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Journal of Clinical Microbiology, May 2000, p. 1791-1796, Vol. 38, No. 5
Pathology and Laboratory Medicine
Service/113, McGuire Veterans Affairs Medical Center, Richmond,
Virginia 23249-0001,1 and Department of
Medical Microbiology and Immunology, Creighton University School of
Medicine, Omaha, Nebraska 681782
Received 21 September 1999/Returned for modification 26 November
1999/Accepted 18 February 2000
AmpC beta-lactamases are cephalosporinases that confer resistance
to a wide variety of Group 1 AmpC beta-lactamases are
cephalosporinases that are poorly inhibited by clavulanic acid
(9). They are clinically significant because they may confer
resistance to a wide variety of Genes for AmpC beta-lactamases have also recently been found on
plasmids that transfer noninducible cephalosporin resistance to
K. pneumoniae (5, 7, 13-15, 29, 31, 39;
A. Bauernfeind, R. Jungwirth, I. Schneider, H. Sahly, and U. Ullmann,
Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C-2,
p. 69, 1998; A. Bauernfeind, S. Schweighart, K. Dornbusch, and H. Giamarellou, Program and Abstr. 30th Intersci. Conf. Antimicrob. Agents
Chemother., abstr. 190, p. 118, 1990; S. Boyer-Mariotte, L. Raskine, B. Hanau, A. Philippon, M. J. Sanson-LE Pors, and G. Arlet, Abstr.
38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C-7, p. 70, 1998), E. coli (3, 19, 30; C. Hoyen, L. B. Rice, and R. A. Bonomo, Abstr. 38th Intersci. Conf.
Antimicrob. Agents Chemother., abstr. C-161, p. 115, 1998) and P. mirabilis (6). These enzymes are believed to have
originated from the chromosomes of Enterobacter,
Citrobacter, and Pseudomonas spp. (9,
35). In a recent survey, K. pneumoniae and E. coli isolates from patients from 8 of 20 intensive care units in
the United States harbored transmissible AmpC-type beta-lactamases
(G. A. Jacoby, P. Han, M. Alvarez, and F. Tenover, Abstr. 35th
Intersci. Conf. Antimicrob. Agents Chemother., abstr. C40, p. 46, 1995). Documentation of these enzymes in seven or more countries in a relatively short time period (since 1989) may portend future problems (4, 21, 20).
Although reported with increasing frequency in case isolates (5,
13-15, 19, 29, 30, 39), the true rate of occurrence of
plasmid-mediated AmpC beta-lactamases in K. pneumoniae,
E. coli, and P. mirabilis remains unknown. Many
laboratories have difficulty detecting these enzymes in clinical
isolates. In a recent study, 28 (74%) of 38 laboratories in
Connecticut reported at least one nonsusceptible result with an
extended-spectrum cephalosporin or aztreonam for an AmpC-producing
strain of E. coli that was known to be resistant to these
agents (34). These data suggest that the standard systems
used in the study failed to detect resistance and that additional
testing was not performed. Current National Committee for Clinical
Laboratory Standards (NCCLS) guidelines for performing in vitro
susceptibility testing (22-24) do not indicate either the
phenotypic screening or confirmatory tests that should be used for
isolates that harbor AmpC beta-lactamases. For this reason, a study was
designed to determine the occurrence of plasmid-mediated AmpC
beta-lactamases in K. pneumoniae, E. coli, and
P. mirabilis at a veterans medical center. The study also
included E. coli isolates that produced high levels of AmpC
enzyme due to chromosome-mediated factors. In addition, we report on a
phenotypic method for the detection of isolates that harbor these enzymes.
Tests for AmpC-producing isolates of K. pneumoniae,
E. coli, and P. mirabilis.
A total of 1,286 consecutive, nonrepeat E. coli (n = 683),
K. pneumoniae (n = 371), and P. mirabilis
(n = 232) isolates were recovered at the McGuire
Veterans Affairs Medical Center (VAMC) during a 14-month period
(November 1995 to January 1997). Isolates were identified with the
Vitek and API 20E systems (bioMerieux Vitek, Hazelwood, Mo.) and were
tested for susceptibility by the standard disk diffusion method
(23). A 30-µg cefoxitin disk (Becton Dickinson
Microbiology Systems, Cockeysville, Md.) was placed on inoculated
Mueller-Hinton agar (Remel, Lenexa, Kans.). By following the NCCLS
criteria for nonsusceptible organisms (24), isolates with
zone diameters less than 18 mm were selected for MIC and beta-lactamase testing.
