Effects of carbon dioxide on growth of proteolytic Clostridium botulinum, its ability to produce neurotoxin, and its transcriptome.

The antimicrobial gas carbon dioxide is frequently used in modified atmosphere packaging. In the present study, the effects of CO2 (10 to 70%, vol/vol) on gene expression (measured using quantitative reverse transcription-PCR and a whole-genome DNA microarray) and neurotoxin formation (measured using an enzyme-linked immunosorbent assay [ELISA]) by proteolytic Clostridium botulinum type A1 strain ATCC 3502 were studied during the growth cycle. Interestingly, in marked contrast to the situation with nonproteolytic C. botulinum types B and E, CO2 had little effect on any of these parameters. At all CO2 concentrations, relative expression of neurotoxin cluster genes peaked in the transition between exponential and stationary phases, with evidence of a second rise in expression in late stationary phase. Microarray analysis enabled identification of coding sequences whose expression profiles matched those of the neurotoxin cluster. Further research is needed to determine whether these are connected to neurotoxin formation or are merely growth phase associated.

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.

First case of type E wound botulism diagnosed using real-time PCR.

Wound botulism is a growing problem among injecting drug users. The condition is often difficult to diagnose, with laboratory confirmation in only 50% of the cases. Here we present a real-time PCR-based method for the diagnosis of wound botulism caused by Clostridium botulinum. The assay includes an internal amplification control which is amplified simultaneously with the genes encoding neurotoxin types A, B, and E. This method was used to detect the first case of wound botulism in an injecting drug user in Sweden. In addition, to the best of our knowledge, this is the first reported case of wound botulism caused by C. botulinum type E.

Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B.

The effects of carbon dioxide, sodium chloride, and sodium nitrite on type B botulinum neurotoxin (BoNT/B) gene (cntB) expression in nonproteolytic Clostridium botulinum were investigated in a tryptone-peptone-yeast extract (TPY) medium. Various concentrations of these selected food preservatives were studied by using a complete factorial design in order to quantitatively study interaction effects, as well as main effects, on the following responses: lag phase duration (LPD), growth rate, relative cntB expression, and extracellular BoNT/B production. Multiple linear regression was used to set up six statistical models to quantify and predict these responses. All combinations of NaCl and NaNO(2) in the growth medium resulted in a prolonged lag phase duration and in a reduction in the specific growth rate. In contrast, the relative BoNT/B gene expression was unchanged, as determined by the cntB-specific quantitative reverse transcription-PCR method. This was confirmed when we measured the extracellular BoNT/B concentration by an enzyme-linked immunosorbent assay. CO(2) was found to have a major effect on gene expression when the cntB mRNA levels were monitored in the mid-exponential, late exponential, and late stationary growth phases. The expression of cntB relative to the expression of the 16S rRNA gene was stimulated by an elevated CO(2) concentration; the cntB mRNA level was fivefold greater in a 70% CO(2) atmosphere than in a 10% CO(2) atmosphere. These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng x ml(-1). unit of optical density(-1) in the 10% CO(2) atmosphere and 126 ng x ml(-1). unit of optical density(-1) in the 70% CO(2) atmosphere.