METHODS article
Front. Microbiol.
Sec. Infectious Agents and Disease
Development of Nanopore amplicon sequencing method for culture-free genotyping of Bacillus anthracis strains directly from environmental samples
Ágnes Nagy 1
Gábor Endre Tóth 2,3
Péter Sály 4,5
Csaba István Pereszlényi 1
Gergely Csaba Babinszky 1
László Makrai 6
Balázs Antal Somogyi 7
Miklós Gyuranecz 8,9,10
1. Hungarian Defence Forces Medical Centre, Budapest, Hungary
2. Bernhard Nocht Institute for Tropical Medicine, Virus Metagenomics and Evolution, Hamburg, Germany
3. Bernhard Nocht Institute for Tropical Medicine, Arbovirus and Entomology Department, Hamburg, Germany
4. HUN-REN Centre for Ecological Research, Institute of Aquatic Ecology, Budapest, Hungary
5. Department of Fishery Research and Development, Institute of Aquaculture and Environmental Safety, Hungarian University of Agriculture and Life Sciences, Gödöllő, Hungary
6. Autovakcina Kft., Budapest, Hungary
7. National Laboratory of Virology, University of Pécs, Pécs, Hungary
8. HUN-REN Veterinary Medical Research Institute, Budapest, Hungary
9. National Laboratory of Health Safety, Budapest, Hungary
10. MolliScience Kft., Biatorbágy, Hungary
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Abstract
Fast and accurate genetic subtyping of pathogens is required to respond appropriately to biological events caused by natural outbreaks or bioattacks involving anthrax. In this study, we developed and validated a culture-free genotyping method that combines a multiplex PCR-based amplicon sequencing method on the Nanopore platform with in silico multiple-locus variable-number tandem repeat analysis (MLVA) of 31 loci to identify an unknown Bacillus anthracis strain directly from environmental samples. The novel method accurately identified repeat numbers for all loci in 12 different MLVA genotype Bacillus anthracis strains analyzed in the study, matching 100% with the reference capillary electrophoresis and Sanger sequencing results. The detection limit of the method, at which all 31 variable-number tandem repeat loci were successfully identified, was found to be 104 CFU spores/sample for pure spore samples and at 106 CFU spores/sample for spiked environmental samples from three matrices (soil, surfaceswab, and muddy water). Specificity tests yielded negative results for samples containing only non-Bacillus anthracis members of the Bacillus cereus group, which produced sequencing reads for 15 loci but were non-specific to Bacillus anthracis. To validate the method, we genotyped 11 Bacillus anthracis strains originating from a historical collection of Hungarian isolates. The MLVA31 typing scheme classified the strains into fourfive groups, threefour of which fell into the A.Br.008/009 Trans-Eurasian (TEA) group within the clade A, and one into the B.Br.CNEVA group within the clade B. The largest group within clade A comprises six strains that are assumed to be members of the dominant Bacillus anthracis population in Hungary. Our results demonstrate that PCR-based amplicon sequencing using the portable MinION device is highly effective for on-site genotyping of pathogens directly from environmental samples. This establishes the NGS-based MLVA genotyping as a valuable tool for biodefense laboratories in preliminary forensic investigations of bioterrorism-related anthrax outbreaks. Furthermore, our results provide new insights into the genetic diversity of Bacillus anthracis in a region (Hungary, Central Europe) that is underrepresented in research and has limited scientific data.
Summary
Keywords
Bacillus anthracis, Central Europe, culture-freegenotyping, environmental samples, genetic diversity, Hungary, MLVA31, Nanopore amplicon sequencing
Received
21 December 2025
Accepted
20 February 2026
Copyright
© 2026 Nagy, Tóth, Sály, Pereszlényi, Babinszky, Makrai, Somogyi and Gyuranecz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Ágnes Nagy
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