ORIGINAL RESEARCH
published: 02 July 2015
doi: 10.3389/ micb.2015.00655
F on ie s in Mic obiology | www. on ie sin.o g 1July 2015 | Volume 6 | A icle 655
Edi ed by:
Yuji Mo i a,
Aichi Gakuin Uni e si y, Japan
Re iewed by:
Jean-Ch is ophe Fa ges,
Uni e si y Lyon 1, F ance
Fabian Cieplik,
Uni e si y Medical Cen e
Regensbu g, Ge many
*Co espondence:
Inmaculada Tomás,
School o Medicine and Den is y.
San iago de Compos ela Uni e si y,
C./ En e ios s/n, 15872,
San iago de Compos ela, Spain
inmaculada. omas@usc
Special y sec ion:
This a icle was submi ed o
An imic obials, Resis ance and
Chemo he apy,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 02 May 2015
Accep ed: 15 June 2015
Published: 02 July 2015
Ci a ion:
P ada-López I, Quin as V,
Casa es-De-Cal MA,
Suá ez-Quin anilla JA,
Suá ez-Quin anilla D and Tomás I
(2015) 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
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
Isabel P ada-López, Víc o Quin as, Ma ia A. Casa es-De-Cal,
Juan A. Suá ez-Quin anilla, Da id Suá ez-Quin anilla and Inmaculada Tomás*
O al Sciences Resea ch G oup, Special Needs Uni , School o Medicine and Den is y, Uni e si y o San iago de
Compos ela, San iago de Compos ela, Spain
Aim: To compa e he immedia e an ibac e ial e ec o wo applica ion me hods (passi e
imme sion and ac i e mou hwash) o wo an isep ic solu ions on he in si u o al bio ilm.
Ma e ial and Me hods: A andomized obse e -masked c osso e s udy was
conduc ed. Fi een heal hy olun ee s wo e a speci ic in ao al de ice o 48 h o o m
a bio ilm in h ee glass disks. One o hese disks was used as a baseline; ano he one
was imme sed in a solu ion o 0.2% Chlo hexidine (0.2% CHX), emaining he hi d in he
de ice, placed in he o al ca i y, du ing he 0.2% CHX mou hwash applica ion. A e
a 2-weeks washou pe iod, he p o ocol was epea ed using a solu ion o Essen ial
Oils (EO). Samples we e analyzed o bac e ial iabili y wi h he con ocal lase scanning
mic oscope a e p e ious s aining wi h LIVE/DEAD®BacLigh ™.
Resul s: The EO showed a be e an ibac e ial e ec compa ed o he 0.2% CHX a e
he mou hwash applica ion (% o bac e ial iabili y =1.16 ±1.00% s. 5.08 ±5.79%,
espec i ely), and was mo e e ec i e in all laye s (p<0.05). In he imme sion, bo h
an isep ics we e signi ican ly less e ec i e (% o bac e ial iabili y =26.93 ±13.11%, EO
s. 15.17 ±6.14%, 0.2% CHX); in he case o EO imme sion, he e we e no signi ican
changes in he bac e ial iabili y o he deepes laye in compa ison wi h he baseline.
Conclusions: The me hod o applica ion condi ioned he an ibac e ial ac i i y o he
0.2% CHX and EO solu ions on he in si u o al bio ilm. The in i o ac i e mou hwash was
mo e e ec i e han he ex i o passi e imme sion in bo h an isep ic solu ions. The e was
mo e pene a ion o he an isep ic inside he bio ilm wi h an ac i e mou hwash, especially
wi h he EO. T ial egis e ed in clinical ials.go wi h he numbe NCT02267239. URL:
h ps://clinical ials.go /c 2/show/NCT02267239.
Keywo ds: an isep ic, chlo hexidine, essen ial oils, imme sion, mou hwash, PL-bio ilm
In oduc ion
The use o o al an isep ics is a ecommended p ocedu e o he chemical con ol o he o al
bio ilms. These o al an isep ics may kill he mic oo ganisms, educe bac e ial i ulence, and e a d
he den al plaque o ma ion. Due o his ac ion on he bac e ia o ming den al bio ilms, a educ ion
o he o al disease is expec ed a e hei applica ion (Co bin e al., 2011).
P ada-López e al. Di e ences be ween o al an isep ic applica ions
Among den al p ac i ione s, he mos commonly p esc ibed
o al an isep ics ha e been hose including in hei o mula ion
Chlo hexidine (CHX) (Va oni e al., 2012) o Essen ial Oils
(EO) (Axelsson, 2004). Al hough he an imic obial e ec i eness
o bo h has been shown in p e ious s udies (Gunsolley, 2010;
Quin as e al., 2015), hey exhibi di e en ai s when ce ain
me hodologies a e ollowed, which, in some cases, may limi
he eliabili y o he esul s. Some o hese di e ences ha e been
ecognized by he scien i ic communi y, who cau iously in e p e
he esul s o he s udies ha ha e used an in i o- o med bio ilm
(Auschill e al., 2004). The use o de e mina e bac e ia o c ea e
a bio ilm in an a i icial en i onmen may esul in a measu e o
an isep ic e ec i eness ha may no be ep esen a i e o he in
si u si ua ion (Auschill e al., 2005).
