CLINICAL MICROBIOLOGY REVIEWS, Jan. 2010, p. 160–201 Vol. 23, No.

1
0893-8512/10/$12.00 doi:10.1128/CMR.00037-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Three Decades of ␤-Lactamase Inhibitors

Sarah M. Drawz1 and Robert A. Bonomo2,3,4,5*
Departments of Pathology,1 Medicine,2 Pharmacology,3 and Molecular Biology and Microbiology,4
Case Western Reserve University School of Medicine, Cleveland, Ohio, and Research Service,
Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio5

INTRODUCTION .......................................................................................................................................................161
MECHANISM OF ACTION OF ␤-LACTAM ANTIBIOTICS .............................................................................161
RESISTANCE TO ␤-LACTAM ANTIBIOTICS .....................................................................................................161
␤-LACTAMASES ........................................................................................................................................................164
Classification ...........................................................................................................................................................164
Class A serine ␤-lactamases .............................................................................................................................164
Class A ESBLs ....................................................................................................................................................164
Class A serine carbapenemases ........................................................................................................................165
Class B metallo-␤-lactamases ...........................................................................................................................165
Class C serine cephalosporinases.....................................................................................................................166
Class D serine oxacillinases ..............................................................................................................................166
␤-Lactamase Hydrolytic Mechanisms..................................................................................................................166
Class A .................................................................................................................................................................166
Class C .................................................................................................................................................................168
Class D .................................................................................................................................................................168
Class B .................................................................................................................................................................168
CIRCUMVENTING ␤-LACTAMASES ....................................................................................................................169
␤-Lactamase Inhibitors: Mechanistic Considerations.......................................................................................169
␤-Lactamase Inhibitors in Clinical Practice ......................................................................................................169
Clavulanic acid, sulbactam, and tazobactam .......................................................................................169
Mechanism of inhibition....................................................................................................................................170
␤-Lactam–␤-lactamase inhibitor combinations: clinical use .......................................................................171
(i) Amoxicillin-clavulanate ............................................................................................................................171
(ii) Ticarcillin-clavulanate .............................................................................................................................172
(iii) Ampicillin-sulbactam..............................................................................................................................173
(iv) Piperacillin-tazobactam ..........................................................................................................................173
Inhibitory Activity of Carbapenems .....................................................................................................................174
INHIBITOR-RESISTANT CLASS A ␤-LACTAMASES........................................................................................176
Epidemiology and Detection of ␤-Lactamase Inhibitor Resistance.................................................................176
Substitutions in Class A Enzymes Conferring Inhibitor Resistance...............................................................178
Arginine 244.........................................................................................................................................................179
Methionine 69......................................................................................................................................................179
Asparagine 276 ....................................................................................................................................................179
Serine 130 ............................................................................................................................................................180
Arginine 275.........................................................................................................................................................180
Lysine 234 ............................................................................................................................................................180
THE PROMISE OF NOVEL ␤-LACTAMASE INHIBITORS .............................................................................180
Monobactam Derivatives .......................................................................................................................................180
ATMO Derivatives ..................................................................................................................................................182
Penems .....................................................................................................................................................................182
BRL 42715 and Syn 1012...................................................................................................................................182
BRL 42715 derivatives........................................................................................................................................183
Oxapenems...........................................................................................................................................................184
Tricyclic carbapenems (trinems) ......................................................................................................................184
1-␤-Methylcarbapenems.....................................................................................................................................184
Penicillin and Cephalosporin Sulfone Derivatives.............................................................................................184
C-2/C-3-substituted penicillin and cephalosporin sulfones ..........................................................................185
C-6-substituted penicillin sulfones ...................................................................................................................185

* Corresponding author. Mailing address: Infectious Diseases Sec-

tion, Louis Stokes Cleveland Department of Veterans Affairs Medical
Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-
3800, ext. 4399. Fax: (216) 231-3482. E-mail: robert.bonomo@med.va
.gov.

160
VOL. 23, 2010 ␤-LACTAMASE INHIBITORS 161

Non-␤-Lactam Inhibitors.......................................................................................................................................186
Boronic acid transition state analogs (TSAs).................................................................................................186
Phosphonates.......................................................................................................................................................187
NXL104.................................................................................................................................................................187
Hydroxamates......................................................................................................................................................187
Other non-␤-lactam inhibitors .........................................................................................................................188
INHIBITION OF METALLO-␤-LACTAMASES ...................................................................................................188
Thiol Derivatives .....................................................................................................................................................189
Pyridine Dicarboxylates .........................................................................................................................................189
Trifluoromethyl Ketones and Alcohols ................................................................................................................189
Carbapenem Analogs..............................................................................................................................................189
Tricyclic Natural Products ....................................................................................................................................189
Succinate Derivatives .............................................................................................................................................189
C-6-Mercaptomethyl Penicillinates ......................................................................................................................190
LESSONS LEARNED ................................................................................................................................................190
A PERSPECTIVE .......................................................................................................................................................191
ACKNOWLEDGMENTS ...........................................................................................................................................191
REFERENCES ............................................................................................................................................................191

INTRODUCTION the bacterial cell wall is essential to maintaining cell shape in a

The development of antibiotics remains one of the most sig-
nificant advances in modern medicine (364). Antibiotics have
saved countless lives and continue to be a mainstay of therapy for
bacterial infections. The clinical success of the first ␤-lactam,
penicillin G (benzylpenicillin [Fig. 1, compound 1), prompted the
search for and development of additional derivatives. This quest
gave rise to the ␤-lactam antibiotics in clinical use today (penicil-
lins, narrow- and extended-spectrum cephalosporins, monobac-
tams, and carbapenems [Fig. 1, compounds 1 to 7) (14). The
common structural feature of these classes of antibiotics is the
highly reactive four-membered ␤-lactam ring.
Unfortunately, ␤-lactamase-mediated resistance to ␤-lactam
antibiotics emerged as a significant clinical threat to these life-
saving drugs. In response to this challenge, two strategies were
advanced to preserve the utility of ␤-lactam antibiotics: (i) dis-
cover or design ␤-lactam antibiotics that are able to evade bacte-
rial enzymatic inactivation conferred by ␤-lactamases, or (ii) in-
hibit ␤-lactamases so the partner ␤-lactam can reach the penicillin
binding proteins (PBPs), the target of ␤-lactam antibiotics.
In this review, we summarize 3 decades of investigation of
␤-lactamase inhibition. This perspective is framed by our back-
ground in clinical infectious diseases. First, we highlight the fun-
damental principles of ␤-lactamase enzymology. We then sum-
marize the salient features of ␤-lactam–␤-lactamase inhibitor
combinations that are used in clinical practice. Next, we define the
problem of resistance to ␤-lactamase inhibitors by explaining the
important changes in class A ␤-lactamases that define this phe-
notype. With this background, we review the ␤-lactamase inhib-
itors that have been developed to this point and discuss the novel
␤-lactamase inhibitors that are hoped to extend the life span of
our current ␤-lactams. We view these agents as extremely impor-
tant to the future of ␤-lactam therapy: inhibitors not only can
preserve our current armamentarium but may also be used as
novel ␤-lactams are introduced into the clinic. Finally, we con-
clude with some “lessons learned.”
MECHANISM OF ACTION OF ␤-LACTAM ANTIBIOTICS
␤-Lactam antibiotics exhibit their bactericidal effects by in-
hibiting enzymes involved in cell wall synthesis. The integrity of
hypertonic and hostile environment (249). Osmotic stability is
preserved by a rigid cell wall comprised of alternating N-acetyl-
muramic acid (NAM) and N-acetylglucosamine (NAG) units.
These glycosidic units are linked by transglycosidases. A pen-
tapeptide is attached to each NAM unit, and the cross-linking of
two D-alanine–D-alanine NAM pentapeptides is catalyzed by
PBPs, which act as transpeptidases (142, 377). This cross-linking
of adjacent glycan strands confers the rigidity of the cell wall.
The ␤-lactam ring is sterically similar to the D-alanine–D-
alanine of the NAM pentapeptide, and PBPs “mistakenly” use
the ␤-lactam as a “building block” during cell wall synthesis
(459). This results in acylation of the PBP, which renders the
enzyme unable to catalyze further transpeptidation reactions
(125). As cell wall synthesis slows to a halt, constitutive pepti-
doglycan autolysis continues. The breakdown of the murein
sacculus leads to cell wall compromise and increased perme-
ability. Thus, the ␤-lactam-mediated inhibition of transpepti-
dation causes cell lysis, although the specific details of penicil-
lin’s bactericidal effects are still being unraveled (20).
RESISTANCE TO ␤-LACTAM ANTIBIOTICS
There are four primary mechanisms by which bacteria can
overcome ␤-lactam antibiotics (14).
(i) Production of ␤-lactamase enzymes is the most common
and important mechanism of resistance in Gram-negative bac-
teria and will be the focus of this review.
(ii) Changes in the active site of PBPs can lower the affinity
for ␤-lactam antibiotics and subsequently increase resistance
to these agents, such as those seen in PBP2x of Streptococcus
pneumoniae (212). Through natural transformation and re-
combination with DNA from other organisms, Neisseria spp.
and Streptococcus spp. have acquired highly resistant, low-
affinity PBPs (39, 313, 459). In a related manner, penicillin
resistance in Streptococcus sanguis, Streptococcus oralis, and
Streptococcus mitis developed from horizontal transfer of a
PBP2b gene from Streptococcus pneumoniae (107, 348). Methicil-
lin resistance in Staphylococcus spp. is also a significant clinical
challenge. While there are many reasons for this resistance, the
␤-lactam resistance phenotype is also conferred by acquisition of
162 DRAWZ AND BONOMO CLIN. MICROBIOL. REV.

FIG. 1. Chemical structures of compounds discussed in the text. Compounds 1 to 7, a representative penicillin (compound 1), an extended-
spectrum cephalosporin (compound 2), a monobactam (compound 3), and carbapenems (compounds 4 to 7). The numbering scheme for
penicillins, cephalosporins, and monobactams is shown. Compounds 8 to 10, ␤-lactamase inhibitors in clinical practice. Compounds 11 to 38,
investigational ␤-lactamase inhibitors: monobactam derivatives (compounds 11 to 14), a penicillin derivative (compound 15), penems (compounds
16 to 20), penam sulfones (compounds 21 to 24), a boronic acid transition state analog (compound 25), non-␤-lactams (compounds 26 to 28), and
metallo-␤-lactamase inhibitors (compounds 29 to 38).

the mecA gene which produces PBP2a (also denoted PBP2⬘) (76). (iii) Decreased expression of outer membrane proteins
PBP2a can assemble new cell wall in the presence of high con- (OMPs) is another mechanism of resistance. In order to access
centration of penicillins and cephalosporins. PBPs on the inner plasma membrane, ␤-lactams must either
VOL. 23, 2010
FIG. 1—Continued.
diffuse through or directly traverse porin channels in the outer
membrane of Gram-negative bacterial cell walls. Some Entero-
bacteriaceae (e.g., Enterobacter spp., Klebsiella pneumoniae, and
Escherichia coli) exhibit resistance to carbapenems based on
loss of these OMPs; the loss of OprD is associated with imi-
penem resistance and reduced susceptibility to meropenem in
the nonfermenter Pseudomonas aeruginosa (169, 187, 236, 286,
306, 395). Resistance to imipenem and meropenem has also
been associated with the loss of the CarO OMP in clinical
isolates of multidrug-resistant Acinetobacter baumannii (278,
346). Point mutations or insertion sequences in porin-encoding
genes can produce proteins with decreased function and thus
lower permeability to ␤-lactams (106). Of note, the disruption
of porin proteins alone is not always sufficient for producing
the resistance phenotype, and typically this mechanism is
found in combination with ␤-lactamase expression (106, 235).
␤-LACTAMASE INHIBITORS 163
(iv) Efflux pumps, as part of either an acquired or intrinsic
resistance phenotype, are capable of exporting a wide range of
substrates from the periplasm to the surrounding environment
(347). These pumps are an important determinant of multidrug
resistance in many Gram-negative pathogens, particularly P.
aeruginosa and Acinetobacter spp. In P. aeruginosa, upregulation
of the MexA-MexB-OprD system, in combination with the or-
ganism’s low outer membrane permeability, can contribute to
decreased susceptibility to penicillins and cephalosporins, as well
as quinolones, tetracycline, and chloramphenicol (224–226, 373,
389). To illustrate, an increase in the carbenicillin MIC from 32
␮g/ml to 1,028 ␮g/ml is associated with overproduction of this
efflux pump (12, 225). Additionally, an upregulated efflux pump
(e.g., AdeABC, an RND-type efflux pump, in A. baumannii) can
augment the carbapenem resistance conferred by a catalytically
poor ␤-lactamase (e.g., OXA-23) (162, 333).
164 DRAWZ AND BONOMO CLIN. MICROBIOL. REV.

TABLE 1. ␤-Lactamase classification schemesa

Bush-Jacoby- Inhibited by
Ambler class Preferred substrates Representative enzyme(s)
Medeiros class clavulanate

A (serine penicillinases) 2a Penicillins ⫹ PC1 from S. aureus

2b Penicillins, narrow-spectrum ⫹ TEM-1, TEM-2, SHV-1
cephalosporins
2be Penicillins, narrow-spectrum and extended- ⫹ SHV-2 to SHV-6, TEM-3 to
spectrum cephalosporins TEM-26, CTX-Ms
2br Penicillins ⫺ TEM-30, SHV-72
2c Penicillins, carbenicillin ⫹ PSE-1
2e Extended-spectrum cephalosporins ⫹ FEC-1, CepA
2f Penicillins, cephalosporins, carbapenems ⫾ KPC-2, SME-1, NMC-A

B (metallo-␤-lactamases) 3 Most ␤-lactams, including carbapenems ⫺ IMP-1, VIM-1, CcrA, and BcII
(B1); CphA (B2); L1(B3)

C (cephalosporinases) 1 Cephalosporins ⫺ AmpC, CMY-2, ACT-1

D (oxacillinases) 2d Penicillins, cloxacillin ⫾ OXA-1, OXA-10

Not classified 4
a
Based on data from reference 57. An updated classification system by Bush and Jacoby is in press (56a) at the time of this writing.

␤-LACTAMASES Class A serine ␤-lactamases. In general, class A enzymes are

The first ␤-lactamase enzyme was identified in Bacillus (Esch-
erichia) coli before the clinical use of penicillin. In a sentinel paper
published nearly 70 years ago, E. P. Abraham and E. Chain
described the B. coli “penicillinase” (1). The enzyme was not
thought to be clinically relevant, since penicillin was targeted to
treat staphylococcal and streptococcal infections, and Abraham,
Chain, and their colleagues were unable to isolate the enzyme
from these Gram-positive organisms (2, 55). It is sobering now to
consider the ramifications of this observation.
Four years later, Kirby successfully extracted these cell-free
“penicillin inactivators” from Staphylococcus aureus, which fore-
shadowed the emergence of a significant clinical problem (202).
The growing number of ␤-lactam antibiotics has since increased
the selective pressure on bacteria, promoting the survival of or-
ganisms with multiple ␤-lactamases (249, 256). Currently, more
than 850 ␤-lactamases are identified (K. Bush, personal commu-
nication). As authors, we speculate that the rapid replication rate,
recombination rates, and high mutation frequency permit bacte-
ria to adapt to novel ␤-lactams by evolution of these ␤-lactamases
(332).
Classification
Two major classification schemes exist for categorizing
␤-lactamase enzymes: Ambler classes A through D, based on
amino acid sequence homology, and Bush-Jacoby-Medeiros
groups 1 through 4, based on substrate and inhibitor profile
(Table 1) (10, 57). A “family portrait” reveals the structural
similarity of class A, C, and D serine ␤-lactamases (Fig. 2).
Class B ␤-lactamases (“a class apart”) are metallo-␤-lactama-
ses (MBLs) (52). MBLs possess either a single Zn2⫹ ion or a
pair of Zn2⫹ ions coordinated to His/Cys/Asp residues in the
active site. In this review, the Ambler classification scheme will be
used.
susceptible to the commercially available ␤-lactamase inhibi-
tors (clavulanate, tazobactam, and [less so] sulbactam), al-
though the K. pneumoniae carbapenemase KPC may be an
important exception to this generalization (319a). The first
plasmid-mediated ␤-lactamase was identified in E. coli in 1963
(and reported in 1965), and was named “TEM” after the pa-
tient from whom it was isolated (100). SHV, another common
␤-lactamase found primarily in K. pneumoniae, was named
from the term “sulfhydryl reagent variable.” Early studies of
SHV-1 showed that p-chloromercuribenzoate inhibited the hy-
drolysis of cephaloridine but not that of benzylpenicillin (251).
TEM and SHV are common ␤-lactamases detected in clinical
isolates of E. coli and K. pneumoniae, pathogens responsible
for urinary tract, hospital-acquired respiratory tract, and
bloodstream infections (60, 145, 372). While SHV-1 and
TEM-1 share 68% sequence homology, the active site of
SHV-1 is approximately 0.7 to 1.2 Å wider than that of TEM-1,
which may have important structural implications, especially
related to the substrate profiles of SHV variants (421).
Although blaTEM and blaSHV may be found on plasmids,
other class A enzymes are encoded on the chromosome (e.g.,
blaPenA from Burkholderia pseudomallei) or on integrons (e.g.,
blaGES-1 from K. pneumoniae and blaVEB-1 in P. aeruginosa and
A. baumannii) (57, 280).
Class A ESBLs. The growing number of ␤-lactamases in E.
coli and K. pneumoniae, as well as the emergence of these
enzymes in other pathogens (e.g., Haemophilus influenzae and
Neisseria gonorrhoeae), led to the development of extended-
spectrum cephalosporins with an oxyimino side chain, carbap-
enems, cephamycins, and monobactams (71, 115, 188, 321).
Upon the introduction of the penems and cephems in the early
1980s, these new agents were effective against many ␤-lactam
resistant bacteria. However, selective pressure quickly fostered
the emergence of extended-spectrum ␤-lactamases (ESBLs),
which could hydrolyze many of the oxyimino-cephalosporins
VOL. 23, 2010
FIG. 2. “Family portrait” of ␤-lactamase enzymes. (A) Class A SHV-1; (B) class B IMP-1; (C) class C E. coli AmpC; (D) class D OXA-1. For
classes A, C, and D, the active-site serine is shown in yellow; for class B, the two Zn2⫹ ions are shown. (Based on PDB entries 1SHV, 1DDK, 2BLS,
and 1M6K, respectively.)
(186, 188).
In a dramatic parallel to the observations of Abraham and
Chain, within 2 years of the introduction of the extended-
spectrum cephalosporins cefotaxime and ceftazidime, novel
ESBLs in E. coli and K. pneumoniae were reported (204).
Interestingly, these “new ␤-lactamases” harbored point muta-
tions in the parent blaTEM-1 and blaSHV-1 genes that led to
single amino acid changes in the ␤-lactamases. Other ESBLs,
such as CTX-M, arose by plasmid transfer from preexisting
chromosomal ESBL genes from Kluyvera spp., which typically
are nonpathogenic commensal organisms (14, 32). CTX-M
ESBLs now represent important enzymes found in isolates
from the community and are the most commonly isolated
ESBLs in many parts of the world, particularly Europe (343).
ESBLs hydrolyze penicillins, narrow- and extended-spec-
trum cephalosporins (including the anti-methicillin-resistant S.
aureus [MRSA] cephalosporin ceftobiprole), and the mono-
bactam aztreonam (11, 321, 357). In contrast, ESBLs cannot
efficiently degrade cephamycins, carbapenems, and ␤-lacta-
mase inhibitors. Since their initial description, more than 200
different ESBLs have been identified, posing a significant risk
to public health and to hospitalized patients in intensive care
units, where infection with an ESBL may lead to significant
morbidity and mortality (up-to-date listings of verified ESBL
sequences are available on the website www.lahey.org/studies
/webt.asp maintained by G. A. Jacoby and K. Bush) (321). The
majority of ESBLs are from the SHV, TEM, and CTX-M
families; less frequently they are derived from BES, GES-1,
␤-LACTAMASE INHIBITORS 165
VEB, and PER enzymes, and sometimes these enzymes do not
belong to any defined family (280).
Class A serine carbapenemases. Class A serine carbapen-
emases include the nonmetallocarbapenamase of class A
(NMC-A), IMI, SME, and KPC. Members of this group of
␤-lactamases can hydrolyze carbapenems as well as cephalo-
sporins, penicillins, and aztreonam (356). These carbapenem-
hydrolyzing enzymes have been identified primarily in Entero-
bacter cloacae, Serratia marcescens, and K. pneumoniae,
bacteria which often harbor multiple resistance determinants,
narrowing the range of treatment options (290, 291, 359, 454,
455). The bla gene for the former two organisms is typically found
on the chromosome, while the K. pneumoniae carbapenemase
blaKPC gene is carried on plasmids containing Tn4401 (292).
MICs of carbapenems in carbapenemase-expressing strains can
vary from moderately increased (2 to 4 ␮g/ml) to resistant (ⱖ 32
␮g/ml) (116b, 356).
Class B metallo-␤-lactamases. Class B enzymes are Zn2⫹-
dependent ␤-lactamases that demonstrate a hydrolytic mech-
anism different from that of the serine ␤-lactamases of classes
A, C, and D (57). Organisms producing these enzymes usually
exhibit resistance to penicillins, cephalosporins, carbapenems,
and the clinically available ␤-lactamase inhibitors (432). Inter-
estingly, the hydrolytic profile of MBLs does not typically in-
clude aztreonam. MBLs likely evolved separately from the
other Ambler classes, which have serine at their active site
(249). The blaMBL genes are located on the chromosome, plas-
mid, and integrons (133, 432). P. aeruginosa, K. pneumoniae,
166 DRAWZ AND BONOMO
and A. baumannii produce class B enzymes encoded by mobile
genetic elements (93, 175, 177). In contrast, Bacillus spp.,
Chryseobacterium spp., and Stenotrophomonas maltophilia pos-
sess chromosomally encoded MBLs, but the majority of these
host pathogens are not frequently responsible for serious in-
fections (432). The role that these ␤-lactamases in Bacillus spp.
and Chryseobacterium spp. will play in the clinical arena is still
unknown.
Class C serine cephalosporinases. Class C AmpC ␤-lacta-
mases include CMY-2, P99, ACT-1, and DHA-1, which are
usually encoded by bla genes located on the bacterial chromo-
some, although plasmid-borne AmpC enzymes are becoming
more prevalent (340). Organisms expressing the AmpC ␤-lac-
tamase are typically resistant to penicillins, ␤-lactam–␤-lacta-
mase inhibitor combinations, and cephalosporins, including
cefoxitin, cefotetan, ceftriaxone, and cefotaxime. AmpC en-
zymes poorly hydrolyze cefepime and are inhibited by cloxacil-
lin, oxacillin, and aztreonam (57). Members of the Enterobac-
teriaceae family, such as Enterobacter spp. and Citrobacter spp.,
are AmpC ␤-lactamase producers that resist inhibition by cla-
vulanate and sulbactam, although Klebsiella spp., Salmonella
spp., and Proteus spp. normally do not harbor chromosomal
blaAmpC genes (19, 184).
Production of chromosomal AmpCs in Gram-negative bac-
teria is at a low level (“repressed”) but can be “derepressed” by
induction with certain ␤-lactams, particularly cefoxitin (14, 24,
148). The mechanism of this regulation has been the subject of
intense investigation (183). Of significant concern is the selec-
tion of mutant bacterial populations that are genetically dere-
pressed for AmpC production, which can cause a dramatic
increase in MICs during the course of ␤-lactam therapy (e.g.,
after 14 days of ceftazidime therapy, a strain of P. aeruginosa
with MICs increased from 1 to 32 ␮g/ml was selected) (184,
193, 233).
Class D serine oxacillinases. Class D ␤-lactamases were
initially categorized as “oxacillinases” because of their ability
to hydrolyze oxacillin at a rate of at least 50% of that of
benzylpenicillin, in contrast to the relatively slow hydrolysis of
oxacillin by classes A and C (98). In bacteria, OXA ␤-lacta-
mases can also confer resistance to penicillins, cephalosporins,
extended-spectrum cephalosporins (OXA-type ESBLs), and
carbapenems (OXA-type carbapenemases). Generally speak-
ing, OXA enzymes are resistant to inhibition by clavulanate,
sulbactam, and tazobactam, (with some exceptions; e.g.,
OXA-2 and OXA-32 are inhibited by tazobactam but not sul-
bactam and clavulanate, and OXA-53 is inhibited by clavu-
lanate) (98, 276, 279, 345). Interestingly, sodium chloride at
concentrations of ⬎50 to 75 mM inhibits some carbapenem-
hydrolyzing oxacillinases (e.g., OXA-25 and OXA-26) (6).
Site-directed mutagenesis studies suggest that susceptibility to
inhibition by sodium chloride is related to the presence of a
Tyr residue at position 144 (161, 279). Presumably, Tyr144 may
facilitate sodium chloride binding better than the Phe residue
found in resistant oxacillinases, although the molecular mech-
anism remains unexplained. Examples of OXA enzymes in-
clude those rapidly emerging in A. baumannii (e.g., OXA-23
and OXA-24/40) and constitutively expressed in P. aeruginosa
(e.g., OXA-50) (141a, 346, 435).
CLIN. MICROBIOL. REV.
␤-Lactamase Hydrolytic Mechanisms
Serine ␤-lactamases acylate ␤-lactam antibiotics, much like
PBPs, and then use strategically positioned water molecules to
hydrolyze the acylated ␤-lactam (265). In this manner, the
␤-lactamase is regenerated and can inactivate additional ␤-lac-
tam molecules. This enzymatic reaction may be represented by
the following equation:
k1 k2 k 3, H 2O
E ⫹ S| ¡ E⫺S O
L; E:S O ¡E ⫹ P (1)
k⫺1
In this scheme, E is a ␤-lactamase, S is a ␤-lactam substrate,
E:S is the Michaelis complex, E ⫺ S is the acyl-enzyme, and P
is the product devoid of antibacterial activity. The rate con-
stants for each step are represented by k1, k⫺1, k2, and k3. k1
and k⫺1 are the association and dissociation rate constants for
the preacylation complex, respectively; k2 is the acylation rate
constant, and k3 is the deacylation rate constant. More com-
plicated branched reaction mechanisms are seen with some
␤-lactamases (129). In order to be uniform, we now define
basic terms that are often used to describe the kinetic behavior
of ␤-lactamases.
The Michaelis constant, Km, is defined as (129)
Km ⫽ k3Ks/共k2 ⫹ k3兲 (2)
where the kinetic constant, Ks, is (k⫺1 ⫹ k2)/k1. The turnover
number, kcat, is a composite rate constant that represents mul-
tiple chemical steps and is defined as (92)
kcat ⫽ k2k3/共k2 ⫹ k3兲 (3)
or
Vmax ⫽ kcat关E兴 (4)
Km, expressed in terms of concentration, represents the rela-
tive affinity of the ES encounter and the rate at which the ES
is converted to P; a large Km value represents poor affinity
(large Ks).
Class A. In Fig. 3, we represent the reaction scheme of a
typical class A ␤-lactamase. In brief, (i) after formation of the
Henri-Michaelis complex, the active-site serine performs a nu-
cleophilic attack on the carbonyl of the ␤-lactam antibiotic that
results in a high-energy tetrahedral acylation intermediate
(‡1); (ii) this intermediate transitions into a lower-energy co-
valent acyl-enzyme following protonation of the ␤-lactam ni-
trogen and cleavage of the C-N bond; (iii) next, an activated
water molecule attacks the covalent complex and leads to a
high-energy tetrahedral deacylation intermediate (‡2); and (iv)
hydrolysis of the bond between the ␤-lactam carbonyl and the
oxygen of the nucleophilic serine regenerates the active en-
zyme and releases the inactive ␤-lactam. Acylation and deacy-
lation require the activation of the nucleophilic serine and
hydrolytic water, respectively. An entire body of experimenta-
tion is devoted to examining the mechanistic roles of the indi-
vidual residues in the active sites of ␤-lactamases; here we
present only several leading insights to provide a foundation
for the discussion of inhibitory pathways.
For class A enzymes, a leading theory holds that Glu166 acts
as the activating base of the hydrolytic water in deacylation
VOL. 23, 2010 ␤-LACTAMASE INHIBITORS 167

