micb-11-00553 Ap il 15, 2020 Time: 19:35 # 1
ORIGINAL RESEARCH
published: 17 Ap il 2020
doi: 10.3389/ micb.2020.00553
Edi ed by:
Sujogya Kuma Panda,
KU Leu en, Belgium
Re iewed by:
Pila Teixei a,
Uni e si y o Minho, Po ugal
Fe nanda Gomes,
Uni e si y o Minho, Po ugal
*Co espondence:
Alex Mi a
[email p o ec ed]
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: 20 Decembe 2019
Accep ed: 13 Ma ch 2020
Published: 17 Ap il 2020
Ci a ion:
Žiemy ˙
e M, Rod íguez-Díaz JC,
Ven e o MP, Mi a A and Fe e MD
(2020) E ec o Dalba ancin on
S aphylococcal Bio ilms When
Adminis e ed Alone o in Combina ion
Wi h Bio ilm-De aching Compounds.
F on . Mic obiol. 11:553.
doi: 10.3389/ micb.2020.00553
E ec o Dalba ancin on
S aphylococcal Bio ilms When
Adminis e ed Alone o in
Combina ion Wi h Bio ilm-De aching
Compounds
Miglë Žiemy ˙
e1, Juan C. Rod íguez-Díaz2, Ma ía P. Ven e o2, Alex Mi a1,3*and
Ma ía D. Fe e 1,3
1Genomics and Heal h Depa men , FISABIO Founda ion, Valencia, Spain, 2Se icio de Mic obiología, Hospi al Gene al
Uni e si a io de Alican e, ISABIAL, Alican e, Spain, 3CIBER Epidemiología y Salud Pública, Mad id, Spain
Mic oo ganisms g own in bio ilms a e mo e esis an o an imic obial ea men and
immune sys em a acks compa ed o hei plank onic o ms. In ac , in ec ions caused
by bio ilm- o ming S aphylococcus au eus and S aphylococcus epide midis a e a la ge
h ea o public heal h, including pa ien s wi h medical de ices. The aim o he cu en
manusc ip was o es he e ec o dalba ancin, a ecen ly de eloped lipoglycopep ide
an ibio ic, alone o in combina ion wi h compounds con ibu ing o bac e ial cell
disagg ega ion, on s aphylococcal bio ilm o ma ion and elimina ion. We used eal-
ime impedance measu emen s in mic o i e pla es o s udy bio ilm g ow h dynamics
o S. au eus and S. epide midis s ains, in he absence o p esence o dalba ancin,
linezolid, ancomycin, cloxacillin, and i ampicin. Fu he expe imen s we e unde aken
o check whe he bio ilm-de aching compounds such as N-ace ylcys eine (NAC) and
icin could enhance dalba ancin e iciency. Real- ime dose– esponse expe imen s
showed ha dalba ancin is a highly e ec i e an imic obial, p e en ing s aphylococcal
bio ilm o ma ion a low concen a ions. Minimum bio ilm inhibi o y concen a ions
we e up o 22 highe compa ed o s anda d E- es alues. Dalba ancin was he only
an imic obial ha could hal new bio ilm o ma ion on es ablished bio ilms compa ed o
he o he ou an ibio ics. The addi ion o NAC dec eased dalba ancin e icacy while
he combina ion o dalba ancin wi h icin was mo e e icien han an ibio ic alone in
p e en ing g ow h once he bio ilm was es ablished. Resul s we e con i med by classical
bio ilm quan i ica ion me hods such as c ys al iole (CV) s aining and iable colony
coun ing. Thus, ou da a suppo he use o dalba ancin as a p omising an imic obial o
ea bio ilm- ela ed in ec ions. Ou da a also highligh ha syne gis ic and an agonis ic
e ec s be ween an ibio ics and bio ilm-de aching compounds should be ca e ully es ed
in o de o achie e an e icien ea men ha could p e en bo h bio ilm o ma ion
and dis up ion.
Keywo ds: dalba ancin, E- es , bio ilm, S aphylococcus, icin, N-ace ylcys eine, xCELLigence
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INTRODUCTION
Inc eased d ug esis ance o bac e ia is signi ican ly educing he
he apeu ic e icacy o an ibio ics (S ewa and Cos e on, 2001).
Commonly, his esis ance is augmen ed in bac e ial bio ilms,
which can be desc ibed as bac e ial communi ies adhe ing
o abio ic o bio ic su aces and encased in a sel -p oduced
ex acellula ma ix (Chung and Toh, 2014;Hall and Mah,
2017). This ma ix is composed o polysaccha ides, p o eins, and
ex acellula DNAs and plays an impo an ole in pe sis en
ch onic in ec ions, esul ing in se ious heal h complica ions.
In ac , bac e ial cells embedded in a ma ix a e up o 1,000
imes mo e esis an o an ibac e ial compounds compa ed
o hei plank onic o m, leading o inc eased mo bidi y and
mo ali y a es o a ious diseases, like hose associa ed wi h
implan able medical de ices (Flemming and Wingende , 2010;
Rod igues, 2011).
