Isolation And Characterization Of Bacteriophages And The Potential To Disrupt Multi-drug Resistant Pseudomonas Aeruginosa Biofilms

Pseudomonas aeruginosa is an opportunistic pathogen that can cause serious problems in clinical settings,and the abuse of antibiotics has led to a wide spread of drug-resistant in environment.Pseudomonas aeruginosa biofilms are thought to be the underlying cause of many chronic and recurrent infectious diseases.Antibiotics have been found to be ineffective in biofilm-growing bacteria due to inaccessibility of drug molecules permeating into the inner surface of the biofilms.For this reason,there is an urgent need for the discovery and the development of new antimicrobial agents.Bacteriophage as a novel antibacterial agent is a promising therapeutic approach that is featured by safety,no poisoning,high efficiency,low cost and short development cycle.This study takes the multiple-drug resistant Pseudomonas aeruginosa as the host to screen the lytic bacteriophage and examine the potential of bacteriophage on multiple-drug resistant Pseudomonas aeruginosa biofilms.Main work in this study is as follows:(1)Three lytic bacteriophages of Pseudomonas aeruginosa,namely G1,LS1 and C1,are morphologically obtained and observed as the bacteriophages with tails.Among them,G1 and LS1 belonged to Myoviridae,while C1 belonged to Podoviridae;G1 has the latent period of 20 mins,with the burst size of 230 PFU/infected cell,while the latent period of LS1 and C1 are about 30 min and the burst size are 77 PFU/infected cell and 86 PFU/infected cell.The most suitable pH value of G1 within 4.0~11.0,while the most suitable pH value of LS1 and C1 respectively within 5.0~5.0,6.0~11.0.(2)Genomic analysis indicated that all these bacteriophages are all double-stranded linear DNA.G1 genome is composed of 87,646 bp,with a G+C content of 54.7%,3 tRNA genes,and only 26 functional genes are predicted with known functions among total 156 open reading frames;LS1 genome is composed of 66,093 bp,with a G+C content of 55.7%%,and 35 genes were predicted to have specific functions among 92 open reading frames(ORFs);The C1 genome is 43,133 bp,with a GC content of 53.6%,59 open reading frames are predicted,27 functional genes are predicted.No integrase,virulence factors and antibiotic resistance gene were annotated in three bacteriophages genome.(3)The potential of bacteriophage LS1 to disrupt Pseudomonas aeruginosa biofilms was assessed by scanning electron microscopy,crystal violet staining and bacterial counts.Results of scanning electron microscopy showed that LS1 can obviously disrupt antibiotic-resistant Pseudomonas aeruginosa biofilms,and results of bacterial counts indicated that the bacteriophage LS1 inhibited the formation of Pseudomonas aeruginosa biofilms with a proportion of 99% and decreased the proportion of Pseudomonas aeruginosa biofilm cells in preformed biofilms by 85 %.Additionally,we found that Pseudomonas aeruginosa biofilms can develop resistant to the bacteriophage LS1 at 24 h.