BACKGROUND
probiotics are defined by the food and agricultural organization of the united nations as "live microorganisms which when administered in adequate amounts confer a health benefit on the host"  <cit> ."
the effectiveness of probiotics is strain-specific, and each strain may contribute to host health through different mechanisms.
probiotics can prevent or inhibit the proliferation of pathogens, suppress production of virulence factors by pathogens, or modulate the immune response.
l. reuteri is a promising therapy for the amelioration of infantile colic, alleviation of eczema, reduction of episodes of workplace illness, and suppression of h. pylori infection  <cit> .
l. reuteri is considered an indigenous organism of the human gastrointestinal tract and is present on the mucosa of the gastric corpus, gastric antrum, duodenum, and ileum  <cit> .
biofilms or adherent structured microbial communities in the oral cavity and respiratory tract are well-characterized and are associated with respiratory infections, dental caries, and periodontitis  <cit> .
in contrast, biofilm-like communities of the gastrointestinal and female urogenital tracts containing beneficial lactobacilli may have a protective role.
in bacterial vaginosis, indigenous lactobacilli are replaced with pathogenic biofilms consisting of gardnerella vaginalis and other bacteria  <cit> .
probiotic l. reuteri can displace g. vaginalis biofilms and could potentially re-establish protective biofilms in the female urogenital tract  <cit> .
due to artifactual removal of biofilms by traditional fixatives during specimen processing, studies of gastrointestinal biofilms are sparse.
using non-aqueous fixatives and special techniques, several groups have documented the presence of intestinal biofilms in the mammalian intestine  <cit> .
the bacterial composition of biofilm-like communities in the mucus layer or the mucosa-associated microbiota , differs from the composition of the fecal microbiota, and the human mam includes lactobacilli  <cit> .
changes in the composition and architecture of the mam through diet or disease may affect overall health status  <cit> .
denser biofilms were found in patients with inflammatory bowel disease  when compared to healthy controls, and 60% of the biofilm mass was comprised of the commensal bacteroides fragilis  <cit> .
these studies indicate a need to understand the contributions of individual strains and species to the aggregate function of gastrointestinal biofilms.
this report describes the ability of an established commensal and probiotic organism, l. reuteri, to form biofilms in vitro and perform potentially beneficial functions as biofilms.
two basic probiotic functions that depend on secreted factors were studied in the context of biofilms.
first, modulation of innate immunity was investigated by studying regulation of human tnf production.
in prior studies, supernatants from planktonic l. reuteri cultures reduced production of the pro-inflammatory cytokine, tnf  <cit> , and tnf suppression was important in alleviating inflammation in a murine model of ibd  <cit> .
probiotic l. reuteri biofilms have not been examined for tnf inhibition.
secondly, production of the antimicrobial compound β-hydroxy-propionaldehyde, known as reuterin, was evaluated in order to assess anti-pathogenic properties of l. reuteri biofilms.
RESULTS
probiotic lactobacillus reuteri forms biofilms
various human isolates of l. reuteri were grown in 96-well polystyrene plates and retention of crystal violet was used to assess relative biofilm densities .
all strains of l. reuteri adhered to polystyrene, but strains differed with respect to relative densities as measured by absorbance spectrophotometry.
l. reuteri strains atcc pta  <dig> and atcc pta  <dig>  formed aggregates with greater optical densities than l. reuteri strains atcc  <dig> and cf48-3a .
the differences between strains were also observed in cell counts.
the bacterial densities  in biofilms of atcc pta  <dig> and atcc pta  <dig> were roughly 10-fold greater than the bacterial densities of atcc  <dig> and cf48-3a biofilms .
l. reuteri biofilms were stained with acridine orange and observed by confocal microscopy .
monospecies biofilms of atcc  <dig> were  <dig> μm  thick.
the thickness of l. reuteri biofilms was assessed at  <dig> and  <dig> hours.
no differences in biofilm thickness were observed.
consistent with this study, other researchers demonstrated formation of only thin biofilms  when l. reuteri biofilms were cultured on plastic coupons for  <dig> hours  <cit> .
l. reuteri biofilms modulate human tnf production
to test the immunomodulatory properties of l. reuteri biofilms, supernatants from the biofilms were added to human monocytoid thp- <dig> cells in the presence and absence of lps.
