Ci a ion: Gan, C.; Langa, E.;
Valenzuela, A.; Balles e o, D.;
Pino-O ín, M.R. Syne gis ic Ac i i y
o Thymol wi h Comme cial
An ibio ics agains C i ical and High
WHO P io i y Pa hogenic Bac e ia.
Plan s 2023,12, 1868. h ps://
doi.o g/10.3390/plan s12091868
Academic Edi o : Despina Vokou
Recei ed: 9 Ma ch 2023
Re ised: 28 Ap il 2023
Accep ed: 29 Ap il 2023
Published: 2 May 2023
Copy igh : © 2023 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/).
plan s
A icle
Syne gis ic Ac i i y o Thymol wi h Comme cial An ibio ics
agains C i ical and High WHO P io i y Pa hogenic Bac e ia
C is ina Gan , Elisa Langa, An onio Valenzuela, Diego Balles e o and M. Rosa Pino-O ín *
Facul y o Heal h Sciences, Uni e sidad San Jo ge, 50830 Villanue a de Gállego, Za agoza, Spain;
[email p o ec ed] (C.G.); [email p o ec ed] (E.L.)
*Co espondence: [email p o ec ed]
Abs ac :
The use o syne gis ic combina ions be ween na u al compounds and comme cial an ibio ics
may be a good s a egy o igh agains mic obial esis ance, wi h ewe side e ec s on human, animal
and en i onmen al, heal h. The an imic obial capaci y o ou compounds o plan o igin ( hymol and
gallic, salicylic and gen isic acids) was analysed agains 14 pa hogenic bac e ia. Thymol showed he
bes an imic obial ac i i y, wi h MICs anging om 125
µ
g/mL ( o Acine obac e baumannii,Pas eu ella
ae ogenes, and Salmonella yphimu ium) o 250
µ
g/mL ( o Bacillus sub ilis,Klebsiella ae ogenes,Klebsiella
pneumoniae,Se a ia ma cescens,S aphylococcus au eus, and S ep ococcus agalac iae). Combina ions o
hymol wi h eigh widely used an ibio ics we e s udied o iden i y combina ions wi h syne gis ic
e ec s. Thymol showed syne gis ic ac i i y wi h chlo amphenicol agains A. baumannii (c i ical
p io i y by he WHO), wi h s ep omycin and gen amicin agains S aphylococcus au eus (high p io i y
by he WHO), and wi h s ep omycin agains S ep ococcus agalac iae, dec easing he MICs o hese
an ibio ics by 75% o 87.5%. The kine ics o hese syne gies indica ed ha hymol alone a he syne gy
concen a ion had almos no e ec on he maximum achie able popula ion densi y and e y li le
e ec on he g ow h a e. Howe e , in combina ion wi h an ibio ics a he same concen a ion,
i comple ely inhibi ed g ow h, con i ming i s ole in acili a ing he ac ion o he an ibio ic. The
ime–kill cu es indica ed ha all he combina ions wi h syne gis ic e ec s we e mainly bac e icidal.
Keywo ds:
hymol; an ibio ics; syne gy; S aphylococcus au eus;S ep ococcus agalac iae;Acine obac e
baumannii; na u al p oduc
1. In oduc ion
The disco e y o an ibio ics (ABXs) was a ue e olu ion o public heal h, and has
sa ed millions o li es. Howe e , hei excessi e consump ion and i a ional use ha e led
o hei dispe sion in he en i onmen and he eme gence o ABX- esis an bac e ia [
1
]. The
Wo ld Heal h O ganiza ion (WHO) has decla ed ha he eme gence o mul id ug- esis an
(MDR) pa hogens is one o he g ea es h ea s o global heal h, ood secu i y, and de el-
opmen [
2
]. In ecen decades, he consump ion o ABXs has con inued o g ow. Be ween
2000 and 2010, ABX d ug consump ion inc eased by 36% ( om 54,083,964,813 s anda d
uni s in 2000 o 73,620,748,816 s anda d uni s in 2010) wo ldwide, wi h aminoglycosides as
one o he mos used [
3
]. This eno mous quan i y o ABXs, once consumed, passes in o he
was ewa e whe e, in he bes o cases, i eaches sewage ea men plan s ha do no elimi-
na e hese esidues [
4
], and discha ge hem in o wa e cou ses. Le els in he ng/L ange
ha e been de ec ed in e luen s con aining high concen a ions o mos ABXs ha we s udy
he e, e.g., chlo amphenicol (CHL) [
5
]. Some o hese was es become pa o he sludge om
was ewa e ea men plan s and end up being applied o soils as e ilize s [
6
]. ABXs ha e
been de ec ed in soils a di e en concen a ions anging om ng/kg o mg/kg [
7
]. Fo
example, a concen a ion o 5.6 ng/kg o s ep omycin (STM) was epo ed in US sandy
loam soil a e he addi ion o manu e [
8
]. All his leads o a la ge dissemina ion o ABX
esidues in he en i onmen , which will acili a e he selec i e p essu e and he sp ead o
Plan s 2023,12, 1868. h ps://doi.o g/10.3390/plan s12091868 h ps://www.mdpi.com/jou nal/plan s
Plan s 2023,12, 1868 2 o 22
esis ance genes [
9
]. Resis ance can a ise om mu a ions ha al e he bac e ial molecula
a ge s o he ABX. The di icul y in ea ing in ec ions due o MDR pa hogens makes i
u gen o sea ch o new an imic obial subs ances wi h di e en mechanisms o ac ion
capable o p oducing less esis ance and, i possible, wi h ewe side e ec s on human,
animal, and en i onmen al heal h, in line wi h he “One Heal h” s a egy. The WHO has
also de eloped an ac ion plan o comba MDR s ains, and one o he key poin s is he
de elopmen o new an imic obial p oduc s [
10
]. The e o e, many s udies ha e ocused on
p ospec ing na u al p oduc s o ind new po en ial an imic obial agen s [11].
