J Antimicrob Chemother 2011; 66: 543 – 549
doi:10.1093/jac/dkq481 Advance Access publication 21 December 2010
IncA/C plasmids mediate antimicrobial resistance linked to virulence
genes in the Spanish clone of the emerging Salmonella enterica
serotype 4,[5],12:i:2
Patricia Garcı́a 1, Beatriz Guerra 2, Margarita Bances 3, M. Carmen Mendoza 1 and M. Rosario Rodicio 1*
1
Departamento de Biologı́a Funcional (Área de Microbiologı́a), Universidad de Oviedo, 33006-Oviedo, Asturias, Spain; 2Department of
Biological Safety, Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, D-12277 Berlin, Germany; 3Laboratorio de Salud
Pública, Consejerı́a de Salud y Servicios Sanitarios, 33001-Oviedo, Asturias, Spain
*Corresponding author. Área de Microbiologı́a, Facultad de Medicina, Universidad de Oviedo, Julián Claverı́a 6, 33006-Oviedo, Spain.
Tel: +34-985103562; Fax: +34-985103148; E-mail: [email protected]
Received 13 August 2010; returned 9 October 2010; revised 12 November 2010; accepted 17 November 2010
Objectives: To broaden knowledge of the molecular bases and genetics of multidrug resistance in clinical
isolates of Salmonella enterica serotype 4,5,12:i:2 belonging to the Spanish clone.
Methods: The relatedness of the isolates was determined by phage typing and XbaI-PFGE. Resistance genes,
integrons and transposable elements were identified by PCR amplification and sequencing. Plasmids were
characterized by alkaline lysis, S1-PFGE, conjugation, replicon typing and Southern blot hybridization.
Results: The isolates were closely related and resistant to five to seven antimicrobials (ampicillin, chloramphenicol, gentamicin, streptomycin/spectinomycin, sulphonamides, trimethoprim and tetracycline, arranged in
different combinations). Most of the responsible genes were provided by a conventional class 1 integron
with the dfrA12-orfF-aadA2 variable region, an atypical class 1 integron containing sul3 next to the estX-pspaadA2-cmlA1-aadA1 variable region and a truncated Tn1721 transposon carrying tet(A). A defective Tn21
with the mer operon and ISVsa3 associated with sul2 were also detected. All resistance genes and mobile
genetic elements were located on large, non-conjugative and highly variable plasmids carrying one (A/C) or
two (A/C and N) replicons, as well as virulence genes of pSLT.
Conclusions: IncA/C plasmids are responsible for multidrug resistance in an increasing number of relevant
human and animal bacterial pathogens, and hence are regarded as an important threat to public health.
Those found in the Spanish clone of Salmonella 4,5,12:i:2 constitute a relevant example of short-term evolution, and could have been involved in the successful adaptation of this pathogen.
Keywords: monophasic Typhimurium, multiple drug resistance, class 1 integrons, sul3 integrons, plasmid evolution
Introduction
Salmonella enterica serotype 4,[5],12:i:2 (Salmonella 4,[5],12:i:2)
represents an emerging serotype antigenically related to Salmonella Typhimurium (1,4,[5],12:i:1,2) but lacking the second phase
flagellar antigen encoded by the fljB gene.1 – 3 Since the
mid-1990s, Salmonella 4,[5],12:i:2 has been increasingly isolated from human clinical samples, animal species, particularly
food-producing animals, and foods from different countries in
Europe, North and South America, Asia and Africa.4 – 7 The antimicrobial resistance profiles of Salmonella 4,[5],12:i:2 isolates
recovered around the world vary considerably, ranging from pansusceptible to multidrug resistant (MDR). Overall, Salmonella
4,[5],12:i:2 isolates from North and South America (i.e. USA
and Brazil) are typically pan-susceptible or resistant to only a
few antimicrobial drugs.8,9 In contrast, the majority of the Salmonella 4,[5],12:i:2 isolates from Europe show two predominant
MDR patterns connected with the so-called European and
Spanish clones. The European clone is circulating in many
countries, including France, Italy, the UK, Germany, Denmark,
the Netherlands, Luxembourg, Poland and Spain, has pigs as
the main reservoir of infection, but has also been recovered
from cattle and poultry.6,10 – 13 The relatedness of these isolates
has been established through PFGE and multiple-locus
variable-number tandem-repeat analysis (MLVA); they were
mainly ascribed to definitive phage types (DTs) 193 and 120,
and were resistant to ampicillin, streptomycin, sulphonamides
and tetracycline, encoded by blaTEM, strA-strB, sul2 and tet(B)
# The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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543
Garcı́a et al.
