In vitro activity of gatifloxacin alone and in combination with cefepime, meropenem, piperacillin and gentamicin against multidrug-resistant organisms

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1 Advance Access published April 14, 2003 Journal of Antimicrobial Chemotherapy DOI: /jac/dkg238 In vitro activity of gatifloxacin alone and in combination with cefepime, meropenem, piperacillin and gentamicin against multidrug-resistant organisms Maria Agnes Dawis 1 *, Henry D. Isenberg 2, Kenneth A. France 3 and Stephen G. Jenkins 2 1 Division of Pediatric Infectious Diseases, 2 Department of Pathology, 3 Center for Clinical Laboratories, Mount Sinai School of Medicine, New York, NY, USA Received 16 October 2002; returned 17 December 2002; revised 3 March 2003; accepted 4 March 2003 Objectives: To study the in vitro interaction of gatifloxacin in combination with gentamicin and with the β-lactams cefepime, meropenem and piperacillin against clinical isolates of Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Burkholderia cepacia, extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae, vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). Methods: The activity of each drug alone was determined by an agar dilution method. Chequerboard synergy testing was then performed against all the isolates. Time kill assays were done on selected isolates to assess correlation with the chequerboard results. Results: Synergy was demonstrated with the following combinations at achievable serum concentrations: gatifloxacin/piperacillin for 80% and gatifloxacin/cefepime for 60% of S. maltophilia; gatifloxacin/gentamicin for 60%, and gatifloxacin/cefepime for 50% of ESBLproducing K. pneumoniae, and in all drug combinations for 50 70% of P. aeruginosa. Indifference was noted for the majority of B. cepacia and VRE isolates. Antagonism at therapeutic serum levels was observed with gatifloxacin/piperacillin against a single isolate of B. cepacia. No distinct trend in drug interaction was seen with the different drug combinations against MRSA. Time kill analyses against selected isolates confirmed the synergic activity of the following drug combinations seen in the chequerboard assays: gatifloxacin/cefepime and gatifloxacin/ piperacillin against P. aeruginosa, gatifloxacin/gentamicin against B. cepacia, and gatifloxacin/ gentamicin and gatifloxacin/meropenem against ESBL-producing K. pneumoniae. Conclusions: Gatifloxacin was synergic with the β-lactams piperacillin, cefepime and meropenem, and with gentamicin against some drug-resistant pathogens. Some of the time kill analyses against P. aeruginosa, B. cepacia and ESBL-producing K. pneumoniae were in accordance with chequerboard results. Time kill analyses against S. maltophilia did not confirm the synergy seen in chequerboard testing. Keywords: synergy, fluoroquinolones, gatifloxacin, susceptibility testing Introduction Combination therapy is often beneficial for patients with serious infections caused by drug-resistant pathogens.the use of combination therapy can broaden the spectrum of antibacterial activity, minimize the emergence of resistant bacterial variants and can sometimes result in synergic interaction, thereby exhibiting antibacterial activity greater than would be expected from each drug individually *Correspondence address. Mount Sinai School of Medicine, One Gustave Levy Place, c/o Dr Betsy Herold, Mailbox 1657, New York, NY 10029, USA. Tel: ; Fax: ; agnesdawis@pol.net Present address: Long Island Jewish Medical Center, New Hyde Park, NY, USA. Present address: Carolinas Medical Center, Charlotte, NC, USA.... Page 1 of The British Society for Antimicrobial Chemotherapy

2 M. A. Dawis et al. Gatifloxacin is an FDA-approved synthetic broadspectrum fluoroquinolone useful for infections caused by both Gram-negative and -positive bacteria. It inhibits both bacterial DNA gyrase and topoisomerase IV. 2,3 The structure of gatifloxacin differs from the earlier fluoroquinolones by the presence in the C-8 position of a methoxy group that enhances antibacterial activity against Gram-positive bacteria, 4 improves its activity against DNA gyrase mutants of Escherichia coli 5 and has the potential to reduce the rate of development of resistance to fluoroquinolones in general. 3 The methoxy group in the C-8 position also diminishes photosensitivity reactions. 