Metabolic profiling of bacterial co-cultures reveals intermicrobiome interactions and dominant species.

In animal production, the use of probiotic microorganisms has increased since the ban on antibiotic growth promoters in 2006. The added microorganisms interact with the microbiome of the animals, whereby the probiotic activity is not fully understood. Several microorganisms of the genus Bacillus are already known for their probiotic activity and are applied as feed supplements to increase the health status of the animals. They are thought to interact with Escherichia coli, one of the most abundant bacteria in the animal gut. In biotechnological applications, co-culturing enables the regulation of bacterial interaction or the production of target metabolites. The basic principles of multi-imaging high-performance thin-layer chromatography (HPTLC) with upstream cultivation were further developed to analyze the metabolic profiles of three axenic bacilli cultures compared to their co-cultures with E. coli DSM 18039 (K12). The comparative profiling visualized bacteria's metabolic interactions and showed how the presence of E. coli affects the metabolite formation of bacilli. The characteristic metabolic profile images showed not only the influence of microbiomes but also of inoculation, cultivation and nutrients on the commercial probiotic. The formation of antimicrobially active metabolites, detected via three different planar bioassays, was influenced by the presence of other microorganisms, especially in the probiotic. This first application of multi-imaging HPTLC in the field of co-culturing enabled visualization of metabolic interactions of bacteria via their produced chemical as well as bioactive metabolite profiles. The metabolic profiling provided evidence of bacterial interactions, intermicrobiome influences and dominant species in the co-culture.

Bacillus subtilis spores in probiotic feed quantified via bacterial metabolite using planar chromatography.

Interest in probiotics in animal production has increased due to the European ban on antibiotic growth promoters in 2006. Bacillus subtilis DSM 29784 (B. s. 29784) is one such probiotic feed additive used in poultry. Cell counting has been the most common tool for feed analysis, besides flow cytometry and quantitative polymerase chain reaction. However, quantification of the active probiotic in feed is challenging, since results are influenced by cultivation conditions, viable but non-culturable bacteria and the high contents of feed ingredients. This study presents the first quantitative analysis of a metabolite generated by B. s. 29784 spores in feed to draw conclusions on the amount of active dried spores in the feed. Thus, it is the first quantification of active probiotic bacteria at the trace level in feed based on metabolite production but not cell counting. To generate the calibration standards, solutions with different amounts of dried B. s. 29784 spores were cultured under the same conditions as the feed sample, ensuring independence from growth performance. Upstream cultivation, metabolite extraction and high-performance thin-layer chromatography analysis were proven to be highly reliable and reproducible. The repeatability of the method (RSD 1.9%) and the recovery (111% ± 21% in feed additive matrix, 96% ± 13% in ionized feed matrix) were excellent. The variations during cultivation occurred due to the complex spore germination process and presence of other microbes in the feed. This new procedure, detecting only those cells that produced the metabolite of interest, has several advantages as it takes into account bacterial viability, cultivation conditions, spore germination process, growth behavior and the influence of the nutrient-rich feed matrix. It truly reflects the activity of the probiotic in the feed product, allows side-by-side comparison of characteristic metabolite patterns and nutrient consumptions to understand the metabolism of dried spores in matrix.

Strain-specific quantification of probiotic Bacillus subtilis in feed by imaging high-performance thin-layer chromatography.

The European Union has banned the use of antibiotic growth promoters in animal production, which has led to increased use of probiotic microorganisms. These feed additives result in higher costs for farmers, which is why the demand for a quality control system to quantify probiotics in feeds has increased in recent years. Imaging high-performance thin-layer chromatography (HPTLC) was proven to be a robust method for determining the probiotic Bacillus subtilis DSM 29784 strain based on the production of selective bacterial metabolites and thus characteristic metabolite pattern. However, to quantify the specific probiotic strain in the feed, identification of a strain-specific metabolite not produced by genetically very similar bacteria is necessary. Compared to five bacteria with high genetic similarity, a strain-specific metabolite was formed in the probiotic bacteria by a two-step cultivation procedure. Among others, antimicrobial properties were found for this metabolite, which indicated probiotic activity. The hyphenation of normal-phase HPTLC with reversed-phase high-performance liquid chromatography diode array detection and high-resolution mass spectrometry allowed the preliminary assignment of this strain-specific metabolite to the molecular formula C35H44N6O2 (580.3527 Da). This metabolite, produced each time via an upstream cultivation process to generate the standard levels, was used for the quantification of probiotic active cells in the feed. Data on selectivity, linearity, detection limit, recovery, and precision have shown the good performance of the method.

