Tigecycline Resistance Genes Research Articles

Genomic characterization of tigecycline-resistant Escherichia coli and Klebsiella pneumoniae isolates from hospital sewage.

The tigecycline-resistant Enterobacterales have emerged as a great public concern, and the mobile tet(X) variants and tmexCD-toprJ efflux pump are mainly responsible for the spread of tigecycline resistance. Hospital sewage is considered as an important reservoir of antimicrobial resistance, while tigecycline resistance in this niche is under-researched. In this study, five Escherichia coli and six Klebsiella pneumoniae strains were selected from a collection of tigecycline-resistant Enterobacterales for further investigation by antimicrobial susceptibility testing, conjugation, whole-genome sequencing, and bioinformatics analysis. All five E. coli strains harbored tet(X4), which was located on different plasmids, including a novel IncC/IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid structure. In addition, tet(X4)-bearing plasmids were able to transfer by conjugation and be stabilized in the recipient in the absence of antibiotics. tmexCD1-toprJ1 was identified in two K. pneumoniae (LZSFT39 and LZSRT3) and it was carried by a novel multidrug-resistance transposon, designated Tn7368, on a novel IncR/IncU hybrid plasmid. In addition, we found that two K. pneumoniae (LZSFZT3 and LZSRT3) showed overexpression of efflux genes acrB and oqxB, respectively, which was most likely to be caused by mutations in ramR and oqxR. In conclusion, the findings in this study expand our knowledge of the genetic elements that carry tigecycline resistance genes, which establishes a baseline for investigating the structure diversity and evolutionary trajectories of human, animal, and environmental tigecycline resistomes.

Characterization of a Tigecycline-, Linezolid- and Vancomycin-Resistant Clinical Enteroccoccus faecium Isolate, Carrying vanA and vanB Genes.

Increasing incidence of Enterococcus faecium resistant to key antimicrobials used in therapy of hospitalized patients is a worrisome phenomenon observed worldwide. Our aim was to characterize a tigecycline-, linezolid- and vancomycin-resistant E. faecium isolate with the vanA and vanB genes, originating from a hematoma of a patient hospitalized in an intensive care unit in Poland. Antimicrobial susceptibility (a broad panel) was tested using gradient tests with predefined antibiotic concentrations. The complete genome sequence was obtained from a mixed assembly of Illumina MiSeq and Oxford Nanopore's MinION reads. The genome was analyzed with appropriate tools available at the Center for Genomic Epidemiology, PubMLST and GenBank. Transferability of oxazolidinone, tigecycline and vancomycin resistance genes was investigated by conjugation, followed by PCR screen of transconjugants for antimicrobial resistance genes and plasmid rep genes characteristic for the donor and genomic sequencing of selected transconjugants. The isolate was resistant to most antimicrobials tested; susceptibility to daptomycin, erythromycin and chloramphenicol was significantly reduced, and only oritavancin retained the full activity. The isolate represented sequence type 18 (ST18) and carried vanA, vanB, poxtA, fexB, tet(L), tet(M), aac(6')-aph(2''), ant(6)-Ia and ant(6')-Ii. The vanA, poxtA and tet(M) genes located on ~ 40-kb plasmids were transferable by conjugation yielding transconjugants resistant to vancomycin, linezolid and tigecycline. The substitutions in LiaS, putative histidine kinase, SulP, putative sulfate transporter, RpoB and RpoC were potential determinants of an elevated daptomycin MIC. Comparative analyses of the studied isolate with E. faecium isolates from other countries revealed its similarity to ST18 isolates from Ireland and Uganda from human infections. We provide the detailed characteristics of the genomic determinants of antimicrobial resistance of a clinical E. faecium demonstrating the concomitant presence of both vanA and vanB and resistance to vancomycin, linezolid, tigecycline and several other compounds and decreased daptomycin susceptibility. This isolate is a striking example of an accumulation of resistance determinants involving various mechanisms by a single hospital strain.

Integrative conjugative elements mediate the high prevalence of tmexCD3-toprJ1b in Proteus spp. of animal source.

