Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School, University of Birmingham, Birmingham B15 2TT, UK
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Abstract |
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Introduction |
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The factors affecting the accumulation of quinolones by Escherichia coli and other Gram-negative bacteria have been studied, but less is known about accumulation by S. aureus. The physical properties of quinolones (e.g. relative hydrophobicity, charge or molecular mass) are important for penetration into the Gram-negative bacterial cell. Increasing molecular mass, hydrophobicity and negative charge hinder penetration of antibiotics into Gramnegative organisms through the porin channels, although hydrophobic molecules appear to enter via the lipopolysaccharide or across the lipid bilayer.3,4 Gram-positive bacteria do not possess an outer membrane, and so lack outer membrane proteins and lipopolysaccharide. Accumulation by S. aureus is thought to take place by simple diffusion across the cytoplasmic membrane5,6 but an active efflux mechanism has been identified in staphylococci which reduces accumulation. The norA locus codes for the protein NorA, which appears to straddle the cell membrane and act as an active efflux pump of hydrophilic quinolones.7 NorA-mediated increased efflux has been described for quinolone-resistant S. aureus.8 Mutations in regulatory genes controlling the expression of NorA have been shown to result in overexpression of the gene,9,10 and a regulatory locus, arlR-arlS, has recently been described that is involved in the control of norA expression.11
Asuquo & Piddock12 compared the kinetics of quinolone accumulation in S. aureus with that into E. coli and Pseudomonas aeruginosa. They concluded that accumulation was more rapid, and that quinolones were accumulated to a higher concentration by S. aureus than by Gram-negative organisms. No correlation was found between the antibacterial activity of each agent and the amount of quinolone accumulated in S. aureus, which confirmed earlier findings.13,14 Takenouchi et al.15 investigated the relationship between the structure and hydrophobicity of quinolones and their activity against a NorA-overproducing mutant of S. aureus and its parent strain. They found that decreased activity of the quinolones correlated with the bulkiness of the C7 substituent and the bulkiness and hydrophobicity of the C8 substituent, but not with hydrophobicity. It has been proposed that for Gram-positive organisms, high molecular weight is not a barrier to penetration.2,12
The proportion of charged and uncharged microspecies of quinolones has been shown to affect accumulation in the bacterial cell.16 Quinolone species with no net charge (uncharged HQ0 and zwitterion HQ± forms) penetrate the cytoplasmic membrane of S. aureus more readily than charged species (HQ+ and HQ).17,18 Alovero et al.19 found that accumulation of sulphanilyl fluoroquinolones into S. aureus was decreased three-fold in the presence of magnesium ions and increased at lower pH. They suggested that compounds like the sulphanilyl fluoroquinolones, which have only one ionizable group, are accumulated in the cell more favourably than are zwitterionic quinolones,18 and that this may be important for their enhanced in vitro activity against S. aureus, since the inhibition of DNA gyrase is similar for both groups of agents.
The aim of this study was to determine whether the physical properties of quinolones affect their accumulation and play a role in antibacterial activity against S. aureus, by comparing the accumulation of 10 structurally similar fluoroquinolone molecules, with differing antibacterial activities and hydrophobicities. The effect of the NorA efflux pump inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP) on accumulation of the 10 agents was also determined.
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Materials and methods |
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S. aureus F77 (NCTC type strain 8532) and E. coli I113 (NCTC 10418) were obtained from the National Collection of Type Cultures, Colindale, UK. S. aureus F145 was a laboratory mutant derived from F77, which was ciprofloxacin resistant but susceptible to nalidixic acid. SA-1199 (wild type), SA-1199B (grlA mutation and constitutive overproducer of NorA) and SA-1199-3 (inducible overproducer of NorA) were kindly donated by G. Kaatz, Washington State University.20 Ten structurally related fluoroquinolones were provided by Parke DavisWarner Lambert (Ann Arbor, MI, USA). All other antibacterial agents were provided as gifts from the respective manufacturers. Bacteriological media were supplied by Unipath (Basingstoke, UK) and chemicals by BDH or Sigma (Poole, UK).
