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Assessment of bacterial viability: a comprehensive review on
recent advances and challenges
Shravanthi S. Kumar and Asit Ranjan Ghosh*
Graphical abstract
Representation of various methods for the determination of bacterial viability.
Abstract
Assessing bacterial contamination in environmental samples is critical in determining threats to public health. The classical
methods are time-consuming and only recognize species that grow easily on culture media. Viable but non-culturable (VBNC)
bacteria are a possible threat that may resuscitate and cause infections. Recent dye-based screening techniques employ
nucleic acid dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA), along with many fluorescent dyes,
which are an effective alternative for viability assessment. The measurement of cellular metabolism, heat flow and ATP
production has also been widely applied in detection approaches. In addition, RNA-based detection methods, including
nucleic acid sequence-based amplification (NASBA), have been applied for bacterial pathogen determination. Stable isotope
probing using
13
C,
15
N and
18
O, which are mobilized by microbes, can also be used for effective viability assessment. Future
detection tools, such as microarrays, BioNEMS and BioMEMS, which are currently being validated, might offer better
microbial viability detection.
Received 18 December 2018; Accepted 13 February 2019; Published 7 March 2019
Author affiliation: Department of Integrative Biology, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore-632014, Tamil
Nadu, India.
*Correspondence: Asit Ranjan Ghosh, asitranjanghosh@vit.ac.in
Keywords: Viability; VBNC; vPCR; MVT; Membrane integrity.
Abbreviations: ahpC, alkyl hydroperoxide reductase subunit C; AMV, avian myeloblastosis virus; DMSO, dimethyl sulfoxide; DOC, deoxycholate; DVC,
direct viable count; EMA, ethidium monoazide; GST, glutathione S- transferase; IMC, isothermal microcalorimeter; IMS, immunomagnetic separation;
LOD, limit of detection; MVT, molecular viability test; NASBA, nucleic acid sequence-based amplification; PI, propidium iodidE; PMA, propidium monoa-
zide; (p)ppGp, guanosine 3¢, 5¢-bispyrophosphatse; RpoS, RNA polymerase sigma S; SIP, stable isotope probing; VBNC, viable but non-culturable.
REVIEW
Kumar and Ghosh,Microbiology
DOI 10.1099/mic.0.000786
000786 ã2019 The Authors
1
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INTRODUCTION
Determining microbial viability is crucial in the field of
microbiology and yet the criteria for determining viability
remain ambiguous. The viability of microbes is key in deter-
mining the safety of food and drinking water and has severe
implications in the fields of environmental and medical
microbiology [1]. Bacterial pathogens are the most common
causes of infectious diseases worldwide. The common bacte-
rial infectious diseases, such as diarrhoea, cholera, dysentery
and pneumonia, are a major threat to many socio-economi-
cally disadvantaged countries, such as Asia–Pacific (APAC)
and African countries, and hence the detection and elimina-
tion of their causative agents or pathogens should be the
primary focus of research [2].
Growth and division is a widespread and accepted parame-
ter for the detection of bacterial viability. The culturabilty of
bacteria is generally ascertained by determining their ability
to propagate in liquid nutrient medium or in solid culture
medium, with them appearing as visible colonies. Culture-
based methods have proven to be efficient and cost-effective
over many years. However, assessing the viability of meso-
philic heterotropic bacteria by agar plate methods requires
overnight incubation and furthermore this technique does
not provide any information about bacterial physiology.
Intact cell membrane, metabolic activity and reproducibility
are the three accepted general parameters for the assessment
of the viability of microbes [3]. Culture-based methods only
account for one of these parameters, i.e. reproducibility.
Absence of growth in culture-based methods does not
always mean non-viability, it may also be interpreted as: (i)
incorrect culture medium; (ii) stress or damage to cells lead-
ing to a dormant state or injured state; (iii) low population
density, due to which there has been no observable growth;
and (iv) slow-growing cells and hence no visible growth [4].
However, many bacterial species have been found to exist in
the viable but non-culturable (VBNC) state. The VBNC
state is characterized by the loss of culturability on routine
agar media and thus reduces the chance of detection by the
conventional plate count technique. This leads to the under-
estimation of total viable cells in environmental and clinical
samples and in turn may prove to be a threat to public
health. Pathogens in the VBNC state are known to retain
virulence and can be resuscitated to initiate infection under
favourable conditions [5]. A number of foodborne out-
breaks have been associated with the resuscitation of patho-
gens that were present in the VBNC state [6, 7]. Many
bacterial human pathogens known to cause disease have
been proven to exist in the VBNC state, with each species
having different induction conditions, resuscitation factors
and mechanisms [8]. Hence the detection of pathogens in
the VBNC state is crucial for the prevention of outbreaks. A
proper detection system is still required to eliminate or
reduce this gap in pathogen detection, and this should be
more efficient and practicable than the traditional system
that has been followed until now.
This article discusses the properties associated with the
VBNC state of bacteria; its induction and resuscitation con-
ditions; and the factors responsible for maintaining it. The
article further assesses alternative methods to overcome the
drawbacks of culture-based methods in determining bacte-
rial viability and discusses the possible application of these
methods in different environmental conditions. The advan-
ces and challenges associated with different molecular meth-
ods, including dye-based as well as cellular property-based
methods, are discussed to aid in the selection of the most
suitable technique.
