bacteria and their by - products play an essential role in the establishment and development of pulpal and periapical alterations . once bacteria gain access to the complex root canal system , they organize in communities known as biofilms . although the bacterial biofilm associated with periradicular infections is mostly constituted of obligatory anaerobic organisms , some facultative anaerobes like enterococcus faecalis are involved in persistent diseases . this type of infection can not be eliminated by host defense mechanisms , and needs to be treated by chemical  mechanical procedures . irrigation by chemical solutions should be aimed to eliminate both the smear layer  an organic and inorganic layer formed during instrumentation  and the biofilm . as the latter is a more difficult structure to remove compared with bacteria in a planktonic state , the irrigating solutions must exert antimicrobial activity as well as be capable of removing the exopolymeric substance ( eps ) to guarantee disruption of the biofilm . it is a bisbiguanide with proven antimicrobial activity and substantivity in contaminated root canals ( for a review , see ref . another irrigant in this group is alexidine ( alx ) , which differs chemically from chx because of the presence of two hydrophobic ethylhexyl groups in its structure ; it provides for faster bactericidal activity and bacterial permeabilization . also , 1% alx can be as effective against e. faecalis infection as 2% chx in bovine dentin blocks , and it has been demonstrated that its interactions with sodium hypochlorite ( naocl ) do not produce precipitates . n - acetylcysteine ( nac ) is a potent thiol - containing antioxidant and a mucolytic agent widely used in medical treatment of chronic bronchitis and acetaminophen overdose . its principal mechanism of action consists of reducing the production of extracellular polysaccharide , thereby disrupting mature biofilms and reducing adhesion of bacteria on surfaces . however , its antimicrobial activity against biofilms appears to depend on the bacterial strain . it is very effective against certain gram - negative bacilli , such as klebsiella pneumoniae , and some gram - positive cocci . recently , nac was found to be effective against e. faecalis biofilm . as a mucolytic agent thus , the aim of this study was to evaluate the efficacy of alx alone and associated with nac , in eradicating two e. faecalis strain biofilms after 1 and 5 min of exposure time . single biofilms of e. faecalis strain atcc 29212 and e. faecalis strain d1 isolated from a failed endodontic treatment were grown in the mbec - high - throughput ( mbec - htp ) device ( innovotech , edmonton , alberta , canada ) , previously described by ceri et al . and adapted for e. faecalis . the top half is a lid with 96 polystyrene pegs , and the bottom half is a fluted trough that guides inoculated growth medium across the pegs when the device is placed on a rocker . we inoculated the trough with approximately 110 colony forming units ( cfus ) per ml of each e. faecalis strain suspended in 22 ml of brain heart infusion ( bhi ; scharlau chemie s.a . , the device was then placed on a rocking table ( model swing sw 8 10000 - 00015 ; ovan , badalona , spain ) and incubated at 37  c for 24 h under aerobic conditions at five rocks per minute . biofilms forming on the lid of the mbec - htp device were rinsed once with 0.9% saline solution for 2 min to remove loosely adherent planktonic bacteria . to verify biofilm formation , four pegs were removed from each device , placed in 200  l 0.9% saline and sonicated on a water - table sonicator ( model 5510e - mt ; branson , danbury , ct , usa ) for 10 min . the disrupted biofilms were diluted serially from 10 to 10and plated for viable cell counting . this growth control was used to evaluate the initial number of bacteria formed in the biofilm in each assay . an initial solution of 2% alx in 30% ethanol ( ph 2.63 ) was prepared . nine twofold dilutions were made of this solution in sterile distilled water up to a concentration of 0.007 8% alx in 0.117% ethanol . the action of ethanol on the biofilms at the concentrations used in the alx solutions was previously determined , showing that ethanol achieved a reduction of 0.5 logarithmic units at all the concentrations tested , which is considered trivial . to prepare the nac combined with alx , we first established the biofilm eradicating ability of nac at ph 11 with naoh ( merck , darmstadt , germany ) and at ph 2.63 without naoh , both for 1 min . nac ( acofarma , terrassa , barcelona , spain ) was dissolved in ethanol at 20% to obtain a final concentration of 100 mgml . all the dilutions were carried out using sterile distilled water and were stored at room temperature until use , for no more than 60 min . to prepare the combinations , dilutions of alx were mixed 11 with nac 100 mgml . the susceptibility tests were done in a microtiter plate known as the challenge plate ( nunclon delta surface ; nunc , roskilde , denmark ) . the dilutions of the irrigating solutions were placed along the length of the challenge plate , allowing the first and the last wells of each row to serve as the sterility and growth controls , respectively . the peg lid was submerged in the challenge plate ( for 1 and 5 min ) . after exposure , the biofilms were rinsed twice by placing the lid on two additional microtiter plates containing 180 ml of 0.9% saline solution for 2 min to remove loosely adherent planktonic bacteria , and they were then placed in a microtiter recovery