- Email: [email protected]
Assessment of the in vitro synergy of daptomycin plus linezolid against multidrug-resistant enterococci
Journal of Global Antimicrobial Resistance 2 (2014) 306–308
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Assessment of the in vitro synergy of daptomycin plus linezolid against multidrug-resistant enterococci Jose´ M. Entenza, Marlyse Giddey, Jacques Vouillamoz, Philippe Moreillon, Stefano Mancini * Department of Fundamental Microbiology, Biophore Building, University of Lausanne, CH-1015 Lausanne, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 August 2014 Accepted 19 September 2014
The widespread incidence of enterococci resistant to ampicillin, vancomycin and aminoglycosides, the first-line anti-enterococcal antibiotics, has made the treatment of severe enterococcal infections difficult and alternatives should be explored. We investigated the activity of daptomycin combined with linezolid against three Enterococcus faecalis and four Enterococcus faecium strains resistant to standard drugs used for therapy. Minimum inhibitory concentrations (MICs) were determined by the broth dilution method. Drug interactions were assessed by the checkerboard and time-kill methods. Synergy was defined by a fractional inhibitory concentration index (FICI) of 0.5 or a 2 log10 CFU/mL killing at 24 h with the combination in comparison with killing by the most active single agent. Indifference was defined by a FICI > 0.5–4.0 or a 1–2 log10 CFU/mL killing compared with the most active single agent. MICs of daptomycin were 2–4 mg/mL for E. faecalis and 2–8 mg/mL for E. faecium. MICs of linezolid were 1–2 mg/ mL for all bacteria. In the checkerboard assay, five isolates showed synergism (FICI < 0.5) and two showed indifference (FICIs of 0.53 and 2). Killing studies revealed synergy of daptomycin plus linezolid against four isolates (2.2–3.7 log10 CFU/mL kill) and indifference (1.1–1.6 log10 CFU/mL kill) for the other three strains. Antagonism was not observed. In conclusion, the combination of daptomycin and linezolid had a synergistic or indifferent effect against multidrug-resistant enterococci. Additional studies are needed to explore the potential of this combination for severe enterococcal infections when first-line antibiotic combinations cannot be used. ß 2014 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: Daptomycin Linezolid Synergy Enterococcus faecalis Enterococcus faecium
1. Introduction The standard of care for treatment of severe enterococcal infections, e.g. endocarditis and meningitis, where synergistic activity is necessary, includes a combination of a b-lactam (e.g. ampicillin) or a glycopeptide (e.g. vancomycin) and an aminoglycoside [1,2]. However, the emergence of enterococcal strains resistant to ampicillin and vancomycin and exhibiting high-level aminoglycoside resistance (HLAR) renders such treatment combinations useless in some patients because the synergistic effect is abolished [3]. This highlights the need to find other combinations of antibiotics for effective enterococcal killing. Treatment of deep-seated enterococcal infections with daptomycin in combination with another active agent, in order to achieve synergy and bactericidal activity, is a suggested alternative
* Corresponding author. Tel.: +41 21 692 5612; fax: +41 21 692 5605. E-mail address: [email protected] (S. Mancini).
