FpvA-mediated ferric pyoverdine uptake in Pseudomonas aeruginosa: identification of aromatic residues in FpvA implicated in ferric pyoverdine binding and transport.

A number of aromatic residues were seen to cluster in the upper portion of the three-dimensional structure of the FpvA ferric pyoverdine receptor of Pseudomonas aeruginosa, reminiscent of the aromatic binding pocket for ferrichrome in the FhuA receptor of Escherichia coli. Alanine substitutions in three of these, W362, W391, and F795, markedly compromised ferric pyoverdine binding and transport, consistent with a role of FpvA in ferric pyoverdine recognition.

Optimization of meropenem minimum concentration/MIC ratio to suppress in vitro resistance of Pseudomonas aeruginosa.

Suppression of resistance in a dense Pseudomonas aeruginosa population has previously been shown with optimized quinolone exposures. However, the relevance to beta-lactams is unknown. We investigated the bactericidal activity of meropenem and its propensity to suppress P. aeruginosa resistance in an in vitro hollow-fiber infection model (HFIM). Two isogenic strains of P. aeruginosa (wild type and an AmpC stably derepressed mutant [MIC = 1 mg/liter]) were used. An HFIM inoculated with approximately 1 x 10(8) CFU/ml of bacteria was subjected to various meropenem exposures. Maintenance doses were given every 8 h to simulate the maximum concentration achieved after a 1-g dose in all regimens, but escalating unbound minimum concentrations (C(min)s) were simulated with different clearances. Serial samples were obtained over 5 days to quantify the meropenem concentrations, the total bacterial population, and subpopulations with reduced susceptibilities to meropenem (>3x the MIC). For both strains, a significant bacterial burden reduction was seen with all regimens at 24 h. Regrowth was apparent after 3 days, with the C(min)/MIC ratio being < or = 1.7 (time above the MIC, 100%). Selective amplification of subpopulations with reduced susceptibilities to meropenem was suppressed with a C(min)/MIC of > or = 6.2 or by adding tobramycin to meropenem (C(min)/MIC = 1.7). Investigations that were longer than 24 h and that used high inocula may be necessary to fully evaluate the relationship between drug exposures and the likelihood of resistance suppression. These results suggest that the C(min)/MIC of meropenem can be optimized to suppress the emergence of non-plasmid-mediated P. aeruginosa resistance. Our in vitro data support the use of an extended duration of meropenem infusion for the treatment of severe nosocomial infections in combination with an aminoglycoside.

Mutations in PA2491 (mexS) promote MexT-dependent mexEF-oprN expression and multidrug resistance in a clinical strain of Pseudomonas aeruginosa.

Disruption of the PA2491 gene in a mini-Tn5-tet insertion mutant of a clinical isolate of Pseudomonas aeruginosa increased expression of the mexEF-oprN multidrug efflux genes and decreased production of outer membrane protein OprD, concomitant with enhanced resistance to chloramphenicol, quinolones, and imipenem, which was reminiscent of previously described nfxC mutants. PA2491 encodes a probable oxidoreductase previously shown to be positively regulated by the MexT positive regulator of mexEF-oprN expression (T. Kohler, S. F. Epp, L. K. Curty, and J. C. Pechere, J. Bacteriol. 181:6300-6305, 1999). Spontaneous multidrug-resistant mutants of the P. aeruginosa clinical isolate hyperexpressing mexEF-oprN and showing reduced production of OprD were readily selected in vitro, and all of them were shown to carry mutations in PA2491, highlighting the probable significance of such mutations as determinants of MexEF-OprN-mediated multidrug resistance in vivo.

The Pseudomonas aeruginosa tonB gene encodes a novel TonB protein.

The Pseudomonas aeruginosa tonB gene was cloned by complementation of the tonB mutation of Pseudomonas putida strain TE516 (W. Bitter, J. Tommassen & P.J. Weisbeek, 1993, Mol Microbiol 7, 117-130). The gene was 1025 bp in length, capable of encoding a protein of 36860 Da. As with previously described TonB proteins, the P. aeruginosa TonB (TonBp.a.) was rich in Pro residues (18.1%) and contained Glu-Pro/Lys-Pro repeats. Unlike previously described TonB proteins, however, TonBp.a. lacked an N-terminal membrane anchor (signal) sequence and contained, instead, a predicted internal signal/anchor sequence, expected to yield an atypical N-terminal cytoplasmic domain in this protein. TonB proteins are essential components in iron-siderophore uptake in bacteria, apparently functioning as energy transducers in coupling the energized state of the cytoplasmic membrane to outer-membrane receptor function. As expected, tonB derivatives of P. aeruginosa were defective in siderophore-mediated iron acquisition. tonB gene expression was inducible by iron-limitation, consistent with the identification of a Fur consensus binding sequence upstream of the gene. TonBp.a. showed substantially greater similarity to the Escherichia coli TonB protein than the Pseudomonas putida protein (31% identity vs. 20% identity) and tonBp.a. was able to complement deficiencies in the acquisition of ferric enterobactin and vitamin B12, and sensitivity to phage phi 80 of an E. coli tonB strain. The larger size of TonBp.a. and its ability to function in both E. coli and P. putida make it a unique TonB protein whose characterization should enhance our understanding of TonB function in bacteria.