In vitro activity of tigecycline in combination with gentamicin against biofilm-forming Staphylococcus aureus

Available online at www.sciencedirect.com

Diagnostic Microbiology and Infectious Disease 68 (2010) 1 – 6 www.elsevier.com/locate/diagmicrobio

Bacteriology

In vitro activity of tigecycline in combination with gentamicin against biofilm-forming Staphylococcus aureus☆ Kevin W. McConeghy, Kerry L. LaPlante⁎ Department of Pharmacy Practice, University of Rhode Island, Veterans Affairs Medical Center (151), Providence, RI 02908, USA Infectious Diseases Research Laboratory, Providence Veterans Affairs Medical Center, Providence, RI 02908, USA Received 8 January 2010; accepted 19 April 2010

Abstract We investigated the activity of tigecycline in combination with gentamicin for the treatment of biofilm-forming methicillin-resistant and sensitive Staphylococcus aureus in an in vitro pharmacodynamic model. Tigecycline monotherapy demonstrated bacteriostatic activity throughout 48 h (−0.24 ± 0.17 log10 CFU/mL), whereas tigecycline in combination with gentamicin demonstrated significant (P b 0.002) kill (−3.66 ± 0.26 log10 CFU/mL) at 48 h. The addition of gentamicin to tigecycline significantly improved the killing activity of tigecycline in biofilm-forming S. aureus. © 2010 Elsevier Inc. All rights reserved. Keywords: Biofilm; Combination; Gentamicin; Methicillin-resistant Staphylococcus aureus; Tigecycline

1. Introduction Biofilms provide a slime-like glycocalyx matrix for populations of bacteria and confer increased protection against antimicrobials in addition to facilitating adherence to medical devices. Currently, there are limited data evaluating the activity of tigecycline and gentamicin alone or in combination against biofilm-producing staphylococcus (Doan et al., 2006; Donlan, 2001; Raad et al., 2007). Tigecycline is a novel glycylcycline antimicrobial chemically similar to minocycline. It demonstrates broad-spectrum activity against Gram-positive, Gram-negative, and anaerobic organisms (Doan et al., 2006). Tigecycline has also been reported to be active against methicillin-resistant Staphylococcus aureus (MRSA) bacteria that are known biofilmproducing organisms (Donlan, 2001; Raad et al., 2007). Gentamicin, a commonly used aminoglycoside, is often added to therapy when MRSA is present (Fowler et al., 2006). Tigecycline and gentamicin both bind to similar sites on the 30S ribosomal subunit where they disrupt protein ☆

This work has been presented in part at the American College of Clinical Pharmacy, St. Louis, MO, October 26 to 27, 2006 Abstract #3. ⁎ Corresponding author. Tel.: +1-401-273-7100 x 2339 (office); fax: +1-401-457-3305. E-mail address: [email protected] (K.L. LaPlante). 0732-8893/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.04.011

translation (Olson et al., 2006; Yoshizawa et al., 1998). However, gentamicin's activity against biofilm-forming staphylococcus is conflicting (Alt et al., 2004; Curtin et al., 2003; Gagnon et al., 1994; van de Belt et al., 2001). We investigate the activity of tigecycline and gentamicin alone and in combination against biofilm-forming methicillin-sensitive S. aureus (MSSA) and MRSA using an in vitro pharmacodynamic model (IVPD). 2. Materials and methods 2.1. Bacterial isolates A well-characterized biofilm-producing reference strain of MSSA (ATCC 35556) was evaluated along with 2 randomly selected known biofilm-producing clinical isolates (MRSA L31 and L198) previously obtained from patients with catheter-related bloodstream infections at the Providence Veterans Affairs Medical Center, Providence, RI. MRSA L31 is a blood isolate susceptible to aminoglycosides, tetracyclines, and trimethoprim/sulfamethoxazole but resistant to fluoroquinolones and macrolides. This isolate also has a vancomycin MIC of 2 μg/mL and was α-hemolysin negative. MRSA L198 demonstrated the same phenotype with the exception of clindamycin susceptibility and was positive for α-hemolysin production. MRSA

2

K.W. McConeghy, K.L. LaPlante / Diagnostic Microbiology and Infectious Disease 68 (2010) 1–6

