In Vitro Activity of Trimethoprim/Sulfamethoxazole Against Stenotrophomonas maltophilia (Sm) Using a Whole Blood Assay

1. In Vitro Activity of Trimethoprim/Sulfamethoxazole Against Stenotrophomonas maltophilia (Sm) Using a Whole Blood Assay Sandy J. Close, Pharm.D. and Steven J. Martin, Pharm.D., BCPS, FCCM College of Pharmacy, University of Toledo, 2801 W. Bancroft St., Toledo, Ohio 43606 Background: S.maltophila is an opportunistic pathogen in the ICU. TMP/SMX is a drug-of-choice for this organism, producing good clinical outcomes. Due to the bacteristatic activity of TMP/SMX against S.maltophilia, it is difficult to use in vitro testing to predict appropriate doses and dosing regimens for TMP/SMX. We hypothesize the immune system plays an important role enhancing the activity of TMP/SMX in vivo. The objective of this study was to determine if the addition of single donor, whole anticoagulated human blood (the immune component) to time kill or pharmacokinetic model assays would more accurately predict the in vivo effect of TMP/SMX against S.maltophilia infections. Methods: Time kill was performed with TMP/SMX alone (peak concentration 5/95 mg/ml), blood alone (75% v/v), T/S + blood, and control over 24 hours against 3 TMP/SMX-susceptible strains of S.maltophilia. The starting inoculum was 105 cfu/ml. A 2-compartment PK model was used to simulate a 7.5 mg/kg Q12H dose (peak 5.67mg/ml, t1/2 9.6 hrs). PK models were run using control, blood, TMP/SMX, and TMP/SMX + blood similar to time kill testing. All experiments were performed in duplicate. Samples were obtained for all assays at 0, 6, 12 and 24 hours for colony counts and PK. MICs were performed pre/post model runs. Results: Time kill and pharmacokinetic models produced similar results. TMP/SMX alone was bacteristatic against all isolates. Blood alone produced a 2.5 log reduction in colony count at 6 hrs with regrowth by 24 hrs. TMP/SMX + blood was similar to blood at 6 hrs, however at 24 hrs the combination was synergistic with a reduction of colony counts to lowest limit of detection (log 2.3) and no regrowth. Conclusions: Immune system activity plays an important role in the activity of TMP/SMX in clinical S.maltophilia infections. Traditional time kill or pharmacokinetic model in vitro testing does not accurately predict TMP/ SMX in vivo activity. Performing these in vitro tests with the addition of whole anticoagulated human blood produces a more accurate simulation of in vivo conditions. Stenotrophomonas maltophilia is a common opportunistic pathogen in immunocompromised and critically ill patients. The majority of infections caused by S. maltophilia are respiratory. While colonization with this organism is common, the mortality rate is significant from true infections. S. maltophilia is resistant to most beta-lactam, aminoglycoside, and fluoroquinolone antibiotics. Trimethoprim/sulfamethoxazole (TMP/SMX) is the drug-of-choice for this organism. There are few data regarding the pharmacokinetics or optimal dosing strategies of TMP/SMX for these infections. Dosages from 8 to 15 mg/kg/day (TMP) given in two to four divided doses have been recommended, to treat serious infections but without clinical validation.1-4 In vitro susceptibility tests for S. maltophilia have shown varying activity of TMP/SMX both alone and in combination with other agents.4-6 Lack of reliability of the results of in vitro testing makes application to the clinical setting difficult. We hypothesized that adding a component of the human immune system to traditional in vitro testing methods may help to more accurately describe the activity of TMP/SMX against S. maltophilia. ABSTRACT BACKGROUND METHODS Three clinical S. maltophilia strains were used in testing. TMP-SMX (TMP Sigma lot #68H1403, SMX Sigma lot #107H1111) were used for all testing. Mueller-Hinton broth (Difco) and Mueller-Hinton agar plates (Remel) were used to maintain the organisms, and for colony counts. Microdilution broth MICs were determined according to the guidelines of the National Committee for Clinical Laboratory Standards.7 MICs were performed before all experiments, and following the model runs for organisms with residual growth at 24 hours. Time kill assays were performed with TMP/SMX alone (peak concentration 5/95 mg/ml), blood alone (75% v/v), TMP/SMX + blood, and control over 24 hours against each of the three TMP/SMX-susceptible strains of SM. The starting inoculum was 105 cfu/ml. Whole anticoagulated human blood was obtained from a single donor on the day of each experiment. An in vitro two compartment glass infection model was used to simulate human pharmacokinetics. In this model, an outer “central” compartment simulates central blood circulation, and an inner “peripheral” or infection compartment simulates tissue infection. Each organism was tested in the model. Models were given a 7.5 mg/kg Q12H dose (peak 5.67mg/ml, t1/2 9.6 hrs). Models were run using control, blood, TMP/SMX, and TMP/SMX + blood similar to time kill testing. All experiments were performed in duplicate. Samples were obtained for all assays at 0, 6, 12 and 24 hours for colony counts and drug concentrations. MICs were performed pre/post model runs.  

