Matrix Assisted Laser DesorptionIonization and Time Flight mass spectrometry

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Clinical

Microbiology

Identification

of

pathogenic

and

nonpathogenic

Leptospira

species

of

Brazilian

isolates

by

Matrix

Assisted

Laser

Desorption/Ionization

and

Time

Flight

mass

spectrometry

Daniel

Karcher

,

Rafaella

C.

Grenfell

,

Andrea

Micke

Moreno

,

Luisa

Zanolli

Moreno

,

Silvio

Arruda

Vasconcellos

_,

_Marcos

_B.

_Heinemann

_,

_Joao

_N.

_de

_Almeida

_Junior

_,

Luiz

Juliano

,

Maria

A.

Juliano

a_{UniversidadeFederaldeSãoPaulo,EscolaPaulistaMedicina,DepartamentodeBiofísica,SãoPaulo,SP,Brazil}

b_{UniversidadedeSãoPaulo,InstitutodeMedicinaTropicaldeSãoPauloLaboratóriodeMicologiaMédicaDivisãodeLaboratórioCentral}

–LIM-03,HospitaldasClínicasdaFaculdadedeMedicinadaUniversidadedeSãoPaulo,SãoPaulo,SP,Brazil

c_{UniversidadedeSãoPaulo,FaculdadedeVeterináriaeZootecnia,DepartamentoMedicinaVeterináriaPreventivaeSaúdeAnimal,São}

Paulo,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20November2016 Accepted21March2018 Availableonline13April2018 AssociateEditor:RoxanePiazza

Keywords: Leptospira Brazil Identification Massspectrometry MALDI-TOF

a

b

s

t

r

a

c

t

MatrixAssistedLaserDesorption/IonizationandTimeofFlightmassspectrometry (MALDI-TOFMS)is apowerfultool forthe identificationofbacteria throughthedetectionand analysisoftheirproteinsorfragmentsderivedfromribosomes.Slightsequencevariationsin conservedribosomalproteinsdistinguishmicroorganismsatthesubspeciesandstrain lev-els.CharacterizationofLeptospiraspp.by16SRNAsequencingiscostlyandtime-consuming, andrecentstudieshaveshownthatcloselyrelatedspecies(e.g.,Leptospirainterrogansand Leptospirakirschneri)maynotbediscriminatedusingthistechnology.Herein,wereportan in-houseLeptospirareferencespectradatabaseusingLeptospirareferencestrainsthatwere validatedwithacollectionofwell-identifiedBrazilianisolateskeptintheBacterial Zoono-sisLaboratoryattheVeterinaryPreventiveMedicineandAnimalHealthDepartmentatSao PauloUniversity.Inaddition,L.interrogansandL.kirschneriweredifferentiatedusingan in-depthmassspectrometryanalysiswithClinProToolsTM_{software.Inconclusion,our} in-housereferencespectradatabasehasthenecessaryaccuracytodifferentiatepathogenic andnon-pathogenicspeciesandtodistinguishL.interrogansandL.kirschneri.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mails:ljuliano@terra.com.br,juliano.epm@hotmail.com(L.Juliano). 1 _{Theseauthorscontributedequallytotheexecutionofthiswork.}

