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 bla HRV-1 and bla HRVM-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 bla HRV-1 and bla HRVM-1 and catalytic kinetics of purified HRV-1 and HRVM-1 showed reduced susceptibility to penicillin, narrow-and extendedspectrum cephalosporins, and carbapenems. These genes were also found in bacterial metagenomes, indicating that they were harbored by actively infecting phages.

Bacteriophages infecting major groups of freshwater heterotrophic bacteria have been rarely isolated, hampering analyses of freshwater viromes. Here, we report the isolation and genomic characterization of P19250A, the first phage that infects the LD28 clade, an abundant freshwater methylotrophic bacterial group. P19250A was isolated from Lake Soyang, an oligotrophic reservoir, using an LD28 strain as a host. Morphological and genomic analyses revealed that P19250A is a lytic siphovirus with a ∼38.6-kb genome. To analyze the distribution of P19250A genome within its habitat, six seasonal viral metagenome (virome) samples were prepared from Lake Soyang. Through binning analysis of freshwater viromes, P19250A was shown to be the most highly assigned freshwater phage that infects heterotrophic bacteria (up to 8.21%) in five viromes. Furthermore, when freshwater virome data collected worldwide were analyzed, P19250A genome also showed high abundance, especially in Lough Neagh, UK, where P19250A genome was recorded as the most abundant bacteriophage. From metagenome analysis, the proportion of P19250A-assigned reads showed seasonal fluctuation following the abundance of the LD28 clade in Lake Soyang. These results showed that P19250A would be an essential resource for analyses of freshwater viromes, and also suggest that phages of other abundant freshwater bacteria need to be isolated for better understanding of freshwater viruses.

A high number of viral metagenomes have revealed countless genomes of putative bacteriophages that have not yet been identified due to limitations in bacteriophage cultures. However, most virome studies have been focused on marine or gut environments, thereby leaving the viral community structure of freshwater lakes unclear. Because the lakes located around the globe have independent ecosystems with unique characteristics, viral community structures are also distinctive but comparable. Here, we present data on viral metagenomes that were seasonally collected at a depth of 1 m from Lake Soyang, the largest freshwater reservoir in South Korea. Through shotgun metagenome sequencing using the Illumina MiSeq platform, 3.08 to 5.54-Gbps of reads per virome were obtained. To predict the viral genome sequences within Lake Soyang, contigs were constructed and 648 to 1,004 putative viral contigs were obtained per sample. We expect that both viral metagenome reads and viral contigs would contribute in comparing and understanding of viral communities among different freshwater lakes depending on seasonal changes.

Bacteriophages of freshwater environments have not been well studied despite their numerical dominance and ecological importance. Currently, very few phages have been isolated for many abundant freshwater bacterial groups, especially for the family Comamonadaceae that is found ubiquitously in freshwater habitats. In this study, we report two novel phages, P26059A and P26059B, that were isolated from Lake Soyang in South Korea, and lytically infected bacterial strain IMCC26059, a member of the family Comamonadaceae. Morphological observations revealed that phages P26059A and P26059B belonged to the family Siphoviridae and Podoviridae, respectively. Of 12 bacterial strains tested, the two phages infected strain IMCC26059 only, showing a very narrow host range. The genomes of the two phages were different in length and highly distinct from each other with little sequence similarity. A comparison of the phage genome sequences and freshwater viral metagenomes showed that the phage populations represented by P26059A and P26059B exist in the environment with different distribution patterns. Presence of the phages in Lake Soyang and Lake Michigan also indicated a consistent lytic infection of the Comamonadaceae bacterium, which might control the population size of this bacterial group. Taken together, although the two phages shared a host strain, they showed completely distinctive characteristics from each other in morphological, genomic, and ecological analyses. Considering the abundance of the family Comamonadaceae in freshwater habitats and the rarity of phage isolates infecting this family, the two phages and their genomes in this study would be valuable resources for freshwater virus research.

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