Isolation and Characterization of a Myoviridae MJ1 Bacteriophage Against Multi-Drug Resistant Escherichia coli 3

Abstract

Background: Antibiotic resistance in Escherichia coli, a member of the Enterobacteriaceae, is of particular concern because it is the most common (Gram-negative) pathogen causing nosocomial and community infections. Researchers are now considering the use of phages for the control of various antibiotic-resistant bacterial infections.

Objectives: The purpose of this study was to isolate and characterize a novel pathogenic/lytic phage that targets multi-drug resistant (MDR) E. coli 3, and to investigate its effectiveness at lysing this bacterium.

Materials and methods: A clinical strain of E. coli 3 was identified based on its 16S rRNA sequencing and its antibiotic resistance profile was determined by the disc diffusion method. A bacteriophage was isolated from wastewater and its various characteristics, such as host range, heat tolerance, pH stability, one step growth, total protein content, and genome size, were determined. The antibacterial property of the phage was determined against log-phase bacterial planktonic cells at 37°C.

Results: The bacteriophage, designated MJ1, was isolated by testing against a clinical MDR E. coli 3 strain. The MJ1 phage showed a wide range of heat and pH stability. The phage morphology, determined by transmission electron microscopy, revealed a structure comprised of a head (108 ± 0.2 nm long by 128 ± 0.5 nm wide) and a contractile tail (123 ± 0.5 nm long by 15 - 26 nm wide). These features placed the MJ1 phage in the family Myoviridae and the order Caudovirales. Eleven structural proteins (17 to 200 kDa) for this phage were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A double stranded DNA, approximately 32 kb, in size was detected for this phage on agarose gels. The phage efficacy against E. coli 3 planktonic cells was also investigated. The MJ1 phage demonstrated a very good capability to reduce the numbers of E. coli 3 planktonic cells, as determined by a change in the bacterial growth (an optical density decrease at 600 nm from 0.40 to 0.12).

Conclusions: MJ1 phage has much potential for use in phage therapy and other applications.

Keywords: Bacteriophage; Contractile; Escherichia coli; Myoviridae; Nosocomial; Planktonic.

Figure 1.. Plaque Assay for MJ1 Phage

A, Spot test for the detection of MJ1 phage; and B, a higher dilution (10-6) of phage titer showing clear plaques with an average diameter of 0.8 mm.

Figure 2.. Stability Assays for MJ1 Phage

A, Stability of the MJ1 phage treated at different temperatures for 60 min; and B, Stability of the MJ1 phage after treatment at different pH values overnight at 37°C. All data are the means of 3 determinations ± standard deviations (SD).

Figure 3.. One Step Growth Experiment

The latent time and burst size of the MJ1 phage were deduced from the curve with a triphasic pattern. All values are the means of 3 determinations ± SD.

Figure 4.. Transmission Electron Micrographs of the Purified MJ1 Phage; Scale Bars, 200 nm

Figure 5.. Results of Agarose Gel Electrophoresis and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

A, The MJ1 phage genome, as detected by agarose (0.6%) gel electrophoresis. Lane 1 shows a high range DNA ladder (Gene Ruler) and lane 2 shows a band of phage DNA having a size of approximately 32 kb; B, Lane 1 shows a 1 kb DNA ladder (New England Biolabs) and lane 2 shows the MJ1 phage DNA restriction analysis with EcoR1; C, Image shows the SDS-PAGE analysis of the MJ1 phage structural proteins; lane 1 shows broad range protein molecular weight markers (Precision Plus Protein^TM , Bio-Red) and lane 2 shows the MJ1 phage proteins.

Figure 6.. Bacterial Reduction Assay

Effect of the MJ1 phage on bacterial growth of an E. coli 3 log-phase culture, compared with a control culture (having no phage). All values are the means of 3 determinations.