Antimicrobial susceptibility and fluctuations in clonal complexes of serogroup 6 Streptococcus pneumoniae isolates collected from children in Beijing, China, between 1997 and 2016

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

Clinical

Microbiology

Antimicrobial

susceptibility

and

ﬂuctuations

in

clonal

complexes

of

serogroup

6 Streptococcus

pneumoniae

isolates

collected

from

children

in

Beijing,

China,

between

1997

and

2016

Wei

Shi

1

_,

_Ying

_Liu

1

_,

_Qinghong

_Meng,

_Lin

_Yuan,

_Wei

_Gao,

_Kaihu

_Yao

∗

CapitalMedicalUniversity,NationalCenterforChildren’sHealth,BeijingChildren’sHospital,BeijingPediatricResearchInstitute, NationalKeyDisciplineofPediatrics(CapitalMedicalUniversity),NationalClinicalResearchCenterforRespiratoryDiseases,Ministryof Education,KeyLaboratoryofMajorDiseasesinChildren,BeijingKeyLaboratoryofPediatricRespiratoryInfectionDiseases,Beijing,China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15August2017 Accepted10February2018 Availableonline14March2018 AssociateEditor:AgnesFigueiredo

Keywords: Streptococcuspneumoniae Children Antimicrobialsusceptibility Clonalcomplex(CC) Sequencetype(ST)

a

b

s

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Thisstudyexaminedtheantimicrobialsusceptibilitypatternsandclonalcomplex(CC) char-acteristicsofserogroup6StreptococcuspneumoniaeisolatescollectedfromchildreninBeijing, China,between1997and2016.SerotypesweredeterminedusingtheQuellungreaction, andtheantimicrobialsusceptibilityprofilesoftheisolatesweredeterminedusingthe disc-diffusion methodor byE-test.Sequencetypes(STs)wereassignedbasedonmultilocus sequencetyping.Atotalof250isolateswereexamined,with55.2%,30.0%,12.8%,and2.0% ofisolatesidentifiedasserotypes6A,6B,6C,and6D,respectively.Alloftheisolateswere sus-ceptibletolevofloxacinandvancomycin,andthenon-suceptibitilityratetopenicillinwas 41.6%.Eighty-twodistinctSTs,assignedto13CCsand28singletons,wereidentified.CC982 wasthemostprevalentCCamongstserotype6Aisolates(34%),followedbyCC9789and CC3173.Amongstserotype6Bisolates,CC90andCC4542werethemostcommon,accounting for25.3%and14.7%ofisolatesrespectively.Overthestudyperiod,theprevalenceofCC982, CC4542,andCC4536isolatesshowingsusceptibilitytopenicillinandcefuroximedecreased, andtheproportionofCC3173,CC9789,CC855,andCC902isolatesshowingnon-susceptibility tothesetwoantibioticsincreased.

creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Corresponding}_author.

E-mail:[email protected](K.Yao).

1 _These_authors_contributed_equally_to_this_work.

Introduction

Streptococcuspneumoniae,themostcommoncauseofbacterial

pneumonia,cancauseaseriesofinvasiveandnon-invasive diseases,andseriouslythreatensthehealthofchildren world-wide. UsingtheDanishtypingsystembasedondifferences in the polysaccharidecapsule, morethan 90 S.pneumoniae

https://doi.org/10.1016/j.bjm.2018.02.004

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serotypes have been detected. Serogroup 6, comprising serotypes 6A,6B, 6C,and 6D,isoneofthe mostprevalent serogroupsworldwide.1–3

The ﬁrst pneumococcal conjugate vaccine (PCV), PCV7, offered protection against seven serotypes (4, 6B, 9V, 14, 18C,19F,and 23F)andwaslicensed in2000.By2015,PCV7 had been replaced with the higher valent vaccines PCV10 (PCV7+1, 5, and 7F) and PCV13 (PCV10+19A, 6A, and 3), whichwereintroducedinover130countries.4,5_Research_into

theefﬁcacyofPCVsshowedthattheirwidespread availabil-ity decreasednasopharyngeal carriage ratesand infections causedbyvaccineserotypes.6–8However,aserotype replace-mentphenomenonappearedwiththeintroductionofPCVs, wherebytheprevalenceofnon-vaccineserotypesincreased withconcomitant decreasesinvaccineserotypes.9–12 _While

there is no universal immunization program for PCVs in China, PCV7 was licensed in 2008 by the private sector, and was withdrawn at the end of 2014. Despite this, an investigation performed in 2011 showed that the vaccina-tion coverage of PCV7 in children under 2 years old was only 9.91%.13 _PCV13 _was _not _introduced _in _China _until

