ABSTRACT
Background An emerging pathogen is Enterococcus faecium resistant to both linezolid and vancomycin (LRVRE). Antimicrobial combinations may be required for therapy and need to be evaluated. The combination of daptomycin and rifampin has demonstrated good in vitro activity against gram-positive bacteria, including E faecium. Telavancin, a newer lipoglycopeptide, has shown in vitro activity against E faecium. We evaluated the combination of telavancin and rifampin and compared the results to the combination of daptomycin and rifampin used previously on the same isolates.
Methods Twenty-four genetically unique (by pulsed-field gel electrophoresis), clinical LRVRE isolates were collected in the United States from 2001-2004. Etest minimal inhibitory concentrations (MICs) (μg/mL) were 0.064-8 for telavancin, 1-4 for daptomycin, and 0.012 to >32 for rifampin. In vitro synergy testing was performed in triplicate by an Etest MIC:MIC ratio method, and summation fractional inhibitory concentration (ΣFIC) was calculated: synergy ≤0.5; indifference >0.5-4; and antagonism >4.
Results The Etest method showed synergy (ΣFICs of 0.1-0.5) with telavancin + rifampin in 20/24 (83%) isolates and indifference (ΣFICs of 0.6-0.8) in 4/24 (17%) isolates. Similarly, the daptomycin + rifampin combination showed synergy (ΣFICs of 0.1-0.5) in 21/24 (88%) isolates and indifference (ΣFICs of 0.6-1.0) in 3/24 (12%) isolates by the Etest method. No antagonism was found.
Conclusions In vitro synergy with both combinations (rifampin + telavancin or daptomycin) was 83% and 88%, respectively, by Etest against these LRVRE isolates. Although both daptomycin and telavancin in combination with rifampin showed a high incidence of synergistic activity, further in vitro synergy testing with this combination should be performed against additional E faecium isolates. In vitro synergy may or may not translate into in vivo effectiveness.
INTRODUCTION
Vancomycin is one of the most widely used antibiotics in the United States for the treatment of serious gram-positive infections. Since the frequency of vancomycin-resistant organisms has increased, the use of vancomycin is being replaced by other antibiotics, such as linezolid, daptomycin, quinupristin/dalfopristin, and tigecycline.1 One emerging pathogen is Enterococcus faecium resistant to both linezolid and vancomycin (LRVRE). Telavancin, a newer lipoglycopeptide, was recently approved for the treatment of complicated skin and skin structure infections caused by Staphylococcus aureus, streptococci, and vancomycin-susceptible Enterococcus faecalis.2 In a study by Mendes et al,3 telavancin showed good activity (minimal inhibitory concentrations [MICs] ≤1 μg/mL) against 27 (100%) strains of VanB phenotype (variable levels of inducible resistance to vancomycin only) E faecium but was less active (10.5% with MICs ≤1 μg/mL) against 392 VanA-type strains (inducible high-level resistance to vancomycin as well as to teicoplanin). Antimicrobial combinations may be required for therapy against these organisms and need to be evaluated. The combination of daptomycin and rifampin showed in vitro synergy by an agar diffusion Etest method (68%) against vancomycin-resistant E faecium (VRE),4 by a checkerboard method (57%) also against VRE,5 and by an Etest MIC:MIC method (88%) against linezolid-resistant VRE.6 In another in vitro study, Lin et al7 reported 65% synergy with the combination of telavancin and rifampin against methicillin-resistant S aureus.
METHODS
In our current investigation, we examined synergistic activity with telavancin when combined with rifampin against LRVRE and compared results to the combination of daptomycin + rifampin tested previously on the same isolates.
Microorganisms and Media
Twenty-four genetically unique clinical E faecium isolates resistant to both linezolid (MICs 8 to >256 μg/mL) and vancomycin (MICs >256 μg/mL) were collected throughout the United States from 2001-2004 and identified using the Vitek system (bioMérieux, Inc., Durham, NC). E faecalis ATCC 29212 was included as a quality control strain. Fingerprinting of isolates was performed by pulsed-field gel electrophoresis. Testing for VanA and VanB phenotypes was not performed. Media (Becton-Dickinson Microbiology Systems, Sparks, MD) included Mueller-Hinton II Broth and Mueller-Hinton II Agar (MHA) plates (for Etest MICs and synergy tests) and trypticase soy agar with 5% sheep blood plates (for subcultures of isolates).
MIC Determination
MICs were performed by Etest (bioMérieux, Inc., Durham, NC) in triplicate following manufacturer's guidelines, and the mean value was reported (Figure 1). Telavancin MICs ranged from 0.064-8 μg/mL; daptomycin MICs were 1-4 μg/mL; and rifampin MICs were 0.012 to >32 μg/mL (Table). The 2012 Clinical and Laboratory Standards Institute (CLSI) interpretive standards for enterococci are rifampin ≤1 susceptible, 2 intermediate, ≥4 resistant; daptomycin ≤4 susceptible.8 The Food and Drug Administration breakpoint for telavancin for E faecalis is ≤1 susceptible. No breakpoints are available for telavancin and E faecium.
