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A bacteriophage detection tool for viability assessment of Salmonella cells.

15 Feb 2014-Biosensors and Bioelectronics (Elsevier)-Vol. 52, pp 239-246

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.

AbstractSalmonellosis, one of the most common food and water-borne diseases, has a major global health and economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions and the potential to preserve virulence in dormant states when cells are viable but non-culturable (VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. 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. Free PVP-SE1 phages in solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor recognition of heat-killed cells. 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. The phage probe was then tested in a magnetoresistive biosensor platform allowing the quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity. Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods.

Topics: Bacteriophage (52%)

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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A bacteriophage detection tool for viability assessment
of Salmonella cells
E. Fernandes
a,b
, V.C. Martins
b,
n
, C. Nóbrega
c,d
, C.M. Carvalho
a,b
, F.A. Cardoso
e
, S. Cardoso
e
,
J. Dias
b
, D. Deng
b
, L.D. Kluskens
a
, P.P. Freitas
b,e
, J. Azeredo
a
a
IBBInstitute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho Campus de Gualtar, 4700-057 Braga, Portugal
b
International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga, 4715-330 Braga, Portugal
c
Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4700-057 Braga, Portugal
d
ICVS/3B's, PT Government Associated Laboratory, Braga/Guimarães, Portugal
e
INESC-MNInstituto de Engenharia de Sistemas e Computadores-Microsistemas e Nanotecnologias and INInstitute of Nanoscience and Nanotechnology,
PT Government Associated Laboratory, Rua Alves Redol 9, 1000-029 Lisbon, Portugal
article info
Article history:
Received 19 July 2013
Received in revised form
26 August 2013
Accepted 28 August 2013
Available online 7 September 2013
Keywords:
Bacteriophage
Salmonella
Cell viability
Viable but non-culturable bacteria
Magnetoresistive biochip
Magnetic nanoparticles
abstract
Salmonellosis, one of the most common food and water-borne diseases, has a major global health and
economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions
and the potential to preserve virulence in dormant states when cells are viable but non-culturable
(VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to
detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between
dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel
bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. Salmo-
nella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, ow-cytometry and
epiuorescence microscopy, and further assayed with a biosensor platform. Free PVP-SE1 phages in
solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor
recognition of heat-killed cells. This ability was conrmed 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. The phage probe was then tested in a magnetoresistive biosensor platform allowing the
quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity.
Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that
does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-
negative and false-positive results of ten associated with most detection methods.
& 2013 Elsevier B.V. All rights reserved.
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. To reduce the incidence and economic burden of
foodborne diseases, the World Health Organization (WHO) has
been enforcing the establishment of a surveillance program to
assure the safety of alimentary products along the food chain
from farm to fork (WHO, 2005). Such actions have stimulated
R&D activities seeking for new methods for microbial detection, in
particular bioanalytical technologies (Nugen and Baeumner, 2008;
Velusamy et al., 2010). In contrast to the actual culture-based
methods, biosensors have started to offer great advantages due to
their faster and more sensitive response (Boehm et al., 2007;
Ivnitski et al., 1999). However, they still suffer from a notorious
drawback: the false-negative results (i.e. the failure to detect a
virulent pathogen when present). The occurrence of false-
negatives is often attributed to technological limitations, such as
low sensitivity, matrix interferences and/or inhibitions. Also, many
bacteria are reported to enter in a dormant state where they can
be hardly distinguished from live and dead cells. Nevertheless,
they keep their virulence and the ability to resuscitate when in
favorable conditions (Oliver, 2005, 2010). In such a state, bacteria
will not grow in standard solid culture media, and as a result will
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/bios
Biosensors and Bioelectronics
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bios.2013.08.053
n
Corresponding author. Tel.: þ 351 253140112; fax: þ 351 253140119.
E-mail addresses: elisabete.fernandes@deb.uminho.pt (E. Fernandes),
veronica.romao@inl.int, veronicamartins@ist.utl.pt (V.C. Martins),
claudianobrega@ecsaude.uminho.pt (C. Nóbrega),
carlacarvalho@deb.uminho.pt (C.M. Carvalho), fcardoso@inesc-mn.pt
(F.A. Cardoso), scardoso@inesc-mn.pt (S. Cardoso), joaodias5@gmail.com (J. Dias),
dengd@mit.edu (D. Deng), kluskens@deb.uminho.pt (L.D. Kluskens),
paulo.freitas@inl.int (P.P. Freitas), jazeredo@deb.uminho.pt (J. Azeredo).
Biosensors and Bioelectronics 52 (2014) 239246

