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

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.

Interaction of Salmonella with E. coli and Proteus spp. in Biofilm Formation

“…But in SAL 1+P combination, highest biofilm formation at 48hr, as a different observation than other interactions' maximal points, which may be due to increased extracellular matrix production of Proteus in some extended time durations [40]. The lower absorbance value at 96hr could be affected by entering bacterial biofilm cells into a viable but non-culturable state under nutrientdepleted conditions [36,37]. Collectively this repeated increment of dual biofilms may be due to the rapid growth of Salmonella, E. coli biofilm in extended time points and more surface coverage with irregular complex biofilm structure and higher exopolymer production [51].…”

“…Rodríguez-Melcón and his team also have investigated the increasing biofilm-forming trend of Salmonella species from 48hr to 72hr. Apart from that, the progression of biofilm formation since 2 to 4 days is affected by their increasing pellicle forming ability with extending incubation time [25], which supports current findings of increasing biofilm formation of SAL 1 isolate at 72hr.The minimum biofilm-forming ability of SAL 1, SAL 3 and Proteus could be affected by entering bacterial biofilm cells into viable but nonculturable stage [36,37] followed by repeat increment at extended post-incubation could also be happened their stress adaptation technique [38]. These VBNC can be investigated using standard plating techniques [39], not by microtiter plate assay.…”

Effect of sunlight and salinity on the survival of pathogenic and non‐pathogenic strains of Vibrio parahaemolyticus in water microcosms

“…Sunlight exposure was accompanied by the transformation of Vibrio cells into VBNC, which is considered a natural defense mechanism for bacteria to withstand under stressful conditions (Salive et al, 2020;Yoon et al, 2019) and recovers once environmental conditions become more favorable (Fern andez-Delgado et al, 2015;Wei & Zhao, 2018).…”

Physics Comes to the Aid of Medicine—Clinically-Relevant Microorganisms through the Eyes of Atomic Force Microscope

“…As demonstrated, VBNC bacteria reduced their size and changed the morphology from bacillary to coccoid. At the same time, no significant alterations were observed in the presence of acid and oxidant compounds, which provide new data for the purpose of further improvement of food safety [ 149 ].…”

“…Coupling AFM with biochemical profiles of bulk populations using fatty acid methyl ester profiling (FAME) allowed for a better understanding the persistence of this pathogen within environmental matrices [148]. Due to AFM analysis, it was possible to better characterize the morphological alterations of Salmonella bacteria entering the viable but non-culturable state (VBNC) upon exposition to stress conditions routinely present in food environments, i.e., low temperatures and high concentrations of sodium chloride [149]. As demonstrated, VBNC bacteria reduced their size and changed the morphology from bacillary to coccoid.…”