In vivo assessment of potential probiotic Lactobacillus salivarius strains: evaluation of their establishment, persistence, and localisation in the murine gastrointestinal tract

The enteric flora comprise approximately 95% of the total number of cells in the human body. Numerous studies have investigated potentially beneficial members of this microbial community due to their ability to elicit immune responses while also protecting against microbial pathogens. We have previously reported on the isolation and identification, from surgically-resected segments of the human gastrointestinal tract (GIT), of potential probiotic lactic acid bacteria (LAB). These bacterial strains exhibit potentially beneficial probiotic traits in vitro such as bile tolerance in the absence of deconjugation; gastric acid resistance; and adherence to epithelial cell lines. The objective of this study was to administer two strains of the previously-isolated LAB to mice over a period of 7 or 14 days in order to assess their ability to establish themselves within specific regions of the GIT. Throughout this feeding period, and for 4 days following cessation of feeding, the numbers of total culturable lactobacilli and of the administered LAB present in faeces were monitored. Spontaneous rifampicin resistant derivatives (50 mg:ml) of Lactobacillus salivarius subsp. salivarius UCC1(LM5) and Lb. salivarius subsp. salivarius UCC118(LM2) were generated to facilitate enumeration of the strains in GIT and faecal samples. Each potential probiotic strain was individually administered to Balb:c mice at a daily concentration of approximately 4.0 109 CFU. After 1 day of feeding, strains UCC1(LM5) and UCC118(LM2) were recovered from murine faeces at Log10 6.95 (1.18) CFU:g and Log10 6.33 (0.37) CFU:g, respectively. Interestingly, UCC118(LM2), which was originally isolated from the ileal-caecal region of the human GIT, was found to have become established in the corresponding region of the murine GIT regardless of the length of the feeding period. UCC118(LM2) was also found to persist in faeces for a period of up to 3 days following cessation of feeding. Administration of UCC1(LM5) and UCC118(LM2) did not result in any significant changes in the levels of indigenous bacteria culturable from faeces. In conclusion, human isolate Lb. salivarius subsp. sali6arius UCC118(LM2) was found to effectively colonise, and survive transit through, the murine gastrointestinal tract. Lactobacillus salivarius subsp. sali6arius UCC118 has been deposited at The National Collections of Industrial and Marine Bacteria (NCIMB) and accorded the accession number NCIMB40829

The effect of vitamin K1 supplemention for 12 months on bone mineral density and indices of vitamin K status and bone turnover in adult Crohn s disease patients

Adult patients with Crohn’s disease (CD), even those in remission, have been shown to have higher circulating under-g-carboxylated osteocalcin (ucOC) concentrations, a sensitive marker of vitamin K nutritional status(1), compared to age- and sex-matched healthy control subjects(2,3). Increased concentrations of ucOC in CD patients in these studies appear to be positively and negatively associated with the rate of bone turnover(3) and bone mineral density (BMD) at some sites(2), respectively. The aim of our study was to investigate whether supplementation with vitamin K1 (1000 mg/d) for 12 months had a positive effect on the rate of bone turnover and BMD in CD patients. We have previously shown that this level of supplementation maximally suppresses the degree of ucOC in CD patients(4)

Microbially modified bile acids (DCA & CDCA) oscillate clock controlled genes in synchronised Caco-2 cells.

<p>Caco-2 cells were synchronized via serum starvation followed by a serum shock and treated with bile acids at 100 μM or with their corresponding bile salts. The cells were harvested for every 6h for a total of 48 hours. The relative expression levels of clock-regulated genes were measured using qRT-PCR and plotted in the graph versus time. The red colour graph represents the vehicle, pink represents the TDCA, brown represents TCDCA, blue represents DCA and green represents CDCA. Data represents pooled results from three independent biological replicates with two technical replicates.</p

Model by which microbially-modified bile acids may influence expression of circadian genes.

