BACKGROUND
probiotics, defined by a working group of the international life sciences institute europe  as "a viable microbial food supplement, which beneficially influences the health of the host"  <cit> , have become the focus of considerable research interest in recent years  <cit> .
live probiotic organisms  have been shown to reduce the symptoms of inflammatory conditions such as inflammatory bowel disease  and irritable bowel syndrome   <cit> .
this is believed to occur via the localized stimulation of anti-inflammatory cytokines  as a result of beneficial alterations to the microbiota  <cit> .
proof of principle and efficacy has been demonstrated with the inert food organism lactococcus lactis which has been engineered to secrete il- <dig> locally within the gut in murine models of ibd in order to alleviate symptoms of gastrointestinal inflammation  <cit> .
however, despite their obvious clinical potential, these strains are often poorly adapted to conditions encountered in the upper gastrointestinal tract and delivery of viable organisms from the mouth to the large intestine remains a major hurdle to their use in human therapies  <cit> .
listeria monocytogenes is a gram-positive intracellular foodborne pathogen capable of withstanding a variety of hostile environmental conditions, including the numerous stresses encountered during the production, preparation, and storage of food  <cit> .
following consumption l. monocytogenes effectively survives the extreme conditions encountered during gastric passage, including the low ph of the stomach, low oxygen content and elevated osmolarity and bile salts associated with the upper small intestine  <cit> .
bile in particular represents a key challenge to bacteria that survive and transit the stomach and enter the small intestine.
listeria has been isolated from the human gallbladder  <cit> , indicating an inherent ability to tolerate high concentrations of bile.
previously sleator et al.,  <cit> , identified a novel bile resistance mechanism designated bile, which when disrupted resulted in a bile sensitive phenotype.
bile, which functions by excluding bile from the cell, has also been shown to facilitate improved gastrointestinal transit in mouse models of infection and as such contributes to the gastrointestinal phase of l. monocytogenes infection.
interestingly, not many homologues of the bile operon have been identified in any of the genomes of the probiotic or commensal organisms sequenced to date.
the only homology found was busa  of l. lactis, which encodes a glycine betaine uptake system.
this system is osmotically inducible as was believed to be the case with bile.
however, sleator et al,  have shown that bile does not play a role in osmotolerance, but in fact a major role in bile tolerance.
herein, we demonstrate that heterologous expression of the listerial bile resistance mechanism bile improves bile resistance in vitro as well as enhancing gastrointestinal persistence and clinical efficacy of the probiotic strain b. breve ucc <dig>  furthermore, expression in l. lactis enhanced bile tolerance and in vivo survival and may have applications in targeted vaccine or drug delivery by this organism.
methods
bacterial strains, plasmids, and culture conditions
the strains and plasmids used in this study are listed in table  <dig>  escherichia coli strain dh10b  used as a cloning host, was grown aerobically at 37°c in luria-bertani  medium  <cit> .bifidobacterium breve ucc <dig>  a human commensal originally isolated from an infant nursling stool, was anaerobically cultured in reinforced clostridial medium  or de man, rogosa and sharpe medium  supplemented with  <dig> %  l-cysteine hcl.
anaerobic conditions were maintained using an anaerocult oxygen-depleting system  in an anaerobic chamber.
lactococcus lactis nz <dig> strain was grown in m <dig> medium supplemented with  <dig> %  glucose  at 30°c.
l. monocytogenes egd-e  <cit>  was cultured aerobically at 37°c in brain heart infusion  medium.
oxoid ltd.,  supplied all growth media.
for solid media,  <dig> % agar was added.
when appropriate, antibiotics were added at the following final concentrations: for e. coli, b. breve, and l. lactis, chloramphenicol  <dig> μg ml- <dig>   <dig> μg ml- <dig> and  <dig> μg ml- <dig> respectively.
porcine bile  was obtained from sigma .
the required quantity of bile was solubilised in water and autoclaved.
genetic manipulations
plasmid dna was isolated from e. coli using a qiaprep spin miniprep kit according to the manufacturer's instructions .
genomic dna was isolated from l. monocytogenes egd-e using a genelute bacterial genomic dna kit  as recommended by the manufacturer.
transformation of l. lactis was achieved according to the protocol of de vos et al.
<cit> .
standard procedures were used for dna manipulation in e. coli  <cit> .
restriction endonucleases , t <dig> dna ligase , and 2× pcr mixture  were used as recommended by the manufacturers.
oligonucleotide primers were synthesized by sigma genosys .
pcr products required for cloning were generated with kod hot-start high-fidelity dna polymerase  using  <dig> ng l. monocytogenes genomic dna.
plasmid construction
analysis of the bile operon in l. monocytogenes egd-e  uncovered the presence of two open reading frames  bilea  and bileb , oriented in the same direction and overlapping by five nucleotides.
pcr primers with incorporated xbai  and psti  restriction enzyme sites  were used to amplify the complete bile operon from the chromosome of l. monocytogenes egd-e. the resultant  <dig>  kb pcr product was digested with xbai and psti and subsequently ligated into similarly digested pnz <dig> using t <dig> dna ligase .
the resultant plasmid, containing bile under the transcriptional control of its own promoter, was designated pnz8048-bile+ as in table  <dig>  both pnz8048-bile+ and pnz8048-bile-  initially introduced into e. coli dh10b as a cloning host.
plasmid dna was extracted from successful transformants, was sequenced and subsequently transformed into the bifidobacterial strain ucc <dig> and lactococcal strain nz <dig> yielding ucc2003-bile+and nz9000-bile+ respectively.
strains harbouring pnz <dig>  which was used as a negative control, were named ucc2003-bile- and nz9000-bile- respectively.
chloramphenicol was added to plates as a selective marker.
electroporation
electroporation of plasmid dna into e. coli and l. lactis was performed essentially as previously described.
electro-transformation of b. breve with plasmid dna was performed as described by macconaill et al.