[1,4]. A good candidate with anti-enterococcal activity to be potentially combined with daptomycin is linezolid. Although daptomycin-non-susceptibility and linezolid resistance among clinical isolates of enterococci have been sporadically reported [5,6], both agents remain generally active in vitro against enterococci, with susceptibility rates >90% [7]. The objective of this study was to evaluate the in vitro antimicrobial activity of daptomycin in combination with linezolid toward a number of Enterococcus faecalis and Enterococcus faecium isolates resistant to ampicillin and vancomycin or/and with HLAR, where combination therapy with these drugs is not possible. 2. Materials and methods 2.1. Micro-organisms and anti-microbial agents A total of seven enterococcal clinical isolates (three E. faecalis and four E. faecium) were tested. Five strains (one E. faecalis and four E. faecium) showed resistance to ampicillin, two (one E. faecalis
http://dx.doi.org/10.1016/j.jgar.2014.09.004 2213-7165/ß 2014 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
J.M. Entenza et al. / Journal of Global Antimicrobial Resistance 2 (2014) 306–308
and one E. faecium) were resistant to vancomycin, and six (three E. faecalis and three E. faecium) showed HLAR. Unless otherwise stated, enterococci were routinely streaked on brain–heart infusion (BHI) agar plates (Difco Laboratories, Detroit, MI) and were cultured aerobically at 37 8C for 24 h. A single bacterial colony was then inoculated into 10 mL of BHI broth and grown statically to exponential phase to an optical density at 600 nm (OD600) of 0.5. Bacterial stocks were kept at 70 8C in BHI broth supplemented with 10% (v/v) glycerol. Daptomycin, linezolid, gentamicin and vancomycin were purchased as commercially available human-injectable products. Ampicillin was from Sigma (Buchs, Switzerland) and gentamicin was from Essex Chemie AG (Luzern, Switzerland).
307
Table 1 Minimum inhibitory concentrations (MICs) of daptomycin (DAP), linezolid (LZD), ampicillin (AMP), vancomycin (VAN) and gentamicin (GEN) for the tested enterococci. Strain
MIC (mg/mL) DAP
LZD
Enterococcus faecalis 4 HH22 V583 2 1110 2
2 2 2
Enterococcus faecium BR 4 UB 2 D366 4 1693 8
1 2 2 2
AMP 32 0.25 1
>128 >128 16 >128
VAN
GEN
2 64 2
>2000 >2000 >2000
2 2 32 2
>2000 >2000 8 >2000
2.2. Susceptibility studies, antibiotic interactions and synergy testing Minimum inhibitory concentrations (MICs) were determined in BHI broth according to Clinical and Laboratory Standards Institute (CLSI) guidelines [8]. To test susceptibility to daptomycin, the growth medium was supplemented with Ca2+ to a final concentration of 50 mg/mL. The MIC was defined as the lowest concentration of drug that inhibited bacterial growth following incubation for 18 h at 37 8C. Enterococcus faecalis ATCC 29212 was used as a quality control strain. Antibiotic interactions were assessed by the checkerboard method in 96-well microtitre plates (Nalge Nunc International, Roskilde, Denmark). Wells were inoculated with 105 CFU/mL and the plates were incubated for 24 h at 37 8C. Fractional inhibitory concentrations (FICs) were calculated as the MIC of drug A or B in the combination/MIC of drug A or B alone, and the FIC index (FICI) was obtained by summing up the FIC values. FICIs of 0.5 were interpreted as synergistic, those of >0.5–4.0 were considered indifferent and those of >4.0 were considered as antagonistic [9]. Drug-induced killing and synergy between daptomycin and linezolid was further assessed by time-kill assays using previously described methods [10]. Each isolate was exposed to daptomycin and linezolid alone or combined at concentrations equal to the MIC, which are drug levels usually achievable in human serum during standard therapy. Tubes were incubated at 37 8C for 24 h. Cultures were sampled at 0, 6 and 24 h, serially diluted and plated onto BHI agar plates. After 24 h of incubation at 37 8C, the number of viable colonies was enumerated. Results represent the mean of three independent experiments performed on different days. Synergy was defined as a 2 log10 decrease in colony counts at 24 h by the combination compared with the most active single agent, with the number of surviving organisms in the combination 2 log10 CFU/mL below the starting inoculum. Indifference was defined as <2 but >1 log10 CFU/mL kill in comparison with the most active single agent. Antagonism was defined as 1 log10 CFU/ mL increase in bacterial growth in comparison with the less active single agent. Bactericidal activity was defined as a 3 log10 decrease in colony count from the initial inoculum. 