Evaluation of efflux pump inhibitors of MexAB- or MexXY-OprM in Pseudomonas aeruginosa using nucleic acid dyes.

BACKGROUND: Increased expression of efflux pumps is an important mechanism of antibiotic resistance in Pseudomonas aeruginosa, and treatment with inhibitors of active efflux pumps seems an attractive strategy to combat with multidrug resistance. Assays using ethidium bromide (EtBr), which accumulates by binding to nucleic acids, are often employed to assess the efficacy of efflux pump inhibitors (EPIs). However, few studies have reported on assays using other nucleic acid dyes. OBJECTIVE: We used different classes of EPIs for MexAB- or MexXY-OprM to measure the accumulation of various fluorescent dyes, including SYBR Safe, AtlasSight, and GelGreen. METHODS: Escherichia coli MG1655ΔacrBΔtolC strain harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of P. aeruginosa were constructed. Then, the accumulation of the above-mentioned nucleic acid dyes and EtBr was measured to assess the efflux ability in the presence and absence of EPIs (MexAB-OprM-specific inhibitor of pyridopyrimidine derivative [ABI-PP], berberine, non-specific inhibitor of phenylalanine-arginine ß-naphthylamide [PAßN], and protonophore of carbonyl cyanide m-chlorophenyl hydrazone [CCCP]). RESULTS: Decreased accumulations of nucleic acid dyes were observed in strains with pABM or pXYM compared with the parental strain. ABI-PP or berberine addition significantly increased the accumulation of any nucleic acids in the strains with the specific pumps. PAßN or CCCP addition showed increased accumulation of almost all dye in strains with pABM or pXYM. However, the inhibition patterns of EPIs differed according to the nucleic acid dyes used. CONCLUSIONS: Accumulation assays for EPIs were suitable to evaluate EPI candidates using various nucleic acid dyes.

Complementation of the Fo c subunit of Escherichia coli with that of Streptococcus mutans and properties of the hybrid FoF1 ATP synthase.

The c subunit of Streptococcus mutans ATP synthase (FoF1) is functionally exchangeable with that of Escherichia coli, since E. coli with a hybrid FoF1 is able to grow on minimum succinate medium through oxidative phosphorylation. E. coli F1 bound to the hybrid Fo with the S. mutans c subunit showed N,N'-dicyclohexylcarbodiimide-sensitive ATPase activity similar to that of E. coli FoF1. Thus, the S. mutans c subunit assembled into a functional Fo together with the E. coli a and b subunits, forming a normal F1 binding site. Although the H(+) pathway should be functional, as was suggested by the growth on minimum succinate medium, ATP-driven H(+) transport could not be detected with inverted membrane vesicles in vitro. This observation is partly explained by the presence of an acidic residue (Glu-20) in the first transmembrane helix of the S. mutans c subunit, since the site-directed mutant carrying Gln-20 partly recovered the ATP-driven H(+) transport. Since S. mutans is recognized to be a primary etiological agent of human dental caries and is one cause of bacterial endocarditis, our system that expresses hybrid Fo with the S. mutans c subunit would be helpful to find antibiotics and chemicals specifically directed to S. mutans.

[Sensitivity surveillance of Pseudomonas aeruginosa isolates for several antibacterial agents in Gifu and Aichi prefecture (2008)].

We investigated the susceptibility to antibacterial agents of 334 strains of Pseudomonas aeruginosa isolated from medical facilities in Gifu and Aichi prefectures from May to September 2008. For the beta-lactams, meropenem (MEPM) and doripenem (DRPM) gave the lowest MIC50 at 0.5 microg/mL, and tazobactam/piperacillin (TAZ/PIPC) gave the highest susceptible rate of the breakpoint by Clinical and Laboratory Standards Institute (CLSI) at 93.1%. For the quinolones, ciprofloxacin (CPFX) gave the lowest MIC50 at 0.25 microg/mL, followed by pazufloxacin (PZFX) at 0.5 microg/mL, and levofloxacin (LVFX) at 1 microg/mL, and susceptible rate was 76.0% for CPFX and 73.4% for LVFX. Susceptible rates to amikacin (AMK) and tobramycin (TOB) of aminoglycocides and colistin (CL) of polypeptides were 98.2%, 97.6% and 96.4%. In 334 strains, IMP-1 MBL producing P. aeruginosa was 1 strain, and the strain showed resistance to all antibacterial agents except AMK and CL used in this study. The strains isolated from urine were lower susceptible rate in comparison with those from sputum, notably the susceptible rate to CPFX from urine was less over 30% than those from sputum. Because the results of the susceptibility test against P. aeruginosa were different in each area, it is important for us to pay attention to the susceptibility to antibacterial agents and the emergence of resistance in the clinical strains through continuous susceptibility surveillance.