JAC Advance Access originally published online on December 14, 2005
Journal of Antimicrobial Chemotherapy 2006 57(2):344-348; doi:10.1093/jac/dki446
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Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Enterobacteriaceae other than Escherichia coli
1 Center for Infectious Diseases and Travel Medicine, Department of Medicine, University Hospital, Freiburg, Germany; 2 Section of Infectious Diseases, Hacettepe University, Ankara, Turkey; 3 Institute of Environmental Medicine and Hospital Epidemiology, University Hospital, Freiburg, Germany
* Correspondence address. Medizinische Universitätsklinik, Hugstetter Strasse 55, D-79106 Freiburg, Germany. Tel: +49-761-270-1819; Fax: +49-761-270-1820; E-mail: kern{at}if-freiburg.de
Received 30 August 2005; returned 28 October 2005; revised 2 November 2005; accepted 11 November 2005
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Objectives: 1-(1-Naphthylmethyl)-piperazine (NMP) has been shown to reverse multidrug resistance (MDR) in Escherichia coli overexpressing RND type efflux pumps, but there is no data on its activity in Enterobacteriaceae other than E. coli.
Methods: The antimicrobial susceptibilities of laboratory strains and 167 clinical isolates of Enterobacteriaceae to a variety of antimicrobial agents were determined in the absence and presence of NMP and, for comparison, of Phe-Arg-ß-naphthylamide (PAßN), another putative efflux pump inhibitor (EPI). A 4-fold or greater reduction of the MIC after EPI addition was considered significant.
Results: NMP consistently reduced the MIC of linezolid in Citrobacter freundii, Enterobacter aerogenes and Klebsiella pneumoniae clinical isolates. Significant effects of NMP addition in >50% of tested isolates were also seen for levofloxacin, tetracycline and chloramphenicol in E. aerogenes, and for levofloxacin and tetracycline in K. pneumoniae, whereas no or minor effects were observed in Serratia marcescens. MDR reversal by NMP was more likely in isolates with decreased susceptibility to fluoroquinolones. In most fluoroquinolone-resistant strains the activity was sufficient to render isolates drug-susceptible at clinically achievable concentrations. The activity of PAßN was different from that of NMP, suggesting different modes of action of the two putative EPIs.
Conclusion: NMP has moderate activity in reversing MDR in many but not all members of the Enterobacteriaceae family including clinical isolates. Its effects on resistance reversal depend on bacterial species and drug, and are different from those seen with PAßN.
Keywords: multidrug resistance , fluoroquinolones , nosocomial pathogens
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Introduction |
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Multidrug resistance (MDR) has been an emerging problem among many bacterial pathogens, notably among nosocomial Gram-negative bacteria. An important mechanism of MDR in Gram-negative bacteria is enhanced efflux of chemically unrelated agents, thereby diminishing access of drugs to their intracellular targets.1 MDR phenotypes can be selected both in vitro and in vivo in a variety of bacterial species by exposure to fluoroquinolones, a relatively new class of highly potent antibacterial compounds that are now being widely used in human and veterinary medicine. This type of MDR has often been linked with overexpression of resistance-nodulation-cell division (RND) type tripartite efflux pumps.1
A new development is the application of putative efflux pump inhibitors (EPIs) that are capable of at least partially reversing the MDR phenotype of Gram-negative bacteria by mechanisms not clearly understood.2 One of these compounds with EPI activity in Pseudomonas aeruginosa is Phe-Arg-ß-naphthylamide (PAßN).3,4 Several arylpiperidines and other compounds capable of reversing MDR in defined Enterobacteriaceae have also been described.5–8 Recently, 1-(1-naphthylmethyl)-piperazine (NMP) has been shown to reverse MDR in Escherichia coli overexpressing RND type efflux pumps, but not in pump-deficient mutants.9 NMP increased the intracellular concentration of ethidium bromide and levofloxacin, suggesting efflux pump inhibition as the mechanism of action. In the present work we evaluated the potency of NMP to reverse MDR in several species of Enterobacteriaceae other than E. coli, including clinical isolates.
