Ratiometric fluorescence detection of pathogenic bacteria resistant to broad-spectrum β-lactam antibiotics

Article abstract of DOI:10.1002/anie.201107810

Identification of bacteria: A methoxyimino cephalosporin derivative containing a pair of fluorescence resonance energy transfer (FRET) fluorophores was synthesized. This probe displays selective cleavage toward different types of β-lactamases, thereby pro

Full text of DOI:10.1002/anie.201107810

Angewandte
Chemie
DOI: 10.1002/anie.201107810
b-Lactam Antibiotics
Ratiometric Fluorescence Detection of Pathogenic Bacteria Resistant
to Broad-Spectrum b-Lactam Antibiotics
Junxiang Zhang, Yang Shen, Sarah L. May, Daniel C. Nelson, and Shuwei Li*
b-Lactam antibiotics have been the frontline drugs to fight
bacterial infections because of their high efficacy and low
toxicity ever since they were first discovered in the 1920s.
These agents confer their antimicrobial ability by inhibiting
transpeptidases, also known as penicillin binding proteins
(PBPs), the activity of which is required for bacterial cell wall
synthesis. To date, hundreds of b-lactam compounds have
been synthesized and evaluated for their antibacterial proper-
ties, including derivatives of penicillin, cephalosporin, mono-
bactam, and carbapenem.[1] As one of the primary strategies
to survive in the presence of these antibiotics, bacterial cells
can acquire b-lactamases, a family of highly efficient enzymes
that are able to destroy b-lactam structural moieties before
they interact with PBPs.[2] Hence, inactivation or inhibition of
potential b-lactamases is a key consideration for the treat-
ment of bacterial infections.
There are many types of b-lactamases, each with distinct
expression patterns and catalytic mechanisms. One of the
most commonly encountered b-lactamases is TEM-1.[3] Pro-
duction of this plasmid-harboring serine hydrolase accounts
for most cases of penicillin resistance in Gram-negative
bacteria, such as Escherichia coli and Klebsiella pneumoniae.
AmpC, also a serine hydrolase, is an inducible chromosomally
encoding enzyme that is often observed in Enterobacter and
Citrobacter species. On the other hand, metallo-b-lactamases,
like the newly isolated NDM-1, require a zinc ion in their
active site for enzymatic activity.[4]
active site of TEM-1, thereby conferring resistance to TEM-
1.[5] Unfortunately, these “next generation” antibiotics can
still be efficiently cleaved by newly emerging b-lactamases
such as NDM-1.[6] In addition, bacteria can readily develop
antibiotic resistances by evolving novel mechanisms to extend
the effectiveness of their current repertoire of b-lactamases.
For example, as few as one or two point mutations to key
residues of TEM-1 can noticeably improve its ability to
degrade methoxyimino cephalosporins. These TEM-1
mutants with enhanced activity are often referred to as
extend-spectrum b-lactamases (ESBLs).[7] Similarly, hyper-
expression of AmpC also causes its hosts to become more
refractory to broad-spectrum cephalosporins.[8]
Since the development of bacterial drug resistance is
tightly associated with the misuse/overuse of antibiotics, it
would be desirable to reserve broad-spectrum antibiotics (e.g.
third- and fourth-generation cephalosporins) for multidrug-
resistant infections only and avoid using them on bacterial
strains sensitive to narrow-spectrum agents (e.g. ampicillin).
To achieve this goal, a diagnostic must be capable of
determining bacterial susceptibility toward various b-lactam
drugs, so a clinical decision on the selection of antibiotics can
be made accordingly. This task is currently achieved by
standard methods like the E-test and the double-disc test.[9]
These approaches usually take 48–72 h, or longer for slow-
growing bacteria like Mycobacterium tuberculosis, which is
often too slow to offer therapeutic guidance. A new Cica-b-
test, based on a chromogenic b-lactam derivative, is able to
shorten the diagnostic period to 24 h. However, its sensitivity
has been reported to be low.[10] PCR and other DNA-based
diagnostic methods are both rapid and sensitive, yet they are
not functional assays and can only detect resistance mecha-
nisms derived from well-characterized genes.[11] As a result,
novel approaches with better sensitivity, requiring less time,
and capable of detecting multidrug-resistant bacteria are still
actively sought to prolong the useful lifespan of broad-
spectrum b-lactam antibiotics.
