An adaptable and non-invasive method for tracking Bifidobacterium animalis subspecies lactis 420 in the mouse gut.

Probiotic strains from the Bifidobacterium or Lactobacillus genera improve health outcomes in models of metabolic and cardiovascular disease. Yet, underlying mechanisms governing these improved health outcomes are rooted in the interaction of gut microbiota, intestinal interface, and probiotic strain. Central to defining the underlying mechanisms governing these improved health outcomes is the development of adaptable and non-invasive tools to study probiotic localization and colonization within the host gut microbiome. The objective of this study was to test labeling and tracking efficacy of Bifidobacterium animalis subspecies lactis 420 (B420) using a common clinical imaging agent, indocyanine green (ICG). ICG was an effective in situ labeling agent visualized in either intact mouse or excised gastrointestinal (GI) tract at different time intervals. Quantitative PCR was used to validate ICG visualization of B420, which also demonstrated that B420 transit time matched normal murine GI motility (~8 hours). Contrary to previous thoughts, B420 did not colonize any region of the GI tract whether following a single bolus or daily administration for up to 10 days. We conclude that ICG may provide a useful tool to visualize and track probiotic species such as B420 without implementing complex molecular and genetic tools. Proof-of-concept studies indicate that B420 did not colonize and establish residency align the murine GI tract.

The effect of early probiotic exposure on the preterm infant gut microbiome development.

Premature birth, especially if born before week 32 of gestation, is associated with increased risk of neonatal morbidity and mortality. Prophylactic use of probiotics has been suggested to protect preterm infants via supporting a healthy gut microbiota (GM) development, but the suggested strains and doses vary between studies. In this study, we profiled the GM of 5, 10 and 30-day fecal samples from two cohorts of preterm neonates (born <30 weeks of gestation) recruited in the same neonatal intensive care unit. One cohort (n = 165) was recruited from September 2006 to January 2009 before probiotics were introduced in the clinic. The second cohort (n = 87) was recruited from May 2010 to October 2011 after introducing Lacticaseibacillus rhamnosus GG and Bifidobacterium animalis ssp. lactis BB-12 supplementation policy. Through V3-V4 region 16S rRNA gene amplicon sequencing, a distinct increase of L. rhamnosus and B. animalis was found in the fecal samples of neonates supplemented with probiotics. During the first 30 days of life, the preterm GM went through similarly patterned progression of bacterial populations. Staphylococcus and Weissella dominated in early samples, but was gradually overtaken by Veillonella, Enterococcus and Enterobacteriaceae. Probiotic supplementation was associated with pronounced reduction of Weissella, Veillonella spp. and the opportunistic pathogen Klebsiella. Potential nosocomial pathogens Citrobacter and Chryseobacterium species also gradually phased out. In conclusion, probiotic supplementation to preterm neonates affected gut colonization by certain bacteria, but did not change the overall longitudinal bacterial progression in the neonatal period.Abbreviations: GM: Gut microbiota; ASV: Amplicon sequence variant; NEC: Necrotizing enterocolitis; DOL: Days of life; NICU: Neonatal intensive care unit; ESPGHAN: European Society for Pediatric Gastroenterology, Hepatology and Nutrition; Db-RDA: Distance-based redundancy analysis; PERMANOVA: Permutational multivariate analysis of variance; ANCOM: Analysis of compositions of microbiomes; LGG: Lacticaseibacillus (former Lactobacillus) rhamnosus GG; BB-12: Bifidobacterium animalis ssp. lactis BB-12; DGGE: Denaturing Gradient Gel Electrophoresis.

Effect of Probiotic Use on Antibiotic Administration Among Care Home Residents: A Randomized Clinical Trial.

