Synergistic interaction between pH and NaCl in the limits of germination and outgrowth of Clostridium sporogenes and Group I Clostridium botulinum vegetative cells and spores after heat treatment.

Group I Clostridium botulinum and Clostridium sporogenes are physiologically and genetically closely related. Both are widely distributed in the environment and can cause foodborne botulism. In this work, a physiological study was conducted with 37 isolates from spoiled canned food and five referenced strains of C. sporogenes (three isolates) and Group I C. botulinum (two isolates). Growth limits of vegetative cells were established as a function of pH and NaCl concentration in PYG modified medium (PYGm) at 30 °C for 48 days. The heat resistance of the spores was studied for 2 min and 10 min at 102 °C and 110 °C. This physiological study (pH, NaCl growth limits and heat resistance) allowed the selection of 14 isolates of C. sporogenes (twelve isolates) and Group I C. botulinum (two isolates) representative of the diversity found. This panel of 14 selected isolates (11 isolated from spoiled canned food and three reference strains), were whole genome sequenced, but no association of physiological and genetic characteristics could be detected. Finally, we studied the ability of spores to germinate and grow from 5 isolates (four C. sporogenes and one Group I C. botulinum), under stress conditions generated by pH and NaCl following a low intensity heat treatment. The accumulation of these 3 stresses creates synergies that will strongly reduce the probability of spore growth in pH and salt conditions where they usually proliferate. The effect is progressive as the conditions become drastic: the number of decimal reduction observed increases translating a probability of growth which decreases. This study provides a better understanding of the behaviour of C. sporogenes and Group I C. botulinum isolates and shows how the combination of pH, NaCl and heat treatment can help prevent or minimise foodborne botulism outbreaks.

Insights into the Structure and Protein Composition of Moorella thermoacetica Spores Formed at Different Temperatures.

The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores.

Temperature impacts the sporulation capacities and spore resistance of Moorella thermoacetica.

Temperatures encountered in cannery allow growth of thermophilic spore-forming bacteria, including the strictly anaerobe Moorella thermoacetica, which grows optimally from 55 °C to 65 °C and is the main cause of spoilage of low-acid canned foods (LACFs) at high temperature. Resistance to wet-heat, biocides and UV-C of spores formed at different temperatures was assessed either for a selection of M. thermoacetica strains or for the strain M. thermoacetica ATCC 39073. Spores formed at 45 °C were significantly more sensitive to wet-heat than spores produced at 55 °C, while spores produced at 65 °C were as heat-resistant as spores produced at 55 °C. Spores of M. thermoacetica ATCC 39073 produced at 45 °C were significantly less resistant to peracetic acid than spores formed at 55 °C, while no difference in sensitivity to H2O2 or to UV-C treatment was observed whatever the sporulation temperature. However, both types of treatment enabled at least a 3.3 log CFU/mL reduction of M. thermoacetica ATCC 39073 spores. M. thermoacetica spores thus showed higher resistance properties when sporulation temperature was close to optimal growth temperature. These findings suggest food spoilage due to M. thermoacetica species could be controllable by holding temperatures below optimal growth temperature from the blanching step to the can filling step.

Genotypic and phenotypic characterization of foodborne Geobacillus stearothermophilus.

Geobacillus stearothermophilus is the main thermophilic spore former involved in flat sour spoilage of canned foods. Three typing methods were tested and applied to differentiate strains at intra-species level: panC sequence analysis, REP-PCR and M13-PCR. panC gene was highly conserved within the studied strains, suggesting a low intra-specific diversity. This was supported by REP-PCR primary assays and M13-PCR results. M13-PCR profile analysis succeeded in differentiating six closely related groups (at 79% threshold similarity) among 127 strains from a range of spoiled canned food products and from different canneries. Phenotypic traits were investigated among 20 selected strains representing groups and origins. Ranges of growth under different temperatures (from 40 °C to 70 °C), pH (from 5.0 to 6.5), NaCl concentrations (from 1 to 5%) and sporulation conditions poorly differed between strains, but wet heat resistance of spores showed a 20-fold variation between strains. Furthermore, in this study, strains that belonged to the same M13-PCR genetic group did not share phenotypic characteristics or common origin. The work emphasizes a low diversity within the G. stearothermophilus species but data from this study may contribute to a better control of G. stearothermophilus spoilage in canned food.

Absence of oxygen affects the capacity to sporulate and the spore properties of Bacillus cereus.