FIG. 3. Proposed reaction mechanism for a penicillin ␤-lactam substrate and a class A serine ␤-lactamase enzyme in which Glu166 participates
in activating a water molecule for both acylation and deacylation (additional evidence exists to suggest that this acylation scheme may exist in
competition with a mechanism where Lys73 acts as the general base to activate Ser70) (261). Dashed lines represent hydrogen bonds, and the
number labels are included to help the reader appreciate the general order of events, not as absolute and discrete steps. Following activation of
the hydroxyl group, Ser70 performs a nucleophilic attack on the carbonyl group of the ␤-lactam antibiotic, resulting in a high-energy acylation
intermediate. Protonation of the ␤-lactam nitrogen leads to cleavage of the C-N bond and formation of the covalent acyl-enzyme, which adopts
a lower energy state. Attack by a catalytic water leads to a high-energy deacylation intermediate, with subsequent hydrolysis of the bond between
the ␤-lactam carbonyl and the oxygen of Ser70. Deacylation regenerates the active enzyme and releases the inactive ␤-lactam.

(166, 392). In contrast, the acylation mechanism is less clear,
and several hypotheses and lines of evidence exist. The first
notion proposes that Glu166, via a water molecule, deproto-
nates the Ser70 hydroxyl before addition to the ␤-lactam bond.
This Glu166 general base theory is supported by quantum
mechanical/molecular mechanical (QM/MM) calculations and
the protonated status of Glu166 in the ultra-high-resolution
(0.85-Å) structure of TEM-1 in complex with an acylation
transition state analog (TSA) (Research Collaboratory for
Structural Bioinformatics Protein Data Bank [PDB] entry
1M40) (163, 265). Surprisingly, substitutions at residue 166 do
not prevent acylation (139, 146, 207, 392).
A second view of acylation maintains that Lys73 can serve as
the general base deprotonating Ser70 (166, 261, 401). Recent
QM/MM studies by Meroueh et al. demonstrate that the path-
way involving Lys73 as the activating base is energetically fa-
vorable and may exist in competition with the pathway where
Glu166 serves as the activating residue (261). Additional ideas
on the mechanism of acylation have been proposed, including
both (i) a role for the C-3 or C-4 substrate carboxylate and
Ser130 in activating Ser70 and (ii) a general acid pathway
where protonation of the ␤-lactam nitrogen is the primary
event (13, 103). Further clarification of the acylation mecha-
nism may prove useful for the design of inhibitors that capi-
talize on the enzymes’ native machinery.
Highly conserved structural motifs in class A enzymes in-
clude four polar regions: (i) the active-site pocket Ser70-Xaa-
Xaa-Lys73, (ii) the “⍀-loop” residues 163 to 178, (iii) Ser130-
Asp131-Asn132, and (iv) Lys234-Thr(Ser)235-Gly236. The
roles of residues in the “SDN” loop comprised of Ser130-
Asp131-Asn132 are related to maintaining the structure of the
active site cavity, enzyme stability, and stabilization of the
enzyme transition state, respectively (182, 305). The functional
explanation for the conservation of the Lys234-Thr(Ser)235-
Gly236 triad is less well understood. Removal of the hydroxyl
group from the middle residue has little impact on penicillin
hydrolysis in TEM (but more on the catalytic efficiency of
cephalosporins), and despite what may be expected from crys-
tal structure alignments, the study of modified ␤-lactams ar-
gues against an interaction between the ␤-lactam carboxylate
and the Thr(Ser) hydroxyl group (112, 181). In SHV, the
Thr235Ala substitution lowers MICs of both penicillins and
cephalosporins (174). Interestingly, the expression of the struc-
turally conserved SHV Thr235Ser variant has no effect on
␤-lactam MICs but does lead to an increased susceptibility to
the piperacillin-tazobactam combination (174).
The most common mechanism for transformation of a
broad-spectrum ␤-lactamase to an ESBL is point mutations
that result in amino acid sequence changes near the enzyme
active site, facilitating hydrolysis of oxyimino-cephalosporins
(186, 341). However, the specific amino acid replacements and
resistance mechanisms vary between enzymes (124). In TEM,
168 DRAWZ AND BONOMO
changes at Ambler positions Arg164 (-His, -Ser), Gly238 (-Ser,
-Ala), and Glu240 (-Lys) result in variants that confer the
ESBL phenotype (40; www.lahey.org/studies/webt.asp). Sev-
eral crystallographic studies demonstrated that the “new”
TEM active site is expanded or remodeled, compared to
TEM-1, to accommodate the larger side chain of expanded-
spectrum cephalosporins (83, 206, 298, 304). The crystal struc-
ture of SHV-2 (Gly238Ser) at 0.91 Å resolution (PDB 1N9B),
compared to SHV-1, showed a displacement in the b3 ␤-strand
containing residues 238 to 242 which created an expanded
␤-lactam binding site but preserved the positioning of the es-
sential catalytic residues (124, 298). Similarly, the PER-1
ESBL active site is expanded by a novel fold of the ⍀-loop and
the insertion of four residues after Lys240 (417).
In contrast, the crystal structure of the acyl-enzyme inter-
mediate of the ESBL Toho-1 in complex with cefotaxime did
not resemble the enlarged active sites seen with the ESBL
TEM and SHV ␤-lactamases. Rather, the Toho-1 enzyme fa-
cilitates cephalosporin binding through movement of the
⍀-loop toward the active site, interactions between conserved
residues Asn104 and Asp240 and the bulky side chain of cefo-
taxime, and contacts with Ser237 that help position the ␤-lac-
tam carbonyl in the oxyanion hole (176, 382). The high-reso-
lution crystal structures of four additional CTX-M
␤-lactamases confirmed that the active sites of these enzymes
were not enlarged compared to those of the narrow-spectrum
TEM-1 and SHV-1 (83, 85). Instead, the structural basis for
the extended substrate profiles in the CTX-M enzyme family
appears to be specific amino acid (i.e., Ser237 and Asp104)
interactions with the oxyimino-cephalosporins and increased
mobility of the b3 ␤-strand (83). Overall, this increased mobil-
ity and activity “costs the enzyme” thermal stability, a theme
recapitulated by the evolution of many resistance phenotypes
(383).
Point mutations are not the only mechanism for the ESBL
phenotype; other alterations in enzyme regulation produce
resistance, such as promoter sequence changes leading to in-
creased enzyme expression or alterations in outer membrane
porins (293, 302).
At this time, the biochemical basis for class A serine carbapen-
emase activity is an area of active investigation. Current evidence
suggests that this property rests upon a remodeling of the active site.
Unlike class A ESBLs (with expanded or enhanced mobility of the
b3 ␤-strand), the active sites of class A carbapenemases such as
KPC-2, NMC-A, and SME-1 reveal that there are multiple alter-
ations in the spatial positions of catalytic residues (200, 338, 388, 402).
We are just beginning to understand how this remodeling allows
enhanced carbapenemase activity compared to that of other class A
enzymes.
Class C. The prevailing evidence regarding the class C hy-
drolytic mechanism suggests that Tyr150 behaves as a general
base by increasing the nucleophilicity of Ser64 for acylation
(299). Early studies of the deacylation mechanism indicated
that Tyr150 also acts as the catalytic base, accepting a proton
from the deacylating water (111, 299). For example, QM/MM
calculations suggested that Tyr150 interacts with Lys67 in a
conjugate base manner (136). However, the crystal structure of
the E. coli AmpC in complex with a deacylation transition state
analog revealed that Tyr150 remains protonated throughout
the reaction and therefore is unlikely to be the anionic base
CLIN. MICROBIOL. REV.
FIG. 4. Schematic representation of the Zn2⫹-binding site of
dinuclear subclass B1 metallo-␤-lactamases such as B. cereus BcII.
that deprotonates the catalytic water for hydrolysis (84). In-
stead, deacylation may proceed by “substrate-activated cataly-
sis” whereby the nitrogen on the ␤-lactam acts as a base and
transiently accepts the proton from the hydrolytic water, while
the Tyr150 proton helps stabilize the water’s developing neg-
ative charge (51, 84). The crystallographic evidence and mu-
tagenesis studies do not rule out a role of Lys67 in the coor-
dinate base mechanism, and further studies exploring the role
of Lys67 are anticipated (84, 271).
Class D. Our understanding of the hydrolytic mechanism of
class D ␤-lactamases is based on the careful study of OXA-10,
-13, and -1 (253, 310, 334, 379, 398, 399). This class of enzymes
is unique because of the direct role of carboxylation of the
active-site Lys70. The carbamic acid on Lys70 can ionize to
yield a carbamate that hydrogen bonds with the nucleophilic
Ser67 residue. In this manner, the carboxylated Lys70 may
serve as the general base by activating both Ser67 for acylation
and the hydrolytic water for deacylation (144, 252, 253).
Class B. In contrast to the serine ␤-lactamases described
above, class B includes Zn2⫹-dependent enzymes that follow a
different hydrolytic mechanism. In general, MBLs use the -OH
group from a water molecule that is coordinated by Zn2⫹ to
hydrolyze the amide bond of a ␤-lactam. MBLs are divided
into three classes based on their Zn2⫹ dependency, i.e.,
whether they (i) are fully active with either one or two ions
(subclass B1, e.g., IMP-1, VIM-2, BcII, and CcrA), (ii) require
two ions (subclass B3, e.g., L1), or (iii) employ one ion and are
inhibited by binding of an additional ion (subclass B2, e.g.,
CphA) (130, 164, 165, 325, 326). Crowder and colleagues com-
piled the recent structural and mechanistic data and summa-
rized the common features for the three subclasses of MBLs
(96). MBLs utilizing two Zn2⫹ ions for hydrolysis, such as the
subclass B3 S. maltophilia L1, coordinate the ␤-lactam sub-
strate by the carboxylate and carbonyl groups, bridged by a
hydroxide ion. After substrate binding, one of the Zn2⫹ ions, in
conjunction with enzyme residues, polarizes the ␤-lactam car-
bonyl for attack by the -OH group, which is hydrogen bonded
to deprotonated Asp120. Nucleophilic attack by the -OH
group creates a tetrahedral species which rapidly collapses into
an intermediate in which the ␤-lactam nitrogen is anionic.
Protonation of the nitrogen leads to product formation. The
source of the proton is not certain, and it may come from
Asp120 or a water molecule.
The B1 enzyme Bacillus cereus BcII is active in both its
mononuclear and dinuclear forms, and in the resting state, the
Zn2⫹-bound -OH group is hydrogen bonded to the
deprotonated Asp120 as well as several other active-site resi-
dues (Fig. 4) (96, 440). After attack by this -OH group, the
VOL. 23, 2010
breakdown of the tetrahedral intermediate requires protona-
tion of the ␤-lactam nitrogen; the source of this proton is under
investigation. In the case of the CphA B2 MBL from Aeromo-
nas hydrophila, a second bound Zn2⫹ ion is inhibitory. The
proposed mechanism includes a water molecule activated by
either His118 or Asp120, rather than a Zn2⫹-bound -OH
group; the singular Zn2⫹ appears to help coordinate the ␤-lac-
tam nitrogen (132, 451).
CIRCUMVENTING ␤-LACTAMASES
␤-Lactamase Inhibitors: Mechanistic Considerations
A successful strategy for combating ␤-lactamase-medi-
ated resistance is the use of agents designed to bind at the
active site, which are frequently ␤-lactams. This strategy can
take two forms: (i) create substrates that reversibly and/or
irreversibly bind the enzyme with high affinity but form
unfavorable steric interactions as the acyl-enzyme or (ii)
develop mechanism-based or irreversible “suicide inhibi-
tors” (53). Examples of the former are extended-spectrum
cephalosporins, monobactams, or carbapenems which form
acyl-enzymes and adopt catalytically incompetent conforma-
tions that are poorly hydrolyzed (Fig. 1, compounds 2, 3, and
4 to 7, respectively). For reversible inhibition, the reaction
can be described as was shown above in equation 1, where S
represents the (very) slowly hydrolyzed substrate. An equi-
librium constant, Ki, can be calculated from the pre-steady-
state rate constants, k⫺1/k1, and yields an estimate of affinity.
Irreversible “suicide inhibitors” can permanently inactivate
the ␤-lactamase through secondary chemical reactions in the
enzyme active site. Equation 5 represents a general mechanism
of irreversible inhibitors (I) leading to permanent enzyme in-
activation (E ⫺ I*):
k1 k2 k3
E⫹I | ¡ E⫺I O
L; E:I O ¡ E ⫺ I* (5)
k⫺1
Examples of these inactivators are the commercially avail-
able class A inhibitors clavulanic acid, sulbactam, and
tazobactam (Fig. 1, compounds 8 to 10). As will be described
below, these types of inactivators often display additional
pathways to inhibition. Irreversible inhibitors can be char-
acterized by first-order rate constants for inhibition (kinact,
the rate of inactivation achieved with an “infinite” concen-
tration of inactivator) and KI values (the concentration of
inactivator which yields an inactivation rate that is half the
value of kinact) (54, 92). While KI closely approximates the
meaning of Km for enzyme substrates, depending on the
individual rate constants comprising the reaction, the KI
may or may not equal the equilibrium constant Ki (⫽ k⫺1/k1)
determined under pre-steady-state conditions.
The 50% inhibitory concentration (IC50) measures the
amount of inhibitor required to decrease enzyme activity to
50% of its uninhibited velocity. While an IC50 can reflect an
inhibitor’s affinity or kcat/kinact ratio, these parameters are not
always congruent; e.g., an inhibitor can have a very poor “af-
finity” and acylate the enzyme slowly but still yield a low IC50
because of very low deacylation rates.
␤-LACTAMASE INHIBITORS 169
␤-Lactamase Inhibitors in Clinical Practice
Clavulanic acid, sulbactam, and tazobactam. Clavulanic
acid, the first ␤-lactamase inhibitor introduced into clinical
medicine, was isolated from Streptomyces clavuligerus in the
1970s, more than 3 decades ago (360). Clavulanate (the salt
form of the acid in solution) showed little antimicrobial
activity alone, but when combined with amoxicillin, clavu-
lanate significantly lowered the amoxicillin MICs against S.
aureus, K. pneumoniae, Proteus mirabilis, and E. coli (47).
Sulbactam and tazobactam are penicillinate sulfones that
were later developed by the pharmaceutical industry as syn-
thetic compounds in 1978 and 1980, respectively (117, 121).
All three ␤-lactamase inhibitor compounds share struc-
tural similarity with penicillin; are effective against many
susceptible organisms expressing class A ␤-lactamases (in-
cluding CTX-M and the ESBL derivatives of TEM-1,
TEM-2, and SHV-1); and are generally less effective against
class B, C, and D ␤-lactamases (53, 60, 67, 341). The activity
of an inhibitor can be evaluated by the turnover number (tn)
(also equivalent to the partition ratio [kcat/kinact]), defined as
the number of inhibitor molecules that are hydrolyzed per
unit time before one enzyme molecule is irreversibly inac-
tivated (58). For example, S. aureus PC1 requires one clav-
ulanate molecule to inactivate one ␤-lactamase enzyme,
while TEM-1 needs 160 clavulanate molecules, SHV-1 re-
quires 60, and B. cereus I requires more than 16,000 (53, 69,
113, 123, 411). For comparison, sulbactam tns are 10,000 and
13,000 for TEM-1 and SHV-1, respectively (180, 410).
The low KIs of the inhibitors for class A ␤-lactamases (nM to
␮M), the ability to occupy the active site “longer” than ␤-lactams
(high acylation and low deacylation rates), and the failure to be
hydrolyzed efficiently are integral to their efficacy (158). Clavu-
lanate, sulbactam, and tazobactam differ from ␤-lactam antibiot-
ics as they possess a leaving group at position C-1 of the five-
membered ring (sulbactam and tazobactam are sulfones, while
clavulanate has an enol ether oxygen at this position). The better
leaving group allows for secondary ring opening and ␤-lactamase
enzyme modification. Compared to clavulanate, the unmodified
sulfone in sulbactam is a relatively poor leaving group, a property
reflected in the high partition ratios for this inhibitor (e.g., for
TEM-1, sulbactam tn ⫽ 10,000 and clavulanate tn ⫽ 160) (179,
180). Tazobactam possesses a triazole group at the C-2 ␤-methyl
position. This modification leads to tazobactam’s improved IC50s,
partition ratios, and lowered MICs for representative class A and
C ␤-lactamases (36, 58, 60).
The efficacy of the mechanism-based inhibitors can vary
within and between the classes of ␤-lactamases (Table 2).
For class A, SHV-1 is more resistant to inactivation by
sulbactam than TEM-1 but more susceptible to inactivation
by clavulanate (328). Comparative studies of TEM- and
SHV-derived enzymes, including ESBLs, found that the
IC50s for clavulanate were 60- and 580-fold lower than those
for sulbactam against TEM-1 and SHV-1, respectively (328).
In our opinion, the explanations for these differences in
inactivation chemistry are likely subtle, yet highly important,
differences in the enzyme active sites. For example, Thom-
son et al. compared the atomic structure models of TEM-1
and SHV-1 and found that the distance between Val216 and
Arg244, residues responsible for positioning of the water
170 DRAWZ AND BONOMO CLIN. MICROBIOL. REV.