https://doi.org/10.1016/j.bjm.2018.03.005

Introduction

LeptospirosisisamammalianzoonosiscausedbyLeptospira strains belonging to the order Spirochaetales. Mammals, including humans, are affected bydifferent clinical mani-festations,dependingonthe virulence,motility,and ability ofthe leptospiralpathogen to surviveinthe host. Suscep-tibility to infection is dependent on age, genetic factors and skin integrity during the infection. Leptospira biology andleptospirosisphysiopathologywerecomprehensively pre-sentedanddiscussedinarecentpublication.1_Theantigenic diversityamongserovarsdifferentiatespathogenic(Leptospira interrogans) and non-pathogenic or saprophytic (Leptospira biflexa)species.2 _{Atleast22specieshavebeenclassified by} moleculartechniques.2–4_{Themicroscopicagglutinationtest} (MAT)isthemostcommonlyuseddiagnosticmethodinthe clinic; however, limitations have been previously reported anddiscussed.3,5_{ThecharacterizationofLeptospiraspp.using} moleculartechniquessuchas16SRNAsequencingiscostly andtime-consuming,6_{especiallytakingintoaccountthelarge} numberofmicroorganismsidentifiedintheclinicalpractice. Thismethoddependsononeorseveraltargetgenes,however thedataforallthepeptideswithamassrangeof2–20kDa couldbecollectedusingMALDI-TOFMSasdemonstratedby Xiaoetal.7_{onmolecularfingerprintingofpathogenicand} non-pathogenic Leptospira. MALDI-TOF MS is a well-established techniquefortherapidcharacterizationofbacteria, andits useiscontinuouslyincreasing.8_{Thistechnologycan} differen-tiatemicroorganisms’speciesbytheanalysisandcomparison ofproteinsorproteinfragmentsderivedfromribosomes.Itis importanttonotethatslightsequencevariationsinconserved ribosomalproteinsare sufficienttodistinguish microorgan-ismsatthesubspeciesandstrainlevels.8_{MALDI-TOFMShas} been proposed tobe a powerful tool for the identification ofLeptospiraatthespecieslevel.6–8,10_However,the misiden-tification ofL.interrogansas L.kirschneri byMALDI-TOFMS hasbeendescribed,and potentialbiomarkersto differenti-ate these species have been investigated.10 _{In the present} paper,wefocusedon(i) thecharacterizationofpathogenic andnon-pathogenicLeptospiraspeciesofaLeptospira Brazil-iancollectionusingMALDI-TOFMSaftercreatinganin-house databaseand(ii)the differentiationofL.interrogansfrom L. kirschneribyin-depthmassspectrometryanalysis.

Material

and

methods

Leptospirastrainsandisolates

Thirty-onereferenceleptospiralstrainsand22fieldisolates belongingtopathogenic(Leptospirainterrogans,Leptospira borg-petersenii,Leptospirakirschneri,LeptospiranoguchiiandLeptospira santarosai)andnon-pathogenic(Leptospirabiflexa)specieswere analyzed.TheLeptospiraisolateswererecoveredfrombovine, dog,human,Rattusnorvegicus,andRattusrattusurinesamples takenfromSaoPaulo,RiodeJaneiroandLondrina(Table1). ThestrainsandisolatesweremaintainedintheLaboratoryof BacterialZoonosis–SchoolofVeterinaryMedicineandAnimal Science/UniversityofSaoPaulo(USP)andstoredinFletcher

semi-solidmedium(FletcherMediumBase,DifcoTM_,NJ,USA) at30◦_{C.Thespeciesofthefieldisolateswerepreviously}

iden-tifiedby16SrRNAsequencing(datanotshown).

