Distinct phenotypic and genomic signatures underlie contrasting pathogenic potential of Staphylococcus epidermidis clonal lineages

micb-10-01971 Augus 27, 2019 Time: 12:8 # 1 ORIGINAL RESEARCH published: 27 Augus 2019 doi: 10.3389/ micb.2019.01971 Edi ed by: Ludmila Chis ose do a, Uni e si y o Washing on, Uni ed S a es Re iewed by: Ka s en Becke , Uni e si y o Müns e , Ge many Vas i Chaudh y, Uni e si y o Tübingen, Ge many *Co espondence: Ma ia Mi agaia [email p o ec ed] Special y sec ion: This a icle was submi ed o E olu iona y and Genomic Mic obiology, a sec ion o he jou nal F on ie s in Mic obiology Recei ed: 02 Ap il 2019 Accep ed: 12 Augus 2019 Published: 27 Augus 2019 Ci a ion: Espadinha D, Sob al RG, Mendes CI, Mé ic G, Sheppa d SK, Ca iço JA, de Lencas e H and Mi agaia M (2019) Dis inc Pheno ypic and Genomic Signa u es Unde lie Con as ing Pa hogenic Po en ial o S aphylococcus epide midis Clonal Lineages. F on . Mic obiol. 10:1971. doi: 10.3389/ micb.2019.01971 Dis inc Pheno ypic and Genomic Signa u es Unde lie Con as ing Pa hogenic Po en ial o S aphylococcus epide midis Clonal Lineages Diana Espadinha1,2, Ri a G. Sob al3, Ca a ina Inês Mendes4, Guillaume Mé ic5, Samuel K. Sheppa d5,6, João A. Ca iço4, He mínia de Lencas e2,7 and Ma ia Mi agaia1* 1Labo a o y o Bac e ial E olu ion and Molecula Epidemiology, Ins i u o de Tecnologia Química e Biológica An ónio Xa ie , Uni e sidade No a de Lisboa, Oei as, Po ugal, 2Labo a o y o Molecula Gene ics, Ins i u o de Tecnologia Química e Biológica An ónio Xa ie , Uni e sidade No a de Lisboa, Oei as, Po ugal, 3Labo a o y o Molecula Mic obiology o Bac e ial Pa hogens, UCIBIO/REQUIMTE, Faculdade de Ciências e Tecnologia, Uni e sidade No a de Lisboa, Cos a de Capa ica, Po ugal, 4Molecula Mic obiology and In ec ion Uni , Ins i u o de Medicina Molecula , Faculdade de Medicina de Lisboa, Uni e sidade de Lisboa, Lisbon, Po ugal, 5The Milne Cen e o E olu ion, Uni e si y o Ba h, Ba h, Uni ed Kingdom, 6MRC CLIMB Conso ium, Ba h, Uni ed Kingdom, 7Labo a o y o Mic obiology and In ec ious Diseases, The Rocke elle Uni e si y, New Yo k, NY, Uni ed S a es Backg ound: S aphylococcus epide midis is a common skin commensal ha has eme ged as a pa hogen in hospi als, mainly ela ed o medical de ices-associa ed in ec ions. No ewo hy, in ec ion a es by S. epide midis ha e he endency o ise s eeply in nex decades oge he wi h medical de ices use and immunocomp omized popula ion g ow h. S aphylococcus epide midis popula ion s uc u e includes wo majo clonal lineages (A/C and B) ha p esen con as ing pa hogenic po en ials. To add ess his dis inc ion and explo e he basis o inc eased pa hogenici y o A/C lineage, we pe o med a de ailed compa a i e analysis using phylogene ic and in eg a ed pangenome-wide-associa ion s udy (panGWAS) app oaches and compa ed he lineages’s pheno ypes in in i o condi ions mimicking ca iage and in ec ion. Resul s: Each S. epide midis lineage had dis inc pheno ypic signa u es in skin and in ec ion condi ions and di e ed in genomic con en . Combina ion o pheno ypic and geno ypic da a e ealed ha bo h lineages we e well adap ed o skin en i onmen al cues. Howe e , hey appea o occupy di e en skin niches, pe o m dis inc biological unc ions in he skin and use di e en mechanisms o comple e he same unc ion: lineage B s ains showed e idence o specializa ion o su i al in mic oae obic and lipid ich en i onmen , cha ac e is ic o hai ollicle and sebaceous glands; lineage A/C s ains showed e idence o adap ion o di e se osmo ic and pH condi ions, po en ially allowing hem o occupy a b oade and mo e supe icial skin niche. In in ec ion condi ions, A/C s ains had an ad an age, ha ing he po en ial o bind blood-associa ed hos ma ix p o eins, o m bio ilms a blood pH, esis an ibio ics and mac ophage acidi y and o p oduce p o eases. These ea u es we e obse ed o be a e in he lineage B s ains. PanGWAS analysis p oduced a ca alog o pu a i e S. epide midis i ulence ac o s and iden i ied an epidemiological molecula ma ke o he mo e pa hogenic lineage. F on ie s in Mic obiology | www. on ie sin.o g 1Augus 2019 | Volume 10 | A icle 1971 micb-10-01971 Augus 27, 2019 Time: 12:8 # 2 Espadinha e al. S. epide midis Clonal Lineages Pa hogenici y Conclusion: The p e alence o A/C lineage in in ec ion is p obably ela ed o a highe me abolic and genomic e sa ili y ha allows apid adap a ion du ing ansi ion om a commensal o a pa hogenic li es yle. The pu a i e i ulence and pheno ypic ac o s associa ed o A/C lineage cons i u e a eliable amewo k o u u e s udies on S. epide midis pa hogenesis and he inding o an epidemiological ma ke o he mo e pa hogenic lineage is an asse o he managemen o S. epide midis in ec ions. Keywo ds: S. epide midis, pan genome, GWAS, clonal lineages, pa hogen, commensal BACKGROUND S aphylococcus epide midis is one o he mos abundan commensal bac e ia o heal hy human skin and mucosa. This o ganism has eme ged in ecen decades as an impo an oppo unis ic pa hogen, being he main cause o nosocomial in ec ions associa ed o indwelling medical de ices such as pe iphe al o cen al in a enous ca he e s (CVCs) (O o, 2009). In ec ions by S. epide midis usually occu due o a b each in he skin ba ie esul ing om he inse ion o he medical de ices, allowing S. epide midis o pene a e he hos issues. The p og ession o in ec ion a e skin pene a ion depends on he abili y o S. epide midis o apidly change om a commensal o a pa hogenic s a e. As skin commensals, S. epide midis su i e and g ow unde nu ien limi a ion, a a low empe a