Serum Metabolome Analysis Identified Amino-Acid Metabolism Associated With Pain in People With Symptomatic Knee Osteoarthritis - A Cross-Sectional Study.

Osteoarthritis (OA) is the most common arthritis affecting synovial joints such as knees and hips of millions of people globally. Usage-related joint pain and reduced function are the most common symptoms experienced by people with OA. To improve pain management, there is a need to identify validated biomarkers predicting therapeutic responses in targeted clinical trials. Our study aimed to identify the metabolic biomarkers for pain and pressure pain detection thresholds (PPTs) in participants with knee pain and symptomatic OA using metabolic phenotyping. Metabolite and cytokine measurements were done on serum samples using LC-MS/MS (liquid gas chromatography integrated magnetic resonance mass spectrometry) and Human Proinflammatory panel 1 kit respectively. Regression analysis was done in a test (n = 75) and replication study (n = 79) to investigate the metabolites associated with current knee pain scores and pressure pain detection thresholds (PPTs). Meta-analysis and correlation were done estimating precision of associated metabolites and identifying relationship between significant metabolites and cytokines respectively. Acyl ornithine, carnosine, cortisol, cortisone, cystine, DOPA, glycolithocholic acid sulphate (GLCAS), phenylethylamine (PEA) and succinic acid were found to be significantly (FDR <.1) associated with pain scores in meta-analysis of both studies. IL-10, IL-13, IL-1ß, IL2, IL8 and TNF-α were also found to be associated with the significant metabolites. Significant associations of these metabolites and inflammatory markers with knee pain suggests that targeting relevant pathways of amino acid and cholesterol metabolism may modulate cytokines and these could be targeted as novel therapeutics development to improve knee pain and OA management. PERSPECTIVE: Foreseeing the global burden of knee pain in Osteoarthritis (OA) and adverse effects of current pharmacological therapies, this study is envisaged to investigate serum metabolites and molecular pathways involved in knee pain. The replicated metabolites in this study suggests targeting amino-acid pathways for better management of OA knee pain.

The Vaginal Microbial Signatures of Preterm Birth Delivery in Indian Women.

Background: The incidence of preterm birth (PTB) in India is around 13%. Specific bacterial communities or individual taxon living in the vaginal milieu of pregnant women is a potential risk factor for PTB and may play an important role in its pathophysiology. Besides, bacterial taxa associated with PTB vary across populations. Objective: Conduct a comparative analysis of vaginal microbiome composition and microbial genomic repertoires of women who enrolled in the Interdisciplinary Group for Advanced Research on Birth Outcomes - A DBT India Initiative (GARBH-Ini) pregnancy cohort to identify bacterial taxa associated with term birth (TB) and PTB in Indian women. Methods: Vaginal swabs were collected during all three trimesters from 38 pregnant Indian women who delivered spontaneous term (n=20) and preterm (n=18) neonates. Paired-end sequencing of V3-V4 region of 16S rRNA gene was performed using the metagenomic DNA isolated from vaginal swabs (n=115). Whole genome sequencing of bacterial species associated with birth outcomes was carried out by shotgun method. Lactobacillus species were grown anaerobically in the De Man, Rogosa and Sharpe (MRS) agar culture medium for isolation of genomic DNA and whole genome sequencing. Results: Vaginal microbiome of both term and preterm samples reveals similar alpha diversity indices. However, significantly higher abundance of Lactobacillus iners (p-value All_Trimesters<0.02), Megasphaera sp (p-value1st_Trimester <0.05), Gardnerella vaginalis (p-value2nd_Trimester= 0.01) and Sneathia sanguinegens (p-value2nd_Trimester <0.0001) were identified in preterm samples whereas higher abundance of L. gasseri (p-value3rd_Trimester =0.010) was observed in term samples by Wilcoxon rank-sum test. The relative abundance of L. iners, and Megasphaera sp. were found to be significantly different over time between term and preterm mothers. Analyses of the representative genomes of L. crispatus and L. gasseri indicate presence of secretory transcriptional regulator and several ribosomally synthesized antimicrobial peptides correlated with anti-inflammatory condition in the vagina. These findings indicate protective role of L. crispatus and L. gasseri in reducing the risk of PTB. Conclusion: Our findings indicate that the dominance of specific Lactobacillus species and few other facultative anaerobes are associated with birth outcomes.

Gut Microbial Profile Is Associated With Residential Settings and Not Nutritional Status in Adults in Karnataka, India.

