In Vitro and In Vivo Antibacterial Activity of Gliotoxin Alone and in Combination with Antibiotics against Staphylococcus aureus

oxins A icle In Vi o and In Vi o An ibac e ial Ac i i y o Glio oxin Alone and in Combina ion wi h An ibio ics agains S aphylococcus au eus Pa icia Es eban 1, Se gio Red ado 2, Lau a Comas 2, M. Pila Domingo 2, M. Isabel Millán-Lou 3, C is ina Se al 4,5, Sonia Alga a e 4, Concepción Lopez 3, An onio Rezus a 3, Julian Pa do 1,5,6, Maykel A ias 2,* and E a M. Gal ez 2,*   Ci a ion: Es eban, P.; Red ado, S.; Comas, L.; Domingo, M.P.; Millán-Lou, M.I.; Se al, C.; Alga a e, S.; Lopez, C.; Rezus a, A.; Pa do, J.; e al. In Vi o and In Vi o An ibac e ial Ac i i y o Glio oxin Alone and in Combina ion wi h An ibio ics agains S aphylococcus au eus.Toxins 2021,13, 85. h ps://doi.o g/10.3390/ oxins13020085 Recei ed: 20 Decembe 2020 Accep ed: 21 Janua y 2021 Published: 23 Janua y 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Fundacion Ins i u o de In es igacion Sani a ia A agon (IIS A agon), Biomedical Resea ch Cen e o A agon (CIBA), 50009 Za agoza, Spain; [email p o ec ed] (P.E.); pa dojim@uniza .es (J.P.) 2Ins i u o de Ca boquımica ICB-CSIC, 50018 Za agoza, Spain; se gio- [email p o ec ed] (S.R.); [email p o ec ed] (L.C.); [email p o ec ed] (M.P.D.) 3Depa men o Mic obiology, Hospi al Uni e si a io Miguel Se e , IIS A agón, 50009 Za agoza, Spain; [email p o ec ed] (M.I.M.-L.); [email p o ec ed] (C.L.); a ezus a@uniza .es (A.R.) 4Depa men o Mic obiology, Uni e si y Clinic Hospi al Lozano Blesa, 50009 Za agoza, Spain; [email p o ec ed] (C.S.); [email p o ec ed] (S.A.) 5Depa men o Mic obiology, Pedia ics, Radiology and Public Heal h, Uni e si y o Za agoza, 50009 Za agoza, Spain 6A agon I+D Founda ion (ARAID), 50018 Za agoza, Spain *Co espondence: [email p o ec ed] (M.A.); [email p o ec ed] (E.M.G.) Abs ac : Mul id ug- esis an bac e ia such as me hicillin- esis an S aphylococcus au eus (MRSA) is one o he majo causes o hospi al-acqui ed and communi y in ec ions and pose a challenge o he human heal h ca e sys em. The e o e, i is impo an o ind new d ugs ha show ac i i y agains hese bac e ia, bo h in mono he apy and in combina ion wi h o he an imic obial d ugs. Glio oxin (GT) is a myco oxin p oduced by Aspe gillus umiga us and o he ungi o he Aspe gillus genus. Some e idence sugges s ha GT shows an imic obial ac i i y agains S. au eus in i o , albei i s e icacy agains mul id ug- esis an s ains such as MRSA o ancomycin-in e media e S. au eus (VISA) s ainsis no known. This wo k aimed o e alua e he an ibio ic e icacy o GT as mono he apy o in combina ion wi h o he he apeu ics agains MRSA in i o and in i o using a Caeno habdi is elegans in ec ion model. Keywo ds: glio oxin; S aphylococcus au eus; MRSA; VISA; ancomycin; syne gism; esis ance; an ibio ic Key Con ibu ion: Glio oxin shows an ibac e ial e icacy agains me hicillin- esis an S aphylococ- cus au eus. 1. In oduc ion S aphylococcus au eus is a G am-posi i e bac e ium o en p esen as pa o he no mal mic obio a o he human body [1–3]. I is an oppo unis ic pa hogen ha a e su passing he skin ba ie can cause a a ie y o sys emic and pyogenic in ec ions, acu e and ch onic in ec ions, and oxin-media ed synd omes [ 4 ]. S. au eus in ec ions ange om skin and so issue in ec ions o se e e nec o izing pneumonia, li e- h ea ening endoca di is, o bac e emia in adul s and child en [ 5 – 8 ]. Du ing he mode n an ibio ic e a, S. au eus has e ol ed he abili y o acqui e esis ance o mos an ibio ics. Me hicillin- esis an S aphylococcus au eus (MRSA) eme ged in he 1960s [ 9 ], being a p e alen and impo an bac e ium ha has sp ead globally and has become a leading cause o bo h nosocomial and communi y-acqui ed bac e ial in ec ions [ 10 ]. MRSA in ec ions a e associa ed wi h highe mo ali y a es han in ec ions caused by me hicillin-suscep ible S. au eus (MSSA) Toxins 2021,13, 85. h ps://doi.o g/10.3390/ oxins13020085 h ps://www.mdpi.com/jou nal/ oxins Toxins 2021,13, 85 2 o 16 s ains [ 11 ]. Vancomycin is a glycopep ide an ibio ic ha inhibi s cell wall biosyn hesis and emains a d ug o choice o he ea men o se e e MRSA in ec ions [ 12 , 13 ]. Howe e , in he las 20 yea s, clinical isola es o ancomycin in e media e- esis an S. au eus (VISA, minimum inhibi o y concen a ion (MIC) = 4–8 µ g/mL) wi h educed suscep ibili y o ancomycin ha e eme ged, which is associa ed wi h pe sis en in ec ions, ea men ailu e, and poo clinical ou comes. Addi ionally, al hough less equen ly, S. au eus s ains wi h comple e esis ance o ancomycin (VRSA, MIC ≥ 16 µ g/mL) ha e been desc ibed [ 14 – 16 ]. These mul id ug- esis an o ganisms pose a challenge o he cu en human heal h ca e sys em. The e o e, he de elopmen o new ea men s a egies o comba esis an S. au eus in ec ions is necessa y. Renewed e o s a e needed o esea ch and de elopmen o new an ibio ics, o he combina ion o hese wi h exis ing ea men s o which bac e ial esis ances a e eme ging. One o he lines o an ibio ics disco e y is he explo a ion o he an ibac e ial ac i i y o na u al p oduc s. Mic obial seconda y me aboli es ha e been s udied o hei po en ial bene i s o humans and ha e p