ABSTRACT
Background: Klebsiella pneumoniae is an important pathogenic bacterium, frequently isolated from nosocomial samples, that exhibits wide antimicrobial resistance profiles, including third generation cephalosporins (3GC), aminoglycosides and quinolones. The resistance to 3GC is mainly due to the synthesis of extended spectrum beta lactamases (ESBL), encoded by conjugative plasmids. Aim: To investigate the potential transference of resistance to 3GC from nosocomial strains of K. pneumoniae to other clinical strains of various species of Enterobacteriaceae. Material and methods: The mating experiments were carried out in liquid media and three nosocomial strains of K. pneumoniae were used as donors. These strains were ESBL-producers and resistant to, at least, one of the 3GC assayed. One strain of Citrobacter freundii, Salmonella typhimurium, Serratia marcescens and Escherichia coli, isolated from clinical specimens, were used as recipients. The presence of bla genes was investigated by PCR. Results: The three nosocomial strains of K. pneumoniae were able to transfer the resistance to 3GC and the genes encoding the ESBL to the susceptible recipient strains of enterobacteria. The frequency of transference was as high as 3.2 x 10-2 transconjugants/recipient cell when the strain of Citrobacter freundii was used as recipient. All transconjugants exhibited high level of resistance to the 3GC assayed. Conclusions: Strains of K. pneumoniae isolated from Chilean hospitals are able to disseminate the ESBL genes to clinical strains of others species of Enterobacteriaaceae.
Subject(s)
Humans , Anti-Bacterial Agents/pharmacology , Cephalosporin Resistance/genetics , Cephalosporins/pharmacology , Klebsiella pneumoniae/enzymology , Transformation, Bacterial/genetics , beta-Lactamases/biosynthesis , Citrobacter freundii/drug effects , Citrobacter freundii/enzymology , Escherichia coli/drug effects , Escherichia coli/enzymology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Microbial Sensitivity Tests , Polymerase Chain Reaction , Salmonella typhimurium/drug effects , Salmonella typhimurium/enzymology , Serratia marcescens/drug effects , Serratia marcescens/enzymology , beta-Lactamases/geneticsABSTRACT
This paper describes an efficient bacterial transformation system that was established for the preparation of competent cells, plasmid preparation, and for the storage in bacterial stocks in our laboratory. Using this method, a number of different plasmids have been amplified for further experiments. Competent cells for bacterial transformation were prepared by the calcium chloride method with an optimum concentration of 75 mM. Three different strains of Escherichia coli that were tested are DH5 alpha, TG1 and XL1 blue, and the most efficient strain being XL1 blue. The optimal optical density (OD600) range for competent cell preparation varied for each of the strains investigated, and for XL1 blue it was 0.15-0.45; for TG1 it was 0.2-0.5; and for DH5 alpha it was 0.145-0.45. The storage time of competent cells and its correlation to transformation efficiency has been studied, and the result showed that competent cells can be stored at 20 ºC for 7 days and at 70 ºC for 15 days. Three critical alterations to previous methods have been made, which are the changing of the normal CaCl2 solution to TB solution, the changing of the medium from LB to S.O.C., and addition of DMSO or PEG8000 during transformation of competent cells with plasmids. Changing the medium from LB to S.O.C., resulted in much faster growth of transformants, and the transformation efficiency was increased. Addition of DMSO or PEG8000 raised transformation efficiencies by 100-300 fold. Our improved bacterial transformation system can raise the transformation efficiency about 103 times, making it becoming a highly efficient bacterial transformation system.
Subject(s)
Escherichia coli/growth & development , Escherichia coli/genetics , Plasmids/genetics , Cell Culture Techniques/methods , Transformation, Bacterial/genetics , DNA, Bacterial/genetics , Buffers , Cloning, Molecular/methods , Electroporation , Solutions , Temperature , Time Factors , Transformation, GeneticABSTRACT
Between July 1996 to June 1997 a total of 531 Stool samples from cases of acute diarrhoea, dysentry, colitis admitted to Kasturba Medical College Hospital, Manipal were investigated. Shigella were isolated from 29 samples (5.46%). Out of them, Shigella flexneri were 16 (55.17%), Shigella boydii 8 (27.58%), Shigella dysentriae 3 (10.34%) and Shigella sonnei 2 (6.89%). Five strains of Shigella flexneri and one strain of Shigella dysentriae were found to show resistance to Nalidixic acid and the newer fluoroquinolones viz., Ciprofloxacin, Norfloxacin and Ofloxacin. The in vitro 'R' factor transfer study showed that the resistance was not transferable.