A new variant of the colistin resistance gene MCR-1 with co-resistance to ß-lactam antibiotics reveals a potential novel antimicrobial peptide.

The emerging and global spread of a novel plasmid-mediated colistin resistance gene, mcr-1, threatens human health. Expression of the MCR-1 protein affects bacterial fitness and this cost correlates with lipid A perturbation. However, the exact molecular mechanism remains unclear. Here, we identified the MCR-1 M6 variant carrying two-point mutations that conferred co-resistance to ß-lactam antibiotics. Compared to wild-type (WT) MCR-1, this variant caused severe disturbance in lipid A, resulting in up-regulation of L, D-transpeptidases (LDTs) pathway, which explains co-resistance to ß-lactams. Moreover, we show that a lipid A loading pocket is localized at the linker domain of MCR-1 where these 2 mutations are located. This pocket governs colistin resistance and bacterial membrane permeability, and the mutated pocket in M6 enhances the binding affinity towards lipid A. Based on this new information, we also designed synthetic peptides derived from M6 that exhibit broad-spectrum antimicrobial activity, exposing a potential vulnerability that could be exploited for future antimicrobial drug design.

Prediction of Antibiotic Resistance Evolution by Growth Measurement of All Proximal Mutants of Beta-Lactamase.

The antibiotic resistance crisis continues to threaten human health. Better predictions of the evolution of antibiotic resistance genes could contribute to the design of more sustainable treatment strategies. However, comprehensive prediction of antibiotic resistance gene evolution via laboratory approaches remains challenging. By combining site-specific integration and high-throughput sequencing, we quantified relative growth under the respective selection of cefotaxime or ceftazidime selection in ∼23,000 Escherichia coli MG1655 strains that each carried a unique, single-copy variant of the extended-spectrum ß-lactamase gene blaCTX-M-14 at the chromosomal att HK022 site. Significant synergistic pleiotropy was observed within four subgenic regions, suggesting key regions for the evolution of resistance to both antibiotics. Moreover, we propose PEARP and PEARR, two deep-learning models with strong clinical correlations, for the prospective and retrospective prediction of blaCTX-M-14 evolution, respectively. Single to quintuple mutations of blaCTX-M-14 predicted to confer resistance by PEARP were significantly enriched among the clinical isolates harboring blaCTX-M-14 variants, and the PEARR scores matched the minimal inhibitory concentrations obtained for the 31 intermediates in all hypothetical trajectories. Altogether, we conclude that the measurement of local fitness landscape enables prediction of the evolutionary trajectories of antibiotic resistance genes, which could be useful for a broad range of clinical applications, from resistance prediction to designing novel treatment strategies.

Comparative genomic analyses of Polymyxin-resistant Enterobacteriaceae strains from China.

BACKGROUND: Mobile colistin resistance like gene (mcr-like gene) is a new type of polymyxin resistance gene that can be horizontally transferred in the Enterobacteriaceae. This has brought great challenges to the treatment of multidrug-resistant Escherichia coli and K. pneumoniae. RESULTS: K. pneumoniae 16BU137 and E. coli 17MR471 were isolated from the bus and subway handrails in Guangzhou, China. K. pneumoniae 19PDR22 and KP20191015 were isolated from patients with urinary tract infection and severe pneumonia in Anhui, China. Sequence analysis indicated that the mcr-1.1 gene was present on the chromosome of E. coli 17MR471, and the gene was in the gene cassette containing pap2 and two copies of ISApl1.The mcr-1.1 was found in the putative IncX4 type plasmid p16BU137_mcr-1.1 of K. pneumoniae 16BU137, but ISApl1 was not found in its flanking sequence. Mcr-8 variants were found in the putative IncFIB/ IncFII plasmid pKP20191015_mcr-8 of K. pneumoniae KP20191015 and flanked by ISEcl1 and ISKpn26. CONCLUSION: This study provides timely information on Enterobacteriaceae bacteria carrying mcr-like genes, and provides a reference for studying the spread of mcr-1 in China and globally.

Carriage of distinct blaKPC-2 and blaOXA-48 plasmids in a single ST11 hypervirulent Klebsiella pneumoniae isolate in Egypt.

