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In 2019, transferable plasmid-borne tet (X3)/ tet (X4) genes conferring high-level tigecycline resistance were identified in Enterobacteriaceae and Acinetobacter . These transferable tet (X) variants are associated with different plasmids and could further spread and disseminate through associated mobile plasmids, raising public concern. In this study, we report the identification and characterization of two novel plasmids carrying tet (X4) in Escherichia coli isolated from retail chicken meat. We believe that our study contributes to our understanding of the genetic environment of tet (X4) and their transferability and capability of recombination and integration with other resistance genes in plasmids.

The emergence and spread of the novel mobile Tet(X) tetracycline destructases confer high-level tigecycline and eravacycline resistance in Escherichia coli and Acinetobacter spp. and pose serious threats to human and animal health. Therefore, a rapid and robust Tet(X) detection assay was urgently needed to monitor the dissemination of tigecycline resistance. We developed a rapid and simple assay to detect Tet(X) producers in Gram-negative bacteria based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This MALDI Tet(X) test was based on the inactivation of tigecycline by a Tet(X)-producing strain after a 3-h incubation of bacterial cultures with tigecycline. Culture supernatants were analyzed using MALDI-TOF MS to identify peaks corresponding to tigecycline (586 ± 0.2 m/z) and a tigecycline metabolite (602 ± 0.2 m/z). The results were calculated using the MS ratio [metabolite/(metabolite + tigecycline)]. The sensitivity of the MALDI Tet(X) test with all 216 test strains was 99.19%, and specificity was 100%. The test can be completed within 3 h. Overall, the MALDI Tet(X) test is an accurate, rapid, cost-effective method for the detection of Tet(X)-producing E. coli and Acinetobacter spp. by determining the unique peak of an oxygen-modified derivative of tigecycline., (Copyright © 2020 Cui, Zheng, Tang, Zhong, Cui, Lian, Fang, He, Wang, Chen, He, Wang, Liu, Liao and Sun.)

Objectives: As the spread of antimicrobial resistance genes becomes an increasing global threat, improved understanding of genetic structure and transferability of the resistant plasmids becomes more critical. The newly description of several plasmid-mediated tet(X) variant genes, tet(X3), tet(X4) and tet(X5), poses a considerable risk for public health. This study aimed to investigate the recombination event that occurred during the conjugation process of a tet(X4)-bearing plasmid., Methods: A Tet(X4)-producing Escherichia coli isolate, 2019XSD11, was subjected to susceptibility testing, S1-PFGE and whole genome sequencing. The genetic features of plasmids and the recombination event were analysed by sequence comparison and annotation. We performed electrotransformation assay to further test the transferability of the tet(X4)-bearing plasmid., Results: A novel type of fusion tet(X4)-bearing plasmid was discovered from the transconjugant, plasmid p2019XSD11-TC2-284 (∼280kbp). The sequence of this plasmid consisted of a hybrid episome of two plasmids p2019XSD11-190 (∼190kbp) harbouring tet(X4) and p2019XSD11-92 (∼92kbp) harbouring bla CTX-M-55 originated from 2019XSD11. The two plasmids were concatenated by IS26 elements. Analyses of the genetic constitution of the plasmids essential for transmission showed the plasmid p2019XSD11-190 lacked an intact type IV secretion system. Beyond this, the origin of transfer region and relaxase genes in plasmid p2019XSD11-190 had no sequence similarity with those in plasmid p2019XSD11-92., Conclusions: The fusion of the two plasmids probably formed through IS26 homologous recombination. Such recombination events presumably play an important role in the dissemination of the tet(X4). Molecular surveillance of tet(X) variant genes and genetic structures warrants further investigation to evaluate the underlying public health risk., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Recent emergence of high-level tigecycline resistance mediated by Tet(X3/X4) in Enterobacteriaceae undoubtably constitutes a serious threat for public health worldwide. Antibiotic adjuvant strategy makes antibiotic more effective against these resistant pathogens through interfering intrinsic resistance mechanisms or enhancing antibiotic actions. Herein, we screened a collection of drugs to identify compounds that are able to restore tigecycline activity against resistant pathogens. Encouragingly, we discovered that anti-HIV agent azidothymidine dramatically potentiates tigecycline activity against clinically resistant bacteria. Meanwhile, addition of azidothymidine prevents the evolution of tigecycline resistance in E. coli and the naturally occurring horizontal transfer of tet(X4). Evidence demonstrated that azidothymidine specifically inhibits DNA synthesis and suppresses resistance enzyme activity. Moreover, in in vivo infection models by Tet(X4)-expression E. coli, the combination of azidothymidine and tigecycline achieved remarkable treatment benefits including increased survival and decreased bacterial burden. These findings provide an effective regimen to treat infections caused by tigecycline-resistant Escherichia coli.

Objectives: The recently discovered tigecycline-inactivating enzyme Tet(X4) can confer high-level tigecycline resistance on its hosts, which makes it a public health concern. This study focused on isolation and screening of Tet(X4)-positive Enterobacteriaceae from the gut microbiota of a cohort of healthy individuals in Singapore., Methods: MinION and Illumina sequencing was performed to obtain the complete genome sequences of Escherichia coli 2EC1-1 and 94EC. Subsequently, 109 human faecal samples were screened retrospectively for eravacycline-resistant Enterobacteriaceae strains, which were further tested for tet(X4) by PCR. The taxonomy of the isolated strains was determined by 16S rRNA gene PCR and Sanger sequencing., Results: Comparative genomic analysis of E. coli 2EC1-1 and 94EC revealed that both carry tet(X4), which is encoded by IncI1-type plasmids p2EC1-1 and p94EC-2, respectively. Retrospective screening of faecal samples collected from 109 healthy individuals showed that the faecal carriage rate of Tet(X4)-producing Enterobacteriaceae is 10.1% (95% CI = 5.1%-17.3%), suggesting that tet(X4) is widely distributed in the gut microbiota of healthy individuals in Singapore., Conclusions: To the best of our knowledge, this is the first report on the prevalence of tet(X4) in the gut microbiota of a healthy human cohort, as well as the first description of this resistance mechanism outside of China. Our findings suggest that surveillance of tet(X4) in community settings is vital to monitor the spread of this resistance mechanism., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Public health interventions to control the recent emergence of plasmid-mediated tigecycline resistance genes rely on a comprehensive understanding of its epidemiology and distribution over a wide range of geographical scales. Here we analysed an Escherichia coli collection isolated from pigs and chickens in China in 2018, and ascertained that the tet(X4) gene was not present at high prevalence across China, but was highly endemic in northwestern China. Genomic analysis of tet(X4)-positive E. coli demonstrated a recent and regional dissemination of tet(X4) among various clonal backgrounds and plasmids in northwestern China, whereas a parallel epidemic coincided with the independent acquisition of tet(X4) in E. coli from the remaining provinces. The high genetic similarity of tet(X4)-positive E. coli and human commensal E. coli suggests the possibility of its spreading into humans. Our study provides a systematic analysis of the current epidemiology of tet(X4) and identifies priorities for optimising timely intervention strategies.

