Genetic characterization of mcr-1-bearing plasmids to depict molecular
mechanisms underlying dissemination of the colistin resistance
determinant
Ruichao Li
1,2
†, Miaomiao Xie
1
†, Jinfei Zhang
1
, Zhiqiang Yang
1
, Lizhang Liu
1
, Xiaobo Liu
1
, Zhiwei Zheng
1
,
Edward Wai-Chi Chan
2
and Sheng Chen
1,2
*
1
Shenzhen Key Lab for Food Biological Safety Control, Food Safety and Technology Research Center, Hong Kong PolyU Shen Zhen
Research Institute, Shenzhen, P. R. China;
2
The State Key Lab of Chirosciences, Department of Applied Biology and Chemical Technology,
The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR
*Corresponding author. Tel: +852-34008795; E-mail: sheng.chen@polyu.edu.hk
†These authors contributed equally to the work.
Received 27 May 2016; returned 13 July 2016; revised 25 July 2016; accepted 29 August 2016
Objectives: To analyse and compare mcr-1-bearing plasmids from animal Escherichia coli isolates, and to inves-
tigate potential mechanisms underlying dissemination of mcr-1.
Methods: Ninety-seven ESBL-producing E. coli strains isolated from pig farms in China were screened for the
mcr-1 gene. Fifteen mcr-1-po sitive strains were subjected to molec ular characteriz ation and bioinformatic
analysis of the mcr-1-bearing plasmids that they harboured.
Results: Three major types of mcr-1-bearing plasmids were recovered: IncX4 (≏33 kb), IncI2 (≏60 kb) and IncHI2
(≏21 6–280 kb), among whi ch the In cX4 and IncI2 plasmids were found to harbour the mcr-1 gene only,
whereas multiple resistance elements including bla
CTX-M
, bla
CMY
, bla
TEM
, fosA, qnrS, floR and oqxAB were detected,
in various combinations, alongside mcr-1 in the IncHI2 plasmids. The profiles of mcr-1-bearing plasmids in the
test strains were highly variable, with coexistence of two mcr-1-bearing plasmids being common. However, the
MIC of colistin was not affected by the number of mcr-1-carrying plasmids harboured. Comparative analysis of
the plasmids showed that they contained an mcr-1 gene cassette with varied structures (mcr-1-orf,ISApl1-mcr-
1-orf and Tn6330), with the IncHI2 type being the most active in acquiring foreign resistance genes. A novel trans-
poson, Tn6330, with the structure ISApl1-mcr-1-orf-ISApl1 was found to be the key element mediating transloca-
tion of mcr-1 into various plasmid backbones through formation of a circular intermediate.
Conclusions: The mcr-1 gene can be disseminated via multiple mobile elements including Tn6330, its circular
intermediate and plasmids harbouring such elements. It is often co-transmitted with other resistance determi-
nants through IncHI2 plasmids. The functional mechanism of Tn6330, a typical composite transposon harbour-
ing mcr-1, should be further investigated.
Introduction
The continuous emergence of novel antibiotic resistance-encoding
genetic elements among the major bacterial pathogens in recent
years has undermined current efforts to devise new antimicrobial
strategies, and poses an enormous threat to public health.
1,2
Polymyxins, including polymyxin B and colistin, are cationic anti-
microbial peptides which act on G ram-negative bacteria by dis-
rupting the outer and inner membranes.
3,4
Polymyxins were
discovered in the late 1940s but deemed unsuitable for treatment
of bacterial infections because of their neurotoxic effects; how-
ever, emergence of MDR Gram-negative bacteria has prompted a
renewed interest in this old antibiotic, which is currently regarded
as a possible last-resort agent to eradicate MDR organisms.
5,6
Resistance to colistin has been reported among different bacterial
species, the underlying mechanism of which is mainly intrinsic in
nature. On the other hand, acquired resistance due to modifica-
tions of the bacterial outer membrane, efflux pumps and capsular
polysaccharides have also been reported.
3,7
Recently, pioneering
work performed by Liu et al.
8
described the recovery of a conjuga-
tive plasmid-med iated polymyxin resistance gene mcr-1,which
encodes an enzyme belonging to the phosphoethanolamine
transferase enzyme family, from both animals and human.