not be detected as colony forming units (CFU), the gold-standard
detection method. Dormant bacteria have therefore been called
viable but non-culturable (VBNC) cells. The VBNC physiological
state is reported for several pathogenic bacteria and occurs under
the inuence of different cellular stress conditions, in particular in
the presence of disinfectant agents (Khamisse et al., 2012). This
represents a major problem in food facilities because VBNC
bacteria may persist and contaminate food, regardless of the
disinfection treatments (Firmesse et al., 2012).
Most of the methods used for VBNC detection involve the use of
uorescent probes in the characterization of the cell physiological
activity (Breeuwer and Abee, 2000; Joux and Lebaron, 2000).
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 bacterial
membrane potential (Deere et al., 1995) or membrane integrity by
uorophores penetration (Caron, 1998) can be quoted. Although
most physiological probes allow evaluation at the single-cell level,
they are time-consuming and do not provide information on the
identity of the assayed cells.
DetectionmethodsbasedonDNAanalysis(e.g.PCRPolymerase
Chain Reaction) (Keer and Birch, 2003; Lu et al., 2009)orow
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. In PCR methods, either the reverse
transcriptase (RT)-PCR, which detects mRNA, a biomolecule that
has a half-life of about 35 min after cell death (Keer and Birch,
2003), or new DNA-intercalating dyes such as ethidium mono-
azide and propidium monoazide that block the amplication of
DNA from dead cells (Lu et al., 2009) are often used. A possible
drawback in these PCR-based methods is the effect that the length
of the PCR amplicon may have on the efciency of removal of the
dead cell signal (Banihashemi et al., 2012).
The same principle of actuation is the basis to other viability
indicator dyes, mostly uorescent molecules widely used in
epiuorescence microscopy and ow cytometry. Cell membrane
diffusion probes and DNA-intercalating dyes are used for tagging
cells with damaged (dead cells) or undamaged (viable cells)
membranes. For instance the commercially available kit LIVE/
DEAD
s
BacLight
from Molecular Probes, also used in this work,
offers a combination of two dyes: a green uorochrome (SYTO9)
able to enter all cells (used to assess total cell counts) and a red
uorochrome (propidium iodidePI) that selectively enters com-
promised cells. These methods can fairly easily distinguish
between viable and dead cells (Banihashemi et al., 2012; Weaver,
1997). However, since they rely on the membrane integrity as a
discrimination factor, intermediate states are generally misclassi-
ed or simply identied as an unknown and poorly character-
ized population. For this reason, in ow cytometry, the way to
circumvent this limitation is through a well-dened gating strat-
egy that is highly dependent on how accurate the positive and
negative controls are for the dened cell populations (e.g. viable,
compromised and dead).
In this work, a bacteriophage is used to discriminate between
VBNC cells and dead cells. Phages are viruses that infect only
bacteria, while being innocuous to humans. They have been
recently considered very interesting biorecognition elements and
biodetection tools due to their high specicity to bacteria, robust-
ness, great stability (even under adverse environmental condi-
tions) and extended shelf-life (Edgar et al., 2006; Santos et al.,
20 10). These characteristi cs, combined with their innocuous nature
andlowproductioncosts,ledtheFoodandDrugAdministration
(FDA)toapprovesomephage-baseddiagnostic protocols for patho-
gen detection (e.g. Mycobacterium tuberculosis, Yersinia pestis,
Bacillus anthracis, and Staphylococcus aureus; Schoeld et al.,
2012). Signicant 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). Studies on
the utilization of phages for the detection of VBNC bacteria are still
limited and have been applied only to E. coli O157:H7 (Awais et al.,
2006; Oda et al., 2004). Those studies either lack the capability to
directly discriminate VBNC from dead cells (Awais et al., 2006)or
do not even mention the ability to detect the VBNC physiological
state (Tlili et al., 2013).
In this work, a broad spectrum virulent phage (PVP-SE1) from
the Myoviridae family was used as a biorecognition element to
distinguish viable and VBNC cells from dead Salmonella Enteritidis
cells. After determining the best bactericidal and bacteriostatic
compound to induce the VBNC state to Salmonella cells, various
tests on the phage ability to discriminate the different Salmonella
cell physiological states (viable, VBNC and heat killed cells) were
made. A phage-based magnetoresistive biochip was developed
where phages are immobilized at surface probe sites, and magnetic
nanoparticles functionalized with anti-Salmonella specic antibodies
are used as labels. A portable electronic platform was used to acquire
the data (Freitas et al., 2012; Martins et al., 2009, 201 0).
2. Materials and methods
2.1. Media and buffers
Luria Bertani (LB) agar plates were prepared by adding either
1.2% or 0.6% of agar to the liquid LB medium to get standard agar
or soft agar medium, respectively. Phosphate buffer (PB; 100 mM
NaH
2
PO
4
, 100 mM Na
2
HPO
4
, pH 7.4); PB Tw20 (PB with 0.02% (v/v)
of Tween 20); SM buffer (100 mM NaCl, 8 mM MgSO
4
,50mM
TrisHCl, pH 7.5); MOPS buffer (100 mM 3-(N-morpholino) pro-
panesulfonic acid, pH 5.7); TE buffer (10 mM TrisHCl, 1 mM
EDTA, pH 7.4); and Bovine serum albumin (BSA), 1% (w/v) in water
were used, where all reagents were acquired from Sigma.
2.2. Bacteriophages and bacterial strains
PVP-SE1 was isolated from a Regensburg (Germany) waste-
water 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 techni-
que as described by Sambrook and Russell (2001) and resus-
pended 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
veried 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
h ypochl orite (commer cial bleachstock concentration 5%) at different
concentrations. A single colon y of Salmonella (S1 400) was inoculated
in 20 mL of LB broth and incubated overnight at 37 1C/200 rpm.
E. Fernandes et al. / Biosensors and Bioelectronics 52 (2014) 239246240

