Diagnostic Microbiology and Infectious Disease 42 (2002) 75–78
In vitro synergy testing of levofloxacin, ofloxacin, and ciprofloxacin in combination with aztreonam, ceftazidime, or piperacillin against Pseudomonas aeruginosa Susan L. Pendlanda,*, Chad R. Messickb, Rose Jungc a
The University of Illinois at Chicago, Department of Pharmacy Practice, Microbiology Research Laboratory, Chicago, IL, USA b Veterans Affairs Cooperative Studies Program, Clinical Research Pharmacy Coordinating Center, Albuquerque, NM, USA c The University of Colorado Health Sciences Center, Department of Pharmacy Practice, Denver, CO, USA Received 6 June 2001; accepted 2 October 2001
This work was presented at the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, Louisiana 1996.
Abstract The synergistic potential of levofloxacin, ofloxacin and ciprofloxacin combined with aztreonam, ceftazidime, or piperacillin was compared using 24 strains of Pseudomonas aeruginosa with varying susceptiblity profiles. Levofloxacin and ciprofloxacin demonstrated similar in vitro activity, with ofloxacin demonstrating less activity compared to the other agents. Predominately additive effects were seen with all combinations, with no significant differences detected between the fluoroquinolone agents. © 2002 Elsevier Science Inc. All rights reserved.
1. Introduction Infections due to Pseudomonas aeruginosa remain a serious therapeutic problem. Combination therapy is advocated to prevent the rapid emergence of resistance, with regimens demonstrating synergy desired. Many synergy studies have been conducted with ciprofloxacin against P. aeruginosa (Davies & Cohen, 1985; Meyer & Liu, 1988; Stratton et al., 1989; Bustamante et al., 1990). A few studies have examined the synergistic potential of ofloxacin (Klepser et al., 1995; Mayer & Nagy, 1999) and levofloxacin (Flynn et al., 1996; Visalli et al., 1998). None of the studies have involved a direct comparison of the three agents. The purpose of this study was to compare the synergistic activity of the levofloxacin, ofloxacin, and ciprofloxacin against P. aeruginosa. Levofloxacin and ofloxacin powders were supplied by the R.W. Johnson Pharmaceutical Research Institute (Raritan, NJ). Ciprofloxacin, aztreonam, ceftazidime, and piperacillin powders were purchased from the United States Pharmacopeia (Rockville, MD). Stock solutions were pre* Corresponding author. Tel.: ⫹1-312-996-8639; fax: ⫹1-312-4131797. E-mail address: [email protected]
pared per National Committee for Clinical Laboratory Standards (NCCLS) guidelines (NCCLS, 2000) and stored in polypropylene vials at ⫺70°C. After stock solutions were thawed, serial two-fold dilutions were prepared in appropriate diluents. Final concentrations (ug/mL) tested were levofloxacin 0.25–32, ofloxacin 0.5–32, ciprofloxacin 0.125–16, aztreonam 1–128, ceftazidime 0.5– 64, and piperacillin 4 –512. Mueller-Hinton Medium (Difco, Detroit, MI) was used for agar dilution susceptibility and synergy testing. Each petri dish for minimum inhibitory concentration (MIC) testing was prepared by mixing 19 mL of molten medium with 1 mL of antibiotic. Each plate for synergy testing was prepared by mixing 18 mL of medium with 1 mL of each antibiotic. The total volume in each petri plate was 20 mL. All antibiotic-containing media were prepared 1 day prior to testing and stored in plastic bags at 4°C. The P. aeruginosa organisms tested included 23 clinical isolates and 1 control strain (ATCC 27853). The isolates were obtained from the Microbiology Laboratory at the University of Illinois at Chicago Medical Center (Chicago, IL). Isolates were selected in an effort to study an equal mixture of sensitive and resistant strains. The organisms were incubated in tryptic soy broth on a platform shaker at 35°C for 2 to 6 h to obtain log phase growth. Bacterial
0732-8893/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 1 ) 0 0 3 1 2 - 1
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Table 1 Summary of P. aeruginosa Susceptibility Data (n ⫽ 24) Antibiotic a
Levofloxacin Ofloxacina Ciprofloxacinb Piperacillinc Ceftazidimed Aztreonamd
MIC range (g/mL)
Sensitive % (#)
Intermediate % (#)
Resistant % (#)
0.5–⬎32 0.5–⬎32 0.125–⬎16 0.5–⬎512 1–64 1–64
38% (9) 12.5% (3) 37.5% (9) 71% (17) 75% (18) 25% (6)
8% (2) 25% (6) 12.5% (3) — 12.5% (3) 42% (10)
54% (13) 62.5% (15) 50% (12) 29% (7) 12.5% (3) 33% (8)
Interpretative criteria: a sensitive ⱕ2, intermediate 4, resistant ⱖ8. sensitive ⱕ1, intermediate 2, resistant ⱖ4. c sensitive ⱕ64, resistant ⱖ128. d sensitive ⱕ8, intermediate 16, resistant ⱖ32. b
