Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human.

Abstract

Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes.
A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of sul genes, the reservoir and plasmid present.
A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the three reservoirs (pigs, pig carcasses and humans) were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged from 33 to 160-kb in size and belonged to nine different incompatibility (Inc) groups: FII, FIB, I1, FIA, B/O, FIC, N, HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%), while IncI1 was the most common type in sul1 and sul3-carrying plasmids (33% and 45%, respectively). Multireplicons were found associated with all three sul genes.
Sul genes were distributed widely in E. coli isolated from pigs and humans with sul2 being most prevalent. Sul-carrying plasmids belonged to diverse replicon types, but most of detected plasmids were conjugative enabling horizontal transfer. IncFII seems to be the dominant replicon type in sul2-carrying plasmids from all three sources.

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RESEARCHOpen Access
Prevalence and characterization of plasmids
carrying sulfonamide resistance genes among
Escherichia coli from pigs, pig carcasses and
human
Shuyu Wu1,2,4, Anders Dalsgaard2, Anette M Hammerum3, Lone J Porsbo1, Lars B Jensen1*
Abstract
Background: Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize
plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a
specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes.
Methods: A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their
susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes
among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on
presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease
digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids.
Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of
sul genes, the reservoir and plasmid present.
Results: A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the
three reservoirs (pigs, pig carcasses and humans) were 65%, 45% and 12% for sul2, sul1, and sul3, respectively.
Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin
and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying
plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged
from 33 to 160-kb in size and belonged to nine different incompatibility (Inc) groups: FII, FIB, I1, FIA, B/O, FIC, N,
HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%), while IncI1 was the most common type
in sul1 and sul3-carrying plasmids (33% and 45%, respectively). Multireplicons were found associated with all three
sul genes.
Conclusions: Sul genes were distributed widely in E. coli isolated from pigs and humans with sul2 being most
prevalent. Sul-carrying plasmids belonged to diverse replicon types, but most of detected plasmids were
conjugative enabling horizontal transfer. IncFII seems to be the dominant replicon type in sul2-carrying plasmids
from all three sources.
Background
High prevalence of sulfonamide resistance has been
observed in Gram-negative bacteria from animals and
humans all over the world [1-5]. In Denmark,
sulfonamides are one of the most frequently used anti-
microbials to treat diseases in pigs and sulfonamide
resistance prevalence in Escherichia coli is relatively high
along the food chain from pigs to pork and humans
[3,5,6]. In contrast to the many different resistance
genes described for other classes of antimicrobials, so
far there are only three genes (sul1, sul2 and sul3)
which have been identified encoding sulfonamide
* Correspondence: lboj@food.dtu.dk
1Department of Microbiology and Risk Assessment, National Food Institute,
Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg,
Denmark
Full list of author information is available at the end of the article
Wu et al. Acta Veterinaria Scandinavica 2010, 52:47
http://www.actavetscand.com/content/52/1/47
© 2010 Wu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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resistance [4,7]. Sul1 has almost exclusively been found
on large conjugative plasmids and on class 1 integrons
[3,6,8,9]. Sul2 was previously considered to be located
on small non-conjugative plasmids, but recently the
gene has also been found on a wide range of large con-
jugative plasmids [1,8] and have been linked to the pre-
valence of streptomycin resistance [3]. Sul3 was
originally described from pigs in Switzerland in 2003
and has since been reported in animals as well as
humans in many countries. A recent publication has
linked sul3 to non-classic class 1 integrons [10]. How-
ever, knowledge about the structure and circulation of
plasmids carrying sul3 has yet not been well described
yet [7].
Genetic localization of sul genes on efficient mobile
genetic structures probably contribute to the wide
spread of sulfonamides resistance. Plasmids unavoidably
play an important role in carrying and mobilizing sul
genes [8]. The identification of plasmids associated with
specific sul genes therefore helps understanding the
mobilization capability of sul-genes among different bac-
terial species and enables determination of how sulfona-
mide resistance disseminates in different environments.
