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JAC Advance Access originally published online on January 12, 2007
Journal of Antimicrobial Chemotherapy 2007 59(2):246-253; doi:10.1093/jac/dkl489
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Combined effect of pH and concentration on the activities of gentamicin and oxacillin against Staphylococcus aureus in pharmacodynamic models of extracellular and intracellular infections

Pierre Baudoux, Nathalie Bles{dagger}, Sandrine Lemaire, Marie-Paule Mingeot-Leclercq, Paul M. Tulkens and Françoise Van Bambeke*

Unité de Pharmacologie cellulaire et moléculaire, Université catholique de Louvain, Brussels, Belgium

* Corresponding author. Tel: +32-2-764-73-78; Fax: +32-2-764-73-73; E-mail: vanbambeke{at}facm.ucl.ac.be

Received 8 July 2006; returned 1 September 2006; revised 19 September 2006; accepted 8 November 2006


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BACKGROUND: Staphylococcus aureus survives in acid media, including phagolysosomes. Conflicting in vitro/in vivo data exist on its susceptibility to antibiotics in such environments.

METHODS: Oxacillin and gentamicin activities against methicillin-susceptible S. aureus ATCC 25923 were compared extracellularly (broth; different pH) and assessed intracellularly (THP-1 macrophages), using a pharmacological approach (antibiotic concentrations: 0.01–1000 x MIC). Antibiotic cellular contents were determined by microbiological assay.

RESULTS: MICs and MBCs increased 72-fold for gentamicin, and decreased 8-fold for oxacillin between pH 7.4 and 5.0. Plots of log10 colony-forming unit changes at 24 h versus log10 of antibiotic concentration followed sigmoidal shapes, allowing calculation of EC50 (relative potency) and apparent Emax (relative efficacy) in all conditions. In broth, the EC50 of gentamicin rose 316-fold and that of oxacillin decreased 15-fold with unchanged apparent Emax [–5 log (limit of detection)] between pH 7.4 and 5. Intracellularly, EC50s were similar to those observed extracellularly at pH 7.4, but Emax values were much lower (–1 log) for both antibiotics. Calculations based on the assumed pH in phagolysosomes (5.4) and on local accumulation of antibiotics (gentamicin, 23-fold; oxacillin, 0.05-fold) suggest that the contrasting effects of acid pH on relative potencies of gentamicin and oxacillin could be almost exactly compensated for by differences in accumulation.

CONCLUSIONS: The weak activity of gentamicin and oxacillin towards intraphagocytic S. aureus compared with extracellular forms is not related to an overall decrease of their relative potencies but to impaired efficacy, suggesting the need for new approaches to improve the eradication of intracellular S. aureus.

Keywords: acid pH , ß-lactams , aminoglycosides , pharmacodynamics , antibiotic accumulation


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Staphylococcus aureus is a widespread pathogenic bacterium capable of surviving and multiplying in hostile environments. It shows a high tolerance to variations in pH,1 which confers an advantage for colonizing body sites characterized by a mildly acidic pH, like skin, mouth, vagina, urine and abscesses,2 where it may cause severe infections. Staphylococcus aureus is also able to survive and thrive intracellularly in acidic compartments such as the phagolysosomes of phagocytic cells,3,4 and this property is considered important to explain the recurrent and relapsing character of many staphylococcal infections.5–8

An acid environment is known to impair the activity of many antibiotics. For macrolides and aminoglycosides, lowering the pH markedly increases their MICs,9–11 which has been considered as a main reason for treatment failures in infections affecting tissues or biological fluids where pH is acidic,12 and for poor efficacy against intracellular forms of S. aureus.11,13 Yet, acid pH may have the opposite effect for other antibiotics, as evidenced by decreased MICs of ß-lactams at acidic versus neutral pH.11 Understanding these contrasting effects of pH on the activity of aminoglycosides and ß-lactams against S. aureus may prove critical for a correct evaluation of their potential usefulness in the treatment of infections where intracellular survival is likely to play a critical role. In the present work, we have used gentamicin and oxacillin to systematically compare the influence of pH towards S. aureus in broth and to examine their activities against its intracellular forms. We used a pharmacological model14 in which bacteria and cells were exposed to a wide range of drug concentrations for up to 24 h, allowing us to obtain detailed information on dose–effect relationships, while being able to draw microbiological and clinically relevant conclusions.


