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In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene
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ARTICLE IN PRESS International Journal of Antimicrobial Agents xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short Communication
In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene夽 Francis F. Arhin ∗ , Ingrid Sarmiento, Gregory Moeck The Medicines Company, 7170 Frederick Banting, St Laurent, Quebec, Canada H4S 2A1
a r t i c l e
i n f o
Article history: Received 17 February 2014 Accepted 31 March 2014 Keywords: Oritavancin mecC MRSA
a b s t r a c t Meticillin-resistant Staphylococcus aureus (MRSA) is routinely detected by amplification of the mecA gene. Recently, MRSA isolates harbouring a novel mec gene (mecC) that is not detected by mecA amplification have been reported. In this study, the activities of the lipoglycopeptide oritavancin as well as the comparators vancomycin, daptomycin and linezolid against 14 mecC MRSA isolates were studied by broth microdilution minimum inhibitory concentration (MIC) and time–kill assays at clinically relevant concentrations of each antibacterial agent. Oritavancin, vancomycin, daptomycin and linezolid MIC90 values (MIC required to inhibit 90% of the isolates) against the mecC isolates were 0.06, 1, 1 and 2 mg/L, respectively. In time–kill assays, oritavancin at concentrations reflective of its free peak in plasma of patients receiving a single 1200 mg intravenous dose and the level 24 h thereafter was bactericidal against all isolates tested, attaining 3 log kill relative to the starting inoculum between 5 min and 15 min. Vancomycin both at its free peak and free trough concentrations was also bactericidal against all isolates, attaining bactericidal activity between 6 h and 24 h. Daptomycin was bactericidal only at its free peak concentration, attaining bactericidal activity between 30 min and 4 h against the tested isolates. Linezolid was bacteriostatic (<3 log kill relative to the starting inoculum) against the tested isolates. Oritavancin’s in vitro activity against mecC MRSA isolates was indistinguishable from that against mecA MRSA isolates both in MIC and time–kill assays. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Introduction Infections caused by meticillin-resistant Staphylococcus aureus (MRSA) have been documented both in healthcare and community settings and continue to challenge healthcare systems. Traditionally, MRSA isolates are identified by amplification of a portion of the mecA gene [1]. Recently, isolates that are phenotypically resistant to meticillin but that are mecA amplification-negative have been described [2]. Further analysis of some of these isolates indicated that they harbour a novel mec gene that is only ca. 70% homologous to the mecA gene. This gene has been designated mecC [3] and has been detected both in human and zoonotic MRSA isolates [4,5]. Although to date no mecC MRSA isolate has been reported
夽 Previously presented in part at the 23rd European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 27–30 April 2013, Berlin, Germany [abstract P1594]. ∗ Corresponding author. Tel.: +1 514 332 1008x1700; fax: +1 514 332 6033. E-mail address: [email protected] (F.F. Arhin).
in the USA, mecC isolates from 13 European nations and from 14 different host species have been described [5]. Since conventional detection of MRSA by amplification of the mecA gene alone will not detect mecC-carrying MRSA, revision of the guidelines for MRSA identification has been suggested [2,5]. Multiplex PCR that identifies both mecA and mecC has been described and evaluated [6,7]. Oritavancin is a semi-synthetic lipoglycopeptide with rapid, concentration-dependent bactericidal activity against Grampositive bacteria including drug-resistant pathogens such as MRSA. Oritavancin has three distinct mechanisms of action: disruption of bacterial membrane integrity; and inhibition of both the transglycosylation and transpeptidation steps of cell wall synthesis [8]. Oritavancin administered as a single once-only dose was noninferior to 7–10 days of treatment with twice-daily vancomycin in two recently completed phase 3 studies [SOLO I (NCT01252719), 2011–2012; and SOLO II (NCT01252732), 2011–2013] in adults with acute bacterial skin and skin-structure infection (ABSSSI) [9].
http://dx.doi.org/10.1016/j.ijantimicag.2014.03.015 0924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Please cite this article in press as: Arhin FF, et al. In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.015
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Table 1 Concentrations of antibiotics tested in time–kill assays. Antimicrobial agent
Representative human dose
Free fraction
fCmax (mg/L)a
fCmin (mg/L)a
Oritavancinb Vancomycind Daptomycine Linezolidf
1200 mg 1000 mg 4 mg/kg 600 mg
0.15 0.54 0.08 0.69
16 16 4 8
2c 4 0.5 2
fCmax , maximum (peak) concentration of non-protein-bound drug in plasma following the approved or recommended dose; fCmin , lowest concentration of nonprotein-bound drug in the approved dosing interval. a Accounting for serum protein binding as noted in package inserts for each drug. b Oritavancin 1200 mg intravenously, single dose (http://clinicaltrials.gov/ ct2/show/NCT01252719 and http://clinicaltrials.gov/ct2/show/NCT01252732). c fCmin is not relevant for oritavancin since it is administered as a single 1200 mg dose for treatment of acute bacterial skin and skin-structure infection; hence, its estimated free concentration at 24 h following administration of the single dose was used as a representative concentration. d Vancomycin 1000 mg intravenously every 12 h (Vancocin® package insert). e Daptomycin 4 mg/kg intravenously every 24 h (Cubicin® package insert). f Linezolid 600 mg orally every 12 h (Zyvox® package insert).
