brazilian journal of microbiology47(2016)410–413
h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /
Medical
Microbiology
Matrix
Assisted
Laser
Desorption/Ionization
Time
of
Flight
Mass
Spectrometry
for
identification
of
Clostridium
species
isolated
from
Saudi
Arabia
Mohammed
Suliman
AlMogbel
∗MolecularDiagnosticandPersonalizedTherapeuticsUnit,CollegeofAppliedMedicalSciences,UniversityofHa’il,Hail,SaudiArabia
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Articlehistory: Received25May2015 Accepted20August2015 Availableonline2March2016 AssociateEditor:AfonsoLuísBarth
Keywords: Clostridiumspecies MALDI–TOF-MS VITEK2 16SrRNA
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TheaimofthisstudywastoidentifydifferentClostridiumspp.isolatedfromcurrencynotes fromtheHa’ilregionofSaudiArabiainSeptember2014usingMALDI–TOF-MS.Clostridium spp.wereidentifiedbyBrukerMALDI–TOF-MSandcomparedwithVITEK2.The confir-mationofthepresence ofdifferentClostridiumspp.wasperformedbydeterminingthe sequenceofthe16SribosomalRNAgene.Inthisstudy,144Clostridiumspp.wereisolated. Amongthesespecimens,MALDI–TOF-MScouldidentify88.8%(128/144)oftheisolatesto thespeciesleveland92.3%(133/144)tothegenuslevel,whereas,VITEK2identified77.7% ofthe(112/144)isolates.Thecorrectidentificationofthe144isolateswasperformedby sequenceanalysisofthe500bp16SrRNAgene.ThemostcommonClostridiumspp. identi-fiedwereClostridiumperfringens(67.36%),Clostridiumsubterminale(14.58%),Clostridiumsordellii (9%)andClostridiumsporogenes(9%).Theresultsofthisstudydemonstratethat MALDI–TOF-MSisarapid,accurateanduserfriendlytechniquefortheidentificationofClostridiumspp. Additionally,MALDI–TOF-MShasadvantagesoverVITEK2intheidentificationoffastidious micro-organisms,suchasClostridiumspp.Incorporatingthistechniqueintoroutine micro-biologywouldleadtomoresuccessfulandrapididentificationofpathogenicanddifficult toidentifymicro-organisms.
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
ManyClostridiumspp.arecapableofcausinginvasive infec-tions inhumans, someof whichcould be serious and life threatening, such as myonecrosis and bacteremia. The abilityofClostridiumspp.tocauseseriousinfections results
∗ Correspondingauthor.
E-mail:[email protected]
predominantlyfromtheproductionofharmfultoxins.1
Pro-ductionofpotenttoxinsbyClostridiumspp.,particularlybyC. botulinum,C.perfringens,C.tetaniandC.difficile,leadstosevere diseasessuchasbotulism,gasgangrene,tetanusand pseu-domembranous colitis.2–4 Clostridium spp. are fastidious in
nature,andtheirisolation,cultureandidentificationina rou-tinediagnosticmicrobiologylaboratoryarecomplicatedand
http://dx.doi.org/10.1016/j.bjm.2016.01.027
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
brazilian journal of microbiology47(2016)410–413
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timeconsuming.Thereareseveralreasonsthatthe identifi-cationofClostridiumspp.inaroutinemicrobiologylaboratory isdifficult,includingtherequirementforaspecificanaerobic system,suchasananaerobicjarfortheculture,anextended incubationperiod,andanoccasionallossofisolatesduring subculturebecauseofoxygensensitivity.Phenotypicand bio-chemicalmethodsrequire timebecausetheproceduresare lengthy,andattimes,theyfailtodistinguishbetweenclosely relatedspp.PCR-basedmolecular methodsandsequencing are expensive and difficult to use for routine diagnostic procedures,andtheyrequirecommittedtechnicalexpertise.5
Recently,manytechnological improvementsto methods fortheidentificationofmicro-organisms,suchas MALDI–TOF-MS, have successfully been incorporated in microbiology laboratoriesglobally.Comparedwithconventionalmethods, MALDI–TOF-MSisauseful,rapid,accurateandsimple tech-nique for the correct identification of micro-organisms.6
Several studies have highlighted the advantages and per-formanceofMALDI–TOF-MSincluding,rapidity,lowsample volumerequirementsandlowreagentcostscomparedwith currentlyavailablemethods. Many studies usingthis tech-nology,whichispredominantlyusedfortheidentificationof aerobicbacteria, haveled tothis technologybeingused in manyclinicallaboratoriesworldwide.Veryfewstudieshave been conducted on the use of MALDI–TOF-MS to identify anaerobicbacteria.Theaimofthisstudywastoidentifythe Clostridiumspp.obtainedfromcurrencynotesinSaudiArabia usingMALDI–TOF-MS.
