Multilocus sequence typing of Clostridium difficile

Abstract

A robust high-throughput multilocus sequence typing (MLST) scheme for Clostridium difficile was developed and validated using a diverse collection of 50 reference isolates representing 45 different PCR ribotypes and 102 isolates from recent clinical samples. A total of 49 PCR ribotypes were represented overall. All isolates were typed by MLST and yielded 40 sequence types (STs). A web-accessible database was set up (http://pubmlst.org/cdifficile/) to facilitate the dissemination and comparison of C. difficile MLST genotyping data among laboratories. MLST and PCR ribotyping were similar in discriminatory abilities, having indices of discrimination of 0.90 and 0.92, respectively. Some STs corresponded to a single PCR ribotype (32/40), other STs corresponded to multiple PCR ribotypes (8/40), and, conversely, the PCR ribotype was not always predictive of the ST. The total number of variable nucleotide sites in the concatenated MLST sequences was 103/3,501 (2.9%). Concatenated MLST sequences were used to construct a neighbor-joining tree which identified four phylogenetic groups of STs and one outlier (ST-11; PCR ribotype 078). These groups apparently correlate with clades identified previously by comparative genomics. The MLST scheme was sufficiently robust to allow direct genotyping of C. difficile in total stool DNA extracts without isolate culture. The direct (nonculture) MLST approach may prove useful as a rapid genotyping method, potentially benefiting individual patients and informing hospital infection control.

FIG. 1.

The relative positions (to scale) of the seven housekeeping loci chosen for MLST on the C. difficile strain 630 chromosome, together with three genes of the pathogenicity locus (PaLoc) detected by PCRs referred to in this study and the genes encoding the binary toxin cdtA and cdtB.

FIG. 2.

Neighbor-joining tree constructed using the concatenated sequences (3,501 nucleotides) of the seven loci used in MLST. Bootstraps were generated using 1,000 replicates, and low values were removed for clarity. STs are shown in bold. The PCR ribotype(s) found in association with each ST are indicted. The STs cluster into four groups, designated 1 to 4, with bootstraps greater than 80 and one outlier (ST-11). These correspond to groups defined previously by microarray analysis of whole genomes (39) which were designated HA1 (human and animal 1), hypervirulent (containing the 027 strain), HA2, and the A⁻ B⁺ clade (also containing other toxigenic types). The total number of variable sites was 103/3,501 (2.9%), and if the outlier ST-11 was excluded, the number was 59/3,501 (1.7%). ST-1 (indicated by *¹) was associated with PCR ribotype 027 and also with a single PCR ribotype 036 isolate. DNA profiles for PCR ribotypes 036 and 027 are very similar and differ by only a single band.

FIG. 3.

Flow diagram summarizing the time required to perform the laboratory work and sequence data analysis in high-throughput MLST.