A majo cause o medical de ice-associa ed and ch onic
in ec ions, esul ing in bo h economical and clinical bu den,
is he bio ilm o ma ion capaci y o S aphylococcus au eus and
S aphylococcus epide midis bac e ia (O o, 2013;Moo meie and
Bayles, 2017). This capaci y, in addi ion o he widesp ead
dissemina ion o me hicillin- esis an S. au eus (MRSA) and
S. epide midis (MRSE), emphasizes he necessi y o in es iga e
new an imic obial compounds and combine di e en ea men
s a egies o inc easing he he apeu ic po en ial o con en ional
an ibio ics (Bja nshol e al., 2013). Fo ins ance, se e al agen s
o cell de achmen and b eaking down o bio ilm ma ix
ha e al eady been epo ed. Some o hem clea e he essen ial
componen s o he bio ilm ma ix, like polysaccha ides, p o eins,
o ex acellula DNAs, des oying i s a chi ec u e (Kaplan, 2010;
Fleming and Rumbaugh, 2017). Ficin, a non-speci ic ig ee plan
p o ease, belongs o his g oup o an i-bio ilm compounds and
is able o dispe se s aphylococcal bio ilms ia enzyma ic lysis
(Baidamshina e al., 2017). O he s employ mic obial signals ha
dispe se bac e ial cells embedded inside he bio ilm exopolyme ic
ma ix, like ni ic oxide in Pseudomonas bio ilms o ce ain
quo um sensing inhibi o s (B ackman and Coenye, 2015;Zhu
e al., 2019). O he an i-bio ilm agen s, like N-ace yl-L-cys eine
(NAC), besides he abili y o impai ma ix a chi ec u e, ha e also
an imic obial p ope ies agains di e en pa hogenic bac e ia,
making his molecule an in e es ing ool o con on bio ilms
(Dinicola e al., 2014;Blasi e al., 2016;Cos a e al., 2017).
In addi ion, a combina ion o bio ilm-de aching compounds
oge he wi h an ibio ics could ep esen an al e na i e s a egy
o he e ec i e ea men o bio ilm-associa ed in ec ions.
Dalba ancin is a new lipoglycopep ide class an ibio ic used
agains many g am-posi i e pa hogens including s aphylococcal
s ains in clinical p ac ice (Chen e al., 2007). I is also a long-
ac ion an ibio ic ha in e e es wi h bac e ial cell wall syn hesis
and does no equi e equen adminis a ion, allowing weekly
dosing and ea lie pa ien discha ge om he hospi al (Sel ze
e al., 2003). Al hough dalba ancin has been p oposed as a
p omising agen in bio ilm-media ed in ec ions, suscep ibili y
o his an ibio ic has mainly been es ed using adi ional
mic obiological es s such as mic odilu ion o aga -based es s.
Howe e , i is well es ablished ha bac e ia beha e di e en ly in a
plank onic s a e o when o ming bio ilms, and he e is cu en ly
limi ed in o ma ion on i s e icacy on bio ilm-embedded bac e ia,
wi h only a ew s udies a ailable (Meeke e al., 2016;Kna l e al.,
2017;Di Pila o e al., 2020), some o hem in animal models
(Da ouiche and Mansou i, 2005;Baldoni e al., 2013). In some
cases, he e icacy o dalba ancin o ancomycin has been shown
o be low, wi h less han an o de -o -magni ude dec ease in iable
coun s o s aphylococci (Kussmann e al., 2018). Thus, ecen
wo k has p oposed he combina ion o an ibio ics and bio ilm-
de aching compounds o ea bio ilm-media ed in ec ions (Chen
e al., 2013;Roy e al., 2018), bu his s a egy has cu en ly no
been es ed wi h dalba ancin. In addi ion, he e is con lic ing
e idence abou he compa a i e e icacy o dalba ancin and
o he an ibio ics o common clinical use in s aphylococcal
in ec ions, such as ancomycin (Da ouiche and Mansou i, 2005;
Kussmann e al., 2018).
Recen ly, we e alua ed bio ilm inhibi ion and induc ion in
S. au eus and S. epide midis s ains using 10 con en ional
an ibio ics and sugges ed ha impedance-based eal- ime cell
analysis (RTCA) could acili a e de e mina ion o an ibio ic
sensi i i y when bac e ia g ow in bio ilms, esul ing in as e and
mo e accu a e assays and he e o e mo e e icien an imic obial
he apy (Fe e e al., 2017b). The aim o he cu en s udy was
o desc ibe he e ec i eness o dalba ancin o p e en in i o
bio ilm o ma ion o s aphylococcal s ains (bo h sensi i e
and me hicillin- esis an isola es) and compa e i s e ec wi h
o he an ibio ics ha a e equen ly used in clinical p ac ice
agains indwelling de ice- ela ed in ec ions. Ou bio ilm g ow h
measu emen s we e pe o med by impedance-based cell analysis
and con i med by mo e classical es s such as c ys al iole
(CV) s aining and coun ing o colony- o ming uni s (CFUs).
In addi ion, he e ec o wo bio ilm-disagg ega ing molecules,
NAC and icin, was es ed in combina ion wi h he an ibio ic,
o e alua e he po en ial syne gy o a combined he apy o ea
s aphylococcal bio ilm in ec ions.
MATERIALS AND METHODS
Bac e ial S ains and G ow h Condi ions
Supplemen a y Table S1 lis s bac e ial s ains used o his
s udy. S aphylococcal s ains we e g own on yp ic soy aga
(TSA) pla es and yp ic soy b o h (TSB) a 37◦C a 120 .c. .
S. epide midis s ain 43040 was isola ed a he Mic obiology
Depa men o he Uni e si y o Elche (Spain), MRSA s ains
we e isola ed a he Mic obiology Depa men o he Alican e
Gene al Hospi al (Spain) om a ca he e ip in pa ien s diagnosed
wi h indwelling de ice- ela ed bac e emia. S. au eus CETC 240
(S. au eus ssp. au eus Rosenbach 1884) is a bio ilm-posi i e s ain
isola ed by FDA, which is me hicillin suscep ible, and a e e ence
s ain ecommended o es an ibio ic esis ance.
RTCA-Based Bio ilm Analysis
Real- ime bio ilm analysis was pe o med using xCELLigence
RTCA SP equipmen (ACEA Biosciences) acco ding o he
manu ac u e ’s ins uc ions. Fo bio ilm o ma ion assays,
bac e ial s ains we e g own o e nigh in TSB and dilu ed wi h
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e e al. Dalba ancin E ec on Bio ilms
il e -s e ilized TSB supplemen ed wi h 0.25% o D-glucose (TSB-
glu). The expe imen s we e pe o med as p e iously desc ibed
by Fe e e al. (2017b). Impedance da a we e egis e ed a 10-
min ime in e als o 20 h, and hey we e ans o med in o cell
index (CI) alues, which accu a ely co ela e wi h bio ilm mass
(Fe e e al., 2017a,b).
