Detection of Carbapenemase-Producing Organisms with a Carbapenem-Based Fluorogenic Probe

Article abstract of DOI:10.1002/anie.201612495

Antibiotic resistance has become a major challenge to public health worldwide. This crisis is further aggravated by the increasing emergence of bacterial resistance to carbapenems, typically considered as the antibiotics of last resort, which is mainly due to the production of carbapenem-hydrolyzing carbapenemases in bacteria. Herein, the carbapenem-based fluorogenic probe CB-1 with an unprecedented enamine–BODIPY switch is developed for the detection of carbapenemase activity. This reagent is remarkably specific towards carbapenemases over other prevalent β-lactamases. Furthermore, the efficient imaging of live clinically important carbapenemase-producing organisms (CPOs) with CB-1 demonstrates its potential for the rapid detection of antibiotic resistance and timely diagnosis of CPO infections.

Full text of DOI:10.1002/anie.201612495

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International Edition: DOI: 10.1002/anie.201612495
German Edition: DOI: 10.1002/ange.201612495
Antibiotic Resistance Hot Paper
Detection of Carbapenemase-Producing Organisms with
a Carbapenem-Based Fluorogenic Probe
Wuyu Mao, Lingying Xia, and Hexin Xie*
Abstract: Antibiotic resistance has become a major challenge
to public health worldwide. This crisis is further aggravated by
the increasing emergence of bacterial resistance to carbape-
nems, typically considered as the antibiotics of last resort,
which is mainly due to the production of carbapenem-hydro-
lyzing carbapenemases in bacteria. Herein, the carbapenem-
based fluorogenic probe CB-1 with an unprecedented enam-
ine–BODIPY switch is developed for the detection of carba-
penemase activity. This reagent is remarkably specific towards
carbapenemases over other prevalent b-lactamases. Further-
more, the efficient imaging of live clinically important
carbapenemase-producing organisms (CPOs) with CB-1 dem-
onstrates its potential for the rapid detection of antibiotic
resistance and timely diagnosis of CPO infections.
methods for CPO detection can be mainly grouped into three
categories, namely phenotypic, genotypic, and enzymatic-
activity-based methods. In clinical microbiology laboratories,
phenotypic, culture-based tests are the most broadly
employed methods, which may be due to their cost efficiency
and practicability. Widely used phenotypic tests for the
detection of CPOs include the modified Hodge test
[
8]
[9]
(MHT), the epsilometer test (E-test), the double disc
[10]
[11]
synergy test (DDST), and the Carba NP test. However,
most of the current phenotypic methods for CPO detection
are largely limited by the low specificity and sensitivity, and
the final results are usually only available after 24–48 hours,
or even later. Polymerase chain reaction (PCR) based
genotypic methods, on the other hand, can circumvent these
limitations, enabling the selective detection of CPOs within
a short period of time with primers for specific carbapene-
mase genes. Moreover, the application of real-time quantita-
tive reverse transcriptase PCR (rt qRT-PCR) can even enable
the detection of the mRNA expression of carbapenemase-
[
1]
C
arbapenems, such as imipenem, doripenem, ertapenem,
and meropenem, are traditionally considered as the anti-
[
2]
biotics of last resort for the treatment of severe infections
owing to their broad spectrum of activity against both Gram-
positive and Gram-negative bacterial pathogens and, more
importantly, their exceptional stability to most b-lactamases
[
12]
encoding genes. However, in spite of their high sensitivity
and accuracy, PCR-based genotypic methods suffer from high
costs and their inability to detect unknown carbapenemase
genes, which makes them less practical for daily screening
tests.
(
bla, b-lactam-hydrolyzing enzymes), including most
extended-spectrum b-lactamases (ESBls). Unfortunately,
however, bacteria with carbapenem resistance have become
increasingly prevalent throughout the world in recent
b-Lactamase activity tests offer valuable information on
the antibiotic resistance of bacteria. Fluorescent b-lactamase
probes, benefiting from high sensitivity, operational simplic-
ity, and relatively low costs, have attracted considerable
[1–3]
years.