<cit> .
essentially, mid-logarithmic-phase cells  were chilled on ice for  <dig> min, followed by centrifugation.
the cell pellet was washed twice and resuspended in  <dig>  m sucrose- <dig> mm citrate buffer .
cells were incubated on ice for  <dig> min, and this was followed by electro-transformation with a bio-rad gene pulser ii apparatus under the following conditions:  <dig> μf,  <dig> Ω and  <dig>  kv cm- <dig>  modified rogosa medium was added to the cells, and the mixture was incubated anaerobically at 37°c for  <dig>  hr prior to plating onto rca  containing the appropriate antibiotic.
plasmid stability study
b. breve ucc <dig> colonies containing pnz8048-bile+ and pnz8048-bile- and l. lactis nz <dig> colonies containing pnz8048-bile+and pnz8048-bile- were first cultured in mrs broth containing  <dig> μg ml- <dig> chloramphenicol and gm <dig> broth with  <dig> μg ml- <dig> respectively.
cells were then sub-cultured in fresh mrs and gm <dig> broth without antibiotic selection every  <dig> hrs for a total of  <dig> generations.
vector segregation stability was monitored by plating isolated colonies every  <dig> hrs.
<dig> colonies were replica plated onto mrscm <dig> and gm17cm <dig>  b. breve was incubated anaerobically at 37°c and l. lactis incubated aerobically at 30°c for  <dig> hrs.
the percentage of loss of the test plasmid in the population was then calculated.
transcriptional analysis
total rna was isolated from b. breve ucc <dig> and l. lactis nz <dig> cells grown to an optical density at  <dig> nm  of  <dig>  using the macaloid acid method as described by ventura et al.
and then treated with dnase .
reverse transcription for cdna was mediated by thermostable superscript reverse transcriptase  using 10– <dig> μg rna as a template in a  <dig> μl reaction mixture containing  <dig> μl 5× superscript iii buffer,  <dig> ng of random primers p <dig>   <dig> μl  <dig> mm dithiothreitol,  <dig>  μl of deoxynucleoside triphosphate mix and the superscript enzyme , which was used according the manufacturer's instructions to produce cdna.
the cdna generated was used as a template for reverse transcription -pcrs performed with primers rt-pcrf and rt-pcrr.
in all cases, control pcr reactions were used to ensure the complete removal of dna from rna preparations prior to reverse transcription.
resistance to bile
to determine the ability of strains to survive at sub-lethal bile concentrations, the b. breve and l. lactis cultures were grown to stationary phase and were subsequently inoculated  into mrs and gm <dig> broth containing 1%  porcine bile.
viable cell counts were performed at intervals by serial dilution in one-quarter-strength ringer's solution and plating onto rcmcm <dig> or gm17cm <dig> respectively.
animal studies
female balb/c  mice  were used for in vivo studies.
mice were housed in pathogen free conditions in a dedicated facility and were fed standard laboratory feed and water ad libtium.
all animal procedures were performed according to the university's ethical guidelines.
inoculations were carried out essentially as described by sleator et al.,  <cit> .
probiotic gastrointestinal persistence studies
prior to commencement of the trials , the absence of background microflora in murine faecal pellets was confirmed by plating onto the two selective agars containing antibiotic.
for the study five mice were used per bacterial strain.
each mouse was administered  <dig> μl of bifidobacteria or lactococcus  by oral pipette for  <dig> consecutive days, leading to colonization of the gastrointestinal tract.
gastrointestinal persistence of both strains was tracked by measuring the excretion of viable b. breve in fresh faecal samples collected for  <dig> days post feeding and l. lactis for  <dig> days.
fresh samples were weighed, homogenized in pbs , diluted and plated onto rca or gm <dig> with the appropriate level of chloramphenicol.
at days nineteen  and four  post dosing the mice were sacrificed by cervical dislocation, intestines and caeca were excised.
the organs were homoginized in  <dig> mls pbs, serially diluted and the bifidobacterial and lactococcal numbers in the intestines and caeca of dosed animals were determined by spread-plating homogenates onto rcacm <dig> and gm17cm <dig> plates.
listeria challenge
animal feeding trials were performed to determine whether the probiotic strains harbouring pnz8048-bile+ conferred protection against subsequent infection with l. monocytogenes.
balb/c mice were fed bifidobacteria  by oral pipette for  <dig> consecutive days before oral infection with l. monocytogenes egd-e::ppl2lux  on day  <dig>  three days post infection, the mice were sacrificed by cervical dislocation, livers and spleens were excised, homogenized in  <dig> mls pbs, and serial dilutions were plated onto bhicm agar, which was followed by overnight incubation at 37°c.
the resulting colonies were used to calculate the number of bacteria per organ.
statistical analysis
numerical results were expressed as arithmetic means +/- standard deviations of the means.
cfu determinations were converted to log <dig> values, and then the arithmetic means and standard deviations were calculated.
error bars in the figures represent standard deviations.
student's t test was performed to determine the statistical significance.
