there is a significant 
 medical need to better understand the interactions of small intestinal 
 epithelial cells with intestinal pathogens , which contribute to and 
 exacerbate a number of diseases including chronic diarrhea , gastroenteritis , and necrotizing enterocolitis . the virulent effects of intestinal pathogens 
 are dependent on their ability to colonize and invade the intestinal 
 mucosa , usually by adhering to and penetrating the epithelial layer . antibiotics have typically been the first line of treatment for intestinal 
 infections , yet with the increasing problem of antibiotic resistance 
 in clinical practice , there has been a need to explore alternative 
 antimicrobial therapies . a potential therapy or prophylactic against 
 microbial pathogenesis is the use of probiotic strains of bacteria , 
 including lactobacilli , bifidobacteria , and commensal escherichia 
 coli ( e.g. , nissle 1917 ) , which have been shown in a variety 
 of animal models to confer beneficial effects to the intestinal mucosa 
 by inhibiting pathogen colonization and invasion , and by modulating 
 the host immune response . biomimetic tissue models can provide a rapid and cheap alternative 
 platform to study the interactions of probiotics with intestinal pathogens . 
 at their simplest , these models are typically made up of 2-d confluent 
 monolayers of epithelial cell types , such as caco-2 , ht-29 , or hela , 
 which are incubated with microbes for short - term monitoring of epithelial however , these models do not fully emulate what happens in vivo , particularly in regard to the physical three - dimensional 
 space that the cells inhabit , despite it being well - known that bacterial 
 colonization is greatly dependent on their 3-d niche . in response , some researchers have developed elegant microfluidic 
 models that create a three - dimensional microenvironment and use flow mechanisms to allow simulation of biofilm formation and peristalsis . in addition , the nasa - developed rotating wall vessels ( rwv ) have 
 enabled prolonged 3-d culture of both mammalian cell types and bacterial 
 populations . this device has been optimized 
 to produce laminar flow to enable the growth of intestinal organoids 
 in suspension culture in conjunction with bacteria to simulate an 
 enteric infection in a fluidic setting . however , thus far the specific 
 three - dimensional surface topography of the intestine has been poorly 
 recreated , i.e. , via re - creation of the intestinal villi . epithelial 
 cells typically become more differentiated and polarized while moving 
 along the crypt  villus axis , and they subsequently express 
 different apical and basolateral receptors . it has been shown in vitro by previous researchers that many strains of bacteria 
 will preferentially adhere to epithelial cells in different stages 
 of differentiation . for instance , salmonella , enteropathogenic e. coli , and listeria all target receptors 
 such as microvilli on differentiated cells residing on the villi , whereas yersinia pseudotuberculosis and pseudomonas have been shown to preferentially adhere to 
 unpolarized , less differentiated cells which are 
 found in the crypt regions in real intestines . salmonella has been shown to interact with the microvilli of polarized enterocytes 
 and induce membrane ruffling through cytoskeleton reorganization , 
 allowing them to penetrate the epithelial layer . two of the drawbacks to traditional ( 2-d ) models is that they rarely 
 allow for both undifferentiated and differentiated epithelial cell 
 types to be cultured at the same time and they completely ignore the 
 physical dimensions typical of villus structures . we have previously 
 demonstrated that small sections of synthetic 3-d intestine can be 
 synthesized from collagen , silicon , and poly lactic - glycolic acid ( plga ) with realistic villus geometries , which can 
 be used to support the growth and differentiation of epithelial cell 
 types in a manner similar to real intestinal tissue . in this study , 
 we aimed to show that these intestinal models can also be used to 
 evaluate the therapeutic potential of two intestinal probiotics ( lactobacillus gasseri and e. coli nissle 
 1917 ) against two intestinal pathogens ( salmonella typhimurium and pseudomonas aeruginosa ) in a more realistic 
 physiological setting . three scenarios of bacterial adhesion were 
 tested : displacement , competition , and inhibition . displacement refers 
