Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School, University of Birmingham, Birmingham B15 2TT, UK
Received 21 May 2003; returned 17 June 2003; revised 11 July 2003; accepted 16 July 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: Bacteroides fragilis was grown in Wilkins Chalgren broth (Oxoid Ltd, UK) in a MKII anaerobic workstation (Don Whitley, Shipley, UK). Susceptibility testing was performed, according to the agar doubling dilution method, using Wilkins Chalgren agar supplemented with 5% horse blood. A fluorometric assay was used to measure the accumulation of quinolones (10 mg/L) by B. fragilis.
Results: The activity of the agents for B. fragilis NCTC 9343/ATCC 25285 was clinafloxacin > garenoxacin > levofloxacin = gatifloxacin > moxifloxacin > ciprofloxacin. A weak correlation was observed between the molecular size of the free form and the MIC, the steady state concentration (SSC) and the initial rate of accumulation, but not for the hydrophobicity of each agent. In the presence of reserpine, the SSC of all agents increased. The addition of CCCP had no effect upon garenoxacin or clinafloxacin accumulation, but significantly increased the SSC of ciprofloxacin, moxifloxacin, gatifloxacin and levofloxacin. Verapamil increased the SSC of garenoxacin, whereas sodium orthovanadate had no effect on the concentration of accumulated garenoxacin.
Conclusions: These data suggest that there is probably more than one type of efflux pump in B. fragilis that exports quinolones.
Keywords: efflux pump inhibitors, antibiotic accumulation, quinolones
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Garenoxacin (1-cyclopro-pyl-8-(difluoromethoxy)-7-[(1R)-1-methyl-2,3-dihydro-1H-5-isoindolyl]-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid methanesulphonate monohydrate) is a new des-F(6)- quinolone and, as such, lacks the fluorine at position 6, a characteristic differentiating it from existing agents in this group. Garenoxacin has a broad spectrum of antibacterial activity, including good activity against anaerobes, exceptional activity against Gram-positive bacteria and the potential to inhibit clinically relevant quinolone-resistant pathogens in vivo.7,1013 The results of in vitro anaerobe studies suggest a potential place for garenoxacin in the treatment of anaerobic infections.7,14 The excellent anti-anaerobic activity of garenoxacin, together with its broad spectrum of activity against Enterobacteriaceae,15 makes it a promising alternative for empirical therapy in mixed aerobicanaerobic infections.
Multidrug efflux pumps can confer fluoroquinolone resistance in bacteria, as well as mutations within the quinolone-resistance-determining regions of Topoisomerase II (gyrA and gyrB) and Topoisomerase IV (parC and parE) genes.16 Miyamae et al.2 and Ricci & Piddock17 both found that accumulation of norfloxacin by B. fragilis was increased in the presence of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), a proton motive force inhibitor, suggesting the presence of an active fluoroquinolone efflux pump(s) in this species. Miyamae et al.18 further reported the cloning and characterization of a gene conferring norfloxacin resistance in Bacteroides thetaiotaomicron. The gene was designated bexA and the encoded protein, BexA, belongs to the multidrug and toxic compound extrusion (MATE) family of efflux transporters.
The aim of this study was to investigate the accumulation of six quinolones by NCTC 9343/ATCC 25285, the type strain of B. fragilis. The hydrophobicity and molecular mass of the agents, and their affect upon accumulation and any role in antibacterial activity against B. fragilis, were also investigated. Finally, the effect of known efflux pump inhibitors on the accumulation of quinolones by B. fragilis was also determined.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
B. fragilis NCTC 9343/ATCC 25285 (Z14) was used throughout this study. Garenoxacin was obtained from Bristol-Myers Squibb (Wallingford, CT, USA), ciprofloxacin and moxifloxacin from Bayer AG (Wuppertal, Germany), gatifloxacin from Grunenthal GmBH (Aachen, Germany), levofloxacin from Aventis (Strasbourg, France) and clinafloxacin from Parke Davis Warner-Lambert (MI, USA). All quinolones used were provided as gifts from the respective manufacturers, and made up and used according to their instructions. The partition coefficient between 1-octanol and 0.1 M sodium phosphate buffer pH 7 was determined for each quinolone, as described by Asuquo & Piddock.19 Reserpine, CCCP, verapamil, sodium orthovanadate and Phe-Arg-ß-naphthylamide (MC-207110) were all obtained from Sigma Chemical Company (Poole, UK).