April 2017, and serogroup 6 pneumococcal strains remain prevalent.14–16

Theaimofthecurrentstudywastoexaminethe antimicro-bialsusceptibilityproﬁlesandclonalcomplexcharacteristics ofserogroup6S.pneumoniaeisolatescollectedfromchildrenin Beijingbetween1997and2016,andtoexploretherelationship betweenantibioticresistanceandclonalcomplex.

Materials

and

methods

Bacterialstrains

A total of 1865 S. pneumoniae isolates were cultured from nasopharyngealspecimensor bronchoalveolarlavage spec-imens collected from children visiting Beijing Children’s Hospitalbetween1997and2016.ThehospitalisaNational Children’sMedicalCenter,withmorethan3million outpa-tientvisitsand70,000hospitalizationseveryyear.Amongthe 1865isolates,250wereserogroup6,andthepercentagerate was13.4%.Thedistributionofthe250serogroup6isolatesby yearwasasfollows:1997,34isolates;1998,6isolates;1999, 6isolates;2000,12isolates;2001,8isolates;2002,4isolates; 2003,6isolates;2004,19isolates;2005,11isolates;2008,6 iso-lates;2009,10isolates;2010,9isolates;2013,41isolates;2014, 32isolates;2015,23isolates;2016,23isolates.Becauseofthe lackofpersonnel,theisolatescollectingworkwasbrokenoff in2006–2007and2011–2012.Alloftheisolateswereidentiﬁed asserotype6A,6B,6C,or6DbyQuellungtestusingantiserum providedbyStatensSerumInstitute(Copenhagen,Denmark). TheEthicsCommitteeofBeijingChildren’sHospital afﬁli-atedtoCapitalMedicalUniversityexemptedthisstudyfrom reviewbecauseitfocusedonthebacterialisolatesanddidnot includepatientdata.

Antimicrobialsusceptibilitytesting

The minimum inhibitory concentrations (MICs) of peni-cillin, amoxicillin-clavulanic acid, ceftriaxone, cefuroxime,

erythromycin, imipenem, levoﬂoxacin, and vancomycin for each of the isolates were determined using E-test strips (AB Biodisk, Solna, Sweden). Disc diffusion tests (Oxoid) were performed to ascertain antimicrobial sus-ceptibilitiestotetracycline,sulfamethoxazole-trimethoprim, and chloramphenicol. The results were interpreted in accordance with the Clinical and Laboratory Standards Institute 2016 guidelines, and both oral breakpoints (sus-ceptible, ≤0.06mg/L; intermediate, 0.12–1mg/L; resistant, ≥2mg/L) and parenteral breakpoints(susceptible, ≤2mg/L; intermediate, 4mg/L; resistant, ≥8mg/L) were used for penicillin.17 S. pneumoniae ATCC49619 was used as quality control.

Multilocussequencetypinganalysis

Isolateswerecharacterizedusingmultilocussequencetyping (MLST).ChromosomalDNAwasextractedfromovernight cul-turesofS.pneumoniaeisolatesgrownontrypticasesoyagar supplementedwith5%sheepblood(OxoidLtd,Basingstoke, England) using a SiMax Genomic DNA Extraction Kit (SBS Genetech Co., Ltd, China) according to the manufacturer’s instructions. Sevenhousekeepinggenes(aroE,gdh,gki, recP, spi,xpt,andddl)were ampliﬁedvia polymerasechain reac-tion from thechromosomalDNA asdescribedpreviously.18

TheproductsweresenttoBGI(Beijing,China)forsequencing from bothstrands.Theresultingsequenceswerecompared with thoseofall known allelesateach ofthe loci,as well as withthesequence types(STs)recorded inthe pneumo-coccal MLST database (https://pubmlst.org/spneumoniae/). NewallelesandallelicproﬁlesweresubmittedtotheMLST database forname assignment.eBURST v3software

(avail-able at http://eburst.mlst.net/) was used to evaluate the

relationships amongthe isolates, and to assign strains to a clonalcomplex (CC) using the stringentgroup deﬁnition of six of seven shared alleles.19 _STs _sharing _six _identical

alleles out of the seven MLST loci were assigned to the sameCC.