lps was added to the thp- <dig> cells to stimulate production of pro-inflammatory tnf by thp- <dig> cells.
l. reuteri strains that produced tnf inhibitory factors as planktonic cultures   demonstrated similar abilities to suppress tnf production when cultured as biofilms .
when tnf inhibitory factors were obtained directly from l. reuteri biofilms grown in 24-well polystyrene plates, atcc pta  <dig> and atcc pta  <dig> also inhibited tnf production by 60% and 50%, respectively, when compared to the media control .
supernatants of l. reuteri atcc pta  <dig> biofilms cultured in a flow cell inhibited tnf by 73% compared to the media control .
l. reuteri strains that did not suppress human tnf in planktonic phase   lacked tnf-inhibitory capabilities when supernatants were obtained from the same strains cultured as biofilms .
surprisingly, supernatants from atcc  <dig> and cf48-3a biofilms did not induce tnf production by thp- <dig> cells in the absence of lps  as the supernatants from planktonic cultures did .
interestingly, the ability of probiotic l. reuteri to regulate human tnf production is strain-specific, and strain-specific tnf inhibition was maintained whether l. reuteri strains were cultured as planktonic cells or biofilms.
the relative abilities to suppress human tnf in monocytoid cells were directly correlated with relative abilities to aggregate and form biofilms on polystyrene surfaces .
l. reuteri cultured as planktonic cells and biofilms produced the antimicrobial factor, reuterin
antimicrobial activities of l. reuteri were assessed by examining supernatants of planktonic and biofilm cultures for reuterin.
planktonic cells and biofilms of l. reuteri produced reuterin, although differences in reuterin production were evident among strains.
planktonic cultures of atcc pta  <dig>  atcc pta  <dig>  attc  <dig> and cf48-3a produced  <dig> ,  <dig> ,  <dig> , and  <dig>  mm of reuterin, respectively.
when reuterin quantities were normalized to initial cfu/ml, planktonic cultures of atcc pta  <dig> and atcc pta  <dig> produced  <dig>  and  <dig>  mmol reuterin/ <dig> cells, respectively, and atcc  <dig> and cf48-3a produced  <dig>  and  <dig>  mmol reuterin/ <dig> cells, respectively .
for biofilms cultured in multiwell plates, the four wild type l. reuteri strains atcc pta  <dig>  atcc pta  <dig>  attc  <dig> and cf48-3a produced  <dig> ,  <dig> ,  <dig> , and  <dig>  mm of reuterin, respectively.
after normalization of reuterin quantities to bacterial cell counts, atcc pta  <dig>  atcc pta  <dig>  cf48-3a, and atcc  <dig> produced  <dig> ,  <dig> ,  <dig> , and  <dig>  mmol of reuterin/ <dig> cells, respectively, when cultured as biofilms in multiwell plates .
trends in reuterin production were consistent with planktonic and biofilm cultures of atcc pta  <dig> and atcc  <dig> producing lower quantities of reuterin than strains atcc  <dig> and cf48-3a.
interestingly, the relative abilities of l. reuteri strains to produce reuterin were inversely correlated with relative abilities to aggregate and adhere to polystyrene .
previous studies indicated that planktonic cultures of human-derived l. reuteri strains used in this study were relatively resistant to the antimicrobial effects of reuterin , when compared to other bacterial species including closely related lactobacilli .
however, since the cell viabilities of planktonic cultures decrease as reuterin accumulates  <cit> , the quantities of reuterin produced by planktonic cultures were normalized to the initial cfu/ml.
reuterin was detected after biofilms were incubated in glycerol for  <dig>   <dig>  and  <dig> hours .
cell viabilities of biofilms after reuterin production exceeded 92% , indicating that the biofilms were relatively resistant to the quantities of reuterin produced by l. reuteri biofilms.
discussion
two hallmark features of probiotic function, modulation of cytokine and reuterin production, were examined in this study.
commensal-derived probiotic l. reuteri strains formed biofilms, and thesebiofilms retained the probiotic functions observed with planktonic cultures.
single species biofilms composed of anti-inflammatory l. reuteri strains atcc pta  <dig> and atcc pta  <dig> secreted factors that suppressed tnf production by lps-activated monocytoid cells.
in contrast, biofilms comprised of immunostimulatory probiotic strains atcc  <dig> and cf48-3a lacked the ability to stimulate human tnf production by human cells in the absence of lps activation.
