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
Original
article
Prevalence
and
molecular
analysis
of
multidrug-resistant
Acinetobacter
baumannii
in
the
extra-hospital
environment
in
Mthatha,
South
Africa
Yaw
Anane
A
a,
Teke
Apalata
b,∗,
Sandeep
Vasaikar
a,b,
Grace
Emily
Okuthe
c,
Sandile
Songca
daWalterSisuluUniversity,FacultyofHealthSciences,DepartmentofLaboratoryMedicine&Pathology,EasternCapeProvince,South Africa
bNelsonMandelaCentralHospital,NationalHealthLaboratoryServices(NHLS),DivisionofMedicalMicrobiology,Mthatha,SouthAfrica cWalterSisuluUniversity,DepartmentofBiological&EnvironmentalSciences,EasternCapeProvince,SouthAfrica
dUniversityofKwaZulu-Natal,CollegeofAgricultureEngineeringandScience,SchoolofChemistryandPhysics,Durban,SouthAfrica
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received10March2019 Accepted4September2019 Availableonline7November2019
Keywords: Acinetobacterbaumannii Carbapenemase-encodinggenes Extra-hospital Multidrug-resistance ISAba1 intI1
a
b
s
t
r
a
c
t
Introduction:ThepresenceofAcinetobacterbaumanniioutsidehospitalsremainsunclear.This studyaimedtodeterminetheprevalenceofmultidrug-resistance(MDR)A.baumanniiinthe extra-hospitalenvironmentinMthatha,SouthAfricaandtoinvestigatethefrequencyof carbapenemase-encodinggenes.
MaterialandMethods:FromAugust2016toJuly2017atotalof598abattoirsamplesand689 aquaticsampleswerecollectedandanalyzedpresumptivelybyculturalmethodsforthe presenceofA.baumanniiusingCHROMagarTMAcinetobactermedium.Speciesidentification wasperformedbyautoSCAN-4(DadeBehringInc.,IL)andconfirmedbythedetectionof theirintrinsicblaOXA-51gene.ConfirmedMDRA.baumanniiisolateswerescreenedforthe presenceofcarbapenemase-encodinggenes,ISAba1insertionsequenceandintegraseintI1. Results:Intotal,248(19.3%)Acinetobacterspecieswereisolated.Acinetobacter.baumanniiwas detectedin183(73.8%)ofwhich85(46.4%)and98(53.6%)wererecoveredfromabattoirand aquaticrespectively.MDRA.baumanniiwasdetectedin56.5%(48/85)abattoirisolatesand 53.1%(52/98)aquaticisolates.Isolatesshowedhighresistancetoantimicrobialsmost fre-quentlyusedtotreatAcinetobacterinfectionssuchaspiperacillin/tazobactam;abattoir(98% ofisolatesresistant),aquatic(94%ofisolatesresistant),ceftazidime(84%,83%),ciprofloxacin (71%,70%),amikacin(41%,42%),imipenem(75%,73%),andmeropenem(74%,71%).Allthe isolatesweresusceptibletotigecyclineandcolistin.AlltheisolatescarriedblaOXA-51-like.The blaOXA-23wasdetectedin32(66.7%)abattoirisolatesand11(21.2%)aquaticisolates.The
∗ Correspondingauthor.
E-mailaddress:tapalata@wsu.ac.za(T.Apalata). https://doi.org/10.1016/j.bjid.2019.09.004
1413-8670/©2019PublishedbyElsevierEspa ˜na,S.L.U.onbehalfofSociedadeBrasileiradeInfectologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
blaOXA-58-likewaspositivein7(14.6%)and4(7.7%)abattoirandaquaticisolates,respectively. BothgroupsofisolateslackedblaOXA-24-like,blaIMP-type,blaVIM-type,blaNDM-1,blaSIM,blaAmpC, ISAba1andinI1.IsolatesshowedhighlevelofMultipleAntibioticResistanceIndex(MARI) rangingfrom0.20-0.52.
Conclusion: Extra-hospitalsourcessuchasabattoirandaquaticenvironmentsmaybea vehicleofspreadofMDRA.baumanniistrainsinthecommunityandhospitalsettings.