8
Following this discovery, a nu mber of studies have reported the
presence of mcr-1 in different species of Enterobacteriaceae
which exhibited MDR phenotypes recovered from farmed animals,
# The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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doi:10.1093/jac/dkw411
J Antimicrob Chemother 2017; : 393–40172
Advance Access publication 28 September 2016
393
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food samples and clinical settings around the world.
9,10
The emer-
gence of mcr-1 has been traced back to Escherichia coli strains iso-
lated in the 1980s when colistin was first introduced into
veterinary practice in China,
11
indicating that this gene has existed
in food animals for a considerable period.
Plasmids harbouring mcr-1 have been reported to include the
IncX4, IncI2, IncP, IncFII and IncHI2 types.
9,12 –15
The prevalence
of mcr-1 in plasmids harboured by different bacterial species high-
lights its potential of being transferred horizontally. Notably,
mcr-1 was often found to be located downstream of ISApl1,
13
which is an IS belonging to the IS30 family.
16
The close genetic
association between ISApl1 and mcr-1 indicates that ISApl1
may play a pivotal role in the dissemination of mcr-1. Data regard-
ing the compl ete sequences of mcr-1-bearing plasmids and the
role of ISApl1 in mediating transposition of the mcr-1 gene are
scarce. In this study, we aimed to investigate if the mcr-1 gene
was prevalent in farm settings, and the range of plasmids which
harboured such a resistance gene. By characterizing the
mcr-1-bearing plasmids in E. coli isolated from far med animals
in different parts of China, we discovered a mcr-1-bearing trans-
posable element which can form a circular intermediate that
plays a key role in genetic translocation, and hence transmission
of the mcr-1 element among a wide range of potential human
pathogens.
Materials and methods
Bacterial strains and identification
Cefotaxime-resistant E. coli strains were isolated from faeces of healthy
pig s on six farms located in six provinces in China, namely Guangdong,
Fujian, Jiangsu, Sh andong, Henan and Liaoning, during the period
September 2014– March 2015, by using MacConkey a gar plates supple-
mented with 4 mg/L cefotaxime. The strains were identified as E. coli by
MALDI-TOF MS using a Bru ker MicroFlex LT mass spectrometer (Bruker
Daltonics) and confirmed using API20E strips (bioMe
´
rieux, Inc.).
Antimicrobial susceptibility tests
MICs of 14 antimicrobial agents as listed in Table S1 (available as
Supplementary data at JAC Online) were determined using the agar dilu-
tion method; results were inte rpreted according t o CL SI recommenda-
tions. E. coli strain ATCC 25922 was used as a quality control strain.
Prevalence of mcr-1-positive E. coli strains
All E. coli strains were screened for the presence of the mcr-1 gene by PCR
using primers as previously described.
8
The PCR products were purified and
sequenced by Sanger sequencing to confirm the genetic identity.
XbaI-PFGE, S1-PFGE and Southern hybridization
E. coli strains that carried the mcr-1 gene were subjected to further char-
acterization. PFGE of XbaI-digested genomic fragments was pe rformed
to assess the genetic relatedness of isolates, using the CHEF-MAP-PER
System (Bio-Rad); genomic DNA of the Salmonella enterica
var. Braenderup H9812 strain restricted with XbaI was used as the refer-
ence standard. Cluster analysis of PFGE patterns was typically performed
by the BioNumerics (Applied Maths) system. S1 nuclease-PFGE was per-
forme d to characterize the plasmid profiles; the location of mcr-1 was
identified by Southern hybridization wi th digoxigenin-labelled mc r-1
probe according to the manufacturer’s instructions for the DIG-High
Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics).
