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.

Evidence for quorum sensing in Clostridium botulinum 56A.

AIMS: Experiments were designed to detect quorum-sensing signals produced by Clostridium botulinum. METHODS AND RESULTS: Clostridium botulinum 56A cell-free supernatants obtained at the end of lag phase, the mid-exponential phase and early stationary phase of growth were assayed for bioluminescence in the Vibrio harveyi quorum-sensing assay system. Twelve and 16-h culture supernatants induced bioluminescence in the auto-inducer 2 (AI-2) but not the auto-inducer 1 (AI-1) assay. Intra-species quorum sensing was also assayed as the ability of the supernatants to promote spore germination and outgrowth in a microtitre plate system. Spore populations exposed to C. botulinum supernatant from the end of lag phase became positive for growth sooner than controls. CONCLUSIONS: The influence of cell-free supernatant on ungerminated spores and detection of bioluminescence in the AI-2 assay are evidence for a signalling molecule(s) and provide a first step in characterizing C. botulinum quorum sensing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that spores do not behave independently of each other and may explain the inocula size effects observed in challenge studies. Whether AI-2 production in C. botulinum serves as an inter-species signal or as a detoxification mechanism remains to be determined.

Acid-tolerant Listeria monocytogenes persist in a model food system fermented with nisin-producing bacteria.

AIMS: To investigate the induction of the acid tolerance response (ATR) in Listeria monocytogenes and to assess the persistence of the pathogen in broth fermented using a nisin-producing starter culture. METHODS AND RESULTS: Lactic, acetic and hydrochloric acids were used to induce the ATR in L. monocytogenes growing at early exponential phase. Cells were then challenged in medium acidified to pH 3.5 with the same acid. Only lactic acid induced a detectable ATR. ATR+ cells maintained their initial numbers after 1 h exposure while ATR- were reduced by c. 4 log10 CFU. ATR+ or ATR- cells were also inoculated in M17G broth fermented with nisin-producing (nis+) or control (nis-) Lactococcus lactis. When exposed to nisin, the numbers of ATR+ cells were c. 2 log10 CFU higher than non detectable ATR- cells at day 3. In the absence of nisin (nis- culture), L. monocytogenes was recovered from all ATR+ and ATR- samples after 30 days. In contrast, no L. monocytogenes were recovered from any nis+ATR- samples but four of five nis+ATR+ samples were positive for L. monocytogenes after 30 days. CONCLUSIONS: The ATR confers cross-resistance to nisin for at least 30 days in a system fermented by nisin-producing bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The cross-resistance induced by the ATR should be considered for the safety of foods fermented with bacteriocin-producing cultures.

Computer simulation of Clostridium botulinum strain 56A behavior at low spore concentrations.

It is generally assumed that spore behavior is independent of spore concentration, but recently published mathematical models indicate that this is not the case. A Monte Carlo simulation was employed in this study to further examine the independence assumption by evaluating the inherent variance in spore germination data. All simulations were carried out with @Risk software. A total of 500 to 4,000 iterations were needed for each simulation to reach convergence. Lag time and doubling time from a higher inoculum concentration were used to simulate the time to detection (TTD) at a lower inoculum concentration under otherwise identical environmental conditions. The point summaries of the simulated and observed TTDs were recorded for the 26 simulations, with kinetic data at the target inoculum concentration. The ratios of the median (R(m) = median(obs)/median(sim)) and 90% range (R(r) = 90% range(obs)/90% range(sim)) were calculated. Most R(m) and R(r) values were greater than one, indicating that the simulated TTDs were smaller and more homogeneous than the observed ones. R(r) values departed farther from one than R(m) values. Ratios obtained when simulating 1 spore with 10,000 spores deviated the farthest from one. Neither ratio was significantly different from the other when simulating 1 spore with 100 spores or simulating 100 spores with 10,000 spores. When kinetic data were not available, the percent positive observed at the 95th percentile of the simulated TTDs was obtained. These simulation results confirmed that the assumption of independence between spores is not valid.

Multimethod assessment of commercial nisin preparations.

