Antimicrobial susceptibility, virulence determinant carriage and molecular characteristics of Staphylococcus aureus isolates associated with skin and soft tissue infections

www .e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Antimicrobial

susceptibility,

virulence

determinant

carriage

and

molecular

characteristics

of

Staphylococcus

aureus

isolates

associated

with

skin

and

soft

tissue

infections

Fangyou

Yu

_,

_Yunling

_Liu

_,

_Jinnan

_Lv

_,

_Xiuqin

_Qi

_,

_Chaohui

_Lu

_,

_Yu

_Ding

_,

_Dan

_Li

_,

Huanle

Liu

_,

_Liangxing

_Wang

a_{DepartmentofLaboratoryMedicine,TheFirstAffiliatedHospitalofWenzhouMedicalUniversity,Wenzhou,China} b_{DepartmentofRespiratoryMedicine,TheFirstAffiliatedHospitalofWenzhouMedicalUniversity,Wenzhou,China}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21May2015

Accepted10August2015

Availableonline25September2015

Keywords:

Staphylococcusaureus

Skinandsofttissueinfections

Molecularcharacteristic

Virulencegenes

a

b

s

t

r

a

c

t

Abetterunderstandingoftheantimicrobialsusceptibility,carriageofvirulence

determi-nantsandmolecularcharacteristicsofStaphylococcusaureusisolatesassociatedwithskin

andsofttissueinfections(SSTIs)mayprovidefurtherinsightsrelatedtoclinicaloutcomes

withtheseinfections.FromJanuary2012toSeptember2013,atotalof128non-duplicate

S.aureusisolateswererecoveredfrompatientswithSSTIs.All128S.aureusSSTIisolates

carriedatleastfivevirulencegenestested.Virulencegenesdetectedamongatleast70%

ofalltestedisolatesincludedhld(100%),hla(95.3%),icaA(96.9%),clf(99.2%),sdrC(79.7%),

sdrD(70.3%),andsdrE(72.7%).TheprevalenceofMRSAisolateswith10virulencegenes

tested(54.4%,31/56)wassignificantlyhigherthanthatamongMSSAisolates(35.2%,25/71)

(p<0.05).Thepositiveratesofseb,sen,sem,sdrEandpvlamongMRSAisolateswere

sig-nificantlyhigherthanamongMSSAisolates(p<0.05).ST7andST630accountingfor10.9%

werefoundtobethepredominantSTs.Themostprevalentspatypewast091(8.6%).

MRSA-ST59-SCCmecIVwasthemostcommonclone(12.3%)amongMRSAisolateswhereasamong

MSSAisolatesthedominantclonewasMSSA-ST7(15.5%).Sixmainclonalcomplexes(CCs)

werefound,includingCC5(52.3%),CC7(11.7%),CC59(8.6%),CC88(6.3%),CC398(4.7%),and

CC121(3.1%).AhighercarriageofsebandsecwasfoundamongCC59isolates.In

compari-sontoCC5andCC7isolates,thosewiththehighestcarriagerates(>80.0%)ofsdrCandsdrD,

CC59isolateshadlowerprevalenceofthesetwovirulencegenes.AllCC59isolateswere

susceptibletogentamicinandtrimethoprim/sulfamethoxazole,whileCC5andCC7isolates

hadresistanceratestothesetwoantimicrobialsof25.4%and20.9%,and40.0%and40.0%,

respectively.Theresistanceratesfortetracycline,clindamycin,anderythromycinamong

CC5isolateswerelowerthanamongCC7andCC59isolates.Inconclusion,themolecular

∗ _{Correspondingauthor.}

E-mailaddress:wangliangxin2014@163.com(L.Wang).

c _{Theseauthorscontributedequallytothiswork.}

http://dx.doi.org/10.1016/j.bjid.2015.08.006

typingofS.aureusSSTIisolatesinthepresentstudyshowedconsiderableheterogeneity.

ST7 andST630becameprevailingclones. DifferentS.aureus clonescausingSSTIswere

associatedwithspecificantimicrobialresistanceandvirulencegeneprofiles.

©2015ElsevierEditoraLtda.Allrightsreserved.

Introduction

Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA), is an important human pathogen

respon-sible for many infectious diseases including skin and soft

tissueinfections(SSTIs),foreign-bodyinfections,pneumonia,

septicarthritis,endocarditis,osteomyelitis,sepsis,and

blood-streaminfectionsinbothhospitalandcommunitysettings.1

Theabilityofthisclinicallyimportantpathogento

success-fully persistwithinthe hostsislargely dueto thecarriage

ofa battery of virulence factors which promote adhesion,

acquisition of nutrients,and evasionof hostimmunologic

responses.2,3 _{Some S. aureus isolates also produce one or}

moreadditionalexoproteins,suchastoxicshocksyndrome

toxin-1(TSST-1),staphylococcalenterotoxins(SEs),exfoliative

toxins (ETs),and leukocidins.2–4 _{Recently, Panton-Valentine}

leukocidin(pvl)encodedbytwocontiguousandcotranscribed

genes(lukS-PVandlukF-PV)isanimportantvirulencefactor

forcommunity-acquiredMRSA(CA-MRSA)affecting

individ-ualswithoutapparentriskfactorsforhospitalacquisition.5,6

S.aureusisthe mostcommonbacterialpathogenidentified

from SSTIs.7 _{SSTIs caused by MRSA is associated with a}

highincidenceoftreatmentfailureandrecurrence.8 _A

bet-terunderstandingoftheantimicrobialsusceptibility,carriage

ofvirulencedeterminants,and molecularcharacteristics of

S. aureus isolates associated with SSTIs may provide

fur-therinsightsrelatedtoclinicaloutcomesoftheseinfections.