u e (<37◦C) and pH (∼4.5–6.4) (Schmid- Wend ne and Ko ing, 2006) and a di e se osmo ic p essu es esul ing om he p oduc ion/e apo a ion o swea and luc ua ions in en i onmen al humidi y (Wilson, 2005). Fu he mo e, hey ha e o cope wi h cell desquama ion, and oxida i e s ess esul ing om UV exposu e (Kammeye and Lui en, 2015). Once in he bloods eam, upon skin ba ie b each, he en i onmen al landscape changes d ama ically and S. epide midis suddenly ace a nu ien - ich and alkaline en i onmen wi h a highe empe a u e, p o-in lamma o y molecules and eac i e oxygen species (ROS) gene a ed by immune cells (Aki a e al., 2006;Weiss and Schaible, 2015) and e en ual an ibio ic p essu e (Cio u e al., 2017). Howe e , he ac o s o S. epide midis ha con ibu e o he ansi ion om heal h o disease s a e a e no comple ely unde s ood. One o he ac o s hough o be c ucial o ansi ion om skin o blood is he o ma ion o bio ilms on he su ace o medical de ices, which can be composed o a mesh o p o eins, exopolysaccha ides and ex acellula DNA (Qin e al., 2007; O o, 2009). These bio ilms can con e p o ec ion agains he hos immune sys em (Vuong e al., 2004) and esis ance o an ibio ics (Fa be e al., 1990;Kha do i e al., 1995;Singh e al., 2010), making in ec ions ex emely di icul o ea (O o, 2009). O he mechanisms ha ha e been shown o be impo an o S. epide midis pa hogenici y include he abili y o e ade human inna e immuni y, namely h ough p ocesses in ol ed in esis ance o an imic obial pep ides (AMP) (Cheung e al., 2010). Besides he ica ope on, which is di ec ly in ol ed in bio ilm o ma ion (Heilmann e al., 1996) and he inse ion sequence IS256, shown o modula e bio ilm o ma ion and an ibio ic esis ance (Kozi skaya e al., 2004), o he i ulence ac o s ha e been p oposed. These include he p ima y a achmen o hos ex acellula ma ices and in e cellula agg ega ion, oxins, p o eases and lipases, all sugges ed o be implica ed in in asi e po en ial (O o, 2012). Whole genome sequence analysis (Conlan e al., 2012;Me ic e al., 2015) has shown ha he S. epide midis popula ion was composed o wo main phylogene ic clus e s: lineage A/C, con aining mos o he isola es om coloniza ion and in ec ion and lineage B, comp ising mainly coloniza ion isola es. In spi e o hei clinical ele ance, he ac o s associa ed o he success o A/C s ains, bo h as colonize s and pa hogens, a e no ully unde s ood. He e we hypo hesize ha he success o A/C s ains as colonize s and as pa hogens is associa ed wi h an inc eased abili y o adap o en i onmen al cons ain s imposed by bo h coloniza ion and in ec ion s a es. To add ess his, we analyzed he genomes o ep esen a i e isola es om A/C and B clus e s and cha ac e ized hei pheno ypic ai s in condi ions ha mimic he coloniza ion and in ec ion scena ios. RESULTS AND DISCUSSION S. epide midis Popula ion S uc u e Is Composed o Two Clus e s Wi h Di e en Pa hogenic Po en ial In p e ious s udies we ha e cha ac e ized a collec ion o 1714 S. epide midis om di e en isola ion da es, geog aphic and clinical o igins (Mi agaia e al., 2007;Rolo e al., 2012). Based on mul ilocus sequence yping (MLST) da a we selec ed a collec ion o 83 isola es ha ep esen he di e si y o s ains in e ms o gene ic backg ounds (71 sequence ypes, be ween 1996 and 2001). To unde s and how isola es o di e en o igins we e ela ed, we gene a ed a pan genome based on he anno a ed genomes om 82 S. epide midis s ains unde s udy (one genome was excluded om he genomic analysis due o possible con amina ion, see Ma e ials and Me hods) and compa ed he genomic con en o he s ains acco ding o hei isola ion sou ce. The pangenome ob ained had a o al size o 5.0 Mbp wi h 31.3% o GC con en , encoding a o al o 6682 genes. F om hese, app oxima ely one hi d (n= 1653) comp ised he co e genome (p esen in all s ains in his s udy), wi h he emainde (5029) comp ising he accesso y genome. Gene accumula ion cu es ha plo pan genome size as a unc ion o he numbe o genomes sequenced, sugges an open pan genome ha is cha ac e is ic o popula ions F on ie s in Mic obiology | www. on ie sin.o g 2Augus 2019 | Volume 10 | A icle 1971 micb-10-01971 Augus 27, 2019 Time: 12:8 # 3 Espadinha e al. S. epide midis Clonal Lineages Pa hogenici y FIGURE 1 | Maximum-likelihood ee o he 82 S. epide midis s ains. The ee was gene a ed by RaxML (S ama akis e al., 2012) embedded in Gubbins (C ouche e al., 2015), based on he co e gene alignmen wi h emo al o he ecombining egions. In blue a e depic ed he coloniza ion isola es, in ed he in ec ion isola es and in g ay a e he isola es o which no epidemiological in o ma ion was a ailable. (A–C) Co espond o S. epide midis clonal clus e s, acco ding o Me ic e al. (2015). ha unde go equen ho izon al gene ans e (HGT) (Me ic e al., 2015) (Supplemen a y Figu e S1). Phylogene ic econs uc ion, based on he Maximum- likelihood (ML) ee de i ed om he co e genome co ec ed o ecombining egions, con i med ha he S. epide midis popula ion is di ided in o h ee clus e s (Figu e 1). Consis en wi h p e ious s udies (Me ic e al., 2015), Clus e s A (66%) and C (6%) we e joined in a single clus e and we e analyzed oge he in u he analysis. The emainde o he isola es belonged o clus e B (28%). The A/C clus e con ained mainly nosocomial isola es (n= 49/59, 83%) whe eas he B clus e con ained a highe p opo ion o isola es om heal hy people in he communi y (n= 13/23, 56%) (p= 0.0007), con i ming p e ious indings by Conlan e al. (2012) o a much smalle and geog aphically delimi ed isola e collec ion. A/C clus e isola es almos equally o igina ed om in