6 In an in vitro study comparing the activity of gatifloxacin with those of ciprofloxacin and ofloxacin, gatifloxacin was more active against Stenotrophomonas maltophilia, equipotent against Burkholderia cepacia, but demonstrated less activity than ciprofloxacin against Pseudomonas aeruginosa. 7 Gatifloxacin, ciprofloxacin, levofloxacin and moxifloxacin inhibited members of the family Enterobacteriaceae comparably. 8,9 Gatifloxacin had good activity against Streptococcus pneumoniae, methicillinsusceptible Staphylococcus aureus and Enterococcus faecalis. 9 Gatifloxacin is more potent than ciprofloxacin against methicillin-resistant S. aureus (MRSA), 9 although the clinical significance of this still needs to be investigated. The primary objective of this study was to examine the in vitro activity of gatifloxacin when combined with the β-lactam agents cefepime, meropenem and piperacillin, and with gentamicin against S. maltophilia, P. aeruginosa, B. cepacia, extended-spectrum β-lactamase (ESBL)- producing strains of Klebsiella pneumoniae, MRSA and vancomycin-resistant Enterococcus faecium (VRE). The activity of each drug alone against these bacteria was also examined. The above pathogens are established causes of nosocomial infections in an appreciable number of medical facilities Combination therapy, primarily against the Gram-negative pathogens, may play a crucial role in eradicating these organisms in patients with sepsis caused by these bacteria. For the Gram-positive organisms, knowledge of the absence of in vitro antagonism between the antimicrobial combinations provides potentially useful information about the possible risks associated with initiating empirical antimicrobial therapy when infections involve multiple pathogens that may include antibiotic-resistant Gram-positive species. Materials and methods Bacterial isolates Ten clinical isolates each of S. maltophilia, P. aeruginosa, B. cepacia, ESBL-producing K. pneumoniae, VRE and MRSA from patients at the Mount Sinai Medical Center were tested in this study. All organisms were collected within 2 months prior to testing, with the exception of eight B. cepacia and four S. maltophilia isolates that were recovered from our collection maintained at 70 C in skimmed milk. Isolates were selected based on their highly resistant antimicrobial susceptibility testing profile. Identification of the stock isolates was reconfirmed with the API 20NE System (biomérieux, Hazelwood, MO, USA). Agar dilution MIC determinations Standard laboratory powders of gatifloxacin (Bristol-Myers Squibb, New Brunswick, NJ, USA), cefepime (Elan Pharmaceuticals, San Diego, CA, USA), meropenem (AstraZeneca, Wilmington, DE, USA), piperacillin (Lederle, Puerto Rico) and gentamicin (Sigma Aldrich, St Louis, MO, USA) were used in the study. The MICs for all isolates of each drug alone was determined by the agar dilution method in accordance with NCCLS guidelines 13 using cation-supplemented Mueller Hinton agar (Becton Dickinson, Baltimore, MD, USA) before chequerboard testing. Agar dilution plates for all antimicrobials were prepared 24 h before inoculation and stored overnight at 4 C, except for dilutions of meropenem, which were prepared on the day of inoculation. Drug-free plates were used as growth controls. Purity of isolates was checked throughout the study by examination of colony morphology and Gram staining. Inoculum was prepared by the direct colony suspension method as follows: three to five identical colonies were selected after h of incubation on 5% sheep blood trypticase soy agar (TSA) plates (Becton Dickinson) and suspended in 0.9% NaCl to a density equivalent to a 0.5 McFarland standard. The suspension was further diluted to obtain a final inoculum of 10 4 cfu/spot of the Steers replicator. The density of each inoculum was determined by dilution studies. Plates were incubated at 35 C for h for the Gram-negative organisms and for 24 h for MRSA and VRE. Standard quality control strains of P. aeruginosa ATCC and S. aureus ATCC were included in each run. Chequerboard synergy testing The agar dilution method served to determine the activity of gatifloxacin in combination with cefepime, meropenem, piperacillin and gentamicin. Dilutions ranging from 256 to 0.03 mg/l were tested for all drug combinations. Organisms and agar dilution plates were prepared as described above for the MIC determination. The fractional inhibitory concentration (FIC) index was calculated using the formulae previously published: 14 FIC index = (Ac/Aa) + (Bc/Ba), where A and B are the two drugs being tested, Aa and Ba are the MICs obtained when each drug was tested alone, and Ac and Bc are the concentrations of each compound at the lowest effective combination. Synergy (FIC index 0.5) was defined as a fourfold or greater decrease in MIC of both drugs in combination compared with the drugs tested individually. Partial synergy Page 2 of 9

