Abstract
A total of 124 Pseudomonas aeruginosa strains were tested for synergy between levofloxacin and cefpirome, ceftazidime, gentamicin, and meropenem. Checkerboards yielded synergistic fractional inhibitory concentration (FIC) indices (≤0.5) with 25 of 496 possible combinations. All other FIC indices were >0.5 to 2 (additive or indifferent), with no antagonism. Time-kill studies with 12 strains showed that levofloxacin (0.06 to 0.5 μg/ml) was synergistic with cefpirome, ceftazidime, gentamicin, and meropenem in 10, 9, 4, and 11 strains, respectively.
Standard therapy for Pseudomonas aeruginosa infections includes broad-spectrum cephalosporins, such as cefpirome (not available in the United States) and ceftazidime; aminoglycosides, such as gentamicin; and carbapenems, such as imipenem and meropenem (3, 5–7, 10, 11, 13–17). Levofloxacin, the l-isomer of ofloxacin, is also active against this organism (9, 19, 20). The current study investigated the activity of levofloxacin, alone and in combination with cefpirome, ceftazidime, gentamicin, and meropenem, against 124 P. aeruginosa strains with different susceptibilities to the latter four agents.
One hundred twenty-four strains of P. aeruginosa, recently isolated from clinical specimens and identified by conventional methodology (12), were tested. Strains resistant to cephalosporins and meropenem only were obtained from David Livermore (Central Public Health Laboratories, London, United Kingdom). Strains included 30 susceptible to ceftazidime, cefpirome, gentamicin, and meropenem; 26 resistant to ceftazidime only; 21 resistant to gentamicin only; 24 resistant to meropenem only; and 23 with various susceptibility patterns. Laboratory powders of known potency were obtained from their various manufacturers.
MICs of each agent alone were determined by broth microdilution testing according to standard National Committee for Clinical Laboratory Standards (NCCLS) methodology (18). Breakpoints for ceftazidime and gentamicin were those recommended by NCCLS (18). Breakpoints used for meropenem were identical to those of imipenem (18), as recently approved (but not yet published) by NCCLS. No cefpirome breakpoints are available. Strains with intermediate susceptibility (18) to ceftazidime, gentamicin, and meropenem were classified as resistant. Less than 5% of resistant strains were intermediate to ceftazidime and gentamicin, but 48% were intermediate to meropenem: all of the latter, however, were resistant (MICs of ≥16 μg/ml) to imipenem. Additionally, because serious P. aeruginosa infections caused by strains with intermediate resistance are treated as if fully resistant, we elected to combine the two groups.
Checkerboard synergy was performed as described previously (2). Fractional inhibitory concentrations (FICs) were calculated as (MIC of drug A or B in combination)/(MIC of drug A or B alone), and the FIC index was obtained by adding the FIC values. FIC indices were interpreted as synergistic if values were ≤0.5, additive or indifferent if >0.5 to 4.0 and antagonistic if >4.0 (1, 2, 8).
Three strains from each of the above four susceptibility groups were tested by time-kill as described previously (1, 2). All compounds were tested alone, and levofloxacin was tested in combination with cefpirome, ceftazidime, gentamicin, and meropenem. Viability counts were performed at 0, 6, 12, and 24 h. Drug carryover was addressed by dilution, as described previously (1, 2). In view of regrowth in many strains (which could have been selected in vitro) after 24 h, synergy was defined as a ≥2-log decrease in the viable count of the combination at 12 h compared to the more active of the two agents alone (8).
Results of microbroth MIC testing of each agent alone for the four organism groups as well as the miscellaneous group are presented in Table 1. As can be seen, high-level resistance to levofloxacin (≥8 μg/ml) was only seen in gentamicin-resistant strains; in other strains, MICs at which 90% of the isolates are inhibited (MIC90s) were ≤4 μg/ml.
TABLE 1.
Broth microdilution MIC50s and MIC90s of each agent alone
| Group (n) | MIC (μg/ml)a
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Levofloxacin
|
Cefpirome
|
Ceftazidime
|
Gentamicin
|
Meropenem
|
||||||
| 50% | 90% | 50% | 90% | 50% | 90% | 50% | 90% | 50% | 90% | |
| Susceptible (30)b | 0.5 | 4 | 8 | 16 | 2 | 8 | 2 | 4 | 1 | 2 |
| Resistant | ||||||||||
| Ceftazidime (26) | 2 | 4 | 64 | >128 | 128 | >128 | 0.5 | 4 | 1 | 4 |
| Gentamicin (21) | 8 | >32 | 8 | 16 | 8 | 8 | 32 | >64 | 1 | 4 |
| Meropenem (24) | 1 | 2 | 8 | 8 | 4 | 8 | 1 | 4 | 8 | 16 |
| Miscellaneous (23)c | 4 | 32 | 128 | >128 | 128 | >128 | 32 | 64 | 4 | 32 |
50% and 90%, MIC50R and MIC90, respectively.
