ABSTRACT
INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43 percent, tobramycin 38 percent, and amikacin 24 percent. Of the genes examined, aac (6')-II (36 percent) was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2")-I, aph (3')-VI, and aac (6')-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a signific ant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.
Subject(s)
Female , Humans , Male , Acetyltransferases/genetics , Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Kanamycin Kinase/genetics , Nucleotidyltransferases/genetics , Pseudomonas aeruginosa/genetics , Aminoglycosides/metabolism , Anti-Bacterial Agents/metabolism , DNA, Bacterial/genetics , Drug Resistance, Bacterial/drug effects , Iran , Pseudomonas aeruginosa/drug effectsABSTRACT
Transgenic Robinia pseudoacacia plants were obtained by Agrobacterium tumefaciens mediated gene transfer. Agrobacterium strain LBA4404 harbouring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes was co-cultivated with hypocotyl segments of in vitro raised seedlings of Robinia. Parameters important for high efficiency regeneration and transformation rates included type of explant, pre-conditioning of explants and appropriate length of co-cultivation period with Agrobacterium. A transformation frequency 16.67% was obtained by 48 hr of pre-conditioning followed by 48 hr of co-cultivation. Transformed tissue was selected by the ability to grow on kanamycin containing medium. Successful regeneration was followed after histochemical GUS assay for the detection of transgenic tissue. This transformation procedure has the potential to expand the range of genetic variation in Robinia.
Subject(s)
Glucuronidase/genetics , Kanamycin Kinase/genetics , Plants, Genetically Modified/genetics , Plasmids , Recombinant Fusion Proteins/metabolism , Agrobacterium tumefaciens/physiology , Robinia/enzymology , Seedlings/enzymology , Transformation, Genetic , TransgenesABSTRACT
Transgenic mice were produced to study the expression of amino-3' glycosyl phosphotransferase gene (neomycin resistance gene) in the embryonic fibroblast cells. A 1.9 Kb linear fragment of neomycin resistance gene under the control of pPGK promoter was microinjected into the pronucleus of mouse embryos. Out of 64 potential founders born, 5 were identified to be transgenic by the polymerase chain reaction (PCR) and southern hybridization. Multiple mice from first and second generation from two transgenic founders (N-10 and N-32) were analysed to determine the germline transmission. It was found to be 24.6 and 71.4% in first and second generation respectively. Results were also further confirmed by RT-PCR, sequencing and in vitro bioassays.