Overexpression of the multidrug efflux pump SmeDEF impairs Stenotrophomonas maltophilia physiology

OBJECTIVES: The use of antibiotics for the treatment of infectious diseases has led to important changes in the structure of pathogenic bacterial populations. However, these changes could be buffered if the expression of antibiotic resistance genes were to lead to the counter-selection of antibiotic-resistant strains in antibiotic-free environments. To test the effect of antibiotic resistance on bacterial fitness, we analysed the effect of the overproduction of the multidrug efflux pump SmeDEF on the physiology of Stenotrophomonas maltophilia. SmeDEF confers resistance to antibiotics belonging to different structural families, and its overexpression is associated with an antibiotic resistance phenotype in clinical isolates of S. maltophilia. RESULTS: Two S. maltophilia isogenic strains were analysed: the wild-type strain D457 and strain D457R, which is a SmeDEF overproducer. In co-culture experiments, under non-selective pressure the wild-type strain displaced the mutant strain D457R. Metabolic profiling showed that SmeDEF overproduction leads to several changes in S. maltophilia metabolism. Using a Dictyostelium discoideum model of bacterial virulence, we found overexpression of SmeDEF to be associated with a reduction in S. maltophilia virulence. CONCLUSIONS: Together, these data indicate that overexpression of the multidrug efflux pump SmeDEF causes a metabolic burden for S. maltophilia.     

The biocide triclosan selects Stenotrophomonas maltophilia mutants that overproduce the SmeDEF multidrug efflux pump

The possibility that triclosan selects Stenotrophomonas maltophilia mutants overexpressing the multidrug resistance pump SmeDEF is analyzed. Five out of 12 triclosan-selected mutants were less susceptible to antibiotics than the wild-type strain and overproduced SmeDEF. Results are discussed in relation to current debates on the potential selection of antibiotic-resistant bacteria by household biocides.     

Contribution of integrons, and SmeABC and SmeDEF efflux pumps to multidrug resistance in clinical isolates of Stenotrophomonas maltophilia

OBJECTIVES: The contribution of integrons and efflux pumps to multidrug resistance in Stenotrophomonas maltophilia was evaluated. MATERIALS AND METHODS: Ninety-three S. maltophilia clinical isolates were studied. PCR and direct sequencing were used to detect the presence of integrons. Real-time PCR was performed to assess and quantify the expression of the Sme efflux pumps of S. maltophilia. RESULTS: Class 1 integrons were detected in 22% of clinical isolates and carried cassettes conferring resistance mainly to aminoglycosides and trimethoprim. The small multidrug resistance gene, smr, was found on class 1 integrons in six isolates. Thirty-one percent of the isolates overexpressed the smeDEF gene, as compared with a control strain, and 59% overexpressed the smeABC gene. Extrusion of ciprofloxacin and meropenem was specific to the SmeABC and SmeDEF pumps, respectively. CONCLUSION: SmeABC and SmeDEF efflux pumps play important roles in resistance of S. maltophilia to ciprofloxacin and meropenem.     

Contribution of resistance-nodulation-division efflux pump operon smeU1-V-W-U2-X to multidrug resistance of Stenotrophomonas maltophilia

KJ09C, a multidrug-resistant mutant of Stenotrophomonas maltophilia KJ, was generated by in vitro selection with chloramphenicol. The multidrug-resistant phenotype of KJ09C was attributed to overexpression of a resistance nodulation division (RND)-type efflux system encoded by an operon consisting of five genes: smeU1, smeV, smeW, smeU2, and smeX. Proteins encoded by smeV, smeW, and smeX were similar to the membrane fusion protein, RND transporter, and outer membrane protein, respectively, of known RND-type systems. The proteins encoded by smeU1 and smeU2 were found to belong to the family of short-chain dehydrogenases/reductases. Mutant KJ09C exhibited increased resistance to chloramphenicol, quinolones, and tetracyclines and susceptibility to aminoglycosides; susceptibility to β-lactams and erythromycin was not affected. The expression of the smeU1-V-W-U2-X operon was regulated by the divergently transcribed LysR-type regulator gene smeRv. Overexpression of the SmeVWX pump contributed to the acquired resistance to chloramphenicol, quinolones, and tetracyclines. Inactivation of smeV and smeW completely abolished the activity of the SmeVWX pump, whereas inactivation of smeX alone decreased the activity of the SmeVWX pump. The enhanced aminoglycoside susceptibility observed in KJ09C resulted from SmeX overexpression.     

The Efflux Pump SmeDEF Contributes to Trimethoprim-Sulfamethoxazole Resistance in Stenotrophomonas maltophilia

Trimethoprim-sulfamethoxazole (co-trimoxazole) is one of the antimicrobials of choice for the treatment of Stenotrophomonas maltophilia infections. The analysis of mutants either lacking or overexpressing the efflux pump SmeDEF shows that this efflux pump contributes to intrinsic and acquired co-trimoxazole resistance in S. maltophilia. Since SmeDEF can extrude a variety of antibiotics, selection with such antimicrobials, including quinolones, might also select for S. maltophilia co-trimoxazole resistance.     

