A Molecular Approach for Investigation of the Prevalence and Neurotoxin Formation of Clostridium botulinum in Food Safety Assessment

Abstract: In the development of new food products, more knowledge is needed about the occurrence and quantity of food-borne pathogens in the food chain to be able to implement effective control measures and assure food safety. In addition, improved understanding of how environmental factors and food preservatives effect the microbial virulence expression in foods will enable the formulation of new strategies for food preservation and risk assessment. In this thesis, novel tools based on PCR methodology have been developed for the investigation of the prevalence of Clostridium botulinum in primary production and for monitoring the botulinum neurotoxin (BoNT) gene expression in the presence of common food preservatives. A semiquantitative enrichment PCR method was designed for the detection of low numbers of spores of C. botulinum types B, E and F in faecal samples from pigs and cattle. The method was able to detect 10 spores of C. botulinum type B per gram faeces with > 95% detection probability. The enrichment PCR was used in two surveys to determine the incidence and quantity of C. botulinum spores in pigs and cattle at slaughterhouses in Sweden. The results revealed that the prevalence was high; 62% in pigs and 73% in cattle regarding C. botulinum type B, with a higher incidence during winter than summer. Less than 4 spores per gram faeces were found in 71% of the positive samples in pigs and in 64% of the positive samples from cattle. Quantitative reverse transcription-PCR (qRT-PCR) methods were developed to monitor the relative BoNT gene expression in C. botulinum types B and E. The bont expression followed the growth cycle in both C. botulinum type B and type E with a maximum in expression as the bacteria entered the stationary growth phase. The relative levels of bontB expression in different strains of C. botulinum type B was found to be well correlated to the neurotoxin formation and the toxicity. The bontB specific qRT-PCR method was used to study the effect of CO2, NaCl and NaNO2 on the neurotoxin gene expression in non-proteolytic C. botulinum. The combination of 2.5% NaCl with 37.5 ppm or 75 ppm NaNO2 resulted in no observed growth of C. botulinum. In addition, 1.25% NaCl in combination with NaNO2 was found to have an inhibitory growth effect of the bacteria. As opposed, the relative bontB expression and the BoNT/B formation remained unchanged. An elevated CO2 concentration was found to have a stimulatory effect on the bontB expression. A 5-fold increase was observed at 70% CO2 atmosphere compared with 10% CO2. This finding was also confirmed when analyzing the BoNT/B formation with ELISA.