[health-vn] Meropenem-Clavulanate Is Effective Against Extensively Drug-Resistant Mycobacterium tuberculosis

[Vern Weitzel]

http://www.sciencemag.org/cgi/content/full/323/5918/1215?ijkey=VhYbFM5CLCF9k&keytype=ref&siteid=sci
See tables in Internet web page.

Science 27 February 2009:
Vol. 323. no. 5918, pp. 1215 - 1218
DOI: 10.1126/science.1167498
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REPORTS
Meropenem-Clavulanate Is Effective Against Extensively Drug-Resistant 
Mycobacterium tuberculosis
Jean-Emmanuel Hugonnet,1 Lee W. Tremblay,1 Helena I. Boshoff,2 Clifton E. Barry, 
3rd,2 John S. Blanchard1*
β-lactam antibiotics are ineffective against Mycobacterium tuberculosis, being 
rapidly hydrolyzed by the chromosomally encoded blaC gene product. The 
carbapenem class of β-lactams are very poor substrates for BlaC, allowing us to 
determine the three-dimensional structure of the covalent BlaC-meropenem 
covalent complex at 1.8 angstrom resolution. When meropenem was combined with 
the β-lactamase inhibitor clavulanate, potent activity against laboratory 
strains of M. tuberculosis was observed [minimum inhibitory concentration 
(MICmeropenem) less than 1 microgram per milliliter], and sterilization of 
aerobically grown cultures was observed within 14 days. In addition, this 
combination exhibited inhibitory activity against anaerobically grown cultures 
that mimic the "persistent" state and inhibited the growth of 13 extensively 
drug-resistant strains of M. tuberculosis at the same levels seen for 
drug-susceptible strains. Meropenem and clavulanate are Food and Drug 
Administration–approved drugs and could potentially be used to treat patients 
with currently untreatable disease.

1 Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris 
Park Avenue, Bronx, NY 10461, USA.
2 Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, 
National Institute of Allergy and Infectious Diseases, National Institutes of 
Health, Bethesda, MD 20892, USA.


* To whom correspondence should be addressed. E-mail: blanchar at aecom.yu.edu

Tuberculosis is perhaps the most persistent human disease caused by an 
infectious bacterium, Mycobacterium tuberculosis. The death toll remains 
extremely high, despite the introduction of modern multidrug chemotherapy in the 
1960s, with between 1.6 and 2 million fatalities annually. An increasing 
percentage of human clinical isolates are drug-resistant or multidrug-resistant 
strains that threaten the ability to treat the disease (1). The continued use of 
multidrug therapy has caused an even more dire problem: strains of M. 
tuberculosis resistant to all first-, second-, and third-line agents. In a 
recent study from South Africa, 54 of 54 patients infected with such highly 
resistant strains died with a mean survival time from diagnosis of 16 days (2).

Since the discovery of penicillin in 1929 (3), the β-lactam class of antibiotics 
has included some of the most clinically important antibacterial agents. The 
development of broad-spectrum derivatives of penicillin, such as the 
cephalosporins and olivanic acid (4), coupled with their low inherent toxicity 
have made them the drugs of choice for the treatment of both Gram-negative and 
Gram-positive bacterial infections. This class, however, has never provided a 
compound useful in the treatment of tuberculosis, and β-lactams are only rarely 
used in the treatment of this disease. One important reason for the lack of 
efficacy was found in the genome sequence of M. tuberculosis, which contains a 
single, highly active, chromosomally encoded class A (Ambler) β-lactamase (5). 
Recently a genetic knockout of the blaC-encoded β-lactamase showed that strains 
lacking this enzyme were more sensitive to β-lactams (6). This suggested that 
the chemical recapitulation of the genetic knockout could similarly resensitize 
the organism to existing β-lactam antibiotics.

We recently cloned and expressed the M. tuberculosis blaC gene and reported a 
detailed enzymatic characterization (7). BlaC exhibits an exceptionally broad 
substrate specificity, hydrolyzing penicillins at nearly the diffusion-limited 
rate, all classes of cephalosporins, and, unexpectedly for a class A 
extended-spectrum β-lactamase, imipenem and meropenem, both carbapenems. Equally 
unexpected, the enzyme was only transiently inhibited by the β-lactamase 
inhibitors sulbactam and tazobactam, penicillanic acid sulfones with potent 
inhibitory activity against other class A β-lactamases. However, clavulanic acid 
is the only Food and Drug Administration (FDA)–approved β-lactamase inhibitor 
that irreversibly inhibits BlaC, suggesting that clavulanic acid may 
recapitulate the genetic knockout, rendering M. tuberculosis susceptible to 
β-lactam antibiotics.