EVALUATION OF THE CULTURE-DEPENDENT
TECHNIQUE
Culture-based detection of microbial contamination
involves a pre-enrichment step wherein the sample is inocu-
lated in a non-selective medium. This helps in the prolifera-
tion of pathogens that are present in low numbers, stressed
or injured, and improves detection [9]. Further inoculation
in several selective media allows the growth of the target
organism while suppressing others, and this is followed by
enumeration and biochemical screening. Culture-based
detection cannot be applied to all bacterial species, as in the
case of spirochaete, Treponema pallidum, the causative
agent of syphilis, or Mycobacterium leprae, the pathogen for
leprosy, which has remained unculturable to date and is
undetectable by culture-based methods in vitro and is there-
fore propagated through rabbits and armadillos for research
[10]. Furthermore, many bacterial species may not grow in
culture media when (i) growth requirements and incubation
conditions are not fulfilled by artificial media; (ii) competi-
tion persists for nutrients among many bacterial species in a
mixed culture, inhibiting growth; (iii) the presence of bacte-
riocin from other bacteria in mixed culture leads to inhibi-
tion of growth of the target species; and (iv) signalling
molecules and beneficial interactions essential for growth of
target species are absent [11]. Such bacterial species are
termed unculturable and cannot be detected by culture-
based methods. Culture-based methods operate through
trial and error, and species are ruled out one after the other
based on their biochemical and physiological properties
until a final answer is reached [12]. Interpreting the results
of culture-based methods requires technical skills, especially
when there are non-conforming results or there is a new
strain with non-conforming properties. Ascertaining the
presence of a pathogen in samples takes many days using
culture-dependent methods; for example, it takes 7–10 days
to obtain a positive detection result for Campylobacter spe-
cies [13].
Viable but non-culturable: a limitation of the
culture-dependent technique
Microbes that fail to cultivate on routine media on which
they normally grow and yet are still alive are said to be in
the VBNC state [14]. The VBNC state was first reported by
Xu et al. [15]. Many species of bacteria when under stress
may enter a dormant state to survive and do not grow on
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culture media but retain metabolic activity and may return
to their virulent state in the presence of nutrition or after
the removal of stress. The factors that induce VBNC are
starvation [16], oxidative stress [17], oxygen limitation [18],
osmotic pressure, chemicals/disinfectants, low pH, tempera-
ture fluctuations, pulsed electric field and aerosolization
[19]. Bacteria in the VBNC state may be assumed to be
non-viable by standard culture methods, but differ vastly
from dead bacteria. VBNC bacteria deviate from conven-
tional microbial growth characteristics and hence cannot be
detected by traditional plating methods. VBNC cells have
an intact cell membrane and maintain the membrane
potential, unlike dead cells, whose membranes have disinte-
grated [20]. There is a constant debate as to whether the
VBNC state is just cells adapting to non-favourable condi-
tions or in fact cells in the state preceding death [21].The
VBNC cells of different species have been extensively stud-
ied and all the results suggest that the characteristics of
VBNC cells are vastly different from those of dead cells
(Table 1). Further, studies have shown that VBNC cells can
be resuscitated under favourable conditions [22]. Tradi-
tional culture methods are not favourable for the determina-
tion of VBNC cells, hence alternative methods to detect
VBNC cells are very important for the detection of contami-
nation in various environmental conditions.
Induction of VBNC cells
VBNC cells that are pathogenic and known to cause human
diseases pose a major threat as they can be avirulent and
undetected by conventional diagnostic methods and
subsequently resuscitate and cause infections. Therefore
understanding the mechanisms involved in the induction of
the VBNC state in bacteria as well as subsequent resuscita-
tion is vital for the prevention of bacterial infections. Bacter-
ia from diverse species and different environmental
conditions have been known to enter the VBNC state under
various stressors, which suggests that this state is a wide-
spread mechanism adopted by bacteria to overcome unfav-
ourable conditions [23]. Gene expression during stressful
conditions and during the VBNC state can help us to deter-
mine which genes influence the induction of the VBNC
state. RpoS, a stress regulator, is one such gene which was
found to regulate the VBNC state [23].
RpoS is a sigma factor that is responsible for the bacterial
survival in the stationary phase as well as in stressful condi-
tions [19]. RpoS controls the expression of a group of spe-
cific genes by interaction with RNA polymerase (RNAP)
and is known to regulate 10 % of the Escherichia coli genome
[24]. RpoS levels are low in the growth conditions of bacte-
rial cells, and as the cells enter the stationary phase or stress-
ful conditions there is an increase in RpoS levels, which
induces an RpoS response [24]. Many regulatory pathways
have been studied that are known to signal the RpoS
response, among which (p)ppGpp (guanosine 3´, 5´- bis-
pyrophosphate) has been found to be indicative [25]. With
an increase in (p)ppGpp there is increase in rpoS expression
and a reduction in RpoS degradation, as well as an increase
in RpoS activity [23]. RelA, a monofunctional alarmone
synthase, and SpoT, a bifunctional synthase as well as
Table 1. Characteristics of VBNC cells
Serial
no.
Traits Description Reference
1 Cell
morphology
VBNC cells have intact cell membranes and have high membrane potential. Dwarfing of cells or miniaturisation; rod-
shaped cells become coccus. Peptidoglycan become more cross-linked, lipoprotein content is enriched and glycan
strands reduce in length, which may lead to changes to the shape of the cell
[20, 155–
158]
2 Energy usage Total cellular content of carbohydrate and lipid and polyhydroxybutrate is reduced, which suggest their use as energy
sources
[159]
3 Translation Continued uptake of amino acids and incorporation into proteins [160]
4 Cellular ATP ATP levels remain higher than those of normal cells [161, 162]
5 Respiration Reduced respiration rate and metabolic rate [161, 163]
6 Genomic
integrity
Chromosomal DNA integrity is retained even after the loss of culturability [164]
7 Gene
expression
Gene expression profiles are different compared to those of culturable cells. A study of V. cholera VBNC cells showed
upregulation of genes related to regulatory functions, cellular processes, energy metabolism, transport and binding, and
downregulation of genes related to protein synthesis and stress response
[23, 165–
167]
8 Virulence Most species have been reported to be avirulent in the VBNC state, but they express virulence when resuscitated under
favourable conditions. Listeria monocytogenes,Vibrio harveyi,Vibrio vulnificus,Vibrio alginolyticus 1 and Vibrio
parahaemolyticus 66 have been reported to express virulence after resuscitation under favourable conditions
[39, 168–
170]
9 Stress
tolerance
VBNC cells are tolerant to stress, such as antibiotics, heavy metals, high temperature, high salinity, ethanol and acid. This
may be because of the lower metabolic rate and increased cross-linking of peptidoglycan. VBNC cells have been shown
to be more resistant to sonication [171], high temperature [172], low salinity, low pH, ethanol, chlorine and antibiotics
than cells in growing in the exponential phase [173]
[14, 174–
176]
10 Resuscitation VBNC cells resuscitate rapidly in host cells [169, 170,
177]
11 Adhesion The adhesion property changes in VBNC cells; some species, such as C. jejuni, retain their adhesion property, but species
such as V. cholera show reduced adhesion properties, and
E. faecalis is unable to attach and form biofilm
[178–180]
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hydrolase, regulate the (p)ppGpp in beta and gamma pro-
teobacteria [26]. Regulation of (p)ppGpp regulates RpoS
and accumulation of (p)ppGpp leads to enhanced stress
resistance in VBNC cells [23].