Following the pre-inoculation, 1 mL was transferred to 15 mL of
fresh LB broth and incubated for approximately 23h at 371C/
200 rpm until the optical density at 600 nm (OD
600
) reached 0.5
0.7 (concentration at 1.0 10
7
CFU/mL). Cells were washed and
resuspended in PB. From the stock solution, aliquots of 500
μ
Lof
bacteria were transferred to 1.5 mL microtubes and centrifuged
(4 1C, 2370g for 15 min), the supernatant was removed and 1 mL of
each of the following concentrations of bleach was added to
the bacterial pellet: 0.01000%, 0.00875%, 0.00750%, 0.00625%,
0.00500%, 0.00250%, and 0.00125% (v/v). The serial dilutions of
bleach were done with milli-Q water. Untreated cells were
incubated with milli-Q water only. 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. The number of culturable cells (after
bleach treatment) was determined based on colony counting and
expressed in colony forming units (CFU).
2.5. Determination of cell viability
Cell viability was assessed after submitting bacteria to different
bleach concentrations using the LIVE/DEAD
s
BacLight
Bacterial
Viability and Counting Kit (Molecular Probes). SYTO9 and PI dyes
were used, accor dingl y to manufacturer's instructions. Upon staining,
cells were analyzed either by epiuorescence micros cop y (OLYMPUS
BX51 EXTREMO microscope) or by ow cytometry (BD LSRII ow
cytometer using F A CS DIVA softw are for acquisition; BD Biosciences).
For absolute cell quantication, 6
μ
m diameter microspheres were
used at a known concentration in the ow cytometry acquisition.
Flow cytometry data was analyzed using the FlowJo software (Tree
Star,Ashland,OR).
2.6. Phage lysis time and adsorption studies
Salmonella cell suspensions were treated with bleach as pre-
viously described or heat-killed after 10 min at 70 1C in a thermo-
block for 1.5 mL microtubes. 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 af ter infection (time 0) and
after 20 min and 40 min of phage inoculation, followed by 10-fold
dilution in MOPS and centrifugation at 10,00 0g 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 concent ration at each t ime point by the initial phage
concentration.
To assess the phage lysis time viable exponential phase grown
Salmonella cells were used. The procedure adopted was identical
to the adsorption assays except that the samples were taken from
cell cultures infected with phages from 0 to 80 min (every 10 min),
immediately plated (without being centrifuged) and their concen-
tration determined by the double-layer agar plate method in LB
medium.
2.7. Phage immobilization on Au surfaces
Cr 5 nm/Au 40 nm thin lm layers were sputtered (Kenosistec
sputtering tool) over a silicon wafer. The wafer was then spin-
coated with a photoresist (PR) polymer (AZ1505 AZ Electronic
Materials) for surface protection and diced in 7 7mm
2
dies using
an automatic dicing saw (Disco, DAD3350). The PR protective layer
was removed prior to surface utilization by a dedicated solvent
(microstrip 3001, Fujilm, 65 1C for 2 h). Substrates were then
rinsed with isopropanol (IPA) and milli-Q water and dried under a
nitrogen stream. To further remove any PR residues and other
organic contaminants, the Cr/Au substrates were exposed to
ultraviolet light/ozone plasma (Novascan Technologies Inc.,
PSDP-UVT series, IA, USA) for 15 min at 50 1C. The gold surface
was then functionalized with a heterobifunctional linker, the
