An outer membrane protein of 50 kDa (OprK) was overproduced in a siderophore-deficient mutant of Pseudomonas aeruginosa capable of growth on iron-deficient minimal medium containing 2,2'-dipyridyl (0.5 mM). The expression of OprK in the mutant (strain K385) was associated with enhanced resistance to a number of antimicrobial agents, including ciprofloxacin, nalidixic acid, tetracycline, chloramphenicol, and streptonigrin. OprK was inducible in the parent strain by growth under severe iron limitation, as provided, for example, by the addition of dipyridyl or ZnSO4 to the growth medium. The gene encoding OprK (previously identified as ORFC) forms part of an operon composed of three genes (ORFABC) implicated in the secretion of the siderophore pyoverdine. Mutants defective in ORFA, ORFB, or ORFC exhibited enhanced susceptibility to tetracycline, chloramphenicol, ciprofloxacin, streptonigrin, and dipyridyl, consistent with a role for the ORFABC operon in multiple antibiotic resistance in P. aeruginosa. Sequence analysis of ORFC (oprK) revealed that its product is homologous to a class of outer membrane proteins involved in export. Similarly, the products of ORFA and ORFB exhibit homology to previously described bacterial export proteins located in the cytoplasmic membrane. These data suggest that ORFA-ORFB-oprK (ORFC)-dependent drug efflux contributes to multiple antibiotic resistance in P. aeruginosa. We propose, therefore, the designation mexAB (multiple efflux) for ORFAB.

OprJ, overproduced in nfxB multidrug-resistant strains of Pseudomonas aeruginosa, and OprK, overproduced in the multidrug-resistant strain K385, were demonstrated to be immunologically cross-reactive using an OprJ-specific monoclonal antibody. Treatment of the purified proteins with trypsin or chymotrypsin yielded virtually indistinguishable digestion patterns, and the N-terminal sequence of two trypsin fragments was identical for both proteins, indicating that OprJ and OprK share identity. The N-terminal amino acid sequences were used to facilitate cloning of the oprJ gene on a 5kbp Kpnl fragment and a 10 kbp BamHl fragment. Nucleotide sequencing of portions of these fragments revealed that oprJ was the terminal gene in a putative three-gene operon, mexC-mexD-oprJ. The predicted mexC-mexD-oprJ gene products exhibit homology to the MexA-MexB-OprM components of the multidrug-resistance efflux pump of P. aeruginosa (43-46% identity). Consistent with an implied role for mexC-mexD-oprJ in drug efflux, the mexC-mexD-oprJ-hyperexpressing strain K385 showed reduced accumulation of a variety of antibiotics as compared with its parent strain, and this drug 'exclusion' was abrogated by energy inhibitors. The mexC and oprJ products are putative lipoproteins of a molecular mass of 40,707 and 51,742 Da, respectively, while mexD was predicted to encode a protein of 111 936 Da. Sequencing upstream of mexC revealed the presence of the nfxB gene transcribed divergently from the efflux genes. Overproduction of OprJ and the attendant multiple-antibiotic resistance of strain K385 was shown to result from a point mutation in nfxB, resulting in a H87-->R change in the predicted NfxB polypeptide. OprJ overproduction and multidrug resistance in K385 was reversed by the cloned nfxB gene, suggesting that nfxB encodes a repressor of mexC-mexD-oprJ expression. Consistent with this, the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli. nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression. These data indicate that the multidrug resistance of nfxB strains is due to overexpression of an efflux operon, mexC-mexD-oprJ, encoding components of a second efflux pump in P. aeruginosa.

The region upstream of the multiple antibiotic resistance efflux operon mexA-mexB-oprM in Pseudomonas aeruginosa was sequenced, and a gene, mexR, was identified. The predicted MexR product contains 147 amino acids with a molecular mass of 16,964 Da, which is consistent with the observed size of the overexpressed mexR gene product. MexR was homologous to MarR, the repressor of MarA-dependent multidrug resistance in Escherichia coli, and other repressors of the MarR family. A mexR knockout mutant showed a twofold increase in expression of both plasmid-borne and chromosomal mexA-reporter gene fusions compared with the MexR+ parent strain, indicating that the mexR gene product negatively regulates expression of the mexA-mexB-oprM operon. Furthermore, the cloned mexR gene product reduced expression of a plasmid-borne mexA-lacZ fusion in E. coli, indicating that MexR represses mexA-mexB-oprM expression directly. Consistent with the increased expression of the efflux operon in the mexR mutant, the mutant showed an increase (relative to its MexR+ parent) in resistance to several antimicrobial agents. Expression of a mexR-lacZ fusion increased threefold in a mexR knockout mutant, indicating that mexR is negatively autoregulated. OCR1, a nalB multidrug-resistant mutant which overproduces OprM, exhibited a greater than sevenfold increase in expression of a chromosomal mexA-phoA fusion compared with its parent. Introduction of a mexR knockout mutation in strain OCR1 eliminated this increase in efflux gene expression and, as expected, increased the susceptibility of the strain to a variety of antibiotics. The nucleotide sequences of the mexR genes of OCR1 and its parental strain revealed a single base substitution in the former which would cause a predicted substitution of Trp for Arg at position 69 of its mexR product. These data suggest that MexR possesses both repressor and activator function in vivo, the activator form being favored in nalB multidrug-resistant strains.

