Detection and molecular characterization of β-lactamase genes in clinical isolates of Gram-negative bacteria in Southern Ecuador

brazjinfectdis2016;20(6):627–630

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

Communication

Detection

and

molecular

characterization

of

␤-lactamase

genes

in

clinical

isolates

of

Gram-negative

bacteria

in

Southern

Ecuador

Diana

Yessenia

Calva

Delgado

_,

_Zorayda

_Patricia

_Toledo

_Barrigas

_,

Sofía

Genoveva

Ochoa

Astutillo

_,

_Ana

_Paulina

_Arévalo

_Jaramillo

_,

_Alessio

_Ausili

a_{UniversidadTécnicaParticulardeLoja(UTPL),DepartamentodeCienciasdelaSalud,Loja,Ecuador} b_{SecretaríaNacionaldeEducaciónSuperior,Ciencia,TecnologíaeInnovación(SENESCYT),Quito,Ecuador}

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Articlehistory:

Received7April2016 Accepted12July2016 Availableonline30July2016

Keywords: ␤-Lactamresistance ␤-Lactamase ESBL Genotypiccharacterization

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Thisworkperformedaphenotypicandgenotypiccharacterizationof79clinicalisolatesof

EnterobacteriaceaeandPseudomonadaceaecollectedinhospitalsofSouthernEcuadorin2013. Ourresultsshowedahighincidenceof␤-lactamasesandESBLswithblaTEMandblaCTX-M

as theprevalentgenes,respectively.By directsequencingofPCR amplicons,the differ-ent␤-lactamasesandvariantsofthegeneswerealsodistinguished.Ourresultsrevealed apredominanceofTEM-1␤-lactamaseandthepresenceofdifferentCTX-Mvariantswitha prevalenceofCTX-M-15.TwoinfrequentCTX-MvariantsinSouthAmericawerealso iden-tified.Tothebestofourknowledge,thisisoneofthefirststudiesdescribingthegenetic characteristicsof␤-lactamasesinEcuador.

©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Sincethediscoveryofantibiotics,manypathogenswhichhad been considereddefeated havecontinued todevelop resis-tancetothesedrugs.Theemergenceofresistant microorgan-ismstoantibioticsisaproblemthathasextendedglobally.1,2 Thisphenomenoniswidespreadinhospitalsmainlybecause ofthe large and ofteninappropriate use ofantimicrobials. However, due to the bacteria’s ability to exchange genetic information,environmentalandnosocomialpathogenshave developedthesameresistancemechanisms,sincemost resis-tance genes have their probable origin in environmental microorganisms.3 _{Resistant pathogens to most antibiotics}

∗ _{Correspondingauthor.}

E-mailaddress:[email protected](A.Ausili).

1 _{Theseauthorscontributedequallytothiswork.}

havealready beendescribed,but inparticularit isofgreat impact the resistance to ␤-lactams. Resistance to these antimicrobialagentsisduetotheproductionof␤-lactamases, and the genes encoding these enzymes have been found worldwidemainlyinGram-negativebacteria,prevalentlyin

EnterobacteriaceaeandPseudomonasspp.4,5

Among ␤-lactamases, extended-spectrum ␤-lactamases (ESBLs),AmpC-type ␤-lactamases,and carbapenemasesare of great concern. In particular ESBLs are the most abun-dantinEnterobacteriaceae,withmorethan600naturalvariants (http://www.lahey.org/Studies/), and with a prevalence of

http://dx.doi.org/10.1016/j.bjid.2016.07.001

1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Table1–Resistanceratefortheclinicalisolates.