L198 demonstrated the same phenotype with the exception of clindamycin susceptibility and was positive for α-hemolysin production. 2.2. Antimicrobial agents Tigecycline (lot no. RB5663) and gentamicin (lot no. 01651120) analytical powder was provided by Wyeth Pharmaceuticals (Pearl River, NY) and Sigma-Aldrich (St. Louis, MO), respectively. 2.3. Susceptibility testing Traditional MICs of study antimicrobial agents were determined by broth microdilution according to the Clinical and Laboratory Standards Institute (2009a, 2009b). 2.4. Quantification of biofilm formation Quantification of biofilm formation was conducted using a previously described colorimetric microtiter plate assay (Christensen et al., 1985; LaPlante and Mermel, 2007; Stepanovic et al., 2007). Adherent bacteria were stained with crystal violet, and the optical density (OD) at 570 nm (OD570) of stained adherent bacterial films was read using a spectrophotometer (Synergy 2; Bio-Tek Instruments, Winooski, VT). The OD of bacterial films was classified into the following categories based on multiples of the OD readings previously described by Stepanovic et al. (2000). Results were averaged and standard deviations were calculated. 2.5. Screening for synergy or antagonism Fractional inhibitory concentration tests evaluating tigecycline and gentamicin in combination were conducted for all 3 isolates and consistently demonstrated indifference between the agents (Bonapace et al., 2002). Time–kill assays were also performed, which did not demonstrate synergy or antagonism. 2.6 In vitro pharmacodynamic model A previously described 1-compartment IVPD provided exposure of bacteria to changing concentrations of tigecycline and gentamicin (Blaser, 1985; Zinner et al., 1985). Prior to each experiment, several colonies from an overnight growth on tryptic soy agar (TSA) were added to Mueller– Hinton broth (Difco Laboratories, Sparks, MD) supplemented with 25 mg/L calcium and 12.5 mg/L magnesium (SMHB) to obtain a suspension corresponding to a 0.5McFarland standard to produce an initial starting inoculum of 106 CFU/mL. Each model was placed in a 37 °C water bath for the duration of the experiment, with a magnetic stir bar to produce continuous mixing of medium. A peristaltic pump (Masterflex; Cole-Parmer Instrument, Chicago, IL) was used to continually replace antibiotic-containing medium with fresh SMHB (at a rate to simulate the halflife [t1/2] of the respective antibiotics). For combination regimen experiments, the elimination rate was set for the drug with the shortest half-life; the drug with the longer half-

life was supplemented (Blaser, 1985). All model simulations were conducted over 48 h performed in duplicate. Area under the curve (AUC)/MIC has been the suggested pharmacodynamic parameter for predicting antimicrobial efficacy of tigecycline (Doan et al., 2006). At steady-state, a 100-mg loading dose followed by 50 mg of tigecycline infused over 1 h every 12 h yields an AUC of 3.06 μg h/mL by serum concentration (Muralidharan et al., 2005). Tigecycline is distributed extensively into tissues; therefore, serum concentrations may underestimate total drug exposure at the actual site of infection (Tombs, 1999). A single 100-mg dose of tigecycline achieves a mean AUC of 9 μg h/ mL in lung tissue; it has also been demonstrated that an AUC/MIC of 20 to 25 achieves 95% probability of microbiologic cure (MacGowan, 2008). We therefore targeted an AUC of 9 μg h/mL to simulate tissue concentration (Meagher et al., 2005; Tombs, 1999). Gentamicin was administered to simulate 1.3 mg/kg every 12 h (Cpeak, 6; trough, 0.4 μg/mL; half-life, 3 h). It should also be noted that tigecycline and gentamicin exhibit moderate protein binding, and therefore, total concentrations of drug were simulated ([Package insert] Tygacil (tigecycline) for injection). 2.7. Pharmacokinetic analyses For the pharmacokinetic analysis in the in vitro model, 1-mL samples from each model were collected at 0, 0.5, 1, 2, 4, 6, 24, 30, and 48 h and stored at −80 °C until ready for analysis. Tigecycline concentrations were determined by a previously described and validated high-performance liquid chromatography method (Dandekar et al., 2003). Gentamicin concentrations were determined by a homogeneous particle-enhanced turbidimetric immunoassay (Architect, Multigent® ; Abbott Diagnostics, Abbott Park, IL) at the Providence Veterans Affairs Medical Center. The gentamicin assay has a range of detection of 0.3 to 10.0 μg/mL and a between-day sample precision and percentage coefficient of variation of 1.35% and b2.75%, respectively. 2.8. Pharmacodynamic analysis For the pharmacodynamic analysis, samples from each model were collected (stated above) and serially diluted in cold 0.9% sodium chloride. Bacterial counts were determined by plating 20-μL aliquots of each diluted sample on TSA. All samples were serially diluted before plating in order to minimize antibiotic carryover with a lower limit of detection for this method of 2 log10 CFU/mL (Laplante, 2006). Plates were incubated at 37 °C for 24 h and colony counts were performed. Reductions in log10 CFU/mL over 48 h were determined by plotting time–kill curves. Synergy was defined as a 2-log10 decrease in CFU per milliliter between the combination and its most active constituent after 24 h and when the number of surviving organisms in the presence of the combination was 2 log10 CFU/mL below the starting inoculums. Bacteriostatic activity was defined as ≤3 log10

