title: Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes.
abstract: BACKGROUND
  Clarithromycin (CLR) is the key drug in eradication therapy of Helicobacter pylori (H. pylori) infection, and widespread use of CLR has led to an increase in primary CLR-resistant H. pylori. The known mechanism of CLR resistance has been established in A2146G and A2147G mutations in the 23S rRNA gene, but evidence of the involvement of other genetic mechanisms is lacking. Using the MiSeq platform, whole-genome sequencing of the 19 clinical strains and the reference strain ATCC26695 was performed to identify single nucleotide variants (SNVs) of multi-drug resistant efflux pump genes in the CLR-resistant phenotype.
  
  
  RESULTS
  Based on sequencing data of ATCC26695, over one million sequencing reads with over 50-fold coverage were sufficient to detect SNVs, but not indels in the bacterial genome. Sequencing reads of the clinical isolates ranged from 1.82 to 10.8 million, and average coverage ranged from 90.9- to 686.3-fold, which were acceptable criteria for detecting SNVs. Utilizing the conventional approach of allele-specific PCR, point mutations in the 23S rRNA gene were detected in 12 clinical resistant isolates, but not in 7 clinical susceptible isolates. All sequencing reads of CLR-resistant strains had a G mutation in an identical position of the 23S rRNA gene. In addition, genetic variants of four gene clusters (hp0605-hp0607, hp0971-hp0969, hp1327-hp1329, and hp1489-hp1487) of TolC homologues, which have been implicated in multi-drug resistance, were examined. Specific SNVs were dominantly found in resistant strains.
  
  
  CONCLUSIONS
  Gene clusters of TolC homologues are involved in CLR susceptibility profiles in individual H. pylori strains. Whole-genome sequencing has yielded novel understanding of genotype-phenotype relationships.
authors.affiliation: Department of Internal Medicine, Division of Gastroenterology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Iwamoto
authors.affiliation: Department of Internal Medicine, Division of Gastroenterology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan ; Department of Medical Pharmaceutics, Kobe Pharmaceutical University, 4-19-1 Motoyama-kita, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Toshihito
authors.initials: T
authors.lastname: Tanahashi
authors.affiliation: Department of Internal Medicine, Division of Gastroenterology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: Rina
authors.initials: R
authors.lastname: Okada
authors.affiliation: Division of Internal Medicine, Okinawa Prefectural Chubu Hospital, 281 Miyazato, Uruma-city, Okinawa 904-2293, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Yoshida
authors.affiliation: Division of Internal Medicine, Okinawa Prefectural Chubu Hospital, 281 Miyazato, Uruma-city, Okinawa 904-2293, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Kikuchi
authors.affiliation: Division of Internal Medicine, Okinawa Prefectural Chubu Hospital, 281 Miyazato, Uruma-city, Okinawa 904-2293, Japan.
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Keida
authors.affiliation: Department of Hepatology, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Murakami
authors.affiliation: Department of Internal Medicine, Division of Gastroenterology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: Lin
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Internal Medicine, Division of Gastroenterology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Internal Medicine, Division of Gastroenterology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Nishiumi
authors.affiliation: Department of Internal Medicine, Division of Gastroenterology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Yoshida
authors.affiliation: Department of Internal Medicine, Division of Gastroenterology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Azuma
keywords: Clarithromycin; Helicobacter pylori; Multidrug efflux; TolC homolog; Whole-genome sequencing