Inactivation of non-proteolytic Clostridium botulinum type E in low-acid foods and phosphate buffer by heat and pressure.

The effect of high pressure thermal (HPT) treatments on the inactivation of spores of non-proteolytic type E Clostridium botulinum TMW 2.990 was investigated at high pressures (300 to 600 MPa) and elevated temperatures (80 to 100 °C) in four low-acid foods (steamed sole, green peas with ham, vegetable soup, braised veal) and imidazole phosphate buffer (IPB). In addition, corresponding conventional thermal treatments at ambient pressure were performed to expose possible synergisms of pressure and temperature on spore inactivation. In general, spore count reduction was more efficient by combining pressure and temperatures < 100 °C and the overall process duration could be shortened due to accelerated heating rates (adiabatic effect). Processing at 90 °C and 600 MPa resulted in inactivation below the detection limit after 5 min in all foods except steamed sole. Traditional thermal processing of spores at 90 °C for 10 min, on the other hand, did not result in an estimated 6-log reduction. Additional HPT treatments in steamed sole and IPB did not reveal pronounced food matrix dependent protective effects. Here, varying pressure levels did not appear to be the driving force for spore count reduction in steamed sole at any temperature. By applying a Weibull distribution on destruction kinetics of isobaric/isothermal holding times, 6D-values were calculated. Compression and decompression phase (1 s pressure holding time) had a considerable impact on spore count reduction (max. -2.9 log units) in both, foods and buffer. Hence, compression and decompression phases should directly be included into the total lethal effect of HPT treatments to avoid prolonged holding times and overprocessing.

Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement.

UNLABELLED: Group II nonproteolytic Clostridium botulinum (gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies with C. botulinum are cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain, bont/E was exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use of pyrE as a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia. IMPORTANCE: The nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophic Clostridium botulinum In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia.

Differential effects of sporulation temperature on the high pressure resistance of Clostridium botulinum type E spores and the interconnection with sporulation medium cation contents.

High pressure thermal (HPT) processing can be used to improve traditional preservation methods and increase food safety and durability, whereas quality related characteristics can be largely maintained. Clostridium (C.) botulinum type E is a non-proteolytic, psychrotrophic, toxin-producing spore former, commonly associated with aquatic environments in temperate regions of the northern hemisphere. Sporulation in nature is likely to occur under varying conditions including temperature and nutrient availability, which might affect resistance properties of resulting spores. In our study, we determined the effect of sporulation temperature (13-38 °C) on the resistance of three Clostridium botulinum type E strains to differently intense HPT treatments (200 MPa at 40 and 80 °C, and 800 MPa at 40 and 80 °C). Furthermore, the effect of cations on sporulation temperature-mediated alterations in HHP resistance was investigated. Results indicate that low and high sporulation temperatures can increase and decrease sporal HPT resistance, respectively, in a treatment-dependent (pressure level, treatment temperature) manner, whereas the trends observed are largely unaffected by pressure dwells (1 s-10 min). Furthermore, results show that the cation content of the sporulation medium (Ca(2+), Mg(2+), Mn(2+)) marginally influences and partially counteracts effects on the HPT resistance of spores grown at low and elevated temperatures, respectively. This suggests that sporulation temperature and medium cations provoke changes in some common spore resistance structures. Sporulation conditions can markedly affect spore resistance properties and, thus, should be considered for the experimental setup of worst case studies aiming to evaluate the effectiveness of food processes in terms of the inactivation of C. botulinum type E spores.

Effect of sporulation medium and its divalent cation content on the heat and high pressure resistance of Clostridium botulinum type E spores.

Clostridium (C.) botulinum type E belongs to the non-proteolytic physiological C. botulinum group II and produces the highly potent Botulinum neurotoxin E (BoNT/E) even at refrigerated temperatures. As C. botulinum type E spores are highly prevalent in aquatic environments, seafood and fishery products are commonly associated with this organism. Hydrostatic high pressure (HHP) treatments, or treatments combining HHP with elevated temperatures (HHPT), can be used to improve traditional preservation methods and increase food safety, quality and durability. In this study, we assessed the effect of different sporulation media and cation concentration on the heat resistance, HHP resistance, and HHPT resistance of spores from three C. botulinum type E strains. SFE (sediment fish extract) sporulation media yielded the most resistant spores, whereas, in M140 media, the least resistant spores were produced. Furthermore our results indicate that the divalent cation content (Ca(2+), Mg(2+) and Mn(2+)) plays a role in the differential development of C. botulinum type E spore resistance to heat, HHP and HHPT in different media. Calcium cations confer heat and HPPT resistance to spores, while high amounts of magnesium cations appear to have a negative effect. Manganese cations in low concentrations are important for the development resistance to HPP and HPPT treatments, but not heat alone. This study provides valuable information on the nature of non-proteolytic C. botulinum type E spores grown in different media. The data provided here can be useful to the food industry and to researchers when considering spore properties in food safety risk assessment and the experimental design of future inactivation studies.

Quantitative real-time reverse transcription-PCR analysis reveals stable and prolonged neurotoxin cluster gene activity in a Clostridium botulinum type E strain at refrigeration temperature.

