A novel inhibitor of multidrug efflux pumps in Staphylococcus aureus

Simon Gibbons1,*, Moyosoluwa Oluwatuyi1 and Glenn W. Kaatz2

1 Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX, UK; 2 Division of Infectious Diseases, Department of Internal Medicine, School of Medicine, Wayne State University and the John D. Dingell Department of Veterans Affairs Medical Center, Detroit, MI 48201, USA

Received 16 September 2002; returned 8 October 2002; revised 11 October 2002; accepted 15 October 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
GG918, a synthetic inhibitor of P-glycoprotein-mediated mammalian tumour multidrug resistance, was found to be equipotent to reserpine in enhancing the in vitro activity of norfloxacin and ciprofloxacin against strains of Staphylococcus aureus expressing distinct efflux-related multidrug resistance pumps. Four- to eight-fold reductions in MICs of these fluoroquinolones were observed for SA-1199B, a strain that overexpresses NorA (the major S. aureus multidrug transporter), and SA-K2068, which possesses a multidrug efflux-related pump distinct from NorA. Neither inhibitor potentiated the activity of newer fluoroquinolones such as levofloxacin or moxifloxacin by more than two-fold, and this effect was observed only in SA-1199B and SA-K2068. GG918 and reserpine exposure resulted in two- to four-fold reductions in norfloxacin and ciprofloxacin MICs in a fluoroquinolone-susceptible control strain and in strains expressing the MsrA and TetK proteins, which mediate efflux-related resistance to macrolides and tetracyclines, respectively, suggesting inhibition of as yet uncharacterized pumps for which norfloxacin and ciprofloxacin are substrates. In the MsrA- and TetK-expressing strains no more than a two-fold augmentation of erythromycin or tetracycline activity was observed with either inhibitor, suggesting minimal, if any, inhibitory activity against these efflux proteins. Using GG918 as a lead compound, a structure–activity evaluation may reveal a more potent and broader spectrum inhibitor of S. aureus antibiotic efflux pumps.

Keywords: multidrug efflux, GG918, Staphylococcus aureus


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Multidrug (MDR) efflux is an increasingly reported phenomenon and has been described for many organisms, including bacteria, fungi and protozoa, and as a mechanism of resistance in mammalian tumour cells.1 Bacteria possess a wide array of drug efflux proteins and a number of clinically relevant species, most notably Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, utilize these transporters as part of their resistance strategy.2 Many of these efflux mechanisms export an extensive range of structurally unrelated antibiotics from the cell, resulting in a reduced intracellular concentration and thus reduced susceptibility.

Examples of efflux-related resistance mechanisms that have been described for S. aureus include those conferred by QacA and NorA, which are MDR transporters, and the more specific MsrA and TetK transport proteins.36 These export proteins were originally described to efflux quaternary ammonium salts (antiseptics), fluoroquinolones, macrolides and tetracyclines, respectively, although these efflux proteins, especially QacA and NorA, actively export a broad array of structurally dissimilar drugs from the bacterial cell.

GG918 (Figure 1) is a synthetic compound that was originally discovered as part of a screening programme designed to identify inhibitors of mammalian P-glycoprotein (P-gp). P-gp is an ABC-type transporter that exports numerous anti-neoplastic agents from cancer cells, making them drug resistant.1,7 It has been shown that co-administration of GG918 with paclitaxel significantly increases the systemic exposure to this anti-neoplastic agent.8 Toxicities associated with GG918 were not observed in this study and the mean maximal serum concentration of the compound was 0.43 ± 0.27 mg/L. The mean area under the plasma concentration–time curve of paclitaxel after oral administration of 1 g of GG918 was comparable to that achieved with oral paclitaxel in combination with another P-gp inhibitor, cyclosporin A.9 Unlike cyclosporin A, GG918 has no known immunosuppressive activity and may be a better candidate for clinical use as a P-gp inhibitor.



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Figure 1. Structure of GG918.

