Biology subjects

Murugan, D.

Publications and source records attributed to Murugan, D..

7 recordsLinked to original sources

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

T cell Egress via Lymphatic Vessels Limits the Intratumoral T cell Repertoire in Melanoma

Antigen-specific CD8+ T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet, the mechanisms of lymphocyte transit remain poorly defined. We find that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression on effector CD8+ T cells. Only high affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8+ T cells exit the tumor, thereby limiting tumor control. CXCR4 inhibition and loss of lymphatic-specific CXCL12 boosts T cell retention and enhances response to therapeutic immune checkpoint blockade. Strategies that limit T cell egress, therefore, provide a new tool to boost immunotherapy response. One-Sentence SummaryLymphatic vessel-mediated, antigen-dependent CD8+ T cell egress limits T cell accumulation in melanomas and impairs anti-tumor immunity.

immunology↗

bioRxiv2022

An open protocol for modeling T Cell Clonotype repertoires using TCRβ CDR3 sequences

T cell receptor (TCR) repertoires can be profiled using next generation sequencing (NGS) to monitor dynamical changes in response to disease and other perturbations. Several strategies for profiling TCRs have been recently developed with different benefits and drawbacks. Genomic DNA-based bulk sequencing, however, remains the most cost-effective method to profile TCRs. The major disadvantage of this method is the need for multiplex target amplification with a large set of primer pairs with potentially very different amplification efficiencies. One approach addressing this problem is by iteratively adjusting the concentrations of the primers based on their efficiencies, and then computationally correcting any remaining bias. Yet there are no standard, publicly available protocols to process and analyze raw sequencing data generated by this method. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both approaches, we show that the concordance between bulk clonality metrics obtained from using the commercial kits and that developed herein is high. Therefore, we suggest the method presented here as an inexpensive and non-commercial alternative for measuring and monitoring adaptive dynamics in TCR clonotype repertoire.

bioinformatics↗

bioRxiv2022

Re-sensitising XDR biofilms using a combination of bacteriophage cocktails and colistin: a natural approach

Persistent antibiotic use results in the rise of antimicrobial resistance with limited or no choice for multidrug resistant (MDR) and extensively drug resistant (XDR) bacteria. This necessitates a need for alternative therapy to effectively combat clinical pathogens that are resistant to last resort antibiotics. The study investigates hospital sewage as a potential source of bacteriophages to control MDR/XDR bacterial pathogens. 81 samples were screened for phages against selected clinical pathogens. 10 phages were isolated against A. baumannii, 5 phages against K. pneumoniae and 16 phages obtained against P. aeruginosa. The novel phages were observed to be strain-specific with a complete growth inhibition of up to 6 hrs. Phage plus colistin combinations further reduced the MBEC of colistin up to 16 folds. Notably, cocktail of phages exhibited supreme efficacy with complete killing at 0.5-1 {micro}g/ml colistin concentrations. Thus, phages specific to clinical strains has a higher edge in treating nosocomial pathogens with their proven anti-biofilm efficacy. In addition, analysis of phage genomes revealed close phylogenetic relations with phages reported from Europe, China and other neighbouring countries. This study serves as a reference and can be extended to other antibiotics and phage types to assess optimum synergistic combinations to combat various drug resistant pathogens in the ongoing AMR crisis.

microbiology↗

bioRxiv2020

The influence of biofilm formation on carbapenem resistance in clinical Klebsiella pneumoniae infections: phenotype vs genome-wide analysis

Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenem-resistant (CR) K. pneumoniae are on the rise in India. The biofilm forming ability of K. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in the present study.K. pneumoniae isolates from patients admitted in critical care units with catheters and ventilators were included. K. pneumoniae (n = 72) were tested for antimicrobial susceptibility as recommended by CLSI 2019 and subjected to 96-well microtitre plate biofilm formation assay. Based on optical density at 570 nm isolates were graded as strong, moderate and weak biofilm formers. Subset of strong biofilm formers were subjected to whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Biofilm formation was compared for an association with the carbapenem susceptibility and with patient outcome. Statistical significance, correlations and graphical representation were performed using SPSS v23.0.Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptible in vitro exhibited higher MICs in biofilm structure. The biofilm forming ability had a significant association with the morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (P<0.05) resulted in fewer ‘average days alive’ for the patient (3.33) in comparison to those negative for biofilms (11.33). Phylogenetic analysis including global isolates revealed the clear association of sequence types with genes for biofilm mechanism and carbapenem resistance. Carbapenemase genes were found specific to each clade. The known hypervirulent clone-ST23 with wcaG, magA, rmpA, rmpA2 and wzc with a lack of mutation for hyper-capsulation might be poor biofilm formers. Interestingly, ST15, ST16, ST307 and ST258 – reported global high-risk clones were wcaJ negative indicating the high potential of biofilm forming capacity. Genes wabG and treC for CPS, bcsA and pgaC for adhesins, luxS for quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA, fimH, mrkD) and pili (pilQ, ecpA).This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors for K. pneumoniae biofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections by K. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and antimicrobial resistance genetic factors will serve to enhance our understanding on biofilm mechanisms in K. pneumoniae.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genomics↗

bioRxiv2020

Phylogenetic and evolutionary analysis reveals the recent dominance of ciprofloxacin resistant S. sonnei and local persistence of S. flexneri clones in India

Shigella is the second leading cause of bacterial diarrhea worldwide. Recently S. sonnei seems to be replacing S. flexneri in low and middle-income countries undergoing economic development. Despite this, studies focusing on these species at genomic level remain largely unexplored. Here we compared the genome sequences of S. flexneri and S. sonnei isolated from India with the publically available genomes of global strains. Our analysis provide evidence for the long term persistence of all PGs of S. flexneri and the recent dominance of ciprofloxacin-resistant S. sonnei lineage in India. Within S. flexneri PGs, majority of the study isolates belonged to PG3 within the predominance of serotype 2. For S. sonnei, the current pandemic involves globally distributed MDR clones that belong to Central Asia lineage III. The presence of such epidemiologically-dominant lineages in association with stable AMR determinants results in the successful survival in the community.

genomics↗

bioRxiv2019

First hybrid complete genome of Aeromonas veronii reveals chromosome-mediated novel structural variant mcr-3.19 from human clinical specimen

BackgroundRecent findings substantiate the origin of plasmid-mediated colistin resistance gene mcr-3 from Aeromonads. The present study aimed to screen the plasmid-mediated colistin resistance among 30 clinical multidrug resistant (MDR) Aeromonas spp.\n\nResultsThe presence of mcr-1, mcr-2, mcr-3, and mcr-4 were screened by PCR, which revealed mcr-3 in a colistin susceptible isolate (FC951). All other isolates were negative for mcr genes. Sequencing of FC951 revealed that mcr-3 (mcr-3.19) identified was different from previously reported variants and had 95.62 and 95.28% nucleotide similarity with mcr-3.3 and mcr-3.10 gene. A hybrid assembly using IonTorrent and MinION reads revealed structural genetic information of mcr-3.19 with an insertion of ISAs18 within the gene. Due to this, mcr-3.19 was non-expressive which makes FC951 susceptible to colistin. Further, in silico sequence and protein structural analysis confirmed the new variant. To the best of our knowledge, this is the first report on novel mcr-3 variant (mcr-3.19).\n\nConclusionsThe significant role of mcr-like genes in different Aeromonas species remains unknown and needs additional investigation to understand the insights on colistin resistance mechanism.

microbiology↗