Submit Manuscript | http://medcraveonline.com Introduction Pseudomonas aeruginosa is frequently a crucial pathogen in hospital acquired and nosocomial infections. It is estimated that mortality rates fluctuated from 18–61% of nosocomial infections 1 and 21% in hospital, increasingly to 54.5% infected with multi- drug resistant strains. 2 The increasing multi-resistant strains to many classes of antibiotic such as beta-lactams, aminoglycosides and fluoroquinolones, imipenem, quinolones and third generation cephalosporins resisted against all relevant treatment and threated to patient’s life. 2,3 Bacteriophages (phage) are often known as predators of their bacterial hosts that complete their evolution by lysis bacterial cell even bacteria can resist against to phage infection. 4,5 Nowadays, with globally increasing emergence of multi-drug resistance, phage therapy has renewed many optimisms for alternative methods or combined both antibiotic and phage as a strategy to combat antibiotic resistant strains (AMR). 6,7 This study aims to isolate phage in environment and apply this mixture of phage-phage cocktail for controlling multi–drug resistant Pseudomonas aeruginosa isolated from many sources. Material and methods Bacterial isolation and their susceptible profiles All samples including stool from diarrheal patient, river water, tap water and ice water and food from local market were collected from provinces and city in the southern region of Vietnam. Then, strains were isolated by using specific agar and molecular test which certified with ISO 15189: 2012 approval. Disk diffusion test was applied to determine susceptibility profile of isolate. All strains resisted to at least one antibiotic such as meropenem, trimethoprim/sulfamethoxazole, Ceftriaxone, Ceftazidime, Augmentin, Gentamycin and Amikacin that was chosen for further test. Phage isolation Pseudomonas aeruginosa ATCC 27853 and Pseudomonas aeruginosa isolated from patients, soil was applied as indicator to phage isolation. Based on double layer agar methods (8), 300 ml of bacterial indicator solution (10 8 CFU/ml) mixed with 100ml samples and 2.6ml of top agar (0.5% bacteriological agar), then poured onto TSA (ThermoFisher products). Following this, the plates were left to balance and dry on the bench for 25 minutes before inverted to incubate at 37 º C overnight. Any determined plaques were harvested by sterile needle and resuspended into 3ml LB media contained indicator, then shaked in 3 hours. Next steps, all mixture filtered through membrane 0.2µm to collect pure lysate as phage. The whole procedure was repeated at least three times to obtain the single clone of phage. All phage was stored at 4 º C for further test. Transmission electron microscopy (TEM) of phage After collecting the phage, each phage pellet was collected at high titre and sent to TEM service centre for processing the image capture. Phage cocktail High titre of each phage was prepared with some mixture (M) as Table 1. The high purify phage was prepared in Tryptic Soy Broth– TSB (ThermoFisher product) buffer and used immediately. The ratio between phage to bacteria was employed from 1.000 to 10.000 for these experiments. Three clones of phage with different diameter (D) were selected to make a matrix to check the effectiveness of lytic phage (Table 1). Table 1 A matrix used to test the effective of phage cocktail Strains (CFU/ml) Phage inoculum (PFU) D≤1mm D=1–2mm D≥2mm M1 10 7 10 2 10 2 10 2 M2 10 4 10 2 10 2 M3 10 2 10 4 10 2 M4 10 2 10 2 10 4 J Microbiol Exp. 2021;9(3):72‒76. 72 ©2021 Tai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially. Application of bacteriophage cocktail to control multi-drug resistant Pseudomonas aeruginosa Volume 9 Issue 3 - 2021 Diep The Tai, Nguyen Anh Quang, Nguyen Thi Ngoc Nhi, Le Ha Duc Anh Department of Microbiology and Immunology, Pasteur Institute in Ho Chi Minh city,Viet Nam Correspondence: Diep The Tai, Department of Microbiology and Immunology, Pasteur Institute in Ho Chi Minh city,Viet Nam, Tel (84-28) 38230352, Fax (84-28) 38231419, Email Received: May 30, 2021 | Published: June 07, 2021 Abstract Multi–drug resistant Pseudomonas aeruginosa, a significant pathogen threats the public health with high mortality. The potential of phage cocktail was designed to lysis various bacterial sources. The candidate phage was isolated from soil, river water, tap water, food and human stool which belongs to Siphoviridae and Podoviridae family. The results identified that phage cocktails inhibited, lysed multi–drug resistant Pseudomonas aeruginosa in 30 minutes with 3 to 4 log CFU reduction. In addition, these cocktails showed effectiveness to bacterial strains isolated from wide sources including environment, food, and human. This renewed approach is contributed to overcome the dramatical increase of antibiotic resistance. Keywords: multi–drug resistant bacteria, Pseudomonas aeruginosa, antimicrobial resistance, phage cocktails, phage isolations Journal of Microbiology & Experimentation Research Article Open Access