Statisticalanalysis

Antimicrobial susceptibility and MLST data were ana-lyzed using WHONET 5.6 software as recommended by the World Health Organization (http://www.who.int/

drugresistance/whonetsoftware/en/).The 2 _and/or _Fisher’s

exact testswere used foranalysis ofcategorical data, and statisticalsigniﬁcancewassetatp≤0.05.

Results

Serotypingandantimicrobialsusceptibilitytesting

Among the 250 isolates, the rates ofserotypes 6A, 6B, 6C, and 6Dwere55.2%(138/250),30.0% (75/250),12.8% (32/250), and 2.0% (5/250), respectively. The results ofantimicrobial susceptibilitytesting,includingMICvalues,forthedifferent serotypesareshowninTable1.Allofthestrainswere suscep-tibletolevoﬂoxacinandvancomycin,andnoresistancewas observed toward amoxycillin-clavulanic acid, ceftriaxone,

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Table1–SusceptibilityandminimuminhibitoryconcentrationvaluesoftheStreptococcuspneumoniaeisolates(grouped byserotype).

Antimicrobial Parameter Isolates

Total(n=250) 6A(n=138) 6B(n=75) 6C(n=32) 6D(n=5) Penicillina _S ₁₄₆_(58.4%) ₈₅_(61.6%) ₃₂_(42.7%) ₂₄_(75.0%) ₅_(100.0%) I 94(37.6%) 49(35.5%) 41(54.7%) 4(12.5%) 0 R 10(4.0%) 4(2.9%) 2(2.7%) 4(12.5%) 0 Amoxycillin-clavulanic acidb S 244(97.6%) 137(99.3%) 72(96.0%) 30(93.8%) 5(100.0%) I 6(2.4%) 1(0.7%) 3(4.0%) 2(6.2%) 0 Cefuroxime S 156(62.4%) 87(63.0%) 40(53.3%) 24(75.0%) 5(100.0%) I 20(8.0%) 13(9.4%) 6(8.0%) 1(3.1%) 0 R 74(29.6%) 38(27.5%) 29(38.7%) 7(21.9%) 0 Ceftriaxoneb _S ₂₄₃_(97.2%) ₁₃₅_(97.8%) ₇₄_(98.7%) ₂₉_(90.6%) ₅_(100.0%) I 7(2.8%) 3(2.1%) 1(1.3%) 3(9.4%) 0 Imipenemb _S ₂₂₉_(91.6%) ₁₂₉_(93.5%) ₆₉_(92.0%) ₂₆_(81.2%) ₅_(100.0%) I 21(8.4%) 9(6.5%) 6(8.0%) 6(18.8%) 0 Erythromycinb _S ₉_(3.6%) ₈_(5.8%) ₁_(1.3%) ₀ ₀ R 241(96.4%) 130(94.2%) 74(98.7%) 32(100.0%) 5(100.0%) Tetracycline S 14(5.6%) 6(4.3%) 8(10.7%) 0 0 I 3(1.2%) 0 2(2.7%) 1(3.1%) 0 R 233(93.2%) 132(95.7%) 65(86.7%) 31(96.9%) 5(100.0%) Trimethoprim/ sulfamethoxazole S 34(13.6%) 19(13.8%) 12(16.0%) 3(9.4%) 0 I 18(7.2%) 13(9.4%) 4(5.3%) 1(3.1%) 0 R 198(79.2%) 10676.8%) 59(78.7%) 28(87.5%) 5(100.0%) Chloramphenicolb _S ₂₁₄_(85.6%) ₁₂₀_(87.0%) ₅₈_(77.3%) ₃₁_(96.9%) ₅_(100.0%) R 36(14.4%) 18(13.0%) 17(22.7%) 1(3.1%) 0

Note:All250isolatesweresusceptibletolevoﬂoxacinandvancomycin.

a _Oral_breakpoints_{(susceptible,}_≤0.06_mg/L;_{intermediate,}_0.12–1_mg/L;_resistant,_≥2_mg/L)_were_used_here. b _No_intermediate_or_resistant_isolates_were_identiﬁed.

or imipenem. However, 96.4% (241/250) of isolates were resistant to erythromycin, with 236 isolates showing MIC values >256mg/L. None of the isolates were resistant to parenteralpenicillin,buttheratesofintermediateresistance andresistancetooralpenicillinreached37.6%(94/250)and 4.0% (10/250), respectively. Thus, penicillin resistance was deﬁnedaccordingtotheoralpenicillinbreakpoints.In addi-tion, high rates of resistance to tetracycline (93.2%) were observed.

