PoLish fournal of lvl iclobiology2013, Vo] .62 , No 4 , 359 364

ORIC]NAL PAPI]R

Eiological Evaluation of Quaternary bis Anrmonium lśalrland Cetylpyridinum Bromide Against S, epidermidis Biofilm

JoANNA STEl.AŃsKA'-' ANNA PIETRUcZUK PADZIKl, N,lAl{'fA sl.RUGAr, N1A(]ll1l l]()IiKo\Vsli l .

ancl STEFAN TYSKIT r

' Departnrent of Pharntaceutical Microbiology, Medical Universitv of \\rar.s.ir. r)r,!rrtrjrDePartment of Medicinal Chemistr1', Medical UniVcrsity of \Ąrarslrl ' ' l)i l]. i l l .. l

'Stuclent Scientitlc Society at Department of Pharilaceutical Micr.obiology,, N4eclical Unir,:r-.,: \.i)l \\,i1.\,r\\rl)cpartment ofAntibiotics and Microbiology, National \' ledicines lnstiture, \\riLrs:rrr l,0iłrrrl

Submitted 25 January 2013, accepted lU October 2013

A b s t r a c t

e t f i c i e l r c Y | a n g c d J i o n 5 9 ' 7 o / o t o 8 8 - 4 % 1 . o I t e s

strphylococcLr l b iot iLrrs, but a.y o lused concenLrat ions caused a tota l eradicat ion ofbacter ia l b iof i lm.

I(ey rr,old..błcterialbiof i lm,s'aphyLococcusepidermidis,qLLater.naryammonilrmconpounds

Introduction

NIany stlains of Gran positive bacteria are dan-gerous plrthogens causing severe hospital infections.Apart fi 'orn infections of clean wounds, the special.rttention sholrld be paid to infections occurring inpatieDts wlto have received implants, t.e. valves, vascular and bone implants, joint prostheses, ol tenclonProstheseS nlrrde of bionratelials (\Ąlójcikowka-Maclre t r l1 . , 2002; Ch l . i s tense l l ' l 982; Gó tz ,2oo2| \4ack e t c l . ,2006). t)ue to the ilcorrect use of antiseptics beforearrd a1łet. sulgeIy, co]onisation of the surgical woundby the rnicrobes oliginating from the hospital envirollment, occurs quite frequentiy. Microbes also canbe transrnitted from the hands of the medical person-nel, ol transfer red from the patier.rti skin, and causean endogenic infection. Another source of potential

Pirthogens łre n-ricl obes that colonise the applied vas-cular ard urinary catheters, drainage tubes, respirationaids as welj trs other therapeutic and diagnostic equip-ment (Trautner et a\.,2004; Pascual,2002). The mostcommon etiologic factors that bring about infectionsof operated tissne are Staphylococcus aureus, Eschericacoli, Pseur1omonąs spp., Klebsiella spp.' Enterobacter

spp., B cteroides spp.. Carlrlrrlri ,tl lrrr'rł;s. irr oiijtoi)cclic patients, clue to a broad LLsc oi bitrl l irtcl i l ls, :;-i,),,of infbction 1s causer-1 by St,iplr,t irit 'r ' i1.. . l)i(/.r /'t i i i j,and 10-20% by Staphy|ococtus rtlrlcł.s ('Ąrcitl ie cll i l ' ,2005; Husa in e tn l . , 1993; Huebner L ' r .71 . , 1990) . 1n fał ,S, ąureus is an in]portant carrse oi.i l l l .ection asso.i. ltędwith use of metal i inplants, bone irncl ioiitt pr()sthescsand cause a so f t t j ssLLe i l t t -ec i t , r t : , r r l l i l r ' l j . .7 r r r , r ' ł i l , / r s r sseen more o l ien in po l ) me l " - . r i : ,oa i . t i ! r . i t i , ta r ion5 ( ( i i l t /200f) ' stąpblocotcli.s 17irlr,l l; i ir l i: l :1r..lrr' i l lg i ls i l i l l l . i lt i l isoften very lesistant b convelltioli l l .t it i i l-)iotics, .lntiscft i cs and d i s in fec tants (Br id ie r r , t r r i . , 2011. Sr r r i th . / ( r1 . ,2008) . The va lues o fmin i r ra l in l r ib i to l r ' .oncenf ta t ion(MIC) and ninimal bactericici lLi conccntration (NlBC)of antibiotics to bacteria grou'l 1n ltiofi lnt, ntirl ' bv upto 1000 fold higher compłrerl rvith Lractcti i l in plankton ic fo rm (Ho iby e t a| . ,2010) . Cu i fent lY r ' ro thcrap icsthat target microbial biofl ln cxistr thrreti)re, uc\v i]lttibiofi lm agents, treatnents and sffntegies irie neeclcrl.The discovery of new antinlicroL)iżtl agents. i}cti\'e l]()tonly against free-srvimming m icloor giirn isurs (planktonic)but also against biotl lms, lcplcsents itn i lrportiutLtask (Pro jan e ta l . ,2002: Ka l i ranen c r{ r1 . ,2o0t i ) . Wi ththis aim, we focused on qulttc.ltrti l arnlnoniun salts

