A = 0.16 chitosans with medium (56.8 kDa) and higher M_w (98.3 kDa), a better (out)growth inhibition was observed compared to low M_w (10.6 kDa) chitosan. The same trend was not evident with chitosans of 0.48 acetylation, where the difference in activity between the low (19.6 kDa) and high M_w (163.0 kDa) chitosans was only minor. In a spore test concentration corresponding to 10²-10³ CFU/ml (spore numbers relevant to food), less chitosan was needed to suppress (out)growth compared to higher spore numbers (equivalent to 10⁸ CFU/ml), as expected. No major differences in chitosan susceptibility between three different strains of B. cereus were detected. Our results contribute to a better understanding of chitosan activity towards bacterial spore germination and (out)growth.     

A new method for rapid and quantitative detection of the Bacillus cereus emetic toxin cereulide in food products by liquid chromatography-tandem mass spectrometry analysis

The Bacillus cereus emetic toxin cereulide causes foodborne intoxication, which may occasionally result in severe disease, and even death. To differentially diagnose the emetic-type of foodborne disease caused by B. cereus and assess the safety of commercial food, we developed a rapid method to quantitate cereulide. This method was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for the extraction of cereulide from food using a normal-phase silica gel cartridge. The limits of detection and quantification were 0.1 and 0.5 ng of cereulide ml⁻¹, respectively. Spiked cereulide was reproducibly recovered with over 67% efficiency from nine diverse foods implicated in cereulide food poisoning. The recovery rate, reproducibility, and intermediate precision for this single laboratory validation using boiled rice were 87.1%, 4.4%, and 7.0%, respectively. Further, we detected a wide range of cereulide concentrations in leftover food and vomitus samples from two emetic foodborne outbreaks. LC-MS/MS analysis correlated closely with those acquired using the HEp-2 cell assay, and quantitated cereulide from 10 food samples at least five times faster than the bioassay. This new method will provide clinicians with an improved tool for more rapidly and quantitatively determining the presence of cereulide in food and diagnosing food poisoning caused by cereulide.     

Influence of the sporulation temperature on the impact of the nutrients inosine and l-alanine on Bacillus cereus spore germination

The effect of sporulation temperature on Bacillus cereus spore germination triggered by the nutrient germinants inosine and l-alanine was investigated. The germination (expressed as % A(600nm) fall) of heat-activated spores of B. cereus strain ATCC14579 produced at 20 and 37 degrees C, and of psychrotrophic strains LM9 and D15 produced at 15 and 37 degrees C was followed in a germination buffer containing inosine at concentrations between 0.01 and 10mmoll(-1), or l-alanine between 1 and 100mmoll(-1). Spores wet-heat resistance at 90 degrees C was also determined. Spores of the three strains produced at the lowest temperatures generally showed a higher germination capacity in response to both inosine and in l-alanine than those produced at 37 degrees C, and were also more susceptible to heat. Low sporulation temperature is confirmed as a detrimental factor to B. cereus spore wet-heat resistance, and conversely gave spores with a sensitivity to the nutrient germinants inosine and l-alanine higher than that of spores formed close to the maximal sporulation temperature.     

Attachment strength and biofilm forming ability of Bacillus cereus on green-leafy vegetables: cabbage and lettuce