Nisin is a GRAS preservative effective against several Gram-positive organisms including Listeria monocytogenes. Commercial preparations are usually fermentation products containing 2.5% pure nisin along with insoluble material which, in this study, was found to influence the quantification and activity of nisin under different conditions. Commercially available samples of nisin were tested for efficacy using various methods, such as well diffusion, time to turbidity, and GUS (where a reporter compound is induced in response to nisin). SDS-PAGE detected a single peptide band, corresponding with the molecular weight of nisin. Protein quantified using the Bradford method indicated that the carrier of some samples was proteinaceous. Though the activity of commercially available nisin preparations is indicated on the label, end users should determine the effect of changing their source of nisin.

Time-to-detection, percent-growth-positive and maximum growth rate models for Clostridium botulinum 56A at multiple temperatures.

We previously developed models for the influence of inoculum size on the growth kinetics (time-to-detection and maximum growth rate) and percent-growth-positive samples of Clostridium botulinum 56A with factors of inoculum size (1, 100, and 10,000 spores/sample). pH (5.5. 6.0 and 6.5) and sodium chloride concentration (0.5%, 2% and 4%) at 30 degrees C. In this present study, data were collected at two more temperatures (15 and 22 degrees C), making the final design a complete 3 X 3 X 3 X 3 factorial with a total of 81 conditions. Growth was followed hourly as change in A620. The Gompertz equation was fit to the growth data, and the parameters derived were used to calculate the maximum growth rate and time-to-detection. Linear regression with polynomial terms was used to analyze the effect of environmental factors on time-to-detection and maximum growth rate. Logistic regression with polynomial terms was used to analyze the data for percent-growth-positive. Despite the fact that the variance is larger in this extended data set (which includes two temperatures that are further away from the optimum), the inoculum size effect is clearly demonstrated. When inoculum size increased, the percent-growth-positive samples increased and the time-to-detection decreased. When the inoculum was 1000 spores/sample or higher, little additional effect on time-to-detection was observed. Inoculum size might influence results through simple probability or quorum sensing. Our results show that the observed effect of inoculum size from the previous report at a single temperature is not restricted to a specific growth condition, but rather a general phenomenon. The maximum growth rate was independent of inoculum levels, confirming our previous results.

Enterocin P selectively dissipates the membrane potential of Enterococcus faecium T136.

Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (DeltaPsi) and the intracellular ATP pool of the indicator organism, the pH gradient (DeltapH) component of the proton motive force (Deltap) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes.

Bacteriocins: safe, natural antimicrobials for food preservation.

Bacteriocins are antibacterial proteins produced by bacteria that kill or inhibit the growth of other bacteria. Many lactic acid bacteria (LAB) produce a high diversity of different bacteriocins. Though these bacteriocins are produced by LAB found in numerous fermented and non-fermented foods, nisin is currently the only bacteriocin widely used as a food preservative. Many bacteriocins have been characterized biochemically and genetically, and though there is a basic understanding of their structure-function, biosynthesis, and mode of action, many aspects of these compounds are still unknown. This article gives an overview of bacteriocin applications, and differentiates bacteriocins from antibiotics. A comparison of the synthesis. mode of action, resistance and safety of the two types of molecules is covered. Toxicity data exist for only a few bacteriocins, but research and their long-time intentional use strongly suggest that bacteriocins can be safely used.

Nisin depletes ATP and proton motive force in mycobacteria.

This study examined the inhibitory effect of nisin and its mode of action against Mycobacterium smegmatis, a non-pathogenic species of mycobacteria, and M. bovis-Bacill Carmette Guerin (BCG), a vaccine strain of pathogenic M. bovis. In agar diffusion assays, 2.5 mg ml(-1) nisin was required to inhibit M. bovis-BCG. Nisin caused a slow, gradual, time- and concentration-dependent decrease in internal ATP levels in M. bovis-BCG, but no ATP efflux was detected. In mycobacteria, nisin decreased both components of proton motive force (membrane potential, Delta Psi and Delta pH) in a time- and concentration-dependent manner. However, mycobacteria maintained their intracellular ATP levels during the initial time period of Delta Psi and Delta pH dissipation. These data suggest that the mechanism of nisin in mycobacteria is similar to that in food-borne pathogens.