OxyR is a transcriptional regulator that is known to regulate
oxidative stress-related genes and was first discovered in
Salmonella typhimurium by Christman et al. [27]. E. coli in
the VBNC state has been shown to have decreased superox-
ide dismutase (SOD) activity leading to oxidative damage,
which suggests that the oxidative stress response is involved
in the induction and maintenance of the VBNC state [28].
Antioxidative enzymes such as alkyl hydroperoxide reduc-
tase subunit C (ahp C) and glutathione S-transferase (GST)
have been shown to influence the induction of the VBNC
state in Vibrio species [29, 30]. In turn, OxyR regulates ahp
C in response to increased ROS levels [31] and GST in
response to organic peroxides [32], hence regulating the
induction and maintenance of the VBNC state [23].
Resuscitation of VBNC cells
The reversal of the changes leading to the formation of
VBNC cells through the removal of stressors is termed
resuscitation [33]. The resuscitation of non-culturable bac-
teria was first reported in Salmonella enteritidis after supple-
mentation with heart infusion broth [34]. Studying the
resuscitation properties is tedious, as it is difficult to differ-
entiate between cells that have resuscitated from the VBNC
state and residual normal cells that are under the limit of
detection [35].
Resuscitation has been proven in many human pathogens
under different conditions, e.g. Salmonella enteritidis after
incubation with catalase in M9 minimal media [36] and
Campylobacter jejuni in embryonated chicken eggs [23].
The conditions for resuscitation are different for each bac-
teria and the removal of stress induction alone may not lead
to resuscitation. VBNC cells of haemolytic E. coli were able
to resuscitate in the presence of amino acids but VBNC cells
of E. coli O157:H7 could not be resuscitated under the same
conditions, which suggests that resuscitation conditions dif-
fer among strains of same species [37]. Temperature
increase, sodium pyruvate [5], amino acids [37] and Tween
20 [38] have been reported to mediate the resuscitation of
VBNC cells. Resuscitated bacterial cells of Listeria monocy-
togenes [39] and Salmonella typhi [33] have been shown to
exhibit virulence and cause infections and death in mice.
CULTURE-INDEPENDENT TECHNIQUES
Fluorescent dyes
Cell viability can be detected by using the characteristics of
viable cells, such as enzyme activity, which may result in
substrate uptake and cleavage, cell energy and integrity of
the cell membrane [40]. These properties of viable cells can
be detected using a microscope or flow cytometry with the
help of fluorescent dyes. Some of the dyes used in viability
detection are listed in Table 2.
Membrane integrity using nucleic acid dyes
Membrane integrity is an accepted biomarker for viable
cells, as cells with compromised membrane are approaching
death or already dead [41]. There are numerous ways to
detect the membrane integrity of cells and in turn the viabil-
ity of cells. The structure of the cell membrane and its com-
position vary widely among bacteria, hence analysis of
viability using membrane integrity as a biomarker is chal-
lenging in mixed cultures or in environmental samples [40].
Polar stains do not permeate into viable cells with intact
membranes but penetrate into dead cells with compromised
membranes [42]. There are two types of dye that can be
used to determine membrane integrity. (i) Dyes that can
permeate into intact and compromised cells, such SYTO 9,
Hoechst 33 342 and acridine orange. (ii) Dyes that can only
permeate compromised cells, such as propidium iodide,
ethidium homodimer, SYTOX Blue, SYTOX Green, YOYO-
1, TOTO-1 and TOPRO3 (77). Cell-permeant dyes bind to
the DNA of live cells, while cell-impermeant dyes bind to
the DNA of dead cells. Dual-staining kits such as the LIVE/
DEAD BacLight Bacterial Viability kit use two fluorophores,
SYTO9 and propidium iodide (PI). The distinction between
live and dead is based on membrane integrity, as PI, a red
fluorescent nucleic acid stain, only enters cells with compro-
mised membranes and SYOT 9 enters all cells and binds to
DNA. SYTO 9 is displaced by PI as PI has a stronger affinity
constant (3.710
5
M) towards nucleic acid than SYTO9
(1.810
5
M) [42].
Membrane potential
There is an electric potential across the bacterial membrane
that is called the membrane potential; this is due to the K+,
Na+, Clgradient that is maintained for active transport in
viable bacteria. The increase of the membrane potential is
termed hyperpolarization and the decrease is termed depo-
larization. In the event of cell membrane rupture due to
stress, the membrane potential reduces to zero and there is
a free flow of inorganic ions across the membrane [43]. The
membrane potential maintains proton motive force, which
in turn plays a role in ATP generation, chemotaxis and
other important cellular mechanisms [44]. Carbocyanine
dyes such as 3,30-dihexyloxacarbocyanine iodide [DiOC6
(3)] and rhodamine (rh123) are cationic lipophilic dyes that
bind to the inner membrane of bacteria that are negatively
charged, whereas oxonol dyes such as bis-(1,3-dibutylbarbi-
turic acid) and trimethine oxonol [DiBAC4(3)], which are
lipophilic and anionic cannot enter cells with a membrane
potential and can only be accumulated in dead cells whose
membrane potential has decreased (depolarization) [45]. A
combination of these dyes can be used in flow cytometry to
determine viable and dead cells.
