Extensive growth and growth boundary model for non-proteolytic Clostridium botulinum - Evaluation and validation with MAP and smoked foods.

The growth inhibiting effect of lactic acid bacteria (LAB) on non-proteolytic Clostridium botulinum was studied. LAB had no significant effect on growth of C. botulinum and their effect was not included in the model to be evaluated. An available cardinal parameter growth and growth boundary model for non-proteolytic C. botulinum (Koukou et al., 2021; https://doi.org/10.1016/j.ijfoodmicro.2021.109162) was evaluated using a total of 822 time-to-toxin (TTT) formation data extracted from the scientific literature for seafood, poultry, vegetables and meat products. These data included smoked products and food stored in air, vacuum or modified atmosphere packaging (MAP) with added CO2. The available extensive model predicted TTT formation without bias (Bf-TTT value = 0.99) and with a reasonable accuracy (Af-TTT value = 1.76). The model was successfully validated for seafood and poultry products. This study substantially increased the range of applicability of the available growth and growth boundary model for non-proteolytic C. botulinum. The performed evaluation showed this model can be used to predict environmental conditions to prevent growth in seafood and poultry products including smoked fish and MAP foods. It is expected that this validated model will contribute to product development and innovation including new sodium reduced foods.

The synergic interaction between environmental factors (pH and NaCl) and the physiological state (vegetative cells and spores) provides new possibilities for optimizing processes to manage risk of C. sporogenes spoilage.

Clostridium sporogenes has been widely used as a surrogate for proteolytic C. botulinum for validating thermal processes in low-acid cans. To limit the intensity of heat treatments, industrials must use other ways of control as an association of acidic and saline environment after a low heat treatment. The probability of growth of pH (7-4.4), sodium chloride concentration (0-11%) and heat treatment (80°C-10 min; 100°C-1.5 min and 5.2 min) were studied on C. sporogenes PA 3679 spores and vegetative cells. Vegetative cells or heat-treated spores were inoculated in PYGm broth at 30 °C for 48 days in anaerobic conditions. Vegetative cells growth (pH 4.6-pH 4.5; 7%-8% NaCl) range is larger than the spore one (pH 5.2-pH 5.0; 6%-7% NaCl). Spores germination and outgrowth rage is decreased if the spores are heat-treated at 100 °C for 1.5 min (pH 5.5-5.3; 4%-5% NaCl) and 5.2 min (pH 5.7-5.3; 4%-5% NaCl). The C. sporogenes PA 3679 spores germination and outgrowth is impacted by their physiological state. The synergic interaction between environmental factors (pH and NaCl) and the physiological state (vegetative cells and spores) opening new possibilities for optimizing food formulation processes to manage the risks of C. sporogenes spoilage.

Cardinal parameter growth and growth boundary model for non-proteolytic Clostridium botulinum - Effect of eight environmental factors.

A new cardinal parameter growth and growth boundary model for non-proteolytic C. botulinum was developed and validated for fresh and lightly preserved seafood and poultry products. 523 growth rates in broth were used to determine cardinal parameter values and terms for temperature, pH, NaCl/water activity, acetic, benzoic, citric, lactic and sorbic acids. The new growth and growth boundary model included the inhibiting interactive effect between these factors and it was calibrated using growth curves from 10 challenge tests with unprocessed seafood. For model evaluation, 40 challenge tests with well characterized fresh and lightly preserved seafood were performed. Comparison of these observed growth curves and growth rates (µmax-values) predicted by the new model resulted in a bias factor (Bf) of 1.12 and an accuracy factor (Af) of 1.40. Furthermore, the new model was evaluated with 94 growth rates and 432 time to toxin formation data extracted from the scientific literature for seafood, poultry, meat, pasta and prepared meals. These data included responses for 36 different toxigenic strains of non-proteolytic C. botulinum. The obtained Bf-/Af-values were 0.97/2.04 for µmax-values and 0.96/1.80 for time to toxin formation. The model correctly predicted 93.8% of the growth responses with 5.6% being fail-safe and <1% fail-dangerous. A cocktail of four non-toxin producing Clostridium spp. isolates was used to develop the new model and these isolates had more than 99.8% 16S rRNA gene similarity to non-proteolytic C. botulinum (Group II). The high number of environmental factors included in the new model makes it a flexible tool to facilitate development or reformulation of seafood and poultry products that do not support the growth of non-proteolytic C. botulinum. Further, evaluation of the new model with well characterized products is desirable particularly for meat, vegetables, pasta and prepared meals as well as for dairy products that was not included in the present study.

