Applications Of Phage And Associated Technologies To Treat Infections By Pandrug-resistant Pathogens

Infections are the main complication of severe burn injury.And it causes not only wound healing delay but also sepsis in severely burn,which will finally lead to multiple organ failure and death.Due to physical damage of the body surface barrier,declining of systemic immune function and invasion of pathogens,the susceptibility to infection increased greatly.As a result,prevention and treatment of burn infections has always been a hot and difficult area of severe burn injury [1,2].With the long-term use of clinical antibiotics,treatment of infectious diseases faces a huge challenge rised by the the increasing antibiotic resistance [3].Acinetobacter baumannii[3,4],Pseudomonas aeruginosa[5-8],Staphylococcus aureus[3,9-11] and Klebsiella pneumoniae[10] are the four domain pathogens in burn ward recently.New anti-bacterial approaches are urgently needed as the research and exploring of traditional antibiotics seems very difficult and slow.In this study,we first carried out molecular epidemiological investigation of Acinetobacter baumannii in our hospital burn ward.Based on this,three more works were done as follows: 1)analysis of phage endolysin in the applications of anti-infections field;2)preliminary analysis of the phage safety and effectiveness;3)analysis of CRISPR in the field of anti-bacterial applications.The results are summarized below:1.A total of 81 A.baumannii isolates were isolated from 3 years and most of them are multi-drug resistant.Except for Polymyxin B,minocycline,and sulbactam/cefoperazone,the resistance rate against left 16 antibiotics was as high as 90%.According with the dug-resistant phenotype,the prevalence of antibiotic resistant genes was also very high.BlaOXA-51-like gene,blaOXA-23-like gene,AmpC,ISAba1-AmpC,PER and SIM were the six most prevalent genes.The prevalence of the six genes were 96%(78/81),93%(75/81),86%(70/81),73%(59/81),73%(59/81)and 52%(42/81),respectively.The 81 isolates were grouped into 28 ST types,including 10 existing ST types and 18 new ST types.Except for five new ST types(ST457,STn2,STn6,STn11 和 STn14),all the left 23 STs belong to one colonal complex(CC),CC92.CC92 was also the most prevalent clonal complex over the world.2.We further analyzed all the Carbapenem-Resistant Acinetobacter baumannii and a total of 248 strains were collected.Wound surfaces,sputa,catheters,and bloods were the four dominant isolating sources,and these covered 94% of all the isolates.The prevalence of OXA-23 increased significantly from 39% in 2012 to 77% in 2013 and 73% in 2014.The OXA-23 gene prevalence altered significantly among the different STs.For ST195 and ST191,only 10% of these isolates carried OXA-23.However,90 percent of ST369 strains carried it.The dominant ST(ST368)remained unchanged during the 3-year study period.However,the ratio decreased from 56% in 2012 to 30% in 2013.The ST population changed year by year,with some minor types diminishing or switching to another minor type.Majority of the 248 isolates displayed a relatively close evolutionary relationship in the phylogenetic tree.These results indicate the wide clonal spreading of CRAB in the Chinese burn unit.3.The concentration of endotoxin in the phage solution decreased more than 400-folds,from 7.03 * 105 EU/ml to 1.70 * 103 EU/ml.From the results of LDH assay,no cytotoxincity was identified after long term treatment of high tilter phage.The phage can also save Hela cell after infection by Acinetobacter baumannii for 2 hours.4.Based on our previous finding,we find a new endolysin of A.baumannii.We further cloned and purified the endolysin PlyAB1,with molecular weight of 21 kDa.PlyAB1 exhibits a marked lytic activity against A.baumannii AB1.Within 30 min,the OD600 of AB1 decreased from 1.05 to 0.2 by PlyAB1 treatment.The viable bacterial counts results showed that the number of A.baumannii AB1 decreased from 1.4?×?107 CFU/ml to 4.1?×?106 CFU/ml in 30 minutes.PlyAB1 displayed a highly lytic effect against all of the 48 hospital-derived pandrug-resistant A.baumannii isolates that were tested.5.Finally,we tried to explore CRISPR technology in the field as anti-microbials.We use CRISPR to inhibit the expression of prtR gene in Pesudonmonas aeruginosa(P.aeruginosa).The CRISPR system was successfully transferred into P.aeruginosa.More than 103-folds decrease in P.aeruginosa was observed after the transformation of recombinant vector PHERDB20T-dCas9-prtR sgRNA.These preliminary results also confirm the potential applications of CRISPR technology as antibacterials.In conclusion,we first carried out molecular epidemiological investigation of A.baumannii in our hospital burn ward.Most of these isolates showed multi-drug resistant phenotype and carried resistant genes.Majority of them belong to CC92.We analyzed the cytotoxicity of the phage with Hela cell and THP-1 cell.We further used animal model to confirm the phage therapy result.We also indentify and characterized one endolysin PlyAB1,which show rapid litic results on clinical isolates.Finnaly,we transfer the CRISPR-cas9 system into P.aeruginosa and explored it roles as potential antimicrobials.