Esterase substrates
Esterase substrates diffuse into cells because of their neutral
charge and are converted into fluorescent end products by
the action of intracellular esterases. Therefore viable cells
with the ability to produce esterase can convert the susbtrate
into fluorescence-emitting products, whereas in dead cells it
remains unhydrolyzed and inactive. [46]. This method
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Table 2. Dyes used to determine viability and their mechanism of action
Name Structure Mechanism of action Reference
Cyanine
dyes
Cyanine dyes belong to polymethine groups and
are non-fluorescent dyes that show fluorescence
after binding to nucleic acids. They are
impermeant to living cells and stain cells with
compromised membrane and dead cells
[181, 182]
Calcein Calcein AM (acetoxymethyl), which is non-
fluorescent, converts to a fluorescent calcein due
to hydrolysis by esterases present inside cells. It
is a cell-permeant dye and stains live cells but is
not retained in dead or compromised cells
[183]
Fluorescein
diacetate
FDA is a cell-permeant dye that is converted into
non-fluorescent fluorescein by the action of
intracellular hydrolysis by cells. This helps to
determine enzymatic activity as well as
membrane integrity during viability detection
[48]
Rhodamine
dyes
Rhodamine is a cationic lipophilic dye and is cell-
permeable and taken up by cells with active
transmembranal potential. Rhodamine 123 and
rhodamine B are the commonly used rhodamine
dyes in viability studies
[184]
Propidium
Iodide
PI is a red fluorescent stain that binds with DNA
and its fluorescence is enhanced after binding.
PI is used to stain dead or compromised cells as
it only enters cells with compromised
membranes and hence cannot be taken up by
live cells. PI is generally used in combination
with another dye that stains viable cells for dual
staining
[42]
Ethidium
monoazide
EMA is a DNA intercalating agent that binds to
the DNA of compromised or dead cells and
upon photolysis inhibits PCR amplification of
DNA from dead or compromised cells. It is used
in combination with PCR-based methods to
amplify the DNA of live cells in a technique
known as viability PCR
[185]
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indicates enzymatic activity and the membrane integrity of
cells. Calcein, fluorescein diacetate and carboxyfluorescein
diacetate are some of the commercially available esterase
susbstrates used in viability studies. Esterase substrates such
as calcein AM (an acetoxymethyl derivative of calcein) con-
verts to a fluorescent end product in the event of acetoxy-
methyl ester hydrolysis by intracellular esterases [47].
Similarly, fluorescein diacetate and carboxyfluorescein diac-
etate also convert to fluorescein in the presence of intracel-
lular esterases and are retained within the cell and are
visualized by flow cytometry [48].
Viability PCR
PCR-based methods are ideal for the detection and quantifi-
cation of specific micro-organisms in food, clinical samples
and environmental samples. PCR-based methods are rapid,
sensitive and robust, but the inability to differentiate
between viable and non-viable microbes leads to the overes-
timation of or false positives for the microbe of interest in
samples. [49]. Combining PCR with DNA intercalating
dyes has been proven to be efficient in determining viability
and has revolutionized microbial detection by overcoming
the drawbacks of PCR methods alone. The DNA intercalat-
ing dyes only penetrate bacterial cells that have compro-
mised cell membrane and interfere with the DNA
amplification of non-viable cells during PCR, hence viability
PCR (vPCR) determines the viability of cells based on mem-
brane integrity [Fig. 1] [50]. vPCR was introduced with
ethidium monoazide (EMA) being used as the DNA inter-
calating dye by Nogva et al. [51]. A second-generation alter-
native DNA intercalating dye, propidium monoazide,
(PMA) was used with PCR to detect viability and its efficacy
was proven by Nocker et al. [52].
The cell membrane is a barrier to DNA intercalating dyes in
viable cells, whereas they can enter membrane-compro-
mised non-viable cells. DNA intercalating dyes possess an
azido group that converts to a reactive nitrene radical on
photolysis. The nitrene group cross-links with the DNA of
the membrane-compromised cells, inducing structural
change in the nucleotide angle, and because of this the DNA
polymerase does not bind to the DNA, resulting in there
being no elongation, which leads to signal reduction [53].
Furthermore, the DNA cross-linkage with the nitrene group
causes it to be insoluble in water and it is eliminated in the
DNA extraction steps as a part of the cell debris [54]. The
remaining unbound intercalating dye is inactivated by reac-
tion with water molecules, forming hydroxylamine, and is
unable to further bind to DNA [52]. Consequently the via-
ble cells, which do not allow intercalating dye to penetrate
because of their intact membranes, are unaffected by inacti-
vated dye after cell lysis during the DNA extraction process
[55].
The use of EMA has been shown to lead to the loss of DNA
from live cells as well during the DNA extraction process.
Ethidium bromide, which is structurally similar to EMA, is
taken up by viable cells [50]. EMA having a single positive
charge is considered to be the reason for its easy penetration
into all cells [41]. EMA can permeate and causes loss of
DNA in the live cells of bacterial species such as Anoxybacil-
lus flavithermus,Bacillus licheniformis and Geobacillus stear-
othermophilus, which are common milk powder
Table 2. cont.
Name Structure Mechanism of action Reference
Propidium
monoazide
PMA similar to EMA is also a DNA-binding
photoreactive dye that suppresses the
amplification of DNA from dead cells during
PCR amplification
[185]
Tetrazolium
salts
Tetrazolium salts are heterocyclic compounds that
can permeate into intact viable cells and are
converted into water-soluble formazan, which is
coloured and can be quantified. Tetrazolium
salts are reduced within the cell by the
oxidoreductases and dehydrogenases present in
viable cells
[186]
Resazurin Resazurin is a redox indicator and a blue non-
fluorescent dye. Resazurin is reduced to
fluorescent resorufin, which is pink, by the
oxidoreductases present in viable cells and
hence is used as a vital dye
[187]
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contaminants [56]. EMA is also cytotoxic towards the viable
cells of bacterial species such as Staphylococcus aureus,
L. monocytogenes,Micrococcus luteus,Mycobacterium
avium and Streptococcus sobrinus [55]. However, Pseudo-
monas syringae species have shown EMA resistance and
stain moderately with EMA, leading to DNA loss during
extraction. The varying ability of EMA to permeate through
the bacterial membranes of different species limits its appli-
cation in viability determination [55].
