Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires\' disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella. The management of engineered water systems to prevent Legionnaires\' disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water samples using a \"viability based flow cytometry-cell sorting and qPCR\" (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water samples. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar; however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires\' disease.

Mycobacterium avium complex (MAC) is a group of environmentally-transmitted pathogens of great public health importance. This group is known to be harbored, amplified, and selected for more human-virulent characteristics by amoeba species in aquatic biofilms. However, a quantitative microbial risk assessment (QMRA) has not been performed due to the lack of dose response models resulting from significant heterogeneity within even a single species or subspecies of MAC, as well as the range of human susceptibilities to mycobacterial disease. The primary human-relevant species and subspecies responsible for the majority of the human disease burden and present in drinking water, biofilms, and soil are M. avium subsp. hominissuis, M. intracellulare, and M. chimaera. A critical review of the published literature identified important health endpoints, exposure routes, and susceptible populations for MAC risk assessment. In addition, data sets for quantitative dose-response functions were extracted from published in vivo animal dosing experiments. As a result, seven new exponential dose response models for human-relevant species of MAC with endpoints of lung lesions, death, disseminated infection, liver infection, and lymph node lesions are proposed. Although current physical and biochemical tests used in clinical settings do not differentiate between M. avium and M. intracellulare, differentiating between environmental species and subspecies of the MAC can aid in the assessment of health risks and control of MAC sources. A framework is proposed for incorporating the proposed dose response models into susceptible population- and exposure route-specific QMRA models.