ABSTRACT: Tigecycline is considered one of the “last resort antibiotics” for treating complex infections caused by multidrug-resistant (MDR) bacteria, especially for combating clinical resistant strains that produce carbapenemases. However, the tet(X4) gene, which carried by different plasmids can mediate high levels of bacterial resistance to tigecycline, was first reported in 2019. Here, we report the emergence of the plasmid-mediated tet(X4) in avian environment of Sichuan Province. A total of 21 tet(X4)-positive Escherichia coli (E. coli) strains were isolated and identified from avian samples in selected regions, with an isolation rate of 1.6% (21/1,286), and all of them were MDR strains. Multilocus Sequence Typing (MLST) method was used to classify the 21 tet(X4)-positive E. coli into the ST206, ST761, ST155, ST1638, ST542, and ST767 types, which also belong to the 3 phylogenetic subgroups A, B1, and C. Tet(X4) is located on mobile plasmids that can be efficiently and stably propagated. The results of fitness cost experiments showed that tet(X4)-positive plasmids may incur some fitness cost to host bacteria, but different tet(X4)-positive plasmids bring about differential fitness costs. Whole-genome sequencing further confirmed the tet(X4) gene can be located on IncX1-type plasmids and the core genetic structures are ISVsa3-rdmc-tet(X4) or rdmc-tet(X4)-ISVsa3, the former is a 7 copies tandem repeat structure. In this study, we isolated and identified tet(X4)-positive E. coli from the avian origin in Sichuan, analyzed the mobility of the tet(X4) by conjugational transfer and S1-PFGE, and evaluated the biological characteristics of the tet(X4)-positive plasmid using the results of conjugational frequency, plasmid stability, and fitness costs. Finally, combined with the third-generation whole-genome sequencing analysis, the molecular transmission characteristics of the tet(X4) were preliminarily clarified, providing a scientific basis for guiding veterinary clinical use in this area, as well as risk assessment and prevention of the transfer and spread of tigecycline resistant strains or genes.

We reported the complete nucleotide sequence of a tet (X4)-carrying plasmid, pSTB20-1T, from a tigecycline-resistant Escherichia coli isolate in China. Sequence analysis indicated that pSTB20-1T contains a hybrid plasmid backbone and a tet (X4)-containing multidrug resistance region, likely originated through recombination of multiple plasmids. tet (X4) was flanked by two IS CR2 , which may be responsible for tet (X4) mobilization. The occurrence and transmission of this novel hybrid plasmid may exacerbate the spread of the clinically significant tet (X4) gene., (Copyright © 2019 American Society for Microbiology.)

Tigecycline is one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria 1 . Tigecycline resistance has sporadically occurred in recent years, primarily due to chromosome-encoding mechanisms, such as overexpression of efflux pumps and ribosome protection 2,3 . Here, we report the emergence of the plasmid-mediated mobile tigecycline resistance mechanism Tet(X4) in Escherichia coli isolates from China, which is capable of degrading all tetracyclines, including tigecycline and the US FDA newly approved eravacycline. The tet(X4)-harbouring IncQ1 plasmid is highly transferable, and can be successfully mobilized and stabilized in recipient clinical and laboratory strains of Enterobacteriaceae bacteria. It is noteworthy that tet(X4)-positive E. coli strains, including isolates co-harbouring mcr-1, have been widely detected in pigs, chickens, soil and dust samples in China. In vivo murine models demonstrated that the presence of Tet(X4) led to tigecycline treatment failure. Consequently, the emergence of plasmid-mediated Tet(X4) challenges the clinical efficacy of the entire family of tetracycline antibiotics. Importantly, our study raises concern that the plasmid-mediated tigecycline resistance may further spread into various ecological niches and into clinical high-risk pathogens. Collective efforts are in urgent need to preserve the potency of these essential antibiotics.

The plasmid-mediated high-level tigecycline resistance gene, tet (X4), was detected in seven Escherichia coli isolates from pork in two Chinese provinces. Two isolates belonged to the epidemic spreading sequence type ST101. Tet (X4) was adjacent to IS Vsa3 and concurrent with floR in all seven isolates. In addition to IncFIB, the replicon IncFII was found to be linked to tet (X4). This report follows a recent detection of tet (X3)/(X4) in E. coli from animals and humans in China.

The recent emergence of plasmid-mediated tigecycline resistance genes, tet (X3) and tet (X4), in animals and humans in China would pose a foreseeable threat to public health. To illustrate this paradigm shift in tigecycline resistance, here, covering the period 2008-2018, we retrospectively analysed a national strain collection of Escherichia coli (n = 2254), obtained from chickens and pigs, in six representative provinces of China. The gene tet (X4) was identified in five pig isolates collected in 2016 and 2018 from the provinces of Sichuan (3/15, 2018), Henan (1/25, 2018) and Guangdong (1/28, 2016), but not in the isolates prior to 2016. None of the isolates was detected harbouring tet (X3). All tet (X4)-positive E. coli exhibited high levels of tigecycline resistance (MICs, 16-64 mg/L), and two were confirmed as colistin resistant, harbouring chromosome-borne mcr-1 gene. The gene tet (X4) was detected on a plasmid in all five isolates, whereas a co-location of tet (X4) on the chromosome of one isolate was observed. Diverse host strains and novel plasmids related to the tet (X4) gene were observed. Our timely findings of the recent emergence of tet (X4) gene in food animal support the rapid surveillance and eradication of this gene before it is established.

