Evaluation of ruminal methane and ammonia formation and microbiota composition as affected by supplements based on mixtures of tannins and essential oils using Rusitec.

BACKGROUND: Dietary supplements based on tannin extracts or essential oil compounds (EOC) have been repeatedly reported as a promising feeding strategy to reduce the environmental impact of ruminant husbandry. A previous batch culture screening of various supplements identified selected mixtures with an enhanced potential to mitigate ruminal methane and ammonia formation. Among these, Q-2 (named after quebracho extract and EOC blend 2, composed of carvacrol, thymol, and eugenol) and C-10 (chestnut extract and EOC blend 10, consisting of oregano and thyme essential oils and limonene) have been investigated in detail in the present study with the semi-continuous rumen simulation technique (Rusitec) in three independent runs. For this purpose, Q-2 and C-10, dosed according to the previous study, were compared with a non-supplemented diet (negative control, NC) and with one supplemented with the commercial EOC-based Agolin® Ruminant (positive control, PC). RESULTS: From d 5 to 10 of fermentation incubation liquid was collected and analysed for pH, ammonia, protozoa count, and gas composition. Feed residues were collected for the determination of ruminal degradability. On d 10, samples of incubation liquid were also characterised for bacterial, archaeal and fungal communities by high-throughput sequencing of 16S rRNA and 26S ribosomal large subunit gene amplicons. Regardless of the duration of the fermentation period, Q-2 and C-10 were similarly efficient as PC in mitigating either ammonia (-37% by Q-2, -34% by PC) or methane formation (-12% by C-10, -12% by PC). The PC was also responsible for lower feed degradability and bacterial and fungal richness, whereas Q-2 and C-10 effects, particularly on microbiome diversities, were limited compared to NC. CONCLUSIONS: All additives showed the potential to mitigate methane or ammonia formation, or both, in vitro over a period of 10 d. However, several differences occurred between PC and Q-2/C-10, indicating different mechanisms of action. The pronounced defaunation caused by PC and its suggested consequences apparently determined at least part of the mitigant effects. Although the depressive effect on NDF degradability caused by Q-2 and C-10 might partially explain their mitigation properties, their mechanisms of action remain mostly to be elucidated.

Shaping the phycosphere: Analysis of the EPS in diatom-bacterial co-cultures.

The phycosphere is a unique niche that fosters complex interactions between microalgae and associated bacteria. The formation of this extracellular environment, and the associated bacterial biodiversity, is heavily influenced by the secretion of extracellular polymers, primarily driven by phototrophic organisms. The exopolysaccharides (EPS) represent the largest fraction of the microalgae-derived exudates, which can be specifically used by heterotrophic bacteria as substrates for metabolic processes. Furthermore, it has been proposed that bacteria and their extracellular factors play a role in both the release and composition of the EPS. In this study, two model microorganisms, the diatom Phaeodactylum tricornutum CCAP 1055/15 and the bacterium Pseudoalteromonas haloplanktis TAC125, were co-cultured in a dual system to assess how their interactions modify the phycosphere chemical composition by analyzing the EPS monosaccharide profile released in the culture media by the two partners. We demonstrate that microalgal-bacterial interactions in this simplified model significantly influenced the architecture of their extracellular environment. We observed that the composition of the exo-environment, as described by the EPS monosaccharide profiles, varied under different culture conditions and times of incubation. This study reports an initial characterization of the molecular modifications occurring in the extracellular environment surrounding two relevant representatives of marine systems.

Rapid Detection of Brettanomyces bruxellensis in Wine by Polychromatic Flow Cytometry.

Brettanomyces bruxellensis is found in several fermented matrices and produces relevant alterations to the wine quality. The methods usually used to identify B. bruxellensis contamination are based on conventional microbiological techniques that require long procedures (15 days), causing the yeast to spread in the meantime. Recently, a flow cytometry kit for the rapid detection (1-2 h) of B. bruxellensis in wine has been developed. The feasibility of the method was assessed in a synthetic medium as well as in wine samples by detecting B. bruxellensis in the presence of other yeast species (Saccharomyces cerevisiae and Pichia spp.) and at the concentrations that produce natural contaminations (up to 105 cells/mL), as well as at lower concentrations (103-102 cells/mL). Wine samples naturally contaminated by B. bruxellensis or inoculated with four different strains of B. bruxellensis species together with Saccharomyces cerevisiae and Pichia spp., were analyzed by flow cytometry. Plate counts were carried out in parallel to flow cytometry. We provide evidence that flow cytometry allows the rapid detection of B. bruxellensis in simple and complex mixtures. Therefore, this technique has great potential for the detection of B. bruxellensis and could allow preventive actions to reduce wine spoilage.

