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Journal of Clinical Microbiology, May 2001, p. 1985-1988, Vol. 39, No. 5
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1985-1988.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Evidence of In Vivo Transfer of a Plasmid Encoding the
Extended-Spectrum 
-Lactamase TEM-24 and Other Resistance Factors
among Different Members of the Family
Enterobacteriaceae
Catherine
Neuwirth,1,*
Eliane
Siebor,1
Andre
Pechinot,1
Jean-Marie
Duez,1
Michele
Pruneaux,1
Frederic
Garel,1
Antoine
Kazmierczak,1 and
Roger
Labia2
Laboratoire de Bactériologie,
Hôpital Universitaire du Bocage, 21034 Dijon
Cedex,1 and CNRS, UBO, MNHN, 29000 Quimper,2 France
Received 10 October 2000/Returned for modification 25 January
2001/Accepted 7 March 2001
 |
ABSTRACT |
The epidemiological study of several multidrug-resistant
Enterobacteriaceae isolated from five patients demonstrated
in vivo dissemination of a 100-kb plasmid encoding the
extended-spectrum 
-lactamase TEM-24 from a clonal strain of
Enterobacter aerogenes to different strains of
Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris,
Proteus mirabilis, and Serratia marcescens.
| |
TEXT |
In France, plasmid-mediated
extended-spectrum beta-lactamases (ESBLs) have been mostly described
from strains of Klebsiella pneumoniae (1, 2, 3, 6, 7,
15), but more recently infections caused by strains of
Enterobacter spp. producing the TEM-24 ESBL have increased
(5, 14, 24). The same phenomenon was observed in our
University Hospital (2,000 beds, in Dijon, France). In 1996, 1997, and
1998 we isolated, respectively, 16, 37, and 50 Enterobacter
aerogenes strains producing TEM-24 among totals of 78, 70, and 70 nonrepetitive ESBL-producing strains. All these strains were analyzed
by pulsed-field gel electrophoresis (PFGE). During our continuous
survey we found that five patients were cocolonized or coinfected with
different multidrug-resistant species of enterobacteria. Following the
use of imipenem, two strains of Proteus mirabilis and
E. aerogenes resistant to this molecule were recovered from
one patient. We report here the epidemiological study and the
-lactamase characterization of all the strains isolated from the
five patients.
The origins of the strains are given in Table
1. The detection of ESBL production was
performed by the double-disk synergy test (19) but with a
quarter of the disk containing third-generation cephalosporin for
Proteus sp. (9).
Analyses of chromosomal DNAs by PFGE were performed as described
previously (15) but with a pulse range from 40 to 5 s
for 20 h at 180 V for strains of E. aerogenes, K. pneumoniae, Serratia marcescens, and Escherichia
coli. For P. mirabilis and Proteus vulgaris,
we used a pulse range from 25 to 5 s for 20 h at 180 V (Fig.
1 and 2). A single
profile was found for the strains of E. aerogenes, similar
to that of the epidemic strain described in 1996 (24). For
the other enterobacteria, strains from the same species (ESBL or not
ESBL producing) isolated from the same patient shared concordant PFGE
patterns, suggesting their clonal origin. Nevertheless, the strains of
the same species isolated from the five patients were not related. This
result excluded the possibility that resistant strains of K. pneumoniae, E. coli, or P. vulgaris were disseminated
between the patients or that there was a common source of
contamination.
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FIG. 1.
PFGE of total DNAs from E. aerogenes
(lanes A to E), K. pneumoniae (lanes F to H), S. marcescens (lanes I to K), and E. coli
(lanes L to O) cut by XbaI. Lane A, patient 1 isolate; lane
B, patient 3 isolate; lane C, patient 2 isolate; lane D, patient 4 isolate; lane E, patient 5 isolate; lane F, patient 1 isolate; lane G,
patient 3 isolate; lane H, patient 2 isolate; lanes I and J, patient 5 isolate; lane K, unrelated strain; lane L, patient 1 isolate; lane M,
patient 3 isolate; lanes N and O, patient 4 isolate.
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FIG. 2.
PFGE of total DNAs from P. vulgaris (lanes A
to F) and P. mirabilis (lanes G to L) cut by
SmaI. Lane A, patient 1 isolate; lane B, patient 2 isolate;
lanes C to F, unrelated strains; lanes G and H, patient 4 isolate;
lanes I to L, unrelated strains.
