1 Laboratory of Experimental Infection, Infectious Diseases Service, Hospital Universitari de Bellvitge, Universitat de Barcelona, Barcelona, Spain; 2 Microbiology Department, IDIBELL-Hospital Universitari de Bellvitge, Universitat de Barcelona, Barcelona, Spain
Received 24 December 2004; returned 20 February 2005; revised 1 July 2005; accepted 28 July 2005
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Abstract |
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Methods: Four strains of S. aureus with different glycopeptide susceptibility (MICs of vancomycin from 1 to 8 mg/L) were used. In vitro experiments were performed by means of timekill curves while we used the mouse peritonitis model for in vivo evaluation.
Results: Combinations of glycopeptides and ß-lactams showed synergy in in vitro timekill curves against the four staphylococcal strains, the highest efficacy being detected against the glycopeptide-intermediate S. aureus (GISA) strain (MIC = 8 mg/L) (log 24 h = 3.19 cfu/mL for vancomycin at 1/2 x MIC and oxacillinat 1/64 x MIC versus 0.56 cfu/mL for vancomycin alone at 1/2 x MIC). On the other hand, no significant increase in efficacy was observed in vivo in the experimental model. The efficacy of the combinations decreased in correlation to the decreasing susceptibility of the strains to glycopeptides, showing only residual activity against the GISA strain (
log 24 h = 1.42 cfu/mL for vancomycin and cloxacillin versus 1.22 cfu/mL for vancomycin).
Conclusions: In the in vivo setting we were unable to demonstrate the synergism between glycopeptides and ß-lactams observed in vitro; nor did combinations show antagonism against any of the strains. Though the usefulness of these combinations cannot be totally ruled out in highly specific clinical conditions, it seems unlikely that they will provide a serious therapeutic alternative in most hGISA and GISA infections in the coming years.
Keywords: vancomycin , animal models , GISA , heterogeneous resistance
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Introduction |
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Among these antimicrobial strategies, the combination of glycopeptides and ß-lactams has been reported to be a promising alternative to glycopeptide monotherapy.13,15,16 It was based on the initial observation of Sieradzki et al.17,18 that in vitro highly vancomycin-resistant strains recover ß-lactam susceptibility and that combinations of glycopeptides and ß-lactams show additive or synergistic effects against vancomycin-susceptible methicillin resistant S. aureus (VS-MRSA), hGISA and GISA isolates.13,15,16,1923 Climo et al.13 confirmed this phenomenon of synergism between vancomycin and ß-lactams in an in vivo experimental model of endocarditis caused by GISA isolates in rabbits. However, contradictory observations have been recorded in subsequent in vitro studies including an antagonistic effect of these combinations at particular sub-MIC ß-lactam concentrations or a false synergy caused by inappropriate testing methods.21,2426 What is more, no further in vivo evidence has confirmed this synergistic effect.
In order to broaden our knowledge of the role of the combinations of glycopeptides and ß-lactams in the therapy of hGISA and GISA infections, we evaluated their activity in vitro by means of timekill curves and studied their efficacy in vivo with a mouse model of peritonitis using four S. aureus strains with different susceptibilities to glycopeptides, including vancomycin-susceptible methicillin-susceptible S. aureus (VS-MSSA), VS-MRSA, hGISA and GISA isolates.
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Materials and methods |
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Four clinical strains of S. aureus with variable degrees of susceptibility to glycopeptides were included in in vitro and in vivo studies. Strain A (HUB 954), VS-MSSA; strain B (HUB 284), VS-MRSA; strain C (HUB 783) hGISA, belonging to the Iberian clone, growing on 4 mg/L vancomycin MuellerHinton plates with a frequency of sub-populations of 3.6 x 106 cfu/mL, as previously described1 (this strain was equivalent to widely described Mu-3 heteroresistant strain); and strain D (Mu50, ATCC 700699), GISA. Strains A, B and C were isolated in our hospital, and strain D, reported as the first GISA strain by Hiramatsu et al.6 was isolated in Japan in 1997. MICs to vancomycin, teicoplanin, cloxacillin and cefotaxime of the four strains were determined by macrodilution according to NCCLS guidelines27 (Table 1).