MLST

Weidentiﬁed82distinctSTsamongthe250isolates,which wereassignedusingeBURSTanalysisto13CCsand28 single-tons(Fig.1).CC982wasthepredominantCC,accountingfor 19.2%(48/250)ofisolates,followedbyCC3173(9.6%,24/250), CC9789 (9.2%, 23/250), CC2912 (8.8%, 22/250), CC90 (7.6%, 19/250),CC2754(6.0%,15/250),CC4542(5.2%,13/250),CC4536 (5.2%,13/250),CC855(4.0%,10/250),CC902(3.6%,9/250),CC386 (3.6%,9/250),CC3113(2.8%,7/250),and CC473(1.2%,3/250). Overall,these13CCsaccountedfor86.0%(215/250)ofthe iso-lates.

ThedistributionofCCsaccordingtoserotypesisshownin

Table2.EachserotypehadapredominantCCthatincluded

themajorityoftheisolates.CC982wasthemostprevalentCC amongstserotype6Aisolates,accountingfor30.4%(42/138) ofisolates, followed byCC9789 (15.9%, 22/138) and CC3173 (15.2%, 21/138).For serotype 6B isolates, CC90 and CC4542 werethemostcommonCCs,accountingfor25.3%(19/75)and 14.7%(11/75)ofisolates,respectively.CC2912accountedfor20 (62.5%)serotype6Cisolates,whilealloftheﬁveserotype6D isolatesbelongedtoCC982.Interestingly,all ofthe CC4536, CC855,and CC3113isolateswere identiﬁedasserotype 6A, whereasCC90andCC386onlycontainedserotype6Bisolates.

RelationshipbetweenantimicrobialsusceptibilityandCC

Table3summarizestheanalysisofantibioticresistance

pro-ﬁlesaccordingtoCCs(Table3).Antibioticresistanceproﬁlesof isolatesbelongingtothesameserotypevariedaccordingtothe CC.Amongsttheserotype6Aisolates,allofthosebelonging toCC982andCC4536weresusceptibletopenicillin,andthe ratesofsusceptibilitytocefuroximewere alsohigh (CC982, 100%;CC4536, 92.3%).Onthecontrary,mostofthe CC3173, CC9789,andCC855isolateswerenon-susceptibletopenicillin andcefuroxime,withnon-susceptibilityratesreaching100%, 73.9%,and100%forpenicillinand100%,73.9%,and80.0%for cefuroxime,respectively.

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4314 4213 4541 4543 4544 6046 6196 6878 7767 8619 8742 10082 10087 10089 10095 10103 8623 8913 13041 473 1876 3113 10101 10104 8738 4173 8621 4542 7755 13155 902 7760 855 99 135 147 242 95 8526 4538 4537 282 744 2766 4174 10384 13025 13027 386 10099 7762 8916 8616 9790 9789 9784 6340 3263 4540 13036 10388 9783 982 4536 4539 90 8617 6339 3387 7756 10105 2763 2754 3173 13154 13028 13026 9791 10107 8618 2912 10392

Fig.1–Distributionofsequencetypes(STs)amongsttheclonalcomplexesofStreptococcuspneumoniae.Theimagedepicts eBURSTanalysisofthemultilocussequencetypingdatageneratedfromallofthe250Streptococcuspneumoniaeisolates analyzedinthisstudy.STsthatarelinkedbyalinebelongtothesamecluster.Circlesizesareproportionaltothenumber ofisolateswithintheST.

Of the serotype 6B isolates, all of the CC4542 isolates were susceptible to penicillin and cefuroxime, but CC902 isolates had a non-susceptibility rate of 100% for these twoantibiotics.Noneofthe CC90isolateswere susceptible to penicillin, and showed a cefuroxime resistance rate of 89.5%.

FluctuationofserotypesandCCsbetweenyears

For effective comparison, the study period was divided intosixranges:1997–1999,2000–2002,2003–2005,2008–2010, 2013–2014,and2015–2016.Thedistributionofserotypesand CCs in the different periods is shown in Fig. 2. A steady increaseintheprevalenceofserotype6Ccouldbeseenfrom 2008, whilethe prevalenceof serotype 6A, which was the most common serotype, decreased over the study period (2₌_11.963,_p_<_0.05).

