摘要

本研究的目的是表征流感嗜血杆菌对氟喹诺酮类，大环内酯类和亚胺培南耐药机制，评估AcrAB-TolC介导的耐药程度，并确定核心基因组多位点序列分型（cgMLST）方案通过使用全基因组测序来检测流感嗜血杆菌。发现GyrA（Ser84和Asp88），ParC（Ser84）和ParE（Asp420）中的四个氨基酸取代与MIC密切相关。我们未发现PBP3中与亚胺培南抗性相关的三个高度保守的氨基酸基序周围的任何氨基酸取代。所有分离株都具有ermB基因。羰基氰化物间氯苯腙（CCCP）将FIPR-6的亚胺培南MIC降低了两倍，GE47和GE88菌株降低了四倍。对于红霉素，MIC降低了两倍。我们发现六个FQR分离株聚为两组。 FQR-1_FQR-3_FQR-5簇中不同基因座的数量为6，而FQR-2和FQR-4在21个基因座中不同。 FQR-1_FQR-3_FQR-5和FQR-2_FQR-4簇彼此相距较远，并与从NCBI下载的19个基因组，8个与亚胺培南异源抗性的菌株和在丹麦分离的4个单倍体环丙沙星菌株进行比较。我们证实GyrA，ParC和ParE中的特定氨基酸取代与喹诺酮抗性有关。另外，抗性程度与这些氨基酸取代的数量有关。我们提供了有力的证据表明药物外排是流感嗜血杆菌中亚胺培南和红霉素耐药的重要机制之一。

The aims of the present study were to characterize the mechanisms of resistance to fluoroquinolones, macrolides, and imipenem in Haemophilus influenzae, to assess the extent of the AcrAB-TolC-mediated resistance, and to define a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae by using whole-genome sequencing. Four amino acid substitutions in GyrA (at Ser84 and Asp88), ParC (at Ser84), and ParE (at Asp420) were found to be closely associated to the MICs. We did not find any amino acid substitution surrounding the three highly conserved amino acid motifs in PBP3 related to imipenem resistance. All the isolates possessed the ermB gene. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the MIC of imipenem by twofold for FQR-6 and fourfold for GE47 and GE88 strains. For erythromycin, the MICs were decreased by twofold. We found that the six FQR isolates were clustered in two groups. The number of different loci within FQR-1_FQR-3_FQR-5 cluster was 6, while FQR-2 and FQR-4 differed for 21 loci. FQR-1_FQR-3_FQR-5 and FQR-2_FQR-4 clusters were distant among each other and compared to 19 genomes downloaded from NCBI, to 8 strains heteroresistant to imipenem, and to 4 strains monoresistant to ciprofloxacin isolated in Denmark. We confirmed that specific amino acid substitutions in GyrA, ParC, and ParE are implicated in quinolone resistance. Additionally, the degree of resistance is related to the number of these amino acid substitutions. We provide robust evidence that drug efflux is one of the substantial mechanisms of imipenem and erythromycin resistance in H. influenzae.

https://www.ncbi.nlm.nih.gov/pubmed/30145620