CLINICAL TRIAL
published: 23 No embe 2017
doi: 10.3389/ micb.2017.02162
F on ie s in Mic obiology | www. on ie sin.o g 1No embe 2017 | Volume 8 | A icle 2162
Edi ed by:
Sa a Ma ía So o,
ISGlobal, Spain
Re iewed by:
Jens K e h,
O egon Heal h and Science Uni e si y,
Uni ed S a es
Thomas Thu nhee ,
Uni e si y o Zu ich, Swi ze land
*Co espondence:
Inmaculada Tomás
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
In ec ious Diseases,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 02 Augus 2017
Accep ed: 20 Oc obe 2017
Published: 23 No embe 2017
Ci a ion:
Quin as V, P ada-López I, Ca ei a MJ,
Suá ez-Quin anilla D, Balsa-Cas o C
and Tomás I (2017) In Si u
An ibac e ial Ac i i y o Essen ial Oils
wi h and wi hou Alcohol on O al
Bio ilm: A Randomized Clinical T ial.
F on . Mic obiol. 8:2162.
doi: 10.3389/ micb.2017.02162
In Si u An ibac e ial Ac i i y o
Essen ial Oils wi h and wi hou
Alcohol on O al Bio ilm: A
Randomized Clinical T ial
Vic o Quin as1, Isabel P ada-López1, Ma ía J. Ca ei a2, Da id Suá ez-Quin anilla1,
Ca los Balsa-Cas o1and Inmaculada Tomás1*
1O al Sciences Resea ch G oup, Depa men o Su ge y and Medical Su gical Special ies, School o Medicine and Den is y,
Heal h Resea ch Ins i u e o San iago (IDIS), Uni e sidade de San iago de Compos ela, San iago de Compos ela, Spain,
2Cen o Singula de In es igación en Tecnoloxías da In o mación, Heal h Resea ch Ins i u e o San iago (IDIS), Uni e sidade
de San iago de Compos ela, San iago de Compos ela, Spain
Cu en ly, he e is li le e idence on he in si u an ibac e ial ac i i y o essen ial oils (EO)
wi hou alcohol. This s udy aimed o e alua e in si u he subs an i i y and an iplaque
e ec on he plaque-like bio ilm (PL-bio ilm) o wo solu ions, a adi ional o mula ion ha
con ains EO wi h alcohol (T-EO) and an alcohol- ee o mula ion o EO (A -EO). Eigh een
heal hy adul s pe o med a single mou hwash o : T-EO, A -EO, and s e ile wa e (WATER)
a e wea ing an indi idualized disk-holding splin o 2 days. The bac e ial iabili y (BV)
and hickness o he PL-bio ilm we e quan i ied a baseline, 30 s, and 1, 3, 5, and 7 h
pos - insing (Tes 1). Subsequen ly, each olun ee wo e he splin o 4 days, applying
wo daily mou hwashes o : T-EO, A -EO, and WATER. The BV, hickness, and co e ing
g ade (CG) o he PL-bio ilm we e quan i ied (Tes 2). Samples we e analyzed by con ocal
lase scanning mic oscopy a e s aining wi h he LIVE/DEAD®BacLigh TM solu ion. To
conduc he compu a ions o he BV au oma ically, a Ma lab oolbox called Den ius
Bio ilm was de eloped. In es 1, bo h EO an isep ics had a simila an ibac e ial e ec ,
educing BV a e a single inse compa ed o he WATER, and keeping i below baseline
le els up o 7 h pos - inse (P<0.001). The mean hickness o he PL-bio ilm a e insing
was no a ec ed by any o he EO o mula ions and anged om 18.58 o 20.19 µm.
A e 4 days, he T-EO and A -EO solu ions we e signi ican ly mo e e ec i e han he
WATER, educing he BV, hickness, and CG o he PL-bio ilm (P<0.001). Al hough,
bo h EO an isep ics p esen ed a simila bac e icidal ac i i y, he A -EO inses led o mo e
signi ican educ ions in he hickness and CG o he PL-bio ilm han he T-EO inses
( hickness =7.90 s. 9.92 µm, P=0.012; CG =33.36 s. 46.61%, P=0.001). In
conclusion, bo h essen ial oils an isep ics had e y high immedia e an ibac e ial ac i i y
and subs an i i y in si u on he 2-day PL-bio ilm a e a single mou hwash. In he 4-day
PL-bio ilm, bo h essen ial oils o mula ions demons a ed a e y good an iplaque e ec in
si u, al hough he alcohol- ee o mula pe o med be e a educing he bio ilm hickness
and co e ing g ade.
Keywo ds: an i-in ec i e agen s, local, bio ilm, den al plaque, essen ial oils, mic oscopy, luo escence
This Clinical T ial was egis e ed a clinical ials.go wi h he numbe NCT03146390 URL: h ps://clinical ials.
go /c 2/show/NCT03146390.
Quin as e al. Essen ial Oils Ac i i y on Bio ilm
INTRODUCTION
The accumula ion o bac e ial bio ilms on oo h su aces esul s
in wo o he mos p e alen in ec ious diseases—ca ies and
pe iodon i is. Al hough p e en ion and con ol o hese diseases
can be achie ed by he daily mechanical emo al o bio ilms,
many people a e ei he unable o unwilling o p ac ice hese
p ocedu es as egula ly o as e icien ly as necessa y. The e is,
he e o e, g ea in e es in he possibili y o using chemicals
o eplace o augmen , mechanical p e en i e, and he apeu ic
p ocedu es (Ma sh and B adshaw, 1993; en Ca e and Ma sh,
1994; Newman, 1996).
The ac i e ing edien s p esen in he mou hwashes ha a e
mos commonly used in he o al ca i y include: chlo hexidine,
combina ions o essen ial oils (EO), iclosan, ce ylpy idinium
chlo ide, and a ious me al sal s such as zinc compounds and
s annous luo ide. O all o hese, chlo hexidine mou hwashes
a e conside ed o be he gold s anda d, as hey ha e hus a
been he mos e ec i e in mic obiological and clinical s udies
(McDonnell and Russell, 1999; Tomás e al., 2008; on Ohle
e al., 2010). Howe e , hei well-known undesi able seconda y
e ec s, mainly a e egula use (Van S ydonck e al., 2012),
ha e led o he scien i ic communi y explo ing he exis ence
o e ec i e al e na i es, especially when con inuous daily use is
equi ed. Acco dingly, EO ha e been ound o be as e ec i e
as chlo hexidine a con olling gingi al in lamma ion a e 6
mon hs o use, al hough he la e pe o ms be e a educing
plaque le els (Van Leeuwen e al., 2011; Neely, 2012).
