for a successful bacterial colonization of the oral cavity , adherence mechanisms are essential , otherwise the bacteria get washed away and swallowed by the salivary flow . oral bacteria have evolved several mechanisms to withstand the salivary flow and succeed in adhesion to and subsequently form biofilm on surfaces of the oral cavity . streptococcus gordonii has been considered to play an important role in cariogenesis because it readily colonizes the clean tooth surfaces and is capable of forming biofilm . among the oral bacteria , s. gordonii appears to have highest affinity to hard surfaces of the oral cavity . s. gordonii biofilm forms an important component of human dental plaque by virtue of its ability to adhere to tooth surfaces . formation of dental plaque precedes cariogenesis ; thus , interfering with s. gordonii adhesion and biofilm formation of hard tissue is likely to improve the oral health . dietary agents that interfere with adhesion of and biofilm formation by bacteria has been the focus of intensive research because such natural agents are likely to be nontoxic to the host . furthermore , the identified active components can be used as supplement to oral health hygiene product negating the necessity to adhere to a particular diet . perhaps most important advantage of searching dietary agents is that approval of clinical trials would be easier to obtain , as toxicity is not an issue . in this respect , cranberry juice and isolated fractions / constituents which inhibit adhesion of bacteria to various surfaces phenolic compounds of cranberry were shown to prevent adherence of uropathogen to animal cells [ 4 , 5 ] . the cranberry components were also shown to reduce the risk of cardiovascular disease , periodontal disease , and inhibit host inflammatory response . earlier studies demonstrated a high - molecular - weight mass , nondialyzable material ( ndm ) prepared from cranberries to contain polyphenolic compounds that inhibited the secretion of proteolytic enzymes by periodontopathogens , adhesion of a number of bacterial species [ 10 , 11 ] , and were also shown to interfere with coaggregation of oral bacterial species and biofilm formation by streptococcus mutans [ 3 , 12 ] . the polyphenol fraction of cranberry was reported to decrease the hydrophobicity of streptococcal species [ 12 , 13 ] .   in the present study , we sought to determine the ability of the high - molecular - weight component from cranberry ( ndm ) to interfere with biofilm formation by s. gordonii in general and in particular on dental composites and titanium discs . we hypothesize that the cranberry ndm will have a beneficiary role by interfering with streptococcal biofilm formation on dental materials . prevention or reduction of oral bacterial load on the surfaces of the oral cavity will have a beneficial role in improving the oral health . ndm was obtained as described previously   from concentrated cranberry juice made from the american cranberry , vaccinium macrocarpon , and provided by ocean spray cranberries , inc . briefly , ndm was obtained after lyophilization of the material retained in a dialysis bag ( 12,000 molecular weight cutoff ) following extensive dialysis . the retentate of the bag designated ndm , is soluble in water up to 4  mg / ml , devoid of proteins , carbohydrates , and fatty acids , and was found to exhibit tannin - like properties suggests that it is rich in phenolic compounds ( e.g. , proanthocyanidins ) . further analysis performed by ocean spray inc . revealed that this fraction is devoid of sugars , acids , and nitrogen and contains 0.35% anthocyanins ( 0.055% cyanidin-3-galactoside , 0.003% cyanidin-3-glucoside , 0.069% cyanidin-3-arabinoside , 0.116% peonidin-3-galactoside , 0.016% peonidin-3-glucoside , and 0.086% peonidin-3-arabinoside ) and 65.1% proanthocyanidins [ 9 , 12 ] . streptococcus gordonii challis ( atcc , rockville , md ) was grown in trypticase soy broth ( tsb ; difco labs ) for 48 hours at 37c . cells were washed in pbs and resuspended to contain 5  10 cells / ml . following the treatment of bacteria with ndm , s. gordonii cells were incubated with 50  l of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-{(phenylamino)carbonyl}-2h - tetrazolium hydroxide ( xtt ) for 4 hours at 37c . in this assay , the tetrazolium salt xtt is cleaved to an orange - colored formazan product by mitochondrial dehydrogenase in viable cells . at the end of incubation period , the absorbance of the resulting supernatant was measured at 490  nm using an elisa reader ( bio rad laboratories ) . prior to the measurement of number of cells in the biofilm , a standard curve was prepared with known numbers of bacteria . following the treatment of bacteria with ndm , s. gordonii cells were incubated with 50  l of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-{(phenylamino)carbonyl}-2h - tetrazolium hydroxide ( xtt ) for 4 hours at 37c . in this assay , the tetrazolium salt xtt is cleaved to an orange - colored formazan product by mitochondrial dehydrogenase in viable cells . at the end of incubation period , the absorbance of the resulting supernatant was measured at 490  nm using an elisa reader ( bio rad laboratories ) . prior to the measurement of number of cells in the biofilm , a standard curve was prepared with known numbers of bacteria . we also used the crystal violet staining method to assess the effect of ndm on the bacterial biofilm formation in a 96-well microtiter plate . adherent bacteria in the wells were fixed with methanol for 15  min , extensively washed with distilled water , and then stained with 0.4% crystal violet ( 100  l ) for 15  min . wells were rinsed with distilled water and dried at 37c for 2  h. after adding 100  l of 95% ( v / v ) ethanol to each well , the plate was shaken to release the stain . all assays were run in triplicate , and the means  sd of three independent experiments were calculated . freshly cultured bacterial suspension containing 5  10 cells / ml was prepared , from which 0.1  ml suspension was placed in each of a 96-well microtiter plate . an equal volume of serially diluted ndm ( 5.0 to 400  g / ml ) in phosphate - buffered saline ( pbs ; 0.1  m na2hpo4 , 0.1  m kh2po4 , 0.15  m nacl , ph 7.4 ) was added to each well containing the bacteria and incubated for 24 hours at 37c . after the incubation , unattached bacteria were washed off by rinsing the plate with pbs . the microtiter plate was replenished with fresh tsb media ( 0.2  ml ) supplemented with 0.2% sucrose and incubated for an additional 24 hours . at the end of incubation , nonadherent cells were removed and the metabolic activity of the bacteria in the biofilm was assessed by xtt assay and by staining with crystal violet , as described above . for control purposes , 2.0  ml of standard bacterial suspension was immersed in a beaker of boiling water for 5 minutes and then treated with xtt reagent as the experimental cells . espe composite material was molded into a 6  mm disc and cured for 10 seconds as recommended by the manufacturer . in a similar manner , amalgam discs ( 6  mm ) an aliquot ( 0.5  ml ) of standard bacterial suspension was incubated with cranberry ndm ( 5 to 200  g / ml ) for 60  min at 37c . bacteria were then added to the discs placed in a 24-well culture dish and further incubated for 24 hours at 37c . after gentle washing to remove the nonadherent bacteria , discs were incubated with 0.5  ml of tsb supplemented with 0.2% sucrose for an additional 48 hours to facilitate biofilm formation . at the end of incubation , discs were rinsed once and then incubated with xtt reagent to assay for their metabolic activity . bacteria were stained using the live / dead baclight kit ( molecular probes - invitrogen , carlsbad , ca ) . this stain distinguishes live cells from dead bacteria based on membrane integrity and two nucleic acid stains . the green fluorochrome ( syto 9 ) can penetrate intact membranes , while the larger red fluorochrome ( propidium iodide ) penetrates only compromised membranes of dead bacteria . cells treated with ndm ( 25  g / ml for 60 minutes ) or not ( control ) were stained with the dye for 15 minutes in dark . bacterial cells ( 5  10 cells ) were pelleted , and the pellet was resuspended in 1.0  ml of ndm ( 25  g / ml ) and incubated for 60  min . the cells were stained with live / dead kit ( molecular probes - invitrogen , carlsbad , ca ) fluorescent dyes , calcein am , and ethidium homodimer diluted according to the manufacturer 's recommendation for 15 minutes in dark at room temperature . similar numbers of bacteria were incubated with 70% isopropanol for 45 minutes to generate dead cells , which were also stained similarly ( dead cell control ) . the effect of ndm on bacterial cells was analyzed by facscan flow cytometry ( becton dickinson ) using 520  20  nm excitation for measuring calcein green fluorescence emission and ethidium homodimer red fluorescence emission using 615  30  nm . the data was processed with flowmax software ( partec ) , and electronic gating with the software was used to separate positive signals from noise . between 7,500 and 50,000 events were acquired using linear amplification for forward and side scatter and logarithmic amplification for