GENETIC ANALYSIS OF CLOSTRIDIUM PERFRINGENS: INSIGHT INTO EVOLUTION OF VIRULENCE

Clostridium perfringens is an important pathogen in veterinary and medical fields. Understanding epidemiology of C. perfringens diseases and evolution of virulence within C. perfringens necessitates an efficient, time and cost effective strain typing method. Multiple-locus variable-number tandem repeat analysis (MLVA) has been applied to typing of other pathogens and we describe here the development of a MLVA scheme for C. perfringens. We characterized five VNTR loci, and screened 112 C. perfringens isolates to evaluate typability, reproducibility, and discriminatory power of the scheme. All isolates were assigned a MLVA genotype and the technique has excellent reproducibility, with a numerical index of discrimination of 0.995. Thus, MLVA is an efficient tool for C. perfringens strain typing, and being PCR based makes it rapid, easy, and cost effective. In addition, it can be employed in epidemiological, ecological, and evolutionary investigations of the organism.Virulence of this species is not fully understood and it does seem that distribution of the toxin/enzyme genes is erratic within the population. We used the MLVA scheme to investigate evolution of virulence and population structure of this species. Analysis of the phylogenetic signal indicates that acquisition of the major toxin genes and other plasmid-borne toxin genes is a recent evolutionary event, and their maintenance is essentially a function of the selective advantage they confer to strains carrying them in certain micro-niches under different conditions. In addition, it indicates the ability of virulent strains to cause disease in different hosts. More interestingly, there is evidence that certain normal flora strains are virulent when they gain access to a different host species. Analysis of the population structure indicates that recombination events are the major tool that shapes the population and this panmixia is interrupted with frequent clonal expansion that mostly corresponds to disease processes. Signature of positive selection was detected in the alpha toxin gene, suggesting the possibility of adaptive alleles on the other chromosomally-encoded determinants. Finally, C. perfringens proved to have a dynamic population, and availability of more genome sequences, use of comparative proteomics and of animal models would provide more insight into the pathogenicity of this organism