Moleculartypinghasprovedtobeanimportanttoolto

inves-tigate MRSA epidemiology. Pulsed-field gel electrophoresis

(PFGE)patterns,SCCmectyping,spatyping,andmulti-locus

sequencetyping(MLST)havebeenprovenusefulfor

monitor-ingtheevolutionaryprocessofpandemicMRSAclones.1_In

China,ST239-MRSA-IIIisapredominantMRSAcloneamong

adults, while ST59-MRSA-IV is the most prevalent clone

amongchildren.9,10 _{Inapreviousstudyweinvestigatedthe}

molecular typing of S. aureus isolated from patients with

SSTIsatourhospitalfromDecember2002toJune2008and

foundthatST239,ST1018,ST59,ST7,andST88werethemost

prevalentsequencetypes.11_{Ashiftofimportantcloneshas}

been observedin several studies.12–14 _{A reportfrom China}

found arapid change of MRSA over a 15-year period at a

tertiary care hospital,when the ST239-MRSA-III-t037 clone

wasreplacedbythe emergingST239-MRSA-III-t030clone.15

Understandingtheshiftofimportantclonesatthelocaland

internationallevelsisofgreatsignificance.Tounderstandthe

shiftofS.aureusclonesassociatedwithSSTIs, the present

study aimed toinvestigate the antimicrobial susceptibility,

carriageofvirulencedeterminants,andmolecular

character-isticsofS.aureusisolatesassociatedwithSSTIsatthehospital

in2012–2013.

Materials

and

methods

CollectionofclinicalisolatesandS.aureusconfirmation

From January2012 toSeptember 2013, a total of128

non-duplicateS.aureusisolates(singleisolateperpatient)were

collectedatTheFirstAffiliatedHospitalofWenzhouMedical

University,Chinafrompussamplesofhospitalizedpatients

withSSTIs.Lesionsrequiringincision anddrainageorwith

spontaneouslydrainingpurulentfluid,carbuncles,furuncles,

boils,cellulitiswithpurulentdrainage,chroniculcer,anddeep

woundswereincluded.S.aureusisolatesfrompatientswith

SSTIs with clinical signs and symptoms of infection such

as increased white blood cell counts, fever, local redness,

swelling,andexudatewereconsideredinvasiveisolatesand

includedforinvestigation.IsolateswereidentifiedasS.aureus

usingGramstain,positivecatalaseandcoagulasetestresults,

and Vitek microbiology analyzer (bioMérieu, Marcyl’Etoile,

France).S.aureusATCC25923wasusedasacontrolstrain.

Ethicsstatement

Thisstudy wasapprovedbytheInstitutionalEthics Review

BoardofTheFirstAffiliatedHospitalofWenzhouMedical

Uni-versity. All patients providedwritten informed consentfor

thisstudy.Thewritteninformedconsentswerealsoobtained

fromthenextofkin,caretakers,orguardiansonbehalfofthe

minors/childrenenrolled.

Antimicrobialsusceptibilitytesting

S. aureus susceptibility to penicillin (10 units),

eryth-romycin (15␮g), clindamycin (2␮g), rifampicin (5␮g),

tetracycline(30␮g), linezolid (30␮g), mupirocin (5␮g),

quin-upristin/dalfopristin(15␮g),trimethoprim/sulfamethoxazole

(1.25/23.75␮g), gentamicin (10␮g), ciprofloxacin (5␮g),

Chloramphenicol (30␮g), and nitrofurantoin (300␮g) were

determined using disc diffusion test recommended by the

Clinical and Laboratory Standards Institute (CLSI).16 _All

discs were obtainedfrom OxoidLtd. Vancomycin MICs for

S.aureusisolatesweredeterminedbyagardilutionmethod.

Interpretivestandardsfortheantimicrobialsusceptibilitytest

and D-test fortested S.aureusisolateswere in accordance

with the guidelines provided byCLSI.16 _{Susceptibility ofS.}

aureustomupirocinwasdeterminedbydiscdiffusion,with

azonediameter≥14mmona5␮gdiscindicating

suscepti-bilityasdescribedpreviously.17,18 _{S.aureusATCC25923 and}

EscherichiacoliATCC25922wereusedasreferencestrainsfor

DNAextraction

S.aureusisolatestestedwereculturedonbloodagarovernight

at 35◦C. Then, three to four bacterial colonies were

sus-pendedandincubated in150␮Lsteriledistilled waterwith

lysostaphin(1mg/mL)(Sangon,China)at37◦Cfor1h.Finally,

DNAwasextractedfollowingtheinstructionsoftheGenomic

DNAExtractionkit(Sangon,China).TheextractedDNAwas

storedat−20◦CandpreparedforPCRdetection.

IdentificationofMRSAisolatesandpvldetection

AmultiplexPCRprotocolwasusedforsimultaneous

ampli-fication of mecA, 16S rRNA, and pvl genes as described

previously.19_{MRSAisolatesharbouringmecAwereconfirmed}

usingMRSAN315andpvl-positiveMRSAisolateidentifiedin

ourpreviousstudyaspositivecontrolstrains.20

Detectionofvirulencegenes

Virulencegenes,includingtoxins(sea,seb,sec,sed,seg,seh,sei,

sej,seo,sen,sem,edin,hla,hlb,hld,hlg,tst,eta,etb),adhesins

(clfA, cna, sdrC, sdrD, and sdrE), icaA and arcA were

identi-fiedusingPCRassayswithprimersandconditionspreviously

described.21,22_{PCRprimersusedforPCRassayswereshown}

inTable1.

S.aureusisolatesharbouringvirulencegenesdetermined

inourpreviousstudywereusedaspositivecontrolstrainsfor

detectingvirulencegenes.20

SCCmectyping

SCCmec typing of MRSA isolates was performed using a

battery ofmultiplex PCRs as described previously.23 MRSA

isolateswithunanticipatedfragmentsorlacking fragments

bymultiplexPCRweredefinedasnon-typeable(NT). MRSA

NCTC10442(SCCmecI),MRSAN315(SCCmecII),MRSA85/2082

(SCCmecIII),MRSAJCSC4744(SCCmecIV),andMRSAWZ153

(SCCmecV)wereusedascontrolstrainsforSCCmectyping.

spatyping

The spa variable repeat region from each S. aureus isolate

was amplified using simplex PCR oligonucleotide primers

aspreviouslydescribed.24,25 _{Followingtheirpurificationand}

sequencing,spatypeswereassignedusingthespadatabase

website(http://www.ridom.de/spaserver).

