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To study the role of Qnr-like pentapeptide repeat proteins (PRPs) from several Gram-positive species with quinolone resistance in vitro.A PCR-based strategy was used to clone and express genes coding for Qnr-like PRPs in Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Clostridium perfringens, C. difficile, Bacillus cereus and B. subtilis in Escherichia coli DH10B. MIC values of nalidixic acid and fluoroquinolones were determined for reference strains and E. coli DH10B harbouring recombinant plasmids containing genes coding for PRPs.Amino acid identity of Qnr-like PRPs in Gram-positive strains compared with that of the plasmid-mediated quinolone resistance determinants QnrA1, QnrB1 and QnrS1 was in the range of 16% to 22%. Recombinant plasmids coding for Qnr-like PRPs conferred reduced susceptibility to fluoroquinolones (in the range of 0.016 to 0.064 mg/L for ciprofloxacin) and nalidixic acid (from 6 to 12 mg/L), depending on the antimicrobial agent and PRP. The PRP from B. subtilis showed no protective effect.The PRPs analysed conferred a reduced susceptibility phenotype in E. coli; the data provide further evidence of the possible roles in quinolone resistance of PRPs from different Gram-positive species. These Gram-positive species may constitute a reservoir for Qnr-like quinolone resistance proteins.