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Materials and methods |
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Bacterial strains
Clinical non-duplicate isolates of Citrobacter spp., Klebsiella pneumoniae, Enterobacter spp. and Serratia marcescens were obtained from different clinical microbiology laboratories. Most isolates were from patients admitted to intensive care units. No attempts were made to investigate in detail the nature of MDR phenotypes among the clinical test isolates. Thirty out of the 167 isolates (18%) were fluoroquinolone-resistant (FQR, MIC of levofloxacin 
8 mg/L), and 52 had an MIC of levofloxacin >1 mg/L. Reference strains of Enterobacteriaceae were from the ATCC. Laboratory strains included Citrobacter freundii CFAS0 that was obtained from C. freundii ATCC 8090 by inactivating acrB using the phage lambda-based Red/ET homologous recombination system (Gene Bridges, Dresden, Germany).10 Enterobacter aerogenes EAEP298 was derived from a clinical isolate by tolC inactivation and was kindly donated by Jean-Marie Pagès.11 E. coli 261, a gift from Jing Chen, carried sdeXY which codes for an RND type MDR efflux pump of S. marcescens.12
Chemicals and media
NMP was obtained from Chess GmbH (Mannheim, Germany), and PAßN and pyronin Y were purchased from Sigma-Aldrich (Steinheim, Germany). Luria–Bertani (LB) broth and agar were obtained from Oxoid (Basingstoke, UK). Ethidium bromide was from Merck (Darmstadt, Germany).
Susceptibility testing
Susceptibilities to a panel of different antibiotics were studied by microbroth dilution in the presence or absence of NMP or PAßN, in accordance with NCCLS performance and interpretive guidelines. Custom microtitre plates containing selected antimicrobials at increasing concentrations were purchased from Merlin Diagnostics (Bornheim, Germany). A 4-fold or greater reduction in the MIC values after addition of NMP or PAßN was considered significant.
Pyronin Y whole cell accumulation assays
Cells were grown overnight on LB agar plates and diluted in 1 mL of PBS + 0.4% glucose (pH 7.4) until an optical density at 600 nm of 
1 was reached. The cells were then transferred to a 96-well plate and NMP was added. Thereafter, pyronin Y was added to a final concentration of 5 or 10 mg/L, and the relative fluorescence intensity was measured over time in a Safire (Tecan, Crailsheim, Germany) fluorescence plate-reader (excitation 545 nm, emission 570 nm). This assay was similar to a previously described ethidium bromide accumulation test,9,13 but, unlike with ethidium bromide, the fluorescence of pyronin Y is quenched after binding to RNA, and decreasing fluorescence correlates with increasing intracellular dye concentration (and efflux inhibition). The use of pyronin Y instead of ethidium bromide as fluorescent dye offered the advantage to include PAßN as EPI, which showed no or only minor effects on the reduction of ethidium bromide MIC in reference strains of Enterobacteriaceae other than E. coli (Table 1), E. coli clinical isolates (W. V. Kern and P. Steinke, unpublished observations) and P. aeruginosa.3
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Results and discussion |
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Intrinsic antibacterial activity of NMP and PAßN
At the concentration needed to reduce the MIC of levofloxacin and other agents by at least 4-fold in E. coli overexpressing acrAB or acrEF (between 50 and 100 mg/L),9 NMP had no measurable growth inhibitory effects (data not shown) even in the pump-deficient strains C. freundii CFAS0, E. aerogenes EAEP298 and E. coli 262. This was unlike PAßN that had increased activity in pump-deficient strains (MIC 
100 mg/L) compared with wild-type parental strains and clinical isolates (MIC > 400 mg/L). In clinical isolates, the intrinsic antibacterial activity of NMP was similar to that seen in reference strains (MIC 400 mg/L).
EPI activity in reference and laboratory strains
When used at a concentration of 25 mg/L, NMP had minor effects on the MIC of the test drugs in reference strains (data not shown)—similar to that seen earlier in E. coli.9 At a concentration of 100 mg/L consistent effects were observed on ethidium bromide resistance reversal (Table 1). Effects on the MIC of the other agents were variable. Effects were generally less significant in S. marcescens and E. coli 261 (which carried the S. marcescens specific sdeXY pump gene) than in the other species (Table 1). Notably, in pump-deficient strains, NMP was unable to reduce the MICs of the test agents except that of rifampicin, whereas PAßN obviously was more toxic presumably owing to its enhanced intrinsic activity in RND type pump-deficient versus pump-competent strains. There was an exceptional interaction across all strains between PAßN and rifampicin in the sense of a highly sensitizing effect of PAßN that was largely independent of RND pump and tolC inactivation (Table 1).
There were small but reproducible effects of PAßN at a concentration of 25 mg/L on pyronin Y MICs in the tested Enterobacteriaceae strains that were not seen with ethidium bromide (Table 1). This provided the opportunity to assess the effects of both putative EPIs on intracellular substrate accumulation. Consistent with the observations of MIC changes there were measurable effects of both putative EPIs on pyronin Y fluorescence in Citrobacter, suggesting that the MIC changes after EPI addition were in fact related to changes in the intracellular substrate concentration (Figure 1). In E. aerogenes EAEP289, changes in the intracellular pyronin Y accumulation after NMP addition could also be demonstrated, but these changes were smaller, and a definite effect on increased pyronin Y accumulation after addition of PAßN was uncertain (Figure 1).