Materials
and
methods
Studydesignandbacterialisolation
Inthisstudy,144Clostridiumspp.wereisolatedinsteriletubes from 320currency notes(1-Riyal)collected separately from theHa’ilregioninSeptember2014.Thenoteswerecollected insteriletubestoavoidcrosscontaminationandthen trans-ferred into newsteriletubes containing sterilebrainheart infusion(BHI)broth.Thetubeswerevortexedfor30sfollowed byincubationinashakerincubatorfor4hat37◦C.Thetubes werevortexedagainfor30sandincubatedat37◦Covernight. Thesamplesweresubculturedonbloodagar(BA)plates con-taining50gof metronidazole and 10ggentamicin discs (Oxoid,UK).Theplateswere incubated for48–72h at37◦C usinganaerobicjars(Oxoid,UK).Allcoloniesthatwere sus-ceptibletometronidazole andresistant togentamicinwere selectedandsubculturedontwoseparatebloodagarplates andincubatedaerobicallyandanaerobically.Allisolatesthat grewanaerobicallyandnotaerobicallyweredesignated anaer-obicbacteriaandwereselectedforfurtheridentification.
IdentificationofbacterialisolatesbyMALDI–TOF-MS
IsolateswereidentifiedbyMALDI–TOF-MS(BrukerDaltonics, Bremen,Germany)usingaformicacid-baseddirect,on-plate preparationmethod.6 Inthismethod,onemicroliterof70%
formic acid per well was deposited onto the MALDI–TOF MSsteelanchorplate(BigAnchor96-wellplate;Bruker Dal-tonics).Thecolonieswere spread into theformicacid and
allowed to dry. The dried mixture was overlain with 2l ofmatrixsolution(␣-cyano-4-hydroxycinnamicacid(HCCA); BrukerDaltonics),dissolvedin50%acetonitrile,47.5%water, and 2.5% trifluoroacetic acid and allowed to dry prior to analysisusingaMALDIBiotyper.AMicroFlexLTmass spec-trometer(Bruker Daltonics) wasused forthe analysis.The spectrawereanalyzedusingBrukerBiotyper3.0software.The manufacturer-recommendedcutoffscoreswereusedfor iden-tification,withscoresof≥2.000indicatingidentificationtothe specieslevel,scoresbetween1.700and1.999indicating iden-tificationtothegenuslevel,andscoresof<1.700indicatingno identification.Theisolatesproducingscores of<1.700were retestedonce,and thehighestscorewasusedforthe final analysis.
IdentificationofbacterialisolatesbyVITEK2
Additionally, the bacterial isolates were identified using a VITEK2(bioMérieux,France)accordingtothemanufacturer’s guidelinesforanaerobicidentification.
Identificationofbacterialisolatesbythe16SrRNAgene
sequence
The identification of the isolates was performed with the 16S rRNA gene sequence. The DNA of the bacterial iso-lates was extracted, and amplification of the 510bp of 16S rRNA gene wasperformed according tothe previously described method, using the universal primers 27F (5 -AGAGTTTGATCMTGGCTCAG-3) and 519R (5 -GWATTACCG-CGGCKGCTG-3).7Thesequencingofthe510bpPCRproduct
wasperformedusingtheaboveforwardandreverseprimers onanAppliedBiosystems3500GeneticAnalyzeraccordingto the manufacturer’sinstructions.Thesequenceresultswere analyzedusingGenBank(http://www.ncbi.nlm.nih.gov).
