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The swine pathogen Actinobacillus pleuropneumoniae possesses a 75-kDa outer membrane protein (OMP), FhuA, the receptor for ferrichrome, a hydroxamate-type siderophore. Polyclonal serum to FhuA reacted with OMP preparations from 12 serotypes of A. pleuropneumoniae under conditions of iron repletion and restriction. Reverse transcription-PCR confirmed that A. pleuropneumoniae fhuA expression is not upregulated in response to low iron levels. An A. pleuropneumoniae fhuA deletion mutant was generated and showed abolishment of ferrichrome uptake.

ABSTRACTThe swine pathogen Actinobacillus pleuropneumoniaepossesses a 75-kDa outer membrane protein (OMP), FhuA, the receptor for ferrichrome, a hydroxamate-type siderophore. Polyclonal serum to FhuA reacted with OMP preparations from 12 serotypes of A. pleuropneumoniaeunder conditions of iron repletion and restriction. Reverse transcription-PCR confirmed that A. pleuropneumoniae fhuAexpression is not upregulated in response to low iron levels. An A. pleuropneumoniae fhuAdeletion mutant was generated and showed abolishment of ferrichrome uptake.

Pseudomonas aeruginosa is an important opportunistic human pathogen. Certain strains can transmigrate across epithelial cells, and their invasive phenotype is correlated with capacity to cause invasive human disease and fatal septicemia in mice. Four multidrug efflux systems have been described in P. aeruginosa, however, their contribution to virulence is unclear. To clarify the role of efflux systems in invasiveness, P. aeruginosa PAO1 wild-type (WT) and its efflux mutants were evaluated in a Madin-Darby canine kidney (MDCK) epithelial cell monolayer system and in a murine model of endogenous septicemia. All efflux mutants except a ΔmexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT. In particular, a ΔmexAB-oprM deletion strain was compromised in its capacity to invade or transmigrate across MDCK cells, and could not kill mice, in contrast to WT which was highly invasive (P < 0.0006) and caused fatal infection (P < 0.0001). The other mutants, including ΔmexB and ΔmexXY mutants, were intermediate between WT and the ΔmexAB-oprM mutant in invasiveness and murine virulence. Invasiveness was restored to the ΔmexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT. We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.

ABSTRACTPseudomonas aeruginosabiofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological changes that occur during the transition from the planktonic to biofilm mode of growth. The resistance ofP. aeruginosabiofilms to numerous antimicrobial agents that are substrates subject to active efflux from planktonic cells suggests that efflux pumps may substantially contribute to the innate resistance of biofilms. In this study, we investigated the expression of genes associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM and MexCD-OprJ, throughout the course of biofilm development. Using fusions to gfp, we were able to analyze spatial and temporal expression of mexAandmexCin the developing biofilm. Remarkably, expression ofmexAB-oprMand mexCD-oprJwas not upregulated but rather decreased over time in the developing biofilm. Northern blot analysis confirmed that these pumps were not hyperexpressed in the biofilm. Furthermore, spatial differences in mexAB-oprMandmexCD-oprJexpression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the contribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to P. aeruginosabiofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in the antibiotic-resistant phenotype of P. aeruginosabiofilms.

Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance during antibiotic therapy. Much of this resistance is promoted by highly homologous three-component efflux systems of broad substrate specificity, of which four have been identified to date. These include MexA-MexB-OprM and MexX-MexY-OprM, which are expressed constitutively in wild type cells and, thus, provide for intrinsic multidrug resistance, and MexC-MexD-OprJ and MexE-MexF-OprN, whose expression so far has only been seen in acquired multidrug resistant mutant strains. Additional homologues of these efflux systems are identifiable in the recently released genome sequence, though their roles, if any, in antimicrobial efflux are unknown. These tripartite pumps are composed of an integral cytoplasmic membrane drug-proton antiporter of the resistance-nodulation-cell division (RND) family of exporters, a channel-forming outer membrane efflux protein (or outer membrane factor (OMF) and a periplasmic membrane fusion protein (MFP) that links the other two. In addition to a number of antimicrobials of clinical significance, these pumps also export dyes, detergents, disinfectants, organic solvents and acylated homoserine lactones involved in quorum-sensing. While the natural functional of these pumps remains undefined, the fact that they contribute to antimicrobial resistance in P. aeruginosa makes them reasonable targets for therapeutic intervention.