o ided nume ous pha maceu ical p oduc s. Fungi ha e he abili y o p oduce a wide a ie y o seconda y me aboli es, gene ally dependen on he s age o ungal de elopmen and en i onmen al ac o s [ 17 ]. Aspe gillus umiga us and o he ungi belonging o he Aspe gillus genus a e especially capable o p oducing a g ea di e si y o compounds. In ac , hey sec e e mo e han 226 seconda y me aboli es, including epipoly hiodioxopipe azines (ETPs), whose bes s udied membe is glio oxin (GT) [18]. GT (Figu e 1) is cha ac e ized by he p esence o an in e nal disul ide b idge in a pipe azine ing, which seems o be necessa y o mos o he biological p ope ies o his compound [ 19 ]. This myco oxin exe s i s oxic ac ion by gene a ing eac i e oxygen species due o he sul u con ained in he molecule, being able o al e na e be ween a comple ely educed di hiol o m and a disul ide o m (Figu e 2) [20]. Toxins 2021, 13, x FOR PEER REVIEW 2 o 16 mo ali y a es han in ec ions caused by me hicillin-suscep ible S. au eus(MSSA) s ains [11]. Vancomycin is a glycopep ide an ibio ic ha inhibi s cell wall biosyn hesis and e- mains a d ug o choice o he ea men o se e e MRSA in ec ions [12,13]. Howe e , in he las 20 yea s, clinical isola es o ancomycin in e media e- esis an S. au eus (VISA, minimum inhibi o y concen a ion (MIC) = 4–8 μg/mL) wi h educed suscep ibili y o ancomycin ha e eme ged, which is associa ed wi h pe sis en in ec ions, ea men ail- u e, and poo clinical ou comes. Addi ionally, al hough less equen ly, S. au eus s ains wi h comple e esis ance o ancomycin (VRSA, MIC ≥ 16 μg/mL) ha e been desc ibed [14–16]. These mul id ug- esis an o ganisms pose a challenge o he cu en human heal h ca e sys em. The e o e, he de elopmen o new ea men s a egies o comba esis an S. au eus in ec ions is necessa y. Renewed e o s a e needed o esea ch and de elop- men o new an ibio ics, o he combina ion o hese wi h exis ing ea men s o which bac e ial esis ances a e eme ging. One o he lines o an ibio ics disco e y is he explo a- ion o he an ibac e ial ac i i y o na u al p oduc s. Mic obial seconda y me aboli es ha e been s udied o hei po en ial bene i s o humans and ha e p o ided nume ous pha - maceu ical p oduc s. Fungi ha e he abili y o p oduce a wide a ie y o seconda y me- aboli es, gene ally dependen on he s age o ungal de elopmen and en i onmen al ac- o s [17]. Aspe gillus umiga us and o he ungi belonging o he Aspe gillus genus a e es- pecially capable o p oducing a g ea di e si y o compounds. In ac , hey sec e e mo e han 226 seconda y me aboli es, including epipoly hiodioxopipe azines (ETPs), whose bes s udied membe is glio oxin (GT) [18]. GT (Figu e 1) is cha ac e ized by he p esence o an in e nal disul ide b idge in a pipe azine ing, which seems o be necessa y o mos o he biological p ope ies o his compound [19]. This myco oxin exe s i s oxic ac ion by gene a ing eac i e oxygen spe- cies due o he sul u con ained in he molecule, being able o al e na e be ween a com- ple ely educed di hiol o m and a disul ide o m (Figu e 2) [20]. Figu e 1. Glio oxin (GT)s uc u e is cha ac e ized by a pipe azine ing con aining a disul ide b idge. Image ob ained om Wikimedia Commons. Figu e 1. Glio oxin (GT) s uc u e is cha ac e ized by a pipe azine ing con aining a disul ide b idge. Image ob ained om Wikimedia Commons. Toxins 2021,13, 85 3 o 16 Toxins 2021, 13, x FOR PEER REVIEW 3 o 16 Figu e 2. Redox cycle be ween he educed (di hiol, le ) and oxidized (disul ide, igh ) o ms o GT. The oxida ion o GT and p esumably o he epipoly hiodioxopipe azines (ETPs) gene a es eac i e oxygen species since i is capable o educing molecula oxygen. [18]. Fu he mo e, educed GT can eac wi h o he accessible hiol g oups on p o eins, inac i a ing, inhibi ing, o dena u ing hem [18]. Some in i o s udies show ha he e- ac i e oxygen species p oduced by GT a e capable o causing DNA damage [21–23]. The e is cu en ly limi ed scien i ic e idence on he use o GT as an an ibio ic. The an ibac e ial ac ion o GT has been s udied agains Esche ichia coli and S. au eus. I is sug- ges ed ha GT could cause damage o E. coli DNA [24] and ha i exhibi s an ibio ic e ec o bo h mic oo ganisms, being mo e e ec i e agains S. au eus [25,26]. He e we ha e ex ended hese s udies and used a MRSA/VISA s ain o s udy he an ibac e ial ac i i y o GT alone and in combina ion wi h ancomycin. We ound ha GT has a po en in i o an ibio ic e ec agains his s ain and ha i s combina ion wi h an- comycin shows a e y good syne gy a lowe concen a ions han single compounds. We also ca ied ou an in i o s udy using Caeno habdi is elegans as a model o MRSA in ec- ion, whe e he combined ea men demons a ed a signi ican highe su i al pe cen - age han he ea men wi h ancomycin as mono he apy. The e o e, ou indings e eal a no el app oach o ake ad an age o he an ibio ic ac i i y o GT and i s po en ia ing e ec when combined wi h ancomycin. Thus, a g ea e an ibio ic e ec is ob ained wi h a lowe concen a ion o ancomycin, and he e o e he possible side e ec s and he ap- pea ance o esis ance due o he use o ancomycin could be educed. 