The global public health sector is confronted with the intensifying issue of antimicrobial resistance, especially the advent of multi-drug-resistant, extensively drug-resistant, and pan-drug-resistant strains. These challenges have narrowed down current treatment options for bacterial infections, thereby calling for urgent strategies to curb the extension of bacterial resistance. The exploration of non-antibiotic alternatives plays a pivotal role in addressing the issue of drug resistance in the modern era. Antimicrobial peptides (AMPs) are considered as promising candidates for the next generation of antibiotic drugs, which mostly are cationic peptides that bind to negatively charged bacterial cell membranes, thereby disrupting the cellular morphology and causing bacterial death. In addition to extracellular targeting mechanisms, AMPs encompass intracellular targeting mechanisms and anti-inflammatory mechanisms. They can be employed both individually or in combination with other antibiotics to amplify the antibacterial effect. However, despite extensive preclinical investigation, the majority of AMP candidates have failed to translate into clinically viable therapeutics. In this review, particular attention is given to both clinical successes and failures, with an emphasis on the roles of pharmacokinetics, dosing constraints, and indication selection. We further evaluate the current impact and limitations of computational and AI-guided AMP design, highlighting the persistent gap between in vitro discovery and in vivo efficacy. By integrating mechanistic insights with clinical and developmental considerations, this review aims to provide a conceptual framework for understanding why most AMPs fail clinically and what principles may enable the development of translationally successful peptide-based antimicrobials.

Bordetella bronchiseptica, long regarded as a veterinary pathogen, is now emerging as a zoonotic threat to humans, particularly in immunocompromised individuals. We report a sentinel event involving a synchronized B. bronchiseptica outbreak in swine and their human caretaker, providing a unique opportunity to examine cross-species transmission and adaptation at the genomic level. Comparative genomics revealed that the human-adapted isolate (RL57) and its swine progenitor (XX35) share an identical chromosome, with XX35 harbouring an extra conjugative plasmid. Remarkably, RL57 did not simply lose this plasmid; instead, the entire plasmid was integrated into the chromosome via site-specific recombination. This integration allowed permanent retention of plasmid-encoded virulence and fitness genes, after which the plasmid was discarded to eliminate its replicative burden - a "capture-and-discard" mechanism of evolution. Following plasmid loss, the RL57 strain exhibited hypervirulence, faster growth, enhanced thermotolerance, and increased biofilm formation, indicating successful adaptation to the human host. Plasmid loss paradoxically rewired bacterial metabolism: sulfur assimilation and sulfonate utilization pathways were upregulated to fuel host adaptation. Strikingly, despite a collapse in transcription of specific metabolic modules, translational compensation maintained high protein levels, driving robust biofilm formation and thermal tolerance. These findings reveal a previously unrecognized evolutionary strategy in which plasmid integration followed by subsequent plasmid loss amplifies pathogenicity and host adaptability. Finally, we propose a One Health surveillance triad - metagenomic tracking of plasmid-chromosome dynamics, recombination hotspot screening, and metabolic shift monitoring - to proactively identify and mitigate such zoonotic events.

In response to the critical challenge of colistin resistance in animal production, we examined the occurrence and genetic features of mcr-1-positive E. coli isolates collected from livestock in Hunan province, China, between 2016 and 2021. A total of 1,358 animal samples from chickens and pigs were collected and screened. Monitoring data indicates that nineteen mcr-1-positive E. coli isolates (1.40

Aldo-keto reductase 1B1 (AKR1B1), also named as aldose reductase, is an enzyme implicated in metabolic regulation and signaling transduction in human cancers, but little is known in canine mammary tumors (CMTs). This study investigated expression in CMT tissues, functional role in tumor progression, and potential of AKR1B1 as a circulating biomarker. AKR1B1 expression was evaluated by immunohistochemistry in paired CMT and adjacent normal mammary tissues, and CMT-U27 cells with lentiviral AKR1B1 overexpression or shRNA-mediated AKR1B1 knockdown were used for investigation of cell proliferation, migration, and Western blot analysis of PI3K/AKT signaling and cell-cycle regulators. Results showed that AKR1B1 was markedly overexpressed in CMT tissues compared to adjacent normal glands. Targeted expression of AKR1B1 in CMT-U27 cells promoted proliferation, migration, accompanied by increase of phosphorylated AKT (Thr308) and downregulation of P21 and P27, whereas AKR1B1 knockdown led to opposite effects. Magnetic-particle chemiluminescence immunoassay was developed to measure serum AKR1B1 concentrations, and results showed serum AKR1B1 was significantly higher in Canines with CMT (4.65 ± 0.37 ng/mL; n = 50) than in healthy controls (2.27 ± 0.15 ng/mL; n = 56). Receiver operating characteristic analysis demonstrated impressive diagnostic performance with AUC = 0.8386 (95% CI 0.7597-0.9174), sensitivity at 74.0% and specificity at 83.9% at a cutoff of 3.08 ng/mL. These findings indicate that AKR1B1 acts as a tumorigenic driver via PI3K/AKT-mediated oncogenesis in CMT and a serum biomarker for non-invasive diagnosis of CMT and monitoring of disease progression.

