In vitro effect photodynamic therapy with differents photosensitizers on cariogenic microorganisms

RESEARCH ARTICLE Open Access In i o e ec pho odynamic he apy wi h di e en s pho osensi ize s on ca iogenic mic oo ganisms P. So ia-Lozano 1 , Y. Gilabe e 2,3 , MP Paz-C is obal 3 , L. Pé ez-A iaga 3 , V. Lampaya-Pé ez 3 , J. Apo a 4 , V. Pé ez-Laguna 3 , I. Ga cía-Luque 5 , MJ Re illo 1 and A. Rezus a 1,3,6* Abs ac Backg ound: An imic obial pho odynamic he apy has been p oposed as an al e na i e o supp ess subgingi al species. This esul s om he balance among S ep ococcus sanguis,S ep ococcus mu ans and Candida albicans in he den al bio ilm. No all he pho osensi ize s ha e he same pho odynamic e ec agains he di e en mic oo ganims. The objec i e o his s udy is o compa e in i o he pho odynamic e ec o me hylene blue (MB), ose Bengal (RB) and cu cumin (CUR) in combina ion wi h whi e ligh on he ca iogenic mic oo ganism S. mu ans, S. sanguis and C. albicans. Resul s: Pho odynamic he apy wi h MB, RB and CUR inhibi ed 6 log 10 he g ow h o bo h bac e ia bu a di e en concen a ions: 0.31–0.62 μg/ml and 0.62–1.25 μg/ml RB we e needed o pho oinac i a e S. mu ans and S. sanguis, espec i ely; 1.25–2.5 μg/ml MB o bo h species; whe eas highe CUR concen a ions (80–160 μg/ml and 160–320 μg/ml) we e equi ed o ob ain he same educ ion in S. mu ans and S. sanguis iabili y espec i ely. The minimal ungicidal concen a ion o MB o 5 log10 CFU educ ion (4.5 McFa land) was 80–160 μg/ml, whe eas o RB i anged be ween 320 and 640 μg/ml. Fo CUR, e en he maximum s udied concen a ion (1280 μg/ml) did no each ha inhibi ion. Incuba ion ime had no e ec in all expe imen s. Conclusions: Pho odynamic he apy wi h RB, MB and CUR and whi e ligh is e ec i e in killing S. mu ans and S. sanguis s ains, al hough MB and RB a e mo e e icien han CUR. C. albicans equi ed highe concen a ions o all pho osensi ize s o ob ain a ungicidal e ec , being MB he mos e icien and CUR ine ec i e. Backg ound The human o al ca i y is colonized by a highly di e se communi y o bac e ia [1]. Den al ca ies is a ch onic, in- asi e disease in ol ing demine aliza ion o he oo h ollowed by des uc ion o he o ganic phase o he den ine [2] and i is he consequence o he in e ac ion be ween o al mic o lo a, die , den i ion and o al en i - onmen [3]. S ep ococci a e he main colonize s o o al su aces and cons i u e 70 % o he cul i able bac e ia exis ing in he human den al plaque [4]. In ac , S. mu ans is he mos p e alen mic oo ganism o he plaque and he p ima y pa hogenic agen esponsible o ca ies disease [5], whe eas S. sanguis is hough o play a benign, i no a bene icial, ole in he o al ca i y [6]. On he o he hand, C. albicans is a commensal ungal species com- monly colonizing human mucosal su aces [7]. False a e al. [8] hypo hesize ha S. mu ans-C. albicans associ- a ion may enhance S. mu ans in ec ion and modula e he de elopmen o hype i ulen bio ilms on oo h su - aces, which will in u n in luence he onse and se e i y o den al ca ies in i o. Fo his eason, S. mu ans,S. sanguis and C. albicans should be included in any s udy abou human den al plaque mic oo ganisms. Pho odynamic he apy (PDT) has been ad oca ed as an al e na i e o an imic obial agen s o supp ess subgingi al species [9] due o he ex ensi e and inapp op ia e use o an imic obial agen s which g adually led o he de elop- men o pe asi e esis ance [10]. An imic obial PDT * Co espondence: [email p o ec ed] 1 Depa men o Mic obiology, Hospi al Uni e si a io Miguel Se e , Za agoza, Spain 3 Heal h Science Ins i u e o A agón, Za agoza, Spain Full lis o au ho in o ma ion is a ailable a he end o he a icle © 2015 So ia-Lozano e al. Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. So ia-Lozano e al. BMC Mic obiology (2015) 15:187 DOI 10.1186/s12866-015-0524-3 (aPDT) is a echnique ha u ilizes eac i e oxygen species (ROS) p oduced by non- oxic dye o pho osensi ize (PS) molecules in he p esence o low in ensi y isible ligh o kill mammalian o mic obial cells [11]. Due o his mech- anism, I is hypo hesized ha bac e ia will no be easily able o de elop esis ance o PDT [12]. Mo e han 400 compounds wi h pho osensi izing p ope ies a e known, including dyes, d ugs, chemicals and many na u al subs ances [13]. Me hylene blue (MB), a well-known dye wi h high ligh abso p ion a 665 nm, is e ec i e in aPDT, showing abili y o kill no only G am posi i e and G am nega i e bac e ia bu also ungi [14–17]. Rose Bengal (RB) is a xan hene dye cha ac e ized by ligh abso p ion a wa eleng hs (λ)o 450–600 nm, used o he diagnosis o eye diseases [18]. F om an an imic obial poin o iew, RB has shown a good p o ile o pho oinac i a e mic oo gan- isms [19–22]. Cu cumin (CUR) is an in ensely yellow pigmen , isola ed om hizomes o Cu cuma longa, wi h a peak o ligh abso p ion a 430 nm [23]. Among i s many biological ac i i ies a e i s an i-ca cinogenic, an ioxidan , an iin lamma o y, an imic obial p ope ies and i s hypoglycemic e ec s in humans [24, 25]. Some s udies ha e shown i s capaci y o e ec i ely pho oi- nac i a e in i o C. albicans [26, 27]. The e a e many pape s explo ing he aPDT e ec o di e en pho osensi ize s (PSs) in almos all kind o mi- c obial species [28–31]. Howe e , only ew o hem com- pa e he e icacy o se e al pho osensi ize s on di e en mic oo ganism [32]. The aim o his s udy was o compa e he pho oinac i- a ion e ec o h ee PSs, MB, RB and CUR, on S. mu ans,S. sanguis and C. albicans. Resul s Pho oinac i a ion o bac e ial suspensions Unde he expe imen al condi ions, PDT wi h MB, RB and CUR inhibi ed 6 log10 he g ow h o bo h s ains o bac e ia eaching a bac e icidal e ec . Howe e , less concen a ion o RB han o he o he PSs was needed o kill S ep ococcus spp. Whe eas his bac e icidal e - ec was achie ed o S. mu ans wi h a concen a ion o RB as low as 0.31–0.62 μg/ml, highe MB concen a ion (1.25–2.5 μg/ml) was needed o each he same educ ion. In he case o S. sanguis, he RB and MB concen a ions needed o ob ain he same bac e icidal e ec we e qui e simila o hose used o S.mu ans (0.62–1.25 μg/ml and 1.25–2.5 μg/ml, espec i ely). Much highe concen a ions o CUR we e necessa y o ob ain he same educ ion ei- he o S. mu ans o S. sanguis (Table 1). Rega ding he e ec o he incuba ion ime o S ep o- coccus cells wi h he PSs, one hou hal ed he minimal concen a ion o MB o RB necessa y o a ain 6 log10 educ ion espec o an incuba ion ime lowe han 1 min (<1 min), especially o S. sanguis (Table 1). In he case o CUR, his e ec was only obse ed o S. sanguis. No signi ican addi ional bene i was achie ed using 3 h o incuba ion (Table 1). Compa ing he pho odynamic e ec o MB o S. mu ans and S. sanguis suspensions, using he op imal