RESULTS
improving tolerance to porcine bile
the bile system was amplified from l. monocytogenes and cloned into the plasmid vector pnz <dig> under the control of its native promoter.
the plasmid was electroporated into both b. breve ucc <dig> and l. lactis nz <dig> .
to establish the segregational stability of the pnz <dig> constructs a plasmid stability assay was performed over approximately  <dig> generations.
both constructs  were capable of replication on b. breve ucc <dig> and were found to exhibit considerable stability  in the absence of selective pressure .
the constructs were also capable of replication in l. lactis nz <dig> and were found to exhibit considerable stability  in the absence of selective pressure.
proof that the operon was heterologously expressed in both bacterial strains was evident as a single mrna transcript was obtained by reverse transcription polymerase chain reaction  analysis  also in vitro studies showing increased bile resistance supports this by indicating that the bile resistance locus was functional.
due to difficulty in obtaining human bile; porcine bile was used to examine the bile tolerance of b. breve bile+ and l. lactis bile+.
porcine bile is considered an acceptable substitute because the salt/cholesterol, phospholipids/cholesterol and glycine/taurine ratios resemble the composition of human bile  <cit> .
we utilised levels of porcine bile  that were lethal for the bacterial species examined and are likely to approximate in vivo levels in regions of the small intestine  where bile is most concentrated  <cit> .
it was observed that the presence of bile rendered the engineered l. lactis strain considerably more resistant to the initial kill by porcine bile relative to the wild-type .
the engineered strain demonstrated a  <dig> -log enhanced resistance to bile over the  <dig> minute course of the kill curve .
in contrast whilst b. breve bile+ demonstrated a similar initial kill to the wild-type  the engineered strain was subsequently better equipped to survive in porcine bile.
at  <dig> minutes and  <dig> minutes post-exposure the b. breve bile+ strain demonstrated  <dig>  log enhanced survival .
similar results were seen in experiments carried out using bovine bile  .
gastrointestinal persistence of l. lactis bile+ and b. breve bile+
in order to test whether the elevated bile tolerance of the engineered strains would influence survival in the gi tract, we utilized a mouse model to follow survival in vivo, using an approach previously outlined by sheehan et al.
<cit> .
to facilitate gastrointestinal colonization by b. breve and l. lactis strains, each balb/c mouse was orally administered  <dig> ×  <dig> cfu for three successive days.
gut persistence was monitored by faecal carriage with samples taken every  <dig> h for  <dig> days in the case of l. lactis and every  <dig> h for  <dig> days for b. breve.
l. lactis bile+ demonstrated significantly enhanced in vivo survival relative to the control over the course of the study .
both bile+ and bile- strains were detectable in faeces at approximately the same levels at  <dig> hours post inoculation.
however, the wild-type was undetectable at day  <dig> post-inoculation.
this reflects the naturally poor rate of gastrointestinal survival for l. lactis also observed in other studies  <cit> .
in contrast l. lactis bile+was detectable at high levels in murine faeces at days  <dig> and  <dig> post-inoculation.
neither bile+ nor bile- strains engineered l. lactis were detectable at day  <dig> post-inoculation.
these data demonstrate significant  enhancement of gastrointestinal survival of l. lactis through rational biological engineering.
b. breve ucc <dig> transiently colonized the murine gi tract at levels of approximately  <dig> cfu/g faeces irrespective of the presence of bile.
however, at day  <dig> post inoculation a significant  difference began to emerge between the engineered b. breve ucc <dig> bile+ strain and the bile-, with the bile+ strain demonstrating elevated persistence in faeces compared to the wildtype, which declined significantly .
at the final day of the experiment  the bile+ strain persisted at levels of approximately  <dig>  ×  <dig> cfu/g faeces whereas the wild-type had declined to  <dig> ×  <dig> cfu/g faeces.
this is a more substantial difference than that observed by sheehan et al.
<cit>  using a similar approach which involved cloning the betl osmotolerance locus into b. breve.
at day  <dig> mice were examined directly for the presence of b. breve ucc <dig> in the small intestine, caecum and large intestine .
we confirmed a 2-log difference between the bile+ and wt strains in the small intestine with the engineered strain present at significantly higher levels.
this finding is important given that this region is associated with the highest levels of bile in the gi tract  <cit> .
interestingly sheehan et al.