Currently little is known about plasmid backbones asso-
ciated with various sul genes especially in different
reservoirs, e.g. animals and human [1,9]. The aims of
this study were therefore to investigate the prevalence of
the sulfonamide resistance genes (sul1, sul2 and sul3) in
E. coli through the food chain from pigs to slaughter
(pig carcasses) and humans and to assess the genetic
diversity of plasmid, based on size and replicon, involved
in the mobility of various sul genes from the three
sources.
Methods
Bacterial strain collection
A total of 501 E. coli isolates collected from three
sources were included in this study: 150 isolates were
obtained in 2007 from fecal samples of Danish pigs as
part of the Danish Integrated Antimicrobial Resistance
Monitoring and Research Program (DANMAP) [5]; 275
isolates were obtained from pig carcasses in a Danish
pig slaughterhouse in November 2007 [11]; and 76 iso-
lates were obtained from stool samples of Danish
healthy humans between May and June 2008, of which
only two had been travelling abroad within three
months before sampling [12].
Sampling and strain isolation for E. coli from pig feces
has been described previously [5,11,12]. Only one isolate
was collected from each pig fecal and human stool sam-
ple. Multiple isolates were collected from each pig car-
cass sample, but only representative isolates with
distinct pulsed-field gel electrophoresis (PFGE) patterns
were included in the characterization of plasmids.
Antimicrobial susceptibility testing
Antimicrobial resistance profiles for all isolates were
determined by an automated microdilution method
(Trek Diagnostic Systems, East Grinstead, UK) for 17
antimicrobial agents included in DANMAP [5]. The
results were interpreted in accordance with EUCAST
guidelines [13], where the Minimum Inhibitory Concen-
tration (MIC) breakpoint for sulfamethoxazole resistance
is > 256 mg/L.
Detection of sul genes and integrons
Multiplex PCR was performed on boiled cell lysates of
the sulfonamide-resistant isolates. Primers developed by
Kerrn et al. [14] were used for detection of sul1 and
sul2 and a new primer-set for sul3 was designed for this
study(sul3-F:5’-CAGATAAGGCAATTGAG-
CATGCTCTGC-3’, sul3-R: 5’-AGAATGATTTCCGT-
GACACTGCAATCATT-3’). PCR was performed in a
20 μL mixture, including 2 μL template DNA, 3 μL 10×
reaction buffer (Ampliqon, Herlev, Denmark); 1 μL
MgCl2(25 mM; Ampliqon); 0.25 μL dNTPs; 0.25 μL
Taq Polymerase (50 mM, Ampliqon); 2.5 μL forward
and reverse primer mixture (volume for sul1:sul2:sul3 =
1:1:2). Amplification was carried out by heating for 5
min at 94°C, followed by 30 cycles at 94°C for 1 min,
68°C for 1 min and 72°C for 2 min, followed by 72°C
for 10 min. The sizes of the amplicon were 433 bp for
sul1, 293 bp for sul2 and 569 bp for sul3. Presences of
class 1 and class 2 integrons were detected by primers
for integrase 1 and integrase 2 described previously
[14,15]. Positive and negative controls were included in
all PCR arrays.
Plasmid-mediated sulfonamide resistance transferability
A proportion of sulfonamide-resistant E. coli isolates
(18 from humans, 18 from pig feces and 21 from pig
carcasses) representing different sul genes were
selected for further plasmid characterization. Conjuga-
tion experiments were performed at 37°C by filter mat-
ing assay using E. coli K12-J53 (nalidixic acid-resistant)
as recipient and transconjugants were selected on
Mueller-Hinton agar plates supplemented 256 mg/L
sulfamethoxazole and 40 mg/L nalidixic acid.
When donor strains were resistant to nalidixic acid or
resistance plasmid transfer failed in the mating experi-
ments or when plasmid co-transfer occurred, electro-
poration was done to obtain recipients with single
plasmid carrying sul genes. Plasmid DNA was purified
by the QIAGEN Plasmid Mini kit (Qiagen, Hilden,
Germany) as described by the manufacturer. Purified
plasmids were used to transform ElectroMAX™DH10B™-
competent cells (Invitrogen, Paisley, United Kingdom)
using a Bio-Rad MicroPulser (Bio-Rad Laboratories, CA,
USA) under standard conditions (1.8 kv, 180 Ω and
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25 μF). Transformants were selected on Mueller-Hinton
agar plates supplemented with 256 mg/L sulfamethoxa-
zole and confirmed to harbor corresponding sul genes
by multiplex PCR assay as described earlier.