BACKGROUND: Carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) causes serious infections with significant morbidity and mortality. However, the epidemiology and transmission mechanisms of CR-hvKP and the corresponding carbapenem-resistant plasmids require further investigation. Herein, we have characterized an ST11 K. pneumoniae strain EBSI041 from the blood sample encoding both hypervirulence and carbapenem resistance phenotypes from a patient in Egypt. RESULTS: K. pneumoniae strain EBSI041 showed multidrug-resistance phenotypes, where it was highly resistant to almost all tested antibiotics including carbapenems. And hypervirulence phenotypes of EBSI041 was confirmed by the model of Galleria mellonella infection. Whole-genome sequencing analysis showed that the hybrid plasmid pEBSI041-1 carried a set of virulence factors rmpA, rmpA2, iucABCD and iutA, and six resistance genes aph(3')-VI, armA, msr(E), mph(E), qnrS, and sul2. Besides, blaOXA-48 and blaSHV-12 were harboured in a novel conjugative IncL-type plasmid pEBSI041-2. The blaKPC-2-carrying plasmid pEBSI041-3, a non-conjugative plasmid lacking the conjugative transfer genes, could be transferred with the help of pEBSI041-2, and the two plasmids could fuse into a new plasmid during co-transfer. Moreover, the emergence of the p16HN-263_KPC-like plasmids is likely due to the integration of pEBSI041-3 and pEBSI041-4 via IS26-mediated rearrangement. CONCLUSION: To the best of our knowledge, this is the first report on the complete genome sequence of KPC-2- and OXA-48-coproducing hypervirulent K. pneumoniae from Egypt. These results give new insights into the adaptation and evolution of K. pneumoniae during nosocomial infections.

Prevalence of mcr-1 in Colonized Inpatients, China, 2011-2019.

In response to the spread of colistin resistance gene mcr-1, China banned the use of colistin in livestock fodders. We used a time-series analysis of inpatient colonization data from 2011-2019 to accurately reveal the associated fluctuations of mcr-1 that occurred in inpatients in response to the ban.

Characterization of a Plasmid-Encoded Resistance-Nodulation-Division Efflux Pump in Klebsiella pneumoniae and Klebsiella quasipneumoniae from Patients in China.

Two multidrug-resistant (MDR) mcr-1-harboring Klebsiella pneumoniae isolates from patients with urinary tract infections and one MDR Klebsiella quasipneumoniae isolate from a patient with bloodstream infection were identified to carry tmexCD1-toprJ1 The addition of the efflux pump inhibitor reduced the tigecycline MIC against all three isolates by 8- to 16-fold. pKQBSI104-1 was transferred from K. quasipneumoniae to Escherichia coli J53 via conjugation. The tmexCD1-toprJ1-carrying plasmids pKP15ZE495-1 (102,569 bp) and pKQBSI104-1 (121,996 bp) were completely sequenced and analyzed.

Prevalence, genomic characteristics, and transmission dynamics of mcr-1-positive Salmonella enterica Typhimurium from patients with infectious diarrhea.

BACKGROUND: Previous studies reported the prevalence of mcr-1 among clinical infected Salmonella isolates in China. However, the transmission dynamics of mcr-1 in different ecological niches were not well investigated. Our objective is to exhibit the transmission dynamics of mcr-1 in Salmonella. METHODS: 598 Salmonella isolates were recovered from ten hospitals; besides 936 pig faces and 167 pork samples were collected from January 2015 to December 2017 in Guangzhou, China. PCR and sequencing were used to identify mcr-1-positive Salmonella. Antimicrobial susceptibility testing was performed with 16 antimicrobials. Conjugation, S1-PFGE, and Southern blot were used to determine the transferability and location of mcr-1. Whole-genome sequencing was used to investigate pangenome, phylogeny, plasmid, and transposon. RESULTS: Eleven mcr-1-positive Salmonella isolates were identified from patients with infectious diarrhea. Five pig fecal samples and three pork samples contained mcr-1-positive Salmonella isolates. All isolates were multi-drug resistant. The mcr-1 genes were located on ∼210-250 kb IncHI2-pST3 plasmids, and 12 mcr-1 genes were transferable. All isolates were assigned to ST34 or its genetically closed STs. The distribution of the core-genome network was significantly correlated with source distributions. The accessory genes-based network demonstrated that the diverse clonal complexes could share highly similar accessory genomes. CONCLUSIONS: The prevalence of mcr-1-positive Salmonella among different sources was low. Clonal transmission could not be the main reason for the expansion of mcr-1-positive Salmonella, but be attributed to the horizontal transfer of IncHI2-pST3 plasmid. Continuous surveillance on Salmonella should be performed to investigate the response of colistin banning in food-producing animals by mcr-1-positive Salmonella populations.