OBJECTIVE:Aldo-keto reductase 1B10 (AKR1B10) is overexpressed in hepatocellular carcinoma (HCC). This study aimed to evaluate its diagnostic efficacy for early and alpha-fetoprotein (AFP)-negative HCC, and its potential role in assessing radical resection and early recurrence. METHODS:A large-scale multicentre clinical study enrolled 1 352 subjects from three medical centres, including HCC, benign liver diseases, and non-hepatocyte cancers. Serum samples were collected for AKR1B10 and AFP testing, and clinical and imaging data were collected for analysis. RESULTS:Serum AKR1B10 markedly increased in HCC patients to 1419.51 ± 89.09 pg/mL, compared to 177.96 ± 9.78 pg/mL in healthy controls. Receiver operating characteristic (ROC) curve analysis showed that for HCC diagnosis, AKR1B10 had an Area under the curve (AUC) of 0.866, sensitivity of 73.10%, and specificity of 95.24%, compared to AFP with 0.750, 64.27%, and 82.14%, respectively. In early HCC, AKR1B10 yielded an AUC of 0.800, a sensitivity of 65.8%, and a specificity of 91.67%, better than AFP (0.717, 59.83%, and 88.1%, respectively). AKR1B10 was positive in 69.27% of AFP-negative HCC cases and showed an AUC of 0.852, a sensitivity of 72.2%, and a specificity of 90.48%. In patients with HCC undergoing curative resection, serum AKR1B10 levels declined to normal within 3-5 days. At 1-3 months after surgery, serum AKR1B10 had a concordance of 94.41% with imaging data, compared to 70.63% for AFP. CONCLUSION:AKR1B10 is a useful serum marker for the diagnosis of early and AFP-negative HCC and shows potential in assessing curative resection and early recurrence.

Gelsemium elegans (G. elegans) is widely recognized as one of the most toxic plants globally, particularly harmful to humans. Some reports indicate that it is non-toxic to pigs and even has a growth-promoting effect; however, the underlying reasons for this paradox remain unclear. Gelsenicine is the main toxic component of G. elegans. This study characterized gelsenicine-induced toxicity using electrophysiological recordings, molecular dynamic simulations, c-Fos immunostaining, and multi-omics technologies. Additionally, we conducted a comprehensive analysis comparing the toxic effects of gelsenicine across various animal species through examinations of tissue distribution, blood gas analysis, metabonomics, and behavioral tests. We demonstrated that gelsenicine-induced hypoxia leads to respiratory depression in mice by enhancing the effect of gamma-aminobutyric acid (GABA) on GABA receptors (GABARs). Glycine significantly ameliorated hypoxia and improved the survival of gelsenicine-poisoned mice. Under gelsenicine-induced hypoxic conditions, N-methyl-D-aspartate (NMDA) receptor function and mitochondrial energy metabolism processes were perturbed, resulting in neuronal excitotoxicity. Finally, we confirmed that pigs could tolerate hypoxia and were resistant to gelsenicine toxicity due to high concentrations of circulating glycine and low levels of NMDA receptors (NMDARs) in the hippocampus. These findings suggest that hypoxic protection should be considered as a potential therapeutic strategy for gelsenicine poisoning. Our study contributes to preventing potential risks posed by G. elegans poisoning to human and animal health.

Escherichia coli strain 22a1303, carrying both mcr-1.1/IncX4 and mcr-3.5/IncP1 plasmids, was isolated from an ongoing antimicrobial resistance monitoring program. We were concerned that the dual presence of the mcr genes could mediate more complex transmission of resistance, so we further analyzed 116 known E. coli strains carrying both genes in the public database. We found that E. coli 22a1303 carrying the mcr-3/dgkA combination and mcr-1 gene, which should mediate high levels of resistance, was sensitive to polymyxin with a MIC value of only 1mg/L. Conjugation experiment demonstrated that the two plasmids can be transferred from the donor strain 22a1303 to the recipient strain E. coli J53 at the same time, and the colistin resistance of transconjugants was increased by 8-fold compared with E. coli 22a1303.

