BLEE General Editado PDF

MI65CH23-Bush ARI 10 August 2011 10:13
ANNUAL
REVIEWS Further Epidemiological Expansion,
Click here for quick links to
Annual Reviews content online,
including:
Structural Studies, and
• Other articles in this volume
• Top cited articles Clinical Challenges of New
• Top downloaded articles
Annu. Rev. Microbiol. 2011.65:455-478. Downloaded from www.annualreviews.org
• Our comprehensive search

β-Lactamases from
by Universidad de Buenos Aires on 10/04/12. For personal use only.
Gram-Negative Bacteria
Karen Bush1 and Jed F. Fisher2
1
Biology Department, Indiana University, Bloomington, Indiana 47401;
email: karbush@indiana.edu
2
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame,
Indiana 46556; email: jed.f.fisher.57@nd.edu
Annu. Rev. Microbiol. 2011. 65:455–78 Keywords

First published online as a Review in Advance on β-lactam, carbapenemase, cephalosporin, cephalosporinase, resistance
July 6, 2011
The Annual Review of Microbiology is online at Abstract

micro.annualreviews.org
β-Lactamase evolution presents to the infectious disease community
This article’s doi:
a major challenge in the treatment of infections caused by multidrug-
10.1146/annurev-micro-090110-102911
resistant gram-negative bacteria. Because over 1,000 of these naturally
Copyright c 2011 by Annual Reviews.
occurring β-lactamases exist, attempts to correlate structure and func-
All rights reserved
tion have become daunting. Although new enzymes in the extended-
0066-4227/11/1013-0455$20.00
spectrum β-lactamase (ESBL) families are frequently identified, the
older CTX-M-14 and CTX-M-15 enzymes have become the most
prevalent ESBLs in global surveillance. Carbapenemases with either
serine-based or zinc-facilitated hydrolysis mechanisms are posing some
of the most critical problems. Most geographical regions now report
KPC serine carbapenemases and the metallo-β-lactamases VIM, IMP,
and NDM-1, even though NDM-1 was only recently identified. The
rapid emergence of these newer enzymes, with multiple β-lactamases
appearing in a single organism, makes the design of new β-lactamase in-
activators or β-lactamase-stable β-lactams all the more difficult. Com-
bination therapy will likely be required to counteract the continuing
evolution of these insidious enzymes in multidrug-resistant pathogens.
455
ability to hydrolyze the β-lactam bond in vir-

Contents tually all β-lactam-containing molecules (46).
Today β-lactamases are either directly or
INTRODUCTION . . . . . . . . . . . . . . . . . . 456
indirectly responsible for most of the multidrug
NOMENCLATURE . . . . . . . . . . . . . . . . . 456
resistance observed in gram-negative bacteria
Extended-Spectrum β-Lactamase
in both hospital and community isolates.
Nomenclature . . . . . . . . . . . . . . . . . . 460
Although many enteric and nonfermentative
EPIDEMIOLOGY . . . . . . . . . . . . . . . . . . . 461
gram-negative isolates produce a chromosomal
Serine Noncarbapenemases . . . . . . . . . 461
β-lactamase, it is the transferable β-lactamases
Carbapenemases . . . . . . . . . . . . . . . . . . . 462
that have created greater havoc. The genetic
Metallo-β-Lactamases . . . . . . . . . . . . . 462
elements that encode the hundreds of ac-
Multiple β-Lactamases . . . . . . . . . . . . . 463
quired β-lactamases now seen in the medical
Environmental β-Lactamases . . . . . . . 463
setting are often associated with mobilizable
RECENT PROGRESS IN THE
resistance factors responsible for decreased

STRUCTURAL AND
susceptibility to multiple antimicrobial families
MECHANISTIC STUDY OF
including the aminoglycosides, tetracyclines,
β-LACTAMASES . . . . . . . . . . . . . . . . . 464
and fluoroquinolones. Because physicians have
Mechanism of the Serine
relied heavily on cephalosporins, β-lactamase-
β-Lactamases . . . . . . . . . . . . . . . . . . . 464
inhibitor combinations, and carbapenems as
Structural Evolution of the Serine
broad-spectrum antibiotics to treat infections
β-Lactamases Toward Expanded
caused by gram-negative bacteria (80), the loss
β-Lactam Resistance . . . . . . . . . . . . 465
of these agents due to β-lactamase-mediated
STRUCTURAL DEVELOPMENT
inactivation has almost created a therapeu-
OF β-LACTAMS RESISTANT
tic vacuum, with few agents remaining for
TO THE β-LACTAMASES AND
therapeutic intervention (66).
NEW β-LACTAMASE
In this review, the epidemiology of some of
INHIBITORS . . . . . . . . . . . . . . . . . . . . . 469
the more deleterious β-lactamases produced by
CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 469
gram-negative pathogens is explored, together
with the challenges involved in the nomencla-
ture of these enzymes. In addition, sophisti-
cated structural studies are presented in an at-
tempt to understand the mechanism of action
INTRODUCTION of some of the newer enzymes. Finally, ther-
β-Lactamase-mediated antibiotic resistance apeutic strategies to counteract the hydrolytic
has probably been investigated more thor- activity of the various classes of β-lactamases
oughly than other resistance mechanisms are presented.
throughout the history of bacterial infectious
diseases. As early as 1940, β-lactamase activity
was described as a penicillin-inactivating mech- NOMENCLATURE
anism that threatened the future use of this crit- Inclusive nomenclature systems for β-
ical class of β-lactam antibiotics (1). Although lactamases historically have been based either
stable penicillins, cephalosporins, and car- on amino acid sequences as proposed by
bapenems with increasing stability to hydrolysis Ambler (2) or on functional characteristics as
were developed to circumvent the inactivating originally defined by Richmond & Sykes (104).
β-Lactamase: an activity of these enzymes, this strategy has been Although it is simpler and more precise to de-
enzyme that
a double-edged sword. As new β-lactams were fine a β-lactamase by its amino acid sequence,
hydrolyzes a β-lactam
bond introduced (Figure 1 summarizes the struc- this designation gives little information to
tures), new β-lactamases emerged with the the clinical world about how the β-lactamase
456 Bush · Fisher

β-lactams
Penicillin Cephalosporin Carbapenem Monobactam
H3C CO2
H3C
NH3 O NH
H N OH
H H H H H2N
N H3C CO2
HO S CH3 N S
S H3C H3C
O S
N CH3 N O N N N O
O O N
H2N O H
CO2 CO2 CO2 N CH3
Amoxicillin Ceftazidime Imipenem O S
NMe2 N O N
CO2 OCH3 OH H2N O SO3
N
H H H H H H CH3 NH Aztreonam
N S N S
CH3 S H3C
O N CH3 S
S N O N OAc N
O O O
CO2 H2 N CO2
CO2
Ticarcillin Cefotaxime Meropenem

Clinically used and exploratory β-lactamase inhibitors

O
N O
O O O O N O O
O S CH3 S CH2 N HO H2N
OH N S CH3
N S N
N N CH3 N CH3 N CH3
O O O N O N
CO2 CO2 CO2 CO2 O OS O3
O
CO2
Clavulanate Sulbactam Tazobactam BLI-489 Nottingham BMCL11-36c NXL104
Figure 1
Representative structures within the subclasses of the β-lactams.
contributes to resistance. Unfortunately, taken on the attributes of expanded-spectrum

many new β-lactamases are described only on cephalosporin and carbapenem hydrolysis
the basis of susceptibility data and sequence (groups 2de and 2df) (93). Carbapenems can
information, ignoring such influences as now be hydrolyzed efficiently by β-lactamases
permeability or efflux mutations that affect from molecular classes A, B, and D, thus
the sensitivity of an organism, and that may making therapeutic options even more limited
produce a relatively benign β-lactamase with (139).
an amino acid substitution that has little effect Figure 2 exemplifies the increasing num-
on enzymatic activity. bers of over 1,000 β-lactamases that can be
An updated functional classification based accounted for in the literature or by the attribu-
on hydrolytic and inhibitory profiles of key β- tions of unique β-lactamase names according Extended-spectrum
lactamases, with expanded functional group- to the Web site that monitors β-lactamase β-lactamase (ESBL):
ings, was recently published by Bush & Jacoby amino acid sequences (46). The naming of a β-lactamase that
(12). As seen in Table 1, a summary of the most β-lactamases can be somewhat capricious, as hydrolyzes oxyimino
cephalosporins and
prevalent functional groups demonstrates in- reviewed in detail by Jacoby (48), with three- or
monobactams in
creasing broad-spectrum activity. In addition to four-letter enzyme names that may refer to ge- addition to penicillins
the previously well-described penicillinases and ographical locations (OHIO-1), patient names and early
cephalosporinases (14) are extended-spectrum (TEM-1), or substrate specificities (IMP). The cephalosporins and
β-lactamases (ESBLs) with less efficient inac- most abundant β-lactamases are in functional that is inhibited by
clavulanic acid or
tivation by the classical β-lactamase inhibitors group 2, molecular class A, with more than 550
tazobactam
(group 2ber) (124), and cloxacillinases that have enzymes. Among these enzymes are at least 40
www.annualreviews.org • New β-Lactamases 457

MI65CH23-Bush
ARI
458
Bush
·
10 August 2011
Table 1 Functional grouping of major β-lactamases aligned with molecular assignmentsa
Fisher
Bush-Jacoby group (12) Molecular class Defining characteristic(s) Selected enzymes
10:13
1 C Hydrolyzes cephalosporins and cephamycins, generally Escherichia coli and Pseudomonas aeruginosa AmpC,
with higher kcat values than penicillins CMY-2, FOX-1, MIR-1, P99
Not inhibited by CLA and TZB
High affinity for aztreonam
1e C Hydrolysis of penicillins, cephamycins, expanded-spectrum GC1, CMY-37
cephalosporins, monobactamsb
Not inhibited by CLA and TZB
2a A Efficient hydrolysis of penicillins PC1 and other staphylococcal penicillinases
Inhibited by CLA and TZB
2b A Efficient hydrolysis of penicillins and early cephalosporins SHV-1, TEM-1, TEM-2, TLE-1 (TEM-90)
(cephaloridine, cefazolin, cephalothin)
Inhibited by CLA and TZB
2be A Hydrolysis of penicillins, expanded-spectrum ESBLsb : CTX-M-15, CTX-M-44 (Toho-1),
cephalosporins, monobactams PER-1, SFO-1, SHV-5, TEM-10, TEM-26,
Inhibited by CLA and TZB VEB-1
2br A Efficient hydrolysis of penicillins and early cephalosporins IRTs: TEM-30, TEM-76, TEM-103, SHV-10,
Not well inhibited by CLA SHV-26
2ber A Hydrolysis of penicillins, expanded-spectrum CMTs: TEM-50, TEM-68, TEM-89
cephalosporins, monobactams
Less efficiently inhibited by CLA and TZB
2c A Efficient hydrolysis of carbenicillin PSE-1, CARB-3
Inhibited by CLA
2d D Efficient hydrolysis of cloxacillin or oxacillin OXA-1, OXA-10
Not always inhibited by CLA
MI65CH23-Bush
ARI
2de D Hydrolysis of penicillins and expanded spectrum ESBLs: OXA-11, OXA-15

cephalosporins
10 August 2011
2df D Hydrolysis of carbapenems and cloxacillin or oxacillin OXA-23, OXA-48

