Search Results (335)

Understanding the interaction between plants and rhizosphere microorganisms is critical for the development of biofertilizers. Fluorescent labeling of rhizosphere microorganisms serves as a key strategy to track their behavior during plant–microbe coculture. However, most newly isolated strains are novel and lack available molecular tools for such studies. In this research, Planococcus dechangensis NEAU-ST10-9^T (P. dechangensis NEAU-ST10-9^T), a salt-tolerant strain, was obtained from the China General Microbiological Culture Collection Center (CGMCC). It significantly increased maize root length by approximately 1.56-fold. To investigate the underlying mechanism, a donor strain (Ec102) and a shuttle plasmid (pAS104) were engineered to mediate conjugation with P. dechangensis NEAU-ST10-9^T and drive GFP overexpression in the bacterium, generating the genetically labeled strain Pd103. The fluorescence intensity (expressed as GFP/OD₆₀₀, arbitrary units) of Pd103 increased with bacterial growth and was approximately tenfold higher than that of the wild-type strain after 16 h of culture. Following inoculation onto maize seeds, confocal microscopy analysis revealed that Pd103 colonized the epidermis and endodermis of maize roots. These results indicated that P. dechangensis NEAU-ST10-9^T could invade maize roots and promote maize seedling growth. In summary, we have successfully established a robust fluorescence labeling and tracking system tailored for P. dechangensis NEAU-ST10-9^T, which constitutes a valuable tool for elucidating the cellular and molecular mechanisms governing its plant–microbe interaction. Full article

Carbapenemase-producing Enterobacterales (CPE) is a global threat. IMP-6, a prevalent carbapenemase in western Japan, is mostly disseminated via CTX-M-2 extended-spectrum β-lactamase (ESBL) co-producing Enterobacterales. However, the existence and characteristics of Enterobacterales harboring bla_IMP-6 without bla_CTX-M-2 remain unclear. We analyzed the phenotypic and genetic characteristics of clinical bla_IMP-6-harboring Enterobacterales isolates, focusing on those lacking bla_CTX-M-2. Overall, 220 bla_IMP-6-harboring isolates collected from 76 Japanese hospitals between 2014 and 2021 were characterized by antimicrobial susceptibility, presence of CTX-M-type ESBLs, plasmid incompatibility, plasmid transfer experiments, and genome sequencing and analysis. Among these, 203 co-harbored bla_CTX-M-2 group, with 90% of them demonstrating high conjugation frequency and broad-spectrum resistance to β-lactams. Of the remaining 17 isolates, nine lacked bla_CTX-M, while eight co-harbored bla_CTX-M-1 group (n = 2) or bla_CTX-M-9 group (n = 6). Eleven isolates carried nontransferable plasmids with genetic structures distinct from those of bla_IMP-6 and bla_CTX-M-2 co-encoding plasmids, including eight non-incompatibility N plasmids. Fifteen isolates carried only bla_IMP-6-encoding plasmids; two carried plasmids with bla_IMP-6 and bla_CTX-M (bla_CTX-M-27 or bla_CTX-M-65). This novel study revealed that bla_IMP-6 can exist without bla_CTX-M-2 on diverse, often nontransferable plasmids, suggesting distinct, lower dissemination pathways compared to those of epidemic bla_CTX-M-2 co-carrying plasmids and highlighting previously overlooked plasmids that necessitate close monitoring. Full article

