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Antimicrobial Agents and Chemotherapy, January 2000, p. 222-225, Vol. 44, No. 1
Service de Bactériologie, Hôpital
Tenon,1 Service de Bactériologie,
Hôpital Lariboisière,2
Service de Microbiologie, Hôpital St.
Louis,3 and Service de
Bactériologie, Hôpital Cochin,4
Paris, France
Received 23 February 1999/Returned for modification 31 May
1999/Accepted 9 October 1999
The genetic organization of the gene coding for DHA-1 and the
corresponding ampR gene was determined by PCR mapping.
These genes have been mobilized from the Morganella
morganii chromosome and inserted into a complex
sulI-type integron, similar to In6 and In7. However, they
are not themselves mobile cassettes. This integron probably includes a
specific site for recombination allowing the mobilization of diverse
resistance genes, as observed for blaCMY-1 and
blaMOX-1.
Recently, a strain of
Salmonella enterica serovar Enteritidis was shown to have a
plasmid-mediated AmpC The plasmid pSAL-1 DNA isolated from the Escherichia coli
HB101 transconjugant was partially digested with Sau3A and
ligated into the BamHI site of pACYC184. Recombinant plasmid
pSAL-2ind was introduced into E. coli JM101, and
the transformants had the same AmpC susceptibility pattern as the donor
strain, Salmonella serovar Enteritidis, and its
transconjugant E. coli HB101(pSAL-1) (5). The
whole sequence (2,126 bp) of ampC and ampR genes
and their intercistronic region was reported and analyzed previously (5). In this work, the recombinant plasmid
pSAL-2ind (4.2 kb) was entirely sequenced and analyzed with
the BLASTN program at the National Center for Biotechnology Information
(1) (Fig. 1). A segment of 1.4 kb located 319 bp downstream from ampR showed the 3' end of
ORF4 (deletion of the first 72 bp of the gene qacE
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Copyright © 2000, American Society for Microbiology. All rights reserved.
A Novel Integron in Salmonella enterica Serovar
Enteritidis, Carrying the blaDHA-1 Gene and
Its Regulator Gene ampR, Originated from
Morganella morganii
ABSTRACT
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-lactamase, named DHA-1, conferring resistance
to cephamycins and extended-spectrum cephalosporins (5). An
ampC gene and its regulator gene ampR in the
opposite orientation were identified on a conjugative plasmid, pSAL-1
(95 kb), and the ampR gene was shown to be functional
(5). DHA-1 was the first plasmid-encoded AmpC -lactamase
found to be inducible. Both the ampC and ampR
genes originate from a region of 2.6 kb in the Morganella
morganii chromosome (between 98 and 100% identity) where they
have the same configuration (4, 5, 14). The aim of this work
was to describe the genetic organization of this plasmid-encoded and
inducible AmpC -lactamase.
1), the
whole gene sulI, and the 5' end of ORF5, with 100%
identity; these three open reading frames are typical of the 3'
conserved segment (3'-CS) of a class 1 integron (9).
Furthermore, a segment of 0.2 kb 170 bp downstream from ampC
is 100% identical to a region characteristic of integrons In6 and In7
(17, 18). Both these integrons are unusual in that they have
a partial duplication of the 3'-CS, and they have a common segment of
2.1 kb, located between the two sulI genes, which includes
ORF341, of unknown function. Different resistance genes (cat
in In6 and dfrA10 in In7) are present downstream from ORF341
(17). E. coli JM101(pSAL-2ind) was
susceptible to sulfonamides despite the presence of a sulI gene. As on pSAL-2ind, the sulI gene of the
second copy of 3'-CS in In6 and In7 is not expressed, probably due to
the absence of the promoter (in the 5' end of the 3'-CS). The high
degree of identity between regions of pSAL-2ind and the
integrons In6 and In7 suggests that the ampC gene is part of
a complex integron structure.
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FIG. 1.
Structure of integron pSAL-1 (9.5 kb) obtained by
genetic analysis of the recombinant plasmid pSAL-2ind and
PCR mapping of the upstream region. (A) Sequence reported here (8,558 bp). (B) Recombinant plasmid pSAL-2ind (4.2 kb). (C)
Sequence (2,126 bp) previously reported (5). Open circle,
59-bp element.
PCR primers corresponding to sequences of In7 were used to map the
unknown region of pSAL-1 (Table 1).
Genomic DNA was extracted from E. coli HB101(pSAL-1) by the
method described by Grimont and Grimont (7). PCRs were
carried out in 100-µl volumes containing 1× PCR buffer (ATGC
Biotechnologie, Noisy-le-Grand, France); 200 mM (each) dATP, dCTP,
dGTP, and dTTP (Pharmacia Biotech, Uppsala, Sweden); 0.5 mM (each)
primer (Oligo-express, Paris, France) (sequences in Table 1); and 5 mM
template DNA and 2 U of Taq DNA polymerase (ATGC
Biotechnologie). To amplify the DNA in the thermal cycler (Perkin-Elmer
Cetus, St. Quentin-en-Yvelines, France), we used a three-step profile
for 40 cycles: denaturation for 30 s at 94°C, annealing for
30 s at variable temperature according to primers, and extension
for 30 s at 72°C. The denaturation for the first cycle and the
extension for the last cycle lasted 3 min. The temperatures used for
annealing were as follows: fragment A, 66°C; fragment B, 62°C;
fragment C, 58°C; fragment F, 60°C; fragment D, 58°C; fragment G,
62°C; fragment E, 58°C.
