Michael T. Sweeney* and Gary E. Zurenko. Infectious Diseases Biology, Pharmacia Corporation, Kalamazoo, Michigan 49007

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2003, p Vol. 47, No /03/$ DOI: /AAC Copyright 2003, American Society for Microbiology. All Rights Reserved. In Vitro Activities of Linezolid Combined with Other Antimicrobial Agents against Staphylococci, Enterococci, Pneumococci, and Selected Gram-Negative Organisms Michael T. Sweeney* and Gary E. Zurenko Infectious Diseases Biology, Pharmacia Corporation, Kalamazoo, Michigan Received 25 November 2002/Returned for modification 27 January 2003/Accepted 26 February 2003 The activities of linezolid, an oxazolidinone antibacterial agent active against gram-positive organisms, alone and in combination with 35 antimicrobial agents were tested in vitro against methicillin-sensitive (n 1 to 2 strains) and methicillin-resistant (n 8to10)Staphylococcus aureus strains; vancomycin-sensitive (n 6) and vancomycin-resistant (n 6to8)Enterococcus faecalis strains; vancomycin-sensitive (n 5) and vancomycin-resistant (n 6) Enterococcus faecium strains; penicillin-sensitive (n 2 to 5), penicillin-intermediate (n 5 to 6), and penicillin-resistant (n 5to6)Streptococcus pneumoniae strains; Escherichia coli (n 6); and Klebsiella pneumoniae (n 6). The fractional inhibitory concentration indices of linezolid in combination with other antimicrobial agents for the organisms tested were generated on checkerboard broth microdilution plates prepared by a semiautomated method. Of 1,380 organism-drug combinations, 1,369 (99.2%) combinations of linezolid with 28 antimicrobial drugs were indifferent, 9 combinations (0.65%) of linezolid with 6 drugs (amoxicillin, erythromycin, imipenem, sparfloxacin, teicoplanin, and tetracycline) were synergistic, and 2 combinations (0.15%) of linezolid with 2 drugs (ofloxacin and sparfloxacin) were antagonistic. Overall, the in vitro data demonstrated that linezolid combined with other antimicrobial agents primarily produces an indifferent response, with infrequent occurrences of synergism and antagonism. Antimicrobial combination therapy may be used to ensure coverage against all pathogens in a potentially mixed infection, or specific combinations of agents known to have synergistic interactions may be used in settings in which the pathogens are known. Antimicrobial combinations are considered to be synergistic if the effect of the combination is greater than the effect of either agent alone or greater than the sum of the effects of the individual agents. Antagonism results if the combination provides an effect less than the effect of either agent alone or less than the sum of the effects of the individual agents. Indifference results if the combination provides an effect equal to the effect of either agent alone. A common laboratory method used to determine synergism, antagonism, and indifference uses fractional inhibitory concentrations (FICs) and FIC indices. The checkerboard technique has been one of the traditional methods used to determine FIC indices (10). Linezolid is an antimicrobial agent from the oxazolidinone class and is especially active against multidrug-resistant grampositive organisms such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis and Enterococcus faecium, and penicillin-resistant Streptococcus pneumoniae. Linezolid has also been shown to have activity against certain gram-negative and anaerobic bacteria (7, 11, 13, 14, 19, 23). This drug was tested in vitro alone and in combination with 35 antimicrobial agents against multiple bacterial species by using checkerboard broth microdilution plates prepared by a semiautomated method. FIC indices were generated and analyzed for synergism, antagonism, and indifference. * Corresponding author. Mailing address: Pharmacia Corporation, 301 Henrietta St., , Kalamazoo, MI Phone: (269) Fax: (269) michael.t.sweeney@pharmacia.com. The objective of this study was to determine the effects of the interaction of linezolid when it was combined with other antimicrobial agents and tested against multiple strains of drug-sensitive and -resistant S. aureus, E. faecalis, E. faecium, and S. pneumoniae. Selected strains of Escherichia coli and Klebsiella pneumoniae were tested as well. (This study was presented in part at the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., 26 to 29 September 1999; the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 17 to 30 September 2000; and the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 16 to 19 December 2001.) MATERIALS AND METHODS Bacterial strains. Bacterial