One Health Genomic Perspective on Pseudescherichia vulneris: A Neglected Reservoir of Last-Resort Resistance Genes

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Ballaben, Julia M. Cabrera, Leandro M. Moreira, Mick Chandler, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7879921/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Antimicrobial resistance (AMR) is a critical global threat, often driven by horizontal gene transfer mediated by mobile genetic elements (MGEs) such as plasmids, transposons, and integrons. Among Enterobacterales, IncHI2/IncHI2A plasmids are of particular concern, as they combine broad host range, conjugative potential, and mosaic architecture enriched with ARGs, biocide tolerance, and heavy-metal resistance. This study provides the first systematic comparative genomics of Pseudescherichia vulneris , an underrecognized yet genomically versatile species at the human–animal–environment interface. All 30 publicly available genomes were analyzed to reconstruct the pangenome, resistome, virulome, and associated MGEs. The pangenome was open, reflecting ongoing diversification and strong potential for horizontal gene acquisition. Resistomes were highly heterogeneous, ranging from minimal repertoires in most animal and environmental isolates to multidrug-resistance profiles in hospital-associated and occasional animal genomes. Clinically significant determinants, including bla KPC−2 , bla KPC−3 , bla CTX−M−9 , and mcr-9 , were frequently linked to MGEs. bla KPC alleles were mobilized by Tn 4401 -like elements, while mcr-9 occurred either within IncHI2/IncHI2A plasmids or integrated into chromosomal contexts, underscoring diverse mobilization routes. In contrast, the virulome was comparatively conserved, dominated by motility, chemotaxis, and siderophore systems, unlike pathogenic Enterobacterales that carry broad MGE-associated virulence factors. Co-occurrence analyses showed modular independence between resistance and virulence, with limited overlaps shaped by ecological origins, suggesting that resistome content may adapt to distinctive environments. Collectively, these findings establish P. vulneris as a reservoir and conduit of last-resort resistance genes, reinforcing its relevance for One Health surveillance and highlighting the urgent need for its systematic inclusion in global antimicrobial resistance monitoring frameworks. Antimicrobial Resistance Transposable Elements Comparative Genomics Horizontal Gene Transfer One Health Figures Figure 1 Figure 2 Figure 3 1. Introduction Antimicrobial resistance (AMR) is escalating worldwide and now constitutes a critical threat to public health. Its rise is driven by the widespread use of antibiotics and biocides in clinical and agricultural settings, exacerbated by shortcomings in water, sanitation, and hygiene infrastructure (Ho et al., 2024 ). At the core of this crisis is the horizontal gene transfer (HGT) of antimicrobial-resistance genes (ARGs) mediated by mobile genetic elements (MGEs): plasmids, transposons, insertion sequences, and integrons. Integrons can capture and assemble a wide diversity of ARGs into cassette arrays, but they are not self-mobile. Instead, they are frequently mobilized when embedded within transposons or plasmids, thereby accelerating ARG exchange across ecological boundaries (Ross et al., 2021 ). Recognizing the interconnectedness of humans, animals, and the environment, mediated by HGT and MGEs, underscores the indispensability of the One Health framework for AMR surveillance (Djordjevic et al., 2024 ). For instance, among plasmid incompatibility groups, dual-replicon IncHI2/IncHI2A plasmids are particularly concerning. Their large size, broad host range, abundant cargo of MGEs and efficient conjugal transfer enable them to act as backbones for complex resistance islands carrying ARGs, heavy-metal tolerance loci, and biocide-resistance determinants (Algarni et al., 2024 ; Castañeda-Barba et al., 2023 ). Notably, IncHI2/IncHI2A plasmids frequently harbor mcr-9 , a phosphoethanolamine transferase that can confer inducible resistance to colistin, often a last-resort antibiotic (Song et al., 2024 ). Unlike mcr-1 , which is mobilized by the composite transposon Tn 6330 (Snesrud et al., 2018), mcr-9 alleles are embedded in diverse genomic arrangements. They may be bordered by IS 1 or IS 6 family members or captured within class 1 integrons, and their expression often depends on the qseC / qseB regulatory system (Cui et al., 2024 ; Faccone et al., 2020 ; Simoni et al., 2021 ). This inducibility complicates phenotypic surveillance, as mcr-9 carriers can appear colistin-susceptible under standard laboratory conditions. With additional complexity introduced by neighboring MGEs that restructure the genomic context around mcr-9 , its association with IncHI2/IncHI2A plasmids positions these genes as particularly insidious drivers of antimicrobial resistance dissemination across clinical, veterinary, and environmental settings. In this broader context, Pseudescherichia vulneris , formerly known as Escherichia vulneris , emerges as a relevant yet underexplored ARG reservoir. This species, which exhibits substantial genomic divergence from E. coli (Alnajar and Gupta, 2017 ), has been isolated from humans, animals, plants, and contaminated soils, supporting its ecological versatility (Gao et al., 2023 ; Tran and Lee, 2024 ; Zilli et al., 2023 ). Such a broad habitat range, coupled with marked genome plasticity, suggests an enhanced capacity to capture and rearrange mobile genetic elements. For instance, a recently described multidrug-resistant P. vulneris strain from a healthy domestic cat harbors a dual-replicon IncHI2/IncHI2A plasmid densely packed with ARGs, together with metal-tolerance genes and putative virulence factors, integrated in a mosaic architecture shaped by distinct MGEs (Cabral et al., 2025 ). Yet P. vulneris remains virtually absent from routine AMR surveillance, a knowledge gap that may obscure its role as a genetic conduit within microbial communities. More recently, the emergence of a P. vulneris strain carrying bla NDM−5 on an IncX3 plasmid from a veterinary hospital environment (Guangzhou, China) was reported, marking the first detection of a carbapenemase-producing P. vulneris in such settings (Cai et al., 2025 ). This finding reinforces the view that the species acts as an environmental bridge for clinically significant ARGs, including carbapenemases, within the One Health continuum. Therefore, it is possible that P. vulneris functions as an environmental intermediary, silently accumulating clinically important ARGs and virulence determinants and subsequently disseminating them to co-occurring Enterobacterales. High-risk genes such as bla CTX−M , bla KPC , and mcr -9 are likely acquired and maintained within modular platforms, including MGEs and, most prominently, IncHI2/IncHI2A plasmids, reflecting multiple niche-specific events rather than clonal expansion. In this sense, P. vulneris may provide a living example of how underexplored taxa can serve as reservoirs and conduits for the mobilome, embodying the genetic promiscuity that underpins emergence of antimicrobial resistance. Adopting a One Health perspective, this study aims to (i) characterize the resistome and virulome of P. vulneris across ecological and geographical niches, (ii) delineate the structural organization of associated MGEs, (iii) assess its potential for horizontal transmission, and (iv) clarify its overlooked role within the One Health framework. 2. Material and Methods 2.1 Genomes retrieval All publicly available assemblies classified as Pseudescherichia vulneris (including the NCBI reference genome, GCA_902164725.1) were downloaded from GenBank in January 2025. For isolates lacking assemblies, raw sequencing reads labelled “ P. vulneris ” were retrieved from the Sequence Read Archive (SRA). After quality control (see below), a total of 30 high-quality genomes were retained for downstream analyses. 2.2 Read processing, de-novo assembly and annotation Raw reads were quality-filtered with fastp v0.20.1 (Chen et al., 2018 ) using parameters -q 20, ‐l 50, with adapter trimming enabled. Filtered reads were assembled de novo with MEGAHIT v1.2.9 (Li et al., 2016 ) under default parameters. Assembly quality was evaluated with CheckM v1.2.2 (Parks et al., 2015 ). (lineage-specific workflow), and contigs < 500 bp or with coverage < 5× were removed. Gene prediction and functional annotation for all genomes, both newly assembled and downloaded, were generated de novo with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP Docker build 2025-03-15). 2.3 Detection of antimicrobial-resistance and virulence genes Protein-coding sequences were screened with ABRicate v1.0.1 ( https://github.com/tseemann/abricate ) against five curated AMR databases: NCBI AMRFinderPlus (Feldgarden et al., 2021 ), CARD (Alcock et al., 2023 ), ResFinder (Bortolaia et al., 2020 ), ARG-ANNOT (Gupta et al., 2014 ), MEGARES (Doster et al., 2020 ), retaining hits with ≥ 80 % identity and coverage. Plasmid replicon types were identified with PlasmidFinder v2.1 (Carattoli et al., 2014 ). Virulence-associatedgenes (VAGs) were detected with ABRicate against VFDB (Zhou et al., 2025 ). Presence/absence matrices were built for 33 non-redundant ARGs and 29 VAGs observed in ≥ 2 genomes. 2.4 Mobile genetic-element (MGE) annotation MGEs, including ISs, Tn, and In, were manually annotated following TnCentral curation guidelines (Ross et al., 2021 ) using SnapGene v6.2 ( https://www.snapgene.com/ ) and a TnCentral-based custom library. Particular attention was given to the genetic contexts surrounding high-risk loci, containing bla KPC , bla CTX-M , and mcr-9 variants. Flanking IS and structural features were confirmed by manual inspection. 2.5 Pangenome reconstruction and phylogeny The pangenome was generated using Panaroo v1.2.9 (Tonkin-Hill et al., 2020 ) (--clean-mode strict, core threshold 95 %) and analyzed with pansripe v0.3.0 ( https://github.com/gtonkinhill/panstripe ) , both with default parameters. BUSCO analysis was performed on 30 P. vulneris genomes, along with Escherichia coli str. K-12 substr. MG1655 (NC_000913.3) as the outgroup. BUSCO groups detected in all P. vulneris genomes were considered core genes, resulting in 64 single-copy orthologs. The BUSCO_phylogenomics pipeline ( https://github.com/jamiemcg/BUSCO_phylogenomics ) was used to construct a core-gene supermatrix. A maximum-likelihood phylogenetic tree was inferred from this supermatrix using IQ-TREE v2.1.3 (Minh et al., 2020 ) under the GTR + F + I + G4 substitution model, as determined by ModelFinder. Branch support was assessed with 1,000 ultrafast bootstrap replicates, and the tree was rooted using the E. coli outgroup. 2.6 Co-occurrence and correlation analysis Binary presence/absence matrices for the 62 marker genes (33 ARGs, 29 VAGs) across 30 genomes were analyzed in Python 3.9. Absolute co-occurrence counts and Spearman’s ρ were calculated with SciPy v1.10.1. Jaccard distances were converted to similarity scores (1 – distance) using scipy.spatial.distance.jaccard. Gene pairs detected in fewer than five genomes were excluded. Heatmaps were rendered with Matplotlib v3.7.1 and Seaborn v0.11.2, with color intensity indicating correlation magnitude; absolute co-occurrence values were plotted alongside. 2.7 Visualization of genomic structures Circular plasmid maps and linear MGE schematics were generated in SnapGene® v6.2 and Proksee (Grant et al., 2023 ) then refined in Inkscape v1.2. Heatmaps and presence/absence matrices in the final manuscript were compiled in R v4.2.2 (Team, 2023 ) using base functions and ggplot2 for consistent aesthetics. 