Search

There is a major public health threat posed by antibiotic resistance around the world. Tigecycline overcomes the resistance mechanisms of traditional tetracyclines and is often seen as the final resort in combating infections caused by bacteria resistant to multiple drugs. However, the introduction of new mobile tet(X) tetracycline destructases is leading to a notable rise in tigecycline resistance. Therefore, a rapid detection method is needed to monitor the spread of tigecycline resistance. In this study, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect tet(X) in bacterial isolates was developed. This method utilized the analysis by LC-MS/MS of metabolite ratios to determine the presence of tet(X). Bacterial suspensions were co-incubated with tigecycline for 1 h, where tet(X) destructase inactivated tigecycline, making a particular metabolite with a 16-Da change in mass. The characterized quantitative ion pairing of tigecycline in the ESI positive mode was observed at 586.1 → 569.1 m/z. The oxygenated tigecycline detection was established at 602.2 → 529.1 m/z. A model was established using 35 tet(X)-positive and 15 tet(X)-negative Enterobacteriaceae strains in this study to optimize the cutoff value. Applying the model to analyze 70 bacterial isolates, the sensitivity of the LC-MS/MS test was 98.9% compared to polymerase chain reaction (PCR), and specificity was 100%. This method is rapid and easy to operate, providing results within 1 h, making it more suitable for routine use in clinical microbiology laboratories.

IntroductionThe dissemination of strains producing tetracyclines monooxygenase Tet(X) from breeding farms to the natural environment poses a potential threat to public health.MethodsAntimicrobial susceptibility testing and WGS were performed to identify resistance phenotypes and genotypes. Cloning experiments, sequence alignment, and homology modeling were used to characterize the function and formation mechanisms of the recombinant variant. The mobilization potential of Tet(X) was assessed by collinearity analysis, conjugation experiments, and phylogenetic analysis.ResultsThree tet(X)-producing Elizabethkingia meningoseptica strains were isolated from bullfrog breeding ponds, the sewage outlet, and downstream river in Zhejiang Province, China. These strains carry a novel Tet(X) variant, differing from Tet(X6) by seven residues, and possess the ability to degrade tetracyclines. Interestingly, the novel Tet(X) is a recombinant variant formed by homologous recombination of Tet(X6) and the C-terminal of Tet(X2). Further analysis revealed that Tet(X6) formed several Tet(X) variants, including Tet(X5), through homologous recombination. The novel tet(X) gene is located on a circularizable integrative and conjugative element (ICEEmeChn3), with ISwz1 participating in the recombination of its multi-drug resistance region, potentially facilitating the mobilization and recombination of tet(X) in early hosts. These three strains were clonally transmitted and shared a close genetic relationship (SNP

The emergence and widespread of tigecycline resistance undoubtedly poses a serious threat to public health globally. The exploration of combination therapies has become preferred antibacterial strategies to alleviate this global burden. In this study, tigecycline-resistant tet(X4)-positive Escherichia coli were selected for adjuvant screening. Interestingly, 9-aminominocycline (9-AMC), one of the tigecycline metabolites, exhibits synergistic antibacterial activity with tigecycline using checkerboard assay. The efficacy in vitro and in vivo was evaluated, and the synergistic mechanism was further explored. The results suggested that 9-AMC combined with tigecycline could inhibit the growth of antibiotic resistant bacteria, efficiently retard the evolution of tet(X4) gene and narrow the drug mutant selection window. In addition, the combination of tigecycline and 9-AMC could destroy the normal membrane structure of bacteria, inhibit the formation of biofilm, remarkably reduce the level of intracellular ATP level, and accelerate the oxidative damage of bacteria. Furthermore, 9-AMC is more stable in the bind of Tet(X4) inactivating enzyme. The transcriptomics analysis revealed that the genes related to the 9-AMC and tigecycline were mainly enriched in ABC transporters. Collectively, the results reveal the potentiation effects on tigecycline and the probability of 9-AMC as a novel tigecycline adjuvant against tet(X4)-positive Escherichia coli, which provides new insights for adjuvant screening.

IntroductionThe 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.MethodsIn 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.ResultsOverall, 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.DiscussionThe 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.

ObjectivesTigecycline is recognized as one of the last-line antibiotics to treat serious bacterial infection caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). The plasmid-borne gene tet(X4) mediates high resistance to tigecycline. However, the prevalence and genetic context of tet(X4) in K. pneumoniae from various sources are not fully understood. Here, we investigated the prevalence of tet(X4)-positive K. pneumoniae and characterized the genetic context of tet(X4)-bearing plasmids in K. pneumoniae isolates.MethodsPolymerase chain reaction (PCR) was used to detect the tet(X4) gene. The transferability of the tet(X4)-carrying plasmids was tested by conjugation assays. The Galleria mellonella infection model was used to test virulence of tet(X4)-positive strains. Whole-genome sequencing and genome-wide analysis were performed to identify the antimicrobial resistance and the virulence genes, and to clarify the genetic characteristics of the tet(X4)-positive isolates.ResultsAmong 921 samples, we identified two tet(X4)-positive K. pneumoniae strains collected from nasal swabs of two pigs (0.22%, 2/921). The two tet(X4)-positive isolates exhibited high minimum inhibitory concentrations to tigecycline (32–256 mg/L) and tetracycline (256 mg/L). The plasmids carrying the tet(X4) gene can transfer from the donor strain K. pneumoniae to the recipient strain Escherichia coli J53. Genetic analysis of the complete sequence of two tet(X4)-carrying plasmids pTKPN_3-186k-tetX4 and pTKPN_8-216k-tetX4 disclosed that the tet(X4) gene was flanked by delta ISCR2 and IS1R, which may mediate the transmission of the tet(X4) gene.ConclusionThe prevalence of tet(X4)-positive K. pneumoniae among different sources was low. ISCR2 and IS1R may contribute to the horizontal transfer of tet(X4) gene. Effective measures should be taken to prevent the transmission of tet(X4)-producing K. pneumoniae in humans or animals.

Introduction: The dissemination of strains producing tetracyclines monooxygenase Tet(X) from breeding farms to the natural environment poses a potential threat to public health., Methods: Antimicrobial susceptibility testing and WGS were performed to identify resistance phenotypes and genotypes. Cloning experiments, sequence alignment, and homology modeling were used to characterize the function and formation mechanisms of the recombinant variant. The mobilization potential of Tet(X) was assessed by collinearity analysis, conjugation experiments, and phylogenetic analysis., Results: Three tet (X)-producing Elizabethkingia meningoseptica strains were isolated from bullfrog breeding ponds, the sewage outlet, and downstream river in Zhejiang Province, China. These strains carry a novel Tet(X) variant, differing from Tet(X6) by seven residues, and possess the ability to degrade tetracyclines. Interestingly, the novel Tet(X) is a recombinant variant formed by homologous recombination of Tet(X6) and the C-terminal of Tet(X2). Further analysis revealed that Tet(X6) formed several Tet(X) variants, including Tet(X5), through homologous recombination. The novel tet (X) gene is located on a circularizable integrative and conjugative element (ICE EmeChn3 ), with IS wz1 participating in the recombination of its multi-drug resistance region, potentially facilitating the mobilization and recombination of tet (X) in early hosts. These three strains were clonally transmitted and shared a close genetic relationship (SNP < 62) with a clinically-sourced strain isolated from the same province., Discussion: To our knowledge, this is the first report of homologous recombination between Tet(X) variants with differing activities. These clonal strains provide evidence of the transmission of tet (X)-positive strains from aquaculture sewage to the natural environment, highlighting the need to strengthen the monitoring and management of this emerging farming model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jin, Jia, Jin, Zhu, Wang, Sun and Cui.)

The prevalence of plasmid-mediated tigecycline resistance gene tet(X4) is presenting an increasing trend. Once tet(X4)-bearing plasmids are captured by multidrug-resistant bacteria, such as blaNDM and mcr-coharboring bacteria, it will promote bacteria to develop an ultra-broad resistance spectrum, limiting clinical treatment options. However, little is known about the destiny of such bacteria or how they will evolve in the future. Herein, we constructed a multidrug-resistant bacteria coharboring tet(X4), blaNDM-5, and mcr-1 by introducing a tet(X4)-bearing plasmid into a blaNDM-5 and mcr-1 positive E. coli strain. Subsequently, the stability of tet(X4) and the plasmid was measured after being evolved under tigecycline or antibiotic-free circumstance. Interestingly, we observed both tet(X4)-bearing plasmids in tigecycline treated strains and non-tigecycline treated strains were stable, which might be jointly affected by the increased conjugation frequency and the structural alterations of the tet(X4)-positive plasmid. However, the stability of tet(X4) gene showed different scenarios in the two types of evolved strains. The tet(X4) gene in non-tigecycline treated strains was stable whereas the tet(X4) gene was discarded rapidly in tigecycline treated strains. Accordingly, we found the expression levels of tet(X4) gene in tigecycline-treated strains were several times higher than in non-tigecycline treated strains and ancestral strains, which might in turn impose a stronger burden on the host bacteria. SNPs analysis revealed that a myriad of mutations occurred in genes involving in conjugation transfer, and the missense mutation of marR gene in chromosome of tigecycline treated strains might account for the completely different stability of tet(X4)-bearing plasmid and tet(X4) gene. Collectively, these findings shed a light on the possibility of the emergence of multidrug resistant bacteria due to the transmission of tet(X4)-bearing plasmid, and highlighted that the antibiotic residues may be critical to the development of such bacteria.

