Unité de Pharmacologie cellulaire et moléculaire, Université catholique de Louvain, Brussels, Belgium
Received 7 September 2004; returned 3 October 2004; revised 10 February 2005; accepted 14 February 2005
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Abstract |
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Methods: Quantitative measurements of cfu changes in broth and in THP-1 macrophages (post-phagocytosis) over time (5 and 24 h) at concentrations spanning from sub-MICs to Cmax (maximal concentration typically observed in patients' serum upon administration of conventional doses); morphological studies using an electron microscope; evaluation of drug stability (HPLC), protein binding (equilibrium dialysis) and measurement of drug cellular accumulation (microbiological assay).
Results: Ertapenem was unable to control L. monocytogenes growth in THP-1 macrophages at all concentrations and times tested, even under conditions where ampicillin and meropenem were bactericidal. This behaviour could not be ascribed to drug instability, protein binding or lack of cell accumulation in comparison with ampicillin or meropenem. Ertapenem, ampicillin and meropenem were equally effective at reducing the post-phagocytosis inoculum of S. aureus (1 log cfu), and caused conspicuous changes in the morphology of intracellular bacteria consistent with their lysis. These effects were obtained, however, only at large multiples (100-fold or more) of the MIC maintained over 24 h. Because of the high intrinsic antimicrobial potency of the ß-lactams studied, these concentrations were below the Cmax.
Conclusions: Ertapenem will probably be ineffective against intraphagocytic forms of L. monocytogenes for reasons that remain to be discovered. Conversely, ertapenem could be an alternative to ampicillin and meropenem against intraphagocytic S. aureus since its longer half-life may allow high concentrations to be maintained for more prolonged times.
Keywords: L. monocytogenes , S. aureus , THP-1 macrophages , ertapenem , ampicillin , meropenem
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Introduction |
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Materials and methods |
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L. monocytogenes. We used a haemolysin-producing strain EGD and followed exactly the methods described previously,12 except that determination of MBCs used a 106 cfu/mL inoculum.
S. aureus. We used a non-ß-lactamase-producing strain (ATCC 25923) following the methods described previously.14
Cells, cell infection and assessment of intracellular activities of antibiotics
THP-1 myelomonocytic cells15
were used throughout our experiments. For L. monocytogenes, we followed a method described previously12
with addition of gentamicin (1 mg/L; 1 x MIC) for control cultures (no ß-lactam added) if maintained for more than 5 h.13
For S. aureus, we used an adaptation of the method described previously for J774 macrophages14
taking into account that THP-1 cells spontaneously grow in suspension. Opsonization was performed with non-decomplemented, freshly thawed human serum diluted 1:10 in serum-free culture medium (RPMI 1640). Phagocytosis was performed at a 4:1 bacteria/macrophage ratio. Elimination of non-phagocytosed bacteria, and collection of cells at the end of the experiment were, therefore, made by centrifugation at room temperature [1300 rpm; 8 min; Eppendorf 5810R Centrifuge equipped with a A-4-62 rotor (Eppendorf Gerätgebau GmbH, Engeldorf, Germany)].
Determination of cellular carbapenem accumulation
Cells were collected and washed free from culture medium by three successive centrifugations in ice-cold phosphate-buffered saline (PBS), resuspended in distilled water and subjected to sonication (10 s at 50 watts; Labsonic L, Braun Biotech International GmbH, Melsungen, Germany). Ertapenem was then assayed by a microbiological method (to avoid the necessity of extraction, and thereby obtaining the needed sensitivity), using Escherichia coli as test organism and following the general procedure described previously16 [lowest limit of detection; 0.25 mg/L for ertapenem and 1 mg/L for meropenem; typical linearity: up to 6070 mg/L (R2=0.998 for ertapenem and 0.989 for meropenem)]. Cell proteins were measured in parallel using the FolinCiocalteu/biuret method,17 and the results used to compute the apparent cellular concentration of antibiotics based on a volume ratio of 5 µL of cell volume per mg protein, as in our previous publications dealing with THP-1 cells.12,18
Electron microscopy
Cells were infected as described above, except that the initial inoculum was increased to 20 bacteria per macrophage. This larger inoculum did not modify the intracellular activity of the ß-lactams studied. Sample handling was then performed as described previously.18
Stability studies of carbapenems
Stability in culture media was evaluated by HPLC to provide us with positive identification of the molecules studied (in comparison with genuine standards) and higher reproducibility compared with bioassays. Assay was based on a published method,19 but extraction was performed three times in succession with pooling of the upper phases in order to improve drug recovery [typical values: 82.3 ± 0.5% (n=3) for ertapenem; 100.0 ± 0.1% for meropenem; a single extraction of ertapenem yielded only 16.6 ± 0.9% (n=3) recovery]. Chromatography was made through a Lichrosphere 100 RP-18 column (25 cm x 4 mm, 5 µm; Merck AG, Darmstadt, Germany). Elution was made with acetonitrile/25 mM phosphate buffer pH 6.5 (v/v, 7:93) for both ertapenem and meropenem, with typical retention times of 8 and 5 min, respectively [linearity (for standards): 0.09200 mg/L (R2=0.999) and 0.19200 mg/L (R2=0.999)].