EO a e composed o a wide a ie y o p oduc s. As a
consequence, hei an imic obial ac i i y is ela ed o hei
composi ion, con igu a ion, amoun , and possible in e ac ions
(Lis-Balchin e al., 1998). The adi ional o mula ion con aining
EO (T-EO) (Lis e ine R
Men olTM, Johnson & Johnson)1
a e a complex mix o phenolic compounds combined wi h
a ious EO: 0.092% eucalyp ol, 0.064% hymol, 0.06% me hyl-
salicyla e, and 0.042% men hol. All o hese a e included in
a hyd oalcoholic ehicle con aining be ween 21.6 and 26.9%
alcohol (Fine, 1988). As a esul , T-EO con ains e hanol, which
is a chemical compound used o dissol e and s abilize he
nume ous subs ances p esen in he inse. The concen a ion
o e hanol p esen in he T-EO inses is mo e han 20%. Such
amoun s ha e been ound o be su icien o dissol e he EO,
bu insu icien when i comes o ha ing a di ec an ibac e ial
e ec (Sissons e al., 1996; Ma che i e al., 2009). In ac , he
manu ac u e p esen s he alcohol con ained in he inse (21.6%)
as being, in e alia, an inac i e ing edien in i s o mula. O e he
yea s, he use o e hanol in mou hwashes, as well as hei e ec s
on he su aces o composi e es o a ions (Penugonda e al., 1994)
and hei possible ole in he de elopmen o o opha yngeal
cance , ha e been discussed (Smigel, 1991; Llewelyn, 1994).
A di ec cause-and-e ec co ela ion be ween he de elopmen
1Lis e ine Cool Min Composi ion. A ailable online a : h ps://www.lis e ine.com/
mou hwash/an isep ic/lis e ine-cool-min -mou hwash (Accessed on Sep embe
14, 2017). Lis e ine Cool Min Ze o alcohol composi ion. A ailable online a :
h ps://www.lis e ine.com/p oduc s/lis e ine-ze o/lis e ine-cool-min -ze o-
alcohol#p oduc -o e iew (Accessed on No embe 1, 2017).
o o opha yngeal ca cinoma and he use o alcohol-con aining
inses has no been demons a ed (Moazzez e al., 2011; Bagán
e al., 2012; Gandini e al., 2012), and p obably ne e will
be (a leas by epidemiological s udies; Lachenmeie , 2012).
Howe e , i is conside ed desi able o elimina e e hanol om
daily mou hwashes, especially o pedia ic popula ions and
pa ien s a highe isk o o al cance (McCullough and Fa ah,
2008; La Vecchia, 2009). Fu he mo e, he ac ha he alcohol
is p esen has mean ha some clinical p ac i ione s do no
p esc ibe he T-EO due o his con o e sy (Vlachojannis e al.,
2013). All o his has led o he de elopmen o a new alcohol-
ee o mula ion o EO (A -EO) (Lis e ine R
Ze oTM, Johnson &
Johnson)1.
The composi ion o A -EO is he same ega ding hei ac i e
ing edien s (eucalyp ol, hymol, me hyl salicyla e, and men hol),
bu sodium luo ide has been added o he mix u e. Some
di e ences a e ound in hei inac i e ing edien s. These a e
based on he alcohol con ained in he T-EO solu ion, which is
no p esen in he A -EO inse, and he p esence o p opylene
glycol, sodium lau yl sul a e and suc alose in he A -EO solu ion,
bu no he T-EO inse.
Two di e en concep s should be aken in o accoun o
measu e he e icacy o an isep ics agains den al plaque: he
subs an i i y and he an iplaque e ec . The subs an i i y o an
o al an isep ic is de ined as he p olonged adhe ence o o al
su aces ( ee h and mucosa) and i s slow elease a e ec i e
doses which gua an ee he con inua ion o he an imic obial
ac i i y (Manau Na a o and Guasch Se a, 2003). The mo e
subs an i i y an o al an isep ic has, he be e . To s udy his in
si u, he mos popula models a e hose ha analyze he e ec
ha a single mou hwash has on a bio ilm o mo e han 24 h
(Ga cía-Caballe o e al., 2013; Quin as e al., 2015b).
The second concep ha should be s udied, he an iplaque
e ec , is de ined as he capaci y ha an agen has o p e en
he o ma ion o bac e ial agg ega es (plaque) on o al su aces.
To s udy his in si u e ec , models s a om a baseline sample
wi h le els o plaque nea o ze o in o de o assess he powe
o he an isep ic when i comes o educing he o ma ion
o bac e ial plaque (no mally den al plaque) compa ed o he
con ol. A 6-mon h clinical s udy using a de e mina e an iplaque
agen is necessa y o ag an an isep ic as e ec i e (Council on
Den al The apeu ics, 1986). Howe e , in he li e a u e, he e is
an es ablished model o 4 days o plaque eg ow h wi h which
au ho s can assess he inhibi o y ac i i y ha di e en agen s
ha e; u he mo e, his de e mines he ela i e e icacy o he
di e en o mula ions as being p edic i e o he an iplaque e ec
o an an isep ic (Singh e al., 2013; Quin as e al., 2015a).
In addi ion, ano he impo an me hodological aspec in he
in si u s udy o an o al an isep ic is he need o conse e he o al
bio ilm in ac a all s ages, namely he o ma ion, collec ion and
analysis o he o al samples. The goal is o no in e e e wi h he
delica e h ee-dimensional s uc u e o he o al bio ilm, which
has been p o ed o be essen ial in e ms o he esis ance o
he e ec s o an an imic obial agen (Wood e al., 2000; Bey h
e al., 2010). Fo hese easons, he use o in ao al disks held
in specially designed appa a us o bio ilm o ma ion combined
wi h he applica ion o con ocal lase scanning mic oscopy has
F on ie s in Mic obiology | www. on ie sin.o g 2No embe 2017 | Volume 8 | A icle 2162
Quin as e al. Essen ial Oils Ac i i y on Bio ilm
p o ed o be ex emely aluable when i comes o analyzing he
o al bio ilm in i s in ac , hyd a ed na u al s a e (Dong e al., 2010;
Gosau e al., 2010; Gu e al., 2012; Ga cía-Caballe o e al., 2013;
Quin as e al., 2015a,b).
As A -EO solu ion has come o he ma ke , i seems
app op ia e o compa e hei an ibac e ial e ec s o hose o
adi ional mou hwashes. Al hough, he e a e some s udies
e alua ing hese e ec s o T-EO and A -EO (Ma che i e al.,
2009, 2011, 2017a,b; Cha les e al., 2012; Pizzo e al., 2013; Ulku
e al., 2013), none o hem ha e assessed and compa ed hei
subs an i i y and an iplaque impac in an in si u model o a non-
des uc u ed o al bio ilm (PL-bio ilm). Fo his eason, he aim o
he p esen s udy was o compa e he in si u an ibac e ial ac i i y
(immedia e e ec , subs an i i y and an iplaque e ec ) o EO wi h
and wi hou alcohol on he PL-bio ilm.
MATERIALS AND METHODS
This esea ch is a andomized, double-blind, c osso e s udy
o he an ibac e ial and an iplaque e icacy o wo a ailable EO
solu ions: a adi ional o mula o EO wi h alcohol (T-EO) and an
alcohol- ee o mula o EO (A -EO). The suppo ing CONSORT
checklis is a ailable as suppo ing in o ma ion (Supplemen a y
Table 1). The s udy ecei ed he app o al o he Clinical Resea ch
E hics Commi ee o Galicia (numbe 2014/008) and was
egis e ed a clinical ials.go wi h he numbe NCT03146390.
