TY - JOUR
AU - LaPlante, Kerry, L.
AB - Abstract Purpose The activity of linezolid and vancomycin lock solutions against biofilm-producing strains of Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis was studied. Methods Two strains each of methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), and S. epidermidis, and 1 strain of vancomycin-susceptible E. faecalis and vancomycin-resistant E. faecalis were tested against vancomycin and linezolid to assess prevention of biofilm formation and eradication of these pathogens within a formed biofilm. Activity was also tested in a 72-hour in vitro central venous catheter (CVC) model. After 24 hours of biofilm growth in a CVC, a lock solution containing vancomycin (2 or 5 mg/mL) or linezolid (1 or 2 mg/mL) alone or in combination with heparin sodium (5,000 units/mL with benzyl alcohol 0.45%) was instilled and incubated at 35 °C for 72 hr. Heparin and 0.9% sodium chloride injection were also tested. Results Linezolid and vancomycin prevented biofilm formation below the minimum inhibitory concentration for 88% and 25% of isolates tested, respectively. The addition of preservative-containing heparin decreased the activity of vancomycin and linezolid lock solutions against all strains. Vancomycin 2- and 5-mg/mL lock solutions had the most activity against MSSA and E. faecalis strains (p < 0.01). Linezolid 2 mg/mL was the most active lock solution against the MRSA strains tested (p < 0.01). There were no significant differences in vancomycin or linezolid lock solution activity against S. epidermidis. Conclusion Heparin reduced activity of vancomycin and linezolid lock solutions against S. aureus, S. epidermidis, and E. faecalis biofilms. While linezolid or vancomycin lock solution reduced overall biofilm burden, it did not completely eradicate the bacteria at tested concentrations. biofilms, catheter-related infections, central venous catheters, heparin, linezolid, vancomycin Key Points Linezolid prevented biofilm formation for 7 of 8 strains tested in a microtiter assay, while vancomycin prevented biofilm in 2 of 8 strains tested. Neither vancomycin nor linezolid eradicated formed biofilms of Staphylococcus aureus, S. epidermidis, or Enterococcus faecalis at the concentrations tested. In the catheter lock model, the addition of heparin was associated with a reduction in the antimicrobial activity of both linezolid and vancomycin. Staphylococcal and enterococcal infections are major problems in hospital settings, especially among patients with indwelling devices.1 These infections are often caused by biofilm-producing bacterial strains, which are difficult to eradicate and frequently progress from intraluminal catheter colonization to bloodstream infection.2,3 When this occurs, the catheter is often removed. However, in some circumstances, an alternative venous access site is lacking, and a high-concentration antibiotic lock solution is used as in situ catheter salvage therapy. Although vancomycin is commonly used in combination with heparin as a lock solution, it is important to assess other antimicrobial agents with activity against biofilm-forming pathogens as alternative agents for use in lock solutions. Linezolid has demonstrated inhibitory effects on biofilm formation and has recently become available in a generic i.v. formulation.4,5 Eradication of biofilm-forming pathogens with lock solutions containing linezolid has had variable results.6,–10 Thus, we evaluated the activity of linezolid and vancomycin in preventing biofilm formation, on a formed biofilm, and in a 72-hour in vitro catheter lock model. The catheter lock model more closely mimics the clinical situation of a catheter-associated infection by using clinically applicable concentrations of antibiotics and central venous catheters (CVCs). Methods Bacterial strains. Well-characterized, biofilm-producing reference isolates of Staphylococcus aureus (ATCC35556, methicillin susceptible), S. epidermidis (RP62A and ATCC35984), and Enterococcus faecalis (ATCC29212, vancomycin susceptible) were evaluated.11,12 A stable, slime-negative mutant strain from the wild-type S. epidermidis RP62A (M7) served as a biofilm-negative control during biofilm prevention and eradication testing.13 We also evaluated 1 clinical urine isolate of S. epidermidis (L369D), 1 clinical vancomycin-resistant E. faecalis from tissue (L2022), and 3 S. aureus clinical blood isolates (methicillin-susceptible S. aureus [MSSA] L2 and methicillin-resistant S. aureus [MRSA] L32 and L83) from Providence Veterans Affairs Medical Center.14 These isolates were previously tested for biofilm formation by our laboratory.14,–16 All cultures were incubated at 35 °C. Lock solutions. Linezolid pharmacy solutiona and analytical powderb (final linezolid lock concentrations of 1 and 2 mg/mL) and vancomycin pharmacy solutionc (final vancomycin lock concentrations of 2 and 5 mg/mL) were evaluated. Heparin sodium pharmacy solution (10,000 units/mL with 0.9% benzyl alcohol preservatived, diluted to a final concentration of 5,000 units/mL and 0.45% benzyl alcohol) was used in the catheter lock model. Vancomycin was reconstituted in 0.9% sodium