(277g) DNA Recovery from Enteric Bacteria for Rapid Diagnostic Genotyping

There is a great need to rapidly identify the organisms in blood infections, and assess their resistance to certain antibiotics, so the correct antibiotic treatment can be implemented from the beginning of therapy. We are developing a microfluidic device that can reduce this diagnostic time from more than 24 hours to under an hour. As part of this process, we need to recover the DNA from bacteria that are captured from infected blood. Bacteria that often contain carbapenam-resistance genes are enteric bacteria, such as E. coli, Klebsiella pneumoniae, and Enterobacter cloacae. While E. coli is relatively easy to chemically lyse in a microfluidic device, E. cloacae is fairly difficult. Because the clinician cannot determine a priori which bacteria will be present, we have developed chemical lysis procedures that work on all 3 of these bacteria, and potentially on even more species.

Following lysis, the bacteria DNA, both genomic and plasmidic, is collected on silica-coated magnetic beads with a recovery efficiency that depends on the type of bead and the adsorption and elution buffers. With the right chemistry, we have been able to recover about 80% of the DNA. Our presentation describes procedures and chemistry for optimal DNA recover from these medically significant bacteria.