 to the ability of the probiotic to physically remove an intestinal 
 pathogen after it has established an adhesive niche on the epithelial 
 cells . competition refers to the ability of the probiotic to compete 
 with the pathogen for adhesive binding sites on the epithelial cells , 
 assuming that the starting inoculum is the same concentration . inhibition 
 refers to the ability of the probiotic to establish an adhesive niche 
 on the epithelial cells , and then retain this niche it once exposed 
 to pathogen , thereby blocking the pathogen from binding . briefy , micromolding 
 techniques were used to create agarose molds of 500 m deep , 
 high aspect ratio holes from a poly(methyl methacrylate ) ( pmma ) template . plga ( 100 mg / ml in chloroform , from lactel absorbable polymers , birmingham , 
 al ) was mixed with a porogen ( sodium bicarbonate , 400 mg / ml ) and homogenized 
 for 2 min . porogen solution under vacuum , followed 
 by freezing at 20 c overnight and then immersion in 
 precooled ethanol for 12 h to extract the chloroform . the scaffolds 
 were then immersed in warm distilled water for 24 h to dissolve the 
 porogen , and sterilized with 70% ethanol for 24 h prior to use . prior 
 to cell seeding , the plga scaffolds were placed into a custom designed 
 scaffold - insert kit from previously reported methods , and then soaked overnight in coculture media which was 
 added to both basolateral and apical compartments . caco-2 cells ( atcc , 
 manassas , va ) passage 1825 , were expanded and maintained in 
 tissue culture medium [ dmem with 10% fetal bovine serum ( fbs ) , 1 
 antimycotic  antibiotic , and 1% nonessential amino acids ] ( all 
 from invitrogen , long island , ny ) . cells were maintained in a humidified 
 37 c incubator with 5% co2 , with regular passage 
 12 times a week and medium change every 2 days . caco-2 were 
 removed from culture flasks with 0.25% ( v / v ) trypsin , 0.02% edta solution 
 in pbs and seeded onto the transwell insert scaffolds at a concentration 
 of 1  10 cells / ml and grown for 21 days , and 1  
 10 cells / ml and cultured for 4 days to produce differentiated 
 and undifferentiated monolayers , respectively . medium was added to 
 both the basolateral and apical compartments and replaced every 2 
 days thereafter , and antibiotics were removed from the tissue culture 
 medium the night before bacterial seeding . caco-2 cells were maintained as 
 in section 2.2.cells were seeded onto the plga 
 scaffolds at a concentration of 1  10 cells / ml . 
 medium was added to both the basolateral and apical compartments after 
 a 30 min cell attachment period , and replaced every 2 days thereafter . experiments were performed at 21 days post caco-2 seeding to enable 
 cell differentiation along the crypt  villus axis , and antibiotics 
 were removed from the tissue culture medium the night before bacterial 
 seeding . salmonella typhimurium 14038 ( st ) , pseudomonas 
 aeruginosa 15692 ( pao1 ) , and lactobacillus gasseri 33323 ( lab ) were from atcc , manassas , va . escherichia coli nissle 1917 ( nissle ) was obtained from a commercial preparation 
 of the probiotic mutaflor as described previously . overnight cultures of nissle , st , and pao1 were grown in 
 lb medium , and lab were grown in difco lactobacilli mrs medium ( all 
 from bd , franklin lakes , nj ) . the bacterial adhesion assay was performed 
 as previously described , but with some 
 modifications . a preculture of bacteria was grown for 16 h , before 
 diluting 1:50 in fresh medium and grown back to midexponential phase 
 for a further 1.5 h. bacteria were then adjusted to a final concentration 
 of 5  10 cells / ml in an even mixture of bacterial 
 medium and dmem ( no antibiotics ) , and 1 ml of bacterial suspension 
 was then added to the apical surface of the caco-2-covered transwells , 
 with incubation at 37 c for 2 h. nonadhered bacteria were removed 
 by washing twice in pbs , and cells were removed from the scaffolds 
 by incubating with 500 l of trypsin  edta at 37 c 
 for 10 min . the reaction was blocked with 500 l of dmem containing 
 fbs , and serial 10-fold dilutions were plated onto mrs agar for lab , 
 and macconkey agar ( emd millipore , billerica , ma ) for st , pao1 , and 
 nissle , with incubation for 24 h at 37 c . macconkey agar selects 
 for lactose - fermenting nissle ( pink colonies ) against non - lactose - fermenting 
 st and pao1 ( yellow - brown colonies ) . the bacterial adhesion assay was performed as in section 2.5 , but bacteria were seeded onto the apical surface 