Here, we report the genetic features and evolutionary mechanisms of two tet (M)-bearing plasmids (pTA2 and pTA7) recovered from swine Escherichia coli isolates. The genetic profiles of pTA2 and pTA7 and corresponding transconjugants were accessed by S1 nuclease pulsed-field gel electrophoresis and Southern hybridization, followed by whole genome sequencing and bioinformatics analysis. The biological influences of pTA2 and pTA7 were determined by stability and direct competition assays. Both pTA7 and pTA2 had the IncR backbone sequences but differed in the multidrug resistance region (MDR). The MDR of pTA2 consisted of sul3 , tet (M), qnr S1, bleO , oqxAB , floR , aadA1 , cmlA1 , aadA2 , and tet (A)- tetR (A) in addition to 22 insertion sequences. Notably, pTA2 carried the novel complex Tn 7124 (IS 26 -ctp-lp- tet (M)-hp-IS 406 tnp-IntI4-IS 26) harboring tet (M). The fragment carrying tet (M) (IS 26-ctp- lp- tet (M)-IS 406 tnp-ctp-aadA1 - cmlA1 - aadA2 - dfrA12- Int I1), named Tn 6942-like , and the two resistance modules IS Vsa3 - VirD2 - floR - lysR and tet (A)- tetR (A) were located in the MDR of pTA7. Both pTA2 and pTA7 were highly stable in E. coli DH5α cells with no fitness cost to the host or disadvantage in growth competition. These results indicate that transposons carrying tet (M) continuously integrate via mediation with an insertion sequence, which accelerates the transmission of tet (M) in E. coli isolates through integration of other drug-resistant genes, thereby posing a potential serious threat to the efficacy of clinical treatment. [ABSTRACT FROM AUTHOR]

Despite scattered studies that have reported mutations in the tet(A) gene potentially linked to tigecycline resistance in clinical pathogens, the detailed function and epidemiology of these tet(A) variants remains limited. In this study, we analyzed 64 Escherichia coli isolates derived from MacConkey plates supplemented with tigecycline (2 μg/mL) and identified five distinct tet(A) variants that account for reduced sensitivity to tigecycline. In contrast to varied tigecycline MICs (0.25 to 16 μg/mL) of the 64 tet(A)-variant-positive E. coli isolates, gene function analysis confirmed that the five tet(A) variants exhibited a similar capacity to reduce tigecycline sensitivity in DH5α carrying pUC19. Among the observed seven non-synonymous mutations, the V55M mutation was unequivocally validated for its positive role in conferring tigecycline resistance. Interestingly, the variability in tigecycline MICs among the E. coli strains did not correlate with tet(A) gene expression. Instead, a statistically significant reduction in intracellular tigecycline concentrations was noted in strains displaying higher MICs. Genomic analysis of 30 representative E. coli isolates revealed that tet(A) variants predominantly resided on plasmids (n = 14) and circular intermediates (n = 13). Within China, analysis of a well-characterized E. coli collection isolated from pigs and chickens in 2018 revealed the presence of eight tet(A) variants in 103 (4.2%, 95% CI: 3.4–5.0%) isolates across 13 out of 17 tested Chinese provinces or municipalities. Globally, BLASTN analysis identified 21 tet(A) variants in approximately 20.19% (49,423/244,764) of E. coli genomes in the Pathogen Detection database. These mutant tet(A) genes have been widely disseminated among E. coli isolates from humans, food animals, and the environment sectors, exhibiting a growing trend in tet(A) variants over five decades. Our findings underscore the urgency of addressing tigecycline resistance and the underestimated role of tet(A) mutations in this context.

ABSTRACT: Objectives: Tetracyclines are used in veterinary medicine for livestock. Tigecycline, a semisynthetic tetracycline derivative, is a last-resort antimicrobial used to treat multidrug-resistant Gram-negative bacterial infections. The prevalence of variants of the mobile tigecycline resistance gene tet(X) in livestock is increasing worldwide. However, the prevalence of Tet(X) among livestock in Japan is unclear. This study was conducted to clarify the prevalence of Tet(X) in pigs in Japan, focusing on isolation and molecular characterisation of plasmid-mediated tet(X)-positive Escherichia coli through retrospective analysis. Methods: We retrospectively screened for tigecycline-resistant E. coli strains isolated from pigs. The tigecycline-resistant strain and tet(X)-harbouring plasmid were characterised. Results: The IncW plasmid harbouring the tet(X) variant [previously named as tet(X6)] was detected in one E. coli isolate from pigs (0.8%; 1/120) in 2012. The tet(X) plasmid was transferable by conjugation to the E. coli ML4909 recipient strain. Some mobile genetic elements (TnAs3 and ISVsa3) were observed in the region surrounding tet(X). The tet(X)-harbouring plasmid shared a conserved backbone with IncW plasmid R388, which is a broad-host-range plasmid. Conclusion: The emergence and spread of tet(X) variants in Enterobacterales poses a public-health concern. To the best of our knowledge, this is the first report of the emergence of an IncW plasmid harbouring tet(X). Using tetracyclines in livestock exerts selective pressure on the tet(X) plasmid; therefore, prudent use of tetracyclines is required.

This study focused on antibiotic resistance genes as emerging contaminants with potential global human health implications. Intensive livestock farming has been identified as a major contributor to the spread of resistant bacteria and genes. The study examined antimicrobial-resistant Escherichia coli and tetracycline-resistant genes in raw milk from commercial dairy farms in Kano State. Out of 300 registered farms, 54 (18 %) were purposively sampled for the study. A total of 313 milk samples were collected and processed through enrichment and inoculation on selective media for Escherichia coli isolation. The antibiogram pattern of the isolated Escherichia coli strains was assessed using the disk diffusion method. The results revealed resistance to various antimicrobial agents, with no resistance to quinolones but high resistance to ampicillin (100 %), erythromycin (73.3 %), and tetracycline (46.7 %), among others. The multiplex polymer-ase chain reaction was conducted on all Escherichia coli isolates to detect tet genes (tet A, B, C, D, and M), and one isolate carried the tet M resistance gene, while six (40 %) others carried the tet A resistance gene. The study concludes that a significant proportion of the cultured Escherichia coli strains were resistant to one or more tested antibiotics, indicating a potential public health threat associated with Escherichia coli contamination in raw milk. We recommend implementing robust regulatory policies governing the use and sales of antimicrobials in animal production. Furthermore, we suggest further investigation into other resistant genes that these isolates might carry to better understand the extent of antibiotic resistance in the region.