Pore-forming peptides are promising antimicrobial and anticancer agents due to their membrane selectivity and biodegradability. Our prior work identified peptide M159, which permeabilized synthetic phosphatidylcholine liposomes without mammalian cell toxicity. Here, we report that the D-type variant (D-M159) induces apoptosis in HeLa cells under starvation. To explore its anticancer mechanism, we analyzed D-M159 cytotoxicity, intracellular uptake, and apoptotic pathways via flow cytometry, confocal microscopy, and Western blot. Calcium dynamics and mitochondrial function were examined via specific labeling and functional assays. Results revealed that D-M159 exhibited starvation-dependent, dose-responsive cytotoxicity and triggered apoptosis in HeLa cells. Mechanistic studies indicated that D-M159 entered the cells via caveolin-dependent and caveolae-dependent endocytosis pathways and induced endoplasmic reticulum stress in HeLa cells by up-regulating proteins such as ATF6, p-IRE1, PERK, GRP78, and CHOP. Meanwhile, D-M159 promoted the expression of IP3R1, GRP75, and VDAC1, which led to mitochondrial calcium iron overload, decreased mitochondrial membrane potential, and increased reactive oxygen species (ROS) generation, thereby activating the mitochondrial apoptotic pathway and inducing the aberrant expression of Bax, Bcl-2, Caspase-9, and Caspase-3. This study showed that D-M159 synergistically induced apoptosis in starved HeLa cells through endoplasmic reticulum stress and mitochondrial dysfunction, demonstrating its potential as a novel anticancer agent.

Multi-drug-resistant Aeromonas hydrophila infections are becoming increasingly threatening, and the development of novel antimicrobial drugs is indispensable. Herein, we demonstrate that this novel peptide is highly active against colistin-resistant A. hydrophila strain and shows sustained killing efficacy in vivo. Mechanistic studies showed that D-Q7 interacted with phosphatidylglycerol and lipopolysaccharide in the bacterial cell membrane, with an increase in intracellular ROS as well as a decrease in ATP level, ultimately leading to cell membrane disruption and bacterial death. Importantly, our study identified gene3832 as a potential regulator of membrane permeability, which may act as a potential modulator of bacterial susceptibility to D-Q7. The role of gene3832 was further confirmed by gene knockout and complementation assays. Consistently, we observed that gene3832 was also involved in biofilm formation in the colistin-resistant A. hydrophila strain. Collectively, our study provides an effective antimicrobial strategy with potential targets for the treatment of drug-resistant A. hydrophila infection.IMPORTANCEAs an environmental, zoonotic pathogen, Aeromonas hydrophila remains a major pathogenic bacterium, bringing large economic losses and eco-environmental pressure during the event of large-scale infection. Currently, the occurrence of colistin-resistant A. hydrophila poses a threat to public health owing to the lack of effective prevention and therapeutics. D-Q7 is a D-type antimicrobial peptide (AMP) with potent sterilization activity against gram-negative ESKAPE pathogens; it is thus of considerable interest to evaluate whether D-Q7 represents a promising therapeutic candidate against this pathogen. Consequently, we found that D-Q7 was a potent antibacterial agent that killed colistin-resistant A. hydrophila 23-c-23 in vitro and in a mouse epicutaneous model of 23-c-23 infection. In addition, we found that gene3832, as a potential transmembrane autotransporter, is related to bacterial resistance to D-Q7. Importantly, our study here will help guide the future design and optimization of novel AMPs to combat colistin-resistant A. hydrophila.

Escherichia coli (E. coli) is a zoonotic bacterium widespread in the environment, highly transmissible, and responsible for significant economic losses and millions of cases of illness annually. The rise of multidrug-resistant (MDR) strains has rendered last-line antibiotics such as polymyxin and meropenem ineffective, making the development of new antibiotics urgent. Although D-CONGA-Q7 has broad-spectrum bactericidal activity, its underlying mechanism remains poorly understood. In this study, we used in vitro and in vivo experiments to demonstrate that D-CONGA-Q7 effectively kills both antibiotic-sensitive and multidrug-resistant strains of E. coli. D-CONGA-Q7 disrupts the cell membranes of Gram-negative bacteria, and the treatment of E. coli strain LN175 with D-CONGA-Q7 resulted in a significant up-regulation of the Mlac gene, suggesting that D-CONGA-Q7 may interact with phospholipids in the cell membrane. Furthermore, in treating K88-induced bacterial enteritis in the small intestine, D-CONGA-Q7 significantly reduced intestinal inflammation. In conclusion, this study provides a novel approach to combat drug-resistant E. coli.

