Constitutive overexpression of the active efflux system MexXY/OprM is a major cause of resistance to aminoglycosides, fluoroquinolones, and cefepime in clinical strains of Pseudomonas aeruginosa. Upregulation of this pump often results from mutations occurring in mexZ, the local repressor gene of the mexXY operon. In this study, analysis of MexXY-overproducing mutants selected in vitro from reference strain PAO1Bes on amikacin (at a concentration 1.5-fold higher than the MIC) led to identification of a new class of mutants harboring an intact mexZ gene and exhibiting increased resistance to colistin and imipenem in addition to aminoglycosides, fluoroquinolones, and cefepime. Reverse transcription-quantitative PCR (RT-qPCR) experiments on a selected clone named PAOW2 demonstrated that mexXY overexpression was independent of mexZ and the PA5471 gene, which is required for drug-dependent induction of mexXY. Furthermore, the transcript levels of the oprD gene, which encodes the carbapenem-selective porin OprD, were found to be reduced drastically in PAOW2. Whole-genome sequencing revealed a single mutation resulting in an M59I substitution in the ParR protein, the response regulator of the ParRS two-component regulatory system (with ParS being the sensor kinase), which is required for adaptive resistance of P. aeruginosa to polycationic peptides such as colistin. The multidrug resistance phenotype was suppressed in PAOW2 by deletion of the parS and parRS genes and conferred to PAO1Bes by chromosomal insertion of the mutated parRS locus from PAOW2. As shown by transcriptomic analysis, only a very small number of genes were expressed differentially between PAOW2 and PAO1Bes, including the lipopolysaccharide (LPS) modification operon arnBCADTEF-ugd, responsible for resistance to polycationic agents. Exposure of wild-type PAO1Bes to different polycationic peptides, including colistin, was shown to result in increased mexY and repressed oprD expression via ParRS, independent of PA5471. In agreement with these results, colistin antagonized activity of the MexXY/OprM substrates in PAO1Bes but not in a ΔparRS derivative. Finally, screening of clinical strains exhibiting the PAOW2 resistance phenotype allowed the identification of additional alterations in ParRS. Collectively, our data indicate that ParRS may promote either induced or constitutive multidrug resistance to four different classes of antibiotics through the activation of three distinct mechanisms (efflux, porin loss, and LPS modification).
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.
From May 1997 to December 2001, a serotype O:6 multidrug-resistant strain of Pseudomonas aeruginosa colonized or infected 201 patients in the University Hospital of Besançon (France). The susceptibility profile of this epidemic clone to fluoroquinolones and aminoglycosides was relatively stable during the outbreak but showed important isolate-to-isolate variations (up to 64-fold) in the MICs of β-lactams. Analysis of 18 genotypically related isolates selected on a quaterly basis demonstrated alterations in the two DNA topoisomerases II and IV (Thr83→Ile in GyrA and Ser87→Leu in ParC) and production of an ANT(2")-I enzyme. Although constitutively overproduced in these bacteria, the MexXY efflux system did not appear to contribute significantly to aminoglycoside resistance. β-Lactam resistance was associated with derepression of intrinsic AmpC β-lactamase (with isolate-to-isolate variations of up to 58-fold) and sporadic deficiency in a 46-kDa protein identified as the carbapenem-selective porin OprD. Of the 18 isolates, 14 were also found to overproduce the efflux system MexAB-OprM as a result of alteration of the repressor protein MexR (His107→Pro). However, complementation experiments with the cloned mexR gene demonstrated that MexAB-OprM contributed only marginally to β-lactam and fluoroquinolone resistance. Of the four isolates exhibiting wild-type MexAB-OprM expression despite the MexR alteration, two appeared to harbor secondary mutations in the mexA-mexR intergenic region and one harbored secondary mutations in the putative ribosome binding site located upstream of the mexAB oprM operon. In conclusion, this study shows that many mechanisms were involved in the multiresistance phenotype of this highly epidemic strain of P. aeruginosa. Our results also demonstrate that the clone sporadically underwent substantial genetic and phenotypic variations during the course of the outbreak, perhaps in relation to local or individual selective drug pressures.