2. Resul s 2.1. An imic obial Suscep ibili y o S. au eus agains GT 2.1.1. E ec o GT on S. au eus G ow h As a esul o an ibiog ams, he mean o he inhibi ion halos ob ained o each im- p egna ed disk was calcula ed (Figu e 3). All an imic obials used as con ols p oduced an inhibi ion halo wi hin he ange es ablished by he Eu opean Commi ee on An imic obial Suscep ibili y Tes ing (EUCAST 2020) speci ic o each s ain. The inhibi ion halo o he wa e and me hanol disks was 0 mm in all cases. Thus, hese esul s show ha he ehicle used o sol e GT, me hanol, does no exe any an imic obial ac ion on any o he mic o- o ganisms unde he es ed condi ions. Inhibi ion halos we e clea ly obse ed a ound GT disks in an ibiog ams, indica ing ha GT is capable o nega i ely a ec ing he g ow h o hese s ains. I is impo an o poin ou he g ow h inhibi ion e ec o GT on MRSA, whe e he inhibi ion halo ob ained was sligh ly highe han he halo o he MSSA s ain. Figu e 2. Redox cycle be ween he educed (di hiol, le ) and oxidized (disul ide, igh ) o ms o GT. The oxida ion o GT and p esumably o he epipoly hiodioxopipe azines (ETPs) gene a es eac i e oxygen species since i is capable o educing molecula oxygen [18]. Fu he mo e, educed GT can eac wi h o he accessible hiol g oups on p o eins, inac i a ing, inhibi ing, o dena u ing hem [ 18 ]. Some in i o s udies show ha he eac i e oxygen species p oduced by GT a e capable o causing DNA damage [21–23]. The e is cu en ly limi ed scien i ic e idence on he use o GT as an an ibio ic. The an ibac e ial ac ion o GT has been s udied agains Esche ichia coli and S. au eus. I is sugges ed ha GT could cause damage o E. coli DNA [ 24 ] and ha i exhibi s an ibio ic e ec o bo h mic oo ganisms, being mo e e ec i e agains S. au eus [25,26]. He e we ha e ex ended hese s udies and used a MRSA/VISA s ain o s udy he an ibac e ial ac i i y o GT alone and in combina ion wi h ancomycin. We ound ha GT has a po en in i o an ibio ic e ec agains his s ain and ha i s combina ion wi h ancomycin shows a e y good syne gy a lowe concen a ions han single compounds. We also ca ied ou an in i o s udy using Caeno habdi is elegans as a model o MRSA in ec- ion, whe e he combined ea men demons a ed a signi ican highe su i al pe cen age han he ea men wi h ancomycin as mono he apy. The e o e, ou indings e eal a no el app oach o ake ad an age o he an ibio ic ac i i y o GT and i s po en ia ing e ec when combined wi h ancomycin. Thus, a g ea e an ibio ic e ec is ob ained wi h a lowe concen a ion o ancomycin, and he e o e he possible side e ec s and he appea ance o esis ance due o he use o ancomycin could be educed. 2. Resul s 2.1. An imic obial Suscep ibili y o S. au eus agains GT 2.1.1. E ec o GT on S. au eus G ow h As a esul o an ibiog ams, he mean o he inhibi ion halos ob ained o each im- p egna ed disk was calcula ed (Figu e 3). All an imic obials used as con ols p oduced an inhibi ion halo wi hin he ange es ablished by he Eu opean Commi ee on An imi- c obial Suscep ibili y Tes ing (EUCAST 2020) speci ic o each s ain. The inhibi ion halo o he wa e and me hanol disks was 0 mm in all cases. Thus, hese esul s show ha he ehicle used o sol e GT, me hanol, does no exe any an imic obial ac ion on any o he mic oo ganisms unde he es ed condi ions. Toxins 2021,13, 85 4 o 16 Toxins 2021, 13, x FOR PEER REVIEW 4 o 16 Figu e 3. An imic obial ac i i y o GT agains me hicillin-suscep ible S aphylococcus au eus (MSSA) and me hicillin- e- sis an S. au eus/ ancomycin in e media e- esis an S. au eus (MRSA/VISA) s ains. (a) S. au eus ATCC 29213 s ain an i- biog am. (b) S. au eus ATCC 700699 s ain an ibiog am. A disk con aining he me hanol solu ion (MET) wi h s e ile dis- illed wa e is loca ed on op o he pla es. The disk con aining he con ol an ibio ic is placed on he le side o he pla e: (a) ce oxi in (FOX) 30 μg and (b) usidic acid (FUS) 10 μg. On he igh side, he disk imp egna ed wi h GT (10 μg) is placed. In he case o esis an S. au eus (b), he e is a ou h disk a he bo om o he pla e, whe e FOX 30 μg disk is loca ed. n ≥ 3. (c) Table con aining he mean ± s.d. o he g ow h inhibi ion halos (diame e in mm), as well as he minimum inhibi o y concen a ion (MIC) (μg/mL) calcula ed as desc ibed in Ma e ials and Me hods o GT o each s ain. (d) Table con aining he mean ± s.d. o he g ow h inhibi ion halos (diame e in mm), as well as he MIC (μg/mL) calcula ed as desc ibed in Ma e ials and Me hods o an ibio ic con ols o each s ain: cloxacillin (CLX), ancomycin (VAN) and FUS. n = 3. 