Multi-locussequencetyping(MLST)

MLSTtypingofS.aureusisolateswasperformedusing

amplifi-cationofinternalfragmentsofthesevenhousekeepinggenes

ofS.aureusasdescribed previously.26_{Followingpurification}

andsequencingofthesegenes,thesequenceswerecompared

withtheexistingsequencesavailableontheMLSTwebsitefor

S.aureus(http://saureus.mlst.net),andSTsweredetermined

accordingtotheallelicprofiles.NovelSTsweredepositedin

theMLSTdatabase(http://saureus.mlst.net/).

Statisticalanalysis

Differencesbetweengroupswereassessedbyusingthe

chi-square test. The software SPSS 13.0 was used to perform

calculations. p-Valuesof<0.05 were consideredstatistically

significant.

Results

and

discussion

Antimicrobialsusceptibility

Among 128S.aureus isolates,57 (44.5%)were identifiedas

MRSAdetermined bycefoxitindisc diffusiontestand were

positiveformecA.TheMRSAprevalenceinthepresentstudy

waslowerthanthe54.1%reportedbetweenDecember2002

and June 2008.11 _{The resistance rates forS. aureus, MRSA,}

and MSSA isolates to antimicrobials are listed in Table 2.

Allisolatestestedweresusceptibletovancomycin,linezolid,

dalfopristin/quinupristin,andnitrofurantoin.Of128S.aureus

isolates,72.7%(93/128)withresistancetothreeormoreclasses

of antimicrobial agents tested were defined as

multidrug-resistantisolates.Onlytwoisolatestestedweresusceptibleto

allantimicrobialagentstested.Twenty-three(18.0%)isolates

wereonlyresistanttopenicillin.Ten(7.8%)isolateswere

con-comitantlyresistanttotwoantimicrobialagentstested

(peni-cillinandanotherantimicrobialagent).ResistanceratesofS.

aureus,MRSA,andMSSAisolatestopenicillin,clindamycin,

and gentamicin were above 60%, whereas to tetracycline,

gentamicin, ciprofloxacin, trimethoprim/sulfamethoxazole,

chloramphenicol and rifampicinwere lessthan 40%.Three

isolateswerepositiveforD-test,indicatingthatresistanceof

theseisolatestoclindamycinwasinducible.OnlyoneMRSA

isolatewasresistanttomupirocinasitexhibitednozoneof

inhibition.

Virulencegeneprofiling

TheinvasivenessofS.aureuslargelydependsonthecarriage

ofabatteryofvirulencefactors.2,3 _{AllS.aureusSSTIisolates}

inthepresentstudyharbouredatleastfivevirulencegenes

tested.FrequenciesofvirulencegenesareshowninTable3.

Virulencegenesweredetectedamongatleast70%ofalltested

isolatesincludedhld(100%),hla(95.3%),icaA(96.9%),clf(99.2%),

sdrC(79.7%),sdrD(70.3%),andsdrE(72.7%).Lessthan10%of

theisolatestestedcarriedeta(7.0%),sed(6.3%),seh(7.0%),tst

(4.7%),andedin(5.5%).Multipleisolatesharbouredpvl(12.5%),

sea(35.9%),seb(14.8%),sec(21.1%),sei(21.1%),seg(26.6%),sem

(30.5%),sen(31.3%),seo(27.3%),hlb(22.7%),hlg(18.8%),andcna

(32.0%).AllS.aureusisolatestestedwerenegativeforsej,etb,

and arcA.Fifty-six(43.75%,56/128) isolatesharbouredmore

than10testedvirulencegenes,amongwhichtwoisolates

har-boured16genes,sevenisolates14genes,13isolates13genes,

sixisolates12genes,10isolates11genes,and18isolates10

genes.OfMRSAisolates,54.4%(31/56)harbouredmorethan

10testedvirulencegenes,whichwassignificantlyhigherthan

thatamongMSSAisolates(35.2%,25/71)(p<0.05).

Staphylococcal enterotoxins (SEs), including five major

classicalantigenictypesofSEs(SEAtoSEE)andnewly

Table1–PCRprimersusedforPCRassays.

PCRproduct Primerdescription Primersequence Refs.