3. Results 3.1. Minimum inhibitory concentrations MICs for the test isolates are shown in Table 1. Daptomycin MICs for E. faecalis ranged from 2 mg/mL to 4 mg/mL, irrespective of their resistance patterns to other drugs. Therefore, daptomycin MICs for E. faecalis fell in the susceptible range for all of these organisms according to the CLSI interpretation standards for enterococci (MIC cut-off of 4 mg/mL) [5]. Daptomycin MICs for E. faecium ranged from 2 mg/mL to 8 mg/mL. One isolate (E. faecium 1693) was non-susceptible to daptomycin (MIC = 8 mg/mL). All enterococcal isolates were susceptible to
linezolid (MIC 2 mg/mL). For each test performed, the MIC of the quality control organism was within the acceptable range. 3.2. Fractional inhibitory concentration indices and time-kill assays In the checkerboard assays, synergism (FICI < 0.5) between daptomycin and linezolid occurred against all tested E. faecalis strains and against E. faecium BR and D366. Indifference occurred against E. faecium UB (FICI = 0.53) and 1693 (FICI = 2) isolates. No antagonism was detected. The rates of killing of daptomycin and linezolid, alone or in combination, against the tested enterococcal strains are displayed in Fig. 1. Daptomycin alone, at concentrations of 1 MIC, exhibited bacteriostatic activity within the first 6 h, followed by growth resumption. Linezolid alone, at concentrations of 1 MIC, was also bacteriostatic. Growth resumption at 24 h was only observed in one isolate (E. faecalis HH22) during exposure to either drug, possibly due to a decrease in drug concentration over time, as resistant derivatives were not obtained. Indeed, no changes in MICs were observed after 24 h of exposure to either drug when compared with the original inoculum (data not shown). The combination of daptomycin and linezolid had a synergistic effect (2.2–3.7 log10 CFU/mL kill compared with linezolid alone, the most active agent, and 2.1–2.77 log10 CFU/mL kill of the starting inoculum) against E. faecalis HH22 and V583 and E. faecium BR and D366. Indifference (1.1–1.6 log10 CFU/mL kill) was observed against strains E. faecalis 1110 and E. faecium UB and 1693. Antagonism was not detected. Bactericidal activity, defined as a 3 log10 CFU/mL reduction in colony count from the starting inoculum, was apparent only for strain E. faecium D366. 4. Discussion In the present in vitro study, a synergistic or indifferent activity of the combination of daptomycin and linezolid was observed against multidrug-resistant E. faecalis and E. faecium strains both using checkerboard and time-kill assays. Synergy studies with the combination of daptomycin and linezolid are scarce. A previous study by Parra-Ruiz et al. [11] demonstrated that the combination of daptomycin with linezolid was synergistic and bactericidal for meticillin-resistant Staphylococcus aureus (MRSA) in an in vitro biofilm model. Moreover, Steed et al. [12] reported a synergistic activity of this combination in an in vitro model of endocardial vegetations. In the case of enterococci, currently there is only one case report describing successful treatment with this combination for E. faecium meningitis [13]. The synergistic activity of daptomycin and linezolid observed in vitro against enterococci in the present
308
J.M. Entenza et al. / Journal of Global Antimicrobial Resistance 2 (2014) 306–308
E. faecium BR 10
8
8
Log cfu/mL
Log cfu/mL
E. faecalis HH22 10
6 4 2 0
0
6
12
18
This work was supported in part by research grants from the Swiss National Science Foundation [grants 310030-125325 and 310030-143799/1].
6 4 2 0
24
Competing interests 0
6
Time (hours)
8
8
Log cfu/mL
Log cfu/mL
10
6 4 2 6
12
18
Not required. References
2 0
6
Log cfu/mL
Log cfu/mL
4 2 12
12
18
24
18
E. faecium D366
10
6
6
Ethical approval
Time (hours)
E. faecalis 1110
0
None declared.