TABLE 2. Kinetic properties of representative ␤-lactamasesa

Clavulanate Sulbactam Tazobactam
Ambler
␤-Lactamase IC50 KI IC50 KI IC50 Reference(s)
class KI (␮M) tn tn tn
(nM) (␮M) (nM) (␮M) (nM)

TEM-1 A 0.1 60 160 1.6 900 10,000 0.01 97 140 73, 180, 283
SHV-1 A 1 12 60 8.6 12,000 13,000 0.07 150 5 58, 153, 410, 411
SHV-5 A 20 1,800 80 137
PC1 A 30 1 80 27 1 47, 58, 69
CTX-M-2 A 200 2,100 20 18
CcrA B ⬎500,000 ⬎500,000 ⬎500,000 400,000 4,000 58
P99 C ⬎100,000 ⬎500,000 5,600 8.5 50 58
CMY-2 C 4,365 101 50 116a
OXA-1 D 1,800 4,700 380 1400 196, 328
OXA-2 D 1,400 0.1 140 10 211, 328
a
The definition of KI may not be uniform from laboratory to laboratory. See the cited references for the methods used in each determination.

molecule important in the inactivation mechanism of clavu-
lanate, was more than 2 Å greater in SHV-1 than in TEM-1
(411). This increased distance may be too great for coordi-
nation of a water molecule, suggesting that the strategic
water is positioned elsewhere in SHV-1 and may be re-
cruited into the active site with acylation of the substrate or
inhibitor. This variation underscores the notion that mech-
anism-based inhibitors may undergo different inactivation
chemistry even in highly similar enzymes (410, 411).
Mechanism of inhibition. Evidence from X-ray crystallog-
raphy, UV difference spectra, isoelectric focusing, mass
spectrometry (MS), and Raman microscopy suggests that
the inactivators of class A ␤-lactamases undergo complex
reaction schemes with multiple branch points after forma-
tion of the acyl-enzyme (45, 78, 79, 157, 309, 396). As rep-
resented in equations 5 and 6, the acyl-enzyme intermediate
can (i) undergo a reversible change that generates a tran-
siently inhibited enzyme, a tautomer (E ⫺ T); (ii) lead to
permanent inactivation as a covalent acyl-enzyme species
(E ⫺ I*); or (iii) regenerate the active enzyme via hydrolysis
(E ⫹ P):
E⫺T
k4
/ k⫺4
k5
E⫺I 3 E ⫹ P
k3
2 of the tazobactam intermediate and the enzyme active site may
E ⫺ I*
The functional inhibition of the enzyme is determined by the
relative rates (k3, k4, k⫺4, and k5) of each of these pathways
and in particular by the formation of the E ⫺ I* species (53).
Figure 5 shows a more detailed mechanism describing clav-
ulanate inhibition of PC1, TEM-1, or SHV-1 (78, 79, 81, 122,
179, 180). Kinetic and mass spectrometry analysis of inactiva-
tion mechanisms, combined with crystallographic studies,
suggests that clavulanate, sulbactam, and tazobactam follow
similar reaction pathways, beginning with formation of an acyl-
enzyme species (48, 81, 307, 308). After acylation, opening of
the five-membered ring leads to formation of a transient imine
intermediate. This imine species is likely the common inter-
mediate preceding the chemical conversions that lead to tran-
sient enzyme inhibition (44, 179, 197). Raman spectroscopy of
SHV/inhibitor crystals show that the imine species then rear-
ranges to form enamine intermediates (197, 307). This en-
amine intermediate, in either the trans or cis conformation,
represents a second important intermediate in the inactivation
mechanism (69, 81, 368). Depending on the properties of the
enzyme and inhibitor, the reaction will ultimately proceed to
deacylation or irreversible (prolonged) inactivation. In the case
of deacylation of the enamine intermediate, the acyl-enzyme
undergoes decarboxylation and ester bond hydrolysis, regen-
erating the active ␤-lactamase, albeit very slowly (260).
The duration of transient inhibition is determined, in part,
by the stability of the intermediate species. Preceding acylation
of the inhibitor, a persistent noncovalent Michaelis complex
may account for inhibition of enzyme activity (196). Following
formation of the acyl-enzyme, stabilization of the enamine
intermediate is a significant factor in prolonged enzyme inhi-
bition. Crystals of tazobactam in complex with the SHV
Glu166Ala variant (PDB 1RCJ) showed that tazobactam
formed stoichiometric amounts of the trans-enamine (308).
The trans-enamine intermediate of tazobactam, as opposed to
the trans-enamine intermediates of clavulanate and sulbactam,
(6) may be stabilized by intra- and intermolecular interactions.
These interactions between the sulfone and triazolyl moieties
explain, in part, tazobactam’s potent in vitro and in vivo inhi-
bition of many serine ␤-lactamases (36, 60, 308, 312).
Data from Raman crystallography also support the hypoth-
esis that compared to clavulanate and sulbactam, tazobactam
more readily forms the trans-enamine intermediate (197). In
this case, Raman spectroscopy allows for the identification of
reaction intermediates and calculation of their rates of decay
and accumulation by examining single crystals in solution.
Studies of SHV-1 in complex with each of the mechanism-
based inhibitors revealed that tazobactam forms a predomi-
nant population of trans-enamine, as opposed to clavulanate
and sulbactam, which form a mixture of trans-enamine and the
VOL. 23, 2010
FIG. 5. Proposed mechanism of inhibition of class A ␤-lactamases by clavulanate, showing the different acyl-enzyme fragmentation products
(expressed in daltons), that have been experimentally observed. (Based on data from references 48, 108, 197, 411, and 453.)
more chemically labile cis-enamine and imine species (197).
Analysis of the inactivation reactions of PC1, TEM, and
SHV ␤-lactamases using mass spectrometry has identified a
series of intermediates beyond the acyl-enzyme, imine, and
enamine (48, 81, 311, 396, 411). The terminal end products of
inhibition with these mechanism-based inhibitors suggest that
a covalent modification of the active-site Ser70 persists
through the inhibition process. Mass adducts (in daltons) cor-
respond to different inactivated enzyme species; e.g., (i) Ser70-
Ser130 cross-linked enzyme or propynyl enzyme (⌬ ⫹ 52 ⫾ 3),
(ii) aldehyde (⌬ ⫹ 70 ⫾ 3), (iii) hydrated aldehyde (⌬ ⫹ 88 ⫾
3), and (iv) decarboxylated trans-enamine (⌬ ⫹ 156 ⫾ 3) are
some of the products (Fig. 5).
␤-Lactam–␤-lactamase inhibitor combinations: clinical use.
Generally, the inhibitors do not inactivate PBPs, but notable
exceptions include (i) the intrinsic activities of sulbactam
against Bacteroides spp., Acinetobacter spp., and N. gonor-
rhoeae; (ii) clavulanate action against Haemophilus influenzae
and N. gonorrhoeae; and (iii) tazobactam inhibition of PBPs in
Borrelia burgdorferi (53, 167, 222, 264, 285, 423, 424, 447). As
these “antibacterial effects” are relatively weak, the inhibitors
are always combined with ␤-lactam antibiotics for clinical use.
Currently, there are five ␤-lactam–␤-lactamase inhibitor for-
mulations available. Amoxicillin-clavulanate, ticarcillin-clavu-
lanate, ampicillin-sulbactam, and piperacillin-tazobactam are
available in the United States. Cefoperazone-sulbactam is used
in several European countries, Japan, and India but is not
available in the United States, and therefore we will not in-
clude this combination in this review. Table 3 summarizes
selected clinical features of these available combinations.
Importantly, as in clinical usage, the ␤-lactamase inhibitors
are combined with ␤-lactams for in vitro susceptibility testing.
The composition of these formulations can have a major im-
␤-LACTAMASE INHIBITORS 171
pact on susceptibility results, which may or may not reflect
clinical efficacy. We present to the reader the following exam-
ple. The ticarcillin-clavulanate preparation for parental admin-
istration is 3.0 g-0.1 g, while ampicillin-sulbactam is at a 2.0
g-1.0 g ratio. However, both combinations are tested at a 2:1
ratio in vitro, and since clavulanate has higher “gram-for-gram”
potency than sulbactam, clavulanate may “test better” in MIC
profiling. However, clinically ampicillin-sulbactam may retain
efficacy because of the relatively large amount of inhibitor
delivered relative to the ␤-lactam and relative to the amount of
␤-lactamase in the cell. This scenario illustrates an important
issue in susceptibility testing and its correlate to clinical effi-
cacy.
(i) Amoxicillin-clavulanate. Amoxicillin-clavulanate was the
first ␤-lactam–␤-lactamase inhibitor combination introduced
into clinical practice, in 1981 in the United Kingdom and in
1984 in the United States, and remains the only combination
available for oral use. The activity of amoxicillin against sus-
ceptible bacteria that do not possess a ␤-lactamase (e. g.,
streptococci, enterococci, E. coli, and Listeria spp.) is not im-
proved by the use of clavulanate. However, the addition of
clavulanate significantly expands amoxicillin’s spectrum to in-
clude penicillinase-producing S. aureus, H. influenzae, Morax-
ella catarrhalis, Bacteroides spp., N. gonorrhoeae, E. coli, Kleb-
siella spp., and P. mirabilis (59, 387). The oral availability of
amoxicillin-clavulanate makes it well suited for the outpatient
clinic, and this ␤-lactam–␤-lactamase inhibitor combination
exerted its greatest impact on the treatment of community-
acquired respiratory infections (59). Additionally, amoxicillin-
clavulanate is sometimes used as an oral equivalent (“step-
down therapy”) of ampicillin-sulbactam or ticarcillin-
clavulanate in the treatment of skin, soft tissue, abdominal, and
gynecological infections. Intravenous formulations of amoxicil-
172 DRAWZ AND BONOMO CLIN. MICROBIOL. REV.

TABLE 3. Selected clinical features of available ␤-lactam–␤-lactamase inhibitor combinations

␤-Lactam–␤- Route of
Proven clinical efficacy for
lactamase Trade name; administration, t1/2, h (% Mean peak serum Approved clinical
susceptible causative
inhibitor manufacturer formulations, and protein bound) levels (␮g/ml) indications
organism(s):
composition dosing intervalsa

Piperacillin- Zosyn; Wyeth i.v., 2.0 g/0.25 g q6h, Piperacillin and 1 h after 3.375-g dose: Appendicitis E. coli, Bacteroides spp.
tazobactam 3.0 g/0.375 g q6h, 4.0 tazobactam, piperacillin, 106; Skin and soft tissue S. aureus
g/0.5 g q6h 0.7–1.2 (30) tazobactam 10.7 infections, including
diabetic foot infections
Postpartum endometritis E. coli
or pelvic inflammatory
disease
Community-acquired H. influenzae
pneumonia
Hospital-acquired, S. aureus, A. baumannii,
ventilator-acquired H. influenzae, K.
pneumonia pneumoniae, P.
aeruginosab

Ampicillin- Unasyn; Pfizer i.v./i.m., 1.0 g/0.5 g q6h, Ampicillin, 1.0 After 15-min infusion Skin and skin structure S. aureus, E. coli,
sulbactam 2.0 g/1.0 g q6h (28); of 1-g/0.5-g dose: infections Klebsiella spp., P.
sulbactam, ampicillin, 40-71; mirabilis, B. fragilis,
1.0 (38) sulbactam, 21-40 Enterobacter spp., A.
calcoaceticus
Intra-abdominal E. coli, Klebsiella spp.,
infections Bacteroides spp.,
Enterobacter spp.
Gynecological infections E. coli, Bacteroides spp.

Amoxicillin- Augmentin; Pediatric oral Amoxicillin, 1.3 1 h after 250-mg/ Otitis media S. pneumoniae, H.
clavulanate GlaxoSmithKline suspension, 600 mg/ (18); 62.5-mg dose: influenzae, Moraxella
42.9 mg/5 ml q12hc clavulanate, amoxicillin, 6.9; catarrhalis
Chewable tablet and 1.0 (25) clavulanate, 1.6 Community-acquired H. influenzae, M.
oral suspension, 125 pneumonia or acute catarrhalis, H.
mg/31.25 mg q8h, bacterial sinusitis parainfluenzae, K.
200 mg/28.5 mg pneumoniae, methicillin-
q12h, 250 mg/62.5 susceptible S. aureus, S.
mg q8h, 400 mg/57.0 pneumoniae
mg q12h
Tablet, 250 mg/125 mg Sinusitis H. influenzae, M.
q8h, 500 mg/125 mg catarrhalis
q8-12h, 875 mg/125
mg q12h
Extended-release Skin and skin structure S. aureus, E. coli,
tablet, 1,000 mg/62.5 infections Klebsiella spp.
mg q12h Urinary tract infections E. coli, Klebsiella spp.,
Enterobacter spp.

Ticarcillin- Timentin; i.v., 3.0 g/0.1 g q4-6h Ticaracillin, 1.1 1 h after 3.1-g dose: Septicemia Klebsiella spp., E. coli, S.
clavulanate GlaxoSmithKline (45); ticarcillin, 131; aureus, P. aeruginosa
clavulanate, clavulanate, 1.8 Lower respiratory tract S. aureus, H. influenzae,
1.1 (25) infections Klebsiella spp.
Bone and joint infections S. aureus
Skin and skin structure S. aureus, Klebsiella spp.,
infections E. coli
Urinary tract infections E. coli, Klebsiella spp., P.
aeruginosa, Citrobacter
spp., Enterobacter
cloacae, Serratia
marcescens, S. aureus
Gynecologic infections Prevotella melaninogenicus,
Enterobacter spp., E.
coli, K. pneumoniae, S.
aureus, S. epidermidis
Intra-abdominal E. coli, K. pneumoniae, B.
infections fragilis
a
i.v., intravenous; i.m., intramuscular; q6h, every 6 h.
b
When treated with piperacillin-tazobactam, hospital-acquired and ventilator-acquired pneumonia caused by P. aeruginosa should be administered with an
aminoglycoside.
c
The dose of amoxicillin-clavulanate in the pedatric oral suspension is 45 mg/kg q12h based on information available in the material supplied by the manufacturer.