isolates from human infections were used in the present study. The isolates were maintained frozen in the vapor phase of a liquid nitrogen freezer and plated on Trypticase soy agar plates supplemented with 5% sheep blood. The quality control strains S. aureus ATCC (UC 9218), E. faecalis ATCC (UC 9217), and S. pneumoniae ATCC 6305 (UC 9912) were originally acquired from the American Type Culture Collection (Manassas, Va.). Antimicrobial agents. The following antibiotic powders were used in the experiments and were obtained from the indicated sources: amoxicillin, ampicillin, bacitracin, cefoxitin, erythromycin, fusidic acid, gentamicin, methicillin, metronidazole, nalidixic acid, norfloxacin, novobiocin, ofloxacin, rifampin, tetracycline, and vancomycin, Sigma Chemical Company (St. Louis, Mo.); ceftazidime, cephalothin, chloramphenicol, clindamycin, and oxacillin, U.S. Pharmacopeia (Rockville, Md.); ampicillin-sulbactam and trovafloxacin, Pfizer (New York, N.Y.); amoxicillin-clavulanic acid, GlaxoSmithKline (Research Triangle Park, N.C.); cefdinir and sparfloxacin, Parke-Davis (Ann Arbor, Mich.); aztreonam and gatifloxacin, Bristol-Myers Squibb (Princeton, N.J.); cefotaxime, Calbiochem (La Jolla, Calif.); imipenem, Merck (Whitehouse Station, N.J.); ciprofloxacin, Miles (Kankakee, Ill.); teicoplanin, Aventis Pharmaceuticals (Bridgewater, N.J.); and cefpodoxime, linezolid, and neomycin, Pharmacia Corporation (Kalamazoo, Mich.). 1902

2 VOL. 47, 2003 IN VITRO ACTIVITIES OF LINEZOLID AND OTHER AGENTS 1903 TABLE 1. MICs of the antibacterial agents used in combination for gram-positive organisms MIC ( g/ml) Drug class and drug S. aureus (n 10 12) a E. faecalis (n 12 14) b E. faecium (n 11) c S. pneumoniae (n 12 17) d Range 50% 90% Range 50% 90% Range 50% 90% Range 50% 90% Amoxicillin e Amoxicillin-clavulanic acid 0.25/ /2 0.5/0.25 4/2 Ampicillin Ampicillin-sulbactam 0.5/0.25 4/2 2/1 4/2 Methicillin Oxacillin Cefdinir Cefotaxime Cefoxitin Cefpodoxime Ceftazidime Cephalothin Ciprofloxacin Difloxacin Gatifloxacin Nalidixic acid Norfloxacin Ofloxacin Sparfloxacin Trovafloxacin Linezolid Bacitracin Chloramphenicol Clindamycin Erythromycin Gentamicin Imipenem Rifampin Teicoplanin Tetracycline Vancomycin a One to 2 methicillin-sensitive isolates and 8 to 10 methicillin-resistant isolates. b Six vancomycin-sensitive isolates and six to eight vancomycin-resistant isolates. c Five vancomycin-sensitive isolates and six vancomycin-resistant isolates. d Two to five penicillin-sensitive isolates, five to six penicillin-intermediate isolates, and five to six penicillin-resistant isolates. e, not determined. Susceptibility and FIC testing. Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) was used for susceptibility testing of S. aureus, enterococci, and enteric bacteria; Mueller-Hinton broth supplemented with 2% lysed horse blood was used for susceptibility testing of S. pneumoniae. The MICs of each drug were determined by broth microdilution according to the standards of the National Committee for Clinical Laboratory Standards (17). Robotics were used to create and inoculate microdilution checkerboard plates. For each linezolid-drug combination tested, a 96-well deep-well plate (Beckman- Coulter, Fullerton, Calif.) was filled with Mueller-Hinton broth containing 1% Alamar Blue (Trek Diagnostics, Westlake, Ohio) by the Multidrop instrument (Labsystems, Helsinki, Finland). Alamar Blue is a colorimetric redox indicator used as an aid for the visual reading of checkerboard plates (package insert; Trek Diagnostics) and had no effect on organism growth (3). Test drugs were diluted twofold from column 1 to column 7 of each deep-well mother plate by the Biomek 2000 instrument (Beckman-Coulter). Column 8 contained no test drug. Twofold dilutions of linezolid were then added to rows 1 to 7 of each deep-well plate containing a specific test drug. Row 8 contained no linezolid. A total of 25 l was transferred by the Multimek 96 instrument (Beckman-Coulter) from each well of each mother plate to a daughter plate containing 165 l of medium. Twelve daughter plates could be created from each mother plate. The Biomek instrument was used to inoculate the daughter plates with 10 l of a 1:10-diluted culture equal to a 0.5 McFarland standard (approximately CFU/ml) from bacterial growth on 18- to 24-h-old blood agar plates, for a final organism concentration of approximately CFU/ml. The plates were incubated at 35 C for 18 2h. The plates were read visually by observing a reduction (a color change from blue to purple or pink, indicating organism growth) or no reduction [a blue