J Infect Chemother (2013) 19:549–559
DOI 10.1007/s10156-013-0640-7
REVIEW ARTICLE
The ABCD’s of b-lactamase nomenclature
Karen Bush
Received: 16 May 2013 / Published online: 5 July 2013
 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2013
Abstract b-Lactamases can be named on the basis of
molecular characteristics or functional properties. Molec-
ular classes A, B, C, and D define an enzyme according to
amino acid sequence and conserved motifs. Functional
groups 1, 2, and 3 are used to assign a clinically useful
description to a family of enzymes, with subgroups des-
ignated according to substrate and inhibitor profiles. In
addition, other designations are used to define the func-
tionality of specific subgroups, such as extended-spectrum
b-lactamases, or ESBLs, and inhibitor-resistant TEM, or
IRT, b-lactamases. None of these systems provides an
unambiguous description of this versatile set of enzymes. A
proposed classification system involving microbiological,
molecular, and biochemical properties is described, based
on the traditional classes A, B, C, and D and functional
groups 1, 2, and 3 designations.
Keywords b-Lactamase
ESBL
Carbapenemase

Nomenclature
Introduction
b-Lactamases, the enzymes that inactivate b-lactam anti-
biotics, serve as a major cause for resistance to this
exceedingly valuable set of antibacterial agents [1]. The
number of unique, naturally occurring b-lactamases now
exceeds 1,300, making this set of enzymes perhaps one of
the most numerous enzyme families that has been studied
K. Bush (&)
Department of Molecular and Cellular Biochemistry,
Indiana University Bloomington, Simon Hall 102B,
212 S. Hawthorne Dr., Bloomington, IN 47405, USA
e-mail: karbush@indiana.edu
in detail [2]. The clinical impact of these enzymes is
particularly critical because of the reliance on cephalo-
sporins and carbapenems for the treatment of serious
infections, especially in healthcare settings. Yet, it is
the use of antibiotics that has driven the evolution of
b-lactamase-producing pathogens to the point where mul-
tiple b-lactamase-encoding genes are produced in a single
organism, together with resistance determinants for many
other antimicrobial classes [3].
Although combinations of b-lactamases, together with
other resistance determinants that are co-transferred, can
lead to pan-resistance [3], the more likely case, at least for
now, is that there will be at least one agent, or combination
of agents, that may be useful for the treatment of a mul-
tidrug-resistant organism [4, 5]. This situation assumes that
clinical testing is done accurately and with sufficient
insight into characteristic phenotypic behavior as to predict
what kind of resistance factor(s) may be present. Thus, the
antibiogram should provide clues as to a resistance mech-
anism that may be contributing to the susceptibility profile
for that organism. In the process of this analysis, it is
important to think about b-lactamases in terms of func-
tional behavior that will allow clinicians to make informed
selections of appropriate antibiotics for therapy. Useful
information concerning transferability and enzymatic
properties would assist in these decisions. If a clinician
were told that a ceftazidime-resistant clinical isolate of
Proteus mirabilis produced a plasmid-encoded clavulanate-
sensitive b-lactamase rather than a chromosomal non-
inhibitable cephalosporinase [6], selection of a b-lactamase
inhibitor combination might be preferred as a therapeutic
option instead of a carbapenem. However, molecular
resistance profiling is more frequently accomplished by
polymerase chain reaction (PCR) analyses for suspected
bla genes, resulting in limited information about
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functionality for most b-lactamases. As is shown in this
review, a combination of molecular and functional
approaches may provide a stronger basis for describing
new b-lactamases and their role in clinical resistance.
Historical classification schemes
Functional classifications
Early research on b-lactamases focused on penicillinases
from gram-positive bacilli [7, 8], extracellular enzymes
that were studied in depth for their biochemical properties.
Other than occasional epidemiological reports, b-lacta-
mases were viewed as easily accessible enzymes for bio-
chemical analyses, whose clinical resistance played a
secondary, or nonexistent, role. As b-lactamases from
gram-negative bacteria were recognized more frequently in
the clinical setting, more intensive efforts were made to
understand the physiological role played by these enzymes.
