Abstract
Atypical Brucella spp., which include Brucella inopinata and related lineages, have emerged as unconventional pathogens with an expanding host range, particularly among exotic amphibians. A captive waxy monkey treefrog (Phyllomedusa bicolor) presented with a focal ulcerative skin lesion above its right eye. Cytology revealed mixed inflammation associated with a bacterial infection, and 16S rRNA sequencing identified the isolate as a member of the Brucella genus. Whole-genome sequencing using hybrid PacBio–Illumina platforms generated a high-quality draft genome. Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values demonstrated the highest similarity to B. inopinata (98.08% ANI; 83.50% dDDH). Core genome phylogenetic analysis further confirmed close clustering with atypical Brucella spp., which are clearly distinct from classical zoonotic lineages. This case represents the first detection of an atypical B. inopinata-like Brucella strain in an amphibian in the Republic of Korea.
1 Introduction
Brucella spp. are facultative intracellular bacteria that cause brucellosis in a wide range of warm-blooded mammals, including livestock and humans (1). Classical Brucella spp. including Brucella abortus, B. melitensis, B. suis, and B. canis are well-recognized causative agents of brucellosis. Brucellosis typically manifests with non-specific systemic symptoms such as fever, sweating, and myalgia. This disease is transmitted through direct contact with infected animals or their secretions, consumption of unpasteurized dairy products, and exposure to contaminated water, soil, meat, or other environmental sources. Since Brucella spp. can persist within host cells and cause chronic infections, treatment often requires prolonged, multi-drug regimens to achieve complete resolution (2). In the Republic of Korea, outbreaks and clinical cases have been predominantly associated with B. abortus and B. melitensis, both of which can infect humans (3).
In recent years, a growing number of genetically divergent and ecologically distinct Brucella strains, collectively referred to as atypical Brucella spp., have been identified. These strains do not belong to the classical mammalian pathogenic species (4, 5). While classical Brucella spp. show well-defined host specificity and are primarily associated with warm-blooded mammals, atypical Brucella spp. exhibit a much broader host range with diverse environmental niches and cold-blooded hosts, including amphibians, reptiles, fish, and soil (6). Amphibians, particularly frogs, have increasingly been recognized as key reservoirs of atypical Brucella spp. (7–11). The isolation of B. inopinata, a species capable of infecting humans, from frogs suggests that these amphibians may represent a newly emerging source of potential public health risk (9, 10).
Genetic analysis of Brucella spp. is particularly challenging because the genus exhibits remarkable genomic homogeneity (12). This high level of homogeneity has fueled long-standing debates not only about species delineation within Brucella spp., but also about its taxonomic distinction from closely related genera such as Ochrobactrum spp. Recent genome-based taxonomic revisions have proposed the inclusion of former Ochrobactrum spp. within an expanded Brucella spp., although this reclassification remains debated (13). Novel Brucella spp. continue to be delineated based on their molecular characteristic, host preference, and pathogenicity (14). However, the limited genetic diversity becomes even more problematic when distinguishing atypical Brucella spp., making whole-genome approaches such as average nucleotide identity (ANI), DNA–DNA hybridization (DDH), and core genome phylogeny essential for classification (6, 12). ANI provides a quantitative measure of mean nucleotide-level similarity between genomes and is widely used to define species boundaries, with a ≥95%–96% threshold generally indicating the same species (15). DDH, the gold standard for bacterial species delineation, evaluates the extent of hybridization between genomic DNA strands and remains a valuable reference point, with a ≥70% similarity supporting species-level relatedness (16). Core genome phylogeny, which infers evolutionary relationships based on the shared set of conserved genes across strains, has proven to be a robust approach for identifying fine-scale evolutionary differences within the genus (17). These whole-genome approaches provide the resolution needed to distinguish atypical Brucella spp. and define their taxonomic position.