chloride injection. The catheter lock model was also tested with 0.9% sodium chloride injection alone. Linezolid analytic powder was used with heparin when necessary, since the maximum available pharmaceutical concentration of linezolid is 2 mg/mL, and the dilution of linezolid with heparin solution would result in a lower concentration. The antibiotic and heparin concentrations used were chosen because they are the final concentrations of standard i.v. antibiotic solutions mixed with commercially available or pharmacy-prepared heparin solutions, thus simplifying the preparation of the lock solutions. Susceptibility testing in planktonic organisms. Conventional minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations were determined in duplicate using a standard microdilution method in cation-adjusted Mueller-Hinton brothe and an inoculum of 5.5 log10 colony-forming units (CFU)/mL, as described by the Clinical and Laboratory Standards Institute.17,18 Prevention of biofilm formation in the presence of antibiotics. Biofilm formation was quantified using a previously described crystal violet assay.15,16,19 Increasing concentrations of linezolid and vancomycin (range, 0–256 mg/L) were evaluated. Supplemented tryptic soy brothf (STSB) with 1% dextrose, 2% sodium chloride, calcium chloride (25 mg/mL), and magnesium sulfate (12.5 mg/L)—an inoculum of ~5.5 log10 CFU/mL—was dispensed into wells containing serial dilutions of antibiotic and then incubated at 35 °C for 24 hours to form biofilm.14,19 After incubation, the liquid was gently aspirated, and each well was rinsed 3 times to remove planktonic bacteria and air-dried. Adherent bacteria were stained with crystal violet and measured using a spectrophotometer.g The optical density of each well was determined photometrically at 570 nm, and this determination was conducted in quadruplicate. Results were averaged. The concentration that prevented biofilm formation was determined graphically by comparing the mean optical density of each antibiotic concentration to the mean optical density of the media control (no biofilm). Antimicrobial susceptibility in established biofilms. We previously described a modified version of the Calgary biofilm device to determine the antimicrobial susceptibility of bacteria embedded in a 24-hour biofilm and the minimum biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC).14,19 All isolates were grown for 24 hours using a pin-lid deviceh and were run in quadruplicate.14,19,20 Briefly, a 0.5 McFarland standard from 24-hour tryptic soy agari (TSA) plates was diluted in STSB without added magnesium to a starting inoculum of ~107 CFU/mL. This inoculum was validated by determining viable counts on TSA plates. Inoculated broth in the pin-lid device was incubated at 35 °C for 24 hours on a rocking table.j The pin-lid was then rinsed 3 times in 1× phosphate buffered saline (PBS) to remove planktonic bacteria; placed into fresh, uninoculated broth containing twofold serial dilutions of antibiotic; and incubated for 24 hours at 35 °C. The MBIC was defined as the lowest concentration of antibiotic that inhibited seeding of the biofilm. This was determined to be the lowest antibiotic concentration without visual planktonic bacterial growth in the media after incubation. To obtain the MBEC, the pins were aseptically rinsed in 1× PBS, sonicated at 45 Hz in a fresh 96-well plate for 10 minutes on a low-output sonicator in order to disperse bacteria from the pin surface, and then incubated for 24 hours at 35 °C. MBECs were determined by analyzing visual growth in the fresh media. Antimicrobial activity in a CVC model. Catheter lock solutions tested included vancomycin (2 or 5 mg/mL) and linezolid (1 or 2 mg/mL) alone and in combination with heparin sodium 5,000 units/mL (plus benzyl alcohol 0.45% as a preservative), heparin sodium 5,000 units/mL with benzyl alcohol, and 0.9% sodium chloride injection. These solutions were evaluated in a previously described in vitro catheter lock model using triple-lumen polyurethane CVCs.19,21,22,k Two sets of catheters were processed (Figure 1). For all catheters, at 0 hour, fresh inocula of ~106 CFU/mL planktonic bacteria in STSB were injected into each CVC lumen.19 Catheter lumens were then clamped and incubated for 24 hours at 35 °C for biofilm development. Contents were then drained aseptically. One set of baseline catheters was processed at this time for inoculum quantification to determine the biofilm development inside the catheter lumens, as previously described.22 The other set was injected and incubated with lock solution (for an additional 72 hours) before processing for quantification. Figure 1 Open in new tabDownload slide Photo of a triple-lumen central venous catheter. Dashed lines represent approximate locations of cut catheter segments during processing. Figure 1 Open in new tabDownload slide Photo of a triple-lumen central venous catheter. Dashed lines represent approximate locations of cut catheter segments during processing. Recovery of organisms. Quantification of the inoculum was determined at the endpoint (24-hour biofilm formation or 72-hour lock solution incubation). Each