Assessing the Relative Concentration Efficiency and Spatial–Temporal Distribution of Plasmid pBI143 in Raw Wastewater

Plasmid pBI143 has recently been identified as being highly abundant in the human gut microbiome, suggesting potential as a fecal water quality indicator and normalization marker in wastewater-based surveillance. We evaluated the relative concentration efficiency and spatial–temporal distribution in raw wastewater to inform its development as a marker. The results showed significantly higher (p < 0.01) enrichment in raw wastewater solids (mean of 9.2 × 10⁷ GC/mL) than in liquid fractions [mean of 2.3 × 10⁶ genome copies (GC)/mL]. The relative concentration efficiencies were 28% for nanotrap particles, 23% for Amicon ultrafiltration, 3.6% for pH drop and filtration, 4% for skim milk flocculation, and 0.04% for poly(ethylene glycol) precipitation compared to direct wastewater extraction. DNase pretreatment reduced pBI143 levels by 90.3%, indicating that it is mainly extracellular in raw wastewater. Over 21 days, pBI143 levels remained stable and were consistently a mean of 1.3 log₁₀ higher than other fecal indicators (Carjivirus, PMMoV, and HF183). In raw wastewater from eight states (n = 16), pBI143 concentrations averaged 2.2 × 10⁶ GC/mL (95% confidence interval of 1.7–2.7 × 10⁶) and pBI143 was detected in all samples. The findings support pBI143’s potential as a fecal indicator and normalization marker, though further validation is needed to confirm its specificity to humans.