Growth characteristics of virulent Bacillus anthracis and potential surrogate strains

The objectives of this study were to compare generation and lag times of virulent Bacillus anthracis strains with those of other Bacillus strains, to identify possible surrogates for growth studies, and to determine if the B. cereus module of the U.S. Department of Agriculture Pathogen Modeling Program (PMP) had predictive value for B. anthracis. Growth characteristics of B. anthracis, B. cereus, B. mycoides, and B. subtilis strains in brain heart infusion broth at pH 6.5, 6.0, and 5.5 were determined by absorbance measurements. Growth curves of B. anthracis Sterne and B. cereus strains appeared similar, and the generation times for strain Sterne fell within the PMP's 95% confidence interval for B. cereus. However, the virulent B. anthracis strains Vollum and Pasteur had shorter generation times than the avirulent Sterne strain and most other surrogates and were lower than the PMP's 95% confidence interval for B. cereus. Growth curves of B. cereus ATCC 9818 and B. subtilis ATCC 6633 were more similar to those of virulent B. anthracis strains, but all potential surrogates had significantly different generation times and lag times under some conditions.     

Combined effect of nisin and carvacrol at different pH and temperature levels on the viability of different strains of Bacillus cereus.

The influence of pH and temperature on the bactericidal action of nisin and carvacrol on vegetative cells of different Bacillus cereus strains was studied. The five strains tested showed significant differences in sensitivity towards nisin, at pH 7.0 and 30 degrees C. Carvacrol concentrations of 0.3 mmol l(-1) had no effect on viability of B. cereus cells. When the same carvacrol concentration was combined with nisin, however, it resulted in a greater loss of viability of cells than when nisin was applied alone. The concentration of carvacrol played an important role on the bactericidal effect of nisin and, therefore, on the synergistic action of both compounds combined. At lower pH values (6.30 and 5.75), nisin was more active against B. cereus cells than at pH 7.0 at 30 degrees C, with a different sensitivity of the strains tested. The combined effect of nisin and carvacrol was found to be significantly different at pH 7.0 and 5.75. When the temperature was 8 degrees C, nisin was significantly less active against B. cereus IFR-NL 94-25 than at 30 degrees C, both at pH 7.0 and 6.30. At 8 degrees C, there was a significant increased effect of nisin at lower pH values. Also at this low temperature, a synergistic effect between nisin and carvacrol on B. cereus cells was observed at the pHs tested. This study indicates the potential of nisin and carvacrol at lower pHs to be used for preservation of minimally processed foods.     

Decontamination of fluid milk containing Bacillus spores using commercial household products

Although commercial sanitizers can inactivate bacterial spores in food processing environments, relatively little data exist as to the decontamination of products and surfaces by consumers using commercial household products. Should a large scale bioterrorism incident occur in which consumer food products were contaminated with a pathogenic sporeformer such as Bacillus anthracis, there may be a need to decontaminate these products before disposal as liquid or solid waste. Studies were conducted to test the efficacy of commercial household products for inactivating spores of Bacillus cereus (used as a surrogate for B. anthracis) in vitro and in fluid milk. Validation of the resistance of the B. cereus spores was confirmed with B. anthracis spores. Fifteen commercial products, designed as either disinfectants or sanitizers or as potential sanitizers, were purchased from retail markets. Products selected had one of the following active compounds: NaOCl, HCl, H2O2, acetic acid, quaternary ammonium compounds, ammonium hydroxide, citric acid, isopropanol, NaOH, or pine oil. Compounds were diluted in water (in vitro) or in 2% fat fluid milk, and spores were exposed for up to 6 h. Products containing hypochlorite were most effective against B. cereus spores. Products containing HCl or H2O2 also reduced significant numbers of spores but at a slower rate. The resistance of spores of surrogate B. cereus strains to chlorine-containing compounds was similar to that of B. anthracis spores. Therefore, several household products on the market may be used to decontaminate fluid milk or similar food products contaminated by spores of B. anthracis.     