K.W. McConeghy, K.L. LaPlante / Diagnostic Microbiology and Infectious Disease 68 (2010) 1–6

decrease or ≤1 log10 increase in CFU/mL; bactericidal activity is defined as ≥3 log10 decrease in CFU/mL. The AUC, AUC/MIC, and Cmax were determined utilizing the trapezoidal method for noncompartmental analysis (Microsoft Excel 2007, ©2006 Microsoft, [Redmond, Washington]). 2.9. Resistance To assess the development of resistance, 100-μL samples were collected at 24 and 48 h and were placed on TSA containing 2-, 4-, and 8-fold the MIC of the respective antibiotic. In addition, MIC testing was conducted using an E-test with samples evaluated directly from the model to prevent the passing of bacteria on antibiotic-containing plates. Plates were examined for growth after 24 and 48 h of incubation at 37 °C. 2.10. Statistical analysis Changes in bacterial growth (CFU/g) at 4, 8, 24, and 48 and time to 99.9% kill were compared by 2-way analysis of variance with Tukey's post-hoc test. A P value of ≤0.05 was considered significant. All statistical analyses were performed using SPSS Statistical Software (Release 15; SPSS, Chicago, IL). 3. Results The MICs for tigecycline were ≤0.125 μg/mL for all 3 isolates. The MICs for gentamicin were 0.125 μg/mL for ATCC 35556 and MRSA L31 and 0.5 μg/mL for MRSA L198. Regrowth in the models was captured and tested for resistance by E-testing, in duplicate through the 48 h model, but no significant changes in MIC were detected during the study. The biofilm formation of 2 clinical MRSA isolates obtained from patients was quantified (Fig. 1). Both isolates were robust biofilm producers (OD, 570 nm) and produced slightly less (12–23%) biofilm than the well characterized biofilm-forming isolate (ATCC 35556). In the IVPD model (Fig. 2), tigecycline monotherapy demonstrated bacteriostatic activity against all biofilm-

3

forming S. aureus isolates at 48 h (average decrease, 0.24 ± 0.17 log10 CFU/mL). Gentamicin monotherapy demonstrated a 3.09 ± 1.0 log10 CFU/mL decrease at 48 h. Tigecycline and gentamicin combination demonstrated significant activity against biofilm-producing staphylococcus, which reached the limit of detection by the 24-h time point. Specifically, tigecycline combination therapy demonstrated a significant increase in kill for blood isolate MRSA L31 at 24 h (mean difference, 3.8; 95% confidence interval [CI], 2.02–5.58 log10 CFU/mL; P b 0.02) and 48 h (mean difference, 3.85; 95% CI, 3.4–4.2 log10 CFU/mL; P b 0.0001). Similar activity was observed for ATCC 35556 at 24 h (mean difference, 3.3; 95% CI, 1.95–4.65 log10 CFU/ mL; P b 0.001) and 48 h (mean difference, 3.5; 95% CI, 2.7–4.30 log10 CFU/mL; P b 0.0001). Of note, MRSA clinical isolate L198, a more robust biofilm producer (Fig. 1), demonstrated regrowth in the presence of clinical concentrations of gentamicin and tigecycline alone after 24 h (Fig. 2C). We performed noncompartmental pharmacokinetic analysis and found tigecycline and gentamicin concentrations to be within 15% of targeted parameters. A tigecycline 100-mg loading dose with 50-mg q12 gave a Cpeak of 2.21 ± 0.15, an AUC 0–24 h of 14.31 ± 0.31 μg h/mL, and an AUC/MIC of 114 ± 2.5 μg h/mL in our model, which is similar to other in vitro pharmacokinetic reports (Garrison and Nuemiller, 2006). Gentamicin was within 98% of target with a calculated Cmax of 5.9 μg/mL and a trough of 0.33 μg/ mL. Pharmacokinetic analyses demonstrate that targeted concentrations were attained and no MIC shift was noted.