' Corresponding author: ] ' stcta l iska, I)ępart]nent of Pharmaceut ica l lv l i . robio log},. Nledica l Unirr l . : l r l ] l \\ ' . l l s l l \ ' ] () .7( i 5 l r ' '( )2 '()07 Wa|saw' Pol l l ld; Phone: +48 22 6280822; fax| +48 22 62t l35l; e lnai l : joanna'stefanskł@1'u,n.edi ' i ' ]

.r(i0

thiit fcl lfcscrl . l . i11ss of biologicall,v active substances,

l ' r , i r l l . l t . t i l l t j l l i ł i ]5 ' ] i ] t in r i c rob ia l agent (Ho ibr ' e Ia l . ,l { ) l0 r ( lh . r i c i r L i r r l . , f0 l l ) . Qu i l te rn i l rv ant r ' l ton i L l I r . )i,,rnf ,ru nris rr i L' . l b r'().\(l spcctlLln'r tractericidcs rr,iclely'ttscd its rlDlis.l)tias. (l isi l ltealion i1ltc1 PrcsetYation agellts( l i i r s rc l , l00 l ) .

- lh t ' . r c t i v i t r , o i quatc rnarY ammonium

.()nrpourds . esP . . i i r l l y thc r -o l c o f long cha in aga ins t(i i lL'rcut bactcri. l. h.rs been previouslv observed. The

I'ositiYcll chłrqctl tlurirter-nirIy uitrogen rvith the polarherrcl glrrups ol .rcirl i, phospholipids cause pertrrrbation()l c\1()Plżrstl ljC i l l ld l l ltter lrrcmbratle l ipid bilavels. Longc l r i i i n r ' \ r . r t i r r l i b . rc tc ! ia l ac t i v i tv aga ius t both Grant -positivc .incl (iranr-ncgativc bacteria as rvell irs againstsorue p i r t l rosc r l i c spcc ies o f fung i and protozoa (Ance ,l r | l t ' t r i l . . ] (X) - l r lou i l l rou c t r l l . , 2007; iVIc l ]a in e tn l . , 2004)

I}c i r iDr o l th i s s tudy w i rs to e\an l i l te the ac t i y i tyol l i\ '(] (lLl. lten'r.ir '\ i lnutoni!lnt salts asainst a gloup ofS L prrlr'r łrlr/i-s stIir ts isolatcd flol1') paticnts I.rospitalizedin thc eiiricrrl htrsr.it,rl i lt \\'arsir\\'. ' l ltese

stri l ins qrorling,rs iri111li1,v',, rrs r|cll rls ir bioii lnt stntcturc, r.r'cre unclercvrrlLrlti()n. Onc ot tltc testccl contpouncls \!as hexa-ilccllpr,r' icl iniLuri blontide (cetylpl'ridinunt bromide),witlclv l inoln suirstrrnce rvith alttimiclobial propertics rrirlclv Lrscal as.ln .l l lt iscptic. lhe secotrd substance\\ . r s r n( \v lv s rn t l t . s i zed der i l a t i vc : b i s [2-hvdroxv ,j ( l . ; , 3 , 9 , 1 { ) p c n l . u l l e t h v l ' . 3 , 5 d j o x o - + - a z a t r i c ) , -c lo I 5 .1 . 1 .0( l . ( r ) idc r S cn , .1 -v loxv) -prop1,1 I -d imethv l -