Aim: To examine the ciprofloxacin susceptibility of 106 Pseudomonas aeruginosa eye isolates from the United Kingdom, Denmark, India, the United States, and Australia, and to determine the molecular mechanisms of resistance.
Methods: Ciprofloxacin susceptibility was tested by an agar dilution method; genomic DNA corresponding to the quinolone target genes gyrA and parC, and the regulatory genes mexR and nfxB controlling drug efflux systems, was amplified by PCR and sequenced; multilocus enzyme electrophoresis was performed to examine the genetic relation among resistant strains.
Results: Three out of 90 keratitis isolates (3.3%), one from the United Kingdom and two from India, exhibited MIC values of 16 mg/l or 32 mg/l. The UK isolate had a mutation in gyrA (Thr83Ile), whereas the two Indian isolates showed mutations in both gyrA (Thr83Ile) and parC (Ser87Leu). The remaining isolates from keratitis, endophthalmitis, contact lens associated red eye (CLARE), and contact lens storage cases showed MIC values below 1 mg/l. Several allelic forms of gyrA and a single variation in the mexR gene product were detected in 10 ciprofloxacin susceptible strains.
Conclusions: The vast majority of eye isolates of P aeruginosa from European countries are fully susceptible to ciprofloxacin and the concentration of ciprofloxacin eye drops used for local treatment (3000 mg/l) exceeds MIC values for strains recorded as resistant. Mutations in more than one target gene were associated with higher MIC values.
Pseudomonas aeruginosa; ciprofloxacin; keratitis
Quinolone antibiotics constitute a clinically successful and widely used class of broad-spectrum antibiotics; however, the emergence and spread of resistance increasingly limits the use of fluoroquinolones in the treatment and management of microbial disease. In this study, we evaluated the quantitative contributions of quinolone target alteration and efflux pump expression to fluoroquinolone resistance in Pseudomonas aeruginosa. We generated isogenic mutations in hot spots of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, and parC and inactivated the efflux regulator genes so as to overexpress the corresponding multidrug resistance (MDR) efflux pumps. We then introduced the respective mutations into the reference strain PA14 singly and in various combinations. Whereas the combined inactivation of two efflux regulator-encoding genes did not lead to resistance levels higher than those obtained by inactivation of only one efflux regulator-encoding gene, the combination of mutations leading to increased efflux and target alteration clearly exhibited an additive effect. This combination of target alteration and overexpression of efflux pumps was commonly observed in clinical P. aeruginosa isolates; however, these two mechanisms were frequently found not to be sufficient to explain the level of fluoroquinolone resistance. Our results suggest that there are additional mechanisms, independent of the expression of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and/or MexXY-OprM efflux pump, that increase ciprofloxacin resistance in isolates with mutations in the QRDRs.
Carbapenems are important agents for the therapy of infections due to multidrug-resistant Pseudomonas aeruginosa; the development of carbapenem resistance hampers effective therapeutic options. To assess the mechanisms leading to resistance, 33 clinical isolates with differing degrees of carbapenem susceptibility were analyzed for the expression of the chromosomal β-lactamase (ampC), the porin that is important for the entry of carbapenems (oprD), and the proteins involved in four efflux systems (mexA, mexC, mexE, and mexX). Real-time reverse transcriptase PCR was performed using primers and fluorescent probes for each of the target genes. The sequencing of regulatory genes (ampR, mexR, nalC, nalD, mexT, and mexZ) was also performed. Diminished expression of oprD was present in all imipenem- and meropenem-resistant isolates but was not required for ertapenem resistance. Increased expression of ampC was not observed in several isolates that were overtly resistant to carbapenems. Increased expression of several efflux systems was observed in many of the carbapenem-resistant isolates. Increased efflux activity correlated with high-level ertapenem resistance and reduced susceptibility to meropenem and aztreonam. Most isolates with increased expression of mexA had mutations affecting nalC and/or nalD. Two isolates with mutations leading to a premature stop codon in mexZ had markedly elevated mexX expressions, although mutations in mexZ were not a prerequisite for overexpression. β-Lactam resistance in clinical isolates of P. aeruginosa is a result of the interplay between diminished production of oprD, increased activity of ampC, and several efflux systems.