Antimicrobial resistance (AMR) in Escherichia coli (E. coli) from food-producing animals constitutes a substantial public health concern. This study characterized antimicrobial resistance profiles, phylogenetic diversity, virulence-gene distribution, and plasmid-borne extended-spectrum β-lactamase (ESBL) determinants of E. coli isolates recovered from water buffaloes with subclinical mastitis. Among the 54 ESBL-producing E. coli isolates, all were resistant to ampicillin and cefotaxime. High resistance rates were also observed for cephalothin (75.9%), trimethoprim–sulfamethoxazole (74.0%), ceftiofur (70.4%), florfenicol (68.5%), and cefazolin (63.0%). Lower resistance was recorded for colistin sulfate (40.7%), enrofloxacin (33.3%), and gentamicin (25.9%). Phylogenetic analysis of ESBL producers identified phylogroup B1 (42.6%) as predominant, followed by groups A (29.6%) and D (25.9%). Multilocus sequence typing (MLST) revealed that ST50 (20.4%) was the most common sequence type, and serogroup O150 was dominant (70.4%). Virulence genes, such as iss (81.5%), astA (59.3%), and espP (38.9%), were frequently detected among ESBL isolates. ESBL genes were predominantly bla_CTX-M-1 (27.8%) in all isolates, while the narrow-spectrum β-lactamase genes bla_TEM-1 (55.6%) and bla_OXA-10 (14.8%) were also commonly co-detected. Bioinformatic analysis predicted that all ESBL genes were associated with plasmid-derived contigs, with the predicted plasmid size ranging from approximately 32 to 187 kb and belonging to IncFIB, IncFIA, IncI1, IncFIA + I1, and IncFII replicon types. Conjugation frequencies ranged from 4.8 × 10⁻⁷ to 4.1 × 10⁻², and plasmids were predicted to carry additional resistance genes mediating resistance to chloramphenicol (floR), sulfonamides (sul1, sul3), tetracyclines (tet(A) and tet(B)), and trimethoprim (dfrA1, dfrA12). The co-carriage of ESBL genes with additional antimicrobial resistance and virulence determinants suggests the potential role of water buffaloes as reservoirs of clinically relevant resistance traits that may disseminate through horizontal gene transfer. Full article

Wastewater treatment plants represent a primary source of environmental dissemination of multidrug-resistant (MDR) bacteria, underscoring the urgent need for in-depth investigation of these organisms. While the resistome of MDR bacteria has been extensively studied, there remains a critical gap in understanding the role of plasmid-borne genes encoding adaptive metabolic functions. We isolated two MDR strains from municipal wastewater, Klebsiella sp. KOS9 and Pseudomonas veronii Yu15, both exhibiting resistance to antibiotics, including ampicillin, cefazolin, kanamycin, streptomycin, erythromycin, chloramphenicol, tetracycline, and ciprofloxacin. The plasmids of these strains harbored genes encoding aliphatic amidases, as well as antibiotic resistance genes (ARGs) and enzymes involved in glycogen and dTDP-L-rhamnose biosynthesis, which may contribute to virulence. In Klebsiella sp. KOS9 a single acetamidase operon, was found on the megaplasmid, along with copper and silver resistance genes. P. veronii Yu15 harbored an operon containing the acetamidase and formamidase genes on the chromosome, as well as a phylogenetically distant acetamidase operon on the conjugative megaplasmid. Both strains exhibit acetamidase activity and P. veronii Yu15 was able to utilize acetamide and formamide as sole nitrogen sources. The occurrence of ARGs and adaptive accessory genes on plasmids likely enhances the competitiveness and environmental flexibility of these MDR bacteria. Full article

