Molecular characterization of fluoroquinolones, macrolides, and imipenem resistance in Haemophilus influenzae: analysis of the mutations in QRDRs and assessment of the extent of the AcrAB-TolC-mediated resistance

Abstract

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.

Isolation of Drug-Resistant Gallibacterium anatis from Calves with Unresponsive Bronchopneumonia, Belgium

“…We detected >20 different resistance genes in the genomes of the G. anatis isolates in our study, including determinants conferring resistance to aminoglycosides, phenicols, macrolides, sulphonamides, trimethoprim, tetracyclines, penicillins, and quinolones. Although many of these resistance genes have been described previously in Pasteurellaceae obtained from either animals or humans (43,44), we detected various other resistance genes not previously reported in G. anatis or bovine Pasteurellaceae. Moreover, 4 resistance genes have so far never been described in Pasteurellaceae at all, namely aadA23, bla CARB-8 , tet(Y) and qnrD1.…”

Monitoring quinolone resistance due to Haemophilus influenzae mutations (2012–17)

“…While MIC values have been reported to increase as the number of QRDR mutations increases (13–21), only one isolate was found to have two mutations in the present study. The isolate 2017-2 showed reduced susceptibility to all fluoroquinolones except for STFX, a finding supporting the above notion (Table 1).…”

“…Isolate 2014-5 showed a high MIC value (0.5 µg/ml) for not only MXF but also CIP and LVX, although this isolate had no mutations in QRDRs of both GyrA and ParC enzymes (Table 1). A factor presumably involved in this is a drug efflux pump (acrAB) (21–22) or H. influenzae -specific porin (23–24), the protein structure of which is similar to that of porin (OmpF); however, solid evidence was not obtained from the present study, and this point remains to be addressed in future studies.…”

Fluoroquinolone resistance in Haemophilus influenzae from nursing home residents in Taiwan: correlation of MICs and mutations in QRDRs

“…A major weakness in the results from these studies was the low number of ‘resistant’ isolates (Georgiou et al ; Biedenbach and Jones ; Pérez‐Vázquez et al ; Yoshizumi et al ; Li et al ; Puig et al ; Fuursted et al ; Cherkaoui et al ). In addition, there were few H. influenzae isolates with high‐level resistance (MICs for levofloxacin or moxifloxacin of >16 μg ml −1 ) reported (Yoshizumi et al ; Li et al ; Kuo et al ; Shoji et al ; Puig et al ; Fuursted et al ; Cherkaoui et al ). Because of the inclusion of more isolates with high‐level resistance to fluoroquinolones, our study provides statistical evidence of the association of MIC levels and the numbers and positions of QRDR mutations, as well as their correlation with nalidixic acid inhibition zone diameters.…”

“…Fluoroquinolone resistance is often caused by mutations in the quinolone‐resistance determining regions (QRDRs) of two principal target enzymes, DNA gyrase, which is partially encoded by subunit genes, gyrA and gyrB, and DNA topoisomerase IV, encoded by parC and parE . The sequences of several fluoroquinolone‐resistant H. influenzae isolates showed amino acid changes at positions 84 and/or 88 in gyrA and/or at the analogous positions (84, 88) in parC (Georgiou et al ; Pérez‐Vázquez et al ; Yoshizumi et al ; Li et al ; Kuo et al ; Shoji et al ; Puig et al ), some studies reported mutations in parE (Fuursted et al ; Cherkaoui et al ). However, since the majority of resistant H. influenzae isolates previously described in QRDR studies had only reduced susceptibility or few numbers of high‐level resistance to fluoroquinolones, no statistical association could be made between mutations and levels of fluoroquinolone resistance (Georgiou et al ; Pérez‐Vázquez et al ; Yoshizumi et al ; Li et al ; Kuo et al ; Shoji et al ; Puig et al ; Fuursted et al ; Cherkaoui et al ).…”