Bac e ia li ing in den al plaque de elop ela ionships o ming
an ex acellula ma ix which is a highly esis an h ee-
dimensional (3-D) s uc u e. This associa ion makes he bio ilm
bac e ia om 10 o 1000 imes mo e esis an o an an isep ic
(Fine e al., 2001; Da ies, 2003). Gi en his, he handling o
he in si u o med bio ilm is an impo an cha ac e is ic ha
s udies in ol ing o al bio ilms should ake in o accoun . In
some s udies he 3-D s uc u e o he o al bio ilm is al e ed
when he sample is collec ed (Pan e al., 2000; Fine e al.,
2005) o du ing he analysis p ocess (Jen sch e al., 2002; Vi ko
e al., 2005). The dis o ion o he 3-D s uc u e p obably
in luences he quan i ica ion o he an isep ic e ec i eness. To
a oid his, speci ic o al de ices ha e been designed o allow
he o ma ion o a non-dis u bed bio ilm, simila o he den al
plaque, which has been called plaque-like bio ilm (PL-bio ilm)
(P ada-López e al., 2015a; Quin as e al., 2015). Fu he mo e, he
use o con ocal lase scanning mic oscope (CLSM) has allowed
he possibili y o analyse he in si u bio ilm in hei na u al
hyd a ed s a e wi hou losing i s complex s uc u e (A weile
e al., 2004). The CLSM has been used in combina ion wi h
dual li e/dead s aining solu ions. This has gi en o in es iga o s
he possibili y o analyse he iabili y o a non-dis u bed in si u
o al bio ilm, be o e and a e he applica ion o he an isep ics.
Among he di e en a ailable s aining solu ions, he SYTO 9
and p opidium iodide has been one o he mos success ully
employed combina ions o luo och omes o isualizing den al
plaque (Fuchsloche Hellemann e al., 2013; Hannig e al., 2013;
Tawakoli e al., 2013; P ada-López e al., 2015a; Quin as e al.,
2015).
Recen ly, some au ho s ha e s a ed ha he me hodology o
he applica ion o a gi en an isep ic could be an impo an ac o
which migh condi ion he esul s on o al an isep ic e ec i eness
(Quin as e al., 2015). Commonly, in s udies wi h undis u bed
bio ilm, he applica ion o he an isep ic has been an ex i o
imme sion o he sample in o he solu ion (Zau a-A i e e al.,
2001; Dong e al., 2010; Gosau e al., 2010; on Ohle e al., 2010).
Howe e , in ecen se ies abou he bac e ial e ec o CHX and
EO, he pa icipan s ha e unde gone an ac i e mou hwash wi h
Abb e ia ions: 0.2% CHX, solu ion o 0.2% o Chlo hexidine; EO, Essen ial Oils;
PL-bio ilm, plaque-like bio ilm; IDODS, in ao al de ice o o e laid disk-holding
splin s; Im-0.2%CHX, imme sion in 0.2% Chlo hexidine; Im-EO, imme sion in
Essen ial Oils; Mw-0.2%CHX, mou hwash wi h 0.2% Chlo hexidine; Mw-EO,
mou hwash wi h Essen ial Oils.
he an isep ic in i o (Ga cía-Caballe o e al., 2013; Quin as e al.,
2015), di e ing om he p e iously desc ibed esul s, mainly in
ega d o he EO ac i i y.
Based on hese p e ious indings, he au ho s o he p esen
s udy in ended o assess i he me hodology o he an isep ic
applica ion migh condi ion he ob ained esul s in e ms o
bac e ial iabili y o he PL-bio ilm. The e o e, he objec i e o
he p esen s udy was o compa e he immedia e e ec o wo
an isep ic applica ion me hods, using sepa a e solu ions o 0.2%
CHX and EO applied ei he by a passi e imme sion o an ac i e
mou hwash (ex i o s. in i o exposu e).
Ma e ial and Me hods
The p esen s udy was designed as a andomized, obse e -
masked, c osso e s udy. The immedia e e ec o 0.2% CHX
and EO solu ions was es ed using hem sepa a ely in imme sion
and mou hwash applica ion on an in si u model o PL-bio ilm
g ow h. The suppo ing CONSORT checklis is a ailable as
suppo ing in o ma ion (supplemen a y Table 1). This p ojec
go he app o al o he Clinical Resea ch E hics Commi ee o
Galicia (numbe 2012/393) and egis e ed in clinical ials.go
wi h he numbe NCT02267239. URL: h ps://clinical ials.go /
c 2/show/NCT02267239.
Selec ion o he S udy G oup
To calcula e an a p io i sample size, he ollowing s a is ical
c i e ia we e es ablished: an e ec size o 0.35, an alpha e o o
0.05 and a s a is ical powe o 87%. Assuming hese c i e ia and
using he epea ed measu es ANOVA es , a sample size o 15
subjec s was equi ed. The sample size calcula ion was pe o med
using he p og am G*Powe 3.1.5. The pa icipan s we e
ec ui ed among den al s uden s a he Facul y o Medicine and
Den is y o San iago de Compos ela (Spain), whe e olun ee
en olmen was asked by esponding o ad e isemen s o he
pa icipa ion in a esea ch s udy a he acul y hall. All o hese
olun ee s we e e ised by he same ained clinician o ensu e
hey ul illed all inclusion and exclusion c i e ia. The olun ee s
chosen me he same inclusion and exclusion c i e ia o p e ious
publica ions o ou g oup (Ga cía-Caballe o e al., 2013; P ada-
López e al., 2015a; Quin as e al., 2015). The inclusion c i e ia
we e he ollowing: being sys emically heal hy adul olun ee s
be ween 20 and 45 yea s old, who p esen ed a good o al heal h
s a us: 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) (WHO, 1997) and an absence o un ea ed ca ies
a he beginning o he s udy. The ollowing exclusion c i e ia
we e applied: smoke o o me smoke , 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 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 phase, a ull mou h
scaling wi h ul asonic ins umen s and ee h polishing wi h
ubbe cup a e den al disclosu e was pe o med by he same
ained clinician on all selec ed pa icipan s (Figu e 1). W i en
in o med consen was ob ained om all pa icipan s in he
s udy.
