Evaluation of plasmid pBI143 for its optimal concentration methods, seasonal impact, and potential as a normalization parameter in wastewater-based epidemiology.

Plasmid pBI143, abundant in the human gut, is a promising human-specific fecal marker. However, studies on its optimal concentration methods, seasonal variations, and potential as a normalization parameter for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), remain limited. Among the three concentration methods compared, polyethylene glycol (PEG) precipitation and centrifugation demonstrated comparable efficiencies (9.3 ± 0.6 and 9.2 ± 0.6 log copies/L, respectively; n = 8 each), outperforming membrane filtration (8.0 ± 0.6 log copies/L; n = 8). PEG precipitation was further applied to quantify pBI143, together with other human-specific fecal markers (crAssphage and pepper mild mottle virus (PMMoV)), in 52 wastewater samples collected weekly over a one year from a wastewater treatment plant in Yamanashi Prefecture, Japan, by quantitative polymerase chain reaction. The higher pBI143 concentrations (9.6 ± 0.5 log copies/L) compared to PMMoV (8.2 ± 0.2 log copies/L) and crAssphage (8.0 ± 0.2 log copies/L) highlighted its potential as a robust marker for human fecal contamination. Unlike PMMoV and crAssphage that remained stable across seasons, pBI143 showed seasonal fluctuations, especially during summer and autumn, suggesting its greater sensitivity to environmental conditions. The study evaluated the suitability of pBI143, crAssphage, and PMMoV for normalizing SARS-CoV-2 concentrations in wastewater; however, non-normalized SARS-CoV-2 concentrations showed the highest correlation with COVID-19 cases (ρ = 0.74), whereas normalization reduced this correlation (PMMoV-normalized, ρ = 0.72; crAssphage-normalized, ρ = 0.70; and pBI143-normalized, ρ = 0.50), likely due to differences in the persistence and structural properties of the markers, indicating that these markers are less effective for SARS-CoV-2 normalization. This study underscores the promising utility of pBI143 in wastewater surveillance but highlights the need for further research across diverse regions to validate its applicability.