 of plga scaffolds in the inset kits , instead of transwells . the following 
 scenarios of bacterial adhesion were tested : probiotic displacing 
 pathogen ( 2 h incubation with st or pao1 followed by 2 h incubation 
 with nissle or lab , at either 1:1 or 3:1 probiotic to pathogen ratios ) ; 
 probiotic inhibiting pathogen adhesion ( 2 h incubation with nissle 
 or lab followed by 2 h incubation with st or pao1 , at either 1:1 or 
 3:1 probiotic to pathogen ratios ) ; probiotic competing with pathogen 
 ( 2 h incubation with mixture of nissle or lab and st or pao1 , at either 
 1:1 or 3:1 probiotic to pathogen ratios ) . the invasive ability of st and pao1 was assessed using the gentamicin 
 protection assay . bacterial adhesion scenarios 
 to the caco-2 surface were set up as described in section 2.4 . nonadhered bacteria were removed by washing 
 twice in pbs , followed by incubation with 1 ml of gentamicin ( 150 
 g / ml in dmem ) for 1 h at 37 c to 
 kill the adhered extracellular bacteria . dead bacteria were removed 
 by washing twice in pbs , followed by an incubation with 500 l 
 of 0.1% triton x-100 for 15 min at 37 c to lyse the caco-2 and 
 release the intracellular ( invaded ) bacteria . serial fold dilutions 
 and plating were then employed as described in section 2.4 . to measure teer values of the caco-2 monolayers 
 before and after bacterial treatments , the medium was aspirated from 
 the insets , replaced with fresh dmem both basolaterally and apically , 
 and incubated at 37 c for 15 min . teer was measured with an 
 evom2 epithelial voltohmmeter with stx3 electrodes ( world precision 
 instruments , sarasota , fl ) . electrodes were placed on the apical and 
 basolateral sides of the inset kits , and the resistance was corrected 
 for surface area ( 0.5 mm ) and expressed as cm . the intrinsic resistance ( scaffold ) was subtracted from the total 
 resistance ( scaffold and caco-2 cells  bacteria ) to give the 
 monolayer resistance . mtt 
 assays were used to assess caco-2 cell viability in the presence of 
 bacteria using a vybrant mtt assay proliferation kit ( invitrogen ) 
 according to the manufacturer s instructions with some modifications . 
 briefly , bacterial adhesion scenarios to the caco-2 surface were set 
 up as described in section 2.4 and extracellular 
 bacteria were removed with gentamicin as described in section 2.5 . after washing twice in pbs , caco-2 were removed 
 from the scaffold surface by a 10 min incubation with 500 l 
 of trypsin  edta at 37 c . cells were centrifuged for 5 
 min at 150 g , resuspended in dmem , and adjusted to 
 a cell density of 1  10 cells / ml . a 100 l 
 suspension of each cell sample was added to a 96 well plate and incubated 
 for 36 h at 37 c with 5% co2 . the wells were replaced 
 with fresh medium containing 10 l of 12 mm mtt stock solution 
 and incubated for 4 h at 37 c , followed by incubation with 100 
 l of sds  hcl solution for a further 4 h. the absorbance 
 was read at 570 nm , and cell viability was assessed against control 
 samples of intestinal scaffolds that had not been exposed to bacteria , 
 and expressed as % cell survival . fluorescence in situ hybridization ( fish ) enabled visualization of the adherent bacteria 
 on the caco-2 monolayers , using the following probes : ttt cat ctg 
 gtg caa gca cc ( lab ) ; tct cgg cct tga aac ccc ( pao1 ) aat cac ttc acc 
 tac gtg ( st ) ; tt - fish - cac cgt agt gcc tcg tca ( nissle ) . intestinal 
 cell - coated plga scaffolds were fixed with 4% paraformaldehyde for 
 20 min at room temperature , then dehydrated by submerging in 50% ethanol 
 for 3 min , 80% ethanol for 3 min , and then 100% ethanol for 3 min . samples were incubated overnight in a humidified chamber at 45 c 
 in hybridization buffer ( 0.01% sds , 20 mm tris - hcl , 900 mm nacl , and 
 30% formamide in pbs ) containing 5 ng/l fish probe . samples 
 were then incubated at 45 c for 25 min with washing buffer ( 450 
 mm nacl , 20 mm tris , 5 mm edta , and 0.01% sds in dh2o ) , 
 followed by a further two washes to remove nonspecific binding of 
 the probe . samples were scanned using a leica sp2 confocal microscope ( leica 
 microsystems , buffalo grove , il ) with z - series capability . three - dimensional 
 rendering images and sections were assembled with volocity 5.0 software 
 ( perkinelmer , waltham , ma ) and imagej . teer , all cell counts , and mtt assays were performed 
 in triplicate , and data are presented as means  sd . statistical 