SamplepreparationforMALDI-TOFanalysis

The strains and isolateswere grownand diluted (1:25)for seven days at 30◦_{C in}

Ellinghausen-McCullough-Johnson-Harris medium(EMJHDifcoTM_{,NJ, USA),andbacterial cells} werecountedusingaPetroffHaussercountingchamber(HS Hausser Scientific, Horsham, PA) bydark field microscopy. Leptospira cultureswerecentrifuged at11,000×gfor10min atroomtemperature,andthepelletwaswashedtwicewith 3mLof phosphate buffered saline (PBS) and suspendedin steriledeionized watertoa finalbacterialconcentrationof 1×108_{organismspermL.Ethanol/formicacidprotein} extrac-tionwasperformedbyadditionof300␮Lofthecultureinto 900␮Lofethanol(99.8%,PA)followedvortexingand10-min ofincubation. This inactivationprocedure was followedby a 10-mincentrifugation at11,000×g atroom temperature, the supernatantwasremoved,andthepelletwas airdried untiltheethanolwascompletelyevaporated.Thisprocesswas repeatedandthematerialwasthendissolvedin30␮Lof70% formicacid(Sigma–Aldrich)followedbytheadditionof30␮L ofacetonitrile(FlukaAnalyticalSigma–Aldrich,Munich, Ger-many).Centrifugationwasperformedat11,000×gfor2min atroomtemperature.Twomicrolitersoftheclearsupernatant werespottedona384targetpolishedsteelplate(Bruker Dal-tonikGmbH,Bremen,Germany)andallowedtodry.Following this, the driedspot was overlaid with2␮Lofmatrix solu-tion,asaturatedsolutionof␣-Cyano-4-hydroxycinnamicacid (HCCA,99%BrukerDaltonikGmbH,BremenorSigma–Aldrich, Munich,Germany)(10mg/mL)inacetonitrile(FlukaAnalytical Sigma–Aldrich)and0.1%trifluoraceticacid(1:2)(TFA-Reagent PlusW99%,Sigma–Aldrich).Finally,sampleswereallowedto dry atroomtemperature.Escherichia coliDH5␣ wasusedas apositivecontrol,andanon-inoculatedmatrixsolutionwas used asa negativecontrol.During data acquisition,it was observedthatsomeisolatesunderwentosmoticlysisin deion-izedwater,whichwascorrectedbyreplacingsteriledeionized waterbysalinesolution(0.85%NaCl)bufferedwithSorensen’s solution(69mMNa2HPO4/8mMNaH2PO4,pH7.6).9This solu-tion has lower osmolarity than PBS, but kept cells intact withoutinterferingwiththeionizationofthebacterial pro-teinsaswellasthemassfingerprintofourpreviouslydata thatweregeneratedinsalinesolution.Additionalmass spec-trawerethenobtainedwithfreshculturepassagestoensure theminimumnumberofspectraforthegenerationofsingle MainSpectrumProfiles(MSP).

InstrumentsettingsforMALDI-TOFMSanalysis

Table1–LeptospirastrainsusedasreferenceforMALDI-TOFMSmeasurements.

Specie Serogruop Serovar Strain Pathogenicity

Leptospiraborgpetersenii Ballum Castellonis Castellon3

Pathogenic

Leptospiraborgpetersenii Celledoni Whitcombi Whitcombi

Leptospiraborgpetersenii Javanica Javanica VeldratBatavia46

Leptospiraborgpetersenii Mini Mini Sari

Leptospiraborgpetersenii Sejroe Hardjo Hardjobovis

Leptospiraborgpetersenii Tarassovi Tarassovi Perepelitsin

Leptospirainterrogans Australis Australis Ballico

Leptospirainterrogans Australis Bratislava JezBratislava

Leptospirainterrogans Autumnalis Autumnalis AkiyamiA

Leptospirainterrogans Bataviae Bataviae VanTienen

Leptospirainterrogans Canicola Canicola HondUtrechtIV

Leptospirainterrogans Djasiman Sentot Sentot

Leptospirainterrogans Hebdomadis Hebdomadis Hebdomadis

Leptospirainterrogans Icterohaemorrhagiae Copenhageni M-20

Leptospirainterrogans Icterohaemorrhagiae Icterohaemorrhagiae RGA

Leptospirainterrogans Pomona Kennewicki Fromm

Leptospirainterrogans Pomona Pomona Pomona

Leptospirainterrogans Pyrogenes Pyrogenes Salinem

Leptospirainterrogans Sejroe Hardjo Hardjoprajitno

Leptospirainterrogans Sejroe Wolffi 3705

Leptospirakirshneri Autumnalis Butembo Butembo

Leptospirakirshneri Cynopteri Cynopteri 3522C

Leptospirakirshneri Grippotyphosa Grippotyphosa MoskovaV

Leptospiranoguchi Panama Panama CZ214K

Leptospirasantarosai Shermani Shermani 1342K

Leptospirabiflexa Andamana Andamana CH11

Non-pathogenic

Leptospirabiflexa Andamana Bovedo Bovedo

Leptospirabiflexa Doberdo Rufino RPE

Leptospirabiflexa Garcia Garcia Garcia

Leptospirabiflexa Nazare Nazare Nazaré

Leptospirabiflexa Seramanga Patoc Patoc-1

CollectionattheBacterialZoonosesLaboratory,DepartmentofVeterinaryPreventiveandAnimalHealthofSchoolofVeterinaryMedicineand AnimalScience,SãoPauloUniversity,Brazil.

