In recent decades, Neisseria gonorrhoeae has developed resistance to several antimicrobial classes. Molecular epidemiology approaches are useful for detecting emerging, often resistant, gonococcal clones. In this study, 67 N. gonorrhoeae isolates from different anatomic sites, collected over 8 years in Italy, were analyzed by whole genome sequencing (WGS). WGS was performed using the Illumina NextSeq 500 platform. Phylogenetic analysis was based on core single nucleotide polymorphism (SNP) and core genome multilocus sequence typing (cgMLST). N. gonorrhoeae multi-antigen sequence typing (NG-MAST), MLST, and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) were carried out in silico using WGS data. Antimicrobial susceptibility against a four-drug panel was evaluated using a gradient diffusion method. Overall, gonococci clustered in accordance with NG-MAST, MLST, NG-STAR, and antimicrobials susceptibility profiles, but not with the site of isolation, HIV status, and patient sexual orientation. Phylogenetic analysis identified nine clades: two of them were the predominant and including gonococci of G1407 and G2400 genogroups.
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References
1.
NewmanL., RowleyJ., HoornS.V, WijesooriyaN.S, UnemoM., LowN., StevensG., GottliebS., KiarieJ., and TemmermanM.2015. Global estimates of the prevalence and incidence of four curable 390 sexually transmitted infections in 2012 based on systematic review and global reporting. PLoS One. 10:e0143304.
UnemoM., and JensenJ.S.2017. Antimicrobial-resistant sexually transmitted infections: gonorrhea and Mycoplasma genitalium. Nat. Rev. Urol. 14:139–152.
6.
Public Health England. 2018. UK case of Neisseria gonorrhoeae with high-level resistance to azithromycin and resistance to ceftriaxone acquired abroad. Health Protection Report, Volume, 12, Number 11.
IsonC.A., HusseyJ., SankarK.N, EvansJ., and AlexanderS.2011. Gonorrhoea treatment failures to cefixime and azithromycin in England, 2010. Euro. Surveill. 16:19833.
9.
UnemoM., GolparianD., SyversenG., VestrheimD.F, and MoiH.2010. Two cases of verified clinical failures using internationally recommended first-line cefixime for gonorrhoea treatment, Norway, 2010. Euro. Surveill. 15:19721.
10.
UnemoM., GolparianD., NicholasR., OhnishiM., GallayA., and SednaouieP.2012. High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure. Antimicrob. Agents. Chemother. 56:1273–1280.
11.
AllenV.G., MitterniL., SeahC., RebbapragadaA., MartinI.E, LeeC., SiebertH., TownsL., MelanoR.G, and LowD.E.2013. Neisseria gonorrhoeae treatment failure and susceptibility to cefixime in Toronto, Canada. JAMA. 309:163–170.
12.
StarninoS., StefanelliP.; and Neisseria gonorrhoeae Italian Study Group. 2009. Azithromycin-resistant Neisseria gonorrhoeae strains recently isolated in Italy. J. Antimicrob. Chemother. 63:1200–1204.
13.
CarannanteA., VaccaP., GhisettiV., LatinoM.A, CusiniM., MatteelliA., VocaleC., PrignanoG., LeliC., OberP., AntonettiR., PolettiF., and StefanelliP.2017. Genetic resistance determinants for cefixime and molecular analysis of gonococci isolated in Italy. Microb. Drug. Resist. 23:247–252.
14.
UnemoM., GolparianD., and ShaferW.M.2014. Challenges with gonorrhea in the era of multi-drug and extensively drug resistance are we on the right track? Expert. Rev. Anti. Infect. Ther. 12:653–656.
15.
OhnishiM., GolparianD., ShimutaK., SaikaT., HoshinaS., IwasakuK., NakayamaJ., KitawakiJ., and UnemoM.2011. Is Neisseria gonorrhoeae initiating a future era of untreatable gonorrhea?: detailed characterization of the first strain with high-level resistance to ceftriaxone. Antimicrob. Agents. Chemother. 55:3538–3545.
16.
GratrixJ., BergmanJ., EganC., DrewsS.J, ReadR., and SinghA.E.2013. Retrospective review of pharyngeal gonorrhea treatment failures in Alberta, Canada. Sex. Transm. Dis. 40:877–879.
17.
ShaferW.M., BalthazarJ.T, HagmanK.E, and MorseS.A.1995. Missense mutations that alter the DNA-binding domain of the MtrR protein occur frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to fecal lipids. Microbiology, 141:907–911.
18.
ItoM., DeguchiT., MizutaniK.S, YasudaM., YokoiS., ItoS., TakahashiY., IshiharaS., KawamuraY., and EzakiT.2005. Emergence and spread of Neisseria gonorrhoeae clinical isolates harboring mosaic-like structure of penicillin-binding protein 2 in Central Japan. Antimicrob. Agents. Chemother. 49:137–143.
