Real-time PCR for diagnosis of botulism and quantification of neurotoxin gene expression in Clostridium botulinum

University dissertation from Divison of Medical Microbiology

Abstract: The objectives of the work presented in this thesis have been to develop molecular methods for (i) detection of Clostridium botulinum in food and clinical samples, and (ii) monitoring the expression of the neurotoxin gene, cnt, during different growth phases and under different growth conditions. For the diagnosis of botulism, a real-time PCR-based method including an internal amplification control for C. botulinum types A, B and E was developed. The method was used to diagnose the first case of wound botulism in an injecting drug user in Sweden, which was also the first case of wound botulism reported to be caused by C. botulinum type E. It was previously assumed that only proteolytic C. botulinum could cause infectious botulism. However, this case revealed that wound botulism can be caused by nonproteolytic C. botulinum. When developing new food products it is important to consider not only the occurrence and quantity of pathogens in the food chain, but how the processing and subsequent storage and handling will affect them. Especially for food designed to have a long shelf-life, understanding the influence of environmental factors, food preservatives, and type of packaging on microbial growth and virulence expression is very important. Quantitative reverse transcription PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to monitor the neurotoxin expression in C. botulinum types A, B, and E. The relative cnt expression varied with growth phase in all three types, reaching a maximum as the late exponential phase was changing into stationary phase. However, for the proteolytic type A, a second increase could be seen in late stationary phase in contrast to type E. The same behaviour has earlier been observed for proteolytic C. botulinum type B with a second increase in cntB mRNA when the bacteria reached the death phase. For the nonproteolytic C. botulinum type E the cntE expression declined rapidly after the peak in the transition between exponential and stationary phase. The cntE mRNA half-life was calculated to be approximately nine minutes. When investigating the effect of carbon dioxide on growth and neurotoxin expression, we found that while proteolytic type A was not affected in either regard, the nonproteolytic types B and E were. Their growth was slowed, but the relative cnt expression was increased with an elevated CO2 concentration, with fivefold higher levels of cntB mRNA and type B toxin being detected at 70% CO2 than at 10%. For type E the increase in expression at 70% CO2 was two-fold greater, when compared with 10% CO2. For the proteolytic strain Hall A microarrays were also used to follow the genome-wide expression profiles. Comparison between the results from the qRT-PCR and the microarrays shows that both methods seem valid, as the results are similar. For example, the expression of the positive regulator gene, cntR, followed the same pattern as cntA, but at a much lower level. In conclusion, our findings, confirmed with both qRT-PCR and ELISA, shed a new cautionary light on the potential risks of botulism associated with nonproteolytic C. botulinum and the use of modified atmosphere packaging. In addition, our PCR method was successfully used on clinical samples and proved to be a valuable complement to standard methods.