3. Results 3.1 Genomic Landscape, Isolate Distribution and Accessory-Genome Diversity A search of GenBank (May 2025) retrieved 32 records annotated as P. vulneris (18 assembled genomes and 14 raw read sets). After quality control, two datasets (ERR11550397 and SRR18495312), were excluded after taxonomic verification, as they corresponded to E. coli and Franconibacter spp., respectively. The final genomic catalogue comprised 30 high-quality P. vulneris genomes (Table S1 ). Assembly contiguity varied considerably. Four genomes were nearly complete, each comprising two contigs (e.g., GCA_022049045.1, GCA_026651835.1, and GCA_900450975.1) (one chromosome and one plasmid), while one (e.g., CP166292) contained three contigs (one chromosome and two plasmids). The remaining 26 assemblies (~ 87%) were flagged as draft-quality, ranging from moderately fragmented (e.g., GCA_032069025 with 26 contigs) to highly fragmented (e.g., GCA_032096375 with 1,321 contigs). Most assemblies nonetheless exhibited robust quality metrics, with N50 values > 200 kb; for example, GCA_037145055 and GCA_037145235 reached N50 values of 527 kb and 552 kb, respectively. Isolate sources were predominantly clinical, followed by urban and animal-related environments, with only one genome originating from a plant host and another from an environmental sample. A core-genome alignment of 64 conserved genes was used to construct a maximum-likelihood phylogeny (Fig. 1 ), which resolved five major clades, each showing distinct gene gain and loss (accessory-genome) profiles. Rarefaction curves did not reach saturation, indicating that the P. vulneris pangenome remains open (Figure S1 ). Across the dataset, a total of 100 distinct ARGs were detected (Table S2 ), spanning aminoglycoside, β-lactam, quinolone, macrolide, tetracycline, and sulfonamide classes. In parallel, 29 virulence-associated genes (VAGs) were identified (Table S3 ), encompassing modules for motility (flagellar and chemotaxis systems), iron acquisition ( fepG , entB ), biofilm formation ( csgB , csgG ), and immune evasion. Plasmid replicon typing identified nine incompatibility groups across the 30 genomes, with IncHI2/IncHI2A being the most prevalent, detected in nine hospital-associated isolates. Beyond this group, plasmid diversity in P. vulneris was limited: most genomes carried zero to two replicon types, and replicons such as Col(pHAD28), IncFII(K), Col440I, and Col(IMGS31) appeared only sporadically. Notably, IncF-type plasmids, considered as major drivers of ARG dissemination in Enterobacterales, were rare, detected in only two genomes, both from hospital-associated isolates. 3.2 Diversity, Distribution, and Genomic Context of Clinically Relevant ARGs A curated panel of 33 clinically and epidemiologically relevant ARGs was screened across the 30 genomes (Fig. 1 ). The most prevalent were catA (n = 28), fosA and qacH (n = 25 each). Other recurrent loci included sul1 (n = 9), mcr-9 (n = 8), tetA , ant(3ʺ)-Ia , and qnrA (n = 7 each), as well as bla CTX-M-9 , bla KPC-2 , ant(2ʺ)-Ia , dfrA16 (n = 6 each), and bla KPC-3 (n = 3). While some isolates carried extensive resistomes encompassing ESBLs, carbapenemases, quinolone resistance determinants, and multiple aminoglycoside-modifying enzymes, most animal isolates from Madagascar, as well as several environmental or plant-associated genomes, harbored minimal repertoires, typically limited to fosA , tetA , and qacH . This disparity underscores how resistome size and composition vary sharply according to ecological context. Such variation is largely driven by MGEs, as inspection of genetic neighborhoods revealed structures consistent with known ARG mobilization pathways. Diverse IS elements flanked carbapenemase, ESBL, together with class 1 integrons assembling modular resistance cassettes in IncHI2/IncHI2A plasmids (e.g., aac(6’)-Ib3, aac(6’)-Ib-cr5, bla OXA-1 , catB3, qacE and sul1 previously shown by Cabral et al., 2025 and bla IMP-26 (Figure S2 A)). These results highlight how P. vulneris integrates clinically relevant ARGs within mosaic MGE architectures that may bridge human, animal, and environmental compartments. As an emblematic example, a Tn 4401 -like element was linked to the dissemination of both bla KPC-2 and bla KPC-3 (Fig. 2 A). This element was identified exclusively in hospital-related isolates, including six from the United Kingdom that carried a hypothetical gene located upstream of bla KPC-2 , and three from North America carrying bla KPC-3 . In the North American isolates, the element occurred within a genomic context compatible with plasmid backbones (apparently not IncHI2/IncHI2A), including tra and vir genes and proximity to a plasmid repA , indicating that the bla KPC mobilization may also occur through conjugation (Figure S2 B). By contrast, in the United Kingdom isolates it was not possible to resolve the broader genomic context, except for lineage SRR17302001, where the element was clearly embedded within the bacterial chromosome, indicating that Tn 4401 -like transposons can mediate chromosomal integration of carbapenemase genes, providing an additional route for their long-term stabilization beyond plasmid-borne maintenance. As a contrasting example, six genomes carried bla CTX-M-9 which is driven by the IS Ecp1 resident promoter. Unlike other ARGs, this locus was not associated with transposons, integrons, or other recognizable MGEs. Its conserved arrangement across isolates suggests possible chromosomal integration or stabilization within plasmid loci; however, given the fragmented nature of several assemblies, it is not possible to unambiguously determine whether these contexts correspond to chromosomal or plasmid regions, and no clear evidence of recent mobilization was detected. In the seven genomes carrying qnrA1 , two main organizational patterns were observed. In five genomes (SRR3654271, SRR17302001, SRR18030938, SRR18031180, and SRR18031317), qnrA1 was consistently positioned upstream of ampR , followed by hypA in the reverse orientation and an SMR family transporter in the same orientation as qnrA1 and ampR . This four-gene configuration ( qnrA1–ampR–hypA–SMR ) exhibited highly conserved synteny across the assemblies. By contrast, two genomes (SRR9697017 and SRR18032359) displayed a truncated arrangement in which qnrA1 was adjacent to ampR , but the contigs terminated at the ampR locus, precluding resolution of downstream genes (Figure S2 C). These cases most likely reflect assembly incompleteness rather than genuine structural variation, given the consistent qnrA1–ampR linkage in all seven genomes. Furthermore, seven genomes carried the tetracycline resistance genes tetA and tetR . Among them, only the complete genome of P. vulneris G3 (CP166292) provided a fully resolved context, where tetA and tetR were embedded within a degenerated Tn 3 -family element. In contrast, the six draft genomes (SRR3654271, SRR17302001, SRR18030938, SRR18031180, SRR18031317, and SRR18032359) consistently displayed a shorter arrangement consisting of tetR and tetA , flanked by two reverse orientation IS 6 -family transposases with an intervening small hypothetical ORF (Figure S2 D). The recurrent conservation of this IS6– tetR – tetA module across multiple genomes suggests a common organizational pattern. 3.2.2. mcr-9 Plasticity in Pseudescherichia vulneris: Dissemination through IncHI2/IncHI2A Plasmids and Alternative Genomic Contexts The mcr-9 gene emerged as the only member of the mcr family present in P. vulneris . All genomes carrying mcr-9 are from hospital-related isolates. For instance, mcr-9.2 corresponded to from the United Kingdom (SRR3654271, SRR17302001, SRR18030938, SRR18031180, SRR18031317, SRR18032359), whereas mcr-9.1 was exclusively detected in plasmid-bearing isolates from China (CP086374.1) and in one Australian strain (SRR9697017). In CP086374.1, the mcr -9.1 locus was clearly plasmid-borne within an IncHI2A backbone (Fig. 2 B). By contrast, the genomic context of mcr -9.1 in SRR9697017 could not be fully resolved due to short-read assembly limitations. Nonetheless, this genome carried a single IncHI2A replicon with the same incompatibility group and plasmid sequence type (ST01) as the Chinese plasmid, suggesting that mcr -9.1 is likely embedded in a comparable genetic environment. The two isolates, CP086374.1 (China) and CP166293 (Brazil), contained fully assembled IncHI2A plasmids, enabling direct comparisons of gene content and organization (Fig. 2 B). Both shared a conserved backbone but diverged markedly in accessory regions containing ARG and MGEs: the Brazilian plasmid carried 15 ARGs including those uniquely present in this context ( bla CTX-M-15 , bla OXA-1 , tetA , sul2 , catA1, ant(3”)-Ia, sul2, aph(3”)-Ib, dfrA14 ), whereas the Chinese plasmid encoded 16 ARGs, featuring a distinct set of resistance genes ( mcr-9.1 , bla IMP-26 , bla DHA-1 , aac(6”)-IIc, aac(3)-IIg, ereA2, mphA, mrx, dfrA19, qacEΔ1 , sul1 , tetD ). These differences highlight independent acquisition events and reinforce the modular, mosaic nature of IncHI2A plasmids also occurring in P. vulneris . 3.4 Co-occurrence Patterns of ARGs and VAGs Building on the gene- and plasmid-level analyses, we next explored how ARGs and VAGs are distributed in relation to one another across the 30 genomes. Co-occurrence analysis revealed non-random associations that point to modular resistance–virulence architectures rather than isolated acquisition events. 3.4.1 ARG Gene Repertoire and Co-occurrence The co-occurrence analysis of the 33 acquired ARGs identified in 30 P. vulneris genomes revealed a structured, non-random distribution of resistance determinants (Fig. 3 A). Core modules such as catA , qacH , and fosA were repeatedly detected across multiple isolates. High-risk genes ( bla CTX-M-9 , bla KPC-2 , and mcr-9 ) were each present in eight genomes, with six isolates (20%) carrying all three simultaneously. Additional associations included frequent pairing of mcr-9 with sul1 and ant(3”)-Ia , linkage of tetA with qnrA , and co-occurrence of bla KPC-2 with bla CTX-M-9 . Moreover, dfrA16 and ant(2”)-Ia often clustered alongside β-lactamases and mcr-9 . Notably, several genomes combined carbapenemases and mcr-9 with qac efflux pumps, pointing to the convergence of antibiotic resistance with tolerance to disinfectants and the potential for cross-selection in clinical, veterinary, and environmental contexts. 3.4.2 Virulence Gene Repertoire and Co-occurrence In parallel, the distribution of 29 VAGs was evaluated across the dataset, covering structural and regulatory components of the flagellar machinery, chemotaxis, siderophore transport, curli fimbriae, stress response, and metal resistance. A clear dichotomy between core and accessory components was observed. Genes such as flhC , fliN , flgC , flgG , and flgH were present in ≥ 96% of genomes, defining a conserved virulence backbone. In contrast, accessory genes including clpK1 , nlpI , tlrA , terC , and Z1307 were present in ≤ 50% of genomes, with clpK1 detected in only one isolate. Genes encoding curli fimbriae ( csgB , csgG ) displayed intermediate prevalence. Co-occurrence and hierarchical clustering analysis (Fig. 3 B) revealed a prominent module composed of flagellar and chemotaxis genes ( fliA , fliG , fliH , fliM , fliN , cheB , cheW , cheY ). A partially overlapping cluster comprised siderophore biosynthesis genes ( entB , fepG ) together with curli fimbriae genes ( csgB , csgG ). Accessory loci such as clpK1 , nlpI , tlrA , terC , and Z1307 showed sporadic co-occurrence and weak connectivity. 3.4.3 Integrative ARG–VAG Co-occurrence To investigate potential cross-domain associations, we integrated ARG and VAG datasets into a combined co-occurrence analysis (Fig. 3 C). Overall, overlap between resistance and virulence modules was limited. The chloramphenicol resistance gene catA displayed strong co-occurrence with a cluster of motility and cell envelope–associated genes ( fliJ–nlpI , including fliI and motA ). Similarly, qacH showed moderate co-association with the same cluster, while fosA exhibited weaker associations. The tellurite resistance gene terC showed broader connectivity, co-occurring with multiple ARGs including mcr-9 , sul1 , qnrA , bla KPC-2 , dfrA16 , tetA , ant(3”)-Ia , bla CTX-M-9 , and ant (2”)-Ia . By contrast, clinically significant resistance genes such as bla KPC , bla IMP-26 , and bla CTX-M showed little to no overlap with virulence determinants. Their Jaccard index values, generally below 0.3, indicate that these genes are rarely found in the same genomes as virulence factors, suggesting that in P. vulneris resistance and virulence evolve largely independently rather than being co-selected within the same mobile platforms. Discussion Despite being long overlooked in antimicrobial resistance (AMR) research, Pseudescherichia vulneris displays a genomic architecture that warrants close attention. Analysis of all thirty genomes currently available, reveal an open pangenome, marked heterogeneity of resistomes and virulomes, and a recurrent association of high-risk ARGs with transmissible IncHI2/IncHI2A plasmids. These broad-host-range plasmids frequently carry multiple ARG classes together with disinfectant, biocide, and heavy-metal tolerance loci, and are widely distributed among Enterobacterales worldwide (Alnajar and Gupta, 2017 ). Although this is not the first report of IncHI2 or IncHI2A plasmids in P. vulneris (Cabral et al., 2025 ), our findings demonstrate that they can occur with notable frequency in this lineage, positioning the species as an additional reservoir of resistance genes. Given their conjugative potential (Fang et al., 2016 ) these plasmids provide a robust platform for the long-term maintenance and horizontal transmission of last-resort resistance determinants across distinct Enterobacterales species from different ecological compartments (Algarni et al., 2024 ). Beyond plasmid carriage, P. vulneris harbors diverse ARG repertoires shaped by integron cassettes and transposons directly associated with gene mobilization. For instance, bla KPC-2 and bla KPC-3 were embedded in Tn 4401 -like elements, which are recognized for mobilizing bla KPC genes at high frequency (Cuzon et al., 2011 ), whereas bla IMP-26 , qacEΔ1 , and