Isolation and molecular characterisation of lytic bacteriophages specific to Escherichia coli isolates from neonatal calf diarrhoea

Bacteriophages (phages) are the most widespread organisms on Earth and can be found in every ecosystem ever discovered. In recent years, advances in sequencing and bioinformatics have provided important insights into phage diversity, taxonomy, host specificity, population structure and genomic characteristics. Neonatal calf diarrhoea, especially due to Escherichia coli infections, causes serious economic losses in cattle breeding worldwide and in our country, and the increasing antimicrobial resistance problem and the difficulties experienced in its treatment increase the need for new and alternative treatment methods. The increasing interest in phage therapy and the generally narrow host range of phages make it necessary to continuously isolate and characterise new phages in order to obtain effective phages against clinically important pathogens. In this study, we aimed to isolate and characterise lytic bacteriophages effective against enteropathogenic E. coli strains isolated from calves with neonatal diarrhoea from various environmental sources and to establish a coliphage collection using next generation sequence analysis technology and bioinformatic approaches. As a result of molecular characterisation of 102 E. coli strains isolated from calves with neonatal diarrhoea, STEC (29.4%), ETEC (10.8%) and EPEC (2%) strains were identified in the pathotype distribution. In addition, 10 hybrid strains (7 STEC/ETEC, 1 ETEC/EPEC, 2 STEC/ETEC/EPEC) were identified. In the lytic activity analyses of 27 coliphages isolated from environmental samples, it was shown that 30% of the phages were able to infect ETEC-1, 26% ETEC-2, 15% ETEC-3, 69% STEC and 8% EPEC+ETEC pathotypes, among which 10 phages had multi-pathotype infection capacity. In the in vitro model established to determine phage-host interaction and population growth dynamics, bacterial growth dynamics were monitored by optical density (OD) measurements in culture studies with phages isolated from 5 different E. coli strains. Centroid Index, a metric system sensitive to OD changes, was used to evaluate phage-host proliferation kinetics. By selecting 12 phages with high lytic activity, their therapeutic efficacy at three different MOI levels (0.1, 1, 10) was evaluated and it was determined that pH 6.3-7.0 range was optimal for both bacterial growth and phage activity. In addition, phages were found to have thermal stability ranging between 30ºC and 60ºC. Analysis of the genome sequences of the five selected phages revealed that these phages have linear dsDNA with genome sizes ranging between 44 and 51bp and GC content ranging between 54.55% and 54.67%. Taxonomically, five phages were identified as belonging to the order Caudoviricetes and the genus Dhillonvirus. The genomes of these phages contain between 58 and 71 ORFs encoding putative and known functional proteins. In addition, the genomes contain core genes associated with basic molecular processes such as DNA replication, transcription regulation, nucleotide metabolism, phage structural formation (capsid and tail) and lysis, and a possible phage defence system anti-CRISPR protein. No undesirable features such as integrase, antimicrobial resistance, virulence or toxin genes were found in any genome. This study revealed that the E. coli phages analysed in this study have a high level of genomic and proteomic similarity with other known Escherichia phages. The findings obtained during the experimental process revealed the therapeutic potential of selected phages by evaluating phage-host bacterial interactions through population dynamics. Accordingly, candidate phages were identified for the development of phage therapy protocols with optimised efficacy and safety for the treatment of neonatal calf diarrhoea. By presenting data on coliphage diversity in different environmental samples, it contributed to the establishment of a characterised coliphage collection that can be used in calves with neonatal diarrhoea. Key Words: E.coli, Phage, Neonatal Calf Diarrhoea, One Health