Many na u al p oduc s om plan s, especially essen ial oils (EOs), ha e been explo ed
o he ea men and p e en ion o MDR bac e ia [
12
,
13
]. Un o una ely, na u al p oduc s
usually ha e weake an ibio ic ac i i y han common ABXs; he e o e, i is di icul o hem
o e ec i ely eplace cu en ABXs in clinical p ac ice. Howe e , some plan -de i ed an-
imic obial compounds ha e been shown o syne gis ically enhance an ibio ic ac i i y [
14
].
The syne gis ic in e ac ion o na u al compounds wi h al eady a ailable ABXs may allow
o he combina ion o be as e ec i e as he ABX alone, and while main aining he use o
comme cial ABXs, i lowe s he minimum inhibi o y concen a ion (MIC) o bo h he ABX
and he na u al p oduc [15].
The use o lowe concen a ions o bo h agen s o e s impo an oppo uni ies in
he sea ch o al e na i es o he ea men o in ec ious diseases, as combina ions wi h
syne gis ic e ec s may educe he p obabili y o he eme gence o bac e ial esis ance [16]
while ha ing e ec i e pha macological esul s [
17
]. Mo eo e , i may in ol e a educ ion
in ABX oxici y [
18
] wi h ewe side e ec s compa ed o hose de i ed om high doses o
syn he ic d ugs [19].
Thymol (2-isop opyl-5-me hylphenol, THY) is one o he main phenolic mono e penes
ound in EOs ex ac ed om plan s belonging o he Lamiaceae amily, such as hose o he
gene a Thymus,Ocimum,O iganum,Sa u eja,Thymb a, and Mona da [
20
–
23
]. I has a molec-
ula weigh o 150.22 g/mol and a solubili y o 900 mg/L [
24
] and
logP (o/w) = 3.3 [25]
,
which indica es ha i is a sligh ly wa e -soluble compound. Mo eo e , i s pKa = 10.6 [
26
],
which indica es ha i is a molecule ha a physiological pH 7.4 will be non-ionized. Essen-
ial oils o hese plan s ha e demons a ed an imic obial p ope ies p ima ily a ibu ed
o hei main componen s, THY [
20
,
21
] among hem. THY exhibi s b oad bioac i i y [
27
];
especially, i s an imic obial ac i i y has been quan i a i ely assessed on Esche ichia coli,
S aphylococcus au eus,Lis e ia monocy ogenes, and Bacillus sub ilis [
28
–
31
]. Howe e , he
an imic obial e ec o THY in combina ion wi h comme cial ABXs has been much less
explo ed. O he au ho s [
14
] ha e s udied he in e ac ion o THY wi h ampicillin, bac-
i acin, e y h omycin, and penicillin in ou ABX- esis an bac e ia, inding syne gis ic
e ec s in se e al cases, as wi h Salmonella yphimu ium combined wi h ampicillin, e acy-
cline, penicillin, o e y h omycin. O he au ho s ha e ound syne gis ic ac i i y be ween
THY and o he ABXs, such as ancomycin agains E. coli [
31
], and an ibio ilm ac i i y in
combina ions o THY wi h h ee aminoglycosides agains Klebsiella pneumoniae [
32
]. These
s udies indica e ha THY p esen s a ou able cha ac e is ics o be used in combina ion
wi h ABXs in he ea men o in ec ious diseases, bu hei in e ac ion wi h mos o he
ABXs used, as well as he syne gis ic e ec s on he nume ous pa hogenic bac e ia o majo
clinical in e es , ha e no ye been s udied. The Eu opean Commission conside s THY a
low- isk p oduc in consump ion, and i is es ed o use as a ood la ou ing. The Food
and D ug Adminis a ion (FDA) has u he classi ied THY as “gene ally sa e” [27].
Ano he in e es ing g oup o plan seconda y me aboli es a e he hyd oxybenzoic
acids, which a e phenolic compounds cha ac e ized by an a oma ic ing wi h an acid g oup
and one o mo e hyd oxyl g oups. Among he ep esen a i es o his chemical amily a e
salicylic acid (2-hyd oxybenzoic acid, SA), gen isic acid (2,5-dihyd oxybenzoic acid, GEA),
and gallic acid (3,4,5- ihyd oxybenzoic acid, GA). SA is a na u al p oduc ha is equen ly
used in cosme ics because o i s abili y o p omo e ex olia ion and o i s an i-in lamma o y
and opical an ibac e ial ac i i y [
33
]. An ibac e ial ac i i y agains a ious bac e ial s ains,
such as E. coli and S. au eus [
34
], has also been demons a ed. Simila o SA, GEA also
Plan s 2023,12, 1868 3 o 22
exhibi s an imic obial ac i i y agains bo h G am-posi i e and nega i e bac e ia [
35
], and
has an ia hy hmic, an i heuma ic, analgesic, and an i-in lamma o y p ope ies [
33
]. GA
has an ioxidan , an imelanogenic [
36
], and an imic obial p ope ies, wi h demons a ed
ac i i y agains En e ococcus aecalis,S. au eus,E. coli, and Pseudomonas ae uginosa among
o he s [37].