Classification of b-lactamases into functional groups has
existed since at least 1968 when Sawai et al. [9] separated
sets of species-specific chromosomal b-lactamases from
gram-negative enteric bacteria into those enzymes with
cephalosporinase or penicillinase activities. This work was
followed by Richmond and Sykes [10], who published their
classical compilation of a large set of chromosomal and ‘‘R
factor-encoded’’ b-lactamases that were being recognized
as major contributors to penicillin and cephalosporin
resistance in gram-negative bacteria in 1973. At that time,
these enzymes were characterized primarily based on
functional and biochemical characterizations, as little
sequence information was known for b-lactamases from
gram-negative pathogens.
More recent functional classification schemes have been
based on the Richmond and Sykes strategy of using bio-
chemical substrate and inhibition profiles to define b-lac-
tamases according to enzymatic properties. Bush [11]
updated the Richmond and Sykes scheme in 1989, using
the limited biochemical and structural data that were
available for major b-lactamases described in the literature.
At that time, few enzymes were characterized with full
kinetic descriptions of hydrolysis profiles, so that the data
set was somewhat incomplete. However, this scheme
served as the basis for one of the most frequently cited
functional classification schemes described below [12].
Molecular classification
Molecular classification was initiated in 1980 after the
amino acid sequences of four b-lactamases had been solved
through protein sequencing. The penicillinases from
Staphylococcus aureus PC1 [8], Bacillus cereus 569/H
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[13], Bacillus licheniformis 749/C [7], and Escherichia
coli/R6K, R-TEM [14] served as the basis for defining the
molecular class A b-lactamases [15]. The combination of
partial sequence of the zinc-containing b-lactamase II from
B. cereus, together with its widely variant mechanistic
properties, allowed Ambler to define class B as a second
structural class of b-lactamases [15], although the amino
acid sequence of this metallo-b-lactamase (MBL), or of
any MBL, was not completely solved until 1985 [16]. At
that time, b-lactamase II was the only MBL that had been
recognized, probably because of the lack of selection
pressure from carbapenems that were not approved for
clinical use until late in 1985 [17]. Shortly following
Ambler’s definitions of structural classes, Jaurin and
Grundstrom sequenced the ampC gene from E. coli K-12,
and demonstrated significant molecular differences
between the AmpC cephalosporinase and b-lactamases in
classes A and B, thus giving rise to the class C cephalo-
sporinase designation [18]. The final molecular category
was provided by Huovinen et al. [19], who demonstrated
that the PSE-2 serine b-lactamase, later named OXA-10,
differed sufficiently from the other three classes to warrant
a separate designation of class D.
Characteristic amino acid motifs for the class A and C
serine b-lactamase sequences were initially defined by
Joris et al. [20], with class D motifs later identified as the
families of class D enzymes increased [21, 22]. Typical
sequence motifs and molecular sizes for the serine b-lac-
tamase classes are shown in Table 1.
Class C cephalosporinases are the largest of the serine
enzymes with approximately 360 amino acids in their
coding sequences, compared to generally less than 310
amino acids for class A and class D enzymes. All three
classes contain the active site serine in a motif with the Ser-
X-X-Lys sequence, although each serine may be numbered
differently [18, 19, 21–25]. Two other conserved sequences
across all three classes are the (Ser/Tyr)-X-Asn motif and
the Lys-(Thr/Ser)-Gly motif, each appearing at different
distances from the active site serine in each class. Class A
enzymes have a conserved glutamic acid at position 166,
whereas class D enzymes have a conserved sequence of
Ser-X-Val, beginning with serine at position 118 [22]. In
addition to their use in defining molecular relatedness of
novel b-lactamases, conserved motifs have been explored
in attempts to assign functional roles important to each
class [25, 26].
