Antimicrobial Agents Research Group, Department of Infection, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
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Abstract |
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Introduction |
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The higher concentrations of antibiotics, including quinolones, accumulated by bacterial cells in the presence of the PMF inhibitor carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) have been interpreted to suggest that an active efflux mechanism is inhibited, and in 1988 Cohen et al. 7 proposed that fluoroquinolones were transported out of E. coli by an energy-dependent, carrier-mediated efflux system energized by the PMF, which has been shown to be acrAB (a multiple-antibiotic efflux pump that is inhibited by CCCP) linked to TolC. 8 In both S. aureus 9 and P. aeruginosa 10,11 active fluoroquinolone efflux systems have been identified. However, the CCCP effect upon quinolone accumulation observed for wild-type S. aureus has also been interpreted for pefloxacin derived data to result from acidification of the intra-membrane space leading to enhanced binding of positively-charged pefloxacin to the inner leaflet of the negatively-charged phospholipid. 12 These authors demonstrated a CCCP effect at pH 6.6 in phosphatidylglycerol liposomes which are devoid of proteins but no effect was seen at the physiological pH of 7.4. In 1993, Nikaido & Thanassi 6 re-interpreted the data for Furet et al., 12 and proposed that this observation was due to a collapse in the pH gradient of the cytoplasmic membrane. It has also been shown that fluoroquinolones do not promote their own accumulation into cells of E. coli. 13
The aim of the present study was to investigate further the effects of pH, temperature, CCCP and increasing external quinolone concentration on the accumulation of 15 agents by E. coli, S. aureus and P. aeruginosa.
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Materials and methods |
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E. coli KL16 Hfr thi-1 relA1 spoT1/Str s, 14 S. aureus NCTC 8532 and P. aeruginosa NCTC 10662 were used throughout. All antimicrobials were obtained as gifts from their manufacturers: amifloxacin and nalidixic acid (Sterling-Winthrop, Guildford, UK); ciprofloxacin and enrofloxacin (Bayer, Newbury, UK); enoxacin and PD117596-2 (Warner Lambert/Parke Davis, Eastleigh, UK); norfloxacin (Merck Sharp and Dohme, Hoddesdon, UK); pefloxacin (May and Baker, Dagenham, UK) ofloxacin (Hoechst, Hounslow, UK); rufloxacin and MF961 (Mediolanum Farmaceutici, Milan, Italy); lomefloxacin (Searle, High Wycombe, UK); fleroxacin (Hoffman La Roche, Welwyn Garden City, UK); temafloxacin (Abbott Laboratories Ltd, Queensborough, UK); and tosufloxacin (Lederle Laboratories, Pearl River, NY, USA). The antimicrobials were used according to the manufacturers' instructions. All other chemicals were purchased from the Sigma Chemical Co. (Poole, UK).
Hydrophobicity of quinolones
The partition coefficent (Papp), after 2 h incubation, between the aqeuous and organic (n-octanol) phase for each agent has been reported previously. 15 The effect of pH upon the partition coefficient of nalidixic acid, a non-fluorinated, hydrophobic quinolone and ciprofloxacin, a fluorinated hydrophilic analogue, was determined as described by Asuquo & Piddock. 15 Sodium phosphate buffer has an optimum functional pH range of 6.57.5 and could not be used for either partition coefficient or accumulation assays performed outside this range, so partition coefficients of quinolones were not investigated at pH 5.6, 6.0 or 8.5 in phosphate buffer. It was also difficult to find a buffer with an optimum functional range between pH 5.6 and 8.5. Consequently, the partition coefficients of these quinolones were investigated in Bis-Tris and Bis-Tris propane buffers because of their apparent compatibility in parallel with those in sodium phosphate buffer between pH 6.5 and 7.5. The buffers were adjusted with sodium hydroxide and hydrochloric acid to obtain the appropriate pH.
