JAC Advance Access originally published online on May 30, 2006
Journal of Antimicrobial Chemotherapy 2006 58(1):108-111; doi:10.1093/jac/dkl216
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Consistent rates of kill of Staphylococcus aureus by gentamicin over a 6-fold clinical concentration range in an in vitro pharmacodynamic model (IVPDM)
Antibiotic Pharmacodynamic Research Institute, Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota Minneapolis, MN 55455, USA
*Correspondence address. Weaver Densford Hall 9-157, University of Minnesota College of Pharmacy, 308 Harvard Street SE, Minneapolis, MN 55455, USA. Tel: +1-612-624-2183; Fax: +1-612-626-5082; E-mail: rotsc001{at}umn.edu
Received 7 September 2005; returned 25 January 2006; revised 1 February 2006; accepted 8 May 2006
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Abstract |
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Objectives: To compare the effect of a 6-fold range in gentamicin concentration on the bacterial killing of Staphylococcus aureus.
Methods: Six 24 h duplicate experiments were performed using an in vitro pharmacodynamic model (IVPDM) which was inoculated with 106 cfu/mL S. aureus (ATCC 29213) and subjected to desired initial gentamicin concentrations of 0, 5, 10, 15 and 20 mg/L. A 2 h half-life was emulated for gentamicin. Samples were drawn at 0.5, 1, 1.5, 2, 3, 4, 6, 9 and 24 h to quantify cfu/mL and gentamicin concentration. These samples were subjected to serial saline dilution to prevent antibiotic carryover and to produce a countable number of colonies. Pre- and post-gentamicin MIC values were performed for S. aureus. Duplicate 24 h kill curves were generated for each experiment and assessed for statistical difference (two-way ANOVA) between the slopes of the kill curves and time to 3 log kill.
Results: Kill curve slopes were analysed out to the 2 h time point and no statistical difference was found between the different concentrations (P > 0.05). Time to 3 log kill was not significantly different between the concentrations. Post-exposure gentamicin MIC values were within one tube dilution of the pre-exposure MIC value (0.25 mg/L).
Conclusions: These data demonstrate that clinical gentamicin concentrations kill S. aureus with equivalent effectiveness and that the use of higher doses of aminoglycosides would probably not improve bacterial kill rates.
Keywords: single daily dose , adjunctive aminoglycosides , S. aureus , pharmacodynamics
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Introduction |
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Staphylococcus aureus is one of the most prevalent pathogens in the United States1 and is often associated with skin infections, infective endocarditis and bacteraemia.2 A common strategy for treating S. aureus bacteraemias and endocarditis is to use an antibiotic combination of a semi-synthetic penicillin or a glycopeptide combined with an aminoglycoside, most often gentamicin, as adjunct therapy.3 This antibiotic combination has been found to decrease the duration of bacteraemia and fever but does not affect mortality.3 Despite the common usage of gentamicin in clinical practice, the bacterial kill relationship of gentamicin concentration to S. aureus is unknown. This raises the question, would there be any benefit to dosing the gentamicin in a single daily dose (SDD) approach (7 mg/kg every 24 h) or instead continuing to use the multi daily dose (MDD) strategy (80 mg every 8 h or 1.5 mg/kg every 8 h)?
Aminoglycosides are known concentration-dependent killers of Gram-negative organisms.4 However, the relationship between concentration and rate of bacterial kill of gentamicin with Gram-positive organisms such as S. aureus is unknown. A study addressing this same question against enterococci found little value using more aggressive aminoglycoside dosing strategies.5
A typical gentamicin peak serum concentration achieved with conventional dosing is 5–8 mg/L. When SDD is used, a much higher gentamicin peak of 20–25 mg/L is desired.6
The purpose of this work was to determine whether higher clinical concentrations of gentamicin are more effective in killing S. aureus.