The major mechanism of resistance to carbapenem
among Gram-negative bacteria is associated with the acquis-
ition/expression of carbapenemases, a group of specific
b-lactamases that are capable of hydrolyzing carbapenems,
as well as nearly all other b-lactam antibiotics. Since the first
[
13]
attention in this area. However, the majority of b-lactamase
probes are currently based on the core structure of cepha-
losporin and can be hydrolyzed by prevailing b-lactamases,
lacking specificity to carbapenemases. Rao and co-workers
recently reported elegantly designed carbapenemase-selec-
tive probes with a stereochemically altered cephalosporin as
[
4]
identification of a carbapenemase in 1993, a number of
carbapenemases, including Ambler class A b-lactamases
(
e.g., KPC, GES), Ambler class B b-lactamases (e.g., IMP,
VIM, NDM), and Ambler class D b-lactamases (e.g., OXA),
[
5]
[14]
have been isolated. The growing incidence of these carba-
the enzyme recognition motif. Nevertheless, these probes
[
6]
penemase-producing organisms (CPOs; e.g., Klebsiella,
Pseudomonas, Escherichia, Acinetobacter) poses a severe
threat to global public health.
structurally differ from carbapenems, and different substitu-
1
ents (R ) on the probes lead to specificity to different
carbapenemases (Figure 1b). We envisaged that a fluorogenic
substrate derived directly from carbapenem would match the
hydrolysis specificity of carbapenemases better and thus
provide higher detection accuracy. Herein, we wish to report
a carbapenem-based fluorogenic probe for the specific
detection of carbapenemases in CPOs.
Carbapenems, unlike cephalosporins, feature trans sub-
stituents on the b-lactam ring, which prevent hydrolysis by
most b-lactamases; these antibiotics can only be hydrolyzed
by carbapenemases. Inspired by the cephalosporin-based
fluorescent probes for the detection of b-lactamase activity,
we designed our carbapenemase-specific probe with the
carbapenem core structure as the enzymatic recognition
Early detection represents one of the most important
[
5a,7]
approaches to prevent the rapid spread of CPOs.
Current
[
*] W. Mao, L. Xia, Prof. Dr. H. Xie
State Key Laboratory of Bioreactor Engineering
Shanghai Key Laboratory of New Drug Design
School of Pharmacy
East China University of Science and Technology
Shanghai 200237 (P.R. China)
E-mail: xiehexin@ecust.edu.cn
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201612495.
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Scheme 1. Synthesis of the carbapenemase-specific fluorogenic probe
CB-1. Reagents and conditions: a) TBSCl, imidazole, DMF, RT, 5 h,
9
8%; b) Rh (OAc) , ZnCl , CH Cl , reflux, 1.5 h; c) TMP, DIPEA, Tf O,
2
4
2
2
2
2
À508C, 1.5 h; d) tributyl(vinyl)tin, P(2-furyl) , Pd (dba) , ZnCl , NMP,
3
2
3
2
3
1
08C, 10 h, 91% from 2; e) 5, Pd(OAc) , P(o-Tol) , TEA, DMF, 808C,
2 3
6 h, 24%; f) NH ·HF , NMP, DMF, RT, 36 h, 79%; g) Zn, THF/PB
4
2
(pH 6.0), 208C, 27%. dba=dibenzylideneacetone,
DIPEA=diisopropylethylamine, NMP=N-methyl-2-pyrrolidone,
PB=phosphate buffer, PNB=para-nitrobenzyl, TBS=tert-butyldime-
thylsilyl, TEA=triethylamine, TMP=2,2,6,6-tetramethylpiperidine,
Tf =trifluoromethanesulfonyl.
after 40 min, resulting in a sharp absorption peak at l =
503 nm, which matches that of unsubstituted BODIPY.
Accordingly, the probe solution turned from purple into
dark purple and eventually into pink. In the meantime,
fluorescence spectra of the probe were also recorded before
and after treatment with the enzyme (Figure 2b). CB-1 in
PBS is essentially non-fluorescent upon excitation at both l =
503 nm and 544 nm, and the addition of IMP-1 triggered the
turn-on of fluorescence at 512 nm with over 200-fold
enhancement. The high fluorescence turn-on ratio of CB-
1 is particularly valuable for the detection of low-abundance
enzymes.