in- cuba ion o each PS, lowe concen a ions we e needed o each he bac e icidal e ec o S. sanguis han o S. mu ans (Fig. 1). Howe e , no di e ences we e obse ed using RB as PS. Pho oinac i a ion o C. albicans Table 1 shows he minimum ungicidal concen a ion (MFC) o each PS s a ing om C. albicans 4.5 McFa land. Unde he expe imen al condi ions, PDT wi h MB, RB bu no wi h CUR inhibi ed 5 log 10 he g ow h o C. albicans, being he needed concen- a ions o MB smalle han he RB ones (Fig. 2). An inc ease in he incuba ion ime wi h he PS was only bene icial o MB, because 3 h hal ed he concen a- ion needed o each a 5 log10 educ ion in C. albi- cans espec o sho e imes (Table 1). Discussion Den al ca ies may be a disease well sui ed o PDT [2]. Ou in es iga ion showed ha PDT using MB o RB and a whi e lamp can kill ca iogenic mic oo ganisms, such as S. mu ans,S. sanguis and C. albicans. In con as , e en hough PDT wi h CUR eaches he same bac e i- cidal e ec , much highe concen a ions we e needed and i was no e ec i e agains yeas s. PDT e icacy depends on he mic oo ganism, he PS and he ligh used. Acco ding o ou esul s, RB showed highe an imic obial pho odynamic e ec o Table 1 Minimal ange concen a ion o educe 6 log10 o S. mu ans and S. sanguis and 5 log10 o C. albicans P e-i adia ion Incuba ion ime (h) MB RB CUR S. mu ans ATCC 35668 <1 min 1.25–2.5 μg/ml 0.31–0.62 μg/ml 80–160 μg/ml 1 h 0.62–1.25 μg/ml 0.15–0.31 μg/ml 160–320 μg/ml 3 h 0.62–1.25 μg/ml 0.31–0.62 μg/ml 160–320 μg/ml S. sanguis ATCC 10556 <1 min 1.25–2.5 μg/ml 0.62–1.25 μg/ml 160–320 μg/ml 1 h 0.31–0.62 μg/ml 0.15–0.31 μg/ml 40–80 μg/ml 3 h 0.15–0.31 μg/ml 0.15–0.31 μg/ml 40–80 μg/ml C. albicans ATCC 1023 <1 min 80–160 μg/ml 320–640 μg/ml >1280 μg/ml 1h 80–160 μg/ml >1280 μg/ml >1280 μg/ml 3h 40–80 μg/ml >1280 μg/ml >1280 μg/ml I adia ion wi h me al halide lamp, λ420–700 nm, luence 37 J.cm −2 So ia-Lozano e al. BMC Mic obiology (2015) 15:187 Page 2 o 8 S ep ococcus spp han he o he PSs s udied, whe eas MB was be e o Candida spp. CUR always showed he lowes an imic obial ac i i y; his could be due o he ac ha he highe peaks o he spec um emis- sion o he lamp co espond be e o he abso p ium spec a o RB and MB han CUR. Table 2 shows ha compa ed wi h p e ious s udies using aPDT wi h MB, RB and CUR o S. mu ans, ou pa ame e s seem o be mo e e icien , especially conside ing he pe cen age o bac e ial g ow h inhibi ion o 99.9999 %. Rega ding he an imic obial e ec o MB-PDT on he iabili y o S. mu ans, A aujo e al. [33] needed 25 μg/ml MB o each 73 % inhibi ion. Ne e heless, ou esul s a e no comple ely compa able because hey used ed ligh , which co esponds wi h he maximum spec um abso bance o MB. Rega ding RB, s udies ca ied ou by Cos a e al. [34] show ha he concen a ion needed o a ain 6.86 log10 CFU/mL educ ion o S. mu ans was 2.02 μg/ml, using a LED lamp wi h λ 440–460 nm and a luence o 95 J/cm 2 .Compa ing o ou esul s hey needed highe concen a ions o PS o each a simila e ec , pe haps because he λo hei lampwaslesscon enien hanou s oexci eRB;an- o he eason could be he use o dis illed wa e as dis- sol en , whe eas hey used phospha e-bu e ed saline because, based in he s udy o Nuñez e al. [35], a sig- ni ican di e ence in he same aPDT expe imen can be p omo ed only by he use o di e en s dissol en s. Acco ding o his s udy, CUR needs much highe