2.1.2. Minimum Inhibi o y Concen a ion o GT and An ibio ics agains S. au eus In Figu e 3c, he alues ob ained o he MIC o GT in he wo s ains o S. au eus a e indica ed. I is impo an o no e ha he MIC o he MRSA s ain (2 μg/mL) was lowe han he MIC o he MSSA s ain (4 μg/mL), which is consis en wi h he esul s o he an ibiog ams, whe e he inhibi ion halo o he s ain MRSA was g ea e han ha o MSSA o he same condi ions o he expe imen . As shown in Figu e 3d, all an imic obials used as a con ol o he S. au eus ATCC 29132 s ain esul ed in a MIC consis en wi h he ange es ablished by EUCAST: Rou ine and ex ended in e nal quali y con ol o MIC de e mina ion and disk di usion as ecom- mended by EUCAST, e sion 10.0, 2020, h p://www.eucas .o g. Conce ning he S. au eus ATCC 700699 s ain, he suscep ibili y ob ained wi h he di e en an ibio ics used as con- ols (Figu e 3d) co obo a ed ha i was a MRSA (shows esis ance o cloxacillin) and VISA (MIC = 4 μg/mL o ancomycin) s ain. Figu e 3. An imic obial ac i i y o GT agains me hicillin-suscep ible S aphylococcus au eus (MSSA) and me hicillin- esis an S. au eus/ ancomycin in e media e- esis an S. au eus (MRSA/VISA) s ains. ( a )S. au eus ATCC 29213 s ain an ibiog am. ( b )S. au eus ATCC 700699 s ain an ibiog am. A disk con aining he me hanol solu ion (MET) wi h s e ile dis illed wa e is loca ed on op o he pla es. The disk con aining he con ol an ibio ic is placed on he le side o he pla e: ( a ) ce oxi in (FOX) 30 µ g and ( b ) usidic acid (FUS) 10 µ g. On he igh side, he disk imp egna ed wi h GT (10 µ g) is placed. In he case o esis an S. au eus ( b ), he e is a ou h disk a he bo om o he pla e, whe e FOX 30 µ g disk is loca ed. n ≥ 3. ( c ) Table con aining he mean ± s.d. o he g ow h inhibi ion halos (diame e in mm), as well as he minimum inhibi o y concen a ion (MIC) ( µ g/mL) calcula ed as desc ibed in Ma e ials and Me hods o GT o each s ain. ( d ) Table con aining he mean ± s.d. o he g ow h inhibi ion halos (diame e in mm), as well as he MIC ( µ g/mL) calcula ed as desc ibed in Ma e ials and Me hods o an ibio ic con ols o each s ain: cloxacillin (CLX), ancomycin (VAN) and FUS. n= 3. Inhibi ion halos we e clea ly obse ed a ound GT disks in an ibiog ams, indica ing ha GT is capable o nega i ely a ec ing he g ow h o hese s ains. I is impo an o poin ou he g ow h inhibi ion e ec o GT on MRSA, whe e he inhibi ion halo ob ained was sligh ly highe han he halo o he MSSA s ain. 2.1.2. Minimum Inhibi o y Concen a ion o GT and An ibio ics agains S. au eus In Figu e 3c, he alues ob ained o he MIC o GT in he wo s ains o S. au eus a e indica ed. I is impo an o no e ha he MIC o he MRSA s ain (2 µ g/mL) was lowe han he MIC o he MSSA s ain (4 µ g/mL), which is consis en wi h he esul s o he an ibiog ams, whe e he inhibi ion halo o he s ain MRSA was g ea e han ha o MSSA o he same condi ions o he expe imen . As shown in Figu e 3d, all an imic obials used as a con ol o he S. au eus ATCC 29132 s ain esul ed in a MIC consis en wi h he ange es ablished by EUCAST: Rou ine and ex ended in e nal quali y con ol o MIC de e mina ion and disk di usion as ec- Toxins 2021,13, 85 5 o 16 ommended by EUCAST, e sion 10.0, 2020, h p://www.eucas .o g. Conce ning he S. au eus ATCC 700699 s ain, he suscep ibili y ob ained wi h he di e en an ibio ics used as con ols (Figu e 3d) co obo a ed ha i was a MRSA (shows esis ance o cloxacillin) and VISA (MIC = 4 µg/mL o ancomycin) s ain. 2.2. An imic obial Syne gy o GT in Combina ion wi h An i-S aphylococcal D ugs As shown in Figu es 4and 5, se e al o he es ed combina ions o GT wi h an ibi- o ics we e ound o be syne gis ic, and he an imic obial ac i i y o single compounds was inc eased. Toxins 2021, 13, x FOR PEER REVIEW 5 o 16 2.2. An imic obial Syne gy o GT in Combina ion wi h An i-S aphylococcal D ugs As shown in Figu es 4 and 5, se e al o he es ed combina ions o GT wi h an ibio ics we e ound o be syne gis ic, and he an imic obial ac i i y o single compounds was in- c eased. Figu e 4. In i o syne gy assay esul s o MSSA ATCC 29213 s ain. The e ec o GT and di e en an ibio ics as mono- he apy o in combina ion was es ed agains S. au eus as desc ibed in Ma e ialsand Me hods. (a) Combina ion o GT (GT) and cloxacillin (CLX). (b) Combina ion o GT and ancomycin (VAN). (c) Combina ion o GT + linezolid (LZD). (d) Com- bina ion o GT + usidic acid (FUS). Fo each panel, he concen a ion o each compound and he mean ± s.d. o he pe - cen age o bac e ial g ow h ob ained in he expe imen s a e indica ed. The symbols co espond o he di e en le els o syne gy as indica ed in Ma e ials and Me hods, calcula ed using he CalcuSyn so wa e, as indica ed in Ma e ials and Me hods: (±) Nea ly addi i e, (+) Sligh syne gism, (++) Mode a e syne gism, (+++) Syne gism, (++++) S ong syne gism, and (+++++) Ve y s ong syne gism. MIC o GT = 4 μg/mL o S. au eus ATCC 29213. n = 3. Figu e 4. In i o syne gy assay esul s o MSSA ATCC 29213 s ain. The e ec o GT and di e en an ibio ics as mono he apy o in combina ion was es ed agains S. au eus as desc ibed in Ma e ials and Me hods. ( a ) Combina ion o GT (GT) and cloxacillin (CLX). ( b ) Combina ion o GT and ancomycin (VAN). ( c ) Combina ion o GT + linezolid (LZD). ( d ) Combina ion o GT + usidic acid (FUS). Fo each panel, he concen a ion o each compound and he mean ± s.d. o he pe cen age o bac e ial g ow h ob ained in he expe imen s a e indica ed. The symbols co espond o he di e en le els o syne gy as indica ed in Ma e ials and Me hods, calcula ed using he CalcuSyn so wa e, as indica ed in Ma e ials and Me hods: ( ± ) Nea ly addi i e, (+) Sligh syne gism, (++) Mode a e syne gism, (+++) Syne gism, (++++) S ong syne gism, and (+++++) Ve y s ong syne gism. MIC o GT = 4 µg/mL o S. au eus ATCC 29213. n= 3. Toxins 2021,13, 85 6 o 16 Fo he MSSA s ain, he esul s in Figu e 4show syne gis ic GT ac i i y wi h all he an ibio ics es ed excep o