Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, December 2003, p. 3945-3949, Vol. 47, No. 12
0066-4804/03/$08.00+0 DOI: 10.1128/AAC.47.12.3945-3949.2003
Copyright © 2003, American
Society for
Microbiology. All Rights Reserved.
Molecular Epidemiology of orf513-Bearing Class 1 Integrons in Multiresistant Clinical Isolates from Argentinean Hospitals
Sonia M. Arduino,1* Mariana Catalano,1 Betina E. Orman,1 Paul H. Roy,2,3 and Daniela Centrón1
Facultad
de Medicina de la Universidad de Buenos Aires, (1121) Capital Federal,
Argentina,1
Département de
Biochimie, Faculté des Sciences et de Génie,
Université Laval, Sainte-Foy, Québec, Canada G1K
7P4,2
Centre de Recherche en
Infectiologie, Centre de Recherche du CHUL, Sainte-Foy,
Québec, Canada G1V
4G23
Received 12 March 2003/
Returned for modification 23 May 2003/
Accepted 4 September 2003

ABSTRACT
The
spread of orf513-bearing class 1 integrons is associated
with
blaCTX-M-2 in gram-negative clinical isolates in
Argentina,
with In35 being the most frequently found integron
(74%). Among
65 isolates without
blaCTX-M-2,
only one harbored a novel orf513-bearing
class 1 integron with the
dfrA3b gene. The finding of orf513
not associated with class 1
integrons in two gram-positive strains
indicates the widespread
occurrence of this putative site-specific
recombinase.

TEXT
Multiple-antibiotic resistance is common in clinical isolates
from
Argentina. Although integrons belonging to all classes
have
been found in clinical and environmental strains of
multiresistant
bacteria
(
21), class 1 integrons
predominate among gram-negative
microorganisms
(
10) and have been
recently described in gram-positive
bacteria
(
4,
17). Class 1 integrons
are composed of three DNA
segments, two that are conserved and one that
includes the antibiotic
resistance gene cassettes of various lengths
and sequences.
The 5' conserved segment (5'-CS)
includes the
intI1 gene, and
downstream of the last gene
cassette, most of the studied class
1 integrons contain at least part
of the 3' conserved segment
(3'-CS) formed by the
qacE
1 gene,
sul1, and orf5, of unknown
function
(
14). Most class
1 integrons are found on defective transposons
related to
Tn
402 that keep the
tniA and
tniB
1 genes. Several
contain one or two
insertion sequences between the conserved
segments and the
transposition genes.
A novel group of orf513-bearing class 1
integrons, also called unusual class 1 integrons, of which pDGO100 is
the prototype was first described in 1990
(6). These integrons begin
with typical class 1 integron structures with one or more gene
cassettes located between the 5'-CSs and the 3'-CSs.
These 3'-CSs, called the first 3'-CSs, include only the
first 1,355 bases of a typical 3'-CS
(6). In all studied
orf513-bearing class 1 integrons, the first 3'-CSs end at the
same point, 24 nucleotides (nt) after the stop codon of the
sul1 gene, as described for In6 and In7
(16). Following the first
3'-CSs, there is a common region which includes orf513 (GenBank
accession number
L06418) and
a region unique to each orf513-bearing class 1 integron. The unique
regions differ in length and sequence, containing the antibiotic
resistance gene dfrA10 (In7), catII (In6),
blaDHA-1 (pSAL-1), blaCTX-M-9
(In60), or blaCTX-M-2 (In35 and InS21)
(1,
5,
11,
16,
20). Adjacent to the
unique regions are the second 3'-CSs with different deletions
in the 5' ends
(1).
In Argentina,
CTX-M-2 is by far the most frequent extended-spectrum
ß-lactamase, comprising 69% of all extended-spectrum
ß-lactamases found among clinical isolates in Argentinean
hospitals (M. Galas, M. Rapoport, F. Pasteran, R. Melano, A. Petroni,
P. Ceriana, A. Rossi, and the WHONET Group, Abstr. 39th Intersci. Conf.
Antimicrob. Agents Chemother., abstr. 1474, 1999). A previous study
with a small number of isolates reported that
blaCTX-M-2 is always located at the same sequence
position in orf513-bearing class 1 integrons with different arrays of
cassettes in the variable regions
(1). Nevertheless, genes
encoding some different enzymes of the CTX-M family have been
identified located near other genetic elements such as ISEcp1,
IS26, and IS903C
(2,
3,
7,
12) in isolates from
Europe and Asia. The goals of the present study were (i) to examine the
orf513-related structures and look for their association with
resistance genes and (ii) to identify the different arrangements of
cassettes in the variable regions of orf513-bearing class 1
integrons.