The aim o his s udy is o explo e combina ions o na u al p oduc s o plan o igin
wi h comme cial ABXs in sea ch o he ones wi h syne gis ic e ec s and wi h lowe e-
qui ed doses o he ABX. Fo his pu pose: (1) he MIC o ou na u al p oduc s and eigh
widely consumed ABXs a e s udied agains 14 mic obial s ains esponsible o nume ous
human and e e ina y diseases and ood spoilage; (2) om he na u al p oduc s wi h he
lowes MIC, combina ions wi h ABXs a e s udied o iden i y syne gis ic combina ions.
Fo his pu pose, bac e icidal and bac e ios a ic syne gis ic e ec s a e quan i ied and he
g ow h kine ics and ime–kill cu es o bac e ia exposed o he mos p omising na u al
p oduc /ABX combina ions a e analysed. Bac e ial ypes we e selec ed based on hei
clinical in e es , as hey cause some o he mos common in ec ions oday [
38
,
39
], and on
hei po en ial se e i y and abili y o gene a e esis ance, acco ding o he WHO’s lis o
p io i y pa hogens [40].
2. Resul s
2.1. An imic obial P ope ies o Na u al P oduc s
The an ibac e ial ac i i y o THY, GA, SA, and GEA agains 14 mic oo ganisms is shown
in Table 1. THY had s ong an imic obial e ec s (See Ma e ial and Me hods o he quali a i e
e alua ion o he an imic obial ac i i y o he na u al p oduc s es ed) agains se en ou
o he nine G am-nega i e bac e ia es ed and agains h ee ou o he i e G am-posi i e
bac e ia, a concen a ions below o equal o 500
µ
g/mL. The lowes MICs we e 125
µ
g/mL
o he G am-nega i e Acine obac e baumannii,Pas eu ella ae ogenes, and S. yphimu ium. The
alues o he a io be ween he minimum bac e icidal concen a ion (MBC) and he MIC o
THY showed ha he ac i i y was bac e icidal in all cases (MBC/MIC
≤
4) [
41
–
43
]. THY had
highe MBC/MIC a ios o he G am-posi i e cocci.
SA was he second mos bioac i e na u al p oduc , showing weak an ibac e ial ac i i y
agains 12 ou o he 14 bac e ia es ed, wi h MIC alues be ween 1000 and 5000
µ
g/mL.
P. ae ogenes was he mos sensi i e s ain o his compound (MIC = 625
µ
g/mL); he same
s ain was also he mos sensi i e wi h THY and GA. GEA and GA exhibi ed low o no
an ibac e ial ac i i y, wi h MIC alues anging be ween 1250 and 5000
µ
g/mL, and be ween
2500 and 5000
µ
g/mL, espec i ely. Acco ding o he MBC/MIC index, all h ee acids
exhibi ed bac e icidal ac i i y.
The MICs o he ABXs a e gi en in Table 2. These concen a ions will be used o calcula e
he ac ional inhibi o y concen a ion index (FIC
I
) in he combina ions wi h syne gis ic e ec s.
2.2. Syne gies be ween Thymol and An ibio ics
The FIC
I
s o he combina ions o THY wi h he ABXs om he checke boa d es a e
shown in Table 3. The co esponding isobolog ams o he combina ions ha showed one
o mo e in e ac ions wi h a FIC
I≤
0.5 a e shown in Figu e 1. Among he 30 combina-
ions o THY wi h he ABXs, ou showed syne gism (FIC
I≤
0.5), 14 showed addi i i y
(0.5 < FIC
I≤
1), and 12 showed no in e ac ion (1 < FIC
I
< 2). None o he combina ions
showed an agonis ic e ec s (FICI≥2).
Two o he mos p onounced esul s we e ob ained wi h he combina ion o THY
and STM agains S. au eus, and THY and CHL agains A. baumannii, bo h showing a
signi ican syne gis ic e ec (FIC
I
= 0.375) and achie ing an ABX dose educ ion om
62.5 o 7.8 µg/mL (ABX dose educ ion o 87.5%). Two o he e y p omising esul s, wi h
a ou - old dose educ ion o ABX (75% dose educ ion), we e obse ed wi h THY and
gen amycin (GTM) agains S. au eus (FIC
I
= 0.375), and THY and STM agains S. agalac iae
(FIC
I
= 0.5). Fo all he o he combina ions es ed, he e we e ei he addi i e e ec s o no
in e ac ion o he compounds (Table 3).
Plan s 2023,12, 1868 4 o 22
Table 1. Sensi i i y o he mic oo ganisms o he na u al p oduc s examined.