The present study was designed to investigate the ability of six Bacillus cereus strains to attach and form biofilm on cabbage and lettuce surfaces. These six strains were; a reference strain DSMZ 345 and five biofilm-producing strains (aquatic strains; TUB8, TUB30, TUB31, TUB32 and TUB33) isolated from drinking-water distribution network. Hydrophobicity, biofilm formation ability, attachment strength (S_R) of spores and vegetative cells of the six B. cereus strains were also determined. Due to their high hydrophobicity, spores of all strains had high ability to attach polystyrene and did not affect by dilution of tryptone soy broth (TSB, 1:20 v/v) in the in vitro experiment. Significant (p < 0.05) enhancement in vitro biofilm formation by vegetative cells of B. cereus was recorded in the diluted TSB. The highest biofilm formation on cabbage and lettuce surfaces was obtained by spores and vegetative cells of all tested strains on the 4^th hour of the incubation period. These populations were significantly (p < 0.05) increased by elongating incubation time from 4 h to 24 h except DSMZ 345 and TUB8. Biofilm formation behavior obtained by B. cereus spores and vegetative cells on the polystyrene surface was different compared with that recorded on produce surface. The S_R of both spores and vegetative cells of the studied strains to the lettuce surface was higher than that of the cabbage surface. The hydrophobicity, biofilm formation and S_R of spores and vegetative cells of the biofilm-producing strains were higher than that of the reference strain DSMZ 345. Scanning electron microscopy (SEM) exposed random distribution of cells either on the surface or cut edge, without clear obvious affinity for the surface structures. Increasing in the presence of large clusters of cells on leaf surfaces was demonstrated after 4 and 24 h.In conclusion, use of aquatic environmental isolates is more useful for studying biofilm formation than the reference strain. Lettuce surface supported the attachment of B. cereus spores and vegetative cells compared with the cabbage surface. Further investigations are required to improve our knowledge of biofilm formation mechanisms by the human pathogenic microorganisms, especially by using the environmental and clinical isolates. To ensure safety level of green-leafy vegetables, biofilm formation after harvest should be considered as critical control point during handling of these vegetables.     

Biodiversity of psychrotrophic bacteria of the Bacillus cereus group collected on farm and in egg product industry

The aim of the present study was (i) to type, by genotypic and phenotypic methods, a collection of psychrotrophic bacteria belonging to the Bacillus cereus group collected in a farm and in 6 egg breaking companies during a period covering a warm and a cold season, and (ii) to characterize the growth potential in liquid whole egg, and the sanitary risk potential (cytotoxic activity on Caco-2 cells and adhesion on stainless steel) of each isolate of the collection. The investigation of specific psychrotrophic and mesophilic signatures together with the study of ability to grow at 6 °C and/or at 43 °C on optimal agar medium allowed highlighting twelve profiles, the major one corresponding to the species Bacillus weihenstephanensis (46.2% of the collection). The diversity of the profiles depended on the season and on the origin of the isolates. All the isolates were able to grow at the same level in liquid whole egg and in optimal medium, even at low temperature. Under the same conditions, the cytotoxic activity depended on the isolate, the medium and the temperature. At 10 °C, no isolate was cytotoxic in liquid whole egg and only one, belonging to the Bacillus weihenstephansensis species, in the optimal medium. All the isolates were able to adhere on stainless steel at various levels, from 2.6 ± 0.2 log cfu/cm² to 4.9 ± 0.1 log cfu/cm². A large majority (80.8%) was strongly adhering and could lead to the formation of biofilms in industrial equipments.     

Bacteriophages BCP1-1 and BCP8-2 require divalent cations for efficient control of Bacillus cereus in fermented foods

Bacillus cereus is a foodborne bacterial pathogen that causes diarrhea and vomiting. In this study, the usefulness of bacteriophages to eradicate B. cereus from fermented foods was investigated. A total of 13 phages were isolated from Korean fermented food products, and 2 (BCP1-1 and BCP8-2) were further characterized. Transmission electron microscopy (TEM), restriction enzyme digestion pattern analysis, and SDS-PAGE of the structural proteins suggest that both phages belong to the family Myoviridae, containing approximately 150 kbp-long genomes. The host ranges of both phages were limited to B. cereus group species (12/13), as they were not able to lyse other Gram-positive or negative strains including Bacillus subtilis. Purified phages were used to inhibit B. cereus growth in a model fermented food system, cheonggukjang, a fast-fermented soybean paste product. BCP1-1 and BCP8-2 were able to effectively eradicate B. cereus from the food only if divalent cations (Ca²⁺, Mg²⁺, or Mn²⁺) were added to the medium. Further studies reveal that divalent cations are essential for phage adsorption, while a monovalent cation (Na⁺) is required for the post-adsorption phase of phage infection. Taken together, our findings imply that a phage could be an ideal anti-bacterial agent for use in fermented food products that require the presence of beneficial microflora and, during phage application, optimization of phage reaction conditions is critical for the successful utilization of phage biocontrol.     