A selective chromogenic agar that distinguishes Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis

A selective and differential plating medium, R & F anthracis chromogenic agar (ACA), has been developed for isolating and identifying presumptive colonies of Bacillus anthracis. ACA contains the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-choline phosphate that upon hydrolysis yields teal (blue green) colonies indicating the presence of phosphatidylcholine-specific phospholipase C (PC-PLC) activity. Among seven Bacillus species tested on ACA, only members of the Bacillus cereus group (B. anthracis, B. cereus, and B. thuringiensis) produced teal colonies (PC-PLC positive) having cream rings. Examination of colony morphology in 18 pure culture strains of B. anthracis (15 ATCC strains plus AMES-1-RIID, ANR-1, and AMED-RIID), with one exception, required 48 h at 35 to 37 degrees C for significant color production, whereas only 24 h was required for B. cereus and B. thuringiensis. This differential rate of PC-PLC synthesis in B. anthracis (due to the truncated plcR gene and PlcR regulator in B. anthracis) allowed for the rapid differentiation on ACA of presumptive colonies of B. anthracis from B. cereus and B. thuringiensis in both pure and mixed cultures. Effective recovery of B. anthracis from a variety of matrices having both high (soil and sewage) and low microbial backgrounds (cloth, paper, and blood) spiked with B. anthracis ANR-1 spores suggests the probable utility of ACA plating for B. anthracis recovery in a diversity of applications.     

Rapid identification of Bacillus cereus based on the detection of a 28.5-kilodalton cell surface antigen

Conventional procedures for the identification of suspect Bacillus cereus isolated on mannitol-egg yolk-polymyxin (MYP) agar may need several days. To facilitate the identification of the bacterium, an enzyme-linked immunosorbent assay (ELISA) was developed. The assay was based on the detection of a 28.5-kDa cell surface antigen of B. cereus. Bacterial colonies grown on MYP agar or nutrient agar were suspended in phosphate-buffered saline (pH 7.2) containing 0.1% Teepol. The cell suspensions were heated at 100 degrees C for 5 min and added to the microtiter plates coated with antibodies against the 28.5-kDa antigen. After washing, the same antibodies labeled with horseradish peroxidase were used as secondary antibodies to reveal the signal of antigen-antibody reaction. For 38 strains of B. cereus and 127 strains of non-B. cereus bacteria (including 79 isolates of Bacillus spp.) tested, the sensitivity and specificity of the ELISA were 100 and 88.2%, respectively. Strains producing false-positive results were members of the B. cereus group (i.e., Bacillus anthracis, Bacillus mycoides, and Bacillus thuringiensis), which are genetically and biochemically similar to B. cereus. Similar ELISA results were obtained by using antibodies against another cell surface antigen with a molecular mass of 20 kDa. If members of the B. cereus group were recognized as a single species, the sensitivity and specificity of the ELISA were 100 and 99.1%, respectively. The ELISA could be used as a rapid method for presumptive identification of B. cereus grown on MYP agar.     

Efficacy of enterocin AS-48 against bacilli in ready-to-eat vegetable soups and purees

The broad-spectrum bacteriocin enterocin AS-48 was tested for biopreservation of ready-to-eat vegetable foods (soups and purees) against aerobic mesophilic endospore-forming bacteria. By adding AS-48 (10 microg/ml), Bacillus cereus LWL1 was completely inhibited in all six vegetable products tested (natural vegetable cream, asparagus cream, traditional soup, homemade-style traditional soup, vegetable soup, and vichyssoise) for up to 30 days at 6, 15, and 22 degrees C. A collection of strains isolated from spoiled purees showed slightly higher resistance to AS-48 in the order Paenibacillus sp. > Bacillus macroides > B. cereus, although they were also completely inhibited in natural vegetable cream by AS-48 at 10 microg/ml. However, cocktails of five or eight strains composed of B. cereus (three strains), B. macroides (two strains), and Paenibacillus sp., Paenibacillus polymyxa, and Paenibacillus amylolyticus showed higher bacteriocin resistance with AS-48 of up to 50 microg/ml required for complete inactivation in natural vegetable cream stored at 22 degrees C. Repetitive extragenic palindromic sequence-based PCR (REP-PCR) analysis showed that paenibacilli (along with some B. cereus) was the predominant survivor in the cocktails after bacteriocin treatment. To increase the effectiveness of enterocin AS-48, the bacteriocin was tested (at 20 microg/ml) against the eight-strain cocktail in natural vegetable cream in combination with other antimicrobials. The combination of AS-48 and nisin had a slight but significant additive effect. Bactericidal activity was greatly enhanced by phenolic compounds (carvacrol, eugenol, geraniol, and hydrocinnamic acid), achieving a rapid and complete inactivation of bacilli in the tested puree at 22 degrees C.     