4
0
24
8
0
24
6
Time (hours) 10
18
E. faecium UB
E. faecalis V583
0
12
Time (hours)
10
0
Funding
8 6 4 2 0
24
0
6
Time (hours)
12
18
24
Time (hours)
E. faecium 1693
Control DAP LZD DAP+LZD
Log cfu/mL
10 8 6 4 2 0
0
6
12
18
24
Time (hours)
Fig. 1. Time-kill assays evaluating the activity of daptomycin (DAP) and linezolid (LZD), alone or in combination, against Enterococcus faecalis (left panel) and Enterococcus faecium (right panel). All anti-microbial agents are at the minimum inhibitory concentration. Data are the mean S.D. of three experiments.
study is consistent with the findings of this in vivo report. Despite the fact that the combination of daptomycin and linezolid provided synergy, the mechanism responsible for this synergy is unknown. Based on its ability to penetrate and depolarise the cell membrane, daptomycin might possibly increase the access of linezolid to its target on the ribosome. When the combination of a b-lactam or vancomycin and an aminoglycoside is not possible, treatment of severe enterococcal infections (particularly endocarditis and meningitis) depends on the susceptibility phenotype of the strain. Synergy between daptomycin and other antibiotics, including rifampicin, quinupristin/dalfopristin, doxycycline, fosfomycin and tigecycline, has been reported both in vitro and in vivo for enterococci [14– 19]. Combining daptomycin with linezolid might constitute another interesting therapeutic regimen for the treatment of severe multidrug-resistant enterococcal infections when first-line combinations cannot be used. Additional studies in animal models are needed to further test the efficacy of this combination. Besides the in vivo studies, the effect of the daptomycin plus linezolid combination on the emergence of resistance to either drug deserves further investigation.
[1] Arias CA, Contreras GA, Murray BE. Management of multidrug-resistant enterococcal infections. Clin Microbiol Infect 2010;16:555–62. [2] Pintado V, Cabellos C, Moreno S, Meseguer MA, Ayats J, Viladrich PF. Enterococcal meningitis: a clinical study of 39 cases and review of the literature. Medicine (Baltimore) 2003;82:346–64. [3] Hollenbeck BL, Rice LB. Intrinsic and acquired resistance mechanisms in enterococcus. Virulence 2012;3:421–33. ˜ oz P, Marin M, Segado A, Roda J, Valerio M, et al. Group for the [4] Cero´n I, Mun ˜ o´n Hospital management of infective endocarditis of the Gregorio Maran (GAME) efficacy of daptomycin in the treatment of enterococcal endocarditis: a 5 year comparison with conventional therapy. J Antimicrob Chemother 2014;69:1669–74. [5] Kelesidis T, Humphries R, Uslan DZ, Pegues DA. Daptomycin nonsusceptible enterococci: an emerging challenge for clinicians. Clin Infect Dis 2011;52: 228–34. [6] Ntokou E, Stathopoulos C, Kristo I, Dimitroulia E, Labrou M, Vasdeki A, et al. Intensive care unit dissemination of multiple clones of linezolid-resistant Enterococcus faecalis and Enterococcus faecium. J Antimicrob Chemother 2012;67:1819–23. [7] Jones RN, Flonta M, Gurler N, Cepparulo M, Mendes RE, Castanheira M. Resistance surveillance program report for selected European nations (2011). Diagn Microbiol Infect Dis 2014;78:429–36. [8] Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Second Informational Supplement, Document M100-S22. Wayne, PA: CLSI; 2012. [9] Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 2003;52:1. [10] Entenza JM, Veloso TR, Vouillamoz J, Giddey M, Majcherczyk P, Moreillon P. In vivo synergism of ceftobiprole and vancomycin against experimental endocarditis due to vancomycin-intermediate Staphylococcus