Antimicrobial resistance in Neisseria gonorrhoeae: history, molecular mechanisms and epidemiological aspects of an emerging global threat

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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 /

Review

Antimicrobial

resistance

in

Neisseria

gonorrhoeae:

history,

molecular

mechanisms

and

epidemiological

aspects

of

an

emerging

global

threat

Ana

Paula

Ramalho

da

Costa-Lourenc¸o,

Késia

Thaís

Barros

dos

Santos,

Beatriz

Meurer

Moreira,

Sergio

Eduardo

Longo

Fracalanzza,

Raquel

Regina

Bonelli

∗ InstituteofMicrobiology,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,RJ,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received9November2016 Accepted5June2017 Availableonline12July2017 AssociateEditor:MarinaBaquerizo

Keywords:

Neisseriagonorrhoeae Surveillance

Resistancemechanisms

a

b

s

t

r

a

c

t

Neisseriagonorrhoeaeistheagentofgonorrhea,asexually transmittedinfectionwithan estimatefromTheWorldHealthOrganizationof78millionnewcasesinpeopleaged15–49 worldwideduring2012.Ifleftuntreated,complicationsmayincludepelvicinﬂammatory diseaseandinfertility.Antimicrobialtreatmentisusuallyeffective;however,resistancehas emergedsuccessivelythroughvariousmolecularmechanismsforalltheregularlyused ther-apeuticagentsthroughoutdecades.Detectionofantimicrobialsusceptibilityiscurrentlythe mostcriticalaspectforN.gonorrhoeaesurveillance,howeverpoorlystructuredhealth sys-temsposedifﬁculties.Inthisreview,wecompileddatafromworldwidereportsregarding epidemiologyandantimicrobialresistanceinN.gonorrhoeae,andhighlighttherelevanceof theimplementationofsurveillancenetworkstoestablishpoliciesforgonorrheatreatment. ©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Neisseria

gonorrhoeae

infections:

symptoms,

surveillance

and

treatment

Thegonococcaldisease

N.gonorrhoeaeistheetiologicalagentofgonorrhea,thesecond mostfrequentlyreportedsexuallytransmittedinfection(STI) intheworld.Thisbacteriumtypicallycolonizesandinfects

∗ _{Corresponding}_author.

E-mail:raquel.bonelli@micro.ufrj.br(R.R.Bonelli).

thegenitaltractinmenandwomen,butmaybefoundin addi-tionalbodysitessuchastherectalandoropharyngealmucosa, withorwithoutclinicallyevidentinfection.1

Gonorrheaisusuallysymptomaticinmen,mostoftenas urethritis,withpainorburningsensationduringurination, urethraldischarge,andpainfultesticles.Incontrast,women develop symptomatic gonococcal cervicitis less frequently, presenting a slight increase invaginal discharge, and rare vaginalbleedingunrelatedtoperiods,orpainandaburning

http://dx.doi.org/10.1016/j.bjm.2017.06.001

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sensationwhenurinating.Theabsenceofsymptomsinmen and women can lead tosustained infections.Complicated gonorrheamaycauseinfertility.2

Surveillanceprogramsaroundtheworld

AreportfromtheWorldHealthOrganization(WHO)indicated theoccurrenceof78millionnewcasesofgonococcal infec-tion inpeople aged15–49worldwideduring2012.3 _Despite

thehighrateofincidence,justafewcountries,includingthe UnitedStates(U.S.),Canada,membersoftheEuropeanUnion (EU),and Australiaconduct broadsurveillanceprogramson gonorrhea.Additionally,somecountriesfromLatin-America and the Caribbeanregion (LAC), and countries from West-ernPacificRegion(WPR)andSouthEastAsianRegion(SEAR) joined the Gonococcal Antimicrobial Surveillance Program (GASP)thatwasconductedbyWHOintheearly1990s.Since antimicrobialresistanceisthemainchallengeassociatedwith thismicroorganism,publishedreportsemphasizethe antimi-crobialsusceptibilityprofileofisolates.However,analysisof published data can be challenging, considering the break-pointstodefineresistancevaryacrossdifferentsurveillance programs.Regardlessofthislimitation,resistanceisclearly anemergingphenomenon.

UnitedStates

Inthe U.S.,the CentersforDiseaseControland Prevention (CDC) supportsthe Gonococcal Isolate Surveillance Project (GISP).Thisprogramanalyzestheﬁrst25–30N.gonorrhoeae iso-latescollectedfrommenwithgonococcalurethral-syndrome in sentinel laboratories located in ﬁve regions of the U.S. monthly.Surveillanceincludesanalysisofdemographicand clinicaldata,andantimicrobialsusceptibility.4_In_recent_years,

thesouthernregionreportedthehighestrateofgonorrhea, reaching131.4casesper100,000individualsin2014.5_The_CDC

estimates820,000newgonorrheacasesperyearthroughout thecountry.6

TheSTISurveillanceNetworkintheU.S.indicatedhigher incidence rates of gonorrhea in men who have sex with men (MSM) of any age, followed by men who have sex with women (MSW), and women, in 2014.5 The network alsoreportedthatthe riskofgonococcalinfection declines withage,withmostcasesoccurringinindividualsunderthe ageof24.5_In_regards_to_{antimicrobial}_resistance,_GISP_data

has shown that nearly 30% of the isolates obtained from MSM,and 12%ofthoseobtainedfromMSW wereresistant tociproﬂoxacin, witha minimum inhibitoryconcentration (MIC)≥1␮g/mLin2014.7 _Irrespective_of_the_sexual_partners

gender,inthatsameyear,0.7%oftheisolates(n=38)presented adecrease inceﬁximesusceptibility(MIC≥0.25␮g/mL),and 2.5%showedazithromycinMICalert values(MIC≥2␮g/mL)

(Table1).7_Moreover,_a_genomic_epidemiology_study_with₂₃₆

GISPisolates obtained in2009–2010 hasshown that anN. gonorrhoeaeclusterwithdecreasedsusceptibilityto extended-spectrumcephalosporins(ESC)spreadpredominantlyamong MSMduringthoseyears.8

Canada

TheNational SurveillanceProgram(NSP)inCanada, imple-mentedin1985,providesdatafromdifferentprovincesacross thecountry regardingN.gonorrhoeaeantimicrobial suscepti-bility.Provincialpublichealthlaboratories(PL)sendresistant isolatesorisolatesnotsubmittedtoantimicrobial suscepti-bilitytestingtotheNationalMicrobiologyLaboratory(NML). In2014,2101of3089N.gonorrhoeaeisolatesculturedinthe PL were sent to NML. Interestingly,contrasting with other countries,NSPdatashowedaconsistentdiminishingtrendin theESCreducedsusceptibilityrates(from7.6%in2011to3.1% in 2014).However,regarding azithromycin,resistancerates rosefrom0.4%in2011to2.3%in2014.9

Europe

InEurope,theextentofN.gonorrhoeaesurveillancevariesin differentcountries,accordingtonationalpublichealth poli-cies.However,datacompiledfrom21countriescomposingthe EuropeanGASP(Euro-GASP)conductedbytheEuropeanCDC (ECDC)reported 50,001casesin2013.10 Euro-GASPhastwo surveillancemodules:onedecentralized,basedonthe com-municationofantimicrobialsusceptibilitytest(AST)resultsto ECDC;andtheothercentralized.Inthiscase,participating lab-oratoriessendisolatestothePublicHealthEngland(London) forAST.10

In the mostrecent Euro-GASPreport, each country was requiredtomakeacontributionof100–200isolates(depending onthenumberofgonorrhoeaecasesdetectedbythenational protocols),obtainedduringApril/MayandOctober/November 2013. With this sampling strategy, the number of isolates includedinthestudy,comparedtothetotalreportedcases ineachcountry,variedfromlessthan1%(inconsequenceof high incidencelevels orveryefﬁcientsurveillance)tomore than 100% (whenunder-reporting from thenational proto-colsoccurred).Forinstance,theUnitedKingdom(U.K.),with a well-established national surveillance program, reported themostcases,32,377in2013,followedbytheNetherlands (n=4171),Spain(n=3314),andHungary(n=1526);while Portu-galreportedonly116casesinthesameyear,andGermanydid notprovideanyinformationonthismatter.Notwithstanding suchdifferences,thesesixcountriescontributedtothe Euro-GASPanalysiswithsimilarnumberofisolates,varyingfrom 88inHungaryto240intheU.K.Consideringsucha heteroge-neouscollection,datacompiledshowedinEurope,inaddition totheU.S.,MSMasthemaingroupatriskofdeveloping gon-orrhea.However,incontrasttothepredominantagegroupin theU.S.,mostcasesreportedforEuropeancitizenswerewith individualsovertheageof25.10

Concerningdetectionofresistance,theEuro-GASPadopts breakpointssetbyTheEuropeanCommitteeon Antimicro-bialSusceptibilityTesting(EUCAST),whicharelowerthanthe values adopted bythe U.S.4,11,12 _With_this _caveat _in_mind,

theEuro-GASPreportedciproﬂoxacinandazithromycin resis-tanceratesof53%and5%,respectively,among1994isolates studiedintheprogramduring2013.10_In_contrast,_for_ceﬁxime,

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Table1–BreakpointsoralertvaluesadoptedfortestingsusceptibilityofNeisseriagonorrhoeaetokeyantimicrobialsby CLSI,GISP,NSP,EUCASTandAGSPandselectedresistancerates.