©2019PublishedbyElsevierEspa ˜na,S.L.U.onbehalfofSociedadeBrasileirade Infectologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Acinetobacterbaumanniihasemergedoverthefewlastdecades asacauseofhealthcare-associatedinfectionswiththis organ-ismassociatedwithincreasedpatient morbidity, mortality, and treatment costs.1–3 Its clinical significance has been
propelledbyits remarkableabilitytoupregulateor acquire resistancedeterminantmakingitoneoftheorganisms threat-ening the current antibiotic era.4,5 The possession of this
arrayofresistancemechanismshavemadethisorganismto beable toresistalmost all availableantibiotics inhospital environmentandhaspositiontheorganismasimportant can-didatefortheevaluationofreservoirsofantibioticresistance intheenvironmentoreveninhumansubjects.6
Acinetobac-ter.baumanniihavetheabilitytosurviveinbothmoistand dryconditionsformanyweeksorevenmonths,hencethis organism is widely distributed in natural and nosocomial environments,humanskin,aswellasmucosalsurfaces facil-itatingitsspread.7–9Thecross-transmissionofthisorganism
frompatienttopatientandthepossibilityofoutbreak exten-sionbypatienttransferhavebeen demonstrated.10 Despite
hospitalecology being intensively studied, its ecology out-sidethehospitalremainsunclearorpoorlydefinedimpeding effective prevention of transmission.2,11 Several
investiga-torshavesuspectedthatthesurvivalofA.baumanniiinthe environment(inparticularinwater)couldcontributetothe transmissionoftheorganism duringoutbreaks.12 Itiswell
establishedthatmembersofthegenusAcinetobacteris ubiq-uitousbut difficultyinunequivocallyidentifythisorganism hasmadetheubiquityinnatureofA.baumanniiawidespread misconception.1,9Thereforetheexistenceofanextra-hospital
reservoirofA.baumanniiandtheimplicationofthispotential reservoirintheoccurrenceofcertaininfections duetothis organismarestillcontroversial.1,2Additionally,theprevalence
andantimicrobialresistanceofA.baumanniioutsidethe hos-pitalsettinghasbeenpoorlyinvestigated.13Thereseemsto
beacontinuousinfluxofnovelstrainsintotheclinicalsetting withthepotentialofnewinfectiousfeatures.11Studieshave
shownthatinfectionscausedbyextra-hospitalstrainsofA. baumanniiareresponsibleforcommunity-acquiredinfections mainlyintropicalandsubtropicalareas.5,14 Thissuggestsa
sourceofthispathogenoutsideofthehospitalandtherefore thesignificanceofenvironmentalisolatesinthe epidemiol-ogyofA. baumanniiisofagreatconcernworldwide.There is no clear evidence about the way of introduction of A. baumannii into hospital environment, its propagation from hospitalsettingstothenaturalenvironmentanditsnatural
habitatoutsidehospitals.15Alsounlikeclinicalstrains,there
are limited reportsusingmolecularanalysis toexplorethe extra-hospitalepidemiologyofA.baumannii.11,16Thepresence
ofA.baumanniiintheaquaticenvironmentraisestheissueof publichealthrisksincepeopleareinconstantcontactwith theaquaticenvironment.17Hospitalwastewaterisdischarged
intomunicipalsewagesystemwithoutpriortreatment, con-tributingtowidespreadcontaminationofnaturalwaterswith emergingpathogenicbacteriathatcancarryMDRgenes.18In theaquaticenvironment,bacteriainteractwithvarious organ-isms,someofwhicharefishusedforconsumptionaswellas commercialorrecreationalpurposes.19Mostoftheantibiotics
usedinthehospitalsendupcontaminatingourlocaldams. Theindiscriminateuseofantibioticsinlivestockproduction asgrowthpromotersortocontrolinfectiousdiseasesandthe treatmentshavebeenlinkedwiththedisseminationof resis-tant bacteria which can be transferredto people via food, directcontactwithinfectedanimalorthroughtheabattoir.In astudyconductedinScotland,pigsandcattleslaughteredfor humanconsumptionandcomingfromdifferentfarmswere sampled(faeces,skin,nostrilandear)forA.baumannii isola-tion.TheprevalenceofA.baumanniicarriagewas1.2%.The16 A.baumanniiisolatedweregroupedintothreedifferent clus-ters,buthaddifferentpulsed-fieldgelelectrophoresis(PFGE) patterns comparedwithhumanisolatesofthethreemajor EuropeanclonesEci,ECII,andECIII.20Recentreportshavealso
described thepresenceofcarbapenem-resistantA. bauman-niiinanimals.TheOXA-23carbapenemasehasbeenfound inAcinetobacterfromcattle,horses,andcats.21–23TheNDM-1
hasalsobeenreportedinAcinetobacterspp.fromfoodanimals (chicken and pigfarms) in China.24,25 However,knowledge
aboutcarbapenemase-producingA.baumanniiof environmen-talorigin(aquaticandabattoirincaseofthisstudy)remains verylimited,makingitdifficulttoassessitsimpactonpublic health.InSouthAfrica,A.baumanniihasbeenisolatedfrom hospital environments; nevertheless onlyfew studies have reportedthepresenceofA.baumanniiintheaquatic environ-ment,meatoffoodanimalsandtheenvironmentwherethese meatscomefrom.Theseareasareasourceofwaterandfood andthereforecanbeoftransmissionofthisorganisminto hos-pitalsettingsormayhorizontallytransferresistancegenesto otherorganisms.
Studyobjectives
Basedonthesepremises,thepresentstudywascarriedoutto determinetheprevalenceofMDRA.baumanniiinaquaticand
abattoirenvironments,theirsusceptibilitytoantimicrobials andtodeterminethefrequencyofcarbapenemase-encoding genes.
