Plasmid-mediated Qnr and AAC(6)-Ib-cr have been recognized as new molecular mechanisms affecting fluoroquinolone (FQ) resistance. C316, an Escherichia coli strain demonstrating resistance to various FQs, was isolated in Japan. Resistance to FQs was augmented in an E. coli CSH2 transconjugant, but PCR failed to detect qnr genes, suggesting the presence of novel plasmid-mediated FQ resistance mechanisms. Susceptibility tests, DNA manipulation, and analyses of the gene and its product were performed to characterize the genetic determinant. A novel FQ-resistant gene, qepA, was identified in a plasmid, pHPA, of E. coli C316, and both qepA and rmtB genes were mediated by a probable transposable element flanked by two copies of IS26. Levels of resistance to norfloxacin, ciprofloxacin, and enrofloxacin were significantly elevated in E. coli transformants harboring qepA under AcrBTolC-deficient conditions. QepA showed considerable similarities to transporters belonging to the 14-transmembrane-segment family of environmental actinomycetes. The effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on accumulation of norfloxacin was assayed in a qepA-harboring E. coli transformant. The intracellular accumulation of norfloxacin was decreased in a qepA-expressing E. coli transformant, but this phenomenon was canceled by CCCP. The augmented FQ resistance level acquired by the probable intergeneric transfer of a gene encoding a major facilitator superfamily-type efflux pump from some environmental microbes to E. coli was first identified. Surveillance of the qepA-harboring clinical isolates should be encouraged to minimize further dissemination of the kind of plasmid-dependent FQ resistance determinants among pathogenic microbes.Fluoroquinolones (FQs) are synthetic chemical agents and among the most commonly prescribed antimicrobials because of their broad-spectrum antimicrobial activity. Extensive clinical and agricultural use of FQs has led to high rates of resistance to these agents among pathogenic microbes (6, 9). The most common mechanism for resistance to FQs among pathogenic microbes is the mutation of chromosomal genes encoding DNA gyrase and/or topoisomerase IV (12, 13). Changes in the expression of efflux pumps and porin proteins are also a common FQ resistance mechanism in bacteria (1), but no plasmid-mediated FQ efflux pump has been documented to date. Four chromosome-dependent efflux systems responsible for FQ resistance have so far been reported, e.g., the resistance nodulation division family, AcrAB-TolC in Escherichia coli (7,28) and MexAB-OprM in Pseudomonas aeruginosa (24); the major facilitator superfamily (MFS), NorA of Staphylococcus aureus (34); the multidrug and toxic compound extrusion family, NorM of Vibrio parahaemolyticus (19); and the ATP-binding cassette family, VcaM of non-O1 Vibrio cholerae (14).Two molecular groups have been identified as plasmid-mediated FQ resistance mechanisms to date. Qnr peptides, QnrA (30), QnrB (15), and QnrS (11), were identified from Klebsiella pneumoniae, Klebsiell...

Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for adults. Penicillins are the drugs of first choice for the treatment of GBS infections, since GBS have been regarded to be uniformly susceptible to penicillins so far. Here we characterize the first strains of GBS with reduced penicillin susceptibility (PRGBS) identified in Japan. Fourteen PRGBS strains were clinically isolated from the sputa of elderly patients from 1995 to 2005; and the MICs of penicillin, oxacillin, and ceftizoxime ranged from 0.25 to 1 g/ml, 2 to 8 g/ml, and 4 to 128 g/ml, respectively. Moreover, some strains were also insusceptible to ampicillin, cefazolin, cefepime, and cefotaxime. All the PRGBS isolates tested possessed a few amino acid substitutions adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively) of PBP 2X, and the amino acid substitutions could be classified into two types, Q557E and V405A. Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum suggested that the amount of PBP 2X among the 14 PRGBS isolates was reduced, although the 2 ATCC strains produced a significant amount of PBP 2X. The introduction of PRGBS-derived PBP 2X genes into penicillin-susceptible strains through allelic exchange elevated their penicillin insusceptibility, suggesting that these altered PBP 2X genes are responsible for the penicillin insusceptibility in PRGBS strains. In this study, we characterized for the first time PRGBS strains on a molecular basis, although several reports have so far mentioned the existence of ␤-lactam-insusceptible GBS from a phenotypic standpoint.