The tigecycline resistance gene tet(X4) was widespread in various bacteria. However, limited information about the plasmid harboring the tet(X4) gene spread among the different species is available. Here, we investigated the transmission mechanisms of the tet(X4) gene spread among bacteria in a pig farm. The tet(X4) positive Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter hormaeche were identified in the same farm. The whole genome sequencing (WGS) analysis showed that the K. pneumoniae belonged to ST727 (n = 11) and ST3830 (n = 1), E. cloacae and E. hormaeche belonged to ST524 (n = 1) and ST1862 (n = 1). All tet(X4) genes were located on the IncHI1 plasmids that could be conjugatively transferred into the recipient E. coli C600 at 30°C. Moreover, a fusion plasmid was identified that the IncHI1 plasmid recombined with the IncN plasmid mediated by ISCR2 during the conjugation from strains B12L to C600 (pB12L-EC-1). The fusion plasmid also has been discovered in a K. pneumoniae (K1L) that could provide more opportunities to spread antimicrobial resistance genes. The tet(X4) plasmids in these bacteria are derived from the same plasmid with a similar structure. Moreover, all the IncHI1 plasmids harboring the tet(X4) gene in GenBank belonged to the pST17, the newly defined pMLST. The antimicrobial susceptibility testing was performed by broth microdilution method showing the transconjugants acquired the most antimicrobial resistance from the donor strains. Taken together, this report provides evidence that IncHI1/pST17 is an important carrier for the tet(X4) spread in Enterobacteriaceae species, and these transmission mechanisms may perform in the environment.

The emergence and widespread of tigecycline resistance undoubtedly poses a serious threat to public health globally. The exploration of combination therapies has become preferred antibacterial strategies to alleviate this global burden. In this study, tigecycline-resistant tet (X4)-positive Escherichia coli were selected for adjuvant screening. Interestingly, 9-aminominocycline (9-AMC), one of the tigecycline metabolites, exhibits synergistic antibacterial activity with tigecycline using checkerboard assay. The efficacy in vitro and in vivo was evaluated, and the synergistic mechanism was further explored. The results suggested that 9-AMC combined with tigecycline could inhibit the growth of antibiotic resistant bacteria, efficiently retard the evolution of tet (X4) gene and narrow the drug mutant selection window. In addition, the combination of tigecycline and 9-AMC could destroy the normal membrane structure of bacteria, inhibit the formation of biofilm, remarkably reduce the level of intracellular ATP level, and accelerate the oxidative damage of bacteria. Furthermore, 9-AMC is more stable in the bind of Tet(X4) inactivating enzyme. The transcriptomics analysis revealed that the genes related to the 9-AMC and tigecycline were mainly enriched in ABC transporters. Collectively, the results reveal the potentiation effects on tigecycline and the probability of 9-AMC as a novel tigecycline adjuvant against tet (X4)-positive Escherichia coli, which provides new insights for adjuvant screening., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sun, Zhang, Ma, Ali, Wu and Li.)

The emergence of pathogens is conferring resistance to last-resort therapies such as tigecycline, colistin, and carbapenems, limiting the therapeutic options, and raising concerns about the emergence of new “superbugs.” This study reports the first incident of a blaNDM–5 and tet(X4) co-harboring Escherichia coli with resistance to carbapenem and tigecycline recovered as the causative agent of a urinary tract infection in a 94-year-old patient. The E. coli strain ECCL209 carries multiple resistance genes [i.e., blaTEM–1B, blaNDM–5, blaCMY–2, aadA22, florR, erm(B), mph(A), erm(42), lnuG, qnrS1, and sul2] and exhibits resistance to almost all clinically used antibiotics. MLST analysis found that the strain belongs to ST648, considered a worldwide high-risk pandemic clone. Moreover, multiple plasmid incompatibility types were detected, i.e., IncHI1A, IncHI1B, IncFII, IncFIA, IncFIB, IncQ1, Col, and IncX4. Genetic analysis revealed that blaNDM–5 and tet(X4) genes were localized on two hybrid plasmids with multiple replicons. Continuous monitoring studies are suggested to quantify the antimicrobial resistance and assess the dissemination of such superbugs into a human healthcare setting.

To investigate tetracycline resistance and resistant genotype in Riemerella anatipestifer, the tetracycline susceptibility of 212 R. anatipestifer isolates from China between 2011 and 2017 was tested. The results showed that 192 of 212 (90.6%) R. anatipestifer isolates exhibited resistance to tetracycline (the MICs ranged from 4 to 256µg/ml). The results of PCR detection showed that, 170 of 212 (80.2%) R. anatipestifer isolates possessed the tet(X) gene. Other genes, including tet(A), tet(M), tet(Q), tet(O), tet(B), and tet(O/W/32/O), were found at frequencies of 20.8, 4.7, 1.4, 0.9, 0.9, and 0.5%, respectively. However, tet(C), tet(E), tet(G), tet(K), and tet(W) were not detected in any isolate. In these tet gene positive strains, 31 (14.6%), 2 (0.9%), 5 (2.4%), 1 (0.5%), 3 (1.4%) were detected containing tet(A)/tet(X), tet(M)/tet(O), tet(M)/tet(X), tet(O)/tet(X), and tet(Q)/tet(X) simultaneously, respectively. One isolates, R131, unexpectedly contained three tet genes, i.e., tet(M), tet(O), and tet(X). Sequence analysis of the tet gene ORFs cloned from R. anatipestifer isolates confirmed that tet(A), tet(B), tet(M), tet(O), tet(Q) and an unusual mosaic tet gene tet(O/W/32/O) were present in R. anatipestifer. The MIC results of R. anatipestifer ATCC 11845 transconjugants carrying tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes exhibited tetracycline resistance with MIC values ranging from 4 to 64µg/ml. Additionally, the tet(X) gene could transfer into susceptible strain via natural transformation (transformation frequencies of ~10-6). In conclusion, the tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes were found and conferred tetracycline resistance in R. anatipestifer isolates. Moreover, the tet(X) is the main mechanism of tetracycline resistance in R. anatipestifer isolates. To our knowledge, this is the first report of tet(A), tet(B), tet(M), tet(O), tet(Q), and mosaic gene tet(O/W/32/O) in R. anatipestifer. [ABSTRACT FROM AUTHOR]

With the large-scale use of antibiotics, antibiotic resistant bacteria (ARB) continue to rise, and antibiotic resistance genes (ARGs) are regarded as emerging environmental pollutants. The new tetracycline-class antibiotic, tigecycline is the last resort for treating multidrug-resistant (MDR) bacteria. Plasmid-mediated horizontal transfer enables the sharing of genetic information among different bacteria. The tigecycline resistance gene tet(X) threatens the efficacy of tigecycline, and the adjacent ISCR2 or IS26 are often detected upstream and downstream of the tet(X) gene, which may play a crucial driving role in the transmission of the tet(X) gene. Since the first discovery of the plasmid-mediated high-level tigecycline resistance gene tet(X4) in China in 2019, the tet(X) genes, especially tet(X4), have been reported within various reservoirs worldwide, such as ducks, geese, migratory birds, chickens, pigs, cattle, aquatic animals, agricultural field, meat, and humans. Further, our current researches also mentioned viruses as novel environmental reservoirs of antibiotic resistance, which will probably become a focus of studying the transmission of ARGs. Overall, this article mainly aims to discuss the current status of plasmid-mediated transmission of different tet(X) genes, in particular tet(X4), as environmental pollutants, which will risk to public health for the “One Health” concept.

The rapid dissemination of plasmid-mediated tet(X) genes in Acinetobacter species has compromised the clinical effectiveness of tigecycline, one of the last-resort antibiotics. However, the classification strategy and homology group of tet(X)-positive Acinetobacter spp. plasmids remain largely unknown. In this study, we classified them by genome-based replicon typing, followed by analyses of structural characteristics, transferability and in vivo effect. A total of 34 plasmids distributed in at least nine Acinetobacter species were collected, including three tet(X3)-positive plasmids and one tet(X6)-positive plasmid from our genome sequencing results. Among them, there were 28 plasmids carrying Rep_3 superfamily replicase genes and classified into six homology groups, consisting of GR31 (82.1%), GR26 (3.6%), GR41 (3.6%), GR59 (3.6%), and novel groups GR60 (3.6%) and GR61 (3.6%). Our tet(X3)-positive plasmids pYH16040-1, pYH16056-1, and pYH12068-1 belonged to the dominant GR31 group, whereas the tet(X6)-positive plasmid pYH12068-2 was unclassified. Structurally, all tet(X)-positive GR31 plasmids shared similar plasmid replication (repB), stability (parA and parB) and accessory modules [tet(X) and sul2], and 97.6% of plasmid-mediated tet(X) genes in Acinetobacter species were adjacent to ISCR2. Conjugation and susceptibility testing revealed pYH16040-1, pYH16056-1, and pYH12068-2, carrying plasmid transfer modules, were able to mediate the mobilization of multiple antibiotic resistance. Under the treatment of tigecycline, the mortality rate of Galleria mellonella infected by pYH16040-1-mediated tet(X3)-positive Acinetobacter spp. isolate significantly increased when compared with its plasmid-cured strain (p < 0.0001). The spread of such plasmids is of great clinical concern, more effects are needed and will facilitate the future analysis of tet(X)-positive Acinetobacter spp. plasmids.