The possibility of discriminating within the Bacillus cereus group using gyrB sequencing and PCR-RFLP

Based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion), we have examined the possibility of differentiating members of the Bacillus cereus group. Fragments of the gyrB gene (362 bp) from pure cultures of 12 B. cereus, 25 B. thuringiensis, 25 B. mycoides and two B. anthracis strains were amplified and subsequently digested with Sau3A1. Furthermore, a majority of the amplicons were sequenced directly to verify the PCR-RE results. The results obtained suggest that only the B. mycoides generates specific fragments following PCR-RE. Conversely, it was not possible to discriminate between the B. cereus and the B. thuringiensis strains using the methods described.     

Thermal resistance of spores from virulent strains of Bacillus anthracis and potential surrogates

The objective of this study was to determine the thermal resistance of spores of Bacillus anthracis and potential surrogates. The heat resistance of spores suspended in buffer (pH 7.0 or 4.5), milk, or orange juice was determined at 70, 80, and 90 degrees C. D-values for B. anthracis strains Sterne, Vollum, and Pasteur ranged from < 1 min at 90 degrees C to approximately 200 min at 70 degrees C and were lower under acidic than under neutral conditions. The D-values for B. anthracis spores fell within the range obtained for spores from eight strains of Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus subtilis. However, there were significant differences (P < 0.001) among the D-values of the strains. The z-values in pH 7.0 buffer and milk averaged approximately 10.5 degrees C and were not significantly different among strains (P < 0.05). The z-values in pH 4.5 buffer and orange juice averaged 12.9 and 13.9 degrees C, respectively, significantly (P < 0.05) higher than those obtained in milk or in pH 7.0 buffer. The significance of this difference was driven by large differences among a few strains. The z-values for B. anthracis strain Pasteur were twice as high in the acid media than in the neutral media. This study confirms that B. anthracis spores are not unusually heat resistant and that spores from validated Bacillus species are appropriate surrogates for thermal resistance studies.     

Influence of nisin on the resistance of Bacillus anthracis sterne spores to heat and hydrostatic pressure

The influence of nisin on the heat and pressure resistance of Bacillus anthracis Sterne spores was examined. The decimal reduction times (D-value) of spores in milk (2% fat) at 80, 85, and 90 degrees C were determined. In the absence of nisin, the D-values were 30.09, 9.30, and 3.86 min, respectively. The D-values of spores heated in the presence of nisin (1 mg/ml) were not significantly different (P = 0.05). However, spores heated in the presence of nisin had a 1- to 2-log reduction in viability, after which the death kinetics became similar to those of spores in the absence of nisin. The z-values all were 11.2 degrees C regardless of the presence or absence of nisin. The pressure sensitivity of B. anthracis Sterne spores in the presence and absence of nisin also was determined. Spores treated with nisin were 10 times more pressure sensitive than were spores subjected to pressure in the absence of nisin under the conditions used in this study.     

Differentiation and characterization by molecular techniques of Bacillus cereus group isolates from poto poto and dégué, two traditional cereal-based fermented foods of Burkina Faso and Republic of Congo