The major diffusion channel in the outer membrane of Haemophilus influenzaetype b (Hib) is porin (341 amino acids; Mr37 782). The Hibporin gene was cloned and overexpressed in Bacillus subtilis. Recombinant Hib porin (Bac porin), having aggregated into inclusion bodies, was purified under denaturing conditions and subsequently refolded. To compare Bac porin that is intrinsically devoid of lipooligosaccharides versus native Hib porin, the properties of Bac porin were assessed by the following four criteria: circular dichroism spectroscopy, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by an anti-Hib porin monoclonal antibody. We conclude that in the absence of lipooligosaccharides, Bac porin was refolded into a functional form which closely resembled the structure of Hib porin.

The major diffusion channel in the outer membrane of Haemophilus influenzaetype b (Hib) is porin (341 amino acids; Mr37 782). The Hibporin gene was cloned and overexpressed in Bacillus subtilis. Recombinant Hib porin (Bac porin), having aggregated into inclusion bodies, was purified under denaturing conditions and subsequently refolded. To compare Bac porin that is intrinsically devoid of lipooligosaccharides versus native Hib porin, the properties of Bac porin were assessed by the following four criteria: circular dichroism spectroscopy, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by an anti‐Hib porin monoclonal antibody. We conclude that in the absence of lipooligosaccharides, Bac porin was refolded into a functional form which closely resembled the structure of Hib porin.

ABSTRACTThe mexCD-oprJand mexAB-oprMoperons encode components of two distinct multidrug efflux pumps inPseudomonas aeruginosa. To assess the contribution of individual components to antibiotic resistance and substrate specificity, these operons and their component genes were cloned and expressed in Escherichia coli. Western immunoblotting confirmed expression of the P. aeruginosaefflux pump components in E. colistrains expressing and deficient in the endogenous multidrug efflux system (AcrAB), although only the ΔacrABstrain, KZM120, demonstrated increased resistance to antibiotics in the presence of the P. aeruginosaefflux genes. E. coliKZM120 expressing MexAB-OprM showed increased resistance to quinolones, chloramphenicol, erythromycin, azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significantly, several β-lactams, which is reminiscent of the operation of this pump in P. aeruginosa. This confirmed previous suggestions that MexAB-OprM provides a direct contribution to β-lactam resistance via the efflux of this group of antibiotics. An increase in antibiotic resistance, however, was not observed when MexAB or OprM alone was expressed in KZM120. Thus, despite the fact that β-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these agents. E. coliKZM120 expressing MexCD-OprJ also showed increased resistance to quinolones, chloramphenicol, macrolides, SDS, and crystal violet, though not to most β-lactams or novobiocin, again somewhat reminiscent of the antibiotic resistance profile of MexCD-OprJ-expressing strains ofP. aeruginosa. Surprisingly, E. coliKZM120 expressing MexCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antibiotic resistance. MexCD-mediated resistance, however, was absent in a tolCmutant of KZM120, indicating that MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosamultidrug efflux pumps are functional and retain their substrate specificity in E. coli.

