In-silico analysis of Covid-19 genome sequences of Indian origin: Impact of mutations in identification of SARS-Co-V2.

Covid-19 disease caused by SARS-CoV-2 is still being transmitted in developed and developing countries irrespective of healthcare setups. India with 1.3 billion people in the world is severely affected by Covid-19 with 11.3 million cases and 157 000 deaths so far. We have assessed the mismatches in WHO recommended rRT-PCR assays primer and probe binding regions against SARS-CoV-2 Indian genome sequences through in-silico bioinformatics analysis approach. Primers and probe sequences belonging to CN-CDC-ORF1ab from China and HKU-ORF1b from Hong Kong targeting ORF1ab gene while NIH-TH-N from Thailand, HKU-N from Hong Kong and US-CDCN-2 from USA targeting N genes displayed accurate matches (>98.3%) with the 2019 novel corona virus sequences from India. On the other hand, none of the genomic sequences displayed exact match with the primer/probe sequences belonging to Charité-ORF1b from Germany targeting ORF1ab gene. We think it will be worthwhile to release this information to the clinical and medical communities working in Indian Covid-19 frontline taskforce to tackle the recently emerging Covid-19 outbreaks as of March-2021.

Comparative genomics of ocular Pseudomonas aeruginosa strains from keratitis patients with different clinical outcomes.

The several virulence and drug-resistance mechanisms of Pseudomonas aeruginosa responsible for poor clinical outcomes in keratitis patients remain largely unknown. Here, we investigated the distribution of virulence factors and drug resistance by genes, mutations, efflux-pump systems of P. aeruginosa strains from keratitis patients with different clinical outcomes, included of whole-genome sequenced and annotated our five P. aeruginosa strains. Of the large number of virulence genes detected in all the genomes, MDR/XDR strains carry exoU and non-MDR strains carry exoS exotoxin of the type III secretion system, considered as main contributors of keratitis pathogenesis. However, several strain-specific virulence and resistance genes were detected in keratitis strains with poor outcome. Mainly, the flagellar genes fliC and fliD detected in the exoS carrying strains, reported to alter the host immune response, might impact the clinical outcome. This study may provide new insights into the genome of ocular strains and requires further functional studies.

Mechanisms of Fluoroquinolone and Aminoglycoside Resistance in Keratitis-Associated Pseudomonas aeruginosa.

Global emergence of multidrug resistant (MDR) strains limits therapeutic efficacy in Pseudomonas aeruginosa corneal ulcers. Identifying the primary causal factors of resistance shall improve clinical management. In this study, we sought to identify the underlying mechanisms of fluoroquinolone and aminoglycoside resistance in MDR, non-MDR, and drug susceptible P. aeruginosa (n = 19) strains obtained from keratitis patients. Phenotypic assays were performed to study the bacterial growth kinetics, efflux, permeability, and biofilm formation. Mutational alteration of target genes (DNA sequencing), relative expression of efflux system genes (real time PCR), and detection of aminoglycoside modifying enzyme (AME) genes (PCR) were done by molecular methods. We repeatedly found the mutations in quinolone resistance determining region of fluoroquinolone target genes, gyrA and parC, and the presence of AME genes, aph(3″)-I and aph(6)-I, in all MDR isolates. Furthermore, the MDR isolates were largely characterized by slower growth, cytotoxic type III secretion system genotype, better biofilm-forming ability, and the presence of additional AME genes. The non-MDR isolates were resensitized upon inhibition of active efflux or enhancement of membrane permeability. Altogether this study highlights target gene alteration and enzymatic drug modification as the major mechanisms of quinolone and aminoglycoside resistance in P. aeruginosa keratitis isolates.