Antimicrobial MICbreakpointoralertvalues(␮g/mL)a _Resistance_or_Reduced_{Susceptibility}_rates_in_the mostrecentreportsofselectedGonococcal SurveillancePrograms(GISP,2014;NSP,2014;

EURO-GASP,2013;AGSP,2014)

CLSI GISPb _NSP

EUCAST/EURO-GASP

AGSP

Ciproﬂoxacin ≥1.0 ≥1.0 ≥1.0 >0.06 ≥1.0 GISP:30%inMSMand12%inMSW

EURO-GASP:53% AGSP:36% Ceftriaxone S≤0.25c _AV_≥_0.125 _≥0.125 _>0.125 _0.06–0.125d _NSP:_3.1% AGSP:54%(RS) Ceﬁxime S≤0.25c _AV_≥_0.25 _≥0.25 _>0.125 _ND _GISP:_0.7%_(RS) NSP:3.1% EURO-GASP:4.7%

Azithromycin ECV≥2.0e _AV_≥_2.0 _≥2.0 _>0.5 _≥1.0 _GISP:_2.5%

NSP:2.3% EURO-GASP:5% AGSP:2.5%

a _Breakpoints_deﬁne_resistance,_except_otherwise_described,_adopted_as_reference_by_the_Gonococcal_Surveillance_Programs_listed. b _GISP_proposes_minimum_inhibitory_{concentration}_(MIC)_alert_values_(AV)_for_ceftriaxone,_ceﬁxime_and_{azithromycin.}

c _CLSI_does_not_propose_resistance_breakpoint_for_ceftriaxone_and_ceﬁxime.

d _AGSP_deﬁnes_ceftriaxone_decreased_{susceptibility}_for_isolates_with_MIC₌_0.06–0.125_␮g/mL.

e _CLSI_indicates_that_isolates_with_MIC_≥2.0_␮g/mL_for_azithromycin_have_mutational_and/or_acquired_resistance_mechanisms,_and_deﬁnes_as epidemiologicalcutoffvalue.

CLSI,ClinicalLaboratoryStandardsInstitution;S,susceptible;ECV,epidemiologicalcutoffvalue;ND,notdetermined;GISP,GonococcalIsolate SurveillanceProject;NSP,NationalSurveillancePrograminCanada;EUCAST,EuropeanCommitteeonAntimicrobialSusceptibilityTesting; EURO-GASP,EuropeanGonococcalAntimicrobialSurveillanceProgram;AGSP,AustralianGonococcalSurveillanceProgram;MSM,menwho havesexwithmen;MSW,menwhohavesexwithwomen.

theGISP.Still,ceﬁximeresistanceinEuropewashigherthan intheU.S.,with93conﬁrmedcases(4.7%oftotalisolates)in

2013(Table1).10

Australia

The Australian Gonococcal Surveillance Program (AGSP) showed a different epidemiological trend between remote regionsandeasternstates(Victoria,NewSouthWales,and Queensland)in2012.Inremoteregions,notiﬁcationrateswere higherbutstablecomparedtopreviousyears,about933cases per 100,000 individuals, with low antimicrobial resistance rates.Incontrast,intheeasternregion,whereaninternational community predominates,the incidence ofgonorrhea was lowerbuthadgrownsince2009(reaching38.5per100,000 indi-viduals),andtheisolatesshowedhigherresistancelevels.13

Consideringdata from the whole country,and MIC break-pointof≥1␮g/mLforbothciproﬂoxacinandazithromycin,14

theAGSPreportedresistanceratesof36%and2.5%forthese drugs, respectively.13 _Moreover, _Australia _does _not_conduct

surveillanceforceﬁxime,butdetected5.4%(n=258)reduced susceptibility to ceftriaxone (MIC 0.06–0.125␮g/mL) among 4804isolatesin2014.13_As_a_reference_for_comparison,_with

aslightdifferencetakenintoaccountinbreakpointsadopted, thispercentagerateismorethantentimeshigherthanthat observedbyGISP(0.4%)in2014,orEuro-GASP(0.1%)in2013

(Table1).7,10

Asia

SurveillanceinN.gonorrhoeaeresistancehasbeenconducted inWPRandSEARsince1992and2007,respectively,through

aWHO-GASPinitiative,undertheAustralianHealth Depart-ment supervision. According to a GASP-WPR/GASP-SEAR report,9744N.gonorrhoeaeisolatesfrom19countrieswere sub-mittedtoantimicrobialsusceptibilitytestingin2010.Among these isolates,quinoloneresistanceorreduced susceptibil-itywaswidespread,reachingratesover90%in11countries. Azithromycinresistanceratesvariedlargely,from0to1%in countriessuchasCambodiaandIndiato34%inMongolia. Dif-ferentrateswerealsoreportedfordecreasedsusceptibilityto ceftriaxone:1.3%inSingapore,10.8%inIndia,20.3%inJapan, 29.3%inKorea,and55.8%inChina.15

Africa

AccordingtoaWHOreportpublishedin2015,astableand efﬁcientsurveillanceprogramforSTIinAfricaRegionhasnot beenimplementedyet.16_In_2013,_a_good_review_about

gono-coccal antimicrobialresistancestudies performed inAfrica demonstrated thatthesmallnumberofisolatestestedand thelackofstandardizationinthesamplingstrategyadoptedin differentcountriesmakemeasuringandcomparisonsof resis-tanceratesdifﬁcult.17_In_a_general_perspective,_data_obtained

between 2004 and 2012 in the African continent demon-stratedariseintheantimicrobialresistance,especiallywith quinolones, and emphasizesthe need forimproving infra-structureandlaboratorynetworktoperformsurveillancein thatregion.17,18

LatinAmerica&Caribbean

In addition to Asia and Africa, WHO-GASP has also been implemented in Latin America and the Caribbean region

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(GASP-LAC) sincethe 1990s.19 _WHO-GASP-LAC _published _a

reportin2013that gatheredinformationonthe antimicro-bialsusceptibilityofN.gonorrhoeaein23countriesfrom1990 to2011.Among12,730isolatestestedforciprofloxacin sus-ceptibility,arisingtrendforresistancewasobservedduring theseyears.Ciprofloxacinresistanceratesstayedbelow5% until2004,rangedtomorethan15%in2006,finallyreaching valuesofgreaterthan40% in2010.ResistantN.gonorrhoeae isolates toazithromycin were not detected until2000 and onwards, with the exception of Cuba, where 10 N. gonor-rhoeaeazithromycinresistantisolatesweredetectedbetween 1995and1998.Azithromycinresistanceratesremainedabove 6%from2000to2009, rosetomorethan25% in2008,and reduced down to 1% in 2010. Among 5171 isolates tested forceftriaxone between 2007and 2011, 20 (0.4%) N. gonor-rhoeae isolatesobtainedfrom Argentina,Brazil,Chile, Cuba andUruguayexhibitedreducedsusceptibilitytoceftriaxone (MIC≥0.125␮g/mL).19

Brazil

Brazil,thelargestandeconomicallymostrelevantcountryin SouthAmerica,doesnothaveanationalgonococcal surveil-lance program. Over the last 20 years, three attempts of establishinglaboratorynetworkswithafocusinN.gonorrhoeae resistancewereconductedbytheBrazilianHealthMinistry (BHM). The ﬁrst one occurred in 1996 as an invitation of PanAmericanHealthOrganization(PAHO)/WHO.Thesecond one,initiatedin2007,wasdenominatedasSengonoProject, and engaged sevenpreviouslyestablished laboratoriesand researchgroups.Bothattemptsfailed,sincenoreportsofthe obtainedresultshavebeenpublished.In2013,anewedition oftheSengonoProjectwasinitiated.Theprojectprovided pre-liminaryresultspublishedasaBHMcommunicationnotein theBHMwebsitein2016.20

The BHM estimated the occurrence of 9,285,000 cases per year amongst the age group of 15–49, in 2015. Unfor-tunately, this number comes from compiled data obtained insmallstudiesperformedwithdifferentsubpopulationsin sevenBrazilianstates,from2002to2012.Thedatainclude cohortsamplingreportsandasymptomaticinfections, differ-entlyfromcasesnotiﬁedinsurveillanceprogramsassociated withHealthCareSystems.21_According_to_statistics_currently

posted onthe BHMwebsite,WHO estimates1,541,800 new gonorrheainfectionsperyearinBrazil.However,no informa-tionaboutthedatasourcefortheestimationswereprovided.22

Inregardstoantimicrobialresistance,areportpresenting GASP-LACoutcomesfor2936isolatesobtainedinBrazilfrom 2000 to 2009 indicated that during this time ciproﬂoxacin resistance rates were lower than 6%. Azithromycin resis-tance ratesvariedfrom 22% (9/41)in2004to 6%(7/110)in 2007,withoneadditionalresistantisolatedetectedin2009. However,itisnotclearifsuchdataarerepresentativeforthe whole country,since no information about the geographic origin ofthe isolatesor samplingstrategy isavailable.The same report described seven isolates obtained in Manaus in 2007 exhibiting decreased susceptibility to ceftriaxone (MIC>0.25␮g/mL).23 _Beyond _this_report, _a_small _number _of

studies were performed sporadically throughout different

regionsofthecountry.During2004and2005,ﬁveciproﬂoxacin resistantisolates(8%)weredetectedamong65N.gonorrhoeae isolatesrecoveredfrompatientswithurethritisinSãoPaulo.24

