Effects of Carbon Dioxide on Neurotoxin Gene Expression in Non-proteolytic 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 growth of, and neurotoxin production by, non-proteolytic 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 formation of type E neurotoxin. Expression of cntE was similar in two strains, with relative expression peaking in the transition between exponential and stationary phase. In stationary phase, cntE mRNA expression declined rapidly. The cntE mRNA half-life was calculated to be approximately nine minutes. Neurotoxin formation occurred in late exponential 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 formation of extracellular neurotoxin were two-fold higher with a headspace carbon dioxide concentration of 70% (v/v) compared to 10% (v/v). This finding sheds a new, cautionary light on the potential risks of botulism associated with the use of modified atmosphere packaging.