Mic oo ganism
Thymol Gallic Acid Salicylic Acid Gen isic Acid
MIC MBC MBC/MIC MIC MBC MBC/MIC MIC MBC MBC/MIC MIC MBC MBC/MIC
Acine obac e baumannii ATCC 19606 125 250 2 5000 5000 1 1250 1250 1 2500 2500 1
Bacillus sub ilis
ATCC 6633 250 500 2 5000 5000 1 1250 1250 1 5000 5000 1
En e ococcus aecalis ATCC 19433 >1000 >1000 - >5000 >5000 - 1250 1250 1 2500 2500 1
Esche ichia coli
ATCC 25922 500 500 1 >5000 >5000 - 1250 >1250 - 5000 >5000 -
Klebsiella ae ogenes ATCC 13048 250 250 1 5000 5000 1 1250 1250 1 5000 5000 1
Klebsiella pneumoniae
C6 250 250 1 >5000 >5000 - 1250 1250 1 5000 5000 1
Lis e ia monocy ogenes ATCC 7644 >1000 >1000 - >5000 >5000 - 1250 1250 1 2500 2500 1
Pas eu ella ae ogenes ATCC 27883 125 125 1 5000 5000 1 625 >1250 - 1250 1250 1
P o eus mi abilis
ATCC 35659 - - - 5000 5000 1 >1250 >1250 - 5000 5000 1
Pseudomona ae uginosa ATCC 27853 >1000 >1000 - 5000 >5000 - >1250 >1250 - 5000 >5000 -
Salmonella yphimu ium ATCC 13311 125 125 1 5000 5000 1 1250 1250 1 2500 2500 1
Se a ia ma cescens ATCC 13880 250 250 1 5000 5000 1 1250 1250 1 2500 2500 1
S aphylococcus au eus ATCC 9144 250 1000 4 >5000 >5000 - 1250 >1250 - 5000 >5000 -
S ep ococcus agalac iae ATCC 12386 250 1000 4 2500 2500 1 1250 1250 1 1250 1250 1
Concen a ion is gi en in µg/mL; -: insu icien da a o es no ca ied ou due o incompa ibili y wi h sol en s.
Table 2. MIC alues (µg/mL) o he comme cial an ibio ics ha we e examined.
Mic oo ganism Amoxicillin Ampicillin Chlo amphenicol E y h omycin Gen amycin Penicillin G S ep omycin Te acycline
A. baumannii 250 250 62.5 15.6 15.6 500 250 0.8
B. sub ilis 0.3 0.3 1.9 0.5 7.8 1.3 15.6 1.6
E. coli 7.8 7.8 7.8 250 31.3 - 125 0.8
K. ae ogenes >500 >500 31.3 62.5 0.8 - 3.9 2
K. pneumoniae 250 125 7.8 62.5 3.1 - 7.8 0.5
P. ae ogenes >500 >500 7.8 >500 6.3 - 7.8 7.8
S. agalac iae 0.2 0.2 15.6 0.5 7.8 0.2 62.5 0.2
S. au eus 0.6 0.2 31.3 0.6 15.6 1.3 62.5 62.5
S. ma cescens 125 125 125 250 6.3 - 0.5 125
S. yphimu ium 3.9 3.9 15.6 31.3 0.8 - 31.3 0.5
-: no es ed.
Plan s 2023,12, 1868 5 o 22
Table 3. FICI alues o hymol—an ibio ics combina ions.
Mic oo ganism Comme cial ABX MIC THY in
Combina ion
MIC ABX in
Combina ion FICI* In e p e a ion
A. baumannii
AMO 62.5 125 1 Addi i i y
AMP 62.5 62.5 0.75 Addi i i y
CHL 31.3 7.8 0.375 Syne gy
ERY 125 15.6 2 No in e ac ion
GTM 62.5 1 0.56 Addi i i y
PEN 125 500 2 No in e ac ion
STM 62.5 125 1 Addi i i y
B. sub ilis STM 125 7.8 1 Addi i i y
E. coli
ERY 250 7.8 0.53 Addi i i y
GTM 250 1.9 0.56 Addi i i y
STM 250 7.8 0.56 Addi i i y
K. ae ogenes CHL 15.6 15.6 0.56 Addi i i y
ERY 250 62.5 2 No in e ac ion
K. pneumoniae
AMO 250 250 2 No in e ac ion
AMP 250 125 2 No in e ac ion
ERY 250 62.5 2 No in e ac ion
S. agalac iae CHL 250 15.6 2 No in e ac ion
STM 62.5 15.6 0.5 Syne gy
S. au eus
CHL 250 31.3 2 No in e ac ion
GTM 31.3 3.9 0.375 Syne gy
STM 62.5 7.8 0.375 Syne gy
TC 250 62.5 2 No in e ac ion
S. ma cescens
AMO 250 125 2 No in e ac ion
AMP 250 125 2 No in e ac ion
CHL 125 62.5 1 Addi i i y
ERY 125 125 1 Addi i i y
TC 250 125 2 No in e ac ion
S. yphimu ium
CHL 62.5 3.9 0.75 Addi i i y
ERY 62.5 15.6 1 Addi i i y
STM 62.5 15.6 1 Addi i i y
Concen a ion is gi en in µg/mL; * FICI alues a e calcula ed acco ding Equa ion (1).
As shown in Figu e 1a, he syne gy o THY and GTM agains S. au eus p esen ed wo
poin s o syne gis ic in e ac ion, wi h FIC
I
alues o 0.375 and 0.5 (in bo h cases, he ABX
concen a ion was educed o 3.9
µ
g/mL). The combina ion o THY wi h STM (Figu e 1b,c)
showed only one poin o syne gis ic in e ac ion when es ed agains bo h S. au eus and S.
agalac iae (poin s abo e o below he lowe do ed line). The educ ion o STM concen a ion
was g ea e in he case o S. au eus. Figu e 1d shows how he combina ion o THY and
CHL p oduced wo in e ac ion poin s wi h FIC
I
= 0.375, one wi h a educ ion o CHL o
7.8
µ
g/mL and he o he o 15.6
µ
g/mL. In he cases whe e wo combina ions had he same
FICI, he one wi h he highes ABX educ ion in i s MIC was chosen o he kine ic es s.
Plan s 2023,12, 1868 6 o 22
Plan s 2023, 12, x FOR PEER REVIEW 5 o 23
FICI ≤ 1), and 12 showed no in e ac ion (1 > FICI < 2). None o he combina ions showed
an agonis ic effec s (FICI ≥ 2).