sea sea-Up sea-Dn TTGCAGGGAACAGCTTTAGGCAATC TGGTGTACCACCCGCACATTGA 21 seb seb-Up seb-Dn GACATGATGCCTGCACCAGGAGA AACAAATCGTTAAAAACGGCGACACAG 21 sec sec-Up sec-Dn CCCTACGCCAGATGAGTTGCACA CGCCTGGTGCAGGCATCATATC 21 sed sed-Up sed-Dn GAAAGTGAGCAAGTTGGATAGATTGCGGCTAG CCGCGCTGTATTTTTCCTCCGAGAG 21 see see-Up see-Dn TGCCCTAACGTTGACAACAAGTCCA TCCGTGTAAATAATGCCTTGCCTGAA 21 seg seg-Up seg-Dn TGCTCAACCCGATCCTAAATTAGACGA CCTCTTCCTTCAACAGGTGGAGACG 21 seh seh-Up seh-Dn CATTCACATCATATGCGAAAGCAGAAG GCACCAATCACCCTTTCCTGTGC 21 sei sei-Up sei-Dn TGGAGGGGCCACTTTATCAGGA TCCATATTCTTTGCCTTTACCAGTG 21 sej sem sen seo sej-Up sej-Dn sem-Up sem-Dn sen-Up sen-Dn seo-Up seo-Dn CTCCCTGACGTTAACACTACTAATAACCC TATGGTGGAGTAACACTGCATCAAAA CTATTAATCTTTGGGTTAATGGAGAAC TTCAGTTTCGACAGTTTTGTTGTCAT ATGAGATTGTTCTACATAGCTGCAAT AACTCTGCTCCCACTGAAC AGTTTGTGTAAGAAGTCAAGTGTAGA ATCTTTAAATTCAGCAGATATTCCATCTAAC 21 22 22 22 tst tst-Up tst-Dn AGCCCTGCTTTTACAAAAGGGGAAAA CCAATAACCACCCGTTTTATCGCTTG 21 eta eta-Up eta-Dn CGCTGCGGACATTCCTACATGG TACATGCCCGCCACTTGCTTGT 21 etb hla hlb hld hlg etb-Up etb-Dn hla-Up hla-Dn hlb-Up hlb-Dn hld-Up hld-Dn hlg-Up hlg-Dn GAAGCAGCCAAAAACCCATCGAA TGTTGTCCGCCTTTACCACTGTGAA CTGATTACTATCCAAGAAATTCGATTG CTTTCCAGCCTACTTTTTTATCAGT GTGCACTTACTGACAATAGTGC GTTGATGAGTAGCTACCTTCAGT AAGAATTTTTATCTTAATTAAGGAAGGAGTG TTAGTGAATTTGTTCACTGTGTCGA GTCAYAGAGTCCATAATGCATTTAA CACCAAATGTATAGCCTAAAGTG 21 22 22 22 22 sdrC sdrC-Up sdrC-Dn CGCATGGCAGTGAATACTGTTGCAGC GAAGTATCAGGGGTGAAACTATCCACAAATTG 21 sdrD sdrD-Up sdrD-Dn CCACTGGAAATAAAGTTGAAGTTTCAACTGCC CCTGATTTAACTTTGTCATCAACTGTAATTTGTG 21 sdrE sdrE-Up sdrE-Dn GCAGCAGCGCATGACGGTAAAG GTCGCCACCGCCAGTGTCATTA 21 cna cna-Up cna-Dn TTCACAAGCTTGGTATCAAGAGCATGG GAGTGCCTTCCCAAACCTTTTGAGC 21 clfA clfA-Up clfA-Dn ATTGGCGTGGCTTCAGTGCTTG GCTTGATTGAGTTGTTGCCGGTGT 21 arcA arcA-Up arcA-Dn CACGTAACTTGCTAGAACGAG GAGCCAGAAGTACGCGAG 21 icaA icaA-Up icaA-Dn TCAGACACTTGCTGGCGCAGTC TCACGATTCTCTCCCTCTCTGCCATT 21

Additionally, SEs are also associated with other diseases

such as allergy sensitization, asthma, chronic obstructive

pulmonary disease, scarlet fever, glomerulonephritis, and

vasculitis.28–31 _{The genes encoding these SEs but sej were}

foundamongS.aureusSSTIisolates,withdifferentcarriage

proportionsrangingfrom6.3%to35.9%inthisinvestigation.In

particular,thepositiveratesforseb,sen,andsemamongMRSA

isolatesweresignificantlyhigherthanamongMSSAisolates

(p<0.05).TheSdrproteinsencodedbythetandemlyarrayed

sdrC,sdrD,andsdrEaremicrobialsurfacecomponentswhich

recognizeadhesivematrixmoleculesandhavedifferentroles

in S.aureuspathogenicity.32 _{Strong correlations betweenS.}

aureusinvasivenessandthepresenceofoneoftheallelic

vari-ants ofthe sdrEgene, aswell ascarriage ofthe sdrDgene

andboneinfectionscausedbyS.aureus,havebeenreported

previously.33,34_{Ourpreviousstudyshowedthat95.5%(85/89)}

ofS.aureusisolatesresponsibleforbloodstreaminfection

Table2–AntimicrobialresistanceprofilesofMRSA, MSSA,andS.aureusisolates.

MRSA (n=57) R(%) MSSA(n=71) R(%) S.aureus (n=128) R(%) Tetracycline 36.8 28.2 32.0 Gentamicin 31.6 14.1 21.9 Penicillin 96.5 97.2 96.9 Oxacillin 100 0.0 44.5 Clindamycin 71.9 66.2 68.8 Erythromycin 71.9 67.6 69.5 Ciprofloxacin 36.8 15.5 25.0 Linezolid 0 0 0 Rifampicin 10.5 0 4.7 Trimethoprim/ sulfamethoxazole 28.1 19.7 23.4 Nitrofurantoin 0 0 0 Cefaclor 40.4 1.4 18.8 Chloroamphenicol 15.8 5.6 10.2 Imipenem 17.5 0 7.8 Mupirocin 1.8 0 0.8 Dalfopristin/ quinupristin 0 0 0 Vancomycin 0 0 0

Table3–ThefrequenciesofvirulencegenesamongS. aureus,MRSAandMSSAisolates.

S.aureus (n=128) (%) MRSA (n=57) (%) MSSA (n=71) (%) p-valuesa sea 35.9 42.1 31.0 >0.05 seb 14.8 21.1 9.9 <0.05 sec 21.1 21.1 21.1 sed 6.3 1.8 9.9 >0.05 seg 26.6 31.6 22.5 >0.05 seh 7.0 3.5 9.9 >0.05 sei 21.1 21.1 21.1 sej 0 0 0 sen 31.3 43.9 21.1 <0.05 sem 30.5 43.9 19.7 <0.05 seo 27.3 26.3 28.2 >0.05 tst 4.7 5.3 4.2 >0.05 eta 7.0 1.8 11.3 >0.05 etb 0 0 0 clfA 99.2 98.2 100 >0.05 cna 32.0 26.3 36.6 >0.05 sdrC 79.7 89.5 71.8 >0.05 sdrD 70.3 70.2 70.4 >0.05 sdrE 72.7 80.7 66.2 <0.05 icaA 96.9 96.5 97.2 >0.05 arcA 0 0 0 pvl 12.5 15.8 9.9 <0.05 hla 95.3 100 91.5 >0.05 hlb 22.7 26.3 19.7 >0.05 hld 100 100 100 hlg 18.8 17.5 19.7 >0.05 edin 5.5 0 9.9 >0.05

a _{MRSAgroupwascomparedwithMSSAgroup.}

thantwosdrloci.35_{Similarly,120(93.8%)of128SSTIisolatesin}

thepresentstudywerefoundtoharbouratleastonesdrlocus

(sdrC,sdrD,orsdrE),with18(14.1%),39(30.5%),and63(49.2%)

harbouringone,two,orthreeoftheseloci,respectively.The

positiverateofsdrEamongMRSAisolateswassignificantly

higherthanamongMSSAisolates.S.aureusisolates

produc-ing TSST-1 encoded by the tst gene have been associated

withtoxicshocksyndrome,staphylococcalscarletfever,and

neonataltoxicshock-likeexanthematousdiseases.3_However,

thepresentstudyfoundthatonly4.7%ofS.aureusSSTIisolates

harbouredtst.Ourpreviousstudyfoundthattheprevalence

ofhlbamongS.aureusisolatesassociatedwithbloodstream

infectionwas67.4%.35_{However,hlbwasonlyidentifiedamong}

22.7%ofS.aureusSSTIisolates,whilethepositivityratesofhla

andhldwere95.3%and100%,respectively.