clamped catheter system was removed from the incubator, the clamps were opened, and the catheter fluid was removed and discarded. Each open lumen was flushed by inserting a sterile needle and adding 1 mL of fresh, sterile 0.9% sodium chloride solution. The flush was collected in a sterile test tube for analysis. To optimize the yield of viable bacteria, the flushed 0.9% sodium chloride was sonicated at 60 Hz for 1 minute and mixed in a Vortex mixer for 15 seconds, as described elsewhere.19 In addition, in triplicate, 3-cm pieces of the catheter were sonicated and mixed in a Vortex mixer. The flushed, sonicated 0.9% sodium chloride solution and the 0.9% sodium chloride solution from the cut segments were each serially diluted 10-fold and plated on TSA plates. Plates were incubated at 35 °C overnight. The limit of detection for each flushed, sonicated, and vortexed culture is 2.0 log10 CFU/mL.23,24 Drug stability and compatibility. The combination of vancomycin or linezolid with heparin has been reported compatible and stable for up to 72 hours at 35 °C.25,26 We also evaluated each catheter lock solution for physical and chemical compatibility by testing for particulate formation, color change, and gas evolution at 0, 24, 48, and 72 hours. The 72-hour dwell time was selected because it is commonly used in clinical settings with hemodialysis catheters.19 Catheter lock solutions were randomly selected for concentration analysis at 0 and 72 hours of incubation. Each solution was incubated at 35 °C to determine stability at the approximate temperature of an in situ intravascular catheter. Vancomycin concentrations were determined using homogeneous particle-enhanced turbidimetric immunoassayl at the Providence Veterans Affairs Medical Center.27 This assay has a detection range of 0.5–80.0 μg/mL and intraday and interday coefficients of variation of <2% and <5%, respectively. Linezolid concentrations were determined by high performance liquid chromatography at the University of Florida, Gainesville, as described previously.28 Data analysis. Each catheter was used to test one lock solution–bacterial strain combination in triplicate. Baseline catheter results were also determined in triplicate. The log10 CFU/mL values from the flushed 0.9% sodium chloride and cut catheter segment were added together to yield a total log10 CFU/mL result. This allowed for quantifying the total biofilm remaining in the catheter and for combining catheter lumens of different gauges. These triplicate total results were subtracted from the triplicate total log10 CFU/mL of the baseline catheters for each strain to determine the activity (reduction in log10 CFU/mL; n = 9 per strain) of the lock solution. Activity was compared between groups using 1-way analysis of variance followed by Tukey’s post-hoc test for multiple comparisons. All statistical analyses were performed using SPSS statistical software, release 20 (SPSS, Chicago, IL). The a priori level of significance was set at 0.05. Results Biofilm formation in the presence of antibiotics. Linezolid and vancomycin prevented biofilm production at concentrations below the MIC of each isolate for 7 (88%) of 8 and 2 (25%) of 8 biofilm-producing isolates tested, respectively (Table 1). Biofilm mass was decreased with increasing concentrations of linezolid and vancomycin (0.5–256 mg/L). Table 1 Minimum Inhibitory and Bactericidal Concentrations for Planktonic Bacteria and Biofilmsa Bacterial Strain Biofilmb Planktonic Bacteria, mg/mL Biofilm Prevention, mg/L MBIC, mg/mL MBEC, mg/mL Linezolid Vancomycin MIC MBC MIC MBC Linezolid Vancomycin Linezolid Vancomycin Linezolid Vancomycin M7 None 1 8 1 1 …c … … … … … ATCC35556 Strong 1 16 1 2 0.5 2 2 8 >256 >256 L2 clinical Strong 2 32 2 2 1 2 4 16 >256 >256 L32 clinical Moderate 2 32 1 2 0.5 1 64 32 >256 >256 L83 clinical Moderate 2 32 2 2 1 1 2 8 >256 >256 ATCC35984 Strong 1 16 2 4 0.5 4 1 32 >256 >256 L369D clinical Strong 1 4 2 2 0.125 0.5 2 8 >256 >256 ATCC29212 Moderate 1 16 2 16 1 4 2 8 >256 >256 L2022 clinical Weak 2 8 256 256 0.5 >256 1 >256 >256 >256 Bacterial Strain Biofilmb Planktonic Bacteria, mg/mL Biofilm Prevention, mg/L MBIC, mg/mL MBEC, mg/mL Linezolid Vancomycin MIC MBC MIC MBC Linezolid Vancomycin Linezolid Vancomycin Linezolid Vancomycin M7 None 1 8 1 1 …c … … … … … ATCC35556 Strong 1 16 1 2 0.5 2 2 8 >256 >256 L2 clinical Strong 2 32 2 2 1 2 4 16 >256 >256 L32 clinical Moderate 2 32 1 2 0.5 1 64 32 >256 >256 L83 clinical Moderate 2 32 2 2 1 1 2 8 >256 >256 ATCC35984 Strong 1 16 2 4 0.5 4 1 32 >256 >256 L369D clinical Strong 1 4 2 2 0.125 0.5 2 8 >256 >256 ATCC29212 Moderate 1 16 2 16 1 4 2 8 >256 >256 L2022 clinical Weak 2 8 256 256 0.5 >256 1 >256 >256 >256 a MIC = minimum inhibitory concentration, MBC = minimum bactericidal concentration, MBIC = minimum biofilm inhibitory concentration, MBEC = minimum biofilm eradication concentration. b Biofilm was previously quantified14 in a microtiter plate assay15 and qualified as none, weak, moderate, or strong using M7 as the biofilm-negative control.13,16 c Test not conducted. Open in new tab Table 1 Minimum Inhibitory and Bactericidal Concentrations for Planktonic Bacteria