Chemiluminescent Carbapenem-Based Molecular Probe for Detection of Carbapenemase Activity in Live Bacteria

Article abstract of DOI:10.1002/chem.202000217

Carbapenemase-producing organisms (CPOs) pose a severe threat to antibacterial treatment due to the acquisition of antibiotic resistance. This resistance can be largely attributed to the antibiotic-hydrolyzing enzymes that the bacteria produce. Current carbapenem “wonder drugs”, such as doripenem, ertapenem, meropenem, imipenem, and so on, are resistant to regular β-lactamases, but susceptible to carbapenemases. Even worse, extended exposure of bacteria to these drugs accelerates the spread of resistance genes. In order to preserve the clinical efficacy of antibacterial treatment, carbapenem drugs should be carefully regulated and deployed only in cases of a CPO infection. Early diagnosis is therefore of paramount importance. Herein, we report the design, synthesis, and activity of the first carbapenemase-sensitive chemiluminescent probe, CPCL, which may be used to monitor CPO activity. The design of our probe enables enzymatic cleavage of the carbapenem core, which is followed by a facile 1,8-elimination process and the emission of green light through rapid chemical excitation. We have demonstrated the ability of the probe to detect a number of clinically relevant carbapenemases and the successful identification of CPO present in bacterial cultures, such as those used for clinical diagnosis. We believe that our use of “turn-on” chemiluminescence activation will find significant application in future diagnostic assays and improve antibacterial treatment.

Full text of DOI:10.1002/chem.202000217

A Journal of
Accepted Article
Title: Chemiluminescence Carbapenem-based Molecular Probe for
Detection of Carbapenemase Activity in Live Bacteria
Authors: Doron Shabat, Sayantan Das, Julian Ihssen, Lukas Wick, and
Urs Spitz
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
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To be cited as: Chem. Eur. J. 10.1002/chem.202000217
Link to VoR: http://dx.doi.org/10.1002/chem.202000217
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Chemistry - A European Journal
RESEARCH ARTICLE
Chemiluminescence Carbapenem-based Molecular Probe for
Detection of Carbapenemase Activity in Live Bacteria
Sayantan Das[a], Julian Ihssen[b], Lukas Wick[b], Urs Spitz*[b], and Doron Shabat*[a]
Abstract: Carbapenemase producing organisms (CPOs) pose a
severe threat to antibacterial treatment due to the acquisition of
antibiotic resistance. The resistance can largely be attributed to the
antibiotic hydrolysing enzymes that the bacteria produce. Current
carbapenem wonder drugs like doripenem, ertapenem, meropenem,
and imipenem among others, are resistant to regular β-lactamases,
but susceptible to carbapenemases. Even worse, extended exposure
of bacteria to these drugs accelerates the spread of resistance genes.
In order to preserve the clinical efficacy of antibacterial treatment,
carbapenem drugs should be carefully regulated and only in cases of
negative bacteria.[6] Since then, several carbapenem based drugs
including meropenem, doripenem, and ertapenem have emerged
as ‘last line agents’ or ‘antibiotics of last resort’ in the treatment of
life-threatening infections involving multiresistant strains of
bacteria.[7] However, the increased exposure to carbapenems,
has resulted in the appearance of carbapenem-resistant bacteria,
and resistant strains of Enterobacteriaceae, Pseudomonas and
Acinetobacter have now been isolated worldwide.[7] Again the
bacterial resistance mechanism primarily relies on the ability of
the organism to secrete carbapenem hydrolyzing enzymes –
a CPO infection. Early diagnosis is therefore of paramount importance. carbapenemases.[8]
Here, we report the design, synthesis, and activity of the first
carbapenemase sensitive chemiluminescent probe CPCL that can
monitor CPO activity. The design of our probe enables enzymatic
cleavage of the carbapenem core which is followed by a facile 1, 8-
elimination process and the emission of green light through rapid
chemiexcitation. We demonstrated the ability of the probe to detect a
number of clinically relevant carbapenemases and the successful
identification of CPO present in bacterial cultures such as those used
for clinical diagnosis. We believe that our use of ‘Turn-On’
chemiluminescence activation will find significant application in future
diagnostic assays and improvement of antibacterial treatment.
