Phages drive bacterial diversity, profoundly influencing microbial communities, from microbiomes to the drivers of global biogeochemical cycling. Aiming to broaden our understanding of Escherichia coli (MG1655, K-12) phages, we screened 188 Danish wastewater samples and isolated 136 phages. Ninety-two of these have genomic sequences with less than 95% similarity to known phages, while most map to existing genera several represent novel lineages. The isolated phages are highly diverse, estimated to represent roughly one-third of the true diversity of culturable virulent dsDNA Escherichia phages in Danish wastewater, yet almost half (40%) are not represented in metagenomic databases, emphasising the importance of isolating phages to uncover diversity. Seven viral families, Myoviridae, Siphoviridae, Podoviridae, Drexlerviridae, Chaseviridae, Autographviridae, and Microviridae, are represented in the dataset. Their genomes vary drastically in length from 5.3 kb to 170.8 kb, with a guanine and cytosine (GC) content ranging from 35.3% to 60.0%. Hence, even for a model host bacterium, substantial diversity remains to be uncovered. These results expand and underline the range of coliphage diversity and demonstrate how far we are from fully disclosing phage diversity and ecology.

Bacteriophage therapy and application of phages for biocontrol necessitate acquisition of suitable phages. The exclusivity of phage–host relations and the risk of phage resistance instigate a need to rapidly isolate and characterize novel phages and continually build sizeable phage libraries. Current methods for phage isolation are both laborious and time consuming, suitable for the isolation of a limited number of phages. The high-throughput screening method for phages upscales and organizes enrichment of phages for fast isolation and identification of potentially hundreds of distinct phages against single hosts. This enables screening of hundreds of samples, in multiple simultaneous setups with varying parameters, increasing the likelihood of isolating multiple distinct phages specific for the given conditions. The efficiency of the method is emphasized by our screening of 200 environmental samples, resulting in the identification of an abundance of unique phage species virulent to Escherichia coli, Salmonella enterica, Enterococcus faecalis, and Pseudomonas aeruginosa.

Glacier melting and altered precipitation patterns influence Arctic freshwater and coastal ecosystems. Arctic rivers are central to Arctic water ecosystems by linking glacier meltwaters and precipitation with the ocean through transport of particulate matter and microorganisms. However, the impact of different water sources on the microbial communities in Arctic rivers and estuaries remains unknown. In this study we used 16S rRNA gene amplicon sequencing to assess a small river and its estuary on the Disko Island, West Greenland (69°N). Samples were taken in August when there is maximum precipitation and temperatures are high in the Disko Bay area. We describe the bacterial community through a river into the estuary, including communities originating in a glacier and a proglacial lake. Our results show that water from the glacier and lake transports distinct communities into the river in terms of diversity and community composition. Bacteria of terrestrial origin were among the dominating OTUs in the main river, while the glacier and lake supplied the river with water containing fewer terrestrial organisms. Also, more psychrophilic taxa were found in the community supplied by the lake. At the river mouth, the presence of dominant bacterial taxa from the lake and glacier was unnoticeable, but these taxa increased their abundances again further into the estuary. On average 23% of the estuary community consisted of indicator OTUs from different sites along the river. Environmental variables showed only weak correlations with community composition, suggesting that hydrology largely influences the observed patterns.

Extensive use of antibiotics in human healthcare as well as in agricultural and environmental settings has led to the emergence and spread of antibiotic-resistant bacteria, rendering many infections increasingly difficult to treat. Coupled with the limited development of new antibiotics, the rise of antimicrobial resistance (AMR) has caused a major health crisis worldwide, which calls for immediate action. Strengthening AMR surveillance systems is, therefore, crucial to global and national efforts in combating this escalating threat. This review explores the potential of metagenomics, a sequenced-based approach to analyze entire microbial communities without the need for cultivation, as a transformative and rapid tool for improving AMR surveillance strategies as compared to traditional cultivation-based methods. We emphasize the importance of monitoring mobile genetic elements (MGEs), such as integrons, transposons, plasmids, and bacteriophages (phages), in relation to their critical role in facilitating the dissemination of genetic resistance determinants via horizontal gene transfer (HGT) across diverse environments and clinical settings. In this context, the strengths and limitations of current bioinformatic tools designed to detect AMR-associated MGEs in metagenomic datasets, including the emerging potential of predictive machine learning models, are evaluated. Moreover, the controversial role of phages in AMR transmission is discussed alongside the potential of phage therapy as a promising alternative to conventional antibiotic treatment.

In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.

Deazaguanine DNA modifications are widespread in phages, particularly in those with pathogenic hosts. Pseudomonas phage iggy substitutes ∼16.5% of its genomic 2′-deoxyguanosine (G) with dPreQ₀, and the iggy deazaguanine transglycosylase (DpdA) is unique in having a strict GA target motif, not observed previously. The iggy PreQ₀ modification is shown to provide protection against both restriction endonucleases and Cas9 (when present in PAM), thus expanding our understanding of the deazaguanine modification system, its potential, and diversity. Phage iggy represents a new genus of Pseudomonas phages within the Queuovirinae subfamily; which have very little in common with other published phage genomes in terms of nucleotide similarity (<10%) and common proteins (<2%). Interestingly, shared similarity is concentrated in dpdA and preQ₀ biosynthesis genes. TEM imaging confirmed a siphovirus morphology with a prolate icosahedral head and a non-contractile flexible tail with one long central tail spike. The observed protective effect of the deazaguanine modification on the iggy DNA may contribute to its broad within-species host range. Phage iggy was isolated on Pseudomonas aeruginosa PAO1, but also infects PDO300, PAK, PA14, as well as 10 of 27 tested environmental isolates and 13 of 20 tested clinical isolates of P. aeruginosa from patients with cystic fibrosis.