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In vitro activity of oritavancin against community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA) and daptomycin-non-susceptible S. aureus (DNSSA)
International Journal of Antimicrobial Agents 36 (2010) 69–72
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
In vitro activity of oritavancin against community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA) and daptomycin-non-susceptible S. aureus (DNSSA) Louis D. Saravolatz ∗ , Joan Pawlak, Leonard B. Johnson St John Hospital and Medical Center and Wayne State University School of Medicine, Detroit, MI, USA
a r t i c l e
i n f o
Article history: Received 9 November 2009 Accepted 26 February 2010 Keywords: Oritavancin Lipoglycopeptide MRSA
a b s t r a c t Isolates of community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA), vancomycinintermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA) and daptomycin non-susceptible S. aureus (DNSSA) are increasing in frequency and new antistaphylococcal therapies are needed. Microdilution testing using Mueller–Hinton broth was used to determine the minimal inhibitory concentrations (MICs) of oritavancin and nine additional antimicrobial agents against 92 CA-MRSA, 23 VISA, 7 DNSSA and 10 VRSA isolates. Minimal bactericidal concentrations were also determined. Pulsed-field gel electrophoresis (PFGE) was performed. Staphylococcal cassette chromosome mec (SCCmec) typing as well as assays for Panton–Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) genes were performed. Oritavancin was more bactericidal than any of the other comparators against CA-MRSA and demonstrated excellent activity against VRSA and VISA. © 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Oritavancin, derived from chloroeremomycin, is a semisynthetic lipoglycopeptide with potent in vitro activity against Grampositive organisms, including meticillin-resistant Staphylococcus aureus (MRSA). The multiple mechanisms of action of oritavancin include inhibition of cell wall synthesis, disruption of the transmembrane potential, and partial inhibition of RNA synthesis [1]. Oritavancin is active against a variety of Gram-positive organisms. It has been shown to perturb the cell membrane to achieve enhanced killing of S. aureus. In addition, it has demonstrated killing of S. aureus both in planktonic and biofilm states. With the significant rise of MRSA isolates in the community and its decreasing susceptibility to vancomycin, there is a need to develop additional antistaphylococcal agents [2]. This study evaluated oritavancin and nine comparator agents against communityassociated MRSA (CA-MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA) and daptomycin non-susceptible S. aureus (DNSSA). To characterise the isolates, staphylococcal cassette chromosome mec (SCCmec) typing as well
∗ Corresponding author. Present address: 19251 Mack Avenue, Suite 333, Grosse Pointe Woods, MI 48236, USA. Tel.: +1 313 343 3362; fax: +1 313 343 7784. E-mail address: [email protected] (L.D. Saravolatz).
as assays for Panton–Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) genes were performed. 2. Methods 2.1. Isolates A collection of 132 MRSA strains was selected for evaluation. CA-MRSA strains (n = 92) were isolated from patients admitted to St John Hospital and Medical Center (Detroit, MI) from July 2003 to May 2007. There were no duplicate isolates from patients. These 92 strains were identified as culture-positive for MRSA within 48 h of admission, in accordance with the definition of CA-MRSA described by the US Centers for Disease Control and Prevention (CDC) [3]. Cultures were performed on samples collected from blood (n = 26), respiratory specimens (n = 33), wound or tissue (n = 32) and catheter tip (n = 1). Patients with culture-positive MRSA after 48 h of hospitalisation were excluded to avoid the inclusion of nosocomial MRSA strains. DNSSA (n = 7) and vancomycin-heteroresistant S. aureus (hVISA) (n = 3) strains were obtained from blood isolates collected from patients at St John Hospital and Medical Center. hVISA isolates were identified using a modified population analysis profile method. VISA (n = 20) and VRSA (n = 10) isolates were obtained through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) programme supported under NIAID/NIH Contract
0924-8579/$ – see front matter © 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2010.02.023
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Table 1 Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) (in mg/L) of oritavancin and comparators.
