It is generally believed that the Pseudomonas aeruginosa biofilm matrix itself acts as a molecular sieve or sink that contributes to significant levels of drug resistance, but it is becoming more apparent that multidrug efflux pumps induced during biofilm growth significantly enhance resistance levels. We present here a novel transcriptional regulator, PA3898, which controls biofilm formation and multidrug efflux pumps in P. aeruginosa. A mutant of this regulator significantly reduced the ability of P. aeruginosa to produce biofilm in vitro and affected its in vivo fitness and pathogenesis in Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA3898 modulates essential virulence genes/pathways, including multidrug efflux pumps and phenazine biosynthesis. Chromatin immunoprecipitation sequencing (ChIP-seq) identified its DNA binding sequences and confirmed that PA3898 directly interacts with promoter regions of four genes/operons, two of which are mexAB-oprM and phz2. Coimmunoprecipitation revealed a regulatory partner of PA3898 as PA2100, and both are required for binding to DNA in electrophoretic mobility shift assays. PA3898 and PA2100 were given the names MdrR1 and MdrR2, respectively, as novel repressors of the mexAB-oprM multidrug efflux operon and activators for another multidrug efflux pump, EmrAB. The interaction between MdrR1 and MdrR2 at the promoter regions of their regulons was further characterized via localized surface plasmon resonance and DNA footprinting. These regulators directly repress the mexAB-oprM operon, independent of its well-established MexR regulator. Mutants of mdrR1 and mdrR2 caused increased resistance to multiple antibiotics in P. aeruginosa, validating the significance of these newly discovered regulators.

The purpose of this study was to examine expression and regulation of 6 multidrug efflux systems, including MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY, MexJK, and MexVW, in 13 non-cystic fibrosis (CF) clinical isolates of Pseudomonas aeruginosa. These isolates displayed a high level of resistance to many clinically important antibiotics. Some isolates simultaneously overexpressed up to 4 different Mex systems, as determined by quantitative real-time reverse transcription PCR. None of the isolates overexpressed MexCD-OprJ, and only 1 isolate overproduced MexJK. All the isolates overexpressed MexXY, while overexpression of MexEF-OprN and MexVW was common. DNA sequencing analysis of regulatory genes showed that no clear correlation could be established among (i) the presence of mutations, (ii) the type of mutations, (iii) the expression level of the Mex systems, and (iv) resistance to antibiotic substrates. The results suggest that the concomitant overexpression of some Mex systems may superimpose their antimicrobial drug efflux capabilities, contributing to the multidrug resistance phenotype in the P. aeruginosa non-CF clinical isolates. The existence of uncharacterized regulators for the Mex systems was signified.

This study aimed to examine aminoglycosides (AMGs) resistance mechanisms, including the AMG-modifying enzyme genes, mexXY, rplY, nuoG, and galU, in the Pseudomonas aeruginosa non-cystic fibrosis (CF) isolates in Thailand. One hundred P. aeruginosa isolates from non-CF patients were examined for susceptibility to AMGs and for the presence of 10 AMG-modifying enzyme genes. Thirty randomly selected isolates were tested for transcription of mexXY and nuoG and mutations in rplY and galU. All the P. aeruginosa isolates exhibited simultaneous resistance to at least 4 AMGs. High resistance rates to amikacin (92%), gentamicin (95%), streptomycin (99%), and tobramycin (96%) were observed, and all isolates were resistant to kanamycin, neomycin, and spectinomycin. Nine AMG-modifying enzyme genes were detected, including aadA1 (84%), aadB (84%), aadA2 (67%), ant(2″)-Ia (72%), strA-strB (70%), aph(3')-IIb (57%), aac(3')-Ia (40%), and aac(6')-IIa (27%). None of the isolates harbored aac(6')-IIb. Of 30 isolates tested, all but 1 isolate expressed MexXY. Two isolates did not express nuoG. Six isolates carried an amino acid change in RplY, but none of the isolates harbored mutation in galU. The results indicated that the AMG-modifying enzyme genes were widespread among the P. aeruginosa non-CF isolates. The MexXY efflux pump and inactivation for rplY played a role in AMG resistance but disruption of nuoG or galU did not.