Background: High infectious disease burden and uncontrolled antibiotic usage across human, animal, and environmental contaminants make antimicrobial resistance (AMR) a growing public health problem in Africa. Mobile genetic elements (MGEs) such plasmids, transposons, integrons, conjugative elements, and phages help spread AMR via horizontal gene transfer (HGT) across human, animal, food, and environmental sources. Despite growing evidence for antibiotic resistance genes (ARGs), Africa lacks a one-health-focused synthesis of mobile genetic element-mediated AMR. Objective: This systematic review and meta-analysis aimed to consolidate information on MGEs and ARGs in AMR dissemination throughout Africa’s one health interface. Methods: The literature was searched using PubMed, Scopus, and ScienceDirect. Observational. molecular epidemiology, whole genome sequencing (WGS), and metagenomic investigations of MGE-associated AMR in Africa were eligible. The study selection, data extraction, and quality assessment were performed by two independent reviewer and quality was graded using ROBVIS 2 utilizing Rayyan software. Narrative synthesis, random-effect meta-analysis, subgroup analysis, and meta-regression were utilized. Results: A total of 109 studies were included, with 91 studies contributing to the meta-analysis. MGEs reported were plasmids (71.7%) and integrons (54.8%). ARGs carried by MGEs were blaCTMX-M-15 (78.6%), Sul2 (69.6%), blaTEM (59.1%), and tetA (49.9%). Horizontal gene transfer was seen in 259 instances; however, transmission was unclear. In 442 observations, transmission pathways across human, animal, and environmental interfaces showed AMR prevalence of 75.1% in human, 98.0% in human–animal, and 61.3% in one health interface. Whole-genome sequencing was the most frequently used method for detecting MGEsThe pooled pathogen and AMR prevalence rates were 73.3% (95% CI: 60.5–83.7%) and 94% (95% CI: 85–98%), with significant heterogeneity (I² = 97.8% and 97.4%, respectively). The prevalence of Escherichia coli was 93% and Salmonella enterica 85% in subgroup analysis. Fluoroquinolones, aminoglycosides, and beta-lactams were prevalent in humans (89.7%) and human–animal interactions (98.0%) according to AMR Class. Conclusions: Horizontal gene transfer has propagated MGE-mediated antimicrobial resistance across human, animal, and environmental interfaces in Africa. To combat AMR in Africa, coordinated, genomics-informed One Health surveillance and antibiotic stewardship are needed. Due to variability and publication bias, these data should be considered cautiously. Pooled data may only show descriptive patterns, and not necessarily precise continent-wide prevalence estimates. Full article

Patients undergoing cervical conization for cervical intraepithelial neoplasia grade II (CIN II) may develop postoperative bloodstream infections (BSIs), and multidrug-resistant (MDR) Escherichia coli isolates with distinct resistance profiles can complicate antimicrobial management. In this case-based study, two E. coli strains, HBFY-1 and HBFY-2, were recovered from blood cultures obtained from a 38-year-old CIN II patient with postoperative fever. The isolates were characterized by antimicrobial susceptibility testing, whole-genome sequencing, conjugation assays, and comparative genomics against publicly available genomes matched by sequence type and serotype. Fever occurred on postoperative day 2. HBFY-1 was fluoroquinolone-resistant; belonged to ST744/O101:H9; carried the extended-spectrum β-lactamase (ESBL) gene bla_CTX-M-27; was phenotypically confirmed as an ESBL producer; and grouped within a multi-source near-neighbor clade consistent with a conserved fluoroquinolone-associated resistance backbone in ST744/O101:H9. HBFY-2 was carbapenem-resistant; belonged to ST48/O113:H32; carried bla_NDM-5 on an IncY-associated plasmid bin; was phenotypically confirmed as a metallo-carbapenemase producer; and did not harbor any ESBL gene. Within the matched ST48/O113:H32 dataset, bla_NDM-5 was detected only in HBFY-2, which clustered within an Asia-enriched lineage, including China-derived human and swine genomes. The bla_CTX-M-27-associated and bla_NDM-5-associated elements were transferred to E. coli C600 at frequencies of 5.3 × 10⁻² and 4.6 × 10⁻⁶, respectively, and transfer of the bla_NDM-5-associated element imposed no detectable growth penalty under the tested conditions. As this study is based on a single clinical case, the findings should be interpreted cautiously, yet they still highlight the potential value of integrating susceptibility testing with rapid genomic characterization for identifying mobilizable carbapenem-resistance platforms. Full article