Integrative conjugative elements (ICEs) are important mobile elements that are associated with the dissemination of antibiotic resistance genes (ARGs) in Proteus. Recent studies demonstrated that the tigecycline resistance gene cluster tmexCD-toprJ has emerged in ICEs of Proteus. However, the prevalence of tmexCD-toprJ positive Proteus from animal sources is unclear. To cover the gap, a total of 762 Proteus spp. were isolated from animal source from six provinces of China to identify the tmexCD-toprJ positive isolates. Eight tmexCD-toprJ positive isolates were identified, with the tmexCD-toprJ positive rate of 1.05% against all Proteus spp. and 2.79% against ICE-bearing Proteus spp. The tmexCD-toprJ gene cluster in these Proteus spp. were tmexCD3-toprJ1b and all of them were carried by ICEs. Genetic structure analysis showed that tmexCD3-toprJ1b-bearing ICEs were complicated and plastic, but the tmexCD3-toprJ1b was specifically integrated into variable region III (VRIII) of ICEs with the help of integrases. Furthermore, we found that the umuC gene, which is presented in VRIII of ICEs and other genetic structures of many other bacterial genomes, was a hotspot for the integration of tmexCD-toprJ-bearing potential mobile elements. In summary, our results suggested that Proteus were important reservoirs for tmexCD-toprJ due to the high prevalence of ICEs. Therefore, continuous surveillance of ICEs-associated ARGs in Proteus is necessary and significant for controlling their future transmission. IMPORTANCE The emergence and spread of tmexCD-toprJ have greatly weakened the function of tigecycline. Although studies have demonstrated the significance of Proteus as carriers for tmexCD-toprJ, the epidemic mechanism and characteristics of tmexCD-toprJ in Proteus remain unclear. Herein, we deciphered that the umuC gene in VRIII of SXT/R391 ICEs was a hotspot for the integration of tmexCD3-toprJ1b-bearing mobile genetic elements by genomic analysis. The mobilization and dissemination of tmexCD3-toprJ1b in Proteus were mediated by highly prevalent ICEs. Furthermore, the co-occurrence of tmexCD3-toprJ1b-bearing ICEs with other chromosomally encoded multidrug resistance gene islands warned that the chromosomes of Proteus are significant reservoirs of ARGs. Overall, our results provide significant insights for the prevention and control of tmexCD3-toprJ1b in Proteus.

Characterisation of a Novel Tigecycline Resistance Gene tet(X22) and its Coexistence with blaNDM-1 in a Pseudomonas caeni Isolate

ObjectivesThe emergence of pathogens that are resistant to both tigecycline and carbapenem poses a threat to public health globally. Continuous emergence of novel tet(X) variants accelerates the tigecycline resistance crisis. This study aimed to characterise the novel tigecycline resistance gene tet(X22) and its coexistence with carbapenem resistance gene blaNDM-1 in Pseudomonas caeni. MethodsThis P. caeni isolate co-harbouring tet(X22) and blaNDM-1 was systematically investigated using antimicrobial susceptibility testing, conjugation assays, genome sequencing, bioinformatic analyses, cloning of tet(X22) and functional analysis, and protein structure prediction. ResultsThe carbapenem-resistant and tigecycline-resistant P. caeni isolate CE14 was obtained from chicken faeces in 2022. CE14 carried multiple antibiotic resistance genes, including the novel tet(X22) and blaNDM-1. Tet(X22) exhibited 64.72–90.48% amino acid identity with other variants [Tet(X) to Tet(X21)]. Cloning of the gene tet(X22) and protein structure prediction revealed that Tet(X22) confers resistance to tetracyclines, including tigecycline. tet(X22) and blaNDM-1 were located in two multidrug-resistant regions of the chromosome. ConclusionsThe occurrence of the novel ISCR2-flanked tet(X22) in P. caeni suggests that the tet(X) variant has adapted to new hosts and may widely spread to further expand the host range. The future global spread of such pathogens co-harbouring tet(X) and blaNDM variants needs to be continuously monitored according to the One Health approach.