color, indicating no growth of organism due to inhibition by a drug(s)]. The MIC of each drug was determined. For wells along the growth-no growth interface, FICs were determined by the following formula: (MIC of linezolid in combination/ MIC of linezolid alone) (MIC of drug x in combination/mic of drug x alone), where x is any of the drugs used in combination with linezolid. The average FIC index was calculated from individual FICs by the formula (FIC 1 FIC 2... FIC n )/n, where n is the total number of individual wells per plate for which FICs were calculated. Synergism was defined as an average FIC index 0.5, antagonism was defined as an average FIC index 4.0, and indifference was defined as an average FIC index from 0.5 to 4.0 (2, 10). Assay reproducibility for synergism was monitored by using a combination of amoxicillin and clavulanic acid with a specific beta-lactamase-positive S. aureus strain (10). Assay reproducibility for antagonism was monitored by using a combination of chloramphenicol and sparfloxacin with a specific vancomycinresistant E. faecalis strain (18). RESULTS Susceptibility results. MIC ranges, the MICs at which 50% of isolates are inhibited (MIC 50 s), and the MIC 90 s of linezolid and drugs used alone and in combination with linezolid for

3 1904 SWEENEY AND ZURENKO ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Ranges of MICs of antimicrobial agents used in combination for gram-negative organisms Drug class and drug E. coli (n 6) MIC range ( g/ml) K. pneumoniae (n 6) Methicillin Cefdinir Cefotaxime Cefoxitin Ceftazidime Cephalothin Ciprofloxacin Difloxacin Nalidixic acid Norfloxacin Ofloxacin Sparfloxacin Trovafloxacin Linezolid 8 8 Aztreonam Bacitracin Fusidic acid Gentamicin Metronidazole Neomycin Novobiocin Teicoplanin Tetracycline gram-positive organisms are shown in Table 1. The ranges of MICs of linezolid for the gram-positive organisms included in the study were 0.25 to 4 g/ml for S. aureus, 2to4 g/ml for E. faecalis, 1to4 g/ml for E. faecium, and 0.25 to 2 g/ml for S. pneumoniae. The ranges of MICs of linezolid and the drugs used alone and in combination with linezolid for gram-negative organisms are shown in Table 2. The MICs of linezolid for E. coli and K. pneumoniae were 8 g/ml. FICs. The activities of linezolid and the other antimicrobial agents from the in vitro checkerboard interactions against the gram-positive organisms are summarized in Table 3. FIC results are only for those strains for which FICs could be calculated from on-scale results. Minimum and maximum FICs and interpretations for the activities of linezolid in combination with 26 of 28 antimicrobials against methicillin-resistant and -sensitive S. aureus strains predominantly showed indifference. Linezolid combined with two drugs produced four determinations of synergism; linezolid plus amoxicillin was synergistic against three methicillin-resistant S. aureus strains (FIC index range for synergism, 0.37 to 0.49), and linezolid plus imipenem was synergistic against one methicillin-sensitive S. aureus strain (FIC index, 0.4). No antagonism was determined. FIC index interpretations for the activities of linezolid in combination with 19 of 22 antimicrobials against vancomycinresistant and -sensitive E. faecalis strains predominantly showed indifference. Linezolid plus teicoplanin was synergistic against a vancomycin-sensitive E. faecalis strain (FIC index, 0.46). Linezolid plus ofloxacin was antagonistic against a vancomycinsensitive E. faecalis strain (FIC index, 4.1), and linezolid plus sparfloxacin was antagonistic against a vancomycin-resistant E. faecalis strain (FIC index, 4.4). FIC index interpretations for the activities of linezolid in combination with 19 of 21 antimicrobials against vancomycinresistant and -sensitive E. faecium strains predominantly showed indifference. Linezolid plus imipenem was synergistic against a vancomycin-resistant E. faecium strain (FIC index, 0.49), and linezolid plus tetracycline was synergistic against a vancomycin-resistant E. faecium strain (FIC index, 0.5). FIC interpretations for the activities of linezolid in combination with 21 of 22 antimicrobials against penicillin-resistant, -intermediate, and -sensitive S. pneumoniae strains predominantly showed indifference. Linezolid plus erythromycin was synergistic against a penicillin-intermediate S. pneumoniae strain (FIC index, 0.48). No antagonism was determined. The activities of linezolid and the other antimicrobial agents from the in vitro checkerboard interactions against E. coli and K. pneumoniae are also summarized in Table 3. Minimum and maximum FIC