Quantitative real-time PCR analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater

Survival of Host-Associated Bacteroidales Cells and Their Relationship with Enterococcus spp., Campylobacter jejuni, Salmonella enterica Serovar Typhimurium, and Adenovirus in Freshwater Microcosms as Measured by Propidium Monoazide-Quantitative PCR

“…Significant concentrations of dissolved DNA are found in marine water, freshwater, and sediments, potentially complicating the interpretation of information from qPCR data due to the relatively long persistence of target DNA (23). The PMA-qPCR method could avoid the issue of persistent DNA and provide a relatively fast, simple, and inexpensive means to discriminate intact/dead cells in mixed populations (2,22,42). However, some limitations of PMA-qPCR have been reported, such as the interference of higher particle concentration (2) and highly complex chemical and biological matrices in environmental samples.…”

“…We employed PMA-qPCR methods to discriminate intact cells and dead cells or free DNA of C. jejuni and S. Typhimurium. Even though PMA concentration and exposure time used for these experiments were not optimized for the bacterial pathogens, diverse bacterial species have been tested with PMA-qPCR in earlier studies and the signal from dead cells and free DNA was successfully removed (2,5,21,23,42). The slightly higher Salmonella cell inactivation rates in sunlight exposure compared to those in dark conditions could be explained by the lag period or increase in number of gene copies, which can cause a steep inactivation phase after the lag periods (33).…”

“…For example, a ratio close to unity indicates that fecal contamination occurred recently based on the reasonable assumption that bacterial DNA is more persistent compared with the relatively faster decay of cells in the natural environment. In addition, inhibition of PMA activity after concentration of biomass by filtration and concurrent higher levels of total suspended solids should be considered in field monitoring (42). Even though very little co-occurrence of fecal Bacteroidales detection was observed for E. coli O157, Salmonella, and Campylobacter among samples in a previous study using only (q)PCR (49), PMA-qPCR analysis for both Bacteroidales and waterborne pathogens could be informative to identify the sources of waterborne pathogens due to similar decay rates in the environment.…”

“…However, little is known regarding the persistence of hostassociated Bacteroidales cells and their genetic markers in freshwater. In two studies, Bacteroidales RNA was measured as an equivalent of whole cells using fluorescent in situ hybridization (FISH) (29,42), but it is difficult to compare FISH results with qPCR results because the number of rRNA gene operons in fecal Bacteroidales is unknown. Furthermore, the relationship of Bacteroidales with FIB or waterborne pathogens needs to be established.…”

“…PMA in combination with PCR or qPCR has been successfully applied to identify intact pathogens in a simple matrix (21,23) and in environmental samples (2,5,38,42). Basically, PMA is a DNA-intercalating dye with an azide group and will penetrate only the membrane of dead cells with compromised cell walls/membrane and then bind their DNA or attach to free DNA.…”