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Occurrence and Detection of AmpC Beta-Lactamases
among Escherichia coli, Klebsiella pneumoniae,
and Proteus mirabilis Isolates at a Veterans Medical
Center
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactam drugs and that may thereby create
serious therapeutic problems. Although reported with increasing frequency, the true rate of occurrence of AmpC beta-lactamases in
Escherichia coli, Klebsiella pneumoniae, and
Proteus mirabilis remains unknown. We tested a total of
1,286 consecutive, nonrepeat isolates of these three species and found
that, overall, 45 (3.5%) yielded a cefoxitin zone diameter less than
18 mm (screen positive) and that 16 (1.2%) demonstrated AmpC bands by
isoelectric focusing. Based on the species, of 683 E. coli,
371 K. pneumoniae, and 232 P. mirabilis
isolates tested, 13 (1.9%), 28 (7.6%), and 4 (1.7%), respectively,
demonstrated decreased zone diameters and 11 (1.6%), 4 (1.1%), and 1 (0.4%), respectively, demonstrated AmpC bands. Cefoxitin resistance
was transferred for all but 8 (E. coli) of the 16 AmpC
producers. We also describe a three-dimensional extract test, which was
used to detect phenotypically isolates that harbor AmpC beta-lactamase.
Of the 45 cefoxitin-resistant isolates, the three-dimensional extract
test accurately identified all 16 AmpC producers and 28 of 29 (97%)
isolates as non-AmpC producers. Interestingly, most (86%) isolates in
the latter group were K. pneumoniae isolates. These data
confirm that, at our institution, E. coli, K. pneumoniae, and P. mirabilis harbor plasmid-mediated
AmpC enzymes.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactam drugs, including
-methoxy--lactams, such as cefoxitin, narrow-, expanded-, and
broad-spectrum cephalosporins, -lactam-beta-lactamase inhibitor
combinations, and aztreonam. Genes for AmpC beta-lactamases are
commonly found on the chromosomes of several members of the family
Enterobacteriaceae, including Enterobacter,
Shigella, Providencia, Citrobacter
freundii, Morganella morganii, Serratia
marcescens, and Escherichia coli (17).
Chromosomal expression is typically inducible except in E. coli and Shigella spp., in which it is usually
constitutive and minimal (17, 27). Occasional isolates of
E. coli (1 to 2%) (17) may produce large amounts
of AmpC enzyme (27) and have a phenotype resembling that of
a derepressed AmpC mutant Enterobacter sp. DNA sequencing
data for five hyperproducing E. coli isolates showed that
the ampC gene was preceded by a strong promoter, which
resulted in increased transcription (25). Although chromosomal genes for group 2b beta-lactamases are common in
Klebsiella pneumoniae (17), genes for AmpC
beta-lactamases are notably absent. The first example of a
chromosomally encoded AmpC-type beta-lactamase in Proteus
mirabilis was reported only recently (8).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Beta-lactamases. Isolates were tested for AmpC activity by a three-dimensional extract method, which was an adaptation of procedures described previously for the detection of extended-spectrum beta-lactamases (ESBLs) (36, 41). Briefly, 50 µl of a 0.5 McFarland bacterial suspension prepared from an overnight blood agar plate was inoculated into 12 ml of tryptic soy broth and the culture was grown for 4 h at 35°C. The cells were concentrated by centrifugation, and crude enzyme preparations were made by freezing-thawing the cell pellets five times. The surface of a Mueller-Hinton agar plate (Remel) was inoculated with one each of two E. coli strains (ATCC 25922 and ATCC 11775) as described for the standard disk diffusion method (23); a 30-µg cefoxitin disk was placed on the inoculated agar. With a sterile scalpel blade, a slit beginning 5 mm from the edge of the disk was cut in the agar in an outward radial direction. By using a pipet, 25 to 30 µl of enzyme preparation was dispensed into the slit, beginning near the disk and moving outward. Slit overfill was avoided. The inoculated media were incubated overnight at 35°C. Enhanced growth of the surface organism at the point where the slit intersected the zone of inhibition was considered a positive three-dimensional test result and was interpreted as evidence for the presence of AmpC beta-lactamase (see Fig. 1). To test the extracts of P. mirabilis, MacConkey agar was also used to suppress the (swarming) growth of unlysed cells, which occasionally interfered with interpretation of results. E. coli strains which contained plasmid derivatives of the FOX-1, LAT-2, and MIR-1 AmpC beta-lactamases (Bush group 1) were tested as positive controls.