atcc  <dig> and cf48-3a produced greater quantities of reuterin than atcc pta  <dig> and atcc pta  <dig> when the bacteria were cultured as planktonic cells or biofilms.
human breast milk-derived strains  differed with respect to relative propensities to form biofilms, and these strains demonstrated different biological properties in the context of biofilms.
lactic acid bacteria secrete factor that inhibit cytokine production by immune cells  <cit> , and this report established that probiotic biofilms cultured in a variety of conditions produced factor that suppress tnf production by lps activated human monocytes/macrophages.
previous studies in this lab have demonstrated that the tnf-inhibitory factors are detected in mid-log to stationary phase cultures  <cit> .
stationary phase cultures yield the most consistent tnf-inhibitory activities .
modulation of the mucosal immune system by intestinal commensal bacteria may have important implications for immune homeostasis and biofilm formation  <cit> .
intestinal bacteria such as l. reuteri may stimulate or suppress innate immune responses via several mechanisms including modulation of pro-inflammatory cytokines.
l. reuteri strains in this study can be divided into two subsets, immunosuppressive  and immunostimulatory strains , and each subset has potential therapeutic value.
tnf inhibitory strains of l. reuteri reduced inflammation in a h. hepaticus-induced murine model of inflammatory bowel disease  <cit> .
by contrast, stimulation of the mucosal innate immune system may be associated with enhanced protection against enteric infections.
interestingly, mucosal inflammation has been associated with enhanced biofilm densities in the intestine  <cit> .
the pro-inflammatory cytokine tnf promotes the proliferation of e. coli, and secretory iga increased agglutination of e. coli, an initial step in biofilm development  <cit> .
although, these experiments were performed with monospecies biofilms in vitro, the data raise questions regarding events that occur in complex microbial communities in vivo.
when not attached to a surface, immunostimulatory l. reuteri strains may stimulate host immune responses and promote commensal biofilm formation, particularly in neonates.
when l. reuteri biofilms are established, probiotic strains may have a diminished ability to stimulate tnf, effectively suppressing the formation of dense, complex multispecies biofilms in the mucus layer.
because such complex, dense biofilms have been associated with inflammation and disease  <cit> , the ability of probiotics to differentially regulate production of immunomodulatory factors in the context of planktonic and biofilm lifestyles may be an important probiotic feature.
alternatively, the tnf stimulatory factor may be produced by l. reuteri biofilms and not detected in the experimental conditions used in this study.
in contrast to immunostimulatory l. reuteri strains, anti-inflammatory probiotics may form denser biofilms in vivo that thwart pathogenic biofilm formation by preventing harmful host:pathogen interactions and overgrowth of commensal bacteria in the intestine.
as an example of pathogen inhibition, other lactobacilli suppressed the binding of staphylococcus aureus to epithelial cells  <cit> .
reuterin is a potent anti-pathogenic compound produced by l. reuteri and capable of inhibiting a wide spectrum of microorganisms including gram-positive bacteria, gram-negative bacteria, fungi, and protozoa  <cit> .
maximum reuterin production by l. reuteri occurs during late log and stationary phase cultures .
reuterin and other anti-pathogenic factors may be important for maintaining a healthy gut microbiota by preventing intestinal overgrowth by other commensal and pathogenic microorganisms.
recently, the addition of l. reuteri atcc  <dig> or reuterin to the intestinal microbiota was shown to reduce the e. coli population in an in vitro fermentation model  <cit> .
thus, antimicrobial compounds like reuterin may have a fundamental role in shaping and modeling the composition and spatial architecture of the gastrointestinal microbiota.
l. reuteri biofilms produced reuterin, indicating that probiotic l. reuteri may be protective against pathogens in either the planktonic or biofilm state.
interestingly, strains that produce relatively high quantities of reuterin are immunostimulatory when cultured as planktonic cells.
in vivo, immunostimulation by l. reuteri may promote colonization and biofilm formation of commensal lactobacilli, and reuterin could prevent opportunistic bacteria from establishing a niche.
hypothetically, once the immunostimulatory strains are established on the mucosal surface, tnf stimulation is diminished, and higher quantities of reuterin are produced.
elevated quantities of reuterin adjacent to the mucosa may effectively alter surrounding commensal microbial populations and prevent colonization and adherence by pathogenic bacteria.
