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. 2025 Nov 18;46(6):1375–1386. doi: 10.24272/j.issn.2095-8137.2025.225

Global genomic insights into the antimicrobial resistance of Escherichia marmotae

Ke-Xin Zhang 1,#, Xiao-He Hu 1,#, Jiang Chang 1,#, Yu Song 1, Min Yue 1, Guo-Ping Zhao 1,2, Biao Tang 1,*
PMCID: PMC12940786  PMID: 41224679

Abstract

Escherichia marmotae has recently emerged as a zoonotic pathogen of growing global significance due to its capacity to acquire and disseminate antimicrobial resistance (AMR). Despite increasing scientific attention, the global distribution, genomic diversity, and evolutionary dynamics of AMR within this species remain largely unknown. This study conducted the first comprehensive global assessment of E. marmotae population structure, distribution, phylogeny, and resistance profiles using 273 high-quality genomes spanning 1950–2025 from the National Center for Biotechnology Information (NCBI) database. Isolates originated from 21 countries, with a pronounced geographic skew toward New Zealand (52.0%), and were predominantly isolated from environmental (61.5%) and animal (30.8%) sources. Phylogenetic reconstruction uncovered extensive cross-species and transboundary transmission, with ST133 representing the most dominant lineage. The overall antimicrobial resistance gene (ARG) carriage rate was 11.7%, including clinically significant β-lactamases such as blaKPC and blaCTX-M. Resistance to aminoglycoside and β-lactam was most frequently observed. Plasmids were identified in 75.8% of isolates, particularly of the IncFIB and IncFII types. Notably, blaKPC-2 was identified in E. marmotae for the first time, predominantly embedded within IS1182- blaTEM-1B-blaKPC-2-IS1182-associated plasmid structures, highlighting its high potential for interspecies gene exchange. This global genomic atlas of E. marmotae offers key insights into the geographic dissemination and interspecies dynamics of AMR within this emerging zoonotic bacterium.

Keywords: Escherichia marmotae, Antimicrobial resistance, Global scale, Genomic characterization

INTRODUCTION

Escherichia marmotae, a Gram-negative bacillus first isolated from the feces of Himalayan marmots (Marmota himalayana) in 2015, was subsequently validated as a distinct species through formal taxonomic classification (Liu et al., 2015). Since its discovery, E. marmotae has been increasingly detected across a broad range of ecological compartments, including wild boar populations, cervid carcasses, and bovine agricultural environments (Nüesch-Inderbinen et al., 2022; Ocejo et al., 2020; Walk et al., 2009). A pivotal case series in 2021 was the first to document invasive infections caused by E. marmotae in humans, manifesting as acute sepsis, postoperative sepsis, spondylodiscitis, and urinary tract infections (Sivertsen et al., 2022). Moreover, the recovery of strain F12YCO47 from fecal samples of asymptomatic individuals demonstrated its capacity for silent intestinal colonization (Ho et al., 2025). These findings collectively implicate E. marmotae as an emerging opportunistic pathogen capable of nosocomial transmission. Its confirmed presence in human, animal, and environmental reservoirs establish a tripartite ecological distribution, reinforcing its classification as a zoonotic threat that requires coordinated surveillance under the One Health paradigm.

Antimicrobial resistance (AMR) poses a mounting global health challenge, primarily attributed to the widespread overuse and misuse of antimicrobial agents (Djordjevic et al., 2024; Hernando-Amado et al., 2019). Within this context, the AMR profile of E. marmotae has received growing attention (Binsker et al., 2024; Nüesch-Inderbinen et al., 2022; Ocejo et al., 2020; Sinha et al., 2024). Commensal strains isolated from wild boars, beef cattle, and other animal hosts have frequently exhibited resistance to multiple antimicrobial classes, including β-lactams (e.g., blaCTX-M, blaTEM-1B, blaSHV12), tetracyclines (e.g., tet(A), tet(B)), sulfonamides (e.g., sul1, sul2), trimethoprim (e.g., dfrA14) (Binsker et al., 2024; Nüesch-Inderbinen et al., 2022; Ocejo et al., 2020). The detection of clinically significant antimicrobial resistance genes (ARGs) encoding extended-spectrum β-lactamases (ESBLs) in E. marmotae raises considerable concern, highlighting both its potential to pose as a significant AMR threat and its capacity to function as a reservoir for resistance determinants contributing to global AMR dissemination (Nüesch-Inderbinen et al., 2022). Nonetheless, the resistance landscape of E. marmotae remains insufficiently explored, leaving major gaps in current understanding.

