Towards standardized quantitative microbial risk assessment in the environment using metagenomics

Abstract

As one of the leading global public health challenges, microbial antibiotic resistance in the environment is an environmental issue with medical concerns. Through the One Health cycle, antibiotic resistance genes (ARGs) present in the environmental microbiome can interact with pathogens through horizontal gene transfer (HGT), breading the emergence and dissemination of antibiotic-resistant pathogens. As such, monitoring and quantifying these microbial pollutants in the environment are essential for assessing the associated microbial risks. The metagenomics approach has drastically enhanced the ability to characterize and quantify these microbial pollutants in a high-throughput manner. Thus, this thesis focused on exploiting the potential of metagenomics in achieving rapid comprehensive microbial risk assessment that is quantitative, reliable (fixated on viable cells), and reproducible for complex environmental samples.

In achieving quantitative metagenomics, a Nanopore-based spike-in facilitated absolute quantification (SAQ) methodology was developed to quantify pathogens and ARGs in wastewater treatment plants (WWTPs). Results generated using Nanopore were in high agreement with Illumina-based and culture-based methods, and 1-hr sequencing time was enough for accurate quantification of dominant pathogens. Additionally, high log removals of pathogens (2.23 logs) and ARGs (1.98 logs) by WWTPs were observed. However, such removal was not complete, and a few pathogens and ARGs could not be effectively removed.

A viability assay was integrated into the previous SAQ methodology to identify the human health risk of exposure to pathogens and ARGs in beaches to achieve a reliable microbial risk assessment with a focus on only viable cells. Results from culture-based and this new method were in high agreement. Adopting this approach, a screening-level quantitative microbial risk assessment (QMRA) was performed. QMRA results demonstrated that Vibrio cholerae contributed to greater health risks than fecal indicator bacteria (FIB), suggesting that the indigenous pathogens in beaches could introduce high health risks to humans, more than the regularly monitored FIB. Additionally, ARGs in beaches under the influence of sewage discharge were of high risks. Results suggested that this SAQ-enabled site-specific QMRA could be an alternative to the conventionally used FIB monitoring to indicate microbial risks.

For benchmarking and controlling technical variabilities and thus ensuring reproducible metagenomic studies, a well-homogenized environmental reference material (ERM) was established using activated sludge (AS) which has very high bacteria biodiversity. By using different pre-analytical metagenomic assays, lists of prokaryotic genera with highly reproducible abundances were prepared from this ERM for the wide adoption of this ERM to benchmark technical variabilities in environmental microbiome studies. In addition, results from the comparison of biological and technical variabilities showed that benchmarking technical variabilities in studies involving samples of small differences (e.g., in longitudinal studies) is also important.

To enhance the detection of class 1 integrons (CL1), a critical vehicle carrying and spreading ARGs in the environment, a new polymerase chain reaction (PCR) primer was developed. The new primer revealed more ARGs and novel integron structures than the previous one, and CL1s in the environment were of much greater diversity than those currently found in the whole genome database.