Clostridium perfringens sporulation, germination and outgrowth in food: a functional genomics approach

At the heart of foodborne disease caused by Clostridium perfringenslays its ability to form spores. The ubiquitous presence of C. perfringensis due to the fact that spores are resilient and are able to survive harsh environmental conditions. As a result, spores of C. perfringenscan be found in many food ingredients and are able to survive a range of processing steps in the food industry. Subsequent spore germination in foods - followed by outgrowth - can lead to product spoilage and foodborne illness. Yet, not all C. perfringensstrains can cause foodborne illness; only certain C. perfringensstrains that produce enterotoxin (CPE) have this ability. Consumption of high levels (more than 105CFU/g) of such CPE producing strains can lead to diarrhea when cells sporulate, concomitant with release of the spores and CPE in the gut. One of the aims of this thesis was to obtain better insight in genes that play a role in the process of sporulation and germination of C. perfringenscompared with those in the better studied Bacillusspecies. This was achieved through in silicoanalysis of germination genes in the genomes of Clostridiumand Bacillusspecies. Overall, it was found that the numbers of gergenes, encoding germinant receptors of the GerA family, are lower in clostridia than in bacilli. Moreover, various Clostridiumspecies are predicted to produce cortex-lytic enzymes that are different from the ones encountered in bacilli. So far, studying gene function and regulation in clostridia has been hampered by the lack of genetic tools, but novel insights in genes putatively involved in sporulation and germination were obtained through whole genome transcriptome analysis during sporulation. The majority of previously characterized C. perfringensgermination genes showed significant upregulated expression profiles in time during sporulation. Such upregulated expression profiles during sporulation were also observed for other genes, including C. perfringenshomologs of Bacillussporulation and germination genes. A comprehensive homology search revealed that approximately half of the upregulated genes are conserved within a broad range of sporeforming Firmicutes; these genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior. Food borne disease by C. perfringenscan only be caused by strains that carry the cpegene, encoding the enterotoxin. This gene can be carried on the chromosome (C-cpe) or on a plasmid (P-cpe). To characterize C. perfringensstrains present in foods, isolates from a nationwide survey were subjected to 16S rDNA sequence analysis, multi locus sequence typing (MLST) and toxin gene profiling. This revealed that the current standard cultivation method gives false-positive results in ~30% of cases (i.e.species other than C. perfringenswere identified). Of the confirmed C. perfringensisolates, only ~10% carried the cpegene. For these cpe-positive strains, the gene was predominantly carried on the chromosome, but P-cpestrains were also found. MLST analysis showed that the C-cpestrains are evolutionarily distant from cpe-negative and P-cpestrains (with the latter two associated with the gut). Overall, these results highlight a need for improved methods to detect C. perfringenswith higher specificity, ideally simultaneously allowing for discrimination between C-cpe, P-cpeand cpe-negative strains. The growth potential of cpe-negative, C-cpeor P-cpe strains was further assessed in a model food. Spores were obtained from 15 diverse strains and spore heat resistance was the highest for C-cpestrains. Spores of the individual strains were inoculated in raw minced beef prior to vacuum packaging and heating, and their germination and growth potential was assessed during storage at 12°C and 25°C. This showed lower outgrowth potential of C-cpestrains than of cpe-negative and P-cpestrains at 12°C, suggesting that the cpe-negative and P-cpestrains may have a competitive advantage over C-cpestrains when these are present in products at low storage temperatures (refrigeration abuse temperatures). Overall, the C-cpestrains produce spores that are relatively heat resistant and if such spores survive (insufficient) heat treatments, they may be able to multiply rapidly if cooling regimes are not appropriate. Once the product reaches temperatures below 12°C, growth will be limited. Therefore, rapid cooling is very important and a critical control point. The P-cpestrains, on the other hand, produce spores that are rather heat sensitive and these will normally be inactivated even by relatively low heat treatments. However, if such strains are introduced as a post-processing contamination, for instance by food handlers, and the product is held for prolonged times at abuse refrigeration conditions, they may pose a risk for food borne infections. The post-processing hygienic measures in combination with adequate cooling may therefore be the most critical with respect to control of P-cpestrains. Overall, this thesis has provided new insights in the genes involved in germination of C. perfringensversus the well-known Bacillusspecies, and potential new candidate genes that play a role in sporulation and germination of spore forming Firmicutes were identified. It was apparent that cultivation methods that are currently available for C. perfringensrendered a large percentage of false positives. Further genetic analysis of C. perfringensisolates obtained from foods showed that C-cpestrains – which are usually associated with food - belong to a cluster that is distinct from P-cpeand cpe-negative strains. Strains belonging to the C-cpeand P-cpeclusters showed different heat resistance characteristics and outgrowth potential in a model food system at low temperatures. This points to different critical control points to prevent foodborne outbreaks due to C. perfringens; for C-cpestrains an adequate heat treatment is required to inactivate spores and rapid cooling is needed to prevent outgrowth in the event any spores survived. For P-cpestrains, spores are rather sensitive to heat treatment, but it is particularly important to prevent post-heating contamination and maintain low storage temperatures of products.