lin-clavulanate are available in Europe and have been studied
primarily for their efficacy against ESBL-producing E. coli bac-
teremia (239, 370).
(ii) Ticarcillin-clavulanate. When introduced in 1985, ticar-
cillin-clavulanate was the first ␤-lactam–␤-lactamase inhibitor
combination available for parenteral administration. Similar to
the case for amoxicillin, the addition of clavulanate does not
increase activity against pathogens for which ticarcillin alone is
effective, such as non-␤-lactamase-producing Haemophilus
spp., E. coli, Proteus spp., Enterobacter spp., Morganella spp.,
Providencia spp., and P. aeruginosa. However, the combination
of ticarcillin and clavulanate does increase activity against
␤-lactamase-producing staphylococci, E. coli, H. influenzae,
Klebsiella spp., Proteus spp., Pseudomonas spp., Providencia
VOL. 23, 2010
spp., N. gonorrhoeae, Moraxella catarrhalis, and Bacteroides spp.
(59, 127, 319). Surprisingly, ticarcillin-clavulanate exhibits ac-
tivity against the multidrug-resistant, non-lactose-fermenting
S. maltophilia, and the use of this combination in addition to
other antimicrobials (e.g., aztreonam or trimethoprim-sulfa-
methoxazole) leads to synergistic killing (118, 275). This phe-
nomenon may represent a very interesting opportunity to study
the activity of ticarcillin-clavulanate against S. maltophilia.
Clavulanate induces the expression of chromosomally medi-
ated AmpC ␤-lactamases in many Enterobacteriaceae and can
antagonize the antibacterial effects of ticarcillin as a partner
␤-lactam (8, 203, 231, 237). This ticarcillin antagonism was
observed in laboratory checkerboard studies with E. cloacae
and Morganella morganii strains (237). However, the impact of
␤-lactamase induction in the clinic is very hard to measure.
When testing or evaluating antibiotics in preclinical or clinical
trials, ␤-lactamase induction may bear importance as an even-
tual predictor of efficacy. In our opinion, the ability of ␤-lac-
tamase inhibitors to induce cephalosporinase production
should be carefully examined and defined before clinical trials
are performed.
In response to the potential for increased AmpC production
due to ␤-lactam inducers (such as clavulanate, but including
also cefoxitin, imipenem, and ceftazidime) as well as the rela-
tively poor performance of amoxicillin-clavulanate and ticar-
cillin-clavulanate against ESBL expressors, Livermore et al.
and Nikaido et al. have proposed the combination of clavu-
lanate with a cephalosporin, such as cefepime or cefpirome,
that is more stable against AmpC enzymes (239, 288).
Cefepime-clavulanate and cefpirome-clavulanate combina-
tions, with a constant concentration of 4 ␮g/ml clavulanate,
were effective at ⱕ1 ␮g/ml against ESBL-producing Enterobac-
teriaceae (239). The combination of cefepime and clavulanate
enjoys a particular advantage due to the inability of class C
enzymes to hydrolyze cefepime and the ability of clavulanate to
inhibit ESBLs. Currently, there are important obstacles to
combining these agents (i.e., all three agents are at the end of
patent protection, and the required trials of the formulations
would be very expensive), but provided that the clinical re-
search can be performed, these combinations may be an at-
tractive alternative to carbapenem treatment of ESBL-produc-
ing organisms. We hasten to add that attention should be paid
to the pharmacokinetics and pharmacodynamics of these novel
combinations before randomized controlled trials are per-
formed, as it is not possible to “fix” a concentration of clavu-
lanate in vivo.
(iii) Ampicillin-sulbactam. Ampicillin shows activity against
most streptococci, enterococci, Listeria spp., and strains of S.
aureus, H. influenzae, E. coli, P. mirabilis, Salmonella spp., and
Shigella spp. that are devoid of ␤-lactamases (an important
exception are ampicillin-resistant strains of H. influenzae that
do not contain a ␤-lactamase and often have substitutions in
PBP sequences) (194, 257). In combination with sulbactam, the
activity extends to ␤-lactamase-containing S. aureus, H. influ-
enzae, M. catarrhalis, E. coli, Proteus spp., Klebsiella spp., and
anaerobes (59, 362, 444). When the combination of ampicillin
at 2.0 g and sulbactam at 1.0 g was marketed in 1987, this
broad-spectrum activity made ampicillin-sulbactam ideal ther-
apy for polymicrobial infections such as abdominal and gyne-
cological surgical infections, aspiration pneumonia, odonto-
␤-LACTAMASE INHIBITORS 173
genic abscesses, and diabetic foot infections. The early clinical
success of this combination (and susceptibility of many patho-
gens to ampicillin-sulbactam and ticarcillin-clavulanate) estab-
lished confidence in the role of ␤-lactam–␤-lactamase inhibitor
therapy. Unfortunately, the resistance to ampicillin-sulbactam
among clinical isolates of E. coli is increasing (199).
Sulbactam is not well absorbed orally and must be admin-
istered parenterally (126). However, the intravenous ampi-
cillin-sulbactam combination is well tolerated, with very few
reported side effects (7, 66).
(iv) Piperacillin-tazobactam. Piperacillin in combination
with tazobactam became available in the United States in 1993.
Piperacillin is a broad-spectrum penicillin that is bactericidal
against many Gram-positive and Gram-negative aerobes and
anaerobes (140). As a single agent, piperacillin demonstrates
activity against P. aeruginosa, pneumococci, streptococci,
anaerobes, and Enterococcus faecalis, and this activity is re-
tained in combination with tazobactam (232). Clinicians must
remember that the addition of tazobactam does not always
increase susceptibility of P. aeruginosa and other Gram-nega-
tive bacilli expressing AmpC ␤-lactamases (140). However,
tazobactam does extend piperacillin’s activity against most
␤-lactamase producing strains of Enterobacteriaceae, H. influ-
enzae, N. gonorrhoeae, and M. catarrhalis and has the potential
to lower MICs against these strains expressing ESBLs (59,
192). The in vitro inhibition of CMY-type ␤-lactamases by
tazobactam is reported, but the clinical relevance of this phe-
notype is still uncertain (34, 116a).
Several recent retrospective studies of clinical outcomes
from E. coli and K. pneumoniae ESBL-producing isolates
argue for the efficacy of piperacillin-tazobactam in the treat-
ment of these infections (135, 336, 370, 420). Gavin et al.
showed that when in vitro testing revealed susceptibility to
piperacillin-tazobactam (ⱕ16/4 ␮g/ml), successful outcomes
were seen in 10 out of 11 non-urinary tract infections (135).
An examination of 43 bloodstream infections caused pri-
marily by CTX-M ESBL-producing E. coli isolates revealed
that piperacillin-tazobactam, amoxicillin-clavulanate (i.v.),
or carbapenem treatment led to lower mortality than with
cephalosporin or fluoroquinolone treatment (9% versus
35%) (370). Another study, including infections caused by
SHV- and TEM-type ESBL-producing K. pneumoniae, es-
tablished that empirical therapy with ␤-lactam–␤-lactamase
inhibitor combinations was validated by in vitro susceptibil-
ity testing in 75% of cases, compared to 0% for oxymino-
cephalosporin treatment (420). Furthermore, initial treat-
ment with an adequate antibiotic was associated with lower
mortality rates (37%) than inadequate empirical therapy
(67%).While this analysis did not stratify the outcome data
based on ESBL type, other authors have reported that
TEM-type ESBLs are more susceptible to piperacillin-ta-
zobactam than are SHV-type ESBLs (185, 361).
Several observational studies have strongly suggested that
the replacement of extended-spectrum cephalosporins with
piperacillin-tazobactam can help lower the rates of infection
from Gram-negative ESBL producers and vancomycin-resis-
tant Enterococcus spp. (16, 43, 215, 216, 323, 355, 365). While
the mechanism for decreasing ESBL-producing isolates is not
clear, it must be noted that the efficacy of ␤-lactam–␤-lacta-
mase inhibitor combinations may be reduced for organisms
174 DRAWZ AND BONOMO
producing multiple ESBLs, particularly if they also have an
AmpC ␤-lactamase (42, 321, 381). For this reason, we caution
against the use of piperacillin-tazobactam for the treatment of
ESBL-producing pathogens.
So, why do the in vitro susceptibility data and the clinically
cited data suggest that piperacillin-tazobactam can be used
in the treatment of ESBL producers? The reasons for this
contradiction include that piperacillin, as a partner ␤-lactam
for ␤-lactamase inhibitors, is relatively resistant to hydroly-
sis by certain plasmid-mediated ␤-lactamases (e.g., TEM)
compared to amoxicillin, ampicillin, or ticarcillin. This sub-
strate stability may be due, in part, to piperacillin’s lower
affinity for and lower turnover (kcats) by these enzymes and
also piperacillin’s greater affinity for bacterial PBPs (234).
We also propose that the pharmacokinetics and pharmaco-
dynamics of the 3.375-g and 4.5-g formulations lead to ex-
tended periods of time when free drug concentrations
(⬎100 ␮g/ml piperacillin in serum) are greater than the
MIC for ⱖ50% of the dosing interval for many nosocomial
pathogens (242). It is likely that the high serum concentra-
tions combined with the superior activity of piperacillin and
the relatively “better” inhibitory activity of tazobactam ver-
sus clavulanate or sulbactam is enough to inhibit a majority
of ␤-lactamases in a complex ␤-lactamase background (class
A and C ␤-lactamases) (328). However, this efficacy is not
always clinically certain, and the physician must be aware
that the susceptibility pattern of an infecting Gram-negative
pathogen does not always reveal the number and complexity
of the ␤-lactamases present. Moreover, the formulation that
is tested in the lab may not mirror what is administered to
patients.
Inhibitory Activity of Carbapenems
It is our opinion that consideration of carbapenems, al-
though they are not explicitly ␤-lactamase inhibitors, is es-
sential to a review of ␤-lactamase inhibition. These thien-
amycin derivatives are among the “last line of defense”
against many multidrug-resistant pathogens. An increasing
body of evidence suggests that carbapenems are effective
not just for their broad-spectrum antibacterial activity but
also for their multiple ␤-lactamase-inhibitory mechanisms.
The insight gleaned from the ability of carbapenems to
inhibit serine ␤-lactamases may serve as a useful starting
point for intelligent inhibitor design. Also, while we await
the development and release of novel ␤-lactamase inhibi-
tors, it behooves us to wisely implement the ␤-lactams cur-
rently in our armamentarium. With this in mind, a discus-
sion of the inhibitory activity of carbapenems follows.
Meropenem, imipenem, ertapenem, and doripenem (dorip-
enem was released in the U.S. and European markets in 2007)
are often reserved for the most difficult-to-treat Gram-negative
infections (Fig. 1, compounds 4 to 7) (320). Although resis-
tance to carbapenems is rare in the clinic, several carbapenem-
hydrolyzing serine ␤-lactamases are identified, such as KPC-2,
NMC-A, SME-1, and IMI-1 (358, 455). These enzymes may be
found in organisms that also produce additional ␤-lactamases,
conferring broad substrate profiles. For example, E. cloacae
isolates can produce IMI-1 or NMC-A, which hydrolyze car-
bapenems, and also TEM and AmpC-type enzymes (359).
CLIN. MICROBIOL. REV.
FIG. 6. Imipenem tautomers hypothesized to form after acylation
of carbapenems by serine ␤-lactamases. The deacylation rate of the
⌬1-pyrroline is significantly lower than that of the ⌬2-pyrroline, which
is thought to play a large role in the inhibitory activity of these com-
pounds (407, 458).
In addition to their broad antibacterial spectrum, carbapenems
are also effective inhibitors, or “slow substrates,” of most serine
␤-lactamases.
The stability of thienamycins to ␤-lactamase hydrolysis was
noted in early studies of imipenem, including low kcat values
for the class A ␤-lactamase of B. cereus and a class C P.
aeruginosa ␤-lactamase (150, 208, 270, 366). Charnas and
Knowles observed biphasic kinetics when RTEM was acylated
by carbapenem compounds; an initial burst was followed by a
slower steady-state reaction, strongly suggestive of a branched
reaction pathway (80, 114). Further work by Mobashery’s
group demonstrated that acylation of TEM-1 by imipenem
occurs readily and is followed by ⌬2- to ⌬1-tautomerization of
the pyrroline double bond in the acyl-enzyme species (Fig. 6)
(407). The rate of deacylation differs for the tautomers (the ⌬1
tautomer is approximately 10-fold slower than the ⌬2 tau-
tomer), and it was initially accepted that molecular rearrange-
ments to the more stable (i.e., more slowly deacylated) species
accounted for enzyme inhibition. Site-directed mutagenesis
studies of TEM-1 Arg244Ser showed that the guanidinium
group on Arg244 provides directly, or indirectly via a water
molecule, the necessary proton for tautomerization to the ⌬1-
pyrroline acyl-enzyme (407, 458). Incidentally, this water ap-
pears to be the same molecule that serves as the proton donor
necessary for clavulanate inhibition in class A enzymes (179).
Elucidation of the crystal structures of several ␤-lactamases
in complex with carbapenems has offered additional insight
into the versatile inhibition mechanism of these compounds.
Maveyraud et al. found that in the structure of TEM-1 com-
plexed with imipenem (PDB 1BT5), the ester carbonyl oxygen
of the acyl-enzyme was not located in the oxyanion hole (255).
This approximately 180° rotation of the carbonyl was also seen
in the crystal structure of acyl-enzyme species of imipenem
complexed via Ser64 with the AmpC ␤-lactamase (PDB 1LL5)
(21). In contrast, the SHV-1/meropenem crystal demonstrated
two conformations of the intermediate acyl species, one where
the meropenem carbonyl oxygen of the acyl-enzyme is in the
oxyanion hole and one where it is not (Fig. 7) (295). Proper
position in this oxyanion hole, the binding pocket for the ␤-lac-
tam carbonyl, is important for both initial enzyme acylation
and hydrolysis of the ester bond (277, 425). A conformation
which places the ␤-lactam carbonyl outside of this electrophilic
center may be delayed in hydrolysis.
Computational models of TEM-1 with imipenem demon-
strated that the covalent adduct formed first after acylation did
have its carbonyl in the oxyanion hole (255). However, this
conformation created a steric clash between the hydroxyethyl
group and residues 129 to 131, which forces the rearrangement
VOL. 23, 2010 ␤-LACTAMASE INHIBITORS 175

FIG. 7. Molecular representation of SHV-1/meropenem acyl-enzyme, based on PDB coordinates 2ZD8. The conformation with the carbonyl
of the acyl-serine bond in the oxyanion hole (formed by NH amides of Ser70 and Ala237) is colored teal, while the conformation with the carbonyl
flipped out is colored purple. Residues 244, 130, and 105, which demonstrate significant orientation changes compared to the apo SHV-1
␤-lactamase, are labeled. Glu166 and Asn132, important for making hydrogen bonds with the deacylation water (the approximate location is
represented as a red sphere) and C-6 hydroxyethyl substituent, respectively, are shown. The large R⬘ carbamoyl-pyrrolidinyl group of meropenem
was disordered and thus is only partially illustrated.

of the acyl-enzyme rotating the carbonyl outside the oxyanion
hole. Thus, while producing a conformational change in the
enzyme, the C-6 hydroxyethyl substituent also facilitates rear-
rangement of the carbapenem to a more slowly deacylated
form. Subsequent examination of the TEM Asn132Ala variant
demonstrated that replacement of the residue relieved the steric
encumbrance of the TEM-1/imipenem acyl-enzyme and allowed
the carbonyl to rotate back into the oxyanion hole (437). Further-
more, the TEM-1/imipenem structure indicated that hydrogen
bonds were formed between the hydroxyl group of the C-6 ␣-1R-
hydroxyethyl substituent on imipenem and both the Glu166-as-
sociated hydrolytic water and the side chain oxygen of Asn132
(255). Similarly, the SHV-1/meropenem and class D OXA-13/
meropenem structures (PDB 2ZD8 and 1H8Y, respectively) re-
vealed that the putative deacylation water molecule has an addi-
tional hydrogen-bonding interaction with the -OH group of
meropenem’s C-6 ␣-1R-hydroxyethyl substituent (295, 334). This
interaction weakens the nucleophilicity and/or changes the direc-
tion of the lone pair of electrons of the water molecule and results
in poor turnover of the carbapenems.
As individual hydrogen atoms are difficult to resolve by
X-ray crystallography, the structural data for ␤-lactamases
and carbapenems have not been able to discriminate be-
tween the ⌬1- and ⌬2-pyrroline tautomers. In contrast, Ra-
man spectroscopy allows the monitoring of specific bond
character and compound reactivity. Recent studies of Ra-
man difference spectra in SHV-1 reacted with meropenem,
ertapenem, and imipenem propose the correlation that the
⌬1-pyrroline tautomer corresponds to the acyl-enzyme spe-
cies with the carbonyl outside the oxyanion hole and the
⌬2-pyrroline tautomer to the acyl-enzyme with the carbonyl
inside the electrophilic center (195). Further, this work sug-
gests that in crystal form the ⌬1- and ⌬2-tautomers are
present in equal amounts but that ⌬1-pyrroline predomi-
nates in solution. This ratio is consistent with the kinetic
data implicating the ⌬1-tautomer as the primary inhibitory
species.
The recent structure of OXA-1, a class D monomeric ␤-lac-
tamase, inactivated by doripenem provided further support for
the role of carbapenems as inactivators of serine ␤-lactamases
(PDB 3ISG) (379a). In this structure by Schneider and col-
leagues, although the carbonyl oxygen is positioned in the
oxyanion hole, water molecules are absent. The carbamate of
Lys70 forms a hydrogen bond to the C-6 hydroxyethyl group,
and Lys212 and Thr213 form a salt bridge and hydrogen bond
to doripenem. Furthermore, the bond angles of the acyl-en-
zyme suggest the ⌬1-tautomer is formed.
In addition to the imipenem-induced conformation change
of class A and C ␤-lactamases, the (albeit slow) turnover of
carbapenems leads to a stable enzyme-product species that
occupies the ␤-lactamase active site for relatively longer than
the enzyme-products of other ␤-lactams, such as penicillin
(whose dissociation is limited by diffusion for class A enzymes)
(149, 254). However, the deacylation rate was still lower than
the dissociation rate for imipenem, suggesting that acyl-en-
zyme rearrangement accounts most significantly for inhibition.
But notably, the presence of hydrolyzed inhibitor in the active
site can contribute to enzyme inhibition.
176 DRAWZ AND BONOMO
INHIBITOR-RESISTANT CLASS A ␤-LACTAMASES
Within several years of the introduction of clavulanate com-
binations for clinical use, resistance to amoxicillin-clavulanate
and ticarcillin-clavulanate was observed in isolates of E. coli
and K. pneumoniae (247, 248, 376, 446). In 1987, Martinez et
al. reported resistance rates in E. coli in Madrid hospitals and
estimated that of amoxicillin-resistant strains, 20 to 30% were
co-amoxicillin-clavulanate resistant (248). Investigations in the
late 1980s demonstrated that this phenotype may result from
production of ␤-lactamases not susceptible to the inhibitors
(e.g., AmpCs from Enterobacter spp. or P. aeruginosa or me-
tallo-␤-lactamases) or enzyme hyperproduction (68). Hyper-
production of a ␤-lactamase can be mediated by mutations in
the promoter region of the gene and/or high copy numbers of
plasmids carrying the bla gene; both scenarios were identified
for the TEM-1 enzyme (247, 248, 446, 448, 449).
Additional resistance mechanisms were concurrently be-
ing examined in research laboratories. In 1989, Oliphant
and Struhl studied clavulanate and sulbactam resistance in
TEM-1 by introducing a series of random substitutions into
the DNA segment which coded for the enzyme’s active site
(300). Through mutagenesis of a 17-amino-acid segment of
the TEM-1 active site (Arg61 to Cys77) and functional se-
lection assays, they found that enzymes with an Ile, Leu, or
Val substitution at Met69 had increased resistance to ampi-
cillin-clavulanate and ampicillin-sulbactam. This foreshad-
owed an important amino acid substitution in inhibitor-
resistant (IR) TEM.
Three years later, Zafaralla et al. engineered by site-directed
mutagenesis the Arg244Ser variant of TEM-1 to explore the
role of this key amino acid and its interaction with the peni-
cillin C-3 carboxylate (457). This work advanced the earlier
hypothesis by Moews et al. that Arg244 played an important
role in the recognition of the conserved substrate C-3/C-4 carbox-
ylate (179, 267). Further studies by Mobashery’s laboratory also
revealed that Arg244Ser was more resistant to inactivation by
clavulanate (179). Interestingly, the first IR class A ␤-lactamase
was isolated from a clinical isolate of E. coli in the same year. The
enzyme was determined to be related to TEM-1 or TEM-2 and
hence was designated inhibitor-resistant TEM (IRT-1) (29, 427).
Sequencing information revealed an Arg-to-Cys or -Ser substitu-
tion at residue 244 (23, 427).
Since that time in the early 1990s, the number of clinically
identified IRTs has expanded to include more than 35 unique
enzymes (Table 4) (www.lahey.org/studies/webt.asp). In addi-
tion to amoxicillin-clavulanate-resistant E. coli, IRTs have
been identified in Klebsiella, Proteus, Shigella, and Citrobacter
spp. (385). IR SHVs have also been isolated from K. pneu-
moniae, with a total of five IR SHVs presently described (Table
5) (109). Operationally, inhibitor resistance refers to resistance
to amoxicillin-clavulanate and may, but does not necessarily,
include resistance to sulbactam and tazobactam (410). More-
over, we stress that this is a “relative resistance”; provided that
sufficient clavulanate concentrations are used, these IR en-
zymes can be inactivated.
Currently, reports describing non-TEM, non-SHV family IR
enzymes are becoming more common. The CTX-M ESBLs,
which typically hydrolyze penicillins and extended-spectrum
cephalosporins (preferentially cefotaxime), have not yet shown
CLIN. MICROBIOL. REV.
significant resistance to the ␤-lactamase inhibitors (32, 68).
Present studies indicate that the KPC-2 ␤-lactamase is not
inactivated by clavulanate, sulbactam, and tazobactam, as in-
dicated by high MICs and turnover numbers of 2,500, 1,000,
and 500, respectively (319a). The ability of KPC-2 to avoid
inactivation by the currently available inhibitors may represent
a novel class of highly IR class A enzymes. The rapid emer-
gence of Enterobacteriaceae harboring these carbapenemases is
a significant public health concern (290).
Epidemiology and Detection of ␤-Lactamase
Inhibitor Resistance
Resistance to ␤-lactam–␤-lactamase inhibitor combinations
challenges the ability to successfully treat serious urinary tract,
respiratory tract, and bloodstream infections (154, 219, 376).
The prevalence of organisms expressing inhibitor-resistant en-
zymes varies throughout the world. In 1993, a French study of
2,972 E. coli isolates from urinary tract infections (UTIs) found
that 25% and 10% of hospital and community isolates, respec-
tively, showed amoxicillin-clavulanate MICs of ⬎16/2 ␮g/ml
(160). Characterization of these isolates, including MIC pro-
files (amoxicillin-clavulanate MIC90 of ⬎1,042 ␮g/ml and
cephalosporin MIC of ⬍32 ␮g/ml), isoelectric focusing, and
DNA-DNA hybridization, suggested that 27.5% and 45% of
hospital and community isolates, respectively, were TEM-1
derived and had substitutions at previously described amino acid
positions that confer the IRT phenotype. In this survey, 4.9% of
E. coli isolates from UTIs were IRT producing. In 1998, a geri-
atric department of a French hospital reported an outbreak of
amoxicillin-clavulanate-resistant isolates which all produced the
same IR TEM ␤-lactamase, TEM-30 (Arg244Ser) (141). In a
study published in 2000, Leflon-Guibout et al. determined the
molecular mechanism of amoxicillin-clavulanate resistance (de-
fined by MICs of ⬎16/2 ␮g/ml) in E. coli isolates from three
French hospitals from 1996 to 1998 (218). The overall resistance
rate was 5%, and the majority of these resistant organisms were
from patients with respiratory tract infections. Production of IRTs
was implicated in resistance in 30 to 41% of the isolates over the
time period, which was only slightly less frequent than the hyper-
production of chromosomal AmpC enzymes. Two independent
Spanish studies reported production of IRTs in 5.4% and 9.5% of
amoxicillin-clavulanate-resistant E. coli isolates (266, 331).
The first IRT identified in the United States was not reported
until 2004 in a 14-month survey of E. coli isolates from a north-
eastern tertiary care clinical microbiology laboratory (198). Kaye
et al. found that 24% of the 283 isolates classified as ampicillin-
sulbactam resistant by disk diffusion and MIC testing were also
resistant to amoxicillin-clavulanate as determined by disk diffu-
sion zone diameters of ⱕ13 mm. Of the amoxicillin-clavulanate-
resistant isolates, 83% were from community-acquired infections.
Many of these 69 isolates were recovered from UTIs of outpa-
tients, and two expressed the IR TEM-34 (Met 69Val) and one
the TEM-122 (Arg275Gln) enzyme. Incidentally, also reported in
2004 was a KPC-2-producing K. pneumoniae isolate from New
York City that contained IRT-2 (41).
Isolates producing IRTs are more frequently reported in
Europe than in the United States. The explanation for this
discrepancy is not clear and probably reflects a combination
of environmental, methodological, and clinical practice fac-
VOL. 23, 2010 ␤-LACTAMASE INHIBITORS 177

TABLE 4. Clinically identified inhibitor-resistant TEM (IRT) ␤-lactamasesa

Amino acid at positionb:
␤-Lactamase
21 39 69 104 127 130 165 182 221 238 244 261 265 275 276

TEM-1 Leu Gln Met Glu Ile Ser Trp Met Leu Gly Arg Val Thr Arg Asn
TEM-30 (IRT-2) Ser
TEM-31 (IRT-1) Cys
TEM-32 (IRT-3) Ile Thr
TEM-33 (IRT-5) Leu
TEM-34 (IRT-6) Val
TEM-35 (IRT-4) Leu Asp
TEM-36 (IRT-7) Val Asp
TEM-37 (IRT-8) Ile Asp
TEM-38 (IRT-9) Val Leu
TEM-39 (IRT-10) Leu Arg Asp
TEM-40 (IRT-11) Ile
TEM-44 (IRT-13) Lys Ser
TEM-45 (IRT-14) Leu Gln
TEM-51 (IRT-15) His
TEM-54 Leu
TEM-58 Ser Ile
TEM-59 (IRT-17) Lys Gly
TEM-65 (IRT-16) Lys Cys
TEM-67 Ile Lys Cys
TEM-73 (IRT-18) Phe Cys Met
TEM-74 (IRT-19) Phe Ser Met
TEM-76 (IRT-20) Gly
TEM-77 (IRT-21) Leu Ser
TEM-78 (IRT-22) Val Arg Asp
TEM-79 Gly
TEM-80 (IRT-24) Leu Val Asp
TEM-81 Leu Val
TEM-82 Val Gln
TEM-83 Leu Cys Gln
TEM-84 Asp
TEM-103 (IRT-28) Leu
TEM-122 Gln
TEM-145 Met His
TEM-159 Phe Ile
TEM-160 Lys Val Thr
a
Based on data from www.lahey.org/studies/webt.asp.
b
Unless otherwise indicated, the amino acid is the same as in the non-IR TEM-1.

tors. The phenomenon does not appear to be related to
large differences in the use of ␤-lactam–␤-lactamase inhib-
itor combinations, as these agents are widely used in both
Europe and the United States and likely produce similar
selective pressures on bacteria (68, 159).
The detection of IRTs presents a significant number of op-
erational challenges. Disagreements between the results of
MIC testing and disk diffusion assays are often seen, especially
among isolates with intermediate resistance phenotypes (68,
198, 301). Currently, an international standard for the amount
of ␤-lactamase inhibitor that should be combined with the
partner ␤-lactam for detection of IR enzymes is not in use, and
different combinations can significantly alter the assigned results
(301, 409). For example, the use of the 2:1 ratio for amoxicillin-
clavulanate appears to underestimate the presence of IR ␤-lac-
tamases compared to employing the fixed concentration of 2
␮g/ml clavulanate (409). Further, organisms producing low levels
of IR enzymes may be undetectable at the 2:1 ratio (302). As a

TABLE 5. Clinically identified inhibitor-resistant SHV ␤-lactamasesa

Amino acid at positionb:
␤-Lactamase
35 69 130 140 146 187 192 193 234 238 240

SHV-1 Leu Met Ser Ala Ala Ala Lys Leu Lys Gly Glu
SHV-10 Gly Arg Asn Val Ser Lys
SHV-26 Thr
SHV-49 Ile
SHV-56 Gln Arg
SHV-72 Val Arg
a
Based on data from www.lahey.org/studies/webt.asp.
b
Unless otherwise indicated, the amino acid is the same as in the non-IR SHV-1.
178 DRAWZ AND BONOMO CLIN. MICROBIOL. REV.