3 In vitro activity of gatifloxacin alone and in combination was defined as a four-fold or greater decrease in MIC with one agent with a two-fold decrease in the other agent (FIC >0.5 but <1). Additivity was defined as a two-fold drop in MIC with both agents (FIC = 1). Indifference was noted when there was no change in MIC whether the agents were tested alone or in combination (FIC >1 but <4) and results were interpreted as antagonistic when there was a four-fold increase in MIC for both agents when the drugs were tested in combination as compared with results when each drug was tested alone (FIC 4). Interpretation of the drug interactions was based on achievable serum concentrations of the drugs. Broth macrodilution MIC determinations and time kill assays Based on results of the chequerboard testing, isolates against which various drug combinations displayed synergy were selected for the time kill studies. Two isolates each of S. maltophilia and B. cepacia and one isolate each of ESBLproducing K. pneumoniae and P. aeruginosa were challenged with the various antibiotic combinations. Prior to performing the time kill analyses, MIC determinations using broth macrodilution method were performed against the selected isolates. Two-fold dilutions of antibiotic were prepared using cation-supplemented Mueller Hinton broth (Becton Dickinson) with a final volume of 1 ml. A control tube containing broth without antimicrobial agent was used for each organism tested. The inoculum was prepared from organisms grown for 4 6 h in broth and diluted to obtain a suspension containing the desired initial inoculum of cfu/ml in the growth control tube. Inoculum count verification plates were prepared for each isolate to be tested. Tubes were incubated at 35 C for h. The MIC was defined as the lowest concentration of antibiotic that completely inhibited the growth of the organism. Strains of E. coli ATCC and P. aeruginosa ATCC were used as control organisms for each antibiotic tested. Drug concentrations used for the time kill assays were based on three criteria: (i) concentrations likely to produce synergy as seen in chequerboard testing; (ii) concentrations that were within clinically achievable serum levels for each drug; and (iii) concentrations that were no more than twice the MIC of each drug. Time kill assays were performed in 10 ml of cation-supplemented Mueller Hinton broth (Becton Dickinson). Each assay included a growth control tube with no antibiotic. The inoculum was prepared from organisms grown for 4 6 h in cation-supplemented Mueller Hinton broth and diluted to obtain the desired initial inoculum of cfu/ml in the growth control tube. The antibioticcontaining tubes and the growth control tubes were incubated at 35 C and sampled at 0, 2, 4, 8 and 24 h from the time of inoculation for colony viability counts. Aliquots of 0.1 ml from serial 10-fold dilutions of each tube were spread onto TSA agar plates in duplicate, incubated at 35 C for 48 h for S. maltophilia and B. cepacia, and for 24 h for the other organisms, at which time colonies were enumerated. Colony counts were performed only on plates with colonies. Data were analysed based on viability counts at 24 h. Antimicrobial agents were considered bactericidal at a given concentration if they reduced the original inoculum by 99.9% (>3 log 10 cfu/ml) for each time period and bacteriostatic if the inoculum was reduced by 0 3 log 10 cfu/ml. The interpretation of results of antimicrobial interaction using time kill methodology was based on the discussion by Eliopoulos & Moellering. 