Identification of tmexC3-tmexD3-toprJ1b in an XDR Providencia rettgeri clinical isolate co-producing NDM-1 and OXA-10 carbapenemases

Emergence of carbapenemase and tigecycline resistance genes in pathogens threaten the efficacy of last-resort antibiotics. Spread and convergence of such resistance genes should be paid high attention to. This study reported an extensively drug-resistant (XDR) Providencia rettgeri clinical strain co-harboring carbapenemase genes blaNDM-1, blaOXA-10 and the tmexCD3-toprJ1b gene cluster. Phenotype and genotype of P. rettgeri Pre20-95 were investigated by antimicrobial susceptibility testing, conjugation assay, stability testing and whole genome sequencing. Bioinformatics tools were used to uncover the genetic structures of its multidrug-resistant (MDR) plasmid pPre20-95-1 and SXT/R391 integrative and conjugative element ICEPreChn20-95. P.rettgeri strain Pre20-95 was isolated from a human clinical infection and displayed an extensively drug-resistant (XDR) phenotype. Whole genome sequencing (WGS) analysis identified a pPrY2001-like MDR plasmid namely pPre20-95-1 co-harboring blaNDM-1 and blaOXA-10 genes in Pre20-95. The multidrug resistance region of pPre20-95-1 was comprised of a Tn6625-derived module and a ∆Tn1696 structure, and blaNDM-1 and blaOXA-10 were located in a composite Tn structure consisting of insertion sequences ISCR1 and ISAba125 and an In125-like class 1 integron, respectively. Furthermore, the novel RND efflux pump gene cluster tmexCD3-toprJ1b was identified on the SXT/R391 ICE ICEPreChn20-95 of its chromosome, and reverse PCR showed that it could form a circular intermediate for transmission. Our findings highlight further dissemination of tmexCD3-toprJ1b gene cluster into a clinical isolate of P. rettgeri and convergence with multiple carbapenemase genes, which increases the risk of the emergence of XDR strains and threatens the treatment of Enterobacterales bacterial infections.

Emergence of novel tigecycline resistance gene tet(X5) variant in multidrug-resistant Acinetobacter indicus of swine farming environments

Antibiotic-resistant bacteria are emerging all the time, but the continued emergence of novel resistance genes and genetic structures is even more alarming. Tigecycline is currently the important last barrier in the treatment of multidrug-resistant (MDR) infections. tet(X), a resistance gene to tigecycline, is the most prevalent and constantly emerging novel variants. In this research, we characterized two MDR Acinetobacter indicus strains to tigecycline that were identified and analyzed by antimicrobial susceptibility testing, conjugation transfer, whole genome sequencing (WGS) and bioinformatics analysis, and gene function analysis. The results showed that three tet(X) variants were carried in BDT201, including tet(X6) on the chromosome, tet(X3) on the plasmid pBDT201–2, and a novel tet(X5) variant adjacent to the ISAba1 elements on the plasmid pBDT201–3. The novel Tet(X5) variant showed 98.7% amino acid identity with Tet(X5) and was named Tet(X5.4). By expressing tet(X5.4) gene, the tigecycline minimum inhibitory concentration (MIC) values for Escherichia coli JM109 increased 32- fold (from 0.13 to 4 mg/L). BDT2076 contained tigecycline and carbapenems resistance genes, such as tet(X3), blaOXA-58, blaNDM-3, and blaCARB-2. The continuous emergence of MDR bacteria and resistance genes is a global environmental health issue that can not be ignored and therefore needs to pay more urgent attention to it.