an)n lon iL rur c l l i r r r i c l c (c luatc rnar l b i s a l rn ton iuut sa l t ) .(,cnerirlJi usctl rlLralcll l irLv irlnmoniunl salts ar.e slightlysolLrble in tr atcr. Nerrlv obtained quater narl ' bis ammo-niLrm salt appliecl lo the tests, has a hygroscopic proper-Lic5 i lttd its lcrl hiqh solubil itf in the \\'ater, so mav beusr . ' i l i r i l hout i rdd i t ion o fe thano l to the tcs tcd so lu t io t r .

Sr rrtht'ris ntr,ihorl irutl blsic antitl icrobiirl activitv ol-tlris irqcnt \\'cre fr.\' ioLrsll dcscribecl (Struga eral., 2008).

Ilxperimental

\ latcr i . r ls and Nle lhods

( lhcnr i ca l s . ( . c t l lpv r i i l i num bronr ide (S iema) i sir l inou'n cation ic q uittenli lrv anl ntoltiLlnt conrpounclu i th an l in r i c r .ob i r r i . r c l i v i t i es .

St'nthcsizccl rlLLiitcf nil l.\ ' bis alrl r.lton ium salt u,asobt.tircrl in l irLu stc1.s. I.irst tbe starting conrpouncl( l , J ,3 .4 ,5 -pc 'n t ln rc t h l l cvc loprentad icne) rvas heatec il ith maleic anhvclricle. The obtaitred compoLlnd \\'assLrbjcc1ct1 to thc reaclion with hvdloxylłmine hydro-chloliclc in \virt!'r solulion.' l l le ploduct r.vas alkylatedrvith 2 chloronletvl oxirane and thcn it tvas condensedr l r th t l in rc thv lan l inc . l l esu l t i l tg compounc l \vas t rans-folnrccl into (l ir.rterr'ri lrv antnroniul' l l salt bv HCI satu-l i r t c ( l n rc thrn{r l I f c . i t l ] ] fn t .

Bacteriirl strains. l\ye|tv threc clinical Stn/r.1lo-torirr-r c/rirlcrini,l is irncl onc rcf-erence strain.S. cpi./cr-

r l rd i s ATCC I22f8 rve lc L rsc r l i r r th i r s l r r r l r . Thc e l ln i c r r lstrains Werę isolatecl tr.onl tbllorr.ing locrrtiolls: blorld(n = 20) , ca theter (n = 2) anc l venous ca theter (n = I ) .S. epiderntidis stlain ,łTCC l222{] rr'as ttsed in biofi lIl l. lssay as a negative control (biotiLr non-producingstrain),The antibiotic susceptibil itv of the tested strainswas deter mine d bv the standarrcl Cl.Sl cl isk dil i isionmethod (CLSI, 2006a). 1'rventl ' one ol' cl inical str ainswele me thicil l in-resistant (MRSll). Fitteen strains wereresistant to er1'thromvcin, eight - to clindanrvcin, five

to gentamicin, two - to alnjkacil l, seven to co-tri-moxazole, six - to ciprofloxacin, thre'e to tetrtcvcliDc.A l l s l ra ins uere suscept ib l c to l inezo l i c l .

Microorganisms rvere dcrivecl l i 'om the collcction o1'Departnent of Pharmaceutical l\l icrobiologr', lVledicalUniversitv of Warsau', Poland.

Antibacterial susceptibil ity testing - MIC method.l he miIl inrunl concęntrations of Lrollr testc'd qtlaternaIyanrmoniLlnr compoultds inhibiting grorrth ol S. cpi,dcrnidis straitts (lV{lCs) were detelrninecl by rcfi 'reuccbroth d i lu t ion rnethoc l s r r s ing \ luc l l e l H i r r ton l l B r i , th .according to Clinical and Laboratory Stirndards Insti-tute guidelines (CLSI, 2006b).