Rapid Fulminant Progression and Mortality Secondary to Aeromonas dhakensis Septicemia with Hepatitis B Virus Infection Following the Ingestion of Snakehead Fish in Mainland China: A Case Report.

Aeromonas dhakensis is an important ubiquitous Gram-negative and freshwater bacterium detected in different reservoirs. It can cause invasive diseases in humans. Herein, we report the first case in Mainland China of a fulminant death of a 29-year-old man as a result of a new, unexpected association between septicemic A. dhakensis and hepatitis B viral infection (HBV). Herein, the patient died from multiple organ failure 5 d postadmission after the ingestion of Snakehead Fish meal. The isolated bacterium was initially misidentified as Aeromonas hydrophila using VITEK-2, while whole-genome sequencing (WGS) revealed that the isolate is A. dhakensis. WGS revealed the occurrence of three antimicrobial genes of resistance: imiH, cphA2, and blaOXA-12; besides, major virulence factors were detected. In silico, multilocus sequence typing (MLST) showed that our A. dhakensis 17FW001 belonged to a novel sequence type (ST557). A comparative genomic analysis of our isolate with nine selected Aeromonas species was done, which elucidated the pathogenicity of our A. dhakensis. In conclusion, we reported for the first time the association between A. dhakensis and HBV in Mainland China. We revealed that septicemic A. dhakensis could result in severe adverse clinical outcomes that end up with unexpected fulminant death especially when it is accompanied with HBV and sheds light on the virulence of A. dhakensis and the high rate of its misdiagnosis that requires to urgently consider screening of all cases of A. dhakensis for HBV in the future. Besides, caution should be taken while dealing with snakeheads which act as a vector for A. dhakensis.

Identification of a Novel Plasmid Carrying mcr-4.3 in an Acinetobacter baumannii Strain in China.

Here, we identified mcr-4.3 in Acinetobacter baumannii, which had not been previously observed to carry an mcr gene. The mcr-4.3-harboring A. baumannii strain AB18PR065 was isolated from pig feces from a slaughterhouse in Guangdong Province of China. The mcr-4.3-carrying pAB18PR065 is 25,602 bp in size and could not be transferred in conjugation, transformation, and electroporation experiments, as we did not find any conjugation-related genes therein. pAB18PR065 harbors two copies of type II toxin-antitoxin systems, which are functional in plasmid stabilization and maintenance. pAB18PR065 shares similarity only with one recently identified plasmid, pAb-MCR4.3 (35,502 bp), from a clinical A. baumannii strain. It is likely that the emergence of pAb-MCR4.3 was due to the insertion of an 11,386-bp, ISAba19-based, composite transposon into pAB18PR065. These data indicate that mcr-4.3 was captured by an A. baumannii-original plasmid via horizontal gene transfer.

Transmission of mcr-1-Producing Multidrug-resistant Enterobacteriaceae in Public Transportation in Guangzhou, China.

Objectives: mcr-1-mediated colistin resistance in bacteria is concerning, as colistin is used in treating multidrug-resistant bacterial infections. And mcr-1-producing bacteria have been identified in multiple sources. Up to 248 million people use public transportation daily in China, however; public transportation hasn't been studied as a potential source of community-based transmission of mcr-1. Herein we investigated mcr-1-producing isolates from public transportation and explored the genomic characteristics of them. Methods: Surface samples were collected from public transportation in Guangzhou, China, from October 2016 to April 2017. Polymerase chain reaction was performed to detect mcr-1 gene, plasmid replicon type and phylogenetic group. Minimum inhibitory concentrations (MICs) were determined by microdilution method. S1-nuclease digestion and pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting were performed with mcr-1-harboring plasmids. Whole-genome sequencing was performed with mcr-1-producing isolates. Results: Of the 737 samples with bacterial growth, 26 isolates were positive for mcr-1, including 23 Escherichia coli and 3 Klebsiella pneumoniae isolates. The E. coli isolates belonged to phylogroups A and B1. Most mcr-1-producing isolates were resistant to ampicillin (25), cefotaxime (21), fosfomycin (16), and gentamicin (15). S1-PFGE, Southern blotting and replicon typing showed that mcr-1 was mainly located on ~33.3 kb to ~220 kb IncX4, IncI2 and IncHI2 plasmids in E. coli, while located on ~33.3 kb untyped plasmid in K. pneumoniae. Several sequence types (ST), including ST2253, ST101, ST10 complex and ST37, were revealed. Between 53 and 66 (mean = 61.8) resistance genes were identified among mcr-1-producing isolates. Conclusions: Public transportation may serve as a source of mcr-1-producing bacteria.

