Moon et al. Microbiome (2020) 8:75 https://doi.org/10.1186/s40168-020-00863-4 RESEARCH Open Access Freshwater viral metagenome reveals novel and functional phage-borne antibiotic resistance genes Kira Moon1, Jeong Ho Jeon2, Ilnam Kang1, Kwang Seung Park2, Kihyun Lee3, Chang-Jun Cha3, Sang Hee Lee2* and Jang-Cheon Cho1* Abstract Background: Antibiotic resistance developed by bacteria is a significant threat to global health. Antibiotic resistance genes (ARGs) spread across different bacterial populations through multiple dissemination routes, including horizontal gene transfer mediated by bacteriophages. ARGs carried by bacteriophages are considered especially threatening due to their prolonged persistence in the environment, fast replication rates, and ability to infect diverse bacterial hosts. Several studies employing qPCR and viral metagenomics have shown that viral fraction and viral sequence reads in clinical and environmental samples carry many ARGs. However, only a few ARGs have been found in viral contigs assembled from metagenome reads, with most of these genes lacking effective antibiotic resistance phenotypes. Owing to the wide application of viral metagenomics, nevertheless, different classes of ARGs are being continuously found in viral metagenomes acquired from diverse environments. As such, the presence and functionality of ARGs encoded by bacteriophages remain up for debate. Results: We evaluated ARGs excavated from viral contigs recovered from urban surface water viral metagenome data. In virome reads and contigs, diverse ARGs, including polymyxin resistance genes, multidrug efflux proteins, and β-lactamases, were identified. In particular, when a lenient threshold of e value of ≤ 1×e−5 and query coverage of ≥ 60% were employed in the Resfams database, the novel β-lactamases blaHRV-1 and blaHRVM-1 were found. These genes had unique sequences, forming distinct clades of class A and subclass B3 β-lactamases, respectively. Minimum inhibitory concentration analyses for E. coli strains harboring blaHRV-1 and blaHRVM-1 and catalytic kinetics of purified HRV-1 and HRVM-1 showed reduced susceptibility to penicillin, narrow- and extended- spectrum cephalosporins, and carbapenems. These genes were also found in bacterial metagenomes, indicating that they were harbored by actively infecting phages. (Continued on next page) * Correspondence: [email protected]; [email protected] 2National Leading Research Laboratory of Drug Resistance Proteomics, Department of Biological Sciences, Myongji University, 116 Myongjiro, Yongin, Gyeonggi-do 17058, Republic of Korea 1Department of Biological Sciences, Inha University, Incheon 22212, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Moon et al. Microbiome (2020) 8:75 Page 2 of 15 (Continued from previous page) Conclusion: Our results showed that viruses in the environment carry as-yet-unreported functional ARGs, albeit in small quantities. We thereby suggest that environmental bacteriophages could be reservoirs of widely variable, unknown ARGs that could be disseminated via virus-host interactions. Keywords: Bacteriophage, Viral metagenome, Virome, Antibiotic resistance gene, Minimum inhibitory concentration, β-lactamase, River Background diverse environments based on PCR of viral DNA. How- Continually emerging antibiotic resistance of pathogenic ever, due to the limitations of PCR to detect unreported bacteria is a significant threat to global health. The or variances of ARGs, shotgun metagenomics that pro- World Health Organization (WHO) reported that from duce large amount of genomic information without a 2017 to 2018, approximately 1.1 million patients from confined scope has been used to screen for ARGs. 69 monitoring countries were infected with antibiotic re- The rapid development of viral metagenomics over the sistant pathogens [1], and this number is expected to last decade has led to several viral metagenome (virome) rise. Antibiotic resistant bacteria are a compelling health analyses of various environments, including oceans, issue because they make antibiotic treatments obsolete, freshwater, and clinical samples [10–14], revealing a even leading to the death of the patient. Bacteria natur- great number of novel viral sequences, including ARGs ally produce antimicrobial substances to disrupt neigh- [15]. Phage-associated ARGs were found in clinical vir- boring competitors, and accordingly, targeted bacteria omes [16, 17] where both bacteria and phages are heav- develop antibiotic resistance genes (ARGs) to defend ily exposed to antibiotics, and in marine and river against such mechanisms. Excessive use of antibiotics in viromes [18–20], indicating that ARGs are commonly medical practice has been implicated to accelerate devel- carried by phages in natural environments. Although a opment and dissemination of antibiotic resistance high proportion of virome reads was found to be encod- among bacterial populations in clinical and natural envi- ing ARGs, those genes remain uncertain whether they ronments. ARGs can be spread from one bacterial cell to were collected from phage genomes or bacterial genome another through horizontal gene transfer (HGT) [2]. remnants [21]. Contrast to ARG detection frequency in HGT of ARGs is commonly mediated by conjugation raw virome reads, only a few ARGs have been discovered that requires physical contact between bacterial cells via in the viral contigs assembled from virome reads [22]. pili or adhesins [3]. Conversely, transduction of ARGs Also, in a recent study, none of the four ARGs selected mediated by bacteriophages (phages) is rare but remains from viral contigs expressed antibiotic resistance when a significant dissemination route of ARGs. cloned into E. coli [21], leading to the conclusion that Although phages are the smallest and simplest bio- numbers of phage-carried ARGs might be overestimated. logical entities on earth, they have significant ecological Nevertheless, as ARGs continue to be detected in phage roles as bacterial population controllers through lytic in- isolates and viromes from diverse environments [18, 23– fections and contributors to bacterial gene diversifica- 26], whether phages are important reservoirs of ARGs or tion. During infection, phages randomly incorporate not is still debated. bacterial genome fragments into phage capsids or Because only a small proportion of phages are specu- genomes as they hijack the hosts’ genome replication lated to encode ARGs, phage-associated ARGs are often machinery. Consequently, those genome fragments can neglected in ARG researches, leaving them as unsuper- be transferred to subsequent bacterial host genomes as vised resistome to threat public health. Although occur- phages infect other host cells. Some phages are known rences are rare, it is evident that phages mediate ARG to have wide host range, a few with even of different transduction between bacterial cells [6] and contribute bacterial orders [4], thereby functioning as a bridge of to ARG dissemination [27]. Therefore, in our present genetic exchange between different bacterial popula- study, viral metagenomes were thoroughly studied to tions. Therefore, phage-associated ARGs are considered screen for distribution of ARGs within phage popula- especially significant due to their possibility for wide dis- tions, search for bona fide ARGs carried by phage ge- semination [5, 6]. Since phages are not directly influ- nomes, and verify phenotypes and functionality of the enced by antibiotics, the presence of ARGs is best phage-encoded ARGs. Using viromes constructed from evaluated by identifying nucleic acids in phage particles. urban rivers, we performed a functional assay of ARGs ARGs conferring resistance to lactamase [7], quinolone recovered from viral contigs and reported the first con- [8], and vancomycin [9] antibiotics were detected across vincing evidence of two new types of β-lactamases Moon et al. Microbiome (2020) 8:75 Page 3 of 15 derived from uncultured phages. Discovery of these pre- taxonomically assigned to bacterial genes, largely derived viously unreported ARGs in the freshwater virome sug- from Proteobacteria (Fig. S3). Along with phage- and gests environmental phages could be an important prophage-related functions, virome reads were also an- reservoir of unexplored ARGs. notated as diverse auxiliary metabolic genes (AMGs) [30], such as enzymes for carbohydrate metabolism, res- Results piration,