ec ion (n= 28/59, 47%) and coloniza ion (n= 31/59, 53%) sou ces, while B clus e isola es we e mos ly om coloniza ion (17/23, 70%) (p= 0.127) e en hough his di e en dis ibu ion did no each s a is ical signi icance. These esul s sugges ha al hough bo h A/C and B s ains a e ca ied asymp oma ically, A/C s ains a e, appa en ly, mo e implica ed in in ec ion e en s. This obse a ion is co obo a ed by mul iple s udies whe ein S. epide midis om in ec ion we e in hei majo i y om A/C clus e (which comp ises clonal complex 2 as de ined by MLST) (Mi agaia e al., 2007;Wide s om e al., 2009; Go don e al., 2012;Hellma k e al., 2013;Che i i e al., 2014). E idence o he Clonal Dissemina ion o A/C Clus e Isola es An impo an cha ac e is ic o some oppo unis ic pa hogens is he abili y o dissemina e in di e en hos s and en i onmen s. This can lead o b oad dis ibu ion o closely ela ed s ains. The mean numbe o SNP di e ences be ween he co e genomes o A/C and B clus e s was 39889, which co espond o 1.6% o he a e age genome size o S. epide midis. Wi hin each clus e , he a e age numbe o SNPs was simila o s ains o clus e B (a e age numbe o SNP di e ences = 16786, anging be ween 31 and 36796) and o s ains o clus e A/C (a e age numbe o SNP di e ences = 17696 nucleo ides, anging be ween 1 and 37617). The lowe numbe o di e ences (<100 SNPs) we e ound be ween 11 pai s o A/C isola es and only one pai in clus e B, sugges ing ha A/C isola es a e mo e clonal han B isola es. Fu he mo e, hese pai s included isola es om di e en coun ies, isola ed ei he om he hospi al o he communi y, demons a ing he dissemina ion o his clus e h ough hese se ings. In con as wi h o he known nosocomial pa hogens, including S. au eus (Ruimy e al., 2009;G undmann e al., 2010), we ound no e idence o geog aphic clus e ing in S. epide midis, since each b anch in he ML ee, co esponding ei he o A/C o B clus e s (conside ing a maximum o 50 SNPs di e ence), con ained isola es om mul iple coun ies (see Supplemen a y Table S1). Fu he mo e, he e we e eigh pai s o s ains om di e en coun ies ha , wi h one excep ion, belonged o he A/C clus e ha di e ged in less han 100 SNPs and ha we e isola ed wi hin he same yea . This was he case o s ains om Denma k and Po ugal (68 SNPs; DEN161, 966N), Denma k and Iceland (60 SNPs; DEN116, ICE122) and A gen ina and Hunga y (17 SNPs; AGT24, HUR105), among o he s (see Supplemen a y Table S1). These examples sugges ex ensi e and apid geog aphic dissemina ion o s ains belonging o he A/C clus e , likely p omo ed by skin- o-skin con ac be ween a ele s. This is consis en wi h p e ious s udies ha hypo hesized b oad geog aphic dissemina ion o his species (Mi agaia e al., 2002; Wide s om e al., 2006). E idence o Ecological Isola ion and Dis inc Biological Func ions o A/C and B Clus e s The success o a bac e ial clone may be in luenced by i s capaci y o adap o di e en en i onmen s and his is equen ly accomplished by he acquisi ion o exogenous gene ic elemen s F on ie s in Mic obiology | www. on ie sin.o g 3Augus 2019 | Volume 10 | A icle 1971 micb-10-01971 Augus 27, 2019 Time: 12:8 # 4 Espadinha e al. S. epide midis Clonal Lineages Pa hogenici y (Uhlemann e al., 2012;Wal he e al., 2018), which will compose he accesso y genome. To assess di e ences in gene con en o he wo S. epide midis clus e s, we analyzed and quan i ied accesso y genome a ia ion. The ma ix o p esence/absence o all he genes in he genomes is ep esen ed in Supplemen a y Table S2. The majo i y o A/C and B pan genomes was composed o accesso y genes (A/C: 74%, 3723/5029; B: 67%, 3381/5029). Among his accesso y genome, 1648 genes we e exclusi e o A/C clus e and hese genes a ied in equency om 93% o 2% and 1306 genes we e exclusi e o clus e B and anged om 100 o 4%, sugges ing high genome plas ici y and clus e -speci ic unc ions. The numbe o accesso y genes sha ed be ween s ains o he same clus e (3723 in A/C and 3381 genes in B) is 1.8–1.6 highe han he numbe o genes sha ed be ween he wo clus e s (2075 genes). This di e ence could be due o he exis ence o a ba ie o gene ic ans e be ween hem. Howe e , in con as o S. au eus, his appa en ly low equency o gene ic ans e is no e iden ly associa ed wi h he p esence o Res ic ion/Modi ica ion (R-M) sys ems (Wald on and Lindsay, 2006;Co aglia e al., 2010) and/o he clus e ed egula ly in e spaced sho palind omic epea s (CRISPR) (Ma a ini and Son heime , 2008). The e was no clea associa ion o a hsdR/hsdM sys em o any o he clus e s and cas and csm genes we e equally dis ibu ed among s ains o he wo clus e s [A/C: 12% and B: 17% (p>0.05)] (Supplemen a y Table S2). An al e na i e explana ion o he lowe le el o gene ic exchange be ween he clus e s, in his appa en ly ecombinogenic species (Mi agaia e al., 2007;Me ic e al., 2015), migh be he exis ence o opism o each o hese lineages o speci ic skin niches (G ice e al., 2009;Oh e al., 2014) ha could ha e lead o some ecological isola ion o he wo gene ic clus e s. Ac ually, skin mic obio a composi ion was p e iously shown o a y acco ding o he en i onmen al cha ac e is ics o he skin ecological niche sampled (Cos ello e al., 2009;G ice e al., 2009). To es he hypo hesis o ecological isola ion we ha e examined he se en MLST genes, which cons i u e a good ep esen a ion o he co e genes, ei he in e ms o genome dis ibu ion and gene ic di e si y (Gomes e al., 2005;Mi agaia e al., 2007). Da a showed an appa en allelic seg ega ion, whe e a oE gene p esen ed no common alleles be ween he clus e s and he emaining genes (a cC, g ,mu S, py R, pi,yqiL), comp