Plasmid sequencing and bioinformatics analyses
Plasmids extracted from the 15 bacterial strains using the QIAGEN Plasmid
Midi Kit were used to prepare the sequencing libraries, which were con-
structed by the NEBNext
w
Ultra
TM
II DNA Library Prep Kit for Illumin a
w
(NEB) and sequenced by the NextSeq 500 Illumina platform, with
2×150 bp paired-end reads. De novo assembly of the reads was con-
ducted with SOAPdenovo2,
17
followed by the use of ResFinde r
17
and
PlasmidFinder
18
to identify resistance genes and plasmid types among
the scaffolds. Construction of complete plasmid seque nces was accom-
plished by using avail able plasmi d reference sequences to align and
assemble the contigs, a nd confirmed by mappin g paired-end reads to
the finished complete sequences. In an attempt to obtain the complete
gene map of mcr-1-bearing plasmids that in silico analysis failed to pro-
duce, PacBio RSII single-molecule , real-time (SMRT) sequencing was
performedtocreatelibrariesof20kbattheWuhanInstituteof
Biotechnology, Wuhan, China. The library preparation work was conducted
according to the instructions of the manuf acturer, Pacific Biosciences.
Illumina contigs were joined together by using the PacBio long contigs
after assembly with HGAP 3.0. The annotations of the plasmid sequences
were conducted by RAST
19
and edited manually. Alignments with highly
homologous complete plas mid sequences available in NCBI for t hese
three plasmid types were performed by using the BR IG tool.
20
Comparison of four complete mcr-1 IncHI2 plasmids was performed
and visualiz ed with Easyfig.
21
The representative mcr-1-bearing plasmid
sequences pECJS-59-244, pECJS-B60-267, pECJS-61-63 and pECGD-8-33
were submitted to NCBI with the accession numbers KX084394,
KX254341, KX254342 and KX254343, respectively.
Detection of circular intermediates
Based on the knowledge that the IS30 family ISs could form DNA circular
intermediates
22
and that ISApl1 exhibits similarity with the IS30 family
members (39% identity with IS30 transposase from E. coli NP_414790 in
protein sequence), a set of reverse primers targeting mcr-1 was designed
to investigate the potential of the ISApl1-mcr-1 segment to circularize
(Table S2). The PCR produ cts were sequenced by Sanger sequen cing to
obtain a complete map of the circular intermediate.
Results
Characterization of mcr-1-positive, ESBL-producing E. coli
Among the 97 cefotaxime-resistant E. coli strains isolated from
faecal samples of pigs from various farms, 35 isolates (36%)
were found to harbour the mcr-1 gene. These mcr-1-p ositive
strains, which were isolated from pigs in farms located in five geo-
graphically diverse provinces of China, were shown to exhibit
genetically divergent PFGE types (Figure S1). However, strains col-
lected from the same province were generally genetically more
related than those obtained from other provinces (data not
shown). The MICs of colistin were either 4 or 8 mg/L for all the
mcr-1-positive strains, with the majority exhibiting resistance to
multiple antimicrobial agents except meropenem (Table S1). To
obtain a comprehensive view of the genetic features of
mcr-1-bearing plasmids in these isolates, 15 mcr-1-positive E.
coli strains of different PFGE types were selected for further char-
acterization. S1-PFGE and Southern hybridization analysis showed
that each of these isolates carried multiple plasmids, among
which some were found to harbour more than one mcr-1-bearing
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plasmid. The size of pla smids observed in th ese isolates ranged
from ≏33 to ≏280 kb, with three major categories being observ-
able: ≏33, ≏60 and ≏216–280 kb. Most of the strains were found
to harbour one ≏33 or 60 kb plasmid, with some, such as strains
FJ-B42 and JS-B65, carrying both. Some strains, such as JS-B60,
JS-64, JS-B73 and JS-59, were also fo und to carry one
mcr-1-bearing plasmid of ≏216–280 kb in size (Table
1).
Genetic characterization of plasmids harbouring the
mcr-1 gene
Plasmids extracted from the 15 mcr-1-positive strains were sub-
jected to nucleotide sequencing using the Illumina platform.
Raw reads were subjected to de novo assembly to obtain contigs
for each sample. BLASTN ana lysis against the resistance gene
database, using the Illumina contigs, showed that plasmids recov-
ered from these 15 strains comprised multiple drug resistance
determinants (Table S3); this finding was consistent with the
MDR phenotypes observable among the test strains (Table S2). In
addition to mcr-1, plasmids derived from most of the strains were
found to harbour multiple resistance elements, including but not
limited t o bla
CTX-M
, bla
CMY
, bla
TEM
, fosA, qnrS, floR and oqxAB.