Encouraged by these results, we conducted further inves-
tigations into the enzymatic hydrolysis of CB-1. We first
monitored the reaction process by high performance liquid
chromatography (HPLC). As shown in the Supporting
Information, Figure S1, after incubation with IMP-1 for
5 min, a major intermediate with broad absorption at about
540 nm was detected, which was further suggested by liquid
chromatography mass spectrometry (LC-MS) to be the
b-lactam-hydrolyzed compound I. This dienamine disap-
peared completely after 60 min, giving rise to a small
amount of fluorescent aldehyde-substituted BODIPY III
(Figure S1b; confirmed by LC-MS and HPLC analysis with
standard compound as reference) and a mixture of more polar
compounds. All of these compounds gave rise to the
characteristic absorption of unsubstituted BODIPY, implying
that the vinyl conjugation of BODIPY had been disrupted,
even though the exact structures of these compounds remain
to be determined at the present stage. These results further
supported our initial hypothesis.
Figure 1. Design of carbapenemase-specific fluorescent probes.
moiety. Furthermore, considering that most carbapenems
lack leaving groups, we employed an alkenyl-linked boron
dipyrromethene (BODIPY) dye as the activatable fluoro-
phore. We envisaged that this carbapenem–BODIPY probe
(
CB-1) would initially be non-fluorescent upon excitation at
[15]
l = 503 nm (absorbance peak of unsubstituted BODIPY),
and carbapenemase-specific hydrolysis of the probe would
generate the labile dienamine I, which undergoes further
spontaneous isomerization or degradation processes that
disrupt the conjugation between carbapenem and BODIPY,
so that the strong green fluorescence of BODIPY is observed
(
Figure 1c).
To test the feasibility of our hypothesis, we synthesized
CB-1 from the commercially available meropenem inter-
mediate 1 (Scheme 1). Upon protection with tert-butyldime-
thylsilyl chloride, the resulting compound 2 was treated with
rhodium(II) acetate followed by trifluoromethanesulfonic
anhydride in the presence of a base to afford 2-triflated
[16]
carbapenem 3. A Stille coupling reaction was employed to
introduce the vinyl group, furnishing key intermediate 4 in
[
17]
9
1% yield over three steps. The BODIPY core was then
[
18]
incorporated in a Heck coupling. Finally, removal of the
[
19]
TBS and PNB protecting groups provided probe CB-1.
With CB-1 in hand, we first examined its absorbance in
phosphate buffered saline (PBS, pH 7.4). A broad absorption
peak at l = 544 nm as well as two minor peaks at 341 nm and
4
33 nm were observed (Figure 2a). Upon addition of 100 nm
of carbapenemase IMP-1, a slight red shift was observed after
min. However, all of these characteristic bands disappeared
Next, we probed the specificity of CB-1 towards carba-
penemases over other b-lactamases. The addition of as little as
1
2
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.1 nm of IMP-1 resulted in an over 20-fold enhancement of
fluorescence intensity within 1 h, while the presence of 100 nm
of TEM-1 (the most common b-lactamase found in Gram-
negative bacteria, class A bla) induced an only marginal
increase in fluorescence under identical conditions (Fig-
ure 3a). To gain more information on the enzymatic specific-
ity of CB-1, more clinically important b-lactamases were
examined. As expected, all of the carbapenemases, including
IMP-1, VIM-27 (class B bla), NDM-1 (class B bla), KPC-3
(class A bla), and OXA-48 (class D bla), generated significant
fluorescence while, impressively, other b-lactamases (TEM-1,
TEM-3, and CTX-M-9) yielded only negligible fluorescence
even at a concentration of 100 nm (Figure 3b), which is as
much as 1000 times higher than the concentration of most
aforementioned carbapenemases. These results demonstrate
the exceptionally high selectivity of CB-1, which enables the
reliable detection of carbapenemases in the presence of other
prevalent b-lactamases.
