Optimization of <i>Shigella</i> and <i>Campylobacter</i> Detection from Milk and Water and Differentiation of Viable Organisms Using Viability PCR

Campylobacter and Shigella are leading causes of enteric infections, especially in low-resource settings, and are transmitted through contaminated food, milk, and water. Detecting these pathogens in environmental samples and differentiating between viable and nonviable bacteria is critical for understanding their transmission and for risk assessment. This study optimized and evaluated laboratory methods for detecting viable Campylobacter and Shigella in water and raw cow milk using viability polymerase chain reaction (PCR). To optimize sample processing and extraction, seven water concentration methods, two water DNA extraction protocols, eight milk concentration methods, and nine milk DNA extraction protocols were compared. Water and milk were spiked with known amounts of bacteria, concentrated, and then subjected to DNA extraction and quantitative polymerase chain reaction (qPCR). For concentrating milk, centrifugation at 16,000×g for 20 min at room temperature using 1.5-mL samples yielded optimal results, while for water, the concentrating pipette approach was selected. For viability PCR, concentrated samples were treated with propidium monoazide (PMAxx) prior to DNA extraction. The results demonstrated exceptional linearity (0.992 to 0.999) and precision across various concentrations, with detection limits ranging from 3.75×102 to 4.54 CFU/mL for Shigella and 4.67×103 to 9.84×101 CFU/mL for Campylobacter. The assays had a within-run variance from 1.9% to 2.7% (repeatability) and between-run variance from 1.6% to 5.2% (reproducibility). The PMAxx-treated dead bacteria showed delayed cycle threshold (Ct) values compared to the viable bacteria, representing an inhibition of the detection of dead bacterial DNA by approximately 94% in milk and 99.6% in water. Viability PCR was successful in differentiating live from dead bacteria, offering a robust method for tracking the transmission of these pathogens in water and milk. These results support the application of viability PCR for more precise public health interventions.