(Chest. 2001;119:391S-396S.)
© 2001
American College of Chest Physicians
Evolution and Clinical Importance of Extended-Spectrum ß-Lactamases*
Louis Rice, MD
*
From the Cleveland Veterans’ Affairs Medical Center, Cleveland, OH.
Correspondence to: Louis Rice, MD, Medical Service III (w), Cleveland Veterans’ Affairs Medical Center, 10701 East Blvd, Cleveland, OH 44106
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Abstract
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In the process of evolution, bacteria have acquired well-developed
mechanisms of resistance to an extensive array of hostile substances.
This time-tempered system of defense is so intricate and adaptable that
contemporary medicine has been hard-pressed to maintain an advantage.
In this article, the processes responsible for bacterial resistance to
extended-spectrum cephalosporins are reviewed. Particular emphasis is
placed on the extended-spectrum ß-lactamases that have emerged to
provide bacteria with formidable resistance to modern drugs. Avoidance
of this problem requires limitations on extended-spectrum cephalosporin
usage. While carbapenems are clearly the treatment of choice for
infections caused by these pathogens, empirical use of
ß-lactam/ß-lactamase inhibitors such as piperacillin/tazobactam has
been associated with reduction in the prevalence of cephalosporin
resistance.
Key Words: bacteria • ß-lactamases • carbapenems • cephalosporin resistance • resistance, ß-lactam
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Introduction
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The
evolution of bacteria through time has permitted the elaboration of a
host of diverse species. These many species have generated a
corresponding diversity of resistance mechanisms that fight naturally
occurring antibiotic challenges, such as those generated by molds,
yeasts, and actinomycetes. As a result, even bacteria that have never
been exposed to commercial antibiotics can nonetheless bear resistance
genes to these drugs.1
Thus, it is not surprising that the
widespread clinical use of commercial antibiotics has mobilized latent
mechanisms to neutralize their impact. This process has been further
facilitated by the variety of genetic elements that can harbor
resistance genes, including the bacterial chromosome, transferable
plasmids, and transposons. Resistance is facilitated by interspecies
transfer through conjugation (DNA transfer during bacterial mating),
transformation (incorporation of free DNA carrying resistance genes
from the environment), or transduction (transfer of genetic material
between pathogenic species by bacteriophage). Gene transfer may occur
across a very broad host range, such as between Gram-negative and
Gram-positive bacteria.1
2
Given the above scenario, there is not only a plethora of pathogenic
species that are potentially resistant to antibiotics, but the means
employed to cause resistance are correspondingly varied. Each pathogen
may require a specific countervailing strategy when resistant strains
emerge in a clinical setting. In this discussion, we will focus on
mechanisms responsible for resistance in Klebsiella
pneumoniae to extended-spectrum cephalosporins such as
ceftazidime, with particular emphasis on the extended-spectrum
ß-lactamases (ESBLs) that have emerged with use of these drugs,
mediating an increased incidence of outbreaks of resistant organisms in
ICUs nationwide.3
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ß-Lactam Resistance
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Beginning with the introduction of penicillin half a century ago,
the ß-lactams have remained the largest antibiotic class of clinical
relevance, comprising four major families: the penicillins,
cephalosporins, carbapenems, and monobactams.4
5
These
antibiotic classes continue to be the objects of directed chemical
modifications in order to modulate their antimicrobial activity (Fig 1
).
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Figure 1. Basic core structures of ß-lactam antibiotics.
Substitutions are indicated by the R1 groups. Structural
differences among these antibiotics confer differences in
susceptibility to ß-lactamases. From Livermore5
with
permission.