This study evaluated the activity of oritavancin and comparators against 14 mecC-carrying MRSA isolates obtained from skin and skin-structure infections (SSSIs). Two MRSA isolates carrying the mecA gene were also included in the study. Materials and methods Antibacterial agents Oritavancin was obtained from The Medicines Company (Parsippany, NJ). Vancomycin was purchased from Sigma–Aldrich (St Louis, MO; product # V2002). Daptomycin was obtained as a pharmaceutically available powder. Linezolid was synthesised inhouse. Bacterial isolates S. aureus isolates used in this study were: ATCC 29213 [quality control (QC) strain, vancomycin-susceptible, meticillin-susceptible S. aureus (MSSA)]; ATCC 43300 (mecA MRSA); ATCC 33591 (mecA MRSA); and 14 isolates of mecC MRSA from SSSIs. The mecC isolates were obtained from Staten Serum Institut (Copenhagen, Denmark). Minimum inhibitory concentration (MIC) determination MICs were determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) methodology [10]. Oritavancin was dissolved in 0.002% polysorbate-80 (Sigma–Aldrich, St Louis, MO) in water; 0.002% polysorbate-80 was maintained throughout oritavancin dilution and MIC assays. Tests with daptomycin included CaCl2 at a concentration of 50 mg/L [10]. All MICs were determined at least four times independently. The modal MIC was reported as final. All test results for the QC strain were within CLSI acceptable ranges. Antibiotic concentration selection for time–kill assays Oritavancin serum protein binding is estimated at ca. 85% in animal species and humans [11]. Oritavancin concentrations were therefore chosen to approximate its free peak [fCmax ; defined as the maximum (peak) concentration of non-protein-bound drug] in plasma following administration of a single 1200 mg dose [12] (Table 1). Since oritavancin free trough levels (fCmin ; defined as the lowest concentration of non-protein-bound drug in the dosing interval) are not relevant for the clinically used single dose, an estimate of the free oritavancin concentration 24 h after the
Table 2 Summary of oritavancin and comparator minimum inhibitory concentrations (MICs) for Staphylococcus aureus isolates tested in this study. Isolate
ATCC 29213 ATCC 33591 ATCC 43300 73617 75814 57147 73829 78651 77950 77431 76918 70611 73415 73348 77964 78085 77601
Phenotype
MSSA MRSA (mecA) MRSA (mecA) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC)
MIC (mg/L) ORI
VAN
DAP
LZD
0.06 0.06 0.06 0.03 0.06 0.03 0.03 0.06 0.03 0.03 0.03 0.06 0.06 0.06 0.06 0.06 0.03
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0.5 1 0.5 1 0.5 0.5 0.5 0.5 0.5 1 0.5 0.5 1 0.5 0.5 1 0.5
2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2
ATCC, American Type Culture Collection; ORI, oritavancin; VAN, vancomycin; DAP, daptomycin; LZD, linezolid; MSSA, meticillin-susceptible S. aureus; MRSA, meticillin-resistant S. aureus.
1200 mg dose was used. Doubling dilution concentrations of comparator agents were likewise chosen to approximate their fCmax and fCmin levels in plasma when administered at approved dosages for ABSSSI, using pharmacokinetic data and protein binding values from their respective package inserts [daptomycin (Cubicin® ), linezolid (Zyvox® ) and vancomycin (Vancocin® )] (Table 1). Time–kill assays Time–kill assays followed guideline M26-A of the CLSI [13] with the following changes. Exponential-phase cells grown in cation-adjusted Mueller–Hinton broth (CAMHB) (Becton Dickinson, Franklin Lanes, NJ) were diluted to ca. 1 × 106 CFU/mL and were challenged with oritavancin or comparators in CAMHB in a 96-well deep-well plate (Corning Inc., Lowell, MA). For assays with oritavancin, CAMHB was supplemented with 0.002% polysorbate80 [10]; for assays with daptomycin, CAMHB was supplemented with 