IAI Accepts, published online ahead of print on 26 October 2009

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Infect. Immun. doi:10.1128/IAI.00838-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Characterization of Virulence Plasmid Diversity Amongst Clostridium perfringens Type B Isolates

Sameera Sayeed, Jihong Li, and Bruce A. McClane^*

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh PA, USA 15261; Science Department, Marywood University, Scranton, PA 18509; Australian Research Council Centre of Excellence in Structural and Functional Microbial Genetics, Department of Microbiology, Monash University, Victoria, Australia

^* To whom correspondence should be addressed. Email: bamcc{at}pitt.edu.

The important veterinary pathogen Clostridium perfringens type B is unique for producing the two most lethal C. perfringens toxins, i.e., toxin and toxin. Our recent study (Miyamoto et. al., J Bacteriol., 2008, 190: 7178-7188) showed that most, if not all, type B isolates carry a 65 kb, toxin-encoding plasmid. However, this toxin plasmid did not possess the cpb gene encoding toxin, suggesting type B isolates carry at least one additional virulence plasmid. Therefore, the current study used Southern blotting of pulsed-field gels to localize the cpb gene to 90 kb plasmids in most type B isolates, although a few isolates carried a 65 kb cpb plasmid distinct from their etx plasmid. Overlapping PCR analysis then showed that the gene encoding the recently discovered TpeL toxin is located 3 kb downstream of the plasmid-borne cpb gene. As shown earlier for their toxin-encoding plasmid, the toxin-encoding plasmids of type B isolates were found to carry a tcp locus, suggesting they are conjugative. Additionally, IS1151-like sequences were identified upstream of the cpb gene in type B isolates. These IS1151-like sequences may mobilize the cpb gene based upon detection of possible cpb-containing circular transposition intermediates. Most type B isolates also possessed a third virulence plasmid that carries genes encoding urease and lambda toxin. Collectively, these findings indicate that type B isolates are among the most plasmid-dependent of all C. perfringens isolates for virulence, as they usually carry three potential virulence plasmids.