genes located on a chromosomal island.6,13,14 The Salmonella
isolates belonging to the Spanish (Salmonella 4,5,12:i:2) clone
were first detected in 1997, ranking among the top eight most
common serotypes recovered from clinical samples in Spain in
subsequent years.4,15 Most of these isolates are phage type
U302, though DT208 and DT193 have also been reported, have
been mainly linked to pigs and pork products,4,16 and are
usually resistant to ampicillin (blaTEM-1), chloramphenicol
(cmlA1), gentamicin [aac(3)-IV], streptomycin/spectinomycin
(aadA2), sulphonamides (sul1), trimethoprim (dfrA12)+
tetracycline [tet(A)].1,17,18 In all Spanish Salmonella 4,5,12:i:2
isolates analysed so far, dfrA12 and aadA2, together with an
open reading frame (ORF) of unknown function (orfF), are
present as gene cassettes in the 1900 bp variable region of a
class 1 integron termed InI. A second integron, with a 150 bp
variable region without gene cassettes, was also found.18 The
two integrons, together with all other resistance genes mapped
on large, non-conjugative plasmids of 200 kb (here termed
pUO-STmRV1, plasmid of the University of Oviedo Salmonella
Typhimurium monophasic Resistance and Virulence 1, formerly
pUO-SVR3) or 150 kb (pUO-STmR1, formerly pUO-SR4), positive
and negative for the spv locus, respectively. The spv locus is the
hallmark of virulence plasmids specific to certain serovars of
S. enterica, including Salmonella Typhimurium, and codes for proteins involved in intracellular multiplication.19 Given the interest in
Salmonella 4,[5],12:i:2 as a worldwide emergent pathogen of
clinical significance, the aim of the present study was to
broaden our knowledge of the molecular bases and genetics of
MDR in a collection of isolates assigned to the Spanish clone.
Materials and methods
Bacterial isolates, serotyping, phage typing and PFGE
analysis
Nineteen Salmonella 4,5,12.i:2 isolates recovered in Asturias (Spain) from
2000 to 2003 were analysed in this study (see Table 1). They were associated with sporadic cases of human salmonellosis with no epidemiological correlation, and recorded at the ‘Laboratorio de Salud Pública’ (LSP) of
Asturias. The oldest Salmonella 4,5,12:i:2 isolates identified in Asturias
(LSP 389/97 and LSP 272/98, carrying pUO-STmRV1 and pUO-STmR1,
respectively),18 Salmonella Typhimurium LT2 and Salmonella Ohio LSP
325/9420 were used as positive or negative control in different experiments (PFGE typing, PCR amplifications, plasmid analysis and Southern
blot hybridizations).
The serotype and phage type of the isolates were determined at the
Spanish National Reference Centre for Salmonella (Centro Nacional de
Microbiologı́a, Madrid, Spain) or at the German National Reference Laboratory (Federal Institute for Risk Assessment, Berlin, Germany), using
standard techniques. XbaI-PFGE analyses were carried out following the
Pulsenet protocol (www.pulsenet.com). Changes in one or more fragments were used to establish different XbaI profiles. The similarity
between profiles was calculated by the Jaccard coefficient (S), and clustering was performed by the unweighted pair group method of analysis
with arithmetic averages (UPGMA), using the software Program MVSP
(Multivariate Statistics Package for PCs; RockWare Inc., Golden, CO, USA).
Profiles with S≥0.70 were considered as members of the same cluster.
Antimicrobial susceptibility testing
All isolates were tested for susceptibility to ampicillin, chloramphenicol,
gentamicin, kanamycin, streptomycin, sulphonamides, tetracycline and
544
trimethoprim by the disc diffusion assay on Mueller– Hinton agar using
commercially available discs (Oxoid, Madrid, Spain).21 Breakpoints were
scored following the interpretative criteria of the CLSI.22 In addition,
MICs of amikacin, amoxicillin, amoxicillin/clavulanate, ceftazidime,
ceftazidime/clavulanate, cefotaxime, cefuroxime, cefoxitin, cefepime,
ciprofloxacin, colistin, fosfomycin, imipenem, meropenem, nalidixic acid,
nitrofurantoin, piperacillin/tazobactam and tobramycin were determined
by the automatic Wider system (Francisco Soria Melguizo SA, Madrid,
Spain) at the Monte Naranco Hospital of Asturias, Spain. Wider is a
computer-assisted image-processing system for susceptibility testing
that uses MIC panels containing lyophilized antimicrobial agents.
PCR screening of resistance genes, integrons, insertion
sequences and transposons
Specific resistance genes were detected by PCR amplification, using previously described primers and conditions as well as primers designed for
the present work (Table S1, available as Supplementary data at JAC
Online). Assays were performed at least twice for each isolate. The
primer pairs used for blaTEM, cmlA1-like and aadA1-like genes are not
each specific for a single gene, but the identity of the obtained amplicons
was established through sequencing (Secugen, Madrid, Spain). PCR amplifications were also performed for detection and characterization of
conventional class 1 integrons, class 1 integrons with an atypical 3′ conserved segment (3′ CS) containing sul3, the insertion sequence ISVsa3
and transposons (Tn21 and Tn1721) (Table S1).
