Aims The effect of amplicon length on the ability of propidium monoazide‐PCR (PMA‐PCR) to reliably quantify viable cells without interference from dead cells was tested on heat‐ and ultraviolet (UV)‐killed Salmonella enterica and Campylobacter jejuni, two important enteric pathogens of concern in environmental, food and clinical samples. Methods and Results PMA treatment followed by quantitative PCR (qPCR) amplification of short DNA fragments (<200 bp) resulted in incomplete signal inhibition of heat‐treated Salm. enterica (3 log reduction) and Camp. jejuni (1 log reduction), whereas PCR amplification of a long DNA fragment (1·5 and 1·6 kb) completely suppressed the dead cell signal. PMA pretreatment of UV‐irradiated cells did not affect PCR amplification, but long‐amplicon PCR was shown to detect only viable cells for these samples, even without the addition of PMA. Conclusions The long‐amplicon PMA‐PCR method was effective in targeting viable cells following heat and UV treatment and was applicable to enteric pathogens including Salmonella and Campylobacter that are difficult to enumerate using culture‐based procedures. Significance and Impact of the Study PCR amplicon length is important for effective removal of the dead cell signal in PMA pretreatment methods that target membrane‐damaged cells, and also for inactivation mechanisms that cause direct DNA damage.
A cell viability assay was applied to measure bacterial enteric pathogens in a river in southern Ontario, Canada that is used as a source of drinking water. Pathogen concentrations were measured using both propidium monoazide (PMA)-quantitative polymerase chain reaction (PCR) and quantitative PCR (qPCR) without PMA pretreatment to compare viable and total (live and dead) cells.The pathogens evaluated were Salmonella enterica, thermophilic Campylobacter, and Escherichia coli O157:H7, and the suspected enteric pathogen Arcobacter butzleri was also investigated.Results showed that for all strains dead cells were detected in few river water samples, and the difference between total and viable cell concentrations for each pathogen group was always less than 0.5 log. A. butzleri was detected at concentrations 2-3 log higher than the other pathogens.S. enterica, Campylobacter, and E. coli O157:H7 were detected at low concentrations at one sample location and at higher concentrations at a second sampling location. Results from this study show qPCR with PMA pretreatment can provide reliable enumeration of viable pathogens in surface water, and that dead cells were rarely present in water used as a source for drinking water treatment.
The decay rates of enteric waterborne pathogens were evaluated following the introduction of Yersinia enterocolitica, Salmonella enterica, Campylobacter jejuni and Arcobacter butzleri into river water at different temperatures (5, 15 and 25°C) for a period of 28 days. To improve the accuracy of the results a molecular viability assay, long amplicon propidium monoazide-polymerase chain reaction (PMA-PCR), was used to quantify the viable cell concentration and results from PCR with and without PMA were compared. As well, the effect of background microbiota was assessed for Y. enterocolitica and S. enterica by inoculating cells into sterile and non-sterile river water. Cell persistence was improved by up to 4 log for Y. enterocolitica and 4.5 log for S. enterica in sterile river water compared to natural river water, showing that the autochthonous biological activity in river water can accelerate the die-off of introduced bacteria. Results also showed that low temperature significantly improved the persistence of all four target bacteria in non-sterile river water. There was a more rapid decline in cell concentration in samples with PMA pretreatment; therefore using PMA-PCR analysis can provide more reliable data on viable/active enteric bacteria in aquatic microcosms and allows for improved assessment of pathogens in the environment.
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