con- cen a ion han RB and MB o pho oinac i a e bac e ia. This esul ag ees wi h hose ob ained by A aujo e al. [23] who, using a concen a ion o 1500 μg/ml and blue LEDs lamp (λ450 nm, luence 5.7 J/cm 2 ), eached 60 % inhibi ion o S. mu ans in plank onic cul u es. Howe e , o he s udies ob ained 95 % educ ion o S. mu ans in- s ead o 6 logs using only 0.73 μg/ml CUR, which could be explained by he highe luence used (72 J/cm 2 ), he λ o he lamp (blue ligh ) and he lowe pe cen age o bac- e icidal ac i i y ob ained. Few s udies compa e he e icacy o di e en PSs o pho oinac i a e o al mic oo ganisms. Rolim e al. [36] showed ha MB, oluidine blue o ho, malachi e g een, e y h osine, eosin and RB, using a ed LEDs lamp o he o me and a blue one o he la e , we e pho oac i e in i o agains S. mu ans, bu only oluidine blue e- duced 99.9 % o he mic oo ganism. In his s udy, hey also ind a bac e icidal e ec o RB on S. mu ans wi hou ligh . Ne e heless, in ou s udy no an imic obial e ec s we e obse ed when he s ains we e exposed ei he o he dyes o he ligh sou ce sepa a ely. Conside ing ha he ca iogenic po en ial o S. sanguis is deemed low compa ed o ha o he S. mu ans, he numbe o epo s using aPDT o kill S. sanguis is lowe han hose o S. mu ans. Chan e al. [37] demons a ed Fig. 1 Pho odynamic e ec o MB and RB on S. sanguis and S. mu ans depending on hei concen a ion (Incuba ion ime wi h he PS <1 min and i adia ion using a me al halide lamp wi h a luence o 37 J/cm 2 ) Fig. 2 Pho odynamic e ec o MB and RB on C. albicans depending on hei concen a ion (incuba ion ime o 1 h o MB and <1 min o RB, and i adia ion using a me al halide lamp whi a luence o 37 J/cm 2 ) So ia-Lozano e al. BMC Mic obiology (2015) 15:187 Page 3 o 8 ha PDT wi h MB was able o ob ain a educ ion o 99–100 % on cul u es o S. sanguis.Howe e , heyused highe concen a ions (100 μg/ml) han we used bu lowe luence (21.2 J/cm 2 ) o a diode lase (665 nm). Pe ei a e al. [32] only ob ained a 9.9 % inhibi ion wi h 5μg/ml RB and hey used a highe luence. The e o e, ou esul s show MB as he mos e icien bac e icidal PS o S. sanguis. Acco ding o he p esen in es iga ion, MB has be e an i ungal p o ile han RB and CUR. O he au ho s [14, 15, 38], show ha MB–aPDT is endowed wi h an i ungal po en ial agains C. albicans, whe eas Cos a e al. [18] show ha RB only each a 1,97 log10 educ ion in C. albicans. Compa ing he h ee PSs, he p esen s udy shows ha MB is he mos e ec i e PS on C. albicans while RB was sligh ly supe io o S. mu ans. Den al ca ies esul om in e ac ions among di e en ca iogenic mic oo ganisms, so using o combining di e en PSs could imp o e he e icacy o aPDT. In his sense, he use o a whi e ligh lamp ha co e s all he abso p ion spec um o mos PSs can e icien ly exci e hem, mak- ing PDT easie o pe o m and a oiding he necessi y o using a lamp o each PS. Howe e , a ligh sou ce wi h an emission spec um ha co esponds o he maximum abso p ion spec um o each PS heo e ic- ally de e mines a highe e icacy [39]. Red ligh sou ces (630–700 nm) ha e been used ex ensi ely in PDT due o hei ela i ely long wa eleng hs, which can e ec i ely pene a e biological issues and ac i- a es some o he mos e ec i e PSs, such as pheno- hiazines and po phy ins. Addi ionally, o he s udies ha e also shown ha blue ligh (380–520 nm) is an a - ac i e op ion o PDT, because blue ligh