Towards a mechanistic understanding of microalgae-bacteria interactions: integration of metabolomic analysis and computational models.

Interactions amongst marine microalgae and heterotrophic bacteria drive processes underlying major biogeochemical cycles and are important for many artificial systems. These dynamic and complex interactions span the range from cooperative to competitive, and it is the diverse and intricate networks of metabolites and chemical mediators that are predicted to principally dictate the nature of the relationship at any point in time. Recent advances in technologies to identify, analyze, and quantify metabolites have allowed for a comprehensive view of the molecules available for exchange and/or reflective of organismal interactions, setting the stage for development of mechanistic understanding of these systems. Here, we (i) review the current knowledge landscape of microalgal-bacterial interactions by focusing on metabolomic studies of selected, simplified model systems; (ii) describe the state of the field of metabolomics, with specific focus on techniques and approaches developed for microalga-bacterial interaction studies; and (iii) outline the main approaches for development of mathematical models of these interacting systems, which collectively have the power to enhance interpretation of experimental data and generate novel testable hypotheses. We share the viewpoint that a comprehensive and integrated series of -omics approaches that include theoretical formulations are necessary to develop predictive and mechanistic understanding of these biological entities.

Characterization of the microbial community in ripened Pecorino Toscano cheese affected by pink discoloration.

Pink discoloration defect can cause economic losses for cheese producers due to the impossibility to sell the defected cheese, but few knowledge is currently available on the causes of this defect. To gain more insight on the causes that lead to the formation of pink discoloration in Pecorino Toscano cheese with the Protected Designation of Origin (PDO) status, the bacterial community in defected and not defected cheese was characterized by high-throughput sequencing of bacterial 16S rRNA gene. The bacterial community in the defected cheese significantly differed compared to the control. The relative abundance of the genera Acidipropionibacterium, Enterococcus, Escherichia/Shigella, Lactobacillus, Lentilactobacillus and Propionibacterium was higher in the cheese with pink discoloration defect. The concentration of short chain fatty acids and of lactic acid in cheese was measured and a shift towards the production of propionate in the cheese with pink discoloration defect was observed. Furthermore, the possible involvement of microbially produced vitamin B12 in the formation of pink discoloration was not supported by the data, since a tendency to a lower concentration of vitamin B12 was measured in the defected cheese compared to the control.

Scaling down the microbial loop: data-driven modelling of growth interactions in a diatom-bacterium co-culture.

An intricate set of interactions characterizes marine ecosystems. One of the most important is represented by the microbial loop, which includes the exchange of dissolved organic matter (DOM) from phototrophic organisms to heterotrophic bacteria. Here, it can be used as the major carbon and energy source. This interaction is one of the foundations of the entire ocean food-web. The carbon fixed by phytoplankton can be redirected to bacteria in two main ways; either (i) bacteria feed on dead phytoplankton cells or (ii) DOM is actively released by phytoplankton (a process resulting in up to 50% of the fixed carbon leaving the cell). Here, we have set up a co-culture of the diatom Phaeodactylum tricornutum and the chemoheterotrophic bacterium Pseudoalteromonas haloplanktis TAC125 and used this system to study the interactions between these two representatives of the microbial loop. We show that the bacterium can thrive on diatom-derived carbon and that this growth can be sustained by both diatom dead cells and diatom-released compounds. These observations were formalized in a network of putative interactions between P. tricornutum and P. haloplanktis and implemented in a model that reproduces the observed co-culture dynamics, revealing an overall accuracy of our hypotheses in explaining the experimental data.

Polyphenols and Organic Acids as Alternatives to Antimicrobials in Poultry Rearing: A Review.