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Materials and methods |
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Model description
Experiments were performed in an in vitro pharmacodynamic model (IVPDM) described previously.7 The pharmacodynamic model consisted of a sealed glass chemostat filled with cation-adjusted Mueller–Hinton broth (CAMHB; Difco, Detroit, MI, USA). Experiments were run in duplicate. Each chemostat had a volume of 
275 mL. An initial desired inoculum of 106 cfu/mL of S. aureus was used for each experiment. Chemostats were placed in a water bath maintained at 37°C and agitated using a magnetic stir bar. The desired starting peak concentration of antibiotic was achieved using a single bolus dose of antibiotic at the start of each experiment. In order to simulate desired rates of drug elimination, the drug-free medium was pumped using a SciQ 400 pump (Watson Marlow, Norway) into the chemostats, thus displacing an equal volume of the drug-containing medium into the waste beaker. The pump rate (Ko mL/h) was set to simulate a gentamicin half-life (t1/2) of 2 h (Ko = 0.693/t1/2 x volume of chemostat).
Antibiotic
Gentamicin was purchased from the Sigma Chemical Co. (Victoria, Australia). Gentamicin stock solutions, with a concentration of 1 mg/mL, were frozen at –80°C.
Bacteria
Methicillin-susceptible S. aureus (ATCC 29213) was used for all experiments.8
Susceptibility testing
Pre- and post-exposure gentamicin MICs were determined using 96-well microtitre trays and broth microdilution techniques. All wells were inoculated with 105–106 cfu/mL. MICs were determined in accordance with NCCLS9 guidelines. The study organism, S. aureus (ATCC 29213), was also used as the control strain. We did not expect to see an inoculum effect with gentamicin.10
Time–kill experiments
Six 24 h experiments including a growth control and five duplicate experiments exhibiting four different desired peak concentrations (Cmax) of gentamicin (5, 10, 15 and 20 mg/L) (Table 1) were used to examine the effect of different clinical concentrations on killing of S. aureus.
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Samples were taken from the experiment at time 0, which was prior to the introduction of gentamicin, and every half hour for the first 2 h. Additional post-antibiotic levels were obtained at 3, 4, 6, 9 and 24 h. Gentamicin carryover was prevented by serial saline dilution. Following incubation for 24 h at 37°C, bacterial colonies were counted and recorded on data sheets. The accuracy range for colony counting in our lab is 30–300 colonies per plate. The lower limit of accuracy in our lab was 300 cfu/mL. To achieve countable colonies we used serial saline dilutions. Colony counts < 300 cfu/mL were not included in our data analysis.
Samples collected at time points 0.5 and 4 h were frozen at –80°C to be analysed at a later date to confirm desired peak concentration and half-life for gentamicin.
Analysis
Time–kill curves were constructed and compared for each gentamicin experiment. Bacterial kill curve slopes were calculated by linear regression using Graph Pad Prism 4 software (Graph Pad Software Inc., San Diego, CA, USA) at 0–2 h of gentamicin exposure to examine the possibility of a concentration-dependent effect. The slopes were then compared for statistical significance using a two-way ANOVA (Prism 4 Graph Pad, Graph Pad Software Inc.).
Gentamicin concentrations were determined using an Axsym System (Abbott Laboratories, Abbot Park, IL, USA) commercial fluorescence polarization immunoassay. The range of detectable concentrations for this test was 0.3–10 mg/L (r = 0.994). Samples in which the anticipated concentration would exceed this range were diluted. Two control values (3 and 8 mg/L) were used in this assay (coefficient of variation values for these controls were 3.7% and 3.6%, respectively).
Time to 3 log kill (T3K) was assessed by using slopes obtained from linear regression out to the 2 h time point. Time was calculated using the equation y = mx + b. T3K values for each experiment were then compared by using a two-way ANOVA (Prism 4). Statistical significance was defined as P < 0.05.
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Results |
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Susceptibility testing
The pre-exposure MIC for S. aureus 29213 was 0.25 mg/L. Post-exposure MICs were either identical to pre-exposure MICs or increased by one tube dilution.
Pharmacokinetics
The target peak concentrations for the experiment were 5, 10, 15 and 20 mg/L. The desired half-life was 2 h. The actual peaks were 3.2, 6.6, 11 and 14 mg/L with measured half-lives of 2.2, 2.0, 2.1 and 2.2 h, respectively. Because we failed to obtain a measured peak of 20 mg/L, an additional experiment was performed. The actual peak concentration for that experiment was 19.3 mg/L with a half-life of 2.5 h.
