Characterization of two lytic bacteriophages isolated from urban surface water in Romania targeting multidrug-resistant Escherichia coli

Abstract

BackgroundThe global rise of multidrug-resistant (MDR) bacteria represents a critical public health threat, and Romania ranks amongst the most affected countries in Europe. As conventional therapy increasingly fails, bacteriophage therapy has re-emerged as a promising alternative to antibiotics. Urban rivers, contaminated with resistant bacterial strains, represent an underexplored and accessible reservoir for the isolation of lytic phages with therapeutic potential. MethodsTwo bacteriophages, 17M_Ec17_D and 22C_Ec22_D, were isolated from the Dambovita River, Bucharest, Romania, using MDR E. coli as host bacteria. Phage characterization included plaque morphology, transmission electron microscopy, and host range assessment by spot assay against 30 MDR E. coli isolates. Whole genome sequencing was performed on Illumina MiSeq and Oxford Nanopore Technologies MinION platforms, followed by bioinformatic analysis including taxonomic classification, lifestyle prediction, and functional annotation. ResultsBoth phages formed clear plaques and were classified as Kayfunavirus (17M_Ec17_D, Podoviridae-like) and Kagunavirus (22C_Ec22_D, Siphoviridae-like) with nucleotide similarities of 89.2% and 71.4% to their closest relatives, respectively, suggesting both are candidates for novel species. Host range analysis revealed lytic activity against 13% and 10% of tested MDR isolates, with complementary infection profiles. Genomic analysis confirmed a strictly lytic lifestyle for both phages, supported by the presence of holin and spanin genes and the absence of lysogenic modules, antibiotic resistance genes, and virulence factors. ConclusionsTo the best of our knowledge, this is the first study conducted in Romania to isolate and genomically characterize lytic bacteriophages targeting MDR E. coli. The characterized phages represent safe therapeutic candidates whose complementary host ranges suggest potential application as part of phage cocktail to broaden antimicrobial coverage against MDR infections.