Antimicrobial susceptibility of Treponema pallidum subspecies pallidum: an in-vitro study

www. helance .com/mic obe Published online Oc obe 9, 2023 h ps://doi.o g/10.1016/S2666-5247(23)00219-7 1 A icles An imic obial suscep ibili y o T eponema pallidum subspecies pallidum: an in- i o s udy Lau en C Tan alo, Nicole A P Liebe man, Cla a Pé ez-Mañá, Cla a Suñe , Ma i Vall Mayans, Ma ia Ubals, Camila González-Bei as, Alicia Rod íguez-Gascón, And és Canu , Fe nando González-Candelas, John Muelle , Kenne h Tapia, Alexande L G eninge , Lo enzo Giacani, O iol Mi jà Summa y Backg ound The inc easing incidence o syphilis and he limi a ions o i s -line ea men wi h penicillin, pa icula ly in neu osyphilis, neona al syphilis, and p egnancy, highligh he need o expand he he apeu ic epe oi e o e ec i e managemen o his disease. We assessed he in- i o e icacy o 18 an ibio ics om se e al classes on T eponema pallidum subspecies pallidum (T pallidum), he syphilis bac e ia. Me hods Using he in- i o cul u e sys em o T pallidum, we exposed he pa hogen o a concen a ion ange o each es ed an ibio ic. A e a 7-day incuba ion, he eponemal bu den was e alua ed by quan i a i e PCR a ge ing he T pallidum p0574 gene. The p ima y ou come was he minimum inhibi o y concen a ion (MIC) a which he quan i a i e PCR alues we e no signi ican ly highe han he inoculum wells. We also in es iga ed he suscep ibili y o mac olide- esis an s ains o high concen a ions o azi h omycin, and he possibili y o de eloping esis ance o linezolid, a p oposed candida e o syphilis ea men . Findings Amoxicillin, ce iaxone, se e al o al cephalospo ins, edizolid, and dalba ancin exhibi ed an i- eponemal ac i i y a concen a ions achie able in human plasma ollowing egula dosing egimens. The expe imen s e ealed a MIC o amoxicillin a 0·02 mg/L, ce iaxone a 0·0025 mg/L, cephalexin a 0·25 mg/L, ce e ame and ce ixime a 0·0313 mg/L, ce u oxime a 0·0156 mg/L, edizolid a 0·0625 mg/L, spec inomycin a 0·1 mg/L, and dalba ancin a 0·125 mg/L. The MIC o zoli lodacin and balo loxacin was 2 mg/L. E apenem, isoniazid, py azinamide, and me onidazole had ei he a poo o no e ec . Azi h omycin concen a ions up o 2 mg/L (64 imes he MIC) we e ine ec i e agains s ains ca ying mu a ions associa ed o mac olide esis ance. Exposu e o sub he apeu ic doses o linezolid o 10 weeks did no induce pheno ypic o geno ypic esis ance. In e p e a ion Cephalospo ins and oxazolidinones a e po en ial candida es o expanding he cu en he apeu ic epe oi e o syphilis. Ou indings wa an es ing e icacy in animal models and, i success ul, clinical assessmen o e icacy. Funding Eu opean Resea ch Council. Copy igh © 2023 The Au ho (s). Published by Else ie L d. This is an Open Access a icle unde he CC BY-NC-ND 4.0 license. In oduc ion Acco ding o WHO, he global bu den o syphilis anges be ween 18 million and 36 million cases, wi h an incidence o 5·6–11·0 million new in ec ions pe yea in adul s.1–3 Al hough mos o hese cases occu in low- income and middle-income coun ies, he e has also been a s eady esu gence o syphilis o e he pas wo decades in high-income coun ies (unpublished).4–6 Penicillin is he p e e ed ea men o syphilis;7 howe e , neu osyphilis equi es in a enous in usions o injec ions e e y 4 h o up o 14 days, neona al syphilis also equi es in a enous in usions e e y 8 h o 12 h o 10 days,8 and no ea men op ions exis o p egnan women alle gic o penicillin in whom doxycycline is con aindica ed because o e a ogenici y and who a e a isk o congeni al ansmission. Fu he mo e, he e a e empo al sho ages in p oduc ion,9 and u al a eas o en do no ha e specialised pe sonnel o adminis e penicillin in amuscula injec ions and p ope ly s o e he d ug.10 The a o emen ioned scena io o e s a compelling a gumen o esea ch endea ou s o b oaden he he apeu ic op ions o syphilis (unpublished). A majo ba ie o es ing al e na i e an ibio ics o syphilis was he inabili y o cul u e he causa i e agen o he disease, T eponema pallidum subspecies pallidum (T pallidum). In a ecen b eak h ough in June, 2018, howe e , con inuous long- e m cul u e o T pallidum was achie ed by cocul i a ion wi h abbi epi helial cells in a mic oae ophilic a mosphe e.11 As a esul , i is now possible o es T pallidum suscep ibili y o an imic obial agen s o de e mine he minimum inhibi o y concen a ions (MICs) associa ed wi h each compound. Using his sys em, we had p e iously shown ha eponemal g ow h was inhibi ed by penicillin G a concen a ions o 0·003 mg/L o mo e and by linezolid concen a ions o 0·5 mg/L o mo e.12 The same s udy Lance Mic obe 2023 Published Online Oc obe 9, 2023 h ps://doi.o g/10.1016/ S2666-5247(23)00219-7 Depa men o Medicine, Di ision o Alle gy and In ec ious Diseases (L C Tan alo BS, P o L Giacani PhD) and Depa men o Labo a o y Medicine and Pa hology (N A P Liebe man PhD, A L G eninge MD), Uni e si y o Washing on, Sea le, WA, USA; Clinical Pha macology Uni , Hospi al Uni e si a i Ge mans T ias i Pujol, Ins i u de Rece ca Ge mans T ias i Pujol, Badalona, Spain (C Pé ez-Mañá PhD); Depa men o Pha macology, The apeu ics and Toxicology, Uni e si a Au ònoma de Ba celona, Ba celona, Spain (C Pé ez-Mañá); Sexually T ansmi ed In ec ions and Skin Neglec ed T opical Diseases Sec ion, Figh In ec ious Diseases Founda ion, Hospi al Ge mans T ias i Pujol, Ba celona, Spain (C Suñe PhD, M Vall Mayans PhD, M Ubals MD, C González-Bei as PhD, O Mi jà MD); Uni e si a de Vic- Uni e si a Cen al de Ca alunya, Vic, Spain (C Suñe , O Mi jà); Pha macokine ic, Nano echnology, and Gene The apy G oup (Pha maNanoGene), Facul y o Pha macy, Uni e si y o he Basque Coun y UPV/EHU, Vi o