Complete Genome Sequences of Novel Bovine T4, rv5-Like, and Dhillonviruses Effective against Escherichia coli O157

Enterohemorrhagic Escherichia coli (EHEC) isolates of serotype O157:H7 are serious foodborne zoonotic pathogens and prime targets for biocontrol using bacteriophages. We report on the complete genome sequences of 11 novel lytic bacteriophages, representing three viral genera, isolated from cattle in Hungary that target E. coli O157 strains.

T he role of bacteriophages as alternative antimicrobial agents has gained renewed interest in the past years (1). Enterohemorrhagic Escherichia coli (EHEC) strains, particularly of the O157:H7 serotype, are serious foodborne zoonotic pathogens, and several phages which show effective lysis of these have previously been characterized (2,3). Presently, there are authorized products for use against EHEC as biocontrol in foodstuffs (EcoShield [4]). Nevertheless, a strategy for characterizing new phage isolates that target the eradication of these pathogens from various environments remains prudent.
Here, we present the genome sequences of 11 new lytic bacteriophages (Table 1). Nine phages originated from fecal samples of healthy cattle, the main reservoir of EHEC (5), were isolated from dairy and meat-producing farms in Hungary, using the EHEC O157:H7 strain Sakai for propagation as described earlier (3). Two of the phages were isolated from a sample taken from the cattle farm environment using the same protocol. Briefly, after precultivation of the samples in 10 volumes of tryptic soy broth with bile salts overnight at 37°C under aerobic conditions, the bacterium-free filtrate was spotted onto layered Luria-Bertani agar plates with the propagating strain. All phages were active against the reference strains EHEC O157:H7 Sakai (6) and EDL933 (7); the T4-like and rV5-like phages lysed several bovine E. coli O157 strains representing EHEC, enteropathogenic E. coli (EPEC), and atypical pathotypes isolated earlier in Hungary, as well (8).
Phage DNA from active lytic stocks was isolated using the phenol-chloroform method (9) and concentrated with the DNA Clean and Concentrator kit (Zymo Research, CA, USA) according to the manufacturer's instructions. Genomic DNA sequencing libraries were prepared using the Nextera XT kit (Illumina, Eindhoven, Netherlands). Sequencing was performed using the NextSeq midoutput reagent kit v2.5 (2 Â 150 bp) on an Illumina NextSeq 500 machine. Coverage was between 71Â and 3,830Â, with an average of 1,298Â (Table 1). Quality control and clipping of the reads were performed within the framework of the ASA 3 P pipeline (10). ASA 3 P quality control/read clipping includes the programs FastQC (https://github.com/s-andrews/FastQC), FastQ Screen (https://www .bioinformatics.babraham.ac.uk/projects/fastq_screen), and Trimmomatic (11). The types of genome termini were determined using PhageTerm with default parameters (12). All phages depicted a linear topology (Table 1). Assembly was performed with SPAdes These phages were isolated from the same environmental sample.
c These phages were isolated from the same bovine fecal sample.
d These phages were isolated from the same bovine fecal sample.
3.10.1 (13). Homology searches were conducted against GenBank with the megaBLAST algorithm, available at the NCBI website; annotation was performed with the RAST server using the RASTtk algorithm with default parameters (14). The sequencing showed that some stocks contained multiple phages; therefore, purification of single plaques followed, and PCR for marker genes with primers specifically designed (data not shown) was used to separate single-phage stocks. Phages originating from the same sample are marked as such in Table 1 Phages within the same genus were very uniform. The T4-like phages differed in only a 1-bp gap. Of the rV5-like phages, vb_EcoM_bov10K2, vb_EcoM_bov11CS3, and vb_EcoM_bov25_3 were 100% identical except for a 1-bp gap, while vb_EcoM_bov22_2 differed from them in only 2 single nucleotide polymorphisms (SNPs). The dhillonviruses differed at most by 11 SNPs, with a gap of 30 or 89 bp. The genome of vb_EcoS_ bov22_1 was assembled as a partial genome sequence, with a different start position compared to the other dhillonviruses.
Data availability. The nucleotide sequences of the phages are deposited in GenBank under the accession numbers MT884006 through MT884015 and MT951623.