Comparison of stress conditions to induce viable but non-cultivable state in Salmonella.

Salmonella can enter on the viable but non-culturable state (VBNC), characterized by the loss of ability to grow in routine culture media hindering detection by conventional methods and underestimation of the pathogen. Despite advances in research done so far, studies comparing conditions that lead Salmonella into the VBNC state are scarce. The main objective of this study was to evaluate different stresses to induce Salmonella to the VNBC state. Osmotic (1.2 M NaCl), acid (peracetic acid, 5.66 mg/mL) and oxidative (hydrogen peroxide, 1.20 mg/mL) stress were used at 4 °C to induce Salmonella enterica serovars Enteritidis and Typhimurium to the VBNC state. The culturability loss was monitored in the brain heart infusion (BHI) broth and agar, and the viability was determined by fluorescence microscopy, using the Live/Dead® kit, and by flow cytometry. Besides, the morphological characterization by atomic force microscopy (AFM) was performed. Storage in 1.2 M NaCl at 4 °C induced the VBNC state in Salmonella cells for periods longer than 121 days, and the percentage of viable cells has reached above 80.9%. More aggressive stress conditions promoted by peracetic acid and hydrogen peroxide induced the VBNC state in periods of, at most 0.14 day, and resulted in percentages of 8.5% to 45.5% viable cells, respectively. The counts of viable cells in the flow cytometer corroborate the results obtained by microscopic counts. The VBNC cells obtained in 1.2 M NaCl at 4 °C showed morphological changes, reducing the size and changing the morphology from bacillary to coccoid. No morphological change was observed on the cells stressed by acid or oxidant compounds.