Ultrasensitive Detection of Salmonella and Listeria monocytogenes by Small-Molecule Chemiluminescence Probes

Article abstract of DOI:10.1002/anie.201904719

Detection of Salmonella and L. monocytogenes in food samples by current diagnostic methods requires relatively long time to results (2–6 days). Furthermore, the ability to perform environmental monitoring at the factory site for these pathogens is limited due to the need for laboratory facilities. Herein, we report new chemiluminescence probes for the ultrasensitive direct detection of viable pathogenic bacteria. The probes are composed of a bright phenoxy-dioxetane luminophore masked by triggering group, which is activated by a specific bacterial enzyme, and could detect their corresponding bacteria with an LOD value of about 600-fold lower than that of fluorescent probes. Moreover, we were able to detect a minimum of 10 Salmonella cells within 6 h incubation. The assay allows for bacterial enrichment and detection in one test tube without further sample preparation. We anticipate that this design strategy will be used to prepare analogous chemiluminescence probes for other enzymes relevant to specific bacteria detection and point-of-care diagnostics.

Full text of DOI:10.1002/anie.201904719

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International Edition: DOI: 10.1002/anie.201904719
German Edition: DOI: 10.1002/ange.201904719
Analytical Methods
Hot Paper
Ultrasensitive Detection of Salmonella and Listeria monocytogenes by
Small-Molecule Chemiluminescence Probes
Michal Roth-Konforti+, Ori Green+, Mario Hupfeld, Lars Fieseler, Nadine Heinrich,
Julian Ihssen, Raffael Vorberg, Lukas Wick, Urs Spitz,* and Doron Shabat*
Abstract: Detection of Salmonella and L. monocytogenes in
food samples by current diagnostic methods requires relatively
long time to results (2–6 days). Furthermore, the ability to
perform environmental monitoring at the factory site for these
pathogens is limited due to the need for laboratory facilities.
Herein, we report new chemiluminescence probes for the
ultrasensitive direct detection of viable pathogenic bacteria.
The probes are composed of a bright phenoxy-dioxetane
luminophore masked by triggering group, which is activated by
a specific bacterial enzyme, and could detect their correspond-
ing bacteria with an LOD value of about 600-fold lower than
that of fluorescent probes. Moreover, we were able to detect
a minimum of 10 Salmonella cells within 6 h incubation. The
assay allows for bacterial enrichment and detection in one test
tube without further sample preparation. We anticipate that this
design strategy will be used to prepare analogous chemilumi-
nescence probes for other enzymes relevant to specific bacteria
detection and point-of-care diagnostics.
and Listeria monocytogenes.[3] The share of tests for these
bacteria in the global food testing market is 40% and 31%,
respectively.[4] Both bacteria are important opportunistic
pathogens, which cause zoonotic diseases, for example,
listeriosis and salmonellosis. The bacteria cannot be trans-
mitted directly from human to human. Instead, fecal–oral
infection takes place almost exclusively after the consumption
of contaminated food. Every year, Salmonella is estimated to
cause circa one million foodborne illnesses in the US, with
19000 hospitalizations and 380 deaths.[5] Human pathogenic
Listeria, L. monocytogenes, is also widely distributed with
infections resulting in a high mortality rate (greater than
20%).[6]
Detection of Salmonella and L. monocytogenes from food
samples is currently performed by applying ISO-certified
reference methods (ISO 6579 and ISO 11290, respective-
ly).[7–9] These methods apply different enrichment cultures
and plating on selective agar media for the detection of
bacteria. Presumptive colonies have to be confirmed after the
initial isolation. In case of L. monocytogenes, a safe negative
result is available after 96 h (4 days). Positive results are
available after 96–144 h (4–6 days), depending on the growth
of the bacteria. In case of Salmonella a negative result is
available after 66 h (< 3 days), while a positive result is
available after 114 h (< 5 days). Rapid alternative methods
such as immunoassays or DNA amplification (PCR in
particular) have improved initial time to results but typically
still require bacterial pre-enrichment of 16–48 h[10] while the
detection time is reduced to a range from hours to few
minutes.[11–13] As these molecular methods detect DNA or cell
surface antigens (genotypic), a confirmatory test to show
bacterial viability (phenotypic) is needed before taking