Isolate #12 Etest minimal inhibitory concentrations (MICs): telavancin (TLV) MIC = 6 μg/mL (left) and rifampin (RI) MIC >32 μg/mL (right).
Telavancin (TLV), Daptomycin (DAP), and Rifampin (RI) Etest Minimal Inhibitory Concentrations (MICs) (μg/mL) and MIC:MIC Synergy Testing
Synergy Testing
In vitro synergy testing was performed in triplicate by an Etest MIC:MIC ratio method.6 All MHA plates were inoculated with a suspension of organism equivalent to a 0.5 McFarland standard. Telavancin and rifampin Etest strips were applied to different sections of an MHA plate. The agar was marked adjacent to the previously determined MIC value on each strip. The strips were removed after 1 hour of incubation at room temperature. A new telavancin strip was placed on the area of each previously removed rifampin strip so the telavancin MIC corresponded with the mark of the rifampin MIC. Rifampin strips were applied in reciprocate fashion to the area of the previous telavancin strip. The same procedure was used for the daptomycin + rifampin combination. The resulting combination ellipses were read after 24 hours of incubation at 35°C (Figure 2).
Isolate #12 Etest synergy test: telavancin (TLV) minimal inhibitory concentration (MIC) (after combination with rifampin) = 0.50 μg/mL (left). Rifampin (RI) MIC (after combination with telavancin) = 2 μg/mL (right). Summation fractional inhibitory concentration = 0.1 (synergy).
To evaluate the effect of the combination in the Etest method, the fractional inhibitory concentration (FIC) was calculated for each antibiotic in each combination:
FIC of telavancin = MIC of telavancin in combination ÷ MIC of telavancin alone, and
FIC of rifampin = MIC of rifampin in combination ÷ MIC of rifampin alone.
The total (summation) fractional inhibitory concentration (ΣFIC) for each isolate was calculated according to the formula ΣFIC = FIC telavancin + FIC rifampin.
To calculate ΣFICs, high off-scale MICs (>32 μg/mL) were converted to the next 2-fold dilution (64 μg/mL), and final FIC values were rounded up to the nearest tenth (ie, 0.06 rounded to 0.1). The mean ΣFIC was used to interpret results of the Etest synergy method. Synergy was defined as ΣFIC ≤0.5, indifference as ΣFIC >0.5-4, and antagonism as ΣFIC >4.9
RESULTS
The Etest synergy method demonstrated synergy (ΣFICs of 0.1-0.5) with telavancin + rifampin against 20/24 (83%) of these linezolid-resistant VRE isolates and indifference (ΣFICs of 0.6-0.8) in 4/24 (17%) (Table). Similarly, the daptomycin + rifampin combination showed synergy (ΣFICs of 0.1-0.5) in 21/24 (88%) isolates and indifference (ΣFICs of 0.6-1.0) in 3/24 (12%) isolates by the Etest method (Table). Three isolates were indifferent (ΣFICs of 0.6, 0.7, 0.7) with the telavancin + rifampin combination but synergistic (ΣFICs of 0.4, 0.4, 0.2) with daptomycin + rifampin. Two isolates were synergistic (ΣFICs of 0.2, 0.4) with telavancin + rifampin but indifferent (ΣFICs of 0.9, 1.0) with daptomycin + rifampin (Table). Testing with both combinations revealed synergy in 23/24 (96%) of isolates. The one isolate interpreted as indifferent by both combinations (rifampin + telavancin or daptomycin with ΣFICs of 0.8 and 0.6) suggests a possible additive (ΣFICs of >0.5-1) effect (Table).9 No antagonism with either combination was found.
DISCUSSION
Telavancin inhibits bacterial cell wall synthesis by interfering with the polymerization and cross-linking of peptidoglycan as well as increasing the permeability of the cell membrane. Telavancin might enhance the entry of rifampin, which specifically inhibits bacterial RNA polymerase.10,11 Daptomycin also disrupts the bacterial cell membrane, which may also enhance the entry of rifampin.12
CONCLUSIONS
In vitro synergy was demonstrated with rifampin + telavancin or daptomycin against 83% and 88%, respectively, of the linezolid- and vancomycin-resistant E faecium isolates. Further Etest synergy testing, as well as time-kill assay with the combination of telavancin and rifampin should be performed against additional E faecium isolates. In vitro synergy may or may not translate into in vivo effectiveness.
This article meets the Accreditation Council for Graduate Medical Education and the American Board of Medical Specialties Maintenance of Certification competencies for Patient Care and Medical Knowledge.
ACKNOWLEDGMENTS
The authors thank Royanne Vortisch for her laboratory assistance; Pat Pankey for laboratory management; and Richard Goering, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE, for performing the pulsed-field gel electrophoresis on the E faecium isolates.
Footnotes
The authors have no financial or proprietary interest in the subject matter of this article.
The results included in this study were presented at the Ninth Annual Ochsner Research Night, New Orleans, LA, abstr. 2, May 8, 2012, and at the 112th General Meeting American Society for Microbiology, San Francisco, CA, abstr. C123, June 17, 2012.
- © Academic Division of Ochsner Clinic Foundation
