Antimicrobial Activities of Gatifloxacin against Nosocomial Isolates of Stenotrophomonas maltophilia Measured by MIC and Time-Kill Studies

Abstract

We determined in vitro activities of gatifloxacin and seven other drugs against 100 isolates of Stenotrophomonas maltophilia using the agar gradient diffusion (Etest) method. Percentages of susceptible isolates were as follows: trimethoprim-sulfamethoxazole, 90%; gatifloxacin, 71%; levofloxacin, 57%; ticarcillin-clavulanic acid, 54%; ceftazidime, 49%; ciprofloxacin, 29%; cefepime, 21%; and piperacillin-tazobactam, 20%. Time-kill studies of three isolates indicated that gatifloxacin was bactericidal at times as early as 3 h of incubation when tested at concentrations equivalent to twice the MIC (two isolates) and 4 times the MIC (one isolate).

Stenotrophomonas maltophilia is a nonfermentative gram-negative bacillus that is now emerging as one of the leading causes of nosocomial infections, especially in intensive-care units, where it is often second only to Pseudomonas aeruginosa in terms of the number of gram-negative bacterial pathogens recovered. This organism usually affects immunocompromised, ventilator-dependent, or debilitated patients, causing pneumonia, bacteremia, urinary tract infection, or other conditions (3, 5, 6, 12). Other risk factors include exposure to broad-spectrum antibiotics, prolonged hospitalization, underlying neoplasia, and use of intravascular devices (3, 6, 12). S. maltophilia is not considered a component of the normal flora of humans, and most systemic infections are thought to occur through exogenous routes that typically involve contaminated catheters, needles, or medical devices (3, 6, 12).

Management of S. maltophilia infections can be difficult due to its inherent multidrug resistance that affects many β-lactams, aminoglycosides, and most other drug classes to some extent as a result of enzymatic destruction of drug by β-lactamases or decreased outer membrane permeability (2). The drug of choice for use against S. maltophilia is trimethoprim-sulfamethoxazole followed by ticarcillin-clavulanic acid. Combination therapy with both agents may be synergistic, but resistance to each also occurs (11). Trimethoprim-sulfamethoxazole is only bacteriostatic for the majority of S. maltophilia isolates; therefore, higher doses are usually used, increasing the potential for toxicity. Hypersensitivity to sulfonamides in some patients limits the use of this drug, as does the occasional development of resistance (11).

Previous in vitro studies of fluoroquinolones suggest that these agents should be considered as potential treatment alternatives, and they are sometimes used, albeit few data regarding clinical efficacy have been reported (11). In vitro fluoroquinolone activity varies according to the agents used, with ciprofloxacin activity being generally lower than those of others such as levofloxacin, gatifloxacin, trovafloxacin, and moxifloxacin (1, 2, 4, 6, 7, 9, 10, 11). In view of the increasing use of fluoroquinolones to treat serious nosocomial infections and concerns about diminishing antimicrobial susceptibilities to these agents over time, we performed an in vitro evaluation of one of the newest fluoroquinolones, gatifloxacin, in comparison with ciprofloxacin, levofloxacin, and five other agents against 100 nonduplicate isolates of S. maltophilia obtained from, patients hospitalized at the University of Alabama at Birmingham Medical Center during 1998 and 1999.

Organisms were identified using the MicroScan WalkAway 96 (Dade MicroScan, West Sacramento, Calif.). Bacterial isolates were stored frozen at −70°C until testing for susceptibility. Fifty-nine organisms were obtained from intensive-care-unit patients. The primary body sites of isolation were as follows: respiratory tract (54 isolates), urine (15 isolates), wounds (6 isolates), and others (25 isolates). Growth from overnight cultures on Trypticase soy agar with 5% sheep blood (Remel, Lenexa, Kans.) was suspended in 3 ml of saline to a 0.5 McFarland turbidity standard and used to inoculate 150-mm-diameter Mueller-Hinton agar plates (Remel), streaking in three directions to yield confluent growth. Etest strips (AB BIODISK, Solna, Sweden) were applied according to the manufacturer's instructions in a radial fashion. Drugs tested included gatifloxacin, ciprofloxacin, levofloxacin, trimethoprim-sulfamethoxazole, piperacillin-tazobactam, ticarcillin-clavulanate, cefepime, and ceftazidime. Agar plates were incubated aerobically at 35°C for 24 h. MICs were read where complete inhibition of growth intersected the strips, according to the manufacturer's instructions, by using a magnifying glass. All MICs were rounded up to the next twofold dilution value for recording purposes. MICs were interpreted according to, NCCLS criteria for non-Enterobacteriaceae (8). MIC breakpoints used for gatifloxacin were ≤2, 4, and 8 μg/ml to designate susceptible, intermediate, or resistant isolates, respectively. P. aeruginosa ATCC 27853 was used for quality control.