We studied 130 nonredundant multiresistant clinical
isolates collected during nosocomial outbreaks at different hospitals
in Buenos Aires, Argentina, between 1993 and 2000. Of these, 100 were
gram-negative bacterial isolates resistant to ß-lactams and
aminoglycosides and were divided into
blaCTX-M-2-positive (n = 35) and
blaCTX-M-2-negative (n = 65)
isolates (Table
1). Thirty were gram-positive bacterial isolates with diverse
mechanisms of resistance: Enterococcus faecium,
resistant to vancomycin (n = 8); beta-hemolytic
Streptococcus, resistant to tetracycline and erythromycin
(n = 6); Staphylococcus aureus, resistant to
methicillin (n = 8); and coagulase-negative
Staphylococcus, resistant to methicillin (n =
8). Isolates were identified by using the API systems (Biomerieux SA,
Marcy-l'Etoile, France) and conventional biochemical tests.
Susceptibility to antimicrobial agents in all the isolates was
determined by the E-Test method (AB Biodisk, Solna, Sweden) according
to the guidelines proposed by the manufacturer (Table
1). Bacterial DNA was
extracted using standard techniques
(13). Isolates were
subjected to PCR analysis with internal primers for detection of the
blaCTX-M-2 gene, orf513, class 1 integrons, and
orf513-bearing class 1 integrons. The characterization of the different
arrays of cassettes in the variable regions was performed by PCR
mapping (Table
2) (8), and several PCR
products obtained were sequenced to confirm the data.
All the
isolates carrying
blaCTX-M-2 harbored
orf513-bearing
class 1 integrons and, as described previously
(
1), this gene
was always
found located at the same position in these structures,
with different
arrays of cassettes in the variable regions.
The cassette array
aacA4-
blaOXA-2-orfD was identified in 26
isolates
(74%) harboring
blaCTX-M-2. In the
remaining nine isolates,
the following cassettes were characterized:
aadA1 in four isolates
(11%),
aadB-
aadA1 in two isolates (6%), and
aacA4,
aacA4-
aadA1,
and orfD in one isolate
each (Fig.
1). The presence of different
types of cassettes was not correlated with
the bacterial species.
The arrangement of cassettes most frequently
found,
aacA4-
blaOXA-2-orfD,
has been
recently described (
1,
5). The sequence reported
for
the
blaOXA-2 gene in InS21 of a
Salmonella
enterica serovar
Infantis isolate from the province of Santa Fe,
Argentina, is
that of a pseudogene (GenBank file AJ311891)
(
5). In contrast,
the
blaOXA-2 gene sequenced in In35 of pMAR-12
(
1), identical
to that
reported previously (GenBank file M95287)(
15), was active
as
demonstrated by the presence of a band of pI 7.7 showing
ß-lactamase
activity in isoelectric focusing experiments. Also,
other
blaOXA-2 sequences in
Klebsiella
pneumoniae and
Salmonella enterica serovar
Typhimurium
isolates were identical to that of the complete gene
reported
previously (M95287)
(
15). The orf513-bearing
class 1 integron
In35 carrying the
blaCTX-M-2 gene
was found located in different
conjugative plasmids, as shown by
different restriction patterns
obtained with
HindIII (data not
shown).
All the strains without
blaCTX-M-2
carried at least one class
1 integron (data not shown). In addition,
23% (15 of 65) of
the isolates carried class 2 integrons. Only
one of the 65 isolates
studied (1.5%) harbored a novel
orf513-bearing class 1 integron
that was characterized in a
Citrobacter freundii isolate and
was termed In38, with the
aacA4-blaOXA-4 cassette array within
the variable
region. This novel genetic structure (Fig.
2) has,
like others already described, a common region that includes
orf513.