URL: h ps://clinical ials.go /c 2/show/NCT03146390.
The “a p io i” sample size calcula ion was pe o med using
he p og am G∗Powe 3.1.5 (Faul e al., 2007). The ollowing
s a is ical c i e ia we e es ablished: (1) an e ec size o 0.7; (2)
an alpha e o o 0.05; and (3) a s a is ical powe o 80%. A
sample size o 19 subjec s was equi ed by hese c i e ia and
he applica ion o he Wilcoxon signed- ank es o analyze he
di e ences in he mic oscopic pa ame e s be ween wo insing
p o ocols.
The pa icipan s we e selec ed among den al s uden s
a he School o Medicine and Den is y o San iago de
Compos ela (Uni e sidade de San iago de Compos ela, Spain),
whe e olun ee en ollmen was sough by in i ing esponses
o ad e isemen s displayed in he acul y hall asking o
pa icipa ion in a esea ch s udy. All hese olun ee s we e
assessed by he same ained clinician o ensu e ha hey
ul illed all he inclusion and exclusion c i e ia ha we e applied
in ou g oup’s p e ious publica ions (Ga cía-Caballe o e al.,
2013; P ada-López e al., 2015a,b; Quin as e al., 2015a,b). The
inclusion c i e ia we e he ollowing: sys emically heal hy adul
olun ee s aged be ween 20 and 45 wi h a good o al heal h
s a us, namely a minimum o 24 pe manen ee h wi h no
e idence o gingi i is o pe iodon i is (Communi y Pe iodon al
Index sco e =0; Wo ld Heal h O ganiza ion, 2013) and an
absence o un ea ed ca ies a he s a o he s udy. The
ollowing exclusion c i e ia we e applied: smoke o o me
smoke , he p esence o den al p os heses o o hodon ic de ices,
an ibio ic ea men , o ou ine use o o al an isep ics in he
p e ious 3 mon hs, and he p esence o any sys emic disease
ha could al e he p oduc ion o composi ion o sali a. Be o e
he s a o each es o expe imen , a ull mou h scaling wi h
ul asonic ins umen s and ee h polishing wi h a ubbe cup a e
den al disclosu e we e pe o med by he same ained clinician
on all he selec ed pa icipan s (Figu e 1). W i en in o med
consen was ob ained om all he olun ee s. To achie e he
aims o he s udy, all he pa icipan s pe o med wo di e en
es s.
To es he an ibac e ial ac i i y o he wo EO solu ions, an
in si u model o PL-bio ilm g ow h was used. An indi idualized
he moplas ic splin called in ao al disk-holding splin (IDODS)
(Ga cía-Caballe o e al., 2013; P ada-López e al., 2013, 2015a,b,c,
2016; Quin as e al., 2015a,b) wi h a capaci y o hold a o al o six
glass disks was made o each o he olun ee s.
Tes 1 (Subs an i i y)
The i s expe imen consis ed o e alua ing he immedia e
an ibac e ial e ec and subs an i i y o he T-EO and A -EO
solu ions. The olun ee s wo e an IDODS o 48 h o enable
g ow h o he PL-bio ilm. They we e allowed o emo e i om
he o al ca i y only du ing meals and o pe o m o al hygiene
measu es (when i had o be s o ed in a p o ided opaque
con aine in humid condi ions). In o de o no dis u b he
o ma ion o he PL-bio ilm, he olun ee s could no use any
oo hpas e o mou hwash as a complemen o he mechanical
emo al o bac e ial plaque.
A e 48 h, he glass disks we e wi hd awn one by one om
he splin om each olun ee ( om igh o le in a dis al-
mesial di ec ion) a baseline, 30 s, and 1, 3, 5, 7h a e pe o ming
he di e en mou hwashes. These mou hwashes we e p ac iced
unde he in es iga o ’s supe ision wi h he IDODS p esen in
he o al ca i y, and hey we e:
1A) A single, 30-s mou hwash wi h 20 mL o s e ile wa e
(nega i e con ol) (M-WATER).
-OR
1B) A single, 30-s mou hwash wi h 20 mL o a adi ional
EO o mula ion (Lis e ine R
Men olTM, Lis e ine R
, Johnson &
Johnson, Mad id, Spain) (M-T-EO).
-OR
1C) A single, 30-s mou hwash wi h 20 mL o an alcohol-
ee EO solu ion (Lis e ine R
ZEROTM, Lis e ine R
, Johnson &
Johnson, Mad id, Spain) (M-A -EO).
On he day o he expe imen , he olun ee s we e no allowed
o ea o d ink du ing he es s. Collec ion o he di e en
PL-bio ilm samples s a ed a 11:50 a.m. (baseline sample) and
inished a 7:00 p.m. ( he inal sample was ob ained 7 h a e using
he mou hwash).
Using an in e ne -based, balanced andomiza ion sys em
(Dallal)2, which indica ed he mou hwash ha each subjec
should use i s , second and hi d, all he olun ee s used he
h ee mou hwashes, wi h a es pe iod o 2 weeks be ween each
applica ion (Figu e 1).
Tes 2 (An iplaque E ec )
The second expe imen consis ed o e alua ing he an iplaque
e ec o bo h EO o mulas. Du ing 96 h, each olun ee wo e
he splin s wi h he glass disks, wi hd awing hem om hei
o al ca i y only du ing meals ( hey we e s o ed in an opaque
2A ailable online a : www. andomiza ion.com (Accessed on Sep embe 03, 2017).
F on ie s in Mic obiology | www. on ie sin.o g 3No embe 2017 | Volume 8 | A icle 2162
Quin as e al. Essen ial Oils Ac i i y on Bio ilm
FIGURE 1 | P o ocol o he s udy.
con aine in humid condi ions) and o pe o m o al hygiene
p ocedu es in ol ing only he mechanical emo al o bac e ial
plaque wi h wa e , wi hou he use o any oo hpas e o
mou hwash.
Using he pe mi ed mechanical o al hygiene measu es
(wi hou he IDODS), he olun ee s pe o med he di e en
p o ocols based on he manu ac u e s’ ins uc ions (wi h he
IDODS in he o al ca i y) o e 4 days in he mo ning (8.30)
F on ie s in Mic obiology | www. on ie sin.o g 4No embe 2017 | Volume 8 | A icle 2162
Quin as e al. Essen ial Oils Ac i i y on Bio ilm
a e b eak as and a nigh (22.00) a e dinne . These p o ocols
we e:
2A) A 30-s mou hwash wi h 20 mL o s e ile wa e (nega i e
con ol) (4D-WATER).
-OR
2B) A 30-s mou hwash wi h 20 mL o a adi ional EO
o mula ion (Lis e ine R
Men olTM, Lis e ine R
, Johnson &
Johnson, Mad id, Spain) (4D-T-EO).