Three isolates for which gatifloxacin MICs were 0.125, 1, and 2 μg/ml, were chosen for time-kill studies. Isolates for which these MICs were obtained were selected because they are considered susceptible in vitro and the MICs are equivalent to or below achievable concentrations of the drug in serum. Organisms grown overnight on blood agar plates were diluted in 3 ml of saline to a 0.5 McFarland turbidity standard. The bacterial suspension (0.05 ml) was further diluted in 5 ml of Mueller-Hinton broth (Remel) to yield a concentration of approximately 10⁶ CFU/ml, verified by plate counts. Gatifloxacin powder was dissolved and prepared for in vitro testing according to instructions from the manufacturer (Bristol-Myers Squibb, Princeton, N.J.) and in compliance with NCCLS guidelines (8). The diluted suspension (0.05 ml) was inoculated into tubes containing 5 ml of Mueller-Hinton broth and gatifloxacin concentrations corresponding to the MIC and 0.5, 2, 4, and 8 times the MIC for each isolate. An additional control tube was inoculated with bacteria without gatifloxacin. Broths were incubated aerobically at 35°C for 24 h. Aliquots (0.1 ml of broth) were removed from each tube, and serial dilutions were plated onto Trypticase soy agar with 5% sheep blood after 0, 3, 6, 12, and 24 h of incubation. Colony counts were performed after 48 h of incubation at 35°C. Bactericidal activity was defined as a ≥3-log₁₀-unit (99.9%) reduction compared with the initial inoculum (2).

Results of susceptibility tests are shown in Table 1. The distribution of gatifloxacin MICs is shown in Fig. 1.

TABLE 1.

Susceptibilities of 100 S. maltophilia isolates to eight antimicrobial agents

Drug	MIC (μg/ml)a			% Susceptible isolates
	Range	50%	90%
Trimethoprim-sulfamethoxazole	0.125–>32	0.5	2	90
Gatifloxacin	0.064–>32	1	16	71
Levofloxacin	0.064–>32	2	32	57
Ciprofloxacin	0.016–>32	4	>32	29
Ticarcillin-clavulanic acid	1–>256	16	>256	54
Piperacillin-tazobactam	1–>256	>256	>256	20
Cefepime	0.064–>256	32	>256	21
Ceftazidime	0.25–>256	16	>256	49

^a50% and 90%, MIC₅₀ and MIC₉₀. 

FIG. 1.

MIC distribution for gatifloxacin tested against 100 S. maltophilia isolates.

Trimethoprim-sulfamethoxazole, with 90% of isolates testing susceptible and with MICs at which 50 and 90% of isolates were inhibited (MIC₅₀ and MIC₉₀) of 0.5 and 2 μg/ml, respectively, for the trimethoprim component, was the drug to which the largest number of isolates were susceptible. Gatifloxacin was second, with 71% of isolates susceptible and with a MIC₅₀ and MIC₉₀ of 1 and 16 μg/ml, respectively. An additional 16 isolates were intermediately resistant to gatifloxacin (MIC = 4 μg/ml), and the remaining 13 were fully resistant (MIC ≥ 8 μg/ml). Other than these two agents, only levofloxacin and ticarcillin-clavulanate had more than 50% of isolates testing susceptible. Gatifloxacin was twofold more potent than levofloxacin and at least fourfold more potent than ciprofloxacin. Among 10 strains resistant to trimethoprim-sulfamethoxazole, 3 (30%) were susceptible to gatifloxacin, in comparison to 2 each for levofloxacin, ciprofloxacin, piperacillin-tazobactam, and ceftazidime, and 1 for ticarcillin-clavulanate and cefepime.

Time-kill studies (Table 2) showed that gatifloxacin was bactericidal against S. maltophilia after as few as 3 h of incubation at concentrations equivalent to twice the MIC (two isolates) and 4 times the MIC (one isolate). No regrowth (defined as an increase of ≥2 log₁₀ CFU/ml) of any of the three isolates tested occurred after 24 h with any concentration of gatifloxacin greater than the MIC. An initial decrease in the colony count was observed in testing at a concentration equivalent to 0.5 times the MIC, but bactericidal activity was not detected and there was evidence of regrowth of two isolates after 24 h of incubation.

TABLE 2.

Time-kill results for gatifloxacin tested against three S. maltophilia isolates

Concn of drug	No. of isolates against which gatifloxacin showed bactericidal activity at:
	3 h	6 h	12 h	24 h
0.5× MIC	0	0	1	0
MIC	0	1	1	1
2× MIC	2	2	2	2
4× MIC	2	3	3	3

Previous in vitro studies have indicated reasonably good in vitro activity of gatifloxacin against S. maltophilia (1, 10), but none has assessed the bactericidal activity of gatifloxacin using time-kill methodology. One recent publication (2) showed that levofloxacin, but not ciprofloxacin, was bactericidal, indicating, however, that all fluoroquinolones may not behave in the same way against this organism. Interpretation of the time-kill data requires some knowledge of the pharmacokinetics of gatifloxacin, but relatively few studies have addressed this issue. Wise et al. (13) reported a mean peak level of 4.1 μg/ml for gatifloxacin at 1.8 h after a 400-mg oral dose given to nine healthy persons. The mean terminal elimination half-life of gatifloxacin in plasma measured in that study was 6.8 h (range, 6.3 to 8.4 h). The area under the plasma concentration curve up to the last measurable concentration (AUC_last) and the AUC extrapolated to infinity (AUC_0–∞) were 27.9 and 31.4 mg · hr/liter, suggesting that gatifloxacin may potentially be useful for treating S. maltophilia strains that show in vitro susceptibility.