For decades antibiotics have been used in poultry rearing to support high levels of production. Nevertheless, several problems have arisen because of the misuse of antibiotics (i.e., antibiotic resistance, residues in animal products, environmental pollution). Thus, the European Union (EU) as well as the European Food Safety Authority (EFSA) promote action plans to diminish the use of antibiotics in animal production. Alternatives to antibiotics have been studied. Polyphenols (PPs) or organic acids (OAs) seem to be two accredited solutions. Phenolic compounds, such as phenols, flavonoids, and tannins exert their antimicrobial effect with specific mechanisms. In contrast, short chain fatty acids (SCFAs) and medium chain fatty acids (MCFAs), the OAs mainly used as antibiotics alternative, act on the pathogens depending on the pKa value. This review aims to collect the literature reporting the effects of these substances applied as antimicrobial molecules or growth promoter in poultry feeding (both for broilers and laying hens). Organic acids and PPs can be used individually or in blends, exploiting the properties of each component. Collected data highlighted that further research needs to focus on OAs in laying hens' feeding and also determine the right combination in blends with PPs.

Competitiveness for Nodule Colonization in Sinorhizobium meliloti: Combined In Vitro-Tagged Strain Competition and Genome-Wide Association Analysis.

Associations between leguminous plants and symbiotic nitrogen-fixing rhizobia are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Although this symbiosis is in part species specific, different rhizobial strains may colonize the same nodule. Some rhizobial strains are commonly known as better competitors than others, but detailed analyses that aim to predict rhizobial competitive abilities based on genomes are still scarce. Here, we performed a bacterial genome-wide association (GWAS) analysis to define the genomic determinants related to the competitive capabilities in the model rhizobial species Sinorhizobium meliloti. For this, 13 tester strains were green fluorescent protein (GFP) tagged and assayed versus 3 red fluorescent protein (RFP)-tagged reference competitor strains (Rm1021, AK83, and BL225C) in a Medicago sativa nodule occupancy test. Competition data and strain genomic sequences were employed to build a model for GWAS based on k-mers. Among the k-mers with the highest scores, 51 k-mers mapped on the genomes of four strains showing the highest competition phenotypes (>60% single strain nodule occupancy; GR4, KH35c, KH46, and SM11) versus BL225C. These k-mers were mainly located on the symbiosis-related megaplasmid pSymA, specifically on genes coding for transporters, proteins involved in the biosynthesis of cofactors, and proteins related to metabolism (e.g., fatty acids). The same analysis was performed considering the sum of single and mixed nodules obtained in the competition assays versus BL225C, retrieving k-mers mapped on the genes previously found and on vir genes. Therefore, the competition abilities seem to be linked to multiple genetic determinants and comprise several cellular components. IMPORTANCE Decoding the competitive pattern that occurs in the rhizosphere is challenging in the study of bacterial social interaction strategies. To date, the single-gene approach has mainly been used to uncover the bases of nodulation, but there is still a knowledge gap regarding the main features that a priori characterize rhizobial strains able to outcompete indigenous rhizobia. Therefore, tracking down which traits make different rhizobial strains able to win the competition for plant infection over other indigenous rhizobia will improve the strain selection process and, consequently, plant yield in sustainable agricultural production systems. We proved that a k-mer-based GWAS approach can efficiently identify the competition determinants of a panel of strains previously analyzed for their plant tissue occupancy using double fluorescent labeling. The reported strategy will be useful for detailed studies on the genomic aspects of the evolution of bacterial symbiosis and for an extensive evaluation of rhizobial inoculants.

Insight into phenotypic and genotypic differences between vaginal Lactobacillus crispatus BC5 and Lactobacillus gasseri BC12 to unravel nutritional and stress factors influencing their metabolic activity.

