Detection and characterization of pCT-like plasmid vectors
for bla
CTX-M-14
in Escherichia coli isolates from humans, turkeys and
cattle in England and Wales
M. O. Stokes
1,2
*, J. L. Cottell
3
, L. J. V. Piddock
3
,G.Wu
4
, M. Wootton
5
, D. J. Mevius
6
, L. P. Randall
1
, C. J. Teale
1
,
M. D. Fielder
2
and N. G. Coldham
1
1
Department of Bacteriology, Animal Health and Veterinary Laboratories Agency, Addlestone, UK;
2
Faculty of Science, Engineering and
Computing, Kingston University, Kingston, UK;
3
School of Immunity and Infection, The College of Medicine and Dental Sciences,
University of Birmingham, Birmingham, UK;
4
Center for Epidemiology and Risk Analysis (CERA), Animal Health and Veterinary
Laboratories Agency, Addlestone, UK;
5
Public Health Wales, University Hospital of Wales, Cardiff, UK;
6
Department of Bacteriology and
TSEs, Central Veterinary Institute of Wageningen, Lelystad, The Netherlands
*Corresponding author. Tel: +44-1932-357246; Fax: +44-1932-347706; E-mail: m.stokes@vla.defra.gsi.gov.uk
Received 2 December 2011; returned 19 January 2012; revised 9 March 2012; accepted 14 March 2012
Objectives: To detect and characterize Escherichia coli strains and pCT-like plasmids implicated in the dissem-
ination of the CTX-M-14 gene in animals and humans, in England and Wales.
Methods: UK CTX-M-14-producing E. coli (n¼ 70) from cattle (n¼ 33), turkeys (n¼ 9), sheep (n¼ 2) and humans
(n¼ 26) were screened using multiplex PCR for the detection of a previously characterized plasmid, pCT. Isolates
found to be carrying two or more pCT genetic markers were further analysed using PFGE. Their antimicrobial-
resistance genes and virulence genes were also determined. These plasmids were transferred to Salmonella
enterica serotype Typhimurium 26R and further examined for incompatibility type, genetic environment of
the bla
CTX-M-14
gene, size, restriction fragment length polymorphism (RFLP) and nikB sequence.
Results: The 25 E. coli isolates carrying pCT genetic markers generated 19 different PFGE profiles, and 23 iso-
lates had different virulence and antimicrobial-resistance gene patterns. One isolate from cattle was a verotoxi-
genic E. coli (‘VTEC’); the rest were commensal or extra-intestinal pathogenic E. coli. pCT-like plasmids with
similar molecular characteristics (size, replicon type, RFLP pattern, pCT markers and genetic environment of
the bla
CTX-M-14
gene) were detected in 21/25 of the field isolates, which comprised those from cattle (n¼ 9),
turkeys (n¼ 8) and humans (n¼ 4). All pCT-like plasmids were conjugative, and most were IncK (n¼ 21) and
had the same local genetic environment flanking the bla
CTX-M-14
gene (n¼ 23). RFLP analysis demonstrated
≥75% similarity among most plasmids (n¼ 22).
Conclusions: pCT-like plasmids were common vectors for horizontal dissemination of 30% of the bla
CTX-M-14
genes to different E. coli isolates from humans, cattle and turkeys.
Keywords: plasmids, CTX-M-14, IncK, pCT, ESBLs, antimicrobial resistance
Introduction
Third- and fourth-genera tion cephalosporins are commonly used for
treating infections in humans and animals caused by Enterobacter-
iaceae. Howev er, their efficacy is being compromised by extended-
spectrum b-lactamases (ESBLs) and cephamycinases (AmpC)
produced by those bacteria. Since the early 1990s bla
CTX-M
genes
hav e become the most common plasmid-borne ESBL genes,
aiding their horizontal transmission among different Escherichia
coli strains and other species of Enteroba cteriaceae.
1–3
Plasmids
of numerous incompatibility groups encoding various bla
CTX-M
genes have been found in bacterial species from human and
veterinary sources acr oss the globe.