F on ie s in Mic obiology | www. on ie sin.o g 2July 2015 | Volume 6 | A icle 655
P ada-López e al. Di e ences be ween o al an isep ic applica ions
FIGURE 1 | P o ocol o he s udy.
In ao al De ice o O e laid Disk-Holding Splin s
(IDODS) o Bio ilm in Si u Fo ma ion
Some in si u models o he g ow h o he bio ilm ha e been
p e iously desc ibed (Ne uschil e al., 1998; Auschill e al.,
2001, 2005; A weile e al., 2004). A e hei conside a ion,
an indi idualized splin o a lowe hemi-a ch was c ea ed o
each olun ee , ollowing he same p o ocol gi en in p e ious
s udies (Ga cía-Caballe o e al., 2013; P ada-López e al., 2015a,b;
F on ie s in Mic obiology | www. on ie sin.o g 3July 2015 | Volume 6 | A icle 655
P ada-López e al. Di e ences be ween o al an isep ic applica ions
Quin as e al., 2015). The In ao al De ice o O e laid Disk-
holding Splin s (IDODS) wo n by he olun ee s held h ee glass
disks (6 mm in diame e , 1 mm hickness); hese we e polished
a 800 g i . This splin has been al eady desc ibed in p e ious
own wo ks (P ada-López e al., 2015a,b; Quin as e al., 2015)
(Figu e 2).
The IDODS wi h he glass disks was wo n by he subjec s
o 48 h (2 days) o a o g ow h o he PL-bio ilm. They
we e allowed o wi hd aw i om he o al ca i y only du ing
meals and o pe o m o al hygiene measu es (while i had o
be s o ed in a p e iously p o ided opaque con aine in humid
condi ions). In o de o no o dis u b he g owing o he PL-
bio ilm, 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.
Applica ion o he 0.2% CHX and EO o
PL-Bio ilm
The sample analysis was di ided in o wo phases, each o
ollowing he applica ion p o ocol o he an isep ic. The i s
consis ed o wi hd awing he glass disks one by one om he
splin (Figu e 1) a e he olun ee had wo n i o 48 h. The
dis al o hese disks was used as a baseline sample. The second
disk unde wen one o wo p o ocols:
A single, 30-s imme sion in 1 mL o 0.2% Chlo hexidine
(O aldine Pe io R
, Johnson & Johnson, Mad id, Spain) (Im-0.2%
CHX).
-OR-
A single, 30-s imme sion in 1 mL o Essen ial Oils in a
hyd oalcoholic solu ion (Lis e ine Men ol, Lis e ine R
, Johnson
& Johnson, Mad id, Spain) (Im-EO).
Nex , he second phase o he s udy was conduc ed.
The las disk in he splin , placed in he o al ca i y, was
wi hd awn a e he olun ee pe o med he ollowing unde
supe ision:
FIGURE 2 | In ao al iew o he In ao al De ice O e laid Disk-holding
Splin (IDODS).
A single, 30-s mou hwash wi h 10 mL o 0.2% Chlo hexidine
(O aldine Pe io R
, Johnson & Johnson, Mad id, Spain)
(Mw-0.2% CHX), ollowing he ins uc ions o he manu ac u e .
-OR-
A single, 30-s mou hwash wi h 20 mL o Essen ial Oils in a
hyd oalcoholic solu ion (Lis e ine Men ol, Lis e ine R
, Johnson
& Johnson, Mad id, Spain) (Mw-EO), ollowing he ins uc ions
o he manu ac u e .
Using an in e ne -based balanced andomiza ion sys em
(www. andomiza ion.com) ha indica ed he an isep ic each
subjec would use i s and second, as well as he hemi-a ch (le
o igh ) selec ed o he imme sion and mou hwash. All subjec s
pe o med he wo es s wi h a es pe iod o 14 days in-be ween
(Figu e 1).
Collec ion o he Samples o PL-Bio ilm
On he day o he expe imen , he olun ee s we e no allowed
o ea o d ink du ing he cou se o he es s. PL-bio ilm samples
collec ion was done indi idually (samples we e aken om jus
one olun ee pe day), s a ing a 8.30 AM ( i s baseline sample
and imme sions) and inished a 9.30 AM (mou hwash).
Immedia ely a e he glass disks we e wi hd awn om he
splin s, hey we e subme ged in 100 µL o luo escence solu ion
LIVE/DEAD R
BacLigh ™and kep in a da k chambe a oom
empe a u e o 15 min. A single in es iga o , masked o he
s udy design, pe o med he mic oscopic obse a ion 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.
P ocessing o he PL-Bio ilm Samples
In he p esen se ies, he same p o ocol desc ibed by Quin as
e al. (2015) was ollowed o e alua e he di e en ields wi hin
he disks. Fou selec ed ields (conside ed as ep esen a i e o he
whole sample), which we e in he cen al pa o each disk we e
e alua ed; hei mean measu es o he hickness and bac e ial
iabili y ep esen ed he whole sample hickness and bac e ial
iabili y, espec i ely. The maximum bio ilm hickness o each
ield was di ided in o h ee equi alen zones o same sized laye s:
ou e laye (laye 1), middle laye (laye 2) and inne laye
(laye 3).
The cap u e o he da a was done wi h he same se ings in
all cases, acco ding o p e iously p esen ed pa ame e s (Quin as
e al., 2015) (Figu e 3).
Quan i ica ion o bac e ial iabili y was also done as
p e iously p esen ed (Quin as e al., 2015) using he
cy o luo og aphic analysis (Leica Con ocal So wa e) o XY
images. 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). Squa e cap u e masks we e
used o measu e he a ea occupied (µm2) by he pixels in each
channel, de e mining he o al a ea occupied by he bio ilm and
he co esponding pe cen age o iabili y. The in ensi y ange
was conside ed a posi i e signal i i was be ween 100 and 255.
