Genotyping of Campylobacter jejuni using seven single-nucleotide polymorphisms in combination with flaA short variable region sequencing

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Abstract
This investigation describes the development of a generally applicable, bioinformatics-driven, single-nucleotide polymorphism (SNP) genotyping assay for the common bacterial gastrointestinal pathogenCampylobacter jejuni. SNPs were identifiedin silicousing the program ‘Minimum SNPs’, which selects for polymorphisms providing the greatest resolution of bacterial populations based on Simpson's index of diversity (D). The high-DSNPs identified in this study were derived from the combinedC. jejuni/Campylobacter colimultilocus sequence typing (MLST) database. Seven SNPs were found that provided aDof 0.98 compared with full MLST characterization, based on 959 sequence types (STs). The seven high-DSNPs were interrogated using allele-specific real-time PCR (AS kinetic PCR), which negates the need for expensive labelled primers or probes and requires minimal assay optimization. The total turnaround time of the SNP typing assay was approximately 2 h. Concurrently, 69C. jejuniisolates were subjected to MLST and flagellin A short variable region (flaASVR) sequencing and combined with a population of 84C. jejuniandC. coliisolates previously characterized by these methods. Within this collection of 153 isolates, 19flaASVR types (D=0.857) were identified, compared with 40 different STs (D=0.939). When MLST andflaASVR sequencing were used in combination, the discriminatory power was increased to 0.959. In comparison, SNP typing of the 153 isolates alone provided aDof 0.920 and was unable to resolve a small number of unrelated isolates. However, addition of theflaASVR locus to the SNP typing procedure increased the resolving power to 0.952 and clustered isolates similarly to MLST/flaASVR. This investigation has shown that a seven-memberC. jejuniSNP typing assay, used in combination with sequencing of theflaASVR, efficiently discriminatesC. jejuniisolates.