ising be ween 11 and 20 alleles, had a maximum o wo alleles sha ed by he wo clus e s. Fu he mo e, we looked o genes ha we e signi ican ly associa ed o each clus e using a pan genome wide-associa ion app oach, ha sco es genes o associa ions wi h speci ic epidemiological ea u es. In pa icula , 166 genes we e iden i ied o ha e a s ong posi i e associa ion (Benjamini–Hochbe g p<0.05) wi h clus e A/C (see Supplemen a y Table S3) and 244 genes wi h a s ong associa ion wi h clus e B (see Supplemen a y Table S4). Tables 1,2include he genes among hese, which ha e unc ions ha we e ei he p esen exclusi ely in one o he clus e s o ha we e p esen in bo h bu in signi ican ly di e en equencies. S ains o he A/C clus e we e en iched o genes in ol ed in p ocesses such as bio ilm o ma ion and adhesion o hos ma ix p o eins, p o eolysis, esis ance o an ibio ics and adap a ion o low pH. Con e sely, s ains om clus e B we e en iched o genes in ol ed in he de oxi ica ion o o ma e and o maldehyde, oxida i e s ess esponse, hos in e ac ion h ough ype VII sec e ion sys em, lipid me abolism and cell wall biosyn hesis. This is consis en wi h S. epide midis clus e s A/C and B pe o ming dis inc biological unc ions and possibly occupying di e en skin niches. S. epide midis o A/C and B Clus e s Ha e Adap ed o Skin En i onmen Condi ions Using Di e en S a egies Skin is he i s ba ie o he human body agains ex e nal agg essions and con ains molecules ha de i e om he me abolism o skin cells and ou mic obio a, bu also om he en i onmen (Wilson, 2005). On he skin, S. epide midis ha e o deal wi h nu ien limi a ion, ha sh and a iable en i onmen al condi ions and mechanic s esses. Pe spi a ion is one o he cen al con ibu o s o he composi ion o skin milieu and a majo physiological unc ion ha assis s in he mo egula ion, skin su ace hyd a ion and immune de ense. Pe spi a ion is composed mainly o wa e , bu con ains se e al o he me aboli es ha con ibu e o i s mul iple unc ions, such as an imic obial pep ides (AMP), immunoglobulins, na u al mois u izing ac o s (lac a e, u ea, elec oly es, amino acids), i amins, me als and sal s (Wilson, 2005). To unde s and i he S. epide midis clus e s di e in hei abili y o adap o skin we ha e compa ed hei abili y o g ow and p oduce bio ilm in condi ions ha mimic he skin en i onmen (acidic pH and inc eased sal concen a ions). Mo eo e , we looked in mo e de ail o he genes ha we e associa ed by he pan genomic wide-associa ion app oach o each lineage and ha could p o ide an ad an age in he skin en i onmen al condi ions. G ow h and Bio ilm Fo ma ion a Acidic pH S aphylococcus epide midis belonging o A/C clus e appea o ha e a dis inc i e abili y o adap o he wo acidic pH alues es ed (pH 4.5 and pH 5.5), as s ains om he A/C clus e eached, in a e age, highe cell densi ies in s a iona y phase (∼12% highe in bo h cases) and p esen ed ei he a g ea e (pH4.5: µA/C= 0.335; µB= 0.274, p= 0.043) o simila a e age g ow h a e (pH 5.5: µA/C= 0.626; µB= 0.594, p= 0.273) when compa ed o s ains om he B clus e (see Figu e 2A and Table 3). Addi ionally, s ains belonging o he A/C clus e we e associa ed o a gene in ol ed in he a ginine deiminase me abolism (a cD, an a ginine/o ni hine an ipo e ) (see Table 1) usually ca ied by he a ginine ca abolic mobile elemen (ACME). Al hough a genomic copy o a cD gene wi h a simila unc ion is known o be ubiqui ous in he S. epide midis genome, his ex a copy oge he wi h he emaining a c ope on wi hin ACME was p e iously sugges ed o be impo an o pH homeos asis in S. epide midis and o con ibu e o addi ional ole ance o low-pH in communi y-associa ed S aphylococcus au eus USA300 clone (Thu low e al., 2013;Lindg en e al., 2014). O he unc ions associa ed o A/C clus e s ains ha can e en ually con ibu e o he obse ed inc eased g ow h a e is he la in-con aining oxido educ ase (YwqN), he YdjH suga kinase; and he hexose- 6-phospha e:phospha e an ipo e (UhpT) (Pa k e al., 2015), F on ie s in Mic obiology | www. on ie sin.o g 4Augus 2019 | Volume 10 | A icle 1971 micb-10-01971 Augus 27, 2019 Time: 12:8 # 5 Espadinha e al. S. epide midis Clonal Lineages Pa hogenici y TABLE 1 | Genes posi i ely associa ed o clus e A/C (Benjamini–Hochbe g p<0.05 and OR >1). Pu a i e unc ions Gene anno a ion Func ional anno a ion F equency in clus e A/C % F equency in clus e B % Hyd olysis g oup_1452 Pu a i e hyd olase 92 35 Adhesion and Bio ilm sd F Se ine-aspa a e epea -con aining p o ein F 68 9 sd I Pu a i e su ace p o ein 63 9 icaA∗∗ Poly-be a-1,6-N-ace yl-D-glucosamine syn hase 37 4 icaB∗∗ Poly-be a-1,6-N-ace yl-D-glucosamine N-deace ylase 37 4 icaC∗∗ Pu a i e poly-be a-1,6-N-ace yl-D-glucosamine expo p o ein 37 4 icaD∗∗ Poly-be a-1,6-N-ace yl-D-glucosamine syn hesis 37 4 icaR∗∗ Bio ilm ope on icaADBC HTH- ype nega i e ansc ip ional egula o 37 4 Nickel and Cobal up ake nm R∗HTH- ype ansc ip ional egula o Nm R 37 0 A ginine deiminase a cD∗∗ A ginine/o ni hine an ipo e 55 4 Penicillin esis ance blaI Penicillinase ep esso 68 17 blaZ Be a-lac amase 60 22 Coppe esis ance mco∗∗ Mul icoppe oxidase mco 68 30 P o eolysis splF∗∗ Se ine p o ease SplF 78 35 Ca bohyd a e me abolism gno∗∗ Glucona e 5-dehyd ogenase 62 22 ywqN∗∗ Pu a i e NAD(P)H-dependen FMN-con aining oxido educ ase YwqN 37 4 ydjH∗Pu a i e suga kinase YdjH 37 4 g oup_1735 Hexose-6-phospha e:phospha e an ipo e uhpT 34 0 Thiamine up ake hiQ Thiamine impo ATP-binding p o ein ThiQ 80 30 Cys eine biosyn hesis cysL HTH- ype ansc ip ional egula o CysL 88 43 ∗Only one gene/allele o ha unc ion and unc ion is