Multiple antibiotic resistance in Stenotrophomonas maltophilia: involvement of a multidrug efflux system

Clinical strains of Stenotrophomonas maltophilia are often highly resistant to multiple antibiotics, although the mechanisms of resistance are generally poorly understood. Multidrug resistant (MDR) strains were readily selected by plating a sensitive reference strain of the organism individually onto a variety of antibiotics, including tetracycline, chloramphenicol, ciprofloxacin, and norfloxacin. Tetracycline-selected MDR strains typically showed cross-resistance to erythromycin and fluoroquinolones and, in some instances, aminoglycosides. MDR mutants selected with the other agents generally displayed resistance to chloramphenicol and fluoroquinolones only, although two MDR strains (e.g., K1385) were also resistant to erythromycin and hypersusceptible to aminoglycosides. Many of the MDR strains expressed either moderate or high levels of a novel outer membrane protein (OMP) of ca. 50 kDa molecular mass, a phenotype typical of MDR strains of Pseudomonas aeruginosa hyperexpressing drug efflux systems. Indeed, the 50-kDa OMP of these S. maltophilia MDR strains reacted with antibody to OprM, the outer membrane component of the MexAB-OprM MDR efflux system of P. aeruginosa. Similarly, a ca. 110-kDa cytoplasmic membrane protein of these MDR strains also reacted with antibody to the MexB component of the P. aeruginosa pump. The outer and cytoplasmic membranes of several clinical S. maltophilia strains also reacted with the anti-OprM and anti-MexB antibodies. N-terminal amino acid sequencing of a cyanogen bromide-generated peptide of the 50-kDa OMP of MDR strain K1385, dubbed SmeM (Stenotrophomonas multidrug efflux), revealed it to be very similar to a number of outer membrane multidrug efflux components of P. aeruginosa and Pseudomonas putida. Deletion of the L1 and L2 beta-lactamase genes confirmed that these enzymes were responsible for the bulk of the beta-lactam resistance of K1385 and its parent. Still, overexpression of the MDR efflux mechanism in an L1- and L2-deficient derivative of K1385 did yield a modest increase in resistance to a few beta-lactams. These data are consistent with the MDR efflux mechanism(s) playing a role in the multidrug resistance of S. maltophilia.     

嗜麦芽窄食单胞菌对氟喹诺酮类药物主动外排机制的研究

目的 探讨嗜麦芽窄食单胞菌耐药株是否存在主动外排作用,以揭示嗜麦芽窄食单胞菌对氟喹诺酮类药物(FQS)的耐药机制.方法 应用琼脂稀释法检测嗜麦芽窄食单胞菌耐药株和敏感株在加入主动外排泵抑制剂羰基氰氯苯腙(CCCP)后对环丙沙星、左氧氟沙星的最低抑菌浓度(MIC) 变化情况.结果 55株嗜麦芽窄食单胞菌对FQS 耐药率较低,CCCP在体外能增强FQS抗菌活性,主动外排机制既存在于FQS 耐药株,也存在于FQS 敏感株, 对耐药株的影响更大.结论 嗜麦芽窄食单胞菌对FQS耐药与主动外排泵有关,泵抑制剂可部分降低这种耐药性.     

The phenolic diterpene totarol inhibits multidrug efflux pump activity in Staphylococcus aureus

The phenolic diterpene totarol had good antimicrobial activity against effluxing strains of Staphylococcus aureus. Subinhibitory concentrations reduced the MICs of selected antibiotics, suggesting that it may also be an efflux pump inhibitor (EPI). A totarol-resistant mutant that overexpressed norA was created to separate antimicrobial from efflux inhibitory activity. Totarol reduced ethidium efflux from this strain by 50% at 15 microM (1/4x MIC), and combination studies revealed marked reductions in ethidium MICs. These data suggest that totarol is a NorA EPI as well as an antistaphylococcal antimicrobial agent.     

A multidrug efflux pump inhibitor reduces fluoroquinolone resistance in Pseudomonas aeruginosa isolates

In general, resistance to fluoroquinolones (FQs) in gram-negative bacteria is acquired either by mutations in DNA gyrase and topoisomerase IV or by active export of the agents via antibiotic efflux pumps. Reduced porin expression is also proposed to be another mechanism leading to resistance. In this study, interaction between levofloxacin, ofloxacin, and ciprofloxacin with MC-207,110 (multidrug efflux pump inhibitor) was investigated by a checkerboard assay using Pseudomonas aeruginosa. Levofloxacin, ofloxacin, and ciprofloxacin were tested at different concentrations (0.06-64 microg/ml) and MC-207,110 was tested at a concentration range of 4-128 microg/ml. In the presence of MC-207,110 (at 128, 64, 32, 16 microg/ml) resistance to FQs was inhibited significantly and MIC values were decreased, except at 8 and 4 microg/ml of MC-207,110. When MC-207,110 was used, resistance of P. aeruginosa to FQs in vitro was inhibited significantly, suggesting that MC-207,110 may be useful for use in clinical treatment protocols to overcome FQs resistance.     