ia-Gas eiz, Spain (P o A Rod íguez-Gascón PhD); Bioa aba, Mic obiology, In ec ious Disease, An imic obial Agen s, and Gene The apy G oup, Vi o ia- Gas eiz, Spain (P o A Rod íguez-Gascón, A Canu MD); Mic obiology Se ice, A aba Uni e si y Hospi al, Osakide za Basque Heal h Se ice, Vi o ia-Gas eiz, Spain (A Canu ); Join Resea ch Uni In ec ion and Public Heal h, FISABIO-Uni e si a de A icles 2 www. helance .com/mic obe Published online Oc obe 9, 2023 h ps://doi.o g/10.1016/S2666-5247(23)00219-7 showed no an i- eponemal ac i i y o moxi loxacin (up o 2 mg/L), and clo azimine (up o 2 mg/L). Addi ionally, a s udy based on he same cul i a ion sys em showed a MIC o 0·1 mg/L o doxycycline,13 and a sepa a e publica ion epo ed low MIC alues o ou penicillin de i a i es and ou cephalospo ins.14 O e all, he e is a la ge knowledge gap conce ning he an ibac e ial ac i i y o a wide ange o d ug classes in wild- ype s ains o T pallidum. In his s udy, we es ed a b oad ange o an ibio ics belonging o he classes o aminopenicillins, cephalo- spo ins, ca bapenems, luo oquinolones, oxazol idinones, lipoglycopep ides, aminoglycosides, mac olides, an i- mycobac e ials, an ipa asi ics, and spi opy imidine ione agains T pallidum. Fu he mo e, gi en ha p e ious wo k showed he e icacy o linezolid o syphilis ea men ,12 we p opaga ed T pallidum in sub he apeu ic concen a ions o linezolid o 10 weeks o assess he po en ial o selec ion o a esis an s ain. Me hods S udy design and T pallidum s ains Fo his in- i o s udy, we used h ee T pallidum s ains (SS14, UW330B, and Chicago C) o pla e inocula ion o pe o m an ibio ic es ing. The SS14 s ain was used o es all an imic obials, whe eas UW330B and Chicago C we e used o es azi h omycin only. We aimed o es a leas one US Food and D ug Adminis a ion-app o ed d ug om each class and subclass o an ibio ics. Two p io i isa ion c i e ia we e applied o selec ing he an ibio ics o es ing: i s , pha macological p ope ies ha would make an an ibio ic sui able o epu posing o ea syphilis; and second, an ibio ics used o o he common condi ions, ega dless o hei pha macological p ope ies, o gain a be e unde s anding o hei po en ial e ec s on syphilis. The de ailed o igin o he T pallidum s ains used in his s udy (appendix p 2) and he an ibio ic selec ion p ocess (appendix p 2) ha e been p o ided. In- i o cul i a ion o T pallidum P ocedu es o he in- i o cul i a ion o T pallidum we e done as desc ibed p e iously.11 B ie ly, wo se s o cul u es we e p epa ed, one o he suscep ibili y assay and one o he bac e icidal and eco e y assay. The suscep ibili y assay in ol ed es ing d ug concen a ion in 96-well pla es (8 × 12 o ma ; Co ning, NY, USA). Each d ug concen a ion was ea ed as a sepa a e expe imen al g oup and es ed eigh imes in eigh eplica e wells. Fi e con ol g oups València, València, Spain (P o F González-Candelas PhD); Ins i u e o In eg a i e Sys ems Biology, Uni e si a de València-CSIC, Pa e na, Spain (P o F González-Candelas); CIBER Epidemiology and Public Heal h, Mad id, Spain (P o F González-Candelas); Inno i a Special y The apeu ics, Wal ham, MA, USA (J Muelle PhD); Depa men o Medicine, Di ision o Alle gy and In ec ious Diseases, and Depa men o Global Heal h, Uni e si y o Washing on, Sea le, WA, USA (K Tapia MSc, L Giacani); Vaccine and In ec ious Disease Di ision, F ed Hu chinson Cance Resea ch Cen e , Sea le, WA, USA (A L G eninge ); School o Medicine and Heal h Sciences, Uni e si y o Papua New Guinea, Po Mo esby, Papua New Guinea (O Mi jà) Co espondence o: D Lo enzo Giacani, Depa men o Global Heal h, Uni e si y o Washing on, Sea le, WA 98104, USA [email protected] on.edu Resea ch in con ex E idence be o e his s udy On Sep 1, 2022, be o e submi ing ou s udy, we sea ched he PubMed da abase o a icles published om incep ion o Sep 1, 2022, epo ing an ibio ics wi h in- i o an i- eponemal ac i i y. Ou sea ch using he key e ms “syphilis”, OR “T eponema pallidum”, AND “suscep ibili y es ing” wi h no language es ic ions, e ie ed 19 publica ions. Mos o hese a icles ocused on azi h omycin esis ance-con e ing mu a ions. Howe e , wo a icles had used a issue-cul u e sys em es ablished in 2018, which acili a ed long- e m mul iplica ion o T eponema pallidum subspecies pallidum (T pallidum) o s udying i s d ug suscep ibili y p o ile. Ou own s udy, done by ou eam, con i med he an i- eponemal ac i i y o penicillin and linezolid as e idenced by he minimum inhibi o y concen a ion (MIC) alue es ima e, whe eas moxi loxacin and clo azimine did no exhibi ac i i y. Ano he g oup o esea che s also epo ed he an i- eponemal ac i i y o doxycycline. Concu en ly, while ou manusc ip was unde going pee e iew, a sepa a e publica ion in June, 2023 sc eened 100 β-lac ams and epo ed he MIC alues o ou penicillin de i a i es and ou cephalospo ins, indica ing hei po en ial o ea ing syphilis. Added alue o his s udy This s udy holds impo an alue by p o iding d ug- suscep ibili y measu emen s o all commonly used classes o an ibio ics, dis inguishing i om p e ious s udies ha ei he ocused on a limi ed numbe o compounds o sc eened nume ous compounds om a single an ibio ic amily, speci ically β-lac ams. No ably, all ou s udies, including ou s, used simila cul u e me hods wi h sligh a ia ions in ou come measu emen ools. Ou esul s ega ding β-lac ams demons a e a p omising alignmen and eplicabili y wi h he indings o he o he esea ch g oup, as bo h se s o da a exhibi MIC alues in he same o de o magni ude. Mo eo e , ou s udy p io i ised eadily a ailable comme cialised compounds ha ha e po en ial o quick ansla ion in o clinical p ac ice i posi i e esul s a e