-OR
2C) A 30-s mou hwash wi h 20 mL o an alcohol- ee
EO solu ion (Lis e ine R
ZEROTM, Lis e ine R
, Johnson &
Johnson, Mad id, Spain) (4D-A -EO).
The collec ion o he samples was ca ied ou indi idually a
8 a.m. in he mo ning so ha hose o each olun ee we e
analyzed on di e en days. I was de e mined ha a minimum
o 10 h should ha e elapsed since he las use o he mou hwash
he p e ious nigh .
In his es , mou hwashes ca ied ou by he olun ee s we e
no supe ised, bu hey we e ins uc ed o use a measu ed
olume o he alloca ed solu ion. To assess he subjec ’s
compliance wi h he insing p o ocol, he bo les con aining he
inse we e weighed be o e hey we e gi en o he olun ee s. A e
he 4-day pe iod, hey we e asked o b ing he bo les back wi h
he emaining mou hwash, and hese we e weighed again.
Using an in e ne -based, balanced andomiza ion sys em
(Dallal), which indica ed he ising p o ocol ha each subjec
should use i s , second and hi d, all he olun ee s used he
h ee egimes, wi h a es pe iod o 2 weeks be ween each p o ocol
(Figu e 1).
P ocessing o he Samples o he
PL-Bio ilm
As he glass disks we e emo ed om he splin , hey we e
immedia ely imme sed in 100 µL o a luo escence solu ion
o LIVE/DEAD R
BacLigh TM and kep in a da k chambe
a oom empe a u e o 15 min. Mic oscope obse a ions
we e pe o med by a single in es iga o who was unawa e
o he s udy design using a Leica TCS SP2 lase scanning
spec al con ocal mic oscope (Leica Mic osys ems Heidelbe g
GmbH, Mannheim, Ge many) wi h an HCX APOL 63x/0.9
wa e -imme sion lens.
Fou ields conside ed o be ep esen a i e o he en i e y o
he samples we e selec ed by an obse e who was blind o he
s udy’s condi ions. Fluo escence emission was de e mined in a
se ies o XY images in which each image co esponded o each
o he Z posi ions (dep h). The op ical sec ions we e scanned
in 1 µm sec ions om he su ace o he bio ilm o i s base,
measu ing he maximum hickness o he ield and subsequen ly
he mean hickness o he bio ilm o he co esponding sample.
The maximum bio ilm hickness o each ield was di ided in o
h ee zones o equi alen laye s: he ou e laye (laye 1), he
middle laye (laye 2), and he inne laye (laye 3).
The cap u e o he da a was done wi h he same se ings in
all cases. The spa ial scan mode (XYZ) and he 1,024 ×1,024
pixels scan o ma esolu ion we e used. The A gon-ion and
DPSS lase we e used a a 13 and 78% o maximum in ensi y,
espec i ely. The alues o he pinhole, zoom and scan speed
we e 121.58 mic ons, 1 and 400 Hz, espec i ely. The only alues
ha we e di e en depending on he sample we e he o se
( ange be ween −1 and 1%) and PMT gain which was di e en
o channel ed and g een, being in gene al e ms, highe o
g een han o ed ( es and posi i e con ol), due o he ac ha
he e was mo e p esence o ed han g een signal, being o he
nega i e con ol he opposi e. These alues we e always adjus ed
o ge a good quali y cap u e wi hou backg ound noise, a oiding
excessi e sa u a ion o he b igh es pixels o he image. As he
echnician was blinded o he expe imen , hey we e ad ised o
make he adjus men s always consis en wi h wha was seeing by
he objec i e o he mic oscope, ob aining an image which was
he closes as possible o eali y.
The quan i ica ion o bac e ial iabili y (BV) in he se ies o
XY images was de e mined using a cy o luo og aphic analysis
(Leica con ocal so wa e). In his analysis, he images o each
luo och ome we e de ined as “channels” (SYTO 9 occupies
he g een channel and P opidium Iodide he ed channel). To
conduc he compu a ions au oma ically, a Ma lab oolbox called
Den ius Bio ilm was de eloped. The main p og am eads all he
images om an expe imen , o ganized in a olde ee, wi h he
image olde a he op, he expe imen olde below his and
all he pa ien olde s a he bo om. The p og am au oma ically
compu es he numbe o disks, ields and 1 µm sec ions om he
images s o ed in each pa ien ’s olde .
The p og am conside s he pa ame e s ixed by expe s: he
BV is cha ac e ized by a high alue in he g een channel (o e
100, wi h a ange be ween 0 and 255) and a low alue in he
ed channel (below 100). Bac e ia a e conside ed no o be iable
i he alues a e high in he ed channel (o e 100) and low
in he g een channel (below 100). Values ha a e high in bo h
channels (o e 100) a e isually o ange and a e conside ed non-
iable bac e ia. The p og am coun s he numbe o pixels unde
hese condi ions o compu e he BV pe cen age o each 1 µm
sec ion ( iable bac e ia/ iable bac e ia +non- iable bac e ia ×
100). De e mina ion o he mean BV pe cen age in each ield
equi ed sec ions wi h a minimum bio ilm a ea o 250 µm2
(∼4,750 pixels).
The p og am also conside s he case whe e epi helial nuclei
a e p esen . These a e cha ac e ized by compac ed a eas wi h a
size ha is g ea e han he bac e ia, and hese ed poin s mus
no be coun ed as non- iable bac e ial popula ion. To elimina e
hese pixels, he p og am dis ega ds epi helial cells, which a e
cha ac e ized by: ha ing a high alue in he ed channel, an
a ea >200 pixels, compac egions wi h a solidi y >0.7, and a
minimum alue o he mean in ensi y o 180. These pa ame e s
we e ixed using a aining se . Wi h his me hodology, he e
could be some misde ec ions, bu he e ec on he BV was e y
low, as wha was impo an was he elimina ion o la ge a eas
wi h a high in ensi y.
All he esul s ob ained o each sec ion, ield and
disk om each pa ien we e s o ed in a wo kshee o be
analyzed by he esea che s. The BV pe cen age was also
s o ed be o e and a e elimina ing he epi helial nuclei
and hei p ope ies o localize hem o e he image.
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Quin as e al. Essen ial Oils Ac i i y on Bio ilm
The mean BV pe cen age o he bio ilm was calcula ed
o he co esponding sample and o each bio ilm
laye .
In Tes 2, apa om he hickness and BV, he co e ing g ade
(CG) was also assessed. This pa ame e is he pe cen age o he
su ace subs a e co e ed by he bio ilm. The cy o luo og am
i sel was used o his pu pose. F om he maximum p ojec ion
(supe posi ion o all cap u ed planes) o each o he analyzed
ields, he CG pe cen age was ob ained by calcula ing he sum
o he bac e ial mass ( iable and non- iable) wi h ega d o
he o al su ace o he ield (% posi i e wi hin he o al
a ea).
S a is ical Analysis
The s a is ical analyses we e pe o med using he R
so wa e (R Co e Team, 2016). The Shapi o–Wilk
es was pe o med o analyze he dis ibu ion o
he quan i a i e a iables associa ed wi h he PL-
bio ilm ( hickness, BV and CG), showing mos ly hese
mic oscopic pa ame e s a non-no mal dis ibu ion in bo h
es s.