Antibiotic susceptibility testing
The MICs of the 10 related fluoroquinolones (PD compounds) were determined by the microbroth doubling dilution method; MICs of all other agents were deter- mined by the agar doubling dilution technique as described previously.12,21
Hydrophobicity and fluorescence of PD quinolones
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.12 The excitation (ex) and emission (
em) maxima of the 10 PD compounds were determined in 0.1 M glycine pH 3 and in 50 mM phosphate buffer at pH 6, 7 and 8 (with and without boiling for 10 min) using a scanning spectrophotometer, and compared with other quinolones. The amount of fluorescence emitted by 0.1 mg/L quinolone in 0.1 M glycine pH 3 and in 50 mM phosphate buffer at pH 6, 7 and 8 was measured in a LS30 luminescence spectrophotometer (Perkin Elmer, Beaconsfield, UK).
Quinolone accumulation
The accumulation of each quinolone was determined between three and six times, using the modified fluorescence method.12 To investigate the role of the NorA efflux pump on accumulation of the PD agents by SA-1199, CCCP was added to a parallel set of tubes to a final concentration of 100 µM. In addition, the accumulation assays were repeated and, instead of using 0.1 M glycine pH 3 to lyse the cells, the cell pellet was resuspended in 50 mM phosphate buffer pH 7 and boiled for 10 min. The concentration of quinolone accumulated was determined by spectrofluorimetry using the appropriate excitation and emission maxima for the agent.
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Results |
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The chemical structures of the 10 PD quinolones were compared with those of common fluoroquinolones and nalidixic acid (Table I) for similarities in the R1, R7 and R8 substituents. All 10 PD compounds had only hydrogen at the R8 position (like ciprofloxacin, norfloxacin, temafloxacin and tosufloxacin), and all had a pyrrolidinyl group at position R7 similar to that of tosufloxacin. At the R1 position, PD112956 and PD114456 had an ethyl substituent like norfloxacin, and PD124979 and PD130390 had a cyclopropyl ring like ciprofloxacin and sparfloxacin. Both PD138086 and PD164534 had a 2,4-difluorophenyl substituent at the R1 position like temafloxacin and tosufloxacin. PD135498 and PD135671 had a t-butyl substituent at R1, PD164413 a cyclobutyl and PD164532 an isopropyl group (Table I
).
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The antibacterial activity of the 10 PD agents against the five strains of S. aureus was compared. Six PD agents (PD124979, PD130390, PD135498, PD135671, PD138086 and PD164534) and tosufloxacin were more active against each S. aureus strain than the other four PD agents (PD112956, PD114456, PD164413 and PD164532), nalidixic acid, ciprofloxacin, norfloxacin and temafloxacin (Table II). The MICs of pairs of agents that had identical groups at the R1 position but that differed only in the side chains on the pyrrolidinyl ring at position R7, were very similar: PD112956 and PD114456 (ethyl group at R1); PD124979 and PD130390 (cyclopropyl ring at R1); PD135498 and 135671 (t-butyl group at R1), PD138086 and PD164534 (2,4-difluorophenyl group at R1) (Tables I and II
). The structures of PD112956, PD135498 and PD164532 differed only in the R1 substituent and showed varying activities against S. aureus. PD135498 was the most active of these three agents with a t-butyl group at R1 (MICs 0.031 mg/L), PD112956 was less active (ethyl group; MICs 0.2516 mg/L), while PD164532 was the least active with an isopropyl group at R1 (MICs 0.532 mg/L).
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Physical properties of quinolones
The partition coefficient (Papp) determined for each PD agent (Table I) ranged from 0.119 (logP 0.924; PD114456) to 1.052 (logP 0.022; PD13548). The fluorescence excitation and emission maxima at pH 3, 6, 7 and 8 were determined for each PD agent and the comparative fluorescence at each pH was measured in arbitrary units. The excitation maxima differed by no more than 6 nm at different pH values for any of the PD compounds. The emission maxima for all the PD agents shifted to a shorter wavelength by approximately 2550 nm, as the pH increased. Six of the PD agents emitted more than twice the fluorescence at pH 7 than at pH 3 (PD114456, PD124979, PD130390, PD138086, PD164413 and PD164534). The remaining four agents (PD112956, PD135498, PD135671 and PD164532) fluoresced equally well at both pH values.