Allspectrawere analyzedbystandardpattern-matching algorithmusingtheMALDIBiotyperTM _{3.1software(Bruker}

Daltonics), and the raw spectra were compared with the reference spectra of the Bruker library (database version 3.3.1, 5627reference spectra)with defaultsettings. TheID criteria used was the recommended by the manufacturer: – a score ≥2.000 indicated species, a score between 1.700 and 1.999 indicated genus level and a score <1.700 was interpreted as no ID. For MainSpectra (MSP) and dendro-gramconstruction,flat-liners and bad qualityspectrawere removedand additionalmeasurementswere carriedout to obtain20spectrafromeachisolate/strain.Spectrawerethen loaded into BiotyperTM _{3.1 software (Bruker Daltonics) for}

MSPcreationand dendrogramclustering construction with thedefaultsettings(distance measure:correlation;linkage: average; scoreoriented). Each spot was measured in 1000-shotstepsforatotalof4000lasershots.Preparationofthe BTSandcalibrationwereperformedfollowingthe manufac-turer’sinstructions:calibrationwassuccessfulwhenproteins of the mass spectra were in a range of ±200 parts per million(ppm).

In-housedatabaseanddendrogramconstruction

Foreachofthe31strains,30individualspectrawereusedto createaMSP.Flat-linersandbadqualityspectrawereremoved, and additional measurements were carried out to obtain 30 spectrafrom each isolate/strain.TheMSP wasobtained usingMALDI-Biotypersoftware(BrukerDaltonics,Germany) andthenloadedintotheBrukerDaltonicsdatabase(version 3.1.2.0).SoftwaresettingsforMSPcreationweresetto max-imalmasserrorofeachsinglespectrum:2000;desiredmass errorfortheMSP:200;desiredpeakfrequencyminimum(%): 25;andmaximaldesiredpeaknumberoftheMSP:70. Den-drogramclusteringwasconstructedwiththedefaultsetting of160(distancemeasure:correlation;linkage:average;score oriented).

Determiningtheefficiencyofthedatabasesearchwith Leptospirafieldisolates

wereexpressedinlogscorevalues,withvalues≥2 indicat-ingreliablespeciesidentificationandvaluesfrom1.7to2.00 indicatingreliablegenusidentification.

DifferentiationofLeptospirainterrogansandLeptospira

kirschneriusingClinProToolsTM

ClinProToolsTM _{(BrukerDaltonics)generatesmultiple} math-ematicalalgorithmstogeneratepatternrecognitionmodels for the classification and prediction of different classes (e.g., L. interrogans class 1, L. kirschneri class 2) from mass spectrometry-basedprofilingdata.Variousspectraofthe dif-ferentserovars(03serovartoL.kirschneriand12 serovarto L.interrogans)wereusedforeachclass,seekingto standard-izethedataforspeciesdistinction.Moreover,ClinProToolsTM provides a list of peaks sorted according to the statistical significance to differentiate between both classes.12 _Thus, torecognizemassspectrapatternsandbiomarkersbetween L.interrogansandL.kirschneri,spectrapeakanalysismodels with ClinProToolsTM _{software v.3.0 (Bruker Daltonics) were} created from anadditional 210mass spectraof11 L. inter-rogans (15 high-quality mass spectra per isolate) and 3 L. kirschneri(15high-qualitymassspectraperisolate)isolates. Spectrawerepretreatedwitharesolutionof800ppm,amass rangeof2000–20,000Da,atophatbaselinesubtractionwith 10%minimalbaselinewidth,enablingnullspectraexclusion, recalibrationwith500ppmmaximalpeakshiftand30%match celebrant peaks. ClinProToolsTM _{models (Bruker Daltonics)} were generated using three algorithms: Genetic Algorithm (GA),SupervisedNeuralNetwork(SNN),andQuickClassifier (QC).Foreachmodel,therecognitioncapability(RC)andcross validation(CV)percentageweregeneratedtodemonstratethe reliabilityandaccuracyofthemodel.RCandCVpercentages areindicatorsofthemodel’sperformanceandserveasuseful predictorsofthemodel’sabilitytoclassifytestisolates.We alsocarriedoutprincipalcomponentanalysis(PCA)included inClinProToolssoftwareaimingtovisualizehomogeneityand heterogeneityofthe proteinspectraofL.interrogansand L. kirschneri.Principalcomponentanalysis(PCA)andtheresults wereshownin3Dscoreplot.