A predictive growth model for Clostridium botulinum during cooling of cooked uncured ground beef.

A dynamic model to predict the germination and outgrowth of Clostridium botulinum spores in cooked ground beef was presented. Raw ground beef was inoculated with a ten-strain C. botulinum spore cocktail to achieve approximately 2 log spores/g. The inoculated ground beef was vacuum packaged, cooked to 71 °C to heat shock the spores, cooled to below 10 °C, and incubated isothermally at temperatures from 10 to 46 °C. C. botulinum growth was quantified and fitted into the primary Baranyi Model. Secondary models were fitted to maximum specific growth rate and lag phase duration using Modified Ratkowsky equation (R2 0.96) and hyperbolic function (R2 0.94), respectively. Similar experiments were also performed under non-isothermal (cooling) conditions. Acceptable zone prediction (APZ) analysis was conducted on growth data collected over 3 linear cooling regimes from the current study. The model performance (prediction errors) for all 22 validation data points collected in the current work were within the APZ limits (-1.0 to +0.5 log CFU/g). Additionally, two other growth data sets of C. botulinum reported in the literature were also subjected to the APZ analysis. In these validations, 20/22 and 10/14 predictions fell within the APZ limits. The model presented in this work can be employed to predict C. botulinum spore germination and growth in cooked uncured beef under non-isothermal conditions. The beef industry processors and food service organizations can utilize this predictive microbial model for cooling deviations and temperature abused situations and in developing customized process schedules for cooked, uncured beef products.

Influence of reduced levels or suppression of sodium nitrite on the outgrowth and toxinogenesis of psychrotrophic Clostridium botulinum Group II type B in cooked ham.

Outgrowth and toxinogenesis of Clostridium botulinum Group II (non-proteolytic) type B were studied in cooked ham prepared with different NaNO2 (ranging from 0 to 80 mg/kg) and sodium chloride (NaCl, ranging from 12 to 19 g/kg) incorporation rates. Cured ground pork batters were inoculated with a cocktail of 3 strains of C. botulinum Group II type B at 3.5 log10 CFU/g, portioned and samples of 50 g were vacuum packed then cooked and cooled based on thermal processing employed by the meat processing industry. These cooked ham model samples were stored under reasonably foreseeable conditions of use and storage i.e. for 14 days at 4 °C, followed by a cold chain break for 1 h at 20 °C then up to 33 days at 8 °C. Storage times and temperatures were used to mimic those commonly encountered along the supply chain. Enumeration of C. botulinum and detection of the botulinum neurotoxin type B (BoNT/B) were performed in triplicate at different storage times. Under these experimental conditions, incorporation rates of NaNO2 ≥ 30 mg/kg prevented the outgrowth and toxinogenesis of C. botulinum Group II type B in the cooked ham model, regardless of the NaCl concentrations tested. In contrast, total removal of nitrite allowed outgrowth and toxin production during storage of the processed meat product. Results showed that the maximum ingoing amount of nitrite (i.e. 150 mg/kg) that may be added according to the EU legislation (Regulation (EC) No 1333/2008) can be reduced in cooked ham while still ensuring control of C. botulinum Group II type B. According to the multiple factors that could affect C. botulinum behavior in processing meat products, outgrowth and toxin production of C. botulinum should be evaluated on a case by case basis, depending on the recipe, manufacturing process, food matrix and storage conditions.

High prevalence of Clostridium botulinum in vegetarian sausages.