TABLE 6. Clinically identified complex mutants of TEM (CMT) ␤-lactamasesa

Amino acid at positionb:
␤-Lactamase
39 69 104 130 164 237 238 240 244 265 275 276 284

TEM-1 Gln Met Glu Ser Arg Ala Gly Glu Arg Thr Arg Asn Ala
TEM-50 (CMT-1) Leu Lys Ser Asp
TEM-68 Ser Lys Met Leu
TEM-89 (CMT-3) Lys Lys Gly Ser
TEM-109 (CMT-5) Leu Lys His Cys
TEM-121 (CMT-4) Lys Lys Ser Thr Lys Ser
TEM-151 Val His Asp Gly
TEM-152 Val His Lys Asp
TEM-154 Leu Ser
TEM-158 (CMT-9) Leu Ser Asp
a
Based on data from www.lahey.org/studies/webt.asp.
b
Unless otherwise indicated, the amino acid is the same as in the non-IR TEM-1.

result of the methodological complications of identifying IR
␤-lactamase producers, the actual IRT prevalence may be under-
estimated (68). Definitive identification requires specific enzyme
kinetic testing, determination of isoelectric points, and molecular
characterization (60, 72, 77). Routine susceptibility tests may not
accurately detect strains expressing IRTs. A direct correlation
between the level of resistance (MIC) and the predicted clinical
outcome is not yet known.
Substitutions in Class A Enzymes
Conferring Inhibitor Resistance
The amino acid substitutions associated with clavulanate
resistance are different from those that produce the ESBL
phenotype, and thus IRTs have unique hydrolytic profiles com-
pared to ESBLs. For example, the E. coli isolates that contain
IRTs may be more susceptible to cephalosporins, carbapen-
ems, and monobactams (35). However, among TEM and SHV
␤-lactamases, substitutions that confer an IR phenotype are
sometimes found in pairs or are combined with amino acid
changes that also increase the ability of the enzyme to hydro-
lyze oxyimino-cephalosporins. These variants are designated
complex mutants of TEM (CMTs) and may foreshadow the
emergence of a new class of versatile ␤-lactamases with even
broader hydrolytic and resistance profiles (Table 6) (369).
Table 7 summarizes kinetic properties of representative
class A IR enzymes that have been identified clinically or
generated in the laboratory. IRTs, when derived from mutated
wild-type bla genes, typically display a reduction in ␤-lactam
catalytic efficiency (kcat/Km), mediated by lower kcat and in-
creased Km values for penicillins compared to the wild-type
enzymes (35). IR ␤-lactamases have increased KIs and IC50s
for clavulanate and sulbactam; smaller increases are observed
for tazobactam. In general, the MICs for organisms expressing
IR enzymes are lowered for penicillins and increased for cla-
vulanate and sulbactam. MICs may remain in the susceptible
range for tazobactam when obtained with the combination of
piperacillin-tazobactam. This susceptibility is likely due to pip-
eracillin’s greater potency and rapid cell entry (see above) as
well as the relative preservation of tazobactam’s KI (36). Of
significant interest is the recent description of the kinetic prop-
erties of SHV-72 (Ile8Phe, Ala146Val, Lys234Arg) (258). In
contrast to most other IR enzymes, this unique IR SHV dem-
onstrates increased catalytic efficiency for penicillins.
Amino acid substitutions in TEM and SHV result in resis-
tance to clavulanate by two primary mechanisms: (i) alter-
ations in the geometry of shape of the oxyanion hole and (ii)
changes in the position of Ser130 or Arg244 (33, 73, 101, 120,
408, 411, 416). The individual residues that are most commonly
substituted in IR TEM enzymes are Arg244, the nearby
Asn276 and Arg275, Met69, and the active-site Ser130 (www
.lahey.org/studies/webt.asp) (Fig. 8).

TABLE 7. Kinetic properties of representative class A inhibitor-resistant and wild-type enzymesa

Clavulanate Sulbactam Tazobactam
␤-Lactamase Reference(s)
⫺1 ⫺1
KI (␮M) kinact (s ) tn KI (␮M) kinact (s ) tn KI (␮M) kinact (s⫺1) tn

TEM-1 0.1 0.02 160 1.6 0.002 10,000 0.01 ⬎0.02 140 73, 180
TEM Met69Leu 1.65 0.002 2,800 22 0.0009 7,000 0.20 0.004 2,900 73
TEM Ser130Gly 105 0.003 1,600 220 0.03 1,900 95 0.04 630 408
TEM Arg244Ser 33 44 0.00001 7,800 179, 180
TEM Asn276Asp 15.6 0.01 250 378
TEM Met69Leu, Asn276Asp 27 49 0.6 386
SHV-1 1 0.04 60 8.6 0.056 13,000 0.07 0.1 5 153, 410, 411
SHV Arg244Ser 63 0.09 50 240 0.13 100 410, 411
SHV Ser130Gly 47 0.03 40 4.2 0.06 5 153
OHIO-1 0.4 0.01 80 17 0.001 40,000 0.30 0.01 400 229
OHIO Met69Ile 10 0.002 2,000 68 0.0004 200,000 18 0.0016 2,000 229
a
The definition of KI may not be uniform from laboratory to laboratory. See the cited references for the methods used in each determination.
VOL. 23, 2010
FIG. 8. Molecular representation of TEM-1 active site, showing
residues that are most frequently implicated in the development of
inhibitor-resistant TEM enzymes. Based on PDB coordinates 1TEM.
Arginine 244. To date, 12 IRT variants with substitutions at
residue 244 have been reported. The guanidinium group
(CH5N3⫹) of Arg244 contributes to substrate affinity through
hydrogen bonding to the conserved ␤-lactam carboxylate
(436). Structural and computational studies of the mechanism
of clavulanate resistance in TEM-1 reveal that a hydrogen on
the N␩2 atom of the guanidinium group of Arg244 and the
carbonyl oxygen atom of the backbone of Val216 coordinate a
water molecule (Fig. 9) (179, 411, 436, 457). When clavulanate
is bound, this water molecule is the likely donor of a proton
that saturates the double bond at the C-2 position, facilitating
the opening of the five-membered ring, formation of an inter-
mediate, and eventual ␤-lactamase inactivation (179). Loss of
the guanidinium group of Arg244 displaces this water molecule
and may explain the inhibitor resistance and catalytic impair-
ment that is observed in the TEM Arg244 variants. Without
protonation of the double bond, elimination of oxygen at C-1
is thermodynamically unfavored, which may lead to increased
inhibitor turnover (179).
Interestingly, we have not found a clinical report of an SHV
enzyme that has the 244 substitution. However, mutagenesis of
SHV at Arg244 revealed that the 244Leu and -Ser substitu-
tions at this residue also confer resistance to amoxicillin-clav-
ulanate (138, 411). The biochemical correlates of these clav-
ulanate-resistant phenotypes are different for SHV and TEM
FIG. 9. Schematic representation of the proposed Michaelis
preacylation complex of TEM-1 and clavulanate.
␤-LACTAMASE INHIBITORS 179
and are related to slight differences in the inactivation mech-
anisms for these enzymes (179, 410, 411, 436, 457).
Methionine 69. Met69 is the most commonly substituted res-
idue in inhibitor-resistant enzyme isolates, appearing either as a
single substitution or in combination with additional amino acid
replacements in 15 of the 36 presently defined IRTs. Despite the
proximity to enzyme nucleophilic Ser70, the Met69 side chain is
oriented away from the catalytic serine and not part of the sub-
strate binding site (191). Residue 69 forms the back wall of the
oxyanion hole or “electrophilic center” that polarizes the ␤-lac-
tam for nucleophilic attack by Ser70 and, in the case of inhibitors,
possibly Ser130 as well (206). The residue is not highly conserved
in class A ␤-lactamases, and TEM can tolerate many substitutions
at the site and still retain activity against most substrates (10, 101).
The SHV Met69Phe, -Lys, and -Tyr variants have increased MICs
and hydrolytic activity for the extended-spectrum ceftazidime
(155). While the residue displays some flexibility in its role in
substrate binding, certain substitutions in TEM and SHV confer
resistance to the inhibitors.
The TEM variants, Met69Ile, -Val, and -Leu, each show an
approximately 10-fold increase in KI and 10-fold decrease in
kinact, leading to a 100-fold decrease in inhibitory efficiency
(kinact/KI) (73). Evidence from X-ray crystallography and mo-
lecular dynamic studies has revealed that each substitution
introduces subtle active-site changes that perturb inhibitor rec-
ognition and enzyme catalysis (262, 438). Similarly, SHV
Met69Ile, -Val, and -Leu show increased clavulanate MICs
and IC50s (155). Reduced inhibitor acylation efficiency in SHV
was elucidated by observed changes in the oxyanion hole in the
crystal structures of SHV Met69Val/Glu166Ala in complex
with clavulanate, sulbactam, and tazobactam (PDB 2H0T,
2H0Y, and 2H10, respectively) (416).
Asparagine 276. Fourteen TEM variants with substitutions
at Asn276 have been discovered; seven demonstrate an IR
phenotype. Ambler position 276 remains unchanged in cur-
rently identified SHV variants. Despite its relative distance (8
to 10 Å) from the enzyme active site, the crystallographic
structure of TEM Asn276Asp (PDB 1CK3) reported by
Swaren et al. confirmed the importance of the electrostatic
environment surrounding residue 276 (400). In TEM, amino
acid 276 is located on the enzyme’s C-terminal ␣-helix H11,
which lies behind the ␤-sheet including Arg244. The
Asn276Asp substitution creates new interactions with Arg244
and significant displacement of ␣-helices H1, H10, and H11.
The Asp at residue 276 is displaced only 0.75 Å compared to
the wild-type Asn, but this movement results in a tighter at-
traction between Arg244 and 276Asp.
This enhanced interaction has two major effects (378, 400).
First, there is an effective “neutralization” of the Arg244 con-
tribution. This decreased positive charge may lower the local
affinity for the C-3 carboxylate of clavulanate and penicillins
(400). Charge changes in the active site may have a more
profound consequence for the inhibitors, which are positioned
as small penicillin substrates and rely heavily on the interac-
tions with Arg244 for binding affinity (179, 378, 457). Second,
the TEM Asn276Asp variant’s increased clavulanate kcat val-
ues may be explained by the displacement of the water mole-
cule necessary for secondary ring opening of clavulanate, as
discussed above for TEM Arg244 variants (378, 400).
Similar to the case for Arg244, IR SHV 276 variants have
180 DRAWZ AND BONOMO
not yet reached clinical attention (411). Molecular modeling of
SHV Asn276Asp showed that novel electrostatic interactions
are formed between Arg244N␩2 and both 276AspO␦1 and
O␦2, reminiscent of the TEM Asn276Asp crystal structure
(108). Additional data from both kinetic studies and timed
electrospray ionization mass spectrometry (ESI-MS) of clavu-
lanate-inhibited SHV-1 and SHV Asn276Asp suggest that the
Asn276Asp phenotype is produced by decreased affinity for
the C-3 carboxylate of clavulanate and not by a change in the
reaction mechanism. These results are consistent with the no-
tion advanced by Thomson et al., suggesting that clavulanate
inactivation in SHV may not rely on a water molecule coordi-
nated by Arg244 and Val216, as in TEM enzymes (191, 210,
411). These important differences in TEM and SHV mecha-
nisms support a divergent evolution of these enzymes vis-a-vis
substrate and inhibitor profiles.
Serine 130. Ser130 is a conserved amino acid among all class
A ␤-lactamases but is replaced by a Gly residue in two IRTs
(TEM-59 and -76) and one IR SHV enzyme (SHV-10). The
multiple roles of this residue include anchoring ␤-lactams to
and stabilizing the active site through hydrogen bonding with
the C-3/C-4 carboxylate of the inhibitors and substrates and
facilitating proton transfer to the ␤-lactam nitrogen during
acylation, leading to ␤-lactam ring opening (179, 180, 214,
426). Ser130 also serves as a second nucleophile which attacks
the imine intermediate and becomes covalently cross-linked to
Ser70 in the terminal inactivation of the mechanism-based inhib-
itors (209). Removing the cross-linking residue seems a clear way
to overcome inactivation, but inhibitors, and particularly tazobac-
tam, retain efficacy against the Ser130Gly enzyme. Based on these
mechanistic roles, how does the Ser130Gly variant maintain hy-
drolytic activity and why does it confer resistance to inhibitors
(408)? In response to the first part of the question, evidence from
X-ray crystallography of SHV and TEM Ser130Gly (PDB 1TDL
and 1YT4, respectively) confirmed that a relocated water mole-
cule, initially predicted by Helfand et al., compensates for the loss
of the Ser130 hydroxyl group by donating a proton to the ␤-lac-
tam nitrogen (153, 157, 180, 408, 438). The decreased catalytic
efficiencies seen in the TEM and SHV variants are likely a result
of perturbations caused by the repositioned water molecule.
These perturbations vary somewhat between SHV and TEM
Ser130Gly (e.g., reorientation of Ser70 and Lys73 in SHV and
TEM Ser130Gly, respectively), but both involve changes in hy-
drogen bonding networks in the active site, which are modified to
accommodate the new water molecule (397, 408).
In regard to the second part of the question concerning
inhibitor resistance, the data on Ser130Gly variants argue that
cross-linking is not essential for enzyme inactivation. Despite
increased MICs for the inhibitors, the turnover numbers for
clavulanate and tazobactam by SHV Ser130Gly are equivalent
to those by the wild-type enzyme (153). This “paradox of in-
hibitor resistance” illustrates that despite replacement of a key
catalytic residue, SHV Ser130Gly is still capable of inactivation
by the mechanism-based inhibitors (153, 157, 311, 397). The
resistant phenotype presents primarily in whole-cell assays,
where bacteria are exposed to a limited inhibitor concentra-
tion. Structural perturbations created by the Ser130Gly substi-
tution discourage formation of the preacylation complex (in-
creased inhibitor KIs), which ultimately leads to fewer
inactivation events (157). Thus, while the enzymatic machinery
CLIN. MICROBIOL. REV.
for inhibition is intact, the decreased accumulation of acyl-
enzyme intermediates and inactivation products can lead to
inhibitor resistance in vivo.
Arginine 275. Similar to observations regarding Arg244 and
Asn276, the Arg275 variant has been identified only in TEM.
Before description of the IR variants TEM-103 and TEM-122,
which possess only the Arg275Leu and -Gln changes, respec-
tively, substitution at 275 was seen only in combination with
the Met69Val and -Leu substitutions (9, 198). Hence, the con-
tribution of Arg275 to the IR phenotype was not appreciated,
and rigorous kinetic and structural studies of this variant have
not yet been completed. However, from crystal structures of
the wild-type enzymes and a better understanding of properties
at nearby Arg244 and Asn276, Chaibi et al. postulated that
substitutions at Arg275 (like those at Asn276) disrupt local
electrostatic interactions and displace the water molecule that
is key to inactivation by clavulanate (74).
Lysine 234. The recent definition of two clinical IR SHV
␤-lactamases with Lys234Arg substitutions draws attention to
this important active-site residue (110, 258). Previously, TEM
or SHV variants obtained from the clinic had not been iden-
tified as having changes at this highly conserved class A resi-
due. Site-directed mutagenesis of Lys234 to Arg in TEM-1
leads to a 10-fold-decreased affinity for penicillins, presumably
because of the hydrogen-bonding role of Lys234 to the C-3
carboxylate (220) (Fig. 3). However, for the clinically isolated
SHV-72 enzyme (Ile8Phe, Ala146Val, Lys234Arg), Km values
for penicillins were not significantly changed and IC50s for
clavulanate increased 10-fold compared to those of SHV-1
(258). Interestingly, the kcat values for the penicillins were
increased, up to 5.8-fold for ticarcillin. Molecular dynamics
simulations show that Lys234Arg induces movement of the
Ser130 oxygen away from Ser70. Mendonca et al. proposed
that the mechanism of IR in SHV-72 may be due to movement
of Ser130 based on its role in both hydrolysis and terminal
inactivation by cross-linking with Ser70 (258).
THE PROMISE OF NOVEL ␤-LACTAMASE INHIBITORS
This section reviews compounds that have demonstrated
favorable inactivation properties against ␤-lactamases and in-
troduces newer compounds showing promise as “second-gen-
eration” inhibitors. Table 8 provides Ki (or KI) values and IC50s
for representative inhibitors against the different ␤-lactamase
classes to illustrate the challenge of achieving broad-spectrum
inhibition.
Monobactam Derivatives
Monocyclic, N-sulfonated ␤-lactams are a family of antibi-
otic compounds produced by bacteria (178, 404, 405). Struc-
ture-activity studies of these monobactams led to the develop-
ment of the synthetic compound aztreonam, which interacted
with the PBPs of a wide range of aerobic Gram-negative bac-
teria, including P. aeruginosa, and was introduced into clinical
practice in 1984 (151, 168, 403). In addition, aztreonam (Fig. 1,
compound 3) resists hydrolysis by many plasmid-mediated
␤-lactamases such as TEM-1 and -2, OXA-2, and SHV-1, be-
having as a very poor substrate with low affinity (e.g., Km of 2.9
VOL. 23, 2010 ␤-LACTAMASE INHIBITORS 181

TABLE 8. Kinetic properties of selected inhibitors against different ␤-lactamase Ambler classesa

Kinetic Value (␮M) for Ambler class:

Inhibitor (compound no. in Fig. 1) Reference(s)
parameter A B C D

Aztreonam (3) Km or Ki 200 ⬎1,000 0.0019 56, 217, 337

BAL30072 siderophore monobactam (14) IC50 1.7 ⬎100 0.09 134
ATMO-penicillin (15) IC50 0.90 418
BRL 42715 methylidene penem (16) IC50 0.008 0.008 0.004 87
BLI-489 methylidene penem IC50 0.009 0.062 294
Novel tri- and bicyclic methylidene IC50 0.0004–0.0031 0.014–0.260 0.0015–0.0048 0.012 25, 441
penems (including 17 and 18)
LK-157 tricyclic carbapenems (20) IC50 0.055 0.062 344
Ro 48-1220 penam sulfone (21) IC50 0.05–1.07 24–200 0.21–3.4 0.10–0.43 367, 422
LN-1-255 penam sulfone (23) KI or IC50 0.100 0.026 0.70 63, 322; Drawz et al.,
unpublished data
Boronic acid meta-carboxyphenyl Ki 3.9 0.001 272, 411
cephalosporin analog (25)
NXL104 (26) IC50 0.008 0.080 31
J-111,225 1-␤-methylcarbapenem (33) Ki 11.1 0.18 0.93 281
Phosphonates Ki 0.08–76 0.016–12.1 0.094 4, 244
C-6-mercaptomethyl penicillnate (38) IC50 6.8 0.10 10.5 61
a
The definition of KI may not be uniform from laboratory to laboratory. See the cited references for the methods used in each determination.

