Although Moraxella catarrhalis is known to be susceptible to macrolides, highly macrolideresistant M. catarrhalis isolates have recently been reported in Japan and China. In this study, we investigated the prevalence of macrolide-resistant M. catarrhalis isolates in Tokyo and Chiba, Japan, and studied the mechanisms underlying their resistance. Specifically, we determined the susceptibility of 593 clinical isolates (collected between December 2011 and May 2014) to erythromycin, using the disk diffusion method. For isolates with erythromycin resistance, we identified the MICs of seven antimicrobial agents, including macrolides, and used PFGE to analyse the clonal spread. We also performed sequencing analysis to investigate macrolide-resistance targets. Thirteen isolates (2.2 %) were found to be resistant to erythromycin, showing a high MIC 90 to erythromycin, clarithromycin, clindamycin and azithromycin. However, those isolates, in addition to 156 randomly selected erythromycinsusceptible strains, were susceptible to amoxicillin-clavulanate, cefixime and levofloxacin. The 13 highly macrolide-resistant isolates were classified into 10 clades and harboured three or four A2058T-mutated 23S rRNA alleles. Three highly macrolide-resistant isolates also exhibited mutations in ribosomal proteins L4 (V27A and R161C) and L22 (K68T). To the best of our knowledge, we have demonstrated for the first time that, whilst the prevalence of macrolideresistant M. catarrhalis isolates is low in clinical settings in Japan, genetically diverse isolates with high-level macrolide resistance due to the acquisition of an A2058T mutation in the 23S rRNA have already spread. Our study therefore lays the basis for epidemiological studies of macrolide-resistant M. catarrhalis clinical isolates.

BackgroundHigh-level macrolide-resistant Moraxella catarrhalis strains have been isolated; however, the underlying mechanism has not been well elucidated. We investigated the role of mutations in the 23S rRNA gene and the L4 and L22 ribosomal proteins using spontaneous erythromycin-resistant mutants and transformants.Materials and methodsThe erythromycin-susceptible M. catarrhalis ATCC25238 and clinical isolate Mc19 were used as parental strains. To obtain spontaneous erythromycin-resistant mutants, in vitro stepwise selection was performed using brain–heart infusion agar plates containing various concentrations of erythromycin. The role of the mutations identified in the spontaneous mutants was validated using transformation experiments.ResultsWe obtained two spontaneous mutants with high-level resistance to erythromycin, S25-32-af10 and S19-256-af10, from ATCC25238 and Mc19, respectively. S25-32-af10 exhibited mutations of Q61R in L4 and Insertion98SRADRIS in L22. S19-256-af10 exhibited three C2611T-mutated alleles in the 23S rRNA gene and G65A in L4. Transformants with single mutations identified in S25-32-af10 or S19-256-af10 showed higher erythromycin and azithromycin minimum inhibitory concentrations (MICs) than those of each parental strain. However, transformants with multiple mutations identified in S25-32-af10 or S19-256-af10 showed macrolide MICs similar to those of each parental strain.ConclusionOur results provide the first evidence suggesting that Q61R in L4 and Insertion98SRADRIS in L22 are involved in the synergistic acquisition of high-level resistance to both 14- and 15-member macrolides, and that C2611T in the 23S rRNA gene and G65A in L4 also synergistically contribute toward conferring high-level 14-member macrolide resistance to M. catarrhalis.

We report on a restriction fragment-length polymorphism (HpyCH4III) assay for profile analysis of 23S rRNA gene A2058T-mutated alleles associated with high-level macrolide resistance in Moraxella catarrhalis. Our assay results were supported by DNA sequencing analysis, allowed for simultaneous testing of many strains, and produced results from pure-cultured colonies within 4 h.