Overthe study period,the percentageofCC982 isolates decreasedfrom37.0%in1997–1999to2.2%in2015–2016.The percentageofCC982,CC4542, and CC4536isolatesshowing highsusceptibilitytopenicillinandcefuroximedecreasedover thestudyperiod,withnosusceptibleisolatesidentiﬁedafter 2010, whilethe proportionof CC3173, CC9789, CC855, and CC902 isolatesshowingnon-susceptibilityto penicillinand cefuroximeincreased.

Discussion

This study investigated the antibiotic susceptibility pro-ﬁles and ﬂuctuations inthe dominantCCs ofserogroup 6

S.pneumoniaestrainsisolatedfromchildreninBeijing,the cap-italcityofChina,between1997and2016.Ofthe250isolates examined,55.2%,30.0%,12.8%,and 2.0%were identiﬁedas serotypes6A,6B,6C,and6D,respectively.Althoughthe major-ityoftheisolatesbelongedtoserotype6A,theprevalenceof thisserotypedecreasedoverthestudyperiod.Researchfrom countries withhigh rates ofPCV7 vaccinationhave shown serotypereplacementofserogroup6.Signiﬁcantdecreasesin theprevalenceofserotype6Aand6Bstrainswereobservedin theUKbetween2006/2007and2010/2011,coincidingwiththe additionofPCV7totheUKNationalImmunizationProgram inSeptember2006,anditsreplacementwithPCV13inApril 2010.11_A_study_from_Israel_conducted_from₁₉₉₉_to_2014,

pre-andpost-PCVimplementation,analyzedtheimpactofPCVs ontheprevalenceofserogroup6carriage,andtheincidence of pneumococcal conjunctivitis, otitis media, and invasive diseasescausedbyserogroup6.Theresearchersfoundthat thecarriageratesofserotype6Aand6Bisolates,alongwith cases of conjunctivitis,otitis media, and invasive diseases causedbyserotype6Aand6B,decreasedremarkablyinthe post-PCV7 period. However, this decrease was offset by a signiﬁcantincreaseintheprevalenceofserotype6Cisolates, althoughthiswashaltedaftertheimplementationofPCV13.20 Severalotherstudiesreportasimilarphenomenon,whereby theprevalenceof6Aand6Bisolatesdecreasedwiththe intro-ductionofPCV7orPCV13,buttheproportionof6Cisolates increaseduntiltheintroductionofPCV13.21,22_The_efﬁcacy_of

PCV13againstserotype6Cisolatesresultsfromtheextensive structuraland immunological similarity between serotypes 6Aand6C.Theinclusionof6AinPCV13resultsinahigher concentrationofanti-6Cantibodies thanthose obtainedby

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T able 2 – Distr ib ution of clonal complex es/sequence types of ser ogr oup 6 Str eptococcus pneumoniae isola tes by ser otype. Ser otypes No . CC982 CC3173 CC9789 CC2912 CC90 CC2754 CC4542 CC4536 CC855 CC902 CC386 CC3113 CC473 Others 6A 138 42 (30.4%) 21 (15.2%) 22 (15.9%) 2 (1.4%) 0 6 (4.3%) 1 (0.7%) 13 (9.4%) 10 (7.2%) 1 (0.7%) 0 7 (5.1%) 2 (1.4%) 11 (8.0%) 6B 75 0 3 (4.0%) 1 (1.3%) 0 19 (25.3%) 7 (9.3%) 11 (14.7%) 0 0 8 (10.7%) 9 (12.0%) 0 1 (1.3%) 16 (21.3%) 6C 32 1 (3.1%) 0 0 20 (62.5%) 0 2 (6.3%) 1 (3.1%) 0 0 0 0 0 0 8 (25.0%) 6D 5 5 (100%) 0 0 0 0 0 0 0 0 0 0 0 0 0 T otal 250 48 24 23 22 19 15 13 13 10 9 9 7 3 35 %, Number of ser otypes di vided by n umber of clonal comple xes/sequence types.

PCV7.23 _We_also_detected_an_increase_of_serotype_6C_in_the

current study, although there were not many 6C isolates overall.