This study aimed to investigate the prevalence and characterization of tet(X4) in Escherichia coli isolates from a pig farm in Shanghai, China, and to elucidate tet(X4) dissemination mechanism in this swine farm. Forty-nine (80.33%) E. coli strains were isolated from 61 samples from a pig farm and were screened for the presence of tet(X). Among them, six (12.24%) strains were positive for tet(X4) and exhibited resistance to tigecycline (MIC ≥ 16 mg/L). They were further sequenced by Illumina Hiseq. Six tet(X4)-positive strains belonged to ST761 with identical resistance genes, resistance profiles, plasmid replicons, and cgMLST type except that additional ColE10 plasmid was present in isolate SH21PTE35. Isolate SH21PTE31, as a representative ST761 E. coli strain, was further sequenced using Nanopore MinION. The tet(X4) in SH21PTE31 was located on IncFIA18/IncFIB(K)/IncX1 hybrid plasmid pYUSHP31-1, highly similar to other tet(X4)-carrying IncFIA18/IncFIB(K)/IncX1 plasmids from ST761 E. coli and other E. coli lineages in China. These IncFIA18/IncFIB(K)/IncX1 plasmids shared closely related multidrug resistance regions, and could reorganize, acquire or lose resistance modules mediated by mobile elements such as ISCR2 and IS26. Phylogenetic analysis were performed including all tet(X4)-positive isolates obtained in this pig farm combined with 43 tet(X4)-positive E. coli from pigs, cow, pork, wastewater, and patients with the same ST from NCBI. The 50 tet(X4)-carrying E. coli ST761 isolates from different areas in China shared a close phylogenetic relationship (0-49 SNPs). In conclusion, clonal transmission of tet(X4)-positive E. coli ST761 has occurred in this swine farm. E. coli ST761 has the potential to become a high-risk clone for tet(X4) dissemination in China.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Shao, Wu, Li, He, Chen, Sun, Yang, Feng, Zhang, Yan, Dai, Tian and Zhong.)

The prevalence of plasmid-mediated tigecycline resistance gene tet(X4) is presenting an increasing trend. Once tet(X4)-bearing plasmids are captured by multidrug-resistant bacteria, such as blaNDM and mcr-coharboring bacteria, it will promote bacteria to develop an ultra-broad resistance spectrum, limiting clinical treatment options. However, little is known about the destiny of such bacteria or how they will evolve in the future. Herein, we constructed a multidrug-resistant bacteria coharboring tet(X4), blaNDM-5, and mcr-1 by introducing a tet(X4)-bearing plasmid into a blaNDM-5 and mcr-1 positive E. coli strain. Subsequently, the stability of tet(X4) and the plasmid was measured after being evolved under tigecycline or antibiotic-free circumstance. Interestingly, we observed both tet(X4)-bearing plasmids in tigecycline treated strains and non-tigecycline treated strains were stable, which might be jointly affected by the increased conjugation frequency and the structural alterations of the tet(X4)-positive plasmid. However, the stability of tet(X4) gene showed different scenarios in the two types of evolved strains. The tet(X4) gene in nontigecycline treated strains was stable whereas the tet(X4) gene was discarded rapidly in tigecycline treated strains. Accordingly, we found the expression levels of tet(X4) gene in tigecycline-treated strains were several times higher than in non-tigecycline treated strains and ancestral strains, which might in turn impose a stronger burden on the host bacteria. SNPs analysis revealed that a myriad of mutations occurred in genes involving in conjugation transfer, and the missense mutation of marR gene in chromosome of tigecycline treated strains might account for the completely different stability of tet(X4)-bearing plasmid and tet(X4) gene. Collectively, these findings shed a light on the possibility of the emergence of multidrug resistant bacteria due to the transmission of tet(X4)-bearing plasmid, and highlighted that the antibiotic residues may be critical to the development of such bacteria. [ABSTRACT FROM AUTHOR]

Many novel tigecycline-inactivating enzymes encoded by tet(X) variants from different bacteria were discovered since the plasmid-mediated tet(X3) and tet(X4) genes conferring high-level resistance to tigecycline in Enterobacterales and Acinetobacter were reported. However, there have been no comprehensive studies of the prevalence of different tet(X) variants in poultry farms. In this study, we collected 45 chicken fecal samples, isolated tet(X)-positive strains, and performed antimicrobial susceptibility testing, conjugation assay, whole-genome sequencing, and bioinformatics analysis. A total of 15 tet(X)-bearing strains were isolated from 13 samples. Species identification and tet(X) subtyping analysis found that the 15 strains belonged to eight different species and harbored four different tet(X) variants. Genomic investigation showed that transmission of tet(X) variants was associated with various mobile genetic elements, and tet(X4) was the most prevalent variant transferred by conjugative plasmids. Meanwhile, we characterized a plasmid co-harboring tet(X6) and blaOXA–58 in Acinetobacter baumannii. In summary, we demonstrated that different tet(X) variants were widely disseminated in the chicken farming environment and dominated by tet(X4). This finding expands the understanding of the prevalence of tet(X) among different animal sources, and it was advocated to reduce the usage of antibiotics to limit the emergence and transmission of novel tet(X) variants in the poultry industry.

The emergence of plasmid-mediated tigecycline resistance gene tet(X4) poses a challenging threat to public health. Based on the analysis of tet(X4)-positive plasmids in the NCBI database, we found that the IncX1-type plasmid is one of the most common vectors for spreading tet(X4) gene, but the mechanisms by which these plasmids adapt to host bacteria and maintain the persistence of antibiotic resistance genes (ARGs) remain unclear. Herein, we investigated the underlying mechanisms of how host bacteria modulate the fitness cost of IncX1 plasmids carrying tet(X4) gene. Interestingly, we found that the tet(X4)-bearing IncX1 plasmids encoding H-NS protein imposed low or no fitness cost in Escherichia coli and Klebsiella pneumoniae; instead, they partially promoted the virulence and biofilm formation in host bacteria. Regression analysis revealed that the expression of hns gene in plasmids was positively linked to the relative fitness of host bacteria. Furthermore, when pCE2::hns was introduced, the fitness of tet(X4)-positive IncX1 plasmid pRF55-1 without hns gene was significantly improved, indicating that hns mediates the improvement of fitness. Finally, we showed that the expression of hns gene is negatively correlated with the expression of tet(X4) gene, suggesting that the regulatory effect of H-NS on adaptability may be attributed to its inhibitory effect on the expression of ARGs. Together, our findings suggest the important role of plasmid-encoded H-NS protein in modulating the fitness of tet(X4)-bearing IncX1 plasmids, which shed new insight into the dissemination of tet(X4) gene in a biological environment.

IntroductionThis study aimed to investigate the genetic factors promoting widespread Q6 dissemination of tet(X4) between Escherichia coli and to characterize the genetic contexts of tet(X4).MethodsWe isolated E. coli from feces, water, soil and flies collected across a large-scale chicken farm in China in 2020. Antimicrobial susceptibility testing and PFGE typing were used to identify tigecycline resistance and assess clonal relationships among isolates. Plasmids present and genome sequences were analyzed by conjugation, S1 pulsed-field gel electrophoresis (PFGE), plasmid stability testing and whole-genome sequencing.ResultsA total of 204 tigecycline-resistant E. coli were isolated from 662 samples. Of these, we identified 165 tet(X4)-carrying E. coli and these strains exhibited a high degree of multidrug resistance. Based on the geographical location distribution of the sampled areas, number of samples in each area and isolation rate of tigecycline-resistant strains and tet(X4)-carrying isolates, 72 tet(X4)-positive isolates were selected for further investigation. Tigecycline resistance was shown to be mobile in 72 isolates and three types of tet(X4)-carrying plasmids were identified, they were IncHI1 (n = 67), IncX1 (n = 3) and pO111-like/IncFIA(HI1) (n = 2). The pO111-like/IncFIA(HI1) is a novel plasmid capable of transferring tet(X4). The transfer efficiency of IncHI1 plasmids was extremely high in most cases and IncHI1 plasmids were stable when transferred into common recipient strains. The genetic structures flanked by IS1, IS26 and ISCR2 containing tet(X4) were complex and varied in different plasmids.DiscussionThe widespread dissemination of tigecycline-resistant E. coli is a major threat to public health. This data suggests careful use of tetracycline on farms is important to limit spread of resistance to tigecycline. Multiple mobile elements carrying tet(X4) are in circulation with IncHI1 plasmids the dominant vector in this setting.

