Spread of extended-spectrum beta-lactamase producing Escherichia coli isolates in Swedish broilers mediated by an incI plasmid carrying blaCTX-M-1.

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by  Borjesson, Stefan

Börjessonetal.ActaVeterinariaScandinavica2013,55:3
http://www.actavetscand.com/content/55/1/3
BRIEF COMMUNICATION Open Access
Spread of extended-spectrum beta-lactamase
Escherichia coli
producing isolates in Swedish
broilers mediated by an incI plasmid carrying
bla
CTX-M-1
Stefan Börjesson1*, Björn Bengtsson1, Cecilia Jernberg2 and Stina Englund1
Abstract
Background: The already high and increasing occurrence of extended-spectrum beta-lactamases (ESBL) producing
Escherichia coliin European broiler populations is of concern due to the fact thatthird and fourth generation
cephalosporins are deemed critically important inhuman medicine. In Sweden 34% of thebroilers carry ESBL/
pAmpC producing E. coli intheir gut, despitethe absence of a known selection pressure such as antimicrobial
usages. The aim of the current study was to characterise a selection ofE. coli strains carrying thebla , to
CTX-M-1
determine if thespread was due to a specific clone.
Findings: Ten isolates carryingbla from Swedish broilers belonged to eight different multi-locussequence
CTX-M-1
types withthreeisolates belonging to ST155. The ST155 isolateswere identical as assessed byPFGE. The bla
CTX-M-1
was inall isolates carried on a plasmid of replicon type incI, which also transferred resistance to tetracycline and
sulfamethoxazole.
Conclusion: The occurrence of ESBL-producing E. coli inthe Swedish broilers is notdue to theemergenceof a
single clone, but rather thespread of a specific incI plasmid carryingbla .
CTX-M-1
Keywords: Extended spectrum betalactamases, ESBL, Broiler,CTX-M-1,Escherichia coli
Findings recent study from the Netherlands concluded that identi-
Theincreasing trend ofextended-spectrum beta-lactamases calESBLproducingE.coliisolates,plasmidsandgenesoc-
(ESBL) and plasmid-mediated AmpC (pAmpC) producing curred both in broiler, meat and humans [4]. It has been
Escherichia coli in food-producing animals worldwide is suggestedthatthereasonfortheincreasingoccurrenceof
worrisome [1,2] due to the significance of third and fourth ESBL and pAmpC producing Enterobacteriaceae in broi-
generation cephalosporins in human medicine. The lers is due to the use of cephalosporins [5]. However, the
European Food Safety Authority concluded in a recent total sales of third generation cephalosporins devoted to
report that there exist evidence that food-producing Swedish animals in 2009 constituted ~20 kg, 0.001% of
animals can be a reservoir for ESBL and pAmpC pro- total sales of veterinary antimicrobials, and they were
ducing Enterobacteriaceae and the corresponding genes mainly prescribed to horses and cattle [6]. Therefore it
and that transmission has occurred between food- was highly surprising when in 2010 34% of broilers in
producing animals and humans [3]. The report also high- Sweden were found to carry ESBL or pAmpC producing
lighted that the high and increasing prevalence in broiler E. coli usingselective cultivation methods[7]. Therewere
and broiler meat is of particular concern. In addition, a only two genes identified bla and bla , with
CTX-M-1 CMY-2
bla clearly dominating. This genotype composition
CMY-2
differs from that in most other European countries [3].
*Correspondence:[email protected] Other European countries than Sweden generally have a
1DepartmentofAnimalhealthandAntimicrobialstrategies,National
higher diversity of genotypes on their broiler farms and a
VeterinaryInstitute(SVA),UppsalaSE-75189,Sweden
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Börjessonetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Börjessonetal.ActaVeterinariaScandinavica2013,55:3 Page2of3
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muchlowerprevalenceofblaCMY-2,butblaCTX-M-1appear Table1EscherichiacoliisolatescarryingblaCTX-M-1
to be common in broilers in most countries. A selection obtainedfrombroilerchickenscharacterizedbymulti
of E. coli isolates carrying bla was characterised and locussequencetypes(MLST),Pulsedfieldgel
CMY-2
compared to human clinical isolates in a previous study electrophoresis(PFGE)andresistancepatterns
[8].Itwasshownthattheoccurrenceof bla wasnot determinedusingVetMICGN-momicrodilutionplates
CMY-2
due to the spread of a single clone and that transmission MLST(clonalcomplex) ResistancepatternA No.of
isolates
tohumansappearsverylimitedandrestrictedtotheplas-
ST57(CC350) SuTc 1
mids. In the current study we characterised a selection of
bla E.coliisolatesfromSwedishbroilersandcom- ST135(N.D.B) SuTc 1C
CTX-M-1
paredtheresultsinaEuropeancontext. ST155(CC150) SuTc 3D
Ten isolates carrying blaCTX-M-1 from Swedish broilers ST219(N.D.) SuTc 1E
(collected February-May 2010) were obtained through ST602(CC446) SmSuTc 1C
the Swedish program for monitoring antimicrobial re-
ST752(CC10) SmSuTc 1
sistance in the veterinary field (SVARM) [7]. Isolates
ST1594(N.D.) SuTc 1
were obtained by culturing ceacal samples on MacCon-
key agar with 1 mg/L cefotaxime. All isolates were ST1640(CC350) SmSuTc 1
multi-resistant, i.e. resistant to three or more antimicro- ADeterminedinapreviousstudy[7],Su=sulphamethoxazole,Tc=tetracycline,
Sm=streptomycinBN.D.=notdeterminedCAssignedtothesamePFGE-cluster
bial classes. The isolates were characterised using Pulsed
basedonacut-offvalueof≥80%DIdenticalpulsedfieldgelelectrophoresis
field gel electrophoresis (PFGE) [9], Multilocus sequence (PFGE)patternsENon-typeablebyPFGEusingXbaI.