Linezolid represents a critical last-resort treatment for severe multidrug-resistant (MDR) Gram-positive bacterial infections. Rising linezolid resistance in Enterococcus isolates threatens its efficacy; this study characterized the molecular features and transfer potential of plasmid-encoded linezolid resistance genes optrA and poxtA in a linezolid-resistant Enterococcus asini isolate from chickens. An E. asini strain was isolated during a surveillance program focusing on drug-resistant Gram-positive bacteria in poultry. PCR screened linezolid resistance genes, conjugation and plasmid stability assays evaluated gene transferability and stability, and whole-genome sequencing (WGS) was performed using both the Illumina and Nanopore platforms. We present the first detection of optrA and poxtA genes in E. asini recovered from chicken feces in China. Sequence analysis of the complete genome showed that poxtA and optrA were situated on two distinct plasmids. The poxtA positive plasmid, pHNGXN23C145Ea-1, also carried multiple resistance genes, including tet(S), fexB, erm(B), ant(6)-Ia, aph(3′)-III. Furthermore, the poxtA gene was flanked by IS1216E mobile elements. The optrA bearing plasmid, pHNGXN23C145Ea-2, harbours a common genetic array of ‘IS1216E fexA-optrA-erm(A)-IS1216E’. Conjugation experiments indicated that neither the poxtA- nor the optrA-bearing plasmid was transferred to recipient strains, which was consistent with sequence analysis showing that both plasmids lacked intact conjugative transfer regions. Stability assays confirmed that poxtA and optrA remained highly stable in the absence of selective pressure. Notably, this discovery was made in a livestock sample, despite the non-use of linezolid in food animals, suggesting that such niches may act as silent reservoirs for resistance genes, which could persist and potentially transfer to clinically relevant MDR pathogens. Full article

Antimicrobial resistance (AMR) represents a global health crisis, driven largely by the mobility of resistance determinants through mobile genetic elements (MGEs). These include plasmids, integrons, insertion sequences, transposons, integrative and conjugative elements (ICEs), and prophages, which together facilitate horizontal gene transfer (HGT) across bacterial species and ecosystems. This review aims to provide a comprehensive synthesis of current knowledge on the types, mechanisms, ecological drivers, and impacts of MGEs in the dissemination of antibiotic resistance genes (ARGs). Methods involved critical evaluation of recent genomic, epidemiological, and ecological studies, alongside case studies of clinically significant resistance outbreaks. Findings highlight how MGEs function as hubs for ARG capture, recombination, and stabilization, enabling the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) pathogens. We also explored their interactions with ecological pressures such as antibiotics, heavy metals, and biocides, as well as their role in One Health transmission pathways. The significance of this study lies in linking molecular insights with applied strategies, including genomic surveillance, MGE-targeted inhibitors, phage therapy, and CRISPR-based interventions. Understanding MGEs is essential for designing effective interventions to mitigate AMR and protect global health. Full article

Gut dysbiosis, defined as a disruption in the structure or function of the intestinal microbiota, is increasingly recognized as a key contributor to inflammatory, metabolic, and neuropsychiatric diseases. Conventional interventions such as broad-spectrum antibiotics, generic probiotics, and fecal microbiota transplantation (FMT) often show limited and inconsistent efficacy because they lack specificity, durability, and robust safety controls. In contrast, recent advances in DNA-based technologies are reshaping the therapeutic landscape by enabling targeted, programmable, and mechanistically informed modulation of the gut ecosystem. This review presents an integrated overview of three major domains driving this shift: CRISPR-based systems that selectively delete, silence, or reprogram microbial genes; synthetic biology-driven live therapeutics engineered to sense disease-associated cues and execute controlled responses; and metagenomics-informed strategies that tailor interventions to patient-specific microbial gene profiles and functional deficits. Additionally, we examine the continued evolution of FMT toward DNA-optimized workflows and defined microbial consortia that offer safer, more standardized alternatives to crude donor material. Across these domains, we discuss delivery platforms (including bacteriophages, conjugative plasmids, extracellular vesicles, and synthetic nanoparticles), and compare their efficiency, specificity, and scalability. We further highlight how DNA-guided interventions interface with host immunity—shaping Treg/Th17 balance, mucosal barrier function, and inflammatory signaling—while also analyzing ecological and evolutionary risks, biocontainment strategies, and regulatory classification gaps that will govern clinical translation. Together, these developments signal a transition from empirical microbiome manipulation to rational ecosystem engineering. DNA-guided therapies hold strong promise for precise and personalized management of gut-related diseases, but their success will depend on rigorous ecological risk assessment, long-term monitoring, and adaptive regulatory frameworks alongside continued technological innovation. Full article

