Biosynthetic Enhancement of the Detection of Bacteria by the Polymerase Chain Reaction

Abstract

Molecular viability testing (MVT) was previously reported to specifically detect viable bacterial cells in complex samples. In MVT, brief nutritional stimulation induces viable cells, but not non-viable cells, to produce abundant amounts of species-specific ribosomal RNA precursors (pre-rRNA). Quantitative polymerase chain reaction (qPCR) is used to quantify specific pre-rRNAs in a stimulated aliquot relative to a non-stimulated control. In addition to excluding background signal from non-viable cells and from free DNA, we report here that MVT increases the analytical sensitivity of qPCR when detecting viable cells. Side-by-side limit-of-detection comparisons showed that MVT is 5-fold to >10-fold more sensitive than standard (static) DNA-targeted qPCR when detecting diverse bacterial pathogens (Aeromonas hydrophila, Acinetobacter baumannii, Listeria monocytogenes, Mycobacterium avium, and Staphylococcus aureus) in serum, milk, and tap water. Sensitivity enhancement may come from the elevated copy number of pre-rRNA relative to genomic DNA, and also from the ratiometric measurement which reduces ambiguity associated with weak or borderline signals. We also report that MVT eliminates false positive signals from bacteria that have been inactivated by moderately elevated temperatures (pasteurization), a condition that can confound widely-used cellular integrity tests that utilize membrane-impermeant compounds such as propidium iodide (PI) or propidium monoazide (PMA) to differentiate viable from inactivated bacteria. MVT enables the sensitive and specific detection of very small numbers of viable bacteria in complex matrices.

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

“…The effects of the PMA treatment under different cell densities were determined using qPCR assays targeting between the 5′ terminus of the mature 16S rRNA and the external transcribed spacer (ETS1) derived from viable and heat‐killed cells, as described previously (Cangelosi et al ., 2010; Weigel et al ., 2013; Do et al ., 2014). For a 100 μM concentration, there was a 2.7 to 3.9 log reduction, and there was a 1.8 to 1.9 reduction in a 50 μM concentration of PMA (Fig.…”

“…This will result in an overestimation of VBNC cells. As an alternative to this viability method, pre‐rRNA has been successfully applied to discriminate viable cells from chlorine‐, serum‐, UV‐ and low‐temperature pasteurization (63 °C for 45 min)‐killed cells of A. hydrophila , P. aeruginosa, L. monocytogenes , M. avium , E. coli , E. faecalis and S. enterica (Cangelosi et al ., 2010; Weigel et al ., 2013, 2017; Do et al ., 2014). Despite the successful application of the pre‐rRNA analysis, its efficacy for VBNC detection has remained problematic under environmental stressors.…”

“…This procedure was repeated in triplicate. The results of the pre‐rRNA experiments were expressed as a positive in Δ C t by subtracting the C t value of the nutritionally stimulated samples from the non‐stimulated samples, as described in a previous study (Do et al ., 2014). Based on Do et al ., if there is a Δ C t greater than one, it can be concluded that pre‐rRNA was synthesized (Do et al ., 2014).…”

“…To overcome this limitation, recent studies have proposed detecting microbial rRNA precursors (pre‐rRNA) as an alternative to rapidly detecting viable cells (Cangelosi et al ., 2010; Weigel et al ., 2013, 2017; Cangelosi and Meschke, 2014; Do et al ., 2014; Spooner et al ., 2016). Pre‐rRNA fractions in growing bacteria cells are significant in the total RNA, and they are much easier to detect than mRNAs as they synthesize rRNA precursors (pre‐rRNA) immediately in response to the nutritional stimulation of bacterial cells (Cangelosi et al ., 2010).…”

“…Pre‐rRNA fractions in growing bacteria cells are significant in the total RNA, and they are much easier to detect than mRNAs as they synthesize rRNA precursors (pre‐rRNA) immediately in response to the nutritional stimulation of bacterial cells (Cangelosi et al ., 2010). The method of quantifying pre‐rRNA precursors has been evaluated for differentiating viable cells from dead cells that have been inactivated by chlorine, serum exposure, pasteurization and UV irradiation (Cangelosi et al ., 2010; Weigel et al ., 2013, 2017; Do et al ., 2014). …”