50 mg/L CaCl2 [10]. Viable cell count was determined by serial dilution plating, including the use of 25 g/L charcoal suspension to limit antibiotic carry-over. Bactericidal activity was defined as a ≥3 log decrease in cell count at 24 h relative to the initial inoculum [13]. All data points in time–kill assays were the mean of duplicate determinations within an experiment. Time–kill assays were repeated at least twice independently. Results The oritavancin MIC against the mecC isolates ranged from 0.03 mg/L to 0.06 mg/L and was within a doubling dilution of the oritavancin MIC for the QC (MSSA) and mecA MRSA isolates (all 0.06 mg/L) (Table 2). The vancomycin MIC was 1 mg/L for all isolates tested. Daptomycin and linezolid MICs were 0.5–1 mg/L and 1–2 mg/L, respectively, for all isolates. Oritavancin, vancomycin, daptomycin and linezolid MIC90 values (MIC required to inhibit 90% of the isolates) against the mecC isolates were 0.06, 1, 1 and 2 mg/L, respectively. In time–kill assays, oritavancin at both its fCmin and fCmax (Table 3) was rapidly bactericidal, attaining 3 log kill within 5–15 min for all tested S. aureus isolates, regardless of mec genotype. Vancomycin was likewise bactericidal at both its fCmin and fCmax , achieving 3 log kill within 6–24 h against all tested isolates. Daptomycin at its fCmin had little to no antibacterial effect or was
Please cite this article in press as: Arhin FF, et al. In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.015
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Table 3 Activity of oritavancin and comparator agents against Staphylococcus aureus isolates in time–kill assays.a Isolate (phenotype)
Oritavancin (fCmin )
Oritavancin (fCmax )b
Vancomycin (fCmin )
Vancomycin (fCmax )
Daptomycin (fCmin )
Daptomycin (fCmax )
Linezolid (fCmin )
Linezolid (fCmax )
ATCC 29213 (MSSA) ATCC 33591 (mecA) ATCC 43300 (mecA) 73617 (mecC) 75814 (mecC) 57147 (mecC) 73829 (mecC) 78651 (mecC) 77950 (mecC) 77431 (mecC) 76918 (mecC) 70611 (mecC) 73415 (mecC) 73348 (mecC) 77964 (mecC) 78085 (mecC) 77601 (mecC)
10–15 min
5–10 min
6–24 h
6–24 h
+3.9
2–4 h
+2.2
−2.8
10–15 min 5–10 min 10–15 min 5–10 min 5–10 min 5–10 min 10–15 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 10–15 min 5–10 min
5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min
6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h
6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h
+3.8 +3.6 +3.4 +3.6 +2.7 +2.1 +1.6 +0.9 +1.9 +1.5 +1.5 +1.5 +2.6 +1.2 +1.5 +0.1
30 min–1 h 1–2 h 1–2 h 1–2 h 1–2 h 1–2 h 30 min–1 h 1–2 h 1–2 h 1–2 h 1–2 h 2–4 h 30 min–1 h 1–2 h 2–4 h 30 min–1 h
+2.4 +2.6 +3.4 +2.2 +1.6 +1.2 +1.7 +0.9 +2.3 +1.8 +1.8 +1.2 +2.5 +1.2 +1.5 +0.5
−2.0 −2.6 −1.8 −1.3 −2.3 −1.8 −1.4 −1.2 −0.5 −0.5 +1.5 +1.5 +1.4 +0.2 −0.2 −1.2
fCmax , maximum (peak) concentration of non-protein-bound drug in plasma following the approved or recommended dose; fCmin , lowest concentration of non-protein-bound drug in the approved dosing interval; MSSA, meticillin-susceptible S. aureus; MRSA, meticillin-resistant S. aureus. a Time to bactericidal activity is indicated for drugs that achieved bactericidal activity (≥3 log decrease in cell density relative to the initial inoculum). Change in cell density (in log CFU/mL) at 24 h relative to the starting inoculum is indicated for drugs with bacteriostatic activity (0 to <3 log kill at 24 h relative to the initial inoculum) and for drugs with little to no inhibition of growth relative to growth control at 24 h. b For oritavancin, fCmax and fCmin are anticipated in patients receiving a single 1200 mg dose. Since fCmin is irrelevant for the single oritavancin dose, an estimate of the free oritavancin concentration at 24 h from this 1200 mg dose was used in place of the fCmin .