Plasmid analysis and Southern hybridizations
Plasmids were detected by alkaline lysis and S1-PFGE.23,24 Plasmid profiles were transferred onto nylon membranes25 and sequentially hybridized with probes specific for spvC (used as a marker of pSLT virulence
genes) and for antimicrobial resistance determinants. The probes were
obtained from LSP 389/97 [blaTEM-1, cmlA1, aac(3)-IV, aadA2, sul1, sul2,
sul3, tet(A) and dfrA12],18 LSP 325/94 (strA-strB)20 and LT2 (spvC), and
prepared with the commercial PCR DIG Labelling Mix (Roche Diagnostic,
Barcelona, Spain). The Inc/rep type of the detected plasmids was investigated by multiplex PCR,26 followed by simplex PCR, Southern blot hybridization and sequencing. The probes were obtained from the reference
strains of Carattoli et al.,26 which were also used as controls for PCR
amplifications. Conjugation experiments were performed in Luria–
Bertani broth at 308C and 378C, with Salmonella 4,5,12:i:2 isolates
acting as donors and Escherichia coli K-12 J53 (rifampicin resistant) as
recipient. Transconjugants were selected on eosin– methylene blue
agar (Pronadisa, Madrid, Spain), containing rifampicin (50 mg/L) plus
either chloramphenicol (30 mg/L) or tetracycline (30 mg/L). pUO-STmRV1
variants detected in the present work were labelled n1 – n16.
Results
Typing of Salmonella 4,5,12:i:2 isolates
The most frequent phage types were U302 (46%) and DT193
(37%). The control strains LSP 389/97 and LSP 272/98 are
U302, like most of the early monophasic isolates belonging to
the Spanish clone.4,16 XbaI-PFGE discriminated 15 profiles
(labelled X1 –X15; Figure 1), each generated from one or two isolates. The X1 profile was exclusively associated with LSP 389/97,
while X2 was shared by LSP 272/98 and two of the new isolates
under study. All the identified profiles formed a single cluster at
S¼ 0.70 (not shown), indicating a close relationship between the
isolates.
JAC
Resistance– virulence plasmids in Salmonella 4,5,12:i:2
XbaI-PFGE profile
S
6
3 4
5
7 8 9 10 13 14 11 12 15 2 1 S
kb
L
kb
1135
668.9
452.7
398.4
244.4
216.9
138.9
104.5
76.8
54.7
33
28.8
20.5
582
485
436.5
339.5
291
242.5
194
145.5
97
48.5
Figure 1. Macrorestriction profiles generated from Salmonella 4,5,12:i:2 isolates by XbaI-PFGE. S¼DNA from S. enterica serovar Braenderup H9812
digested with XbaI and L¼Lambda Ladder PFG Marker (New England Biolabs); both used as size standards. The distribution of the isolates into
XbaI-PFGE profiles is shown in Table 1.
Genetic bases of antimicrobial drug resistance
The analysed isolates were resistant to four or more antimicrobial agents, and were hence regarded as MDR. The observed
resistances were combined into nine profiles (R1 – R9), taking
into account the phenotypes (7) and resistance gene combinations (9) (Table 1). The most frequent profile was R1, which
corresponded to the heptaresistant phenotype of the control
LSP 389/97,18 and was also shown by 10 other isolates. The
R2–R9 profiles resulted from the absence of one or two resistances
of the major phenotype. Overall, resistances to streptomycin/
spectinomycin (encoded by aadA2 and/or aadA1) and sulphonamides (sul1, sul2 and sul3) were shared by all isolates, with the
others being variably represented. The isolates were susceptible
to other tested antimicrobials.
Involvement of integrons and transposons in MDR of the
Salmonella 4,5,12:i:2 Spanish clone
All isolates under study carried conventional class 1 integrons, as
revealed by PCR amplification of the intI1 and qacED1 genes
characteristic of these integrons. The 150 and 1900 bp variable regions expected for In0 and InI were detected in 19 and
16 isolates, respectively, and the dfrA12-orfF-aadA2 gene array
of InI was revealed by nested PCR amplification. In addition,
PCR mapping in conjunction with sequencing demonstrated the
presence of an unusual class 1 integron, containing sul3
instead of sul1 in the 3′ CS and a type III variable region
(4900 bp/estX-psp-aadA2-cmlA1-aadA1, with estX and psp
encoding a putative esterase and phosphoserine transferase,
respectively)27 in 18 of the isolates under study and in the two
control strains (Figure 2). Overall, three class 1 integron profiles
were identified (Table 1): In0, InI and type III In-sul3 (15
isolates); In0 and InI (1 isolate); and In0 and type III In-sul3
(3 isolates).
With regard to transposable elements: (i) the 19 isolates were
positive for ISVsa3, an insertion sequence that has been reported
next to sul2 in plasmids of the A/C incompatibility group (see
below); (ii) the 19 isolates also carried a defective Tn21-like
transposon, being PCR positive for the tnpR (resolvase) gene
and the entire mer operon (mercury resistance), but negative
for the tnpA (transposase) gene; (iii) all tet(A)-positive isolates
contained a defective Tn1721-like transposon, yielding the
expected amplicons with primer pairs targeting the tnpR and
tnpA genes, but not the mcp (putative methyl-accepting chemotaxis protein) gene, specific to this transposon; and (iv) amplification of the tnpA and tnpR genes of Tn3 failed even in isolates
containing blaTEM-1.