sul1 were carried within class 1 integrons. Similarly, qnrA1 was consistently linked to ampR , and in most genomes also to hypA and a SMR family transporter, forming a conserved arrangement previously reported in Enterobacterales and often associated with IS CR1 and class 1 integrons (Gomaa Elsayed et al., 2024 ; Jacoby et al., 2015 ). In addition, tet(A) and tetR were invariably associated with flanking IS 6 -family transposases, resembling IS 26 -mediated pseudo-transposons described in Enterobacterales, where these elements promote gene capture, recombination, and stabilization (Blake et al., 2025 ; Cabral et al., 2025 ). By contrast, bla CTX-M-9 showed conserved synteny without recognizable MGEs, suggesting chromosomal integration or stable plasmid maintenance. Together, these observations reflect two contrasting modes of resistance maintenance in P. vulneris : dynamic mobilization via transposons and integrons versus apparent long-term stabilization. Interestingly, several hospital-associated genomes carried high-burden resistance profiles that included bla CTX-M-9 and mcr-9 , whereas most animal and environmental isolates encoded only baseline determinants such as fosA , tetA , and qacH . This contrast suggests that P. vulneris adapts its resistome according to ecological context, accumulating clinically significant ARGs in hospital environments while maintaining minimal repertoires in non-clinical settings. Such ecological plasticity reinforces the need to consider P. vulneris within a One Health surveillance framework, as its ability to shift resistome content across environments positions it as a silent but relevant intermediary in AMR dissemination. Notably, the triad bla KPC-2/3 , bla CTX-M-9 , and mcr-9 occurred together in six genomes (20%), highlighting convergence of resistance to carbapenems, ESBLs, and colistin within single isolates. This co-occurrence mirrors plasmid-borne dissemination of ESBLs and carbapenemases in Klebsiella pneumoniae (Andrade et al., 2018 ). Moreover, the geographic distribution of mcr-9 in this dataset aligns with global surveys, with mcr-9.1 predominating in Asia and the Americas and mcr-9.2 more common in Europe (Song et al., 2024 ). The recurrent association of mcr-9 with IncHI2/IncHI2A plasmids underscores its dissemination potential across diverse Enterobacterales lineages, while occasional chromosomal integration highlights multiple routes of mobilization and maintenance. Compared to classical Enterobacterales , the resistome of P. vulneris appears comparatively simpler and seems to rely predominantly on plasmid acquisition. While K. pneumoniae and E. coli typically combine multiple resistance layers—including chromosomal mutations ( gyrA, parC ), plasmid-borne ESBLs ( bla CTX-M ), diverse carbapenemases (KPC, NDM, OXA-48-like), and multiple mcr alleles (Jana et al., 2017 ; Kerek et al., 2025 )—and Enterobacter spp. possess inducible chromosomal AmpC β-lactamases supplemented by plasmid-borne determinants (De Maayer et al., 2025 ; Teixeira et al., 2025 ), P. vulneris shows no intrinsic β-lactamase arsenal. Instead, it appears to rely on acquisition of IncHI2/IncHI2A plasmids carrying bla KPC-2/3 and mcr-9 as its principal route to clinically relevant resistance. This plasmid-dependent and comparatively “lighter” resistome mirrors its opportunistic ecology and suggests that its epidemiological impact becomes significant only when high-risk plasmids converge within the same host genome. The integrative co-occurrence analysis further highlighted modular independence between resistance and virulence. Core virulence modules, including motility and chemotaxis, were consistently conserved, while siderophore transport and curli fimbriae formed a secondary cluster. Clinically critical resistance determinants ( bla KPC , bla IMP-26 , bla CTX-M , mcr-9 ) rarely overlapped with virulence loci, in contrast to E. coli ExPEC lineages where IncF plasmids often co-carry resistance and adhesins such as fimH (Carattoli, 2013 ; Johnson and Nolan, 2009 ). Only limited bridges were observed, notably catA and qacH with motility or envelope clusters, and terC spanning multiple ARGs. This pattern mirrors modularity reported in environmental Enterobacterales (Manandhar et al., 2022 ), suggesting that resistance and virulence in P. vulneris evolve under distinct selective pressures. In addition, P. vulneris virulence content contrasts with that of major pathogens. E. coli (ExPEC) typically harbors dense pathogenicity islands encoding adhesins ( fimH, papG ), toxins ( hlyA, cnf1 ), siderophores (aerobactin, salmochelin), and secretion systems (T3SS, T6SS) (Biggel et al., 2020 ; Desvaux et al., 2020 ). K. pneumoniae often acquires hypervirulent plasmids with regulators ( rmpA/rmpA2 ), potent siderophores ( iuc, iro, ybt ), and genotoxins such as clb (Hetta et al., 2025 ; Rahmat Ullah et al., 2024 ). Enterobacter spp., particularly the E. cloacae complex, maintain robust repertoires enriched in adhesion, biofilm, and immune evasion (Manandhar et al., 2022 ). By contrast, P. vulneris displays a fragmented and ecologically oriented virulome, largely restricted to motility, chemotaxis, environmental sensing, and basic adhesion. The absence of pathogenicity islands or horizontally acquired high-virulence modules suggests an environmentally adapted, opportunistic lifestyle. In line with our observations, Cai et al. ( 2025 ) recently described the emergence of bla NDM-5 -carrying P. vulneris and Pantoea dispersa isolated from a veterinary hospital environment in China, expanding the known diversity of carbapenemase contexts in this species. Their detection of an IncX3 plasmid carrying bla NDM-5 further supports the conclusion that P. vulneris can acquire and maintain distinct plasmid backbones for disseminating high-risk ARGs. Together with our findings, these data confirm that P. vulneris is not a passive environmental commensal but an adaptable vector linking clinical, veterinary, and environmental reservoirs within the One Health network. By systematically analyzing all genomes currently available for P. vulneris , this study establishes a much-needed foundation for understanding its mobilome and resistance potential. Although the dataset remains small (30 genomes) and biased toward hospital contexts, with fragmented assemblies and missing metadata, these constraints primarily affect resolution of mobile element boundaries rather than the broader conclusions. The absence of phenotypic data for colistin susceptibility further highlights gaps that future studies should address. Nonetheless, the findings are consistent with broader surveys showing that ARG–virulence co-localization is more common in pathogens associated with human and animal hosts than in environmental speciess (Pan et al., 2020 ), and that horizontal transfer is shaped by both genetic compatibility and ecological connectivity (Lund et al., 2025 ). Paradoxically, such gaps explain why P. vulneris has long been overlooked in AMR research. Despite this neglect, our analyses uncovered high-risk ARGs, stabilized mcr-9 loci, and IncHI2/IncHI2A plasmids with mosaic architectures, underscoring that P. vulneris is unlikely to be only a passive commensal. Conclusions Pseudescherichia vulneris emerges as an underrecognized yet genomically versatile member of Enterobacterales, equipped with a broad mobilome and capable of harboring clinically significant resistance determinants. Its pangenome remains open, reflecting ongoing diversification, while the resistome spans from baseline determinants ( fosA, tetA, qacH ) in non-clinical isolates to multidrug-resistance profiles in hospital-associated and occasional animal strains. Importantly, bla KPC-2/3 , bla CTX-M-9 , and mcr-9 were recurrently detected, with mcr-9 embedded in IncHI2/IncHI2A plasmids and, in some cases, chromosomal contexts, underscoring multiple routes of mobilization and stabilization. In contrast to epidemic pathogens such as Escherichia coli and Klebsiella pneumoniae , P. vulneris lacks an intrinsic β-lactamase arsenal or high-virulence pathogenicity islands. Its resistome appears comparatively “lighter” and plasmid-dependent, suggesting that its epidemiological relevance becomes critical only when high-risk plasmids converge within the same genome. This opportunistic profile reflects an environmentally adapted lifestyle but also highlights its capacity to act as a silent reservoir for last-resort resistance genes. From a One Health perspective, the recurrent association of high-risk ARGs with transmissible IncHI2/IncHI2A plasmids—structures enriched in disinfectant, biocide, and heavy-metal tolerance loci—represents a durable chassis for cross-sector dissemination. Recognizing P. vulneris in genomic surveillance is therefore essential, as expanding monitoring to include this neglected lineage will improve the anticipation of multidrug-resistant platforms bridging clinical, veterinary, and environmental compartments. Declarations CRediT AUTHORSHIP CONTRIBUTION STATEMENT Anelise Stella Ballaben : Writing - original draft, Visualization, Resources, Methodology, Investigation, Formal analysis, Data curation. Julia M. Cabrera : Methodology, Writing – review & editing. Mick Chandler: Writing – review & editing, Data curation. Leandro M. Moreira: Writing – review & editing. Alessandro M. Varani: Writing – review & editing, Methodology, Investigation, Validation, Formal analysis, Data curation. FUNDING A.S.B and J.M.C were supported by a post-doctoral and doctoral fellowship from FAPESP [grant #2023/08702-6 and #2023/10686-9, respectively]. CONFLICT OF INTEREST None to declare. ACKNOWLEDGEMENTS We thank the National Council for Scientific and Technological Development (CNPq), Brazil and the São Paulo State Research Foundation (FAPESP, São Paulo). References Alcock, B.P., Huynh, W., Chalil, R., Smith, K.W., Raphenya, A.R., Wlodarski, M.A., Edalatmand, A., Petkau, A., Syed, S.A., Tsang, K.K., Baker, S.J.C., Dave, M., Mccarthy, M.C., Mukiri, K.M., Nasir, J.A., Golbon, B., Imtiaz, H., Jiang, X., Kaur, K., Kwong, M., Liang, Z.C., Niu, K.C., Shan, P., Yang, J.Y.J., Gray, K.L., Hoad, G.R., Jia, B., Bhando, T., Carfrae, L.A., Farha, M.A., French, S., Gordzevich, R., Rachwalski, K., Tu, M.M., Bordeleau, E., Dooley, D., Griffiths, E., Zubyk, H.L., Brown, E.D., Maguire, F., Beiko, R.G., Hsiao, W.W.L., Brinkman, F.S.L., Van Domselaar, G., Mcarthur, A.G., 2023. CARD 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database. 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Presence/absence matrix of 100 ARGs across 30 genomes, grouped by antimicrobial class. Genome identifiers correspond to those listed in Supplementary Table 1. Only hits with ≥80% identity and ≥80% coverage across at least one reference database (AMRFinderPlus, CARD, ResFinder, ARG-ANNOT, or MEGARes) were retained. TableS3.xlsx Supplementary Table 3. Virulence-associated genes (VAGs) identified in P. vulneris Presence/absence matrix of 29 predicted VAGs, including genes associated with motility, iron acquisition, biofilm formation, stress response, and immune evasion. Genes are categorized as core (detected in ≥95% of genomes) or accessory (detected in <95% of genomes). FigureS1.png Supplementary Figure 1. Rarefaction curve of the Pseudescherichia vulneris pangenome (n = 30). The solid line represents the mean accessory-gene diversity, and the shaded area shows standard deviation across permutations. The absence of saturation suggests an open pangenome. FigureS2.jpg Supplementary Figure 2. Genetic contexts of selected ARGs identified in Pseudescherichia vulneris . (A) Class 1 integron-associated resistance regions. Representative arrangements of resistance loci within IncHI2/IncHI2A plasmid (CP086375.1), showing modular cassettes composed of bla IMP-26 , qacEΔ1, and sul1 . (B) Tn 4401-like elements carrying bla KPC-3 . The element was located within regions compatible with plasmid backbones, adjacent to tra and vir genes and near a plasmidic repA , suggesting potential conjugative mobilization. (C) qnrA1 genomic contexts. Five assemblies (SRR3654271, SRR17302001, SRR18030938, SRR18031180, SRR18031317) displayed a conserved four-gene configuration ( qnrA1–ampR–hypA–SMR ), while two (SRR9697017 and SRR18032359) showed truncated contigs containing qnrA1 and ampR only. (D) tetR – tetA genes. The complete plasmid of strain G3 (CP166292) is shown as reference, revealing tetR and tetA embedded within two inverse oriented IS 6 elements The draft genomes (SRR3654271, SRR17302001, SRR18030938, SRR18031180, SRR18031317, and SRR18032359) displayed similar and conserved arrangement consisting of tetR and tetA , and fragments of both IS 6. 