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Timekill curves were performed according to NCCLS guidelines.28 Bactericidal activity was evaluated by determining the killing rate for a bacterial isolate by an antimicrobial agent. Aliquots of S. aureus were cultured at 37°C in MuellerHinton broth tubes at an inoculum size of 105 cfu/mL and antibiotics were then added at chosen concentrations. For each strain, vancomycin, teicoplanin, oxacillin and cefotaxime were tested for a range of concentrations according to their MICs and their achievable concentration in human serum. Glycopeptides were tested at concentrations of 2 x MIC, 1 x MIC and 1/2 x MIC while ß-lactams at 2, 1, 1/4, 1/8 and 1/16 x MIC for strain A, and 1, 1/4, 1/16, 1/64 and 1/128 x MIC for strains B, C and D. Growth control was assessed with an extra tube without antibiotic in all experiments. At 0, 4, 8 and 24 h of incubation, aliquots of 100 µL were taken from each tube to perform direct and 10-fold dilutions, which were cultured on trypticase soy agar (TSA) plates at 37°C for 24 h. Quantitative results were then obtained by performing bacterial counts. To avoid carryover antimicrobial agent interference, the sample was placed on the plate in a single streak down the centre, allowed to absorb into the agar until the plate surface appeared dry, and the inoculum was then spread over the plate. Synergy of a combination of antibiotics was defined as a decrease of at least 2 log10 cfu/mL at 24 h compared with the single most active agent. An antibiotic or combination was considered bactericidal when it obtained a reduction of at least 3 log10 cfu/mL.
Preparation of inoculum used in mice peritonitis
Colonies from fresh overnight cultures on 5% blood agar plates were resuspended and grown for 46 h at 37°C in trypticase soy broth (TSB). Immediately before inoculation, cultures in TSB were centrifuged and resuspended in sterile saline, adjusted to an optical density of 0.5 McFarland (108 cfu/mL) and then diluted to the appropriate size. Inoculum sizes from 5 x 106 to 5 x107 cfu/mL were finally used in mice experiments.
Mouse peritonitis model and sample processing
The animal studies were approved by the Ethics Committee for Animal Experiments at the University of Barcelona. The model was based on a previously described protocol.14,29,30 Inbred, female, C57BL/6 mice (6 weeks;
1416 g) were used (Harlan Int. Ibérica, S.A., Barcelona, Spain). Mice were kept ten to a cage and they had food and water ad libitum. After a 1 week quarantine, inoculation was performed by intraperitoneal injection of 0.5 mL of the inoculum via a 26-gauge syringe. The inoculum consisted of a 5 x 106 to 5 x 107 cfu/mL staphylococcal suspension with 5% (w/v) mucin in sterile saline. In every set of experiments, 4 h after inoculation a group of mice were killed as controls (total n
13) and antibiotic therapy was initiated (Hour 0). The rest of the mice were randomized to different therapeutic regimens (total n
6 per therapy) or the control group (total n
12). Antibiotic treatment or placebo was administered for 24 h following schedules described below. A set of 48 h therapy experiments, using vancomycin alone and combined with cloxacillin, were performed with strains C and D to determine if a more prolonged treatment period may involve changes in the efficacy of monotherapies or combinations. At each time point (0, 24 and 48 h), mice were anaesthetized intraperitoneally with 30 µL of ketamine/xylazine (5:1) and peritoneal washes were performed by injecting 2 mL of sterile saline intraperitoneally followed by a 1 min external massage of the abdomen. Immediately, 0.1 mL of blood was withdrawn by cardiac puncture to perform qualitative blood cultures and animals were then killed by cervical dislocation. Next, the abdomen was opened using an aseptic technique and 0.2 mL of peritoneal fluid (PF) was recovered. PF samples were diluted and plated (0.1 mL) on 5% blood TSA plates to perform direct and 10-fold dilution cultures. To avoid carryover antimicrobial agent interference, the sample was placed on the plate in a single streak down the centre, allowed to absorb into the agar until the plate surface appeared dry, and the inoculum was then spread over the plate. The detection limit using this method was 101 cfu/mL. In order to assess bacteraemia, blood samples from cardiac puncture were grown in TSB at 37°C for 24 h and then 100 µL of broth was cultured on TSA plates (37°C for 24 h) to check for S. aureus growth. Mortality of control and therapeutic groups was recorded at 24 h of therapy.