exclusi e in clus e ; ∗∗Only one gene/allele o ha unc ion and is en iched in he clus e bu he unc ion is no exclusi e in clus e . which lead ei he o he p oduc ion o educing powe (NADH) o he biosyn hesis o majo cell componen s o o he escue o al e na i e suga sou ces. The wo g oups could no be dis inguished by hei abili y o p oduce bio ilm a pH 5.5 (ODA/C= 1.538 s. ODB= 1.536, p= 0.994), since almos all s ains, i espec i e o hei gene ic backg ound, p oduced bio ilm in his condi ion, sugges ing ha bio ilm is impo an o he g ow h o his commensal a he su ace o he skin (see Table 3 and Figu e 2C). G ow h in Osmo ic S ess Condi ions The be e capaci y o he s ains om he A/C clus e o wi hs and pe spi a ion, osmo ic s ess and desicca ion condi ions equen ly imposed a he skin su ace was appa en om hei capaci y o g ow a sodium chlo ide concen a ions cha ac e is ic o he skin (0.15M NaCL) (Schi ek e al., 2001) and in high salini y s ess (2M NaCl) (P ice-Whelan e al., 2013). Whe eas a a physiological concen a ion (0.15M NaCl), s ains om clus e A/C and B had equi alen a e age g ow h a es (µA/C= 0.701 s. µB= 0.670, p= 0.411) (see Table 3), a osmo ic s ess condi ions (2M NaCl), he medium g ow h a e o A/C clus e s ains was signi ican ly highe (µA/C= 0.339 s. µB= 0.298, p= 0.011; see Figu e 2B). Fu he mo e, hey showed a much sho e lag phase (A/C: 361 min s. B: 404 min), and eached highe inal ODs (14% highe ) (see Table 3). The esul s sugges ha , in con as o S. epide midis s ains o B clus e , s ain om A/C clus e a e well adap ed o a ange o salini y concen a ions sugges ing imp o ed su i al in a b oade ange o niches. We ound ha di e en genes indi ec ly in ol ed on esis ance o osmo ic s ess we e speci ically associa ed o ei he clus e A/C o B. This is he case o genes which we e associa ed o A/C clus e ha a e in ol ed in he p oduc ion o polysaccha ide in e cellula adhesion (PIA) (icaADBCR ope on), which h ough he p oduc ion o bio ilm induce p o ec ion agains as osmo ic changes by dec easing he a e o wa e loss and ehyd a ion (Robe son and Fi es one, 1992;de Go au e al., 2009) (see Table 1). Fu he mo e, genes associa ed o B clus e ha could induce osmop o ec ion include hose in ol ed in: pep idoglycan c oss-linking (dacA - a pbp4 homolog, yodJ) ha p o ides ex a igidi y o he bac e ial cell wall (Wyke e al., 1981;Loskill e al., 2014); and he up ake o manni ol (m lA, m lR), a known osmoly e (Ke s e al., 1996;Zahid e al., 2015). The absence o m lD, esponsible o manni ol deg ada ion, sugges s ha s ains o B clus e use manni ol mainly as an osmoly e a he han as a ca bon o ene gy sou ce (da Cos a e al., 1998). F on ie s in Mic obiology | www. on ie sin.o g 5Augus 2019 | Volume 10 | A icle 1971 micb-10-01971 Augus 27, 2019 Time: 12:8 # 6 Espadinha e al. S. epide midis Clonal Lineages Pa hogenici y TABLE 2 | Genes posi i ely associa ed o clus e B (Benjamini–Hochbe g p<0.05 and OR >1). Pu a i e unc ions Gene anno a ion Func ional anno a ion F equency in clus e B % F equency in clus e A/C % Fo ma e de oxi ica ion g oup_862 g oup_863 Pu a i e o ma e dehyd ogenase Pu a i e o ma e dehyd ogenase 91 22 0 0 Fo maldehyde assimila ion/de oxi ica ion hxlR_1 HTH- ype ansc ip ional ac i a o HxlR 22 0 Coppe esis ance cueR∗HTH- ype ansc ip ional egula o 22 0 Oxida i e s ess esponse g oup_3122 (c O)∗∗(?) Glycosyl-4,40-diaponeu ospo enoa e acyl ans e ase 22 0 c P∗∗ Diapolycopene oxygenase 22 0 c Q∗∗ 4,40-diaponeu ospo enoa e glycosyl ans e ase 22 0 c M∗∗ Dehyd osqualene syn hase 22 0 c N∗∗ Dehyd osqualene desa u ase 22 0 Hos -in e ac ion and Vi ulence esaA∗ESAT-6 sec e ion accesso y ac o 65 0 esaB∗ESAT-6 sec e ion accesso y ac o 65 0 essC∗∗ ESAT-6 sec e ion machine y p o ein 65 0 esxA∗ESAT-6 sec e ion sys em ex acellula p o ein A 65 0 essB∗∗ g oup_2414∗∗ ESAT-6 sec e ion machine y p o ein ESAT-6 sec e ion machine y p o ein 39 30 0 0 yezG_1 yezG_2 yezG_3 yezG_5 Pu a i e an i oxin Pu a i e an i oxin Pu a i e an i oxin Pu a i e an i oxin 52 48 43 26 0 0 0 2 Ca bohyd a e me abolism eA∗T ehalose-6-phospha e hyd olase 26 0 m lR∗T ansc ip ional egula o M lR 22 0 m lA∗PTS sys em manni ol-speci ic EIICB componen 22 0 Lipid me abolism g oup_3907∗NADP-dependen 7-alpha-hyd oxys e oid dehyd ogenase 48 0 Benzoa e deg ada ion ligI∗2-py one-4,6-dica baxyla e hyd olase 26 0 T anspo sys ems g oup_1444 ABC anspo e ATP-binding p o ein Na A 43 0 Cell wall biosyn hesis dacAD-alanyl-D-alanine ca boxypep idase DacA 39 2 yodJ∗∗∗ Pu a i e ca boxypep idase YodJ 35 2 T ansposable elemen s bin3_3 Tn552 DNA-in e ase 43 0 in - n Tn916 ansposase In -Tn 87 34 ∗Only one gene/allele o ha unc ion and unc ion is exclusi e in clus e ; ∗∗Mo e han one gene/allele o ha unc ion bu unc ion is exclusi e in clus e ; ∗∗∗Only one gene/allele o ha unc ion and is en iched in he clus e bu he unc ion is no exclusi e in clus e . Al hough s ains o bo h clus e s ca y genes ha a e associa ed o esis ance o osmo ic s ess, o e all, he p ocesses used by s ains o he A/C clus e appea o be mo e e icien han hose o s ains om he B clus e , as illus a ed by i s highe g ow h a e in i o. Gene ic T ai s Associa ed o E asion o Skin Inna e Immuni y Skin is con inuously exposed o commensal mic oo ganisms and challenged o de end he human body om in asion by pa hogens. To deal wi h his ex ensi e mic obial exposu e, skin issues p oduce an imic obial pep ides (AMPs) and ee a y acids ha a ge he bac e ial cell wall o cell memb ane s uc u es (Kenny e al., 2009;Gallo and Hoope , 2012). To su i e, bac e ia ha e de eloped mul iple s a egies, including he p