人型支原体ParE基因突变与耐氟喹诺酮类药物的关系

目的探讨人型支原体对氟喹诺酮类药物的耐药机制。方法自临床分离的8株耐氟喹诺酮类药物的人型支原体（Mh）,对其Pa正基因PCR扩增后进行测序分析,与基因库中的野生型菌株MHPG21基因序列比对,分析ParE基因突变位点与菌株耐氟喹诺酮类药物的关系。结果与野生株MHPG21对比,6株检出ParE基因所编码的氨基酸残基发生D426-N变异。结论Mh临床分离株对氟喹诺酮类药物的耐药可能与ParE基因所编码的氨基酸残基D426→N变异有关。     

Evidence for active drug efflux in fluoroquinolone resistance in Clostridium hathewayi

BACKGROUND: Most fluoroquinolones have shown limited effectiveness against anaerobic bacteria. Evidence for a multidrug efflux pump, like those involved in fluoroquinolone resistance in some other bacteria, was investigated in Clostridium hathewayi. METHODS:A parent strain of C.hathewayi was isolated from human intestinal microflora on a medium with a low concentration of norfloxacin and a mutant strain was selected from it on a medium with a high concentration of norfloxacin. Fluoroquinolone sensitivity, drug accumulation, and the effects of different concentrations of fluoroquinolones on the kinetics of growth in the presence and absence of efflux pump inhibitors were measured. RESULTS: Both strains were resistant to several fluoroquinolones and dyes. The pump inhibitor reserpine increased the sensitivity of both strains to some drugs; it affected the growth kinetics and the efflux of norfloxacin and ethidium bromide. CONCLUSION: The efflux of fluoroquinolone appears to be one reason for fluoroquinolone resistance inC. hathewayi.     

Characterization of Mycoplasma hominis mutations involved in resistance to fluoroquinolones.

Fluoroquinolone-resistant mutants of Mycoplasma hominis were selected in vitro from the PG21 susceptible reference strain either by multistep selection on increasing concentrations of various fluoroquinolones or by one-step selection on agar medium with ofloxacin. The quinolone resistance-determining regions (QRDR) of the structural genes encoding the A and b subunits of DNA gyrase were amplified by PCR, and the nucleotide sequences of eight multistep-selected resistant strains were compared to those of susceptible strain PG21. Four high-level resistant mutants that were selected on norfloxacin or ofloxacin contained a C-to-T transition in the gyrA QRDR, leading to substitution of Ser-83 by Leu in the GyrA protein. Analysis of the sequence of the gyrB QRDR of the eight multistep-selected mutants did not reveal any difference compared to that of the gyrB QRDR of the reference strain M. hominis PG21. Similar analyses of eight one-step-selected mutants did not reveal any base change in the gyrA and gyrB QRDRs. These results suggest that in M. hominis, like in other bacterial species, a gyrA mutation at Ser-83 is associated with fluoroquinolone resistance.     

Inhibition of NorA efflux pump of Staphylococcus aureus by anacardic acids isolated from the cashew nutshell liquid of Anacardium occidentale L.

The rising of diseases caused by multidrug-resistant bacteria has encouraged researchers to explore more antimicrobial substances, as well as chemicals capable of potentiating the action of existing ones against multidrug-resistant bacteria. Anacardium occidentale produces a fruit known as cashew nut, filled with a dark, almost black, caustic, and flammable liquid called cashew nutshell liquid (CNSL). The goal of the study was to evaluate the intrinsic antimicrobial activity of the major compounds present in CNSL, called anacardic acids (AA), as well as their possible modulatory action as an adjuvant of Norfloxacin against a Staphylococcus aureus strain overproducing the NorA efflux pump (SA1199B). Microdilution assays were performed to determine the minimum inhibitory concentration (MIC) of AA against different microbial species. Norfloxacin and Ethidium Bromide (EtBr) resistance modulation assays were performed in the presence or absence of AA against SA1199-B. AA showed antimicrobial activity against Gram-positive bacterial strains tested but no activity against Gram-negative bacteria or yeast strains. At subinhibitory concentration, AA reduced the MIC values for Norfloxacin and EtBr against the SA1199-B strain. Furthermore, AA increased the intracellular accumulation of EtBr in this NorA overproducer strain, indicating that AA are NorA inhibitors. Docking analysis showed that AA probably modulates Norfloxacin efflux by spatial impediment at the same binding site of NorA.     