This region starts 24 nt after the
sul1 gene stop
codon in the
first 3'-CS and ends with the same segment of 28
nt described
for In6 and In35
(
1,
19). The unique region
located between
the common region that includes orf513 and the second
3'-CS
is an open reading frame of 714 nt that starts 123 nt
downstream
of the end of the common region and shows no similarity to
any
reported sequence. The first 47 amino acids of the product of
this
uncharacterized open reading frame have 96% identity with
the
N-terminal protein sequence of dihydrofolate reductase type
IIIb from
an isolate of
Shigella sonnei (PIR accession number
A37174)
(
18),
and the
corresponding gene has been named the
dfrA3b gene.
The
last 96 nt of the unique region and the following partial second
3'-CS
of this orf513-bearing class 1 integron have 100%
identity with
the sequence of In6 reported by Valentine et al. (GenBank
accession
number
U04278)
(
19). No duplications of
the common region were
observed at the beginning of the unique region,
as described
for In6. Deletions in the second 3'-CSs have been
described
to be different in length, but in the case of this new
structure
the second 3'-CS starts at the same point as that
described
for In6 (Fig.
3) (
19).
The entire
gene of orf513 was detected in one
Enterococcus faecium
isolate
and in one group G
Streptococcus isolate. The
analysis of the
sequence revealed that it was identical to that of the
orf513
gene described in pDGO100 (GenBank accession number
L06418).
These
isolates did not harbor either
int1 or the
sulI
gene, and the
putative association of orf513 with resistance genes was
not
determined. This finding indicates the widespread occurrence
of
this putative site-specific recombinase in the bacterial
population and
demonstrates that it is not associated solely
with class 1 integrons.
Further analysis to determine the environment
of orf513 in these
gram-positive isolates is in progress in
our laboratory.
It is
noteworthy that only 5 of 39 multiresistant nonfermenting isolates, one
Acinetobacter and four Pseudomonas aeruginosa
isolates, harbored orf513-bearing class 1 integrons. In
this regard, one possible explanation is that chromosomal resistance
mechanisms such as efflux pumps are more common than plasmid-mediated
resistance factors in these genera in this bacterial
population.
In conclusion, almost all orf513-bearing class 1
integrons are associated with blaCTX-M-2 in the
gram-negative bacterial population under study and the
sequences adjacent to the
blaCTX-M-2 gene are conserved
in all the studied isolates. As has been described for class 1
integrons (9), it seems
that once located in these orf513-bearing class 1 integrons, the whole
genetic structures are transferred among different plasmids, thus
enabling them to be disseminated. Therefore, the capture of the
blaKLUA-1 gene from the chromosome of Kluyvera
ascorbata by an as yet unknown mechanism that possibly involves
orf513 has taken place once, and since that event, the genehas spread through different plasmids under selection due to
antimicrobial pressure. These findings may explain the unusual
distribution of ß-lactamases among the bacterial population in
Argentina.
Nucleotide sequence accession
number.
The sequence of In38
has been submitted to GenBank under accession number
AY162283.

ACKNOWLEDGMENTS
We
are grateful to P. Jeric for providing some of the gram-positive
isolates.
This work was supported by a grant from ANPCyT, PICT99
07064, Buenos Aires, Argentina, to M.C. and
D.C.

FOOTNOTES
* Corresponding
author. Mailing address: Dept. Microbiología, Facultad de
Medicina, Universidad de Buenos Aires, Paraguay 2155, P-12, (1121)
Capital Federal, Argentina. Phone: 54-11-5950-9500. Fax:
54-11-4508-3705. E-mail:
Sonia.Arduino{at}crchul.ulaval.ca.


REFERENCES
1 - Arduino,
S. M., P. H. Roy, G. A. Jacoby,
B. E. Orman, S. A. Piñeiro, and D.
Centrón. 2002. blaCTX-M-2 is
located in an unusual class 1 integron (In35) which includes Orf513.Antimicrob. Agents Chemother.
46:2303-2306.[Abstract/Free Full Text]
2 - Cao,
V., T. Lambert, and P. Courvalin. 2002. ColE1-like
plasmid pIP843 of Klebsiella pneumoniae encoding
extended-spectrum ß-lactamase CTX-M-17. Antimicrob.
Agents Chemother.
46:1212-1217.[Abstract/Free Full Text]
3 - Chanawong,
A., F. H. M'Zali, J. Heritage, J.-H. Xiong,
and P. M. Hawkey. 2002. Three cefotaximases,
CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in
the People's Republic of China. Antimicrob. Agents
Chemother.
