Clostridium difficile in Retail Meat Products, USA, 2007

To determine the presence of Clostridium difficile, we sampled cooked and uncooked meat products sold in Tucson, Arizona. Forty-two percent contained toxigenic C. difficile strains (either ribotype 078/toxinotype V [73%] or 027/toxinotype III [NAP1 or NAP1-related; 27%]). These findings indicate that food products may play a role in interspecies C. difficile transmission

Mycobacteriosis (tuberculosis, TB)

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Genome Sequencing and Analysis of a Type A Clostridium perfringens Isolate from a Case of Bovine Clostridial Abomasitis

Clostridium perfringens is a common inhabitant of the avian and mammalian gastrointestinal tracts and can behave commensally or pathogenically. Some enteric diseases caused by type A C. perfringens, including bovine clostridial abomasitis, remain poorly understood. To investigate the potential basis of virulence in strains causing this disease, we sequenced the genome of a type A C. perfringens isolate (strain F262) from a case of bovine clostridial abomasitis. The ∼3.34 Mbp chromosome of C. perfringens F262 is predicted to contain 3163 protein-coding genes, 76 tRNA genes, and an integrated plasmid sequence, Cfrag (∼18 kb). In addition, sequences of two complete circular plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), and two incomplete plasmid fragments, pF262A (48.5 kb) and pF262B (50.0 kb), were identified. Comparison of the chromosome sequence of C. perfringens F262 to complete C. perfringens chromosomes, plasmids and phages revealed 261 unique genes. No novel toxin genes related to previously described clostridial toxins were identified: 60% of the 261 unique genes were hypothetical proteins. There was a two base pair deletion in virS, a gene reported to encode the main sensor kinase involved in virulence gene activation. Despite this frameshift mutation, C. perfringens F262 expressed perfringolysin O, alpha-toxin and the beta2-toxin, suggesting that another regulation system might contribute to the pathogenicity of this strain. Two complete plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), unique to this strain of C. perfringens were identified

Identification of Novel Pathogenicity Loci in Clostridium perfringens Strains That Cause Avian Necrotic Enteritis

Type A Clostridium perfringens causes poultry necrotic enteritis (NE), an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s) not present in commensal type A isolates. We used high-throughput sequencing (HTS) technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs) were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb), NELoc-2 (11.2 kb) and NELoc-3 (5.6 kb). The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes ∼85 and ∼70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne

Identification of a Second Arcanobacterium pyogenes Neuraminidase and Involvement of Neuraminidase Activity in Host Cell Adhesion

Arcanobacterium pyogenes, a common inhabitant of the upper respiratory and urogenital tracts of economically important animals, such as cattle and swine, is also an opportunistic pathogen associated with suppurative infections in these animals. A. pyogenes expresses neuraminidase activity encoded by the nanH gene, and previously, construction of a nanH mutant of A. pyogenes BBR1 indicated that a second neuraminidase is present in this strain. A 5,112-bp gene, nanP, was cloned and sequenced, and this gene conferred neuraminidase activity on an Escherichia coli host strain. The predicted 186.8-kDa NanP protein exhibited similarity to a number of bacterial neuraminidases and contained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases. As expected, insertional inactivation of the nanP gene in A. pyogenes BBR1 resulted in a mutant with reduced neuraminidase activity. However, insertional inactivation of the nanP gene in the nanH mutant strain resulted in a complete lack of neuraminidase activity. Like NanH, NanP was localized to the A. pyogenes cell wall. However, unlike the nanH gene, which was present in 100% of the strains examined, nanP was present in only 64.2% of the isolates (n = 53). A. pyogenes adheres to HeLa cells, and a nanP mutant displayed a wild-type adhesion phenotype with these cells. In contrast, the ability of a nanH nanP double mutant to bind to HeLa cells was reduced by 53%. The wild-type adhesion phenotype was restored by providing nanP in trans. These data indicate that the neuraminidases of A. pyogenes play a role in adhesion of this organism to host epithelial cells

Widespread Distribution of a Tet W Determinant among Tetracycline-Resistant Isolates of the Animal Pathogen Arcanobacterium pyogenes

Tetracycline resistance is common among isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes. The tetracycline resistance determinant cloned from two bovine isolates of A. pyogenes was highly similar at the DNA level (92% identity) to the tet(W) gene, encoding a ribosomal protection tetracycline resistance protein, from the rumen bacterium Butyrivibrio fibrisolvens. The tet(W) gene was found in all 20 tetracycline-resistant isolates tested, indicating that it is a widely distributed determinant of tetracycline resistance in this organism. In 25% of tetracycline-resistant isolates, the tet(W) gene was associated with a mob gene, encoding a functional mobilization protein, and an origin of transfer, suggesting that the determinant may be transferable to other bacteria. In fact, low-frequency transfer of tet(W) was detected from mob(+) A. pyogenes isolates to a tetracycline-sensitive A. pyogenes recipient. The mobile nature of this determinant and the presence of A. pyogenes in the gastrointestinal tract of cattle and pigs suggest that A. pyogenes may have inherited this determinant within the gastrointestinal tracts of these animals

Immunization with Genetic Toxoids of the Arcanobacterium pyogenes Cholesterol-Dependent Cytolysin, Pyolysin, Protects Mice against Infection

Pyolysin (PLO), a cholesterol-dependent cytolysin expressed by Arcanobacterium pyogenes, is an important host-protective antigen. However, this molecule is toxic and requires inactivation prior to its use as a vaccine. Three genetically toxoided, nonhemolytic PLO molecules, HIS-PLO.F(497), HIS-PLO.ΔP(499), and HIS-PLO.A(522), were found to be nontoxic, and vaccinated mice were protected from infection, indicating the potential of these toxoids as vaccines. Furthermore, in a mouse model of infection, A. pyogenes carrying the F(497) mutation was as attenuated as a PLO-deficient strain, indicating that the cytolytic activity of PLO is important in virulence