Afewyearslater,ciproﬂoxacinresistancereached17%among 152isolatesobtainedbetween2006and 2010from patients withgonorrheainRiodeJaneiro.25_More_recently_{(2011–2012),}

among 201 N. gonorrhoeae isolates obtained from patients withurethritis and cervicitisinaHealthService Facilityin MinasGerais,21%wereresistanttociproﬂoxacinand5%were resistanttoazithromycin.26

Neisseriagonorrhoeaegenotyping

Understanding clonal relationships among N. gonorrhoeae resistant isolates is an important strategy to control the spread and increase of resistance. There are two main DNAsequence-basedtypingmethodsperformedin gonococ-cus withdata storedon publicwebsite. TheN.gonorrhoeae multiantigen sequence type (NG-MAST) is speciﬁc to this speciesand analysistwovariable loci,porBandtbpB,which encode oneof the two porins expressed byN. gonorrhoeae (PIB porin), and the B subunit of binding transferrin pro-tein, respectively (http://www.ng-mast.net/). NG-MAST is a convenienttoolformicro-epidemiologicalstudiesduetoits discriminatory property.27 _The _other _method _is _multilocus

sequence typing (MLST),available for the genderNeisseria. Thisapproachisbasedontheanalysisofsevenhousekeeping genes, and is appropriate to track the spread of interna-tionalclones(http://pubmlst.org/neisseria/).Bothtypingtools havedemonstratedthatinfectionsmayoccurinclusters,and resistantclonesmayspreadacrosscontinents.28_In_addition

to those methods, whole genome sequencing has brought new epidemiologic information in studies of transmission pathways.8,29

Currenttreatmentrecommendations

Published guidelines usually state policies for syndromic treatment of gonorrhea,deﬁned assymptomatic urethritis inmen,andmucopurulentcervicitisinwomen,evenwhile a microbiological diagnostic is not possible.6,21 _The

treat-ment recommendationsvary slightly,especiallyconcerning antimicrobialdoses,andare describedinTable2.TheBHM proposestwodifferentstrategiesforBrazil.Formost territo-ries,ciprofloxacinshouldbeprescribedwithacombinationof azithromycin.However,studiesshowingciprofloxacin resis-tance ratesover5%inSãoPaulo, MinasGerais, andRiode Janeiro,encourageciprofloxacintobereplacedbyceftriaxone inthosestates.21_This_{recommendation}_placed_Brazil_in_the

vastgroupofcountries(U.S.,Europeancountries, Australia, and others)thatrecommend acombinationof ceftriaxone-azithromycin fortherapy.6,30–33 _The_WHO _Guideline _for_the

treatmentofN.gonorrhoeaebasedondatafromhigh, middle-and low-income countries, publishedin2016,recommends ceftriaxoneorceﬁximeplusazithromycinprimarilyfor treat-mentofgenitalandanorectalgonococcalinfections(Table2).3

Over the last 80 years, N. gonorrhoeae has devel-oped oracquired resistancemechanismsforsulfonamides, penicillins, tetracycline, ciproﬂoxacin, and more recently azithromycinandceftriaxone,makingthismicroorganisma

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Table2–Antimicrobialtherapyrecommendedforgonococcalinfection.

Country/region/ organization

Therapy Specialsituations Year guideline published

Reference

Europe CRO500mgIM+AZM2gPO IfCROisunavailable,orantimicrobial injectionisimpossible,orpatientrefusesto takethemedication:CFX400mgPO+AZM 2gPO

IfAZMisunavailable,orpatientcannottake oralmedication:CRO500mgIM

IncaseofESCresistance,orifpatientis allergictopenicillinorcephalosporin:SPC2g IM+AZM2gPO

2013 29

U.S. CRO250mgIM+AZM1gPO IfCROisunavailable:CFX400mgPO+AZM 1gPO

2015 6 Canada CRO250mgIM+AZM1gPOor

CFX800mgPO

IfCROisunavailable:SPC2gIM+AZM1gPO Ifpatientisallergictocephalosporin:AZM2g PO

2013 32

Brazil CIP500mgPO+AZM500mgPOor CRO500mgIM+AZM500mgPO

Ifpatientisallergictocephalosporin:AZM2g PO

Ifpatientis<18year-oldorpregnant:CRO 500mgIM+AZM500mgPO

2015 20

Australia CRO500mgIM+AZM1gPO None 2016 30 WHO CRO250mgIM+AZM1gPOor

CFX400mgPO+AZM1gPO

Iflocalrecentdataconﬁrmsusceptibility,one antimicrobialinsingledoseisapossibility: CRO250mgIMorCFX400mgPOorSPC2gIM

2016 3

AZM,azithromycin;CFX,ceﬁxime;CIP,ciproﬂoxacina;CRO,ceftriaxone;SPC,spectinomycin;ESC,extendspectrumcephalosporin.

candidatetocauseanuntreatabledisease(Fig.1).34–36_In_the

nextsection,wedescribetheevolutionofN.gonorrhoeae resis-tanceinahistoricalandmolecularperspective,andprovide thebasisforunderstandingwhythisperceptionﬁtsamong thethreatsofthepost-antibioticera.

Evolution

of

antimicrobial

resistance

Sulfonamides,penicillinandtetracycline:threeobsolete choices

Sulfonamides

Sulfonamideswereintroducedasatherapyforgonorrhea dur-ingthe 1930s.37 _However,_by_1944,_a _rate_of_75%_treatment

failurewithsulphathiazoleorsulphapyridinewerereported amongst World War II soldiers in the Italian and Sicilian campaign.38_This_class_of_{antimicrobials}_acts_as_a_competitive

substratetothedihydropteroatesynthetase(DHPS)enzyme inthefolicacid biosynthesis.Inthegonococcus,resistance isachievedbyincreasedproductionoftheusualsubstrate, p-aminobenzoicacid,orsynthesisofamutatedDHPSwithlow afﬁnitytotheantimicrobial.39

Inthe1960s,inordertoimprovetheefﬁcacyof sulfona-midetotreatuncomplicatedgonorrhea,acombinedtherapy withtrimethoprim was proposed asan option.40 _This

sec-ondcompoundinhibitsan additionalreactioninthe same metabolicpathwaycatalyzedbythedihydrofolatereductase (DHFR)enzyme.However,N.gonorrhoeaeDHRFhaslowafﬁnity fortrimethoprim,andmayalsobegeneticallymodiﬁed, mak-ingthebacterialesssensitivetothisantimicrobial.41_Still,_the

synergiccombinationofsulfonamideswithtrimethoprimwas

usedtotreatgonorrhea inhighand multi-dosetherapeutic schemesuntilthe1970s.42,43

Penicillin

Penicillin, successfullyusedfordecades,wasintroducedas antimicrobialtreatmentforgonorrhea in1943, primarilyin casesofsulfonamidetreatmentfailure.44_However,_penicillin

decreasedsusceptibilityingonococcusstartinginthe1960s.45

From then until the 1980s, the efﬁcientpenicillin dose for gonococcus infectionsincreased24-folds,from 200,000Uto 4.8millionU.Meanwhile,penicillinMICofclinicalisolates var-iedfrom≤0.015␮g/mLto2.0␮g/mL.39

As a␤-lactam antimicrobial, penicillininhibits bacterial cell wall synthesis by binding to transpeptidase enzymes called penicillin-binding proteins (PBP) in the periplasm.46

Accordingly,overtheﬁrst50yearsofuse,penicillinresistance mechanisms ingonococcus were related todecreased sus-ceptibilitybycumulativechromosomalmutationsindifferent genesrelatedtocellwallbiosynthesis(penAand ponA1),or structuresaffectingtheperiplasmicdrugconcentration(penB, penCandmtrR).

PBP2 is an important PBP in N. gonorrhoeae. Mutations suchasanaspartateinsertionafterthe345position,anda variable number of substitutions inthe proteinc-terminal have been detected in isolates with penicillin decreased susceptibility.47,48_Such_new_alleles_are_reported_as_numbered

penA variants.49–55 _A _single _base _mutation _in _PBP1, _called

ponA1 (TtoCin1261bp,resultinginthe alterationL421P), decreasestheacylationwiththe␤-lactam.56

Mutations in the porB gene are known as penB muta-tions. Speciﬁc substitutions G120K and A121D have been characterizedtoaffecttopenicillinand tetracyclineMICin

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Cefixime or ceftriaxone Azithromycin Ciprofloxacin Tetracycline Penicillin Sulfonamide 1930 1940 1950 1960 1970 1980 1990 2000 2010 2020

Fig.1–EvolutionofNeisseriagonorrhoeaeresistancetoantimicrobials.Colorchangesindicateeventsthatimpactedthelevel ofresistancealongthetimeeachantimicrobialwasused.Sulfonamide:introduction,resistancereported,combineduse withtrimethoprim;penicillinandtetracycline:introduction,chromosomally-mediatedresistancereported,

plasmid-mediatedresistancereported;ciproﬂoxacin:introduction,resistancereported;azithromycin:introduction, resistancereported,highlevelresistancereported;ceﬁximeorceftriaxone:introduction,reducedsusceptibilityreported.

