Biological characteristics and genome of a C3-morphotype phage against multidrug-resistant Escherichia coli

Abstract:

[Objective] The emergence of multidrug-resistant bacteria represents a serious challenge to public health security. In this study, we isolated the phages against multidrug-resistant Escherichia coli and studied their biological and genomic features, aiming to provide a theoretical foundation for the development of phage therapies against drug-resistant bacteria. [Methods] We used the double plate method to isolate E. coli phages from sewage. We employed phosphotungstic acid staining and transmission electron microscopy to observe the phage morphology, and subsequently assessed the biological properties, including host range, thermal and pH stability, one-step growth curve, and in vitro antibacterial effect of the phages. Furthermore, we conducted an in vivo bacteriostasis test to evaluate the protective effects of the phages on Galleria mellonella larvae infected with multidrug-resistant E. coli N1203-1Af. Finally, we carried out whole genome sequencing to analyze the genomic characteristics of the phages. [Results] Five E. coli phages (pEC-S163-2.1, pEC-S163-2.2, pEC-M1167-5Ar.1, pEC-m1291-2DR.1, and pEC-N1203-2Af.1) were isolated. The phage pEC-N1203-2Af.1 presented a C3 morphotype rare among short-tailed phages and was characterized by a long head with the length 2–3 times that of the width. Furthermore, pEC-N1203-2Af.1 demonstrated lytic activity against three out of the 15 tested E. coli strains. Ten minutes after infection, the phage entered an exponential growth phase and exhibited stable activity within the temperature range of –20 to 50 ℃ and pH 4.0–10.0. After treatment with pEC-N1203-2Af.1 at the multiplicity of infection (MOI) levels of 100, 1, and 0.01 for 48 h, the survival of G. mellonella larvae infected with E. coli N1203-2Af reached over 70% (7/10). The genome of phage pEC-N1203-2Af.1 had a total length of 77 334 bp and the G+C content of 42.18%. Notably, the phage did not carry any drug resistance gene or virulence factor. Functional gene prediction revealed a total of 121 CDS in the phage genome, with CDS53–CDS64 encoding the phage structure and lysis module. [Conclusion] The phage pEC-N1203-2Af.1 against multidrug-resistant E. coli exhibits potent antibacterial activity and stable biological characteristics. Our findings suggest that pEC-N1203-2Af.1 may represent a new member of the Kuravirus genus, characterized by a rare C3 morphotype that may be attributed to the main head protein encoded by CDS63. Notably, the distal trimeric protein of bacteriophages with a C3 morphotype exhibits less than 50% consistency across different regions, which indicates that the bacteriophages with this morphotype may have evolved in diverse directions to adapt to varying environmental conditions.