aureus. Antimicrob Agents Chemother 2011;55:3977–84. ˜ a-Monje A, Herna´ndez-Quero J. Activity of [11] Parra-Ruiz J, Bravo-Molina A, Pen linezolid and high-dose daptomycin, alone or in combination, in an in vitro model of Staphylococcus aureus biofilm. J Antimicrob Chemother 2012;67: 2682–5. [12] Steed ME, Vidaillac C, Rybak MJ. Novel daptomycin combinations against daptomycin-nonsusceptible methicillin-resistant Staphylococcus aureus in an in vitro model of simulated endocardial vegetations. Antimicrob Agents Chemother 2010;54:5187–92. [13] Le J, Bookstaver PB, Rudisill CN, Hashem MG, Iqbal R, James CL, et al. Treatment of meningitis caused by vancomycin-resistant Enterococcus faecium: highdose and combination daptomycin therapy. Ann Pharmacother 2010;44: 2001–6. [14] Rand KH, Houck H. Daptomycin synergy with rifampicin and ampicillin against vancomycin-resistant enterococci. J Antimicrob Chemother 2004;53:530–2. [15] Pankey G, Ashcraft D, Patel N. In vitro synergy of daptomycin plus rifampin against Enterococcus faecium resistant to both linezolid and vancomycin. Antimicrob Agents Chemother 2005;49:5166–8. [16] Schutt AC, Bohm NM. Multidrug-resistant Enterococcus faecium endocarditis treated with combination tigecycline and high-dose daptomycin. Ann Pharmacother 2009;43:2108–12. [17] Knoll BM, Hellmann M, Kotton CN. Vancomycin-resistant Enterococcus faecium meningitis in adults: case series and review of the literature. Scand J Infect Dis 2013;45:131–9. [18] Descourouez JL, Jorgenson MR, Wergin JE, Rose WE. Fosfomycin synergy in vitro with amoxicillin, daptomycin, and linezolid against vancomycin-resistant Enterococcus faecium from renal transplant patients with infected urinary stents. Antimicrob Agents Chemother 2013;57:1518–20. [19] Rice LB, Eliopoulos CT, Yao JD, Eliopoulos GM, Moellering Jr RC. In vivo activity of the combination of daptomycin and fosfomycin compared with daptomycin alone against a strain of Enterococcus faecalis with high-level gentamicin resistance in the rat endocarditis model. Diagn Microbiol Infect Dis 1992;15:173–6.
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Assessment of the in vitro synergy of daptomycin plus linezolid against multidrug-resistant enterococci Jose´ M. Entenza, Marlyse Giddey, Jacques Vouillamoz, Philippe Moreillon, Stefano Mancini * Department of Fundamental Microbiology, Biophore Building, University of Lausanne, CH-1015 Lausanne, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 August 2014 Accepted 19 September 2014
The widespread incidence of enterococci resistant to ampicillin, vancomycin and aminoglycosides, the first-line anti-enterococcal antibiotics, has made the treatment of severe enterococcal infections difficult and alternatives should be explored. We investigated the activity of daptomycin combined with linezolid against three Enterococcus faecalis and four Enterococcus faecium strains resistant to standard drugs used for therapy. Minimum inhibitory concentrations (MICs) were determined by the broth dilution method. Drug interactions were assessed by the checkerboard and time-kill methods. Synergy was defined by a fractional inhibitory concentration index (FICI) of 0.5 or a 2 log10 CFU/mL killing at 24 h with the combination in comparison with killing by the most active single agent. Indifference was defined by a FICI > 0.5–4.0 or a 1–2 log10 CFU/mL killing compared with the most active single agent. MICs of daptomycin were 2–4 mg/mL for E. faecalis and 2–8 mg/mL for E. faecium. MICs of linezolid were 1–2 mg/ mL for all bacteria. In the checkerboard assay, five isolates