In vitro activities of antimicrobial combinations against Stenotrophomonas (Xanthomonas) maltophilia.

Stenotrophomonas (Xanthomonas) maltophilia is inherently resistant to multiple antimicrobial agents. In order to investigate the in vitro potential of combinations of antimicrobial agents, we obtained 230 epidemiologically unrelated clinical isolates from seven hospitals across Canada and from Northwestern Memorial Hospital in Chicago. Ticarcillin-clavulanate combined with ciprofloxacin or trimethoprim-sulfamethoxazole were assayed for synergy against 31 ticarcillin-resistant strains of S. maltophilia by using microtiter checkerboard panels and against 20 strains by using time-kill methodology. The combination of ciprofloxacin with ceftazidime was also evaluated by time-kill studies. Ticarcillin-clavulanate plus trimethoprim-sulfamethoxazole demonstrated synergy by checkerboard panels, with fractional inhibitory concentration indices ranging from 0.033 to 0.49, and by time-kill studies for all 20 strains tested. Synergy between ticarcillin-clavulanate plus ciprofloxacin was found by the checkerboard method for 24 of 31 strains (77%), with fractional inhibitory concentration indices ranging from 0.188 to 0.75. A correlation between synergy by the checkerboard method and the reference time-kill study method was not observed for ticarcillin-clavulanate plus ciprofloxacin, with results for 3 of 10 strains being nonconcordant. Synergy with both ticarcillin-clavulanate plus ciprofloxacin and ceftazidime plus ciprofloxacin by the time-kill method was found to correlate with ciprofloxacin MICs of <32 micrograms/ml and zone diameters of >15 mm on Mueller-Hinton agar. Evaluation of these combinations in vivo may be warranted.     

Levofloxacin in vitro activity and time-kill evaluation of Stenotrophomonas maltophilia clinical isolates

The in vitro activity of levofloxacin and eight other antimicrobial agents against 60 clinical isolates of Stenotrophomonas maltophilia was determined by an agar dilution method using 10(4) and 10(6) cfu/spot inocula. At the lower inoculum, 85.0% of the isolates were susceptible to levofloxacin but only 58.3% were susceptible to ofloxacin; at the higher inoculum, 78.3% were susceptible to levofloxacin and 36.7% to ofloxacin. In time-kill studies, levofloxacin exerted bactericidal activity within 4 h. With ofloxacin and ciprofloxacin bacterial regrowth was observed after 8 h. Levofloxacin may represent an alternative drug in the treatment of infections caused by S. maltophilia.     

In vitro susceptibility of Stenotrophomonas maltophilia to various antimicrobial combinations.

Stenotrophomonas maltophilia has emerged as a significant pathogen in compromised patients, causing infections which are difficult to treat. Clinical isolates from patients in the Tucson area were tested against single and combination antibiotics using three testing methods. Ticarcillin/clavulanate, trimethoprim/sulfamethoxazole and trovafloxacin provided comparable inhibitory activity, in vitro. Ciprofloxacin, imipenem and ticarcillin were active less often. Agreements between disk diffusion and broth microdilution results were poor for ciprofloxacin and trimethoprim/sulfamethoxazole; however, agreement was > or = 90% for the other drugs tested. Major or very major errors were observed with ticarcillin, ticarcillin/clavulanate, and trovafloxacin. The addition of aztreonam to ticarcillin/clavulanate enhanced the activity compared to ticarcillin/clavulanate alone using the double-disk diffusion, broth microdilution (checkerboard), and time-kill testing methods. Trovafloxacin exhibited good activity by all three methods, with bactericidal activity at > or = 2x MIC. These results indicate that the newer fluoroquinolones or the triple combination of ticarcillin/clavulanate plus aztreonam may be potential options for treatment of infection caused by S. maltophilia in patients who are intolerant to or fail trimethoprim/sulfamethoxazole therapy.     