It is of great medical importance to prevent b-lactamases
from hydrolyzing b-lactam drugs. One way to counteract
these enzymes is to concurrently administer both a b-lactam
drug and a b-lactamase inhibitor. Several prescription drugs
utilize this strategy, including Unasyn (ampicillin plus sulbac-
tam) and Augmentin (amoxicillin plus clavulanate). Another
general approach is to develop b-lactams that are not
hydrolyzable by b-lactamases. Many third- and fourth-gen-
eration cephalosporins, in which a methoxyimino group
attaches to the b-lactam ring, are too large to fit into the
Currently, many fluorogenic b-lactamase substrates are
available, which allow the detection of b-lactamases with high
sensitivity and in real time.[12] Yet, most of these molecules are
hydrolyzable by all types of b-lactamases; only a few show
selective cleavage toward different b-lactamases.[13] So exist-
ing b-lactamase substrates cannot distinguish enzymes with
extended substrate profiles from those with narrow-spectrum.
Herein, we describe the synthesis of a novel fluorescent b-
lactamase substrate (C-2; Figure 1). This compound contains
a methoxyimino side chain, so it is resistant to TEM-1, but
should be susceptible to enzymes like NDM-1 and over-
expressed AmpC. Indeed, C-2 (50 mm) only degraded slightly
when incubated with highly purified TEM-1 (1 mg). By
[*] Dr. J. X. Zhang, Y. Shen, S. L. May, Prof. D. C. Nelson, Prof. S. W. Li
Institute for Bioscience and Biotechnology Research
University of Maryland College Park
9600 Gudelsky Drive, Rockville, MD 20850 (USA)
E-mail: sli@umd.edu
Prof. S. W. Li
Department of Chemistry and Biochemistry
University of Maryland College Park (USA)
Prof. D. C. Nelson
Department of Veterinary Medicine
University of Maryland College Park (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201107810.
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Figure 1. Structure of C-1, C-2, and their hydrolyzed products. C-1
(50 mm) is degraded by 1 mg TEM-1 completely within 30 s. By
contrast, C-2 (50 mm) is only slightly hydrolyzed under the same
conditions after 4 h. lex =440 nm. RFU=relative fluoresence unit.
Scheme 1. Synthesis of C-2 (or C-1). a) Ph2CN2, TsOH. b) Trt-Gly-OH,
HBTU, HOBt. c) 5% TFA, 10% TIS. d) Orange peel acetylesterase,
PBS buffer, pH 5.5. e) Ph2CN2, MeCN, pH 3. f) Trt-Gly-OSu. g) 4-nitro-
phenyl chloroformate. h) F-1, DIPEA. i) 5% TFA, 10% TIS. j) F-2,
DIPEA. DIPEA=N,N-diisopropylethylamine, HBTU=2-(1H-benzotria-
zol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphat, Bt=benzotria-
zole, TFA=trifluoroacetic acid, TIS=triisopropylsilane, Trt=trityl,
contrast, its counterpart without this bulky methoxyimino
group (C-1) was instantly degraded under the same condition.
Consequently, C-2 should easily distinguish b-lactamases Ts =4-toluenesulfonyl.
capable of hydrolyzing methoxyimino cephalosporins from
those lacking this activity.