There has been increasing research on atypical Brucella spp., yet no such pathogens have been detected in the Republic of Korea. This case represents the first report in the Republic of Korea of an atypical Brucella spp., specifically a B. inopinata-like strain isolated from skin lesions on an exotic frog (Phyllomedusa bicolor).
2 Case description
An adult captive waxy monkey treefrog (P. bicolor) was referred to the university hospital (Figure 1). Physical examination suggested that a focal ulcerative skin lesion on the dorsal aspect of the right eye had progressed, exposing the underlying muscle. Cytology of the lesion revealed mixed-type inflammation associated with bacterial infection, and bacterial culture and antibiotic susceptibility testing were subsequently conducted. Based on 16S ribosomal RNA gene sequencing results, bacteria isolated from the ulcer were identified as Brucella sp. The pathogen was considered susceptible to doxycycline based on large inhibition zones of 32.1 mm, and the animal was treated accordingly. After 1 month, the lesion showed a noticeable reduction in size, and by 3 months, it had diminished to a level consistent with what we expected under spontaneous healing.
Figure 1
To determine the taxonomic position of Brucella sp. isolated in this case, phylogenetic analysis was conducted (Figure 2). A neighbor-joining tree based on 16S rRNA gene sequences demonstrated that the isolate clearly belongs to Brucella spp. The isolate was clustered most closely with atypical Brucella spp., including B. inopinata. In contrast, classical zoonotic Brucella spp. such as B. abortus, B. melitensis, and B. suis form a separate branch. These findings suggested that the isolate in this case was more closely related to atypical Brucella spp. than to classical pathogenic species.
Figure 2
Whole-genome sequencing of the isolate was performed using a commercial hybrid sequencing service provided by Ebiogen Inc. (Seoul, Republic of Korea) using combined PacBio and Illumina platforms. The resulting high-quality reads were assembled and curated to generate a draft genome suitable for genomic analyses. De novo assembly and error correction were performed using the Microbial Genome Analysis application implemented in SMRT Link v13.1.0.221970 following the manufacturer’s recommended workflow (18). Hybrid assembly produced two contigs (3.37 Mb; N50 = 2.13 Mb), and quality metrics, including a 99.99% mapping rate and 99.19% BUSCO completeness, confirmed the high accuracy and completeness of the genome, indicating that downstream comparative analysis was appropriate. Assembly validation was performed using Jellyfish, GenomeScope, Inspector, pbmm2, Pilon, BUSCO, and BLAST (19–24). More than 3,000 coding sequences were predicted and functionally annotated using Prokka (25), with functional annotations further refined using EggNOG (26) and InterProScan (27).
Subsequent genomic analyses were conducted to determine the genomic relatedness of the isolate to other Brucella spp. ANI was assessed using the OrthoANIu algorithm implemented in EZbioCloud (15). Genome assemblies of representative Brucella spp. had ANI values ranging from 97.50 to 98.08%. The highest ANI value was observed in B. spp. BO2 (98.10%), B. inopinata (98.08%), and B. spp. BO3 (97.92%), atypical Brucella species, whereas slightly lower ANI values were observed with classical Brucella spp. B. microti (97.87%) and B. canis (97.76%). These results indicated that the isolate in this study was most similar to atypical Brucella spp. at the genomic level rather than to classical pathogenic species.
Digital DDH (dDDH) was performed using the Genome-to-Genome Distance Calculator to evaluate genomic relatedness between the isolate and Brucella spp. (16). The isolate showed the highest similarity to B.spp. BO2 (83.80%), B. inopinata (83.50%), B. spp. BO3 (83.10%), followed by B. microti (81.90%), while classical zoonotic species such as B. abortus, B. melitensis, B. suis, B. canis, and B. ovis showed slightly lower values (79.30–81.20). These results indicated that the isolate was more closely related to atypical Brucella spp. at the genomic level.