To e alua e an imic obial e iciency on bac e ial bio ilms,
i e an ibio ics wi h di e en mechanisms o ac ion we e
es ed: linezolid (Acco dpha ma), ancomycin (P ize ),
cloxacillin (No mon), i ampicin (Ma i), and dalba ancin
(Angelini). One hund ed mic oli e s o each an ibio ic dilu ed
in TSB-glu ( wo old dilu ions o inal concen a ions om
32 o 0.0625 mg/L) was used as backg ound o impedance
measu emen s. Fu he , 100 µl o bac e ial cell suspension
(OD600 = 0.175) was added, eaching a inal op ical densi y
o 0.0875. This op ical densi y co esponds o 107–108cells,
depending on he s ain. The lowes an ibio ic concen a ion
equi ed o inhibi bac e ial g ow h wi h a CI alue ≤0.05 was
conside ed as he minimum bio ilm inhibi o y concen a ion
(MBIC) (Bja nshol e al., 2013;Fe e e al., 2017b).
To es he an ibio ic e ec on al eady- o med bac e ial
bio ilms, he expe imen s we e pe o med as p e iously desc ibed
(Fe e e al., 2017a). B ie ly, 100 µl o cell suspension
(OD600 = 0.153) was used as backg ound. Then, 75 µl o TSB-
glu was added o each well, eaching a inal OD600 = 0.0875,
and bio ilms we e g own o 6 h (S. au eus 240), 7 h (S. au eus
MRSA4), o 9 h (S. epide midis 43040), co esponding o
he exponen ial phase o bio ilm g ow h o each s ain a
which ime he an ibio ics we e added (25 µl o each dilu ion,
eaching inal concen a ions om 32 o 0.0625 mg/L o each
es ed an ibio ic). A e he addi ion o an ibio ics, CI was
moni o ed o a u he 20 h. Two eplica es o each an ibio ic
concen a ion sample and wo nega i e con ols we e included in
each expe imen .
An ibio ilm Compounds Assays
Fo RTCA expe imen s, icin (Sigma) a concen a ions o 10,
100, and 1,000 mg/L and NAC (Sandoz) a inal concen a ions
o 0.5, 1, 2, 4, and 8 g/L we e used alone and in combina ion
wi h dalba ancin (0.5, 4, and 32 mg/L). In sho , icin and NAC
we e dilu ed in TSB-glu o he co esponding concen a ions, and
100 µl o each dilu ion was used as backg ound when an i-bio ilm
subs ances we e added a he beginning o he expe imen .
A e he backg ound was measu ed, 100 µl o cell suspensions
(OD600 = 0.175) was added in o he co esponding wells, and
bio ilm o ma ion was moni o ed o 20 h.
When NAC and icin we e added a he exponen ial bio ilm
g ow h phase, 100 µl o co esponding cell suspensions was
used as backg ound as desc ibed abo e. A e ha , 75 µl o he
TSB-glu was added, eaching a inal OD600 o 0.0875. When
bac e ial bio ilm g ow h eached an exponen ial g ow h phase,
25 µl o he di e en concen a ions es ed o NAC o icin
and hei combina ions wi h dalba ancin was added in o he
co esponding wells. A e he addi ion o bio ilm-de aching
compounds, bio ilm g ow h was egis e ed o 20 h mo e. Two
eplica es o each condi ion and hei espec i e con ols we e
es ed in each expe imen .
The e ec o NAC and icin on plank onic bac e ial g ow h was
also measu ed, by means o an abso bance pla e eade In ini e
M200 (Tecan, Du ham, NC, Uni ed S a es). B ie ly, o e nigh
bac e ial cul u es we e dilu ed o OD600 = 0.175, and 100 µl o
each cell suspension was added in o he co esponding wells o
96-well pla es. Then, 100 µl o bio ilm-de aching subs ances was
added o he inal concen a ions o 10, 100, and 1,000 mg/L o
icin and 0.5, 1, 2, 4, 8, 16, and 32 g/L o NAC. Nine y-six-well
pla es we e incuba ed a 37◦C wi h o bi al shaking a 120 pm,
and bac e ial plank onic g ow h dynamics we e moni o ed o
20 h. Two eplica es o each concen a ion we e included as well
as hei espec i e con ols.
MIC De e mina ion
To de e mine minimum inhibi o y concen a ions (MICs) o he
es ed an ibio ics on S. au eus and S. epide midis s ains on solid
media, he E- es (bioMe ieux) me hod was used acco ding o
he manu ac u e ’s ins uc ions, ollowing Baldoni e al. (2013).
B o h mic odilu ion assays we e pe o med in acco dance wi h
he Eu opean Commi ee on An imic obial Suscep ibili y Tes ing
(EUCAST, 2018). MBIC (o BIC) was calcula ed ollowing
Bja nshol e al. (2013) and Fe e e al. (2017b).
Bio ilm Quan i ica ion
In o de o de e mine dalba ancin and icin e ec alone and in
combina ion on p e o med MRSA4 bio ilms, 175 µl o bac e ial
suspension (OD600 = 0.0875) was inocula ed in TSB-glu and
g own in 96-well la -bo om Ibidi ibiT ea µ-pla es 89626 (Ibidi,
Ge many). These pla es a e coa ed wi h a hin polyme laye in
o de o assu e be e bio ilm a achmen . A e 7 h o g ow h,
25 µl o dalba ancin, icin, o he combina ion o hese wo
compounds was added, eaching inal concen a ions o 32 mg/L
and 1 g/L, and he bio ilms we e g own o an addi ional 24 h.
