MinION Nanopore Sequencing Enables Correlation between Resistome Phenotype and Genotype of Coliform Bacteria in Municipal Sewage.

Yu Xia,Yu Deng,An-Dong Li,Li-Guan Li,Tong Zhang,Xiao-Tao Jiang

doi:10.3389/fmicb.2017.02105

Abstract

Wastewater treatment plants (WWTPs) functioned as the intersection between the human society and nature environment, are receiving increasingly more attention on risk assessment of the acquisition of environmental antibiotic resistance genes (ARGs) by pathogenetic populations during treatment. However, because of the general lack of robust resistome profiling methods, genotype, and resistance phenotype is still poorly correlated in human pathogens of sewage samples. Here we applied MinION sequencing to quantify the resistance genes of multiple antibiotic resistant (MAR) coliform bacteria, a common indicator for human enteric pathogens in sewage samples. Our pipeline could deliver the results within 30 h from sample collection and the resistome quantification was consistent to that based on the Illumina platform. Additionally, the long nanopore reads not only enabled a simultaneous identification of the carrier populations of ARGs detected, but also facilitated the genome reconstruction of a representative MAR strain, from which we identified an instance of chromosomal integration of environmental resistance gene obtained by plasmid exchange with a porcine pathogen. This study demonstrated the utilization of MinION sequencing in quick monitoring and simultaneous phylogenetic tracking of environmental ARGs to address potential health risk associated with them.

Highlights

With the spread of antibiotic resistant pathogens and the related increase of human morbidity and mortality, antibiotic resistance genes (ARGs) have been considered as emerging environmental pollutants by World Health Organization (2014)
Genotype resistance prediction based on Nanopore technology was found to be reliable as long match to reference sequences could be achieved and the quantification based on MinION sequencing was largely consistent to that based on Illumina sequencing platform
Difficulties remain in assigning phylogeny for ARG-containing nanopore reads as marker genes may not be located in close genomic proximity of ARGs

Summary

Introduction

With the spread of antibiotic resistant pathogens and the related increase of human morbidity and mortality, antibiotic resistance genes (ARGs) have been considered as emerging environmental pollutants by World Health Organization (2014). Though some researchers found the core resistome of wastewater treatment system was different from that of human pathogens (Munck et al, 2015), wastewater treatment plants (WWTPs) are receiving increasingly more social and scientific attention on risk assessment of the acquisition of environmental ARGs by clinical pathogens during the treatment processes (Finley et al, 2013; Yang et al, 2014). Among the microbial communities of WWTPs, coliform bacteria are the most commonly used indicator for sanitary quality of water before and after treatment and fecal coliforms have frequently been surveyed as indicator of potential presence of human enteric pathogens (Meng et al, 2015). Investigation on the resistome (composition of ARGs) of MAR coliforms would add valuable genetic information to facilitate the risk assessment of environmental ARGs in wastewater treatment processes

Methods

Results

Conclusion