fluorescence . samples were measured in triplicate , and selected samples were controlled with epifluorescence microscopy to confirm the bacterial nature of stain . bacterial samples were stained with the live / dead baclight bacterial viability kit ( invitrogen l-13152 ) , a rapid epifluorescence staining method as specified by the manufacturer . the bacteria were incubated for 15 minutes in dark and then examined for the difference in live and dead cells between ndm - treated and control s. gordonii by confocal microscope . images were analyzed by using comos software ( bio rad ) , and green and red images were merged and formatted on confocal assistant software ( bio rad ) . data are expressed as the means  standard deviations of three independent experiments with triplicate samples in each experiment . to investigate the effect of ndm on metabolic activity of s. gordonii in biofilm , bacteria were treated with serially diluted ndm and then measured their metabolic activity by xtt metabolic assay . cranberry ndm inhibited s. gordonii metabolic activity in a dose - dependent manner ( table 1 ) . a 50% reduction in bacterial metabolic activity was seen when treated with 7.5  g / ml of ndm , while 50  g / ml of ndm inhibited by 96% . total inhibition in metabolic activity colony forming units of the ndm treatment of bacteria revealed that viability was not affected at any of the concentrations tested suggesting that the inhibition of metabolic activity was not related to ndm toxicity to bacteria ( data not shown ) . the results ( figure 1 ) show that the time of treatment and concentration of ndm affected the s. gordonii metabolic activity . pretreatment of bacteria with 2.5  g / ml of ndm for 15 minutes reduced bacterial metabolic activity as estimated by xtt assay by 20% of the bacteria when compared to the untreated bacteria ( control ) . bacteria treated with 25  g / ml ndm showed metabolic reduction of 66% ( figure 1 ) . higher concentrations of ndm ( 100  g / ml ) completely abolished the metabolic activity ( data not shown ) . contrary to the reduction in metabolic activity of s. gordonii by ndm treatment , there was no reduction in biofilm mass when stained with crystal violet ( figure 2 ) . biofilm mass was reduced by only 8% of the control when bacteria were treated with 100  g / ml . there was no significant difference in biofilm mass between the groups treated with different concentrations of ndm . the data suggests that the cranberry ndm at relatively low concentrations does not appear to inhibit biofilm formation as estimated by the total mass but the metabolic activity of the formed biofilm was affected by ndm . in figures 1 and 2 , one ml aliquots of standard bacterial suspension in triplicate was incubated with varying concentrations of ndm ( 5.0 to 200  g / ml ) for 15 , 30 , 45 , and 60 minutes . after the incubation , bacteria were centrifuged and the pellet was washed with pbs once and suspended in 0.2  ml of tsb culture media supplemented with 0.2% glucose for an additional 48 hours and measured their metabolic activity in the biofilm by xtt . another parallel set of bacterial cells treated in a similar way was stained with crystal violet , and the optical density was measured at 550  nm . for control purposes , 1.0  ml of standard bacterial suspension was immersed in a beaker of boiling water for 5 minutes and then treated as above with xtt and crystal violet . next , we investigated the effect of cranberry ndm on preformed s. gordonii biofilm and assayed metabolic activity by xtt and biofilm mass by crystal violet method . the results ( table 2 ) demonstrate that the ndm failed to detach the bacteria from the preformed biofilm mass , even at a concentration of 3  mg / ml . on the other hand , it is of interest to note that the bacterial metabolic activity was reduced by ndm treatment ( table 2 ) . ndm at a concentration of 100  g / ml for 60 minutes reduced the metabolic activity by 50% , but the same concentration had no effect on biofilm mass . increasing the length of treatment of preformed biofilm up to 8 hours also showed similar results ( data not shown ) . the results ( figure 3 ) demonstrate that the ndm treatment of s. gordonii showed a reduction in metabolic activity of bacteria adherent to both titanium and polystyrene surfaces . greater than 50% reduction in metabolic activity was seen with 10  g / ml concentration of ndm . higher concentration of ndm ( > 25  g / ml ) reduced the metabolic activity of the adherent bacteria by greater than 98% . in contrast , ndm at the same concentrations reduced total bacterial mass by only 58 percent , when stained with crystal violet ( data not shown ) , consistent with the data shown in figure 2 . we tested the ndm effect on metabolic activity of biofilm created on amalgam , composite , and hydroxyl apatite discs by xtt assay as described previously . the results ( table 3 ) of the study showed that ndm treatment inhibited s. gordonii biofilm metabolic activity on the two dental restorative materials as well as on polystyrene surface . 