Expression of the multidrug efflux system MexC-MexD-OprJ in nfxB mutants of Pseudomonas aeruginosa contributes to resistance to fluoroquinolones and the “fourth-generation” cephems (cefpirome and cefozopran), but not to most β-lactams, including the ordinary cephems (ceftazidime and cefoperazone). nfxB mutants also express a second multidrug efflux system, MexA-MexB-OprM, due to incomplete transcriptional repression of this operon by the mexR gene product. To characterize the contribution of the MexC-MexD-OprJ system to drug resistance in P. aeruginosa, a site-specific deletion method was employed to remove the mexA-mexB-oprM region from the chromosome of wild-type and nfxB strains of P. aeruginosa. Characterization of mutants lacking the mexA-mexB-oprM region clearly indicated that the MexC-MexD-OprJ efflux system is involved in resistance to the ordinary cephems as well as fluoroquinolones and the fourth-generation cephems but not to carbenicillin and aztreonam. Rabbit polyclonal antisera and murine monoclonal antibody against the components of the MexA-MexB-OprM system were prepared and used to demonstrate the reduced production of this efflux system in the nfxB mutants. Consistent with this, transcription of the mexA-mexB-oprM operon decreased in an nfxB mutant. This reduction appears to explain the hypersusceptibility of the nfxB mutant to β-lactams, including ordinary cephems.
The MexXY components of the MexXY-OprM multidrug efflux system of Pseudomonas aeruginosa are encoded by a MexZ repressor-regulated operon that is inducible by antibiotics that target the ribosome. Mutant strains disrupted in a gene, PA5471, were shown to be compromised for drug-inducible mexXY expression and, therefore, MexXY-OprM-mediated antimicrobial resistance. The PA5471 gene was inducible by the same ribosome-targeting agents that induce mexXY expression. Moreover, vector-driven expression of cloned PA5471 was sufficient to promote mexXY expression and MexXY-mediated resistance in the absence of antibiotic exposure, consistent with PA5471 directly or indirectly activating mexXY expression following its own upregulation in response to antibiotics. The requirement for PA5471 for mexXY expression and antimicrobial resistance was, however, obviated in mutants lacking the MexZ repressor of mexXY expression, suggesting that PA5471 directly or indirectly modulates MexZ activity in effecting mexXY expression. While the recruitment of PA5471 and MexXY in response to ribosome disruption by antimicrobials is consistent with their genes playing a role in protecting cells from the adverse consequences of disrupting the translation process, reminiscent of trans-translation, these genes appear to operate independently in their contribution to resistance: mutants defective in trans-translation showed a much more modest (twofold) decrease in resistance to ribosome-targeting agents than those lacking PA5471 or MexXY, and this decrease was observed whether functional PA5471/MexXY was present or not.
At least four broad-spectrum efflux pumps (Mex) are involved in elevated intrinsic antibiotic resistance as well as in acquired multidrug resistance in Pseudomonas aeruginosa. Substrate specificity of the Mex pumps has been shown to be determined by the cytoplasmic membrane component (MexB, MexD, MexF, and MexY) of the tripartite efflux pump system. Alignment of their amino acid sequences with those of the homologous AcrB and AcrD pump proteins of Escherichia coli showed conservation of five charged amino acid residues located in or next to transmembrane segments (TMS). These residues were mutated in the MexF gene by site-directed mutagenesis and replaced by residues of opposite or neutral charge. MexF proteins containing combined D410A and A411G substitutions located in TMS4 were completely inactive. Similarly, the substitutions E417K (next to TMS4) and K951E (TMS10) also caused loss of activity towards all tested antibiotics. The substitution E349K in TMS2 resulted in a MexF mutant protein which was unable to transport trimethoprim and quinolones but retained partial activity for the transport of chloramphenicol. All mutated MexF proteins were expressed at comparable levels when tested by Western blot analysis. It is concluded that charged residues located in or close to TMS are essential for proper function of MexF.