ated with each fluoroquinolone antibiotic. Within each grouping, the responses were treated as correlated observations and analyzed with a repeated measures approach. When the numbers were too sparse to use the log-linear model, the McNemar’s test for paired data were applied, and the exact p-value determined from the Binomial distribution. A p-value of ⬍0.05 was considered statistically significant. A summary of the susceptibility results is presented in Table 1. MICs for the P. aeruginosa control strain were within acceptable limits (data not shown). Levofloxacin and ciprofloxacin demonstrated similar in vitro activity, with ofloxacin being less active than the other agents. These results are in agreement with other reports in the literature (Diekema et al., 1999; Flynn et al., 1996; Klepser et al., 1995). The results of the checkerboard tests are listed in Table 2. Predominately additive effects were observed with the various combinations of antibiotics tested. For the statistical analysis, the data were ultimately classified into two categories: synergy/additive and indifferent. Synergy and additive results were combined into one category because of the small number of synergistic combinations obtained. Antagonism was omitted as a category since it never occurred. There was no significant difference in synergistic activity between the fluoroquinolones when combined with aztreonam, ceftazidime or piperacillin. Bustamante et al. (1990) tested ciprofloxacin plus aztreonam or ceftazidime against multi-resistant isolates of P. aeruginosa. They reported the frequency of synergy to be dependent on antibiotic susceptibilities, with ⬎ 50% synergy seen with ciprofloxacin-resistant/aztreonam-sensitive
suspensions were diluted in sterile normal saline with the turbidity of each suspension adjusted to match a 0.5 McFarland standard using a spectrophotometer at a wavelength of 625 nm. The suspensions were further diluted 1:100 in sterile saline to obtain an inoculum of 1 ⫻ 106 CFU/mL. The checkerboard procedure was performed using the agar dilution susceptibility procedure (NCCLS, 2000). A replicator device (Craft Machine Inc., Chester, PA) was used to inoculate approximately 8 uL of each bacterial suspension onto the agar plates, resulting in a final inoculum of approximately 1 ⫻ 104 CFU/spot. Agar plates without antibiotics were used as a growth control. All tests were performed in duplicate, and all plates were incubated at 35°C for 18 h. The MIC was read as the lowest concentration of antimicrobial agent(s) at which complete inhibition of growth occurred. MICs were determined for each agent alone and in combination. Synergy was determined by calculating the fractional inhibitory concentration (FIC) index (Eliopoulos, 1996). The FIC index was obtained by adding the FIC of the fluoroquinolone and the FIC of the beta lactam antibiotic. The FIC of each fluoroquinolone was calculated by dividing the MIC of the fluoroquinolone/beta lactam combination by the MIC of the fluoroquinolone alone. The FIC of each beta lactam was calculated by dividing the MIC of the beta lactam/fluoroquinolone combination by the MIC of the beta lactam alone. Results for synergy were defined as ⱕ0.5 synergy, ⬎0.5 to 1.0 additive, ⬎1.0 to 4.0 indifferent, and ⬎4.0 antagonism. The log-linear model was used to determine differing rates of synergy for the various combinations. The data were grouped into the combinations associ-
Table 2 Checkerboard data for Pseudomonas aeruginosa. Data are presented as number (%) of isolates Interpretation (FIC Index)
Synergy (ⱕ0.5) Additive (⬎0.5–1) Indifferent (⬎1–4) Antagonism (⬎4)
1 (5) 15 (71) 5 (24) 0
0 15 (71) 6 (29) 0
0 13 (62) 8 (38) 0
0 20 (87) 3 (13) 0
2 (9) 18 (78) 3 (13) 0
2 (9) 19 (86) 1 (5) 0
2 (9) 21 (91) 0 0
2 (9) 19 (82) 2 (9) 0
0 22 (96) 1 (4) 0
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strains versus ⬍5% with ciprofloxacin-sensitive/aztreonamresistant strains. Contrary to results with the aztreonamresistant strains, they found that 75% of ciprofloxacin-sensitive/caftazidime-resistant strains demonstrated synergy. We did not find that enhanced activity was dependent on susceptibility patterns. Seventy-five percent of our isolates were intermediate or resistant to aztreonam. The addition of any fluoroquinolone lowered the MIC of aztreonam resulting in synergistic or additive activity in the majority of strains, regardless of fluoroquinolone susceptibility (data not shown). Most of our isolates were sensitive to ceftazidime. Again we did not detect any differences in the incidence of synergy based on susceptibility patterns of ceftazidime or fluoroquinolones (data not shown). Two separate studies have examined the synergistic potential