Comparison of various in vitro susceptibility methods for testing Stenotrophomonas maltophilia

A total of 57 clinical isolates were screened by disk diffusion for a related pharmacodynamic study. Testing was performed using National Committee for Clinical Laboratory Standards guidelines, except that results were interpreted at 16 to 18 h and 48 h. Of the 57 isolates, 19 were randomly chosen for additional comparative susceptibility testing of five methods (disk diffusion, Etest, Alamar colorimetric broth microdilution, Vitek, and MicroScan) and an in-house broth microdilution method. The two diffusion methods (disk and Etest) had the closest correlation. The commercial broth microdilution methods and the in-house microdilution method generated inconsistent results for all agents except trimethoprim-sulfamethoxazole. Vitek compared poorly with both diffusion and microbroth dilution methods. The most significant discrepancies were evident with all methods when the incubation period was extended to 48 h. When results were interpreted at 48 h, the incidence of resistance for all bactericidal agents was approximately double the resistance observed at 16 to 18 h. The bacteriostatic agents, trimethoprim-sulfamethoxazole and doxycycline, demonstrated the greatest in vitro activity and were least influenced by extended incubation with diffusion methods. Because correlative in vivo and in vitro studies have not revealed an effective therapeutic regimen for serious S. maltophilia infections, susceptibility results with all testing methods should be interpreted with caution when choosing therapy for patients with life-threatening infections. Susceptibility testing for this heterogeneous group remains controversial and routine testing, with the possible exception of doxycycline (or minocycline) and trimethoprim-sulfamethoxazole, should be avoided. Our data support that if testing is done with bactericidal agents, consideration should be given to interpretation after 48-h incubation.     

Antibiotic susceptibility profile of Xanthomonas maltophilia. In vitro activity of beta-lactam/beta-lactamase inhibitor combinations

The susceptibility of 42 strains of Xanthomonas (Pseudomonas) maltophilia to 37 antibiotics (mainly beta-lactams, aminoglycosides, and fluorinated quinolones) was tested. Xanthomonas maltophilia was resistant to most beta-lactams, with ceftazidime, moxalactam, and ICI-194008 being the most active ones. Aminoglycosides had a very modest activity, with quinolones showing only moderate activity against this species. Trimethoprim/sufamethoxazole was effective against all strains tested. We also tested the synergy of several beta-lactam/beta-lactamase inhibitors against X. maltophilia. Only aztreonam/clavulanic acid at 3:1, 1:1 and, mainly, 2:1 combinations had synergistic activity, decreasing the rate of resistance from 92.8% for aztreonam alone to 32.4% for aztreonam-clavulanic acid at 1:1 and 0% for aztreonam-clavulanic acid at 2:1.     

In vivo efficacies of beta-lactam-beta-lactamase inhibitor combinations against a TEM-26-producing strain of Klebsiella pneumoniae.

We examined the efficacies of the beta-lactam-beta-lactamase inhibitor combinations ampicillin-sulbactam and piperacillin-tazobactam in the treatment of intra-abdominal abscesses caused by a TEM-26-producing strain of Klebsiella pneumoniae. At lower doses, both combinations reduced abscess colony counts by more than 3 log10 CFU/g from that of untreated controls, but treatment with these drugs was inferior to treatment with imipenem. Increasing the doses of the combinations resulted in a further decrease in abscess CFU to a level where both were similar to imipenem in efficacy. These results suggest that the beta-lactam-beta-lactamase inhibitor combinations ampicillin-sulbactam and piperacillin-tazobactam may be viable alternatives for the treatment of serious infections caused by susceptible extended-spectrum beta-lactamase-producing strains of K. pneumoniae.     

In vitro activities of membrane-active peptides alone and in combination with clinically used antimicrobial agents against Stenotrophomonas maltophilia

The in vitro activities of buforin II, cecropin P1, and magainin II, alone and in combination with six clinically used antimicrobial agents, against 12 clinical isolates of Stenotrophomonas maltophilia were investigated. Antimicrobial activities were measured by MIC and time-kill studies. The isolates were susceptible to the peptides at concentrations in the range of 0.50 to 16 microg/ml. Synergy was observed when the peptides were combined with polymyxin E, meropenem, ceftazidime, piperacillin, and clarithromycin.     

