Biology subjects

Neeravi, A. R.

Publications and source records attributed to Neeravi, A. R..

5 recordsLinked to original sources

bioRxiv2026

Development and validation of a genome-informed multiplex PCR for specific detection of typhoidal Salmonella serovars

Enteric fever caused by Salmonella enterica serovars Typhi and Paratyphi A, B and C remains a major public health burden in endemic regions. Existing molecular assays frequently demonstrate limited specificity due to cross-reactivity with non-typhoidal Salmonella (NTS). In this study, we developed and validated a genomics-informed multiplex PCR assay capable of simultaneously differentiating all four typhoidal Salmonella serovars. A curated dataset of 3,239 Salmonella genomes, including S. Typhi (n=361), S. Paratyphi A (n=453), S. Paratyphi B (n=511), S. Paratyphi C (n=62), and NTS genomes (n=1,853), was used for comparative genomic analysis. Thirty published PCR targets were evaluated in silico, followed by pangenome and SNP analyses to identify discriminatory loci for mismatch amplification mutation assay (MAMA)-based primer design. Candidate primers were validated using in silico PCR, BLASTn analysis, and laboratory testing against a panel of typhoidal Salmonella, clinical NTS isolates, and non-Salmonella bacterial pathogens. In silico evaluation demonstrated substantial cross-reactivity among many published targets, whereas SNP-informed primer design targeting staG (S. Typhi), SPA0152 (S. Paratyphi A), SPAB_03490 (S. Paratyphi B), and SPC_0571 (S. Paratyphi C) achieved predicted specificities of 98-100% while retaining high analytical sensitivity (>97%) across target genomes. Combined with a pan-Salmonella invA target, the multiplex assay precisely identified all target serovars in vitro with minimal cross-reactivity. These findings demonstrate that genomics-informed SNP-based primer design enables reliable multiplex differentiation of typhoidal Salmonella serovars and provides a scalable framework for improving enteric fever diagnosis and surveillance in endemic settings. ImportanceTyphoidal Salmonella serovars remain major causes of enteric fever in endemic regions, yet molecular differentiation from non-typhoidal Salmonella (NTS) remains challenging because of extensive genomic conservation and cross-reactivity of commonly used diagnostic targets. In this study, we combined large-scale comparative genomics of 3,239 Salmonella genomes with SNP-informed primer design to develop a multiplex PCR assay capable of simultaneously differentiating all four typhoidal serovars (S. Typhi, S. Paratyphi A, B, and C) from NTS and other non-Salmonella pathogens. Unlike conventional gene-content-based assays, this approach incorporated lineage-specific SNPs and mismatch amplification strategies to improve specificity while maintaining high analytical sensitivity. In silico evaluation demonstrated high diagnostic performance across diverse global lineages, while in vitro testing confirmed accurate serovar-level discrimination with minimal cross-reactivity. These findings demonstrate the value of population-scale genomics for molecular assay development and provide a scalable framework for improving diagnosis and surveillance of enteric fever in endemic settings.

molecular biology↗

bioRxiv2022

ompC/F mutations drive XDR phenotype and lineage defining super clones of E. coli: Sequential events and consequences

Multi-drug resistant Escherichia coli is an increasing public health problem. Though, PBP3 insertions with blaNDM, blaCMY and blaOXA-48 like is restricted to South-East Asia with few reports from USA. The study suggests ompC/F variants as a core factor to classify ESBL (E), non-ESBL (NE), and ESBL with PBP3 and carbapenemases (EPBP3) clones. EPBP3 results in treatment complication, as most of the time, E. coli with PBP3 insertions co-carries blaNDM (87.5%), blaCMY (96.3%) and blaOXA-48 like (88.8%) implicating it as a predisposing factor for carbapenemase gene acquirement. Cefiderocol and cefepime/zidebactam are the choice against EPBP3 E. coli. Evolutionary BEAST analysis revealed consecutive events of YRIN and YRIK insertions in PBP3 gene leading to a surge in MDR E. coli clones. Further, emergence of the super clones STs 410, 405, 167 and 617 featuring these phenotypes is a major threat for developing and developed countries, which needs close monitoring. ImportanceThe manuscript describes various E. coli resistant genotypes across the globe and their importance in the choice of antimicrobial for treatment. The study identified six clades based on ompC and ompF mutations with a strong correlation to PBP3 insertions co-carried with beta-lactamases including blaNDM. Though, the ompC and ompF mutations were reported to precede the acquisition of carbapenemases in E. coli, clade segregation based on AMR genes as observed in this study reveals the ompC and ompF genes as a potential biomarker for AMR clade identification in E. coli. Currently, cefiderocol and cefepime/zidebactam seems to be the only choice to cover the AMR mechanism mediated by PBP3 insertions. Further, emergence of the super clones STs 410, 405, 167 and 617 featuring these PBP3 phenotypes is a major threat for developing and developed countries, which needs close monitoring.

genomics↗

bioRxiv2022

Genomic rearrangements of mobile genetic elements associated with carbapenem resistance of Acinetobacter baumannii