The emergence of whole-genome sequencing (WGS) has transformed AMR research by enabling precise identification of genetic determinants responsible for resistance phenotypes (Gessese et al., 2025). Beyond delineating the genomic architecture of resistant pathogens, WGS facilitates the detection, comparison, and global sharing of ARG profiles across human, animal, and environmental sectors. This cross-sectoral interoperability supports real-time surveillance of ARG emergence, prevalence, transmission, and evolutionary dynamics, aligning with the One Health objectives and informing targeted public health responses (Djordjevic et al., 2024; One Health High-Level Expert Panel (OHHLEP) et al., 2022).

An increasing number of E. marmotae genomes have been made publicly available, including 273 high-quality assemblies deposited in the National Center for Biotechnology Information (NCBI) database as of 9 July 2025. This dataset provides an opportunity for a global-scale genomic investigation. Based on these data, the present study aimed to: (1) map the spatiotemporal distribution of E. marmotae isolates across countries and sources; (2) resolve its phylogenetic structure and dominant sequence types; (3) identify and contextualize high-risk ARGs and explore their potential transmission mechanisms; and (4) evaluate the global role of E. marmotae in AMR dissemination under the One Health framework. This integrative analysis offers critical insights into the ecological and evolutionary dynamics of AMR in an emerging zoonotic bacterium.

MATERIALS AND METHODS

Data collection and quality control

A total of 273 E. marmotae whole-genome sequences were retrieved from the NCBI database as of 9 July 2025 (Supplementary Table S1). Genome assemblies were subjected to stringent quality control, retaining only those with GC content between 49% and 52%, N50 values exceeding 30 000, sequence lengths ranging from 4 to 6 Mbp, fewer than 500 contigs, and average nucleotide identity (ANI) scores above 95% as determined by FastANI analysis (https://github.com/ParBLiSS/FastANI) (Richter and Rosselló-Móra, 2009) (Supplementary Figure S1). Associated metadata, including geographic origin, sample collection date, and sample source, were extracted from NCBI. Genomes lacking complete metadata were excluded from downstream analyses (Supplementary Figure S1 and Table S1).

Genomic annotation and functional profiling

Multilocus sequence typing (MLST) was conducted using MLST v.2.23.0 (https://github.com/tseemann/mlst) based on the previously established protocols (Larsen et al., 2012). Population relationships were resolved through minimum spanning trees constructed with the MSTree V2 algorithm in GrapeTree v.2.2.0 (Zhou et al., 2018). ARGs were identified using ResFinder v.4.7.2 (Bortolaia et al., 2020), while plasmids were detected using PlasmidFinder v.2.1 (Carattoli et al., 2014). Virulence factors (VFs) were predicted using the ABRicate v.1.0.1 tool with the Virulence Factor Database (VFDB; last accessed 14 October 2020) (Zhou et al., 2025). Gene sequence alignments were visualized using Easyfig v.2.2.5 (Sullivan et al., 2011). Visualization charts were generated using R v.4.2.2.

Phylogenetic analysis

Core genome single-nucleotide polymorphisms (SNPs) were identified using Snippy v.4.6.0 (https://github.com/tseemann/snippy). Maximum-likelihood (ML) phylogenetic reconstruction was conducted with IQ-TREE v.1.6.12 (Nguyen et al., 2015) under the best-fit substitution model selected by ModelFinder, with 1 000 bootstrap replicates to assess nodal support. Recombinant regions were detected and removed using Gubbins v.3.2.1 (Croucher et al., 2015). Population structure was resolved via Bayesian hierarchical clustering using RhierBAPs v.1.0.1 (Tonkin-Hill et al., 2018). The resulting phylogenetic tree was annotated and rendered using the Interactive Tree of Life (iTOL) platform (Letunic & Bork, 2021).