Importance: Probiotics are frequently used by residents in care homes (residential homes or nursing homes that provide residents with 24-hour support for personal care or nursing care), although the evidence on whether probiotics prevent infections and reduce antibiotic use in these settings is limited. Objective: To determine whether a daily oral probiotic combination of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp lactis BB-12 compared with placebo reduces antibiotic administration in care home residents. Design, Setting, and Participants: Placebo-controlled randomized clinical trial of 310 care home residents, aged 65 years and older, recruited from 23 care homes in the United Kingdom between December 2016 and May 2018, with last follow-up on October 31, 2018. Interventions: Study participants were randomized to receive a daily capsule containing a probiotic combination of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp lactis BB-12 (total cell count per capsule, 1.3 × 1010 to 1.6 × 1010) (n = 155), or daily matched placebo (n = 155), for up to 1 year. Main Outcomes and Measures: The primary outcome was cumulative antibiotic administration days for all-cause infections measured from randomization for up to 1 year. Results: Among 310 randomized care home residents (mean age, 85.3 years; 66.8% women), 195 (62.9%) remained alive and completed the trial. Participant diary data (daily data including study product use, antibiotic administration, and signs of infection) were available for 98.7% randomized to the probiotic group and 97.4% randomized to placebo. Care home residents randomized to the probiotic group had a mean of 12.9 cumulative systemic antibiotic administration days (95% CI, 0 to 18.05), and residents randomized to placebo had a mean of 12.0 days (95% CI, 0 to 16.95) (absolute difference, 0.9 days [95% CI, -3.25 to 5.05]; adjusted incidence rate ratio, 1.13 [95% CI, 0.79 to 1.63]; P = .50). A total of 120 care home residents experienced 283 adverse events (150 adverse events in the probiotic group and 133 in the placebo group). Hospitalizations accounted for 94 of the events in probiotic group and 78 events in the placebo group, and deaths accounted for 33 of the events in the probiotic group and 32 of the events in the placebo group. Conclusions and Relevance: Among care home residents in the United Kingdom, a daily dose of a probiotic combination of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp lactis BB-12 did not significantly reduce antibiotic administration for all-cause infections. These findings do not support the use of probiotics in this setting. Trial Registration: ISRCTN Identifier:16392920.

Influenza infection elicits an expansion of gut population of endogenous Bifidobacterium animalis which protects mice against infection.

BACKGROUND: Influenza is a severe respiratory illness that continually threatens global health. It has been widely known that gut microbiota modulates the host response to protect against influenza infection, but mechanistic details remain largely unknown. Here, we took advantage of the phenomenon of lethal dose 50 (LD50) and metagenomic sequencing analysis to identify specific anti-influenza gut microbes and analyze the underlying mechanism. RESULTS: Transferring fecal microbes from mice that survive virulent influenza H7N9 infection into antibiotic-treated mice confers resistance to infection. Some gut microbes exhibit differential features to lethal influenza infection depending on the infection outcome. Bifidobacterium pseudolongum and Bifidobacterium animalis levels are significantly elevated in surviving mice when compared to dead or mock-infected mice. Oral administration of B. animalis alone or the combination of both significantly reduces the severity of H7N9 infection in both antibiotic-treated and germ-free mice. Functional metagenomic analysis suggests that B. animalis mediates the anti-influenza effect via several specific metabolic molecules. In vivo tests confirm valine and coenzyme A produce an anti-influenza effect. CONCLUSIONS: These findings show that the severity of influenza infection is closely related to the heterogeneous responses of the gut microbiota. We demonstrate the anti-influenza effect of B. animalis, and also find that the gut population of endogenous B. animalis can expand to enhance host influenza resistance when lethal influenza infection occurs, representing a novel interaction between host and gut microbiota. Further, our data suggest the potential utility of Bifidobacterium in the prevention and as a prognostic predictor of influenza.

Characterization of potentially probiotic lactic acid bacteria and bifidobacteria isolated from human colostrum.