showed synergism (FICI < 0.5) and two showed indifference (FICIs of 0.53 and 2). Killing studies revealed synergy of daptomycin plus linezolid against four isolates (2.2–3.7 log10 CFU/mL kill) and indifference (1.1–1.6 log10 CFU/mL kill) for the other three strains. Antagonism was not observed. In conclusion, the combination of daptomycin and linezolid had a synergistic or indifferent effect against multidrug-resistant enterococci. Additional studies are needed to explore the potential of this combination for severe enterococcal infections when first-line antibiotic combinations cannot be used. ß 2014 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: Daptomycin Linezolid Synergy Enterococcus faecalis Enterococcus faecium
1. Introduction The standard of care for treatment of severe enterococcal infections, e.g. endocarditis and meningitis, where synergistic activity is necessary, includes a combination of a b-lactam (e.g. ampicillin) or a glycopeptide (e.g. vancomycin) and an aminoglycoside [1,2]. However, the emergence of enterococcal strains resistant to ampicillin and vancomycin and exhibiting high-level aminoglycoside resistance (HLAR) renders such treatment combinations useless in some patients because the synergistic effect is abolished [3]. This highlights the need to find other combinations of antibiotics for effective enterococcal killing. Treatment of deep-seated enterococcal infections with daptomycin in combination with another active agent, in order to achieve synergy and bactericidal activity, is a suggested alternative
* Corresponding author. Tel.: +41 21 692 5612; fax: +41 21 692 5605. E-mail address: [email protected] (S. Mancini).
[1,4]. A good candidate with anti-enterococcal activity to be potentially combined with daptomycin is linezolid. Although daptomycin-non-susceptibility and linezolid resistance among clinical isolates of enterococci have been sporadically reported [5,6], both agents remain generally active in vitro against enterococci, with susceptibility rates >90% [7]. The objective of this study was to evaluate the in vitro antimicrobial activity of daptomycin in combination with linezolid toward a number of Enterococcus faecalis and Enterococcus faecium isolates resistant to ampicillin and vancomycin or/and with HLAR, where combination therapy with these drugs is not possible. 2. Materials and methods 2.1. Micro-organisms and anti-microbial agents A total of seven enterococcal clinical isolates (three E. faecalis and four E. faecium) were tested. Five strains (one E. faecalis and four E. faecium) showed resistance to ampicillin, two (one E. faecalis
http://dx.doi.org/10.1016/j.jgar.2014.09.004 2213-7165/ß 2014 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
J.M. Entenza et al. / Journal of Global Antimicrobial Resistance 2 (2014) 306–308
and one E. faecium) were resistant to vancomycin, and six (three E. faecalis and three E. faecium) showed HLAR. Unless otherwise stated, enterococci were routinely streaked on brain–heart infusion (BHI) agar plates (Difco Laboratories, Detroit, MI) and were cultured aerobically at 37 8C for 24 h. A single bacterial colony was then inoculated into 10 mL of BHI broth and grown statically to exponential phase to an optical density at 600 nm (OD600) of 0.5. Bacterial stocks were kept at 70 8C in BHI broth supplemented with 10% (v/v) glycerol. Daptomycin, linezolid, gentamicin and vancomycin were purchased as commercially available human-injectable products. Ampicillin was from Sigma (Buchs, Switzerland) and gentamicin was from Essex Chemie AG (Luzern, Switzerland).
