Mobilization of qnrB2 and ISCR1 in plasmids.
ABSTRACT The DNA sequences of two IncHI2 plasmids, pEC-IMP and pEC-IMPQ, from metallo-beta-lactamase-producing Enterobacter cloacae clinical isolates were determined. The two conjugative plasmids are almost identical, but pEC-IMPQ carries an additional segment containing an orf513 (ISCR1), a truncated 3' conserved sequence, and a qnrB2. Comparative analyses provide support for the proposed ISCR1-mediated gene mobilization.
- SourceAvailable from: Ying-Tsong Chen[Show abstract] [Hide abstract]
ABSTRACT: Two plasmids carrying blaKPC-2 isolated from carbapenem-resistant Escherichia coli (CR-EC) and carbapenem-resistant Klebsiella pneumoniae (CR-KP), respectively, were completely sequenced. The CR-KP strain was selected from an outbreak in 2012, and the CR-EC strain was the first blaKPC-2-carrying E. coli identified in the same carbapenem resistance monitoring programme in Taiwan. Antimicrobial susceptibility tests, multilocus sequence typing (MLST) and the conjugal transfer of plasmids were performed. Complete sequencing of the plasmids was performed using a shotgun approach. The CR-EC and CR-KP strains in this study were determined to be ST410 and ST11, respectively, by MLST. From CR-EC, we identified a 145 kb conjugative plasmid that carries blaKPC-2, blaCMY-2, blaCTX-M-3 and blaTEM-1. The plasmid is a chimera composed of three regions related to IncI, IncN and RepFIC replicons. From CR-KP, we identified an 86.5 kb plasmid, pKPC-LK30, which carries blaKPC-2 and blaSHV-11. The plasmid is very similar to two blaKPC-2-carrying IncFIIK plasmids, but lacks one of the replication origins and cannot conjugate. The differences in cross-species transferability of the two plasmids can be explained by genetic differences between their backbones and could have resulted in the confined blaKPC-2-carrying CR-KP outbreak in Taiwan. Plasmid pKPC-LKEc is the first blaKPC-2-carrying plasmid identified from CR-EC in Taiwan. With relatively high transferability it should be closely monitored.Journal of Antimicrobial Chemotherapy 10/2013; · 5.34 Impact Factor
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ABSTRACT: The gene for New Delhi metallo-β-lactamase 1 (NDM-1) has been reported to be transmitted via plasmids which are easily transferable and capable of wide distribution. We report the isolation of two NDM-1 producing strains and possible in vivo transfer of blaNDM-1 in a patient. Clinical samples were collected for bacterial culture and antibiotic susceptibility testing from a patient during a 34-day hospitalization. The presence of blaNDM-1 was detected by PCR and sequencing. Plasmids of interest were sequenced. Medical records were reviewed for evidence of association between the administration of antibiotics and the acquisition of the NDM-1 resistance. A NDM-1 positive Raoultella planticola was isolated from blood on the ninth day of hospitalization without administration of any carbapenem antibiotics and a NDM-1 positive Escherichia coli was isolated from feces on the 29th day of hospitalization and eight days after imipenem administration. The blaNDM-1 was carried by a 280 kb plasmid pRpNDM1-1 in R. planticola and a 58 kb plasmid pEcNDM1-4 in E. coli. The two plasmids shared a 4812 bp NDM-1-ISCR1 element which was found to be excisable from the plasmid as a free form and transferrable in vitro to a NDM-1 negative plasmid from E. coli. blaNDM-1 was embedded in an ISCR1 complex class 1 integron as a novel 4812 bp NDM-1-ISCR1 element. The element was found to be able to self excise to become a free form, which may provide a new vehicle for NDM-1 dissemination. This mechanism could greatly accelerate the spread of NDM-1 mediated broad spectrum β-lactam resistance.PLoS ONE 01/2014; 9(3):e89893. · 3.53 Impact Factor
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ABSTRACT: Four nontyphoidal Salmonella strains with resistance to extended-spectrum cephalosporins and nonclassical quinolone resistance phenotype were studied. Two S. Give were isolated from pediatric patients with acute gastroenteritis, and two S. Heidelberg were recovered from raw chicken meat. Phenotypic characterization included antimicrobial susceptibility testing and detection of extended-spectrum β -lactamases (ESBLs) by the double-disc synergy method. The detection of quinolone resistance-determining regions (QRDR) of gyrA, gyrB, and gyrC genes, bla ESBLs genes, and plasmid-mediated quinolone resistance (PMQR) determinants was carried out by molecular methods. Plasmid analysis included Southern blot and restriction patterns. Transferability of resistance genes was examined by transformation. bla TEM-1 + bla SHV-12 genes were detected in S. Give SG9611 and bla TEM-1 + bla CTX-M-2 in the other three strains: S. Give SG9811, S. Heidelberg SH7511, and SH7911. Regardless of origin and serovars, the qnrB19 gene was detected in the 4 strains studied. All determinants of resistance were localized in plasmids and successfully transferred by transformation. This study highlights the circulation of qnrB19 associated with bla TEM-1, bla SHV-12, and bla CTX-M-2 in S. Give and S. Heidelberg in Venezuela. The recognition of factors associated with increasing resistance and the study of the molecular mechanisms involved can lead to a more focused use of antimicrobial agents.International Journal of Microbiology 01/2013; 2013:628185.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2009, p. 1235–1237
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 53, No. 3
Mobilization of qnrB2 and ISCR1 in Plasmids?