A e ha , he supe na an was disca ded, bac e ial cells we e
washed using phospha e bu e saline (PBS, pH = 7.4) o emo e
unadhe ed cells, and bac e ial bio ilms we e s ained using 0.1%
CV as p e iously desc ibed (S epano ic e al., 2000). Bio ilm
mass was quan i ied by an abso bance pla e eade , In ini e M200
(Tecan, Du ham, NC, Uni ed S a es), a 610 nm.
Viable Coun Assay
To assess he numbe o iable unadhe ed, plank onic bac e ia
and bio ilm-embedded bac e ia, 175 µl o MRSA4 suspension
(OD600 = 0.0875) was g own o 7 h in iplica e in he
xCELLigence sys em. A e ha , 25 µl o icin and dalba ancin
a he co esponding concen a ions was added as desc ibed
abo e, and he bio ilms we e cul i a ed o an addi ional 24 h.
The supe na an was hen collec ed, and se ial dilu ions we e
p epa ed, using 100 µl o each dilu ion o pla ing on o TSA
pla es in iplica e.
To e alua e iable cell numbe in bac e ial bio ilms, bio ilms
we e ca e ully insed using PBS bu e o elimina e non-adhe ed
cells, esuspended wi h 200 µl o PBS and sonica ed o 5 min in
o de o dis up bio ilm ma ix, and he se ial dilu ions o each
sample we e pla ed on TSA pla es in iplica e as desc ibed abo e
and incuba ed a 37◦C o e nigh . A e ha , CFUs we e coun ed,
a e aged, and exp essed as log10.
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S a is ical Analysis
To s udy di e ences in he bio ilm CI alues, eg ession analysis
was pe o med by a linea model, using he unc ion lm (lib a y
s a s) in he R s a is ical package (Calcagno, 2013) be ween 10 and
20 h o bio ilm o ma ion ime. Fo bio ilm inhibi ion/induc ion
analyses o CFUs and CV s aining, expe imen s we e pe o med
in iplica e wi h h ee independen epea s in each expe imen .
S a is ical signi icance was assessed using S uden ’s - es , whe e
∗p≤0.0 and ∗∗∗p≤0.001.
RESULTS
Dalba ancin E ec on S aphylococcal
Bio ilm Fo ma ion
Fi s ly, we e alua ed dalba ancin’s e ec on s aphylococcal
bio ilm o ma ion by eal- ime impedance analysis when he
an ibio ic was added oge he wi h he bac e ial inoculum. Mos
o he es ed S. au eus and S. epide midis s ains had simila
bio ilm g ow h dynamics wi h compa able CI (co esponden o
FIGURE 1 | Dalba ancin e ec on bac e ial bio ilm o ma ion in
S aphylococcus au eus 240 (A),S. au eus MRSA 4 (B), and S aphylococcus
epide midis 43040 (C) s ains. The cell index (CI) alues a e measu ed by
impedance in an xCELLigence equipmen and co ela e wi h o al bio ilm
mass. Red as e isks indica e dalba ancin- ee con ols. Dalba ancin was
added om he beginning o he expe imen a concen a ions om 0.0625 o
32 mg/L as indica ed in he legend. Da a a e he means o wo eplica es.
bio ilm mass) alues. MRSA4 was he s ain wi h he highes
bio ilm o ma ion capaci y (up o 26% highe CI han ha o
MRSA2 a 20 h), while he lowes CI alues we e obse ed o
MRSA1 (Figu e 1 and Supplemen a y Figu e S1). The eal-
ime dose– esponse expe imen s showed ha dalba ancin is a
highly e ec i e an imic obial and could p e en bac e ial bio ilm
o ma ion a low concen a ions. The MBICs o he es ed S
au eus s ains we e be ween 0.5–1 and 2 mg/L o S. epide midis
s ain 43040 (Figu e 1 and Supplemen a y Figu e S2). Table 1
shows he alues o MBIC and MIC (as measu ed by E- es s) o
dalba ancin o S. epide midis and S. au eus s ains, indica ing
ha he MBIC is up o 22 imes highe compa ed o he g ow h
o he same s ains on aga pla es.
Dalba ancin E ec on Bio ilm Fo ma ion
Compa ed o O he An ibio ics
To u he e alua e he dalba ancin e ec on bio ilm o ma ion
in S. au eus and S. epide midis s ains, we compa ed he
e ec o dalba ancin on bio ilm o ma ion o ou an ibio ics
wi h di e en mechanisms o ac ion, all o hem commonly
used in clinical p ac ice: ancomycin, linezolid, cloxacillin, and
i ampicin. Fo hese expe imen s, we selec ed he MRSA4
isola e, which showed he highes CI alues compa ed o he
o he MRSA s ains (Supplemen a y Figu e S2), oge he wi h
me hicillin-suscep ible S. au eus s ain 240 and he clinical isola e
o S. epide midis 43040. Figu es 2A–C show he pe cen age o
bio ilm o ma ion inhibi ion/induc ion ela i e o he an ibio ic-
ee con ol o each s ain (co esponding o 100% in he
g aphs). Al hough he an ibio ic e ec appea ed o be s ain
speci ic, dalba ancin and i ampicin p e en ed bio ilm o ma ion
in a dose-dependen manne , showing highe bio ilm inhibi ion
a es han ancomycin, linezolid, and cloxacillin. Cloxacillin was
only e ec i e agains S. au eus s ain 240 and could pa ially
inhibi bio ilm o ma ion a some o he es ed concen a ions in
S. epide midis 43040. Howe e , none o he es ed concen a ions
o his an ibio ic could elimina e p e o med bio ilm comple ely
in his s ain o in MRSA4. In ac , he exposu e o MRSA4
o low concen a ions o cloxacillin (0.06–0.125 mg/L) was no
only ine ec i e bu in ac also p omo ed bio ilm o ma ion
up o 20% compa ed o he un ea ed con ol (Figu e 2B).
