The Genotypes Of P.aeruginosa In Chronic Infections & The Evolution Of P.aeruginosa In Ventilator-associated Pneumonia Patients

Background:The opportunistic pathogen Pseudomonas aeruginosa(P.aeruginosa)is one of the leading causes of nosocomial infections worldwide.Among the various types of infections caused by P.aeruginosa,the lung infections are attracting the most concern.P.aeruginosa was reported to be the most frequently isolated bacteria from the respiratory tract and one of the major causes of ventilator-associated pneumonia(VAP).The ability of P.aeruginosa to establish perisitent infections in VAP patients is suggested to that P.aeruginosa could undergo a series of genetic mutations that often leads to its better adaptability to the host environment.Furthermore,the emergence of epidemic P.aeruginosa clonal linages has further facilitated the dissemination and persistence of P.aeruginosa lung infections among VAP patients.Therefore,it is valuable to investigate the adaptation mechanism of P.aeruginosa during chronic VAP infection.The whole genome sequencing techniques have made it possible to perform investigations on within-host evolution,genetic adaption for P.aeruginosa.Objective:1.To analyze the genotypes of P.aeruginosa isolated from inpatients with chronic infection more than 30 days in 1~stt affiliated hospital of Guangxi Medical University..2.To investigate the characteristics of genome,evolutionary dynamics,genetic adaption and parallel evoltion of P.aeruginosa during chronic VAP infection,and to investigate the virulence production of P.aeruginosa from VAP patients in vitro and in vivo.Methods:1.From December 2013 to June 2014,we collected the P.aeruginosa strains which caused human infection more than 30 days,and extracted DNA from these strains.The genetic relationships between these strains were determined by RAPD-PCR method.2.P.aeruginosa isolates were longitudinally sampled from the airways of VAP patients.Pacbio and Illumina sequencing platforms were used to obtain the genomes of P.aeruginosa strains.Core genome alignment and phylogenetic analysis was carried out using Parsnp v1.1.2 and FastTree v2,respectively.Genomic islands of P.aeruginosa genome were predicted by Island Viewer 3server.Comparison of PA_D1 genome against other five full P.aeruginosa genomes was done by BLAST search using BLAST Ring Image Generator 0.95.Antibiotic resistance genes were identified by the ResFinder 2.1 server,and nucleotide differences were detected and evaluated by the software CLC genomic Workbench 8.5.3.We also determined the quantification of elastase and pyoverdine production for P.aeruginosa strains from VAP patients in vitro,and applied P.aeruginosa strains to a murine silicone implant model for evaluating the virulence of P.aeruginosa strains.Results1.From December 2013 to June 2014,a total of 132 isolated P.aeruginosa strains,which caused infection more than 30 days,were collected from 30patients.All of these P.aeruginosa strains were distinguished into 26 kinds of RAPD type.The P.aeruginosa strains with genotype F and H were responsible for the main hospital-acquired-P.aeruginosa-infection outbreaks,which were involved with 9 VAP patients and 5 buring patients,respectively.2.Twenty-five P.aeruginosa isolates were longitudinally sampled from the airways of four VAP patients.The the genomes of all 25 isolates were closely related to each other and form a monophyletic group.Alignment of the PA_D1 genome with 5 other P.aeruginosa genomes using BLAST Ring Image Generator revealed that it harbours several strain-specific genomic regions with low sequence identities to the other 5genomes.We identified 31 GIs from in the genome of PA_D1 strains,and found that that most of the GIs in the PA_D1 genome correlate well with the strain-specific regions,suggesting that these strain-specific regions were probably acquired through horizontal gene transfer events.We found that the 25 isolates strains harbour the same set of antibiotic resistance genes.We also found that most of the predicted antibiotic resistance genes are in the GIs of the PA_D1 genome with low sequence identities to the other 5 genomes,which suggests that horizontal gene transfer is important for the acquisition of antibiotic resistance genes in our isolates.We found that almost all detected SNPs are non-synonymous.This provided the direct evidence that strong positive selective forces have dominated the P.aeruginosa genomes in the lungs of VAP patients during VAP.We identified three obvious parallel evolution events:the evolution of mutations in the mpl gene was identified in the isolates from patient 1,2 and 3;the evolution of mutations in the lasR gene was found in isolates from patient 1,3,and 4;and the evolution of pyoverdine-deficient mutants was identified in the isolates from patients 2 and 3.3.We also found that P.aeruginosa strains isolated at later stages of VAP infections produce less elastase in vitro,and are likely to be less virulent in vivo,than their earlier isolated counterparts.Conclusions:From December 2013 to June 2014,there were 26 kinds of RAPD type in P.aeruginosa strains,which caused infection more than 30 days.The genotype F strains were responsible for the main hospital-acquired-infection outbreaks in VAP patiens.We identified a novel P.aeruginosa strain isolated from VAP patient.These isolates belonging to this lineage could undergo rapid adaptive evolution after infectioning the host that leads to defective quorum sensing,reduced production of siderophore pyoverdine and virulence factor elastase,and attenuated virulence in vivo.