Resuscitation of Seventeen-year Stressed Salmonella typhimurium

Abstract

Abstract Salmonella enterica serovar typhimurium was stressed by incubation in seawater microcosms for seventeen years. The microcosms were prepared in such a way as to allow progressive evaporation of the water. Despite being introduced into the sterile seawater at very high concentrations, the Salmonella rapidly declined to levels undetectable by plate counts on nutrient agar. After two years of starvation, about half of the seawater volume had evaporated from each microcosm, and salt crystals appeared. Inoculation of the salty suspension did not result in any culturable strains in selective and non-selective media. However, incubation of samples in nutrient-rich broth, without supplemental growth factors, allowed resuscitation of stressed cells, yielding colonies that remained viable for extended periods of time. After three years the total volume of water had evaporated from each microcosm, and only salt crystals remained. These microcosms were then incubated at room temperature for seventeen years. Resuscitation of VBNC Salmonella from the salt crystals was conducted in vitro and in vivo; recovery occurring after incubation in both nutrient broth and in mice. Recovery occurred in the Salmonella administered orally into the mice, but not in those administered by intra-peritoneal injection. The identity of initial, stressed and revived strains was confirmed by DGGE analysis and sequencing of 16S RNA gene fragments.