The number of known carbapenemases has been steadily
increasing since the discovery of Non-Metallo Carbapenemase of
Class A (NMCA) in 1994.[9] All the known carbapenemases and
hence the carbapenemase producing organisms (CPOs) present
a major challenge to the continued clinical efficacy of existing
carbapenem antibiotics. In order to prevent bacterial resistance,
these drugs must be provided in a careful and controlled fashion,
and only administered to patients with a confirmed CPO infection.
For this reason, an early detection of CPO infection is of crucial
importance.
The current armory of methods used to detect the presence of
carbapenemase lacks any universal test that can detect all the
different classes of carbapenemase. Although a number of
phenotypic tests are used in routine laboratory practice,[10–15] all
the current methods suffer from major limitations with respect to
assay duration (1-2 days), specificity, and sensitivity.[16] Genotypic
tests, for example Polymerase Chain Reaction (PCR)-based
techniques and the more advanced real-time Reverse
Transcriptase-PCR (RT-PCR) methods, do permit the specific
identification of a member of the carbapenemase family.[17,18]
However, the PCR techniques are both costly and require highly
skilled personnel for accurate results, which limits their
applicability. Furthermore, the assays are unable to identify new
enzyme variants. A recently introduced advanced MALDI-TOF
based detection method also suffers from severe limitations
regarding the interpretation of the resulting complex peak
patterns.[19] In addition, the results may be inconclusive because
different antibacterial resistance mechanisms, or enzymatic
hydrolysis, can interfere with the assay.
Introduction
Since the discovery of penicillin several decades ago, β-lactam
antibiotics have been considered to be a ‘wonder drug’, effective
against a wide range of bacterial infections.[1] However, bacteria
have the unique ability to develop multi-drug-resistance when
repeatedly exposed to an array of widely used antibiotics.[2] Out of
several mechanisms of antibiotic bacterial resistance,[3] the most
common is the hydrolysis of β-lactam drugs by β-lactamases
(Bla).[4] For example, extended spectrum β-lactamases (ESBLs)
can hydrolyze penicillins and nearly all third-generation
cephalosporins.[5] The emergence of multi-resistant bacterial
strains has resulted in a global crisis with respect to antibacterial
treatment with common antibiotics. In the 1970s, scientists
discovered revolutionary carbapenem based drugs, with a very
broad spectrum of activity against both Gram-positive and Gram-
Because of these difficulties, considerable efforts have been
invested in searching for new improved methods that are sensitive,
specific, rapid, and cost effective. Following the successful
development of β-lactamase responsive chromogenic and
fluorogenic molecular probes for the identification of bacterial
pathogens,[20–29] scientists are now actively seeking similar
methods for the specific detection of carbapenemase and CPOs.
Our group has recently achieved a significant breakthrough in the
field of chemiluminescence sensing and imaging, with the
discovery of phenoxy-dioxetane luminophores, which are suitable
for use under physiological conditions.[30–33] These next
generation phenoxy-dioxetane chemiluminescent luminophores
[a]
Dr. S. Das, Prof. D. Shabat
School of Chemistry, Faculty of Exact Sciences
Tel Aviv University
Tel Aviv 69978 Israel
E-mail: chdoron@post.tau.ac.il
Dr. J. Ihssen, Dr. L. Wick, Dr. U Spitz
BIOSYNTH CARBOSYNTH
Rietlistr. 4 Postfach 125 9422 Staad, Switzerland.