Breast milk is the main source of nutrition for infants; it contains considerable microflora that can be transmitted to the infant endogenously or by breastfeeding, and it plays an important role in the maturation and development of the immune system. In this study, we isolated and identified lactic acid bacteria (LAB) from human colostrum, and screened 2 strains with probiotic potential. The LAB isolated from 40 human colostrum samples belonged to 5 genera: Lactobacillus, Bifidobacterium, Streptococcus, Enterococcus, and Staphylococcus. We also isolated Propionibacterium and Actinomyces. We identified a total of 197 strains of LAB derived from human colostrum based on their morphology and 16S rRNA sequence, among them 8 strains of Bifidobacterium and 10 strains of Lactobacillus, including 3 Bifidobacterium species and 4 Lactobacillus species. The physiological and biochemical characteristics of strains with good probiotic characteristics were evaluated. The tolerances of some of the Bifidobacterium and Lactobacillus strains to gastrointestinal fluid and bile salts were evaluated in vitro, using the probiotic strains Bifidobacterium lactis BB12 and Lactobacillus rhamnosus GG as controls. Among them, B. lactis Probio-M8 and L. rhamnosus Probio-M9 showed survival rates of 97.25 and 78.33% after digestion for 11 h in artificial gastrointestinal juice, and they exhibited growth delays of 0.95 and 1.87 h, respectively, in 0.3% bile salts. These two strains have the potential for application as probiotics and will facilitate functional studies of probiotics in breast milk and the development of human milk-derived probiotics.

Enhancing Immunomodulatory Function of Red Ginseng Through Fermentation Using Bifidobacterium animalis Subsp. lactis LT 19-2.

Removal of sugar moieties from ginsenosides has been proposed to increase their biological effects in various disease models. In order to identify strains that can increase aglycone contents, we performed a screening using bacteria isolated from the feces of infants focusing on acid tolerance and ß-glucosidase activity. We isolated 565 bacteria and selected Bifidobacterium animalis subsp. lactis LT 19-2 (LT 19-2), which exhibited the highest ß-glucosidase activity with strong acid tolerance. As red ginseng (RG) has been known to exert immunomodulatory functions, we fermented RG using LT 19-2 (FRG) and investigated whether this could alter the aglycone profile of ginsenosides and improve its immunomodulatory effect. FRG increased macrophage activity more potently compared to RG, demonstrated by higher TNF-α and IL-6 production. More importantly, the FRG treatment stimulated the proliferation of mouse splenocytes and increased TNF-α levels in bone marrow-derived macrophages, confirming that the enhanced immunomodulatory function can be recapitulated in primary immune cells. Examination of the molecular mechanism revealed that F-RG could induce phosphorylations of ERK, p38, JNK, and NF-κB. Analysis of the ginsenoside composition showed a decrease in Rb1, Re, Rc, and Rb3, accompanied by an increase in Rd, Rh1, F2, and Rg3, the corresponding aglycone metabolites, in FRG compared to RG. Collectively, LT 19-2 maybe used as a probiotic strain to improve the bioactivity of functional foods through modifying the aglycone/glycoside profile.

Optimisation of a droplet digital PCR for strain specific quantification of a probiotic Bifidobacterium animalis strain in poultry feed.

The use of probiotics in animal nutrition to provide health benefits is widely accepted. Bifidobacterium animalis (BAN) is an example of a commonly used beneficial strain. BAN is applied in a multi-strain feed additive for poultry. As part of the increased demand for tracking and tracing of feed additives within modern quality management, it is crucial to determine the quantity of the active strain after mixing the probiotic product into feed. A real-time PCR protocol, already developed some years ago, was replaced with a Droplet Digital PCR (ddPCR) assay, as this third generation PCR method is known for higher precision, sensitivity and does not require standard curves. Each sample is partitioned into thousands of small subsamples that are measured individually and an absolute result value for each sample is extrapolated via Poisson distribution. The following parameters were evaluated for the ddPCR assay: optimal annealing temperature (59 °C), concentration of primers (500 nM) and probe (400 nM), and PCR cycle number (50 cycles). The linearity of the optimised ddPCR assay was tested with BAN DNA extracted from pure culture. The obtained standard curve was linear (R2 = 0.9982) and the efficiency (E) of the method was 99.98%. To finalise the development, the Limit of Blank (LoB = 9.17 × 102 copies g-1), Limit of Detection (LoD = 1.15 × 103 copies g-1) and Limit of Quantification (LoQ = 1.57 × 103 copies g-1) for the assay were determined using poultry feed free of BAN and feed spiked with different concentrations of the strain. A BAN strain-specific, probe-based ddPCR assay for the quantification in poultry feed was developed.