10:13
2e A Efficient hydrolysis of cephalosporins CepA

Inhibited by CLA and TZB but not by aztreonam
2f A Hydrolysis of carbapenems, cephalosporins, penicillins, IMI-1, KPC-2, KPC-3, SME-1, GES-2
and cephamycins
Poorly inhibited by CLA, low inhibition by TZB
3a B Hydrolysis of all β-lactams except monobactams IMP-1, L1, NDM-1, VIM-1
Inhibited by EDTA and metal ion chelators, not inhibited
by CLA and TZB
3b B Preferential hydrolysis of carbapenems CphA, Sfh-1
Inhibited by EDTA and metal ion chelators, not inhibited
by CLA and TZB
a
Adapted from References 12 and 14.
b
Expanded-spectrum β-lactams are usually defined as those cephalosporins and monobactams that contain a side chain containing an aminothiazoleoxime moiety extending from the β-lactam
ring. These include the cephalosporins cefotaxime, ceftriaxone, ceftazidime, cefepime and cefpodoxime, and the monobactam aztreonam. The enzymes in groups 1e, 2be, and 2de are known as
ESBLs.
Abbreviations: CLA, clavulanic acid; CMT, complex mutant TEM; ESBL, extended spectrum β-lactamase; IRT, inhibitor-resistant TEM; TZB, tazobactam.
www.annualreviews.org • New β-Lactamases

459
600
Group 2/class A
500
Number of naturally occurring enzymes
400
300
200 Group 2/class D

Group 1/class C
100
Group 3/class B
0
1975 1981 1987 1993 1999 2005 2011
Year
Figure 2
The number of naturally occurring β-lactamases identified from the main functional groups and molecular
classes as indicated by the year in which the structures were provided to the curators of the β-lactamase Web
site (46), or by the year in which the enzymes were reported in the literature (12), adapted with permission
(copyright c American Society for Microbiology, 2010).
variants in the TEM and SHV families that have enzymes compared with the OXA, CTX-M,
one to three amino acid substitutions but confer CMY, VIM, and KPC families that at least
no additional resistance phenotype compared doubled in number since 2005. As sequencing
with the parent TEM-1 or SHV-1 enzyme continues to be inexpensive and rapid, it is ex-
(46). On the basis of percentage increase in pected that the number of unique β-lactamases
growth since 2005, class D (group 2d) and class will expand to represent additional naturally
C (group 1) β-lactamases represent the most occurring homologs of common enzymes that
Metallo-β-lactamase rapidly growing classes of enzymes, with in- may have no obvious selective advantage.
(MBL): a β-lactamase creases of at least 130%, as shown in Figure 3.
that contains at least
Curiously, the number of TEM variants, the
one zinc atom at the
active site to facilitate second most-prevalent family of β-lactamases,
has grown the slowest in the past five years,
Extended-Spectrum β-Lactamase
hydrolysis of carbapen-
ems and most β-lactam perhaps owing to an approaching saturation
Nomenclature
antibiotics except of mutable amino acids that confer resistance Disagreements about appropriate nomencla-
for monobactams, ture have arisen recently, particularly related
without affecting the fitness of the enzyme.
resulting in resistance
to most β-lactams in Relatively small increases in numbers of to the definition of ESBLs. Historically, these
producing organisms variants were also seen in the IMP family enzymes were included as a subset of the group
of metallo-β-lactamases (MBLs) and SHV 2b β-lactamases that could efficiently hydrolyze
460 Bush · Fisher

Increase (%)
OXA 130
TEM 16
SHV 51
CTX-M 100
CMY 190
IMP 26
VIM 125
GES 2010 89
2005
KPC 270
0 50 100 150 200
Number of β-lactamases identified
Figure 3
The increased numbers of natural variants in β-lactamase families from 2005 to 2010, based on families with
at least 10 enzymes as enumerated by December 31, 2010 (46).
cephalosporins and monobactams with an oxy- spectrum cephalosporins and monobactams,

imino substituent at the C-7 (cephalosporin) but not carbapenems (13). Many clinical
or C-3 (monobactam) position of the β-lactam microbiology laboratories now include ESBL
ring (14) (Figure 1). In the updated Bush & testing routinely for multidrug-resistant en-
Jacoby scheme (12), the ESBL designation was teric bacteria, with a recommendation that car-
expanded to include rare class C cephalospori- bapenems may be useful therapeutic agents.
nases (group 1e) and a subset of OXA en- Thus, an expansion of the definition of ESBLs
zymes (group 2de) with traditional ESBL hy- to include carbapenemases would only confuse
drolytic profiles. These designations are similar the situation, not improve it.
to those proposed in 2008 by Livermore (65),
who suggested an expansion of the ESBL ter- EPIDEMIOLOGY
minology to include OXA-related ESBLs and Although all β-lactamases have the potential to
unusual plasmid-encoded AmpC cephalospori- confer resistance to the specific substrates that
nases with increased cefepime-hydrolyzing are most efficiently hydrolyzed, enzymes that
activity. may be transferred among species are the great- Carbapenemase:
a β-lactamase that
In 2009, a group of eight primarily Euro- est medical concern (74). The mobile elements
hydrolyzes most
pean β-lactamase investigators proposed that on which they reside can rapidly spread within β-lactam antibiotics,
the ESBL terminology be extended beyond an individual (120), within a hospital (133), or especially
the functional group 2be enzymes (14) to within the nonhospitalized community (136), carbapenems, thereby
include all transferable AmpC cephalospori- carrying with them resistance determinants for conferring
carbapenem resistance
nases and carbapenemases, including both ser- most antibacterial drug classes (134). Several
to the producing
ine and metallo-β-lactamases (36). Their rea- recent review articles have described the ap- organism
soning was that their nomenclature would pearance and dissemination of these enzymes in
Expanded-spectrum
provide a simpler vocabulary for health care detail (11, 47, 66, 139). cephalosporins:
professionals and politicians. A similar argu- cephalosporins with a
Serine Noncarbapenemases
ment also was made by Lee et al. (60). A C7 side chain
rebuttal to this expanded ESBL definition Representative key acquired β-lactamases containing an
described below have emerged as the major oxyimino group,
was presented by a group of 17 β-lactamase
including cefotaxime,
investigators from Europe and the United causes for many challenging gram-negative
ceftriaxone,
States who argued that the ESBL terminol- infections. Plasmid-encoded class C/group 1 ceftazidime, and
ogy has become well established to include CMY enzymes that now appear in Escherichia cefepime
transferable enzymes that hydrolyze expanded- coli with high frequencies (117) are also found in

animals in the food chain and can be transferred producing Klebsiella pneumoniae was accompa-
between these animals and humans (144). The nied by a decrease in the prevalence of problem-
most prominent class A/group 2be ESBLs atic VIM-type MBLs in that facility (32, 120).
are now the CTX-M enzymes (41), with the
global emergence of a CTX-M-15-producing
E. coli clone, strain ST131 O25:H4 (95). Class Metallo-β-Lactamases
D ESBLs are also prevalent, especially in MBLs continue to thrive globally in the clin-
nonfermentative bacteria, such as Pseudomonas ical environment, with outbreaks of VIM and
aeruginosa (83), and in Acinetobacter spp., where IMP-producing pathogens reported through-
OXA enzymes not only are intrinsic but may out Europe and the Asia-Pacific region (139).
also be acquired through class 1 integrons or The MBL SPM-1, once thought to be confined
insertion sequences (101). Although organisms to Brazil, has now escaped to Europe, after a
producing these acquired AmpC cephalospori- Swiss patient initially treated in Brazil was iden-
nases and ESBLs should still be susceptible tified with an SPM-1-producing P. aeruginosa
to carbapenems, non-β-lactamase-mediated isolate (108).
resistance to carbapenems may also emerge, as In 2009, a novel MBL was described with
documented in a recent study demonstrating biochemical characteristics resembling those

that AmpC-type β-lactamases help to promote of the related IMP-1 and VIM-2 enzymes, but
the emergence of carbapenem resistance in with only 32% sequence identity to VIM-1
porin-deficient clinical isolates (71). (150). This enzyme was initially identified from
northern European strains that originated in
patients from India or patients who had trav-
Carbapenemases eled to India for medical procedures (56). The
Acquired carbapenemases, including both ser- naming of this enzyme as NDM-1 (New Delhi
ine carbapenemases and MBLs, have become metallo-β-lactamase), following one of the
one of the most discussed resistance factors in common conventions for naming β-lactamases
the past two years. Serine carbapenemases, ini- based on the city of origin of the index patient
tially recognized as species-specific chromoso- (48), has become a controversial issue. This
mally encoded enzymes, were virtually dormant is somewhat curious, as several other recent
until 1994 (139). By the late 1990s, plasmid- MBLs have been named according to the lo-
encoded carbapenemases began to emerge cation of their original isolates, including VIM
when the KPC enzymes were first identified (Verona integron-borne metallo-β-lactamase)
in the northeastern United States (149). To- (58), SPM (São Paulo metallo-β-lactamase)
day, the KPC and GES families of plasmid- (125), and SIM-1 (Seoul IMipenemase) (61).
encoded serine carbapenemases include at least Recent surveillance studies have identified
10 unique KPC amino acid sequences and 17 NDM-1 in clinical isolates from India as
unique GES variants (46) that have spread early as 2006 (16). Within a short period,
throughout the world in both fermentative and NDM-1 MBL was identified in most parts
nonfermentative bacteria. KPC-producing En- of the world beyond India, Pakistan, Sweden,
terobacteriaceae, Acinetobacter spp., and P. aerug- and the United Kingdom, where the first
inosa isolates have been discussed in several re- producing strains were isolated. Organisms
cent review articles that highlight the clinical producing this enzyme have now appeared in
problems caused by these multidrug-resistant Australia (100), Denmark (in a patient from
gram-negative pathogens (11, 84, 139), many of Bosnia and Herzegovina) (39), Germany (in a
which are treatable by only colistin/polymyxins, patient previously hospitalized in Serbia) (38),
and possibly tigecycline (66, 120). In the com- Montenegro (6), Singapore (55), Taiwan (146),
petitive environment of the intensive care unit and the United States (17). Epidemiologically,
of a Greek hospital, the emergence of KPC-2- these reports included strains from patients
462 Bush · Fisher

who had recently been in India or Pakistan, as in a colonizing E. coli isolate that coproduced
well as from patients who had never traveled CTX-M-15, and the penicillinases TEM-1,
outside their home countries in the Balkans or OXA-1, and OXA-2 (99); (c) characterization
northern Europe (6, 38, 39). of an NDM-1-producing K. pneumoniae isolate
Although the NDM-1 gene is carried by that produced genes encoding the CTX-M-15,
promiscuous plasmids that have now appeared SHV-11, and TEM-1 enzymes (39); and
in many genera of Enterobacteriaceae and nonfer- (d ) confirmation of genes encoding the
mentative bacteria (38), a German E. coli isolate NDM-1, OXA-23, and OXA-51 enzymes in
appears to have blaNDM-1 inserted into the chro- three Indian isolates of Acinetobacter baumannii
mosome (99). NDM-1-producing pathogens (51). Effective therapy therefore must include
have been identified in the popular news me- agents that can treat organisms producing both
dia as “superbugs” because many of them are serine and metallo-β-lactamases; detection
multidrug-resistant organisms causing infec- of each of these is critical (82). Methodology
tions resistant to all antibiotics except colistin, relying upon synergistic activity between a
and possibly tigecycline (56), similar to many key substrate and a selective inhibitor in broth
KPC-producing pathogens (102). Although dilution, disk diffusion, or commercial systems
these infections may have fatal outcomes (38), now exists to detect organisms with acquired
a number of NDM-1-producing bacteria have β-lactamases, including AmpC, ESBLs, and
been identified as colonizing bacteria that have MBLs (102, 134). The most definitive detection
not caused clinically recognizable disease (55, systems rely on PCR to amplify a β-lactamase-
63, 99, 146, 150). However, the presence of encoding gene, a technique expected to develop
these organisms in the New Delhi water supply into bedside diagnostics using multiplex PCR
and seepage ditches (140) indicates that these to identify β-lactamase families (145).
organisms have the potential to spread unno-
ticed throughout the community without caus-
ing infection in most of the recipients, so de- Environmental β-Lactamases
tection is important in the medical community Unusual environmental niches have provided
(82). the sources for novel β-lactam-hydrolyzing
enzymes. The highly thermophilic bacterium
Thermus thermophilus HB8 produces the protein
Multiple β-Lactamases TTHA1623, which is related structurally to the
Because of the increasing numbers of β- superfamily of classical MBLs (148). The iso-
lactamases per clinical isolate that are now lated protein binds two iron atoms that can be
appearing, not only is the selection of ther- replaced by zinc to mimic the folding in MBLs.
apy more challenging, but it is also more Although the protein apparently does not hy-
difficult to detect the causative resistance drolyze β-lactams, it may provide insight into
factors in multidrug-resistant pathogens. MBL evolution in organisms that need to re-
Some of the more insidious organisms produce spond to the threat of environmental β-lactams
combinations of ESBLs and carbapenemases, in more temperate environments.
sometimes with both serine and metallo-β- The unusual class A OIH-1 penicilli-
lactamases in the same strains. Examples of nase (129, 130) is produced by the alka-
these include (a) isolation of a Greek strain liphilic and salt-tolerant gram-positive bac-
of K. pneumoniae that produced the relatively terium Oceanobacillus iheyensis, which grows off
innocuous TEM-1, the plasmid-encoded the coast of Japan at a depth of 1,050 m in
CMY-2 cephalosporinase, the ESBL CTX- an environment where no β-lactams would
M-15, and two carbapenemases, the MBL be expected (130). When expressed in E. coli,
VIM-19 and the serine carbapenemase KPC-2 OIH-1 conferred resistance to all penicillins but
(102); (b) identification of the NDM-1 MBL piperacillin, was inactivated by clavulanic acid