46:630-637.[Abstract/Free Full Text]
4 - Clark,
N. C., O. Olsvik, J. M. Swenson, C. A.
Spiegel, and F. Tenover. 1999. Detection of a
streptomycin/spectinomycin adenylyltransferase gene (aadA) in
Enterococcus faecalis. Antimicrob. Agents
Chemother.
43:157-160.[Abstract/Free Full Text]
5 - Di
Conza, J., J. Ayala, P. Power, M. Mollerach, and G. Gutkind.2002
. Novel class 1 integron (InS21) carrying
blaCTX-M-2 in Salmonella enterica serovar
Infantis. Antimicrob. Agents Chemother.
46:2257-2261.[Abstract/Free Full Text]
6 - Hall,
R. M., and H. W. Stokes. 1990. The
structure of a partial duplication in the integron of plasmid pDGO100.Plasmid
23:76-79.[CrossRef][Medline]
7 - Karim,
A., L. Poirel, S. Nagarajan, and P. Nordmann. 2001.
Plasmid-mediated extended-spectrum ß-lactamase (CTX-M-3-like)
from India and gene association with insertion sequence ISEcp1.FEMS Microbiol. Lett.
201:237-241.[Medline]
8 - Lévesque,
C., L. Piché, C. Larose, and P. H. Roy. 1995.
PCR mapping of integrons reveals several novel combinations of
resistance genes. Antimicrob. Agents Chemother.
39:185-191.[Abstract]
9 - Martinez
Freijo, P., A. C. Fluit, F.-J. Schmitz, J. Verhoef, and
M. E. Jones. 1999. Many class 1 integrons
comprise distinct stable structures occurring in different species of
Enterobacteriaceae isolated from widespread geographic regions
in Europe. Antimicrob. Agents Chemother.
43:686-689.[Abstract/Free Full Text]
10 - Peters,
E. D. J., M. A. Leverstein-van Hall,
A. T. A. Box, J. Verhoef, and A. C.
Fluit. 2001. Novel gene cassettes and integrons.Antimicrob. Agents Chemother.
45:2961-2964.[Abstract/Free Full Text]
11 - Sabaté,
M., F. Navarro, E. Miró, S. Campoy, B. Mirelis, J. Barbé,
and G. Prats. 2002. Novel complex sul1-type
integron in Escherichia coli carrying
blaCTX-M-9. Antimicrob. Agents
Chemother.
46:2656-2661.[Abstract/Free Full Text]
12 - Saladin,
M., V. T. B. Cao, T. Lambert, J.-L. Donay, J.-L.
Herrmann, Z. Ould-Hocine, C. Verdet, F. Delisle, A. Philippon, and G.
Arlet. 2002. Diversity of CTX-M ß-lactamases
and their promoter regions from Enterobacteriaceae isolated in
three Parisian hospitals. FEMS Microbiol. Lett.
209:161-168.[Medline]
13 - Sambrook,
J., E. F. Fritsch, and T. Maniatis. 1989.
Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor,
N.Y.
14 - Stokes,
H. W., and R. M. Hall. 1989. A
novel family of potentially mobile DNA elements encoding site-specific
gene-integration functions: integrons. Mol. Microbiol.
3:1669-1683.[Medline]
15 - Stokes,
H. W., and R. M. Hall. 1992. The
integron In1 in plasmid R46 includes two copies of the OXA-2 gene
cassette. Plasmid
28:225-234.[CrossRef][Medline]
16 - Stokes,
H. W., C. Tomaras, Y. Parsons, and R. M. Hall.1993
. The partial 3'-conserved segment
duplications in the integrons In6 from pSa and In7 from pDGO100 have a
common origin. Plasmid
30:39-50.[CrossRef][Medline]
17 - Tauch,
A., S. Gotker, A. Puhler, J. Kalinowski, and G. Thierbach.2002
. The 27.8-kb R-plasmid pTET3 from Corynebacterium
glutamicum encodes the aminoglycoside adenyltransferase gene
cassette aadA9 and the regulated tetracycline efflux system
Tet 33 flanked by active copies of the widespread insertion sequence
IS6100. Plasmid
48:117-129.[CrossRef][Medline]
18 - Thomson,
C. J., N. Barg, and S. G. B. Amyes.1990
. N-terminal amino acid sequence of the novel type
IIIb trimethoprim-resistant plasmid-encoded dihydrofolate reductase
from Shigella sonnei. J. Gen.