indices and interpretations for linezolid in combination with 21 of 22 antimicrobials predominantly showed indifference. Linezolid plus sparfloxacin was synergistic against a K. pneumoniae strain (FIC index, 0.46). No antagonism was determined. DISCUSSION Linezolid is a new antimicrobial agent belonging to the oxazolidinone class which is primarily active against gram-positive pathogens. Many studies have found instances of improved efficacies of certain antibiotics when they are combined with antibiotics of other classes. Since there is clinical interest in the use of combinations of antimicrobial agents to improve the spectrum of drug activity, studies with linezolid combined with other antimicrobial agents were performed to determine if synergism, antagonism, or indifference would be the predominant response when the combinations were tested against gram-positive and gram-negative isolates. To date, there have been few articles related to studies of the activities of linezolid in combination with other drugs. Di Pentima et al. (5) reported results on the in vitro activities of linezolid in combination with rifampin, vancomycin, and ciprofloxacin against four Flavobacterium meningosepticum isolates in which FIC indices indicated additivity or indifference for these combinations. In that study, FIC indices ranged from 0.74 to 1.5 for linezolid plus rifampin, 0.75 to 1 for linezolid plus vancomycin, and 0.62 to 1 for linezolid plus ciprofloxacin. No antagonism was reported. Hirschl et al. (12) also reported predominant indifference and occurrences of partial synergy and synergy for linezolid in combination with amoxicillin, clarithromycin, and metronidazole against Helicobacter pylori strains. FIC indices ranged from 0.31 to 2.5 for linezolid plus amoxicillin, 0.38 to 2.5 for linezolid plus clarithromycin, and 0.5 to 2.12 for linezolid plus metronidazole. No antagonism was reported. Sisson et al. (20) evaluated the in vivo pharmacokinetics of linezolid in combination with aztreonam and determined that the combination did not alter the disposition of either drug under single-dose conditions. Finally, Allen et al.

4 VOL. 47, 2003 IN VITRO ACTIVITIES OF LINEZOLID AND OTHER AGENTS 1905 Drug class and drug used in combination with linezolid TABLE 3. Results of checkerboard testing of the activities of linezolid drug combinations against the organisms a FIC index range (no. of isolates) S. aureus b E. faecalis c E. faecium d S. pneumoniae e E. coli-k. pneumoniae f Amoxicillin g (10) j Amoxicillin-clavulanic acid (10) Ampicillin (12) (11) (17) Ampicillin-sulbactam (10) Methicillin (10) (14) (11) (14) (12) Oxacillin (10) Cefdinir (10) (12) (11) (12) (12) Cefotaxime (10) (12) (11) (12) (12) Cefoxitin (10) (12) (11) (12) (12) Cefpodoxime (17) Ceftazidime (10) (12) (11) (12) (12) Cephalothin (10) (12) (11) (12) (12) Ciprofloxacin (10) (12) (11) (17) (12) Difloxacin (10) (12) (11) (17) (12) Gatifloxacin (10) (12) Nalidixic acid (10) (12) (11) (17) (12) Norfloxacin (10) (12) (11) (17) (12) Ofloxacin (10) h (12) (11) (17) (12) Sparfloxacin (10) h (12) (11) (17) i (12) Trovafloxacin (10) (12) (11) (17) (12) Aztreonam (12) Bacitracin (10) (12) (11) (12) (12) Chloramphenicol (12) (17) Clindamycin (10) (17) Erythromycin (11) i (17) Fusidic acid (12) Gentamicin (11) (12) (11) (12) Imipenem i (10) (12) i (11) Metronidazole (12) Neomycin (12) Novobiocin (12) Rifampin (12) (12) (11) Teicoplanin (10) i (12) (11) (12) (12) Tetracycline (10) (12) i (11) (12) (12) Vancomycin (12) (12) (11) (17) a Results for FIC indices of 0.5 are synergistic, those at for FIC indices of 0.5 to 4.0 are indifferent, and those for FIC indices of 4.0 are antagonistic. b Includes methicillin-sensitive (n 1 to 2 strains) and methicillin-resistant (n 8 to 10) strains. c Includes vancomycin-sensitive (n 6) and vancomycin-resistant (n 6 to 8) strains. d Includes vancomycin-sensitive (n 5) and vancomycin-resistant (n 6) strains. e Includes penicillin-sensitive (n 2 to 5), -intermediate (n 5 to 6), and -resistant (n 5 to 6) strains. f Includes E. coli (n 6) and K. pneumoniae (n 6) strains. g Drug combination resulted in synergism for three strains. h Drug combination resulted in antagonism for one strain. i Drug combination resulted in synergism for one strain. j, not determined. (1) reported that linezolid in combination with cefepime, doxycycline, quinupristin-dalfopristin, and vancomycin improved or enhanced the killing of isolates of staphylococci and enterococci. The results of this checkerboard study predominantly showed indifference when linezolid was combined with 35 different antimicrobial