cloxacillin. In he case o he combina ion wi h ancomycin, a d as ic dec ease in he g ow h pe cen age o all combina ions was obse ed, esul ing in s ong and e y s ong syne gism. In he es ed combina ions o GT wi h linezolid, he educ ion o he g ow h pe cen age was obse ed o a lesse ex en , bu again, all he concen a ions es ed showed syne gis ic e ec s— om nea ly addi i e o e y s ong syne gism, he bes combina ion being 1 2 MIC o GT (2 µ g/mL) plus concen a ions om 0.13 o 2 µ g/mL o linezolid. GT in combina ion wi h usidic acid showed syne gy when using 0.07 µ g/mLo usidic acid wi h 1 and 2 µ g/mL o GT. I also showed syne gy when combining 2 µ g/mL o GT wi h 0.01 µ g/mL o usidic acid, whe e a la ge educ ion in he g ow h pe cen age was seen. No ably, such a low concen a ion o usidic acid only showed a ound 70% g ow h inhibi ion. Finally, 2 µ g/mL GT showed s ong syne gism in combina ion wi h 0.02 and 0.04 µg/mL o usidic acid. Toxins 2021, 13, x FOR PEER REVIEW 6 o 16 Fo he MSSA s ain, he esul s in Figu e 4 show syne gis ic GT ac i i y wi h all he an ibio ics es ed excep o cloxacillin. In he case o he combina ion wi h ancomycin, a d as ic dec ease in he g ow h pe cen age o all combina ions was obse ed, esul ing in s ong and e y s ong syne gism. In he es ed combina ions o GT wi h linezolid, he educ ion o he g ow h pe cen age was obse ed o a lesse ex en , bu again, all he con- cen a ions es ed showed syne gis ic e ec s— om nea ly addi i e o e y s ong syne - gism, he bes combina ion being½ MIC o GT (2 μg/mL) plus concen a ions om 0.13 o 2 μg/mL o linezolid. GT in combina ion wi h usidic acid showed syne gy when using 0.07 μg/mLo usidic acid wi h 1 and 2 μg/mL o GT. I also showed syne gy when com- bining 2 μg/mL o GT wi h 0.01 μg/mL o usidic acid, whe e a la ge educ ion in he g ow h pe cen age was seen. No ably, such a low concen a ion o usidic acid only showed a ound 70% g ow h inhibi ion. Finally, 2 μg/mL GT showed s ong syne gism in combina ion wi h 0.02 and 0.04 μg/mL o usidic acid. Figu e 5. In i o syne gy assay esul s o MRSA/VISA ATCC 700699 s ain. The e ec o GT and di e en an ibio ics as mono he apy o in combina ion was es ed agains S. au eus as desc ibed in Ma e ials and Me hods.(a) Combina ion o GT (GT) and ancomycin (VAN). (b) Combina ion o GT + linezolid (LZD). (c) Combina ion o GT + usidic acid (FUS). Fo each panel, he concen a ion o each compound and he mean ± s.d. o he pe cen age o bac e ial g ow h ob ained in he expe imen s a e indica ed. The symbols co espond o he di e en le els o syne gy as indica ed in Ma e ials and Me hods, calcula ed using he CalcuSyn so wa e as indica ed in Ma e ials and Me hods: (++) Mode a e syne gism, (+++) Syne gism, and (++++) S ong syne gism. MIC o GT = 2 μg/mL o S. au eus ATCC 700699. n = 3. Rega ding he MRSA s ain, syne gis ic e ec s o GT in combina ion wi h ancomy- cin, linezolid, and usidic acid we e demons a ed (Figu e 5). In his case, usidic acid a Figu e 5. In i o syne gy assay esul s o MRSA/VISA ATCC 700699 s ain. The e ec o GT and di e en an ibio ics as mono he apy o in combina ion was es ed agains S. au eus as desc ibed in Ma e ials and Me hods. ( a ) Combina ion o GT (GT) and ancomycin (VAN). ( b ) Combina ion o GT + linezolid (LZD). ( c ) Combina ion o GT + usidic acid (FUS). Fo each panel, he concen a ion o each compound and he mean ± s.d. o he pe cen age o bac e ial g ow h ob ained in he expe imen s a e indica ed. The symbols co espond o he di e en le els o syne gy as indica ed in Ma e ials and Me hods, calcula ed using he CalcuSyn so wa e as indica ed in Ma e ials and Me hods: (++) Mode a e syne gism, (+++) Syne gism, and (++++) S ong syne gism. MIC o GT = 2 µg/mL o S. au eus ATCC 700699. n= 3. Rega ding he MRSA s ain, syne gis ic e ec s o GT in combina ion wi h ancomycin, linezolid, and usidic acid we e demons a ed (Figu e 5). In his case, usidic acid a Toxins 2021,13, 85 7 o 16 0.04 µ g/mL showed syne gy when combined wi h GT a hal he MIC o his s ain (1 µ g/mL). GT a 1 µ g/mL p esen ed mode a e syne gism when combined wi h 1 µ g/mL o linezolid and syne gism when combined wi h 2 µ g/mL (linezolid MIC o his s ain). Fo he MRSA s ain, he combina ion o compounds wi h he g ea es syne gis ic e ec s was GT plus ancomycin. Syne gy was obse ed when combining 0.5 µ g/mL o an- comycin wi h 1 µ g/mL o GT, al hough he g ow h pe cen age exceeded 20%. A highe concen a ions o ancomycin, a e y s ong syne gism and g ea e educ ion in he g ow h pe cen age we e ound when combining 1 µ g/mL o GT wi h 1 µ g/mL o ancomycin and when combining 0.5–1 µg/mL o GT wi h 2 µg/mL o ancomycin. 