Class B MBLs, all of which contain at least one active site
zinc atom, are easy to classify on a functional basis, as these
enzymes are readily inhibited by metal ion chelators. On a
molecular level, the MBLs have been divided into three
subclasses, based initially on analyses of crystal structures
of eight of these enzymes and subdivided, in part, according
to the amino acids involved in zinc ligands [27, 28]. Because
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Table 1 Distinguishing characteristics of the three major molecular classes of serine b-lactamases
Molecular class Molecular size (kDa) Characteristic amino acid motifs References

A B31 S70TSK –a S130DN E166XXLN K234TG [20, 23, 24]

(SXXK) (SXN)
C [35 S64TSK – Y150AN – K315TG [18, 25]
(SXXK) (YXN) (KSG)
Db B31 S70TFK S118XV Y144GN – K216TG [19, 21, 22]
(FGN) (KSG)
The most common motifs are shown in bold; alternative sequences are shown in parentheses, with X representing sequences that can accom-
modate multiple substitutions
a
No corresponding motif
b
Numbering according to Ref. [22]

Table 2 Zinc ligands in the four subclasses of class B metallo-b-lactamases

B subclass Typical enzymes Substrate preference Number of Consensus amino acids at active site References
active site
Zn2? atoms Ligands Ligands
to Zn1 to Zn2

B1a BCII, IMP family, All b-lactams except 2 His116 Asp120 [27, 28]
VIM family, SPM-1, CcrA monobactams His118 Cys221
His196 His263
B1b NDM family a
All b-lactams except 2 His116 Asp120 [29]
monobactams His118 Cys221
His196 His263
B2 CphA, Sfh-1 Carbapenems 1 NAa Asp120 [27, 28, 31]
Cys221
His263
B3 L1, FEZ-1, CAU-1 All b-lactams except 2 His/Gln116 Asp120 [27, 28]
monobactams His118 His121
His196 His263
a
Defined to be in a new subclass of B1 because of low sequence homology between NDM-1 and other B1 MBLs [10]
b
Not applicable; binding of Zn in this site results in enzyme inhibition of the CphA MBL [11]

of the wide diversity in structural homology among the
various MBLs, even within a designated subclass, func-
tional as well as molecular characteristics were also used to
subdivide the various enzymes into subclasses B1, B2, and
B3 [28]. After these original assignments had been descri-
bed, the structurally dissimilar NDM-1 MBL emerged, with
a unique sequence that had only 32.4 % identity to any
known MBL, suggesting a second B1 subclass [29, 30].
These four subclasses and the characteristic amino acids
involved in binding to active site zinc ions are shown in
Table 2, together with substrate preferences. A notable
structural feature of subclasses B1 and B3 is that they have
two Zn2? involved in b-lactam hydrolysis, whereas subclass
B2 apparently utilizes only one functional Zn2? for maximal
enzymatic activity [31]. Functional differences of these
subclasses are discussed next.
Alignment of functional and molecular nomenclature
By the mid-1990s, gene sequencing had become available
to a large number of research laboratories, so that the
sequencing of bla genes was relatively accessible to many
in the b-lactamase community. The epidemic of ESBLs
that began in earnest in the late 1980s spawned an out-
pouring of novel b-lactamase sequences [32]. Many of the
new ESBLs were fully characterized with respect to both
structural and functional characteristics [33–35], so that it
was possible to try to align structures with function among
the growing number of unique b-lactamases. Although the
1989 functional classification scheme had attempted such
an alignment, the amount of structural information avail-
able was minimal compared to the situation just 6 years
later when Bush, Medeiros, and Jacoby proposed a more

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complete nomenclature in which functional groups were
associated with molecular classes [12]. Functionality was
based on key b-lactam substrates and inhibitors that would
define a hydrolytic and inhibitor profile for each group of
enzymes. In addition, the functional groups were closely
aligned with molecular classes, with the hopes that, in the
future, one might be able to define the properties of a new
b-lactamase based on structural information. Key sub-
strates included penicillins (benzylpenicillin, carbenicillin,
and cloxacillin), early cephalosporins (cephaloridine or
cephalothin) in contrast to expanded-spectrum cephalo-
sporins (cefotaxime or ceftazidime), the monobactam
aztreonam, and carbapenems (usually imipenem). Inhibi-
tors included EDTA that could be used to identify MBLs
and clavulanic acid that was used primarily to identify
group 2 serine b-lactamases in contrast to group 1 cepha-
losporinases that did not respond well to this inhibitor.
Initially, relative hydrolysis rates were used as the basis for
group assignments, with most literature data normalized to
either benzylpenicillin or cephaloridine [12]. Novel
enzymes were often not purified to homogeneity so that full
kinetic data were not available for analysis.