sulfo-LC-SPDP (sulfosuccinimidyl 6-[3-(2-pyridyldithio)-pr opiona-
mido] hexanoate). A droplet of 20 m L of sulfo-LC-SPDP at 1 mg/mL
in PB was placed over the gold substrate covering the entire
surface (1 h at RT). The substrates were then rinsed with PB and
milli-Q water and blow-dried with a nitrogen gun. A phage
solution in MOPS buffer ( 1 10
10
phages/mL) or a polyclonal
anti-Salmonella antibody (PA1-20811, Thermo Scientic) in PB
solution (200 mg/mL) was then spotted in discrete areas of the
substrate ( 0.5 mL spots) and allowed to immobilize for 2 h at RT.
The excess of phage was removed by rinsing with MOPS buffer and
a solution of BSA at 1% (w/v) in TE buffer (dispensed over the
whole surface and allowed to react for 1 h to block the free gold
areas). After rinsing the excess of BSA in PB, a Salmonella
Enteritidis sample was dispensed over the functionalized surface
and allowed to react for 40 min. The unbound cells were washed-
out by substrate dipping and rinsing in PB. The functionalization
protocol was performed at RT inside a humidied Petri dish to
prevent evaporation of the spotted solutions.
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 dedi-
cated microfabrication process (Martins et al., 2009) and wire-
bonded to a PCB chip-carrier . Wires were protected with silicone gel.
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. Brie y, the MR chip
architecture comprises two distinct sensing areas arranged in
two columns. Each column is composed of 3 groups of 5 U-
shaped 2.5 80
μ
m
2
spin-valve sensors. One of the sensors acts as
reference (no phage probe attached).
The MR-biochip functionalization follows the same protocol
previously described for Cr/Au substrates. A 1 mL droplet of
Salmonella-specic phage was spotted over the left column of
sensors (12 sensors) and a non-specic phage (Campylobacter
phage) on the right column of sensors (12 sensors). After functio-
nalization, the MR-chip was introduced on the portable reading
platform (Germano et al., 2009) developed at INESC-ID, and the
microuidic system sealed. The sensors were biased by a 1 mA
current while an external magnetic eld (3 mT DC bias eld and
1.35 mT
rms
AC eld at 211 Hz) was applied (these elds will be
used later to magnetize the MNPs labels when present). The
baseline signal was rst acquired for 10 min with PB inside the
microuidic channel (0.5 mL total volume). Test solutions with
Salmonella Enteritidis at 1.0 10
8
cells/mL were introduced
inside the channel with the aid of a syringe pump (New Era
NE-30 0), and allowed to settle down for 40 min (phage-Salmonella
recognition). The unbound bacteria were washed-out by rinsing
with a solution of PB Tw20 at a ow rate of 5 mL/min. The
antibody-conjugated MNPs (prepared as described below) were
then introduced inside the channel and allowed to settle for
30 min while signal acquisition was recorded for all sensors
sequentially (Salmonell a-antibo dyMNP recogn ition ). The unbou nd
MNPs were washed-out with PB Tw20 at a ow rate of 50 mL/min
for 5 min. The difference between the signal acquired after
washing and the baseline signal (
Δ
V
binding
) is proportional to the
number of cells bound to the sensor surface. For sensor to sensor
and chip to chip comparison purposes the
Δ
V
binding
signal was
E. Fernandes et al. / Biosensors and Bioelectronics 52 (2014) 239246 241

