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
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labo a o y de ec ion, and clinical implica ions. Clin. Mic obiol. Re . 2010,23, 99–139. [C ossRe ]
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