The major alignments are shown in Table 3, where class
A b-lactamases fall into functional groups beginning with
the number 2 (except for group 2d); class B enzymes are in
functional group 3; class C cephalosporinases are aligned
with functional group 1; and class D enzymes are in
functional group 2d, a group that was originally defined as
b-lactamases which could efficiently hydrolyze oxacillin or
cloxacillin [12]. Subgroups were assigned according to
functional differences based on substrate profiles and
response to clavulanic acid. The rationale for these
assignments is described in previous references [12, 36]. In
2010, Bush and Jacoby [36] expanded the 1995 classifi-
cation by adding additional subgroups to accommodate
recently described b-lactamases as shown in Table 3.
Descriptors of the functional groups are provided in the
current review in Table 3 and include summaries of IC50
data for clavulanic acid and substrate profiles based on kcat
values for hydrolysis of various substrate classes, using
kinetic data not available when the 1995 scheme was
introduced. The 2010 classification included additional
subgroups 1e, 2ce, 2de, and 2df in an attempt to fit novel
enzymes into functional groups consistent with both
structural homologies and functional activities. The sub-
groups ending in ‘‘e’’ included three subgroups of b-lac-
tamases with enhanced hydrolysis of expanded-spectrum
cephalosporins similar to class A ESBLs, but with struc-
tural features that aligned them with class C (subgroup 1e,
the extended-spectrum AmpC, or ESAC enzymes [57]),
class A (subgroup 2ce), or class D (subgroup 2de). Simi-
larly, the class D b-lactamases with carbapenem-hydro-
lyzing activities were assigned to the new subgroup 2df to
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differentiate them from the class A serine carbapenemases
in subgroup 2f. Although there is logic in these assign-
ments, it is sometimes difficult to define precisely where a
novel enzyme should be placed functionally.
Nomenclature based on ESBL functionality
In addition to the molecular–structural nomenclature just
described, a number of other names abound in the literature
to describe b-lactamases in language that makes sense to a
practicing clinician. Very soon after the first epidemics of
cefotaxime-resistant or ceftazidime-resistant Enterobacte-
riaceae were reported to be caused by variants of the
common TEM and SHV b-lactamases, the term ‘‘extended-
spectrum b-lactamases’’ began to appear [58, 59], and was
quickly abbreviated to ESBL [60]. ESBLs were known
initially as plasmid-encoded group 2be TEM- and
SHV-derived enzymes that could confer resistance by
hydrolyzing expanded-spectrum cephalosporins such as
cefotaxime and ceftazidime, as well as aztreonam, but
some have also included the common chromosomal K1
b-lactamase from Klebsiella oxytoca that hydrolyzed
aztreonam similar to the TEM and SHV ESBLs [11, 12]. In
the 1995 functional classification, enzymes, including K1,
were assigned to group 2be, ‘‘the extended-spectrum
b-lactamases,’’ if ‘‘hydrolysis rates for the extended-spec-
trum b-lactam antibiotics, ceftazidime, cefotaxime, or
aztreonam, were [10 % that for benzylpenicillin’’ [12].
These early enzymes were more responsive to inhibition by
clavulanic acid, tazobactam, or sulbactam than the parent
enzymes [58], resulting in the use of clavulanate as a
diagnostic tool to identify the presence of an ESBL in a
resistant isolate [61]. Another common characteristic of
ESBLs was their inability to hydrolyze carbapenems, so
that carbapenem therapy was utilized extensively in areas
with high rates of infections caused by ESBL-producing
organisms [62]. From recent analyses of the proliferation of
new b-lactamases, group 2 enzymes, particularly those
families that include ESBL activity, are the most rapidly
growing set of b-lactamases [2].