The relative expression levels of six botulinum neurotoxin cluster genes in a group II Clostridium botulinum type E strain grown at 10 or 30 degrees C were investigated using quantitative real-time reverse transcription-PCR. An enzyme-linked immunosorbent assay was used to confirm neurotoxin expression. Distinct mRNA and toxin production patterns were observed at the two temperatures. The average relative mRNA levels at 10 degrees C were higher than (ntnh and p47), similar to (botE), or lower than (orfx1, orfx2, orfx3) those at 30 degrees C. The maximum botE expression levels and average neurotoxin levels at 10 degrees C were 45 to 65% of those at 30 degrees C. The relative mRNA levels at 10 degrees C declined generally slowly within 8 days, as opposed to the rapid decline observed at 30 degrees C within 24 h. Distinct expression patterns of the six genes at the two temperatures suggest that the type E neurotoxin cluster genes are transcribed as two tricistronic operons at 30 degrees C, whereas at 10 degrees C monocistronic (botE or orfx1 alone) and bicistronic (ntnh-p47 and orfx2-orfx3) transcription may dominate. Thus, type E botulinum neurotoxin production may be involved with various temperature-dependent regulatory events. In light of group II C. botulinum type E being a dangerous food-borne pathogen, these findings may be important in terms of the safety of refrigerated packaged foods of extended durability.

Effects of carbon dioxide on neurotoxin gene expression in nonproteolytic Clostridium botulinum Type E.

Carbon dioxide is an antimicrobial gas commonly used in modified atmosphere packaging. In the present study, the effects of carbon dioxide on the growth of and neurotoxin production by nonproteolytic Clostridium botulinum type E were studied during the growth cycle. Quantitative reverse transcription-PCR and an enzyme-linked immunosorbent assay were used to quantify expression of the type E botulinum neurotoxin gene (cntE) and the formation of type E neurotoxin. The expression levels of cntE were similar in two strains, with relative expression peaking in the transition between exponential phase and stationary phase. In stationary phase, cntE mRNA expression declined rapidly. The cntE mRNA half-life was calculated to be approximately 9 minutes. Neurotoxin formation occurred in late exponential phase and stationary phase. High carbon dioxide concentrations delayed growth by increasing the lag time and decreasing the maximum growth rate. The effects of carbon dioxide concentration on relative neurotoxin gene expression and neurotoxin formation were significant. Expression of cntE mRNA and the formation of extracellular neurotoxin were twofold higher with a headspace carbon dioxide concentration of 70% (vol/vol) compared to 10% (vol/vol). This finding sheds a new, cautionary light on the potential risks of botulism associated with the use of modified atmosphere packaging.

Persistence of Clostridium botulinum neurotoxin type E in tissues from selected freshwater fish species: implications to public health.

Rainbow trout (Oncorhynchus mykiss), round gobies (Neogobius melanostomas), yellow walleye (Stizostedion vitreum), and yellow perch (Perca flavescens) were given Clostridium botulinum neurotoxin type E (BoNT/E) at four doses (0, 800, 1500, and 4000 mouse lethal doses). BoNT/E was sought in the fish tissues at death or at the conclusion of the experiment (10 days after treatment). Fish were divided into a "fillet" (axial musculature) and a "nonfillet" sample before testing for BoNT/E toxicity with a mouse bioassay. BoNT/E was detected in all species. The percentage of positive BoNT samples ranged across the species and doses from 0 (trout, perch, and walleye) to 17% (round goby) in fillet tissues and from 0 (perch) to 92% (round goby) in nonfillet tissues. The lack of positive fillet samples in three key commercial fish species suggests that the public health implications of eating these fish are minimal. However, the presence of toxin in the nonfillet compartment of a high proportion of fish supports the hypothesis that live intoxicated fish are a vehicle for the transfer of BoNT/E to fish-eating birds, which are then in turn, intoxicated.

Expression of botulinum neurotoxins A and E, and associated non-toxin genes, during the transition phase and stability at high temperature: analysis by quantitative reverse transcription-PCR.

Production of botulinum neurotoxin A (BoNT/A) and associated non-toxic proteins (ANTPs), which include a non-toxic non-haemagglutinin (NTNH/A) as well as haemagglutinins (HAs), was found previously to be dependent upon an RNA polymerase alternative sigma factor (BotR/A). Expression of the botR/A, bont/A and antp genes, monitored by reverse transcription and real-time PCR analysis, occurred concomitantly at the transition between the exponential and stationary growth phases of Clostridium botulinum A. The botR/A expression level was about 100-fold less than those of the bont/A and antp genes. Therefore, BotR/A is an alternative sigma factor controlling the botulinum A locus genes during the transition phase. The highest toxin concentration was released into the culture supernatant 12 h after maximum expression of the botR/A, bont/A and antp genes, without any apparent bacterial lysis. Toxin levels were then stable over 5 days in cultures at 37 degrees C, whereas a dramatic decrease in lethal activity was observed between 24 and 48 h in cultures at 44 degrees C. High temperature did inhibit transcription, since expression levels of the botR/A, bont/A and antp genes were similar in cultures at 37 and 44 degrees C. However, incubation at 44 degrees C triggered a calcium-dependent protease that degraded BoNT/A and NTNH/A, but not HAs. In C. botulinum E, which contains no gene related to botR, the bont/E and p47 genes were also expressed during the transition phase, and no protease activation at 44 degrees C was evident.