PMA, analogously to propidium iodide (PI), with an azide
group added to phenanthridine ring and with a double posi-
tive charge, is considered to be a second-generation DNA
intercalating dye. Like propidium iodide, PMA does not
enter live cells and is more specific in differentiating viable
and non-viable cells [57]. PMA, with a double positive
charge, permeates less and does not permeate into slightly
compromised cells and requires a higher concentration to
attain the same result as EMA, as observed in species such
as Legionella pneumophilia [58]. Some of the amplification
techniques used after PMA/EMA treatment are listed in
Table 3.
RNA-based methods
Nucleic acid sequence-based amplification (NASBA)
NASBA is a continuous self-sustained nucleic acid amplifi-
cation method that was developed in 1991 by J. Compton
[59]. NASBA has been applied to study bacterial viability to
determine the antibiotic sensitivity of mycobacterial species
[60, 61]. NASBA is an RNA amplification process that takes
place in isothermal conditions and without any equipment
[59]. The short life of mRNA makes it a more appropriate
target to represent the living population of microbes in a
sample than DNA. Avian myeloblastosis virus (AMV)
reverse transcriptase, RNaseH and T7 RNA polymerase are
the enzymes used to simultaneously amplify RNA targets,
resulting in 10
9
-fold amplification [62]. The process
involves the use of two primers, one of which is comple-
mentary to the target RNA sequence, while the other
behaves as a recognition sequence for T7 RNA polymerase.
The reaction mixture also contains dNTPs and NTPs. The
RNA target is amplified by the AMV reverse transcriptase
enzyme with the help of a primer targeting the RNA
sequence to form cDNA [63]. The RNaseH digests the
remaining RNA and the second primer binds to the cDNA
and converts it into double-stranded DNA using AMV
reverse transcriptase, which acts as a transcriptionally active
promoter [64]. T7 RNA polymerase then transcribes many
copies of target RNA to form dsDNA and the cyclic process
continues [59]. The process is carried out at 41 C as the
DNA remains stable and does not denature, which results in
the amplification of DNA instead of target RNA [Fig. 2]
[63]. DNA does not interfere in the cycle and there is no
requirement to use an RNA extraction procedure or DNase
to reduce DNA contamination, as in the case of RT-PCR
[1]. Many studies have used NASBA to determine the via-
bility of pathogens, such as Haemophilus influenzae,Neisse-
ria meningitidis and Streptococcus pneumoniae, which are
associated with bacterial meningitis [65], V. cholerae in
water sources [66], E. coli in drinking water [67] and Salmo-
nella spp. in food samples [68]. Recent studies have
explored the use of NASBA as a point-of-care (POC) system
for the rapid and easy determination of viability to measure
antimicrobial resistance, as it eliminates the need for a ther-
mal cycler [69]. However, RNA stability may be compro-
mised during sample collection and storage. This drawback
can be overcome by adding RNase inhibitors such as guani-
dine thiocyanate to ensure the preservation of RNA integ-
rity [70].
Molecular viability test (MVT)
Due to its shorter average half-life, mRNA has been used to
detect bacterial metabolic responses and viability [71].
Fig. 1. Mechanism involved in vPCR when using the DNA intercalating dyes PMA/EMA. The dye enters compromised/dead cells, binds
covalently to the DNA upon phtoactivation and stops the amplification of DNA from dead cells.
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However, because of its short life span and unstable nature
it is a challenge to use mRNA as a marker to determine via-
bility. rRNA has a half-life of days, which is much longer
than mRNA and accounts for 90 % of the total cellular
RNA, hence it allows better viability determination in
microbes [72–74]. However, rRNA has been reported to
persist in dead bacterial cells [75, 76]. rRNA precursors
called pre-rRNA have been been reported as a target to
detect viability in several studies [75–77]. Mature rRNA is
formed by enzymatic removal of leader and tail fragments
of pre-rRNA [57] and makes up more than 25 % of the total
rRNA of the cell in growing bacteria [78]. Pre-rRNA is syn-
thesized in bacteria immediately in response to nutritional
stimulus and this synthesis stops when growth slows and
the cells become non-viable [79]. This makes pre-rRNA the
ideal target for viability detection using RT-PCR [Fig. 3].
The sensitivity of MVT is high due to the release many cop-
ies of pre-rRNA after cell lysis [57]. Thus MVT uses the
ability to synthesize macromolecule in the presence of nutri-
tion [80]. Cells that can catalyze the process of RNA synthe-
sis and also possess membrane integrity can respond to the
nutritional stimulus by producing pre-rRNA [57]. MVT has
been used to detect the pre-rRNA upshift in viable bacteria
in different environments, such as water sources [75],
human serum [76], and milk [80]. MVT has also been
proven to be effective in determining the viable cells of bac-
terial species such as E. coli,A. hydrophila,Enterococcus fae-
calis [77], Acinetobacter baumannii,Pseudomonas
aeruginosa,S. aureus and Mycobacterium tuberculosis [76].
However, MVT cannot be applied to viruses due to their
lack of ribosomes.
Cellular and metabolic properties
Adenosine triphosphate (ATP)
ATP is the energy currency in all living organisms and so is
an excellent biomarker for viability microbes, as ATP syn-
thesis is arrested immediately after cell death [81]. ATP
concentration within a cell varies depending on growth con-
ditions and under standard growth conditions intracellular
ATP concentration can be compared to the cell concentra-
tion in suspension. Luciferin–luciferase, which is responsi-
ble for bioluminescence in fireflies, influenced the
development of the ATP detection system [82].
D luciferin+O
2
+ATP+Mg
+2
fi
firefly luciferase
oxyluciferin
+AMP+ PPi+light.
The light intensity correlates directly with the ATP concen-
tration and can therefore be interpreted as the presence of
viable microbes. ATP has been used as a biomarker to detect
microbial contamination in various environments, such as
clean room facilities [83], simulated Martian conditions
[84], hospital water sources [85], hospital surfaces and
instruments [86, 87], aquatic environments [88] and soil
[89].