Large plasmids are responsible for MDR in the Salmonella
4,5,12:i:2 Spanish clone
As shown in Figure 3, all isolates under study carried large plasmids, ranging in size from 150 to 200 kb. These plasmids
carried either IncA/C (68%) or IncA/C and IncN (32%) replicon(s),
with those present in LSP 389/97 and LSP 272/98 (pUO-STmRV1
and pUO-STmR1) belonging to the latter group. An IncI1-Ig
plasmid of 100 kb co-existed with an IncA/C –IncN plasmid in
a single isolate (Figure 3), while small cryptic plasmids were
found in all isolates (not shown). The nucleotide sequences of
the IncA/C amplicons generated from the 19 isolates and the
two control strains were identical, and exhibited 5 and 22
nucleotide substitutions with respect to the A/C1 and A/C2 subgroups represented by plasmids RA1 and p2039, respectively.28
This results in one (Asn to Thr) and two (Ser to Thr and Gln to
His) changes in the A/C1 and A/C2 RepA proteins. None of the
IncA/C+IncN plasmids could be transferred into E. coli by conjugation under the conditions used.
Results from Southern blot hybridizations mapped all the
resistance genes, integrons and transposons to either the IncA/C
545
Strain/isolation
year
389/97
272/98
Phage
type
U302
U302
XbaI-PFGE
profile
X1
X2
Resistance
profile
R1
RV1 A/C-N
R2
[AMP-AMX]-CHL-[GEN-TOB]-[STR-SPE]-SUL –TMP
blaTEM-1-cmlA1-aac(3)-IV-[aadA1-aadA2]-[sul1-sul2-sul3]-dfrA12
In0 + InI + type III-In-sul3
tnpR (Tn21)+ mer
tnpA +tnpR (Tn1721)
R1 A/C-N
[AMP-AMX]-CHL-[GEN-TOB]-[STR-SPE]-SUL-TET-TMP
blaTEM-1-cmlA1-aac(3)-IV-[aadA1-aadA2]-[sul1-sul2-sul3]-tet(A)-dfrA12
In0 + InI + type III-In-sul3
tnpR (Tn21)+ mer
tnpA +tnpR (Tn1721)
[AMP-AMX]-CHL-[GEN-TOB]-[STR-SPE]-SUL-TMP
blaTEM-1-cmlA1-aac(3)-IV-[aadA1-aadA2]-[sul1-sul2-sul3]-dfrA12
blaTEM-1-cmlA1-aac(3)-IV-[aadA1-aadA2-strA-strB]-[sul1-sul2-sul3]-dfrA12
In0 + InI + type III-In-sul3
tnpR (Tn21)+ mer
tnpA +tnpR (Tn1721)
RV1-v8 A/C
RV1-v9 A/C-N
cmlA1-aac(3)-IV-[aadA1-aadA2-strA-strB]-[sul1-sul2-sul3]-dfrA12
In0 + InI + type III-In-sul3
tnpR (Tn21)+ mer
tnpA +tnpR (Tn1721)
RV1-v10 A/C-N
RV1-v11 A/C
RV1-v12 A/C-Ne
R6
[AMP-AMX]-CHL-[GEN-TOB]-[STR-SPE]-SUL
blaTEM-1-cmlA1-aac(3)-IV-[aadA1-aadA2]-[sul1-sul2-sul3]
In0 + InI + type III-In-sul3
tnpR (Tn21)+ mer
tnpA +tnpR (Tn1721)
RV1-v13 A/C
R7
[AMP-AMX]-CHL-[STR-SPE]-SUL-TET
blaTEM-1-cmlA1-[aadA1-aadA2]-[sul1-sul2-sul3]-tet(A)
In0 + InI + type III-In-sul3
tnpR (Tn21)+ mer
tnpA +tnpR (Tn1721)
RV1-v14 A/C
R8
[AMP-AMX]-CHL-[GEN-TOB]-[STR-SPE]-SUL-TET
blaTEM-1-cmlA1-aac(3)-IV-[aadA1-aadA2-strA-strB]-[sul1-sul2-sul3]-tet(A)
In0 + InI + type III-In-sul3
tnpR (Tn21)+ mer
tnpA +tnpR (Tn1721)
RV1-v15 A/C
R9
[AMP-AMX]-[GEN-TOB]-[STR-SPE]-SUL-TET-TMP
blaTEM-1-aac(3)-IV-[aadA1-aadA2]-[sul1-sul2-sul3]-tet(A)-dfrA12
In0 + InI
tnpR (Tn21)+ mer
tnpA +tnpR (Tn1721)
RV1-v16 A/C
X6
X3
X3
X4
X7
X5
X6
X9
X10
X12
R1
R1
R1
R1
R1
R1
R1
R1
R1
R1
503/02
66/01
U302
DT193
X14
X2
R2
R3
247/00
80/01
1142/03
U310
DT193
U302
X2
X5
X15
R4
R4
R5
262/02
718/02
73/01
a
U302
U302
DT193
X11
X13
X11
X8
pUO-STmb Inc
groupc
In0 + InI + type III-In-sul3
tnpR (Tn21)+ mer
tnpA +tnpR (Tn1721)
U302
U302
U302
NTd
DT193
U302
U302
DT193
DT193
NT
DT193
Integrons and transposon
genes
[AMP-AMX]-CHL-[GEN-TOB]-[STR-SPE]-SUL-TET-TMP
blaTEM-1-cmlA1-aac(3)-IV-[aadA1-aadA2]-[sul1-sul2-sul3]-tet(A)-dfrA12
21/00
37/00
40/00
151/00