of levofloxacin against P. aeruginosa. Neither study included comparisons with other fluoroquinolones. Flynn et al. (1996) studied the combination of levofloxacin plus aztreonam, ceftazidime, or piperacillin. They found levofloxacin plus aztreonam to be synergistic for 16% of isolates and additive for 5%. Seventy-six percent of the isolates demonstrated indifference, with another 3% reported as antagonistic. They reported synergy, additivity, and indifference in 24%, 0%, and 76% of strains with levofloxacin/ ceftazidime compared to 22%, 8%, and 70% of strains, respectively, with levofloxacin/piperacillin. Their study was designed with predicted concentrations that would demonstrate synergy within clinically relevant ranges of antibiotics. As a result, the majority of their isolates gave indeterminate results. Visalli et al. (1998) also examined the activity of levofloxacin in combination with ceftazidime. They reported 12% synergy with levofloxacin-sensitive/ceftazidime-resistant strains versus 0% synergy in multi-sensitive strains. The 12% incidence of synergy in their study contrasted sharply with the 75% reported by Bustamante et al. (1990) with ciprofloxacin-sensitive/ceftazidime-resistant strains. In comparison, 2 (9%) isolates in our study demonstrated synergy with the combination of ceftazidime and ciprofloxacin or ofloxacin. Synergy was not observed with the combination of levofloxacin and ceftazidime. Both strains exhibiting synergy with ofloxacin were susceptible to ceftazidime. Synergistic combinations with ciprofloxacin included 1 ceftazidime-susceptible and 1 ceftazidime-resistant strain. Studies comparing combination therapy with ciprofloxacin versus ofloxacin have been previously described. Klepser et al. (1995) compared the serum bactericidal activities of ciprofloxacin and ofloxacin alone and in combination with ceftazidime or piperacillin in 12 healthy volunteers against 3 strains of multi-sensitive P. aeruginosa. As monotherapy, ciprofloxacin demonstrated greater killing than ofloxacin. The combination of ciprofloxacin and piperacillin also demonstrated greater killing than the ofloxacin/piperacillin combination. However, the combination of ofloxacin and ceftazidime was equivalent to the ciprofloxacin/ceftazidime combination. They were the first to demon-
strate that the advantage ciprofloxacin exhibits as monotherapy against P. aeruginosa may not be maintained when using combination therapy. Mayer and Nagy (1999) also reported similar FIC indices with the combination of ceftazidime and ciprofloxacin or ofloxacin against 3 strains of P. aeruginosa. We were not able to discern any differences in the synergistic potential of ciprofloxacin, ofloxacin or levofloxacin when combined with either ceftazidime or piperacillin against 24 strains of P. aeruginosa having various susceptibility profiles. This may be a result of differences in methodology or in the strains studied. There are known limitations in using checkerboard methodology and these have been well described in the literature (Cappelletty & Rybak, 1996). Use of the agar dilution checkerboard assay did not permit the differentiation of inhibitory effects versus bactericidal activity. However, the goal of the study was to compare the synergistic potential of the three fluoroquinolones and the agar dilution methodology provided a simple and expensive way to test a large number of clinical strains. There have been many reports in the literature on the in vitro synergism observed with ciprofloxacin and beta lactam antibiotics. While fewer studies have been conducted with ofloxacin and levofloxacin, the combination of these fluoroquinolones with beta lactam antibiotics has also resulted in enhanced activity. While in vitro data do not always correlate with in vivo efficacy, the available in vitro and clinical data obtained with aminoglycosides and beta lactam antibiotics would suggest that the enhanced in vitro activity observed with fluoroquinolone-␤ lactam combinations may be beneficial. In summary, we compared the activity of the three fluoroquinolones to determine if there were differences in the synergistic potential of the different agents. No significant differences were found between ciprofloxacin, ofloxacin, and levofloxacin when combined with aztreonam, ceftazidime, or piperacillin against 23 clinical strains of P. aeruginosa.
Acknowledgments We thank Jennifer Woodward and Seonyoung Ryu for technical assistance and Bruce Lindgren for performing the statistical analysis. This study was funded by a grant from R.W. Johnson Pharmaceutical Research Institute.
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