Preparation of spheroplasts from E. coli KL16 and accumulation of quinolones
Late exponential phase cells (A530 of 0.80.9) were harvested by centrifugation at 7000g for 10 min at 20°C. The pellet was resuspended in approximately one-third of the original volume in phosphate-buffered saline (PBS) and re-centrifuged. The pellet was resuspended in one-tenth of the original volume in 30 mM TrisHCl in 20% sucrose (Trissucrose), pH 8.0. Disodium-ethylene diamine acetic acid pH 8.0 was added to the suspension to achieve a final concentration of 10 mM, followed by 20 g/L freshly prepared lysozyme to obtain a final concentration of 0.3 g/L. The mixture was incubated on a magnetic stirrer at room temperature with constant, gentle mixing by a magnetic stirring bar. After 30 min of incubation, spheroplast formation was verified by a decrease in osmotic fragility by >80% in distilled water as compared with Trissucrose and by a change in shape of the cells from rods to spheres as viewed under a 100x objective lens (oil immersion) of a light microscope. Spheroplasts were centrifuged at 3003g for 15 min at 20°C, washed in Trissucrose and re-centrifuged at 3003g at 20°C for 15 min. For quinolone accumulation assays, spheroplasts were concentrated 20-fold (A530 = 20) in the transport assay buffer (50 mM sodium phosphate pH 6.0, 20 mM lithium lactate, 20% sucrose, 1 mM magnesium sulphate) at room temperature and were immediately used in accumulation assays as described below.
Quinolone accumulation
The accumulation of all quinolones into E. coli and S. aureus was measured by
a fluorimetric uptake assay as described previously
15
but modified as follows: cells were harvested at 25°C and concentrated 20-fold. The cell or
spheroplast suspension (3 mL) was allowed to equilibrate with mixing for 10 min at 37°C.
From the cell suspensions two samples (0.5 mL; time-zero samples) were withdrawn into
ice-cold phosphate buffer (1 mL) on ice and the cells harvested immediately by centrifugation at
12,000g for 3 min at 4°C. The spheroplasts were cooled quickly and centrifuged at
1°C. Quinolone was added to the remaining tubes to attain final concentrations of 10, 25,
50, 75 and 100 mg/L. At timed intervals two samples were withdrawn into buffer and centrifuged
as for the antibiotic-free control. The harvested cells were resuspended in 0.1 M
glycineHCl, pH 3.0, and incubated at room temperature overnight, then centrifuged at
4°C for 5
min. The supernatant was re-centrifuged to remove any cell debris. The concentration of
quinolone in the supernatant was estimated by fluorimetry using a luminescence
spectrophotometer LS30 (Perkin Elmer, Beaconsfield, UK) at the appropriate excitation and
emission wavelengths of each agent.
15
The antibiotic-free control time-zero value was subtracted from the values obtained for each
timed sample. All experiments were performed twice on at least two separate occasions. In
previous studies
16
it has been determined by Student's t-test and analysis of variance that the
standard deviation of the values for a particular time point, regardless of whether the samples are
duplicates from the same assay tube or from an assay on a different day, are 10% of
the mean value. The same was found to be true in the present study.
For those agents for which the initial rate data suggested that the accumulation pathway was saturated, the experiment was carefully repeated by studying the kinetics, sampling every 10 s up to 1 min, then every 30 s up to 5 min, at each concentration and then analysing the data with the computer software Enzpack (Biosoft, Cambridge, UK). To investigate the role of an active efflux pump for 15 quinolones, CCCP was added to a parallel set of tubes to a final concentration of 100 µM.
The effect of pH upon the concentration of quinolone accumulated by E. coliand S. aureus was investigated with ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, lomefloxacin and nalidixic acid in Isosensitest broth. The appropriate pH was achieved by adjusting the pH of Isosensitest broth with sodium hydroxide or hydrochloric acid.
The effect of temperature (ranging from 0 to 60°C) upon accumulation into E. coliand S. aureuswas investigated with ciprofloxacin, enrofloxacin and nalidixic acid. In addition, the viability of E. coliand S. aureus was determined at 37, 50 and 60°C.
For P. aeruginosa the procedure did not employ centrifugation of the cells until after exposure to quinolones, nor did it expose the cultures to cold temperatures, as both conditions have previously been shown to affect the concentration of fluoroquinolone accumulated by this species. 15 Exponentially growing cells (100 mL, A470 of 0.60.7) in Isosensitest broth plus 0.5% glucose with mixing were maintained at 37°C. At time zero, 10 mL of the culture was removed to serve as the control. Quinolone was added to the remaining 90 mL of culture to the appropriate final concentration and at timed intervals 10 mL was withdrawn to ice-cold tubes embedded on ice, and centrifuged at 3003g at 4°C for 10 min in a precooled centrifuge. The pellets were resuspended in 1 mL of ice-cold 30 mM sodium phosphate buffer containing 0.5% glucose and re-centrifuged at 10,000g at 4°C for 3 min, and the supernatant discarded. Quinolone extraction and estimation were as above.
The amount of fluorescence, in arbitrary units, of each fluoroquinolone at 0.1 mg/L in lysis medium (50 mM phosphate buffer at pH 6, 7 and 8 and in 0.1 M glycine, pH 3) was determined in an LS30 luminescence spectrophotometer.