Single-peakanalysis

Foreachpeak,theAUCforthediscriminationofthegroups wasdirectlyobtainedfromtheClinProToolsTM_{v.3.0software} (BrukerDaltonics).ForthefivepeakswiththehighestAUC,the detection performanceswere verified usingFlexAnalysisTM v.3.4(BrukerDaltonics). Aftersmoothing and baseline sub-traction,themasslistsforeachisolatewereobtainedusing thecentroidalgorithmwithasignal-to-noise(SN)threshold of0.5andamaximumof500peaksandexportedtoMicrosoft Excel.TheSNratiosofthepeakswithatoleranceof1000ppm were exported toSPSS version 18.0.ROC (Receiver Operat-ingCharacteristic)curveswereconstructed,andtheiroptimal cutoffvalueswere determined withthemaximum Youden index.11

Results

Referencespectrawerecreated forall31 leptospiralstrains andappliedasunassignedMSPsinthecommercially avail-ableMALDIBiotyperTM_{databasespectralibrary,whichlacks} leptospiralproteinprofiles(Fig.1).TheMSPswereclustered accordingtopathogenicityintheMALDI-TOFMSdendrogram, and the pathogenic species (red) are clearly differentiated fromthenonpathogenicLeptospiraspecies(green)(Fig.2). Sim-ilarly, the pathogenic L. borgpeterseniiand L. interrogansare locatedinseparateclusters,but,asexpected,poor discrim-inationwasobtainedforL.interrogansandL.kirschneri.

RepresentativemassspectraofL.interrogansand L. borg-peterseniiobtainedbydirectanalysisandbyproteinextraction protocolareshowninFig.1.InAandC,massspectrawere obtainedwithoutproteinextractionandpeakswithlow inten-sitywereobserved.Incontrast,BandDshowhigherquality massspectraobtainedafterproteinextractionprotocol,with peakswithhigherintensity.

The 22 field isolates belonging to L. biflexa, L. interro-gans and L. santarosai had the correct species assigned by MALDI-TOFMS,andallisolatesshowedscorevaluesover2.0 (Table2),whereit ispossibletoidentifyall isolatesbythe correctspeciesIDfollowingourin-housedatabase. ThePCA reproduces through different statistical teststhe reduction ofseveral variablesofaset ofdata,whereeach point rep-resentsaspectrumandeachcolorrepresentsagroupingof similardata.Fig.3ApresentsthePCAforL.interrogansspecies inredandL.kirschneriingreen,thereisaperceptible distinc-tion betweenthetwogroupsevenwithsomecloserpoints showing that the PCAanalysis does not guarantee aclear separationbetweenthespecies.BpresentsthePCAforthe serovarsthatformedtheclassL.kirschneriinA,aclear sepa-rationbetweentheserovarsisobserved.CpresentsthePCA fortheserovarsthatformtheclassL.interrogans,whichshows thatthegrouprepresentingL.interrogansserovarBataviaecan becompletelyseparated,sincetheotherclusterscannotbe separated.

The three classification models from ClinProToolsTM showed RCvalues ≥90%inthe discrimination ofL. interro-gansandL.kirschneri.Thebestresultswereprovidedbythe GAmodel,withRCandCVvaluesof100%and97%, respec-tively.DetailsofthesevaluesareshowninTable3.Thestrain distributionmapsbasedontheGAmodelshowthatL. inter-rogans andL.kirschnericanbedistinguishedbased ontheir peptide massfingerprints,thebest separatingpeaksofthe currentstatisticsortorderaredisplayedinFig.4.