Difference in the Gut Microbiome between Ovariectomy-Induced Obesity and Diet-Induced Obesity.

During menopausal transition, the imbalance of estrogen causes body weight gain. Although gut microbiome dysbiosis has been reported in postmenopausal obesity, it is not clear whether there is any difference in the microbiome profile between dietary-induced obesity and postmenopausal obesity. Therefore, in this study, we analyzed intestinal samples from ovariectomized mice and compared them with those of mice with high-fat diet-induced obesity. To further evaluate the presence of menopause-specific bacteria-gene interactions, we also analyzed the liver transcriptome. Investigation of the 16S rRNA V3-V4 region amplicon sequence profile revealed that menopausal obesity and dietary obesity resulted in similar gut microbiome structures. However, Bifidobacterium animalis was exclusively observed in the ovariectomized mice, which indicated that menopausal obesity resulted in a different intestinal microbiome than dietary obesity. Additionally, several bacterial taxa (Dorea species, Akkermansia muciniphila, and Desulfovibrio species) were found when the ovariectomized mice were treated with a high-fat diet. A significant correlation between the above-mentioned menopause-specific bacteria and the genes for female hormone metabolism was also observed, suggesting the possibility of bacteria-gene interactions in menopausal obesity. Our findings revealed the characteristics of the intestinal microbiome in menopausal obesity in the mouse model, which is very similar to the dietary obesity microbiome but having its own diagnostic bacteria.

Discovery of α-l-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42.

Enzymes of the glycoside hydrolase family 42 (GH42) are widespread in bacteria of the human gut microbiome and play fundamental roles in the decomposition of both milk and plant oligosaccharides. All GH42 enzymes characterized so far have ß-galactosidase activity. Here, we report the existence of a GH42 subfamily that is exclusively specific for α-l-arabinopyranoside and describe the first representative of this subfamily. We found that this enzyme (BlArap42B) from a probiotic Bifidobacterium species cannot hydrolyze ß-galactosides. However, BlArap42B effectively hydrolyzed paeonolide and ginsenoside Rb2, plant glycosides containing an aromatic aglycone conjugated to α-l-arabinopyranosyl-(1,6)-ß-d-glucopyranoside. Paeonolide, a natural glycoside from the roots of the plant genus Paeonia, is not hydrolyzed by classical GH42 ß-galactosidases. X-ray crystallography revealed a unique Trp345-X12-Trp358 sequence motif at the BlArap42B active site, as compared with a Phe-X12-His motif in classical GH42 ß-galactosidases. This analysis also indicated that the C6 position of galactose is blocked by the aromatic side chains, hence allowing accommodation only of Arap lacking this carbon. Automated docking of paeonolide revealed that it can fit into the BlArap42B active site. The Glcp moiety of paeonolide stacks onto the aromatic ring of the Trp252 at subsite +1 and C4-OH is hydrogen bonded with Asp249 Moreover, the aglycone stacks against Phe421 from the neighboring monomer in the BlArap42B trimer, forming a proposed subsite +2. These results further support the notion that evolution of metabolic specialization can be tracked at the structural level in key enzymes facilitating degradation of specific glycans in an ecological niche.

Selective method for identification and quantification of Bifidobacterium animalis subspecies lactis BB-12 (BB-12) from the gastrointestinal tract of healthy volunteers ingesting a combination probiotic of BB-12 and Lactobacillus rhamnosus GG.