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08:04:02","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":157687,"visible":true,"origin":"","legend":"","description":"","filename":"c9179399431743f6bc8c91e420d0ab411structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7879921/v1/52207fb1973eb50198e4ef84.xml"},{"id":95178939,"identity":"917b0f37-dced-4a22-9533-8cb0c730230d","added_by":"auto","created_at":"2025-11-05 08:04:02","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172192,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7879921/v1/b24e1beab438cf80d14bd176.html"},{"id":95178927,"identity":"74d66464-3b75-4387-97c2-6a6578fdbdb4","added_by":"auto","created_at":"2025-11-05 08:04:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2929578,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogeny, gene gain–loss dynamics, and distribution of resistance genes and plasmid replicons in Pseudescherichia vulneris. From left to right: a maximum-likelihood phylogeny of 30 P. vulneris genomes reconstructed from 64 core genes using IQ-TREE. Branch colors correspond to panstripe analysis, with gradients indicating relative rates of gene gain (yellow) and loss (dark shades); the gain–loss scale is shown in the figure legend. Terminal labels include isolate identifiers with year and country of isolation, and are accompanied by a color strip denoting the ecological source (clinical/human, animal, environmental, urban, plant, or unknown). To the right of the tree, a red–white heatmap depicts the presence (red) or absence of 33 clinically and epidemiologically relevant ARGs. Further to the right, a gray-scale matrix shows the presence of ten plasmid replicon types, with shading indicating detected replicon groups. Together, the visualization highlights phylogenetic structure, ecological origins, and the heterogeneous distribution of ARGs and plasmid backbones across the species.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7879921/v1/00f9413726e1227872da09a5.jpg"},{"id":95178925,"identity":"45ac6069-744e-4f0b-a164-6d2d9fe83ba0","added_by":"auto","created_at":"2025-11-05 08:04:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4269415,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic structures of Tn\u003cem\u003e4401\u003c/em\u003e transposons carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e alleles and comparative organization of IncHI2A plasmids in Pseudescherichia vulneris.\u003cstrong\u003e\u003cbr\u003e\n(A)\u003c/strong\u003e Comparative analysis of Tn\u003cem\u003e4401\u003c/em\u003e structures using SnapGene. Six isolates harbored bla\u003csub\u003eKPC-2\u003c/sub\u003e, whereas two carried bla\u003csub\u003eKPC-3\u003c/sub\u003e. The H272Y substitution, which differentiates the two alleles, is highlighted. Notably, bla\u003csub\u003eKPC-3\u003c/sub\u003e–positive isolates contained an exclusive upstream open reading frame encoding a hypothetical protein not observed in bla\u003csub\u003eKPC-2\u003c/sub\u003e contexts. \u003cstrong\u003e(B) \u003c/strong\u003eCircular maps of the two fully resolved IncHI2A plasmids identified in P. vulneris. Despite sharing a conserved backbone, they displayed marked diversity in accessory resistance regions. The plasmid from China carried mcr-9 together with multiple ARGs, whereas the plasmid from a domestic animal in Brazil lacked mcr-9, underscoring independent acquisition events.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7879921/v1/6993600603284aa4bbbb9e5c.jpg"},{"id":95228405,"identity":"af2dd2da-ae29-42a4-8316-5b4206c7c12d","added_by":"auto","created_at":"2025-11-05 16:33:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4026015,"visible":true,"origin":"","legend":"\u003cp\u003eIntegrative co-occurrence analyses of ARGs and VAGs in Pseudescherichia vulneris. \u003cstrong\u003e(A) \u003c/strong\u003ePairwise co-occurrence matrix of 33 clinically relevant ARGs across 30 genomes, based on absolute frequency of co-detection. Clustering highlights distinct modules, including the recurrent association of catA, qacH, and fosA \u003cstrong\u003e(B) \u003c/strong\u003eCo-occurrence matrix of 29 VAGs, revealing a conserved core module comprising motility and chemotaxis genes (e.g., flhA, fliM, cheY) and a more variable distribution of accessory factors such as clpK1 and tlrA. \u003cstrong\u003e(C) \u003c/strong\u003eCross-domain co-occurrence matrix between ARGs and VAGs, measured by Jaccard similarity. Most ARG–VAG pairs showed minimal overlap, but catA, qacH, and fosA exhibited moderate associations with structural and motility-related genes, suggesting ecologically driven co-distribution. The tellurite-resistance gene terC displayed broad connectivity, co-occurring with multiple ARGs (mcr-9, sul1, bla\u003csub\u003eCTX-M-9,\u003c/sub\u003e dfrA16), underscoring its potential role in multidrug-resistance platforms. Color scales indicate absolute co-occurrence frequency (\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e) or Jaccard similarity values (\u003cstrong\u003eC\u003c/strong\u003e), with hierarchical clustering applied to both rows and columns.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7879921/v1/038b89f3de91b73f010537ac.jpg"},{"id":95312268,"identity":"a2b31027-17a1-4b5e-ba39-86a5304fef2f","added_by":"auto","created_at":"2025-11-06 15:48:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12224354,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7879921/v1/d6a3e2e2-2a61-4236-84f7-b803087b6141.pdf"},{"id":95226647,"identity":"0f5cc058-29e3-4434-8fbc-f02f9f0d5a5b","added_by":"auto","created_at":"2025-11-05 16:31:32","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9344,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 1. Metadata and assembly characteristics of the 30 Pseudescherichia vulneris genomes analyzed. Includes NCBI accession numbers, assembly status (complete or draft), year and country of isolation, host/source of origin, and taxonomic identification method.\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7879921/v1/dda3044d6175f77132034971.xlsx"},{"id":95228506,"identity":"6c54f7f9-ea39-4e90-bee4-0bb4c3b694f7","added_by":"auto","created_at":"2025-11-05 16:33:50","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19971,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2. Antimicrobial resistance genes (ARGs) identified in P. vulneris. Presence/absence matrix of 100 ARGs across 30 genomes, grouped by antimicrobial class. Genome identifiers correspond to those listed in Supplementary Table 1. Only hits with ≥80% identity and ≥80% coverage across at least one reference database (AMRFinderPlus, CARD, ResFinder, ARG-ANNOT, or MEGARes) were retained.\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7879921/v1/c528ecca8188a435cbcd9ccc.xlsx"},{"id":95227099,"identity":"434038d0-1360-4e12-9be9-bd803eb28c6a","added_by":"auto","created_at":"2025-11-05 16:32:06","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12925,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 3. Virulence-associated genes (VAGs) identified in P. vulneris Presence/absence matrix of 29 predicted VAGs, including genes associated with motility, iron acquisition, biofilm formation, stress response, and immune evasion. Genes are categorized as core (detected in ≥95% of genomes) or accessory (detected in \u0026lt;95% of genomes).\u003c/p\u003e","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7879921/v1/00f5fb0e5869afe12550613d.xlsx"},{"id":95178923,"identity":"311ae445-032c-4d58-8827-38ef6e284cdf","added_by":"auto","created_at":"2025-11-05 08:04:02","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":32207,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 1. Rarefaction curve of the \u003cem\u003ePseudescherichia vulneris\u003c/em\u003e pangenome (n = 30). The solid line represents the mean accessory-gene diversity, and the shaded area shows standard deviation across permutations. The absence of saturation suggests an open pangenome.\u003c/p\u003e","description":"","filename":"FigureS1.png","url":"https://assets-eu.researchsquare.com/files/rs-7879921/v1/158c9de2f79a9915ff88808c.png"},{"id":95178938,"identity":"f54b9aad-446c-45c1-933a-a31f8dc410a8","added_by":"auto","created_at":"2025-11-05 08:04:02","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":6729601,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 2.\u0026nbsp; Genetic contexts of selected ARGs identified in \u003cem\u003ePseudescherichia vulneris\u003c/em\u003e.\u0026nbsp;\u0026nbsp; \u003cstrong\u003e(A) \u003c/strong\u003eClass 1 integron-associated resistance regions. Representative arrangements of resistance loci within IncHI2/IncHI2A plasmid (CP086375.1), showing modular cassettes composed of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP-26\u003c/sub\u003e, \u003cem\u003eqacEΔ1, \u003c/em\u003eand\u003cem\u003e sul1\u003c/em\u003e. \u003cstrong\u003e(B) \u003c/strong\u003eTn\u003cem\u003e4401-like\u003c/em\u003e elements carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-3\u003c/sub\u003e. The element was located within regions compatible with plasmid backbones, adjacent to \u003cem\u003etra\u003c/em\u003e and \u003cem\u003evir\u003c/em\u003e genes and near a plasmidic \u003cem\u003erepA\u003c/em\u003e, suggesting potential conjugative mobilization. \u003cstrong\u003e(C) \u003c/strong\u003e\u003cem\u003eqnrA1\u003c/em\u003e genomic contexts. Five assemblies (SRR3654271, SRR17302001, SRR18030938, SRR18031180, SRR18031317) displayed a conserved four-gene configuration (\u003cem\u003eqnrA1–ampR–hypA–SMR\u003c/em\u003e), while two (SRR9697017 and SRR18032359) showed truncated contigs containing \u003cem\u003eqnrA1\u003c/em\u003e and \u003cem\u003eampR\u003c/em\u003e only. \u003cstrong\u003e(D) \u003c/strong\u003e\u003cem\u003etetR\u003c/em\u003e–\u003cem\u003etetA genes.\u003c/em\u003e The complete plasmid of strain G3 (CP166292) is shown as reference, revealing \u003cem\u003etetR\u003c/em\u003e and \u003cem\u003etetA\u003c/em\u003e embedded within two inverse oriented IS\u003cem\u003e6\u003c/em\u003e elements The draft genomes (SRR3654271, SRR17302001, SRR18030938, SRR18031180, SRR18031317, and SRR18032359) displayed similar and conserved arrangement consisting of \u003cem\u003etetR\u003c/em\u003e and \u003cem\u003etetA\u003c/em\u003e, and fragments of both IS\u003cem\u003e6.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7879921/v1/d888ae4f06de90e5ec14f9bb.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"One Health Genomic Perspective on Pseudescherichia vulneris: A Neglected Reservoir of Last-Resort Resistance Genes","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAntimicrobial resistance (AMR) is escalating worldwide and now constitutes a critical threat to public health. Its rise is driven by the widespread use of antibiotics and biocides in clinical and agricultural settings, exacerbated by shortcomings in water, sanitation, and hygiene infrastructure (Ho et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). At the core of this crisis is the horizontal gene transfer (HGT) of antimicrobial-resistance genes (ARGs) mediated by mobile genetic elements (MGEs): plasmids, transposons, insertion sequences, and integrons. Integrons can capture and assemble a wide diversity of ARGs into cassette arrays, but they are not self-mobile. Instead, they are frequently mobilized when embedded within transposons or plasmids, thereby accelerating ARG exchange across ecological boundaries (Ross et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recognizing the interconnectedness of humans, animals, and the environment, mediated by HGT and MGEs, underscores the indispensability of the One Health framework for AMR surveillance (Djordjevic et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor instance, among plasmid incompatibility groups, dual-replicon IncHI2/IncHI2A plasmids are particularly concerning. Their large size, broad host range, abundant cargo of MGEs and efficient conjugal transfer enable them to act as backbones for complex resistance islands carrying ARGs, heavy-metal tolerance loci, and biocide-resistance determinants (Algarni et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Casta\u0026ntilde;eda-Barba et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, IncHI2/IncHI2A plasmids frequently harbor \u003cem\u003emcr-9\u003c/em\u003e, a phosphoethanolamine transferase that can confer inducible resistance to colistin, often a last-resort antibiotic (Song et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUnlike \u003cem\u003emcr-1\u003c/em\u003e, which is mobilized by the composite transposon Tn\u003cem\u003e6330\u003c/em\u003e (Snesrud et al., 2018), \u003cem\u003emcr-9\u003c/em\u003e alleles are embedded in diverse genomic arrangements. They may be bordered by IS\u003cem\u003e1\u003c/em\u003e or IS\u003cem\u003e6\u003c/em\u003e family members or captured within class 1 integrons, and their expression often depends on the \u003cem\u003eqseC\u003c/em\u003e/\u003cem\u003eqseB\u003c/em\u003e regulatory system (Cui et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Faccone et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Simoni et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This inducibility complicates phenotypic surveillance, as \u003cem\u003emcr-9\u003c/em\u003e carriers can appear colistin-susceptible under standard laboratory conditions. With additional complexity introduced by neighboring MGEs that restructure the genomic context around \u003cem\u003emcr-9\u003c/em\u003e, its association with IncHI2/IncHI2A plasmids positions these genes as particularly insidious drivers of antimicrobial resistance dissemination across clinical, veterinary, and environmental settings.