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Scientific
Publications - Work Done by Microbiology Reader
Chemotherapy 2003;49:287–293 Postantibiotic and Sub-MIC Effects of Benzylpenicillin against Streptococcus pneumoniae with Different Susceptibilities for PenicillinInga Odenholt, Ingegerd Gustafsson, Elisabeth Löwdin ABSTRACT Background: The purpose of the study was to examine whether penicillin-susceptible and nonsusceptible strains of Streptococcus pneumoniae exhibited different pharmacodynamic responses to benzylpenicillin. Methods: The postantibiotic effects (PAEs) and the postantibiotic sub-MIC effects (PA SMEs) were investigated by optical density against strains of S. pneumoniae with different susceptibilities to benzylpenicillin. To validate the data, the PAE and PA SME of one susceptible and one resistant strain were also tested with the viable count method. The post-MIC effects (PMEs) were studied in an in vitro kinetic model, simulating human pharmacokinetics with a half-life of 1 h and a time above MIC of approximately 20% of 24 h. Results: There were no differences with respect to the PAEs, PA SMEs and PMEs of benzylpenicillin for the various strains of S. pneumoniae, irrespective of their susceptibility to penicillin. For both some of the susceptible and resistant strains investigated, longer PA SMEs at 0.2 and 0.3 x MIC were noted, indicating that these parameters might be more dependent on the type of strain rather than on the susceptibility status. Conclusion: No differences in the pharmacodynamic response after similar drug exposure were seen for S. pneumoniae strains with different penicillin susceptibility. Key Words:
INTRODUCTION Streptococcus pneumoniae is a leading cause of infectious diseases and is still associated with high morbidity and mortality worldwide [1]. Until relatively recently, S. pneumoniae was considered so uniformly sensitive to penicillin that sensitivity tests were not usually performed. Today, this is different in that many countries in Europe and the rest of the world are reporting a steady increase of S. pneumoniae not susceptible to penicillin [2, 3]. During the last decade, the spread of non-penicillinsusceptible pneumococci with increased MICs has created a clinical treatment problem, especially in infections where therapeutic concentrations are difficult to achieve, such as in meningitis [4]. This has raised the question whether penicillin may still be the drug of choice in the treatment of diseases caused by this pathogen. Clinical and animal studies indicate that by optimizing the dosing of penicillin, it may still be possible to use penicillin for treatment of S. pneumoniae with decreased susceptibility [5–11]. Pharmacodynamic studies of antibiotics have rendered findings of great interest in recent years. Results from in vitro and animal studies have shown that different classes of antibiotics behave differently with regards to antibacterial activity [7–9, 12]. One of the pharmacodynamic parameters most studied is the postantibiotic effect (PAE), which describes the suppression of bacterial growth after short exposure of bacteria to antimicrobials [13]. The PAE has been offered as one of many explanations for the success of intermittent dosing with drugs that exhibit short half-lives. Another explanation involves the postantibiotic sub-MIC effect (PA SME), which also takes into account the effect of subinhibitory concentrations after exposure to suprainhibitory concentrations [14–17]. The post-MIC effect (PME) describes a similar phenomenon, but instead of using static concentrations, an in vitro kinetic model is used, where different pharmacokinetics can be simulated [18, 19]. The purpose of the present study was to examine whether penicillin-susceptible and -resistant strains of S. pneumoniae exhibited different pharmacodynamic responses to penicillin with respect to the PAE, PA SME and PME.
MATERIALS AND METHODS Antibiotic Benzylpenicillin was provided from Astra Zeneca (Södertälje, Sweden). The antibiotic was obtained as a reference powder with known potency and dissolved in distilled water prior to each experiment.
Bacterial Strains and Media The strains used in the present study were four penicillin-sensitive strains of S. pneumoniae (A 2000, 5070, 5015, 5038) and four penicillin-resistant strains (2151, 32475, 40932, 43647). Strains A 2000 and 2151 were clinical isolates belonging to a Spanish-Icelandic clone obtained from the Department of Microbiology, Reykjavik, Iceland. They were multiresistant isolates of serotype 6B, with a similar susceptibility profile except for penicillin. The other susceptible strains (5070, 5015, 5038) were obtained from the Department of Clinical Microbiology, Uppsala, Sweden, and the other three resistant strains (32475, 40932, 43647) were clinical isolates obtained from Centre Hospitalier Intercommunal Créteil (France). The strains were grown in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich., USA) for 6 h at 37 °C in 5% CO2, resulting in approximately 5 X 108 CFU/ml.