14 Synergy was interpreted as a 2 log 10 decrease in viable count with the combination at 24 h compared with the most active single drug. Indifference was defined as a <10-fold decrease in viable count at 24 h with the combination, compared with the most active single antimicrobial alone. Antagonism was defined as 2 log 10 increase in colony count at 24 h with the combination, compared with the most active single drug alone. 14 Results The MICs of the antimicrobials for the different organisms are summarized in Table 1. MIC 50 and MIC 90 values represent the concentrations at which 50% and 90% of strains, respectively, were inhibited. The gatifloxacin MICs for S. maltophilia ranged from 0.5 to 4 mg/l, the lowest values of the agents tested. More than 50% of these isolates were resistant to cefepime (MIC 50 = 32 mg/l), meropenem (MIC 50 = 256 mg/l) and piperacillin (MIC 50 = 256 mg/l). At least 50% of the P. aeruginosa isolates tested were susceptible to all of the antibiotics tested based on established breakpoints, 15 except for gatifloxacin, where the MIC 50 was 4 mg/l (susceptible breakpoint 2 mg/l). ESBL-producing K. pneumoniae were very susceptible to meropenem, with 90% of the isolates inhibited at mg/l, but were resistant to piperacillin with an MIC 50 of 256 mg/l. The MICs for the majority of B. cepacia, MRSA and VRE isolates were above achievable serum levels for all of the agents. The meropenem MIC was 8 mg/l for nine of the 10 strains of B. cepacia. Results of the chequerboard synergy testing are summarized in Table 2. The combination of gatifloxacin/piperacillin exhibited synergy for 80% and gatifloxacin/cefepime for 60% of S. maltophilia, whereas gatifloxacin/gentamicin showed partial synergy for 80% of the strains tested. Indifference was noted with gatifloxacin/meropenem for all isolates of S. maltophilia. For P. aeruginosa, all drug combinations displayed synergy against at least 50% of the isolates, and gatifloxacin/meropenem was synergic against 70%. All drug combinations were indifferent against at least 50% of B. cepacia isolates, with gatifloxacin/piperacillin antagonistic with one isolate. Gatifloxacin/gentamicin was synergic Page 3 of 9

4 M. A. Dawis et al. Table 1. Agar dilution MICs for the organisms tested MIC (mg/l) Species Antibiotic range MIC 50 MIC 90 S. maltophilia gatifloxacin cefepime 4 > meropenem piperacillin 32 > >256 gentamicin 2 > P. aeruginosa gatifloxacin cefepime meropenem piperacillin gentamicin B. cepacia gatifloxacin 1 > cefepime 1 > >256 meropenem piperacillin gentamicin 0.5 > ESBL-producing K. pneumoniae gatifloxacin 1 > cefepime meropenem piperacillin 32 > >256 gentamicin MRSA gatifloxacin cefepime 4 >256 >256 >256 meropenem piperacillin 2 > gentamicin VRE gatifloxacin 4 > cefepime 256 >256 >256 >256 meropenem 256 >256 >256 >256 piperacillin >256 >256 >256 gentamicin 8 >256 >256 >256 against 60% and gatifloxacin/cefepime against 50% of ESBL-producing K. pneumoniae. Indifference was noted with all drug combinations for at least 80% of VRE. Synergy was seen against only 20 30% of MRSA with all drug combinations. For the time kill analyses, two isolates each of S. maltophilia and B. cepacia and one isolate each of ESBLproducing K. pneumoniae and P. aeruginosa were selected as test organisms for the various antibiotic combinations. Determination of the MICs for the selected isolates using the broth macrodilution method revealed that in general, MICs for the isolates were higher by a two-fold or less doubling dilution as determined using the agar dilution method (data not shown). Figures 1 4 represent the time kill analyses on the selected isolates. Concentrations tested for each combination were predicted on those that were likely to produce synergy based on chequerboard testing, concentrations that were within clinically achievable serum levels for each drug and concentrations that were no more than twice the MIC of each drug. Figure 1 represents the time kill curves for S. maltophilia. At the concentrations tested, none of the combinations showed synergy. Time kill responses of P. aeruginosa are depicted in Figure 2. Gatifloxacin at 2 mg/l in combination with cefepime at 8 mg/l, and gatifloxacin at 2 mg/l in combination with piperacillin at 64 mg/l, resulted in at least a 2 log 10 decrease in viable colonies, and by definition were synergic. The effect of these combinations was bacteriostatic. Increasing the dose of gatifloxacin to 4 mg/l in combination with cefepime at 8 mg/l and in combination with piperacillin at 64 mg/l