Recent literature has introduced the emerging technology of check details molecular AST [16–19] in which quantitative PCR is used to monitor the growth of bacterial cultures in the presence of antibiotic agents. They are based on the amplification of the rpoB gene; the 16S ribosomal locus universally found in the bacterial genome. The technology is based on the premise that the growth kinetics of bacteria in culture can be monitored by measuring the increasing amounts genomic DNA. In this fashion, MICs may be determined on the same day as the initial inoculation rather than an overnight incubation. H 89 molecular weight The kinetics of increasing PCR signal from a growing culture in the
presence of an antibiotic can be used to determine whether a pathogen is resistant or susceptible to the agent. Furthermore, one group reports a workflow in
which molecular AST can be performed on bacteria harvested directly from blood culture using serum separation tubes, identifying the pathogen with species specific qPCR probes, and producing a molecular AST result in a single day . Our group has previously reported a novel methodology termed Enzyme Template Generation and Amplification (ETGA) that enables universal, sensitive and quantitative measurement of bacterial proliferation via measurement of endogenous DNA polymerase activity . In this report, we demonstrate that molecular AST and MIC Doramapimod determination can be performed via ETGA-mediated monitoring of DNA polymerase activity. We compare the functionality of ETGA AST to
PCR-based molecular AST using gene-specific qPCR assays (gsPCR) against either S. aureus or E. coli. We also show that ETGA AST can be used to determine MICs from bacteria harvested directly however from spiked blood cultures. Methods Bacterial strains, cultivation, and antibiotics tested The following strains were used in this study: Escherichia coli ATCC 25922, methicillin susceptible Staphylococcus aureus ATCC 29213, and methicillin resistant Staphylococcus aureus NRS241. All strains were propagated on Brain-Heart Infusion Agar (Teknova, Hollister, CA). The S. aureus strains, both methicillin resistant and susceptible, were tested for susceptibility against oxacillin and vancomycin (Sigma Aldrich, St. Louis, MO). The E. coli strain was tested for susceptibility against ciprofloxacin and tetracycline (Sigma Aldrich, St. Louis, MO). Macrodilution broth method for the determination of antimicrobial susceptibility The macrobroth dilution method and the interpretive standards for determining the antimicrobial susceptibility of a microorganism to an antimicrobial agent are published by the Clinical and Laboratory Standards Institute [6, 8].