The tigecycline resistance gene tetX has an expensive fitness cost based on increased outer membrane permeability and metabolic burden in Escherichia coli

Livestock-derived tetX-positive Escherichia coli with tigecycline resistance poses a serious risk to public health. Fitness costs, antibiotic residues, and other tetracycline resistance genes (TRGs) are fundamental in determining the spread of tetX in the environment, but there is a lack of relevant studies. The results of this study showed that both tetO and tetX resulted in reduction in growth and an increased in the metabolic burden of E. coli, but the presence of doxycycline reversed this phenomenon. Moreover, the protection of E. coli growth and metabolism by tetO was superior to that of tetX in the presence of doxycycline, resulting in a much lower competitiveness of tetX-carrying E. coli than tetO-carrying E. coli. The results of RNA-seq showed that the increase in outer membrane proteins (ompC, ompF and ompT) of tetX-carrying E. coli resulted in increased membrane permeability and biofilm formation, which is an important reason for fitness costs. Overall, the increased membrane permeability and metabolic burden of E. coli is the mechanistic basis for the high fitness cost of tetX, and the spread of tetO may limit the spread of tetX. This study provides new insights into the rational use of tetracycline antibiotics to control the spread of tetX.

Pseudomonas Acts as a Reservoir of Novel Tigecycline Resistance Efflux Pump tmexC6D6-toprJ1b and tmexCD-toprJ Variants.

Several variants of the plasmid-carried tigecycline resistance gene cluster, tmexCD-toprJ, have been identified. This study characterized another novel variant, tmexC6D6-toprJ1b, located on the chromosome of environmental-origin Pseudomonas mendocina. TMexC6D6-TOprJ1 mediates resistance to multiple drugs, including tigecycline. The promoter activity of tmexC6D6-toprJ1b and negative transcriptional repression by the upstream regulator tnfxB6 are crucial for the expression of tmexC6D6-toprJ1b. tmexC6D6-toprJ1b was found in the plasmids or chromosomes of different Pseudomonas species from six countries. Two genetic backgrounds, class 1 integrons and int-carrying integrase units, were found adjacent to the tmexC6D6-toprJ1b gene cluster and might mediate the transfer of this novel efflux pump gene cluster in Pseudomonas. Further phylogenetic analysis revealed Pseudomonas as the major reservoir of tmexCD-toprJ variants, warranting closer monitoring in the future. IMPORTANCE Tigecycline is one of the treatment options for serious infections caused by multidrug-resistant bacteria, and tigecycline resistance has gained extensive attention. The emergence of a transferable tigecycline resistance efflux pump gene cluster, tmexCD-toprJ, severely challenged the efficiency of tigecycline. In this study, we identified another novel tmexCD-toprJ variant, tmexC6D6-toprJ1b, which could confer resistance to multiple classes of antibiotics, including tigecycline. Although tmexC6D6-toprJ1b was found only in Pseudomonas species, tmexC6D6-toprJ1b might spread to Enterobacteriaceae hosts via mobile genetic elements resembling those of other tmexCD-toprJ variants, compromising the therapeutic strategies. Meanwhile, novel transferable tmexCD-toprJ variants are constantly emerging and mostly exist in Pseudomonas spp., indicating Pseudomonas as the important hidden reservoir and origin of tmexCD-toprJ variants. Continuous monitoring and investigations of tmexCD-toprJ are urgent to control its spread.

Low prevalence of mobilized resistance genes blaNDM, mcr-1, and tet(X4) in Escherichia coli from a hospital in China.

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.

Global distribution and genomic characteristics of tet(X)-positive Escherichia coli among humans, animals, and the environment

