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Complete Sequences of mcr-1-Harboring Plasmids from ExtendedSpectrum--Lactamase- and Carbapenemase-Producing
Enterobacteriaceae
Aiqing Li,a Yong Yang,a Minhui Miao,a Kalyan D. Chavda,b José R. Mediavilla,b Xiaofang Xie,a Ping Feng,a
Barry N. Kreiswirth,b Liang Chen,b Hong Dua
Yi-Wei Tang,c
Department of Clinical Laboratory, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, Chinaa; Public Health Research Institute Tuberculosis Center,
New Jersey Medical School, Rutgers University, Newark, New Jersey, USAb; Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, New
York, USAc
T
he plasmid-mediated colistin resistance gene, mcr-1, has recently been reported from animals and hospitalized patients in
China (1). Since then, mcr-1 has been found in ⬃20 countries on
four different continents (2). Alarmingly, mcr-1 has also been
identified in several multidrug-resistant bacteria, including extended-spectrum--lactamase (ESBL)-producing and carbapenemase-producing Enterobacteriaceae (CPE) (3–9). However,
knowledge regarding the structure of mcr-1-harboring plasmids is
limited. Here we completely sequenced four mcr-1-harboring
plasmids (three of which are identical), isolated from two ESBLproducing Escherichia coli and two carbapenemase-producing
Klebsiella pneumoniae clinical isolates (4).
In a recent study, we identified mcr-1 in two ESBL-producing
E. coli (SZ01 and SZ02) and two carbapenemase-producing K.
pneumoniae (SZ03 and SZ04) clinical isolates from a tertiary hospital in eastern China (4). SZ01, SZ02, and SZ04 carry ESBL gene
blaCTX-M-55, while SZ03 and SZ04 harbor carbapenemase gene
blaNDM-5. Multilocus sequence typing (MLST) (10, 11) showed
that the two E. coli isolates, SZ01 and SZ02, belong to two unrelated sequence types (STs) (ST2448 and ST2085), while the two K.
pneumoniae strains (isolated from the same patient) both belong
to ST25. The mcr-1-harboring plasmids from all four isolates were
subsequently transferred to recipient strain E. coli J53 AZr via conjugation, along with the blaNDM-5-harboring plasmids from SZ03
and SZ04. Susceptibility testing revealed that the four mcr-1-harboring E. coli transconjugants were resistant to colistin but not to
any of the other antimicrobial agents tested. The two blaNDM-5
transconjugants were resistant to all -lactams, except for aztreonam, but remained susceptible to other classes of antimicrobial
agents (data not shown). The mcr-1- and blaNDM-5-harboring
plasmids from these transconjugants were extracted and subjected
to sequencing using the Illumina MiSeq platform (12). The sequencing reads were assembled de novo using SPAdes (13), and
gaps were closed by standard PCR and Sanger sequencing as described previously (12).
The mcr-1-harboring plasmids from SZ01, SZ03, and SZ04
(subsequently named pMCR1-IncX4) were all identical, belonging to the IncX4 incompatibility group, and were 33,287 bp in
length with a G⫹C content of 41.8%. The backbone of pMCR1-
July 2016 Volume 60 Number 7
IncX4 is similar to that of other IncX4 plasmids, including pJIE143
(GenBank accession no. JN194214) (14), pBS512_33 (CP001059),
pCROD2 (FN543504) (15), and pSH146_32 (JX258655) (16).