Pharmacodynamics
Figure 1 displays the kill curve data and bacterial regrowth over the 24 h period of the experiment. Although our peak concentrations fell short of the predicted values, we still achieved a 6-fold range that would allow us to discern the concentration relationship that best predicted eradication of S. aureus by gentamicin.
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Kill curve slopes were used to analyse the rate of bacterial kill by different concentrations of gentamicin in this experiment. A steeper slope indicates a more rapid rate of kill as shown in the decline of bacterial cfu/mL. From the time of initial gentamicin exposure to the 2 h time point, the difference in the slopes of the kill curves between concentration levels was not statistically significant (P > 0.05). This indicates that higher concentrations of gentamicin do not kill S. aureus at a faster rate or eliminate a greater portion of the bacterial burden than do lower concentrations.
A 3 log kill (99.9% reduction in cfu/mL) was achieved within the range of 1.2–1.61 h for all experiments. This difference was not statistically different (P > 0.05) between either the models or the different gentamicin concentrations.
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Discussion |
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Aminoglycosides remain useful antibiotics but are known to cause nephrotoxicity and ototoxicity.6 With the advent of single daily dosing, many supporters believed that this new regimen could lead to not only better clinical outcomes, but also reduced toxicity.11–21 While this theory is yet to be proven definitively in a large, well-designed human trial, many smaller studies have been inconclusive in their assessment of this new dosing strategy for aminoglycosides.22,23
The kill curve data in our experiment showed an insignificant difference in slopes when analysed out to the 2 h time point. Since our experiment covered over a 6-fold range in concentrations with no statistical difference in rate of kill, this would lead us to believe that increased clinical concentrations of gentamicin are not more effective in killing S. aureus than lower concentrations. In addition, the T3K, which occurred within the first 2 h of the experiments, was also not statistically different between the experiments.
A limitation of the present study is whether or not this data can be extrapolated to mimic a more clinically relevant scenario. Gentamicin is not recommended as monotherapy for any infection caused by S. aureus. Rather gentamicin is used as an adjunct to agents with good efficacy against Gram-positive infections such as vancomycin or nafcillin.24 Clinically the combination has only been shown to slightly reduce the duration of bacteraemia and fever.3
Another issue with the study was the difficulty in achieving the desired peak concentration of gentamicin for each experiment. Because the model is a closed system, when drug was injected at the beginning of the experiment, medium was displaced into the waste beaker. We believe that this may have resulted in partial drug elimination even before the pump was turned on. In retrospect, a more concentrated stock solution of gentamicin would have resulted in a smaller volume displacement when gentamicin was injected.
This experiment was designed to answer a fundamental question of whether increasing gentamicin concentration kills S. aureus more effectively? The results of this experiment show that the bacterial kill relationship appears to be similar over a range of clinical concentrations with lower concentrations resembling MDD demonstrating comparable bacterial kill rate to higher SDD concentrations.
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None to declare.
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Acknowledgements |
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No outside sponsor.
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References |
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1 Pfaller MA, Jones RN, Doern GV, et al. (1998) Bacterial pathogens isolated from patients with bloodstream infection: frequencies of occurrence and antimicrobial susceptibility patterns from the SENTRY antimicrobial surveillance program (United States and Canada, 1997). Antimicrob Agents Chemother 42:1762–70.
2 Crossley KB and Archer GL. (1997) The Staphylococci in Human Disease (Churchill Living Stone Inc., New York).
3 Crouch M. (2002) Infective endocarditis. In Dipiro JT (Ed.). Pharmacotherapy a Pathophysiologic Approach, 5th edition (McGraw-Hill, New York) pp. 1905.
4 Moore RD, Lietman PS, Smith CR. (1987) Clinical response to aminoglycoside therapy: importance of the ratio of peak concentration to minimal inhibitory concentration. J Infect Dis 155:93–9.[Medline]
5 Ross GH, Hovde LB, Ibrahim YH, et al. (2001) In vitro pharmacodynamic analysis of single daily dosing versus conventional dosing of gentamicin administered with penicillin against Enterococcus faecalis. Pharmacotherapy 21:1479–85.[Medline]
6 Micromedex. Drug Dex Evaluation of Gentamicin www.micromedex.com (31 August 2005, date last accessed).
7
Zabinski RA, Vance-Bryan K, Krinke AJ, et al. (1993) Elimination of the activity of temafloxacin versus Bacteroides fragilis using an in vitro pharmacodynamic system. Antimicrob Agents Chemother 37:1377–9.