Material
and
methods
Studydesignandsettings
ThiswasaCross-sectionalprospectivedescriptivestudy con-ductedbetweentheperiodofAugust2016andJuly2017at: 1 TheUmzikanturedmeatabattoir,aprivateabattoirlocated
in SouthRidge Park, O.R.Tambo districtmunicipality in Mthatha,EasternCape.ThisredmeatabattoirisatypeE5 abattoir.Itoperates asanabattoirandmeatwholesalers anditistheonlyoperationalabattoirinMthatha.Abattoirs differgreatlyinthescaleoftheiroperations.UsingtheEU definitionoflivestockunits,thescaleisgradedaccording tothenumberofbeasttheyaredesignedtohandle (with-outsomehealthrisk)perweekasfollows:E:-toslaughter fiveunits, D:-toslaughter 15units, C:-toslaughter50 units,B:-toslaughter200units,andA:-toslaughter1000 units(exportingtype).InitsbasicformoneEULivestock Unit(ELU)equals:onecowbeast,twocalves,fivepigsor10 sheep.26
2 TheMthatha dam(coordinates: 31◦332S28◦4424E), an earth-filltypedamwhichimpoundstheMthathariverat alocationabouteightkilometersupstreamofthecity of MthathaintheORTamboDistrictMunicipalityofthe East-ernCapeProvince.Thedamwasestablishedin1977and serves mainlyfor municipal and industrialpurposes. Its hazardpotentialhasbeenrankedhigh.Thestoragecapacity oftheDamis253,674,000cubicmeters.Itservesasthemain sourceofrawwatersupply(65.9mL/day)fortheThomhil WTWwhichtreatsandsupplieswatertothecityandits environswithatotalpopulationofabout750,000.
Samplecollectionfromabattoirenvironment
Fivehundredandninety-eightsampleswererecoveredfrom the hides, from structural and work surfaces (equipment, floors,doors,knives,saws,handlingpanelsfromthe slaugh-ter area)within theabattoir and from carcassesand meat atallstagesofprocessing(fromflayingtoevisceration, split-tingandcooling).Ateachsite,anareaof10cm2wassampled usingasterileswabmoistenedwithphysiologicalsaline. Ani-malsampleswerealsocollected from recentlyslaughtered animals destinedforhuman consumption. Carcasseswere sampledbysystematicrandomsampling technique.Swabs weretakenaccordingtothemethoddescribedbythe inter-national organization for standardization.27 The abdomen
(flank),thorax(lateral),crutch,breast(lateral),werethe sam-plingsites.Swabbingwasperformedapproximatelysixhours aftercleaninganddisinfectionandpriortoslaughter activi-tiesintheabattoir.Thesampleswerecooledandtransported tothe laboratoryforplatingwithin10haftersampling.For meat samples, 25g of meat were cut aseptically into very small pieces and homogenized by using a stomacher bag
(Interscience,SaintNom,France)andthensuspendedin ster-ilewater.
Samplecollectionfromaquaticenvironment
Overall,689sampleswerecollectedfromtheMthathadam. Thedamwassampledfivetimeswithinthestudyperiodand sampleswerecollectedusingaseptictechniquesinthe morn-ings(between08:00and10:00hlocaltime).Duplicatewater sampleswerecollectedatmarginsandinthemiddleofthe dam,about 15cmdownthe watersurface in100ml steril-ized bottles. Thecollectedwatersampleswere placedina coolerboxwithtemperaturemaintainedbetween4and10◦C usingicepacks.Thesesampleswereimmediatelytransported tothe WalterSisuluUniversity laboratoryand subjectedto serialten-folddilutionsinsteriledistilled water.Forty-nine fishesbelongingtodifferentspecieswerecollectedaseptically and immediatelytransportedinathermalbag tothe labo-ratory wheretheyweresacrificed.Samplesfromskin, gills, intestines,andeyeswerecollected.Thebodysurfaceandgills werecarefullywashed,atfirstunderthestreamoftapwater and then using70% ethylalcoholtoremovenormal exter-nalbacterialflora.Then,thefishweredisinfectedwith70% ethanolandafteropeningthebodycavity,samplesfromthe kidneysandintestineweretaken.Thesampleswerediluted 1:1inPBSandhomogenized.
Bacterialcultivationandidentification
Samples were presumptively identifiedbydirect plating onChromogenicCHROMagarTMAcinetobacter(CHROMagarTM; Paris, France) with supplement Ref. CR102 which allows thegrowthofcarbapenem-resistantisolatesofAcinetobacter species,incubatedat37◦Cinaerobicconditionsandexamined after24hforthegrowthoftypicalredcoloniesofAcinetobacter species.CHROMagarTM Acinetobacterwaspreparedaccording to the manufacturer’s description. Cefsulodin sodium salt hydrate (Sigma-Aldrich) was added at 15mg/L to suppress the growth of Pseudomonas and Aeromonas species. Gram-positive bacteria and yeastsare inhibited. Confirmation of presumptivecolonieswasperformedbyinspectionofcolony morphologyon theseparateplates.Theplateswere exam-ined forthe growth oftypicalred colonies ofAcinetobacter species. One typicalcolony representative of each type of morphology and shape was further sub-cultured on tripti-casesoyagar (TSA)andcharacterizedbasedonphenotypic tests:Gram-stain,catalaseandoxidasetests.Gram-negative coccobacilli,oxidase-negative, andcatalase-positiveisolates were presumptively identified to the genus Acinetobacter. SpeciesidentificationwerecarriedoutusingGram-negative IDtype2panel(REF.B1017-27)oftheMicroScan autoSCAN-4 automated System (Dade Behring Inc., Deerfield, IL). A high percentage(≥95%)was utilizedasthe acceptance cri-terion for identification by MicroScan. Suspected colonies were also further verified using the Acinetobacter specific primer set Ac436F and Ac676r to amplify the 16S rRNA gene.ConfirmationofA.baumanniiisolateswascarriedout by polymerase chain reaction analysis of the presence of inherentBlaOXA-51-likegenes.IsolateslackingtheBlaOXA-51-like gene wereinvestigatedfurtherbysequence analysisofthe 16S rRNA gene. Primers were ordered from TIB Molbiol, Germany. All the strains were freshly suspended in skim
milk (Merck, Germany) containing 15% glycerol in sterile glass,screw-cappedvials and storedat-80◦Cuntil further use.