We have previously shown that meropenem was an extremely slow substrate for M. 
tuberculosis BlaC, being hydrolyzed five orders of magnitude slower than 
ampicillin. A more detailed investigation of the kinetics of meropenem 
hydrolysis under near stoichiometric enzyme concentrations revealed a 
steady-state burst with a magnitude dependent on the concentration of BlaC (Fig. 
1A). The reaction of meropenem with the enzyme to form the acyl-enzyme 
intermediate (acylation half-reaction) is fast relative to hydrolysis of the 
substrate (deacylation). Extrapolation of the final, linear rate to zero time 
revealed that enzyme acylation was stoichiometric with meropenem. At a single 
catalytic enzyme concentration, the linear rates yielded plots typical of 
Michaelis-Menten kinetics (Fig. 1B), with Michaelis constant Km = 3.4 ± 0.7 µM 
and turnover number kcat = 0.08 ± 0.01 min–1. Because of its extremely slow 
turnover rate, we investigated the possibility that meropenem could act as an 
inhibitor of BlaC and whether it was possible to trap the covalently acylated 
form of the enzyme. Meropenem acts as a slow, tight-binding inhibitor of the 
hydrolysis of the chromogenic β-lactam nitrocefin by BlaC. The time courses of 
nitrocefin hydrolysis are nonlinear in the presence of meropenem, and an 
analysis of these data yielded an inhibition constant (Ki) value of 16 ± 2 µM 
and a Ki*value of 1.1 ± 0.8 µM (fig. S1, A and B). The ability of meropenem to 
act as an inhibitor of BlaC in addition to being a very poor substrate for BlaC 
added to its potential as an active partner with clavulanate.



	Fig. 1. Kinetics of BlaC with meropenem. (A) Time courses of meropenem 
hydrolysis with various concentrations of BlaC. Enzyme concentrations are 
reported on the right. (B) Michaelis-Menten kinetics of BlaC with meropenem at 
single enzyme concentration (0.8 µM). (C) Mass spectra of enzyme-meropenem 
species. The 25+ charge state ions are shown. The mass reported for each peak 
was calculated as described in (20) from the two ions with m/z values of 
1165.946 and 1167.702. [View Larger Version of this Image (22K GIF file)]

The rapid acylation and slow deacylation of BlaC by meropenem suggested that we 
could observe the covalently bound species by Fourier transform ion cyclotron 
resonance (FTICR) mass spectrometry. A freshly prepared solution of BlaC and 
meropenem displayed a peak corresponding to the mass of the covalently acylated 
BlaC-meropenem complex [charge/mass (m/z) = 29,167.5] and a second peak whose 
mass corresponded to the mass of the covalently acylated BlaC-meropenem complex 
–44 (m/z = 29,123.6) (Fig. 1C). After 7 min of incubation, both these peaks 
decreased in intensity with the corresponding appearance of the free enzyme. 
Small-molecule mass spectrometry revealed the presence of two species, one with 
the expected mass for hydrolyzed meropenem (m/z = 402) and another with a mass 
44 mass units smaller (m/z = 358, fig. S2). Hydrolysis of meropenem in 1 N NaOH 
followed by mass spectrometry revealed only the presence of hydrolyzed 
meropenem. Together, these experiments suggest that, after β-lactam ring 
opening, the covalently bound meropenem partitions between direct hydrolysis and 
enzyme-catalyzed decomposition of the C6 hydroxyethyl substituent, yielding 
acetaldehyde (m/z = 44). The proposed chemical mechanism is discussed below.

The mass spectrometry results suggested that soaking of crystals of BlaC with 
meropenem followed by vitrification would trap the meropenem complex at the 
active site. Crystals of BlaC were prepared as previously described (8). 
Crystals soaked for 90 min with 50 mM meropenem containing 20% glycerol were 
vitrified and analyzed by x-ray diffraction at the Brookhaven National 
Laboratory Synchrotron Radiation Source. Crystals were present in the same space 
group as we previously reported for the BlaC-clavulanate complex, and 
diffraction data to 1.8 Å resolution were used to solve the structure (final 
model: Rwork = 0.152 and Rfree = 0.192, table S1) with molecular replacement 
methods and the structure of the BlaC-clavulanate complex (9). Clear electron 
density that was contiguous with the β-hydroxyl side chain of Ser70 (Ambler 
numbering is used throughout for residue identification) was observed in the 
active site (Fig. 2, A and B). The carbonyl oxygen of the covalent 
enzyme-meropenem ester is oriented to interact with the main chain amide of 
Ser70 and Thr253, residues comprising the "oxyanion" hole. When compared to the 
structure of the BlaC-clavulanate complex, a number of active site 
rearrangements are evident. Most notably, the -amino group of Lys73 and the 
Ser130 side chain hydroxyl no longer hydrogen-bond to the Ser70 ester oxygen. 
The Lys73 -amino group and the carboxyl side chain of Glu166 interact with the 
meropenem C8 hydroxyl group, whereas the Ser130 side chain hydroxyl group 
hydrogen bonds to the nitrogen of the pyrroline ring (fig. S3). The pyrroline 
ring shows a collinear relationship of C6, N1, C2, and C3, requiring that the 
double bond originally present between C2 and C3 has isomerized, as first 
proposed by Knowles (10). The structure clearly shows that the thioether sulfur 
atom at the C3 position is in the S configuration requiring protonation of the 
re face of the C2-C3 double bond. We thus propose a chemical mechanism for 
meropenem hydrolysis by BlaC as shown in Fig. 2C. The electron density beyond 
the thioether sulfur atom is weak and discontinuous, so the modeled 
configuration of the terminal pyrazole ring is uncertain at this time. Whether 
double bond isomerization is concerted with β-lactam ring opening is also 
unclear, although we draw it as concerted (Fig. 2C).