 
Previous work on inhibitors of MDR pumps in S. aureus includes the screening of a synthetic library against the NorA MDR transporter.10 These inhibitors acted in a synergic manner with ciprofloxacin and dramatically suppressed the emergence of ciprofloxacin-resistant S. aureus upon in vitro exposure to the drug. Other inhibitors of MDR pumps in S. aureus include the anti-hypertensive plant alkaloid reserpine, the porphyrin pheophorbide A, 5'-methoxyhydnocarpin D (a flavonolignan) and selected flavones.11 The antimicrobial activity of berberine, a natural antibiotic found in some plants and also a NorA substrate, was found to be potentiated by low concentrations of 5'-methoxyhydnocarpin D by inhibition of its efflux.12 Identification of effective inhibitors of NorA and other S. aureus efflux pumps could restore the clinical utility of pump substrates, which prompted the current investigation of the activity of GG918 as an inhibitor of such pumps.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains and media

S. aureus RN4220 containing plasmid pUL5054, which carries the gene encoding the MsrA macrolide efflux protein, and strain CD-1281, which possesses the TetK tetracycline efflux protein, were generously provided as gifts from J. Cove (University of Leeds, UK) and C. Dowson (University of Warwick, UK), respectively. SA-1199B, which overexpresses the NorA MDR efflux protein, SA-K2068, which exhibits an MDR efflux phenotype conferred by a pump distinct from NorA, and S. aureus ATCC 25923 were also used.13,14 All strains were cultured on nutrient agar (Oxoid, Basingstoke, UK) before determination of MICs. Cation-adjusted Mueller–Hinton broth (MHB; Oxoid) was used for susceptibility tests.

Antibiotics and chemicals

Tetracycline, norfloxacin and erythromycin were obtained from Sigma (Poole, UK). Ciprofloxacin, levofloxacin and moxifloxacin were obtained from their respective manufacturers. GG918 was provided by GlaxoSmithKline (Stevenage, UK).

Susceptibility tests

MICs were determined at least in duplicate by microdilution techniques according to the NCCLS guidelines, using S. aureus 25923 as a quality control strain.15 The effects of GG918 and reserpine (final concentrations 10 and 20 mg/L, respectively) on MICs were also determined. Both of these compounds were dissolved in DMSO before dilution into MHB for use in MIC determinations. The highest concentration of DMSO remaining after dilution (25%, v/v) caused no inhibition of bacterial growth (data not shown).

Ethidium efflux

Ethidium bromide (EtBr) is a substrate for many Gram-positive MDR pumps, including NorA. The efficiency of efflux pumps for which EtBr is a substrate can be assessed fluorometrically by the loss of fluorescence over time from cells loaded with EtBr. SA-1199B and SA-K2068 were loaded with EtBr as previously described, and the effect of varying concentrations of reserpine and GG918 on EtBr efflux was determined to generate a dose–response profile for each compound.16 Results were expressed as percentage reduction of the total efflux observed for test strains in the absence of inhibitors.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Susceptibility data for test strains in the presence and absence of inhibitors are shown in Table 1. Neither reserpine nor GG918 by themselves had inhibitory activity against any test strain at the concentrations employed (data not shown). The presence of either compound resulted in at least a four-fold reduction in norfloxacin MICs for all strains possessing efflux-related resistance phenotypes, regardless of that phenotype. With respect to ciprofloxacin, a four-fold or greater potentiation of activity was observed only with SA-1199B and SA-K2068; no more than two-fold MIC changes were observed for the other test strains. The inhibitors only minimally augmented the activity of levofloxacin and moxifloxacin, which are more recently developed fluoroquinolones with improved potency compared with ciprofloxacin against S. aureus.


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Table 1.  MICs of test strains (mg/L)
 
The presence of GG918 or reserpine resulted in no more than a two-fold reduction in erythromycin and tetracycline MICs for all strains. The equivalence in activity regardless of the efflux-related resistance trait present suggests that the modest MIC reductions observed are not related to specific inhibition of MsrA or TetK.

Overall, the effects of GG918 and reserpine on susceptibility data were equivalent for all test strains. In general, the activities of norfloxacin and ciprofloxacin were the most potentiated. This was especially true for SA-1199B, which overexpresses NorA, and SA-K2068, which possesses a novel non-NorA MDR pump. Both norfloxacin and ciprofloxacin are quite hydrophilic molecules with small substituents at the C7 and C8 positions, characteristics that make them more favourable substrates for NorA, and probably for the SA-K2068 efflux pump.17 The lack of significant activity of either inhibitor on MICs of levofloxacin and moxifloxacin for strains bearing these pumps may relate to molecular hydrophobicity in the case of levofloxacin and structural features in the case of both compounds, characteristics that may reduce recognition and transport.