Structure–activity relationships of various ESBL fami-
lies have identified mutational changes correlated with
increased cephalosporin or monobactam hydrolysis. In the
early TEM and SHV variants, an ESBL phenotype was
associated with enzymes that had substitutions at amino
acids 104, 164, 238, or 240 [35, 63]. However, as we now
know, substitutions at these positions do not always confer
an ESBL phenotype, leading to potential mislabeling of
novel enzymes as ESBLs based on sequence analysis rather
than functional activity. For example, the recently identi-
fied ‘‘weak ESBL’’ TEM-187 had kcat values of 12.5, 2.6,
and 2.6 s-1 for cefotaxime, ceftazidime, and aztreonam,
respectively, but conferred no clinical resistance to these
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Table 3 Alignment of b-lactamase classification schemes, based on Refs. [12, 13], using arbitrary hydrolysis and inhibition parameters as
activity assessments
Molecular Functional Penicillinase Cephalosporinase ESBL Carbapenemase Monobactamase Clavulanic Representative References
class group activitya activityb activityc activity activity acid enzymes
sensitived

A 2a Y N N N N Y PC1 [12, 37]

2b Y Y N N N Y TEM-1, SHV-1 [12, 38]
2be Y Y Y N Y Y CTX-M-14, -15 [38]
2br Y Y N N N N TEM-30, SHV-10 [39, 40]
2ber Y Y Y N V V TEM-50, TEM-121 [41, 42]
2c Y N N N N Y PSE-4, CARB-3 [12, 43]
2ce Y N Ne N N Y RTG-4 [44]
2e Y Y Y N V Y SFO-1, FEC-1, L2 [45, 46]
2f Y Yf Y Y Y Y KPC-2. SME-1f [47]
B 3ag Y Y Y Y N N IMP, VIM, NDM, L1 [29]
3b Y N N Y N N CphA [48]
C 1 N Y N N N N AmpC, ACT-1 [38, 49]
1e N Y Y N N N GC1, CMY-37 [50, 51]
D 2d Y N N N N V OXA-1, OXA-10 [12, 52]
2de Y Y V N N V OXA-11, OXA-15 [53]
2df Y N Y Nh N N OXA-23, OXA-48 [54, 55]

Activity data that are inconsistent with the published distinctive substrate profiles are shown in bold and underlined
a
Y = kcat [ 5 s-1; N = kcat \ 5 s-1; V = variable within the functional group
b
Hydrolysis of cephaloridine or cephalothin
c
Based on hydrolysis of cefotaxime, ceftazidime, or cefepime
d
Y = IC50 \ 2 lM; N C 2 lM; V = variable among the functional group
e
In spite of kcat values generally B1 s-1, resistance to cefepime and cefpirome is seen in producing organisms
f
SME enzymes compared to KPC carbapenemases have lower hydrolysis rates and lower catalytic efficiencies for expanded-spectrum cephalosporins, resulting in
clinical susceptibility to those cephalosporins in SME-producing S. marcescens [47, 56]
g
Includes subclasses B1 and B3
h
In spite of kcat values generally B1 s-1, these enzymes confer resistance to carbapenem

agents, with minimum inhibitory concentrations (MICs) for
each antibiotic of 0.06, 0.5, and \0.06 mg/l, respectively,
in the parent Proteus mirabilis clinical isolate [64]. This
enzyme had four amino acid mutations, Leu21Phe,
Ala184Val, Thr265Met, and the common Arg164His sub-
stitution found in many TEM ESBLs (http://www.lahey.
org/Studies/), but the combination of mutations apparently
subdued full ESBL behavior in the producing organism.
The obvious question arises as to how to define ESBLs
in today’s environment. Hydrolysis of one aminothiazole
oxime-substituted b-lactam does not predict hydrolysis of
similarly substituted cephalosporins or monobactams by
that ESBL; this is demonstrated in Fig. 1 where kcat values
with the CTX-M-3 ESBL are more than four orders of
magnitude larger for cefotaxime and aztreonam compared
to ceftazidime hydrolysis [65]. Similarly, different ESBLs
demonstrate a wide range of kcat values for the expanded-
spectrum b-lactams. Ceftazidime hydrolysis can vary more
than 6,000 fold when kcat values for the K1 or CTX-M-3
enzymes are compared to the TEM-10 ESBL (Fig. 1).
Frère and colleagues [66] summed up the situation quite
appropriately: ‘‘…we believe that, when carefully deter-
mined, the catalytic properties of class A b-lactamases will
present a continuum, where only the extremes will fall into
clearly distinct groups.’’