and tazobactam, but did not affect sensitivity to MBLs. All β-lactamase classes now present ei-
expanded-spectrum cephalosporins, carbapen- ther immediate or near-future clinical impact.
ems, or aztreonam (129). Neither clavulanate nor tazobactam possesses
β-Lactamase-producing organisms from clinically meaningful activity against class C
other aquatic sources including rivers, lakes, and class D β-lactamases, and both are useless
and wastewater streams have been reported against the MBLs. How can the historical
from Brazil (101) and China (64). In Italy, the experience with class A serine-dependent β-
novel IMP-22 MBL was identified from an iso- lactamases be brought to bear against the class
late collected upstream from the city sewage C and class D enzymes? What can be done to
treatment plant (96). The Seine River in Paris inhibit the MBLs, for which the current struc-
has provided a rich source of β-lactamases. An tural foundation for inhibitor design is fragile?
Aeromonas allosaccharophila isolate expressed the
novel PER-6 ESBL (33), and at least three
Mechanism of the Serine
isolates of Pseudomonas fluorescens produced a

unique class A/group 2f carbapenemase named β-Lactamases
BIC-1, an enzyme displaying 68% and 59% The catalytic mechanism of the class A serine
amino acid identity with the serine carbapen- β-lactamases involves sequential opening of
emases SFC-1 from Serratia fonticola and KPC- the β-lactam by the active-site serine to give
2, respectively. Perhaps more alarming are the a serine acyl-enzyme intermediate, which is
results from a 2009 surveillance study of the then deacylated from the serine by hydrolysis
Seine in which genes encoding ESBLs were to complete enzyme turnover. This hydrolysis
identified in 71% of the ceftazidime-resistant event—which for many β-lactams and many
Aeromonas isolates, including genes that en- β-lactamases is efficient—sets the mechanism
coded the clavulanic acid–inhibitable VEB-1a, of the serine β-lactamases apart from the
SHV-12, PER-1, PER-6, TLA-2, and GES- penicillin-binding proteins (PBPs), which are
7 enzymes (35). Because these genes are often closely evolutionarily related but are poorly
present on mobilizable elements with transfer- capable of hydrolytic deacylation (76, 135).
able resistance determinants for other antibi- The β-lactam inactivators clavulanate and
otic classes, community water sources provide tazobactam act first as substrates to acylate
a rich environment for the facile exchange of the active-site serine, with concomitant
antibiotic resistance. opening of their β-lactam ring. However,
their acyl-enzymes undergo intramolecular
rearrangements in competition with hydrolytic
RECENT PROGRESS IN THE deacylation to give new acyl-enzymes that no
STRUCTURAL AND longer engage the hydrolytic residues of the
MECHANISTIC STUDY OF active site (27). These stable acyl-enzymes
β-LACTAMASES coincide with β-lactamase inactivation. Like-
The impetus behind the clinical introduction of wise, newer β-lactams—such as the oxyimino
β-lactam/β-lactamase inhibitor combinations, cephalosporins and the carbapenems—often
exemplified by the amoxicillin-clavulanate are poor substrates of the class A β-lactamases
and piperacillin-tazobactam pairings, was the because of their structural ability to form stable
gram-negative prevalence of Ambler class acyl-enzymes without rearrangement.
A/Bush-Jacoby group 2b serine-dependent Hence there is intimate coupling of β-
β-lactamases (exemplified by the TEM-1 lactam structure to β-lactamase mechanism,
enzyme). The β-lactamase landscape sub- inhibition, and resistance development. Clin-
sequently has changed dramatically to now ical introduction of β-lactam/β-lactamase in-
include powerful class A, class C, and class hibitor combinations resulted in either the
D serine-dependent β-lactamases and class B overproduction of group 1 or group 2
464 Bush · Fisher

β-lactamases (97) or the appearance of Lys-234

inhibitor-resistant group 2br β-lactamases, wat-2 2H
Me
2H
largely the result of a point mutation(s) of N
O 2.03 HO
active-site residues that disfavored the delete- 2H
N Thr-235
2H 2H O
rious aspects of the rearrangements of their 2H 2.08
acyl-enzymes (37). The active-site develop- Ser-230 2.05
ments that have followed represent extraordi- O 2H Lys-73
nary adaptive enzyme evolution (3, 53, 109). In 2.32
each instance, the labyrinth of catalytic residues 2H
Ser-70
N wat-1
2H
2.41 Asn-170
within the active site is preserved, as their sur- 2H
O 1.75
2H
2H O 2.26 O
rounding residues are altered to enable greater 2.20
2H
resistance ability. O 2H N
1.87 2H
A key observation toward the understanding 2H
N O 2.40
of the catalytic labyrinth is made from compar- Asn-132 2H O

isons of the active sites of the serine β-lactamase
and PBPs. The PBPs are the transpeptidase and
Glu-166
carboxypeptidase catalysts of bacterial cell wall Figure 4
biosynthesis, the molecular targets of the β- A schematic of the hydrogen-bond network (hydrogen bonds are shown as
lactam antibiotics, and the evolutionary prede- dashed lines, with hydrogen bond distances in angstroms shown in gray
cessors of the serine β-lactamases. PBPs use a numbers) within the active site of the R274N/R276N pd-Toho-1 Class A
lysine-serine dyad to accomplish acyl transfer β-lactamase, as determined from 2.1 Å resolution neutron diffraction obtained
at neutral pH and at ambient temperature (PDB accession codes 2xr0 for the
from the central amide of the . . .D-Ala-D-Ala
X-ray structure and 2xqz for the neutron structure; 127). The solution of the
terminus of the peptidoglycan stem, followed neutron structure was facilitated by D2 O exchange of the hydrogens on the
by acyl transfer from the serine to an amino heteroatoms: These exchangeable hydrogens are labeled 2 H. The
acid of a second peptidoglycan. β-Lactams are R274N/R276N mutations facilitated crystallization of the enzyme and are not
efficient antibiotics owing to the stability of relevant to this hydrogen-bond network. Glu-166 is the basic residue in this
apoenzyme structure. Adapted from figure 3 of Reference 127.
their PBP acyl-enzymes. Mutation of the PBP
active site to disfavor β-lactam acylation is
the primary mechanism of gram-positive resis-
β-lactamase serine confers mechanistic versa-
tance and now contributes to β-lactam resis-
tility compared with the simpler lysine-serine
tance in a few gram-negative bacteria, notably
dyad of the PBPs. Either the lysine or the glu-
Haemophilus influenzae (132) and Neisseria gon-
tamate of the β-lactamase may be used to en-
orrhoeae (143). The outstanding difference be-
able serine reaction with the β-lactam struc-
tween the PBPs and the serine β-lactamases
ture, and with the glutamate providing catalyti-
is the addition to the serine β-lactamases of
cally meaningful rates of hydrolytic deacylation.
a glutamate residue, located on a short pep-
tide strand (the –loop) that transverses the
active site (9). The primary role of the glu-
tamate in hydrolytic deacylation of the acyl- Structural Evolution of the Serine
enzyme was identified by its mutagenesis to a β-Lactamases Toward Expanded
glutamine, creating a deacylation-defective β- β-Lactam Resistance
lactamase. The interplay of this glutamate has The β-lactam antibiotics of the greatest clinical
been visualized through high-resolution neu- value against infection caused by gram-negative
tron diffraction of D2 O-exchanged crystals of pathogens are the β-lactamase inhibitor com-
an ESBL serine β-lactamase (126, 127). The binations, the oxyimino cephalosporins, and
glutamate is the most basic residue within the the carbapenems. Dramatic structural evo-
active site (Figure 4). The ability of a ly- lution of all four classes of the β-lactamases
sine and glutamate to act cooperatively on the toward these latter sets of β-lactams has

happened. We summarize in this section the possible (81). The high-resolution structure of
outcome of this evolution for the three serine the latter enzyme with its D240G mutation
β-lactamase classes, those of the class A (no- and kinetic studies of other CTX-M-44 mu-
tably the CTX, GES, IMI, KPC, NMC, and tants (116) suggest a basis for the ESBL activity.
SME enzymes), class C (AmpC, encompassing Gly-240 allows breathing motions of the pro-
the ACC, ACT, MIR, CMY, DHA, FOX, and tein domains that permit substantial side chain
MOX plasmid-mediated clusters), and class D reorganization, which in turn enables engage-
(OXA) β-lactamases. ment of the oxyimino cephalosporin as a sub-
strate and, following its hydrolysis, its departure
Class A. The advantages of the oxyimino as a hydrolyzed β-lactam (24).
cephalosporins include β-lactamase stability Carbapenem-hydrolyzing β-lactamases are
against group 2b TEM and SHV-1 enzymes found within the class A and class C serine
and excellent activity against non-ESBL or β-lactamases, as well as the class B MBLs.
carbapenemase-producing enteric bacteria. Al- The dominant class A carbapenemases are the
though the newest TEM/SHV variants attain KPC-prefixed enzymes, named for their initial
ESBL status largely by allele recombination (3), (and continuing) association with K. pneumoniae
they have been joined by the widespread class A, (142). Additional families of serine carbapen-
group 2be family of β-lactamases termed CTX- emases include GES, IMI, NMC, and SME
M (active on CefoTaXime, isolated in Munich), enzymes. The term carbapenemase is an incom-
whose spectrum also includes many oxyimino plete descriptor of their catalytic ability: The
cephalosporins, especially cefotaxime (7). Many serine carbapenem-hydrolyzing β-lactamases
CTX-M enzymes typically have low reactiv- are broad-spectrum enzymes that upon acqui-
ity toward ceftazidime and retain susceptibility sition decrease bacterial susceptibility to all β-
to clavulanate and tazobactam β-lactamase in- lactam classes (8, 43, 84). Moreover, expansion
hibitor combinations. Concurrently, the CTX- especially within the KPC and GES classes is
M enzymes continue their diversification with occurring rapidly (88). Many of these enzymes
additional clinical compromise of ceftazidime are mediocre catalysts, but the level of catalytic
reported (86). activity that they attain is sufficient that when
The clinically most important CTX-M combined with other genetic adaptation (such
ESBL is CTX-M-15, derived from CTX-M- as deletion of the specific porin used for entry
3 by a D240G mutation. The replacement of of the β-lactam), clinical resistance is attained
the active-site (but noncatalytic) Asp-240 by (54). Likewise, although many of these enzymes
glycine extends the substrate spectrum to both are susceptible to clavulanate and tazobactam
ceftazidime and aztreonam (18). Several crys- inactivation (31), newer variants attain suffi-
tal structures of CTX-M enzymes have been cient levels of hydrolysis toward these inactiva-
solved, including those of the clinically un- tors to likewise attain inhibitor resistance (92).
common but not atypical CTX-M-44 (Toho- Efforts to correlate the molecular basis
1) enzyme (115) and the clinically encountered for these developments to protein structure
CTX-M-9 enzyme (20, 23). Neutron diffrac- have focused on study of the NMC (123),
tion study of the former enzyme provides the SME, KPC-2 (52), and GES-1 (119) enzymes.
best evidence of the hydrogen-bond interac- A TEM-like protein fold that incorporates
tions within its resting state (126, 127). A cor- an active-site disulfide as a signature motif
relation exists between the substrate kcat /Km is seen. This disulfide bridge connects the
(the catalytic efficiency of an enzyme for a helix containing the nucleophilic serine to an
given substrate) and the thermal stability of adjacent strand that defines one side of the
the acyl-enzyme intermediate of the CTX-M- active site. Additional features contributing to
44, suggesting that rapid evaluation of the sub- catalysis are important residue replacements,
strate spectrum of the CTX-M enzymes may be especially the appearance of asparagine at
466 Bush · Fisher