typing(MLST)(http://mlst.ucc.ie/)andPolymeraseChain
Reaction (PCR) based plasmid replicon typing [10]. For The results show that the occurrence of ESBL produ-
comparison of PFGE pattern DICE coefficient and cing E. coli in the Swedish broiler population is, like the
UPGMAclusteranalysiswasperformed,withpositiontol- pAmpC producing E. coli [8], not due to one specific
erance and optimisation both set at 1%. Transferability of clonebutappearstobe connectedtoaspecificincIplas-
bla was tested through conjugation to E. coli mid also carrying resistance to sulfamethoxazole and
CTX-M-1
HMS174. Transformation was performed on transconju- tetracycline.Itis well-established thatbla onIncI
CTX-M-1
gants PCR positive for multiple plasmid replicon types. plasmids are frequently isolated both in the poultry in-
Plasmid DNA was extracted according to the protocol by dustry and in human clinical settings in Europe [2]. In a
Sambrook and Russell [11] with minor modifications and study from the Netherlands it was also the IncI plasmids
transformed into ElectroMax™DH10B™ (Gibco Invitrogen, carrying bla that dominated and the plasmids
CTX-M-1
Carlsbad, CA, USA) according to the manufacturer’s were found to be identical in isolates of human and
instructions, voltage changed to 1250 V. Transconjugants broiler origin [4]. Furthermore, genetically related
and transformants were tested for antimicrobial sus- bla -IncI plasmids from human and broiler iso-
CTX-M-1
ceptibility using VetMIC GN-mo microdilution plates lates have also been described in France [13]. It is there-
with epidemiological cut-off value set by EUCAST fore possible that the Swedish broiler population can be
(SVA, Uppsala, Sweden), for ESBL genes [12] and plas- a potential source for these plasmids and genes in
mid replicon types. human clinical settings. However, to establish if this is
The 10 isolates belonged to eight different STs, with the case, further and more extensive studies should be
ST155 (3 isolates) being the most common (Table 1). carriedout.
Nine isolates were typeable using PFGE and 1 was non- Because the incI plasmid carries phenotypical resist-
typeable. All isolates were able to transfer bla In ancetotetracyclineandsulfamethoxazole, usageofthese
CTX-M-1.
addition to the ESBL phenotype, all isolates transferred antibiotics, in addition to use of cephalosporins and
resistance to sulfamethoxazole and tetracycline and one other B-lactam antibiotics, may facilitate the spread of
also transferred streptomycin resistance. It was found the plasmid. Previous studies have also suggested that
that the transconjugants contained plasmids belonging co-resistance has played a role inthe rapid emergence of
to the IncI replicon type. However, five transconjugants ESBL producing bacteria[2]. However,theoccurrencein
were also PCR positive for multiple plasmids replicon Sweden of ESBL, nor pAmpC, producing E. coli cannot
types and these five were subjected to transformation. be explained by antibiotic usage because this usage is
Theresulting transformantswere allpositivefor the IncI very limited. For example, in 2011 only 6 of 3185 broiler
replicon type and resistant to sulfamethoxazole and flocks were treated with antibiotics and no cephalospor-
tetracycline. Transformation on a transconjugant posi- ins were prescribed [14]. A likely explanation to the high
tive for IncI verified that the IncI plasmid carried occurrence can be a top-down transmission from
bla and sulfamethoxazole and tetracycline resist- imported grand-parent birds. The potential for transmis-
CTX-M-1
ancephenotype. sion of cephalosporin-resistant E. coli, along with
Börjessonetal.ActaVeterinariaScandinavica2013,55:3 Page3of3
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Competinginterests humansinFrance,2003to2008.AntimicrobAgentsChemother2010,
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Authors’contributions ResistanceMonitoring(SVARM)2011.Uppsala:NationalVeterinaryInstitute
SBparticipatedinthedesignofthestudy,carriedouttheanalysesand (SVA);2012.
draftedthemanuscript.BBparticipatedinthedesignofthestudyand 15. BortolaiaV,BisgaardM,BojesenAM:Distributionandpossible
assistedandinthedraftingofthemanuscript.CJcarriedoutthePFGE- transmissionofampicillin-andnalidixicacid-resistantEscherichiacoli
analyses.SEparticipatedinthedesignofthestudyhelpedtodraftthe withinthebroilerindustry.VetMicrobiol2010,142:379–386.
manuscriptandcarriedouttheanalyses.Allauthorshaveread,participated 16. PersoonsD,HaesebrouckF,SmetA,HermanL,HeyndrickxM,MartelA,Catry
inandapprovedthefinalmanuscript. B,BergeAC,ButayeP,DewulfJ:Riskfactorsforceftiofurresistancein
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Acknowledgments
AnthonyÖsterdahl,SVA,Uppsalaisacknowledgedforcommentsand doi:10.1186/1751-0147-55-3
linguisticrevisionofthemanuscript.ThisworkwassupportedbyThe Citethisarticleas:Börjessonetal.:Spreadofextended-spectrumbeta-
lactamaseproducingEscherichiacoliisolatesinSwedishbroilers
SwedishCivilContingenciesAgency.
mediatedbyanincIplasmidcarryingbla .ActaVeterinaria
CTX-M-1
Scandinavica201355:3.
Authordetails
1DepartmentofAnimalhealthandAntimicrobialstrategies,National
VeterinaryInstitute(SVA),UppsalaSE-75189,Sweden.2Departmentof
Preparedness,SwedishInstituteforCommunicableDiseaseControl
(Nobelsväg18),Solna17182,Sweden.
Received:31October2012Accepted:15January2013
Published:21January2013
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