gonococcus.57 _Since_these_positions_are_presumably_located

ina region that formsa pore restriction zone, it was ﬁrst hypothesizedthatthesesubstitutionsaffectthepenetration ofpenicillin acrossthe outer membrane.57,58 _However,

fur-therstudiesdemonstratedthatpenBmutationsaffecttheMIC ofthisdrugonlywhenMtrC-MtrD-MtrEefﬂuxpumpis over-expressedinthegonococcus,throughapossiblesynergistic mechanism.59

The multiple transferable resistance (mtr) system of N. gonorrhoeaeencodesproteinsthatresemble bacterial/efﬂux-transportmoleculesoccurringinotherbacterialspecies.In thegonococcus,itsexpressionisaffectedbytwo transcrip-tionalregulators,MtrR(repressor)andMtrA(activator),and impactsonthesusceptibilityofthemicroorganismtoa vari-etyofhydrophobicsubstances,includingantimicrobialssuch as penicillin, tetracycline and macrolides, and detergent-like fattyacids.58,60 _Many _different_mechanisms _have_been

describedtoaffecttheexpressionofmtrCDE.Theseinclude mutationsleadingtodramaticaminoacidchangesA39Tor G45Dinthehelix-turn-helixDNAbindingstructureofMtrR,61

and asinglebase pair(bp) deletion (T:A)or adinucleotide insertion(TT:AA)withinthemtrRpromoter,whichmayimpair mtrR expression.60–62 _Other _changes _are _increased _mtrCDE

expressionrelatedtoamutationoutsidethemtrRlocus,61_and

acytosinetotiminemutation120bpupstreammtrC,which generatesasecondpromotertothegeneinsensitivetothe repressiveeffectofmtrR.61,63

PilQisanimportantgonococcaloutermembrane compo-nent,memberofsecretinproteinfamily,andinvolvedinType IVpilusformation.PilQandTypeIVpilusformtogether an SDS-resistantmultimericcomplex,whichactsasaporefor antibioticsandsmall molecules.ThereplacementG666L in thepilQgene,knownaspilQ2mutationorpenC,hindersthe creationofthePilQmultimeric,disruptingtheporeformed, andhencedecreasingtheinﬂuxofpenicillin.56,64

Throughsimultaneousoccurences,chromosomally medi-atedmechanismsleadtoapenicillinMICupto4␮g/mLin

gonococcus, which istwice the MIC reported in infections onlytreatablewithveryhigh penicillindoses.39,56 _However,

duringthe 1970s,N.gonorrhoeaeisolatespresentingMIC up to128␮g/mLemerged,65,66 _and_ended _the_penicillin _era_for

gonorrheatreatment.Thenewresistancemechanismwasa plasmid-mediated␤-lactamase(bla)genetypeTEM(bla-TEM),

and the isolatesbecame known as penicillinase-producing N.gonorrhoeae(PPNG).66,67 _N._gonorrhoeae_bla

TEMcarried

plas-midsaregeneticallyrelated,butpresentdifferentsizesand insertion/deletion sites, and are named according to their epidemiologicalorigin.68,69_The_most_frequently_described

bla-plasmidsinN.gonorrhoeaeareAsia(7426bp),Africa(5588bp), and Rio/Toronto (5154bp). Nevertheless, other types, such as Nimes (6798bp), New Zealand (9309bp), Johannesburg (4865bp), and Australian (3269bp) have been identiﬁed in gonococci.65–67,69–74

Tetracycline

Tetracycline wasintroducedasatreatmentoptionfor gon-orrheainpatients allergictopenicillininthe1950s.34 _This

antimicrobial agent affects protein synthesis by binding to the 30S ribosomal subunit.75 _Similar _to _penicillin

resis-tance,tetracyclineresistancegraduallyevolved.Infact,some of the chromosomal mutations related to penicillin resis-tance, such as penB and mtr overexpression, impair the tetracycline activity, raising the MIC to1␮g/mL.39,47

More-over, an additional single amino acid alteration V57M in theribosomalprotein10S(rpsJ1)isenoughtoelevate tetra-cycline MIC to≥2␮g/mL,76 _the _current _CLSI_breakpoint_for

resistance.12

Theﬁrst gonococcus isolatesshowingtetracycline high-levelresistance(MIC24–32␮g/mL)weredetectedintheU.S.in 1985.77,78_The_TetM_protein_was_identiﬁed_as_the_mechanism

responsible for the resistance phenotype, protecting the ribosomefromtetracyclinebinding.39 _In_N._gonorrhoeae,_tetM

isconservativelycarriedbytwoconjugativeplasmids(Dutch and American), similar in size, but probably evolutionarily

(7)

unrelated. Endonuclease restriction-fragment patterns of bothplasmidsdemonstrate largedifferences,andeventhe sequencesoftherespectivetetMencodedarenotidentical.79

Given the spread of resistance, the quinolone era in the gonorrheatreatmentstartedwhentetracyclinewasnolonger recommendedbythemid-1980s.80

Thequinoloneeraingonorrheatreatment

Ciprofloxacinwasdevelopedin1983andwasintroducedto theU.K.andU.S.marketsinthesecondpartofthe1980s.81 Initially, gonorrhea treatment using ciprofloxacin was per-formedwith a singledose of 250mg.However, because of earlyreportedcasesofdecreasedsusceptibility,thefirstCDC recommendation forciprofloxacintherapy forthis STIwas 500mg in a single dose.80,82 _Although _isolates _exhibiting

reducedsusceptibility(MIC≥0.25␮g/mL)hadbeen detected in London before 1989,82 _and _with _many _therapeutic

fail-ures reported during the 1990s,83–85 _{ciproﬂoxacin} _therapy

was continued to be used at the same dosage through-outthe world,foradditional10–25years dependingonthe country.

QuinolonesaffecttheactivityofDNAgyraseand topoiso-meraseIV,twotopoisomerasesessentialforDNAreplication, transcription,recombination,andrepair.Thisclassof antimi-crobialagentsactsbyformingadrug–enzyme–DNAcomplex, withfurtherreleaseofdouble-strand-DNAbreaks.86N. gonor-rhoeaeresistancetociproﬂoxacinismediatedbymutationsin thequinoloneresistance-determiningregion(QRDR),located nearthe topoisomerasesDNAbindingsite.Suchmutations inﬂuencesusceptibilityofisolatescumulatively.87_Mutations

leadingtoasingleaminoacidchangeinGyrApositions91or 95leadtointermediaryresistancelevel(MIC0.1–0.5␮g/mL); however,threeormoreaminoacidchangesinGyrA(at pos-itions91,95and102)andParC(87and91)and/orParE(439) proteinsmayleadtoMIC≥32␮g/mL.25,88

Onedecadeafterbeingrecommendedasthefirstchoice forgonorrheatreatmentintheU.S.,ciprofloxacintreatment wasabandonedintheAsianWesternPacific,becauseofhigh resistancerates. In Japan, forexample,the resistance per-centagewas6%in1993–1994androseto24%in1997–1998. Unfortunately,noinformationaboutthephylogenyof resis-tantstrainswasavailable.89_By _then,_{ciprofloxacin-resistant}

gonococcusalsoemerged inEurope.Studies conﬁrmedthe geneticcorrelationamongtheisolates,highlightingtheclonal patternofciproﬂoxacinresistantN.gonorrhoeaespread,mainly duetosexualnetworks.90–92 _In_a_survey_study_conducted_in

Brazilfrom2006to2010,23ofthe25resistantciproﬂoxacin N. gonorrhoeae isolates were clustered into two clonal groups.25

IntheU.S.,thegonococcalciprofloxacinresistancereached thewestcoastofthecountryandthen spreadthroughthe restoftheterritory.Thefirstciprofloxacinresistanceisolates (MIC1–16␮g/mL) weredetectedinHawaii in1999,affecting 9.5%oftheisolates.93_Soon_afterwards,_the_same_trend_was

seeninCalifornia(4.9%in2001),whichmadetheCDC with-drawtheciproﬂoxacinrecommendationtreatmentinthose states.94_{Ciproﬂoxacin}_was_removed_from_guidelines_in_Asia,

Europe,andtheU.S.intheearlytomid-2000s.95

Extendspectrumcephalosporinsandazithromycin:the currentrecommendedantimicrobialstotreatgonorrhea

Azithromycin

Azithromycinwasincludedasapossibletherapyfor gonor-rheainthebeginningofthe1980s.95_This_macrolide_interacts

withthePsiteofthe50Sribosomalsubunit,impairing the peptidyltransferasepolypeptidechainelongation.96

Differentmechanismsmayimpactazithromycinactivityin N.gonorrhoeae.Oneofthemistheoverexpressionoftheefﬂux pumpmtrCDEdirectedbythesamemolecularmechanisms reported to decrease the susceptibility of N. gonorrhoeaeto penicillin,increasingtheazithromycinMICto0.5␮g/mL.62,97

Anadditionalmechanism ofazithromycin reduced suscep-tibility is the occurrenceofmutations inthe L4 ribosomal protein.97,98_This_protein_is_in_close_contact_with_the

peptidyl-transferase region in the V domain of the 23S rRNA, and mutationsleading to dramaticaminoacid changes,as the reportedG70Dinitsextendedloop,mayindirectlyimpactthe rRNAconformation.97,99–101

Whenmutationsoccurdirectlyinthis23SrRNAdomain, resistancetoazithromycinemerges.Inthiscase,twomain mechanisms are described. A single adenine methylation, promoted by plasmid mediated erm enzymes in the 2058 position of the peptidyl transferase V domain, prevents antimicrobialtargetbinding,andrisestheazithromycinMIC to 1–4␮g/mL.95,102,103 _Moreover, _the _speciﬁc _{substitutions}

