Quantitative 16S rRNA Gene Amplicon Sequencing for Comprehensive Pathogenic Bacterial Tracking in a Municipal Wastewater Treatment Plant

Quantification
of bacterial pathogens in the environment is crucial
for determining their potential risk of pathogenicity and infection.
Here, we applied quantitative sequencing (qSeq) based on the 16S rRNA
gene with spike-in internal standards for comprehensive pathogen quantification
in a municipal wastewater treatment plant (WWTP). A novel bacterial
pathogen database based on biosafety levels (BSLs) was constructed
for rapid pathogen identification. Pathogen taxonomies were obtained
from the constructed database using a basic local alignment search
tool, and amounts of pathogens were quantified by regression analysis
of spike-in internal standards. The total pathogen concentrations
were gradually decreased from 6.8 ± 4.9 × 107 copies/mL in the influent to 1.6 ± 1.4 × 105 copies/mL in the effluent. BSL2 pathogens (e.g., Aeromonas spp., Mycobacterium spp., Klebsiella spp., and pathogenic Escherichia spp.) were detected
more than 4.0 × 103 copies/mL at the effluent. The
genus Arcobacter (BSL1) showed the highest abundance
throughout the WWTP (8.8 ± 8.1 × 104 to 2.5 ±
3.5 × 107 copies/mL). BSL2 Mycobacterium spp. noticeably increased at the anaerobic treatment (2.3 ×
106 copies/mL) and showed least removal at the effluent.
The qSeq analysis with the pathogen database provided a holistic view
of the microbial pathogen dynamics and ecology in the WWTP.