307
Table 1 Minimum inhibitory concentrations (MICs) of daptomycin (DAP), linezolid (LZD), ampicillin (AMP), vancomycin (VAN) and gentamicin (GEN) for the tested enterococci. Strain
MIC (mg/mL) DAP
LZD
Enterococcus faecalis 4 HH22 V583 2 1110 2
2 2 2
Enterococcus faecium BR 4 UB 2 D366 4 1693 8
1 2 2 2
AMP 32 0.25 1
>128 >128 16 >128
VAN
GEN
2 64 2
>2000 >2000 >2000
2 2 32 2
>2000 >2000 8 >2000
2.2. Susceptibility studies, antibiotic interactions and synergy testing Minimum inhibitory concentrations (MICs) were determined in BHI broth according to Clinical and Laboratory Standards Institute (CLSI) guidelines [8]. To test susceptibility to daptomycin, the growth medium was supplemented with Ca2+ to a final concentration of 50 mg/mL. The MIC was defined as the lowest concentration of drug that inhibited bacterial growth following incubation for 18 h at 37 8C. Enterococcus faecalis ATCC 29212 was used as a quality control strain. Antibiotic interactions were assessed by the checkerboard method in 96-well microtitre plates (Nalge Nunc International, Roskilde, Denmark). Wells were inoculated with 105 CFU/mL and the plates were incubated for 24 h at 37 8C. Fractional inhibitory concentrations (FICs) were calculated as the MIC of drug A or B in the combination/MIC of drug A or B alone, and the FIC index (FICI) was obtained by summing up the FIC values. FICIs of 0.5 were interpreted as synergistic, those of >0.5–4.0 were considered indifferent and those of >4.0 were considered as antagonistic [9]. Drug-induced killing and synergy between daptomycin and linezolid was further assessed by time-kill assays using previously described methods [10]. Each isolate was exposed to daptomycin and linezolid alone or combined at concentrations equal to the MIC, which are drug levels usually achievable in human serum during standard therapy. Tubes were incubated at 37 8C for 24 h. Cultures were sampled at 0, 6 and 24 h, serially diluted and plated onto BHI agar plates. After 24 h of incubation at 37 8C, the number of viable colonies was enumerated. Results represent the mean of three independent experiments performed on different days. Synergy was defined as a 2 log10 decrease in colony counts at 24 h by the combination compared with the most active single agent, with the number of surviving organisms in the combination 2 log10 CFU/mL below the starting inoculum. Indifference was defined as <2 but >1 log10 CFU/mL kill in comparison with the most active single agent. Antagonism was defined as 1 log10 CFU/ mL increase in bacterial growth in comparison with the less active single agent. Bactericidal activity was defined as a 3 log10 decrease in colony count from the initial inoculum. 3. Results 3.1. Minimum inhibitory concentrations MICs for the test isolates are shown in Table 1. Daptomycin MICs for E. faecalis ranged from 2 mg/mL to 4 mg/mL, irrespective of their resistance patterns to other drugs. Therefore, daptomycin MICs for E. faecalis fell in the susceptible range for all of these organisms according to the CLSI interpretation standards for enterococci (MIC cut-off of 4 mg/mL) [5]. Daptomycin MICs for E. faecium ranged from 2 mg/mL to 8 mg/mL. One isolate (E. faecium 1693) was non-susceptible to daptomycin (MIC = 8 mg/mL). All enterococcal isolates were susceptible to
linezolid (MIC 2 mg/mL). For each test performed, the MIC of the quality control organism was within the acceptable range. 3.2. Fractional inhibitory concentration indices and time-kill assays In the checkerboard assays, synergism (FICI < 0.5) between daptomycin and linezolid occurred against all tested E. faecalis strains and against E. faecium BR and D366. Indifference occurred against E. faecium UB (FICI = 0.53) and 1693 (FICI = 2) isolates. No antagonism was detected. The rates of killing of daptomycin and linezolid, alone or in combination, against the tested enterococcal strains are displayed in Fig. 1. Daptomycin alone, at concentrations of 1 MIC, exhibited bacteriostatic activity within the first 6 h, followed by growth resumption. Linezolid alone, at concentrations of 1 MIC, was also bacteriostatic. Growth resumption at 24 h was only observed in one isolate (E. faecalis