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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, September 2000, p. 2534-2536, Vol. 44, No. 9
Department of Microbiology, School of
Medicine,1 and University Hospital V. Macarena,2 Seville, and Area of
Microbiology, Department of Biology, and IMEDEA (CSIC-UIB), University
of Illes Balears, Palma de Mallorca,3 Spain
Received 15 November 1999/Returned for modification 19 March
2000/Accepted 26 May 2000
Forty clonally related clinical isolates of Escherichia
coli from hospitalized patients were resistant to cefoxitin
(MICs, >256 µg/ml) and ceftazidime (MICs, 32 to 256 µg/ml) and
were intermediate or resistant to cefotaxime (MICs, 16 to 128 µg/ml)
but susceptible to both cefepime (MICs, 0.5 to 2 µg/ml) and imipenem
(MICs, 0.125 to 0.25 µg/ml). Resistance to Most Escherichia coli
strains do not produce clinically relevant levels of the chromosomally
encoded AmpC Forty-four cefoxitin-resistant (MICs, >32 µg/ml) isolates of
E. coli obtained from clinical samples (January to December
1994) have been studied. Forty isolates were obtained from inpatients, and four were obtained from outpatients. Identification and preliminary susceptibility testing were performed using commercial panels (panels
6P; Pasco). All 40 isolates from inpatients showed similar patterns of
resistance to
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Activities of Imipenem and Cephalosporins against Clonally
Related Strains of Escherichia coli Hyperproducing
Chromosomal
-Lactamase and Showing Altered Porin
Profiles
ABSTRACT
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-lactams was related to
high-level production of AmpC -lactamase and loss of OmpF porin.
TEXT
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-lactamase (4). Gene amplification or
mutations at either the promoter and/or the attenuator of the
structural -lactamase gene result in AmpC hyperproduction (2,
5, 11, 16, 20). This causes increased resistance to penicillins,
cephalosporins, and -lactam--lactamase inhibitor combinations.
-Lactams penetrate into gram-negative bacteria throughout
nonspecific porins (17). Two major porins have been
described in E. coli: OmpF and OmpC (24). Loss of nonspecific porins in E. coli and other
Enterobacteriaceae are related to increased levels of
resistance to -lactams, particularly when combined with production
of an efficient -lactamase(s) (1, 3, 8-10, 13, 21).
Zwitterionic cephalosporins (cefepime, cefpirome, etc.) are more active
against organisms producing increased levels of inducible chromosomal
AmpC -lactamase than are older oxyimino-cephalosporins (cefotaxime,
ceftriaxone, ceftazidime, etc.) (7). We have, however,
scarce information on the activity of zwitterionic cephalosporins
against E. coli hyperproducing AmpC. In this study, we have
evaluated the in vitro activities of imipenem and cephalosporins
against clonally related clinical isolates of E. coli
hyperproducing chromosomal -lactamase and the pattern of porin
expression in these isolates.
-lactams, fluoroquinolones, co-trimoxazole, and
aminoglycosides. A variety of resistance patterns were observed in
isolates from outpatients, none of them being identical to that of
isolates from inpatients (data not shown). Genomic DNA from the 44 clinical isolates and from strain ATCC 25922 was separated after
digestion with XbaI by pulsed-field gel electrophoresis (PFGE) as previously described (14). All 40 isolates from
inpatients presented the same PFGE pattern, in contrast to the 4 isolates from outpatients and strain ATCC 25922, which showed patterns unrelated to each other and to that of isolates from inpatients (Fig.
1). The activities of cefoxitin (Sigma,
Madrid, Spain), cefotaxime (Sigma), ceftriaxone (Sigma), ceftazidime
(Glaxo, Madrid, Spain), cefepime (Bristol-Myers Squibb, Madrid, Spain),
and imipenem (Merck Sharp and Dhome, Madrid, Spain) against all 44 isolates were determined by microdilution (15). MIC ranges
and MICs at which 90% of isolates were inhibited, respectively, for
the 40 isolates from inpatients were as follows (in micrograms per
milliliter): >256 and >256 (cefoxitin); 0.5 to 2 and 2 (cefepime), 16 to 128 and 32 (both cefotaxime and ceftriaxone), 32 to 256 and 128 (ceftazidime), and 0.125 to 0.5 and 0.5 (imipenem). All strains
were resistant to cefoxitin and ceftazidime, intermediate or resistant
to cefotaxime and ceftriaxone, and susceptible to both cefepime and
imipenem (15).
View larger version (94K):
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FIG. 1.
PFGE profiles of E. coli clinical isolates
hyperproducing chromosomal -lactamase. Lanes 1, 2, 7, and 8:
markers. Lanes 3, 4, 5, 6, 9, 10, 11, and 12: isolates HUS4/94,
HUS7/94, HUS23/94, HUS31/94, HUS34/94, HUS36/94, HUS42/94, and
HUS47/94, respectively.