normalized to each sensor baseline signal (V
sensor
) at the measure-
ment conditions, as explained by Martins et al. (2009).
2.9. Antibody-conjugated MNPs preparation
Commercial 250 nm Protein A modied MNPs (Nanomag,
Micromod) were used. 1 mL of MNPs stock solution (4.9 10
11
particles/mL) was transferred to a microtube, placed in a magnetic
concentrator (Dynal-biotech) to remove the supernatant, and
washed twice in 100 mL of PB Tw20. Then, 1 mL of anti-Salmonella
polyclonal antibody (stock concentration at 1 mg/mL, PA1-20811,
Thermo Scientic) was added to the MNPs in a total volume of 5 mL
of PB Tw20 and allowed to react for 45 min at 200 rpm and RT.
An afnity reaction links the Protein A of the MNPs to the Fc region
of the antibody. The unbound antibody was removed by the same
magnetic separation procedure. The functionalized MNPs were
nally resuspended in 5 mL of PB Tw20 and injected over the chip.
2.10. Statistical analysis
All data are represented as mean7 SD (standard deviation). For
Figs. 2 and 3, means were compared using two-way ANOVA
followed by the Bonferroni post hoc test. Differences were con-
sidered signicant whenever po 0.05 and represented as
n
for
po 0.05,
nn
for po 0.001 and
nnn
for po 0.0001.
3. Results and discussion
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 rst
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 com-
pounds, known to induce the VBNC state in Salmonella cells, were
tested (data not shown). From those, sodium hypochlorite (com-
mercial bleach) was found to be the most efcient in producing
VBNC cells.
Eight different concentrations of bleach, ranging from 0% to
0.01% (v/v), were tested. As shown in Fig. 1A (linear curve), the
break-point, dened as the concentration at which 100% of the
cells lose their ability to form colonies in solid, non-selective
medium, was found to be 0.006%. For bleach concentrations below
the break-point, at least a fraction of the population is still viable
and able to grow in solid medium, while above this concentration
no colonies are observed. However, when exposed to fresh liquid
medium under adequate growth conditions all tested concentra-
tions of bleach, even above the break-point, showed cell growth
(Supplementary data, Fig. S2.1). This observation, complemented
with results from other analytical methods, such as uorescence
microscopy and ow cytometry (Fig. 1B and C), indicates that
bleach concentrations above 0.006% can induce Salmonella Enter-
itidis cells to enter a VBNC state.
In ow cytometry assays, as previously explained, using the
LIVE/DEAD BacLight kit, live bacteria with intact membranes are
supposed to appear green and dead bacteria with damaged
membranes to emit red. However, in bleach-treated cells, inter-
mediate colors, from yellow to orange, were also observed under
an epiuorescence microscope (Fig. 1B) indicating the existence of
other cell states where membranes present different degrees of
damage (compromised cells).
In order to quantitatively determine the relative and absolute
proportion of the different cell populations (classied as live, dead
or compromised), ow cytometry analyses were conducted for the
different cell samples (Fig. 1A bars and
1C). Untreated bacteria
appeared mostly as live cells (SYTO9þ ,PI ), while treated
samples, with increasing bleach concentrations, were either com-
promised or dead (Fig. 1A bars and
1C).
Additionally, to correlate ow cytometry data with the cell's
metabolic activity in each physiological state the PrestoBlue
TM
assay from Life Technologies was used. The compound resazurin is
effectively reduced (from blue to pink) only by enzymatic activity
in viable cells. Populations treated with up to 0.01% of bleach,
despite not being culturable, were still able to reduce the resazurin
reagent, while heat-killed cells presented no activity (Supplementary
data, Fig. S2.1).
The VBNC state has been associated to a survival mechanism of
the bacteria upon exposure to harsh but sub-lethal conditions
(Mizunoe et al., 1999). This represents a major problem to water
treatment factory plants and other industrial facilities that typi-
cally use 0.51% (v/v) bleach as a disinfectant in combination with
traditional cell cultures as an inspection method. These results
prompted us to study whether the Salmonella-specic phage PVP-
SE1 would be able to detect bacteria at the VBNC state.
3.2. Prole of phage adsorption to cells in different
physiological states
The PVP-SE1 is a virulent phage able to lyse bacterial cells. In a
solid state biosensing system this could be a limitation on the
recognition of live organisms. Such characteristic has refrained
researchers from including these phages in their detection systems
(Santos et al., 2010). In this work, the phage inoculation conditions
(i.e. buffer pH and ionic strength) were studied in order to avoid
Salmonella cells from lysing within the assay time frame. Fig. 2A
shows percentual phage adsorption to Salmonella Enteritidis vs
time in solution to assess both the lysis time or latent period and
adsorption rate. For the lysis time studies, the bacterial cells were
lysed and the total number of phages was quantied by the double
layer agar technique (Sambrook and Russell, 2001). The plaque
forming units (PFU) are directly related to the number of phages in
the sample (Fig. 2B and C). It was observed that no cell lysis
occurred up to 80 min after phage inoculation. This unusual large
period for cell infection and burst may be related to the fact that
the phage is not present in ideal infection conditions. For biosen-
sing purposes this observation opens novel opportunities to
explore the potential of lytic phages while circumventing their
greatest limitation (Carvalho et al., 2010b).
After optimization of non-lysing conditions, the phage ability to
recognize different cell physiological states (viable, VBNC and
dead) was evaluated. For this purpose VBNC cells were prepared
by treatment with bleach at 0.01% and dead cells prepared by
heating at 70 1C for 10 min (where no bacteria could be recovered
after resuscitation assays). Results conrmed 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. It was found that the phage could
efciently adsorb to both viable and VBNC Salmonella cell suspen-
sions. Furthermore, viable cells and VBNC cells were both
promptly recognized by the phage ( o 20 min) while dead cells
presented a lower adsorption rate. Although there is a slight
increase in the adsorption (19%) to dead cells with time of
inoculation (from 20 to 40 min), the adsorption to viable cells
(78%) is more efcient. This indicates that phages may have
biological mechanisms which link them preferably to viable cells.
This observation is expected to have a major impact in the existing
bioanalytical eld once phages can be used as a bioelement to
signicantly reduce the number of false-positive and false-
negative results presented by traditional molecular detection
systems.
E. Fernandes et al. / Biosensors and Bioelectronics 52 (2014) 239246242

