Transferecia de de ß-lactamasas de espectro extendido desde cepas hospitalarias de Klebsiella pneumoniae a otras especies de enterobacterias / Transference of extended-spectrum ß-lactamases from nosocomial strains of Klebsiella pneumoniae to other species of Enterobacteriaceae

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.

Geobacillus stearothermophilus LV cadA gene mediates resistance to cadmium, lead and zinc in zntA mutants of Salmonella entérica serovar Typhimurium

Salmonella entérica serovar Typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus LV exhibit a hypersensitive phenotype to cadmium chloride. Deletion of the ORF STM3576 from the Salmonella genome resulted in cadmium, lead and zinc sensitivity, confirming that this ORF is a homologue of the zntA gene. The observed sensitivity was reverted upon expression of the G. stearothermophilus LV cadA gene. These results indicate that the cadA gene product is involved in Cd, Pb and Zn resistance as a classical P-type ATPase and strongly suggest that the observed hypersensitive phenotype to these metals can be related to the function of the host ·zntA gene product.

Rapid identification of Salmonella typhimurium in stools by using 4-methylumbelliferone derivatives.

A total of 150 stool samples were collected from the Paediatric ward and screened for the presence of Salmonella typhimurium. A comparison of standard biochemical tests and rapid fluorescence method was done. The fluorescence method was found to be rapid, less time consuming and easy to perform whereas identification of Salmonella typhimurium using standard biochemical tests taken 18 to 24 hours.

Circular dichroism studies of the coenzyme environment in the active sites of mutant forms of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex.

The circular dichroism has been used to evaluate the effect of mutation on the environment of the pyridoxal phosphate coenzyme in the active site of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Seven mutant forms of the alpha 2 beta 2-complex with single amino acid replacements at residues 87, 109, 188, 306, and 350 of the beta-subunit have been prepared by site-directed mutagenesis, purified to homogeneity, and characterized by absorption and circular dichroism spectroscopy. Since the wild type and mutant alpha 2 beta 2 complexes all exhibit positive circular dichroism in the coenzyme absorption band, pyridoxal phosphate must bind asymmetrically in the active site of these enzymes. However, the coenzyme may have an altered orientation or active site environment in five of the mutant enzymes that display less intense ellipticity bands. The mutant enzyme in which lysine 87 is replaced by threonine has very weak ellipticity at 400 nm. Since lysine 87 forms a Schiff base with pyridoxal phosphate in the wild type enzyme, our results demonstrate the importance of the Schiff base linkage for rigid or asymmetric binding. Although the mutant enzymes display spectra in the presence of L-serine that differ from that of the wild type enzyme, addition of alpha-glycerol 3-phosphate converts the spectra of two of the mutant enzymes to that of the wild type enzyme. We conclude that this alpha-subunit ligand may produce a conformational change in the alpha-subunit that is transmitted to the mutant beta-subunits and partially corrects conformational alterations in the mutant enzymes.

Role of oxygen dependent mechanisms in virulence of Salmonella typhimurium.

Catalase and superoxide dismutase (SOD) activities of five strains of S. typhimurium, namely SF 1546 LT2 71, SF 1835 C5 50, 386 SF 1591 Ra 20, SF 1567 Rd1, and an Indian strain were determined and correlated with their virulence profile. All five Salmonella strains exhibited catalase and SOD activities. No correlation was observed between either SOD and/or catalase activity and LD50 values of the isolates. Oxygen free radical generation elicited by macrophages, in the presence of virulent and avirulent salmonellae was also not statistically significant (P greater than 0.05), although the virulent species significantly resisted the macrophage bactericidal activity (P less than 0.05). It appears, therefore, that oxygen-dependent bactericidal mechanisms may not be important in phagocytic killing of S. typhimurium.