position 132 and mutation at position 170 terionic cephalosporins (cefepime) and newer
(proximal to the catalytic Glu-166 residue). carbapenems (ertapenem and doripenem)
The effect of mutation at position 170 was remain poor AmpC substrates (71, 103).
ESAC: expanded-
evaluated in the GES subclass using imipenem Although the class C and class A β- spectrum AmpC
as substrate. Changing the Gly-170 of GES-1 lactamases share a serine-dependent acylation/
(not a carbapenemase: kcat /Km for imipenem, deacylation mechanism, the architecture of
3 × 103 M−1 s−1 ) to Asn-170 (GES-2) im- the active site of the one class is distinctly dif-
proves kcat /Km values by an order of magnitude; ferent from that of the other (21, 78). A con-
further mutation to Ser-170 (GES-5) provides sequence is poor inhibition of many AmpC
a carbapenemase showing a kcat /Km value of enzymes by clavulanate and tazobactam. Cu-
3 × 103 M−1 s−1 , sufficient to impart imipenem riously, point mutations that improve the hy-
resistance (30). Comparison of the detailed drolytic ability of a CMY-AmpC toward ce-
kinetics for GES-5 and GES-1 shows the fotaxime diminish its ability toward the car-
G170S mutation to improve both the rate of bapenems and toward sulbactam as an inacti-
serine acylation (by 5,000-fold) and deacylation vator (28). In contrast to the significant expan-
(rate-limiting, improved by 16-fold). A struc- sion of substrate spectrum accomplished by se-
tural basis for the advantage of Ser-170 relative quential point mutations at specific active-site
to either Gly-170 or Asn-170 is not obvious. residues within the class A active site, class C
enzymes appear to require concurrent muta-
Class C. The expanded-spectrum cefotaxime tion of at least two active-site residues (59).
and ceftazidime cephalosporins are increas- For example, a single Gly214Glu mutation in
ingly compromised by plasmid-encoded class the -loop of CMY-2 AmpC gives CMY-32,
C, group 1 extended-spectrum AmpC enzymes isolated from a clinical E. coli strain. CMY-32
(47, 59). The plasmid-encoded expanded- achieves an incremental improvement in activ-
spectrum AmpC (ESAC) β-lactamases ity against cefotaxime (E. coli CMY-32, MIC =
includes five clusters: CMY-2 (nominal Cit- 64 μg ml−1 ) compared with CMY-2 (MIC =
robacter freundii ), MIR-1 and ACT-1 (nominal 16 μg ml−1 ). Two changes observed for AmpC
Enterobacter), DHA-1 (nominal Morganella ADC-33 (isolated from a clinical A. bauman-
morganii ), ACC, and MOX-1 (also called nii strain), Arg replacing Pro210 and duplica-
CMY-1) and FOX-prefixed clusters. AmpC tion of an Ala residue at position 215 of the
β-lactamases historically were chromosomal -loop, likely account for its high-level activity
cephalosporinases in P. aeruginosa and many against ceftazidime, cefotaxime, cefepime, cef-
Enterobacteriaceae (including Enterobacter spp., pirome, and aztreonam, but not carbapenems
C. freundii, and Serratia marcescens) but are now (105). Multiple mutations of ADC-7, many of
also plasmid-dispersed among the Enterobac- which are distal from the active site, account
teriaceae (77). This development coincides to for the ADC-51 and ADC-53 enzymes isolated
change from a chromosomal gene governed from a ceftazidime-resistant A. baumannii clin-
by a weak promoter to a plasmid with strong ical isolate. Among these mutations in ADC-
constitutive expression. Although the AmpC 51 is an active-site Val208Ala change in the -
β-lactamases are not particularly effective to- loop, and in ADC-53 an Asn283Ser mutation
ward carbapenems or monobactams, the ability in the H-10 helix (106).
of the most active AmpC enzymes (CMY-2,
ACT-1, ADC-33, and DHA-1) to hydrolyze Class D. A primary factor in the recent emer-
the expanded-spectrum cephalosporins, the gence of Acinetobacter spp. as a major gram-
carbapenem imipenem (71), or the monobac- negative pathogen is its expression of broad-
tam aztreonam (105) is sufficient, when spectrum class D and class B β-lactamases
combined with porin and efflux manipulation, (34), a circumstance closely paralleled in
to attain clinical levels of resistance (50). Zwit- P. aeruginosa (139). In both bacteria, class D

(OXA-prefixed) β-lactamase expression abets Class B. Dramatic expansion in β-lactamase

porin manipulation and efflux transporters to diversity and capability has occurred within the
attain carbapenem resistance. This synergism metallo-β-lactamases as well (70). The appear-
requirement reflects the modest in vitro car- ance of NDM-1 in 2009—an acquired MBL
bapenemase ability of the OXA-β-lactamases. with neither genetic nor structural precedent,
Nonetheless, the high-level expression of these and hydrolytically capable toward all β-lactams
carbapenem-hydrolyzing class D β-lactamases save aztreonam (150)—is significant for its sud-
as a multidrug-resistant phenotype (94), cou- denness and for its immediate clinical relevance
pled with the extraordinary ability of the OXA (75). As a catalyst of metal-dependent β-lactam
class to rapidly mutate toward expansion of its hydrolysis, NDM-1 is only the newest resis-
catalytic spectrum (42), provides the basis for tance enzyme summoned from the metallo-
their present clinical concern (141). hydrolase superfamily (73, 87, 128). The
The molecular mechanism of the serine class MBLs, chromosomally located in some gram-
D enzymes is distinct from that of class A and negative bacteria such as Stenotrophomonas
class C. Catalytic activation of serine by the class maltophilia and some gram-positive bacteria
D enzymes requires prior reaction of the amine such as Bacillus cereus, share a common fold with
of an active lysine with carbon dioxide. The high sequence diversity. The largest subfamily
functional group so formed, a carbamate anion, (B1) includes the common VIM and IMP
is the catalytic base for serine activation. This enzymes, whereas the B2 enzymes are strict
seeming mechanistic peculiarity is also encoun- carbapenemases with poor hydrolysis of other
tered with the gram-positive β-lactam sensor β-lactams.
proteins BlaR1 and MecR1 (19) for gene regula- Of the four β-lactamase structural classes,
tion of β-lactamase (BlaR1) and PBP 2b expres- the MBLs are arguably the most enigmatic with
sion in methicillin-resistant S. aureus (MecR1), respect to the relationship of structure (of both
respectively. For these sensors, decarboxylation substrate and protein) to mechanism (91). The
of the lysine (the molecular reverse of its forma- dilemma arises from the complexity of the ac-
tion) after serine acylation is involved in the sig- tive site. The MBL active site accommodates
nal transduction. Accordingly, hydrolytic de- two metal ions in separate but proximal sites.
acylation of these sensors is slow, whereas for One site is occupied by a Zn(II) ion under the
the OXA-β-lactamases the carbamate is pre- typical circumstance of in vitro isolation (57).
served and hydrolytic deacylation is catalytic. Occupancy of the second site by a second Zn(II)
The kcat value for oxacillin by OXA-10, for ex- ion is catalytically advantageous for the B1 and
ample, is 100 s−1 (138). The bases for this differ- B3 subclass enzymes and coincides with the
ence and the related issue of inhibitor design— protein crystallization conditions used for these
the class D enzymes are poorly inhibited by enzymes. A current challenge to MBL mech-
clavulanate and sulbactam (131)—are a current anistic study is identifying in vitro conditions
objective of protein (4, 62, 110, 138) and crys- that coincide with the in vivo circumstance of
tallographic (25, 111, 112) studies. Apart from their catalysis. Among the confounding ques-
a catalytically advantageous insertion of the hy- tions are determining which metal site is pref-
drophobic valine for the hydrophilic threonine erentially occupied and determining the metal
proximal to the catalytic lysine, to modulate the ion mobility within the active site (10, 44, 113).
lysine basicity to favor the CO2 reaction (138), Moreover, as may also be true for the serine
no clear hypothesis has emerged. Rather, what enzymes, the MBL mechanism may vary in re-
is observed is an enzyme class capable of ex- sponse to the substantially different β-lactam
ploring, and tolerating, diversity within its ac- substrate structures. Although several mecha-
tive site. Whereas the result may be a narrow- nisms have been proposed (22, 118, 147), these
spectrum breadth, the activity nonetheless en- mechanisms pose uncertainty for understand-
compasses the clinically used β-lactams. ing the role of active-site mutations (15, 72).
468 Bush · Fisher

The most reliable association between enzyme and pharmacological synergy with ceftazidime
properties and clinical correlation remains, as (29, 69). Initial mechanistic evaluation of
always, microbiological. NXL-104 with the TEM and P99 class A
β-lactamases indicates enzyme inactivation
via formation of a catalytically inert covalent
STRUCTURAL DEVELOPMENT complex (122). This complex presumably arises
OF β-LACTAMS RESISTANT TO from diversion of an acyl-enzyme intermediate.
THE β-LACTAMASES AND NEW NXL104 is being developed in combination
β-LACTAMASE INHIBITORS with either ceftazidime or ceftaroline (26, 68),
expanding the antimicrobial activity of these
Several reviews give the status of both
agents to include many multidrug-resistant
clinically efficacious and exploratory β-
gram-negative bacteria that produce either
lactamase inhibitors (5, 27, 114). Current
ESBLs or serine (KPC) carbapenemases (121).
efforts toward new β-lactamase inhibitors are
In contrast to the serine β-lactamases, known

divided between serine-catalyzed enzymes
inhibitors of the MBL enzymes, typically
and metallo-catalyzed enzymes. The func-
with metal-chelating substructures, have only
tional group arrays that lead to nonhydrolytic