3.3. Phage performance as a biorecognition element
The phage's validation as a biorecognition tool will be centered
on its immobilization on a solid sensing surface. 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. Viable, VBNC and dead cell suspensions were
incubated for 40 min with the immobilized phage. The degree of
phage's recognition was evaluated through a semi-quantitative
optical method to measure the cell's surface density over the
phage spot area. Fig. 3A shows relative surface density of captured
cell for varying populations of viableþVBNC cells (with increasing
number of dead cells). Though cell density results cannot be
directly compared in absolute terms with other quantitative data
(e.g. cytometry and MR-sensor) the observed results follow the
same trend as that shown in Fig. 1A (bars plot). 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 ow
cytometry analysis (Fig. 1Abars plot).
Also important to notice is that even after 40 min of incubation,
dead cells were completely undetected by the phage as opposed to
adsorption results for cells in solution (Fig. 2 triangles). A possible
explanation could be that phages bound weakly to dead cells are
easily released along the several washing steps performed in solid
surface experiments.
Fig. 1. Induction of VBNC physiological state in Salmonella. Assessment of the physiological state of Salmonella Enteritidis cells af ter treatment with different concentrations of
bleach by three different evaluation methods: (A) CFU counts on standard solid culture media (black line) compared to ow 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 10
8
cells/mL. Results presented for the ow cytometry analysis are the mean of
independent triplicates from three independent experiments (n¼ 9). (B) Epiuorescence 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 ow 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 ow 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 gure legend, the
reader is referred to the web version of this article.)
E. Fernandes et al. / Biosensors and Bioelectronics 52 (2014) 239246 243