CA-MRSA (n = 92) MIC50 MIC90 MIC range Breakpoint %S MBC50 MBC90 MBC range VISA (n = 20) MIC50 MIC90 MIC range Breakpoint %S MBC50 MBC90 MBC range VRSA (n = 10) MIC50 MIC90 MIC range Breakpoint %S MBC50 MBC90 MBC range
ORI
VAN
DAP
LZD
Q/D
SXT
TIG
MIN
RIF
TEI
0.06 0.12 0.015–0.25 N/A N/A 0.12 0.25 0.03–0.5
1 1 0.5–2 ≤2 100 1 1 0.5–2
0.5 1 0.25–1 ≤1 100 0.5 1 0.25–2
2 2 1–4 ≤4 100 >8 >8 4 to >8
0.25 0.5 0.12–0.5 ≤1 100 0.5 >8 0.25 to >8
2.4/0.12 9.5/0.5 1.2/0.06 to >76/4 ≤38/2 98 2.4/0.12 19/1 1.2/0.06 to >76/4
0.12 0.25 0.06–0.5 ≤0.5 100 >2 >2 0.5 to >2
0.12 0.5 0.06–8 ≤4 97 >16 >16 0.5 to >16
0.008 0.008 0.004–0.015 ≤1 100 0.5 1 0.015–2
0.5 0.5 0.25–1 ≤8 100 0.5 1 0.25–1
1 2 0.25–2 N/A N/A 1 2 0.5–4
4 8 4–8 ≤2 0 4 8 4–8
2 4 1–8 ≤1 25 2 4 1–8
1 2 0.5–2 ≤4 100 4 >8 2 to >8
0.5 1 0.25–2 ≤1 95 1 >8 0.25 to >8
4.8/0.25 >76/4 1.2/0.06 to >76/4 ≤38/2 75 4.8/0.25 >76/4 2.4/0.12 to >76/4
0.25 0.25 0.06–0.5 ≤0.5 100 0.5 >2 0.12 to >2
0.12 0.5 0.03–2 ≤4 100 8 >16 0.06 to >16
0.015 >4 0.004 to >4 ≤1 55 1 >4 0.03 to >4
4 8 1–16 ≤8 90 4 16 2–32
0.5 1 0.25–1 N/A N/A 1 2 0.5–2
>64 >64 32 to >64 ≤2 0 >64 >64 64 to >64
0.5 1 0.5–1 ≤1 100 1 1 0.5–1
2 2 1–4 ≤4 100 >8 >8 8 to >8
0.5 0.5 0.5 ≤1 100 8 >8 1 to >8
2.4/0.12 38/2 1.2/0.06 to >76/4 ≤38/2 90 2.4/0.12 >76/4 2.4/0.12 to >76/4
0.25 0.5 0.06–1 ≤0.5 90 1 >2 0.25 to >2
0.12 2 0.06–2 ≤4 100 >16 >16 8 to >16
0.008 0.008 0.004 to >4 ≤1 90 0.12 1 0.008 to >4
32 >32 8 to >32 ≤8 20 >32 >32 8 to >32
ORI, oritavancin; VAN, vancomycin; DAP, daptomycin; LZD, linezolid; Q/D, quinupristin/dalfopristin; SXT, sulfamethoxazole/trimethoprim; TIG, tigecycline; MIN, minocycline; RIF, rifampicin; TEI, teicoplanin; CA-MRSA, community-associated meticillin-resistant Staphylococcus aureus; VISA, vancomycin-intermediate S. aureus; VRSA, vancomycinresistant S. aureus; MIC50/90 , MIC for 50% and 90% of the organisms, respectively; %S, percent susceptible; MBC50/90 , MBC for 50% and 90% of the organisms, respectively; N/A, not applicable (oritavancin breakpoints have not been assigned).
Pulsed-field gel electrophoresis (PFGE) was performed on the CA-MRSA isolates using restriction enzyme SmaI [4]. PFGE patterns were visually compared with S. aureus USA100 to USA800 (NARSA, Herndon VA.) and categorised. Strains were assigned to a specific pulsed field group only when all the bands matched. A multiplex polymerase chain reaction (PCR) procedure was used to determine the SCCmec type of all the isolates [5]. PVL genes lukS-PV and lukF-PV were identified by PCR [6]. The presence of the arginine catabolic mobile element (ACME) was determined by PCR detection of the arcA locus [7]. PCR testing was performed on all isolates. DNA used for the PCR assays was extracted from isolates using a Qiagen QIAamp DNA Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s directions. Controls used for PCR were S. aureus ATCC BAA-44, ATCC BAA-41, ATCC BAA-39, NRS-384, ATCC49775, NCTC-8325 and ATCC12228.
Therapeutics Corp. (Cambridge, MA). Plates were stored at −70 ◦ C and used within 3 months of preparation. Oritavancin was tested in the presence of polysorbate 80 at a concentration of 0.002% to prevent binding of the antibiotic to plastic surfaces. For testing of daptomycin, Mueller–Hinton broth was supplemented with calcium to a final concentration of 50 mg/L. Microtitre plates were inoculated with 105 colony-forming units/mL and were incubated in air at 35 ◦ C for 18–24 h. Minimal inhibitory concentration (MIC) values were determined in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [8]. MICs were read visually as the lowest drug concentration well with no visible bacterial growth. Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were used to monitor quality control for all the antibiotics with each run. Quality control results were in the acceptable range for both organisms and all antibiotics. Minimal bactericidal concentration (MBC) values of all 10 antibiotics for all the isolates were also determined according to CLSI guidelines [9]. Ten microlitres from each well showing no visible growth were subcultured onto a blood agar plate (Remel Inc., Lenexa, KS) and incubated in air at 35 ◦ C from 24 h to 48 h. The MBC was defined as the lowest concentration of antimicrobial agent that reduced the initial inoculum by 99.9% based on colony counts from the growth control well.