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Three principal mechanisms cause resistance to
ß-lactams6
: (1) a reduction in the affinity of the drug
targets (penicillin-binding proteins) via amino-acid substitution, a
phenomenon occurring in both Gram-positive and Gram-negative
bacteria7
; (2) in Gram-negative species, alteration in
outer-membrane permeability that denies passage to the ß-lactams
(eg, downregulation of an outer-membrane porin confers
imipenem resistance in Pseudomonas
aeruginosa)8
; and (3) in both Gram-positive and
Gram-negative bacteria, and of principal clinical importance for
Gram-negative rods, such as K pneumoniae, the production of
ß-lactamases that inactivate the drug through hydrolysis of the
ß-lactam ring.7
9
ß-Lactamases are produced by a wide variety of bacteria, including
aerobic Gram-positive or Gram-negative and anaerobic species. They can
be encoded by genes on chromosomes or plasmids.4
6
In
Gram-negative species, ß-lactamases are found in the periplasmic
space, between the cell wall and the outer membrane; in Gram-positive
bacteria, which lack outer membranes, they are excreted. There are many
types of ß-lactamases, which vary both in their ability to inactivate
a given ß-lactam as well as in their susceptibility to inhibitors
such as clavulanate, sulbactam, and tazobactam.6
ß-Lactamases are classified according to their amino-acid sequences
(as presented in Table 1 )9
and by their functional characteristics as defined by
their substrate and inhibitor profiles.10
The parent enzymes of class A, particularly the TEM (named after a
little girl) and sulfhydryl variable (SHV) series expressed
predominantly by Escherichia coli and K
pneumoniae,4
are of special interest in the
generation of ESBL variants. These enzymes are plasmid encoded and thus
are readily transferred to other bacteria. For the most part, they are
constitutively produced, not inducible, and are penicillinases,
demonstrating little activity against cephalosporins. Furthermore, they
are subject to inhibition by clinically available inhibitors.
TEM enzymes can confer dramatic levels of resistance (Table 2 ). In the presence of TEM-1, which may be considered the progenitor
enzyme of the TEM series, resistance to ampicillin is increased
> 100-fold in E coli, whereas this enzyme is completely
inactive against ceftazidime, a third-generation cephalosporin.
However, when the extended-spectrum variant TEM-26 is expressed by
K pneumoniae, a minimum inhibitory concentration (MIC) of
256 mg/mL for ceftazidime follows (again, far in excess of a clinically
useful concentration).11
12
One cautionary note, however, is that in assessing the implications of
such tables of MICs, the interpretations are not always
straightforward; in some instances, they may be confused by the
phenomenon of an inoculum effect (Table 3
).13
Ceftazidime, by virtue of its bulk and charge, has
difficulty accumulating in the periplasmic space and tends to be more
readily hydrolyzed by ESBLs compared with other third-generation
cephalosporins, resulting in a high MIC even at the standard inoculum
of 105 cfu/mL. In the case of cefotaxime,
however, the same inoculum for a TEM-26-producing strain yields an MIC
of 1, within the susceptible range. Increasing the inoculum 100-fold,
however, causes the cefotaxime MIC to increase more than two orders of
magnitude, reflecting the fact that ESBL-producing strains will show
higher in vitro resistance as the number of organisms
increases. As a result, in clinical situations where there is a high
local concentration of organisms, drugs with nominal MICs that are
within the attainable range may nonetheless prove ineffective.
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Origin of Extended-Spectrum ß-Lactamases
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ESBLs find their origins in genes that are already present in many
bacteria. Approximately 25 to 30% of E coli, for instance,
are resistant to ampicillin, in most cases as a result of their
possessing a plasmid carrying the TEM-1 ß-lactamase
gene.14
Similarly, one study suggests that virtually all
Klebsiella species carry an SHV-1-like ß-lactamase gene on their
chromosome.15
Various mechanisms, whether involving
alterations in the promoter or translocation of the gene to a plasmid,
can mediate high-level expression that facilitates the emergence of
ESBL-producing variants.
