aggregatibacter actinomycetemcomitans ( aa ) is a gram - negative coccobacillus , facultative anaerobe , closely associated with the aetiology of severe periodontitis , but it is also implicated in different human systemic diseases , such as infective endocarditis and , rarely , in brain abscesses , osteomyelitis , or low birth weight [ 14 ] . in particular , aa virulence is strongly linked to the production of the leukotoxin ( ltxa ) , homologous to repeats - in - toxin ( rtx ) family . although not all aa strains can determine an agp , high levels of ltxa have been always detected in agp related to aa infection . clones with different leukotoxin - producing abilities have been identified , and this intraspecies diversity of aa referred to specific genotypes named 652 , jp2 , and , more recently , y4 [ 1 , 57 ] . clinically , it is one of the microorganisms responsible for both aggressive periodontitis ( agp ) and chronic periodontitis ( chp ) . these affections are chronic inflammations of the periodontal tissues related to the presence of subgingival bacteria that occur in severe forms in 520% of adult populations , according to what was recently reported by the world health organization [ 810 ] . it has been recognized that both regular supportive periodontal therapy and the domiciliary oral hygiene , related to supragingival plaque control through a host modulation of periodontal bacteria , are essential for obtaining a good clinical outcome . in this context , the use of a local chemical antiseptic able to inhibit the plaque biofilm formation could be fundamental to support the mechanical therapy [ 1113 ] . at the same time , as previously stated , aa plays an important role as an etiological agent of systemic diseases when it disseminates in the bloodstream . oral bacteria could promote a transient bacteraemia , during all the dental procedures , with variable severity . this variability could be related to both the kind of treatment and the level of communication established between oral cavity and bloodstream . in this study , daycare ( curaden healthcare , via g. parini , 19-saronno va ) and listerine ( johnson & johnson consumer healthcare , morris plains , nj , usa ) are both based on the same essential oils combination ( menthol 0,042% , thymol 0,064% , methyl salicylate 0,06% , and eucalyptol 0,0092% ) but differ in terms of the absence of alcohol ( daycare ) or the presence of alcohol with a concentration of 26,2% ( listerine ) . they show both bacteriostatic and bactericidal activities that are related to cell wall disruption and enzymatic inhibition . dentosan ( johnson & johnson consumer healthcare , morris plains , nj , usa ) is a chlorhexidine ( chx- ) based mouthrinse , and it is recognised as the most effective chemical agent , usable in the oral cavity , for biofilm inhibition . it exists in three different concentrations ( 0.20% , 0.12% , and 0.05% ) related to its clinical use . for this analysis , the percentage of chx chosen is 0.12% , that is , the concentration commonly used for biofilm modulation in long - term therapies [ 12 , 15 , 16 ] . this determines an increased permeability at low concentration while , at high concentration , it causes a precipitation of bacterial cytoplasm with bactericidal effect [ 15 , 16 ] . the aim of this work is to evaluate these commercial mouthwashes activity in order to prevent aa biofilm formation . in particular , their antimicrobial effect has been tested by calculating the minimum inhibitory concentration ( mic ) , the minimum bactericidal concentration ( mbc ) , and the minimal eradication biofilm concentration ( mebc ) . additionally , the bactericidal effect of tested mouthwashes will be evaluated in relation to the exposure time . therefore , aa viability will be estimated after 10 , 20 , 30 , 40 , 50 , and 60 seconds of exposure to each mouthwashes at the mbc dilution . the results will allow to estimate the effect on planktonic growth ( pg ) and on biofilm development ( bd ) of essential oil - based mouthwashes with or without alcohol , and it will be compared with chx activity . this study was performed using a strain of aggregatibacter actinomycetemcomitans dsm 11123   from the deutsche sammlung von mikroorganismen und zellkulturen gmbh ( german collection of microorganisms and cell cultures ) . the bacteria were grown in anaerobic difficile agar ( microbiol , uta , cagliari , italy ) at 37c for 24 hours with a co2 concentration of 5% . after the incubation , the aa was suspended in vials containing schaedler broth to obtain a concentration with a turbidity equivalent to the no . 