Core genome-based phylogeny was reconstructed using the Codon Trees pipeline at the Bacterial and Viral Bioinformatics Resource Center (28). The resulting phylogeny revealed a distinct separation between classical pathogenic Brucella spp. (B. abortus, B. melitensis, B. suis, B. canis, and B. ovis) and atypical Brucella spp. (Figure 3). The isolates in this study clustered strongly with B. inopinata and were clearly separated from the classical Brucella spp. clade. This core-genome phylogeny corroborated the ANI and dDDH results, indicating that the isolate was most closely related to atypical Brucella spp. at the genomic level. Whole-genome sequence-based phylogenetic analysis clearly demonstrated that the isolate was most closely related to B. inopinata, supporting its classification within the atypical Brucella group.
Figure 3
3 Discussion
Newly reported atypical Brucella spp. identified across diverse host species have emerged as a potential public health concern (7). Over the past decade, these non-classical Brucella spp. have been isolated from mammals (including humans), fish, amphibians and even environmental sources, suggesting that their distribution and host adaptability may be far wider than previously recognized (6, 10). Atypical Brucella spp. show high genomic homogeneity, which necessitates whole-genome sequencing for accurate identification; misclassification can lead to clinical issues in both human and veterinary medicine (5, 12).
In this case, whole-genome sequencing-based phylogenetic analysis revealed that the isolate clustered closely with B. inopinata and related atypical Brucella spp., with the closest relationship observed with BO2, supporting its classification as a B. inopinata-like organism. Brucella inopinata-like strains have been isolated from humans presenting with brucellosis-like symptoms. Reported cases exhibited clinical signs such as fever, fatigue, and osteoarticular lesions, or, in more severe cases, polyadenopathies accompanied by multiple pulmonary consolidations, and were generally acquired through exposure to infected animals (29, 30). Reported human cases highlight their zoonotic potential, yet atypical Brucella spp. often evade classical diagnostic expectations, leading to delayed recognition and prolonged treatment (1, 5, 29). Atypical Brucella spp. are particularly concerning because they exhibit marked genomic homogeneity, making them difficult to distinguish from one another and from classical Brucella spp. using conventional diagnostic methods (12). It has been suggested that unique traits from atypical Brucella spp. could be transferred to classical species through mobile genetic elements, posing a public health concern (1). New genotypes and atypical strains continue to be discovered across diverse hosts and environments, indicating that the diversity of this group remains underrecognized. Such findings heighten the potential zoonotic risk, as genetically cryptic strains may circulate undetected and enter human or animal populations (6, 7, 10).
Although atypical Brucella spp. have been detected in a variety of animal hosts, the repeated isolation of B. inopinata-like strains from frogs has attracted particular attention (5, 10, 29). In frogs, infections caused by B. inopinata and B. inopinata-like strains have been associated with clinical signs such as lethargy and skin lesions, and have been isolated from frogs found dead without prior clinical observations (5, 31, 32). Given that B. inopinata-like strains have also been recovered from humans with brucellosis-like illnesses, individuals who handle amphibians such as veterinarians, animal keepers, and exotic-pet industry workers may be at increased risk of exposure. The recurrent detection of B. inopinata-like strains in anurans suggests that frogs may serve as potential reservoirs or transmission intermediaries for atypical Brucella spp. (8, 10, 11).
In this case, a B. inopinata-like Brucella strain was isolated from a skin lesion of a waxy monkey treefrog (P. bicolor). The B. inopinata-like strain was rapidly identified from the skin lesion, enabling timely treatment and recognition of the potential risk of transmission to humans or other animals. Antimicrobial susceptibility testing further allowed the initiation of appropriate antimicrobial therapy, and the affected frog ultimately recovered. However, as with classical pathogenic Brucella spp. infections, treatment of atypical Brucella spp., including B. inopinata-like strains, often require prolonged management (2). In this case, full recovery was achieved only after approximately 3 months. This outcome showed that the possibility of transmission to other animals or humans cannot be fully excluded even when appropriate precautions are followed. The difficulty of determining the timing and route of exposure increases when infected animals are found dead. The fact that B. inopinata-like strains can cause disease in humans indicates that individuals working with frogs should exercise caution (29).