action. Furthermore, a point-of-care test is difficult to achieve
as transfer of samples after pathogen enrichment requires
a biosafety laboratory. Outsourcing pathogen tests to external
service laboratories adds transport time to the availability of
results. Lateral flow assays (LFAs) are also widely used as
a rapid detection method for various monitoring and diag-
nostic purposes, including bacterial detection.[14]
A
mongst known human pathogenic bacteria there are
several that can lead to foodborne illness, resulting from the
consumption of contaminated food. Most of such illnesses are
infections, caused by a variety of pathogenic bacteria, viruses
and parasites.[1] Owing to the widespread occurrence and
hazard of food-borne pathogenic bacteria, there is an obvious
need to detect and identify the source, both as either a food
contaminant or after animal/human infection.[2] Two major
pathogens responsible for food contamination are Salmonella
[*] M. Roth-Konforti,[+] O. Green,[+] D. Shabat
Department of Organic Chemistry, School of Chemistry, Faculty of
Exact Sciences, Tel Aviv University
Tel Aviv 69978 (Israel)
E-mail: chdoron@post.tau.ac.il
M. Hupfeld
Nemis Technologies AG
Zurich (Switzerland)
L. Fieseler, N. Heinrich
Institute of Food and Beverage Innovation
Zurich University of Applied Sciences
Wꢀdenswil (Switzerland)
Food manufacturers today rely on random sample testing
of finished products. Hence, faster methods exhibiting better
sensitivity and specificity, which can be performed at the point
of care, are desired in food processing (environmental
monitoring). Significant effort has gone into the development
of detection methods, to allow for fast, accurate, and cost-
efficient detection and identification of food-borne patho-
genic bacteria. Considering all requirements, specifically the
need for phenotypic testing, todayꢀs most widespread meth-
J. Ihssen, R. Vorberg, L. Wick, U. Spitz
BIOSYNTH
Rietlistr. 4, Postfach 125, 9422 Staad (Switzerland)
E-mail: urs.spitz@bluewin.ch
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201904719.
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ods for pathogen detection and enumeration remain selective
The light-emission mechanism of our phenoxy-dioxetane
chromogenic and fluorogenic probes.[6,15–18]
chemiluminescence probes is initiated by the removal of
a triggering responsive group, which serves as the substrate of
a specific bacterial enzyme (Figure 1A). Once the trigger is
cleaved, spontaneous 1,6-elimination of a spacer occurs to
yield a phenolate intermediate. This phenolate decomposes
through a chemiexcitation process, to emit intense green light.
Probe CLSP is composed of phenoxy-dioxetane masked with
a C8-ester group, which is a known substrate of a Salmonella
esterase.[16] Similarly, probe CLLP is composed of phenoxy-
dioxetane masked with a myo-inositol 1-phosphate group;
a known substrate of the virulence factor phosphatidylinosi-
tol-specific phospholipase C (PI-PLC), which is produced
only by the pathogenic L. monocytogenes (Figure 1B).[38]
The synthesis of the Salmonella probe, CLSP, was
performed as described in the Figure 2. In brief, an esterifi-
cation reaction between caprylic acid and 4-hydroxybenzyl
alcohol afforded compound 1a, which was then converted to
its iodine derivative 1b. The latter was reacted with phenol 1c
to produce ether 1d. Oxidation of the enol-ether function of
1d with singlet oxygen afforded the corresponding dioxetane
CLSP.
Chemiluminogenic assays for enzymatic detection have
been shown to be orders of magnitude more sensitive than
equivalent fluorescence detection assays.[19–21] Recently, our
group has explored new approaches for amplifying chemilu-
minescence light intensity[22–24] under physiological condi-
tions.[25–34] A remarkable enhancement of light emission was
obtained by simply improving the emissive nature of the
excited species, formed during the chemiexcitation of
Schaapꢀs dioxetanes.[35–37] Phenoxy-dioxetane probes, bearing
conjugated electron-withdrawing substituent at their ortho
position, could release benzoate derivative, during their
chemiexcitation, which was found to be highly emissive under
aqueous conditions.[29] These new phenoxy-dioxetane lumi-
nophores exhibited light emission intensity of up to 3000-fold
greater than that of the original Schaapꢀs dioxetanes. As such,
we sought to utilize our chemiluminescence luminophores to
design probes for detection of Salmonella and L. monocyto-
genes. Herein, we report new efficient chemiluminescence
probes for the ultrasensitive direct detection of two types of
pathogenic bacteria.