Figu e 1. Isobolog ams (blue solid line) o THY in e ac ions wi h (a) GTM; (b) STM; (c) STM; (d)
CHL ha include syne gis ic effec s. The THY concen a ion is ep esen ed on he x-axis and he
diffe en ABX concen a ions on he y-axis. The MIC alues a e loca ed on he espec i e axes (poin s
whe e he isobolog am in e sec s he coo dina e axes). The s aigh “addi ion line” (uppe dashed
line), allows o he dis inc ion o addi i e effec s (abo e he s aigh line o in i s immedia e icini y)
om syne gis ic effec s (conca e isoboles below he line). I also has a line ep esen ing he syne gy
edge (lowe doed line). The poin s abo e o below he lae line ep esen syne gis ic combina-
ions.
Table 3. FICI alues o hymol—an ibio ics combina ions.
Mic oo ganism Comme cial ABX
MIC THY in
Combina ion
MIC ABX in
Combina ion FICI * In e p e a ion
A. baumannii
AMO 62.5 125 1 Addi i i y
AMP 62.5 62.5 0.75 Addi i i y
CHL 31.3 7.8 0.375 Syne gy
ERY 125 15.6 2 No in e ac ion
GTM 62.5 1 0.56 Addi i i y
PEN 125 500 2 No in e ac ion
STM 62.5 125 1 Addi i i y
B. sub ilis STM 125 7.8 1 Addi i i y
E. coli
ERY 250 7.8 0.53 Addi i i y
GTM 250 1.9 0.56 Addi i i y
STM 250 7.8 0.56 Addi i i y
K. ae ogenes CHL 15.6 15.6 0.56 Addi i i y
ERY 250 62.5 2 No in e ac ion
K. pneumoniae
AMO 250 250 2 No in e ac ion
AMP 250 125 2 No in e ac ion
ERY 250 62.5 2 No in e ac ion
Figu e 1.
Isobolog ams (blue solid line) o THY in e ac ions wi h (
a
) GTM; (
b
) STM; (
c
) STM; (
d
) CHL
ha include syne gis ic e ec s. The THY concen a ion is ep esen ed on he x-axis and he di e en
ABX concen a ions on he y-axis. The MIC alues a e loca ed on he espec i e axes (poin s whe e
he isobolog am in e sec s he coo dina e axes). The s aigh “addi ion line” (uppe dashed line),
allows o he dis inc ion o addi i e e ec s (abo e he s aigh line o in i s immedia e icini y) om
syne gis ic e ec s (conca e isoboles below he line). I also has a line ep esen ing he syne gy edge
(lowe do ed line). The poin s abo e o below he la e line ep esen syne gis ic combina ions.
2.3. Syne gy Kine ics S udy and Time–Kill Cu es
Figu es 2a, 3a, 4a and 5a illus a e he g ow h kine ics o he syne gis ic combina ions
(blue line). The g ow h kine ics o ABXs alone ( ed lines) and THY alone (g een lines),
a di e en concen a ions, a e also shown. The cu es ha e a g ea e colou in ensi y a
highe concen a ions ( he da kes cu e is he MIC concen a ion and he ligh es one
ep esen s he syne gis ic concen a ion) o bo h ABX and THY. The con ol is ep esen ed
by a black line. Cmax, , and Tm50 alues a e included in a able below he g aphs o be e
cha ac e ize he g ow h kine ics cu es. Figu es 2b, 3b, 4b and 5b show he ime–kill cu es
ha p esen he mo ali y o he bac e ia along he g ow h kine ics. As can be seen in all o
he g ow h kine ics cu es o he syne gies (as well as hose o he espec i e MICs o ABXs
and THY), he e was comple e g ow h inhibi ion, so hese cu es a e plo ed ho izon ally
on he x-axis.
The kine ic s udy o THY and GTM syne gy agains S. au eus is shown in Figu e 2.
T ea men wi h GTM a he syne gis ic concen a ion caused a dec ease in he g ow h
a e o S. au eus (Figu e 2a), causing a delay in he exponen ial g ow h phase. Al hough
exposu e o THY alone a he syne gis ic concen a ion had li le e ec on he g ow h a e
( , Tm50) o on he maximum g ow h (Cmax) o he bac e ia, i con ibu ed o enhancing
he e ec o he ABX when combined, as he syne gis ic combina ion p oduced a o al
inhibi ion o g ow h o e he 24 h s udied. Figu e 2b shows how a 6 h, he combina ion
(blue line) had al eady killed a la ge pa o he bac e ial popula ion, esul ing in a educ ion
in he bac e ial popula ion o app oxima ely 5 log
10
CFU/mL compa ed o he con ol,
and 2.9 log
10
CFU/mL compa ed o GTM. This con i ms he bac e icidal e ec o he
combina ion and i s syne gis ic e ec .
Plan s 2023,12, 1868 7 o 22
Plan s 2023, 12, x FOR PEER REVIEW 7 o 23
effec o he ABX when combined, as he syne gis ic combina ion p oduced a o al inhibi-
ion o g ow h o e he 24 h s udied. Figu e 2b shows how a 6 h, he combina ion (blue
line) had al eady killed a la ge pa o he bac e ial popula ion, esul ing in a educ ion in
he bac e ial popula ion o app oxima ely 5 log10 CFU/mL compa ed o he con ol, and
2.9 log10 CFU/mL compa ed o GTM. This confi ms he bac e icidal effec o he combina-
ion and i s syne gis ic effec .
