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These were constructed using Clonal Frame (Didelot and Falush 2007), and ancestry times were calculated in years using isolate dates and previous estimate of the molecular clock (Didelot et al. Polymorphisms between four pairs of isolates were mapped along the whole M120 genome (Sebaihia et al. The presence of the Pa Loc and therefore the genetic potential for toxin expression had been assessed previously by PCR assay for the presence of toxin genes.

2006) by aligning their de novo assemblies against M120 using Mu MMER version 3.25 (Kurtz et al. The distribution of polymorphisms in the Pa Loc region was plotted using DNAplotter (Carver et al. The Pa Loc region and insertion sites were studied in detail, sequences being translated and annotated using Artemis genome browser and annotation tool (Rutherford et al. Sequence comparisons were performed using the Artemis Comparison Tool (Carver et al. Analysis of the Pa Loc genes was performed using MEGA 5.2 (Tamura et al. BLAST searches of genes and predicted translation products against Gen Bank were used to identity their putative functions. Isolates which were PCR positive for = 5]) were genotyped by MLST; a total of nine STs were identified, eight of which were novel (table 1).

A subset ( = 8) that did not match any of the previously determined banding patterns available in Australia was referred to the United Kingdom Anaerobe Reference Unit (University Hospital of Wales, Heath Park, Cardiff, Wales, UK) for comparison against their library.

The most parsimonious scenario involves separate gains of the AThe two Pa Loc variants were each acquired independently approximately 1,300 years ago.The aim of this study was to explore the evolutionary history of the Pa Loc in clade 5. 2011), and a lack of genes conferring mobility (Braun and von Eichel-Streiber 1999) suggest that it is stably integrated. Transfer of the Pa Loc to nontoxigenic strains and resultant toxigenic conversion has been demonstrated experimentally (Brouwer et al. Because of the large size of the toxin-encoding genes, analysis of genetic polymorphism within the Pa Loc has frequently been confined to toxinotyping, a method based on PCR and restriction endonuclease digests which infers nucleotide sequence variation indirectly (Rupnik et al. Within clade 5, toxinotypes are largely congruent with genotype (Stabler et al.Phylogenetic analyses and annotation of clade 5 Pa Loc variants and adjoining genomic regions were undertaken using a representative collection of toxigenic and nontoxigenic strains. The symptoms of CDI are caused by two closely related toxins, designated A and B, and encoded by the pathogenicity locus (Pa Loc), a 19 kb chromosomally integrated DNA sequence (Braun et al. However, nontoxigenic strains exist which intermingle phylogenetically with toxigenic strains of clades 1, 4, and 5, suggesting multiple gain and loss events (Elliott et al. 2012); for example, the 078 Pa Loc is designated toxinotype V. 2012), have been proposed to impact on the severity of clinical disease.infection are caused by two closely related toxins, Tcd A and Tcd B, which are encoded by the 19.6 kb Pathogenicity Locus (Pa Loc).The Pa Loc is variably present among strains, and in this respect it resembles a mobile genetic element.

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