3 mcfarland standard ( about 10  cfu / ml ) then diluted to 1/100 ( obtaining a 10  cfu / ml ) using a spectrophotometer at 620  nm ( dms100s , varian , nh , usa ) . to analyze antibacterial activity , 200  l of each previously described suspended strain was added to eleven wells of a 96-well plate and incubated for 48  h incubation at 37c with a co2 concentration of 5% . before incubation , each mouthwashe was added to the bacterial suspension following a scalar concentration pair to d = 1/2 ( where d is the dilution factor and n is equal to a number from 1 to 11 following wells sequence ) . after incubation , the pg was evaluated with spectrophotometric method using a microtiter plate reader with a lecture at 620  nm ( slt - spectra ii , slt instruments , germany ) . in order to establish when the mouthwashes reach the bactericidal concentration , whenever the spectrophotometric analysis showed an absorbance value equal to 0.000 for pg ( equivalent to the absence of bacterial growth ) , subcultures in difficult anaerobic agar all the analyses were performed three times in order to be able to do an analysis of the variance ( anova ) . once the mbc was identified , the time required for each mouthwash to reach the bactericidal effect was tested . each product was therefore placed in 6 tubes containing a bacteria concentration equal to 10  cfu / ml . sequentially , a sample was performed from each tube every 10 seconds , and a subculture in anaerobic difficile agar was prepared in order to determine the residual bacterial viability in relation with six different exposure times ( from 10 to 60 seconds ) . to perform bd analysis , the method of the microtiter plate biofilm production assay , the medium was removed and the microtiter plate wells were washed three times , with 200  l of pbs ( 0.1  m , ph 7.4 ) buffer using a pipette , and allowed to dry for 15  min . the microtiter wells were stained with 200  l of 0.4% crystal violet for 15  min . at room temperature . the unbound crystal violet stain was removed , and the wells were washed three times with 200  l of pbs buffer . the wells were air - dried for 15  min , and the crystal violet in each well was solubilized by adding 200  l of 33% acetic acid . at the end of the coloration , the biofilm was evaluated using the microtiter plate reader ( slt - spectra ii , slt instruments , germany ) at 620  nm .   to analyze the different effect that each mouthwash had on bd and pg , the absorbance values were replaced with comparing values from 0 to 1 and , in particular , the value 1 was always correlated to the absorbance value obtained with the dilution equal to 1/2 . to perform the comparison between different mouthwashes effect , antiplanktonic and antibiofilm activities were analyzed separately using the raw absorbance values . to compare the results obtained from different moutwashes and from different bacterial state ( planktonic or biofilm ) , the absorbance raw values were converted . to perform this conversion each value were transformed into a number from 0 to 1 using as 1 the absorbance value related to the dilution equal to 1/2 ( i.e. , if the 1/2abs raw value is equal to 1.29 it become 1 , so all the previous raw value ( r ) related to the same mouthwash and the same bacterial state will be converted in this way : x = r/1.29 ) . statistical analysis was performed using the two - way anova procedure with a dedicated software ( minitab , version 15.1.1.0 for windows , minitab inc . from the statistical analysis , it was possible to highlight how bacterial viability is always related to the dilution factor ( p < 0,0005 ) . it is noticed that dentosan is the most effective mouthwash against both pg and bd without any specific activity ( p : 0,192 ) . listerine inhibits the bd at a minor dilution despite the dilution necessary to have the antiplanktonic activity , obtaining mebc at the left of the mbc / mic ( 1/2 against 1/2 ) . however , a statistic relevance that highlighted a difference between its antiplanktonic and the antibiofilm effects was not found ( p : 0,095 ) ( table 1 ) . bd and pg inhibition by daycare shows a peculiar behavior , whereas dentosan and listerine have only one dilution exponent of difference between mebc and mbc / mic ( resp . , 1/2 against 1/2 for dentosan and 1/2 against 1/2 for listerine ) for daycare two exponents of difference ( 1/2 against 1/2 ) with a p - value statistically relevant could be noticed ( p value : 0,032 ) ( table 1 ) .   by the analysis of antiplanktonic and antibiofilm activities separately , it was possible to determine some differences between mouthwashes in terms of raw abs values . in both of the antibacterial effects , there is a statistical difference related to the mouthwash examined ( p : 0,002 for anti - planktonic effect and p : 0,017 for antibiofilm activity ) . for mbc / mic activity , the worst mouthwash is listerine with an optically determined bacterial growth , yet with a concentration of 1/2 , followed by daycare at 1/2 . the best is dentosan that inhibited bacterial growth until a dilution of 1/2 ( table 1 ) . in the antibiofilm evaluation , listerine was the first mouthwash in which it was possible to measure the bd with an mebc at a dilution of 1/2 . although the listerine does not have a high mebc , it showed to have a good biofilm inhibition until the higher dilution tested ( 1/2 ) with abs values minor then registered for dentosan . as for the