Strategies Of Prevention And Control Of Nosocomial Infections From Pseudomonas Aeruginosa

Pseudomonas aeruginosa(P.aeruginosa)is an important opportunistic nosocomial pathogen and particularly important in hospital acquired pneumoniae.Although antibiotic therapy is available for treatment for this infection,drug resistant isolates that pose a major threat to patients chronically colonized with P aeruginosa have emerged.The development of an effective vaccine against P.aeruginosa would therefore facilitate the prevention and treatmentfor such infections.Based on the resistant situation in Zhenjiang areas,and in our effort to develop a more effective DNA vaccine against P. aeruginosa.We used an adaptor molecule,myeloid differentiation factor 88(MyD88),that stimulated signal transduction to microbial components in cells,and enhanced immune response induced by mutil-eitope gene of P.aeruginosa.Objective:(1)To investigate the antimicrobial susceptibility of P. aeruginosa,isolated from Zhenjiang area to 13 antibiotics and, to identify the structure and dissemination of class I integron.(2)To clone and detect P.aeruginosa OprF gene;The effect of P.aeruginosa vaccine was analysesd by detecting the specific antibodies in serum samples through ELISA method which was established with purified recombinant OprF.protein coated 96-well plates.(3)To design the pseudomonas aeruginosa epitope-MyD88 gene vaccine and study its immunogenicity in Balb/c mice.Methods:(1)K-B test was used to determine the resistant rate of 71 strains of P.aeruginosa.DNA template was extracted by boiling method,and then PCR method was utilized to detect class I integron.(2)The OprF gene was amplified from genomic DNA of P.aeruginosa and the recombinant plasmid pMD18-T-OprF was constructed by using T-A cloning.After double digestion with BamHâ… and Hindâ…¢,the OprF gene was subcloned into prokaryotic expression vector pET32a to construct expression plasmid pET32a-OprF which was expressed in E.coli BL21(DE3)cells with induction by IPTG.Then the recombinat protein was analyzed with SDS-PAGE and Western blotting.ELISA assay plates were coated with purified OprF protein for detecting P.aeruginosa antibodies in serum samples.(3)The epigene was designed and synthesized,containing three B cell epitopes of OprF and one foreign 'promiscuous' T cell epitope by overlap extension PCR, and tPA signal encoding sequence was amplified by PCR method and then was inserted to the 5' terminus of the epigene to construct tPA-OprF DNA fragment.The DNA fragment and MyD88 gene was cloned into expression vector pIRES to construct recombinant plasmid pIRES-tPA-OprF:MyD88.Balb/c mice were inoculated with pIRES-tPA-OprF:MyD88 plasmid,and the serum antibody titer was measured by ELISA.The number of viable bacteria in lung homogenate was examined after intratracheal challenge with P.aeruginosa.Results:The resistant rates were between 18.3%to 77.5%.The prevalence of classâ… integron was 38%.These integrons include 5 gene cassettes(aadB,aac(6)-â…¡,PSE-â… ,dfrA17 and aadA5),the dfrA17 and aadA5 gene cassette were often found.(2)The DNA sequence analysis confirmed that the cloned OprF gene was according to Genbank date.The OprF fusion protein was proved by SDS-PAGE and Western blot.Then,an ELISA was developed to detect the specific IgG of P.aeeuginosa in sera from Balb/c mice.(3)The recombinant plasmid pIRES-tPA-OprF:MyD88 was successfully constructed.Mice were immunized with this epigene vaccine,and the result showed that the higher level of antigen specific antibody was detected,and resulted in a fewer number of bacteria in the lung as compared with control plasmid treatment.Conclusion:(1)The resistant rates of P.aeruginosa to 13 drugs were different,and the resistant rates in positive strains of integron were obviously higher than that in negative strains, which indicated that the integron may play an important role in multidrug-reisistant of P.aeruginosa.(2)OprF gene was cloned and expressed in E.coli system effectively;The purified protein acted as antigen was used to coat ELISA assay plates for detecting the specific IgG of P.aeruginosa in sera from vaccine-immunized Balb/c mice.(3)The construction of the recombinant expression plasmid pIRES-tPA-OprF:MyD88 was proved to be successful and it could elicit dramatic immune response when it was applied as the epitope vaccine in experimental animal.It could be used as a potential candidate of preventive vaccine of P.aeruginosa.