Simultaneous overexpression of the MexAB-OprM and MexXY efflux systems was demonstrated by real-time reverse transcription-PCR and immunoblotting experiments for 12 multiresistant clinical isolates of Pseudomonas aeruginosa. DNA sequencing analysis showed that nine of these strains (named agrZ mutants) harbored mutations in mexZ, the product of which downregulates the expression of the mexXY operon. In addition, 8 of the 12 strains exhibited mutations in genes known to control transcription of the mexAB-oprM operon. Four of them were nalB mutants with alterations in the repressor gene mexR, three of them appeared to be nalC mutants deficient in gene PA3721 and overexpressing gene PA3720, and one strain was a nalB nalC double mutant. For MexAB-OprM as well as for MexXY, no clear correlation could be established between (i) the types of mutations, (ii) the expression level of mexA or mexX, and (iii) resistance to effluxed antibiotics. Finally, three isolates, named agrW mutants, overproduced MexXY and had an intact mexZ gene, and four strains overproduced MexAB-OprM and had intact mexR and PA3721 genes (nalD mutants). These data show that clinical isolates are able to broaden their drug resistance profiles by coexpressing two Mex efflux pumps and suggest the existence of additional regulators for MexAB-OprM and MexXY.
This study investigates the role of active efflux system MexXY in the emergence of aminoglycoside (AG) resistance among cystic fibrosis (CF) isolates of Pseudomonas aeruginosa. Three genotypically related susceptible and resistant (S/R) bacterial pairs and three other AG-resistant CF strains were compared to four non-CF strains moderately resistant to AGs. As demonstrated by immunoblot experiments, pump MexY was strongly overproduced in all of the resistant bacteria. This MexXY upregulation was associated with a 2- to 16-fold increase in the MICs of AGs in the S/R pairs and lower intracellular accumulation of dihydrostreptomycin. Alterations in mexZ, the repressor gene of operon mexXY, were found in all of the AG-resistant CF isolates and in one non-CF strain. Complementation of these bacteria with a plasmid-borne mexZ gene dramatically reduced the MICs of AGs, thus highlighting the role played by MexXY in the development of moderate resistance in CF patients. In contrast, complementation of the three non-CF strains showing wild-type mexZ genes left residual levels of resistance to AGs. These data indicate that a locus different from mexZ may be involved in overproduction of MexXY and that other nonenzymatic mechanisms contribute to AG resistance in P. aeruginosa.
Pan-aminoglycoside-resistant Pseudomonas aeruginosa mutants expressing the mexXY components of the aminoglycoside-accommodating MexXY-OprM multidrug efflux system but lacking mutations in the mexZ gene encoding a repressor of this efflux system and in the mexXY promoter have been reported (S. Fraud and K. Poole, Antimicrob. Agents Chemother. 55:1068–1074, 2011). Genome sequencing of one of these mutants, K2966, revealed the presence of a mutation within the predicted promoter region of the rplU-rpmA operon encoding ribosomal proteins L21 and L27, consistent with an observed 2-fold decrease in expression of this operon in the mutant relative to wild-type P. aeruginosa PAO1. Moreover, correction of the mutation restored rplU-rpmA expression and, significantly, reversed the elevated mexXY expression and pan-aminoglycoside resistance of the mutant. Reduced rplU-rpmA expression was also observed in a second mexXY-expressing pan-aminoglycoside-resistant mutant, K2968, which, however, lacked a mutation in the rplU-rpmA promoter region. Restoration of rplU-rpmA expression in the K2968 mutant following chromosomal integration of the rplU-rpmA operon derived from wild-type P. aeruginosa failed, however, to reverse the elevated mexXY expression and pan-aminoglycoside resistance of this mutant, although it did so for K2966, suggesting that the mutation impacting rplU-rpmA expression in K2968 also impacts other mexXY-related genes. Increased mexXY expression owing to reduced rplU-rpmA expression in K2966 and K2968 was dependent on PA5471, whose expression was also elevated in these mutants. Thus, mutational disruption of ribosome function, by limiting expression of ribosomal constituents, promotes recruitment of mexXY and does so via PA5471, reminiscent of mexXY induction by ribosome-disrupting antimicrobial agents. Interestingly, reduced rplU-rpmA expression was also observed in a mexXY-expressing pan-aminoglycoside-resistant clinical isolate, suggesting that ribosome-perturbing mutations have clinical relevance in the recruitment of the MexXY-OprM aminoglycoside resistance determinant.