and Biofilmsa Bacterial Strain Biofilmb Planktonic Bacteria, mg/mL Biofilm Prevention, mg/L MBIC, mg/mL MBEC, mg/mL Linezolid Vancomycin MIC MBC MIC MBC Linezolid Vancomycin Linezolid Vancomycin Linezolid Vancomycin M7 None 1 8 1 1 …c … … … … … ATCC35556 Strong 1 16 1 2 0.5 2 2 8 >256 >256 L2 clinical Strong 2 32 2 2 1 2 4 16 >256 >256 L32 clinical Moderate 2 32 1 2 0.5 1 64 32 >256 >256 L83 clinical Moderate 2 32 2 2 1 1 2 8 >256 >256 ATCC35984 Strong 1 16 2 4 0.5 4 1 32 >256 >256 L369D clinical Strong 1 4 2 2 0.125 0.5 2 8 >256 >256 ATCC29212 Moderate 1 16 2 16 1 4 2 8 >256 >256 L2022 clinical Weak 2 8 256 256 0.5 >256 1 >256 >256 >256 Bacterial Strain Biofilmb Planktonic Bacteria, mg/mL Biofilm Prevention, mg/L MBIC, mg/mL MBEC, mg/mL Linezolid Vancomycin MIC MBC MIC MBC Linezolid Vancomycin Linezolid Vancomycin Linezolid Vancomycin M7 None 1 8 1 1 …c … … … … … ATCC35556 Strong 1 16 1 2 0.5 2 2 8 >256 >256 L2 clinical Strong 2 32 2 2 1 2 4 16 >256 >256 L32 clinical Moderate 2 32 1 2 0.5 1 64 32 >256 >256 L83 clinical Moderate 2 32 2 2 1 1 2 8 >256 >256 ATCC35984 Strong 1 16 2 4 0.5 4 1 32 >256 >256 L369D clinical Strong 1 4 2 2 0.125 0.5 2 8 >256 >256 ATCC29212 Moderate 1 16 2 16 1 4 2 8 >256 >256 L2022 clinical Weak 2 8 256 256 0.5 >256 1 >256 >256 >256 a MIC = minimum inhibitory concentration, MBC = minimum bactericidal concentration, MBIC = minimum biofilm inhibitory concentration, MBEC = minimum biofilm eradication concentration. b Biofilm was previously quantified14 in a microtiter plate assay15 and qualified as none, weak, moderate, or strong using M7 as the biofilm-negative control.13,16 c Test not conducted. Open in new tab Antimicrobial susceptibility in established biofilms Linezolid and vancomycin did not produce any effect at concentrations below the MIC against bacteria embedded in an established 24-hour biofilm (Table 1). Linezolid inhibited seeding of formed biofilm below the MIC for 1 isolate with weak biofilm, while vancomycin did not inhibit seeding at concentrations below the MIC for any of the isolates tested. Linezolid and vancomycin did not eradicate formed biofilms at concentrations of 256 mg/L or lower. These results are within approximately 2 dilutions of previous work14; this difference is generally acceptable and may be due to variability in biofilm growth. Drug stability and compatibility. Vancomycin plus heparin solution and linezolid plus heparin solution were evaluated for compatibility over 72 hours of incubation at 35 °C. All mixtures demonstrated physical compatibility. There was no visible haze, particulate formation, color change, or gas evolution at 72 hours. Concentrations before incubation were within 6% of the targets. After incubation, linezolid concentrations increased approximately 30% and vancomycin concentrations increased approximately 16%. This may be due to losses in volume from evaporation during incubation. Antimicrobial activity in a catheter model. Results of the catheter lock models were combined for the 2 strains each of MSSA, MRSA, S. epidermidis, and E. faecalis. Baseline catheter results after 24-hour biofilm development were similar for all strains tested at ~107–108 CFU/mL and within 5% of previously reported results using the same strains.22 Vancomycin 2 mg/mL demonstrated more activity against MSSA than all other lock solutions tested (p < 0.01; Figure 2, panel A). Combination with heparin decreased activity of the antibiotic lock solutions; however, the activity of vancomycin 2 mg/mL only was significantly reduced with the addition of heparin (mean difference, −0.96 log CFU/mL; 95% confidence interval, −1.63 to 0.29 log CFU/mL; p < 0.01). Figure 2 Open in new tabDownload slide Killing activity (expressed as the mean ± S.E. of the mean [indicated by error bars] change in log10 colony-forming units [CFU]/mL) at 72 hours in catheters containing (A) methicillin-susceptible Staphylococcus aureus (2 strains, ATCC35556 and L2), (B) methicillin-resistant S. aureus (2 strains, L32 and L83), (C) S. epidermidis (2 strains, ATCC35984 and L369D), and (D) Enterococcus faecalis (vancomycin-susceptible E. faecalis [ATCC29212] and vancomycin-resistant E. faecalis [L2022]). NS = 0.9% sodium chloride injection, Hep = heparin sodium 5,000 units/mL (with benzyl alcohol 0.45%), VAN2 = vancomycin 2 mg/mL, VAN2H = vancomycin 2 mg/mL plus heparin, VAN5 = vancomycin 5 mg/mL, VAN5H = vancomycin 5 mg/mL plus heparin, LZD1 = linezolid 1 mg/mL, LZD1H = linezolid 1 mg/mL plus heparin, LZD2 = linezolid 2 mg/mL, LZD2H = linezolid 2 mg/mL plus heparin. Figure 2 Open in new tabDownload slide Killing activity (expressed as the mean ± S.E. of the mean [indicated by error bars] change in log10 colony-forming units [CFU]/mL) at 72 hours in catheters containing (A) methicillin-susceptible Staphylococcus aureus (2 strains, ATCC35556 and L2), (B) methicillin-resistant S. aureus (2 strains, L32 and L83), (C) S. epidermidis (2 strains, ATCC35984 and L369D), and (D) Enterococcus faecalis (vancomycin-susceptible E. faecalis [ATCC29212] and vancomycin-resistant E. faecalis [L2022]). NS = 0.9% sodium chloride injection, Hep = heparin sodium 5,000 units/mL (with benzyl alcohol 