The Laws And Mechanisms Of Bacteriophage Controlling Pseudomonas Aeruginosa Persisters

Pseudomonas aeruginosa（P.aeruginosa）is a common food or waterborne pathogen whose intrinsic antibiotic resistance leads to high mortality rates in infected patients.Persisters are a tolerant subpopulation that survives antibiotic treatment and a phenotypic variant of the bacteria.P.aeruginosa persisters which is the main reason why chronic infections are difficult to cure,can cause recurrent urinary tract infections,burn skin infections and lung infections.If antibiotics are added repeatedly,persisters can quickly evolve into antibiotic-resistant bacteria.Strains with high persistence levels rapidly replace strains with low persistence levels,resulting in tolerance at the population level.Tolerance,in turn,increases the chances of spreading antibiotic-resistant mutations,paving the way for the rapid evolution of resistance.Current therapeutic approaches for persistent bacteria include chemical methods（e.g.,antibiotic combination therapy and new drug development）,physical methods（e.g.,nanomaterials and LED lighting control）,and biological methods（e.g.,phage therapy）.The widespread use of antibiotics in agriculture,animal husbandry,and clinical settings has caused problems with drug-resistant bacteria in the environment and hospital,such as disrupting the community structure of bacterial populations and threatening human public health.When physical methods such as nanomaterials are used to control persisters during patient treatment,they may affect cellular homeostasis in vivo and require strict control of their biosafety.Bacteriophages（phages）are promising approaches for controlling infections with normal bacteria as well as drug-resistant bacteria.Phages have significant advantages over these traditional antimicrobial methods,such as phage specificity and self-limiting properties.But when bacteria enter a persistent state,their cell membranes change and their metabolic level decreases,are the phages still able to infest the bacteria?Lambda phage（λphage）can lysate Escherichia coli（E.coli）,and the expression of bacteriophage lysate gene leads to the death of bacteria when the persisters of E.coli turn into bacteria in normal growth state.Based on this,it is speculated that the two phage strains currently screened can infect P.aeruginosa.The phage is adsorbated on the persisters.When the persisters returns to normal growth state,the expression of phage related genes will be up-regulated and the persisters will be lysed,finally achieving the purpose of eliminating the persisters.To test this conjecture,we conducted a study on the control pattern and mechanism of phage on P.aeruginosa persisters.This will help us to further understand the interaction between phages and retention bacteria and the evolutionary process,and secondly,to better understand the mechanism of phages’role in controlling the growth of persisters.Here,we explored the process and mechanism of infestation using high-titer phages to infest P.aeruginosa persisters（MOI=100）screened with oxfloxacin.The results revealed that phages are able to infect specific bacteria and therefore can be used as biological control agents to control bacterial-induced diseases in animals,plants and humans.In this study,two homologous phages（named PPAY and PPAT）infecting P.aeruginosa were isolated and characterized.The results of phage spot studies on plates showed that the phage spots of PPAT were in the shape of transparent dots.In contrast,the phage spots of PPAY were translucent plaques with a halo.Transmission electron microscopy results showed that PPAT（65 nm）and PPAY（60 nm）strains were similar in size and both morphologically consisted of an icosahedral head and a shorter tail,belonging to the short-tailed phage family（Podoviridae）.One-step growth curves showed a latency of 20 min and a burst time of 30 min for PPAT and PPAY.The burst size was higher for PPAT（953 PFU/cell）than for PPAY（457 PFU/cell）.Similarly,the adsorption rate constant at 5 min was higher for PPAT(5.97x10^-7 m L/min)than for PPAY(1.32x10^-7 m L/min).Next,whole-genome sequencing of the phage was performed,in which the genomes of PPAT and PPAY were 54,888 bp and 50,154 bp,respectively.Homology and evolutionary analyses showed that PPAT and PPAY are closely related to PA11.The presence of tail fibronectin in PPAY,but not in PPAT,may explain the ring effect of PPAY phagocytosis.In conclusion,both PPAT and PPAY screened in this paper are a new lytic phage infecting P.aeruginosa,and both are homologous.Both phage strains showed growth inhibitory effects on bacteria,which could help in the treatment of bacterial infections and could be used for research and clinical purposes.In this project,a screening model of stable P.aeruginosa persisters was established,and the persisters were screened by using ofloxacin antibiotic with a screening time of 12 h and the persistence rate of about 0.01%.It was observed by transmission electron microscopy that the persisters were generally longer and had more surface protein content than normal bacteria,and were more likely to form biofilms.The transcriptome analysis showed that after P.aeruginosa PAO1 entered the persistence,the persisters formed biofilm by affecting the community-sensing signal molecule C4-HSL,pel polysaccharide synthesis,alginate production,flagellar movement,etc.,to build a"protective barrier"for the persisters.In addition,bacterial energy metabolism was reduced,and the outer membrane proteins were regulated to resist antibiotic penetration into the cell and increase the chance of survival.The expression of genes such as PA0610（prt N）in P.aeruginosa was significantly higher than that of normal bacteria,which promoted the production of binding proteins that regulated bacterial growth and metabolism and adapted to environmental stress.Among them,prt N encoded the pyocyanin activator protein,which regulated the production of pyocyanin and was a key gene affecting the persistence rate of persisters.Phages could cleave most of the persisters（The amount of bacteria decreased from about 10⁵ CFU/m L to about 10² CFU/m L）,but it took longer time to lyse normal bacteria.Phages were adsorbed on the persisters,and when antibiotics were removed,they could also kill the persisters.After the first burst,the phage adsorbed on the persisters without phages infection,and carried out a new round of infection process.Compared with the initial,the survival rate of PAO1 persisters（the amount of bacteria on the plate）after the addition of PPAT for 12 h,was about 5×10^-5,and the survival rate of PAO1 persisters（the amount of bacteria on the plate）after the addition of PPAY for 12 h,was about 1×10^-4.Compared with the initial growth,the survival rate of PAO1?prt N persisters（the amount of bacteria on the plate）after the addition of PPAT for 12 h,was about 1×10^-3,and the survival rate of PAO1?prt N persisters（the amount of bacteria on the plate）after the addition of PPAY for 12 h,was about 0.5×10^-3.During the control of PAO1 persisters by PPAT,mainly through the reduction of ATP to promote deep persistence and the increase of intracellular catalase content to promote the replication process,finally the cell membrane damaged,and the bacteria were lysed and died.The control of PAO1 persisters by PPAY was mainly through the reduction of ATP to promote deep persistence,finally the cell membrane damaged,and the bacteria are lysed and died.Phages had poor control on the PAO1?prt N persisters.PAO1?prt N persisters may affect the adsorption of phage by regulating the production of pyocyanin and the synthesis of alginate and flagella.The prt N gene of bacteria may affect the evolution of phage-resistance and play an important role in the coevolution of bacteria and phages.With the increase of persistence,the control level of PPAT on PAO1?prt N persisters decreased,and the control of PAO1?prt N persisters by PPAY depended on the increase of hydrogen peroxide produced by the increase of SOD,which enhanced the damage to cell membrane.Phages could effectively control the growth of persisters,infect and cleave them or adsorb on them,and promote the replication process of progeny phages by increasing the expression of intracellular catalase and other genes,thus killing the persisters.In this study,the control effect and related mechanism of phages on P.aeruginosa were analyzed.The results provide theoretical and technical support for the effective control of drug-resistant persisters and chronic infection.