S1 nuclease digestion of plasmids and PFGE
S1 nuclease digestion followed by PFGE was performed
to visualize and determine the molecular size of plas-
mids [14]. Agarose plugs were prepared according to
the PulseNet protocol [16] except the concentration of
the cells was adjusted to 0.83-0.85 on a Dade Micoscan
turbidity meter (Dade Behring, CA, USA). Slices of
plugs were digested with 5 U of S1 nuclease (Promega,
Madison, WI, USA) for 45 min at 37°C. The plasmids
were separated in a 1% SeaKem Gold agarose (Cambrex,
East Rutherford, NJ, USA) gel using the CHEF DR III
System (Bio-Rad, Hercules, CA) in 0.5× Tris-borate
EDTA at 14°C and an angle of 120 at 6 V/cm gradient
with pulse ramping from 6.8 to 38.4 s over 18 h.
Plasmid replicon type determination
Plasmids from parental and transformant/transconjugant
strains were assigned to 18 incompatibility (Inc) groups
by PCR-based replicon typing (PBRT) of total DNA
using previously described primers and conditions [17].
IncQ was screened as described previously [18]. The
presence of IncX1 was investigated using the primers
(IncX1-F: 5’-GCAGATTGATTCACGTGAAG-3’, IncX1-
R 5’-CCTCTGAAACCGTATGGTATTC-3’). All positive
results generated by multiplex PCR were confirmed
using single primer pair.
Results and discussion
Prevalence of sulfonamide resistant E. coli and sul genes
A total of 109 sulfonamide resistant E. coli isolates were
detected and prevalences of sulfonamide resistance were
17% for pig feces (26/150), 18% from pig carcasses (49/
275) and 45% from humans stool (34/76) (Table 1). The
prevalence of sul1 gene varied from 23% to 29% in E.
coli from the three sources. The sul2 gene was the most
prevalent sulfonamide resistance gene found in all of the
three sources (> 40%). The sul3 gene was present alone
in 9% of pig feces and 10% of pig carcasses, while in
human isolates it was detected simultaneously with sul2
in two E. coli isolates. The presence of two sul genes
was detected in E. coli from all sources (18% from pig
feces, 20% from pig carcasses and 31% from humans).
A higher prevalence of sul2 than sul1 and sul3 in E.
coli from animals and humans has been observed in pre-
vious studies in Denmark as well as in other countries
[3,6,14,19,20]. In the UK, sulfonamide resistance in
human E. coli persists undiminished with sul2 maintain-
ing dominant despite that the human usage of sulfona-
mides had been terminated decades ago[1,2]. We found
that sul3 occurred with the lowest prevalence of the
three sul genes among all E. coli from the three sources,
which correspondsto
[3,6,9,21,22]. The higher prevalence of combined sul
genes in human E. coli isolates compared with food ani-
mal isolates was also observed in earlier studies [3,6].
This is likely due to the acquisition of additional genes
from different sources (animals, food and environment),
as well as the selective pressure by the human’s con-
sumption of antimicrobial agents.
Sulfonamides are widely used in Danish veterinary
practice. Combined formulations of sulfonamide/tri-
methoprim have been frequently used in Danish pig
production [5]. This may explain why sulfonamide resis-
tance occurs with high prevalence in E. coli isolates
from Danish pigs. From the previous epidemiological
study of the human isolates included in this investiga-
tion no strong correlation between prevalence of sulfo-
namide resistance in isolates of human origin and
human usage of sulfonamides could be found.
