Genome
Note
Complete
genome
of
a
methicillin-resistant
Staphylococcus
vitulinus
from
Danish
ground
beef
meat
carrying
a
mecA2
resistance
gene
and
a
novel
ccr
allotype
Esmée
Lauren
Looman
a,1,
Paula
van
Tienen
a,1,
Duncan
Y.K.
Ng
b,
Sharmin
Baig
b,
Anaëlle
Fait
c,
Søren
Overballe-Petersen
b,
Paal
S.
Andersen
b,
Marc
Stegger
b,*
a
ErasmusUniversityMedicalCenter,Rotterdam,TheNetherlands
b
DepartmentofBacteria,ParasitesandFungi,StatensSerumInstitut,Copenhagen,Denmark
cDepartmentofVeterinaryandAnimalSciences,FacultyofHealthandMedicalSciences,UniversityofCopenhagen,Copenhagen,Denmark
ARTICLE INFO
Articlehistory: Received2May2020
Receivedinrevisedform28August2020 Accepted9September2020
Availableonline8October2020
Keywords: Staphylococcusvitulinus Resistance SCCmec mecA mecA2 Whole-genomesequencing ABSTRACT
Objectives:Toreportthecompletegenomesequenceofamethicillin-resistantStaphylococcusvitulinus
fromgroundbeeftoallowcomparisonwithotheravailableS.vitulinusgenomesandtoinvestigateits
SCCmecelement.
Methods: Meat samples from grocery stores in Denmark were examined for the presence of
staphylococcalspeciesbyplatingonselectiveplates.Onecolonyisolatedfrombeefwasidentifiedas
S.vitulinusbyMALDI-TOFandgenomesequencedusingacombinationofIlluminaandOxfordNanopore
technologies.PhylogeneticandinsilicoresistomeanalyseswereperformedforallavailableS.vitulinus
genomes.
Results:TheclosedgenomeofS.vitulinusTienloo1isolatehadachromosomesizeof2,628,028bpand
containedasinglenovel2,380bpplasmidbasedonahybridassembly.ItcarriedmecAastheonly
resistancemarker.Theisolatewasfoundnottocarryanyimmuneevasionclustergenes,whichhavebeen
putativelyassociatedtohumanorigin.ComparisonwithallpubliclyavailableS.vitulinusdraftgenomes
showedadiversepopulationandrevealedthatonlytheDanishbeefisolatecontainedamecgenein
additiontoaccrgenecomplex.Additionally,thesingleccrCgenewithintheisolatewasnovelanddistant
fromthemecA2gene.
Conclusion:Thisisolate,Tienloo1,fromagroundbeefmeatsamplerepresentsthefirstcompletegenome
ofS.vitulinusfoundtocarryamecA2geneandanovelccrallotypeinitsSCCmecelementthatisdistinct
fromallpubliclyavailabledraftS.vitulinusgenomes.
©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobial
Chemotherapy.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.
org/licenses/by-nc-nd/4.0/).
Retailmeatisoftencontaminatedbybacteria,includingvarious Staphylococcusspecies,ofwhichsomemaybedisease-causingin humans[1–3].Theresistanceprofiles,andinparticularthe beta-lactamresistanceofthesespecies,areofspecialinterestinrelation to human disease and public health surveillance. Beta-lactam resistance is well described in staphylococci, as well as in Staphylococcusvitulinus[4].
Inanongoingstudyonthepresenceofstaphylococcalspeciesin Danishretailmeat,samplesofbeef,chickenandporkmeatwere purchased at various grocery stores in Copenhagen, Denmark. Within1dayafterpurchasingandpriortoexpirationdates,50gof eachmeatsamplewashomogenisedfor2minin100mLbuffered peptonewater(BPW),usingtheSewardStomacher400Laboratory Blender(SewardLimited,WestSussex,UK).Subsequently,1mLof thesamplewasenrichedin9mLtrypticsoybroth(TSB)at37Cfor 24hbeforeplating500
m
LonSA-Selectplates(Bio-Rad,Hercules, CA, USA). After culturing overnight, species identification was performed by MALDI-TOF. One colony isolated from beef was identified as S. vitulinus. For short-read sequencing, DNA was purifiedwiththeQiagenBloodand TissueDNAkit followedby paired-end sequencing using the Illumina NextSeq sequencing* Correspondingauthorat:DepartmentofBacteria,ParasitesandFungi,Statens SerumInstitut,Artillerivej5,DK-2300Copenhagen,Denmark.
E-mailaddress:mtg@ssi.dk(M.Stegger).