Addi ionally, nei he linezolid no ancomycin appea ed o be
e ec i e agains S. au eus and S. epide midis bio ilm de elopmen
a low concen a ions, and bo h es ed an ibio ics induced bio ilm
g ow h a concen a ions <4 mg/L in s ain MRSA4. I is
impo an o unde line ha he MBIC o all es ed an ibio ics is
conside ably highe han ha es ima ed by he adi ional E- es
me hod (Supplemen a y Table S2).
Dalba ancin E ec on Es ablished
Bio ilms
I is known ha some an ibio ics ha e a limi ed e icacy o
pene a e in es ablished bac e ial bio ilms (Je e son e al., 2005).
Fo his eason, we u he es ed he e ec o dalba ancin
on al eady- o med s aphylococcal bio ilms. In ou expe imen al
se ing, we conside ed es ablished bio ilms hose which we e
on exponen ial bio ilm g ow h phase (Fe e e al., 2017a), ha
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TABLE 1 | Compa ison be ween dalba ancin MBICs a 20 h o g ow h, as
de e mined by impedance measu emen s, and MICs measu ed by s anda d
E- es .
S ain MBIC (mg/L) MIC (mg/L)
S aphylococcus epide midis 43040 2 0.094
S aphylococcus au eus 240 0.5 0.064
MRSA1 1 0.125
MRSA2 0.5 0.094
MRSA3 0.5 0.023
MRSA4 0.5 0.125
MRSA5 0.5 0.125
is, be ween 6 and 9 h depending on he s ain, as p e ious
wo k has shown ha an exopolyme ic ma ix is ully o med by
ha ime (Fe e e al., 2017b;Gu ié ez e al., 2017). Figu e 3
shows bio ilm g ow h dynamics when di e en dalba ancin
concen a ions we e added a he exponen ial bio ilm g ow h
phase. The da a indica e ha bio ilm elimina ion was ne e
achie ed, bu high concen a ions o his an ibio ic (8–32 mg/L)
we e able o educe o ully p e en new bio ilm o ma ion
and i s u he de elopmen in all he es ed s ains. Mo eo e ,
dalba ancin a 32 mg/L concen a ion was able o dec ease he CI
alues o e 40% compa ed o an ibio ic- ee con ols a e 20 h o
inocula ion (Figu es 2D–F). The po en ial e ec o dalba ancin
was e iden in he me hicillin- esis an isola e MRSA4, whe e
all es ed concen a ions esul ed in bio ilm g ow h educ ion
and pa ial elimina ion. Addi ionally, a s ain-dependen e ec
was obse ed a low concen a ions: whe eas a concen a ion o
0.50 mg/L p e en ed new bio ilm g ow h in S. epide midis 43040,
concen a ions lowe han 4 mg/L in S. au eus 240 u ned ou o
be ine ec i e (Figu es 2D–F,3).
Dalba ancin E ec on Es ablished
Bio ilms Compa ed o O he An ibio ics
To e alua e he po en ial e ec o dalba ancin in a
compa a i e way, we pe o med dose– esponse expe imen s
using con en ional an ibio ics on al eady- o med bio ilms
(Figu es 2D–F). In con as o dalba ancin, exposu e o
es ablished MRSA4 bio ilms o ancomycin, linezolid, cloxacillin,
and i ampicin had no inhibi o y e ec a he maximum es ed
concen a ion o 32 mg/L. In addi ion, lowe doses o hese
FIGURE 2 | Concen a ion-dependen e ec o linezolid, ancomycin, cloxacillin, i ampicin, and dalba ancin on bio ilms in S aphylococcus au eus 240, S. au eus
MRSA4, and S aphylococcus epide midis 43040 s ains. (A–C) The an ibio ics we e added a he beginning o he expe imen oge he wi h he bac e ial inoculum.
(D–F) The an ibio ics we e added when bio ilms we e al eady o med, a hei exponen ial g ow h phase. All cha s indica e bio ilm o ma ion a 20 h o g ow h
exp essed as he pe cen age o cell index (CI) compa ed wi h he con ol wi hou an ibio ic. Values below 100% indica e bio ilm inhibi ion, whe eas alues o e 100%
indica e bio ilm induc ion, in compa ison wi h he bio ilm mass achie ed in he absence o each an ibio ic.
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FIGURE 3 | Dalba ancin e ec on es ablished s aphylococcal bio ilms in
S aphylococcus au eus 240 (A),S. au eus MRSA4 (B), and S aphylococcus
epide midis 43040 (C) s ains. Dalba ancin was added a he exponen ial
g ow h phase o he bio ilm (ma ked by black a ows) a 6 h (S. au eus 240),
7h(S. au eus MRSA4), o 9 h (S. epide midis 43040) a he concen a ions
shown in he legend. Black as e isks indica e he an ibio ic- ee cell con ol.
Da a a e he means o wo eplica es.
an ibio ics (<32 mg/L) inc eased bio ilm o ma ion in his
s ain. The da a indica e ha dalba ancin was he only e ec i e
an imic obial showing a s ong bio ilm inhibi ion capaci y o
his s ain. Dalba ancin also hal ed new bio ilm o ma ion a
8–32 mg/L in S. au eus 240, while he o he es ed an ibio ics
esul ed in inc eased bio ilm g ow h a hese concen a ions.
In he case o S. epide midis s ain 43040, bo h i ampicin
and cloxacillin we e able o dec ease bio ilm g ow h a he
concen a ions ≤8 mg/L (Figu e 2F). Su p isingly, highe
concen a ions o hese an ibio ics (8–32 mg/L) had a limi ed
e ec in S. epide midis 43040. The leas e ec i e an ibio ic on
p e o med bio ilm g ow h inhibi ion was ancomycin. This
an ibio ic induced bio ilm o ma ion o all es ed s ains (>30%
ela i e o he an ibio ic- ee con ols) a 20 h o bio ilm g ow h.