Antimicrobialsusceptibilitytesting
Antimicrobial susceptibility testing was performed by MicroScanautoSCAN-4 System.Theantibioticstestedwere amikacin (AMK), amoxicillin/clavulanic acid (AMC), ampi-cillin/sulbactam (AMS), ampicillin (AMP), cefazolin (CEP), cefepime(FEP), cefotaxime (CTX), ceftazidime(CAZ), ceftri-axone(CRO),cefuroxime(CXM),cefoxitin(FOX),cephalothin (CEPH), ciprofloxacin (CIP), gentamicin (GEN), imipenem (IMP),meropenem (MEM),levofloxacin(LVX), nitrofurantoin (NIT), tetracycline (TEC), tobramycin (TOB), trimetho-prim/sulfamethoxazole(SXT),piperacillin/tazobactam(TZP), piperacillin(PRL),colistin(CST),andtigecycline(TGC) accord-ing to the CLSI guidelines v27.28 Non-susceptibility was
defined as the combination of resistance and intermedi-ate resistance. MDR A. baumannii isolates were defined as acquirednon-susceptibilitytoatleastoneagentinthreeor moreantimicrobialcategories.29Escherichiacoli(ATCC#25922)
andPseudomonasaeruginosa(ATCC#27853)wereincludedas qualitycontrolstrains.
Multipleantibioticresistanceindex(MARI)
The MARI was calculated and interpreted according to Krumperman(1983)astheratioofthenumberofantibiotics to which the isolateswere resistant (a) to the total num-berofantibioticstowhichtheisolateswereexposed(b),i.e: MARI=a/b.
TheMARIofindividualA.baumanniifromthetwodifferent sourceswascalculated.BacteriahavingMARI(>0.2)originate fromahighrisksourceofcontaminationwhereseveral antibi-oticsareused.MARIvalueof≤0.2indicatesstrainoriginated fromsourceswhereantibioticsareseldomorneverused.30
Investigationofcarbapenemase-encodinggenes,ISAba1 andintI1byPCR
Genomic DNA from an overnight culture on tryptic soy broth ofA. baumanniiwas extracted using the MagNaPure Compact® nucleic acid isolation kit I (REF. 03730964001, Roche diagnostics, Mannheim, Germany) according to the manufacturer’s protocol.Thepresenceofthe genes encod-ing Ambler class D Serine-Carbapenemase (blaOXA-51-like, blaOXA-23-like, blaOXA-24-like, andblaOXA-58-like),Ambler classB Metallo--lactamases (blaIMP-1, blaVIM, blaSIM and blaNDM-1) and Ambler classC blaAmpC were analyzed byPCR. ISAba1 andintI1werealsodetermined.ThepresenceoftheISAba1 upstreamblaOXA-23-likeandblaOXA-51-likegenestopromotegene expressionwereperformedusingISAba1forward/blaOXA-23-like reverseorblaOXA-51-likereverseprimers.31Thedetailsofprimer sequencesusedforPCRamplificationoftheresistantgenes arelistedinTable1.
Ethical
considerations
Ethical approval for this project was obtained from the Walter Sisulu University Research Ethics and Biosafety Committee [Reference number: 019/2016]. Also written permission was sought from the authorities of the two studysites.For theprotectionofanimalsusedforscientific purposes the experiments described complied with the guidelines of the EuropeanUnionCouncil http://ec.europa. eu/environment/chemicals/labanimals/legislationen.htm (Directive2010/63/EU).
Statistical
analysis
The data were analyzed using the Statistical Package for Social Sciences (SPSS, IBM, version 23.0 Armonk, NY). The frequency of some parameters including A. bauman-nii susceptibility and also resistant genes were compared between abattoir and aquatic A. baumannii by Chi-square or Fisher’s exact test. P-values <0.05 were considered as significant.
Results
AntimicrobialsusceptibilitytestingandMARI
A total of 1287 samples were collected from the abattoir and aquatic sources from which 248 (19.3%) Acinetobacter species were isolated. A. baumannii was the predominant species (n=183, 73.8%) of which 85 (46.4%)and 98 (53.6%) were recovered from abattoir and aquatic, respectively. Of these a total of 100 MDR A. baumannii isolates, includ-ing 56.5% (48/85) abattoir and 53.1% (52/98) aquatic, were selected foranalysis. Table2shows thedistribution ofthe identified Acinetobacterspecies according tothe sourcesof samples.AllMDRisolatesshowedasusceptiblephenotypein responsetocolistinandtigecycline.Amongabattoirisolates, all(100%)wereresistanttotrimethoprim-sulfamethoxazole, and >70% were resistant to cephalosporins (ceftazidime, cefazolin, cephalothin, cefotaxime, ceftriaxone, cefepime, cefuroxime, and cefoxitin), gentamycin and ciprofloxacin. Carbapenem resistantwas 75%againstimipenem and 74% againstmeropenem.However,therewereconsiderable differ-ences inresistanceratesandpatternsbetweentheisolates recoveredfromthetwostudyareas.Chi-squaretestrevealed thattherewerenosignificantdifferencesinresistancerates among different sources of isolates (p<0.253). The resis-tanceratesagainstimipenem,meropenem,ciprofloxacin,and gentamycin were 73%, 71%,70%, and 75%, respectively, in isolates recovered from aquaticsource,whereas resistance rateswere57–94%againstotherantimicrobialagentsexcept foramikacin (42%).Fig.1showstheresistance characteris-ticsoftheseisolates.TheA.baumanniiexhibited25antibiotic resistantpatternswithhighlevelofMARI(>0.2)rangingfrom 0.20–0.52.