bacteriostatic against the tested isolates. Daptomycin at its fCmax was bactericidal against all isolates, achieving 3 log kill between 30 min and 4 h. Linezolid at both its fCmin and fCmax had little to no effect or was bacteriostatic against all tested isolates. Discussion With reports of antibiotic resistance on the rise, infections caused by MRSA continue to challenge healthcare providers. Directed therapy to treat these infections relies upon correct identification of MRSA as the aetiological agent. Whereas identification of the mecA gene by PCR has represented the gold standard for confirmation of MRSA [1], the emergence of mecC MRSA, which cannot be identified using the conventional mecA PCR test, has created a diagnostic challenge. Modifications to the guidelines for MRSA identification have now been suggested [2,3] and the latest European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines include information for identifying MRSA carrying the mecC gene (http://www.eucast.org/fileadmin/ src/media/PDFs/EUCAST files/Resistance mechanisms/EUCAST detection of resistance mechanisms v1.0 20131211.pdf). Identification of such mecC isolates of MRSA highlights the need to evaluate existing and investigational therapies used for the treatment of MRSA. In this study, oritavancin demonstrated potent in vitro activity against a panel of mecC isolates from SSSIs. Its MIC90 of 0.06 mg/L was identical to the values reported both for MRSA and MSSA isolates in a US surveillance initiative [14] and also the oritavancin MICs of the two mecA MRSA isolates tested in the current study. This suggests that oritavancin is equipotent against mecAcarrying and mecC-carrying MRSA in vitro. By MIC90 comparisons, oritavancin was 16–32-fold more potent than the comparators vancomycin, daptomycin and linezolid against the tested mecC MRSA isolates. This observation corroborates findings from a US surveillance initiative in which oritavancin was found to be 8–32-fold more potent in vitro than these same comparators against MRSA [14]. The rapid bactericidal activity of oritavancin against mecC MRSA isolates reported here extends previous observations of its rapidly bactericidal activity against MSSA and mecA MRSA [15]. Whereas
the in vitro activities of vancomycin, daptomycin and linezolid were also similar for the mecA-carrying and mecC-carrying MRSA isolates, the rate of cell killing by oritavancin was more rapid than that of the comparators. In conclusion, oritavancin is equally active both against mecC MRSA and mecA MRSA isolates in MIC and time–kill assays. Acknowledgments The authors thank Dr R. Skov and the Staphylococcus Laboratory, Staten Serum Institut (Copenhagen, Denmark) for providing the mecC MRSA isolates. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twenty-fourth informational supplement, document M100-S24. Wayne, PA: CLSI; 2014. [2] García-Álvarez L, Holden MT, Lindsay H, Webb CR, Brown DF, Curran MD, et al. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect Dis 2011;11:595–603. [3] Ito T, Hiramatsu K, Tomasz A, de Lencastre H, Perreten V, Holden MT, et al. Guidelines for reporting novel mecA gene homologues. Antimicrob Agents Chemother 2012;56:4997–9. [4] Petersen A, Stegger M, Heltberg O, Christensen J, Zeuthen A, Knudsen LK, et al. Epidemiology of methicillin-resistant Staphylococcus aureus carrying the novel mecC gene in Denmark corroborates a zoonotic reservoir with transmission to humans. Clin Microbiol Infect 2013;19:E16–22. [5] Paterson GK, Harrison EM, Holmes MA. The emergence of mecC methicillinresistant Staphylococcus aureus. Trends Microbiol 2014;22:42–7. [6] Pichon B, Hill R, Laurent F, Larsen AR, Skov RL, Holmes M, et al. Development of a real-time quadruplex PCR assay for simultaneous detection of nuc, Panton–Valentine leucocidin (PVL), mecA and homologue mecALGA251 . J Antimicrob Chemother 2012;67:2238–41. [7] Stegger M, Andersen PS, Kearns A, Pichon B, Holmes MA, Edwards G, et al. Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecALGA251 . Clin Microbiol Infect 2012;18:395–400. [8] Zhanel GG, Schweizer F, Karlowsky JA. Oritavancin: mechanism of action. Clin Infect Dis 2012;54:S214–19. [9] Corey R., Perez A., Moeck G., Jiang H., Good S., SOLO I Investigators. A singledose of oritavancin (ORI) is comparable to 7–10 days of vancomycin (VAN) in
Please cite this article in press as: Arhin FF, et al. In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.015
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the treatment of acute bacterial skin and skin structure infections (ABSSSI): the SOLO I study. In: 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC); 10–13 September 2013, Denver, CO. Washington, DC: ASM Press; 2013 [abstract L-204]. [10] Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, document M7-A9. 9th ed. Wayne, PA: CLSI; 2012. [11] Arhin FF, Belley A, McKay GA, Beaulieu S, Sarmiento I, Parr Jr TR, et al. Assessment of oritavancin serum protein binding across species. Antimicrob Agents Chemother 2010;54:3481–3. [12] Belley A, Arhin FF, Sarmiento I, Deng H, Rose W, Moeck G. Pharmacodynamics of a simulated single 1,200-milligram dose of oritavancin in an in vitro
pharmacokinetic/pharmacodynamic model of methicillin-resistant Staphylococcus aureus infection. Antimicrob Agents Chemother 2013;57:205–11. [13] National Committee for Clinical and Laboratory Standards. Methods for determining bactericidal activity of antimicrobial agents; approved guideline, document M26-A. Wayne, PA: NCCLS; 1999. [14] Mendes RE, Farrell DJ, Sader HS, Jones RN. Oritavancin microbiologic features and activity results from the surveillance program in the United States. Clin Infect Dis 2012;54:S203–13. [15] McKay GA, Beaulieu S, Arhin FF, Belley A, Sarmiento I, Parr Jr T, et al. Time–kill kinetics of oritavancin and comparator agents against Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. J Antimicrob Chemother 2009;63:1191–9.
