Microbial Risk Assessment Across Multi-Environments Based on Metagenomic Absolute Quantification with Cellular Internal Standard

The risk posed by microorganisms in diverse environments has emerged as a significant concern. Despite this, existing microbial risk assessment frameworks often lack comprehensiveness and systematicness. To tackle this constraint, we developed a cellular spike-in (one Gram-positive and one Gram-negative bacteria) method that enables absolute quantification of microorganisms in various environmental compartments. This method was rigorously evaluated for reproducibility, accuracy, and applicability. Furthermore, we investigated biases that might arise from DNA extraction to sequencing under different cell lysis conditions for both types of bacteria, and importantly, demonstrated that this spike-in absolute quantification method could correct such biases. We then applied this method to a range of samples to determine the absolute abundance of various microorganisms, pathogens, and antibiotic resistance genes (ARGs) across eight different sample types, including influent, effluent, primary sludge, activated sludge, marine water, marine bathing beach water, marine fishery water, and river water. Based on the results, we evaluated and compared the treatment efficiencies in terms of pathogens and ARGs in five WWTPs of different operational modes. Finally, we integrated the absolute abundances of 1) total pathogens and key pathogens used for cumulative pathogenic possibility calculation in the framework of Quantitative Microbial Risk Assessment (QMRA); 2) Risk Rank1&2 ARGs and high-risk ARGs associated with ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) +EV (E.coli and Vibrio spp.); 3) two most common fecal indicator bacteria (FIBs), namely Escherichia coli and Enterococci; and 4) plasmids and other mobile genetic elements (MGEs). This innovative method enables us to effectively compare the microbial risk across distinct environments, while also establishing a benchmark for microbial risk assessment and paving the way for more accurate and reliable risk assessment framework.