previousobservations
Transferability of sul genes
A total of 57 sulfonamide-resistant E. coli isolates (18
from pig feces, 21 from pig carcasses and 18 from
humans) were included for conjugation assay and plas-
mid analysis. Conjugative plasmids were successfully
transferred from 42 isolates (82%), including 22 isolates
where the sul plasmids were co-transferred with another
plasmid (data not shown). Electro-transformation was
conducted to ensure transconjugants containing only
Table 1 Prevalence of sul1, sul2 and sul3 genes in sulfonamide-resistant E. coli isolated from pig feces, pig carcasses
and human stools
Origin No. of isolates testedNo. of isolates with sul genes (percentage)
sul1 only
sul2 only
sul3 only two sul genes
8 (31%)a
10 (20%)b
6 (18%)c
Human stool
Pig carcass
Pig feces
26
49
34
6 (23%)
13 (27%)
10 (29%)
12 (46%)
21 (43%)
15 (44%)
0
5 (10%)
3 (9%)
aSix strains were positive for both sul1 and sul2; two strains contained both sul2 and sul3.
bSeven strains contained both sul1 and sul2; one strain contained sul1 and sul3; and two strains harbored sul2 and sul3.
cSix strains contained both sul1 and sul2.
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one plasmids encoding for sul-resistance. This was
performed for the 22 isolates containing multiple conju-
gative plasmids, including 9 E. coli isolates with non-
conjugative plasmids and 6 isolates showing resistance
to nalidixic acid. The results showed, that 53/57 trans-
conjugants/transformants obtained a single sul gene,
while the remaining four isolates (all from humans) con-
tained single plasmids of different sizes carrying both
sul1 and sul2 genes (Table 2).
Additional phenotypically expressed resistances were
co-transferred with sulfonamide resistance by 55 plas-
mids (96%), resulting in diverse resistance patterns
(Table 2) indicating possible co-selection for all three
tested sulfonamide resistance genes. Overall, the most
frequently co-transferred resistances were to streptomy-
cin (54%), ampicillin (51%) and trimethoprim (46%).
These three resistances were co-transferred more fre-
quently by sul2 plasmids than those by sul1 and sul3
plasmids with highest frequency for streptomycin and
ampicillin resistance (both 79%), indicating that the use
of these antimicrobials possibly contribute to the predo-
minance of the sul2 gene via co-selection, which corro-
borates previous studies by Bean et al. [1,2].
Tetracycline resistance was co-transferred in 16 plas-
mids carrying sul1 and/or sul2 genes. Chloramphenicol
resistance was found to be co-transferred in 7 plasmids
with 6 of them being sul3 associated. The strong link
between chloramphenicol resistance and sul3 presence
has already been reported for Salmonella and E. coli
[9,23].
The presence of intI1 was detected in sul1 (71%), sul2
(17%) and sul3 (100%) -carrying plasmids from all three
sample reservoirs (Table 2). Two out of 28 intI1-carry-
ing plasmids could not be transferred by conjugation.
Only one of 57 isolates carrying the sul2 plasmid con-
tained intI2. It is interesting to note, that all sul3 plas-
mids were conjugative and harbored intI1 as recently
detected [9]. The association of sul3-carrying conjuga-
tive plasmids with integrons in E. coli isolates from pigs
(carcass or feces) could probably facilitate the further
spread of this gene to other bacteria and reservoirs via
food chain.
Plasmid analysis and replicon typing
S1 nuclease digestion followed by PFGE showed that the
15 plasmids carrying single sul1 genes ranged from 33-
160 kb in size. Replicon typing demonstrated seven dif-
ferent incompatibility groups (I1, N, FIB, FIA, HI1, FII
and B/O) for the plasmids (Table 2). IncI1 was the most
common group, accounting for 5 intI1-containing con-
jugative plasmids (from pig feces and carcasses). Out of
the six sul1 plasmids originating from human speci-
mens, four belonged to FI or FII type (three plasmids
had multireplicons FIA-FIB or FII-FIB), one plasmid
belonged to B/O and one plasmid could not be assigned.
This replicon profile in human E. coli isolates seems dif-
ferent from those found in E. coli from pigs which
mostly belonged to I1 and N. Due to the limited num-
ber of strains obtained from each source, a firm conclu-
sion on whether specific replicon profiles are associated
with pigs or humans would require the testing of addi-
tional strains.