Over the past 2 decades, many variants have in fact been generated. In
1982, when ceftazidime first became available, TEM-1, TEM-2, and SHV-1
were known; at present, we know of > 60 TEM-producing variants, most
of them ESBLs, as well as at least 20 new SHVs. As shown in Table 4
, minor amino-acid substitutions in TEMs can dramatically alter their
ability to confer resistance to third-generation cephalosporins.
Alterations in three positions of the TEM-1 enzyme, a protein that
contains 263 amino acids, can readily generate ESBL activity. To
generate TEM-12, a one-nucleotide change converts the arginine at
position 162 to a serine; an additional one-nucleotide change,
resulting in a glutamate-to-lysine change in position 237 or 102,
yields TEM-10 or TEM-26, respectively. Consequently, the ceftazidime
MICs change from the very low value associated with TEM-1 to 256 mg/mL
for TEM-26, the most potent of all the ESBLs against ceftazidime. Thus,
a situation arises in which essentially all Klebsiella species and one
third of E coli are only one or two mutations away from
elaborating ESBLs. Given the extraordinarily rapid rate of bacterial
reproduction and the possibility that the enzyme coding sequence can be
located on high-copy number plasmids, this results in an extremely high
probability of such variants being produced in the clinical setting.
The survival of such ESBL-producing variants is strictly dependent on
their being given a selective advantage. Both TEM-1 and SHV-1 have
optimal structures as penicillinases,16
so that the change
in specificity to a cephalosporin-resistant ESBL makes the organism
somewhat less resistant to penicillins. Thus, it is only in the context
of cephalosporin use that the resistant variants emerge. For any given
patient, the evolution of resistance may follow a unique path,
including not only TEM and SHV alterations but also interactions of
these altered enzymes with other cellular components.
Table 5
shows an example of this complexity, in which alterations in membrane
proteins also contribute to the development of ceftazidime resistance.
The patient began with an E coli infection that was
ampicillin resistant, owing to the presence of the TEM-1 gene, but was
ceftazidime sensitive. Within a month, a ceftazidime-resistant strain
had emerged, without the elaboration of an ESBL. Instead, resistance
was effected through acquisition of a heavily expressed SHV-1 and the
concomitant elimination of one of the outer-membrane porins, reducing
access of the antibiotic to the periplasmic space. The combination of
TEM-1 and SHV-1, each relatively inactive against ceftazidime, together
with the diminished uptake of the drug, was sufficient to generate
resistance. Over the next several weeks (by December 23), the SHV-1
gene had mutated to generate SHV-8, by itself sufficient to confer
resistance, so that within a few days (by December 27), the
bacteria had restored production of the missing membrane porin, given
that its elimination was no longer required to resist ceftazidime.
Presumably, its restoration would then benefit the bacteria, since
membrane proteins normally would be expected to serve a variety of
functions unrelated to antimicrobial transit.
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Treatment of K pneumoniae Outbreaks
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The literature dealing with responses to ESBL-expressing K
pneumoniae is not extensive, but from several documented accounts,
a definite pattern is beginning to emerge. While there is evidence that
these organisms can spread from hospital to hospital (making
infection-control measures relevant to their containment), their
emergence is first and foremost a response to the use of antibiotics
(specifically, extended-spectrum cephalosporins such as ceftazidime and
ceftriaxone).17
Consequently, an essential component to
the control of these outbreaks has included the reduction and even
elimination of cephalosporin use and concomitant substitution with
alternative drugs. While no substitute strategy has proved uniformly
effective, two approaches have shown promise. The first strategy
involves the use of carbapenems such as imipenem, while the second
involves the combination of a ß-lactam antibiotic together with a
ß-lactamase inhibitor, specifically piperacillin/tazobactam.
