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Enhancing bile tolerance improves survival and persistence of Bifidobacterium and Lactococcus in the murine gastrointestinal tract.

Watson D, Sleator RD, Hill C, Gahan CG - BMC Microbiol. (2008)

Bottom Line: In vitro bile tolerance of both strains was significantly enhanced (P < 0.001), following heterologous expression of the Listeria monocytogenes bile resistance mechanism BilE.Strains harbouring bilE were also recovered at significantly higher levels (P < 0.001), than control strains from the faeces and intestines of mice (n = 5), following oral inoculation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland. watson_d@hotmail.com

ABSTRACT

Background: 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 (GI) delivery in the host. Herein we outline a patho-biotechnology strategy to improve gastric delivery and host adaptation of a probiotic strain Bifidobacterium breve UCC2003 and the generally regarded as safe (GRAS) organism Lactococcus lactis NZ9000.

Results: In vitro bile tolerance of both strains was significantly enhanced (P < 0.001), following heterologous expression of the Listeria monocytogenes bile resistance mechanism BilE. Strains harbouring bilE were also recovered at significantly higher levels (P < 0.001), than control strains from the faeces and intestines of mice (n = 5), 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.

Conclusion: 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.

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Related in: MedlinePlus

(A) Flow diagram for the expression of the bilEAB operon, indicating the listerial bilE operon (B) RT-PCR analysis of the heterologous expression of the bilEAB bile resistance loci in B. breve and L. lactis (a) Control PCRS using cDNA template and the 16S RNA specific primers Bif16SF, Bif16SR, Lac16SF and Lac16SR to confirm the presence of the correct template. (b) bilE specific primers RTPCR F and RTPCR R amplify a 500 bp fragment from both UCC3003-bilE+ and NZ9000-bilE+ cDNA, while as expected no product was obtained when the cDNA of the control strains UCC3003-bilE- and NZ9000-bilE- were used as template.
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Figure 2: (A) Flow diagram for the expression of the bilEAB operon, indicating the listerial bilE operon (B) RT-PCR analysis of the heterologous expression of the bilEAB bile resistance loci in B. breve and L. lactis (a) Control PCRS using cDNA template and the 16S RNA specific primers Bif16SF, Bif16SR, Lac16SF and Lac16SR to confirm the presence of the correct template. (b) bilE specific primers RTPCR F and RTPCR R amplify a 500 bp fragment from both UCC3003-bilE+ and NZ9000-bilE+ cDNA, while as expected no product was obtained when the cDNA of the control strains UCC3003-bilE- and NZ9000-bilE- were used as template.

Mentions: The BilE system was amplified from L. monocytogenes and cloned into the plasmid vector pNZ8048 under the control of its native promoter. The plasmid was electroporated into both B. breve UCC2003 and L. lactis NZ9000 (see Table 1). To establish the segregational stability of the pNZ8048 constructs a plasmid stability assay was performed over approximately 50 generations. Both constructs (pNZ8048-bilE+ and pNZ8048-bilE-) were capable of replication on B. breve UCC2003 and were found to exhibit considerable stability (98.55 +/- 0.63%) in the absence of selective pressure (data not shown). The constructs were also capable of replication in L. lactis NZ9000 and were found to exhibit considerable stability (100%) 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 (RT-PCR) analysis (Fig. 2) 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 [20]. We utilised levels of porcine bile (1% w/v) that were lethal for the bacterial species examined and are likely to approximate in vivo levels in regions of the small intestine (e.g. the duodenum) where bile is most concentrated [20]. 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 (empty pNZ8048). The engineered strain demonstrated a 2.5-log enhanced resistance to bile over the 20 minute course of the kill curve (Fig. 3A). In contrast whilst B. breve bilE+ demonstrated a similar initial kill to the wild-type (after 5 minutes) the engineered strain was subsequently better equipped to survive in porcine bile. At 15 minutes and 20 minutes post-exposure the B. breve bilE+ strain demonstrated 2.5 log enhanced survival (Fig. 3B). Similar results were seen in experiments carried out using bovine bile (oxgall) (data not shown).