0.45%), VAN2 = vancomycin 2 mg/mL, VAN2H = vancomycin 2 mg/mL plus heparin, VAN5 = vancomycin 5 mg/mL, VAN5H = vancomycin 5 mg/mL plus heparin, LZD1 = linezolid 1 mg/mL, LZD1H = linezolid 1 mg/mL plus heparin, LZD2 = linezolid 2 mg/mL, LZD2H = linezolid 2 mg/mL plus heparin. Linezolid 2 mg/mL was more active against MRSA than all other lock solutions tested (p < 0.01; Figure 2, panel B). Only linezolid 2 mg/mL and vancomycin 2 mg/mL were significantly more active than heparin (p < 0.02). The activity of linezolid 1 and 2 mg/mL was significantly reduced by the addition of heparin (p < 0.01). All antibiotic lock solutions were significantly more active against S. epidermidis than heparin or 0.9% sodium chloride injection (p < 0.01; Figure 2, panel C). There were no significant differences between the linezolid or vancomycin lock solutions tested. However, we noted that the largest mean reductions in CFU per milliliter were seen against these bacterial strains, perhaps related to the amount of biofilm produced. We also noted a reduction in antibiotic activity when the lock solution was combined with heparin versus the lock solution alone; however, these differences were not significant. Vancomycin 2 and 5 mg/mL demonstrated greater activity against E. faecalis than all other lock solutions tested (p < 0.01; Figure 2, panel D). Only vancomycin 2 and 5 mg/mL and linezolid 1 mg/mL demonstrated significantly greater activity than heparin (p < 0.01). All antibiotics demonstrated greater activity alone than in combination with heparin (p < 0.02). None of the heparin-containing lock solutions demonstrated significantly different activity compared with heparin alone. Discussion In this study, we used a previously described catheter modeling system19,21,–23 with drug concentrations and lock times commonly used in clinical settings to compare the activity of linezolid and vancomycin in a catheter lock solution. The drug concentrations used were 500–2,000 times the MIC for linezolid and 1,000–5,000 times the MIC for vancomycin (except against the vancomycin-resistant E. faecalis [VRE] strain, which was approximately 20 times the MIC). This may account for the reduction of bacteria in a formed biofilm by linezolid and vancomycin lock solutions, as well as the activity of vancomycin against a VRE strain. A lower antibiotic concentration (2 mg/mL versus 5 mg/mL) demonstrated greater activity against some bacteria in this study. Linezolid and vancomycin do not demonstrate true concentration-dependent killing29,30; therefore, our results were not surprising. Notably, many of these differences were not statistically significant. In the cases where there was greater antimicrobial activity at lower concentrations, we hypothesize that increases beyond a certain concentration may not provide additional killing. This concentration-independent killing has been demonstrated previously for linezolid.31 It should be noted, however, that antibiotics tested in this study did not eradicate these biofilms at the concentrations and dwell times tested. The baseline catheters with 24-hour biofilm development yielded approximately 108 CFU/mL. The mean lock solution activity was ~2–3 log10 CFU/mL, which would leave ~105–106 CFU/mL remaining in the catheter lumen, highlighting the difficulty in eradicating formed biofilms. However, the activity of a lock solution against formed biofilm may increase with repeated lock removal and instillation,32 as occurs in clinical practice. Heparin locks and 0.9% sodium chloride solutions also demonstrate ~1–2 log10 CFU/mL reductions; this may be explained by the force of the lock solution instillation and removal, as bacteria removed in lock solution were not quantified. There are conflicting in vitro data in the literature regarding the ability of linezolid to eliminate staphylococci embedded in a biofilm.7,10,33,–38 Similar to our results, many studies have found a decrease in bacterial counts but could not demonstrate eradication.4,9,39 Some studies that found linezolid to be ineffective in eliminating biofilms used concentrations of <1 mg/mL.10,33 In our study, linezolid at 1 and 2 mg/mL did not eliminate formed biofilms. Other studies did not quantify biofilm formation.7,35,36 In addition, some studies did not evaluate the antimicrobial activity of lock solutions containing heparin.7,35 We found differences in activity of linezolid lock solutions between MRSA and MSSA strains, which may be strain dependent or due to differences in the amount of biofilm produced. Differences between our study and others that evaluated lock solutions may also reflect different methodologies, catheter lock dwell times, or drug concentrations or the use of preservative-containing versus nonpreservative-containing heparin. Even at high antibiotic concentrations, the addition of heparin with benzyl alcohol attenuated the activity of both linezolid and vancomycin lock solutions. Heparin has been shown to stimulate staphylococci biofilm formation by affecting quorum sensing and increasing cell-to-cell interactions.40,41 Alcohols, including the benzyl alcohol preservative in heparin solution, have also been reported to