Spore formation in Bacillus subtilis biofilms

Spore formation by a Bacillus strain (Bacillus subtilis SpoIVFB-GFP) engineered with a green fluorescent protein (GFP) fused to a polytopic membrane protein (SpoIVF) that fluoresces during sporulation was observed. Biofilms of B. subtilis SpoIVFB-GFP containing ca. 8 log CFU/ml vegetative cells and spores below the lower detection limit (i.e., <1 log CFU/ ml) were allowed to develop on glass wool (37 degrees C). These biofilms were subsequently exposed to nutrient limitation to stimulate spore formation, which was monitored for fluorescence by confocal scanning laser microscopy. Sporulation in corresponding planktonic cells was also monitored for comparative purposes. Planktonic B. subtilis SpoIVFB-GFP cells began fluorescing after 5 h, while B. subtilis SpoIVFB-GFP biofilm cells began fluorescing after 30 h. Results suggested that an existing biofilm of vegetative B. subtilis cells may be stimulated to form spores when exposed to conditions of nutrient limitation. From a practical point of view, it may be suggested that a window of time does exist before sporulation occurs in attached Bacillus biofilms highlighting the need for shorter operating runs between cleaning and sanitation of food-processing equipment surfaces.     

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis

Chlorine, ClO2, and a commercial raw fruit and vegetable sanitizer were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5 to 11.0) ClO2 (200 microg/ml) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log CFU/ml respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log CFU/ml resulting from treatment with 200 microg/ml of chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5- and 0.4-log CFU/ml reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO2 (85 microg/ml), acidified (pH 3.4) ClO2 (85 microg/ml), and a mixture of ClO2 (85 microg/ml) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log CFU/ml, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/ml of organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO2 (400 microg/ml) for 30 min reduced populations by 4.6 and 5.2 log CFU/ml, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO2.     

Survival of Bacillus cereus vegetative cells and spores during in vitro simulation of gastric passage

The enteric pathogen Bacillus cereus must survive gastric passage in order to cause diarrhea by enterotoxin production in the small intestine. The acid resistance and the survival after gastric passage were assessed by in vitro experiments with acidified growth medium and gastric simulation medium with B. cereus NVH 1230-88 vegetative cells and spores. First, batch incubations at constant pH values for 4 h, which represented different physiological states of the stomach, showed that spores were resistant to any gastric condition in the pH range of 2.0 to 5.0, while vegetative cells were rapidly inactivated at pH values of ≤4.0. Second, a dynamic in vitro gastric experiment was conducted that simulated the continuously changing in vivo conditions due to digestion dynamics by gradually decreasing the pH from 5.0 to 2.0 and fractional emptying of the stomach 30 to 180 min from the start of the experiment. All of the B. cereus spores and 14% (± 9%) of the vegetative cells survived the dynamic simulation of gastric passage.     

Physiology of dairy-associated Bacillus spp. over a wide pH range

Bacillus species isolated from alkaline wash solutions used for cleaning in place in South African dairy factories have been suggested to contaminate product contact surfaces of dairy processing equipment and result in post-pasteurization spoilage of milk and milk products. Growth and attachment of such Bacillus isolates under alkaline and acidic conditions have not been previously described. Therefore, the in vitro growth temperature and pH ranges, attachment abilities and hydrophobicity, and enzyme production capabilities of four Bacillus isolates (tentatively identified as B. subtilis115, B. pumilus122, B. licheniformis137 and B. cereus144) previously isolated from the alkaline wash solutions in a South African dairy were examined. Growth pH ranges were determined in buffered Standard One-like Nutrient Broth and in unbuffered 1% Milk Medium at pH values ranging from 3 to 12. Growth and attachment to stainless steel surfaces and production of protease and lipase enzymes were determined in 1% Milk Medium at pH 4, 7 and 10. Colony hydrophobicity of each isolate by the Direction of Spreading Method (DOS) was also determined at pH 4, 7 and 10. In addition, Arrhenius plots were used to examine the growth temperature ranges of the isolates. All isolates grew at pH values ranging from 4.5 to 9.5 in buffered Standard One-like Nutrient Broth, and from pH 4 to 10 in 1% Milk Medium. All isolates also attached to stainless steel at pH 4, 7 and 10 in 1% Milk Medium. Generally the attachment of B. subtilis115, B. pumilus122 and B. lichenformis137 to stainless steel surfaces was enhanced at pH 4 and 10, compared to pH 7. By contrast, the best attachment of B. cereus144 cells to stainless steel surfaces was at pH 7. Planktonic and attached cells of all isolates produced proteolytic enzymes at pH 7 and 10, but not at pH 4. Similarly, planktonic and attached cells of B. subtilis115, B. pumilus122 and B. licheniformis137 produced lipolytic enzymes at pH 7 and 10, and weak lipolysis was observed at pH 4. The Bacillus cereus144 isolate showed no lipolytic activity at pH 10. All isolates exhibited low hydrophobic properties at all pH values even though attachment to stainless steel at the same pH values occurred. None of the isolates grew below 11 degrees C or above 56 degrees C, and optimum growth temperatures were in the high mesophilic range (36-44 degrees C).     