<cit>  did not demonstrate enhanced survival of the betl+ strain in the small intestine.
we also determined significantly enhanced levels of the engineered strain in the caecum and large intestine .
overall, the approximately  <dig> log increased persistence of the engineered strain in the murine intestine mirrored the results of the faecal persistence studies.
effect of engineered bile+ b. breve on oral listeria challenge
administration of b. breve ucc <dig> has previously been demonstrated to reduce splenic levels of l. monocytogenes following oral infection of mice  <cit> .
we hypothesized that the elevated colonisation of b. breve bile+ in the small intestines of mice  may further enhance this protective effect since l. monocytogenes invades at this site.
to test this hypothesis we administered mice with either wild-type or bile+ b. breve ucc <dig> on three consecutive days and challenged with oral l. monocytogenes infection at day  <dig> post-inoculation.
at the  <dig> day time point we again detected higher levels of the engineered bile+ strain relative to the wild-type in mouse faeces .
following oral infection with l. monocytogenes we determined that oral dosing with the engineered bile+ b. breve strain significantly enhanced clearance of l. monocytogenes as assessed by pathogen load in the liver .
a similar  trend was seen when the numbers of listeria in the spleens of the infected mice was determined .
although differences in infectious burden were small the data indicate improved probiotic efficacy of engineered b. breve.
discussion and
CONCLUSIONS
foodborne pathogens are capable of rapid adaptation to diverse environmental niches associated with survival in the external environment and transit through the gastrointestinal tract of the host  <cit> .
in contrast, the autochthonous gastrointestinal microbiota demonstrates a high degree of niche-specialisation  <cit> .
this specialisation is an impediment to the delivery of so-called probiotic strains to the human gi tract as these strains must survive gastric acid in the stomach and elevated osmolarity and bile acids in the small intestine before establishing a presence in the large bowel  <cit> .
the term 'patho-biotechnology' was recently coined to highlight the beneficial biotechnological uses of molecular systems derived from pathogens  <cit> .
this concept was recently applied to improve the osmotolerance of potential probiotic strains through rational molecular engineering  <cit> .
here we implement the patho-biotechnology concept to improve the in vivo and in vitro bile resistance of a potential probiotic strain, b. breve ucc <dig>  as well as the food isolate l. lactis, through cloning and expression of a bile tolerance system in these organisms.
bile acids are amphipathic molecules synthesized from cholesterol that are produced in the liver, stored interdigestively in the gall bladder and released into the duodenum where their main function is the breakdown of dietary fats.
bile also plays a vital role in the physiochemical defences of the host through direct degradation of bacterial membranes  <cit> .
selection of probiotic strains for use in humans has typically been based upon intrinsic acid and bile resistance traits  <cit> .
however, members of the lactobacillus and bifidobacteria spp are known to demonstrate low levels of innate bile tolerance when compared to pathogens such as l. monocytogenes  <cit> .
we recently described the presence of a novel bile exclusion system  in l. monocytogenes which permits the active exclusion of bile from the cell in a manner similar to multi drug efflux pumps of gram negative bacteria such as e. coli.
in listeria elimination of bile results in a 5-log reduction in tolerance of bile in vitro and has a significant impact upon virulence potential  <cit> .
bile is not widely distributed in gram positive organisms; however homology was found with the osmotically inducible busa of l. lactis.
sleator et al.,  <dig>  showed that bile is involved in bile exclusion and not in osmotolerance as previously believed.
here cloning and expression of bile in b. breve and l. lactis resulted in a  <dig>  to  <dig> -log increased tolerance of porcine bile.
similarly these engineered strains demonstrate improved resistance to bovine bile in vitro .
these data provide proof of concept that bile tolerance in these strains can be significantly enhanced through rational genetic manipulation.
wild-type l. lactis survives poorly in the murine gi tract  <cit> .
however, l. lactis has been proposed as a vehicle for delivery of oral vaccines and biotherapeutic agents  <cit> .
indeed, an l. lactis strain engineered to produce human il- <dig> has recently been investigated in human clinical trials for the therapy of crohn's disease  <cit> .
we show that l. lactis expressing cloned bile demonstrates significantly enhanced survival in the gi tract of mice  relative to the wild-type l. lactis strain.
the results indicate that molecular engineering approaches may have the potential to enhance the proposed biomedical applications of l. lactis.
survival of b. breve in the murine gi tract was also significantly enhanced through expression of bile.
the engineered strain was detected at significantly higher levels than the wild-type in faeces  as well as in the small intestine, caecum and colon of inoculated animals.
bifidobacterium species are known to only transiently colonise the gi tract when administered as a probiotic therapeutic in experimental animals  <cit>  and humans  <cit> .
here we show that increased bile tolerance can significantly impact on survival and colonisation.
it is likely that enhanced persistence will also enhance efficacy in therapeutic models.
indeed, in support of this we demonstrate that bifidobacteria with increased bile tolerance are capable of reducing oral infection with l. monocytogenes.
whilst the levels of enhanced protection are low  they are statistically significant.
we recently demonstrated that cloning and expression of the betaine uptake system betl in b. breve ucc <dig> can significantly enhance acid and salt tolerance of the engineered strain and promote survival in the mouse gi tract  <cit> .
we determined moderately higher rates of survival in vivo when strains are engineered to enhance bile tolerance.
indeed, we find greater colonisation of the small intestine through enhanced bile tolerance of b. breve, a finding that may have significant biomedical consequences since the small intestine is the key site of immune sampling in the gi tract.
in addition, we have extended the earlier study to demonstrate an application in the gras organism l. lactis.
this is particularly significant given that l. lactis heterologously expressing the cytokine il- <dig> locally within the gut in murine models of ibd have previously been shown to alleviate symptoms of gastrointestinal inflammation  <cit> .
thus, any attempt to increase the git persistence of this strain may be important for the future development of novel vaccine and drug delivery systems.
collectively this work outlines the application of the patho-biotechnology concept to enhance the robustness and efficacy of potential probiotic organisms.
whilst this approach is unlikely to gain immediate acceptance by regulatory bodies given the genetically modified nature of the constructed strains, we propose that this work represents a further proof-of-concept that may inform future studies to enhance delivery of live organisms, vaccines or biotherapeutic agents to the gi tract  <cit> .
authors' contributions
dw, rds, cgmg and ch participated in the design and coordination of the study and the drafting of the manuscript.
dw and rds carried out the main experimental work.
all authors have read and approved the final manuscript.