41/00
417/00
411/01
576/01
578/01
3/02
84/01
Resistance phenotype/genotype
a
CHL-[GEN-TOB]-[STR-SPE]-SUL-TET-TMP
cmlA1-aac(3)-IV-[aadA1-aadA2]-[sul1-sul2-sul3]-tet(A)-dfrA12
RV1-v1
RV1-v1
RV1-v1
RV1-v2
RV1-v2
RV1-v3
RV1-v4
RV1-v5
RV1-v6
RV1-v7
A/C-N
A/C-N
A/C-N
A/C
A/C
A/C
A/C
A/C
A/C
A/C
Antimicrobial abbreviations: AMP/AMX, ampicillin/amoxicillin; CHL, chloramphenicol; GEN/TOB, gentamicin/tobramycin; STR/SPE, streptomycin/spectinomycin; SUL, sulphonamides; TET, tetracycline; and TMP, trimethoprim.
pUO-STm: plasmid University of Oviedo-Salmonella Typhimurium monophasic.
c
Plasmid incompatibility groups tested by multiplex PCR were: HI1, HI2 and I1-Ig ; X, L/M and N; FIA, FIB and W; and A/C, T and FIIA. Positive results were confirmed by simplex PCR, Southern blot hybridization and sequencing.
d
NT, not typeable with the available phage library.
e
An R plasmid of the IncI1-Ig group was also present in this isolate.
b
Garcı́a et al.
546
Table 1. Phenotypic and molecular properties of Salmonella 4,5,12:i:2 isolates analysed in this work
JAC
Resistance– virulence plasmids in Salmonella 4,5,12:i:2
intI1
estX
psp aadA2
cmlA1
aadA1
qacH IS440
sul3
orf1
IS15D /26''
Figure 2. Structure of the type III sul3 integron carried by strain LSP 389/97 of the Salmonella 4,5,12:i:2 Spanish clone, as determined by PCR
mapping and sequencing.
S 1 2 3 4
5 6 7 8 9 10 11 12 13 14 15
kb
S 16 17 18 19 20 21 L
kb
244.4
216.9
242.5
138.9
104.5
145.5
194
97
48.5
Figure 3. Plasmid analysis of Salmonella 4,5,12:i:2 isolates by S1-PFGE. Lanes 1 –21; 21/00 (n1), 37/00 (n1), 40/00 (n1), 151/00 (n2), 247/00 (n10),
417/00 (n3), 41/01 (n2), 66/01 (n9), 73/01 (n16), 80/01 (n11), 84/01 (n13), 411/01 (n4), 576/01 (n5), 578/01 (n6), 3/02 (n7), 262/02 (n14), 503/02
(n8), 718/02 (n15), 1142/03 (n12), 389/97 (pUO-STmRV1) and 272/98 (pUO-STmR1). For additional properties of the control pUO-STmRV1 and its
variants (n) see Table 1. S¼DNA from S. enterica serovar Braenderup H9812 digested with XbaI and lane L¼Lambda Ladder PFG Marker (New
England Biolabs); both used as size standards.
or the IncA/C –IncN plasmids, which also hybridized with spvC,
used as representative of pSLT virulence genes. Fifty four
percent of the IncA/C plasmids (7/13) and 50% of the IncA/C –
IncN plasmids (3/6) conferred the R1 heptaresistant profile
previously associated with pUO-STmRV1. Differences in size
identified seven variants (n1 – n7) within this group but a correlation between size and presence of one or two replicons was
not observed. Nine other variants (n8– n16) differed from
pUO-STmRV1 not only in size but also in resistance profile
(Table 1). It is also of note that aac(3)-IV hybridized not only
with several pUO-STmRV1 variants, but also with the IncI1-Ig
plasmid co-resident with one of them (n12).
Discussion
In Salmonella 4,5,12:i:2 isolates of the Spanish clone all genes
responsible for the MDR phenotype mapped on IncA/C plasmids,
with about one-third of them also being positive for the IncN
replicon. In contrast, the genes responsible for the tetra-resistant
phenotype of the European clone are chromosomally located.14
MDR plasmids belonging to the IncA/C family have emerged in a
large number of relevant human and animal bacterial pathogens,
and are therefore regarded as an important threat to public health.