In Tes 1 (subs an i i y) and Tes 2 (an iplaque e ec ),
he F iedman es was used o in a-mou hwash and in e -
mou hwash compa isons using all he PL-bio ilm samples
(including di e en ia ing be ween he h ee bio ilm laye s). In
bo h es s, he Wilcoxon signed- ank es was used o pai wise
compa isons (wi h Bon e oni adjus men ) o he in a- and
in e -mou hwash esul s (including di e en ia ing be ween he
h ee bio ilm laye s). The signi icance le el es ablished was a
P<0.05. In he Tes 1, he Bon e oni-co ec ed P- alues applied
we e <0.003 and <0.016, and in he Tes 2, his alue was
<0.016.
RESULTS
A o al o 30 olun ee s we e e alua ed o ob ain he calcula ed
sample size (n=19). When his numbe o pa icipan s mee ing
he inclusion and exclusion c i e ia was achie ed, he en ollmen
p ocess was ended. A o al o 11 subjec s we e ineligible as hey
did no mee all o he inclusion c i e ia. All he pa icipan s
pe o med bo h o he es s, al hough a subjec was excluded a e
pe o ming he Tes 1 o an unexpec ed e en (Supplemen a y
Table 2, CONSORT low diag am). No ad e se e ec s we e
epo ed by hem a any s age o he expe imen . Eigh een
subjec s comple ed he insing p o ocols sa is ac o ily in bo h
es s. In Tes 2, he e u ns o each p oduc sugges ed good
compliance wi h he ins uc ions.
Tes 1 (Subs an i i y)
In luence o a Single Mou hwash o T-EO and A -EO
on he Thickness o he PL-Bio ilm
Nei he he T-EO no he A -EO an isep ics could educe he
hickness o he PL-bio ilm o 48 h a e a single applica ion.
Thei baseline hicknesses we e 21.81 ±5.28 µm and 20.71 ±
4.13 µm, espec i ely. A e a single mou hwash, he hicknesses
we e sligh ly educed (20.19 ±3.62 µm and 18.58 ±3.14 µm,
espec i ely), bu did no achie e s a is ical signi icance.
In luence o a Single Mou hwash o T-EO and A -EO
on he Bac e ial Viabili y o he PL-Bio ilm
The mean BV a baseline anged be ween 63.99 ±19.7%
and 79.54 ±5.31% o all he h ee ising p o ocols, wi h
no s a is ical di e ences be ween hem. Bo h EO o mula ions
achie ed simila esul s a all he ime poin s measu ed. In ac , no
di e ences we e ound be ween hem om he immedia e sample
(30 s) o he 7-h sample (Figu e 2). The EO o mula ions we e
FIGURE 2 | Pe cen ages o bac e ial iabili y o he PL-bio ilm in baseline condi ions, a 30 s, 1, 3, 5, and 7 h a e a single mou hwash wi h s e ile wa e (M-WATER),
wi h essen ial oils wi h alcohol (M-T-EO), and wi h essen ial oils wi hou alcohol (M-A -EO). *S a is ically signi ican di e ences in ega d o he 30-s sample (P<0.003).
+S a is ically signi ican di e ences in ega d o he baseline (P<0.003).
F on ie s in Mic obiology | www. on ie sin.o g 6No embe 2017 | Volume 8 | A icle 2162
Quin as e al. Essen ial Oils Ac i i y on Bio ilm
e ec i e a educing he BV a e a single mou hwash wi h espec
o hei espec i e baseline le els (BV a 30 s o M-T-EO and
M-A -EO =6.53 ±7.60% and 4.13 ±3.89%, espec i ely; P<
0.001). These esul s we e s a is ically lowe han hose om he
M-WATER (62.39 ±8.17%; P<0.001). Bo h solu ions we e able
o keep he BV unde baseline le els o 7 h (BV a 7 h o M-T-EO
and M-A -EO =18.20 ±9.38% and 20.10 ±10.27%, espec i ely;
P<0.001). Again, hese indings we e s a is ically lowe han
hose om he M-WATER (BV a 7 h =76.78 ±4.40%; P<0.001;
Figu es 2,3).
In e ms o BV eco e y conce ning he 30-s sample,
signi ican eco e y was no achie ed un il 7 h a e he use o an
M-T-EO inse (BV a 30 s s. a 7 h o M-EO =6.53 ±7.60% s.
18.20 ±9.38%; P<0.001). Howe e , o he M-A -EO solu ion,
FIGURE 3 | Rep esen a i e images o he ob ained bac e ial iabili ies a 30 s, 3 and 7 h a e a single mou hwash wi h s e ile wa e (M-WATER), adi ional essen ial
oils (M-T-EO), and alcohol- ee essen ial oils (M-A -EO).
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Quin as e al. Essen ial Oils Ac i i y on Bio ilm
signi ican eco e y was iden i ied in he 3-h sample (BV a 30 s
s. a 3 h o M-A -EO =4.13 ±3.89% s. 12.35 ±8.86%; P<
0.001; Figu e 2).
When i comes o di e en ia ing be ween he h ee bio ilm
laye s, he wo EO an isep ics had lowe BV le els in all he laye s
(Table 1). No signi ican di e ences we e ound in BV o he
same bio ilm laye be ween he EO o mula ions, wi h he ou e
laye s being gene ally mo e iable han he inne ones in all he
samples. The e we e no signi ican di e ences be ween he h ee
laye s o he M-T-EO inse (BV a 30 s by laye s =6.67 ±6.80%
s. 5.63 ±8.15% s. 7.29 ±9.17%, espec i ely; P>0.016) o
be ween he deepe laye s o he M-A -EO inse (BV a 30 s by
laye s 2 and 3 =3.22 ±3.24% s. 3.51 ±4.94%, espec i ely; P>
0.016; Table 1).
Tes 2 (An iplaque E ec )
In luence o a 4-Day P o ocol o T-EO and A -EO
Mou hwashes on he Thickness and Co e ing G ade
o he PL-Bio ilm
The A -EO inses we e mo e e ec i e han he T-EO o mula ion
a educing he hickness o he o al bio ilm a e 4 days o use
( hickness o 4D-T-EO s. 4D-A -EO =9.92 ±2.87 µm s. 7.90
±2.91 µm; P=0.012), bu bo h solu ions we e mo e powe ul
han he nega i e con ol ( hickness o 4D-WATER =22.76 ±
6.21 µm; P<0.001; Table 2 and Figu e 4).
The A -EO inses we e mo e e ec i e han he T-EO solu ion
a educing he CG o he o al bio ilm a e 4 days o use (CG o
4D-T-EO s. 4D-A -EO =46.61 ±19.12% s. 33.36 ±12.01%,
espec i ely; P=0.001). The wo EO solu ions we e signi ican ly
mo e e ec i e han he nega i e con ol a educing he CG
(CG o 4D-WATER =73.92 ±17.49%; P<0.001; Table 2 and
Figu e 5).
