الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
ESKAPE are bacteria with MDR which are capable toescape from the activity of antibiotics. They are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosaandEnterobacter.These pathogens can cause serious infections to patients undergoing organ transplant or chemotherapy, neonates, elders, and ICU residents or generally immunocompromised individuals.These infections lead to raised treatment cost, extended hospital stay and an increased possibility of further infections, in addition to their fatal effect.
Several infection control programs, treatments, and immunization approaches were analyzed to limit the infection with those pathogens.Enterococciare one of the leading causesof nosocomial infections including devices associated bacteremiasuch ascatheterassociated urinary tract infection. Even though the infections due to Enterococcus faecalisare more common, the infections due to Enterococcus faeciumare more arduous to treat owing to the increase of multiple antibiotic resistant traits.
This study aimed to focus on the molecular studying of some E. faeciumantigens that can be usedaspossiblevaccine candidates and to developthese antigens bygenetic recombinant techniques. The selection of these antigens was based mainly on their subcellular localization as surface exposed proteins and their attribution to the virulence of E. faecium. E. faeciumisolatewas obtained as a pure isolate from routine microbiology analysis lab at Faculty of Medicine-Alexandria University, and this was the only used isolate.
The identity of this isolate was confirmed by Gram stain, MALDI-TOF technology and sequencing of its 16S rRNA.
The encapsulation of E. faeciumisolate was confirmed using Antony‟s staining technique.PCR amplification of eight genes of possibly immunogenic proteinswas performed using gene specific designed primers where all the selectedgenes weresuccessfully amplified.
Knowing that these eight genessequences are highly conserved among different E. faeciumspecies also are promising candidates to vaccination.
Only five of the eight genes (fms3, fms22, swpA, saga,and the surface protein) weresuccessfully cloned in pQE31and transformed intoE. coliM15 (pREP4).
After the cloning of these genes, they were subjected to IPTG protein induction, efficiently expressed and then purified, excluding fms22which was expressed in low yield and consequently eliminated from this study.In-vivotrials were carriedoutusing mice as the animal modelwhereIFA and BCG were used as adjuvants to study the antigenicity of the recombinant proteinsfor the sake of vaccine production against E. faecium.
Comparison of bacterial count in heart and kidneyswas chosen to be the end point of the animal trialtodefine the validity of the vaccine.
Summary, Conclusion and Recommendations Before starting the animal trial, a pre-immunization E. faecium whole cell and cell lysate ELISA were done to determine the mice basal IgG against E. faeciumthat will facilitate the comparison between the antibody titer before and after immunization.
By performing a dot-blot assay, the natural immunogenicity of the expressed proteins was assessed revealing two possibilities, either the low immunogenicity of these antigens or the low expression level of such antigens in E. faeciumused isolate.
The level ofboth totalIgGand IgG1 in immunized micewas detected by indirect ELISA and itwas significant compared to the non-immunized group. However, the level of IgG2a was not significant in all trials.
Despite the presence of high antibody response, there was no protection in mice treated with antigens by comparing the bacterial count in both heart and kidneysof antigen-treated groups and negative control and this was confirmed by both opsonophagocytic assay and E. faeciumwhole cell indirect ELISA.Indirect ELISA and dot-blot using irrelevant His-tagged proteins showed that the main response of the immune system to produce antibody was not against the His-tagand confirmedthe presence of interaction between the raised antibody and the recombinant proteins.
Upon sequencing of the genes after cloning and alignment using Nucleotide BLAST search tool, the results of BLAST-N confirmed the identity of the selected inserts and the high conservation between different bacterial subspecies of E. faecium.
Finally, the presence of polysaccharide capsule surrounding E. faeciumisolate might contribute tothe prevention ofinteraction between the raised antibodies and their antigens by masking these antigens.
To our knowledge, these vaccination trials, excluding SagA,are novelsince no previous studies were performed using these antigens as vaccines for the protection against E. faecium infection