Accumulation of garenoxacin by Bacteroides fragilis compared with that of five fluoroquinolones

Vito Ricci and Laura Piddock*

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Garenoxacin is a novel des-F(6)-quinolone with good anti-anaerobe activity. The accumulation of garenoxacin and five other quinolones in the presence and absence of a variety of efflux pump inhibitors, including carbonyl cyanide m-chlorophenyl hydrazone (CCCP: 100 µM), verapamil (25 µM), reserpine (20 mg/L), sodium orthovanadate (50 µM) and Phe-Arg-ß-naphthylamide (MC-207110) (20 mg/L) was investigated.

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteroides fragilis is the anaerobic organism isolated most frequently from patients with intra-abdominal abscesses.1 Bacteroides species are involved in 60% of cases of anaerobic intra-abdominal infections following a perforated appendix or surgery on the gastrointestinal tract, and of the organisms isolated, B. fragilis accounts for 30%–60% of the causative agents.2 B. fragilis infections, like most anaerobic infections, are treated usually with combinations of antibiotics such as metronidazole, clindamycin and a third-generation cephalosporin. However, investigators have reported metronidazole-resistant B. fragilis,35 and the number of strains resistant to clindamycin and most cephalosporins is also increasing.6 Fluoroquinolones have been suggested as possible alternative treatment, as the new generation of fluoroquinolones have good in vitro activity against a broad spectrum of anaerobes including B. fragilis.79

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 aerobic–anaerobic 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains, antimicrobial agents, growth conditions and susceptibility testing

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.


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Table 1. Physical properties of each agent
 
Statistical analysis

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 Student’s t-test. A P value of <0.05 was considered significant. Correlation coefficients (r) were calculated using the CORREL statistical function in Excel.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Activity and accumulation of the six quinolones by B. fragilis

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).


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Table 2. Susceptibility of B. fragilis to the six quinolones ± five efflux pump inhibitors
 
As found previously with norfloxacin, all agents accumulated rapidly, and had kinetics typical of these agents seen formerly with aerobic bacteria: an initial rapid linear phase of accumulation, achieving a steady state concentration (SSC) within 5 min of exposure (data not shown). Ciprofloxacin, which had the least anti-anaerobic activity, accumulated to the highest concentration, achieving an SSC of 95.5 ± 3.8 ng ciprofloxacin/mg dry cells (Table 3). The agent with the greatest anti-anaerobic activity, clinafloxacin, accumulated to the lowest concentration, achieving an SSC of 14.4 ± 2.0 ng clinafloxacin/mg dry cells. The rate of accumulation during the initial rapid linear phase of accumulation also differed between the quinolones (Table 3). During the initial period, ciprofloxacin and garenoxacin accumulated at the slowest rate, 0.159 and 0.144 ng quinolone/mg cells/s, respectively. The other agents accumulated at a more rapid rate, 0.21–0.36 ng quinolone/mg dry cells/s. There was no correlation between the Papp and the SSC (r = 0.52) or the initial rate (r = 0.51). However, the molecular mass of the free form of each agent influenced both the initial rate (r = 0.85) and the SSC (r = 0.84), such that those agents with the lowest mass accumulated fastest and to the highest concentration.


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Table 3. Initial rate and steady state concentrations of the fluoroquinolones alone and in the presence of the various efflux pump inhibitors
 
Effect of known efflux pump inhibitors on the concentration of fluoroquinolone accumulated by B. fragilis

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The physical properties of antibiotics (e.g. relative hydrophobicity, charge or molecular mass) are important for penetration into the Gram-negative bacterial cell. In the present study, ciprofloxacin, which has the lowest molecular mass, had the poorest activity for B. fragilis NCTC 9343/ATCC 25285, whereas those quinolones with high molecular masses, such as clinafloxacin and garenoxacin, had greater activity. Other studies have also shown a weak correlation between molecular mass and activity,19,21,22 but have suggested that a more important factor to take into consideration may not be the molecular mass but the three-dimensional shape of the fluoroquinolone molecule. A better relationship was observed between the molecular mass of the free form of each agent and the SSC: some agents with the lowest molecular mass achieved the highest SSC. Similar findings have been made for aerobic and microaerophilic bacteria.19,23 There was no correlation between hydrophobicity (Papp) and MIC, or hydrophobicity and SSC, suggesting that the hydrophobicity of an agent is not important for the activity against, or accumulation by, B. fragilis. Similar findings have been made previously for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus,19 but not for Streptococcus pneumoniae.23

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 proton–drug 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 enzyme–DNA 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
 
We are grateful to Bristol-Myers Squibb for funding this study.


    Footnotes
 
* Corresponding author. Tel: +44-121-414-6966; Fax: +44-121-414-3599; E-mail: l.j.v.piddock{at}bham.ac.uk Back


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 Results
 Discussion
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