Characterization of plasmids which mediate resistance to multiple antibiotics in gram-negative bacteria of nosocomial origin

BACKGROUND: The genetic and molecular mechanisms involved in antimicrobial resistance of 10 strains of gramnegative bacilli (1 Serratia marcescens; 2 Escherichia coli; 1 Proteus mirabilis; 4 Klebsiella pneumoniae; 1 Enterobacter cloacae y 1 Alcaligenes faecalis), isolated from adult patients with nosocomial pulmonary infection at the in-patient facilities of the University Hospital of Los Andes, Mérida, Venezuela, have been studied. METHODS: The antimicrobial susceptibility was determined by minimum inhibitory concentrations using the dilution method in agar. The study of extrachromosomal genes was carried out by conjugation, bacterial infection with the bacteriophage M13 and curing of plasmid by acridine orange. The plasmids were isolated by alkaline lysis and analysis of restriction endonuclease digestion was carried out separately using the enzymes EcoRI and HindIII. A DNA probe, derived from the region which encodes the TEM-1 beta-lactamase of the plasmid pBR322 was used for dot-blot hybridization tests. RESULTS: All of the gramnegative bacilli showed resistance to ampicillin, carbenicillin and cephalothin (> 128 micrograms/ml) and 3 strains also showed resistance to gentamicin (> 64 micrograms/ml). Genetic and molecular procedures showed the presence of conjugative plasmids of approximately 54 kb in all the 10 strains. The restriction patterns obtained by using EcoRI and HindIII indicated common DNA fragments in most of the plasmids studied. The dot-blot hybridization tests confirmed homology between the plasmids and the DNA probe used (TEM-1 beta-lactamase). CONCLUSIONS: In this study, the gramnegative bacteria of nosocomial origin harbored self-transferable plasmids of approximately 54 kb, which mediate resistance to gentamicin and encode a beta-lactamase of the TEM group.