ABSTRACT: Objectives: The mobile tigecycline resistance gene tet(X4), conferring resistance to all tetracyclines, is largely reported from China, however the global spread of such a novel resistance mechanism is a concern for preserving the efficacy of these last-resort antibiotics. The aim of our study was to determine the genetic basis of resistance in a tigecycline-resistant Escherichia coli strain (2-326) isolated from sewage in Bergen, Norway, using whole-genome sequencing (WGS). Methods: WGS was carried out using Illumina MiSeq-based sequencing. In vitro conjugation assays were performed to determine the potential of isolate 2-326 to transfer tigecycline resistance to other strains. Results: Escherichia coli isolate 2-326 belongs to pathogenic sequence type 167 (ST167) and carries several clinically important antibiotic resistance genes including tet(X4), blaCTX-M-14, dfrA12, sul2, qnrS1 as well as several aminoglycoside resistance genes. Tigecycline resistance along with resistance to tetracycline, sulfamethoxazole, chloramphenicol and azithromycin was transferred to green fluorescent protein (GFP)-encoding E. coli strain CV601-GFP by conjugation. Conclusion: To the best of our knowledge, this is the first report of E. coli carrying mobile tet(X4) gene from Norway. Our study demonstrates the ongoing spread of new mechanisms of resistance against last-resort antibiotics and the need for surveillance of such resistance factors in the population in order to mitigate their spread.

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.

Despite scattered studies that have reported mutations in the tet(A) gene potentially linked to tigecycline resistance in clinical pathogens, the detailed function and epidemiology of these tet(A) variants remains limited. In this study, we analyzed 64 Escherichia coli isolates derived from MacConkey plates supplemented with tigecycline (2 μg/mL) and identified five distinct tet(A) variants that account for reduced sensitivity to tigecycline. In contrast to varied tigecycline MICs (0.25 to 16 μg/mL) of the 64 tet(A)-variant-positive E. coli isolates, gene function analysis confirmed that the five tet(A) variants exhibited a similar capacity to reduce tigecycline sensitivity in DH5α carrying pUC19. Among the observed seven non-synonymous mutations, the V55M mutation was unequivocally validated for its positive role in conferring tigecycline resistance. Interestingly, the variability in tigecycline MICs among the E. coli strains did not correlate with tet(A) gene expression. Instead, a statistically significant reduction in intracellular tigecycline concentrations was noted in strains displaying higher MICs. Genomic analysis of 30 representative E. coli isolates revealed that tet(A) variants predominantly resided on plasmids (n = 14) and circular intermediates (n = 13). Within China, analysis of a well-characterized E. coli collection isolated from pigs and chickens in 2018 revealed the presence of eight tet(A) variants in 103 (4.2%, 95% CI: 3.4–5.0%) isolates across 13 out of 17 tested Chinese provinces or municipalities. Globally, BLASTN analysis identified 21 tet(A) variants in approximately 20.19% (49,423/244,764) of E. coli genomes in the Pathogen Detection database. These mutant tet(A) genes have been widely disseminated among E. coli isolates from humans, food animals, and the environment sectors, exhibiting a growing trend in tet(A) variants over five decades. Our findings underscore the urgency of addressing tigecycline resistance and the underestimated role of tet(A) mutations in this context. [ABSTRACT FROM AUTHOR]

tet(X4) is the critical resistance gene for tigecycline degradation that has been continually reported in recent years. In particular, pathogenic bacteria carrying tet(X4) are a severe threat to human health. However, information describing Escherichia coli coharboring tet(X4) with virulence genes is limited. Here, we isolated an E. coli strain coharboring tet(X4) and the heat-stable toxin gene astA from a dead piglet. The strain named 812A1-131 belongs to ST10. The genome was sequenced using the Nanopore and Illumina platforms. The virulence genes astA and tet(X4) are located on the chromosome and in the IncHI1-type plasmid p812A1-tetX4-193K, respectively. The plasmid could be conjugatively transferred to recipient E. coli J53 with high frequency. In vivo experiments showed that strain 812A1-131 is pathogenic to Galleria mellonella and could colonize the intestines of mice. In summary, pathogenic E. coli could receive a plasmid harboring the tet(X4) gene, which can increase the difficulty of treatment. The prevalence and transmission mechanisms of pathogenic bacteria coharboring the tet(X4) gene need more attention.

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.

ABSTRACT Emergence of pathogens harboring multiple resistance genes incurs great concerns. Cooccurrence of mobile resistance genes conferring resistance to tigecycline, colistin, and carbapenems in Escherichia coli has not been investigated. This study aimed to characterize three E. coli isolates coharboring tet(X4), mcr-1, and blaNDM-5. Isolates coharboring tet(X4), mcr-1, and blaNDM-5 were identified and characterized by PCR, Sanger sequencing, antimicrobial susceptibility testing, conjugation assays, Illumina sequencing, nanopore sequencing, and bioinformatic analysis. Three E. coli isolates carrying tet(X4), mcr-1, and blaNDM-5 were identified from pigeons in China. They were resistant to almost all antimicrobials except enrofloxacin. tet(X4) and blaNDM-5 could be conjugated into E. coli C600, but mcr-1 was nontransferable in three isolates. Three isolates belonged to sequence type 6775 (ST6775), and clonal dissemination of isolates carrying tet(X4), mcr-1, and blaNDM-5 existed in the pigeon farm. Genetic analysis revealed that mcr-1 mediated by the Tn6330 was located on the chromosome, tet(X4) was located on the IncFII plasmid, and blaNDM-5 was located on the IncX3 plasmid. We first characterized the E. coli isolates carrying tet(X4), mcr-1, and blaNDM-5 simultaneously. Relevant measures should be taken to decrease the prevalence of pathogens carrying tet(X4), mcr-1, and blaNDM-5. IMPORTANCE Tigecycline and colistin are regarded as vital antimicrobials to treat multidrug-resistant (MDR) bacterial infections, such as that caused by carbapenemase-producing Enterobacteriaceae (CPE). Cooccurrence of mobile resistance genes conferring resistance to last-resort antimicrobials in E. coli remains unknown. Here, we characterized E. coli strains coharboring tet(X4), mcr-1, and blaNDM-5 phenotypically and genetically. Resistance genes tet(X4), mcr-1, and blaNDM-5 were located on transposons or plasmids that were mobile genetic elements related to the capture, accumulation, and dissemination of such important resistance genes. The emergence of E. coli isolates carrying tet(X4), mcr-1, and blaNDM-5 highlights the importance of monitoring the coexistence of novel mobile resistance genes in different settings with a One Health approach. Risk of transmission of such MDR pathogens from animals to humans should be evaluated comprehensively.