biofilms are relatively resistant to several antimicrobial agents when compared to planktonic cultures  <cit> .
the enhanced resistance of biofilms to antimicrobial compounds may explain, in part, the resistance of l. reuteri biofilms to reuterin and elevated amounts of reuterin produced by these biofilms, as described in this study.
while the growth conditions used for the flow cell and planktonic cultures differed, similar probiotic activities by each l. reuteri strain were observed.
tnf inhibitory activities and reuterin production of l. reuteri were also consistent when biofilms  and planktonic cells were cultured using the same growth conditions.
although these experiments were conducted with biofilms grown in vitro on abiotic surfaces, biofilms with probiotic function may be important for delivery of beneficial effects in the mammalian host.
a mutant strain of l. crispatus, unable to bind mucus and adhere to the colonic mucosa, did not have a protective effect in a murine colitis model compared to the wild type aggregating strain even when the bacteria were continuously supplied to mice  <cit> .
mucus-binding ability may be important for probiotics to adhere to the mucosal surface and form biofilms within the intestine.
defects in cell surface features may affect biofilm formation and the abilities of probiotics to persist and colonize the intestine in vivo.
l. reuteri strain 100– <dig> deficient in alanylation of cell surface lipoteichoic acids proliferated similarly to wild type cells as planktonic bacteria, but failed to compete effectively and form complex biofilms in vivo  <cit> .
in the context of established intestinal microbial communities, probiotic biofilms may be more effective at long-term colonization and restoring missing functions in disease states.
CONCLUSIONS
in conclusion, probiotic strategies for the prevention and treatment of disease may require discovery and development of strains that form effective biofilms.
if biofilm formation facilitates long-term colonization and persistence in the intestine, biofilms that retain probiotic functions may be important for sustained efficacy in vivo.
the human gastrointestinal microbiota is a complex ecosystem that is shaped and maintained by multiple host and microbial factors.
changes in the spatial distribution, community architecture, or composition of the gastrointestinal microbiota may alter intestinal physiology and immunity, including susceptibility to infection.
probiotics in biofilm-like communities may be essential for long-term remodeling of the composition and function of the intestinal microbiome.

 probiotics are defined by the food and agricultural organization of the united nations as "live microorganisms which when administered in adequate amounts confer a health benefit on the host"." the effectiveness of probiotics is strain-specific, and each strain may contribute to host health through different mechanisms. probiotics can prevent or inhibit the proliferation of pathogens, suppress production of virulence factors by pathogens, or modulate the immune response. l. reuteri is a promising therapy for the amelioration of infantile colic, alleviation of eczema, reduction of episodes of workplace illness, and suppression of h. pylori infection. l. reuteri is considered an indigenous organism of the human gastrointestinal tract and is present on the mucosa of the gastric corpus, gastric antrum, duodenum, and ileum. biofilms or adherent structured microbial communities in the oral cavity and respiratory tract are well-characterized and are associated with respiratory infections, dental caries, and periodontitis. in contrast, biofilm-like communities of the gastrointestinal and female urogenital tracts containing beneficial lactobacilli may have a protective role. in bacterial vaginosis, indigenous lactobacilli are replaced with pathogenic biofilms consisting of gardnerella vaginalis and other bacteria. probiotic l. reuteri can displace g. vaginalis biofilms and could potentially re-establish protective biofilms in the female urogenital tract. due to artifactual removal of biofilms by traditional fixatives during specimen processing, studies of gastrointestinal biofilms are sparse. using non-aqueous fixatives and special techniques, several groups have documented the presence of intestinal biofilms in the mammalian intestine. the bacterial composition of biofilm-like communities in the mucus layer or the mucosa-associated microbiota, differs from the composition of the fecal microbiota, and the human mam includes lactobacilli. changes in the composition and architecture of the mam through diet or disease may affect overall health status. denser biofilms were found in patients with inflammatory bowel disease when compared to healthy controls, and 60% of the biofilm mass was comprised of the commensal bacteroides fragilis. these studies indicate a need to understand the contributions of individual strains and species to the aggregate function of gastrointestinal biofilms. this report describes the ability of an established commensal and probiotic organism, l. reuteri, to form biofilms in vitro and perform potentially beneficial functions as biofilms. two basic probiotic functions that depend on secreted factors were studied in the context of biofilms. first, modulation of innate immunity was investigated by studying regulation of human tnf production. in prior studies, supernatants from planktonic l. reuteri cultures reduced production of the pro-inflammatory cytokine, tnf, and tnf suppression was important in alleviating inflammation in a murine model of ibd. probiotic l. reuteri biofilms have not been examined for tnf inhibition. secondly, production of the antimicrobial compound β-hydroxy-propionaldehyde, known as reuterin, was evaluated in order to assess anti-pathogenic properties of l. reuteri biofilms.  