Quinolone accumulation
The steady-state concentrations (SSCs) of the PD quinolones accumulated by wild-type S. aureus SA-1199 did not correlate with the MICs of the agents, e.g. PD138086 (MIC 0.03 mg/L) was accumulated to approximately one-third the concentration of PD164532 (MIC 1 mg/L) by SA-1199 (Table II). Similarly, the SSCs of drugs accumulated by SA-1199 or F77 bore no correlation with the structure of the R1 or R7 substituent. For example, the SSCs of some agents with identical substituents at R1 accumulated by SA-1199 were broadly similar (e.g. for PD138086 and PD164534, which have a 2,4-difluorophenyl group at R1, the SSCs were 30 and 25 ng/mg, respectively; and for PD135498 and PD135671, which have a t-butyl group, the SSCs were 52 and 42 ng/mg). In contrast, other agents, such as PD112956 and PD114456, which have an ethyl group at R1, have SSCs of 14 and 54 ng/mg, while PD124979 and PD130390 (cyclopropyl group) have SSCs of 40 and 62 ng/mg, respectively. The concentrations of PD112956, PD135498 and PD164532 accumulated (which were structurally identical apart from the substituents at the R1 position), were quite different: 14, 52 and 87 ng/mg, respectively.
For all 10 PD agents, the presence of CCCP increased the concentration of quinolone accumulated by S. aureus SA-1199 (Table II). The accumulation of agents with an ethyl group at R1 (PD112956 and PD114456) and with a cyclopropyl group at this position (PD124979 and PD130390), was increased by CCCP approximately three- and two-fold, respectively. Although the SSC was increased 2.7-fold by the presence of the NorA efflux inhibitor CCCP for the least hydrophobic agent (PD114456; logP 0.924) compared with 1.3-fold for the most hydrophobic agent (PD135498; logP 0.022), there was no apparent correlation between logP and the fold increase of the SSC for the other agents in the presence of CCCP.
Six of the 10 PD agents emitted 50% less fluorescence at pH 3 than at pH 7. However, only for PD164534 was the accumulation by F77 significantly lower after cell lysis at pH 3 (46 ng/mg) than at pH 7 (121 ng/mg) (Table I). Accumulation of the 10 PD agents by SA-1199 was determined only using cell lysis at pH 3, but none of the six agents that emitted less fluorescence at pH 3 were accumulated to lower concentrations compared with the other agents.
There was no apparent relationship between the accumulation of the 10 PD agents, measured following cell lysis in pH 3 glycine or in pH 7 phosphate buffer, with boiling, and the logP of each agent for S. aureus F77 (r = 0.042 and r = 0.320, respectively, where r is the correlation coefficient) or E. coli I113 (r = 0.162, r = 0.339; data not shown). A weak positive correlation (r = 0.399) was shown between accumulation into S. aureus F77 and molecular mass after cell lysis at pH 7. However, the omission of the data point for PD135671 yielded a correlation equation of coefficient of 0.745 (Figure 1). However, there was no apparent relationship between accumulation and molecular mass for S. aureus following lysis in pH 3 glycine (r = 0.361), or E. coli I113 following cell lysis at either pH 3 or pH 7 (r = 0.151, r = 0.492), nor between accumulation and MIC for S. aureus F77 (r = 0.290, r = 0.115) or E. coli I113 (r = 0.233, r = 0.534) with lysis at either pH (data not shown).
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Discussion |
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The concentration of quinolone accumulated did not correlate with the MIC for S. aureus F77 or SA-1199, as some agents that were accumulated to a high concentration also had a high MIC for S. aureus. This is in agreement with earlier studies.2,12,26 While the MIC data demonstrated that the R1 group was important for antibacterial activity, there was no clear relationship between the chemical structure and the concentration accumulated for the 10 agents. The data concur with earlier studies which have concluded that while quinolone structure does affect the interaction with the target, it has little influence on the concentration of the agent accumulated.2,14
The concentration of all the PD agents accumulated by SA-1199 was increased in the presence of CCCP, suggesting that NorA was inhibited. However, the concentration of those agents with an ethyl or cyclopropyl group at the R1 position accumulated by SA-1199 was two- to three-fold higher in the presence of CCCP, which may suggest a link between quinolone structure and the affinity for the NorA efflux pump. Piddock et al.26 reported variation in the fold increase of the concentration of quinolone accumulated by E. coli in the presence of CCCP for different agents, and proposed that the variance could reflect different affinities of an efflux pump for each quinolone. McCaffrey et al.6 found that CCCP had little effect on the accumulation of ofloxacin and fleroxacin by S. aureus, while others have shown increased activity or accumulation of the hydrophilic quinolones, norfloxacin and ciprofloxacin, in the presence of efflux inhibitors.7,8,27,28 Takenouchi et al.15 demonstrated an increase in norfloxacin accumulation by SA-1199 and SA-1199B in the presence of CCCP, while the accumulation of CS-940 was not affected. The variability in the reported effects upon efflux by CCCP and other efflux inhibitors appears to be dependent on the quinolone and the bacterial strain used and may reflect the affinity of the quinolone as a substrate for NorA, where those quinolones that are a poor substrate for the carrier protein are expelled from the bacterial cell less efficiently.15
Although NorA has been proposed to have a greater affinity for hydrophilic quinolones,7 in this study, the fold increase of the SSC for SA-1199 in the presence of CCCP did not correlate with logP. Some studies have found a correlation between the concentration of fluoroquinolone accumulated and molecular hydrophobicity for S. aureus,2,6,7 but none was observed in the present study or others.12 It remains to be established whether accumulation is determined primarily by hydrophobicity or by quinolone structure. Further investigation of how the quinolone structure affects affinity for the NorA pump would be interesting.