\u003c/p\u003e\u003cp\u003eIn this broader context, \u003cem\u003ePseudescherichia vulneris\u003c/em\u003e, formerly known as \u003cem\u003eEscherichia vulneris\u003c/em\u003e, emerges as a relevant yet underexplored ARG reservoir. This species, which exhibits substantial genomic divergence from \u003cem\u003eE. coli\u003c/em\u003e (Alnajar and Gupta, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), has been isolated from humans, animals, plants, and contaminated soils, supporting its ecological versatility (Gao et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Tran and Lee, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zilli et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Such a broad habitat range, coupled with marked genome plasticity, suggests an enhanced capacity to capture and rearrange mobile genetic elements. For instance, a recently described multidrug-resistant \u003cem\u003eP. vulneris\u003c/em\u003e strain from a healthy domestic cat harbors a dual-replicon IncHI2/IncHI2A plasmid densely packed with ARGs, together with metal-tolerance genes and putative virulence factors, integrated in a mosaic architecture shaped by distinct MGEs (Cabral et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Yet \u003cem\u003eP. vulneris\u003c/em\u003e remains virtually absent from routine AMR surveillance, a knowledge gap that may obscure its role as a genetic conduit within microbial communities.\u003c/p\u003e\u003cp\u003eMore recently, the emergence of a \u003cem\u003eP. vulneris\u003c/em\u003e strain carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;5\u003c/sub\u003e on an IncX3 plasmid from a veterinary hospital environment (Guangzhou, China) was reported, marking the first detection of a carbapenemase-producing \u003cem\u003eP. vulneris\u003c/em\u003e in such settings (Cai et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This finding reinforces the view that the species acts as an environmental bridge for clinically significant ARGs, including carbapenemases, within the One Health continuum.\u003c/p\u003e\u003cp\u003eTherefore, it is possible that \u003cem\u003eP. vulneris\u003c/em\u003e functions as an environmental intermediary, silently accumulating clinically important ARGs and virulence determinants and subsequently disseminating them to co-occurring Enterobacterales. High-risk genes such as \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e, and \u003cem\u003emcr\u003c/em\u003e-9 are likely acquired and maintained within modular platforms, including MGEs and, most prominently, IncHI2/IncHI2A plasmids, reflecting multiple niche-specific events rather than clonal expansion. In this sense, \u003cem\u003eP. vulneris\u003c/em\u003e may provide a living example of how underexplored taxa can serve as reservoirs and conduits for the mobilome, embodying the genetic promiscuity that underpins emergence of antimicrobial resistance. Adopting a One Health perspective, this study aims to (i) characterize the resistome and virulome of P. vulneris across ecological and geographical niches, (ii) delineate the structural organization of associated MGEs, (iii) assess its potential for horizontal transmission, and (iv) clarify its overlooked role within the One Health framework.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Genomes retrieval\u003c/h2\u003e\u003cp\u003eAll publicly available assemblies classified as \u003cem\u003ePseudescherichia vulneris\u003c/em\u003e (including the NCBI reference genome, GCA_902164725.1) were downloaded from GenBank in January 2025. For isolates lacking assemblies, raw sequencing reads labelled \u0026ldquo;\u003cem\u003eP. vulneris\u003c/em\u003e\u0026rdquo; were retrieved from the Sequence Read Archive (SRA). After quality control (see below), a total of 30 high-quality genomes were retained for downstream analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Read processing, \u003cem\u003ede-novo\u003c/em\u003e assembly and annotation\u003c/h2\u003e\u003cp\u003eRaw reads were quality-filtered with fastp v0.20.1 (Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) using parameters -q 20, ‐l 50, with adapter trimming enabled. Filtered reads were assembled de novo with MEGAHIT v1.2.9 (Li et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) under default parameters. Assembly quality was evaluated with CheckM v1.2.2 (Parks et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). (lineage-specific workflow), and contigs\u0026thinsp;\u0026lt;\u0026thinsp;500 bp or with coverage\u0026thinsp;\u0026lt;\u0026thinsp;5\u0026times; were removed. Gene prediction and functional annotation for all genomes, both newly assembled and downloaded, were generated \u003cem\u003ede novo\u003c/em\u003e with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP Docker build 2025-03-15).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Detection of antimicrobial-resistance and virulence genes\u003c/h2\u003e\u003cp\u003eProtein-coding sequences were screened with ABRicate v1.0.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tseemann/abricate\u003c/span\u003e\u003cspan address=\"https://github.com/tseemann/abricate\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e against five curated AMR databases: NCBI AMRFinderPlus (Feldgarden et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), CARD (Alcock et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), ResFinder (Bortolaia et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), ARG-ANNOT (Gupta et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), MEGARES (Doster et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), retaining hits with \u0026ge;\u0026thinsp;80 % identity and coverage. Plasmid replicon types were identified with PlasmidFinder v2.1 (Carattoli et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Virulence-associatedgenes (VAGs) were detected with ABRicate against VFDB (Zhou et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Presence/absence matrices were built for 33 non-redundant ARGs and 29 VAGs observed in \u0026ge;\u0026thinsp;2 genomes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Mobile genetic-element (MGE) annotation\u003c/h2\u003e\u003cp\u003eMGEs, including ISs, Tn, and In, were manually annotated following TnCentral curation guidelines (Ross et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) using SnapGene v6.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.snapgene.com/\u003c/span\u003e\u003cspan address=\"https://www.snapgene.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e and a TnCentral-based custom library. Particular attention was given to the genetic contexts surrounding high-risk loci, containing \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M\u003c/sub\u003e, and \u003cem\u003emcr-9\u003c/em\u003e variants. Flanking IS and structural features were confirmed by manual inspection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Pangenome reconstruction and phylogeny\u003c/h2\u003e\u003cp\u003eThe pangenome was generated using Panaroo v1.2.9 (Tonkin-Hill et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (--clean-mode strict, core threshold 95 %) and analyzed with pansripe v0.3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/gtonkinhill/panstripe\u003c/span\u003e\u003cspan address=\"https://github.com/gtonkinhill/panstripe\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, both with default parameters. BUSCO analysis was performed on 30 \u003cem\u003eP. vulneris\u003c/em\u003e genomes, along with \u003cem\u003eEscherichia coli\u003c/em\u003e str. K-12 substr. MG1655 (NC_000913.3) as the outgroup. BUSCO groups detected in all \u003cem\u003eP. vulneris\u003c/em\u003e genomes were considered core genes, resulting in 64 single-copy orthologs. The BUSCO_phylogenomics pipeline (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/jamiemcg/BUSCO_phylogenomics\u003c/span\u003e\u003cspan address=\"https://github.com/jamiemcg/BUSCO_phylogenomics\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to construct a core-gene supermatrix. A maximum-likelihood phylogenetic tree was inferred from this supermatrix using IQ-TREE v2.1.3 (Minh et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) under the GTR\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G4 substitution model, as determined by ModelFinder. Branch support was assessed with 1,000 ultrafast bootstrap replicates, and the tree was rooted using the \u003cem\u003eE. coli\u003c/em\u003e outgroup.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Co-occurrence and correlation analysis\u003c/h2\u003e\u003cp\u003eBinary presence/absence matrices for the 62 marker genes (33 ARGs, 29 VAGs) across 30 genomes were analyzed in Python 3.9. Absolute co-occurrence counts and Spearman\u0026rsquo;s ρ were calculated with SciPy v1.10.1. Jaccard distances were converted to similarity scores (1 \u0026ndash; distance) using scipy.spatial.distance.jaccard. Gene pairs detected in fewer than five genomes were excluded. Heatmaps were rendered with Matplotlib v3.7.1 and Seaborn v0.11.2, with color intensity indicating correlation magnitude; absolute co-occurrence values were plotted alongside.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Visualization of genomic structures\u003c/h2\u003e\u003cp\u003eCircular plasmid maps and linear MGE schematics were generated in SnapGene\u0026reg; v6.2 and Proksee (Grant et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) then refined in Inkscape v1.2. Heatmaps and presence/absence matrices in the final manuscript were compiled in R v4.2.2 (Team, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) using base functions and ggplot2 for consistent aesthetics.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Genomic Landscape, Isolate Distribution and Accessory-Genome Diversity\u003c/h2\u003e\u003cp\u003eA search of GenBank (May 2025) retrieved 32 records annotated as \u003cem\u003eP. vulneris\u003c/em\u003e (18 assembled genomes and 14 raw read sets). After quality control, two datasets (ERR11550397 and SRR18495312), were excluded after taxonomic verification, as they corresponded to \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eFranconibacter\u003c/em\u003e spp., respectively. The final genomic catalogue comprised 30 high-quality \u003cem\u003eP. vulneris\u003c/em\u003e genomes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAssembly contiguity varied considerably. Four genomes were nearly complete, each comprising two contigs (e.g., GCA_022049045.1, GCA_026651835.1, and GCA_900450975.1) (one chromosome and one plasmid), while one (e.g., CP166292) contained three contigs (one chromosome and two plasmids). The remaining 26 assemblies (~\u0026thinsp;87%) were flagged as draft-quality, ranging from moderately fragmented (e.g., GCA_032069025 with 26 contigs) to highly fragmented (e.g., GCA_032096375 with 1,321 contigs). Most assemblies nonetheless exhibited robust quality metrics, with N50 values\u0026thinsp;\u0026gt;\u0026thinsp;200 kb; for example, GCA_037145055 and GCA_037145235 reached N50 values of 527 kb and 552 kb, respectively.\u003c/p\u003e\u003cp\u003eIsolate sources were predominantly clinical, followed by urban and animal-related environments, with only one genome originating from a plant host and another from an environmental sample. A core-genome alignment of 64 conserved genes was used to construct a maximum-likelihood phylogeny (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which resolved five major clades, each showing distinct gene gain and loss (accessory-genome) profiles. Rarefaction curves did not reach saturation, indicating that the \u003cem\u003eP. vulneris\u003c/em\u003e pangenome remains open (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAcross the dataset, a total of 100 distinct ARGs were detected (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), spanning aminoglycoside, β-lactam, quinolone, macrolide, tetracycline, and sulfonamide classes. In parallel, 29 virulence-associated genes (VAGs) were identified (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), encompassing modules for motility (flagellar and chemotaxis systems), iron acquisition (\u003cem\u003efepG\u003c/em\u003e, \u003cem\u003eentB\u003c/em\u003e), biofilm formation (\u003cem\u003ecsgB\u003c/em\u003e, \u003cem\u003ecsgG\u003c/em\u003e), and immune evasion.