BLASTn analysis showed that pMCR1-IncX4 has a query coverage of
87% and maximal 97% identity to pSH146_32, isolated from a Salmonella enterica Heidelberg strain from a porcine diagnostic specimen from Minnesota in 2002 (16), and a query coverage of 77%
and maximal 99% identity to pJIE143, isolated from an E. coli
ST131 strain from Australia in 2006 (14) (Fig. 1). Plasmid
pMCR1-IncX4 possesses a replication region highly similar to the
one on pJIE143 (Fig. 1), including the identical replication initiation protein gene pir, vegetative origins oriV-␣ and oriV-, and
highly similar oriV-␥ (one less iteron). The region from traM to
oriV-␥, encompassing the majority of the transfer region and including the taxABC and pilX operons, shares ⬎99.9% nucleotide
identity with pMCR1-IncX4 and pJIE143 (Fig. 1). However, the
region in pMCR1-IncX4 between taxD and the histone-like nucleotide-structuring protein gene hns, where colistin resistance
gene mcr-1 is located, is absent in pJIE143. In contrast, this region
is highly similar to that of pSH146_32, except for the insertion of
an mcr-1-pap2 element and an IS26 element in pMCR1-IncX4
(Fig. 1). A 2,610-bp mcr-1-pap2 fragment (nucleotides [nt] 2339
to 4948 in pMCR1-IncX4) was inserted into a hypothetical gene
(locus tag pSH146_32_13). Interestingly, insertion element
ISApl1, initially found to be associated with mcr-1 in pHNSHP45
(1), was not present in pMCR1-IncX4.
Received 10 March 2016 Accepted 3 April 2016
Accepted manuscript posted online 18 April 2016
Citation Li A, Yang Y, Miao M, Chavda KD, Mediavilla JR, Xie X, Feng P, Tang Y-W,
Kreiswirth BN, Chen L, Du H. 2016. Complete sequences of mcr-1-harboring
plasmids from extended-spectrum--lactamase- and carbapenemase-producing
Enterobacteriaceae. Antimicrob Agents Chemother 60:4351–4354.
doi:10.1128/AAC.00550-16.
Address correspondence to Liang Chen, chen11@njms.rutgers.edu, or
Hong Du, hong_du@126.com.
A.L., Y.Y., and M.M. contributed equally to this work.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Here we completely sequenced four mcr-1-haboring plasmids, isolated from two extended-spectrum--lactamase (ESBL)-producing Escherichia coli and two carbapenemase-producing Klebsiella pneumoniae clinical isolates. The mcr-1-harboring plasmids from an E. coli sequence type 2448 (ST2448) isolate and two K. pneumoniae ST25 isolates were identical (all pMCR1IncX4), belonging to the IncX4 incompatibility group, while the plasmid from an E. coli ST2085 isolate (pMCR1-IncI2) belongs
to the IncI2 group. A nearly identical 2.6-kb mcr-1-pap2 element was found to be shared by all mcr-1-carrying plasmids.
Li et al.
(A) IncX4
oriVα
oriTα
oriTβ
oriVβ
pSH146_32
oriVα
oriTα
oriTβ
dnaJ
pilX3-4
pilX2
pilX1
actX
taxC
taxA
trbM
taxB
pilX11
pilX10
pilX9
pilX8,7
pilX6
pilX5
pir
taxD
hns
hhs
topB
oriVγ
(32 Kb)
oriVβ
pMCR1_IncX4
pJIE143
oriVα
oriTα
oriTβ
dnaJ
pilX3-4
pilX2
pilX1
actX
taxC
taxA
trbM
taxB
pilX11
pilX10
pilX9
pilX8,7
pilX6
pilX5
IS26
hns
hhs
topB
pap2
mcr-1
pir
taxD
oriVγ
(33 Kb)
oriVβ
dnaJ
pilX3-4
pilX2
pilX1
actX
taxC
taxA
taxB
pilX11
pilX10
pilX9
pilX8,7
pilX6
pilX5
trbM
hns
hhs
topB
parA
blaCTX-M-15
taxD
(B) IncI2
topB
ydiA
nuc
rci
topB
ydiA
nuc
rci
ybbK
ISApl1
mcr-1
pap2
mcr-1
pap2
traJ
traA
nikB
nikB
pilV
pilU
pilT
pilS
pilR
pilQ
pilP
pilO
pilN
yfdA
traK
traJ
traI
traH
traG
traE
traD,C
traB
pilM
pilL
traL
nikA
IS683
mok
yaec
parA
IS200
mok
yaec
parA
dnaJ
traL
nikA
repA
(64 Kb)
repA
pHNSHP45
ybbK
traJ
traA
traE
traD,C
traB
pilM
pilL
traK
traJ
traI
traH
traG
pilV
pilU
pilT
pilS
pilR
pilQ
pilP
pilO
pilN
yfdA
eaa
(65 Kb)
hicA,B
pMCR1_IncI2
FIG 1 Structures of plasmids pMCR1-IncX4 and pMCR1-IncI2. (A) Comparison of IncX4 plasmids pSH146_32 (GenBank accession no. JX258655),
pMCR1_IncX4 (KU761327, this study), and pJIE143 (JN194214); (B) comparison of IncI2 plasmids pHNSHP45 (KP347127) and pMCR1_IncI2 (KU761326,
this study). Colored arrows represent open reading frames, with dark blue, yellow, green, red, purple, and orange arrows representing replication genes, mobile
elements, plasmid transfer genes, the mcr-1 gene, other antimicrobial resistance genes, and plasmid backbone genes, respectively. Blue shading denotes regions
of shared homology among different plasmids.