This study was performed to evaluate the effect of anaerobiosis on the formation of Bacillus cereus spores and their resulting properties. For this purpose, an appropriate sporulation medium was developed (MODs). Sporulation of 18 strains from different phylogenetic groups of B. cereus was studied in MODs medium in aerobiosis and anaerobiosis. In anaerobiosis, sporulation ability was weaker and more heterogeneous than in aerobiosis. Among tested strains, B. cereus AH187 produced the highest level of spores in anaerobiosis. This strain was therefore chosen to study spore properties. Spores produced in anaerobiosis were more resistant to wet heat at 90 °C, 92.5 °C, 95 °C, 97.5 °C and 100 °C. For example, D90 were 21,09 ± 1.70 and 81.87 ± 2.00 for aerobiosis and anaerobiosis conditions, respectively. Spores produced in anaerobiosis have a z-value of 7.70 °C compared with 10.52 °C for spores produced in aerobiosis. Spores produced in anaerobiosis were also more resistant to 1 M NaOH, 1 M nitrous acid and pulsed light at fluences of 0.34 J cm(-2) and 0.49 J cm(-2). No difference in resistance to UV-C, 5% hydrogen peroxide or 0.25 mM formaldehyde was observed between these two conditions. In the presence of L-alanine, spores produced in anaerobiosis germinated more efficiently than spore produced in aerobiosis. No difference in germination was observed with inosine as inducer. No difference in the size of spores produced in the different conditions was observed by transmission electron microscopy. However, spores obtained under anaerobic conditions had a damaged exosporium, or in some cases a completely detached exosporium, unlike spores produced under aerobic conditions. This study shows that few spores are formed under anaerobic condition; nevertheless, this condition has an impact on the spore properties of B. cereus AH 187 strain. Spores obtained under anaerobic condition were more resistant to heat and to some chemical compounds. This is an important feature, considering the risk associated with the presence of this pathogen in thermally processed and packaged food in absence of oxygen.

Spores of Bacillus cereus strain KBAB4 produced at 10 °C and 30 °C display variations in their properties.

Spores of the psychrotrophic Bacillus cereus KBAB4 strain were produced at 10 °C and 30 °C in fermentors. Spores produced at 30 °C were more resistant to wet heat at 85 °C, 1% glutaraldehyde, 5% hydrogen peroxide, 1M NaOH and pulsed light at fluences between 0.5 and 1.75 Jcm(-2) and to a lesser extent to monochromatic UV-C at 254 nm. No difference in resistance to 0.25 mM formaldehyde, 1M nitrous acid and 0.025 gl(-1) calcium hypochlorite was observed. Spores produced at 10 °C germinated more efficiently with 10 mM and 100 mM l-alanine than spores produced at 30 °C, while no difference in germination was observed with inosine. Dipicolinic acid (DPA) content in the spore was significantly higher for spores prepared at 30 °C. Composition of certain fatty acids varied significantly between spores produced at 10 °C and 30 °C.

Convergence of Bigelow and Arrhenius models over a wide range of heating temperatures.

The heat resistance of the bacterial spores of Moorella thermoacetica, Clostridium sporogenes, Geobacillus stearothermophilus and Bacillus coagulans was determined over a wide range of temperatures using the capillary method and thermoresistometer Mastia. The results showed that the two experimental methods gave similar heat resistance values excepted for Geobacillus stearothermophilus. The effect of temperature on thermal resistance was evaluated using the Arrhenius and Bigelow models. The fit of the heat sensitivity parameters of the Arrhenius and Bigelow models on the heat resistance parameter values obtained over a wide temperature range was equally good. Despite the apparent mathematical incompatibility of the two equations, it is recognized that they yield the same goodness of fit. This paper finds a mathematical reason for this convergence and explains why inside a temperature range of at least 100 °C, no significant difference in the quality of fit between these two models can be found.

Spore-forming bacteria responsible for food spoilage.

This review explores the main spore-forming bacteria involved in the spoilage of various processed foods. Bakery products are specifically spoiled by Bacillus species, the dominant one being Bacillus amyloliquefaciens, while different Clostridium species classically contaminate refrigerated vacuum-packed meats. These two genera have also been isolated from milk products, even when milk is pasteurized, sterilized, dehydrated or fermented, according to heat treatment and storage temperature. Finally, the most heat-resistant microorganisms are isolated in low-acid canned foods, the three predominant species being Geobacillus stearothermophilus, Moorella thermoacetica and Thermoanaerobacterium spp.

Contamination pathways of spore-forming bacteria in a vegetable cannery.