As seen in Table 3, ESBL-type activities have been
assigned to enzymes in multiple functional groups, i.e.,
those ending in ‘‘e,’’ although not all these subgroups had
enzymes that demonstrated a kcat value C‘5 s-1 for at least
one expanded-spectrum cephalosporin. However, some of
these enzymes that did not fulfill the biochemical definition
for an ESBL were capable of causing clinical resistance in
the producing organism. In alignment with this approach,
Livermore had also proposed an expanded definition of
ESBLs that coincides with the designations in Table 3,
indicating that all b-lactamases with the ability to hydro-
lyze and confer resistance to aminothiazole-oxime-con-
taining b-lactams should receive an ESBL designation
[67]. Although there has been a small effort to expand the
designation of ESBLs to include carbapenemases and
AmpC cephalosporinases [68], this approach has not been
widely accepted [69]. As seen in Fig. 1, hydrolysis of

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Fig. 1 Variance of kcat values
for aminothiazole-oxime-
containing b-lactam antibiotics
and imipenem with traditional
extended-spectrum b-
lactamases (ESBLs) and
carbapenemases. CTX
cefotaxime, CAZ ceftazidime,
ATM aztreonam, IPM imipenem
carbapenems is quite distinct with respect to ESBLs com-
pared to carbapenemases, i.e., no ESBLs hydrolyze imi-
penem at a measurable rate, whereas expanded-spectrum b-
lactams can be hydrolyzed at varying rates by both ESBLs
and carbapenemases. With the increased attention to tra-
ditional ESBL production in the diagnostic world, the
separation of ESBLs from carbapenemases remains an
important distinction in the clinical laboratory, allowing
carbapenems, or possibly b-lactamase-inhibitor combina-
tions, to be utilized for ESBL-associated infections but not
for carbapenemase-producing pathogens. In fact, using the
current CLSI and EUCAST breakpoints for expanded
spectrum cephalosporins and carbapenems, a recent study
showed that it was possible to separate many ESBL-pro-
ducing bacteria from KPC or VIM carbapenemase-pro-
ducing isolates using ESBL combination disks or the
VITEK2 [70], thus supporting distinctive nomenclature for
the two groups of enzymes.
Ambiguity of classification schemes
In spite of decades of attempts to provide useful b-lacta-
mase nomenclature, no classification scheme is perfect.
Any effort to define cleanly differentiated functional
groups for 1,300 unique b-lactamases is going to meet with
substantial difficulty, particularly when many of these
enzymes are described only in the context of an amino acid
sequence, and perhaps a phenotype reported for an organ-
ism that may also produce multiple b-lactamases with
different functional characteristics in a background of porin
defects or upregulated efflux pumps.
Initial attempts to classify enzymes according to
‘‘penicillinase’’ and ‘‘cephalosporinase’’ designations were
complicated when new sets of substrates were used for
classification purposes. Thus, carbapenemases, although
well studied in the 1960s, would never have been identi-
fied as such until imipenem entered the clinical arena
20 years later. Other attempts to classify b-lactamases on
the basis of chromosomally or plasmid-encoded enzymes
have been thwarted; for example, variants of the common
chromosomal SHV-1 found in many K. pneumoniae
strains have been identified both as plasmid-encoded (58)
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and chromosomal enzymes [71]. Class C enzymes that were
initially identified as species-specific AmpC cephalospo-
rinases now appear quite promiscuously as plasmid-enco-
ded enzymes in most species of the Enterobacteriaceae
with little regard to their origin [72]. The plasmid-mediated
AmpC enzymes fall into five major families that are closely
related to species-specific AmpC enzymes from the fol-
lowing organisms: Aeromonas spp. (CMY-1-related
enzymes, MOX and FOX families), Citrobacter freundii
(CMY-2-related enzymes, LAT-1, CFE-1), Enterobacter
spp. (MIR family and ACT family), Hafnia alvei (ACC
family), and Morganella morganii (DHA family) [72].
Nomenclature confusion has arisen in the past when
different groups assigned disparate names to similar
enzymes before sequence information was available. One
early example was the family of CARB enzymes, ‘‘car-
benicillin-hydrolyzing penicillinases,’’ that included a set
of Pseudomonas-specific enzymes (PSE) b-lactamases [73,
74], with members found in both class A (group 2c) [75]
and class D (group 2d) [19]. At this time, there are 14
CARB enzymes with two different molecular origins and
functional characteristics (http://www.lahey.org/Studies/),
including the RTG-4 (CARB-10) enzyme with extended-
spectrum characteristics [44], making analysis of structure–
function relationships a bit difficult. The CTX-M family
provides another example of different investigators who
independently identified novel enzymes with no clear
parental lineage at the time of discovery. Thus, the MEN-1
[76] and the Toho-1 enzymes [77], described in the liter-
ature before the identification of CTX-M-1, were subse-
quently shown to be members of the CTX-M family of
enzymes [78]. MEN-1 is identical to the enzyme called
CTX-M-1 [78] and Toho-1, together with Toho-2 [79],
eventually became CTX-M-44 and CTX-M-45 (http://
www.lahey.org/Studies/).