BACKGROUND:Citrinin (CTN) is a mycotoxin that is difficult to eliminate and easy to ingest. Chronic exposure to CTN can lead to inflammatory bowel disease (IBD). The herb Koumine has strong anti-inflammatory activity and is considered a candidate for the treatment of IBD. PURPOSE:To investigate the effect of Koumine on IBD induced by CTN exposure and its mechanism of action. RESULTS:This study demonstrated that Koumine effectively attenuates CTN-induced inflammatory damage in the mouse intestine and IPEC-J2 cells. Furthermore, Koumine suppressed CTN-induced upregulation of the IP3R1-GRP75-VDAC1 complex, mitochondrial calcium overload, elevated mitochondrial reactive oxygen species (mtROS) levels, and subsequent pyroptosis. Specific overexpression of mtROS counteracted the therapeutic effect of Koumine on CTN exposure-induced pyroptosis but did not alter mitochondrial calcium levels. Silencing GRP75 ameliorated CTN-induced mitochondrial calcium overload and pyroptosis. Notably, siGRP75 addition did not further enhance the therapeutic effect of Koumine. CONCLUSIONS:Koumine ameliorates CTN-induced intestinal inflammation by mediating mtROS production via the IP3R1-GRP75-VDAC1 complex. Koumine is a potential agent for the treatment of intestinal inflammation induced by mycotoxin exposure such as CTN.

Aldo-keto reductase 1B1 (AKR1B1) is a highly conserved, NADPH-dependent oxidoreductase that plays pivotal roles across multiple metabolic pathways. Although mounting evidence highlights its diverse contributions to disease progression and its potential as a therapeutic target, the literature remains fragmented with respect to how AKR1B1's metabolic functions intersect with pathogenesis. This review article summarizes the key metabolic functions of AKR1B1 under physiological conditions and discusses its pathological involvement in a spectrum of disorders, including diabetic complications, inflammatory diseases, and cancer. In addition, we review the progress of clinical research on AKR1B1 inhibitors over the past two decades, outlining the achievements and persistent challenges. By integrating insights from metabolism and diseases, this work provides a foundation and inspires new research efforts to advance the understanding of AKR1B1.

Multi-drug resistance of bacteria producing extended-spectrum β-lactamase (ESBL) is a public health challenge. Thus, this study aimed to investigate the antimicrobial susceptibility of ESBL-producing Escherichia coli (ESBL-EC) in Hunan Province, China. A total of 1366 fecal samples were collected from pig, chicken, and cattle farms over a six-year period, which were assessed using strain isolation, 16S rRNA identification, polymerase chain reaction, drug sensitivity testing, whole-genome sequencing, and bioinformatics analysis. The results showed an overall prevalence of 6.66% for ESBL-EC strains, with ESBL positivity extents for pigs, chickens, and cattle isolates at 6.77%, 6.54%, and 12.5%, respectively. Most ESBL-EC isolates were resistant to cefotaxime, tetracycline, and trimethoprim-sulfamethoxazole; however, all the isolates were susceptible to meropenem, with relatively low resistance to amikacin and tigecycline. Various multi-locus sequence types with different origins and similar affinities were identified, with ST155 (n = 16) being the most common subtype. Several types of resistance genes were identified among the 91 positive strains, with beta-lactamase blaCTX-M-55 being the most common ESBL genotype. IncFIB was the predominant plasmid type. Widespread use of antibiotics in animal farming may increase antibiotic resistance, posing a serious threat to the health of farmed animals and, thus, to human food security and health.