However, it should be noted that there is still a slight possibility
that some of the resistance genes (Table S3) were not plasmid
borne, and were presumably locate d i n the chromosome since
the plasmid DNA used for high-throughput sequencing could be
contaminated by chromosomal DNA, a phenomenon confirmed
by other sequencing projects in our laboratory.
Six IncX4, seven IncI2 and seven IncHI2 plasmids were con-
firmed to harbour the mcr-1 gene among the 15 test strains, five
of which were found to contain two mcr-1-bearing plasmids
(Table
1). The c omplete plasmid sequences of ≏33 and ≏60 kb
plasmids could be obtained by in silico analysis of the Illumina
sequencing data using the corresponding reference sequences
(pHNSHP45 and pECJS-B65-33). The ≏33 kb plasmid, pECGD-
8-33, obtained from sample GD-8, was shown to belong to the
IncX4 type, comprising 55 coding sequences (CDSs) with a size of
33307 bp, and an overall GC content of 41.84%. The complete
plasmid sequence of the ≏60 kb plasmid, pECJS-61-63, obtained
from sample JS-61, was shown to belong to the IncI2 type and
comprise 88 CDSs, with a size of 63656 bp and a GC content of
42.64%. The complete plasmid sequences of the 216–280 kb plas-
mids could not be generated from the Illumina sequencing data.
Two representative plasmids, from samples JS-59 and JS-B60,
respectively, were subjected to PacBio sequencing to obtain the
complete sequence. The complete sequence of a 216–280 kb cat-
egory plasmid, namely pECJS-59-244, which was derived from
sample JS-5 9, was obtained. I t wa s shown that th is plasmid
belonged to the IncHI2 type, contained 321 CDSs, and had a size
of 243572 bp, of w hich the GC content was 46.10%. Another
IncHI2 plasmid, pECJS-B60-267, which was obtained from sample
JS-B60, was found to be 267486 bp in size. These two IncHI2-type
plasmids were compared with the contigs of IncHI2 plasmids in
other samples (Figure
1). IncHI2-type plasmids have been reported
as genetic elements mediating the transmission of MDR genes
(Table S4) . Results of comparative analysis showed that a wide
range of resistance genes and genetic elements could be found
in a mosaic MDR region of IncHI2-type plasmids, including inte-
grons,ISsandvariousresistancegenecassettes.Themapof
mcr-1-bearing plasmids could be completed s uccess fully by
using references of similar types to assist assembly of contigs of
different samples, with the excepti on of I ncHI2-type plasmids,
Table 1. The sizes and profiles of mcr-1-bearing plasmids harboured by 15 ESBL-producing E. coli strains
Strain
mcr-1
plasmids (kb)
a
Other
plasmids (kb)
a
Plasmids with complete
sequence
b
Plasmids with
scaffolds
b
mcr-1 locus
c
Circular
form
GD-8 33 104; 90; 60 pECGD-8-33(IncX4) — mcr-1-orf –
HN-15 60 120; 90; 78 pECHN-15–61(IncI2) — ISApl1-mcr-1-orf –
FJ-44 250, 33 560; 78; 60 pECFJ-44-33(IncX4) pECFJ-44-250 mcr-1-orf –
JS-56 60 33 pECJS-56-62(IncI2) — mcr-1-orf –
JS-59 244 90 pECJS-59-244(IncHI2) — Tn6330 +
JS-61 230, 60 104; pECJS-61-63(IncI2) pECJS-61-230 mcr-1-orf;Tn6330 +
JS-63 230, 60 104; 33 pECJS-63-63(IncI2) pECJS-63-230 mcr-1-orf –
SD-112 33 104; 78 pECSD-112-33(IncX4) — mcr-1-orf –
SD-137 60 138; 104; 33 pECSD-137-60(IncI2) — mcr-1-orf –
FJ-B42 60, 33 138; 104 pECFJ-B42-33(IncX4) — mcr-1-orf –
pECFJ-B42-63(IncI2) ISApl1-mcr-1-orf
FJ-B44 33 480; 270; 78; 60 pECFJ-B44-33(IncX4) — mcr-1-orf –
JS-B60 250 560 pECJS-B60-267(IncHI2) — ISApl1-mcr-1-orf –
JS-B65 60, 33 90; 40 pECJS-B65-33(IncX4) — mcr-1-orf –
pECJS-B65-60(IncI2) mcr-1-orf
JS-64 280 560; 104; 60 — pECJS-64-280 Tn6330 +
JS-B73 230 100 — pECJS-B73-230 Tn6330 +
a
The size of the plasmids was determined according to the S1-PFGE results.