Different responses of planktonic and attached Bacillus subtilis and Pseudomonas fluorescens to sanitizer treatment.

Three commercial sanitizers containing iodophor (I), peracetic acid/ hydrogen peroxide (PAH), or chlorhexidine gluconate (CG) were evaluated in vitro against planktonic and sessile Bacillus subtilis or Pseudomonas fluorescens cells grown in Standard One Nutrient Broth. Sessile cells were attached to stainless steel or polyurethane test surfaces. Planktonic and attached cells of both bacteria were enumerated by plate counts after sanitizer treatment for 1, 3, or 5 min. Sessile cells were dislodged from test surfaces by shaking them with beads. Cell morphologies were monitored by scanning electron microscopy (SEM). Attached B. subtilis and P. fluorescens cells on both surface types were less susceptible to all three sanitizers than their planktonic counterparts. PAH, I, and CG were equally effective against planktonic P. fluorescens cells, which were reduced by 99.999% after 1, 3, and 5 min exposure. PAH was the only sanitizer effective against attached P. fluorescens cells on both surface types; it reduced counts by < or = 99.9% after 1, 3, and 5 min exposure. PAH was also the most effective sanitizer against planktonic B. subtilis cells, reducing counts by 99.9% after 1, 3, and 5 min. Sessile B. subtilis cells on both surface types were the least susceptible to all sanitizers; counts were reduced by only 99.5% or less after exposure to PAH for 5 min. SEM revealed that planktonic and attached cells of both bacteria exhibited symptoms of surface roughness, indentations, and shape distortions after treatment with any of the sanitizers.     

Detection of Bacillus spores using PCR and FTA filters

Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens. We have explored the utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores. Isolated spores from several Bacillus spp., B. subtilis, B. cereus, and B. megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR. Spore preparations were examined microscopically to ensure that the presence of vegetative cells, if any, did not yield misleading results. PCR primers SRM86 and SRM87 targeted a conserved region of bacterial rRNA genes, whereas primers Bsub5F and Bsub3R amplified a product from a conserved sequence of the B. subtilis rRNA gene. With the use of the latter set of primers for nested PCR, the sensitivity of the PCR-based assay was increased. Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR. FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples.     

Thermal inactivation of Bacillus cereus and Clostridium perfringens vegetative cells and spores in pork luncheon roll

The aim of this study was to design a thermal treatment(s) for pork luncheon roll, which would destroy Bacillus cereus and Clostridium perfringens vegetative cells and spores. B. cereus and C. perfringens vegetative and spore cocktails were used to inoculate luncheon meat. Samples were subjected to different temperatures and removal times. The decimal-reduction times (D-values) were calculated by linear regression analysis (D = -1/slope of a plot of log surviving cells versus time). The log(10) of the resulting D-values were plotted against their corresponding temperatures to calculate (-1/slope of the curve) the thermal resistance (z-values) of each cocktail. The D-values for vegetative cells ranged from 1 min (60 degrees C) to 33.2 min (50 degrees C) for B. cereus and from 0.9 min (65 degrees C) to 16.3 min (55 degrees C) for C. perfringens. The D-values for B. cereus spores ranged from 2.0 min (95 degrees C) to 32.1 min (85 degrees C) and from 2.2 min (100 degrees C) to 34.2 min (90 degrees C) for C. perfringens. The z-values were calculated to be 6.6 and 8.5 degrees C for B. cereus vegetative and spores, respectively, and 7.8 and 8.4 degrees C for C. perfringens vegetative cells and spores, respectively. The D-values of B. cereus and C. perfringens suggest that a mild cook of 70 degrees C for 12s and 1.3 min would achieve a 6 log reduction of B. cereus and C. perfringens vegetative cells, respectively. The equivalent reduction of B. cereus and C. perfringens spores would require the pork luncheon meat to be heated for 36 s at 105 and 110 degrees C, respectively. The results of this study provide the thermal inactivation data necessary to design a cooking protocol for pork luncheon roll that would inactivate B. cereus and C. perfringens vegetative cells and spores. The data may also be used in future risk assessment studies.     