pvlhasbeencloselyassociatedwithCA-MRSAinfections

and there is astrong epidemiological association between

carriage of pvlgenes and successfulCA-MRSA lineages.1,36

Infections causedbypvl-positive S. aureusisolatesare

pre-dominantlyrepresentedbyskinandsoft-tissueinfection.5,6

AmongS.aureusisolatescausingSSTIinourhospitalbetween

December2002andJune2008,theoverallpositivityratesof

pvlgenes23.4%(26/111),andamongMRSAandMSSAisolates

therateswere,21.7%(13/60)and25.5%(13/51),respectively.11

Comparedwithourpreviousstudy,theoverallpositivityrates

ofpvlgenesinthepresentstudy(12.5%)waslower,aswere

theratesamongMRSA(15.8%)andMSSA(9.9%)isolates,

indi-catingthatthereisadecreasedtrendintheprevalenceofpvl

genesamongS.aureusSSTIisolatesatourhospital.

Moleculartyping

Molecular typing of S. aureusisolates tested are shown in

Table4.Among57MRSAisolates,24,14,13,andthree

har-boured SCCmec types III, IV, II, and V, respectively. Three

isolateswereclassifiedasnon-typeable.

A total of 28 STs were identified among 128 S. aureus

isolates. ST7and ST630accounting for10.9% (14/128 each)

were found to be the predominant STs, followed by ST59

(8.6%, 11/128), ST5 (6.3%, 8/128), ST88 (6.3%, 8/128), ST1

(5.5%, 7/128), ST965(5.5%, 7/128), ST398(4.7%, 6/128each),

ST25 (4.7%,6/128),andST188(3.9%,5/128).ST239,ST6,and

ST121accountedforfour isolateseach.ST1463andST1821

accountedforthreeisolateseach.ST8andST1349accounted

for two isolates each. The remaining STs including ST12,

ST15,ST118,ST692,ST789,ST1281,ST1920,ST2259,ST2832,

ST2833, and ST72 were identified in only oneisolate. The

STsofnineisolateswerenotidentified.TwonovelSTs

char-acterized asST2832and 2833wereidentified intwoMRSA

isolates and have been deposited in the MLST database

(http://saureus.mlst.net/).SixteenPVL-positiveisolateswere

distributedamongninedifferentSTsincludingST88(five

iso-lates),ST59(threeisolates),andST121(twoisolates).ST239

andST5werethemostdominantSTsinChina.9,37

Interest-ingly,inthepresentstudy,thesetwopredominantSTswere

foundtobeminorclones,whileST630andST7,seldomnoted

inChineseisolatespreviously,werethemajorclonesamong

S.aureusSSTIisolates.AnotherstudyfromChinafoundST398

Table4–MolecularcharacteristicsofS.aureusSSTIsisolates.

CC(no.) STs(no.) spatypes(no.) MRSA(no.) MSSA(no.) SCCmec(no.)

5(60) ST630(14) t377(7) 2 5 III(2) t4047(2) 2 t030(1) 1 III t4549(1) 1 t4047(2) 2 II(1),IV(1) t5554(1) 1 ST5(8) t2460(2) 2 II(2) t311(2) 2 II(2) t4352(1) 1 II t002(1) 1 t548(1) 1 t535(1) 1 ST965(7) t062(7) 7 III(4),II(2),NT(1) ST25(6) t349(2) 2 t227(2) 2 III(2) t8170(1) 1 t078(1) 1 ST188(5) t189(4) 1 3 II t1858(1) 1 III ST239(4) t030(3) 3 III(3) t2270(1) 1 III(1) ST6(4) t701(3) 3 t10519(1) 1 III ST1462(3) t189(3) 3 ST1821(3) t377(1) 1 t2196(1) 1 t4549(1) 1 II ST8(2) t008(1) 1 t377(1) 1 ST15(1) t062 1 ST118(1) t189(1) 1 ST1920(1) t286(1) 1 ST72(1) t148 1 ST1(7) t127(7) 2 5 III(2) 7(15) ST7(14) t091(8) 2 6 III(2) t796(5) 5 t2828(1) 1 IV ST789(1) t091(1) 1 III 59(11) ST59(11) t437(8) 7 1 IV(6),V(1) t163(3) 2 1 IV(1),NT(1) 88(8) ST88(8) t5348(3) 1 2 IV t1764(1) 1 II t5351(1) 1 t2788(1) 1 t7637 1 V t1376 1 IV 398(6) ST398(6) t571(4) 1 3 IV t011(1) 1 t034(1) 1 121(4) ST121(4) t159(3) 2 1 III(1),IV(1) t2091(1) 1

S.aureus SSTI isolates.38 _{In contrast to our previous study}

whereST239wasthemostprevalentSTaccountingfor21.6%

(24/111) of S. aureus SSTI isolates,11 _{this ST accounted for}

only3.1%(4/128)inthepresentstudy.Interestingly,ST630not

foundinour previousstudyturnedout tobethe

predomi-nantSTinthepresentstudy,whileST1018,thesecondmost

prevalentSTinourpreviousstudy,wasnotfound.

Fifty-twospatypeswereidentifiedamongthe128isolates.