Mean MARIofisolatesfrom bothsourcesare shown in Fig.2.ThemajorityoftheA.baumanniifromtheabattoirsource (31isolates,64.6%)wereresistanttoatleasteightantibiotics
Table1–ListofprimersusedforPCRamplificationofresistantgenes.
Gene Oligonucleotidesequence Fragmentsize(bp) Location References
16SrRNA F:5’TTTAAGCGAGGAGGA GG3’
R:5’ATTCTACCATCCTCT CCC3’ 240 16SrRNA 32 OXA-23-like F: 5’GATCGGATTGGA-GAACCAGA3’ R: 5’ATTTCTGACCGCATTTCCAT3’ 501 blaOXA-23 33
OXA-24-like F:5’GGTTAGTTGGCCCCCTTA
AA3’
R: 5’AGTTGAGC-GAAAAGGGGATT3’
246 blaOXA-24 33
OXA-51-like F:5’TAATGCTTTGATCGGCCT
TG3’ R:5’TGGATTGCACTTCATCTT GG3’ 353 blaOXA-51 33 OXA-58-like F: 5’AAGTATTGGGGCTTGTGCTG-3’ R: 5’CCCCTCTGCGCTCTACATAC3’ 599 blaOXA-58 33 IMP-1 F: 5’GATGGTATGGTGGCTCTTGT3’ R: 5’TTAATTTGCCGGACTTAGGC3’ 448 BlaIMP 34 NDM-1 F:5’ATTAGCCGCTGCATTGAT3’ R: 5’CATGTCGAGATAGGAAGTG3’ 154 blaNDM 35 VIM-like F: 5’ACTCACCCCCATGGAGTTTT3’ R: 5’ACGACTGAGCGATTTGT-GTG3’ 815 blaVIM 36
SIM-1-like F:5’TAATGCTTTGATCGGCCT
TG3’ R:5’TGGATTGCACTTCATCTT GG3’ 353 blaSIM 33 AmpC F:ACAGAGGAGCTAATCATGCG R:GTTCTTTTAAACCATATACC 1243 blaampC 37 ISAba1 F:5-CACGAATGCAGAAGTTG-3 R:5-CGACGAATACTATGACAC-3 599 ISAba1 38
ISAba1/blaOXA-23-like F:AATGATTGGTGACAATGAAG R:ATTTCTGACCGCATTTCCAT
1433 ISAba1/blaOXA-23 38
ISAba1/blaOXA-51-like F:AATGATTGGTGACAATGAAG R:TGGATTGCACTTCATCTTGG
1252 ISAba1/blaOXA-51 31
intI1 F:5’CAGTGGACATAAGCC TGTTC3’
R:5’CCCGACGCATAGACT GTA3’
160 Integrasegene(intI1) 39
whilstthemajorityofA.baumanniifromtheaquaticsource(21 isolates,40.4%)wereresistanttosixantibiotics(Table3). Moleculardetectionofcarbapenemase-encodinggenesin A.baumannii
The two groups of MDR isolates were analyzed for carbapenemase-encoding resistance genes (blaOXA-23-like, blaOXA-24-like,blaOXA-51-like,blaOXA-58-like,blaIMP-1,blaSIM,blaVIM, blaampC andblaNDM-1).TheblaOXA-51 isintrinsicallyfoundin A. baumannii and was detected in all abattoir and aquatic isolates which is consistent with the blaOXA-51-like gene as anintrinsiccarbapenemase. Atotal of32(66.7%) ofthe 48 abattoirisolatescarriedblaOXA-23,whereas11(21.2%)ofthe52
aquaticisolateswereblaOXA-23positive.TheblaOXA-58-likewas detectedinseven(14.6%)andfour(7.7%)abattoirandaquatic isolatesrespectively.There werenoblaOXA-24-like, blaIMP-type, blaVIM-type, blaNDM-1, blaSIM, and blaampC genes amongboth groupsofisolates.AllisolateslackedISAba1andntI1. Accord-ing toChi-squaretest, thereweresignificant differencesin the detectionofresistantgenesamongdifferentsourcesof isolates(p<0.001).
Discussion
Overthepasttwodecades,Acinetobacterspecieshavebecome virulent pathogens, responsible for nosocomial infections
Table2–DistributionoftheidentifiedAcinetobacterspeciesaccordingtothesourcesofsamples.