The 25 sul2-carrying plasmids ranged from 33-120 kb
in size. Five different incompatibility groups (FII, B/O,
FIB, I1 and FIA) were demonstrated (Table 2). Among
them FII was the predominant replicon type (52%) and
distributed equally in E. coli from the three sources. This
is in contrast to a study in the UK where the FII replicon
was not detected in 33 sul2-carrying plasmids in E. coli
isolated from hospitals [1]. The 15 FII plasmids were
similar in size (33-55 kb) and none contained intI1. How-
ever, the plasmids were associated with various resistance
phenotypes and conjugation capabilities. It is possible
that they represent the same plasmid backbone circulat-
ing in pigs and humans. The B/O replicon type was
found in plasmids from E. coli isolated from both human
and pig, but with different resistance profiles, indicating
they are different plasmids sharing the same backbone.
FIA, FIB, and I1 replicons were also found to be asso-
ciated with sul2 genes. Three sul2-carrying plasmids
were negative for all the tested Inc groups. For the four
plasmids carrying both sul1 and sul2 genes, two harbored
the FIB replicon alone, one harbored FIB-FII and one
had FIA-FIB-FII multireplicons.
Overall, IncF (FIA, FIB, FIC and FII) seemed to be
the most common replicon associated with sul2,
accounting for 72% sul2-carrying plasmids (21/29).
IncF plasmids, which are considered having a narrow
host range and being conjugative, seemed to be well
adapted to E. coli as it was found in more than 50% of
E. coli strains from different sources (especially FIB
and FII) [24,25]. Localization on common plasmids
such as IncF could indicate transmission between the
three reservoirs studied.
The 11 sul3-carrying plasmids ranged from 33-115 kb
in size. Replicon typing detected five different incompat-
ibility groups: I1, FII, FIB, FIC and X1 (Table 2). IncI1
was found to be the most prevalent (45%) and all were
conjugative and associated with class 1 integrons. The
IncI1 plasmids had similar sizes, but differed in the
resistance profiles. FII, X1 and FIB-FIC were also found
to be associated with the sul3 gene. Two sul3-carrying
plasmids were negative for all the tested Inc groups.
When comparing plasmid size and the resistance phe-
notype, we found that E. coli isolates with larger sized
plasmids did not always show resistance to a higher
number of different antimicrobials. For instance, the 120
kb-sized plasmid contained in isolate 70-141-6 was only
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Table 2 Characterization of sul genes and plasmids in 57 E. coli isolates.
sul gene located
on plasmids
Strain IDSample sourceInc groupPlasmid size
(kb)
Co-transferred resistanceConjugative Integrase
sul1
sul1
sul1
sul1
sul1
sul1
sul1
sul1
sul1
sul1
sul1
sul1
sul1
sul1
sul1
sul1
sul1
70-7-3
70-10-1
70-13-2
70-58-1
v3-s1-8-4
v2-f2-2
v2-s3-3-10
v2-s3-2-3
3847
v4-s1-20-3
70-7-2
3796
3880
3849
3846
70-138-3
3806
pig feces
pig feces
pig feces
pig feces
pig carcass
pig carcass
pig carcass
pig carcass
human stool
pig carcass
pig feces
human stool
human stool
human stool
human stool
pig feces
human stool
I1
I1
I1
I1
I1
N
N
FIB
FIB
HI1
FII
FIA, FIB
FIA, FIB
FIB, FII
B/O
Unknown
Unknown
60
78
140
78
78
33
33
105
80
160
45
95
78
80
55
85
40
TET TMP
STR SPE TET
STR SPE
SPE TET
TET TMP
TET TMP
TET TMP
SPE STR
AMP SPE STR
SPE STR TET
AMP
AMP AUG GEN TMP
TMP
STR
AMP AUG
STR TMP
AMP TET TMP
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
Yes
No
Yes
IntI1
IntI1
IntI1
IntI1
IntI1
IntI1
IntI1
IntI1
IntI1
IntI1
IntI1
IntI1
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
sul2
