1 Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, 2939 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: multidrug efflux, GG918, Staphylococcus aureus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 concentrationtime 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.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 MuellerHinton 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 doseresponse 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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.
|
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 |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Marshall, N. J. & Piddock, L. J. V. (1997). Antibacterial efflux systems. Microbiologia 13, 285300.[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, 25965.[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, 15359.[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, 120714.[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, 1636.[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, 4595602.[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, 427.[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, 246875.
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, 24048.
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: structureactivity relationships. Journal of Medicinal Chemistry 44, 2618.[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, 14337.
13 . Kaatz, G. W., Seo, S. M. & Ruble, C. A. (1993). Efflux-mediated fluoroquinolone resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 37, 108694.[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, 8338.
15 . National Committee for Clinical Laboratory Standards. (1999). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Villanova, PA, USA.
16
.
Kaatz, G. W., Seo, S. M., OBrien, 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, 14046.
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, 183542.[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, 122540.[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, 492831.[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, 9889.