The emergence and spread of carbapenemase genes, colistin resistance genes mcr-1, and tigecycline resistance gene tet(X) represent a significant threat to clinical therapy and public health. In this study, we investigated the presence of carbapenemase genes, mcr-1, and tet(X) in 298 Escherichia coli strains obtained from a teaching hospital in China. In total, eight (2.68%), six (2.01%), and one (0.34%) E. coli isolates carried blaNDM, mcr-1, and tet(X4), respectively. The blaNDM gene was located on IncX3 (n = 4), F2:A-:B- (n = 3), and F2:A1:B1 (n = 1) plasmids, with high similarity to multiple plasmids belonging to the same incompatibility type from Enterobacteriaceae. Six MCR-producing strains contained mcr-1-carrying IncI2 plasmids, organized similarly to other mcr-1-bearing IncI2 plasmids from animals in China. The blaCTX−M−55/64/132/199 gene located within a typical transposition unit (ISEcp1-blaCTX−M-orf477Δ) was inserted near dnaJ to generate 5-bp direct repeats in four mcr-1-positive plasmids. The tet(X) and another four resistance genes [aadA2, tet(A), floR, and Δlnu(F)] were co-located on an IncX1 plasmid, highly similar to other tet(X4)-carrying IncX1 plasmids from Escherichia and Klebsiella of animal or food origin, except that the conjugative transfer region of IncX1 plasmids was absent in our plasmid. Although a low prevalence of blaNDM, mcr-1, and tet(X) was observed in E. coli from patients in this study, their dissemination associated with some successful pandemic plasmids is of great concern. The continued surveillance of these crucial resistance genes in patients should be strengthened.

ObjectivesCarbapenems, colistin, and tigecycline are critically important antibiotics in clinics. After the global appearance of blaNDM and mcr mediating the resistance to carbapenems and colistin, respectively, tigecycline becomes the last-resort drug against severe human infections caused by multidrug-resistant bacteria. Recently, a mobile tigecycline resistance gene tet(X4) has been identified in Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii that causes high resistance to tigecycline and other tetracyclines. In this study, the prevalence of tet(X4) in E. coli isolates from duck and goose farms in Southeast China was identified and characterized.MethodsFeces, soil, sewage, and dust samples were collected from duck and goose farms along with the southeast coast provinces of China. Antimicrobial susceptibility testing and polymerase chain reaction screening were performed to investigate the phenotype and genotype of tigecycline resistance. Conjugation, S1 pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing were used to determine the transferability, genetic location, and the genomic characteristics of tet(X4).ResultsIn total, 1,716 samples were collected, and 16 isolates (0.9%) recovered from Guangdong, Shandong, and Jiangsu were positive for tet(X4) gene with tigecycline minimum inhibitory concentrations ≥16 mg/L. Notably, among these tet(X4)-positive E. coil isolates, seven of them were from the environment samples (soil and sewage). PFGE and multilocus sequence typing demonstrated that ST3997 was the most prevalent sequence type (eight isolates, 50%) in Jiangsu province. By conjugation assays, 11 isolates were able to transfer tet(X4) plasmid to E. coli C600 recipient, and these plasmids belonged to IncHI1 and IncX1 detected by sequence analysis. tet(X4) was found adjacent to an insertion sequence ISCR2 downstream and a catD gene upstream for all isolates. In addition, multiple-drug resistance to tigecycline, chlortetracycline, ampicillin, florfenicol, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and fosfomycin was profiled in most of the tet(X4)-positive isolates.ConclusionThe identification of tet(X4) harboring E. coli strains in duck farms and their surrounding environment enlarges our knowledge of the variety and prevalence of tigecycline resistance. The prevalence of tet(X4) raises concern for the use of tetracyclines in animal farming, and the tet(X4) gene should be listed as primary gene for resistance surveillance.

The emergence and prevalence of tigecycline-resistant Klebsiella pneumoniae have seriously compromised the effectiveness of antimicrobial agents in the treatment of infections. To explore the role of the plasmid-borne tet(A) gene in tigecycline resistance in carbapenem-resistant K. pneumoniae (CRKP), a total of 63 CRKP isolates were collected from a tertiary hospital in Hangzhou, China. The minimum inhibitory concentration (MIC) of tigecycline, mutation rate of tet(A) gene, genetic surroundings of tet(A)-carrying transmissible plasmid and the contribution of tet(A) mutation to tigecycline resistance were analyzed using antimicrobial susceptibility test, whole-genome sequencing, tigecycline resistance evolution experiment, and plasmid conjugation experiment. Our results showed that 52.4% (33 isolates) of the test isolates carried the tet(A) gene; among them, 75.8% (25 isolates) exhibited a tigecycline non-susceptible phenotype (MIC = 4 mg/L). Three clonal groups (cluster I, cluster II, and cluster III) were identified in these tet(A)-bearing isolates. All 17 isolates belonged to serotype KL21 (cluster I), which differed by only 13 SNPs, suggesting a clonal spread of tet(A)-positive ST11 K. pneumoniae with serotype KL21 occurred in the sampling hospital. The induction of tigecycline resistance experiments showed that 71.4% of strains evolved tet(A) mutations and developed a high-level tigecycline resistance. Eight amino acid substitutions were identified in these mutants. The most common amino acid substitution was A370V, followed by S251A and G300E. Twelve isolates carrying tet(A) mutants succeeded in the filter mating experiment with a conjugation efficiency of 10–3–10–8. Tigecycline MICs in E. coli EC600 transconjugants with a mutated tet(A) were 2 to 8-fold higher than those in E. coli EC600 transconjugants with a wild-type tet(A). One ColRNAI/IncFII type and two IncFII type tet(A)-bearing conjugative plasmids were identified in this study, including a class 1 integron containing multiple antibiotic resistance genes, i.e., tet(A), qnrS1, blaLAP–2, catA2, sul2, and dfrA14. Our study revealed the wide-spread situation of plasmid-borne tet(A) gene in clinical CRKP, and mutation of tet(A) is a potential driven force that lead to tigecycline resistance.

The purpose of this study was to characterize the complete sequence of a novel plasmid carrying tigecycline resistance gene tet(X) and carbapenemase gene blaOXA-58 from a swine Acinetobacter sp. strain SH19PTT10. Minimal inhibitory concentration (MIC) was performed using microbroth dilution method. The isolate SH19PTT10 was highly resistant (16 mg/L) to tigecycline, and also exhibited resistance to ampicillin, streptomycin, tetracycline, chloramphenicol, florfenicol, ciprofloxacin, and sulfamethoxazole/trimethoprim. Although SH19PTT10 harbored blaOXA-58, it was susceptible to cefotaxime and meropenem. The genome sequence of SH19PTT10 was determined using PacBio single-molecule real-time sequencing. Plasmid pYUSHP10-1 had a size of 174,032 bp and showed partial homology to several plasmids found in Acinetobacter isolates. It contained two repA genes, putative toxin-antitoxin systems (HipA/HipB, RelE/RelB, and BrnT/BrnA), partitioning genes (parA and parB), and heavy metal resistance-associated genes (copA/copB, nrp, and czcA/czcD) but the transfer region or proteins was not found. pYUSHP10-1 carried 16 resistance genes, mainly clustered in two mosaic multiresistance regions (MRRs). The first MRR contained sul3, qacI-aadA1-clmA1-aadA2-blaCARB-2-dfrA16 cassette, aac(3)-IId, and blaOXA-58. The blaOXA-58 gene was associated with ISAba3, as previously described. The second MRR is the tet(X) region (ISAcsp12-aph(3')-Ia-IS26-ΔxerD-tet(X)-res-ISCR2-sul2) related to the corresponding region in other tet(X)-bearing plasmids. The pdif sites, as well as mobile elements, play an important role in mobilization of DNA modules and plasmid evolution. Coexistence of numerous resistance genes on a single plasmid may contribute to the dissemination of these genes under pressure posed by different agents, which may explain the presence of clinically crucial resistance genes tet(X) and blaOXA-58 in livestock. Thus, rational drug use and continued surveillance of tet(X) and blaOXA-58 in livestock are warranted.

The objective of this study was to explore the genetic and biological features of the tet(M)-harboring plasmid pTS14 in Salmonella enterica strain S14 isolated from a chicken fecal sample. Plasmid pTS14 was identified by conjugation, S1-pulsed-field gel electrophoresis (PFGE), Southern hybridization, and plasmid sequencing. The biological characteristics of pTS14 were assessed via stability, growth kinetics, and starvation survival experiments. Strain S14, belonging to ST3007, harbored a 119-kb tet(M)-bearing IncF2:A1:B1 conjugative plasmid pTS14. The plasmid pTS14 contained a novel transposon Tn6709 with the genetic structure IS26-tnpA1-tnpA2-Δorf13-LP-tet(M)-tnpX-ΔtnpR-IS26, and the resistance genes tet(B), tet(D), strAB, sul2, and blaTEM–1b. In addition, pTS14 was found to be highly stable in the recipient strain E. coli J53. The transconjugant TS14 exhibited a higher survival ratio than E. coli J53 under permanent starvation-induced stress. The tet(M)-bearing IncF2 epidemic plasmid lineage may accelerate the dissemination of tet(M) and other genes by coselection, which could constitute a potentially serious threat to clinical treatment regimens.