Characterization of emeA, a norA Homolog and Multidrug Resistance Efflux Pump, in Enterococcus faecalis

We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the Enterococcus faecalis V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene emeA (enterococcal multidrug resistance efflux).     

Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efflux pump YhiV (MdtF)

OBJECTIVES: YhiV (MdtF) is an resistance nodulation division (RND) type efflux pump in Escherichia coli with significant homology to AcrB but usually expressed at a low level in clinical isolates. When overexpressed the pump confers decreased susceptibility to a variety of substances including erythromycin and ethidium bromide (EtBr). We characterized two mutants of E. coli E12 (DeltaacrB DeltaacrF) overexpressing yhiV that showed surprising differences in their spectrum of multidrug resistance (MDR). METHODS: The two mutants obtained after repeated exposure of E. coli E12 to levofloxacin were tested for antimicrobial susceptibility to a variety of agents and for intracellular accumulation of selected pump substrates. Gene expression was studied by quantitative RT-PCR, and yhiV was sequenced. Gene inactivation and replacement were done by phage lambda-based homologous recombination. RESULTS: Mutant DKO20/1 overexpressed yhiV, showed a wild-type yhiV sequence and had >2-fold increased MICs of fluoroquinolones, novobiocin, macrolides/ketolides, EtBr, oxacillin and Phe-Arg-beta-naphthylamide (PAbetaN, a putative efflux pump inhibitor) compared with the E12 parent. A second mutant, strain DKO1/17 that had the Val-610-->Phe point mutation in YhiV differed from DKO20/1 by faster growth, >2-fold increased MICs of linezolid and tetracycline, but >2-fold decreased MICs of PAbetaN, azithromycin and telithromycin. Inactivation of yhiV in DKO1/17 and reintroduction of the wild-type and mutant yhiV sequence confirmed that the differing MICs of most of the drugs were associated with the observed single point mutation. Intracellular drug accumulation studies with linezolid and PAbetaN were consistent with the MIC results. CONCLUSIONS: The region around amino acid Val-610 in YhiV appears to be involved in determining recognition and efficiency of export of a number of MDR efflux pump substrates. This single point mutation in the periplasmic loop of the pump can increase resistance to a given drug such as a fluoroquinolone while decreasing resistance to another one.     

Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy

Whole-cell assays were implemented to search for efflux pump inhibitors (EPIs) of the three multidrug resistance efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isolates of Pseudomonas aeruginosa. Secondary assays were developed to identify lead compounds with exquisite activities as inhibitors. A broad-spectrum EPI which is active against all three known Mex efflux pumps from P. aeruginosa and their close Escherichia coli efflux pump homolog (AcrAB-TolC) was discovered. When this compound, MC-207,110, was used, the intrinsic resistance of P. aeruginosa to fluoroquinolones was decreased significantly (eightfold for levofloxacin). Acquired resistance due to the overexpression of efflux pumps was also decreased (32- to 64-fold reduction in the MIC of levofloxacin). Similarly, 32- to 64-fold reductions in MICs in the presence of MC-207,110 were observed for strains with overexpressed efflux pumps and various target mutations that confer resistance to levofloxacin (e.g., gyrA and parC). We also compared the frequencies of emergence of levofloxacin-resistant variants in the wild-type strain at four times the MIC of levofloxacin (1 microg/ml) when it was used either alone or in combination with EPI. In the case of levofloxacin alone, the frequency was approximately 10(-7) CFU/ml. In contrast, with an EPI, the frequency was below the level of detection (<10(-11)). In summary, we have demonstrated that inhibition of efflux pumps (i) decreased the level of intrinsic resistance significantly, (ii) reversed acquired resistance, and (iii) resulted in a decreased frequency of emergence of P. aeruginosa strains that are highly resistant to fluoroquinolones.     