agents and tested against drug-sensitive and -resistant organisms. In the evaluation of the activities of 1,380 linezolid-drug combinations against the organisms tested, 1,369 combinations (99.2%) were indifferent. From nine determinations of synergism (0.65%), linezolid plus amoxicillin resulted in three cases of synergism against strains of methicillin-resistant S. aureus. Linezolid in combination with ofloxacin and sparfloxacin resulted in low levels of antagonism (0.15%) against two strains of E. faecalis. Differences in the results of checkerboard assays for the detection of in vitro synergy between antimicrobial agents have been demonstrated. Mackay et al. (15) tested the same checkerboard on 3 separate days to test reproducibility and found no statistically significant differences. However, they did conclude that all combinations tested showing synergism according to the FIC index at 24 h also showed synergism by time-kill assays

5 1906 SWEENEY AND ZURENKO ANTIMICROB. AGENTS CHEMOTHER. at 24 h but that the correlation between synergy at 2 or 5haccording to the FIC index and by the time-kill assay at the same time points was poor. Certain problems have been associated with the checkerboard microdilution method itself, such as variability in the inoculum as a potential source of error as well as the selection of inappropriate antibiotic concentrations (9). Checkerboard assays are laborious and time-consuming. However, the robotic instruments used in the experiments described here allowed large numbers of strains and drug combinations to be tested. In our experiments, the results for each linezolid-drug combination that initially demonstrated synergism or antagonism were successfully obtained upon retesting, while combinations that showed indifference were not further evaluated. Additionally, inherent assay variability appeared to be kept to a minimum, as determined from the results for the control plates for synergism (100% synergism with amoxicillinclavulanate plates with the appropriate organism) and control plates for antagonism (100% antagonism with chloramphenicol-ciprofloxacin plates with the appropriate organism). These control plates, which were used during each linezolid-drug combination experiment, were included to ensure the accuracy of the assay. Also of concern was the interpretation of results from the literature from studies that used the checkerboard method, since numerous definitions have been assigned for synergism, antagonism, additivity, and indifference. Cappelletty and Rybak (4) reported synergism as a fourfold decrease in MICs, and synergism has been defined as an FIC index of 0.5, with marked synergism being an eightfold decrease in MICs and FIC indices of In our experiments, synergism was defined as an FIC index of 0.5. Antagonism has been defined as an FIC index 1, 1, 2, or 4. From these different values, a result may be defined as indifferent or antagonistic, depending on the FIC index value chosen. In our experiments, antagonism was defined as an FIC index 4. On the basis of information in the literature and the recommended interpretations for FIC indices, it is our belief that the FIC interpretations that we selected adequately categorized the linezolid-drug interactions (2, 10). Even with the variabilities known to be inherently associated with synergy studies, such as the 1-dilution (twofold) variability associated with the performance of serial dilutions in the microdilution plate system, checkerboard methods nonetheless indicate that certain combinations of antibiotics are more useful than others against organisms (6, 8, 16, 21, 22). The major value of this study is the demonstration that the combination of linezolid with a particular beta-lactam, quinolone, or other antibacterial agent primarily results in indifference and rarely results in antagonism. This is important, since there are limitations in the spectrum of activity of linezolid against certain organisms such as gram-negative and anaerobic bacteria. Animal and clinical studies are needed to establish whether therapy with linezolid in combination with another agent in selected clinical situations such as endocarditis, osteomyelitis, nosocomial and community-acquired pneumonia, and skin infections may be beneficial. REFERENCES 1. Allen, A. P., R. Cha, and M. J. Rybak In vitro activities of quinupristindalfopristin and cefepime, alone and in combination with various antimicrobials, against multidrug-resistant staphylococci and enterococci in an in vitro pharmacodynamic model. Antimicrob. 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