Atividade antimicrobiana in vitro de uma combinação de óleos vegetais de caju e mamona e de óleos essenciais de cravo, eugenol, timol e vanilina contra bactérias Gram-negativas e Gram-positivas no rúmen de bovinos

A preocupação pública com o uso rotineiro de antibióticos e ionóforos na dieta de ruminantes aumentou devido ao surgimento de bactérias resistentes aos antibióticos que podem representar riscos à saúde humana. Assim, esforços têm sido empregados para o desenvolvimento de compostos alternativos para substituir antibióticos e ionóforos na dieta de ruminantes. Este estudo foi realizado para avaliar os efeitos in vitro de uma mistura contendo óleos vegetais de caju e mamona e óleos essenciais de cravo-da-índia, eugenol, timol e vanilina sobre a atividade das bactérias Gram-negativas e Gram-positivas presentes no rúmen. O experimento foi elaborado de forma que cada bactéria fosse exposta às doses de 1,5, 3, 5 e 6,0 mg/mL da mistura, com seis repetições. As bactérias foram cultivadas em meio M2 de Hobson em tubos Hungate. A atividade antimicrobiana foi avaliada em espectrofotômetro a 600 nm. As leituras foram realizadas 0, 8, 12 e 24 horas após a inoculação a 39º C. As quatro concentrações (1,5, 3,0, 4,5 e 6,0 mg/mL) da mistura de óleos vegetais e essenciais inibiram o crescimento da Prevotella albensis, Prevotella bryantii, Prevotella ruminicola e Anaerovibrio lipolyticus 8 e 12 horas após a incubação. Para Ruminococcus albus e Ruminococcus flavefaciens a adição da mistura de óleos essenciais nas concentrações de 3,0 e 4,5 mg/mL resultou em maior impacto na dinâmica de crescimento, com redução na densidade óptica após 12 h de incubação. Em conclusão, os resultados mostram que a ação combinada dos óleos vegetais e essenciais influencia o crescimento de bactérias gram-negativas e gram-positivas e pode ser usado como um modulador ruminal. Deste modo, o estudo contribui com novas informações sobre a ação combinada dos óleos vegetais e essenciais como agentes antimicrobianos na produção de ruminantes.

In silico Structural, Functional and Phylogenetic Analyses of cellulase from Ruminococcus albus

Background Cellulose is the primary component of the plant cell wall and an important source of energy for the ruminant and microbial protein synthesis in the rumen. Cell wall content is digested by anaerobic fermentation activity mainly of bacteria belonging to species Fibrobacter succinogenes, Ruminicoccus albus, Ruminococcus flavefaciens, and Butyrivibrio fibrisolvens. Bacteria belonging to the species Ruminococcus albus contain cellulosomes that enable it to adhere to and digest cellulose, and its genome encodes cellulases and hemicellulases. This study aimed to perform an in silico comparative characterization and functional analysis of cellulase from Ruminococcus albus to explore physicochemical properties and to estimate primary, secondary, and tertiary structure using various bio-computational tools. The protein sequences of cellulases belonging to 6 different Ruminococcus albus strains were retrieved using UniProt. In in silico composition of amino acids, basic physicochemical characteristics were analyzed using ProtParam and Protscale. Multiple sequence alignment of retrieved sequences was performed using Clustal Omega and the phylogenetic tree was constructed using Mega X software. Bioinformatics tools are used to better understand and determine the 3D structure of cellulase. The predicted model was refined by ModRefiner. Structure alignment between the best-predicted model and the template is applied to evaluate the similarity between structures. Results In this study are demonstrated several physicochemical characteristics of the cellulase enzyme. The instability index values indicate that the proteins are highly stable. Proteins are dominated by random coils and alpha helixes. The aliphatic index was higher than 71 providing information that the proteins are highly thermostable. No transmembrane domain was found in the protein, and the enzyme is extracellular and moderately acidic. The best tertiary structure model of the enzyme was obtained by the use of Raptor X, which was refined by ModRefiner. Raptor X suggested the 6Q1I_A as one of the best homologous templates for the predicted 3D protein structure. Ramachandran plot analysis showed that 90.1% of amino acid residues are within the most favored regions. Conclusions This study provides for the first time insights about the physicochemical properties, structure, and function of cellulase, from Ruminococcus albus, that will help for detection and identification of such enzyme in vivo or in silico.