 probiotics, defined by a working group of the international life sciences institute europe as "a viable microbial food supplement, which beneficially influences the health of the host", have become the focus of considerable research interest in recent years. live probiotic organisms have been shown to reduce the symptoms of inflammatory conditions such as inflammatory bowel disease and irritable bowel syndrome. this is believed to occur via the localized stimulation of anti-inflammatory cytokines as a result of beneficial alterations to the microbiota. proof of principle and efficacy has been demonstrated with the inert food organism lactococcus lactis which has been engineered to secrete il- locally within the gut in murine models of ibd in order to alleviate symptoms of gastrointestinal inflammation. however, despite their obvious clinical potential, these strains are often poorly adapted to conditions encountered in the upper gastrointestinal tract and delivery of viable organisms from the mouth to the large intestine remains a major hurdle to their use in human therapies. listeria monocytogenes is a gram-positive intracellular foodborne pathogen capable of withstanding a variety of hostile environmental conditions, including the numerous stresses encountered during the production, preparation, and storage of food. following consumption l. monocytogenes effectively survives the extreme conditions encountered during gastric passage, including the low ph of the stomach, low oxygen content and elevated osmolarity and bile salts associated with the upper small intestine. bile in particular represents a key challenge to bacteria that survive and transit the stomach and enter the small intestine. listeria has been isolated from the human gallbladder, indicating an inherent ability to tolerate high concentrations of bile. previously sleator et al.,, identified a novel bile resistance mechanism designated bile, which when disrupted resulted in a bile sensitive phenotype. bile, which functions by excluding bile from the cell, has also been shown to facilitate improved gastrointestinal transit in mouse models of infection and as such contributes to the gastrointestinal phase of l. monocytogenes infection. interestingly, not many homologues of the bile operon have been identified in any of the genomes of the probiotic or commensal organisms sequenced to date. the only homology found was busa of l. lactis, which encodes a glycine betaine uptake system. this system is osmotically inducible as was believed to be the case with bile. however, sleator et al, have shown that bile does not play a role in osmotolerance, but in fact a major role in bile tolerance. herein, we demonstrate that heterologous expression of the listerial bile resistance mechanism bile improves bile resistance in vitro as well as enhancing gastrointestinal persistence and clinical efficacy of the probiotic strain b. breve ucc furthermore, expression in l. lactis enhanced bile tolerance and in vivo survival and may have applications in targeted vaccine or drug delivery by this organism. methods bacterial strains, plasmids, and culture conditions the strains and plasmids used in this study are listed in table escherichia coli strain dh10b used as a cloning host, was grown aerobically at 37°c in luria-bertani medium.bifidobacterium breve ucc a human commensal originally isolated from an infant nursling stool, was anaerobically cultured in reinforced clostridial medium or de man, rogosa and sharpe medium supplemented with % l-cysteine hcl. anaerobic conditions were maintained using an anaerocult oxygen-depleting system in an anaerobic chamber. lactococcus lactis nz strain was grown in m medium supplemented with % glucose at 30°c. l. monocytogenes egd-e was cultured aerobically at 37°c in brain heart infusion medium. oxoid ltd., supplied all growth media. for solid media, % agar was added. when appropriate, antibiotics were added at the following final concentrations: for e. coli, b. breve, and l. lactis, chloramphenicol μg ml- μg ml- and μg ml- respectively. porcine bile was obtained from sigma. the required quantity of bile was solubilised in water and autoclaved. genetic manipulations plasmid dna was isolated from e. coli using a qiaprep spin miniprep kit according to the manufacturer's instructions. genomic dna was isolated from l. monocytogenes egd-e using a genelute bacterial genomic dna kit as recommended by the manufacturer. transformation of l. lactis was achieved according to the protocol of de vos et al.. standard procedures were used for dna manipulation in e. coli. restriction endonucleases, t dna ligase, and 2× pcr mixture were used as recommended by the manufacturers. oligonucleotide primers were synthesized by sigma genosys. pcr products required for cloning were generated with kod hot-start high-fidelity dna polymerase using ng l. monocytogenes genomic dna. plasmid construction analysis of the bile operon in l. monocytogenes egd-e uncovered the presence of two open reading frames bilea and bileb, oriented in the same direction and overlapping by five nucleotides. pcr primers with incorporated xbai and psti restriction enzyme sites were used to amplify the complete bile operon from the chromosome of l. monocytogenes egd-e. the resultant kb pcr product was digested with xbai and psti and subsequently ligated into similarly digested pnz using t dna ligase. the resultant plasmid, containing bile under the transcriptional control of its own promoter, was designated pnz8048-bile+ as in table both pnz8048-bile+ and pnz8048-bile- initially introduced into e. coli dh10b as a cloning host. plasmid dna was extracted from successful transformants, was sequenced and subsequently transformed into the bifidobacterial strain ucc and lactococcal strain nz yielding ucc2003-bile+and nz9000-bile+ respectively. strains harbouring pnz which was used as a negative control, were named ucc2003-bile- and nz9000-bile- respectively. chloramphenicol was added to plates as a selective marker. electroporation electroporation of plasmid dna into e. coli and l. lactis was performed essentially as previously described. electro-transformation of b. breve with plasmid dna was performed as described by macconaill et al.. essentially, mid-logarithmic-phase