Visualization of probiotics via epifluorescence microscopy and fluorescence in situ hybridization (FISH).

Aerobic plate counts, the standard for bacterial enumeration in the probiotic industry, can be biased towards fast-growing organisms that replicate on synthetic media and can significantly underestimate total bacterial abundance. Culture-independent approaches such as fluorescence in situ hybridization (FISH) hold promise as a means to rapidly and accurately enumerate bacteria in probiotic products. In addition, FISH has the potential to more accurately represent bacterial growth dynamics in the environment in which products are applied without imposing additional growth constraints that are required for enumeration via plate counts. In this study, we designed and optimized three new FISH probes to visualize and quantify Bacillus amyloliquefaciens, Bacillus pumilus, and Bacillus licheniformis within probiotic products. Microscopy-based estimates were consistent or higher than label claims for Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus in both a direct fed microbial (DFM) product as well as a crop microbial biostimulant (CMB) product. Quantification with FISH after a germination experiment revealed the potential for this approach to be used after application of the product.

Dysregulated FXR-FGF19 signaling and choline metabolism are associated with gut dysbiosis and hyperplasia in a novel pig model of pediatric NASH.

To investigate the role of bile acids (BAs) in the pathogenesis of diet-induced nonalcoholic steatohepatitis (NASH), we fed a "Western-style diet" [high fructose, high fat (HFF)] enriched with fructose, cholesterol, and saturated fat for 10 wk to juvenile Iberian pigs. We also supplemented probiotics with in vitro BA deconjugating activity to evaluate their potential therapeutic effect in NASH. Liver lipid and function, cytokines, and hormones were analyzed using commercially available kits. Metabolites, BAs, and fatty acids were measured by liquid chromatography-mass spectrometry. Histology and gene and protein expression analyses were performed using standard protocols. HFF-fed pigs developed NASH, cholestasis, and impaired enterohepatic Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling in the absence of obesity and insulin resistance. Choline depletion in HFF livers was associated with decreased lipoprotein and cholesterol in serum and an increase of choline-containing phospholipids in colon contents and trimethylamine-N-oxide in the liver. Additionally, gut dysbiosis and hyperplasia increased with the severity of NASH, and were correlated with increased colonic levels of choline metabolites and secondary BAs. Supplementation of probiotics in the HFF diet enhanced NASH, inhibited hepatic autophagy, increased excretion of taurine and choline, and decreased gut microbial diversity. In conclusion, dysregulation of BA homeostasis was associated with injury and choline depletion in the liver, as well as increased biliary secretion, gut metabolism and excretion of choline-based phospholipids. Choline depletion limited lipoprotein synthesis, resulting in hepatic steatosis, whereas secondary BAs and choline-containing phospholipids in colon may have promoted dysbiosis, hyperplasia, and trimethylamine synthesis, causing further damage to the liver.NEW & NOTEWORTHY Impaired Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling and cholestasis has been described in nonalcoholic fatty liver disease (NAFLD) patients. However, therapeutic interventions with FXR agonists have produced contradictory results. In a swine model of pediatric nonalcoholic steatohepatitis (NASH), we show that the uncoupling of intestinal FXR-FGF19 signaling and a decrease in FGF19 levels are associated with a choline-deficient phenotype of NASH and increased choline excretion in the gut, with the subsequent dysbiosis, colonic hyperplasia, and accumulation of trimethylamine-N-oxide in the liver.

A comparison of methods for enumerating bacteria in direct fed microbials for animal feed.

Aerobic plate counts are the standard enumeration method for probiotic-containing products. This counting method is limited by the ability of many cells to enter a viable but non-culturable (VBNC) state upon exposure to stressful conditions like dehydration and heating commonly used in probiotic product preparation. Alternative enumeration methods are available including flow cytometry (FC) which counts total live/dead cells by assessing cellular integrity and/or metabolic activity, and quantitative polymerase chain reaction (qPCR) in which enumeration is correlated with the quantity of a nucleic acid target. These three methods were compared for enumerating three lactic acid bacteria (LAB): Pediococcus acidilactici, Pediococcus pentosaceus, and Lactobacillus plantarum, and a Bacillus subtilis related strain in twenty samples of a mixed probiotic product ranging in age from one to 825 days post-production. Flow cytometry and qPCR enumerations were similar and much higher compared to plate counts at later storage times, suggesting that some strains in the population were entering the VBNC state and were only countable by FC and qPCR. We propose the use of FC and/or qPCR as an alternative to plate counts for more accurate enumeration of bacteria in probiotic products.

Short communication: Typing and tracking Bacillaceae in raw milk and milk powder using pyroprinting.

Contamination of fluid and processed milk products with endospore-forming bacteria, such as Bacillaceae, affect milk quality and longevity. Contaminants come from a variety of sources, including the dairy farm environment, transportation equipment, or milk processing machinery. Tracking the origin of bacterial contamination to allow specifically targeted remediation efforts depends on a reliable strain-typing method that is reproducible, fast, easy to use, and amenable to computerized analysis. Our objective was to adapt a recently developed genotype-based Escherichia coli strain-typing method, called pyroprinting, for use in a microbial source-tracking study to follow endospore-forming bacillus bacteria from raw milk to powdered milk. A collection of endospores was isolated from both raw milk and its finished powder, and, after germination, the vegetative cells were subject to the pyroprinting protocol. Briefly, a ribosomal DNA intergenic transcribed spacer present in multiple copies in Bacillaceae genomes was amplified by the PCR. This multicopy locus generated a mixed PCR product that was subsequently subject to pyrosequencing, a quantitative real-time sequencing method. Through a series of enzymatic reactions, each nucleotide incorporation event produces a photon of light that is quantified at each nucleotide dispensation. The pattern of light peaks generated from this mixed template reaction is called a pyroprint. Isolates with pyroprints that match with a Pearson correlation of 0.99 or greater are considered to be in the same group. The pyroprint also contains some sequence data useful for presumptive species-level identification. This method identified groups with isolates from raw milk only, from powdered milk only, or from both sources. This study confirms pyroprinting as a rapid, reproducible, automatically digitized tool that can be used to distinguish bacterial strains into taxonomically relevant groups and, thus, indicate probable origins of bacterial contamination in powdered milk.

Pyroprinting: a rapid and flexible genotypic fingerprinting method for typing bacterial strains.

Bacterial strain typing is commonly employed in studies involving epidemiology, population ecology, and microbial source tracking to identify sources of fecal contamination. Methods for differentiating strains generally use either a collection of phenotypic traits or rely on some interrogation of the bacterial genotype. This report introduces pyroprinting, a novel genotypic strain typing method that is rapid, inexpensive, and discriminating compared to the most sensitive methods already in use. Pyroprinting relies on the simultaneous pyrosequencing of polymorphic multicopy loci, such as the intergenic transcribed spacer regions of rRNA operons in bacterial genomes. Data generated by sequencing combinations of variable templates are reproducible and intrinsically digitized. The theory and development of pyroprinting in Escherichia coli, including the selection of similarity thresholds to define matches between isolates, are presented. The pyroprint-based strain differentiation limits and phylogenetic relevance compared to other typing methods are also explored. Pyroprinting is unique in its simplicity and, paradoxically, in its intrinsic complexity. This new approach serves as an excellent alternative to more cumbersome or less phylogenetically relevant strain typing methods.