Drug Resistance Analysis And Screening Of Dominant Antigens Of Pseudomonas Aeruginosa

Objective:To investigate the specimen sources and clinical distribution of Pseudomonas aeruginosa isolates in the Second Hospital of Tianjin Medical University,then to study the resistance of Pseudomonas aeruginosa to commonly used antibiotics and compare the resistance between specimen sources so as to provide evidence for rational use of antibiotics in clinic.To identify epitopes of Pseudomonas aeruginosa dominant antigens that can stimulate the body to produce antibodies and analyze the cellular location and function of antigens,and to obtain vaccines that had potential value.Methods:346Pseudomonas aeruginosa isolates were collected from the Second Hospital of Tianjin Medical University from August in2012to August in2013,then the strains were studied based on the specimen sources and clinical distribution.Antimicrobial susceptibility test was done by Broth dilution method test,and the drug resistance of these strains to antibiotics was identified.These strains were divided into group I and group II based on isolated locations,and then were divided into ICU group and non-ICU group.28serum were collected with the patients infected with Pseudomonas aeruginosa in7-10days from the Second Hospital of Tianjin Medical University and Tianjin Fourth Hospital from March2012to December2012,30adult serum were collected from the Second Hospital of Tianjin Medical University in healthy volunteers. The sera collected from controls and patients infected by Pseudomonas aeruginosa were used to screen the immunodominant of mimic epitopes of PA by a random12-peptide phage display through one negative selection panning and three rounds biopanning.The positive phages clones selected by ELISA were sequenced and then compared with the published sequence of strain PAOl.And the cellular location and function of antigens were analysed.Results:1.More than half of346Pseudomonas aeruginosa strains were isolated from sputum specimens,followed by urine and secretions,accounting for19.7%and 6.6%,respectively, blood and other specimens had smaller proportions of2.0%and6.6%.346Pseudomonas aeruginosa were mainly distributed in Western medicine (35,10.1%), urology (34,9.8%), the cadre ward (32,9.2%),ICU (31,9.0%), pediatrics (31,9.0%),department of Infectious Diseases(30,8.7%), department of Respiratory Medicine(29,8.4%),other departments such as neurosurgery, renal medicine, cardiology, surgery, cardiac surgery, neurology, etc.(the proportion between3%-5%) were less scattered.2.The drug sensitivity test results showed that piperacillin had the highest resistance rate,near30%, followed by cefoperazone, ceftazidime, cefepime, aztreonam, ticarcillin/clavulanic acid, gentamicin and piperacillin/tazobactam,with the resistance rates between15%to25%while others were below15%.3.The antimicrobial resistance rates of PA to cefepime in Department A was higher than Department B.The antimicrobial resistance rates of PA from group ICU and non-ICU group had no difference.4.After four rounds of biopanning,a significant enrichment of phages were achieved. The inserts fron30positive phages clones were devided into12peptides based on different amino acids sequence.Through comparison with PAO1genomic sequences, we got673kinds of proteins,whose cellular localization were cytoplasm,cytoplasmic membrane,periplasm,outer membrane,extracellular and unknown,and the proportions were49.3%,21.8%,5.9%,6.1%,1.5%and15.3%respectively. Gene products could be classified into26categories,and the genes in each part of the cells were in accordance with its function.The genes that had higher frequency were as follows:pvdD, PA0758, phzFl. The antigens of extracellular and outer membrane protein belonged to Pseudomonas aeruginosa dominant antigens.Conclusions:346Pseudomonas aeruginosa strains were mainly isolated from sputum specimens,followed by urine.The strains were mainly distributed in Western medicine and urology.Piperacillin had higher resistance rates to Pseudomonas aeruginosa.The antimicrobial resistance rates of PA from different places were not in agreement.Group ICU and non-ICU group had no difference in antimicrobial resistance rates. The antigens of extracellular and outer membrane protein belonged to Pseudomonas aeruginosa dominant antigens.We speculated that Pyoverdine、OprE、 YfiB and Pel could be used for vaccine research, unbiased phage display peptide library technology provided a new approach for vaccine research.