ob ained in clinical ials. Addi ionally, we conside ed ele an pha macokine ic and pha macodynamic aspec s o aid in he in e p e a ion o MIC esul s and p o ide a mo e comp ehensi e unde s anding o he e icacy o an ibio ics’ e icacy, dosing egimen, and po en ial ea men ou comes. We iden i ied compounds ha exhibi ed an i- eponemal ac i i y in i o a achie able concen a ions in human plasma, such as amoxicillin, ce iaxone, o al cephalospo ins, edizolid, and dalba ancin. In addi ion, we ha e done long- e m p opaga ion o T pallidum in sub he apeu ic concen a ions o linezolid, p e iously shown o be ac i e, and we ha e seen no e idence o selec ion o pheno ypic o geno ypic esis ance. Implica ions o all he a ailable e idence The cumula i e expe ience om a ious s udies, including ou own, o e s a aluable new app oach o a deepe unde s anding o T pallidum d ug suscep ibili y and has impo an implica ions o he ield. The esul s o ou s udy show he clinical po en ial o se e al app o ed d ugs, including β-lac ams and oxazolidinones, ha could be epu posed o ea ing syphilis and o he eponemal in ec ions. See Online o appendix A icles www. helance .com/mic obe Published online Oc obe 9, 2023 h ps://doi.o g/10.1016/S2666-5247(23)00219-7 3 wi hou an ibio ics we e included, each es ed in eigh eplica e wells. Fou con ol g oups we e ha es ed a di e en imes (a day 0, day 1, day 4, and day 7) a e inocula ion, and one g oup con aining he an ibio ic sol en (dime hyl sul oxide o wa e ) ins ead o he es d ug was ha es ed on day 7. Range es ed (mg/L) P ima y MIC Seconda y MIC MBC (mg/L) D ug plasma concen a ions* P ima y MIC (mg/L)† Seconda y MIC (mg/L)† Cmin (mg/L) Dose adminis e ed o he Cmin calcula ion Unbound ac ion Na u al penicillins Benza hine penicillin G 0·0001–0·06‡ No es ed in his s udy 0·003‡ 0·003‡ 0·012§ 1·2 million uni s single dose, IM 0·55–0·72 Aminopenicillins Amoxicillin 0·0025–0·16 0·02 0·01 0·01 >0·2¶ 500 mg single dose, PO 0·83 Cephalospo ins Ce iaxone 0·00063–1 0·0025 0·0025 0·0025 29·7 1000 mg/24 h, IM 0·50 Cephalexin 0·0625–8 0·25 0·25 0·25 0·30¶ 1000 mg single dose, PO 0·85–0·90 Ce e ame 0·0039–0·25 0·0313 0·0625 0·0625 >0·3 500 mg/12 h, PO 0·78 Ce u oxime 0·0039–0·25 0·0156 0·0156 0·0156 0·20¶ 250 mg single dose, PO 0·50 Ce ixime 0·0039–0·25 0·0313 0·0313 0·0313 0·08 400 mg/24 h, PO 0·34 Ca bapenems E apenem 0·00375–2 >2 >2 >2 0·8 1 g/24 h, IV 0·05 Te acyclines Doxycycline 0·004–2·5|| 0·1|| No de e mined in his s udy 0·1|| >1 100 mg/24 h, PO 0·07–0·18 Fluo oquinolones Moxi loxacin 0·06–2‡ No de e mined in his s udy 2‡ >2‡ 0·4–0·6 400 mg/24 h, PO 0·50 Balo loxacin 0·25–16 2 2 >2 0·23 100 mg/12 h, PO ·· Mac olides Azi h omycin 0·0313–2 <0·0313** 0·125** <0·0313** 0·05 250 mg/24 h, PO 0·5–0·9 Oxazolidinones Linezolid 0·0156–2 0·5 0·125 0·125 6·2 600 mg/12 h, PO 0·69 Tedizolid 0·0078–0·5 0·0625 0·313 0·0156–0·0313 0·41 200 mg/24 h, PO 0·10–0·30 Lipoglycopep ides Dalba ancin 0·0039–0·25 0·125 0·125 0·125 19·5†† 1500 mg single dose, IV 0·07 Aminoglycosides Spec inomycin 0·02–2 0·1 0·1 0·25 15¶ 2000 mg single dose, IM ·· An imycobac e ials Isoniazid 0·0078–0·5 >0·5 >0·5 >0·5 Unde ec able 300 mg/24 h, PO ·· Py azinamide 1·0–64 >64 >64 >64 7 1500 mg/24 h, PO ·· Clo azimine 0·06–2‡ No de e mined in his s udy 1‡ 1‡ 0·02§§ 200 mg single dose, PO ·· An ipa asi ics I e mec in 0·125–40 MIC h eshold una ained‡‡ MIC h eshold una ained‡‡ MIC h eshold una ained‡‡ 0·01§§ 12 mg single dose, PO ·· Ni oimidazoles Me onidazole 0·0313–2 >2 >2 >2 11·8 500 mg/8 h, PO 0·8 Spi opy imidine ione Zoli lodacin 0·250–4 2 1 2 1§§ 3000 mg single dose, PO ·· Cmin=minimum blood plasma concen a ion. IM=injec ion in o a muscle. IV=injec ion in o a ein. MBC=minimum bac e icidal concen a ion. MIC=minimum inhibi o y concen a ion. PO=o al adminis a ion. *The appendix (p 7) p o ides he li e a u e sou ces used as a e e ence o pha macokine ic in o ma ion. †The p ima y MIC was de ined as he lowes an ibio ic dilu ion a which he p0574 qPCR alues we e no signi ican ly highe han he inoculum wells (day 0 con ol g oup), as p e iously de ined by Edmondson and colleagues.13 A seconda y MIC was de ined as he lowes an ibio ic dilu ion a which he p0574 qPCR alues we e signi ican ly lowe han he posi i e con ol wells (day 7 con ol g oup), which mo e closely ollows he b o h dilu ion p ocedu e. ‡Haynes and colleagues.12 §22 days a e single dose adminis a ion. ¶8 h a e adminis a ion. ||Edmondson and colleagues.13 **Only o suscep ible s ains. ††168 h a e adminis a ion. ‡‡Because o i e mec in oxici y o S 1Ep cells. §§24 h a e adminis a ion. Table : MIC and li e a u e plasma concen a ion alues A icles 4 www. helance .com/mic obe Published online Oc obe 9, 2023 h ps://doi.o g/10.1016/S2666-5247(23)00219-7 Wells we e seeded wi h 3 × 10³ abbi S 1Ep cells in 150 μL minimum essen ial media and incuba ed o e nigh . The nex day, minimum essen ial media was emo ed and 150 μL T pallidum cul u e media 2 (TpCM2; equilib a ed o e nigh in a 34°C igas incuba o ) was added o 3 h o acclima e cells o low oxygen. A e emo ing TpCM2, 148·5 μL o a 3·3 × 10⁵ T pallidum cells pe mL inoculum was added o each well (5 × 10⁴ T pallidum cells pe well). An ibio ic solu ions (1·5 µL) we e added om 100- imes concen a ed s ocks wi hou al e ing olume ollowed by incuba ion a 34°C in a igas incuba o un il ha es . The es ed concen a ion ange o each d ug is epo ed ( able) along wi h he key mic obiological and pha macokine ic alues o es ed an ibio ics and he s anda d o ca e benza hine penicillin G. Expe imen al wells wi h a ying an ibio ic concen a ions we e ha es ed a e a week o DNA quan i ica ion. Con ol