These plasmids share a highly conserved backbone, which comprises the origin of replication, the specific repA gene, the partition
parAB genes and the genes encoding a type IV secretion-like
system involved in mating pair formation during conjugation.29 – 32
Despite the latter, the conjugative capability of IncA/C plasmids
varies considerably, and conjugation frequencies ranging from
1022 to 1028, as well as no plasmid transfer, have been
reported.29,31,33,34 In the present work, none of the IncA/
C+IncN plasmids could be conjugated into E. coli under the
applied conditions, but pUO-STmRV1 could be mobilized from
LSP 389/97 into the same recipient (P. Garcı́a and M. R. Rodicio,
unpublished results). Experiments aiming to identify the transfer
function(s) impaired in plasmids of the pUO-STmRV1 group are in
progress.
IncA/C plasmids proved to be highly efficient in recruiting antimicrobial resistance traits, and it has been suggested that recent
MDR members of the family could have evolved from a common
ancestor through stepwise integration of horizontally acquired
resistance gene arrays into the conserved backbone.29,31 These
gene arrays have a mosaic structure, and are composed of resistance genes and mobile genetic elements, such as insertion
sequences, transposons and integrons. Particularly, sul2 next to
a truncated ISVsa3 element has been found at various locations
in most of the IncA/C plasmids sequenced so far,31,32,35 and this
conserved array appears to be also present in all IncA/C+IncN
plasmids carried by the Salmonella 4,5,12,:i:2isolates of the
Spanish clone. The sul2 gene was already present in pRA1, the
first known member of the IncA/C family, isolated in 1971
from the fish pathogen Aeromonas hydrophila (formerly Aeromonas liquefaciens), and is regarded as the first antimicrobial resistance gene acquired by the IncA/C backbone.31,36 The mer operon
and a gene coding for a tetracycline efflux protein, are also represented in the IncA/C plasmids sequenced so far, although the
latter belongs to different classes. For instance, pRA1 and the
IncA/C plasmids of Yersinia pestis and Photobacterium damselae
contain tet(D) and the IncA/C plasmid of Yersinia ruckeri contains
547
Garcı́a et al.
tet(B), while the tetracycline resistance determinant of the
Salmonella 4,5,12,:i:2 Spanish clone is tet(A), carried also by
the IncA/C plasmids that are responsible for resistance to thirdgeneration cephalosporins in MDR isolates of Salmonella
Newport.29,32
Of particular interest is the presence of three class 1 integrons
in the IncA/C plasmids of the Salmonella 4,5,12,:i:2 Spanish
clone, which supply most of the resistance genes responsible
for MDR in these isolates (aadA1, aadA2, cmlA1, dfrA12, sul1
and sul3). The sul3 integron with the estX-psp-aadA2cmlA1-aadA1 variable region was first reported in E. coli isolates
collected from diarrhoeic swine in the USA.37 Later on, it was also
detected in Salmonella Typhimurium from Portugal, and termed
sul3 integrons of type III, to distinguish it from two other types of
sul3 integron with different gene arrays.25 Interestingly, two Salmonella Typhimurium DT104 isolates from the south of Portugal
shared the predominant heptaresistant profile of the Salmonella
4,5,12,:i:2 Spanish clone, as well as a conventional class integron
with the dfrA12-orfF-aadA2 variable region and a type III In-sul3.
These isolates contained non-conjugative plasmids of 220 kb
that hybridized with sul3,27 and could be related to those
found in the monophasic Spanish clone.
Salmonella 4,[5],12:i:2 has demonstrated its evolutionary
success, becoming one of the most prevalent serotypes worldwide.38 The driving forces for this success remain unknown but,
in the Spanish clone, properties conferred by the large IncA/
C+IncN plasmids, including MDR and additional virulence
functions, could have played a role. These plasmids constitute
a relevant example of short-term evolution,39 with 16 variants
(n1– n16) represented in 19 closely related contemporary isolates. Plasmid instability was in part connected with loss/acquisition of resistance genes carried or not by class 1 integrons or
transposons. However, variations in size, as yet not associated
with phenotypic properties, have also occurred. The molecular
bases for the intrinsic instability of the IncA/C+IncN plasmids
characteristic of the Salmonella 4,5,12:i:2 Spanish clone, as
well as its possible impact in adaptation of the host, are interesting issues that deserve further attention.
Acknowledgements
We are grateful to Dr A. Carattoli (Instituto Superiore di Sanitá, Rome,
Italy) for kindly supplying the control strains for determination of
plasmid incompatibility groups. We also thank the personnel of the
Spanish National Reference Center for Salmonella, Centro Nacional de
Microbiologı́a (CNM), Madrid, Spain, and the German National Reference
Laboratory for Salmonella, Federal Institute for Risk Assessment, Berlin,
Germany, especially A. Echeita, A. Schroeter and J. Ledwolorz, for the
phage typing of Salmonella isolates, and F. Vázquez (Hospital Monte
Naranco, Oviedo, Spain) for the MIC tests.