<p>Bile acids are synthesised from cholesterol and conjugated with taurine or glycine in the liver and stored in the gall bladder. Upon food intake, the bile salts are released into the duodenum and aid in fat digestion and adsorption. Gut microbes in the intestinal lumen deconjugate bile salts to yield unconjugated bile acids. Recent work from other groups has demonstrated that diet, antibiotics and probiotics may influence this microbial activity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167319#sec012" target="_blank">discussion</a>). Within the ileal enterocytes, unconjugated bile acids influence the amplitude and periodicity of circadian gene expression. Nearly 95% of bile salts and bile acids are reabsorbed in the terminal ileum and transported back to the liver via the hepatic portal circulation. Upon reaching the liver, the bile acids further influence circadian gene expression profiles.</p

Unconjugated bile acids (DCA & CDCA) influence expression of clock related genes in murine peripheral organs.

<p>C57BL/6 mice were given vehicle (Corn oil) or DCA or CDCA each at 9 μmol/kg bodyweight dissolved in corn oil via oral gavage. The mice were gavaged three times (t = 0, t = 24h and t = 48h) with corn oil or bile acids and were fasted for 3h prior to harvesting of tissues from (A, B) Ileum, (C, D) Colon and (E, F) Liver. All tissues were harvested within the same 45 minute window during the light phase to minimize variation between subjects (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167319#sec002" target="_blank">Materials & Methods</a>). Total RNA was isolated from tissues and mRNA expression was measured using qRT-PCR. The expression of clock-regulated genes was analysed. Data are plotted relative to β-actin expression. The white bar represents the results from vehicle, grey bar represents the DCA treatment group and the black bar represents the CDCA group. Error bars denote SEM. Statistical significance determined by one way ANOVA (*P<0.05, **P<0.01, ***P<0.001), n = 4.</p

Unconjugated bile acids (DCA and CDCA) influence expression of genes encoding input regulators of clock-regulated genes in murine peripheral organs.

<p>C57BL/6 mice were given vehicle (Corn oil) or DCA or CDCA each at 9 μmol/ kg bodyweight dissolved in corn oil via oral gavage. The mice were gavaged three times (t = 0, t = 24h and t = 48h) with corn oil or bile acids and were fasted for 3h prior to harvesting of tissues from (A, B) Ileum, (C, D) Colon and (E, F) Liver. All tissues were harvested within the same 45 minute window during the light phase to minimize variation between subjects (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167319#sec002" target="_blank">Materials & Methods</a>). Total RNA was isolated from tissues and mRNA expression was measured using qRT-PCR. The expression of clock-controlled genes was analysed. Data are plotted relative to β-actin expression. The white bar represents the results from vehicle, grey bar represents the DCA treatment group and the black bar represents the CDCA group. Error bars denote SEM. Statistical significance was determined by one way ANOVA (*P<0.05, **P<0.01, ***P<0.001), n = 4.</p

Delta weight gain over the eight week intervention period.

<p>(A) Bac⁺ intervention, when compared to Bac^- intervention, causes a significant reduction in weight gain in diet induced obese mice at weeks 2–4 (early intervention period) but this does not persist with time. (B) Vancomycin treatment results in a two phase reduction in weight gain in diet induced obese mice. In phase one (early; weeks 1–4) a significant reduction in weight gain relative to the initial start weight is observed. In the second phase, diet induced obese mice receiving vancomycin gain weight relative to the initial start weight but weight change continues to be significantly less than that in diet induced obese controls. Data represented as mean SEM n = 9–10 *p<0.05.</p

Microbial distribution at phylum level.

<p>Phylum level microbial distribution in all data sets at intervention week 2 and week 8. The pie charts represent total percentage read number for the corresponding colour coded phylum (<i>n</i> = 9–10 per group).</p

Microbial distribution at family level.

<p>Family level microbial distribution in all data sets at intervention week 2 and week 8. The pie charts represent total percentage read number for the corresponding colour coded family (<i>n</i> = 9–10 per group).</p

Total bacterial number observed in all treatment groups at both time points.

<p>Quantitative PCR reveals that the changes occurring are qualitative not quantitative as no significant difference is observed between time points. Total bacterial numbers calculated as copies of 16 S rRNA/g wet stool. Statistical significant difference between treatment groups is denoted by ***. p value based on kruskal wallis analysis with statistical significant determined as p≤0.05. Error bars represent the standard error of the mean.</p