Misleading nomenclature can also result from historical
precedence in naming a family of enzymes that are struc-
turally related but which eventually demonstrate muta-
tional changes resulting in functional differentiation. Class
D OXA enzymes, first identified as penicillinases with the
ability to hydrolyze oxacillin [80], now include more than
340 structural variants (http://www.lahey.org/Studies/).
J Infect Chemother (2013) 19:549–559
Although the OXA enzymes were first known as ‘‘oxacil-
linases,’’ many newer class D enzymes exhibit little
hydrolysis of the oxacillin-type penicillins [22]; however,
all the OXA enzymes that have been studied microbio-
logically or biochemically can hydrolyze amino- and car-
boxy-penicillins, such as amoxicillin and ticarcillin,
respectively [22]. Subgroups of these enzymes include the
group 2de ESBLs such as OXA-11 and the group 2df
carbapenemases that are particularly represented by OXA-
23- and OXA-48-related enzymes in Acinetobacter spp.
[22]. Inhibitory activity of clavulanic acid is quite variable
among the OXA enzymes, with IC50 values ranging from
0.009 to 100 lM [12], although all are inhibited by NaCl
[22]. These extreme differences in substrate and inhibitor
spectra among, and within, subgroups are related to the
wide diversity of blaOXA sequences, such that enzymes in
this highly diverse family, such as OXA-60, can exhibit as
little as 19 % amino acid identity with other class D
enzymes [81], although some enzymes differ only by single
amino acid substitutions [22].
Another limiting designation that is in the literature
includes the inhibitor resistant TEM (IRT) enzymes in
functional subgroup 2br. These enzymes originally
included only derivatives of the TEM-1 b-lactamase [82],
but now include at least one SHV variant, SHV-10 [40]. It
is likely that an inhibitor-resistant CTX-M variant will
eventually be identified that also falls into this subgroup,
so that a TEM-related designation will be even less use-
ful. A final example of perhaps misleading nomenclature
is the ‘‘CMT,’’ or complex mutant TEM, enzyme group
(group 2ber), with both reduced affinity for clavulanic
acid and increased hydrolysis of aminothiazole-oxime-
containing b-lactams compared to the parent TEM-1
enzyme. Enzymes in this group have amino acid substi-
tutions that are represented in both ESBLs and inhibitor-
resistant TEM b-lactamases [42]. This nomenclature is
based heavily on the phenotypic response of the produc-
ing organisms rather than on well-defined enzymatic
properties, resulting in functional assignments that may
become subjective.
Several apparently anomalous enzyme assignments are
shown in Table 3, with the bolded enzyme groups con-
taining at least some family members that exhibit bio-
chemical properties not fully aligned with the guidelines
set for either functional or molecular assignments [36].
Some of these variations are caused by the arbitrary defi-
nitions set in this current review: inhibition being signifi-
cant if the IC50 value was\2 lM and hydrolysis defined as
being important if a substrate was hydrolyzed with a kcat
value [5 s-1 without taking into account enzyme affinity
for the substrate. Although the kinetic parameter of kcat/Km
is more indicative of the efficiency of an enzyme, many
b-lactamases have such high Km values for the expanded-
555
spectrum cephalosporins that the catalytic efficiency ratio
becomes meaningless in terms of physiological b-lactam
concentrations achievable in a bacterial cell [83]. When
enzymes have Km values much lower than physiological b-
lactam concentrations, the maximal hydrolysis rate is
proportional to kcat, thereby justifying the use of this
parameter for classification purposes. Examples of b-lac-
tamases that do not align clearly with their subgroup
description based on the parameters outlined in Table 3
include the following:
(a) Subgroup 2ber, the inhibitor-resistant ESBL sub-
group, with TEM-121 (CMT-4) that readily hydro-
lyzed ceftazidime but not cefotaxime (kcat values of
40 and 0.5 s-1, respectively) and was weakly inhib-
ited by clavulanic acid and tazobactam (IC50 values of
1 and 0.3 lM, respectively) [42];
(b) Subgroup 2ce, the CARB-type RTG-1 (CARB-10)
enzyme conferring resistance to cefepime and cefpi-
rome, but exhibiting a kcat value of 0.15 s-1 for
cefpirome, and kcat/Km values of 0.001 and
0.0005 lM-1s-1 for cefepime and cefpirome, respec-
tively [44];
(c) Subgroup 2df class D carbapenemases such as OXA-
23 with kcat values for imipenem, meropenem, and
doripenem of 2.1 (or 0.92), 0.13, and 0.20 s-1,
respectively [38].
