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Background: Campylobacter is a well-known bacterial pathogen for triggering acute gastroenteritis in humans both in developed and developing countries. This organism is highly resistant to fluoroquinolones. Macrolides are very much useful for the treatment of campylobacteriosis when clinical therapy is necessary. However, increasing resistance to azithromycin, a potent macrolide has been reported in Campylobacter in recent years. Macrolide resistance in Campylobacter is found mainly due to point mutation in V region of 23S rRNA. Results: We have developed a PCR based assay, which can detect the azithromycin resistant and sensitive Campylobacter strains utilizing mutation responsible for the phenotype. This PCR was validated using 359 Campylobacter strains isolated from diarrhoeal patients at Kolkata, India. Antimicrobial resistance through disk diffusion method was also performed on these strains as a gold standard. Studies through sequencing analysis further confirmed the PCR result. Conclusion: This study describes a simple and rapid method for detection of mutation conferring macrolide resistance with additional feature of identification of sensitive strains. [ABSTRACT FROM AUTHOR]

Background: Worldwide, noroviruses are the leading cause of acute gastroenteritis (AGE) in people of all age groups. In India, norovirus rates between 1.4 to 44.4% have been reported. Only a very few complete norovirus genome sequences from India have been reported. Objective: To perform full genome sequencing of noroviruses circulating in India during 2017–2021, identify circulating genotypes, assess evolution including detection of recombination events. Methodology: Forty-five archived norovirus-positive samples collected between October 2017 to July 2021 from patients with AGE from two hospitals in Kolkata, India were processed for full genome sequencing. Phylogenetic analysis, recombination breakpoint analysis and comprehensive mutation analysis were also performed. Results: Full genome analysis of norovirus sequences revealed that strains belonging to genogroup (G)I were genotyped as GI.3[P13]. Among the different norovirus capsid-polymerase combinations, GII.3[P16], GII.4 Sydney[P16], GII.4 Sydney[P31], GII.13[P16], GII.16[P16] and GII.17 were identified. Phylogenetic analysis confirmed phylogenetic relatedness with previously reported norovirus strains and all viruses were analyzed by Simplot. GII[P16] viruses with multiple residue mutations within the non-structural region were detected among circulating GII.4 and GII.3 strains. Comprehensive mutation analysis and selection pressure analysis of GII[P16] viruses showed positive as well as negative selection sites. A GII.17 strain (NICED-BCH-11889) had an untypeable polymerase type, closely related to GII[P38]. Conclusion: This study highlights the circulation of diverse norovirus strains in eastern India. These findings are important for understanding norovirus epidemiology in India and may have implications for future vaccine development. [ABSTRACT FROM AUTHOR]

Background: Metagenomic analysis of the gut microbiome and resistome is instrumental for understanding the dynamics of diarrheal pathogenesis and antimicrobial resistance transmission (AMR). Metagenomic sequencing of 20 diarrheal fecal samples from Kolkata was conducted to understand the core and variable gut microbiota. Five of these samples were used for resistome analysis. The pilot study was conducted to determine a microbiota signature and the source of antimicrobial resistance genes (ARGs) in the diarrheal gut. Results: 16S rRNA amplicon sequencing was performed using Illumina MiSeq platform and analysed using the MGnify pipeline. The Genome Taxonomy Database (GTDB-Tk) was used for bacterial taxonomic identification. Diarrheal etiology was determined by culture method. Phylum Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were consistently present in 20 samples. Firmicutes was the most abundant phylum in 11 samples. The Bacteroidetes/Firmicutes ratio was less than 1 in 18 samples. 584 genera were observed. 18 of these were present in all the 20 samples. Proteobacteria was the dominant phylum in 6 samples associated with Vibrio cholerae infection. Conservation of operational taxonomic units (OTUs) among all the samples indicated the existence of a core microbiome. Asymptomatic carriage of pathogens like Vibrio cholerae and Helicobacter pylori was found. Signature of Candidate phyla or "microbial dark matter" occurred. Significant correlation of relative abundance of bacterial families of commensals and pathogens were found. Whole-genome sequencing (WGS) on Illumina MiSeq system and assembly of raw reads using metaSPAdes v3.9.1 was performed to study the resistome of 5 samples. ABRicate was used to assign ARG function. 491 resistance determinants were identified. In 80% of the samples tetracycline resistance was the most abundant resistance determinant. High abundance of ARGs against β-lactams, aminoglycosides, quinolones and macrolides was found. Eschericia sp. was the major contributor of ARGs. Conclusions: This is the first comparative study of the gut microbiome associated with different diarrheal pathogens. It presents the first catalogue of different bacterial taxa representing the core and variable microbiome in acute diarrheal patients. The study helped to define a trend in the gut microbiota signature associated with diarrhea and revealed which ARGs are abundantly present and the metagenome-assembled genomes (MAGs) contributing to AMR. [ABSTRACT FROM AUTHOR]

