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Timilsina, S.

Publications and source records attributed to Timilsina, S..

6 recordsLinked to original sources
bioRxiv2025

Harnessing Lytic Phages for Biofilm Control in Carbapenem-Resistant Klebsiella pneumoniae Causing Urinary Tract Infection

BackgroundKlebsiella pneumoniae is a major opportunistic pathogen with rising multidrug resistance and biofilm-related infections. Molecular and phage characterization is crucial to understand resistance mechanisms and explore alternative therapies such as phage therapy. MethodsWe performed whole-genome sequencing and antibiotic susceptibility testing of hospital-isolated Klebsiella pneumoniae (KP6697). MLST, plasmid replicon analysis, and resistance gene identification were conducted using bioinformatics. Phage isolation, electron microscopy-based morphological and biofilm analysis, and evaluation of lytic activity, stability, and host range were performed. Phage genome sequencing and annotation identified functional genes. ResultsThe host strain Klebsiella pneumoniae (KP6697) was multidrug-resistant, exhibiting resistance to 18 of 22 tested antibiotics, and genome analysis identified ST16 with eight plasmid replicons and 23 resistance genes, including blaCTX-M-15, blaNDM-5, and blaOXA-181. Functional annotations revealed extensive metabolic versatility and a rich repertoire of genes for biofilm formation, quorum sensing, secretion systems, and stress response. A lytic phage, Phage_KP6697_Omshanti, was isolated and classified as a Caudoviricetes member with a 45.3kb genome encoding lysis, replication, and structural genes. It demonstrated short latency, high burst size, thermal and pH stability, and broad host range against CRKP and other MDR strains. Importantly, microscopy confirmed its ability to inhibit and degrade biofilms at multiple stages, highlighting strong therapeutic potential. ConclusionComprehensive analysis of carbapenem-resistant K. pneumoniae (KP6697) revealed multidrug resistance and strong biofilm formation. The lytic phage Phage_KP6697_Omshanti, with depolymerase and endolysin activity, disrupted biofilms, and its stability, high burst size, and genomic traits suggest potential as an anti-CRKP agent, especially with antibiotics IMPORTANCEKlebsiella pneumoniae is increasing multidrug resistance and robust biofilm formation pose severe clinical challenges, limiting treatment options. Understanding the molecular basis of its resistance and exploiting bacteriophages with strong biofilm-disrupting properties provide promising alternative therapeutic strategies. This study highlights the isolation and genomic characterization of a lytic phage with potent anti-biofilm activity against carbapenem-resistant K. pneumoniae, underscoring its potential in combating resistant infections.

microbiology↗
bioRxiv2024

Intercontinental dissemination and diversification of Xanthomonas perforans in tomato production

Emerging and re-emerging plant diseases continue to present multifarious threats to global food security. Considerable recent efforts are therefore being channeled towards understanding the nature of pathogen emergence, their spread and evolution. Xanthomonas euvesicatoria pv. perforans (Xep), one of the causal agents of bacterial spot of tomato, rapidly emerged and displaced other bacterial spot xanthomonads in tomato production regions around the world. In less than three decades, it has become a dominant xanthomonad pathogen in tomato production systems across the world and presents a model for understanding diversification of recently emerged bacterial plant pathogens. Although Xep has been continuously monitored in Florida since its discovery, the global population structure and evolution at the genome-scale is yet to be fully explored. The objectives of this work were to determine genetic diversity globally to ascertain if different tomato production regions contain genetically distinct Xep populations, to examine genetic relatedness of strains collected in tomato seed production areas in East Asia and other production regions, and to evaluate variation in type III effectors, which are critical pathogenicity and virulence factors, in relationship to population structure. We used genome data from 270 strains from 13 countries for phylogenetic analysis and characterization of Xop effector gene diversity among strains. Our results showed notable genetic diversity in the pathogen. We found genetically similar strains in distant tomato production regions, including seed production regions, and diversification over the past 100 years, which is consistent with intercontinental dissemination of the pathogen in hybrid tomato production chains. Evolution of the Xep pangenome, including the acquisition and loss of type III secreted effectors, is apparent within and among phylogenetic lineages. The apparent long-distance movement of the pathogen, together with variants that may not yet be widely distributed, poses risks of emergence of new variants in tomato production.

microbiology↗
bioRxiv2019

Potential soil transmission of a novel Candidatus Liberibacter strain detected in citrus seedlings grown in soil from a huanglongbing infested citrus grove