Carbapenems and tigecycline are two important classes of antimicrobial agents to treat the infections caused by Enterobacterales. Here, we reported a plasmid carrying both blaIMP–26 and tet(A) variant in clinical Klebsiella pneumoniae KP-1572. MIC results showed that K. pneumonia KP-1572 was resistant to a wide range of antimicrobials. The blaIMP–26 and tet(A) variant were located on an identical plasmid, which was indicated by S1-PFGE and southern blotting hybridization and can be successfully transferred by electroporation. Whole-plasmid sequencing and analysis revealed that a 142,993-bp-sized plasmid, designated pIMP1572, contains an IncFIIk backbone and a variable region harboring blaIMP–26 and tet(A) variant. The plasmid pIMP1572 was apparently originated from a tet(A)-carrying IncFIIk plasmid but with a deletion length of 6,216-bp and a multiple drug resistance region (MDRR) insertion of 25,259 bp. The plasmid pIMP1572 in the present study represents the first report of the IncFIIk plasmid co-carrying blaIMP and tet(A) variant, which should be monitored.

Chlamydia suis is a swine pathogen that has also recently been found to cause zoonotic infections of the human eye, pharynx, and gastrointestinal tract. Many strains contain a tetracycline class C gene [tet(C)] cassette that confers tetracycline resistance. The cassette was likely originally acquired by horizontal gene transfer from a Gram-negative donor after the introduction of tetracycline into animal feed in the 1950s. Various research groups have described the capacity for different Chlamydia species to exchange DNA by homologous recombination. Since over 90% of C. suis strains are tetracycline resistant, they represent a potential source for antibiotic-resistance spread within and between Chlamydiaceae species. Here, we examined the genetics of tet(C)-transfer among C. suis strains. Tetracycline-sensitive C. suis strain S45 was simultaneously or sequentially co-infected with tetracycline-resistant C. suis strains in McCoy cells. Potential recombinants were clonally purified by a harvest assay derived from the classic plaque assay. C. suis strain Rogers132, lacking transposases IS200 and IS605, was the most efficient donor, producing two unique recombinants detected in three of the 56 (5.4%) clones screened. Recombinants were found to have a minimal inhibitory concentration (MIC) of 8-16 μg/mL for tetracycline. Resistance remained stable over 10 passages as long as recombinants were initially grown in tetracycline at twice the MIC of S45 (0.032 μg/mL). Genomic analysis revealed that tet(C) had integrated into the S45 genome by homologous recombination at two unique sites depending on the recombinant: a 55 kb exchange between nrqF and pckG, and a 175 kb exchange between kdsA and cysQ. Neither site was associated with inverted repeats or motifs associated with recombination hotspots. Our findings show that cassette transfer into S45 has low frequency, does not require IS200/IS605 transposases, is stable if initially grown in tetracycline, and results in multiple genomic configurations. We provide a model for stable cassette transfer to better understand the capability for cassette acquisition by Chlamydiaceae species that infect humans, a matter of public health importance.

Annotations of non-pathogenic bacterial genomes commonly reveal putative antibiotic resistance genes and the potential risks associated with such genes is challenging to assess. We have examined a putative tetracycline tet(L) gene (conferring low level tetracycline resistance), present in the majority of all publicly available genomes of the industrially important operational group Bacillus amyloliquefaciens including the species B. amyloliquefaciens, Bacillus siamensis and Bacillus velezensis. The aim was to examine the risk of transfer of the putative tet(L) in operational group B. amyloliquefaciens through phylogenetic and genomic position analysis. These analyses furthermore included tet(L) genes encoded by transferable plasmids and other Gram-positive and -negative bacteria, including Bacillus subtilis. Through phylogenetic analysis, we could group chromosomally and plasmid-encoded tet(L) genes into four phylogenetic clades. The chromosomally encoded putative tet(L) from operational group B. amyloliquefaciens formed a separate phylogenetic clade; was positioned in the same genomic region in the three species; was not flanked by mobile genetic elements and was not found in any other bacterial species suggesting that the gene has been present in a common ancestor before species differentiation and is intrinsic. Therefore the gene is not considered a safety concern, and the risk of transfer to and expression of resistance in other non-related species is considered negligible. We suggest a subgrouping of the tet(L) class into four groups (tet(L)1.1, tet(L)1.2 and tet(L)2.1, tet(L)2.2), corresponding with the phylogenetic grouping and tet(L) from operational group B. amyloliquefaciens referred to as tet(L)2.2. Phylogenetic analysis is a useful tool to correctly differentiate between intrinsic and acquired antibiotic resistance genes.

Tet(M)-type proteins confer resistance to tetracycline and related antibiotics by interacting with the ribosome. Genes encoding Tet(M) have been found in a range of bacteria, including Escherichia coli. In the current study, conjugation experiments were performed between seven different tetracycline-resistant, azide-susceptible E. coli strains isolated from ducks and tetracycline-sensitive, azide-resistant E.coli J53. Transconjugants were obtained from two of the strains at a frequency of 1.2 × 10−8. PCR, southern blotting and sequencing demonstrated that tet(M) in the transconjugants was located on a ~50 kb IncHI2-type plasmid and was part of a composite transposon, designated Tn6539. This transposon is flanked by two IS26 elements in opposite orientation and contains the Tn3ΔtnpA+Δorf13-lp-tet(M)+gamma delta+tnpX+ΔtnpR sequences. The Δorf13-lp-tet(M) sequence was a highly conserved genetic fragment in E. coli harboring tet(M) and mainly located in the composite transposons flanked by IS6-family elements. In summary, Tn6539 is a new composite transposon capable of horizontal transfer of tet(M) among E. coli isolates.

Many novel tigecycline-inactivating enzymes encoded by tet (X) variants from different bacteria were discovered since the plasmid-mediated tet (X3) and tet (X4) genes conferring high-level resistance to tigecycline in Enterobacterales and Acinetobacter were reported. However, there have been no comprehensive studies of the prevalence of different tet (X) variants in poultry farms. In this study, we collected 45 chicken fecal samples, isolated tet (X)-positive strains, and performed antimicrobial susceptibility testing, conjugation assay, whole-genome sequencing, and bioinformatics analysis. A total of 15 tet (X)-bearing strains were isolated from 13 samples. Species identification and tet (X) subtyping analysis found that the 15 strains belonged to eight different species and harbored four different tet (X) variants. Genomic investigation showed that transmission of tet (X) variants was associated with various mobile genetic elements, and tet (X4) was the most prevalent variant transferred by conjugative plasmids. Meanwhile, we characterized a plasmid co-harboring tet (X6) and bla OXA–58 in Acinetobacter baumannii. In summary, we demonstrated that different tet (X) variants were widely disseminated in the chicken farming environment and dominated by tet (X4). This finding expands the understanding of the prevalence of tet (X) among different animal sources, and it was advocated to reduce the usage of antibiotics to limit the emergence and transmission of novel tet (X) variants in the poultry industry. [ABSTRACT FROM AUTHOR]

The tetracycline resistance gene tet(W) encodes a ribosomal protection protein that confers a low level of tetracycline resistance in the probiotic bacterium Bifidobacterium animalis subsp. lactis. With the aim of assessing its phylogenetic origin and potential mobility, we have performed phylogenetic and in silico genome analysis of tet(W) and its flanking genes. tet(W) was found in 41 out of 44 examined B. animalis subsp. lactis strains. In 38 strains, tet(W) was flanked by an IS5-like element and an open reading frame encoding a hypothetical protein, which exhibited a similar GC content (51–53%). These genes were positioned in the same genomic context within the examined genomes. Phylogenetically, the B. animalis subsp. lactis tet(W) cluster in a clade separate from tet(W) of other species and genera. This is not the case for tet(W) encoded by other bifidobacteria and other species where tet(W) is often found in association with transferable elements or in different genomic regions. An IS5-like element identical to the one flanking the B. animalis subsp. lactis tet(W) has been found in a human gut related bacterium, but it was not associated with any tet(W) genes. This suggests that the IS5-like element is not associated with genetic mobility. tet(W) and the IS5 element have previously been shown to be co-transcribed, indicating that co-localization may be associated with tet(W) expression. Here, we present a method where phylogenetic and in silico genome analysis can be used to determine whether antibiotic resistance genes should be considered innate (intrinsic) or acquired. We find that B. animalis subsp. lactis encoded tet(W) is part of the ancient resistome and thereby possess a negligible risk of transfer.