Minimum Inhibitory Concentrations The MICs for all investigated strains were determined in fluid media by a macrodilution technique in triplicate on different occasions according to the guidelines of the National Committee for Clinical Laboratory Standards [20]. Twofold serial dilutions of benzylpenicillin were added to broth and inoculated with a final inoculum of approximately 105 CFU of the test strain per milliliter and incubated at 37°C in 5% CO2 for 20 h. The MIC was defined as the lowest concentration of the antibiotic allowing no visible growth. The MICs for all strains were also determined spectrophotometrically in the BioScreen C. Twofold serial dilutions of benzylpenicillin in broth saturated with CO2 were made in microplates containing 400-µl wells, and the test strains were added to give a bacterial density of approximately 105 CFU/ml. The plates were then incubated in the BioScreen C (see below). The MIC was defined as the lowest concentration that prevented growth, as measured by optical density (OD). The lowest detectable level of OD for S. pneumoniae corresponded to approximately 5 X 105 CFU/ml.
Antibiotic Concentrations The concentrations of benzylpenicillin in the kinetic experiments were determined by a microbiological agar diffusion method, using Bacillus stearothermophilus ATCC (American Type Culture Collection) 3032 as the test organism. A standardized inoculum of spore suspension was mixed with tryptone-glucose agar (pH 7.4) and poured into plates. After the plates were dried, 0.03-ml volumes of all samples and standards, diluted in Todd-Hewitt broth, were placed in agar wells at a volume of 0.01 ml. The assays were performed in triplicate and the plates were incubated overnight at 56° C. The lower limit of detection was 0.03 mg/l. The coefficient of variation was approximately 10%. The concentrations of benzylpenicillin were determined in all the in vitro kinetic experiments.
Determination of the PAEs and PA SMEs in BioScreen C The BioScreen C (Lab Systems, Finland) is a computerized incubating turbidometric reader, where growth curves are monitored continuously, which is useful to follow the growing part of the PAE and PA SME determinations. The machine can process 200 wells in the same experiment. All bacterial strains were exposed to 10 X MIC for 2 h in 5% CO2. To eliminate the antibiotic, the cultures were washed three times, centrifuged each time for 10 min at 1,400 g and thereafter diluted 10–1 in fresh medium, giving a residual concentration of benzylpenicillin in the tube of approximately 0.01%. The unexposed control strains were washed similarly but were also diluted 10–2, 10–3, 10–4 and 10–5 in order to obtain an inoculum close to the exposed strains, which was less than 0.5 log10 CFU/ml in the experiments. Viable counts of the exposed cultures were measured before antibiotic exposure, after 2 h of induction and after washing. Viable counts of the controls were also measured at the start of the experiments, before and after washing and dilution at 2 h. Both the exposed strains and the different dilutions of the controls were then transferred in a volume of 40 µl and inoculated into microtiter wells with 360 µl of Todd-Hewitt broth saturated with CO2 and incubated in the BioScreen C at 35° C. Growth curves were measured automatically as OD at 540 nm in the computer every 10 min for 20 h. Earlier experimental studies showed that the growth of the controls with different inocula is parallel. In the experiments, where the exposed culture and the control do not quite match in inoculum, a control curve for each strain and experiment can therefore be constructed with the same initial inoculum as the corresponding exposed strain. The PAE was calculated as the difference in time for the exposed cultures and the corresponding control to grow up to a defined point (A50) on the OD curve, where A50 is defined as 50% of the maximal OD of the control [21–24]. The PA SMEs for the same strains were determined as follows. The postantibiotic phase was induced as described above, and the controls were diluted (10–2, 10–3, 10–4 and 10–5) in the same way as in the PAE experiments. Viable counts were also used as described above. The strains in the postantibiotic phase were then exposed to 0.1, 0.2 and 0.3 X MIC of benzylpenicillin in carbon dioxide-saturated Todd-Hewitt broth and incubated in the BioScreen C. Growth curves were monitored automatically for 20 h. The PA SME was defined as the difference in time for the cultures exposed to sub- MICs in the postantibiotic phase and the corresponding control with the same inoculum as the preexposed culture to reach A50 (defined as described above) [21–24]. All experiments were performed in triplicate.