resulted in enhanced activity. For the combination of gatifloxacin and piperacillin, growth at 2 and 4 h followed the growth curve with either gatifloxacin alone or piperacillin alone. This may suggest that the addition of one of either drug may have prevented the emergence of resistant subpopulations of P. aeruginosa. The combination of gatifloxacin/gentamicin at achievable serum concentrations showed indifference. Meropenem at 0.06 mg/l with gatifloxacin at 4 mg/l failed to inhibit the growth of the organism tested. In Figure 3, the combination gatifloxacin (2 mg/l)/ gentamicin (8 mg/l) showed synergic activity and was bactericidal against B. cepacia. Indifference was seen with the combination of piperacillin with gatifloxacin against another isolate of B. cepacia. For ESBL-producing K. pneumoniae (Figure 4), the combinations of gatifloxacin (2 mg/l)/gentamicin (4 mg/l) and gatifloxacin (2 mg/l)/meropenem (0.25 mg/l) both followed the growth curve with gatifloxacin alone at 2 and 4 h. At 24 h, however, these two combinations resulted in a >2 log 10 reduction in the number of viable colonies, indicating synergy. As in the earlier case with the P. aeruginosa isolate, it may be that the presence of either of the drug in the combination may have prevented the emergence of resistant subpopulations. Nevertheless, the combinations were lethal against this particular isolate. Discussion Gatifloxacin is a recently developed antibacterial agent differing from earlier fluoroquinolones by the presence of a methoxy group at the C-8 position. 5,6 The presence of the methoxy group has conferred improved antibacterial activity against both Gram-positive and Gram-negative organisms, 7 9 making gatifloxacin a broad-spectrum antimicrobial agent applicable in many clinical settings. The inhibitory activity of gatifloxacin against S. aureus topoisomerase IV and E. coli DNA gyrase has been studied previously. 2 Results of another study examining the effect of fluoroquinolone structure against gyrase mutants of E. coli has shown that for most of Page 4 of 9

5 In vitro activity of gatifloxacin alone and in combination Table 2. Chequerboard testing of antibiotic interactions against different organisms a,b Interaction GAT/FEP GAT/MEM GAT/PIP GAT/GEN S. maltophilia synergy partial synergy additivity indifference P. aeruginosa synergy partial synergy additivity indifference B. cepacia synergy partial synergy additivity indifference antagonism ESBL-producing K. pneumoniae GAT, gatifloxacin; FEP, cefepime; MEM, meropenem; PIP, piperacillin; GEN, gentamicin. a Values represent the number of isolates for which a particular drug interaction was demonstrated. b Antimicrobial interactions reported were based on achievable serum concentration for each drug. the mutants, gatifloxacin (C-8-methoxy derivative) was more lethal than C-8-H compounds such as ciprofloxacin and AM This study also suggested that in E. coli the C-8-methoxy group improved the inhibition of topoisomerase IV when the gyrase allele demonstrates sufficient resistance. 5 Several in vitro studies have been performed to examine the susceptibilities of both Gram-negative and -positive clinical isolates to gatifloxacin. 7 9,16,17 Overall, the growth of S. maltophilia, P. aeruginosa and VRE isolates tested in this study was inhibited by gatifloxacin concentrations within the ranges described in earlier studies. 8,9,16,17 In a study comparing the activity of ciprofloxacin, gatifloxacin and levofloxacin against 210 P. aeruginosa isolates from the urinary tract, there were no significant differences in susceptibility patterns of the three fluoroquinolones tested. 18 Notable is the enhanced activity of gatifloxacin against S. maltophilia in comparison with older fluoroquinolones such as ciprofloxacin. 7,8,17 The MICs of gatifloxacin for B. cepacia, ESBLproducing K. pneumoniae and MRSA isolates in this study synergy partial synergy additivity indifference VRE synergy partial synergy additivity indifference MRSA synergy partial synergy additivity indifference were generally higher than those previously reported in studies involving these organisms. 