2.3. Mu an P e en ion Concen a ion o GT and Vancomycin Alone o in Combina ion agains S. au eus Nex , we de e mined he mu an p e en ion concen a ion (MPC) o GT and an- comycin alone o in combina ion. These expe imen s we e only ca ied ou wi h S. au eus ATCC 700699, GT, and ancomycin since GT has been shown o be an an i-s aphylococcal compound agains his me hicillin- esis an and ancomycin-in e media e s ain and shows syne gy in combina ion wi h ancomycin. I should also be no ed ha he s udy o MRSA and VISA s ains is mo e ele an o clinical and pha maceu ical se ings. All MPC de e mina ions we e made in iplica e and he esul s we e iden ical. Table 1 e lec s he MPCs o an imic obials alone and in combina ion wi h ancomycin agains he MRSA s ain. Table 1. Mu an P e en ion Concen a ion ob ained o GT, ancomycin, and combina ion o bo h agains S. au eus ATCC 700699. n= 3. Compound Indi idually Combina ion Vancomycin 64 0 32 Glio oxin 0 256 2 Mu an P e en ion Concen a ion (µg/mL). The MPC o ancomycin was hal ed when i was combined wi h a e y low concen- a ion o GT, speci ically he MIC o GT o his s ain (2 µg/mL). 2.4. In Vi o An ibio ic E icacy o GT as Mono he apy and in Combina ion wi h Vancomycin agains S. au eus in C. elegans Model The an ibio ic e icacy o GT as mono he apy and in combina ion wi h ancomycin agains MRSA was e alua ed using a C. elegans in ec ion model. The ea men s selec ed o his su i al s udy we e hose ha esul ed in syne gis ic ac i i y in he in i o expe imen s o MRSA. The synch oniza ion me hod was op imized o ob ain a la ge numbe o nema odes a he same de elopmen al s age o he su i al assays. Fo his pu pose, cul u e pla es wi h a high con en o g a id wo ms and eggs we e used. The bes pe o mance was ob ained when 2 pla es we e synch onized, dis ibu ing he M9 bu e wi h he wo ms in se e al 15 mL alcon ubes so ha hey con ained a pelle o abou 3 mm a e cen i uga ion. The addi ion o a bleaching solu ion o his pelle is su icien i he exposu e ime is con olled. A signi ican su i al a e educ ion was obse ed in C. elegans in ec ed wi h MRSA using liquid-based assays (Figu e 6), ob aining a ound 40% su i al. This educ ion was diminished when GT and ancomycin ea men s we e used bo h in mono he apy and in combina ion. Su i al cu es we e ob ained using he G aphPad P ism p og am (Figu e 6). I was obse ed ha he syne gis ic e ec seen in i o o wo o he combina ions p oduced a highe pe cen age o su i al o he nema odes compa ed o he e ec o he an imic obials in mono he apy. The combina ion o 1 2 MIC o ancomycin (2 µ g/mL) wi h 1 4 MIC o GT (0.5 µ g/mL) esul ed in 74.55% su i al o nema odes, being 58.58% o GT and 52.11% o ancomycin a he same concen a ions in mono he apy. On he o he hand, he combina ion o 1 4 o ancomycin MIC (1 µ g/mL) wi h 1 2 o GT MIC (1 µ g/mL) esul ed Toxins 2021,13, 85 8 o 16 in 74.80% su i al o nema odes. The same concen a ions in mono he apy esul ed in 59.60% su i al o GT and 51.79% o ancomycin. S a is ical analysis o he su i al assay e ealed ha he esul s we e s a is ically signi ican when compa ing he GT plus ancomycin combina ion wi h he in ec ed con ol and wi h he e ec obse ed a he same concen a ion o ancomycin in mono he apy. Toxins 2021, 13, x FOR PEER REVIEW 8 o 16 o ancomycin a he same concen a ions in mono he apy. On he o he hand, he com- bina ion o ¼ o ancomycin MIC (1 μg/mL) wi h ½ o GT MIC (1 μg/mL) esul ed in 74.80% su i al o nema odes. The same concen a ions in mono he apy esul ed in 59.60% su i al o GT and 51.79% o ancomycin. S a is ical analysis o he su i al as- say e ealed ha he esul s we e s a is ically signi ican when compa ing he GT plus ancomycin combina ion wi h he in ec ed con ol and wi h he e ec obse ed a he same concen a ion o ancomycin in mono he apy. Figu e 6. Su i al cu es o in i o assay wi h Caeno habdi iselegans MRSA in ec ion model ha was ea ed wi h GT, ancomycin, and combina ions o bo h. The su i al pe cen age o he nema odes is ep esen ed o each ea men and o he in ec ed con ol e e y 24 h o 7 days. (a) Resul s o mono he apy ea men wi h ½ MIC o ancomycin, ¼ MIC o GT, and he combina ion o hese wo concen a ions. (b) Resul s o mono he apy ea men wi h ¼ MIC o ancomycin, ½ MIC o GT, and he combina ion o hese wo concen a ions. *, **, *** S a is ically signi ican di e ences (p< 0.05, 0.01, o 0.001, espec i ely) ega ding he an ibio ic combina ion pe o med as indica ed in Ma e ials and Me hods. n = 4. The oxic e ec o GT and ancomycin alone o in combina ion was also s udied in C. elegans a he concen a ions ha had been es ed in he in ec ion expe imen s (Figu e 7). The su i al pe cen age o ancomycin was 93.00% and 94.74% o 2 and 1 μg/mL, e- spec i ely. Rega ding GT alone, su i al was sligh ly educed, 85.22% and 88.98% o 1 and 0.5 μg/mL, espec i ely, al hough i was no signi ican ly di e en o ancomycin. Mo eo e , wi h espec o combina ions, 89.74% su i al was shown o 2 μg/mLo an- comycin plus 0.5 μg/mL o GT. Addi ionally, 86.87% o nema odes su i ed in he case o he combina ion o 1 μg/mL o ancomycin wi h 1 μg/mL o GT. Thus, hese esul s indi- ca e ha GT migh show a sligh oxic e ec a he es ed concen a ions, al hough his e ec is no signi ican ly di e en om ha obse ed in ancomycin. Figu e 6. Su i al cu es o in i o assay wi h Caeno habdi is elegans MRSA in ec ion model ha was ea ed wi h GT, ancomycin, and combina ions o bo h. The su i al pe cen age o he nema odes is ep esen ed o each ea men and o he in ec ed con ol e e y 24 h o 7 days. ( a ) Resul s o mono he apy ea men wi h 1 2 MIC o ancomycin, 1 4 MIC o GT, and he combina ion o hese wo concen a ions. ( b ) Resul s o mono he apy ea men wi h 1 4 MIC o ancomycin, 1 2 MIC o GT, and he combina ion o hese wo concen a