AIM: To develop a novel validated method for the isolation of Bifidobacterium animalis ssp. lactis BB-12 (BB-12) from faecal specimens and apply it to studies of BB-12 and Lactobacillus rhamnosus GG (LGG) recovered from the healthy human gastrointestinal (GI) tract. METHODS AND RESULTS: A novel method for isolating and enumerating BB-12 was developed based on its morphologic features of growth on tetracycline-containing agar. The method identified BB-12 correctly from spiked stool close to 100% of the time as validated by PCR confirmation of identity, and resulted in 97-104% recovery of BB-12. The method was then applied in a study of the recovery of BB-12 and LGG from the GI tract of healthy humans consuming ProNutrients® Probiotic powder sachet containing BB-12 and LGG. Viable BB-12 and LGG were recovered from stool after 21 days of probiotic ingestion compared to baseline. In contrast, no organisms were recovered 21 days after baseline in the nonsupplemented control group. CONCLUSIONS: We demonstrated recovery of viable BB-12, using a validated novel method specific for the isolation of BB-12, and LGG from the GI tract of healthy humans who consumed the probiotic supplement. SIGNIFICANCE AND IMPACT OF THE STUDY: This method will enable more detailed and specific studies of BB-12 in probiotic supplements, including when in combination with LGG.

Spray-drying process preserves the protective capacity of a breast milk-derived Bifidobacterium lactis strain on acute and chronic colitis in mice.

Gut microbiota dysbiosis plays a central role in the development and perpetuation of chronic inflammation in inflammatory bowel disease (IBD) and therefore is key target for interventions with high quality and functional probiotics. The local production of stable probiotic formulations at limited cost is considered an advantage as it reduces transportation cost and time, thereby increasing the effective period at the consumer side. In the present study, we compared the anti-inflammatory capacities of the Bifidobacterium animalis subsp. lactis (B. lactis) INL1, a probiotic strain isolated in Argentina from human breast milk, with the commercial strain B. animalis subsp. lactis BB12. The impact of spray-drying, a low-cost alternative of bacterial dehydration, on the functionality of both bifidobacteria was also investigated. We showed for both bacteria that the spray-drying process did not impact on bacterial survival nor on their protective capacities against acute and chronic colitis in mice, opening future perspectives for the use of strain INL1 in populations with IBD.

Diversity of the subspecies Bifidobacterium animalis subsp. lactis.

Strains of Bifidobacterium animalis subsp. lactis are well-known health-promoting probiotics used commercially. B. animalis subsp. lactis has been isolated from different sources, and little is known about animal isolates of this taxon. The aim of this study was to examine the genotypic and phenotypic diversity between B. animalis subsp. lactis strains different animal hosts including Cameroon sheep, Barbary sheep, okapi, mouflon, German shepard and to compare to BB12, food isolates and the collection strain DSM 10140. Ten strains of B. animalis subsp. lactis from different sources were characterised by phenotyping, fingerprinting, and multilocus sequence typing (MLST). Regardless of origin, MLST and phylogenetic analyses revealed a close relationship between strains of B. animalis subsp. lactis with commercial and animal origin with the exception of isolates from ovine cheese, mouflon and German Shepard dog. Moreover, isolates from dog and mouflon showed significant differences in fermentation profiles and peptide mass fingerprints (MALDI-TOF). Results indicated phenotypic and genotypic diversity among strains of B. animalis subsp. lactis.

A peptidome-based phylogeny pipeline reveals differential peptides at the strain level within Bifidobacterium animalis subsp. lactis.

Bifidobacteria are gut commensal microorganisms belonging to the Actinobacteria group. Some specific strains of Bifidobacterium animalis subsp. lactis are used in functional foods as they are able to exert health-promoting effects in the human host. Due to the limited genetic variability within this subspecies, it is sometimes difficult for a manufacturer to properly track its strain once included in dairy products or functional foods. In this paper, we present a peptidome-based analysis in which the proteomes of a set of B. animalis subsp. lactis strains were digested in silico with human gut endopeptidases. The molecular masses were compared along all the strains to detect strain-specific peptides. These peptides may be interesting towards the development of methodologies for strain identification in the final product.

Suppression subtractive hybridisation and real-time PCR for strain-specific quantification of the probiotic Bifidobacterium animalis BAN in broiler feed.