 differences were determined by using a student s unpaired t test , with p values of less than 0.05 
 being considered significant (  = 0.05 ) . for experiments in 
 which we had 12 scenarios of adhesion we used a bonferroni correction 
 and divided  by 12 and hence regarded p values 
 of less than 0.004 as statistically significant . we also used a bonferroni 
 correction on plate counts ; however , in this scenario , since we separated 
 the probiotics into 2 separate experiments that looked at both adhesion 
 and invasion , we divided  by 6 . fish and confocal microscopy was used to determine the location 
 of the four strains of bacteria to the caco-2 monolayers on intestinal 
 plga scaffolds ( figure 1 ) . the majority of the nissle ( figure 1a ) and pao1 ( figure1c ) cells 
 located to the base of the scaffold where the undifferentiated cells 
 resided , whereas most of the lab and st and primarily located to the 
 tips of the villi , on the differentiated cells . figure 2 shows colony counts from 2-d cultures of caco-2 ( no villi ) , 
 cultured on polyester transwell inserts for 4 days and 21 days , which 
 produce undifferentiated and differentiated monolayers , respectively . colony counts were normalized to caco-2 cell 
 number to account for differences in cell density in the two data 
 sets . for both lab and st , the highest number of cells adhered to 
 the 21 day differentiated caco-2 cultures , and for both nissle and 
 pao1 the highest counts were on the 4 day undifferentiated cultures . figure 3 shows the location of the bacteria 
 on the intestinal scaffolds when used in a 1:1 pathogen / probiotic 
 ratio . the images show that nissle and st have a different adhesive 
 niche ( figure 3a ) , compared to nissle and pao1 , 
 which share a similar adhesive niche in the crypt region ( figure 3b ) . in contrast , lab and st are located in similar 
 positions with the differentiated cells on the villus ( figure 3c ) , whereas the majority of lab and pao1 were in 
 different positions . confocal microscopy of plga scaffolds cultured for 21 
 days with caco-2 ( blue ) , and 2 h with bacteria ( red and green ) . a 
 = nissle , b = lab , c = st , and d = pao1 . 20 magnification shows 
 full coverage of scaffolds with caco-2 and bacteria ( a - d1 ) with a 
 zoomed in 40 magnification enabling visualization of individual 
 bacteria ( a - d2 ) , and 3-d rendering shows bacteria adhering selectively 
 to different locations along the crypt  villus axis on an individual 
 villus measuring 500 m ( a - d3 ) . confocal microscopy of plga scaffolds cultured for 21 days with caco-2 
 ( blue ) , and 2 h with a 1:1 concentration of probiotic ( red ) and pathogen 
 ( green ) . a = nissle and st , b = nissle and pao1 , c = lab and st , and 
 d = lab and pao1 . the adhesive and invasive activity of st and the potential therapeutic 
 effects of the probiotics were assessed through colony counts for 
 adhesion and invasion , measurements of tight junction integrity through 
 teer , and caco-2 cell survival using an mtt assay . the probiotics 
 were assessed for their ability to displace st from the scaffolds , 
 inhibit the adhesion of st , or compete with the pathogen for colonization 
 space , as well as how these scenarios affected the invasion of st 
 into the caco-2 cells . figure 4a shows that 
 lab reduced the adhesion of st to the caco-2 through displacement , 
 inhibition , and competition . reduction in adhesion through displacement 
 and competition appeared to be a dose - dependent event , with a greater 
 reduction being obtained by using a 3:1 ratio of lab to st . using 
 the bonferroni post test it was found that , with a 3:1 ratio of lab 
 to st , a significant reduction of st adhesion occurred via displacement . a 3:1 ratio of lab used in the displacement assay also gave the greatest 
 reduction in invasion into the caco-2 monolayers . similarly , the reduction 
 of st using nissle as the probiotic appeared to be a dose - related 
 event , and none of the 1:1 adhesion scenarios were significant . however , 
 in contrast to lab , which was most effective at displacing st , nissle 
 was most effective against st when used as a high - dose pretreatment 
 to inhibit adhesion . colony counts from a 2 h incubation of caco-2 cultures 
 with st , with results showing adhesion to the caco-2 surface , or invasion 
 into the caco-2 cells , expressed as log cfu / ml . the colony 
 counts of st in isolation were compared to colony counts of st when 
