Bacteriophage Isolation, Purification, and Characterization Techniques Against Ubiquitous Opportunistic Pathogens

Abstract

Healthcare‐associated infection with “ESKAPE” pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) is a global health crisis due to their extensive intrinsic antibiotic resistance and the ability to quickly acquire resistance determinants. Alternative treatment options are required to combat this crisis, and one possibility is the use of bacteriophages, or viruses that strictly infect the pathogenic bacteria. Currently, there is a renaissance in research and development into the use of phages to target multi‐, extensively, and pan‐resistant bacterial infections in humans, known as phage therapy. Using A. baumannii as an example, this article describes the isolation and purification of bacteriophages from sewage and soil samples, as well as general methods used in phage research such as precipitation of phages using polyethylene glycol, host range analysis, single‐cell burst size determination, DNA extraction, and restriction fragment length polymorphism analysis. © 2022 National Research Council Canada. Current Protocols © 2022 Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Industry. Basic Protocol 1: Isolation of bacteriophages against A. baumannii from sewage samples Alternate Protocol 1: Isolation of bacteriophages against A. baumannii from soil samples Support Protocol 1: Titering a bacteriophage stock Basic Protocol 2: Purification of phage to an axenic working stock Support Protocol 2: Liquid propagation of bacteriophage Basic Protocol 3: Host range analysis using the spot plate method Basic Protocol 4: Single burst size analysis Alternate Protocol 2: One‐step growth curve Basic Protocol 5: Precipitation of bacteriophage using PEG 6000 Basic Protocol 6: DNA extraction from dsDNA bacteriophages Basic Protocol 7: Restriction fragment length polymorphism analysis of novel phage genomes

Localization of G1A1a Allergenic Domain Destroyed by Thermal Processing

“…Then, we centrifuged the bacterial solution at 4 °C and 9,000 rpm for 10 min. Then, we took the supernatant and mixed it evenly in the following volume phage ratio: chloroform = 15:1 . We added the remaining supernatant with final concentrations of 1 M NaCl and 10% PEG 8,000.…”

“…Then, we took the supernatant and mixed it evenly in the following volume phage ratio: chloroform = 15:1. 31 We added the remaining supernatant with final concentrations of 1 M NaCl and 10% PEG 8,000. We dissolved the solution and placed it overnight in a refrigerator at 4 °C, then centrifuged it at 10,000g for 10 min, and collected the bacteriophage precipitates.…”

Engineered endolysin of Klebsiella pneumoniae phage is a potent and broad-spectrum bactericidal agent against “ESKAPEE” pathogens

“…Phage ФKp84B was isolated from untreated wastewater collected at the Second Hospital of Nanjing using the standard double-layered plating technique ( Peters et al, 2022 ). The wastewater underwent filtration through a 0.22 μm membrane, followed by overnight incubation after mixing with the bacterial culture of Klebsiella pneumoniae 84 (Kp 84).…”

Characterization of Virulent T4-Like Acinetobacter baumannii Bacteriophages DLP1 and DLP2

“…Bacteriophages DLP1 and DLP2 were isolated from an Ottawa, Canada sewage sample using AB5075 cm using established protocols [ 18 ]. Axenic stocks of DLP1 and DLP2 were generated by three successive rounds of a modified streak-for-isolation technique.…”

“…Restriction fragment length polymorphism (RFLP) analysis [ 1 , 16 , 18 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ] was performed on 1 µg of DLP1, DLP2, and Escherichia phage Lambda (control) gDNA. The DNA was subjected to a panel of 16 restriction enzymes: Eco RI, Bam HI, Bg III, Eco 321, Hind III, Kpn I, Sma I, Pst I, Sa II, Nde I, Not I, Xho l, Xba I, Msp I, Hpa I, and Pae I (FastDigest, Thermo Scientific, Waltham, MA, USA).…”