In the Republic of Korea, surveillance and research on Brucella spp. infections have focused primarily on classical pathogenic species that affect livestock and humans. The potential risks associated with atypical Brucella lineages have not been previously recognized (3). This lack of recognition is concerning because atypical Brucella spp. display substantial genomic diversity and can infect a broad range of non-traditional hosts, including amphibians. The Republic of Korea has seen a steady increase in the importation of exotic frog species. This increased movement of amphibians across borders poses the risk of introducing novel or previously unrecognized pathogens into the country (33). As the importation of exotic frogs into the Republic of Korea continues to increase, awareness and monitoring of the potential risks associated with atypical Brucella spp. are warranted.
In this case, we report the first identification of atypical Brucella spp. in an amphibian in the Republic of Korea. This finding highlights the need to broaden national surveillance beyond classical pathogenic Brucella spp. and to apply genomic methods that can distinguish atypical strains. The detection of this organism in an exotic amphibian also raises the possibility of spillover to other captive animals, native wildlife, and people who handle amphibians. This case emphasizes the importance of recognizing atypical Brucella spp. as emerging pathogens and of implementing careful monitoring to reduce public and animal health risks.
Statements
Data availability statement
The 16S rRNA gene sequence generated in this study was deposited in the NCBI GenBank database under accession number PX945274.1. Other datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Ethics statement
Written informed consent was obtained from the participants for the publication of this case report.
Author contributions
MK: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft. ZH: Data curation, Methodology, Writing – review & editing. J-IH: Conceptualization, Data curation, Methodology, Project administration, Supervision, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This study was supported by the National Institute of Wildlife Disease Control and Prevention as a “Specialized Graduate School Support Project for Wildlife Diseases Specialists”.
Acknowledgments
The authors wish to express gratitude to the staff of the Laboratory of Exotic and Wildlife Medicine, and the students of Jeonbuk National University for their continued support in conducting this research.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that Generative AI was not used in the creation of this manuscript.
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References
1.
El-SayedAAwadW. Brucellosis: evolution and expected comeback. Int. J Vet Sci Med. (2018) 6:S31–5. doi: 10.1016/j.ijvsm.2018.01.008,
2.
QureshiKAParvezAFahmyNAAbdel HadyBHKumarSGangulyAet al. Brucellosis: epidemiology, pathogenesis, diagnosis and treatment-a comprehensive review. Ann Med. (2023) 55:2295398. doi: 10.1080/07853890.2023.2295398,
3.
ShinISRohSGGillBCKimYSHwangKW. Assessment of brucellosis-causing pathogens with an emphasis on the prevalence of Brucella melitensis in the Republic of Korea: insights from a decade of pathogen surveillance (2014–2023), a retrospective study. Osong Public Health Res Perspect. (2024) 15:489–96. doi: 10.24171/j.phrp.2024.0134,
4.
RajendhranJ. Genomic insights into Brucella. Infect Genet Evol. (2021) 87:104635. doi: 10.1016/j.meegid.2020.104635,
5.
UfermannCMHofreuterDGadicherlaAKSpröerCBunkBOehmeRet al. A new member of the novel, non-core Brucella clade: an exotic frog isolate closely related to atypical Brucella isolates from recent human brucellosis cases in Australia. BMC Microbiol. (2025) 25:790. doi: 10.1186/s12866-025-04479-2,
6.
OcchialiniAHofreuterDUfermannCMAl DahoukSKöhlerS. The retrospective on atypical Brucella species leads to novel definitions. Microorganisms. (2022) 10:813. doi: 10.3390/microorganisms10040813,
7.