Clostridium botulinum is a significant food safety concern due to its ability to produce highly potent neurotoxin and resistant endospores. Vegetarian sausages have become a popular source of plant protein and alternative for meat products. While vegetarian sausages have not been linked to botulism, numerous outbreaks due to preserved vegetables suggest a frequent occurrence of C. botulinum spores in the raw material. The product formulation of vegetarian sausages involves limited NaCl and preservatives, and shelf-lives may be several months. The safety of vegetarian sausages thus relies mainly on heat treatment and chilled storage. The main food safety concern is C. botulinum Group II that can grow and produce toxin at refrigeration temperatures. Here we show a high overall prevalence (32%) of C. botulinum in 74 samples of vegetarian sausages from seven producers. Both Groups I and II strains and genes for neurotoxin types A, B, E and F were detected in the products. The highest cell counts (1200 spores/kg) were observed for C. botulinum Group II in products with remaining shelf-lives of 6 months at the time of purchase. We conclude that vacuum-packaged vegetarian sausage products frequently contain C. botulinum spores and may possess a high risk of C. botulinum growth and toxin production. Chilled storage below 3°C and thorough reheating before consumption are warranted.

Inactivation of Bacillus and Clostridium Spores in Coconut Water by Ultraviolet Light.

Bacterial spores are generally more resistant than vegetative bacteria to ultraviolet (UV) inactivation. The UV sensitivity of these spores must be known for implementing UV disinfection of low acid liquid foods. UV inactivation kinetics of bacterial spores in coconut water (CW) and distilled sterile water was studied. Populations of Bacillus cereus and Clostridium sporogenes dormant spores were reduced by more than 5.5 log10 at the UV-C photon fluence of 1142 µE·m-2 and 1919 µE·m-2 respectively. C. sporogenes spores showed higher UV-C resistance than B. cereus, with the photon fluence 300 µE·m-2 required for one log inactivation (D10) and 194 µE·m-2, respectively. No significant difference was observed in D10 values of spores suspended in the two fluid types (p > 0.05). The inactivation kinetics of microorganisms were described by log linear models with low root mean square error and high coefficient of determination (R2 > 0.98). This study clearly demonstrated that high levels of inactivation of bacterial spores can be achieved in CW. The baseline data generated from this study will be used to conduct spore inactivation studies in continuous flow UV systems. Further proliferation of the technology will include conducting extensive pilot studies.

Proteomic analysis of four Clostridium botulinum strains identifies proteins that link biological responses to proteomic signatures.

Microorganisms alter gene and protein expression in response to environmental conditions to adapt and survive. Whereas the genetic composition of a microbe represents an organism's biological potential, the proteins expressed provide a functional readout of the organism's response to the environment. Understanding protein expression patterns in response to specific environmental conditions furthers fundamental knowledge about a microbe, which can be especially useful for understudied organisms such as Clostridium botulinum examined herein. In addition, protein expression patterns that reproducibly occur in certain growth conditions hold potential in fields such as microbial forensics, in which determination of conditions in which an unknown possible biothreat sample had been grown may be important. To investigate the identity and reproducibility of protein profile patterns for varied strains, we defined the proteomic profiles of four Group I strains of Clostridium botulinum, a Category A biothreat agent and the organism responsible for the production of the botulinum neurotoxin (BoNT), in two different culture media grown for five days. The four C. botulinum strains produced one of three neurotoxins (BoNT/A, /B, or /F), and their protein profiles were compared to that of a fifth non-toxigenic strain of C. sporogenes. These strains each had DNA sequences available to assist in accurate protein identification. Differing culture growth phase, bacterial strain, and growth medium resulted in reproducible protein profiles, which were used to calculate relative protein abundance ratios as an internally normalized metric of microbial growth in varying conditions. The resulting protein profiles provide functional information about how four Group I C. botulinum strains and a C. sporogenes strain respond to the culture environment during growth and explores the feasibility of using these proteins to characterize unknown samples.

Growth of non-toxigenic Clostridium botulinum mutant LNT01 in cooked beef: One-step kinetic analysis and comparison with C. sporogenes and C. perfringens.