Figure 1. A) General structure and chemiexcitation pathway of probes designed for the detection of bacterial enzymes. B) Molecular structures of
chemiluminescence probes CLSP and CLLP for detection of Salmonella and Listeria monocytogenes, respectively.
Figure 2. General synthetic scheme for CLSP.
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Figure 3. General synthetic scheme for CLLP.
The synthesis of the Listeria probe, CLLP, was performed
as described in the Figure 3. A reaction between penta-
protected inositol derivative 2a and 4-hydroxybenzaldehyde
under the indicated conditions afforded phosphate 2b. The
aldehyde functionality of 2b was reduced to its benzylalcohol
derivative 2c, which was further reacted with mesyl-chloride
to give compound 2d. The latter was reacted through an SN2
reaction with phenol 2e to afford ether 2 f. Compound 2 f was
treated with lithium iodide to selectively remove the methyl
phosphonate protecting group. Additional alkaline hydroly-
sis, to remove the methyl from the acrylate substituent,
yielded compound 2g. Oxidation of the enol-ether function of
2g with singlet oxygen afforded the corresponding dioxetane
CLLP.
The chemiluminescence emission profile of CLSP over
time was measured in physiological buffer, in the presence
and in the absence of porcine liver esterase (PLE). The total
light emission signal observed upon activation with PLE was
about 300-fold higher than that obtained in the absence of the
enzyme (Figure 4). This result is remarkable, especially given
the fact that CLSP is composed of an activated phenolic ester,
which suffers of a relatively high rate of background
hydrolysis. Similarly, the chemiluminescence emission profile
of CLLP was measured in the presence and in the absence of
PI-PLC. The total light emission signal upon activation by PI-
PLC was almost 700-fold greater than the background signal.
In addition, both CLSP and CLLP show a logarithmic
correlation between probe concentration and enzyme con-
centration, enabling quantitative enzyme detection (see
Supporting Information, Figures S1 and S2).
Figure 4. Chemiluminescence kinetic profiles of CLSP or CLLP [10 mm]
in PBS (pH 7.4, 10% DMSO) in the presence or absence (control) of
porcine liver esterase [1 UmLÀ1] or PI-PLC [0.5 UmLÀ1], respectively, at
room temperature (top). Relative total light units (RLU) emitted,
normalized to that of the control (bottom).
produced by its corresponding specific bacterial strain.
Therefore, probes CLSP or CLLP were added to cultures of
several appropriate strains of bacteria, preincubated at 378C
for 6 h. For the Salmonella detection, CLSP was tested with
the following bacterial strains: Escherichia coli ATCC 25922
(E. coli), Citrobacter freundii ATCC 8090, Salmonella Typhi-
murium (D) ATCC 14028 (S. Typhimurium), Salmonella
Enteritidis (D) ATCC 13076 (S. Enteritidis 1) and Salmonella
Enteritidis RKI 05/07992 (S. Enteritidis. 2). Both E. coli and
C. freundii served as negative controls, as they do not possess
Next, we sought to evaluate the ability of the CLSP and
CLLP probes to detect Salmonella and L. monocytogenes,
respectively. In order to identify one type of bacteria among
others, it is particularly important to find out whether the
chemiluminescent signal of the probe can be selectively
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a C8-esterase. Moreover, these bacteria may be present in
Furthermore, the obtained light emission intensity, allowed
stool samples tested for Salmonella, as they occur in the
intestinal tracts of animals and humans.[39,40] Additionally,
some serotypes of E. coli can cause serious food poisoning in
their hosts, and are occasionally responsible for product
recalls owing to food contamination, similarly to Salmonel-
la.[41]
For L. monocytogenes detection, CLLP was added to
cultures of the following bacterial strains: E. coli ATCC
25922, Listeria innocua (6a) ATCC 33090, L. monocytogenes
(4b) ATCC 19115 (L. monocytogenes 1) and Listeria mono-
cytogenes ATCC 7644 (L. monocytogenes 2). Both E. coli and
L. innocua served as negative controls, as they do not possess
a PI-PLC enzyme.[38] Moreover, they may be present in food
samples tested for L. monocytogenes, as L. innocua occurs in
food sources, and some serotypes of E. coli may as well.