The synthesis of C-2 started from cefotaxime, a widely- between F-1 and F-2 fluorophores in its structure. When C-
used cephalosporin drug containing a characteristic 2-amino- 2 is hydrolyzed by a b-lactamase, this FRET will disappear as
thiazol-4-yl-methoxyimino structure that is shared by many one fluorophore moves away from the other. As a result, the
third-generation antibiotics (Scheme 1). Once its free car- degradation of C-2 is accompanied by the decrease of
boxylate was masked by forming benzhydryl ester, cefotax- fluorescence at 590 nm and the increase of fluorescence at
ime was coupled with a trityl-protected glycine (Trt-Gly-OH) 520 nm (Figure 2A). This design allows us to follow the
to give I-1. The benzhydryl and trityl groups on I-1 were hydrolysis of C-2 based on the ratio of fluorescence intensity
removed by 5% TFA, and its acetyl was then removed by an at two wavelengths. Compared to other methods that only
acetylesterase fleshly prepared from orange peel to yield I-2. measure absolute fluorescence intensity, the ratiometric
This cleavage of benzhydryl and trityl before enzymatic approach represents unique advantages. The ratio of two
treatment is necessary because I-1 itself does not dissolve in fluorescence signals is independent on the amount of
aqueous solution required by the acetylesterase. The carbox- substrate present, but proportional to the activity of the
ylate on I-2 was masked again by benzhydryl and its amine enzymes. In addition, because fluorescence variations caused
was coupled to trityl-protected glycine N-hydroxysuccinimide by instruments and other unpredictable factors are cancelled
ester (Trt-Gly-OSu) under mild conditions. Then, its free out, the quantitative determination of enzyme activity is more
hydroxy group was activated by reacting with 4-nitrophenyl accurate and reliable, which in turn improves the sensitivity of
chloroformate to provide I-3. After I-3 was coupled with a detection. Using this substrate, we tested b-lactamase activity
fluorescent compound F-1, its benzhydryl and trityl groups from several bacterial species, including Enterobacter cloacae
were removed, which exposed an amine to couple with (AmpC positive, inducible), Klebsiella pneumoniae (NDM-1
another fluorescent compound F-2 to offer the final product positive), and Enteric group 137 (ESBL positive). The
C-2. The synthesis of C-1 followed the same scheme, but enzymatic activity was monitored by measuring time-depen-
started from 7-aminocephalosporanic acid (7-ACA) instead. dent fluorescence changes at 520 and 590 nm. We plotted
C-2 is a fluorescent molecule with a single emission peak (F590/F520)/(F590/F520
) (y axis) versus time (x axis) of these
S
at 590 nm when it is excited at 440 nm. This is caused by samples. Here F520 and F590 are the fluorescence intensities at
efficient fluorescence resonance energy transfer (FRET) 520 and 590 nm. F590/F520 is the ratio of the sample containing
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Whether this trend can be extended to all bacterial species at
this level of sensitivity remains to be determined.
Even though AmpC (when hyper-expressed), NDM-1,
and ESBLs all have high activity hydrolyzing C-2 and other
methoxyimino cephalosporins, they use different mechanisms
and can be inhibited by different small molecules. For
example, phenylboronic acid is a potent inhibitor of AmpC.
Likewise, EDTA can chelate zinc ions from NDM-1, thereby
inactivating this metallo-b-lactamase. Most ESBLs derived
from TEM-1 remain susceptible to clavulanate although some
ESBL mutants also acquire new capabilities to resist clav-
ulanate and other TEM-1 inhibitors. By combining C-2 with
these substances, we should be able to determine the subclass
of b-lactamase present to provide more information for
therapeutic determination. As expected, different classes of
b-lactamases showed distinct responses when incubated with
these inhibitors, suggesting the type of a b-lactamase from an
untested bacterial strain could be quickly assigned (Fig-
ure 2D).
Finally, we tested whether C-2 can be used to detect
bacteria from blood directly. Microbial infection in the
circulatory system, which is usually a sterile environment,
can have serious consequences. Bacterial cells can easily
spread to other parts of the body to establish new colonies and
cause secondary infections. The immune system can also elicit
whole-body inflammatory responses, leading to life-threat-
ening sepsis and septic shock conditions. Therefore, detecting
blood-borne bacterial cells is of clinical importance. As shown
in Figure 3, we were able to detect the same bacterial species
expressing various classes of b-lactamases in the presence of
Figure 2. Quantitative determination of b-lactamase activity from dif-
ferent bacterial species. A) Time-dependent fluoresence change at a
10 min interval when C-2 was incubated with E. cloacae (inducible
AmpC) cell lysate. B) Time course of the b-lactamase activity from
Enterobacter cloacae (inducible AmpC), Klebsiella pneumoniae (NDM-
1), Enteric group 137 (ESBL), and Escherichia coli (TEM-1). RR
(relative ratio)=(F590/F520)/(F590/F520)S. C) Minimal number of cells
(5ꢀ105 to 1ꢀ106) required for detecting b-lactamase activity above
background (RR<0.95, horizontal dash line) dertermined by a set of
serial dilution experiments. D) Inhibition profiles of different classes of
b-lactamases against various inhibitors. EDTA, ethylenediaminetetra-
acetic acid; PhB(OH)2, phenylboronic acid.