Susceptibilities of 123 strains of Xanthomonas maltophilia to eight beta-lactams (including beta-lactam-beta-lactamase inhibitor combinations) and ciprofloxacin tested by five methods.

This study evaluated the susceptibility of 123 Xanthomonas maltophilia strains to ticarcillin, ticarcillin-clavulanate, ampicillin, amoxicillin-clavulanate, ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, imipenem, and ciprofloxacin by Kirby-Bauer disk, E test, and Sensititre dehydrated microdilution MIC and conventional agar dilution MIC methodology. Intermediate susceptibility breakpoints for members of the family Enterobacteriaceae were used. When results were analyzed as MICs for 50 and 90% of the strains tested and percentages of strains susceptible at the breakpoint, good correlation between the methods was observed, with ticarcillin-clavulanate clearly the most active beta-lactam by all four methods. However, when the various methods were compared with the agar dilution methodology by regression analysis, poor r2 values (0.3 to 0.7) were obtained for compounds with sufficient on-scale values to permit analysis. When the number of strains with log2 ratios of reference agar dilution MICs to test MICs of +3 to -3 were analyzed, correlation was also poor, with many major and very major discrepancies for all methods tested. Results obtained with time-kill studies of nine strains with discrepant ticarcillin-clavulanate MICs appeared to correlate best when compared at 24 h with agar dilution MICs. The concentration of ticarcillin-clavulanate required to reduce the colony count by > or = 2 log10 reduction values for eight of nine strains compared with that for growth controls was < or = 16.0/2.0 micrograms/ml at 6 h and ranged from 16.0/2.0 micrograms/ml to 128.0/2.0 micrograms/ml at 24 h. The susceptibility method of choice for X. maltophilia has not yet been standardized, but time-kill studies correlated best with agar dilution MICs.     

In vitro susceptibility of Stenotrophomonas maltophilia isolates: comparison of disc diffusion, Etest and agar dilution methods

The disc diffusion, Etest and agar dilution techniques were compared to evaluate the antimicrobial susceptibility profile of 70 Stenotrophomonas maltophilia isolates to seven antimicrobial agents. The S. maltophilia isolates were consecutively collected from May 2000 to May 2002 from individual patients, who were hospitalized in a private Brazilian hospital. The antimicrobial susceptibility tests were carried out and interpreted according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. The Etest was carried out according to the manufacturer's instructions. There was good agreement among the distinct susceptibility testing results for chloramphenicol, doxycycline, gatifloxacin, trimethoprim-sulfamethoxazole and ticarcillin-clavulanate, suggesting that the disc diffusion and Etest methods are reliable for testing this group of antimicrobials against S. maltophilia. In contrast, a weak correlation was found between the disc diffusion and agar dilution techniques for testing polymyxin B and colistin with unacceptable very major error rates (18.1% and 22.7% for polymyxin B and colistin, respectively). Trimethoprim- sulfamethoxazole (MIC50, 0.06 mg/L; 98.5% susceptible) and gatifloxacin (MIC50, 0.12 mg/L; 98.5% susceptible) were the most potent antimicrobial agents tested against S. maltophilia isolates. In contrast, the worst in vitro activity was found for ticarcillin-clavulanate (MIC50, 16 mg/L; 59.1% susceptible). Although our results confirm that trimethoprim-sulfamethoxazole, gatifloxacin and doxycycline have an excellent in vitro activity against S. maltophilia, further clinical studies are necessary to evaluate the clinical efficacy of these compounds for the treatment of S. maltophilia infections, since no randomized controlled trials have been carried out and no correlation between the clinical response and susceptibility testing results has been reported.     