mM for TEM-2) (403). With class C enzymes, such as E.
cloacae P99, aztreonam forms a stable acyl-enzyme that is very
slowly hydrolyzed (low kcat) (128, 403). Aztreonam also exhib-
its very low Ki values for chromosomally encoded cephalospo-
rinases (e.g., 1.9 nM for P99) and serves as a transient inacti-
vator (56, 128). In vivo, where periplasmic ␤-lactamase
concentrations can be in the low mM range, clinically signifi-
cant catalytic efficiencies may be obtainable (128).
Further modification of the monobactam nucleus was em-
barked upon to capitalize on the inhibitory activity of aztreo-
nam. The 1,5-dihydroxy-4-pyridone monobactam Syn 2190
(Fig. 1, compound 11) possesses a novel C-3 side chain that
may utilize the iron uptake pathway to enter Gram-negative
bacteria. Syn 2190 displayed IC50s in the nM range for cepha-
losporinases (e.g., 6 nM for E. cloacae P99) and synergy with
ceftazidime and cefpirome in MIC testing of clinical strains,
including AmpC-derepressed variants of P. aeruginosa (15,
289). In addition, Syn 2190 in combination with ceftazidime or
cefpirome (at a 1:1 ratio) showed improved efficacy compared
to ceftazidime-tazobactam in murine models of systemic infec-
tions with cephalosporin-resistant P. aeruginosa. Syn 2190 has
also shown utility for the challenging clinical task of identifying
production of plasmid-mediated AmpC ␤-lactamase in Kleb-
siella spp. In combination with cefotetan, Syn 2190 achieved
100% specificity and 91% sensitivity for AmpC producers (28).
However, Syn 2190 has lower affinity than tazobactam for class
A enzymes and could not restore susceptibility to cephalospo-
rins in bacteria expressing these enzymes. This limitation
against class A enzymes may partly explain the absence of
further development of this compound.
The design of bridged monobactam derivatives was guided
by the crystal structure of the monobactam aztreonam in com-
plex with the class C ␤-lactamase from Citrobacter freundii
(PDB 1FR1) (152, 299). Studies of the structure demonstrated
that before deacylation, aztreonam had to rotate around the
C-3–C-4 bond to allow a hydrolytic water access to the ester
bond. This rearrangement requires breaking and reforming of
the active-site hydrogen bond network and leads to rate-limit-
ing deacylation (205). The introduction of a bridge between
C-3 and C-4 limits this rotation and stabilizes the acyl-enzyme
against hydrolysis by AmpC enzymes (99, 171).
Heinze-Krauss et al. synthesized and evaluated of a panel of
bridged monobactams that exhibited very favorable inhibition
of the class C C. freundii and P. aeruginosa ␤-lactamases (IC50s
of as low as 10 nM) but lower affinities for the class A TEM-3
(IC50s of ⬎100 ␮M) (152). At sufficiently high concentrations,
class A ␤-lactamases were acylated rapidly, but the rate of
deacylation was higher, leading to high hydrolysis rates and low
active-site occupancy. Comparison of the crystal structures of
class A TEM-1, PC1, the ␤-lactamase from Bacillus lichenifor-
mis 749/C, and the class C ␤-lactamase from C. freundii re-
vealed that in class A enzymes, hydrolysis occurs on the oppo-
site side of the bridged monobactam ester by a water molecule
activated by hydrogen bond networks not present in class C
enzymes (152, 166, 191, 267, 299, 392). Rotation about the
C-3–C-4 bond is less critical to the inhibition mechanism in
class A enzymes, and restricting this rotation does less to sta-
bilize the acyl-enzyme. For example, the bridged monobactam
Ro 48-1256 (Fig. 1, compound 12) (at 4 and 8 ␮g/ml) effec-
tively potentiated the activity of imipenem against both induc-
ible and derepressed class C-expressing P. aeruginosa isolates
(238). The activities of piperacillin and ceftazidime were also
potentiated by the combination with Ro 48-1256 against the
strains expressing elevated quantities of AmpC enzyme. How-
ever, Ro 48-1256 did not improve the activity of piperacillin,
ceftazidime, or carbapenems against organisms expressing
class A, B, or D ␤-lactamases.
Modification of the monobactam structure at the ␤-lactam
nitrogen has had some success against class A ␤-lactamases
(50, 171). Analysis of a series of N-sulfonyloxy-␤-lactam
monobactam derivatives demonstrated rapid acylation of the
TEM-1 active site and resistance to deacylation, as evident
from turnover numbers ranging from 2 to 8 (50). Additional
analysis of one of these derivatives (Fig. 1, compound 13)
revealed that following enzyme acylation in the class A car-
bapenemase NMC-A, the tosylate group is eliminated and
gives rise to two acyl-enzyme species (273). These inhibited en-
zyme species are trapped in local energy minima which yield low
182 DRAWZ AND BONOMO
deacylation rates. Based on molecular simulations, Mourey et al.
postulated that the acyl-enzyme alternates between two different
conformations that move in and out of the oxyanion hole (273).
This motion requires breaking and reforming of hydrogen bonds
between the acyl-enzyme ester and carbonyl oxygen atoms and
the oxyanion hole backbone nitrogens and Ser130 oxygen atom.
In both conformations, the water implicated in the deacylation
reaction is positioned poorly for hydrolysis, offering a structural
explanation for the irreversible inhibition.
A series of monobactam derivatives was recently developed
to target pathogens harboring multiple ␤-lactamases. Some of
these monobactam analogues act as very effective bactericidal
agents for difficult-to-treat Gram-negative pathogens, while
others are bridged monobactams, such as BAL29880, that are
inhibitors of AmpC enzymes (99, 102, 116a, 317). Additional
derivatives include monobactams bearing a siderophore side
chain which can enhance cell entry through bacterial iron up-
take systems (49). Of this siderophore type, BAL19764 and
BAL30072 (Fig. 1, compound 14) have potent antibacterial
activity against carbapenem-resistant P. aeruginosa, Acineto-
bacter spp., S. maltophilia, and S. marcescens as well as against
Gram-negative bacilli expressing VIM and IMP ␤-lactamases
(37, 102). Additionally, BAL19764 is stable to hydrolysis by
MBLs (314). Further, these siderophore compounds are po-
tent enzyme inhibitors; BAL30072 has low ␮M IC50s for
TEM-3 (1.7 ␮M), and the AmpCs from C. freundii and P.
aeruginosa (0.09 and 15 ␮M, respectively) (134). By combining
BAL19764 or BAL30072 with clavulanate and a bridged
monobactam (all at a fixed concentration of 4 ␮g/ml), the
MICs of the siderophore compounds were improved for a
range of Gram-negative bacteria (including P. aeruginosa) pro-
ducing AmpCs as well as carbapenemases or overexpression of
efflux systems (315, 316). However, the activity of carbapenems
is still superior to that of these new monobactams for many
ESBL-producing isolates (e.g., K. pneumoniae CTX-M and
SHV-5, E. coli, and E. cloacae) (38, 102, 173). The dual anti-
bacterial/inhibitory action of these monobactam derivatives
makes them important leads, and the further development of
the siderophore compound BAL30072 seems likely.
ATMO Derivatives
Certain cephems adopt catalytically incompetent conforma-
tions in the active site, effectively “trapping” the enzyme.
Evidence from the crystal structure of an AmpC enzyme com-
plexed with ceftazidime (PDB 1IEL) suggested that the pres-
ence of the 2-amino-4-thiazolyl methoxyimino (ATMO) R1
side group, which is common among extended-spectrum ceph-
alosporins (e.g., cefotaxime and ceftazidime), destabilized the
formation of the deacylation transition state (350). Synthesis of
inhibitors containing this ATMO side chain on a penicillin
(Fig. 1, compound 15) and carbacephem backbone led to
AmpC IC50s of 900 nM and 80 nM, respectively (418). Subse-
quent examination of the crystal structure of the ATMO-pen-
icillin in complex with the AmpC (PDB 1LLB) showed that it
resembled the acyl-enzyme formed by ceftazidime. The
ATMO groups were located in very similar positions, in both
cases leading to steric hindrance between the deacylating water
and the ␤-lactam ring nitrogen, accounting for inhibition. This
design highlights the potential of incorporating destabilizing
CLIN. MICROBIOL. REV.
groups onto conserved substrate skeletons, which will facilitate
recognition by the enzyme (418).
Penems
BRL 42715 and Syn 1012. Methylidene penems are potent
inhibitors in which the inhibitory properties of the penem
structure are enhanced by introduction of a double bond at C-6
and a sulfide at penem position 1 (260). These compounds
offer advantages over the traditional ␤-lactamase inhibitors,
including (i) a large R1 side chain that may aid in cell perme-
ability and contribute to active-site affinity and (ii) a novel
inactivation mechanism. The parent compound from this
group, BRL 42715 C-6-(N1-methyl-1,2,3-triazolylmethyl-
ene)penem (Fig. 1, compound 16), is an effective inhibitor of
class A, C, and D ␤-lactamases (119, 250). IC50s of BRL 42715
were 10- to 100-fold lower than those of clavulanate, tazobac-
tam, and sulbactam for class A TEM-1 and SHV-1, class C P99,
class D OXA-1, and the S. aureus ␤-lactamase NCTC 11561
(87). However, BRL 42715 was a substrate for class B metallo-
␤-lactamases from Aeromonas hydrophila and S. maltophilia
and for BcII (250). In susceptibility testing, BRL 42715 con-
centrations of 0.25 ␮g/ml or lower were able to restore amoxi-
cillin MICs to ⬍16 ␮g/ml for TEM-1- and OXA-1-producing
organisms (87). Further, amoxicillin-BRL 42715 combinations
were more effective than amoxicillin-clavulanate for E. coli, K.
pneumoniae, H. influenzae, S. aureus, and N. gonorrhoeae
strains producing plasmid-mediated ␤-lactamases (mostly
TEM and SHV expressors) and for K. oxytoca, Proteus vulgaris,
and P. mirabilis strains producing chromosomal ␤-lactamases.
Kinetic studies of BRL 42715 and NCTC 11561, TEM-1, and
P99 showed rapid, irreversible inactivation with an inhibition
stoichiometry of 1:1, or turnover numbers of 0 (119).
Initial studies to elucidate the BRL 42715 inactivation product
using UV difference spectroscopy for both enzyme- and base-
catalyzed hydrolysis of the methylidene penem suggested the
presence of a dihydrothiazepine rearrangement product (46, 250).
Accordingly, mass spectrometry studies demonstrated that the
methylidene penem inhibition pathway does not involve fragmen-
tation in the active site (119, 250, 406). When TEM-1, P99, and
the class A ␤-lactamase from B. cereus I were inactivated by BRL
42715, spectra showed a mass increases equivalent to the molec-
ular masses of the inhibitor plus the enzyme (119). The crystal
structure of the class C ␤-lactamase E. cloacae 908R in complex
with BRL 42715 (PDB 1Y54) confirmed the formation of a stable
seven-membered thiazepine ring covalently attached to Ser64
(263). Studies of both BRL 42715 and related penems support the
mechanism involving formation of an imine intermediate from
the acyl-enzyme, which then undergoes rearrangement, and even-
tually endo-trig cyclization to form the seven-membered thiaz-
epine ring end product of inactivation for class A, C, and D
enzymes (Fig. 10) (250, 263, 294, 428).
Crystallization of BLI-489, a related heterocyclic O C-6-substi-
tuted penem with IC50s of 9.0 and 6.2 nM for SHV-1 and class C
GC1, respectively (see below), revealed the common seven-mem-
bered thiazepine ring when complexed with the SHV-1 and GC1
enzymes (PDB 1ONG and 1ONH, respectively) (294). Interest-
ingly, the orientation of the ring in SHV-1 and GC1 differed by
180° about the bond to the acylated serine. This rotameric pref-
erence may be due to the relative “openness” of the class C
VOL. 23, 2010
FIG. 10. Proposed reaction mechanisms for the inactivation of a serine ␤-lactamase by BRL 42715, showing formation of the seven-membered
thiazepine ring (A), and by LN-1-255, showing intermolecular capture by the pyridyl nitrogen leading to a bicyclic aromatic intermediate (B).
(Based on data from references 263, 294, 322, and 428.)
binding site at the bottom of the b3 ␤-sheet. Crystallization of a
tricyclic 6-methylidene derivative in complex with SHV-1 and
GC1 (PDB 1Q2P and 1Q2Q, respectively) also showed the com-
mon seven-membered thiazepine ring covalently attached to
Ser70 and Ser64, respectively (429). The SHV-1/penem complex
was similar to what was observed with BLI-489, where the ring
had R stereochemistry at the new C-7 moiety (294). However, in
the GC1/penem complex, both the R and S chiral forms of the
intermediate were found.
Taken together, these findings suggest that the 6-methylidene
penems’ acyl-enzyme stability is likely due to the low occupancy
or disorder of the hydrolytic water molecule and, in part, to the
conjugation of the ester with the ring system. The nucleophilic
attack of the double bond at C-6 appears to be important to the
inhibition mechanism of the methylidene penems, and these
novel inhibitors seem to share a similar inactivation mechanism
for different ␤-lactamase classes (429).
Most ␤-lactamases appear to follow this linear pathway to
inactivation by the penem compounds, i.e., formation of an
acyl-enzyme and rearrangement to the stable dihydrothia-
zepine ring adduct (119, 250). However, some class A en-
zymes, such as the ␤-lactamases from Staphylococcus albus
(Staphylococcus epidermidis) and K. pneumoniae K1, exhibit
more complex kinetics (46, 250). A branched pathway leads
to an acyl-enzyme hydrolysis product, which also rapidly
rearranges to the dihydrothiazepine species.
While BRL 42715 and the structurally related investiga-
tional penem Syn 1012 showed promising in vitro kinetics
against a large spectrum of Gram-positive and Gram-negative
isolates, they were relatively unstable in human and mouse
plasma (342). Further, serum levels of BRL 42715 and Syn
1012 were undetectable 1 h after intravenous administration in
a rabbit model. Thus, the successful elements of BRL 42715
have been applied to additional drug development efforts di-
rected at improving in vivo stability (see below).
␤-LACTAMASE INHIBITORS 183
BRL 42715 derivatives. Derivatives of BRL 42715, including
tricyclic and bicyclic heterocyclic methylidene penems, have been
the focus of a large body of recent structure-activity studies and
continue to show promising inhibition properties in investiga-
tional studies. The bicyclic heterocycles are synthesized as
6-methylidene penems attached to thiophene, imidazole, and pyr-
azole rings (428). Extension of the second heterocyclic ring yields
the tricyclic derivatives, which show improved stability in solution
and increased lipophilicity in in vivo studies (429). As a group,
these compounds have proven to be potent inhibitors of class A,
C, and D ␤-lactamases (25, 156, 335, 428, 441).
A panel of one tricyclic and six bicyclic penem compounds
displayed IC50s of 0.4 to 3.1 nM for TEM-1 (class A), 7.8 to 72
nM for IMI-1 (class A), 1.5 to 4.8 nM for AmpC (class C), and
14 to 260 nM for a CcrA (class B) (441). Compared to tazobac-
tam IC50s, these penems were 100- to 56,000-fold more active
against TEM-1 and the AmpC enzymes. Each of the seven
penems, at a constant concentration of 4 ␮g/ml, was combined
with piperacillin, which significantly lowered MICs in ESBL
producers and restored susceptibility in piperacillin-resistant
Enterobacter spp., Citrobacter spp., and Serratia spp. In a mouse
model of infection caused by E. coli and Enterobacter aerogenes
class A (including ESBL) and C ␤-lactamase-producing organ-
isms, piperacillin-penem combinations decreased the overall
50% effective dose (ED50) of piperacillin from 1.6- to 8-fold
(441). However, class B metallo-␤-lactamase producers were
not reported for susceptibility tests or the murine model, and
they warrant further attention for clinical translation.
Of the panel of tricyclic and bicyclic penem compounds
tested, the bicyclic BLI-489 was chosen as a lead compound to
be combined with piperacillin (335). With 4 ␮g/ml of BLI-489,
92% of non-piperacillin-susceptible clinical strains (including ex-
pressors of representative ␤-lactamases from all four classes) had
piperacillin–BLI-489 MICs of ⱕ16 ␮g/ml, in contrast to 66% for
piperacillin-tazobactam. Furthermore, the BLI-489 combination
184 DRAWZ AND BONOMO
demonstrated improved activity over piperacillin-tazobactam
against ESBL- and AmpC-expressing strains, as well as against a
panel of staphylococcal and streptococcal isolates. Notably, BLI-
489 did not improve the activity of piperacillin against the entero-
cocci and penicillin-intermediate and -resistant strains of S. pneu-
moniae (which may reflect that the piperacillin resistance
mechanism is not ␤-lactamase mediated) (335). While these prac-
tical studies help to bring closer the possible clinical introduction
of these novel inhibitors, only a small number of the isolates were
KPC or IRT producers. These KPC- and IRT-producing strains
also expressed up to three other ␤-lactamases, which complicates
the interpretation of activity against these enzymes. Thus, one
cannot yet endorse the efficacy of this inhibitor combination for
these relevant resistance threats.
Two of these bicyclic investigational penems (Fig. 1, com-
pounds 17 and 18), containing either a dihydropyrazolo[1,5-
c][1,3]thiazole or a dihydropyrazolo[5,1-c][1,4]thiazine moiety,
are extremely effective inactivators of the class D OXA-1 ␤-lac-
tamase, with nM affinity and low turnover values compared to
tazobactam (tn ⫽ 0 and 350, respectively) (25). Additionally,
these penems demonstrate efficacy with IR class A ␤-lactama-
ses. Against the SHV Arg244Ser variant, the penem inhibitors
showed significantly lower KI values than clavulanate and sul-
bactam (penems 1 and 2, 1.0 to 4.4 ␮M; clavulanate and sul-
bactam, 63 and 240 ␮M, respectively) and extremely low turn-
over numbers (tn ⫽ 0, 50, and 100 for the penems, clavulanate,
and sulbactam, respectively) (410).
Oxapenems. Oxapenems are derivatives of clavulanic acid,
possessing an oxygen atom at penem position 1. Their synthesis
was first described in the 1970s, and Cherry et al. introduced an
oxapenem that had improved in vitro activity over clavulanate
against the E. cloacae AmpC and staphylococcal ␤-lactamases
(86). However, the compound was unstable and did not have
activity in whole-cell assays. In 1993, Pfaendler et al. improved
the stability of the compounds by adding bulky substituents at
the C-2 position (339). The lead compound from this panel of
oxapenems, the zwitterionic AM-112 (Fig. 1, compound 19),
has shown potent ␤-lactamase-inhibitory properties, particu-
larly for class C and D enzymes (189, 190). Against class A
enzymes, IC50s were 10- to 20-fold higher for AM-112 com-
pared to clavulanate (190). However, the IC50s of AM-112 for
the class C enzymes from E. cloacae, S. marcescens, and P.
aeruginosa, as well as OXA-1 and OXA-5 from E. coli, were
1,000- to 100,000-fold lower than those of clavulanate. AM-112
in combination with ceftazidime at 1:1 or 1:2 reduced ceftazi-
dime MICs against class A ESBL producers and the class C
enzyme hyperproducers. For the class D enzyme producers
and P. aeruginosa strains tested, AM-112 did not enhance the
activity of ceftazidime. The susceptibility results were similar
for AM-112’s protection of cefepime, ceftriaxone, and ce-
foperazone (189). In a mouse sepsis model, ceftazidime pro-
tection was maintained against E. cloacae P99 and K. pneu-
moniae SHV-5 (190). The preclinical development of AM-112
has been led by Amura Ltd., and it warrants further examina-
tion as a potential multi-class ␤-lactamase inhibitor.
Tricyclic carbapenems (trinems). Rational structure-based
design informed the synthesis of novel tricyclic carbapenems
built by the fusion of a cyclohexane ring onto a carbapenem
scaffold (90, 91). LK-157 (Fig. 1, compound 20) was derived by
introduction of a methoxy substituent at position C-4 and
CLIN. MICROBIOL. REV.
shows nM affinity for both the class A TEM-1 and class C E.
cloacae 908R ␤-lactamases (431). The IC50s of clavulanate and
LK-157 for TEM-1 were similar (0.030 ␮M and 0.055 ␮M,
respectively), but that of LK-157 was over 2000-fold better for
E. cloacae AmpC (136.2 ␮M and 0.062 ␮M, respectively)
(344). In combination with ampicillin, LK-157 (at 30 ␮g/ml)
restored susceptibility for the AmpC-overexpressing E. cloacae
P99 strain in MIC testing (431). Against a large panel of
bacteria producing class A ESBLs (excepting KPC and
CTX-M) and class C ␤-lactamases, LK-157 in combination
with cephalosporins showed improved potency compared to
clavulanate, tazobactam, and sulbactam (324). The lowest
MICs (ranging from ⱕ0. 025 to 1.6 ␮g/ml) were achieved for
cefepime or cefpirome in combination with LK-157 at 4 ␮g/ml.
Despite these promising data with class A and C ␤-lactamase-
producing Gram-negative bacteria, LK-157 behaves as a sub-
strate with the carbapenem-hydrolyzing enzymes BcII and
NMC-A (431). Further, while the inhibitor displays nM affinity
for and rapidly inactivates the class D OXA-10 ␤-lactamase,
the acyl-enzyme is unstable and rapidly hydrolyzed (431).
The crystal structure of LK-157 in complex with the E. clo-
acae P99 (PDB 2Q9N), together with spectroscopic data for
reaction intermediates, suggests that after deacylation at the
active-site Ser64, the C-4 methoxy group is eliminated (344,
431). Further, the catalytic water molecule presumed to be
responsible for deacylation in class C enzymes is not observed
in the AmpC/LK-157 crystal structure. Rotation about the
opened ␤-lactam ring after acylation likely leads to displace-
ment of this water molecule and subsequent enzyme inhibition
(344). The C-10 ethyldiene group is probably not bulky enough
to induce the conformational change seen with inhibition of
class A enzymes by carbapenems, but resonance stabilization
of the acyl-enzyme through the acyl carbonyl and ethyldiene
group may contribute to stability of the intermediate (255,
344). Trinems are worthy of attention for the potential to serve
as leads for additional structure-based inhibitor design, and
Lek Pharmaceuticals continues to investigate LK-157. Devel-
oped for parental use, the half-life (t1/2) of LK-157 in rats is
similar to those of other ␤-lactams after intravenous adminis-
tration (354). Additional studies have focused on developing
an oral agent with improved permeability over LK-157, and
while prodrug esters have good solubility, the intestinal per-
meability remains low.
1-␤-Methylcarbapenems. A panel of carbapenem deriva-
tives bearing a methyl group at C-1 and various substituents at
C-2 were screened for inhibitory activity against the MBL
IMP-1 (282). The study revealed a compound, J-110,441, that
had potent inhibitory activity against several MBLs but also
low Ki values for class A TEM and class C E. cloacae enzymes
(2.54 ␮M and 0.037 ␮M, respectively). Susceptibility testing of
the AmpC-expressing E. cloacae demonstrated an MIC de-
crease from 64 to 4 ␮g/ml with imipenem and J-110,411 at 4
