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.     

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.     

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.     

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.     

Incidence, diversity and toxin gene characteristics of Bacillus cereus group strains isolated from food products marketed in Belgium

The major objectives of this study were to determine the incidence, diversity and characteristics of Bacillus cereus group spp. isolated from food products marketed in Belgium. The food products investigated in this study included cooked pasta, lasagna, béchamel sauce, bolognaise sauce, fresh minced beef, fresh-cut vegetables and raw basmati rice. B. cereus group spp. were detected in 56.3% (324 of 575) of the samples giving rise to 380 strains. The highest incidence (100%) occurred in the raw basmati rice. Although only 10 (2.6%) of the 380 isolates were determined to be psychrotolerant (able to grow at ≤ 7 °C), 25 (6.2%), 189 (49.7%) and 334 (87.9%) isolates were able to grow at mild temperature abuse conditions of 8 °C, 9 °C and 10 °C, respectively. The large diversity of the isolates obtained (overall and between isolates obtained from the same product type) was highlighted by the results of the (GTG)₅ PCR fingerprinting of 80 selected isolates. Sixty-one of these 80 isolates belonged to 15 distinct clusters (≥ 85% Pearson correlation) whereas the remaining 19 were each clustered separately. Further diversity was also found in the distribution of toxin genes as 16 different profiles were observed in the 80 selected isolates. Whilst none of 80 selected strains harboured the ces gene required for the production of the emetic toxin cereulide, 42 strains (52.5%) carried all seven genes required for the production of the diarrhoeal enterotoxins: haemolytic BL, non-haemolytic enterotoxin and cytotoxin K. The results of this study highlight not only the omnipresence but also the highly diverse ecology of B. cereus spp. within and across several food product types available on the retail market in Belgium. They should also provide the impetus for more studies to enable detailed risk assessment studies to be performed.     

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.     

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.     

Bacillus cereus spores during housing of dairy cows: factors affecting contamination of raw milk

The contamination of raw milk with Bacillus cereus spores was studied during the indoor confinement of dairy cattle. The occurrence of spores in fresh and used bedding material, air samples, feed, feces, and the rinse water from milking equipment was compared with the spore level in bulk tank milk on 2 farms, one of which had 2 different housing systems. A less extensive study was carried out on an additional 5 farms. High spore concentrations of >100 spores/L in the raw milk were found on 4 of the farms. The number of spores found in the feed, feces, and air was too small to be of importance for milk contamination. Elevated spore contents in the rinse water from the milking equipment (up to 322 spores/L) were observed and large numbers of spores were found in the used bedding material, especially in free stalls with >5 cm deep sawdust beds. At most, 87,000 spores/g were found in used sawdust bedding. A positive correlation was found between the spore content in used bedding material and milk (r = 0.72). Comparison of the genetic fingerprints obtained by the random amplified polymorphic DNA PCR of isolates of B. cereus from the different sources indicated that used bedding material was the major source of contamination. A separate feeding experiment in which cows were experimentally fed B. cereus spores showed a positive relationship between the number of spores in the feed and feces and in the feces and milk (r = 0.78). The results showed that contaminated feed could be a significant source of spore contamination of raw milk if the number of spores excreted in the feces exceeded 100,000/g.     

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.     

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.     

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.     

Optimization of pressure-induced germination of Bacillus sporothermodurans spores in water and milk

Bacillus sporothermodurans produces highly resistant endospores that can survive ultra-high-temperature treatment in milk. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk sterility. In this work, the rate of spore germination of B. sporothermodurans LTIS27 was measured in distilled water after high-pressure treatments with varying pressure (50–600 MPa), treatment temperature (20–50 °C), pressure-holding time (5–30 min) and post-pressurization incubation time (30–120 min) at 37 °C or 4 °C. The results showed that pressure-induced germination was maximal (62%) after a treatment at 200 MPa and 20 °C and increased with pressure-holding time and post-pressurization incubation time. Treatment temperature had no significant effect on germination. A central composite experimental design with three factors (pressure, pressure-holding time, and post-pressurization incubation time) using response surface methodology was used to optimize the germination rate in distilled water and in skim milk. No factor interaction was observed. Germination was induced at lower pressure and was faster in milk than in distilled water, but complete germination was not reached. The optimum germination obtained with experimental data was 5.0 log cfu/mL in distilled water and 5.2 log cfu/mL in milk from 5.7 log cfu/mL of spores initially present in the suspension. This study shows the potential of using high hydrostatic pressure to induce the germination of B. sporothermodurans spores in milk before a heat treatment.     

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.     