In luence o a 4-Day P o ocol o T-EO and A -EO
Mou hwashes on he Bac e ial Viabili y o he
PL-Bio ilm
The T-EO and A -EO o mula ions a e 4 days o use showed
simila e ec i eness in main aining he BV a signi ican ly lowe
le els han he nega i e con ol (BV o 4D-T-EO and 4D-A -EO
s. 4D-WATER =26.27 ±14.61% and 31.08 ±16.52% s. 51.35
±5.38%, espec i ely; P<0.001; Table 2). In e ms o he BV by
laye s, he ou e laye s had signi ican ly mo e BV han he inne
ones in all he expe imen s. The T-EO and A -EO o mula ions
we e signi ican ly mo e e ec i e a educing he BV o laye s 1
and 2 han he nega i e con ol, wi h laye 3 unal e ed (BV o
laye 1 =40.10 ±17.31% and 39.81 ±19.09% s. 82.47 ±7.58%,
espec i ely; BV o laye 2 =24.32 ±16.16% and 30.73 ±17.06%
s. 51.76 ±13.53%, espec i ely; P<0.001 o all compa isons;
Table 3).
DISCUSSION
Me hodology App oach
This is he i s s udy o compa e he an ibac e ial ac i i y in i o
ha T-EO and A -EO solu ions ha e on he non-des uc u ed
o al bio ilm. F om a me hodological pe spec i e, o pe o m all
he BV compu a ions au oma ically h ough a Ma lab oolbox
called Den ius Bio ilm ensu es ha he expe imen ’s indings a e
accu a e, quick o ob ain, eliable, and epea able, which is e y
impo an when i comes o compa ing esul s and ex ac ing
obus conclusions. In he subs an i i y s udy ( es 1), mean BV
da a o each subjec we e ob ained om analyzing ∼160 sec ions
o 1 µm (a ound 40 sec ions pe ield X ou ields X one disk) o
each e alua ed momen (six- ime momen s: baseline, 30 s, 1, 3,
5, and 7 h). In he an iplaque s udy ( es 2), when he nega i e
con ol was applied, he mean BV alues o each subjec we e
ob ained om e alua ing ∼1,200 sec ions (a ound 50 sec ions
pe ield X ou ields X six disks); when an isep ic solu ions we e
used, he mean BV esul s o each subjec we e de i ed om he
analysis o ∼360 sec ions (a ound 15 sec ions pe ield X ou
ields X six disks).
In he p esen se ies, he BV was assessed by he Li e/Dead R
BacLigh TM luo escence assay. This solu ion s ains he bac e ia
in ed o g een depending on he pe meabili y o hei
memb ane (p opidium iodide which s ains he cell in ed only
i he memb ane pe meabili y is al e ed). Gi en ha he es ed
an isep ics ac mos ly a his cellula elemen , his iabili y
me hod may be sui able o his ype o s udy. Al hough, i s ly
concei ed as a echnique only alid o BV assessmen o single
species model (In i ogen communica ion), i s simplici y and
good esul s also in in si u s udies con aining mul iple bac e ial
species (Boulos e al., 1999; Ihalin e al., 2003; Tomás e al., 2009;
Bey h e al., 2010; Dong e al., 2010; Gosau e al., 2010; Ga cía-
Caballe o e al., 2013; Tawakoli e al., 2013; P ada-López e al.,
2015a,b; Quin as e al., 2015a,b) has p oduced he manu ac u e
o make a ecommenda ion also o mul iple species models
(Li e/Dead R
BacLigh TM Use Manual)3.
Despi e his, he e has been some discussion abou he
eliabili y o his echnique (Hannig e al., 2010; Tawakoli e al.,
2013; Ne uschil e al., 2014), mainly due o he ac ha no
only ed and g een bac e ia appea in he analysis, bu also
o ange egions appea wi h “unknown” in e p e a ion (Be ney
e al., 2007). Hannig e al. (2010) conside ed ha li e/dead
s aining me hods we e eliable when e alua ing an imic obial
agen s ac i i y. Howe e , hey con inued o ask he ques ion
abou “how dead is dead?” due o se e al s ages o i ali y which
ha e been discussed and desc ibed in he li e a u e ( iable and
cul u able, iable bu non-cul u able, do man , non- iable and
p e-ly ic, and a i al dead bac e ia). The exac di e en ia ion o
hese s ages is s ill one o he g ea es challenges in mode n
mic obiology (Decke , 2001).
Se e al a emp s ha e been made o compa e he Li e/Dead R
BacLigh TM wi h he gold s anda d - adi ional plaque cul u es
- (Boulos e al., 1999; Ihalin e al., 2003; Tomás e al., 2009;
Tawakoli e al., 2013). One o hese was pe o med se e al yea s
ago by he au ho s (Tomás e al., 2009) in a s udy on he in
si u subs an i i y o he chlo hexidine on he sali a y bac e ia.
The mean BV ob ained was compa ed wi h plaque cul u es.
A good co ela ion was obse ed be ween bo h echniques in
he baseline and immedia e samples. Howe e , his co ela ion
3Li e/Dead R
BacLigh TM Use Manual. A ailable online a : h ps:// ools.
he mo ishe .com/con en /s s/manuals/mp07007.pd (Accessed on Sep embe 30,
2017).
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Quin as e al. Essen ial Oils Ac i i y on Bio ilm
TABLE 1 | Bac e ial iabili y in 2-day PL-bio ilm unde basal condi ions and in he samples collec ed a 30 s and 1, 3, 5, and 7 h a e a single mou h inse wi h: s e ile
wa e , adi ional essen ial oils solu ion, and alcohol- ee essen ial oils solu ion.