Background: In the Bengal Delta, research has shown that climate and cholera are linked. One demonstration of this is the relationship between interannual ocean-atmospheric oscillations such as the El Niño Southern Oscillation (ENSO) and the Indian Ocean Dipole (IOD). What remains unclear in the present literature is the nature of this relationship in the specific context of Kolkata, and how this relationship may have changed over time. Results: In this study, we analyse the changing relationship between ENSO and IOD with cholera in Kolkata over recent (1999–2019) and historical (1897–1941) time intervals. Wavelet coherence analysis revealed significant non-stationary association at 2–4 year and 4–8 year periods between cholera and both interannual timeseries during both time intervals. However, coherence was notably weakened in the recent interval, particularly with regards to ENSO, a result supported by a complementary SARIMA analysis. Similar coherence patterns with temperature indicate it could be an important mediating factor in the relationship between cholera and oscillating climate phenomena in Kolkata. Conclusions: This study reveals a shifting relationship between cholera and climate variables (ENSO and IOD) in Kolkata, suggesting a decoupling between environmental influences and cholera transmission in recent years. Our results therefore do not suggest that an intensification of ENSO is likely to significantly influence cholera in the region. We also find that the relationship between cholera and interannual climate variables is distinct to Kolkata, highlighting the spatial heterogeneity of the climate-cholera relationship even within the Bengal Delta. [ABSTRACT FROM AUTHOR]

The gut microbiome derived short chain fatty acids perform multitude of functions to maintain gut homeostasis. Here we studied how butyrate stymie enteric bacterial invasion in cell using a simplistic binary model. The surface of the mammalian cells is enriched with microdomains rich in cholesterol that are known as rafts and act as entry points for pathogens. We showed that sodium butyrate treated RAW264.7 cells displayed reduced membrane cholesterol and less cholera-toxin B binding coupled with increased membrane fluidity compared to untreated cells indicating that reduced membrane cholesterol caused disruption of lipid rafts. The implication of such cellular biophysical changes on the invasion of enteric pathogenic bacteria was assessed. Our study showed, in comparison to untreated cells, butyrate-treated cells significantly reduced the invasion of Shigella and Salmonella, and these effects were found to be reversed by liposomal cholesterol treatment, increasing the likelihood that the rafts' function against bacterial invasion. The credence of ex vivo studies found to be in concordance in butyrate fed mouse model as evident from the significant drift towards a protective phenotype against virulent enteric pathogen invasion as compared to untreated mice. To produce a cytokine balance towards anti-inflammation, butyrate-treated mice produced more of the gut tissue anti-inflammatory cytokine IL-10 and less of the pro-inflammatory cytokines TNF-α, IL-6, and IFN-γ. In histological studies of Shigella infected gut revealed a startling observation where number of neutrophils infiltration was noted which was correlated with the pathology and was essentially reversed by butyrate treatment. Our results ratchet up a new dimension of our understanding how butyrate imparts resistance to pathogen invasion in the gut. [ABSTRACT FROM AUTHOR]

Background: Eradication of Helicobacter pylori provides the most effective treatment for gastroduodenal diseases caused by H. pylori infection. Clarithromycin, a member of the macrolide family, still remains the most important antibiotic used in H. pylori eradication treatment. But the increasing prevalence of clarithromycin resistant H. pylori strains due to point mutations in the V region of the 23S rRNA, poses a great threat in treating the ailing patients. So, we aimed for PCR-mediated rapid detection of the point mutation at 2143 position of 23S rRNA gene in H. pylori that is relevant to clarithromycin resistance from culture and simultaneously from biopsy specimens to avoid the empirical treatment. Results: Newly developed PCR assay using DNA of pure culture detected point mutation in 23S rRNA gene in 21 (8.04%) of 261 clinical strains tested. The agar dilution method showed that all these 21 strains were resistant to clarithromycin indicating the perfect match of the PCR based results. Additionally, the sequencing study also identified the A to G mutation at 2143 position in 23S rRNA gene of the resistant strains only. Consequently, the newly developed Nested-ASP-PCR dealing directly with 50 biopsy specimens demonstrated 100% sensitivity and specificity with the findings of agar dilution method taken as Gold standard. Bioinformatics based analysis such as accessibility analysis and dot plot clearly stated that the base pairing probability has increased due to mutation. Computational studies revealed that the point mutation confers more stability in secondary structure due to conversion of loop to stem. Furthermore, interaction studies showed binding affinity of the CLR to the mutant type is weaker than that to the wild type. Conclusion: This assay outlines a rapid, sensitive and simple approach to identify point mutation that confers clarithromycin resistance as well as clarithromycin sensitive strains, providing rapid initiation of effective antibiotic treatment. Additionally, it is simple to adopt for hospital based diagnostic laboratories to evaluate the degree of regional clarithromycin resistance from biopsy specimens itself. Furthermore, in silico studies provide evidence or a signal that the prevalence of clarithromycin resistance may rise in the near future as a result of this point mutation. [ABSTRACT FROM AUTHOR]