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In vitro Activity Against Stenotrophomonas maltophilia: Single Versus Combination Agents
Eljim P. Tesoro*
Steven J. Martin‡
Susan L. Pendland*
†The University of
This study was presented in part in poster form at the
37th Interscience Conference on Antimicrobial Agents and Chemotherapy
KEY WORDS: Stenotrophomonas maltophilia, antibiotics, in vitro, cotrimoxazole, ticarcillin-clavulanate, ofloxacin, doxicycline
The activities of cotrimoxazole, ticarcillin-clavulanate, ofloxacin, and doxycycline were studied against four isolates of Stenotrophomonas maltophilia using time-kill assays at simulated peak serum concentrations. Against three susceptible strains, ofloxacin and ticarcillin-clavulanate were bactericidal alone; in combination, they were synergistic and bactericidal against one ticarcillin-clavulanate—resistant strain. The addition of co-trimoxazole to ticarcillin-clavulanate was also synergistic and bactericidal against the ticarcillin-clavulanate resistant strain. Of note, the addition of doxycycline to ticarcillin-clavulanate or ofloxacin produced antagonism in susceptible strains.
Stenotrophomonas maltophilia has been implicated in an increasing number of opportunistic infections, especially in immunocompromised patients.1 Overuse of broad-spectrum antibiotics and prolonged antibiotic prophylaxis may correlate with the increased selective growth of this pathogen.1,2 Unfortunately, this gram-negative nosocomial pathogen is intrinsically resistant to commonly used antibiotics.3 Decreased outer membrane permeability and the production of two types of beta-lactamases have rendered most cephalosporins, aminoglycosides, and carbapenems ineffective as treatment options.4
Cotrimoxazole is considered the drug-of-choice for S. maltophilia infection.5 There have been reports of additional benefits when using combination therapy, especially with cotrimoxazole and ticarcillin clavulanate.6,7 This study examined the activity of several antibiotics, alone and in combinations against S. maltophilia.
Four clinical isolates of S. maltophilia were obtained from the microbiology laboratory at the University of Illinois at Chicago Medical Center (UICMC; Chicago, IL). Macrobroth minimum inhibitory concentrations (MICs) were determined according to NCCLS guidelines for cotrimoxazole, ticarcillin-clavulanate, doxycycline, and ofloxacin.8 Time-kill studies were performed in accordance with NCCLS guidelines;9 however, simulated peak serum concentrations were used instead of multiples of the MIC, because the latter are unlikely to be achievable in vivo against resistant strains. Antibiotic concentrations (mg/mL) tested were co-trimoxazole (trimethoprim:sulfamethoxazole), 4:76; ofloxacin, 4; ticarcillin/clavulanate, 190/2; doxycycline, 4. Antibiotics were then added singly and in the following combinations: ticarcillin-clavulanate + co-trimoxazole, ticarcillin-clavulanate + ofloxacin, ticarcillin-clavulanate + doxycycline, co-trimoxazole + doxycycline, ofloxacin + doxycycline.
After inoculation, aliquots of each mixture were diluted and plated using an automatic spiral plater (WASP, Bioscience International, Frederick, MD) at 0, 2, 4, 6, 8, and 24 hours. Plates were incubated at 35˚C for 24 hours, and colonies counted using an automatic colony counter (Protos, Bioscience International, Frederick, MD). All experiments were performed in duplicate. Antibiotic-free controls were included in all experiments. The lower limit of detection was 1.3 log10 CFU/mL. Bactericidal activity was defined as a ³ 3 log10 decrease in CFU/mL from time-zero.9 Synergy was defined as a ³ 2 log10 decrease in CFU/mL at 24 hours with combination agents as compared with the single most active agent.9 Antagonism was defined as a ³ 2 log10 increase in CFU/mL when in combination as opposed to the single most active agent.9
MIC data for the four clinical isolates are shown in Table 1. All strains were susceptible to cotrimoxazole and doxycycline. Strain No. 57 was resistant to ticarcillin-clavulanate and intermediately susceptible to ofloxacin. Strain No. 86 was intermediately susceptible to ticarcillin-clavulanate.
The activities of the various agents against the 4 strains are shown in Figure 1. Cotrimoxazole alone had no effect in time-kill assays; growth was similar to that of the antibiotic-free controls. Doxycycline alone was bacteriostatic. Ticarcillin-clavulanate and ofloxacin were rapidly bactericidal against all isolates except strain No. 57, which grew similar to controls against both agents.
Synergistic activity was observed with ticarcillin-clavulanate when combined with ofloxacin or cotrimoxazole against the strain (No. 57) intermediately susceptible to ofloxacin and resistant to ticarcillin-clavulanate. Other combinations had similar effects to either drug alone (bacteriostatic) against this strain. Against the sensitive strains, the combinations of ticarcillin-clavulanate + ofloxacin, ticarcillin-clavulanate + co-trimoxazole, and doxycycline + co-trimoxazole showed similar bacteriostatic activity to either drug alone. Finally, the combinations of doxycycline + ticarcillin-clavulanate and doxycycline + ofloxacin were antagonistic against three susceptible strains and two susceptible strains, respectively.