Annotations of non-pathogenic bacterial genomes commonly reveal putative antibiotic resistance genes and the potential risks associated with such genes is challenging to assess. We have examined a putative tetracycline tet(L) gene (conferring low level tetracycline resistance), present in the majority of all publicly available genomes of the industrially important operational group Bacillus amyloliquefaciens including the species B. amyloliquefaciens, Bacillus siamensis and Bacillus velezensis. The aim was to examine the risk of transfer of the putative tet(L) in operational group B. amyloliquefaciens through phylogenetic and genomic position analysis. These analyses furthermore included tet(L) genes encoded by transferable plasmids and other Gram-positive and -negative bacteria, including Bacillus subtilis. Through phylogenetic analysis, we could group chromosomally and plasmid-encoded tet(L) genes into four phylogenetic clades. The chromosomally encoded putative tet(L) from operational group B. amyloliquefaciens formed a separate phylogenetic clade; was positioned in the same genomic region in the three species; was not flanked by mobile genetic elements and was not found in any other bacterial species suggesting that the gene has been present in a common ancestor before species differentiation and is intrinsic. Therefore the gene is not considered a safety concern, and the risk of transfer to and expression of resistance in other non-related species is considered negligible. We suggest a subgrouping of the tet(L) class into four groups (tet(L)1.1, tet(L)1.2 and tet(L)2.1, tet(L)2.2), corresponding with the phylogenetic grouping and tet(L) from operational group B. amyloliquefaciens referred to as tet(L)2.2. Phylogenetic analysis is a useful tool to correctly differentiate between intrinsic and acquired antibiotic resistance genes. [ABSTRACT FROM AUTHOR]

Objectives: Tigecycline is recognized as one of the last-line antibiotics to treat serious bacterial infection caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). The plasmid-borne gene tet (X4) mediates high resistance to tigecycline. However, the prevalence and genetic context of tet (X4) in K. pneumoniae from various sources are not fully understood. Here, we investigated the prevalence of tet (X4)-positive K. pneumoniae and characterized the genetic context of tet (X4)-bearing plasmids in K. pneumoniae isolates., Methods: Polymerase chain reaction (PCR) was used to detect the tet (X4) gene. The transferability of the tet (X4)-carrying plasmids was tested by conjugation assays. The Galleria mellonella infection model was used to test virulence of tet (X4)-positive strains. Whole-genome sequencing and genome-wide analysis were performed to identify the antimicrobial resistance and the virulence genes, and to clarify the genetic characteristics of the tet (X4)-positive isolates., Results: Among 921 samples, we identified two tet (X4)-positive K. pneumoniae strains collected from nasal swabs of two pigs (0.22%, 2/921). The two tet (X4)-positive isolates exhibited high minimum inhibitory concentrations to tigecycline (32-256 mg/L) and tetracycline (256 mg/L). The plasmids carrying the tet (X4) gene can transfer from the donor strain K. pneumoniae to the recipient strain Escherichia coli J53. Genetic analysis of the complete sequence of two tet (X4)-carrying plasmids pTKPN&#95;3-186k-tetX4 and pTKPN&#95;8-216k-tetX4 disclosed that the tet (X4) gene was flanked by delta IS CR2 and IS 1R , which may mediate the transmission of the tet (X4) gene., Conclusion: The prevalence of tet (X4)-positive K. pneumoniae among different sources was low. IS CR2 and IS 1R may contribute to the horizontal transfer of tet (X4) gene. Effective measures should be taken to prevent the transmission of tet (X4)-producing K. pneumoniae in humans or animals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yang, He, Wu, Qin, Chen, Feng, Zhao, Xu, Guo, Tian, Dai, Yan and Qin.)

The prevalence of plasmid-mediated tigecycline resistance gene tet (X4) is presenting an increasing trend. Once tet (X4)-bearing plasmids are captured by multidrug-resistant bacteria, such as bla NDM and mcr -coharboring bacteria, it will promote bacteria to develop an ultra-broad resistance spectrum, limiting clinical treatment options. However, little is known about the destiny of such bacteria or how they will evolve in the future. Herein, we constructed a multidrug-resistant bacteria coharboring tet (X4), bla NDM-5 , and mcr-1 by introducing a tet (X4)-bearing plasmid into a bla NDM-5 and mcr-1 positive E. coli strain. Subsequently, the stability of tet (X4) and the plasmid was measured after being evolved under tigecycline or antibiotic-free circumstance. Interestingly, we observed both tet (X4)-bearing plasmids in tigecycline treated strains and non-tigecycline treated strains were stable, which might be jointly affected by the increased conjugation frequency and the structural alterations of the tet (X4)-positive plasmid. However, the stability of tet (X4) gene showed different scenarios in the two types of evolved strains. The tet (X4) gene in non-tigecycline treated strains was stable whereas the tet (X4) gene was discarded rapidly in tigecycline treated strains. Accordingly, we found the expression levels of tet (X4) gene in tigecycline-treated strains were several times higher than in non-tigecycline treated strains and ancestral strains, which might in turn impose a stronger burden on the host bacteria. SNPs analysis revealed that a myriad of mutations occurred in genes involving in conjugation transfer, and the missense mutation of marR gene in chromosome of tigecycline treated strains might account for the completely different stability of tet (X4)-bearing plasmid and tet (X4) gene. Collectively, these findings shed a light on the possibility of the emergence of multidrug resistant bacteria due to the transmission of tet (X4)-bearing plasmid, and highlighted that the antibiotic residues may be critical to the development of such bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xiao, Liu, Chen, Peng, Li, Liu and Wang.)

Introduction: This study aimed to investigate the genetic factors promoting widespread Q6 dissemination of tet (X4) between Escherichia coli and to characterize the genetic contexts of tet (X4)., Methods: We isolated E. coli from feces, water, soil and flies collected across a large-scale chicken farm in China in 2020. Antimicrobial susceptibility testing and PFGE typing were used to identify tigecycline resistance and assess clonal relationships among isolates. Plasmids present and genome sequences were analyzed by conjugation, S1 pulsed-field gel electrophoresis (PFGE), plasmid stability testing and whole-genome sequencing., Results: A total of 204 tigecycline-resistant E. coli were isolated from 662 samples. Of these, we identified 165 tet (X4)-carrying E. coli and these strains exhibited a high degree of multidrug resistance. Based on the geographical location distribution of the sampled areas, number of samples in each area and isolation rate of tigecycline-resistant strains and tet (X4)-carrying isolates, 72 tet (X4)-positive isolates were selected for further investigation. Tigecycline resistance was shown to be mobile in 72 isolates and three types of tet (X4)-carrying plasmids were identified, they were IncHI1 (n = 67), IncX1 (n = 3) and pO111-like/IncFIA(HI1) (n = 2). The pO111-like/IncFIA(HI1) is a novel plasmid capable of transferring tet (X4). The transfer efficiency of IncHI1 plasmids was extremely high in most cases and IncHI1 plasmids were stable when transferred into common recipient strains. The genetic structures flanked by IS1, IS26 and ISCR2 containing tet (X4) were complex and varied in different plasmids., Discussion: The widespread dissemination of tigecycline-resistant E. coli is a major threat to public health. This data suggests careful use of tetracycline on farms is important to limit spread of resistance to tigecycline. Multiple mobile elements carrying tet (X4) are in circulation with IncHI1 plasmids the dominant vector in this setting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Zhang, Cai, Lu, Sun, Cao, Xu, Webber and Jiang.)

The emergence and spread of carbapenemase genes, colistin resistance genes mcr-1 , and tigecycline resistance gene tet (X) represent a significant threat to clinical therapy and public health. In this study, we investigated the presence of carbapenemase genes, mcr-1 , and tet (X) in 298 Escherichia coli strains obtained from a teaching hospital in China. In total, eight (2.68%), six (2.01%), and one (0.34%) E. coli isolates carried bla NDM , mcr-1 , and tet (X4), respectively. The bla NDM gene was located on IncX3 ( n = 4), F2:A-:B- ( n = 3), and F2:A1:B1 ( n = 1) plasmids, with high similarity to multiple plasmids belonging to the same incompatibility type from Enterobacteriaceae. Six MCR-producing strains contained mcr-1 -carrying IncI2 plasmids, organized similarly to other mcr-1 -bearing IncI2 plasmids from animals in China. The bla CTX-M-55/64/132/199 gene located within a typical transposition unit (IS Ecp1 - bla CTX-M - orf477 Δ) was inserted near dnaJ to generate 5-bp direct repeats in four mcr-1 -positive plasmids. The tet (X) and another four resistance genes [ aadA2, tet (A), floR , and Δ lnu (F)] were co-located on an IncX1 plasmid, highly similar to other tet (X4)-carrying IncX1 plasmids from Escherichia and Klebsiella of animal or food origin, except that the conjugative transfer region of IncX1 plasmids was absent in our plasmid. Although a low prevalence of bla NDM , mcr-1 , and tet (X) was observed in E. coli from patients in this study, their dissemination associated with some successful pandemic plasmids is of great concern. The continued surveillance of these crucial resistance genes in patients should be strengthened., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sun, Sun, Jiang, Mei, Wang, Wang, Kong, Jiao and Wang.)