Undernutrition is a leading contributor to disease and disability in people of all ages. Several studies have reported significant association between nutritional status and gut microbiome composition but other factors such as demographic settings may also influence the adult microbiome. The relationship between undernourishment and gut microbiome in adults has not been described to date. In this study, we compared the gut microbiome in fecal samples of 48 individuals, from two demographic settings (rural and urban slum) in Karnataka, India using 16S rRNA sequencing. Nutritional status was assessed based on BMI, with a BMI of < 18.5 kg/m2 classified as undernourished, and a BMI in the range 18.5-25 kg/m2 as nourished. We analyzed 25 individuals from rural settings (12 undernourished and 13 nourished) and 23 individuals from urban slum settings (11 undernourished and 12 nourished). We found no significant difference in overall gut microbial diversity (Shannon and Unweighted UniFrac) between undernourished and nourished individuals in either geographical settings, however, microbial taxa at the phylum level (i.e., Firmicutes and Proteobacteria) and beta diversity (unweighted UniFrac) differed significantly between the rural and urban slum settings. By predicting microbial function from 16S data profiling we found significant differences in metabolic pathways present in the gut microbiota from people residing in different settings; specifically, those related to carbohydrate and lipid metabolism. The weighted sum of the KEGG Orthologs associated with carbohydrate metabolism (Spearman's correlation coefficient, ρ = -0.707, p < 0.001), lipid metabolism (Spearman's correlation coefficient, ρ = -0.330, p < 0.022) and biosynthesis of secondary metabolites (Spearman's correlation coefficient, ρ = -0.507, p < 0.001) were decreased in the urban slum group compared to the rural group. In conclusion, we report that the geographical location of residence is associated with differences in gut microbiome composition in adults. We found no significant differences in microbiome composition between nourished and undernourished adults from urban slum or rural settings in India.

Vaginal Microbiome of Pregnant Indian Women: Insights into the Genome of Dominant Lactobacillus Species.

The trillions of microorganisms residing in the human body display varying degrees of compositional and functional diversities within and between individuals and contribute significantly to host physiology and susceptibility to disease. Microbial species present in the vaginal milieu of reproductive age women showed a large personal component and varies widely in different ethnic groups at the taxonomic, genomic, and functional levels. Lactobacillus iners, L. crispatus, L. gasseri, L. jensenii, and L. johnsonii are most frequently detected bacterial species in the vaginal milieu of reproductive age women. However, we currently lack (i) an understanding of the baseline vaginal microbiota of reproductive age Indian women, (ii) the extent of taxonomic and functional variations of vaginal microbiota between individuals and (iii) the genomic repertoires of the dominant vaginal microbiota associated with the Indian subjects. In our study, we analyzed the metagenome of high vaginal swab (HVS) samples collected from 40 pregnant Indian women enrolled in the GARBH-Ini cohort. Composition and abundance of bacterial species was characterized by pyrosequencing 16S rRNA gene. We identified 3067 OTUs with ≥ 10 reads from four different bacterial phyla. Several species of lactobacilli were clustered into three community state types (CSTs). L. iners, L. crispatus, L. gasseri, and L. jensenii are the most frequently detected Lactobacillus species in the vaginal environment of Indian women. Other than Lactobacillus, several species of Halomonas were also identified in the vaginal environment of most of the women sampled. To gain genomic and functional insights, we isolated several Lactobacillus species from the HVS samples and explored their whole genome sequences by shotgun sequencing. We analyzed the genome of dominant Lactobacillus species, L. iners, L. crispatus, L. gasseri, and L. paragesseri to represent the CSTs and identify functions that may influence the composition of complex vaginal microbial ecology. This study reports for the first time the vaginal microbial ecology of Indian women and genomic insights into L. iners, L. crispatus, L. gasseri, and L. paragesseri commonly found in the genital tract of reproductive age women.

Molecular Insights into Antimicrobial Resistance Traits of Commensal Human Gut Microbiota.

Antimicrobial resistance (AMR) among bacterial species that resides in complex ecosystems is a natural phenomenon. Indiscriminate use of antimicrobials in healthcare, livestock, and agriculture provides an evolutionary advantage to the resistant variants to dominate the ecosystem. Ascendency of resistant variants threatens the efficacy of most, if not all, of the antimicrobial drugs commonly used to prevent and/or cure microbial infections. Resistant phenotype is very common in enteric bacteria. The most common mechanisms of AMR are enzymatic modifications to the antimicrobials or their target molecules. In enteric bacteria, most of the resistance traits are acquired by horizontal gene transfer from closely or distantly related bacterial population. AMR traits are generally linked with mobile genetic elements (MGEs) and could rapidly disseminate to the bacterial species through horizontal gene transfer (HGT) from a pool of resistance genes. Although prevalence of AMR genes among pathogenic bacteria is widely studied in the interest of infectious disease management, the resistance profile and the genetic traits that encode resistance to the commensal microbiota residing in the gut of healthy humans are not well-studied. In the present study, we have characterized AMR phenotypes and genotypes of five dominant commensal enteric bacteria isolated from the gut of healthy Indians. Our study revealed that like pathogenic bacteria, enteric commensals are also multidrug-resistant. The genes encoding antibiotic resistance are physically linked with MGEs and could disseminate vertically to the progeny and laterally to the distantly related microbial species. Consequently, the AMR genes present in the chromosome of commensal gut bacteria could be a potential source of resistance functions for other enteric pathogens.