Please cite this article in press as: Arhin FF, et al. In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.015
ARTICLE IN PRESS International Journal of Antimicrobial Agents xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short Communication
In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene夽 Francis F. Arhin ∗ , Ingrid Sarmiento, Gregory Moeck The Medicines Company, 7170 Frederick Banting, St Laurent, Quebec, Canada H4S 2A1
a r t i c l e
i n f o
Article history: Received 17 February 2014 Accepted 31 March 2014 Keywords: Oritavancin mecC MRSA
a b s t r a c t Meticillin-resistant Staphylococcus aureus (MRSA) is routinely detected by amplification of the mecA gene. Recently, MRSA isolates harbouring a novel mec gene (mecC) that is not detected by mecA amplification have been reported. In this study, the activities of the lipoglycopeptide oritavancin as well as the comparators vancomycin, daptomycin and linezolid against 14 mecC MRSA isolates were studied by broth microdilution minimum inhibitory concentration (MIC) and time–kill assays at clinically relevant concentrations of each antibacterial agent. Oritavancin, vancomycin, daptomycin and linezolid MIC90 values (MIC required to inhibit 90% of the isolates) against the mecC isolates were 0.06, 1, 1 and 2 mg/L, respectively. In time–kill assays, oritavancin at concentrations reflective of its free peak in plasma of patients receiving a single 1200 mg intravenous dose and the level 24 h thereafter was bactericidal against all isolates tested, attaining 3 log kill relative to the starting inoculum between 5 min and 15 min. Vancomycin both at its free peak and free trough concentrations was also bactericidal against all isolates, attaining bactericidal activity between 6 h and 24 h. Daptomycin was bactericidal only at its free peak concentration, attaining bactericidal activity between 30 min and 4 h against the tested isolates. Linezolid was bacteriostatic (<3 log kill relative to the starting inoculum) against the tested isolates. Oritavancin’s in vitro activity against mecC MRSA isolates was indistinguishable from that against mecA MRSA isolates both in MIC and time–kill assays. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Introduction Infections caused by meticillin-resistant Staphylococcus aureus (MRSA) have been documented both in healthcare and community settings and continue to challenge healthcare systems. Traditionally, MRSA isolates are identified by amplification of a portion of the mecA gene [1]. Recently, isolates that are phenotypically resistant to meticillin but that are mecA amplification-negative have been described [2]. Further analysis of some of these isolates indicated that they harbour a novel mec gene that is only ca. 70% homologous to the mecA gene. This gene has been designated mecC [3] and has been detected both in human and zoonotic MRSA isolates [4,5]. Although to date no mecC MRSA isolate has been reported
夽 Previously presented in part at the 23rd European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 27–30 April 2013, Berlin, Germany [abstract P1594]. ∗ Corresponding author. Tel.: +1 514 332 1008x1700; fax: +1 514 332 6033. E-mail address: [email protected] (F.F. Arhin).
in the USA, mecC isolates from 13 European nations and from 14 different host species have been described [5]. Since conventional detection of MRSA by amplification of the mecA gene alone will not detect mecC-carrying MRSA, revision of the guidelines for MRSA identification has been suggested [2,5]. Multiplex PCR that identifies both mecA and mecC has been described and evaluated [6,7]. Oritavancin is a semi-synthetic lipoglycopeptide with rapid, concentration-dependent bactericidal activity against Grampositive bacteria including drug-resistant pathogens such as MRSA. Oritavancin has three distinct mechanisms of action: disruption of bacterial membrane integrity; and inhibition of both the transglycosylation and transpeptidation steps of cell wall synthesis [8]. Oritavancin administered as a single once-only dose was noninferior to 7–10 days of treatment with twice-daily vancomycin in two recently completed phase 3 studies [SOLO I (NCT01252719), 2011–2012; and SOLO II (NCT01252732), 2011–2013] in adults with acute bacterial skin and skin-structure infection (ABSSSI) [9].
http://dx.doi.org/10.1016/j.ijantimicag.2014.03.015 0924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Please cite this article in press as: Arhin FF, et al. In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.015
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Table 1 Concentrations of antibiotics tested in time–kill assays. Antimicrobial agent
Representative human dose
Free fraction
fCmax (mg/L)a
fCmin (mg/L)a
Oritavancinb Vancomycind Daptomycine Linezolidf
1200 mg 1000 mg 4 mg/kg 600 mg
0.15 0.54 0.08 0.69
16 16 4 8
2c 4 0.5 2
fCmax , maximum (peak) concentration of non-protein-bound drug in plasma following the approved or recommended dose; fCmin , lowest concentration of nonprotein-bound drug in the approved dosing interval. a Accounting for serum protein binding as noted in package inserts for each drug. b Oritavancin 1200 mg intravenously, single dose (http://clinicaltrials.gov/ ct2/show/NCT01252719 and http://clinicaltrials.gov/ct2/show/NCT01252732). c fCmin is not relevant for oritavancin since it is administered as a single 1200 mg dose for treatment of acute bacterial skin and skin-structure infection; hence, its estimated free concentration at 24 h following administration of the single dose was used as a representative concentration. d Vancomycin 1000 mg intravenously every 12 h (Vancocin® package insert). e Daptomycin 4 mg/kg intravenously every 24 h (Cubicin® package insert). f Linezolid 600 mg orally every 12 h (Zyvox® package insert).
