Antibiotic resistance genes carried by microbial communities in drinking water systems

Abstract

The overuse and misuse of antibiotics not only for human therapy but also for livestock breeding have led to the emergence and prosperity of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) and attracted great concerns worldwide. Drinking water distribution system (DWDS) considered as important means to remove pathogens for drinking water supply has been discovered as reservoir of ARGs. However, the comprehensive profiles of ARGs distributed in DWDS or the elimination efficiency of ARGs by drinking water treatment process or the hosts of ARGs distributed in DWDS has not been well studied yet. Thus, the major objectives of this study were (1) to reveal the wide-spectrum profiles of ARGs in DWDS; (2) to investigate the elimination efficiency of ARGs using traditional drinking water treatment process; (3) to develop a metagenomic assembly based method for identification of ARG-carrying genomes; (4) to explore the impacts of chlorination treatment on the shift of ARGs among microbial community.

To scan for the occurrence, abundance and diversity of ARGs in DWDS, the metagenomic sequencing approach combined with structured ARG database was established. ARGs were found enriched by 81%-109% after traditional drinking water treatment processes, while the diversity of ARGs greatly decreased by 28%-51%. Acridine, chloramphenicol, and polymyxin related ARGs were fully removed during drinking water treatment.

A metagenomic assembly based method for identifying ARG-carrying genomes in environmental samples was developed, could be applied to 1) identify host of ARGs, 2) to quantify their abundance in samples based on coverage, 3) to explore the antibiotic resistome shared among bacteria, and 4) to determine the correlations of ARGs with associated genetic elements.

Moreover, the broad-spectrum profiles of ARGs were detected in tap water samples collected from six countries or regions, including mainland China, Hong Kong, Macau, Taiwan, South Africa, Singapore and United States, respectively. Totally, 16 ARG types and 183 ARG subtypes were detected. The most dominant ARG types were bacitracin, multidrug and aminoglycoside resistance genes. 8 subtypes were generalists which existed in all samples, including bacitracin undecaprenyl diphosphatase, bacitracin undecaprenol kinase, multidrug efflux protein, multidrug HAE1-family protein, multidrug mexF, beta-lactam TEM-2, macrolide macB, and beta-lactam TEM-15.

The culture-based isolation approach combined with antimicrobial susceptibility test and high-throughput sequencing technique revealed that 66.8% ARG subtypes decreased after chlorination treatments, while, only 4.5% ARG subtypes were enriched after chlorination. Tetracycline resistance genes were effectively removed by chlorination treatment (>78%). This study is the first application of high-throughput sequencing technique combined with traditional culture-based isolation approach to detect ARGs of viable microorganisms after chlorination process.

The established class 1 integrase database and the assembly of gene cassettes carried by integrons were applied to investigate the abundance of intI1 in diverse environments and the arrangement of gene cassettes. Class 1 integrase intI1 genes were detected at abundance of 0 –1.3 × 〖10〗^(-1)copy of intI1/cellin drinking water samples. Aminoglycoside resistance genes were most frequently observed on gene cassettes, carried by 56.5% assembled ACGCs, followed by trimethoprim resistance genes and beta-lactam resistance genes. This is the first study to utilize metagenomic assembly based approach to scan the arrangements of ARGs-carrying gene cassettes and explore the ARG types carried by gene cassettes of class 1 integrons.