Analysis of the Gut Microbiome of Rural and Urban Healthy Indians Living in Sea Level and High Altitude Areas.

The diversity and basic functional attributes of the gut microbiome of healthy Indians is not well understood. This study investigated the gut microbiome of three Indian communities: individuals residing in rural and urban (n = 49) sea level Ballabhgarh areas and in rural high altitude areas of Leh, Ladakh in North India (n = 35). Our study revealed that the gut microbiome of Indian communities is dominated by Firmicutes followed by Bacteroidetes, Actinobateria and Proteobacteria. Although, 54 core bacterial genera were detected across the three distinct communities, the gut bacterial composition displayed specific signatures and was observed to be influenced by the topographical location and dietary intake of the individuals. The gut microbiome of individuals living in Leh was observed to be significantly similar with a high representation of Bacteroidetes and low abundance of Proteobacteria. In contrast, the gut microbiome of individuals living in Ballabhgarh areas harbored higher number of Firmicutes and Proteobacteria and is enriched with microbial xenobiotic degradation pathways. The rural community residing in sea level Ballabhgarh areas has unique microbiome characterized not only by a higher diversity, but also a higher degree of interindividual homogeneity.

An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples.

To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed.

Effect of LexA on Chromosomal Integration of CTXϕ in Vibrio cholerae.

UNLABELLED: The genesis of toxigenic Vibrio cholerae involves acquisition of CTXϕ, a single-stranded DNA (ssDNA) filamentous phage that encodes cholera toxin (CT). The phage exploits host-encoded tyrosine recombinases (XerC and XerD) for chromosomal integration and lysogenic conversion. The replicative genome of CTXϕ produces ssDNA by rolling-circle replication, which may be used either for virion production or for integration into host chromosome. Fine-tuning of different ssDNA binding protein (Ssb) levels in the host cell is crucial for cellular functioning and important for CTXϕ integration. In this study, we mutated the master regulator gene of SOS induction, lexA, of V. cholerae because of its known role in controlling levels of Ssb proteins in other bacteria. CTXϕ integration decreased in cells with a ΔlexA mutation and increased in cells with an SOS-noninducing mutation, lexA (Ind(-)). We also observed that overexpression of host-encoded Ssb (VC0397) decreased integration of CTXϕ. We propose that LexA helps CTXϕ integration, possibly by fine-tuning levels of host- and phage-encoded Ssbs. IMPORTANCE: Cholera toxin is the principal virulence factor responsible for the acute diarrheal disease cholera. CT is encoded in the genome of a lysogenic filamentous phage, CTXϕ. Vibrio cholerae has a bipartite genome and harbors single or multiple copies of CTXϕ prophage in one or both chromosomes. Two host-encoded tyrosine recombinases (XerC and XerD) recognize the folded ssDNA genome of CTXϕ and catalyze its integration at the dimer resolution site of either one or both chromosomes. Fine-tuning of ssDNA binding proteins in host cells is crucial for CTXϕ integration. We engineered the V. cholerae genome and created several reporter strains carrying ΔlexA or lexA (Ind(-)) alleles. Using the reporter strains, the importance of LexA control of Ssb expression in the integration efficiency of CTXϕ was demonstrated.

A novel, broad-range, CTXΦ-derived stable integrative expression vector for functional studies.

CTXΦ, a filamentous vibriophage encoding cholera toxin, uses a unique strategy for its lysogeny. The single-stranded phage genome forms intramolecular base-pairing interactions between two inversely oriented XerC and XerD binding sites (XBS) and generates a functional phage attachment site, attP(+), for integration. The attP(+) structure is recognized by the host-encoded tyrosine recombinases XerC and XerD (XerCD), which enables irreversible integration of CTXΦ into the chromosome dimer resolution site (dif) of Vibrio cholerae. The dif site and the XerCD recombinases are widely conserved in bacteria. We took advantage of these conserved attributes to develop a broad-host-range integrative expression vector that could irreversibly integrate into the host chromosome using XerCD recombinases without altering the function of any known open reading frame (ORF). In this study, we engineered two different arabinose-inducible expression vectors, pBD62 and pBD66, using XBS of CTXΦ. pBD62 replicates conditionally and integrates efficiently into the dif of the bacterial chromosome by site-specific recombination using host-encoded XerCD recombinases. The expression level of the gene of interest could be controlled through the PBAD promoter by modulating the functions of the vector-encoded transcriptional factor AraC. We validated the irreversible integration of pBD62 into a wide range of pathogenic and nonpathogenic bacteria, such as V. cholerae, Vibrio fluvialis, Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Gene expression from the PBAD promoter of integrated vectors was confirmed in V. cholerae using the well-studied reporter genes mCherry, eGFP, and lacZ.