Fourteen-Genome Comparison Identifies DNA Markers for Severe-Disease-Associated Strains of Clostridium difficile

Abstract

Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising ϳ3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxin A-negative and B-positive (A-B؉) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains.

Structure of Clostridium difficile PilJ Exhibits Unprecedented Divergence from Known Type IV Pilins

“…The 12 sequences that diverge from that of R20291 contain two distinct clusters, 1 of 4 identical sequences, 3 of which stem from ribotype 078 (NAP7/8) strains, and another of 3 identical sequences including 2 017 ribotypes. This same clustering pattern, a large NAP1 cluster, a smaller NAP7/8 cluster, and a small number of sequences falling between these two consensus sequences, was observed previously in a genome-wide comparison of 14 C. difficile strains (48). The lone exception to this rule is the sequence of CIP 107932, which clusters with those of the NAP7/8 strains despite the overall genetic similarity between CIP 107932 and NAP1 strains.…”

Genome Analysis of Clostridium difficile PCR Ribotype 014 Lineage in Australian Pigs and Humans Reveals a Diverse Genetic Repertoire and Signatures of Long-Range Interspecies Transmission

“…To identify a core set of orthologous genes for microevolutionary analysis, de novo assembled RT014 genomes were analyzed using three independent orthology-calling algorithms; COGtriangles (COG), OrthoMCL (OMCL), and bidirectional best-hit (BDBH), all implemented in the program GET_HOMOLOGUES v2.0.6 (Contreras-Moreira and Vinuesa, 2013), following the approaches of previous studies of the C. difficile core genome (Scaria et al, 2010; Forgetta et al, 2011; Treangen et al, 2014). To ensure confidence in clustering of homologous and not paralogous gene families the following stringent conditions were applied; (i) minimum BLAST pairwise alignment coverage of 90%, (ii) minimum BlastP sequence identity of 95%, (iii) E -value threshold of 1e −10 , and (iv) inparalogs were excluded.…”

“…The C. difficile genome (~4.3 Mbp) contains a high proportion of mobile genetic elements (11% in strain 630) and the species displays a large pan-genome estimated at over 9000 coding sequences (CDS), but remarkably shows an ultra-low level of genome conservation (as low as 16%; Sebaihia et al, 2006; Scaria et al, 2010; Forgetta et al, 2011; Treangen et al, 2014). …”

Cefoperazone-treated mice as an experimental platform to assess differential virulence ofClostridium difficilestrains

“…However, the emergence of epidemic strains of C. difficile with apparently increased virulence (e.g., NAP1/BI/027) has opened new questions about the specific determinants of C. difficile virulence in different isolates. 27 While important insights into the pathogenesis of CDI have been gained from experimental infection of Syrian hamsters, 14,28 one limitation of this model is that animals infected with toxigenic C. difficile strains generally have a uniformly fatal course, reducing the ability to detect differences in virulence. 19,20 In this report, we present a model of CDI that should be useful to show relative differences in the pathogenicity of C. difficile strains.…”