Mutations in genes mexR and nalC have previously been shown to drive overexpression of the MexAB-OprM multidrug efflux system in Pseudomonas aeruginosa. A transposon insertion multidrug-resistant mutant of P. aeruginosa overproducing MexAB-OprM was disrupted in yet a third gene, PA3574, encoding a probable repressor of the TetR/AcrR family that we have dubbed NalD. Clinical strains overexpressing MexAB-OprM but lacking mutations in mexR or nalC were also shown to carry mutations in nalD. Moreover, the cloned nalD gene reduced the multidrug resistance and MexAB-OprM expression of the transposon mutant and clinical isolates, highlighting the significance of the nalD mutations vis-à-vis MexAB-OprM overexpression in these isolates.
MexXY is an inducible efflux system that contributes to the natural resistance of Pseudomonas aeruginosa to antibiotics. Experiments involving real-time PCR after reverse transcription in reference strain PAO1 showed concentration-dependent induction of gene mexY by various ribosome inhibitors (e.g., chloramphenicol, tetracycline, macrolides, and aminoglycosides) but not by antibiotics acting on other cellular targets (e.g., β-lactams, fluoroquinolones). Confirming a functional link between the efflux system and the translational machinery, ribosome protection by plasmid-encoded proteins TetO and ErmBP increased the resistance of a ΔmexAB-oprM mutant of PAO1 to tetracycline and erythromycin, respectively, as well as the concentrations of both drugs required to induce mexY. Furthermore, spontaneous mutations resulting in specific resistance to dihydrostreptomycin or spectinomycin also raised the minimal drug concentration for mexXY induction in strain PAO1. While strongly upregulated in a PAO1 mutant defective in gene mexZ (which codes for a putative repressor of operon mexXY), gene mexY remained inducible by agents such as tetracycline, chloramphenicol, and spectinomycin, suggesting additional regulatory loci for mexXY. Altogether, these data demonstrate physiological interplays between MexXY and the ribosome and are suggestive of an alternative function for MexXY beyond antibiotic efflux.
Pseudomonas aeruginosa expresses a low level of the MexAB-OprM efflux pump and shows natural resistance to many structurally and functionally diverse antibiotics. The mutation that has been referred to previously as nfxC expresses an additional efflux pump, MexEF-OprN, exhibiting resistance to fluoroquinolones, imipenem, and chloramphenicol and hypersusceptibility to β-lactam antibiotics. To address the antibiotic specificity of the MexEF-OprN efflux pump, we introduced a plasmid carrying the mexEF-oprN operon into P. aeruginosa lacking the mexAB-oprM operon. The transformants exhibited resistance to fluoroquinolones, trimethoprim, and chloramphenicol but, unlike most nfxC-type mutants, did not show β-lactam hypersusceptibility. The transformants exhibited additional resistance to tetracycline. In the next experiment, we analyzed the MexEF-OprN pump subunit(s) responsible for substrate selectivity by expressing MexE, MexF, OprN, and MexEF in strains lacking MexA, MexB, OprM, and MexAB, respectively. The MexEF-OprM/ΔMexAB transformants exhibited MexEF-OprN-type pump function that rendered the strains resistant to fluoroquinolones and chloramphenicol but did not change susceptibility to β-lactam antibiotics compared with the host strain. The MexAB-OprN/ΔOprM, MexAF-OprM/ΔMexB, and MexEB-OprM/ΔMexA mutants exhibited antibiotic susceptibility indistinguishable from that in the mutant lacking both types of efflux pumps. The results imply that the MexEF-OprM pump selects substrates by a MexEF functional unit. Interestingly, OprN did not link functionally with the MexAB complex, despite the fact that OprM interacted functionally with MexEF.
