Novel nonsense mutation in the katA gene of a catalase-negative Staphylococcus aureus strain

brazilian journal of microbiology47(2016)177–180

ht t p : / / w w w . b j m i c r o b i o l . c o m . b r /

Medical

Microbiology

Novel

nonsense

mutation

in

the

katA

gene

of

a

catalase-negative

Staphylococcus

aureus

strain

夽

Jaime

Lagos

_,

_Pedro

_Alarcón

_,

_Dona

_Benadof

_,

_Soledad

_Ulloa

_,

_Rodrigo

_Fasce

_,

Javier

Tognarelli

_,

_Carolina

_Aguayo

_,

_Pamela

_Araya

_,

_Bárbara

_Parra

_,

_Berta

_Olivares

_,

Juan

Carlos

Hormazábal

_,

_Jorge

_Fernández

a_{MolecularGeneticsSubdepartment,Chile} b_{VirologySubdepartment,Chile}

c_{InfectionDiseaseSubdepartment,Chile}

d_{PublicHealthInstitute,ClinicalLaboratory,HospitalRobertodelRío,Chile}

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Articlehistory:

Received4November2014 Accepted8August2015

AssociateEditor:AgnesMarieSá Figueiredo Keywords: Staphylococcusaureus Catalase-negative Catalasegene katA

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We reportthefirstdescriptionofararecatalase-negativestrainofStaphylococcusaureus

inChile.Thisnewvariantwasisolatedfrombloodandsynovialtissuesamplesofa pedi-atricpatient.Sequencinganalysisrevealedthatthiscatalase-negativestrainisrelatedto ST10strain,whichhasearlierbeendescribedinrelationtoS.aureuscarriers.Interestingly, sequenceanalysisofthecatalasegenekatArevealedpresenceofanovelnonsensemutation thatcausesprematuretranslationaltruncationoftheC-terminusoftheenzymeleading toalossof222aminoacids.Ourstudysuggeststhatlossofcatalaseactivityinthisrare catalase-negativeChileanstrainisduetothisnovelnonsensemutationinthekatAgene, whichtruncatestheenzymetojust283aminoacids.

©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Staphylococcusaureus,oneofthe mostimportantpathogens known to mankind, is a prominent member of the genus

Staphylococcus. Most members of this genus are catalase-positivewithnotableexceptionsbeingS.saccharolyticusandS. aureussubsp.anaerobius,bothofwhicharecatalase-negative andareknowntogrowvigorouslyinanaerobicconditions.1,2

Presence or absence ofcatalase enzymeis widely used to facilitateidentificationoftheorganismatthegenusleveland someauthorshaveevensuggestedthatcatalasemightbean

夽_{Sponsorships:PublicHealthInstituteofChile.}

∗ _{Correspondingauthorat:SubdeptoGenéticaMolecular,InstitutodeSaludPúblicadeChile,Marathon1000, ˜Nu ˜noa,Santiago,Chile.}

E-mail:jofernand@gmail.com(J.Fernández).

importantcontributortovirulencegivenitsabilityto decom-pose hydrogen peroxide, a reactive oxygen intermediate indispensable for the bactericidal activity ofphagocytes.3,4

Althoughseveralcasesofhumaninfectioncausedby catalase-negativeS.aureus(CNSA)havebeenreported,4–7_themolecular

basisforlossofcatalaseactivityremainspoorlystudied. Anumberofstudieshavesuggestedthatthelossof cata-laseenzymaticactivityislinkedtoeithera5-bpdeletionor apoint mutationinthe katAgene.7–9 _{Inthe presentstudy,}

http://dx.doi.org/10.1016/j.bjm.2015.11.012

1517-8382/©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

178

wereport the isolation and characterization ofa catalase-negativeclinicalstrainofS.aureuscollectedfrombloodand synovialtissuesamplesofapediatricpatient.