	Fig. 2. (A) Overall structure of BlaC displayed in rainbow from N term (blue) 
to the C term (red), with the meropenem adduct displayed as a surface mesh. (B) 
Fo–Fc omit density (green) contoured at 4.0 surrounds the covalent meropenem 
adduct formed at the Ambler active-site residue Ser70. Structure figures were 
produced using Pymol (www.pymol.org). (C) Proposed chemical mechanism for the 
BlaC-catalyzed reaction with meropenem. [View Larger Version of this Image (50K 
GIF file)]

The combination of clavulanate and amoxicillin has previously been shown to 
inhibit the growth of M. tuberculosis strains (11). Because amoxicillin is one 
of the best substrates of BlaC, complete inactivation of BlaC would be required 
to maintain inhibitory concentrations of the antibiotic. More recently, the 
addition of clavulanate to susceptible and multidrug-resistant strains of M. 
tuberculosis has been shown to potentiate the effects of all classes of 
β-lactams (12, 13). We determined the minimum inhibitory concentration (MIC) 
values of M. tuberculosis H37Rv in 7H9 medium at 37°C by penicillins, 
cephalosporins, and carbapenems in the absence and presence of 2.5 µg ml–1 
clavulanate. The addition of clavulanate had only a modest effect on the MIC 
values of ampicillin and amoxicillin but a significant effect on the MIC values 
of cephalothin, imipenem, and meropenem (table S2). On the basis of the low MIC 
value of meropenem in the presence of clavulanate (0.32 µg ml–1) and its low 
rate of hydrolysis by BlaC, we selected this carbapenem for detailed analysis. 
When various combinations of meropenem and clavulanate were added daily for 5 
consecutive days to cultures of M. tuberculosis Erdman under aerobic growth 
conditions, the colony-forming units per milliliter (CFU ml–1) dropped rapidly 
until complete sterilization was obtained after 9 to 12 days (Fig. 3A). Although 
the number and identity of the cell wall cross-linking transpeptidase targets of 
meropenem in M. tuberculosis are not known (see below), it is clear that a 
combination of clavulanate and meropenem rapidly sterilizes actively growing 
aerobic cultures of M. tuberculosis.



	Fig. 3. Killing curves of M. tuberculosis after exposure to β-lactams and 
clavulanate. (A) Aerobic growth using the microdilution method. Meropenem and 
clavulanate were added at 2 µg ml–1 + 1 µg ml–1 (), 2 µg ml–1 + 2 µg ml–1 (), 4 
µg ml–1 + 1 µg ml–1 (), and 4 µg ml–1 + 2 µg ml–1 (), respectively, for 5 
consecutive days. (B) Meropenem is cidal for nonreplicating anaerobic M. 
tuberculosis. Hypoxically adapted M. tuberculosis H37Rv was treated under 
anaerobic conditions with twofold dilutions of meropenem (0.19 to 12.5 µg ml–1) 
in the presence or absence of 2.5 µg ml–1 clavulanate. Isoniazid (0.16 to 1.0 µg 
ml–1) and metronidazole (4.6 to 73 mM) served as negative and positive controls, 
respectively. Survival was determined by measurement of ATP amounts in surviving 
bacteria during aerobic outgrowth of 100-fold diluted cells at either 1 week 
(white bars) or 2 weeks (shaded bars) of treatment or by enumeration of CFUs 
(inset) after 2 weeks of compound exposure. [View Larger Version of this Image 
(14K GIF file)]

