Exploring the Remarkable Diversity of Culturable Escherichia coli Phages in the Danish Wastewater Environment

Abstract

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.

Systematic exploration ofEscherichia coliphage-host interactions with the BASEL phage collection

“…The Felixounavirus genus in the Ounavirinae subfamily of Myoviridae comprises a group of phages with genomes of 84-91 kb and characteristically straight lateral tail fibers of which Salmonella phage Felix O1 has been most well studied [30,90] (Figs 11A and 11B). Previous work aimed at the isolation of E. coli phages differed greatly in the reported abundance of Felixounavirus phages, ranging from no detection [23] over a moderate number of isolates in most studies [25,91] to around one fourth of all [24]. Across our phage sampling experiments we found a single Felixounavirus phage, JohannRWettstein (Bas61), which is rather distantly related to Felix O1 within the eponymous genus ( Fig 11B).…”

“…This strain, E. coli K-12 MG1655 ΔRM, therefore lacks the O-antigen glycan barrier (see below), all restriction systems, as well as the RexAB and PifA Abi systems (see Materials and Methods as well as S1 Text). E. coli K-12 ΔRM was subsequently used to isolate hundreds of phages from environmental samples such as river water or compost, but mostly from the inflow of different sewage treatment facilities (Fig 2A and Previous bacteriophage isolation studies had already provided deep insight into the diversity of tailed, lytic E. coli phages in samples ranging from sewage over diverse natural environments to infant guts or blood and urine of patients in tertiary care [23][24][25][26][27][28][29]. Despite the wide diversity of known E. coli phages [30], nearly all phage isolates reported in these studies belonged to five major groups and were either myoviruses of 1) Tevenvirinae or 2) Vequintavirinae subfamilies and close relatives, 3) large siphoviruses of the Markadamsvirinae subfamily within Demerecviridae, or small siphoviruses of 4) diverse Drexlerviridae subfamilies or 5) the genera Dhillonvirus, Nonagvirus, and Seuratvirus of the Siphoviridae family.…”

“…Despite the wide diversity of known E. coli phages [30], nearly all phage isolates reported in these studies belonged to five major groups and were either myoviruses of 1) Tevenvirinae or 2) Vequintavirinae subfamilies and close relatives, 3) large siphoviruses of the Markadamsvirinae subfamily within Demerecviridae, or small siphoviruses of 4) diverse Drexlerviridae subfamilies or 5) the genera Dhillonvirus, Nonagvirus, and Seuratvirus of the Siphoviridae family. Podoviruses of any kind were rarely reported, and if, then were mostly Autographiviridae isolated using enrichment cultures that are known to greatly favor such fast-growing phages [24,31]. This pattern does not seem to be strongly biased by any given strain of E.coli as isolation host because a large, very thorough study using diverse E. coli strains reported essentially the same composition of taxonomic groups [23].…”

“…While few representatives like FritzSarasin (Bas04) or PeterMerian (Bas05) are highly resistant, most Drexlerviridae are very sensitive to any kind of RM systems sometimes including the type I machineries that are largely unable to target any other phage that we tested ( Fig 4D). Previous work suggested that Drexlerviridae might employ DNA methyltransferases as a defense strategy against host restriction [24], and these phages indeed encode variable sets of N6-adenine and C5-cytosine methyltransferases. However, we found no obvious pattern that would link DNA methyltransferases or any other specific genomic features to restriction resistance / sensitivity.…”

“…To classify our phage isolates on the species level, we generated pairwise whole-genome alignments using progressiveMauve implemented in Geneious Prime 2021.0.1 [134] with genomes of related phages as identified in our phylogenies. From these alignments, the nucleotide sequence identity was determined as the query coverage multiplied by the identity of aligned segments [24]. When the genomes were largely syntenic and showed >95% nucleotide sequence identity, we classified our isolates as the same species as their close relative [141], at least if this phage has been assigned to a species by the ICTV [142].…”