Concentrations of tested colnpoLluds in l iclLricl lrc-clium ranged fl 'om 0.0625 to 256 frg x ml. I, cct,vlp,vridinum bromide was dissolved iu 500/o ethanol, qua-telnarl bis amnonium salt - in disti]latcd r 'łter' T1rcfinal inoculum ofall nricroorgirnisnrs studied wrs aboLrtl0 'CFUxmL ' r (co lony fo rnt ing un i t s per mL) . MICSwere read after l8 h incubation at 35'C. 'Ihe MIC valuesdetermined in susceptibil it), test on planktonic bacteriarvere applied also during staphvlococcal biofi lnr susccp,tibil ity dc teUr.r in at ion.

S. epidermidisbiofilm susceptibiliq' testing - MTTmethod. All strains were liept fr-ozcn at -70'C in Bl.{lb ro th supp lenrentec l u i th l0% g lvcero l , L rn t i l use . Thestr'. l ins u'ere first grown frr;rn a flozen stock on BLllagaf plates bY incubatiorr cluring 24 ir at,17'C. Bactcrial colonies rvere harvestecl, anci sr.rspended in 0.85%NaCl to prepare cells suspensions for-biofi lm produc,tiotl. T}e clensity of all cell suspetrsiol-ls \Ą.as adjustcdto the value of -3.2 units in McFarlancl scale, l 'hich tasequa l to 1o 'qCFUxmL ' . Each inocu lunr was t l i l u tedI to l0 with fresh BHI broth. Individual rvells ol polv-styrene, sterile 96-rvell tissue culture plates (KartellS.p.A., ivledlab, Italy), u,ere fi l led up with 200 prl ol 'diluted cultures and incubatcd for' 24 h at 37"C. Addi-tionally 200 pl of BHI medium was added to sor.ne wells,as control of steril ity and non-specific nedia binding.After incubation planl<tonic cells were rcntovecl anclbacterial biofi lm lavcr rvas trcated with qLraternary bisammonium salt at concentt atiolts t anging lrom 2.0 to128.0 prgxmL-r . f ' o r comparat i r ,e i rnd qua l i t l ' cont ro l

Pul.Poses, other lvells lvith biofi ln] lvere 1lęatccl tt' ithquaternirr)'ammonium derivati\c - cetvl;rin.cl in iu nr

Stelrrńska |' tr nl'

Eradication of staphylococcai biofi lms 3 6 i

bromidum at concentl'ations from 0.125 to 8.0 pg xx mL r. The plates were incubated with these com-pounds for 18 h at 37'C. Following incubation period,the analyzed compounds were removed from thelnicroplate wells and they were washed u.ith sterilephosphate bufl-er saline (PBS). Alive bacterial cellswere stained with 3-(4,5-dimethyl2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (NITT; 0.5olo in PBS)for 2 hours at 37"C. In parallel, appropriate bacterialbiofilm not treated with these two compounds was usedas a control. After 2h ofstaining, solution was removedand biofilm was solubilized by 150 pI DMSO with 25 glglicyne bufl-er (0.1 M x L ', pH 10.2) treatment. Solutionabsorbance was measured at 554 nm wavelength usinga PowerWave XS (BioTek) spectrophotometer.

Comparing the average results ofabsorbance (4,,u)of the control biofilm with the biofilm treated withtested compounds, the P €Icentage ofkill ing efficiencyfor each concentration and for each strain, was calcu-Iated by the following formula:

A , con t ro l b i o f i im - A , . ,t e r l .d Lr.ofr lnl

fe f .en laoe oI Ń l l l l l ] c en] . l c11 .v=' A . - con t r , r l b ro f r l n r

Results

The MIC values for the cetylpyridinum blonidefor planktonic cells ranged from 0.l25ggxml-r to8.0 [g x mL I. For the almost halfofstrains (1 1/24-467o)the MIC value was 2.0 pg x mL r. For the referencesstraln S. epidermidls (AfCC 12228) this value was0.125 ggxrrl '. Only for the one tested strain MICvalue was 8.0 pg x nrL-', For the quaternary bis ammo-nium salt MIC values for the planktonic cells ranged

f rom l6 .0pgxmL ' (on ly e t t " - r c le rences s t ra in) to

128.0 pgx mL ) (14/24-58.301).'fhe

average value of absolbancc rvas cirlculated tbr

each strain, after treatment of tested conlpounds.