1,4,5
The first UK veterinary
CTX-M isolate was reported in 2004.
6
This E. coli was isolated
from ca ttle and was found to harbour a 93.6 kb IncK plasmid
that encoded bla
CTX-M-14
. This plasmid has been designated ‘pCT’
and sequenced, which has allowed the design of a multiplex PCR
assay for the detection of similar plasmids.
7,8
Whilst the
bla
CTX-M-14
gene remains uncommon in UK human isolates, it has
been found to be prevalent in E. coli from cattle and turke y s.
9,10
In Spain, Fr ance and Asia the bla
CTX-M-14
gene is frequently detected
in human E. coli isolates.
11 –13
The aim of this study was to investigate the mode of dissem-
ination of bla
CTX-M-14
in bacteria isolated from cattle, turkeys and
# Crown copyright 2012.
J Antimicrob Chemother 2012; 67: 1639–1644
doi:10.1093/jac/dks126 Advance Access publication 18 April 2012
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humans in England and Wales, by molecular typing of
CTX-M-14-producing E. coli and their plasmids.
Materials and methods
Bacterial strains
All CTX-M-14-producing E. coli isolates of veterinary origin in the Animal
Health and Veterinary Laboratories Agency (AHVLA) culture collection
(n¼ 44) were selected and grown on Chromagar CTX or Luria– Bertani
minus glucose (LB2G) agar plates with 2 mg/L cefotaxime at 378C. The
isolates from cattle (C, n¼ 33) and sheep (S, n¼ 2) were obtained
through routine surveillance or diagnostic submissions, while those
from turkeys (T, n¼ 9) were obtained during a study on the prevalence
of CTX-M in poultry;
10
with the exception of C8 and C9, all were from dif-
ferent farms and they consisted of eight faecal isolates and one caecum
isolate from healthy animals.
10
All human (H) CTX-M-14 isolates were iso-
lated from urine samples (except one unknown) obtained from individual
patients from Wales (n¼ 18) and England (n¼ 8) through hospital or
community submissions. Field isolates producing CTX-M-1 isolated from
cattle (n¼ 7), turkeys (n¼ 5) and chickens (n¼ 15), CTX-M-3 from cattle
(n¼ 1) and chicken (n¼ 1) and CTX-M-15 from cattle (n¼ 8) were grown
as described above, and used in this study to compare the specific asso-
ciation of pCT-like plasmids for the bla
CTX-M-14
gene.
Analysis of E. coli isolates
The clonal relatedness of CTX-M-14 E. coli field isolates was examined by
XbaI PFGE and antimicrobial and virulence DNA arrays. PFGE was carried
out following the CDC PulseNet protocol and the results were analysed
using the Dice coefficient (Bionumerics 5.10, Applied Maths), with a
cut-off of 85% similarity used to define distinct clusters.
14
DNA array
(Alere Inc.) analysis was performed on E. coli field isolates and Salmonella
enterica serotype Typhimurium 26R transconjugants, as previously
described.
15
This version of the array has 153 probes for antimicrobial re-
sistance and 120 for virulence genes.
16
GeneSpring (Agilent Technologies)
was used for cluster analysis of the gene array data.
PCR analysis of plasmids
Suitable positive and negative controls were included for all PCRs. The
bla
CTX-M
sequence type was confirmed by PCR, using primers described
by Batchelor et al.,
17
and DNA sequence analysis. Investigation of the
bla
CTX-M-14
genetic context and the presence of the insertion sequence
ISEcp1, the pCT multiplex PCR testing for sigma factor, shufflon recombin-
ase, pilN and pCT008-009 and the nikB sequencing for transconjugants
were all performed as described by Cottell et al.
7
(see Table 1). IncK rep-
licon typing was carried out as described by Carattoli et al.,
18
but with a
new forward primer 5
′
-CAGGATCCGGGAAGTCAGAAAAC-3
′
, designed from
the pCT sequence (FN868832.1).
