Explorative Study on Isolation and Characterization of a Microviridae G4 Bacteriophage, EMCL318, against Multi-Drug-resistant Escherichia coli 15-318

Abstract

Bacteriophages screened and isolated from sewage water samples exhibited antibacterial activities against multi-drug-resistant Escherichia coli strains. Five different water samples from Canadian habitats such as Kamloops Wastewater Treatment Center, Domtar, the Pacific Ocean, Bisaro Anima Cave, and alkali ponds, were used in this study. Four Enterobacteriaceae strains including one non-resistant and three clinical multi-drug Escherichia coli strains (E. coli 15-102, E. coli 15-124, and E. coli 15-318) were selected as target bacteria to screen for the bacteriophages from these collected water samples. Seeded agar assay technique was implemented for the screening. It was found that only sewage water sample exhibited a significant number of plaques count with the E. coli 15-318 (1.82 × 10² plaques/plate) cells in comparison to E. coli non-resistant strain K12 (8 plaques/plate). The phage did not produce plaques in the E. coli 15-124 and E. coli 15-102 strains. The bacteriophage, designated EMCL318, was isolated, purified, characterized, and identified to belong to the G4 species of the Family Microviridae, GenBank accession number MG563770. This is an explorative study conducted in order to reveal the viruses as alternative potentials to fight against emerging and existing multi-drug-resistant infectious diseases.

Keywords: Multi-drug-resistant organisms; alternative therapeutic measures; bacteriophages; phage therapy.

Figure 1

Plaques formation (as indicated with the black arrow heads) on (A) E. coli 15-318 strain, (B) Non-resistant E. coli strain K12.

Figure 2

SDS-PAGE of the purified phage. The major band was identified as G4 F protein using mass spectrometry. The J and G proteins are denoted with asterisks (*) since they are only tentatively identified from the molecular weight.

Figure 3

Cryo-EM structure of the purified phage. On the left is the 9.3 Å image reconstruction of the phage with the surface colored according to the radius. On the right is the image reconstruction with the structure of G4 [19] shown in the electron density envelope.

Figure 4

Phylogenetic relatedness among F proteins derived from different Microviridae. Neighbor-joining tree based on the deduced amino acid sequences from the F proteins using the CLC Sequence Viewer 7 platform (selected alignment by CanadianF alignment). The bootstrap test was performed with 100 replicates. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of amino acid substitutions per site. The protein EMCL318 retrieved highlighted in red.