Poto poto (a maize sourdough) and dégué (a pearl millet-based food) are two traditional African fermented foods. The molecular biology of toxigenic and pathogenic bacteria found in those foods is largely unknown. The purpose of this study was to study the phylogenetic relatedness and toxigenic potential of 26 Bacillus cereus group isolates from these traditional fermented foods. The relatedness of the isolates was evaluated with repetitive element sequence-based PCR (REP-PCR) and 16S rDNA sequencing analysis. A multiplex real-time PCR assay targeting the lef and capC genes of B. anthracis pXO1 and pXO2 plasmids and the sspE chromosomal gene of B. cereus and B. anthracis also was carried out. Melting curve analysis of the sspE amplification product was used to differentiate B. cereus from B. anthracis, and the presence of the B. cereus enterotoxin genes was determined with PCR amplification. Isolates had 15 different REP-PCR profiles, according to which they could be clustered into four groups. 16S rDNA sequencing analysis identified 23 isolates as B. cereus or B. anthracis and three isolates as B. cereus or Bacillus sp. Multiplex real-time PCR amplification indicated the absence of the lef and capC genes of B. anthracis pXO 1 and pXO2 plasmids, and melting curve analysis revealed amplification of the 71-bp sspE product typical of B. cereus in all isolates instead of the 188-bp amplicon of B. anthracis, confirming the identity of these isolates as B. cereus. Four isolates had amylolytic activity. All isolates had lecithinase activity and beta-hemolytic activity. Enterotoxin production was detected in two isolates. The emetic toxin gene was not detected in any isolate. The nheB toxin gene was detected in 19 isolates by PCR amplification; one of these isolates also contained the hblD (L1) gene. The cytotoxin K cytK-1 gene was not detected, but the cytK-2 gene was clearly detected in six isolates.     

2种乳链菌肽天然变异体抑菌活性的研究

以利斯特氏菌和蜡状芽孢杆菌为材料 ,研究了 2种乳链菌肽天然变异体nisinA和nisinZ的抑菌活性。结果表明 ,nisinA和nisinZ对被试的 2株利斯特氏菌及蜡状芽孢杆菌1 189具有相同的抑菌效果。而对另一株蜡状芽孢杆菌 1 350 ,nisinZ比nisinA显示较强的抑菌活性。不同的培养基对这 2种乳链菌肽天然变异体的抑菌活性具有不同的影响     

Bacillus cereus food poisoning and its toxins

The genus Bacillus includes members that demonstrate a wide range of diversity from physiology and ecological niche to DNA sequence and gene regulation. The species of most interest tend to be known for their pathogenicity and are closely linked genetically. Bacillus anthracis causes anthrax, and Bacillus thuringiensis is widely used for its insecticidal properties but has also been associated with foodborne disease. Bacillus cereus causes two types of food poisoning, the emetic and diarrheal syndromes, and a variety of local and systemic infections. Although in this review we provide information on the genus and a variety of species, the primary focus is on the B. cereus strains and toxins that are involved in foodborne illness. B. cereus produces a large number of potential virulence factors, but for the majority of these factors their roles in specific infections have not been established. To date, only cereulide and the tripartite hemolysin BL have been identified specifically as emetic and diarrheal toxins, respectively. Nonhemolytic enterotoxin, a homolog of hemolysin BL, also has been associated with the diarrheal syndrome. Recent findings regarding these and other putative enterotoxins are discussed.     

Effects of chitosan and a low-molecular-weight chitosan on Bacillus cereus and application in the preservation of cooked rice

Shrimp chitosan with 95% deacetylation and low-molecular-weight chitosan (LMWC) isolated from chitosan hydrolysate were investigated for their effects on the growth of Bacillus cereus and for use in the preservation of cooked rice. Four strains of Bacillus cereus were used: standard strain BCRC 10603 and three isolates (nos. 1 through 3) from cooked rice. The antibacterial activity of chitosan against B. cereus was greatly decreased when the reaction pH was changed from 6.0 to 7.0, but LMWC activity was less affected by this pH change. The susceptibility of B. cereus cells to chitosan decreased with increasing of cell age, in accordance with the relative electronegativity of the cell surface. B. cereus spores were more sensitive to LMWC and chitosan than were vegetative cells. Addition of 80 ppm LMWC and chitosan in sterile saline (pH 7.0) greatly reduced the D-value for the tested four strains at 90 degrees C from 30.77 to 46.51 min to 7.47 to 10.17 min and 4.68 to 7.91 min, respectively, and at 100 degrees C from 1.95 to 2.56 min to 0.89 to 0.93 min and 0.72 to 0.80 min, respectively. Addition of 2,000 ppm LMWC to raw rice water before steam cooking effectively inhibited increases in total aerobic bacteria and B. cereus in cooked rice stored at 37 and 18 degrees C.     

