Abstract
Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔCt<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 Gram-negative and 2 Gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours.
Highlights
Current molecular diagnostic technologies for septicemia have primarily focused on pathogen identification as a means to optimize antimicrobial therapy for patients
The sample is spun-down and the unseen bacterial cell pellet is washed in reticulocyte saline (RS) buffer
Susceptibility testing of septicemia relies on either extended multi-day phenotypic methods that jeopardize patient survival or molecular genotyping techniques that are limited by the number of resistance cassettes the system can detect and that are unable to detect newly emergent resistance genes
Summary
Current molecular diagnostic technologies for septicemia have primarily focused on pathogen identification as a means to optimize antimicrobial therapy for patients. Compounding the issue of genotyping for resistance is the fact that the number of potential resistance genes scale with each pathogen, straining the limits of current diagnostic technology and economic feasibility. Despite this constraint, molecular diagnostic systems have demonstrated species identification in less than 24 hours, a drastic improvement in comparison to the gold standard culture-based susceptibility and Gram staining-based identification methods that yield results in 24 to 72 hours [3,4,6,7]. Even though literature agrees that molecular diagnostic detection rapidly decreases the time to sepsis diagnosis, much debate over the accuracy of pathogen identification and the appropriateness of the method for prescribing antimicrobial therapy remains [1,2,8,9,10,11,12]
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