Synergistic Effect of Electrolyzed Water and Citric Acid Against Bacillus Cereus Cells and Spores on Cereal Grains

ABSTRACT:  The effects of acidic electrolyzed water (AcEW), alkaline electrolyzed water (AlEW), 100 ppm sodium hypochlorite (NaClO), and 1% citric acid (CA) alone, and combinations of AcEW with 1% CA (AcEW + CA) and AlEW with 1% CA (AlEW + CA) against Bacillus cereus vegetative cells and spores was evaluated as a function of temperature (25, 30, 40, 50, or 60 °C) and dipping time (3 or 6 h). A 3-strain cocktail of Bacillus cereus cells or spores of approximately 10⁷ CFU/g was inoculated in various cereal grains (brown rice, Job's tear rice, glutinous rice, and barley rice). B . cereus vegetative cells and spores were more rapidly inactivated at 40 °C than at 25 °C. Regardless of the dipping time, all treatments reduced the numbers of B . cereus vegetative cells and spore by more than 1 log CFU/g, except the deionized water (DIW), which showed approximately 0.7 log reduction. The reductions of B . cereus cells increased with increasing dipping temperature (25 to 60 °C). B . cereus vegetative cells were much more sensitive to the combined treatments than spores. The effectiveness of the combined electrolyzed water (EW) and 1% CA was considerable in inhibiting B . cereus on cereal grains. The application of combined EW and CA for controlling B . cereus cells and spores on cereal grains has not been previously reported. Therefore, the synergistic effect of EW and CA may provide a valuable insight on reducing foodborne pathogens on fruits, vegetables, and cereal grains.     

Inhibition of Bacillus cereus in milk fermented with kefir grains

The effects of kefir-fermented milk were tested against a toxigenic strain of Bacillus cereus. The incubation of milk with B. cereus spores plus 5% kefir grains prevented spore germination and growth of vegetative forms. In contrast, when 1% kefir grains was used, no effects were observed. The presence of metabolically active kefir grains diminished titers of nonhemolytic enterotoxin A, as assessed by enzyme-linked immunosorbent assay. During fermentation, kefir microorganisms produce extracellular metabolites such as organic acids, which could play a role in the inhibition of spore germination and growth of B. cereus, although the effect of other factors cannot be ruled out. Results of the present study show that kefir-fermented milk is able to antagonize key mechanisms involved in the growth of B. cereus as well as interfere with the biological activity of this microorganism.     

Spore formation by Bacillus cereus in broth as affected by temperature, nutrient availability, and manganese