Effect of Lactococcus garvieae, Lactococcus lactis and Enterococcus faecalis on the behaviour of Staphylococcus aureus in microfiltered milk

The effect of four strains of Lactococcus garvieae, three strains of Lactococcus lactis and one strain of Enterococcus faecalis on Staphylococcus aureus SA15 growth in microfiltered milk was evaluated. Lactococcus and Enterococcus strains were co-cultured with S. aureus in microfiltered milk and in medium buffered at pH 6.8. All Lactococcus and Enterococcus strains were able to inhibit S. aureus growth after 6h of incubation. Inhibition by L. lactis and E. faecalis strains could be partially attributed to the decrease in pH below 6.0 as it has been observed in medium buffered at pH 6.8. L. garvieae strains were the most effective to inhibit S. aureus growth without acidification. Inhibition of S. aureus could not be attributed neither to production of lactate, acetate or nor to antistaphylococcal substance. Amino acids competition was not involved in the inhibition by L. garvieae as addition of valine, isoleucine, threonine, methionine and phenylalanine did not suppress the inhibition of S. aureus.     

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.     

婴幼儿配方乳粉及婴幼儿米粉中的蜡样芽胞杆菌流行情况、耐药性及分子特征研究

目的 了解流通环节市售婴幼儿配方乳粉和婴幼儿米粉中的蜡样芽胞杆菌流行情况、抗生素耐药性和毒力基因等分子特征。方法 采用GB 4789.14—2014《食品卫生微生物学检验 蜡样芽胞杆菌检验》对样品中的蜡样芽胞杆菌进行检测与生化鉴定。进一步通过抗生素敏感实验和全基因组测序对11株代表性分离株进行研究,获得耐药表型及耐药、毒力基因等分子特征信息。结果 婴幼儿配方乳粉和婴幼儿米粉中蜡样芽胞杆菌的检出率分别为11.97%（56/468）和20.74%（28/135）。11株代表性蜡样芽胞杆菌均为多重耐药菌株,全基因组测序结果发现11株分离株共携带8个耐药基因和14个毒力基因,其中主要携带磷霉素、β-内酰胺类抗生素抗性基因,以及肠毒素基因（hbl和nhe）,另发现1株携带ces基因簇蜡样芽胞杆菌菌株。11株分离株具有遗传多样性,11株分离株分属10个ST型。结论 婴幼儿配方乳粉和婴幼儿米粉中的蜡样芽胞杆菌检出率较低,但代表菌株的耐药谱和遗传特征具有多样性,应进一步加强监测及采取有效措施进行控制,以保障相关食品的安全性。     

Identification and safety evaluation of Bacillus species occurring in high numbers during spontaneous fermentations to produce Gergoush, a traditional Sudanese bread snack

Gergoush is a naturally fermented Sudanese Bread snack produced in three fermentation steps (primary starter, adapted starter and final dough), followed by three baking steps for a half to one hour at above 200 °C. This study examines the microbiota of two sets of fermentations performed at a traditional production site in Khartoum, Sudan in 2006 and 2009, respectively. In 2006 four different milk/legume based primary starters (faba bean, chick pea, lentil and white bean) were sampled in order to enumerate and identify the Bacillus at species or group level. In 2009 specific focus was on the enumeration and safety evaluation of the dominant Bacillus cereus group species occurring during various Gergoush productions (including the three fermentations steps and after baking). In 2006, the primary starters contained Bacillus spp. in the order of between 7.7 and 8.1 log₁₀ CFU/g. Species identifications were performed by M13-PCR typing using the Escherichia coli phage M13 derived primer PM13 combined with internally transcribed 16-23S rRNA PCR, 16S rRNA gene and gyrA or gyrB gene sequencing, and selected phenotypic tests. Depending on the legume used, 40-68% of the isolates were identified as B. cereus sensu lato, 16-27% as Bacillus licheniformis, 8-32% as Bacillus subtilis and 4-20% as Bacillus sonorensis. During the second set of fermentation trials performed in 2009, the Bacillus spp. and B. cereus occurred in numbers of between 7.7-9.9 and 6.1-7.8 log₁₀ CFU/g, respectively, while no bacteria were detected after baking. A total of 180 B. cereus sensu lato isolates from four different primary starters, adapted starters and final doughs were further identified as B. cereus sensu stricto (118 isolates) and Bacillus thuringiensis (62 isolates). The safety of Gergoush was evaluated based on the counts and toxin gene profiles of the dominant B. cereus species. Considering that no bacteria survived the baking process, and that the cereulide synthetase gene cesB involved in the production of the heat stable emetic toxin cereulide was not detected in any of the investigated B. cereus isolates, the results indicate, that Gergoush produced at the traditional production site is safe for human consumption. This study is the first to identify the Bacillus of Gergoush to species level, and it is the first to perform a safety evaluation of the product, based on the dominant B. cereus species.     

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.