␮g/ml. Further work with these carbapenem derivatives has
focused on their potential as MBL inhibitors and will be dis-
cussed below (see Inhibition of Metallo-␤-Lactamases).
Penicillin and Cephalosporin Sulfone Derivatives
Following the success of sulbactam and tazobactam, medic-
inal chemists have focused much effort on the development of
VOL. 23, 2010
penicillin and cephalosporin sulfones with functionalities that
may improve inhibitor efficiency. One of the first series of
investigational sulfones, the C-7 alkylidene cephems, had lead
compounds that were potent inhibitors of class C ␤-lactamases
(65). Subsequent reports have explored the roles of C-2 and
C-6 penam and C-3 cephem substituents. The findings for each
will be discussed in the context of the derivative group.
C-2/C-3-substituted penicillin and cephalosporin sulfones.
␤-Lactamase inhibitors with C-2 substitutions have shown ef-
ficacy against TEM- and SHV-type enzymes, including ESBLs
(367, 422). The acrylonitrile derivative Ro 48-1220 (Fig. 1,
compound 21) achieves an IC50 for TEM-1 (0.08 ␮M) that is
comparable to that of clavulanate (0.10 ␮M), but the IC50 for
SHV-1 (0.27 ␮M) is slightly higher than that of clavulanate
(0.05 ␮M) (422). Inhibition with Ro 48-1220 was as effective as
tazobactam for TEM-1 and SHV-1, with IC50s within 0.03 ␮M
for both enzymes. Most class C enzymes tested were inhibited
at lower concentrations of Ro 48-1220 than of tazobactam
(IC50 range approximately 2- to 30-fold lower) (367, 422). The
IC50s for X. maltophilia and Bacteroides fragilis class B enzymes
were 24 and 200 ␮M, in comparison to 4 and ⬎10 mM for
tazobactam (367). Ro 48-1220 (at 4 ␮g/ml) performed well in
susceptibility testing, protecting the activity of ceftriaxone and
ceftazidime against class C- and ESBL-producing organisms
(422). Central to the potency of this sulfone inhibitor was the
low enzyme recovery rate compared to tazobactam, especially
for class C ␤-lactamases from C. freundii and E. coli (367). UV
absorption spectroscopy and X-ray crystallography revealed
the identity of a linear enamine formed by elimination of the
SO2 group, ring opening, and rearrangement into a linear
conjugated system (367). This particularly stable intermediate
presumably accounted for enhanced inhibitor potency.
The design of the C-2-substituted penicillin sulfone SA2-13
(Fig. 1, compound 22) drew on the insight that the tazobactam
imine is the common intermediate, or “gatekeeper,” in both
permanent interaction and enzyme regeneration (58, 309, 453).
If the more stable enamine, the trans-enamine, can be favored,
then the reaction will be “trapped” in transient inhibition. In
fact, IR enzymes do exhibit decreased trans-enamine forma-
tion, as in the case of the SHV Met69Val/Glu166Ala variant
reacted with tazobactam and clavulanate (416). SA2-13 in-
cludes a negatively charged carboxyl group attached to a linker
which is designed to stabilize the trans-enamine conformation
in close proximity to conserved active-site residues. Interest-
ingly, the crystal structure of SA2-13 in complex with SHV-1
(PDB 2H5S) showed an additional salt bridge with Lys234 and
hydrogen bonds with Ser130 and Thr235. Compared to SHV-1
inhibited by tazobactam, SA2-13 had a 10-fold-lower deacyla-
tion rate, presumably mediated by the stabilization of the trans-
enamine species (309). However, the dissociation constant for
SA2-13 was 17-fold lower than that for tazobactam (0.1 versus
1.7 ␮M, respectively), and there was 47% 24-hour recovery of
activity of the SA2-13/SHV-1 mixture versus complete inacti-
vation with tazobactam.
The 6-alkylidene-2⬘-substituted penicillin sulfone LN-1-255
(Fig. 1, compound 23), attains nM KIs for the class A SHV-1 and
ESBL SHV-2 and class D oxacillinase-, ESBL-, and even carbap-
enemase-type OXA enzymes (63, 322) (S. M. Drawz et al., un-
published data). Moreover, LN-1-255 restores the activity of pip-
eracillin against E. coli IR SHV enzyme producers and of
␤-LACTAMASE INHIBITORS 185
ceftazidime and cefpirome against E. coli and K. pneumoniae
expressing CTX-M, OXA, and CMY ␤-lactamases (63, 322).
Meropenem MICs were lowered from 32 to 1 ␮g/ml with 4 ␮g/ml
of LN-1-255 against S. marcescens possessing SME-1. The C-3
hydrophobic catechol moiety of this investigational compound
appears to improve both its entry into cells via siderophore iron
channels and its affinity for ␤-lactamase active sites (75).
X-ray crystallography of LN-1-255 in complex with SHV-1
(PDB 3D4F) revealed that the inhibitor’s C-6 (heteroaryl)
alkylidene group plays an important role in the formation of a
planar bicyclic aromatic intermediate (322). The pyridyl nitro-
gen of the C-6 substituent acts as a base and promotes intramo-
lecular capture, leading to the formation of a bicyclic species
(Fig. 10B). The acyl-enzyme ester carbonyl of the intermediate
is resonance stabilized by the conjugated ␲ system. Addition-
ally, the carbonyl group of the intermediate is positioned out-
side the oxyanion hole. The decreased deacylation rates of this
species are likely due to resonance stabilization and the loca-
tion of the carbonyl, at an increased distance from the hydro-
lytic water and improperly positioned for nucleophilic attack
(322).
By expanding the five-membered penicillin sulfone design to
a six-membered cephalosporin sulfone, Buynak et al. devel-
oped synthetic methodologies allowing modification of C-3
substituents (62, 64). The different functional groups intro-
duced at C-3 had significant impact on whether the inhibitor
was selective for either class A or class C ␤-lactamases, with
some features, such as electronegativity, increasing inhibition
of both classes. DVR-II-41S (Fig. 1, compound 24) has both
the C-7 (heteroaryl)alkylidene group of LN-1-255 and a C-3
vinyl substituent, similar to nitrocefin, an excellent substrate
for nearly all classes of ␤-lactamases (62, 64). This cephalo-
sporin sulfone demonstrated IC50s of 90 nM for class A TEM-1
and 10 nM for class C GC1. The crystal structure of GC1/
DVR-II-41S (PDB 1GA0) revealed a bicyclic aromatic system
reminiscent of the LN-1-255 intermediate (94). Additional sta-
bility results from the placement of the inhibitor’s anionic
sulfinate group between Tyr105 and the acyl-enzyme bond,
likely blocking the approach of the deacylating water molecule.
C-6-substituted penicillin sulfones. C-6-substituted penicil-
linates are nM to low ␮M inhibitors of serine ␤-lactamases,
with the sulfone oxidation state at the penam thiazolidine
sulfur showing improved affinities over the sulfide state (27, 61,
374, 375). The stereochemistry and specific functionality of the
C-6 substituents can affect the specificity of inhibition. For
example, ␤ isomer alkenyl derivatives, as opposed to ␣ isomer
derivatives, show improved IC50s for class B ␤-lactamases, and
derivatives with thiazole rings, particularly with fluorine,
amino, or hydroxyl groups, show potency for class A ␤-lacta-
mases (374). Further, compounds with a mercaptomethyl
group at C-6, as opposed to hydroxymethyl, are better inhibi-
tors of class B enzymes, while C-6-mercapto-penicillinates are
relatively less active inhibitors of classes A, B, and C (61).
The C-6-hydroxyakylpenicillinates have been employed as
tools for studying the mechanism of ␤-lactamase hydrolysis and
inhibition (143). The crystal structure of 6-␣-hydroxymeth-
ylpenicillanate complexed to TEM-1 (PDB 1TEM) suggests
that while C-6-substituted penicillin sulfones are acylated in
the same way as ␤-lactam substrates, the hydroxymethyl group
at C-6 impedes the approach of the hydrolytic water and pro-
186 DRAWZ AND BONOMO
longs the lifetime of the acyl-enzyme species, effectively inhib-
iting the enzyme (254). However, the same compound acted as
a substrate for the class A NMC-A. Crystal structure determi-
nation of NMC-A complexed with 6-␣-[(1-hydroxy-1-methyl)
ethyl]penicillanate (PDB 1BUL) showed that the repositioning
of residue Asn132 in NMC-A, compared to TEM-1, enlarged
the substrate binding cavity and allowed Asn132 to make new
hydrogen bonds with the C-6 substituent (274). These interac-
tions permit approach by the hydrolytic water and facilitate
deacylation of the acyl-enzyme intermediate for the 6-␣-hy-
droxymethylpenicillanate and C-6-substituted carbapenems
such as imipenem in NMC-A. Upon introduction of a bulkier
C-6 hydroxypropyl group, deacylation was impeded, in a mech-
anism very similar to that for the inhibition of TEM-1 by
imipenem (407).
Generalization of these and other findings leads to the con-
clusion that when the hydrolytic water molecule approaches
the acyl-enzyme from the “␣ direction,” as with class A en-
zymes, ␣-hydroxyalkylpenicillinates are effective inhibitors.
Conversely, ␤-hydroxyalkylpenicillinates inhibit the “␤ ap-
proach” in class C enzymes (143). A group at Wyeth Research
found that a 6-␤-hydroxymethyl penicillin sulfone has the best
activity against both class A and C enzymes, with IC50s of 8 nM
for TEM-1 and 1.2 ␮M for AmpC (26). Presumably, the in-
hibitor worked like tazobactam or sulbactam in class A en-
zymes, but the specific crowding of the ␤-face of the ester
prevents approach of the hydrolytic water in class C ␤-lacta-
mases (51). The class D OXA-10 enzyme showed inhibition by
both C-6 ␣- and ␤-hydroxyisopropylpenicillinates (252). While
only the ␤-isomer could be crystallized with OXA-10 (PDB
1K54), this structure and computational models of the ␣-iso-
mer suggest related, but different, mechanisms of inactivation
based on the C-6 stereochemistry of the inhibitor. However,
both mechanisms involved displacing the hydrolytic water nec-
essary for deacylation.
Non-␤-Lactam Inhibitors
Boronic acid transition state analogs (TSAs). In the 1970s,
boronic acid compounds were described as forming reversible,
dative covalent bonds with serine proteases and inhibiting
these enzymes by assuming tetrahedral reaction intermediates
(30, 230). Kiener and Waley then applied the chemistry to the
class A ␤-lactamase from B. cereus and showed that boronic
acid derivatives, as well as the phenyl and m-aminophenyl
derivatives, were mM inhibitors of the enzyme (201). Further
study by other groups demonstrated that these compounds,
which lack the ␤-lactam motif, form adducts that resemble the
geometry of the tetrahedral transition state of the ␤-lactamase
hydrolytic reaction (22, 82, 95) (Fig. 11). By modifying these
transition state analogs to contain the R1 side chains of natural
substrates, affinities in the nM range for both class A TEM-1
and ESBL-type SHV and class C enzymes have been deter-
mined (70, 392, 394, 412). Weston et al. used the crystallo-
graphic structure of E. coli AmpC ␤-lactamase in complex with
m-aminophenylboronic acid (PDB 3BLS) as a model for de-
signing boronic acid-based inhibitors specifically for the class C
active site (425, 443). The compound showing the greatest
affinity was benzo[b]thiophene-2-boronic acid, which had a Ki
CLIN. MICROBIOL. REV.
FIG. 11. Scheme illustrating the interactions of a serine ␤-lacta-
mase with a cefotaxime substrate (A) compared to a cefotaxiime-like
boronic acid TSA (B). Notice that the reaction with the inhibitor is
reversible.
for the E. coli AmpC of 27 nM.
In addition to their demonstrated potential as effective in-
hibitors, these compounds have been used to probe the impor-
tant interactions between the inhibitor and the enzyme active
site, thereby informing the design of future TSAs. Using the
individual crystal structures for nine TSAs and four ␤-lactams
complexed with the E. coli AmpC ␤-lactamase, computational
analyses have generated consensus binding pockets in the
AmpC active site (352). The functionalities recognized by cer-
tain residues led to the development of new glycylboronic acid
TSAs, including a compound containing the R1 side chain of
cephalothin. One of these cephalothin analogs achieved a Ki of
1 nM for the E. coli AmpC (Fig. 1, compound 25), a 300-fold
improvement over the parent compound, which lacked the
meta-carboxylphenyl substituent (272). The substituent was de-
signed to resemble both the dihydrothiazine ring and the C-4
carboxylate of the cephalosporin nucleus. In the crystal struc-
ture of this cephalothin meta-carboxyphenyl analog complexed
with an AmpC (PDB 1MY8), the inhibitor carboxylate inter-
acted with Asn289, a residue not previously implicated in struc-
tural or modeling studies as interacting with the cephalosporin’s
C-4 carboxylate. Asn289 is not a highly conserved residue
among class C ␤-lactamases, but the low Ki is maintained when
different AmpCs with other residues at position 289 are tested
(e.g., Kis of 29 nM for E. cloacae P99 and 11 nM for A.
baumannii (107a, 241, 371). The versatility of this inhibitor may
reflect an important flexibility in AmpC active sites that could
affect the recognition of both substrates and inhibitors.
High-affinity inhibitors will be clinically useful only if they
can effectively permeate the bacterial cell membrane and re-
store susceptibility to partner ␤-lactams. The boronic acid de-
rivatives enter both S. aureus producing class A ␤-lactamases
and Enterobacteriaceae such as E. coli, C. freundii, E. cloacae,
and P. aeruginosa expressing class C ␤-lactamases, and they
effectively protect ampicillin and ceftazidime (70, 272, 349,
430, 436). Boronates have not yet been developed for clinical
use in combination with a ␤-lactam, in part because of con-
cerns about the toxicity of boron. The safety of boron-contain-
ing compounds is currently being addressed by clinical studies
sponsored by Anacor Pharmaceuticals. Anacor develops boron
compounds for medical uses and has current trials for boron-
VOL. 23, 2010
based topical treatments of fungal, bacterial, and anti-inflam-
matory diseases, as well as research stage data on a systemic
antibacterial agent.
Phosphonates. Phosphonate monoester derivatives can
acylate the active-site serines of class A, C, and D ␤-lactama-
ses, leading to effective inhibition (4, 223, 243, 244, 353). These
compounds exhibit branched kinetic pathways, in some cases
reflecting inhibition by the products that are formed. Acyl
phosphonates inhibit class C enzymes, while cyclic acyl phos-
phonates inhibit both class A and C ␤-lactamases. For diacyl
phosphonates, hydrophobic substituents decrease the inhibi-
tor’s Ki value, and the acylation rates of these compounds
follow the order class D ⬎ class C ⬎ class A (243, 244).
Majumdar et al. posit that this relative inhibitor efficiency
reflects the general hydrophobicity of the enzyme active sites
and that rational design of diacyl phosphate side chains could
lead to nM inhibition of all serine ␤-lactamase classes.
Crystal structures with these phosphonic acids have also offered
important insight into the reaction mechanisms of class C en-
zymes (297). The ability of the E. cloacae GC1 ␤-lactamase to
hydrolyze extended-spectrum cephalosporins was elucidated by
trapping of the enzyme in complex with a phosphonate cefo-
taxime transition state analog (297). A three-residue insertion
into the ⍀-loop of the enzyme, compared to the C. freundii ␤-lac-
tamase/transition state analog complex, allowed for a better fit of
cefotaxime’s side chain in the enzyme active site and facilitated
the activation of the hydrolytic water molecule. The clinical po-
tential of phosphonates has been limited by their poor stability in
aqueous solution and susceptibility to phosphodiesterases.
NXL104. NXL104 (also known as AVE1330A) (Fig. 1,
compound 26) is a bridged diazabicyclo[3.2.1]octanone non-
␤-lactam inhibitor with very promising activity against class
A, C, and D ␤-lactamases (31, 116, 240, 390). The majority
of the published work on NXL104 describes MIC testing,
although IC50s of 8, 38, and 80 nM have been reported for
class A TEM-1 and KPC-2 and class C P99 enzymes, respec-
tively (31, 390). A remarkable property of NXL104 is the
prolonged deacylation rate. The 50% enzyme recovery time
point was 7 days for both TEM-1 and P99 (compared to 7
min for TEM-1 and clavulanate and 290 min for P99 and
tazobactam), suggesting the presence of a very stable and
long-lived intermediate species (31). The formation of a
covalent complex has been supported by an ESI-MS study of
TEM-1 and NXL104 (391). Currently, the only reported
crystal structure of the inhibitor is in complex with CTX-M-
15, and it shows opening of the NXL104 ring upon acylation,
interaction with several conserved active-site residues, and
displacement of the putative deacylation water by approxi-
mately 1 Å (104).
With ceftazidime as a partner ␤-lactam in a 4:1 ratio
(ceftazidime at 4 ␮g/ml and NXL104 at 1 ␮g/ml), NXL104
lowered the MICs for a series of Enterobacteriaceae strains
at least 8-fold compared to those of ceftazidime alone, to ⱕ4
␮g/ml for E. coli, K. pneumoniae, Citrobacter diversus, and P.
mirabilis strains and to 2 ␮g/ml for E. cloacae and Serratia
sp. strains (31). Against class A ␤-lactamase producers (pri-
marily TEM and SHV types), the ceftazidime-NXL104 com-
bination was comparable to ceftazidime-clavulanate but
superior to amoxicillin-clavulanate and piperacillin-tazobac-
tam. Ceftazidime-NXL104 was more potent than piperacil-
␤-LACTAMASE INHIBITORS 187
lin-tazobactam for class C enzyme-producing Enterobacteri-
aceae, including both ceftazidime-resistant and -susceptible
strains of C. freundii and E. cloacae. The NXL104 concen-
tration of 4 ␮g/ml restored MICs into the susceptible range
for a collection of clinical P. aeruginosa isolates (21% non-
susceptible to ceftazidime versus 6% nonsusceptible to
ceftazidime-NXL104) (380). In in vivo murine studies, in-
cluding models of sepsis with CTX-M Enterobacteriaceae
producers, pneumonia with AmpC and SHV-11 K. pneu-
moniae producers, and thigh infection and sepsis with
KPC-2, TEM-1, and SHV-11 K. pneumoniae producers, the
ceftazidime-NXL104 combination has demonstrated prom-
ising therapeutic efficacy (221, 259, 442).
NXL104 also enhanced the activity of cefotaxime in a study
of Enterobacteriaceae expressing CTX-M, KPC, or OXA-48,
where 4 ␮g/ml of the inhibitor lowered MICs to ⱕ1 ␮g/ml. For
isolates with ertapenem resistance due to combinations of
␤-lactamases (ESBLs or AmpCs) and impermeability, the
MICs were still ⱕ2 ␮g/ml. While cefotaxime-NXL104 lowered
the MIC of one S. marcescens isolate expressing SME-1 from
0.5 to 0.12 ␮g/ml, MICs for K. pneumoniae and E. coli express-
ing class B IMP and VIM metallo-␤-lactamases were not low-
ered by either cephalosporin with NXL104.
Two recent studies examined more closely the ability of
NXL104 to restore susceptibility to ␤-lactams for KPC en-
zyme-producing clinical isolates (116, 390). Stachyra et al.
demonstrated that NXL104 at 4 ␮g/ml restored susceptibility
of six KPC-2 or -3 producers to piperacillin, ceftazidime, ceftri-
axone, and imipenem (390). Endimiani et al. tested NXL104 at
various concentrations (1, 2, and 4 ␮g/ml) against a panel of 42
K. pneumoniae isolates with ⱖ3 bla genes each and MIC90s of
ⱖ128 ␮g/ml for all ␤-lactams tested (116). All of these K.
pneumoniae strains were susceptible to NXL104 at 2 ␮g/ml
with cefotaxime, ceftazidime, cefepime, and aztreonam and at
4 ␮g/ml with piperacillin. Collectively, these data suggest that
NXL104, in combination with several cephalosporins, aztreo-
nam, and even imipenem, may be an excellent candidate for
restoring susceptibility to difficult-to-treat emerging carbapen-
emase- and ESBL-producing organisms.
NXL104, manufactured by Novexel Inc., is currently in
phase II clinical trials in combination with ceftazidime for
treatment of complicated urinary tract and intra-abdominal
infections (www.clinicaltrials.gov). Additionally, Forest Labo-
ratories Inc. intends to begin phase I clinical trials with
NXL104 in combination with the novel broad-spectrum ceph-
alosporin ceftaroline (which is currently in phase III trials as
monotherapy for complicated skin infections and community-
acquired pneumonia). The results of clinical trials are awaited.
Hydroxamates. O-Aryloxycarbonyl hydroxamate is an irre-
versible inhibitor of the class C E. cloacae P99 ␤-lactamase that
utilizes a mechanism of action which differs from those of the
currently available inhibitors (Fig. 1, compound 27) (330, 450).
The proposed inhibition mechanism involves rate-limiting
acylation of the hydroxamate. However, the inhibitor is stabi-
lized not by the traditional oxyanion hole comprised of the
backbone nitrogens of Ser64 and Ser318 but rather by the side
chains of Tyr150 and Lys315 (330). As with clavulanate and
tazobactam, the acyl-enzyme proceeds to either hydrolysis or
inactivation. The inactivation mechanism leads to aminolysis of
Lys315 and a covalent cross-linking of Ser64 and Lys315, as
188 DRAWZ AND BONOMO
FIG. 12. Molecular representation of cross-linked active-site resi-
dues Ser64 and Lys315 (shown in yellow) formed after aminolysis of an
O-aryloxycarbonyl hydroxamate inhibitor in E. cloacae P99 ␤-lacta-
mase. (Based on PDB 2P9V and data from references 330 and 450.)
seen in the crystal structure of the inhibited P99 (Fig. 12) (PDB
2P9V) (450). TEM-2 and OXA-1 ␤-lactamases were also in-
hibited by O-aryloxycarbonyl hydroxamates, and the MBL
GIM-1 was inhibited by a “reverse” hydroxamate (hydroxy-
lamino replacing hydroxylamine) conjugated to a cephalospo-
rin nucleus (131, 330). Additional mechanistic studies of hy-
droxamates and their derivatives, as well as in vitro activity
data, will be of much interest.
Other non-␤-lactam inhibitors. Work by Conrath et al. ex-
plored the ␤-lactamase-inhibitory potential of soluble frag-
ments derived from the variable region of heavy-chain drom-
edary antibodies (89). IC50s of 35 ␮M and 300 ␮M were
obtained for TEM-1 and the B. cereus MBL BcII, but the
development of this strategy would require innovative tech-
niques such as increasing the permeativity of these 15-kDa
polypeptides to outer membranes of Gram negative ␤-lacta-
mase producers.
Inhibitors with a sulfonamide core and varied substituents
were developed by structure-based optimization (351, 413).
This non-␤-lactam design approach is intended to avoid both
hydrolysis by ␤-lactamases and the upregulation of ␤-lactama-
ses induced by some ␤-lactam-based inhibitors. Starting from a
map of binding “hot spots” developed from crystal structures
of 13 different ligands in complex with the E. coli AmpC ␤-lac-
tamase, 200,000 commercially available small molecules were
docked into the active site of the enzyme by computer simu-
lation (351, 352). Based on these results, lead compounds were
screened in vitro, and the molecule with the highest affinity (26
␮M) was crystallized in complex with the AmpC (PDB 1L2S)
(Fig. 1, compound 28). The structure closely resembled the
prediction of the docking simulation, revealing the plasticity of
