Department of Clinical Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
Department of Microbiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan.
J Antimicrob Chemother. 2023 May 3;78(5):1225-1230. doi: 10.1093/jac/dkad074.
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.
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.
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.
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.
2019 年，从一名儿科患者中分离出一株高水平氟喹诺酮耐药溶血嗜血杆菌（左氧氟沙星 MIC=16mg/L）。本研究旨在确定溶血嗜血杆菌的喹诺酮耐药性是否可转移至流感嗜血杆菌，并鉴定溶血嗜血杆菌高水平喹诺酮耐药的机制。
通过使用来自高水平氟喹诺酮耐药溶血嗜血杆菌 2019-19 株的基因组 DNA 或经 PCR 扩增的喹诺酮靶基因，对流感嗜血杆菌进行水平基因转移试验。通过定点突变鉴定赋予喹诺酮耐药性的氨基酸。
添加溶血嗜血杆菌 2019-19 的基因组 DNA 后，在含有喹诺酮的琼脂平板上获得了耐药菌落。值得注意的是，在左氧氟沙星琼脂上生长的流感嗜血杆菌表现出与溶血嗜血杆菌相同的耐药水平。测序分析表明，流感嗜血杆菌的 gyrA、parC 和 parE 被溶血嗜血杆菌的取代，提示两株菌之间发生了水平转移。当依次添加喹诺酮靶基因片段时，添加 parE 以及 gyrA 和 parC 有助于高水平耐药。特别是，ParE 的第 439 位和第 502 位氨基酸的取代与高水平耐药有关。
这些发现表明，喹诺酮耐药性可以在种间转移，并且 ParE 的第 439 位和第 502 位氨基酸的取代以及 GyrA 和 ParC 中的氨基酸取代都有助于高水平的氟喹诺酮耐药。