A 2-year and 7-month old child complaining of a sore knee was admitted to the Hospital Pediátrico Roberto del RíoinSantiago,Chile.Thepreliminarydiagnosiswasoneof the infectious arthritis; therefore, both blood and synovial tissuesampleswereanalyzedusingroutinemicrobiological techniques.10

The isolated Staphylococcal strain was received at the PublicHealthInstituteofChileandanalyzedasperroutine microbiological laboratory protocols.10 _{The studies}

under-takenincluded ananalysisofspecificculturingconditions, macroscopic and microscopic appearance as well as cata-lase activity assay.10 _{Antibiotic susceptibility testing was}

conductedonMueller-Hinton agar usingthe disk diffusion method as per guidelines issuedby CLSI.11 _{The 16S rRNA}

gene of the novel strain was amplified by PCR using the primerpair:Belt45CGGTCGACAGAGGTTTGATCCTGGCTCAG 3 and 1500R 5 GGTTACCTTGTTACGACTT 3, as described earlier in the literature.12,13 _{MLST was performed using a}

standardizedinternational scheme asdescribed byEnright etal.14ForamplificationofthecompletekatAgenesequence, the previously enumerated primers, cat1 and cat2, were employed.15 _{Amplicons of the 16S rRNA, katA and MLST}

housekeepinggeneswerepurifiedandsequencedonanABI 310 DNA automated sequencer (Applied Biosystems). For sequencing the complete katA gene sequence, the follow-ing set ofprimers was used: (a) cat1; (b)cat2; (c) CAT-FW 5 GTGCCCGAGCAACACCCACCCATTACA3;and(d)CAT-RV5 TCAGCGCACGTCGAACCTGTCGAG3. All the sequence data generated were assembled and edited electronically using Bioedit7.2.0andChromasLite2.1.1software.DNAsequences of the housekeeping genes were submitted to the MLST database (http://saureus.mlst.net/) in order to obtain the sequencetype(ST).

Theisolatedstrain(denominatedCHI-2609)wasgrownon 5%sheepblood trypticasesoyagar (TSA) underboth aero-bicaswell asanaerobic conditions.Positive cultureresults wereobtainedunderaerobicatmosphericconditions.Opaque, smooth, creamy and ␤-hemolytic golden-yellow colonies wereobservedfollowingovernightincubationintheculture medium.Gramstainingoftheculturepreparationsshowed thattheisolatedstrainwascontainingofGram-positivecocci clusters.Interestingly,the catalasetests,boththe 3%H2O2

slidetest aswell asthe nutrient broth tubetest with30% H2O2,wererepeatedlynegative.Theisolatewaspositivefor

coagulaseactivitywhentestedusingtherabbitplasma coag-ulasetest.CHI-2609differsfromS.saccharolyticusandS.aureus

subsp. anaerobius by virtue of its clumpingfactor, positive ureaandVogesProskauertestaswellaswithrespecttoacid productionfromtrehalose,mannose,sucroseandmaltose.10

CHI-2609 strain is also deficient in the ability to ferment mannitol,andissensitivetomethicillin,vancomycin, eryth-romycin,andclindamycin.

Toconfirmthattheisolatedstrain,CHI-2609,wasS.aureus

subsp.aureus,nucleotideanalysisofthe16SrRNAgenewas conducted.Theresultsdemonstrated100%sequenceidentity withthe16SRNAsequenceofATCC12600(GenBank acces-sionnumberAJ000472)typestrain.TheMLSTtypingrevealed

anewalleleandSTthathadneverbeenreportedearlier.The novelSTwassubmittedtotheMLSTdatabaseandwasgiven thedesignationST3145.TherelationshipbetweenST3145and previouslyknown MLSTdatabase STs wasexamined using concatenatedsequencesofthesevenMLSTlocisoastoenable theconstructionofaneighbor-joiningtree(datanotshown). ST3145 was observed tocluster with ST10, ST145, ST1162, ST1936,ST1951andST2289,alloftheseareknowntodiffer from eachotheratonlyasingleMLSTlocus.ST10has pre-viouslybeendescribedbySakwinskaetal.,16_andBlumental

etal.,17_{inS.aureuscarriers.}

In ordertodecipher the mechanismresponsible forthe lossofcatalaseactivity,thecompletekatAgeneofCHI-2609 was amplified and sequenced (Genbank accession num-berKM036425).Nucleotidesequenceanalysisdemonstrated 98% identity with the corresponding sequence of the S. aureussubsp.aureustypestrainATCC12600(GenBank acces-sion number AJ000472). The two sequences differed in 30 nucleotidesofwhich25weresynonymousmutations(Table1). Oftheremaining5nucleotides,4weremissensemutations identifiedas:(a)C322TsubstitutionleadingtoaR108Cchange; (b)C923TsubstitutionleadingtoaT308Mchange;(c)C1165T substitutionleadingtoaH389Ychange;and(d)A1442G sub-stitutionleadingtoaK481Rchange.Ofgreatinterestwasthe 5th mutation:asingle-basesubstitution ofG>Aatposition 852,whichresultedinaTGGtoTGAalterationatthe284th codonleadingtoaprematureterminationofthetranslation. Theamplificationandsequencingstepswererepeatedthrice so astoconfirmthe presenceofthe unusualG852A muta-tionbeyonddoubt.SequenceidentitybetweentheCHI-2609