probiotic lactobacillus reuteri forms biofilms various human isolates of l. reuteri were grown in 96-well polystyrene plates and retention of crystal violet was used to assess relative biofilm densities. all strains of l. reuteri adhered to polystyrene, but strains differed with respect to relative densities as measured by absorbance spectrophotometry. l. reuteri strains atcc pta and atcc pta formed aggregates with greater optical densities than l. reuteri strains atcc and cf48-3a. the differences between strains were also observed in cell counts. the bacterial densities in biofilms of atcc pta and atcc pta were roughly 10-fold greater than the bacterial densities of atcc and cf48-3a biofilms. l. reuteri biofilms were stained with acridine orange and observed by confocal microscopy. monospecies biofilms of atcc were μm thick. the thickness of l. reuteri biofilms was assessed at and hours. no differences in biofilm thickness were observed. consistent with this study, other researchers demonstrated formation of only thin biofilms when l. reuteri biofilms were cultured on plastic coupons for hours. l. reuteri biofilms modulate human tnf production to test the immunomodulatory properties of l. reuteri biofilms, supernatants from the biofilms were added to human monocytoid thp- cells in the presence and absence of lps. lps was added to the thp- cells to stimulate production of pro-inflammatory tnf by thp- cells. l. reuteri strains that produced tnf inhibitory factors as planktonic cultures demonstrated similar abilities to suppress tnf production when cultured as biofilms. when tnf inhibitory factors were obtained directly from l. reuteri biofilms grown in 24-well polystyrene plates, atcc pta and atcc pta also inhibited tnf production by 60% and 50%, respectively, when compared to the media control. supernatants of l. reuteri atcc pta biofilms cultured in a flow cell inhibited tnf by 73% compared to the media control. l. reuteri strains that did not suppress human tnf in planktonic phase lacked tnf-inhibitory capabilities when supernatants were obtained from the same strains cultured as biofilms. surprisingly, supernatants from atcc and cf48-3a biofilms did not induce tnf production by thp- cells in the absence of lps as the supernatants from planktonic cultures did. interestingly, the ability of probiotic l. reuteri to regulate human tnf production is strain-specific, and strain-specific tnf inhibition was maintained whether l. reuteri strains were cultured as planktonic cells or biofilms. the relative abilities to suppress human tnf in monocytoid cells were directly correlated with relative abilities to aggregate and form biofilms on polystyrene surfaces. l. reuteri cultured as planktonic cells and biofilms produced the antimicrobial factor, reuterin antimicrobial activities of l. reuteri were assessed by examining supernatants of planktonic and biofilm cultures for reuterin. planktonic cells and biofilms of l. reuteri produced reuterin, although differences in reuterin production were evident among strains. planktonic cultures of atcc pta atcc pta attc and cf48-3a produced,,, and mm of reuterin, respectively. when reuterin quantities were normalized to initial cfu/ml, planktonic cultures of atcc pta and atcc pta produced and mmol reuterin/ cells, respectively, and atcc and cf48-3a produced and mmol reuterin/ cells, respectively. for biofilms cultured in multiwell plates, the four wild type l. reuteri strains atcc pta atcc pta attc and cf48-3a produced,,, and mm of reuterin, respectively. after normalization of reuterin quantities to bacterial cell counts, atcc pta atcc pta cf48-3a, and atcc produced,,, and mmol of reuterin/ cells, respectively, when cultured as biofilms in multiwell plates. trends in reuterin production were consistent with planktonic and biofilm cultures of atcc pta and atcc producing lower quantities of reuterin than strains atcc and cf48-3a. interestingly, the relative abilities of l. reuteri strains to produce reuterin were inversely correlated with relative abilities to aggregate and adhere to polystyrene. previous studies indicated that planktonic cultures of human-derived l. reuteri strains used in this study were relatively resistant to the antimicrobial effects of reuterin, when compared to other bacterial species including closely related lactobacilli. however, since the cell viabilities