19.
LindbergR., FredlundH., NicholasR., and UnemoM.2007. Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: association with genetic polymorphisms in penA, mtrR, porB1b, and ponA. Antimicrob. Agents. Chemother. 51:2117–2122.
20.
ChisholmS.A., DaveJ., and IsonC.A.2010. High-level azithromycin resistance occurs in Neisseria gonorrhoeae as a result of a single point mutation in the 23S rRNA genes. Antimicrob. Agents. Chemother. 54:3812–3816.
MartinI.M.C., IsonC.A, AanensenD.M, FentonK.A, and SprattB.G.2004. Rapid sequence-based identification of gonococcal transmission clusters in a large metropolitan area. J. Infect. Dis. 189:1497–1505.
ShimutaK., UnemoM., NakayamaS., Morita-IshiharaT., DorinM., and KawahataT., OhnishiM.; and Antibiotic-Resistant Gonorrhea Study Group. 2013. Antimicrobial resistance and molecular typing of Neisseria gonorrhoeae isolates in Kyoto and Osaka, Japan, 2010 to 2012: intensified surveillance after identification of the first strain (H041) with high-level ceftriaxone resistance. Antimicrob. Agents. Chemother. 57:5225–5232.
DemczukW., SidhuS., UnemoM., WhileyD.M, AllenV.G, DillonJ.R, ColeM., SeahC., TrembizkiE., TreesD.L, KershE.N, AbramsA.J, de VriesH.J, van DamA.P, MedinaI., BharatA., MulveyM.R, Van DomselaarG., and MartinI.2017. Neisseria gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR): a novel antimicrobial resistance multilocus typing scheme for tracking the global dissemination of N. gonorrhoeae strains. J. Clin. Microbiol. 55:1454–1468.
27.
EzewudoM.N., JosephS.J, Castillo-RamirezS., DeanD., Del RioC., DidelotX., DillonJ.A, SeldenR.F, ShaferW.M, TuringanR.S, UnemoM., and ReadT.D.2015. Population structure of Neisseria gonorrhoeae based on whole genome data and its relationship with antibiotic resistance. PeerJ, 3:e806.
28.
SpiteriG., ColeM., UnemoM., HoffmannS., IsonC., and van de LaarM.2013. The European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP)—a sentinel approach in the European Union (EU)/European Economic Area (EEA). Sex. Transm. Infect. 89:16–18.
29.
EUCAST. 2018. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) Breakpoints tables for interpretation of MICs and zone diameters, Version 8.0, 2018. Available at www.eucast.org
30.
UnemoM., GolparianD., Sánchez-BusóL., GradY., JacobssonS., OhnishiM., LahraM.M, LimniosA., SikoraA.E, WiT., and HarrisS.R.2016. The novel 2016 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations: phenotypic, genetic and reference genome characterization. J. Antimicrob. Chemother. 71:3096–3108.
JoshiN.A., and FassJ.N.2011. Sickle: a sliding-window, adaptive, quality-based trimming tool for FastQ files (Version 1.33) (Software). Available at https://github.com/najoshi/sickle
33.
SimpsonJ.T., Kim WongK., ShaunD., JackmanS.D, JacquelineE., ScheinJ.E, StevenJ.M, JonesS.J.M, and BirolI.2009. ABySS: a parallel assembler for short read sequence data. Genome Res. 19:1117–1123.
34.
GardnerS.N., SlezakT., and HallB.G.2015. kSNP3.0: SNP detection and phylogenetic analysis of genomes without genome alignment or reference genomes. Bioinformatics, 31:2877–2878.
35.
TamuraK., and NeiM.1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10:512–526.
36.
KumarS., StecherG., and TamuraK.2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33:1870–1874.
37.
TamuraK., NeiM., and KumarS.2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A, 101:11030–11035.
38.
JolleyK.A., and MaidenM.C.2010. BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics, 11:595.
39.
HusonD.H., and BryantD.2006. Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 23:254–267.
40.
LeeS.G., LeeH., JeongS.H, YongD., ChungG.T, LeeY.S, ChongY., and LeeK.2010. Various penA mutations together with mtrR, porB and ponA mutations in Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone. J. Antimicrob. Chemother. 65:669–675.
41.
CarannanteA., CusiniG.M, MatteelliA., Dal ConteI., GhisettiV., D'AntuonoA., CavriniF., AntonettiR., and StefanelliP.2012. Cefixime and ceftriaxone susceptibility of Neisseria gonorrhoeae in Italy from 2006 to 2010. Clin. Microbiol. Infect. 18:558–564.
42.