Themostprevalentspatypewast091(8.6%,11/128),followed

byt062(7.0%,9/128),t377(7.0%,9/128),t437(7.0%,9/128),t189

(6.3%,8/128),t127(4.7%,6/128),t796(3.9%,4/128),t571(3.1%,

3/128),t4047(3.1%,3/128),andt030(3.1%,3/128).Othertypes

identifiedincludedt159,t163,t5348,andt701(threeisolates

each). In previous reportsof Chinese isolates, t30and t37,

typicallyassociatedwithST239,werethemostprevalentspa

types.9,15_{Theproportionsoft37andt30wereextremelylow,}

aswellastheproportionsofST239andST5inthe present

Fifty-seven MRSA isolates were distributed in different

clones. 12.3% (7/57) of MRSA isolates belonged to

MRSA-ST59-SCCmec IV-t 437/163, which was the most common

cloneinthepresentstudy.Likewise,inastudyfrom China

ST59-MRSA-IVa-t437wasfoundtobethepredominantclone

amongCA-MRSAisolatesassociatedwithSSTIsinchildren.39

OurpreviousstudyconductedbetweenDecember 2002and

June2008foundthatST239-MRSA-SCCmecIIIaccountingfor

30.2% (19/63) was the most prevalent clone among MRSA

SSTIisolates, followedbyST1018-MRSA-SCCmecIII

account-ing for 15.9% (10/63).11 _{However,only three MRSA isolates}

were ST239-MRSA-SCCmec III and there was no

ST1018-MRSA-SCCmecIII isolate in the present study. Among 71

MSSA isolates, the dominant clone was MSSA-ST7 (15.5%,

11/71),followedbyMSSA-ST630(12.7%,9/71),MSSA-ST398and

MSSA-ST1(7.0%,5/71each),andMSSA-ST25andMSSA-ST88

(5.6%,4/71each).MRSA-ST398isolatesareusuallyassociated

withinfectionsinbothanimalsandhumans.40,41_ST398MSSA

isolatesofhumanoriginareusuallylinkedtot571.42 _Inthe

presentstudy,fourofsixST-398S.aureusisolates,including

threeMSSAandoneMRSAisolate,werelinkedtot571.

Taken together, our data showed that S.aureus isolates

associatedwithSSTIsweregeneticallydiverseandthemain

clonesassociatedwithSSTIsaregoingthrougharapidshiftin

ourhospital.

Comparisonofantimicrobialresistanceandmolecular typingamongthemajorclonalcomplexes(CCs)

Clustering analysis by eBURST v3 showed that six clonal

complexes(CCs)werefound(Table4),includingCC5(52.3%,

67/128),CC7(11.7%,15/128),CC59(8.6%,11/128),CC88(6.3%,

8/128),andCC398(4.7%,6/128).Thedistributionofsome

vir-ulencegenes,especiallyenterotoxin genes,were correlated

with different MRSA lineages.43,44 _{In the present study, a}

highercarriage ofseb and secwas foundamongCC59

iso-lates,whileahighercarriageofenterotoxingeneswerefound

amongCC5isolates(Table5).AllCC7andCC59isolateswere

notcarryingsehandseo,while11.8%and34.2%ofCC5isolates

werefoundtocarrythesetwogenes,respectively.Although

theproportionsofsebandsecamongCC5and CC7isolates

werelow,thepositivityratesofthesetwoenterotoxingenes

amongCC59 isolateswere 54.5% (seb) and 63.6% (sec). The

prevalenceofcnaamongCC5isolateswas38.8%,whilethis

virulencegenewasidentifiedinnoneoftheCC7and CC59

isolates.Comparedwiththehighercarriagerates(>80.0%)of

sdrCandsdrDamongCC5andCC7isolates,thepositivityrates

oftwosdrlociweresignificantlyloweramongCC59isolates,

especiallysdrDprevalenceof9.1%.Interestingly,allCC59

iso-latescarriedsdrE,whileonly26.7%ofCC7isolateswerefound

tocarrysdrE.Thecarriageratesofpvland hlbamongCC59

isolatesweresignificantlyhigherthanthoseratesamongCC5

andCC7isolates(p<0.05).Thesedifferencesincarriagerates

ofvirulencegenesamongdifferentCCisolatessuggestedthat

differentS.aureuslineagesassociatedwithSSTIshavespecific

patternsofvirulencegenes.

The predominant MRSA clones in China were

associ-atedwithspecificantimicrobialresistanceprofiles.45 _Inthe

presentstudy,although81.8%ofCC59isolateswereMRSA,all

isolates were susceptible to gentamicin, rifampicin, and

Table5–Specificantimicrobialresistanceandvirulence geneprofilesofmajorCCs.

CC5(n=67) (%) CC7(n=15) (%) CC59(n=11) (%) Virulencegenes sea 37.3 23.3 27.3 seb 7.5 13.3 54.5 sec 19.4 0 63.6 sed 10.4 6.7 0 seg 34.3 0 27.3 seh 11.8 0 0 sei 29.8 6.7 0 sen 44.8 6.7 27.3 sem 40.3 6.7 9.1 seo 34.3 0 0 tst 7.5 0 0 eta 9 6.7 9.1 clfA 100 100 100 cna 38.8 0 0 sdrC 89.6 93.3 54.5 sdrD 82.1 100 9.1 sdrE 82.1 26.7 100 icaA 95.5 100 100 pvl 4.5 0 27.3 hla 91.0 93.3 100 hlb 26.9 0 45.5 hld 100 100 100 hlg 10.4 13.3 27.3 edin 4.5 0 0 Antimicrobialagents MRSA 43.3 26.7 81.8 Tetracycline 25.4 66.7 45.5 Gentamycin 25.4 40.0 0 Penicillin 97 93.3 100 Clindamycin 59.7 73.3 81.8 Erythromycin 62.7 73.3 81.8 Ciprofloxacin 35.8 6.7 18.2 Rifampicin 7.5 0 0 Trimethoprim/ sulfamethoxazole 20.9 40 0 Chloroamphenicol 9.0 0 27.3 Mupirocin 0 6.7 0

trimethoprim/sulfamethoxazole (Table 5). However,

the resistance rates for gentamicin and

trimetho-prim/sulfamethoxazole were respectively 25.4% and 20.9%

amongCC5isolates,and40.0%and40.0%amongCC7isolates

(Table5).Theresistanceratesfortetracycline,clindamycin,

anderythromycinamongCC5isolateswererelatively lower

relativetoCC7andCC59isolates(Table5).Astheprevalence

ofMRSAamongCC7isolateswaslowerthanCC5andCC59

isolates, the resistancerates of some antimicrobialagents

amongCC7isolateswerelowerthanotherCCisolates,suchas

ciprofloxacinandchloramphenicol.However,onlyoneisolate

withresistancetomupirocininthisinvestigationbelonged

toCC7.

