At this time point the signal moved both above and below the 3.33 cycle breakpoint at several dilutions of drug, and a MIC Metabolism inhibitor was unable to be determined. These results provide evidence that ETGA can be used to generate a reliable MIC for AST analysis by as much as 16 hours sooner than traditional AST methods, and functions in a similar fashion to molecular
AST analysis using gsPCR assays. Molecular AST MIC determination of bacteria from positive blood cultures Beuving and colleagues [19, 20] have demonstrated that molecular AST can be performed on bacteria harvested directly from positive blood cultures by collecting the microbes from the culture using a SST. Such a method could produce a reliable MIC for a series
of antibiotics against a pathogenic microbe without the need for its isolation, thereby further reducing the time required to obtain a reliable result. The same methodology was applied to the following ETGA experiments. Blood cultures were spiked with MSSA, MRSA, or E. coli, allowed to be called positive in a BACTEC 9050 incubator, the bacteria were harvested with an SST, and molecular AST was performed as previously described in the materials and methods. The results and comparison of the molecular analyses to the macrobroth dilution selleck method are shown in Table 1 and Additional file 1: Table S2. Analysis was carried out as before using both molecular methods at the four and six hour incubation time points. ETGA analysis produced MIC values that were mostly in agreement with the macrobroth method and correlated with the CLSI interpretation. selleck products However, one discrepancy (Table 1, footnote b) was observed at the four hour time point of the MRSA versus vancomycin series. While the MIC was determined to be less than 0.25 μg/mL, the 16 μg/mL culture, produced a signal with a Ct value greater than 3.33 cycles above the baseline. This isolated result was neither supported by the results from the other cultures in the series, its paired gsPCR reactions,
nor the results from the six hour time point. The result is most likely indicative of an operator error. Such a result can occur when performing standard AST dilution methods. CLSI and similar AST protocols provide guidelines for interpreting such results aminophylline and repeating the testing, if necessary [6, 7]. The gsPCR analysis produced similar results to the ETGA analysis (Table 1) with two important discrepancies that require attention. The first is MRSA versus oxacillin at the six hour time point (Table 1, superscript c). Using the gsPCR method, the MIC was called at 2 μg/mL. Based on CLSI interpretation, this MIC value represents a susceptible phenotype. The expected phenotype, however, is resistant, and this is verified by the macrobroth method, the ETGA method at four and six hours, and the gsPCR method at 4 hours.