The mcr-1-harboring plasmid from E. coli strain SZ02,
pMCR1-IncI2, belongs to the IncI2 group, the same plasmid incompatibility group as pHNSHP45, the first plasmid reported to
harbor mcr-1 (1). Plasmid pMCR1-IncI2 is 64,964 bp in length
and harbors 83 predicted open reading frames (ORFs), with a
G⫹C content of 42.7%. The plasmid backbone of pMCR1-IncI2
is similar to that of other IncI2 plasmids, such as pSH146_65
(GenBank accession no. JN983044)(16), pBK15692 (KC845573)
(17), and pHNSHP45 (KP347127) (1). BLASTn search results
showed that pMCR1-IncI2 exhibits only 86% query coverage and
97% overall identity to pHNSHP45. In addition, sequence alignment of both plasmids identified ⬎1,400 nucleotide differences
(single nucleotide polymorphisms [SNPs]), suggesting that
pMCR1-IncI2 and pHNSHP45 are distinct, a finding that suggests
that the mcr-1 gene may be repeatedly acquired. Further analysis
showed that the mcr-1 gene in pMCR1-IncI2 integrated downstream of the nikB gene, in the same location as in pHNSHP45.
Similar to the analysis of pMCR1-IncX4, the mcr-1-associated
ISApl1 element was not found in pMCR1-IncI2 (Fig. 1).
Thus far, mcr-1 has been found in different plasmid incompatibility groups, including IncI2 (1, 9), X4 (6, 18), HI2 (19), and P
(18). However, little is known regarding the mechanism whereby
this gene can be mobilized between different plasmids. We therefore compared the mcr-1 neighboring regions of pHNSHP45,
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pMCR1-IncI2, and pMCR1-IncX4, as well as additional mcr-1harboring contig sequences from the NCBI WGS database (20,
21) (Fig. 2). The comparison identified a nearly identical 2,600-bp
region (nt 2349 to 4948 on pMCR1-IncX4) shared by all sequences examined, encompassing the mcr-1 and pap2 (encoding a
putative PAP family transmembrane protein) genes. Our analysis
suggests that the 2.6-kb mcr-1-pap2 element has been horizontally
transferred into different plasmid backbones (Fig. 2). Further inspection of the upstream and downstream junctions of this mcr1-pap2 element failed to identify any direct or inverted repeat
sequences. In pHNSHP45, ISApl1 is inserted directly upstream of
the mcr-1-pap2 element, and a 25-bp inverted reverse repeat is
located adjacent to the 2.6-kb element. The 25-bp invert repeat was
also identified at the same position in strain 2013LSAL02374, which
contains a contig with a sequence from an IncP plasmid (Fig. 2).
Consistent with the analysis of pMCR1-IncI2 and pMCR1-IncX4,
ISApl1 was not always associated with mcr-1, and it was absent in
several contigs belonging to the IncI2, IncX4, and IncP plasmids (Fig.
2). One possible explanation regarding the transfer mechanism of
mcr-1 is that the mcr-1-pap2 element was initially translocated by the
integration of ISApl1 (20), and the latter was subsequently lost following integration. Nevertheless, the exact mechanism underpinning
mcr-1 transfer requires additional study.