Additionally, as mentioned above, E. coli may also be present
in stool samples.[40–42]
Both probes were also added to sterile saline, and the
luminescence amounted was used to normalize the measured
signal obtained for each bacterial strain. The signal intensities,
obtained by each sample with the appropriate probe, are
shown in Figure 4. Probe CLSP was able to differentiate
between the E. coli and C. freundii controls and the Salmo-
nella strains, with a signal enhancement of up to 693-fold for
the Salmonella strains compared to the sterile control.
CLSP to visually distinguish between Salmonella (S. Enter-
itidis RKI 05/07992; S. Enteritidis 2) and E. coli (E. coli
ATCC 25922), using a high-resolution digital camera (Fig-
ure 5B.
Similarly, probe CLLP was able to differentiate between
the E. coli and L. innocua controls and the L. monocytogenes
strains, with a signal-to-noise ratio of up to 10000-fold higher
for the L. monocytogenes strains than that of the sterile
control. Remarkably, the signal intensity produced by the
pathogenic L. monocytogenes strains was about three orders
of magnitude greater than that of the non-pathogenic
L. innocua. This enables us to differentiate between the
pathogenic and non-pathogenic Listeria within a short time
after addition of CLLP (Figure 5D).
With chemiluminescence probes CLSP and CLLP in
hand, we next sought to compare their ability to detect
Salmonella and L. monocytogenes with that of currently
existing fluorescence methods. 4-Methylumbelliferyl capry-
late (MUCAP) and 4-methylumbelliferyl myo-inositol 1-
phosphate (MUMIP) are two commercially available fluoro-
genic probes for the detection of Salmonella and L. mono-
cytogenes, respectively.[16,42] MUCAP and MUMIP are mostly
used in chromogenic nutrient agar plates for confirmatory
testing.[18,43] The mode of action of these probes is based on
removal of a triggering enzyme-labile group by the Salmo-
Figure 5. A) Relative Luminescence (RLU) emitted from cultures of different bacterial strains (inoculated with 1ꢁ105 CFUmLÀ1, incubation time
6 h) after the addition of CLSP [10 mm] (normalized to that of CLSP, which was added to sterile growth medium), 27–30 min after addition of the
probe. B) Images of Salmonella (S. Enteritidis RKI 05/07992; S. Enteritidis 2) and E. coli (E. coli ATCC 25922) tube cultures (incubated overnight,
ca. 2ꢁ109 CFUmLÀ1) after the addition of CLSP [20 mm] taken with a high-resolution, high quantum efficiency digital camera, with white light
illumination turned on (left) and off (right). C) RLU emitted from cultures of different bacterial strains (inoculated with 1ꢁ105 CFUmLÀ1
,
incubation time 6 h) after the addition of CLLP [10 mm] (normalized to that of CLLP, which was added to sterile growth medium), 8 min after
addition of the probe D) Images of L. monocytogenes ATCC 7644 (L. monocytogenes 2) and E. coli (E. coli ATCC 25922) tube cultures (incubated
overnight, ca. 2ꢁ109 CFUmLÀ1) after the addition of CLLP [67 mm] with white light illumination turned on (left) and off (right).
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nella C8 esterase or by the PI-PLC enzyme to release
a fluorescent coumarin derivative (4-methylumbelliferone).
This fluorophore produces a measurable fluorescence signal
(lmax = 460 nm) with an excitation at wavelength of 360 nm.
Figure 6 shows the signal-to-noise ratios obtained by each
chemiluminescence probe (CLSP and CLLP) and by their
fluorogenic commercial analogues (MUCAP and MUMIP)
probe concentrations 1/10 of that required for the fluores-
cence commercial analogues).
