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.

Antibacterially active substituted anilides of carboxylic and sulfonic acids.

Anilides of carboxylic and sulfonic acids were prepared and tested for antimicrobial activity. While these anilides were ineffective against Gram-negative organisms, there was a good correlation between chemical structure and biological activity against Gram-positive species. Both the nature and position of the benzene ring substituents and the length of the carbon side chain affected the activity and specificity of the compounds. The highest activity was observed when the acyl or sulfuryl moiety had a C7-C9 side chain attached. The CONH and SO2NH bridging groups were equally effective. The attachment of COOH or COOCH3 groups in the omega-position did not effect activity, but the substitution of the acidic proton of the sulfonamide group by an alkyl group rendered the compound inactive. Six compounds, which were substituted anilides of sulfonic acids, fatty acids, or the analagous alpha-methylene-substituted acids, were bacteriostatic at 10 ppm against Bacillus cereus, Staphylococcus aureus, Streptococcus faecalis, and Lactobacillus plantarum. One of these compounds, 2-hydroxy-5-nitroanilide of alpha-methylenedecanoic acid, was bactericidal at 1 ppm.

Molecular characterization of the replicon of the Pediococcus pentosaceus 43200 pediocin A plasmid pMD136.

The pediocin A-encoding plasmid of Pediococcus pentosaceus 43200, pMD136, was characterized by restriction enzyme analysis. Analysis of its replicon was facilitated by the construction of a probe vector consisting of the Escherichia coli plasmid pSP72 and the cat gene from Staphylococcus aureus plasmid pC194. The replication region of pMD136 was localized on a 1.6-kb EcoRI/BglII fragment. Sequencing analysis revealed a non-coding region, repA, spanning the first 440 bp, followed by an open reading frame, repB, encoding a putative protein of 390 amino acids. The non-coding region contained two sets of 6-bp and two sets of 22-bp direct repeats and two sets of inverted repeats upstream of the open reading frame. Strong homology of the isolated replicon was found to theta-type replicons of Lactococcus lactis plasmids. Segregational stability assay suggested at least two regions as potentially involved in the stabilization of pMD136. The plasmid's strong homology to other theta-type replicons and its relatively high stability suggest that pMD136 belongs to the widespread family of theta-replication plasmids.

Measurement and synthesis of insoluble and soluble dextran by Streptococcus mutans.

Total and insoluble dextransucrase activities of 10 strains of oral streptococci were measured by a modified filter disk assay. Strains that were nonadherent to hard surfaces had only low levels of insoluble dextransucrase activity. A physical rather than metabolic mechanism is suggested to explain the decreased insoluble and increased soluble activities observed when dextran T-10 is added to the media.

Bicarbonate inhibition of Saccharomyces cerevisiae and Hansenula wingei growth in apple juice.

The ability of sodium bicarbonate to inhibit growth of Saccharomyces cerevisiae and Hansenula wingei in apple juice was investigated. Sodium bicarbonate at concentrations of 0.06, 0.12, and 0.24 M was added to pasteurized apple juice that was then inoculated with 10(3) or 10(5) cfu/ml of either yeast. Growth of both yeasts was inhibited by 0.12 M sodium bicarbonate when incubation was at 4 degrees C; 0.24 M sodium bicarbonate caused a slow die off of yeast. At 18 degrees C, H. wingei became more sensitive and died in the presence of 0.12 M sodium bicarbonate, but S. cerevisiae became resistant to 0.24 M sodium bicarbonate. These results could not be attributed to bicarbonate-induced pH elevation or sodium. Potassium and ammonium bicarbonate were also inhibitory, implicating bicarbonate ion as the antimicrobial agent.

Evidence that dissipation of proton motive force is a common mechanism of action for bacteriocins and other antimicrobial proteins.

While bacteriocins from lactic acid bacteria (LAB) have generated tremendous interest among food microbiologists, they are not unique. The biosphere is awash with antimicrobial proteins such as colicins, defensins, cecropins, and magainins. These proteins share many characteristics. They are low molecular weight, cationic, amphiphilic, tend to aggregate and are benign to the producing organism. In cases where the mode of action has been investigated, the cell membrane appears to be the site of action. There is increasing evidence that bacteriocins from many bacterial genera also share these characteristics. After a brief introduction on the significance of LAB bacteriocins, this review provides some background on proton motive force. Current studies of mechanisms for various bacteriocins are reviewed. Evidence is then introduced that bacteriocins produced by lactic acid bacteria act by the common mechanism of depleting proton motive force. The role and importance of energized membranes in this process is examined. These observations are linked to literature which demonstrates that many other classes of antimicrobial proteins act by the same mechanism. Questions regarding the role of receptor proteins and the physical mechanism by which PMF is depleted remain unresolved.

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.

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.

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.

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.

Dependence of Clostridium botulinum gas and protease production on culture conditions.

Reports that Clostridium botulinum toxin can sometimes be detected in the absence of indicators of overt spoilage led to a systematic study of this phenomenon in a model system. Media with various combinations of pH (5.0 to 7.0) and glucose (0.0 to 1.0%) were inoculated with vegetative cells of C. botulinum 62A and incubated anaerobically at 35 degrees C. Although growth and toxin production occurred at all pH and glucose combinations, accumulation of gas was delayed or absent in media with low pH, low glucose levels, or both. Other proteolytic C. botulinum strains gave similar results. Trypsin activation was required to detect toxin in some low pH cultures. The trypsinization requirement correlated with low proteolytic activity in the cultures. Proteolytic activity of the strains examined was 5- to 500-fold lower in botulinal assay medium than in cooked meat medium. The results indicate that the absence of gas accumulation does not preclude the presence of botulinal toxin and that proteolytic cultures grown under adverse conditions may require trypsinization for the detection of toxin.