\u003c/p\u003e\u003cp\u003ePlasmid replicon typing identified nine incompatibility groups across the 30 genomes, with IncHI2/IncHI2A being the most prevalent, detected in nine hospital-associated isolates. Beyond this group, plasmid diversity in \u003cem\u003eP. vulneris\u003c/em\u003e was limited: most genomes carried zero to two replicon types, and replicons such as Col(pHAD28), IncFII(K), Col440I, and Col(IMGS31) appeared only sporadically. Notably, IncF-type plasmids, considered as major drivers of ARG dissemination in Enterobacterales, were rare, detected in only two genomes, both from hospital-associated isolates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Diversity, Distribution, and Genomic Context of Clinically Relevant ARGs\u003c/h2\u003e\u003cp\u003eA curated panel of 33 clinically and epidemiologically relevant ARGs was screened across the 30 genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The most prevalent were \u003cem\u003ecatA\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;28), \u003cem\u003efosA\u003c/em\u003e and \u003cem\u003eqacH\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;25 each). Other recurrent loci included \u003cem\u003esul1\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;9), \u003cem\u003emcr-9\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;8), \u003cem\u003etetA\u003c/em\u003e, \u003cem\u003eant(3ʺ)-Ia\u003c/em\u003e, and \u003cem\u003eqnrA\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;7 each), as well as \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-9\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e, \u003cem\u003eant(2ʺ)-Ia\u003c/em\u003e, \u003cem\u003edfrA16\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;6 each), and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-3\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\u003cp\u003eWhile some isolates carried extensive resistomes encompassing ESBLs, carbapenemases, quinolone resistance determinants, and multiple aminoglycoside-modifying enzymes, most animal isolates from Madagascar, as well as several environmental or plant-associated genomes, harbored minimal repertoires, typically limited to \u003cem\u003efosA\u003c/em\u003e, \u003cem\u003etetA\u003c/em\u003e, and \u003cem\u003eqacH\u003c/em\u003e. This disparity underscores how resistome size and composition vary sharply according to ecological context.\u003c/p\u003e\u003cp\u003eSuch variation is largely driven by MGEs, as inspection of genetic neighborhoods revealed structures consistent with known ARG mobilization pathways. Diverse IS elements flanked carbapenemase, ESBL, together with class 1 integrons assembling modular resistance cassettes in IncHI2/IncHI2A plasmids (e.g., \u003cem\u003eaac(6\u0026rsquo;)-Ib3, aac(6\u0026rsquo;)-Ib-cr5, bla\u003c/em\u003e\u003csub\u003eOXA-1\u003c/sub\u003e, \u003cem\u003ecatB3, qacE\u003c/em\u003e and \u003cem\u003esul1\u003c/em\u003e previously shown by Cabral et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP-26\u003c/sub\u003e(Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA)). These results highlight how \u003cem\u003eP. vulneris\u003c/em\u003e integrates clinically relevant ARGs within mosaic MGE architectures that may bridge human, animal, and environmental compartments.\u003c/p\u003e\u003cp\u003eAs an emblematic example, a Tn\u003cem\u003e4401\u003c/em\u003e-like element was linked to the dissemination of both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-3\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This element was identified exclusively in hospital-related isolates, including six from the United Kingdom that carried a hypothetical gene located upstream of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e, and three from North America carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-3\u003c/sub\u003e. In the North American isolates, the element occurred within a genomic context compatible with plasmid backbones (apparently not IncHI2/IncHI2A), including \u003cem\u003etra\u003c/em\u003e and \u003cem\u003evir\u003c/em\u003e genes and proximity to a plasmid \u003cem\u003erepA\u003c/em\u003e, indicating that the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e mobilization may also occur through conjugation (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). By contrast, in the United Kingdom isolates it was not possible to resolve the broader genomic context, except for lineage SRR17302001, where the element was clearly embedded within the bacterial chromosome, indicating that Tn\u003cem\u003e4401\u003c/em\u003e-like transposons can mediate chromosomal integration of carbapenemase genes, providing an additional route for their long-term stabilization beyond plasmid-borne maintenance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs a contrasting example, six genomes carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-9\u003c/sub\u003e which is driven by the IS\u003cem\u003eEcp1\u003c/em\u003e resident promoter. Unlike other ARGs, this locus was not associated with transposons, integrons, or other recognizable MGEs. Its conserved arrangement across isolates suggests possible chromosomal integration or stabilization within plasmid loci; however, given the fragmented nature of several assemblies, it is not possible to unambiguously determine whether these contexts correspond to chromosomal or plasmid regions, and no clear evidence of recent mobilization was detected.\u003c/p\u003e\u003cp\u003eIn the seven genomes carrying \u003cem\u003eqnrA1\u003c/em\u003e, two main organizational patterns were observed. In five genomes (SRR3654271, SRR17302001, SRR18030938, SRR18031180, and SRR18031317), \u003cem\u003eqnrA1\u003c/em\u003e was consistently positioned upstream of \u003cem\u003eampR\u003c/em\u003e, followed by \u003cem\u003ehypA\u003c/em\u003e in the reverse orientation and an SMR family transporter in the same orientation as \u003cem\u003eqnrA1\u003c/em\u003e and \u003cem\u003eampR\u003c/em\u003e. This four-gene configuration (\u003cem\u003eqnrA1\u0026ndash;ampR\u0026ndash;hypA\u0026ndash;SMR\u003c/em\u003e) exhibited highly conserved synteny across the assemblies. By contrast, two genomes (SRR9697017 and SRR18032359) displayed a truncated arrangement in which \u003cem\u003eqnrA1\u003c/em\u003e was adjacent to \u003cem\u003eampR\u003c/em\u003e, but the contigs terminated at the \u003cem\u003eampR\u003c/em\u003e locus, precluding resolution of downstream genes (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). These cases most likely reflect assembly incompleteness rather than genuine structural variation, given the consistent \u003cem\u003eqnrA1\u0026ndash;ampR\u003c/em\u003e linkage in all seven genomes.\u003c/p\u003e\u003cp\u003eFurthermore, seven genomes carried the tetracycline resistance genes \u003cem\u003etetA\u003c/em\u003e and \u003cem\u003etetR\u003c/em\u003e. Among them, only the complete genome of \u003cem\u003eP. vulneris\u003c/em\u003e G3 (CP166292) provided a fully resolved context, where \u003cem\u003etetA\u003c/em\u003e and \u003cem\u003etetR\u003c/em\u003e were embedded within a degenerated Tn\u003cem\u003e3\u003c/em\u003e-family element. In contrast, the six draft genomes (SRR3654271, SRR17302001, SRR18030938, SRR18031180, SRR18031317, and SRR18032359) consistently displayed a shorter arrangement consisting of \u003cem\u003etetR\u003c/em\u003e and \u003cem\u003etetA\u003c/em\u003e, flanked by two reverse orientation IS\u003cem\u003e6\u003c/em\u003e-family transposases with an intervening small hypothetical ORF (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD). The recurrent conservation of this IS6\u0026ndash;\u003cem\u003etetR\u003c/em\u003e\u0026ndash;\u003cem\u003etetA\u003c/em\u003e module across multiple genomes suggests a common organizational pattern.\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2. \u003cem\u003emcr-9 Plasticity in Pseudescherichia vulneris: Dissemination through IncHI2/IncHI2A Plasmids and Alternative Genomic Contexts\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003emcr-9\u003c/em\u003e gene emerged as the only member of the \u003cem\u003emcr\u003c/em\u003e family present in \u003cem\u003eP. vulneris\u003c/em\u003e. All genomes carrying \u003cem\u003emcr-9\u003c/em\u003e are from hospital-related isolates. For instance, \u003cem\u003emcr-9.2\u003c/em\u003e corresponded to from the United Kingdom (SRR3654271, SRR17302001, SRR18030938, SRR18031180, SRR18031317, SRR18032359), whereas \u003cem\u003emcr-9.1\u003c/em\u003e was exclusively detected in plasmid-bearing isolates from China (CP086374.1) and in one Australian strain (SRR9697017). In CP086374.1, the \u003cem\u003emcr\u003c/em\u003e-9.1 locus was clearly plasmid-borne within an IncHI2A backbone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). By contrast, the genomic context of \u003cem\u003emcr\u003c/em\u003e-9.1 in SRR9697017 could not be fully resolved due to short-read assembly limitations. Nonetheless, this genome carried a single IncHI2A replicon with the same incompatibility group and plasmid sequence type (ST01) as the Chinese plasmid, suggesting that \u003cem\u003emcr\u003c/em\u003e-9.1 is likely embedded in a comparable genetic environment.\u003c/p\u003e\u003cp\u003eThe two isolates, CP086374.1 (China) and CP166293 (Brazil), contained fully assembled IncHI2A plasmids, enabling direct comparisons of gene content and organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Both shared a conserved backbone but diverged markedly in accessory regions containing ARG and MGEs: the Brazilian plasmid carried 15 ARGs including those uniquely present in this context (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-1\u003c/sub\u003e, \u003cem\u003etetA\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e, \u003cem\u003ecatA1, ant(3\u0026rdquo;)-Ia, sul2, aph(3\u0026rdquo;)-Ib, dfrA14\u003c/em\u003e ), whereas the Chinese plasmid encoded 16 ARGs, featuring a distinct set of resistance genes (\u003cem\u003emcr-9.1\u003c/em\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP-26\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eDHA-1\u003c/sub\u003e, \u003cem\u003eaac(6\u0026rdquo;)-IIc, aac(3)-IIg, ereA2, mphA, mrx, dfrA19, qacEΔ1\u003c/em\u003e, \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003etetD\u003c/em\u003e). These differences highlight independent acquisition events and reinforce the modular, mosaic nature of IncHI2A plasmids also occurring in \u003cem\u003eP. vulneris\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Co-occurrence Patterns of ARGs and VAGs\u003c/h2\u003e\u003cp\u003eBuilding on the gene- and plasmid-level analyses, we next explored how ARGs and VAGs are distributed in relation to one another across the 30 genomes. Co-occurrence analysis revealed non-random associations that point to modular resistance\u0026ndash;virulence architectures rather than isolated acquisition events.\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1 ARG Gene Repertoire and Co-occurrence\u003c/h2\u003e\u003cp\u003eThe co-occurrence analysis of the 33 acquired ARGs identified in 30 \u003cem\u003eP. vulneris\u003c/em\u003e genomes revealed a structured, non-random distribution of resistance determinants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Core modules such as \u003cem\u003ecatA\u003c/em\u003e, \u003cem\u003eqacH\u003c/em\u003e, and \u003cem\u003efosA\u003c/em\u003e were repeatedly detected across multiple isolates. High-risk genes (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-9\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e, and \u003cem\u003emcr-9\u003c/em\u003e) were each present in eight genomes, with six isolates (20%) carrying all three simultaneously. Additional associations included frequent pairing of \u003cem\u003emcr-9\u003c/em\u003e with \u003cem\u003esul1\u003c/em\u003e and \u003cem\u003eant(3\u0026rdquo;)-Ia\u003c/em\u003e, linkage of \u003cem\u003etetA\u003c/em\u003e with \u003cem\u003eqnrA\u003c/em\u003e, and co-occurrence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-9\u003c/sub\u003e. Moreover, \u003cem\u003edfrA16\u003c/em\u003e and \u003cem\u003eant(2\u0026rdquo;)-Ia\u003c/em\u003e often clustered alongside β-lactamases and \u003cem\u003emcr-9\u003c/em\u003e. Notably, several genomes combined carbapenemases and \u003cem\u003emcr-9\u003c/em\u003e with \u003cem\u003eqac\u003c/em\u003e efflux pumps, pointing to the convergence of antibiotic resistance with tolerance to disinfectants and the potential for cross-selection in clinical, veterinary, and environmental contexts.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2 Virulence Gene Repertoire and Co-occurrence\u003c/h2\u003e\u003cp\u003eIn parallel, the distribution of 29 VAGs was evaluated across the dataset, covering structural and regulatory components of the flagellar machinery, chemotaxis, siderophore transport, curli fimbriae, stress response, and metal resistance.