High Rates of Human Fecal Carriage of mcr-1-Positive Multidrug-Resistant Enterobacteriaceae Emerge in China in Association With Successful Plasmid Families.

Background: mcr-1-mediated colistin resistance in Enterobacteriaceae is concerning, as colistin is used in treating multidrug-resistant Enterobacteriaceae infections. We identified trends in human fecal mcr-1-positivity rates and colonization with mcr-1-positive, third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae in Guangzhou, China, and investigated the genetic contexts of mcr-1 in mcr-1-positive 3GC-R strains. Methods: Fecal samples were collected from in-/out-patients submitting specimens to 3 hospitals (2011-2016). mcr-1 carriage trends were assessed using iterative sequential regression. A subset of mcr-1-positive isolates was sequenced (whole-genome sequencing [WGS], Illumina), and genetic contexts (flanking regions, plasmids) of mcr-1 were characterized. Results: Of 8022 fecal samples collected, 497 (6.2%) were mcr-1 positive, and 182 (2.3%) harbored mcr-1-positive 3GC-R Enterobacteriaceae. We observed marked increases in mcr-1 (0% [April 2011] to 31% [March 2016]) and more recent (since January 2014; 0% [April 2011] to 15% [March 2016]) increases in human colonization with mcr-1-positive 3GC-R Enterobacteriaceae (P < .001). mcr-1-positive 3GC-R isolates were commonly multidrug resistant. WGS of mcr-1-positive 3GC-R isolates (70 Escherichia coli, 3 Klebsiella pneumoniae) demonstrated bacterial strain diversity; mcr-1 in association with common plasmid backbones (IncI, IncHI2/HI2A, IncX4) and sometimes in multiple plasmids; frequent mcr-1 chromosomal integration; and high mobility of the mcr-1-associated insertion sequence ISApl1. Sequence data were consistent with plasmid spread among animal/human reservoirs. Conclusions: The high prevalence of mcr-1 in multidrug-resistant E. coli colonizing humans is a clinical threat; diverse genetic mechanisms (strains/plasmids/insertion sequences) have contributed to the dissemination of mcr-1, and will facilitate its persistence.

Dissemination and Mechanism for the MCR-1 Colistin Resistance.

Polymyxins are the last line of defense against lethal infections caused by multidrug resistant Gram-negative pathogens. Very recently, the use of polymyxins has been greatly challenged by the emergence of the plasmid-borne mobile colistin resistance gene (mcr-1). However, the mechanistic aspects of the MCR-1 colistin resistance are still poorly understood. Here we report the comparative genomics of two new mcr-1-harbouring plasmids isolated from the human gut microbiota, highlighting the diversity in plasmid transfer of the mcr-1 gene. Further genetic dissection delineated that both the trans-membrane region and a substrate-binding motif are required for the MCR-1-mediated colistin resistance. The soluble form of the membrane protein MCR-1 was successfully prepared and verified. Phylogenetic analyses revealed that MCR-1 is highly homologous to its counterpart PEA lipid A transferase in Paenibacili, a known producer of polymyxins. The fact that the plasmid-borne MCR-1 is placed in a subclade neighboring the chromosome-encoded colistin-resistant Neisseria LptA (EptA) potentially implies parallel evolutionary paths for the two genes. In conclusion, our finding provids a first glimpse of mechanism for the MCR-1-mediated colistin resistance.

Diverse Secondary Metabolites from the Marine-Derived Fungus Dichotomomyces cejpii F31-1.

By adding l-tryptophan and l-phenylalanine to GPY medium, twenty-eight compounds, including amides, polyketides, a sesquiterpenoid, a diterpenoid, a meroterpenoid, diketopiperazines, ß-carbolines, fumiquinazolines, and indole alkaloids, were discovered from the marine-derived fungus Dichotomomyces cejpii F31-1, demonstrating the tremendous biosynthetic potential of this fungal strain. Among these compounds, four amides dichotomocejs A-D (1-4), one polyketide dichocetide A (5), and two diketopiperazines dichocerazines A-B (15 and 16) are new. The structures of these new compounds were determined by interpreting detailed spectroscopic data as well as calculating optical rotation values and ECD spectra. Obviously, Dichotomomyces cejpii can effectively use an amino acid-directed strategy to enhance the production of nitrogen-containing compounds. Dichotomocej A (1) displayed moderate cytotoxicity against the human rhabdomyosarcoma cell line RD with an IC50 value of 39.1 µM, and pityriacitrin (22) showed moderate cytotoxicity against the human colon carcinoma cell line HCT116 with an IC50 value of 35.1 µM.