The vaginal microbiota, normally characterized by lactobacilli presence, is crucial for vaginal health. Members belonging to L. crispatus and L. gasseri species exert crucial protective functions against pathogens, although a total comprehension of factors that influence their dominance in healthy women is still lacking. Here we investigated the complete genome sequence and comprehensive phenotypic profile of L. crispatus strain BC5 and L. gasseri strain BC12, two vaginal strains featured by anti-bacterial and anti-viral activities. Phenotype microarray (PM) results revealed an improved capacity of BC5 to utilize different carbon sources as compared to BC12, although some specific carbon sources that can be associated to the human diet were only metabolized by BC12, i.e. uridine, amygdalin, tagatose. Additionally, the two strains were mostly distinct in the capacity to utilize the nitrogen sources under analysis. On the other hand, BC12 showed tolerance/resistance towards twice the number of stressors (i.e. antibiotics, toxic metals etc.) with respect to BC5. The divergent phenotypes observed in PM were supported by the identification in either BC5 or BC12 of specific genetic determinants that were found to be part of the core genome of each species. The PM results in combination with comparative genome data provide insights into the possible environmental factors and genetic traits supporting the predominance of either L. crispatus BC5 or L. gasseri BC12 in the vaginal niche, giving also indications for metabolic predictions at the species level.

Correlation of Breed, Growth Performance, and Rumen Microbiota in Two Rustic Cattle Breeds Reared Under Different Conditions.

The use of rustic cattle is desirable to face challenges brought on by climate change. Maremmana (MA) and Aubrac (AU) are rustic cattle breeds that can be successfully used for sustainable production. In this study, correlations between two rearing systems (feedlot and grazing) and the rumen microbiota, the lipid composition of rumen liquor (RL), and the growth performance of MA and AU steers were investigated. Bacterial community composition was characterized by high-throughput sequencing of 16S rRNA gene amplicons, and the RL lipid composition was determined by measuring fatty acid (FA) and the dimethyl acetal profiles. The main factor influencing bacterial community composition was the cattle breed. Some bacterial groups were positively correlated to average daily weight gain for the two breeds (i.e., Rikenellaceae RC9 gut group, Fibrobacter and Succiniclasticum in the rumen of MA steers, and Succinivibrionaceae UCG-002 in the rumen of AU steers); despite this, animal performance appeared to be influenced by short chain FAs production pathways and by the presence of H2 sinks that divert the H2 to processes alternative to the methanogenesis.

Symbiotic and Nonsymbiotic Members of the Genus Ensifer (syn. Sinorhizobium) Are Separated into Two Clades Based on Comparative Genomics and High-Throughput Phenotyping.

Rhizobium-legume symbioses serve as paradigmatic examples for the study of mutualism evolution. The genus Ensifer (syn. Sinorhizobium) contains diverse plant-associated bacteria, a subset of which can fix nitrogen in symbiosis with legumes. To gain insights into the evolution of symbiotic nitrogen fixation (SNF), and interkingdom mutualisms more generally, we performed extensive phenotypic, genomic, and phylogenetic analyses of the genus Ensifer. The data suggest that SNF likely emerged several times within the genus Ensifer through independent horizontal gene transfer events. Yet, the majority (105 of 106) of the Ensifer strains with the nodABC and nifHDK nodulation and nitrogen fixation genes were found within a single, monophyletic clade. Comparative genomics highlighted several differences between the "symbiotic" and "nonsymbiotic" clades, including divergences in their pangenome content. Additionally, strains of the symbiotic clade carried 325 fewer genes, on average, and appeared to have fewer rRNA operons than strains of the nonsymbiotic clade. Initial characterization of a subset of ten Ensifer strains identified several putative phenotypic differences between the clades. Tested strains of the nonsymbiotic clade could catabolize 25% more carbon sources, on average, than strains of the symbiotic clade, and they were better able to grow in LB medium and tolerate alkaline conditions. On the other hand, the tested strains of the symbiotic clade were better able to tolerate heat stress and acidic conditions. We suggest that these data support the division of the genus Ensifer into two main subgroups, as well as the hypothesis that pre-existing genetic features are required to facilitate the evolution of SNF in bacteria.

Metal-Resistance in Bacteria: Why Care?

Heavy metal resistance is more than the tolerance one has towards a particular music genera [...].

Editorial for the Special Issue: Macro and Microorganism Interactions.

The knowledge of symbiotic, parasitic, and commensal interactions between macro and microorganisms is fundamental to explaining their coexistence, ecology, and productivity [...].

