Introduction

Clostridium perfringens, a gram-positive, spore-forming rod, is an important cause of histotoxic and enteric disease in humans and other animals. Although ubiquitously distributed in soils throughout the world, C. perfringens is also found as part of the normal animal intestinal flora. Like all pathogenic Clostridia, C. perfringens is renowned for toxin production with an arsenal of over 15 different toxins. The presence of genes encoding the four ''major lethal'' toxins (a-toxin, p-toxin, e-toxin, and i-toxin) is used to classify C. perfringens isolates into one of the five types (A-E) (Table 1). Type A strains are largely responsible for human gas gangrene and other histotoxic infections, whereas type C strains secrete p-toxin and can cause enteritis nectroticans in undernourished individuals. Although not used in the typing scheme, the C. perfringens enterotoxin (CPE) is responsible for C. perfringens type A food poisoning, as well as nonfood-borne gastrointestinal (GI) illness. This chapter describes the current status of C. perfringens genomics, as well as molecular methods (where available) for diagnosing human infections caused by this bacterium.

C. PERFRINGENS: GENOMIC INSIGHTS Genome Projects

Early physical maps indicated that the C. perfringens genome size varies between isolates (3-3.7 Mb).[1] These studies also mapped the location of chromosomal virulence factors (such as a-toxin and perfringolysin O), the key two-component regulator VirR/VirS, as well as some metabolic and sporulation-related genes.[2] Those physical mapping results have recently been confirmed by genome sequencing projects with two type A C. perfringens (strain 13 and ATCC 13124).[3,4]

As typical of clostridial species, the strain 13 genome was found to be AT-rich (71% overall AT content).[4] No high GC content chromosomal regions were detected, suggesting the absence of chromosomal pathogenicity islands.[4] Many sporulation and germination genes with homologues in Bacillus subtilis were identified; however, over 80 genes mediating these two processes in B. subtilis are absent from strain 13.[4] Genomic analysis of strain 13 identified some potential new virulence factors and putative two-component regulatory systems that could be important for pathogenesis.1-4-1 DNA microarrays and proteomic platforms based on strain 13 have been developed, and their use is now contributing to our understanding of regulatory and metabolic pathways.[5]

Extrachromosomal Virulence Factors

Many C. perfringens virulence factors, including toxins such as CPE, p-toxin, e-toxin, i-toxin, and p2-toxin, as well as urease, can be encoded by genes located on large (~ 100 kb) virulence plasmids.[6,7] Interestingly, at least one toxin gene, cpe, can reside on either the chromosome or large plasmids (further discussion below).[7] Some virulence genes colocalize to the same plasmid, including the urease and cpe genes in various C. perfringens types, as do defective cpe and i-toxin genes in type E isolates.[7,8] To date, only one virulence plasmid (pCP13 carrying the gene encoding p2-toxin) has been se-quenced.[4] The best studied C. perfringens virulence plasmids (other than pCP13) are those carrying cpe sequences in types A (discussed below) and E strains.[8,9] The absence of chromosomal clonality in isolates possessing any of the plasmid-borne toxin genes suggests that these plasmids (or their toxin genes) are mobile. The cpe plasmid was recently shown to transfer between C. perfringens isolates via conjugation, confirming horizontal transfer for that toxin gene.[10] One insertion element, IS1151, has been linked to multiple toxin genes, suggesting that this element may also contribute to toxin gene mobility.[7,9]

Polymerase Chain Reaction-Based Toxin Typing of C. perfringens

A rapid multiplex polymerase chain reaction (PCR) assay has been developed for determining the toxin type (A-E)

Table 1 C. perfringens toxin types

Toxin

Diseasesa

Genotypes

Human

Veterinary plc plc, cpeh plc, cpb2 plc, cpb2, cpe

Gangrene

GI disease (food poisoning,0 antibiotic-associated diarrhea,d sporadic diarrhead)

Diarrhea (dogs, pigs, etc.) Necrotic enteritis (fowl)

Dysentery (lambs) Enterotoxemia (sheep)

pic, cpb pic, cpb, cpb2 pic, cpb, cpb2, cpe pic, cpb, cpe

Enteritis necroticans

Necrotic enteritis (piglets, foals, etc.)

Enterotoxemia (sheep)

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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