sou ces can be used in combina ion wi h many PSs, such as RB, eosin, e y h osine, and CUR o pho oinac i a e o al mic oo ganisms [36]. Fo his eason, one limi a ion o ou s udy is he use o he same lamp o pho oac i a e he h ee PSs, whose emission spec um ma ches qui e accu a ely wi h he maximum abso p ium spec a o RB and MB bu no wi h CUR. This could in luence he bad esul s ob ained wi h he la e . Acco ding o ou da a, he minimal bac e icidal o un- gicidal concen a ion was educed in some expe imen s wi h a p e-i adia ion incuba ion ime o 1 h. Howe e , conside ing ha he inc ease in he concen a ion was only o one o wo dilu ions, he di icul y o main ain he apeu ic concen a ions o he PS in he high low con- di ions wi hin he o al ca i y (due o sali a and/o gingi al c e icula luid) o a long pe iod o ime [40] do no sup- po he use o incuba ion ime in he clinical se ing. And ade e al. [26] using CUR and Rezus a e al. [41] wi h hype icin concluded ha none incuba ion ime enhance he pho oinac i a ion o plank onic cul u es o C. albicans. Addi ionally, al hough he ad e se e ec s o blood and sali a could be a oided wi h he help o den al dams, no p e-incuba ion ime is mo e com o able o he pa ien s and mo e e icien o doc o s. Conclusions The pho odynamic e icacy o each PS a ies acco ding o he a ge mic oo ganism. The combina ion o di e - en PS and whi e ligh could be a p omising app oach o ea hose in ec ions caused by a combina ion o mic o- o ganisms, such as ca ies. Table 2 Summa y o he in i o PDT s udies using me hylene blue, ose Bengal o cu cumin on S. mu ans, S. sanguis and C. albicans PS Concen a ion Inhibi ion λFluence (μg/ml) (%) (nm) (J/cm 2 ) S. mu ans A aújo e al. [33]MB25 73 ND ND Ou s udy MB 2.5 999.999 420–700 37 Cos a e al. [34] RB 2.02 999.999 440–460 95 Ou s udy RB 0,62 999.999 420–700 37 A aújo e al. [23] CUR 1500 99.9 450 5.7 Paschoal e al. [44] CUR 1473.5 60 450 72 Manoil e al. [45] CUR 0.73 95 % 360–550 542 Ou s udy CUR 160 999.999 420–700 37 S. sanguis Chan e al. [37] AM 100 99–100 632.8 21.2 Ou s udy AM 2.5 999.999 420–700 37 Pe ei a e al. [32] RB 5 9.9 455 95 Ou s udy RB 0.62 999.999 420–700 37 Ou s udy CUR 1500 999.999 420–700 37 Ma iello e al. [46] TB 200 84.32 660 10 C. albicans Souza e al. [15] AM 100 99.9 660 39,5 Peloi e al. [14] 4 AM 22,5 95,14 663 6 Souza e al. [16] AM 100 88,6 685 28 Ou s udy AM 160 99.999 420–700 37 Cos a e al. [18] RB 23 9.9 455 95 Demido a e al. [47] RB 200 99,9999 525–555 80 Ou s udy RB >1280 <99.999 420– 700 37 And ade e al. [26] CUR 7,3 89.5 455 5,28 Do igo e al. [48] CUR 14,8 85 440–460 18 Ou s udy CUR >2460 <99.999 420–700 37 PS pho osen ize , MB me hylene blue, RB ose bengal, CUR cu cumin, TB oluidine blue o ho, ND no da a So ia-Lozano e al. BMC Mic obiology (2015) 15:187 Page 4 o 8 Ma e ials and me hods Chemicals Me hylene Blue (MB) and Cu cumin (CUR) we e pu - chased om Sigma-Ald ich and Rose Bengal om Fluka. Sabou aud Dex ose Aga (SB) and Columbia Blood Aga (BA) we e pu chased om Oxoid. Mic oo ganisms and g ow h condi ions S. mu ans ATCC 35668, S. sanguis ATCC 10556 and C. albicans ATCC 10231 s ains we e ob ained om he Ame ican Type Cul u e Collec ion (ATCC; Rock ille, MD). McFa land scale is ecommended o pe o mance o suscep ibili y es ing by CLSI [42] and EUCAST [43]. The yeas s we e g own ae obically o e nigh in SB medium a 35 °C. S ock inoculum suspensions we e p e- pa ed in dis illed wa e and adjus ed o op ical densi ies co esponding o 4.5 McFa land o i e logs educ ion assays. Cell iabili y was assessed coun ing he numbe o colony- o ming uni s (CFU), de eloped on SB a e an incuba ion pe iod o 24 h a 35 °C. S. mu ans and S. sanguis we e g own ae obically in BA medium a 35 °C o 48 h. S ock inoculum suspen- sions we e p epa ed in dis illed wa e and adjus ed o op ical densi ies co esponding o 0.5 McFa land o six logs educ ion assays. Cell iabili y was assessed coun - ing he numbe o CFU, de eloped on BA a e an incu- ba ion pe iod o 48 h o S. mu ans and 24 h o S. sanguis a 35 °C. Pho osensi ize solu ions S ock MB, RB and CUR solu ions we e p epa ed in dis- illed wa e and dilu ed ei he wi h bidis illed wa e o he desi ed concen a ion immedia ely p io o use. The concen a ions used anged om 0.1–12800 μg/ml. All solu ions we e p epa ed and handled unde ligh - es ic ed condi ions. Ligh sou ce In o de o co e he spec um abso p ion o he 3 PSs (Fig. 3), MB, RB and CUR (maximal abso p ion λa 665, 557 and 430 nm espec i ely), we used a me al halide lamp emi ing a 420–700 nm (Fig. 4) wi h an i adiance o 90 mW/cm 2 , being he speci ic i adiance a he max- imal abso p ium λo each PS : 292 μW/cm 2 a 557 nm, 300 μW/cm 2 a 665 nm and 186 μW /cm 2 a 430 nm. Mic oo ganisms suspensions wi h he di e en PSs p e- pa ed in o a 96 wells mic o i e pla e, wi h a well diame e o 6 mm, we e i adia ed o 6 min and 51 s a a dis ance o 10 cm. The ligh beam diame e was 21 cm and he Fig. 3 Spec um abso p ion o ose Bengal a, me hylene blue band cu cumin c So ia-Lozano e al. BMC Mic obiology (2015) 15:187 Page 5 o 8 luence used 37 J/cm 2 . The e ec i e ligh ecei ed on he dishes esul s om in eg a ing he powe o he lamp o all he e ec i e wa eleng hs o each PS. Pho odynamic ea men s o mic oo ganisms Suspensions wi h he desi ed McFa land alue o e e y mic oo ganism we e p epa ed in bidis illed wa e . 90 μL o hese ini ial suspensions was d opped in o di e en wells o a mic o i e pla e and 10 μL o he di e en PS solu ions we e added. The inal PS concen a ion in he expe imen s used anged om 0.01–1280 μg/ml. The pla es we e hen main ained in he da k o di e en pe iods o ime (<1 min, 1 and 3 h) o e alua e he in lu- ence o con ac ime wi h he PS on he ou come o he pho odynamic ea men s. A e wa ds, mic oo ganisms we e subjec ed o illumina ion (37 J/cm 2 ). Fungal and bac e ial cul u es g own unde he same condi ions wi h and wi hou PS, ei he kep in he da k o illumina ed, se ed as con ols. A e pho odynamic ea men s, samples and con ols we e incuba ed a 35 °C o 24 h, in case o C. albicans and S. sanguis expe imen s, and o 48 h in case o S. mu ans. The an imic obial e ec was de e mined by coun ing he numbe o CFU pe millili e. A c i e ion o 5 log10 uni dec ease om he s a ing inoculum was adop ed o de ine ungicidal ac i i y, and a mo e s ingen c i e ion o 6 log10 uni o bac e icidal ac i i y, due o he di e ences in cell size and mass be- ween Candida spp and S ep ococcus spp ( he cell con- cen a ion o C. albicans used was 10 imes lowe (>10 5 ) han ha used o he wo bac e ial species (>10 6 )). All expe imen s we e ca ied ou a leas i e imes. Abb e ia ions MB: Me hylene blue; RB: Rose bengal; CUR: Cu cumin; CFU: Colony o ming uni s; PDT: Pho odynamic he apy; aPDT: An imic obial pho odynamic he apy; PS: Pho osensi ize ; SB: Sabou aud dex ose aga ; BA: Columbia blood aga . Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Au ho s’con ibu ions PSL concei ed o he s udy, conduc ed all expe imen s and d a ed he manusc ip . YG pa icipa ed in he design o he s udy, in he in e p e a ion o da a o he wo k and she d a ed he manusc ip , he co e le e and he answe s o he e iewe ’s epo s. LPA and VLP conduc ed some expe imen s and helped o d a he manusc ip . VPL is s udying he pho odynamic e ec in co-cul u es o C. albicans and S. mu ans and she checked ou esul s. She also e ised he manusc ip and con ibu ed o he esolu ion o e iewe ’s epo s. IGL has con ibu ed o he in e p e a ion o da a o he wo k and o he esolu ion o bio ilms ques ions and in he discussion o he inal manusc ip because she wo ks wi h us in he p ojec CTQ 2013–48767-C3-2-R om he Spanish Minis y o Science and Inno a ion s uding he e ec o pho odynamic he apy on bio ilms. MPPC pa icipa ed in he design o he s udy, analyzed he da a, pe o med he Figs. 1 and 2 and he e ised he manusc ip pa icula ly he me hodology and he ma e ials. JAA pe o med he Figs. 3 and 4 and he w o e e e y hing ela ed ligh pa ame e s bo h in he manusc ip and in he answe s o he e iewe ’s epo s. MJR con ibu ed o he design o he wo k, and e ising and co ec ing i , pa icula ly he backg ound and he discussion. AR concei ed o he s udy and d a ed he manusc ip . All au ho s ead and app o ed he inal manusc ip . Fig. 4 Rela i e emission cu e o he me al halide lamp So ia-Lozano e al. BMC Mic obiology (2015) 15:187 Page 6 o 8 Acknowledgemen s This s udy was suppo ed by g an s CTQ2013-48767-C3-2-R om he Spanish Minis y o Science and Inno a ion, Spain and Eu opean Regional De elopmen Fund. Au ho de ails 1 Depa men o Mic obiology, Hospi al Uni e si a io Miguel Se e , Za agoza, Spain. 2 Depa men o De ma ology, Hospi al San Jo ge, Huesca, Spain. 3 Heal h Science Ins i u e o A agón, Za agoza, Spain. 4 Depa men o Applied Physics. Facul y o Science, Uni e si y o Za agoza, Za agoza, Spain. 5 Depa men o Mic obiology, Uni e si y o Se illa, Se illa, Spain. 6 Depa men o Mic obiology, Uni e si y o Za agoza, Za agoza, Spain. Recei ed: 6 No embe 2014 Accep ed: 21 Sep embe 2015 Re e ences 1. Vahabi S, Fek azad R, Ay emlou S, Tahe i S, Zangeneh N. 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Ma iello FD, Coelho AA, Ma ins OP, Ma iello RD, Fe ão Júnio JP. In i o e ec o pho odynamic he apy on Agg ega ibac e ac inomyce emcomi ans and S ep ococcus sanguinis. B az Den J. 2011;22(5):398–403. 47. Demido a TN, Hamblin MR. E ec o cell-pho osensi ize binding and cell densi y on mic obial pho oinac i a ion. An imic ob Agen s Chemo he . 2005;49(6):2329–35. 48. Do igo LN, Pa a ina AC, Ca mello JC, Machado AL, B une i IL, Bagna o VS. Suscep ibili y o clinical isola es o Candida o pho odynamic e ec s o cu cumin. Lase s Su g Med. 2011;43(9):927–34. Submi you nex manusc ip o BioMed Cen al and ake ull ad an age o : • Con enien online submission • Tho ough pee e iew • No space cons ain s o colo figu e cha ges • Immedia e publica ion on accep ance • Inclusion in PubMed, CAS, Scopus and Google Schola • Resea ch which is eely a ailable o edis ibu ion Submi you manusc ip a www.biomedcen al.com/submi So ia-Lozano e al. BMC Mic obiology (2015) 15:187 Page 8 o 8