Efficacy of an antibiotic therapy was defined as the decrease in the number of cfu (log cfu/mL) in PF after 24 h of therapy.
Therapeutic experiments
Pharmacokinetic studies to select the appropriate dose regimens resulting in serum concentrations similar to those in humans were described in a previous study.14 All antimicrobials were administered subcutaneously. Mice infected with either strain A, B, C, or D were randomized to receive vancomycin (30 mg/kg every 4 h) or teicoplanin (40 mg/kg in a single daily dose) as monotherapy or in combination with cloxacillin (160 mg/kg every 2 h) or cefotaxime (200 mg/kg every 2 h) or control group with saline serum as placebo. Average peak and trough levels of vancomycin and teicoplanin in serum were 44 and 0.6 mg/L (at 15 min and 4 h), and 148 and 1.15 mg/L (at 1 and 24 h), respectively; average peak and trough levels of cloxacillin and cefotaxime were 163 and 0.4 mg/L (at 15 min and 2 h), and 162 and 0.2 mg/L (at 10 min and 2 h), respectively. Results of experiments regarding monotherapies were previously reported.14
The possibility of a change in MIC for strains C and D (hGISA and GISA) to glycopeptides by selection of vancomycin or teicoplanin-resistant sub-populations during experimental therapy was investigated by performing a new determination of MIC on the colonies growing in PF at 24 h of therapy.
Pharmacodynamic analysis
We evaluated the efficacy of the particular combination of vancomycin and cloxacillin according to the method described by Mouton et al.31 We calculated the pharmacodynamic indices (PDIs) for vancomycin and cloxacillin on the basis of previously reported pharmacokinetic studies.14 Different PDIs were based on different susceptibilities of the strains involved in the study. The efficacy of the single therapy was then described as a function of the PDI most appropriate for each agent using a linear regression model. For cloxacillin t < MIC is widely accepted as the best PDI to predict efficacy; for vancomycin, we found that AUC/MIC was the parameter with the best correlation with efficacy (a multiple regression analysis with different PDIs was used to determine which was the more appropriate PDI to predict efficacy of the single therapy; data not shown). Alternatively, a multiple regression analysis was used to determine the contribution of each PDI to the efficacy of the combination therapy. A prediction of the efficacy of the combination was then calculated using the linear regression models for both single-agent therapies by means of a linear combination, and the predicted values were compared with the experimentally observed values.
Statistics
Analysis of variance (ANOVA) with Scheffé post hoc test was used to analyse multiple bacterial count comparisons between therapeutic and control groups. Comparisons between strains were made by using ANOVA (with Tukey post hoc test). Two-tailed Fisher's exact test was used for categorical data (survival, bacteraemia). A P value of <0.05 was considered statistically significant for all tests. SPSS statistical program was used for calculation of the linear and multiple regression models of the pharmacodynamic analysis.
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Results |
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The most significant timekill curves of the four strains involved in the study are shown in Figure 1.
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Strain B
Glycopeptides achieved a minimum bacterial count reduction of 12 log cfu/mL at 24 h when tested alone at concentrations above the MIC (slightly better for vancomycin), their activity being only bacteriostatic. Cefotaxime was bactericidal at 1 x MIC, showing better activity than oxacillin, which was not effective at that concentration. The addition of the ß-lactams at 1 x MIC to vancomycin at concentrations above the MIC did not improve the activity of the glycopeptide. On the other hand, the combination of teicoplanin at 1 x MIC with oxacillin at 1 x MIC showed additive effect, without being bactericidal; the addition of cefotaxime did not improve the activity achieved by the ß-lactam alone. Combinations of teicoplanin at 1/2 x and 1/4 x MIC with both cefotaxime and oxacillin at sub-MIC concentrations up to 1/64 x MIC were synergistic; since the activity of vancomycin alone at 1/2 x MIC remained moderate, only combinations with vancomycin at 1/4 x MIC showed synergism. All combinations reached only bacteriostatic activity.
Strain C
Vancomycin and teicoplanin alone were bacteriostatic at concentrations above the MIC and retained a slight activity at 1/2 x MIC, showing a similar profile to strain B curves. Oxacillin and cefotaxime were only bacteriostatic at 1 x MIC concentration when tested alone. Combination of both ß-lactams at that concentration with glycopeptides above the MIC did not result in any improvement. Combinations of both glycopeptides at 1/4 x MIC concentrations with oxacillin or cefotaxime showed synergism for a range of concentrations up to 1/64 x MIC for both ß-lactams; only the ones with vancomycin at 1/4 x MIC and oxacillin at 1/4 x and 1/16 x MIC were bactericidal.