b
Plasmids for which complete sequences have been obtained. Complete sequences were not available in five IncHI2 mcr-1-bearing plasmids, for which
only the assembly scaffolds were generated. The replicon types of samples were confirmed by PCR using specific primers listed in Table S2.
c
The mcr-1 locus for pECFJ-44-250 and pECJS-63-230 was unavailable.
Characterization of mcr-1-bearing plasmids
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which contained a large MDR region with numerous ISs. As a result,
complete sequences of six IncX4-type, seven IncI2-type and two
IncHI2-type plasmids were obtained using this approach (Table 1).
Plasmids belonging to each of these three types obtained from
this study, and genetically similar mcr-1-bearing plasmids
reported previously, were subjec ted to comparative analysis.
Figure 1. Sequence alignment of I ncHI2 mcr-1 plasmids and WGS contigs in six samples. pECJS-59-244 was used as reference to compare with
pHNSHP45-2 (GenBank no. KU341381), pECJS-B60-267 and other WGS data involving IncHI2 contigs detectable in mcr-1-positive strains. The outer
circle with red arrows denotes annotation of reference plasmid. Among the five mcr-1 IncHI2 strains (JS-44, JS-61, JS-63, JS64 and B73) without
complete plasmid sequences, JS-44 exhibits sequence similarity with the reference, but alignment was not successful because of the overall low
sequence homology. Note that Tn6330 is only present in plasmids pECJS-59-244, pECJS-61-230, pECJS-64-280 and pECJS-B73-230. The number of
ISApl1 repeats is not depicted in this figure. One MDR region was observable in the backbone of all IncHI2 plasmi ds; det ailed information o f the
mcr-1 location in the complete sequences of IncHI2 plasmids is denoted in Figure S2. Information about the IncHI2 plasmids tested in this study is
provided in Table
1. This figure is available in colour in the online version of JAC and in black and white in the print version of JAC.
Li et al.
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The ≏33 kb, IncX4-type plasmids harboured by E. coli strains iso-
lated from different geographical locations of C hina as wel l as
other countr ies were fo und to be almost iden tical (Figure
2).
Apart from mcr-1, no other resistance genes were detectable; in
addition, no ISApl1 elements were found to flank the mcr-1 gene.
A genetically homologous, yet mcr-1-negative plasmid of this type,
namely pSH146_3 2, was previously isolated from a Salmonella
Heidelberg strain (JX258655), suggesting that the mcr-1-orf
gene cassette was most likely introduced into this plasmid back-
bone to form a mcr-1-bearing plasmid of this type (Figure
2).
Figure 2. Sequence alignment of IncX4-type mcr-1-bearing plasmids. pESTMCR, which was recovered in Estonia with GenBank no. KU743383, was used
as reference to match with other I ncX4-type plasmids with (six plasmids in this stu dy and pmcr1_In c X4 , KU761327) and withou t t he mcr-1 gene
(pSH146_32, JX258655). The outer circle with red arrows denotes the anno tation of reference sequence. pSH146_32 exhibits a lower degree of
sequence homology to the reference sequence when compared with other plasmids, and is depicted by a grey circle instead of the light pink colour
chosen to represent this plasmid. The figure shows that the six IncX4 plasmidstestedinthisstudyandtwopreviouslyreportedIncX4plasmids
(pESTMCR and pmcr1_IncX4) share an extremely high degree of sequence homology. Information about the IncX4 plasmids tested in this study is
provided in Table
1. This figure is available in colour in the online version of JAC and in black and white in the print version of JAC.
Characterization of mcr-1-bearing plasmids
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