Analysis of results for resting str ins durirrs experi

ment lvith cetylpyridinr.ur brontidt, allorr,ecl to classil,r '

these strains as lorv, intermediltc łnd high trioii lm pro

ducers, depending on absorbancc r'łlLLe. .l

he absot bance

levels (A.,.,) were assumed sul.ricctivelt' b.v rttthors in

order to classiĘ the analysecl stt.ains i11t() ] t]IotlPs ofdit

ferent biofi lm production levcl. l: iIsl uloup: high biofi lmproducers - absorbance values irbovc 1.5; second gilorrp:

intermediate biofi lm produccls - .tbsorbrnce 1.0 1.5,

and third group: lorv biofi lm procluccrs - irbsotbartcc

below L0. Only f'erv (3/24) strains wcre classifiecl its high

biofi lm producers, whereas I J ot tc:itccl strains clisplaved

Iow bioti lm production phenoty pc. I Irc seconcl group oi

eight strains, includingS. epidtrnidis AI CC 12228, pte

viously described as non-biotl lnr producer [23 ]. Average

absorbance value for this slrairr rvas 1,172.

llte percentage of kil l ing eilricierlcy was calculatetl

fo r each s t ra in . In 0 .125ggxr l r l . Lcot tce ! r t r i r l i on o t '

cetylpyridinum bromide, the ;'crccrttage oikil l ing effi '

cienc;' rvas 43.60/o for tlre first grrlLIi l of st|Ji l ls, ó]';l,,,fot second group, and 44'7.]ó l i lr thirtl grou1''. ln the

highest concentration 8.0 prg x l l lI, ' t lris eł}iciencr. rvas

48.3ok, 64.10/o and 47.Oul, respcctivcll. \lcr.l iunr valitcs

of kil l ing efficiency ratrgecl l.rLltrl ]6'6% to 64'l9ó lor

all tested concentrations in thlcc gtoul's of striritts.' lhe

most sensitive tbr cetylpfridinunr bromicle rvete

strains from intermediate irioli lnr procluccrs group(48.6-64'1oń oI kil l ing ef|iciclrc1,)'

, l1lc l l]ost !.esisti}l lt

were bacterial strains from thirtl grr:up low biofi lnrproducers (26.6 to 47.0o/o ol l i i l l ing cll-icien+). Iror'

3 .0E

I' ó ? D

't 0

| . l , l

\ li: i-r l:',i

, j i l C ! r

la Arcc12228

0 5

Concentration [Pg x mL ']

F i g ' l ' Va l ueso fb i oń lmabso rbanceo fS ' ep i de , .m id i s s t r a i n sg l oups : l o r vb i o l i l n rp rod t l c c | s (7 \ ' . . . l ' ( ) ) .medium biol i lm produccrs ( A... Lo- 1.5), high biofim producers (A... > 1.5), strain 340/0S and rei i fcn.cs stfnin A l 'CC 1 222..,

after treatment with cetylpyridinum bromide.

0.0

Quaternary bis ammonium salt

IŁ

Tł- { , l l

J i

T

-T, -1, J{ l"l* .Jd l . rJ

162 stefańska J. ef al.

Strain 340/08

Quaternary bis amonium salt

,r , LOW

. |\,,IEDIUI\,1

T HIGH

I 340/08

B Arcc1222\

2 0

1 . 5

E

4 t )

3 5

3 0

1 . 0

0 5

c 02 .0 4 .0 8 .0 16 .0 32 .4 64 .0 128 .0

Concentration [Pg x mL-']

t i ig. l . \/ i l lues ofbiofi lm absorbance of S. epiderrr idis st lains gloups: low biofiJrn producers (A-,, < 1.0),l l lcdiLlnl biof i|nl pr'oc1ucels (A'.. 1'0. l '5), hi8h biońm Producers (A- . > 1'5)' strain 340/08 and leferences strni l] Al.Cc l2228'

after treatlnent with ouaterl)arvbis anlnlonium salt.

