(109e) Rapid and Specific Genotyping for Blood Infection Identification

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. Conventional identification of species and resistance profiling currently takes more than 24 hours.

Our research team has put together a diagnostic process to identify specific bacterial species and the presence of targeted antibiotic resistance genes within 1 hr. The process consists of several unit operations, each of which has been developed individually and is currently being linked into a complete process. The sequence of unit operations follows. First bacteria are separated from blood using a centrifugal sedimentation process requiring only about 2 minutes. Separation efficiency is about 70% even down to 6 CFU/mL blood. Next the bacteria are concentrated by filtration, lysed, and their DNA collected on silica-coated magnetic beads and eluted with an overall recovery efficiency of about 50%. The DNA is reduced in size using sonication. This DNA is run through a capture column having complementary sequences unique to the species and to the antibiotic resistance gene. Sections of captured DNA are then fluorescently labeled with 4 different colors of fluorescing molecules. The bacterial DNA is thermally released from the capture/labeling column in a 3-5 µL volume, which then flows through an optofluidic silicon chip for single molecule detection. Positive fluorescent signal indicates the presence of the specific bacterial species and/or resistance gene. These unit operations have been validated individually for the species E. coli, K. pneumoniae and E. cloacae and the carbapenam resistance genes NDM, KPC, IMP and VIM. Linking these unit operations in sequence is much more challenging, and this presentation shows the progress and challenges of building and successfully implementing the entire diagnostic procedure.