Dual-substrate plate diffusion assay for proteases.

A plate diffusion assay for endopeptidases was developed. Proteases applied to plates containing 1% casein, 1% gelatin, and 1.5% agar caused distinct zones reminiscent of immunoprecipitation bands. The diameter of the zones was linearly proportional to the log of the enzyme activity applied over a range from 0.01 to greater than 100 IU/ml.

Interaction of pH and NaCl on culture density of Clostridium botulinum 62A.

Clostridium botulinum 62A growth rates declined with decreasing pH and increasing salt levels. Lysis rates, however, were affected only by pH. Due to competition between growth and lysis rates, an accurate assessment of interactive effects was obtained only when optical density determinations were made at multiple intervals.

Characterization of a halo-acid-tolerant variant of Clostridium botulinum B-aphis.

Clostridium botulinum B-aphis spores plated on medium containing 4% salt at pH 6.0 yielded colonies at a frequency of ca. 1 in 10(6). A subculture of one of these colonies, designated strain Ba410, was compared with the parent strain, B-aphis, for a variety of traits. After 7 days of incubation at 37 degrees C, strain Ba410 grew in medium containing 7% NaCl, whereas strain B-aphis could not grow in salt concentrations greater than 5%. The strains also differed in cellular and colonial morphology. After exponential growth in the basal medium was completed, lysis of both strains was pH dependent; in media containing salt, lysis of Ba410 cells was pH independent. Strain Ba410 was more proteolytic than strain B-aphis in conditions of low pH and high salt, so that its toxin could be detected by the mouse assay. In a medium containing alanine and cysteine, the germination rate of B-aphis was 0.77% min-1, whereas that of Ba410 was 0.14% min-1; 2% salt inhibited the germination of Ba410 but not B-aphis.

Quantitation of pH- and salt-tolerant subpopulations from Clostridium botulinum.

Plating efficiencies of Clostridium botulinum 62A spores on media with variable pH (7.0 to 5.5) and salt (0, 1, 2, and 3%) levels revealed that only a very small subpopulation could give rise to colonies. The relative size of this subpopulation decreased by orders of magnitude with decreasing pH and increasing salt concentrations. Strong interactions of pH with salt were noted. For example, on a medium containing 2% salt at pH 5.5, colonies could be formed from only 1 in 100,000 spores. Proper monitoring of medium anaerobiosis was critical in obtaining reproducible results.

Metabiotic effect of Bacillus licheniformis on Clostridium botulinum: implications for home-canned tomatoes.

The metabiotic effect of Bacillus licheniformis on Clostridium botulinum was examined. B. licheniformis elevated the pH of a model system with an initial pH of 4.4 so that C. botulinum grew and produced toxin. Toxin production was observed when spores from both species were coinoculated at levels as low as 10 spores per ml. When pint jars of tomatoes were used, canner size contributed to a 10,000-fold difference in the lethality of a boiling water bath process on B. licheniformis spores. Botulinal toxin was not detected in pH-elevated jars of tomatoes containing C. botulinum spores.

Effect of plating medium on heat activation requirement of Clostridium botulinum spores.

Clostridium botulinum 62A and ATCC 25763 spores required heat activation for maximum colony formation when plated on reinforced clostridial agar (BBL Microbiology Systems) but not when plated on botulinum assay medium. Spores from strains B-aphis and 53B did not exhibit heat activation when plated on either medium.

Mechanism by which ammonium bicarbonate and ammonium sulfate inhibit mycotoxigenic fungi.

In this study we examined the mechanism by which ammonium bicarbonate inhibits mycotoxigenic fungi. Elevated extracellular pH, alone, was not responsible for the antifungal activity. Although conidia of Penicillium griseofulvum and Fusarium graminearum had internal pH (pHi) values as high as 8.0 in buffer at an external pH (pHo) of 9.5, their viability was not markedly affected. The pHi values from conidia equilibrated in glycine-NaOH-buffered treatments without ammonium bicarbonate or ammonium sulfate were similar to values obtained from buffered treatments containing the ammonium salts. Thus, inhibition did not appear to be directly related to increased pHi. Ammonium sulfate in buffered media at pH greater than or equal to 8.7 was as inhibitory as ammonium bicarbonate, but was completely ineffective at pH less than or equal to 7.8. The hypothesis that free ammonia caused the fungal inhibition was tested by using ammonium sulfate as a model for ammonium bicarbonate. Viability, expressed as log CFU/ml, and percent germination of P. griseofulvum and F. graminearum decreased dramatically as the free ammonia concentration increased. Germination rate ratios (the germination rate in buffered ammonium sulfate divided by the germination rate in buffer alone) decreased linearly as the free ammonia concentration increased, further establishing NH3 as the toxic agent. Ammonium bicarbonate inhibits fungi because the bicarbonate anion supplies the alkalinity necessary to establish an antifungal concentration of free ammonia.