2.3. Antimicrobials and in vitro susceptibility testing
3. Results
In vitro susceptibility testing was performed with the following antimicrobials: oritavancin (0.004–8 mg/L); linezolid (0.25–8 mg/L); tigecycline (0.015–2 mg/L); vancomycin (0.12–32 mg/L); quinupristin/dalfopristin (Q/D) (0.06–8 mg/L); daptomycin (0.06–8 mg/L); sulfamethoxazole/trimethoprim (SXT) (0.6/0.03–76/4 mg/L); teicoplanin (0.06–32 mg/L); minocycline (0.3–16 mg/L); and rifampicin (0.02–4 mg/L). Microtitre dilution plates containing cation-adjusted Mueller–Hinton broth and the antimicrobials were prepared and supplied by Targanta
SCCmec types observed for the 92 CA-MRSA strains were IVa (40), II (38), IV (13) and not typeable (1). For the 20 VISA isolates, the SCCmec types found were II (18), IVd (1) and I (1). All three hVISA strains typed as SCCmec type II. VRSA SCCmec types were II (6), IV (2) and not typeable (2). DNSSA SCCmec types were II (4), IVa (2) and III (1). The 13 CA-MRSA and 2 VRSA isolates that typed as SCCmec type IV could not be classified into the SCCmec type IV subgroups a, b, c or d. Although SCCmec type IVa was the most commonly found among CA-MRSA isolates (43%), there were still 41% of isolates identified
#HHSN272200700055C. VISA isolates were cultured from blood (n = 12), wound (n = 3) and peritoneal fluid, bone, cerebrospinal fluid, bile and sputum (1 each). VRSA isolates were cultured from wound (n = 6), catheter site (n = 1), urine (n = 1), nephrostomy tube (n = 1) and unknown (n = 1). 2.2. Molecular typing
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Table 2 Geometric mean minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) (in mg/L) of oritavancin and comparators against daptomycin non-susceptible Staphylococcus aureus (n = 7).
MIC50 MIC90 Geometric mean MIC MIC range Breakpoint %S MBC50 MBC90 Geometric mean MBC MBC range
ORI
VAN
DAP
LZD
Q/D
SXT
TIG
MIN
RIF
TEI
0.25 0.5 0.2 0.06–0.5 N/A N/A 0.5 1 0.37 0.12–1
2 2 1.64 1–2 ≤2 100 2 2 2 2
4 4 4 4 ≤1 0 8 8 5.94 4–8
2 2 1.48 1–2 ≤4 100 >8 >8 10.7 2 to >8
0.25 0.5 0.25 0.12–0.5 ≤1 100 1 >8 1.8 0.12 to >8
2.4/0.12 >76/4 5.6/0.40 1.2/0.06 to >76/4 ≤38/2 71 4.8/0.25 >76/4 11.65/0.60 2.4/0.12 to >76/4
0.12 0.25 0.12 0.06–0.25 ≤0.5 100 >2 >2 0.99 0.12 to >2
0.06 4 0.12 0.03–4 ≤4 100 >16 >16 26 8 to >16
>4 >4 0.37 0.004 to >4 ≤1 43 >4 >4 1.8 0.06 to >4
1 2 1.2 1–2 ≤8 100 2 2 1.8 1–2
ORI, oritavancin; VAN, vancomycin; DAP, daptomycin; LZD, linezolid; Q/D, quinupristin/dalfopristin; SXT, sulfamethoxazole/trimethoprim; TIG, tigecycline; MIN, minocycline; RIF, rifampicin; TEI, teicoplanin; MIC50/90 , MIC for 50% and 90% of the organisms, respectively; %S, percent susceptible; MBC50/90 , MBC for 50% and 90% of the organisms, respectively; N/A, not applicable (oritavancin breakpoints have not been assigned).
as SCCmec type II and 14% as SCCmec type IV. SCCmec type II was most commonly identified among VISA, VRSA and DNSSA strains, but other SCCmec types were also found among these strains. PFGE results showed that the 40 CA-MRSA isolates that typed as SCCmec type IVa were indistinguishable from USA300. Fifteen of the CAMRSA strains that typed as SCCmec type II were related to USA100 and two of the CA-MRSA SCCmec type II strains were related to USA600. The remaining 35 isolates could not be classified. Only the 40 CA-MRSA SCCmec type IVa isolates and the 2 DNSSA SCCmec type IVa isolates were PVL- and ACME-positive. Since the activity of oritavancin against PVL-positive and PVLnegative CA-MRSA was similar, with MIC90 values (MIC for 90% of the strains) of 0.12 mg/L and 0.25 mg/L, respectively, the results were combined (Table 1). The MBC90 (MBC for 90% of the strains) for oritavancin was 0.25 mg/L both for PVL-positive and PVL-negative CA-MRSA. Among CA-MRSA isolates, rifampicin had the lowest MIC90 of all agents tested and oritavancin had the lowest MBC90 . Against the CA-MRSA isolates, oritavancin was eight-fold more active than vancomycin by MIC90 determination and four-fold more active than vancomycin by MBC90 determination. The MICs of the VRSA and VISA isolates were higher for oritavancin compared with oritavancin MICs for the CA-MRSA strains. The relative order of activity for VISA MIC90 values was: tigecycline > minocycline > Q/D > oritavancin, linezolid > daptomycin > vancomycin, teicoplanin > SXT. However, when focusing on MBC results, oritavancin was most active (Table 1). The MIC range for oritavancin against hVISA was 0.12–0.25 mg/L. Among VRSA isolates, the relative order of activity by MIC90 was rifampicin > tigecycline, Q/D > oritavancin, daptomycin > linezolid, minocycline > teicoplanin, vancomycin (Table 1). For the DNSSA isolates, geometric mean MIC and MBC results were determined since there were only seven isolates. For these isolates, the relative order of geometric mean MIC activity was tigecycline, minocycline > oritavancin > Q/D > rifampicin > teicoplanin > linezolid > vancomycin > daptomycin > SXT (Table 2). However, when evaluating bactericidal activity, oritavancin was the most active agent against the DNSSA isolates. 4. Discussion The increase in isolates of S. aureus with resistance to meticillin and decreased susceptibility to vancomycin has created an urgent need for the development of new antistaphylococcal agents. In this study, oritavancin demonstrated potent activity against S. aureus. The agent’s long half-life provides a pharmacokinetic advantage compared with other antistaphylococcal agents. The rapid bactericidal activity against strains of CA-MRSA may also provide an
