High-Level Quinolone-Resistant<i>Haemophilus haemolyticus</i>in Pediatric Patient with No History of Quinolone Exposure

Background Quinolone-resistant bacteria are known to emerge via the accumulation of mutations in a stepwise manner. Recent studies reported the emergence of quinolone low-susceptible Haemophilus influenzae ST422 isolates harbouring two relevant mutations, although ST422 isolates harbouring one mutation were never identified. Objectives To investigate if GyrA and ParC from quinolone low-susceptible isolates can be transferred horizontally and simultaneously to susceptible isolates. Methods Genomic DNA was extracted from an H. influenzae isolate harbouring amino acid substitutions in both gyrA and parC and mixed with clinical isolates. The emergence of resistant isolates was compared, and WGS analysis was performed. Results By adding the genomic DNA harbouring both mutated gyrA and parC, resistant bacteria exhibiting recombination at gyrA only or both gyrA and parC loci were obtained on nalidixic acid and pipemidic acid plates, and the frequency was found to increase with the amount of DNA. Recombination events in gyrA only and in both gyrA and parC occurred with at least 1 and 1–100 ng of DNA, respectively. The genome sequence of a representative strain showed recombination events throughout the genome. The MIC of quinolone for the resulting strains was found to be similar to that of the donor. Although the recombination efficacy was different among the various strains, all strains used in this study obtained multiple genes simultaneously. Conclusions These findings indicate that H. influenzae can simultaneously obtain more than two mutated genes. This mechanism of horizontal transfer could be an alternative pathway for attaining quinolone resistance.

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Contribution of amino acid substitutions in ParE to quinolone resistance in Haemophilus haemolyticus revealed through a horizontal transfer assay using Haemophilus influenzae

Tanaka

Wajima

Nakaminami

et al. 2023

Journal of Antimicrobial Chemotherapy

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Background In 2019, a high-level quinolone-resistant Haemophilus haemolyticus strain (levofloxacin MIC = 16 mg/L) was isolated from a paediatric patient. In this study, we aimed to determine whether the quinolone resistance of H. haemolyticus could be transferred to Haemophilus influenzae and to identify the mechanism underlying the high-level quinolone resistance of H. haemolyticus. Methods A horizontal gene transfer assay to H. influenzae was performed using genomic DNA or PCR-amplified quinolone-targeting genes from the high-level quinolone-resistant H. haemolyticus 2019-19 strain. The amino acids responsible for conferring quinolone resistance were identified through site-directed mutagenesis. Results By adding the genomic DNA of H. haemolyticus 2019-19, resistant colonies were obtained on agar plates containing quinolones. Notably, H. influenzae grown on levofloxacin agar showed the same level of resistance as H. haemolyticus. Sequencing analysis showed that gyrA, parC and parE of H. influenzae were replaced by those of H. haemolyticus, suggesting that horizontal transfer occurred between the two strains. When the quinolone-targeting gene fragments were added sequentially, the addition of parE, as well as gyrA and parC, contributed to high-level resistance. In particular, amino acid substitutions at both the 439th and 502nd residues of ParE were associated with high-level resistance. Conclusions These findings indicate that quinolone resistance can be transferred between species and that amino acid substitutions at the 439th and 502nd residues of ParE, in addition to amino acid substitutions in both GyrA and ParC, contribute to high-level quinolone resistance.

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High-Level Quinolone-ResistantHaemophilus haemolyticusin Pediatric Patient with No History of Quinolone Exposure

Cited by 6 publications

References 35 publications

Emergence of Quinolone Low-Susceptible Haemophilus influenzae Harboring the Mutated Quinolone Targeting Gene of Haemophilus haemolyticus

Emergence of Quinolone Low-Susceptible Haemophilus influenzae Harboring the Mutated Quinolone Targeting Gene of Haemophilus haemolyticus

Alternative quinolone-resistance pathway caused by simultaneous horizontal gene transfer in Haemophilus influenzae

Contribution of amino acid substitutions in ParE to quinolone resistance in Haemophilus haemolyticus revealed through a horizontal transfer assay using Haemophilus influenzae

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