of planktonic cultures decrease as reuterin accumulates, the quantities of reuterin produced by planktonic cultures were normalized to the initial cfu/ml. reuterin was detected after biofilms were incubated in glycerol for and hours. cell viabilities of biofilms after reuterin production exceeded 92%, indicating that the biofilms were relatively resistant to the quantities of reuterin produced by l. reuteri biofilms. discussion two hallmark features of probiotic function, modulation of cytokine and reuterin production, were examined in this study. commensal-derived probiotic l. reuteri strains formed biofilms, and thesebiofilms retained the probiotic functions observed with planktonic cultures. single species biofilms composed of anti-inflammatory l. reuteri strains atcc pta and atcc pta secreted factors that suppressed tnf production by lps-activated monocytoid cells. in contrast, biofilms comprised of immunostimulatory probiotic strains atcc and cf48-3a lacked the ability to stimulate human tnf production by human cells in the absence of lps activation. atcc and cf48-3a produced greater quantities of reuterin than atcc pta and atcc pta when the bacteria were cultured as planktonic cells or biofilms. human breast milk-derived strains differed with respect to relative propensities to form biofilms, and these strains demonstrated different biological properties in the context of biofilms. lactic acid bacteria secrete factor that inhibit cytokine production by immune cells, and this report established that probiotic biofilms cultured in a variety of conditions produced factor that suppress tnf production by lps activated human monocytes/macrophages. previous studies in this lab have demonstrated that the tnf-inhibitory factors are detected in mid-log to stationary phase cultures. stationary phase cultures yield the most consistent tnf-inhibitory activities. modulation of the mucosal immune system by intestinal commensal bacteria may have important implications for immune homeostasis and biofilm formation. intestinal bacteria such as l. reuteri may stimulate or suppress innate immune responses via several mechanisms including modulation of pro-inflammatory cytokines. l. reuteri strains in this study can be divided into two subsets, immunosuppressive and immunostimulatory strains, and each subset has potential therapeutic value. tnf inhibitory strains of l. reuteri reduced inflammation in a h. hepaticus-induced murine model of inflammatory bowel disease. by contrast, stimulation of the mucosal innate immune system may be associated with enhanced protection against enteric infections. interestingly, mucosal inflammation has been associated with enhanced biofilm densities in the intestine. the pro-inflammatory cytokine tnf promotes the proliferation of e. coli, and secretory iga increased agglutination of e. coli, an initial step in biofilm development. although, these experiments were performed with monospecies biofilms in vitro, the data raise questions regarding events that occur in complex microbial communities in vivo. when not attached to a surface, immunostimulatory l. reuteri strains may stimulate host immune responses and promote commensal biofilm formation, particularly in neonates. when l. reuteri biofilms are established, probiotic strains may have a diminished ability to stimulate tnf, effectively suppressing the formation of dense, complex multispecies biofilms in the mucus layer. because such complex, dense biofilms have been associated with inflammation and disease, the ability of probiotics to differentially regulate production of immunomodulatory factors in the context of planktonic and biofilm lifestyles may be an important probiotic feature. alternatively, the tnf stimulatory factor may be produced by l. reuteri biofilms and not detected in the experimental conditions used in this study. in contrast to immunostimulatory l. reuteri strains, anti-inflammatory probiotics may form denser biofilms in vivo that thwart pathogenic biofilm formation by preventing harmful host:pathogen interactions and overgrowth of commensal bacteria in the intestine. as an example of pathogen inhibition, other lactobacilli suppressed the binding of staphylococcus aureus to epithelial cells. reuterin is a potent anti-pathogenic compound produced by l. reuteri and capable of inhibiting a wide spectrum of microorganisms including gram-positive bacteria, gram-negative bacteria, fungi, and protozoa. maximum reuterin production by l. reuteri occurs during late log and stationary phase cultures. reuterin and other anti-pathogenic factors may be important for maintaining a healthy gut microbiota by preventing intestinal overgrowth by other commensal and pathogenic microorganisms. recently, the addition of l. reuteri atcc or reuterin to the intestinal microbiota was shown to reduce the e. coli population in an in vitro fermentation model. thus, antimicrobial compounds like reuterin may have a fundamental role in shaping and modeling the composition and spatial architecture of the gastrointestinal microbiota. l. reuteri biofilms produced reuterin, indicating that