Isolates with decreased cefoxitin zone diameters were tested for the presence of beta-lactamases by isoelectric focusing (IEF) of cell extracts as described previously (10). The cells were grown in 50 ml of tryptic soy broth (Becton Dickinson Microbiology Systems) for 4 h and were washed in 0.1 M phosphate buffer (pH 7). The centrifuged cells were resuspended in 300 µl of phosphate buffer and were frozen at
70°C. The cells were sonicated with a Branson Cell
Disruptor 200 (Branson Ultrasonics Corp., Danbury, Conn.) for 10 s
and were then cooled with ice for 10 s; this cycle was repeated
four times. Cellular debris was removed by centrifugation. The
quantities of proteins in the preparations were not determined. Enzyme
activity on the focused gels was detected with molten agar containing
nitrocefin (50 µg/ml). Filter paper strips moistened with one of two
different inhibitors at 1 mM were briefly applied to the focused gel
surface prior to the addition of the molten agar (33). AmpC
beta-lactamases are inhibited by cloxacillin, and preparations with IEF
patterns that demonstrated the loss of a nitrocefin band after
application of a cloxacillin-moistened strip were interpreted to
contain AmpC enzyme and show AmpC activity by IEF.
All isolates that demonstrated AmpC activity by IEF were tested for the
ability to transfer resistance to recipient strains. Each donor strain
was tested by filter mating with two or more of the following recipient
E. coli strains: CGSC 1867, C600, and 26R793. The selective
medium contained 400 µg of sodium azide per ml and 4 µg of
aztreonam per ml, 512 µg of nalidixic acid per ml and 25 µg of
cefoxitin per ml, or 512 µg of rifampin per ml and either 4 µg of
aztreonam per ml or 50 or 75 µg of cefoxitin per ml.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Occurrence of AmpC-producing organisms. Of the 1,286 isolates that were tested, 45 (3.5%) yielded cefoxitin zone diameters less than 18 mm (screen positive), and 16 of these (1.2%) demonstrated AmpC bands by IEF. Based on the species, of 683 E. coli, 371 K. pneumoniae, and 232 P. mirabilis isolates tested, 13 (1.9%), 28 (7.6%), and 4 (1.7%), respectively, demonstrated decreased zone diameters and 11 (1.6%), 4 (1.1%), and 1 (0.4%), respectively, demonstrated AmpC bands.
All 16 AmpC-producing isolates yielded a positive three-dimensional test result with at least one of the two surface organisms. For most isolates, the growth patterns of both surface organisms were similar and relatively easy to interpret (Fig. 1A). The extract of one E. coli isolate, however, inhibited the growth of one surface organism uniformly along the entire length of the slit (Fig. 1B) and thereby interfered with interpretation of the test result. In contrast, no inhibition was observed along the slit with the second surface organism (Fig. 1C). MacConkey agar markedly suppressed growth from unlysed Proteus cells (Fig. 1D), and its use allowed easier interpretation of test results (Fig. 1E). Positive test results were seen with extracts of the three control strains. An extract of only 1 of the 29 non-AmpC-producing isolates that yielded decreased cefoxitin zone diameters was associated with a positive three-dimensional test result. The extract source was a K. pneumoniae isolate that harbored two ESBLs (of the SHV type). The positive three-dimensional test result was partially reversed when a disk containing clavulanic acid (Augmentin; Becton Dickinson Microbiology Systems) was added to the extract (140 µl) prior to injection into the slit.
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-lactam alone.
In contrast, for 11 of the 16 AmpC producers, the addition of 4 µg of
Ro 48-1220 per ml to cefoxitin decreased the MIC at least fourfold
relative to the MIC of the drug alone (Table 1). Fourfold or greater
differences in MICs were achieved for the remaining five isolates, one
E. coli isolate (M752) and four K. pneumoniae
isolates, in the presence of higher concentrations of the inhibitor Ro
48-1220 (8 and 32 µg of Ro 48-1220 per ml, respectively) (data not
shown). Fourfold or greater differences in MICs were also obtained with
ceftriaxone and 4 µg of the inhibitor Ro 48-1220 per ml for the same
11 isolates compared with those of cefoxitin (data not shown).