moderate activity against the MBLs and hence
diversion of serine-derived β-lactamase acyl-
still remain exploratory structures (88).
enzymes are known, and their incorporation
A strategy with a more immediate prospect
into new β-lactam guises is a proven strategy.
is that of β-lactamase-resistant β-lactams (89),
The 6-alkylidene penem BLI-489 (137) and
which have been used for the past 30 years,
the 6-(hydroxymethyl)penam Nottingham
resulting in the oxyimino cephalosporins
BMCL11-36c (85) are good examples (see
and the carbapenems. The new β-lactams
structures in Figure 1). BLI-489 is a potent
conceptually derive from the monobactam
inhibitor of all serine β-lactamase classes (98)
aztreonam, showing gram-negative activity
with low propensity toward resistance devel-
and intrinsic resistance to MBL hydrolysis.
opment (107) but decreased activity against
The newer monobactams, many of which
bacteria producing multiple β-lactamases.
contain siderophores to allow uptake by
The 6-(hydroxymethyl)penam, an exploratory
bacterial iron-transport systems, improve
structure, shows excellent in vitro activity
these characteristics owing to broader PBP
against both class A and class C enzymes.
inhibition combined with access to siderophore
Replacement of the β-lactam substructure
transporters. One example is BAL-30072, an
with a hydrolytically inert but serine-reactive
O-sulfate of a monocyclic N-hydroxy-β-lactam
cyclobutanone attained respectable levels of
(termed a sulfactam). BAL-30072 is bacte-
class D inhibition (49).
ricidal against a breadth of gram-negative
New lactam templates conferring β-
pathogens (79, 90). A second new monocyclic
lactamase inhibition have appeared. An un-
β-lactam is the monocarbam MC-1, which also
precedented three-component mixture known
possesses siderophore substructure and features
as BAL30376, which contains the N-sulfonic
N-(sulfonyl amino)carbonyl activation of the
acid of a bicyclic β-lactam and is formulated
β-lactam (40). MC-1 is active in vitro against
with clavulanate as a companion β-lactamase
P. aeruginosa at an MIC of 0.5 μg ml−1 (45).
inhibitor and a siderophore-substituted
monobactam, shows activity against many
ESBL-, AmpC-, and MBL-expressing gram- CONCLUSIONS
negative bacteria (67). A second new structure Although the evolutionary origin of what
is the bicyclic imidazolidinone-N-sulfate, we now describe as mechanisms for an-
NXL104 (121), which shows excellent in tibiotic resistance is uncertain—for advan-
vitro activity against serine class A and class tage to the bacterium within an ecologi-
C enzymes (including KPC carbapenemases) cal niche? for intraspecies or interspecies

communication?—the burgeoning of antibi- The expansion of the substrate capabilities of

otic resistance is the predictable evolutionary the β-lactamases is irrevocable. As the clinical
response to the extensive human use of antibi- use of β-lactams has progressed from penicillins
otics. As the therapeutic β-lactam has changed to advanced cephalosporin and carbapenem
in response to increasing resistance, new β- structures, the β-lactamases have followed.
lactamases have been mobilized from obscure While our strategy on this game board is
bacteria in obscure ecological niches and al- still one of offense, the prospects for effective
tered to better embrace these new β-lactams as β-lactam monotherapy are of low probability.
prey. As the β-lactamases so evolve, they often Our best hope for the future is for β-lactamase
leave behind their hydrolytic ability toward inhibitor combinations that will allow broad-
older β-lactams, thereby requiring the bacteria spectrum applications against the multitude
to secure an ensemble of β-lactamases to meet of existing enzymes that are already poised to
the antibiotic challenges. The dilemma is ours. evolve into even more effective catalysts.
SUMMARY POINTS
1. More than 1,000 naturally occurring β-lactamases have been identified, frequently as
the result of the facile transfer of mobile elements from one gram-negative organism
to another; yet, only a few of these β-lactam-hydrolyzing enzymes have become promi-
nent resistance determinants worldwide. The most common acquired β-lactamases are
the AmpC cephalosporinases (CMY family), the ESBL CTX-M-14 and CTX-M-15 en-
zymes, the serine carbapenemases (KPC enzymes), and the metallo-β-lactamases (NDM-
1, VIM, and IMP families).
2. Gram-negative bacteria that produce acquired carbapenemases are often resistant to
most, if not all, β-lactams. In addition, they are resistant to other antibiotic classes
(such as aminoglycosides, tetracyclines, and fluoroquinolones) because of the concerted
movement of mobile resistance elements.
3. Environmental β-lactamases are a source of some of the newer β-lactamases that are
appearing in both commensal and nosocomial isolates.
4. Many organisms now produce multiple β-lactamases from different functional groups,
making it more difficult to synthesize new β-lactams to counteract the evolving challenges
to these agents.
5. Our ability to interpret structural and mechanistic experiments on these β-lactamases
in terms of resistance development is primitive. Nonetheless, as we better understand
the genetic development of the β-lactamases, i.e., where are they coming from and what
next may they present us with, we secure better guidance for the empirical development
of β-lactamase-resistant structures.
FUTURE ISSUES
1. As new β-lactamase variants threaten our antibacterial armamentarium, will medicinal
chemistry be able to provide effective β-lactamase-stable antibiotics or β-lactamase inac-
tivators? In particular, what are the capabilities of the new classes of serine β-lactamase
inactivators? Can synergistic fusion between natural product MBL inhibitors and the
current exploratory MBL inhibitor structures be achieved?
470 Bush · Fisher

2. Will further understanding of environmental sources for new β-lactamase provide

prospective guidance for new antibacterial development?
3. As multiple β-lactamases are increasingly found in clinical isolates, can we combine
improved bedside diagnostic techniques with molecular and microbiological studies to
guide clinical therapy? Will we be able to correlate the β-lactamase-encoding genes in
these strains with strain fitness, perhaps leading to a better understanding of mutations
that might be selected in environmental, as well as nosocomial, isolates?
DISCLOSURE STATEMENT
K.B. receives retirement compensation from Bristol-Myers Squibb, Wyeth (Pfizer), and Johnson
& Johnson. She has served as a consultant or member of a Scientific Advisory Board for Ana-
cor Pharmaceuticals, Inc., Basilea Pharmaceutica Ltd., Cempra Pharmaceuticals, Cubist Phar-
maceuticals, Inc., Medivir, Merck, Novexel, Protez Pharmaceuticals, Inc., and Shionogi & Co.,
Ltd.
LITERATURE CITED
1. Abraham EP, Chain E. 1940. An enyzme from bacteria able to destroy penicillin. Nature 146:837
2. Ambler RP. 1980. The structure of β-lactamases. Philos. Trans. R. Soc. Lond. Biol. Sci. 289:321–31
3. Barlow M, Fatollahi J, Salverda M. 2009. Evidence for recombination among the alleles encoding TEM
and SHV β-lactamases. J. Antimicrob. Chemother. 63:256–59
4. Baurin S, Vercheval L, Bouillenne F, Falzone C, Brans A, et al. 2009. Critical role of tryptophan 154 for
the activity and stability of class D β-lactamases. Biochemistry 48:11252–63
5. Bebrone C, Lassaux P, Vercheval L, Sohier JS, Jehaes A, et al. 2010. Current challenges in antimicrobial
chemotherapy: focus on β-lactamase inhibition. Drugs 70:651–79
6. Bogaerts P, Verroken A, Jans B, Denis O, Glupczynski Y. 2010. Global spread of New Delhi metallo-
β-lactamase 1. Lancet Infect. Dis. 10:831–32
7. Bonnet R. 2004. Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob.
Agents Chemother. 48:1–14
8. Bonnin RA, Nordmann P, Potron A, Lecuyer H, Zahar JR, Poirel L. 2011. Carbapenem-hydrolyzing
GES-type extended-spectrum β-lactamase in Acinetobacter baumannii. Antimicrob. Agents Chemother.
55:349–54
9. Bos F, Pleiss J. 2008. Conserved water molecules stabilize the ω-loop in class A β-lactamases. Antimicrob.
10. Breece RM, Hu Z, Bennett B, Crowder MW, Tierney DL. 2009. Motion of the zinc ions in catalysis by
a dizinc metallo-β-lactamase. J. Am. Chem. Soc. 131:11642–43
11. Bush K. 2010. Alarming β-lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae. Curr.
Opin. Microbiol. 13:558–64
12. Bush K, Jacoby GA. 2010. Updated functional classification of β-lactamases. Antimicrob. Agents
Chemother. 54:969–76
13. Bush K, Jacoby GA, Amicosante G, Bonomo RA, Bradford P, et al. 2009. Comment on: redefining
extended-spectrum β-lactamases: balancing science and clinical need. J. Antimicrob. Chemother. 64:212–
13
14. Bush K, Jacoby GA, Medeiros AA. 1995. A functional classification scheme for β-lactamases and its
correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211–33
15. Castanheira M, Deshpande LM, Mendes RE, Rodriguez-Noriega E, Jones RN, Morfin-Otero R. 2011.
Comment on: role of changes in the L3 loop of the active site in the evolution of enzymatic activity of
VIM-type metallo-β-lactamases. J. Antimicrob. Chemother. 66:684–85

16. Castanheira M, Deshpande LM, Mathai D, Bell JM, Jones RN, Mendes RE. 2011. Early dissemination
of NDM-1- and OXA-181-producing Enterobacteriaceae in Indian hospitals: report from the SENTRY
Antimicrobial Surveillance Program, 2006–2007. Antimicrob. Agents Chemother. 55:1274–78
17. Centers for Disease Control and Prevention. 2010. Detection of Enterobacteriaceae isolates carrying
metallo-β-lactamase – United States, 2010. MMWR Morb. Mortal. Wkly. Rep. 59:750
18. Celenza G, Luzi C, Aschi M, Segatore B, Setacci D, et al. 2008. Natural D240G Toho-1 mutant con-
ferring resistance to ceftazidime: biochemical characterization of CTX-M-43. J. Antimicrob. Chemother.
62:991–97
19. Cha J, Mobashery S. 2007. Lysine Nζ -decarboxylation in the BlaR1 protein from Staphylococcus aureus
at the root of its function as an antibiotic sensor. J. Am. Chem. Soc. 129:3834–35
20. Chen Y, Bonnet R, Shoichet BK. 2007. The acylation mechanism of CTX-M β-lactamase at 0.88 Å
resolution. J. Am. Chem. Soc. 129:5378–80
21. Chen Y, Minasov G, Roth TA, Prati F, Shoichet BK. 2006. The deacylation mechanism of AmpC
β-lactamase at ultrahigh resolution. J. Am. Chem. Soc. 128:2970–76
22. Crowder MW, Spencer J, Vila AJ. 2006. Metallo-β-lactamases: novel weaponry for antibiotic resistance
in bacteria. Acc. Chem. Res. 39:721–28
23. Delmas J, Chen Y, Prati F, Robin F, Shoichet BK, Bonnet R. 2008. Structure and dynamics of CTX-M
enzymes reveal insights into substrate accommodation by ESBLs. J. Mol. Biol. 375:192–201
24. Delmas J, Leyssene D, Dubois D, Birck C, Vazeille E, et al. 2010. Structural insights into substrate
recognition and product expulsion in CTX-M enzymes. J. Mol. Biol. 400:108–20
25. Docquier JD, Benvenuti M, Calderone V, Giuliani F, Kapetis D, et al. 2010. Crystal structure of the
narrow-spectrum OXA-46 class D β-lactamase: relationship between active-site lysine carbamylation
and inhibition by polycarboxylates. Antimicrob. Agents Chemother. 54:2167–74
26. Drawz SM, Bethel CR, Doppalapudi VR, Sheri A, Pagadala SR, et al. 2010. Penicillin sulfone inhibitors
of class D β-lactamases. Antimicrob. Agents Chemother. 54:1414–24
27. Drawz SM, Bonomo RA. 2010. Three decades of β-lactamase inhibitors. Clin. Microbiol. Rev. 23:160–201
28. Endimiani A, Doi Y, Bethel CR, Taracila M, Adams-Haduch JM, et al. 2010. Enhancing resistance to
cephalosporins in class C β-lactamases: impact of Gly214Glu in CMY-2. Biochemistry 49:1014–23
29. Endimiani A, Hujer KM, Hujer AM, Pulse ME, Weiss WJ, Bonomo RA. 2011. Evaluation of ceftazidime
and NXL104 in two murine models of infection due to KPC-producing Klebsiella pneumoniae. Antimicrob.
30. Frase H, Shi Q, Testero SA, Mobashery S, Vakulenko SB. 2009. Mechanistic basis for the emergence of
catalytic competence against carbapenem antibiotics by the GES family of β-lactamases. J. Biol. Chem.
284:29509–13
31. Frase H, Toth M, Champion MM, Antunes NT, Vakulenko SB. 2011. Importance of position 170 in
the inhibition of GES-type β-lactamases by clavulanic acid. Antimicrob. Agents Chemother. 55:1556–62
32. Giakoupi P, Maltezou H, Polemis M, Pappa O, Saroglou G, et al. 2009. KPC-2-producing Klebsiella
pneumoniae infections in Greek hospitals are mainly due to a hyperepidemic clone. Eurosurveillance 14(21)
33. Girlich D, Poirel L, Nordmann P. 2010. PER-6, an extended-spectrum β-lactamase from Aeromonas
allosaccharophila. Antimicrob. Agents Chemother. 54:1619–22
34. Girlich D, Poirel L, Nordmann P. 2010. First isolation of the blaOXA-23 carbapenemase gene from an
environmental Acinetobacter baumannii isolate. Antimicrob. Agents Chemother. 54:578–79
35. Girlich D, Poirel L, Nordmann P. 2011. Diversity of clavulanic acid-inhibited extended-spectrum β-
lactamases in Aeromonas sp. from the Seine River, Paris, France. Antimicrob. Agents Chemother. 55:1256–61
36. Giske CG, Sundsfjord AS, Kahlmeter G, Woodford N, Nordmann P, et al. 2009. Redefining extended-
spectrum β-lactamases: balancing science and clinical need. J. Antimicrob. Chemother. 63:1–4
37. Gniadkowski M. 2008. Evolution of extended-spectrum β-lactamases by mutation. Clin. Microbiol. Infect.
14(Suppl. 1):11–32
38. Gottig S, Pfeifer Y, Wichelhaus TA, Zacharowski K, Bingold T, et al. 2010. Global spread of New Delhi
metallo-β-lactamase 1. Lancet Infect. Dis. 10:828–29
39. Hammerum AM, Toleman MA, Hansen F, Kristensen B, Lester CH, et al. 2010. Global spread of New
Delhi metallo-β-lactamase 1. Lancet Infect. Dis. 10:829–30
472 Bush · Fisher