E-Mail:urs.spitz@bluewin.ch
[b]
Supporting information for this article is given via a link at the end of
the document
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10.1002/chem.202000217
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RESEARCH ARTICLE
exhibit up to 3000-fold greater light emission than that of the
original Schaap's dioxetanes, in aqueous media.[30,34] This
discovery has already found use in a number of applications that
require rapid and sensitive detection of various enzymes and
analytes.[35–43] In addition, the intense light emitted from the probe
can be used to effectively image transfected and native enzymatic
activity in live cells.[30,44] Another important milestone was
achieved with the development of chemiluminescence probes for
ultrasensitive detection of the two deadliest food-borne
pathogenic bacteria, Salmonella and Listeria monocytogenes.[45]
Our continued interest in the use of small molecular
chemiluminescent probes to identify a specific target has now
motivated us to design and evaluate a novel carbapenem
chemiluminescence probe. Here, we report the first
chemiluminescence carbapenemase specific probe (CPCL) for
detection of carbapenemase and CPO activity.
Results and Discussion
Recent developments in probe design for detection of
carbapenemase activity have included an example based on
coumarin as a fluorescent dye, and modified cephalosporin as
triggering substrate (Figure 1A).[46] In addition, an unprecedented
enamine BODIPY switch, cleverly introduced by the Xie group,
enabled the development of a highly efficient fluorogenic probe
for the selective detection of carbapenemase over other β-
lactamase activity (Figure 1B, Scheme 1).[47]
Figure 1. (A, B) Recent examples of carbapenemase fluorogenic probes; (C) Our carbapenemase specific chemiluminescence probe, CPCL, and its chemiexcitation
activation mechanism.
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Figure 2. Synthesis of the carbapenemase-specific chemiluminescence probe CPCL.
The same group also reported a coumarin based fluorogenic
probe that could selectively detect metallo-β-lactamases (Figure
1B, Scheme 2).[48] These examples have generated the essential
inspiration needed to develop a chemiluminescence turn-on
probe for the detection of carbapenemase.
Bla-1 (Figure 3A). SPM-1 (São Paulo metallo-β-lactamase) is a
broad-spectrum β-lactamase that also possesses
carbapenemase activity.[53] It is therefore expected to be able to
hydrolyze the carbapenem moiety of CPCL and to activate the
chemiexcitation mechanism of the probe. In contrast, Bla-1 is a
regular penicillinase, which is not likely to be able to hydrolyze
carbapenem.[54,55] A solution of CPCL in PBS, pH 7.4 without any
β-lactamase was used as control. Indeed, as anticipated,
incubation of CPCL with SPM-1 produced an intense
chemiluminescence signal that was clearly distinct from the
control result (Figure 3A). This chemiluminescence signal was
observed immediately after the addition of SPM-1, reached a
The structure of our probe, CPCL, and its chemiluminescence
activation pathway are depicted in Figure 1C. Probe CPCL
consists of an enzyme recognizable carbapenem core attached
through a 4-hydroxymethylphenyl self-immolative linker to a
dioxetane-based
chemiluminescence
luminophore.
The
introduction of this 4-hydroxymethylphenyl linker, provides
convenient access to attach the carbapenem moiety to the
dioxetane luminophore. We envisioned that upon hydrolysis of the
β-lactam core by carbapenemase, there would be a spontaneous
1, 8-elimination to generate phenolate-dioxetane I. This species
rapidly decomposes through a chemiexcitation process to form
the excited benzoate ester II. While this excited benzoate ester
decays to its ground state III, the energy is concurrently released
in the form of a green photon. This light emission can be
measured as chemiluminescence, which correlates with the
carbapenemase activity.
maximum within
5 minutes, and then gradually decayed.
Incubation of CPCL with Bla-1, however, produced only a minor
increase in signal compared to that of the control. Figure 3B
clearly shows the 55-fold light enhancement obtained upon
activation of CPCL by SPM-1. We similarly evaluated the ability
of CPCL to detect several more clinically prevalent
carbapenemases; the Ambler class-B carbapenemse, VIM-15
(Verona integron–encoded metallo-β-lactamase),[56] two class-A
carbapenemases,
KPC-1
(Klebsiella
pneumoniae
carbapenemase),[57] and NMCA[9] (See Supporting Information).