To quantify and compare distinct transmission events and to assess the transmission risk of different subpopulations, SNP-based thresholds were applied: pairwise distances of ≤5 SNPs were defined as clonal transmission, while distances between 6 and 49 SNPs were classified as closely related transmission events (Watt et al., 2025).

RESULTS

Global distribution and source composition of E. marmotae genomes

A total of 281 non-duplicate E. marmotae whole-genome sequences were retrieved from public repositories on 9 July 2025. After applying stringent quality control filters, 273 genomes were retained for downstream analysis (Supplementary Figure S1). These isolates spanned 21 countries, with New Zealand (52.0%, 142/273) contributing the largest proportion (Figure 1A). The earliest available genome, NCTC:8196, dated to 1950 and was derived from the United Kingdom, while a second isolate, dated to 1976, lacked geographic metadata but was submitted by the European Bioinformatics Institute (EBI). Only two genomes predated 2000, with 75 obtained between 2000 and 2019 and 192 collected between 2020 and 2025. Temporal peaks in collection occurred in 2020 (93 isolates) and 2021 (79 isolates), while four isolates lacked collection date metadata (Figure 1B).

Figure 1.

Figure 1

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Geographic location, temporal patterns, and sources of Escherichia marmotae

A: Global strain distribution. B: Collection time and source analysis. C: Sankey diagram of collection countries, times, and sources.

Environmental sources accounted for the majority of isolates (61.5%, 168/273), including feces (n=77), water (n=44), sediment and soil (n=26), biofilm (n=16), leafy greens (n=2), montane grasslands (n=1), and pooled sediment from farm floors (n=2). Animal-derived isolates comprised 30.8% (84/273) (Figure 1B; Supplementary Table S1). Source distribution varied significantly by country. In New Zealand, the United States, and the United Kingdom, isolates were predominantly of environmental origin, whereas the majority of Tunisian strains were derived from animal sources. Although animal and environmental reservoirs dominated the global dataset, human-associated strains were also detected in Norway, the United Kingdom, the United States, and other regions (Figure 1C).

Prevalence patterns and phylogenetic structure of E. marmotae

A total of 45 known sequence types (STs) were identified among the 273 E. marmotae isolates (Figure 2A), with ST133 emerging as the most prevalent (15.4%, 42/273). Five STs exceeded a 4% frequency threshold, including ST133, ST5260, ST2559, ST8370, and ST7347. Geographically, New Zealand exhibited the greatest ST diversity, accounting for 64.44% (29/45), followed by the United Kingdom (26.68%, 12/45) and Germany (24.44%, 11/45) (Supplementary Figure S2). Several STs exhibited clear geographic clustering (Supplementary Figure S2), with ST133 showing the broadest geographic detection in seven out of 21 countries (33.33%). ST5260 exhibited moderate geographic dissemination, appearing in five countries (23.81%). Temporally, ST133 displayed remarkable persistence over a 22-year period (2001–2022) (Figure 2A), while overall ST diversity increased over time (Figure 2A). A decline in diversity in 2022 (n=18), 2023 (n=1), and 2025 (n=1) likely reflected reduced sampling density rather than true contraction. Cross-host transmission was evident for ST133, ST5260, and several other lineages, spanning environmental, animal, and human-associated origins (Figure 2B).

Figure 2.

Figure 2

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Molecular epidemiology of Escherichia marmotae sequence types (STs)

A: Distribution of STs, color-coded by collection year. B: Minimum spanning tree of global E. marmotae, color-coded by collection source.

