Comparison of stress conditions to induce viable but non-cultivable state in Salmonella
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FOOD MICROBIOLOGY - RESEARCH PAPER
Comparison of stress conditions to induce viable but non-cultivable state in Salmonella Andres Felipe Vanegas Salive 1 & Cláudia Vieira Prudêncio 2 & François Baglinière 3 & Leandro Licursi Oliveira 4 & Sukarno Olavo Ferreira 5 & Maria Cristina Dantas Vanetti 3 Received: 3 September 2019 / Accepted: 10 March 2020 # Sociedade Brasileira de Microbiologia 2020
Abstract 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. Keywords Viable but non-culturable . Stress conditions . Salmonella enterica . Survival
Introduction Despite efforts to reduce the presence of Salmonella in foods, this pathogen remains a leading cause of foodborne Responsible Editor: Mariza Landgraf. * Maria Cristina Dantas Vanetti [email protected] 1
Agricultural Microbiology Laboratory Ecofarm, Ibague, State of Tolima, Colombia
2
Center of Biological and Health Sciences, Federal University of Western of Bahia, Barreiras, Bahia, Brazil
3
Department of Microbiology, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil
4
Department of Biology, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil
5
Department of Physics, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil
bacterial illness in humans worldwide [1, 2]. In the food processing chain, Salmonella is subjected to various unfavorable environmental conditions that, if not result in a cell death, can lead to the formation of injured and stre
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