S. pneumoniae MLST is based on sequence data from

standardized fragments ofseven housekeepinggenes. The MLST classification reveals important insights into the geographic spread of successful pathogenic clones, as well as the emergence of associations between STs and serotypes,combinationsthatcanbetracedbacktoserotype switching events. Among S. pneumoniae isolates, antimi-crobial resistance associated with specific pneumococcal serotypes, even CCs/STs, most often reflects the spread of a few internationally disseminated clones recognized by the Pneumococcal Molecular Epidemiology Network (PMEN;http://www.pneumogen.net/pmen/).Theseclonesare frequently associatedwith disease,widelydistributed geo-graphically, and usually resistant toone or moreclinically relevantantibiotics.24

Data from the currentstudy showed ﬂuctuations inthe dominance of particular CCs. The proportion of CCs with highratesofnon-susceptibilitytopenicillinandcefuroxime, suchas CC3173,CC9789, CC855,andCC902,increased dur-ing the study period. On the contrary,CC982, CC4542, and CC4536, all of which show high rates of susceptibility to penicillin and cefuroxime, decreased or even disappeared. Previous studies have also shown ﬂuctuation of CCs/STs. Forexample,researchesfromUnitedStates,25_Canada26_and

Russia27_showed_that_amongst_serotype_19A_isolates,_CC320,

which has a multidrug-resistant phenotype with high ␤-lactamMICs,replacedCC230,whichisassociatedwithhigh susceptibilitytopenicillin.Ourpreviousresearchesfrom Bei-jing,Chinaindicatedthattheglobally-disseminatedresistant cloneCC271,whichhasahighrateofnon-susceptibilityto ␤-lactamantibiotics,tooktheplaceofCC983amongstserotype 19F pneumococcus strains,28 _while _amongst _serotype _23F

isolates, ST342 was replacedby ST81,which demonstrates higher␤-lactamresistance.29_Also_our_study_on_serotype₁₄

strainsfromBeijing,ChinareveledthatCC876overtookCC875 as the dominant CC, with CC876 isolates showing higher non-susceptibility rates to␤-lactam antibiotics than CC875 isolates.30 _All_of_these_examples _of_clonal_shift_phenomena

wereassumedtobecausedbyantibioticpressure,asCCs/STs withhighratesofantibioticresistancereplacedthosewithlow resistance.

Taken together,theseobservationsunderlinethe impor-tance of further long-term surveillance of S. pneumoniae

based on serotype and genotype, combined with antimi-crobial susceptibility proﬁle analysis, in the hopes of preventing infections caused by this important human pathogen.

Ethical

statement

Thepneumococcalisolatesinvolvedinthisstudy were col-lected previouslyduringaseriesofepidemiologicalsurveys onthisbacterium.TheEthicsCommitteeofBeijingChildren’s HospitalafﬁliatedtoCapitalMedicalUniversityexemptedthis studyfromreviewbecauseitfocusedonthebacterialisolates anddidnotincludepatientdata.

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b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 891–899

Table3–SusceptibilityandminimuminhibitoryconcentrationvaluesoftheStreptococcuspneumoniaeisolatesgroupedbyclonalcomplex.