Inhibition of antibiotic efflux in bacteria by the novel multidrug resistance inhibitors biricodar (VX-710) and timcodar (VX-853)

Inhibitors of mammalian multidrug efflux, such as the plant alkaloid reserpine, are also active in potentiating antibiotic activity by inhibiting bacterial efflux. Based on this precedent, two novel mammalian multiple drug resistance inhibitors, biricodar (VX-710) and timcodar (VX-853), were evaluated for activity in a variety of bacteria. Both VX-710 and VX-853 potentiated the activity of ethidium bromide (EtBr), a model efflux substrate, against three clinically significant gram-positive pathogens: Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae. Similar to reserpine, VX-710 and VX-853 directly blocked EtBr efflux in S. aureus. Furthermore, these compounds were effective in lowering the MICs of several clinically used antibiotics, including fluoroquinolones, suggesting that VX-710 and VX-853 are representatives of a new class of bacterial efflux inhibitors with the potential for use in combination therapy.     

Relative contributions of the AcrAB,MdfA and NorE efflux pumps to quinolone resistance in Escherichia coli

Quinolones are widely used, broad-spectrum antimicrobial agents. In screens for genes that, when overexpressed, allow Escherichia coli to grow on otherwise lethal concentrations of the fluoroquinolone norfloxacin, the ydhE gene was identified. We have shown that ydhE encodes a multidrug efflux pump with a narrower substrate range than that of its closest homologue, encoded by norM, and named the gene norE. The relative contributions to drug resistance of NorE compared with the two other known E. coli quinolone pumps, AcrAB and MdfA, have been defined. Overexpression of each of the three pumps separately resulted in roughly similar levels of quinolone resistance, whereas simultaneous overexpression of norE or mdfA in combination with acrAB gave synergic increases in quinolone resistance. The level of quinolone resistance mediated by efflux pumps seems to be constrained to an approximately 10-fold maximum, even with increased production of the pumps. We measured the drug resistance of an isogenic set of strains containing the various permutations of single, double and triple drug efflux pump mutants. The DeltanorE and DeltamdfA mutants were somewhat more susceptible to fluoroquinolones than the parent strain, and acrAB mutants were four- to six-fold more susceptible. Mutants lacking two or all three efflux pumps were not significantly more susceptible to fluoroquinolones than those lacking only one of the three pumps.     

Evaluation of efflux pump inhibitors of MexAB- or MexXY-OprM in Pseudomonas aeruginosa using nucleic acid dyes

BackgroundIncreased expression of efflux pumps is an important mechanism of antibiotic resistance in Pseudomonas aeruginosa, and treatment with inhibitors of active efflux pumps seems an attractive strategy to combat with multidrug resistance. Assays using ethidium bromide (EtBr), which accumulates by binding to nucleic acids, are often employed to assess the efficacy of efflux pump inhibitors (EPIs). However, few studies have reported on assays using other nucleic acid dyes.ObjectiveWe used different classes of EPIs for MexAB- or MexXY-OprM to measure the accumulation of various fluorescent dyes, including SYBR Safe, AtlasSight, and GelGreen.MethodsEscherichia coli MG1655ΔacrBΔtolC strain harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of P. aeruginosa were constructed. Then, the accumulation of the above-mentioned nucleic acid dyes and EtBr was measured to assess the efflux ability in the presence and absence of EPIs (MexAB-OprM-specific inhibitor of pyridopyrimidine derivative [ABI-PP], berberine, non-specific inhibitor of phenylalanine-arginine β-naphthylamide [PAβN], and protonophore of carbonyl cyanide m-chlorophenyl hydrazone [CCCP]).ResultsDecreased accumulations of nucleic acid dyes were observed in strains with pABM or pXYM compared with the parental strain. ABI-PP or berberine addition significantly increased the accumulation of any nucleic acids in the strains with the specific pumps. PAβN or CCCP addition showed increased accumulation of almost all dye in strains with pABM or pXYM. However, the inhibition patterns of EPIs differed according to the nucleic acid dyes used.ConclusionsAccumulation assays for EPIs were suitable to evaluate EPI candidates using various nucleic acid dyes.     

