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Journal ArticleDOI

A bacteriophage detection tool for viability assessment of Salmonella cells.

TL;DR: 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.

Summary (3 min read)

1. Introduction

  • The ingestion of food, its derivatives and water contaminated with microbial pathogens (e.g. Escherichia coli, Campylobacter sp. or Salmonella sp.) is responsible for about 2.2 million deaths annually.
  • “Dormant” bacteria have therefore been called viable but non-culturable (VBNC) cells.
  • Significant progress has been reported in the phage-based detection of foodborne and waterborne pathogens (Hagens and Loessner, 2007; Singh et al., 2012; Smartt et al., 2012).

2.2. Bacteriophages and bacterial strains

  • PVP-SE1 was isolated from a Regensburg wastewater plant in the context of a European Project (Phagevet-P).
  • Salmonella Enteritidis strain S1400 was used as host (Sillankorva et al., 2010).
  • Campylobacter coli phage vB_CcoM-IBB_35, isolated from poultry intestines, was used as negative control (Carvalho et al., 2010a).

2.3. Phage propagation and buffer exchange

  • The phages were produced using the double layer agar technique as described by Sambrook and Russell (2001) and resuspended in SM buffer.
  • Exchange of SM buffer by MOPS buffer was needed to avoid the presence of amine groups from SM buffer, which may interfere with the surface chemistry adopted for phage immobilization on solid substrates.
  • Buffer exchange was made using a Vivaspin 500 centrifugal concentrator (MW 100 kDa).
  • Following the buffer exchange the concentration of phage was verified using the double layer agar technique.

2.4. Induction of Salmonella into viable but non-culturable (VBNC) state

  • Bacteria were induced to enter the VBNC state by using sodium hypochlorite (commercial bleach—stock concentration 5%) at different concentrations.
  • The serial dilutions of bleach were done with milli-Q water.
  • The samples were mixed at 200 rpm for 1 min at room temperature.
  • Following chlorination, the suspensions were centrifuged at 3420xg for 10 min at 4 1C and washed twice with cold PB.

2.5. Determination of cell viability

  • Cell viability was assessed after submitting bacteria to different bleach concentrations using the LIVE/DEADs BacLight™ Bacterial Viability and Counting Kit (Molecular Probes).
  • SYTO9 and PI dyes were used, accordingly to manufacturer's instructions.
  • Upon staining, cells were analyzed either by epifluorescence microscopy (OLYMPUS BX51 EXTREMO microscope) or by flow cytometry (BD LSRII flow cytometer using FACS DIVA software for acquisition; BD Biosciences).
  • For absolute cell quantification, 6 μm diameter microspheres were used at a known concentration in the flow cytometry acquisition.
  • Flow cytometry data was analyzed using the FlowJo software (Tree Star, Ashland, OR).

2.6. Phage lysis time and adsorption studies

  • 1 mL of each Salmonella sample was infected with PVP-SE1 phage at a multiplicity of infection (MOI) of 0.001, which refers to the number of phages that were added per cell.
  • Samples were taken immediately after infection (time 0) and after 20 min and 40 min of phage inoculation, followed by 10-fold dilution in MOPS and centrifugation at 10,000g for 10 min.
  • The supernatant was 10-fold serially-diluted in MOPS and plated to assess the concentration of PFU (plaque forming unit).
  • The phage adsorption fraction was calculated by dividing the PFU concentration at each time point by the initial phage concentration.
  • To assess the phage lysis time viable exponential phase grown Salmonella cells were used.

2.7. Phage immobilization on Au surfaces

  • Cr 5 nm/Au 40 nm thin film layers were sputtered (Kenosistec sputtering tool) over a silicon wafer.
  • The wafer was then spincoated with a photoresist (PR) polymer (AZ1505 AZ Electronic Materials) for surface protection and diced in 7 7 mm2 dies using an automatic dicing saw (Disco, DAD3350).
  • Substrates were then rinsed with isopropanol (IPA) and milli-Q water and dried under a nitrogen stream.
  • The gold surface was then functionalized with a heterobifunctional linker, the sulfo-LC-SPDP (sulfosuccinimidyl 6-[3′-(2-pyridyldithio)-propionamido] hexanoate).
  • Spot pictures were taken with an optical stereomicroscope (Nikon SMZ 1500) equipped with a CCD camera and analyzed using the image processing software ImageJ.