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A large plasmid of about 100 kb was isolated by the method of Birnboim
and Doly from the ESBL-producing strains (4). The restriction patterns obtained after digestion of the plasmid by EcoRI were very similar. The plasmid was easily transferred
from E. aerogenes, K. pneumoniae, P. vulgaris, P. mirabilis,
and S. marcescens to E. coli K-12 C600,
which is resistant to sodium azide (selection with 256 µg of sodium
azide per ml and 8 µg of netilmicin per ml) or from E. coli to K. pneumoniae 10031, which is resistant to
rifampin (selection with 100 µg of rifampin per ml and 8 µg of
netilmicin per ml). Resistance to -lactams was cotransferred with
resistance to aminoglycosides (amikacin, kanamycin, netilmicin,
tobramycin), sulfonamides, and chloramphenicol. The MICs of -lactams
(Table 2) were determined in
Mueller-Hinton broth by a microdilution method for the clinical
strains and their transconjugants. The levels of resistance were very
similar among the transconjugants. For the P. mirabilis
strains, extended-spectrum cephalosporin MICs were very low. This
may explain why it was difficult to detect ESBL production in this
species. P. mirabilis and E. aerogenes
strains isolated from patient 4 in February and in March were resistant
to imipenem (respectively, MICs of 8 and 16 to 32 µg/ml). Isoelectric
focusing was performed as previously reported (24). The
-lactamase activity was located in the gels by an iodine starch
procedure (20). A -lactamase with a pI of 6.5 was
detected in all the ESBL-producing strains as well as in their
transconjugants. PCR was performed on plasmids extracted from the
transconjugants and from the non-ESBL-producing P. mirabilis strain which was resistant to imipenem with primers J (forward, 5'-CTTATTCCCTTTTTTGCGGC-3') and E (reverse,
5'-GGTCTGACAGTTACCAATGC-3') (8) at positions
236 and 1079 of the TEM family gene -lactamase according to
Sutcliffe numbering (25). The sequence of the gene encoding the -lactamase with a pI of 5.4 produced by the P. mirabilis strain was identical to that of TEM-1b (16,
25), and the sequence of the gene encoding the -lactamase
with a pI of 6.5 was identical to that of the extended-broad-spectrum
-lactamase TEM-24b (8, 17).
These results demonstrate clearly that there has been an in vivo
transfer of the plasmid encoding ESBL TEM-24 from E. aerogenes to K. pneumoniae, E. coli, P. mirabilis, S. marcescens, and P. vulgaris. In all cases, the
ESBL was first detected in the strain of E. aerogenes
and only later in other species in a site colonized by E. aerogenes. For patients 1, 2, and 3 we unfortunately did not keep
the non-ESBL-producing species of Enterobacteriaceae isolated before the E. aerogenes strain. The transfer
probably occurred in the wound of patient 2 because the resistant
strains of K. pneumoniae and P. vulgaris were
never found in the stools. For the two other patients, we isolated the
non-ESBL-producing strain (E. coli and P. mirabilis for patient 4, S. marcescens for
patient 5) from the same site as we did the identical TEM-24-producing strain. The transfer in vivo of plasmid has already been described (12, 21, 22, 24), but each report concerned only one
patient. This study, the first one describing five patients, proves
that the spread of plasmid is no longer exceptional and can concern species for which the ESBL TEM-24 had not yet been described, like
P. vulgaris.
The analysis of the outer membrane proteins of the strains of P. mirabilis and E. aerogenes resistant to imipenem
and isolated from patient 4 was carried out as previously reported
(Fig. 3) (18, 23). A band
of 40 kDa was not detected in the strain of E. aerogenes that is resistant to imipenem as already reported (10, 11, 13). In the P. mirabilis strain
resistant to imipenem all the major bands were present. The resistance
was probably due to some modifications in the penicillin-binding
proteins, which we already described for this species
(23). This report is the first description of the
selection of two different species of Enterobacteriaceae
resistant to imipenem in a single patient following treatment with
imipenem. If such strains were to be more often isolated, there would
soon be no medical therapies available.
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FIG. 3.
Outer membrane protein profiles of P. mirabilis and E. aerogenes. Lane A, P. mirabilis ATCC 29906; lane B, P. mirabilis
imipenem-resistant strain from patient 4; lane C, E. aerogenes imipenem-susceptible strain from patient 4; lane D,
E. aerogenes imipenem-resistant strain from patient 4. Molecular mass standards in kilodaltons are given on the
left.