plate with 180 ml of bhi per well and sonicated 10 min . the disrupted biofilms were diluted serially from 10 to 10 in 0.9% saline and 10  l aliquots were plated for viable cell counting . the effect of each test agent on the biofilm was determined by calculating the percentage of viable bacteria kill as follows : [ 1(mean cfuirrigant / mean cfuinitial bacterial number)]100% the term eradication was used to denote the death of 100% of the bacterial population . when the percentage varied from 100% , the student 's t - test was used , previously subjecting data to the anscombe transformation . single biofilms of e. faecalis strain atcc 29212 and e. faecalis strain d1 isolated from a failed endodontic treatment were grown in the mbec - high - throughput ( mbec - htp ) device ( innovotech , edmonton , alberta , canada ) , previously described by ceri et al . and adapted for e. faecalis . the top half is a lid with 96 polystyrene pegs , and the bottom half is a fluted trough that guides inoculated growth medium across the pegs when the device is placed on a rocker . we inoculated the trough with approximately 110 colony forming units ( cfus ) per ml of each e. faecalis strain suspended in 22 ml of brain heart infusion ( bhi ; scharlau chemie s.a . , the device was then placed on a rocking table ( model swing sw 8 10000 - 00015 ; ovan , badalona , spain ) and incubated at 37  c for 24 h under aerobic conditions at five rocks per minute . biofilms forming on the lid of the mbec - htp device were rinsed once with 0.9% saline solution for 2 min to remove loosely adherent planktonic bacteria . to verify biofilm formation , four pegs were removed from each device , placed in 200  l 0.9% saline and sonicated on a water - table sonicator ( model 5510e - mt ; branson , danbury , ct , usa ) for 10 min . the disrupted biofilms were diluted serially from 10 to 10and plated for viable cell counting . this growth control was used to evaluate the initial number of bacteria formed in the biofilm in each assay . an initial solution of 2% alx in 30% ethanol ( ph 2.63 ) was prepared . nine twofold dilutions were made of this solution in sterile distilled water up to a concentration of 0.007 8% alx in 0.117% ethanol . the action of ethanol on the biofilms at the concentrations used in the alx solutions was previously determined , showing that ethanol achieved a reduction of 0.5 logarithmic units at all the concentrations tested , which is considered trivial . to prepare the nac combined with alx , we first established the biofilm eradicating ability of nac at ph 11 with naoh ( merck , darmstadt , germany ) and at ph 2.63 without naoh , both for 1 min . nac ( acofarma , terrassa , barcelona , spain ) was dissolved in ethanol at 20% to obtain a final concentration of 100 mgml . all the dilutions were carried out using sterile distilled water and were stored at room temperature until use , for no more than 60 min . to prepare the combinations , dilutions of alx were mixed 11 with nac 100 mgml . the susceptibility tests were done in a microtiter plate known as the challenge plate ( nunclon delta surface ; nunc , roskilde , denmark ) . the dilutions of the irrigating solutions were placed along the length of the challenge plate , allowing the first and the last wells of each row to serve as the sterility and growth controls , respectively . the peg lid was submerged in the challenge plate ( for 1 and 5 min ) . after exposure , the biofilms were rinsed twice by placing the lid on two additional microtiter plates containing 180 ml of 0.9% saline solution for 2 min to remove loosely adherent planktonic bacteria , and they were then placed in a microtiter recovery plate with 180 ml of bhi per well and sonicated 10 min . the disrupted biofilms were diluted serially from 10 to 10 in 0.9% saline and 10  l aliquots were plated for viable cell counting . the effect of each test agent on the biofilm was determined by calculating the percentage of viable bacteria kill as follows : [ 1(mean cfuirrigant / mean cfuinitial bacterial number)]100% the term eradication was used to denote the death of 100% of the bacterial population . when the percentage varied from 100% , the student 's t - test was used , previously subjecting data to the anscombe transformation . the nac at ph 11 had no antimicrobial effect and at ph 2.63 showed limited antimicrobial effect , with a mean of reduction of 1.14 of logarithmic units . the results of the antimicrobial activity of alx alone and associated with nac against both e. faecalis strain biofilms are shown in table 1 . globally , the two strains tested showed very similar sensitivity to alx alone and combined with nac . after 1-min contact time , alx eradicated the biofilms at all concentrations except for 0.007 8% for e. faecalis atcc 29212 and 0.015 6%0.007 8% for e. faecalis d1 . when combined with nac , similar results were obtained regarding eradication / concentration , the exception being alx at 0.015 6% against e. faecalis atcc 29212 ; when used alone , it achieved eradication but not when combined . after 5-min contact time , alx alone and combined with nac eradicated all enterococci biofilms . this study aimed to evaluate the antimicrobial activity of alx alone and combined with nac against two e. faecalis strain biofilms . the presence of eps is one mechanism that may explain why bacteria in the form of biofilm are less sensitive to antimicrobial agents than planktonic forms . eps acts as a barrier limiting the penetration by the agent . in this sense , the association of an