Development of Viability PCR for Water Quality Assessment and Disinfection Quantification

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Ahmed, Tahmina, Civil Engineering - School of Engineering and Applied Science, University of Virginia
Smith, James, EN-CEE, University of Virginia
Taniuchi, Mami, MD-INMD Infectious Dis, University of Virginia

Campylobacter and Shigella are prevalent enteric pathogens transmitted via contaminated food, milk, and water, necessitating robust detection methods to differentiate viable from non-viable bacteria. This dissertation evaluates laboratory techniques for detecting these pathogens in water and raw cow milk and distinguishing between live and dead bacteria using viability polymerase chain reaction (vPCR). To optimize sample processing, seven water concentration methods and nine milk concentration methods were compared, alongside respective DNA extraction protocols. Concentrated samples were subjected to DNA extraction and qPCR after spiking with known bacterial amounts. For milk, centrifugation at 16,000 x g for 20 minutes yielded optimal results, while the concentrating pipette approach was selected for water. Initially, the samples were split into two, where one was treated with propidium monoazide (PMAxx), a DNA intercalating dye, while the other was left untreated. PMAxx penetrates the compromised cell membrane of dead microorganisms, intercalating with the DNA, blocking PCR amplification, and delaying the threshold cycle (Ct). Viability PCR with PMAxx effectively differentiated live from dead bacteria, exhibiting exceptional linearity (0.992 to 0.999) and precision with detection limits ranging from 3.75x102 to 4.54 CFU/ml for Shigella and 4.67x103 to 9.84x101 CFU/ml for Campylobacter. The PMAxx-treated dead bacteria showed delayed Ct values than the viable bacteria, representing the removal of dead bacterial DNA signal of approximately 94% in milk and 99.6% in water.
The study also developed a viability PCR assay for Cryptosporidium oocysts in water, demonstrating significant differentiation between live and dead oocysts. The differences in the threshold cycle (dCt) were compared for heat-killed and live oocysts spiked in synthetic groundwater (SGW) for both PMAxx-treated and untreated qPCR. The PMAxx-treated heat-killed oocysts showed delayed Ct values compared to the viable oocysts, with an average dCt of ≥ 6, representing about 99% removal of dead DNA signal. Disinfection kinetics of silver, copper, chlorine, and their combinations against Cryptosporidium were evaluated utilizing the vPCR. At the tested concentrations, both silver and copper individually exhibited a log reduction (LR) of 0.44 at 24 hours. However, the combination of copper and chlorine showed no disinfection efficacy. Similarly, a commercial prototype point-of-use water purification device that releases silver and copper ions into solution (MadiDrop+Cu) performed better disinfection against Cryptosporidium oocysts in water. The contact time of disinfection interventions was identified as crucial for effective disinfection.
Field assessments in Dhaka's urban slums highlighted the effectiveness of implementing MadiDrop+Cu for tap water treatment. Bathing water samples collected from 40 households were analyzed to determine the efficacy of water-treatment interventions. Out of the samples tested, three were found to be positive for Campylobacter spp. by vPCR. The presence of chloride ions in the tap water may be responsible for lower viable pathogen detection by vPCR. The study revealed a correlation between silver concentration and pathogen die-off, emphasizing the importance of monitoring silver and copper concentrations for intervention efficacy. Despite variations in silver release from MadiDrops, median silver concentrations remained within the ranges considered effective for disinfection. Furthermore, spiking Shigella sonnei into synthetic groundwater demonstrated significant pathogen die-off with MadiDrop and MadiDrop+Cu. Overall, this dissertation contributes valuable insights into effective detection and disinfection methods for controlling waterborne pathogens, highlighting the need for further research to optimize interventions and assess their real-world efficacy.

PHD (Doctor of Philosophy)
viability PCR, PMA, milk, Campylobacter, Shigella, Cryptosporidium, point-of-use water treatment technology
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