microorganisms exist as free - floating cells or , more often , in a community of cells attached to a substrate . this sessile form of life is referred to as a biofilm . by definition ,
a biofilm is a community of cells attached to either a biotic or an abiotic surface enclosed in a complex exopolymeric substance .
biofilms allow microorganisms to trap nutrients and withstand hostile environmental conditions by quorum sensing ( qs ) .
qs is a widespread and well - known cell - to - cell communication phenomena that regulates biofilm formation and virulence behaviors.2 , 3 , 4 qs also involves chemical communication among bacteria including formation , secretion , detection , and reaction to molecules known as autoinducers .
several serious infections are reported to be a result of biofilm formation , which leads to chronic diseases in most cases .
these persistent infections are a challenge for public health on a global scale , because they reduce the effectiveness of treatments and increase morbidity , mortality , and health - care costs .
streptococcus mutans is an impotent pathogen and is a common cause of oral infections such as dental caries .
s.  mutans effectively utilizes dietary sucrose to synthesize large amounts of exopolysaccharides , which play an important role in the accumulation , adhesion , and plaque matrix formation of microorganisms .
the ability of a microorganism to form a biofilm on a host tissue surface is an important step in the development of infection . because of poor hygiene
, various pathogenic microorganisms cause infections . at present , a number of antibiotics are used for treating these infections .
however , because these antibiotics are associated with significant side effects , there is increased attention toward using natural , biologically active herbal compounds as an alternative medicine.7 , 8 both embelin and piperine are natural compounds that are found in embelia ribes and piper longum , respectively .
they are reputed medicinal herbs and their various parts have been used as a traditional cure in the asian system of medicine ( indian , chinese , and malaysian ) for treating a variety of disease conditions.9 , 10 , 11 , 12 the objective of this study was to identify the effects of natural compounds such as embelin and piperine on biofilm formation .
s.  mutans was isolated from dental caries or plaque from patients in the outpatient department of peoples dental academy ( bhopal , madhya pradesh , india ) .
the bacterium was isolated from both male and female patients ( mean age , 20 years ) .
bacterial samples were cultured on the following media : brain heart infusion ( bhi ) agar medium ( himedia laboratories , india ) in a 5% co2-enriched atmosphere and mutans - sanguis agar medium ( himedia laboratories , india ) .
biochemical tests were performed to identify the bacterial strains . among the 20 samples from patients having dental caries , 30 isolates
the compounds were dissolved in dimethyl sulfoxide ( dmso , mark , germany ) at a concentration of 10  mg / ml . quantification of s.  mutans isolates ' biofilm formation was carried out using the microtiter plate method . to assay biofilm formation of the s.  mutans
isolates , an overnight culture of each isolate was grown in bhi broth ( himedia laboratories , india ) for 1820 hours at 37c .
approximately 1  ml of each overnight culture was transferred to 10  ml of sterile bhi broth with the addition of 1% sucrose for biofilm production .
the suspensions were adjusted using the same bhi medium to 0.5 on the mcfarland turbidity standard as measured by absorbance ( 0.080.1 at 625  nm ) in a spectrophotometer ( shimadzu , australia ) , corresponding to approximately 10  cfu / ml . then , from each culture , a 250-l volume was transferred into the wells of a microtiter plate ( himedia laboratories ) .
after 24 hours , the planktonic suspension and nutrient solution were aspirated and each well was washed three times with 300  l of sterile physiological saline .
the remaining attached bacteria were fixed with 250  l of 96% ethanol / well and , after 15 minutes , the plates were emptied and left to dry .
each well was then stained for 5 minutes with 200  l of 2% crystal violet ( cv gram stain , merck , germany ) .
the stain was rinsed off by placing the plates under running tap water . after drying the stained plates , biofilms were visible as purple rings on the sides of each well .
the quantitative analysis of biofilm formation was performed by adding 200  l of 33% ( v / v ) glacial acetic acid ( merck ) per well . the optical density ( od ) of the stain
was then measured at 492  nm using an enzyme - linked immunosorbent assay reader ( lisa , germany ) as described previously .