Owing to its resistance to hydrolysis by ESBLs, imipenem is
particularly indicated when an outbreak of ESBL-producing K
pneumoniae has already materialized.18
19
Ciprofloxacin has been shown to be effective when used in place of
imipenem,19
but such a substitution may not be useful
since it has been reported that resistance to fluoroquinolines such as
ciprofloxacin can occur in a substantial fraction of ESBL
strains.20
While imipenem is the most effective therapy
for eliminating an outbreak of ESBL organisms, its use is also not
without risks, as there have been two reported instances of nosocomial
imipenem-resistance outbreaks involving Acinetobacter
baumanii21
and P
aeruginosa.22
As an alternative approach, we have used piperacillin/tazobactam as a
substitute for ceftazidime at the Cleveland Veterans Affairs Medical
Center, where ceftazidime-resistant ESBL-producing K
pneumoniae had increased steadily during the early
1990s.23
Although piperacillin/tazobactam is not indicated
as the drug of choice to eliminate an outbreak of ESBL-containing
organisms, as a significant number of species are resistant to
ß-lactam/ß-lactamase inhibitor combinations,24
this
drug combination has proved particularly helpful as an empiric
alternative to ceftazidime for treating infections in locales where
ESBL-containing organisms are known to be present and for decreasing
the likelihood of new outbreaks. Over 4 years of extensive use of this
combination, results have indicated not only a reduction in
ESBL-producing K pneumoniae but also a reduction from 25%
to 5% of piperacillin/tazobactam-resistant variants of this organism.
Moreover, there has been no selection for resistant variants in other
pathogens such as Pseudomonas species, despite its relatively heavy
use. The incidence of vancomycin-resistant Enterococcus has also
remained notably low.
While confirmation in further settings is necessary, these data taken
together suggest that the use of piperacillin/tazobactam is appropriate
for routine substitution of the extended-spectrum cephalosporins,
whereas the carbapenems may be reserved for situations in which
outbreaks of ESBL-containing organisms nonetheless materialize. In
addition, it must be emphasized that returning to the use of
cephalosporins is an unattractive option, as the molecular logic
described in Table 4
will again impose itself, meaning that only one or
two nucleotide substitutions are needed to generate another outbreak.
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Discussion
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Dr. Joseph Lynch:
How often do plasmid-mediated resistances
get transferred to other Enterobacteriaceae besides K
pneumoniae? How widespread is fluoroquinolone resistance, and
is it increasing?
Dr. Louis Rice:
There are many plasmids capable of being
transferred. In vitro, they are readily transferred to
E coli and many other species. While Klebsiella is by
far the most common source, they have also been found in E
coli, as well as species of Proteus, Salmonella, Serratia, and
Enterobacter. The fluoroquinolone correlation appears to be getting
progressively stronger as well, although it is unclear why. George
Jacoby and colleagues28
recently reported on
plasmid-mediated fluoroquinolone resistance on the same plasmid as the
ESBL.
Dr. Ronald Jones:
We have been monitoring these problems, and
over the past year have observed a major change in endemic rates of
ESBLs both in North and South America. For example, one species,
Proteus mirabilis, rarely showed ESBL variants before
1997. In 1998, we found a 10-fold increase in ESBL-containing strains,
involving the same ESBLs formerly present in Klebsiella species and
E coli. In short, once the genetic background is in
place, plasmids are readily transferred between species. We observed
one especially dramatic example of this in one South American
institution, which imposed an infection control program that resulted
in a significant reduction (from 40% to 25%) of K
pneumoniae ESBL-containing strains, but as a result of an
exclusive focus on this genus to the exclusion of other enteric
bacilli, the frequency of the same ESBL variants in P
mirabilis quadrupled. This and other institutions are now also
seeing disappearance of efficacy of fourth-generation cephalosporins
against Amp C enzyme-producing species, as these ESBL-containing
isolates develop further levels of coresistance, some also in
fluoroquinolone-resistant strains.
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Footnotes
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Abbreviations: ESBL = extended-spectrum
ß-lactamase; MIC = minimum inhibitory concentration; SHV = sulfhydryl
variable
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Abstract
Introduction