Production of antimicrobial metabolites by strains of Lactobacillus or Lactococcus co-cultured with Bacillus cereus in milk

During co-culture of Lactobacillus (five strains) or Lactococcus (two strains) with Bacillus cereus, organic acids and other potentially antimicrobial metabolites are produced. Lactic acid was produced at very different rates by the lactic acid bacteria (LAB) and the final concentrations varied much, however, the crucial point of rapid pH reduction during the initial hours of fermentation coincides with lactic acid production. Moderate amounts of acetic acid were produced during fermentation and the final concentrations were much smaller compared to lactic acid. According to these experiments, production of diacetyl, carbon dioxide and ethanol was considered too small to contribute to inhibition of B. cereus. The inhibitory substance produced by the LAB strains was not sensitive to proteinase K, trypsin or pepsin, so it was not likely that the LAB strains produced bacteriocins antagonistic against B. cereus. The strains that produced lactic acid fastest inhibited B. cereus best. Increased concentrations of lactic and acetic acid and carbon dioxide were also observed after co-culture with B. cereus compared to growth of the LAB strains alone, which indicates that B. cereus stimulates the biosynthetic capacities of the LAB strains.     

Effective use of nisin to control Bacillus and Clostridium spoilage of a pasteurized mashed potato product

Heat-resistant spore-forming bacteria such as Bacillus and Clostridium can survive and grow in cooked potato products. This situation represents both a public health problem and an economic problem. The natural food preservative nisin is used in heat-treated foods to prevent the growth of such bacteria. A cocktail of Clostridium sporogenes and Clostridium tyrobutyricum spores was inoculated into cooked mashed potatoes, which were vacuum packed, pasteurized, and incubated at 8 and 25 degrees C. The shelf life of the mashed potatoes at 25 degrees C was extended by at least 58 days with the addition 6.25 microg of nisin per g. At 8 degrees C, in control samples not containing nisin, the natural contaminant Bacillus grew, but the inoculated Clostridium strains did not until the temperature was raised to 20 degrees C after 39 days. No bacterial growth occurred in nisin-containing samples. The shelf life of the mashed potatoes was extended by at least 30 days with 6.25 microg of nisin per g. In trials involving a cocktail of Bacillus cereus and Bacillus subtilis strains, 6.25 microg of nisin per g extended the shelf life of mashed potato samples that were not vacuum packed by at least 27 days at 8 degrees C. At 25 degrees C, 25 microg of nisin per g extended shelf life by a similar period. Shelf life extension was also observed at lower nisin levels. Microbiological analysis of the mashed potato ingredients showed that a high spore level was associated with the onion powder. It is emphasized that the preservative and the ingredients must be well mixed to ensure good nisin efficacy. Nisin remained at effective levels after pasteurization, and good retention was observed throughout the shelf life of the mashed potatoes.     

Prevalence and Fate of Bacillus cereus in African Traditional Cereal-Based Foods Used as Infant Foods

The objective of the present work was to estimate the prevalence of Bacillus cereus group species in traditional cereal-based lactic acid-fermented slurries and nonfermented flours used to prepare infant foods in an African context. High counts on mannitol-egg yolk-polymixin agar medium were determined for the fermented slurries (median, 4.5 × 10(4) CFU/ml of slurry) compared with the nonfermented flours, most of whose counts were lower than 10(-1) CFU/g. Virulence genes were characterized in 60 isolates from 26 traditional cereal-based foods in Ouagadougou (Burkina Faso). Seventy-two and 38 % of isolates were positive for the complete set of genes coding for hemolysin BL and nonhemolytic enterotoxin, respectively, suggesting a high enterotoxigenic potential for these foodborne isolates. No potentially emetic toxin-producing strains were detected. Because of the high counts found for fermented slurries, survival tests with vegetative cells inoculated in fermented slurries were performed, which showed that growth of B. cereus was inhibited. This result suggests that fermentation in traditional production units is presumably not adequately controlled, enabling growth during any unit operations before fermentation, or even during the fermentation step, when the process was poorly controlled. However, adding nisin (0.1 mg/ml) enabled a 5-log reduction in the B. cereus population in 5 h, suggesting that the use of nisin could be a way to upgrade the hygienic quality of this type of food.     