This study evaluated the activity of oritavancin and comparators against 14 mecC-carrying MRSA isolates obtained from skin and skin-structure infections (SSSIs). Two MRSA isolates carrying the mecA gene were also included in the study. Materials and methods Antibacterial agents Oritavancin was obtained from The Medicines Company (Parsippany, NJ). Vancomycin was purchased from Sigma–Aldrich (St Louis, MO; product # V2002). Daptomycin was obtained as a pharmaceutically available powder. Linezolid was synthesised inhouse. Bacterial isolates S. aureus isolates used in this study were: ATCC 29213 [quality control (QC) strain, vancomycin-susceptible, meticillin-susceptible S. aureus (MSSA)]; ATCC 43300 (mecA MRSA); ATCC 33591 (mecA MRSA); and 14 isolates of mecC MRSA from SSSIs. The mecC isolates were obtained from Staten Serum Institut (Copenhagen, Denmark). Minimum inhibitory concentration (MIC) determination MICs were determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) methodology [10]. Oritavancin was dissolved in 0.002% polysorbate-80 (Sigma–Aldrich, St Louis, MO) in water; 0.002% polysorbate-80 was maintained throughout oritavancin dilution and MIC assays. Tests with daptomycin included CaCl2 at a concentration of 50 mg/L [10]. All MICs were determined at least four times independently. The modal MIC was reported as final. All test results for the QC strain were within CLSI acceptable ranges. Antibiotic concentration selection for time–kill assays Oritavancin serum protein binding is estimated at ca. 85% in animal species and humans [11]. Oritavancin concentrations were therefore chosen to approximate its free peak [fCmax ; defined as the maximum (peak) concentration of non-protein-bound drug] in plasma following administration of a single 1200 mg dose [12] (Table 1). Since oritavancin free trough levels (fCmin ; defined as the lowest concentration of non-protein-bound drug in the dosing interval) are not relevant for the clinically used single dose, an estimate of the free oritavancin concentration 24 h after the
Table 2 Summary of oritavancin and comparator minimum inhibitory concentrations (MICs) for Staphylococcus aureus isolates tested in this study. Isolate
ATCC 29213 ATCC 33591 ATCC 43300 73617 75814 57147 73829 78651 77950 77431 76918 70611 73415 73348 77964 78085 77601
Phenotype
MSSA MRSA (mecA) MRSA (mecA) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC) MRSA (mecC)
MIC (mg/L) ORI
VAN
DAP
LZD
0.06 0.06 0.06 0.03 0.06 0.03 0.03 0.06 0.03 0.03 0.03 0.06 0.06 0.06 0.06 0.06 0.03
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0.5 1 0.5 1 0.5 0.5 0.5 0.5 0.5 1 0.5 0.5 1 0.5 0.5 1 0.5
2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2
ATCC, American Type Culture Collection; ORI, oritavancin; VAN, vancomycin; DAP, daptomycin; LZD, linezolid; MSSA, meticillin-susceptible S. aureus; MRSA, meticillin-resistant S. aureus.
1200 mg dose was used. Doubling dilution concentrations of comparator agents were likewise chosen to approximate their fCmax and fCmin levels in plasma when administered at approved dosages for ABSSSI, using pharmacokinetic data and protein binding values from their respective package inserts [daptomycin (Cubicin® ), linezolid (Zyvox® ) and vancomycin (Vancocin® )] (Table 1). Time–kill assays Time–kill assays followed guideline M26-A of the CLSI [13] with the following changes. Exponential-phase cells grown in cation-adjusted Mueller–Hinton broth (CAMHB) (Becton Dickinson, Franklin Lanes, NJ) were diluted to ca. 1 × 106 CFU/mL and were challenged with oritavancin or comparators in CAMHB in a 96-well deep-well plate (Corning Inc., Lowell, MA). For assays with oritavancin, CAMHB was supplemented with 0.002% polysorbate80 [10]; for assays with daptomycin, CAMHB was supplemented with 50 