wells wi hou an ibio ics we e ha es ed a 1 day, 4 days, and 7 days, while a con ol wi h sol en alone was ha es ed a e 1 week. T eponemal bu den was assessed using quan i a i e PCR a ge ing he T pallidum-speci ic p0574 gene (sensi i i y o abou en eponemal genomes pe eac ion).13 A second se o pla es was p epa ed o he bac e icidal and eco e y assay. T eponemes exposed o he d ug concen a ion we e subcul u ed in o an ibio ic- ee eco e y pla es. These pla es we e incuba ed o 7 mo e days be o e DNA ex ac ion. In addi ion o he egula con ols o T pallidum cul u es, we assessed whe he he es ed an imic obials showed oxici y on abbi S 1Ep cells (cocul u ed wi h T pallidum), which a e essen ial o he adequa e su i al and g ow h o spi oche es in i o. A de ailed desc ip ion o ou expe imen s on cell cul u e, ha es , and DNA ex ac ion and quan i ica ion, and cy o oxici y assays is p o ided (appendix pp 2–4). T pallidum incuba ion wi h a sub he apeu ic concen a ion o linezolid To in es iga e whe he p olonged exposu e o a sub he apeu ic concen a ion o linezolid could selec o a less suscep ible o esis an T pallidum s ain, o induce gene ic changes associa ed wi h linezolid esis ance, he SS14 s ain was g own o an ex ended du a ion in six-well cul u e pla es wi h and wi hou linezolid added o he cell cul u e media. P elimina y s udies12 showed ha linezolid was e ec i e a limi ing eponemal g ow h a a concen a ion o 0·5 mg/L o highe . In his expe imen , TpCM2 media con aining a concen a ion o 0·2 mg/L o linezolid was used o 2 weeks o p opaga ion o exe an ibio ic p essu e. The a ionale and calcula ions suppo ing he selec ed pe iod o an ibio ic p essu e (appendix p 4–5) and he p o ocol used o whole-genome sequencing o iden i y linezolid esis ance mu a ions (appendix p 5) ha e been p o ided. Ou comes The p ima y MIC o each expe imen was de ined as he lowes an ibio ic dilu ion a which he p0574 quan i a i e PCR alues we e no signi ican ly highe han he inoculum wells (day 0, con ol g oup), as p e iously de ined by Edmondson and colleagues.15 Addi ionally, we analysed a seconda y MIC, which was de ined as he lowes an ibio ic dilu ion a which he p0574 quan i a i e PCR alues we e signi ican ly lowe han he posi i e con ol (day 7, con ol g oup). The seconda y MIC de ini ion was mo e simila o he b o h dilu ion p ocedu e and was used p e iously by Haynes and colleagues.12 The minimum bac e icidal con cen a ion (MBC) was de ined as he lowes concen a ion a which he e was no bac e ial g ow h a e subcul u ing in o he an ibio ic- ee media. The sample size consis ed o eigh eplica es pe d ug concen a ion and con ol g oup. Each sample ep esen ed a echnical eplica e om he same sou ce mix u e. Rigo ous expe imen al condi ions minimised subs an ial a ia ions among samples. S a is ical analysis The s a is ical analyses ha we did o his s udy a e epo ed in de ail (appendix p 6). We used he K uskal– Wallis mean- ank es o compa e he dis ibu ion o quan i a i e PCR alues among independen g oups wi h di e en an ibio ic concen a ions and con ol g oups. Dunn’s es was used o pai wise compa isons be ween speci ic an ibio ic g oups and con ol g oups a day 0 o day 7. A alse disco e y a e Benjamin–Hochbe g co ec ion (p<0·05) was applied o mul iple com- pa isons. In he cy o oxici y expe imen , abso bance was compa ed be ween he an ibio ic and con ol g oups, wi h he median blank alue o media-con aining wells sub ac ed om he expe imen al eadings. Role o he unding sou ce The unde o he s udy had no ole in s udy design, da a collec ion, da a analysis, da a in e p e a ion, w i ing o he epo , o he decision o submi he A icle o publica ion. Resul s T eponemal g ow h in he absence o an imic obial agen s p og essed as expec ed (ie, a consis en inc ease in he numbe o p0574 copies de ec ed, anging om 1·5 o 3·0 loga i hmic uni s inc ease ( igu e 1A–K). O e all, no di e ences we e seen when compa ing eponemal g ow h in he an ibio ic- ee con ol g oup wells a day 7 o he H₂O and dime hyl sul oxide wells, despi e some a iabili y in yield. All p alues o Dunn’s es we e highe han 0·13, excep o isoniazid (p=0·032). Se e al o he an imic obials es ed in his s udy showed a p ima y MIC o T pallidum a concen a ions achie able in human plasma ( able), including amoxicillin (0·02 mg/L), ce iaxone (0·0025 mg/L), cephalexin (0·25 mg/L), A icles www. helance .com/mic obe Published online Oc obe 9, 2023 h ps://doi.o g/10.1016/S2666-5247(23)00219-7 5 ce e ame and ce ixime (0·0313 mg/L), ce u oxime (0·0156 mg/L), edizolid (0·0625 mg/L), spec inomycin (0·1 mg/L), and dalba ancin (0·125 mg/L; igu e 1 A–F, H, I,J). The MIC alues o hese an ibio ics we e o he same o de o lowe han concen a ions achie ed in humans a e he adminis a ion o s anda d dose egimens ( able 1). Balo loxacin and zoli lodacin p esen ed inhibi o y ac i i y a 2 mg/L ( igu e 1G, K). Isoniazid, py azinamide, and me onidazole had no e ec on T pallidum iabili y a he es ed concen a ions ( igu e 1L, M, O), whe eas e apenem educed eponemal g ow h a concen a ions highe han 0·3 mg/L compa ed o he day 7 con ol g oup, bu none o he concen a ions up o 2 mg/L me he c i e ia o he p ima y MIC ( igu e 1P). I e mec in ( igu e 1N) only had appa en e icacy agains T pallidum, bu a s eadily declining me abolic ac i i y o S 1Ep cells no ed upon exposu e o Figu e 1: T eponema pallidum suscep ibili y o an imic obials Non-an ibio ic con ol wells ep esen eponemal g ow h in he absence o an ibio ic om day 0 (inoculum) o day 7 a e pla e inocula ion. DMSO and H₂O ba s a e ela i e o S 1Ep cell cul u es o which he compound sol en was added ins ead o he es ed an ibio