Antimicrobial Susceptibility Studies
Comparison of various in vitro susceptibility methods for testing Stenotrophomonas maltophilia

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Abstract

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.

Introduction

Stenotrophomonas (formerly Xanthomonas) maltophilia has emerged over the last decade as an important nosocomial pathogen. Hospitalized patients may become colonized with S. maltophilia; however, evidence exists that it is a major opportunistic pathogen in immunocompromised patients Arpi et al 1994, Elting and Bodey 1990, Irifune et al 1994, Khadori et al 1990, Laing et al 1995, Marshall et al 1989, Muder et al 1987, Vartivarian et al 1994a, Victor et al 1994, Zuravleff and Yu 1982. Factors which predispose patients to colonization and infection include broad-spectrum therapy, especially beta-lactam agents, long-term central venous catheterization, and hospitalization for greater than one week Arpi et al 1994, Elting and Bodey 1990, Khadori et al 1990, Laing et al 1995, Marshall et al 1989, Villarino et al 1992. Major disease syndromes include pneumonia, bacteremia, soft tissue infections, and urinary tract infections Elting and Bodey 1990, Irifune et al 1994, Khadori et al 1990, Laing et al 1995, Marshall et al 1989, Vartivarian et al 1994b, Victor et al 1994, Zuravleff and Yu 1982.

S. maltophilia is typically resistant to most beta-lactam antibiotics and aminoglycosides Chow et al 1988, Cullman 1991, Garcia-Rodriguez et al 1991, Garrison et al 1996, Neu et al 1989, Vartivarian et al 1994a. Resistance is attributed to several mechanisms: outer membrane protein alterations associated with efflux or reduced permeability and the production of two inducible beta-lactamases Bonfiglio and Livermore 1991, Cullman 1991, Paton et al 1994. Earlier released quinolones demonstrate variable in vitro susceptibility and their clinical effectiveness is questionable Chow et al 1988, Neu et al 1989, Vartivarian et al 1994b. Newer quinolone agents may have better efficacy (Visalli et al. 1997). Trimethoprim-sulfamethoxazole is frequently recommended for the treatment of serious S. maltophilia infections. Chloramphenicol, doxycycline, and minocycline have in vitro activity but the efficacy of these agents needs to be established by further clinical data Hohl et al 1991, Irifune et al 1994. Ticarcillin-clavulanate also appears to have excellent in vitro activity, but no controlled therapeutic trials have been done and isolates develop resistance rapidly (Garrison et al. 1996).

To date, no reliable guidelines are in place for S. maltophilia susceptibility testing. As the frequency of serious nosocomial infections caused by S. maltophilia increases, the resistance encountered with this organism forces laboratories to revisit the issue of appropriate susceptibility testing methods. Previous investigators have demonstrated the inconsistencies of disk diffusion susceptibility testing compared to agar dilution or broth microdilution Hohl et al 1991, Ringertz et al 1989. Especially problematic were agents such as ciprofloxacin and doxycycline Hohl et al 1991, Pankuch et al 1994, Ringertz et al 1989. Trailing endpoints and the presence of “inner colonies” within the zone of inhibition are problems associated with beta-lactam testing using broth microdilution and diffusion methods, respectively (Pankuch et al. 1994). Recent experience with the Etest (AB Biodisk, Solna, Sweden) demonstrated few very major or major errors compared to agar dilution. Resistant microcolonies were seen primarily when testing the aminoglycosides (Yao et al. 1995).

As a companion study to a recently published pharmacodynamic evaluation (Garrison et al. 1996), this study was conducted with two primary objectives. First, to characterize the impact of prolonged (48 h) versus standard (16 to 18 h) incubation on disk diffusion susceptibility of 57 clinical isolates of S. maltophilia. Secondly, to compare five commonly utilized susceptibility methods to an in-house microdilution MIC method. The five specific methods include: standard disk diffusion, Etest, Alamar broth microdilution method (Alamar/Sensititre, Sacramento, CA, USA), Vitek AMS GNS-F3, F6 (bioMerieux, Hazlewood, MO, USA), and MicroScan broth microdilution method (Dade International Corp., Sacramento, CA, USA).

Section snippets

Bacterial isolates

The 57 original isolates were obtained from patients at one of the participating institutions and other centers throughout the United States. Isolates were stored in brain heart infusion broth with 10% glycerol (Difco laboratories, Detroit, MI, USA) at −70°C until use. Prior to testing, isolates were removed from a permanent save vial and passed three times on Columbia sheep blood agar plates (Remel, Lenexa, KS, USA). Fifty-seven clinical isolates of S. maltophilia were tested by disk diffusion

Results

Disk susceptibility testing of the five quality control isolates of P. aeruginosa demonstrated no significant change in zone diameters measured at 16 to 18 h versus 48 h. Average reduction in zone diameter between the two incubation periods was: ceftazidime 1.2 mm, ciprofloxacin 1.4 mm, gentamicin 1.0 mm, and ticarcillin-clavulanate 2.0 mm. These slight reductions did not change the interpretive result of the control isolates. In addition, no inner zone colonies were seen.

Table 1 is a summary

Discussion

In the disk diffusion portion of our evaluation, 57 strains were tested using NCCLS performance standards, including the recommended incubation period. The tests were reinterpreted after an additional 24-h period. Pronounced changes in susceptibility were seen with respect to beta-lactams, aminoglycosides, and ciprofloxacin when plates were read at 48 h. Approximately two-thirds of the isolates appeared susceptible to these agents after 16 to 18 h, but at 48 h, the percentage of strains

Acknowledgements

The authors thank both AB Biodisk and the Alamar/Sensititre companies for donating Etest strips and Alamar panels, respectively, for a portion of this study. We also thank Steve Rittenhouse, Julia Moody, and Jeffrey Phillips for their assistance in providing S. maltophilia isolates. ????, Hawkey et al 1993

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