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Results |
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The Pappof nalidixic acid in phosphate buffer hardly varied with pH, whereas in Bis-Tris and Bis-Tris propane the Papp of this agent ranged from 17.98 at pH 5.6 to 0.184 at pH 8.5 (Figure 1). There was a decrease in the hydrophobicity of nalidixic acid as the pH increased from 5.6 to 8.5. The data obtained in sodium phosphate buffer and Bis-Tris at pH 6.5 were comparable, and with that obtained at pH 7.0 in Bis-Tris propane and pH 7.2 in phosphate buffer. However, at pH 7.5 the partition coefficient of this agent in phosphate buffer was more than double that obtained in Bis-Tris propane. The partition coefficients of ciprofloxacin in phosphate buffer did not show a dependence on pH and varied from -0.121 at pH 7.5 to 0.062 at pH 6.8. In Bis-Tris and Bis-Tris propane, the hydrophilicity of this agent increased with an increase in pH from 0.017 at 5.6, to -0.081 at pH 8.5.
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The concentration of ciprofloxacin and enrofloxacin accumulated by E. coli increased with an increase in temperature from 0°C to 40°C, then decreased at 50°C and 60°C (Figure 3a). The concentration of enrofloxacin accumulated was lower at 60°C. The concentration of nalidixic acid accumulated increased from 0°C to 30°C, but decreased above this temperature. For S. aureus, the concentration of ciprofloxacin and enrofloxacin accumulated increased from 0°C to 40°C and then decreased above this temperature (Figure 3b). The decrease in the accumulated concentrations of ciprofloxacin and enrofloxacin at 60°C was small compared with the decrease in the concentration of nalidixic acid accumulated. After 5 min at 50°C the number of cells was half that at 37°C, and the lower cell numbers correlated with the apparent lower accumulation of these agents, indicating that cell death, and not the effect of temperature upon the accumulation process per se, was responsible.
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The concentration of 15 quinolones, either alone or combined with 100µM CCCP, accumulated after 5 min exposure for spheroplasts and intact cells in phosphate buffer was determined. For fluoroquinolones the concentrations accumulated by spheroplasts (Table II, column 2) ranged between 17% and 73% of the concentration accumulated by intact cells Table II, column 1). It was hypothesized that the lower concentrations of fluoroquinolone accumulated by spheroplasts was due to active efflux across the cytoplasmic membrane into the transport medium, whereas intact cells would efflux via the cytoplasmic membrane, followed by a periplasm-spanning protein and an outer membrane protein. Addition of CCCP to spheroplasts (to inhibit the putative efflux system) increased the concentration accumulated (Table II, column 5), but did not give rise to concentrations similar to those seen with with intact cells in the absence of CCCP (Table II, column 1). CCCP also increased the concentration of all agents accumulated by intact cells (Table II, column 4). For seven of the agents the increase in the concentration accumulated by intact cells in the presence of CCCP was approximately three-fold or higher than in the absence of CCCP (Table II, column 8); however, the percent increase for spheroplasts in the presence of CCCP (Table II, column 9) was lower than that seen for intact cells for half of the drugs tested.
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Effect of external concentration of 15 quinolones upon accumulation by E. coli, S. aureus and P. aeruginosa
The concentrations of each agent accumulated after exposure of the bacteria to 10 mg/L were similar to those obtained previously 15 and are typical values for each quinolone and these species. For intact cells of E. coli and 12/15 quinolones, and for S. aureus and ten agents, with an increase in drug concentration there was a linear increase in the concentration accumulated (data not shown). For spheroplasts of E. coli a linear increase was observed for ten agents. For three agents and E. coli spheroplasts, despite repeated experiments the data could not be analysed by the computer software.
P. aeruginosa behaved marked differently, with apparent saturation of the accumulation pathway for 13 agents (Table III). Only for two quinolones (MF961 and PD117596) was a linear increase observed.
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Discussion |
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For P. aeruginosa the data suggest that the apparent decrease in the accumulation of quinolones after attaining a peak concentration reported previously 15 and also observed by Li et al. 10 may have been the result of leaky cell envelopes injured by low temperature and centrifugation. 20 For the present study the method was modified so that the organism was not centrifuged or exposed to low temperatures before the accumulation assay; this had no effect upon the maximum concentration attained, but substantially reduced the amount of quinolone loss. We recommend that, when measuring the accumulation of any antibiotic into P. aeruginosa, this modification be included if appropriate, otherwise erroneous data may be obtained.
