Rapid molecular characterization of Clostridium difficile and assessment of populations of C. difficile in stool specimens

Abstract

Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h. A subset of historical and recent C. difficile isolates (n = 31) was also analyzed by pulsed-field gel electrophoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type 1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied the populations of C. difficile in patient stool specimens (n = 23). Our results indicated that 13% of individual stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization results for multiple bacterial isolates (n = 10). Direct molecular analysis of stool specimens gave results that correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid, informative, and less costly than the testing of multiple patient stool isolates.

FIG. 1.

Design for amplification and pyrosequencing of the tcdC gene. A diagram of a portion of the tcdC gene used to detect the 1-bp, 18-bp, and 39-bp tcdC gene deletions found in strains of Clostridium difficile is shown. The sequence from NCBI accession number DQ870674 (C. difficile strain ATCC 43594) was used, and the forward and reverse primer locations are shaded in gray. Deletion locations are shown in boldface and in brackets, with the 1-bp and 18-bp deletions being underlined. The 39-bp deletion is shown with a dotted line over it. Sequencing primers S1, S2, and S3 are boxed and have arrows indicating the direction of sequencing.

FIG. 2.

Molecular characterization of Clostridium difficile from patient specimens (1980 to 2008) by multiplex real-time PCR, sequence analysis of the tcdC gene, and PFGE analysis. ^a, the tcdA and tcdB genes were detected by real-time PCR; ^b, the cdtA and cdtB genes were detected by real-time PCR; ^c, CldS is the abbreviation used in our database to designate a Clostridium difficile subtype; ^d, NAP is the abbreviation used for North American pulsed-field type; ^e, this strain, received by our laboratory in 1980, was a control strain provided by the CDC for our C. difficile cytotoxin assay.