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Dispatch
NmcA Carbapenem-hydrolyzing
Enzyme in Enterobacter cloacae in North America1
Sudha Pottumarthy,* Ellen Smith Moland,† Stefan Juretschko,* Susan
R. Swanzy,* Kenneth S. Thomson,† and Thomas R. Fritsche*
*University of Washington School of Medicine, Seattle, Washington, USA:
and †Creighton University Medical Center, Omaha, Nebraska, USA
Suggested citation for this article: Pottumarthy
S, Moland ES, Juretschko S, Swanzy SR, Thomson KS, Fritsche TR. NmcA
carbapenem-hydrolyzing enzyme in Enterobacter cloacae in North
America. Emerg Infect Dis [serial online] 2003 Aug [date cited].
Available from: URL: http://www.cdc.gov/ncidod/EID/vol9no8/03-0096.htm
An imipenem-resistant
Enterobacter cloacae isolate was recovered from the blood of
a patient with a hematologic malignancy. Analytical isoelectric focusing,
inhibitor studies, hydrolysis, induction assays, and molecular sequencing
methods confirmed the presence of a NmcA carbapenem-hydrolyzing enzyme.
This first report of NmcA detected in North America warrants further
investigation into its distribution and clinical impact.
Carbapenem resistance mediated by acquired carbapenemases is a growing
concern worldwide (1–3). Carbapenems are the most potent
of β-lactam antibiotics with a broad spectrum of activity against
aerobic and anaerobic bacterial pathogens. These antibiotics are also
the most reliable of the β-lactams for treatment of infections caused
by organisms that produce extended-spectrum β-lactamases (ESBLs)
or AmpC β-lactamases (4,5). Intrinsic resistance
does occur, however, in a number of species, including Bacillus cereus,
Stenotrophomonas maltophilia, Flavobacterium sp. and Chryseobacterium
sp., Aeromonas hydrophila, Legionella gormanii, and Janthinobacterium
lividum, because of the presence of metallo-β-lactamases (1).
Acquired carbapenemases, on the other hand, are a heterogeneous mixture
of enzymes that belong to Ambler class A, B, or D enzymes and may be plasmid
or chromosomally encoded (1,2). Here we describe the
characterization of imipenem resistance attributable to an acquired class
A carbapenem-hydrolyzing enzyme, NmcA, in an Enterobacter cloacae
isolate.
The Study
A 33-year-old man with acute myeloid leukemia was admitted to a hospital
in Seattle, Washington, with neutropenic fever; he was initially treated
with ceftazidime. His therapy was changed to acyclovir and imipenem because
of unremitting fevers and oral lesions positive for herpes simplex virus
type 1, whereupon his fever defervesced immediately. The fever recurred
2 weeks after treatment was begun, and imipenem-resistant E. cloacae
was isolated from the blood. After the results of susceptibility testing
were obtained, his therapy was changed to levofloxacin, and he exhibited
a good clinical response.
The E. cloacae isolate was susceptible to piperacillin, piperacillin-tazobactam,
ceftazidime, ceftriaxone, cefepime, ciprofloxacin, gentamicin, aztreonam,
and trimethoprim-sulfamethoxazole and was resistant to ampicillin, amoxicillin-clavulanic
acid, cefazolin, and cefoxitin. By both disk diffusion and E-test methods
(AB Biodisk, Solna, Sweden), the zones of inhibition around the imipenem,
meropenem, and ertapenem disks were indistinct, with inner colonies extending
up to the disk or the highest concentration on the E-test strip (MIC >32
μg/mL) and were interpreted as resistant (Figure
1). Addition of 10 μL of 1,000 μg/mL clavulanic acid to
the imipenem, meropenem, and ertapenem disks resulted in clearly defined
and enlarged zones of inhibition and loss of the inner colonies (Figure
2). Using either cefoxitin or imipenem as inducers by the disk induction
method resulted in the formation of blunted or D-shaped zones of inhibition
to meropenem and ertapenem (6). We performed a carbapenem
bioassay by incubating 100 μL of a 50 μg/mL solution of each
carbapenem (imipenem, meropenem, or ertapenem) with 100 μL of either
crude β-lactamase extract or phosphate-buffered saline for 90 min
at room temperature. The inactivating capacity of the enzyme was then
tested by a disk diffusion assay that demonstrated hydrolysis of imipenem,
meropenem, and ertapenem (7).