A2143Gand/orC2599Tpresentinonetofourrrlgenealleles, encodingthe 23S RNA, resultinvariedazithromycinMICs. Mutationsinasinglealleleusuallydonotsigniﬁcantlyimpact

theMIC103,104_however,_MICs_of₉₆_␮g/mL_or_>256_␮g/mL_were

reportedforisolatesshowingC2599TorA2143Gmutationsin thefouralleles,respectively.105,106

N. gonorrhoeae exhibiting a high level of resistance to azithromycin has emergedin the pastfew years.The ﬁrst caseoccurredinArgentinain2001.107_Since_then,_the

detec-tion of high level azithromycin resistance has occurred in different countries, such as Scotland, England, Italy, U.S. and China.108–110 _An_outbreak _with _isolates _exhibiting

MIC≥256␮g/mLaffectedﬁvemenandthreewomen,all het-erosexual, inEngland, between November 2014and March 2015.Sevenisolateswere from thesame NG-MASTST9768 andpresentedtheA2143Gsubstitutioninallfour23S encod-inggenealleles.111_Nonetheless,_other_studies_{characterizing}

collections ofisolatespresenting highMIC toazithromycin demonstratedthatsuchresistancefrequentlyoccurin non-clonallyrelatedisolates,withavarietyofNG-MASTSTsbeing detectedinasamecountryorregion.104,106,112_In_this_case_it

isworthmentioningthattheoccurrenceofNG-MASTST649 presenting similar azithromycin MIC (≥256␮g/mL) and 23S mutationsinthreedifferentcontinents.104,106,109

Ceftriaxone

The ESC ceftriaxone presents high activity against gram-negative bacteria, such as N. gonorrhoeae, by high afﬁnity bindingtoPBP2.113_Ceftriaxone_introduction_as_a

monother-apy for gonococcal infections was driven by the rise of ciproﬂoxacin resistancerates.35 _Ceﬁxime,_an _oral_ESC, _was

in use before ceftriaxone; however, susceptibility to this antimicrobialdecreasedveryquickly.Forexample,resistance

(8)

ratesreached30%insixhospitalsinthecentralterritoryof Japaninthebeginningofthe2000s.49,114_{Subsequently,}

treat-mentfailures withceﬁximemonotherapy werereported in Europe.115–117

ESCresistancemightbepromotedbyalterationsinpenB, mtrR,andpenCgene,althoughmutationsinpenAgene,which encodes PBP2, seem tobe the main ceftriaxone resistance determinant. Thealtered penA gene can result from point mutations, or from genetic recombination between com-mensalNeisseriaspp.colonizingotherhumansites,thelast generating a mosaic-like structure.95,118 _The _altered _PBP2

presentsdiminishedafﬁnitytoceftriaxone.

Resistance to ceftriaxone is characterized by MIC>0.5␮g/mL by CDC5 _and _>0.125_␮g/mL _by _EUCAST,11

and detection of resistant isolates is still rare, with one reportedcaseineach Japan,Franceand Spain.55,119,120 _ESC

resistance has been related to the presence of different patterns of PBP2. Nevertheless, studies have associated the reduced susceptibility to the clonal spread of speciﬁc lineages.55,119–121_In_Japan,_the_resistant_isolate_exhibited_MIC

8␮g/mLand4␮g/mLtoceﬁximeandceftriaxone,respectively, presentingPBP2patternhighlysimilartothePBP2X,withfour additionalmutatedaminoacidsresiduesA311V,T316P,A328T, andT484S.55 _This_isolate_was_assigned _to_ST7363_by_MLST,

the same ST of previous ceﬁxime-resistant N. gonorrhoeae isolatescirculatinginJapan.54_The_isolates_detected_in_France

and Spain were included inthe same NG-MAST ST1407, a successfulclone circulating in Europeand associated with decreasedsusceptibility toESC. Theisolates exhibitedMIC 4␮g/mLand1.5␮g/mLtoceﬁxime,and2␮g/mLand1.5␮g/mL to ceftriaxone in France and Spain, respectively. The PBP2 XXXIVpatternwithoneadditionalaminoacidsubstitution A501Pwasdetectedintheisolatesfrombothcountries.119,120

Theoccurrenceofthiscloneisepidemiologically relatedto MSM.

TheemergenceofESCresistanceingonococcusandthe absenceofanewperspectivefortreatmentofgonorrheahave motivatedthe dualtherapy protocols withceftriaxone and azithromycin.Accordingtoagenciesperformingsurveillance programs,thisstrategymaydeceleratetheincreaseof resis-tanceratestotheseantimicrobials.30,122

Concluding

remarks

Currently, comparing N. gonorrhoeae resistance rates and epidemiological trends amongcountries is a difﬁcult task, consideringthedifferentbreakpointsandsamplingstrategies thathavebeenadoptedworldwide.Dataobtainedin differ-entcountriesemphasizethatsurveillanceisindeedessential topreservethepossibilityoftreatmentbasedonsyndromic diagnosisforgonorrhea.

Theimplementationofsuchsurveillanceprogramsmay pose some challenges. For instance, in Brazil, the lack of interactionbetweenresearchcentersandtheofﬁcialhealth systemsfacilitiesfacemajorobstacles.Moreover,continuing institutionalandﬁnancialassistanceisnecessarytoobtain, maintainand analyze N.gonorrhoeaeisolates inastandard wayforconsecutiveyears.Collectingantimicrobial suscepti-bilitytestingresultsfromdiagnosticlaboratoriesmayprove

usefultohaveanideaofcurrentresistancerates.However, such approachis notapplicable toepidemiological studies sincepatients’dataandeventheisolatesarenotavailablefor furtherstudies

In conclusion, given the particularities of the disease, whichiscommunitybased,andthedifﬁcultiesofobtainand preserveN.gonorrhoeaeisolates,extensiveorganizationis nec-essarytoimplementadequatesurveillanceprograms.Inthis sense,webelievethatagoodstrategywouldbetoestablish associationsbetweenSTImedicalcentersandaccredited lab-oratories that canreport resultstoa governmentalagency responsible for standardizedprotocols,centralize data and publishregular reports.Theinternationalagencies collabo-ration mayprovenecessaryinachieving anefﬁcientglobal action,especiallyinlowincomecountries.

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s

1.ChanPA,RobinetteA,MontgomeryM,etal.Extragenital

infectionscausedbyChlamydiatrachomatisandNeisseria

gonorrhoeae:areviewoftheliterature.InfectDisObstet Gynecol.2016;2016:5758387.

2.IsonCA.BiologyofNeisseriagonorrhoeaeandtheclinical

pictureofinfection.In:GrossGE,TyringSK,eds.Sexually

TransmittedInfectionsandSexuallyTransmittedDiseases.Berlin

Heidelberg:Springer;2011:77–90.

3.WHOGuidelinesfortheTreatmentofNeisseriagonorrhoeae. Geneva:WorldHealthOrganization;2016

http://www.ncbi.nlm.nih.gov/books/NBK379221/.

4.CDC.GonococcalIsolatesSurveillanceProjectProtocol2016.A Atlanta:CDC;2016

http://www.cdc.gov/std/gisp/gisp-protocol-many-2016.pdf.

Accessed02.06.17.

5.CDC.SexuallyTransmittedDiseaseSurveillance,NationalProﬁle;

2014https://www.cdc.gov/std/stats14/gonorrhea.htm.

Accessed01.06.17.

6.CDC.SexuallyTransmittedDiseasesTreatmentGuidelines;2015

http://www.cdc.gov/mmwr/pdf/rr/rr6403.pdf.Accessed

02.06.17.

7.CDC.GonococcalIsolatesSurveillanceProjectProﬁles;2014

http://www.cdc.gov/std/gisp2014/gisp-2014-text-ﬁg-tables. pdf.Accessed02.06.17.

8.GradYH,KirkcaldyRD,TreesD,etal.Genomic

epidemiologyofNeisseriagonorrhoeaewithreduced

susceptibilitytoceﬁximeintheUSA:aretrospective

observationalstudy.LancetInfectDis.2014;14(3):220–226.

9.CanadaPHAof,CanadaPHAof.NationalSurveillanceof AntimicrobialSusceptibilitiesofNeisseriagonorrhoeae–Annual Summary;2014https://www.canada.ca/en/public-health/ services/publications/drugs-health-products/national-

surveillance-antimicrobial-susceptibilities-neisseria-gonorrhoeae-annual-summary-2014.html.Published

05.02.16,Accessed01.06.17.

10.ECDC.GonococcalAntimicrobialSusceptibilitySurveillancein Europe2014.Stockholm:ECDC;2016http://ecdc.europa.eu/

en/publications/Publications/gonococcal-antimicrobial-susceptibility-surveillance-Europe-2014.pdf.Accessed

02.06.17.

11.EUCAST.TheEuropeanCommitteeonAntimicrobial SusceptibilityTesting.BreakpointTablesforInterpretationof MICsandZoneDiameters.Version7.0.EUCAST;2017

http://www.eucast.org/clinicalbreakpoints/.Accessed

(9)

12.CLSI.ClinicalandLaboratoryStandardsInstitute.Performance StandardsforAntimicrobialSusceptibilityTesting.

Twenty-SeventhInformationalSupplementM100-S27.Wayne:

CLSI;2017.

13.LahraMM,EnriquezRP.NationalNeisseriaNetwork.