Characterization of the microbial community composition in Italian Cinta Senese sausages dry-fermented with natural extracts as alternatives to sodium nitrite.

Nitrite is widely used in meat products as a multifunctional additive, combining flavour and colour properties with antioxidant and antimicrobial effects. However, nitrite may form reaction products (i.e., nitrosamine) that are potentially carcinogenic to humans. The meat industry, in response to consumers' demands for nitrite-free products, is seeking natural alternatives to nitrite, such as plant-based extracts. Three types of dry-fermented sausages were manufactured: NIT, containing 30 ppm of sodium nitrite; GSE, containing grape seed extract and olive pomace hydroxytyrosol; and CHE, containing chestnut extract and olive pomace hydroxytyrosol. Next-generation sequencing (NGS) was used to analyse microbial consortia, which were correlated with physical and chemical parameters. The prokaryotic community composition was similar among treatments, with a high relative abundance of Staphylococcus xylosus and Lactobacillus sakei, collectively accounting for 87% of the total community. However, significant differences were observed in both operational taxonomic unit (OTU) presence/absence and relative abundance. Ten genera varied in abundance between treatments. The increase in Lactobacillaceae in CHE may explain the reduced pH levels detected in these samples. In conclusion, NGS analysis showed that the prokaryotic community composition was similar in GSE and NIT, while CHE varied in both the composition and relative abundance of different taxa.

The metabolic shift in highly and weakly virulent Dickeya solani strains is more affected by temperature than by mutations in genes encoding global virulence regulators.

Global warming may shortly increase the risk of disease development on plants. Significant differences in the metabolic activity screened with Phenotype Microarray at 22°C and 28°C were observed between D. solani strains with high and low virulence level. Highly virulent D. solani was characterized by a higher number of metabolized compounds and a faster metabolism and was more tolerant to non-favorable pH and osmolarity. Metabolic phenotyping showed for the first time that the mutation in pecT gene, which encodes a global repressor of virulence, affects several pathways of the basic cell metabolism. PecT mutants had a higher maceration capacity of potato tissue and showed a higher pectinolytic activity than the wild-type strains. On the contrary, mutation in expI gene, which encoded the signaling molecules synthase crucial for quorum sensing, had an insignificant effect on the cell metabolism, although it slightly reduced the potato tissue maceration. The ability to utilize most of the tested compounds was higher at 28°C, while the survival at non-favorable pH and osmolarity was higher at 22°C. These results proved that the temperature of incubation had the most significant impact on the D. solani metabolic profiles.

Olive fruit fly rearing procedures affect the vertical transmission of the bacterial symbiont Candidatus Erwinia dacicola.

BACKGROUND: The symbiosis between the olive fruit fly, Bactrocera oleae, and Candidatus Erwinia dacicola has been demonstrated as essential for the fly's larval development and adult physiology. The mass rearing of the olive fruit fly has been hindered by several issues, including problems which could be related to the lack of the symbiont, presumably due to preservatives and antibiotics currently used during rearing under laboratory conditions. To better understand the mechanisms underlying symbiont removal or loss during the rearing of lab colonies of the olive fruit fly, we performed experiments that focused on bacterial transfer from wild female flies to their eggs. In this research, eggs laid by wild females were treated with propionic acid solution, which is often used as an antifungal agent, a mixture of sodium hypochlorite and Triton X, or water (as a control). The presence of the bacterial symbiont on eggs was evaluated by real-time PCR and scanning electron microscopy. RESULTS: DGGE analysis showed a clear band with the same migration behavior present in all DGGE profiles but with a decreasing intensity. Molecular analyses performed by real-time PCR showed a significant reduction in Ca. E. dacicola abundance in eggs treated with propionic acid solution or a mixture of sodium hypochlorite and Triton X compared to those treated with water. In addition, the removal of bacteria from the surfaces of treated eggs was highlighted by scanning electron microscopy. CONCLUSIONS: The results clearly indicate how the first phases of the colony-establishment process are important in maintaining the symbiont load in laboratory populations and suggest that the use of products with antimicrobial activity should be avoided. The results also suggest that alternative rearing procedures for the olive fruit fly should be investigated.