antimicrobial substance  alx  with an agent of proven mucolytic activity or a potential eps disruptor such as nac should enhance antimicrobial efficacy against biofilms . to carry out these objectives , the mbec - htp device was used . it permits the formation of 96 statistically equivalent biofilms on polystyrene surfaces and moreover is easy to use , quick and gives reproducible results . in this device , two single e. faecalis strain biofilms were grown for 24 h , an ideal time period for obtaining adequate biofilm density . the strains studied were e. faecalis atcc 29212 , a strain of reference used in antimicrobial susceptibility studies and a strain isolated from a failed endodontic treatment , e. faecalis d1 . alexidine has been used as an antiseptic in mouthwashes and as a disinfectant in contact lens solutions , although in the latter , it has been associated with a fusarium keratitis outbreak . in this study , all alx dilutions eradicated the biofilms after 5-min contact time , whereas at 1 min , the concentrations that achieved 100% kill of e. faecalis atcc 29212 and e. faecalis d1 biofilms were 0.015 6% and 0.031% , respectively . these results evidence that alx effectiveness is both time- and concentration - dependent , as is chx , and support the use of irrigants at an optimal time of exposure to ensure the killing of microbial biofilms . the activity of alx would be comparable to that of naocl , which eradicated biofilms of e. faecalis grown in the mbec - htp device at a concentration of 0.006 25% after 1 min of exposure . however , in view of the fact that alx is a cationic molecule similar to chx , the results obtained with alx are noteworthy , far superior to the results obtained with chx in a previous study using the same methodology . such diverse efficacy may be attributed to structural differences , conditioning dissimilar mechanisms of action . both solutions disrupt the integrity of the bacterial cytoplasmic membrane , causing leakage of the intracellular contents . however , alx has greater affinity for lipoteicoic acids than chx , which might lead to faster permeabilization of the bacterial membrane . consequently , the bactericidal action would be more rapid , and particularly evident in an exposure period not only of 5 min but of as little as just 1 min . although its antimicrobial activity against planktonic bacteria has been demonstrated , its effect on biofilms appears to depend on the microorganism in question . in fact , aslam and darouiche demonstrated an increase of non - viable cells in nac - treated staphylococcus aureus biofilms , in contrast with a minimal to null anti - biofilm activity against e. faecalis biofilms . we found that the antibiofilm activity of nac is ph - dependent and very scarce with one of exposure , though exposure times of 1 week have served to demonstrate its activity against e. faecalis . our results indicate that the combination of nac+alx does not improve the results of alx alone . eradication was obtained with 0.015 6% and 0.031% alx ( for each of the strains ) , and when combined with nac it was achieved at 0.031% after 1 min of contact time . its ability to disrupt the eps in biofilms may condition its efficacy in detaching bacteria adhered to a surface , which would favor a higher bacterial biofilm recovery . as a result , a greater antimicrobial concentration to kill them would be needed . overall , no differences were obtained in the sensitivity of the two e. faecalis ( reference and clinical isolate ) strains to the irrigating solutions tested , despite the fact that strain d1 may have undergone environmental stress in a treated root canal , favoring the expression of virulence factors . the similar behavior of the two strains , likewise observed in a previous study using the same methodology , might be due to the fact that they are strains of the same species . within the limitations of this in vitro study , alx showed antimicrobial properties against the two e. faecalis strain biofilms tested at very low concentrations , and its combined use with nac was not seen to enhance its activity . taking into account that sensitivity is species - dependent , future research using other species and multispecies biofilms grown in the complex root canal system and under strictly controlled clinical conditions should be carried out to support these findings .
the purpose of this study was to assess the efficacy of alexidine ( alx ) , alone and combined with n - acetylcysteine ( nac ) , in eradicating two enterococcus faecalis strain biofilms . 
 the biofilms of e. faecalis atcc 29212 and the clinical isolate e. faecalis d1 were grown in the mbec - high - throughput device for 24 h and were exposed to five twofold dilutions of alx ( 2%0.007 8% ) alone and combined with 100 mgml1 nac , for 1 and 5 min . 
 eradication was defined as 100% kill of biofilm bacteria . 
 the student 's t - test was used to compare the efficacy of the associations of the two irrigants . 
 after 1-min contact time , alx eradicated the biofilms at all concentrations except for 0.007 8% and 0.015 6%0.007 8% with e. faecalis atcc 29212 and e. faecalis d1 , respectively . 
 similar results for eradication and concentration were obtained when it was combined with 100 mgml1 nac . 
 after 5 min of contact time , alx alone and combined with nac eradicated all enterococci biofilms . 
 alx showed antimicrobial properties against the two e. faecalis strain biofilms tested at very low concentrations , and its combined use with nac was not seen to enhance its activity .