\u003c/p\u003e\u003cp\u003eA clear dichotomy between core and accessory components was observed. Genes such as \u003cem\u003eflhC\u003c/em\u003e, \u003cem\u003efliN\u003c/em\u003e, \u003cem\u003eflgC\u003c/em\u003e, \u003cem\u003eflgG\u003c/em\u003e, and \u003cem\u003eflgH\u003c/em\u003e were present in \u0026ge;\u0026thinsp;96% of genomes, defining a conserved virulence backbone. In contrast, accessory genes including \u003cem\u003eclpK1\u003c/em\u003e, \u003cem\u003enlpI\u003c/em\u003e, \u003cem\u003etlrA\u003c/em\u003e, \u003cem\u003eterC\u003c/em\u003e, and \u003cem\u003eZ1307\u003c/em\u003e were present in \u0026le;\u0026thinsp;50% of genomes, with \u003cem\u003eclpK1\u003c/em\u003e detected in only one isolate. Genes encoding curli fimbriae (\u003cem\u003ecsgB\u003c/em\u003e, \u003cem\u003ecsgG\u003c/em\u003e) displayed intermediate prevalence.\u003c/p\u003e\u003cp\u003eCo-occurrence and hierarchical clustering analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) revealed a prominent module composed of flagellar and chemotaxis genes (\u003cem\u003efliA\u003c/em\u003e, \u003cem\u003efliG\u003c/em\u003e, \u003cem\u003efliH\u003c/em\u003e, \u003cem\u003efliM\u003c/em\u003e, \u003cem\u003efliN\u003c/em\u003e, \u003cem\u003echeB\u003c/em\u003e, \u003cem\u003echeW\u003c/em\u003e, \u003cem\u003echeY\u003c/em\u003e). A partially overlapping cluster comprised siderophore biosynthesis genes (\u003cem\u003eentB\u003c/em\u003e, \u003cem\u003efepG\u003c/em\u003e) together with curli fimbriae genes (\u003cem\u003ecsgB\u003c/em\u003e, \u003cem\u003ecsgG\u003c/em\u003e). Accessory loci such as \u003cem\u003eclpK1\u003c/em\u003e, \u003cem\u003enlpI\u003c/em\u003e, \u003cem\u003etlrA\u003c/em\u003e, \u003cem\u003eterC\u003c/em\u003e, and \u003cem\u003eZ1307\u003c/em\u003e showed sporadic co-occurrence and weak connectivity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.4.3 Integrative ARG\u0026ndash;VAG Co-occurrence\u003c/h2\u003e\u003cp\u003eTo investigate potential cross-domain associations, we integrated ARG and VAG datasets into a combined co-occurrence analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Overall, overlap between resistance and virulence modules was limited.\u003c/p\u003e\u003cp\u003eThe chloramphenicol resistance gene \u003cem\u003ecatA\u003c/em\u003e displayed strong co-occurrence with a cluster of motility and cell envelope\u0026ndash;associated genes (\u003cem\u003efliJ\u0026ndash;nlpI\u003c/em\u003e, including \u003cem\u003efliI\u003c/em\u003e and \u003cem\u003emotA\u003c/em\u003e). Similarly, \u003cem\u003eqacH\u003c/em\u003e showed moderate co-association with the same cluster, while \u003cem\u003efosA\u003c/em\u003e exhibited weaker associations. The tellurite resistance gene \u003cem\u003eterC\u003c/em\u003e showed broader connectivity, co-occurring with multiple ARGs including \u003cem\u003emcr-9\u003c/em\u003e, \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003eqnrA\u003c/em\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e, \u003cem\u003edfrA16\u003c/em\u003e, \u003cem\u003etetA\u003c/em\u003e, \u003cem\u003eant(3\u0026rdquo;)-Ia\u003c/em\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-9\u003c/sub\u003e, and ant\u003cem\u003e(2\u0026rdquo;)-Ia\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eBy contrast, clinically significant resistance genes such as \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP-26\u003c/sub\u003e, and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M\u003c/sub\u003e showed little to no overlap with virulence determinants. Their Jaccard index values, generally below 0.3, indicate that these genes are rarely found in the same genomes as virulence factors, suggesting that in \u003cem\u003eP. vulneris\u003c/em\u003e resistance and virulence evolve largely independently rather than being co-selected within the same mobile platforms.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite being long overlooked in antimicrobial resistance (AMR) research, \u003cem\u003ePseudescherichia vulneris\u003c/em\u003e displays a genomic architecture that warrants close attention. Analysis of all thirty genomes currently available, reveal an open pangenome, marked heterogeneity of resistomes and virulomes, and a recurrent association of high-risk ARGs with transmissible IncHI2/IncHI2A plasmids. These broad-host-range plasmids frequently carry multiple ARG classes together with disinfectant, biocide, and heavy-metal tolerance loci, and are widely distributed among Enterobacterales worldwide (Alnajar and Gupta, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Although this is not the first report of IncHI2 or IncHI2A plasmids in \u003cem\u003eP. vulneris\u003c/em\u003e (Cabral et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), our findings demonstrate that they can occur with notable frequency in this lineage, positioning the species as an additional reservoir of resistance genes. Given their conjugative potential (Fang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) these plasmids provide a robust platform for the long-term maintenance and horizontal transmission of last-resort resistance determinants across distinct Enterobacterales species from different ecological compartments (Algarni et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBeyond plasmid carriage, \u003cem\u003eP. vulneris\u003c/em\u003e harbors diverse ARG repertoires shaped by integron cassettes and transposons directly associated with gene mobilization. For instance, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-3\u003c/sub\u003e were embedded in Tn\u003cem\u003e4401\u003c/em\u003e-like elements, which are recognized for mobilizing \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e genes at high frequency (Cuzon et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), whereas \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP-26\u003c/sub\u003e, \u003cem\u003eqacEΔ1\u003c/em\u003e, and \u003cem\u003esul1\u003c/em\u003e were carried within class 1 integrons. Similarly, \u003cem\u003eqnrA1\u003c/em\u003e was consistently linked to \u003cem\u003eampR\u003c/em\u003e, and in most genomes also to \u003cem\u003ehypA\u003c/em\u003e and a SMR family transporter, forming a conserved arrangement previously reported in Enterobacterales and often associated with IS\u003cem\u003eCR1\u003c/em\u003e and class 1 integrons (Gomaa Elsayed et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Jacoby et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In addition, \u003cem\u003etet(A)\u003c/em\u003e and \u003cem\u003etetR\u003c/em\u003e were invariably associated with flanking IS\u003cem\u003e6\u003c/em\u003e-family transposases, resembling IS\u003cem\u003e26\u003c/em\u003e-mediated pseudo-transposons described in Enterobacterales, where these elements promote gene capture, recombination, and stabilization (Blake et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Cabral et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). By contrast, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-9\u003c/sub\u003e showed conserved synteny without recognizable MGEs, suggesting chromosomal integration or stable plasmid maintenance. Together, these observations reflect two contrasting modes of resistance maintenance in \u003cem\u003eP. vulneris\u003c/em\u003e: dynamic mobilization via transposons and integrons versus apparent long-term stabilization.\u003c/p\u003e\u003cp\u003eInterestingly, several hospital-associated genomes carried high-burden resistance profiles that included \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-9\u003c/sub\u003e and \u003cem\u003emcr-9\u003c/em\u003e, whereas most animal and environmental isolates encoded only baseline determinants such as \u003cem\u003efosA\u003c/em\u003e, \u003cem\u003etetA\u003c/em\u003e, and \u003cem\u003eqacH\u003c/em\u003e. This contrast suggests that \u003cem\u003eP. vulneris\u003c/em\u003e adapts its resistome according to ecological context, accumulating clinically significant ARGs in hospital environments while maintaining minimal repertoires in non-clinical settings. Such ecological plasticity reinforces the need to consider \u003cem\u003eP. vulneris\u003c/em\u003e within a One Health surveillance framework, as its ability to shift resistome content across environments positions it as a silent but relevant intermediary in AMR dissemination.\u003c/p\u003e\u003cp\u003eNotably, the triad \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2/3\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-9\u003c/sub\u003e, and \u003cem\u003emcr-9\u003c/em\u003e occurred together in six genomes (20%), highlighting convergence of resistance to carbapenems, ESBLs, and colistin within single isolates. This co-occurrence mirrors plasmid-borne dissemination of ESBLs and carbapenemases in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (Andrade et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, the geographic distribution of \u003cem\u003emcr-9\u003c/em\u003e in this dataset aligns with global surveys, with \u003cem\u003emcr-9.1\u003c/em\u003e predominating in Asia and the Americas and \u003cem\u003emcr-9.2\u003c/em\u003e more common in Europe (Song et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The recurrent association of \u003cem\u003emcr-9\u003c/em\u003e with IncHI2/IncHI2A plasmids underscores its dissemination potential across diverse \u003cem\u003eEnterobacterales\u003c/em\u003e lineages, while occasional chromosomal integration highlights multiple routes of mobilization and maintenance.\u003c/p\u003e\u003cp\u003eCompared to classical \u003cem\u003eEnterobacterales\u003c/em\u003e, the resistome of \u003cem\u003eP. vulneris\u003c/em\u003e appears comparatively simpler and seems to rely predominantly on plasmid acquisition. While \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e typically combine multiple resistance layers\u0026mdash;including chromosomal mutations (\u003cem\u003egyrA, parC\u003c/em\u003e), plasmid-borne ESBLs (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M\u003c/sub\u003e), diverse carbapenemases (KPC, NDM, OXA-48-like), and multiple \u003cem\u003emcr\u003c/em\u003e alleles (Jana et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kerek et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e)\u0026mdash;and \u003cem\u003eEnterobacter\u003c/em\u003e spp. possess inducible chromosomal AmpC β-lactamases supplemented by plasmid-borne determinants (De Maayer et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Teixeira et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), P. \u003cem\u003evulneris\u003c/em\u003e shows no intrinsic β-lactamase arsenal. Instead, it appears to rely on acquisition of IncHI2/IncHI2A plasmids carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2/3\u003c/sub\u003e and \u003cem\u003emcr-9\u003c/em\u003e as its principal route to clinically relevant resistance. This plasmid-dependent and comparatively \u0026ldquo;lighter\u0026rdquo; resistome mirrors its opportunistic ecology and suggests that its epidemiological impact becomes significant only when high-risk plasmids converge within the same host genome.\u003c/p\u003e\u003cp\u003eThe integrative co-occurrence analysis further highlighted modular independence between resistance and virulence. Core virulence modules, including motility and chemotaxis, were consistently conserved, while siderophore transport and curli fimbriae formed a secondary cluster. Clinically critical resistance determinants (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP-26\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M\u003c/sub\u003e, \u003cem\u003emcr-9\u003c/em\u003e) rarely overlapped with virulence loci, in contrast to \u003cem\u003eE. coli\u003c/em\u003e ExPEC lineages where IncF plasmids often co-carry resistance and adhesins such as \u003cem\u003efimH\u003c/em\u003e (Carattoli, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Johnson and Nolan, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Only limited bridges were observed, notably \u003cem\u003ecatA\u003c/em\u003e and \u003cem\u003eqacH\u003c/em\u003e with motility or envelope clusters, and \u003cem\u003eterC\u003c/em\u003e spanning multiple ARGs. This pattern mirrors modularity reported in environmental \u003cem\u003eEnterobacterales\u003c/em\u003e (Manandhar et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), suggesting that resistance and virulence in \u003cem\u003eP. vulneris\u003c/em\u003e evolve under distinct selective pressures.\u003c/p\u003e\u003cp\u003eIn addition, \u003cem\u003eP. vulneris\u003c/em\u003e virulence content contrasts with that of major pathogens. \u003cem\u003eE. coli\u003c/em\u003e (ExPEC) typically harbors dense pathogenicity islands encoding adhesins (\u003cem\u003efimH, papG\u003c/em\u003e), toxins (\u003cem\u003ehlyA, cnf1\u003c/em\u003e), siderophores (aerobactin, salmochelin), and secretion systems (T3SS, T6SS) (Biggel et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Desvaux et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eK. pneumoniae\u003c/em\u003e often acquires hypervirulent plasmids with regulators (\u003cem\u003ermpA/rmpA2\u003c/em\u003e), potent siderophores (\u003cem\u003eiuc, iro, ybt\u003c/em\u003e), and genotoxins such as \u003cem\u003eclb\u003c/em\u003e (Hetta et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Rahmat Ullah et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eEnterobacter\u003c/em\u003e spp., particularly the \u003cem\u003eE. cloacae\u003c/em\u003e complex, maintain robust repertoires enriched in adhesion, biofilm, and immune evasion (Manandhar et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). By contrast, \u003cem\u003eP. vulneris\u003c/em\u003e displays a fragmented and ecologically oriented virulome, largely restricted to motility, chemotaxis, environmental sensing, and basic adhesion. The absence of pathogenicity islands or horizontally acquired high-virulence modules suggests an environmentally adapted, opportunistic lifestyle.