Results
TheidentificationofdifferentClostridiumspp.fromourstudy using MALDI–TOF-MS is presented in Table 1. The results showedthatof144Clostridiumspp.,MALDI–TOF-MScould cor-rectlyidentify88.8%(128/144)tothespeciesleveland92.3% (133/144)tothegenuslevel.TheClostridiumspp.identifiedat thespecieslevelandgenuslevelwereasfollows:(i)C. perfrin-gens,94.8%(92/97)oftheisolateswereidentifiedtothespecies
Table1–Identificationof144Clostridiumspeciesusing MALDI–TOF-MSatalogscore≥2.000(specieslevel)and
≥1.700(genuslevel).
Species MALDI–TOFMSatlog(score) ≥2.000 ≥1.700 Not reliable Misidentification C.perfringens 92 95 2 0 C.subterminale 17 17 0 4 C.sordelli 7 8 1 4 C.sporogenes 12 13 0 0 Total 128 133 3 8
412
brazilian journal of microbiology47(2016)410–413Table2–ComparisonoftheidentificationofClostridium speciesbyMALDI–TOF-MS,VITEK2and16SrRNA. Species MALDI–TOFMS VITEK2 16SrRNA
C.perfringens 95 78 97
C.subterminale 17 20 21
C.sordellii 8 5 13
C.sporogenes 13 9 13
Total 133 112 144
levelwithascore≥2.0,and98%(95/97)oftheisolateswere identifiedtothegenuslevelwithascoreof1.7–2.0.AmongC. perfringens,2%oftheisolates(2/97)yieldednoreliable identifi-cationwithascore<1.7.(ii)AmongC.subterminale,81%(17/21) oftheisolateswereidentifiedtothespecieslevelwithascore ≥2.0and81%(17/21)oftheisolatestothegenuslevelwitha score1.7–2.0.AmongC.subterminale,19%(4/21)oftheisolates weremisidentified.(iii)AmongC.sordelli,53.8%(7/13)ofthe isolateswereidentifiedtothespecieslevelwithascore≥2.0, and61%(8/13)oftheisolatestothegenuslevelwithascore of1.7–2.0.AmongC.sordelli,7.6%(1/13)oftheisolatesyielded noreliableidentificationwithascore<1.7,and30%(4/13)of theisolatesweremisidentified.(iv)AmongC.sporogenes,92.3% (12/13)oftheisolateswereidentifiedtothespecieslevelwith ascore≥2.0,and100%(13/13)oftheisolateswereidentified tothegenuslevelwithascore1.7–2.0.
TheidentificationofClostridiumspp.fromourstudyusing VITEK2ispresentedinTable2.Theresultsshowedthatof 144Clostridium spp., VITEK2could identify77.7% (112/144) correctly.ThefollowingClostridiumspp.wereidentified:C. per-fringens,80% (78/97),C. subterminale, 95% (20/21),C. sordelli, 38%(5/13)and C.sporogenes, 69%(9/13). Additionally,12.5% (18/144)oftheisolateswerenotidentified,and9.7%(14/144) weremisidentifiedbyVITEK2.
Byusingthesequenceanalysisof16SrRNA,alltheisolates (144/144)weresuccessfullyidentified,and76%(110/144)ofthe Clostridiumisolateswereidentifiedby16SrRNA, MALDI–TOF-MS and VITEK 2 (Fig. 1). Individually, 81% (79/97) of C. perfringenswereidentifiedbyall3methods,and16.5%(16/97) wereidentifiedbyMALDI–TOF-MSand16SrRNAonly.Among C.sporogenes,69%(9/13)wereidentifiedbyallofthemethods,
110 24 6 4 4 3 4 16S rRNA MALDI-TOF-MS VITEK 2
Fig.1–SummaryofClostridiumspp.identifiedbyBruker
MALDI–TOF-MScomparedtoVITEK2identification.The
totalnumberofisolates,n=144.
and 30% (4/13) were identified by MALDI–TOF-MS and 16S rRNAonly.Atotalof30%(4/13)oftheisolatesofC.sordelli were identifiedbyall 3methods,30% (4/13)wereidentified byMALDI–TOF-MSand16S rRNAonly,and7.7%(1/13)were identifiedby16SrRNAandVITEK2only.Ofthestrains,81% (17/21)oftheisolatesofC.subterminalewereidentifiedbyall 3methods,and29%(6/21)were identifiedby16SrRNAand VITEK2.