Bayesian phylogenetic reconstruction delineated the population into seven major lineages (Lineages 1 to 7). Isolates from New Zealand and the United States were distributed across all lineages, suggesting broad genomic heterogeneity. German isolates were concentrated in Lineages 3, 5, 6, and 7, while those from the United Kingdom were present in Lineages 1, 3, 4, 5, 6, and 7. Tunisian isolates were primarily clustered within Lineages 3 and 5 (Figure 3). Environmental strains were broadly distributed across all lineages, whereas animal-derived isolates were predominantly assigned to Lineages 3 and 5. Human-associated genomes were largely confined to Lineages 1 and 3 (Figure 3). Heatmap overlays of VFs and ARGs revealed considerable lineage-specific differences. In particular, Lineage 5 encompassed human and animal strains harboring high levels of both virulence and resistance determinants, while most environmental isolates exhibited reduced VF and ARG burdens. Notably, the cdtA, cdtB, and cdtC genes were present in 45.8% (n=125/273) of isolates (Figure 3), indicating their widespread dissemination.

Figure 3.

Figure 3

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Phylogenetic tree of 273 Escherichia marmotae strains, annotated by geographic distribution, host sources, genetic lineages, and associated ARGs and virulence factors

SNP-based transmission analysis identified 103 clonal transmission events, 66.0% (68/103) of which occurred among isolates from environmental sources, and 40.8% (42/103) of which occurred among animal isolates. All clonal transmission events were confined to a single country, with no cross-border transmission (Figure 4A). When the threshold was relaxed to SNP<50, the number of transmission events increased markedly (Figure 4B). Closely related, non-cross-habitat events were detected with the highest frequency (Environment-Environment, n=192), followed by Animal-Animal (n=56) and the first Human-Human pair. Cross-habitat transmission events were dominated by Environment-Animal interactions (n=58), with an additional 35 Human-to-other-source pairings. Notably, transnational transmission was evident across all event types, with 148 cross-border events involving 14 countries—eight of which contributed less than 2% (Antarctica, Denmark, Poland, Spain, France, Italy, Australia, and Norway). Isolates from New Zealand dominated these events (69.6%, 103/148) (Figure 4C). The smallest SNP distance (n=10) was observed in two isolate pairs within clade A, which spans a 0–38 SNP range and includes genomes collected between 2017 and 2021 (Figures 3, 4D). This clade, which shares an undefined ST, is directly linked to a previously reported clinical isolate (Sivertsen et al., 2022) and encompasses samples derived from three distinct habitats (animal, environment, and human) across three continents (Europe, Oceania, and Antarctica).

Figure 4.

Figure 4

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Transmission dynamics of E. marmotae

A: Inferred transmission events defined by SNP≤5. B: Inferred transmission events defined by 6≤SNP<50. C: Transnational transmission events involving isolates from different countries. D: Temporal distribution of isolation sources and countries for Clade A strains.

For the most common STs, limited genomic similarity was observed among isolates. In ST133 (n=42), only 20.8% (179/861) of pairwise genome comparisons exhibited fewer than 50 SNP differences (Supplementary Table S2). The earliest genome dated to 2005 and the most recent to 2022, demonstrating that this lineage reflects long-term persistence rather than a recent clonal outbreak. ST5260 (n=16) displayed a comparable pattern, with just 24.2% of pairwise comparisons falling below the 50 SNP threshold over a 71-year sampling span (Supplementary Table S2), confirming that it likewise represents an evolutionarily stable lineage rather than a recent monoclonal expansion.

ARG landscape in E. marmotae

Among the 273 analyzed E. marmotae genomes, 11.7% (32/273) harbored acquired ARGs, totaling 96 unique gene copies, corresponding to an average of 0.35 ARGs per strain. These genes conferred resistance to nine antibiotic classes, including aminoglycosides, amphenicols, β-lactams, folate pathway antagonists, fosfomycin, macrolides, quinolones, tetracyclines, and rifamycins. β-lactam resistance genes were most prevalent, detected in 84.4% (27/32) of ARG-positive isolates, followed by aminoglycoside resistance genes in 46.9% (15/32) (Figure 5A). The most frequently encountered ARGs were aph(6)-Id and aph(3'')-Ib (13.5%, 13/96), followed by blaCTX-M-14 (11.5%, 11/96) (Figure 5A).

Figure 5.