oduc ion o ca o enoid pigmen s ha p o ec om ee a y acids and he inac i a ion o AMPs by p o eolysis. We ound ha s ains belonging o he B clus e mo e commonly con ained he ope on in ol ed in s aphyloxan hin biosyn hesis (c OPQMN) ha was desc ibed o con e esis ance o ee a y acids p oduced by skin cells (Chambe lain e al., 1991). The p oduc ion o he ca o enoid was con i med pheno ypically o he g oup o i e s ains ca ying his ope on h ough he obse a ion o p oduc ion o a yellow colo by colonies g own on aga g ow h medium. O he pigmen s had been ound o be syn hesized by S. epide midis (Ogo, 1985), bu his is he i s ime ha s aphyloxan hin ope on is desc ibed in his species. We also ound a se ine-p o ease (SplF) posi i ely associa ed o s ains om clus e A/C (Table 1). S aphylococcal p o eases ha e al eady been implica ed in he mechanism o de ense agains he human inna e immune sys em, as some o hese p o eases ha e he abili y o clea e and inac i a e human AMPs, namely de mcidin (Lai e al., 2007) and LL-37 (Siep awska-Lupa e al., 2004) ha a e p oduced by epi helial cells F on ie s in Mic obiology | www. on ie sin.o g 6Augus 2019 | Volume 10 | A icle 1971 micb-10-01971 Augus 27, 2019 Time: 12:8 # 7 Espadinha e al. S. epide midis Clonal Lineages Pa hogenici y FIGURE 2 | Pe o mance o clus e A/C and B s ains in he g ow h and bio ilm assays. (A) A e age g ow h a es o A/C and B s ains g own in liquid medium a pH 4.5; (B) A e age g ow h a es o A/C and B s ains g own in liquid medium wi h 2M NaCl; (C) A e age bio ilm o ma ion capaci y o A/C and B s ains a pH 5.5; (D) A e age bio ilm o ma ion capaci y o A/C and B s ains a pH 7. and al hough his could be he case also o S. epide midis SplF, his has ye o be p o en. Al oge he , he esul s sugges ha bo h S. epide midis o A/C and B clus e a e equipped o e ade he hos immune sys em and ha could be a majo ad an age o su i al in bo h skin and blood. In e ac ion Wi h he Hos and Pa hogens Commensal bac e ia a e in cons an in e ac ion wi h he su ounding en i onmen and he hos . Sec e ion sys ems a e cen al elemen s in hese in e ac ions bu ha e been in es iga ed mos ly om he pe spec i e o pa hogen-hos in e ac ions. Recen da a sugges ha hese sys ems may play a ole in mu ualis ic ela ionships be ween bac e ia and he hos (Sil e e al., 2007) o in media ion o bac e ial in e ac ions (Basle e al., 2013). We ound ha S. epide midis s ains belonging o B clus e we e associa ed o genes ela ed o ype VII sec e ion sys em (T7SS) (Lai e al., 2007). In pa icula , all he conse ed T7SS s uc u al genes (esaA, esaB, essA, essB, essC) we e co- loca ed in he ch omosome. Mo eo e , he e was conside able s ain- o s ain a ia ion in he numbe and ype o sec e ed oxin/an i oxin-homologs downs eam his ope on (see Table 2). T7SS we e p e iously desc ibed in S. epide midis and we e p oposed o sec e e an inhibi o y oxin agains P opionibac e ium acnes (Ch is ensen e al., 2016), ano he common commensal o he skin. Ye , in S. au eus, he unc ions o T7SS a e b oade , in ol ing no only in aspecies compe i ion (Cao e al., 2016), bu also pe sis ence and pa hogenici y (Bu s e al., 2008;Kneupe e al., 2014;Ko ea e al., 2014). The iden i ica ion o a T7SS in S. epide midis belonging o B clus e is consis en wi h he hypo hesis ha S. epide midis o clus e B and P opionibac e ium may sha e a simila niche in he skin. S. epide midis Gene ic Con en Re lec s Adap a ion o An h opogenic Ac i i ies P e ious s udies ha e shown ha human skin composi ion is also de ined by ou daily ou ines (Bouslimani e al., 2015), including ac i i ies ha imply equen con ac wi h he skin, like hygiene habi s and he applica ion o skin ca e p oduc s and clo hing. Mo eo e , swea has been conside ed o long as a cleanse , allowing he exc e ion o oxic subs ances ha could ha e been abso bed due o acu e o ch onic exposu e o con amina ed en i onmen (ai , wa e , ood) o o consumed p oduc s. We ound ha S. epide midis o ei he A/C o B clus e we e associa ed o six loci ela ed o he up ake, de oxi ica ion o egula ion o me aboli es (glucona e, ehalose, coppe , and o maldehyde) ha acco ding o he Human Me abolome Da abase1(Wisha e al., 2018) a e p esen in he skin issues and ha a e known o be simul aneously p esen in he composi ion o ood, pha maceu ical p oduc s, de e gen s/disin ec an s and/o cosme ics (see Tables 1,2). Deg ada ion o Suga and Suga De i a i es Genes in ol ed in he deg ada ion o suga s o suga de i a i es, namely ehalose ( eA) and glucona e (gno), we e associa ed wi h B and A/C clus e s espec i ely, sugges ing ha s ains om he wo clus e s use hese al e na i e suga s as ca bon and ene gy 1www.hmdb.ca F on ie s in Mic obiology | www. on ie sin.o g 7Augus 2019 | Volume 10 | A icle 1971 micb-10-01971 Augus 27, 2019 Time: 12:8 # 8 Espadinha e al. S. epide midis Clonal Lineages Pa hogenici y TABLE 3 | Pe o mance o clus e A/C and clus e B s ains in skin and blood condi ions. Condi ions es ed Clus e A/C Clus e B p- alue Skin G ow h a pH 5.5 G ow h a e (h−1) 0.626 0.594 0.273 Lag phase (min) 234 212 – Final OD (nm) 1.369 1.231 – G ow h a 2M NaCl G ow h a e (h−1) 0.339 0.298 0.011 Lag phase (min) 361 404 – Final OD (nm) 1.231 1.08 – Bio ilm a pH 5.5 (OD595 nm) 1.538 1.536 0.994 Blood G ow h a pH 7.4 G ow h a e (h−1) 0.788 0.728 0.122 Lag phase (min) 191 212 – Final OD (nm) 1.456 1.389 – Bio ilm a pH 7 (OD595 nm) 1.475 0.377 0.0003 Adhesion o collagen (OD595 nm) 0.185 0.175 0.671 Skin and Blood G ow h a pH 4.5 G ow h a e (h−1) 0.335 0.274 0.043 Lag phase (min) 234 276 – Final OD (nm) 0.591 0.528 – G ow h a 