Extended and global phylogenetic view of the Bacillus cereus group population by combination of MLST, AFLP, and MLEE genotyping data

The Bacillus cereus group of bacteria includes species that can cause food-poisoning or spoilage, such as B. cereus, as well as Bacillus anthracis, the cause of anthrax. In the present report we have conducted a multi-datatype analysis using tools from the HyperCAT database (http://mlstoslo.uio.no/) that we recently developed, combining data from multilocus sequence typing (Tourasse et al., 2010), amplified fragment length polymorphism, and multilocus enzyme electrophoresis typing techniques. We provide a comprehensive snapshot of the B. cereus group population, incorporating 2213 isolates including 450 from food and dairy products, in the form of both phylogenetic supertrees and superclusters of genetically closely related isolates. Our main findings include the detection of phylogenetically separated groups of isolates possibly representing novel evolutionary lineages within the B. cereus group, a putative new branch of B. anthracis, as well as new groups of related strains containing both environmental and clinical isolates. In addition, the multi-datatype analysis revealed to a larger extent than previously recognized that food-borne isolates can share identical genotyping profiles with strains from various other origins. Altogether, the global analysis confirms and extends the results underlining the opportunistic nature of B. cereus group organisms, and the fact that isolates responsible for disease outbreaks and contamination of foodstuffs can originate from various genetic backgrounds.     

Amino acids improve acid tolerance and internal pH maintenance in Bacillus cereus ATCC14579 strain

This study investigated the involvement of glutamate-, arginine- and lysine-dependent systems in the Acid Tolerance Response (ATR) of Bacillus cereus ATCC14579 strain. Cells were grown in a chemostat at external pH (pH_e) 7.0 and 5.5. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted) compared with cells grown at pH 7.0 (unadapted), indicating that B. cereus cells grown at low pH_e were able to induce a marked ATR. Glutamate, arginine and lysine enhanced the resistance of unadapted cells to pH 4.0 acid shock of 1-log or 2-log populations, respectively. Amino acids had no detectable effect on acid resistance in acid-adapted cells. An acid shock at pH 4.0 resulted in a marked drop in internal pH (pH_i) in unadapted cells compared with acid-adapted cells. When acid shock was achieved in the presence of glutamate, arginine or lysine, pH_i was maintained at higher values (6.31, 6.69 or 6.99, respectively) compared with pH_i in the absence of amino acids (4.88). Acid-adapted cells maintained their pH_i at around 6.4 whatever the condition. Agmatine (a competitive inhibitor of arginine decarboxylase) had a negative effect on the ability of B. cereus cells to survive and maintain their pH_i during acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. This adaptation depends on pH_i homeostasis and is enhanced in the presence of glutamate, arginine and lysine. Hence evaluations of the pathogenicity of B. cereus must take into account its ability to adapt to acid stress.     

Purification and characterization of extracellular 1,2-alpha-L-fucosidase from Bacillus cereus

Bacillus cereus isolated from a soil sample, inductively produced alpha-L-fucosidase in culture medium containing porcine gastric mucin (PGM). The production of the enzyme was also weakly induced by L-fucose and D-arabinose, but not by other sugars including glucose. The enzyme was purified 61-fold with an overall recovery of 1.8% from the culture fluid supplemented with PGM by ammonium sulfate precipitation, acetone fractionation, and subsequent column chromatography. The purified enzyme was found homogeneous by SDS-PAGE and its molecular mass was estimated to be approximately 196,000 kDa. Its optimum pH was 7.0 and it was stable in the pH range of 5.0 to 9.0. The enzyme hydrolyzed the alpha-(1-->2)-L-fucosidic linkage in oligosaccharides such as Fucalpha1-2Galbeta1-4Glc (2'-fucosyllactose), Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose I), and the glycoprotein PGM. The enzyme was inactive on p-nitrophenyl alpha-L-fucoside, the alpha-(1-->3)-L-fucosidic linkages in Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose III) and orosomucoid, the alpha-(1-->4)-L-fucosidic linkage in Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose II), and the alpha-(1-->6)-L-fucosidic linkage in thyroglobulin.