With the excessive genome plasticity, Acinetobacter baumannii has the capability to acquire and disseminate antimicrobial resistance genes that are often associated with mobile genetic elements (MGE). Analyzing the genetic environment of resistance genes often provides valuable information on the origin, emergence, evolution and spread of resistance. Thus, we characterized the genomic features of some clinical isolates of carbapenem-resistant A. baumannii to understand the role of diverse MGE and their genetic context that are responsible for the dissemination of carbapenem resistance genes. For this, a total of 17 clinical isolates of A. baumannii obtained from multiple hospitals in India between the years 2018 and 2019 were analysed. Antimicrobial resistance determinants, genetic context of resistance genes and molecular epidemiology were studied using whole genome sequencing. A high prevalence of blaOXA-23 was observed followed by the presence of dual carbapenemase, blaOXA-23 and blaNDM. Three novel Oxford sequence types were identified. Majority of the isolates belonged to dominant clone, IC2 followed by less prevalent clones such as IC7 and IC8. Complex diverse AbaR4 like and AbGRI-like islands belonging to IC2 lineage were identified. To the best of our knowledge, this is the first study that provides a comprehensive profiling of resistance islands along with the MGE, acquired antimicrobial resistance genes and the distribution of clonal lineages of carbapenem resistant A. baumannii from India.

microbiology↗

bioRxiv2020

Mosaic antimicrobial resistance/virulence plasmid in hypervirulent ST2096 Klebsiella pneumoniae in India: The rise of a new superbug?

Hypervirulent K. pneumoniae (HvKp) is typically associated with ST23 clone; however, hvKp is also emerging from clones ST11, ST15 and ST147, which are also multi-drug resistant (MDR). Here, we aimed to characterise nine novel MDR hvKp isolates harbouring mosaic plasmids simultaneously carrying antimicrobial resistance (AMR) and virulence genes. Nine HvKp isolates obtained from hospitalised patients in southern India were characterized for antimicrobial susceptibility and hypervirulence phenotypes. All nine hvKp isolates were subjected to whole genome sequencing (WGS) using Ilumina HiSeq2500 and a subset of four were sequenced using Oxford Nanopore MinION. Among the nine isolates, seven were carbapenem-resistant, two of which carried blaNDM-5 on an IncFII plasmid and five carried blaOXA-232 on a ColKP3 plasmid. The virulence determinants were encoded in a mosaic plasmid ([~]320 Kbp) that formed as a result of its insertion in a IncFIB-IncHI1B plasmid co-integrate. The mosaic plasmid carried AMR genes (aadA2, armA, blaOXA-1, msrE, mphE, sul1 and dfrA14) in addition to rmpA2, iutA and iucABCD virulence genes. Interestingly the mosaic plasmid carried its own type IV-A3 CRISPR-cas system that is likely able to target the acquisition of IncF plasmid with the help of a traL spacer. The convergence of virulence and AMR is the biggest threat among invasive K. pneumoniae infections. However, increasing reports of the presence of mosaic plasmid carrying both AMR and virulence genes suggests MDR-hvKp isolates are no longer confined to selected clones and the containment of such isolates is very challenging. IMPORTANCEKlebsiella pneumoniae is an opportunistic pathogen that commonly associated with hospital-acquired infections in the urinary tract, respiratory tract, lung, wound sites. The organism has gained notoriety by acquiring additional genetic traits to become either hypervirulent (HV) phenotype or multidrug resistant (MDR) phenotype. Though the infections by both these phenotypes were very challenging to treat, the MDR K. pneumonia (MDR-Kp) were remained in the hospital settings while HV K. pneumonia (hvKp) strains were mostly originated from the community settings. In a recent turn of events, the evolution of MDR-Kp and hvKp has converged as both clones found to carry both MDR plasmids and virulence plasmid. These convergent strains are challenging to treat and is associated with higher mortality rate. As the recent hvKp isolates harbour mosaic plasmid encoding both AMR and virulence determinants there is a need to investigate the evolution of these pathogens. The significance of our research is in characterising the novel mosaic plasmid identified in MDR-hvKp isolates that belong sequence type (ST) 2096. Tracking the possible evolution pathway of MDR-hvKPs would greatly help in the proper surveillance and management of this superbugs. RepositoriesThe whole genome sequences of the present study isolates have been deposited in GenBank, NCBI, with accession numbers CP053765 - CP053770, CP053771 - CP053780, CP058798-CP058806, JAARNO010000001.1 - JAARNO010000005.1, JAAQSG000000000, JAARNJ000000000, JAARMH000000000 and JAAQTC000000000

genomics↗

bioRxiv2019

First Indian report on Genome-wide Comparison of Multidrug-Resistant Escherichia coli from Blood Stream Infections

BackgroundMultidrug-resistant (MDR) E. coli with extended-spectrum {beta}-lactamases (ESBLs) is becoming endemic in health care settings around the world. Baseline data on virulence and AMR of specific lineages of E. coli circulating in developing countries like India is currently lacking.\n\nMethodsWhole-genome sequencing was performed for 60 MDR E. coli isolates. Genome-wide analysis was performed at single nucleotide polymorphism (SNP) level resolution to identify the relation between the isolates in context of time, virulence and AMR determinants possessed.\n\nResultsGenome comparison revealed the presence of ST-131 global MDR and ST410 as emerging-MDR clades of E. coli. AMR gene profile for cephalosporin and carbapenem resistance differed between the clades. Genotypes blaCTX-M-15 and blaNDM-5 were common among cephalosporinases and carbapenemases, respectively. For aminoglycoside resistance, rmtB was positive for 31.7% of the isolates, of which 30% were co-harboring carbapenemases. Further, the FimH types and virulence gene profile positively correlated with the SNP based phylogeny, which also revealed the evolution of MDR clones among the study population with temporal accumulation of SNPs. The predominant clone was ST167 (blaNDM lineage) followed by ST405 (global clone ST131 equivalent) and ST410 (fast spreading high risk clone).\n\nConclusionsThis is the first report on the whole genome analysis of MDR E. coli lineages circulating in India. Data from this study will provide public health agencies a baseline portfolio of AMR and virulence in pathogenic E. coli in the region.

genomics↗