Cotrimoxazole and, to a lesser extent, ticarcillin-clavulanate are drugs-of-choice for S. maltophilia; however, there are no controlled trials of therapy for S. maltophilia infection in humans, and mortality remains high.5,10 Muder et al.2 reported 91 cases of S. maltophilia bacteremia in patients with underlying malignancy, immunosuppressive therapy, and other risk factors for opportunistic infection. Despite 94% of patients receiving an antimicrobial agent to which the blood isolate was susceptible, 14-day mortality was 21%.2 Thus, optimal antimicrobial therapy for S. maltophilia remains elusive. The purpose of the present study was to examine several different antibiotics alone and in combination to identify the best in vitro activity. The drugs chosen for the study reflected the formulary at UICMC at the time of this project. The organisms studied were chosen for their variation in susceptibility to the drugs studied.
In the present study, time-kill assays and MIC susceptibility testing did not correlate well with S. maltophilia. Cotrimoxazole showed susceptibility by the broth macrodilution method, but the drug had no effect in time-kill testing. Others have reported similar results.11 This dichotomy may be due to the lack of an intact immune response in vitro.6,12 Doxycycline produced a classic inhibition of growth against all strains throughout the 24-hour study period. Although clinical data with doxycycline are lacking, there are reports suggesting that tetracycline is not efficacious against S. maltophilia.5 However, doxycycline has shown greater in vitro activity than tetracycline against this opportunistic pathogen, and therefore, warranted further study.
The two bactericidal agents, ticarcillin-clavulanate and ofloxacin, were highly active against three of the four isolates tested, with rapid reduction in colony counts to below the lower limits of detection. The potent activity of ticarcillin-clavulanate seen in the present study may be due to the high concentration tested, because others have reported regrowth and the emergence of resistance when tested in an in vitro pharmacodynamic chamber model.13 When tested at a lower concentration (2 mg/mL), ofloxacin has also been reported to exhibit regrowth of S. maltophilia after 8 hours.14 In the present study, ofloxacin was bacteriostatic even against the intermediately susceptible strain. We found ofloxacin to produce the most rapid bacterial killing of any of the single agents tested. Newer fluoroquinolone agents may show even greater promise for treatment of S. maltophilia infections. Recent in vitro data from the SENTRY Antimicrobial Surveillance Program showed cotrimoxazole, ticarcillin-clavulanate, gatifloxacin, and trovafloxacin to be the most active agents against S. maltophilia.(3) In addition, synergistic activity has been reported when gatifloxacin was combined with ceftazidime, ticarcillin-clavulanate, or aztreonam against this opportunistic pathogen.15
In the present study, synergistic activity was difficult to show because of the potent activity of ticarcillin-clavulanate and ofloxacin. Against strain No. 57, which was intermediately susceptible to ofloxacin and resistant to ticarcillin-clavulanate, the combination of ticarillin-clavulanate plus ofloxacin or cotrimoxazole was synergistic. Synergy between cotrimoxazole and ticarcillin-clavulanate in resistant strains has been previously reported.6 Traub et al.7 studied three nonresistant strains (two susceptible and one intermediately-susceptible) and found synergy with only one of the susceptible strains when using the combination of cotrimoxazole and ticarcillin-clavulanate.
The antagonism seen in the present study between doxycycline and ticarcillin-clavulanate or ofloxacin may have been predicted. Both fluoroquinolones and tetracyclines must penetrate the cytoplasmic membrane to inhibit protein and nucleic acid synthesis, respectively. Theoretically, this antagonism may be attributed to competitive inhibition of active transport sites, resulting in a lower intracellular concentration of both agents. However, contrary to our results with ofloxacin and doxycycline, Gradelski et al.15 reported synergy with the combination of gatifloxacin and minocycline against one strain of S. maltophilia. The synergy was seen when sub-MIC concentrations of the agents were tested.
In summary, we found the combination of ticarcillin-clavulanate and cotrimoxazole or ofloxacin to be synergistic and bactericidal for one ticarcillin-clavulanate resistant strain of S. maltophilia. As single agents, ticarcillin-clavulanate and ofloxacin were bactericidal against the three remaining strains. The combination of ticarcillin-clavulanate and cotrimoxazole was also bactericidal at 24 hours, although the rate of kill was reduced against two of the strains. Similar results were seen with the combination of ticarcillin-clavulanate and ofloxacin. The significance of the slowing killing rate with the combinations is unknown. Clinical studies are needed to discern the most effective regimen for infections due to these opportunistic pathogens. However, based on the in vitro data, caution is warranted with the addition of doxycycline to bactericidal agents due to the potential for antagonistic interactions.
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Table 1. MICs (µg/mL) of Cotrimoxazole, Ticarcillin-Clavulanate, Ofloxacin, and Doxycycline Against 4 Clinical Strains of S. maltophilia
No. 19 No. 30 No. 57 No. 86
Co-trimoxazole* <0.25:4.75 0.5:9.5 <0.25:4.75 <0.25:4.75
Ticarcillin-clavulanate† 16:2 16:2 128:2 32:2
Ofloxacin‡ 2 2 4 1
Doxycycline§ 4 2 4 2
*NCCLS breakpoints: 2:38 sensitive, 4:76 resistant
†NCCLS breakpoints: 16:2 sensitive, 32:2–64:2 intermediate, 128:2 resistant
‡NCCLS breakpoints: 2 sensitive, 4 intermediate, 8 resistant
§NCCLS breakpoints: 4 sensitive, 8 intermediate, 16 resistant
Figure 1. Time-kill curves of single versus combination agents against 4 clinical strains of S. maltophilia. (TS = trimethoprim-sulfamethoxazole, TC = ticaricillin-clavulanate, O = ofloxacin, D = doxycycline, Limit = Lower Limit of Detection).
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