The isolation of locally adapted rhizobial strains with high symbiotic activity represents an effective strategy for increasing soybean yield under extreme environmental conditions. In this study, seven novel strains were isolated from nodules of soybeans grown in a greenhouse using field soil from the Lower Volga region. Five genomes were assembled into complete circular chromosomes, whereas two strains yielded near-complete chromosomes containing single repeat-mediated junctions. All strains had putative plasmids that were independently validated as circular by long-read mapping and confirmed by the presence of characteristic replication and conjugation-associated genes. Genome sequences of strains were about 11 Mb, and GC contents were 63.1–63.3%. Comparative genome analyses demonstrated that all strains had average nucleotide identity values of 95.4% with Bradyrhizobium japonicum USDA 6^T and 96.3% with Bradyrhizobium barranii 144S4^T, forming a distinct cluster in phylogenetic trees. No significant differences were detected between B. japonicum and B. barranii that would explain the species boundary. Therefore, it is proposed to unite all novel strains into the subspecies Bradyrhizobium japonicum subsp. saratovii subsp. nov., and all other strains of B. japonicum and B. barranii we suggest dividing into four subspecies: Bradyrhizobium japonicum subsp. japonicum subsp. nov., Bradyrhizobium japonicum subsp. barranii comb. nov., Bradyrhizobium japonicum subsp. apii comb. nov., and Bradyrhizobium japonicum subsp. saratovii subsp. nov. The proposed taxonomic framework expands current knowledge of the biodiversity of soybean symbiotic bacteria and contributes to a better understanding of the distribution and the evolution of bacteria Bradyrhizobium spp. in previously unexplored regions. Full article

The rapid spread of antibiotic resistance through plasmid-mediated conjugation remains a primary global health concern. Despite its critical role in horizontal gene transfer, no approved drugs currently target this process, leaving a critical therapeutic gap. Integration Host Factor (IHF), a DNA-binding protein essential for plasmid replication and mobilization, emerges as a promising yet underexplored target for anti-conjugation strategies. This work aimed to develop a predictive computational model and identify small molecules that disrupt IHF function, thereby reducing plasmid transfer and limiting resistance gene dissemination. A curated dataset of 65 compounds with reported anti-plasmid activity was analyzed using a 3D-QSAR model based on algebraic descriptors computed with QuBiLS-MIDAS. The model was validated through leave-one-out cross-validation (Q² = 0.82), Tropsha’s criteria, and Y-scrambling. Representative compounds were selected via pharmacophore clustering and evaluated through molecular docking at both the DNA-binding site and a predicted allosteric pocket of IHF. The most promising complexes underwent 200 ns molecular dynamics simulations to assess stability and interaction patterns. The QSAR model demonstrated strong predictive performance (R² = 0.90). Docking simulations revealed more favorable binding energies at the allosteric site (up to −12.15 kcal/mol) compared to the DNA-binding site. Molecular dynamics confirmed the stability of these interactions, with allosteric complexes showing lower RMSD fluctuations and consistent binding energy profiles. Dynamic cross-correlation analysis revealed that allosteric ligand binding induces conformational changes in key catalytic residues, including Pro65, Pro61, and Leu66. These alterations may compromise DNA recognition and disrupt the initiation of replication. To our knowledge, this is the first computational study proposing allosteric inhibition of IHF as an anti-conjugation strategy. These findings provide a foundation for experimental validation and the development of novel agents to prevent horizontal gene transfer, offering a promising approach to restoring antibiotic efficacy against multidrug-resistant pathogens. Full article