HH22) during exposure to either drug, possibly due to a decrease in drug concentration over time, as resistant derivatives were not obtained. Indeed, no changes in MICs were observed after 24 h of exposure to either drug when compared with the original inoculum (data not shown). The combination of daptomycin and linezolid had a synergistic effect (2.2–3.7 log10 CFU/mL kill compared with linezolid alone, the most active agent, and 2.1–2.77 log10 CFU/mL kill of the starting inoculum) against E. faecalis HH22 and V583 and E. faecium BR and D366. Indifference (1.1–1.6 log10 CFU/mL kill) was observed against strains E. faecalis 1110 and E. faecium UB and 1693. Antagonism was not detected. Bactericidal activity, defined as a 3 log10 CFU/mL reduction in colony count from the starting inoculum, was apparent only for strain E. faecium D366. 4. Discussion In the present in vitro study, a synergistic or indifferent activity of the combination of daptomycin and linezolid was observed against multidrug-resistant E. faecalis and E. faecium strains both using checkerboard and time-kill assays. Synergy studies with the combination of daptomycin and linezolid are scarce. A previous study by Parra-Ruiz et al. [11] demonstrated that the combination of daptomycin with linezolid was synergistic and bactericidal for meticillin-resistant Staphylococcus aureus (MRSA) in an in vitro biofilm model. Moreover, Steed et al. [12] reported a synergistic activity of this combination in an in vitro model of endocardial vegetations. In the case of enterococci, currently there is only one case report describing successful treatment with this combination for E. faecium meningitis [13]. The synergistic activity of daptomycin and linezolid observed in vitro against enterococci in the present
308
J.M. Entenza et al. / Journal of Global Antimicrobial Resistance 2 (2014) 306–308
E. faecium BR 10
8
8
Log cfu/mL
Log cfu/mL
E. faecalis HH22 10
6 4 2 0
0
6
12
18
This work was supported in part by research grants from the Swiss National Science Foundation [grants 310030-125325 and 310030-143799/1].
6 4 2 0
24
Competing interests 0
6
Time (hours)
8
8
Log cfu/mL
Log cfu/mL
10
6 4 2 6
12
18
Not required. References
2 0
6
Log cfu/mL
Log cfu/mL
4 2 12
12
18
24
18
E. faecium D366
10
6
6
Ethical approval
Time (hours)
E. faecalis 1110
0
None declared.
4
0
24
8
0
24
6
Time (hours) 10
18
E. faecium UB
E. faecalis V583
0
12
Time (hours)
10
0
Funding
8 6 4 2 0
24
0
6
Time (hours)
12
18
24
Time (hours)
E. faecium 1693
Control DAP LZD DAP+LZD
Log cfu/mL
10 8 6 4 2 0
0
6
12
18
24
Time (hours)
Fig. 1. Time-kill assays evaluating the activity of daptomycin (DAP) and linezolid (LZD), alone or in combination, against Enterococcus faecalis (left panel) and Enterococcus faecium (right panel). All anti-microbial agents are at the minimum inhibitory concentration. Data are the mean S.D. of three experiments.
study is consistent with the findings of this in vivo report. Despite the fact that the combination of daptomycin and linezolid provided synergy, the mechanism responsible for this synergy is unknown. Based on its ability to penetrate and depolarise the cell membrane, daptomycin might possibly increase the access of linezolid to its target on the ribosome. When the combination of a b-lactam or vancomycin and an aminoglycoside is not possible, treatment of severe enterococcal infections (particularly endocarditis and meningitis) depends on the susceptibility phenotype of the strain. Synergy between daptomycin and other antibiotics, including rifampicin, quinupristin/dalfopristin, doxycycline, fosfomycin and tigecycline, has been reported both in vitro and in vivo for enterococci [14– 19]. Combining daptomycin with linezolid might constitute another interesting therapeutic regimen for the treatment of severe multidrug-resistant enterococcal infections when first-line combinations cannot be used. Additional studies in animal models are needed to further test the efficacy of this combination. Besides the in vivo studies, the effect of the daptomycin plus linezolid combination on the emergence of resistance to either drug deserves further investigation.
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