advantage over available antibiotics used to treat MRSA including vancomycin, daptomycin, linezolid and tigecycline. The in vitro bactericidal activity of oritavancin was also superior against VISA and DNSSA compared with daptomycin, linezolid, vancomycin and tigecycline. In this study, oritavancin demonstrated excellent antistaphylococcal activity even against isolates with decreased susceptibility or resistance to drugs of the general glycopeptide class such as vancomycin and teicoplanin. Oritavancin demonstrated superior activity compared with drugs that may be used in the treatment of CA-MRSA such as vancomycin, daptomycin, linezolid, SXT and tigecycline. This superiority was sustained in the presence of PVL genes and ACME, both of which have been suggested as virulence factors [7]. Although the majority of CA-MRSA isolates in this study were recent isolates identical to USA300, other isolates were included that are now spreading into the community including SCCmec type II, as has been recently described [10]. The excellent activity of oritavancin against CA-MRSA was found among all molecular types evaluated in this study, supporting the rationale for evaluating this agent in clinical trials against current S. aureus infections. Oritavancin demonstrated good activity against VISA and hVISA, although this was less impressive than its activity against CA-MRSA. Based on this level of activity, one would expect oritavancin to perform well against infections caused by VISA and hVISA, however this will need to be evaluated in clinical trials. This study evaluated the in vitro activity against ten VRSA strains. The activities of oritavancin and daptomycin were both impressive. Based on this activity, one could consider oritavancin in the treatment of VRSA infections. However, to date there has been no clinical experience with using oritavancin in the treatment of VRSA infections. The problem of DNSSA emerged early in clinical trials, with 6 of 19 microbiological failures associated with reduced daptomycin susceptibility [11]. Oritavancin demonstrated excellent in vitro activity against these isolates. In previous studies [12,13], MICs of oritavancin were higher, reflecting the absence of polysorbate 80, which prevents binding of the antibiotic to plastic surfaces. Comparison of those results with this study must be done with caution. There have been two clinical trials of complicated skin and skin-structure infections completed and presented at international meetings [14,15]. To date neither study has been published in a peer-review journal. Although non-inferiority was demonstrated with oritavancin as judged against comparators, these trials were performed when MRSA was less prevalent than seen now. Based on the in vitro activity seen in this study, one would expect oritavancin to be a candidate for study against S. aureus seen currently
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with increasing resistance to currently approved antistaphylococcal agents.
[6]
Acknowledgments Parts of this work were presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC)/Infectious Diseases Society of America (IDSA) 46th Annual Meeting, 25–28 October 2008, Washington, DC (poster C1-187). Funding: This study was supported by a grant from Targanta Therapeutics (Cambridge, MA), which is now a wholly owned subsidiary of The Medicines Company (Parsippany, NJ). Competing interests: LDS has served as a consultant to Targanta Therapeutics Corp., a wholly owned subsidiary of The Medicines Company. All other authors declare no competing interests. Ethical approval: Not required. References [1] Poulakou G, Giamarellou H. Oritavancin: a new promising agent in the treatment of infections due to Gram-positive pathogens. Expert Opin Investig Drugs 2008;17:225–43. [2] Moran GJ, Krishnadasan A, Gorwitz RJ, Fosheim GE, McDougal LK, Carey RB, et al. Methicillin-resistant Staphylococcus aureus infections among patients in the emergency department. N Engl J Med 2006;355:666–74. [3] Said-Salim B, Mathema B, Kreiswirth BN. Community-acquired methicillinresistant Staphylococcus aureus: an emerging pathogen. Infect Control Hosp Epidemiol 2003;24:451–5. [4] McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, Tenover FC. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J Clin Microbiol 2003;41:5113–20. [5] Zhang K, McClure JA, Elsayed S, Louie T, Conly JM. Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chro-
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mosome mec types I to V in methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2005;43:5026–33. Lina G, Piémont Y, Godail-Gamot F, Bes M, Peter MO, Gauduchon V, et al. Involvement of Panton–Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 1999;29: 1128–32. Diep BA, Gill SR, Chang RF, Phan TH, Chen JH, Davidson MG, et al. Complete genome sequence of USA300, an epidemic clone of community-acquired methicillin-resistant Staphylococcus aureus. Lancet 2006;367:731–9. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Eighteenth informational supplement. Document M100-S18. Wayne, PA: CLSI; 2008. Clinical and Laboratory Standards Institute. Methods for determining bactericidal activity of antimicrobial agents; approved guidelines. Document M26-A. Wayne, PA: CLSI; 1999. Johnson LB, Jose J, Yousif F, Pawlak J, Saravolatz L. Prevalence of colonization with community-associated methicillin-resistant Staphylococcus aureus among end-stage renal disease patients and healthcare workers. Infect Control Hosp Epidemiol 2009;30:4–8. Fowler Jr VG, Boucher HW, Corey GR, Abrutyn E, Karchmer AW, Rupp ME, et al. Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphylococcus aureus. N Engl J Med 2006;355:653–65. Biavasco F, Vignaroli C, Lupidi R, Manso E, Facinelli B, Varaldo PE. In vitro antibacterial activity of LY333328, a new semisynthetic glycopeptide. Antimicrob Agents Chemother 1997;41:2165–72. Zeckel ML, Preston DA, Allen BS. In vitro activities of LY333328 and comparative agents against nosocomial Gram-positive pathogens collected in a 1997 global surveillance study. Antimicrob Agents Chemother 2000;44:1370–4. Wasilewski M, Disch D, McGill J, Harris H, O’Riordan W, Zeckel ML. Equivalence of shorter course therapy with oritavancin compared to vancomycin/cephalexin in complicated skin/skin structure infections. In: 41st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). ASM Press: Washington, DC; 2001. Poster UL-18. Giamarellou H, O’Riordan W, Harris H, Porter S, Loutit J. Phase III trial comparing 3–7 days of oritavancin versus 10–14 days of vancomycin/cephalexin in the treatment of patients with complicated skin/skin structure infections (CSSI). In: 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). Washington, DC: ASM Press; 2003. Poster L-739.
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
In vitro activity of oritavancin against community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA) and daptomycin-non-susceptible S. aureus (DNSSA) Louis D. Saravolatz ∗ , Joan Pawlak, Leonard B. Johnson St John Hospital and Medical Center and Wayne State University School of Medicine, Detroit, MI, USA
a r t i c l e
i n f o
Article history: Received 9 November 2009 Accepted 26 February 2010 Keywords: Oritavancin Lipoglycopeptide MRSA
a b s t r a c t Isolates of community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA), vancomycinintermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA) and daptomycin non-susceptible S. aureus (DNSSA) are increasing in frequency and new antistaphylococcal therapies are needed. Microdilution testing using Mueller–Hinton broth was used to determine the minimal inhibitory concentrations (MICs) of oritavancin and nine additional antimicrobial agents against 92 CA-MRSA, 23 VISA, 7 DNSSA and 10 VRSA isolates. Minimal bactericidal concentrations were also determined. Pulsed-field gel electrophoresis (PFGE) was performed. Staphylococcal cassette chromosome mec (SCCmec) typing as well as assays for Panton–Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) genes were performed. Oritavancin was more bactericidal than any of the other comparators against CA-MRSA and demonstrated excellent activity against VRSA and VISA. © 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Oritavancin, derived from chloroeremomycin, is a semisynthetic lipoglycopeptide with potent in vitro activity against Grampositive organisms, including meticillin-resistant Staphylococcus aureus (MRSA). The multiple mechanisms of action of oritavancin include inhibition of cell wall synthesis, disruption of the transmembrane potential, and partial inhibition of RNA synthesis [1]. Oritavancin is active against a variety of Gram-positive organisms. It has been shown to perturb the cell membrane to achieve enhanced killing of S. aureus. In addition, it has demonstrated killing of S. aureus both in planktonic and biofilm states. With the significant rise of MRSA isolates in the community and its decreasing susceptibility to vancomycin, there is a need to develop additional antistaphylococcal agents [2]. This study evaluated oritavancin and nine comparator agents against communityassociated MRSA (CA-MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA) and daptomycin non-susceptible S. aureus (DNSSA). To characterise the isolates, staphylococcal cassette chromosome mec (SCCmec) typing as well
∗ Corresponding author. Present address: 19251 Mack Avenue, Suite 333, Grosse Pointe Woods, MI 48236, USA. Tel.: +1 313 343 3362; fax: +1 313 343 7784. E-mail address: [email protected] (L.D. Saravolatz).
as assays for Panton–Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) genes were performed. 2. Methods 2.1. Isolates A collection of 132 MRSA strains was selected for evaluation. CA-MRSA strains (n = 92) were isolated from patients admitted to St John Hospital and Medical Center (Detroit, MI) from July 2003 to May 2007. There were no duplicate isolates from patients. These 92 strains were identified as culture-positive for MRSA within 48 h of admission, in accordance with the definition of CA-MRSA described by the US Centers for Disease Control and Prevention (CDC) [3]. Cultures were performed on samples collected from blood (n = 26), respiratory specimens (n = 33), wound or tissue (n = 32) and catheter tip (n = 1). Patients with culture-positive MRSA after 48 h of hospitalisation were excluded to avoid the inclusion of nosocomial MRSA strains. DNSSA (n = 7) and vancomycin-heteroresistant S. aureus (hVISA) (n = 3) strains were obtained from blood isolates collected from patients at St John Hospital and Medical Center. hVISA isolates were identified using a modified population analysis profile method. VISA (n = 20) and VRSA (n = 10) isolates were obtained through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) programme supported under NIAID/NIH Contract
0924-8579/$ – see front matter © 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2010.02.023
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Table 1 Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) (in mg/L) of oritavancin and comparators.
CA-MRSA (n = 92) MIC50 MIC90 MIC range Breakpoint %S MBC50 MBC90 MBC range VISA (n = 20) MIC50 MIC90 MIC range Breakpoint %S MBC50 MBC90 MBC range VRSA (n = 10) MIC50 MIC90 MIC range Breakpoint %S MBC50 MBC90 MBC range
ORI
VAN
DAP
LZD
Q/D
SXT
TIG
MIN
RIF
TEI
0.06 0.12 0.015–0.25 N/A N/A 0.12 0.25 0.03–0.5
1 1 0.5–2 ≤2 100 1 1 0.5–2
0.5 1 0.25–1 ≤1 100 0.5 1 0.25–2
2 2 1–4 ≤4 100 >8 >8 4 to >8
0.25 0.5 0.12–0.5 ≤1 100 0.5 >8 0.25 to >8
2.4/0.12 9.5/0.5 1.2/0.06 to >76/4 ≤38/2 98 2.4/0.12 19/1 1.2/0.06 to >76/4
0.12 0.25 0.06–0.5 ≤0.5 100 >2 >2 0.5 to >2
0.12 0.5 0.06–8 ≤4 97 >16 >16 0.5 to >16
0.008 0.008 0.004–0.015 ≤1 100 0.5 1 0.015–2
0.5 0.5 0.25–1 ≤8 100 0.5 1 0.25–1
1 2 0.25–2 N/A N/A 1 2 0.5–4
4 8 4–8 ≤2 0 4 8 4–8
2 4 1–8 ≤1 25 2 4 1–8
1 2 0.5–2 ≤4 100 4 >8 2 to >8
0.5 1 0.25–2 ≤1 95 1 >8 0.25 to >8
4.8/0.25 >76/4 1.2/0.06 to >76/4 ≤38/2 75 4.8/0.25 >76/4 2.4/0.12 to >76/4
0.25 0.25 0.06–0.5 ≤0.5 100 0.5 >2 0.12 to >2
0.12 0.5 0.03–2 ≤4 100 8 >16 0.06 to >16
0.015 >4 0.004 to >4 ≤1 55 1 >4 0.03 to >4
4 8 1–16 ≤8 90 4 16 2–32
0.5 1 0.25–1 N/A N/A 1 2 0.5–2
>64 >64 32 to >64 ≤2 0 >64 >64 64 to >64
0.5 1 0.5–1 ≤1 100 1 1 0.5–1
2 2 1–4 ≤4 100 >8 >8 8 to >8
0.5 0.5 0.5 ≤1 100 8 >8 1 to >8
2.4/0.12 38/2 1.2/0.06 to >76/4 ≤38/2 90 2.4/0.12 >76/4 2.4/0.12 to >76/4
0.25 0.5 0.06–1 ≤0.5 90 1 >2 0.25 to >2
0.12 2 0.06–2 ≤4 100 >16 >16 8 to >16
0.008 0.008 0.004 to >4 ≤1 90 0.12 1 0.008 to >4
32 >32 8 to >32 ≤8 20 >32 >32 8 to >32
ORI, oritavancin; VAN, vancomycin; DAP, daptomycin; LZD, linezolid; Q/D, quinupristin/dalfopristin; SXT, sulfamethoxazole/trimethoprim; TIG, tigecycline; MIN, minocycline; RIF, rifampicin; TEI, teicoplanin; CA-MRSA, community-associated meticillin-resistant Staphylococcus aureus; VISA, vancomycin-intermediate S. aureus; VRSA, vancomycinresistant S. aureus; MIC50/90 , MIC for 50% and 90% of the organisms, respectively; %S, percent susceptible; MBC50/90 , MBC for 50% and 90% of the organisms, respectively; N/A, not applicable (oritavancin breakpoints have not been assigned).
Pulsed-field gel electrophoresis (PFGE) was performed on the CA-MRSA isolates using restriction enzyme SmaI [4]. PFGE patterns were visually compared with S. aureus USA100 to USA800 (NARSA, Herndon VA.) and categorised. Strains were assigned to a specific pulsed field group only when all the bands matched. A multiplex polymerase chain reaction (PCR) procedure was used to determine the SCCmec type of all the isolates [5]. PVL genes lukS-PV and lukF-PV were identified by PCR [6]. The presence of the arginine catabolic mobile element (ACME) was determined by PCR detection of the arcA locus [7]. PCR testing was performed on all isolates. DNA used for the PCR assays was extracted from isolates using a Qiagen QIAamp DNA Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s directions. Controls used for PCR were S. aureus ATCC BAA-44, ATCC BAA-41, ATCC BAA-39, NRS-384, ATCC49775, NCTC-8325 and ATCC12228.
Therapeutics Corp. (Cambridge, MA). Plates were stored at −70 ◦ C and used within 3 months of preparation. Oritavancin was tested in the presence of polysorbate 80 at a concentration of 0.002% to prevent binding of the antibiotic to plastic surfaces. For testing of daptomycin, Mueller–Hinton broth was supplemented with calcium to a final concentration of 50 mg/L. Microtitre plates were inoculated with 105 colony-forming units/mL and were incubated in air at 35 ◦ C for 18–24 h. Minimal inhibitory concentration (MIC) values were determined in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [8]. MICs were read visually as the lowest drug concentration well with no visible bacterial growth. Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were used to monitor quality control for all the antibiotics with each run. Quality control results were in the acceptable range for both organisms and all antibiotics. Minimal bactericidal concentration (MBC) values of all 10 antibiotics for all the isolates were also determined according to CLSI guidelines [9]. Ten microlitres from each well showing no visible growth were subcultured onto a blood agar plate (Remel Inc., Lenexa, KS) and incubated in air at 35 ◦ C from 24 h to 48 h. The MBC was defined as the lowest concentration of antimicrobial agent that reduced the initial inoculum by 99.9% based on colony counts from the growth control well.
2.3. Antimicrobials and in vitro susceptibility testing
3. Results
In vitro susceptibility testing was performed with the following antimicrobials: oritavancin (0.004–8 mg/L); linezolid (0.25–8 mg/L); tigecycline (0.015–2 mg/L); vancomycin (0.12–32 mg/L); quinupristin/dalfopristin (Q/D) (0.06–8 mg/L); daptomycin (0.06–8 mg/L); sulfamethoxazole/trimethoprim (SXT) (0.6/0.03–76/4 mg/L); teicoplanin (0.06–32 mg/L); minocycline (0.3–16 mg/L); and rifampicin (0.02–4 mg/L). Microtitre dilution plates containing cation-adjusted Mueller–Hinton broth and the antimicrobials were prepared and supplied by Targanta
SCCmec types observed for the 92 CA-MRSA strains were IVa (40), II (38), IV (13) and not typeable (1). For the 20 VISA isolates, the SCCmec types found were II (18), IVd (1) and I (1). All three hVISA strains typed as SCCmec type II. VRSA SCCmec types were II (6), IV (2) and not typeable (2). DNSSA SCCmec types were II (4), IVa (2) and III (1). The 13 CA-MRSA and 2 VRSA isolates that typed as SCCmec type IV could not be classified into the SCCmec type IV subgroups a, b, c or d. Although SCCmec type IVa was the most commonly found among CA-MRSA isolates (43%), there were still 41% of isolates identified
#HHSN272200700055C. VISA isolates were cultured from blood (n = 12), wound (n = 3) and peritoneal fluid, bone, cerebrospinal fluid, bile and sputum (1 each). VRSA isolates were cultured from wound (n = 6), catheter site (n = 1), urine (n = 1), nephrostomy tube (n = 1) and unknown (n = 1). 2.2. Molecular typing
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Table 2 Geometric mean minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) (in mg/L) of oritavancin and comparators against daptomycin non-susceptible Staphylococcus aureus (n = 7).
MIC50 MIC90 Geometric mean MIC MIC range Breakpoint %S MBC50 MBC90 Geometric mean MBC MBC range
ORI
VAN
DAP
LZD
Q/D
SXT
TIG
MIN
RIF
TEI
0.25 0.5 0.2 0.06–0.5 N/A N/A 0.5 1 0.37 0.12–1
2 2 1.64 1–2 ≤2 100 2 2 2 2
4 4 4 4 ≤1 0 8 8 5.94 4–8
2 2 1.48 1–2 ≤4 100 >8 >8 10.7 2 to >8
0.25 0.5 0.25 0.12–0.5 ≤1 100 1 >8 1.8 0.12 to >8
2.4/0.12 >76/4 5.6/0.40 1.2/0.06 to >76/4 ≤38/2 71 4.8/0.25 >76/4 11.65/0.60 2.4/0.12 to >76/4
0.12 0.25 0.12 0.06–0.25 ≤0.5 100 >2 >2 0.99 0.12 to >2
0.06 4 0.12 0.03–4 ≤4 100 >16 >16 26 8 to >16
>4 >4 0.37 0.004 to >4 ≤1 43 >4 >4 1.8 0.06 to >4
1 2 1.2 1–2 ≤8 100 2 2 1.8 1–2
ORI, oritavancin; VAN, vancomycin; DAP, daptomycin; LZD, linezolid; Q/D, quinupristin/dalfopristin; SXT, sulfamethoxazole/trimethoprim; TIG, tigecycline; MIN, minocycline; RIF, rifampicin; TEI, teicoplanin; MIC50/90 , MIC for 50% and 90% of the organisms, respectively; %S, percent susceptible; MBC50/90 , MBC for 50% and 90% of the organisms, respectively; N/A, not applicable (oritavancin breakpoints have not been assigned).