Determination of the PAEs and PA SMEs with Viable Counts To validate the data in the BioScreen, the PAE and PA SME of benzylpenicillin were determined by viable counts against S. pneumoniae A 2000 and 2151. After incubation for 6 h, the test strains in the exponential growth phase were diluted 10–1 to obtain a starting inoculum of 107–108 CFU/ml. The strains were then exposed to 10 X MIC (MICs determined with the macrodilution technique) of benzylpenicillin for 2 h at 37°C in 5% CO2. To eliminate the antibiotic, the cultures and the controls were washed, centrifuged and diluted as described above. The difference in inoculum between the unexposed control strains and the exposed strains was less than 1 log10 CFU/ml in the experiments. The cultures with bacteria in the postantibiotic phase and the controls were thereafter divided into four different tubes. In order to determine the PAE, one tube of each culture were reincubated at 37°C in 5% CO2 for another 22 h. Samples were drawn at 0 and 2 h (before and after washing), and at 3, 4, 5, 6, 8, 11 and 24 h and if necessary diluted in phosphate-buffered saline. Three dilutions of each sample were seeded on blood agar plates (Colombia agar base with 5% horse blood; Acumedia Manufactures Inc., Baltimore, Md., USA) and counted for determination of the numbers of CFUs. The sensitivity of the culturing was 5 X 101 CFU/ml. The PAE was defined according to the following formula: PAE = T – C, where T is the time required for the viable counts of the cultures exposed to antibiotic to increase by 1 log10 above the counts observed immediately after washing and C is the corresponding time for the controls [13]. For determination of PA SMEs, the remaining three tubes of the control cultures and the cultures in the postantibiotic phase were exposed to 0.1, 0.2 and 0.3 X MIC of benzylpenicillin, respectively, and reincubated at 37°C in 5% CO2 for another 22 h. Samples were drawn and viable bacteria were determined as described above. The PA SME was defined according to the following formula: PA SME = TPA – C, where TPA is the time required for the cultures previously exposed to antibiotic, which were thereafter exposed to different sub-MICs, to increase by 1 log10 above the counts observed immediately after washing, and C is the corresponding time for the unexposed control [14, 15]. All antibiotic/bacterial combinations were investigated in triplicate.
Determination of the PMEs in an in vitro Kinetic Model A previously described model was used in these experiments [18, 19]. It consists of a spinner flask with a 0.45-µm filter membrane and a prefilter fitted in between the upper and the bottom part. A magnetic stirrer ensures homogenous mixing of the culture and prevents membrane pore blockage. In one of the side arms of the culture vessel, a silicon membrane is inserted to enable repeated sampling. A thin plastic tubing to a vessel containing fresh medium connects the other arm. The medium is removed from the culture flask through the filter in order to prevent bacterial removal, at a constant rate with a pump (P-500, Pharmacia Biotech, Uppsala, Sweden). Fresh sterile medium is sucked into the flask at the same rate by the negative pressure built up inside the culture vessel. The antibiotic was added to the vessel and eliminated at a constant rate according to first-order kinetics: C = Co X e–kt, where Co is the initial antibiotic level, C the antibiotic level at the time t, k the rate of elimination and t the time elapsing since the addition of antibiotic. The medium used was Todd- Hewitt saturated with 5% CO2. The apparatus was thereafter placed in a thermostatic room at 37°C during the experiments. In order to obtain a similar drug exposure as reflected by pharmacodynamic indices (time above MIC: T > MIC, maximum concentration MIC: Cmax/MIC, area under the concentration curve/MIC: AUC/MIC), a target concentration of 1 mg/l for the penicillin-susceptible and 100 mg/l for the penicillin-resistant strains with a simulated half-life of 1 h was used to determine the PMEs. PME was defined as the difference in time for the numbers of CFUs in the kinetic model to increase 1 log10 CFU/ml, calculated from the numbers obtained at the time when the antibiotic concentration had declined to the MIC, and the corresponding time for a control culture, grown in the test tube without antibiotic, to increase 1 log10 CFU/ml [20]. All experiments were performed in triplicate.