cells were chilled on ice for min, followed by centrifugation. the cell pellet was washed twice and resuspended in m sucrose- mm citrate buffer. cells were incubated on ice for min, and this was followed by electro-transformation with a bio-rad gene pulser ii apparatus under the following conditions: μf, Ω and kv cm- modified rogosa medium was added to the cells, and the mixture was incubated anaerobically at 37°c for hr prior to plating onto rca containing the appropriate antibiotic. plasmid stability study b. breve ucc colonies containing pnz8048-bile+ and pnz8048-bile- and l. lactis nz colonies containing pnz8048-bile+and pnz8048-bile- were first cultured in mrs broth containing μg ml- chloramphenicol and gm broth with μg ml- respectively. cells were then sub-cultured in fresh mrs and gm broth without antibiotic selection every hrs for a total of generations. vector segregation stability was monitored by plating isolated colonies every hrs. colonies were replica plated onto mrscm and gm17cm b. breve was incubated anaerobically at 37°c and l. lactis incubated aerobically at 30°c for hrs. the percentage of loss of the test plasmid in the population was then calculated. transcriptional analysis total rna was isolated from b. breve ucc and l. lactis nz cells grown to an optical density at nm of using the macaloid acid method as described by ventura et al. and then treated with dnase. reverse transcription for cdna was mediated by thermostable superscript reverse transcriptase using 10– μg rna as a template in a μl reaction mixture containing μl 5× superscript iii buffer, ng of random primers p μl mm dithiothreitol, μl of deoxynucleoside triphosphate mix and the superscript enzyme, which was used according the manufacturer's instructions to produce cdna. the cdna generated was used as a template for reverse transcription -pcrs performed with primers rt-pcrf and rt-pcrr. in all cases, control pcr reactions were used to ensure the complete removal of dna from rna preparations prior to reverse transcription. resistance to bile to determine the ability of strains to survive at sub-lethal bile concentrations, the b. breve and l. lactis cultures were grown to stationary phase and were subsequently inoculated into mrs and gm broth containing 1% porcine bile. viable cell counts were performed at intervals by serial dilution in one-quarter-strength ringer's solution and plating onto rcmcm or gm17cm respectively. animal studies female balb/c mice were used for in vivo studies. mice were housed in pathogen free conditions in a dedicated facility and were fed standard laboratory feed and water ad libtium. all animal procedures were performed according to the university's ethical guidelines. inoculations were carried out essentially as described by sleator et al.,. probiotic gastrointestinal persistence studies prior to commencement of the trials, the absence of background microflora in murine faecal pellets was confirmed by plating onto the two selective agars containing antibiotic. for the study five mice were used per bacterial strain. each mouse was administered μl of bifidobacteria or lactococcus by oral pipette for consecutive days, leading to colonization of the gastrointestinal tract. gastrointestinal persistence of both strains was tracked by measuring the excretion of viable b. breve in fresh faecal samples collected for days post feeding and l. lactis for days. fresh samples were weighed, homogenized in pbs, diluted and plated onto rca or gm with the appropriate level of chloramphenicol. at days nineteen and four post dosing the mice were sacrificed by cervical dislocation, intestines and caeca were excised. the organs were homoginized in mls pbs, serially diluted and the bifidobacterial and lactococcal numbers in the intestines and caeca of dosed animals were determined by spread-plating homogenates onto rcacm and gm17cm plates. listeria challenge animal feeding trials were performed to determine whether the probiotic strains harbouring pnz8048-bile+ conferred protection against subsequent infection with l. monocytogenes. balb/c mice were fed bifidobacteria by oral pipette for consecutive days before oral infection with l. monocytogenes egd-e::ppl2lux on day three days post infection, the mice were sacrificed by cervical dislocation, livers and spleens were excised, homogenized in mls pbs, and serial dilutions were plated onto bhicm agar, which was followed by overnight incubation at 37°c. the resulting colonies were used to calculate the number of bacteria per organ. statistical analysis numerical results were expressed as arithmetic means +/- standard deviations of the means. cfu determinations were converted to log values, and then the arithmetic means and standard deviations were calculated. error bars in the figures represent standard deviations. student's t test was performed to determine the statistical significance.  improving tolerance to porcine bile the bile system was amplified from l. monocytogenes and cloned into the plasmid vector pnz under the control of its native promoter. the plasmid was electroporated into both b. breve ucc and l. lactis nz. to establish the segregational stability of the pnz constructs a plasmid stability assay was performed over approximately generations. both constructs were capable of replication on b. breve ucc and were found to exhibit considerable stability in the absence of selective pressure. the constructs were also capable of replication in l. lactis nz and were found to exhibit considerable stability in the absence of selective pressure. proof that the operon was heterologously expressed in both bacterial strains was evident as a single mrna transcript was obtained by reverse transcription polymerase chain reaction analysis also in vitro studies showing increased bile resistance supports this by indicating that the bile resistance locus was functional. due to difficulty in obtaining human bile; porcine bile was used to examine the bile tolerance of b. breve bile+ and l. lactis bile+. porcine bile is considered an acceptable substitute because the salt/cholesterol, phospholipids/cholesterol and glycine/taurine ratios resemble the composition of human bile. we utilised levels of porcine bile that were lethal for the bacterial