A study was done to determine the effect of interacting factors on sporulation of Bacillus cereus in broth. Vegetative cells (1.4 to 2.2 log CFU/ml) of B. cereus strain 038-2 (capable of growing at 12 degrees C) and strain F3812/84 (capable of growing at 8 degrees C) were inoculated into 30 ml of tryptic soy broth (TSB), TSB supplemented with manganese (50 microg/ml), diluted (10%) TSB (dTSB), and dTSB supplemented with manganese (50 microg/ml) and incubated at 8, 12, or 22 degrees C for up to 30, 30, or 10 days, respectively. Unheated and heated (80 degrees C for 10 min) cultures were plated on brain heart infusion agar to determine total cell counts (vegetative cells plus spores) and the number of spores produced, respectively. Both strains of B. cereus survived in TSB and dTSB for 30 days at 8 degrees C but did not sporulate. At 12 degrees C, cells grew in TSB to a population of 6.0 +/- 0.8 log CFU/ml, which was maintained for 30 days. Neither strain grew in dTSB at 12 degrees C and survived for at least 30 days. Spores were not produced in any of the test broths at 12 degrees C. At 22 degrees C, cells reached a stationary growth phase between 12 and 24 h in TSB, TSB supplemented with manganese, and dTSB supplemented with manganese, and approximately 1% of the CFU were spores. In dTSB, cell growth and spore formation were retarded at 22 degrees C and a significantly lower number of spores was produced compared with the number of spores produced in TSB, TSB supplemented with manganese, and dTSB supplemented with manganese. The addition of manganese to TSB did not affect cell growth or spore formation, but manganese did enhance sporulation in dTSB. This study provides useful information on spore formation by B. cereus as affected by conditions that may be imposed in liquid milieus on the surface of foods and on food contact surfaces in processing environments.     

Impact of intestinal microbiota and gastrointestinal conditions on the in vitro survival and growth of Bacillus cereus

Ingestion of B. cereus can result in diarrhea, if these bacteria survive gastrointestinal passage and achieve growth and enterotoxin production in the small intestine. The gastrointestinal survival of vegetative cells and spores of the diarrheal food poisoning strain B. cereus NVH 1230-88 was investigated during in vitro batch experiments simulating the stomach, duodenum and ileum using simulation media and competing intestinal microbiota. All spores and approx. 30% of the vegetative B. cereus cells survived the 2 h incubation in gastric medium with pH 4.0. Sterile intestinal medium induced germination of spores and enabled outgrowth of vegetative cells to approx. 7 log CFU/mL. The behavior of B. cereus in the intestinal environment with competing intestinal bacteria was determined by their relative concentrations. Besides the numbers of intestinal bacteria, the nutrition and composition of the intestinal community were also very important for the growth inhibition of B. cereus.     

Presence and significance of Bacillus cereus in dehydrated potato products

Dehydrated potato contains Bacillus cereus at a prevalences of 10 to 40% and at numbers usually less than 10(3) CFU g(-1). B. cereus in dehydrated potato is likely to be present as spores that are able to survive drying of the raw vegetable and may represent a significant inoculum in the reconstituted (rehydrated) product where conditions favor germination of, and outgrowth from, spores. Holding rehydrated mashed potato alone, or as part of another product (e.g., potato-topped pie), at temperatures above 10 degrees C and below 60 degrees C may allow growth of vegetative B. cereus. Levels exceeding 10(4) CFU g(-1) are considered hazardous to human health and may be reached within a few hours if stored inappropriately between these temperatures. Foods incorporating mashed potato prepared from dehydrated potato flakes have been implicated in B. cereus foodborne illness. This review is a summary of the information available concerning the prevalence and numbers of B. cereus in dehydrated potato flakes and the rate at which growth might occur in the rehydrated product.     

Application of gaseous ozone to control populations of Escherichia coli, Bacillus cereus and Bacillus cereus spores in dried figs

The effect of ozonation as a method to reduce Escherichia coli, Bacillus cereus and Bacillus cereus spores in dried figs was investigated. Dried figs were sprinkle inoculated with E. coli, B. cereus and B. cereus spores in sterile bags at a level of 10(7)microorganism g(-1), mixed and allowed to dry for 1h at 25 degrees C prior to ozonation. Inoculated samples were exposed to gaseous ozone in a chamber at 20 degrees C and 70% relative humidity. Ozone concentrations of 0.1, 0.5 and 1.0 ppm up to 360 min were used to inactivate E. coli and B. cereus while 1.0, 5.0, 7.0 and 9.0 ppm ozone concentrations for 360 min were used to treat B. cereus spores. E. coli and B. cereus counts were decreased by 3.5 log numbers at 1.0 ppm ozone concentration for 360 min ozone treatment. Up to 2 log reductions in the number of B. cereus spores were observed above 1.0 ppm ozone concentration at the end of 360 min of ozonation. No significant changes in color, pH and moisture content values of dried figs were observed after the ozonation treatments. No significant changes were found between sweetness, rancidity, flavor, appearance and overall palatability of ozonated and non-ozonated dried figs. Ozonation was found to be effective especially in reduction of vegetative cells in dried figs and a promising method for the decontamination of dried figs.     