RESULTS Minimum Inhibitory Concentrations The MICs of benzylpenicillin for the different strains are shown in table 1. The MICs for the investigated strains were the same with the two methods, with the exception of two strains, where a difference of one dilution step was noted.
Antibiotic Concentrations in the in vitro Kinetic Model The mean ± SD of the antibiotic concentrations in all experiments are shown in figure 1. The observed values were within 1.5% of the target values. The half-life was 1 h (0.9–1.0 h) in all experiments.
The PAEs and PA SMEs When comparing the PAEs and PA SMEs from the viable count method with those from the BioScreen C, almost identical values were found (table 2). The PAEs and PA SMEs of all strains with the BioScreen C are presented in table 3. There was no difference in PAEs between the strains irrespective of the susceptibility status (1.5–2.7 h for the susceptible strains and 1.2–2.1 h for the resistant strains). There was a greater variation in the PA SMEs in that some of the strains were more sensitive to sub-MIC concentrations. However, the variation did not correlate with the susceptibility to penicillin.
The PMEs Table 4 displays the pharmacokinetic/pharmacodynamic indices and the PMEs. The T > MIC in the experiments was approximately 20% for all strains. Also, the other pharmacodynamic indices were relatively similar with a maximal 2-fold variation between the strains. Figure 2 shows the PMEs for S. pneumoniae A 2000 and 2151. With a similar drug exposure, no significant differences in PMEs were noted for any of the investigated strains, irrespective of penicillin susceptibility.
Discussion The goal of antimicrobial therapy is to maximize the bactericidal activity against the infecting pathogen. For optimal therapy, it is important to take both pharmacokinetic and pharmacodynamic parameters into consideration [7–9, 12]. The aim of the present study was to investigate if pharmacodynamic parameters such as the PAE, PA SME and PME were similar for strains of S. pneumoniae with different susceptibilities to penicillin. In this study, we found no significant difference in the PAEs and PA SMEs for the susceptible and resistant strains. These findings are in accordance with the results of Fuursted et al. [25], who did not find any differences in PAE between strains of S. pneumoniae with different susceptibilities to penicillin. Nor did Spangler et al. [26] find any differences in the PAEs of penicillin-susceptible, -intermediate and -resistant strains of S. pneumoniae. However, they reported a difference in PAE between 1 and 6 h that was highly strain dependent. The same authors also tested the effects of sub-MICs on the bacteria in the postantibiotic phase. As with the PAEs, the effects of sub-MICs seemed to vary with the strain type rather than with the susceptibility status [26]. In our study, we also found that the PA SMEs were strain dependent but there was no correlation between longer PA SMEs and the susceptibility to penicillin. The PAEs and PA SMEs in this study were in accordance with earlier studies with a penicillin-susceptible strain of S. pneumoniae [14, 21]. In the in vitro kinetic model, where continuously decreasing concentrations of benzylpenicillin were used and similar T > MIC was simulated, no major differences in PMEs were noted for any of the investigated strains in spite of different susceptibilities to penicillin and disregarding differences in Cmax/MIC and AUC/MIC. In a study of Erlendsdottir et al. [11], penicillin-susceptible, -intermediate and -resistant strains of S. pneumoniae were studied in four different animal models. Also, in this study, using immunocompetent animals, it was shown that the same T > MIC was required for efficacy for both the susceptible and the resistant strain [11]. Dahl-Knudsén et al. [10] also showed that there was a highly significant correlation between log MIC and log ED50 for pneumococci with different penicillin susceptibilities in a mouse peritoneal model. The results from the present and other studies are, however, in contrast to the findings of