HarrisonO.B., ClemenceM., DillardJ.P, TangC.M, TreesD., GradY.H, and MaidenM.C.2016. Genomic analyses of Neisseria gonorrhoeae reveal an association of the gonococcal genetic island with antimicrobial resistance. J. Infect. 73:578–587.
43.
SachdevD., KumariI., BalaM., KumarV., and SalujaD.2017. Mutation pattern in the genome of Neisseria gonorrhoeae and its association with multidrug-resistant isolates from Delhi, India. Indian J. Med. Microbiol. 35:109–112.
44.
JohnsonS.R., GradY., AbramsA.J, PettusK., and TreesD.L.2017. Use of whole-genome sequencing data to analyze 23S rRNA-mediated azithromycin resistance. Int. J. Antimicrob. Agents. 49:252–254.
45.
DemczukW., LynchT., MartinI., Van DomselaarG., GrahamM., BharatA., AllenV., HoangL., LefebvreB., TyrrellG., HorsmanG., HaldaneD., GarceauR., WylieJ., WongT., and MulveyM.R.2015. Whole-genome phylogenomic heterogeneity of Neisseria gonorrhoeae isolates with decreased cephalosporin susceptibility collected in Canada between 1989 and 2013. J. Clin. Microbiol. 53:191–200.
46.
HarrisS.R., ColeM.J, SpiteriG., Sánchez-BusóL., GolparianD., JacobssonS., GoaterR., AbudahabK., YeatsC.A, BercotB., BorregoM.J, CrowleyB., StefanelliP., TripodoF., AbadR., AanensenD.M, and UnemoM.; Euro-GASP study group. 2018. Public health surveillance of multidrug-resistant clones of Neisseria gonorrhoeae in Europe: a genomic survey. Lancet. Infect. Dis. 18:758–768.
47.
CarannanteA., RennaG., Dal ConteI., GhisettiV., MatteelliA., PrignanoG., ImparaG., CusiniM., D'AntuonoA., VocaleC., AntonettiR., GainoM., BusettiM., LatinoM.A, MencacciA., BonannoC., CavaM.C, GiraldiC., and StefanelliP.2014. Changing antimicrobial resistance profiles among Neisseria gonorrhoeae isolates in Italy, 2003 to 2012. Antimicrob. Agents Chemother. 58:5871–5876.
48.
StefanelliP, VescioM.F, LandiniM.P, Dal ConteI., MatteelliA., CristaudoA., GainoM., CusiniM., BarbuiA.M, MencacciA., De NittisR., GhisettiV., StroppianaE., and CarannanteA.; Neisseria gonorrhoeae antimicrobials resistant Study Group. 2017. Time trend analysis (2009–2016) of antimicrobial susceptibility in Neisseria gonorrhoeae isolated in Italy following the introduction of the combined antimicrobial therapy. PLoS One. 12:e0189484.
49.
BignellC., and UnemoM.2013. European STI Guidelines Editorial Board. 2012 European guideline on the diagnosis and treatment of gonorrhoea in adults. Int. J. STD. AIDS. 24:85–92.
50.
MortimerT.D., and GradY.H.2019. Applications of genomics to slow the spread of multidrug-resistant Neisseria gonorrhoeae. Ann. N. Y. Acad. Sci. 1435:93–109.
51.
EndimianiA., GuilarteY.N, TinguelyR., HirzbergerL., SelviniS., LupoA., HauserC., and FurrerH.2014. Characterization of Neisseria gonorrhoeae isolates detected in Switzerland (1998–2012): emergence of multidrug-resistant clones less susceptible to cephalosporins. BMC Infect. Dis. 14:106.
52.
UnemoM., Del RioC., and ShaferW.M.2016. Antimicrobial resistance expressed by Neisseria gonorrhoeae: a major global public health problem in the 21st century. Microbiol. Spectr. 4:EI10-0009-2015.
53.
JacobssonS., GolparianD., ColeM., SpiteriG., MartinI., BergheimT., BorregoM.J, CrowleyB., CrucittiT., Van DamA.P, HoffmannS., JevericaS., KohlP., Mlynarczyk-BonikowskaB., PakarnaG., StaryA., StefanelliP., PavlikP., TzelepiE., AbadR., HarrisS.R, and UnemoM.2016. WGS analysis and molecular resistance mechanisms of azithromycin-resistant (MIC >2 mg/L) Neisseria gonorrhoeae isolates in Europe from 2009 to 2014. J. Antimicrob. Chemother. 71:3109–3116.
54.
BennettJ.S., JolleyK.A, SparlingP.F, SaundersN.J, HartC.A, FeaversI.M, and MaidenM.C.2007. Species status of Neisseria gonorrhoeae: evolutionary and epidemiological inferences from multilocus sequence typing. BMC Biol. 5:35.
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