2. In both time kill testing and pharmacokinetic models, TMP/SMX alone did not exhibit significant activity against three strains of S. maltophilia. The addition of whole anticoagulated human blood to TMP/SMX did show bactericidal activity against these three organisms. One of the main disadvantages of using in vitro laboratory studies to evaluate the potential clinical efficacy of a drug regimen is the inability to take into consideration the innate activity of the human immune system against the organism causing infection. The use of TMP/SMX in treating S. maltophilia infections is an excellent example of this problem, because while TMP/SMX often appears to fail in traditional in vitro testing, it is known to work in the clinical setting. While TMP/SMX has been the drug of choice for S. maltophilia infections, little work has been done to show appropriate dosing regimens. With increasing reports of S. maltophilia isolates that are resistant to TMP/SMX, more work is important to evaluate the possibilities of more effective dosing regimens as well as possible combination regimens with other agents that maintain activity against S. maltophilia such as ticarcillin/clavulanate. The addition of whole anticoagulated human blood to our in vitro model allows for a more accurate simulation of in vivo conditions under which these experiments can be conducted. RESULTS Time kill and pharmacokinetic models produced similar results. Time kill curves and results from pharmacokinetic model runs for all three isolates are shown in the figures below. TMP/SMX alone was bacteristatic against all isolates. Blood alone produced at 2.5 log reduction in colony count at 6 hours with regrowth by 24 hrs. TMP/SMX + blood was similar to blood at 6 hours, however at 24 hours the combination was synergistic with a reduction of colony counts to lowest limit of detection (log 2.3) and no regrowth. DISCUSSION 1 Gales AC, Jones RN, Forward KR, Liñares J, Sader HS, Verhoef J. Emerging Importance of Multidrug-Resistant Acinetobacter Species and Stenotrophomonas maltophilia as Pathogens in Seriously Ill Patients: Geographic Patterson, Epidemiological Features, and Trends in the SENTRY Antimicrobial Surveillance Program. Clin Infect Dis 2001;32(Suppl 2):104-13. 2 Garrison MW, Anderson DE, Campbell DM, Carroll KC, Malone CL, Anderson JD et al. Stenotrophomonas maltophilia: Emergence of Multidrug-Resistant Strains during Therapy and in an In Vitro Pharmacodynamic Chamber Model. Antimicrob Agents Chemother 1996;40:2859-64. 3 Vartivarian S, Anaissie E, Bodey G, Sprigg H, Rolston K. A Changing Pattern of Susceptibility of Xanthomonas maltophilia to Antimicrobial Agents: Implications for Therapy. Antimicrob Agents Chemother 1994;38:624-27. 4 Penzak SR, Abate BJ. Stenotrophomonas (Xanthomonas) maltophilia: A Multidrug-Resistant Nosocomial Pathogen. Pharmacotherapy 1997;17:293-301. 5 Muñoz Bellindo JL, Muñoz Criado S, García García I, Alonso Manzanares MA, Gutiérrez Zufiaurre MN, García-Rodríguez JA. In Vitro Activities of B-Lactam-B-Lacatamase Inhibitor Combinations against Stenotrophomonas maltophilia: Correlation between Methods for Testing Inhibitory Activity, Time-Kill Curves and Bactericidal Activity. Antimicrob Agents Chemother 1997;41:2612-15. 6 Poulos CD, Matsumara SO, Willey BM, Low DE, McGeer A. In Vitro Activities of Antimicrobial Combinations against Stenotrophomonas (Xanthomonas) maltophilia. Antimicrob Agents Chemother 1995;39:2220-23. REFERENCES