In addition to the two aforementioned mcr-1 plasmids, we also
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pir
ISEcp1
oriVγ
(34 Kb)
Sequences of mcr-1-Harboring Plasmids
~2.6 kb
IncI2
(pHNSHP45)
ISApl1 mcr-1 pap2
nikB
topB
Nucleotide sequence accession numbers. The complete nucleotide sequences of plasmids pMCR1-IncI2, pMCR1-IncX4,
and pNDM5-IncX3 have been deposited in GenBank under accession no. KU761326, KU761327, and KU761328, respectively.
ACKNOWLEDGMENTS
IncI2
(pMCR1-IncI2)
(EC13)
(EC7)
nikB
mcr-1 pap2
topB
IncX4
(pMCR1-IncX4)
(2012CEB2196SAL)
(2012CEB4337SAL)
taxD mcr-1 pap2
FUNDING INFORMATION
IncP
(2013LSAL02374) ISApl1 mcr-1 pap2
mcr-1 pap2
FIG 2 Genetic structure of mcr-1-harboring elements in selected plasmids.
Plasmid incompatibility groups are noted on the left, along with the plasmids
and/or strains harboring the genetic structure depicted. Arrows denote open
reading frames (ORF), with red, gray, yellow, and white arrows denoting
mcr-1, pap2, ISApl1, and neighboring genes, respectively. Following BLAST
analysis of NCBI draft genomes, only contigs long enough to unambiguously
identify plasmid replicon groups were included in this figure. The dashed
yellow outline of the IncP ISApl1 ORF therefore indicates that only a partial
sequence is available. The small black arrows adjacent to ISApl1 denote the
inverted reverse repeats (IRR). The GenBank accession numbers of the mcr-1
contig sequences for EC5, EC7, EC13, 2012CEB2196SAL, 2012CEB4337SAL,
2013LSAL02374, and 2013LSAL04524 are JWKF01000084, JWKG01000081,
JUJZ01000081, LKJJ01000031, LKJK01000087, LNCZ01000024, and
LKJD01000010, respectively.
sequenced the two blaNDM-5-harboring plasmids from strains
SZ03 and SZ04, which were found to be identical and subsequently named pNDM5-IncX3. Plasmid pNDM5-IncX3 is 46,161
bp in length, with a G⫹C content of 46.7%, and harbors 58 putative ORFs. The sequence of pNDM5-IncX3 showed 100% query
coverage and overall ⬎99.9% nucleotide identity to blaNDM-5-harboring plasmids pEc1929 (GenBank accession no. KT824791)
(22) and pNDM_MGR194 (KF220657) (23), as well as blaNDM-4harboring pJEG027 plasmid (KM400601) (24) and blaNDM-7-harboring plasmid pKpN01-NDM7 (CP012990) (25). Notably,
IncX3 plasmids harboring different blaNDM variants were frequently found in different hospitals among isolates of different
multilocus sequence types and species in China (22, 26–28), suggesting that IncX3 plasmids are the primary type of vector responsible for the wide dissemination of NDM metallo--lactamases in China. Alarmingly, pNDM5-IncX3 was found to
coexist with pMCR1-IncX4 within the same clinical isolate
(strains SZ03 and SZ04), resulting in resistance to both colistin
and carbapenems (4).
In summary, this study characterizes two mcr-1-harboring
plasmids from ESBL-producing E. coli and carbapenemase-producing K. pneumoniae. The identification of the same plasmid
(pMCR1-IncX4) in isolates of different species (SZ01, SZ03, and
SZ04) suggests that plasmid transfer is contributing to the dissemination of mcr-1 in hospital settings in China. The potential for
further spread of mcr-1-harboring plasmids within multidrugresistant bacterial strains poses significant challenges for successful clinical treatment and infection control strategies.
July 2016 Volume 60 Number 7
This work, including the efforts of Liang Chen, was funded by HHS | NIH
| National Institute of Allergy and Infectious Diseases (NIAID)
(R21AI117338). This work, including the efforts of Barry N. Kreiswirth,
was funded by HHS | NIH | National Institute of Allergy and Infectious
Diseases (NIAID) (R01AI090155). This work, including the efforts of
Hong Du, was funded by National Natural Science Foundation of China
(NSFC) (81572032).
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