ions. *, **, *** S a is ically signi ican di e ences (p< 0.05, 0.01, o 0.001, espec i ely) ega ding he an ibio ic combina ion pe o med as indica ed in Ma e ials and Me hods. n= 4. The oxic e ec o GT and ancomycin alone o in combina ion was also s udied in C. elegans a he concen a ions ha had been es ed in he in ec ion expe imen s (Figu e 7) . The su i al pe cen age o ancomycin was 93.00% and 94.74% o 2 and 1 µ g/mL, e- spec i ely. Rega ding GT alone, su i al was sligh ly educed, 85.22% and 88.98% o 1 and 0.5 µ g/mL, espec i ely, al hough i was no signi ican ly di e en o ancomycin. Mo eo e , wi h espec o combina ions, 89.74% su i al was shown o 2 µ g/mLo an- comycin plus 0.5 µ g/mL o GT. Addi ionally, 86.87% o nema odes su i ed in he case o he combina ion o 1 µ g/mL o ancomycin wi h 1 µ g/mL o GT. Thus, hese esul s indica e ha GT migh show a sligh oxic e ec a he es ed concen a ions, al hough his e ec is no signi ican ly di e en om ha obse ed in ancomycin. Toxins 2021,13, 85 9 o 16 Toxins 2021, 13, x FOR PEER REVIEW 9 o 16 Figu e 7. Toxici y es in non-in ec ed C. elegans ea ed wi h GT, ancomycin, and combina ions o bo h. The su i al pe cen age o he nema odes is ep esen ed o each ea men and o he non- in ec ed con ol e e y 24 h o 7 days. Resul s o mono he apy ea men wi h ½ MIC o ancomycin, ½ MIC o GT, ¼ MIC o ancomycin, ¼ MIC o GT, and he combina ion o wo concen a ions. *, S a is ically signi ican di e ences (p< 0.05) ega ding he non-in ec ed con ol. n = 4. 3. Discussion S. au eus is one o he leading causes o skin and so issue in ec ions in all age g oups. The e is also e idence o an inc ease in communi y-acqui ed me hicillin- esis an S. au eus in ec ions (CA-MRSA). CA-MRSA in ec ions can be di icul o ea due o e- sis ance o an ibio ics, and hei in ec ion a e is on he ise wo ldwide [27]. The e o e, he s udy o new an ibio ics as mono he apy o in combina ion wi h exis ing ea men s is necessa y o be able o deal wi h hese in ec ions. Since i s disco e y, GT has spa ked in e es in exploi ing i s an imic obial ac i i ies, which has been es ed agains i al, ungal, and bac e ial pa hogens [24,28–30]. Ou ind- ings e eal he an imic obial ac i i y o GT agains S. au eus s ains bo h sensi i e and esis an o me hicillin and wi h educed suscep ibili y o ancomycin ( ha is, MRSA/VISA s ains). Rega ding he MSSA s ain, he only ial pe o med wi h he same s ain ha has been ound in he li e a u e [25] esul ed in a MIC = 3.98 μg/mL, a alue p ac ically equi alen o ou s (MIC = 4). Some esea che s ha e s udied he e ec o GT agains di e en MRSA s ains, ob aining a MIC ange be ween 0.5–4 μg/mL [25,31,32], con i ming he alidi y o he esul ound in his s udy o he MRSA ATCC 700699 s ain (MIC = 2 μg/mL). Impo an ly, ou esul s p o ide no el obse a ions and ex end p e i- ous analyses by showing ha GT p esen s ac i i y no only agains MRSA bu agains a MRSA/VISA s ain. The equen use o ancomycin as he main agen o ea MRSA in ec ions is a di ec consequence o he wo ldwide sp ead o mul id ug esis an MRSA clones du ing he las decades. This inc eased selec i e p essu e has esul ed in he eme gence o MRSA isola es wi h educed suscep ibili y o ancomycin and, mo e ecen ly, in he eme gence o VISA s ains wi h a high le el o an ibio ic esis ance[33]. Fo he as majo i y o cases, VISA s ains ha e eme ged in pa ien s wi h MRSA in ec ions unde going p olonged ancomy- cin he apy, which o en ended in ea men ailu e [34–37]. A p esen a la ge numbe o clinical s ains o S. au eus wi h educed suscep ibili y o ancomycin ha e been epo ed, including he e o esis an VISA ( esis an subpopula ions among he o al bac e ial popu- la ion o he s ain, which can be selec ed by ea men ) and VISA [38]. Fo all hese easons, he s ain we selec ed o he assays in his s udy wasATCC 700699, a MRSA and VISA s ain. The esul s ob ained a e no el since he e is no scien i ic e idence o de e mine he e ec o GT o VISA s ains. Al hough he molecula de e mi- nan s o esis ance a e no comple ely de e mined, i is clea ha he sequen ial acquisi ion o poin mu a ions can lead o esis ance [36]. He e we demons a e ha he combina ion o GT wi h ancomycin dec eases i s MPC (MIC o he leas -suscep ible, single- Figu e 7. Toxici y es in non-in ec ed C. elegans ea ed wi h GT, ancomycin, and combina ions o bo h. The su i al pe cen age o he nema odes is ep esen ed o each ea men and o he non-in ec ed con ol e e y 24 h o 7 days. Resul s o mono he apy ea men wi h 1 2 MIC o ancomycin, 1 2 MIC o GT, 1 4 MIC o ancomycin, 1 4 MIC o GT, and he combina ion o wo concen a ions. *, S a is ically signi ican di e ences (p< 0.05) ega ding he non-in ec ed con ol. n= 4. 