Tigecycline, one of the last-resort therapeutic options for complicated infections caused by multidrug-resistant pathogens, especially carbapenem-resistant Enterobacterales and Acinetobacter in recent years. The emergence of antibiotic-resistant bacteria and antibiotic-resistant genes has threatened the effectiveness of antibiotics and public health with the excessive use of antibiotics in clinics. However, the emergence and dissemination of high-level mobile tigecycline-resistance gene tet(X) is challenging for clinical effectiveness of antimicrobial agent. This study aimed to characterize an E. coli strain T43, isolated from an inpatient in a teaching hospital in China. The E. coli T43 was resistant to almost all antimicrobials except colistin and consisted of a 4,774,080 bp chromosome and three plasmids. Plasmids pT43-1 and pT43-2 contained tigecycline-resistance gene tet(X4). Plasmid pT43-1 had a size of 152,423 bp with 51.05% GC content and harbored 151 putative open reading frames. pT43-1 was the largest plasmid in strain T43 and carried numerous resistance genes, especially tigecycline resistance gene tet(X4) and carbapenemase resistance gene blaNDM-5. The tet(X) gene was associated with IS26. Co-occurrence of numerous resistance genes in a single plasmid possibly contributed to the dissemination of these genes under antibiotics stress. It might explain the presence of clinically crucial resistance genes tet(X) and blaNDM-5 in clinics. This study suggested the applicable use of antibiotics and continued surveillance of tet(X) and blaNDM-5 in clinics are imperative. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

ABSTRACT: Objectives: This work is aimed to investigate the prevalence of tet(X4) in healthy individuals and patients and assess risk factors associated with tet(X4)-positive populations. Methods: A total of 662 patients and 120 healthy individuals from three municipal hospitals during August 2021 to September 2021 were selected to investigate the prevalence of tet(X4) in gut microbiota. A further case-control study was conducted to identify the risk factors associated with tet(X4)-positive populations. The tet(X4)-positive isolates were characterised by antimicrobial susceptibility testing, multilocus sequence typing (MLST), whole genome sequencing, and bioinformatics analyses. Results: The prevalence of tet(X4)-positive Enterobacteriaceae in healthy individuals and patients (19.1%, 95% CI: 16.3%–21.8%) was substantially higher than previous studies in China (less than 1%). Patients ranging from 19 to 45 years of age had significantly higher odds of tet(X4)-positive bacterial colonization (OR = 2.545, 95% CI: 1.106–5.856). All tet(X4)-positive Enterobacteriaceae were resistant to tigecycline. In addition, tet(X4)-positive Escherichia coli were highly diverse, with CC10 belonging to the dominant clone. Genome analysis showed that tet(X4) was adjacent to ISVsa3 on the plasmids. Conclusion: Data from this study suggested that geographic region may partly explain the high prevalence of tet(X4)-positive Enterobacteriaceae in healthy individuals and patients. Young and middle-aged populations were associated with the colonization of tet(X4)-positive isolates.

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]

ABSTRACT The emergence of the plasmid-mediated colistin resistance gene mcr-1 and the plasmid-mediated tigecycline resistance gene tet(X4) represents a significant threat to public health. Although mcr-1 and tet(X4) have been reported to coexist in the same isolate, there are no reports on the emergence of plasmids coharboring mcr-1 and tet(X4). In this study, we aimed to investigate the opportunities for the emergence of mcr-1- and tet(X4)-coharboring plasmids and their destiny in Escherichia coli. Two plasmids carrying both mcr-1 and tet(X4) were constructed through conjugation assays and confirmed by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Nanopore long-read sequencing. Seven evolved plasmids carrying mcr-1 and tet(X4) from one of the two plasmids were acquired after continuous evolutionary processes. The fitness effects of mcr-1- and tet(X4)-coharboring plasmids were studied by stability experiments, competition experiments, and growth curve measurements. A plasmid carrying mcr-1 and tet(X4) and conferring no fitness cost to its host strain E. coli C600 emerged after evolution during serial passages of bacteria. We proved that it can be anticipated that mcr-1 and tet(X4) could appear in a single plasmid, and the possibility of occurrence in field strains should be monitored constantly. The originally formed cointegrate plasmids coharboring mcr-1 and tet(X4) could evolve into a plasmid with lower fitness costs. This will undoubtedly accelerate the transmission of mcr-1 and tet(X4) globally. The findings highlighted the great possibility of novel hybrid plasmids positive for mcr-1 and tet(X4), and the risk is worthy of increasing attention and public concern globally. IMPORTANCE Tigecycline and colistin are used as last-resort therapies to treat infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, the emergence of the plasmid-mediated tigecycline resistance gene tet(X4) and the plasmid-mediated colistin resistance gene mcr-1 represents a significant threat to human health. A plasmid coharboring mcr-1 and tet(X4) has not emerged so far, but the potential risk should not be ignored. Plasmids coharboring such vital resistance genes will greatly accelerate the progression of pan-drug resistance among pathogens globally. Therefore, evaluation of the emerging opportunity for the mcr-1- and tet(X4)-coharboring plasmids and their destiny in E. coli is of great significance. We provide important insight into the contributions of intI1, IS26, a truncated ISCR2 (ΔISCR2), and IS4321R during the generation of cointegrate plasmids carrying mcr-1 and tet(X4) and highlight the importance of antimicrobials in the evolution and diversity of mcr-1- and tet(X4)-coharboring plasmids. We show that monitoring of the occurrence of mcr-1-carrying MDR plasmids and tet(X4)-bearing MDR plasmids in the same strain should be strengthened to avoid the formation of mcr-1- and tet(X4)-coharboring plasmids.

