Evaluation of fluorescence-based amplified fragment length polymorphism analysis for molecular typing in hospital epidemiology: comparison with pulsed-field gel electrophoresis for typing strains of vancomycin-resistant Enterococcus faecium.

Fluorescence-based amplified fragment length polymorphism (fbAFLP) is a novel assay based on the fluorescent analysis of an amplified subset of restriction fragments. The fbAFLP assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The ligation of adapters with primer-specific sites coupled with primers containing selective nucleotides allowed the full potential of PCR to be realized while maintaining the advantages of restriction endonuclease analysis. Fluorescence-based fragment analysis with polyacrylamide gel electrophoresis provides the accurate band sizing required for homology assessment. The large number of phylogenetically informative characters obtained by fbAFLP is well suited for cluster analysis and database development. The method demonstrated excellent reproducibility and ease of performance and interpretation. We typed 30 epidemiologically well-characterized isolates of vancomycin-resistant enterococci from an outbreak in a university hospital by fbAFLP. Clustering of fbAFLP data matched epidemiological, microbiological, and pulsed-field gel electrophoresis data. This study demonstrates the unprecedented utility of fbAFLP for epidemiological investigation. Future developments in standardization and automation will set fbAFLP as the "gold standard" for molecular typing in epidemiology.

Comparison of three methods to recover vancomycin-resistant enterococci (VRE) from perianal and environmental samples collected during a hospital outbreak of VRE.

OBJECTIVE: To establish an efficient and sensitive technique for recovering vancomycin-resistant enterococci (VRE) from perianal and environmental samples collected during implementation of control measures for an outbreak of VRE. DESIGN: Perianal and environmental samples were collected in triplicate on sterile swabs. One swab was used to inoculate a selective broth medium containing 6 pg of vancomycin and 8 pg of ciprofloxacin per mL, one to inoculate Campylobacter agar containing 10 microg/mL of vancomycin, and one to inoculate Enterococcosel agar containing 8 microg/mL of vancomycin. SETTING: Samples were collected in the intensive care units of a 600-bed university hospital over a period of 2 months. SAMPLE SELECTION: Patients and their immediate environment were sampled if they resided in a ward with a patient known to be colonized or infected with VRE. RESULTS: Of the 88 perianal samples obtained from 63 patients, 37 were positive for VRE by broth culture, with 36 also recovered on both types of solid media (sensitivity, 97.3%; negative predictive value, 98.1%). Of the initial samples collected from each of the 63 patients, 20 were positive for VRE by all methods. Of the 500 environmental samples cultured, 139 were positive for VRE in broth, with only 33 recovered on Campylobacter agar (sensitivity, 23.7%; negative predictive value, 77.2%) and 22 on Enterococcosel agar (sensitivity, 15.8%; negative predictive value, 75.2%). CONCLUSIONS: Our data indicate that, when performing surveillance cultures during an outbreak of VRE, use of an enrichment broth medium is required to recover VRE contaminating environmental surfaces; however, direct inoculation to selective solid medium is adequate to recover VRE in patient perianal specimens.

Comparison of the Vitek GPS-TB card with disk diffusion testing for predicting the susceptibility of enterococci to vancomycin.

OBJECTIVE: To compare the ability of the Vitek GPS-TB card with disk diffusion testing for determining the susceptibility of enterococci to vancomycin. DESIGN: Vitek susceptibility testing was performed using the GPS-TB card and software version R05.03. Disk diffusion susceptibility testing was performed according to National Committee for Clinical Laboratory Standards guidelines. When discrepancies occurred between the interpretation of Vitek and disk diffusion, both tests were repeated and the epsilometer test (E test) and agar screen containing 6 microgram/mL vancomycin were performed. RESULTS: Of 415 isolates tested, 313 were susceptible to vancomycin and 97 were resistant to vancomycin by both test methods. Two isolates were intermediate by Vitek and resistant by disk diffusion, 2 were intermediate by Vitek and susceptible by disk diffusion, and 1 was susceptible by Vitek and intermediate by disk diffusion. All but 1 of these latter 5 isolates (intermediate by Vitek and susceptible by disk diffusion) were available for retesting. On repeat testing, the 2 isolates that were intermediate by Vitek and resistant by disk diffusion were resistant by both methods, the 1 isolate that was intermediate by Vitek and susceptible by disk diffusion was susceptible by both methods, and the isolate that was susceptible by Vitek and intermediate by disk diffusion was also susceptible by both methods. These results were confirmed by E test and agar screen. CONCLUSION: We found the results of the GPS-TB card compared well with disk diffusion. However, isolates with intermediate results by Vitek should be retested using another method, such as the E test.