Enhancing bile tolerance improves survival and persistence of Bifidobacterium and Lactococcus in the murine gastrointestinal tract.

Watson D, Sleator RD, Hill C, Gahan CG - BMC Microbiol. (2008)

(A) Flow diagram for the expression of the bilEAB operon, indicating the listerial bilE operon (B) RT-PCR analysis of the heterologous expression of the bilEAB bile resistance loci in B. breve and L. lactis (a) Control PCRS using cDNA template and the 16S RNA specific primers Bif16SF, Bif16SR, Lac16SF and Lac16SR to confirm the presence of the correct template. (b) bilE specific primers RTPCR F and RTPCR R amplify a 500 bp fragment from both UCC3003-bilE+ and NZ9000-bilE+ cDNA, while as expected no product was obtained when the cDNA of the control strains UCC3003-bilE- and NZ9000-bilE- were used as template.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577680&req=5

Figure 2: (A) Flow diagram for the expression of the bilEAB operon, indicating the listerial bilE operon (B) RT-PCR analysis of the heterologous expression of the bilEAB bile resistance loci in B. breve and L. lactis (a) Control PCRS using cDNA template and the 16S RNA specific primers Bif16SF, Bif16SR, Lac16SF and Lac16SR to confirm the presence of the correct template. (b) bilE specific primers RTPCR F and RTPCR R amplify a 500 bp fragment from both UCC3003-bilE+ and NZ9000-bilE+ cDNA, while as expected no product was obtained when the cDNA of the control strains UCC3003-bilE- and NZ9000-bilE- were used as template.
Mentions: The BilE system was amplified from L. monocytogenes and cloned into the plasmid vector pNZ8048 under the control of its native promoter. The plasmid was electroporated into both B. breve UCC2003 and L. lactis NZ9000 (see Table 1). To establish the segregational stability of the pNZ8048 constructs a plasmid stability assay was performed over approximately 50 generations. Both constructs (pNZ8048-bilE+ and pNZ8048-bilE-) were capable of replication on B. breve UCC2003 and were found to exhibit considerable stability (98.55 +/- 0.63%) in the absence of selective pressure (data not shown). The constructs were also capable of replication in L. lactis NZ9000 and were found to exhibit considerable stability (100%) 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 (RT-PCR) analysis (Fig. 2) 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 [20]. We utilised levels of porcine bile (1% w/v) that were lethal for the bacterial species examined and are likely to approximate in vivo levels in regions of the small intestine (e.g. the duodenum) where bile is most concentrated [20]. 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 (empty pNZ8048). The engineered strain demonstrated a 2.5-log enhanced resistance to bile over the 20 minute course of the kill curve (Fig. 3A). In contrast whilst B. breve bilE+ demonstrated a similar initial kill to the wild-type (after 5 minutes) the engineered strain was subsequently better equipped to survive in porcine bile. At 15 minutes and 20 minutes post-exposure the B. breve bilE+ strain demonstrated 2.5 log enhanced survival (Fig. 3B). Similar results were seen in experiments carried out using bovine bile (oxgall) (data not shown).

Bottom Line: In vitro bile tolerance of both strains was significantly enhanced (P < 0.001), following heterologous expression of the Listeria monocytogenes bile resistance mechanism BilE.Strains harbouring bilE were also recovered at significantly higher levels (P < 0.001), than control strains from the faeces and intestines of mice (n = 5), following oral inoculation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland. watson_d@hotmail.com

ABSTRACT

Background: 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 (GI) delivery in the host. Herein we outline a patho-biotechnology strategy to improve gastric delivery and host adaptation of a probiotic strain Bifidobacterium breve UCC2003 and the generally regarded as safe (GRAS) organism Lactococcus lactis NZ9000.

Results: In vitro bile tolerance of both strains was significantly enhanced (P < 0.001), following heterologous expression of the Listeria monocytogenes bile resistance mechanism BilE. Strains harbouring bilE were also recovered at significantly higher levels (P < 0.001), than control strains from the faeces and intestines of mice (n = 5), 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus