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Synergy time-kill studies of 47 methicillin-resistant Staphylococcus aureus strains with differing resistance phenotypes showed that combinations of subinhibitory concentrations of ACHN-490 and daptomycin yielded synergy against 43/47 strains at 24 h, while the combination was indifferent against the remaining 4 strains. ACHN-490 and ceftobiprole showed synergy in 17/47 strains tested at 24 h, while 6/47 strains showed synergy for subinhibitory combinations of ACHN-490 and linezolid.
Methicillin-resistant Staphylococcus aureus (MRSA) strains resistant to multiple classes of antibiotics are increasingly encountered in clinical settings (1, 9, 11, 13, 14, 16) and cannot be treated with conventional β-lactams. Most methicillin-resistant strains are resistant to all available quinolones, and vancomycin-hetero-intermediate (hVISA), -intermediate (VISA), and -resistant (VRSA) S. aureus strains have appeared (7, 26). While most multidrug-resistant S. aureus strains are hospital acquired, there has been a worrisome increase during the past few years in the incidence of community-acquired MRSA. Such strains are currently more susceptible to most antibiotics but typically are more virulent than hospital strains (10, 14, 15, 20, 21, 24, 25, 27). Development of S. aureus strains with diminished susceptibility to vancomycin can be attributed at least partially to selective pressure caused by vancomycin use in the community. The increase in the number of infections due to community-acquired MRSA will likely lead to more glycopeptide use in the community setting, thereby increasing the selective pressure for vancomycin resistance. Therefore, an alternative to vancomycin is urgently needed.
ACHN-490 (Fig. (Fig.1)1) is a neoglycoside, a next-generation aminoglycoside, currently in clinical development. ACHN-490 is not affected by most aminoglycoside-modifying enzymes and is highly active against both Gram-negative rods and MRSA (MIC90, 2 μg/ml)(2, 22). While ACHN-490 is currently under development for the treatment of complicated urinary tract infections and acute pyelonephritis as a single agent, use of combination therapy for serious nosocomial infections is becoming the standard of care (3, 23). This prompted us to examine the activity of ACHN-490 activity with other Gram-positive agents against a spectrum of MRSA strains.
In vitro methodology for the detection of synergy between two antibacterials has not been standardized. Although checkerboard analyses have been used extensively in the past, we feel that they are neither as sensitive nor as discriminatory as time-kill analysis to accurately detect synergy (5). In the present study, we examined the synergistic activities of ACHN-490 with daptomycin, ceftobiprole, or linezolid against 47 MRSA strains with various resistotypes by using time-kill methodology.
The strains studied were recent isolates and comprised 2 hVISA isolates (Hershey isolates, screened by the Etest macromethod and confirmed by population analyses), 20 VISA isolates (4 Hershey Medical Center isolates and 16 from the Network on Antimicrobial Resistance in S. aureus [NARSA]), 5 VRSA (3 Michigan isolates, 1 Hershey isolate, and 1 New York City isolate), 10 community-acquired MRSA isolates (9 Panton-Valentine leukocidin [PVL] positive and 1 PVL negative), and 10 hospital-acquired MRSA (including S. aureus ATCC 33591) with differing resistance phenotypes.
ACHN-490 and ceftobiprole susceptibility powders were manufactured by Achaogen, Inc., South San Francisco, CA; daptomycin was obtained from Cubist Pharmaceuticals, Lexington, MA; linezolid was from Pfizer, Inc., New York, NY. Initial drug MICs were validated according to CLSI recommendations (6). MICs were determined by a modification of the broth macrodilution method using 24 h of incubation. Kill kinetics of each drug alone were determined by incubating an initial inoculum of 5 × 105 to 5 × 106 CFU/ml with drug concentrations at the MIC, two dilutions above (2× and 4× the MIC), and three dilutions below the MIC (1/2, 1/4, and 1/8× the MIC) (5). All daptomycin testing was performed in Mueller-Hinton broth with added calcium.
Viability counts were performed at 0, 3, 6, 12, and 24 h of incubation at 35°C in a shaking water bath by plating undiluted and 10-fold serial diluted samples onto trypticase soy with 5% sheep blood agar plates (Becton Dickinson, Inc., Sparks, MD). ACHN-490 was combined with each of the three antimicrobial agents listed above and tested at one and two dilutions below the MIC of each drug. Concentrations in synergy time-kill tests were selected such that one of the two drugs would yield a growth curve similar to that of the drug-free control while the other drug would be more active (5).
We describe our findings here by using the following definitions: (i) synergy, ≥2-log10 decrease in CFU/ml between the combination and its most active constituent after 3, 6, 12, and 24 h, with the number of surviving organisms in the presence of the combination at ≥2 log10 CFU/ml below the starting inoculum; (ii) indifference, between a 2-log10 CFU/ml decrease and a 2-log10 CFU/ml increase in counts between the combination and its most active constituent; (iii) antagonism, a ≥2-log10 CFU/ml increase in counts between the combination and its most active constituent.
Results of antimicrobial susceptibility (6) at 24 h by strain phenotype are presented in Table Table1.1. MIC ranges for all strains tested were as follows: ACHN-490, 0.5 to 8.0 μg/ml; daptomycin, 0.25 to 4.0 μg/ml; ceftobiprole, 0.5 to 4.0 μg/ml; linezolid, 1.0 to 4.0 μg/ml.
Synergy time-kill data obtained with each of the 47 isolates are listed in Table S1 of the supplemental material. When ACHN-490 was combined with daptomycin, synergy was seen at sub-MIC levels of ACHN-490 in 23/47 strains at 3 h, 28/47 at 6 h, 37/47 at 12 h, and 43/47 at 24 h. Of the four strains not showing synergy between ACHN-490 and daptomycin at 24 h, two strains showed synergy at 3 h and 12 h, one showed synergy at 6 h and 12 h, and one strain did not show synergy at any time period tested. Indifference (as defined above) was seen in 24/47 strains at 3 h, 19/47 at 6 h, 10/47 at 12 h, and 4/47 at 24 h, and there were no strains showing antagonism at any of the time points.
The combination of ACHN-490 with ceftobiprole led to synergy at sub-MIC levels of ACHN-490 in 2/47, 7/47, 12/47, and 17/47 strains at 3 h, 6 h, 12 h, and 24 h, respectively. Indifference was observed in 45/47 strains at 3 h, 38/47 at 6 h, 34/47 at 12 h, and 30/47 at 24 h, while antagonism was seen in 0/47 strains at 3 h, 2/47 strains at 6 h, 1/47 at 12 h, and 0/47 at 24 h.
When ACHN-490 was combined with linezolid, synergy was seen at sub-MIC levels of ACHN-490 in 0/47 strains at 3 h and 6 h, 1/47 after 12 h, and 6/47 after 24 h. Indifference was observed in 34/47 strains at 3 h, 20/47 at 6 h, 24/47 at 12 h, and 39/47 at 24 h. However, by contrast with both daptomycin and ceftobiprole, for the ACHN-490 and linezolid combinations, antagonism was observed in 13/47 strains at 3 h, 27/47 strains at 6 h, 22/47 strains at 12 h, and 2/47 strains at 24 h (17). We note that synergy results are typically reported after 24 h and that interpretation of observations at earlier time points is controversial. After 24 h, most strains demonstrated indifference to the ACHN-490/linezolid combination, which supports the potential clinical utility of this combination even in the absence of observed synergy. We did not conduct further investigations into the mechanism of antagonism observed after 24 h in the 2 of the 47 strains tested.
Table Table22 shows a summary of the above results, with the number of strains demonstrating synergy, indifference, and antagonism with each combination at the various time points. The combination of ACHN-490 and daptomycin had the highest number of strains showing synergy at 24 h, followed by ACHN-490 and ceftobiprole, while ACHN-490 and linezolid had the least number of strains showing synergy at 24 h. Figure Figure22 a, b, and c depicts the percentage of organisms against which the antibiotic combinations showed synergistic killing as a function of time. Figure Figure33 is a graphic depiction of synergy observed when ACHN-490 was combined with daptomycin in subinhibitory concentrations and tested against a VISA strain. As can be seen, combination with ACHN 490 led to the daptomycin MIC dropping from nonsusceptible (2 μg/ml) to susceptible (0.5 μg/ml) in the synergistic combination tested. This strain was chosen because daptomycin is not uniformly potent against VISA strains (8, 19) and because combination with ACHN-490 in this strain led to synergy, but this time with achievable concentrations of daptomycin in the combination.
ACHN-490 is active against a broad range of highly resistant Enterobacteriaceae, including those carrying multiple resistance factors, such as extended-spectrum β-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPCs) (12, 18). Unlike marketed aminoglycosides, ACHN-490 is active against strains possessing a wide range of the most clinically relevant aminoglycoside-modifying enzymes (AMEs) (2). ACHN-490 is equipotent to gentamicin against the most common strains of Enterobacteriaceae and staphylococci (S. aureus and coagulase-negative staphylococci) while it demonstrates activity similar to amikacin against Proteus mirabilis and the indole-positive Proteae. Potency is also similar to amikacin on aminoglycoside-susceptible strains of Pseudomonas aeruginosa and Acinetobacter baumannii. The activity in vitro was confirmed in animal studies to define the efficacy and pharmacokinetics (4, 22).
The clinical significance of in vitro synergy by time-kill analysis remains to be confirmed clinically. We feel that, in the absence of new agents, synergy with two well-chosen agents represents the only clinical alternative until new broad-spectrum agents are introduced. In the current study, subinhibitory concentrations of ACHN-490 and daptomycin showed very clear synergy in 46 of the 47 strains tested at time points between 3 and 24 h, irrespective of the resistance phenotype. Daptomycin has been described to select for resistant strains during therapy for systemic infections and is also not active in vitro, with MICs of >1 μg/ml against most VISA strains (although it is uniformly potent against VRSA) (7, 17). The combination between ACHN-490 and daptomycin would seem to be able to greatly improve the spectrum and therapeutic efficacy of daptomycin by removing both of the latter disadvantages. Clinical studies are required to test the validity of this hypothesis.
This study was supported by a grant from Achaogen, Inc., South San Francisco, CA.
Published ahead of print on 9 February 2010.
†Supplemental material for this article may be found at http://aac.asm.org/.
‡The authors have paid a fee to allow immediate free access to this article.