AustralianGonococcalSurveillanceProgrammeannual

report,2015.CommunDisIntellQRep.2017;41(1).E.

14.LahraMM,EnriquezRP.AustralianGonococcalSurveillance

Programme,1Januaryto31March2015.CommunDisIntellQ

Rep.2015;39(2):E297–E298.

15.LahraMM.WHOWesternPaciﬁcandSouthEastAsian

GonococcalAntimicrobialSurveillanceProgramme.

SurveillanceofantibioticresistanceinNeisseriagonorrhoeae

intheWHOWesternPaciﬁcandSouthEastAsianRegions,

2010.CommunDisIntellQRep.2012;36(1):95–100.

16.WHO.BaselineReportonGlobalSexuallyTransmittedInfection Surveillance.WHO;2012http://www.who.int/reproductive

health/publications/rtis/9789241505895/en/.Accessed

02.06.17.

17.NdowaFJ,FrancisJM,MachihaA,Faye-KetteH,Fonkoua

MC.Gonococcalantimicrobialresistance:perspectivesfrom

theAfricanregion.SexTransmInfect.2013;89(suppl

4):iv11–iv15.

18.WHO|Progressreportoftheimplementationoftheglobal strategyforpreventionandcontrolofsexuallytransmitted infections:2006–2015.WHO.http://www.who.int/

reproductivehealth/publications/rtis/progress-report-stis-strategy/en/Accessed02.06.17.

19.DillonJ-AR,TreckerMA,ThakurSD.Gonococcal

AntimicrobialSurveillanceProgramNetworkinLatin

AmericaandCaribbean1990–2011.Twodecadesofthe

gonococcalantimicrobialsurveillanceprograminSouth

AmericaandtheCaribbean:challengesandopportunities.

SexTransmInfect.2013;89(suppl4):iv36–iv41.

20.NotíciasBdaSBlog,Saúde,Ministério.Pesquisarevelaaltas taxasderesistênciaaosantimicrobianosnopaís.Blogda Saúde.http://www.blog.saude.gov.br/gx0dy8Accessed 01.06.17.

21.ProtocoloClínicoeDiretrizesTerapêuticasparaAtenção IntegralàsPessoascomInfecçõesSexualmente Transmissíveis(IST)|DepartamentodeVigilância, PrevençãoeControledasIST,doHIV/AidsedasHepatites Virais. http://www.aids.gov.br/publicacao/2015/protocolo-

clinico-e-diretrizes-terapeuticas-para-atencao-integral-pessoas-com-infeccAccessed02.06.17.

22.DSTnoBrasil|DepartamentodeVigilância,Prevenc¸ãoe ControledasIST,doHIV/AidsedasHepatitesVirais.

http://www.aids.gov.br/pagina/dst-no-brasilAccessed

02.06.17.

23.StarninoS,GASP-LACWorkingGroup,GalarzaP,etal.

Retrospectiveanalysisofantimicrobialsusceptibilitytrends

(2000–2009)inNeisseriagonorrhoeaeisolatesfromcountries

inLatinAmericaandtheCaribbeanshowsevolving

resistancetociproﬂoxacin,azithromycinanddecreased

susceptibilitytoceftriaxone.SexTransmDis.

2012;39(10):813–821.

24.BeldaJuniorW,VelhoPENF,ArnoneM,FagundesLJ.

Emergenceofﬂuoroquinolone-resistantNeisseria

gonorrhoeaeinSãoPaulo,Brazil.BrazJMicrobiol.

2007;38(2):293–295.

25.UeharaAA,AmorinELT,FerreiraMdeF,etal.Molecular

characterizationofquinolone-resistantNeisseriagonorrhoeae

isolatesfromBrazil.JClinMicrobiol.2011;49(12):4208–4212.

26.CostaLMB,PedrosoERP,VieiraNetoV,SouzaVCP,Teixeira

MJB.AntimicrobialsusceptibilityofNeisseriagonorrhoeae

isolatesfrompatientsattendingapublicreferralcenterfor

sexuallytransmitteddiseasesinBeloHorizonte,Stateof

MinasGerais,Brazil.RevSocBrasMedTrop.

2013;46(3):304–309.

27.IlinaEN,OparinaNY,ShitikovEA,BorovskayaAD,Govorun

VM.MolecularsurveillanceofclinicalNeisseriagonorrhoeae

isolatesinRussia.JClinMicrobiol.2010;48(10):3681–3689.

28.UnemoM,DillonJ-AR.Reviewandinternational

recommendationofmethodsfortypingNeisseria

gonorrhoeaeisolatesandtheirimplicationsforimproved

knowledgeofgonococcalepidemiology,treatment,and

biology.ClinMicrobiolRev.2011;24(3):447–458.

29.VidovicS,CaronC,TaheriA,etal.Usingcrude

whole-genomeassembliesofNeisseriagonorrhoeaeasa

platformforstrainanalysis:clonalspreadofgonorrhea

infectioninSaskatchewan,Canada.JClinMicrobiol.

2014;52(10):3772–3776.

30.BignellC,UnemoM,EuropeanSTIGuidelinesEditorial

Board.2012Europeanguidelineonthediagnosisand

treatmentofgonorrhoeainadults.IntJSTDAIDS.

2013;24(2):85–92.

31.Gonorrhoea–AustralianSTIManagementGuidelines.

http://sti.guidelines.org.au/sexually-transmissible-infections/gonorrhoeaAccessed01.06.17.

32.HamasunaR,YasudaM,IshikawaK,etal.Thesecond

nationwidesurveillanceoftheantimicrobialsusceptibility

ofNeisseriagonorrhoeaefrommaleurethritisinJapan,

2012–2013.JInfectChemotherOffJJpnSocChemother.

2015;21(5):340–345.

33.GovernmentofCanadaPHAofC.CanadianGuidelineson SexuallyTransmittedInfections–PublicHealthAgencyof Canada.

http://www.phac-aspc.gc.ca/std-mts/sti-its/cgsti-ldcits/index-eng.phpPublished01.02.13,Accessed02.06.17.

34.UnemoM,ShaferWM.AntibioticresistanceinNeisseria

gonorrhoeae:origin,evolution,andlessonslearnedforthe

future.AnnNYAcadSci.2011;1230:E19–E28.

35.UnemoM,NicholasRA.Emergenceofmultidrug-resistant,

extensivelydrug-resistantanduntreatablegonorrhea.

FutureMicrobiol.2012;7(12):1401–1422.

36.UnemoM,ShaferWM.Futuretreatmentofgonorrhea–

novelemergingdrugsareessentialandinprogress?Expert

OpinEmergDrugs.2015;20(3):357–360.

37.KampmeierRH.Introductionofsulfonamidetherapyfor

gonorrhea.SexTransmDis.1983;10(2):81–84.

38.CampbellDJ.GonorrhoeainN.Africaandcentral

Mediterranean.BrMedJ.1944;2(4357):44.

39.JohnsonSR,MorseSA.AntibioticresistanceinNeisseria

gonorrhoeae:geneticsandmechanismsofresistance.Sex TransmDis.1988;15(4):217–224.

40.CsonkaGW,KnightGJ.Therapeutictrialoftrimethoprimas

apotentiatorofsulphonamidesingonorrhoea.BrJVener

Dis.1967;43(3):161–165.

41.AverettDR,RothB,BurchallJJ,BaccanariDP.Dihydrofolate

reductasefromNeisseriasp.AntimicrobAgentsChemother.

1979;15(3):428–435.

42.LawrenceA,PhillipsI,NicolC.Variousregimensof

trimethoprim-sulfamethoxazoleusedinthetreatmentof

gonorrhea.JInfectDis.1973;128(suppl):673–678.

43.LewisDA.TheGonococcusﬁghtsback:isthistimeaknock

out?SexTransmInfect.2010;86(6):415–421.

44.VanSlykeCJ,ArnoldRC,BuchholtzM.Penicillintherapyin

sulfonamide-resistantgonorrheainmen.AmJPublicHealth

NationsHealth.1943;33(12):1392–1394.

45.MartinJE,LesterA,PriceEV,SchmaleJD.Comparativestudy

ofgonococcalsusceptibilitytopenicillinintheUnited

States,1955–1969.JInfectDis.1970;122(5):459–461.

46.ZapunA,Contreras-MartelC,VernetT.Penicillin-binding

proteinsandbeta-lactamresistance.FEMSMicrobiolRev.

(10)

47.SparlingPF,SarubbiFA,BlackmanE.Inheritanceof

low-levelresistancetopenicillin,tetracycline,and

chloramphenicolinNeisseriagonorrhoeae.JBacteriol.

1975;124(2):740–749.

48.FedarovichA,CookE,TombergJ,NicholasRA,DaviesC.

StructuraleffectoftheAsp345ainsertionin

penicillin-bindingprotein2frompenicillin-resistantstrains

ofNeisseriagonorrhoeae.Biochemistry(Mosc).

2014;53(48):7596–7603.

49.ItoM,DeguchiT,MizutaniK-S,etal.Emergenceandspread

ofNeisseriagonorrhoeaeclinicalisolatesharboring

mosaic-likestructureofpenicillin-bindingprotein2in

CentralJapan.AntimicrobAgentsChemother.

2005;49(1):137–143.