Pseudomonas aeruginosa strain K437 is defective in the production of a 90kDa ferripyoverdine receptor and is unable to grow in an iron-deficient medium in the presence of the non-metabolizable iron chelator 2,2'-dipyridyl (0.25 mM). An attempt to clone the ferripyoverdine receptor gene was made by complementing this growth defect. A number of clones restoring growth of K437 on dipyridyl-containing medium were obtained and several of these restored moderate expression of the 90 kDa receptor. A 5.5 kb xhoI-HindIII fragment derived from one of these clones was similarly capable of complementing the dipyridyl growth defect although it failed to restore expression of the 90 kDa ferripyoverdine receptor. Nucleotide sequencing of the 5.5 kb fragment revealed two large open reading frames (ORFs), designated ORFA and ORFB, which appeared to form an operon and were capable of encoding products of 41 kDa and 112 kDa, respectively. Using a phage T7-based expression system, products of 42 kDa and c. 108 kDa were produced from the cloned DNA, confirming that the ORFs were, indeed, expressed. The cloned ORFAB operon was inducible under conditions of iron limitation in both P. aeruginosa and Escherichia coli. In addition, mutants expressing ORFAB constitutively were constitutive for pyoverdine and ferripyoverdine receptor production suggesting that components of the pyoverdine-mediated iron-transport system are co-regulated with ORFAB. The predicted products of ORFA and ORFB showed significant homology to the Escherichia coli EnvC and EnvD polypeptides which are reportedly involved in septum formation. In addition, the ORFB product showed moderate homology to the CzcA polypeptide identified as a component of a membrane-associated plasmid-encoded cation efflux system in Alcaligenes eutrophus. Using in vitro mutagenesis and gene replacement, ORFA- and ORFB-deficient mutants of K372, the parent strain of K437, were constructed. These mutants were unable to grow on iron-deficient minimal medium containing 0.25 mM dipyridyl although they expressed the ferripyoverdine receptor and were proficient in pyoverdine-mediated iron uptake. Despite the homology of the ORFA and ORFB products to EnvC and EnvD, respectively, the ORFA-ORFB-deficient mutants were not defective in septum formation.(ABSTRACT TRUNCATED AT 400 WORDS)

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. Köhler, S. F. Epp, L. K. Curty, and J. C. Pechére, J. Bacteriol. 181:6300-6305, 1999). Spontaneous multidrugresistant 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.Three-component multidrug efflux systems of the resistancenodulation-division (RND) family are prevalent in gram-negative bacteria, in which they contribute significantly to intrinsic and acquired resistance to a range of clinically important antimicrobial agents (i.e., antibiotics and biocides) (48). In Pseudomonas aeruginosa, an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobial agents (19), seven tripartite RND family efflux systems have been described to date 35,50,51 [25,52,54,64,73] and mexZ [40,69], respectively).The MexEF-OprN system is apparently quiescent in wildtype cells, at least under the usual laboratory growth conditions (32), but it is expressed in nfxC-type multidrug-resistant strains isolated in vitro (14, 32, 42) and in clinics (15,24). nfxC mutants display resistance to fluoroquinolones, chloramphenicol, trimethoprim, and the carbapenem imipenem (14, 32), although resistance to imipenem results not from MexEF-OprN expression (32) but from the concomitant decrease in outer membrane protein OprD in these mutants (14,42). OprD is a basic amino acid-peptide channel and a primary route of entry for carbapenems, such as imipenem, in P. aeruginosa (67, 68), and it is often absent in imipenem-resistant strains of P. aeruginosa (4, 31). In addition to its role in the export of and resistance to antimicrobials, MexEF-OprN also promotes resistance (or tolerance) to organic solvents (36), dyes (16), and biocides, such as triclosan (8). As a result of MexEF-OprN hyperexpression, nfxC mutants also express reduced levels of several quorum-sensing (i.e., homoserine lactone)-dependent extracellular virulence factors (33) and are attenuated for virulence (10), apparently as a result of MexEF-OprN-mediated export of a molecule(s) necessary for homoserine lactone production (33).Unlike the majority of RND-type efflux systems in P. aeruginosa, which are negatively regulated by linked repressor genes, MexEF-OprN expression is positively regulated by the product of the linked mexT gene, a LysR family regulator of mexEFoprN expr...