The sequence of the S. aureus genome has recently been published, and examination of the data reveals the presence of up to 17 open reading frames encoding putative drug transporters.18 The four-fold potentiation of norfloxacin activity by inhibitors in RN4220 (MsrA), CD-1281 (TetK) and ATCC 25923, strains not possessing known quinolone efflux systems, is likely to be related to the inhibition of one or more of these as yet uncharacterized pumps. These data indicate that GG918 and reserpine may have more affinity for MDR-type pumps than for more limited spectrum pumps such as MsrA and TetK, or at least for pumps for which fluoroquinolones are substrates.

The effect of inhibitors on the EtBr efflux capability of SA-1199B and SA-K2068 compared with the effect observed for reserpine is shown in Figure 2. For SA-1199B, concentrations of <=10 µM GG918 were more potent than the same concentrations of reserpine. Both inhibitors were very potent versus SA-K2068, with GG918 appearing more effective at concentrations of <=5 µM. These data indicate that at low concentrations GG918 is more potent than reserpine as an inhibitor of MDR pump-mediated EtBr efflux in S. aureus.



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Figure 2. Effect of inhibitors on ethidium bromide efflux; the data presented are means of duplicate experiments. (a) SA-1199B; (b) SA-K2068; inverted filled triangles, GG918; filled circles, reserpine.

 
The development of efflux pump inhibitors, which could be used in conjunction with existing antibiotics, could extend the useful lifetime of some antibiotics by improving therapeutic efficacy and by suppressing the emergence of resistant variants that might otherwise arise during treatment. The former phenomenon has been evaluated in an animal model of a P. aeruginosa infection caused by a strain overexpressing the MexAB–OprM MDR efflux system, with treatment consisting of levofloxacin plus inhibitor or levofloxacin alone.19 Improved therapeutic efficacy was observed in animals treated with the combination versus those treated with only levofloxacin. The latter phenomenon has been demonstrated for both NorA and PmrA, a recently described Streptococcus pneumoniae MDR transporter, where it has been shown that the combination of reserpine and a fluoroquinolone markedly reduced the emergence of resistant variants in vitro compared with what was observed with the fluoroquinolone alone.10,20 However, the concentrations of reserpine required for the observed effect were not clinically relevant as the potential for adverse effects, such as neurotoxicity, eliminates the use of reserpine as an efflux pump inhibitor in the clinical setting.

GG918 is a first step toward developing an inhibitor active against S. aureus antibiotic efflux pumps, especially NorA. A further effort to identify an even more potent compound with a good toxicity profile and broader spectrum of activity seems reasonable. The combination of a broad-spectrum MDR pump inhibitor with antibiotics that are known pump substrates could reduce the morbidity and mortality that might result from a delay in the institution of effective therapy for serious S. aureus infections.


    Acknowledgements
 
Drs Edet Udo, Jon Cove and Professor Christopher Dowson are thanked for the provision of bacterial strains. GlaxoSmithKline are thanked for the provision of GG918. We thank the Royal Society (Grant No. 21595) for a research grant to S. Gibbons and the EPSRC for a PhD studentship for M. Oluwatuyi.


    Footnotes
 
* Corresponding author. Tel: +44-20-7753-5913; Fax: +44-20-7753-5909; E-mail: simon.gibbons{at}ulsop.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Ling, V. (1997). Multidrug resistance: molecular mechanisms and clinical relevance. Cancer Chemotherapy and Pharmacology 40, Suppl., S3–8.[CrossRef][ISI][Medline]

2 . Marshall, N. J. & Piddock, L. J. V. (1997). Antibacterial efflux systems. Microbiologia 13, 285–300.[Medline]

3 . Littlejohn, T. G., Paulsen, I. T., Gillespie, M. T., Tennent, J. M., Midgley, M., Jones, I. G. et al. (1992). Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus. FEMS Microbiology Letters 74, 259–65.[Medline]