BASAL 30 S 1 H 3 H 5 H 7 H
BACTERIAL VIABILITY (%) OF THE 2-DAY PL-BIOFILM; MEAN ±STANDARD DEVIATION, MEDIAN (INTERQUARTILE RANGE)
M-WATER
Laye 1
(ou e laye )
86.82 ±3.57
87.51 (2.18)
81.86 ±6.63
80.48 (8.15)
85.46 ±5.78
83.31 (6.89)
89.11 ±6.13
91.90 (8.74)
86.26 ±3.76
87.36 (6.29)
90.75 ±3.45
89.54 (4.58)
Laye 2
(middle laye )
82.06 ±5.02
(80.48 (3.87)
69.21 ±7.83
68.53 (8.15)
73.94 ±9.79
75.54 (6.02)
82.75 ±5.98
80.91 (8.06)
77.20 ±8.07
78.34 (11.84)
83.07 ±4.25
84.73 (5.48)
Laye 3
(inne laye )
69.74 ±16.21
73.69 (22.91)
36.08 ±20.97
31.09 (26.52)
44.18 ±14.80
44.34 (30.87)
50.12 ±13.22
56.27 (15.80)
48.95 ±17.78
52.45 (32.48)
56.53 ±12.59
60.34 (9.10)
M-T-EO
Laye 1
(ou e laye )
75.54 ±17.28
75.53 (19.71)
6.67 ±6.80
4.44 (6.06)
9.90 ±17.56
4.16 (4.80)
18.49 ±14.37
16.36 (20.45)
22.35 ±14.73
15.56 (28.93)
35.42 ±19.00
35.55 (31.05)
Laye 2
(middle laye )
67.86 ±21.95
75.91 (32.06)
5.63 ±8.15
2.77 (3.53)
7.97 ±18.16
1.69 (4.65)
8.04 ±10.79
3.68 (7.61)
8.58 ±7.78
5.45 (6.14)
13.26 ±9.09
9.91 (11.96)
Laye 3
(inne laye )
48.56 ±28.37
51.87 (32.67)
7.29 ±9.17
1.72 (12.59)
9.02 ±17.95
1.35 (6.06)
5.81 ±9.26
0.52 (7.15)
4.76 ±7.62
1.37 (4.29)
5.92 ±9.21
2.56 (5.05)
M-A -EO
Laye 1
(ou e laye )
84.74 ±15.33
90.03 (9.74)
5.66 ±5.72
3.88 (6.75)
10.01 ±8.30
8.82 (10.24)
20.74 ±14.73
18.56 (12.87)
27.69 ±18.15
21.85 (21.27)
41.99 ±18.96
46.53 (27.42)
Laye 2
(middle laye )
76.37 ±19.38
81.93 (27.11)
3.22 ±3.24
1.76 (5.33)
4.04 ±4.25
2.46 (4.84)
9.70 ±8.52
7.62 (10.00)
12.67 ±10.22
8.32 (14.72)
14.28 ±11.27
11.35 (14.96)
Laye 3
(inne laye )
51.95 ±31.30
59.06 (56.86)
3.51 ±4.94
2.01 (3.13)
1.23 ±1.18
0.98 (1.30)
6.63 ±8.39
4.23 (7.16)
6.53 ±7.06
3.38 (8.52)
4.02 ±6.50
1.50 (4.27)
BACTERIAL VIABILITY, INTRA-MOUTHWASH ANALYSIS; STATISTICAL SIGNIFICANCE
M-WATER
Laye 1 s. Laye 2 P<0.016 P<0.016 P<0.016 — P<0.016 P<0.016
Laye 1 s. Laye 3 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016
Laye 2 s. Laye 3 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016
M-T-EO
Laye 1 s. Laye 2 P<0.016 — P<0.016 P<0.016 P<0.016 P<0.016
Laye 1 s. Laye 3 P<0.016 — — P<0.016 P<0.016 P<0.016
Laye 2 s. Laye 3 P<0.016 — — P<0.016 — P<0.016
M-A -EO
Laye 1 s. Laye 2 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016
Laye 1 s. Laye 3 P<0.016 — P<0.016 P<0.016 P<0.016 P<0.016
Laye 2 s. Laye 3 P<0.016 — P<0.016 P<0.016 P<0.016 P<0.016
BACTERIAL VIABILITY, INTER-MOUTHWASH ANALYSIS; STATISTICAL SIGNIFICANCE
M-WATER s. M-T-EO
Laye 1 s. Laye 1 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016
Laye 2 s. Laye 2 — P<0.016 P<0.016 P<0.016 P<0.016 P<0.016
Laye 3 s. Laye 3 — P<0.016 P<0.016 P<0.016 P<0.016 P<0.016
M-WATER s. M-A -EO
Laye 1 s. Laye 1 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016
Laye 2 s. Laye 2 — P<0.016 P<0.016 P<0.016 P<0.016 P<0.016
Laye 3 s. Laye 3 — P<0.016 P<0.016 P<0.016 P<0.016 P<0.016
M-T-EO s. M-A -EO
Laye 1 s. Laye 1 — — — — — —
Laye 2 s. Laye 2 — — — — — —
Laye 3 s. Laye 3 — — — — — —
Di e ences be ween he h ee bio ilm laye s, as well as in a-mou h inse and in e -mou h inse compa isons. M-WATER, A single, 30-s mou hwash wi h 20 mL o s e ile wa e ; M-T-EO,
A single, 30-s mou hwash wi h 20 mL o a adi ional essen ial oils solu ion; M-A -EO, A single, 30-s mou hwash wi h 20 mL o an alcohol- ee essen ial oils solu ion. —, no s a is ical
signi icance.
F on ie s in Mic obiology | www. on ie sin.o g 9No embe 2017 | Volume 8 | A icle 2162
Quin as e al. Essen ial Oils Ac i i y on Bio ilm
Pizzo, G., Compila o, D., Di Libe o, B., Pizzo, I., and Campisi, G. (2013). E ec s
o wo essen ial oil mou h inses on 4-day sup agingi al plaque eg ow h: a
andomized c oss-o e s udy. Am. J. Den . 26, 156–160.
Pizzo, G., La Ca a, M., Lica a, M. E., Pizzo, I., and D’Angelo, M. (2008).
The e ec s o an essen ial oil and an amine luo ide/s annous luo ide
mou h inse on sup agingi al plaque eg ow h. J. Pe iodon ol. 79, 1177–1183.
doi: 10.1902/jop.2008.070583
P ada-López, I., Quin as, V., Casa es-De-Cal, M. A., Suá ez-Quin anilla, J.
A., Suá ez-Quin anilla, D., and Tomás, I. (2015a). Ex i o s. in i o
an ibac e ial ac i i y o wo an isep ics on o al bio ilm. F on . Mic obiol. 6:655.
doi: 10.3389/ micb.2015.00655
P ada-López, I., Quin as, V., Donos, N., Ál a ez-Fe nández, M., and Tomás,
I. (2013). “In si u subs an i i y o he essen ial oils in he o al ca i y,” in
Mic obial Pa hogens and S a egies o Comba ing Them: Science, Technology
and Educa ion, ed A. Méndez-Vilas (Badajoz: Fo ma ex Resea ch Cen e ),
1112–1122.
P ada-López, I., Quin as, V., Donos, N., Suá ez-Quin anilla, D., and Tomás, I.
(2015b). Cha ac e is ics o in si u o al bio ilm a e 2 and 4 days o e olu ion.
Quin essence In . 46, 287–298. doi: 10.3290/j.qi.a33402
P ada-López, I., Quin as, V., and Tomás, I. (2015c). The in ao al de ice o o e laid
disk-holding splin s as a new in si u o al bio ilm model. J. Clin. Exp. Den . 7,
e126–e132. doi: 10.4317/jced.52093
P ada-López, I., Quin as, V., Vilaboa, C., Suá ez-Quin anilla, D., and Tomás,
I. (2016). De ices o in si u de elopmen o non-dis u bed o al bio ilm.
A sys ema ic e iew. F on . Mic obiol. 7:1055. doi: 10.3389/ micb.2016.
01055
P a en, J., Ba ne , P., and Wilson, M. (1998). Composi ion and suscep ibili y o
chlo hexidine o mul ispecies bio ilms o o al bac e ia. Appl. En i on. Mic obiol.
64, 3515–3519.
Quin as, V., P ada-López, I., Donos, N., Sua ez-Quin anilla, D., and Tomás, I.