Protein binding studies
We used the equilibrium dialysis technique (cut-off, 60008000 mol. wt; Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) with membranes soaked successively in three baths of water, and three baths of PBS for 15 min each. Preliminary studies showed that equilibration required a minimum of 32 h at 37°C under constant rotation at 8 rpm and without serum. The concentration of antibiotic in the serum-free compartment was measured by HPLC (see method above) at the end of the experiment and compared with the initial concentration to calculate the percentage of bound drug: 100[(concentration in serum-free cell x 2)/(initial concentration)].
Materials
Ertapenem, meropenem and gentamicin were obtained as Invanz®, Meronem® and Geomycine®, respectively (i.e. the registered commercial products for parenteral administration in Belgium), and supplied by Merck Sharp & Dohme BV (Haarlem, The Netherlands), Astra Pharmaceutical (Brussels, Belgium) and GlaxoSmithKline s.a. [Rixensart, Belgium; on behalf of Schering-Plough Belgium (Brussels, Belgium)]. Ampicillin was purchased as the sodium salt from SigmaAldrich (St Louis, MO, USA), and cell culture media and serum were obtained from Gibco Biocult (Paisley, UK). All other reagents were obtained from Merck AG or from SigmaAldrich.
Statistical analyses
Curve fitting analyses were made with GraphPad Prism® software (version 4.0) and group comparisons (Student's t-test, one-way analyses of variance) with Instat Prism® software (version 3.01), both from GraphPad Prism® software, San Diego, CA, USA.
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Results |
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Table 1 shows the MICs and MBCs observed in the present study. With respect to L. monocytogenes, ertapenem showed an MIC similar to ampicillin and was thus 10-fold less potent than meropenem. All three ß-lactams were bacteriostatic towards L. monocytogenes. For S. aureus, MICs were determined both at pH 7.4 and pH 5.5 to mimic the conditions prevailing in the extracellular milieu and in phagolysosomes, respectively. All three ß-lactams had low MICs with no significant difference between them, and with slightly lower values at pH 5.5. MBCs were about two-fold larger than the MICs demonstrating the bactericidal activity of these ß-lactams against the strain of S. aureus used.
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In the first series of experiments, the influence of the incubation time on the activity of antibiotics was evaluated at increasing fractional concentrations observed in the serum of patients receiving conventional doses of the antibiotics tested. The concentrations were chosen as guided by the Cmax reported for the respective antibiotics in the literature (50, 50 and 155 mg/L for ampicillin, meropenem and ertapenem, respectively9,20,21
) and using two incubation time periods (5 and 24 h). Results are shown in Figure 1 in a synoptic fashion comparing the activities seen in broth (mimicking the situation of extracellular bacteria) with those in cells (phagocytosed bacteria) for all three antibiotics and for both types of bacteria. Considering the results globally, two main aspects were striking. First, it appears that most of the antibacterial effects (based on the decrease in cfu) were already obtained at the lowest concentration tested [for meropenem towards phagocytosed L. monocytogenes, however, a gain of 1 log cfu decrease could be obtained at 24 h when increasing the concentration from the lowest to the highest value tested (550 mg/L)]. Secondly, all effects were also time-dependent, with significant gains of activity in all conditions when incubation was prolonged from 5 to 24 h (the effect of ertapenem, however, was only to slow down bacterial growth). Concentrating now on L. monocytogenes, we see that the extracellular activity of all three ß-lactams was essentially bacteriostatic at 5 h, and that only a modest decrease in cfu (0.50.7 log units) was obtained at 24 h at all concentrations tested. Examination of the results obtained for intracellular activity revealed: (i) that intracellular bacterial growth was essentially similar to that in broth; (ii) that meropenem and ampicillin were bacteriostatic at 5 h at all concentrations tested, but achieved a bactericidal effect at 24 h, as previously described;13
and (iii) that, in sharp contrast, ertapenem was unable to decrease the post-phagocytosis inoculum, with bacteria actually growing at a multiplication rate about half of that of controls at all concentrations tested. Moving now to S. aureus, we first see that all three ß-lactams exerted a marked bactericidal effect towards bacteria in broth (
5 log decrease in 24 h) without significant differences between them. Intracellular growth of S. aureus in control cells was first delayed, but eventually proceeded at a rate that allowed to reach in 24 h an increase over the original inoculum of about two-thirds of what was seen in broth (as already reported for J774 macrophages14
). All three ß-lactams were able to significantly reduce the number of cell-associated cfu, but this reduction (0.71.2 log units) was much less pronounced than for bacteria in broth. No marked difference was seen between the three drugs.