Clinical significance of overexpression of multiple RND-family efflux pumps in Bacteroides fragilis isolates

OBJECTIVES: The aim of the present study was to determine correlation between bmeB efflux pump overexpression and resistance to fluoroquinolones and beta-lactams in Bacteroides fragilis clinical isolates (n = 51) and the effects of broad-spectrum efflux pump inhibitors (EPIs) on the MICs of the test antibiotics. METHODS: Susceptibility to garenoxacin, levofloxacin, moxifloxacin, cefoxitin and faropenem +/- EPIs (CCCP, MC-207,110, reserpine and verapamil) was determined. Expression of bmeB efflux pumps was measured, topoisomerase genes were sequenced and beta-lactamase production was determined. RESULTS: Isolates were grouped into categories based on susceptibility patterns, topoisomerase sequence and efflux pump expression. Panel I isolates (19/51, 37.3%) were highly resistant to fluoroquinolones and cefoxitin (resistance to all agents was significantly reduced by EPIs, P < 0.05), had a point mutation in gyrA (C-->T) causing a Ser-82-->Phe substitution, and overexpressed bmeB4 and bmeB15. Panel II isolates (7/51; 13.7%) had intermediate-level resistance to fluoroquinolones and cefoxitin and a GyrA substitution. Panel IIIA isolates (21/51; 41.2%) had intermediate-level fluoroquinolone resistance and high-level cefoxitin resistance [resistance to all agents was significantly reduced by EPIs (P < 0.05)] and overexpressed bmeB4 and bmeB15. Panel IIIB isolates (4/51; 7.8%) had low-level fluoroquinolone resistance and high-level resistance to cefoxitin [cefoxitin resistance was significantly reduced by EPIs (P < 0.05)] and overexpressed bmeB4, bmeB6, bmeB10 and bmeB14. All isolates were beta-lactamase-positive. CONCLUSIONS: These data suggest that bmeB efflux pump overexpression can (i) cause low- to intermediate-level clinically relevant fluoroquinolone resistance; (ii) be coupled with GyrA substitutions to cause high-level fluoroquinolone resistance; (iii) contribute to high-level clinically relevant resistance to beta-lactams.     

多重耐药铜绿假单胞菌外排泵基因表达与耐药表型和耐药程度的关系

目的建立实时荧光定量PCR法检测多重耐药铜绿假单胞菌(Pa)的外排泵基因表达量,探讨不同类型外排泵基因表达与耐药表型和耐药程度的关系。方法收集临床分离的多重耐药Pa共80株,分析其耐药表型;用实时荧光定量PCR检测不同类型外排泵基因的表达量,用外排泵抑制剂CCCP及PAβN进行外排泵基因筛查,并比较其结果。结果 64株(80%)检出了不同类型外排泵基因,包括单独表达MexA 30株(37.5%),MexC 12株(15.0%),MexX 8株(10.0%),同时表达MexA和MexC者10株(12.5%),同时表达MexA和MexX者4株(5.0%)。外排泵抑制剂筛查试验总阳性率分别为CCCP抑制法73.8%,PAβN抑制法72.5%。MexA的表达主要增强对美罗培南、三代头孢菌素、氟喹诺酮类、大环内酯类的耐药,且随表达量的增加而增强。MexC型主要增强对氨基糖苷类、氟喹诺酮类、三代头孢菌素、哌拉西林/他唑巴坦类的耐药性,且随表达量的增加而增强。MexX型外排泵的表达主要增强对氨基糖苷类、大环内酯类、氟喹诺酮类和氨曲南的耐药性。MexA和MexC型同时表达与MexA型一致,并增强了对氟喹诺酮类的耐药。MexA和MexX型同时表达与MexA型一致,并增强了对庆大霉素的耐药而降低了对氟喹诺酮类的耐药性。结论 Pa的外排泵基因的表达量与特定种类的药物耐药程度有关,外排泵基因的高表达参与并加剧了Pa的耐药性。     