70-13-7
70-165-18
70-13-9
70-56-3
70-58-2
v2-f3-2
v2-s1-3-5
v3-s1-2-8
v4-s3-17-3
v2-f3-6
v2-s1-1-10
3783
3816
3837
3859
70-141-6
70-166-11
70-10-6
70-11-5
3841
v2-s1-1-8
3834
v2-s3-1-8
3815
3810
pig feces
pig feces
pig feces
pig feces
pig feces
pig carcass
pig carcass
pig carcass
pig carcass
pig carcass
pig carcass
human stool
human stool
human stool
human stool
pig feces
pig feces
pig feces
pig feces
human stool
pig carcass
human stool
pig carcass
human stool
human stool
FII
FII
FII
FII
FII
FII
FII
FII
FII
FII
FII
FII
FII
FII
FII
FIB
B/O
B/O
B/O
B/O
I1
FIA, FIB
Unknown
Unknown
Unknown
55
45
55
55
40
50
45
55
40
55
55
50
33
33
55
120
80
70
78
55
50
60
45
80
95
AMP
AMP
AMP STR
AMP STR TMP
AMP STR TMP
AMP
AMP STR
AMP STR
AMP STR
AMP STR TET TMP
AMP GEN STR TMP
AMP TMP
AMP STR TET TMP
AMP STR TET TMP
STR
TET
TMP
AMP STR TET
AMP STR SPE TMP
AMP STR
AMP SPE STR TMP
AMP GEN STR TET TMP
AMP STR
AMP SPE STR
STR
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
–a
No
No
–
Yes
Yes
Yes
Yes
Yes
–
–
Yes
Yes
–
IntI2
IntI1
IntI1
IntI1
IntI1
sul1+2
sul1+2
sul1+2
sul1+2
3853
3823
3872
3881
human stool
human stool
human stool
human stool
FIB
FIB
FIB, FII
FIA, FIB, FII
130
139
100
110
AMP CHL STR TET TMP
AMP STR TMP
STR TET TMP
GEN STR TET TMP
No
–
No
No
IntI1
sul3
sul3
sul3
sul3
70-13-1
70-11-1
v2-s3-3-6
v2-s3-2-4
pig feces
pig feces
pig carcass
pig carcass
I1
I1
I1
I1
80
78
78
78
CHL
——
——
CHL SPE STR TMP
Yes
Yes
Yes
Yes
IntI1
IntI1
IntI1
IntI1
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associated with resistance to tetracycline (Table 2).
However, isolates with smaller plasmids of only 33-kb
size (isolates 3816 and 3837) showed resistance to ampi-
cillin, streptomycin, tetracycline and trimethoprim. This
indicates that resistance genes only account for a small
part of the plasmid gene sequence and more studies are
needed to determine the contents and function of such
plasmids.
The findings of diverse incompatibility groups among
various sul genes indicate that sul genes have been able
to transfer into multiple plasmid backbones on multiple
occasions, or that the plasmid backbones have diversi-
fied extensively since the acquisition of sul genes [1].
The IncF in our study was the most prevalent, carrying
FII alone or in combination with FIA or/and FIB or/and
FIC (multireplicon). This suggests that these plasmids
are evolving through replicon sequence divergence,
mosaicism and replicon co-integration in resolution pro-
cess [26]. As human stays on the top of the food chain,
the sul plasmids from healthy human reflects a more
mixed population compared with pig or pork (partly
represented by pig carcasses) also reflecting other reser-
voirs that connected to the farm to fork chain.
Sul-carrying plasmids in pig feces and on pig car-
casses represent a pool of resistance genes that may
transfer to human via the food chain, the most impor-
tant non-human reservoir for transmission of antimicro-
bial resistance to humans. In fact, the colonization of
sulfonamide-resistant E. coli from animal sources
(chicken or pig) into the human gut has been success-
fully demonstrated in both in vitro and in vivo studies
[27,28]. The transfer of the sul2 gene from an E. coli
strain of a pig origin to a sulfonamide-sensitive E. coli
strain of human origin was demonstrated in the intes-
tine of mice and transfer of sul2 has also been detected
among E. coli in the human intestine [15]. By combin-
ing all these date it could be speculated that consump-
tion of pork contaminated with sulfonamide-resistant E.
coli could result in the transfer of sul genes from pigs
to humans.