Screening of a Tn5-Hg insertional library (12,000 clones) constructed in wild-type Pseudomonas aeruginosa strain PAO1 identified four genes (namely, galU, nuoG, mexZ, and rplY) whose disruption individually led to increased resistance to aminoglycosides (means of twofold). Inactivation of these genes was associated with (i) impaired outer membrane uptake, (ii) reduced active transport, (iii) increased MexXY-OprM-mediated active efflux, and (iv) alteration of target of aminoglycosides, respectively. In addition, suppression of the gene rplY, which codes for ribosomal protein L25, was found to result in both moderate upregulation of the efflux system MexXY-OprM and hypersusceptibility to β-lactam antibiotics. Construction of double, triple, and quadruple mutants demonstrated cumulative effects of the different mechanisms on aminoglycoside resistance, with MICs increasing from 16- to 64-fold in the quadruple mutant compared to the wild-type strain PAO1. Altogether, these results illustrate how P. aeruginosa may gradually develop high resistance to these antibiotics via intrinsic (i.e., nonenzymatic) mechanisms, as in cystic fibrosis patients.
Several nalB-type multidrug-resistant mutants of Pseudomonas aeruginosa overexpressed MexAB-OprM and carried mutations in the local regulatory gene, mexR. Others, dubbed nalC types, carried mutations elsewhere and overexpressed MexAB-OprM less extensively than the nalB strains. Available evidence showed that MexR acted solely as repressor. Disruption of the mexR gene at various places suggested that the 5′ end of mexR may be a part of the mexAB-oprM promoter.
A mutant, named 11B, hypersusceptible to aminoglycosides, tetracycline, and erythromycin was isolated after Tn501 insertion mutagenesis of Pseudomonas aeruginosa PAO1. Cloning and sequencing experiments showed that 11B was deficient in an, at that time, unknown active efflux system that contains homologs of MexAB. This locus also contained a putative regulatory gene, mexZ, transcribed divergently from the efflux operon. Introduction of a recombinant plasmid that carries the genes of the efflux system restored the resistance of 11B to parental levels, whereas overexpression of these genes strongly increased the MICs of substrate antibiotics for the PAO1 host. Antibiotic accumulation studies confirmed that this new system is an energy-dependent active efflux system that pumps out aminoglycosides. Furthermore, this system appeared to function with an outer membrane protein, OprM. While the present paper was being written and reviewed, genes with a sequence identical to our pump genes, mexXY of P. aeruginosa, have been reported to increase resistance to erythromycin, fluoroquinolones, and organic cations in Escherichia coli hosts, although efflux of aminoglycosides was not examined (Mine et al., Antimicrob. Agents Chemother. 43:415–417, 1999). Our study thus shows that the MexXY system plays an important role in the intrinsic resistance of P. aeruginosa to aminoglycosides. Although overexpression of MexXY increased the level of resistance to fluoroquinolones, disruption of the mexXY operon in P. aeruginosa had no detectable effect on susceptibility to these agents.
The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems. To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a ΔmexB strain. Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the mexB genes of a representative 46 were sequenced, and 19 unique single mutations were identified. While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops. By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from Escherichia coli, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it. Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition. Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction. As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.