ABSTRACTTonB couples the energized state of the cytoplasmic membrane to the operation of outer membrane receptors responsible for Fe(III) siderophore uptake across the outer membrane of gram-negative bacteria. A tonBmutant of Pseudomonas aeruginosadeficient in iron siderophore uptake was shown in the present study to be hypersusceptible to a wide variety of antibiotics, reminiscent of the phenotype of mutants defective in the mexAB-oprMantibiotic efflux operon. This was not related to influences of atonBmutation on the iron status of the cell, and indeed, intrinsic antibiotic susceptibility and mexAB-oprMexpression were unaffected by iron levels in the growth medium. The presence of tonBon a multicopy plasmid increased the level of resistance of a MexAB-OprM+strain but not that of a MexAB-OprM−strain to a variety of antimicrobial agents.mexAB-oprMexpression was not, however, altered in atonBdeletion mutant, indicating that any influence of TonB on MexAB-OprM-mediated multidrug resistance was at the level of pump activity. Consistent with this, drug accumulation assays revealed that the tonBdeletion mutant exhibited decreased levels of drug efflux. Still, the multidrug resistance of a nalBstrain was not wholly abrogated by a tonBmutation, indicating that it is likely not an essential component of the efflux apparatus. Similarly, elimination of tonBfrom an nfxBstrain only partially compromised MexCD-OprJ-mediated multidrug resistance. Intriguingly, the drug susceptibility of amexAB-oprMdeletion strain was increased following deletion of tonB, suggesting that TonB may also influence antibiotic resistance mediated by determinants other than MexAB-OprM (and MexCD-OprJ). Thus, TonB plays an important role in both intrinsic and acquired antibiotic resistance in P. aeruginosa.

ABSTRACTA Pseudomonas aeruginosastrain carrying an insertion of an ΩHg interposon in the mexBgene (mexB::ΩHg; strain K879) produced markedly reduced but still detectable levels of OprM, the product of the third gene of the mexAB-oprMmultidrug efflux operon. By using alacZtranscriptional fusion vector, promoter activity likely responsible for OprM expression in themexB::ΩHg mutant was identified upstream ofoprM. Introduction of the oprMgene, but not the mexABgenes, into a P. aeruginosamultidrug-susceptible ΔmexAB-oprMmutant increased resistance to quinolones, cephalosporins, erythromycin, and tetracycline. A ΔmexAB-oprMstrain carrying the oprMgene accumulated markedly less antibiotic than the deletion strain without oprM. Antibiotic accumulation by the MexAB−OprM+strain was markedly enhanced upon treatment of cells with the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that MexAB-independent OprM function likely involves an efflux process. Moreover, pretreatment of cells with CCCP prior to the accumulation assay abrogated any differences in accumulation levels between the MexAB−OprM+and MexAB−OprM−strains, indicating that reduced drug accumulation by the OprM+strain (in the absence of CCCP) cannot be due to OprM-mediated reduction in outer membrane permeability. It appears, therefore, that OprM can be expressed and function in a drug efflux capacity independent of MexAB.

ABSTRACTThe MexAB-OprM multidrug efflux system exports a number of antimicrobial compounds, including β-lactams. In an attempt to define more fully the range of antimicrobial compounds exported by this system, and, in particular, to determine whether β-lactamase inhibitors were also accommodated by the MexAB-OprM pump, the influence of pump status (its presence or absence) on the intrinsic antibacterial activities of these compounds and on their abilities to enhance β-lactam susceptibility in intact cells was assessed. MIC determinations clearly demonstrated that all three compounds tested, clavulanate, cloxacillin, and BRL42715, were accommodated by the pump. Moreover, by using β-lactams which were readily hydrolyzed by thePseudomonas aeruginosaclass C chromosomal β-lactamase, it was demonstrated that elimination of themexAB-oprM-encoded efflux system greatly enhanced the abilities of cloxacillin and BRL42715 (but not clavulanate) to increase β-lactam susceptibility. With β-lactams which were poorly hydrolyzed, however, the inhibitors failed to enhance β-lactam susceptibility in MexAB-OprM+strains, although BRL42715 did enhance β-lactam susceptibility in MexAB-OprM−strains, suggesting that even with poorly hydrolyzed β-lactams this inhibitor was effective when it was not subjected to efflux. MexEF-OprN-overexpressing strains, but not MexCD-OprJ-overexpressing strains, also facilitated resistance to β-lactamase inhibitors, indicating that these compounds are also substrates for the MexEF-OprN pump. These data indicate that an ability to inactivate MexAB-OprM (and like efflux systems in other bacteria) will markedly enhance the efficacies of β-lactam–β-lactamase inhibitor combinations in treating bacterial infections.