In conclusion, the molecular characteristic of S. aureus

SSTIisolatesinthepresentstudyshowedconsiderable

het-erogeneity and ST7 and ST630 were the prevalent clones.

Different S. aureus clones causing SSTIs were associated

with specific antimicrobial resistance and virulence gene

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisstudywassupportedbygrantsfromNaturalScienceFund

ofChina(81271906H2002),MedicalScienceFundofZhejiang

Province,China (2012RCA041)and WenzhouMunicipal

Sci-enceandTechnologyBureau,China(Y20130239).

r

e

f

e

r

e

n

c

e

s

1. DavidMZ,DaumRS.Community-associated

methicillin-resistantStaphylococcusaureus:epidemiologyand clinicalconsequencesofanemergingepidemic.Clin MicrobiolRev.2010;23:616–87.

2. BubeckWardenburgJ,PatelRJ,SchneewindO.Surface proteinsandexotoxinsarerequiredforthepathogenesisof Staphylococcusaureuspneumonia.InfectImmun.

2007;75:1040–4.

3. DingesMM,OrwinPM,SchlievertPM.Exotoxinsof Staphylococcusaureus.ClinMicrobiolRev.2000;13:16–34.

4. BunikowskiR,MielkeME,SkarabisH,etal.Evidencefora disease-promotingeffectofStaphylococcusaureus-derived exotoxinsinatopicdermatitis.JAllergyClinImmunol. 2000;105:814–9.

5. GilletY,IssartelB,VanhemsP,etal.Associationbetween Staphylococcusaureusstrainscarryinggenefor

Panton-Valentineleukocidinandhighlylethalnecrotising pneumoniainyoungimmunocompetentpatients.Lancet. 2002;359:753–9.

6. FrancisJS,DohertyMC,LopatinU,etal.Severe

community-onsetpneumoniainhealthyadultscausedby methicillin-resistantStaphylococcusaureuscarryingthe Panton-Valentineleukocidingenes.ClinInfectDis. 2005;40:100–7.

7. RayGT,SuayaJA,BaxterR.Incidence,microbiology,and patientcharacteristicsofskinandsoft-tissueinfectionsina U.S.population:aretrospectivepopulation-basedstudy.BMC InfectDis.2013;13:252.

8. LabrecheMJ,LeeGC,AttridgeRT,etal.Treatmentfailureand costsinpatientswithmethicillin-resistantStaphylococcus aureus(MRSA)skinandsofttissueinfections:aSouthTexas AmbulatoryResearchNetwork(STARNet)study.JAmBoard FamMed.2013;26:508–17.

9. LiuY,WangH,DuN,etal.Molecularevidenceforspreadof twomajormethicillin-resistantStaphylococcusaureusclones withauniquegeographicdistributioninChinesehospitals. AntimicrobAgentsChemother.2009;53:512–8.

10.GengW,YangY,WuD,etal.Community-acquired, methicillin-resistantStaphylococcusaureusisolatedfrom childrenwithcommunity-onsetpneumoniainChina.Pediatr Pulmonol.2010;45:387–94.

11.YaoD,YuFY,QinZQ,etal.Molecularcharacterizationof Staphylococcusaureusisolatescausingskinandsofttissue infections(SSTIs).BMCInfectDis.2010;10:133.

12.Aires-de-SousaM,CorreiaB,deLencastreH.Changing patternsinfrequencyofrecoveryoffivemethicillin-resistant StaphylococcusaureusclonesinPortuguesehospitals: surveillanceovera16-yearperiod.JClinMicrobiol. 2008;46:2912–7.

13.SolaC,CortesP,SakaHA,etal.Evolutionandmolecular characterizationofmethicillin-resistantStaphylococcusaureus

epidemicandsporadicclonesinCordoba,Argentina.JClin Microbiol.2006;44:192–200.

14.ConceicaoT,Aires-de-SousaM,FuziM,etal.Replacementof methicillin-resistantStaphylococcusaureusclonesinHungary overtime:a10-yearsurveillancestudy.ClinMicrobiolInfect. 2007;13:971–9.

15.ChenH,LiuY,JiangX,etal.Rapidchangeof

methicillin-resistantStaphylococcusaureusclonesinaChinese tertiarycarehospitalovera15-yearperiod.Antimicrob AgentsChemother.2010;54:1842–7.

16.CLSI.Performancestandardsforantimicrobialsusceptibility testing,18thinformationalsupplement(M100-S22).Wayne, PA,USA:ClinicalandLaboratoryStandardsInstitute;2012.

17.FuchsPC,JonesRN,BarryAL.Interpretivecriteriafordisk diffusionsusceptibilitytestingofmupirocin,atopical antibiotic.JClinMicrobiol.1990;28:608–9.

18.AnthonyRM,ConnorAM,PowerEG,etal.Useofthe polymerasechainreactionforrapiddetectionofhigh-level mupirocinresistanceinstaphylococci.EurJClinMicrobiol InfectDis.1999;18:30–4.

19.McClureJA,ConlyJM,LauV,etal.NovelmultiplexPCRassay fordetectionofthestaphylococcalvirulencemarker Panton-Valentineleukocidingenesandsimultaneous discriminationofmethicillin-susceptiblefrom-resistant staphylococci.JClinMicrobiol.2006;44:1141–4.

20.YuF,YangL,PanJ,etal.Prevalenceofvirulencegenesamong invasiveandcolonisingStaphylococcusaureusisolates.JHosp Infect.2011;77:89–91.