mg/L CaCl2 [10]. Viable cell count was determined by serial dilution plating, including the use of 25 g/L charcoal suspension to limit antibiotic carry-over. Bactericidal activity was defined as a ≥3 log decrease in cell count at 24 h relative to the initial inoculum [13]. All data points in time–kill assays were the mean of duplicate determinations within an experiment. Time–kill assays were repeated at least twice independently. Results The oritavancin MIC against the mecC isolates ranged from 0.03 mg/L to 0.06 mg/L and was within a doubling dilution of the oritavancin MIC for the QC (MSSA) and mecA MRSA isolates (all 0.06 mg/L) (Table 2). The vancomycin MIC was 1 mg/L for all isolates tested. Daptomycin and linezolid MICs were 0.5–1 mg/L and 1–2 mg/L, respectively, for all isolates. Oritavancin, vancomycin, daptomycin and linezolid MIC90 values (MIC required to inhibit 90% of the isolates) against the mecC isolates were 0.06, 1, 1 and 2 mg/L, respectively. In time–kill assays, oritavancin at both its fCmin and fCmax (Table 3) was rapidly bactericidal, attaining 3 log kill within 5–15 min for all tested S. aureus isolates, regardless of mec genotype. Vancomycin was likewise bactericidal at both its fCmin and fCmax , achieving 3 log kill within 6–24 h against all tested isolates. Daptomycin at its fCmin had little to no antibacterial effect or was
Please cite this article in press as: Arhin FF, et al. In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.015
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Table 3 Activity of oritavancin and comparator agents against Staphylococcus aureus isolates in time–kill assays.a Isolate (phenotype)
Oritavancin (fCmin )
Oritavancin (fCmax )b
Vancomycin (fCmin )
Vancomycin (fCmax )
Daptomycin (fCmin )
Daptomycin (fCmax )
Linezolid (fCmin )
Linezolid (fCmax )
ATCC 29213 (MSSA) ATCC 33591 (mecA) ATCC 43300 (mecA) 73617 (mecC) 75814 (mecC) 57147 (mecC) 73829 (mecC) 78651 (mecC) 77950 (mecC) 77431 (mecC) 76918 (mecC) 70611 (mecC) 73415 (mecC) 73348 (mecC) 77964 (mecC) 78085 (mecC) 77601 (mecC)
10–15 min
5–10 min
6–24 h
6–24 h
+3.9
2–4 h
+2.2
−2.8
10–15 min 5–10 min 10–15 min 5–10 min 5–10 min 5–10 min 10–15 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 10–15 min 5–10 min
5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min 5–10 min
6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h
6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h 6–24 h
+3.8 +3.6 +3.4 +3.6 +2.7 +2.1 +1.6 +0.9 +1.9 +1.5 +1.5 +1.5 +2.6 +1.2 +1.5 +0.1
30 min–1 h 1–2 h 1–2 h 1–2 h 1–2 h 1–2 h 30 min–1 h 1–2 h 1–2 h 1–2 h 1–2 h 2–4 h 30 min–1 h 1–2 h 2–4 h 30 min–1 h
+2.4 +2.6 +3.4 +2.2 +1.6 +1.2 +1.7 +0.9 +2.3 +1.8 +1.8 +1.2 +2.5 +1.2 +1.5 +0.5
−2.0 −2.6 −1.8 −1.3 −2.3 −1.8 −1.4 −1.2 −0.5 −0.5 +1.5 +1.5 +1.4 +0.2 −0.2 −1.2
fCmax , maximum (peak) concentration of non-protein-bound drug in plasma following the approved or recommended dose; fCmin , lowest concentration of non-protein-bound drug in the approved dosing interval; MSSA, meticillin-susceptible S. aureus; MRSA, meticillin-resistant S. aureus. a Time to bactericidal activity is indicated for drugs that achieved bactericidal activity (≥3 log decrease in cell density relative to the initial inoculum). Change in cell density (in log CFU/mL) at 24 h relative to the starting inoculum is indicated for drugs with bacteriostatic activity (0 to <3 log kill at 24 h relative to the initial inoculum) and for drugs with little to no inhibition of growth relative to growth control at 24 h. b For oritavancin, fCmax and fCmin are anticipated in patients receiving a single 1200 mg dose. Since fCmin is irrelevant for the single oritavancin dose, an estimate of the free oritavancin concentration at 24 h from this 1200 mg dose was used in place of the fCmin .