Exogenous ATP is also measured in a cell suspension using
this method, but this can overestimate the viable count and
to overcome this drawback the removal of extracellular ATP
enzymatically is necessary. Extracellular ATP can be
removed by (i) filtration of the cell suspension, or (ii) enzy-
matic hydrolysis of extacellular ATP using enzymes such as
apyrase, which is isolated from potato tubers and converts
ATP to ADP, which does not act as a substrate for lucifer-
ase. Apyrase does not enter into the cell membrane, and
intracellular ATP is not degraded and can be estimated by
cell disruption to release intracellular ATP [90]. After the
Table 3. Different amplification techniques used after PMA/EMA treatment
Serial
no.
Technique used Organism detected Sample studied Reference
1 qPCR S. enteritidis
L. pneumophila, Salmonella typhimurium
NA* [188]
[185]
2 Multiplex PCR Aeromonas, C. jejuni, C. coli, Salmonella, Shigella, enteroinvasive E. coli (EIEC),
Vibrio,Yersinia,L. pneumophila, S. typhimurium and S. aureus
Faecal sample and
environmental water
sample
[189]
[190]
3 Microarray Different molecular operational taxonomic unit (MOTU) corresponding to
different phyla detected
Clean room facilities
housing spacecraft
hardware
[191]
4 DGGE Legionella species Aquatic environment [192]
5 454 pyrosequencing Cyanobacteria and Cryomorphaceae Sea and canal water [193]
6 Ion torrent sequencing Microbial community analysis Water sample [194]
7 Loop-mediated
isothermal
amplification
Salmonella strains NA* [195]
8 Metagenomic library
construction
Eukaryotes, prokaryotes and viruses Cleanroom environment [196]
*Not available.
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removal of exogenous ATP, the cell is lysed and intracellular
ATP is measured by bioluminescence assay [85]. Non-
microbial sources of ATP also interfere with ATP measure-
ment and may lead to false-positive results.
Respiration
Using tetrazolium salts to detect respiration
The dehydrogenase enzyme present in the electron trans-
port system is used as a measure of respiratory activity of
microbial cells. Tetrazolium salts are reduced by the dehy-
drogenase enzyme and converted into formazan, which is a
measure of the total respiratory activity of the cell [Fig. 4].
The quantity of formazan produced is the measure of respi-
ratory activity and is measured colorimetrically. 2,3,5-tri-
phenyl tetrazolium chloride (TTC) [91], 2-(4-iodophenyl)
3-(4-nitro-phenyl)5-phenyl tetrazolium chloride (INT)
[92], 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) [93]
and 3¢-[1-(phenylamino)-carbonyl]3,4- tetrazolium]-bis
(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT),
[94] are some of the tetrazolium salts in wide use for the
measurement of microbial viability.
Resazurin
Resazurin is an oxidation reduction indicator that is purple
in colour and turns pink due to cellular oxidoreductases
[Fig. 4]. Resazurin is itself a weakly fluorescent dye that is
reduced to a highly fluorescent resorufin. It is an oxidation
reduction indicator for aerobic as well as anaerobic respira-
tion. Resaruzin was first reported for the detection of con-
tamination of milk by Pesch and Simmert in 1929 [95].
Resazurin has been used to determine microbial viability for
the determination of the minimum inhibitory concentration
(MIC) of antibiotics [96], antimicrobial susceptibility testing
[97] and drug-resistant bacterial detection [98]. Recent
advances include resazurin-amplified picoarray detection to
quantify E. coli and S. aureus. Using this technique, single
bacteria can be detected by fluorescence using resazurin to
determine the microcolonies formed by single colonies
entrapped in picochambers [99].
Direct viable count (DVC)
The DVC method was first reported by Kogure et al. in con-
text of the quantification of viable bacteria in the marine
environment through incubating samples with nalidixic
acid (antimicrobial agent) and yeast extract (nutrient
source) [100]. Nalidixic acid inhibits DNA synthesis and
the cells continue to use the nutrients to become elongated/
fattened.[101]. These cells are easily visualized by the micro-
scopic method and fluorescent microscopy. However, when
DVC is applied to enumerate mixed microbial communi-
ties, some bacteria may be resistant to the antimicrobial
used. To overcome this drawback, an antibiotic cocktail is
used and its effectiveness has been proven [102]. Another
drawback is the difficulty of differentiating between elon-
gated and fattened cells and cells that have not elongated.
The fattened and elongated cells could be missed during the
count, as they may be smaller than the average population
size in a mixed microbial community. To address this draw-
back, glycine was used to induce spheroplast in viable cells
and this was named the quantitative DVC (qDVC) proce-
dure [101].
Glycine inhibits bacterial growth by disrupting peptidogly-
can synthesis, leading to loose cell walls. The viable cells
Fig. 2. Schematic representation of the process involved in NASBA using the enzymes T7 RNA polymerase, AMV reverse transcriptase
and Rnase H.
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become swollen with loose cell walls, forming unstable
spheroplasts, and are easily lysed by freeze/thaw treatment
[101]. The elongated/fattened cells are metabolically active
and capable of growth. The remaining cells that do not
respond to the substrates can be stained and visualized. The
number of viable cells is obtained by subtracting the stained
cells that remain after DVC treatment and lysing from the
total number of cells [103]. qDVC can be used to detect via-
ble cells in mixed bacterial environments with bacterial cells
of different sizes, as it is independent of bacterial morphol-
ogy. DVC procedures have been widely applied in studying
the marine environment. They have also been used to assess
VBNC Listeria monocytogenes contamination in potable
water [104]. Further, DVC has been applied to enumerate
water-borne bacteria and their viability [105]. Recent stud-
ies have used DVC in combination with fluorescent in situ
hybridization for the detection of bacterial contamination
[106]. In one such study, Tirodimos et al. described the
detection of Helicobacter pylori in river water using antibi-
otic novobiocin, with the resulting swollen cells being used
as rRNA targets for a hybridization probe, as intracellular
rRNA is increased due to nutritional stimulus [107].