ic. In he ba cha , he middle line ep esen s he median p0574 gene copies pe uni o olume om eigh biological eplica es, he leng h o he ba ep esen s he IQR, and he do s ep esen he indi idual alues. *Seconda y MIC: p alues ( o he Dunn’s es ) a e p o ided o he compa ison be ween he lowes an ibio ic dilu ion a which he p0574 qPCR alues we e signi ican ly lowe han he posi i e con ol (day 7 con ol g oup). †P ima y MIC: p alues ( o he Dunn’s es ) a e p o ided o he compa ison be ween he lowes an ibio ic dilu ion a which he p0574 qPCR alues we e no signi ican ly highe han he inoculum wells (day 0 con ol g oup). DMSO=dime hyl sul oxide. MIC=minimum inhibi o y concen a ion. ‡Isoniazid, py azinamide, me onidazole, and e apenem do no ha e p alues because he MIC was una ained (ie, he MIC alue is highe han he highes concen a ion es ed). In he case o i e mec in, we showed ha he educ ion in he g ow h o T pallidum a a concen a ion o 10 mg/L o highe ac ually e lec s oxici y o S 1Ep cells and he e o e canno be conside ed an MIC. 100000 10000 1000 100 10 1 0·1 Day 0 Day 1 Day 4 Day 7 0·00063 mg/L 0·005mg/L 0·00125 mg/L 0·0025 mg/L*† 0·01 mg/L 0·02 mg/L 0·05 mg/L 0·25 mg/L 0·1mg/L 0·5 mg/L 1·0 mg/L No an ibio ic H 2 0 BCe iaxone p<0·001 p=0·059 100000 10000 1000 100 10 Day 0 Day 1 Day 4 Day 7 0·0025 mg/L 0·005mg/L 0·01 mg/L* 0·02 mg/L† 0·04 mg/L 0·08 mg/L 0·16 mg/L No an ibio ic H 2 0 A Amoxicillin p=0·011 p=0·063 No an ibio ic 100000 10000 1000 1 0·1 10 100 Day 0 Day 1 Day 4 Day 7 0·0625 mg/L 0·125mg/L 0·25 mg/L*† 0·5mg/L 1 mg/L 2mg/L 4 mg/L 8 mg/L H 2 0 C Cephalexin p<0·003 p=0·140 100000 10000 1000 100 10 Day 0 Day 1 Day 4 Day 7 0·0039 mg/L 0·0078 mg/L 0·125 mg/L 0·25 mg/L No an ibio ic H 2 0 DCe e ame p<0·001 p=0·082 0·0156 mg/L 0·0625 mg/L* Mean p0574 copie sM ean p0574 copies 10000 1000 100 10 1 Day 0 Day 1 Day 4 Day 7 0·0039 mg/L 0·0078 mg/L 0·0313 mg/L 0·0156 mg/L*† 0·0625 mg/L 0·125 mg/L 0·25 mg/L No an ibio ic H 2 0 E Ce u oxime p<0·001 p=0·296 Mean p0574 copies 10000 1000 100 10 1 Day 0 Day 1 Day 4 Day 7 0·0039 mg/L 0·0078 mg/L 0·0313 mg/L 0·0156 mg/L 0·0625 mg/L 0·125 mg/L*† 0·25 mg/L No an ibio ic H 2 0 I Dalba ancin p=0·005 p=0·131 10000 1000 100 10 1 Day 0 Day 1 Day 4 Day 7 0·0039 mg/L 0·0078 mg/L 0·0313 mg/L*† 0·0156 mg/L 0·0625 mg/L 0·125 mg/L 0·25 mg/L No an ibio ic H 2 0 F Ce ixime p<0·001 p=0·430 10000 1000 100 10 1 Day 0 Day 1 Day 4 Day 7 0·25 mg/L 0·5 mg/L 2 mg/L*† 1 mg/L 4 mg/L 8 mg/L 16 mg/L No an ibio ic H 2 0 G Balo loxacin p=0·031 p=0·099 10000 1000 100 10 1 Day 0 Day 1 Day 4 Day 7 0·0078 mg/L 0·0156 mg/L 0·0625 mg/L† 0·0313 mg/L* 0·125 mg/L 0·25 mg/L 0·5 mg/L 0·0078 mg/L 0·0156 mg/L 0·0625 mg/L 0·0313 mg/L 0·125 mg/L 0·25 mg/L 0·5 mg/L No an ibio ic DMSO H Tedizolid p=0·003 p=0·231 Mean p0574 copies 100000 10000 1000 100 10 Day 0 Day 1 Day 4 Day 7 1 mg/L 2 mg/L 8 mg/L 4 mg/L 16 mg/L 32 mg/L 64 mg/L No an ibio ic H 2 0 M Py azinamide N I e mec in 10000 1000 100 10 Day 0 Day 1 Day 4 Day 7 0·02 mg/L 0·04 mg/L 0·25 mg/L 0·1 mg/L*† 0·5mg/L 1 mg/L 2 mg/L No an ibio ic H 2 0 J Spec inomycin p=0·025 p=0·113 10000 1000 100 10 Day 0 Day 1 Day 4 Day 7 0·25 mg/L 0·5 mg/L 1 mg/L* 2 mg/L † 4 mg/L No an ibio ic DMSO K Zoli lodacin p=0·010 p=0·270 10000 1000 100 10 1 Day 0 Day 1 Day 4 Day 7 No an ibio ic H 2 0 L Isoniazid†† 10000 1000 100 10 Day 0 Day 1 Day 4 Day 7 0·125 mg/L 1·25 mg/L 5 mg/L 2·5 mg/L 10 mg/L 20 mg/L 40 mg/L No an ibio ic H 2 0 O Me onidazole 10000 1000 100 10 Day 0 Day 1 Day 4 Day 7 0·0313 mg/L 0·0625 mg/L 0·25 mg/L 0·125 mg/L 0·5 mg/L 1 mg/L 2 mg/L No an ibio ic DMSO P E apenem 10000 1000 100 1 10 Day 0 Day 1 Day 4 Day 7 0·00375 mg/L 0·075 mg/L 0·3 mg/L 0·15 mg/L 0·6 mg/L 0·8 mg/L 1·2 mg/L 2 mg/L No an ibio ic H 2 0 0·0313 mg/L† A icles 6 www. helance .com/mic obe Published online Oc obe 9, 2023 h ps://doi.o g/10.1016/S2666-5247(23)00219-7 i e mec in (appendix p 8) suppo ed ha his e ec was mos ly caused by oxici y exe ed by his an ipa asi ic on he cells suppo ing T pallidum g ow h, a he han on he pa hogen i sel . In analyses o es ablish he seconda y MIC, which co esponds o he minimum an ibio ic dilu ion a which he p0574 quan i a i e PCR alues we e signi ican ly lowe han he day 7 con ol wells, he cu o concen a ion alues ei he emained he same as he p ima y MIC o we e sligh ly lowe , excep o ce e ame ( igu e 1A–K; able). The esul s o he abo emen ioned compounds we e u he suppo ed by hei MBC, mani es ed as he absence o g ow h when eponemes exposed o hese an ibio ics we e subcul u ed wi hou he an ibio ics ( igu e 2). Subcul u ing esul s demons a ed low Figu e 2: Reco e y assays ollowing an ibio ic emo al In he ba cha , he middle line ep esen s median p0574 gene copies pe uni o olume om eigh biological eplica es, he leng h o he ba ep esen s he IQR, and he do s ep esen he indi idual alues. DMSO=dime hyl sul oxide. MBC=minimum bac e icidal concen a ion. MIC=minimum inhibi o y concen a ion. *MBC. †Isoniazid, py azinamide, me onidazole, and e apenem do no ha e p alues because he MIC was una ained (ie, he MIC alue was highe han he highes concen a ion es ed). In he case o i e mec in, we showed ha he educ ion in he g ow h o T pallidum a a concen a ion o 10 mg/L o highe ac ually e lec s oxici y o S 1Ep cells and he e o e canno be conside ed an MIC. 100000 10000 1000 100 10 1 0·1 Day 7 0·00063 mg/L 0·005 mg/L 0·00125 mg/L 0·0025 mg/L 0·01 mg/L 0·02 mg/L 0·05 mg/L 0·25 mg/L 0·1mg/L 0·1mg/L 0·5 mg/L 1·0 mg/L No an ibio ic H 2 0 B Ce iaxone 100000 10000 1000 100 1 10 100000 100000 10000 1000 100 1 Day 7 0·0025mg/L 0·005mg/L 0·01 mg/L 0·02 mg/L 0·04 mg/L 0·08 mg/L 0·16 mg/L No an ibio ic H 2 0 A Amoxicillin