40. Han S, Zaniewski RP, Marr ES, Lacey BM, Tomaras AP, et al. 2010. Structural basis for effectiveness of
siderophore-conjugated monocarbams against clinically relevant strains of Pseudomonas aeruginosa. Proc.
Natl. Acad. Sci. USA 107:22002–7
41. Hawkey PM, Jones AM. 2009. The changing epidemiology of resistance. J. Antimicrob. Chemother.
64(Suppl. 1):i3–10
42. Higgins PG, Schneiders T, Hamprecht A, Seifert H. 2010. In vivo selection of a missense mutation in
adeR and conversion of the novel blaOXA-164 gene into blaOXA-58 in carbapenem-resistant Acinetobacter
baumannii isolates from a hospitalized patient. Antimicrob. Agents Chemother. 54:5021–27
43. Hirsch EB, Tam VH. 2010. Detection and treatment options for Klebsiella pneumoniae carbapenemases
(KPCs): an emerging cause of multidrug-resistant infection. J. Antimicrob. Chemother. 65:1119–25
44. Hu Z, Periyannan G, Bennett B, Crowder MW. 2008. Role of the Zn1 and Zn2 sites in metallo-β-
lactamase L1. J. Am. Chem. Soc. 130:14207–16
45. Huband MD, Gootz TD, Flanagan ME, Quinn JP, Mullins LM, et al. 2010. In vitro antibacterial
activity of MC-1: a new siderophore-monocarbam conjugate versus recent Gram-negative bacterial
clinical isolates. 50th Intersci. Conf. Antibacterial Agents Chemother. (Boston, MA). Abstract F-2129
46. Jacoby G, Bush K. 2010. β-lactamase classification and amino acid sequences for TEM, SHV and OXA
extended-spectrum and inhibitor resistant enzymes. http://www.lahey.org/Studies/

47. Jacoby GA. 2009. AmpC β-lactamases. Clin. Microbiol. Rev. 22:161–82
48. Jacoby GA. 2006. β-lactamase nomenclature. Antimicrob. Agents Chemother. 50:1123–29
49. Johnson JW, Gretes M, Goodfellow VJ, Marrone L, Heynen ML, et al. 2010. Cyclobutanone analogues
of β-lactams revisited: insights into conformational requirements for inhibition of serine- and metallo-
β-lactamases. J. Am. Chem. Soc. 132:2558–60
50. Kallman O, Giske CG, Samuelsen O, Wretlind B, Kalin M, Olsson-Liljequist B. 2009. Interplay of
efflux, impermeability, and AmpC activity contributes to cefuroxime resistance in clinical, non-ESBL-
producing isolates of Escherichia coli. Microbial Drug Resist. 15:91–95
51. Karthikeyan K, Thirunarayan MA, Krishnan P. 2010. Coexistence of blaOXA-23 with blaNDM-1 and
armA in clinical isolates of Acinetobacter baumannii from India. J. Antimicrob. Chemother. 65:2253–54
52. Ke W, Bethel CR, Thomson JM, Bonomo RA, van den Akker F. 2007. Crystal structure of KPC-2:
insights into carbapenemase activity in class A β-lactamases. Biochemistry 46:5732–40
53. Khersonsky O, Tawfik DS. 2010. Enzyme promiscuity: a mechanistic and evolutionary perspective.
Annu. Rev. Biochem. 79:471–505
54. Kitchel B, Rasheed JK, Endimiani A, Hujer AM, Anderson KF, et al. 2010. Genetic factors associated with
elevated carbapenem resistance in KPC-producing Klebsiella pneumoniae. Antimicrob. Agents Chemother.
54:4201–7
55. Koh TH, Khoo CT, Wijaya L, Leong HN, Lo YL, et al. 2010. Global spread of New Delhi metallo-β-
lactamase 1. Lancet Infect. Dis. 10:828
56. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, et al. 2010. Emergence of a new antibiotic
resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study.
Lancet Infect. Dis. 10:597–602
57. Lassaux P, Traoré DA, Loisel E, Favier A, Docquier JD, et al. 2011. Biochemical and structural charac-
terization of the subclass B1 metallo-β-lactamase VIM-4. Antimicrob. Agents Chemother. 55:1248–55
58. Lauretti L, Riccio ML, Mazzariol A, Cornaglia G, Amicosante G, et al. 1999. Cloning and characteriza-
tion of blaVIM, a new integron-borne metallo-β-lactamase gene from a Pseudomonas aeruginosa clinical
isolate. Antimicrob. Agents Chemother. 43:1584–90
59. Le Turnier S, Nordmann P, Eb F, Mammeri H. 2009. Potential evolution of hydrolysis spectrum for
AmpC β-lactamase of Escherichia coli. J. Antimicrob. Chemother. 63(1):216–18
60. Lee JH, Bae IK, Hee Lee S. 2011. New definitions of extended-spectrum β-lactamase conferring world-
wide emerging antibiotic resistance. Med. Res. Rev. doi:10.1002/med.20210
61. Lee K, Yum JH, Yong D, Lee HM, Kim HD, et al. 2005. Novel acquired metallo-β-lactamase gene,
bla(SIM-1), in a class 1 integron from Acinetobacter baumannii clinical isolates from Korea. Antimicrob.

62. Leonard DA, Hujer AM, Smith BA, Schneider KD, Bethel CR, et al. 2008. The role of OXA-1 β-
lactamase Asp(66) in the stabilization of the active-site carbamate group and in substrate turnover.
Biochem. J. 410:455–62
63. Leverstein-Van Hall MA, Stuart JC, Voets GM, Versteeg D, Tersmette T, Fluit AC. 2010. Global
spread of New Delhi metallo-β-lactamase 1. Lancet Infect. Dis. 10:830–31
64. Li D, Yang M, Hu J, Zhang J, Liu R, et al. 2009. Antibiotic-resistance profile in environmental bacteria
isolated from penicillin production wastewater treatment plant and the receiving river. Microb. Ecol.
11:1506–17
65. Livermore DM. 2008. Defining an extended-spectrum β-lactamase. Clin. Microbiol. Infect. 14(Suppl.
1):3–10
66. Livermore DM. 2009. Has the era of untreatable infections arrived? J. Antimicrob. Chemother. 64(Suppl.
1):i29–36
67. Livermore DM, Mushtaq S, Warner M. 2010. Activity of BAL30376 (monobactam BAL19764 +
BAL29880 + clavulanate) versus gram-negative bacteria with characterized resistance mechanisms.
J. Antimicrob. Chemother. 65:2382–95
68. Livermore DM, Mushtaq S, Warner M, Miossec C, Woodford N. 2008. NXL104 combinations versus
Enterobacteriaceae with CTX-M extended-spectrum β-lactamases and carbapenemases. J. Antimicrob.
69. Livermore DM, Mushtaq S, Warner M, Zhang J, Maharjan S, et al. 2011. Activities of NXL104 combi-
nations with ceftazidime and aztreonam against carbapenemase-producing Enterobacteriaceae. Antimicrob.
70. Maltezou HC. 2009. Metallo-β-lactamases in gram-negative bacteria: introducing the era of pan-
resistance? Int. J. Antimicrob. Agents 33:405.e1–7
71. Mammeri H, Guillon H, Eb F, Nordmann P. 2010. Phenotypic and biochemical comparison of
the carbapenem-hydrolyzing activities of five plasmid-borne AmpC β-lactamases. Antimicrob. Agents
72. Merino M, Perez-Llarena FJ, Kerff F, Poza M, Mallo S, et al. 2010. Role of changes in the L3 loop of
the active site in the evolution of enzymatic activity of VIM-type metallo-β-lactamases. J. Antimicrob.
73. Mir-Montazeri B, Ammelburg M, Forouzan D, Lupas AN, Hartmann MD. 2011. Crystal structure of a
dimeric archaeal cleavage and polyadenylation specificity factor. J. Struct. Biol. 173:191–95
74. Miriagou V, Cornaglia G, Edelstein M, Galani I, Giske CG, et al. 2010. Acquired carbapenemases in
gram-negative bacterial pathogens: detection and surveillance issues. Clin. Microbiol. Infect. 16:112–22
75. Moellering RCJ. 2010. NDM-1—a cause for worldwide concern. N. Engl. J. Med. 363:2377–79
76. Morar M, Wright GD. 2010. The genomic enzymology of antibiotic resistance. Annu. Rev. Genet.
44:25–51
77. Munier GK, Johnson CL, Snyder JW, Moland ES, Hanson ND, Thomson KS. 2010. Positive extended-
spectrum-β-lactamase (ESBL) screening results may be due to AmpC β-lactamases more often than to
ESBLs. J. Clin. Microbiol. 48:673–74
78. Murano K, Yamanaka T, Toda A, Ohki H, Okuda S, et al. 2008. Structural requirements for the stability
of novel cephalosporins to AmpC β-lactamase based on 3D-structure. Bioorg. Med. Chem. 16:2261–75
79. Mushtaq S, Warner M, Livermore D. 2010. Activity of the siderophore monobactam BAL30072 against
multiresistant non-fermenters. J. Antimicrob. Chemother. 65:266–70
80. Mylotte JM. 2006. Nursing home-acquired pneumonia: update on treatment options. Drugs Aging
23:377–90
81. Nitanai Y, Shimamura T, Uchiyama T, Ishii Y, Takehira M, et al. 2010. The catalytic efficiency (kcat /Km )
of the class A β-lactamase Toho-1 correlates with the thermal stability of its catalytic intermediate analog.
Biochim. Biophys. Acta 1804:684–91
82. Nordmann P, Poirel L, Carrër A, Toleman MA, Walsh TR. 2011. How to detect NDM-1 producers.
J. Clin. Microbiol. 49:718–21
83. Nordmann P. 2010. Gram-negative bacteriae with resistance to carbapenems. Med. Sci. 26:950–59
84. Nordmann P, Cuzon G, Naas T. 2009. The real threat of Klebsiella pneumoniae carbapenemase-producing
bacteria. Lancet Infect. Dis. 9:228–36
474 Bush · Fisher

85. Nottingham M, Bethel CR, Pagadala SR, Harry E, Pinto A, et al. 2011. Modifications of the C6-
substituent of penicillin sulfones with the goal of improving inhibitor recognition and efficacy. Bioorg.
Med. Chem. Lett. 21:387–93
86. Novais A, Comas I, Baquero F, Canton R, Coque TM, et al. 2010. Evolutionary trajectories of β-
lactamase CTX-M-1 cluster enzymes: predicting antibiotic resistance. PLoS Pathog. 6:e1000735
87. Oelschlaeger P. 2008. Outsmarting metallo-β-lactamases by mimicking their natural evolution. J. Inorg.
Biochem. 102:2043–51
88. Oelschlaeger P, Ai N, Duprez KT, Welsh WJ, Toney JH. 2010. Evolving carbapenemases: Can medicinal
chemists advance one step ahead of the coming storm? J. Med. Chem. 53:3013–27
89. Page MGP, Heim J. 2009. New molecules from old classes: revisiting the development of beta-lactams.
IDrugs 12:561–65
90. Page MG, Dantier C, Desarbre E. 2010. In vitro properties of BAL30072, a novel siderophore sulfactam
with activity against multiresistant gram-negative bacilli. Antimicrob. Agents Chemother. 54:2291–302
91. Page MI, Badarau A. 2008. The mechanisms of catalysis by metallo β-lactamases. Bioinorg. Chem. Appl.
576297
92. Papp-Wallace KM, Bethel CR, Distler AM, Kasuboski C, Taracila M, Bonomo RA. 2010. Inhibitor
resistance in the KPC-2 β-lactamase, a preeminent property of this class A β-lactamase. Antimicrob.