Antimicrobial Parameter Isolates

CC982(n=48) CC3173(n=24) CC9789(n=23) CC2912(n=22) CC90(n=19) CC2754(n=15) CC4542(n=13) CC4536(n=13) Penicillina _S ₄₈_(100%) ₀ ₆_(26.1%) ₁₇_(77.3%) ₀ ₁₄_(93.3%) ₁₃_(100%) ₁₃_(100%) I 0 22(91.7%) 16(69.6%) 1(4.5%) 18(94.7%) 1(6.7%) 0 0 R 0 2(8.3%) 1(4.3%) 4(18.2%) 1(5.3%) 0 0 0 MIC50 0.032 0.75 0.5 0.032 0.75 0.032 0.016 0.023 MIC90 0.064 1 0.75 1.5 1 0.064 0.032 0.032 Amoxycillin-clavulanic acidb S 48(100%) 24(100%) 22(95.7%) 21(95.5%) 16(84.2%) 15(100%) 13(100%) 13(100%) I 0 0 1(4.3%) 1(4.5%) 3(15.8%) 0 0 0 MIC50 0.016 0.75 0.5 0.016 0.75 0.016 0.016 0.016 MIC90 0.032 1 0.75 0.75 1.5 0.38 0.032 0.032 Cefuroxime S 48(100%) 0 6(26.1%) 17(77.3%) 2(10.5%) 13(86.7%) 13(100%) 12(92.3%) I 0 2(8.3%) 4(17.4%) 1(4.5%) 0 0 0 1(7.7%) R 0 22(91.7%) 13(56.5%) 4(18.2%) 17(89.5%) 2(13.3%) 0 0 MIC50 0.064 2 1.5 0.064 1.5 0.047 0.023 0.032 MIC90 0.125 3 2 3 2 1.5 0.25 0.064 Ceftriaxoneb _S ₄₈_(100%) ₂₀_(83.3%) ₂₃_(100%) ₂₁_(95.5%) ₁₉_(100%) ₁₅_(100%) ₁₃_(100%) ₁₃_(100%) I 0 4(16.7%) 0 1(4.5%) 0 0 0 0 MIC50 0.032 0.75 0.38 0.032 0.5 0.032 0.016 0.023 MIC90 0.064 1.5 0.75 1 1 0.75 0.064 0.047 Imipenemb _S ₄₈_(100%) ₁₇_(70.8%) ₂₁_(91.3%) ₁₇_(77.3%) ₁₄_(73.7%) ₁₅_(100%) ₁₃_(100%) ₁₃_(100%) I 0 7(29.2%) 2(8.7%) 5(22.7%) 5(26.3%) 0 0 0 MIC50 0.012 0.125 0.125 0.016 0.125 0.008 0.016 0.012 MIC90 0.023 0.25 0.125 0.19 0.25 0.023 0.023 0.023 Erythromycinb _S ₁_(2.1%) ₀ ₁_(4.3%) ₀ ₀ ₀ ₀ ₄_(30.8%) R 47(97.9%)24(100%) 22(95.7%)22(100%) 19(100%) 15(100%) 13(100%) 9(69.2%) MIC50 >256 >256 >256 >256 >256 >256 >256 >256 MIC90 >256 >256 >256 >256 >256 >256 >256 >256 Tetracycline S 0 0 1(4.3%) 0 1(5.3%) 2(13.3%) 0 0 I 0 0 0 0 0 0 0 0 R 48(100%) 24(100%) 22(95.7%)22(100%) 18(94.7%) 13(86.7%) 13(100%) 13(100%) Trimethoprim/ sulfamethoxazole S 0 0 1(4.3%) 2(9.1%) 1(5.3%) 0 0 4(30.8%) I 1(2.1%) 3(12.5%) 0 1(4.5%) 0 2(13.3%) 0 3(23.1%) R 47(97.9%) 21(87.5%) 22(95.7%) 19(86.4%) 18(94.7%) 13(86.7%) 13(100%) 6(46.2%) Chloramphenicolb _S ₄₆_(95.8%)₂₄_(100%) ₁₈_(78.3%)22_(100%) ₁₀_(52.6%) ₁₄_(93.3%) ₁₂_(92.3%) ₇_(53.8%) R 2(4.2%) 0 5(21.7%) 0 9(47.4%) 1(6.7%) 1(7.7%) 6(46.2%)

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b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 891–899