B. fragilis was grown anaerobically at 37°C in an atmosphere of 80% N2, 10% H2 and 10% CO2, which was attained in a MKII anaerobic workstation (Don Whitley, Shipley, UK). The organism was grown in Wilkins Chalgren broth (Oxoid Ltd, UK), and susceptibility testing was performed according to the agar doubling dilution method using Wilkins Chalgren agar supplemented with 5% horse blood, as described previously.17 The plates were incubated for 48 h in the anaerobic workstation. All MIC determinations were performed on at least three separate occasions.
Measurement of quinolone accumulation
Accumulation of quinolones (10 mg/L) by B. fragilis was measured using a fluorometric assay, as described by Ricci & Piddock for norfloxacin.17 The fluorescence of each agent was determined at the appropriate excitation and emission wavelengths (Table 1). Although garenoxacin is not fluorinated at the C6 position, this agent is able to fluoresce because of the C8 di-fluoro methoxy moiety.20 The data on accumulation were converted and expressed as ng quinolone/mg dry weight cells. When studying the effect of the efflux pump inhibitors CCCP (100 µM), reserpine (20 mg/L), verapamil (25 µM), sodium orthovanadate (50 µM) and Phe-Arg-ß-naphthylamide (20 mg/L), the inhibitor was added to a parallel set of tubes to the desired final concentration.
|
All experiments were performed in duplicate on three separate days to obtain the mean value, from which the S.D. was calculated. Differences in accumulation values between different experiments were analysed by Students t-test. A P value of <0.05 was considered significant. Correlation coefficients (r) were calculated using the CORREL statistical function in Excel.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The order of activity against B. fragilis NCTC9343 was clinafloxacin > garenoxacin > gatifloxacin = levofloxacin > moxifloxacin > ciprofloxacin (Table 2). There was a weak correlation between the molecular mass and the MIC values (r = 0.76), such that agents with the lowest mass tended to be more active. There was no correlation between hydrophobicity (Papp) and the MIC (r = 0.54) (data not shown).
|
|
Reserpine decreased the MIC of all agents by one to two dilutions. CCCP and Phe-Arg-ß-naphthylamide reproducibly lowered the MICs of ciprofloxacin, moxifloxacin, gatifloxacin and levofloxacin by one to two dilutions, but had no effect upon the activity of garenoxacin or clinafloxacin (Table 2). Verapamil decreased the MIC of ciprofloxacin, moxifloxacin and garenoxacin by one dilution only. The MICs of all six agents were unaffected by sodium orthovanadate.
