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.

, 587 strains of gram-negative bacterial isolates demonstrating resistance to ceftazidime and a combination of sulbactam and cefoperazone were subjected to a disk diffusion screening test using sodium mercaptoacetic acid; 431 strains (73.4%) appeared to produce metallo-␤-lactamase (MBL). Of these 431 strains, 357 were found by PCR to carry genes for IMP-1 type MBL (bla IMP-1 ), while only 7 and 67 strains carried the IMP-2 gene (bla IMP-2 ) and the VIM-2 gene (bla VIM-2 ), respectively. Neither VIM-1 nor SPM-1 type MBL genes were found among the strains tested. Of 431 strains, 427 carried the intI1 gene, and 4 strains carrying both the intI1 and intI3 genes were reidentified as Pseudomonas putida harboring bla IMP-1 . Of these four P. putida strains, three strains and one strain, respectively, were separately isolated from two hospitals located in the same prefecture, and the three strains showed very similar pulsed-field gel electrophoresis patterns. Of 357 bla IMP-1 carriers, 116, 53, 51, 47, and 30 strains were identified as Pseudomonas aeruginosa, Alcaligenes xylosoxidans, P. putida/fluorescens, Serratia marcescens, and Acinetobacter baumannii, respectively. Four strains carrying bla IMP-2 were reidentified as P. putida. Sixty-three P. aeruginosa strains and four P. putida strains carried bla VIM-2 . Of 427 intI1-positive strains, 180, 53, 51, 47, and 35 were identified as P. aeruginosa, A. xylosoxidans, P. putida/fluorescens, S. marcescens, and A. baumannii, respectively. In the present study, it was confirmed that strains carrying bla IMP-1 with a class 1 integron are the most prevalent type in Japan, although several intI3 carriers have also been identified sporadically in this country.

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...

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