3. Discussion S. au eus is one o he leading causes o skin and so issue in ec ions in all age g oups. The e is also e idence o an inc ease in communi y-acqui ed me hicillin- esis an S. au eus in ec ions (CA-MRSA). CA-MRSA in ec ions can be di icul o ea due o esis ance o an ibio ics, and hei in ec ion a e is on he ise wo ldwide [ 27 ]. The e o e, he s udy o new an ibio ics as mono he apy o in combina ion wi h exis ing ea men s is necessa y o be able o deal wi h hese in ec ions. Since i s disco e y, GT has spa ked in e es in exploi ing i s an imic obial ac i i ies, which has been es ed agains i al, ungal, and bac e ial pa hogens [ 24 , 28 – 30 ]. Ou ind- ings e eal he an imic obial ac i i y o GT agains S. au eus s ains bo h sensi i e and esis an o me hicillin and wi h educed suscep ibili y o ancomycin ( ha is, MRSA/VISA s ains). Rega ding he MSSA s ain, he only ial pe o med wi h he same s ain ha has been ound in he li e a u e [ 25 ] esul ed in a MIC = 3.98 µ g/mL, a alue p ac ically equi alen o ou s (MIC = 4). Some esea che s ha e s udied he e ec o GT agains di e en MRSA s ains, ob aining a MIC ange be ween 0.5–4 µ g/mL [ 25 , 31 , 32 ], con- i ming he alidi y o he esul ound in his s udy o he MRSA ATCC 700699 s ain (MIC = 2 µg/mL ). Impo an ly, ou esul s p o ide no el obse a ions and ex end p e i- ous analyses by showing ha GT p esen s ac i i y no only agains MRSA bu agains a MRSA/VISA s ain. The equen use o ancomycin as he main agen o ea MRSA in ec ions is a di ec consequence o he wo ldwide sp ead o mul id ug esis an MRSA clones du ing he las decades. This inc eased selec i e p essu e has esul ed in he eme gence o MRSA isola es wi h educed suscep ibili y o ancomycin and, mo e ecen ly, in he eme gence o VISA s ains wi h a high le el o an ibio ic esis ance [ 33 ]. Fo he as majo i y o cases, VISA s ains ha e eme ged in pa ien s wi h MRSA in ec ions unde going p olonged ancomycin he apy, which o en ended in ea men ailu e [ 34 – 37 ]. A p esen a la ge numbe o clinical s ains o S. au eus wi h educed suscep ibili y o ancomycin ha e been epo ed, including he e o esis an VISA ( esis an subpopula ions among he o al bac e ial popula ion o he s ain, which can be selec ed by ea men ) and VISA [38]. Fo all hese easons, he s ain we selec ed o he assays in his s udy was ATCC 700699, a MRSA and VISA s ain. The esul s ob ained a e no el since he e is no scien i ic e idence o de e mine he e ec o GT o VISA s ains. Al hough he molecula de e mi- nan s o esis ance a e no comple ely de e mined, i is clea ha he sequen ial acquisi ion o poin mu a ions can lead o esis ance [ 36 ]. He e we demons a e ha he combina ion o GT wi h ancomycin dec eases i s MPC (MIC o he leas -suscep ible, single-s epmu an ), Toxins 2021,13, 85 16 o 16 36. Howden, B.P.; Da ies, J.K.; Johnson, P.D.; S inea , T.P.; G ayson, M.L. Reduced ancomycin suscep ibili y in S aphylococcus au eus, including ancomycin-in e media e and he e ogeneous ancomycin-in e media e s ains: Resis ance mechanisms, labo a o y de ec ion, and clinical implica ions. Clin. Mic obiol. Re . 2010,23, 99–139. [C ossRe ] 37. Liña es, J. The VISA/GISA p oblem: The apeu ic implica ions. Clin. Mic obiol. In ec . 2001,7, 8–15. [C ossRe ] 38. Xu, J.; Pang, L.; Ma, X.X.; Hu, J.; Tian, Y.; Yang, Y.L.; Sun, D.D. Pheno ypic and Molecula Cha ac e isa ion o S aphylococcus Au eus wi h Reduced Vancomycin Suscep ibili y De i a ed in Vi o. Open Med. 2018,13, 475–486. [C ossRe ] 39. Pa do, J.; U ban, C.; Gal ez, E.M.; Eke , P.G.; Mülle , U.; Kwon-Chung, J.; Lobigs, M.; Müllbache , A.; Wallich, R.; Bo ne , C.; e al. The mi ochond ial p o ein Bak is pi o al o glio oxin-induced apop osis and a c i ical hos ac o o Aspe gillus umiga us i ulence in mice. J. Cell Biol. 2006,174, 509–519. [C ossRe ] 40. The Eu opean Commi ee on An imic obial Suscep ibili y Tes ing (EUCAST). Disk Di usion Me hod o An imic obial Suscep i- bili y Tes ing, Ve sion 8.0, 2020. A ailable online: h ps://www.eucas .o g/ ileadmin/s c/media/PDFs/EUCAST_ iles/Disk_ es _documen s/2021_manuals/Reading_guide_ _8.0_EUCAST_Disk_Tes _2021.pd (accessed on 23 Janua y 2021). 41. Clinical and Labo a o y S anda ds Ins i u e. Pe o mance S anda ds o An imic obial Disk Suscep ibili y Tes s, 12 h ed.; App o ed S anda d; Supplemen M02-A12; CLSI: Wayne, PA, USA, 2015. 42. Clinical and Labo a o y S anda d Ins i u e. Pe o mance S anda ds o An imic obial Suscep ibili y Tes ing: Twen y-Fi s In o ma ional Supplemen M100-S21; CLSI: Wayne, PA, USA, 2011. 43. Odds, F.C. Syne gy, an agonism, and wha he cheque boa d pu s be ween hem. J. An imic ob. Chemo he . 2003 ,52, 1. [C ossRe ] 44. Chou, T.C. D ug combina ion s udies and hei syne gy quan i ica ion using he Chou-Talalay me hod. Cance Res. 2010 , 70, 440–446. [C ossRe ] [PubMed] 45. Chou, T.; Hayball, M. CalcuSyn o Windows: Mul iple-D ug Dose-E ec Analyze and Manual; Bioso : Camb idge, UK, 1996. 46. Dong, Y.; Zhao, X.; Domagala, J.; D lica, K. E ec o luo oquinolone concen a ion on selec ion o esis an mu an s o Mycobac- e ium bo is BCG and S aphylococcus au eus. An imic ob. Agen s Chemo he . 1999,43, 1756–1758. [C ossRe ] [PubMed] 47. Blondeau, J.M. New concep s in an imic obial suscep ibili y es ing: The mu an p e en ion concen a ion and mu an selec ion window app oach. Ve . De ma ol. 2009,20, 383–396. [C ossRe ] [PubMed] 48. Beanan, M.J.; S ome, S. Cha ac e iza ion o a ge m-line p oli e a ion mu a ion in C. elegans. De elopmen 1992,116, 755–766. 49. Kong, C.; Yehye, W.A.; Abd Rahman, N.; Tan, M.W.; Na han, S. Disco e y o po en ial an i-in ec i es agains S aphylococcus au eus using a Caeno habdi is elegans in ec ion model. BMC Complemen . Al e n. Med. 2014,14, 4. [C ossRe ]