Lister et al. [27], who studied the pharmacodynamics of amoxicillin against pneumococci with different susceptibilities to penicillin in an in vitro kinetic model. They first observed loss of antibacterial effect for the resistant strain 4 h after the concentration of amoxicillin had fallen under the MIC. However, for the sensitive and intermediate strains, regrowth coincided with the MIC level, which is in contrast to the findings in our study. As an explanation for the hypersensitivity of the resistant strains to sub-MICs, it was hypothesized that the extensive alterations in penicillin-binding proteins required to achieve full resistance to penicillin may slow the recovery of penicillin-resistant strains after the drug levels fall below the MIC. In contrast, intermediate resistant strains, which have not altered their penicillin-binding proteins as extensively, may be able to recover much more rapidly during the postantibiotic phase, and thus regrowth is observed shortly after the concentration of the drugs has fallen below the MIC [27]. Also, in a recent study of ours using the same in vitro kinetic model as in this study, it was shown that differences may exist between strains with different antibiotic susceptibilities. A T > MIC of 50% was required to obtain maximal efficacy of amoxicillin against a penicillin-susceptible and a penicillin-intermediate (MIC = 0.25 mg/l) strain of S. pneumoniae. For a strain with an MIC of 2 mg/l, a T > MIC of 60% and also Cmax of 10 X MIC were needed [28]. However, also for amoxicillin and pneumococci, some studies have not been able to show pharmacodynamic differences for strains with different penicillin susceptibilities. Andes and Craig [29], in a thigh infection model in mice, showed that the T > MIC of amoxicillin required for bacteriologic efficacy and survival was similar for both penicillin-susceptible and -resistant S. pneumoniae. They were also able to show that there was an excellent correlation between the MIC of amoxicillin for S. pneumoniae irrespective of the penicillin susceptibility status and the change in log10 CFU/thigh [29]. Obviously, there are still inconsistencies in the literature regarding whether strains with reduced antibiotic susceptibility have different pharmacodynamic properties. Different experimental designs and end points could explain this, and further studies are needed. In conclusion, our study indicates that the PAEs, PA SMEs and PMEs of benzylpenicillin for different strains of S. pneumoniae are similar irrespective of the penicillin susceptibility. For some of the strains, longer PA SMEs were noted. However, the variation did not correlate with the susceptibility to penicillin, indicating that these parameters might be more dependent on the type of strain rather than on the susceptibility status.
REFERENCES 1 Mufson MA: Streptococcus pneumoniae; in Mandell GI, Douglas RG, Bennet JE (eds): Principles and Practice of Infectious Diseases, ed 3. New York, Medical Publications, 1990, pp 1539–1551. 2 Baquero F: Epidemiology and management of penicillin-resistant pneumococci. Curr Opin Infect Dis 1996;9:372–379. 3 Goldstein FW, Acar JF: Antimicrobial resistance among respiratory tract isolates of Streptococcus pnemoniae: Results of a 1992–93 Western Europe and USA collaborative surveillance study. The Alexander Project Collaborative Group. J Antimicrob Chemother 1996;38(suppl A):71–84. 4 Martinez E, Miro´ JM, Almirante B, Aguado JM, Fernadez-Viladrich P, Fernadez-Guerrero ML, Villanueva JL, Dronda F, Moreno-Torrico A, Montejo M, Llinares P, Gatell JM, Spanish Pneumococcal Endocarditis Study Group: Effect of penicillin resistance of Streptococcus pneumoniae on the presentation, prognosis and treatment of pneumococcal endocarditis in adults. Clin Infect Dis 2002:35:130–139. 5 Friedland IR: Comparison of the response to antimicrobial therapy of penicillin-resistant and penicillin-susceptible pneumococcal disease. Pediatr Infect Dis J 1995;14:885–890. 6 Palleres R, Linares J, Vadillo M, Cabellos C, Manresa F, Viladrich PF, Martin R, Gudiol F: Resistance to penicillin and cephalosporin and mortality from severe pneumococcal pneumonia in Barcelona, Spain. N Engl J Med 1995; 333:474–480. 