Modeling the germination kinetics of clostridium botulinum 56A spores as affected by temperature, pH, and sodium chloride.

The germination kinetics of proteolytic Clostridium botulinum 56A spores were modeled as a function of temperature (15, 22, 30 degrees C), pH (5.5, 6.0, 6.5), and sodium chloride (0.5, 2.0, 4.0%). Germination in brain heart infusion (BHI) broth was followed with phase-contrast microscopy. Data collected were used to develop the mathematical models. The germination kinetics expressed as cumulated fraction of germinated spores over time at each environmental condition were best described by an exponential distribution. Quadratic polynomial models were developed by regression analysis to describe the exponential parameter (time to 63% germination) (r2 = 0.982) and the germination extent (r2 = 0.867) as a function of temperature, pH, and sodium chloride. Validation experiments in BHI broth (pH: 5.75, 6.25; NaCl: 1.0, 3.0%; temperature: 18, 26 degrees C) confirmed that the model's predictions were within an acceptable range compared to the experimental results and were fail-safe in most cases.

Sensitivity of nisin-resistant Listeria monocytogenes to heat and the synergistic action of heat and nisin.

Nisin, a bacteriocin produced by some strains of Lactococcus lactis, acts against foodborne pathogen Listeria monocytogenes. A single exposure of cells to nisin can generate nisin-resistant (Nisr) mutants, which may compromise the use of nisin in the food industry. The objective of this research was to compare the heat resistance of Nisr and wild type (WT) Listeria monocytogenes. The synergistic effect of heat-treatment (55 degrees C) and nisin (500 IU ml-1) on the Nisr cells and the WT L. monocytogenes Scott A was also studied. When the cells were grown in the absence of nisin, there was no significant (alpha = 0.05) difference in heat resistance between WT and Nisr cells of L. monocytogenes at 55, 60 and 65 degrees C. However, when the Nisr cells were grown in the presence of nisin, they were more sensitive to heat at 55 degrees C than the WT cells. The D-values at 55 degrees C were 2.88 and 2.77 min for Nisr ATCC 700301 and ATCC 700302, respectively, which was significantly (alpha = 0.05) lower than the D-value for WT, 3.72 min. When Nisr cells were subjected to a combined treatment of heat and nisin, there was approximately a four log reduction during the first 7 min of treatment.

Carbon dioxide and nisin act synergistically on Listeria monocytogenes.

This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nis(r)) cells grown in broth at 4 degrees C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nis(r) cells. Wild-type cells grown in 100% CO(2) were two to five times longer than cells grown in air. Nisin (2.5 microg/ml) did not decrease the viability of Nis(r) cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO(2). There was a quantifiable synergistic action between nisin and CO(2) in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 microg of nisin per ml increased from 3.1 to 12.5 microg/ml over 35 days, but this increase was markedly delayed for cultures in CO(2). This synergism between nisin and CO(2) was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO(2) and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO(2) atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4 degrees C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30 degrees C. Cells grown in the presence of 100% CO(2) and those grown at 4 degrees C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO(2) is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.

Nisin A depletes intracellular ATP and acts in bactericidal manner against Mycobacterium smegmatis.

Nisin is a bacteriocin produced by many strains of Lactococcus lactis. This study examined the effect of nisin on Mycobacterium smegmatis, a non-pathogenic species of Mycobacterium. Nisin had a minimum inhibitory concentration of 8.0 micrograms ml-1 and a minimum inhibitory dose of 7.5 micrograms ml-1 against Myco. smegmatis. Treatment with 25.0 micrograms ml-1 nisin caused partial inhibition of Myco smegmatis; the survivors were nisin-sensitive when tested in a separate experiment. Mycobacterium smegmatis cells exposed to 50.0 micrograms ml-1 of nisin, lost their viability. the effect of nisin on the growth of Myco. smegmatis was both time- and concentration-dependent. Nisin (10.0 micrograms ml-1) caused 97.7 +/- 2.0% reduction in internal ATP and leakage of intracellular ATP out of Myco. smegmatis cells after several hours of treatment. These data suggest that nisin inhibits Myco. smegmatis by the same mechanism by which it inhibits other bacteria and warrants further investigation as a possible antitubercular agent.