In vitro inhibitory and bactericidal activity of cefpiramide and seven antipseudomonal agents against Pseudomonas aeruginosa

Cefpiramide was tested against 493 clinical isolates of Pseudomonas aeruginosa and the results of minimal inhibitory concentration, minimal bactericidal concentration, bactericidal rate, and time-kill synergy studies were compared with those obtained with seven other antipseudomonal agents. The minimal inhibitory concentrations of cefpiramide for P. aeruginosa were comparable to all of the agents tested. Minimal bactericidal concentration results were generally within one twofold dilution of the minimal inhibitory concentration values for all agents tested. Bactericidal rate studies showed that at concentrations of four times the minimal inhibitory concentration, all of the agents produced rapid killing. Results of time-kill synergy studies showed a marked synergistic interaction between cefpiramide and each of three aminoglycosides, gentamicin, tobramycin, and amikacin. These results suggest that cefpiramide may be useful in the therapy of infections due to P. aeruginosa.     

Comparison of different antimicrobial susceptibility testing methods for Stenotrophomonas maltophilia and results of synergy testing

Accurate determination of resistance is important to ensure appropriate antimicrobial therapy in Stenotrophomonas maltophilia infections. This study was undertaken to evaluate the susceptibility results obtained by disc diffusion, E-test, Phoenix system, and reference agar dilution method and also to evaluate the in vitro activity of various antimicrobial combinations against multidrug-resistant S. maltophilia. Susceptibilities to several antimicrobial agents were determined by agar dilution, disc diffusion, and E-test according to the US Clinical Laboratory and Standards Institute (CLSI) guidelines. Results were also evaluated in the in Phoenix system for available agents. Twelve different antibiotic combinations were tested for synergy by the E-test method. Most synergic combinations were confirmed by microdilution checkerboard assay. Tigecycline, trimethoprim/sulfamethoxazole (TMP–SMX) and doxycycline were the most effective drugs against S. maltophilia. Poorest agreement was determined by disc diffusion and E-test against ticarcillin/clavulanate and ciprofloxacin (κ < 0.4), by disc diffusion against colistin (κ < 0.4), and by the Phoenix system against piperacillin/tazobactam (κ < 0.4). Based on these data, disc diffusion seems to be unreliable for ticarcillin/clavulanate, ciprofloxacin, and colistin; E-test for ticarcillin/clavulanate and ciprofloxacin; and the Phoenix system for piperacillin/tazobactam for S. maltophilia susceptibility testing. Synergistic activity was detected predominantly with TMP–SMX + ticarcillin/clavulanate and TMP–SMX + ceftazidime. TMP–SMX + ceftazidime synergy was also supported by the checkerboard method. However, TMP–SMX + ticarcillin/clavulanate combination revealed indifferent effect by the checkerboard assay. As ticarcillin/clavulanate and ciprofloxacin E-test results were beyond the acceptable correlation limits, synergy testing performed with these agents was considered as unreliable. Further studies are required to standardize susceptibility testing, especially for colistin, ticarcillin/clavulanate, and ciprofloxacin for S. maltophilia. TMP–SMX-containing drug combinations seemed to be more synergistic on multidrug-resistant S. maltophilia; however, these results merit further evaluation.     

In vitro activity of micafungin (FK-463) against Candida spp.: microdilution,time-kill,and postantifungal-effect studies

