A comprehensive insight into tetracycline resistant bacteria and antibiotic resistance genes in activated sludge using next-generation sequencing.

Kailong Huang,Junying Tang,Hongqiang Ren,Ke Xu,Xu-Xiang Zhang

doi:10.3390/ijms150610083

Abstract

In order to comprehensively investigate tetracycline resistance in activated sludge of sewage treatment plants, 454 pyrosequencing and Illumina high-throughput sequencing were used to detect potential tetracycline resistant bacteria (TRB) and antibiotic resistance genes (ARGs) in sludge cultured with different concentrations of tetracycline. Pyrosequencing of 16S rRNA gene revealed that tetracycline treatment greatly affected the bacterial community structure of the sludge. Nine genera consisting of Sulfuritalea, Armatimonas, Prosthecobacter, Hyphomicrobium, Azonexus, Longilinea, Paracoccus, Novosphingobium and Rhodobacter were identified as potential TRB in the sludge. Results of qPCR, molecular cloning and metagenomic analysis consistently indicated that tetracycline treatment could increase both the abundance and diversity of the tet genes, but decreased the occurrence and diversity of non-tetracycline ARG, especially sulfonamide resistance gene sul2. Cluster analysis showed that tetracycline treatment at subinhibitory concentrations (5 mg/L) was found to pose greater effects on the bacterial community composition, which may be responsible for the variations of the ARGs abundance. This study indicated that joint use of 454 pyrosequencing and Illumina high-throughput sequencing can be effectively used to explore ARB and ARGs in the environment, and future studies should include an in-depth investigation of the relationship between microbial community, ARGs and antibiotics in sewage treatment plant (STP) sludge.

Highlights

Extensive use and abuse of antibiotics in health protection and agricultural production have led to the emergence of widespread various antibiotic resistance genes (ARGs) and resistant bacteria (ARB) in the environment [1,2], which is thought to pose an ever increasing threat to public health [3]
Increasing evidence suggested that sewage treatment plants (STPs) serve as important reservoirs for environmental tetracycline resistant bacteria (TRB) and resistance genes [9,10,11]
20 mg/L tended to increase the number of operational taxonomic units (OTUs) in the sludge, which agrees with the patterns of Chao 1 and Shannon index (Table 1)

Summary

Introduction

Extensive use and abuse of antibiotics in health protection and agricultural production have led to the emergence of widespread various antibiotic resistance genes (ARGs) and resistant bacteria (ARB) in the environment [1,2], which is thought to pose an ever increasing threat to public health [3]. Increasing evidence suggested that sewage treatment plants (STPs) serve as important reservoirs for environmental tetracycline resistant bacteria (TRB) and resistance genes (tet) [9,10,11]. Both culture-based [9,10,12] and culture-independent approaches [13] have been used to explore the TRB in STPs. Classical microbiological methodology relies on plate counting of coliforms, which makes the assessment results unrepresentative and biased. Growing evidence has shown that next-generation sequencing is a powerful metagenomic tool for comprehensive overview of microbial communities and/or functional genes in various environmental compartments, including soil [16], human gastrointestinal tract [17], sediments [18], and wastewater treatment plants [19]

Methods

Results

Conclusion