Molecular characterization of plasmids
Veterinary and human isolates containing bla
CTX-M-14
plasmids with at
least two pCT genetic markers were transferred by conjugation to
rifampicin-resistant Salmonella Typhimurium 26R, enabling further char-
acterization of plasmids in an identical host background, as described
previously by Randall et al.
10
Sizing of the plasmids was carried out
using S1 nuclease PFGE, with pCT, low-range (2030–194000 bp) and
mid-range (15000–242500 bp) PFGE molecular marker ladders (NEB)
as size calibration standards.
19
Restriction fragment length polymorph-
ism (RFLP) analysis of the plasmids was performed as previously
described.
8
Results
Molecular analysis of veterinary and human E. coli
isolates
A total of 25 (35.7%, n¼ 7 from humans, n¼ 9 from cattle and
n¼ 9 from turkeys) of the 70 CTX-M-14-producing E. coli field iso-
lates were found to encode two or more pCT genetic markers
(sigma factor, shufflon recombinase, pilN and pCT008-009)
(Table 1). These isolates were selected for further study. Of the
non-CTX-M-14-producing veterinary isolates (n¼ 37), two cattle
CTX-M-1-producing isolates had sigma factor and shufflon re-
combinase pCT genetic markers on IncF plasmids, and three
chicken CTX-M-1-producing isolates had the shufflon recombin-
ase pCT genetic markers on Incl1 plasmids. Additionally, two of
the CTX-M-15-producing isolates from cattle had two pCT
genetic markers (sigma factor with pilN, and sigma factor with
shufflon recombinase; results not shown).
The XbaI PFGE for the 25 CTX-M-14-producing E. coli isolates
with pCT markers identified 19 unique clusters at 85% similarity,
and identified H5 and H6 as clones (Figure 1). With the excep-
tions of the human H5 and H6 clones and T5 and T7 from
turkeys, all isolates had different genes for antibiotic resistance
(Figure S1a, available as Supplementary data at JAC Online)
and virulence (Figure S1b, available as Supplementary data at
JAC Online) as determined by microarrays; assigned arrays and
PFGE types are stated in Table 1. This supported the PFGE
profile and demonstrated that the E. coli isolates were clearly un-
related. Analysis of virulence genes (Figure S1b) identified a ver-
otoxigenic E. coli (‘VTEC’) strain (C7) harbouring stx1A, eae, hlyA
and type III secretion systems genes, such as nleA. Other iso-
lates contained genes that were found among extra-intestinal
E. coli (ExPEC) or commensal isolates. The serum-resistance
gene (iss) and the gene for long polar fimbriae (lpf) were found
in a high proportion of isolates (80% and 56%, respectively).
Virulence genes including pic, vat, tsh, nfaE, sat and pfrB, which
are typically found in ExPEC, were identified among this group
of strains. Genes that are often associated with ExPEC, such as
those for iron utilization, iroN and ireA, and those for microcin
production, celb, mchBCF, mcmA and
cma,
were found in
various strains in this study (Figure S1b).
Molecular analysis of plasmids
All bla
CTX-M-14
plasmids harbouring two or more pCT genetic
markers were conjugated into Salmonella Typhimurium 26R. The
pCT PCR showed that 19/25 (76%; human n¼ 4, cattle n¼ 8and
turkey n¼ 7) encoded all four pCT genetic markers, three had
three genetic markers (12%; one each from human, cattle
and turkey), two human isolates had two genetic markers
(8%) and the transconjugant of T4 had one marker (Table 1).
However, pCT markers were in the field isolates harbouring
pMSC2, which contained sigma factor, pMST4 had shufflon recom-
binase, pilN, and pCT008-009 and pMST6 had sigma factor.
