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

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

doi:10.3389/fmicb.2018.02201

Abstract

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 culture-independent 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.

Highlights

Campylobacter species are one of the most common bacterial foodborne pathogens, causing gastrointestinal disorders such as bloody diarrhea, dysentery syndrome, and cramps (Silva et al, 2011)
The objectives of this study were to (1) design new primer set that broadly covers flaA short variable region (SVR) of various Campylobacter species, (2) compare strain discrimination obtained by the flaA SVR sequencing done with the new primers with those obtained by a commonly used flaA restriction fragment length polymorphism (RFLP) method, and (3) apply the flaA SVR sequencing to examine the diversity of Campylobacter spp. in geese fecal samples without bacteria cultivation
A total of 23 Campylobacter strains were isolated from 16 geese fecal samples, and were characterized by flaA RFLP and flaA SVR

Summary

Introduction

Campylobacter species are one of the most common bacterial foodborne pathogens, causing gastrointestinal disorders such as bloody diarrhea, dysentery syndrome, and cramps (Silva et al, 2011). The most common hosts for Campylobacter spp. are avian species, including commercial poultry species (e.g., chickens and turkeys) and waterfowl (e.g., geese; Pacha et al, 1988), they are found in other animals such as cattle, pigs, and dairy cows (Westrell et al, 2009; Silva et al, 2011). Campylobacter species cannot grow but can survive well in various environments such as fresh and coastal waters and sediment after they are released from their primary hosts (e.g., birds and animals). Campylobacter spp. is frequently and abundantly detected in various environmental water samples (Ishii et al, 2014a; Oster et al, 2014; Zhang et al, 2016)

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