Transparency declarations
None to declare.
Supplementary data
Table S1 is available as Supplementary data at JAC Online (http://jac.
oxfordjournals.org/).
References
1 Echeita MA, Herrera S, Usera MA. Atypical, fljB-negative Salmonella
enterica subsp. enterica strain of serovar 4,5,12:i:2 appears to be a
monophasic variant of serovar Typhimurium. J Clin Microbiol 2001; 39:
2981– 3.
2 Garaizar J, Porwollik S, Echeita A et al. DNA microarray-based typing of
an atypical monophasic Salmonella enterica serovar. J Clin Microbiol
2002; 40: 2074– 8.
3 Zamperini K, Soni V, Waltman D et al. Molecular characterization
reveals Salmonella enterica serovar 4,[5],12:i:2 from poultry is a variant
Typhimurium serovar. Avian Dis 2007; 51: 958– 64.
4 Echeita MA, Aladuena A, Cruchaga S et al. Emergence and spread of an
atypical Salmonella enterica subsp. enterica serotype 4,5,12:i:2 strain in
Spain. J Clin Microbiol 1999; 37: 3425.
5 Soyer Y, Switt AM, Davis MA et al. Salmonella 4,5,12:i:2, an emerging
Salmonella serotype that represents multiple distinct clones. J Clin
Microbiol 2009; 47: 3546–56.
6 Hopkins KL, Kirchner M, Guerra B et al. Multiresistant Salmonella
enterica serovar 4,[5],12:i:2 in Europe: a new pandemic strain? Euro
Surveill 2010; 15: pii¼19580.
7 Tennant SM, Diallo S, Levy H et al. Identification by PCR of
non-typhoidal Salmonella enterica serovars associated with invasive
infections among febrile patients in Mali. PLoS Negl Trop Dis 2010; 4:
e621.
8 Agasan A, Kornblum J, Williams G et al. Profile of Salmonella enterica
subsp. enterica (subspecies I) serotype 4,5,12:i:2 strains causing
food-borne infections in New York City. J Clin Microbiol 2002; 40: 1924– 9.
9 Tavechio AT, Ghilardi AC, Fernandes SA. ‘Multiplex PCR’ identification of
the atypical and monophasic Salmonella enterica subsp. enterica
serotype 1,4,[5],12:i:2 in Sao Paulo State, Brazil: frequency and
antibiotic resistance patterns. Rev Inst Med Trop Sao Paulo 2004; 46:
115–7.
10 Mossong J, Marques P, Ragimbeau C et al. Outbreaks of monophasic
Salmonella enterica serovar 4,[5],12:i:2 in Luxembourg, 2006. Euro
Surveill 2007; 12: pii¼719.
11 Meakins S, Fisher IS, Berghold C et al. Antimicrobial drug resistance in
human nontyphoidal Salmonella isolates in Europe 2000– 2004: a report
from the Enter-net International Surveillance Network. Microb Drug Resist
2008; 14: 31– 5.
12 Dionisi AM, Graziani C, Lucarelli C et al. Molecular characterization of
multidrug-resistant
strains
of
Salmonella
enterica
serotype
Typhimurium and monophasic variant (S. 4,[5],12:i:2) isolated from
human infections in Italy. Foodborne Pathog Dis 2009; 6: 711–77.
Funding
13 Hauser E, Tietze E, Helmuth R et al. Pork contaminated Salmonella
enterica serovar 4,[5],12:i:2, an emerging health risk for humans. Appl
Environ Microbiol 2010; 76: 4601 –10.
P. G. is the recipient of a grant from the ‘Fundación para el Fomento en Asturias de la Investigación Cientı́fica Aplicada y la Tecnologı́a’ (FICYT, ref.
BP08-031). This work was supported by project FISS-08-PI080656 of the
‘Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo’, Spain.
14 Lucarelli C, Dionisi AM, Torpdahl M et al. Evidence for a second
genomic island conferring multidrug resistance in a clonal group of
Salmonella Typhimurium and its monophasic variant circulating in Italy,
Denmark and United Kingdom. J Clin Microbiol 2010; 48: 2103 –9.
548
Resistance– virulence plasmids in Salmonella 4,5,12:i:2
15 Echeita MA, Aladueña AM, de la Fuente M et al. Análisis de cepas de
Salmonella spp aisladas de muestras clı́nicas de origen humano en
España. Años 2004–2005 (I). Boletı́n Epidemiológico 2007; 15: 145–56.
Centro Nacional de Epidemiologı́a, Instituto de Salud Carlos III, Spain.
16 de la Torre E, Zapata D, Tello M et al. Several Salmonella enterica
subsp. enterica serotype 4,5,12:i:2 phage types isolated from swine
samples originate from serotype Typhimurium DT U302. J Clin Microbiol
2003; 41: 2395– 400.