21.CampbellSJ,DeshmukhHS,NelsonCL,etal.Genotypic characteristicsofStaphylococcusaureusisolatesfroma multinationaltrialofcomplicatedskinandskinstructure infections.JClinMicrobiol.2008;46:678–84.

22.JarraudS,MougelC,ThioulouseJ,etal.Relationships betweenStaphylococcusaureusgeneticbackground,virulence factors,agrgroups(alleles),andhumandisease.Infect Immun.2002;70:631–41.

23.KondoY,ItoT,MaXX,etal.CombinationofmultiplexPCRs forstaphylococcalcassettechromosomemectype assignment:rapididentificationsystemformec,ccr,and majordifferencesinjunkyardregions.AntimicrobAgents Chemother.2007;51:264–74.

24.KoreenL,RamaswamySV,GravissEA,etal.spatyping methodfordiscriminatingamongStaphylococcusaureus isolates:implicationsforuseofasinglemarkertodetect geneticmicro-andmacrovariation.JClinMicrobiol. 2004;42:792–9.

25.HarmsenD,ClausH,WitteW,etal.Typingof

methicillin-resistantStaphylococcusaureusinauniversity hospitalsettingbyusingnovelsoftwareforsparepeat determinationanddatabasemanagement.JClinMicrobiol. 2003;41:5442–8.

26.EnrightMC,DayNP,DaviesCE,etal.Multilocussequence typingforcharacterizationofmethicillin-resistantand methicillin-susceptibleclonesofStaphylococcusaureus.JClin Microbiol.2000;38:1008–15.

27.ThomasD,ChouS,DauwalderO,etal.Diversityin Staphylococcusaureusenterotoxins.ChemImmunolAllergy. 2007;93:24–41.

28.Semic-JusufagicA,BachertC,GevaertP,etal.Staphylococcus aureussensitizationandallergicdiseaseinearlychildhood: population-basedbirthcohortstudy.JAllergyClinImmunol. 2007;119:930–6.

29.BachertC,GevaertP,ZhangN,etal.Roleofstaphylococcal superantigensinairwaydisease.ChemImmunolAllergy. 2007;93:214–36.

30.WangCC,LoWT,HsuCF,etal.EnterotoxinBisthe

predominanttoxininvolvedinstaphylococcalscarletfeverin Taiwan.ClinInfectDis.2004;38:1498–502.

31.KoyamaA,KobayashiM,YamaguchiN,etal.

GlomerulonephritisassociatedwithMRSAinfection:a possibleroleofbacterialsuperantigen.KidneyInt. 1995;47:207–16.

32.JosefssonE,McCreaKW,NiEidhinD,etal.Threenew membersoftheserine-aspartaterepeatproteinmultigene familyofStaphylococcusaureus.Microbiology.1998;144Pt 12:3387–95.

33.PeacockSJ,MooreCE,JusticeA,etal.Virulentcombinations ofadhesinandtoxingenesinnaturalpopulationsof Staphylococcusaureus.InfectImmun.2002;70:4987–96.

34.TradS,AllignetJ,FrangeulL,etal.DNAmacroarrayfor identificationandtypingofStaphylococcusaureusisolates.J ClinMicrobiol.2004;42:2054–64.

35.YuF,LiT,HuangX,etal.Virulencegeneprofilingand molecularcharacterizationofhospital-acquired

Staphylococcusaureusisolatesassociatedwithbloodstream infection.DiagnMicrobiolInfectDis.2012;74:363–8.

36.RasigadeJP,LaurentF,LinaG,etal.Globaldistributionand evolutionofPanton-Valentineleukocidin-positive methicillin-susceptibleStaphylococcusaureus,1981–2007.J InfectDis.2010;201:1589–97.

37.ZhangW,ShenX,ZhangH,etal.Molecularepidemiological analysisofmethicillin-resistantStaphylococcusaureusisolates fromChinesepediatricpatients.EurJClinMicrobiolInfect Dis.2009;28:861–4.

38.ZhaoC,LiuY,ZhaoM,etal.Characterizationofcommunity acquiredStaphylococcusaureusassociatedwithskinandsoft tissueinfectioninBeijing:highprevalenceofPVL+ST398. PLoSONE.2012;7:e38577.

39.WuD,WangQ,YangY,etal.Epidemiologyandmolecular characteristicsofcommunity-associatedmethicillin-resistant andmethicillin-susceptibleStaphylococcusaureusfrom skin/softtissueinfectionsinachildren’shospitalinBeijing, China.DiagnMicrobiolInfectDis.2010;67:1–8.

40.vanDuijkerenE,IkawatyR,Broekhuizen-StinsMJ,etal. Transmissionofmethicillin-resistantStaphylococcusaureus strainsbetweendifferentkindsofpigfarms.VetMicrobiol. 2008;126:383–9.

41.PanA,BattistiA,ZoncadaA,etal.Community-acquired methicillin-resistantStaphylococcusaureusST398infection, Italy.EmergInfectDis.2009;15:845–7.

42.DavidMZ,SiegelJ,LowyFD,etal.Asymptomaticcarriageof sequencetype398,spatypet571methicillin-susceptible Staphylococcusaureusinanurbanjail:anewlyemerging, transmissiblepathogenicstrain.JClinMicrobiol. 2013;51:2443–7.

43.DiepBA,CarletonHA,ChangRF,etal.Rolesof34virulence genesintheevolutionofhospital-and

community-associatedstrainsofmethicillin-resistant Staphylococcusaureus.JInfectDis.2006;193:1495–503.

44.KimT,YiJ,HongKH,etal.Distributionofvirulencegenesin spatypesofmethicillin-resistantStaphylococcusaureus isolatedfrompatientsinintensivecareunits.KoreanJLab Med.2011;31:30–6.

45.ChengH,YuanW,ZengF,etal.Molecularandphenotypic evidenceforthespreadofthreemajormethicillin-resistant Staphylococcusaureusclonesassociatedwithtwo

characteristicantimicrobialresistanceprofilesinChina.J AntimicrobChemother.2013;68:2453–7.