In Vitro Activities of Telavancin and Vancomycin against Biofilm-Producing Staphylococcus aureus,S. epidermidis,and Enterococcus faecalis Strains

We investigated the activities of telavancin and vancomycin against biofilm-producing Staphylococcus and Enterococcus strains. At clinically attainable concentrations, telavancin was active against bacteria embedded in biofilm (minimal biofilm eradication concentration [MBEC], 0.125 to 2 μg/ml) and inhibited biofilm formation at concentrations below the MIC. Vancomycin did not demonstrate the same activity (MBEC, ≥512 μg/ml) against Staphylococcus aureus and Enterococcus faecalis. Telavancin may have a unique role in biofilm-associated infections.Staphylococci and enterococci account for a large proportion of hospital-acquired infections, especially among patients with indwelling devices (17). These infections are often caused by biofilm-producing strains which are difficult to eradicate and which may cause bacteremia and metastatic infections (30).Telavancin is a new lipoglycopeptide antimicrobial agent with a core chemical structure similar to that of vancomycin yet modified to include a lipophilic side chain (15). Telavancin possesses a second mechanism of action that causes rapid depolarization and loss of the functional integrity of the bacterial membrane (1, 12). These two mechanisms of action may be implicated in the lower range of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) MICs observed with telavancin (MIC range, 0.06 to 1.0 μg/ml) compared to vancomycin (MIC range, 0.5 to 2 μg/ml) (8, 13).Previous reports have suggested that telavancin is more effective than vancomycin against biofilm-forming S. aureus in a pharmacokinetic filter model (9). We compared telavancin and vancomycin activities by using previously described in vitro biofilm activity assays. The first assay evaluated each agent''s activity in a preformed biofilm by using both a modified version and the standard version of the Calgary Biofilm Pin Lid Device (CBPD) (3, 14). The second assay evaluated activity in preventing biofilm formation by planktonic isolates (14).(This work was presented in part at the 48th annual Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 24 to 28 October 2008.)Telavancin and vancomycin analytical powder, freshly prepared each day, was obtained from Astellas Pharmaceuticals, Inc. (Audubon, PA), and Sigma-Aldrich (St. Louis, MO), respectively. Well-characterized biofilm-producing reference strains of S. aureus (ATCC 35556), Staphylococcus epidermidis (RP62A; ATCC 35984), and Enterococcus faecalis (ATCC 29212; vancomycin susceptible) were evaluated. A stable, slime-negative mutant, M7, from wild-type S. epidermidis RP62A served as a control (21). We also evaluated three randomly selected, biofilm-producing clinical isolates (methicillin-susceptible S. aureus [MSSA] L2, and MRSA L32 and L83) previously obtained from patients with catheter-related bloodstream infections at the Providence Veterans Affairs Medical Center.The medium used for biofilm growth was Bacto tryptic soy broth (TSB; Becton Dickinson, Sparks, MD) plus 1% glucose and 2% NaCl (14). All assays were run in quadruplicate, and cultures were incubated at 35°C. Conventional MICs and minimal bactericidal concentrations (MBCs) were determined in duplicate by using Clinical and Laboratory Standards Institute (CLSI) guidelines (5, 6).We used a modified version of the CBPD to determine the antimicrobial susceptibility of bacteria embedded in a 24-h biofilm to determine the minimum biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC) (3). Briefly, a starting inoculum of 7 log₁₀ CFU/ml was established by the direct colony suspension method from a 24-h tryptic soy agar (TSA; Becton Dickinson, Sparks, MD) plate. This inoculum was then standardized with McFarland standards and validated by determination of viable counts on TSA plates. Biofilms developed on the pin lid (Immuno TSP; Nunc, Roskilde, Denmark) submerged in the inoculated broth at 35°C for 24 h on a rocking table (Boekel Shake and Bake; Boekel Scientific; Feasterville, PA). The pin lid was then rinsed three times in 1× phosphate-buffered saline (PBS) to remove sessile bacteria before placement into fresh uninoculated broth containing serial dilutions of each antibiotic and incubated for 24 h at 35°C. The next day, the pin lid was removed and the MBIC was recorded and defined as the last well in which there is no visible growth after incubation in the presence of biofilm and antibiotic. Next, to obtain the MBEC, the pin lid was rinsed in 1× PBS and then sonication (10 min) was performed on a low-output sonicator (45 Hz) in order to disperse the bacteria from the pin surface. After sonication, the broth was vortexed for 30 s. The fluid was serially diluted and plated in duplicate on TSA, and the number of CFU per milliliter was determined. The limit of detection was 2.4 CFU/ml. Viable counts were also obtained by measuring the turbidity at 570 nm (A₅₇₀) on a 96-well plate reader.Quantification of biofilm formation in the presence of telavancin (0 to 16 μg/ml) and vancomycin (0 to 16 μg/ml) was conducted with a previously described colorimetric microtiter plate assay (4, 14, 24). Wells with sterile TSB alone served as negative controls, and the mean optical density (OD) values of these wells was subtracted from the OD values of the test wells.Following incubation, the liquid was gently aspirated and replaced with sterile PBS (pH 7.3). Each well was rinsed three times and air dried. Adherent bacteria were then stained with crystal violet. The OD at 570 nm (OD₅₇₀) of stained adherent bacterial films was read with a spectrophotometer (Synergy 2; Bio-Tek Instruments, Inc., Winooski, VT). The ODs of bacterial films were classified into nonadherent, weakly, moderately, and strongly adherent categories based on multiples of the OD readings as described by Stepanović et al. The test was carried out in quadruplicate. Results were averaged, and standard deviations were calculated (23).Among planktonic bacteria, the ranges of telavancin MICs and MBCs were 0.03 to 0.25 and 0.125 to 1 μg/ml, respectively. The ranges of vancomycin MICs and MBCs were 1 to 2 and 2 to 16 μg/ml, respectively (Table ​(Table1).1). When evaluating the activity of sessile bacteria seeding the medium from formed biofilm, the MBICs of telavancin and vancomycin were 0.25 to 1 and 4 to 16 μg/ml, respectively (Table ​(Table1).1). Overall, the telavancin and vancomycin MBICs for each bacterial isolate were the same as the corresponding MICs or up to 3 dilutions higher.

TABLE 1.

Susceptibility testing results for planktonic and adherent (biofilm) strains