Bacterial pathogens and community composition in advanced sewage treatment systems revealed by metagenomics analysis based on high-throughput sequencing.

Xin Lu,Weigang Liang,Yuan Wang,Yunfei Tan,Xu-Xiang Zhang,Kailong Huang,Bo Liu,Zhu Wang,Junying Tang,Horacio Bach

doi:10.1371/journal.pone.0125549

Abstract

This study used 454 pyrosequencing, Illumina high-throughput sequencing and metagenomic analysis to investigate bacterial pathogens and their potential virulence in a sewage treatment plant (STP) applying both conventional and advanced treatment processes. Pyrosequencing and Illumina sequencing consistently demonstrated that Arcobacter genus occupied over 43.42% of total abundance of potential pathogens in the STP. At species level, potential pathogens Arcobacter butzleri, Aeromonas hydrophila and Klebsiella pneumonia dominated in raw sewage, which was also confirmed by quantitative real time PCR. Illumina sequencing also revealed prevalence of various types of pathogenicity islands and virulence proteins in the STP. Most of the potential pathogens and virulence factors were eliminated in the STP, and the removal efficiency mainly depended on oxidation ditch. Compared with sand filtration, magnetic resin seemed to have higher removals in most of the potential pathogens and virulence factors. However, presence of the residual A. butzleri in the final effluent still deserves more concerns. The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment. Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence.

Highlights

The relative number of the sequences assigned to known pathogens was 3,428 (55.33%) for sewage influent (SI), 3,883 (62.63%) for primary effluent (PE), 102 (1.65%) for activated sludge (AS), 76 (1.23%) for secondary effluent (SE), 83 (1.34%) for filter effluent (FFE) and 56 (0.90%) for final resin effluent (FRE) (S5 Table), and a total of 20 genera containing potentially pathogenic species were detected in the six samples (Fig 1A)
At species level, pyrosequencing showed that 1,422 (6 species, 22.95%), 1,302 (7 species, 21.00%), 23 (7 species, 0.37%), 23 (4 species, 0.37%), 21 (7 species, 0.34%) and 6 (4 species, 0.10%) sequences were closely related to known pathogens in SI, PE, AS, SE, FFE and FRE, respectively (Fig 1B), demonstrating that the sewage treatment processes can effectively reduce the relative abundance of the pathogens
Arcobacter butzleri had the highest abundance in the sewage treatment plant (STP), and the relative abundance ranged from 33.33% to 94.87% of total pathogenic bacteria in the six samples (Fig 1C)

Summary

Objectives

This study aimed to investigate the fates of pathogens and VFs in both conventional and advanced treatment processes of a full-scale STP by joint use of 454 pyrosequencing, Illumina high-throughput sequencing and q-PCR

Methods

Results

Conclusion