Tigecycline is a last-resort drug used to treat serious infections caused by multidrug-resistant bacteria. tet(X4) is a recently discovered plasmid-mediated tigecycline resistance gene that confers high-level resistance to tigecycline and other tetracyclines. Since the first discovery of tet(X4) in 2019, it has spread rapidly worldwide, and as a consequence, tigecycline has become increasingly ineffective in the clinical treatment of multidrug-resistant infections. In this study, we identified and analyzed tet(X4)-positive Escherichia coli isolates from duck farms in Hunan Province, China. In total, 976 samples were collected from nine duck farms. Antimicrobial susceptibility testing and whole-genome sequencing (WGS) were performed to establish the phenotypes and genotypes of tet(X4)-positive isolates. In addition, the genomic characteristics and transferability of tet(X4) were determined based on bioinformatics analysis and conjugation. We accordingly detected an E. coli strain harboring tet(X4) and seven other resistance genes in duck feces. Multi-locus sequence typing analysis revealed that this isolate belonged to a new clone, and subsequent genetic analysis indicated that tet(X4) was carried in a 4608-bp circular intermediate, flanked by ISVsa3-ORF2-abh elements. Moreover, it exhibited transferability to E. coli C600 with a frequency of 10-5. The detection of tet(X4)-harboring E, coli strains on duck farms enhances our understanding of tigecycline resistance dynamics. The transferable nature of the circular intermediate of tet(X4) contributing to the spread of tigecycline resistance genes poses a substantial threat to healthcare. Consequently, vigilant monitoring and proactive measures are necessary to prevent their spread.

Osteoarthritis (OA) is a degenerative joint disease, Increasingly, mitochondrial autophagy has been found to play an important regulatory role in the prevention and treatment of osteoarthritis. Koumine is a bioactive alkaloid extracted from the plant Gelsemium elegans. In previous research, Koumine was found to have potential in improving the progression of OA in rats. However, the specific mechanism of its action has not been fully explained. Therefore, the aim of this study was to investigate whether Koumine can alleviate OA in rats by influencing mitochondrial autophagy. In the in vitro study, rat chondrocytes (RCCS-1) were induced with IL-1β (10 ng/mL) to induce inflammation, and Koumine (50 μg/mL) was co-treated. In the in vivo study, a rat OA model was established by intra-articular injection of 2% papain, and Koumine was administered orally (1 mg/kg, once daily for two weeks). It was found that Koumine effectively reduced cartilage erosion in rats with osteoarthritis. Additionally, it decreased the levels of inflammatory factors such as IL-1β, IL-6, and extracellular matrix (ECM) components MMP13 and ADAMTS5 in chondrocytes and articular cartilage tissue, while increasing the level of Collagen II.Koumine inhibited the production of reactive oxygen species (ROS) in cartilage tissue and increased the number of autophagosomes in chondrocytes and articular cartilage tissue. Additionally, it upregulated the expression of mitochondrial autophagy proteins LC3Ⅱ/Ⅰ, PINK1, Parkin, and Drp1. The administration of Mdivi-1 (50 μM) reversed the enhanced effect of Koumine on mitochondrial autophagy, as well as its anti-inflammatory and anti-ECM degradation effects in rats with OA. These findings suggest that Koumine can alleviate chondrocyte inflammation and improve the progression of OA in rats by activating PINK1/Parkin-mediated mitochondrial autophagy.

为了评价加味健猪散对母猪便秘的缓解程度和对采食量的影响,本试验选取产前30 d无便秘症状的母猪50头随机分为5个组,即对照组(基础日粮)、健猪散组(基础日粮中添加0.5 kg/t健猪散)、加味健猪散低、中和高剂量组(基础日粮中分别添加0.25、0.5和1 kg/t加味健猪散)进行预防试验,饲喂15 d,每天对母猪粪便进行评分,并记录采食历时和采食量;另选取产前30 d有严重便秘症状的母猪40头,随机分为5个组(试验分组同上)进行治疗试验,饲喂5 d,每天对母猪粪便进行评分,并记录采食量.结果显示,预防试验中,与对照组相比,各试验组的便秘母猪头数和采食量均无显著差异(P＞0.05),但采食历时显著缩短(P＜0.05);不同剂量加味健猪散组与健猪散组的采食历时无显著差异(P＞0.05).治疗试验中,对照组、健猪散组、加味健猪散低、中和高剂量组的有效率分别为25.0％、87.5％、87.5％、100％和100％;各试验组的采食量较对照组均显著增加(P＜0.05).结果表明,在饲粮中添加健猪散或加味健猪散虽不能显著性地预防母猪出现便秘,但能治疗母猪便秘,并增加母猪便秘后的采食量,加味健猪散的效果要优于同剂量的健猪散.