Isoelectric focusing (IEF) to determine isoelectric points (pIs) and
general inhibitor characteristics with an ampholine polyacrylamide gel
(pH range 3.5–9.5) on a flatbed apparatus (Multiphor LKB, Bromma, Stockholm,
Sweden) indicated that this organism produced a β-lactamase enzyme
with a pI of 6.9 that was inhibited by clavulanic acid and not by cloxacillin
(8,9). This enzyme hydrolyzed 1 μg/mL imipenem on
the IEF gel overlay. Inducibility of the enzyme by imipenem (4 mg/L) and
cefoxitin (8 mg/L) was confirmed by the broth induction method and IEF
(10). After the broth induction procedure, the uninduced
and induced preparations were serially diluted in 0.1 M potassium phosphate
buffer, pH 7.0, permitting both qualitative and semiquantitative assessment
of enzyme activity among the various β-lactamases in the isolate,
as determined by IEF. The broth induction enzyme preparation from a 100-mL,
3.5-h Mueller-Hinton broth produced a visible but light AmpC band, pI
>9.0. The induction with cefoxitin and imipenem increased the levels
of both the AmpC and the pI 6.9 carbapenem-hydrolyzing enzyme, as visualized
in Figure 3. A cefoxitin-induced preparation of
E. cloacae NOR-1 (that produces NmcA) was included as a control
strain for IEF (11).
Chromosomal DNA was extracted from overnight cultures with the Qiagen
DNeasy Tissue kit (QIAGEN Inc., Valencia, CA). Polymerase chain reaction
(PCR)-amplification with a thermal cycler (MJ Research PTC - 200-DNA Engine,
San Francisco, CA) was used to obtain a 2,122 base pair gene fragment
consisting of the carbapenemase structural gene nmcA and its regulatory
gene nmcR (12,13). The nucleotide sequences of
the primers were 5´- TGC CAG CTT AAT TAT TTT CAG ATT AG -3´
(nmcR positions 32–56) and 5´- ATT TTT TTC ATG ATG AAG TTA
AGC C -3´ (nmcA positions 2,129–2,154). PCR amplification
of the nmcR/nmcA genes showed positive results with both NOR-1
and the strain under study, suggesting that the latter strain harbored
a carbapenem-hydrolyzing enzyme similar to that of NOR-1 (11).
Subsequent sequence analysis of the amplified DNA-region showed a similarity
of 100% (2,122 base pairs) between the sequence of our strain and the
carbapenemase genes, regulatory (nmcR) and structural (nmcA)
genes of the E. cloacae strain NOR-1 (13).
Conclusions
Ambler class A carbapenem-hydrolyzing enzymes are relatively rare and
have been reported to date in only a handful of isolates of Enterobacteriaceae
worldwide (1,2,14–16). Sme-1 was identified
from two Serratia marcescens strains collected in London in 1982,
and IMI-1 was found in two strains of E. cloacae isolated in southern
California in 1984. Both enzymes were retrospectively characterized from
clinical isolates recovered before carbapenems were marketed (17,18).
Subsequently, imipenem-resistant S. marcescens strains were identified
from Minnesota, California, and Boston during a 15-year period, 1985–1999.
The Minnesota isolate produced an enzyme identical to the London Sme-1
enzyme, but the Boston and California strains produced a related Sme-1
enzyme with a single amino-acid variation, named Sme-2. More recently,
a fourth class A β-lactamase, KPC-1, has been described in a strain
of Klebsiella pneumoniae collected in North Carolina (15).
NmcA was the first class A carbapenemase identified in a clinical isolate
of E. cloacae, NOR-1, in 1990, following the introduction of carbapenems
(11). This strain was recovered from the pus of a fistulized
subcutaneous abscess of a hospitalized French patient who had received
one intravenous bolus (500 mg) of imipenem before the strain isolation.
