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.
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ABSTRACT: We investigated the prevalence of plasmid-mediated quinolone resistance (PMQR) qnr genes by the polymerase chain reaction (PCR) in antibiotic-resistant bacteria isolates collected from aquatic environments in Jinan during 2 years (2008.3-2009.11). Genes were identified to variant level by PCR restriction fragment length polymorphism analysis or sequencing. qnrA1, qnrB2, qnrB4, qnrB6, qnrB9, qnrS1, and the new qnrB variant qnrB26 were detected in 31 strains from six genera (Klebsiella spp., Escherichia coli, Enterobacter spp., Proteus spp., Shigella spp., and Citrobacter spp.), four of which contained double qnr genes. Other PMQR genes, aac(6')-Ib-cr and qepA, were found in 12 (38.7%) and 5 (16.1%) of 31 isolates, respectively; while qepA was found in Shigella spp. for the first time. Eight types of β-lactamase genes and eight other types of resistance genes were also present in the 31 qnr-positive isolates. The detection rate for five β-lactamase genes (blaTEM, blaCTX, ampR, blaDHA, and blaSHV) was >45%. Class 1 integrons and complex class 1 integrons were prevalent in these strains, which contained 15 different gene cassette arrays and 5 different insertion sequence common region 1 (ISCR1)-mediated downstream structures. qnrA1, qnrB2, and qnrB6 were present in three ISCR1-mediated downstream structures: qnrA1-ampR, sapA-like-qnrB2, and sdr-qnrB6. We also analyzed the horizontal transferability of PMQR genes and other resistance determinants. The qnr genes and some integrons and resistance genes from 18 (58.1%) of the 31 qnr-positive strains could be transferred to E. coli J53 Azi(R) or E. coli DH5α recipient strains using conjugation or transformation methods. The results showed that a high number of qnr genes were associated with other resistance genes in aquatic environments in Jinan. This suggests that we should avoid over-using antibiotics and monitor aquatic environments to control the spread of antibiotic resistance genes.Microbial drug resistance (Larchmont, N.Y.) 07/2013; · 1.99 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. · 3.73 Impact Factor
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ABSTRACT: Enterobacteriaceae resistant to quinolones frequently arise in animals, being easily disseminated through the food-chain. The aim of this study was to investigate the presence of plasmid-mediated quinolone resistance (PMQR) determinants in Salmonella spp. (n=183) and Escherichia coli (n=180) isolates, collected from food-producing animals and food products among swine, poultry, rabbits and cattle. All isolates were subjected to antimicrobial susceptibility testing and molecular screening of PMQR determinants. β-Lactamase-encoding genes, and the quinolone resistance determining region (QRDR) of gyrA, gyrB, parC and parE genes were also investigated in PMQR-positive isolates. Plasmid characterization was performed by conjugation, followed by replicon-typing. Genetic relatedness of PMQR-positive E. coli was examined by Multilocus Sequence Typing, while Salmonella was previously serotyped. The association of mobile genetic elements and PMQR was investigated through PCR mapping assays. Overall, 4.1% (15/363) isolates harbored qnrB2 (n=3), qnrB19 (n=3), and qnrS1 (n=9) genes. All but one isolate presented one to four mutations in QRDR of gyrA or parC genes, which is consistent with the range of MIC values detected (0.19-64mg/L) for ciprofloxacin; 60% (9/15) of qnr-harboring isolates were non-susceptible to β-lactam antibiotics which was justified by the presence of β-lactamases from TEM (TEM-1, n=8; TEM-135, n=1) and SHV (SHV-108, n=1) families. Analysis of mobile genetic elements revealed that qnr genes were detected nearby relevant genetic elements like intI1, ISEcl2, IS26 and ISCR1 and enclosed in diverse Inc. type plasmids. This study illustrated the existence of Qnr-producing E. coli and Salmonella from food-producing animals, associated to specific mobile elements that might mediate their transference between species and among distinct settings.Veterinary Microbiology 08/2013; · 3.13 Impact Factor
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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