The emergence of tigecycline-resistant bacteria in agri-food chains poses a public health concern. Recently, plasmid-mediated tet(X4) was found to be resistant to tigecycline. However, genome differences between tet(X4)-positive Escherichia coli of human and pork origins are still under-investigated. In this study, 53 pork samples were collected from markets in Jiangsu, China, and 23 tet(X4)-positive isolates were identified and shown to confer resistance to multiple antibiotics, including tigecycline. tet(X4)-positive isolates were mainly distributed in E. coli (n = 22), followed by Klebsiella pneumoniae (n = 1). More than half of the tet(X4) genes were able to be successfully transferred into E. coli C600. We downloaded all tet(X4)-positive E. coli isolates from humans and pork found in China from the NCBI database. A total of 42 known STs were identified, of which ST10 was the dominant ST. The number of ARGs and plasmid replicons carried by E. coli of human origin were not significantly different from those carried by E. coli of pork origin. However, the numbers of insertion sequences and virulence genes carried by E. coli of human origin were significantly higher than those carried by E. coli of pork origin. In addition to E. coli, we analyzed all 23 tet(X4)-positive K. pneumoniae strains currently reported. We found that these tet(X4)-positive K. pneumoniae were mainly distributed in China and had no dominant STs. This study systematically investigated the tet(X4)-positive isolates, emphasizing the importance of the continuous surveillance of tet(X4) in pork. [ABSTRACT FROM AUTHOR]

Efflux is the most common mechanism of tetracycline resistance. Class A tetracycline efflux pumps, which often have high prevalence in Enterobacteriaceae, are encoded by tet(A) and tet(A)-1 genes. These genes have two potential start codons, GTG and ATG, located upstream of the genes. The purpose of this study was to determine the start codon(s) of the class A tetracycline resistance (tet) determinants tet(A) and tet(A)-1, and the tetracycline resistance level they mediated. Conjugation, transformation and cloning experiments were performed and the genetic environment of tet(A)-1 was analysed. The start codons in class A tet determinants were investigated by site-directed mutagenesis of ATG and GTG, the putative translation initiation codons. High-level tetracycline resistance was transferred from the clinical strain of Klebsiella pneumoniae 10-148 containing tet(A)-1 plasmid pHS27 to Escherichia coli J53 by conjugation. The transformants harbouring recombinant plasmids that carried tet(A) or tet(A)-1 exhibited tetracycline MICs of 256-512 µg ml(-1), with or without tetR(A). Once the ATG was mutated to a non-start codon, the tetracycline MICs were not changed, while the tetracycline MICs decreased from 512 to 64 µg ml(-1) following GTG mutation, and to ≤4 µg ml(-1) following mutation of both GTG and ATG. It was presumed that class A tet determinants had two start codons, which are the primary start codon GTG and secondary start codon ATG. Accordingly, two putative promoters were predicted. In conclusion, class A tet determinants can confer high-level tetracycline resistance and have two start codons., (© 2014 The Authors.)

Background: In Escherichia coli the genes involved in the acquisition of tetracycline resistance are mainly tet(A) and tet(B). In addition, tet(M) is the most common tetracycline resistance determinant in enterococci and it is associated with conjugative transposons and plasmids. Although tet(M) has been identified in E. coli, to our knowledge, there are no previous reports studying the linkage of the tet(M) gene in E. coli to different mobile genetic elements. The aim of this study was to determine the occurrence of tet(A), tet(B), and tet(M) genes in doxycycline-resistant E. coli isolates from pigs, as well as the detection of mobile genetic elements linked to tet(M) in E. coli and its possible transfer from enterococci., Results: tet(A) was the most frequently detected gene (87.9%) in doxycycline-resistant isolates. tet(M) was found in 13.1% E. coli isolates. The tet(M) gene was detected in relation with conjugative transposons in 10 out of 36 enterococci isolates analyzed but not in any of E. coli isolates positive for tet(M). Southern blot showed that in E. coli and in most of the enterococci isolates the tet(M) gene was carried on a plasmid. According to the phylogenetic analysis, E. coli contained a new tet(M) allele grouping separately. Mating experiments revealed that tet(M) was carried on a mobile element successfully transferred between enterococci and between enterococci and E. coli., Conclusions: The detection of tet(M) in E. coli isolates from pigs was higher than expected. In our study, tet(M) detected in E. coli seems not to have been transferred from enterococci, although it can not be ruled out that the horizontal transfer of this gene occurred from other intestinal tract bacteria.

Objectives: To report a novel tigecycline resistance gene, tet(X6), and its variants in four bacterial species isolated from chickens and pigs in China.Methods: WGS was conducted to identify the suspected resistance genes in the tigecycline-resistant Myroides phaeus 18QD1AZ29W. Functional cloning, homology modelling and molecular docking were performed to compare the function with other Tet(X) variants. Retrospective screening for tet(X6) was conducted for 80 isolates in our WGS data collection, and all genomic environments of tet(X6)-positive isolates were analysed.Results: The tigecycline-resistant M. phaeus 18QD1AZ29W isolated from a pig farm in Shandong in 2018 was positive for tet(X2) and a novel tet(X) gene, designated tet(X6). Tet(X6) could increase the MICs of all tested tetracyclines/glycylcyclines for Escherichia coli only 2- to 4-fold, which was possibly due to a lower tetracycline binding capacity of Tet(X6) compared with that of other Tet(X) variants. Retrospective screening showed that seven other isolates (7/80, 8.8%), comprising four Proteus spp. and three Acinetobacter spp. from chickens and pigs in Shandong and Guangdong, were positive for three different variants of tet(X6). The analysis of the genomic environment revealed that two tet(X6)-positive isolates from M. phaeus and Proteus cibarius, respectively, contained ISCR2, which may play a role in tet(X6) transmission.Conclusions: This study identified a novel type of tigecycline resistance gene, tet(X6), in Myroides, Acinetobacter and Proteus from chickens and swine. Tet(X6) conferred lower tetracycline/glycylcycline MICs than other Tet(X) variants, and ISCR2 may play a role in the transmission of tet(X6). [ABSTRACT FROM AUTHOR]