Antimicrobialagent-diskconcentration Number(%)ofresistantisolates

␤-Lactamase/ESBL(n=73) AmpC(n=6) Total(n=79)

AMK-30␮g 9(11%) 0(0%) 9(11%) AMC-20/10␮g 19(25) 2(2%) 21(27%) AMP-10␮g 50(63%) 1(1%) 51(65%) SAM-10/10␮g 47(59%) 1(1%) 48(61%) ATM-30␮g 30(38) 1(1%) 31(39%) FEP-30␮g 13(16%) 0(0%) 13(16%) CTX-30␮g 58(73%) 0(0%) 58(73%) FOX-30␮g 17(22%) 2(2%) 19(24%) CAZ-30␮g 18(18%) 0(0%) 18(23%) CIP-5␮g 52(66%) 0(0%) 52(66%) IMP-10␮g 7(9%) 0(0%) 7(9%) MEM-10␮g 0(0%) 0(0%) 0(0%) NET-30␮g 51(65%) 1(1%) 52(66%) PTZ-100/10␮g 4(5%) 0(0%) 4(5%)

Abbreviations: AMK, amikacin; AMC, amoxicillin/clavulanic acid; AMP, ampicillin; SAM, ampicillin/sulbactam; ATM, aztreonam; FEP, cefepime;CTX,cefotaxime;FOX,cefoxitin;CAZ, ceftazidime;CIP,ciprofloxacin;IMP,imipenem;MEM,meropenem; NET,netilmicin;PTZ, piperacillin/tazobactam.

CTX-MfamilythatbecamepredominantoverTEMandSHV duringthefirstdecadeofthe21stcentury.6

Asfarasweknow,thereisalackofdetailedreports char-acterizingthe␤-lactamasespreadinEcuador.7,8_Inthiswork aphenotypicandmolecularcharacterizationof␤-lactamases wasperformedinclinicalpathogensisolatedintwomain pub-lichospitalsoftheLojaprovince,Ecuador.

TheclinicalisolatesofEnterobacteriaceaeandPseudomonas aeruginosawereobtainedfrompatientsofYgnacioMonteros andIESSHospitals.ThesampleswerecollectedbetweenMay andNovember2013,whilethebacterialculturesweregrown inagar-MacConkeyPetridishes,identifiedbytraditional bio-chemical testing and validated by MicrogenTM _GnA+B-ID

system.

Antimicrobial susceptibility testing was performed by Kirby-Bauer disk diffusion procedure according to CLSI guidelines.9 _{The isolates were tested for susceptibility to} amikacin (AMK), amoxicillin/clavulanic acid (AMC), ampi-cillin (AMP),ampicillin/sulbactam(SAM), aztreonam (ATM), cefepime(FEP),cefotaxime(CTX),cefoxitin(FOX),ceftazidime (CAZ),ciprofloxacin(CIP),imipenem(IMP),meropenem(MEM), netilmicin (NET), and piperacillin/tazobactam (PTZ). The investigationofphenotypicproductionofESBLand AmpC-type ␤-lactamase was performed by techniques of double disk synergism (DDST) on Mueller Hinton agar and com-bination disk method. In particular for ESBL detection, DDSTwas performedon agarwithAMC-20/10␮gdisk pos-itioned25mm(center-to-center)awayfromdiskscontaining CAZ-30␮g, CTX-30␮g, FEP-30␮g and ATM-30␮g, and the confirmation combination disk methodwith disks of CTX-30␮g, cefotaxime/clavulanic acid (30/10␮g), CAZ-30␮g and ceftazidime/clavulanic acid (30/10␮g)10_{; for AmpC-type} ␤-lactamase detection, DDST (for constitutive AmpC) was performedwithboronicacidcontainingdisk(30␮g)placedata center-to-centerdistancetoaCAZ-30␮gandaCTX-30␮gdisk of25mm,whileforcombinationdiskmethod(forinducible AmpC),disks ofCTX-30␮g,CAZ-30␮gand ATM-30␮gwere placedclosetoaIMP-10␮g.11,12

ThreemultiplexPCRandonesimplexPCRwereperformed to determine the presence of blaTEM, blaSHV, blaOXA-1-like

and blaCTX-M genes inESBL-producing isolates, and blaACC,

blaFOX,blaMOX,blaDHA,blaLAT,blaCMY,blaMIRandblaACTgenes

inplasmid-mediatedAmpC␤-lactamase-producingisolates. PCR primersand procedures were performedaspreviously described.13

PurificationandsequencingofPCRproductswerecarried out byMacrogenInc. (Korea) andthe typesof␤-lactamase geneswereidentifiedbycomparisonwiththesequencesin GenBank(http://www.ncbi.nlm.nih.gov/genbank/).