De e mina ion o he mean pe cen age o bac e ial iabili y in
each ield equi ed sec ions wi h a minimum a ea o bio ilm o
250 µm2; he mean pe cen age o bac e ial iabili y o he bio ilm
F on ie s in Mic obiology | www. on ie sin.o g 4July 2015 | Volume 6 | A icle 655
P ada-López e al. Di e ences be ween o al an isep ic applica ions
FIGURE 3 | Rep esen a i e images o he PL-bio ilm (“s acked
p ojec ion” o images in he “Z” axis) bac e ial iabili y unde basal
condi ions, a e imme sion and a e mou hwash wi h 0.2%
Chlo hexidine and Essen ial Oils. (They a e images o ep esen a i e ields
o he PL-bio ilm. I is a maximum p ojec ion o all ob ained images in he
plane XY in he Z axis o a same ield. Tha is commonly called “s acked
p ojec ion.” These images do no ep esen no he ou e , he middle o he
inne laye s, hey ep esen all o hem p ojec ed in he same axis).
was calcula ed o he co esponding sample and o each bio ilm
laye .
S a is ical Analysis
The da a on hickness and bac e ial iabili y in he PL-bio ilm,
we e exp essed as mean and s anda d de ia ion o he mean.
The ype o dis ibu ion o he quan i a i e a iables analyzed
was de e mined using he Kolmogo o –Smi no es , ob aining
a no mal dis ibu ion o all alues. Repea ed measu es ANOVA
es and pai wise compa isons (wi h he Bon e oni co ec ion)
we e used o he analysis o in a- and in e -applica ion
esul s o 0.2% CHX and EO and in e -an isep ic solu ion
esul s (including di e en ia ing be ween he 3 bio ilm laye s).
Measu emen s we e s a is ical signi ican i he p alue less
han 0.05. The s a is ical analysis was pe o med by he PASW
S a is ics Base 20 package o Windows (IBM, Mad id, Spain).
Resul s
In luence o he Applica ion o Me hods o 0.2%
CHX and EO on he PL-Bio ilm Thickness
The hicknesses ob ained in bo h baseline disks we e 19.17 and
20.33 µm, be o e applying ei he 0.2% CHX o EO, espec i ely.
A e he Im-0.2% CHX, he hickness was 17.64 µm and
15.77 µm a e he mou hwash. When he applied an isep ic
was he EO, he ob ained hicknesses we e 17.97 and 20.82 µm,
a e imme sion and mou hwash, espec i ely. No signi ican
di e ences we e ound in ei he case.
In luence o he Applica ion Me hods o 0.2%
CHX and EO on he PL-Bio ilm Bac e ial Viabili y
The bac e ial iabili y in he baseline disks was no signi ican ly
di e en be ween he wo se ies o 0.2% CHX and he EO
(72.21 ±10.48% s. 75.72 ±14.33%).
A e he Im-0.2% CHX, he bac e ial iabili y was
signi ican ly educed o 15.17 ±6.14%. In con as , he
bac e ial iabili y a e he Mw-0.2% CHX was 5.08 ±5.79%
(Figu e 4), which was signi ican ly lowe han he Im-0.2% CHX
(p=0.001). In addi ion, bo h esul s di e ed signi ican ly om
hei baseline alues (p<0.001) (Table 1).
In he same way, he Im-EO signi ican ly educed he bac e ial
iabili y o 26.93 ±13.11%. Howe e , a e Mw-EO, he bac e ial
iabili y was educed o 1.16 ±1.00% (Figu e 5), which was
signi ican ly lowe han he Im-EO (p<0.001). Besides, bo h
esul s di e ed signi ican ly om hei baseline alues (p<
0.001) (Table 1).
In he compa ison o he wo an isep ics, he Im-0.2% CHX
ob ained signi ican ly lowe alues o bac e ial iabili y compa ed
wi h he Im-EO (15.17 ±6.14% CHX s. 26.93 ±13.11% EO,
p<0.05). On he o he hand, he Mw-EO achie ed signi ican ly
lowe bac e ial iabili y in compa ison wi h he Mw-0.2% CHX
(1.16 ±1.00% s. 5.08 ±5.79%, p<0.05) (Table 2).
In luence o he Applica ion Me hods o CHX and
EO on he PL-Bio ilm Bac e ial Viabili y by Laye s
When accoun ing o he di e en laye s, he alues o baseline
bac e ial iabili y dec eased p og essi ely o deepe laye s
F on ie s in Mic obiology | www. on ie sin.o g 5July 2015 | Volume 6 | A icle 655
P ada-López e al. Di e ences be ween o al an isep ic applica ions
FIGURE 4 | To al bac e ial iabili y and by PL-bio ilm laye s in he 0.2% Chlo hexidine se ies. (PL-bio ilm, plaque like-bio ilm; 0.2% CHX, 0.2% o
Chlo hexidine; CLSM, con ocal lase scanning mic oscope).
TABLE 1 | In e -applica ion analysis o 0.2% o Chlo hexidine and
Essen ial Oils by laye s.
Applica ion me hod 0.2% Chlo hexidine Essen ial
(in e -applica ion) Oils
TOTAL
Baseline s. imme sion p<0.001 p<0.001
Baseline s. mou hwash p<0.001 p<0.001
Imme sion s. mou hwash p=0.001 p<0.001
LAYER 1
Baseline s. imme sion p<0.001 p<0.001
Baseline s. mou hwash p<0.001 p<0.001
Imme sion s. mou hwash p=0.001 p=0.002
LAYER 2
Baseline s. imme sion p<0.001 p<0.001
Baseline s. mou hwash p<0.001 p<0.001
Imme sion s. mou hwash p=0.001 p<0.001
LAYER 3
Baseline s. imme sion p<0.001 –
Baseline s. mou hwash p<0.001 p<0.001
Imme sion s. mou hwash p=0.046 p<0.001
(baseline sample p io o 0.2% CHX applica ion in laye 1 =
83.91 ±9.51%, laye 2 =78.26 ±9.93%, and laye 3 =54.46 ±
25.43% (Figu e 4 and Table 3); baseline sample p io o EO
applica ion in laye 1 =89.53 ±8.34%, laye 2 =82.12 ±13.26%,
and laye 3 =55.53 ±28.19%) (Figu e 5 and Table 3).