Combined E ec o Dalba ancin and
Bio ilm-De aching Compounds
Fo his analysis, we selec ed an eme ging he apeu ic agen
wi h mucoly ic p ope ies, NAC (Kundukad e al., 2017), and a
na u al plan p o ease, icin, which has ecen ly been desc ibed
as an enzyme wi h unique p ope ies o des oy he bio ilm
ma ix (Baidamshina e al., 2017). Supplemen a y Figu e S3
summa izes he e ec o bo h an i-bio ilm compounds on he
bio ilm o ma ion o S. au eus 240, MRSA4, and S. epide midis
43040 s ains, when added a he momen o inocula ion.
G aphs show ha all es ed NAC concen a ions induced bio ilm
o ma ion in S. epide midis 43040, while 8 g/L was able o sligh ly
diminish bio ilm o ma ion in bo h Sa240 and MRSA4 s ains.
On he o he hand, icin (1,000, 100, o 10 mg/L) showed a
no able e ec on S. au eus bio ilms, inhibi ing hei o ma ion by
47% in Sa240 and 25% in MRSA4 s ains, a e 20 h o bio ilm
g ow h. On he con a y, his compound esul ed in induc ion o
S. epide midis 43040 bio ilm o ma ion. Thus, he e ec o his
de aching compounds is species dependen .
Gi en ha al eady-es ablished bio ilms a e e y di icul
o e adica e, we nex es ed whe he NAC o icin alone o
in combina ion wi h dalba ancin could ha e any e ec on
p e o med s aphylococcal bio ilms. Figu e 4 sums up he e ec
o dalba ancin and bio ilm-de aching compounds sepa a ely and
in combina ion when hey we e added a he exponen ial bio ilm
g ow h phase. Figu es 4A–C ep esen CI alues aken a 10 h o
bio ilm de elopmen , while Figu es 4D–F ep esen hose aken
a 20 h. Dalba ancin alone a he concen a ion o 32 mg/L
( ep esen ed as D32) g ea ly diminished new bio ilm o ma ion
o all he es ed s ains. NAC, when adminis e ed alone on
al eady- o med bio ilms, also had an inhibi o y e ec on all h ee
es ed s ains (Figu e 4). Howe e , when dalba ancin and NAC
we e combined, he inhibi o y e ec was d ama ically hampe ed,
and in S. au eus, i e en led o bio ilm induc ion. This sugges s
ha he combina ion o NAC and dalba ancin is an agonis ic.
Ficin, when adminis e ed alone, had a signi ican e ec in bo h
S. au eus s ains, p e en ing new bio ilm o ma ion up o 52%
compa ed o an un ea ed con ol a bo h 10 and 20 h o bio ilm
g ow h (Figu es 4A–E). Howe e , icin had no inhibi o y e ec
on S. epide midis 43040 bio ilms. In e es ingly, he combina ion
o icin wi h 32 mg/L o dalba ancin on his s ain p oduces
less inhibi ion han he an ibio ic alone (Figu e 4F), sugges ing
a po en ial coun e p oduc i e e ec o bo h compounds. On
he con a y, he combina ion o 32 mg/L dalba ancin and
1,000 mg/L icin in S. au eus MRSA4 led o a signi ican
imp o emen o bio ilm inhibi ion ela i e o he an ibio ic alone
(p<0.05) (Figu e 5A), sugges ing a po en ia ing e ec . Al hough
icin alone p oduced highe bio ilm educ ion (Figu e 5A), he
de ached cells we e iable as hey a e no a ec ed by his molecule
(Figu e 5B). This is con i med by an inc ease in plank onic cells
a e icin adminis a ion (Figu e 6C). Thus, when using icin,
an e ec i e an ibio ic is needed in o de o inac i a e bac e ial
cells which a e de ached as a esul o he enzyme’s ac i i y and o
p e en u he coloniza ion.
E ec o Bio ilm-De aching Compounds
on Plank onic Bac e ial G ow h
To in es iga e i NAC and icin only hold bio ilm-de aching
p ope ies o also ha e a di ec an imic obial e ec on bac e ial
cell g ow h, we u he assessed he e ec o bo h compounds on
plank onic bac e ial g ow h. A e he exposu e o S. au eus 240,
MRSA4, and S. epide midis 4340 o di e en NAC concen a ions
(0.5–32 g L), i was obse ed ha 8 g/L educed bac e ial g ow h
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FIGURE 4 | E ec on he bio ilm g ow h o S aphylococcus au eus 240 (A,D),S. au eus MRSA4 (B,E), and S aphylococcus epide midis s ains (C,F), espec i ely,
o he bio ilm-de aching compounds icin (F) (1,000, 100, 10 mg/L) and N-ace yl-L-cys eine (NAC) (8, 2, 0.5 g/L), and he an ibio ic dalba ancin (D) (32, 4, 0.5 mg/L)
alone o in combina ion. (A),(B) and (C) g aphs co espond o he induc ion o inhibi ion alues obse ed a 10 h o bio ilm o ma ion shown as pe cen age ela i e
o he con ol, while (D),(E) and (F) o 20 h o bio ilm g ow h, espec i ely. On he Xaxis, ze o co esponds o bio ilm mass o an imic obial- ee con ols a 10 and
20 h o bio ilm g ow h on an xCELLigence 96-well pla e.
o e 50%, indica ing ha his compound alone has a s ong
an imic obial e ec . Bac e ial g ow h was ully elimina ed when
he NAC concen a ion eached 32 g/L (MIC o all es ed s ains)
(da a no shown). On he con a y, none o he es ed icin
concen a ions (1,000, 100, and 10 mg/L) a ec ed plank onic
bac e ial g ow h, indica ing ha icin has a p o eoly ic e ec
only on he bio ilm exopolyme ic ma ix, esul ing in an e icien
bio ilm-embedded cell dispe sal (Figu e 5B).