A

B

C

D

8000 8000

L. borgpetersenii serovar castellonis strain castellon 3 (with extraction) L. borgpetersenii serovar castellonis strain castellon 3 (without extraction) L. interrogans serovar autumnalis strain akiyami A (with extraction) L. interrogans serovar autumnalis strain akiyami A (without extraction)

8000

8000

8000 0

0

0

0 2000

2000

2000

2000

2000 3000

4000

4000

4000

4000

4000 5000 7000 9000 10000 11000 m/z

6000

6000

6000

6000

6000

Intens

. [a.u.]

Fig.1–MALDI-TOFMSspectraobtainedbyanalyzingthereferencestrainsofLeptospirainterrogansandLeptospira

borgpeterseniiwithandwithoutextractionasdescribedin“Materialandmethods”section.Thesedatashowtheimportance oftheproteinextractiontoobtainthebetterqualityofspectra.

MSP dendogram

Leptospira biflexa sg. saramanga sv. patoc strain patoc 1 Leptospira biflexa sg. andamana sv. bovedo strain bovedo

Leptospira kirchneri sg. grippotyphosa sv. grippotyphosa strain moskva V Leptospira interrogans sg. autumnalis sv. autumnalis c. akiyami A

Leptospira kirschneri sg. cynopteri sv. cynopteri strain 3522 C Leptospira kirschneri sg. autumnalis sv. butembo strain butembo Leptospira interrogans sg. australis sv. bratislava strain jez bratislava Leptospira interrogans sg. australis sv. australis c. ballico

Leptospira interrogans sg. canicola sv. canicola strain hond uthecht IV Leptospira interrogans sg. pomona sv. pomona strain pomona Leptospira interrogans sg. hebdomadis sv. hebdomadis strain hebdomadis Leptospira interrogans sg. icterohaemorrhagiae sv. copenhageni strain M 20 Leptospira interrogans sg. sejroe sv. hardjo c. hardjoprojitno

Leptospira interrogans sg. sejroe sv. wolfii c. 3705 Leptospira noguchii sg. pamana sv. pamana strain CZ 214K Leptospira borgpetersenii sg. celledoni sv. whitcombi strain whitcombi Leptospira borgpetersenii dg. mini sv.mini strain sari

Leptospira borgpetersenii sg. javanica sv. javanica strain veldrat batavia 46 Leptospira santarosai sg. shemani sv. shemani c. 1342 k

Leptospira biflexa sg. andamana sv. andamanda strain ch-11

Distance level

0 100 200 300 400 500 600

0 0

2

2

0 10

PC1 -10

-20 -2

4

-8 -6

-6 6

-4

-4 4

A

C

5

0 0

PC3

PC3

PC3

PC2 PC2

PC1 PC1

10

10

-10 5

5

-5 -5

-5 -3 -2 -1 0 1 2 3 4

-2

-2 -6

-6 6

-4

-4 4

0

0 0

2

2 4 6 8

B

Leptospira interrogans

Leptospira interrogans sejroe

Leptospira interrogans pyrogenes

Leptospira interrogans pomona

Leptospira interrogans bataviae

Leptospira interrogans copenhageni

Leptospira interrogans autumnalis

Leptospira interrogans bratislava

Leptospira interrogans australis

Leptospira interrogans hebdomadis

Leptospira kirschneri

Leptospira kirschneri autumnalis

Leptospira kirschneri cynopteri

Leptospira kirschneri grippotyphosa

Table2–Identificationresultsof22leptospiralfieldisolatesbyMALDI-TOFMSand16SrRNAgenesequencing.