biofilm formation was scored as follows : nonbiofilm forming ( a492    1 ) ;  + , weak ( 1    a492    2 ) ;  + + , moderate ( 2  <  a492    3 ) ; and  + + + , strong ( a492  > 
the biofilm of s.  mutans clinical isolates was grown as follows : individual sterile culture dishes were filled with 2.5  ml of bhi broth with 1% sucrose .
glass cover slips containing the attached biofilm were removed from the dishes , rinsed briefly with phosphate - buffered saline , and stained for 5 minutes with 0.5% crystal violet .
the minimum inhibitory concentration ( mic ) of the natural compounds embelin and piperine was evaluated on all the isolates by the broth dilution method .
the natural compounds were dissolved in dmso ( initial concentration , 20.0078  mg / ml ) .
each well was inoculated with 5  l of suspension containing 10  cfu / ml of bacteria .
after incubation , 5  l of tested broth was placed on the sterile bhi plates and incubated at respective temperature ( 37c ) .
the mic for bacterial isolates was determined as the lowest concentration of the extracts inhibiting the visual growth of the test cultures on the agar plate .
triplicates were maintained.15 , 16 only those isolates of s.  mutans that were classified as strong biofilm producers were used in the biofilm - inhibition assay .
test compounds were dissolved in dmso ( 10  mg / ml ) , and twofold dilutions were prepared to obtain a final concentration ranging from 2  mg / ml to 0.0078  mg / ml in the wells after the addition of the freshly diluted bhi broth culture containing 10  cfu of the strong biofilm - forming isolates per well .
after incubation at 37c for 24 hours , the microtiter plate was washed , fixed , and biofilms were stained and visualized as explained earlier .
the inhibitory effect of the compounds on biofilm production was calculated by subtracting the media control .
the biofilm inhibitory concentration ( bic ) is the concentration of the natural compound at which the biofilm formation was reduced to an absorbance ( a492 )  <  0.5  od . each assay for bic determination was performed in triplicate .
calculations and statistics were performed using graphpad 5.0 software ( graphpad software inc . , la jolla , ca , usa ) .
the 30 s.  mutans isolates were screened for biofilm formation by the microtiter plate method and the results are shown in fig .  1 .
all 30 isolates were classified based on their biofilm - forming potential as follows : 18 were strong biofilm producers , nine isolates were moderate producers , and three were poor / nonbiofilm producers . the s.  mutans atcc 25175 was included as an assay control and identified to be a moderate biofilm producer .
this observation confirms that the magnitude and intensity of biofilm formation of the 18 isolates were significantly greater than those of the poor / nonbiofilm producers .
1 also shows the quantitative evaluation for identifying and demarcating strong biofilm - producing s.  mutans isolates from moderate- and poor / nonbiofilm - producing isolates .
the biofilm - forming potential of the strong producers at od492 was greater than 1 , whereas that of nonbiofilm - producing isolates at od492 was less than 1 .
the isolate sm03 was identified as the strongest biofilm producer , whereas sm06 and sm27 were identified as moderate and poor / nonbiofilm producers , respectively .
to visualize biofilm formation by the three categories of s.  mutans isolates ( strong , moderate , and poor / nonbiofilm producers ) , the microscopic slide assay was performed and the results are shown in fig .  2 .
the biofilm formation was clearly visible for the strong biofilm producer sm03 , followed by the moderate biofilm producer sm06 at 48 hours .
however , even after 48 hours , the poor / nonbiofilm producer sm27 did not form the biofilm .
the strong biofilm producers also showed strong adherence to the slide , and therefore , we selected one of the strongest biofilm producers ( sm03 isolate ) for evaluating the mic and bic of the natural compounds .
the results of mic and minimum biofilm inhibitory concentration ( mbic ) of the natural compounds analyzed for the strongest biofilm producer isolate sm03 are presented in table  1 . a significant difference in the mic and bic of the natural compounds was noted : the mic of embelin was 0.55    0.02 , whereas that of piperine was 0.33    0.02 , ( mic , piperine < embelin ) .
the mbic of embelin was 0.0620    0.03 , whereas that of piperine was 0.0407    0.03 .
these results confirm that piperine is the most potent antimicrobial and antibiofilm compound ( piperine  >  embelin ) .
the results of biofilm inhibition by embelin and piperine in each strong biofilm - forming s.  mutans isolate are presented in table  2 .