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Bacterial strain and determination of extracellular activity of antibiotics

All experiments were performed using a methicillin-susceptible strain of S. aureus (ATCC 25923) obtained from the American Tissue Cell Collection (Manassas, VA, USA). MICs and MBCs were determined in Mueller–Hinton broth or agar, respectively, adjusted to specific pH values by addition of 2 M HCl or NaOH (the pH being checked before and after incubation). Killing curve experiments were performed as described previously.14

Cell infection and determination of intracellular activity and cellular accumulation of antibiotics

All experiments were conducted with THP-1 macrophages, exactly as described previously.11,14 Cell associated antibiotics were assayed by a microbiological method (disc diffusion), with Bacillus subtilis ATCC 6633 as test-organism and antibiotic medium #11 adjusted at pH 8 for gentamicin, and S. aureus ATCC 25923 and antibiotic medium #11 adjusted at pH 5 for oxacillin. The apparent cellular/extracellular concentration ratio of antibiotics (Cc/Ce) was calculated as previously described.11,14

Analysis of the dose–response curves and statistical analysis

Data from the dose–response experiments were used to derive a pharmacological model based on the Hill equation (response versus log10 of drug concentration, using a slope factor of 1), which allowed calculation of the maximal efficacy, Emax, being the drug concentration causing a maximal effect, and the relative potency, EC50, defined as the drug concentration causing a response half-way between the effect in absence of drug (E0) and Emax; these are two key pharmacological descriptors of the activity of most drugs.15 (Details and application of these to different classes of antibiotics acting on S. aureus have been given earlier.)11 In this analysis, the following points should be borne in mind:

  1. Because antimicrobial effects, like those of all chemotherapeutic agents, consist of a fractional reduction of an original inoculum, the log10 of the colony count reduction needs to be used as descriptor of the response for curve-fitting analysis.
  2. Emax values of antimicrobial agents are—by definition and in contrast to most non-chemotherapeutic agents—negative numbers, since they pertain to decreases in bacterial counts; a larger activity is therefore, strictly speaking, associated with a smaller Emax. Since this is rather counterintuitive, we use the term ‘maximal activity’ to define the maximal reduction of bacterial counts observed.
  3. Our limit of detection is a reduction of approximately –5 log cfu compared with the original inoculum; since this reduction of colony-forming units was always reached for bacteria grown in broth when exposed to increasing concentrations of oxacillin or gentamicin; Emax for extracellular activity had to be arbitrarily set to that value for the purpose of our analysis.11 Emax values given here are, therefore, not meant to describe the maximal activity that could be observed for the drug if other experimental conditions had been used (such as the use of a more concentrated initial inoculum).
All curve fittings and determinations of the Emax and EC50 values were made using GraphPad Prism® version 4.02 for Windows (GraphPad Prism Software, San Diego, CA, USA). The apparent static concentration (drug concentration causing no apparent change compared with the original inoculum) was thereafter determined by graphic intrapolation. Analyses of variance (ANOVA), which compare means by splitting the overall observed variance into different parts, were made with GraphPad Instat® (GraphPad Prism Software); analyses of covariance (ANCOVA), a method testing whether certain factors have an effect after controlling for quantitative predictors, was made with XLSTAT Pro© (version 7.5.2; Addinsoft SARL, Paris, France). Tukey's test for multiple comparisons was used in both cases.

Materials

Gentamicin was procured from Glaxo-SmithKline-Belgium as the commercial product registered for parenteral use (GEOMYCIN®). Oxacillin was purchased from Sigma-Aldrich-Fluka (St Louis, MO, USA) in powder form for microbiological evaluation (potency, 93.9%). Cell culture media and fetal calf serum were purchased from Invitrogen (Paisley, Scotland, UK) and Difco (Sparks, MD, USA). Human serum for opsonization of S. aureus was obtained from healthy volunteers and stored at –80°C as pooled samples until use. Other reagents were purchased from E. Merck AG (Darmstadt, Germany) or Sigma-Aldrich-Fluka.


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Influence of pH on extracellular activities

Figure 1 shows how the MIC and MBC values of gentamicin and oxacillin are affected when determined in media adjusted to pH values ranging from 7.4 to 5.0. Acid pH drastically reduced the activity of gentamicin, the MIC of which was approx. 70 times higher at pH 5.0 than at pH 7.4. This effect was particularly noticeable between pH 6.0 and 5.0, with the MIC increasing from 0.5 to 14.5 mg/L. In contrast, lowering the pH over the same range markedly and almost linearly increased the activity of oxacillin (~10-fold decrease in MIC). The MBCs of both drugs varied in parallel to their MICs over the whole range of pH values investigated, remaining systematically 2–4-fold larger than the corresponding MIC, indicating that the bactericidal character of both antibiotics was fully maintained in spite of the overall decrease or increase of intrinsic activity.


