Y plating. Thedetection protocol used automated nucleic acid extraction cards [29] and the single-step RT-qPCR method described in Materials and Methods. It required 5.5 hours to complete, including a 1.5hour nutritional stimulation. DNA was not quantified and ratios of pre-rRNA to gDNA (P:G) were not calculated. Instead, pre-rRNA in nutritionally stimulated samples was quantified relative to prerRNA in non-stimulated (time 0) controls. The results were expressed as the difference in Ct values between stimulated and non-stimulated aliquots (DCt). Here, DCt values did not consistently increase with increasing 1531364 viable cell GDC-0032 numbers, because the proportions of viable to inactivated cells were roughly similar in each sample. In four experiments conducted as shown in Figure 5, positive DCt values were always observed when viable cell densities were ,100 CFU/mL serum or more (a typical replicate is shown in Figure S3). Therefore, ratiometric pre-rRNA analysis can be applied to samples with physiologically relevant cellFigure 4. Ratiometric pre-rRNA analysis M. bovis BCG cells in serum. Cells were incubated separately in filtered and unfiltered human serum at 37uC for 30 days. The serum-incubated cells were then resuspended in pre-warmed 7H9 broth and samples were taken after 0,1, 2, 4, and 24 hours. P:G ratios were calculated as in Figure 2. Values are means and SDs of nine ratiometric permutations from three technical replicates of each sample type. From a five-point gDNA standard curve, qPCR efficiency was calculated as 1.001. A separate experiment with H37Ra yielded similar results (Figure S2). doi:10.1371/buy GW433908G journal.pone.0054886.gViability Testing by Pre-rRNA AnalysisFigure 5. Ratiometric pre-rRNA analysis of A. baumannii cells in serum by using a rapid semi-automated approach. Serumincubated cells were plated to quantify viable CFU/mL, then serially diluted in serum. Viable CFU/mL values (X-axis) were subsequently calculated from plating results. To initiate nutritional stimulation, dilutions in serum were further diluted 1:10 into TSB or PBS (stimulated and non-stimulated, respectively). After 90 minutes, pre-rRNA was quantified by one-step probe-based q-RT-PCR as described in text. Values are means and SDs of DCt values (non-stimulated minus stimulated) from two replicates of each dilution. Control samples with no bacteria (0 CFU/mL) yielded no RT-qPCR results, and therefore could not be plotted as DCt values. Reaction efficiency could not be calculated for this experiment, because no standard curve was used. A replicate experiment (Figure S3) yielded similar results, showing a limit of detection of #56 viable CFU/mL. doi:10.1371/journal.pone.0054886.gdensities, in a procedure that yields results faster than bacteriological culture.DiscussionThis study asked whether ratiometric pre-rRNA analysis, which was originally developed for analysis of drinking water, could also assess the viability of bacterial cells in human specimens. Although human fluids such as serum are rich in certain nutrients, they are usually limited in some required nutrients, and bacterial growth rates are sub-maximal in such environments. Therefore, we hypothesized that the provision of limiting nutrients would stimulate pre-rRNA synthesis in viable bacteria derived from such samples. The results showed this to be true for all organisms tested. When bacteria were not viable, as seen in P. aeruginosa after incubation in the first lot of serum, nutritional stimulation fai.Y plating. Thedetection protocol used automated nucleic acid extraction cards [29] and the single-step RT-qPCR method described in Materials and Methods. It required 5.5 hours to complete, including a 1.5hour nutritional stimulation. DNA was not quantified and ratios of pre-rRNA to gDNA (P:G) were not calculated. Instead, pre-rRNA in nutritionally stimulated samples was quantified relative to prerRNA in non-stimulated (time 0) controls. The results were expressed as the difference in Ct values between stimulated and non-stimulated aliquots (DCt). Here, DCt values did not consistently increase with increasing 1531364 viable cell numbers, because the proportions of viable to inactivated cells were roughly similar in each sample. In four experiments conducted as shown in Figure 5, positive DCt values were always observed when viable cell densities were ,100 CFU/mL serum or more (a typical replicate is shown in Figure S3). Therefore, ratiometric pre-rRNA analysis can be applied to samples with physiologically relevant cellFigure 4. Ratiometric pre-rRNA analysis M. bovis BCG cells in serum. Cells were incubated separately in filtered and unfiltered human serum at 37uC for 30 days. The serum-incubated cells were then resuspended in pre-warmed 7H9 broth and samples were taken after 0,1, 2, 4, and 24 hours. P:G ratios were calculated as in Figure 2. Values are means and SDs of nine ratiometric permutations from three technical replicates of each sample type. From a five-point gDNA standard curve, qPCR efficiency was calculated as 1.001. A separate experiment with H37Ra yielded similar results (Figure S2). doi:10.1371/journal.pone.0054886.gViability Testing by Pre-rRNA AnalysisFigure 5. Ratiometric pre-rRNA analysis of A. baumannii cells in serum by using a rapid semi-automated approach. Serumincubated cells were plated to quantify viable CFU/mL, then serially diluted in serum. Viable CFU/mL values (X-axis) were subsequently calculated from plating results. To initiate nutritional stimulation, dilutions in serum were further diluted 1:10 into TSB or PBS (stimulated and non-stimulated, respectively). After 90 minutes, pre-rRNA was quantified by one-step probe-based q-RT-PCR as described in text. Values are means and SDs of DCt values (non-stimulated minus stimulated) from two replicates of each dilution. Control samples with no bacteria (0 CFU/mL) yielded no RT-qPCR results, and therefore could not be plotted as DCt values. Reaction efficiency could not be calculated for this experiment, because no standard curve was used. A replicate experiment (Figure S3) yielded similar results, showing a limit of detection of #56 viable CFU/mL. doi:10.1371/journal.pone.0054886.gdensities, in a procedure that yields results faster than bacteriological culture.DiscussionThis study asked whether ratiometric pre-rRNA analysis, which was originally developed for analysis of drinking water, could also assess the viability of bacterial cells in human specimens. Although human fluids such as serum are rich in certain nutrients, they are usually limited in some required nutrients, and bacterial growth rates are sub-maximal in such environments. Therefore, we hypothesized that the provision of limiting nutrients would stimulate pre-rRNA synthesis in viable bacteria derived from such samples. The results showed this to be true for all organisms tested. When bacteria were not viable, as seen in P. aeruginosa after incubation in the first lot of serum, nutritional stimulation fai.