both the substrate and a b-lactamase of interest, whereas
(F590/F520)S is the ratio of the substrate itself under the same
condition. By calculating (F590/F520)/(F590/F520)S, we can com-
pensate variations and compare results measured at different
time points and on various instruments. The value of (F590
F520)/(F590/F520 roughly ranges from 0.2 to 1.0, which
/
)
S
corresponds to the degree of C-2 degradation. If all conditions
are kept constant when testing the activity of several b-
lactamases, a lower value means a more efficient enzyme to
hydrolyze C-2. Clearly, C-2 was able to distinguish bacterial
strains capable of degrading methoxyimino cephalosporin
derivatives from those strains lacking this activity (Fig-
ure 2B).
Clinically, the time period between the specimen collec-
tion and the determination of drug resistance should be as
short as possible. In this aspect, a more sensitive assay would
certainly be favorable. To find out the detection limit of the C-
2 based assay, we performed an experiment in which the b-
lactamase activity was measured on a set of serial dilutions of
Figure 3. Detection of various b-lactamases in the presence of whole
blood. For each b-lactamase, C-2 was incubated with a mixture of
sheep blood and a bacterial cell lysate, then its fluorescence was
recorded.
bacterial cells (Figure 2C). The value of (F590/F520)/(F590/F520
)
S
at 0.95 under this experimental condition was considered
reflecting the minimal b-lactamase activity above the back-
ground. With this criterion, we concluded that 5 ꢀ 105 to 1 ꢀ
106 cells were required to reliably determine whether a
bacterial strain is resistant to methoxyimino cephalosporins.
The activity of b-lactamases in different bacteria is dependent
on many factors such as the subfamily of b-lactamases present
and their relative expression level. Therefore, our result here
only serves as an example to show that C-2 can detect broad-
spectrum b-lactamases from a small number of bacterial cells
that we studied and only in a limited number of species.
blood, but we can no longer use (F590/F520)/(F590/F520
) to
S
compare the enzymatic reactivity from different samples. The
oxy- and deoxy-hemoglobin in blood can absorb visible light
strongly (up to 600 nm), so the fluorescence signal at 520 nm
is almost completely suppressed and that at 590 nm is
attenuated. Therefore, the decrease of fluorescent signal at
590 nm alone is used to estimate the b-lactamase activity in a
sample, which loses the benefits offered by the ratiometric
measurement. The sensitivity is also compromised owing to
this absorption, as well as shorter half-life of the substrate in
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blood. To improve the detection of bacterial cells in blood, we
will develop b-lactam probes incorporating FRET fluoro-
phores at the near-infrared region (800–1000 nm) in future
studies since blood has minimal absorbance in this window.
The Cica-b-test is not compatible with blood test because it
relies on a chromogenic substrate whose color change upon
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We are losing the war to fight bacterial infections. The
recently discovered NDM-1 and its global dissemination is an
alarming example as most bacteria expressing NDM-1 are
only susceptible to a few effective antibiotics that are usually
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will delay the emergence of multidrug-resistant superbugs.
The ratiometric fluorescence measurement reported here is a
quick and sensitive procedure for screening microbes resist-
ant to methoxyimino cephalosporins. This assay would
provide useful advice for antibiotic selection in a timely
fashion and prevent unnecessary use of broad-spectrum
antimicrobial agents. In conjunction with classic microbio-
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Received: November 6, 2011
Published online: January 13, 2012
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Keywords: antibiotics · cephalosporins ·
drug-resistant bacteria · fluorescence · b-lactamases
.
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