93. Park A. 2010. Antibiotics. NDM-1 How dangerous is the mutation? Time, Oct. 4, 176:20
94. Park YS, Lee H, Lee KS, Hwang SS, Cho YK, et al. 2010. Extensively drug-resistant Acinetobacter
baumannii: risk factors for acquisition and prevalent OXA-type carbapenemases—a multicentre study.
Int. J. Antimicrob. Agents 36:430–35
95. Peirano G, Pitout JD. 2010. Molecular epidemiology of Escherichia coli producing CTX-M β-lactamases:
the worldwide emergence of clone ST131 O25:H4. Int. J. Antimicrob. Agents 35:316–21
96. Pellegrini C, Mercuri PS, Celenza G, Galleni M, Segatore B, et al. 2009. Identification of bla(IMP-22)
in Pseudomonas spp. in urban wastewater and nosocomial environments: biochemical characterization of
a new IMP metallo-enzyme variant and its genetic location. J. Antimicrob. Chemother. 63:901–8
97. Perez-Moreno MO, Perez-Moreno M, Carulla M, Rubio C, Jardi AM, Zaragoza J. 2004. Mechanisms
of reduced susceptibility to amoxycillin-clavulanic acid in Escherichia coli strains from the health region
of Tortosa (Catalonia, Spain). Clin. Microbiol. Infect. 10:234–41
98. Petersen PJ, Jones CH, Venkatesan AM, Bradford PA. 2009. Efficacy of piperacillin combined with
the penem β-lactamase inhibitor BLI-489 in murine models of systemic infection. Antimicrob. Agents
99. Pfeifer Y, Witte W, Holfelder M, Busch J, Nordmann P, Poirel L. 2011. NDM-1-producing Escherichia
coli in Germany. Antimicrob. Agents Chemother. 55:1318–19
100. Poirel L, Lagrutta E, Taylor P, Pham J, Nordmann P. 2010. Emergence of metallo-β-lactamase NDM-
1-producing multidrug-resistant Escherichia coli in Australia. Antimicrob. Agents Chemother. 54:4914–16
101. Pontes DS, Pinheiro FA, Lima-Bittencourt CI, Guedes RLM, Cursino L, et al. 2009. Multiple antimi-
crobial resistance of gram-negative bacteria from natural oligotrophic lakes under distinct anthropogenic
influence in a tropical region. Microb. Ecol. 58:762–72
102. Pournaras S, Poulou A, Voulgari E, Vrioni G, Kristo I, Tsakris A. 2010. Detection of the new metallo-
β-lactamase VIM-19 along with KPC-2, CMY-2 and CTX-M-15 in Klebsiella pneumoniae. J. Antimicrob.
103. Queenan AM, Shang W, Flamm R, Bush K. 2010. Hydrolysis and inhibition profiles of β-lactamases
from molecular classes A to D with doripenem, imipenem, and meropenem. Antimicrob. Agents Chemother.
54:565–69
104. Richmond MH, Sykes RB. 1973. The β-lactamases of gram-negative bacteria and their possible physi-
ological role. Adv. Microb. Physiol. 9:31–88
105. Rodriguez-Martinez JM, Nordmann P, Ronco E, Poirel L. 2010. Extended-spectrum cephalosporinase
in Acinetobacter baumannii. Antimicrob. Agents Chemother. 54:3484–88
106. Rodriguez-Martinez JM, Poirel L, Nordmann P. 2010. Genetic and functional variability of AmpC-type
β-lactamases from Acinetobacter baumannii. Antimicrob. Agents Chemother. 54:4930–33

107. Ruzin A, Petersen PJ, Jones CH. 2010. Resistance development profiling of piperacillin in combination
with the novel β-lactamase inhibitor BLI-489. J. Antimicrob. Chemother. 65:252–57
108. Salabi AE, Toleman MA, Weeks J, Bruderer T, Frei R, Walsh TR. 2010. First report of the metallo-β-
lactamase SPM-1 in Europe. Antimicrob. Agents Chemother. 54:582
109. Salverda MLM, de Visser JAG, Barlow M. 2010. Natural evolution of TEM-1 β-lactamase: experimental
reconstruction and clinical relevance. FEMS Microbiol. Rev. 34:1015–36
110. Schneider KD, Bethel CR, Distler AM, Hujer AM, Bonomo RA, Leonard DA. 2009. Mutation of
the active site carboxy-lysine (K70) of OXA-1 β-lactamase results in a deacylation-deficient enzyme.
Biochemistry 48:6136–45
111. Schneider KD, Karpen ME, Bonomo RA, Leonard DA, Powers RA. 2009. The 1.4 Å crystal structure
of the class D β-lactamase OXA-1 complexed with doripenem. Biochemistry 48:11840–47
112. Schneider KD, Ortega CJ, Renck NA, Bonomo RA, Powers RA, Leonard DA. 2011. Structures of the
class D carbapenemase OXA-24 from Acinetobacter baumannii in complex with doripenem. J. Mol. Biol.
406:583–94
113. Selevsek N, Rival S, Tholey A, Heinzle E, Heinz U, et al. 2009. Zinc ion-induced domain organization
in metallo-β-lactamases: a flexible “zinc arm” for rapid metal ion transfer? J. Biol. Chem. 284:16419–31
114. Shahid M, Sobia F, Singh A, Malik A, Khan HM, et al. 2009. β-Lactams and β-lactamase-inhibitors in
current- or potential-clinical practice: a comprehensive update. Crit. Rev. Microbiol. 35:81–108
115. Shimamura T, Ibuka A, Fushinobu S, Wakagi T, Ishiguro M, et al. 2002. Acyl-intermediate structures
of the extended-spectrum class A β-lactamase, Toho-1, in complex with cefotaxime, cephalothin, and
benzylpenicillin. J. Biol. Chem. 277:46601–8
116. Shimizu-Ibuka A, Oishi M, Yamada S, Ishii Y, Mura K, et al. 2011. Roles of residues Cys69, Asn104,
Phe160, Gly232, Ser237, and Asp240 in extended-spectrum β-lactamase Toho-1. Antimicrob. Agents
117. Sidjabat HE, Paterson DL, Qureshi ZA, Adams-Haduch JM, O’Keefe A, et al. 2009. Clinical features and
molecular epidemiology of CMY-type β-lactamase-producing Escherichia coli. Clin. Infect. Dis. 48:739–44
118. Simona F, Magistrato A, Dal Peraro M, Cavalli A, Vila AJ, Carloni P. 2009. Common mechanistic
features among metallo-β-lactamases: a computational study of Aeromonas hydrophila CphA enzyme.
J. Biol. Chem. 284:28164–71
119. Smith CA, Caccamo M, Kantardjieff KA, Vakulenko S. 2007. Structure of GES-1 at atomic resolution:
insights into the evolution of carbapenamase activity in the class A extended-spectrum β-lactamases.
Acta Crystallogr. D 63:982–92
120. Souli M, Galani I, Antoniadou A, Papadomichelakis E, Poulakou G, et al. 2010. An outbreak of infection
due to β-lactamase Klebsiella pneumoniae carbapenemase 2-producing K. pneumoniae in a Greek university
hospital: molecular characterization, epidemiology, and outcomes. Clin. Infect. Dis. 50:364–73
121. Stachyra T, Levasseur P, Pechereau MC, Girard AM, Claudon M, et al. 2009. In vitro activity of the
β-lactamase inhibitor NXL104 against KPC-2 carbapenemase and Enterobacteriaceae expressing KPC
carbapenemases. J. Antimicrob. Chemother. 64:326–29
122. Stachyra T, Pechereau MC, Bruneau JM, Claudon M, Frere JM, et al. 2010. Mechanistic studies of the
inactivation of TEM-1 and P99 by NXL104, a novel non-β-lactam β-lactamase inhibitor. Antimicrob.
123. Swarén P, Maveyraud L, Raquet X, Cabantous S, Duez C, et al. 1998. X-ray analysis of the NMC-A
β-lactamase at 1.64 Å resolution, a class A carbapenemase with broad substrate specificity. J. Biol. Chem.
273:26714–21
124. Thai QK, Bos F, Pleiss J. 2009. The lactamase engineering database: a critical survey of TEM sequences
in public databases. BMC Genomics 10:390
125. Toleman MA, Simm AM, Murphy TA, Gales AC, Biedenbach DJ, et al. 2002. Molecular character-
ization of SPM-1, a novel metallo-β-lactamase isolated in Latin America: report from the SENTRY
antimicrobial surveillance programme. J. Antimicrob. Chemother. 50:673–79
126. Tomanicek SJ, Blakeley MP, Cooper J, Chen Y, Afonine PV, Coates L. 2010. Neutron diffraction studies
of a class A β-lactamase Toho-1 E166A/R274N/R276N triple mutant. J. Mol. Biol. 396:1070–80
476 Bush · Fisher

127. Tomanicek SJ, Wang KK, Weiss KL, Blakeley MP, Cooper J, et al. 2011. The active site protonation
states of perdeuterated Toho-1 β-lactamase determined by neutron diffraction support a role for Glu166
as the general base in acylation. FEBS Lett. 585:364–68
128. Tomatis PE, Fabiane SM, Simona F, Carloni P, Sutton BJ, Vila AJ. 2008. Adaptive protein evolution
grants organismal fitness by improving catalysis and flexibility. Proc. Natl. Acad. Sci. USA 105:20605–10
129. Toth M, Smith C, Frase H, Mobashery S, Vakulenko S. 2010. An antibiotic-resistance enzyme from a
deep-sea bacterium. J. Am. Chem. Soc. 132:816–23
130. Toth M, Vakulenko SB, Smith CA. 2009. Purification, crystallization and preliminary X-ray analysis of
the β-lactamase Oih-1 from Oceanobacillus iheyensis. Acta Crystallogr. F 65:582–85
131. Totir MA, Cha J, Ishiwata A, Wang B, Sheri A, et al. 2008. Why clinically used tazobactam and sulbactam
are poor inhibitors of OXA-10 β-lactamase: Raman crystallographic evidence. Biochemistry 47:4094–101
132. Tristram S, Jacobs MR, Appelbaum PC. 2007. Antimicrobial resistance in Haemophilus influenzae. Clin.
Microbiol. Rev. 20:368–89
133. Tsakris A, Voulgari E, Poulou A, Kimouli M, Pournaras S, et al. 2010. In vivo acquisition of a plasmid-
mediated bla(KPC-2) gene among clonal isolates of Serratia marcescens. J. Clin. Microbiol. 48:2546–49
134. Tsering DC, Das S, Adhiakari L, Pal R, Singh TS. 2009. Extended spectrum β-lactamase detection in
gram-negative bacilli of nosocomial origin. J. Glob. Infect. Dis. 1:87–92