This set of experiments essentially demonstrates the selective
ability of CPCL to detect carbapenemase activity over that of
regular β-lactamase.
CPCL was synthesized from a previously reported enol-ether 1
(Figure 2).[30] The nucleophilic substitution reaction of enol ether
1 with iodide 2[49] yielded boronate 3. Then a crucial Suzuki
coupling reaction[50] with readily synthesizable triflate 4[51] provides
the advanced intermediate 5. Sequential deprotection of TBS and
4-nitrobenzyl (PNB) protecting groups afforded the prefinal
compound 7.[52] The enol ether 7 was then subjected to oxidation
with singlet oxygen to produce the CPCL probe.
Next, we sought to determine the Limit of Detection (LOD, See
supporting information) value of the CPCL probe for
carbapenemase SPM-1. The total light emission obtained by
CPCL for different concentrations of carbapenemase SPM-1, was
plotted to produce a linear correlation (Figure 3C). The LOD value
of CPCL for carbapenemase SPM-1 was then calculated to be as
low as 0.06 mU/mL. In a similar manner, the LOD values were
As a first step, we evaluated the chemiluminescence emission of
the CPCL probe in the presence of the β-lactamases SPM-1 and
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Figure 3. (A) Chemiluminescence kinetic profiles of CPCL (10 μM) in the presence of indicated β-lactamases (SPM-1 and Bla-1, 1.0 unit/mL) in PBS (pH 7.4, 10%
DMSO) at room temperature; Inset shows the image of chemiluminiscence activation of CPCL (100 μM) in presence of SPM-1 (1.0 unit/mL) versus control; (B)
Relative total light units (RLU) emitted from CPCL (10 μM) in the presence of indicated β-lactamases (1.0 unit/mL) in PBS (pH 7.4, 10% DMSO) at room temperature
for 15 min. (C) Linear correlation between the total light emitted from CPCL (10 µM) and concentration of SPM-1 (PBS of pH 7.4, 10% DMSO).
benzoate species II by MS analysis. After a further 15 minutes of
incubation, a sharp decrease in the peak corresponding to phenol
I and a subsequent increase of the peak corresponding to
benzoate II was observed and after a total of 60 minutes, the
majority of phenol I was completely transformed to benzoate II.
Almost, no hydrolysis of probe CPCL was observed in the
absence of carbapenemase SPM-1. This analysis clearly
indicates that probe CPCL is hydrolysed by carbapenemase
SPM-1 through a 1, 8-elimination reaction that produces phenol I.
The latter undergoes rapid chemiexcitation to generate benzoate
II and a green photon.
calculated for KPC-1, VIM-15, and NMCA (See Supporting
information).
In addition, we also synthesized an analogous fluorescent probe
CPFL with 7-hydroxy-coumarin (umbelliferone) as the fluorescent
reporter, in order to compare the efficiency for detection of
carbapenemases (LOD) to CPCL. However, due to the higher
pKa value of hydroxy-coumarin, CPFL was unable to release
umbelliferone in the presence of the carbapenemases (See
Supporting information).[48]
After establishing the sensitivity and specificity of CPCL, we
proceeded to evaluate the ability of our probe to detect live
bacterial cells that can express various carbapenemases.
Imipenem resistant strains of Pseudomonas aeruginosa
(harboring the IMP-2 carbapenemase gene)[58] and Klebsiella
pneumoniae (harboring the KPC-2 carbapenemase gene)[59] were
selected for this purpose. IMP, one of the major types of the class-
B metallo β-lactamases, has been found worldwide. Similarly, the
KPC type belongs to a class-A serine carbapenemase family and
exhibits a broad hydrolysis spectrum against most β-lactam
antibiotics. Both these enzymes are capable of hydrolyzing the β-
lactam ring of carbapenems including imipenem and meropenem,.