Retrospective analysis of 189 nonredundant strains of Pseudomonas aeruginosa sequentially recovered from the sputum samples of 46 cystic fibrosis (CF) patients over a 10-year period (1998 to 2007) revealed that 53 out of 189 (28%) samples were hypersusceptible to the β-lactam antibiotic ticarcillin (MIC ≤ 4 μg/ml) (phenotype dubbed Tichs). As evidenced by trans-complementation and gene inactivation experiments, the mutational upregulation of the efflux system MexXY was responsible for various degrees of resistance to aminoglycosides in a selection of 11 genotypically distinct strains (gentamicin MICs from 2 to 64 μg/ml). By demonstrating for the first time that the MexXY pump may evolve in CF strains, we found that a mutation leading to an F1018L change in the resistance-nodulation-cell division (RND) transporter MexY was able to increase pump-promoted resistance to aminoglycosides, cefepime, and fluoroquinolones twofold. The inactivation of the mexB gene (which codes for the RND transporter MexB) in the 11 selected strains showed that the Tichs phenotype was due to a mutational or functional loss of function of MexAB-OprM, the multidrug efflux system known to contribute to the natural resistance of P. aeruginosa to β-lactams (e.g., ticarcillin and aztreonam), fluoroquinolones, tetracycline, and novobiocin. Two of the selected strains synthesized abnormally low amounts of the MexB protein, and 3 of 11 strains expressed truncated MexB (n = 2) or MexA (n = 1) polypeptide as a result of mutations in the corresponding genes, while 7 of 11 strains produced wild-type though nonfunctional MexAB-OprM pumps at levels similar to or even higher than that of reference strain PAO1. Overall, our data indicate that while MexXY is necessary for P. aeruginosa to adapt to the hostile environment of the CF lung, the MexAB-OprM pump is dispensable and tends to be lost or inactivated in subpopulations of P. aeruginosa.
In this study, we investigated the resistance mechanisms to fluoroquinolones of 85 non-cystic fibrosis strains of Pseudomonas aeruginosa exhibiting a reduced susceptibility to ciprofloxacin (MICs from 0.25 to 2 μg/ml). In addition to MexAB-OprM (31 of 85 isolates) and MexXY/OprM (39 of 85), the MexEF-OprN efflux pump (10 of 85) was found to be commonly upregulated in this population that is considered susceptible or of intermediate susceptibility to ciprofloxacin, according to current breakpoints. Analysis of the 10 MexEF-OprN overproducers (nfxC mutants) revealed the presence of various mutations in the mexT (2 isolates), mexS (5 isolates), and/or mvaT (2 isolates) genes, the inactivation of which is known to increase the expression of the mexEF-oprN operon in reference strain PAO1-UW. However, these genes were intact in 3 of 10 of the clinical strains. Interestingly, ciprofloxacin at 2 μg/ml or 4 μg/ml preferentially selected nfxC mutants from wild-type clinical strains (n = 10 isolates) and from first-step mutants (n = 10) overexpressing Mex pumps, thus indicating that MexEF-OprN represents a major mechanism by which P. aeruginosa may acquire higher resistance levels to fluoroquinolones. These data support the notion that the nfxC mutants may be more prevalent in the clinical setting than anticipated and strongly suggest the involvement of still unknown genes in the regulation of this efflux system.
Mutations in mexR yield a multidrug resistance phenotype in nalB mutants of Pseudomonas aeruginosa as a result of derepression of the mexAB-oprM multidrug efflux operon. MexR produced by several nalB strains carried single amino acid changes that compromised MexR stability or its ability to dimerize. Changes at residues L95 and R21, however, produced a stable MexR protein capable of dimerization and, thus, likely compromised DNA binding.