Figure 1
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  		Figure 1.. MICs and MBCs of gentamicin (left-hand panel) and oxacillin (right-hand panel) as a function of the pH of Mueller–Hinton broth (MIC) or agar (MBC). Values are arithmetical means of four determinations.

 
In order to further understand the influence of pH on antibacterial intrinsic activity, we performed full dose–response studies at the 24 h time point. We checked that acid pH (5.4 vs. 7.4) only slightly reduced the rate of S. aureus growth (3 vs. 2–2.5 log cfu increase in 24 h), without affecting the overall shape of the individual dose–responses. Results are shown in Figure 2. The corresponding pertinent regression parameters, drug response descriptors Emax, EC50 (based on the Hill equation), apparent static concentration and statistical analyses are presented in Table 1. When data are examined using mass values for drug concentration, we see that acid pH decreased the relative potency of gentamicin and increased that of oxacillin (EC50) without modifying their apparent maximal efficacies (Emax). All responses, not only for each antibiotic individually but also when comparing antibiotics, become largely superimposable when data are examined using multiples of the MIC as drug concentration values (Figure 2, lower panels).


Figure 2
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  		Figure 2.. Dose–response curves of the extracellular activity of gentamicin (left-hand panels) and oxacillin (right-hand panels) against Staphylococcus aureus. The graphs show the change in the number of colony units (log cfu) per mL after 24 h of incubation of S. aureus in broth adjusted to different pH values. Data are plotted as a function of the drug concentration expressed in mg/L (upper panels), and in multiples of the MIC of each drug measured at the considered pH (lower panels; see Figure 1 for MIC values). For each diagram, the horizontal dotted lines show a static effect (initial inoculum: 5.94 ± 0.05 log cfu/mL), whereas the vertical dotted lines show the MIC at pH 7.4 for the upper panels, or are set to 0 (corresponding to the log of the MIC at each pH) for the lower panels. The zones highlighted in grey correspond to the serum concentrations ranges (total drug) that can be observed in patients (gentamicin, 1–18 mg/L; oxacillin, 0.5–86 mg/L) after conventional iv administration. Data are means ± SD of three independent experiments (most of the SD bars are smaller than the symbols). The limit of detection was –5 log cfu. Curves were constructed by non-linear regression using the Hill equation. See Table 1 for regression parameters, pharmacological and microbiological descriptors, and statistical analyses.

 


Figure 3
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  		Figure 3.. Dose–response curves of the intracellular activity of gentamicin (left-hand panels) and oxacillin (right-hand panels) against Strptococcus aureus phagocytosed by THP-1 macrophages. The ordinates show the change in the log10 cfu per mg of cell protein after 24 h of incubation compared with the post-phagocytosis inoculum (6.67 ± 0.07 log10 cfu/mg protein; the horizontal dotted line shows, therefore, a static effect. The abscissa shows the drug concentration expressed as follows. Upper panels: actual extracellular concentration (log10 mg/L); the zones highlighted in grey correspond to the serum concentrations ranges (total drug) that can be observed in patients (gentamicin, 1–18 mg/L; oxacillin, 0.5–86 mg/L) after conventional intravenous administration. Lower panels: (i) actual extracellular concentration (closed symbols and continuous lines) expressed as log10 x MIC, with MIC measured at pH 7.4 (filled squares, gentamicin; filled upside-down triangles, oxacillin) or at pH 5.4 (filled circles, gentamicin; filled triangles, oxacillin); (ii) concentrations assumed to prevail (open symbols and dotted lines) in lysosomes (gentamicin; open circles) or in whole cells (oxacillin; open triangles) expressed as log10 x MIC measured at pH 5.4. For all panels, the vertical dotted line shows the MIC at pH 7.4. Data are means ± SD of three independent experiments (most SD bars are smaller than the symbols). The limit of detection was –5 log. Curves were constructed by non-linear regression using the Hill equation. See Table 1 for regression parameters, pharmacological and microbiological descriptors, and statistical analyses.