Ying-Tsong Chen,1†* Tsai-Lien Liao,1† Yen-Ming Liu,1Tsai-Ling Lauderdale,2
Jing-Jou Yan,3and Shih-Feng Tsai1,4,5
Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan1; Division of Clinical Research,
National Health Research Institutes, Miaoli, Taiwan2; Department of Pathology, National Cheng Kung University Hospital, Tainan,
Taiwan3; Genome Research Center and Institute of Biomedical Informatics, National Yang-Ming University, Taipei, Taiwan4;
and Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan5
Received 22 July 2008/Returned for modification 1 November 2008/Accepted 6 December 2008
The DNA sequences of two IncHI2 plasmids, pEC-IMP and pEC-IMPQ, from metallo-?-lactamase-
producing Enterobacter cloacae clinical isolates were determined. The two conjugative plasmids are almost
identical, but pEC-IMPQ carries an additional segment containing an orf513 (ISCR1), a truncated 3?
conserved sequence, and a qnrB2. Comparative analyses provide support for the proposed ISCR1-mediated
Encoding a putative product of 513 amino acids, orf513 was
initially identified adjacent to integrons In6 and In7 (11). To-
gether with noncassette resistance genes, it was commonly
found between truncated and full-length 3? conserved se-
quences (3?-CS) of class 1 integrons (9, 11). Their function
remained mysterious until comparative analyses linked these
so-called common region (CR) elements to a group of IS91-
like insertion sequences (ISs) (13). The IS91-like ISs are a
family of unusual IS elements that differ from most other IS
elements in both structure and mode of transposition. They
can perform rolling-circle (RC) transposition, in which a single
IS element can mobilize the sequences to which it is attached
(4, 12). It was proposed that orf513, later termed insertion
sequence common region 1 (ISCR1), may have mobilized the
nearby sequence and a truncated 3?-CS from one integron to
the 3?-CS of another integron through RC transposition, thus
facilitating the formation of complex class 1 integrons associ-
ated with ISCR1 (13). In addition to this putative recombinase
function, the ISCR1 element has also been shown to play a role
in the expression of nearby genes by providing a promoter
ISCR1 were found to be associated with many antimicrobial
resistance genes, including the plasmid-mediated quinolone
resistance determinant qnr (5) as well as genes encoding resis-
tance to chloramphenicol, trimethoprim, aminoglycosides, and
?-lactams (8, 13, 14). However, lacking the 59 base elements
required for site-specific recombination, these orf513-linked
genes could not have been acquired as gene cassettes. It was
hypothesized that these antimicrobial resistance genes were
added to the 3?-CS of the class 1 integron through comobili-
zation with the nearby ISCR1 from other integrons using RC
transposition and homologous recombination (1, 13).
In a recent study on the prevalence of QnrA, QnrB, and
QnrS among clinical isolates of Enterobacter cloacae, the
association of Qnr with the IMP-8 metallo-?-lactamase
(MBL) was investigated (15). From 56 IMP-8 MBL produc-
ers, eight qnrB-positive, blaIMP-8-positive transconjugants
and four qnrB-negative blaIMP-8-positive transconjugants
were obtained. Restriction pattern analysis on these plas-
mids gave very similar patterns, suggesting the occurrence of
horizontal mobility of qnrB2 (15). To investigate the possi-
ble horizontal transfer mechanisms responsible for qnrB2,
we have conducted complete DNA sequencing and compar-
ative analysis on two of the plasmids, the qnrB2-positive
plasmid pEC-IMPQ and the qnrB2-negative plasmid pEC-
The DNA sequences of the two plasmids were determined
using a whole-genome shotgun approach as described before
(3). The two plasmids are 324,503 bp and 318,782 bp in length
and have a common backbone similar to that of the IncHI2
plasmids (2, 6, 7). The larger one, pEC-IMPQ, carries an
additional segment which contains qnrB2, a truncated 3?-CS,
and an ISCR1. Outside this region, single nucleotide substitu-
tions were found in five positions, and a 20-kb inversion, prob-
ably facilitated by the two flanking IS26s, was detected (Fig.
blaSHV-12, were found in both pEC-IMP and pEC-IMPQ.
Among them, only blaIMP-8was located within an integron.
