A comparative analysis of gut microbial dysbiosis by select antibiotics and DSS to understand the effects of perturbation on the host immunity and metabolism.

AIMS: Balanced gut microbial composition of the host plays a crucial role in maintaining harmony among various physiological processes to maintain physiological homeostasis. Immunity and metabolism are the two physiologies mainly controlled by the gut microbiota. Reports suggested that gut microbial composition and diversity alteration are the leading causes of the host's healthy homeostasis alteration or a diseased state. The extent of gut perturbation depends on the perturbing agents' strength, chemical nature, and mode of action. In the current report, we have studied the effects of different perturbing agents on gut microbial dysbiosis and its impact on host immunity and metabolism. MATERIALS AND METHODS: We studied the perturbation of gut microbial composition and diversity using next-generation sequencing and further investigated the changes in host immune and metabolic responses. KEY FINDINGS: Enrichment or abolition of a particular phylum or genus depended on the perturbing agents. In the current study, treatment with neomycin yielded an increase in the Bacteroidetes phylum. Vancomycin treatment caused a significant rise in Verrucomicrobia and Proteobacteria phyla. The treatment with AVNM and DSS caused a substantial increase in the Proteobacteria phylum. The gut microbial diversity was also lowest in AVNM treated group. The altered gut microbial composition ultimately altered the immune responses at localized and systemic levels of the host. Gut dysbiosis also changed the systemic level of SCFAs. SIGNIFICANCE: This study will help us understand how the enrichment of a particular phylum and genus maintains the host's immune responses and metabolism.

Cost Effective Method for gDNA Isolation from the Cecal Content and High Yield Procedure for RNA Isolation from the Colonic Tissue of Mice.

Microbiome studies are quickly gaining momentum. Since most of the resident microbes (consisting of bacteria, fungi, and viruses) are difficult to culture, sequencing the microbial genome is the method of choice to characterize them. It is therefore important to have efficient methodology for gDNA isolation of gut microbes. Mouse models are widely used to understand human disease etiology while avoiding human ethics-related complications. However, the widely used kit-based methods are costly, and sometimes yields (in terms of quality and quantity) are sub-optimal. To overcome this problem, we developed a straightforward, standardized DNA isolation procedure from mouse cecal content for further microbiome-related studies. The reagents we used to standardize the procedure are readily available even in a not-so-well-equipped laboratory, and the reagents are not expensive. The yield and quality of the DNA are also better than those obtained by the readily available kit-based methods. Additionally, we modified the kit-based method of RNA isolation from the colon tissue sample of the mouse for better yield. Churning the tissue with liquid nitrogen at the beginning of the procedure improves RNA quality and quantity. Graphical abstract.

Differential colitis susceptibility of Th1- and Th2-biased mice: A multi-omics approach.

The health and economic burden of colitis is increasing globally. Understanding the role of host genetics and metagenomics is essential to establish the molecular basis of colitis pathogenesis. In the present study, we have used a common composite dose of DSS to compare the differential disease severity response in C57BL/6 (Th1 biased) and BALB/c (Th2 biased) mice with zero mortality rates. We employed multi-omics approaches and developed a newer vector analysis approach to understand the molecular basis of the disease pathogenesis. In the current report, comparative transcriptomics, metabonomics, and metagenomics analyses revealed that the Th1 background of C57BL/6 induced intense inflammatory responses throughout the treatment period. On the contrary, the Th2 background of BALB/c resisted severe inflammatory responses by modulating the host's inflammatory, metabolic, and gut microbial profile. The multi-omics approach also helped us discover some unique metabolic and microbial markers associated with the disease severity. These biomarkers could be used in diagnostics.

Lactobacillus rhamnosus GG reverses mortality of neonatal mice against Salmonella challenge.

Pathogenic infection is one of the major causes of death in newborns. Antibiotic based therapies are still the major mode of treatment for infection. Increased usage of antibiotics leads to selective evolution of microorganisms and causes diseases in adulthood. Attempts to develop alternatives to antibiotics did not yield much success. A recent viable trend is to identify novel probiotics that could alleviate problems associated with over usage of antibiotics. We screened three different Lactobacillus species to establish their efficacy in neonates in protecting against Salmonella challenge. The methodologies employed are metagenomics, metabonomics, transcriptional profiling, molecular assays and behavioral studies. Among the three probiotics used, only Lactobacillus rhamnosus GG (LGG) treatment of the neonates resulted in rescuing of 80% of the Salmonella-infected mice. We have shown that LGG (MTCC #1408) can prevent Salmonella mediated infection in neonates. In the current report, results from histopathology, gene expression, neutrophil infiltration, metabolite and metataxonomic profiling, and protein level data suggested that LGG treatment of the neonates enhanced anti-inflammatory cytokine expression and increased the gut barrier function. The current report establishes the potential use of LGG in clinical intervention of infectious diseases.