Development and validation of a comparative genomic fingerprinting method for high-resolution genotyping of Campylobacter jejuni

Abstract

Campylobacter spp. are a leading cause of bacterial gastroenteritis worldwide. The need for molecular subtyping methods with enhanced discrimination in the context of surveillance- and outbreak-based epidemiologic investigations of Campylobacter spp. is critical to our understanding of sources and routes of transmission and the development of mitigation strategies to reduce the incidence of campylobacteriosis. We describe the development and validation of a rapid and high-resolution comparative genomic fingerprinting (CGF) method for C. jejuni. A total of 412 isolates from agricultural, environmental, retail, and human clinical sources obtained from the Canadian national integrated enteric pathogen surveillance program (C-EnterNet) were analyzed using a 40-gene assay (CGF40) and multilocus sequence typing (MLST). The significantly higher Simpson's index of diversity (ID) obtained with CGF40 (ID = 0.994) suggests that it has a higher discriminatory power than MLST at both the level of clonal complex (ID = 0.873) and sequence type (ID = 0.935). High Wallace coefficients obtained when CGF40 was used as the primary typing method suggest that CGF and MLST are highly concordant, and we show that isolates with identical MLST profiles are comprised of isolates with distinct but highly similar CGF profiles. The high concordance with MLST coupled with the ability to discriminate between closely related isolates suggests that CFG40 is useful in differentiating highly prevalent sequence types, such as ST21 and ST45. CGF40 is a high-resolution comparative genomics-based method for C. jejuni subtyping with high discriminatory power that is also rapid, low cost, and easily deployable for routine epidemiologic surveillance and outbreak investigations.

Fig 1

Selection of markers for CGF assay. (A) Distribution of the CGF40 target genes around the C. jejuni NCTC11168 genome sequence. Arrowheads in the two innermost circles depict the location and direction of the target genes, and white blocks on the outermost circles depict the 16 hypervariable regions previously identified in the C. jejuni genome (60). The seven MLST loci (aspA, uncA, glyA, pgm, glnA, tkt, and gltA) also are depicted in the figure. This genome visualization was generated using CGView (58). (B) Binary accessory genes identified from comparative genomic analysis were checked for unbiased population frequency and genome distribution. Only genes that fulfilled these criteria and that had available multiple-sequence data were targeted for SNP-free PCR primer design, and they form part of the final set of markers used in the assay described here.

Fig 2

Visualization of the eight 5-plex PCRs that comprise the CGF40 assay used in this study. Expected binary results for three genome-sequenced C. jejuni strains (NCTC 11168, RM1221, and 81-176) are shown (black, present band; white, absent band) along with gel representations of experimental data from the QIAxcel instrument.

Fig 3

Analysis of cluster homogeneity among multiple-isolate clusters. (A) Distribution of average numbers of matching MLST loci among multiple-isolate CGF40 clusters (n = 47). (B) Distribution of average numbers of matching CGF40 loci among multiple-isolate ST clusters (n = 43). The examination of clusters obtained using either method supports the high concordance between the two methods.

Fig 4

Relationship between MLST- and CGF40-based genetic similarity assessments. (A) Distribution of matching MLST loci when CGF was used as the primary method. (B) Distribution of CGF matching loci when MLST was used as the primary method. In both cases, isolates that share a higher number of matching CGF40 loci tend toward higher levels of MLST similarity.