The tigecycline resistance gene tet (X4) was widespread in various bacteria. However, limited information about the plasmid harboring the tet (X4) gene spread among the different species is available. Here, we investigated the transmission mechanisms of the tet (X4) gene spread among bacteria in a pig farm. The tet (X4) positive Escherichia coli , Klebsiella pneumoniae , Enterobacter cloacae and Enterobacter hormaeche were identified in the same farm. The whole genome sequencing (WGS) analysis showed that the K. pneumoniae belonged to ST727 ( n  = 11) and ST3830 ( n  = 1), E. cloacae and E . hormaeche belonged to ST524 ( n  = 1) and ST1862 ( n  = 1). All tet (X4) genes were located on the IncHI1 plasmids that could be conjugatively transferred into the recipient E. coli C600 at 30°C. Moreover, a fusion plasmid was identified that the IncHI1 plasmid recombined with the IncN plasmid mediated by IS CR2 during the conjugation from strains B12L to C600 (pB12L-EC-1). The fusion plasmid also has been discovered in a K. pneumoniae (K1L) that could provide more opportunities to spread antimicrobial resistance genes. The tet (X4) plasmids in these bacteria are derived from the same plasmid with a similar structure. Moreover, all the IncHI1 plasmids harboring the tet (X4) gene in GenBank belonged to the pST17, the newly defined pMLST. The antimicrobial susceptibility testing was performed by broth microdilution method showing the transconjugants acquired the most antimicrobial resistance from the donor strains. Taken together, this report provides evidence that IncHI1/pST17 is an important carrier for the tet (X4) spread in Enterobacteriaceae species, and these transmission mechanisms may perform in the environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ma, Wang, Yang, Su, Li, Bai, Feng, Huang, Yang and Tang.)

The lack of selective markers has been a key problem preventing multistep genetic engineering in filamentous fungi, particularly for industrial species such as the lignocellulose degrading Penicillium oxalicum JUA10-1(formerly named as Penicillium decumbens). To resolve this problem, we constructed a genetic manipulation system taking advantage of two established genetic systems: the Cre-loxP system and Tet-on system in Penicillium oxalicum JUA10-1. This system is simple and efficient. The expression of Cre recombinase was activated by doxycycline since it was controlled by Tet-on system. Using this system, two genes, ligD and bglI, were sequentially disrupted by loxP flanked ptrA. The successful application of this procedure will provide a useful tool for genetic engineering in filamentous fungi. This system will also play an important role in improving the productivity of interesting products and minimizing by-product when fermented by filamentous fungi.

The emergence and prevalence of tigecycline-resistant Klebsiella pneumoniae have seriously compromised the effectiveness of antimicrobial agents in the treatment of infections. To explore the role of the plasmid-borne tet (A) gene in tigecycline resistance in carbapenem-resistant K. pneumoniae (CRKP), a total of 63 CRKP isolates were collected from a tertiary hospital in Hangzhou, China. The minimum inhibitory concentration (MIC) of tigecycline, mutation rate of tet (A) gene, genetic surroundings of tet (A)-carrying transmissible plasmid and the contribution of tet (A) mutation to tigecycline resistance were analyzed using antimicrobial susceptibility test, whole-genome sequencing, tigecycline resistance evolution experiment, and plasmid conjugation experiment. Our results showed that 52.4% (33 isolates) of the test isolates carried the tet (A) gene; among them, 75.8% (25 isolates) exhibited a tigecycline non-susceptible phenotype (MIC = 4 mg/L). Three clonal groups (cluster I, cluster II, and cluster III) were identified in these tet (A)-bearing isolates. All 17 isolates belonged to serotype KL21 (cluster I), which differed by only 13 SNPs, suggesting a clonal spread of tet (A)-positive ST11 K. pneumoniae with serotype KL21 occurred in the sampling hospital. The induction of tigecycline resistance experiments showed that 71.4% of strains evolved tet (A) mutations and developed a high-level tigecycline resistance. Eight amino acid substitutions were identified in these mutants. The most common amino acid substitution was A370V, followed by S251A and G300E. Twelve isolates carrying tet (A) mutants succeeded in the filter mating experiment with a conjugation efficiency of 10–3–10–8. Tigecycline MICs in E. coli EC600 transconjugants with a mutated tet (A) were 2 to 8-fold higher than those in E. coli EC600 transconjugants with a wild-type tet (A). One ColRNAI/IncFII type and two IncFII type tet (A)-bearing conjugative plasmids were identified in this study, including a class 1 integron containing multiple antibiotic resistance genes, i.e., tet (A), qnrS1 , bla LAP– 2, catA2 , sul2 , and dfrA14. Our study revealed the wide-spread situation of plasmid-borne tet (A) gene in clinical CRKP, and mutation of tet (A) is a potential driven force that lead to tigecycline resistance. [ABSTRACT FROM AUTHOR]

The emergence of pathogens is conferring resistance to last-resort therapies such as tigecycline, colistin, and carbapenems, limiting the therapeutic options, and raising concerns about the emergence of new "superbugs." This study reports the first incident of a bla NDM-5 and tet (X4) co-harboring Escherichia coli with resistance to carbapenem and tigecycline recovered as the causative agent of a urinary tract infection in a 94-year-old patient. The E. coli strain ECCL209 carries multiple resistance genes [i.e., bla TEM-1 B , bla NDM-5 , bla CMY- 2 , aadA22, florR, erm (B), mph (A), erm (42), lnuG , qnrS1 , and sul 2] and exhibits resistance to almost all clinically used antibiotics. MLST analysis found that the strain belongs to ST648, considered a worldwide high-risk pandemic clone. Moreover, multiple plasmid incompatibility types were detected, i.e., IncHI1A, IncHI1B, IncFII, IncFIA, IncFIB, IncQ1, Col, and IncX4. Genetic analysis revealed that bla NDM-5 and tet (X4) genes were localized on two hybrid plasmids with multiple replicons. Continuous monitoring studies are suggested to quantify the antimicrobial resistance and assess the dissemination of such superbugs into a human healthcare setting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shafiq, Zeng, Permana, Bilal, Huang, Yao, Algammal, Li, Yuan and Jiao.)

Introduction: Enterococcus spp. have gradually evolved from commensals to causing life-threatening hospital-acquired infections globally due to their inherent antimicrobial resistance ability and virulence potential. Enterococcus spp. recovered from livestock and raw meat samples were characterized using antimicrobial susceptibility testing and whole-genome sequencing. Materials and methods: Isolates were confirmed using the MALDI-ToF mass spectrometer, and antimicrobial susceptibility was determined using the Kirby-Bauer disk diffusion method. Whole genome sequencing was performed on isolates resistant to two or more antibiotics. Bioinformatics analysis was performed to determine sequence types, resistance and virulence gene content and evolutionary relationships between isolates from meat and livestock samples, and other enterococci genomes curated by PATRIC. eBURST analysis was used to assign genomes to clonal complexes. Results: Enterococcus spp. were predominantly E. faecalis (96/236; 41%) and E. faecium (89/236; 38%). Overall, isolates showed resistance to erythromycin (78/236; 33%), tetracycline (71/236; 30%), ciprofloxacin (20/236; 8%), chloramphenicol (12/236; 5%), linezolid (7/236; 3%), ampicillin (4/236; 2%) and vancomycin (1/236, 0.4%). Resistance to two or more antimicrobial agents was detected among 17% (n = 40) Enterococcus spp. Resistance genes for streptogramins [lsa(A), lsa(E), msr(C)], aminoglycosides [aac(6')-Ii, aph(3')-III, ant(6)-Ia, aac(6')-aph(2?), str], amphenicol [cat], macrolides [erm(B), erm(T), msr(C)], tetracyclines [tet(M), tet(L), tet(S)] and lincosamides [lsa(A), lsa(E), lnu(B)] were detected among the isolates. Genes for biofilm formation, adhesins, sex pheromones, cytolysins, hyaluronidase, oxidative stress resistance, quorum-sensing and anti-phagocytic activity were also identified. Potential plasmids with replicon sequences (rep1, rep2, repUS43, repUS47, rep9a, rep9b) and other mobile genetic elements (Tn917, cn_5536_ISEnfa1, Tn6009, ISEnfa1, ISEfa10) were detected. Clinically relevant E. faecium ST32 and ST416 clones were identified in meat samples. Conclusion: The occurrence of antimicrobial-resistant Enterococcus spp. in livestock and raw meat samples, carrying multiple resistance and virulence genes, including known clones associated with hospital-acquired infections, underscores the critical need for employing robust tools like whole genome sequencing. Such tools provide detailed data essential for ongoing surveillance efforts aimed at addressing the challenge of antimicrobial resistance with a focus on one health. [ABSTRACT FROM AUTHOR]

With the large-scale use of antibiotics, antibiotic resistant bacteria (ARB) continue to rise, and antibiotic resistance genes (ARGs) are regarded as emerging environmental pollutants. The new tetracycline-class antibiotic, tigecycline is the last resort for treating multidrug-resistant (MDR) bacteria. Plasmid-mediated horizontal transfer enables the sharing of genetic information among different bacteria. The tigecycline resistance gene tet (X) threatens the efficacy of tigecycline, and the adjacent IS CR2 or IS 26 are often detected upstream and downstream of the tet (X) gene, which may play a crucial driving role in the transmission of the tet (X) gene. Since the first discovery of the plasmid-mediated high-level tigecycline resistance gene tet (X4) in China in 2019, the tet (X) genes, especially tet (X4), have been reported within various reservoirs worldwide, such as ducks, geese, migratory birds, chickens, pigs, cattle, aquatic animals, agricultural field, meat, and humans. Further, our current researches also mentioned viruses as novel environmental reservoirs of antibiotic resistance, which will probably become a focus of studying the transmission of ARGs. Overall, this article mainly aims to discuss the current status of plasmid-mediated transmission of different tet (X) genes, in particular tet (X4), as environmental pollutants, which will risk to public health for the "One Health" concept., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Wen, Wang, Wang, Jia, Chen, Liu, Zhu, Zhao, Wu, Yang, Huang, Ou, Mao, Gao, Sun, Tian and Cheng.)