MühldorferKWibbeltGSzentiksCAFischerDScholzHCZschöckMet al. The role of ‘atypical’ Brucella in amphibians: are we facing novel emerging pathogens?J Appl Microbiol. (2017) 122:40–53. doi: 10.1111/jam.13326,
8.
LatheefSKeyburnABrozIBagnaraABayleyCFrithSet al. Atypical Brucella sp. in captive Australian green tree frogs (Litoria caerulea): clinical features, pathology, culture and molecular characterization. Aust Vet J. (2020) 98:216–21. doi: 10.1111/avj.12925,
9.
HelmickKEGarnerMMRhyanJBradwayD. Clinicopathologic features of infection with novel Brucella organisms in captive waxy tree frogs (Phyllomedusa Sauvagii) and Colorado River toads (Incilius Alvarius). J Zoo Wildl Med. (2018) 49:153–61. doi: 10.1638/2017-0026R1.1,
10.
ScholzHCMühldorferKShiltonCBenedictSWhatmoreAMBlomJet al. The change of a medically important genus: worldwide occurrence of genetically diverse novel Brucella species in exotic frogs. PLoS One. (2016) 11:e0168872. doi: 10.1371/journal.pone.0168872,
11.
EisenbergTHamannHPKaimUSchlezKSeegerHSchauerteNet al. Isolation of potentially novel Brucella spp. from frogs. Appl Environ Microbiol. (2012) 78:3753–5. doi: 10.1128/AEM.07509-11,
12.
FosterJTBeckstrom-SternbergSMPearsonTBeckstrom-SternbergJSChainPSRobertoFFet al. Whole-genome-based phylogeny and divergence of the genus Brucella. J Bacteriol. (2009) 191:2864–70. doi: 10.1128/JB.01581-08,
13.
MorenoEMiddlebrookEAAltamirano-SilvaPAl DahoukSArajGFArce-GorvelVet al. If you’re not confused, you’re not paying attention: ochrobactrum is not Brucella. J Clin Microbiol. (2023) 61:e0043823. doi: 10.1128/jcm.00438-23,
14.
MorenoE. The one hundred year journey of the genus Brucella (Meyer and Shaw 1920). FEMS Microbiol Rev. (2021) 45:fuaa045. doi: 10.1093/femsre/fuaa045,
15.
YoonSHHaSMLimJKwonSChunJ. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek. (2017) 110:1281–6. doi: 10.1007/s10482-017-0844-4,
16.
Meier-KolthoffJPCarbasseJSPeinado-OlarteRLGökerM. TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acids Res. (2022) 50:D801–7. doi: 10.1093/nar/gkab902,
17.
KellerAAnkenbrandMJ. Inferring Core genome phylogenies for Bacteria. Methods Mol Biol. (2021) 2242:59–68. doi: 10.1007/978-1-0716-1099-2_4,
18.
ChinCSAlexanderDHMarksPKlammerAADrakeJHeinerCet al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods. (2013) 10:563–9. doi: 10.1038/nmeth
19.
MarçaisGKingsfordC. A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics. (2011) 27:764–70. doi: 10.1093/bioinformatics/btr011,
20.
VurtureGWSedlazeckFJNattestadMUnderwoodCJFangHGurtowskiJet al. GenomeScope: fast reference-free genome profiling from short reads. Bioinformatics. (2017) 33:2202–4. doi: 10.1093/bioinformatics/btx153,
21.
ChenYZhangYWangAYGaoMChongZ. Accurate long-read de novo assembly evaluation with inspector. Genome Biol. (2021) 22:312. doi: 10.1186/s13059-021-02527-4,
22.
WalkerBJAbeelTSheaTPriestMAbouellielASakthikumarSet al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. (2014) 9:e112963. doi: 10.1371/journal.pone.0112963,
23.
SimãoFAWaterhouseRMIoannidisPKriventsevaEVZdobnovEM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. (2015) 31:3210–2. doi: 10.1093/bioinformatics/btv351,
24.
