High-Throughput flaA Short Variable Region Sequencing to Assess Campylobacter Diversity in Fecal Samples From Birds

Current approach to identify sources of human pathogens is largely dependent on the cultivation and isolation of target bacteria. For rapid pathogen source identification, culture-independent strain typing method is necessary. In this study, we designed new primer set that broadly covers flaA short variable region (SVR) of various Campylobacter species, and applied the flaA SVR sequencing method to examine the diversity of Campylobacter spp. in geese fecal samples (n = 16) with and without bacteria cultivation. Twenty-three Campylobacter strains isolated from the 16 geese fecal samples were grouped similarly by conventional flaA restriction fragment length polymorphism (RFLP) method and by the flaA SVR sequencing method, but higher discriminant power was observed in the flaA SVR sequencing approach. For culture-independent flaA SVR sequencing analysis, we developed and optimized the sequence data analysis pipeline to identify as many genotypes as possible, while minimizing the detection of genotypes generated by sequencing errors. By using this pipeline, 51,629 high-quality flaA sequence reads were clustered into 16 operational taxonomic units (=genotypes) by using 98% sequence similarity and >50 sequence duplicates. Almost all flaA genotypes obtained by culture-dependent method were also identified by cultureindependent flaA SVR MiSeq sequencing method. In addition, more flaA genotypes were identified probably due to high throughput nature of the MiSeq sequencing. These results suggest that the flaA SVR sequencing could be used to analyze the diversity of Campylobacter spp. without bacteria isolation. This method is promising to rapidly identify potential sources of Campylobacter pathogens.