135. Urbach C, Evrard C, Pudzaitis V, Fastrez J, Soumillion P, Declercq JP. 2009. Structure of PBP-A from
Thermosynechococcus elongatus, a penicillin-binding protein closely related to class A β-lactamases. J. Mol.
Biol. 386:109–20
136. Urban C, Bradford PA, Tuckman M, Segal-Maurer S, Wehbeh W, et al. 2008. Carbapenem-resistant
Escherichia coli harboring Klebsiella pneumoniae carbapenemase β-lactamases associated with long-term
care facilities. Clin. Infect. Dis. 46:e127–30
137. Venkatesan AM, Agarwal A, Abe T, Ushirogochi H, Ado M, et al. 2008. 5,5,6-fused tricycles bearing
imidazole and pyrazole 6-methylidene penems as broad-spectrum inhibitors of β-lactamases. Bioorg.
Med. Chem. 16:1890–902
138. Vercheval L, Bauvois C, di Paolo A, Borel F, Ferrer JL, et al. 2010. Three factors that modulate the
activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys70. Biochem.
J. 432:495–504
139. Walsh TR. 2010. Emerging carbapenemases: a global perspective. Int. J. Antimicrob. Agents 36(Suppl.
3):S8–14
140. Walsh TR, Weeks J, Livermore DM, Toleman MA. 2011. Dissemination of NDM-1 positive bacteria in
the New Delhi environment and its implications for human health: an environmental point prevalence
study. Lancet Infect. Dis. 11:355–62
141. Walther-Rasmussen J, Hoiby N. 2006. OXA-type carbapenemases. J. Antimicrob. Chemother. 57:373–83
142. Walther-Rasmussen J, Hoiby N. 2007. Class A carbapenemases. J. Antimicrob. Chemother. 60:470–82
143. Whiley DM, Goire N, Lambert SB, Ray S, Limnios EA, et al. 2010. Reduced susceptibility to ceftriaxone
in Neisseria gonorrhoeae is associated with mutations G542S, P551S and P551L in the gonococcal PBP 2.
J. Antimicrob. Chemother. 65:1615–18
144. Winokur PL, Vonstein DL, Hoffman LJ, Uhlenhopp EK, Doern GV. 2001. Evidence for transfer of
CMY-2 AmpC β-lactamase plasmids between Escherichia coli and Salmonella isolates from food animals
and humans. Antimicrob. Agents Chemother. 45:2716–22
145. Woodford N. 2010. Rapid characterization of β-lactamases by multiplex PCR. Methods Mol. Biol.
642:181–92
146. Wu HS, Chen TL, Chen IC, Huang MS, Wang FD, et al. 2010. First identification of a patient colonized
with Klebsiella pneumoniae carrying blaNDM-1 in Taiwan. J. Chin. Med. Assoc. 73:596–98
147. Wu S, Xu D, Guo H. 2010. QM/MM studies of monozinc β-lactamase CphA suggest that the crystal
structure of an enzyme-intermediate complex represents a minor pathway. J. Am. Chem. Soc. 132:17986–
88
148. Yamamura A, Okada A, Kameda Y, Ohtsuka J, Nakagawa N, et al. 2009. Structure of TTHA1623, a novel
metallo-β-lactamase superfamily protein from Thermus thermophilus HB8. Acta Crystallogr. F 65:455–59

149. Yigit H, Queenan AM, Rasheed JK, Biddle JW, Domenech-Sanchez A, et al. 2003. Carbapenem-resistant
strain of Klebsiella oxytoca harboring carbapenem-hydrolyzing β-lactamase KPC-2. Antimicrob. Agents
150. Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, Walsh TR. 2009. Characterization of a
new metallo-β-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique
genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob. Agents Chemother.
53:5046–54
478 Bush · Fisher

MI65-Frontmatter ARI 11 August 2011 7:34
Annual Review of
Microbiology
Volume 65, 2011 Contents
To the Happy Few

Hiroshi Nikaido p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Regulation of DnaA Assembly and Activity: Taking Directions from
the Genome
Alan C. Leonard and Julia E. Grimwade p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p19
Regulation of Alternative Sigma Factor Use

Sofia Österberg, Teresa del Peso-Santos, and Victoria Shingler p p p p p p p p p p p p p p p p p p p p p p p p p p p p37
Fungal Protein Production: Design and Production
of Chimeric Proteins
Peter J. Punt, Anthony Levasseur, Hans Visser, Jan Wery, and Eric Record p p p p p p p p p p p p p57
Structure and Function of MARTX Toxins and Other Large
Repetitive RTX Proteins
Karla J.F. Satchell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p71
Eukaryotic Picoplankton in Surface Oceans
Ramon Massana p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p91
Life on the Outside: The Rescue of Coxiella burnetii from Its Host Cell
Anders Omsland and Robert A. Heinzen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 111
Molecular Mechanisms of Staphylococcus aureus Iron Acquisition
Neal D. Hammer and Eric P. Skaar p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 129
Protein Quality Control in the Bacterial Periplasm
Melisa Merdanovic, Tim Clausen, Markus Kaiser, Robert Huber,
and Michael Ehrmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 149
Prospects for the Future Using Genomics and Proteomics
in Clinical Microbiology
Pierre-Edouard Fournier and Didier Raoult p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 169
The RpoS-Mediated General Stress Response in Escherichia coli
Aurelia Battesti, Nadim Majdalani, and Susan Gottesman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 189
Bacterial Osmoregulation: A Paradigm for the Study
of Cellular Homeostasis
Janet M. Wood p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 215
vi
Lipoprotein Sorting in Bacteria

Suguru Okuda and Hajime Tokuda p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 239
Ligand-Binding PAS Domains in a Genomic, Cellular,
and Structural Context
Jonathan T. Henry and Sean Crosson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 261
How Viruses and Toxins Disassemble to Enter Host Cells
Takamasa Inoue, Paul Moore, and Billy Tsai p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 287
Turning Hepatitis C into a Real Virus
Catherine L. Murray and Charles M. Rice p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 307
Recombination and DNA Repair in Helicobacter pylori
Marion S. Dorer, Tate H. Sessler, and Nina R. Salama p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 329
Kin Discrimination and Cooperation in Microbes

Joan E. Strassmann, Owen M. Gilbert, and David C. Queller p p p p p p p p p p p p p p p p p p p p p p p p p p 349

Dinoflagellate Genome Evolution
Jennifer H. Wisecaver and Jeremiah D. Hackett p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 369
Motility and Chemotaxis in Campylobacter and Helicobacter
Paphavee Lertsethtakarn, Karen M. Ottemann, and David R. Hendrixson p p p p p p p p p p p p 389
The Human Gut Microbiome: Ecology and Recent
Evolutionary Changes
Jens Walter and Ruth Ley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 411
Approaches to Capturing and Designing Biologically Active Small
Molecules Produced by Uncultured Microbes
Jörn Piel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 431
Epidemiological Expansion, Structural Studies, and Clinical
Challenges of New β-Lactamases from Gram-Negative Bacteria
Karen Bush and Jed F. Fisher p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 455
Gene Regulation in Borrelia burgdorferi
D. Scott Samuels p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 479
Biology of Clostridium difficile: Implications for Epidemiology
and Diagnosis
Karen C. Carroll and John G. Bartlett p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 501
Interactions of the Human Pathogenic Brucella Species
with Their Hosts
Vidya L. Atluri, Mariana N. Xavier, Maarten F. de Jong,
Andreas B. den Hartigh, and Renée M. Tsolis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 523
Contents vii
Metabolic Pathways Required for the Intracellular Survival

of Leishmania
Malcolm J. McConville and Thomas Naderer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 543
Capsules of Streptococcus pneumoniae and Other Bacteria: Paradigms for
Polysaccharide Biosynthesis and Regulation
Janet Yother p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 563
Synthetic Poliovirus and Other Designer Viruses: What Have We
Learned from Them?
Eckard Wimmer and Aniko V. Paul p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 583
Regulation of Antigenic Variation in Giardia lamblia
César G. Prucca, Fernando D. Rivero, and Hugo D. Luján p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 611
Alternative Pathways of Carbon Dioxide Fixation: Insights into the

Early Evolution of Life?
Georg Fuchs p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 631
Index
Cumulative Index of Contributing Authors, Volumes 61–65 p p p p p p p p p p p p p p p p p p p p p p p p p p p 659
Errata
An online log of corrections to Annual Review of Microbiology articles may be found at

http://micro.annualreviews.org/
viii Contents

BLEE General Editado PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

BLEE General Editado PDF

Uploaded by

Copyright:

Available Formats

MI65CH23-Bush ARI 10 August 2011 10:13

• Our comprehensive search

Annu. Rev. Microbiol. 2011. 65:455–78 Keywords

The Annual Review of Microbiology is online at Abstract

ability to hydrolyze the β-lactam bond in vir-

resistance factors responsible for decreased

456 Bush · Fisher

Ticarcillin Cefotaxime Meropenem

Clinically used and exploratory β-lactamase inhibitors

contributes to resistance. Unfortunately, taken on the attributes of expanded-spectrum

www.annualreviews.org • New β-Lactamases 457

Table 1 Functional grouping of major β-lactamases aligned with molecular assignmentsa

2de D Hydrolysis of penicillins and expanded spectrum ESBLs: OXA-11, OXA-15

2df D Hydrolysis of carbapenems and cloxacillin or oxacillin OXA-23, OXA-48

2e A Efﬁcient hydrolysis of cephalosporins CepA

www.annualreviews.org • New β-Lactamases

Number of naturally occurring enzymes

200 Group 2/class D

460 Bush · Fisher

cephalosporins and monobactams with an oxy- spectrum cephalosporins and monobactams,

www.annualreviews.org • New β-Lactamases 461

documented in a recent study demonstrating biochemical characteristics resembling those

462 Bush · Fisher

www.annualreviews.org • New β-Lactamases 463

isolates of Pseudomonas ﬂuorescens produced a

464 Bush · Fisher

β-lactamases (97) or the appearance of Lys-234

of the catalytic labyrinth is made from compar- Asn-132 2H O

www.annualreviews.org • New β-Lactamases 465

466 Bush · Fisher

www.annualreviews.org • New β-Lactamases 467

(OXA-preﬁxed) β-lactamase expression abets Class B. Dramatic expansion in β-lactamase

468 Bush · Fisher

In contrast to the serine β-lactamases, known

tional group arrays that lead to nonhydrolytic

www.annualreviews.org • New β-Lactamases 469

communication?—the burgeoning of antibi- The expansion of the substrate capabilities of

470 Bush · Fisher

2. Will further understanding of environmental sources for new β-lactamase provide

www.annualreviews.org • New β-Lactamases 471

472 Bush · Fisher

extended-spectrum and inhibitor resistant enzymes. http://www.lahey.org/Studies/

www.annualreviews.org • New β-Lactamases 473

474 Bush · Fisher

Agents Chemother. 54:890–97

www.annualreviews.org • New β-Lactamases 475

476 Bush · Fisher

gram-negative bacilli of nosocomial origin. J. Glob. Infect. Dis. 1:87–92

www.annualreviews.org • New β-Lactamases 477

478 Bush · Fisher

Volume 65, 2011 Contents

To the Happy Few

Regulation of Alternative Sigma Factor Use

Lipoprotein Sorting in Bacteria

Kin Discrimination and Cooperation in Microbes

Joan E. Strassmann, Owen M. Gilbert, and David C. Queller p p p p p p p p p p p p p p p p p p p p p p p p p p 349

Metabolic Pathways Required for the Intracellular Survival

Alternative Pathways of Carbon Dioxide Fixation: Insights into the

Georg Fuchs p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 631

Cumulative Index of Contributing Authors, Volumes 61–65 p p p p p p p p p p p p p p p p p p p p p p p p p p p 659

An online log of corrections to Annual Review of Microbiology articles may be found at

You might also like