Strain D
Vancomycin and teicoplanin were on the threshold of bactericidal activity when tested alone at concentration of 2 x MIC; their activity was better than against strains B and C. Oxacillin and cefotaxime tested alone at concentrations above the MIC were also in the range of bactericidal activity, but without reaching a 3 log cfu/mL decrease. The addition of ß-lactams at 1 x MIC to vancomycin at concentrations above the MIC was additive and almost synergistic; their addition to teicoplanin at concentrations above the MIC reached a clear synergistic and bactericidal effect. Combinations of both glycopeptides at concentrations of 1/2 x and 1/4 x MIC with cefotaxime and oxacillin at sub-MIC concentrations up to 1/64 (even at 1/128 x MIC for oxacillin) were synergistic and bactericidal in all cases. Combinations were more active against this strain.
Therapeutic efficacy in the mouse peritonitis model
Mortality and bacteraemia by strains
Mortality of control mice at 24 h differed according to strain: 55% in strain A, 22% in strain B, 62% in strain C and 21% in strain D, reflecting the virulence of the model. At 48 h, mortality was 50% for strain C and 17% for strain D. On the other hand, mortality was 0% in all therapeutic groups with all strains.
Bacteraemia of control and therapeutic groups after 24 h therapy for all strains is shown in Table 2.
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In vivo efficacy after 24 h of therapy with vancomycin alone or combined with cloxacillin against the four S. aureus strains is compared in Figure 2(a), showing a significant decrease for both single and combined therapies when glycopeptide resistance increased. Efficacy of monotherapies and combinations increased from 24 to 48 h of therapy for strains C and D, but no significant differences were observed at this point between monotherapy and the combined treatment (Figure 2b). Multiple regression analysis of the efficacy of the combination therapy as a function of PDIs for vancomycin and cloxacillin showed that only the AUC/MIC for vancomycin could explain that efficacy, being non-significant for the contribution of t > MIC cloxacillin to the model and confirming that the addition of the ß-lactam did not mean any synergistic nor additive effect; thus R2 for the model including both antibiotics PDIs was 0.573, while partial R2 including only AUC/MIC for vancomycin was 0.569. Prediction of efficacy of the combination was closer to the observed efficacy, with a moderate correlation (Figure 3).
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Discussion |
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In the present study, we evaluated the efficacy of these combinations against four strains of S. aureus with different glycopeptide susceptibilities, first in vitro and then in vivo. We used two susceptible strains A and B (VS-MSSA and VS-MRSA) and two heteroresistant and intermediate-resistant strains C and D (hGISA and GISA), which have been associated with clinical failures following glycopeptide therapy;1,6 our hGISA strain (strain C), belonging to the reported Iberian clone, was equivalent to standard Mu3 strain. In timekill curves, a synergistic effect between glycopeptides and ß-lactams was observed for all the S. aureus strains studied at sub-MIC concentrations for both antibiotic groups. The relationship between this synergistic effect and the degree of vancomycin resistance was only partially observed. On the one hand, combinations showed the highest efficacy against strain D (GISA), most of them being synergistic and bactericidal, whereas with strains B (VS-MRSA) and C (hGISA) they were only bacteriostatic. However, with strain A (VS-MSSA), the combinations of glycopeptides and ß-lactams also showed synergy and, in fact, were highly bactericidal with cefotaxime. Similarly, an in vitro synergistic effect between vancomycin and cefepime against some MSSA strains has been described.22
Another controversial aspect regarding the possible synergism of glycopeptide and ß-lactam combination against VS-MRSA, hGISA and GISA strains is the influence of the concentration of ß-lactams on the effect of such combinations.21,24,26 It has been suggested that ß-lactams at or near the MIC may produce a synergistic or additive effect, while lower ß-lactam concentrations combined with vancomycin may be antagonistic. In our study, we observed synergism and/or bactericidal activity with oxacillin at concentrations considerably lower than the MIC (range 416 mg/L). However, our results were not in clear disagreement with those reported by other authors,13,16,26 since oxacillin concentrations at which they observed synergism (referred to as near the MIC) were in the same range as ours (referred to as sub-MIC), depending on the MICs for the strains studied. We selected the test concentrations according to MICs for the strains and the range of achievable human serum levels; consequently, we did not study the effect of concentrations of ß-lactams <4 mg/L and so we cannot rule out a possible antagonistic effect at these low levels. In any case, we conclude that sub-MIC levels of ß-lactams do not necessarily determine antagonism against hGISA and GISA strains, as has been stated elsewhere.21,24,32
As we described previously,14 our mouse peritonitis model was able to compare the efficacy among different antibiotic therapies and also the efficacy of a therapy among strains with different susceptibilities. We selected bacterial counts in PF as the major endpoint for the assessment of antibiotic efficacy, since the differences found in bacteraemia rates between the antibiotic schedules were usually in a narrow range and mortality was 0% for all therapies.14 We used a short period of treatment of 24 h in order to increase the relevance of the model, since this is the critical period of such an acute infection.