2.8. MR-biochip measurement

  • The MR-biochip was produced at INESC MN through a dedicated microfabrication process (Martins et al., 2009) and wirebonded to a PCB chip-carrier.
  • The probe sites on the MR biochip terminate with exposed Cr/Au pads, underneath which lie the magnetoresistive sensors that will detect the magnetic nanoparticle labels.
  • Briefly, the MR chip architecture comprises two distinct sensing areas arranged in two columns.
  • A 1 mL droplet of Salmonella-specific phage was spotted over the left column of sensors (12 sensors) and a non-specific phage (Campylobacter phage) on the right column of sensors (12 sensors).
  • The difference between the signal acquired after washing and the baseline signal is proportional to the number of cells bound to the sensor surface.

2.9. Antibody-conjugated MNPs preparation

  • Commercial 250 nm Protein A modified MNPs (Nanomag, Micromod) were used.
  • The unbound antibody was removed by the same magnetic separation procedure.
  • The functionalized MNPs were finally resuspended in 5 mL of PB Tw20 and injected over the chip.

2.10. Statistical analysis

  • All data are represented as mean7SD (standard deviation).
  • For Figs. 2 and 3, means were compared using two-way ANOVA followed by the Bonferroni post hoc test.

3.1. Induction of VBNC physiological state in Salmonella

  • Since the goal of this work was to prove the phage ability to detect the VBNC state of bacterial cells, a process was first developed capable of affecting cell viability in a controlled manner that would not lead to killing or lysing the entire cell population.
  • For this purpose different bactericidal and bacteriostatic compounds, known to induce the VBNC state in Salmonella cells, were tested (data not shown).
  • When exposed to fresh liquid medium under adequate growth conditions all tested concentrations of bleach, even above the break-point, showed cell growth (Supplementary data, Fig. S2.1).
  • In order to quantitatively determine the relative and absolute proportion of the different cell populations (classified as live, dead or compromised), flow cytometry analyses were conducted for the different cell samples (Fig. 1A bars and 1C).
  • Results confirmed that, despite being present in sub-optimal host infection conditions, the phage adsorption capability was conserved, maintaining its potential to be used as a detection tool.

3.3. Phage performance as a biorecognition element

  • After optimization of the surface chemistry (Supplementary data, Fig. S3.1 and S3.2), the phage was immobilized on an Au surface at discrete areas by manual spotting.
  • Also according to phage adsorption rates in solution, the immobilized phages were able to discriminate between viable and dead cells.
  • This resulted in reduced cell densities for samples with increasing number of dead cells (Fig. 3A) but proportional to the relative concentration of viable plus VBNC cells (compromised population) obtained by flow cytometry analysis (Fig. 1A—bars plot).
  • Identical biorecognition elements may hinder each other's proper attachment.
  • This is a common scenario in standard immunoassays where a labeling antibody may block the epitopes to the capture antibody or vice versa.

3.4. Phage-based magnetoresistive biochip for cell viability assessment

  • The feasibility of developing a “sandwich” phage-based biosensing system and its potential as a cell viability determination tool was assessed making use of an existent magnetoresistive (MR) biochip (Freitas et al., 2012; Martins et al., 2009, 2010) and respective electronic reader (Germano et al., 2009).
  • The biomolecular recognition strategy used on the biochip combines the phage and a magnetically-labeled antibody as recognition and labeling elements, respectively.
  • After the functionalization of the biochip with PVP-SE1 bacteriophage, each cell solution was loaded over the chip surface and incubated.
  • After washing, the magnetic fringe field created by the labels was detected as a variation on the sensor resistance.
  • Fig. 4A (dashed line and black dots) shows the biosensor normalized output for decreasing concentrations of viableþ VBNC cells.