The Degradative Capabilities of New Amycolatopsis Isolates on Polylactic Acid.

Polylactic acid (PLA), a bioplastic synthesized from lactic acid, has a broad range of applications owing to its excellent proprieties such as a high melting point, good mechanical strength, transparency, and ease of fabrication. However, the safe disposal of PLA is an emerging environmental problem: it resists microbial attack in environmental conditions, and the frequency of PLA-degrading microorganisms in soil is very low. To date, a limited number of PLA-degrading bacteria have been isolated, and most are actinomycetes. In this work, a method for the selection of rare actinomycetes with extracellular proteolytic activity was established, and the technique was used to isolate four mesophilic actinomycetes with the ability to degrade emulsified PLA in agar plates. All four strains-designated SO1.1, SO1.2, SNC, and SST-belong to the genus Amycolatopsis. The PLA-degrading capability of the four strains was investigated by testing their ability to assimilate lactic acid, fragment PLA polymers, and deteriorate PLA films. The strain SNC was the best PLA degrader-it was able to assimilate lactic acid, constitutively cleave PLA, and form a thick and widespread biofilm on PLA film. The activity of this strain extensively eroded the polymer, leading to a weight loss of 36% in one month in mesophilic conditions.

Effects of Chestnut Tannin Extract, Vescalagin and Gallic Acid on the Dimethyl Acetals Profile and Microbial Community Composition in Rumen Liquor: An In Vitro Study.

The addition of polyphenol extracts in ruminant diets is an effective strategy to modulate rumen microflora. The aim of this in vitro trial was to study the effects of chestnut tannin extract (CHT), vescalagin (VES) and gallic acid (GAL) on dietary fibre degradability and on the dimethyl acetals (DMA) profile and microbial community composition of rumen liquor. Four diets (basal diet; basal diet plus CHT; basal diet plus VES; basal diet plus GAL) were fermented for 24 h using ewe rumen liquor. At the end of the fermentation, the microbial communities were characterized by sequencing the 16S rRNA gene. The DMA profile was analyzed by gas chromatography. Chestnut tannin extract did not affect fibre degradability, whereas VES and GAL showed a detrimental effect. The presence of CHT, VES and GAL influenced the concentration of several DMA (i.e., 12:0, 13:0, 14:0, 15:0, 18:0 and 18:1 trans-11), whereas the composition of the microbial community was marginally affected. The inclusion of CHT led to the enrichment of the genera Anaerovibrio, Bibersteinia, Escherichia/Shigella, Pseudobutyrivibrio and Streptococcus. The results of this study support the hypothesis that the activity of CHT is due to the synergistic effect of all components rather than the property of a single component.

Dietary supplementation of chestnut and quebracho tannins mix: Effect on caecal microbial communities and live performance of growing rabbits.

The interest in antimicrobial compounds as feed additives is currently increasing. Among different options, tannins seem to have several beneficial effects when employed in animals diet. The present study aimed at investigating the influence on caecal microbial communities of the supplementation of a chestnut and quebracho tannins mix in meat rabbit's diet, also considering animals live performances. Four groups of rabbits were fed with a different diet: a control diet (C); a control diet with coccidiostat (CC), and two experimental diets with 0.3% (T0.3) and 0.6% (T0.6) chestnut and quebracho tannins mix. For microbial analysis, culture-dependent and culture-independent methods were employed. Live performances were not significantly affected by tannins mix supplementations, as well as culturable microbial loads of E. coli, Enterobacteriaceae, Bacteroides spp. and Bifidobacterium spp. C. perfringens was always under the detection limit. A consistent result was obtained by qPCR. As for PCR-DGGE analysis, the Richness and evenness (Shannon-Weiner index) of bacterial communities in caecum resulted significantly higher in control samples (C and CC) than in those from rabbit fed with tannin-containing diets. Sequencing analysis revealed that the phylum Firmicutes was less represented in samples from control groups. As for the methanogen archaeal DGGE, no significant differences were found in richness and diversity among different groups, all dominated by Methanobrevibacter spp.. This work highlights the potential antimicrobial effect of chestnut and quebracho tannins mix in an in vivo system revealed by molecular analysis.