25  g / ml of ndm inhibited approximately 50% of bacterial metabolic activity on all three discs . no measurable inhibition of metabolic activity was seen when bacteria were treated with ndm concentrations greater than 100  g / ml . in contrast , the total bacterial mass of the biofilm bacteria was reduced by only 58% as determined by crystal violet staining ( data not shown ) consistent with the data shown earlier . the data obtained in this study so far suggested that the cranberry ndm inhibited the metabolic function of s. gordonii without being bactericidal . in order to confirm this observation , we stained the biofilm of s. gordonii created on polystyrene with live / dead baclight according to the recommended protocol by the manufacturer ( invitrogen ) . stained bacteria were first viewed by a fluorescent microscope and then by confocal microscope . figure 4 shows that ndm treatment did not appear to cause cell death , and both ndm treated ( figure 4(a ) ) and untreated bacteria ( figure 4(b ) ) appear to contain similar proportion of live and dead bacteria . the results of flow cytometry ( figures 5(a ) and 5(b ) ) indicate that ndm treatment did not change in the ratio of live to dead bacteria consistent with our microscopic observation that the ndm has no cytotoxic effect on s. gordonii cells . early colonization on the tooth surface and subsequent biofilm formation by s. gordonii and their ability to coaggregate with several oral microorganisms result in the formation of dental plaque . the plaque and biofilm formation leads to caries and subsequently leads to periodontal disease if left untreated . constituents of cranberry were demonstrated to decrease the hydrophobicity of streptococcal species [ 12 , 16 ] . cranberry high - molecular - weight component was shown to inhibit secretion of glucosyl and fructosyltransferases by oral streptococci   and coaggregation of bacteria . the high - molecular - weight component of cranberry was shown to be highly soluble in water , lacking proteins , carbohydrates , and fatty acids [ 17 , 18 ] . previous studies have shown that relatively high concentrations of ndm ( e.g. , 0.52  mg / ml ) were required to cause 80% or more reduction of biofilm formation by s. mutans on saliva - coated ha   and by p. gingivalis on polystyrene surfaces . in the present study , we show that lower concentrations of ndm ( 0.050.1  mg / ml ) selectively inhibited the metabolic activity of oral bacteria , s. gordonii . this conclusion is based on ( i ) the effect of ndm on metabolic activity of preformed biofilm that was more profound than on the total biofilm mass as measured by crystal violet stain and ( ii ) the confocal microscopy stain with live / dead stain showing no effect on the ratio of viable / dead s. sangius . the effect of ndm on metabolic activity may indirectly interfere with the ability of the bacteria to adhere and form biofilm onto various dental surfaces as shown in the present study and in other studies [ 7 , 12 , 16 ] . previous studies have shown that ndm affects various physicochemical properties of uropathogenic bacteria [ 4 , 9 ] . the present study shows an effect on metabolic activity , and further studies are needed to see how these two effects are connected . perhaps most important is to study how these effects of relatively low concentrations of ndm on metabolic activity of s. sangius biofilm influences cariogenicity of the bacteria such as acid formation . either way , ndm seems to affect s. sangius adhesion and biofilm formation mainly by inhibiting metabolic activity of the cariogenic bacteria . it is expected that supplementing oral health product such as mouth wash with ndm will affect not only s. mutans total counts as shown previously   but also s. sangius cariogenic activity . our study revealed the beneficial role of cranberry ndm in reducing the s. gordonii metabolic activity in the biofilm created on various dental surfaces such as titanium implant material , amalgam , and composite materials . the potential use of cranberry ndm in oral rinse merits further investigation , since it appears to benefit the health of the oral cavity , by reducing the metabolic activity of s. gordonii .