Acinetobacterspecies isolated
Totalno.of isolates
Abattoirisolates Aquaticisolates
Source Numberof
isolatesfound
Source Numberof isolatesfound
A.baumannii 100 Floors 5 Sediment 17
Doors 1 Watersample 15
Knives 9 Gills 5
Saws 7 Intestines 3
Handlingpanels 2 Eyes 5
Slaughterarea 4 Skin 7
Abdomen(Flank) 7 Thorax(Lateral) 3
Crutch 2
Breast(Lateral) 8
Total 48(56.5%) Total 52(53.1%)
Acinetobacterlwoffii 36 Floors 2 Sediment 5
Doors 3 Watersample 4
Knives 3 Gills 2
Saws 2 Intestines 3
Handlingpanels 2 Eyes 1
Slaughterarea 4 Skin 4
Abdomen(Flank) 1
Total 17(20%) Total 19(19.4%)
Acinetobactercalcoaceticus 25 Floors 3 Sediment 4
Doors 4 Watersample 3
Saws 2 Gills 1
Handlingpanels 1 Intestines 2
Slaughterarea 1 Eyes 1
Skin 3
Total 11(12.9%) Total 14(14.3%)
Acinetobacterpittii 22 Floors 3 Sediment 4
Doors 1 Watersample 3
Handlingpanels 3 Intestines 4
Slaughterarea 1 Skin 2
Breast(Lateral) 1 Total 9(10.6%) Total 13(13.3%) Total 183 85 98 41 98 94 91 94 97 81 84 74 72 91 95 71 85 0 81 75 74 71 71 84 98 58 0 100 42 90 91 81 92 93 79 83 73 72 89 92 70 82 0 75 73 71 72 79 73 94 57 0 92 0 10 20 30 40 50 60 70 80 90 100 PE RCEN T A GE RE SI S T A NC E ANTIBIOTICS AMIKACIN AMPICILLIN AMOXICILLIN/CLUVALANIC ACID AMPICILLIN/SULBACTAM CEFAZOLIN CEFTRIAXONECEFTRIAXONE CIPROFLOXACINLEVOFLOXACIN COLISTIN
GENTAMICINIMIPENEMMEROPENEM NITROFURANTOINTETRACYCLINE
PIPERACILLIN
PIPERACILLIN/TAZOBACTAM TOBRAMYCINTIGECYCLINE
TRIMETHOPRIM/SULFAMETHOX... CEPHALOTHINCEFOTAXIMECEFTAZIDIME
CEFOXITINCEFEPIME
ABATTOIR AQUATIC
Table3–MultipleantibioticresistanceamongA.baumanniiisolatesfromsamplingsites(n=numberofisolates).
Samplingsites Fiveantibiotics(n)(%) Sixantibiotics(n)(%) Sevenantibiotics(n)(%) >Sevenantibiotics(n)(%)
Abattoir 2(4.2%) 10(20.8%) 5(10.4%) 31(64.6%) Aquatic 7(13.5%) 21(40.4%) 6(11.5%) 18(34.6%) 0.34 0.28 0 0.1 0.2 0.3 0.4 0.5 0.6 MA R I n d e x Sampling sites Abattior Aquatic
Fig.2–MeanMultipleAntibioticResistanceIndex(MARI) valuesinAbattoirandAquaticisolates.
andoutbreaks,particularlyinintensivecareunitsandhigh
dependency units (HDUs).2 Majority of published studies
have concentrated on the hospital epidemiology of these organisms2and animalhealthcaresettingsmakingit
diffi-culttodemonstratetheextra-hospitaloriginofA.baumannii. Theintensive use ofantibiotics over the last few decades in human and as growth promoting and as prophylactic agentsin livestockhave resultedin serious environmental andpublichealthproblems,sincethisenhances antimicro-bial selectivepressure. Alsoanthropological activities have greatimpactsonourlocaldams.Theantibioticresidues con-centrationincreasesinhospitalandindustrialeffluentsand theseendupinourlocaldamsleadingtothedevelopment ofantibiotic-resistantbacteria.Grosetal.,studiedthe antibi-oticconcentrationsinaquaticenvironmentsandthemedian concentrationsinsurfaceandgroundwaterwerereportedas 0.030and0.071gL−1,respectively.40Theseconcentrationsare
abovetheMIC/MBCforallantibiotics,accordingtotheBritish SocietyforAntimicrobialChemotherapy.41 A.baumannii
iso-latedfromvariousenvironmentallocationshasbeenlinked withnosocomial spread.42 Theresistant bacteria from the
extra-hospitalenvironmentsmaybetransmittedtohumans, inwhomtheycausediseasethatcannotbetreatedby con-ventionalantibiotics.Inaquaticenvironmenttwohypotheses canarise fromthe detectionofA.baumannii:(1) waterisa normal habitatof A. baumannii, or (2) the presence ofthe bacteriumresultsfromhumanoranimalcontamination.To ourknowledge,thisisthefirststudyinvestigatingthe molec-ular mechanisms accounting for the multidrug-resistance observedinA.baumanniirecoveredfromabattoirandaquatic samplesintheEasternCapeProvinceofSouthAfrica.
