Isolation and Structural and Molecular Identification of Phages against Antibiotic Resistance Gram Negative Bacteria Isolated from Burn Wounds and Study the Efficacy and Cytotoxicity of them on Skin Fibroblast Cells (HFSF-PI 3) and Skin Epithelial Tumor Cells (A-375)
Isolation and Structural and Molecular Identification of Phages against Antibiotic Resistance Gram Negative Bacteria Isolated from Burn Wounds and Study the Efficacy and Cytotoxicity of them on Skin Fibroblast Cells (HFSF-PI 3) and Skin Epithelial Tumor Cells (A-375)
Ladan Rahimzadeh Torabi,1Monir Doudi,2,*Nafiseh Sadat Naghavi,3Ramesh Monajemi,4
1. Department of Microbiology, Falavarjan Branch, Islamic Azad University, Falavarjan 84515/155, Isfahan, Iran 2. Department of Microbiology, Falavarjan Branch, Islamic Azad University, Falavarjan 84515/155, Isfahan, Iran 3. Department of Microbiology, Falavarjan Branch, Islamic Azad University, Falavarjan 84515/155, Isfahan, Iran 4. Department of Biology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran
Introduction: Invasive bacteria find a special place for their rapid growth and multiplication in burn wounds. The presence of resistance of gram-negative bacteria to common antibiotics in patients with burn wounds has become a serious and controversial challenge in hospitals.
Methods: In this study, gram-negative bacteria were isolated from specialized hospitals for various accidents and burns from several cities in Iran and bacteriophages were isolated from municipal wastewater and hospital wastewater in Isfahan. To confirm the sex and species of bacterial isolates, biochemical identification along with molecular identification was performed using 16S rRNA and universal primers F27 and R1492, and designed primers FT1, RT1, FLAMB and RLAMB were used to confirm bacteriophages. In the present study, observe the morphology of isolated phages, study the antibacterial activity of phages, determine the host range, evaluate the rate of phage uptake, determine the stability of phages at different temperatures and different pHs, evaluate the frequency of infection, evaluate the bactericidal rate of phage, draw a single growth chart Phage stage and .... Molecular identification of phage with determination of approximate size of phage genome, study of enzymatic digestion pattern, identification of proteomics pattern, sequencing of part of genome of one of bacteriophages and their cytotoxic effects on two categories of normal human skin fibroblast cells (HFSF-PI 3) and cells Human epithelial tumor tumors (A-375) were presented.
Results: In this study, a total of 9 specific bacteriophages were isolated for 50 clinical strains of burn wound. Based on the results of host range and morphological observations of bacteriophage Pɸ-Bw-Ab specific to Acinetobacter baumannii strain 101IAU_FAL belonging to the siphoviridae family, bacteriophage 1-VbɸAB specific to Acinetobacter baumannii BAH_Glau strain
Plasmaviridae, bacteriophage BwϕP-Ec01 Specific to Escherichia coliADB_66-1 Bacteriophage ɸ PAVb-1 Bacteriophage Pseudomonas aeruginosa NEG_RA1300 Belongs to the family MyoViridae, EnɸP-HO bacteriophages specific to Enterobacteriaceae Hormachi EHO-Iau100 strain belonged to the Inoviridae family, bacteriophages PɸBw-Kp1, PɸBw-Kp2 and PɸBw-Kp3 were specific to Klebsiella pneumoniae strains belonging to the family Iaufa_ladu, respectively. Bacteriophages at pH 7 or neutral had the highest percentage of stability and antibacterial activity and lytic phage activity was significantly reduced at acidic pH 4, 5 and 10. What is certain is that phages at pH with high acidity are not able to survive . In the thermal stability test, the stability of the desired phages decreased with increasing temperature. According to the results, the rate of phage uptake increased with increasing time, and the rate of uptake of bacteriophages also increased. The phage genomes were sensitive to most restriction enzymes. All isolated bacteriophages had different proteomics patterns in SDS. However, protein fragments were observed in the range of 25 to 90 kDa for each phage. Overall LDH results showed that the isolated bacteriophages had no toxic effect on normal human skin fibroblast cell line (HFSF-PI 3) and a relative cytotoxic effect on human skin epithelial tumor cell line A-375. Overall MTT results showed that isolated bacteriophages had no toxic effect on normal human skin fibroblast cell line (HFSF-PI 3) and human skin epithelial tumor cell line A-375.
Conclusion: With various considerations in the field of potential ability of phages and safety studies, the phages studied in this study can be a good choice and a suitable proposal for control and treatment of such resistant pathogens in burn wounds of hospitalized patients.