Robust variation in infant gut microbiome assembly across a spectrum of lifestyles.

Infant microbiome assembly has been intensely studied in infants from industrialized nations, but little is known about this process in nonindustrialized populations. We deeply sequenced infant stool samples from the Hadza hunter-gatherers of Tanzania and analyzed them in a global meta-analysis. Infant microbiomes develop along lifestyle-associated trajectories, with more than 20% of genomes detected in the Hadza infant gut representing novel species. Industrialized infants-even those who are breastfed-have microbiomes characterized by a paucity of Bifidobacterium infantis and gene cassettes involved in human milk utilization. Strains within lifestyle-associated taxonomic groups are shared between mother-infant dyads, consistent with early life inheritance of lifestyle-shaped microbiomes. The population-specific differences in infant microbiome composition and function underscore the importance of studying microbiomes from people outside of wealthy, industrialized nations.

Direct loop-mediated isothermal amplification (LAMP) assay for rapid on-site detection of Bifidobacterium longum subspecies longum, infantis, and suis in probiotic products.

As interest in probiotics increases, the need for accurate description of probiotic compositions present in commercial products is also increasing. Since Bifidobacterium longum used as probiotics is labeled at species or subspecies levels, a detection method for distinguishing B. longum subsp. longum, infantis, and suis is needed. Thus, we designed three LAMP primer sets for B. longum subspecies. Each primer set was specific for the target subspecies. The detection level was 0.2 pg for each target DNA (about 102 CFU/mL). To apply these LAMP assays to on-site detection, a direct DNA extraction method was optimized and combined with LAMP assay. Finally, direct LAMP assays were used to monitor the presence of B. longum subspecies in 16 probiotic products. They could specifically and sensitively detect target subspecies within approximately 45 min. These rapid on-site detection methods are useful for identifying B. longum subspecies in probiotic products.

Evaluation of 2'-Fucosyllactose and Bifidobacterium longum Subspecies infantis on Growth, Organ Weights, and Intestinal Development of Piglets.

Human milk is rich in oligosaccharides that influence intestinal development and serve as prebiotics for the infant gut microbiota. Probiotics and 2'-fucosyllactose (2'-FL) added individually to infant formula have been shown to influence infant development, but less is known about the effects of their synbiotic administration. Herein, the impact of formula supplementation with 2'-fucosyllactose (2'-FL) and Bifidobacterium longum subsp. infantis Bi-26 (Bi-26), or 2'-FL + Bi-26 on weight gain, organ weights, and intestinal development in piglets was investigated. Two-day-old piglets (n = 53) were randomized in a 2 × 2 design to be fed a commercial milk replacer ad libitum without (CON) or with 1.0 g/L 2'-FL. Piglets in each diet were further randomized to receive either glycerol stock alone or Bi-26 (109 CFU) orally once daily. Body weights and food intake were monitored from postnatal day (PND) 2 to 33/34. On PND 34/35, animals were euthanized and intestine, liver and brain weights were assessed. Intestinal samples were collected for morphological analyses and measurement of disaccharidase activity. Dry matter of cecum and colon contents and Bifidobacterium longum subsp. infantis abundance by RT-PCR were also measured. All diets were well tolerated, and formula intake did not differ among the treatment groups. Daily body weights were affected by 2'-FL, Bi-26, and day, but no interaction was observed. There was a trend (p = 0.075) for greater total body weight gain in CON versus all other groups. Jejunal and ascending colon histomorphology were unaffected by treatment; however, there were main effects of 2'-FL to increase (p = 0.040) and Bi-26 to decrease (p = 0.001) ileal crypt depth. The addition of 2'-FL and/or Bi-26 to milk replacer supported piglet growth with no detrimental effects on body and organ weights, or intestinal structure and function.

Flow cytometry: a versatile technology for specific quantification and viability assessment of micro-organisms in multistrain probiotic products.

AIMS: Classical microbiology techniques are the gold standard for probiotic enumeration. However, these techniques are limited by parameters of time, specificity and incapacity to detect viable but nonculturable (VBNC) micro-organisms and nonviable cells. The aim of the study was to evaluate flow cytometry as a novel method for the specific quantification of viable and nonviable probiotics in multistrain products. METHODS AND RESULTS: Custom polyclonal antibodies were produced against five probiotic strains from different species (Bifidobacterium bifidum R0071, Bifidobacterium longum ssp. infantis R0033, Bifidobacterium longum ssp. longum R0175, Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011). Evaluation of specificity confirmed that all antibodies were specific at least at the subspecies level. A flow cytometry method combining specific antibodies and viability assessment with SYTO® 24 and propidium iodide was applied to quantify these strains in three commercial products. Analyses were conducted on two flow cytometry instruments by two operators and compared with classical microbiology using selective media. Results indicated that flow cytometry provides higher cell counts than classical microbiology (P < 0·05) in 73% of cases highlighting the possible presence of VBNC. Equivalent performances (repeatability and reproducibility) were obtained for both methods. CONCLUSIONS: This study showed that flow cytometry methods can be applied to probiotic enumeration and viability assessment. Combination with polyclonal antibodies can achieve sufficient specificity to differentiate closely related strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Flow cytometry provides absolute and specific quantification of viable and nonviable probiotic strains in a very short time (<2 h) compared with classical techniques (>48 h), bringing efficient tools for research and development and quality control.

Determination of certain bacterial groups in gut microbiota and endotoxin levels in patients with nonalcoholic steatohepatitis.

BACKGROUND/AIMS: In recent years, the role of the gut microbiota has emerged in several diseases. Herein we aimed to determine the fecal microbiota, endotoxin levels, and inflammation markers in patients with nonalcoholic steatohepatitis (NASH) and healthy controls. MATERIALS AND METHODS: A total of 46 NASH patients and 38 healthy controls were included. NASH patients were diagnosed according to the steatosis, activity, and fibrosis score/fatty liver inhibition of progression algorithm. 16S rRNA gene-targeted specific primers were used for quantification of certain bacterial groups, and a plasmid library was constructed and sequenced in order to determine dominant Lactobacillus and Bifidobacterium members in patients and controls. RESULTS: Significantly decreased Akkermansia muciniphila and increased Enterobacteriaceae levels were determined in patients compared to healthy controls even after adjusting for the body mass index (BMI) and age. Patients with ≥F2 fibrosis had significantly higher Enterobacteriaceae levels compared to F0-F1 fibrosis. Serum endotoxin and high-sensitivity C-reactive protein levels were significantly higher in patients group. According to the sequencing results, L. reuteri, which was one of the dominant Lactobacillus species in the patient group, could not be detected in healthy controls. Bifidobacterium infantis was found in the patients' feces but not in the controls. CONCLUSION: We demonstrated a BMI and age-independent association between the presence of NASH and levels of A. muciniphila and Enterobacteriaceae as well as increased endotoxin levels. L. reuteri was abundant in the patient group, suggesting that dominant Lactobacillus species should be considered before probiotic treatments.