No an ibio ic 100000 10000 1000 1 10 100 Day 7 0·0625mg/L 0·125mg/L 0·25 mg/L 0·5mg/L 1 mg/L 2mg/L 4 mg/L 8 mg/L H20 C Cephalexin 100000 10000 1000 100 1 10 DMSO Day 7 0·0039 mg/L 0·0078 mg/L 0·125 mg/L 0·25 mg/L No an ibio ic D Ce e ame 0·0156 mg/L 0·0625 mg/L Mean p0574 copies Mean p0574 copies 10 100000 10000 1000001000000 100000 10000 1000 100 1 10 Day 7 0·0039 mg/L 0·0078 mg/L 0·0313 mg/L 0·0156 mg/L 0·0625 mg/L 0·125 mg/L 0·25 mg/L No an ibio ic H 2 0 E Ce u oxime Mean p0574 copies 100000 100000 10000 1000 Day 7 0·0039 mg/L 0·0078 mg/L 0·0313 mg/L 0·0156 mg/L 0·0625 mg/L 0·125 mg/L 0·25 mg/L No an ibio ic H 2 0 I Dalba ancin 10000 100000 100000 1000 100 10 0·1 1 DMSO Day 7 0·0039 mg/L 0·0078 mg/L 0·0313 mg/L 0·0156 mg/L 0·0625 mg/L 0·125 mg/L 0·25 mg/L No an ibio ic F Ce ixime 10000 1000 100 10 1 DMSO Day 7 0·25 mg/L 0·5 mg/L 2 mg/L 1 mg/L 4 mg/L 8 mg/L 16 mg/L No an ibio ic G Balo loxacin 10000 1000 100 10 0·1 1 Day 7 0·0078 mg/L 0·0156 mg/L 0·0625 mg/L 0·0313 mg/L 0·125 mg/L 0·25 mg/L 0·5 mg/L 0·0078 mg/L 0·0156 mg/L 0·0625 mg/L 0·0313 mg/L 0·125 mg/L 0·25 mg/L 0·5 mg/L No an ibio ic DMSO H Tedizolid Mean p0574 copies Day 7 1 mg/L 2 mg/L 8 mg/L 4 mg/L 16 mg/L 32 mg/L 64 mg/L No an ibio ic H 2 0 M Py azinamide† N I e mec in 10000 1000 100 10 Day 7 0·02 mg/L 0·04 mg/L 0·25 mg/L 0·01 mg/L 0·5mg/L 1 mg/L 2 mg/L No an ibio ic H 2 0 J Spec inomycin 1000 100 10 1 Day 7 0·25 mg/L 0·5 mg/L 1 mg/L 2 mg/L 4 mg/L No an ibio ic DMSO K Zoli lodacin 10000 1000 100 Day 7 No an ibio ic H 2 0 L Isoniazid† 10000 1000 100 1 10 DMSO Day 7 0·125 mg/L 1·25 mg/L 5 mg/L 2·5 mg/L 10 mg/L 20 mg/L 40 mg/L No an ibio ic O Me onidazole† 10000 1000 100 10 Day 7 0·0313 mg/L 0·0625 mg/L 0·25 mg/L 0·125 mg/L 0·5 mg/L 1 mg/L 2 mg/L No an ibio ic DMSO P E apenem† 100000 10000 1000 Day 7 0·00375 mg/L 0·075 mg/L 0·3 mg/L 0·15 mg/L 0·8 mg/L 0·6 mg/L 1·2 mg/L 2 mg/L No an ibio ic H 2 0 0·0313 mg/L * * * * ** * * * * * A icles www. helance .com/mic obe Published online Oc obe 9, 2023 h ps://doi.o g/10.1016/S2666-5247(23)00219-7 7 MBC alues o amoxicillin (0·01 mg/L), ce iaxone (0·0025 mg/L), cephalexin (0·25 mg/L), ce e ame (0·0625 mg/L), ce u oxime (0·0156 mg/L), ce ixime and edizolid (0·0313 mg/L), dalba ancin (0·125 mg/L), spec inomycin (0·25 mg/L; igu e 2 A–F, H–J). The MBC o zoli lodacin was 2 mg/L, and o balo loxacin was 4 mg/L ( igu e 2G, K). The absence o e ec was con i med o isoniazid, me onidazole, py azinamide, and e apenem ( igu e 2L, M, O, P). The wa e -soluble e azolium assay which we did o ule ou cy o oxic ac i i y o he an ibio ics o cul u ed cells showed ha none o he es ed concen a ions o cephalospo ins, edizolid and dalba ancin we e oxic o he S 1Ep cells (appendix p 8). Mo eo e , none o he es ed concen a ions o isoniazid, me onidazole, and py azinamide we e cy o oxic o S 1Ep cells (appendix p 8). Al hough i e mec in appa en ly supp essed T pallidum g ow h a concen a ions o 20 mg/L o highe ( igu e 1N), i also a ec ed S 1Ep cell homoeos asis a hese high concen a ions (appendix p 8), sugges ing ha absence o eponemal g ow h was no caused by speci ic ac i i y on he pa hogen. The expe imen s ca ied ou o e alua e T pallidum esis ance o mac olides showed ha azi h omycin was e ec i e agains T pallidum s ains ha did no ha e ei he o he 23S RNA gene mu a ions (A2058G o A2059G) con e ing esis ance o mac olides, such as Chicago C. Fo his s ain, azi h omycin had a p ima y MIC and MBC lowe han 0·031 mg/L ( igu e 3A, B). Azi h omycin, howe e , emained ine ec i e o wo s ains (SS14 and UW330B) ca ying ei he one o he a o emen ioned mu a ions, a leas up o 2·0 mg/L ( igu e 3C–F); he e o e, inc easing mac olide dosage would no be a iable s a egy o o e come he well documen ed and widesp ead gene ic esis ance o T pallidum o his class o compounds. Las ly, we did expe imen s o e alua e whe he T pallidum p opaga ion employing selec i e p essu e wi h linezolid concen a ions o 0·2 mg/L o 2 weeks ollowed by p opaga ion a 0·03 mg/L o 8 addi ional weeks would selec o a less suscep ible (o ully esis an ) s ain. The esul s o he linezolid suscep ibili y assay done wi h he SS14 s ain p opaga ed wi h ( igu e 3G, H) and wi hou ( igu e 3I, J) linezolid, and hei espec i e eponemal eco e y assay g aphs ha e been p esen ed. O e all, suscep ibili y o linezolid emained he same in bo h cases (p ima y MIC alue 0·5 mg/L). Mu a ions mapping o he 23S RNA genes, as well as o he L3, L4, and L22 50S ibosomal p o eins, ha e been es ablished as a linezolid esis ance mechanism.15 These a ge s co espond o p0189 (L3), p0190 (L4), and p0194 (L22) in T pallidum genes. Sequencing o he SS14 s ain p opaga ed in sub he apeu ic linezolid concen a ions did no ha e any gene ic di e ences compa ed wi h he s ain p opaga ed in he absence o an ibio ic, including he RNA-encoding and p o ein-encoding a ge s. Genomic da a a e a ailable on GenBank unde biop ojec PRJNA885511. Discussion On he basis o in- i o cul u e o T pallidum s ains, MIC alues o less han 0·1 mg/L we e demons a ed o amoxicillin, se e al cephalospo ins, edizolid, and dalba ancin. A hese concen a ions, he o ganism showed no g ow h in he p esence o he an ibio ics o a e subcul u ing on an ibio ic- ee media. These MIC alues add o he exis ing da a on penicillin, doxycycline, and linezolid and ep esen aluable in o ma ion o he op imisa ion and expansion o he ea men op ions o syphilis. Al hough he MIC indica es he suscep ibili y o he pa hogen o he an ibio ic, clinical ou comes also depend on achie able d ug concen a ions a he in ec ion si e. Pha macokine ic–pha macodynamic analysis in eg a es bo h an ibio ic exposu e (ie, pha