Toxigenicity testing of clinical isolates of non-typhoidal salmonellae in Vero cell culture & Caenorhabditis elegans model.

The non-typhoidal salmonellae (NTS) are recognized agents of gastroenteritis worldwide. Some of the NTS do not produces cytotoxic changes in tissue culture and not much is known about the endotoxicity of the clinical isolates of NTS (mostly Salmonella enterica serotype Typhimurium and Salmonella enterica serotype Enteritidis). We examined the exotoxic (cytotoxin) and endotoxic activity of clinical isolates of NTS in two assay models namely Vero cell culture and the nematode, Caenorhabditis elegans. Bacteria-free culture supernatants of 40 isolates NTS were tested in 96 well microtitre plate containing confluent monolayers of Vero cells. For the effects on C. elegans, the worms were exposed to bacteria free culture supernatants in 24 well microtitre plate for 24 h and then transferred to OP50 Escherichia coli lawn culture. The endotoxic activity of the live bacterium was studied by feeding the worms in the lawn culture of NTS separately. No cytopathic effect was observed with NTS tested in Vero cell culture assay. Likewise, the worms exposed to the bacteria-free culture supernatants were found active up to 7 days. In the co-culture killing assay, worms were found dead with characteristic stiff and straight appearance by 16(th) day. The worms were alive up to 21 days in OP50 E. coli. Bacteria-free culture supernatants did not have any deleterious effect on worms or in Vero cell culture, suggesting that there is no soluble toxic factor (diffusible toxin) in the culture supernatants. However, live NTS were found to be lethal to the worms; indicating that direct interaction between viable NTS and C. elegans is necessary for killing.

extractability of inner-membrane proteins from Salmonella typhimurium intact cells, spheroplasts and inner-membrane fragments by non-denaturing detergents

The effect of Triton X-100, Na cholate and Tween 80 on the solubilization of integral membrane proteins in intact cells, spheroplasts and inner-membrane fragments of Salmonella typhimurium was studied. The detergents were used in various concentrations [1.6 to 64 mM] and cytochromes b and d were used as marker to monitor the solubilization of membrane-bound proteins. Results showed that no inner-membrane protein solubilization was detected after the treatment of intact cells with detergents. The effect of Na cholate and Tween 80 on spheroplasts and inner-membrane fragments was also negligible in comparison to Triton X-100. Triton X-100 solubilized cytochromes from inner-membrane fragments more efficiently than from spheroplasts. The ratio of total protein solubilization to solubilize cytochromes showed that in spheroplasts this ratio was maximum at 1.6 mM Triton X-100 while it was maximum at 16-32 mM Triton X-100 in inner-membrane fragments. This difference between spheroplasts and inner-membrane fragments may be due to the orientation of the inner- membrane in spheroplasts [right side out] and inner-membrane fragments [in-side out as well as right side out], and to the presence of peripheral proteins attached to cytoplasmic membrane in spheroplasts

Effect of gamma irradiation on chemical and biological properties of lipopolysaccharide from Salmonella typhimurium.

Lipopolysaccharide (LPS) from S. typhimurium on exposure to gamma-radiation resulted in decrease in toxicity and was less mitogenic, Silver stained profiles of irradiated LPS on polyacrylamide gels revealed complete loss of its heteropolysaccharides which was confirmed further by analysing lipid A and LPS from Salmonella minnesota Re mutants on SDS-PAGE. Glucosamine and 2-keto 3-deoxy-octonate(Kdo) contents were significantly decreased on treatment. Lipid A obtained by removal of heteropolysaccharides from LPS was less toxic on exposure to gamma radiations.

Obtención, aislamiento y caracterización de mutantes auxotróficos de salmonella typhinurium / Obtain, isolation and auxotrophics mutants of salmonella typhimurium

Se comprobó el efecto inductor mutagénico de la luz uv, obteniendo mutantes auxotróficos y morfológicos de salmonella typhimurium. El aislamiento de mutantes auxotróficos mediante la técnica de réplica en placa mejora con el uso de penicilina mediante el método de Hollyday, para la identificación de los requerimientos específicos de cada mutante, se caracterizó 3 mutantes auxotróficos de 24 mutantes escogidos al azar. De 24 mutantes auxotróficos, el 30 por ciento resultó ser metronina dependientes, y 25 por ciento revirtieron la características de mutantes auxotróficos.