897

CC855(n=10) CC902(n=9) CC386(n=9) CC3113(n=7) CC473(n=3) Others(n=35) Total(n=250) Penicillina _S ₀ ₀ ₈_(88.9%) ₇_(100%) ₀ ₂₀_(57.1%) ₁₄₆_(58.4%) I 10(100%) 8(88.9%) 1(11.1%) 0 3(100%) 14(40.0%) 94(37.6%) R 0 1(11.1%) 0 0 0 1(2.9%) 10(4.0%) MIC50 0.25 0.5 0.064 0.023 0.25 0.064 0.047 MIC90 0.75 1.5 0.094 0.023 0.38 1 0.75 Amoxycillin-clavulanic acidb S 10(100%) 9(100%) 9(100%) 7(100%) 3(100%) 34(97.1%) 244(97.6%) I 0 0 0 0 0 1(2.9%) 6(2.4%) MIC50 0.5 0.38 0.064 0.016 0.125 0.032 0.032 MIC90 1 0.75 0.094 0.023 0.125 1 0.75 Cefuroxime S 2(20.0%) 0 9(100%) 7(100%) 3(100%) 24(68.6%) 156(62.4%) I 5(50.0%) 5(55.6%) 0 0 0 2(5.7%) 20(8.0%) R 3(30.0%) 4(44.4%) 0 0 0 9(25.7%) 74(29.6%) MIC50 1 1 0.094 0.094 0.38 0.094 0.094 MIC90 1.5 1.5 0.125 0.19 0.5 2 2 Ceftriaxoneb _S ₁₀_(100%) ₉_(100%) ₉_(100%) ₇_(100%) ₃_(100%) ₃₃_(94.3%) ₂₄₃_(97.2%) I 0 0 0 0 0 2(5.7%) 7(2.8%) MIC50 0.38 0.5 0.064 0.125 0.125 0.047 0.064 MIC90 0.5 1 0.125 0.19 0.19 0.75 0.75 Imipenemb _S ₉_(90.0%) ₉_(100%) ₉_(100%) ₇_(100%) ₃_(100%) ₃₄_(97.1%) ₂₂₉_(91.6%) I 1(10.0%) 0 0 0 0 1(2.9%) 21(8.4%) MIC50 0.094 0.094 0.032 0.008 0.032 0.023 0.023 MIC90 0.125 0.125 0.064 0.012 0.032 0.125 0.125 Erythromycinb _S ₀ ₀ ₀ ₀ ₀ ₃_(8.6%) ₉_(3.6%) R 10(100%) 9(100%) 9(100%) 7(100%) 3(100%) 32(91.4%) 241(96.4%) MIC50 >256 >256 >256 >256 >256 >256 >256 MIC90 >256 >256 >256 >256 >256 >256 >256 Tetracycline S 0 2(22.2%) 0 0 2(66.7%) 6(17.1%) 14(5.6%) I 0 1(11.1%) 0 0 0 2(5.7%) 3(1.2%) R 10(100%) 6(66.7%) 9(100%) 7(100%) 1(33.3%) 27(77.1%) 233(93.2%) Trimethoprim/sulfamethoxazoleS 9(90.0%) 0 8(88.9%) 0 3(100%) 6(17.1%) 34(13.6%) I 1(10.0%) 1(11.1%) 0 3(42.9%) 0 3(8.6%) 18(7.2%) R 0 8(88.9%) 1(11.1%) 4(57.1%) 0 26(74.3%) 198(79.2%) Chloramphenicolb _S ₉_(90.0%) ₉_(100%) ₉_(100%) ₇_(100%) ₂_(66.7%) ₂₅_(71.4%) ₂₁₄_(85.6%) R 1(10.0%) 0 0 0 1(33.3%) 10(28.6%) 36(14.4%)

Note:All250isolatesweresusceptibletolevoﬂoxacinandvancomycin.

a _Oral_breakpoints_{(susceptible,}_≤0.06_mg/L;_{intermediate,}_0.12–1_mg/L;_resistant,_≥2_mg/L)_were_used_here. b _No_intermediate_or_resistant_isolates_were_identiﬁed.

(8)

100

a

b

90 80 70 60 50 P ercetange (%) Years Serotype CCs/STs Years P ercetange (%) 40 6A 982 3173 9789 2912 2754 4542 4536 855 386 3113 473 Other 902 90 6B 6C 6D 30 20 10 0 1997-19992000-20022003-20052008-20102013-20142015-2016 1997-19992000-20022003-20052008-20102013-20142015-2016 100 90 80 70 60 50 40 30 20 10 0

Fig.2–Distributionofserotypesandclonalcomplexes/sequencetypesofStreptococcuspneumoniaeisolatesbytimeperiod.

Authors’

contributions

Alloftheauthorsparticipatedinthedesignofthestudy.WS, YL,QM,LY,andWGcollectedtheisolatesandperformedthe antimicrobialsusceptibilitytest.WS,YL,andQMdidtheMLST part.WS,YLandKYcollectedthedata,analyzedthem, inter-pretedtheresults, anddraftedthemanuscript.All authors reviewedandrevisedthemanuscriptandapprovedtheﬁnal version.

Funding

ThisstudywassupportedbytheResearchFundsofProfession QuotaBudgetfromBeijingMunicipalScienceandTechnology Commission[grantnumber2016-bjsekyjs-3]andtheBeijing TalentsFund[grantnumber2015000021469G209].The spon-sorshadnoroleinstudydesign,inthecollection,analysis, andinterpretationofdata,inthewritingofthereport,orin thedecisiontosubmitthearticleforpublication.

Conﬂicts

of

interest

Nonedeclared.

Acknowledgments

WethankTamsinSheen,PhD,fromLiwenBianji,EdanzEditing China(www.liwenbianji.cn/ac),foreditingtheEnglishtextof adraftofthismanuscript.

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