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Finally, seven overlapping PCR fragments allowed us to map the unknown region of the 5.3 kb in pSAL-1 downstream from the sequences corresponding to pSAL-2ind. For each PCR fragment (except A), DNA sequence was determined by the procedure of Sanger et al. (16) with the PCR primers, fluorescent dye-labeled dideoxynucleotides, thermal cycling with Taq polymerase, and the ABI 373A DNA sequencer (Applied Biosystems). The BLASTN program at the National Center for Biotechnology Information was used for the alignment of the DNA sequences (1). The PCR products from pSAL-1 and the clone pSAL-2ind overlap by 9.5 kb (Fig. 1).
With NciI, AluI, and Sau3A, results of restriction patterns of the PCR A product were consistent with those of the integrase gene of sulI-type integrons (data not shown). Only the first 110 bp of the int gene (11%) was sequenced (PCR B) because of the high degree of conservation of this gene in sulI-type integrons (8). Other genes and open reading frames were identified: aadA2, ORF4, sulI, ORF341, ampC, and ampR. These findings are consistent with the susceptibility pattern of E. coli HB101(pSAL-1): resistance to streptomycin, spectinomycin, and sulfonamides. A 2.1-kb region including ORF341 between the first sulI gene and the ampC gene displays 100% identity with the region common to In6 and In7 (17, 18).
The plasmid pSAL-1 includes several characteristic elements of
integrons. First, an int gene encodes a site-specific
recombinase (integrase) (9). Second, the aadA2
gene is suitably oriented to be a cassette and the 59-bp element is
present, downstream from this gene (15). Third, ORF4
(qacE1 gene) and a sulI gene constitute a
partial 3'-CS of the sulI-type integron. The plasmid pSAL-1
thus has a genetic organization similar to those of pSa and pDGO100
(In6 and In7) (17): the presence of a 2.1-kb common region
including ORF341 is associated with a partial duplication of the 3'-CS.
These observations indicate that the ampC and
ampR genes on pSAL-1 were inserted into a complex
sulI-type integron. Three distinct integrons, In6, In7, and
this new one on pSAL-1, have a similar genetic organization, suggesting
that they are descended from a common ancestral integron. The lengths
of the deletions at the 5' end of the second 3'-CS (CS2) are 77 bp in In7, 248 bp in In6, and 180 bp in the new integron (Fig.
2).
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Nevertheless, there is a difference between pSAL-1 and integrons In6 and In7: the origin of the antibiotic resistance genes downstream from ORF341. The genes dfrA10 (resistance to trimethoprim) and cat (resistance to chloramphenicol) have the same orientation as the gene cassette(s) upstream from the first 3'-CS. It is possible that they were cassettes but have lost their mobility due to the deletion of the 59-bp element and the first bases of the adjacent 3'-CS, as suggested by Stokes et al. (17).
The genes ampC and ampR in opposite orientations
are not located between a 5'-CS and a 3'-CS. No 59-bp element is
present adjacent to ampC or ampR. The expression
of the ampC gene was necessarily different from that of
cassette genes (9). To date, only three families of
-lactamases (classification of Ambler [2]) are
known to be encoded by cassettes: class A (PSE or CARB) (12,
15), class B metallo--lactamases
(blaIMP gene) (3), and class D
(OXA-type) (13). This is the first time that a gene encoding
a class C -lactamase has been found inserted in an integron. The
ampC gene is transcribed under the control of its regulatory
gene ampR binding to DNA in the ampC-ampR
intercistronic region, which includes overlapping promoters (5,
11).
It would be interesting to compare the genetic organization of DHA-1
with those of other plasmid-encoded cephalosporinases. Few data are
available. The ampC genes and, in some cases, the neighboring regions have been sequenced. The ampC genes of
the plasmid-encoded -lactamases CMY-1 and MOX-1 (6, 10)
have upstream fragments of 168 and 488 bp, respectively, which are identical to the region common to In6, In7, and pSAL-1 (Fig.
3). Both the ampC gene coding
for CMY-1 and that coding for MOX-1 have the same orientation, like
cassette genes, but no 59-bp element has been found nearby. These
enzymes are not inducible, and they are not associated with an
ampR gene.
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The blaDHA-1 gene and its regulator
ampR gene were probably mobilized from the M. morganii chromosome to the conjugative plasmid of
Salmonella serovar Enteritidis after recombination catalyzed by genes of the integron. The phylogenic origin of MOX-1 and CMY-1 is
unknown. It is possible that ampC genes coding for these
-lactamases were inserted into a sulI-type complex
integron. We suggest that a preferential recombination site is present
at the end of the common region and that this leads to the insertion of
a resistance gene into the complex structure of the integron. Valentine
and colleagues (18) suggested that ORF341 encodes a
recombinase recognizing this specific recombination site.
Nucleotide sequence accession number. The sequence has been deposited in the EMBL database under accession no. AJ237702.
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ACKNOWLEDGMENTS |
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This study was supported in part (C.V.) by a grant from the Fondation pour la Recherche Médicale.
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FOOTNOTES |
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* Corresponding author. Mailing address: Service de Bactériologie, Hôpital Tenon, 4, rue de la Chine, 75970 Paris Cedex 20, France. Phone: (33) (1) 56 01 70 18. Fax: (33) (1) 56 01 61 08. E-mail: service.bacterio{at}tnn.ap-hop-paris.fr.
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