Characterization of CTX-M-140, a Variant of CTX-M-14 Extended-Spectrum ß-Lactamase with Decreased Cephalosporin Hydrolytic Activity, from Cephalosporin-Resistant Proteus mirabilis.

CTX-M-140, a novel CTX-M-type extended-spectrum ß-lactamase (ESBL), was identified in cephalosporin-resistant clinical isolates of Proteus mirabilis CTX-M-140 contained an alanine-to-threonine substitution at position 109 compared to its putative progenitor, CTX-M-14. When it was expressed in an Escherichia coli isogenic background, CTX-M-140 conferred 4- to 32-fold lower MICs of cephalosporins than those with CTX-M-14, indicating that the phenotype was attributable to this single substitution. For four mutants of CTX-M-14 that were constructed by site-directed mutagenesis (A109E, A109D, A109K, and A109R mutants), MICs of cephalosporins were similar to those for the E. coli host strain, which suggested that the alanine at position 109 was essential for cephalosporin hydrolysis. The kinetic properties of native CTX-M-14 and CTX-M-140 were consistent with the MICs for the E. coli clones. Compared with that of CTX-M-14, a lower hydrolytic activity against cephalosporins was observed for CTX-M-140. blaCTX-M-140 is located on the chromosome as determined by I-CeuI pulsed-field gel electrophoresis (I-CeuI-PFGE) and Southern hybridization. The genetic environment surrounding blaCTX-M-140 is identical to the sequence found in different plasmids with blaCTX-M-9-group genes among the Enterobacteriaceae Genome sequencing and analysis showed that P. mirabilis strains with blaCTX-M-140 have a genome size of ∼4 Mbp, with a GC content of 38.7% and 23 putative antibiotic resistance genes. Our results indicate that alanine at position 109 is critical for the hydrolytic activity of CTX-M-14 against oxyimino-cephalosporins.

NDM-1-Producing Citrobacter freundii, Escherichia coli, and Acinetobacter baumannii Identified from a Single Patient in China.

We identified New Delhi metallo-ß-lactamase (NDM-1)-producing Citrobacter freundii GB032, Escherichia coli GB102, and Acinetobacter baumannii GB661 in urine and stool samples from a single patient in China. Plasmid profiling and Southern blotting indicated that blaNDM-1 from GB032 and that from GB102 were likely located on the same plasmid, while blaNDM-1 from GB661 was located on a very large (>400-kb) plasmid. This case underscores the broad host range of blaNDM-1 and its potential to spread between members of the family Enterobacteriaceae and A. baumannii.

CTX-M-137, a hybrid of CTX-M-14-like and CTX-M-15-like ß-lactamases identified in an Escherichia coli clinical isolate.

OBJECTIVES: To characterize a novel CTX-M chimera, CTX-M-137, from Escherichia coli clinical isolates in China. METHODS: Isolates were collected from five hospitals between 22 February 2009 and 20 December 2011. Resistance genes were investigated by PCR. blaCTX-M-137 was cloned and purified for kinetic measurements. Conjugation experiments, S1-PFGE and Southern blotting were performed to study the plasmid harbouring blaCTX-M-137. The genetic environment of blaCTX-M-137 was determined by genomic cloning and sequencing. RESULTS: A total of 247 cephalosporin-resistant E. coli were identified. blaCTX-M group genes were the most prevalent extended-spectrum ß-lactamase (ESBL) genes, with 71 isolates harbouring blaCTX-M-1 group genes and 137 isolates harbouring blaCTX-M-9 group genes. A novel chimera of CTX-M-14-like and CTX-M-15-like ESBLs, designated CTX-M-137, was identified from a 60-year-old man with a urinary tract infection. The N-terminus of CTX-M-137 matched CTX-M-14 and the C-terminus matched CTX-M-15. CTX-M-137 conferred resistance to ceftazidime, cefotaxime and aztreonam. Purified CTX-M-137 showed good hydrolytic activity against ceftazidime and cefotaxime, and was inhibited by clavulanic acid. The blaCTX-M-137 was carried on an ∼83 kb IncI1 plasmid. blaCTX-M-137 was carried on a complete transposition unit ISEcp1-blaCTX-M-137-Δorf477 inserted into yagA, which is part of the IncI1 plasmid backbone. CONCLUSIONS: We identified a novel CTX-M chimera, CTX-M-137, with a CTX-M-14-like N-terminus and a CTX-M-15-like C-terminus. Our findings suggest an ongoing diversification of CTX-M-type ESBLs through recombination events.