Reserpine increased the SSC of all agents (Table 3). Clinafloxacin (P = 0.000464) and garenoxacin (P = 0.0000154) were the most affected. CCCP and Phe-Arg-ß-naphthylamide significantly increased the SSC for ciprofloxacin, moxifloxacin, gatifloxacin and levofloxacin, reflecting the effect upon MIC values. However, in the presence of CCCP the SSC increased more than in the presence of Phe-Arg-ß-naphthylamide. CCCP and Phe-Arg-ß-naphthylamide had no significant effect upon the accumulation of garenoxacin or clinafloxacin. Verapamil increased the SSCs of ciprofloxacin (P = 0.0201), moxifloxacin (P = 0.019) and garenoxacin (P = 0.0154), again reflecting the effect upon MIC values. The SSC of garenoxacin increased most, from 26 ± 5.8 ng garenoxacin/mg dry cells to 49.1 ± 3.9 ng garenoxacin/mg dry cells. The increase in the SSC of clinafloxacin in the presence of verapamil was not statistically significant (P = 0.2975). Sodium orthovanadate had no effect upon the SSC of any agent (Table 3).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To establish what, if any, type of putative efflux pumps operate in B. fragilis, accumulation experiments were performed with five known efflux pump inhibitors, which are thought to inhibit different types of efflux systems. CCCP was used as it is a protonophore that dissipates the proton motive force (pmf) across the cytoplasmic membrane. Efflux pumps that are pmf-driven include the RND pumps AcrAB and MexAB systems found in E. coli and P. aeruginosa, respectively.24 In the presence of CCCP, there was an increase in the concentration of accumulated ciprofloxacin, moxifloxacin, levofloxacin and gatifloxacin, suggesting the existence of at least one pmf-driven efflux pump, for which these fluoroquinolones are substrates. Although CCCP acts upon RND-type pumps that use the pmf, it will also have an impact on other targets in or on the cell, which could indirectly or directly affect fluoroquinolone uptake and/or efflux. Phe-Arg-ß-naphthylamide has been identified as an inhibitor of the RND family of transporters in Gram-negative bacteria.25 It is thought that this inhibitor binds to specific sites in the efflux pump protein, and that some antibiotics interact with those same amino acids, so that when the inhibitor is present efflux of the antibiotic is prevented. Phe-Arg-ß-naphthylamide and CCCP increased the SSC of the same agents, but the effect was much less pronounced with Phe-Arg-ß-naphthylamide. This may be because it is a more specific efflux pump protein inhibitor than CCCP, a general pmf uncoupler.
Reserpine has been shown to inhibit members of the major facilitator (MF) family of transporters, including NorA from S. aureus26 and BmrA from Bacillus subtilis.27 Inhibition of BmrA by reserpine is based on direct interactions with the efflux pump protein. Reserpine is also known to act on RND efflux pumps. Reserpine enhanced the concentration accumulated of all six agents, suggesting that they were all effluxed by an efflux pump belonging to the MF or RND families. CCCP had no effect upon the accumulation of garenoxacin and clinafloxacin, but the effect of reserpine was strong. Pumps of the MF and RND families in other bacteria are both protondrug antiporters; thus it may have been expected that CCCP would affect the accumulation of garenoxacin and clinafloxacin, as was seen with reserpine. One hypothesis to explain these data is that there is a pump that transports clinafloxacin and garenoxacin, and that reserpine also directly interacts with this protein. Similar observations have also been made with S. pneumoniae.23 Verapamil increased the concentration of three agents accumulated, perhaps suggesting that these quinolones are substrates for another efflux pump protein that also belongs to the MF family. However, MIC data suggest that reserpine and verapamil are also inhibitors of B. thetaiotaomicron BexA, a member of the MATE family of efflux transporters. The data in the present study do not allow differentiation between the type of pump protein present, but it is postulated that clinafloxacin and garenoxacin are not substrates of a pump belonging to either the MF or RND families, but possibly a MATE pump. In support of this hypothesis, analysis of the genome of B. fragilis suggests that there are members of both the MF and MATE families in this species (unpublished analyses).
Sodium orthovanadate was used as it is an ATPase inhibitor and inhibits ATP-dependent efflux systems, such as ABC transporters.28 Sodium orthovanadate had no effect upon accumulation of any of the quinolones used in the study, suggesting that no ATP-dependent efflux pumps exist in B. fragilis.