Microbiol.
136:673-677.[Abstract/Free Full Text]
19 - Valentine,
C. R., M. J. Heinrich, S. L. Chissoe, and
B. A. Roe. 1994. DNA sequence of direct
repeats of the sulI gene of plasmid pSa.Plasmid
32:222-227.[CrossRef][Medline]
20 - Verdet,
C., G. Arlet, G. Barnaud, P. H. Lagrange, and A.
Philippon. 2000. A novel integron in Salmonella
enterica serovar Enteritidis, carrying the
blaDHA-1 gene and its regulator gene ampR,
originated from Morganella morganii. Antimicrob. Agents
Chemother.
44:222-225.[Abstract/Free Full Text]
21 - White,
P. A., C. J. McIver, and W. D.
Rawlinson. 2001. Integrons and gene cassettes in the
Enterobacteriaceae. Antimicrob. Agents
Chemother.
45:2658-2661.[Abstract/Free Full Text]
Antimicrobial Agents and Chemotherapy, December 2003, p. 3945-3949, Vol. 47, No. 12
0066-4804/03/$08.00+0 DOI: 10.1128/AAC.47.12.3945-3949.2003
Copyright © 2003, American
Society for
Microbiology. All Rights Reserved.
This article has been cited by other articles:
-
Marquez, C., Labbate, M., Raymondo, C., Fernandez, J., Gestal, A. M., Holley, M., Borthagaray, G., Stokes, H. W.
(2008). Urinary Tract Infections in a South American Population: Dynamic Spread of Class 1 Integrons and Multidrug Resistance by Homologous and Site-Specific Recombination. J. Clin. Microbiol.
46: 3417-3425
[Abstract]
[Full Text]
-
Quiroga, M. P., Andres, P., Petroni, A., Soler Bistue, A. J. C., Guerriero, L., Vargas, L. J., Zorreguieta, A., Tokumoto, M., Quiroga, C., Tolmasky, M. E., Galas, M., Centron, D.
(2007). Complex Class 1 Integrons with Diverse Variable Regions, Including aac(6')-Ib-cr, and a Novel Allele, qnrB10, Associated with ISCR1 in Clinical Enterobacterial Isolates from Argentina. Antimicrob. Agents Chemother.
51: 4466-4470
[Abstract]
[Full Text]
-
Toleman, M. A., Bennett, P. M., Walsh, T. R.
(2006). Common regions e.g. orf513 and antibiotic resistance: IS91-like elements evolving complex class 1 integrons. J Antimicrob Chemother
58: 1-6
[Abstract]
[Full Text]
-
Toleman, M. A., Bennett, P. M., Walsh, T. R.
(2006). ISCR Elements: Novel Gene-Capturing Systems of the 21st Century?. Microbiol. Mol. Biol. Rev.
70: 296-316
[Abstract]
[Full Text]
-
Soler Bistue, A. J. C., Martin, F. A., Petroni, A., Faccone, D., Galas, M., Tolmasky, M. E., Zorreguieta, A.
(2006). Vibrio cholerae InV117, a Class 1 Integron Harboring aac(6')-Ib and blaCTX-M-2, Is Linked to Transposition Genes.. Antimicrob. Agents Chemother.
50: 1903-1907
[Abstract]
[Full Text]
-
Valverde, A., Canton, R., Galan, J. C., Nordmann, P., Baquero, F., Coque, T. M.
(2006). In117, an Unusual In0-Like Class 1 Integron Containing CR1 and blaCTX-M-2 and Associated with a Tn21-Like Element. Antimicrob. Agents Chemother.
50: 799-802
[Abstract]
[Full Text]
-
Valverde, A., Coque, T. M., Sanchez-Moreno, M. P., Rollan, A., Baquero, F., Canton, R.
(2004). Dramatic Increase in Prevalence of Fecal Carriage of Extended-Spectrum {beta}-Lactamase-Producing Enterobacteriaceae during Nonoutbreak Situations in Spain. J. Clin. Microbiol.
42: 4769-4775
[Abstract]
[Full Text]