Candidatus Liberibacter spp. are Alphaproteobacteria associated with plants and psyllid vectors. Most cause plant diseases, including Ca Liberibacter asiaticus (Las) associated with citrus huanglongbing (HLB). Replacing HLB-infected by Las-free citrus trees results in fast re-infection despite psyllid control. To check if HLB could be soil-borne, we performed an insect-free greenhouse-experiment with 130 mandarin seedlings in two citrus-grove soils (A and B), non-autoclaved or autoclaved. Liberibacter-specific 16S-rDNA PCR primers to detect Las were used to search for Ca. Liberibacter spp. in mandarin leaves. Seven plants grown in non-autoclaved soil B showed HLB-like symptoms and tested positive after 2.5 and 8.5 months using three different primer systems: two based on the 16S-rDNA gene (primers HLBas/HLBr and OI2c/OI1) and one based on the rplA/rplJ gene (primers LAA2/LAJ5). DNA segments from these plants amplified by primers OI2c/OI1 were cloned and sequenced; they were 95.9 % similar to Las and 94.8% to Ca. Liberibacter africanus (Laf). The DNA product from Liberibacter-group specific PCR primers for the rplA/rplJ gene was 87.6% similar to that of Las and 78.2% of Laf. As the strain obtained originated from soil and was different from existing Ca. Liberibacter species, this strain may be a new species.

ecology↗
bioRxiv2019

Recently emerged and diverse lineages of Xanthomonas perforans have independently evolved through plasmid acquisition and homologous recombination originating from multiple Xanthomonas species

Xanthomonas perforans is the predominant pathogen responsible for bacterial leaf spot of tomato and X. euvesicatoria of pepper in the southeast United States. Previous studies have indicated significant changes in the X. perforans population collected from Florida tomato fields over the span of two decades including a shift in race, diversification into three genetic groups, and host range expansion to pepper. Recombination originating from X. euvesicatoria was identified as the primary factor driving the diversification of X. perforans in Florida. The aim of this study was to genetically characterize X. perforans strains that were isolated from tomato and pepper plants grown in Alabama and compare them to the previously published genomes available from GenBank. Surprisingly, a maximum likelihood phylogeny coupled with a Bayesian analysis of population structure revealed the presence of two novel genetic groups in Alabama, which each harbored a different transcription activation-like effector (TALE). While one TALE, avrHah1, was associated with adaptation of X. perforans to pepper, the other was identified as a new class within the avrBs3 family, designated here as pthXp1. Examination of patterns of homologous recombination between X. perforans and other closely related Xanthomonas spp. indicated that the lineages identified here emerged in part through recent recombination events originating from xanthomonads associated with diverse hosts of isolation. Our results also suggest that the evolution of pathogenicity to pepper has likely emerged independently within X. perforans and in one lineage, was associated with the recombination-mediated remodeling of the Xps type II secretion and TonB transduction systems.\n\nImportanceThe emergence of novel pathogen lineages has important implications in the sustainability of genetic resistance as a disease management tool in agricultural ecosystems. In this study, we identified two novel lineages of X. perforans in Alabama. While one lineage was isolated from symptomatic pepper plants, confirming the host range expansion of X. perforans, the other lineage was isolated from tomato and acquired a novel transcription activation-like effector, pthXp1. Unlike AvrBs4, PthXp1overcomes Bs4-mediated resistance in tomato, indicating the evolution of this novel lineage towards fitness on this host. Our findings also show that different phylogenetic groups of the pathogen have experienced independent recombination events originating from multiple Xanthomonas species. This suggests a continuous gene flux between related xanthomonads associated with diverse plant hosts which results in the emergence of novel pathogen lineages and associated phenotypes, including host range expansion.

genomics↗
bioRxiv2017

Genome shuffling in a globalized bacterial plant pathogen: Recombination-mediated evolution in Xanthomonas euvesicatoria and X. perforans

Bacterial recombination and clonality underly the evolution and epidemiology of pathogenic lineages as well as their cosmopolitan spread. While the spread of stable clonal bacterial pathotypes drives disease epidemics, recombination leads to the evolution of new bacterial lineages. Recombinant lineages of plant bacterial pathogens are typically associated with colonization of novel hosts and emergence of new diseases. Here, we show that recombination between evolutionarily and phenotypically distinct plant pathogenic lineages has generated new recombinant lineages with unique combinations of pathogenicity and virulence factors. X. euvesicatoria (Xe) and X. perforans (Xp) are two closely related monophyletic species causing bacterial spot disease on tomato and pepper worldwide. We sequenced the genomes of strains representing populations on tomato in Nigeria and found shuffling of secretion systems and effectors such that these strains contain genes from both Xe and Xp. Multiple strains, from populations in Nigeria, Italy, and Florida, USA, exhibited extensive genomewide homologous recombination and both species exhibited dynamic open pangenomes. Our results show that recombination is generating new lineages of bacterial spot pathogens on tomato with consequences for disease management strategies.\n\nImportanceThe Xanthomonas pathogens that cause bacterial spot of tomato and pepper have been model systems for plant-microbe interactions. Two of these pathogens, X. euvesicatoria and X. perforans, are very closely related. Genome sequences of bacterial spot field strains from Nigeria, Italy, and the United States showed varying levels of homologous recombination that changed the amino sequence of effectors, secretion systems, and other proteins. This shuffling of genome content occurred between X. euvesicatoria and X. perforans, while a Nigerian lineage also contained the lipopolysaccharide cluster of a distantly related Xanthomonas species. Gene content varied among strains and the affected genes are important in the establishment of disease, therefore our findings point to global variation in the host-pathogen interaction driven by gene exchange among evolutionarily distinct lineages.

evolutionary biology↗