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EPI activity in clinical isolates
As shown in Table 2, the spectrum of antimicrobial drugs affected by the addition of NMP (100 mg/L) depended on species and drugs. Across the different species, no effects of NMP were seen with streptomycin (as expected), and limited effects were seen with oxacillin, a relatively hydrophobic ß-lactam (data not shown), and clarithromycin. Conversely, NMP consistently reduced the MIC of linezolid in the majority of C. freundii, E. aerogenes and K. pneumoniae clinical isolates, but this interaction did not result in a clinically relevant reduction of the MIC to levels below the breakpoint of resistance. Significant effects of NMP addition in >50% of tested clinical isolates were also seen for levofloxacin, tetracycline and chloramphenicol in E. aerogenes, and for levofloxacin and tetracycline in K. pneumoniae. No or minor effects were observed in S. marcescens (Table 2) including ethidium bromide.
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In many FQR strains (five out of seven Citrobacter isolates, six out of six Enterobacter isolates and three out of three Serratia isolates) the activity of NMP was sufficient to render isolates drug-susceptible at clinically achievable concentrations. However, this was dependent on the initial MIC level. In FQR K. pneumoniae isolates, for example, which were almost all highly FQR (levofloxacin MIC >32 mg/L), addition of NMP was insufficient to decrease the MIC of levofloxacin to <8 mg/L (data not shown). Although clarithromycin was a pump substrate given the MIC changes in pump-deficient strains, the effects of NMP on its MIC were small and usually non-significant (Table 2).
PAßN showed a slightly differing spectrum of activity (Table 2). Particularly strong effects across the species were observed for the interaction of PAßN and rifampicin, similar to those seen in E. coli.9 Apart from these effects, PAßN significantly and more effectively than NMP reduced the resistance to clarithromycin in all species except S. marcescens (Table 2), which showed very minor effects of PAßN on increased drug susceptibility. Effects of PAßN on macrolide resistance were previously described in E. coli and E. aerogenes, and were linked to the AcrAB-TolC system, but PAßN may be additionally active on a pump with high specificity for macrolide and ketolides14 while NMP was found unable to reduce the MIC of ketolides in E. coli.9 It is tempting to speculate that the strong effects of PAßN on macrolide resistance observed here in Enterobacteriaceae other than E. coli are linked to dual inhibition of an RND type efflux pump and a pump that preferentially accommodates macrolides/ketolides. A permeabilizing effect of PAßN, however, which may specifically affect rifampicin and macrolides (as shown for polymyxin B nonapaptide) cannot be excluded.
PAßN at a concentration of 25 mg/L had very limited effects on fluoroquinolone MICs (Table 2), but at a concentration of 100 mg/L it enhanced the effect on the reduction of the levofloxacin MIC to a potency that was similar to NMP (data not shown).
The subgroup of 52 clinical isolates with decreased fluoroquinolone susceptibility (and usually MDR phenotypes) were characterized in general by more significant EPI effects. In this subgroup PAßN even at the higher concentration of 100 mg/L had no effect on the MIC of ethidium bromide. It significantly reduced the pyronin Y MIC in most isolates of C. freundii, but much less so in E. aerogenes, S. marcescens and K. pneumoniae. NMP, in contrast, was more effective than PAßN in reducing the MICs of both dyes (data not shown).
Conclusions
Taken together, NMP showed moderate activity as an MDR reversal agent in Enterobacteriaceae other than E. coli. Resistance reversal by NMP to clinically relevant MIC values was achieved for levofloxacin in a variable proportion of the clinical isolates. The spectrum of activity of NMP differed from that seen with PAßN. PAßN was consistently associated with a large reduction of rifampicin MICs across the species tested, and with large reductions of clarithromycin MICs in all species except S. marcescens. Another noteworthy difference was the differential ability of the two EPIs to reduce the MICs of the two dyes, ethidium bromide and pyronin Y. This suggests the presence of different targets of the two putative EPIs within a given species. Alternative explanations include different binding sites in a given pump, and perhaps other effects such as differential membrane permeability alterations by one of the two compounds.
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None to declare.
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Acknowledgements |
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This study was supported by the Landesstiftung Baden-Württemberg.
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References |
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