We evaluated the in vitro activity of the new echinocandin antifungal micafungin against Candida spp. using microdilution and time-kill methods. Additionally, we examined the postantifungal effect (PAFE) of micafungin. Finally, we evaluated the effect of the addition of serum and plasma on the MIC of micafungin. Four Candida albicans isolates and two isolates of each Candida glabrata, Candida krusei, and Candida tropicalis were selected for testing. The MICs of micafungin were determined in RPMI 1640 medium buffered with morpholinepropanesulfonic acid alone and with the addition of 10, 20, and 50% human serum and plasma. MICs were determined by using two endpoints: a prominent reduction in growth (the MIC at which 80% of isolates are inhibited [MIC(80)]) and complete visual inhibition of growth (MIC(100)). The minimum fungicidal concentration (MFC) of micafungin for each isolate was also determined. Time-kill curves were determined for each isolate in RPMI 1640 medium with micafungin at concentrations ranging from 0.125 to 16 times the MIC(80) to assess the correlation between MIC(80) and fungicidal activity. PAFE studies were conducted with each isolate by using concentrations ranging between 0.25 and 4 times the MIC(80). The MIC(80)s for the test isolates ranged from 0.0039 to 0.25 micro g/ml. Overall, the addition of serum or plasma increased the MIC 6 to 7 doubling dilutions for C. albicans and 3 to 4 doubling dilutions for C. krusei and C. tropicalis. Micafungin time-kill studies demonstrated fungicidal activity at concentrations ranging from 4 to 16 times the MIC(80). Micafungin is very potent agent against a variety of Candida spp., producing fungicidal activity against 7 of 10 isolates tested. A PAFE was observed against all isolates. The PAFE was influenced by the drug concentration, with the highest concentration resulting in the longest observed PAFE in each case. The highest concentration tested, four times the MIC, resulted in a PAFE of more than 9.8 h for 5 of 10 isolates tested (range, 0.9 to > or =20.1 h).     

Differences in the in vitro inhibitory and bactericidal activity of ceftizoxime, cefoxitin, cefotetan, and penicillin G against Bacteroides fragilis group isolates. Comparison of time-kill kinetic studies with MIC values

Nineteen strains of the Bacteroides fragilis group were used to determine the bactericidal activity of ceftizoxime, cefoxitin, cefotetan, and penicillin G with time-kill kinetics studies. Each antimicrobial agent was tested at subinhibitory (1/2 X MIC), inhibitory (1 X MIC), and suprainhibitory (4 X MIC) concentrations. Penicillin G exhibited virtually no sustained bactericidal activity at any of the antimicrobial concentrations tested. At subinhibitory concentrations, ceftizoxime was considerably more bactericidal than cefoxitin or cefotetan: At 12 hr, ceftizoxime killed 89% of the inoculum, whereas cefoxitin and cefotetan killed 35% and 33% of the inoculum, respectively. At inhibitory concentrations, ceftizoxime was again more bactericidal than cefoxitin and cefotetan: At 12 hr, ceftizoxime killed 90% of the inoculum, whereas cefoxitin and cefotetan killed 78% and 73%, respectively. At suprainhibitory concentrations, all three antimicrobial agents showed comparable bactericidal activity at 12 and 24 hr. Ceftizoxime and cefoxitin had somewhat lower killing rates overall against test strains with high MICs (greater than or equal to 32) versus low MICs (less than or equal to 16). However, at subinhibitory concentrations, ceftizoxime killed the B. fragilis group strains with high or low MIC values more effectively than cefotetan killed strains with low MICs. At the highest antibiotic concentrations tested (4 X MIC), only slight differences were seen in the bactericidal activity of the three compounds, regardless of MICs.     

In vitro synergy testing of macrolide-quinolone combinations against 41 clinical isolates of Legionella.

Combination antimicrobial therapy against Legionella species has not been well studied. Several quinolones have activity against Legionella strains, which prompted this in vitro search for a synergistic combination with the macrolides. By a checkerboard assay, erythromycin, clarithromycin, and azithromycin, each in combination with ciprofloxacin and levofloxacin, were tested for synergy against 46 isolates of Legionella. The agar dilution method was employed using buffered charcoal-yeast extract media. A final inoculum of 10(4) CFU per spot was prepared from 24-h growth of each isolate. Plates were incubated at 35 degrees C for 48 h. Synergy, partial synergy, additive effect, or indifference was observed for all combinations of antibiotics tested. There was no antagonism observed. Synergy occurred to a significantly greater extent for the clarithromycin-levofloxacin (P = 0.0001) and azithromycin-levofloxacin (P = 0.003) combinations versus erythromycin-levofloxacin. The azithromycin-ciprofloxacin combination demonstrated significantly greater synergy than did either erythromycin-ciprofloxacin (P = 0.003) or clarithromycin-ciprofloxacin (P = 0.001). The newer macrolides clarithromycin and azithromycin may be more active in combination with a fluoroquinolone than is erythromycin.