Novel molecular assessment method using precursor ribosomal RNA to rapidly quantify hard-to-monitor pathogens in aqueous suspensions: application to ultraviolet disinfection of <i>Mycobacterium avium</i>

Rapidly assessing the viability of <i>Mycobacterium</i> species in drinking water and reuse systems is critical, as these pathogens pose significant monitoring challenges and health risks. Traditional culture methods are slow and biased, often failing to quantify viable but dormant/injured organisms' post-ultraviolet treatment. This study introduces a real-time reverse transcription polymerase chain reaction assay based on precursor ribosomal RNA (pre-rRNA) as a biomarker for bacterial viability. Brief nutritional stimulation detects low concentrations of metabolically active and dormant target pathogens, effectively distinguishing viable from dead organisms. <i>Mycobacterium avium</i> 104 in phosphate-buffered saline suspensions treated with ultraviolet irradiation (254 nm) were assessed using this rapid molecular method. Strong correlations (<i>r</i> > 0.94) with traditional culture-based assessment were observed. It reliably determined log reductions in pre-rRNA of stimulated samples relative to disinfectant dose within a day, significantly faster than the 12-14 days required for culture methods. An inactivation rate constant for <i>M. avium</i> 104 was established, along with a conversion factor (2.75) to convert pre-rRNA to bacterial log reduction. This pre-rRNA assay with nutritional stimulation is promising for rapid and reliable quantification of low concentrations of viable mycobacteria. It can significantly enhance water quality monitoring, accelerate disinfection technology evaluation, and improve public health risk assessment.