The objective of this study was to investigate the growth kinetics of Clostridium botulinum LNT01, a non-toxigenic mutant of C. botulinum 62A, in cooked ground beef. The spores of C. botulinum LNT01 were inoculated to ground beef and incubated anaerobically under different temperature conditions to observe growth and develop growth curves. A one-step kinetic analysis method was used to analyze the growth curves simultaneously to minimize the global residual error. The data analysis was performed using the USDA IPMP-Global Fit, with the Huang model as the primary model and the cardinal parameters model as the secondary model. The results of data analysis showed that the minimum, optimum, and maximum growth temperatures of this mutant are 11.5, 36.4, and 44.3 °C, and the estimated optimum specific growth rate is 0.633 ln CFU/g per h, or 0.275 log CFU/g per h. The maximum cell density is 7.84 log CFU/g. The models and kinetic parameters were validated using additional isothermal and dynamic growth curves. The resulting residual errors of validation followed a Laplace distribution, with about 60% of the residual errors within ±0.5 log CFU/g of experimental observations, suggesting that the models could predict the growth of C. botulinum LNT01 in ground beef with reasonable accuracy. Comparing with C. perfringens, C. botulinum LNT01 grows at much slower rates and with much longer lag times. Its growth kinetics is also very similar to C. sporogenes in ground beef. The results of computer simulation using kinetic models showed that, while prolific growth of C. perfringens may occur in ground beef during cooling, no growth of C. botulinum LNT01 or C. sporogenes would occur under the same cooling conditions. The models developed in this study may be used for prediction of the growth and risk assessments of proteolytic C. botulinum in cooked meats.

Mechanism of bio molecule stabilized selenium nanoparticles against oxidation process and Clostridium Botulinum.

The bio molecules from plant leaf extract utilized in the preparation of selenium material at the nano scale. The selenium ion was reduced to selenium nanoparticles in the presence of molecule residue of the plant leaf extract. The bio molecule stabilized selenium nanoparticles were grown gradually in the reaction mixture. The selenium nanoparticles were characterized using atomic absorption spectroscopy, fourier transform inferred spectroscopy, X-ray diffraction, scanning electronic microscope and transmission electronic microscope. The selenium nanoparticles were synthesized successfully as the nano-crystalline pure hexagonal phase and the size range of 26-41 nm with spherical in shape. The activity and mechanism of nanoparticles suggested that the selenium nanoparticles are causes of leakage of reducing sugars and protein of pathogens membrane cell. The selenium nano are responsible for death and fully inhibited the microbial growth of pathogen. The bio molecule stabilized selenium nanoparticles were also investigated for the antioxidant agent. Selenium nanoparticles showed scavenging activity up to 94.48%. These results recommended that the advantages of using this method for synthesis of selenium nanoparticles with excellent antioxidant and antimicrobial mechanism and activity, which can be used as the antioxidant and antibiotic agent.

Inhibition of Clostridium botulinum in Model Reduced-Sodium Pasteurized Prepared Cheese Products.

The 1986 Food Research Institute-Tanaka et al. model predicts the safety of shelf-stable process cheese spread formulations using the parameters of moisture, pH, NaCl, and disodium phosphate (DSP) to inhibit toxin production by Clostridium botulinum. Although this model is very reliable for predicting safety for standard-of-identity spreads, the effects of additional factors have not been considered. The objective of this study was to create a predictive model to include the interactive effect of moisture, pH, fat, sorbic acid, and potassium-based replacements for NaCl and DSP to reflect modern reduced-sodium recipes. Eighty formulations were identified using a central composite design targeting seven factors: 50 to 60% moisture, pH 5.4 to 6.2, 0 to 0.2% sorbic acid, 10 to 30% fat, 1.7 to 2.4% NaCl, 0.8 to 1.6% DSP, and 0 to 50% potassium replacement for sodium salts. Samples were inoculated with proteolytic C. botulinum spores at 3 log spores per g, hot filled into sterile vials, and stored anaerobically at 27°C. Samples were assayed at 0, 1, 2, 3, 4, 8.5, 17.5, 26, and 40 weeks for the presence of botulinum toxin using the mouse bioassay. A parametric survival model was fit to the censored time-to-toxin data. All linear, quadratic, and pairwise effects were considered for model fit. As hypothesized, the effects of pH, sorbate, moisture, DSP, and NaCl were highly significant (P < 0.001). Fat concentration and potassium replacement effects were significant at P < 0.021 and P < 0.057, respectively. The model consistently predicted the safety failure of the toxic samples, but it also predicted failure for some samples that were not toxic. This model is an adjunct to existing models by adding the factors of potassium salts, fat, and sorbic acid to predict the botulinal safety of prepared process cheese products but is not intended to be a substitute for formulation evaluation by a competent process authority.