Inthecurrentstudy,the prevalenceofA. baumanniiwas 14.2%inabattoirand14.0%inaquaticsamplescollected.Of
these, MDR A.baumannii was recoveredfrom 56.5%(48/85) abattoirisolatesand53.1%(52/98)aquaticisolatesandwere analyzed.Therefore,ourstudyevidencedtheextra-hospital presenceofA.baumanniiinEasternCapeProvinceofSouth Africa.Thisisinagreementwithapreviousstudythatfound 51.2%(85/166)frommeatsamplestobeMDR,9i.e.acquired
resistancetoatleastoneagentinthreeormore antimicro-bialcategoriesamongtheeightdifferentclassesofantibiotics usedtodefineMDR.29Incontrast,astudyconductedby Sten-stromet al. inNkonkobeMunicipalityofthe Eastern Cape Province, SouthAfrica,found that theprevalence of Acine-tobacter calcoaceticuscomplex from soil and watersamples in Alice town was 41%.6 Also previous studies in Senegal,
Scotland, and La Reunion Island isolated A. baumannii as animalcolonizersin5.1%,1.2%,and6.5%,respectively.5,13,20
Thestudy alsorevealed thequantitativechangesin antibi-oticresistanceattwosamplingsites.Ingeneral,susceptibility testing showed that 98% in abattoir isolates and 90% in aquaticisolateswereresistanttoampicillinwhichisusually a non-effectiveantibioticfor thetreatment ofAcinetobacter infections.Ahighincidenceofampicillinresistanceinaquatic environments was reported.43 High resistanceto
trimetho-prim/sulfamethoxazoleinabattoir(100%)andaquaticisolates (92%) is a result of the inherent resistant of A. baumannii tothis compoundthroughthe targetprotein,dihydrofolate reductase, whichhasalowaffinity forthe drug44 andalso
toselectivepressure causedbythe useofthese antimicro-bialagentfortreatmentofinfectionsincattle.TheMARIis a good riskassessment tooland its value (nominally0.20) hasbeenappliedtodifferentiatelow-andhigh-riskregions whereantibioticsareoverused.45Thepresentstudyshowed
MARIrangingfrom0.20to0.52(meanMARI;aquatic,0.28and abattoir,0.34)whichindicatesthattheisolatesemergedfrom high-risksourcesofcontaminationandalsogivesanideaof thenumberofA.baumanniiisolatesshowingantibiotic resis-tanceintheriskzoneofthesusceptibilitystudy.Thisindicates that the study areas wheretheseisolatescame fromwere exposedtoanenvironmentofhigh-riskcontaminationfrom aregion orarea wherethereishigh antibioticuse,30
prob-ablyfrom hospitaland industrialeffluentswhichfindtheir wayinto aquaticenvironmentor the indiscriminateuseof antibioticsinthelivestockslaughteredatthe abattoir.This canleadtohighantibioticresistanceselectivepressure.46The
highresistanceratetociprofloxacin,gentamycin,and tetracy-clinewasnotexpected.Theseantimicrobialsmayhavebeen usedintheseanimalseitherasgrowthpromoter, prophylac-ticorfortreatmentincaseofabattoirisolatesandalsoending inaquaticenvironment.Thisresultreflectstheuseof antimi-crobialagentsinlivestockinSouthAfrica,astetracyclinehas longbeentheantimicrobialmostcommonlyusedinlivestock, accounting forover 30% ofthe total amount of antimicro-bial consumption.47 Furthermorethe high resistant rateto cephalosporinsmay bebecausethis antibioticisfrequently
usedinhospitalsand mighthavefoundtheirwayintothe aquaticenvironmentincaseoftheisolatesfromthedamor mighthavebeenintroducedasagrowthpromoter, prophylac-ticallyorfortreatmentoffoodanimalsthatwereslaughtered atthe abattoir. Thecurrent study showed that all isolates frombothsourcesweresusceptibletocolistinandtigecycline whicharelastlineantibiotics.Colistinandtigecyclineare nor-mallyusedasadrugoflastresortinhospitalswhichlimits thepossibilityofbacteriadevelopingresistanceagainstthese antibiotics.Also,theyarerarelyusedinanimalseitherasa growthpromoter,prophylacticallyorfortreatment.Itisalso noteworthytohighlight thefactthat therewasno statisti-caldifferenceintheresistanceratesofA.baumanniiisolates recoveredfromtheabattoirandfromtheaquaticenvironment (p<0.253). This wide range ofresistance, as demonstrated here,isacauseforconcern.