4. Discussion With the advent of multidrug-resistant organisms and recurring treatment failures in patients with serious bacterial infections caused by biofilm-forming bacteria, new treatment approaches must be evaluated to improve outcomes. In our experiments, tigecycline demonstrated consistent bacteriostatic activity for all 3 isolates, which is consistent with previous studies (Laplante et al., 2006; Mercier et al.,

Fig. 1. Quantitative measurement of clinical MRSA isolates L198 and L31. Controls included a biofilm nonforming staphylococcus (ATCC 12228) and a characterized biofilm-forming S. aureus (ATCC 35556). Results are an average of a quadruplicate run ± standard error of the mean.

4

K.W. McConeghy, K.L. LaPlante / Diagnostic Microbiology and Infectious Disease 68 (2010) 1–6

Fig. 2. In vitro pharmacodynamic models. Results are presented as mean ± standard deviation for the MSSA ATCC 35556 (A) and both MRSA L31 (B) and L198 (C).

K.W. McConeghy, K.L. LaPlante / Diagnostic Microbiology and Infectious Disease 68 (2010) 1–6

2002; Scheetz et al., 2007). We have now demonstrated tigecycline's activity against organisms that are known biofilm producers. Tigecycline plus gentamicin therapy demonstrated significant (P b 0.001) increase in kill versus tigecycline monotherapy for all isolates at 24 and 48 h. Gentamicin demonstrated bactericidal activity with significant kill for 2 of the 3 isolates tested. Because both gentamicin alone and the combination reached the lower limit of detection (2 log10 CFU/mL), we cannot state that there was synergy or an additive effect. MRSA clinical isolate L198, a more robust biofilm producer (Fig. 1), demonstrated significant regrowth in the presence of clinical concentrations of gentamicin alone and tigecycline alone after 24 h (Fig. 2C). Note that this isolate had the highest MIC to gentamicin of the 3 tested (0.5 μg/ mL) but did not demonstrate a significant change in MIC with an E-test after exposure in the model for 48 h. Of notable interest, our in-house data demonstrate that after the first 24 h, bacteria formed as mature biofilm within the IVPD as slime were noted in the glass ports. We therefore hypothesize that bacterial colonies remained sequestered in the biofilm and released (seed) susceptible strains that were captured when taking samples from the model (Costerton et al., 1999). However, regrowth or biofilm was not observed in tigecycline gentamicin combination therapy. Although the combination of tigecycline and gentamicin proved effective in providing bactericidal activity against biofilm-forming clinical isolates, gentamicin monotherapy had a greater decrease in CFU/mL in the first 4 h than the combination of tigecycline and gentamicin (Fig. 2). This postponement of activity was not observed past 24 h. This may be explained by the bacteriostatic nature of tigecycline, which may delay gentamicin's killing effects by preventing the bacteria from undergoing protein synthesis. This has been a hypothesis for observed treatment failures with other antibiotic bactericidal/bacteriostatic combinations (LaPlante et al., 2009; Olsson et al., 1961). Importantly, gentamicin is not used as monotherapy for MRSA because of the risk of resistance developing, and our inclusion of a gentamicin monotherapy arm was only as an experimental control. Overall, we found that tigecycline demonstrates bacteriostatic activity in biofilm-forming S. aureus. We also note that gentamicin and tigecycline did not demonstrate antagonism in our model. This observation provides useful data regarding antimicrobial activity with combination therapy in various infection models. Although previous studies have investigated higher inoculums of bacteria in determining antibiotic activity in vitro, evidence suggests that protein synthesis inhibitors are not necessarily affected by elevated inocula of staphylococci (Laplante and Rybak, 2004). Further investigations could be conducted using organisms with elevated MICs or lower concentrations of antibiotic to characterize any additive or synergistic effects of this antibiotic combination.

