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Antimicrobial Agents and Chemotherapy, June 2001, p. 1879-1881, Vol. 45, No. 6
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.6.1879-1881.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Expression of Multidrug Efflux Pump SmeDEF by
Clinical Isolates of Stenotrophomonas maltophilia
Ana
Alonso and
Jose L.
Martinez*
Departamento de Biotecnología
Microbiana, Centro Nacional de Biotecnología, CSIC, Campus
Universidad Autónoma de Madrid, Cantoblanco, 28049-Madrid, Spain
Received 11 October 2000/Returned for modification 6 February
2000/Accepted 16 March 2001
 |
ABSTRACT |
The presence of the multidrug efflux pump SmeDEF was assessed in a
collection of clinical isolates of Stenotrophomonas
maltophilia. All isolates encoded this pump, as demonstrated by
PCR. Forty-seven percent of the strains overproduced a protein of the
same size that was immunoreactive against an anti-SmeF antibody, and
33% overexpressed the gene semD when they were tested by
reverse transcription-PCR. A correlation between smeDEF
overexpression and antibiotic resistance was observed.
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TEXT |
Stenotrophomonas
maltophilia is an opportunistic pathogen that has been associated
with different human pathologies (5) and that is
considered an archetype of antibiotic-resistant bacteria. It has been
demonstrated that S. maltophilia produces beta-lactamases (10, 13, 14), aminoglycoside acetyltransferases
(7), and erythromycin-inactivating enzymes
(1), with an obvious role in the antibiotic resistance
phenotype of this bacterial species. Like all bacterial species studied
so far (9, 11), S. maltophilia probably
contains several multidrug resistance (MDR) efflux pumps, as
demonstrated by the analysis of multidrug-resistant mutants that can be
obtained upon antibiotic selective pressure (15). In a
recent article, we have described smeDEF, the first MDR pump
so far cloned and characterized in this bacterial species (2). In the present work, we have analyzed both the
presence and the levels of expression of the SmeDEF pump in a
collection of clinical isolates of S. maltophilia and
correlated the results of those analyses with those obtained from the
study of the susceptibilities of such isolates to antibiotics.
The clinical strains of S. maltophilia used in the present
work were isolated from different sources at the Hospital of Mostoles (Madrid, Spain) (Table 1). S. maltophilia strain D457R is a single-step multidrug-resistant
spontaneous mutant that is derived from S. maltophilia D457
and that overexpresses the SmeDEF multidrug efflux system
(3). Bacteria were routinely cultured at 37°C in
Luria-Bertani medium (4). The MICs of antibiotics were
determined in Mueller-Hinton agar (4) by the E-test method
(AB Biodisk), according to the manufacturer's instructions.
To assess the presence of the smeDEF efflux system among
S. maltophilia isolates, chromosomal DNA (obtained with the
Genome DNA Kit; Bio 101) from 15 clinical strains obtained from
different clinical sources was subjected to PCR amplification with
primers specific for the smeD gene. An internal fragment of
150 bp from the smeD gene was amplified with primers 1 (5'-CCAAGAGCCTTTCCGTCAT-3') and 2 (5'-TCTCGGACTTCAGCGTGAC-3'). A total of 100 ng of
chromosomal DNA was used as the template for each PCR. The reaction
mixture (50 µl) contained each deoxynucleotide (dCTP, dGTP, dATP and
dTTP) at a concentration of 0.2 mM, 0.5 µM each primer, 1.5 mM
MgCl2, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 1.0 U of
Taq DNA polymerase. The mixture was heated at 94°C for
90 s, followed by 35 cycles of 30 s at 94°C, 60 s at
58°C, and 90 s at 72°C and, finally, one 10-min extension step
at 72°C before the end of the reaction. PCR products were run on
2.0% agarose gels and stained with ethidium bromide. Figure
1 shows that a PCR product of the
expected size was detected in all strains analyzed. This demonstrates
that the smeDEF system was ubiquitously found in S. maltophilia isolates.
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FIG. 1.
Presence of the smeDEF efflux pump in the
genomes of clinical isolates of S. maltophilia. Lane M,
molecular size markers; lane C , negative control, for which the same
PCR reaction was performed but without the addition of DNA. A band of
the predicted size was observed in all isolates.
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|
It has been described that overexpression of the outer membrane protein
SmeF, the gene of which is a part of the smeDEF operon, is
associated with increased levels of resistance for in vitro-selected multidrug-resistant mutants of S. maltophilia (2,
3). To test whether overexpression of the smeDEF
operon was also associated with increased levels of resistance in
clinical isolates of S. maltophilia, we analyzed both the
expression of the SmeF protein by Western blotting and smeD
RNA by reverse transcription-PCR (RT-PCR). Whole-cell extracts from
clinical S. maltophilia isolates were electrophoresed on
sodium dodedcyl sulfate (SDS)-polyacrylamide gels and immunoblotted as
described previously with a polyclonal antibody raised against the
outer membrane efflux component of SmeDEF, SmeF (2). As
shown in Fig. 2, an immunoreactive band of the same size as SmeF was detected in 7 of 15 clinical isolates (strain D457R is not a clinical isolate but is a mutant derivative that
has been included in the analysis as a control).
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FIG. 2.
Expression of SmeF by clinical isolates of S. maltophilia. Whole-cell protein extracts of S. maltophilia were obtained and electrophoresed in SDS-10%
polyacrylamide gels. Western blot analysis was performed with an
anti-SmeF antibody.
