Gp96 rich lysate as a vaccine candidate against infection with salmonella typhimurium

Glycoprotein 96 is the primary chaperone of the endoplasmic reticulum. Immunization with it induced potent Cytotoxic T lymphocyte responses to intracellular bacteria. S. typhimurium is a facultative intracellular bacterium and acquired resistance against this bacterium mainly depends on activity of Cytotoxic T cells. This study aimed to evaluate the capacity of Glycoprotein 96 rich lysate as a vaccine candidate to induce a protective immune response in mice against a lethal dose challenge with Salmonella typhimurium. Mice were infected with S. typhimurium. Then their spleens and livers were harvested and homogenized and the protein content of whole crude lysate was enriched using ammonium sulfate precipitation. SDS-polyacrylamide gel electrophoresis transfer method was used for enrichment of the protein from crude sample. Immunoblotting was conducted to detect Glycoprotein 96. Isoelectric point was achieved through the use of isoelectric focusing. PBS and whole crude lysate [from uninfected and infected mice] were injected to mice of test group, mice of control-1 group and mice of control-2 group, respectively, on days 0 and 14. Twenty-one days after the last immunization, the LD50 and bacterial loads of livers and spleens were determined. Immunization with Glycoprotein 96 rich lysate isolated from livers and spleens of S. typhimurium-infected mice induced protection against infection by S. typhimurium. Also, the bacterial burden of livers and spleens in mice that received gp96 rich lysate significantly decreased when compared to that of mice in the control groups

[Plasmid profile of pseudomonas aeruginosa and its relation with antibiotic resistance in hospital isolates]

P. aeruginosa is one of the causes of nosocomial infections with an unusual resistance to antibiotics. The source of resistance in this bacterium may be chromosomal or plasmid. The aim of the present study was to investigate the antibacterial susceptibility patterns with the presence of plasmids in P. aeruginosa isolates. In this study, 140 P. aeruginosa isolates were collected from hospitals in Urmia/Iran. The susceptibility patterns were determined against antibiotics. Plasmids were extracted by alkaline lysis method, electrophoresed and investigated by a UV transilluminator. Single digestion of plasmids with EcoR1 and HincII were performed and the restriction patterns were compared using a ladder. The rates of resistances to antibiotics were as follows: gentamicin 49.3%, cephalothin 99.3%, ticarcillin 100%, ceftizoxime 79.3%, co-trimoxazole 97.7%, amikacin 35%, carbenicillin 67.1%, ceftriaxone 65.7%, ciprofloxacin 58.6%, piperacillin 52.8%, imipenem 1.4%, kanamycin 65.7%, ofloxacin 72.1% and ampicillin 100%. In Whole, 65.7% of isolates harbored plasmids. Restriction enzyme analysis of plasmids showed unique pattern for all of plasmid positive isolates. All the plasmid positive isolates were resistant to ceftriaxone and kanamycin. The plasmid source of resistance to ceftriaxone was proved by plasmid elimination and transformation in E. coli DHS alpha and the plastid source of resistance to kanamycin was proved by plasmid elimination. Also there was a significant correlation between the presence of plasmid in isolates and resistance to some of antibiotics. There was a high frequency of plasmids in P. aeruginosa isolates, indicating that plasmids have an important role in transferring of resistance genes in this bacterium

[Role of orfD Pseudomonas aeruginosa H103 gene in glucose uptake]

Pseudomonas aeruginosa is a gram negative non facultative bacterium and one of the members of normal flora in different sites of body in healthy humans. This bacterium can resist in fluids and hospital environments for a long time. Pseudomonas aeruginosa has two systems for glucose uptake: a low affinity oxidative pathway and a high affinity phosphorylative pathway. The orfBCD genes are located over two million base pair upstream of the genes involved in the high affinity uptake system. Although the role of these genes are unknown by now, they may have a role in regulation of glucose uptake. In the present study, the role of orfD gene in glucose uptake in P.aeruginosa has been investigated. orfD fragment were cloned in pUCP20 as vector and the recombinant plasmid transferred into WMA200 strain of P.aeruginosa, a mutant strain of P.aeruginosa with a chromosomal deletion of orfBCD. So we compared the rate of glucose uptake by P.aeruginosaWMA200, P.aeruginosaWMA200/pUCP20/orfD and P.aeruginosa H103 as wild type strain of P.aeruginosa by using labeled glucose under conditions at low substrate concentration and low cell density. Carbohydrate uptake patterns differed considerably among three strains. The wild type is able to uptake glucose at a faster rate than the mutant; however, the mutant complemented with orfD shows an intermediate uptake comparing to the wild type and the mutant.orfD gene has an important role in carbohydrate uptake in P.aeruginosa strains however further studies are required to determine the involved mechanism