In this experimental study, a direct relationship between the increase in MICs of vancomycin and the decrease in the efficacy of glycopeptide and ß-lactam combinations was observed; in a similar way, we have previously reported that the increase in glycopeptide resistance was correlated to a decrease in the efficacy of monotherapies with vancomycin or teicoplanin,14 and also it is in agreement with clinical data on the relevant influence of glycopeptide susceptibility.1,9 The synergistic pattern clearly found in in vitro tests was not observed, since combinations of glycopeptides and ß-lactams did not significantly improve the efficacy of monotherapies with glycopeptides against any strain, although antagonism was not detected either. A confirmation of this point was given, first, by the multiple regression analysis of the efficacy of the combination, which showed that only AUC/MIC of vancomycin could explain the efficacy of the combination and t > MIC for cloxacillin was not significant to the model; second, in Figure 3, in which the predicted and the observed efficacy of the combination were similar or, in other words, the intercept of the regression line between predicted and observed responses was 0, indicating that the combinations have no synergy (as it was previously proposed by Mouton et al.31). Prolonging the treatment up to 48 h did not provide significant additional information about differences in monotherapy and combination activities, although both increased slightly at this time point. Antibiotic doses were selected on the basis of achievable concentrations in humans in clinical practice (according to susceptible strains). At these doses, it seems logical that sub-inhibitory concentrations were found in animal experiments involving resistant strains (B, C and D for ß-lactams, and C and D for glycopeptides); following the same criteria we selected concentrations for in vitro studies which were also in the sub-inhibitory range. The discrepancies observed between in vitro and in vivo experiments using these sub-inhibitory concentrations are difficult to explain, even accepting the differences between both environments.
Why our model was not able to corroborate the in vivo synergism observed by Climo et al.13 using glycopeptides and ß-lactams in a model of endocarditis by GISA could be explained by differences in the experimental design of both studies. Endocarditis is a deep-seated and more difficult-to-treat infection and penetration of vancomycin or teicoplanin in cardiac vegetations is lower than in the peritoneum. In addition, models were based on different animal species and involved distinct treatment schedules. While vancomycin monotherapy failed in endocarditis, it preserves a certain activity in the mouse peritonitis model against the same GISA strain (Mu50); the addition of a ß-lactam resulted in a synergistic combination in the first model and was indifferent in the second one. Whereas the endocarditis model was shown to be adequate to assess the effect of the combinations, it may be that our peritonitis model was not demanding enough, although the observation that slight increases in vancomycin MICs determined a progressive loss of the efficacy of the combined therapy is a differential finding of our model.
In conclusion, while substantial synergism was observed in in vitro timekill studies with combinations of ß-lactams and glycopeptides against VS-MSSA, VS-MRSA, hGISA and especially against the GISA (Mu50) strain, in the mouse peritonitis model only occasional traces of this activity were found. On the other hand, no antagonism was observed either in vitro or in vivo at any sub-MIC ß-lactam concentration. However, the most important issue continues to be the actual potential of these combinations in clinical practice. There is a certain microbiological and experimental basis for a synergistic effect, and combinations may be useful in highly specific clinical conditions, but overall it does not seem that this alternative will play a major role in the therapy of most hGISA and GISA infections in the coming years.
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Acknowledgements |
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