bacteriostatic against the tested isolates. Daptomycin at its fCmax was bactericidal against all isolates, achieving 3 log kill between 30 min and 4 h. Linezolid at both its fCmin and fCmax had little to no effect or was bacteriostatic against all tested isolates. Discussion With reports of antibiotic resistance on the rise, infections caused by MRSA continue to challenge healthcare providers. Directed therapy to treat these infections relies upon correct identification of MRSA as the aetiological agent. Whereas identification of the mecA gene by PCR has represented the gold standard for confirmation of MRSA [1], the emergence of mecC MRSA, which cannot be identified using the conventional mecA PCR test, has created a diagnostic challenge. Modifications to the guidelines for MRSA identification have now been suggested [2,3] and the latest European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines include information for identifying MRSA carrying the mecC gene (http://www.eucast.org/fileadmin/ src/media/PDFs/EUCAST files/Resistance mechanisms/EUCAST detection of resistance mechanisms v1.0 20131211.pdf). Identification of such mecC isolates of MRSA highlights the need to evaluate existing and investigational therapies used for the treatment of MRSA. In this study, oritavancin demonstrated potent in vitro activity against a panel of mecC isolates from SSSIs. Its MIC90 of 0.06 mg/L was identical to the values reported both for MRSA and MSSA isolates in a US surveillance initiative [14] and also the oritavancin MICs of the two mecA MRSA isolates tested in the current study. This suggests that oritavancin is equipotent against mecAcarrying and mecC-carrying MRSA in vitro. By MIC90 comparisons, oritavancin was 16–32-fold more potent than the comparators vancomycin, daptomycin and linezolid against the tested mecC MRSA isolates. This observation corroborates findings from a US surveillance initiative in which oritavancin was found to be 8–32-fold more potent in vitro than these same comparators against MRSA [14]. The rapid bactericidal activity of oritavancin against mecC MRSA isolates reported here extends previous observations of its rapidly bactericidal activity against MSSA and mecA MRSA [15]. Whereas
the in vitro activities of vancomycin, daptomycin and linezolid were also similar for the mecA-carrying and mecC-carrying MRSA isolates, the rate of cell killing by oritavancin was more rapid than that of the comparators. In conclusion, oritavancin is equally active both against mecC MRSA and mecA MRSA isolates in MIC and time–kill assays. Acknowledgments The authors thank Dr R. Skov and the Staphylococcus Laboratory, Staten Serum Institut (Copenhagen, Denmark) for providing the mecC MRSA isolates. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twenty-fourth informational supplement, document M100-S24. Wayne, PA: CLSI; 2014. [2] García-Álvarez L, Holden MT, Lindsay H, Webb CR, Brown DF, Curran MD, et al. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect Dis 2011;11:595–603. [3] Ito T, Hiramatsu K, Tomasz A, de Lencastre H, Perreten V, Holden MT, et al. Guidelines for reporting novel mecA gene homologues. Antimicrob Agents Chemother 2012;56:4997–9. [4] Petersen A, Stegger M, Heltberg O, Christensen J, Zeuthen A, Knudsen LK, et al. Epidemiology of methicillin-resistant Staphylococcus aureus carrying the novel mecC gene in Denmark corroborates a zoonotic reservoir with transmission to humans. Clin Microbiol Infect 2013;19:E16–22. [5] Paterson GK, Harrison EM, Holmes MA. The emergence of mecC methicillinresistant Staphylococcus aureus. Trends Microbiol 2014;22:42–7. [6] Pichon B, Hill R, Laurent F, Larsen AR, Skov RL, Holmes M, et al. Development of a real-time quadruplex PCR assay for simultaneous detection of nuc, Panton–Valentine leucocidin (PVL), mecA and homologue mecALGA251 . J Antimicrob Chemother 2012;67:2238–41. [7] Stegger M, Andersen PS, Kearns A, Pichon B, Holmes MA, Edwards G, et al. Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecALGA251 . Clin Microbiol Infect 2012;18:395–400. [8] Zhanel GG, Schweizer F, Karlowsky JA. Oritavancin: mechanism of action. Clin Infect Dis 2012;54:S214–19. [9] Corey R., Perez A., Moeck G., Jiang H., Good S., SOLO I Investigators. A singledose of oritavancin (ORI) is comparable to 7–10 days of vancomycin (VAN) in
Please cite this article in press as: Arhin FF, et al. In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.015
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the treatment of acute bacterial skin and skin structure infections (ABSSSI): the SOLO I study. In: 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC); 10–13 September 2013, Denver, CO. Washington, DC: ASM Press; 2013 [abstract L-204]. [10] Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, document M7-A9. 9th ed. Wayne, PA: CLSI; 2012. [11] Arhin FF, Belley A, McKay GA, Beaulieu S, Sarmiento I, Parr Jr TR, et al. Assessment of oritavancin serum protein binding across species. Antimicrob Agents Chemother 2010;54:3481–3. [12] Belley A, Arhin FF, Sarmiento I, Deng H, Rose W, Moeck G. Pharmacodynamics of a simulated single 1,200-milligram dose of oritavancin in an in vitro
pharmacokinetic/pharmacodynamic model of methicillin-resistant Staphylococcus aureus infection. Antimicrob Agents Chemother 2013;57:205–11. [13] National Committee for Clinical and Laboratory Standards. Methods for determining bactericidal activity of antimicrobial agents; approved guideline, document M26-A. Wayne, PA: NCCLS; 1999. [14] Mendes RE, Farrell DJ, Sader HS, Jones RN. Oritavancin microbiologic features and activity results from the surveillance program in the United States. Clin Infect Dis 2012;54:S203–13. [15] McKay GA, Beaulieu S, Arhin FF, Belley A, Sarmiento I, Parr Jr T, et al. Time–kill kinetics of oritavancin and comparator agents against Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. J Antimicrob Chemother 2009;63:1191–9.
Please cite this article in press as: Arhin FF, et al. In vitro activities of oritavancin and comparators against meticillin-resistant Staphylococcus aureus (MRSA) isolates harbouring the novel mecC gene. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.015