Biology subjects

Sudarshan, A. S.

Publications and source records attributed to Sudarshan, A. S..

4 recordsLinked to original sources

bioRxiv2026

Accounting for DNA Recovery and Cell Culturability Enhances Quantitative Compatibility of Molecular and Legiolert Assays for Legionella pneumophila

Disagreement between molecular and culture-based assays for Legionella pneumophila detection is widely reported, yet comparisons have largely been based on direct assay-derived concentrations or binary positive/negative outcomes. However, it remains unclear whether molecular-culture disagreement reflects concentration-level incompatibility or unaccounted methodological and physiological differences related to DNA recovery and cell culturability. In this study, we also observed disagreement between molecular and Legiolert assays in source and finished drinking water samples collected from eight full-scale drinking water systems across the United States. Molecular thresholds adjusted for DNA recovery and cell culturability only partially resolved these discrepancies. We therefore developed a probabilistic Monte Carlo framework that incorporates sample-specific DNA recovery and cell culturability to evaluate the quantitative consistency of culturable L. pneumophila concentrations estimated by molecular and Legiolert assays. Quantitatively consistent and inconsistent samples occurred across both binary concordant and discordant classifications, demonstrating that positive/negative agreement poorly reflects concentration-level comparability. Overall, molecular and Legiolert assays showed strong quantitative consistency once sample-specific DNA recovery and cell culturability were considered. A small proportion of persistent inconsistencies at specific sampling sites, coupled with atypical microbial indicators, suggest that sample heterogeneity likely contributed to the remaining discrepancies. These findings demonstrate that integrating DNA recovery and cell culturability enhanced quantitative consistency between molecular and Legiolert assays and supports the use of molecular methods as rapid quantitative tools to complement culture-based L. pneumophila monitoring. SynopsisAccounting for DNA recovery and cell culturability revealed broad quantitative compatibility between molecular and Legiolert assays for Legionella pneumophila in drinking water. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/739452v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1e01cdcorg.highwire.dtl.DTLVardef@86bbc2org.highwire.dtl.DTLVardef@190fc66org.highwire.dtl.DTLVardef@1aa9ec8_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2026

High-accuracy hierarchical rRNA operon profiling resolves genomovar-level taxonomy and microdiversity using Nanopore sequencing.

Genomovar-level and intragenomic diversity cannot be resolved by conventional amplicon sequencing due to the limitation of fragment lengths and read accuracy, while the application of metagenomic profiling to a large number of samples can be resource intensive. Here, we report UltraRes-rrn, an integrated wet-lab and computational workflow for high-accuracy rrn (i.e., 16S- ITS-23S rRNA) operon profiling using Nanopore sequencing. By integrating ultra-long DNA recovery, long-read amplicon sequencing, and unique molecular identifiers (UMI)-based consensus correction, UltraRes-rrn obtains full-length 16S-ITS-23S rRNA operon consensus sequences with mean accuracies exceeding 99.98%. To achieve higher resolution, we propose a hierarchical rRNA operon profiling strategy in which concatenated 16S+23S rRNA genes (16S23S) serve as a primary and the internal transcribed spacer (ITS) provides a secondary marker. The 16S23S marker achieved discrimination at the genomovar level compared to either 16S or 23S rRNA genes alone, which mitigates the ITS-driven over-splitting observed with the full-length rrn operon and allows for larger proportion of data being classified at higher confidence thresholds. Further, ITS variation was strongly structured by tRNA occurrence patterns, suggesting that ITS can capture taxon microdiversity missed by either 16S or 23S rRNA gene sequences alone. The UltraRes-rrn workflow was applied to full-scale nitrogen removal reactors, revealing intraspecies diversity variation driven by different carbon regimes, which would not have been possible with a shorter gene sequence. In summary, UltraRes-rrn enables cost-effective community profiling at genomovar-level resolution in complex ecosystems.

microbiology↗

bioRxiv2025

Gene-centric analysis of Raskinella chloraquaticus reveals a functionally conserved taxonomic group in global drinking water distribution systems

A recent metagenomic survey of drinking water systems revealed that a highly prevalent and dominant uncultured bacterial genus (Raskinella) was represented globally by a single species (Raskinella chloraquaticus). R. chloraquaticus comprises of two sub-species groups, Lineages 1 and 2, the former representing a globally prevalent genomovar. The objective of this study was to perform comparative analysis of the gene content of R. chloraquaticus to characterize the gene-level diversity and determine factors shaping the diversity of this species. Pangenome analysis revealed that R. chloraquaticus possesses a core set of genes that constitute a major portion (87.74%) of the known gene content of the genome. Furthermore, most of the gene diversity of R. chloraquaticus is associated with Lineage 2 organisms, which consists of at least four distinct genomovars. Lineage 1 organisms consist of a higher proportion of identical genes than would have been expected if changes primarily occurred through random mutations and thus is potentially indicative of recombination. In contrast, Lineage 2 organisms appear to have emerged through random mutations and display stronger geographic preference. These results indicate that homologous recombination and geographic isolation likely shape the genetic repertoire of R. chloraquaticus. Further, the high level of gene conservation in R. chloraquaticus may be reflective of highly selective environment in drinking water systems. Thus, R. chloraquaticus may represent a model organism to probe selective pressures shaping the drinking water microbiome.

microbiology↗

bioRxiv2024

New drinking water genome catalog identifies a globally distributed bacterial genus adapted to disinfected drinking water systems.

Genome-resolved insights into the structure and function of the drinking water microbiome can advance the effective management of drinking water quality. To enable this, we constructed and curated thousands of metagenome-assembled and isolate genomes from drinking water distribution systems globally to develop a Drinking Water Genome Catalog (DWGC). The current DWGC disproportionately represents disinfected drinking water systems due to a paucity of metagenomes from non-disinfected systems. Using the DWGC, we identify core genera of the drinking water microbiome including a genus (UBA4765) within the order Rhizobiales that is frequently detected and highly abundant in disinfected drinking water systems. We demonstrate that this genus has been widely detected but incorrectly classified in previous amplicon sequencing-based investigations of the drinking water microbiome. Further, we show that a single genome variant (genomevar) within this genus is detected in >82% of drinking water systems included in this study. We propose a provisional name for this uncultured bacterium as "Raskinella chlorumaquaticus" and describe the genus as "Raskinella" (pending SeqCode approval). Metabolic annotation and modeling-based predictions indicate that this bacterium is capable of necrotrophic growth, able to metabolize halogenated compounds, proliferates in a biofilm-based environment and shows clear indications of disinfection-mediated selection. SynopsisCreation and analysis of a new curated drinking water genome catalog identifies uncharacterized bacterial species that is widely distributed in disinfected drinking water systems.

microbiology↗