With the exception of pMSH5, pMSH6, pMSH7 and pMST4, all of
the plasmids (n¼ 21/25) belonged to the IncK replicon type
(Table 1). All of the bla
CTX-M-14
plasmids (n¼ 23/25), apart from
pMSH5 and pMSH6, had the same ISEcp1-bla
CTX-M-14
-pseudogene
R genetic environment (Table 1). Sequence analysis of nikB
showed that most of the plasmids (n¼ 23/25) had the same
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Table 1. The 25 E. coli isolates with two or more pCT genetic markers used in this study
Plasmid
ID
Host
species Location
Year of
isolation Sample
PFGE, AMR and
virulence types
E. coli field
isolate pCT
PCR multiplex
Salmonella Typhimurium
26R transconjugant pCT
PCR multiplex bla
CTX-M-14
ISEcp1-bla
CTX-M-14
-
pseudogene R
Plasmid
size (kb)
IncK
replicon
type
nikB
sequence
pMSC1 cattle England 2010 faeces 1, 1, 1 S, R, N, P S, R, N, P ++90 ++
pMSC2 cattle Wales 2010 faeces 2, 2, 2 S, R, N, P R, N, P ++85 ++
pMSC3 cattle Wales 2010 faeces 3, 3, 3 S, R, N, P S, R, N, P ++110 ++
pMSC4 cattle England 2010 faeces 4, 4, 4 S, R, N, P S, R, N, P ++90 ++
pMSC5 cattle England 2010 faeces 1, 5, 1 S, R, N, P S, R, N, P ++95 ++
pMSC6 cattle Wales 2006 faeces 5, 6, 5 S, R, N, P S, R, N, P ++95 ++
pMSC7 cattle England 2008 faeces 6, 7, 6 S, R, N, P S, R, N, P ++95 ++
pMSC8 cattle Wales 2006 caecum 7, 8, 7 S, R, N, P S, R, N, P ++95 ++
pMSC9 cattle Wales 2006 faeces 7, 9, 8 S, R, N, P S, R, N, P ++95 ++
pMSH1 human Wales 2007 urine 8, 10, 9 S, R, N, P S, R, N, P ++95 ++
pMSH2 human Wales 2007 urine 9, 11, 10 S, R, N, P S, R, N, P ++95 ++
pMSH3 human Wales 2007 urine 10, 12, 11 S, R, N, P S, R, N, P ++95 ++
pMSH4 human Wales 2007 unknown 11, 13, 12 S, R, N, P S, R, N, P ++105 ++
pMSH5 human Wales 2007 urine 12, 14, 13 S, N S, N + 2 100 2 + (9)
pMSH6 human Wales 2007 urine 12, 14, 13 S, N S, N + 2 100 2 + (9)
pMSH7 human England 2009 urine 13, 15, 14 S, R, N S, R, N ++100 2 +
pMST1 turkey England 2006 faeces 14, 16, 15 S, R, N, P S, R, N, P ++95 ++
pMST2 turkey England 2006 faeces 15, 17, 16 S, R, N, P S, R, N, P ++95 ++
pMST3 turkey England 2006 faeces 16, 18, 17 S, R, N, P S, R, N, P ++95 ++
pMST4 turkey England 2006 faeces 16, 4, 17 S, R, N, P S ++ND 2 + (FI)
pMST5 turkey England 2006 faeces 17, 12, 18 S, R, N, P S, R, N, P ++95
++
pMST6
turkey England 2006 faeces 18, 19, 219 S, R, N, P R, N, P ++95 ++
pMST7 turkey England 2006 faeces 17, 12, 18 S, R, N, P S, R, N, P ++100 ++
pMST8 turkey England 2006 faeces 19, 20, 20 S, R, N, P S, R, N, P ++95 ++
pMST9 turkey England 2006 faeces 19, 21, 21 S, R, N, P S, R, N, P ++95 ++
FI, field isolate used for nikB sequencing; unknown, not recorded.
pCT markers: S, sigma factor (1289 bp); R, shufflon recombinase (945 bp); N, pilN (627 bp); and P, pCT008-009 (428 bp).
ISEcp1-bla
CTX-M-14
-pseudogene R is the genetic environment surrounding bla
CTX-M-14
.
The accession number for the pCT plasmid is FN868832.1.
DH5a pCT was used as a positive control, and Salmonella Typhimurium 26R without plasmid pCT was used as a negative control.