as SCCmec type II and 14% as SCCmec type IV. SCCmec type II was most commonly identified among VISA, VRSA and DNSSA strains, but other SCCmec types were also found among these strains. PFGE results showed that the 40 CA-MRSA isolates that typed as SCCmec type IVa were indistinguishable from USA300. Fifteen of the CAMRSA strains that typed as SCCmec type II were related to USA100 and two of the CA-MRSA SCCmec type II strains were related to USA600. The remaining 35 isolates could not be classified. Only the 40 CA-MRSA SCCmec type IVa isolates and the 2 DNSSA SCCmec type IVa isolates were PVL- and ACME-positive. Since the activity of oritavancin against PVL-positive and PVLnegative CA-MRSA was similar, with MIC90 values (MIC for 90% of the strains) of 0.12 mg/L and 0.25 mg/L, respectively, the results were combined (Table 1). The MBC90 (MBC for 90% of the strains) for oritavancin was 0.25 mg/L both for PVL-positive and PVL-negative CA-MRSA. Among CA-MRSA isolates, rifampicin had the lowest MIC90 of all agents tested and oritavancin had the lowest MBC90 . Against the CA-MRSA isolates, oritavancin was eight-fold more active than vancomycin by MIC90 determination and four-fold more active than vancomycin by MBC90 determination. The MICs of the VRSA and VISA isolates were higher for oritavancin compared with oritavancin MICs for the CA-MRSA strains. The relative order of activity for VISA MIC90 values was: tigecycline > minocycline > Q/D > oritavancin, linezolid > daptomycin > vancomycin, teicoplanin > SXT. However, when focusing on MBC results, oritavancin was most active (Table 1). The MIC range for oritavancin against hVISA was 0.12–0.25 mg/L. Among VRSA isolates, the relative order of activity by MIC90 was rifampicin > tigecycline, Q/D > oritavancin, daptomycin > linezolid, minocycline > teicoplanin, vancomycin (Table 1). For the DNSSA isolates, geometric mean MIC and MBC results were determined since there were only seven isolates. For these isolates, the relative order of geometric mean MIC activity was tigecycline, minocycline > oritavancin > Q/D > rifampicin > teicoplanin > linezolid > vancomycin > daptomycin > SXT (Table 2). However, when evaluating bactericidal activity, oritavancin was the most active agent against the DNSSA isolates. 4. Discussion The increase in isolates of S. aureus with resistance to meticillin and decreased susceptibility to vancomycin has created an urgent need for the development of new antistaphylococcal agents. In this study, oritavancin demonstrated potent activity against S. aureus. The agent’s long half-life provides a pharmacokinetic advantage compared with other antistaphylococcal agents. The rapid bactericidal activity against strains of CA-MRSA may also provide an
advantage over available antibiotics used to treat MRSA including vancomycin, daptomycin, linezolid and tigecycline. The in vitro bactericidal activity of oritavancin was also superior against VISA and DNSSA compared with daptomycin, linezolid, vancomycin and tigecycline. In this study, oritavancin demonstrated excellent antistaphylococcal activity even against isolates with decreased susceptibility or resistance to drugs of the general glycopeptide class such as vancomycin and teicoplanin. Oritavancin demonstrated superior activity compared with drugs that may be used in the treatment of CA-MRSA such as vancomycin, daptomycin, linezolid, SXT and tigecycline. This superiority was sustained in the presence of PVL genes and ACME, both of which have been suggested as virulence factors [7]. Although the majority of CA-MRSA isolates in this study were recent isolates identical to USA300, other isolates were included that are now spreading into the community including SCCmec type II, as has been recently described [10]. The excellent activity of oritavancin against CA-MRSA was found among all molecular types evaluated in this study, supporting the rationale for evaluating this agent in clinical trials against current S. aureus infections. Oritavancin demonstrated good activity against VISA and hVISA, although this was less impressive than its activity against CA-MRSA. Based on this level of activity, one would expect oritavancin to perform well against infections caused by VISA and hVISA, however this will need to be evaluated in clinical trials. This study evaluated the in vitro activity against ten VRSA strains. The activities of oritavancin and daptomycin were both impressive. Based on this activity, one could consider oritavancin in the treatment of VRSA infections. However, to date there has been no clinical experience with using oritavancin in the treatment of VRSA infections. The problem of DNSSA emerged early in clinical trials, with 6 of 19 microbiological failures associated with reduced daptomycin susceptibility [11]. Oritavancin demonstrated excellent in vitro activity against these isolates. In previous studies [12,13], MICs of oritavancin were higher, reflecting the absence of polysorbate 80, which prevents binding of the antibiotic to plastic surfaces. Comparison of those results with this study must be done with caution. There have been two clinical trials of complicated skin and skin-structure infections completed and presented at international meetings [14,15]. To date neither study has been published in a peer-review journal. Although non-inferiority was demonstrated with oritavancin as judged against comparators, these trials were performed when MRSA was less prevalent than seen now. Based on the in vitro activity seen in this study, one would expect oritavancin to be a candidate for study against S. aureus seen currently
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with increasing resistance to currently approved antistaphylococcal agents.
[6]
Acknowledgments Parts of this work were presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC)/Infectious Diseases Society of America (IDSA) 46th Annual Meeting, 25–28 October 2008, Washington, DC (poster C1-187). Funding: This study was supported by a grant from Targanta Therapeutics (Cambridge, MA), which is now a wholly owned subsidiary of The Medicines Company (Parsippany, NJ). Competing interests: LDS has served as a consultant to Targanta Therapeutics Corp., a wholly owned subsidiary of The Medicines Company. All other authors declare no competing interests. Ethical approval: Not required. References [1] Poulakou G, Giamarellou H. Oritavancin: a new promising agent in the treatment of infections due to Gram-positive pathogens. Expert Opin Investig Drugs 2008;17:225–43. [2] Moran GJ, Krishnadasan A, Gorwitz RJ, Fosheim GE, McDougal LK, Carey RB, et al. Methicillin-resistant Staphylococcus aureus infections among patients in the emergency department. N Engl J Med 2006;355:666–74. [3] Said-Salim B, Mathema B, Kreiswirth BN. Community-acquired methicillinresistant Staphylococcus aureus: an emerging pathogen. Infect Control Hosp Epidemiol 2003;24:451–5. [4] McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, Tenover FC. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J Clin Microbiol 2003;41:5113–20. [5] Zhang K, McClure JA, Elsayed S, Louie T, Conly JM. Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chro-
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