4. Conclusions

  • The lytic phage PVP-SE1 was explored as an alternative biorecognition element for bacterial detection and viability assessment.
  • Taking into account the problematic occurrence of false positives associated with DNA-chips and the high production costs, poor stability and cross-reactivity related to immuno-chips, the development of phage-based biochips emerges as a valuable tool.
  • The feasibility to immobilize phages on sensing surfaces and conjugate this biomolecular tool with electronic analytical devices without losing functionality was proven.
  • The combined use of the magnetoresistive sensor with the phage probes allowed a clear detection of viable from dead Salmonella cells.

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Journal ArticleDOI
TL;DR: Various aspects of VBNC bacteria are described, which include their proteomic and genetic profiles under the VB NC state, conditions of resuscitation, methods of detection, antibiotic resistance, and observations on Rpf.
Abstract: Under stress conditions, many species of bacteria enter into starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. Several human pathogenic bacteria have been reported to enter into the VBNC state under these conditions. The pathogenic VBNC bacteria cannot be grown using conventional culture media, although they continue to retain their viability and express their virulence. Though there have been debates on the VBNC concept in the past, several molecular studies have shown that not only can the VBNC state be induced under in vitro conditions but also that resuscitation from this state is possible under appropriate conditions. The most notable advance in resuscitating VBNC bacteria is the discovery of resuscitation-promoting factor (Rpf), which is a bacterial cytokines found in both Gram-positive and Gram-negative organisms. VBNC state is a survival strategy adopted by the bacteria, which has important implication in several fields, including environmental monitoring, food technology, and infectious disease management; and hence it is important to investigate the association of bacterial pathogens under VBNC state and the water/foodborne outbreaks. In this review, we describe various aspects of VBNC bacteria, which include their proteomic and genetic profiles under the VBNC state, conditions of resuscitation, methods of detection, antibiotic resistance, and observations on Rpf.

338 citations


Cites background from "A bacteriophage detection tool for ..."

  • ...” Several recent studies have shown the usefulness of bacteriophages in the detection of VBNC cells present in the bacterial populations (72, 73)....

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Journal ArticleDOI
TL;DR: This review highlights advances in techniques used to engineer phages as vehicles for drug delivery and vaccines, as well as for the assembly of new materials, and discusses existing challenges and opportunities.
Abstract: Soon after their discovery in the early 20th century, bacteriophages were recognized to have great potential as antimicrobial agents, a potential that has yet to be fully realized. The nascent field of phage therapy was adversely affected by inadequately controlled trials and the discovery of antibiotics. Although the study of phages as anti-infective agents slowed, phages played an important role in the development of molecular biology. In recent years, the increase in multidrug-resistant bacteria has renewed interest in the use of phages as antimicrobial agents. With the wide array of possibilities offered by genetic engineering, these bacterial viruses are being modified to precisely control and detect bacteria and to serve as new sources of antibacterials. In applications that go beyond their antimicrobial activity, phages are also being developed as vehicles for drug delivery and vaccines, as well as for the assembly of new materials. This review highlights advances in techniques used to engineer phages for all of these purposes and discusses existing challenges and opportunities for future work.

295 citations

Journal ArticleDOI
TL;DR: This review provides an overview of the biology of the VB NC state, its relationship to food safety, and novel methods developed for the rapid detection and identification of VBNC cells.
Abstract: The viable but non-culturable (VBNC) state is a form of dormancy employed by many bacteria as a method of survival and can be found in nearly any ecological niche. Major characteristics that distinguish dormant cells is their ability to evade detection by routine laboratory culture, to tolerate stressful environments including food pasteurization processes and antibiotics, and to resuscitate within a host and cause disease. Given these defining characteristics, these resilient microbes raise significant concern for the food industry and for the health of those consuming foods harboring these veiled pathogens. This review provides an overview of the biology of the VBNC state, its relationship to food safety, and novel methods developed for the rapid detection and identification of VBNC cells.

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TL;DR: The importance of electrochemical biosensors as simple, reliable, cost-effective, and accurate tools for bacterial detection is emphasized, as well as the most recent advancements in phage-based sensing assays and sensors.

