title: Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7.
abstract: The multiresistant taxonomic outlier Pseudomonas aeruginosa PA7 possesses the conserved efflux genes, mexXY; however these are linked to a unique gene encoding an outer membrane channel, dubbed oprA, that is absent in most P. aeruginosa strains. Using genetic knockouts and single copy chromosomal complementation, we showed that aminoglycoside resistance in PA7 is mediated in part by the MexXY-OprA pump, and intriguingly that MexXY in this strain can utilize either the OprA or OprM outer membrane channel, linked to the mexAB efflux genes. We also identified a small portion of the oprA gene immediately downstream of the mexY gene in PAO1, suggesting that non-PA7 P. aeruginosa strains might have possessed, but lost, the intact mexXY-oprA efflux pump locus. Consistent with this, most of a panel of serotype strains possessed the truncated oprA but the serotype O12 isolate had an intact mexXY-oprA locus, similar to PA7 and the related strain DSM 1128. We also showed that the mexZ repressor gene upstream of mexXY-oprA in PA7 is mutated, leading to overexpression of mexXY-oprA, using sequencing, homologous replacement and real-time quantitative reverse transcriptase PCR. Finally we assessed the contribution of MexXY and aminoglycoside modifying enzymes AAC together to resistance in PA7 and the AAC(6')-Iae-mediated amikacin-resistant clinical isolate IMCJ2.S1, concluding that the effect of the modifying enzymes is enhanced by functional efflux, especially in the presence of divalent cations, to develop high-level aminoglycoside resistance in P. aeruginosa.
authors.affiliation: Department of Microbiology, School of Pharmacy, Aichi Gakuin University, Nagoya, Aichi 464-8650, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Morita
authors.affiliation: Department of Microbiology, School of Pharmacy, Aichi Gakuin University, Nagoya, Aichi 464-8650, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Tomida
authors.affiliation: Department of Microbiology, School of Pharmacy, Aichi Gakuin University, Nagoya, Aichi 464-8650, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kawamura
mesh_terms: D000595:Amino Acid Sequence; D000617:Aminoglycosides / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D026901:Membrane Transport Proteins / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D026901:Membrane Transport Proteins; C402791:MexXY protein, Pseudomonas aeruginosa; C402792:MexZ protein, Pseudomonas aeruginosa