Characterization of ES10 lytic bacteriophage isolated from hospital waste against multidrug-resistant uropathogenic E. coli

Abstract

Escherichia coli is the major causative agent of urinary tract infections worldwide and the emergence of multi-drug resistant determinants among clinical isolates necessitates the development of novel therapeutic agents. Lytic bacteriophages efficiently kill specific bacteria and seems promising approach in controlling infections caused by multi-drug resistant pathogens. This study aimed the isolation and detailed characterization of lytic bacteriophage designated as ES10 capable of lysing multidrug-resistant uropathogenic E. coli. ES10 had icosahedral head and non-contractile tail and genome size was 48,315 base pairs long encoding 74 proteins. Antibiotics resistance, virulence and lysogenic cycle associated genes were not found in ES10 phage genome. Morphological and whole genome analysis of ES10 phage showed that ES10 is the member of Drexlerviridae. Latent time of ES10 was 30 min, burst size was 90, and optimal multiplicity of infection was 1. ES10 was stable in human blood and subsequently caused 99.34% reduction of host bacteria. Calcium chloride shortened the adsorption time and latency period of ES10 and significantly inhibited biofilm formation of host bacteria. ES10 caused 99.84% reduction of host bacteria from contaminated fomites. ES10 phage possesses potential to be utilized in standard phage therapy.

Keywords: E. coli; antibiotic resistance; bacteriophage; biofilm; phage therapy.

Figure 1

Phylogenetic tree of ES10 phage, constructed on the basis of whole genome similarity of ES10 phage with reported phages genome on NCBI.

Figure 2

Proteomic based phylogenetic tree of ES10 phage.

Figure 3

Transmission electron micrograph of ES10 phage. Scale used is of 45 nm.

Figure 4

Evaluation of effect of different media on ES10 phage plaque size (A), effect of different concentrations of soft gar of plaque size of ES10 phage (B), and effect of incubation period on plaque size of ES10 phage (C).

Figure 5

Effect of different temperature on ES10 phage stability (A), effect of pH on ES10 phage stability (B).

Figure 6

Optimal multiplicity of infection of ES10 phage.

Figure 7

Evaluation of latent time and burst size of ES10.

Figure 8

Effect of calcium chloride and magnesium chloride on replication cycle of ES10 phage. Stars are representing the difference between the groups by p value. P is showing the p-value in independent sample t-test. ^***P < 0.001, ^**P < 0.01, ^*P < 0.05.

Figure 9

Effect of ES10 alone and ES10 in combination with calcium chloride and magnesium chloride on host bacteria turbidity reduction (A), and host bacteria biofilm formation inhibition (B).

Figure 10

Effect of incubation period (A), temperature (B), pH (C), and media (D) on turbidity reduction of host bacteria by ES10. Stars are representing the difference between the groups by p value. P is showing the p-value in independent sample t-test. ^***P < 0.001, ^**P < 0.01, ^*P < 0.05.

Figure 11

Reduction in host bacterial colony forming unit by ES10 phage on contaminated fomites.