Figure 5

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ARGs and plasmids in Escherichia marmotae strains

A: Classification of ARGs. B: Geographic variation in ARGs. C: ARG distribution by host source. D: Prevalence of plasmids. E: Proportion of plasmid-carrying and plasmid-free strains.

ARG abundance varied markedly across geographic regions. Tunisian genomes exhibited the highest ARG density, averaging 3.1 ARG copies per strain (Figure 5B). Source-specific comparisons revealed a pronounced stratification: environmental isolates were numerically dominant (61.5%, 168/273) but harbored the lowest ARG diversity (0.04 ARG copies per strain); animal-derived isolates, though fewer in number (30.8%, 84/273), exhibited higher ARG diversity (0.79 ARG copies per strain across six classes); and human-derived isolates, despite their minimal representation (7.0%, 19/273), showed markedly elevated ARG density (1.3 ARG copies per strain) (Figure 5C).

ARG heatmap analysis demonstrated that most genomes encoded limited ARG repertoires, but a subset (particularly of human origin) exhibited multidrug-resistant profiles (Figure 3). Notably, blaCMY-2 was identified in phylogenetically clustered human and animal isolates, indicating potential cross-species transmission.

Plasmid analysis revealed widespread mobilization potential, with 75.8% (207/273) of E. marmotae strains harboring plasmids. Among these, 58.6% (160/273) contained one or two replicon types, with IncFIB (n=63) and IncFII (n=56) being the most frequently detected; IncX1 was found in a single isolate (Figure 5D, E).

The convergence of ARGs and VFs within specific genomes raises concerns over synergistic enhancement of pathogenicity and resistance evolution. VF heatmaps revealed marked heterogeneity in virulence gene distribution across isolates (Figure 3). Of particular concern, strain CNCTC 8131—isolated from a human in Czechia—co-harbored high loads of VFs and ARGs, including the carbapenemase gene blaKPC-2 and cytolethal distending toxin genes cdtA, cdtB, and cdtC, illustrating the emergence of strains carrying both resistance and virulence determinants, which warrants further attention.

Genetic architecture of blaKPC genes in E. marmotae

A human clinical strain, CNCTC 8131, isolated in 2022 from a patient in Czechia, harbored the clinically significant carbapenemase gene blaKPC-2 (Figure 3). Comparative genomic analysis demonstrated that blaKPC-2 was embedded within a conserved genomic island flanked by mobile genetic elements, forming a composite transposon structure (IS1182-blaTEM-1B-blaKPC-2-IS1182). This multidrug resistance cassette co-localized with tandem conjugative transfer modules on an IncN-type plasmid (unnamed1, accession CP146381.1), indicating a strong potential for horizontal gene dissemination. The blaKPC-2 locus was positioned between IS6 and IS1182 mobile genetic elements (Figure 6A). Detailed homology comparisons with blaKPC-2-carrying plasmids from other strains are provided in Supplementary Table S3.

Figure 6.

Figure 6

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Genomic context of carbapenem resistance genes blaKPC-2 and blaKPC-3

A: blaKPC-2 flanking regions. B: blaKPC-3 flanking region.

In a second clinical isolate, strain 6-77, obtained in 2021 from Portugal, the carbapenemase-encoding gene blaKPC-3 was identified within a distinct mobile genetic element hotspot flanked by IS21 and IS1182. Structural reconstruction revealed a composite transposon (IS21-IS21-blaKPC-3-IS1182), suggesting mobilization through insertion sequence-mediated recombination. The blaKPC-3-harboring plasmid (unnamed1) exhibited conserved structural organization with conjugative plasmids from E. coli and Klebsiella pneumoniae, encoding a complete type IV secretion system that enables transmission via bacterial conjugation. These findings suggest that E. marmotae harbors the potential for blaKPC-3 dissemination via plasmid transfer, suggesting a significant public health risk (Figure 6B).

DISCUSSION

Under the One Health framework, a comprehensive AMR atlas of E. marmotae was constructed based on 273 high-quality genomes spanning human, animal, and environment origins across 21 countries and territories between 1950 and 2025. The global detection of AMR-associated E. marmotae isolates in multiple ecological compartments reflects robust environmental adaptability and its capacity to serve as a conduit for ARG dissemination across the animal-environment-human continuum.