In Vivo Competitions between Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminoccus albus in a Gnotobiotic Sheep Model Revealed by Multi-Omic Analyses

ABSTRACT Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens are the three predominant cellulolytic bacterial species found in the rumen. In vitro studies have shown that these species compete for adherence to, and growth upon, cellulosic biomass. Yet their molecular interactions in vivo have not heretofore been examined. Gnotobiotically raised lambs harboring a 17-h-old immature microbiota devoid of culturable cellulolytic bacteria and methanogens were inoculated first with F. succinogenes S85 and Methanobrevibacter sp. strain 87.7, and 5 months later, the lambs were inoculated with R. albus 8 and R. flavefaciens FD-1. Longitudinal samples were collected and profiled for population dynamics, gene expression, fibrolytic enzyme activity, in sacco fibrolysis, and metabolite profiling. Quantitative PCR, metagenome and metatranscriptome data show that F. succinogenes establishes at high levels initially but is gradually outcompeted following the introduction of the ruminococci. This shift resulted in an increase in carboxymethyl cellulase (CMCase) and xylanase activities but not in greater fibrolysis, suggesting that F. succinogenes and ruminococci deploy different but equally effective means to degrade plant cell walls. Expression profiles showed that F. succinogenes relied upon outer membrane vesicles and a diverse repertoire of CAZymes, while R. albus and R. flavefaciens preferred type IV pili and either CBM37-harboring or cellulosomal carbohydrate-active enzymes (CAZymes), respectively. The changes in cellulolytics also affected the rumen metabolome, including an increase in acetate and butyrate at the expense of propionate. In conclusion, this study provides the first demonstration of in vivo competition between the three predominant cellulolytic bacteria and provides insight on the influence of these ecological interactions on rumen fibrolytic function and metabolomic response. IMPORTANCE Ruminant animals, including cattle and sheep, depend on their rumen microbiota to digest plant biomass and convert it into absorbable energy. Considering that the extent of meat and milk production depends on the efficiency of the microbiota to deconstruct plant cell walls, the functionality of predominant rumen cellulolytic bacteria, Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens, has been extensively studied in vitro to obtain a better knowledge of how they operate to hydrolyze polysaccharides and ultimately find ways to enhance animal production. This study provides the first evidence of in vivo competitions between F. succinogenes and the two Ruminococcus species. It shows that a simple disequilibrium within the cellulolytic community has repercussions on the rumen metabolome and fermentation end products. This finding will have to be considered in the future when determining strategies aiming at directing rumen fermentations for animal production.

Regulatory Properties of the ADP-Glucose Pyrophosphorylase from the ClostridialFirmicutesMemberRuminococcus albus

ABSTRACTADP-glucose pyrophosphorylase fromFirmicutesis encoded by two genes (glgCandglgD) leading to a heterotetrameric protein structure, unlike those in other bacterial phyla. The enzymes from two groups ofFirmicutes,BacillalesandLactobacillales, present dissimilar kinetic and regulatory properties. Nevertheless, no ADP-glucose pyrophosphorylase fromClostridiales, the third group inFirmicutes, has been characterized. For this reason, we cloned theglgC andglgD genes fromRuminococcus albus. Different quaternary forms of the enzyme (GlgC, GlgD, and GlgC/GlgD) were purified to homogeneity and their kinetic parameters were analyzed. We observed that GlgD is an inactive monomer when expressed alone but increased the catalytic efficiency of the heterotetramer (GlgC/GlgD) compared to the homotetramer (GlgC). The heterotetramer is regulated by fructose-1,6-bisphosphate, phosphoenolpyruvate, and NAD(P)H. The first characterization of theBacillalesenzyme suggested that heterotetrameric ADP-glucose pyrophosphorylases fromFirmicuteswere unregulated. Our results, together with data fromLactobacillales, indicate that heterotetramericFirmicutesenzymes are mostly regulated. Thus, the ADP-glucose pyrophosphorylase fromBacillalesseems to have distinctive insensitivity to regulation.IMPORTANCEThe enzymes involved in glycogen synthesis fromFirmicuteshave been less characterized in comparison with other bacterial groups. We performed kinetic and regulatory characterization of the ADP-glucose pyrophosphorylase fromRuminococcus albus. Our results showed that this protein that belongs to different groups fromFirmicutes(Bacillales,Lactobacillales, andClostridiales) presents dissimilar features. This study contributes to the understanding of how this critical enzyme for glycogen biosynthesis is regulated in theFirmicutesgroup, whereby we propose that these heterotetrameric enzymes, with the exception ofBacillales, are allosterically regulated. Our results provide a better understanding of the evolutionary relationship of this enzyme family inFirmicutes.