Direct whole-genome sequencing of Coxiella burnetii from environmental samples using a two-step selective enrichment approach
4 affiliations
- 1Saint-Petersburg Pasteur Institute, St. Petersburg, Russia
- 2North-West Anti-Plague Station, St. Petersburg, Russia
- 3Center of Hygiene and Epidemiology in Astrakhan region, Astrakhan, Russia
- 4Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov First Moscow State Medical University, Moscow, Russia
Abstract
Current high-throughput sequencing platforms enable routine whole-genome sequencing of bacteria via clonal amplification from primary samples, followed by DNA extraction, library preparation, and sequencing. This approach enriches bacterial DNA while removing contaminating nucleic acids, allowing efficient genomic sequencing using minimal sequencing resources per sample. However, for fastidious or obligate intracellular bacteria, such as Coxiella burnetii, cultivation is often impractical, leaving pathogen DNA heavily diluted by host and environmental nucleic acids. In such cases, sequencing requires substantially greater throughput per sample, highlighting the need for culture-independent methods to support genomic epidemiology and surveillance. Here, we report a novel two-stage DNA enrichment strategy applied to C. burnetii-contaminated sheep wool samples. Such samples feature very low pathogen biomass and a high host-DNA background. Our approach integrates (i) selective whole-genome amplification of complex DNA extracts, followed by (ii) in-solution hybridization capture using custom double-stranded DNA baits. Critically, we developed a WGA-based method to synthesize whole-genome dsDNA baits from minimal input DNA, enabling efficient bait production even in the absence of cultured isolates. Notably, we demonstrate that baits derived from C. burnetii DNA contaminated with an unrelated host (e.g., chicken) remain effective for enriching C. burnetii from samples derived from a different host species (e.g., sheep), indicating robust cross-host applicability. Using this workflow, we achieved up to 95% C. burnetii enrichment and generated six genomic assemblies suitable for downstream phylogenomic and epidemiological analyses. This cultivation-free, two-stage enrichment framework can be applied for phylogenetics and epidemiology and may significantly expand the feasibility of genomic studies for intracellular and otherwise uncultivable bacterial pathogens. Author summary: While detecting specific bacterial pathogens is straightforward using standard PCR, understanding their evolution and spread requires whole-genome sequencing. However, for hard-to-culture pathogens like Coxiella burnetii, obtaining pure bacterial DNA for sequencing is highly challenging. These bacteria grow inside host cells, resulting in low bacterial DNA yields and high contamination from the host. Consequently, their genomic diversity remains poorly understood. To overcome this, we developed a two-step DNA enrichment method combining selective whole-genome amplification with hybrid capture. This approach enables the sequencing of Coxiella genomes directly from complex biological or environmental samples, bypassing the need for prior bacterial culturing. It efficiently enriches target DNA even when total yields are low and host contamination is high. Additionally, we pioneered the use of Phi29 polymerase to generate hybridization probes, allowing rapid production of abundant probes from minimal starting material. Crucially, we demonstrated that these probes can be synthesized from mixed DNA preparations. For instance, DNA from Coxiella cultures grown in chicken cells can be used to capture Coxiella DNA from mammalian or environmental samples, provided the host species are evolutionarily distant. By eliminating the culturing bottleneck, our method significantly expands the capacity for genomic surveillance and epidemiological studies of difficult-to-cultivate pathogens.
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BibTeX
@article{bezruchko2026direct,
title = {{Direct whole-genome sequencing of Coxiella burnetii from environmental samples using a two-step selective enrichment approach}},
author = {Mikhail Bezruchko and Olga Freylikhman and Erik Khalilov and Elena Vakalova and Denis Kasatkin and Sofia Platova and Ivan Gorokhov and Alina Saitova and Islam Karmokov and Nikolay Tokarevich and Dmitrii Polev and Vladimir Dedkov},
journal = {PLOS Neglected Tropical Diseases},
year = {2026},
month = sep,
doi = {10.1371/journal.pntd.0014716},
url = {https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014716},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Direct whole-genome sequencing of Coxiella burnetii from environmental samples using a two-step selective enrichment approach
AU - Mikhail Bezruchko
AU - Olga Freylikhman
AU - Erik Khalilov
AU - Elena Vakalova
AU - Denis Kasatkin
AU - Sofia Platova
AU - Ivan Gorokhov
AU - Alina Saitova
AU - Islam Karmokov
AU - Nikolay Tokarevich
AU - Dmitrii Polev
AU - Vladimir Dedkov
PY - 2026
DA - 2026/09/08
JO - PLOS Neglected Tropical Diseases
DO - 10.1371/journal.pntd.0014716
UR - https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014716
AB - Current high-throughput sequencing platforms enable routine whole-genome sequencing of bacteria via clonal amplification from primary samples, followed by DNA extraction, library preparation, and sequencing. This approach enriches bacterial DNA while removing contaminating nucleic acids, allowing efficient genomic sequencing using minimal sequencing resources per sample. However, for fastidious or obligate intracellular bacteria, such as Coxiella burnetii, cultivation is often impractical, leaving pathogen DNA heavily diluted by host and environmental nucleic acids. In such cases, sequencing requires substantially greater throughput per sample, highlighting the need for culture-independent methods to support genomic epidemiology and surveillance. Here, we report a novel two-stage DNA enrichment strategy applied to C. burnetii-contaminated sheep wool samples. Such samples feature very low pathogen biomass and a high host-DNA background. Our approach integrates (i) selective whole-genome amplification of complex DNA extracts, followed by (ii) in-solution hybridization capture using custom double-stranded DNA baits. Critically, we developed a WGA-based method to synthesize whole-genome dsDNA baits from minimal input DNA, enabling efficient bait production even in the absence of cultured isolates. Notably, we demonstrate that baits derived from C. burnetii DNA contaminated with an unrelated host (e.g., chicken) remain effective for enriching C. burnetii from samples derived from a different host species (e.g., sheep), indicating robust cross-host applicability. Using this workflow, we achieved up to 95% C. burnetii enrichment and generated six genomic assemblies suitable for downstream phylogenomic and epidemiological analyses. This cultivation-free, two-stage enrichment framework can be applied for phylogenetics and epidemiology and may significantly expand the feasibility of genomic studies for intracellular and otherwise uncultivable bacterial pathogens. Author summary: While detecting specific bacterial pathogens is straightforward using standard PCR, understanding their evolution and spread requires whole-genome sequencing. However, for hard-to-culture pathogens like Coxiella burnetii, obtaining pure bacterial DNA for sequencing is highly challenging. These bacteria grow inside host cells, resulting in low bacterial DNA yields and high contamination from the host. Consequently, their genomic diversity remains poorly understood. To overcome this, we developed a two-step DNA enrichment method combining selective whole-genome amplification with hybrid capture. This approach enables the sequencing of Coxiella genomes directly from complex biological or environmental samples, bypassing the need for prior bacterial culturing. It efficiently enriches target DNA even when total yields are low and host contamination is high. Additionally, we pioneered the use of Phi29 polymerase to generate hybridization probes, allowing rapid production of abundant probes from minimal starting material. Crucially, we demonstrated that these probes can be synthesized from mixed DNA preparations. For instance, DNA from Coxiella cultures grown in chicken cells can be used to capture Coxiella DNA from mammalian or environmental samples, provided the host species are evolutionarily distant. By eliminating the culturing bottleneck, our method significantly expands the capacity for genomic surveillance and epidemiological studies of difficult-to-cultivate pathogens.
SN - 1935-2735
ER -