In recent years, the plasmid-mediated transmission of the tigecycline resistance gene tet(X) in Escherichia coli has received considerable attention. However, studies on the global distribution of tet(X)-positive E. coli remain scarce. Herein, we performed a systematic genomic analysis of 864 tet(X)-positive E. coli isolates from humans, animals and the environment around the world. These isolates were reported in 25 countries and isolated from 13 different hosts. China reported the most tet(X)-positive isolates (71.76 %), followed by Thailand (8.45 %) and Pakistan (5.9 %). Pigs (53.93 %), humans (17.41 %), and chickens (17.41 %) were determined to be important reservoirs of these isolates. The sequence types (STs) of E. coli were highly diverse, with the ST10 clone complex (Cplx) being the most prevalent clone. Correlation analysis revealed a positive association between the antibiotic resistance genes (ARGs) in ST10 E. coli and the presence of insertion sequences and plasmid replicons; however, we found no significant correlation between ARGs and virulence genes. Furthermore, the ST10 tet(X)-positive isolates from multiple sources displayed a high degree of genetic similarity (<200 single-nucleotide polymorphisms [SNPs]) to the mcr-1-positive but tet(X)-negative human-derived isolates, suggesting clonal transmission. The most prevalent tet(X) variant in the E. coli isolates was tet(X4), followed by tet(X6)-v. Genome-wide association study (GWAS) indicated that compared to tet(X4), tet(X6)-v harbored more significantly different resistance genes. Notably, certain tet(X)-positive E. coli isolates from different geographical locations or hosts shared a few SNPs (<200 SNPs), indicating cross-contamination. Therefore, continuous global surveillance of tet(X)-positive E. coli is imperative in the future.

The Prevalence of Multidrug-Resistant Enterobacteriaceae among Neonates in Kuwait.

Increasing numbers of neonates with serious bacterial infections, due to resistant bacteria, are associated with considerable morbidity and mortality rates. The aim of this study was to evaluate the prevalence of drug-resistant Enterobacteriaceae in the neonatal population and their mothers in Farwaniya Hospital in Kuwait and to determine the basis of resistance. Rectal screening swabs were taken from 242 mothers and 242 neonates in labor rooms and wards. Identification and sensitivity testing were performed using the VITEK® 2 system. Each isolate flagged with any resistance was subjected to the E-test susceptibility method. The detection of resistance genes was performed by PCR, and the Sanger sequencing method was used to identify mutations. Among 168 samples tested by the E-test method, no MDR Enterobacteriaceae were detected among the neonates, while 12 (13.6%) isolates from the mothers' samples were MDR. ESBL, aminoglycosides, fluoroquinolones, and folate pathway inhibitor resistance genes were detected, while beta-lactam-beta-lactamase inhibitor combinations, carbapenems, and tigecycline resistance genes were not. Our results showed that the prevalence of antibiotic resistance in Enterobacteriaceae obtained from neonates in Kuwait is low, and this is encouraging. Furthermore, it is possible to conclude that neonates are acquiring resistance mostly from the environment and after birth but not from their mothers.

Efflux Pumps and Different Genetic Contexts of tet(X4) Contribute to High Tigecycline Resistance in Escherichia fergusonii from Pigs.

Tigecycline is a last-resort antibiotic for the treatment of infections caused by multidrug-resistant bacteria. The emergence of plasmid-mediated tigecycline resistance genes is posing a serious threat to food safety and human health and has attracted worldwide attention. In this study, we characterized six tigecycline-resistant Escherichia fergusonii strains from porcine nasal swab samples collected from 50 swine farms in China. All the E. fergusonii isolates were highly resistant to tigecycline with minimal inhibitory concentration (MIC) values of 16-32 mg/L, and all contained the tet(X4) gene. In addition, 13-19 multiple resistance genes were identified in these isolates, revealed by whole-genome sequencing analysis. The tet(X4) gene was identified as being located in two different genetic structures, hp-abh-tet(X4)-ISCR2 in five isolates and hp-abh-tet(X4)-ΔISCR2-ISEc57-IS26 in one isolate. The role of efflux pumps in tigecycline resistance was evaluated by using inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The MIC values of tigecycline showed a 2- to 4-fold reduction in the presence of CCCP, indicating the involvement of active efflux pumps in tigecycline resistance in E. fergusonii. The tet(X4) gene was found to be transferable to Escherichia coli J53 by conjugation and resulted in the acquisition of tigcycline resistances in the transconjugants. Whole-genome multilocus sequence typing (wgMLST) and phylogenetic analysis showed a close relationship of five isolates originating from different pig farms, suggesting the transmission of tet(X4)-positive E. fergusonii between farms. In conclusion, our findings suggest that E. fergusonii strains in pigs are reservoirs of a transferable tet(X4) gene and provide insights into the tigecycline resistance mechanism as well as the diversity and complexity of the genetic context of tet(X4) in E. fergusonii.

