The frequency of BRO ß-lactamase and its relationship to antimicrobial susceptibility and serum resistance in Moraxella catarrhalis.

We investigated the frequency of BRO ß-lactamase and its relationship to antibiotic susceptibility profiles and serum susceptibility. Moraxella catarrhalis clinical isolates (n = 197) were collected from patients with respiratory tract infections in Tokyo between November 2004 and April 2005. Phenotypic and genotypic detection of ß-lactamases was performed. The MICs of 6 antibiotics were determined by Etest, and the serum bactericidal assay was conducted by using the culture-and-spot test. Nearly all (192; 97%) of the clinical isolates were ß-lactamase producers; of these, 182 (95%) were bro-1 and 10 (5%) were bro-2 positive. MIC50, MIC90, and geometric mean MICs of penicillin, amoxicillin, cefixime, and clarithromycin for BRO-1 isolates were significantly higher than for BRO-2 isolates. The frequency of intermediate and full serum resistance was significantly higher in BRO-1 isolates than in BRO-2 isolates (P = 0.0056), but not BRO-negative isolates (P = 0.1333). We provide the first evidence that the presence of BRO-1 in M. catarrhalis is associated with reduced susceptibility to clarithromycin and ß-lactam antibiotics, as well as serum non-sensitive (intermediate and resistant).

Antimicrobial susceptibility and mechanisms of high-level macrolide resistance in clinical isolates of Moraxella nonliquefaciens.

We investigated antimicrobial susceptibility and the molecular mechanism involved in conferring high-level macrolide resistance in 47 clinical isolates of Moraxella nonliquefaciens from Japan. Antimicrobial susceptibility was determined using Etest and agar dilution methods. Thirty-two erythromycin-non-susceptible strains were evaluated for the possibility of clonal spreading, using PFGE. To analyse the mechanism related to macrolide resistance, mutations in the 23S rRNA gene and the ribosomal proteins, and the presence of methylase genes were investigated by PCR and sequencing. The efflux system was examined using appropriate inhibitors. Penicillin, ampicillin, amoxicillin, cefixime, levofloxacin and antimicrobials containing ß-lactamase inhibitors showed strong activity against 47 M. nonliquefaciens isolates. Thirty-two (68.1 %) of the 47 isolates showed high-level MICs to macrolides (MIC ≥128 mg l(-1)) and shared the A2058T mutation in the 23S rRNA gene. The geometric mean MIC to macrolides of A2058T-mutated strains was significantly higher than that of WT strains (P<0.0001). Thirty-two isolates with high-level macrolide MICs clustered into 30 patterns on the basis of the PFGE dendrogram, indicating that the macrolide-resistant strains were not clonal. In contrast, no common mutations of the ribosomal proteins or methylase genes, or overproduction of the efflux system were observed in A2058T-mutated strains. Moreover, of the 47 M. nonliquefaciens strains, 43 (91.5 %) were bro-1 and 4 (8.5 %) were bro-2 positive. Our results suggest that most M. nonliquefaciens clinical isolates show high-level macrolide resistance conferred by the A2058T mutation in the 23S rRNA gene. This study represents the first characterization of M. nonliquefaciens.

Role of Moraxella catarrhalis outer membrane protein CD in bacterial cell morphology and autoaggregation.

Moraxella catarrhalis, an important pathogen in the human respiratory tract, causes otitis media and lower respiratory tract infections. M. catarrhalis outer membrane protein CD (OMPCD) is a major heat-modifiable OMP with demonstrable potential as a vaccine candidate. The gene encoding OMPCD of M. catarrhalis strains was subjected to nucleotide sequence analysis and then inactivated by insertional mutagenesis. The ompCD mutant strains exhibited a modest growth defect in comparison with the wild-type strains. In optical microscopy and scanning/transmission electron microscopy examinations, regarding morphology, the cell size and cell wall of the ompCD mutant strains were significantly larger and thinner, respectively, than those of the wild-type strain. Furthermore, the ompCD mutant strains exhibited significant autoaggregation and increased surface hydrophobicity, in addition to a reduction in the adherence to HEp-2 cells, compared to the wild-type strains. Strains repaired by replacing the mutated ompCD gene exhibited phenotypic characteristics very similar to those of the wild-type strains. These results indicate that M. catarrhalis OMPCD, in addition to its functions related to bacterial growth and adherence to human epithelial cells, plays a very important role in bacterial physiology and pathogenesis, including aspects such as stabilizing bacterial cell morphology and preventing autoaggregation by reducing surface hydrophobicity.

Molecular mechanism of macrolide-lincosamide resistance in Moraxella catarrhalis.

We identified a Moraxella catarrhalis strain with high-level resistance to azithromycin (MIC>256 mg l(-1)), NSH1, isolated from nasopharyngeal swab samples from an inpatient with acute bronchitis in a Japanese hospital in 2011 and determined its mechanism of macrolide-lincosamide resistance. Antimicrobial susceptibility of M. catarrhalis strains was determined using the Etest and agar dilution methods. Mutations in the four 23S rRNA alleles, the ribosomal proteins L4 and L22, and methylase genes erm(B) and erm(F) were tested by PCR and/or sequencing. The efflux system was examined using appropriate inhibitors. Transformation experiments were performed using DNA amplicons of the 23S rRNA gene of M. catarrhalis strain NSH1. This strain showed high-level resistance to erythromycin, clarithromycin, azithromycin, clindamycin (MICs>256 mg l(-1)) and josamycin (MIC = 128 mg l(-1)), and contained the A2058T mutation (Escherichia coli numbering) in four of the 23S rRNA alleles. Mutation of the ribosomal proteins and overproduction of the efflux system were not observed, and methylase genes were not detected. When amplified DNA containing the single A2058T mutation was transformed into M. catarrhalis strains, transformants with three A2058T-mutated 23S rRNA alleles showed high-level resistance to macrolide-lincosamide, similar to strain NSH1. In contrast, transformants with two A2058T-mutated 23S rRNA alleles showed low-level MICs (azithromycin: 0.38-0.5 mg l(-1)). Thus, a single A2058T mutation occurring in at least three 23S rRNA alleles confers high-level resistance to 14-, 15- and 16-membered macrolides and lincosamides in M. catarrhalis possessing four 23S rRNA alleles. This study represents the first evidence, to our knowledge, of this effect in M. catarrhalis.