A e he Im-0.2% CHX p o ocol, he sample showed a
homogeneous dec ease in alue om he baseline si ua ion in all
laye s (Im-0.2% CHX, laye 1 =17.90 ±9.16%, laye 2 =16.23 ±
7.14%, and laye 3 =11.38 ±5.81%, p<0.001 in all cases). In
addi ion, a e he Mw-0.2% CHX p o ocol, he bac e ial iabili y
was mo e educed compa ed o he imme sion p o ocol (Mw-
0.2% CHX, laye 1 =5.22 ±6.16%, laye 2 =5.04 ±6.40%, laye
3=4.97 ±5.00%, p<0.05 compa ing Im-0.2% CHX and i s
baseline) (Figu e 4 and Table 1).
A e he Im-EO p o ocol, he sample showed a gene al
dec ease om he baseline disk in he supe icial laye s (Im-
EO, laye 1 =16.18 ±12.38% and laye 2 =25.60 ±14.51%,
p<0.001 in laye s 1 and 2) bu no in he deepes laye (Im-
EO, laye 3 =39.02 ±17.50%). In con as , he esul s a e he
Mw-EO showed a highly educed bac e ial iabili y in all laye s o
he PL-bio ilm, hus i was signi ican ly mo e e ec i e a educing
bac e ial iabili y han he imme sion p o ocol (Mw-EO, laye 1
=1.62±1.54%, laye 2 =1.12 ±1.16%, and laye 3 =0.72 ±
0.56%; p<0.05 compa ing Im-EO and i s baseline) (Figu e 5
and Table 1).
Compa ing he wo an isep ics, al hough bo h showed a
signi ican educ ion o bac e ial iabili y a e imme sion in all
laye s, in laye 3 he 0.2% CHX solu ion showed mo e educed
bac e ial iabili y han did he EO (laye 3, Im-0.2% CHX s. Im-
EO, 11.38 ±5.81% s. 39.02 ±17.50%; p<0.001). In con as ,
in compa ison wi h he mou hwash applica ion, he Mw-EO
ob ained lowe bac e ial iabili y han he Mw-0.2% CHX, in all
laye s (p<0.05) (Table 2).
Discussion
To he bes o he au ho ’s knowledge, he e a e no published
s udies which compa e he immedia e an ibac e ial e ec o an
o al an isep ic applied using he wo me hods e e ed o in
much o he li e a u e (ex i o passi e imme sion and in i o
ac i e mou hwash) wi hin he same expe imen (using he same
olun ee and PL-bio ilm wi hin one g ow h pe iod).
In he p esen se ies, glass disks we e used ins ead o enamel
ones o se e al easons. The i s , and mo e impo an , is ha
p e ious esea ch (Ne uschil e al., 1998) and own’s (unpublished
da a) e ealed ha he e we e no signi ican di e ences in he
bac e ial iabili y and hickness o he 2-day PL-bio ilm o med
on enamel o polished glass analyzed wi h CLSM. This is a e y
F on ie s in Mic obiology | www. on ie sin.o g 6July 2015 | Volume 6 | A icle 655
P ada-López e al. Di e ences be ween o al an isep ic applica ions
FIGURE 5 | To al bac e ial iabili y and by PL-bio ilm laye s in he Essen ial Oils se ies. (PL-bio ilm, plaque like-bio ilm; EO, Essen ial Oils; CLSM, con ocal
lase scanning mic oscope).
TABLE 2 | In e -an isep ic solu ion analysis be ween 0.2% o
Chlo hexidine and Essen ial Oils by laye s.
Applica ion me hod 0.2% Chlo hexidine s.
(in e -an isep ic solu ion) Essen ial Oils
BASELINE
To al –
Laye 1 –
Laye 2 –
Laye 3 –
IMMERSION
To al p=0.007
Laye 1 –
Laye 2 –
Laye 3 p<0.001
MOUTHWASH
To al p=0.020
Laye 1 p=0.041
Laye 2 p=0.034
Laye 3 p=0.006
impo an ac because i is much easie o ob ain a egula glass
disk in size and hickness han om enamel, being his c ucial
o he cons uc ion o he in ao al splin s and he s abili y o
he disks in o i . Ano he eason is he di icul y o analysing he
bio ilm o med on an enamel disk due o he i egula i y (i is no
a plane su ace) gi ing dis o ed images a he CLSM. In addi ion,
al hough he s e ili y o he enamel disks could be achie ed easily,
he e always could exis he la en isk o p ion diseases, which
would b ing an impo an mo al dilemma.
PL-bio ilm Thickness
The p esen se ies showed a cons an hickness o he PL-bio ilm
a e all applied si ua ions. The applica ion o he an isep ic,
ei he by imme sion o mou hwash, did no change he basal
TABLE 3 | In a-applica ion analysis o 0.2% o Chlo hexidine and
Essen ial Oils by laye s.