Fight or flee, a vital choice for Clostridioides difficile.

Clostridioides difficile is a leading cause of healthcare-associated infections, causing billions of economic losses every year. Its symptoms range from mild diarrhea to life-threatening damage to the colon. Transmission and recurrence of C. difficile infection (CDI) are mediated by the metabolically dormant spores, while the virulence of C. difficile is mainly due to the two large clostridial toxins, TcdA and TcdB. Producing toxins or forming spores are two different strategies for C. difficile to cope with harsh environmental conditions. It is of great significance to understand the molecular mechanisms for C. difficile to skew to either of the cellular processes. Here, we summarize the current understanding of the regulation and connections between toxin production and sporulation in C. difficile and further discuss the potential solutions for yet-to-be-answered questions.

Arginine-Enhanced Antimicrobial Activity of Nanozymes against Gram-Negative Bacteria.

The continuous reduction of clinically available antibiotics has made it imperative to exploit more effective antimicrobial therapies, especially for difficult-to-treat Gram-negative pathogens. Herein, it is shown that the combination of an antimicrobial nanozyme with the clinically compatible basic amino acid L-arginine affords a potent treatment for infections with Gram-negative pathogens. In particular, the antimicrobial activity of the antimicrobial nanozyme is dramatically increased by ≈1000-fold after L-arginine stimulation. Specifically, the combination therapy enhances bacterial outer and inner membrane permeability and promotes intracellular reactive oxygen species (ROS) generation. Moreover, the metabolomic and transcriptomic results reveal that combination treatment leads to the increased ROS-mediated damage by inhibiting the tricarboxylic acid cycle and oxidative phosphorylation, thereby inducing an imbalance of the antioxidant and oxidant systems. Importantly, L-arginine dramatically significantly accelerates the healing of infected wounds in mouse models of multidrug-resistant peritonitis-sepsis and skin wound infection. Overall, this work demonstrates a novel synergistic antibacterial strategy by combining the antimicrobial nanozymes with L-arginine, which substantively facilitates the nanozyme-mediated killing of pathogens by promoting ROS production.

Genomic characterization revealing the high rate of tet(X4)-positive Escherichia coli in animals associated with successful genetic elements.

Introduction: The rapid spread of plasmid-mediated tet(X4) conferring high tigecycline resistance poses a significant threat to public health. Escherichia coli as the most common pathogen which carries tet(X4) has been widely disseminated in China. Thus, comprehensive investigations are required to understand the mechanism of transmission of tet(X4)-positive E. coli. Methods: In this study, a total of 775 nonduplicate samples were collected in Guangdong, China from 2019 to 2020. We screened for tet(X4)-positive E. coli by PCR amplification and species identification. Furthermore, we analyzed the phylogenetics and genetic context of tet(X4)-positive E. coli through whole-genome sequencing and long-reads sequencing. Results: Overall, 146 (18.84%) tet(X4)-positive E. coli were isolated, comprising 2 isolates from humans and 144 isolates from pigs. The majority of tet(X4)-positive E. coli exhibited resistance to multiple antibiotics but all of them were susceptible to amikacin and colistin. Phylogenetic analysis showed that ST877, ST871, and ST195 emerged as the predominant sequence types in tet(X4)-positive E. coli. Further analysis revealed various genetic environments associated with the horizontal transfer of tet(X4). Notably, a 100-kbp large fragment insertion was discovered downstream of tet(X4), containing a replicon and a 40-kbp gene cluster for the bacterial type IV secretion system. Discussion: The high colonization rate of tet(X4)-positive E. coli in animals suggests that colonization as a key factor in its dissemination to humans. Diverse genetic context may contribute to the transfer of tet(X4). Our findings underline the urgent need for controlling the spread of plasmid-mediated tigecycline resistance.