An important problem in tuberculosis therapy is the phenotypic drug resistance 
of populations of organisms that are in a nonreplicative state, termed 
"persistence" (14, 15). The M. tuberculosis L,D-transpeptidase has recently been 
reported to be a target for carbapenems, and this enzyme is thought to be 
expressed as the organism enters into the persistent state, with corresponding 
changes in the nature of peptidoglycan cross-linking (16). There are several in 
vitro models of this state, but the most widely used is the Wayne model (17), 
where organisms that are grown in sealed tubes enter into a viable but 
nonreplicative (NRP) state after 2 weeks because of consumption of available 
oxygen. Combinations of clavulanate and meropenem were tested for their ability 
to sterilize organisms in this state. Drug combinations were added to NRP2 
cultures within an anaerobic chamber, and cellular viability was assessed 1 week 
and 2 weeks later by measuring intracellular adenosine triphosphate (ATP) 
concentrations as well as CFUs. All clavulanate–β-lactam combinations were 
effective in reducing viability, but the decrease was more pronounced with the 
two carbapenems, imipenem and meropenem, than with amoxicillin and cefuroxime 
(fig. S4). In the case of the clavulanate-meropenem combination, we observed 
more than a log kill over a 2-week exposure time, comparable to metronidazole, 
which is shown as a control compound in Fig. 3B (12).

The combination of clavulanate with β-lactams, especially meropenem, was also 
tested for the ability to inhibit the growth of extensively drug-resistant (XDR) 
clinical strains of M. tuberculosis. Thirteen clinical isolates exhibiting the 
XDR phenotype were tested (18). Clavulanate was used at a concentration of 2.5 
µg ml–1, and the MIC values of these strains for meropenem were determined. The 
susceptibility of these strains was experimentally indistinguishable from that 
determined for H37Rv and the Erdman strain, that is, 1 µg ml–1 (Table 1). In 
contrast, substantial variability in the MIC values to ampicillin, amoxicillin, 
cephalothin, and imipenem was observed for these same strains (table S2). The 
clavulanate-meropenem combination is thus equally effective against both 
susceptible and XDR strains.



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  	Table 1. MIC values for β-lactams in the presence of 2.5 µg ml–1 clavulanic 
acid. The XDR strains were a subset of those previously reported (18).

These structural and mechanistic studies of carbapenem interactions with the 
BlaC β-lactamase have revealed properties of specific β-lactam antibiotics that 
can be exploited in the treatment of tuberculosis, including the treatment of 
multidrug- and extensively drug-resistant strains. The structure of the 
meropenem-inactivated acyl-enzyme, in combination with our mechanistic proposal 
for its hydrolysis and the structure of the clavulanate-BlaC complex, provides 
the information necessary to design improved tuberculosis-specific β-lactams 
that could form longer-lived acyl-enzyme intermediates. Among currently approved 
β-lactams, however, meropenem is superior on the basis of its poor activity as a 
substrate for BlaC, ability to transiently inhibit BlaC, and activity against 
nonreplicating organisms. This activity provides experimental evidence that 
peptidoglycan remodeling occurs in M. tuberculosis in the nonreplicating state, 
which may be an important determinant of clinical efficacy.

Ten years ago, a report on the early bactericidal activity of 
amoxicillin-clavulanate in patients with tuberculosis appeared (19), but no 
additional reports have appeared since then. Our studies reveal that clinical 
strain-to-strain variability is observed with combinations of clavulanate with 
penicillins, cephalosporins, and imipenem but not with meropenem. The synergism 
of the clavulanate-meropenem combination and the uniform activity against 
drug-susceptible, laboratory, and XDR clinical strains suggest this combination 
could be useful in the treatment of tuberculosis. Both clavulanate and meropenem 
are FDA-approved drugs, and both clavulanate and meropenem are sufficiently free 
of side effects to be approved for pediatric use in children over 3 months old.


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20. Materials and methods are available as supporting material on Science Online.
21. The authors wish to thank J. Chan and E. Russel for help in the Wayne model 
studies and H. Xiao for assistance in mass spectrometry. This work was supported 
partially by the NIH (AI33696 to J.S.B.); in part by the Intramural Research 
Program of the NIH, National Institute of Allergy and Infectious Diseases; and 
in part by a grant from the Bill and Melinda Gates Foundation and the Wellcome 
Trust through the Grand Challenges in Global Health Initiative. A provisional 
U.S. patent application was filed on 27 May 2008 related to this work. Structure 
coordinates have been deposited as Protein Data Bank identification code 3DWZ.
Supporting Online Material
www.sciencemag.org/cgi/content/full/323/5918/1215/DC1

Materials and Methods

Figs. S1 to S4

Tables S1 and S2

References


Received for publication 21 October 2008. Accepted for publication 6 January 2009.