Use of a Genetic Approach To Evaluate the Consequences of Inhibition of Efflux Pumps in Pseudomonas aeruginosa

Drug efflux pumps in Pseudomonas aeruginosa were evaluated as potential targets for antibacterial therapy. The potential effects of pump inhibition on susceptibility to fluoroquinolone antibiotics were studied with isogenic strains that overexpress or lack individual efflux pumps and that have various combinations of efflux- and target-mediated mutations. Deletions in three efflux pump operons were constructed. As expected, deletion of the MexAB-OprM efflux pump decreased resistance to fluoroquinolones in the wild-type P. aeruginosa (16-fold reduction for levofloxacin [LVX]) or in the strain that overexpressed mexAB-oprM operon (64-fold reduction for LVX). In addition to that, resistance to LVX was significantly reduced even for the strains carrying target mutations (64-fold for strains for which LVX MICs were >4 microg/ml). We also studied the frequencies of emergence of LVX-resistant variants from different deletion mutants and the wild-type strain. Deletion of individual pumps or pairs of the pumps did not significantly affect the frequency of emergence of resistant variants (at 4x the MIC for the wild-type strain) compared to that for the wild type (10(-6) to 10(-7)). In the case of the strain with a triple deletion, the frequency of spontaneous mutants was undetectable (<10(-11)). In summary, inhibition of drug efflux pumps would (i) significantly decrease the level of intrinsic resistance, (ii) reverse acquired resistance, and (iii) result in a decreased frequency of emergence of P. aeruginosa strains highly resistant to fluoroquinolones in clinical settings.     

Role of the Mmr Efflux Pump in Drug Resistance in Mycobacterium tuberculosis

Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated.     

Intrinsic Resistance of Mycobacterium smegmatis to Fluoroquinolones May Be Influenced by New Pentapeptide Protein MfpA

The fluoroquinolones (FQ) are used in the treatment of Mycobacterium tuberculosis, but the development of resistance could limit their effectiveness. FQ resistance (FQ(R)) is a multistep process involving alterations in the type II topoisomerases and perhaps in the regulation of efflux pumps, but several of the steps remain unidentified. Recombinant plasmid pGADIV was selected from a genomic library of wild-type (WT), FQ-sensitive M. smegmatis by its ability to confer low-level resistance to sparfloxacin (SPX). In WT M. smegmatis, pGADIV increased the MICs of ciprofloxacin (CIP) by fourfold and of SPX by eightfold, and in M. bovis BCG it increased the MICs of both CIP and SPX by fourfold. It had no effect on the accumulation of (14)C-labeled CIP or SPX. The open reading frame responsible for the increase in FQ(R), mfpA, encodes a putative protein belonging to the family of pentapeptides, in which almost every fifth amino acid is either leucine or phenylalanine. Very similar proteins are also present in M. tuberculosis and M. avium. The MICs of CIP and SPX were lower for an M. smegmatis mutant strain lacking an intact mfpA gene than for the WT strain, suggesting that, by some unknown mechanism, the gene product plays a role in determining the innate level of FQ(R) in M. smegmatis.     