Surface characteristics of Bacillus cereus and its adhesion to stainless steel

The ability of a Bacillus cereus strain, isolated from spoiled milk, to adhere to the surface of stainless steel chips was evaluated during its growth in diluted tryptic soy broth (DTSB). The number of cells that adhered to the surface increased markedly as the culture reached the end of the log phase and entered stationary phase, and continued to increase with further incubation. The surface properties of cells from the log, stationary, and late stationary phases were measured by hydrophobic interaction chromatography (HIC) and electrostatic interaction chromatography (ESIC). It was found that surface hydrophobicity of B. cereus vegetative cells from the late stationary phase was the highest followed by those from the stationary phase and the log phase cultures. While the vegetative cells prepared from stationary phase and log phase cultures, respectively, had the highest and the lowest surface charges. Adhesion of B. cereus vegetative cells to stainless steel was positively correlated with the cell surface hydrophobicity (R = 0.979). Surface hydrophobicity and surface positive charge noted on the spores harvested from diluted tryptic soy agar (DTSA) and Mn2+-tryptone glucose extract agar were higher than those harvested from the sucrose or lactose-added DTSA. A wide variation in the surface charge values was noted on the surface of various spores prepared from cultures grown on the four different media tested, while their ability to adhere to stainless steel chips in phosphate buffered saline (PBS) showed no significant difference (p > 0.05). Similarly, the number of spores or vegetative cells adhering to stainless steel suspended in PBS, milk or diluted milk (1000 x) did not differ significantly (p > 0.05).     

植酸对枯草芽孢杆菌及其噬菌体的作用研究

通过在培养基中添加不同浓度的植酸,探究了植酸对枯草芽孢杆菌(Bacillus subtilis)0085和0090菌体生长、芽孢形成的影响及对枯草芽孢杆菌噬菌体P85和P90的抑制效果。结果表明,添加0.150%植酸对其噬菌体P85和P90有明显抑制效果,但影响芽孢的形成;添加0.070%植酸既可以在一定程度上抑制噬菌体P85和P90又不会影响枯草芽孢杆菌及芽孢的生长。     

Evaluation of chlorine, chlorine dioxide, and a peroxyacetic acid-based sanitizer for effectiveness in killing Bacillus cereus and Bacillus thuringiensis spores in suspensions, on the surface of stainless steel, and on apples

Chlorine (10 to 200 microg/ml), chlorine dioxide (10 to 200 microg/ml), and a peroxyacetic acid-based sanitizer (40 and 80 microg/ ml) were evaluated for effectiveness in killing spores of Bacillus cereus and Bacillus thuringiensis in suspensions and on the surface of stainless steel and apples. Water and 5% horse serum were used as carriers for spore inoculum applied to the surface of stainless steel coupons, and 5% horse serum was used as a carrier for inoculum applied to apples. Inocula were dried on stainless steel for 5 h and on apples for 22 to 24 h before treating with sanitizers. At the concentrations of sanitizers tested, sensitivities of planktonic B. cereus and B. thuringiensis spores were similar. A portion of the spores surviving treatment with chlorine and, more markedly, chlorine dioxide had decreased tolerance to heat. Planktonic spores of both species were more sensitive to sanitizers than were spores on the surface of stainless steel or apples. At the same concentrations, chlorine was more effective than chlorine dioxide in killing spores in suspension and on stainless steel. The lethality of chlorine dioxide was markedly reduced when inoculum on stainless steel coupons was suspended in 5% horse serum as a carrier rather than water. Chlorine and chlorine dioxide at concentrations of 10 to 100 microg/ml were equally effective in killing spores on apples. Significant reductions of > or = 3.8 to 4.5 log CFU per apple were achieved by treatment with 100 microg/ml of either of the two sanitizers. The peroxyacetic acid sanitizer (40 and 80 microg/ml) was ineffective in killing Bacillus spores in the test systems investigated. Results provide information on the effectiveness of sanitizers commonly used in the food processing industry in killing Bacillus spores in suspension, on a food-contact surface, and on a ready-to-eat food.     

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.     