To further validate that the fluorescence turn-on of
CB-1 is specifically induced by carbapenemase activation,
an inhibition assay was also conducted. Ethylenediaminete-
[20]
traacetic acid (EDTA), an effective inhibitor for all metal-
dependent class B b-lactamases, significantly reduced the
fluorescence enhancement mediated by IMP-1, NDM-1, and
VIM-27, but remained ineffective for serine-dependent KPC-
Figure 2. Optical properties of CB-1. a) UV/Vis spectra of CB-1 (10 mm
in PBS, pH 7.4) before and after incubation with IMP-1 bla (100 nm) at
3
(Figure 3c). On the other side, phenylboronic acid (PBA)
2
58C. Photographs of CB-1 upon incubation with IMP-1 bla are shown
selectively inhibited the activity of KPC-3, leaving all other
metal-dependent carbapenemases unaffected. These data
confirm that the fluorescence intensification of CB-1 is due
to probe hydrolysis by specific carbapenemases.
as insets. b) Fluorescence spectra of CB-1 (1 mm in PBS, pH 7.4) upon
incubation with IMP-1 bla (100 nm) at 258C (l =503 nm). Fluores-
ex
cence images of CB-1 (5 mm) before and after incubation with IMP-
1
bla (100 nm) at l =365 nm are shown as insets.
ex
Figure 3. Fluorescence response of CB-1 to different b-lactamases. a) Time dependence of the fluorescence of CB-1 (5 mm in PBS, pH 7.4) upon
incubation with the indicated b-lactamases. b) Changes in the fluorescence intensity of CB-1 (5 mm in PBS, pH 7.4) upon incubation with a range
of b-lactamases for 30 min. c) Changes in the fluorescence intensity of CB-1 (5 mm in PBS, pH 7.4) upon incubation with the indicated
carbapenemases (0.2 nm) in the presence of inhibitors. d) Time dependence of the fluorescence of CB-1 (5 mm in PBS, pH 7.4) in the presence of
various b-lactamases. e) Time dependence of the absorbance of imipenem (300 mm in PBS, pH 7.4) in the presence of various b-lactamases. Data
were collected after incubation for 30 min. l /l =500/535 nm; error bars indicate Æstandard deviation.
ex em
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CB-1 is a structural analogue of carbapenems. To deter-
mine whether the hydrolysis specificity of the probe matches
that of carbapenems, imipenem was selected as an example.
The hydrolysis of imipenem by b-lactamases was monitored
with a UV/Vis spectrophotometer. The overall hydrolysis
profiles of CB-1 and imipenem are very similar (Figure 3d,e).
Significant hydrolysis can only be mediated by carbapene-
mases although the hydrolysis of imipenem obviously
requires higher enzyme concentrations to be detected.
These results support our hypothesis that the carbapenem-
based fluorogenic reagent delivers more accurate information
on carbapenemase activity.
Sensitivity is critical for a probe applied in microbiology
laboratories or in the clinic. With a microplate reader, the
detection limit of CB-1 for IMP-1 within 30 min was
determined to be 1.1 pm (Figure S2). Analogously, the
detection limits for VIM-27, NDM-1, and KPC-3 were
calculated to be 7.7 pm, 2.7 pm, and 4.0 pm, respectively.
Incubation for prolonged periods of time can further improve
the detection sensitivity (e.g., the detection limit for NDM-
Figure 4. Fluorescence response of CB-1 to various live b-lactamase-
expressing bacteria. a) Fluorescence imaging of the indicated probes
6
upon incubation with transformed E. coli (10 CFUs) in PBS (pH 7.4)
at room temperature for 2 h. 1: VIM-27 E. coli; 2: IMP-1 E. coli; 3: KPC-
1
after 60 min was 1.3 pm).
3
E. coli; 4: TEM-1 E. coli; 5: b-lactamase-negative E. coli; l =365 nm;
ex
Having confirmed the suitability of CB-1 for the detection
to carbapenemases, we wondered whether this probe could be
applied in the screening of CPOs. A series of Escherichia coli
E. coli) transformed with a variety of carbapenemases were
pseudo color. b) Enhanced fluorescence of CB-1 (5 mm in PBS, pH 7.4)
after incubation with a range of antibiotic-resistant bacteria for 2 h.