Characterization of tetracycline resistance mediated by the efflux pump Tap from Mycobacterium fortuitum

OBJECTIVES: The aim of this study was to characterize the efflux pump Tap from Mycobacterium fortuitum, to test its sensitivity to well known efflux inhibitors, to study the interaction between tetracycline and these compounds and to test the ability of these compounds to overcome efflux pump-mediated tetracycline resistance. For all these studies, we produced Tap protein in Mycobacterium smegmatis. METHODS: Antibiotic susceptibility tests, tetracycline uptake/efflux experiments and checkerboard synergy tests. RESULTS: Tetracycline uptake/efflux experiments showed that Tap protein from M. fortuitum uses the electrochemical gradient across the cytoplasmic membrane to extrude tetracycline from the cell. This efflux activity is inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) and reserpine, consistent with the decrease in MIC observed in antibiotic susceptibility testing in the presence of these inhibitors. Accumulation was not inhibited in experiments in which o-vanadate and chlorpromazine (CPZ) were tested. Inhibitor-treated cells used glycerol as a carbon source to re-establish the electrochemical gradient across the membrane and to restore efflux activity. CCCP, reserpine and CPZ reduced the MIC of tetracycline in the M. smegmatis strain expressing the Tap protein, whereas o-vanadate increased the MIC. We also observed synergy between tetracycline and CPZ or reserpine, and antagonism with o-vanadate. CONCLUSIONS: The Tapfor efflux pump uses the electrochemical gradient to extrude tetracycline from the cell. This efflux activity can be inhibited by several compounds. This suggests that similar compounds could be used to overcome antibiotic resistance mediated by efflux pumps.     

Assignment of the substrate-selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa

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 beta-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 beta-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/DeltaMexAB transformants exhibited MexEF-OprN-type pump function that rendered the strains resistant to fluoroquinolones and chloramphenicol but did not change susceptibility to beta-lactam antibiotics compared with the host strain. The MexAB-OprN/DeltaOprM, MexAF-OprM/DeltaMexB, and MexEB-OprM/DeltaMexA 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.     

Molecular mechanisms of decreased susceptibility to fluoroquinolones in avian Salmonella serovars and their mutants selected during the determination of mutant prevention concentrations

OBJECTIVES: Salmonella enterica isolates of six serovars and mutants obtained during determination of mutant prevention concentrations (MPCs) were investigated for mechanisms of decreased susceptibility to fluoroquinolones. METHODS: The quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE genes were sequenced. MIC values were determined in the presence/absence of the efflux pump inhibitors carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or Phe-Arg-beta-naphthylamide (PA beta N). PCR assays for the quinolone resistance genes qnrA, qnrB, qnrS or aac(6')-Ib-cr were applied. The MPC values of ciprofloxacin (MPC(CIP)) were determined for all isolates and selected mutants were investigated for their quinolone resistance mechanisms. RESULTS: In contrast to 11 nalidixic acid-susceptible isolates, 24 nalidixic acid-resistant isolates exhibited single mutations in gyrA (Asp-87 --> Tyr, Gly, Asn or Ser-83 --> Phe, Tyr) or parC (Thr-57 --> Ser). While CCCP had no influence on the MICs, PA beta N decreased the MIC(CIP) values by 1-3 dilution steps and MIC(NAL) values by up to 6 dilution steps. Of the resistance genes investigated, only qnrS was present, in a single Salmonella Infantis isolate. The MPC(CIP) values were 4-64-fold higher than the MICs and ranged between 1-16 and 0.12-1 mg/L, respectively, for isolates resistant or susceptible to nalidixic acid. Only mutants obtained from formerly nalidixic acid-susceptible isolates developed single mutations in gyrA or gyrB. CONCLUSIONS: In field isolates and mutants, target site mutations and efflux seem to be important mechanisms for decreased fluoroquinolone susceptibility. Mutants derived during MPC determination from field isolates already harbouring single-step mutations in gyrA did not exhibit further mutations in any target genes.