Ultra-deep HiFi sequencing reveals rare structural-variant subpopulations in multidrug-resistant Pseudomonas aeruginosa from an LVAD-associated infection.
- 1Laboratory for Pathogenesis of Clinical Drug Resistance and Persistence, School of Public Health, San Diego State University, San Diego, CA, 92182, USA.
- 2Department of Biology, San Diego State University, USA.
Abstract
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen frequently associated with multidrug-resistant infections in clinical settings. The rise of antibiotic-resistant strains and their genomic complexity necessitate advanced tools for comprehensive genetic characterization, particularly in the context of resistance to antibiotic and phage therapies. Here, we use ultra-deep high-fidelity (HiFi) sequencing to analyze strain NRD619, a multidrug-resistant P. aeruginosa isolate that had colonized the driveline of a left ventricular assist device of an 82-year-old male who was identified as a phage therapy candidate. Through HiFi long-read sequencing and bioinformatic analysis, we identified low-frequency heterogeneous structural variants (SVs) and investigated their potential phenotypic consequences through functional annotation and literature-based gene-function analysis. Small variants in known resistance-associated genes, including an early frameshift in mexZ, were consistent with components of the multidrug-resistant phenotype, whereas several other genotype-phenotype associations remained uncertain. Ultra-deep long-read sequencing identified five low-frequency heterogeneous SV signals. A reciprocal 31.7-kb deletion and duplication of a phage-rich region were supported by 3 and 95 reads, respectively; three multi-kilobase insertion candidates were each supported by a single HiFi molecule. Observed variant allele frequencies ranged from 1.2119% to 0.0122%. The insertions intersected genes involved in type IV pili, transport, and stress response, providing plausible hypotheses for effects on biofilm-related behavior, device colonization, and other phenotypes. This study demonstrates how ultra-deep sequencing can uncover cryptic genetic heterogeneity in bacterial pathogens, offering insights into P. aeruginosa population structure in a clinical isolate that are inaccessible to conventional-depth sequencing. Because sequencing followed single-colony recovery and laboratory expansion, the origin and in vivo frequencies of the rarest SVs cannot be established. The detected diversity may reflect structural states accessible in vivo or variation arising during ex vivo propagation; the latter remains relevant because culture-based genomic and phenotypic characterization interrogates the expanded population. Future applications could refine our understanding of multidrug resistance, phage susceptibility, and adaptations influencing host colonization. Chronic bacterial infections are usually represented by a single genome, even though clinicians and microbiologists know genetically diverse subpopulations can coexist within a patient. Using ultra-deep, high-fidelity long-read sequencing of a multidrug-resistant Pseudomonas aeruginosa clinical isolate from a left ventricular assist device-associated infection, we reveal structural heterogeneity distinguished by phage and mobile-element architectures and associated small variants. Coverage-based modeling shows that several of these rare structural states would be poorly sampled at standard sequencing depths, yet they occur in loci linked to virulence, surface interactions, transport, and stress response. Because the isolate underwent single-colony recovery before sequencing, the study cannot determine whether individual rare variants arose in vivo or during ex vivo propagation. Either possibility is informative: the former represents hidden within-host diversity, whereas the latter can alter the population used for culture-based genomic and phenotypic characterization. These findings establish ultra-deep long-read sequencing as a strategy for revealing hidden adaptive potential in chronic and device-associated infection isolates.
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BibTeX
@article{bosco2026ultradeep,
title = {{Ultra-deep HiFi sequencing reveals rare structural-variant subpopulations in multidrug-resistant Pseudomonas aeruginosa from an LVAD-associated infection.}},
author = {Abigail Bosco and Tiffany Luong and Maryam Ahmadi Jeshvaghane and Mahshid Fallahpour and Samuel J Modlin and Dwayne Roach and Faramarz Valafar},
journal = {Current research in microbial sciences},
year = {2026},
month = oct,
doi = {10.1016/j.crmicr.2026.100678},
url = {https://europepmc.org/article/MED/42999899},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Ultra-deep HiFi sequencing reveals rare structural-variant subpopulations in multidrug-resistant Pseudomonas aeruginosa from an LVAD-associated infection.
AU - Abigail Bosco
AU - Tiffany Luong
AU - Maryam Ahmadi Jeshvaghane
AU - Mahshid Fallahpour
AU - Samuel J Modlin
AU - Dwayne Roach
AU - Faramarz Valafar
PY - 2026
DA - 2026/10/25
JO - Current research in microbial sciences
DO - 10.1016/j.crmicr.2026.100678
UR - https://europepmc.org/article/MED/42999899
AB - Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen frequently associated with multidrug-resistant infections in clinical settings. The rise of antibiotic-resistant strains and their genomic complexity necessitate advanced tools for comprehensive genetic characterization, particularly in the context of resistance to antibiotic and phage therapies. Here, we use ultra-deep high-fidelity (HiFi) sequencing to analyze strain NRD619, a multidrug-resistant P. aeruginosa isolate that had colonized the driveline of a left ventricular assist device of an 82-year-old male who was identified as a phage therapy candidate. Through HiFi long-read sequencing and bioinformatic analysis, we identified low-frequency heterogeneous structural variants (SVs) and investigated their potential phenotypic consequences through functional annotation and literature-based gene-function analysis. Small variants in known resistance-associated genes, including an early frameshift in mexZ, were consistent with components of the multidrug-resistant phenotype, whereas several other genotype-phenotype associations remained uncertain. Ultra-deep long-read sequencing identified five low-frequency heterogeneous SV signals. A reciprocal 31.7-kb deletion and duplication of a phage-rich region were supported by 3 and 95 reads, respectively; three multi-kilobase insertion candidates were each supported by a single HiFi molecule. Observed variant allele frequencies ranged from 1.2119% to 0.0122%. The insertions intersected genes involved in type IV pili, transport, and stress response, providing plausible hypotheses for effects on biofilm-related behavior, device colonization, and other phenotypes. This study demonstrates how ultra-deep sequencing can uncover cryptic genetic heterogeneity in bacterial pathogens, offering insights into P. aeruginosa population structure in a clinical isolate that are inaccessible to conventional-depth sequencing. Because sequencing followed single-colony recovery and laboratory expansion, the origin and in vivo frequencies of the rarest SVs cannot be established. The detected diversity may reflect structural states accessible in vivo or variation arising during ex vivo propagation; the latter remains relevant because culture-based genomic and phenotypic characterization interrogates the expanded population. Future applications could refine our understanding of multidrug resistance, phage susceptibility, and adaptations influencing host colonization. Chronic bacterial infections are usually represented by a single genome, even though clinicians and microbiologists know genetically diverse subpopulations can coexist within a patient. Using ultra-deep, high-fidelity long-read sequencing of a multidrug-resistant Pseudomonas aeruginosa clinical isolate from a left ventricular assist device-associated infection, we reveal structural heterogeneity distinguished by phage and mobile-element architectures and associated small variants. Coverage-based modeling shows that several of these rare structural states would be poorly sampled at standard sequencing depths, yet they occur in loci linked to virulence, surface interactions, transport, and stress response. Because the isolate underwent single-colony recovery before sequencing, the study cannot determine whether individual rare variants arose in vivo or during ex vivo propagation. Either possibility is informative: the former represents hidden within-host diversity, whereas the latter can alter the population used for culture-based genomic and phenotypic characterization. These findings establish ultra-deep long-read sequencing as a strategy for revealing hidden adaptive potential in chronic and device-associated infection isolates.
SN - 2666-5174
ER -