Susceptible to cephalosporins, gentamicin, and meropenem.
Resistant to cephalosporins, gentamicin, and meropenem (n = 10); resistant to gentamicin and cephalosporins and susceptible to meropenem (n = 12); gentamicin susceptible, resistant to cephalosporins and meropenem (n = 1).
Checkerboard titration results are listed in Table 2. Synergistic FIC indices (≤0.5) were found in nine strains (7.3%) (three fully susceptible, three resistant to ceftazidime, three miscellaneous) with levofloxacin-cefpirome, eight strains (6.5%) (three ceftazidime resistant, four meropenem resistant, one miscellaneous) with levofloxacin plus ceftazidime, one ceftazidime-resistant strain (0.8%) with levofloxacin-gentamicin, and seven strains (5.6%) (two fully susceptible, two ceftazidime resistant, one meropenem resistant, two miscellaneous) with levofloxacin plus meropenem. All other FIC indices were >0.5 to 2 (additive or indifferent), and no antagonism (FIC indices of >4) was found.
TABLE 2.
Results of checkerboard synergy testinga
| Group (n) | Result for combination with FIC index ofb:
|
|||||||
|---|---|---|---|---|---|---|---|---|
| Levofloxacin + cefpirome
|
Levofloxacin + ceftazidime
|
Levofloxacin + gentamicin
|
Levofloxacin + meropenem
|
|||||
| ≤0.5 | >0.5–4 | ≤0.5 | >0.5–4 | ≤0.5 | >0.5–4 | ≤0.5 | >0.5–4 | |
| Susceptible (30)c | 3 | 27 | 0 | 30 | 0 | 30 | 2 | 28 |
| Resistant | ||||||||
| Ceftazidime (26) | 3 | 23 | 3 | 23 | 1 | 25 | 2 | 24 |
| Gentamicin (21) | 0 | 21 | 0 | 21 | 0 | 21 | 0 | 21 |
| Meropenem (24) | 0 | 24 | 4 | 20 | 0 | 24 | 1 | 23 |
| Miscellaneous (23) | 3 | 20 | 1 | 22 | 0 | 23 | 2 | 21 |
No antagonistic FIC indices (>4) were found).
Results for FIC indices of ≤0.5 are synergistic, and those of >0.5 to 4.0 are additive or indifferent.
Susceptible to all compounds.
The results of time-kill synergy tests are listed in Table 3. Checkerboard titrations with these strains showed that one strain showed synergy with levofloxacin plus ceftazidime, and one showed synergy with levofloxacin plus meropenem. Time-kill synergy assays showed that levofloxacin, at sub-MIC concentrations of 0.06 to 0.5 μg/ml, showed synergy with cefpirome, ceftazidime, gentamicin, and meropenem in 10, 9, 4, and 11 strains, respectively.
TABLE 3.
Comparison of synergy testing by checkerboard and time-kill methodologies
| Strain | MIC (μg/ml)
|
Result for drug combination by methoda
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Levofloxacin | Cefpirome | Ceftazidime | Gentamicin | Meropenem | Levofloxacin + cefpirome
|
Levofloxacin + ceftazidime
|
Levofloxacin + gentamicin
|
Levofloxacin + meropenem
|
|||||
| C | T | C | T | C | T | C | T | ||||||
| Susceptible to all compounds | |||||||||||||
| 1 | 8 | 8 | 2 | 4 | 1 | Ad | Ad | Ad | Sy (0.5/8.0) | Ad | Ad | Ad | Sy (0.5/0.5) |
| 2 | 0.5 | 8 | 2 | 2 | 1 | Ad | Sy (0.125/2) | Ad | Sy (0.125/1) | Ad | Ad | Ad | Sy (0.125/0.25) |
| 3 | 1 | 8 | 4 | 2 | 1 | Ad | Sy (0.125/4) | Ad | Ad | Ad | Ad | Ad | Sy (0.125/0.25) |
| Resistant to cefpirome (≤8.0 μg/ml) and ceftazidime | |||||||||||||
| 4 | 0.5 | 64 | 64 | 0.5 | 1 | Ad | Sy (0.125/16) | Ad | Sy (0.125/32) | Ad | Ad | Ad | Sy (0.125/0.5) |
| 5 | 2 | 64 | 128 | 2 | 1 | Ad | Sy (0.5/16) | Ad | Sy (0.5/16) | Ad | Ad | Ad | Sy (0.5/0.5) |
| 6 | 0.5 | 128 | 128 | 0.5 | 2 | Ad | Sy (0.25/64) | Ad | Sy (0.25/64) | Ad | Sy (0.25/0.25) | Ad | Sy (0.25/1) |
| Resistant to gentamicin | |||||||||||||
| 7 | 2 | 8 | 8 | 64 | 0.5 | Ad | Sy (0.5/8) | Ad | Sy (0.5/2) | Ad | Ad | Ad | Sy (0.5/0.25) |
| 8 | 1 | 8 | 4 | 8 | 4 | Ad | Sy (0.25/4) | Ad | Sy (0.25/1) | Ad | Sy (0.25/1) | Ad | Sy (0.25/0.25) |
| 9 | 0.5 | 8 | 8 | 64 | 0.5 | Ad | Sy (0.06/2) | Ad | Sy (0.06/4) | Ad | Sy (0.06/16) | Ad | Sy (0.06/0.06) |