1
ELLoomanandPvanTienencontributedequallytothiswork.
http://dx.doi.org/10.1016/j.jgar.2020.09.013
2213-7165/©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobialChemotherapy.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
JournalofGlobalAntimicrobialResistance23(2020)221–223
ContentslistsavailableatScienceDirect
Journal
of
Global
Antimicrobial
Resistance
platformandtheir300-cycleV2kit(Illumina,SanDiego,CA,USA). Adaptorsequences wereremovedwith Trimmomatic v0.36 [5]
using the following settings: ILLUMINACLIP:/NexteraPE-PE. fa:2:30:10LEADING:20 TRAILING:20. For long-read sequencing, DNAwas purified from a colony scrape of half a 1
m
L sterile inoculationloopresuspendedin200m
LPBSusinganAgencourt GenfindV2kit(BeckmanCoulter,Brea,CA,USA)scaledto300m
L magnetic beads and a DynaMag-2 Magnet (ThermoFisher, Waltham,MA,USA).ThepurifiedDNAwas appliedtoanR9.4.1 flowcellinaMinIONMk1BusingaLigationSequencingKit1D, SQK-LSK109withNativeBarcodingKit1D,SQK-NBD103(Oxford Nanopore Technologies, Oxford, UK), following the standard protocol starting with 1m
g DNA, omitting the optional ‘DNA fragmentation’andchoosingthe‘LongFragmentBuffer’.Rawreads werebase-calledwithONT’sAlbacorev2.3.4software.Sequencing adaptorswereremovedwithPorechopv0.2.3[6]andreadsquality filteredto at least q8 with NanoFiltv2.2.0 [7] prior to hybrid assemblyusingUnicyclerv0.4.7 [8].The completegenomein a singlecontigwasannotatedusingNCBI’sannotationpipelineand analysedforantimicrobialresistancegenesusingABRicatewith theResFinderdatabase.PhylogeneticanalysisofS.vitulinuswithall availabledraftgenomes fromRefSeq(https://www.ncbi.nlm.nih. gov/refseq/,accessedon23September2019,n=9)wasperformed usingSNPs detected withNASP[9] afterremovalof duplicated regionsusingNUCmerintheTienloo1referencechromosomeand exclusionofpositionswithlessthan10-foldcoverageand<90% unambiguous variant calls. Phylogenetic reconstruction on the resulting SNP alignment was performed using the maximum likelihoodasimplementedinIQ-TREE[10].The S. vitulinus Tienloo1 isolate had a chromosome size of 2,628,028bpwithaGC-contentof32.9%andcontainedasingle novel2,380bpplasmid,whichhadasequencingdepth15times higherthan the chromosome.ResFinder (v3.1) analysisdidnot revealanyresistancegenesotherthanmecAusingathresholdof 90%and80%minimumhitlength.Hence,noresistancemarkerwas foundonthesmallhighcopynumberplasmid.Theisolatedidnot encodegenesforthePanton-Valentineleukocidin(lukS/F-PV)or genes(scn,sakorchp)relatedtotheimmuneevasioncluster(IEC) and human origin in Staphylococcus aureus. The complete and annotated(NCBIProkaryoticGenomeAnnotationPipeline(PGAP)) chromosomeandplasmidareavailableunderGenBankaccession IDsCP051882andCP051881,respectively.
Staphylococcus vitulinus has previously been described in horse samplesfrom Denmark [11], and in chicken and bovine samples from the US [12,13]; this corroborates the livestock-relatedoriginoftheDanishisolate.Furtherinvestigationsintothe staphylococcalcassettechromosome mec (SCCmec)revealed no
readily identifiable direct repeats in the vicinity of a detected mecA2gene,noranyidentifiableccrgene-complexgenes,similar towhathaspreviouslybeendescribed[14].Interestingly,a phenol-solublemodulin (PSM) encodinggene waspresent upstreamof mecA.Thepsm-mecgeneencodesacytolyticamphipathicpeptide toxin that hasbeen shown to influence methicillin resistance, biofilm formation, cell spreading and the expression of other virulence factors [15]. Analyses including all nine available S. vitulinusdraftgenomes revealeda coregenomeof82.6%(2.17 Mb) with a divergent population with 5,800–18,700 SNPs separatingall isolates(see Fig.1).Investigations intomecAand SCCmeccontentinthepubliclyavailableS.vitulinusdraftgenomes showed thattwo-thirds oftheRefSeq isolatescontainedoneto threeccrgenesthatgenerallydifferedinallotypebetweenisolates. However,thegenomeoftheTienloo1isolatepresentedhereisthe onlygenomethatcontainedamecgeneinadditiontoanyccrgene complex.AnalysisofmecAitselfinstaphylococcihasshownthat themecA2allelelikelyemergedinS.vitulinusmorethan50years ago, but alsothat S. vitulinus, despite encoding mecA, displays varyinglevelsofphenotypicresistancetobeta-lactams[16].Our isolatealsoharbouredasinglenovel ccrCgene (<76% sequence similaritytoccrC1and<73%toccrC2)thatwasfound>90kbfrom themecA2gene.GiventhedistancebetweenthemecAgeneandthe novel ccr gene, the latter was perhaps placed the outside the SCCmecelementitself.However,whereastheregioncontainsno indication of insertion sequences (IS), prophage structural elementsorplasmidreplicons,thegeneticcontentbetweenthe twogenesincludesrestriction-modificationsystems,effluxpumps andatoxin-antitoxinsystemthatcombinedcouldbetheresultof horizontalgene transfer.Interestingly, twootherisolates, SNUC 330andF1028,fromCanadaandKoreacontainedasimilarnovel ccrC allotype. In silico analysis revealed a scarce repertoire of resistancegenesinmostisolates(Fig.1).