AltschulSFGishWMillerWMyersEWLipmanDJ. Basic local alignment search tool. J Mol Biol. (1990) 215:403–10. doi: 10.1016/S0022-2836(05)80360-2,
25.
SeemannT. Prokka: rapid prokaryotic genome annotation. Bioinformatics. (2014) 30:2068–9. doi: 10.1093/bioinformatics/btu153,
26.
Huerta-CepasJSzklarczykDForslundKCookHHellerDWalterMCet al. eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Res. (2016) 44:D286–93. doi: 10.1093/nar/gkv1248,
27.
JonesPBinnsDChangHYFraserMLiWMcAnullaCet al. InterProScan 5: genome-scale protein function classification. Bioinformatics. (2014) 30:1236–40. doi: 10.1093/bioinformatics/btu031,
28.
OlsonRDAssafRBrettinTConradNCucinellCDavisJJet al. Introducing the bacterial and viral bioinformatics resource center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic Acids Res. (2023) 51:D678–89. doi: 10.1093/nar/gkac1003,
29.
RouzicNDesmierLCariouMEGayEFosterJTWilliamsonCHDet al. First case of brucellosis caused by an amphibian-type Brucella. Clin Infect Dis. (2021) 72:e404–7. doi: 10.1093/cid/ciaa1082,
30.
TillerRVGeeJELonswayDRGribbleSBellSCJennisonAVet al. Identification of an unusual Brucella strain (BO2) from a lung biopsy in a 52 year-old patient with chronic destructive pneumonia. BMC Microbiol. (2010) 10:23. doi: 10.1186/1471-2180-10-23,
31.
FischerDLorenzNHeuserWKämpferPScholzHCLierzM. Abscesses associated with a Brucella inopinata-like bacterium in a big-eyed tree frog (Leptopelis vermiculatus). J Zoo Wildl Med. (2012) 43:625–8. doi: 10.1638/2011-0005R2.1,
32.
KimuraMUneYSuzukiMParkESImaokaKMorikawaS. Isolation of Brucella inopinata-like Bacteria from White’s and Denny’s tree frogs. Vector-Borne Zoonot Dis. (2017) 17:297–302. doi: 10.1089/vbz.2016.2027,
33.
ParkSJKimEJeonIKooKS. Commercial breeding of non-native amphibians and reptiles in South Korea: trends and potential ecological risks. Korean J Environ Biol. (2025) 43:527–5. doi: 10.11626/KJEB.2025.43.4.527
34.
ThompsonJDGibsonTJPlewniakFJeanmouginFHigginsDG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. (1997) 25:4876–82. doi: 10.1093/nar/25.24.4876,
35.
KumarSStecherGLiMKnyazCTamuraK. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. (2018) 35:1547–9. doi: 10.1093/molbev/msy096,
Summary
Keywords
atypical Brucella, Brucella inopinata-like strain, frog, phylogenetic analysis, whole genome sequencing
Citation
Kim M, Hmohn ZZW and Han J-I (2026) First report of a Brucella inopinata-like atypical Brucella strain isolated from a waxy monkey treefrog (Phyllomedusa bicolor) in the Republic of Korea: Case Report. Front. Vet. Sci. 13:1814429. doi: 10.3389/fvets.2026.1814429
Received
20 February 2026
Revised
15 June 2026
Accepted
30 June 2026
Published
21 July 2026
Volume
13 - 2026
Edited by
Kumaragurubaran Karthik, Tamil Nadu Veterinary and Animal Sciences University, India
Reviewed by
Sevil Erdenliğ Gürbilek, Harran University, Türkiye
Carolina Cárdenas-Amaya, Universidad Veracruzana, Mexico
Updates
Copyright
© 2026 Kim, Hmohn and Han.
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) and the copyright owner(s) 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: Jae-Ik Han, jihan@jbnu.ac.kr
† Present address: Wut Hmohn, Department of Livestock and Aquaculture Research, Yezin, Myanmar
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.