Compa ison o Impedance
Measu emen s Wi h Classical Bio ilm
Quan i ica ion Me hods
To e i y ha obse ed changes in CI a e compa able o
s anda d me hodologies, we pe o med CV s aining and CFUs o
MRSA4 bio ilms un ea ed o ea ed wi h icin and dalba ancin
alone and hei combina ion. Figu es 6A,B ep esen op ical
densi y measu emen s a 24 h o bio ilm g ow h when icin
and dalba ancin we e added. The addi ion o dalba ancin alone
signi ican ly dec eased he numbe o adhe ed bac e ial cells on
96-well pla es. Reduced s aining in he wells was also obse ed
in cases whe e icin was added alone and in combina ion wi h
dalba ancin, con i ming ou p e ious obse a ions o he abili y
o icin o de ach bac e ial cells om bio ilms. We also pe o med
iable cell coun ing in bo h bio ilm and unadhe ed bac e ial
cells in supe na an s (Figu e 6C). CFU coun s showed ha
he iabili y o bio ilm-embedded cells and plank onic cells was
signi ican ly a ec ed by dalba ancin alone. When icin was added
alone, cell iabili y was no a ec ed and a lowe numbe o
bac e ial cells we e obse ed in bio ilms, oge he wi h a highe
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FIGURE 5 | Bio ilm dis up ion by icin alone and in combina ion wi h dalba ancin. (A) Bio ilm ea men by icin [ma ked as F (1,000, 100, and 10 mg/L)] and
dalba ancin [ma ked as D (32 mg/L)], ei he alone o in combina ion (F1000 D32), in me hicillin- esis an S aphylococcus au eus s ain MRSA4. Ficin, dalba ancin, o
bo h was added on es ablished bio ilms a e 7 h o g ow h in an xCELLigence equipmen . (B) E ec o icin on plank onic bac e ial g ow h, measu ed as op ical
densi y in a 96-well pla e. Bac e ia we e g own on TSB supplemen ed wi h 0.25% glucose a 37◦C. Da a a e he means o h ee eplica es.
FIGURE 6 | E ec o 1,000 mg/L icin (ma ked as F1000) and 32 mg/L
dalba ancin (ma ked as D32) on p e o med MRSA4 bio ilms as measu ed by
CV s aining and iable CFU coun ing. (A) MRSA4 bio ilm quan i ica ion by CV
in la -bo om 96-well Ibidi pla es, pe o med by duplica e. (B) Bio ilm
o ma ion capaci y a e di e en ea men s and CV s aining (measu ed as
op ical densi y). Da a show a e age alues om h ee biological eplica es.
(C) Bac e ial iabili y o bio ilm and plank onic MRSA4 cells a e ea men by
icin, dalba ancin, and hei combina ion o 24 h. Da a show he a e age o
log CFUs om h ee eplica es. S a is ical signi icance was assessed by - es ;
as e isks indica e *p≤0.05 and ***p≤0.001.
numbe o plank onic cells compa ed o he un ea ed con ol.
These obse a ions con i m he lack o an imic obial p ope ies
o icin and i s bio ilm-disagg ega ing ac i i y. On he o he hand,
when icin was added oge he wi h dalba ancin, he iabili y o
bo h bio ilm and unadhe ed cells dec eased almos h ee o de s
o magni ude in bio ilm cells and wo o de s o magni ude in
una ached cells, showing a po en ia ing e ec and sugges ing
ha icin inc eases he suscep ibili y o bio ilms o his an ibio ic.
DISCUSSION
Bio ilm- o ming capaci y o s aphylococcal s ains con ibu es
eno mously o he pa hogenesis o implan -associa ed in ec ions,
p o ec ing hese oppo unis ic pa hogens om bo h immune
sys em a ack and an ibio ic ea men (G ies and Kielian, 2017).
Dalba ancin has al eady been desc ibed as an an ibio ic wi h a
po en in i o bac e icidal ac i i y agains many g am-posi i e
pa hogens, including MRSA and MRSE, in a plank onic mode o
g ow h (Chen e al., 2007). Howe e , he e ec o dalba ancin on
bac e ial bio ilms emains unclea as i has only been es ed in a
ew occasions by using s anda d me hods such as CV s aining
o MIC de e mina ions (Fe nández e al., 2016;Kna l e al.,
2017). The impedance-based me hod pe o med in he cu en
manusc ip allows s udying o he dynamics o bio ilm o ma ion
and he e o e he ex en o bio ilm educ ion a di e en ime
poin s, ob ia ing he need o selec o a speci ic endpoin
(Fe e e al., 2017b). In his s udy, we e alua e he dalba ancin
e ec on he pa e n and dynamics o in i o bio ilm g ow h
in one S. epide midis and six S. au eus s ains and compa e i s
e icacy o ou di e en con en ional an ibio ics used in clinical
p ac ice ( ancomycin, cloxacillin, linezolid, and i ampicin).
Ou expe imen s p o e ha dalba ancin and i ampicin we e
he bes he apeu ic agen s agains S. au eus and S. epide midis
bio ilm o ma ion in a dose-dependen manne when added
a he beginning o bio ilm g ow h. In e es ingly, he supe io
e icacy o hese wo an ibio ics is no ela ed o hei mechanisms
o ac ion, as i ampicin inhibi s RNA polyme ase (Campbell
e al., 2001) and dalba ancin in e e es wi h bac e ial cell wall
syn hesis (Chen e al., 2007). Al hough i ampicin has been
used in clinical p ac ice agains s aphylococcal bio ilm- ela ed
in ec ions o almos h ee decades (Zimme li and Sendi, 2019),
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his an ibio ic should be adminis e ed ca e ully because o he
dange o apid eme gence o i ampicin- esis an bac e ia
(Raad e al., 2007;Zhou e al., 2012). Thus, dalba ancin
eme ges as a p omising solu ion in he igh agains
de ice- ela ed in ec ions, con i ming he p omising esul s
ob ained in animal models (Da ouiche and Mansou i, 2005;
Baldoni e al., 2013).