Bacterial antibiotic resistance, especially the emergence of multidrug-resistant (MDR) strains, urgently requires the development of effective treatment strategies. It is always of interest to delve into the mechanisms of resistance to current antibiotics and target them to promote the efficacy of existing antibiotics. In recent years, non-antibiotic compounds have played an important auxiliary role in improving the efficacy of antibiotics and promoting the treatment of drug-resistant bacteria. The combination of non-antibiotic compounds with antibiotics is considered a promising strategy against MDR bacteria. In this review, we first briefly summarize the main resistance mechanisms of current antibiotics. In addition, we propose several strategies to enhance antibiotic action based on resistance mechanisms. Then, the research progress of non-antibiotic compounds that can promote antibiotic-resistant bacteria through different mechanisms in recent years is also summarized. Finally, the development prospects and challenges of these non-antibiotic compounds in combination with antibiotics are discussed.

为了评价加味健猪散对小鼠肠推进、采食量与排便的影响,试验将20只(18±2)g昆明小鼠分为生理盐水组与加味健猪散组,每组10只,分别以生理盐水、1 g/mL加味健猪散水煎液灌胃小鼠0.4 mL;30 min后,再次给每只小鼠灌胃5％活性炭粉末生理盐水混悬液0.2 mL;20 min后处死小鼠取肠管计算活性炭粉末的推进率.另取(18±2)g昆明小鼠120只,雌雄各半,随机按照性别分成雌性生理盐水组、雄性生理盐水组、雌性加味健猪散组和雄性加味健猪散组,每组30只,连续灌胃给药5 d,记录各组小鼠每天的采食量、粪便排出量和粪便粒数.结果表明:与生理盐水组相比,加味健猪散组肠道的活性炭炭末推进率极显著提高(P＜0.01).与生理盐水组相比,加味健猪散组小鼠的平均采食量从给药第2天开始显著增加(P＜0.05);雄性加味健猪散组的平均粪便排出量在试验第2～5天显著增加(P＜0.05);雌性加味健猪散组的平均粪便排出量则在试验第3～5天显著增加(P＜0.05);加味健猪散组平均粪便粒数在试验第3,5天显著增加(P＜0.05).说明加味健猪散能提高小鼠的肠推进率,增加小鼠采食量并促进排便.

Docosahexaenoic acid (DHA) and selenium (Se) are nutrients that confer several health benefits to both humans and animals. Widespread use of DHA in milk powder and health products requires large-scale mass production via Schizochytrium sp., while Se intended for human consumption is produced as organic Se via yeast. However, producing these nutrients on an industrial scale is constrained by various factors. We found that supplementing Schizochytrium sp. with Na2SeO3 (0.5 mg/L) improves its biomass and DHA production and also provides organic Se. De novo assembled transcriptome and biochemical indicators showed that Na2SeO3 promotes forming acetyl coenzyme A and L-cysteine via the glycerol kinase and cysteine synthase pathways, promoting DHA synthesis through the polyketide synthase pathway. However, high doses of Na2SeO3 (5 mg/L) limited the biomass of Schizochytrium sp. and DHA content. This study provided a theoretical basis for the simultaneous production of organic Se and DHA via Schizochytrium sp.

Background: The plasmid−mediated tigecycline resistance gene tet(X4) confers a high level of resistance to tigecycline. The experiment aims to investigate the prevalence and characterization of tet(X4) in Escherichia coli isolates from chicken and pig farms in Hunan province, China. Methods: A total of six tet(X4) positive strains were identified in 257 E. coli derived from chicken samples in Xiangtan city (n = 2), pig samples in Xiangxiang city (n = 1), Chenzhou city (n = 2), and Zhuzhou city (n = 1). The presence of tet(X4) was directly detected by PCR assay, and then the broth dilution method determined the antimicrobial susceptibility profile of the tet(X4)−positive isolates. Genomic locations were identified by whole−genome sequencing (WGS) and bioinformatics. Results: Almost all tet(X4)−positive strains showed high resistance to multidrug, including tigecycline. Resistome analysis revealed many antibiotic resistance genes, including those with resistance to tetracyclines, β−lactams, phenicols, quinolones, lincosamides chloramphenicol, aminoglycosides and sulfamids. These tet(X4)−bearing strains exhibited six distract STs, such as ST10, 202, ST218, ST362, ST2077, ST7068. The plasmid replicon types carrying tet(X4) were the hybrid plasmid IncFIA(HI1)/IncHIA/IncHIB(R27) (5/6) and IncX1 (1/6). Conclusions: The presence of similar genetic environments in E. coli from different cities suggests there may be horizontal transmission pathways promoting the broad spread of drug−resistant genes in Hunan Province, putting great pressure on multidrug resistance monitoring.