these results clearly confirm that the mbic of embelin and piperine had reproducible biofilm inhibitory activity against each of the strong biofilm - producing isolates of s.  mutans .
the comparative effect of minimum biofilm - inhibition concentrations of embelin ( 0.0620    0.03 ) and piperine ( 0.0407    0.03 ) at od492  <  0.5 shows the significant inhibition of biofilm by these compounds .
the mbic and mic values indicate that piperine has better ability to inhibit biofilm formation than embelin .
s.  mutans plays a significant role in dental infection by effectively utilizing sugars and synthesizing large amounts of exopolysaccharides , which plays a vital role in adhesion of bacteria and accumulation of biofilm .
thus , the organized structure and mechanisms of biofilm are responsible for the emergence of drug - resistant bacteria .
the present allopathic formulations used in oral care contain antibiotics , antimicrobial agents , surfactants , and alcohol , but these are not efficient in completely eradicating oral pathogens ; in addition , they were found to be cytotoxic.18 , 19 therefore , there is a growing interest in using plant - derived products against oral pathogens .
thus , in this study , the natural compounds embelin , which is commonly found in e.  ribes , and piperine , which is commonly found in p.  longum  both are widely distributed plant species in india  were chosen for the evaluation of the biofilm - inhibition effect.20 , 21 the objective of this study was to evaluate the biofilm - inhibition effect of natural compounds on clinical isolates of s.  mutans .
thirty clinical isolates were screened ; of these , 18 strong biofilm producers were identified ( fig .  1 ) .
to determine the minimal concentration required for biofilm inhibition , we evaluated the inhibitory effects of embelin and piperine on bacterial growth and biofilm formation by sm03 , which was identified to be the strongest biofilm - forming isolate ( table  1 ) .
the comparative effects of embelin and piperine compounds on growth inhibition and biofilm inhibition are summarized in table  1 .
the biofilm - inhibition concentration was significantly lower than the concentration required for inhibition of bacterial growth .
these data reflect the fact that the mbic used has the potential to inhibit biofilm formation by s.  mutans .
our results also showed that the mbic of piperine was lower than that of embelin .
the compounds might have exhibited these effects by inhibiting the activity of receptors and molecules involved in the quorum sensing pathway , which is required for biofilm formation.16 , 22 , 23 absorbance at 492  nm for biofilm inhibition in the presence of natural compounds suggested the significant potential of biofilm inhibition by piperine and embelin ( piperine  >  embelin ; table  2 ) .
we have presented a comprehensive analysis of the effects ( inhibitory ) of embelin and piperine on strong biofilm - forming isolates of s.  mutans .
. it would be interesting to find out the mode of action or cell - to - cell inhibition action behind this effect .
we conclude that these compounds would be very useful in controlling the biofilm - forming infection of s.  mutans .
 we aimed to evaluate the effects of the natural compounds embelin and piperine on the biofilm - formation property of streptococcus mutans . 
 a total of 30 clinical isolates were identified as s.  mutans and screened for biofilm formation using the microtiter plate method . the strongest biofilm producer ( sm03 ) 
 was used for identifying both minimum inhibitory concentration ( mic ) and minimum biofilm inhibitory concentration ( mbic ) . 
 we subsequently used this concentration against each of the strong biofilm producer isolates at a492  <  0.5 optical density ( od ) . 
 of the 30 isolates screened for biofilm formation , 18 isolates showed strong biofilm formation , 09 isolates showed moderate formation , and 03 isolates showed poor / nonbiofilm formation . 
 the mic of embelin for the strongest biofilm producer ( sm03 ) was 0.55    0.02 , whereas that of piperine was 0.33    0.02 . 
 the mbic of embelin was 0.0620    0.03 , whereas that of piperine was 0.0407    0.03 , which was lower than that of embelin . at od492  <  0.5 , the mbic of both compounds significantly inhibited biofilm formation of all the 18 strong biofilm - forming isolates . 
 the results of this study demonstrate a significant antibiofilm effect of the natural compounds embelin and piperine , which can contribute towards the development of a database for novel drug candidates for treating oral infections caused by s.  mutans . 
Introduction
Material and methods
Results
Discussion
Conclusion
Conflicts of interest