The impedance measu emen s pe o med in he cu en wo k
show ha , once he bio ilm is o med, dalba ancin was he
only es ed an ibio ic which could a es new bio ilm o ma ion
and p e en i s u he de elopmen in me hicillin- esis an
isola e MRSA4 and was e ec i e in Sa240 and Se43040 a he
concen a ions o 8–32 mg/L. The o he es ed an imic obials
no only lacked an inhibi o y e ec on al eady- o med bio ilms,
bu hey also caused an induc ion o bio ilm o ma ion
(Figu es 2D–F). Gi en ha some o hese an ibio ics ha e been
shown o be able o pene a e h ough hick bio ilms, hei
lack o inhibi ion could be due o a low me abolic ac i i y
o bio ilm-embedded cells, which is known o dec ease hei
suscep ibili y o an ibio ics (Je e son e al., 2005;Jacqueline and
Caillon, 2014;Lopa kin e al., 2019). Thus, ou da a sugges
ha dalba ancin may ac on es ablished bio ilms mo e e icien ly
han linezolid and ancomycin, which a e among he mos
common an ibio ics clinically adminis e ed o bio ilm in ec ions
caused by S. au eus and S. epide midis (Choo and Chambe s,
2016). This may be acili a ed by i s mechanism o ac ion,
because his an ibio ic no only inhibi s bac e ial cell wall
syn hesis bu has also an abili y o bind o bac e ial memb anes
(Chen e al., 2013).
Gi en ha e en dalba ancin showed a limi ed e icacy o
e adica e al eady-es ablished bio ilms, an e o was made o
in es iga e i s combined e ec oge he wi h icin and NAC,
which ha e been epo ed o ha e bio ilm-de aching p ope ies.
Unexpec edly, in i o in e ac ions be ween dalba ancin and
bio ilm-de aching compounds in p e o med bio ilms showed a
dose and species-dependen e ec (Figu e 4). Fo example, he
combina ion o NAC a concen a ions o 8, 2, and 0.5 g/L
wi h 32 mg/L o dalba ancin showed a dec eased e iciency in
inhibi ing S. epide midis 43040 bio ilms compa ed o dalba ancin
alone. This indica es ha he combina ion o bio ilm-de aching
and an imic obial compounds should be ca e ully es ed in o de
o p edic i s combined e ec .
We also obse ed ha NAC had a di ec an imic obial e ec
on plank onic cells, while icin did no inhibi bac e ial g ow h
a any o he es ed concen a ions (Figu e 5B). This sugges s
ha icin is able o de ach S. au eus bio ilms by a ge ing only
he bio ilm ma ix s uc u e, in con as o NAC. This should
be conside ed when designing combined ea men s a egies.
Fo ins ance, ou da a demons a e ha icin in combina ion
wi h dalba ancin a inal concen a ions o 1,000 and 32 mg/L,
espec i ely, showed an enhanced e iciency in he e adica ion
o es ablished MRSA4 bio ilms compa ed o dalba ancin alone
(Figu e 5A). Al hough icin alone p o ided an e en g ea e
bio ilm educ ion, i s lack o an imic obial ac i i y implies ha
de ached bio ilm cells wi hou he p esence o an app op ia e
an ibio ic could each he bloods eam and esul in se ious
medical complica ions. The e o e, we p opose he combina ion
o bo h a bio ilm-de aching compound and an e icien an ibio ic
o maximal e iciency.
Al oge he , he obse a ions om he cu en manusc ip
show ha dalba ancin has a s ong ac i i y agains s aphylococcal
bio ilms in i o, making his an ibio ic a p omising agen o
comba bio ilm-media ed in ec ions. Al hough i s e ec on
al eady- o med bio ilms is limi ed, icin appea s o in ensi y
i s e icacy, and he combina ion o dalba ancin wi h his
o o he disagg ega ing compounds should be u he
s udied in he u u e. The di e ences ob ained be ween
aga -g own and bio ilm-g own cul u es unde line ha he
use o app op ia e bio ilm suscep ibili y es s, such as hose
p o ided by impedance measu emen s, may o e a mo e
accu a e al e na i e o he selec ion o as and indi idualized
an ibio ic ea men . Whe he he use o impedance-based
bio ilm suscep ibili y es s allow ea lie discha ge om he
hospi al and lowe a es o ea men ailu e should be clinically
e alua ed in he u u e.
DATA AVAILABILITY STATEMENT
The aw da a suppo ing he conclusions o his a icle will be
made a ailable by he au ho s, wi hou undue ese a ion, o any
quali ied esea che .
AUTHOR CONTRIBUTIONS
AM, JR-D, and MF concei ed and designed he s udy. MŽ
pe o med he expe imen s and analyzed he da a. AM p o ided
eagen s. MV pe o med E- es s. MV and JR-D p o ided bac e ial
s ains. MŽ, AM, and MF d a ed he manusc ip . All au ho s
e ised and app o ed he inal manusc ip .
FUNDING
This wo k was suppo ed by he Spanish Minis y o Science,
Inno a ion and Uni e si ies (g an # RTI2018-102032-B-I00 o
AM and schola ship #FPU17/01302 o MŽ).
ACKNOWLEDGMENTS
We hank ACEA Biosciences o p o iding E-pla es and
assessmen o he use o he xCELLigence equipmen .
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound online
a : h ps://www. on ie sin.o g/a icles/10.3389/ micb.2020.
00553/ ull#supplemen a y-ma e ial
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