5

Acknowledgment The authors gratefully acknowledge Leslie Pierson, Core Laboratory Supervisor, and Michael Kline M.D., Chief of Laboratory Services at the Veterans Affairs Medical Center in Providence, RI, for analysis of the gentamicin samples. They also gratefully acknowledge David P. Nicolau, Pharm. D., FCCP, and Christina Sutherland at the Center for AntiInfective Research and Development at Hartford Hospital (Hartford, CT) for high-performance liquid chromatography analysis of tigecycline concentrations. They thank Fatemeh Akhlaghi, Pharm.D., Ph.D. (College of Pharmacy, University of Rhode Island), for offering guidance on analysis and interpretation of pharmacokinetics. This publication was made possible by RI-INBRE grant no. P20RR016457 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH. References Alt V, Bechert T, Steinrucke P, Wagener M, Seidel P, Dingeldein E, Domann E, Schnettler R (2004) In vitro testing of antimicrobial activity of bone cement. Antimicrob Agents Chemother 48:4084–4088. Blaser J (1985) In-vitro model for simultaneous simulation of the serum kinetics of two drugs with different half-lives. J Antimicrob Chemother 15(Suppl A):125–130. Bonapace CR, Bosso JA, Friedrich LV, White RL (2002) Comparison of methods of interpretation of checkerboard synergy testing. Diagn Microbiol Infect Dis 44:363–366. Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton DM, Beachey EH (1985) Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 22:996–1006. Clinical and Laboratory Standards Institute (CLSI) (2009a) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—eighth edition, M07-A8. Wayne, PA: CLSI. Clinical and Laboratory Standards Institute (CLSI) (2009b) Performance standards for antimicrobial susceptibility testing: 19th informational supplement, M100-S19. Wayne, PA: CLSI. Costerton JW, Stewart PS, EP G (1999) Bacterial biofilms: a common cause of persistent infections. Science 284:1318–1322. Curtin J, Cormican M, Fleming G, Keelehan J, Colleran E (2003) Linezolid compared with eperezolid, vancomycin, and gentamicin in an in vitro model of antimicrobial lock therapy for Staphylococcus epidermidis central venous catheter-related biofilm infections. Antimicrob Agents Chemother 47:3145–3148. Dandekar PK, Tessier PR, Williams P, Nightingale CH, Nicolau DP (2003) Pharmacodynamic profile of daptomycin against Enterococcus species and methicillin-resistant Staphylococcus aureus in a murine thigh infection model. J Antimicrob Chemother 52:405–411. Doan TL, Fung HB, Mehta D, Riska PF (2006) Tigecycline: a glycylcycline antimicrobial agent. Clin Ther 28:1079–1106. Donlan R (2001) Biofilm and device-associated infections. Emerg Infect Dis 7:277–281. Fowler VG Jr, Boucher HW, Corey GR, Abrutyn E, Karchmer AW, Rupp ME, Levine DP, Chambers HF, Tally FP, Vigliani GA, Cabell CH, Link AS, DeMeyer I, Filler SG, Zervos M, Cook P, Parsonnet J, Bernstein

6

K.W. McConeghy, K.L. LaPlante / Diagnostic Microbiology and Infectious Disease 68 (2010) 1–6

