AUTHOR=Zhang Qian , Al-Ghalith Gabriel A. , Kobayashi Mayumi , Segawa Takahiro , Maeda Mitsuto , Okabe Satoshi , Knights Dan , Ishii Satoshi 

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

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.02201

DOI=10.3389/fmicb.2018.02201

ISSN=1664-302X

ABSTRACT=<p>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 <italic>flaA</italic> short variable region (SVR) of various <italic>Campylobacter</italic> species, and applied the <italic>flaA</italic> SVR sequencing method to examine the diversity of <italic>Campylobacter</italic> spp. in geese fecal samples (<italic>n</italic> = 16) with and without bacteria cultivation. Twenty-three <italic>Campylobacter</italic> strains isolated from the 16 geese fecal samples were grouped similarly by conventional <italic>flaA</italic> restriction fragment length polymorphism (RFLP) method and by the <italic>flaA</italic> SVR sequencing method, but higher discriminant power was observed in the <italic>flaA</italic> SVR sequencing approach. For culture-independent <italic>flaA</italic> 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 <italic>flaA</italic> sequence reads were clustered into 16 operational taxonomic units (=genotypes) by using 98% sequence similarity and &gt;50 sequence duplicates. Almost all <italic>flaA</italic> genotypes obtained by culture-dependent method were also identified by culture-independent <italic>flaA</italic> SVR MiSeq sequencing method. In addition, more <italic>flaA</italic> genotypes were identified probably due to high throughput nature of the MiSeq sequencing. These results suggest that the <italic>flaA</italic> SVR sequencing could be used to analyze the diversity of <italic>Campylobacter</italic> spp. without bacteria isolation. This method is promising to rapidly identify potential sources of <italic>Campylobacter</italic> pathogens.</p>