Avaliação dos fatores físico-químicos que controlam o desenvolvimento do Clostridium botulinum em mortadelas comercializadas no estado do Rio de Janeiro / Evaluation of the physical-chemical factors that control the development of Clostridium botulinum in mortadella marketed in the state of Rio de Janeiro

A mortadela é um produto cárneo embutido muito consumido pelos brasileiros. Por ser um alimento embalado à vácuo apresenta condições propícias ao desenvolvimento de esporos bacterianos de patógenos como o Clostridium botulinum. Muitos aditivos usados na produção de carnes industrializadas visam melhorar o sabor, a cor e a aparência do produto, além de estender a sua vida útil. Desta forma, os fatores que garantem a segurança destes produtos são a conservação em baixas temperaturas após sua produção, na comercialização e no transporte; adição dos conservantes nitrato e nitrito de sódio nas concentrações adequadas; e o tratamento térmico adequado durante o seu processamento. Este trabalho teve como objetivo avaliar o risco da presença da toxina botulínica em amostras de mortadela através da análise das características físico químicas do produto. Foram coletadas, no varejo do município do Rio de Janeiro/RJ, dez amostras de mortadelas de cinco diferentes marcas comerciais, que foram analisadas quanto ao pH, atividade de água, teor de umidade, resíduo mineral fixo, temperatura de conservação, data de fabricação e prazo de validade. De acordo com o risco do crescimento do Clostridium botulinum, 100% das amostras apresentaram atividade de água (Aw) acima de 0,955 e pH acima dos valores de 4,5 o que favorece a multiplicação dessa bactéria.(AU)

Avaliação dos fatores físico-químicos que controlam o desenvolvimento do Clostridium botulinum em mortadelas comercializadas no estado do Rio de Janeiro

A mortadela é um produto cárneo embutido muito consumido pelos brasileiros. Por ser um alimento embalado à vácuo apresenta condições propícias ao desenvolvimento de esporos bacterianos de patógenos como o Clostridium botulinum. Muitos aditivos usados na produção de carnes industrializadas visam melhorar o sabor, a cor e a aparência do produto, além de estender a sua vida útil. Desta forma, os fatores que garantem a segurança destes produtos são a conservação em baixas temperaturas após sua produção, na comercialização e no transporte; adição dos conservantes nitrato e nitrito de sódio nas concentrações adequadas; e o tratamento térmico adequado durante o seu processamento. Este trabalho teve como objetivo avaliar o risco da presença da toxina botulínica em amostras de mortadela através da análise das características físico químicas do produto. Foram coletadas, no varejo do município do Rio de Janeiro/RJ, dez amostras de mortadelas de cinco diferentes marcas comerciais, que foram analisadas quanto ao pH, atividade de água, teor de umidade, resíduo mineral fixo, temperatura de conservação, data de fabricação e prazo de validade. De acordo com o risco do crescimento do Clostridium botulinum, 100% das amostras apresentaram atividade de água (Aw) acima de 0,955 e pH acima dos valores de 4,5 o que favorece a multiplicação dessa bactéria.

Mortadella is a sausage widely consumed in Brazil. It is a vacuum packed food, what provides conditions conducive to the development of spores of pathogen bacteria such as Clostridium botulinum. Many additives used in the production of processed meats aim to improve the flavor, color, appearance of the product and extend its shelf-life. The major factors to guarantee the safety of these products are low temperatures stocking, commercialization and transport; addition of preservatives sodium nitrate and sodium nitrite at the appropriate concentrations; and the appropriate heat treatment during processing. This work aimed to evaluate the risk of the presence of botulin toxin in mortadella samples by analyzing the physicochemical characteristics of the product. Ten mortadella samples from five different commercial brands were collected at a market, in the city of Rio de Janeiro, RJ, Brazil. The samples were analyzed for pH, water activity, moisture content, fixed mineral residue, storage temperature, date of manufacture and expiry date. According to the risk of Clostridium botulinum presence, all samples presented water activity (Aw) above 0.955 and pH above 4,5, conditions that favors bacteria proliferation.

Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502.

Botulinum neurotoxin (BoNT), produced by neurotoxigenic clostridia, is the most potent biological toxin known and the causative agent of the paralytic disease botulism. The nutritional, environmental, and genetic regulation of BoNT synthesis, activation, stability, and toxin complex (TC) formation is not well studied. Previous studies indicated that growth and BoNT formation were affected by arginine and glucose in Clostridium botulinum types A and B. In the present study, C. botulinum ATCC 3502 was grown in toxin production medium (TPM) with different levels of arginine and glucose and of three products of arginine metabolism, citrulline, proline, and ornithine. Cultures were analyzed for growth (optical density at 600 nm [OD600]), spore formation, and BoNT and TC formation by Western blotting and immunoprecipitation and for BoNT activity by mouse bioassay. A high level of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold, enhanced growth, slowed lysis, and reduced endospore production by greater than 1,000-fold. Similar effects on toxin production were seen with equivalent levels of citrulline but not ornithine or proline. In TPM lacking glucose, levels of formation of BoNT/A1 and TC were significantly decreased, and extracellular BoNT and TC proteins were partially inactivated after the first day of culture. An understanding of the regulation of C. botulinum growth and BoNT and TC formation should be valuable in defining requirements for BoNT formation in foods and clinical samples, improving the quality of BoNT for pharmaceutical preparations, and elucidating the biological functions of BoNTs for the bacterium.IMPORTANCE Botulinum neurotoxin (BoNT) is a major food safety and bioterrorism concern and is also an important pharmaceutical, and yet the regulation of its synthesis, activation, and stability in culture media, foods, and clinical samples is not well understood. This paper provides insights into the effects of critical nutrients on growth, lysis, spore formation, BoNT and TC production, and stability of BoNTs of C. botulinum We show that for C. botulinum ATCC 3502 cultured in a complex medium, a high level of arginine repressed BoNT expression by ca. 1,000-fold and also strongly reduced sporulation. Arginine stimulated growth and compensated for a lack of glucose. BoNT and toxin complex proteins were partially inactivated in a complex medium lacking glucose. This work should aid in optimizing BoNT production for pharmaceutical uses, and furthermore, an understanding of the nutritional regulation of growth and BoNT formation may provide insights into growth and BoNT formation in foods and clinical samples and into the enigmatic function of BoNTs in nature.

Oxidation-reduction potential and lipid oxidation in ready-to-eat blue mussels in red sauce: criteria for package design.

BACKGROUND: Ready-to-eat in-package pasteurized blue mussels in red sauce requires refrigerated storage or in combination with an aerobic environment to prevent the growth of anaerobes. A low barrier packaging may create an aerobic environment; however, it causes lipid oxidation in mussels. Thus, evaluation of the oxidation-reduction potential (Eh) (aerobic/anaerobic nature of food) and lipid oxidation is essential. Three packaging materials with oxygen transmission rate (OTR) of 62 (F-62), 40 (F-40) and 3 (F-3) cm3 m-2 day-1 were selected for this study. Lipid oxidation was measured by color changes in thiobarbituric acid reactive substances (TBARS) at 532 nm (TBARS@532) and 450 nm (TBARS@450). RESULTS: Significantly higher (P < 0.05) TBARS@532 was found in mussels packaged in higher OTR film. TBARS@450 in mussels packaged with F-62 and F-40 gradually increased during refrigerated storage (3.5 ± 0.5 °C), but remained constant after 20 days of storage for mussels packaged with F-3. The Eh of pasteurized sauce was not significantly affected (P > 0.05) by OTR and remained negative (< -80 mV) during storage. Negative Eh values can support the growth of anaerobes such as Clostridium botulinum. The headspace oxygen concentration was reduced by about 50% from its initial value during pasteurization, and then further declined during storage. The headspace oxygen concentration was higher in trays packaged with higher OTR film. CONCLUSION: Mussels packed with high OTR film showed higher lipid oxidation, indicating that high barrier film is required for packaging of mussels. Pasteurized mussels must be kept in refrigerated storage to prevent growth of anaerobic proteolytic C. botulinum spores under temperature abuse. © 2016 Society of Chemical Industry.