Discussion
The members of the genus Clostridium are found in soil, aquaticsediment, decayingplants, vertebratesandinsects. Additionally,theycompriseanessentialpartoftheanaerobic floraofhumansandarepresentingastrointestinaland vagi-nalflora.Thegenushasbeenreportedfrom othersources, suchaspapercurrencyandretailsurfaces.8,9 Thisresearch,
which is aimedat the rapidand accurate identification of Clostridium species, is crucial because of the capability of theseorganismstoproducetoxins.Thetoxinsproducedby Clostridiumspp.comprisethemajorcausesoftheinfections associated with the species, which may range from local-izedwoundinfectionstosystemiclifethreateningdiseases. The infections caused byClostridium spp. includegas gan-grene,antibioticassociatedcolitis,neutropenicenterocolitis andneurologicalsyndromes,suchastetanusandbotulism.1
Fromatreatmentperspective,itisessentialtoidentify par-ticularbacteriainaroutinemicrobiologylaboratory.Bacterial identificationisnormallybasedonphenotypictests, includ-ing morphology, Gram staining and abiochemical pattern. Althoughsomeofthesetestsareperformedrapidly,complete identification is routinely achieved withinhoursto several daysbasedonthenatureofthemicro-organism.Thus,these conventional,time-consumingproceduresdelayappropriate treatmentofpatients.
AnaerobicbacteriasuchasClostridiumspp.arecomplexin nature,andtheirisolation,cultureandidentificationina rou-tinediagnosticmicrobiologylaboratoryaretediousandtime consuming.Themostreliableandaccurateidentificationof anaerobeshasbeenattributedtosequencingofthe16SrRNA gene.Thistechniquehasbeenshowntobehighlyproficient andisconsideredthe“goldstandard”.Regardlessofthewide acceptabilityof16SrRNAsequenceidentificationofanaerobic bacteria, this techniqueremainsinapplicable formost rou-tinemicrobiologylaboratoriesbecauseitistooexpensiveand time-consumingforroutineuse.Severaltechniqueshavebeen employedforthepurposeofcorrectlyidentifyingor classi-fyinganaerobicbacteria.10 VITEK2andMALDI–TOF-MS are
twooftheautomationmethodsnowwidelyusedinroutine microbiology. Comparedwith the results ofthe traditional method,severalstudieshaveshownpromisingresultsin iden-tifyinganaerobicbacteriabyVITEK2However,forClostridium spp.identification,thissystemremainsunsatisfactory.Inour study,78%oftheClostridiumisolateswereidentifiedbyVITEK 2.Theseresultswereslightlydifferentfromthosepreviously published(44.4%and64.3%)byMoryetal.andBlaironetal.11,12
IdentificationofmicrobesbyMALDI–TOF-MShasbeeninuse formorethan adecadeandhasbeenshowntobearapid, lessexpensiveandaccuratemethodforidentifyingmicrobes,
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includinganaerobic bacteria.12,13 Inourstudy,
MALDI–TOF-MScouldcorrectlyidentify89%(128/144)ofthemicrobesto thespeciesleveland92%(133/144)tothegenuslevel.These resultsareconsistentwiththosepublishedbySchmittetal.14
Inhis study,comprising 179anaerobic bacteria, 70.8% and 91.7%wereidentifiedbyMALDI–TOF-MStothespeciesand genus level, respectively. His results further demonstrated thatamongthe179anaerobicbacteriatested,28isolatesof Clostridiumspeciesyieldedresultsof89%and93%for iden-tificationtothespeciesandgenuslevel,respectively.Garner etal.,15reporteda96%identificationratebyMALDI–TOF-MS
atboththe genusand the specieslevels of108Clostridium isolates.SeveralstudieshavecomparedMALDI–TOF-MSand VITEK2fortheidentificationofanaerobicbacteriato demon-stratewhichtechniqueismoresuitable,andmostofthese studiesclearlyconcludedthattheMALDI–TOF-MStechnique israpid,reliableandmoreaccurate.
Ourresultssuggest thatMALDI–TOF-MSperformsbetter andissuperiortoVITEK2fortheidentificationofClostridium spp.andthatitcould besuccessfullyincorporated intothe routineidentificationoftheclinicalstrainsofClostridiumspp.
Funding
ThisworkwassupportedbytheUniversityofHa’il.
Conflict
of
interest
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