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

Abstract

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.

The Association between Transformation Ability and Antimicrobial Resistant Potential in <i>Haemophilus influenzae</i>

“…differed depending on the strain 13,14) , suggesting that this horizontal transfer ability might be related to the ease of developing quinolone resistance. In addition, as -lactam resistance was also developed by point mutations in targeting genes 20) , development of resistance to -lactams might also be related to horizontal transfer ability.…”

“…Fifty quinolone-susceptible H. influenzae isolates obtained from a Japanese hospital in 2018 were used for this study 21) . For the horizontal gene transfer assay, H. influenzae Rd and 2017-22B, which was suggested to be highly transferable strains in a previous study 13,14) , were used as control strains. The bacterial strains were cultured overnight on chocolate agar at 37 C.…”

“…A previous report on quinolone resistance (low quinolone susceptibility) suggested that horizontal transfer efficiency might vary among clinical isolates 13,14) , indicating that quantifying horizontal transfer ability was possible by utilising an experimental transfer assay for quinolone resistance. Therefore, 50 quinolone-susceptible clinical isolates, which were all isolated as quinolone low-susceptible isolates from a hospital in a year 21) , were tested using a horizontal transfer assay with a mutated gyrA fragment (30 ng).…”

“…The mechanism of low quinolone susceptibility in H. influenzae is known to be developed by point mutations in DNA gyrase, and then in topoisomerase IV, similar to those in other gram-negative bacteria [9][10][11][12] . Further, the horizontal transfer of these target genes for quinolone low susceptibility by transformation was recently reported as an alternative mechanism for the development and spread of quinolone resistance 13,14) .…”