Figures (4)
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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.

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References
More filters

Book
15 Jan 2001
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

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Book
01 Jan 2001
TL;DR: The content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

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

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Journal Article
TL;DR: The ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form is described, as well as the ability of these cells to retain virulence.
Abstract: It had long been assumed that a bacterial cell was dead when it was no longer able to grow on routine culture media. We now know that this assumption is simplistic, and that there are many situations where a cell loses culturability but remains viable and potentially able to regrow. This mini-review defines what the "viable but nonculturable" (VBNC) state is, and illustrates the methods that can be used to show that a bacterial cell is in this physiological state. The diverse environmental factors which induce this state, and the variety of bacteria which have been shown to enter into the VBNC state, are listed. In recent years, a great amount of research has revealed what occurs in cells as they enter and exist in this state, and these studies are also detailed. The ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form is described, as well as the ability of these cells to retain virulence. Finally, the question of why cells become nonculturable is addressed. It is hoped that this mini-review will encourage researchers to consider this survival state in their studies as an alternative to the conclusion that a lack of culturability indicates the cells they are examining are dead.

1,185 citations


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Journal ArticleDOI
TL;DR: The central role of catalase in the VBNC response of some bacteria, including its genetic regulation, is described and a variety of interesting chemical and biological factors have been shown to allow resuscitation, including extracellular resuscitation-promoting proteins, a novel quorum-sensing system and interactions with amoeba.
Abstract: Many bacteria, including a variety of important human pathogens, are known to respond to various environmental stresses by entry into a novel physiological state, where the cells remain viable, but are no longer culturable on standard laboratory media. On resuscitation from this ‘viable but nonculturable’ (VBNC) state, the cells regain culturability and the renewed ability to cause infection. It is likely that the VBNC state is a survival strategy, although several interesting alternative explanations have been suggested. This review describes the VBNC state, the various chemical and physical factors known to induce cells into this state, the cellular traits and gene expression exhibited by VBNC cells, their antibiotic resistance, retention of virulence and ability to attach and persist in the environment, and factors that have been found to allow resuscitation of VBNC cells. Along with simple reversal of the inducing stresses, a variety of interesting chemical and biological factors have been shown to allow resuscitation, including extracellular resuscitation-promoting proteins, a novel quorum-sensing system (AI-3) and interactions with amoeba. Finally, the central role of catalase in the VBNC response of some bacteria, including its genetic regulation, is described.

928 citations


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TL;DR: The conventional methods, analytical techniques and recent developments in food pathogen detection, identification and quantification, with an emphasis on biosensors are described.
Abstract: Food safety is a global health goal and the foodborne diseases take a major crisis on health. Therefore, detection of microbial pathogens in food is the solution to the prevention and recognition of problems related to health and safety. For this reason, a comprehensive literature survey has been carried out aiming to give an overview in the field of foodborne pathogen detection. Conventional and standard bacterial detection methods such as culture and colony counting methods, immunology-based methods and polymerase chain reaction based methods, may take up to several hours or even a few days to yield an answer. Obviously this is inadequate, and recently many researchers are focusing towards the progress of rapid methods. Although new technologies like biosensors show potential approaches, further research and development is essential before biosensors become a real and reliable choice. New bio-molecular techniques for food pathogen detection are being developed to improve the biosensor characteristics such as sensitivity and selectivity, also which is rapid, reliable, effective and suitable for in situ analysis. This paper not only offers an overview in the area of microbial pathogen detection but it also describes the conventional methods, analytical techniques and recent developments in food pathogen detection, identification and quantification, with an emphasis on biosensors.

908 citations


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  • ...Such actions have stimulated R&D activities seeking for new methods for microbial detection, in particular bioanalytical technologies (Nugen and Baeumner, 2008; Velusamy et al., 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.