Figu e 2. Kine ic assays and Cmax, , and Tm50 alues o THY (g eenish cu es) and GTM ( eddish
cu es) alone and in combina ion (blue cu es) agains S. au eus; he da ke he colou o he cu e,
he highe he concen a ion applied o he wo compounds when es ed alone. Black cu es co e-
spond o he con ol. (a) G ow h kine ics assay. -: alues achie ed ou side he s udied ange. E o
ba s a e s anda d de ia ions (n = 4). (b) Time–kill cu es. E o ba s a e s anda d de ia ions (n = 3).
The kine ics o THY and STM agains S. au eus (Figu e 3a) indica es ha bo h p od-
uc s a he syne gis ic concen a ion dec ease he bac e ial g ow h bu hey ha e lile e -
ec on bac e ial Cmax alues. A hal he MIC, bo h p oduc s ma kedly slowed he g ow h
a e o he bac e ia, and nei he p oduc eached he s a iona y phase a e 24 h. In Figu e
3b, we can see ha a 24 h, he combina ion p oduced a dec ease in su i o s o 8.29 log10
Figu e 2.
Kine ic assays and Cmax, , and Tm50 alues o THY (g eenish cu es) and GTM ( eddish
cu es) alone and in combina ion (blue cu es) agains S. au eus; he da ke he colou o he cu e, he
highe he concen a ion applied o he wo compounds when es ed alone. Black cu es co espond
o he con ol. (
a
) G ow h kine ics assay. -: alues achie ed ou side he s udied ange. E o ba s a e
s anda d de ia ions (n = 4). (b) Time–kill cu es. E o ba s a e s anda d de ia ions (n = 3).
Plan s 2023,12, 1868 8 o 22
Plan s 2023, 12, x FOR PEER REVIEW 8 o 23
CFU/mL compa ed o he con ol, and 8.14 log10 CFU/mL compa ed o STM, hus demon-
s a ing he bac e icidal and syne gis ic effec s, espec i ely.
Figu e 3. Kine ic assays and Cmax, , and Tm50 alues o THY (g eenish cu es) and STM ( eddish
cu es) alone and in combina ion (blue cu es) agains S. au eus; he da ke he colou o he cu e,
he highe he concen a ion applied o he wo compounds when es ed alone. Black cu es co e-
spond o he con ol. (a) G ow h kine ics assay. -: alues achie ed ou side he s udied ange. E o
ba s a e s anda d de ia ions (n = 4). (b) Time–kill cu es. E o ba s a e s anda d de ia ions (n = 3).
Figu e 4 shows he kine ics o THY and STM agains S. agalac iae. The syne gis ic
combina ion p oduced a o al inhibi ion o g ow h h oughou he 24 h s udied (Figu e
4a), whe eas bo h p oduc s alone only sligh ly affec ed he Cmax (cu es e y simila o
he con ol). I we look a he syne gy cu e (blue) in Figu e 4b, i can be seen ha he
combina ion was able o kill bac e ia e y quickly (4 h), wi h a educ ion in he bac e ial
popula ion o 3.6 log10 CFU/mL compa ed o he con ol, and 3.17 log10 CFU/mL compa ed
o STM.
Figu e 3.
Kine ic assays and Cmax, , and Tm50 alues o THY (g eenish cu es) and STM ( eddish
cu es) alone and in combina ion (blue cu es) agains S. au eus; he da ke he colou o he cu e, he
highe he concen a ion applied o he wo compounds when es ed alone. Black cu es co espond
o he con ol. (
a
) G ow h kine ics assay. -: alues achie ed ou side he s udied ange. E o ba s a e
s anda d de ia ions (n = 4). (b) Time–kill cu es. E o ba s a e s anda d de ia ions (n = 3).
Plan s 2023,12, 1868 9 o 22
Plan s 2023, 12, x FOR PEER REVIEW 9 o 23
Figu e 4. Kine ic assays and Cmax, , and Tm50 alues o THY (g eenish cu es) and STM ( eddish
cu es) alone and in combina ion (blue cu es) agains S. agalac iae; he da ke he colou o he
cu e, he highe he concen a ion applied o he wo compounds when es ed alone. Black cu es
co espond o he con ol. (a) G ow h kine ics assay. -: alues achie ed ou side he s udied ange.
E o ba s a e s anda d de ia ions (n = 4). (b) Time–kill cu es. E o ba s a e s anda d de ia ions
(n = 3).
The g ow h cu es o A. baumannii a e shown in Figu e 5. The esul s show ha bo h
compounds a sub-MIC concen a ions affec ed he Cmax o he bac e ia in a concen a-
ion-dependen manne (Figu e 5a). The ime–kill cu e o he syne gy (Figu e 5b) e-
ealed a educ ion in he bac e ial popula ion o 8.15 log10 CFU/mL in compa ison o he
con ol, and 7.42 log10 CFU/mL in compa ison o CHL a 24 h.
Figu e 4.
Kine ic assays and Cmax, , and Tm50 alues o THY (g eenish cu es) and STM ( eddish
cu es) alone and in combina ion (blue cu es) agains S. agalac iae; he da ke he colou o he
cu e, he highe he concen a ion applied o he wo compounds when es ed alone. Black cu es
co espond o he con ol. (
a
) G ow h kine ics assay. E o ba s a e s anda d de ia ions (n = 4).
(b) Time–kill cu es. E o ba s a e s anda d de ia ions (n = 3).