The identification of 45 distinct STs revealed a high degree of genetic heterogeneity, indicative of ongoing adaptive diversification. Among these, ST133 and ST5260 emerged as dominant and epidemiologically relevant lineages. ST133 exhibited broad ecological and geographical dissemination, paralleling the epidemiological success of E. coli ST10 and ST69—clinically important lineages with established dual-host adaptability in animals and humans (Stoesser et al., 2016). Forty-one ST133 isolates were identified across seven countries and three host origins, reflecting sustained transmission capacity across both environmental and host boundaries. This lineage displayed temporal continuity from its earliest record in 2001 (Australia) to its most recent detection in 2022 (Tunisia and the Czech Republic), suggesting sustained persistence over two decades. ST133 encompassed a broad spectrum of host types and regional subpopulations, functioning as a globally distributed “pan-host-pan-region” lineage marked by sustained persistence and transmission. In contrast, ST5260 exhibited strong environmental affinity and a more confined geographic range, predominantly isolated from environmental samples in New Zealand (88.9%, 12/16), suggesting pronounced regional clustering and environmental adaptation. Nevertheless, ST5260 isolates were also identified in the United Kingdom, Germany, France, and Norway, indicating its capacity for long-range transmission. Temporally, this lineage was primarily identified between 2014 and 2021, consistent with an emerging trajectory over the past decade. Both ST133 and ST5260 were detected across human, animal, and environmental sources. While ST133 exhibited broad and frequent cross-host dissemination, ST5260 appeared more likely to mediate subtle, cryptic exchange through environmental pathways. These contrasting transmission patterns underscore the potential for different STs to mediate transmission across hosts and sources. The post-2020 expansion of ST diversity, potentially associated with intensified surveillance, emphasizes the need for detailed characterization of ST-associated genomic signatures.

Phylogenetic analysis revealed distinct lineage differentiation and extensive cross-regional dissemination of E. marmotae. Isolates from New Zealand and the United States were distributed across all seven lineages, consistent with sustained transmission and repeated, independent expansion events. German and British isolates were concentrated in Lineages 3, 5, 6, and 7, but also exhibited moderate diversity, reflecting regionally structured evolutionary dynamics within European populations. Tunisian isolates were primarily clustered within Lineages 3 and 5, likely associated with specific animal hosts (e.g., wild birds) and environmental exposure pathways. Lineage 5 was notable for its enrichment of human- and animal-derived stains co-harboring multiple ARGs and VFs, suggesting the presence of high-risk clonal groups with elevated transmission capacity and pathogenic potential. In contrast, environmental isolates across most lineages exhibited lower ARG and VF burdens, indicating a possible reservoir function with reduced immediate virulence but later capacity for resistance gene propagation within ecosystems. Notably, across the dataset, 45.8% (125/273) of isolates carried the cdtA, cdtB, and cdtC genes, which encode the cytolethal distending toxin capable of inducing cell cycle arrest and DNA damage, thereby contributing to pathogenicity in both humans and animals. The widespread distribution of these genes across divergent lineages suggests evolutionary conservation of this pathogenic strategy in E. marmotae and its potential role in host colonization and disease.

This study provides the most expansive global overview to date of AMR dynamics in E. marmotae, establishing a high-resolution genomic framework for future surveillance. Population-level analysis confirmed that E. marmotae serves as a significant ARG reservoir, with human-derived isolates—despite representing a minority—harboring disproportionately high ARG loads. Geographic variation in ARG distribution was also evidence, with the highest average ARG abundance detected in Tunisian isolates. Potential associations with national antibiotic policies and ecological drivers—such as the role of migratory birds as intercontinental vectors—are plausible but remain speculative due to sampling limitations in certain regions. For instance, Tunisia’s historically elevated antimicrobial consumption contrasts with the European Union’s regulatory constraints (Klein et al., 2018), but causality cannot be inferred from these associations alone. The co-detection of high-risk ARGs such as blaKPC-2 and blaCTX-M-14 with mobile genetic elements, including IncFIB plasmids and IS1182, underscores the role of E. marmotae as a cryptic but consequential vector in AMR ecology. These findings support its prioritization in One Health surveillance frameworks to mitigate future resistance dissemination across interconnected human, animal, and environmental domains.