Applica ion me hod 0.2% Chlo hexidine Essen ial
(in a-applica ion) Oils
BASELINE
Laye 1 s. Laye 2 p=0.038 p=0.021
Laye 1 s. Laye 3 p=0.005 p=0.001
Laye 2 s. Laye 3 p=0.006 p=0.001
IMMERSION
Laye 1 s. Laye 2 –p=0.004
Laye 1 s. Laye 3 –p<0.001
Laye 2 s. Laye 3 –p<0.001
MOUTHWASH
Laye 1 s. Laye 2 –p=0.021
Laye 1 s. Laye 3 – –
Laye 2 s. Laye 3 – –
hickness. The p e ious li e a u e suppo his si ua ion, no
ma e he an isep ic solu ion o he me hodology o applica ion
used in he s udies (Zau a-A i e e al., 2001; Dong e al., 2010;
on Ohle e al., 2010; Ga cía-Caballe o e al., 2013; Quin as e al.,
2015), so ha a single applica ion o 0.2% CHX o EO does no
a ec he hickness o a ma u e bio ilm.
PL-bio ilm Bac e ial Viabili y
In he li e a u e, he mean bac e ial iabili y in a 2-day PL-bio ilm
oscilla ed be ween 60 and 77% ( on Ohle e al., 2010; Gu e al.,
2012; Ga cía-Caballe o e al., 2013; P ada-López e al., 2015a;
Quin as e al., 2015), he p esen se ies showed esul s in his
ange (72 and 76% in bo h baseline si ua ions, be o e CHX and
EO applica ions, espec i ely).
The mean bac e ial iabili y a e an Im-0.2% CHX anged
om 0.7 o 35.16% in he p e ious scien i ic li e a u e (Zau a-
A i e e al., 2001; Gosau e al., 2010; on Ohle e al., 2010). Such
F on ie s in Mic obiology | www. on ie sin.o g 7July 2015 | Volume 6 | A icle 655
P ada-López e al. Di e ences be ween o al an isep ic applica ions
wide a ia ions migh be caused by he di e en me hodologies
used in he s udies. These di e ences mainly a ise because o
a ange o ac o s, such as he an isep ic concen a ion o he
ime lapse o applica ion. In hei s udy on PL-bio ilm, on
Ohle e al. (2010) chose a 0.1% CHX concen a ion and hei
imme sion imes a ied be ween 1 and 10 min. This p o ocol
con as s wi h ha ollowed by Zau a-A i e e al. (2001) and
Gosau e al. (2010), who e alua ed a comme cial 0.2% CHX
concen a ion, and selec ed an imme sion ime lapse o 1 min.
Despi e he ob ious me hodological di e ences wi h o he se ies
( ime o exposu e and concen a ion), he esul s o he p esen
s udy in e ms o bac e ial iabili y o he PL-bio ilm a e an
Im-0.2% CHX (app oxima ely a 15%) a e in acco dance wi h
he esul s epo ed in he p e ious li e a u e (Zau a-A i e e al.,
2001; Gosau e al., 2010; on Ohle e al., 2010). Respec o he
applied CHX p o ocol, he manu ac u e ecommenda ions in
e ms o ime o applica ion we e ollowed (30 s).
Rega ding he Mw-0.2% CHX, he e a e ew e alua ions o he
bac e ial iabili y o he PL-bio ilm a e an ac i e mou hwash
wi h his an isep ic (Ga cía-Caballe o e al., 2013; Quin as e al.,
2015). In he p esen se ies, he bac e ial iabili y was nea o
5% which is consis en wi h ha epo ed in p e ious s udies
(Ga cía-Caballe o e al., 2013; Quin as e al., 2015).
Conce ning he mean bac e ial iabili y a e an Im-EO, he
esul s ound in he li e a u e anged om 23 o 31% (Dong
e al., 2010; Gosau e al., 2010). In hese cases, he an isep ic
concen a ion did no a y om one s udy o ano he . This is
p obably one o he easons why he ange is na owe o he EO
han o he 0.2% CHX. Gosau e al. (2010) and Dong e al. (2010)
imme sed a 12 and 48 h-PL-bio ilm o 1 min, espec i ely. The
p esen se ies showed a mean bac e ial iabili y wi hin he named
ange (app oxima ely a 27%).
In ega d o he assessmen o he e icacy o an ac i e Mw-EO,
o he bes o he au ho ’s knowledge, he e is only one s udy in
which he bac e ial iabili y o he PL-bio ilm has been e alua ed
(Quin as e al., 2015). This s udy showed a e y low bac e ial
iabili y 30 s a e he Mw-EO, nea o 1%, which is simila o
he p esen esul s.
Despi e he isible lack o p e ious li e a u e, he esul s
o he p esen se ies, in e ms o bac e ial iabili y, ha e a
clea in e p e a ion, acco ding o he au ho s: doing an ac i e
mou hwash g ea ly educes he bac e ial iabili y o he PL-
bio ilm, mo e so han doing an imme sion wi h he same
an isep ic. When di e en ia ing be ween 0.2% CHX and EO, we
ound doing an Im-0.2% CHX was mo e e ec i e han doing an
Im-EO.
On he o he hand, when a mou hwash was done, he EO
solu ion was mo e e ec i e han he 0.2% CHX. In p e ious
esea ch o he au ho s (Quin as e al., 2015), he immedia e e ec
o he EO s. 0.2% CHX has been al eady p esen ed and discussed
in de ail.
PL-bio ilm Bac e ial Viabili y by Laye s
The dis ibu ion o he bac e ial iabili y in o he PL-bio ilm in
he baseline disks was signi ican ly lowe in he deepes laye s.
This dis ibu ion pa e n o iabili y, in which i al bac e ia
o e lay non- i al bac e ia, has been p e iously desc ibed in o he
in i o bio ilm s udies ha analyzed bac e ial iabili y in laye s
(A weile e al., 2004; Ga cía-Caballe o e al., 2013; P ada-López
e al., 2015a; Quin as e al., 2015).
A e he Im-0.2% CHX, he bac e ial iabili y dec eased
signi ican ly in all laye s. Howe e , he e we e no di e ences
among he laye s (18% ou e , 16% middle and 11% inne laye ).