The kine ics o THY and STM agains S. au eus (Figu e 3a) indica es ha bo h p oduc s
a he syne gis ic concen a ion dec ease he bac e ial g ow h bu hey ha e li le e ec on
bac e ial Cmax alues. A hal he MIC, bo h p oduc s ma kedly slowed he g ow h a e o
he bac e ia, and nei he p oduc eached he s a iona y phase a e 24 h. In Figu e 3b, we
can see ha a 24 h, he combina ion p oduced a dec ease in su i o s o 8.29 log
10
CFU/mL
compa ed o he con ol, and 8.14 log
10
CFU/mL compa ed o STM, hus demons a ing
he bac e icidal and syne gis ic e ec s, espec i ely.
Figu e 4shows he kine ics o THY and STM agains S. agalac iae. The syne gis ic
combina ion p oduced a o al inhibi ion o g ow h h oughou he 24 h s udied (Figu e 4a),
whe eas bo h p oduc s alone only sligh ly a ec ed he Cmax (cu es e y simila o he
con ol). I we look a he syne gy cu e (blue) in Figu e 4b, i can be seen ha he
combina ion was able o kill bac e ia e y quickly (4 h), wi h a educ ion in he bac e ial
Plan s 2023,12, 1868 16 o 22
M07-A9 (2018). In o de o achie e a mo e accu a e measu emen o mic obial g ow h, he
abso bance o each well was also measu ed a 625 nm using a mic opla e eade . Na u al
p oduc ac i i y was classi ied as s ong (<400
µ
g/mL), mode a e (400–800
µ
g/mL), o
weak (>800
µ
g/mL) [
44
]. In addi ion, o na u al p oduc s, he MBC was also s udied; his
is de ined as he lowes concen a ion a which all bac e ia a e killed. Fo i s de e mina ion,
a 10
µ
L aliquo was aken om each non-g ow h column o he incuba ed 96-well pla es and
inocula ed on o an aga pla e. The pla es we e subsequen ly cul u ed o 24 h a he op imal
g ow h empe a u e o each bac e ial s ain (Table S1) and moni o ed o any g ow h. The
MBC/MIC a io de e mines he bac e icidal o bac e ios a ic e ec o he p oduc on a
bac e ium. An imic obial subs ances a e conside ed o ha e bac e icidal ac i i y when
MBC/MIC ≤4 [41–43]; he e o e, in his s udy he same c i e ion was ollowed o THY.
4.4. De e mina ion o he P oduc Combina ion Beha iou
4.4.1. Checke boa d Assays and F ac ional Inhibi o y Concen a ion Index
Selec ion o he combina ions o be examined (among all possible ones) was made
acco ding o he ollowing c i e ia: (1) he na u al p oduc should ha e he s onges
an imic obial ac i i y among he ou es ed; (2) he ABX should ha e a MIC > 10
µ
g/mL
( his inc eases he impo ance o educing i s e ec i e dose han i i was al eady low).
The checke boa d me hod was used o measu e po en ial syne gies [
14
,
122
,
123
] be-
ween THY (d ug A) and he es ed ABXs (d ug B). Fo he mic odilu ion checke boa d es ,
THY was se ially dilu ed e ically om columns 1 o 7 o 96-well mic o i e pla es. The
co esponding ABX was hen se ially dilu ed ho izon ally om ows A o G o he pla e,
bo h p oduc s s a ing wi h a s ock dilu ion co esponding o ou imes he MIC ob ained
o ha p oduc agains a speci ic bac e ium.
Nex , he pla es we e inocula ed wi h bac e ial suspension adjus ed o he McFa land
s anda d p epa ed as discussed in Sec ion 4.3. The pla es we e incuba ed a he op imal
empe a u e o each s ain (Table S1) o 24 h and hen he abso bance (625 nm) was
measu ed o e alua e he bac e ial g ow h in he same way as desc ibed in Sec ion 4.3.
To es he ype o in e ac ion be ween he d ug combina ions, he FIC
I
was calcula ed
o each combina ion, as ollows [123,124]:
FICI=FICA+FICB=MICA+B
MICA
+MICB+A
MICB
(1)
whe e FIC
A
is he MIC o d ug A (na u al p oduc ) in he p esence o he comme cial ABX
(d ug B) (MIC
A+B
) di ided by he MIC o d ug A alone (MIC
A
). FIC
B
is he MIC o d ug B in
he p esence o d ug A (MIC
B+A
) di ided by he MIC o he d ug B alone (MIC
B
). Acco ding
o he Eu opean Commi ee on an imic obial suscep ibili y es ing guidance [
125
], a FIC
I
alue
≤
0.5 indica es syne gy; be ween 0.5 and 1 indica es addi i i y, whe eas om >1
o 2, he e is “no in e ac ion” be ween he agen s; FIC
I
alues
≥
2 imply an agonis ic
e ec s [126,127].
4.4.2. Isobolog ams
An isobolog am (Figu e 1) is a ep esen a ion o he in e ac ion be ween wo sub-
s ances. Isobolog ams ha e been used o display he esul s o he checke boa d es s [
128
].
Unlike he g ow h kine ics o ime–kill cu es, his ep esen a ion allows o he s udy o he
in e ac ion o ABXs and he na u al p oduc a se e al es ed concen a ions. Only isobolo-
g ams ha showed in he checke boa d es one o mo e in e ac ions wi h a
FICI≤0.5
ha e
been plo ed.