7 Craig WA: Choosing an antibiotic on the basis of pharmacodynamics. Ear Nose Throat J 1998;77:7–12. 8 Craig WA: Does the dose matter? Clin Infect Dis 2001;33(suppl 3):S233–S237. 9 Dagan R, Klugman KP, Craig W A, Baquero F: Evidence to support the rationale that bacterial eradication in respiratory tract infection is an important aim of antimicrobial therapy. 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Baltimore, Williams & Wilkins, 1996, pp 296–329. 14 Odenholt-Tornqvist I, Löwdin E, Cars O: Pharmacodynamic effects of subinhibitory concentrations in vitro. Antimicrob Agents Chemother 1991;35:1834–1839. 15 Odenholt-Tornqvist I, Löwdin E, Cars O: Postantibiotic effects and postantibiotic sub-MIC effects of roxithromycin, clarithromycin and azithromycin on respiratory tract pathogens. Antimicrob Agents Chemother 1995;39:221– 226. 16 Licata L, Smith CE, Goldschmidt RM, Barrett JF, Frosco M: Comparison of the postantibiotic sub-MIC effects of levofloxacin and ciprofloxacin on Staphylococcus aureus and Streptococcus pneumoniae. Antimicrob Agents Chemother 1997;41:950–955. 17 Spangler SK, Lin G, Jacobs MR, Appelbaum PC: Postantibiotic effect and postantibiotic sub-MIC effect of levofloxacin compared to those of ofloxacin, ciprofloxacin, erythromycin, azithromycin and clarithromycin against 20 pneumococci. Antimicrob Agents Chemother 1998;42:1253–1255. 18 Löwdin E, Odenholt I, Bengtsson S, Cars O: Pharmacodynamic effects of sub-MICs of benzylpenicillin against Streptococcus pyogenes in a newly developed in vitro kinetic model. Antimicrob Agents Chemother 1996;40:2478– 2482. 19 Löwdin E, Odenholt I, Cars O: In vitro studies of pharmacodynamic properties of vancomycin against Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother 1998;42:2739–2744. 20 National Committee for Clinical Laboratory Standards: Methods for Determining Bactericidal Activity of Antimicrobial Agents; Tentative Guideline M26-T. Villanova, NCCLS, 1992. 21 Löwdin E, Odenholt-Tornqvist I, Cars O: A new method to determine postantibiotic effect and effects of subinhibitory antibiotic concentrations. Antimicrob Agents Chemother 1993; 37:2200–2205. 22 Odenholt-Tornqvist I: Studies on the postantibiotic effect and the postantibiotic sub-MIC effect of meropenem. J Antimicrob Chemother 1993;31:881–892. 23 Odenholt I, Löwdin E, Cars O: Comparative in vitro pharmacodynamics of BO-2727, meropenem and imipenem against gram-positive and gram-negative bacteria. Clin Microbiol Infect 1997;3:73–81. 24 Odenholt I, Löwdin E, Cars O: In vitro pharmacodynamic studies of L-749,345 in comparison with imipenem and ceftriaxone against Gram-positive and Gram-negative bacteria. Antimicrob Agents Chemother 1998;42:2365– 2370. 25 Fuursted K, Dahl-Knudsén J, Barendorf Petersén M, Lykke Poulsen R, Rehm D: Comparative study of bactericidal activities, postantibiotic effects, and effects on bacterial virulence of penicillin G and six macrolides against Streptococcus pneumoniae. Antimicrob Agents Chemother 1997;41:781–784. 26 Spangler SK, Lin G, Jacobs MR, Appelbaum PC: Postantibiotic effect of Sanfetrinem compared with those of six other agents against 12 penicillin-susceptible and -resistant pneumococci. Antimicrob Agents Chemother 1997;41: 2173–2176. 27 Lister PD, Pong A, Chartrand SA, Sanders CC: Rationale behind high-dose amoxicillin therapy for acute otitis media due to penicillin-nonsusceptible pneumococci: Support from in vitro pharmacodynamic studies. Antimicrob Agents Chemother 1997;41:1926–1932. 28 Gustafsson I, Löwdin E, Odenholt I, Cars O: Pharmacokinetic and pharmacodynamic parameters for antimicrobial effects of cefotaxime and amoxicillin in an in vitro kinetic model. Antimicrob Agents Chemother 2001;45: 2436–2440. 29 Andes D, Craig WA: In vivo activities of amoxicillin and amoxicillin-clavulanate against Streptococcus pneumoniae: Application to breakpoint determinations. Antimicrob Agents Chemother 1998;42:2375–2379.
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