increase S. epidermidis and S. aureus biofilm development.42,–44 Data on the effect of heparin on enterococcal biofilms are lacking; however, the changes in linezolid and vancomycin activity after adding heparin sodium 5,000 units/mL suggest that heparin may increase biofilm in E. faecalis as well. We have noted similar results using telavancin 5 mg/mL or vancomycin 5 mg/mL alone or in combination with preservative-containing heparin sodium 2,500 units/mL with the same biofilm-forming strains used in this study.22 However, in the current study, heparin significantly reduced lock solution activity against S. aureus strains, whereas this difference previously was not significant. This finding may be dose related, since we used a lower heparin concentration in the previous study. This study had some limitations. We used a limited number of isolates with differing degrees of biofilm formation. This could make comparisons difficult; however, biofilm formation was confirmed in previous microtiter plate assays and qualified as weak, moderate, or strong according to published criteria16 using S. epidermidis strain M7 as a biofilm-negative control.13,–16 Also, the recovered bacteria from the baseline catheters and vancomycin comparators were similar between the isolates and previous work. Thus, we believe that biofilm formation is consistent among these strains under our study conditions.19,22 While the lock solution dwell times mirrored clinical situations as closely as possible, the inoculation and biofilm development may not occur clinically in a situation where lock solutions are routinely used to prevent catheter-related bloodstream infections. Thus, our results from the catheter lock model cannot be used to predict the prevention of catheter-related infections with a lock solution. Future testing of anticoagulants other than heparin, including EDTA and sodium citrate, may be helpful in determining optimal antibiotic lock therapy. Conclusion Heparin reduced activity of vancomycin and linezolid lock solutions against S. aureus, S. epidermidis, and E. faecalis biofilms. While linezolid or vancomycin lock solution reduced overall biofilm burden, it did not completely eradicate the bacteria at tested concentrations. Disclosures This work was supported in part by an investigator-initiated research grant through Pfizer, makers of linezolid. This material is the result of work supported with the resources of and use of facilities at the Providence Veterans Affairs Medical Center. The views expressed are those of the authors and do not necessarily reflect the position or policy of the U.S. Department of Veterans Affairs. Dr. LaPlante has received research funding from or has served as an adviser or consultant for Cubist, Astellas, Theravance, Forest (Allergan), Davol, Marvao, Melinta, The Medicines Company, and Pfizer. Dr. Luther has received research funding from Cubist and Pfizer. Dr. Mermel has received research funding from or served as a consultant for Theravance, Astellas, CareFusion, Fresenius Medical, and Marvao Medical. Acknowledgments The technical assistance of Suzanne Woodmansee, Kayla Babcock, and Kathryn Daffinee is acknowledged. We gratefully acknowledge Charles Peloquin, Pharm.D., for his analysis of the linezolid samples. Christine Long, B.S., and Clyde Belgrave, M.D., are acknowledged for their analysis of vancomycin samples. Footnotes The authors have declared no other potential conflicts of interest. Additional information. Part of this article was presented as a poster (P-1117) at the 23rd European Congress of Clinical Microbiology and Infectious Diseases, Berlin, Germany, on April 27, 2013. a Pfizer, New York, NY, lots 08E14Z99, 10I24Z49, and 11C03U04. b Pfizer, lot 00184033. c Hospira, Lake Forest, IL, lots NC052936 and 12070DD. d Hospira, lot 24-129-DK. e Difco Laboratories, Sparks, MD. f Becton-Dickinson, Sparks, MD. g Synergy 2, Bio-Tek Instruments, Winooski, VT. h Nunc-TSP, catalog item 445497. i Becton-Dickinson. j Boekel Shake and Bake, Boekel Scientific, Feasterville, PA. k Arrow-Howes, product item 15703, Reading, PA, lot RF 1118084. l PETIA, Architect Multigent, Abbott Diagnostics, Abbott Park, IL. References 1 Mermel LA Allon M Bouza E . Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 update by the Infectious Diseases Society of America . Clin Infect Dis . 2009 ; 49 : 1 – 45 . [Erratum, Clin Infect Dis. 2010; 50:1079. Dosage error in text.] Google Scholar Crossref Search ADS PubMed WorldCat 2 De Freitas LW Neto MM Nascimento MM . Bacterial colonization in hemodialysis temporary dual lumen catheters: a prospective study . Ren Fail . 2008 ; 30 : 31 – 5 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Mermel LA . What is the evidence for intraluminal colonization of hemodialysis catheters? Kidney Int 2014 ; 86 : 28 – 33 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Wu S Yang T Luo Y . Efficacy of the novel oxazolidinone compound FYL-67 for preventing biofilm formation by Staphylococcus aureus . J Antimicrob Chemother . 2014 ; 69 : 3011 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Reiter KC Villa B Paim TG . Inhibition of biofilm maturation by linezolid in methicillin-resistant Staphylococcus epidermidis clinical isolates: comparison with other drugs . J Med Microbiol . 