In vitro and in vivo assessment of Salmonella enterica serovar Typhimurium DT104 virulence

Abstract

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104 has become a widespread cause of human and other animal infection worldwide. The severity of clinical illness in S. enterica serovar Typhimurium DT104 outbreaks has led to the suggestion that this strain possesses enhanced virulence. In the present study, in vitro and in vivo virulence-associated phenotypes of several clinical isolates of S. enterica serovar Typhimurium DT104 were examined and compared to S. enterica serovar Typhimurium ATCC 14028s. The ability of these DT104 isolates to survive within murine peritoneal macrophages, invade cultured epithelial cells, resist antimicrobial actions of reactive oxygen and nitrogen compounds, and cause lethal infection in mice were assessed. Our results failed to demonstrate that S. enterica serovar Typhimurium DT104 isolates are more virulent than S. enterica serovar Typhimurium ATCC 14028s.

FIG. 1

Macrophage survival. Survival of Salmonella strains was determined in BALB/c (ity^s) peritoneal macrophages. Peritoneal macrophages were harvested from mice 4 days after injection with 5 mM sodium periodate and plated at a density of 3 × 10⁵ to 5 × 10⁵/well. They were infected 24 h later at a multiplicity of infection of 5:1 with opsonized S. enterica serovar Typhimurium 14028s, S. enterica serovar Typhimurium DT104 isolates, or macrophage-sensitive mutant phoP S. enterica serovar Typhimurium. Extracellular bacteria were killed using amikacin (100 μg/ml) since the DT104 isolates are resistant to gentamicin. Results are expressed as percent survival and represent the average of two independent assays. Error bars indicate standard deviation.

FIG. 2

Susceptibility to reactive oxygen and nitrogen species. (A and B) Salmonella cultures were grown overnight in LB medium, diluted in saline, and plated (100 μl containing 10⁶ CFU) on M9 minimal plates containing 0.2% glucose. Then 15 μl of 3% hydrogen peroxide (A) or paraquat (1 mM) (B) was spotted onto 6-mm-diameter paper disks, the disks were placed onto the bacterial lawn, and the plates were incubated overnight at 37°C. The zone of inhibition was measured on two axes, averaged, and plotted. (C) Susceptibility to acidified nitrite was determined by adding 20 μl of an overnight culture to 2 ml of acidified LB (pH 5) with or without 20 mM sodium nitrite. Bacteria were incubated at 37°C with aeration for 3 and 6 h, and viable cells were enumerated by plating dilutions onto LB agar.

FIG. 2

Susceptibility to reactive oxygen and nitrogen species. (A and B) Salmonella cultures were grown overnight in LB medium, diluted in saline, and plated (100 μl containing 10⁶ CFU) on M9 minimal plates containing 0.2% glucose. Then 15 μl of 3% hydrogen peroxide (A) or paraquat (1 mM) (B) was spotted onto 6-mm-diameter paper disks, the disks were placed onto the bacterial lawn, and the plates were incubated overnight at 37°C. The zone of inhibition was measured on two axes, averaged, and plotted. (C) Susceptibility to acidified nitrite was determined by adding 20 μl of an overnight culture to 2 ml of acidified LB (pH 5) with or without 20 mM sodium nitrite. Bacteria were incubated at 37°C with aeration for 3 and 6 h, and viable cells were enumerated by plating dilutions onto LB agar.

FIG. 2

Susceptibility to reactive oxygen and nitrogen species. (A and B) Salmonella cultures were grown overnight in LB medium, diluted in saline, and plated (100 μl containing 10⁶ CFU) on M9 minimal plates containing 0.2% glucose. Then 15 μl of 3% hydrogen peroxide (A) or paraquat (1 mM) (B) was spotted onto 6-mm-diameter paper disks, the disks were placed onto the bacterial lawn, and the plates were incubated overnight at 37°C. The zone of inhibition was measured on two axes, averaged, and plotted. (C) Susceptibility to acidified nitrite was determined by adding 20 μl of an overnight culture to 2 ml of acidified LB (pH 5) with or without 20 mM sodium nitrite. Bacteria were incubated at 37°C with aeration for 3 and 6 h, and viable cells were enumerated by plating dilutions onto LB agar.

FIG. 3

Invasion of HEp-2 cells by Salmonella strains. HEp-2 cells were grown to confluence and infected at a multiplicity of infection of 10 to 50 with S. enterica serovar Typhimurium 14028s, S. enterica serovar Typhimurium DT104 isolates, and hilA and invA mutant strains of S. enterica serovar Typhimurium grown overnight in LB without aeration at 37°C. Nonadherent bacteria were removed by washing three times with phosphate-buffered saline; then, RPMI plus 10% fetal calf serum containing amikacin at 100 μg/ml was added and the plates were incubated for an additional 1 h. The cells were washed three times with phosphate-buffered saline and lysed with 1% Triton X-100. Bacterial invasion was determined by plating serial dilutions onto LB agar. The results, expressed as percent invasion, are representative of three independent assays. Error bars indicate standard deviation.