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

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.

Fig 1. Occurrence and abundance of potential pathogens at genus and species levels revealed by 454 pyrosequencing of 16S rRNA gene amplicons.

(A) Relative abundance of potential pathogens at genus level. The effective pyrosequencing reads were classified using MEGAN. Rare genera refer to the taxa with maximum abundance <1% in any sample, including Bacillus, Campylobacter, Helicobacter, Klebsiella, Leptospira, Neisseria and Serratia. (B) Sequence number of potential pathogens at species level under the same sequencing depth (6200 reads). (C) Relative abundance of potential pathogens at species level. All the effective sequences identified as potentially pathogenic species were aligned using the local BLASTN tool. The relative abundance was obtained by normalizing the sequence number of each pathogenic taxon to the total number of all pathogenic taxa in one sample.

Fig 2. Diversity and relative abundance of potential pathogens at genus (A) and species (B) levels revealed by Illumina shotgun sequencing of the metagenomes.

(A) Relative abundance of potentially pathogenic genera. The denoised reads were aligned using MEGAN against 16S rRNA gene Silva database. Rare genera, indicating the taxa with maximum abundance lower than 1% in any sample, include Bordetella, Campylobacter, Corynebacterium, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Salmonella, Serratia, Staphylococcus, Treponema, Vibrio and Yersinia. (B) Heat map illustrating relative abundance (log) of potential pathogens at species level generated by MetaPhlAn. The relative abundance was obtained by normalizing the sequence number of each pathogenic taxon to the total number of all pathogenic taxa in one sample.

Fig 3. Abundance of four potentially pathogenic bacteria in the samples collected from the STP at different time points.

The abundance was determined by quantitative real time PCR and normalized to one nanogram of extracted DNA.

Fig 4. Metagenomic analyses of virulence factors (VFs) at six locations along sewage flow in the STP.

Sequence number (A) and types (B) of virulence factors were calculated based on alignment of the Illumina shotgun sequences (normalized to 9,000,000 reads for each sample) against MvirDB protein database.