Isolation and characterization of lytic bacteriophages from various sources in Addis Ababa against antimicrobial-resistant diarrheagenic Escherichia coli strains and evaluation of their therapeutic potential

Abstract

Background: Escherichia coli is a common fecal coliform, facultative aerobic, gram-negative bacterium. Pathogenic strains of such microbes have evolved to cause diarrhea, urinary tract infections, and septicemias. The emergence of antibiotic resistance urged the identification of an alternative strategy. The use of lytic bacteriophages against the control of pathogenic E. coli in clinics and different environmental setups (waste and drink water management) has become an alternative therapy to antibiotic therapy. Thus, this study aimed to isolate and characterize lytic bacteriophage from various sources in Addis Ababa, tested them against antimicrobial-resistant diarrheagenic E. coli strains and evaluated their therapeutic potential under in vitro conditions.

Methods: A total of 14 samples were processed against six different diarrheagenic E. coli strains. The conventional culture and plaque analysis agar overlay method was used to recover lytic bacteriophage isolates. The phage isolates were characterized to determine their lytic effect, growth characteristics, host range activity, and stability under different temperature and pH conditions. Phage isolates were identified by scanning electron microscope (SEM), and molecular techniques (PCR).

Results: In total, 17 phages were recovered from 84 tested plates. Of the 17 phage isolates, 11 (65%) were Myoviridae-like phages, and 6 (35%) phage isolates were Podoviridae and Siphoviridae by morphology and PCR identification. Based on the host range test, growth characteristics, and stability test 7 potent phages were selected. These phages demonstrated better growth characteristics, including short latent periods, highest burst sizes, and wider host ranges, as well as thermal stability and the ability to survive in a wide range of pH levels.

Conclusions: The promising effect of the phages isolated in this study against AMR pathogenic E. coli has raised the possibility of their use in the future treatment of E. coli infections.

Keywords: Myoviridae; Podoviridae; Siphoviridae; Diarrheagenic E. coli; Lytic bacteriophages.

Fig. 1

Study area geographic map

Fig. 2

Representative bacteriophage plaque images during isolation and purification depicting different morphologies: (A) Clear plaque with elevated halos (B) Large size clear plaques (C) Small sized clear plaques (D) Small pinned clear plaques

Fig. 3

Frequency of lytic phages isolated against the six pathogenic E. coli strains

Fig. 4

Frequency of lytic phages recovered from the sample sites

Fig. 5

Scanning electron microscopy representative images of phage families: (A) Myoviridae like phage (B) Siphoviridae like phage (C) Podoviridae like phage morphology

Fig. 6

Analysis of the separated phages’ host range representative image: The spot lysed by different phages that depict a clear lysis zone on a TSA agar plate

Fig. 7

Representative image of phage plaques in the EOP reference host

Fig 8

One-step growth curves in seven potent phages: The double-layer agar method was used to calculate the phage titers. Latent periods and burst sizes were estimated from the graph in which an arrow line indicated for each phage. A EH-B-A (A1) phage growth curve; (B) EP-M-A growth curve; (C) EP-B-K (E2); (D) EH-B-A (A2); (E) ET-SD-TH; (F) EI-SP-GF; (G) EH-SD-TH phage growth curve. The average of two separate trials is shown by each data point. Standard deviations between the duplicate samples are displayed by error bars

Fig. 9

Stability of E. coli lytic phages at different temperature and pH levels: (A) Phages were incubated at various temperatures between 25 and 90 °C. (B) pH sensitivity test at different pH ranges between 3 and 11. The average of two separate experiments was used to draw the graph. Vertical lines in the graph represent standard deviations

Fig. 10

Gel images of PCR identified phage isolates: (A) Lane-1, 1 kb plus ladder; Lane-2, T5; Lane-3 & 4, T7 and Lane-5 to 8, FO1 phages with 200 bp, 461 bp, and 519 bp respectively (B) Lane-1, 1 kb plus ladder; Lane-2 to 8 all phages were T4 phages with 240 bp size (C) Seven potent phages selected in separate gel; Lane-1, 1 kb plus ladder; Lane − 2 & 3, 519 bp sized FO1 phages of EP-M-A & EP-B-K (E2), Lane-4, 461 bp sized T7 phage EI-SP-GF and Lane- 5 to 8, 240 bp sized phages of EH-SD-TH, EH-B-A (A1), EH-B-A (A2), ET-SD-TH