Characterization of fatty acid composition, spore germination, and thermal resistance in a nisin-resistant mutant of Clostridium botulinum 169B and in the wild-type strain.

The membrane fatty acids, thermal resistance, and germination of a nisin-resistant (Nisr) mutant of Clostridium botulinum 169B were compared with those of the wild-type (WT) strain. In the membranes of WT cells, almost 50% of the total fatty acids were unsaturated, but in those of Nisr cells, only 23% of the fatty acids were unsaturated. WT and Nisr spores contained similar amounts (approximately 23%) of unsaturated fatty acids, but the saturated straight-chain/branched-chain ratio was significantly higher in Nisr spores than in WT spores. These fatty acid differences suggest that Nisr cell and spore membranes may be more rigid, a characteristic which would interfere with the pore-forming ability of nisin. Nisr C. botulinum did not produce an extracellular nisin-degrading enzyme, nor were there any differences in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of coat proteins extracted from WT and Nisr spores, eliminating these as possible reasons for nisin resistance. Nisr spores had thermal resistance parameters similar to those of WT spores. In WT spores, but not in Nisr spores, nisin caused a 40% reduction in thermal resistance and a twofold increase in the germination rate. Because the nisin-induced increase in the germination rate of WT spores occurred only in the presence of a germinant (a molecule that triggers germination), nisin can be classified as a progerminant (a molecule that stimulates germination only in the presence of a germinant).

Analysis of the influence of environmental parameters on Clostridium botulinum time-to-toxicity by using three modeling approaches.

This study used the technique of waiting time modeling to analyze the combined effects of temperature, pH, carbohydrate, protein, and lipid on the time-to-toxicity of Clostridium botulinum 56A. Waiting time models can be used whenever the time to the occurrence of some event is the variable of interest. In the case of the time-to-toxicity data, the variable is the time from the beginning of an experiment until a tube is identified as positive. The statistical analysis used the SAS procedure LIFEREG and included determination of the form of the response surface, identification of the error distribution, and simplification of the response surface. We found that increasing the macromolecule concentration decreased the probability of toxin formation. The probability of toxin formation also decreased at lower temperatures and at pHs further from the optimum. The waiting time modeling approach to developing models for botulinal toxin formation compared favorably with other approaches but had one specific advantage. Waiting time models have the inherent advantage that safety concerns regarding predictions are automatically quantified in the analysis by formally identifying a distribution of times-to-toxicity. The use of this time-to-toxicity distribution permits a customizable margin of safety (e.g., one in a million) not possible with other approaches.

Influence of lipid composition on pediocin PA-1 binding to phospholipid vesicles.

Pediocin PA-1 bound to anionic lipid vesicles with saturated or unsaturated fatty acid chains in a lipid concentration-dependent fashion. Little change in binding parameters was observed for zwitterionic lipid vesicles. Decreasing the anionic lipid content of the vesicles gave a higher relative dissociation constant for the peptide-lipid interactions and further supports the electrostatic interaction model of binding.

Nisin resistance in Listeria monocytogenes ATCC 700302 is a complex phenotype.

Nisin resistance in Listeria monocytogenes ATCC 700302 is a complex phenotype involving alterations in both the cytoplasmic membrane and the cell wall and a requirement for divalent cations. In addition to a lower ratio of C15 to C17 fatty acids than in the wild-type strain (A. S. Mazzotta and T.J. Montville, J. Appl. Microbiol. 82: 32-38, 1997), this nisin-resistant (Nisr) strain contained significantly more zwitterionic phosphatidylethanolamine and less anionic phosphatidylglycerol and cardiolipin. The extraction of cardiolipin was enhanced by a penicillin-lysozyme step to disrupt the cell wall. This study is the first to quantify the phosphatidylethanolamine component of the L. monocytogenes cytoplasmic membrane. While these cytoplasmic membrane changes were induced by nisin, the Nisr strain also showed altered sensitivities to cell wall-acting compounds, even when grown in the absence of nisin, suggesting a constitutive alteration in the strain's cell wall. A model which integrates the roles of the cell membrane, cell wall, and divalent cations is presented. Finally, nisin resistance in L. monocytogenes ATCC 700302 conferred cross-resistance to the class IIa bacteriocin pediocin PA-1 and the class IV leuconocin S.