Gene regulation network in Pseudomonas aeruginosa is complex. With a relatively large genome (6.2 Mb), there is a significant portion of genes that are proven or predicted to be transcriptional regulators. Many of these regulators have been shown to play important roles in biofilm formation and maintenance. In this study, we present a novel transcriptional regulator, PA1226, which modulates biofilm formation and virulence in P. aeruginosa. Mutation in the gene encoding this regulator abolished the ability of P. aeruginosa to produce biofilms in vitro, without any effect on the planktonic growth. This regulator is also essential for the in vivo fitness and pathogenesis in both Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA1226 regulates many essential virulence genes/pathways, including those involved in alginate, pili, and LPS biosynthesis. Genes/operons directly regulated by PA1226 and potential binding sequences were identified via ChIP-seq. Attempts to confirm the binding sequences by electrophoretic mobility shift assay led to the discovery of a co-regulator, PA1413, via co-immunoprecipitation assay. PA1226 and PA1413 were shown to bind collaboratively to the promoter regions of their regulons. A model is proposed, summarizing our finding on this novel dual-regulation system.

ABSTRACT. As study of multidrug efflux pumps is a crucial step for development of efflux pump inhibitors for treatment of Pseudomonas aeruginosa infection, the objective of this study was to examine the contribution of the MexXY multidrug efflux systems and other chromosomal mechanisms in aminoglycoside (AMG) resistance in P. aeruginosa isolated from dogs and cats. Thirteen Pseudomonas aeruginosa isolates from canine and feline infections were examined for contribution of the MexXY multidrug efflux pump and four other chromosomally-encoded genes including PA5471, galU, nuoG and rplY to AMG resistance. All the isolates were resistant to multiple AMGs and expressed mexXY. Deletion of mexXY caused 2-to 16-fold reduction in AMG MICs. Overproduction of MexXY did not fully account for the observed AMG resistance. No good correlations were detected between MexXY transcription level and AMG MICs. While no mutations were found in mexZ, PA5471 expression varied and its impact on MexXY expression and AMG resistance is diverse. No mutations were found in galU. Only two isolates carried a single base change G-367-T in rplY. Complete transcription of nuoG was detected in all the isolates. In conclusion, the MexXY multidrug efflux pump plays a role in AMG resistance in the dog and cat P. aeruginosa isolates, while disruption of nuoG, rplY and galU did not have a significant impact. These results indicate the existence of uncharacterized AMG-resistance mechanisms. Pseudomonas aeruginosa is infamously known as a common cause of chronic and recurrent infections in both humans and animals, of which the most-notably diseases in dogs and cats include otitis externa/media, urinary tract infection and pyoderma [9,10,20]. Treatment of P. aeruginosa infection is challenging, because the pathogen intrinsically exhibits and efficiently develops high resistance to several antimicrobials structurally and functionally unrelated, leading to multidrug resistance [1,24].As many antipseudomonal drugs are available for therapeutic use in dogs and cats, aminoglycosides (AMGs) are considered a vital component of antipseudomonal chemotherapy [22] due to their efficacy, safety and reasonable price. However, panaminoglycoside resistance e.g., gentamicin, spectinomycin, streptomycin and amikacin has been increasingly reported in the P. aeruginosa dog and cat isolates [18,19,23]. Such resistance has been clarified to be predominantly due to a poorly understood mechanism namely "impermeability resistance" as a consequence of diminished drug uptake and/or accumulation [21,31]. The MexXY efflux system, a multidrug efflux pump in the resistance-nodulation-cell-division (RND) family, is involved in the reduced level of AMG accumulation implicated in both impermeability-type and adaptive-type AMG resistance [12,21,22]. The MexXY efflux pump is encoded by the mexXY operon and evidently serves as the major AMG-resistance mediating system in P. aeruginosa clinical isolates. This system additionally confers resistance to nonaminoglycosides, including tetracycline...