2013 ; 62 : 394 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 6 Castagnola E Moroni C Gandullia P . Catheter lock and systemic infusion of linezolid for treatment of persistent Broviac catheter-related staphylococcal bacteremia . Antimicrob Agents Chemother . 2006 ; 50 : 1120 – 1 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Curtin J Cormican M Fleming G . 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 . 2003 ; 47 : 3145 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Fennell JP O’Donohoe M Cormican M . Linezolid lock prophylaxis of central venous catheter infection . J Med Microbiol . 2008 ; 57 : 534 – 5 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Fernandez-Hidalgo N Gavalda J Almirante B . Evaluation of linezolid, vancomycin, gentamicin and ciprofloxacin in a rabbit model of antibiotic-lock technique for Staphylococcus aureus catheter-related infection . J Antimicrob Chemother . 2010 ; 65 : 525 – 30 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Giacometti A Cirioni O Ghiselli R . Comparative efficacies of quinupristin-dalfopristin, linezolid, vancomycin, and ciprofloxacin in treatment, using the antibiotic-lock technique, of experimental catheter-related infection due to Staphylococcus aureus . Antimicrob Agents Chemother . 2005 ; 49 : 4042 – 5 . Google Scholar Crossref Search ADS PubMed WorldCat 11 Wu JA Kusuma C Mond JJ . Lysostaphin disrupts Staphylococcus aureus and Staphylococcus epidermidis biofilms on artificial surfaces . Antimicrob Agents Chemother . 2003 ; 47 : 3407 – 14 . Google Scholar Crossref Search ADS PubMed WorldCat 12 Tong Z Zhang Y Ling J . An in vitro study on the effects of nisin on the antibacterial activities of 18 antibiotics against Enterococcus faecalis . PLoS One . 2014 ; 9 : e89209 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Schumacher-Perdreau F Heilmann C Peters G . Comparative analysis of a biofilm-forming Staphylococcus epidermidis strain and its adhesion-positive, accumulation-negative mutant M7 . FEMS Microbiol Lett . 1994 ; 117 : 71 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 14 LaPlante KL Mermel LA . In vitro activities of telavancin and vancomycin against biofilm-producing Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis strains . Antimicrob Agents Chemother . 2009 ; 53 : 3166 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 15 Christensen GD Simpson WA Younger JJ . Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices . J Clin Microbiol . 1985 ; 22 : 996 – 1006 . Google Scholar PubMed WorldCat 16 Stepanovic S Vukovic D Dakic I . A modified microtiter-plate test for quantification of staphylococcal biofilm formation . J Microbiol Methods . 2000 ; 40 : 175 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 17 Performance standards for antimicrobial susceptibility testing; 24th informational supplement. M100-S24 . Wayne, PA : Clinical and Laboratory Standards Institute ; 2014 . WorldCat COPAC 18 Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, ninth edition. M07-A9 . Wayne, PA : Clinical and Laboratory Standards Institute ; 2012 . WorldCat COPAC 19 LaPlante KL Mermel LA . In vitro activity of daptomycin and vancomycin lock solutions on staphylococcal biofilms in a central venous catheter model . Nephrol Dial Transplant . 2007 ; 22 : 2239 – 46 . Google Scholar Crossref Search ADS PubMed WorldCat 20 Ceri H Olson ME Stremick C . The Calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms . J Clin Microbiol . 1999 ; 37 : 1771 – 6 . Google Scholar PubMed WorldCat 21 Shah CB Mittelman MW Costerton JW . Antimicrobial activity of a novel catheter lock solution . Antimicrob Agents Chemother . 2002 ; 46 : 1674 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 22 Luther MK Mermel LA LaPlante KL . Comparison of telavancin and vancomycin antibiotic lock solutions in the eradication of biofilm-producing staphylococci and enterococci from central venous catheters . Am J Health-Syst Pharm . 2016 ; 73 : 315 – 21 . Google Scholar Crossref Search ADS PubMed WorldCat 23 Luther MK Mermel LA LaPlante KL . Comparison of ML8-X10 (a prototype oil-in-water micro-emulsion based on a novel free fatty acid), taurolidine/citrate/heparin and vancomycin/heparin antimicrobial lock solutions in the eradication of biofilm-producing staphylococci from central venous catheters . J Antimicrob Chemother . 2014 ; 69 : 3263 – 7 . Google Scholar Crossref Search ADS PubMed WorldCat 24 Luther MK Arvanitis M Mylonakis E . Activity of daptomycin or linezolid in combination with rifampin or gentamicin against biofilm-forming Enterococcus faecalis or E. faecium in an in vitro pharmacodynamic model using simulated endocardial vegetations and an in vivo survival assay using Galleria mellonella larvae . Antimicrob Agents Chemother . 2014 ; 58 : 4612 – 20 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Krishnasami Z Carlton D Bimbo L . Management of hemodialysis catheter-related bacteremia with an adjunctive antibiotic lock solution . Kidney Int . 