IncHI1 plasmids mediated the tet(X4) gene spread in Enterobacteriaceae in porcine.

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.

Comprehensive Analysis of Antimicrobial, Heavy Metal, and Pesticide Residues in Commercial Organic Fertilizers and Their Correlation with Tigecycline-Resistant tet(X)-Variant Genes.

With the issue of the antimicrobial additive ban in feed in Chinese animal husbandry, it is important to determine the potential drivers of the spread of the newly discovered tigecycline-resistant tet(X)-variant genes. Here, we investigated the correlations between residues of heavy metals, antimicrobials, and pesticides and the relative abundance of tet(X)-variant genes in 94 commercial organic-fertilizer samples collected from 9 Chinese provinces. A total of 5 heavy metals (mercury, lead, arsenic, chromium, and cadmium), 10 antimicrobials, and 18 pesticides were detected. The tet(X)-variant genes, including tet(X)/(X2), tet(X3), tet(X4), tet(X5), and tet(X6) were detected in 39 (41.5%) samples. Although tet(X)-variant-carrying bacteria were not isolated from these samples, the tet(X4)-carrying plasmids could be captured by exogenous Escherichia coli. Correlation analysis revealed that heavy metals, other than antimicrobials, showed a significant positive association with the relative abundance of the tet(X)-variant genes, especially tet(X3) and tet(X4) (R = 0.346 to 0.389, P < 0.001). The correlation was attributed to the coselection of the tet(X3)/tet(X4) gene on the same plasmid and the conjugation-promoting effect of tet(X3)/tet(X4)-carrying plasmids by subinhibitory concentrations of heavy metals. The heavy metals increased the permeability of the bacterial outer membrane and upregulated the transcription of type IV secretion system (T4SS)-encoding genes on tet(X)-variant-carrying plasmids, therefore enhancing the bacterial conjugation rates. Taken together, our findings have indicated that heavy metals may play an important role in spreading tet(X)-variant genes within the animal manure-related environment. IMPORTANCE An antimicrobial resistance gene (ARG) is considered a novel contaminant for the environment. Most animal feces are usually made into commercial organic fertilizers in China and will pose a threat to the farmland soil and agricultural product if fertilizers harboring clinically significant antimicrobial-resistant (AMR) genes are applied on farmland. This study has indicated that heavy metals may play an important role in the transmission of transferable tigecycline resistance genes [tet(X3) and tet(X4)]. The mechanism was that heavy metals posed a coselection effect of the tet(X3)/tet(X4) gene on the same plasmid and could increase the conjugation ability of tet(X3)/tet(X4)-carrying plasmids. The conjugation-promoting concentrations of heavy metals are lower than the maximal limits defined in the national standard for fertilizers, indicating a high transmission risk of tet(X3)/tet(X4) genes within the animal manure-related environment. The findings in this study will provide scientific evidence for the future development of effective measures to reduce AMR dissemination.

Uncovering the diverse hosts of tigecycline resistance gene tet(X4) in anaerobic digestion systems treating swine manure by epicPCR

The tet(X4) gene is a clinically important tigecycline resistance gene and has shown high persistence in livestock-related environments. However, the bacterial hosts of tet(X4) remain unknown due to the lack of appropriate approaches. Herein, a culture-independent and high-throughput epicPCR (emulsion, paired isolation, and concatenation polymerase chain reaction) method was developed, optimized, and demonstrated for the identification of bacterial hosts carrying tet(X4) from environmental samples. Considering the high sequence similarity between tet(X4) and other tet(X)-variant genes, specific primers and amplification conditions were screened and optimized to identify tet(X4) accurately and link tet(X4) with the 16S rRNA gene, which were further validated using artificially constructed bacterial communities. The epicPCR targeting tet(X4) was applied for the identification of bacterial hosts carrying this resistance gene in anaerobic digestion systems treating swine manure. A total of 19 genera were identified as tet(X4) hosts, which were distributed in the phyla Proteobacteria, Bacteroidota, Firmicutes, and Caldatribacteriota. Sixteen genera and two phyla that were identified have not been previously reported as tet(X4) bacterial hosts. The results indicated that a far more diverse range of bacteria was involved in harboring tet(X4) than previously realized. Compared with the tet(X4) hosts determined by correlation-based network analysis and metagenomic binning, epicPCR revealed a high diversity of tet(X4) hosts even at the phylum level. The epicPCR method developed in this study could be effectively employed to reveal the presence of tet(X4) bacterial hosts from a holistic viewpoint.