Biofilm formation and sporulation by Bacillus cereus on a stainless steel surface and subsequent resistance of vegetative cells and spores to chlorine, chlorine dioxide, and a peroxyacetic acid-based sanitizer

Biofilm formation by Bacillus cereus 038-2 on stainless steel coupons, sporulation in the biofilm as affected by nutrient availability, temperature, and relative humidity, and the resistance of vegetative cells and spores in biofilm to sanitizers were investigated. Total counts in biofilm formed on coupons immersed in tryptic soy broth (TSB) at 12 and 22 degrees C consisted of 99.94% of vegetative cells and 0.06% of spores. Coupons on which biofilm had formed were immersed in TSB or exposed to air with 100, 97, 93, or 85% relative humidity. Biofilm on coupons immersed in TSB at 12 degrees C for an additional 6 days or 22 degrees C for an additional 4 days contained 0.30 and 0.02% of spores, respectively, whereas biofilm exposed to air with 100 or 97% relative humidity at 22 degrees C for 4 days contained 10 and 2.5% of spores, respectively. Sporulation did not occur in biofilm exposed to 93 or 85% relative humidity at 22 degrees C. Treatment of biofilm on coupons that had been immersed in TSB at 22 degrees C with chlorine (50 microg/ml), chlorine dioxide (50 microg/ml), and a peroxyacetic acid-based sanitizer (Tsunami 200, 40 microg/ml) for 5 min reduced total cell counts (vegetative cells plus spores) by 4.7, 3.0, and 3.8 log CFU per coupon, respectively; total cell counts in biofilm exposed to air with 100% relative humidity were reduced by 1.5, 2.4, and 1.1 log CFU per coupon, respectively, reflecting the presence of lower numbers of vegetative cells. Spores that survived treatment with chlorine dioxide had reduced resistance to heat. It is concluded that exposure of biofilm formed by B. cereus exposed to air at high relative humidity (> or =97%) promotes the production of spores. Spores and, to a lesser extent, vegetative cells embedded in biofilm are protected against inactivation by sanitizers. Results provide new insights to developing strategies to achieve more effective sanitation programs to minimize risks associated with B. cereus in biofilm formed on food contact surfaces and on foods.     

Lethality of chlorine, chlorine dioxide, and a commercial produce sanitizer to Bacillus cereus and Pseudomonas in a liquid detergent, on stainless steel, and in biofilm

Many factors that are not fully understood may influence the effectiveness of sanitizer treatments for eliminating pathogens and spoilage microorganisms in food or detergent residues or in biofilms on food contact surfaces. This study was done to determine the sensitivities of Pseudomonas cells and Bacillus cereus cells and spores suspended in a liquid dishwashing detergent and inoculated onto the surface of stainless steel to treatment with chlorine, chlorine dioxide, and a commercial produce sanitizer (Fit). Cells and spores were incubated in a liquid dishwashing detergent for 16 to 18 h before treatment with sanitizers. At 50 microg/ml, chlorine dioxide killed a significantly higher number of Pseudomonas cells (3.82 log CFU/ml) than did chlorine (a reduction of 1.34 log CFU/ml). Stainless steel coupons were spot inoculated with Pseudomonas cells and B. cereus cells and spores, with water and 5% horse serum as carriers. Chlorine was more effective than chlorine dioxide in killing cells and spores of B. cereus suspended in horse serum. B. cereus biofilm on stainless steel coupons that were treated with chlorine dioxide or chlorine at 200 microg/ml had total population reductions (vegetative cells plus spores) of > or = 4.42 log CFU per coupon; the number of spores was reduced by > or = 3.80 log CFU per coupon. Fit (0.5%) was ineffective for killing spot-inoculated B. cereus and B. cereus in biofilm, but treatment with mixtures of Fit and chlorine dioxide caused greater reductions than did treatment with chlorine dioxide alone. In contrast, when chlorine was combined with Fit, the lethality of chlorine was completely lost. This study provides information on the survival and sanitizer sensitivity of Pseudomonas and B. cereus in a liquid dishwashing detergent, on the surface of stainless steel, and in a biofilm. This information will be useful for developing more effective strategies for cleaning and sanitizing contact surfaces in food preparation and processing environments.     

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.