17 Guerra B, Laconcha I, Soto SM et al. Molecular characterisation of
emergent multiresistant Salmonella enterica serotype [4,5,12:i:2]
organisms causing human salmonellosis. FEMS Microbiol Lett 2000;
190: 341–7.
18 Guerra B, Soto SM, Arguelles JM et al. Multidrug resistance is mediated
by large plasmids carrying a class 1 integron in the emergent Salmonella
enterica serotype [4,5,12:i:2]. Antimicrob Agents Chemother 2001; 45:
1305– 8.
19 Rotger R, Casadesús J. The virulence plasmids of Salmonella. Int
Microbiol 1999; 2: 177– 84.
20 Martı́nez N, Rodrı́guez I, Rodicio R et al. Molecular basis and evolution
of multiple drug resistance in the foodborne pathogen Salmonella
enterica serovar Ohio. Foodborne Pathog Dis 2010; 7: 189– 98.
21 Clinical and Laboratory Standards Institute. Performance Standards
for Antimicrobial Disk Susceptibility Tests—Tenth Edition: Approved
Standard M2-A10. CLSI, Wayne, PA, USA, 2009.
22 Clinical and Laboratory Standards Institute. Performance Standards
for Antimicrobial Susceptibility Testing: Nineteenth Informational
Supplement M100-S19. CLSI, Wayne, PA, USA, 2009.
23 Kado CI, Liu ST. Rapid procedure for detection and isolation of large
and small plasmids. J Bacteriol 1981; 145: 1365 –73.
24 Barton BM, Harding GP, Zuccarelli AJ. A general method for detecting
and sizing large plasmids. Anal Biochem 1995; 226: 235– 40.
25 Sambrook J, Russel DW. Molecular Cloning: A Laboratory Manual. Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001.
26 Carattoli A, Bertini A, Villa L et al. Identification of plasmids by
PCR-based replicon typing. J Microbiol Methods 2005; 63: 219– 28.
27 Antunes P, Machado J, Peixe L. Dissemination of sul3-containing
elements linked to class 1 integrons with an unusual 3′ conserved
sequence region among Salmonella isolates. Antimicrob Agents
Chemother 2007; 51: 1545 –8.
JAC
28 Carattoli A, Miriagou V, Bertini A et al. Replicon typing of plasmids
encoding resistance to newer b-lactams. Emerg Infect Dis 2006; 12:
1145– 8.
29 Welch TJ, Fricke WF, McDermott PF et al. Multiple antimicrobial
resistance in plague: an emerging public health risk. PLoS One 2007; 2: e309.
30 Kim MJ, Hirono I, Kurokawa K et al. Complete DNA sequence and
analysis of the transferable multiple-drug resistance plasmids (R
plasmids) from Photobacterium damselae subsp. piscicida isolates
collected in Japan and the United States. Antimicrob Agents Chemother
2008; 52: 606–11.
31 Fricke WF, Welch TJ, McDermott PF et al. Comparative genomics of
the IncA/C multidrug resistance plasmid family. J Bacteriol 2009; 191:
4750–7.
32 Call DR, Singer RS, Meng D et al. blaCMY-2-positive IncA/C plasmids
from Escherichia coli and Salmonella enterica are a distinct component
of a larger lineage of plasmids. Antimicrob Agents Chemother 2010; 54:
590–6.
33 Pan JC, Ye R, Wang HQ et al. Vibrio cholerae O139 multiple-drug
resistance mediated by Yersinia pestis pIP1202-like conjugative
plasmids. Antimicrob Agents Chemother 2008; 52: 3829 –36.
34 Poole TL, Edrington TS, Brichta-Harhay DM et al. Conjugative
transferability of the A/C plasmids from Salmonella enterica isolates
that possess or lack blaCMY in the A/C plasmid backbone. Foodborne
Pathog Dis 2009; 6: 1185 –94.
35 Lindsey RL, Fedorka-Cray PJ, Frye JG et al. Inc A/C plasmids are
prevalent in multidrug-resistant Salmonella enterica isolates. Appl
Environ Microbiol 2009; 75: 1908 –15.
36 Aoki T, Egusa S, Ogata Y et al. Detection of resistance factors in fish
pathogen Aeromonas liquefaciens. J Gen Microbiol 1971; 65: 343–9.
37 Bischoff KM, White DG, Hume ME et al. The chloramphenicol
resistance gene cmlA is disseminated on transferable plasmids that
confer multiple-drug resistance in swine Escherichia coli. FEMS Microbiol
Lett 2005; 243: 285–91.
38 Switt AI, Soyer Y, Warnick LD et al. Emergence, distribution, and
molecular and phenotypic characteristics of Salmonella enterica
serotype 4,5,12:i:2. Foodborne Pathog Dis 2009; 6: 407– 15.
39 Baquero F. From pieces to patterns: evolutionary engineering in
bacterial pathogens. Nat Rev Microbiol 2004; 2: 510– 8.
549