the enzyme active site to recognize functional groups different
from ␤-lactams. This catalyzed the optimization of a novel
inhibitor, and replacement of the chloride substituent with a
CLIN. MICROBIOL. REV.
carboxylate yielded a compound with a 26-fold-improved Ki (1
␮M) (413). In MIC testing, the inhibitor restored the suscep-
tibility to ceftazidime (at a 1:1 ratio) in the AmpC producers E.
cloacae and C. freundii (but not P. aeruginosa) and, in contrast
to clavulanate, did not upregulate ␤-lactamase expression in an
inducible strain of E. cloacae.
␤-Sultams are the sulfonyl analogs of ␤-lactams, and N-acyl
␤-sultams (Fig. 1, compound 29) inactivate the P99 enzyme (318,
419). ESI-MS studies suggest that the ␤-sultams sulfonylate the
serine residue to form a sulfonate ester. Elimination of the sul-
fonate anion leads to C-O bond fission and formation of a dehy-
droalanine at the previous Ser64 residue (419). These compounds
do not show inhibition of class B MBLs, and there are currently
no studies examining their activities in class A or D enzymes.
BLIP is a 165-amino-acid protein isolated from the same
organism that produces clavulanic acid, Streptomyces clavulig-
erus (105). Studies of BLIP’s inhibitory activity have revealed
affinities for class A enzymes that vary from pM to ␮M (the
enzymes with the highest affinities for BLIP are KPC-2 and the
P. vulgaris ␤-lactamases) (147, 393). Many of the individual
class A residues which contribute to BLIP binding have been
identified through crystallography and mutagenesis (363, 456,
460). While the clinical viability of a peptide inhibitor based on
this protein is limited because of protein degradation in vivo,
the high-affinity interactions offer direction for the design of
novel ␤-lactamase inhibitors.
The mixture of vanadate and catechol compounds in aque-
ous solution produces vanadate-catechol complexes which can
inhibit class A TEM-2, class C P99, and class D OXA-1 ␤-lac-
tamases with apparent Kis of 62, 0.58, and 1.5 ␮M, respectively
(5). A crystal structure with the complex has not been attained,
but molecular modeling suggests that the active-site serine is
bound to a penta- or hexa-coordinated vanadium. Vandate-
catechols represent another group of compounds that are use-
ful as tools for novel inhibitory active-site interactions, but lack
clear clinical potential.
INHIBITION OF METALLO-␤-LACTAMASES
We note that designing and/or discovering inhibitors to
MBLs presents special challenges. The diversity of class B
enzyme subclasses (i.e., B1, B2, and B3) and the mechanistic
complexities regarding the role of one or two Zn2⫹ ions have
impeded effective inhibitor design. In addition, indiscrimi-
nately inhibiting or chelating metal ions can have untoward
effects on natural or endogenous metalloenzymes. For exam-
ple, similarities between the active-site architectures of MBLs
and essential mammalian enzymes (e.g., human glyoxalase II)
complicate the design of an inhibitor that would be safe for
human use (97). As a class, the MBLs are resistant to all the
mechanism-based inhibitors of the serine enzymes (the inhib-
itory activity of the methylidene penems against class B ␤-lac-
tamases may be an important exception), and few investiga-
tional inhibitors demonstrate sub-␮M efficacy (335, 441). Here,
we provide a brief survey of the more promising agents in
development.
VOL. 23, 2010
Thiol Derivatives
Due to catalytic dependence on Zn2⫹ ions, the metalloen-
zymes are inhibited by thiols or chelating agents that have an
affinity for the hydrophobic pocket of the enzyme active site
and bind to, or interfere with, the bonding network between
the hydrolytic water and the Zn2⫹ ions (170, 227, 312). Thiol
derivatives, such as thiomandelic acids, typically show low-␮M
Ki inhibition of B1 (e.g., BcII, IMP-1, and VIM-2) and B3 (e.g.,
L1) subclasses but over 100-fold-higher Ki values for the sub-
class B2 CphA MBL (268). Structural analysis of thiol-based
inhibitors complexed with the three MBL subclasses suggests
that the thiol group chelates the Zn2⫹ ions for subclasses B1
and B3 (227). For example, the crystal structure of IMP-1 from
P. aeruginosa bound to a thiol inhibitor (Fig. 1, compound 30)
(PDB 1DD6) showed interactions with active-site residues,
allowing the thiol of the inhibitor to bridge the two catalytic
Zn2⫹ molecules and displace the bridging water important for
hydrolysis (88). A similar inhibition mechanism was resolved
from the crystal structure of the B1 VIM-2 complexed to a
mercaptocarboxylate compound (452). However, this binding
mode was not observed in the crystal structure of the thiolate
D-captopril bound to the subclass B2 CphA from A. hydrophila
(227). Instead, the inhibitor’s main interactions with the en-
zyme were via the thiolate carboxylate group (227).
Mass spectrometric data suggest that mercaptoacetic esters
may act as mechanism-based inhibitors for BcII by generating
mercaptoacetic acid in situ and forming a disulfide linkage with
a cysteine residue in the enzyme active site (327). However, as
with other thiols, the IC50s of these mercaptoacetic esters
varied from low ␮M to mM across MBL subclasses.
Pyridine Dicarboxylates
Recent work demonstrates that pyridine carboxylates, in
particular 2-picolinic and pyridine-2,4-dicarboxylic acids (Fig.
1, compound 31), are competitive inhibitors of CphA (a sub-
class B2 MBL), with Ki values of 5.7 and 4.5 ␮M, respectively
(170). The pyridine carboxylate derivatives showed little inhib-
itory activity for B1 and B3 MBLs. In the crystal structure of
CphA/pyridine-2,4-dicarboxylic acid (PDB 2GKL), the cata-
lytic Zn2⫹ ion is coordinated by the nitrogen and one of the
carboxylate oxygens of the inhibitor (170). The Zn2⫹ ion is also
engaged in electrostatic interactions with residues Asp120,
Cys221, and His263. Hydrophobic interactions with Asn233,
Trp87, and Val67 help to further stabilize the inhibitor in the
active site. The larger and more flexible active sites of subclass
B1 and B3 MBLs may not permit comparable stabilizing con-
tacts with the pyridine carboxylates, offering an explanation for
the B2 specificity.
Trifluoromethyl Ketones and Alcohols
Reactive trifluoromethyl ketones and alcohols, conju-
gated to a penicillin-like phenyl side chain, effectively inhibit
MBLs from B. cereus, P. aeruginosa, S. maltophilia, and A.
hydrophila in vitro, presumably by binding the active-site
Zn2⫹ ion (433, 434). The crystal structure of a biphenyl
tetrazole inhibitor, L-159,061 (Fig. 1, compound 32), in
complex with the B. fragilis enzyme (PDB 1A8T) indicates
␤-LACTAMASE INHIBITORS 189
that the tetrazole portion of the molecule interacts directly
with the active-site Zn2⫹ ions (414). IC50 and Ki values in
the low ␮M range were observed, and the incorporation of
substituents on the phenyl groups and their interactions with
specific residues were essential to the strong binding of the
biphenyl tetrazoles (414). These inhibitors do appear to
enter cells, as the addition of imipenem or penicillin G to
biphenyl tetrazole compounds increased inhibition zones for
an imipenem-resistant B. fragilis isolate (414).
Carbapenem Analogs
1-␤-Methylcarbapenems with cyclic C-2 substituents, such as
J-110,441, achieve sub-␮M Kis for the IMP-1, CcrA, LI, and
BcII MBLs as well as representative class A and C enzymes
(282). For several IMP-1-producing S. marcescens and P.
aeruginosa isolates, imipenem MICs were lowered to 4 ␮g/ml
by the addition of J-110,441. The further development of
J-111,225 included a trans-3,5-disubstituted pyrrolidinylthio
substituent at the C-2 position (Fig. 1, compound 33) (281).
J-111,225 maintained inhibitory activity against IMP-1 (Ki of
0.18 ␮M) and synergy with imipenem but had moderately
improved MICs as a single agent against IMP-1-expressing S.
marcescens and P. aeruginosa (MIC range of 4 to 32 ␮g/ml).
However, J-111,225 acted as a substrate for other chromo-
somally encoded class B MBLs, including CcrA, L1, and BcII.
Tricyclic Natural Products
Natural product screening for inhibitors to BcII led to the
discovery of extracts from Chaetomium funicola which demon-
strated IC50s ranging from 389 to 0.3 ␮M for BcII, P. aerugi-
nosa IMP-1, and B. fragilis CfiA MBLs (329). The “lead com-
pound” of the three tricyclic compounds, SB238569 (Fig. 1,
compound 34), also showed low Ki values for the same en-
zymes (79, 17, and 3.4 ␮M, respectively). In combination with
meropenem, 8 ␮g/ml of SB238569 restored susceptibility to B.
fragilis CfiA. The crystal structure of CfiA and SB236050 (PDB
1KR3) identified important polar (Lys184, Asn194, and
His162) and ring-stacking (Trp49) interactions which likely
contribute to the inhibitory activity. Comparison of this struc-
ture with that of IMP-1/mercaptocarboxylate (PDB 1DD6)
suggests that the binding site created by the loop preceding
Lys184 is shorter in IMP-1 and may leave a larger portion of
the compound exposed to solvent (88). The decreased inhibi-
tory activity of the tricyclic inhibitor for S. maltophilia L1 may
also be explained by active-site variations, including differences
in the positions of the Lys184 and Asn194 equivalents.
Succinate Derivatives
The IMP-1 enzyme is encoded by a transferable blaIMP-1
gene and represents an increasing resistance threat, and thus
the development of inhibitors for this target is a priority. Suc-
cinic acid compounds have been screened as IMP-1 inhibitors,
and several derivatives have restored meropenem susceptibility
of IMP-1-expressing E. coli (269, 415). Compound 35 (Fig. 1)
belongs to a series of 2,3-(S,S)-disubstituted succinic acids
which inhibit IMP-1 with nM IC50s (415). Crystal structure
190 DRAWZ AND BONOMO
analysis revealed that the inhibitors’ carboxylate groups inter-
act with both Zn2⫹ ions and active-site residues while displac-
ing the bridging water (PDB 1JJE and 1JJT) (415). Disubsti-
tuted succinic acids have also been described as inhibitors of
the L1 MBL and display binding modes similar to those in
IMP-1 (284, 303).
IMP-1 is also inhibited by N-arylsulfonyl hydrazone com-
pounds, with IC50s as low as 1.6 ␮M (Fig. 1, compound 36)
(384). Structure-activity relationship studies revealed that
bulky aromatic substituents on both sides of the sulfonyl hy-
drazone backbone improved the activity of these inhibitors.
Liénard et al. demonstrated the ability of dynamic combina-
torial mass spectrometry (DCMS) to identify potent BcII in-
hibitor leads (228). A meso-2,3-dimer-captosuccinic acid was
bound to the MBL active site via one thiol group, while the
second thiol group was linked in a disulfide bond to a thiol-
containing compound from a screening library. ESI-MS re-
vealed which compounds bound the enzyme, and subsequent
structure optimization led to the development of a mercapto-
carboxylate that inhibits BcII with a Ki of 740 nM (Fig. 1,
compound 37).
C-6-Mercaptomethyl Penicillinates
Penicillin derivatives with C-6-mercaptomethyl substituents
(Fig. 1, compound 38) show promising kinetics against class A,
B, and C ␤-lactamases (IC50s of 6.8, 0.10, and 10.5 ␮M, re-
spectively, against representative enzymes) (61). These peni-
cillinates, in combination with piperacillin, lowered MICs
against MBL-producing strains of P. aeruginosa. Building in-
hibitors on the backbone of the natural substrate for all ␤-lac-
tamases, the penicillin nucleus, may help bridge the gap be-
tween these diverse enzyme classes.
LESSONS LEARNED
We dedicate the penultimate section of this review to sum-
marizing the “lessons learned” from our consideration of the
␤-lactamase inhibitors. From the perspective of clinicians, we
propose that several important features must be regarded in
order to optimize the development of ␤-lactamase inactivators.
(i) High affinity for the active site of the target ␤-lactamase.
It is axiomatic that in order for an inhibitor to work efficiently,
it must exhibit high affinity for its target. This principle is
emphasized by the development of clavulanate resistance in
TEM-1 and SHV-1 ␤-lactamases mediated by substitution of
amino acid residues 69, 130, 244, and 276, leading to increased
inhibitor KIs and/or IC50s (73, 153, 378, 411). Particularly
promising are those inhibitors that maintain high affinities
across different groups of ␤-lactamases, such as the C-2-sub-
stituted sulfone LN-1-255, the methylidene penems, and phos-
phonates which achieve nM inhibition of class A, C, and D (4,
25, 63, 245, 322, 441) (Drawz et al., unpublished data).
(ii) Mimicking the “natural substrate.” The available inhib-
itors are effective primarily against class A serine ␤-lactamases,
and the structures of class A ␤-lactamase inhibitors are similar
to those of penicillin, bearing a fused four- or five-membered
ring system. Similarly, data on transition state analogs suggest
that class C ␤-lactamases have high affinity for inhibitors that
CLIN. MICROBIOL. REV.
resemble the natural cephalosporin substrates (272). A guide-
line for inhibitor design may be “penicillin analogs for penicil-
linases, and cephalosporin analogs for cephalosporinases.”
This principle also ensures that the inhibition mechanism fol-
lows the substrate mechanism—save for hydrolysis. While
␤-lactamases continue to develop resistance to all developed
␤-lactams, designing inhibitors that follow the hydrolytic mech-
anism may help to thwart the emergence of resistant isolates,
as the enzyme must find novel ways to preserve function but
evade inhibition. We hasten to add that this approach may not
be practical in all cases.
(iii) Stabilizing interactions in the active site. Stabilizing or
prolonging the acyl-enzyme intermediate is central to success-
ful inactivation by the currently available class A inhibitors (81,
396). Promising novel inhibitors, such as the methylidene pen-
ems (e.g., BRL 42715) and LN-1-255 are designed to capitalize
on and enhance this stabilization. One approach is to prolong
the acylation step (k2). Alternatively, acyl-enzyme intermedi-
ates can be stabilized by multiple interactions in the active site,
as observed for SHV-1 with tazobactam and SA2-13 (308, 309).
The inhibition of NMC-A by the monobactam derivative 13
(Fig. 1) is enhanced by the incorporation of chemical compo-
nents that confer structural flexibility to the inhibitor, allowing
the enzyme-substrate species to adopt multiple conformations
and trap the enzyme in energy minima unfavorable for hydro-
lysis (273, 308, 445). Another viable approach is to design
inhibitors that induce conformational changes in the enzyme
or displace key water molecules important for hydrolysis, such
as seen with the large seven-membered intermediate formed
from the methylidene penems (60, 294, 429).
(iv) Reaction chemistry that slows deacylation and favors
inactivation over inhibitor hydrolysis. Features of an inhibitor
which slow deacylation and drive toward terminal inactivation,
such as acyl-enzyme stabilization (see above) or the presence
of a good leaving group at C-1, contribute to effective inhibi-
tion. Inhibitors that can delay deacylation for longer than 15 to
20 min (koff rate of ⱕ0.001 s⫺1) may outlast the bacterial
generation time, allowing the partner ␤-lactam time to disrupt
the cell wall synthesis necessary for cell division (396, 410). For
example, the bridged monobactams delay deacylation by lim-
iting the C-3–C-4 rotation necessary for approach of the hy-
drolytic water in class C enzymes (152, 238). Additionally,
relatively minor changes introduced by the acylation of mero-
penem by SHV-1, including minor displacements to active-site
residues (overall root mean square deviation [RMSD] of 0.29
Å) and a restructured hydrogen bonding network, lead to per-
sistent complex formation (295).
(v) Rapid cell penetration. ␤-Lactamases are typically found
in the periplasmic space of Gram-negative bacteria, and the
rapidity with which inhibitors can access their target is a pre-
requisite for successful inhibition. Certain ␤-lactams are zwit-
terionic and rapidly penetrate the bacterial outer membrane
(e.g., cefepime). The same principle extends to the inhibitors;
e.g., the enhanced cell entry of LN-1-255 and the novel
monobactams BAL19764 and BAL30072 is likely due to up-
take through siderophore channels (75, 316). Attention must
also be paid to designing “permeable drugs” that are not hin-
dered as they pass through porin channels or quickly returned
into the medium by multidrug efflux pumps, an important
VOL. 23, 2010
resistance determinant independent of ␤-lactamases (see
above) (287).
(vi) Low propensity to induce ␤-lactamase production. Suc-
cessful inhibitors need to avoid inducing expression of the
enzymes that they are designed to inhibit, particularly among
AmpC ␤-lactamases. Examples of this approach include the
non-␤-lactam inhibitors with sulfonamide cores designed to
evade hydrolysis as well as to avoid induction of AmpC enzyme
expression (413).
(vii) Identification of measureable biological correlates of
␤-lactamase inhibition in the cell. Regardless of how attractive
an inhibitor’s laboratory characteristics may be (e.g., nM Ki
values, low IC50s, high kinacts, or low MICs), a successful in-
hibitor must prove its worth in the clinical setting. In vivo
efficacy requires an integration of many complex features, in-
cluding those listed above, partnership with an appropriate
␤-lactam, sufficient serum and periplasm concentrations, and
factors less well understood (e.g., serum protein binding and
individual clearance rates). Many microbiological properties
(time kill, mutant prevention concentration, and synergy) are
important parameters that need to be considered. There is no
certain consensus on how best to identify and measure these
attributes in the early research on candidate inhibitors. Previ-
ous studies with ␤-lactam antibiotics have demonstrated that
MICs may be poorly correlated with the sensitivity of PBPs for
inactivation (213). Finding reliable biological correlates of in-
hibition is a substantial and relevant challenge, and our current
methodological outcome measurements may benefit from re-
examination in this vein.
(viii) In the meantime, use what we have. While scientists
and physicians continue struggling to stay ahead, or at least
abreast, of the abilities of ␤-lactamases, we must utilize intel-
ligently and productively the agents that are currently avail-
able. For example, we can commit to applying the research that
demonstrates the potential of novel combinations of ␤-lactams
and ␤-lactamase inhibitors, such as clavulanate with cefepime,
cefpirome, or meropenem, to expand and enhance inhibitors’
abilities to protect partner ␤-lactams (172, 239). Several sig-
nificant hurdles stand in the way of these novel combinations,
such as corporate relationships affecting the funding of the
trials necessary to demonstrate clinical efficacy, as well as op-
timization of the pharmacokinetics, pharmacodynamics, and
safety of the components. The ␤-lactamase-inhibitory activity
of carbapenems should not be overlooked in the face of in-
creasing ␤-lactam resistance. Carbapenems, for the time being,
remain effective against many difficult-to-treat infections and
teach us that the distinction between an inhibitor and a slow
substrate may be blurred.
A PERSPECTIVE
From our vantage point, the main challenge in ␤-lactamase
inhibitor development is discovering novel inhibitors with ac-
tivity against a broad spectrum of inhibitor-susceptible and
-resistant enzymes from multiple classes (17, 410). New inhib-
itors must also target carbapenemases (serine and metallo-), as
carbapenems are still the most potent ␤-lactams available for
clinical use. Future advances in inhibitor design against versa-
tile ␤-lactamases must incorporate strategies that address each
␤-LACTAMASE INHIBITORS 191
of the key structural features of these diverse proteins, keeping
in mind that mechanism-based inhibitors may engage in
unique reaction chemistry with ␤-lactamases. Compounds with
the chemical features of the “ideal inhibitor,” such as en-
hanced permeability through the cell membrane, affinity for
the active site of the ␤-lactamase, formation of intermediates
that “trap” the enzyme, displacement of critical water mole-
cules, and prolonged time to enzyme recovery, many of which
are illustrated by the investigational complexes highlighted
herein, may have a significant impact on the development of
“second-generation” inhibitors that target resistant ␤-lactamases.
We also acknowledge the magnitude of this challenge. In 30
years of targeted research on ␤-lactamase inactivation, new in-
hibitors have been only slowly introduced into clinical trials.
There have been many starts, but few agents cross the finish line.
This is a testament to the versatility and complexity of ␤-lactama-
ses and the incredible evolutionary ability of the organisms har-
boring them. Is the perfect ␤-lactamase inhibitor an unattainable
goal? Perhaps. Yet, we anticipate with excitement the achieve-
ments in the next 3 decades and suspect that better chemistries
and combinations will ultimately be found.
ACKNOWLEDGMENTS
This work was supported in part by the Department of Veterans
Affairs Merit Review Program and National Institutes of Health grant
1RO1 A1063517-01. R.A.B. is also supported by the Veterans Inte-
grated Service Network (VISN) 10 Geriatric Research, Education, and
Clinical Center (GRECC). S.M.D. was supported in part by NIH grant
T32 GM07250 and the Case Medical Scientist Training Program.
We thank the four anonymous reviewers and A. Endimiani and
M. G. P. Page for careful reading and suggestions regarding this
review.
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Sarah M. Drawz received her bachelor’s de-
gree from Amherst College magna cum
laude. She is currently an M.D./Ph.D. stu-
dent at Case Western Reserve University
School of Medicine in the Department of
Pathology. Her Ph.D. dissertation work fo-
cuses on the inhibition of ␤-lactamase en-
zymes. She plans to return to her medical
studies in the coming year.
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Robert A. Bonomo received his M.D. from
the Case Western Reserve University
School of Medicine. He trained in Inter-
nal Medicine and Infectious Diseases at
University Hospitals of Cleveland and re-
ceived a Certificate of Added Qualifica-
tion in Geriatrics. Dr. Bonomo served as
section chief of the infectious disease di-
vision at the Cleveland Veterans Affairs
Medical Center before becoming Director
of the Veterans Integrated Service Net-
work 10 Geriatric Research, Education, and Clinical Center
(GRECC). He is a Professor of Medicine at Case Western Reserve
University School of Medicine and also holds appointments in the
Departments of Pharmacology and Molecular Biology and Micro-
biology. Dr. Bonomo’s research focuses on microbial resistance to
antibiotics, especially ␤-lactams.