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|
Some outer membrane components of efflux MDR pumps, such as OprM of
Pseudomonas aeruginosa (8, 16) and TolC of
Escherichia coli (6), are associated with more
than one MDR system. In order to assess if SmeF expression was linked
to the expression of the smeDEF operon, we analyzed the
expression of smeD, the first gene of the operon, by RT-PCR
analysis. RNA was obtained with the TriReagent-LS Kit (Molecular
Research Center Inc.) according to the manufacturer's instructions.
Residual DNA was eliminated with RNase-free DNase (Boehringer
Mannheim), followed by acid phenol treatment and RNA precipitation with
ethanol. The RNA concentration and purity were estimated by
spectrophotometric analysis at 260 and 280 nm, and the RNA quality was
analyzed by electrophoresis on 1% agarose gels under denaturing
conditions (12). For RT-PCR analysis, a two-step reaction
was carried out. First, 5 µg of RNA was incubated for 1 h at
37°C with primer 2 and avian myeloblastosis virus (AMV) reverse
transcriptase (U.S. Biochemicals) to obtain cDNA. cDNA was then PCR
amplified with primers 1 and 2 as described above. Amplicons were
analyzed on 2% agarose gels and stained with ethidium bromide. The
same reaction protocol performed for RT-PCR was carried out for each
sample, but without AMV reverse transcriptase, as a control to detect
DNA contamination in the RNA preparations, with negative results in all
cases. The amount of smeD RNA was analyzed from bacteria
grown to the early exponential or stationary growth phase because it
was previously demonstrated that smeDEF expression is
induced at the exponential phase of growth and is very low at the
stationary phase of growth (2). As shown in Fig.
3, an RT-PCR product of the predicted
size was found in 5 of the 15 isolates analyzed. All of the isolates
with a positive RT-PCR result also expressed a protein immunoreactive against the anti-SmeF antiserum, indicating that expression of smeD and SmeF was linked in these strains. Two isolates
(isolates E301 and E759), however, overproduced a protein that was of
the same size as SmeF and that was immunoreactive against the SmeF antibody, but a RT-PCR band was not detected in our analysis. Two
possibilities might explain these results: (i) the immunoreactive band
detected in E301 and E759 was SmeF, but its expression was associated
with other efflux systems in these strains, so that SmeF was expressed
but smeD was not; (ii) the immunoreactive band detected in
E301 and E759 was not SmeF but another protein which shares some
structural features with SmeF and which is then recognized with the
polyclonal anti-SmeF antibody, so that neither SmeF nor smeD
was expressed in these isolates. In any case, it is thus clear that
smeDEF is not expressed by these two strains.
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FIG. 3.
Expression of smeDEF RNA by clinical isolates
of S. maltophilia. The expression of smeDEF was
analyzed by RT-PCR. RNAs obtained at the exponential (lanes a) and
stationary (lanes b) growth phases were analyzed. A clear band of the
predicted size was observed for strains D457R, C357, E729, E923, and
F375; and a very faint band was observed for strain D457. Note that
strain D457R is not a clinical isolate but a multidrug-resistant mutant
derived from strain D457 (3), and it has been included in
the present analysis as a control for smeDEF expression.
Lane M, molecular size markers (from top to bottom, 404, 331, 242, 190, 157, 147, and 112 bp); C+, positive control, for which PCR was
performed with chromosomal DNA from S. maltophilia D457 as
the template; C, negative control, for which PCR was performed
without DNA.
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The expression of smeDEF was correlated with the
susceptibilities to antibiotics of the collection of clinical isolates
described in Table 1. The MICs of tetracycline, chloramphenicol,
erythromycin, and the quinolones were, overall, higher for the strains
in which expression of SmeF was detectable by Western blotting and in
which smeD was detectable by RT-PCR (33% of the isolates)
than for the isolates that did not express the protein at detectable
levels. This relationship was not observed for the MICs of amikacin or ticarcillin-clavulanic acid. Noteworthy increased levels of resistance to tetracycline, chloramphenicol, erythromycin, and the quinolones, with no effect on the MIC of amikacin or ticarcillin-clavulanic acid,
were observed in the multidrug-resistant mutant S. maltophilia strain D457R (which overexpresses SmeF) compared to
its isogenic wild-type S. maltophilia strain D457 (Table
2). The same substrate range was observed
when smeDEF was cloned in a low-copy-number plasmid and
introduced in the heterologous host E. coli
(3).
Our data indicate that smeDEF is overexpressed by 33% of
the clinical isolates analyzed in our work and suggest that its
overexpression might contribute to increased levels of resistance to
tetracycline, chloramphenicol, erythromycin, and quinolones in clinical
isolates of S. maltophilia.
| |
ACKNOWLEDGMENTS |
Thanks are given to Ignacio Alós for the gift of the clinical
isolates used in the present work.
The present research was aided in part by grant 08.2/022/98 from
CAM. A. Alonso was a recipient of a fellowship from Gobierno Vasco.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Biotecnología Microbiana, Centro Nacional de
Biotecnología, CSIC, Campus Universidad Autónoma de
Madrid, Cantoblanco, 28049-Madrid, Spain. Phone: 34-91-5854571. Fax:
34-91-5854506. E-mail: jlmtnez{at}cnb.uam.es.
| |
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Antimicrobial Agents and Chemotherapy, June 2001, p. 1879-1881, Vol. 45, No. 6
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.6.1879-1881.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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