In conclusion, analyses of the now 10 available S. vitulinus genomes showed a diverse population with few resistance markers.However,multipleacquisitionsoftheSCCelementwere observed.Additionally,theIECgeneclusterassociatedtohuman adaptationwasabsentfromthegenomes,therebyhighlightingthe species’non-humanreservoir.
Funding None.
Conflictofinterest Nonedeclared.
Fig.1.Phylogenetictreeofall10availableStaphylococcusvitulinusgenomesequences.Theanalysiswasbasedon39,734SNPsdetectedina2.17Mbcoregenomeacrossthe collection.InformationonisolateID,countryoforigin,samplematerialanddetectedresistancegenesarepresentedforallisolates.Scalebarindicatessubstitutionspersite. 222 E.L.Loomanetal./JournalofGlobalAntimicrobialResistance23(2020)221–223
Ethicalapproval Notrequired. Acknowledgements
WewouldliketothankthetechnicalstaffoftheUniversityof Copenhagen,DepartmentofVeterinaryandAnimalSciencesandat StatensSerumInstitutforprovidinglaboratoryassistance. References
[1]TangY,LarsenJ,KjeldgaardJ,AndersenPS,SkovR,IngmerH. Methicillin-resistantand-susceptibleStaphylococcusaureusfromretailmeatinDenmark. IntJFoodMicrobiol2017;249:72–6.
[2]LarsenJ,PetersenA,SørumM,SteggerM,vanAlphenL,Valentiner-BranthP, etal.Meticillin-resistantStaphylococcusaureusCC398isanincreasingcauseof diseaseinpeoplewithnolivestockcontactinDenmark,1999to2011.Euro Surveill2015;20(37)10.2807/1560-7917.ES.2015.20.37.30021.
[3]Becker K, Heilmann C, Peters G. Coagulase-negative staphylococci. Clin MicrobiolRev2014;27:870–926.
[4]MiragaiaM.FactorscontributingtotheevolutionofmecA-Mediated beta-lactamresistanceinstaphylococci: updateandnewinsightsfromwhole genomesequencing(WGS).FrontMicrobiol2018;9:2723.
[5]BolgerAM,LohseM,UsadelB.Trimmomatic:aflexibletrimmerforIllumina sequencedata.Bioinformatics2014;30:2114–20.
[6]WickRR,JuddLM,GorrieCL,HoltKE.Completingbacterialgenomeassemblies withmultiplexMinIONsequencing.MicrobGenom2017;3:e000132.
[7]DeCosterW,D’HertS,SchultzDT,CrutsM,VanBroeckhovenC.NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 2018;34:2666–9.
[8]WickRR,JuddLM,GorrieCL,HoltKE.Unicycler:resolvingbacterialgenome assembliesfromshortandlongsequencingreads.PLoSComputBiol2017;13: e1005595.
[9]SahlJW,LemmerD,TravisJ,SchuppJM,GilleceJD,AzizM,etal.NASP:an accurate,rapidmethodfortheidentificationofSNPsinWGSdatasetsthat supportsflexibleinputandoutputformats.MicrobGenom2016;2:e000074. [10]NguyenLT,SchmidtHA,vonHaeselerA,MinhBQ.IQ-TREE:afastandeffective stochasticalgorithmforestimatingmaximum-likelihoodphylogenies.Mol BiolEvol2015;32:268–74.
[11]MoodleyA,GuardabassiL.Clonalspreadofmethicillin-resistant coagulase-negativestaphylococciamonghorses,personnelandenvironmentalsitesat equinefacilities.VetMicrobiol2009;137:397–401.
[12]Bhargava K, Zhang Y. Characterization of methicillin-resistant coagulase-negativestaphylococci(MRCoNS)inretailmeat.FoodMicrobiol2014;42:56–60. [13]AdkinsPRF,DufourS,SpainJN,CalcuttMJ,ReillyTJ,StewartGC,etal. Cross-sectionalstudy to identifystaphylococcalspecies isolatedfrom teatand inguinalskinofdifferent-ageddairyheifers.JDairySci2018;101:3213–25. [14]HiramatsuK,ItoT,TsubakishitaS,SasakiT,TakeuchiF,MorimotoY,etal.
Genomic basisfor methicillin resistance in Staphylococcus aureus. Infect Chemother2013;45:117–36.
[15]QinL,McCauslandJW,CheungGY.OttomPSM-Mec-Avirulencedeterminant thatconnectstranscriptionalregulation,virulence,andantibioticresistancein staphylococci.FrontMicrobiol2016;7:1293.
[16]RoloJ,WorningP,BoyeNielsenJ,SobralR,BowdenR,BouchamiO,etal. EvidencefortheevolutionarystepsleadingtomecA-mediatedbeta-lactam resistanceinstaphylococci.PLoSGenet2017;13:e1006674.