Accumulation of all six quinolones by B. fragilis NCTC 9343 was determined, and indicated that the quinolones with the greatest anti-anaerobic activity accumulated to the lowest SSC. However, the quinolone with the least anti-anaerobic activity (ciprofloxacin) accumulated to the highest SSC. Similar data were obtained with five of the six agents and S. pneumoniae.23 This is counter-intuitive as it was hypothesized that the higher the concentration accumulated the greater the antibacterial effect the agent would have. Despite accumulating poorly, there were sufficient levels of clinafloxacin and garenoxacin to allow interaction between drug and target enzyme(s). This greater potency exhibited by clinafloxacin and garenoxacin could be due to their greater ability to enhance stabilization of cleavable complexes. Quinolones inhibit DNA gyrase activity by stabilizing the enzymeDNA complex, termed the cleavable complex. The complex is thought to be bactericidal upon release of the cleaved DNA.29 When the ratio of MIC/SSC is calculated it is observed that, except for ciprofloxacin, the ratio for each quinolone is very similar. The MIC presumably reflects the interaction of the quinolone with the target alone, and for those very active quinolones, which interact at very low concentrations, accumulation has little, if any, impact upon this.
The data from the present study has provided evidence for the presence of active efflux pumps in the B. fragilis type strain NCTC 9343/ATCC 25285, which may belong to the MF, RND and MATE families. It may be that over-expression of one or more such pumps will lead to quinolone resistance. These data also suggest, as with other species, that more than one pump can transport these agents and that each pump has overlapping, but distinct substrates.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Miyamae, S., Nikaido, H., Tanaka, Y. et al. (1998). Active efflux of norfloxacin by Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 42, 211921.
3 . Brogan, O., Garnett, P. A. & Brown, R. (1989). Bacteroides fragilis resistant to metronidazole, clindamycin and cefoxitin. Journal of Antimicrobial Chemotherapy 23, 6602.[ISI][Medline]
4 . Lamothe, F., Fijalkowski, C., Malouin, F. et al. (1986). Bacteroides fragilis resistant to both metronidazole and imipenem. Journal of Antimicrobial Chemotherapy 18, 6423.[ISI][Medline]
5 . Rotimi, V. O., Duerden, B. I., Ede, V. et al. (1979). Metronidazole-resistant Bacteroides from untreated patient. Lancet 1, 833.
6 . Snydman, D. R. & Cuchural, G. J. (1994). Susceptibility variations in Bacteroides fragilis: a national survey. Infectious Disease in Clinical Practice 3, S3443.[ISI]
7
.
Hoellman, D. B., Kelly, L. M., Michael, R. J. et al. (2001). Comparative anti-anaerobic activity of BMS 284756. Antimicrobial Agents and Chemotherapy 45, 58992.
8
.
Milatovic, D., Schmitz, F-J., Brisse, S. et al. (2000). In vitro activities of sitafloxacin (DU-6859a) and six other fluoroquinolones against 8,796 clinical bacterial isolates. Antimicrobial Agents and Chemotherapy 44, 11027.
9
.
Snydman, D. R., Jacobus, N. V., McDermott, L. A. et al. (2000). Comparative in vitro activities of clinafloxacin and trovafloxacin against 1,000 isolates of Bacteroides fragilis group: effect of the medium on test results. Antimicrobial Agents and Chemotherapy 44, 171012.
10 . Davidson, R. J., De Azavedo, J., Bast, D. et al. (2000). The activity of BMS-284756, a novel des-(6)F-quinolone, against ciprofloxacin susceptible and nonsusceptible Streptococcus pneumoniae. In Program and Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 2000. Abstract E1053, p. 175. American Society for Microbiology, Washington, DC, USA.
11
.
Fung-Tomc, J. C., Minassian, B., Kolek, B. et al. (2000). Antibacterial spectrum of a novel des-fluoro(6)quinolone, BMS-284756. Antimicrobial Agents and Chemotherapy 44, 33516.
12 . Lawrence, L. E., Frosco, M., Ryan, B. M. et al. (2000). Bactericidal activity of BMS-284756, a novel des-F(6)-quinolone, against Staphylococcus aureus strains with topoisomerase mutations. In Program and Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 2000. Abstract E1044, p.174. American Society for Microbiology, Washington, DC, USA.