4 0

3 5

F ^ ^

.{l r l

at .l

B4 0

3 5

E

ś 2 ' 5

:i : , ,

concenlraton[Ps 'n rL ' ]

ffi control

S o.lzs

Io t: : 1 0

2 A

4 0

8 .0

concentrationI p s ' m L ' ]

fi conttol

M 2.a

f f iooi lBo:ł,1

| . . . 32a

l : j ' 6 4 O

f i$j rzeo

Fig' 3. \'zrlLres ofbiofilm absorbance ofs' epider|hidis sv^in 340l08, aft€r tleatment with cetylPyridinum bromide (A)

and quaternar) bis ammonium sait (B).

strains fror.n the second and third group the most effec-tive concentration was 8.0 pg x mL-r, while for the firstgroup the most effective was 0.25 pg x mL '. For the all

tested S. epidermidis strains the most effective was thehighest used concentration - 8.0 ggxn]l-r (averageabout 53% of kill ing efficiency) (Fig. 1).

Cetylpyridinum bromide

Strarn 340/08

I hese resuits shorvecl that cetylpyridinum bromide isactivity towards S. epideruidis btofilnt in vitro, regard-less oi the concentratiolt. None of the concentrationsrrscd caused total er adicat ion of staplrylococcal biof ilm,but the clear limjting ellect on the growth was observed.

During the experiment with quaternary bis ammo-nium salt the analyzed strains were again classified tothree biofilrn producers groups, as it was describedabove. In this palt of research some strains were clas-sifled to difi.elent gIoup Łls it rvas pler'iousl1'. ,Ib filstgroup high biofiLr proclucers 9 strains were classified,lo second- internrediate biofilm producers - 9 strains,ancl to thircl - lon'biofilnr producers - 6 strains. Thereferences strain in this part display'ed lorv biol'i lm pro-ducers pherotype (average absorbance value 0.943).

For each strain the percentage of kill ing efficiencvfbr rluaternary bis amntonium salt \\'as also cirlculated.,,\verage kill ing efficiency ranged from 59.7 to 88.4%tbr tested conce ntr ations (fior.n 2.0 to 128.0 [g x mL !)

in allgroups. The interntediate biofilnr producers (74.3to 88.470 of hill ing efficiency) were the most sensitiveto this compound, rvhile the low biofiim producerstrains M'ere the most resistant (59.7 to 80.3olo). Forall tested strains tl.re mosI effective concentration was128,0 pg x n l l (F ig. 2) .

An.torg most of tested strains increased concentra-tiou of quaternar')' bis ammonium salt, causecl higheranti'biotilm eficiency The most lesistant strain forcetylpyridinum bromide and quaternar)' bis ammo-nium sirlt treiltntent q.irs strain 340i08 (Fig.3). Onlvthe highest cot'rcentrations of quaternary bis itll ' lnro-llirLnr sirlt slight clecrease of absorbirnce virlues, antlthr.rs dccleased ofrluantitv oiviable bacterial cells. ForcetvlpyridiDum bromide uas not observed such a cleareffect on bactelil l biofllm.

Discussion

ln this stud)' trr'o quaternary arr]monium salts \Ą'ereexirmined against planktonic cells and biofi lm ofclini-cal Stap hylo co cc tt s e pider m i di s straills. Cetylpyriclinumbr-omiclc is rvidely used as an antiseptic compouncl. Theseconcl analyzecl agent - newly synthesized derivativecalled quaternary bis ammonium salt.

Tbere was no previoLrs reports about activity ofthesetrvo conrpourds agai,lst S. epiderrrldis biofi lm andplanktonic cells. IVIost of publications are focused onothel widely usecl, chemical .urtiseplics - chlorhexidine,triclosan, alcohols, benzalkonium chlolide (Karpanene/c i . , 2008; Cha icb e ta l . , 20 l l ; Russe l , 2003; loannouet ąl.,2007\ ' Dtrr i lrg this sttlcly cetylpyridinunr bromideproved to be more eft'ective compound, comparecl witha newll ' synthesized quaternary bis ammonium salt.Activity of this derivative towards planktonic bacteria

