Metagenomic Analysis of Antibiotic Resistance and Virulence Genes along the Santa Cruz River

Abstract

Antimicrobial resistance is a growing global concern as it is associated with at least 2.8 million infections and 35,000 deaths in the United States alone every year. Globally, it is estimated to result in over 1.2 million deaths annually. Antibiotic resistant bacteria (ARB) are commonly present in sewage and can be disseminated into water through sewage leaks. Since at least 2017, the Santa Cruz River has experienced frequent raw sewage leaks from the International Outfall Interceptor (IOI). To assess the impact these sewage leaks had on this critical water source, sediment samples were taken in triplicate from near the leaks (Nogales) and two locations away from the contamination point (Tubac and Marana) at four different timepoints over the period of one year between October 2019 and October 2020. DNA was extracted from each sample (n=108), and sequenced using Illumina and Oxford Nanopore technologies. The study aimed to understand the impact on microbial communities, pathogens, and levels of antibiotic resistant genes (ARGs) using tools and/or databases including Kraken2 custom and Greengenes databases (taxonomy), ABRicate and DeepARG (ARGs), and Virulence Factor Database (pathogens). Thirty-three samples were chosen from each sequencing technology for further analysis to determine the differences in results between Illumina and Oxford Nanopore and different databases. Results showed that there were no significant differences in number of ARGs, virulence factors, or microbial communities between locations or timepoints using Illumina sequencing reads. There were significant differences between the two sequencing technologies using different databases. Microbial communities differed between the two databases, as the Kraken2 custom database had a greater abundance of unclassified family for Oxford Nanopore sequence reads compared to the Greengenes database. There were significant differences in the Shannon diversity indexes between sequencing technologies. Beta diversity revealed that using the Kraken2 custom database, samples clustered together based on sequencing technology, not sampling location. Overall, the study found no differences in the sediment along the Santa Cruz River in ARGs, pathogens, or microbial communities due to the sewage leaks. However, the study did find that there can be highly significant differences in results depending on the sequencing technology and databases that are used for the analysis, therefore caution must be applied when comparing studies using different approaches.