Isolation and Characterization of Effective Lytic Bacteriophages to Control the Antibiotic Resistant Klebsiella Pneumoniae Infections in Noth-West of Iran

Due to emerging antibiotic-resistant bacteria, finding effective alternative antimicrobials to combat the bacteria is unavoidable. Bacteriophage therapy as a biologic and highly potentiated therapeutic approach was considered recently. This study aimed to isolate lytic bacteriophage(s) against multi-drug resistant clinical isolates of Klebsiella pneumonia and evaluate the morphological and biological characteristics as candidates for phage therapy.

Obtained clinical isolates from Shahid Madani Hospital in Tabriz, were confirmed as K. pneumoniae, and an antibiotic sensitivity test was performed using the standard disk diffusion method. Then, the isolation of lytic bacteriophages against 16 selected antibiotic-resistant bacterial isolates was carried out. Briefly, samples for phage isolating were gained from sewage water and centrifuged. The supernatant was filtered using a 0.45 µm Filter, mixed with fresh tripton soy broth medium and magnesium sulfate and incubated at room temperature. Then, the solution was mixed with chloroform and evaluation of plaque formation was performed using the agar bilayer technique. Genomes of the bacteriophages were subjected to molecular analysis using Hind III and EcoRV enzymes. Host range determination of the isolated bacteriophages was evaluated using spot test and agar bilayer techniques. Three bacteriophages with a broader host range than others were selected for more characterization. One step growth curve of the phages was drawn and their latent period and burst size were investigated. Then, the stability and survivability of the isolated lytic bacteriophages to different temperatures (-20, 37, 50℃), different rates of pH value (3,4,5,7,9,11,12), and also UV radiation were evaluated. Finally, morphological characteristics of bacteriophage with the broadest host range (PKpMa2/20) were investigated using electron microscopy.

36 clinical bacterial isolates were confirmed as K. pneumoniae based on standard biochemical tests. In the antibiotic susceptibility assay, it was revealed that the highest and lowest rates of antibiotic resistance belonged to cefazoline (80%) and tobramycin (26%) respectively. Isolation of bacteriophages against all 16 selected isolates was successfully performed. After the extraction of the genomes of the phages, a high variety of genome sequences was detected according to molecular analysis. In host range determination, PKpMa2/20 had lytic activity against 11 of 15 isolates. In a study of the one-step growth curve of the phages, with a broader host range than other isolated bacteriophages, latent periods were shown 10, 10, and 15 min and their burst size were 350, 210, and 180 pfu/ml respectively. All of the phages showed appropriate stability in temperatures of -20 and 37 ℃. In the temperature of 50℃, the survivability of PKpMa2/20 was higher than two other phages. Also, PKpMa2/20 was stable in the pH range of 3-12; in the pH value of 3, two other phages stability was ceased but PKpMa2/20 showed appropriate lytic activity. In electron microscopy, PKpMa2/20 had a short tail length of 10 nm and a head with a diameter of about 60 nm that possibly belonged to the family Podoviridae. 

Based on appropriate lytic effects, latent period, and burst size of the three isolated bacteriophages in the current study, especially PKpMa2/20, and also, their high stability to different temperatures, pH, and UV radiation, they could be considered as candidates for phage therapy to control resistant strains of K. pneumoniae infections but for the therapeutic use of theses phages, more molecular analysis is needed.