0.15 M NaCl G ow h a e (h−1) 0.701 0.670 0.411 Lag phase (min) 212 212 – Final OD (nm) 1.476 1.470 – An ioxidan capaci y (nmol/µL) 10.20 10.79 0.428 Cell wall cha ge (OD410 nm) 20.10 27.09 0.084 P o eolysis High (no isola es) 29 4 Medium (no isola es) 21 14 0.032 Low (no isola es) 10 5 Penicillin esis ance no. esis an isola es (%) 52 (87%) 12 (48%) no. suscep ible isola es (%) 8 (13%) 11 (9%) 0.002 Oxacillin esis ance no. esis an isola es (%) 37 (62%) 10 (43%) n . suscep ible isola es (%) 23 (38%) 13 (57%) 0.147 Gen amicin esis ance no. esis an isola es (%) 21 (35%) 2 (9%) no. suscep ible isola es (%) 39 (65%) 21 (91%) 0.026 sou ces. The use o glucona e as a ca bon sou ce and i s cen al ole in bac e ial cell di ision and coloniza ion was p e iously desc ibed o o he bac e ia like S ep ococcus suis (Shi e al., 2014) and Esche ichia. coli (Sweeney e al., 1996). T ehalose is no commonly used by S. epide midis as a ca bon sou ce. Ac ually, he absence o ehalose e men a ion has been conside ed a signa u e o S. epide midis used o dis inguish i om o he bac e ial species (Kloos and Schlei e , 1975). A possible o igin o ehalose ound in he skin, besides ood and cosme ics (Oh ake and Wang, 2011), a e bac e ia o he genus P opionibac e ium (also pa o skin na u al lo a), which a e desc ibed o be capable o syn hesizing his suga (Piwowa ek e al., 2018). The esul s sugges once mo e ha P opionibac e ium and S. epide midis o B clus e migh sha e he same niche, possibly hai ollicles and sebaceous egions whe ein P opionibac e ium a e en iched (Scha schmid and Fischbach, 2013) and he ehalose p oduced by P opionibac e ium can be used by S. epide midis as a ca bon and ene gy sou ce. Ha ing he capaci y o me abolize glucona e and ehalose, ei he om an h opogenic o mic obial o igin, can be highly ad an ageous o S. epide midis in an en i onmen o nu ien limi a ion like he skin. Resis ance and Modula ion o Coppe S aphylococcus epide midis s ains belonging o A/C clus e and B clus e we e associa ed o genes in ol ed in coppe egula ion, al hough using di e en mechanisms. While s ains om he A/C clus e we e associa ed o mul icoppe oxidase (mco), an enzyme in ol ed in he oxida ion o Cu+ o Cu2+ (Si hisak e al., 2005), B clus e s ains we e associa ed o cueR, a Me R- amily me allo egula o y ansc ip ional ac i a o ha senses in acellula Cu+and up egula es copA and cueO, wo genes in ol ed in coppe e lux and oxida ion in esponse o inc easing coppe concen a ions (Cha u edi and Hende son, 2014). Coppe is u ilized by bac e ia o se e al essen ial me abolic p ocesses, bu is also c i ical o he main enance o a heal hy skin, namely o he syn hesis and s abiliza ion o ex acellula ma ix skin p o eins and angiogenesis (Raju e al., 1982;Philips e al., 2012). Al hough an impo an enzyme co ac o and signaling molecule, he in acellula le els o ee coppe can be damaging o bac e ia, leading o ROS- ela ed oxida i e s ess de i ed om i s educ ion and oxida ion cycles, eason by which i is equen ly added o cosme ics as a biocide. Coppe oxida ion pe o med by sys ems like Mco and Cue p obably help S. epide midis o igh ly egula e and main ain coppe homeos asis in he skin (Fes a and Thiele, 2012; Cha u edi and Hende son, 2014). De oxi ica ion o Fo maldehyde S aphylococcus epide midis belonging o B clus e we e addi ionally associa ed wi h a gene (hxlR) in ol ed in de oxi ica ion o o maldehyde, a me aboli e ha can be highly dele e ious o human cells. The o maldehyde- esponsi e ansc ip ional ac o hxlR is pa o he o maldehyde de oxi ica ion/assimila ion ia a ibulose monophospha e (RuMP)-dependen pa hway, h ough which o maldehyde is con e ed in o uc ose-6 phospha e and subsequen ly inco po a ed in o he usual ca bon me abolism pa hway (Chen e al., 2016). Fo maldehyde, is ubiqui ously ound in na u e and is an impo an me aboli e o bo h bac e ia and human cells (Bu gos-Ba agan e al., 2017), bu is also one o he mos syn hesized compounds as he esul o an h opogenic ac i i ies. Toxici y o o maldehyde o animals, humans and bac e ia (Tang e al., 2009;Chen e al., 2016) is due o he as eac ion o o maldehyde wi h ee hiol (−SH) and amine g oups (−NH2) on p o eins and DNA (Feldman, 1973;Page and Bu ne , 2003), F on ie s in Mic obiology | www. on ie sin.o g 8Augus 2019 | Volume 10 | A icle 1971 micb-10-01971 Augus 27, 2019 Time: 12:8 # 9 Espadinha e al. S. epide midis Clonal Lineages Pa hogenici y making o maldehyde one o he mos po en p o ein and DNA c oss-linking agen s. The p esence o genes o he de oxi ica ion in S. epide midis s ains belonging o B clus e p obably allowed hem o adap o o maldehyde esul ing ei he om human and bac e ial cells me abolism o om con inuous exposu e o o maldehyde con aining p oduc s (dyes, syn he ic ex iles, disin ec an s, and cosme ics). In his ega d, S. epide midis migh be unc ioning no only o i s own bene i bu also in he bene i o he hos by a oiding ha hese compounds each oxic concen a ions o human cells. S ains o A/C Clus e Ha e a Highe Capaci y o Su i al in Hospi al and In ec ion En i onmen al Condi ions Du ing in ec ion S. epide midis has o adap o a new en i onmen , he bloods eam, whe ein he pH is s abilized a 7.4, he concen a ion o sal is s eady (0.15 M), speci ic hos ma ix p o eins a e p oduced, he immune esponse is exace ba ed, and inhibi o y concen a ions o an ibio ics may be p esen . One o he s a egies o deal wi h such s esses is he a achmen o he su ace o a de ice, ollowed by he de elopmen o he bio ilm, whe e bac e ial cells a e p o ec ed. To e alua e i S. epide midis belonging