Inthisstudy,isolateswerealsotestedmolecularlyforgene similarities.Molecularanalysisofthe48and52MDRA. bau-mannii isolates showed that all isolates from both sources harboured blaOXA-51–like and 32 (66.7%) and 11 (21.2%) iso-lates harbouredblaOXA-23–like genes in abattoir and aquatic isolates, respectively. This indicates that blaOXA-23 was the mostprevalentcarbapenemhydrolyzingclassD-lactamase genefrequently foundincarbapenem-resistant A. bauman-niiisolatescollectedfrom extra-hospitalenvironments. Our studyshowedthatblaOXA-23producersinparticularand car-bapenemase producers in general, may be isolated from extra-hospital environments. Among the hypotheses that could explain the selection of this carbapenemase is the indiscriminate use of penicillins or penicillin–-lactamase inhibitorcombinations.Thiscouldcreateselectivepressure for-lactamasesbecause blaOXA-23 does confer,inaddition todecreased susceptibilitytocarbapenems, ahigh level of resistancetothosecompounds.Itiswellestablishedthat ani-malscanbereservoirsofantimicrobial-resistantbacteria.48
TheseidentifieddeterminantsarenotonlyofconcerninA. baumannii.Rather,thesegenescanbetransferredtoother bac-teriabyhorizontalgenetransferandareofgreatconcernto humanhealth.Therefore,thereistheneedtoemphasizeon strictadherence topersonalandgeneralhygienetoreduce theriskofopportunisticinfectionbyA.baumanniiwhichis difficulttocontrol.CasesofAcinetobacterinfectionhavebeen tracedtoenvironmentduetopoorhygiene,especiallyhand hygiene.1
Thecurrent study showed that all MDR isolates lacked significant antibioticresistance features,such class1 inte-gronsand ISAba1, indicating thatthe class 1integron and ISAba1associatedresistancetraitsarenotimplicatedinMDR ofenvironmentalA.baumannii.Thisisinagreementwitha studyconductedbyHamoudaetal.wherenoclass1integrons andISAba1werefoundinA.baumanniiisolatesfromrecently slaughtered animalsdestinedforhumanconsumptionand collected at major Scottish abattoirs.20 ISAab1 is
consid-eredthe firststepinresistanceevolutioninA.baumannii.31
Theabsenceoftheseresistance-driven mobileelements in these sourcessuggests that no or little antibiotic selective pressure wasappliedto thesepathogens,contrary totheir clinicalcounterpart.Thereforethehighresistancerateto car-bapenemswhichisthedrugofchoicetotreatA.baumannii infectionsinbothabattoirandaquaticisolatescanbepartly
linkedtotheincreasedexpressionlevelofblaOXA-51-like car-bapenemases.Inaddition,itisspeculatedthatISAba1attains fullexpressionunderconditionsofexcessiveselective pres-sure(presenceofcarbapenems);thus,onewouldnotexpect to find this mechanism ofresistance inan environmental isolate.14 However,threeisolates (AB4,AB28and AF3) that
expressed blaOXA-51-like in our study unexpectedly showed imipenem MICs that were lower than 4g/mL, which sug-geststhatimipenemresistanceinsomeA.baumanniistrains maybeprimarilymodulatedbygenesotherthanblaOXA-51-like. ThiscanalsobeduetotheblaOXA-51-likegenesbeingusually silentorpoorlyexpressed,49thusnotconferringaresistance phenotype. Also,the increaseduse ofcarbapenems antibi-oticstotreatfoodproducing animalsmayexplainthe high level of resistance to antibiotics of this class observed in thecurrentstudy.Thepresentresultsprovideevidencethat extra-hospital A. baumannii isolatesserve as a reservoirof multiple antibiotics resistant and hence as potential route fortheentryofMDRpathogensintohumanpopulation.Our results also suggest that the nosocomial pathogen A. bau-mannii iswell adapted todifferent environments, notonly to the hospital setting. This has very important implica-tions forhumanhealth, as infections byMDR are difficult totreatandoftenrequiresexpensiveantibiotics,long term therapy and evenmortality.Furtherstudies shouldbe per-formedascertaintheprevalenceofMDRA.baumanniiinother sources.
Conclusion
Thisstudyhasdemonstratedbymolecularidentificationthat MDRA.baumanniiisactuallypresentoutsidethehospital.The presenceofMDRA.baumanniiintheextra-hospital environ-mentmaybeathreattopublichealthconsideringthatthis may providea vectorforthe spread oftheseopportunistic pathogenintobothcommunityandhospitalsettings environ-ment.Coordinatedapproaches toreduceintegratedhuman healthrisksintheenvironmentaswellascarefulcompliance withthe WHOguidelines50on surveillance,rational
antibi-oticprescribing,andstandardtreatmentguidelinesforboth community-andhospital-acquiredinfectionswilllead appre-ciably towards reducing the ever-rising threat ofantibiotic resistance. However, further studies are needed for better understanding of the mechanism of interactions between thedifferentpotentialreservoirsandhumans.Also,possible impactonthehorizontaltransferofblaOXAgenes,surviving inselectedconditionoroccurrenceofinfectionoutside the hospitalsettingshouldbefurtherinvestigated.
Funding
Funding of the research through the National Research Foundation (NRF)Thuthukagrant UID:107619isgraciously appreciated.Beyondthis,itdidnottakepartintheresearch activitiesofknowledgecreationanddissemination.The Nel-sonMandelaAcademicHospital(NMAH)andtheMicrobiology LaboratoryattheNationalHealthLaboratoryServices(NHLS) attheNMAHarealsoacknowledged.
Availability
of
data
and
materials
Alldatageneratedoranalyzedduringthisstudyareincluded inthispublishedarticle(anditssupplementaryinformation file).
Authors’contributions
Conceivedanddesignedtheexperiments: S.S.P,T.A., S.D.V., A.Y.A. Performed the experiments: S.D.V., A.Y.A. Analysed the data: S.S.P, T.A., S.D.V, A.Y.A, O.G. E. Contributed reagents/materials/analysistools:S.S.P,O.G.E.,S.D.VAnalysed dataand wrotethe manuscript:S.S.P,T.A., S.D.V, A.Y.A.All authorsreadandapprovedthefinalmanuscript.
Ethical
approval
EthicalapprovalforthisprojectwasobtainedfromtheWalter SisuluUniversityResearchEthicsCommittee(Human) [Refer-encenumber:019/2016].
Conflict
of
interests
The authors declare that there is no conflict of interests regardingthepublicationofthispaper.
Acknowledgments
Theauthorswarmlyacknowledgethehost,WalterSisulu Uni-versityandtheco-host,UniversityofKwaZulu-Natal.
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