50.LindbergR,FredlundH,NicholasR,UnemoM.Neisseria

gonorrhoeaeisolateswithreducedsusceptibilitytoceﬁxime

andceftriaxone:associationwithgeneticpolymorphisms

inpenA,mtrR,porB1b,andponA.AntimicrobAgents

Chemother.2007;51(6):2117–2122.

51.WhileyDM,LimniosEA,RayS,SlootsTP,TapsallJW.

DiversityofpenAalterationsandsubtypesinNeisseria

gonorrhoeaestrainsfromSydney,Australia,thatareless

susceptibletoceftriaxone.AntimicrobAgentsChemother.

2007;51(9):3111–3116.

52.PandoriM,BarryPM,WuA,etal.Mosaicpenicillin-binding

protein2inNeisseriagonorrhoeaeisolatescollectedin2008

inSanFrancisco,California.AntimicrobAgentsChemother.

2009;53(9):4032–4034.

53.LeeS-G,LeeH,JeongSH,etal.VariouspenAmutations

togetherwithmtrR,porBandponAmutationsinNeisseria

gonorrhoeaeisolateswithreducedsusceptibilitytoceﬁxime

orceftriaxone.JAntimicrobChemother.2010;65(4):

669–675.

54.OhnishiM,WatanabeY,OnoE,etal.Spreadofa

chromosomalceﬁxime-resistantpenAgeneamong

differentNeisseriagonorrhoeaelineages.AntimicrobAgents

Chemother.2010;54(3):1060–1067.

55.OhnishiM,GolparianD,ShimutaK,etal.IsNeisseria

gonorrhoeaeinitiatingafutureeraofuntreatablegonorrhea?

Detailedcharacterizationoftheﬁrststrainwithhigh-level

resistancetoceftriaxone.AntimicrobAgentsChemother.

2011;55(7):3538–3545.

56.RoppPA,HuM,OleskyM,NicholasRA.MutationsinponA,

thegeneencodingpenicillin-bindingprotein1,andanovel

locus,penC,arerequiredforhigh-levelchromosomally

mediatedpenicillinresistanceinNeisseriagonorrhoeae.

AntimicrobAgentsChemother.2002;46(3):769–777.

57.OleskyM,HobbsM,NicholasRA.Identiﬁcationandanalysis

ofaminoacidmutationsinporinIBthatmediate

intermediate-levelresistancetopenicillinandtetracycline

inNeisseriagonorrhoeae.AntimicrobAgentsChemother.

2002;46(9):2811–2820.

58.MaierTW,ZubrzyckiL,CoyleMB,ChilaM,WarnerP.

GeneticanalysisofdrugresistanceinNeisseriagonorrhoeae:

productionofincreasedresistancebythecombinationof

twoantibioticresistanceloci.JBacteriol.1975;124(2):834–842.

59.OleskyM,ZhaoS,RosenbergRL,NicholasRA.

Porin-mediatedantibioticresistanceinNeisseria

gonorrhoeae:ion,solute,andantibioticpermeationthrough

PIBproteinswithpenBmutations.JBacteriol.

2006;188(7):2300–2308.

60.HagmanKE,PanW,SprattBG,BalthazarJT,JuddRC,Shafer

WM.ResistanceofNeisseriagonorrhoeaetoantimicrobial

hydrophobicagentsismodulatedbythemtrRCDEefﬂux

system.MicrobiolReadEngl.1995;141(Pt3):611–622.

61.WarnerDM,ShaferWM,JerseAE.Clinicallyrelevant

mutationsthatcausederepressionoftheNeisseria

gonorrhoeaeMtrC-MtrD-MtrEEfﬂuxpumpsystemconfer

differentlevelsofantimicrobialresistanceandinvivo

ﬁtness.MolMicrobiol.2008;70(2):462–478.

62.ZarantonelliL,BorthagarayG,LeeEH,VealW,ShaferWM.

Decreasedsusceptibilitytoazithromycinanderythromycin

mediatedbyanovelmtr(R)promotermutationinNeisseria

gonorrhoeae.JAntimicrobChemother.2001;47(5):651–654.

63.OhneckEA,ZaluckiYM,JohnsonPJT,etal.Anovel

mechanismofhigh-level,broad-spectrumantibiotic

resistancecausedbyasinglebasepairchangeinNeisseria

gonorrhoeae.mBio.2011;2(5).

64.ZhaoS,TobiasonDM,HuM,SeifertHS,NicholasRA.The

penCmutationconferringantibioticresistanceinNeisseria

gonorrhoeaearisesfromamutationinthePilQsecretinthat

interfereswithmultimerstability.MolMicrobiol.

2005;57(5):1238–1251.

65.AshfordWA,GolashRG,HemmingVG.

Penicillinase-producing.LancetLondEngl.

1976;2(7987):657–658.

66.PhillipsI.Beta-lactamase-producing,penicillin-resistant

gonococcus.LancetLondEngl.1976;2(7987):656–657.

67.PercivalA,RowlandsJ,CorkillJE,etal.

Penicillinase-producinggonococciinLiverpool.LancetLond

Engl.1976;2(8000):1379–1382.

68.PagottoF,AmanAT,NgLK,YeungKH,BrettM,DillonJA.

Sequenceanalysisofthefamilyofpenicillinase-producing

plasmidsofNeisseriagonorrhoeae.Plasmid.2000;43(1):24–34.

69.MüllerEE,FayemiwoSA,LewisDA.Characterizationofa

novel␤-lactamase-producingplasmidinNeisseria

gonorrhoeae:sequenceanalysisandmoleculartypingofhost

gonococci.JAntimicrobChemother.2011;66(7):1514–1517.

70.YeungKH,DillonJR,PauzéM,WallaceE.Anovel

4.9-kilobaseplasmidassociatedwithanoutbreakof

penicillinase-producingNeisseriagonorrhoeae.JInfectDis.

1986;153(6):1162–1165.

71.VanEmbdenJD,Dessens-KroonM,VanKlingerenB.Anew

beta-lactamaseplasmidinNeisseriagonorrhoeae.JAntimicrob

Chemother.1985;15(2):247–250.

72.GoubyA,BourgG,RamuzM.Previouslyundescribed

6.6-kilobaseRplasmidinpenicillinase-producingNeisseria

gonorrhoeae.AntimicrobAgentsChemother.

1986;29(6):1095–1097.

73.BrettM.Anovelgonococcalbeta-lactamaseplasmid.J

AntimicrobChemother.1989;23(4):653–654.

74.TrembizkiE,BuckleyC,LawrenceA,LahraM,WhileyD,

GRANDStudyInvestigators.Characterizationofanovel

Neisseriagonorrhoeaepenicillinase-producingplasmid

isolatedinAustraliain2012.AntimicrobAgentsChemother.

2014;58(8):4984–4985.

75.ChopraI,RobertsM.Tetracyclineantibiotics:modeof

action,applications,molecularbiology,andepidemiology

ofbacterialresistance.MicrobiolMolBiolRevMMBR.

2001;65(2):232–260,secondpage,tableofcontents.

76.HuM,NandiS,DaviesC,NicholasRA.High-level

chromosomallymediatedtetracyclineresistancein

Neisseriagonorrhoeaeresultsfromapointmutationinthe

rpsJgeneencodingribosomalproteinS10incombination

withthemtrRandpenBresistancedeterminants.

77.CDC.Tetracycline-resistantNeisseriagonorrhoeae–Georgia, Pennsylvania,NewHampshire.https://www.cdc.gov/

mmwr/preview/mmwrhtmL/00000611.htmAccessed

02.06.17.

78.MorseSA,JohnsonSR,BiddleJW,RobertsMC.High-level

tetracyclineresistanceinNeisseriagonorrhoeaeisresultof

acquisitionofstreptococcaltetMdeterminant.Antimicrob

AgentsChemother.1986;30(5):664–670.

79.PachulecE,vanderDoesC.Conjugativeplasmidsof

(11)

https://www.cdc.gov/mmwr//preview/mmwrhtmL/

00001459.htm.Accessed02.06.17.

81.AndersonJE,HobbsMM,BiswasGD,SparlingPF.Opposing

selectiveforcesforexpressionofthegonococcallactoferrin

receptor.MolMicrobiol.2003;48(5):1325–1337.

82.GransdenWR,WarrenCA,PhillipsI,HodgesM,BarlowD.

DecreasedsusceptibilityofNeisseriagonorrhoeaeto

ciproﬂoxacin.LancetLondEngl.1990;335(8680):51.

83.BirleyH,McDonaldP,CareyP,FletcherJ.Highlevel

ciproﬂoxacinresistanceinNeisseriagonorrhoeae.Genitourin

Med.1994;70(4):292–293.

84.TapsallJW,LimniosEA,ThackerC,etal.High-level

quinoloneresistanceinNeisseriagonorrhoeae:areportof

twocases.SexTransmDis.1995;22(5):310–311.

85.DeguchiT,SaitoI,TanakaM,etal.Fluoroquinolone

treatmentfailureingonorrhea.EmergenceofaNeisseria

gonorrhoeaestrainwithenhancedresistanceto

ﬂuoroquinolones.SexTransmDis.1997;24(5):247–250.

86.JacobyGA.Mechanismsofresistancetoquinolones.Clin

InfectDisOffPublInfectDisSocAm.2005;41(suppl 2):S120–S126.

87.GilesJA,FalconioJ,YuengerJD,ZenilmanJM,DanM,Bash

MC.Quinoloneresistance-determiningregionmutations

andportypeofNeisseriagonorrhoeaeisolates:resistance

surveillanceandtypingbymolecularmethodologies.JInfect

Dis.2004;189(11):2085–2093.