o A/C and B clus e s ha e de eloped di e en capaci ies o adap o an in ec ion en i onmen , we compa ed hei abili y o bind collagen I, o g ow and p oduce bio ilm in blood-simila pH and sal condi ions (g ow h: pH 7.4; 0.15 M NaCl; bio ilm: pH 7), o esis oxida i e s ess and o e ade he immune sys em (by measu ing he su ace cha ge and p o eoly ic ac i i y). Mo eo e , we sea ched o speci ic genes wi hin hese lineages ha could be associa ed o a highe in ec ious capaci y. Binding o Hos Ma ix P o eins and Bio ilm Fo ma ion a Blood pH When a medical de ice is in oduced in o he human body, i s su ace becomes coa ed wi h a laye o hos p o eins (Vaudaux e al., 1995; on Ei e al., 2002;A ecubie a e al., 2007;Singh e al., 2012). Fo eign-body associa ed in ec ions a e a ibu ed o he a achmen o bac e ia o hese hos p o eins, such as collagen I om issues, and ib onec in and ib inogen om blood, h ough binding o su ace p o eins (Pa i e al., 1994; Fos e and Hook, 1998) ollowed by he p oduc ion o bio ilm. To unde s and i S. epide midis belonging o he wo gene ic lineages di e ed in hei abili y o cause de ice-associa ed in ec ion, we es ed hei abili y o bind collagen I (see Table 3), he collagen ype mo e abundan in human issues (Singh e al., 2012), a he han he skin. Howe e , ou esul s showed ha binding o collagen was no a dis inc i e ac o con ibu ing o hei di e en in ec ious capaci y (ODA/C= 0.185 s. ODB= 0.175, p= 0.067) (see Supplemen a y Figu e S2A). Mo eo e , we iden i ied wo adhesins o mic obial su ace componen s ecognizing adhesi e ma ix molecules (MSCRAMMs), namely Sd F and Sd I, ha we e associa ed o A/C s ains and ha ha e been desc ibed o be in ol ed in binding o collagen I and ib onec in (Ha o d e al., 2001;Sakinc e al., 2005;A ecubie a e al., 2007), espec i ely. In spi e o he iden i ied associa ion be ween Sd F and A/C clus e , his did no impac di ec ly he pheno ype o collagen binding, p obably due o he exis ence o known edundancy o p o eins o he han Sd F ha bind collagen I. On he o he hand, he compa ison o he wo lineages ega ding he abili y o o m bio ilm a pH 7 (simila o blood’s pH) showed ha A/C s ains p oduced signi ican ly mo e bio ilm (ODA/C= 1.475; ODB= 0.377, p= 0.0003) (see Table 3 and Figu e 2D) and we e simul aneously associa ed o he p esence o he ica ope on, in ol ed in he p oduc ion o polysaccha ide in e cellula adhesion (PIA) (Benjamini–Hochbe g p= 0.029). These esul s sugges ha upon skin dis up ion he s ains ha a e able o p oduce bio ilm and ha su i e be e in blood en i onmen a e hose belonging o A/C clus e ha in addi ion o he abili y o binding collagen I ha e he po en ial o bind ib onec in (mainly ound in he blood a e inju y) and o p oduce PIA-dependen bio ilms a blood’s pH. Resis ance o Hos Immune Sys em Du ing In ec ion Once in he bloods eam, bac e ia a e immedia ely ecognized by monocy es and neu ophils h ough se e al Pa e n ecognizing ecep o s (such as Toll-Like ecep o s, Nod-Like ecep o s and C- ype lec in ecep o s), leading o he exp ession o AMPs, phagocy osis, deg anula ion, espi a o y bu s and killing and he o ma ion o neu ophil ex acellula aps ha cap u e bac e ia inducing a apid in lamma o y esponse (cy okines and chemokines) (Aki a e al., 2006). Bac e ial phagocy osis by mac ophages occu s ia he o ma ion o a memb ane-enclosed phagosome con aining he mic obe in an acidic en i onmen (can be as low as pH 4.5), ex emely ich in bac e icidal enzymes and oxic compounds ( eac i e oxygen and ni ogen species and coppe ) (Weiss and Schaible, 2015). Acco ding o ou da a, S. epide midis s ains ha e de eloped se e al mechanisms ha allow hem o su i e wi hin mac ophages. In pa icula , we obse ed ha al hough A/C s ains had a signi ican ly highe medium g ow h a e a pH 4.5 (pH o mac ophages milieu) (µA/C= 0.335; µB= 0.274, p= 0.043) han B s ains, hey did no a y signi ican ly in hei o al an ioxidan capaci y (see Supplemen a y Figu e S2B). The lack o di e ence in an ioxidan capaci y migh be due o he ac ha bo h A/C and B clus e s we e associa ed o genes ha p o ide esis ance o oxida i e s ess (A/C clus e : mco; B clus e : c OPQMN ope on) and ole ance o coppe (A/C clus e : mco; B clus e : cueR) (see Tables 1,2). Especially, s aphyloxan hin p oduced by c OPQMN was desc ibed in S. au eus o pa icipa e in he sca enging o ee adicals and o ha e impac in i ulence (Claudi z e al., 2006;Mish a e al., 2011). We ha e also compa ed he su ace cha ge and p o eoly ic ac i i y o he wo clus e s, as a measu e o he capaci y o e ade he hos immune sys em. Acco ding o ou da a, s ains o he A/C and B clus e s showed a simila a e age alue o su ace cha ge measu ed by he ela i e pe cen age o unbound cy och ome C, bu he alues ob ained a ied om 0 and 78% among s ains (see Supplemen a y Figu e S2C and Supplemen a y Table S5). In pa icula , closely ela ed s ains in he phylogene ic ee (DEN116 and ICE21, 101 SNPs) showed comple ely dis inc ela i e su ace cha ges (72 and F on ie s in Mic obiology | www. on ie sin.o g 9Augus 2019 | Volume 10 | A icle 1971 micb-10-01971 Augus 27, 2019 Time: 12:8 # 16 Espadinha e al. S. epide midis Clonal Lineages Pa hogenici y Ma a ini, L. A., and Son heime , E. J. (2008). CRISPR in e e ence limi s ho izon al gene ans e in s aphylococci by a ge ing DNA. Science 322, 1843– 1845. doi: 10.1126/science.1165771 May, L., Klein, E. Y., Ro hman, R. E., and Laxmina ayan, R. (2014). T ends in an ibio ic esis ance in coagulase-nega i e s aphylococci in he Uni ed S a es, 1999 o 2012. An imic ob. 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