probiotic l. reuteri may be protective against pathogens in either the planktonic or biofilm state. interestingly, strains that produce relatively high quantities of reuterin are immunostimulatory when cultured as planktonic cells. in vivo, immunostimulation by l. reuteri may promote colonization and biofilm formation of commensal lactobacilli, and reuterin could prevent opportunistic bacteria from establishing a niche. hypothetically, once the immunostimulatory strains are established on the mucosal surface, tnf stimulation is diminished, and higher quantities of reuterin are produced. elevated quantities of reuterin adjacent to the mucosa may effectively alter surrounding commensal microbial populations and prevent colonization and adherence by pathogenic bacteria. biofilms are relatively resistant to several antimicrobial agents when compared to planktonic cultures. the enhanced resistance of biofilms to antimicrobial compounds may explain, in part, the resistance of l. reuteri biofilms to reuterin and elevated amounts of reuterin produced by these biofilms, as described in this study. while the growth conditions used for the flow cell and planktonic cultures differed, similar probiotic activities by each l. reuteri strain were observed. tnf inhibitory activities and reuterin production of l. reuteri were also consistent when biofilms and planktonic cells were cultured using the same growth conditions. although these experiments were conducted with biofilms grown in vitro on abiotic surfaces, biofilms with probiotic function may be important for delivery of beneficial effects in the mammalian host. a mutant strain of l. crispatus, unable to bind mucus and adhere to the colonic mucosa, did not have a protective effect in a murine colitis model compared to the wild type aggregating strain even when the bacteria were continuously supplied to mice. mucus-binding ability may be important for probiotics to adhere to the mucosal surface and form biofilms within the intestine. defects in cell surface features may affect biofilm formation and the abilities of probiotics to persist and colonize the intestine in vivo. l. reuteri strain 100– deficient in alanylation of cell surface lipoteichoic acids proliferated similarly to wild type cells as planktonic bacteria, but failed to compete effectively and form complex biofilms in vivo. in the context of established intestinal microbial communities, probiotic biofilms may be more effective at long-term colonization and restoring missing functions in disease states.  in conclusion, probiotic strategies for the prevention and treatment of disease may require discovery and development of strains that form effective biofilms. if biofilm formation facilitates long-term colonization and persistence in the intestine, biofilms that retain probiotic functions may be important for sustained efficacy in vivo. the human gastrointestinal microbiota is a complex ecosystem that is shaped and maintained by multiple host and microbial factors. changes in the spatial distribution, community architecture, or composition of the gastrointestinal microbiota may alter intestinal physiology and immunity, including susceptibility to infection. probiotics in biofilm-like communities may be essential for long-term remodeling of the composition and function of the intestinal microbiome. 
quantification of the antimicrobial glycerol derivative, reuterin, was assessed in order to document the antipathogenic potential of probiotic biofilms. l. reuteri biofilms differed in the quantities of reuterin secreted in this physiological state. l. reuteri biofilms secreted factors that confer specific health benefits such as immunomodulation and pathogen inhibition. immunomodulatory activities of biofilms were demonstrated by the abilities of specific l. reuteri strains to suppress human tnf production by lps-activated monocytoid cells. future probiotic selection strategies should consider a strain's ability to perform beneficial functions as a biofilm.
 commensal-derived probiotic bacteria inhibit enteric pathogens and regulate host immune responses in the gastrointestinal tract, but studies examining specific functions of beneficial microbes in the context of biofilms have been limited in scope.  lactobacillus reuteri formed biofilms that retained functions potentially advantageous to the host including modulation of cytokine output and the production of the antimicrobial agent, reuterin. immunomodulatory activities of biofilms were demonstrated by the abilities of specific l. reuteri strains to suppress human tnf production by lps-activated monocytoid cells. quantification of the antimicrobial glycerol derivative, reuterin, was assessed in order to document the antipathogenic potential of probiotic biofilms. l. reuteri biofilms differed in the quantities of reuterin secreted in this physiological state.  l. reuteri biofilms secreted factors that confer specific health benefits such as immunomodulation and pathogen inhibition. future probiotic selection strategies should consider a strain's ability to perform beneficial functions as a biofilm. 