Background: The emergence of carbapenem-resistant enterobacteriaceae (CRE) co-harboring the mcr-1.1 gene and carbapenemase-encoding genes poses a severe threat to public health. Urban wastewater treatment plants (WWTPs) act as natural reservoirs and hotspots for the dissemination of antimicrobial resistance genes (ARGs). This study aimed to elucidate the molecular characteristics of CRE carrying mcr-1.1 in urban WWTPs. Methods: Samples were collected from the influent of urban WWTPs in Fengxian, Shanghai, from April 2024 to March 2025. mcr-1.1-positive Escherichia coli (E. coli) isolates were screened using real-time PCR, and their antimicrobial susceptibility was determined via the broth microdilution method. Plasmid conjugation assays were performed with E. coli C600 as the recipient strain. Whole-genome sequencing (WGS) was carried out to analyze the molecular characteristics of mcr-1.1-positive E. coli isolates. Results: A total of 312 samples were collected, and 5 (1.6%) mcr-1.1-positive E. coli isolates were identified. All isolates were multidrug-resistant (MDR) but susceptible to tigecycline (TIG). WGS of strain EC0176 (sequence type 156 [ST156], enteroaggregative E. coli [EAEC]) detected the presence of bla_NDM-5, bla_TEM-1, bla_CTX-M-55, and mcr-1.1 as well as related virulence genes. Further analysis revealed that pEC0176 was an IncHI2-type plasmid co-harboring mcr-1.1, bla_NDM-5, arr-3, aph(4)-Ia, aph(3′)-Ia, aac(3)-IVa, and mph(A). The plasmid pEC0176 harbored similar backbones as p20014-MCR, p2017.03.02CC_1, pSC2017167-mcr-256k, pEC17CM13_MCR and pGDE043-mcr1, including the type IV secretion system (T4SS) and IncHI-type conjugal transfer genes. Conjugation experiments confirmed that pEC0176 could be horizontally transferred into E. coli C600, with an average transfer efficiency of 3.3 × 10⁻². Phylogenetic analysis showed that the MCR-1 protein of EC0176 is closely related to that of two human-derived E. coli strains from China (GenBank accession: AVR64822.1 and WP_076611062.1). Conclusions: To our knowledge, this is the first report of E. coli ST156 carrying an IncHI2-type plasmid co-harboring mcr-1.1 and bla_NDM-5 from urban WWTPs in Fengxian, Shanghai. Our findings underscore the severe status of bacterial antimicrobial resistance and emphasize the necessity of enhancing antimicrobial resistance surveillance in urban WWTPs. Full article

Carbapenem-resistant Enterobacter cloacae Complex (CRECC) has emerged as an important multidrug-resistant pathogen in healthcare settings, although it has historically received less attention than carbapenem-resistant Klebsiella pneumoniae and other major carbapenem-resistant Enterobacterales (CRE). Recent epidemiological reports from several regions indicate increasing detection rates of CRECC in tertiary hospitals, where it is associated with bloodstream infections, pneumonia, urinary tract infections, and prolonged hospitalization. The dissemination of carbapenemase genes, particularly bla_NDM, bla_KPC, and bla_OXA-48-like, carried predominantly on conjugative plasmids (e.g., IncFII, IncX3, IncL), represents the primary resistance mechanism, often accompanied by porin loss and efflux pump overexpression. High-risk clones such as ST171 and ST78 contribute to nosocomial persistence and outbreak potential. Beyond clinical settings, CRECC and related resistance determinants have been reported in companion animals, livestock, food products, wastewater systems, and natural aquatic environments. Although most available studies examine these sectors separately, the recurring detection of genetically related resistance genes and plasmid types suggests potential epidemiological links that warrant integrated surveillance. Environmental reservoirs, particularly hospital effluents and wastewater treatment systems, may facilitate the maintenance and dissemination of resistance genes. This review synthesizes current evidence on the epidemiology, resistance mechanisms, and evolutionary dynamics of CRECC in human, animal, and environmental contexts under a One Health framework. A better understanding of its ecological distribution and genetic plasticity is essential to inform coordinated surveillance strategies and mitigate the public health risks associated with the continued spread of carbapenem resistance. Full article