Mechanistic action of pediocin and nisin: recent progress and unresolved questions.

Nisin and pediocin PA-1 are examples of bacteriocins from lactic acid bacteria (LAB) that have found practical applications as food preservatives. Like other natural antimicrobial peptides, LAB bacteriocins act primarily at the cytoplasmic membranes of susceptible microorganisms. Studies with in vivo as well as in vitro membrane systems are directed toward understanding how bacteriocins interact with membranes so as to provide a mechanistic basis for their rational applications. The dissipation of proton motive force was identified early on as the common mechanism for the lethal activity of LAB bacteriocin. Models for nisin/membrane interactions propose that the peptide forms poration complexes in the membrane through a multistep process of binding, insertion, and pore formation. This review focuses on the current knowledge of: (1) the mechanistic action of nisin and pediocin-like bacteriocins, (2) the requirement for a cell factor such as a membrane protein, (3) the influence of membrane potential, pH, and lipid composition on the specificity and efficacy of bacteriocins, and (4) the roles of specific amino acids and structural domains of the bacteriocins in their action.

Electrostatic interactions, but not the YGNGV consensus motif, govern the binding of pediocin PA-1 and its fragments to phospholipid vesicles.

The purpose of this study was to characterize in detail the binding of pediocin PA-1 and its fragments to target membranes by using tryptophan fluorescence as a probe. Based on a three-dimensional model (Y. Chen, R. Shapira, M. Eisenstein, and T. J. Montville, Appl. Environ. Microbiol. 63:524-531, 1997), four synthetic N-terminal pediocin fragments were selected to study the mechanism of the initial step by which the bacteriocin associates with membranes. Binding of pediocin PA-1 to vesicles of phosphatidylglycerol, the major component of Listeria membranes, caused an increase in the intrinsic tryptophan fluorescence intensity with a blue shift of the emission maximum. The Stern-Volmer constants for acrylamide quenching of the fluorescence of pediocin PA-1 in buffer and in the lipid vesicles were 8.83 +/- 0.42 and 3.53 +/- 0.67 M-1, respectively, suggesting that the tryptophan residues inserted into the hydrophobic core of the lipid bilayer. The synthetic pediocin fragments bound strongly to the lipid vesicles when a patch of positively charged amino acid residues (K-11 and H-12) was present but bound weakly when this patch was mutated out. Quantitative comparison of changes in tryptophan fluorescence parameters, as well as the dissociation constants for pediocin PA-1 and its fragments, revealed that the relative affinity to the lipid vesicles paralleled the net positive charge in the peptide. The relative affinity for the fragment containing the YGNGV consensus motif was 10-fold lower than that for the fragment containing the positive patch. Furthermore, changing the pH from 6.0 to 8.0 decreased binding of the fragments containing the positive patch, probably due to deprotonation of His residues. These results demonstrate that electrostatic interactions, but not the YGNGV motif, govern pediocin binding to the target membrane.

Nisin Resistance in Clostridium botulinum Spores and Vegetative Cells.

The frequencies at which vegetative cells and spores of Clostridium botulinum strains 56A, 62A, 17409A, 25763A, 213B, B-aphis, and 169B formed colonies on agar media containing 0, 10(sup2), 10(sup3), and 10(sup4) IU of nisin per ml at 30(deg)C were determined. Strain 56A had the highest frequencies of nisin resistance, while strains 62A, 169B, and B-aphis had the lowest. For most strains, spores were more resistant than vegetative cells. One exposure to nisin was sufficient to generate stable nisin-resistant isolates in some strains. Stepwise exposure to increasing concentrations of nisin generated stable resistant isolates from all strains. Spores produced from nisin-resistant isolates maintained their nisin resistance. The frequency of spontaneous nisin resistance was reduced considerably by lowering the pH of the media and adding 3% NaCl. Nisin-resistant isolates of strains 56A and 169B also had increased resistance to pediocin PA1, bavaricin MN, plantaricin BN, and leuconocin S.