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17 mm; MIC, 
16 µg/ml) demonstrated no
cloxacillin-inhibited band by IEF, the MICs of several drugs were
determined for these organisms. Table 2
shows the MICs at which 50% of isolates are inhibited
(MIC50s), MIC90s, and MIC100s of
these drugs.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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E. coli, K. pneumoniae, and P. mirabilis are the species in the family
Enterobacteriaceae that are most commonly isolated in the
clinical laboratory (16, 26). However, few studies have
assessed the occurrence of AmpC beta-lactamases among these species.
Gazouli et al. (11) tested 2,133 E. coli isolates
from 10 Greek hospitals and found that 63 (3%) had cefoxitin zone
diameters of 14 mm. Eight isolates lacked an outer membrane protein,
and 55 (2.6%) contained AmpC beta-lactamases on the basis of the
results of hydrolysis and inhibition studies and hybridization tests
with AmpC-specific probes. Cefoxitin resistance was transferred for only a "few" isolates. These results mimic our results for E. coli, wherein 1.9 and 1.6% of the isolates demonstrated decreased susceptibility to cefoxitin and AmpC bands, respectively. Similar data
have not been reported for K. pneumoniae, but our results indicate that this species harbors AmpC enzymes less frequently (1.1%)
than E. coli does. The higher incidence of AmpC
beta-lactamases in E. coli may reflect two modes of
production: hyperproduction of chromosome-mediated AmpC and
plasmid-mediated AmpC beta-lactamases. On a comparative note, these
enzymes were present in E. coli and K. pneumoniae
isolates at our institution less frequently than ESBLs (4 and 19%,
respectively), as reported previously (10). Reports of
plasmid-mediated AmpC in P. mirabilis are rare (6, 42), while the first isolation of a chromosomally encoded AmpC in
this species was reported only in 1998 (8).
The current NCCLS documents do not indicate the screening and
confirmatory tests that should be used for the detection of AmpC
beta-lactamases in K. pneumoniae and E. coli
(24). We used the standard disk diffusion breakpoint for
cefoxitin (zone diameter, <18 mm) to screen isolates and the
three-dimensional extract test as a confirmatory test. Our results
indicate that the disk diffusion test has poor specificity, especially
with K. pneumoniae isolates. Of the 45 isolates with
decreased cefoxitin susceptibility, 29 (64%) were non-AmpC producers,
and 24 (83%) of these were K. pneumoniae. Had we used a
cefoxitin zone diameter of 14 mm as the criterion for the screening
of isolates (11), the number of non-AmpC producers would
have decreased from 29 to 10 (all K. pneumoniae), but we also would have failed to detect 2 (E. coli) of the 16 (13%) AmpC producers. Cefoxitin resistance in non-AmpC producers may
be due to a lack of permeation of porin (28) (see below).
The results of this study underscore the need for reliable laboratory
tests that confirm the presence of AmpC beta-lactamases in clinical isolates.
All 16 of the AmpC-producing and 1 of the 29 non-AmpC-producing isolates were positive by the three-dimensional extract test. An extract of one of the AmpC producers inhibited the growth of one surface organism (Fig. 1B). Because this test is a confirmatory test and the additional cost is minimal, the use of two indicator organisms is recommended. The reason for the false-positive result with the non-AmpC producer was unclear and is the focus of ongoing studies. A limitation of methods used to detect the AmpC enzyme is that an increasing number of clinical isolates have multiple beta-lactamases, which in turn can make inhibition patterns complex and difficult to interpret (37, 38). The isolate with the discrepant extract test result harbored two SHV-type ESBLs. However, group 2be beta-lactamases usually are not active against cephamycins. Interestingly, two other non-AmpC-producing K. pneumoniae isolates also harbored two SHV-type ESBLs each but were negative by the extract test.
Several features of the in vitro susceptibility testing results (Table 1) were worthy of note. The results of screening of two AmpC-producing E. coli isolates (M656 and M683, Table 1) by the disk diffusion method were borderline, with initial cefoxitin zone diameter readings of 17 mm and readings of 18 mm on repeat testing (data not shown). By using the current breakpoint of 18 mm (24) as the cutoff criterion in the screening test, these isolates nearly missed detection. These results also suggest that the true frequency of AmpC producers may be somewhat higher than the 1.2% stated above. The cefoxitin MICs for these isolates were within the susceptible range, as were the MICs of ceftriaxone, cefotaxime, ceftazidime, aztreonam, cefepime, and imipenem (Table 1). Interestingly, Bauernfeind et al. (2) recently isolated a clinically significant strain of K. pneumoniae that harbored a novel type of AmpC beta-lactamase and that also demonstrated a low level of activity against cephamycins (cefoxitin MIC, 4 µg/ml). These data suggest that although screening methods which use cefoxitin in standardized methods to detect AmpC-harboring isolates are useful, they are not perfect.