The reason for lack of dependence on pH of the partition coefficients of nalidixic acid and ciprofloxacin in phosphate buffer compared with Bis-Tris is not certain, although differences in the composition of the two buffers may cause the discrepancy. It is also not certain if the large difference in the data obtained for nalidixic acid in Bis-Tris and Bis-Tris propane is due to a pH effect or to a difference in the buffers. The increase in the hydrophilicity of both agents with an increase in pH in Bis-Tris and Bis-Tris propane, suggests that both agents are potential candidates for the porin pathway of accumulation, since there are more zwitterionic species of the agents at pH around neutrality.
The fluorescence of most agents decreased with alkaline pH, so the increase in the concentration of drug accumulated at alkaline pH is not a result of enhanced fluorescence at these pH. The lower concentrations of quinolones accumulated at acid pH for both E. coli and S. aureus in the present study has been shown previously for pefloxacin 5,12 and norfloxacin, 20 and our data confirm and extend these previous observations. In the present study, unlike that of Valisena et al., 21 there was no significant decrease in the concentration of pefloxacin accumulated at pH 8.5 for S. aureus. The reason for the discrepancy in the data is uncertain, although differences in methodology may be partly responsible.
Several factors may be responsible for the lower concentrations of quinolones accumulated at acidic pH. For instance, differences in ionic binding to cell components such as lipopolysaccharide 5 may account for this observation. Piddock 4 suggested that the effect of pH was related to the acid base equilibrium of the quinolone. At neutral pH, quinolones exist mostly as zwitterions which favour their diffusion through the hydrophilic porin channels of Gram-negative bacteria, whereas at acid pH quinolones exist as a charged species (HQ2+) which does not penetrate the porin channels. These investigations into the effect of pH on the hydrophobicity of nalidixic acid and ciprofloxacin showed that the hydrophobicity of both agents increased as pH increased from acidic to neutral pH, suggesting that at neutral pH more of these agents existed in the zwitterionic form which favour penetration through porin channels. Overall these data suggest that fluoroquinolones penetrate bacteria by simple diffusion. The decrease in the hydrophobicity of nalidixic acid with increased pH and the increased concentration accumulated at acid pH in S. aureus are not surprising given the proposal, by Nikaido & Thanassi, 6 that the uncharged fraction is in a greater proportion for acidic quinolones at acidic pH and that the uncharged species is accumulated. These data also suggest that the decreased hydrophobicity of nalidixic acid at neutral and basic pH may enhance the diffusion of this agent across the outer membrane; this may account for the increase in the concentration of this agent accumulated at pH 7.4 8.5 and higher concentrations accumulated by E. coli, but not S. aureus. If only uncharged species of nalidixic acid mediate permeation, this explains why high concentrations of this agent are required to detect quinolone accumulation in bacteria. It cannot be ruled out that the observed effect is a result of the bactericidal action of nalidixic acid, but in a previous study with this agent and fluoroquinolones, similar data were obtained after a 5 min exposure when most of the cells were shown to be viable. 1
The observed low concentration of quinolones accumulated at low temperatures has been reported previously for pefloxacin 5 and enoxacin 22 and was attributed to a decrease in cell metabolism and membrane fluidity at low temperature. At 60°C, the cell viability was reduced and this accounted for the apparent decrease in the concentration of quinolones accumulated.
Further support for the hypothesis that most quinolones are accumulated by S. aureus and E. coli via a process of energy-independent, simple diffusion was derived from the experiments with a range of quinolone concentrations, showing that for 10/15 and 12/15 quinolones and S. aureus and E. coli, respectively, the concentration accumulated increased in a linear fashion with an increase in the external concentration. For three agents and E. coli, and for five agents and S. aureus, the data indicated that saturation may occur at high concentrations. For E. coli there were insufficient data to draw any conclusions, but for S. aureus there was an apparent relationship between the affinity of a putative transporter and the MIC of the same agents. It is possible that if even higher concentrations of quinolone were investigated than those in the present study that saturation would be observed for more agents. This would especially be the case if the carrier molecule had low affinity for fluoroquinolones, with higher affinity for some agents than for others.
For P. aeruginosa the data implied that for 12/15 agents there was apparent saturation of an accumulation pathway. Although efflux of fluoroquinolones from P. aeruginosa has been demonstrated, and the proteins involved have been identified, 10,11 saturation of an efflux pathway would give rise to apparently higher concentrations of drug accumulated, as when efflux is inhibited by CCCP.