13
.
Takahata, M., Mitsuyama, J., Yamashiro, Y. et al. (1999). In vitro and in vivo antimicrobial activities of T-3811ME, a novel des-F(6)-quinolone. Antimicrobial Agents and Chemotherapy 43, 107784.
14
.
Snydman, D. R., Jacobus, N. V., McDermott, L. A. et al. (2002). In vitro activities of newer quinolones against Bacteroides fragilis group organisms. Antimicrobial Agents and Chemotherapy 46, 32769.
15 . Kirby, J. T., Mutnick, A. H., Jones, R. N. et al. (2002). Geographic variations in garenoxacin (BMS284756) activity tested against pathogens associated with skin and soft tissue infections: report from the SENTRY Antimicrobial Surveillance Program (2000). Diagnostic Microbiology and Infectious Disease 43, 3039.[CrossRef][ISI][Medline]
16 . Piddock, L. J. V. (1999). Mechanisms of fluoroquinolone resistance: an update 19941998. Drugs 58, Suppl. 2, 118.[ISI][Medline]
17
.
Ricci, V. & Piddock, L. J. V. (2000). Accumulation of norfloxacin by Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 44, 23616.
18
.
Miyamae, S., Ueda, O., Yoshimura, F. et al. (2001). A MATE family multidrug efflux transporter pumps out fluoroquinolones in Bacteroides thetaiotaomicron. Antimicrobial Agents and Chemotherapy 45, 33416.
19 . Asuquo, A. E. & Piddock, L. J. V. (1993). Accumulation and killing kinetics of fifteen quinolones for Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 31, 86580.[Abstract]
20
.
Kappel, E. M., Shakibaei, E., Bello, A. et al. (2002). Effects of the des-F(6)-quinolone garenoxacin (BMS-284756), in comparison to those of ciprofloxacin and ofloxacin, on joint cartilage in immature rats. Antimicrobial Agents and Chemotherapy 46, 33202.
21 . Ashby, J., Piddock, L. J. V. & Wise, R. (1985). An investigation of the hydrophobicity of the quinolones. Journal of Antimicrobial Chemotherapy 16, 8058.
22 . Bazile, S., Moreau, N., Bouzard, D. et al. (1992). Relationships among antibacterial activity, inhibition of DNA gyrase, and intracellular accumulation of fluoroquinolones. Antimicrobial Agents and Chemotherapy 36, 26227.[Abstract]
23
.
Piddock, L. J. V. & Johnson, M. M. (2002). Accumulation of ten fluoroquinolones by wildtype and efflux mutant Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 46, 81320.
24 . Poole, K. (2001). Multidrug resistance in Gram-negative bacteria. Current Opinions in Microbiology 4, 5008.[CrossRef]
25
.
Lomovskaya, O., Warren, M. S., Lee, A. et al. (2001). Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrobial Agents and Chemotherapy 45, 10516.
26 . Klyachko, K. A., Schuldiner, S. & Neyfakh, A. A. (1997). Mutations affecting substrate specificity of the Bacillus subtilis multidrug transporter Bmr. Journal of Bacteriology 179, 218993.[Abstract]
27
.
Aeschlimann, J. R., Dresser, L. D., Kaatz, G. W. et al. (1999). Effects of NorA inhibitors on in vitro antibacterial activities and postantibiotic effects of levofloxacin, ciprofloxacin, and norfloxacin in genetically related strains of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 43, 33540.
28 . Paulsen, I. T. & Lewis, K. (2001). Microbial multidrug efflux: introduction. Journal of Molecular Microbiology and Biotechnology 3, 1434.[ISI][Medline]
29 . Drlica, K. & Zhao, X. (1997). DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiology and Molecular Biology Reviews 61, 37792.[Abstract]
30 . Mortimer, P. G. S. & Piddock L. J. V. (1991). Comparison for the methods used for measuring the accumulation of quinolones into Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 28, 63953.[Abstract]