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TL;DR: This review proposes to gather and comment different ligands used for the detection of whole cell bacteria and label-free methods, which enable the user to skip sampling processing steps and decrease the overall test cost.
Abstract: With the aim of getting earlier, sensitive and specific information on the presence –or absence – of bacterial pathogens, biosensors are getting an increasing interest for more than two decades. This is partly due to their reduced format, to the possibility to address several questions with a single device and also to the increasing panel of physical approaches that can be exploited for signal transducing. When designing a biosensor, the choice of the ligand motif remains a key element as it drives the efficiency and sensitivity of the assay. In this review, we propose to gather and comment different ligands used for the detection of whole cell bacteria. Because time is a crucial issue when looking for a pathogen, our attention was focused on whole cell assays and label-free methods, which enable the user to skip sampling processing steps and decrease the overall test cost.

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"A bacteriophage detection tool for ..." refers methods in this paper

  • ...Detection methods based on DNA analysis (e.g. PCR—Polymerase Chain Reaction) (Keer and Birch, 2003; Lu et al., 2009) or flow cytometry (Nebe-von-Caron et al., 2000; Phe et al., 2005; Suller and Lloyd, 1999) have recently been developed to identify the cell's physiological state....

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TL;DR: The developed platform is portable and capable of operating autonomously for nearly eight hours and the noise level of the described platform is one order of magnitude lower than the one presented by the previously used measurement set-up.
Abstract: This paper presents a prototype of a platform for biomolecular recognition detection. The system is based on a magnetoresistive biochip that performs biorecognition assays by detecting magnetically tagged targets. All the electronic circuitry for addressing, driving and reading out signals from spin-valve or magnetic tunnel junctions sensors is implemented using off-the-shelf components. Taking advantage of digital signal processing techniques, the acquired signals are processed in real time and transmitted to a digital analyzer that enables the user to control and follow the experiment through a graphical user interface. The developed platform is portable and capable of operating autonomously for nearly eight hours. Experimental results show that the noise level of the described platform is one order of magnitude lower than the one presented by the previously used measurement set-up. Experimental results also show that this device is able to detect magnetic nanoparticles with a diameter of 250 nm at a concentration of about 40 fM. Finally, the biomolecular recognition detection capabilities of the platform are demonstrated by performing a hybridization assay using complementary and non-complementary probes and a magnetically tagged 20mer single stranded DNA target.

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"A bacteriophage detection tool for ..." refers background or methods in this paper

  • ...…of developing a “sandwich” phage-based biosensing system and its potential as a cell viability determination tool was assessed making use of an existent magnetoresistive (MR) biochip (Freitas et al., 2012; Martins et al., 2009, 2010) and respective electronic reader (Germano et al., 2009)....

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  • ...After functionalization, the MR-chip was introduced on the portable reading platform (Germano et al., 2009) developed at INESC-ID, and the microfluidic system sealed....

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TL;DR: This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization approach with a physiological assay for the direct enumeration of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration.
Abstract: Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 108 nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed.

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"A bacteriophage detection tool for ..." refers methods in this paper

  • ...Among various approaches, the direct viable count (DVC) method combined with nucleic acid staining (Baudart et al., 2002; Besnard et al., 2000), the measurement of respiratory activity (Winding et al., 1994) or other metabolic activities (Duncan et al., 1994; Nybroe, 1995), and the estimation of…...

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TL;DR: In this article, the magnetoresistive (MR) biochip concept has emerged a decade ago and since then considerable achievements were made in the field of bio-analytical assays.

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"A bacteriophage detection tool for ..." refers methods in this paper

  • ...…of developing a “sandwich” phage-based biosensing system and its potential as a cell viability determination tool was assessed making use of an existent magnetoresistive (MR) biochip (Freitas et al., 2012; Martins et al., 2009, 2010) and respective electronic reader (Germano et al., 2009)....

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  • ...A portable electronic platformwas used to acquire the data (Freitas et al., 2012; Martins et al., 2009, 2010)....

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Frequently Asked Questions (1)
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.