To ensure quality management during the production processes of probiotics and for efficacy testing in vivo, accurate tools are needed for the identification and quantification of probiotic strains. In this study, a strain-specific qPCR assay based on Suppression Subtractive Hybridisation (SSH) for identifying unique sequences, was developed to quantify the strain Bifidobacterium animalis BAN in broiler feed. Seventy potential BAN specific sequences were obtained after SSH of the BAN genome, with a pool of closely related strain genomes and subsequent differential screening by dot blot hybridisation. Primers were designed for 30 sequences which showed no match with any sequence database entry, using BLAST and FASTA. Primer specificity was assessed by qPCR using 45 non-target strains and species in a stepwise approach. Primer T39_S2 was the only primer pair without any unspecific binding properties and it showed a PCR efficiency of 80% with a Cq value of 17.32 for 20 ng BAN DNA. Optimised feed-matrix dependent calibration curve for the quantification of BAN was generated, ranging from 6.28 × 10(3)cfu g(-1) to 1.61 × 10(6)cfu g(-1). Limit of detection of the qPCR assay was 2 × 10(1)cfu g(-1) BAN. Applicability of the strain-specific qPCR assay was confirmed in a spiking experiment which added BAN to the feed in two concentrations, 2 × 10(6)cfu g(-1) and 2 × 10(4)cfu g(-1). Results showed BAN mean recovery rates in feed of 1.44 × 10(6) ± 4.39 × 10(5)cfu g(-1) and 1.59 × 10(4) ± 1.69 × 10(4)cfu g(-1), respectively. The presented BAN-specific qPCR assay can be applied in animal feeding trials, in order to control the correct inclusion rates of the probiotic to the feed, and it could further be adapted, to monitor the uptake of the probiotic into the gastrointestinal tract of broiler chickens.

Gut microbiota analysis reveals a marked shift to bifidobacteria by a starter infant formula containing a synbiotic of bovine milk-derived oligosaccharides and Bifidobacterium animalis subsp. lactis†CNCM I-3446.

Non-digestible milk oligosaccharides were proposed as receptor decoys for pathogens and as nutrients for beneficial gut commensals like bifidobacteria. Bovine milk contains oligosaccharides, some of which are structurally identical or similar to those found in human milk. In a controlled, randomized double-blinded clinical trial we tested the effect of feeding a formula supplemented with a mixture of bovine milk-derived oligosaccharides (BMOS) generated from whey permeate, containing galacto-oligosaccharides and 3'- and 6'-sialyllactose, and the probiotic Bifidobacterium animalis subsp. lactis (B. lactis) strain CNCM I-3446. Breastfed infants served as reference group. Compared with a non-supplemented control formula, the test formula showed a similar tolerability and supported a similar growth in healthy newborns followed for 12 weeks. The control, but not the test group, differed from the breast-fed reference group by a higher faecal pH and a significantly higher diversity of the faecal microbiota. In the test group the probiotic B. lactis increased by 100-fold in the stool and was detected in all supplemented infants. BMOS stimulated a marked shift to a bifidobacterium-dominated faecal microbiota via increases in endogenous bifidobacteria (B. longum, B. breve, B. bifidum, B. pseudocatenulatum).

Fermented probiotic beverages based on acid whey.

BACKGROUND: Production of fermented probiotic beverages can be a good method for acid whey usage. The obtained products combine a high nutritional value of whey with health benefits claimed for probiotic bacteria. The aim of the study was to define quality properties of beverages based on fresh acid whey and milk with addition of buttermilk powder or sweet whey powder. METHODS: Samples were inoculated with two strains of commercial probiotic cultures: Lactobacillus acidophilus La-5 or Bifidobacterium animalis Bb-12. After fermentation, samples were stored at refrigerated conditions. After 1, 4, 7, 14 and 21 days sensory characteristics, hardness, acetaldehyde content, titratable acidity, pH acidity and count of bacteria cells were evaluated. RESULTS: Throughout all storage period, the number of bacteria was higher than 8 log cfu/ml in the all samples. Beverages with La-5 strain had higher hardness and acidity, whilst samples with Bb-12 contained more acetaldehyde. Samples with buttermilk powder had better sensory properties than with sweet whey powder. CONCLUSIONS: Obtained products made of acid whey combined with milk and fortified with buttermilk powder or sweet whey powder, are good medium for growth and survival of examined probiotic bacteria strains. The level of bacteria was sufficient to provide health benefits to consumers.