species examined and are likely to approximate in vivo levels in regions of the small intestine where bile is most concentrated. it was observed that the presence of bile rendered the engineered l. lactis strain considerably more resistant to the initial kill by porcine bile relative to the wild-type. the engineered strain demonstrated a -log enhanced resistance to bile over the minute course of the kill curve. in contrast whilst b. breve bile+ demonstrated a similar initial kill to the wild-type the engineered strain was subsequently better equipped to survive in porcine bile. at minutes and minutes post-exposure the b. breve bile+ strain demonstrated log enhanced survival. similar results were seen in experiments carried out using bovine bile. gastrointestinal persistence of l. lactis bile+ and b. breve bile+ in order to test whether the elevated bile tolerance of the engineered strains would influence survival in the gi tract, we utilized a mouse model to follow survival in vivo, using an approach previously outlined by sheehan et al.. to facilitate gastrointestinal colonization by b. breve and l. lactis strains, each balb/c mouse was orally administered × cfu for three successive days. gut persistence was monitored by faecal carriage with samples taken every h for days in the case of l. lactis and every h for days for b. breve. l. lactis bile+ demonstrated significantly enhanced in vivo survival relative to the control over the course of the study. both bile+ and bile- strains were detectable in faeces at approximately the same levels at hours post inoculation. however, the wild-type was undetectable at day post-inoculation. this reflects the naturally poor rate of gastrointestinal survival for l. lactis also observed in other studies. in contrast l. lactis bile+was detectable at high levels in murine faeces at days and post-inoculation. neither bile+ nor bile- strains engineered l. lactis were detectable at day post-inoculation. these data demonstrate significant enhancement of gastrointestinal survival of l. lactis through rational biological engineering. b. breve ucc transiently colonized the murine gi tract at levels of approximately cfu/g faeces irrespective of the presence of bile. however, at day post inoculation a significant difference began to emerge between the engineered b. breve ucc bile+ strain and the bile-, with the bile+ strain demonstrating elevated persistence in faeces compared to the wildtype, which declined significantly. at the final day of the experiment the bile+ strain persisted at levels of approximately × cfu/g faeces whereas the wild-type had declined to × cfu/g faeces. this is a more substantial difference than that observed by sheehan et al. using a similar approach which involved cloning the betl osmotolerance locus into b. breve. at day mice were examined directly for the presence of b. breve ucc in the small intestine, caecum and large intestine. we confirmed a 2-log difference between the bile+ and wt strains in the small intestine with the engineered strain present at significantly higher levels. this finding is important given that this region is associated with the highest levels of bile in the gi tract. interestingly sheehan et al. did not demonstrate enhanced survival of the betl+ strain in the small intestine. we also determined significantly enhanced levels of the engineered strain in the caecum and large intestine. overall, the approximately log increased persistence of the engineered strain in the murine intestine mirrored the results of the faecal persistence studies. effect of engineered bile+ b. breve on oral listeria challenge administration of b. breve ucc has previously been demonstrated to reduce splenic levels of l. monocytogenes following oral infection of mice. we hypothesized that the elevated colonisation of b. breve bile+ in the small intestines of mice may further enhance this protective effect since l. monocytogenes invades at this site. to test this hypothesis we administered mice with either wild-type or bile+ b. breve ucc on three consecutive days and challenged with oral l. monocytogenes infection at day post-inoculation. at the day time point we again detected higher levels of the engineered bile+ strain relative to the wild-type in mouse faeces. following oral infection with l. monocytogenes we determined that oral dosing with the engineered bile+ b. breve strain significantly enhanced clearance of l. monocytogenes as assessed by pathogen load in the liver. a similar trend was seen when the numbers of listeria in the spleens of the infected mice was determined. although differences in infectious burden were small the data indicate improved probiotic efficacy of engineered b. breve. discussion and  foodborne pathogens are capable of rapid adaptation to diverse environmental niches associated with survival in the external environment and transit through the gastrointestinal tract of the host. in contrast, the autochthonous gastrointestinal microbiota demonstrates a high degree of niche-specialisation. this specialisation is an impediment to the delivery of so-called probiotic strains to the human gi tract as these strains must survive gastric acid in the stomach and elevated osmolarity and bile acids in the small intestine before establishing a presence in the large bowel. the term'patho-biotechnology' was recently coined to highlight the beneficial biotechnological uses of molecular systems derived from pathogens. this concept was recently applied to improve the osmotolerance of potential probiotic strains through rational molecular engineering. here we implement the patho-biotechnology concept to improve the in vivo and in vitro bile resistance of a potential probiotic strain, b. breve ucc as well as the food isolate l. lactis, through cloning and expression of a bile tolerance system in these organisms. bile acids are amphipathic molecules synthesized from cholesterol that are produced in the liver, stored interdigestively in the gall bladder and released into the duodenum where their main function is the breakdown of dietary fats. bile also plays a vital role in the physiochemical defences of the host through direct degradation of bacterial membranes. selection of probiotic strains for use in humans has typically been