Isolation and Characterization of Phage Against Drug-Resistant Biofilm Forming Bacteria

Abstract

Bacteriophages are viruses that infect bacteria. They are considered the most abundant life form on Earth and have a significant impact on bacterial populations and their biofilms in various environments, including the human body. Phages have been used for decades as a natural alternative to antibiotics for treating bacterial infections and have shown promising results in laboratory and clinical studies. The current study aimed to investigate the isolation and characterization of bacteriophages targeting biofilm-forming multi-drug resistant strains of E. coli AM 1, Enterobacter cloacae AM 2, and E. coli AM 4. Biofilms, intricate assemblages of bacterial colonies encased in a viscous matrix, play a significant role in the persistence of numerous microbial infections, with estimations suggesting their involvement in over 60% of nosocomial infections and 80% of chronic infections. The objective of the study was to evaluate the efficacy of the identified bacteriophages in controlling both planktonic bacteria and biofilms. The selection of bacterial strains was based on their capacity to form biofilms. Three bacteriophages with the names AM1, AM2, and AM4 were discovered in wastewater, with AM1 infecting E. coli AM 1 and AM2 infecting Enterobacter cloacae AM 2 and AM4 infecting E. coli AM 4. These phages showed limited host ranges, and their temperature and pH stability, latency duration, and burst size per cell were evaluated. Results showed that AM1, AM2, and AM4 displayed heat tolerance within a range of 37-60 °C. AM1 and AM4 were found to be stable within a pH range of 5-11, while AM2 displayed stability between 3-9. The latency periods for these phages were 25 minutes for AM1, 20 minutes for AM2, and 25 minutes for AM4, and their burst sizes per cell were 325, 295, and 312 phages, respectively. The lytic activity of isolated phages was assessed against the suspensions of their host bacteria. Phages AM1, AM2, and AM4 were found to significantly diminish bacterial cultures with an exponential growth phase. After 24, 72, and 120 hours, phages AM1, AM2 and AM4 were successful in lowering the biofilm biomass of each respective host, resulting in diminishes of more than half-fold, 2-fold, and 3-fold, respectively. Furthermore, biofilm was allowed to grow on coverslips for 72 and 120 hours by E. coli AM 1, Enterobacter cloacae AM 2, and E. coli AM 4, and then tested for lysis by their respective phages. A notable decrease in biofilm was observed under the specified DRSML QAU

15 conditions. Amazingly, the reduction of biofilm biomass by treatment with the phage cocktail was similar to that of the individual phages. In conclusion, my research findings indicate that wastewater represents a promising avenue for the discovery of bacteriophages capable of combating newly arising antibiotic-resistant bacteria. These phages can effectively control bacteria in both their planktonic and biofilm forms. Single phages have the potential to eliminate bacterial biofilms, my findings suggest that in a phage cocktail it is not compulsory that the biofilm will be eradicated in more capacity than single phage. Thus, it is proposed that phage cocktails could offer the same potential for the eradication of bacteria.