 


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  		Table 1.. Pertinent regression parametersa (with confidence intervals, CI), and statistical analyses of the dose–response curves illustrated in Figures 2 and 3

 
Intracellular activity and antibiotic accumulation

Dose–response experiments were then performed for both antibiotics against intracellular S. aureus, also using the 24 h time point and the same range of drug concentrations as for the extracellular activities. Data are presented in Figure 3, with the pertinent regression parameters, drug response descriptors Emax, EC50 (based on the Hill equation), apparent static concentration, and the statistical analyses presented in Table 1. This data revealed a considerable loss of maximal efficacy of the antibiotics against intracellular bacteria (Emax) with a slight but significant increase in relative potency (EC50).

The cellular accumulation of gentamicin and oxacillin was then measured in infected cells after 24 h of incubation. Because of lack of sensitivity of our microbiological assay, we had to perform these experiments with cells incubated with large extracellular concentrations of antibiotic. To ascertain that accumulation of gentamicin and oxacillin was linearly related to the extracellular concentration, we measured the cell drug contents at increasing extracellular concentrations (50, 100, 150 and 200 mg/L for gentamicin; 300 and 400 mg/L for oxacillin). This enabled us to calculate the cellular drug content of cells incubated with low concentrations of antibiotics by extrapolation from the values observed at large concentrations. The mean accumulation values, when expressed as the apparent cellular (Cc) to extracellular (Ce) drug concentration ratios, were 0.57 ± 0.20 (SD) for gentamicin and 0.05 ± 0.03 (SD) for oxacillin; there was no evidence that extracellular concentration influenced these values: regression slopes of Cc/Ce versus Ce were 0.0012 (CI: –0.0042 to 0.0019) and 0.0003 (CI: –0.0008 to 0.0013) for gentamicin and oxacillin, respectively. These Cc/Ce ratios were then used to calculate the apparent cellular drug concentration at each extracellular concentration used in our previous experiments.

The data of Figure 3 (upper panels) were then re-plotted taking into account (i) the effect of pH on the MIC (as shown in Figure 1) assuming a pH of 5.4 for phagolysosomes,16 and (ii) the combination of this effect and the apparent local drug concentration. For the latter parameter, we started from the values of apparent cell concentration as explained above, but assumed that (i) cell-associated gentamicin was localized in the phagolysosomes, as is commonly accepted,17,18 and that these vacuoles accounted for approx. 2.5% of the cell volume19 (gentamicin local concentration would then be 40 times larger than deduced from its apparent Cc/Ce ratio); (ii) cell-associated oxacillin would be uniformly distributed, as suggested from previous studies examining the subcellular distribution of 14C-labelled penicillin in macrophages.20 The results of these calculations (Figure 3, lower panels) show that for both gentamicin and oxacillin the effects of pH could be almost entirely compensated for by their respective local accumulation properties. Indeed, when only the acid pH parameter was taken into account, we observed a marked shift of the data over the MIC scale towards lower values for gentamicin and to larger values for oxacillin. Data, however, returned to their original position when local drug concentrations were used to calculate the corresponding multiple of MIC.


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Aminoglycosides and ß-lactams have long been considered poorly efficient against intracellular bacteria, because of their small or slow cellular accumulation (ref. 21 for review), and in the case of aminoglycosides, the confounding effect of the intra-phagosomal acid pH on their activity.22 Yet, both types of antibiotics are used to treat various types of staphylococcal infections.23–26 Most in vitro studies confirmed the inefficacy of ß-lactams and aminoglycosides against intracellular S. aureus but used short-term exposures and limited concentration ranges.11 Studies using sufficiently large concentrations and a 24 h exposure time showed significant activity for different ß-lactams and for gentamicin.11,14 The present data go one step further in offering a rational, mechanistic explanation to the apparent contradiction between these various models.