The MICs of -lactams for the other AmpC-producing isolates tested in
this study were variable but were generally lower than those reported
elsewhere for K. pneumoniae and E. coli isolates that harbor plasmid-mediated AmpC enzymes (Table 1) (7, 11, 17). These results may be due to the screening method, which was
designed to include isolates with borderline susceptibility to
cephamycins. As seen in early reports, several AmpC producers were
resistant to many expanded-spectrum -lactams including cephamycins but were susceptible to "fourth-generation" cephalosporins (e.g., cefepime) and carbapenems. Given that AmpC producers are typically resistant to cephamycins and susceptible to fourth-generation cephalosporins and that ESBL producers are frequently susceptible to
cephamycins and variably resistant to fourth-generation cephalosporins (17), it is of therapeutic interest for a clinical
laboratory to distinguish between these beta-lactamases. These issues
become more significant with the ever increasing number of reports of AmpC- or ESBL-producing organisms for which the MICs of
expanded-spectrum -lactams are low and which are associated with
clinical disease (2, 32).
Tzouvelekis et al. (40) reported that Ro 48-1220, a potent AmpC enzyme inhibitor, at a concentration of 4 µg/ml protected ceftriaxone and ceftazidime against organisms that produced group 1 or 2be beta-lactamases. In our study, this inhibitor at the same concentration protected cefoxitin against most AmpC producers. However, up to eight times greater inhibitor concentration was required to ensure protection against five isolates that harbored at least one other beta-lactamase in addition to AmpC (Table 1). Ro 48-1220 inhibits both extended-spectrum and AmpC beta-lactamases, and this may account for the increased inhibitor concentrations needed to ensure protection against these isolates.
In our study, 64% of all isolates with decreased susceptibility to
cefoxitin failed to harbor an AmpC beta-lactamase. Because most of
these isolates (83%) were K. pneumoniae, MICs were
determined (Table 2) and were compared to the MICs for AmpC-producing
K. pneumoniae. Some overlap in MIC endpoints was seen
between isolates that produced AmpC enzymes and isolates that did not
produce these enzymes (Table 1), thereby making it difficult to
distinguish both groups on the basis of phenotypic results. The
MIC100 data demonstrate that for some non-AmpC producers
cefoxitin MICs are greater than those for the majority of the AmpC
producers. These data corroborate the results of the cefoxitin disk
test, which was used to initially screen isolates for AmpC production;
this test was nonspecific (see above). Cephamycin resistance in
non-AmpC-producing K. pneumoniae strains is often due to
porin-deficient mutants (1, 28). Hernandez-Alles et al.
(12) demonstrated that interruption of a porin gene by
insertion sequences is a common type of mutation that causes the loss
of porin expression and increased cefoxitin resistance in K. pneumoniae. In our study, large differences (3 twofold
dilutions) in MICs between cefpodoxime or ceftriaxone with and without
clavulanic acid were seen for 7 of the 24 Klebsiella
non-AmpC producers, and all differences were attributed to the presence
of ESBLs (data not shown).
In summary, we have demonstrated that at our institution the overall rate of occurrence of relatively high levels of AmpC beta-lactamase production in nonrepeat E. coli, K. pneumoniae, and P. mirabilis isolates was 1.2%. Cefoxitin resistance was transferred for half of the 16 AmpC producers. This is significant in light of recent reports which suggest that these new plasmid-mediated enzymes may create serious therapeutic problems in the future (18, 31). For a relatively large number of cefoxitin-resistant K. pneumoniae isolates (86%), cephamycin resistance was not associated with the AmpC enzyme. The three-dimensional extract test was a reliable method of detection of isolates that harbor the AmpC enzyme.
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ACKNOWLEDGMENTS |
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We thank Patricia A. Bradford for providing E. coli
DH5(pCLL3414), which expressed the ACT-1 -lactamase and which
was used as a standard in IEF testing (7). We also thank
Michael W. Climo for the testing of isolates by pulsed-field gel electrophoresis.
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FOOTNOTES |
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* Corresponding author. Mailing address: Pathology and Laboratory Medicine Service/113, McGuire Veterans Affairs Medical Center, 1201 Broad Rock Blvd., Richmond, VA 23249-0001. Phone: (804) 675-5809. Fax: (804) 675-5518. E-mail: Philip.Coudron{at}med.va.gov.
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Abstract
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