Expansion of the Clostridium perfringens toxin-based typing scheme

Abstract

Clostridium perfringens causes many different histotoxic and enterotoxic diseases in humans and animals as a result of its ability to produce potent protein toxins, many of which are extracellular. The current scheme for the classification of isolates was finalized in the 1960s and is based on their ability to produce a combination of four typing toxins - α-toxin, β-toxin, ε-toxin and ι-toxin - to divide C. perfringens strains into toxinotypes A to E. However, this scheme is now outdated since it does not take into account the discovery of other toxins that have been shown to be required for specific C. perfringens-mediated diseases. We present a long overdue revision of this toxinotyping scheme. The principles for the expansion of the typing system are described, as is a mechanism by which new toxinotypes can be proposed and subsequently approved. Based on these criteria two new toxinotypes have been established. C. perfringens type F consists of isolates that produce C. perfringens enterotoxin (CPE), but not β-toxin, ε-toxin or ι-toxin. Type F strains will include strains responsible for C. perfringens-mediated human food poisoning and antibiotic associated diarrhea. C. perfringens type G comprises isolates that produce NetB toxin and thereby cause necrotic enteritis in chickens. There are at least two candidates for future C. perfringens toxinotypes, but further experimental work is required before these toxinotypes can formally be proposed and accepted.

Keywords: Clostridium perfringens; Disease; Pathogenesis; Terminology; Toxinotyping; Toxins.

Fig. 1. Multiplex PCR analysis of representative C. perfringens type A to G strains

The strains were grown in TGY broth [79] to a turbidity at 600nm between 1.0 and 1.5. Genomic DNA (equivalent to 5mL of culture) was prepared as described previously [80]. DNA preparations were diluted 1 in 50 in sterile distilled water and used as templates in the multiplex toxin PCR based on a previous method [81]. The oligonucleotide primers and their concentrations are listed in Table 2. PCR reactions were prepared using 0.1 units/μL Taq DNA polymerase (Roche) in 1 x supplied buffer (Roche), 2 mM MgSO₄ and 0.4 mM dNTPs. The template constituted 0.1 volumes of the final reaction. PCR was performed with an initial denaturation at 95°C for five minutes, followed by 35 cycles of 95°C for 1 minute, 55°C for 1 minute and 72°C for 1 minute. Amplified products were resolved by electrophoresis through a 1.5% (w/v) TAE agarose gel. The multiplex PCR profiles of the following C. perfringens strains are shown: JIR325 (type A)[82], JGS1984 (type B)[83], CN3717 (type C)[84], JGS4138 (type D)[85], ATCC27324 (type E), SM101 (type F)[86], EHE-NE18 (type G)[87]. Size standards were PCR Markers (Promega).