This study presents the first identification of blaKPC-2 in E. marmotae and offers comparative genomic insights into the architectures of blaKPC-2 and blaKPC-3, representing a critical advancement in global carbapenemase surveillance. Analysis of conserved plasmid backbones revealed blaKPC-2 dissemination via IS6-driven recombination and transposition. Multiple blaKPC-2-harboring plasmids encoded complete conjugative machinery, including traN, traK, virB11, and virB10, supporting autonomous plasmid transfer via conjugation. In contrast, blaKPC-3 was predominantly flanked by IS21 and IS1182, implicating these elements in its mobilization. Notably, high sequence identity was observed in the blaKPC-3 genetic regions of plasmids pWI1-KPC3 and p2-MV931658, indicating IS-mediated horizontal transfer across strains. Conservation of these genetic architectures across phylogenetically distinct hosts suggests that blaKPC-3 transmission is driven by a stable and recurrent mechanism. Key conjugation genes, such as traI, traK, and trbJ, were also encoded, further confirming the intercellular mobility of these plasmids. Collectively, the blaKPC loci were consistently associated with mobile genetic elements, notably insertion sequences (IS1182, ISKpn6, and IS21) that facilitate recombinatorial capture and interspecies transfer. These data demonstrate that blaKPC-2 and blaKPC-3 effectively circulate among bacterial taxa via IS-mediated recombination and conjugative plasmid transfer (Carattoli, 2013; Partridge et al., 2018).

Several factors may act in concert to drive AMR evolution in E. marmotae: At the national level, disparities in antibiotic regulation and usage likely impose divergent selective pressures. Despite potential sampling limitations, isolate-level comparisons across countries and regions revealed substantial variation in both ARG abundance and composition, suggesting that country-specific patterns of antibiotic usage—particularly the types and quantities administered—may influence the selection and propagation of resistant strains. Beyond anthropogenic pressures associated with livestock production, host ecology also adds a layer of complexity. Resistant E. marmotae strains have been isolated from wildlife, particularly migratory birds, which may act as long-distance vectors for the exchange of resistance genes, thereby increasing opportunities for resistant bacteria to emerge and spread. The ecological niche of E. marmotae is also significant due to its close phylogenetic relationship with E. coli—a species known for high environmental prevalence and extensive ARG carriage—raises the likelihood of interspecies gene transfer in shared habitats. Given the near-ubiquitous presence of antimicrobial-resistant E. coli, it represents a potential source of ARGs for E. marmotae in the natural environment. At the molecular level, horizontal gene transfer plays a central role in shaping the E. marmotae resistome. The presence of diverse mobile genetic elements, including plasmids, integrons, and insertion sequences such as ISKpn6 and IS1182 flanking blaKPC-2 and blaKPC-3, highlights the dynamic genetic architecture facilitating ARG mobilization. These elements undoubtedly act as key vectors in horizontal gene transfer and function as significant factors shaping resistance in E. marmotae.

Although originally isolated from marmots, the genomic detection of E. marmotae in cattle (Ocejo et al., 2020) raises concerns over potential foodborne transmission pathways linking wildlife, livestock, and humans. As a wildlife-adapted species, the carriage of multidrug-resistant plasmids by E. marmotae presents a credible risk for horizontal gene transfer across ecological compartments, particularly to clinically relevant pathogens within the animal-environment-human continuum.