JM, Price CS, Forrest GN, Fätkenheuer G, Gareca M, Rehm SJ, Brodt HR, Tice A, Cosgrove SE, Group, S. a. E. a. B. S (2006) Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphylococcus aureus. N Engl J Med 355:653–656. Gagnon RF, Richards GK, Kostiner GB (1994) Time–kill efficacy of antibiotics in combination with rifampin against Staphylococcus epidermidis biofilms. Adv Perit Dial 10:189–192. Garrison MW, Nuemiller JJ (2006) In vitro activity of tigecycline against quinolone-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. Int J Antimicrob Agents 29:191–196. LaPlante KL (2006) In vitro activity of lysostaphin, mupirocin and tea tree oil against clinical Staphylococcus aureus. Diagn Microbiol Infect Dis 57:413–418. LaPlante KL, Rybak MJ (2004) Impact of high-inoculum Staphylococcus aureus on the activities of nafcillin, vancomycin, linezolid, and daptomycin, alone and in combination with gentamicin, in an in vitro pharmacodynamic model. Diagn Microbiol Infect Dis 48:4665–4672. LaPlante KL, Rybak MJ, Leuthner KD, Chin JN (2006) Impact of Enterococcus faecalis on the bactericidal activities of arbekacin, daptomycin, linezolid, and tigecycline against methicillin-resistant Staphylococcus aureus in a mixed-pathogen pharmacodynamic model. Antimicrob Agents Chemother 50:1298–1303. LaPlante KL, Sakoulas G (2009) Evaluating aztreonam and ceftazidime pharmacodynamics with Escherichia coli in combination with daptomycin, linezolid, or vancomycin in an in vitro pharmacodynamic model. Antimicrob Agents Chemother 53:4549–4555. LaPlante KL, Mermel LA (2007) In vitro activity of daptomycin and vancomycin lock solutions on staphylococcal biofilms in a central venous catheter model. Nephrol Dial Transplant 22:2239–2246. MacGowan AP (2008) Tigecycline pharmacokinetic/pharmacodynamic update. J Antimicrob Chemother 62(Suppl 1):i11–i16. Meagher AK, Ambrose PG, Grasela TH, Ellis-Grosse E (2005) Pharmacokinetic/pharmacodynamic profile for tigecycline—a new glycylcycline antimicrobial agent. Diagn Microbiol Dis 52:165–171. Mercier RC, Kennedy C, Meadows C (2002) Antimicrobial activity of tigecycline (GAR-936) against Enterococcus faecium and Staphylococcus aureus used alone and in combination. Pharmacotherapy 22:1517–1523.

Muralidharan G, Micalizzi M, Speth J, Raible D, Troy S (2005) Pharmacokinetics of tigecycline after single and multiple doses in healthy subjects. Antimicrob Agents Chemother 49:220–229. Olson M, Ruzin A, Feyfant E, Rush T, O'Connell J, Bradford P (2006) Functional, biophysical, and structural bases for antibacterial activity of tigecycline. Antimicrob Agents Chemother 50:2156–2166. Olsson R, Kirby JC, Romansky MJ (1961) Pneumococcal meningitis in the adult: clinical, therapeutic, and prognostic aspects in forty-three patients. Ann Intern Med 55:545–549. [Package insert] Tygacil (tigecycline) for injection. Philadelphia, PA 19101: Wyeth Pharmaceuticals Inc. Raad I, Hanna H, Jiang Y, Dvorak T, Reitzel R, Chaiban G, Sherertz R, Hachem R (2007) Comparative activities of daptomycin, linezolid, and tigecycline against catheter-related methicillin-resistant Staphylococcus bacteremic isolates embedded in biofilm. Antimicrob Agents Chemother 51:1656–1660. Scheetz MH, Qi C, Warren JR, Postelnick MJ, Zembower T, Obias A, GA N (2007) In vitro activities of various antimicrobials alone and in combination with tigecycline against carbapenem-intermediate or -resistant Acinetobacter baumannii. Antimicrob Agents Chemother 51:1621–1626. Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40:175–179. Stepanovic S, Vukovic D, Hola V, Di Bonaventura G, Djukic S, Cirkovic I, Ruzicka F (2007) Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS 115:891–899. Tombs NL (1999) Tissue distribution of GAR-936, a broad-spectrum antibiotic, in male rats 39th ICAAC Program and Abstracts. San Francisco (CA): American Society for Microbiology. van de Belt H, Neut D, Schenk W, van Horn JR, van Der Mei HC, Busscher HJ (2001) Staphylococcus aureus biofilm formation on different gentamicin-loaded polymethylmethacrylate bone cements. Biomaterials 22:1607–1611. Yoshizawa S, Fourmy D, Puglisi J (1998) Structural origins of gentamicin antibiotic action. EMBO J 17:6437–6448. Zinner SH, Blaser J, Stone BB, Groner MC (1985) Use of an in-vitro kinetic model to study antibiotic combinations. J Antimicrob Chemother 15 (Suppl A):221–226.