Pseudomonas aeruginosa K437 lacks the ferripyoverdine receptor and, as a result, grows poorly on an iron-deficient minimal medium supplemented with ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and pyroverdine. By using a phagemid-based in vivo cloning system, attempts were made to clone the receptor gene by complementing this growth defect. Several recombinant phagemids carrying P. aeruginosa chromosomal DNA which provided for good growth on EDDHA-pyoverdine-containing medium and which concomitantly restored production of the ferripyroverdine receptor in strain K437 were isolated. These phagemids contained a common 4.6-kb SphI fragment which similarly restored production of the receptor in K437. Nucleotide sequencing of the SphI fragment revealed a single large open reading frame, designated fpvA (ferripyoverdine uptake), of 2439 bp. The predicted translation product of fpvA has a molecular mass of 89,395 Da. N-terminal amino acid sequence analysis of the purified ferripyoverdine receptor confirmed fpvA as the receptor gene. Moreover, it indicated that the receptor is initially synthesized as a precursor with a signal sequence of 27 amino acids which is cleaved to yield the mature protein. The deduced FpvA polypeptide exhibited homology to regions shown to be conserved in TonB-dependent receptor proteins. FpvA also shared strong homology (41.3% identity) with the PupA protein of Pseudomonas putida WCS358. This protein is the receptor for the iron-bound form of pseudobactin, a compound structurally very similar to pyoverdine.

Suppression of resistance in a dense Pseudomonas aeruginosa population has previously been shown with optimized quinolone exposures. However, the relevance to ␤-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 ؋ 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 (>3؋ 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 <1.7 (time above the MIC, 100%). Selective amplification of subpopulations with reduced susceptibilities to meropenem was suppressed with a C min /MIC of >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.Bacterial resistance is a rapidly spreading and serious problem that threatens our therapeutic armamentarium. Given that the drug development process takes many years, it is imperative that the utilities of currently available agents be preserved through the judicious and optimal use of these agents. It has been shown that suboptimal dosing represents a selective pressure that is imposed on the bacteria and that facilitates the emergence of resistance (9, 11). On the other hand, all bacterial subpopulations are killed with optimal dosing, which results in the sustained suppression of both total and resistant populations over time. It has also previously been shown that the emergence of resistance in Pseudomonas aeruginosa could be suppressed by optimizing the exposure of quinolones (11, 28). However, it is less certain if the same is true for the ␤-lactam antibiotics.The pharmacodynamics of ␤-lactams have been relatively well elucidated. The time above the MIC (T Ͼ MIC) of the pathogen has repeatedly been shown to be the pharmacodynamic variable most closely linked to bactericidal activity (2, 21). However, the breakpoint of optimal activity is controversial, and none of the ...

A pyoverdine-deficient strain of Pseudomonas aeruginosa was unable to grow in an iron-deficient minimal medium in the presence of the nonmetabolizable iron chelator ethylene diamine-di(omega-hydroxyphenol acetic acid) (EDDHA), although addition of enterobactin to EDDHA-containing minimal media did restore growth of the pyoverdine-deficient P. aeruginosa. Consistent with the apparent ability of enterobactin to provide iron to P. aeruginosa, enterobactin-dependent 55Fe3+ uptake was observed in cells of P. aeruginosa previously grown in an iron-deficient medium containing enterobactin (or enterobactin-containing Escherichia coli culture supernatant). This uptake was energy dependent, was observable at low concentrations (60 nM) of FeCl3, and was absent in cells cultured without enterobactin. A novel protein with a molecular weight of approximately 80,000 was identified in the outer membranes of cells grown in iron-deficient minimal medium containing enterobactin, concomitant with the induction of enterobactin-dependent iron uptake. A Tn501 insertion mutant lacking this protein was isolated and shown to be deficient in enterobactin-mediated iron transport at 60 nM FeCl3, although it still exhibited enterobactin-dependent growth in iron-deficient medium containing EDDHA. It was subsequently observed that the mutant was, however, capable of enterobactin-mediated iron transport at much higher concentrations (600 nM) of FeCl3. Indeed, enterobactin-dependent iron uptake at this concentration of iron was observed in both the mutant and parent strains irrespective of whether they had been cultured in the presence of enterobactin.(ABSTRACT TRUNCATED AT 250 WORDS)