7 9,16 It does appear in this study that gatifloxacin, like other fluoroquinolones, 19 has less activity against many strains of ESBL-producing K. pneumoniae. Combinations of fluoroquinolones with other antimicrobial agents have been investigated extensively Most studies combining fluoroquinolones with aminoglycosides have shown indifference against members of the Enterobacteriaceae and against P. aeruginosa, whereas fluoroquinolones with antipseudomonal penicillins have been reported to be synergic against 20 50% of P. aeruginosa isolates. 22 In one study using the chequerboard methodology evaluating ciprofloxacin with ticarcillin clavulanate against S. maltophilia, synergy was seen against 24 of 31 strains. 17 To date, there have been three in vitro studies published examining the antimicrobial interaction of gatifloxacin with non-fluoroquinolone compounds against the same species of bacteria tested in this study In one study using both chequerboard and time kill analysis against P. aeruginosa ATCC 27853, there was no synergy or antagonism demon- Page 5 of 9

6 M. A. Dawis et al. Figure 1. S. maltophilia time kill assays. GEN, gentamicin; GAT, gatifloxacin; FEP, cefepime; PIP, piperacillin. strated with the following combinations: gatifloxacin/ amikacin, gatifloxacin/imipenem and gatifloxacin/cefepime. 24 In another published study using time kill analyses, synergy was seen with the following combinations: gatifloxacin/ ticarcillin clavulanate and gatifloxacin/ceftazidime against five of eight S. maltophilia strains; gatifloxacin/cefepime against three of eight, gatifloxacin/piperacillin against seven of eight P. aeruginosa strains; and gatifloxacin/ceftazidime against five of six B. cepacia strains. 25 A recent time kill study against 10 P. aeruginosa isolates combining ceftazidime or cefepime with gatifloxacin, ciprofloxacin, levofloxacin or moxifloxacin indicated that in vitro synergy was demonstrable against 60 80% of isolates tested, and that no significant differences existed among the cephalosporin/ fluoroquinolone combinations. 26 In this study, the chequerboard analysis showed that gatifloxacin in combination with gentamicin or one of the three β-lactam agents demonstrated synergic activity against many strains of S. maltophilia, P. aeruginosa and ESBLproducing K. pneumoniae isolates. Using both chequerboard and time kill methods to assess the antibiotic combinations against these non-fermenters, there was no antagonism between the combined agents tested at concentrations up to 8 MIC for these pathogens (data not shown). By chequerboard analysis, only the gatifloxacin/gentamicin combination demonstrated synergic activity against B. cepacia, VRE and MRSA, and then only for 20% of the strains tested. Using the time kill method in this study, some, but not all, of the combinations tested against the selected isolates confirmed the synergic activity demonstrated with the chequerboard method. This may be because the concentrations tested in the time kill method were not optimal. Using higher concentrations of antimicrobials closer to the maximum achievable serum concentrations may yield more favourable results. Differences in results between chequerboard and time kill method may also stem from the inherent limitation of chequerboard analysis to provide only an all-ornone response at one point in time. Results reported are not quantifiable and may only reflect inhibitory, but not bactericidal, activity. In general, chequerboard assays are considered screening assays to assess possible synergic activity based on bacteriostatic activity, but bactericidal activity may not be appreciated; this can only be assessed by a method such as a killing curve. With the combinations of gatifloxacin/piperacillin against P. aeruginosa, and gatifloxacin/gentamicin and gatifloxacin/ meropenem against ESBL-producing K. pneumoniae (Figures 2b, and 4a and b), it is worthwhile to note that the time kill curves for the drug combinations at 24 h indicate synergy, although the 2 and 4 h kill curves reflect the activity of the more active drug. In these cases, it may be that the addition of the second antimicrobial prevents the emergence of resistant subpopulations of the strains tested. Prevention of emergence of resistance may be clinically as important as synergic bactericidal activity for these difficult to treat pathogens. The significance of the above in vitro findings must be confirmed in the clinical setting, but properly randomized and controlled clinical trials may not be feasible to perform. The concentrations of the antimicrobials used in the time kill Page 6 of 9

