Use of random amplified polymorphic DNA PCR to examine epidemiology of Stenotrophomonas maltophilia and Achromobacter (Alcaligenes) xylosoxidans from patients with cystic fibrosis.

Stenotrophomonas maltophilia and Achromobacter (Alcaligenes) xylosoxidans have been increasingly recognized as a cause of respiratory tract colonization in cystic fibrosis (CF). Although both organisms have been associated with progressive deterioration of pulmonary function, demonstration of causality is lacking. To examine the molecular epidemiology of S. maltophilia and A. xylosoxidans in CF, isolates from patients monitored for up to 2 years were fingerprinted using a PCR-based randomly amplified polymorphic DNA (RAPD-PCR) method. Sixty-one of 69 CF centers screened had 183 S. maltophilia culture-positive patients, and 46 centers had 92 A. xylosoxidans-positive patients. At least one isolate from each patient was genotyped, and patients with > or =10 positive cultures (12 S. maltophilia cultures, 15 A. xylosoxidans cultures) had serial isolates genotyped. In addition, centers with multiple culture-positive patients were examined for evidence of shared clones. There were no instances of shared genotypes among different CF centers. Some patients demonstrated isolates with a single genotype throughout the observation period, and others had intervening or sequential genotypes. At the six centers with multiple S. maltophilia culture-positive patients and the seven centers with multiple A. xylosoxidans-positive patients, there were three and five instances of shared genotypes, respectively. The majority of shared isolates were from pairs who were siblings or otherwise epidemiologically linked. These findings suggest RAPD-PCR typing can distinguish unique CF isolates of S. maltophilia and A. xylosoxidans, person-to-person transmission may occur, there are not a small number of clones infecting CF airways, and patients with long-term colonization may either have a persistent organism or may acquire additional organisms over time.

Comparison of two commercial systems (Vitek and MicroScan-WalkAway) for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Antimicrobial susceptibility testing of cystic fibrosis (CF) isolates of Pseudomonas aeruginosa is difficult because the organisms are often mucoid and slow-growing. This study of 498 CF strains examined the correlation of results derived from two commonly used commercial systems (Vitek, MicroScan-WalkAway) with a reference method for 10 antimicrobials. Correlation to reference results was unacceptably low for all agents and both commercial systems had a high rate of very major (false-susceptible) errors. Although mucoid strains produced a 4.8% greater intermethod error, it was not markedly different than non-mucoid strains for the Vitek System. Overall, these tested commercial systems performed poorly for CF isolates in contrast to earlier reported, high correlations with the reference methods (broth microdilution frozen panels and agar dilution) of the National Committee for Clinical Laboratory Standards, the standardized disk diffusion test, and the Etest (AB BIODISK, Solna, Sweden).

Comparison of agar diffusion methodologies for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Pseudomonas aeruginosa is the most common pathogen infecting the lungs of patients with cystic fibrosis (CF). Improved antimicrobial chemotherapy has significantly increased the life expectancy of these patients. However, accurate susceptibility testing of P. aeruginosa isolates from CF sputum may be difficult because the organisms are often mucoid and slow growing. This study of 597 CF isolates of P. aeruginosa examined the correlation of disk diffusion and Etest (AB BIODISK, Solna, Sweden) results with a reference broth microdilution method. The rates of interpretive errors for 12 commonly used antipseudomonal antimicrobials were determined. The disk diffusion method correlated well (zone diameter versus MIC) for all of the agents tested. However, for mucoid isolates, correlation coefficients (r values) for piperacillin, piperacillin-tazobactam, and meropenem were <0.80. The Etest correlation with reference broth microdilution results (MIC versus MIC) was acceptable for all of the agents tested, for both mucoid and nonmucoid isolates. Category interpretation errors were similar for the disk diffusion and Etest methods with 0.4 and 0.1%, respectively, very major errors (false susceptibility) and 1.1 and 2.2% major errors (false resistance). Overall, both agar diffusion methods appear to be broadly acceptable for routine clinical use in susceptibility testing of CF isolates of P. aeruginosa.

Evaluation of reference dilution test methods for antimicrobial susceptibility testing of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis.

The development of multidrug-resistant Pseudomonas aeruginosa in patients with cystic fibrosis (CF) is most likely a consequence of increasing life expectancy and more prolonged exposure to antibiotics. The optimal method for antibiotic susceptibility testing of CF strains, particularly mucoid P. aeruginosa strains, is unknown. Antimicrobial susceptibilities of 48 CF strains (25 mucoid) and 50 non-CF strains to 12 anti-Pseudomonas agents were tested by both agar dilution and commercially custom-prepared broth microdilution plates (PML Microbiologicals, Portland, Oreg.) in three laboratories simultaneously to determine if broth microdilution could substitute for agar dilution as the reference method in subsequent studies. Comparison of MICs generated by agar dilution and broth microdilution demonstrated correlation coefficients (r) exceeding 0.85 for all agents tested; correlation was excellent for aminoglycosides (r >/= 0.92) and very good for beta-lactam agents including agents paired with a beta-lactamase inhibitor (r >/= 0.87) and for ciprofloxacin (r = 0.86). Correlation was not improved by 48-h readings, but correlation between 24- and 48-h readings ranged between 0.91 and 0.98 for both methods. Interlaboratory variations were minimal, as the percentage of acceptable variations was 94% for both methods, and serious discords were infrequent (<2% of comparisons). However, CF strains were more likely to have serious discords than were non-CF strains (P < 0. 0001), although mucoid strains were not more likely to have serious discords than were nonmucoid strains. In this study, MICs determined by custom-prepared broth microdilution compared favorably with MICs determined by agar dilution. Thus, this broth microdilution assay can serve as a reference method and facilitate future studies to determine the optimal method for antibiotic susceptibility testing of CF strains.

Microbiology of sputum from patients at cystic fibrosis centers in the United States.

During a phase III national collaborative study of aerosolized tobramycin (1 July 1995 through 30 September 1996), the microbiology of specimens from 595 patients at 69 cystic fibrosis (CF) centers was examined. Samples from three screening visits were processed in a single laboratory by means of standardized techniques for identification and susceptibility testing. From 1,753 pretreatment specimens, 5,128 pathogens were isolated (average, 2.9/specimen). Of the 3,936 Pseudomonas aeruginosa isolates, 56.7% were mucoid. The specimens of 125 patients (21.0%) yielded tobramycin-resistant P. aeruginosa (213 isolates); 61 (10.3%), Stenotrophomonas maltophilia; and 52 (8.7%), Alcaligenes xylosoxidans. Isolation of Burkholderia cepacia was an exclusion criterion. Only visit 3 sputum samples were cultured for gram-positive organisms and fungi (n = 465 patients); samples from 201 patients (43.2%) yielded Staphylococcus aureus (18.8% of isolates were oxacillin-resistant), and those from 114 (24.5%) yielded an Aspergillus species. Compared with the Cystic Fibrosis Foundation Patient Registry, the current study identified many more patients colonized with S. maltophilia, A. xylosoxidans, Aspergillus species, and oxacillin-resistant S. aureus, suggesting the utility of standardized processing of CF specimens.