(2015a). An iplaque e ec o essen ial oils and 0.2% chlo hexidine on an in
si u model o o al bio ilm g ow h: a andomised clinical ial. PLoS ONE
10:e0117177. doi: 10.1371/jou nal.pone.0117177
Quin as, V., P ada-López, I., P ados-F u os, J. C., and Tomás, I. (2015b).
In si u an imic obial ac i i y on o al bio ilm: essen ial oils s. 0.2 %
chlo hexidine. Clin O al In es ig 19, 97–107. doi: 10.1007/s00784-014-
1224-3
R Co e Team (2016). R: A Language and En i onmen o S a is ical Compu ing.
Vienna: R ounda ion o s a is ical compu ing. A ailable online a : h p://www.
R-p ojec .o g (Accessed on July 30, 2017).
Robinson, C., S a o d, S., Rees, G., B ookes, S. J., Ki kham, J., Sho e, R.
C., e al. (2006). Plaque bio ilms: he e ec o chemical en i onmen on
na u al human plaque bio ilm a chi ec u e. A ch. O al Biol. 51, 1006–1014.
doi: 10.1016/j.a cho albio.2006.04.010
Rosin, M., Welk, A., Koche , T., Majic-Tod , A., K ame , A., and Pi en,
F. A. (2002). The e ec o a polyhexame hylene biguanide mou h inse
compa ed o an essen ial oil inse and a chlo hexidine inse on bac e ial
coun s and 4-day plaque eg ow h. J. Clin. Pe iodon ol. 29, 392–399.
doi: 10.1034/j.1600-051X.2002.290503.x
Singh, A., Daing, A., and Dixi , J. (2013). The e ec o he bal, essen ial oil and
chlo hexidine mou h inse on de no o plaque o ma ion. In . J. Den . Hyg. 11,
48–52. doi: 10.1111/j.1601-5037.2012.00556.x
Sissons, C. H., Wong, L., and Cu ess, T. W. (1996). Inhibi ion by
e hanol o he g ow h o bio ilm and dispe sed mic ocosm den al
plaques. A ch. O al Biol. 41, 27–34. doi: 10.1016/0003-9969(95)
00103-4
Smigel, K. (1991). High-alcohol mou hwashes a e unde sc u iny. J. Na l. Cance
Ins . 83:751. doi: 10.1093/jnci/83.11.751
Tawakoli, P. N., Al-Ahmad, A., Ho h-Hannig, W., Hannig, M., and Hannig,
C. (2013). Compa ison o di e en li e/dead s ainings o de ec ion and
quan i ica ion o adhe en mic oo ganisms in he ini ial o al bio ilm. Clin. O al
In es ig. 17, 841–850. doi: 10.1007/s00784-012-0792-3
en Ca e, J. M., and Ma sh, P. D. (1994). P ocedu es o es ablishing e icacy o
an imic obial agen s o chemo he apeu ic ca ies p e en ion. J. Den . Res. 73,
695–703. doi: 10.1177/00220345940730031601
Tomás, I., Cousido, M. C., Tomás, M., Lime es, J., Ga cía-Caballe o, L.,
and Diz, P. (2008). In i o bac e icidal e ec o 0.2% chlo hexidine bu
no 0.12% on sali a y obliga e anae obes. A ch. O al Biol. 53, 1186–1191.
doi: 10.1016/j.a cho albio.2008.07.009
Tomás, I., Ga cía-Caballe o, L., Cousido, M. C., Lime es, J., Ál a ez,
M., and Diz, P. (2009). E alua ion o chlo hexidine subs an i i y on
sali a y lo a by epi luo escence mic oscopy. O al Dis. 15, 428–433.
doi: 10.1111/j.1601-0825.2009.01570.x
Ulku , F., A un, T., and Ozdemi , F. (2013). The e ec s o h ee di e en mou h
inses in a 4-day sup agingi al plaque eg ow h s udy. Eu . J. Den . 7, 352–358.
doi: 10.4103/1305-7456.115420
Van Leeuwen, M. P., Slo , D. E., and Van de Weijden, G. A. (2011). Essen ial
oils compa ed o chlo hexidine wi h espec o plaque and pa ame e s o
gingi al in lamma ion: a sys ema ic e iew. J. Pe iodon ol. 82, 174–194.
doi: 10.1902/jop.2010.100266
Van S ydonck, D. A., Slo , D. E., Van de Velden, U., and Van de Weijden, F.
(2012). E ec o a chlo hexidine mou h inse on plaque, gingi al in lamma ion
and s aining in gingi i is pa ien s: a sys ema ic e iew. J. Clin. Pe iodon ol. 39,
1042–1055. doi: 10.1111/j.1600-051X.2012.01883.x
Vlachojannis, C., Ch ubasik-Hausmann, S., Hellwig, E., and Al-Ahmad, A. (2015).
A p elimina y in es iga ion on he an imic obial ac i i y o Lis e ine(R),
i s componen s, and o mix u es he eo . Phy o he . Res. 29, 1590–1594.
doi: 10.1002/p .5399
Vlachojannis, C., Winsaue , H., and Ch ubasik, S. (2013). E ec i eness and
sa e y o a mou hwash con aining essen ial oil ing edien s. Phy o he . Res. 27,
685–691. doi: 10.1002/p .4762
on Ohle, C., Gieseke, A., Nis ico, L., Decke , E. M., DeBee , D., and S oodley, P.
(2010). Real- ime mic osenso measu emen o local me abolic ac i i ies in ex
i o den al bio ilms exposed o suc ose and ea ed wi h chlo hexidine. Appl.
En i on. Mic obiol. 76, 2326–2334. doi: 10.1128/AEM.02090-09
Waale , S. M., Rolla, G., Skjo land, K. K., and Ogaa d, B. (1993). E ec s
o o al insing wi h iclosan and sodium lau yl sul a e on den al
plaque o ma ion: a pilo s udy. Scand. J. Den . Res. 101, 192–195.
doi: 10.1111/j.1600-0722.1993. b01103.x
Wood, S. R., Ki kham, J., Ma sh, P. D., Sho e, R. C., Na ess, B., and
Robinson, C. (2000). A chi ec u e o in ac na u al human plaque bio ilms
s udied by con ocal lase scanning mic oscopy. J. Den . Res. 79, 21–27.
doi: 10.1177/00220345000790010201
Wood, S. R., Ki kham, J., Sho e, R. C., B ookes, S. J., and Robinson, C.
(2002). Changes in he s uc u e and densi y o o al plaque bio ilms
wi h inc easing plaque age. FEMS Mic obiol. Ecol. 39, 239–244.
doi: 10.1111/j.1574-6941.2002. b00926.x
Wo ld Heal h O ganiza ion (2013). O al Heal h Su eys: Basic Me hods. Wo ld
Heal h O ganiza ion.
Zau a-A i e, E., an Ma le, J., and en Ca e, J. M. (2001). Cono ocal mic oscopy
s udy o undis u bed and chlo hexidine- ea ed den al bio ilm. J. Den . Res. 80,
1436–1440. doi: 10.1177/00220345010800051001
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