Interestingly, in an earlier study, Schaapꢀs adamantyli-
dene-dioxetane was applied to prepare a chemiluminescent
probe for PI-PLC enzyme using the same triggering substrate
demonstrated in this work.[44] However, this dioxetane suffers
from an extremely weak light emission efficiency under
aqueous conditions and is completely unusable without
additives at physiological pH values. Therefore, a long
incubation time (up to 5 days) and autoradiography film
had to be used in order to detect a measurable signal. The
CLLP probe described in this study produces a light emission
intensity more than three orders of magnitude higher than the
classic Schaapꢀs adamantylidene-dioxetane under physiolog-
ical conditions without any additives.
To further establish that our probes can be used for
environmental sampling, we tested whether CLSP is able to
detect dry stressed Salmonella recovered from stainless steel
plates. According to certification guidelines for food diag-
nostics, Salmonella presence/absence needs to be detected
from stainless steel surfaces partially at low concentration
(25–75%) and fully at elevated concentrations (100%) in
defined time to results.[45] We plated low or high concen-
trations of Salmonella (200–600 CFU or 1.0–2.0 ꢁ 104 CFU
respectively) and let them dry for 2.5 h. Afterwards, we
recovered the Salmonella bacteria using a standard swab and
transferred them into an enrichment medium. After 16 h of
incubation CLSP was added and the luminescence signal was
measured (Figure 7A).
Light emission from either a low or high amount of
Salmonella showed a typical chemiluminescence kinetic
profile with an increase in signal, which decays over time
(see Supporting Information, Figure S5). At high concentra-
tions (Figure 7B) 100% of the swabs gave a high signal-to-
background ratio (close to 5000:1) and all samples could be
differentiated from the control. For low concentrations
(Figure 7B) 88% of the swab samples produced a high
signal-to-background ratio (close to 2000:1). For 12% of the
swabs, no signal above background could be detected. We
attribute this fact to the typically low recovery rates of swabs,
which we measured to be between 1–2% (data not shown).
This makes it likely that swabbing a low number of bacteria
from a stainless-steel plate will result in some samples not
containing any Salmonella for enrichment and later for
enzyme activity detection.[46] In order to be useful in the food
industry, several strains of Salmonella need to be detected,
therefore we show that CLSP can detect a variety of strains of
Salmonella (see Supporting Information, Figure S6). The
obtained results fill some key certification requirements as
stated by the Association of Official Agricultural Chemists
(AOAC).
Figure 6. A) Signal/noise ratio values for CLSP [10 mm] and MUCAP
[100 mm] plotted against different Salmonella concentrations (S. Enter-
itidis RKI 05/07992; S. Enteritidis 2) using logarithmic scales. B) Sig-
nal/noise ratio values for CLLP [10 mm] and MUMIP [100 mm] plotted
against different L. monocytogenes concentrations (L. monocytogenes
(4b) ATCC 19115) using logarithmic scales.
for different bacteria concentrations (for Salmonella and
L. monocytogenes, respectively). Remarkably CLSP, exhib-
ited a limit of detection (LOD) value of 28.8 ꢁ 103 CFUmLÀ1,
while MUCAP detected Salmonella with an LOD value of
17800 ꢁ 103 CFUmLÀ1. Similarly, CLLP exhibited an LOD
value of 48.8 ꢁ 103 CFUmLÀ1, while MUMIP detected
L. monoctogenes with an LOD value of 31250 ꢁ
103 CFUmLÀ1. The enhanced sensitivity observed by CLSP
and CLLP (625 and 640-fold, respectively) clearly demon-
strate the advantage of our chemiluminescence substrates
versus currently existing fluorescence substrates for bacterial
detection assays.
The performed chemiluminescence assays showed a pos-
itive logarithmic correlation between the signal-to-noise ratio
and bacteria concentration, even at a concentration range as
low as the limit of detection, making this assay suitable for
quantitative as well as qualitative assays (see Figures S3 and
S4). In addition, chemiluminescence assays require signifi-
cantly less substrate than fluorescence assays (the chemilu-
minescence data obtained in Figure 6 were obtained with
Currently many bacterial detection methods are designed
for a qualitative endpoint determination. This has the
disadvantage that potentially highly contaminated samples
can only be detected after the same time as minimal
contaminated samples. A chemiluminescence dynamic mon-
itoring method has the potential to monitor samples right
from the start, thereby detecting highly contaminated samples
earlier. To this end, we inoculated Salmonella cells in different
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Figure 7. A) Either a low amount of Salmonella (200–600 CFU) or high amount (1.0–2.0ꢁ104 CFU) was plated on a stainless-steel plate. After
2.5 h of drying bacteria were partially recovered by swabs and grown in enrichment medium for 16 h. Afterwards, CLSP was added [10 mm] and the
luminescence signal monitored. B) Luminescence signal detected from a Salmonella strain recovered after dry stress on a stainless steel plate.