Phenotypic and Genotypic Characterization of Bacteriocinogenic Enterococci Against Clostridium botulinum.

The present study aimed to characterize Enterococcus faecalis (n = -6) and Enterococcus faecium (n = 1) isolated from healthy chickens to find a novel perspective probiotic candidate that antagonize Clostridium botulinum types A, B, D, and E. The isolated enterococci were characterized based on phenotypic properties, PCR, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). The virulence determinants including hemolytic activity on blood agar, gelatinase activity, sensitivity to vancomycin, and presence of gelatinase (gelE) and enterococcal surface protein (esp) virulence genes were investigated. Also, the presence of enterocin structural genes enterocin A, enterocin B, enterocin P, enterocin L50A/B, bacteriocin 31, enterocin AS48, enterocin 1071A/1071B, and enterocin 96 were assessed using PCR. Lastly, the antagonistic effect of the selected Enterococcus spp. on the growth of C. botulinum types A, B, D, and E was studied. The obtained results showed that four out of six E. faecalis and one E. faecium proved to be free from the tested virulence markers. All tested enterococci strains exhibited more than one of the tested enterocin. Interestingly, E. faecalis and E. faecium significantly restrained the growth of C. botulinum types A, B, D, and E. In conclusion, although, the data presented showed that bacteriocinogenic Enterococcus strains lacking of virulence determinants could be potentially used as a probiotic candidate against C. botulinum in vitro; however, further investigations are still urgently required to verify the beneficial effects of the tested Enterococcus spp. in vivo.

A study on the toxigenesis by Clostridium botulinum in nitrate and nitrite-reduced dry fermented sausages.

Nitrite has been traditionally used to control Clostridium botulinum in cured meat products. However, in the case of dry fermented sausages, environmental factors such as pH, aw and the competitive microbiota may exert a more relevant role than nitrite in the inhibition of the growth and toxin production by C. botulinum. In this challenge test study, two varieties of Mediterranean dry sausages (salchichón and fuet) were inoculated with spores of C. botulinum Group I (proteolytic) and C. botulinum Group II (nonproteolytic). Sausages were prepared with 150 mg/kg of NaNO3 and 150 mg/kg of NaNO2 (maximum ingoing amounts allowed by the European Union regulation), with a 25% and 50% reduction, and without nitrate/nitrite. The initial pH in both products was 5.6, and decreased to values below 5.0 in salchichón and to 5.2 in fuet. Lactic acid bacteria counts reached 8-9 log cfu/g after fermentation. The aw decreased from initial values of 0.96 to about 0.88-0.90 at the end of ripening. Botulinum neurotoxin was not detected in any of the sausages, including those manufactured without nitrate and nitrite. Despite the environmental conditions were within the range for germination and growth of C. botulinum Group I during the first 8 days of the ripening process in fuet and 10-12 days in salchichón, acidity, aw and incubation temperature combined to inhibit the production of toxin, independently of the concentration of curing agents. Although decreasing or even removing nitrate/nitrite from the formula did not compromise safety regarding C. botulinum in the conditions tested in this study, their antimicrobial role should not be underestimated in the case that other hurdles could fail or other ripening conditions were used, and also considering the effect of nitrite on other pathogens.

Clostridium botulinum Toxin Production in Relation to Spoilage of Atlantic Salmon (Salmo salar) Packaged in Films of Varying Oxygen Permeabilities and with Different Atmospheres.