Zau a-A i e e al. (2001) analyzed he bac e ial iabili y in he
di e en laye s a e a 1 min imme sion in 0.2% CHX showing i s
e icacy as well, bu wi h wide anges o alues (ou e laye =16–
42%, middle laye =19–55% and inne laye =21–58%). These
da a we e p obably ob ained due o he small sample size and he
cha ac e is ics o he olun ee s ( h ee hea y plaque- o me s and
h ee ligh plaque- o me s).
On he o he hand, he Mw-0.2% CHX educed he bac e ial
iabili y simila ly in all laye s, being hese indings in acco dance
wi h hose p e iously desc ibed in si u s udies (Ga cía-Caballe o
e al., 2013; Quin as e al., 2015). This educ ion in bac e ial
iabili y ob ained by mou hwash, as eco ded by laye s, was
highe han he ob ained om he imme sion me hod.
In he p esen se ies, when applying he EO an isep ic, he
bac e ial iabili y o he PL-bio ilm was educed in all laye s a e
he imme sion. The ou e laye showed signi ican less iabili y
han he o he wo (16% in he ou e laye s. 26% in he middle
and 39% in he deepes laye ). In he same manne , Dong e al.
(2010), a e a 1-min Im-EO ound less bac e ial iabili y in he
ou e laye (ou e laye =22% s. middle laye =34%, and
inne laye =37%). In bo h s udies, a educ ion in he bac e ial
iabili y was shown, bu a di e en spa ial dis ibu ion in he
bac e ial iabili y compa ed o hei baselines could be seen
(Figu e 5). While in he baseline sample he bac e ial iabili y
dec eased om he ou e o he inne laye s, a e an Im-EO, his
dis ibu ion was in e ed, showing an inc ease in he bac e ial
iabili y om he ou e o he inne laye s. This ac could be
explained by he low capaci y o he EO solu ion o pene a e
ma u e bio ilms, esul ing in a loss o e icacy in he deepes laye s
o he PL-bio ilm. In he con as , his e ec seen a e he Im-EO
was comple ely los a e he Mw-EO. The EO applied ollowing
he manu ac u e ’s ins uc ions (a single mou hwash wi h 20 mL
o 30 s) was clea ly mo e e ec i e in all laye s han he simple
imme sion, achie ing bac e ial iabili y esul s nea o 0% in he
h ee laye s.
The esul s o he p esen se ies con i med ha , o p ope ly
assess he immedia e an ibac e ial e ec o 0.2% CHX and
EO, an in i o ac i e mou hwash ollowing he manu ac u e ’s
ecommenda ions should be done. This se ies has also shown ha
an ac i e mou hwash helped o maximize he e icacy o he 0.2%
CHX solu ion and, mainly, he EO solu ion compa ed o a single
imme sion. In addi ion, he indings o his in es iga ion sugges
cau ious in e p e a ion o he esul s o s udies ha ollowed an
ex i o an isep ic applica ion (imme sion) in PL-bio ilm. This
p e ious li e a u e abou an imic obial ac i i y o bo h 0.2% CHX
and EO elies on in i o and in si u s udies ha do no ollow
p ope me hodologies, p e ending o equa e a simple imme sion
(ex i o) wi h an ac i e mou hwash (in i o). To some ex en ,
his equa ion could be conside ed alid when isola ed bac e ia
a e s udied. Howe e , when alking abou bac e ia associa ed in
a mo e complex s uc u e such as a na u ally- o med bio ilm
F on ie s in Mic obiology | www. on ie sin.o g 8July 2015 | Volume 6 | A icle 655
P ada-López e al. Di e ences be ween o al an isep ic applica ions
( ha is 10–1000 imes mo e esis an han bac e ia in plank onic
phase) (Fine e al., 2001), some hing mo e han he simple
con ac wi h he an isep ic is needed. This highe ac i i y o
he an isep ic when applied as a mou hwash could be due o
he hyd odynamic o ces ha appea in he mou h hank o
he ac ion o he ongue, cheeks and o he muscles o he
o al ca i y ha con ibu e o he mo emen o he mou hwash
h oughou all he su aces o he mou h. This mo emen could
achie e some hing ha he passi e imme sion could no , which
is b eaking o he su ace o ce o he PL-bio ilm, being his
c ucial o he an isep ic pene a ion. This is he same heo y
ollowed in endodon ics wi h he “hyd odynamic ac i a ion” o
he an isep ic in he in e io o he adicula canal by agi a ion
(Welle e al., 1980; Pee e s e al., 2014). This mo emen achie es
o b eak he su ace o ce o small oo canals con ibu ing o
maximize he chemical ac ion o he an isep ic (Pee e s e al.,
2014).
Finally, he au ho s would like o poin ou ano he possible
di e en ia ing a iable which may condi ion he an isep ic
e ec i eness: he empe a u e. In he ex i o expe imen , he
an isep ic was a oom empe a u e (be ween 18 and 20◦C).
Howe e , when he applica ion was in i o, he empe a u e
o he an isep ic solu ion ose by se e al deg ees. In u u e
in es iga ions, i would be in e es ing o s udy he ole ha
he empe a u e migh play in he an imic obial e ec o he
an isep ic.
Conclusion
The me hod o applica ion condi ioned he an ibac e ial ac i i y
o he 0.2% Chlo hexidine and he Essen ial Oils on he
plaque-like bio ilm. The in i o ac i e mou hwash p o ocol
was mo e e ec i e han he ex i o passi e imme sion in bo h
an isep ic solu ions, condi ioning he ob ained esul s. The e
was mo e pene a ion o he an isep ic inside he bio ilm wi h
an ac i e mou hwash, especially when he Essen ial Oils we e
used.