Strainidentification Genomespecies(16SrRNAIdentification) Serogroup MALDI-TOF-MSIdentification

Species Scorevalues

Ranarum L.biflexa Semaranga L.biflexa 2.355

M85/06 L.interrogans L.interrogans 2.565

M46/07 L.interrogans Icterohaemorrhagiae L.interrogans 2.070

M67/07 L.interrogans Icterohaemorrhagiae L.interrogans 2.535

M71/07 L.interrogans Icterohaemorrhagiae L.kirschneri 2.643

M5/90 L.interrogans Icterohaemorrhagiae L.interrogans 2.342

M64/06 L.interrogans Icterohaemorrhagiae L.interrogans 2.542

61H L.kirschneri Pomona L.kirschneri 1.898

M110/06 L.kirschneri L.kirschneri 1.866

16CAP L.meyeri Grippotyphosa L.meyeri 2.828

19CAP L.meyeri Grippotyphosa L.meyeri 3.000

LO9 L.santarosai L.santarosai 2.574

M52/08-12 L.santarosai L.santarosai 2.359

M52/08-19 L.santarosai L.santarosai 1.833

U160 L.santarosai L.santarosai 2.017

U164 L.santarosai Tarassovi L.santarosai 2.093

An776 L.santarosai Bataviae L.santarosai 2.366

10ACAP L.santarosai Grippotyphosa L.santarosai 2.525

6BCAP L.santarosai Grippotyphosa L.santarosai 2.457

21CAP L.santarosai Grippotyphosa L.santarosai 2.614

M4/98 L.santarosai Sejroe L.santarosai 2.370

BOV6 L.santarosai Sejroe L.santarosai 2.434

CollectionattheBacterialZoonosesLaboratory,DepartmentofVeterinaryPreventiveandAnimalHealthofSchoolofVeterinaryMedicineand AnimalScience,SãoPauloUniversity,Brazil.

Table3–Completeresultsderivedfromtheclassificationmodels.

Classificationmodel Crossvalidation(CV)(%)Recognitioncapability(RC)(%) Integrationregionsusedforclassification

Peak#1(Da)Peak#2(Da)Peak#3(Da)Peak#4(Da)Peak#5(Da)

GAa _{97.2 100.0 8057 4671 5472 8084 8305}

SNNb _{55.6 100.0 8057 8097 6710 8084 12,679}

QCc _{92.6 93.7 8057 – – – –}

ResultsobtainedbyanalyzingofClinProTools. a _{GeneticAlgorithm.}

b _{SupervisedNeuralNetwork.} c _{QuickClassifier.}

andsingle-peakanalysisresultsforthe differentiationofL. interrogansfromL.kirschneriare summarizedinTable4and exemplifiedinFig.5.

Discussion

Duringleptospirosisoutbreaks,Leptospiraspecies identifica-tion is an essential step for tracking and controlling the pathogentransmission.Thedeterminationofaserovarmay be insufficient as different species may have the same serovarbut may be distinct intheirability tocause mam-malianinfection.13_{DNAsequencingisanalternativemethod}

Table4–Single-peakanalysisforthediscriminationofL.kirschneriandL.interrogans.

Peaks(m/z) ClinProTools FlexAnalysis Sensitivity(%) Specificity(%)

AUCa _Daveb _PWKWc _PADd _Avee _Avef _AUCg _Cutoff

8059 0.99 3.68 0 0.00226 16.72 20.63 0.99 6.96 98 95

3090 0.92 2.90 <0.0001 0.0000017.81 7.51 0.74 3.93 96 48

3074 0.91 4.14 <0.0000 <0.0000017.13 8.04 0.83 5.41 100 59

3118 0.90 12.03 <0.0001 <0.0000018.38 9.47 0.83 14.00 100 56

6710 0.87 2.85 0 <0.00000114.26 16.36 0.87 3.22 85 86

PeakswiththebestperformancesaccordingtoClinProToolsTM_{andFlexAnalysis}TM_softwares. a _{AUC,areaunderthecurve.}

b _{Dave,differencebetweenthemaximalandtheminimalaveragepeakarea/intensityofthegroups.} c _{PWKW,p-valueofWilcoxon/Kruskal–Wallistest(range:0–1;0Dgood).}

d _{PAD:p-valueofAnderson–Darlingtest,<0.05indicatesdatanotnormallydistributed;givesinformationaboutthenormaldistribution(range:} 0–1;0=notnormallydistributed).