Achieving completeness of multipartite bacterial genomes has been a difficult task, especially in rhizobia. In this study, we performed a deep bioinformatic analysis of the newly re-sequenced genome of Rhizobium favelukesii LPU83^T. This strain was isolated from acid soils in Argentina and is capable of nodulating several leguminous plants, although it is unable to fix nitrogen efficiently in any of them. Oxford Nanopore sequencing allowed us to completely assemble the symbiotic plasmid of the strain, pRfaLPU83b, and we discovered that it harbors three intact prophages and a high density of insertion sequences (ISs). These characteristics show why it is often so difficult to complete the symbiotic plasmids of rhizobial strains and the importance of having long-read sequencing methods. Upon detailed analysis of this replicon, we identified a complete conjugation system with gene structure consistent with quorum sensing-associated systems that may have contributed to the genetic mosaic structure of the strain. Furthermore, we identified in the symbiotic plasmid of R. favelukesii LPU83^T a large proportion of the symbiotic genes previously identified as essential for Biological Nitrogen Fixation (BNF) in symbiosis with alfalfa, with a high percentage of identity with respect to those of Sinorhizobium meliloti 2011. Among the determinants related to BNF, we found genes encoding the HrrP and SapA peptidases in the LPU83 genome, previously described and related to the degradation of nodule-specific cysteine-rich peptides. These peptides are essential for bacteroid differentiation and, therefore, efficient BNF. Our results show that despite having these genes, they are not directly responsible for the inefficient BNF phenotype of LPU83. Full article

The coexistence of antibiotic resistance genes (ARGs), particularly those conferring resistance to last-resort antibiotics, is increasingly present in lesser-studied bacterial species. Tigecycline is currently one of the last important barriers in the treatment of carbapenem-resistant bacterial infections, whose resistance gene, tet(X), is prevalent across multiple bacterial genera, but the coexistence of tet(X3) and tet(X4) in Acinetobacter sp. is rarely observed. Here, we report a strain co-harboring the chromosomal tet(X3) and plasmid-borne tet(X4) isolated from a commercial beef cattle farm in Henan province, China. The strain exhibited resistance to ampicillin, gentamicin, chloramphenicol, sulfamethoxazole, tetracycline, doxycycline, tigecycline, and omadacycline. Based on whole-genome sequencing (WGS), the strain was identified as A. indicus using Average Nucleotide Identity (ANI) and digital DNA–DNA hybridization (dDDH). Chromosomal tet(X3) was identified in the genetic context, ISVsa3-XerD-tet(X3)-res-ISVsa3. The plasmid-located tet(X4) with the genetic context, ISVsa3-abh-tet(X4)-ISVsa3, and 14 additional resistance genes were located in multiple pdif modules. Two different typing methods, the Rep-based strategy (designed for A. baumanii) and MOB-typer, identified the tet(X4)-positive plasmid as GR31 and rep_cluster_1656, respectively. Conjugation assay failed to observe the transfer of the tet(X4)-positive plasmid into recipients, E. coli J53 and Salmonella LGJ2. The co-occurrence of tet(X3) and tet(X4) in Acinetobacter may suggest a risk of rapid dissemination of tigecycline resistance and the hidden presence of numerous undetected bacteria co-carrying high-risk ARGs in the agroecological system, both of which should cause particular concern. Full article