A bacteriophage detection tool for viability assessment of Salmonella cells.

TLDR: This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability and shows the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods.

About: This article is published in Biosensors and Bioelectronics.The article was published on 2014-02-15 and is currently open access. It has received 90 citations till now. The article focuses on the topics: Bacteriophage.

Figures

Fig. 3. Phage vs antibody performance as a biorecognition element. Density of cells specifically captured by phage and antibody spots, covalently immobilized on gold solid surface. (A) The surface coverage was analyzed by ImageJ software. (B) Stereoscope pictures for representative spots of Salmonella cells specifically recognized by phage PVP-SE1 (top) and anti-Salmonella antibody (bottom). Statistical analysis for phage vs antibody results: *po0.05, ***po0.0001, two-way ANOVA.

Fig. 2. Profile of phage adsorption to cells at different physiological states. Studies of bacteriophage PVP-SE1 adsorption to Salmonella Enteritidis in phosphate buffer at room temperature. (A) Adsorption profile of phage to viable, VBNC and dead cells (upper curves) and phage growth curve in viable cells (bottom curve). For phage growth, average values result from triplicates of one single assay and standard deviations are all below 20%. (B) Picture of plaque forming units (PFU) on a culture plate for phage adsorption counts. (C) Zoom-in over PFU on the bacterial lawn. Statistical analysis: ***po0.0001 for viable vs dead, þ þ þpo0.0001 for VBNC vs dead; two-way ANOVA.

Fig. 1. Induction of VBNC physiological state in Salmonella. Assessment of the physiological state of Salmonella Enteritidis cells after treatment with different concentrations of bleach by three different evaluation methods: (A) CFU counts on standard solid culture media (black line) compared to flow cytometry results for relative percentage of cells in the viable (green bars), compromised (yellow bars) and dead (red bars) states. The culturability test was collected from several trials over time and performed in triplicate; thus all CFU counts were normalized to the same initial cell concentration of 2.39 108 cells/mL. Results presented for the flow cytometry analysis are the mean of independent triplicates from three independent experiments (n¼9). (B) Epifluorescence microscopy images of Salmonella Enteritidis cells treated with different concentrations of bleach (0%, left panel; 0.006% middle panel; 0.01% right panel) where the different physiological states may be distinguished based on each cell's coloration (green, viable; yellowish, compromised; red, dead). (C) Representative flow cytometry plots showing the percentage of live, dead and compromised cells present after each treatment. For both assays, depicted in (B) and (C), cells were stained with SYTO 9 (green) and propidium iodide (PI; red); for flow cytometry analysis, the gating strategy adopted was the one described in the LIVE/DEAD BacLight kit's inset (Berney et al., 2007). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Phage-based magnetoresistive biochip for cell viability assessment. Phage-based magnetoresistive biochip measurements performed on an electronic reader. (A) Normalized differential voltage signal for Salmonella samples subjected to different treatments (dashed line and black dots). The acquired sensor signals are differential values (ΔVbinding), calculated from the difference between the sensor baseline (Vsensor) and the signal from the specifically bound MNPs over the sensor (Vparticles) as previously described (Martins et al., 2009). The shaded area in blue on the bottom refers to the average signal from the control sensors using an unspecific phage. Each point and shaded area represents the average value of at least 10 independent sensors with respective associated standard deviation (error bars). Bars represent the concentration of cells and respective cell state (viable, compromised (comp.) and dead) for each cell sample measured on the MR-chip. Cell concentrations were obtained by flow cytometry using the LIVE/DEAD counting Baclight kit, in which each data point results from the analysis of triplicate samples from one representative experiment out of three. (B) Schematic representation of the “sandwich” type biomolecular recognition strategy adopted in the MR-biochip measurements. (C) Picture of the MR-biochip PCB used for the detection of Salmonella cells, shown on a standard cell culture plate.

Frequently asked questions

Q1. What have the authors contributed in "A bacteriophage detection tool for viability assessment of salmonella cells" ?

This work presents and validates a novel bacteriophage ( phage ) -based microbial detection tool to detect and assess Salmonella viability. This ability was confirmed for immobilized phages on gold surfaces, where the phage detection signal follows the same trend of the concentration of viable plus VBNC cells in the sample. Salmonella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, flow-cytometry and epifluorescence microscopy, and further assayed with a biosensor platform.