The rapid dissemination of plasmid-mediated tet (X) genes in Acinetobacter species has compromised the clinical effectiveness of tigecycline, one of the last-resort antibiotics. However, the classification strategy and homology group of tet (X)-positive Acinetobacter spp. plasmids remain largely unknown. In this study, we classified them by genome-based replicon typing, followed by analyses of structural characteristics, transferability and in vivo effect. A total of 34 plasmids distributed in at least nine Acinetobacter species were collected, including three tet (X3)-positive plasmids and one tet (X6)-positive plasmid from our genome sequencing results. Among them, there were 28 plasmids carrying Rep&#95;3 superfamily replicase genes and classified into six homology groups, consisting of GR31 (82.1%), GR26 (3.6%), GR41 (3.6%), GR59 (3.6%), and novel groups GR60 (3.6%) and GR61 (3.6%). Our tet (X3)-positive plasmids pYH16040-1, pYH16056-1, and pYH12068-1 belonged to the dominant GR31 group, whereas the tet (X6)-positive plasmid pYH12068-2 was unclassified. Structurally, all tet (X)-positive GR31 plasmids shared similar plasmid replication ( repB ), stability ( parA and parB ) and accessory modules [ tet (X) and sul2 ], and 97.6% of plasmid-mediated tet (X) genes in Acinetobacter species were adjacent to IS CR2 . Conjugation and susceptibility testing revealed pYH16040-1, pYH16056-1, and pYH12068-2, carrying plasmid transfer modules, were able to mediate the mobilization of multiple antibiotic resistance. Under the treatment of tigecycline, the mortality rate of Galleria mellonella infected by pYH16040-1-mediated tet (X3)-positive Acinetobacter spp. isolate significantly increased when compared with its plasmid-cured strain ( p < 0.0001). The spread of such plasmids is of great clinical concern, more effects are needed and will facilitate the future analysis of tet (X)-positive Acinetobacter spp. plasmids., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Huang, Cui, He, Sun, Liu and Huang.)

This study aimed to investigate the prevalence and characterization of tet (X4) in Escherichia coli isolates from a pig farm in Shanghai, China, and to elucidate tet (X4) dissemination mechanism in this swine farm. Forty-nine (80.33%) E. coli strains were isolated from 61 samples from a pig farm and were screened for the presence of tet (X). Among them, six (12.24%) strains were positive for tet (X4) and exhibited resistance to tigecycline (MIC ≥ 16 mg/L). They were further sequenced by Illumina Hiseq. Six tet (X4)-positive strains belonged to ST761 with identical resistance genes, resistance profiles, plasmid replicons, and cgMLST type except that additional ColE10 plasmid was present in isolate SH21PTE35. Isolate SH21PTE31, as a representative ST761 E. coli strain, was further sequenced using Nanopore MinION. The tet (X4) in SH21PTE31 was located on IncFIA18/IncFIB(K)/IncX1 hybrid plasmid pYUSHP31-1, highly similar to other tet (X4)-carrying IncFIA18/IncFIB(K)/IncX1 plasmids from ST761 E. coli and other E. coli lineages in China. These IncFIA18/IncFIB(K)/IncX1 plasmids shared closely related multidrug resistance regions, and could reorganize, acquire or lose resistance modules mediated by mobile elements such as IS CR2 and IS 26 . Phylogenetic analysis were performed including all tet (X4)-positive isolates obtained in this pig farm combined with 43 tet (X4)-positive E. coli from pigs, cow, pork, wastewater, and patients with the same ST from NCBI. The 50 tet (X4)-carrying E. coli ST761 isolates from different areas in China shared a close phylogenetic relationship (0-49 SNPs). In conclusion, clonal transmission of tet (X4)-positive E. coli ST761 has occurred in this swine farm. E. coli ST761 has the potential to become a high-risk clone for tet (X4) dissemination in China., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Lu, Wang, Jiang, Wu, Pan and Jiao.)

To investigate tetracycline resistance and resistant genotype in Riemerella anatipestifer, the tetracycline susceptibility of 212 R. anatipestifer isolates from China between 2011 and 2017 was tested. The results showed that 192 of 212 (90.6%) R. anatipestifer isolates exhibited resistance to tetracycline (the MICs ranged from 4 to 256 μg/ml). The results of PCR detection showed that, 170 of 212 (80.2%) R. anatipestifer isolates possessed the tet(X) gene. Other genes, including tet(A), tet(M), tet(Q), tet(O), tet(B), and tet(O/W/32/O), were found at frequencies of 20.8, 4.7, 1.4, 0.9, 0.9, and 0.5%, respectively. However, tet(C), tet(E), tet(G), tet(K), and tet(W) were not detected in any isolate. In these tet gene positive strains, 31 (14.6%), 2 (0.9%), 5 (2.4%), 1 (0.5%), 3 (1.4%) were detected containing tet(A)/tet(X), tet(M)/tet(O), tet(M)/tet(X), tet(O)/tet(X), and tet(Q)/tet(X) simultaneously, respectively. One isolates, R131, unexpectedly contained three tet genes, i.e., tet(M), tet(O), and tet(X). Sequence analysis of the tet gene ORFs cloned from R. anatipestifer isolates confirmed that tet(A), tet(B), tet(M), tet(O), tet(Q) and an unusual mosaic tet gene tet(O/W/32/O) were present in R. anatipestifer. The MIC results of R. anatipestifer ATCC 11845 transconjugants carrying tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes exhibited tetracycline resistance with MIC values ranging from 4 to 64 μg/ml. Additionally, the tet(X) gene could transfer into susceptible strain via natural transformation (transformation frequencies of ~10−6). In conclusion, the tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes were found and conferred tetracycline resistance in R. anatipestifer isolates. Moreover, the tet(X) is the main mechanism of tetracycline resistance in R. anatipestifer isolates. To our knowledge, this is the first report of tet(A), tet(B), tet(M), tet(O), tet(Q), and mosaic gene tet(O/W/32/O) in R. anatipestifer.

Objectives: Carbapenems, colistin, and tigecycline are critically important antibiotics in clinics. After the global appearance of bla NDM and mcr mediating the resistance to carbapenems and colistin, respectively, tigecycline becomes the last-resort drug against severe human infections caused by multidrug-resistant bacteria. Recently, a mobile tigecycline resistance gene tet (X4) has been identified in Escherichia coli , Klebsiella pneumoniae , and Acinetobacter baumannii that causes high resistance to tigecycline and other tetracyclines. In this study, the prevalence of tet (X4) in E. coli isolates from duck and goose farms in Southeast China was identified and characterized. Methods: Feces, soil, sewage, and dust samples were collected from duck and goose farms along with the southeast coast provinces of China. Antimicrobial susceptibility testing and polymerase chain reaction screening were performed to investigate the phenotype and genotype of tigecycline resistance. Conjugation, S1 pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing were used to determine the transferability, genetic location, and the genomic characteristics of tet (X4). Results: In total, 1,716 samples were collected, and 16 isolates (0.9%) recovered from Guangdong, Shandong, and Jiangsu were positive for tet (X4) gene with tigecycline minimum inhibitory concentrations ≥16 mg/L. Notably, among these tet (X4)-positive E. coil isolates, seven of them were from the environment samples (soil and sewage). PFGE and multilocus sequence typing demonstrated that ST3997 was the most prevalent sequence type (eight isolates, 50%) in Jiangsu province. By conjugation assays, 11 isolates were able to transfer tet (X4) plasmid to E. coli C600 recipient, and these plasmids belonged to IncHI1 and IncX1 detected by sequence analysis. tet (X4) was found adjacent to an insertion sequence IS CR2 downstream and a cat D gene upstream for all isolates. In addition, multiple-drug resistance to tigecycline, chlortetracycline, ampicillin, florfenicol, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and fosfomycin was profiled in most of the tet (X4)-positive isolates. Conclusion: The identification of tet (X4) harboring E. coli strains in duck farms and their surrounding environment enlarges our knowledge of the variety and prevalence of tigecycline resistance. The prevalence of tet (X4) raises concern for the use of tetracyclines in animal farming, and the tet (X4) gene should be listed as primary gene for resistance surveillance. [ABSTRACT FROM AUTHOR]