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

The emergence of the mobile tigecycline-resistance gene, tet(X4), poses a significant threat to public health. To investigate the prevalence and genetic characteristics of the tet(X4)-positive Escherichia coli in humans, 1101 human stool samples were collected from a tertiary class-A hospital in Beijing, China, in 2019. Eight E. coli isolates that were positive for tet(X4) were identified from clinical departments of oncology (n = 3), hepatology (n = 2), nephrology (n = 1), urology (n = 1), and general surgery (n = 1). They exhibited resistance to multiple antibiotics, including tigecycline, but remained susceptible to meropenem and polymyxin B. A phylogenetic analysis revealed that the clonal spread of four tet(X4)-positive E. coli from different periods of time or departments existed in this hospital, and three isolates were phylogenetically close to the tet(X4)-positive E. coli from animals and the environment. All tet(X4)-positive E. coli isolates contained the IncX1-plasmid replicon. Three isolates successfully transferred their tigecycline resistance to the recipient strain, C600, demonstrating that the plasmid-mediated horizontal gene transfer constitutes another critical mechanism for transmitting tet(X4). Notably, all tet(X4)-bearing plasmids identified in this study had a high similarity to several plasmids recovered from animal-derived strains. Our findings revealed the importance of both the clonal spread and horizontal gene transfer in the spread of tet(X4) within human clinics and between different sources.

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 largescale 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 tigecyclineresistant 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. [ABSTRACT FROM AUTHOR]

A randomized controlled field trial was conducted to evaluate the effects of two sets of treatment strategies on ceftiofur and tetracycline resistance in feedlot cattle. The strategies consisted of ceftiofur crystalline-free acid (CCFA) administered to either one or all of the steers within a pen, followed by feeding or not feeding a therapeutic dose of chlortetracycline (CTC). Eighty-eight steers were randomly allocated to eight pens of 11 steers each. Both treatment regimens were randomly assigned to the pens in a two-way full factorial design. Non-type-specific (NTS) E. coli (n = 1,050) were isolated from fecal samples gathered on Days 0, 4, 12, and 26. Antimicrobial susceptibility profiles were determined using a microbroth dilution technique. PCR was used to detect tet(A), tet(B), and bla CMY-2 genes within each isolate. Chlortetracycline administration greatly exacerbated the already increased levels of both phenotypic and genotypic ceftiofur resistance conferred by prior CCFA treatment (P<0.05). The four treatment regimens also influenced the phenotypic multidrug resistance count of NTS E. coli populations. Chlortetracycline treatment alone was associated with an increased probability of selecting isolates that harbored tet(B) versus tet(A) (P<0.05); meanwhile, there was an inverse association between finding tet(A) versus tet(B) genes for any given regimen (P<0.05). The presence of a tet(A) gene was associated with an isolate exhibiting reduced phenotypic susceptibility to a higher median number of antimicrobials (n = 289, median = 6; 95% CI = 4-8) compared with the tet(B) gene (n = 208, median = 3; 95% CI = 3-4). Results indicate that CTC can exacerbate ceftiofur resistance following CCFA therapy and therefore should be avoided, especially when considering their use in sequence. Further studies are required to establish the animal-level effects of co-housing antimicrobial-treated and non-treated animals together.

Tetracycline resistance is one of the most frequently encountered resistance properties in bacteria of animal origin. The aim of the present study was to investigate the prevalence and diversity of tetracycline resistance (tet) genes among Escherichia coli clinical isolates from diseased ducks in China and to report the identification and sequencing of the tet(M) gene. The susceptibility of 85 Escherichia coli strains to tetracyclines was determined by broth microdilution, and the presence of tet genes was investigated by multiplex PCR. All of the 85 isolates were fully resistant to both oxytetracycline and tetracycline, and 76.5 % were resistant to doxycycline. Seventy-seven of the isolates (90.6 %) encoded multiple tet genes, with 17.6, 38.8 and 34.1 % encoding two, three and four tet genes, respectively, and only 7.1 % encoded a single tet(A) gene. The MICs of oxytetracycline and tetracycline for all isolates ranged from 16 to ≥128 µg ml(-1) with a MIC90 of >128 µg ml(-1), regardless of the type or number of tet genes encoded. Isolates containing tet(M) commonly had more than one tet gene per strain. The doxycycline resistance rate in the tet(M)-positive isolates was significantly higher than in the tet(M)-negative isolates (P<0.05). A full-length tet(M) gene, including the promoter region, was obtained by PCR in seven of the 41 tet(M)-positive isolates and was sequenced and cloned. The cloned tet(M) gene conferred resistance to tetracyclines in the recombinant Escherichia coli host strain. These results revealed that, in these isolates, the prevalence of multiple tet genes was strikingly high and that tet(M) played a role in doxycycline resistance.

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

Twenty-eight Escherichia coli isolates from various animal and environmental sources with defined tetracycline-resistance genotypes for tet(A), tet(B), and tet(C) were tested for their susceptibility to tetracycline by means of both broth microdilution and Etest. All tet(C)-positive isolates had tetracycline minimum inhibitory concentrations clustering around an intermediate susceptibility range of 2 to 16 microg/mL. Detecting tet(C)-positive isolates by means of susceptibility testing may therefore be difficult with use of the current breakpoint for tetracycline of the Clinical and Laboratory Standards Institute guidelines.

An extended range whole-cell tetracycline biosensor strain was constructed by insertion of the tet(M) gene, encoding tetracycline resistance by ribosomal protection, into plasmid pTGFP2, which contains a transcriptional fusion between a tetracycline regulated promoter and the green fluorescent protein gene. Tetracycline, oxytetracycline, chlortetracycline and minocycline all effectively induced the resulting Escherichia coli MC4100/pTGM biosensor and similar dose-response characteristics were recorded by flow cytometry for all four compounds. The novel tetracycline biosensor was responsive to drug concentrations ranging from below 5 ngml(-1) to 16 microgml(-1), which represents a significant improvement of the original version.