Spoilage of low-acid canned food during prolonged storage at high temperatures is caused by heat resistant thermophilic spores of strict or facultative bacteria. Here, we performed a bacterial survey over two consecutive years on the processing line of a French company manufacturing canned mixed green peas and carrots. In total, 341 samples were collected, including raw vegetables, green peas and carrots at different steps of processing, cover brine, and process environment samples. Thermophilic and highly-heat-resistant thermophilic spores growing anaerobically were counted. During vegetable preparation, anaerobic spore counts were significantly decreased, and tended to remain unchanged further downstream in the process. Large variation of spore levels in products immediately before the sterilization process could be explained by occasionally high spore levels on surfaces and in debris of vegetable combined with long residence times in conditions suitable for growth and sporulation. Vegetable processing was also associated with an increase in the prevalence of highly-heat-resistant species, probably due to cross-contamination of peas via blanching water. Geobacillus stearothermophilus M13-PCR genotypic profiling on 112 isolates determined 23 profile-types and confirmed process-driven cross-contamination. Taken together, these findings clarify the scheme of contamination pathway by thermophilic spore-forming bacteria in a vegetable cannery.

A new chemically defined medium for the growth and sporulation of Bacillus cereus strains in anaerobiosis.

A new chemically defined liquid medium, MODS, was developed for the aerobic growth and anaerobic growth and sporulation of Bacillus cereus strains. The comparison of sporulation capacity of 18 strains of B. cereus has shown effective growth and spore production in anaerobiosis..

Comparative subproteome analyses of planktonic and sessile Staphylococcus xylosus C2a: new insight in cell physiology of a coagulase-negative Staphylococcus in biofilm.

Staphylococcus xylosus is a Gram-positive bacterium found on the skin of mammals and frequently isolated from food plants and fermented cheese or meat. To gain further insight in protein determinants involved in biofilm formation by this coagulase-negative Staphylococcus, a comparative proteomic analysis between planktonic and sessile cells was performed. With the use of a protocol previously developed, protein patterns of the cytoplasmic and cell envelope fractions were compared by 2-DE. Following protein identification by MALDI-TOF mass spectrometry and bioinformatic analyses, this study revealed differences in expression levels of 89 distinct proteins with 55 up-expressed and 34 down-expressed proteins in biofilm compared to planktonic cells. Most proteins differentially expressed were related to nitrogen and carbon metabolisms. Besides amino acid biosynthesis and protein translation, protein determinants related to protein secretion were up-expressed in biofilm, suggesting a more active protein trafficking in sessile cells. While up-expression of several enzymes involved in pentose phosphate and glycolytic pathways was observed in biofilm, connections with unexpected metabolic routes were further unravelled. Indeed, this proteomic analysis allowed identifying novel proteins that could be involved in a previously uncovered exopolysaccharide biosynthetic pathway in S. xylosus as well as several enzymes related to polyketide biosynthesis. This findings are particularly relevant considering exopolysaccharide production in S. xylosus is ica-independent contrary to coagulase-negative model strain Staphylococcus epidermidis RP62A.

Proteomic analysis of cell envelope from Staphylococcus xylosus C2a, a coagulase-negative staphylococcus.

Staphylococcus xylosus is a saprophytic bacterium commonly found on skin of mammals but also used for its organoleptic properties in manufacturing of fermented meat products. This bacterium is able to form biofilms and to colonize biotic or abiotic surfaces, processes which are mediated, to a certain extent, by cell-envelope proteins. Thus, the present investigation aimed at evaluating and adapting different existing methods for cell-envelope subproteome analyses of the strain S. xylosus C2a. The protocol selected consisted initially of a lysostaphin treatment producing protoplasts and giving a fraction I enriched in cell wall proteins. A second fraction enriched in membrane proteins was then efficiently recovered by a procedure involving delipidation with a mixture of tributyl phosphate, methanol, and acetone and solubilization with a buffer containing ASB14. Proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 168 protein spots was identified corresponding to 90 distinct proteins. To categorize and analyze these proteomic data, a rational bioinformatic approach was carried out on proteins identified within cell envelope of S. xylosus C2a. Thirty-four proteins were predicted as membrane-associated with 91% present, as expected, within fraction II enriched in membrane proteins: 24 proteins were predicted as membranal, 3 as lipoproteins, and 7 as components of membrane protein complex. Eighteen out of 25 (72%) proteins predicted as secreted were indeed identified in fraction I enriched in cell wall proteins: 6 proteins were predicted as secreted via Sec translocon, and the remaining 19 proteins were predicted as secreted via unknown secretion system. Eighty-one percent (25/31) of proteins predicted as cytoplasmic were found in fraction II: 8 were clearly predicted as interacting temporarily with membrane components. By coupling conventional 2-DE and bioinformatic analysis, the approach developed allows fractionating, resolving, and analyzing a significant and important set of cell envelope proteins from a coagulase-negative staphylococcus, that is, S. xylosus C2a.