We show first of all that antimicrobial responses are always related to concentration, obeying the classical pharmacology described by the Hill equation.27 This is not contradictory to what is commonly assumed as being the key pharmacodynamic properties of ß-lactams (time-dependency) and aminoglycosides (concentration-dependency).28 Our conclusions, indeed, are based on results observed over a much wider range of drug concentrations than usual, and which includes sub-MIC and supra-MIC values. If the observation is limited to the Cmin–Cmax range (as shown in Figures 2 and 3), one sees that oxacillin activity is already almost maximal, and therefore appears to be concentration-independent, whereas gentamicin is fully concentration dependent within the same range. (For oxacillin, which is 90% protein-bound, the Cmin–Cmax zone may need to be shifted to the left of 1 log10 unit, since it is generally agreed that only free concentration is related to activity; our model, unfortunately, does not allow analysis in detail of the effect of extracellular protein binding on intracellular activities,11,14 preventing further examination of this parameter here.) This ‘wide range of drug concentrations’ approach was actually critical to understand how pH affects the activity of antibiotics. The data shows that only relative potencies (EC50) and not apparent maximal efficacies (Emax) are modified by acid pH, but since activity in broth always reached the limit of detection, we cannot exclude that the real Emax is beyond that limit. We therefore suggest that pH acts by modulating the binding and/or target accessibility of gentamicin and oxacillin, and not by making bacteria more or less tolerant to the drugs. Acid pH indeed impairs gentamicin transport into bacteria,29 probably as a result of its larger ionization at pH 5.4 versus pH 7.4 (the pKas of the amino groups of gentamicin being between 5.5 and 9).30 Conversely, the pKa of the carboxylate function in oxacillin (about 2.4)31 is probably too low to markedly modulate the behaviour of the molecule in the 5.4–7.4 pH range. Yet, we know that bringing the pH to 5.5 increases the affinity of penicillin for its binding proteins (PBPs) based on binding studies to Escherichia coli PBPs 1b, 1c, 2 and 3,32 (the main PBPs in S. aureus are PBPs 1, 2, 3 and 4)33 and on direct measurement of penicillin binding to whole cell wall extracts of non-ß-lactamase producing methicillin-susceptible S. aureus (S. Lemaire, F. Van Bambeke, M. P. Mingeot-Leclercq, Y. Ghipczinsky and P. M. Tulkens, unpublished data). A key general conclusion of our studies is therefore that (i) aminoglycosides will exert activity against S. aureus in an acidic environment if their concentration reaches a value that compensates for their decreased relative potency, which is probably what takes place intracellularly through the local accumulation of the drug; and (ii) conversely, the low accumulation of ß-lactams in cells can be compensated for by their commensurate increase in relative potency, making these drugs to appear active in spite of their apparently unfavourable cellular pharmacokinetics. Therefore, we see that intracellular activity of aminoglycosides and ß-lactams cannot be simply deduced from the study of their cellular accumulation only.

Effects of pH and local concentration, however, fail to explain the poor eradicating capabilities of aminoglycosides and ß-lactams towards intracellular S. aureus. We see that decreased maximal efficacy (Emax), rather than a change in apparent potency (EC50), is probably the critical determinant. Observed for many antibiotics with distinct modes of action,11 this effect could result from the selection of pre-existing resistant subpopulations, an inaccessibility of part of the population to the drugs, or an increased tolerance to the drugs. Nevertheless, bacteria collected from cells exposed to large concentrations of gentamicin or oxacillin show an unaltered MIC when retested in broth. Inaccessibility of the drug could result from very local differences in environment not translated into obvious morphological differences. Increased tolerance may result from alteration of the metabolic status of the bacteria, such as formation of the so-called ‘small colony variants’34 that are intrinsically less sensitive to antibiotics.35 Exposure of S. aureus to mild acid pH modifies the expression level of about 400 genes2 in a similar way to heat shock or behaviour in biofilms,36,37 two situations where bacteria are poorly susceptible to antibiotics.38

Although our conditions of drug exposure are remote from those prevailing in patients, our data may nevertheless help a better understanding of how the activity of antibiotics could be improved in the clinical arena. Thus, strategies aiming at increasing the drug relative potencies (resulting in a lower MIC) and/or their concentrations at the site of infection could be useful for optimizing activity. Reducing local MICs by manipulating lysosomal pH proved efficient for increasing the intracellular activity of aminoglycosides in vitro13 but is difficult to exploit in vivo. Selecting molecules with low MICs at acidic pH and optimizing exposure of intracellular bacteria to these drugs by prolonging the time of exposure and using extracellular concentrations as high as possible appear to be straightforward approaches.


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None to declare.


   Footnotes
 
{dagger} Present address: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université libre de Bruxelles, Belgium. Back


   Acknowledgements
 
We thank Mrs M. C. Cambier and Mr M. Vergauwen for their dedicated technical assistance. P. B. and S. L. are respectively boursiers of a programme FIRST Europe Objectif 1 of the Region Wallonne, and of the Belgian Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture (F.R.I.A.). F.V.B. is Maître de Recherches of the Belgian Fonds National de la Recherche Scientifique (F.N.R.S.). This work was supported by the Belgian Fonds de la Recherche Scientifique Médicale (F.R.S.M.; grant no. 3.4.549.00 [EC] F and 3.4.639.04 [EC] F) and by the "STAPHAUR" programme of the Region Wallonne (grant no. EP1A320501R052F/415735).


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