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 mexXYoprA 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 mexXYoprA 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(69)-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. INTRODUCTIONPseudomonas aeruginosa is a common nosocomial pathogen that causes a broad range of infections with a high mortality rate (Mahar et al., 2010; Iida et al., 2010;Lambert et al., 2011). A major factor in its prominence as a pathogen is, in part, its intrinsic resistance to a number of antibacterial agents (Poole et al., 1993;Hancock, 1998;Poole, 2002) and, particularly, the development of increased multidrug resistance in healthcare settings (Giamarellos-Bourboulis et al., 2006;Kirikae et al., 2008;Kallen et al., 2010;Keen et al., 2010). This organism readily acquires resistance via chromosomal mutations (e.g. overexpression of the efflux pump) and lateral gene transfer (e.g. acquisition of metallob-lactamase) (Lister et al., 2009;Poole, 2011). The emergence and spread of multidrug-, extensive drug-and pan-drug-resistant P. aeruginosa infections are very serious and of great concern, as few agents are effective against these organisms. In addition, antibiotic-resistant Gram-negative bacteria, including P. aeruginosa, increase the hospital costs and length of stay associated with healthcare-associated infections (Mauldin et al., 2010).Aminoglycosides such as amikacin, gentamicin and tobramycin are a vital component of antipseudomonal chemotherapy for a variety of infections, particularly pulmonary infections in cystic fibrosis (CF) patients (Poole, 2005(Poole, , 2011. Aminoglycoside resistance in P. aeruginosa has often arisen via acquired aminoglycoside-modifyi...

The TetR Family of Regulators

“…Expression of mexXY is controlled by the TFR MexZ and requires ArmZ (PA5471) (122), which interacts with MexZ (31,123). armZ encodes a homolog of RtcB, an RNA ligase involved in recovery from stress-induced RNA damage (124), and is cotranscribed with PA5470, which encodes a homolog of PrfH.…”

Emergence of antibiotic-resistant extremophiles (AREs)

“…It has also been observed that in P. aeruginosa, the mexZ repressor gene upstream of mexXY-oprA in PA7 is mutated, leading to overexpression of mexXY-oprA. In addition, the effect of modifying enzymes was enhanced by the presence of this efflux pump to develop a high-level aminoglycoside resistance (Morita et al 2012).…”

Innate Aminoglycoside Resistance of Achromobacter xylosoxidans Is Due to AxyXY-OprZ, an RND-Type Multidrug Efflux Pump

“…Nevertheless, AxyXY-OprZ confers a much higher level of resistance to aminoglycosides than MexXY-OprM in wild strains. It has recently been reported for some aminoglycosideresistant strains of P. aeruginosa that mexX and mexY are linked to the oprA gene in the same operon and that MexXY can utilize either OprM or OprA to form drug efflux complexes (18). Such an operon, including an oprA gene, has been also described in Burkholderia pseudomallei, a species intrinsically highly resistant to aminoglycosides due to expression of the AmrAB-OprA efflux pump (19).…”