based upon intrinsic acid and bile resistance traits. however, members of the lactobacillus and bifidobacteria spp are known to demonstrate low levels of innate bile tolerance when compared to pathogens such as l. monocytogenes. we recently described the presence of a novel bile exclusion system in l. monocytogenes which permits the active exclusion of bile from the cell in a manner similar to multi drug efflux pumps of gram negative bacteria such as e. coli. in listeria elimination of bile results in a 5-log reduction in tolerance of bile in vitro and has a significant impact upon virulence potential. bile is not widely distributed in gram positive organisms; however homology was found with the osmotically inducible busa of l. lactis. sleator et al., showed that bile is involved in bile exclusion and not in osmotolerance as previously believed. here cloning and expression of bile in b. breve and l. lactis resulted in a to -log increased tolerance of porcine bile. similarly these engineered strains demonstrate improved resistance to bovine bile in vitro. these data provide proof of concept that bile tolerance in these strains can be significantly enhanced through rational genetic manipulation. wild-type l. lactis survives poorly in the murine gi tract. however, l. lactis has been proposed as a vehicle for delivery of oral vaccines and biotherapeutic agents. indeed, an l. lactis strain engineered to produce human il- has recently been investigated in human clinical trials for the therapy of crohn's disease. we show that l. lactis expressing cloned bile demonstrates significantly enhanced survival in the gi tract of mice relative to the wild-type l. lactis strain. the results indicate that molecular engineering approaches may have the potential to enhance the proposed biomedical applications of l. lactis. survival of b. breve in the murine gi tract was also significantly enhanced through expression of bile. the engineered strain was detected at significantly higher levels than the wild-type in faeces as well as in the small intestine, caecum and colon of inoculated animals. bifidobacterium species are known to only transiently colonise the gi tract when administered as a probiotic therapeutic in experimental animals and humans. here we show that increased bile tolerance can significantly impact on survival and colonisation. it is likely that enhanced persistence will also enhance efficacy in therapeutic models. indeed, in support of this we demonstrate that bifidobacteria with increased bile tolerance are capable of reducing oral infection with l. monocytogenes. whilst the levels of enhanced protection are low they are statistically significant. we recently demonstrated that cloning and expression of the betaine uptake system betl in b. breve ucc can significantly enhance acid and salt tolerance of the engineered strain and promote survival in the mouse gi tract. we determined moderately higher rates of survival in vivo when strains are engineered to enhance bile tolerance. indeed, we find greater colonisation of the small intestine through enhanced bile tolerance of b. breve, a finding that may have significant biomedical consequences since the small intestine is the key site of immune sampling in the gi tract. in addition, we have extended the earlier study to demonstrate an application in the gras organism l. lactis. this is particularly significant given that l. lactis heterologously expressing the cytokine il- locally within the gut in murine models of ibd have previously been shown to alleviate symptoms of gastrointestinal inflammation. thus, any attempt to increase the git persistence of this strain may be important for the future development of novel vaccine and drug delivery systems. collectively this work outlines the application of the patho-biotechnology concept to enhance the robustness and efficacy of potential probiotic organisms. whilst this approach is unlikely to gain immediate acceptance by regulatory bodies given the genetically modified nature of the constructed strains, we propose that this work represents a further proof-of-concept that may inform future studies to enhance delivery of live organisms, vaccines or biotherapeutic agents to the gi tract. authors' contributions dw, rds, cgmg and ch participated in the design and coordination of the study and the drafting of the manuscript. dw and rds carried out the main experimental work. all authors have read and approved the final manuscript. 
collectively the data indicates that bile tolerance can be enhanced in bifidobacterium and lactococcus species through rational genetic manipulation and that this can significantly improve delivery to and colonisation of the gi tract. in vitro bile tolerance of both strains was significantly enhanced, following heterologous expression of the listeria monocytogenes bile resistance mechanism bile. furthermore, a b. breve strain expressing bile demonstrated increased efficacy relative to the wild-type strain in reducing oral l. monocytogenes infection in mice.
 the majority of commensal gastrointestinal bacteria used as probiotics are highly adapted to the specialised environment of the large bowel. however, unlike pathogenic bacteria; they are often inadequately equipped to endure the physicochemical stresses of gastrointestinal delivery in the host. herein we outline a patho-biotechnology strategy to improve gastric delivery and host adaptation of a probiotic strain bifidobacterium breve ucc and the generally regarded as safe organism lactococcus lactis nz  in vitro bile tolerance of both strains was significantly enhanced, following heterologous expression of the listeria monocytogenes bile resistance mechanism bile. strains harbouring bile were also recovered at significantly higher levels, than control strains from the faeces and intestines of mice, following oral inoculation. furthermore, a b. breve strain expressing bile demonstrated increased efficacy relative to the wild-type strain in reducing oral l. monocytogenes infection in mice.  collectively the data indicates that bile tolerance can be enhanced in bifidobacterium and lactococcus species through rational genetic manipulation and that this can significantly improve delivery to and colonisation of the gi tract. 