Study on Microbial Communities Modulation to Mitigate Obesity Related Gut Microbiota Dysbiosis

Abstract

In the last few decades, obesity has become a global health problem affecting adults, teenagers, and children of both genders and has been linked with chronic metabolic diseases. The intestinal microbiota has appeared as a potent and vital endogenic feature that affects energy homeostasis and human health. The idea of gut microbiota dysbiosis mitigation has given a new insight into the treatment of obesity and metabolic diseases. Many options are used to modulate intestinal dysbiosis microbiota, among which probiotics are the most used option. However, it is reported that a single probiotic candidate cannot modulate the complex microbial communities of the gut. In this context, a multi-strains probiotic community developed from individual strains was used for mitigation of obesity-related gut microbiota dysbiosis in simulated CoMiniGut model. Initially, 143 bacterial isolates from 37 Dahi (a traditional fermented milk product of Pakistan) samples were chosen for microscopic, macroscopic, and phenotypic characterization. This returned 112 Gram-positive, immotile, catalase-oxidase negative candidates. Which further were evaluated for acid production, lactose fermentation, and curd formation. Seventy isolates were able to produce acids and utilize lactose. Their identification was confirmed through Rep-Seq, which were then exposed to low pH and high bile salts. Of these, 49 isolates survived, were evaluated for in vitro cell surface hydrophobicity, auto-aggregation, and survival in vitro simulated gastrointestinal fluids. All the 49 isolates presented in vitro cell surface hydrophobicity and auto-aggregation above 50% but only 22 isolates survived in the in vitro simulated gastrointestinal fluids. These isolated were evaluated for antagonistic potential against some common foodborne pathogens (Bacillus subtilis ATCC 19659, Escherichia coli ATCC 25922, Salmonella enterica ATCC 27870, Pseudomonas aeruginosa ATCC 15422, Staphylococcus aureus ATCC 6538, Streptococcus pneumoniae ATCC 49619) and fungi (Geotrichum candidum QAUGC01, Aspergillus flavus QAUAF01 and Candida albican ATTC 90028), antibiotic susceptibility, anti-oxidative and in vitro cholesterol assimilation abilities. The isolates exhibited good antibacterial and antifungal activities and had in vitro cholesterol assimilation activity of 50–99%. These isolates were assessed for hemolytic, deoxyribonuclease, and decarboxylase activities and found negative for the tested characteristics. Two multi-strains communities were prepared from the isolates, one was consisting of 49 strains that survived in acids, bile salts and presented the cell surface hydrophobicity and autoaggregation and was named as multi strains probiotic community 1 (MSPC-1). The second community consisted of 22 strains that along with survival in acids and DRSML QAU Study on Microbial Communities Modulation to Mitigate Obesity Related Gut Microbiota Dysbiosis V bile survived well in the simulated gastrointestinal fluids and was named as multi strains probiotic community 2 (MSPC-2). These communities were evaluated and compared for in vitro synergistic growth, cell surface hydrophobicity, auto-aggregation, antibacterial activity, and cholesterol assimilation. In comparison to MSPC-1 MSPC-2 presented significantly high growth, high in vitro cell surface hydrophobicity (98%), auto-aggregation (83%), antipathogenic activity, and in vitro cholesterol assimilation (88%). The MSPC-1 was excluded from further characterization while survival of MSPC-2 was evaluated in the small intestine (TSI). The MSPC-2 survived the harsh conditions of the small intestine. Whole genomes of all the MSPC-2 isolates were sequenced and analyzed. The size of the genomes ranged from 1.8 to 2.4 Mb, GC contents were between 31 and 52%. On genome mining, it was found that none of the strains is extreme-drug resistant, bacteriocin genes were found in each genome, with no virulence and pathogenic genes. CRISPR genes were also present, indicating the immunity of the isolates to transfer elements. The MSPC-2 was used for the modulation of obesity-related gut microbiota obtained from Pakistani-origin obese volunteers (n=3) using CoMiniGut. It was observed that the probiotic community improved the bacterial count and richness of the experimental group. Alpha diversity was observed to be shifted from 0.7 to 0.8 and from 0.6 to 0.7 respectively when measured through Shannon and Simpson indexes. Beneficial bacterial genera were increased i.e., Bifidobacterium increased from 3% to 16.3%, Coprococcus increased from 0.79 to 1.34%, Lactobacillus from 5.38% to 13.71%, and Prevotella from 1.73 to 903%. Some bacterial genera, that contain mostly pathogenic members were decreased in response to probiotic treatment i.e., Actinomyces decreased from 7.34% to 2.23%, Anaerostipes decreased from 2.70% to 1.61%, Clostridium decreased from 9.68% to 7.47%, Dailister from 7.75% to 0.89%, Enterobacter from 5.15% to 0.08%, Escherichia from 1.07% to 0.01% and Sutterella from 2.31% to 0.12%. Furthermore, looking at the functionality, genes responsible for carbohydrate transport and metabolism, nucleotide transport and metabolism, replication, recombination and repair, translation, ribosomal structure, and biogenesis were increased for obese microbiota in response to probiotic treatment. Among the microbial metabolites, butyrate was specifically improved in both the groups with probiotic treatment. The MSPC-2 can be used as an option for modulation of dysbiosis gut microbiota, after evaluation in the in-vivo trails.