Session: Poster Session: β-Lactamase Detection in the Clinical Laboratory
Saturday, October 25, 2008: 12:00 AM
Room: Hall C
Background: Conventional phenotyping requires large numbers of bacteria, which increases the total time-to-result. We report results for a new method that uses approximately 500 bacterial cells to detect the ESBL phenotype. Methods: A multi-channel fluidic device used computerized microscopy to measure growth of immobilized bacteria. Colonies from 18 ESBL-positive and 36 ESBL-negative Klebsiella spp. and Escherichia coli isolates were re-suspended from agar plates, pre-grown for 2 hours, and then 10 μL of a 7E5 CFU/mL inoculum was delivered to multiple flow cells. Bacteria were concentrated onto a poly-L-lysine-coated glass surface, capturing approximately 200-500 cells in the microscope’s field of view. ESBL detection used separate fluidic channels containing 256 μg/mL of a 3rd generation cephalosporin (3GC) with or without the addition of 4 μg/mL of clavulanic acid (CA). Ceftazidime (CAZ) and cefotaxime (CTX) were the 3GCs tested. A growth control and CA control were run in parallel. The system acquired images every 10 minutes and computed mass of the cell population throughout the test. The ratio of cell mass in the 3GC only to the 3GC-CA combination condition was calculated and the system classified isolates as ESBL-positive if the maximum ratio was greater than a threshold criterion. Results were compared to CLSI confirmatory ESBL disk diffusion test results. Results: The average maximum ratio of ESBL-negative isolates was 1.9 +/- 1.3 sd while the range for the ESBL positive isolates was 13 to 211. The system correctly classified 18 of 18 ESBL-positive and 36 of 36 ESBL-negative isolates, using threshold criterion of 5, in a total test time of 3 hours. Conclusions: Direct measurement of growth of small numbers of immobilized bacteria enabled identification of the ESBL phenotype in Enterobacteriaceae. The method shows promise for rapid testing, with a bacterial sample size that is compatible with direct extraction from clinical specimens.