A "Tet(L)-12" version of Tet(L), a tetracycline efflux protein with 14 transmembrane segments (TMS), was constructed by deletion of two central TMS. Tet(L)-12 catalyzed Na+/H+ antiport and antiport with K+ as a coupling ion as well as or better than wild-type Tet(L) but exhibited no tetracycline-Me2+/H+ antiport in Escherichia coli vesicles.

Tigecycline resistance in bacteria has become a significant threat to food safety and public health, where the development of which is attributed to plasmid-mediated tet(X4) genes. In this study, the genomes of 613 tet(X4)-producing Escherichia coli (E. coli) isolates, available from public databases, are evaluated to determine their international prevalence and molecular characterization. These E. coli isolates have been disseminated in 12 countries across Asia and Europe. It was found that pigs and their products (n = 162) were the most common vehicle, followed by humans (n = 122), chickens (n = 60), and the environment (n = 49). Carbapenems-resistant genes blaNDM-5 (1.3%) and blaNDM-1 (0.2%) were identified, as well as colistin-resistant genes mcr-1.1 (12.6%) and mcr-3.1 (0.5%). It was noted that the tigecycline-resistant gene cluster tmexC-tmexD-toprJ1 was identified in seven (1.1%) isolates. Phylogenomic results indicated that tet(X4)-producing E. coli isolates fell into seven lineages (lineages I, II, III, IV, V, VI, and VII), and international spread mainly occurred in Asian countries, especially China, Pakistan, Singapore, and Malaysia. Four forms of tet(X4) transposon units were found, including the I-type (IS26-tet(X4)-ISCR2), II-type (ΔIS1R-tet(X4)-ISCR2), III-type (ISCR2-tet(X4)-ISCR2), and IV-type (ISCR2-tet(X4)-ΔISCR2). These findings underline further challenges for the spread of E. coli bearing tet(X4) gene. [ABSTRACT FROM AUTHOR]

The effects of mutations in host genes on tetracycline resistance mediated by the Tet(O) and Tet(M) ribosomal protection proteins, which originated in Campylobacter spp. and Streptococcus spp., respectively, were investigated by using mutants of Salmonella typhimurium and Escherichia coli. The miaA, miaB, and miaAB double mutants of S. typhimurium specify enzymes for tRNA modification at the adenosine at position 37, adjacent to the anticodon in tRNA. In S. typhimurium, this involves biosynthesis of N6-(4-hydroxyisopentenyl)-2-methylthio-adenosine (ms2io6A). The miaA mutation reduced the level of tetracycline resistance mediated by both Tet(O) and Tet(M), but the latter showed a greater effect, which was ascribed to the isopentenyl (i6) group or to a combination of the methylthioadenosine (ms2) and i6 groups but not to the ms2 group alone (specified by miaB). In addition, mutations in E. coli rpsL genes, generating both streptomycin-resistant and streptomycin-dependent strains, were also shown to reduce the level of tetracycline resistance mediated by Tet(O) and Tet(M). The single-site amino acid substitutions present in the rpsL mutations were pleiotropic in their effects on tetracycline MICs. These mutants affect translational accuracy and kinetics and suggest that Tet(O) and Tet(M) binding to the ribosome may be reduced or slowed in the E. coli rpsL mutants in which the S12 protein is altered. Data from both the miaA and rpsL mutant studies indicate a possible link between stability of the aminoacyl-tRNA in the ribosomal acceptor site and tetracycline resistance mediated by the ribosomal protection proteins.

The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host.

Objectives: Starting from the observation that Escherichia coli tolC mutations severely reduced the high-level resistance to tetracycline afforded by Tn10- and plasmid-encoded Tet(A) pumps, we studied the mechanism of this susceptibility., Methods: The MIC of tetracycline for MC4100 tolC::Tn10 and several tolC mutants carrying the Tn10 in other sites on the chromosome (thr::Tn10) was determined. The effect of a tolC mutation on the level of expression of Tn10 tet(A) was examined by using a tet(A)::lacZ gene fusion. Influence of tolC mutations on tetracycline efflux and accumulation was quantified by spectrofluorometric assays. The contribution of the AcrAB multidrug efflux system to high-level tetracycline resistance was measured in a Tn10-carrying acrAB null mutant strain., Results: Tn10- and plasmid-encoded Tet(A) conferred 5- to 6-fold lower levels of tetracycline resistance in tolC mutants, as compared with control strain tolC+. Spectrofluorometric analyses showed that this resulted from a decrease in drug efflux in tolC mutants. Chlortetracycline resistance was also compromised by loss of TolC. Mutational loss of the AcrAB multidrug efflux transporter had the same effect as tolC mutations on tetracycline resistance. This indicated that tolC mutations act through inactivation of the AcrAB system., Conclusions: Our results are compatible with the hypothesis that the AcrAB pump is an important component in the development of high levels of resistance to tetracycline in E. coli, perhaps by working in combination with Tet(A).

The emergence of the mobile tigecycline-resistance gene, tet(X4), poses a significant threat to public health. To investigate the prevalence and genetic characteristics of the tet(X4)-positive Escherichia coli in humans, 1101 human stool samples were collected from a tertiary class-A hospital in Beijing, China, in 2019. Eight E. coli isolates that were positive for tet(X4) were identified from clinical departments of oncology (n = 3), hepatology (n = 2), nephrology (n = 1), urology (n = 1), and general surgery (n = 1). They exhibited resistance to multiple antibiotics, including tigecycline, but remained susceptible to meropenem and polymyxin B. A phylogenetic analysis revealed that the clonal spread of four tet(X4)-positive E. coli from different periods of time or departments existed in this hospital, and three isolates were phylogenetically close to the tet(X4)-positive E. coli from animals and the environment. All tet(X4)-positive E. coli isolates contained the IncX1-plasmid replicon. Three isolates successfully transferred their tigecycline resistance to the recipient strain, C600, demonstrating that the plasmid-mediated horizontal gene transfer constitutes another critical mechanism for transmitting tet(X4). Notably, all tet(X4)-bearing plasmids identified in this study had a high similarity to several plasmids recovered from animal-derived strains. Our findings revealed the importance of both the clonal spread and horizontal gene transfer in the spread of tet(X4) within human clinics and between different sources. [ABSTRACT FROM AUTHOR]