Phenothiazines and thioxanthenes inhibit multidrug efflux pump activity in Staphylococcus aureus

Efflux-related multidrug resistance (MDR) is a significant means by which bacteria can evade the effects of selected antimicrobial agents. Genome sequencing data suggest that Staphylococcus aureus may possess numerous chromosomally encoded MDR efflux pumps, most of which have not been characterized. Inhibition of these pumps, which may restore clinically relevant activity of antimicrobial agents that are substrates for them, may be an effective alternative to the search for new antimicrobial agents that are not substrates. The inhibitory effects of selected phenothiazines and two geometric stereoisomers of the thioxanthene flupentixol were studied using strains of S. aureus possessing unique efflux-related MDR phenotypes. These compounds had some intrinsic antimicrobial activity and, when combined with common MDR efflux pump substrates, resulted in additive or synergistic interactions. For S. aureus SA-1199B, which overexpresses the NorA MDR efflux pump, and for two additional strains of S. aureus having non-NorA-mediated MDR phenotypes, the 50% inhibitory concentration (IC(50)) for ethidium efflux for all tested compounds was between 4 and 15% of their respective MICs. Transport of other substrates was less susceptible to inhibition; the prochlorperazine IC(50) for acriflavine and pyronin Y efflux by SA-1199B was more than 60% of its MIC. Prochlorperazine and trans(E)-flupentixol were found to reduce the proton motive force (PMF) of S. aureus by way of a reduction in the transmembrane potential. We conclude that the mechanism by which phenothiazines and thioxanthenes inhibit efflux by PMF-dependent pumps is multifactorial and, because of the unbalanced effect of these compounds on the MICs and the efflux of different substrates, may involve an interaction with the pump itself and, to a lesser extent, a reduction in the transmembrane potential.     

Antifolate activity of epigallocatechin gallate against Stenotrophomonas maltophilia

The catechin epigallocatechin gallate, one of the main constituents of green tea, showed strong antibiotic activity against 18 isolates of Stenotrophomonas maltophilia (MIC range, 4 to 256 microg/ml). In elucidating its mechanism of action, we have shown that epigallocatechin gallate is an efficient inhibitor of S. maltophilia dihydrofolate reductase, a strategic enzyme that is considered an attractive target for the development of antibacterial agents. The inhibition of S. maltophilia dihydrofolate reductase by this tea compound was studied and compared with the mechanism of a nonclassical antifolate compound, trimethoprim. Investigation of dihydrofolate reductase was undertaken with both a trimethoprim-susceptible S. maltophilia isolate and an isolate with a high level of resistance. The enzymes were purified using ammonium sulfate precipitation, gel filtration, and methotrexate affinity chromatography. The two isolates showed similar levels of dihydrofolate reductase expression and similar substrate kinetics. However, the dihydrofolate reductase from the trimethoprim-resistant isolate demonstrated decreased susceptibility to inhibition by trimethoprim and epigallocatechin gallate. As with other antifolates, the action of epigallocatechin gallate was synergistic with that of sulfamethoxazole, a drug that blocks folic acid metabolism in bacteria, and the inhibition of bacterial growth was attenuated by including leucovorin in the growth medium. We conclude that the mechanism of action of epigallocatechin gallate on S. maltophilia is related to its antifolate activity.     

Use of an efflux pump inhibitor to determine the prevalence of efflux pump-mediated fluoroquinolone resistance and multidrug resistance in Pseudomonas aeruginosa

Fluoroquinolone-resistance in Pseudomonas aeruginosa may be due to efflux pump overexpression (EPO) and/or target mutations. EPO can result in multidrug resistance (MDR) due to broad substrate specificity of the pumps. MC-04,124, an efflux pump inhibitor (EPI) shown to significantly potentiate activity of levofloxacin in P. aeruginosa, was used to examine the prevalence of EPO in clinical isolates. MICs were determined for ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin with or without EPI and for other antipseudomonal agents by using broth microdilution against P. aeruginosa isolates from adults (n = 119) and children (n = 24). The prevalence of the EPO phenotype (>/=8-fold MIC decrease when tested with EPI) was compared among subgroups with different resistance profiles. The EPO phenotype was more prevalent among levofloxacin-resistant than levofloxacin-sensitive strains (61%, 48/79 versus 9%, 6/64). EPO was present in 60% of fluoroquinolone-resistant strains without cross-resistance, while it was present at variable frequencies among strains with cross-resistance to other agents: piperacillin-tazobactam (86%), ceftazidime (76%), cefepime (65%), imipenem (56%), gentamicin (55%), tobramycin (48%), and amikacin (27%). The magnitude of MIC decrease with an EPI paralleled the frequency of which the EPO phenotype was observed in different subgroups. EPI reduced the levofloxacin MIC by as much as 16-fold in eight strains for which MICs were 128 microg/ml. Efflux-mediated resistance appears to contribute significantly to fluoroquinolone resistance and MDR in P. aeruginosa. Our data support the fact that increased fluoroquinolone usage can negatively impact susceptibility of P. aeruginosa to multiple classes of antipseudomonal agents.