Duringthestudy,atotalof79isolatesresistantto␤-lactams wererecovered.Themostfrequentisolatedbacterialspecies were Escherichia coli (39) followed by Klebsiella pneumoniae

(9),Klebsiellaoxytoca(7)Enterobacteraerogenes(7),Enterobacter cloacae(5)Proteusmirabilis(3),Pseudomonasaeruginosa(3), Cit-robacterfreundii(3),Proteusvulgaris(2),andEnterobacterkoseri

(1).

Allisolatesshowedantimicrobialresistancetooneormore ␤-lactam antibiotics, and among them,73 were confirmed to be positive for ␤-lactamase/ESBL and six for AmpC ␤-lactamaseproduction(seeTable1fordetails).

ByPCRamplification,itwasfoundthat31%ofthesamples showedthepresenceofone␤-lactamasegene,whilstisolates oftencarriedtwo(28%),three(20%),four(18%),and occasion-allyfive(3%)differentvariantsof␤-lactamaseenzymes(see

Table2fordetails).

ThegenesencodingCTX-Mwerethemostcommonand werefoundin63ofthe73ESBL-producingisolates.In partic-ularthegenesblaCTX-Mbelongingtophylogeneticgroups1,2

and9werefoundin48,4and20isolates,respectively.Itwas alsodetectedthat52oftheESBL-producingisolatescarried

blaTEM, 21blaSHV, and 34blaOXA-1-likegenes. Finally,blaDHA

wasthemostfrequentlyobservedplasmid-mediatedAmpC ␤-lactamasegene(foundinfour isolates),whileoneisolate carriedblaLATandoneblaMOX.Toidentifythespecificblagenes

detectedinthePCRassays,ampliconsDNAsequenceanalyses wereperformedandtheresultsaresummarizedinTable2.

brazj infect dis.2016;20(6):627–630

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Table2–Summaryof␤-lactamaseencodinggenesdetectedinESBL-producingbacteriafromclinicalisolates.

Enzyme Class Family Group Variant Spectrum Combination

␤-Lactamase(79) ␤-Lactamase(73) CTX-M(72) Group1(48) CTX-M-1(5) Extended

CTX-M-3(6) Extended

CTX-M-12(5) Extended

CTX-M-15(30) Extended BL/ESBL3/2(2) CTX-M-55(2) Extended BL/ESBL3/1(7) Group2(4) CTX-M-2(4) Extended BL/ESBL2/2(8) Group9(20) CTX-M-14(16) Extended BL/ESBL2/1(9) CTX-M-65(4) Extended BL/ESBL1/2(4)

TEM(52) TEM-1(51) Broad BL/ESBL1/1(20)

TEM-12(1) Extended BL3(2)

SHV(21) SHV-1(5) Broad BL2(2)

SHV-2(4) Extended BL1(3)

SHV-11(10) Broad ESBL1(16)

SHV-12(2) Extended AmpC(6)

OXA-1-like(34) *OXA-1-like(34) Broad

AmpC(6) DHA(4) DHA-1(4) Broad

LAT(1) CMY-75(1) Broad

MOX(1) MOX(1) Broad

Thenumbersinbracketsrepresentthenumberofisolates(inenzymeandclasscolumns)andoffoundgenes(infamily,group,variantand combinationcolumns).Thelastcolumnrepresentsthedifferentcombinationsof␤-lactamase(BL),ESBLandAmpCgenesfound.*Inthecase ofblaOXA-1-like,itwasnotpossibletodistinguishthedifferentvariantsbydirectsequencingofPCRamplicons.13

Manystudiesontheresistanceto␤-lactamsconductedin SouthAmerica wereperformed usingmainly aphenotypic approach.InEcuadorthefewreportspublishedonthistopic lackalmosttotallyofagenotypicstudy,14–17_{and,tothebest} ofourknowledge,thisisoneofthefirststudiesdescribingthe geneticcharacteristicsof␤-lactamasesinthiscountry.8,9,17

OurdataconfirmedtheprevalenceinLojaprovinceofE.coli

andKlebsiella spp.as␤-lactamase producers,6,18 _{and a} pre-dominanceofESBLproducers,showingthehighestresistance levels mainly against cefotaxime, ciprofloxacin, netilmicin, andampicillin.