Coexistence of tmexCD3-toprJ1b tigecycline resistance genes with two novel bla VIM-2-carrying and bla OXA-10-carrying transposons in a Pseudomononas asiatica plasmid.

Tigecycline and carbapenems are considered the last line of defense against microbial infections. The co-occurrence of resistance genes conferring resistance to both tigecycline and carbapenems in Pseudomononas asiatica was not investigated. P. asiatica A28 was isolated from hospital sewage. Antibiotic susceptibility testing showed resistance to carbapenem and tigecycline. WGS was performed to analyze the antimicrobial resistance genes and genetic characteristics. Plasmid transfer by conjugation was investigated. Plasmid fitness costs were evaluated in Pseudomonas aeruginosa transconjugants including a Galleria mellonella infection model. Meropenem and tigecycline resistant P. asiatica A28 carries a 199, 972 bp long plasmid PLA28.4 which harbors seven resistance genes. Sequence analysis showed that the 7113 bp transposon Tn7389 is made up of a class I integron without a 5'CS terminal and a complete tni module flanked by a pair of 25bp insertion repeats. Additionally, the Tn7493 transposon, 20.24 kp long, with a complete 38-bp Tn1403 IR and an incomplete 30-bp Tn1403 IR, is made up of partial skeleton of Tn1403, a class I integron harboring bla OXA-10, and a Tn5563a transposon. Moreover, one tnfxB3-tmexC3.2-tmexD3b-toprJ1b cluster was found in the plasmid and another one in the the chromosome. Furthermore, plasmid PLA28.4 could be conjugated to P. aeruginosa PAO1, with high fitness cost. A multidrug-resistant plasmid carrying tmexCD3-toprJ1b and two novel transposons carrying bla VIM-2 and bla OXA-10 -resistant genes was found in hospital sewage, increasing the risk of transmission of antibiotic-resistant genes. These finding highlight the necessary of controlling the development and spread of medication resistance requires continuous monitoring and management of resistant microorganisms in hospital sewage.

Genomic and functional analysis of high-level tigecycline resistant Klebsiella michiganensis co-carrying tet(X4) and tmexCD2-toprJ2 from pork

Tigecycline plays an important role in the clinical treatment of infections caused by multidrug-resistant pathogens. The emergence of plasmid-mediated tigecycline resistance genes tet(X) and tmexCD1-tmexJ1 has been reported in a variety of animal and animal-derived foods, and have the potential spread to humans, seriously limiting the choice of clinical medication. Herein, three ST92 Klebsiella michiganensis isolates co-harboring tet(X4) and tmexCD2-toprJ2 were collected from pork samples in Jiangsu Province, China. These K. michiganensis isolates were all multidrug-resistant isolates. Genome analysis showed that tmexCD2-toprJ2 and tet(X4) were located on IncFIB(K) and IncX1 plasmids, respectively. The IncFIB(K) plasmid pMX581-77k is a novel tmexCD2-toprJ2-bearing plasmid. Worryingly, there were only a small number of SNPs between K. michiganensis isolated from pork in this study and K. michiganensis from human sources, with the possibility of clonal transmission. In addition, tet(X4) and tmexCD2-toprJ2 in K. michiganensis were able to stabilize in the absence of antibiotics. The growth curve indicated that the tmexCD2-toprJ2-positive plasmid imposed a burden on the growth of host bacteria. Interestingly, we found that the high-level resistance phenotype to tigecycline in these K. michiganensis isolates was mainly mediated by tet(X4). However, both tet(X4) and tmexCD2-toprJ2 expression were significantly elevated when host bacteria were exposed to tigecycline. This study systematically investigated K. michiganensis co-carrying tet(X4) and tmexCD2-toprJ2, emphasizing the importance for continuous surveillance of tigecycline-resistant K. michiganensis in animal-derived foods.