4 . Ubukata, K., Itoh-Yamashita, N. & Konno, M. (1989). Cloning and expression of the norA gene for fluoroquinolone resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 33, 1535–9.[ISI][Medline]

5 . Ross, J. I., Eady, E. A., Cove, J. H., Cunliffe, W. J., Baumberg, S. & Wootton, J. C. (1990). Inducible erythromycin resistance in staphylococci is encoded by a member of the ATP-binding transport super-gene family. Molecular Microbiology 4, 1207–14.[ISI][Medline]

6 . Guay, G. G., Khan, S. A. & Rothstein, D. M. (1993). The tet(K) gene of plasmid pT181 of Staphylococcus aureus encodes an efflux protein that contains 14 transmembrane helices. Plasmid 30, 163–6.[CrossRef][ISI][Medline]

7 . Hyafil, F., Vergely, C., Du Vignaud, P. & Grand-Perret, T. (1993). In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative. Cancer Research 53, 4595–602.[Abstract]

8 . Malingré, M. M., Beijnen, J. H., Rosing, H., Koopman, F. J., Jewell, R. C., Paul, E. M. et al. (2001). Co-administration of GF120918 significantly increases the systemic exposure to oral paclitaxel in cancer patients. British Journal of Cancer 84, 42–7.[Medline]

9 . Malingré, M. M., Terwogt, J. M., Beijnen, J. H., Rosing, H., Koopman, F. J., van Tellingen, O. et al. (2000). Phase I and pharmacokinetic study of oral paclitaxel. Journal of Clinical Oncology 18, 2468–75.[Abstract/Free Full Text]

10 . Markham, P. N., Westhaus, E., Klyachko, K., Johnson, M. E. & Neyfakh, A. A. (1999). Multiple novel inhibitors of the NorA multidrug transporter of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 43, 2404–8.[Abstract/Free Full Text]

11 . Guz, N. R., Stermitz, F. R., Johnson, J. B., Beeson, T. D., Willen, S., Hsiang, J. et al. (2001). Flavonolignan and flavone inhibitors of a Staphylococcus aureus multidrug resistance pump: structure–activity relationships. Journal of Medicinal Chemistry 44, 261–8.[CrossRef][ISI][Medline]

12 . Stermitz, F. R., Lorenz, P., Tawara, J. N., Zenewicz, L. A. & Lewis, K. (2000). Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin, a multidrug pump inhibitor. Proceedings of the National Academy of Sciences, USA 97, 1433–7.[Abstract/Free Full Text]

13 . Kaatz, G. W., Seo, S. M. & Ruble, C. A. (1993). Efflux-mediated fluoroquinolone resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 37, 1086–94.[Abstract]

14 . Kaatz, G. W., Moudgal, V. V. & Seo, S. M. (2002). Identification and characterization of a novel efflux-related multidrug resistance phenotype in Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 50, 833–8.[Abstract/Free Full Text]

15 . National Committee for Clinical Laboratory Standards. (1999). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Villanova, PA, USA.

16 . Kaatz, G. W., Seo, S. M., O’Brien, L., Wahiduzzaman, M. & Foster, T. J. (2000). Evidence for the existence of a multidrug efflux transporter distinct from NorA in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 44, 1404–6.[Abstract/Free Full Text]

17 . Takenouchi, T., Tabata, F., Iwata, Y., Hanzawa, H., Sugawara, M. & Ohya, S. (1996). Hydrophilicity of quinolones is not an exclusive factor for decreased activity in efflux-mediated resistant mutants of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 40, 1835–42.[Abstract]

18 . Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa, H., Kobayashi, I. et al. (2001). Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357, 1225–40.[CrossRef][ISI][Medline]

19 . Renau, T. E., Leger, R., Flamme, E. M., Sangalang, J., She, M. W., Yen, R. et al. (1999). Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone levofloxacin. Journal of Medicinal Chemistry 42, 4928–31.[CrossRef][ISI][Medline]

20 . Markham, P. N. (1999). Inhibition of the emergence of ciprofloxacin resistance in Streptococcus pneumoniae by the multidrug efflux inhibitor reserpine. Antimicrobial Agents and Chemotherapy 43, 988–9.[Abstract/Free Full Text]