The identification of E. marmotae in human clinical samples—especially strains carrying diverse ARGs—underscores the need for cross-sectoral integration of surveillance systems. Wildlife reservoirs, including marmots, have been confirmed as carriers of acquired ARGs, paralleling patterns observed in ESBL-producing E. coli from wild boars (Nüesch-Inderbinen et al., 2022). Environmental routes, particularly waterborne transmission, critically reinforce the interconnectedness of One Health domains and emphasize the necessity for coordinated intervention. Complicating surveillance efforts is the documented misidentification of E. marmotae as E. coli in clinical settings (Sinha et al., 2024; Sivertsen et al., 2022), which may compromise accurate assessment of human-associated AMR risks and necessitate urgent optimization of diagnostic protocols. This misidentification is largely attributed to the close phylogenetic relationship and overlapping phenotypic features of E. marmotae and E. coli, combined with the limited resolution of traditional biochemical identification methods and incomplete MALDI-TOF MS reference databases. Misannotations were also evident in the present dataset, highlighting the importance of genome-based species identification for accurate AMR surveillance and risk assessment.

This global genomic assessment established E. marmotae as a genetically diverse and transferable reservoir of ARGs, with demonstrated capacity for cross-species and environmental dissemination. Given its close phylogenetic proximity to E. coli, E. marmotae is prone to misidentification, thereby obscuring its contribution to resistant infections and undermining public health surveillance. The dissemination of ARGs via E. marmotae raises the likelihood of difficult-to-treat infections, posing a serious threat to global public health security. To mitigate this risk, E. marmotae should be integrated into One Health surveillance systems, with a focus on interfaces linking wildlife, livestock, and human populations. Surveillance strategies should prioritize detection of high-risk plasmids and ARGs hotspots to interrupt transmission across environmental and host boundaries. Effective AMR containment will require coordinated interventions, including the enforcement of antibiotic stewardship policies in agricultural systems and the expansion of genomic surveillance efforts in high-burden regions.

From a One Health perspective, these findings highlight E. marmotae as an underrecognized but potentially multidrug-resistant zoonotic species. Key surveillance blind spots, such as clinical misidentification, limited sequencing coverage in low-income countries, and insufficient environmental screening, may collectively obscure its true epidemiological relevance. To address these deficiencies, E. marmotae should be included on AMR risk watchlists for high-priority regions, supporting the development of open-access genomic surveillance infrastructure and improving taxonomic and diagnostic protocols in both veterinary and clinical microbiology.

CONCLUSION

This study provides the first comprehensive global genomic characterization of E. marmotae, integrating analyses of its genetic diversity, population structure, and antimicrobial resistance profiles. The findings reveal that E. marmotae is broadly distributed across animal, environmental, and human reservoirs, exhibiting substantial phylogenetic diversity and carrying a wide array of ARGs. These features establish it as both an emerging zoonotic pathogen and a cryptic reservoir of clinically relevant resistance determinants. The identification of high-risk ARGs—such as blaKPC-2 and blaCTX-M-14—within mobile genetic contexts underscores its potential to contribute to resistance dissemination across ecological and host boundaries. Given its capacity for environmental persistence, interspecies transmission, and diagnostic misidentification, E. marmotae represents a critical blind spot in current AMR surveillance. Coordinated One Health strategies that integrate genomic monitoring, improved taxonomic resolution, and targeted surveillance at wildlife-livestock-human interfaces are urgently needed to mitigate the risks associated with this overlooked but increasingly relevant pathogen.

SUPPLEMENTARY DATA

Supplementary data to this article can be found online.

zr-46-6-1375-S1.zip (780.5KB, zip)

Acknowledgments

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHORS’ CONTRIBUTIONS

K.X.Z. analyzed the data, performed statistical analyses, and wrote the manuscript. X.H.H. analyzed the data and performed statistical analyses. J.C. revised the manuscript. Y.S. analyzed the data. M.Y. revised the manuscript. G.P.Z. revised the manuscript and provided funding support. B.T. supervised the study, revised the manuscript, and provided funding support. All authors read and approved the final version of the manuscript.

Funding Statement

This work was supported by the “Leading Goose” R&D Program of Zhejiang Province (2023C03045, 2024C03217), Zhejiang Provincial Natural Science Foundation of China (LY23C180001), and Research Funds of Hangzhou Institute for Advanced Study, UCAS (2023HIAS-Y031)

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