Er.Lclrc,rt irrtt of sl.tphvloe,, ' i t .r l h, ' l i lnrs -r(.l

was comparable with conrnronlY usec.l t[iclosan lnclchlorhexicliue. Hou'ever, Nll(ls valLrcs l it quirtcln.trvbis anlln<lr-l ium salt slrorvcd, thilt it ci ln irlsn irct a \ ar)effective antir.uicrobial aecnt. it iras bccn shorr,n thatcetylpyridinium chloritlc firr c\iririfrlc LLscd in nror.rthrinsing preparations, intel cts strongll ' rr' i l l i SlnIlrr1p1'clts tltL!ldi1s biofi lnt. fhis conpoLLntl tl i l luses sLrrwll inbiofi lm structures and appcilrs to bioaccLurulatc tirrnring a structure that practically eanrrot be rr.łshccl outfr-om biol' iLr (Nlarcotte et rrl., 100-,.

B io f i ln r deve lopmeut i s . i d lnanr i c . rnd r ' . r r i r tb leprocess ' depet l c1ent < ln nra l l l c ] i i l e re l l t c \ l c I l l i l l ł i l ( l ( ) |s .and i t i s near l r 'P r i r c t i ca l \ i ! rpor r i l r i c to o i r tł in i / r l ' i 1 , i )i n a r c p r o d u c i b l c n r a n n e r . i r r o l j i n r i t r c l l l o n r t l r c : . u r r 'strair.r. S. cpldełłł11-r bioii luls \\.rc nr()fc rcsist.lnL toboth ofusecl contpouncls coupulcrl to pl.rnktonic lornlofthe sanle strains. No11e ol.i]]c tt'slcd cot]lpotll l(łs \Viisab le to co inp le teJy er i rd i c . l te thc b io f i ln rs s t rL l c tLL fcsattached to surtace of polvstvr'.ne fl itc \\'cl ls.

Smith and HLurtcr' (Snritlr r'/ (r1.. l(X)ti) concluctcdil study on the ell-ccts oi I u,1, l)cnz.rlkorirulr chkrricle,.l ' l i ,chlorhexidine cliglucon.lte an(l l 't{, tIiclosirn rlgil ir]st l)iof l lm formed by c l in i ca l n ru l l i , . ln rs r . s i s t i rn t i r ( ) l r l t ( s ( ) t '

S' rłtłrett.s i lnd P act.riginostt- ,l

hc sltlrlc.otlct.nll. it l()l]S o1tested conrpounds rvere choscl as Lrscr.l i irr ci isinlcctionin hosp i ta l s . For thcse conrpor rnc l s thc l i i l l i nq c l t i c i cncvrvas: for .S. ałrett.s - fronl lJ9.'t l t() i(X)(ł'. l i)l. I] l].rl lqi.rJosa - about 20olo (Snith ct rl l., 1008). lhe results fronlth i s s tudy shou, , that bac ter i . r l cc l l s iu b io l i lm s t rL rc tLucare ver\, resista!rt, despite tl l i l( c() nacll t r.tl i()ns {)l lestcdcompo l rnc l s le re h ighest th .ur us f ( i in o i r f s l r ( l \ .

L rves t i s i r t io r . r ( ) r l thc 'bac tc r i c id . tL i r c t i v i tv o l th . t cs t rdcompounc ls : ce t l lpvr id in L l n r bronr ide .u r r l i r n . ' r t l l s r n 'thesized derir,ativc: quatcrn.lr\ bis .rnrnroniunr sii lt,against biol.i lnrs oi.l ' cpłlel-i lrŁli. i str.ains,.onfiIl l lccl l l,]el i te ra turcs data (Snr i th e t a l . , 1008; ( lha icb c t a/ . , 201 l ) .Bac ter ia Present in the b io l i l I l l Ł - \ I r ib j t s i sn i l i cłnt l \ 'gleatel [esistance lbr antibactcI.ial i i8Ł'Irts, i l, l c()l l,]Pil l. ison u'ith planktonic tbrms ol thc siinrc siri l ins.

None o f these conrpounr l s caLrsc r l ( ( ) t . l l c r i i ( l i ! . r t r ( )nof s taph l , lococca I b io f i ]n r in l i l t cs tc r . l conccn l r ' . r ( ious .

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