88.LindbäckE,RahmanM,JalalS,WretlindB.Mutationsin

gyrA,gyrB,parC,andparEinquinolone-resistantstrainsof

Neisseriagonorrhoeae.APMISActaPatholMicrobiolImmunol Scand.2002;110(9):651–657.

89.TanakaM,NakayamaH,HaraokaM,SaikaT.Antimicrobial

resistanceofNeisseriagonorrhoeaeandhighprevalenceof

ciproﬂoxacin-resistantisolatesinJapan,1993to1998.JClin

Microbiol.2000;38(2):521–525.

90.deNeelingAJ,vanSanten-VerheuvelM,SpaargarenJ,

WillemsRJ.AntimicrobialresistanceofNeisseriagonorrhoeae

andemergingciproﬂoxacinresistanceintheNetherlands,

1991to1998.AntimicrobAgentsChemother.

2000;44(11):3184–3185.

91.MavroidiA,TzouvelekisLS,TassiosPT,FlemetakisA,

DaniilidouM,TzelepiE.CharacterizationofNeisseria

gonorrhoeaestrainswithdecreasedsusceptibilityto

ﬂuoroquinolonesisolatedinGreecefrom1996to1999.JClin

Microbiol.2000;38(9):3489–3491.

92.SuX,LindI.Molecularbasisofhigh-levelciproﬂoxacin

resistanceinNeisseriagonorrhoeaestrainsisolatedin

Denmarkfrom1995to1998.AntimicrobAgentsChemother.

2001;45(1):117–123.

93.CDC.Fluoroquinolone-resistanceinNeisseriagonorrhoeae,

Hawaii,1999,anddecreasedsusceptibilitytoazithromycin

inN.gonorrhoeae,Missouri,1999.MMWRMorbMortalWkly

Rep.2000;49(37):833–837.

94.CDC.Increasesinﬂuoroquinolone-resistantNeisseria

gonorrhoeae–HawaiiandCalifornia,2001.MMWRMorb MortalWklyRep.2002;51(46):1041–1044.

95.UnemoM,ShaferWM.AntimicrobialresistanceinNeisseria

gonorrhoeaeinthe21stcentury:past,evolution,andfuture.

ClinMicrobiolRev.2014;27(3):587–613.

96.PoehlsgaardJ,DouthwaiteS.Thebacterialribosomeasa

targetforantibiotics.NatRevMicrobiol.2005;3(11):870–881.

97.BelkacemA,JacquierH,GoubardA,etal.Molecular

epidemiologyandmechanismsofresistanceof

azithromycin-resistantNeisseriagonorrhoeaeisolatedin

Franceduring2013–14.JAntimicrobChemother.

2016;71(9):2471–2478.

98.AllenVG,SeahC,MartinI,MelanoRG.Azithromycin

resistanceiscoevolvingwithreducedsusceptibilityto

cephalosporinsinNeisseriagonorrhoeaeinOntario,Canada.

99.UrlaubH,KruftV,BischofO,MüllerEC,Wittmann-Liebold

B.Protein-rRNAbindingfeaturesandtheirstructuraland

functionalimplicationsinribosomesasdeterminedby

cross-linkingstudies.EMBOJ.1995;14(18):4578–4588.

100.Tait-KamradtA,DaviesT,CronanM,JacobsMR,Appelbaum

PC,SutcliffeJ.Mutationsin23SrRNAandribosomalprotein

L4accountforresistanceinpneumococcalstrainsselected

invitrobymacrolidepassage.AntimicrobAgentsChemother.

2000;44(8):2118–2125.

101.ZengelJM,JerauldA,WalkerA,WahlMC,LindahlL.The

extendedloopsofribosomalproteinsL4andL22arenot

requiredforribosomeassemblyorL4-mediatedautogenous

control.RNA.2003;9(10):1188–1197.

102.RobertsMC,ChungWO,RoeD,etal.Erythromycin-resistant

NeisseriagonorrhoeaeandoralcommensalNeisseriaspp.

carryknownrRNAmethylasegenes.AntimicrobAgents

Chemother.1999;43(6):1367–1372.

103.DemczukW,MartinI,PetersonS,etal.Genomic

epidemiologyandmolecularresistancemechanismsof

azithromycin-resistantNeisseriagonorrhoeaeinCanadafrom

1997to2014.JClinMicrobiol.2016;54(5):1304–1313.

104.ChisholmSA,DaveJ,IsonCA.High-levelazithromycin

resistanceoccursinNeisseriagonorrhoeaeasaresultofa

singlepointmutationinthe23SrRNAgenes.Antimicrob

AgentsChemother.2010;54(9):3812–3816.

105.BercotB,BelkacemA,GoubardA,etal.High-level

azithromycin-resistantNeisseriagonorrhoeaeclinicalisolate

inFrance,March2014.EuroSurveillBullEurSurMalTransm

EurCommunDisBull.2014;19(44).

106.StevensK,ZaiaA,TawilS,etal.Neisseriagonorrhoeae

isolateswithhigh-levelresistancetoazithromycinin

Australia.JAntimicrobChemother.2015;70(4):1267–1268.

107.GalarzaPG,AlcaláB,SalcedoC,etal.Emergenceofhigh

levelazithromycin-resistantNeisseriagonorrhoeaestrain

isolatedinArgentina.SexTransmDis.2009;36(12):787–788.

108.ChisholmSA,NealTJ,AlawattegamaAB,BirleyHDL,Howe

RA,IsonCA.Emergenceofhigh-levelazithromycin

resistanceinNeisseriagonorrhoeaeinEnglandandWales.J

AntimicrobChemother.2009;64(2):353–358.

109.KatzAR,KomeyaAY,SogeOO,etal.Neisseriagonorrhoeae

withhigh-levelresistancetoazithromycin:casereportof

theﬁrstisolateidentiﬁedintheUnitedStates.ClinInfectDis

OffPublInfectDisSocAm.2012;54(6):841–843.

110.NiC,XueJ,ZhangC,ZhouH,vanderVeenS.High

prevalenceofNeisseriagonorrhoeaewithhigh-level

resistancetoazithromycininHangzhou,China.JAntimicrob

Chemother.2016;71(8):2355–2357.

111.ChisholmSA,WilsonJ,AlexanderS,etal.Anoutbreakof

high-levelazithromycinresistantNeisseriagonorrhoeaein

England.SexTransmInfect.2016;92(5):365–367.

112.JacobssonS,GolparianD,ColeM,etal.WGSanalysisand

molecularresistancemechanismsof

azithromycin-resistant(MIC>2mg/L)Neisseriagonorrhoeae

isolatesinEuropefrom2009to2014.JAntimicrobChemother.

2016;71(11):3109–3116.

113.BarryPM,KlausnerJD.Theuseofcephalosporinsfor

gonorrhea:theimpendingproblemofresistance.Expert

OpinPharmacother.2009;10(4):555–577.

114.TanakaM,NakayamaH,TunoeH,etal.Aremarkable

reductioninthesusceptibilityofNeisseriagonorrhoeae

isolatestocephemsandtheselectionofantibiotic

regimensforthesingle-dosetreatmentofgonococcal

infectioninJapan.JInfectChemotherOffJJpnSocChemother.

2002;8(1):81–86.

115.UnemoM,GolparianD,SyversenG,VestrheimDF,MoiH.

(12)

recommendedﬁrst-lineceﬁximeforgonorrhoeatreatment,

Norway,2010.EuroSurveillBullEurSurMalTransmEur

CommunDisBull.2010;15(47).

116.UnemoM,GolparianD,StaryA,EigentlerA.FirstNeisseria

gonorrhoeaestrainwithresistancetoceﬁximecausing

gonorrhoeatreatmentfailureinAustria,2011.EuroSurveill

BullEurSurMalTransmEurCommunDisBull.2011;16(43).

117.UnemoM,GolparianD,HestnerA.Ceftriaxonetreatment

failureofpharyngealgonorrhoeaveriﬁedbyinternational

recommendations,Sweden,July2010.EuroSurveillBullEur

SurMalTransmEurCommunDisBull.2011;16(6).

118.AmeyamaS,OnoderaS,TakahataM,etal.Mosaic-like

structureofpenicillin-bindingprotein2Gene(penA)in

clinicalisolatesofNeisseriagonorrhoeaewithreduced

susceptibilitytoceﬁxime.AntimicrobAgentsChemother.

2002;46(12):3744–3749.

119.UnemoM,GolparianD,NicholasR,OhnishiM,GallayA,

SednaouiP.High-levelceﬁxime-andceftriaxone-resistant

NeisseriagonorrhoeaeinFrance:novelpenAmosaicallelein

asuccessfulinternationalclonecausestreatmentfailure.

120.CámaraJ,SerraJ,AyatsJ,etal.Molecularcharacterization

oftwohigh-levelceftriaxone-resistantNeisseriagonorrhoeae

isolatesdetectedinCatalonia,Spain.JAntimicrobChemother.

2012;67(8):1858–1860.

121.LahraMM,RyderN,WhileyDM.Anewmultidrug-resistant

strainofNeisseriagonorrhoeaeinAustralia.NEnglJMed.

2014;371(19):1850–1851.

https://www.cdc.gov/mmwr/preview/mmwrhtmL/rr5912a1. htm.Accessed02.06.17.