Heat flow
All physiochemical activity of microbes is accompanied by
heat flow, which is a biomarker for the viability of cells. The
thermal effect of physiochemical activity can be measured
by isothermal microcalorimetry (IMC) [108]. Microca-
lorimeters measure the heat flow between the reaction vessel
containing the sample and the heat sink, with these being
connected by a thermophile [109]. Commercial isothermal
microcalorimeters can detect heat produced of the order of
0.2 µW. Heat production by a single E. coli cell has been
estimated to be between 1.4–3.5 pW cell
1
. [108]. Consider-
ing an average of 2 pW cell
1
, a value of around 100 000 is
required to produce a notable signal that can be picked up
by a commercial microcalorimeter. A concentration of
about 2.510
4
–110
5
bacteria ml
1
is the detectable limit
for isothermal microcalorimeters, making them more sensi-
tive than spectrophotometers [109]. The heat produced by
bacteria in tropical soils as measured by IMC was correlated
to viable counts of bacteria measured using plate counting
methods [110]. IMC has been used to detect bacterial con-
tamination and infections such as bacterial contamination
of donated blood platelets [111]. Mixed communities of
bacteria were analysed in marine sediments efficiently using
IMC [112]. IMC has also been used to detect the MIC and
efficiency of various antimicrobial compounds, such as sele-
nium compounds, against S. aureus [113], cephalopsorins
against E. coli [114], and also various antibiotics against E.
coli and S. aureus [115]. The application of IMC for rapid
pathogen detection in urine has been carried out to deter-
mine four pathogens simultaneously, as the heat flow curves
are unique for each species, independent of the initial con-
centration of pathogen in the sample [116]. IMC is conve-
nient for use in combination with any quality control and
downstream analysis, as it is a non-destructive technique
and does not require any treatment with dyes or other com-
pounds [41]. IMC allows for real-time microbial viability
studies to determine the effect of antimicrobials and help in
the development of new drugs [117]. However, IMC meas-
ures the net signal of all the chemical and physical processes
occurring inside the ampoule containing the sample, and
unknown phenomena may produce heat that is accounted
for in the net signal, and simultaneous exothermic and
endothermic reactions may lead to incorrect signals [118].
Fig. 3. The illustration describes rRNA synthesis in bacteria . RNA polymerase produces a 30S RNA transcript that is further con-
verted to pre-rRNA subunits with leader and tail sequences that are then removed by endonucleolytic activity to produce mature
rRNA. Primers can be designed to recognize pre-rRNA sequences, which can be amplified by RT-qPCR to measure pre-rRNA upshift in
response to nutritional stimulus, which is a sign of bacterial viability.
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Stable isotope probing (SIP)
SIP involves the utilization of heavy isotopes by microbes
and the incorporation of these isotopes in their DNA.
13
C,
15
N and H
2
18
O are some of the isotopically labelled sub-
strates that are metabolized by microbes [119]. Only meta-
bolically active microbes can metabolize and incorporate
labelled isoptopes and can be separated from unlabelled
microbes. The unlabelled cells essentially include non-viable
microbes that are unable to metabolize the isotopes. DNA
SIP gives the representation of the replicating cells present
in the sample [120]. Labelled DNA can be separated from
non-labelled DNA by cesium chloride density gradient with
ethidium bromide [121].
13
C- DNA is separated from
12
C-
DNA and is retrieved. The fraction containing
13
C- DNA is
representative of the microbes that are metabolically active
and are able to incorporate the labelled isotopes [122]. Cell
division is essential to incorporate labelled isotopes into
DNA and may require a longer incubation time, especially
among slow-growing microbes. To overcome these limita-
tions, RNA SIP is better and more suitable for the detection
of metabolically active cells. RNA has a faster turnover rate,
easily incorporates isotopes and reduces the incubation time
[119]. RNA SIP represents the transcriptional activity of the
cells by incorporating isotopes [123]. Labelled DNA/RNA
detected by SIP is later characterized by gene analysis
through fingerprinting analysis or 16S rDNA sequencing
approaches. With recent advances in sequencing technology
we can obtain the metagenomes or metatranscriptomes of
labelled microbes detected by SIP [124]
FUTURE PROSPECTS
Isothermal nucleic acid amplification
Isothermal reactions must take place at constant and low
temperature to carry out amplification. Nucleic acid ampli-
fication methods such as PCR, although widely used,
require a thermal cycler, which is expensive and is not por-
table. This has motivated research into alternative amplifi-
cation methods that avoid the use of thermal cyclers to
reduce the expense and increase the ease of the process [9].
Conversely, isothermal amplification processes reduce the
need for thermal control and power consumption. Some of
the isothermal nucleic acid amplification techniques used in
viable pathogen detection are listed in Table 4.
Biosensors
The use of biosensors in pathogen detection involves the
use of a biomarker that is specific to the pathogen and inter-
acts with a biological receptor (monoclonal antibody, RNA,
DNA, glycan, enzyme, whole cell). A transducer helps con-
vert the probe–target interaction into a signal and a data
output system to analyse the data. Based on the type of
probe and transducer used, different types of biosensor have
been used to detect pathogens, for example optical and elec-
trochemical biosensors were used to detect E.coli O157:H7
Fig. 4. Oxidoreductases and dehydrogenases present in viable cells reduce tetrazolium salt to water-soluble formazan and resazurin
to fluorescent resorufin, which is detected by a colour change and is a measure of the respiratory activity of viable cells.
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[125], piezoelectric and magnetic biosensors were used to
detect E. coli [126, 127] and immunosensors were used to
detect E.coli O157:H7 [128].
Microarrays
Microarrays consist of capture probes immobilized onto
solid surfaces by specific surface chemistry. The immobiliza-
tion of species-specific DNA probes on the surface of a
transducer helps to hybridize target DNA with the DNA
probe. After washing unbound sample DNA, the resulting
stable double-stranded DNA are detected using optical sig-
nals by a prelabelled target sequence. A pattern of fluores-
cence will appear on the array. Microarray technology has
been used to determine bacterial contamination by patho-
gens such as E.coli, Salmonella species, S. aureus and C.
jejuni from different sources [129–132]. Common food-
borne and water-borne pathogens, such as Shigella species,
Salmonella species, L. monocytogenes and E. coli O157:H7
have been detected simultaneously in a complex food matrix
using a microplate chip in which each microarray was inte-
grated into each well of the microplate [133, 134]. Microar-
rays have also been used efficiently for the determination of
pathogens in complex human samples such as cerebrospinal
fluid for the detection of central nervous system infections
to aid diagnosis and treatment [135]. Microarrays have
proven to be valuable in pathogen determination due to
their superior specificity and sensitivity in various sample
matrices, and because they have detection limits of as low as
10 c.f.u. ml
1
.
