Emi Tanaka1, Takeaki Wajima2, Hidemasa Nakaminami1, Norihisa Noguchi1. 1. Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan. 2. Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan. Electronic address: twajima@toyaku.ac.jp.
Abstract
OBJECTIVES: In recent years, Haemophilus influenzae strains with reduced susceptibility to quinolones have emerged and spread in Japan. In addition, an outbreak of isolates with low quinolone susceptibility among paediatric patients has also been reported. The aim of this study was to determine the molecular characteristics of an H. influenzae ST422 outbreak clone with low quinolone susceptibility isolated from a paediatric patient using whole-genome sequencing. METHODS: The PacBio RS II platform was used for sequencing, and de novo assembly was performed using RS HGAP assembly version 3.0. The assembled sequences were annotated using DFAST version 1.1.15. Prophages were estimated using the PHASTER program. RESULTS: Whole-genome sequencing of H. influenzae ST422 isolate 2018-Y40 revealed that the genome size was 1 957 393bp, comprising 1 926 protein-coding sequences, 19 rRNAs, and 57 tRNAs, with a guanine-cytosine (GC) content of 38.2%. This isolate had no relevant exogenous antimicrobial-resistant genes. However, amino acid substitutions were found in both GyrA and ParC, as well as at the 385th and 526th amino acid residues in penicillin-binding protein 3. In addition, four intact prophage regions and one incomplete prophage region were found. CONCLUSIONS: The whole-genome sequence of H. influenzae 2018-Y40 indicated that this clone emerged as a result of extensive genomic rearrangement by integration of multiple phages. As genomic rearrangement occasionally leads to a new phenotype, this clone could have acquired antimicrobial resistance and diversification via rearrangement events. These findings can form a basis to help clarify the mechanisms of low quinolone susceptibility and the spread of this outbreak clone.
OBJECTIVES: In recent years, Haemophilus influenzae strains with reduced susceptibility to quinolones have emerged and spread in Japan. In addition, an outbreak of isolates with low quinolone susceptibility among paediatric patients has also been reported. The aim of this study was to determine the molecular characteristics of an H. influenzae ST422 outbreak clone with low quinolone susceptibility isolated from a paediatric patient using whole-genome sequencing. METHODS: The PacBio RS II platform was used for sequencing, and de novo assembly was performed using RS HGAP assembly version 3.0. The assembled sequences were annotated using DFAST version 1.1.15. Prophages were estimated using the PHASTER program. RESULTS: Whole-genome sequencing of H. influenzae ST422 isolate 2018-Y40 revealed that the genome size was 1 957 393bp, comprising 1 926 protein-coding sequences, 19 rRNAs, and 57 tRNAs, with a guanine-cytosine (GC) content of 38.2%. This isolate had no relevant exogenous antimicrobial-resistant genes. However, amino acid substitutions were found in both GyrA and ParC, as well as at the 385th and 526th amino acid residues in penicillin-binding protein 3. In addition, four intact prophage regions and one incomplete prophage region were found. CONCLUSIONS: The whole-genome sequence of H. influenzae 2018-Y40 indicated that this clone emerged as a result of extensive genomic rearrangement by integration of multiple phages. As genomic rearrangement occasionally leads to a new phenotype, this clone could have acquired antimicrobial resistance and diversification via rearrangement events. These findings can form a basis to help clarify the mechanisms of low quinolone susceptibility and the spread of this outbreak clone.