macokine ics) and an imic obial ac i i y (ie, pha macodynamics). Howe e , he absence o pha macokine ic–pha macodynamic models o T pallidum hinde s he de ini ion o pha macokine ic and pha macodynamic a ge s ha co ela e wi h clinical e icacy. Consequen ly, we ha e compa ed MIC alues ob ained in ou in- i o model wi h ough plasma concen a ions achie ed in indi iduals ecei ing cu en dosing ecommenda ions o de e mine whe he plasma concen a ions a e g ea e han he MIC du ing he en i e dosing in e al (pe cen age o ime be ween wo doses du ing which he unbound ac ion o he d ug concen a ion emains abo e he MIC). Ou esul s con i m he po en ial e ec i eness o penicillin ( ea men o choice), doxycycline, and ce iaxone ( ea men al e na i es) o p ima y, seconda y, o la en syphilis. O al amoxicillin and o al cephalospo ins migh also be e ec i e ea men op ions, e en when conside ing he ee ac ion (no bound o plasma p o eins), which indica es he concen a ion a he si e o in ec ion, al hough u he s udies a e needed o con i m he clinical u ili y o hese an ibio ics. In he case o neu osyphilis, he di e en ial abili y o hese molecules o pene a e he CNS equi es indi idual conside a ion. Ou s udy p o ides MIC alues o β-lac ams used o ea syphilis, including amoxicillin and ce iaxone. Amoxicillin esul s a e consis en wi h he p obabili y o eaching he clinical e icacy a ge se by EUCAST,16 which is 100% o MIC alues up o 0·5 mg/L wi h he 500 mg pe 8 h egimen. In obse a ional s udies, amoxicillin has been shown o be e ec i e in ea ing ea ly syphilis wi h a 95% success a e.17,18 Ce iaxone is ecommended o ea ea ly syphilis,19 and some e idence om a e ospec i e s udy in ol ing 24 pa ien s sugges ed ha ce iaxone migh be a po en ial op ion o ea ing neu osyphilis.20 The pene a ion o all β-lac am an ibio ics in o he CNS in he absence o meningeal in lamma ion is gene ally poo (ie, ce eb ospinal luid s se um a io o 0·15). Howe e , a daily dose o 1 g o 2 g ce iaxone achie es concen a ions in he CNS o 0·4 mg/L, mo e han 160 imes highe han A icles 8 www. helance .com/mic obe Published online Oc obe 9, 2023 h ps://doi.o g/10.1016/S2666-5247(23)00219-7 No an ibio ic No an ibio ic 10000001000000 100000 10000 1000 100 Mean p0574 copiesMean p0574 copies 1000000 100000 10000 1000 100 10 Day 0 Day 1 Day 4 Day 7 DMSO 0·0313 mg/L 0·0625 mg/L 0·125 mg/L* 0·5 mg/L 0·25 mg/L 1 mg/L 2 mg/L 0·0313 mg/L 0·0625 mg/L 0·125 mg/L 0·5 mg/L 0·25 mg/L 1 mg/L 2 mg/L No an ibio ic 1000000 1000000 100000 10000 1000 100 10 DMSO Day 7 No an ibio ic AChicago C suscep ibili y assay Azi h omycin high-dose es ing BChicago C eco e y assay p=0·001 100000 10000 1000 100 10 Day 0 Day 1 Day 4 Day 7 DMSO 0·0313 mg/L 0·0625 mg/L 0·125 mg/L 0·5 mg/L 0·25 mg/L 1 mg/L 2 mg/L No an ibio ic CUW330B suscep ibili y assay 0·0313 mg/L 0·0625 mg/L 0·125 mg/L 0·5 mg/L 0·25 mg/L 1 mg/L 2 mg/L 100000 10000 100 DMSO Day 7 No an ibio ic G D UW330B eco e y assay 1000 Day 0 Day 4 Day 7 DMSO 0·0313 mg/L 0·0625 mg/L 0·125 mg/L 0·5 mg/L 0·25 mg/L 1 mg/L 2 mg/L 0·0313 mg/L 0·0625 mg/L 0·125 mg/L 0·5 mg/L 0·25 mg/L 1 mg/L 2 mg/L No an ibio ic 1000000 100000 10000 1000 100 DMSO Day 7 No an ibio ic EF SS14 eco e y assaySS14 suscep ibili y assay Mean p0574 copiesMean p0574 copies 1000000 100000 10000 1000 1 100 10 Day 0 Day 4 Day 1 Day 7 DMSO 0·0156 mg/L 0·0313 mg/L 0·0625 mg/L 0·125 mg/L* 0.5 mg/L† 0·25 mg/L 1 mg/L 2 mg/L No an ibio ic Suscep ibili y assay H Reco e y assay p=0·008 p=0·055 1000000 100000 10000 1000 1 100 10 Day 7 DMSO 0·0156 mg/L 0·0313 mg/L 0·0625 mg/L 0·125 mg/L 0·5 mg/L 0·25 mg/L 1 mg/L No an ibio ic †† p=0·006 p=0·108 1000000 100000 10000 1000 1 100 10 Day 0 Day 4 Day 1 Day 7 DMSO 0·0156 mg /L 0·0313 mg /L 0·0625 mg /L 0·125 mg/ L* 0·5 mg/L† 0·25 mg/L 1 mg/L 2 mg/L IJ Reco e y assay 1000000 100000 10000 1000 1 100 10 Day 7 DMSO 0·0156 mg /L 0·0313 mg /L 0·0625 mg /L 0·125 mg/L 0·5 mg/L 0·25 mg/L 1 mg/L 2 mg/L Wi h lizenolid exposu e Linezolid selec i e p essu e es ing Suscep ibili y assay Linezolid selec i e p essu e es ing Wi hou lizenolid exposu e †† Figu e 3: Suscep ibili y and eco e y assays o Chicago C, UW330B, and SS14 o azi h omycin, and suscep ibili y and eco e y assays wi h and wi hou linezolid exposu e Non-an ibio ic con ol wells ep esen eponemal g ow h in absence o an ibio ic om day 0 (inoculum) o day 7 a e pla e inocula ion. DMSO ba s a e ela i e o S 1Ep cell cul u es o which he compound sol en was added ins ead o he es ed an ibio ic. Suscep ibili y and eco e y assays o he SS14 s ain p opaga ed in he p esence o sub he apeu ic concen a ion o linezolid (G, H), o absence o an ibio ic (I, J). In he ba cha , he middle line ep esen s he median p0574 gene copies pe uni o olume om eigh biological eplica es, he leng h o he ba ep esen s he IQR, and he do s ep esen he indi idual alues. DMSO=dime hyl sul oxide. MBC=minimum bac e icidal concen a ion. *Seconda y MIC: p alues ( o he Dunn’s es ) a e p o ided o he compa ison be ween he lowes an ibio ic dilu ion a which he p0574 qPCR alues we e signi ican ly lowe han he posi i e con ol (day 7 con ol g oup). †P ima y MIC: p alues ( o he Dunn’s es ) a e p o ided o he compa ison be ween he lowes an ibio ic dilu ion a which he p0574 qPCR alues we e no signi ican ly highe han he inoculum wells (day 0 con ol g oup). ‡MBC. A icles www. helance .com/mic obe Published online Oc obe 9, 2023 h ps://doi.o g/10.1016/S2666-5247(23)00219-7 9 he MIC alue (0·0025 mg/L) we epo ed. No conclusi e esul s ha e been epo ed wi h ce ixime (87% and 56% cu a i e esul s in he pe -p o ocol and in en ion- o- ea popula ions o a s udy on people wi h ea ly syphilis).21 