Acco ding o he esul s o he p esen s udy, u u e
in es iga ions on o al an isep ics should ake in o accoun he
me hodology o he applica ion. To ob ain a si ua ion as close
as possible o he clinical eali y, he plaque-like bio ilm should
be o med in i o. In addi ion, he an isep ic applica ion should
be in si u, wi h an ac i e mou hwash o , a leas , ake in o
conside a ion he ole ha he mo emen o he solu ion may
ha e in he an isep ic an imic obial ac i i y.
Au ho Con ibu ions
Concep ion and design he expe imen s: IT, JS, DS. Pe o med
he expe imen s: IP, VQ. Analyzed da a: IT, MACDC.
In e p e a ion o he da a: IP, VQ. D a ing and e ising
he manusc ip : IP, VQ, MACDC, IT. Final app o al: IT, JS, DS.
Ag eemen : IP, VQ, MACDC, JS, DS, IT.
Acknowledgmen s
This wo k was suppo ed by p ojec PI11/01383 om Ca los III
Ins i u e o Heal h (Gene al Di ision o E alua ion and Resea ch
P omo ion, Mad id, Spain), which is in eg a ed in Na ional
Plan o Resea ch, De elopmen and Inno a ion (PN I+D+I
2008-2011). This p ojec was co inanced by Eu opean Regional
De elopmen Fund (ERDF 2007-2013). The unde s had no ole
in s udy design, da a collec ion and analysis, decision o publish,
o p epa a ion o he manusc ip .
Supplemen a y Ma e ial
The Supplemen a y Ma e ial o his a icle can be ound
online a : h p://jou nal. on ie sin.o g/a icle/10.3389/ micb.
2015.00655
Re e ences
A weile , N. B., Hellwig, E., Sculean, A., Hein, N., and Auschill, T. M. (2004).
Indi idual i ali y pa e n o in si u den al bio ilms a di e en loca ions in he
o al ca i y. Ca ies Res. 38, 442–447. doi: 10.1159/000079625
Auschill, T. M., A weile , N. B., Ne uschil, L., B ecx, M., Reich, E., and
Sculean, A. (2001). Spa ial dis ibu ion o i al and dead mic oo ganisms in
den al bio ilms. A ch. O al. Biol. 46, 471–476. doi: 10.1016/S0003-9969(00)
00136-9
Auschill, T. M., Hein, N., Hellwig, E., Follo, M., Sculean, A., and A weile ,
N. B. (2005). E ec o wo an imic obial agen s on ea ly in si u bio ilm
o ma ion. J. Clin. Pe iodon ol. 32, 147–152. doi: 10.1111/j.1600-051X.2005.
00650.x
Auschill, T. M., Hellwig, E., Sculean, A., Hein, N., and A weile , N. B. (2004).
Impac o he in ao al loca ion on he a e o bio ilm g ow h. Clin. O al
In es ig. 8, 97–101. doi: 10.1007/s00784-004-0255-6
Axelsson, P. (2004). P e en i e Ma e ials, Me hods and P og ams, 1s Edn. Su ey:
Quin essence.
Co bin, A., Pi s, B., Pa ke , A., and S ewa , P. S. (2011). An imic obial pene a ion
and e icacy in an in i o o al bio ilm model. An imic ob. Agen s Chemo he . 55,
3338–3344. doi: 10.1128/AAC.00206-11
Da ies, D. (2003). Unde s anding bio ilm esis ance o an ibac e ial agen s. Na .
Re . D ug Disco . 2, 114–122. doi: 10.1038/n d1008
Dong, W. L., Zhou, Y. H., Li, C. Z., Liu, H., Shang, S. H., and Pan, B. Q. (2010).
Es ablishmen and applica ion o an in ac na u al model o human den al
plaque bio ilm. Shanghai Kou Qiang Yi Xue 19, 196–201.
Fine, D. H., Fu gang, D., and Ba ne , M. L. (2001). Compa a i e an imic obial
ac i i ies o an isep ic mou h inses agains isogenic plank onic and bio ilm
o ms o Ac inobacillus ac inomyce emcomi ans. J. Clin. Pe iodon ol. 28,
697–700. doi: 10.1034/j.1600-051x.2001.028007697.x
Fine, D. H., Fu gang, D., Sina a, K., Cha les, C., McGui e, A., and Kuma , L. D.
(2005). In i o an imic obial e ec i eness o an essen ial oil-con aining mou h
inse 12 h a e a single use and 14 days’ use. J. Clin. Pe iodon ol. 32, 335–340.
doi: 10.1111/j.1600-051x.2005.00674.x
Fuchsloche Hellemann, C., G ade, S., Heue , W., Di me , M. P., S iesch, M.,
Schwes ka-Polly, R., e al. (2013). Th ee-dimensional analysis o ini ial bio ilm
o ma ion on poly e a luo oe hylene in he o al ca i y. J. O o ac. O hop. 74,
458–467. doi: 10.1007/s00056-013-0174-8
Ga cía-Caballe o, L., Quin as, V., P ada-López, I., Seoane, J., Donos, N.,
and Tomás, I. (2013). Chlo hexidine subs an i i y on sali a y lo a
and plaque-like bio ilm: an in si u model. PLoS ONE 8:e83522. doi:
10.1371/jou nal.pone.0083522
Gosau, M., Hahnel, S., Schwa z, F., Ge lach, T., Reiche , T. E., and Bu ge s, R.
(2010). E ec o six di e en pe i-implan i is disin ec ion me hods on in i o
human o al bio ilm. Clin. O al Implan s Res. 21, 866–872. doi: 10.1111/j.1600-
0501.2009.01908.x
F on ie s in Mic obiology | www. on ie sin.o g 9July 2015 | Volume 6 | A icle 655