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In conclusion, the E. aerogenes strain producing TEM-24
isolated in our hospital represents a serious danger: it spreads very easily and is at the origin of plasmid dissemination among
Enterobacteriaceae.
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Bactériologie, Hôpital Universitaire du Bocage, B.P. 1542, 21034 Dijon Cedex, France. Phone: 33-3 80 29 32 60. Fax: 33-3 80 29 36 67. E-mail: catherine.neuwirth{at}chu-dijon.fr.
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REFERENCES |
| 1.
|
Arlet, C.,
M. Rouveau,
I. Casin,
P. J. M. Bouvet,
P. H. Lagrange, and A. Philippon.
1994.
Molecular epidemiology of Klebsiella pneumoniae strains that produce SHV-4 -lactamase and which were isolated in 14 French hospitals.
J. Clin. Microbiol.
32:2553-2558[Abstract/Free Full Text].
|
| 2.
|
Bermudes, H.,
C. Arpin,
F. Jude,
Z. El-Harrif,
C. Bebear, and C. Quentin.
1997.
Molecular epidemiology of an outbreak due to extended-spectrum -lactamase-producing enterobacteria in a French hospital.
Eur. J. Clin. Microbiol. Infect. Dis.
16:523-527[CrossRef][Medline].
|
| 3.
|
Bingen, E. H.,
P. Desjardins,
G. Arlet,
F. Bourgeois,
P. M. Kurkdjan,
N. Y. Lambert-Zechovsky,
E. Denamur,
A. Philippon, and J. Elion.
1993.
Molecular epidemiology of plasmid spread among extended broad-spectrum -lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital.
J. Clin. Microbiol.
31:179-184[Abstract/Free Full Text].
|
| 4.
|
Birnboim, H. C., and J. Doly.
1979.
Rapid alkaline extraction procedure for screening recombinant plasmid DNA.
Nucleic Acids Res.
7:1513-1523[Abstract/Free Full Text].
|
| 5.
|
Bosi, C.,
A. Davin-Regli,
C. Bornet,
M. Mallea,
J. M. Pages, and C. Bollet.
1999.
Most Enterobacter aerogenes strains in France belong to a prevalent clone.
J. Clin. Microbiol.
37:2165-2169[Abstract/Free Full Text].
|
| 6.
|
Brun-Buisson, C.,
P. Legrand,
A. Philippon,
F. Montralvers,
M. Ansquer, and J. Duval.
1987.
Transferable enzymatic resistance to third-generation cephalosporins during nosocomial outbreak of multiresistant Klebsiella pneumoniae.
Lancet
ii:302-306.
|
| 7.
|
Chanal, C. M.,
D. L. Sirot,
A. Petit,
R. Labia,
A. Morand,
J. L. Sirot, and R. A. Cluzel.
1989.
Multiplicity of TEM-derived -lactamase from Klebsiella pneumoniae strains isolated at the same hospital and relationships between the responsible plasmids.
Antimicrob. Agents Chemother.
33:1915-1920[Abstract/Free Full Text].
|
| 8.
|
Chanal, C.,
M. C. Poupart,
D. Sirot,
R. Labia,
J. Sirot, and R. Cluzel.
1992.
Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 -lactamase genes.
Antimicrob. Agents Chemother.
36:1817-1820[Abstract/Free Full Text].
|
| 9.
|
Chanal, C.,
D. Sirot,
J. P. Romaszko,
L. Bret, and J. Sirot.
1996.
Survey of prevalence of extended-spectrum -lactamase among Enterobacteriaceae.
J. Antimicrob. Chemother.
38:127-132[Abstract/Free Full Text].
|
| 10.
|
Charrel, R. N.,
J. M. Pagès,
P. De Micco, and M. Mallea.
1996.
Prevalence of outer membrane porin alteration in -lactam antibiotic-resistant Enterobacter aerogenes.
Antimicrob. Agents Chemother.
40:2854-2858[Abstract].
|
| 11.
|
Chow, J. W., and D. M. Shlaes.
1991.
Imipenem resistance associated with the loss of a 40 kDa outer membrane protein in Enterobacter aerogenes.
J. Antimicrob. Chemother.
28:499-504[Abstract/Free Full Text].
|
| 12.
|
d'Agata, E.,
L. Venkataraman,
P. De Girolami,
L. Weigel,
M. Samore, and F. Tenover.
1998.