4.4.3. G ow h Kine ics Tes s
Fo a be e in e p e a ion o he bac e ios a ic e ec s o syne gis ic combina ions
( hose wi h a FIC
I≤
0.5), g ow h kine ics es s we e ca ied ou . Bac e ial cul u es we e
adjus ed o he McFa land s anda d, as p e iously desc ibed (Sec ion 4.3). They we e
hen exposed o di e en concen a ions (MIC and suble hal concen a ions) o na u al
Plan s 2023,12, 1868 17 o 22
p oduc s, comme cial ABX, and a combina ion o bo h (acco ding o he esul s ob ained
in he checke boa d es ) in a 96-well mic opla e. They we e hen incuba ed a he co e-
sponding empe a u e o each bac e ium and abso bance measu emen s we e aken e e y
hou o 24 h. The esul s we e plo ed as abso bance s. ime o ob ain g ow h cu es
(Figu es 2a, 3a, 4a and 5a). All expe imen s we e pe o med in quad uplica e. Kine ic
cu es we e i ed o a logis ic model (Equa ion (2)) o sigmoid mic obial g ow h [
129
]
wi h he Excel Sol e add-in (Mic oso 365):
Abso bance =Cmax
1+eb− (2)
whe e Cmax is he ca ying capaci y, meaning he maximum achie able popula ion densi y,
is he in insic a e o he popula ion inc ease, and bis a i ing pa ame e . Cmax, , and
Tm50 ( ime in which hal o he ca ying capaci y is eached) we e calcula ed o cha ac e ize
he kine ics o he di e en cu es (see Figu es 2a, 3a, 4a and 5a).
4.4.4. Time–Kill Cu es
To s udy he bac e icidal p ope ies o he combina ions, ime–kill cu es we e ob-
ained acco ding o Hu e al. [
126
] (Figu es 2b, 3b, 4b and 5b). To his end, bac e ial cul u es
(adjus ed o he McFa land s anda d, as p e iously desc ibed) we e exposed o THY and
ABXs (alone and in combina ion) o a inal olume o 10 mL, a concen a ions o he
selec ed syne gis ic combina ions. Con ol ubes wi hou an imic obial agen s we e also
included. Bac e ial cul u es exposed o he di e en concen a ions we e incuba ed a 37
◦
C
o 24 h. Samples (100
µ
L) we e collec ed a 0, 2, 4, 6, and 24 h. Se ial dilu ions o each
sample we e hen p epa ed om 10
−1
o 10
−7
, and 10
µ
L o each dilu ion was seeded
on aga pla es in iplica e. Following o e nigh incuba ion a 37
◦
C, he colonies we e
coun ed. The esul s we e plo ed as log
10
CFU (y-axis) s. ime (x-axis) o ob ain he
ime–kill cu es. A p oduc was conside ed bac e icidal when he dec ease in he numbe
o su i o s was g ea e han 3 log
10
CFU/mL- old compa ed o he con ol. In addi ion,
syne gy was de ined as a
≥
2 log
10
CFU/mL- old dec ease by he combina ion compa ed o
he mos ac i e single agen [130].
5. Conclusions
In his s udy, he an imic obial p ope ies o THY and h ee o he p oduc s o na -
u al o igin (GA, SA, and GEA) we e assessed on 14 G am-posi i e and G am-nega i e
pa hogenic bac e ia. THY p o ed ac i e agains 10 o hem. THY also showed syne gis ic
e ec s when combined wi h GTM, STM, and CHL, and he eac ion o some o hese agains
bac e ia, we e conside ed as c i ical (A. baumannii) and o high p io i y (S. au eus) by he
WHO, educing he MIC o hese ABXs by 75% o 87.5%. The s udy o he g ow h kine ics
oge he wi h he ime–kill cu es seems o indica e ha he possible ole o THY in he
syne gies is o acili a e he access o he ABX, p obably by al e ing he bac e ial en elope.
The esul s p esen ed in his wo k show ha THY as a p oduc o be explo ed as
an RMA, which may allow o a educ ion in he consump ion o ABXs in clinical and
e e ina y se ings. This could con ibu e o educing hei impac on he en i onmen and
he gene a ion o esis ance, in line wi h he One Heal h s a egy.
The iden i ica ion and cha ac e iza ion o hese syne gies is he i s s ep in a se ies o
be made owa ds a heal hie li e and a sa e en i onmen . Al hough he ABXs a e al eady
ma ke ed and THY is conside ed by he EPA and FDA as a sa e p oduc , he mode o
applica ion ha would be he sa es and mos e ec i e o ea ing human and e e ina ian
in ec ions o hese h ee bac e ia is s ill a challenge.
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a : h ps:
//www.mdpi.com/a icle/10.3390/plan s12091868/s1, Table S1: Mic oo ganisms’ e e ence and
cul u e condi ions acco ding o ATCC da ashee s o each mic oo ganism. Figu e S1: Chemical
s uc u es o he es ed na u al p oduc s.
Plan s 2023,12, 1868 18 o 22
Au ho Con ibu ions:
Concep ualiza ion, M.R.P.-O.; me hodology, C.G. and M.R.P.-O.; alida ion,
C.G., E.L. and M.R.P.-O.; o mal analysis, C.G.; in es iga ion, C.G., A.V. and M.R.P.-O.; esou ces,
M.R.P.-O.; da a cu a ion, C.G. and A.V.; w i ing—o iginal d a p epa a ion, C.G. and M.R.P.-O.;
w i ing— e iew and edi ing, E.L., D.B. and M.R.P.-O.; isualiza ion, C.G. and A.V.; supe ision, E.L.
and M.R.P.-O.; p ojec adminis a ion, M.R.P.-O.; unding acquisi ion, M.R.P.-O. All au ho s ha e
ead and ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by Cá ed a No al ia, Uni e sidad San Jo ge and Founda ion Sabadell.
Da a A ailabili y S a emen : Da a is con ained wi hin he a icle o supplemen a y ma e ial.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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