2002 ; 61 : 1136 – 42 . Google Scholar Crossref Search ADS PubMed WorldCat 26 Bookstaver PB Rokas KE Norris LB . Stability and compatibility of antimicrobial lock solutions . Am J Health-Syst Pharm . 2013 ; 70 : 2185 – 98 . Google Scholar Crossref Search ADS PubMed WorldCat 27 LaPlante KL Woodmansee S . Activities of daptomycin and vancomycin alone and in combination with rifampin and gentamicin against biofilm-forming methicillin-resistant Staphylococcus aureus isolates in an experimental model of endocarditis . Antimicrob Agents Chemother . 2009 ; 53 : 3880 – 6 . Google Scholar Crossref Search ADS PubMed WorldCat 28 LaPlante KL Rybak MJ . 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 . Antimicrob Agents Chemother . 2004 ; 48 : 4665 – 72 . Google Scholar Crossref Search ADS PubMed WorldCat 29 Ryback MJ . The pharmacokinetic and pharmacodynamic properties of vancomycin . Clin Infect Dis . 2006 ; 42 ( suppl 1 ): S35 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 30 Rayner CR Forrest A Meagher AK . Clinical pharmacodynamics of linezolid in seriously ill patients treated in a compassionate use programme . Clin Pharmacokinet . 2003 ; 42 : 1411 – 23 . Google Scholar Crossref Search ADS PubMed WorldCat 31 Alloush HM Salisbury V Lewis RJ . Pharmacodynamics of linezolid in a clinical isolate of Streptococcus pneumoniae genetically modified to express lux genes . J Antimicrob Chemother . 2003 ; 52 : 511 – 3 . Google Scholar Crossref Search ADS PubMed WorldCat 32 Capdevila JA Segarra A Planes AM . Successful treatment of haemodialysis catheter-related sepsis without catheter removal . Nephrol Dial Transplant . 1993 ; 8 : 231 – 4 . Google Scholar PubMed WorldCat 33 El-Azizi M Rao S Kanchanapoom T . In vitro activity of vancomycin, quinupristin/dalfopristin, and linezolid against intact and disrupted biofilms of staphylococci . Ann Clin Microbiol Antimicrob . 2005 ; 4 : 2 . Google Scholar Crossref Search ADS PubMed WorldCat 34 Gander S Hayward K Finch R . An investigation of the antimicrobial effects of linezolid on bacterial biofilms utilizing an in vitro pharmacokinetic model . J Antimicrob Chemother . 2002 ; 49 : 301 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 35 Raad I Hanna H Jiang Y . Comparative activities of daptomycin, linezolid, and tigecycline against catheter-related methicillin-resistant Staphylococcus bacteremic isolates embedded in biofilm . Antimicrob Agents Chemother . 2007 ; 51 : 1656 – 60 . Google Scholar Crossref Search ADS PubMed WorldCat 36 Wiederhold NP Coyle EA Raad II . Antibacterial activity of linezolid and vancomycin in an in vitro pharmacodynamic model of gram-positive catheter-related bacteraemia . J Antimicrob Chemother . 2005 ; 55 : 792 – 5 . Google Scholar Crossref Search ADS PubMed WorldCat 37 Wilcox MH Kite P Mills K . In situ measurement of linezolid and vancomycin concentrations in intravascular catheter-associated biofilm . J Antimicrob Chemother . 2001 ; 47 : 171 – 5 . Google Scholar Crossref Search ADS PubMed WorldCat 38 Reiter KC Sant’Anna FH d’Azevedo PA . Upregulation of icaA, atlE and aap genes by linezolid but not vancomycin in Staphylococcus epidermidis RP62A biofilms . Int J Antimicrob Agents . 2014 ; 43 : 248 – 53 . Google Scholar Crossref Search ADS PubMed WorldCat 39 Smith K Perez A Ramage G . Comparison of biofilm-associated cell survival following in vitro exposure of methicillin-resistant Staphylococcus aureus biofilms to the antibiotics clindamycin, daptomycin, linezolid, tigecycline and vancomycin . Int J Antimicrob Agents . 2009 ; 33 : 374 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 40 Shanks RM Donegan NP Graber ML . Heparin stimulates Staphylococcus aureus biofilm formation . Infect Immun . 2005 ; 73 : 4596 – 606 . Google Scholar Crossref Search ADS PubMed WorldCat 41 Shenep LE Shenep MA Cheatham W . Efficacy of intravascular catheter lock solutions containing preservatives in the prevention of microbial colonization . J Hosp Infect . 2011 ; 79 : 317 – 22 . Google Scholar Crossref Search ADS PubMed WorldCat 42 Milisavljevic V Tran LP Batmalle C . Benzyl alcohol and ethanol can enhance the pathogenic potential of clinical Staphylococcus epidermidis strains . Am J Infect Control . 2008 ; 36 : 552 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 43 Redelman CV Maduakolam C Anderson GG . Alcohol treatment enhances Staphylococcus aureus biofilm development . FEMS Immunol Med Microbiol . 2012 ; 66 : 411 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 44 Luther MK Bilida S Mermel LA . Ethanol and isopropyl alcohol exposure increases biofilm formation in Staphylococcus aureus and Staphylococcus epidermidis . Infect Dis Ther . 2015 ; 4 : 219 – 26 . Google Scholar Crossref Search ADS PubMed WorldCat Copyright © 2017 by the American Society of Health-System Pharmacists, Inc. All rights reserved.
TI - Comparison of linezolid and vancomycin lock solutions with and without heparin against biofilm-producing bacteria
JF - American Journal of Health-System Pharmacy
DO - 10.2146/ajhp150804
DA - 2017-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/comparison-of-linezolid-and-vancomycin-lock-solutions-with-and-without-juemH0b4pY
SP - e193
VL - 74
IS - 9
DP - DeepDyve
ER -