The molecular and clinical epidemiology of Enterobacteriaceae-producing extended-spectrum -lactamase in a tertiary care hospital.
J. Infect.
36:279-285[CrossRef][Medline].
|
| 13.
|
de Champs, C.,
C. Henquell,
D. Guelon,
D. Sirot,
N. Gazuy, and J. Sirot.
1993.
Clinical and bacteriological study of nosocomial infections due to Enterobacter aerogenes resistant to imipenem.
J. Clin. Microbiol.
31:123-127[Abstract/Free Full Text].
|
| 14.
|
de Champs, C.,
D. Sirot,
C. Chanal,
R. Bonnet, and J. Sirot.
2000.
A 1998 survey of extended-spectrum -lactamases in Enterobacteriaceae in France. The French Study Group.
Antimicrob. Agents Chemother.
44:3177-3179[Abstract/Free Full Text].
|
| 15.
|
Gouby, A.,
C. Neuwirth,
G. Bourg,
N. Bouzigues,
M. J. Carles-Nurit,
E. Despaux, and M. Ramuz.
1994.
Epidemiological study by pulsed-field gel electrophoresis of an outbreak of extended-spectrum -lactamase-producing Klebsiella pneumoniae in a geriatric hospital.
J. Clin. Microbiol.
32:301-305[Abstract/Free Full Text].
|
| 16.
|
Goussard, S., and P. Courvalin.
1991.
Sequence of the genes blaT-1B and blaT-2.
Gene
102:71-73[CrossRef][Medline].
|
| 17.
|
Goussard, S., and P. Courvalin.
1999.
Updated sequence information for TEM -lactamase genes.
Antimicrob. Agents Chemother.
43:367-370[Abstract/Free Full Text].
|
| 18.
|
Hopkins, J. M., and K. J. Towner.
1990.
Enhanced resistance to cefotaxime and imipenem associated with outer membrane protein alterations in Enterobacter aerogenes.
J. Antimicrob. Chemother.
25:49-55[Abstract/Free Full Text].
|
| 19.
|
Jarlier, V.,
M. H. Nicolas,
G. Fournier, and A. Philippon.
1988.
Extended broad-spectrum -lactamases conferring transferable resistance to newer -lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns.
Rev. Infect. Dis.
10:867-878[Medline].
|
| 20.
|
Labia, R.,
M. Barthélemy, and J. M. Masson.
1976.
Multiplicité des bêta-lactamases: un problème d'isoenzymes?
C. R. Acad. Sci.
283D:1597-1600.
|
| 21.
|
Marchandin, H.,
C. Carrière,
D. Sirot,
H. Jean-Pierre, and H. Darbas.
1999.
TEM-24 produced by four different species of Enterobacteriaceae, including Providencia rettgeri, in a single patient.
Antimicrob. Agents Chemother.
43:2069-2073[Abstract/Free Full Text].
|
| 22.
|
Marchandin, H.,
H. Jean-Pierre,
C. de Champs,
D. Sirot,
H. Darbas,
P. F. Perigault, and C. Carrière.
2000.
Production of a TEM-24 plasmid-mediated extended-spectrum -lactamase by a clinical isolate of Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
44:213-216[Abstract/Free Full Text].
|
| 23.
|
Neuwirth, C.,
E. Siébor,
J. M. Duez,
A. Péchinot, and A. Kazmierczak.
1995.
Imipenem resistance in clinical isolates of Proteus mirabilis associated with alterations in penicillin-binding proteins.
J. Antimicrob. Chemother.
36:335-342[Abstract/Free Full Text].
|
| 24.
|
Neuwirth, C.,
E. Siébor,
J. Lopez,
A. Péchinot, and A. Kazmierczak.
1996.
Outbreak of TEM-24-producing Enterobacter aerogenes in an intensive care unit and dissemination of the extended-spectrum -lactamase to other members of the family Enterobacteriaceae.
J. Clin. Microbiol.
34:76-79[Abstract].
|
| 25.
|
Sutcliffe, J. G.
1978.
Nucleotide sequence of the ampicillin resistance gene of Escherichia coli pBR322.
Proc. Natl. Acad. Sci. USA
75:3737-3741[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, May 2001, p. 1985-1988, Vol. 39, No. 5
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1985-1988.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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