Molecular And Metagenomic Characterization Of Bacterial Pathogens And Antibiotic Resistance Genes In Complex Environments
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2020
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University of Hawaii at Manoa
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Bacterial pathogens and antibiotic resistant genes (ARGs) in the environment have become important clinical issues. Characterization of pathogens and ARGs in the environment is essential for health risk assessment. The high diversity and usually low concentrations of bacterial pathogens and their ARGs in environmental samples pose challenges to current detection methods. Current molecular methods, including polymerase chain reaction (PCR) and next generation sequencing (NGS), suffer different limitations in specificity, sensitivity, throughput, and/or quantitative capacity. The overall objective of this dissertation is to develop new molecular and metagenomic approaches to characterize bacterial pathogens and ARGs in complex environments, with particular aims in improving specificity, sensitivity and throughput of current molecular methods. Four specific objectives are covered in this dissertation. First, this dissertation examined the overestimation potential of dye-based qPCR method for ARG detection by comparing with specific probe-based qPCR, which was subsequently confirmed through NGS amplicon sequencing. Overestimation of ARGs was observed in dye-based qPCR reaction, which is mainly caused by non-specific amplification. Second, a novel method that combines multiplex PCR with NGS (mPCR-NGS) was developed for simultaneous detection of multiple waterborne pathogens. The multiplex level of mPCR was significantly increased by using NGS to display amplicons of 14 target genes simultaneously. Third, metagenomic sequencing and bioinformatic analyses were used to detect ARGs in pristine soil samples, and a total of 48 ARGs were detected. Fourth, a quantitative metagenomic sequencing methods was developed by spiking non-natural internal DNA standards into DNA samples. Comparison between the quantitative metagenomic sequencing approach and qPCR quantification demonstrated comparable levels of accuracy. The findings in this study help to better understand and improve current molecular methods for the characterization (detection or quantification) of pathogens and ARGs in the environment. The overestimation of ARGs by dye-based qPCR suggests its inappropriateness for ARG detection in environmental sample. The novel mPCR-NGS and quantitative metagenomic sequencing show promises in high throughput detection and quantification of target genes for environmental monitoring.
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Civil engineering, antibiotic resistance genes, metagenomic sequencing, pathogens, qPCR
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