E apenem, a b oad-spec um β-lac am ca bapenem, was o e all ine ec i e agains T pallidum in i o a e a 1-week- long incuba ion. One could hypo hesise ha e apenem has educed inhibi o y ac i i y on he only known T pallidum penicillin-binding p o ein wi h β-lac amase ac i i y, he 47 kDa lipop o ein, compa ed wi h o he β-lac ams.22 Howe e , his hypo hesis equi es u he s udies o be co obo a ed. Addi ional expe imen s done wi h o he ca bapenems showed ha bo h do ipenem and biapenem a e no e ec i e agains T pallidum up o 2 mg/L in i o (p>0·05 s con ol wells wi h no an ibio ic) a e a 1 week-long incuba ion, whe eas imipenem and me openem signi ican ly inhibi T pallidum g ow h a 2 mg/L, bu a e no comple ely eponemicidal. The low MIC alues and ex ended hal -li e o dalba ancin (ie, 145 h) sugges ha a single in usion could main ain high and p olonged plasma concen- a ions, po en ially leading o syphilis cu e. Howe e , his agen migh no ha e a subs an ial impac on CNS in ec ions because o sca ce pene a ion o he blood– b ain ba ie 23,24 in he animal model (2% in abbi wi h non-in lamed meninges and 5% in abbi wi h in lamed meninges). The an i- eponemal ac i i y o zoli lodacin could be explained by compa ing he sequences o he DNA gy ase subuni B (Gy B) p o ein o T pallidum (TP0116) wi h ha o Neisse ia gono hoeae Gy B p o ein (NG1772). Al hough he e is only 51% sequence iden i y be ween hese wo enzymes, conse a ion o key amino-acid esidues in he T pallidum Gy B p e iously iden i ied in he cogna e N gono hoeae enzyme migh accoun o he esul s p esen ed he e, e en hough u he s udies a e needed o e alua e his hypo hesis.25 Ou expe imen did no e eal pheno ypical o gene ic changes po en ially ela ed o de elopmen o esis ance o linezolid ollowing cul u ing o he pa hogen in sub he apeu ic concen a ions o his an ibio ic. The expe imen design ha inco po a ed a 2-week pe iod o ele a ed an ibio ic p essu e ( ollowed by addi ional weeks a lowe p essu e) o selec ion o esis ance mu an s in eponemes was subjec ed o ho ough and me iculous conside a ion (appendix p 4–5), aking in o accoun he usual ange o mu a ion a es o bac e ia, gi en ha he e is no in o ma ion on he ac ual mu a ion a e o T pallidum.26,27 Doing u he expe imen s wi h longe cul i a ion pe iods and highe concen a ions o linezolid could p o ide addi ional alue and insigh s. The pha macodynamic and pha macokine ic-based b eakpoin o linezolid in g am-posi i e bac e ia is 1 mg/L, ep esen ing he highes MIC alue a which he e is a high likelihood o achie ing clinical e icacy.28 In ou s udy he MIC o linezolid was 0·5 mg/L which is lowe ha he pha macodynamic and pha macokine ic b eakpoin . Addi ionally, linezolid has a a ou able CNS pene a ion (38% in abbi model)29 and has been e ec i e in ea ing CNS in ec ions caused by o he bac e ial species. These indings sugges ha i is an excellen candida e o clinical e alua ion in he ea men o syphilis, including neu osyphilis.30 The in- i o cul u e me hod o T pallidum o de e mining an imic obial suscep ibili y is no de oid o echnical limi a ions ha make an ibio ic es ing s ill p ocedu ally complex o his pa hogen. This me hod is simila o he b o h dilu ion p ocedu e commonly used wi h o he bac e ia,31 bu he wo sys ems di e in some impo an ways. Fi s , he p esence o abbi epi helial cells is necessa y o p omo e he long- e m su i al and mul iplica ion o T pallidum; 7 days o incuba ion a e used ins ead o he 16–20 h ypically used in o he bac e ia because he doubling ime is abou 40 h, and bac e ial quan i ica ion by quan i a i e PCR needs o be used ins ead o isual inspec ion o u bidi y, which does no inc ease in pa allel o T pallidum concen a ion in he cul u e media. Howe e , in his and p e ious s udies,12,13 he cul u e me hod o T pallidum yielded ep oducible esul s, and he e o e we belie e ha his assay p o ides an accu a e assessmen o MIC alues. Second, s anda dised me hods o de e mining MICs and MBCs in cul u e we e adap ed o he pu pose o a di e en cul u e me hod al oge he ; he e o e ou esul s need o be in e p e ed in his con ex and based on he p oposed ou come de ini ions. Ano he limi a ion o ou s udy is he po en ial o a mode a e le el o a iabili y in DNA measu emen esul s among eplica es because o he limi ed numbe o a ge o ganisms when g owing T pallidum in e y small olumes, and he mul is ep p ocess in ol ed in molecula de ec ion. This d ug-suscep ibili y es ing me hod is mo e challenging han simply coun ing colonies o less di icul pa hogens on a pe i dish, which is no possible wi h T pallidum. The o ma adop ed in ou s udy, howe e , allowed us o es eigh eplica es o a ious concen a ions o each an ibio ic, esul ing in a mo e p ecise de e mina ion o ede e mina ion o he MIC o se e al an ibio ics, including linezolid, compa ed wi h p e ious me hods used. Ano he limi a ion o ou s udy includes ha suscep ibili y es ing was only conduc ed wi h one T pallidum s ain. Replica ing he es ing wi h se e al s ains om bo h he Nichols and SS14 clades, and geog aphical egions would be mo e labo ious han a no mal MIC, bu i would inc ease he obus ness and gene alisabili y o da a. Addi ionally, we did no include penicillin as an in e nal compa a o in ou assays, ins ead elying on da a om p e ious expe imen s12 ha we e no done simul aneously wi h he cu en s udy. Las ly, his s udy is limi ed o in- i o es ing, which is only he i s s ep owa ds explo ing new he apies o syphilis. None heless, gi en ha he s udied molecules ha e been app o ed o use in humans and hei sa e y is well es ablished, hei epu posing could mo e apidly o he nex s ages o p eclinical and clinical esea ch.