Prevalence and molecular analysis of macrolide-resistant Moraxella catarrhalis clinical isolates in Japan, following emergence of the highly macrolide-resistant strain NSH1 in 2011

Abstract

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.

Use of next generation sequence to investigate potential novel macrolide resistance mechanisms in a population of Moraxella catarrhalis isolates

“…Considering that different non-synonymous mutations may cause the same alternation of amino acid, all the 4770 SNPs analyzed in this study were confirmed after assessment for both synonymous and non-synonymous mutations. In M. catarrhalis , there are four identical copies of rRNA (16S, 23S, and 5S rRNA genes) operons in which the 16S and 23S rRNA genes are interspersed with genes encoding tRNAs for isoleucine and alanine 14 . Among the 4770 SNPs observed to be unique to either susceptible or resistant isolates, 29 were from the rRNA gene, including A2144T, A2330T, and C2480T mutation of 23S rRNA, which were mainly contributed by the resistant isolates ( Table 4 ) as previously noted 7 .…”

“…In 2012, we reported for the first time that ribosomal mutation was crucial for creating macrolide-non-susceptible M. catarrhalis , and that A2330T (position referring to M. catarrhalis 23S rRNA gene, GenBank number NR_103214.1) mutation was related to high-level macrolide-resistance 10 . In the following three years, several studies confirmed that indeed A2330T mutation of the 23S rRNA gene could lead to isolates with high-level macrolide resistance (MIC > 256 g/L) 11 12 13 14 .…”

“…Findings from a recent study in Japan revealed that M. catarrhalis strains with high level macrolide resistance also exhibit mutations in ribosomal proteins L4 (V27A and R161C) and L22 (K68T) 14 . Interestingly and in contrast to M. catarrhalis , mutation of the 23S rRNA gene is usually not the main reason for macrolide-resistance in other bacterial species (such as Streptococcus pneumoniae, Streptococcus pyogenes , etc 15 16 17 ).…”

“…Firstly, we intended to gain further insights into whether A2330T mutation is solely responsible for macrolide resistance in M. catarrhalis , or whether other mechanisms, including methylase, efflux pump, or other genes or mutations 14 , alone or in combination, are involved. Secondly, given that macrolide resistant M. catarrhalis are so different from other macrolide resistant cocci, we assumed that macrolide resistance in M. catarrhalis may be associated with the distinct genomic background of this organism.…”

Bronchiectasis in Children: Current Concepts in Immunology and Microbiology

“…Most M. catarrhalis strains produce β-lactamase; 92% in the PROTEKT study (87). In contrast, M. catarrhalis is almost universally susceptible to azithromycin (87,95,107,108), although macrolide resistance has recently been reported in China (109,110), Japan (111,112), and Pakistan (113). In Australian Indigenous children with bronchiectasis, most M. catarrhalis isolates from the NP (91%) and BAL (100%) were β-lactamase positive; however, azithromycin resistance was not tested (89).…”