Thus, IMP-2 and KPC-2 expressing bacteria can serve as
To better understand the activation and disassembly pattern of
the CPCL probe upon reaction with carbapenemase SPM-1, we
monitored the reaction products by RP-HPLC (Figure 4). CPCL
(100 µM in PBS at pH 7.4) appears in the chromatogram at a
retention time (Rt) of 16.45 minutes. Upon activation by SPM-1
(after 5 min reaction time), two new peaks are observed in the
chromatogram with Rt of 16.39 and 6.88 minutes. A careful
observation of the first peak (Rt = 16.39 minute) revealed that it
has a different UV absorption pattern to that of CPCL. The mass
spectrum (MS) analysis of this peak confirmed a value matching
phenol I (No value fit for CPCL was observed). Similarly, the
second peak at Rt = 6.88 minute, was confirmed to be the
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10.1002/chem.202000217
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RESEARCH ARTICLE
Figure 4. Activation of CPCL (100 μM) in the presence of SPM-1 (1.0 unit/mL) in PBS (pH 7.4, 10% DMSO) at room temperature, monitored by RP-HPLC; Inset
shows the UV absorption spectrum of an intermediate species formed in the activation pathway
models for detection of carbapenemase activity by our CPCL
chemiluminescence probe. A strain of Escherichia coli that does
not harbor any antibiotic resistant gene (imipenem sensitive) was
also examined, as well as a sterile control. Carbapenemase
expressing bacteria were cultivated for 16 h at 37°C on tryptic soy
chemiluminescence output produced by the imipenem resistant P.
aeruginosa and K. pneumoniae. Both cell suspension with 108
CFU/mL could be clearly detected by the CPCL probe. This cell
concentration is easily reached when colonies on an agar plate or
infected body parts are sampled. However 107 CFU/mL
agar plates (TSA) containing the carbapenem antibiotic imipenem. concentrations can also be detected easily by CPCL (See
The imipenem sensitive control strain was cultivated on TSA
without antibiotic. Colonies were resuspended in PBS (pH 7.4)
and light emission was measured in the presence of the CPCL
probe. Figure 5A clearly shows the intense light emission signal
produced by CPCL in the presence of imipenem-resistant
Pseudomonas aeruginosa and Klebsiella pneumoniae. In
contrast, light emission by carbapenemase negative E. coli was
significantly lower and was almost identical to that of the sterile
control. The total light emission produced by each bacterial strain
Supporting Information). To provide further support for the
observed carbapenemase activity by CPCL, we performed
inhibition experiments with known inhibitors. As can be seen in
Figure 5C, significant inhibition was obtained with EDTA for the
metallo- carbapenemase strain IMP-2 P. aeruginosa. The activity
of the non-metallo- carbapenemase strain KPC-2 K. pneumoniae
was completely inhibited by 3-amino-phenylboronic acid inhibitor,
a known inhibitor of serine-carbapenemases. The data obtained
so far provide solid support for the ability of CPCL to detect
carbapenemase activity and thereby, to conclusively confirm the
existence of CPOs in a test sample.
after 20 min is presented in Figure 5B.
A
similar
chemiluminescence assay was carried out in nutrient broth
medium (another widely used non-selective culture medium for
bacterial assays) instead of TSA (See Supporting Information).
The obtained results were very similar, with strong
In comparison to hydrolytically stable probes, the CPCL probe
suffers from higher background signal due to spontaneous
hydrolysis of the beta-lactam ring. However, it should be noted
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10.1002/chem.202000217
Chemistry - A European Journal
RESEARCH ARTICLE
that the sensitivity of clinical diagnostic assays is not as important
as in food safety. The reason being that food pathogens must be
detected at very low levels (1 CFU/mL) where as in clinical
samples the concentration of pathogens is much higher (> 10,000
CFU/mL). In addition, chemiluminescence-based detection assay
is much more matrix independent relative to fluorescence-based
assaying. Clinical samples tend to contain a lot of components
that cause background fluorescence, which render many of these
assays useless. For this reason, chemiluminescence probes,
even those
with high background, are often superior to fluorescence-based
ones in clinical diagnostics.