Assigned PFGE, antimicrobial resistance (AMR) and virulence types are all indicated numerically; for PFGE a cut-off at 85% similarity was applied, and for AMR and virulence genes
profiles, a difference of one gene was used to define new groups.
bla
CTX-M-14
pCT-like plasmids in E. coli from humans and animals
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sequence as pCT (this includes the sequence from the T4 field
isolate). Variations were only seen in plasmids pMSH5 and
pMSH6, which had nine nucleotide substitutions relative to pCT:
nucleotides 81 (TC), 90 (CT), 96 (TG), 129 (AC), 130
(TG), 216 (AG), 222 (CG), 231 (TC) and 240 (AG).
RFLP analysis of the plasmids in the transconjugants with re-
striction enzymes PstI (Figure S2, available as Supplementary
data at JAC Online) and EcoRI (results not shown) revealed
that 22/25 (88%) of the plasmids had 75%–85% similarity in
banding patterns, while the clonal plasmids pMSH5 and pMSH6
had 65% similarity to the other pCT-like plasmids. There was
also a small cluster at the 70% similarity level following EcoRI di-
gestion, which included plasmids pMSC2, pMSC3 and pMSH4. S1
nuclease sizing showed that 15 plasmids were of similar size
to pCT (93.6 kb, 95 kb by S1 nuclease sizing), 6 were larger
(100–110 kb) and 3 were smaller (85–90 kb) (Table 1). Differ-
ences in RFLP patterns were consistent with the varying sizes
of plasmids. The T4 transconjugant had no detectable
plasmid or IncK or nikB sequence, but had the bla
CTX-M-14
gene
in the same genetic environment and the sigma factor genetic
marker. Aminoglycoside (aac6) resistance gene was identified
on pMSH5 and pMSH6 by DNA array. The results of the molecular
typing data demonstrated that 21/25 bla
CTX-M-14
plasmids
shared a high level of similarity to each other and to pCT; this
comprised 30% of all CTX-M-14 E. coli tested.
Discussion
Until now, bla
CTX-M-14
IncK pCT plasmids from the UK have only
been reported in cattle. In this study, bla
CTX-M-14
IncK pCT-like
plasmids were identified in 21/70 (30%) of CTX-M-14 E. coli iso-
lates from humans, cattle and turkeys, of which 17 of the field
isolates were unrelated to each other based on molecular
typing by PFGE, antimicrobial-resistance and virulence microar-
rays. Plasmids were defined as being pCT-like by the presence of
three or more pCT markers: the ISEcp1-bla
CTX-M-14
-pseudogene R
genetic environment, nikB sequence and the IncK replicon type.
Although there were some variations among the pCT-like plas-
mids in terms of RFLP patterns, size or genetic markers, they
shared a similar genetic backbone with pCT. The variations in
pCT-like plasmids are likely to have occurred through mutation
and recombination. Some markers identified in the field isolates
but not in the transconjugants could be located on the chromo-
some or on other plasmids. Also, since no plasmid and only
genes for ISEcp1-bla
CTX-M-14
-pseudogene R and sigma factor
were detected in the transconjugant of T4, it is likely that part
of the plasmid from the T4 field isolate was integrated into the
chromosome of the transconjugant, as all of the pCT markers,
nikB and a plasmid of similar size were detected in the field
isolate. The absence of IncK in pMSH5, pMSH6 and pMSH7 may
be the result of the plasmids belonging to an alternative replicon
type, and along with pMST4, these were not described as pCT-like
plasmids. Only 10.8% (n¼ 4/37) of non-CTX-M-14-producing iso-
lates (CTX-M-1, CTX-M-3 and CTX-M-15) had at least two of the
pCT genetic markers, none of which was IncK, demonstrating a
strong association of bla
CTX-M-14
with pCT and IncK (n¼ 21/25).
Individual pCT markers are not IncK-specific and can be found
in other plasmids, but these markers are designed to work in
combination for detection of pCT-like plasmids.
All pCT-like plasmids in this study were successfully conju-
gated to a Salmonella recipient strain, as was reported previously
for pCT-like plasmids from the outbreak farm.