mbec , the best activity was again the one of dentosan ( 1/2 ) while the worst effect was found for daycare that showed the highest level of abs for biofilm , yet with a dilution of 1/2 ( figure 2 , table 1 ) . in analyzing the raw values of absorbance related to pg and bd separately it was possible to highlight a good activity of daycare against pg in contrast to the worst activity on bd inhibition related to other mouthwashes ( table 1 , figures 1 and 2 ) . all mouthwashes tested presented a good antibacterial activity when they are used at concentration under a dilution of 1/2 . from the exposure time analysis , dentosan and listerine were found to inhibit bacterial viability after only 10 and 20 seconds , respectively , while daycare needs 30 seconds to obtain a totally bactericidal effect . recently , mouthwashes have obtained a great importance as therapeutic keystone against oral infections such as caries , candidiasis , and especially periodontitis . their activity can be applied for pathology prevention , as well as for preventing systemic pathologies linked to transient bacteraemia that is always generated during dental manipulations [ 3 , 14 , 22 ] . a good mouthwash should have a good antibacterial activity against both pg and bd in order to prevent all these conditions . the present analysis is focused on the study of this double effect , fundamental to evaluate the real antibacterial power of each mouthwash . from the results , the huge difference between mouthwashes activity against aa in its planktonic and biofilm forms clearly appears . firstly , alcoholic products behave in a different way from the other two mouthwashes so that , while they have the worst effect against pg , they show at the same time the best antibiofilm activity at high dilution . in accordance with the literature , essential oils with alcohol have the best antibiofilm activity [ 2325 ] . on the base of these results , it seems that an alcoholic mouthwash could be a good choice in preventing plaque formation after periodontal therapies . in contrast , essential oil nonalcoholic mouthwashes show a better antiplanktonic rather than antibiofilm activity . in this case , it could be considered a good choice in order to prevent bacterial systemic dissemination . in particular , they could be useful for treating specific anatomic areas with a high risk of bacteraemia .   at the same time , it is reported how transient bacteraemia could be determined not only during some clinical procedures or in particular conditions but also through subclinical dental infection or daily dental brushing [ 14 , 26 ] . patients with underlying cardiopathy or with oral condition with high risk of oral bacteria dissemination need to be supported by an adequate prophylaxis through local antiseptic products . although aa pg seems to be susceptible to all tested mouthwashes , it becomes necessary to refer to a mouthwash with a strong anti - planktonic activity in case of therapies with an high risk of bacterial dissemination from the oral cavity .   in conclusion , this work acknowledges how both essential oil - based mouthwashes , with or without alcohol , have a good and comparable antibacterial power when they are used pure on aa . in accordance with the literature , it was demonstrated that all of them showed the bactericidal effect within the maximum exposure time in vivo ( 60 seconds ) even though the bacterial response fluctuates significantly after consecutive dilutions of tested antiseptics , as previously described . however , further studies on the chemical impact of alcohol as well as the action of the active ingredients of the referred mouthwashes will be needed to more full investigation . these results suggest that it is necessary for clinicians to understand both mouthwashes molecules and the pathogenic agents involved in order to obtain a therapeutic effect . further studies will be needed to confirm these results in vivo and on others bacterial species .
the aim of this work is to determine the antibacterial activity of three marketed mouthwashes on suspended and sessile states of aggregatibacter actinomycetemcomitans . 
 the efficacy of two commonly used products in clinical practice , containing essential oils as active ingredients ( menthol , thymol , methyl salicylate , and eucalyptol ) in association with or without alcohol , has been evaluated in comparison with a chlorhexidine - based mouthwash . 
 the microtiter plate assay , in order to obtain a spectrophotometric measurement of bacterial responses at growing dilutions of each antiseptic , was used for the study . 
 the analysis revealed that a good antibacterial activity is reached when the abovementioned mouthwashes were used at concentration over a 1/24 dilution and after an exposure time of 30 seconds at least . in conclusion , the alcoholic mouthwash appears to have a better biofilm inhibition than its antiplanktonic activity while the nonalcoholic product demonstrates an opposite effect with a better antiplanktonic behavior .