Conclusions
To our knowledge, this is the first study to assess the
plasmid replicons involved in various sul genes from dif-
ferent reservoirs. The diverse replicon profiles indicate
no clear association between the replicon types and spe-
cific sul genes or sample sources. However, the localiza-
tion of sul genes on wide spread replicons such as IncF
is very likely to contribute to the dissemination of sulfo-
namide resistance. In addition, all sul3-carrying plasmids
were found to be conjugative and associated with class 1
integrons. This underscores the potential of sul3 to
become more widespread in the future.
Acknowledgements
Henrik Hasman from the National Food Institute, Technical University of
Denmark is thanked for his help to design the primer for IncX1 and helpful
discussions during the study. Alessandra Carattoli from Istituto Superiore di
Sanita, Rome, Italy is appreciated for the encouragement and discussion
during the study. Hanne-Dorthe Emborg and Vibeke F. Jensen from the
National Food Institute, Technical University of Denmark are acknowledged
for providing information from the DANMAP and VetStat databases. Karin S.
Pedersen and Frank Hansen are thanked for excellent technical assistance at
SSI. We also thank Hans O. Jørgensen and Inger M. Giversen at the Danish
Armed Forces Health Services and Erling Døssing from the Infirmeriet in
Holstebro for establishing the contact to the recruits. We are grateful to the
recruits without whom the study would not have been possible.
This study was supported by a grant from the EU Marie Curie Program
TRAINAU (MEST-CT-2004-007819).
Author details
1Department of Microbiology and Risk Assessment, National Food Institute,
Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg,
Denmark.2Department of Veterinary Disease Biology, Faculty of Life
Sciences, University of Copenhagen, Stigbøjlen 4, DK-1870 Frederksberg C,
Denmark.3National Center for Antimicrobials and Infection Control, Statens
Serum Institut, 5 Artillerivej, DK-2300 Copenhagen S, Denmark.4Shuyu Wu’s
present address changed to: US-CDC China Office, Suite 403, Dongwai
Diplomatic Building, 23 Dongzhimenwai Dajie, Beijing 100600, China.
Authors’ contributions
WS performed the experiment and was responsible for the data analysis and
writing the manuscript. AD and LJE were involved in the study design and
preparation of the manuscript. AMH and LJP participated in the study
design and data analysis. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Table 2: Characterization of sul genes and plasmids in 57 E. coli isolates. (Continued)
sul3
sul3
sul3
sul3
sul3
sul3
sul3
v4-s3-3-4
V3-s3-2-4
v4-f1-2
v4-s1-20-4
70-8-1
v4-s1-20-6
v2-s1-1-9
pig carcass
pig carcass
pig carcass
pig carcass
pig feces
pig carcass
pig carcass
I1
X1
FII
FIB, FIC
FIB, FIC
Unknown
Unknown
55
115
78
65
78
55
33
CHL
AMP NEO SPE TMP
CHL
SPE
SPE
CHL
CHL
Yes
Yes
Yes
Yes
Yes
Yes
Yes
IntI1
IntI1
IntI1
IntI1
IntI1
IntI1
IntI1
AMP: ampicillin; AUG: amoxillin + clavulanat; CHL: chloramphenicol GEN: gentamicin; SPE: spectinomycin; STR: streptomycin; NEO: neomyxin; SMX:
sulfamethoxazole; TET: tetracycline; TMP: trimethoprim.
aconjugation not conducted.
Wu et al. Acta Veterinaria Scandinavica 2010, 52:47
http://www.actavetscand.com/content/52/1/47
Page 6 of 7

Page 7

Received: 29 March 2010 Accepted: 30 July 2010
Published: 30 July 2010
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doi:10.1186/1751-0147-52-47
Cite this article as: Wu et al.: Prevalence and characterization of
plasmids carrying sulfonamide resistance genes among Escherichia coli
from pigs, pig carcasses and human. Acta Veterinaria Scandinavica 2010
52:47.
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