The bars illustrate the signal/noise ratio after 10 min of incubation. C) Dynamic monitoring of luminescence signal with different starting
concentrations of Salmonella. Salmonella were inoculated in different log concentrations (10–10000 CFUmLÀ1) and placed together with CLSP
(10 mm). Luminescence signal was constantly monitored over 14 h.
concentrations with CLSP and monitored how the lumines-
cence signal developed during bacterial growth (Figure 7C).
As expected, CLSP is stable in an enrichment medium and
yields light signals curves depending on the starting number of
bacteria and time of growth. Higher contamination levels
could be detected in around half the time compared to low
contamination levels. Such experiments may offer the ability
not only to detect specific bacteria but also to give informa-
tion about the initial level of contamination; which can be
valuable to determine if acceptable levels of environmental
bacteria have been breached in a high-risk environment.
In summary, we have developed two new chemilumines-
cence probes, CLSP and CLLP, for the direct detection of two
of the most widely distributed and deadliest food-borne
pathogenic bacteria, Salmonella and Listeria monocytogenes.
The probes were composed of a phenoxy-dioxetane lumino-
phore masked by a triggering group, which is designed for
activation by a specific bacterial enzyme. Remarkably, CLSP
and CLLP were able to distinguish Salmonella or L. mono-
cytogenes strains from other bacteria that may occur in
samples tested for food-borne pathogens. Furthermore,
CLLP was able to differentiate between two strains of
Listeria, the pathogenic L. monocytogenes and non-patho-
genic L. innocua. Similarly, CLSP could visually differentiate
between Salmonella and E. coli strains. The two chemilumi-
nescence probes could detect their corresponding bacteria
with an LOD value of about 600-fold more sensitive than that
of fluorescent probes. Moreover, the Salmonella probe was
able to detect a minimum of 10 pathogenic bacterial cells after
only 6 h of incubation (Tables S2 and S3). As such, the current
report exemplifies the most sensitive luminogenic bacterial
enzymatic assays known to date. We anticipate that the design
strategy presented in this study will be broadly used to
prepare analogous chemiluminescence probes for other
enzymes relevant to selective bacteria detection. Such
chemiluminescence probes could serve as a supportive tech-
nological platform, which is complementary with current
state-of-the-art molecular biology methods.
Acknowledgements
D.S. thanks the Israel Science Foundation (ISF), the Bina-
tional Science Foundation (BSF) for financial support.
Conflict of interest
NEMIS Technologies AG is a Swiss startup developing
diagnostic detection kits for food safety applications. Bio-
synth AG is a chemical synthesis company based in Switzer-
land producing diagnostic molecules for detection applica-
tions.
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Manuscript received: April 16, 2019
Version of record online: && &&
,
&&&&
&&&&
www.angewandte.org
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2019, 58, 2 – 9
These are not the final page numbers!

Angewandte
Research Articles
Chemie
Research Articles
Analytical Methods
Salmon and Lister would be proud: The
development of new chemiluminescence
probes for the direct detection of two of
the most widely distributed and deadliest
food-borne pathogenic bacteria, Salmo-
nella and Listeria monocytogenes, is de-
scribed. The two probes could detect
their corresponding bacteria with a limit
of detection about 600-fold lower than
that of fluorescent probes.
M. Roth-Konforti, O. Green, M. Hupfeld,
L. Fieseler, N. Heinrich, J. Ihssen,
R. Vorberg, L. Wick, U. Spitz,*
D. Shabat*
&&&& — &&&&
Ultrasensitive Detection of Salmonella
and Listeria monocytogenes by Small-
Molecule Chemiluminescence Probes
Angew. Chem. Int. Ed. 2019, 58, 2 – 9
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
&&&&
These are not the final page numbers!