Eight isolates from hospitalized patients, including organisms covering
the whole range of MICs of ceftazidime and cefotaxime for the 40 isolates from inpatients, were further studied. MICs of cephalosporins
and imipenem were determined with Etest strips (AB Biodisk, Solna,
Sweden) in the presence of clavulanic acid (2 µg/ml) or BRL 42715 (4 µg/ml). A clinical strain of Klebsiella pneumoniae
(HUS57/94) producing SHV-5 -lactamase (unpublished results) was used
as a control in these studies. MICs of both cefoxitin and cefotaxime
did not decrease in the presence of clavulanic acid. BRL 42715, however, significantly decreased the MICs of both cefoxitin and
cefotaxime (Table 1). Both -lactamase
inhibitors decreased the MIC of cefotaxime, but not of cefoxitin, for
K. pneumoniae HUS57/94 (data not shown).
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Plasmid DNA was obtained by the alkaline-lysis method (12)
and analyzed by electrophoresis in 0.7% agarose gels. All eight isolates contained a plasmid of 5.1 kb; isolate HUS23/94 additionally contained a plasmid of 8.0 kb. Attempts to transfer resistance to
cephalosporins from isolates HUS31/94, HUS23/94, HUS36/94, and HUS47/94
to E. coli J53-2 (F
pro met
Rifr) by conjugation, transformation (heat shock method),
and electroporation, using ampicillin (50 µg/ml), cefoxitin (10 µg/ml), or cefotaxime (10 µg/ml) as selective agents
(22), consistently failed. Although strains of E. coli producing AmpC-type -lactamase from plasmid have been
described (6), these results suggest that the most probable
cause of -lactam resistance in our isolates is of chromosomal origin.
Isoelectric point (pI) determinations using crude supernatants were
performed by isoelectric focusing using the Phast-System (gel pI range,
3.5 to 9; Pharmacia, Sant Cugat del Vallés, Spain). All eight
isolates produced a -lactamase with a pI of 
9. Isolate HUS23/94
also produced a -lactamase with a pI of 5.4, compatible with TEM-1.
-Lactamase activity was determined spectrophotometrically using
crude supernatants from sonicated cells. One unit of activity was
defined as the amount of enzyme that hydrolyzes 1 µmol of cephaloridine per min at 37°C. Inhibition of -lactamase was
determined after preincubation (10 min at 37°C) of supernatants with
cloxacillin (250 µM) or clavulanic acid (2 µM). Cloxacillin and
clavulanic acid inhibited 88.7 to 99.6% and 0.6 to 8.1% of the
-lactamase activity, except in isolate HUS23/94. Outer membrane
protein profiles from bacteria grown to logarithmic phase in
Mueller-Hinton broth or in nutrient broth were determined by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (12% acrylamide and
6 M urea in the running gel). OmpF porin was absent in the eight
isolates grown in either Mueller-Hinton broth or nutrient broth (Fig.
2).
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Hyperproduction of AmpC causing resistance of clinical isolates of E. coli to oxyimino-cephalosporins has been previously documented (2, 4, 16), but the epidemiological relationship of the strains included in those studies has not been reported. In this study we have shown that 40 isolates from inpatients were clonally related but epidemiologically unrelated to other AmpC-hyperproducing strains (data not shown) cultured from outpatients in the same geographical area. These 40 isolates presumably represent a single strain causing a nosocomial outbreak. Data on patients from whom the isolates were obtained are not available for a complete analysis, thus precluding an adequate epidemiological study.
The isolates described in this report were resistant to
oxyimino-cephalosporins but susceptible to both cefepime and imipenem, two compounds highly stable to AmpC -lactamases from other
enterobacteria (7). This study suggests that both cefepime
and imipenem may represent a therapeutic alternative for infections
caused by AmpC-hyperproducing E. coli strains, as suggested
for other organisms (23). Loss of both OmpF and OmpC porins
in laboratory mutants of E. coli determines an 8- to 16-fold
increase in the MICs of cephalosporins (9). Loss of OmpF
alone in E. coli mutants expressing increased amounts of
AmpC determines a two- to fourfold increase in the MICs of
cephalosporins (10). There is scarce information on the
number and nature of porins expressed by clinical isolates of E. coli, and the relationship of porin expression, if any, with
antimicrobial resistance. Reguera et al. (21) reported that
seven out of eight clinical strains of E. coli resistant to
amoxicillin-clavulanic acid expressed only OmpC or expressed OmpF with
altered electrophoretic mobility. In order to evaluate the role of
porins in antimicrobial resistance, it would be necessary to compare
Omp profiles and susceptibility data of bacteria cultured in the same
medium. OmpF, an important porin for -lactam penetration (18,
19), is not expressed in high-osmolarity media, such as
Mueller-Hinton broth, the most commonly used medium for susceptibility testing. In the isolates herein studied, we have observed that OmpF is
not expressed in Mueller-Hinton broth (as would be expected); nor is it
expressed in the low-osmolarity medium nutrient broth, suggesting that
this porin is actually not expressed by these isolates. This also
suggests that resistance due to cooperation between -lactamase
production and altered permeability demonstrated in laboratory mutants
of E. coli can also be expressed by clinical isolates.
The level of resistance to cephalosporins varied in different (but
clonally related) isolates. It does not seem probable that variations
in -lactamase activity accounts for the differences in
susceptibility to cefotaxime and ceftazidime. This is suggested by both
the poor correlation between enzyme activity and MICs (even if strain
HUS23/94 is not considered) and by the differences of MICs of cefoxitin
and cefotaxime in the presence of BRL 42715. It is possible that other
mechanisms (penicillin-binding protein expression, active efflux, etc.)
contribute to the observed phenotypes of resistance. New studies are in
progress to evaluate these possibilities.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the assistance of Janet Dawson in the preparation of the manuscript.
This work was supported in part by grants from Consolidation of Research Groups, Consejería de Educación, Junta de Andalucía, to L.M.-M., A.P. and E.J.P. and the Comisión Interministerial de Ciencia y Tecnología, Ministerio de Educación, Spain (grant PB96-0197) to V.J.B.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, School of Medicine, University of Seville, Apdo. 914, 41080 Seville, Spain. Phone: 34-95-5008287. Fax: 34-95-4377413. E-mail: lmartin{at}cica.es.
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