Live-dead discrimination analysis, qPCR assessment for opportunistic pathogens, and population analysis at ozone wastewater treatment plants

Reduction of Antibiotic Resistant Bacteria During Conventional and Advanced Wastewater Treatment, and the Disseminated Loads Released to the Environment

“…Here, we focused on the absolute abundance of bacteria in 100 mL of wastewater. For visualizing changes of the relative abundance within the surviving population caused by these advanced wastewater treatments a normalization to 100 ng DNA would be possible and was shown in previous poplications of the group (Alexander et al, 2016 ; Jäger et al, 2018 ).…”

“…A recent study revealed that PMA treatment in wastewater samples is a suitable tool to focus on the viable part of the population. In this study, the authors were focusing on the indicator bacteria E. coli and enterococci and showed no significant differences between the cultivation-based approaches and the PMA-qPCR experiments, but there were significant differences between the culture-based method and qPCR experiments without PMA treatment (Li et al, 2014 ; Jäger et al, 2018 ). Possible wastewater matrix effects on the PMA efficiencies should be controlled with internal standard experiments and the PMA concentrations should become adjusted to the wastewater characteristic of state.…”

“…The wastewater samples were filtered by vacuum filtration on polycarbonate membranes (Ø 47 mm, pore size 0.2 μm, Whatman Nucleopore Track-Etched Membranes, Sigma-Aldrich, Munich, Germany) using 200 to 250 mL of the water samples. By using propidium mono azid (PMA, 25 μM) prior to DNA extraction according to Jäger et al ( 2018 ), the evaluation of disinfection processes can be limited to viable cells with intact cell membranes and an overestimation by molecular biology methods can be avoided (Nocker et al, 2007a , b ). A recent study revealed that PMA treatment in wastewater samples is a suitable tool to focus on the viable part of the population.…”

“…The primer systems and the calculation of the cell equivalents were done based on the already known genome sizes of the retference bacteria and are listed in Supplementary Information Table 1 . The PMA-treatment was performed prior to DNA extraction to consider the viable fraction of the wastewater sample (Jäger et al, 2018 ). The Ct–values from the wastewater samples were adjusted to the corresponding regression line and then normalized to 100 mL of filtered wastewater to show the different reduction efficiencies of absolute abundance within the surviving population of the wastewater samples.…”

Development of PMAxxTM-Based qPCR for the Quantification of Viable and Non-viable Load of Salmonella From Poultry Environment

“…Quantitative PCR (qPCR) can be used for quantifying pathogen load in clinical samples; however, primers targeting a specific region of the nucleic acids will also amplify DNA obtained from the non-viable load present in the population. Viability assays, such as live/dead cell viability assay, trypan blue, BacTiter-Glo TM microbial cell viability assay (Promega, Australia), LIVE/DEAD BacLight bacterial viability kit (ThermoFisher Scientific, Australia) and propidium monoazide (PMA) (Banihashemi et al, 2012;Barbau-Piednoir et al, 2014;Jäger et al, 2018) or ethidium bromide monoazide (Nocker et al, 2006;Wang and Mustapha, 2010) based qPCR can be used for quantifying viable microbial load. PMA is a nucleic acid intercalating dye that is cell impermeant and therefore will diffuse only across the cell membrane of dead microbes.…”