8 Antimicrobial Wildtype Distribution of Microorganisms EUCAST Online Database. www.escmid.org (30 January 2006, date last accessed).
9 National Committee for Clinical Laboratory Standards. (1993) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Third Edition: Approved Standard (NCCLS, Villanova, PA, USA).
10 Soriano F, Santamaria M, Ponte C, et al. (1988) In vivo significance of the inoculum effect of antibiotics on Escherichia coli. Eur J Clin Microbiol Infect Dis 7:410–2.[CrossRef][Web of Science][Medline]
11 Bourget P, Fernandez H, Delouis C, et al. (1991) Pharmacokinetics of tobramycin in pregnant women. Safety and efficacy of a once-daily dose regimen. J Clin Pharm Ther 16:167–76.[Medline]
12
Calandra T, Zinner SH, Viscoli C, et al. (1993) Efficacy and toxicity of single daily doses of amikacin and ceftriaxone versus multiple daily doses of amikacin and ceftazidime for infection in patients with cancer and granulocytopenia. Ann Intern Med 119:584–93.
13 Dudley MN and Zinner SH. (1991) Single daily dosing of amikacin in an in-vitro model. J Antimicrob Chemother 27:15–19.
14 Gilbert DN, Lee BL, Dworkin RJ, et al. (1998) A randomized comparison of the safety and efficacy of once-daily gentamicin or thrice-daily gentamicin in combination with ticarcillin-clavulanate. Am J Med 105:182–91.[CrossRef][Web of Science][Medline]
15
Kafetzis DA, Sianidou L, Vlachos E, et al. (1991) Clinical and pharmacokinetic study of a single daily dose of amikacin in paediatric patients with severe Gram-negative infections. J Antimicrob Chemother 27:105–12.
16 Karachalios GN, Houpas P, Tziviskou E, et al. (1998) Prospective randomized study of once-daily versus twice-daily amikacin regimens in patients with systemic infections. Int J Clin Pharmacol Ther 36:561–4.[Web of Science][Medline]
17
Maller R, Ahrne H, Holmen C, et al. (1993) Once versus twice-daily amikacin regimen: efficacy and safety in systemic gram-negative infections. J Antimicrob Chemother 31:939–48.
18 Murry KR, McKinnon PS, Mitrzyk B, et al. (1999) Pharmacodynamic characterization of nephrotoxicity associated with once-daily aminoglycoside. Pharmacotherapy 19:1252–60.[CrossRef][Web of Science][Medline]
19
Nicolau DP, Freeman CD, Belliveau PP, et al. (1995) Experience with a once-daily aminoglycoside program administered to 2184 adult patients. Antimicrob Agents Chemother 39:650–5.
20 Prins JM, Buller HR, Kuijper EJ, et al. (1993) Once versus thrice daily gentamicin in patients with serious infections. Lancet 341:335–9.[CrossRef][Web of Science][Medline]
21
Rybak MJ, Abate BJ, Kang SL, et al. (1999) Prospective evaluation of the effect of an aminoglycoside dosing regimen on rates of observed nephrotoxicity and ototoxicity. Antimicrob Agents Chemother 43:1549–55.
22 Bakri FE, Pallett A, Smith AG, et al. (1998) Ototoxicity induced by once-daily gentamicin. Lancet North Am Ed 351:1407–8.
23 Koo J, Tight R, Rajkumar V, et al. (1996) Comparison of once-daily versus pharmacokinetic dosing of aminoglycosides in elderly patients. Am J Med 101:177–83.[CrossRef][Web of Science][Medline]
24
Bisno AL, Dismukes WE, Durack DT, et al. (1989) Antimicrobial treatment of infective endocarditis due to viridans streptococci, entercocci, and staphylococci. JAMA 261:1471–7.
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