Table1–Nonsynonymousandsynonymousnucleotide substitutionofkatAgene.

Synonymous Nonsynonymous Aminoacidchange

T30A C322T R103C T33A G852A a A75G C923T T308M G78A C1165T H389Y T90A A1442G K481R T93A C110T T114C T302A G318A G321A T357A G366A A393G T421C G444T G447T A480T T507G A546G T576C T594A T712C T882G C1164T a _Stopcodon.

brazilian journal of microbiology47(2016)177–180

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catalasegeneandthekatAsequencesofS.aureusMSSA476, COL, NCTC 8325 and USA300 was 99.6% for the protein sequence and between 98 and 98.2% for the nucleotide sequence.

Catalaseproductionisabiochemicalfeaturethathas tradi-tionallybeenassociatedwithStaphylococcusspp.sincelong.It isuniversallyusedasascreeningtesttodistinguishbetween twomajorspeciesofGram-positivecocci:Staphylococcusand

Streptococcus.Inthecaseofanatypicalisolatesuchasours, useofthecatalasetestmayleadtoanincorrect identifica-tion,especiallywhentheabilitytofermentanimportantsugar suchasmannitolisabsentasdescribedbyShittuetal.,18_and

Kateeteetal.19

Catalase is an oxidoreductase that allows bacteria to withstandintracellularhydrogenperoxide.Productionof cata-lase has been hypothesized to be a virulence factor in S. aureusbut itsexactrole inthatrespectneedstobefurther elucidated.8,20_{Staphylococciwithacatalase-negative}

pheno-typewerethoughttobelessvirulentbutseveralcasesofCNSA bacteremia,whichhaveonoccasionprovenfatal,havebeen reported.5

TheC-terminalregionofcatalaseappearstobeessential forenzymatic activity. Thestudies carried out on the Bac-teroidesfragilisandS.aureussubsp.anaerobiuscatalasegenes havedemonstrated thatcatalyticactivity isobservedto be lostwhen thelast 21and 50 aminoacids,respectively, are deleted.21 _{Theoccurrenceofaprematuretranslation}

termi-nationinthekatAgeneofS.aureussubsp.aureushasbeen reportedintwostudies.Toetal.4_{foundamutationat}

posi-tion 802whichchangedthe GAA codontoTAAwhichis a stopsignalfortranslation.Ellisetal.7_{reportedtheinsertionof}

thymineafterposition31whichalsoleadstotheinsertionofa prematurestopcodonatposition64.Inourstudy,onCHI-2609 isolates,theinsertionofaprematuretranslationtermination codoninthe katAgeneresultedinan849-bp openreading framethat encoded apolypeptide ofjust 283 aminoacids suggestingthatthelossofthelast222aminoacidsofthe C-terminusgeneareresponsibleforthelackofcatalaseactivity. Toconclude,ourstudyisthefirstreportofarare catalase-negativestrainofS.aureusinChile.Inaddition,wehavealso identifiedahithertounreportedprematuretranslational trun-cationofcatalasethatleadstoalossof222aminoacidsfrom theC-terminus.Inspiteofthepossibilitythatcatalasecould functionasapotentialvirulencefactor,itappearsinourcase thatlackofenzymaticactivitydidnotimpairtheabilityofthe isolatetocausesepticarthritis.Finally,itisimperativethat clinicalmicrobiallaboratorystaffmustbemadeawareofthe existenceofsuchcatalase-negativeS.aureusisolates,whose incidenceandclinicalimplicationsremainunknown.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WethankMaríaIbá ˜nezAravenafromPublicHealthInstitute ofChileforexcellenttechnicalassistance.Thisstudywas sup-portedbyPublicHealthInstituteofChile.

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