It is widely documented that in South America ESBL-producingEnterobacteriaceaehaveoneofthehighestincidence intheworldandCTX-Msarethemostbroadlydistributed.9 InparticularthetypeCTX-M-2isthe mostfrequentinthe SouthernCone,19_{CTX-M-2-group,CTX-M-8,andCTX-M-9in} Brazil,20_{CTX-M-1-groupinColombia,}21_andCTX-M-2, CTX-M-14,CTX-M-15,CTX-M-24,andCTX-M-56inPeruandBolivia.22 Inourstudy,itisdocumentedthat CTX-Menzymes are thedominantESBLsintheLoja province,and amongthem the prevailing types are CTX-M-15 and CTX-M-14. These dataareconsistentwiththeliterature21,22 _asthesetwo vari-antsarepresentandquitefrequentinEcuadorneighboring countries. Interestingly,thetypes CTX-M-55and CTX-M-65 weredetected.Actually,thesevariantsarecommoninEast Asia,23,24 _{and onepossible hypothesis oftheir presence in} Southern Ecuador can be related to the recent immigra-tionfromChinaandthetraderelationshipbetweenEcuador andChina.25_{Asexpected,astrongpresenceofTEM-type} ␤-lactamaseswasalsofound,inparticularTEM-1␤-lactamase. TEM-typepresents awidegeographicaldistribution,and in Ecuadorthesedataarenotparticularlysurprising consider-ingtheincidenceofTEMinneighboringColombia26_andthe tradeandmigratoryflowsbetweenthese twocountries, as wellastherecentintenseEcuadorianimmigrationtoandfrom EuropeandNorthAmericawhereTEMispredominantsuch

asCTX-M-15.Itisworthtomentionthefindingofone iso-late harboringTEM-12. ThisESBLvariant hasbeen already found in SouthAmerica27 _{and it is one ofthe most} com-mon inNorthAmerica.28 _{Inourstudy,the presenceofthe} non-ESBL OXA-1-type, SHV-1 and SHV-11, mainly foundin

Klebsiella (9 isolates) and Proteus (4 isolates), and the ESBL SHV-2andSHV-12,foundinEnterobacter,KlebsiellaandE.coli,

were also detected. The non-ESBL OXA-1-type and SHV-1 areamongthemostwidespreadinEnterobacteriaceae,29_while to the best of our knowledge SHV-11 variant is emergent in South America. Regarding ESBLs, the variants found in this study are among the most common in Latin Amer-ica,indeedSHV-2withSHV-5werethefirstESBLSHV-types describedinEnterobacteriaceae,30_{whileSHV-12ishighly} preva-lent in Mexico, Colombia, and Brazil.31 _{Concerning AmpC} ␤-lactamase,thereisnotsufficientdatatodraw significant conclusionsonitsepidemiology,butitisinterestingtonote thepresenceoftwoCMY-75genesfoundinC.freundii.This variant has been already detected in C. freundii in Spain (EMBL/GenBank/DDBJdatabases:AFK73434.1),butisemergent inSouthAmerica.OntheotherhandDHA-1hasbeen previ-ouslyrecognizedinSouthAmerica,beingquitepredominant inArgentina.32

In conclusion, in this work the predominance of ESBLs in Southern Ecuador was demonstrated witha prevalence of CTX-M-type enzymes and we identified two emergent variantsinSouthAmerica.Ahigh presenceofTEM-type ␤-lactamaseswerealsodetected,beingTEM-1␤-lactamasethe mostfrequentvariantfound.However,therearesome lim-itationsinthis study,includingthe lownumberofisolates andtherestrictedgeographicalareaofsampling.Inorderto minimizetheseproblemsandgathermoreindicative informa-tion onmolecular epidemiologyand geneticcharacteristics of␤-lactamases withinthe entireterritory ofEcuador, fur-therisolatesfromdifferenthealthinstitutionsandregionsof Ecuadorhavetobecollectedandcharacterized.

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Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisworkwassupportedbytheProjectPROYCCSAL1042of UTPLandbythePrometeoProjectoftheSecretariatforHigher Education,ScienceTechnologyandInnovationoftheRepublic ofEcuador.

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