Shelf life of fish packaged under modified atmosphere (MA) is extended, but within the United States, commercial application of MA with impermeable packaging films is restricted due to concerns that botulinum toxin production would precede spoilage when contaminated fish are held at abusive storage temperatures. Use of semipermeable packaging films has been advocated; however, previous studies are inconclusive in determining the oxygen transmission rate (OTR) of a film that is needed to achieve an acceptable margin of safety (i.e., toxin production occurs only after spoilage). This study was conducted to determine the influence of OTR (target OTRs of 3 to 15,000) on the development of spoilage volatiles and toxin in salmon inoculated with type E Clostridium botulinum and subjected to air, vacuum, or 75:25 CO2:N2 MA and storage temperatures of 4, 8, 12, or 16°C. The most dominant headspace volatile peak that was produced during spoilage of samples at 4, 8 or 12°C was a peak, having a Kovats retention index (KI) of 753, and at which external standards of 2- or 3-methyl 1-butanol also eluted. Under anaerobic conditions, both the aerobic microbial populations and the size of the KI 753 spoilage peak were less in inoculated samples compared with uninoculated samples. C. botulinum-inoculated samples that were stored at 12 or 16°C under conditions favorable for anaerobic growth were also characterized by a KI 688 peak. Using a previously developed model that related the percentage of elderly consumers who would prepare a sample having the KI 753 spoilage peak of a specific size, it was determined that for salmon packaged with 3 or 3,000 OTR films under any atmosphere and stored at 12 or 16°C, 2 to 61% of the consumers could potentially prepare toxin-contaminated samples. Hence, when abusive storage conditions are suspected, the fish should not be consumed.

Functional csdA is needed for effective adaptation and initiation of growth of Clostridium botulinum ATCC 3502 at suboptimal temperature.

The activity of RNA helicase csdA (cbo2802) after temperature downshift was compared to its activity at optimal growth temperature, and the effect of sense and antisense oriented insertional inactivation of cbo2802 on the growth of ATCC 3502 at suboptimal temperature was evaluated. The relative cbo2802 transcript level was significantly induced for 30min to 5h after cold shock. In contrast, a significant decrease in the relative transcript level of cbo2802 was observed within the same time frame at 37°C. Inactivation of cbo2802 led to an extensive delay in initiation of exponential growth at 20°C but not at 37°C. In addition, the mean minimum growth temperatures of the mutant strains were higher than those of the wild-type strain. During a 24-hour incubation at 37°C, all strains were motile, whereas at 20°C the mutant strains showed severely impaired motility compared to the wild-type strain. This study shows that a functional csdA is needed for effective adaptation and initiation of growth and motility of Clostridium botulinum ATCC 3502 at suboptimal temperature.

Optimal spore germination in Clostridium botulinum ATCC 3502 requires the presence of functional copies of SleB and YpeB, but not CwlJ.

Germination, the process by which dormant endospores return to vegetative growth, is a critical process in the life cycle of the notorious pathogen Clostridium botulinum. Crucial is the degradation by hydrolytic enzymes of an inner peptidoglycan spore layer termed the cortex. Two mechanistically different systems of cortex lysis exist in spores of Clostridium species. C. botulinum ATCC 3502 harbours the Bacillus-like system of SleB, CwlJ and YpeB cortex lytic enzymes (CLEs). Through the construction of insertional gene knockout mutants in the sleB, cwlJ and ypeB genes of C. botulinum ATCC 3502 and the production of spores of each mutant strain, the effect on germination was assessed. This study demonstrates a reduced germination efficiency in spores carrying mutations in either sleB or ypeB with an approximate 2-fold reduction in heat resistant colony forming units (CFU/OD600) when plated on rich media. This reduction could be restored to wild-type levels by removing the spore coat and plating on media supplemented with lysozyme. It was observed that cwlJ spores displayed a similar germination efficiency as wild-type spores (P > 0.05). An optimal germinant commixture was identified to include a combination of l-alanine with sodium bicarbonate as it resulted in a 32% drop in OD600, while the additional incorporation of l-lactate resulted in a 57% decrease. Studies of the germination efficiency of spores prepared from all three CLE mutants was performed by monitoring the associated decrease in optical density but a germination defect was not observed in any of the CLE mutant strains. This was likely due to the lack of specificity of this particular assay. Taken together, these data indicate that functional copies of SleB and YpeB, but not CwlJ are required for the optimal germination of the spores of C. botulinum ATCC 3502.