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Because the results shown in Figure 1 suggested that ertapenem was unable to block the intracellular growth of L. monocytogenes while being active against intracellular S. aureus, we performed electron microscopic studies to directly examine the morphology of the intracellular bacteria after 24 h of incubation in control cells and in cells incubated with this antibiotic. Figure 2 (a d) shows that both control cells and ertapenem-treated cells contained an abundance of intact L. monocytogenes in various stages of phagocytosis (Figure 2a), division (Figure 2b), and clearly present in the cytosol while being surrounded with actin (Figure 2bd). No apparent differences were seen between control and ertapenem-treated cells. In contrast, L. monocytogenes-infected cells that had been incubated with ampicillin or meropenem for 24 h contained no recognizable Listeria apart from a few structures that looked like highly damaged bacterial cells (not shown). Cells infected with S. aureus and left without antibiotic showed clearly recognizable bacteria, sometimes in a process of division, in what appeared like phagocytic vacuoles (Figure 2e). In contrast to what had been seen with L. monocytogenes, the morphology of S. aureus was clearly altered in ertapenem-treated cells (Figure 2fh), where most sections showed bacterial ghosts (Figure 2f and h), bacterial bodies with evidence of partial loss of electron-dense material (Figure 2g), together with apparently intact bacteria (Figure 2f). Similar images were obtained for meropenem- and ampicillin-treated cells (not shown).
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In order to get more insight into the contrasting behaviour of ertapenem towards intracellular L. monocytogenes and S. aureus on the one hand, and to better analyse the differences in activity seen with ampicillin and meropenem on the other hand, we systematically compared intracellular and extracellular activities over a wider range of extracellular concentrations than in the experiments reported in Figure 1. For this purpose, bacteria or cells were exposed to a range of concentrations from sub-MIC to 1000 x MIC values, allowing for a direct pharmacological comparison between drugs. Results are shown in Figure 3 with activity expressed as a function of multiples of the MIC. This allows for direct comparisons at equipotent concentrations. Considering L. monocytogenes first, all three ß-lactams showed a concentration-dependent activity in broth in a range of one- to 100-fold their MICs. The bacterial responses to meropenem and ampicillin were undistinguishable whereas ertapenem was about four-fold less potent in that range. For intracellular bacteria, both meropenem and ampicillin showed a concentration-dependent activity that reached a bactericidal effect (
2 log decrease in cfu) at high concentrations. Ampicillin was systematically
10-fold more potent than meropenem in the 1100 x MIC range. In contrast, bacterial growth was seen for ertapenem at all concentrations tested, and the function that could be fitted to the data suggested that ertapenem would be unable to achieve even a bacteriostatic effect whatever its extracellular concentration. Moving now to S. aureus, the data show: (i) that a bactericidal effect was obtained against bacteria in broth from low multiples of MIC with four-fold higher potency for ertapenem compared with the two other ß-lactams; (ii) that intracellular activity was, like in broth, concentration-dependent; (iii) but that the reduction of the inoculum was quite limited (
1 log only) in all cases, with no meaningful difference in bacterial response between the three antibiotics.
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The surprising results observed with ertapenem towards intracellular L. monocytogenes prompted us to run a series of controls to check for an artefactual cause. We first examined whether the drug would remain sufficiently stable in the culture medium, based on previous experience with meropenem and imipenem that showed a fast degradation of both drugs when incubated as concentrated solutions in aqueous media at 37°C.22
During incubation in complete culture medium, ertapenem degradation (original concentration 155 mg/L) proceeded according to zero-order kinetics, with 62% of drug remaining intact after 24 h. For meropenem (original concentration 50 mg/L), degradation reached
50% over the same period. We also tested whether the high protein binding of ertapenem reported in human serum23
would play a critical role here. Equilibrium dialysis experiments, showed that, indeed, only 32.1 ± 0.6% (n=3) of total ertapenem was free in complete culture medium (10% fetal calf serum). Reducing the serum concentration to 2% increased this value to 42.6 ± 2.5% (n=3). However, this did not improve the activity against intracellular L. monocytogenes [changes in post-phagocytosis inoculum (log cfu) of 0.63 ± 0.09 versus 0.70 ± 0.09 at 5 h and of 1.83 ± 0.12 versus 1.77 ± 0.19 at 24 h for cells incubated with 2% and 10% serum, respectively].