State of evidence on municipal strategies for health promotion and prevention: a literature and database research (Scoping Review).

BACKGROUND: There are gaps in the research regarding the implementation and evidence of overall strategies for municipal health promotion addressing communities. The aim of this scoping review is to gain initial findings concerning theoretical models, approaches and evidence on strategies of municipal health promotion, which include self-care, mutual aid and healthy environments. The findings can enrich the development of health promotion services. METHODS: A systematic scoping literature analysis was conducted in the databases PubMed, Web of Science, SAGE-Journals, Wiley-Online, ScienceDirect, LIVIVO and WiSo database as well as in a German project database. Evaluation studies and research reports on strategies in municipal health promotion were included and analysed qualitatively. RESULTS: According to our predefined inclusion and exclusion criteria, 15 hits were included. Capacity building, planning and the establishment of structures for health promotion were identified as theory-based models and approaches. None of the publications included showed clear evidence of the effects of municipal health promotion measures in terms of classically medically defined evidence. CONCLUSIONS: The use of evidence-based theoretical models and approaches is no guarantee for the success of strategies for municipal health promotion. Challenges with regard to evidence are the execution of study designs corresponding to higher evidence classes and the isolation of effects of health promotion measures in complex environments. TRIAL REGISTRATION: This scoping review was not registered beforehand.

Effects of the Probiotic Activity of Bacillus subtilis DSM 29784 in Cultures and Feeding Stuff.

The European Union banned the usage of antibiotic growth promoters in animal production. The probiotic microorganism of the genus Bacillus appeared to be an attractive candidate to replace antibiotics. The Bacillus subtilis DSM 29784 is one of these strains. To date, the probiotic effect has not been completely understood, but it is supposed that the effect depends on metabolites of the microorganism. Imaging high-performance thin-layer chromatography (HPTLC) is a powerful tool to visualize differences in the metabolite profile of bacteria with high genetic similarity to allow a better understanding of the probiotic effect. In comparison to other bacteria, especially these bacterial cells were more robust to harsh cultivation conditions and produced a higher level of antioxidants or bioactive substances such as surfactin. HPTLC enabled the comparison of pure cell cultures to the spore cultivation in the feed, and the results explain and support the probiotic effect.

Imaging high-performance thin-layer chromatography as powerful tool to visualize metabolite profiles of eight Bacillus candidates upon cultivation and growth behavior.

Imaging high-performance thin-layer chromatography (HPTLC) was explored with regard to its ability to visualize changes in the metabolite profile of bacteria. Bacillus subtilis has become a model organism in many fields. The increasing interest in these bacteria is driven by their attributed probiotic activity. However, growth behavior and metabolism of Bacillus species have a considerable influence on their activity and secondary metabolite profile. On the HPTLC plate, cultivation broths of Bacillus species (B. subtilis, B. licheniformis, B. pumilus and B. amyloliquefaciens) and some B. subtilis strains of high genetic similarity up to 99.5% were applied directly and compared with their respective liquid-liquid extracts. The latter as well as the cultivation in a minimal medium reduced the matrix load and improved the zone resolution. Cultivation parameters such as nutrient supply, cultivation temperature, cultivation time and rotational speed (oxygen level) as well as medium change were shown to have a considerable influence on the growth behavior and resulting metabolite profiles. Imaging HPTLC turned out to be an efficient and affordable tool to visualize such influences of cultivation parameters on the metabolite profiles. It converts the complexity of reaction processes occurring during cell cultivation in easy-to-understand images, which are helpful to figure out factors of influence and understand activity changes. The results highlighted that optimal cultivation conditions need to be found for the intended bacterial application, and in particular, these conditions have to be kept constant. It must be ensured that small variations in cultivation parameters of bacteria do not change the specified (probiotic) effect on the health of animals and humans. The HPTLC metabolite profiles represented the cultivation conditions of specific bacteria and were found to be a proof of the activity of distinct bacteria. In addition, HPTLC can also be used to optimize and streamline the culture media. The quality control of cultivation or fermentation processes can benefit from such a powerful tool, as a picture is worth a thousand words.