7 In vitro activity of gatifloxacin alone and in combination Figure 2. P. aeruginosa time kill assays. FEP, cefepime; GAT, gatifloxacin; PIP, piperacillin; GEN, gentamicin; MEM, meropenem. Figure 3. B. cepacia time kill assays. GEN, gentamicin; GAT, gatifloxacin; PIP, piperacillin. analyses were all within clinically achievable levels. This emphasizes the potential beneficial value of these combinations for treatment of seriously ill patients with infections caused by the pathogens tested, especially when there is a paucity of other therapeutic options. Each clinical case needs to be individualized, however. Both the chequerboard and time kill approaches demonstrated that the bacteria in this investigation, chosen on the basis of their general in vitro unresponsiveness to usual therapeutic agents, reacted favourably to combinations of gatifloxacin, a recently released fluoroquinolone, with an aminoglycoside as well as with several β-lactams. If clinical experience supports these Page 7 of 9

8 M. A. Dawis et al. Such guidance may lessen the selective pressures existing in many medical facilities with constantly increasing resistant microbial populations challenging the rapid recovery of patients. In summary, chequerboard analysis showed that gatifloxacin is synergic or partially synergic with the β-lactams cefepime, meropenem and piperacillin, as well as with gentamicin against many drug-resistant pathogens. Antagonism was only seen with a single isolate of B. cepacia when tested with the combination of gatifloxacin and piperacillin. Indifference was noted with all drug combinations for at least 80% of VRE. Synergy was seen against only 20 30% of MRSA with all drug combinations. Time kill analyses of the different combinations correlated with chequerboard results with some of the selected isolates tested. The clinical impact of these findings remains to be further elucidated. Figure 4. ESBL-producing K. pneumoniae time kill assays. GEN, gentamicin; GAT, gatifloxacin; MEM, meropenem; FEP, cefepime. observations, synergy studies to evaluate appropriate combinations of antimicrobial agents may guide therapy for unresponsive infectious complications. Greater efforts to simplify the analytical approach of antimicrobial combinations, based on dilutions and/or multiples of achievable blood levels, are needed to promote greater appreciation of rational antimicrobial combination therapeutic regimens. Acknowledgements This study was presented in part at the 101st Annual Meeting of the American Society for Microbiology, Orlando, FL, May 19 24, 2001, and was supported by grants from Bristol- Myers Squibb, Wallingford, CT and Elan Pharmaceuticals, San Diego, CA, USA. References 1. Eliopoulos, G. M. & Eliopoulos, C. T. (1988). Antibiotic combinations: should they be tested? Clinical Microbiology Reviews 1, Takei, M., Fukuda, H., Yasue, T., Hosaka, M. & Oomori, Y. (1998). Inhibitory activities of gatifloxacin (AM-1155), a newly developed fluoroquinolone, against bacterial and mammalian type II topoisomerases. Antimicrobial Agents and Chemotherapy 42, Zhao, X., Xu, C., Domagala, J. & Drlica, K. (1997). DNA topoisomerase targets of the fluoroquinolones: a strategy for avoiding bacterial resistance. Proceedings of the National Academy of Sciences, USA 94, Zhao, X., Wang, J. Y., Xu, C., Dong, Y., Zhou, J., Domagala, J. et al. (1998). Killing of Staphylococcus aureus by C-8-methoxy fluoroquinolones. Antimicrobial Agents and Chemotherapy 42, Lu, T., Zhao, X. & Drlica, K. (1999). Gatifloxacin activity against quinolone resistant gyrase: allele-specific enhancement of bacteriostatic and bactericidal activities by the C-8-methoxy group. Antimicrobial Agents and Chemotherapy 43, Marutani, K., Matsumoto, M., Otabe, Y., Nagamuta, M., Tanaka, K., Miyoshi, A. et al. (1993). Reduced phototoxicity of a fluoroquinolone antibacterial agent with a methoxy group at the 8 position in mice irradiated with long-wavelength UV light. Antimicrobial Agents and Chemotherapy 37, Fung-Tomc, J., Minassian, B., Kolek, B., Washo, T., Huczko, E. & Bonner, D. (2000). In vitro antibacterial spectrum of a new broad-spectrum 8-methoxy fluoroquinolone, gatifloxacin. Journal of Antimicrobial Chemotherapy 45, Page 8 of 9

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