e _{Ave,area/intensityaverageofagroupfromLeptospirakirschneri.} f _{Ave,area/intensityaverageofagroupfromLeptospirainterrogans.}

g _{AUCsandsignal-to-noisecutoffvalueswereobtainedfromanROCcurveconstructedusingSPSSVersion18.0andFlexAnalysis.}

byothercentersthatalsoconstructedinhouseLeptospiraMSP databases.Moreover,allfieldisolateshadthecorrectspecies assigned,withscores above2.0,which ensuresthe quality ofour MSP database forLeptospira species ID.The distinc-tionofL.interrogansandL.kirschneriusingClinProToolsTM_and single-peakanalysisisalsonoteworthy.AlthoughMALDI-TOF MShasalreadybeensuccessfullyappliedinLeptospiragenus andspeciesidentification,6–8 _{themisidentificationofclosely} relatedspecies,suchasL.interrogansandL.kirschneri,hasalso beenreportedandrepresentsanimportantchallengeinthe implementationofMALDI-TOFMSforLeptospiraidentification. Hereweobservedthatwithproperanalysis,Leptospiraspecies canbedistinguishedbasedontheirpeptidemassfingerprints. The ClinProToolsTM _{software is a biomarker analyzer} that has been widely applied in microbiology, providing a rapidandcost-savingmethodforepidemiologicalclustering, strain typing and subspeciesidentification.14–16 _{Using both} ClinProToolsTM_{andsingle-peakanalysiswithFlexAnalysis}TM has provided higher discriminatory power to detect bio-markerpeaks.14–17 _{Ourresultscorroboratepreviousfindings} thatoneisolatebiomarkerwith8000–8100Dacaneffectively distinguishthe closelyrelatedpathogenicspeciesL. interro-gansfromL.kirschneri.10_{Indeed,wefurtherdescribedtheSN} cut-offvaluethathastobeadoptedtoaccuratelydifferentiate thesetwotaxabyasimpleinspectionofthemassspectrum. Recently,L.kirschneriserovarMosdokwas,forthefirsttime, linkedtohumanleptospirosisinthe southernhemisphere; therefore,rapidspeciesIDusingMALDI-TOFMSmaybethe firststeptoimplementcontrolstrategies.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest,evenduringthe itemproofs.

Pk 6,711 Da

Pk 8,057 Da

8.0 9.0

0.0 0.0 1.0

1.0 2.0

2.0 3.0

3.0 4.0

4.0 5.0

5.0 6.0

7.0

6.0 7.0 0.5

0.5 1.5

1.5 2.5

2.5 3.5

3.5 4.5

4.5 5.5

5.5 6.5

6.5 7.5

8.5

Fig.4–StraindistributionmapcorrespondingtoLeptospira interrogans(red)andLeptospirakirschneri(green).Thex-axis showsthepeakarea/intensityvalueswithrespecttothe mostrelevantpeak(8057Da)todistinguishLeptospira interrogans(red)fromLeptospirakirschneri(green).The

y-axisshowsthepeakarea/intensityvalueswithrespectto thepeakwith(6711Da)fromLeptospirainterrogans(red)and

Leptospirakirschneri(green).Theellipsesrepresentthe spectrawithgreaterdistinctionbetweenthetwogroups, whereasprominentpeaksinthex-axisandy-axis.

Acknowledgements

0

0 1000

1000 2000

7950 8000 8150 _m/z

Leptospira interrogans

Leptospira kirschneri

8250 8200

8100 8050

200 400 600 800 3000 4000

8098

Intens

. [a.u.]

Intens

. [a.u.]

8059

Fig.5–RepresentativespectraofLeptospirainterrogansandLeptospirakirschineri.Therepresentativepeaksthatallow differentiationofthestrainsinthespectraareshowed,inredforL.interrogans,andinblueforL.kirschneri.Thepeakwith

m/z=8059inL.interroganswedetectedasshowninTable3byClinProToolsanalysis.Thepeakm/z=8098inL.kirschineri

waspreviouslyidentifiedbyRettingeretal.10

eTecnologico(CNPq—Projects– 443978-2014-0and 467478-2014-7).L.Z.M.isrecipientofaPhDfellowshipfromFAPESP (2013/17136-2).

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