The present strain was also isolated from the patient during therapy with
imipenem. Similar to the NOR-1 isolate, this strain displayed decreased
susceptibility to carbapenems (imipenem and meropenem) while remaining
fully susceptible to extended-spectrum cephalosporins (1).
The strain also had decreased susceptibility to ertapenem. Although the
zones of inhibition around the carbapenem disks were poorly defined, leading
us to question the purity of the isolate, repeated testing confirmed our
initial observations. The carbapenem-hydrolyzing enzyme was inhibited
by clavulanic acid, as shown by the disk diffusion method and confirmed
by the IEF overlay method, similar to the other class A carbapenem-hydrolyzing
enzymes (1,2). We found that adding of clavulanic acid
to the carbapenem disks, similar to the method used for National Committee
for Clinical Laboratory Standards ESBL confirmatory testing, was a simple
and helpful diagnostic approach. Because other investigators have defined
the hydrolytic spectrum of the NmcA enzyme, we have not established whether
the NmcA enzyme was solely responsible for the carbapenem resistance in
our isolate (11,13).
Class A carbapenem-hydrolyzing β-lactamases are usually coproduced
with other β-lactamases, conferring distinct biochemical advantages
to the parent strain (3). The carbapenem-hydrolyzing
enzyme IMI-1 was produced along with AmpC and TEM-type enzymes; Sme-1
was co-produced along with a chromosomal AmpC (3,18).
Our strain was similar to NOR-1 and produced an AmpC enzyme of pI >9.0
along with the carbapenem-hydrolyzing enzyme of pI 6.9. The inducibility
of NmcA in this strain of E. cloacae by cefoxitin and imipenem
was evident by disk induction and confirmed by broth induction with IEF,
similar to the NOR-1 strain. Expression of NmcA has been shown to be inducible
because of the presence of a LysR-type regulatory gene, Nmc-R, which precedes
the blaNMC-A gene (1). NmcR mediates biosynthesis
of the carbapenemase enzyme at the basal state, which is further increased
when β-lactam-mediated induction occurs (13). Naas
et al. have shown that NmcR acts as a positive regulator for carbapenemase
biosynthesis even in the absence of an inducer (unlike Amp-R, which is
a negative regulator of the cephalosporinase expression in the AmpC-AmpR
system) and deletion of NmcR results in a sharp decrease in carbapenem
MIC and loss of ß-lactamase inducibility (13).
A recent study has shown that AmpD from E. cloacae NOR-1 is involved
in the regulation of expression of both ß-lactamases (NmcA and AmpC)
(19). These findings suggest that different structural
genes may be under the control of identical regulatory systems and that
nucleotide substitutions in AmpD could lead to the stable co-expression
of the NmcA together with overexpression of the AmpC enzyme in E. cloacae
(19).
This is the first report of the isolation of the NmcA carbapenem-hydrolyzing
enzyme from a clinical isolate of E. cloacae in North America.
The importance of this finding for clinicians and laboratorians is illustrated
by the following: the NmcA enzyme is able to hydrolyze carbapenems; the
NmcA enzyme occurred in E. cloacae (a common nosocomial pathogen);
the NmcA enzyme was difficult to detect; the NmcA enzyme is inducible;
and the expression of NmcA is coregulated with AmpC. Accurately identifying
the different mechanisms of carbapenem resistance will help determine
the epidemiology, risk factors, and therapeutic options in each case.
The continued emergence of novel resistance mechanisms to carbapenems
worldwide reemphasizes the need not only for prudent carbapenem use but
also for prudent antibiotic use in general.
Dr. Pottumarthy,
a Fellow of the Royal College of Pathologists of Australasia, is currently
a senior fellow in laboratory medicine at the University of Washington
Medical Center, Seattle. Her research interests include antimicrobial
susceptibility testing, approaches, and methods; antimicrobial resistance
detection; and ß-lactamases.
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1This work was presented in part at the 13th
Annual European Congress of Clinical Microbiology and Infectious Diseases,
Glasgow, Scotland, 2003.
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