| Resistant to meropenem | |||||||||||||
| 10 | 1 | 8 | 2 | 4 | 8 | Ad | Ad | Ad | Ad | Ad | Ad | Ad | Ad |
| 11 | 2 | 8 | 8 | 2 | 16 | Ad | Sy (0.25/8) | Sy | Sy (0.25/4) | Ad | Sy (0.25/0.25) | Ad | Sy (0.25/2) |
| 12 | 0.125 | 8 | 1 | 1 | 16 | Ad | Sy (0.25/2) | Ad | Ad | Ad | Ad | Sy | Sy (0.25/0.125) |
C, checkerboard titration; T, time-kill; Sy, synergistic; Ad, additive or indifferent. Values in parentheses are MICs and indicate the lowest concentration (micrograms per milliliter) of each compound that yielded sustained bactericidal activity (≥100-CFU/ml drop) at 12 h compared to that of the more active drug.
Levofloxacin yields MICs for all organisms which are 1 to 2 dilutions lower than those for ofloxacin (9, 19, 20). Our study confirms these findings. Of note in our study were the higher levofloxacin MICs for strains resistant to gentamicin only. Recently, NCCLS has approved breakpoints of ≤2.0 μg/ml (susceptible), 4.0 μg/ml (intermediate), and ≥8.0 μg/ml (18). Recent studies have documented MIC50s of 0.5 to 1.0 μg/ml and MIC90s of 2.0 to 8.0 μg/ml for P. aeruginosa (9, 20). Of broad-spectrum cephalosporins with activity against P. aeruginosa, cefpirome has been reported to have a MIC50 of 2.0 to 16.0 μg/ml and a MIC90 of 8.0 to 16.0 μg/ml (5, 10, 14). Gargalianos et al. (10) have demonstrated the range of cefpirome MICs to be 1.0 to 16.0 μg/ml in P. aeruginosa strains with increased non-β-lactamase-mediated resistance to carbenicillin, plasmid-mediated β-lactamase production, and partially derepressed chromosomal β-lactamase expression. Two strains with totally derepressed chromosomal β-lactamase expression yielded cefpirome MICs of 16.0 and 32.0 μg/ml, respectively. In all of the latter resistance groups, ceftazidime MIC ranges were <0.5 to 32.0 μg/ml (10).
Ceftazidime MICs for P. aeruginosa generally correspond with those of cefpirome (3, 5, 10, 14). This was also the case in our study. Although a small percentage of P. aeruginosa strains are resistant to ceftazidime, widespread use of this compound in the United States has not led to a significant rise in ceftazidime resistance (3). Although gentamicin was originally very active against P. aeruginosa strains, resistance is common in most hospital settings (6, 11, 15, 16).
Meropenem, a recently developed parenteral carbapenem, is very active against P. aeruginosa, with MIC50s of 0.25 to 0.5 μg/ml and MIC90s of 1.0 to 4.0 μg/ml for imipenem-susceptible strains. The in vitro activity of meropenem is greater than that of imipenem (7, 13, 16). Against a series of P. aeruginosa strains with well-characterized resistance mechanisms, meropenem retained high-level activity against strains with the more common types of resistance mechanisms known to affect other β-lactams. Resistance to meropenem may not arise as readily in P. aeruginosa as it does with most other β-lactams (16).
Our findings that time-kill tests for synergy were more discriminatory than the checkerboard methodology reflect findings by our group and others for other organisms (1, 2, 4). Our study shows that levofloxacin, in sub-MIC concentrations of ≤0.5 μg/ml, was synergistic at 12 h, when combined with cefpirome, ceftazidime, or meropenem in 9 to 11 strains, and had lower synergy rates when combined with gentamicin. Clinical studies are necessary to test the validity of these in vitro findings, as well as the significance of regrowth after 24 h.
Acknowledgments
This study was supported by a grant from Hoechst-Marion-Roussel, Clinical Pharmacology and Anti-infectives, Romainville, France.
We thank D. Livermore for provision of some strains.
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