A colony blot immunoassay for the rapid identification of Bacillus cereus

A colony blot immunoassay was developed for the rapid identification of Bacillus cereus using antibodies against the 28.5-kDa cell-surface antigen of B. cereus. Suspect colonies from plates were blotted onto a Whatman #541 membrane, dried, and fixed by UV irradiation. The membrane was then immersed in an anti-B. cereus antibody-horseradish peroxidase conjugate for 60 min. After washing and reacting with 4-chloro-1-naphthol and H2O2, the appearance of purple spots indicated the presence of B. cereus. This assay effectively identified 61 of 62 B. cereus strains tested. Among 38 non-B. cereus strains, which were other Bacillus spp. (19 genera), 36 gave true-negative results, and 2 showed false-positive results. The sensitivity and specificity for B. cereus were 98.4 and 94.7%, respectively. The present assay is easy to use, and the rapid identification of B. cereus can be completed in 2.5 h.     

Bacillus cereus,Bacillus thuringiensis and Bacillus mycoides differentiation using a PCR-RE technique

A method was developed to differentiate between Bacillus cereus, Bacillus mycoides and Bacillus thuringiensis using the polymerase chain reaction combined with a restriction endonuclease (PCR-RE) technique. This fast and simple protocol, applied to pure culture strains, was developed using the gyrB DNA sequence, as previously proposed by other authors. Strains from international collections were used to optimize the method which was then applied to the identification of strains isolated from food samples. Amplifications were specific for the B. cereus group. Only Staphylococcus aureus gave the same size PCR product, but it was easily differentiated from strains in the B. cereus group by using restriction analysis, based on digestion with the RsaI, Sau3AI and EcoRI endonucleases. Specific amplifications and good differentiations were obtained using pure strains, suggesting the possibility of using the method described to identify the B. cereus group directly in food samples.     

婴幼儿配方乳粉加工环境中蜡样芽孢杆菌多位点序列分型

以分离自婴幼儿配方乳粉加工环境中蜡样芽孢杆菌为研究对象,利用多位点序列分型(MLST)技术对蜡样芽孢杆菌的多样性和系统进化关系进行探究。结果表明,84株蜡样芽孢杆菌共分成24个ST型,分别是ST-24、ST-26、ST-32、ST-62、ST-144、ST-374、ST-999、ST-1119、ST-1243、ST-1284、ST-1333、ST-1334、ST-1335、ST-1336、ST-1337、ST-1338、ST-1339、ST-1340、ST-1341、ST-1342、ST-1343、ST-1344、ST-1345及ST-1347,其中ST-999(22.62%)、ST-1343(15.48%)、ST-1335(7.14%)与ST-1345(7.14%)是该婴幼儿配方乳粉加工环境中的优势ST型。同时发现了3个新的等位基因pur-242,pyc-198,ilv-276与14个新的ST型ST-1333、ST-1334、ST-1335、ST-1336、ST-1337、ST-1338、ST-1339、ST-1340、ST-1341、ST-1342、ST-1343、ST-1344、ST-1345和ST-1347。系统发育分析表明24个ST型与B.cereus、B.anthracis和B.thuringiensis这三个种显示了更近的系统发育关系,与蜡样芽孢杆菌群体中的另外8个种的亲缘关系较远。     

Influence of food matrix on inactivation of Bacillus cereus by combinations of nisin, pulsed electric field treatment, and carvacrol.

Carvacrol was used as a third preservative factor to enhance further the synergy between nisin and pulsed electric field (PEF) treatment against vegetative cells of Bacillus cereus. When applied simultaneously with nisin (0.04 microg/ml), carvacrol (0.5 mM) enhanced the synergy found between nisin and PEF treatment (16.7 kV/cm, 30 pulses) in potassium-N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) buffer. The influence of food ingredients on bactericidal activity was tested using skimmed milk that was diluted to 20% with sterile demineralized water. The efficacy of PEF treatment was not affected by the presence of proteins, and results found in HEPES buffer correlated well with results in milk (20%). Nisin showed less activity against B. cereus in milk. Carvacrol was not able to enhance the synergy between nisin and PEF treatment in milk, unless used in high concentrations (1.2 mM). This concentration in itself did not influence the viable count. Carvacrol did act synergistically with PEF treatment in milk, however not in HEPES buffer. This synergy was not influenced by proteins in milk, as 5% milk still allows synergy between carvacrol and PEF treatment to the same extent as 20% milk.