Microarray identification of Clostridium difficile core components and divergent regions associated with host origin

Abstract

Clostridium difficile is a gram-positive, spore-forming enteric anaerobe which can infect humans and a wide variety of animal species. Recently, the incidence and severity of human C. difficile infection has markedly increased. In this study, we evaluated the genomic content of 73 C. difficile strains isolated from humans, horses, cattle, and pigs by comparative genomic hybridization with microarrays containing coding sequences from C. difficile strains 630 and QCD-32g58. The sequenced genome of C. difficile strain 630 was used as a reference to define a candidate core genome of C. difficile and to explore correlations between host origins and genetic diversity. Approximately 16% of the genes in strain 630 were highly conserved among all strains, representing the core complement of functional genes defining C. difficile. Absent or divergent genes in the tested strains were distributed across the entire C. difficile 630 genome and across all the predicted functional categories. Interestingly, certain genes were conserved among strains from a specific host species, but divergent in isolates with other host origins. This information provides insight into the genomic changes which might contribute to host adaptation. Due to a high degree of divergence among C. difficile strains, a core gene list from this study offers the first step toward the construction of diagnostic arrays for C. difficile.

FIG. 1.

Comparative genomic analysis of 73 strains of C. difficile isolated from various hosts. Two arrays with dye swap for each strain were averaged and analyzed with GACK software. The CDS were arranged according to the numbering of the C. difficile 630 genes, with CD0001 at the top and CD3680 at the bottom, followed by CDS from the plasmid pCD630 (CDP01 to CDP11). Each column represents an isolate, and each row corresponds to a specific CDS. The status of each CDS is indicated by color as follows: red, present/conserved; green, absent; and gray, divergent. The positions of the putative conjugative transposon (CTn1 to CTn7), prophages, and the genomic island are indicated.

FIG. 2.

Hierarchical clustering of 73 strains of C. difficile based on the microarray data. The strain numbers, host origins, toxinotypes, PFGE types, presence of binary toxin, tcdC deletion observed, and tcdC stop codon detected by sequencing are indicated in addition to the number of divergent CDS compared to that of reference strain 630. n.d., not determined.

FIG. 3.

Dendrogram of C. difficile isolates based on PFGE results. The group numbers based on CGH clustering and PFGE types are indicated.

FIG. 4.

Hierarchical clustering of C. difficile isolates based on the microarray data with CDS from strain QCD-32g58. Each column represents a specific CDS, and each row corresponds to a strain. The clustering method is based on the Pearson absolute with both rows and columns. The status of each CDS is indicated by color as follows: red, present/conserved; green, absent; and gray, divergent.

FIG. 5.

Distribution of the known or putative virulence-related CDS among C. difficile strains. Each row corresponds to a CDS, and each column in each panel represents a test strain. The status of each CDS is indicated by color as follows: red, present/conserved; green, absent; and gray, divergent. The designations of these CDS are indicated on the right. Intergenic regions are not shown.

FIG. 6.

Patterns of CDS potentially associated with antibiotic resistance among C. difficile strains. Each row corresponds to a CDS, and each column in each panel represents a test strain. The status of each CDS is indicated by color as follows: red, present/conserved; green, absent; and gray, divergent. The designations of these CDS are indicated on the right. Intergenic regions are not shown.