8
Therefore, conju-
gation was the most likely mechanism for the spread of pCT-like
plasmids to diverse E. coli strains recovered from humans, cattle
and turkeys. This demonstrates that pCT-like plasmids are im-
portant vectors for the dissemination of the bla
CTX-M-14
gene.
Similar reports from Korea show that IncF bla
CTX-M-14
plasmids
have a role in disseminating the bla
CTX-M-14
gene between
E. coli that have no major clonal relationship based on either
PFGE or multilocus sequence typing.
13
This mechanism for CTX-M-14 dissemination by pCT-like plas-
mids is in contrast to that for the spread of CTX-M-15, where the
pandemic E. coli clone O25:ST131 plays a dominant role.
20 – 22
This successful E. coli clone has been found to disseminate
bla
CTX-M-15
and other bla
CTX-M
genes on various plasmids in the
UK and across Europe.
21 – 23
In addition, mobile elements, such
as integrons and transposons, also play important roles in
mobilizing resistance genes onto different plasmids and
chromosomes.
24 – 26
The horizontal transmission of plasmids between unrelated
E. coli and Salmonella in animals has been shown previously.
8,27
Sever al reports ha v e commented on the transmission of plasmids
H5
100
80
60
H6
H1
H3
H4
T3
C3
C7
C4
C2
H2
C8
C9
C6
C1
C5
H7
T4
T1
T2
T6
T5
T7
T8
T9
Figure 1. XbaI PFGE of the 25 E. coli isolates containing bla
CTX-M-14
pCT-like plasmids isolated from humans, cattle and turkeys. Salmonella
Braenderup was used as the control to align the banding profiles.
Similarities in banding patterns were calculated using the Dice
coefficient (Bionumerics 5.10). C, cattle isolates; H, human isolates; T,
turkey isolates.
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between human and veterinary isolates. These include IncN
bla
CTX-M-1
plasmids in unrelated E. coli isolates from humans and
pigs and the IncI1 bla
CTX-M-1
,IncHI2bla
CTX-M-2
and IncHI2
bla
CTX-M-9
plasmids in E. coli and Salmonella isolates from
humans and poultry across E ur ope.
28 –31
The detection of similar pCT-like plasmids in human and vet-
erinary isolates from England and Wales suggests that these
plasmids are effective vectors for the dissemination of bla
CTX-M
among different animal host species that may be linked by the
food chain or the environment. Food has previously been found
to be contaminated with quinolone-resistant ESBL bla
CTX-M-14
E. coli and those carrying bla
CTX-M-1
IncI1 plasmids that were
also found in broilers across Europe.
29,32,33
Several routes exist
for the transmission of bla
CTX-M
genes from humans to
animals, and these include land flooded with sewage-
contaminated water and various wildlife vectors, including rats
and gulls.
34 – 36
The presence of the pCT-like plasmids in over
45% of bla
CTX-M-14
-producing animal isolates examined suggests
a reservoir of these vectors in food-producing animals in the UK.
Such plasmids may be maintained through the prophylactic and
therapeutic use of third- and fourth-generation cephalosporins in
veterinary medicine.
28,37
Further work is needed to identify the
contribution of animal reservoirs to the dissemination of
bla
CTX-M-14
IncK pCT-like plasmids through the food chain to
humans. In conclusion, pCT-like plasmids were shown to be im-
portant vectors for the horizontal dissemination of the bla
CTX-M-14
gene to clonally unrelated E. coli isolates from humans, cattle
and turkeys.
Acknowledgements
We would like to thank Neil Woodford, Martin Woodward, Manal AbuOun,
Fabrizio Lemma, Heather Wearing, Monique Toszeghy, Meenaxi Sharma,
Robert Horton, Hannah Reeves, Christine Boinett and Irene Freire-Martin
for their help.
Funding
This work was funded by the Department for Environment, Food and
Rural Affairs (project grants VM2207 and OD2028).
Transparency declarations
None to declare.
Supplementary data
Figures S1 and S2 are available as Supplementary data at JAC Online
(http://jac.oxfordjournals.org/).
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