Carbapenemase detection by PCR, which is
a genotype
technique has also its own disadvantages. For example, to
perform a PCR test, one would need the proper primer in hand.
Carbapenemases are evolving rapidly and many new genetic
variants continuously emerge. Our proposed chemiluminescence
phenotypic testing is capable of detecting previously unknown
carbapenemases. Such detection is not possible with the PCR
technique. Hence, genotypic and phenotypic testing should
always be used in combination. In addition, PCR testing takes at
least several hours (even when done on already grown clinical
isolates) and requires a rather laborious workup with a need of
certain expertise, such as DNA extraction and RT-PCR.[60–62] On
the contrary, our chemiluminescence testing can be performed
within 15 min without the need of a skilled expertise and tests are
possible in simple, low-cost bench top luminometers.
Conclusions
In summary, we have developed the first chemiluminescence
probe for the detection of carbapenemase and carbapenemase-
producing organisms. The unique structural design of CPCL
enables the probe to rapidly disassemble via an efficient 1, 8-
elimination process upon hydrolysis of the β-lactam ring by a
carbapenemase. This disassembly process results in the release
of a luminophore that undergoes chemiexcitation to produce an
extremely bright light emission signal that enables a rapid and
selective detection of carbapenemase, in less than 10 minutes. In
practice, a variety of clinically important carbapenemases were
successfully detected using a micro plate reader. Probe CPCL
was also able to detect carbapenemase transfected bacteria,
cultured in the standard non-selective media widely used in
clinical diagnostics. Although the existing design and
chemiluminescence efficacy of the probe may be further improved,
we believe that the data presented here will be of great value in
developing future diagnostic assays for the rapid and accurate
detection of CPO infection.
Acknowledgements
We thank Dr. Mario Hupfeld of Nemis Technologies AG,
Switzerland for helpul suggestions during revision of the
manuscript.
Keywords: Chemiluminescecne • Carbapenemase • Molecular
Figure 5. (A)
Chemiluminescence of colonies from agar plates:
Probes • Dioxetanes
Chemiluminescence kinetic profiles of CPCL (10 μM, PBS pH 7.4, 5% DMSO)
in the presence of live β-lactamase expressing bacteria cells (5*108 CFU/mL) ;
(B) Relative total light emission within 20 min from CPCL (10 μM) in the
presence of live β-lactamase expressing bacteria cells (5*108 CFU/mL) min. (C)
Relative total light emission within 15 min from CPCL (10 μM) in PBS (pH 7.4,
5% DMSO) upon incubation with live β-lactamase expressing bacteria cells (109
CFU/mL) in the absence or presence of inhibitors (EDTA and 3-
aminophenylboronic acid, 3-APB, 10 mM). The data correspond to the average
of three independent biological experiments, error bars represent standard
deviations.
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This article is protected by copyright. All rights reserved.

10.1002/chem.202000217
Chemistry - A European Journal
RESEARCH ARTICLE
RESEARCH ARTICLE
Sayantan Das, Julian Ihssen, Lukas
Wick, Urs Spitz*, and Doron Shabat*
Page No. – Page No.
Chemiluminescence Carbapenem-
based Molecular Probe for Detection
of Carbapenemase Activity in Live
Bacteria
Design, synthesis, and activity of the first carbapenemase sensitive
chemiluminescent probe CPCL is described. The ability of the probe to detect a
number of clinically relevant carbapenemases as well as live bacteria with
carbapenemase genes (CPO) are successfully demonstrated.
This article is protected by copyright. All rights reserved.