The blaTEM-1and blaSHV-12genes were associated with Tn3
and IS26, respectively. There were also other resistance genes,
including those encoding a tetracycline efflux pump and its
regulator, tetAR; dihydropteroate synthetase genes sul1 and
sul2; chloramphenicol acetyltransferase genes catA2 and catB3;
hipBA genes encoding putative multidrug tolerance proteins; a
dihydrofolate reductase gene, dfrA19; a putative rifampin
ADP-ribosyl transferase gene; a putative aminoglycoside 3?-
phosphotransferase gene; and several aminoglycoside acetyl-
transferase genes, aac3, aacA4, aac6, and aac(6?)-IIc. Most of
these antimicrobial resistance genes are located in the four
integrons of both of the plasmids (Fig. 1a). Several gene clus-
ters responsible for heavy metal resistance were also identified.
The extra qnrB2-containing region of pEC-IMPQ is located
after the 3?-CS of a class 1 integron (Fig. 1b). This integron,
which is present in both plasmids, contains a blaIMP-8MBL
* Corresponding author. Mailing address: 35 Keyan Rd., Zhunan
Town, Miaoli County 350, Taiwan. Phone: 886-37246166, ext. 35345.
Fax: 886-37586459. E-mail: firstname.lastname@example.org.
† These authors contributed equally to this article.
?Published ahead of print on 15 December 2008.
gene, aminoglycoside acetyltransferase gene aacA4, chloram-
phenicol acetyltransferase gene catB3, quaternary ammonium
transporter gene qacE?1, and dihydropteroate synthetase gene
sul1. In pEC-IMP, an ISCR1 and dihydrofolate reductase gene
dfrA19 were identified downstream of sul1. In pEC-IMPQ, the
duplication of the ISCR1 and the 3? end of the nearby sul1
were identified. A qnrB2, a truncated qacE?1, and another sul1
were identified between the duplicated ISCR1 (Fig. 1b).
In pEC-IMPQ, the 5.8-kb sequence at approximately bp
149926 to 155739 flanked by the duplications is identical to a
recently described qnrB2-containing sequence found between
two ISCR1s on a plasmid from a Salmonella enterica serovar
Keurmassar strain (5). This suggests that the region in com-
mon that includes qnrB2 was derived from the same immediate
ancestor (Fig. 1b). The two sul1 genes differ at one position, as
indicated in the figure.
On the basis of sequence analyses and the proposed mobi-
lizing function of ISCR1, a model was made (Fig. 1b). In this
model, a circular intermediate that carries the qnrB2-truncated
qacE?1-sul1 was produced by RC replication initiated from
FIG. 1. (a) Schematic diagram of the plasmids pEC-IMPQ and pEC-IMP. The qnrB2-containing region in pEC-IMPQ is depicted in a white
box. The inversion is indicated by dotted lines. The five nucleotide substitutions are indicated by black arrowheads. The four class 1 integrons are
depicted in gray boxes. The other features indicated are as follows: repHIA and repHI2, replication origins; ter, tellurite resistance gene cluster; pbr,
lead resistance gene cluster; mer, mercury resistance gene cluster; ars, arsenic resistance gene cluster; aph, putative aminoglycoside phosphotrans-
ferase gene; ereA2*, erythromycin esterase pseudogene; and arr, putative rifampin ADP-ribosyl transferase gene. The ISCR1-containing regions
are detailed in panel b. (b) The genetic contexts near the extra region found in pEC-IMPQ. The repeats of the redundant ISCR1 in pEC-IMPQ
are marked. The dotted line indicates the 5.8-kb region identical to a previously reported plasmid from a Salmonella enterica serovar Keurmassar.
The proposed circular intermediate of an aberrant RC replication carrying the qnrB2-truncated qacE?1-sul1 is shown above. The proposed
integration event between this intermediate and pEC-IMP are marked (the big ?). The intI1 integrase genes of the class 1 integrons are
crosshatched. The resistance genes that comobilized with the ISCR1 are shown in gray. Other resistance genes are shown in white. The single
nucleotide differences of the sul1 genes are indicated (T and A) at the positions of variation. Truncated orf genes are marked with an asterisk after
the gene name. The str genes encode streptomycin resistance proteins.
1236 CHEN ET AL.ANTIMICROB. AGENTS CHEMOTHER.
the replication origin oriIS of the ISCR1 element. The circular
intermediate was then inserted into pEC-IMP by homologous
recombination somewhere between the 3? moiety of the sul1
gene and the oriIS. This would explain the formation of the
so-called complex class 1 integron of pEC-IMPQ, in which the
qnrB2-containing extra region was found between two CRs
(Fig. 1b). It is, however, also possible that the pEC-IMP was
created from pEC-IMPQ by the deletion of this extra region
via excision between the two repeat regions (Fig. 1b).
In summary, the major difference between two related plas-
mids isolated from E. cloacae was a qnrB2-containing region
flanked by two ISCR1s. Our comparative analyses provide sup-
port for the proposed ISCR1-mediated gene mobilization.
Nucleotide sequence accession numbers. The annotated
DNA sequences of plasmids pEC-IMPQ and pEC-IMP have
been submitted to the GenBank database under accession
numbers EU855788 and EU855787.
The project was supported in part by a grant from the National
Science Council (NSC 97-3112-B-400-005) and intramural grants from
the National Health Research Institutes (MG-097-PP12, CL-096-
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