RFI=relative fluorescence intensity. NDM-1-Kp: NDM-1 Klebsiella
pneumoniae (ATCC BAA2146); VIM-1-Kp: VIM-1 Klebsiella pneumoniae
(
(
(
NCTC 13440); MDR-Ab: multidrug-resistant Acinetobacter baumannii
ATCC BAA1605); OXA-48-Kp: OXA-48 Klebsiella pneumoniae (NCTC
first evaluated (Figure 4a). All carbapenemase-encoded
E. coli incubated with CB-1 showed intense fluorescence
whereas the TEM-1 E. coli, as well as b-lactamase-negative
E. coli, exhibited little fluorescence. As a control, the non-
13442); TEM-3-E. coli: TEM-3 Escherichia coli (NCTC 13351); CTX-M-9-
Ec: CTXM-9 Enterobacter cloacae (NCTC 13464); SHV-18-Kp: SHV-18
Klebsiella pneumoniae (ATCC 700603); TEM-1-E. coli: TEM-1 Escherichia
coli (ATCC 35218); E. coli: b-lactamase-negative Escherichia coli
[
13g]
specific fluorogenic probe CDC-1
was used, which
(
LMG194). l /l =500/535 nm; the data correspond to the average
responds to all b-lactamase-expressing microbes by emitting
strong fluorescence. Inspired by these results, we further
tested clinically important CPOs with CB-1. Four strains of
CPOs, as well as four strains of other antibiotic-resistant
ex em
of three independent experiments. CFUs=colony-forming units.
bacteria and antibiotic-susceptible E. coli, were selected for Acknowledgements
this study. As expected, CB-1 showed excellent specificity in
CPO detection. Only CPOs generated substantial fluores-
cence whereas other microbes hardly enhanced the fluores-
cence intensity (Figure 4b). The ability to distinguish CPOs
from other bacterial strains is of great importance for
microbiological investigations or clinical applications.
In summary, we have developed a highly selective probe
with a carbapenem core structure for the detection of
carbapenemase activity. This fluorogenic reagent reproduces
the enzymatic hydrolysis profiles of carbapenems, and
This study is financially supported by the National Natural
Science Foundation of China (21472048), the Fundamental
Research Funds for the Central Universities, the “Thousand
Plan” Youth Program, and the East China University of
Science & Technology. We thank Prof. Jianghong Rao at
Stanford University for kindly providing b-lactamase plas-
mids.
fluorescence enhancements by factors of over 200 can be Conflict of interest
observed upon specific activation by carbapenemases.
CB-1 benefits from its green fluorescence, reducing potential
interference from the autofluorescence of bacteria or the
culture medium. While further refining may still be necessary,
this new carbapenem-based fluorogenic probe holds great
promise for the timely and accurate detection of CPOs in
microbiology laboratories or the diagnosis of CPO-infected
diseases. Moreover, this study has demonstrated the unpre-
cedented use of caged enamine–BODIPY derivatives as
molecular switches with fluorescence turn-on, which might
inspire a general strategy for the design of BODIPY-based
fluorogenic imaging reagents.
The authors declare no conflict of interest.
Keywords: antibiotic resistance · carbapenemases ·
fluorescent probes · b-lactamases
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Manuscript received: December 23, 2016
Revised: January 27, 2017
Final Article published: && &&, &&&&
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Antibiotic Resistance
W. Mao, L. Xia, H. Xie*
&&&& — &&&&
Detection of Carbapenemase-Producing
Organisms with a Carbapenem-Based
Fluorogenic Probe
Lighting up resistance: A fluorogenic
probe for the detection of carbapenemase
activity has been developed. The utiliza-
tion of carbapenem as the enzyme rec-
ognition motif in this reagent leads to
remarkable specificity to carbapene-
mases over other prevailing b-lacta-
mases, enabling the rapid detection of
carbapenemases.
6
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Angew. Chem. Int. Ed. 2017, 56, 1 – 6
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