\u003c/p\u003e\u003cp\u003eIn line with our observations, Cai et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) recently described the emergence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-5\u003c/sub\u003e-carrying \u003cem\u003eP. vulneris\u003c/em\u003e and \u003cem\u003ePantoea dispersa\u003c/em\u003e isolated from a veterinary hospital environment in China, expanding the known diversity of carbapenemase contexts in this species. Their detection of an IncX3 plasmid carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-5\u003c/sub\u003e further supports the conclusion that \u003cem\u003eP. vulneris\u003c/em\u003e can acquire and maintain distinct plasmid backbones for disseminating high-risk ARGs. Together with our findings, these data confirm that \u003cem\u003eP. vulneris\u003c/em\u003e is not a passive environmental commensal but an adaptable vector linking clinical, veterinary, and environmental reservoirs within the One Health network.\u003c/p\u003e\u003cp\u003eBy systematically analyzing all genomes currently available for \u003cem\u003eP. vulneris\u003c/em\u003e, this study establishes a much-needed foundation for understanding its mobilome and resistance potential. Although the dataset remains small (30 genomes) and biased toward hospital contexts, with fragmented assemblies and missing metadata, these constraints primarily affect resolution of mobile element boundaries rather than the broader conclusions. The absence of phenotypic data for colistin susceptibility further highlights gaps that future studies should address. Nonetheless, the findings are consistent with broader surveys showing that ARG\u0026ndash;virulence co-localization is more common in pathogens associated with human and animal hosts than in environmental speciess (Pan et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and that horizontal transfer is shaped by both genetic compatibility and ecological connectivity (Lund et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Paradoxically, such gaps explain why \u003cem\u003eP. vulneris\u003c/em\u003e has long been overlooked in AMR research. Despite this neglect, our analyses uncovered high-risk ARGs, stabilized \u003cem\u003emcr-9\u003c/em\u003e loci, and IncHI2/IncHI2A plasmids with mosaic architectures, underscoring that \u003cem\u003eP. vulneris\u003c/em\u003e is unlikely to be only a passive commensal.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u003cem\u003ePseudescherichia vulneris\u003c/em\u003e emerges as an underrecognized yet genomically versatile member of Enterobacterales, equipped with a broad mobilome and capable of harboring clinically significant resistance determinants. Its pangenome remains open, reflecting ongoing diversification, while the resistome spans from baseline determinants (\u003cem\u003efosA, tetA, qacH\u003c/em\u003e) in non-clinical isolates to multidrug-resistance profiles in hospital-associated and occasional animal strains. Importantly, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2/3\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-9\u003c/sub\u003e, and \u003cem\u003emcr-9\u003c/em\u003e were recurrently detected, with \u003cem\u003emcr-9\u003c/em\u003e embedded in IncHI2/IncHI2A plasmids and, in some cases, chromosomal contexts, underscoring multiple routes of mobilization and stabilization.\u003c/p\u003e\u003cp\u003eIn contrast to epidemic pathogens such as \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eP. vulneris\u003c/em\u003e lacks an intrinsic β-lactamase arsenal or high-virulence pathogenicity islands. Its resistome appears comparatively \u0026ldquo;lighter\u0026rdquo; and plasmid-dependent, suggesting that its epidemiological relevance becomes critical only when high-risk plasmids converge within the same genome. This opportunistic profile reflects an environmentally adapted lifestyle but also highlights its capacity to act as a silent reservoir for last-resort resistance genes.\u003c/p\u003e\u003cp\u003eFrom a One Health perspective, the recurrent association of high-risk ARGs with transmissible IncHI2/IncHI2A plasmids\u0026mdash;structures enriched in disinfectant, biocide, and heavy-metal tolerance loci\u0026mdash;represents a durable chassis for cross-sector dissemination. Recognizing \u003cem\u003eP. vulneris\u003c/em\u003e in genomic surveillance is therefore essential, as expanding monitoring to include this neglected lineage will improve the anticipation of multidrug-resistant platforms bridging clinical, veterinary, and environmental compartments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT AUTHORSHIP CONTRIBUTION STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnelise Stella Ballaben\u003c/strong\u003e: Writing - original draft, Visualization, Resources, Methodology, Investigation, Formal analysis, Data curation. \u003cstrong\u003eJulia M. Cabrera\u003c/strong\u003e: Methodology, Writing – review \u0026amp; editing. \u003cstrong\u003eMick Chandler:\u003c/strong\u003e Writing – review \u0026amp; editing, Data curation. Leandro M. Moreira: Writing – review \u0026amp; editing. \u003cstrong\u003eAlessandro M. Varani:\u0026nbsp;\u003c/strong\u003e Writing – review \u0026amp; editing, Methodology, Investigation, Validation, Formal analysis, Data curation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.S.B and J.M.C were \u0026nbsp; supported by a post-doctoral and doctoral fellowship from FAPESP [grant #2023/08702-6 and #2023/10686-9, respectively].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the National Council for Scientific and Technological Development (CNPq), Brazil and the São Paulo State Research Foundation (FAPESP, São Paulo).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlcock, B.P., Huynh, W., Chalil, R., Smith, K.W., Raphenya, A.R., Wlodarski, M.A., Edalatmand, A., Petkau, A., Syed, S.A., Tsang, K.K., Baker, S.J.C., Dave, M., Mccarthy, M.C., Mukiri, K.M., Nasir, J.A., Golbon, B., Imtiaz, H., Jiang, X., Kaur, K., Kwong, M., Liang, Z.C., Niu, K.C., Shan, P., Yang, J.Y.J., Gray, K.L., Hoad, G.R., Jia, B., Bhando, T., Carfrae, L.A., Farha, M.A., French, S., Gordzevich, R., Rachwalski, K., Tu, M.M., Bordeleau, E., Dooley, D., Griffiths, E., Zubyk, H.L., Brown, E.D., Maguire, F., Beiko, R.G., Hsiao, W.W.L., Brinkman, F.S.L., Van Domselaar, G., Mcarthur, A.G., 2023. 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Crit Rev Microbiol 0, 1\u0026ndash;35. https://doi.org/10.1080/1040841X.2025.2492156\u003c/li\u003e\n\u003cli\u003eLi, D., Luo, R., Liu, C.M., Leung, C.M., Ting, H.F., Sadakane, K., Yamashita, H., Lam, T.W., 2016. MEGAHIT v1.0: A fast and scalable metagenome assembler driven by advanced methodologies and community practices. Methods. https://doi.org/10.1016/j.ymeth.2016.02.020\u003c/li\u003e\n\u003cli\u003eLund, D., Parras-Molt\u0026oacute;, M., Inda-D\u0026iacute;az, J.S., Ebmeyer, S., Larsson, D.G.J., Johnning, A., Kristiansson, E., 2025. Genetic compatibility and ecological connectivity drive the dissemination of antibiotic resistance genes. Nature Communications 16, 1\u0026ndash;13. https://doi.org/10.1038/s41467-025-57825-3\u003c/li\u003e\n\u003cli\u003eManandhar, S., Nguyen, Q., Nguyen Thi Nguyen, T., Pham, D.T., Rabaa, M.A., Dongol, S., Basnyat, B., Dixit, S.M., Baker, S., Karkey, A., 2022. Genomic epidemiology, antimicrobial resistance and virulence factors of Enterobacter cloacae complex causing potential community-onset bloodstream infections in a tertiary care hospital of Nepal. JAC Antimicrob Resist 4. https://doi.org/10.1093/jacamr/dlac050\u003c/li\u003e\n\u003cli\u003eMinh, B.Q., Schmidt, H.A., Chernomor, O., Schrempf, D., Woodhams, M.D., Von Haeseler, A., Lanfear, R., Teeling, E., 2020. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol 37. https://doi.org/10.1093/molbev/msaa015\u003c/li\u003e\n\u003cli\u003ePan, Y., Zeng, J., Li, L., Yang, J., Tang, Z., Xiong, W., Li, Y., Chen, S., Zeng, Z., 2020. Coexistence of Antibiotic Resistance Genes and Virulence Factors Deciphered by Large-Scale Complete Genome Analysis. mSystems 5. https://doi.org/10.1128/msystems.00821-19\u003c/li\u003e\n\u003cli\u003eParks, D.H., Imelfort, M., Skennerton, C.T., Hugenholtz, P., Tyson, G.W., 2015. 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Front Vet Sci 10. https://doi.org/10.3389/fvets.2023.1328331\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Antimicrobial Resistance, Transposable Elements, Comparative Genomics, Horizontal Gene Transfer, One Health","lastPublishedDoi":"10.21203/rs.3.rs-7879921/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7879921/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial resistance (AMR) is a critical global threat, often driven by horizontal gene transfer mediated by mobile genetic elements (MGEs) such as plasmids, transposons, and integrons. Among Enterobacterales, IncHI2/IncHI2A plasmids are of particular concern, as they combine broad host range, conjugative potential, and mosaic architecture enriched with ARGs, biocide tolerance, and heavy-metal resistance. This study provides the first systematic comparative genomics of \u003cem\u003ePseudescherichia vulneris\u003c/em\u003e, an underrecognized yet genomically versatile species at the human\u0026ndash;animal\u0026ndash;environment interface. All 30 publicly available genomes were analyzed to reconstruct the pangenome, resistome, virulome, and associated MGEs. The pangenome was open, reflecting ongoing diversification and strong potential for horizontal gene acquisition. Resistomes were highly heterogeneous, ranging from minimal repertoires in most animal and environmental isolates to multidrug-resistance profiles in hospital-associated and occasional animal genomes. Clinically significant determinants, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;3\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;9\u003c/sub\u003e, and \u003cem\u003emcr-9\u003c/em\u003e, were frequently linked to MGEs. \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e alleles were mobilized by Tn\u003cem\u003e4401\u003c/em\u003e-like elements, while \u003cem\u003emcr-9\u003c/em\u003e occurred either within IncHI2/IncHI2A plasmids or integrated into chromosomal contexts, underscoring diverse mobilization routes. In contrast, the virulome was comparatively conserved, dominated by motility, chemotaxis, and siderophore systems, unlike pathogenic Enterobacterales that carry broad MGE-associated virulence factors. Co-occurrence analyses showed modular independence between resistance and virulence, with limited overlaps shaped by ecological origins, suggesting that resistome content may adapt to distinctive environments. Collectively, these findings establish \u003cem\u003eP. vulneris\u003c/em\u003e as a reservoir and conduit of last-resort resistance genes, reinforcing its relevance for One Health surveillance and highlighting the urgent need for its systematic inclusion in global antimicrobial resistance monitoring frameworks.\u003c/p\u003e","manuscriptTitle":"One Health Genomic Perspective on Pseudescherichia vulneris: A Neglected Reservoir of Last-Resort Resistance Genes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 08:03:57","doi":"10.21203/rs.3.rs-7879921/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-26T11:59:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-16T00:06:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-10T03:33:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225850069304767869222456173788743523623","date":"2025-12-02T17:58:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6897386106889507909103476678597669209","date":"2025-11-19T03:44:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-24T04:43:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-18T14:50:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-17T16:24:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Current Microbiology","date":"2025-10-16T16:54:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"288098ec-5a83-4c80-8550-5c389e741bd7","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T19:08:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-05 08:03:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7879921","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7879921","identity":"rs-7879921","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}