We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.Aminoglycosides such as kanamycin, gentamicin, and neomycin bind to the A site of the 16S rRNA of the bacterial 30S ribosomal subunit and subsequently block growth through interference with protein synthesis (25). These agents have been used for the treatment of a broad range of life-threatening infections due to both gram-positive and gram-negative bacteria in human and veterinary medicine (18, 37). However, bacteria have acquired various aminoglycoside resistance mechanisms, such as through the production of aminoglycoside-modifying enzymes (acetyltransferase, nucleotidyltransferase, and phosphotransferase), the reduction of antibiotic penetration on the outer membrane protein, the acquisition of reduced affinity by changing key nucleotides within the 16S rRNA, and augmented excretion by an efflux pump system (5,25,36,42).In 2003, a plasmid-mediated 16S rRNA methyltransferase, which confers a high level of resistance to various clinically important aminoglycosides, was reported to be involved as part of a novel aminoglycoside resistance mechanism in pathogenic gram-negative rods (16, 53). At present, five types of plasmidmediated 16S rRNA methyltransferase genes, rmtA, rmtB, rmtC, rmtD, and armA, have been found worldwide in members of the family Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. (4,7,10,11,17,20,34,48,(50)(51)(52). Also, these genes are mediated by bacterium-specific recombination systems, such as transposons, and are easily translocated to other DNA target sites (17,19,47,49).The 16S rRNA methyltransferases conferring aminoglycoside resistance are supposed to have evolved as a self-defense mechanism in aminoglycoside-producing actinomyce...

We evaluated the in vitro activity of fosfomycin against a total of 192 CTX-M ␤-lactamase-producing Escherichia coli strains isolated in 70 Japanese clinical settings. Most of the isolates (96.4%) were found to be susceptible to fosfomycin. On the other hand, some of the resistant isolates were confirmed to harbor the novel transferable fosfomycin resistance determinants named FosA3 and FosC2, which efficaciously inactivate fosfomycin through glutathione S-transferase activity.

Macrolide-resistant Mycoplasma pneumoniae (MR M. pneumoniae) has been isolated from clinical specimens in Japan since 2000. A comparative study was carried out to determine whether or not macrolides are effective in treating patients infected with MR M. pneumoniae. The clinical courses of 11 patients with MR M. pneumoniae infection (MR patients) treated with macrolides were compared with those of 26 patients with macrolide-susceptible M. pneumoniae infection (MS patients). The total febrile days and the number of febrile days during macrolide administration were longer in the MR patients than in the MS patients (median of 8 days versus median of 5 days [P ‫؍‬ 0.019] and 3 days versus 1 day [P ‫؍‬ 0.002], respectively). In addition, the MR patients were more likely than the MS patients to have had a change of the initially prescribed macrolide to another antimicrobial agent (63.6% versus 3.8%; odds ratio, 43.8; P < 0.001), which might reflect the pediatrician's judgment that the initially prescribed macrolide was not sufficiently effective in these patients. Despite the fact that the febrile period was prolonged in MR patients given macrolides, the fever resolved even when the initial prescription was not changed. These results show that macrolides are certainly less effective in MR patients.Mycoplasma pneumoniae is a common pathogen causing community-acquired respiratory tract infection mainly in children and young adults. Macrolides are generally considered to be the first-choice agents for treatment of M. pneumoniae infection. Although tetracyclines and fluoroquinolones are effective against M. pneumoniae, these agents are not recommended for children because of their toxicity. Tetracyclines can cause depression of bone growth, permanent gray-brown discoloration of the teeth, and enamel hypoplasia when given during tooth development. Although the clinical importance of fluoroquinolones has not been demonstrated, they produce cartilage erosion in young animals. Thus, these agents should be given only when there is no alternative (15).As reported by Lucier et al. (9) and Okazaki et al. (14), an A-to-G transition or A-to-C transversion at position 2063 or 2064 of domain V of the M. pneumoniae 23S rRNA gene results in resistance to macrolide antibiotics. We have previously reported the isolation of macrolide-resistant (MR) M. pneumoniae from ca. 20% of clinical specimens collected from pediatric patients in Japan (11). Most of those isolates were highly resistant to 14-membered ring macrolides (MIC, Ͼ256 g/ml) and moderately resistant to 15-and 16-membered ring macrolides.Even in the cases of patients infected with MR M. pneumoniae, some pediatricians had the impression that there was a good response to macrolide therapy (11). There is a similar debate about the management of infection due to pneumococci. As noted in The Infectious Diseases Society of America (IDSA) guidelines for community-acquired pneumonia management (10), despite the increase of resistant isolates, a corresponding increase has not been seen in...