Characteristics of tet(X4)-Producing Escherichia coli in Chicken and Pig Farms in Hunan Province, China.

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. 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. 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). 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.

Contamination of Proteus mirabilis harbouring various clinically important antimicrobial resistance genes in retail meat and aquatic products from food markets in China.

Proteus mirabilis is an opportunistic pathogen frequently associated with nosocomial infection and food poisoning cases. Contamination of P. mirabilis in retail meat products may be important transmission routes for human infection with P. mirabilis. In this study a total of 89 P. mirabilis strains were isolated from 347 samples in 14 food markets in China and subjected to whole-genome sequencing. Phylogenetic analysis showed that all 89 strains were divided into 81 different clones (SNPs >5), indicating high genetic diversity of P. mirabilis in food markets. Antimicrobial susceptibility testing showed that 81 (91.01%) strains displayed multidrug resistance profiles. Seventy-three different resistance genes (or variants) were found, including various clinically important antimicrobial resistance genes aac(6')-Ib-cr (77.53%), bla CTX-M (39.33%), fosA3 (30.34%), as well as multiresistance gene cfr (4.50%), tigecycline resistance gene cluster tmexCD3-toprJ1 (4.50%) and carbapenemase gene bla NDM-1 (1.12%). Diverse genetic elements including Tn7 transposon, plasmid, SXT/R391 integrative conjugative element were associated with the horizontal transfer of cfr. tmexCD3-toprJ1 and bla NDM-1 were located on ICEPmiChnJZ26 and Salmonella genomic island 1, respectively. Our study emphasized high contamination of P. mirabilis harbouring various clinically important antimicrobial resistance genes in retail meat and aquatic products, which might be an important issue in terms of food safety and human health.

Antimicrobial Resistance Surveillance of Tigecycline-Resistant Strains Isolated from Herbivores in Northwest China.

There is no doubt that antimicrobial resistance (AMR) is a global threat to public health and safety, regardless of whether it’s caused by people or natural transmission. This study aimed to investigate the genetic characteristics and variations of tigecycline-resistant Gram-negative isolates from herbivores in northwest China. In this study, a total of 300 samples were collected from various provinces in northwest China, and 11 strains (3.67%) of tigecycline-resistant bacteria were obtained. In addition, bacterial identification and antibiotic susceptibility testing against 14 antibiotics were performed. All isolates were multiple drug-resistant (MDR) and resistant to more than three kinds of antibiotics. Using an Illumina MiSeq platform, 11 tigecycline-resistant isolates were sequenced using whole genome sequencing (WGS). The assembled draft genomes were annotated, and then sequences were blasted against the AMR gene database and virulence factor database. Several resistance genes mediating drug resistance were detected by WGS, including fluoroquinolone resistance genes (gyrA_S83L, gyrA_D87N, S83L, parC_S80I, and gyrB_S463A), fosfomycin resistance genes (GlpT_E448K and UhpT_E350Q), beta-lactam resistance genes (FtsI_D350N and S357N), and the tigecycline resistance gene (tetR N/A). Furthermore, there were five kinds of chromosomally encoded genetic systems that confer MDR (MarR_Y137H, G103S, MarR_N/A, SoxR_N/A, SoxS_N/A, AcrR N/A, and MexZ_K127E). A comprehensive analysis of MDR strains derived from WGS was used to detect variable antimicrobial resistance genes and their precise mechanisms of resistance. In addition, we found a novel ST type of Escherichia coli (ST13667) and a newly discovered point mutation (K127E) in the MexZ gene of Pseudomonas aeruginosa. WGS plays a crucial role in AMR control, prevention strategies, as well as multifaceted intervention strategies.