Expression of the mexXY multidrug efflux operon in wild type Pseudomonas aeruginosa is substantially enhanced by the ribosome-targeting antimicrobial spectinomycin (18-fold) and this is wholly dependent upon the product of the PA5471 gene. In a mutant strain lacking the mexZ gene encoding a repressor of mexXY gene expression, expression of the efflux operon increases modestly (5-fold) and is still responsive (18-fold) to spectinomycin. Spectinomycin induction of mexXY expression in the mexZ mutant is, however, independent of PA5471 suggesting that PA5471 functions as an anti-repressor (dubbed ArmZ for anti-repressor MexZ) that serves only to modulate MexZ's repressor activity, with additional gene(s)/gene product(s) providing for the bulk of the antimicrobial-inducible mexXY expression. Consistent with PA5471/ArmZ functioning as a MexZ anti-repressor, an interaction between MexZ and ArmZ was confirmed using a bacterial 2-hybrid assay. Mutations compromising this interaction (P68S, G76S, R216C, R221W, R221Q, G231D and G252S) were identified and localized to one region of an ArmZ structural model that may represent a MexZ-interacting domain. Introduction of representative mutations into the chromosome of P. aeruginosa reduced (P68S, G76S) or obviated (R216C, R2211W) antimicrobial induction of mexXY gene expression, rendering the mutants pan-aminoglycoside-susceptible. These data confirm the importance of an ArmZ-MexZ interaction for antimicrobial-inducible mexXY expression and intrinsic aminoglycoside resistance in P. aeruginosa.
In order to define the contributions of the mechanisms for carbapenem resistance in clinical strains of Pseudomonas aeruginosa, we investigated the presence of OprD, the expressions of the MexAB-OprM and MexEF-OprN systems, and the production of the β-lactamases for 44 clinical strains. All of the carbapenem-resistant isolates showed the loss of or decreased levels of OprD. Three strains overexpressed the MexAB-OprM efflux system by carrying mutations in mexR. These three strains had the amino acid substitution in MexR protein, Arg (CGG) → Gln (CAG), at the position of amino acid 70. None of the isolates, however, expressed the MexEF-OprN efflux system. For the characterization of β-lactamases, at least 13 isolates were the depressed mutants, and 12 strains produced secondary β-lactamases. Based on the above resistance mechanisms, the MICs of carbapenem for the isolates were analyzed. The MICs of carbapenem were mostly determined by the expression of OprD. The MICs of meropenem were two- to four-fold increased for the isolates which overexpressed MexAB-OprM in the background of OprD loss. However, the elevated MICs of meropenem for some individual isolates could not be explained. These findings suggested that other resistance mechanisms would play a role in meropenem resistance in clinical isolates of P. aeruginosa.
Streptococcus pneumoniae is the leading cause of community-acquired pneumonia (CAP). High global incidence of macrolide and penicillin resistance has been reported, whereas fluoroquinolone resistance is uncommon. Current guidelines for suspected CAP in patients with co-morbidity factors and recent antibiotic therapy recommend initial empiric therapy using one fluoroquinolone or one macrolide associated to other drugs (amoxicillin, amoxicillin/clavulanate, broad-spectrum cephalosporins). Resistance to fluoroquinolones is determined by efflux mechanisms and/or mutations in the parC and parE genes coding for topoisomerase IV and/or gyrA and gyrB genes coding for DNA gyrase. No clinical cases due to fluoroquinolone-resistant S. pneumoniae strains have been yet reported from Italy.
A 72-year-old patient with long history of chronic obstructive pulmonary disease and multiple fluoroquinolone treatments for recurrent lower respiratory tract infections developed fever, increased sputum production, and dyspnea. He was treated with oral levofloxacin (500 mg bid). Three days later, because of acute respiratory insufficiency, the patient was hospitalized. Levofloxacin treatment was supplemented with piperacillin/tazobactam. Microbiological tests detected a S. pneumoniae strain intermediate to penicillin (MIC, 1 mg/L) and resistant to macrolides (MIC >256 mg/L) and fluoroquinolones (MIC >32 mg/L). Point mutations were detected in gyrA (Ser81-Phe), parE (Ile460-Val), and parC gene (Ser79-Phe; Lys137-Asn). Complete clinical response followed treatment with piperacillin/tazobactam.
This is the first Italian case of community-acquired pneumonia due to a fluoroquinolone-resistant S. pneumoniae isolate where treatment failure of levofloxacin was documented. Molecular analysis showed a group of mutations that have not yet been reported from Italy and has been detected only twice in Europe. Treatment with piperacillin/tazobactam appears an effective means to inhibit fluoroquinolone-resistant strains of S. pneumoniae causing community-acquired pneumonia in seriously ill patients.