Microfluidics
Biological nano- and microelectromechanical systems
incorporate many laboratory processes on a chip for the
rapid and convenient detection of pathogens using semicon-
ductor technology. Microfluidic chips have been proven to
be a promising tool in pathogen detection due to their flexi-
bility for automation, miniaturization and multiplexing
[136]. Several microfluidic systems fabricated from nano-
materials have been developed for DNA extraction [137,
138] as well as nucleic acid amplification [139]. Such micro-
fluidic devices have been used to extract DNA and for PCR
reaction to detect E. coli, Streptococcus mutans and Staphy-
lococcus epidermidis [140]. Microfluidic chips have recently
been fabricated to carry out loop-mediated isothermal
amplification (LAMP) for the rapid detection of multiple
pathogens causing pneumonia, which can be carried out in
90 min to detect an abundance of nucleic acid of as low as
10 copies [141]. Microfluidic devices combining LAMP and
gold nanoparticles have also been developed for the detec-
tion of Salmonella spp. in food samples, with a limit of
detection of as low as 10 c.f.u. ml
1
[142]. Further advances
in fabrication methods, miniaturization and data analysis
could lead to a miniaturized standalone laboratory tech-
nique, enabling its use in low-resource settings for pathogen
detection. Three-dimensional printing of microfluidic sys-
tems could further reduce the costs and production time
[143].
DISCUSSION
Culture-based diagnosis of pathogens has many lacunae
that can be addressed using alternative molecular methods
that are more reliable and less time-consuming. However,
the major difficulty in pathogen detection remain distin-
guishing between viable and dead cells, and failure to do
this accurately results in overestimation or underestimation,
leading to the risk of contamination.
There are various methods to determine the viability of
microbial communities, but much validation is required
when these methods are employed. These methods need to
be developed further for their application to determine the
viability of different microbes in different environmental
conditions. The practicality of the assay and the interpreta-
tion of results are important factors when considering dif-
ferent techniques to apply to determine viability [41].
Membrane integrity is compromised when electroporation
is used to allow the entry of DNA into cells. A study in
which the proton motive force of Camplyobacter was abol-
ished showed the entry of EtBr into bacteria, which is a
marker for dead cells and compromises the use of mem-
brane integrity as a marker for viability [144]. The use of
nucleic acid-based determination of viability is highly
dependent on environmental conditions, which may influ-
ence viability determination [1]. Several factors are import-
ant and influence the application of viability staining using
Table 4. Isothermal nucleic acid amplification techniques used in pathogen detection
Serial
no.
Technique used Species detected Reference
1 Nucleic acid sequence-based amplification V. cholerae
H. influenzae, N. meningitidis
[66]
[65]
2 Loop-mediated isothermal amplification E. coli [197]
[198]
3 Helicase-dependent amplification Trichomonas vaginalis
E. coli
[199]
[200]
4 Strand displacement amplification V. cholerae [201]
5 Rolling-circle amplification M. tuberculosis
V. cholerae
[202]
[203]
6 Recombinase polymerase amplification E. coli, Klebsiella pneumoniae, Proteus mirabilis, P. aeruginosa and E. faecalis [204]
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PI and SYTO9, such as the bleaching effect of SYTO9, the
binding affinities of SYTO9 and background fluorescence
[42]. Loss of DNA has been reported due to the use of EMA
for viability determination and results in the underrepresen-
tation of viability [54], whereas reports have shown that
PMA does not completely eliminate signal from dead cells,
leading to false positives [2]. Bacterial inactivation by UV,
solar disinfection, low-temperature pasteurization and anti-
biotics do not cause membrane disintegration, and because
of this membrane integrity cannot be used to establish via-
bility, hence the manner in which cell death occurs plays an
important role in deciding which method of viability deter-
mination should be employed [57]. Due to the diversity of
cell types, it is not feasible to apply a single universal
method or technique to determine viability; using several
viability factors will give a better representation of viable
cells [145]. Each method has its limitations and may cause
species biases and careful consideration of which method
should be applied is important.
Limit of detection (LOD) is another factor that influences
the selection of viability determination methods. An enrich-
ment step is required during pathogen detection, as the
LOD is high for most detection methods. To overcome this
issue, immunomagnetic separation (IMS) can be used as a
preparatory step. The IMS method is widely used for the
selective concentration and isolation of target organisms in
a complex matrix. It decreases the time required for detec-
tion, as there is no need for an enrichment step for pathogen
detection [146]. Further, IMS improves pathogen detection
by removing signal from inhibitory agents present in the
sample matrix [147]. IMS involves the coating of antibodies
or ligands targeting pathogens on super paramagnetic
beads, thus forming immunomagnetic beads that target bac-
teria in a complex matrix, forming a microbe–bead complex
[148]. The microbe–bead complex can be concentrated
from the matrix by applying an external magnetic field. IMS
has been used in combination with PCR [149, 150], flow
cytometry with vital stains [150, 151], bioluminescence
[152], stable isotope probing [153] and enzyme-linked
immunosorbent assay (ELISA) [154], and can be explored
as a enrichment step with other viability determination
methods.
CONCLUSION
The development of strategies and systems to obtain rapid
and efficient detection of pathogenic contamination and
reduce the proliferation of pathogens are of the utmost
importance to prevent health hazards. Detection methods
for contamination must be rapid, cost-effective, versatile
and able to test large numbers of many different analytes
from various sample matrices. There are many detection
methods, however, and methods can be selected based on
the type of sample, matrix characteristics, and the equip-
ment and chemicals required, as well as technical profi-
ciency. Further validation and standardization of these
methods is required to improve their sensitivity, specificity
and repeatability.
Funding information
All the funds and facilities for the research work were provided by VIT
University, Vellore.
Acknowledgements
The authors would like to acknowledge Vellore Institute of Technology,
Vellore for their continuous support and providing facilities for
research work.
Conflicts of interest
The authors declare that there are no conflicts of interest.
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