In previous studies in this laboratory it had been observed that some agents were accumulated to low concentrations in bacteria, and that some of these agents also fluoresced poorly. To address the question of whether the low SSCs were an artefact of the method using lysis at pH 3, the fluoresence was determined at a range of pHs. In addition, the SSC was determined for S. aureus F77 after cell lysis at both pH 3 and pH 7. Although six of the 10 PD quinolones emitted less than half the fluorescence at pH 3 than at pH 7, the differences observed in the fluorescence were not reflected in differences in the measured accumulation using the two methods of cell lysis. This is as would be expected, as standard solutions used for calibration were prepared in the same pH medium used for cell lysis for each method. The preparation of quinolone standard solutions in the lysis buffer should negate any differences due to low fluorescence at pH 3. However, any errors in measuring the concentration of quinolone in the lysed cell solution would be greater where less fluorescence is emitted. Therefore, assuming cell lysis was complete under both conditions used, the measurement of accumulation should be more accurate at pH 7, where 6/10 PD agents were more fluorescent, than at pH 3.
The accumulation of the PD agents by S. aureus bore no relationship to either logP or the MIC. However, previous workers have reported a positive correlation between quinolone accumulation and logP for S. aureus.2,6 Asuquo & Piddock12 found no direct relationship between hydrophobicity or molecular mass and accumulation or with antibacterial activity. In addition, Bazile et al.2 found that a more bulky and hydrophobic quinolone (BMY 40397; logP 1.05) was accumulated to a high concentration by S. aureus, demonstrating that molecular mass is not a limiting factor for accumulation. In the present study, a positive correlation (r = 0.745) was shown between accumulation of 9/10 PD agents in S. aureus F77 and molecular mass following cell lysis at pH 7. The cause of this apparent association observed when cell lysis was carried out at pH 7 and not pH 3, is not clear, but may be a consequence of the differential fluorescence of the agents at the two pHs.
Recently, it has been proposed that the activity of quinolones against S. aureus is dependent on the hydrophobicity and/or bulkiness of the substituents at R7 and R8, and not the molecular hydrophobicity.15 Bulkiness of the R7 substituents of the PD agents used in this study was estimated by comparison with the values determined by Takenouchi et al.15 for identical or similar substituents. No correlation was observed between either the bulkiness of the R7 group and SSCs in F77, or the MIC increment ratio of SA-1199B and SA-1199 for this range of quinolones (data not shown).
Nikaido & Thanassi18 discussed the dynamics of quinolone accumulation during the initial rapid phase and suggested that for resistant strains that actively efflux fluoro-quinolones from the cell (such as S. aureus expressing norA), a higher rate of influx may enhance antibacterial activity. Re-analysis of the data from Asuquo & Piddock12 shows that for S. aureus there is a direct relationship between the rate of accumulation of fluoroquinolones during the initial rapid phase and hydrophobicity (r = 0.699; Figure 2), suggesting that the initial rate of influx is dependent on hydrophobicity. Further study of the complex relationship of quinolone accumulation by S. aureus, particularly during the initial rapid phase, with hydrophobicity, molecular mass and structure is warranted, and will aid the development of new quinolone antibacterial agents with enhanced activity against S. aureus.
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Acknowledgments |
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Notes |
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References |
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Received 25 May 2000; returned 6 September 2000; revised 23 October 2000; accepted 12 November 2000