Contribution of Type II Topoisomerase Mutations to Fluoroquinolone Resistance in Enterococcus faecium from Japanese Clinical Setting.

High-level fluoroquinolone resistance is conferred by the mutation of conserved serine and acidic amino acids in the quinolone resistance-determining region (QRDR) of the A subunits of the type II topoisomerases, DNA gyrase (GyrA) and topoisomerase IV (ParC). In Japan, fluoroquinolone-resistant Enterococcus faecium continues to emerge in clinical settings. We analyzed 131 Japanese E. faecium clinical isolates for susceptibility to levofloxacin (LVFX), and QRDR mutational status. The bacterial collection had a high percentage of resistance (79%) and showed elevated drug minimal inhibitory concentrations (MICs). Eighty-three isolates had single or combined mutations in gyrA and/or parC; all were resistant to LVFX. A strong correlation was evident between log-transformed MICs and the total number of QRDR mutations (r = 0.7899), confirming the involvement of QRDR mutations in drug resistance, as previously described. Three-dimensional modeling indicated that the amino acid change(s) in QRDR could disrupt the interaction between the enzymes and drugs: the most common cause of quinolone resistance. Interestingly, eight isolates had a single mutation on gyrA and exhibited significantly reduced susceptibility. These data imply that either DNA gyrase or topoisomerase IV can be the primary target of fluoroquinolones, although topoisomerase IV is commonly thought to be the primary target in gram-positive bacteria.

Characterization of Enterococcus faecium with macrolide resistance and reduced susceptibility to quinupristin/dalfopristin in a Japanese hospital: detection of extensive diversity in erm(B)-regulator regions.

Cross-resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics is mainly mediated by the erm (erythromycin ribosome methylation) genes that encode 23S rRNA methylases in enterococi, and various mechanisms are involved in the streptogramin B resistance. Prevalence of MLSB resistance and its genetic mechanisms were analyzed for a total of 159 strains of Enterococcus faecium isolated from clinical specimens in a university hospital in Japan from 1997 to 2006. Resistance to erythromycin (EM) and clindamycin was detected in 88.1% and 89.9% of all the strains examined, respectively, and expression of resistance was totally constitutive. Although none of the strain was resistant to quinupristin/dalfopristin (Q/D), 28 strains (17.6%) showed intermediate resistance to Q/D (MIC: 2 µg/ml). The erm(B) gene was detected in 139 strains (87.4%), and msrC was found in all the strains examined, whereas no other known MLSB resistance genes were identified. The erm(B) regulator region (RR) containing a coding region of the leader peptide was classified into 13 genetic variations (L1-L3, M, S1-S7, D, and R genotypes) in 56 strains. However, no relatedness was identified between the erm(B) RR genotype and EM resistance, or reduced susceptibility to Q/D, although most of Q/D-intermediate strains were assigned to the L1, L2, and S1 genotypes. Q/D-intermediate strains were classified into five multiple-locus variable-number tandem-repeat analysis (MLVA) types, including four types of clonal complex (CC)-C1, five sequence types (STs), including four STs of CC-17, and several resistance gene/virulence factor profiles. The present study revealed the occurrence of Q/D-intermediate E. faecium, which are composed of heterogeneous strains in Japan, and more genetic diversity in the erm(B) RRs than those reported previously.