 treated with probiotic lao1 ( a ) or nissle ( b ) ; dp = displacement , 
 i = inhibition of adhesion , and c = competition . significance was 
 assessed with an unpaired t test , followed by a bonferroni 
 correction post test to determine significance across the multiple 
 scenarios ( p < 0.008 ) . figure 5a shows that , in isolation , 
 st reduces the teer of caco-2 monolayers compared to a bacteria - free 
 control . both lab and nissle increased teer values for caco-2 cells 
 growing on the scaffolds in a dose - dependent manner , despite the presence 
 of st . this increase was significant for all three scenarios of adhesion 
 with a 3:1 ratio of probiotic to pathogen . in addition , the percentage 
 cell survival significantly increased with the addition of lab and 
 nissle ( figure 5b ) . applying a 3 probiotic 
 treatment to the caco-2 before the addition of st promoted the highest 
 level of cell survival , with no difference to control samples without 
 st ( i.e , 100% cell survival ) . the displacement and competition scenarios 
 at a 3:1 ratio enabled over 80% of the cells to survive , with no significant 
 difference between the two . nissle appeared to promote higher levels 
 of cell survival compared to lab at lower concentrations ( 1:1 ) however , 
 with a significant difference in all three scenarios tested . teer values 
 ( a ) and % cell survival from mtt assay ( b ) of caco-2 monolayer plga 
 scaffolds after incubation for 2 h with the pathogen st , and then 
 a series of treatments with a 3:1 or 1:1 ratio of probiotic lab or 
 nissle . dp = displacement , i = inhibition of adhesion , and c = competition . significance was assessed by comparing teer and % cell survival to 
 samples with pathogen only ( no probiotic ) using an unpaired t test , followed by a bonferroni correction post test to 
 determine significance across the multiple scenarios ( p < 0.004 ) . the bonferroni significances were plotted on the graphs 
 ( a  e = lowest to highest significance ) . the adhesive and invasive potential 
 of pao1 and the subsequent therapeutic effects of the probiotics were 
 assessed using colony counts , teer , and an mtt assay . it was found 
 that lab significantly lowered the number of adhered st to the caco-2 
 in all scenarios tested except the 1:1 inhibition assay ( figure 6a ) . invasion was not significantly reduced in any 
 case , even with higher dose of lab . it was found that nissle significantly 
 lowered the number of adhered st to the apical surface of the caco-2 
 in most scenarios tested , except the 1:1 competition assay . in contrast 
 to lab , nissle managed to significantly reduce invasion into the caco-2 
 cells in every scenario with a high dose of probiotic ( figure 6b ) . colony counts from a 2 h incubation of caco-2 cultures 
 pao1 , with results showing adhesion to the caco-2 surface , or invasion 
 into the caco-2 cells , expressed as log cfu / ml . the colony 
 counts of pao1 in isolation were compared to colony counts of st when 
 treated with probiotic lao1 ( a ) or nissle ( b ) ; dp = displacement , 
 i = inhibition of adhesion , and c = competition . significance was 
 assessed with an unpaired t test , followed by a bonferroni 
 correction post test to determine significance across the multiple 
 scenarios ( p < 0.008 ) . as with the pathogen st , figure 7a 
 shows that , in isolation , pao1 reduced the teer of caco-2 monolayers 
 compared to a bacteria - free control , and that both probiotics raised 
 teer values when used in conjunction with pao1 . again , the rise in 
 teer was dose - dependent : higher concentrations of probiotic had a 
 more significant effect across all scenarios tested . displacement 
 and competition treatments raised the teer more than inhibition , however , 
 there was little difference between the two strains of probiotic . however , there was a difference in how the two probiotics affected 
 cell survival . although the percentage cell survival was significantly 
 improved in all scenarios tested , treatments with nissle fared better 
 than lab in all cases except a 1:1 competition ( figure 7b ) . teer values ( a ) and % cell survival from mtt assay ( b ) of caco-2 
 monolayer plga scaffolds after incubation for 2 h with the pathogen 
 pao1 , and then a series of treatments with 3:1 or 1:1 ratio of probiotic 
 lab or nissle . dp = displacement , i = inhibition of adhesion , and 
 c = competition . significance was assessed by comparing teer and % 
 cell survival to samples with pathogen only ( no probiotic ) using an 
 unpaired t test , followed by a bonferroni correction 
 post test to determine significance across the multiple scenarios 
 ( p < 0.004 ) . the bonferroni significances were 
 plotted on the graphs ( a  e = lowest to highest significance ) . biomimetic tissue cell models can be used to simplify studies on 
 bacterial  host cell interactions to determine the effects of 
 one or two specific factors . in this study , we looked at the effects 
 of intestinal epithelial cell differentiation on the adhesion and 
 invasion of two pathogens , and the subsequent potentially therapeutic 
 or prophylactic effects of two well - known probiotics . it has been 
 shown extensively in the literature that the differentiation of caco-2 
 is a growth related process that closely mimics the differentiation 
 profiles of small intestinal epithelium in vivo , with undifferentiated cells during exponential growth that turn 
 into polarized and differentiated cells at postconfluence . in the 
 small intestine , villus 
 axis , with dividing nondifferentiated cells residing near the crypt 
 regions that move up the villi to become more differentiated cells . we have shown in our previous studies that we can mimic this phenomenon 
 using a 3-d villus scaffold to support the directional growth and 
 differentiation of caco-2 . nondifferentiated intestinal epithelial cells express basolateral 
 markers over the entire surface ( including cadherins , integrins , etc . ) whereas differentiated epithelial cells also display apical markers , 
 including brush border enzymes and microvilli . it has been shown in 
 previous in vivo studies that certain strains of 
 bacteria will preferentially adhere to epithelial cells in different 
 stages of differentiation . the pathogen salmonella has been shown to interact with the apical surface of differentiated 
 epithelial cells in the intestine , which is accompanied by a degeneration 
 of microvilli upon invasion . in contrast , some strains of pseudomonas including 
 pao1 may preferentially adhere to and invade nondifferentiated cells . also , through wound - healing experiments , it 
 was shown that higher levels of pseudomonas interact 
 with cells that had reverted back to their nondifferentiated state . it has been suggested that pseudomonas may preferentially 
 interact with basolateral receptors , although it is worth noting that 
 this phenomenon has not been as widely characterized as salmonella . we have shown that , using both traditional 2-d transwell inserts 
 and our biomimetic model , the ecological niche of both st and pao1 
 resembles that of previously reported methods , with the majority of 
 adhesion at differentiated cells at the tips of the villi and undifferentiated 
 cells near the crypt region , respectively . in addition , the higher 
 levels of lab adhesion to the differentiated cells at the tips of 
 the villi were in agreement with the literature . some strains of e. coli have been found 
 to interact mainly with differentiated cell types ; however , in our 3-d model we found that the majority of 
 nissle was located near the undifferentiated cells at the base of 
 the villi , and this could potentially be due to the variation in adhesins 
 expressed by different strains of e. coli ( nissle 
 for example has no s - fimbriae and inactive p - fimbriae ) . as a further demonstration of the different bacterial 
 niches in the small intestine , we also showed that two strains of 
 bacteria could be cultured in a 3-d model in different locations along 
 the crypt  villus axis . our hypothesis was that the therapeutic 
 potential of probiotics against intestinal pathogens may be altered 
 based on the differences in ecological niche . in our 3-d intestinal 
 model , we found that both probiotics tested successfully reduced the 
 adhesion and invasion of st into caco-2 monolayers in a dose - dependent 
 manner , in good agreement with the literature , which has shown extensively 
 that lab and nissle can be used to displace , compete with , and inhibit st adhesion and 
 invasion . further , we found that probiotics were most effective at 
 different stages of st infection . although both treatments worked , 
 lab appeared to be more successful at displacing the pathogen once 
 it had already adhered to the caco-2 cells as opposed to inhibiting 
 st adhesion through a preincubation . in contrast , with nissle the 
 opposite was true , which was surprising as we predicted that the location 
 of nissle in the crypt regions may reduce the likelihood of st inhibition 
 as they were shown to have different adhesive niche , but this suggests 
 that factors other than steric hindrance play a role in its therapeutic 
 effects . the colony counts were supported by results from an mtt assay , 
 which showed that cell survival after incubation with st was highest 
 after a 2 h preincubation with nissle . as well as having therapeutic 
 affects against st , strains of lactobacillus have 
 also been shown by previous researchers to display antimicrobial activity 
 against enteroinvasive pseudomonas strains in vitro , and we have shown in this study 
 that , in a 3-d setting , even though lab and pao1 occupy a different 
 adhesive niche along the crypt  villus axis , the probiotic is 
 still able to inhibit the adhesion of pao1 to the apical surface of 
 the cells . this shows that , in the same way as nissle with st , steric 
 hindrance is not essential to the antimicrobial activity of the probiotic . there is very little in the literature regarding the use of nissle 
 to treat a pseudomonas intestinal infection , although 
 there is some evidence that it can be used to treat urinary tract 
 infections from pseudomonas through bactericidal 
 activity . our results suggest that nissle 
 can be used to effectively lower the adhesion and invasion of pao1 
 into epithelial cells in a dose - dependent manner , particularly through 
 inhibition and displacement . interestingly , nissle was the only probiotic 
 that significantly prevented the invasion of pao1 into the caco-2 
 cells , as well as promoting greater caco-2 cell survival for an infection 
 with pao1 over lab across all scenarios of adhesion tested , apart 
 from a 1:1 competition scenario . we have demonstrated the feasibility of developing 
 an in vitro artificial intestine from a biocompatible 
 polymer , which can be molded into villous shapes to mimic the topography 
 of the small intestine , providing a platform for the differentiation 
 of epithelial cell types , and the subsequent adhesion / invasion of 
 pathogenic bacteria . we showed that strains of bacteria can live on 
 epithelial cells that are in different stages of differentiation , 
 and that this alters where they reside on the crypt we showed that , in this 3-d environment , probiotics exert their 
 effects through different mechanisms . for example , lab was more effective 
 at displaying pathogenic bacteria once it had colonized , and nissle 
 was more effective at preventing attachment . with further experimentation , 
 we believe that this system could provide a platform for more specific 
 targeting of probiotics to certain intestinal pathogens , for example 
 to determine which probiotics are best to be taken routinely as an 
 inhibitory measure , and which can be used for elimination of an infection 
 once established .
biomimetic in vitro intestinal models are becoming useful tools for 
 studying host  microbial interactions . in the past , these models 
 have typically been limited to simple cultures on 2-d scaffolds or 
 transwell inserts , but it is widely understood that epithelial cells 
 cultured in 3-d environments exhibit different phenotypes that are 
 more reflective of native tissue , and that different microbial species 
 will preferentially adhere to select locations along the intestinal 
 villi . 
 we used a synthetic 3-d tissue scaffold with villous features 
 that could support the coculture of epithelial cell types with select 
 bacterial populations . 
 our end goal was to establish microbial niches 
 along the crypt  villus axis in order to mimic the natural microenvironment 
 of the small intestine , which could potentially provide new insights 
 into microbe - induced intestinal disorders , as well as enabling targeted 
 probiotic therapies . 
 we recreated the surface topography of the small 
 intestine by fabricating a biodegradable and biocompatible villous 
 scaffold using poly lactic - glycolic acid to enable the culture of 
 caco-2 with differentiation along the crypt  villus axis in 
 a similar manner to native intestines . 
 this was then used as a platform 
 to mimic the adhesion and invasion profiles of both salmonella and pseudomonas , and assess the therapeutic potential 
 of lactobacillus and commensal escherichia 
 coli in a 3-d setting . 
 we found that , in a 3-d environment , lactobacillus is more successful at displacing pathogens , 
 whereas nissle is more effective at inhibiting pathogen adhesion .