For many of these weaker enzymes to be detected,
reduced susceptibility alerted investigators to the possi-
bility of more than one resistance mechanism in the pro-
ducing organism. For example, carbapenemase-producing
organisms may harbor feeble enzymes that have poor
carbapenem hydrolysis rates, but the organisms also may
have porin defects or increased efflux mechanisms [55,
84]. Unfortunately, enzyme nomenclature cannot capture
these coordinated mechanisms responsible for clinical
resistance.
b-Lactamase nomenclature in the future
It is clear that functional group assignments based solely on
biochemical or kinetic properties are not always going to
be helpful in determining actual contributions of these
enzymes to clinical resistance. Isolated enzymes may not
be stable, so that kcat values may be underestimated, or
large quantities of catalytically inefficient enzymes pro-
duced in an organism with a porin deficiency may be
sufficient to confer clinical resistance to a poor substrate.
Most of the enzymes described in the literature have been
identified because they were associated with some clinical
signal, either through susceptibility testing or through
failed therapy. Therefore, functional analyses need to
incorporate some measure of microbiological effects in the
123
556
Fig. 2 Flow chart of activities
leading to b-lactamase
classification assignments. Note
that enzyme purification and
characterization activities can
occur using either the original
clinical isolate or a
transformant. Bla beta-
lactamase
original clinical isolate. This aspect has not been fully
addressed by previous classification criteria.
Figure 2 describes a scheme that may be used to define a
novel b-lactamase with respect to both structural and
functional characteristics (see [36]). A reduced microbio-
logical or clinical response to a b-lactam antibiotic in a
wild-type strain of that species that should be susceptible is
the most likely reason to suspect that a b-lactamase may be
involved in lower susceptibility. b-Lactamase production
can often be determined in whole cells with a rapid plate
assay using nitrocefin or another chromogenic cephalo-
sporin [85, 86]. If b-lactamase activity is detected, attempts
should be made to transfer the bla gene to a b-lactamase-
negative strain to determine whether the gene is on a mobile
element. Susceptibility testing of the original strain and any
transformants, using appropriate antibiotics and inhibitor
combinations, will provide strong clues as to what kind of
enzyme may be present. PCR can then be conducted, using
primers for the most likely enzyme families, based on
susceptibility testing. Full sequencing of the bla gene will
then lead to the assignment of the new enzyme to a
molecular class. In parallel, it should be possible to purify
the protein from the transformant or clinical isolate and
determine biochemical properties that will allow final
functional assignment. By using a combination of micro-
biology, genetics, and biochemistry, full characterization of
a new enzyme can be accomplished and a functional
assignment can be confirmed using the criteria listed in
Table 3. If ambiguity about the biochemical assignment
remains, the functional designation should reflect the clin-
ical effect of the enzyme in the original isolate and should
be assigned based on the resistance profile conferred. This
effort completes the cycle of a nomenclature that may
provide useful information to the clinician who must make a
call about appropriate therapy for an infected patient.
123
J Infect Chemother (2013) 19:549–559
Although b-lactamase identification is not a rapid pro-
cess that may guide immediate therapy, many b-lactam-
resistant pathogens carry mobile elements that can spread
readily throughout a hospital unit. Thus, early identification
of a transferable b-lactamase in an index patient may allow
intervention strategies that will prevent further dissemina-
tion of a major resistance determinant.
Conflict of interest The author has no conflict of interest that would
affect the content of this manuscript.
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