Phage Treatment Of Multidrug Resistant Pseudomonas Aeruginosa Infection In Scalded Mice

Pseudomonas aeruginosa?PA?is widely distributed in nature.PA is a very common pathogenic bacteria in nosocomial infection involved multiple organs and tissues.PA occupy the major proportion of burn infection.Growing drug-resistant strains,especially multi-drug resistant strains,result in failed and prolonged treatment of PA infection.In order to find powerful agent against MDR PA infection complicated to scald injure,multi-drug resistant PA and its specific lytic phage were identified and administrated to scald mice.Followed to 16s RNA analysis,the antibiotics sensitivity of clinical PA isolates were tested by serials plate dilution method.We got multidrug resistant Pseudomonas aeruginosa named as PA53 that was resistant to 14 of 18 tested antibiotics,including cefoxitin sodium,ceftriaxone sodium,ceftamet sodium,cefmino sodium,cefazoxime hydrochloride sodium,cefuroxime sodium,lincomycin hydrochloride,clindamycin phosphate,moxifloxacin hydrochloride,levofloxacin hydrochloride,aztreonam,streptomycin sulfate,ampicillin sodium and azithromycin.The lytic phage N323,against PA53,was isolated and purified by double agar method.The biological properties of the lytic phage were analyzed in detail.We observed morphology by transmission electron microscope?TEM?,analyzed structural proteins by SDS-PAGE,tested phage replication by one-step growth curve,labeled lytic efficiency by MOI analysis,and determined phage stability at different pH and temperature by lysis activity measuring.The lytic phage N323 belongs to caudovirales,podoviridae.N323 has an icosahedral symmetry head of 50nm in diameter,and a tail of 15 nm in length.The MOI of phage N323 is 10,and under this condition,the latent period of phage N323 is 10 min,the outbreak time is nearly30min,and the outbreak amount is 200 PFU/cell.The lysis activity is stable under50?but deactivate rapidly at 80?.And phage N323 maintains stable lysis activity between pH 4 and pH 12 for 1 hour.No less than 6 proteins can be observed in SDS-PAGE,they are ranging from 25 to 170 kDa,the concentration of the main protein with molecular weight of 48kDa is the highest.For the scald mice model establishment,we tested serials of burn area and duration to the ICR female mice at 100?.And the minimal lethal dose?100%?of PA53 to the scalded mice model was determined.At 100?,burning 2.5 cm²/10s is suitable to get the stable and survival scald model for PA infection and phage protection.And the minimum lethal dose?100%?of PA53 for the scalded mice is 1.8×10¹¹CFU/mL.The tissue distribution and titers of different administration routes,including intraperitoneal injection?i.p.?,subcutaneous injection?s.c.?and intramuscular injection?i.m.?,were tested by PFU.In order to evaluate the protection of different administration routes,the individual mortality rate of scalded mice infected by minimal LD100 PA53 were calculated after 10MOI N323 administration through intraperitoneal injection?i.p.?,subcutaneous injection?s.c.?and intramuscular injection?i.m.?.The titer of phage in spleen,liver and blood maintained the highest level under intraperitoneal injection.The titer of N323 in spleen,liver and blood by i.p.injection are higher than other two injection routes,which is ranged from 10⁸to10⁶PFU/mg in spleen,from 10⁸to 10⁷PFU/mg,and 10⁶to 10²PFU/mL in blood.The i.p.administrations of 10MOI N323 decreased the mortality rate of scalded mice attacked by minimal LD100 PA53 obviously.The protective efficiency of i.p.administration is stronger than other two administrations obviously.In smmary,the lytic bacteriophage N323 could effectively protect the scalded mice infected with multidrug resistant Pseudomonas aeruginosa PA53,and the protective effect of i.p.is the strongest.