Cellular accumulation of ertapenem
L. monocytogenes-infected and non-infected cells were collected after 5 h and 24 h of incubation with ertapenem (155 mg/L) and subjected to bioassay. The apparent cell antibiotic concentrations (in mg per litre of cell volume) were 83.1 ± 7.1 and 68.6 ± 1.5 at 5 h, and 27.2 ± 9.3 and 37.1 ± 12.2 at 24 h for infected and uninfected cells, respectively.
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Discussion |
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Upon testing for activity over a wide range of concentrations covering values below and above the MIC (i.e. when using a pharmacologically oriented design), all three antibiotics showed clear-cut concentration-related effects towards both extracellular and intracellular bacteria. This may come as a surprise, since ß-lactams have for long been ranked as concentration-independent antibiotics.24 However, we show here that the antibacterial concentration effect actually follows a sigmoidal function. Because the MICs of the ß-lactams studied here are quite low with respect to the organisms tested, almost maximal effects can be observed at antibiotic concentrations corresponding to low (410%) fractional amounts of the maximal concentration tested (chosen based on an estimation of the Cmax in patients). The clinical implication could be that ß-lactam therapy will be optimal, with respect to both extracellular and intracellular bacteria, only if the serum concentration remains at values exceeding the MIC for the whole period of observation (time becoming then the predominant parameter governing activity). Conversely, ß-lactams will be expected to show concentration-dependent effects if their concentration falls closer to the MIC, which will be the case in vivo upon under-dosing or with organisms with high MIC values.
Quite surprisingly, ertapenem, which shares many of the properties of meropenem against Gram-positive organisms,9 was inefficient against the intraphagocytic forms of L. monocytogenes under conditions in which ampicillin and meropenem are active (as demonstrated here and in our previous publications12,13 ). This could not be ascribed to drug instability (in comparison with meropenem) or lack of cell penetration [the concentrations of ertapenem being higher than those of meropenem or of ampicillin (at extracellular concentrations of 50 mg/L as used here)12 ]. Ertapenem is known to be highly protein bound.10 While binding to serum proteins present in the culture fluid is probably unimportant in our model (decreasing the protein concentration did not improve the intracellular activity of ertapenem), the situation may be different within the cell where phagocytosed L. monocytogenes becomes surrounded by a thick layer of actin.18
In contrast, all three ß-lactams were active against intracellular S. aureus. The morphological studies are of particular interest, since they revealed images of bacterial ghosts quite similar to those seen with extracellular S. aureus exposed to penicillin25 or faropenem,26 which are directly related to the binding of the ß-lactams to their targets. ß-Lactams are not expected to accumulate in cells, owing to the presence of a free carboxyl function on all these molecules.1,2,27 Yet, we observe here that the cellular concentration of ertapenem can reach values far above the MIC for S. aureus. When phagocytosed by macrophages, this bacterium is, however, not free in the cytosol but is primarily located in phagolysosomes and related vacuoles,28 which are acidic,29 and where the concentration of ß-lactams is expected to be much lower.27 This may explain why many reports have pointed to the apparent inability of ß-lactams to act against intraphagosomal organisms in general,1,2,30 and S. aureus in particular.31,32 In this context, it must be emphasized that a significant reduction in the intracellular inoculum was obtained here only when (i) the extracellular concentration markedly exceeded the MIC for the offending organism (and was actually close to the maximal concentration tested), and (ii) the incubation time was brought to 24 h. This suggests that large extracellular concentrations and prolonged exposure may compensate for the lack of cellular accumulation, as previously proposed based on studies with methicillin,33 especially since acidic pH does not adversely affect activity. We also cannot exclude the possibility that the activity of ß-lactams towards phagosomal organisms is enhanced by cellular factors, as previously suggested.34,35 However, we have to stress the fact that eradication of S. aureus remained quite limited in our model, as was also noted for fluoroquinolones in similar models.14,36 Translated to the in vivo situation, this may have important consequences in terms of recurrence of the infection and emergence of resistance. These points will need to be specifically addressed in future studies, the aim of which should be to establish what is the true impact or the limitations of the present findings.
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Acknowledgements |
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