Ray Hachem, MD The University of Texas M. D. Anderson Cancer Center 1515 Holcombe Blvd.

1. Ray Hachem, MD The University of Texas M. D. Anderson Cancer Center 1515 Holcombe Blvd. Department of Infectious Diseases, Infection Control and Employee Health Unit 402 Houston, TX 77030 rhachem@mdanderson.org Figure 1. Time kill activity of antibiotics against 10 strains of MRSA embedded in biofilm after 4-hour daily exposure over five days. linezolid (lzd), vancomycin (vanco), tigecycline (tige), minocycline (mino), and rifampin (rifam) E-1352 ACTIVITY OF RIFAMPIN AGAINST MRSA EMBEDDED IN BIOFILM: THE RISK OF RESISTANCE IF USED ALONE AND BENEFIT OF SYNERGY IF USED IN COMBINATION R. Hachem1, H. Hanna1, Y. Jiang1, T. Dvorak1, R. Reitzel1, G. Chaiban1, R. Sherertz2, and I. Raad1. 1The University of Texas M. D. Anderson Cancer Center, Houston, TX and 2Bowman Gray School of Medicine, Winston-Salem, NC ABSTRACT MATERIALS AND METHODS RESULTS (cont.) Background: Recent guidelines have suggested the use of antibiotic catheter lock therapy (ALT) for the prevention and salvage of catheter-related MRSA bacteremia (CR-MRSA). We sought to test the activity of rifampin used alone or in combination with other antibiotics against CR-MRSA isolates embedded in biofilm Methods: Ten CR-MRSA bacteremic isolates from cancer patients were tested. Silicone disks (4-6 replicates per isolate) were colonized with MRSA by sequentially incubating them in plasma, then in Mueller-Hinton broth (MHB) containing 105 CFU/ml of each organism. The disks were subsequently exposed for 4 hrs to antibiotic solutions containing rifampin alone or linezolid, minocycline, rifampin, tigecycline, or vancomycin alone or in combination with rifampin at a concentration of 2 mg/ml or placed in water (control). Disks were then removed, washed and incubated in MHB at 37C overnight. This 4-hr exposure and overnight incubation was repeated daily for 5 days. Every day disks were removed, washed, sonicated and then colony count determined. Results: When rifampin was used alone, it was the least effective in eradicating MRSA from biofilm after 5 days of 4-hr daily exposures (ALT). Seven of the 9 MRSA isolates that were initially highly susceptible to rifampin became resistant to this antibiotic after 5-day ALT exposures. Decreased susceptibility to linezolid was also noted when used alone. Decreased susceptibility to other antibiotics did not occur. However, when rifampin was used in combination with other antibiotics, the combination was synergistically effective in eliminating MRSA colonization in biofilm more rapidly than any of the antibiotics alone. Conclusion: If used alone, rifampin failed to eradicate MRSA embedded in biofilm and is likely to generate rifampin-resistant MRSA. However, when used in combination with other anti-staphylococcal antibiotics, the combination was synergistically effective in eradicating MRSA embedded in biofilm. • The ability of various antibiotics to eradicate MRSA organisms embedded in biofilm was determined by the silicone disk biofilm colonization in vitro model as follows: • Ten CR-MRSA bacteremic isolates from cancer patients were tested. Silicone disks (4-6 replicates per isolate) were colonized with MRSA by sequentially incubating them in plasma, then in Mueller-Hinton broth (MHB) containing 105 CFU/ml of each isolate • Sterile silicone disks were placed in human plasma and incubated, shaking for 24 hours at 37C .The plasma was then replaced with 1mL of bacterial inoculum and incubated shaking for 24 hours at 37ºC. Bacterial inoculum was made by diluting 10 MRSA isolates that had caused CRBSI, to 5.5x105 cells/mL in Muller Hinton Broth (MHB). • The disks were exposed for 4 hrs to antibiotic solutions containing linezolid, minocycline, rifampin, tigecycline, or vancomycin alone or in combination with rifampin at a concentration of 2 mg/ml or placed in water. • Disks were then removed, washed and incubated in MHB at 37ºC overnight. This 4-hr exposure and overnight incubation were repeated daily for 5 days. Disks were removed, washed, sonicated and colony counts determined daily. RESULTS • Seven of the nine MRSA isolates that were initially highly susceptible to rifampin became resistant to this antibiotic after 5-day ALT exposures (Table 1). • When used alone, rifampin was the least effective in eradicating MRSA from biofilm after five days of 4-hour daily exposures (ALT) (Figure 1). • The combination of rifampin with other antibiotics was synergistically effective in eliminating MRSA colonization in biofilm more rapidly than any of the antibiotics along (P<0.05) (Figure 2 and Figure 3). Figure 3. Synergistic time kill activity of rifampin (R) in combination with linezolid (L) against 10 strains of MRSA embedded in biofilm after 4-hour daily exposure over five days. Figure 2. Synergistic time kill activity of rifampin (R) in combination with vancomycin (V) against 10 strains of MRSA embedded in biofilm after 4-hour daily exposure over five days INTRODUCTION Intraluminal colonization of the central venous catheter (CVC) is universal and is often caused by methicillin-resistant staphylococci that embed themselves in the biofilm layer in the lumen of the catheter, resulting in catheter-related bloodstream infections (CRBSI). Recent guidelines have suggested the use of antibiotic catheter lock therapy (ALT) for the prevention and salvage treatment of CRBSI in high-risk patients. However, MRSA organisms that are susceptible to glycopeptides, such as vancomycin in suspension, become resistant to this antibiotic when embedded in biofilm on catheter surfaces. In this current study, we used an in vitro silicone disk biofilm colonization model to determine the activity of rifampin used alone or in combination. CONCLUSIONS When used alone, rifampin failed to eradicate MRSA embedded in biofilm and resulted in rifampin-resistant MRSA. The combination of rifampin with other anti-staphylococcal antibiotics, was synergistically effective in eradicating MRSA embedded in biofilm. Hence improving the limited activity of vancomycin and linezolid in biofilm. Note: L=Linezolid, R=Rifampin Note: V=Vancomycin, R=Rifampin Note: V=Vancomycin, R=Rifampin