The apparent saturation of accumulation observed for all three species, but in particular P. aeruginosa,could result from one or more factors. Firstly, the transport process into the cell could be mediated by a carrier protein, probably located in the cytoplasmic membrane. In addition to apparent saturation, for P. aeruginosa there was also a correlation between the Vmax values and the MIC, those with the greatest velocity also being the most active. The previous lack of data to support the hypothesis that fluoroquinolones enter P. aeruginosa via a carrier-mediated process may be a consequence of the fact that only a few agents have been investigated, and not always because of the high concentrations used in the present study. However, many mutant E. coli, S. aureus and P. aeruginosa have been selected in the last 10 years, and there has been no description of a transport mutant with defective uptake into the cell.
Secondly, the observed effect could be a result of experimental artefacts. For example, calculation of initial rates could have been inaccurate because of the long periods of drug exposure (exposure during accumulation experiment plus centrifugation times); however, at each sampling time the cells were immediately cooled then centrifuged. This has previously been shown to prevent efflux of drug. 23 There is also linearity of the rates of accumulation at the early sampling time points (data not shown).
P. aeruginosa is lysed by chelators such as EDTA, so it is possible that, at high fluoroquinolone concentrations, drug-mediated chelation of divalent cations results in cell lysis with drug release, creating an artifical plateau and apparent Vmax. However, supplementing the medium with 20 mM magnesium chloride did not abolish the phenomenon, and no significant decrease in cell numbers has been observed (data not shown).
The experimental conditions may have been such that only the fluoroquinolone bound within the cell was retained and that free fluoroquinolone was washed out by the washing steps; however, this artefact would be present in all experiments with all three species and the `saturation' data are predominantly for P. aeruginosa. The procedure used for P. aeruginosa differed slightly from that used for the other two species because in previous experiments after reaching a maximum concentration there was significant loss of fluoroquinolone; 10,14 despite this the steady-state concentration from these experiments and the initial rates are remarkably similar to the results of previous experiments with the same method used with S. aureus and E. coli.
In addition, the values for accumulated fluoroquinolone reflect binding to a cellular compartment, and for P. aeruginosa the saturation observed at comparatively low quinolone concentrations may have been due to structural differences in the cell envelope of this species and not saturation of an accumulation process.
Finally, the observed effect could have been due to bactericidal action of the drugs, but a previous study obtained similar data after a 5 min exposure, 15 and as the most microbiologically active agents did not saturate, this is unlikely.
The CCCP-induced increase in the concentration of quinolones accumulated by bacteria has been variously interpreted. Early studies 7,24 proposed that CCCP blocks an energy-dependent efflux system for quinolones, with a consequent increase in the concentration of these agents accumulated. The CCCP effect has also been interpreted to be the result of a perturbation of the outer and the cytoplasmic membranes resulting in the influx of quinolones into bacteria. 5 Such perturbation was proposed to result from a collapse in the pH gradient of the cytoplasmic membrane by CCCP. 6 Evidence for this proposal was derived from the studies of Furet et al., 12 who demonstrated an increase in the concentration of pefloxacin into phosphatidylglycerol liposomes in the presence of CCCP at pH 5.2, but no effect was seen at pH 7.4. The present study demonstrated an increase in the concentration of most quinolones into a susceptible strain of E. coli in the presence of CCCP at pH 7.0, and that this increase was greater for intact cells than for spheroplasts. At the pH typically used in the measurement of accumulation of quinolones, i.e. pH 7.0, it is possible that differences between the external and intracellular pH may be more limited than at pH 5.2, the pH at which pefloxacin accumulation was measured with phosphatidylglycerol liposomes. However, because of the difference in pH between the external and the intracellular compartments, it is likely that most quinolones accumulate in the periplasm of E. coli and cytoplasm of S. aureus by simple diffusion due to a concentration gradient. Therefore, the effect of CCCP observed in the present study at pH 7.0 probably does suggest the presence of an energy-dependent efflux for quinolones in E. coli, most likely via the AcrAB and TolC system. 6 ,23 ,25 The extent of the CCCP effect varied for the quinolone and for the species, perhaps reflecting substrate specificity of the efflux system.
In conclusion, the data for E. coli and S. aureus suggest that accumulation of most quinolones into these bacteria proceeds by simple diffusion. These data also suggest that broad conclusions for the class based upon data obtained with a single drug and one bacterial species are not possible.
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Acknowledgments |
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Notes |
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References |
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Received 23 February 1998; returned 28 May 1998; revised 6 July 1998; accepted 18 August 1998