Plasmid-encoded tet(X) genes that confer high-level tigecycline resistance in Escherichia coli

Abstract

Tigecycline is one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria¹. 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.

Figure 1 |. Map of tet(X4) sampling areas in China.

The tet(X4) sampling areas of pig farms, chicken houses, and hospitals (A, B, C, and D) are denoted with pink, green, and blue circles, respectively. The distribution of tet(X4)-positive E. coli strains in China is shaded in light blue.

Figure 2 |. The activity of Tet(X4) on tetracyclines in vitro and in vivo.

a, Homology modelling of the tetracyclines-inactivating protein Tet(X4). The modelling of Tet(X4) is based on published tigecycline/Tet(X) complex (PDB accession number 4A6N) using the online server Phyre2 (ref. 41) and AutoDock version 4.2.6 (ref. 42). The substrate binding domain (light green), FAD binding domain (pink), C-terminal helix (light blue), and tigecycline (orange) are displayed. The C-terminal and N-terminal are marked in black characters. b, Microbiological degradation assays. The activity of tet(X4) on tetracycline antibiotics degradation is evaluated by measuring the changes of inhibition zones after the addition of supernatant from different cocultures. The experiments are performed in triplicate and repeated three times with similar results. Blank, the supernatant without treatment (only containing tetracyclines) is added; X+, the supernatant from the coculture incubated with the tet(X4) construct, namely E. coli JM109+pBAD24-tet(X4), is added; X-, the supernatant from the coculture incubated with E. coli JM109 containing the empty vector pBAD24 alone is added; TC, tetracycline; CTC, chlortetracycline; OTC, oxytetracycline; DOX, doxycycline; MIN, minocycline; TGC, tigecycline; ERA, eravacycline. c-d, The levels of tetracycline (c) and eravacycline (d) degradation by Tet(X4). Statistical analysis is conducted using unpaired and two sided t test. Individual values of biological replicates (n=6) are shown as dots, while the means (middle lines) and standard deviations are displayed as error bars. Theoretical max indicates the initial tetracycline or eravacycline concentration in the medium prior to inoculation. e, In vivo effects of tigecycline treatment (50 mg/kg per 24 hours) in a murine thigh model. P value is calculated by a two-way ANOVA test. Individual values of animals (n=6) are shown as dots, while the means (middle lines) and standard deviations are displayed as error bars.

Figure 3 |. Characteristics of the IncQ1 tet(X4)-harboring plasmid pLHM10–1-p6.

a, Structure of the index plasmid pLHM10–1-p6. GC skew and GC content are indicated from the inside out. Positions and transcriptional directions of the predicted ORFs are denoted with arrows. Genes associated with the plasmid replication, antimicrobial resistance, heavy metal resistance, mobile element, and conjugative transfer are highlighted in green, pink, dark yellow, blue, and cyan, respectively. Other genes are marked as grey arrows. b, Linear comparison of the representative IncQ1 plasmid sequences. Results of sequence alignment are generated with Easyfig version 2.1 (ref. 43). The arrows represent the position and transcriptional direction of the ORFs. Regions of homology between 73% and 100% are marked by grey shading. c, Conjugation transfer efficiencies of the index plasmid pLHM10–1-p6 into E. coli, K. pneumoniae, and S. Typhimurium strains. Transfer efficiency is calculated based on colony counts of the transconjugant and recipient cells in triplicate, and all data points are displayed, along with mean and standard deviation, respectively. d, Plasmid stability experiment results. All experiments are conducted in triplicate. Error bars denote the means (middle lines) and standard deviations.

Figure 4 |. XbaI-PFGE dendrogram and details about tet(X4)-positive E. coli isolates.

The PFGE assay is conducted successfully for once according to the standard protocol, and then used for the following analysis. The full gel images have also been provided in the supplementary material. PFGE patterns with a cutoff at 85% similarity (the dotted line) are considered to the same PFGE cluster, and indicated as groups A-K, respectively. E. coli strains carrying pLHM10–1-p6-like plasmid are underlined. ^aThese strains are isolated from environmental samples in pig farms. ^bTGC, tigecycline; TC, tetracycline; SXT, sulfamethoxazole/trimethoprim; FFC, florfenicol; CTX, cefotaxime; CS, colistin; CIP, ciprofloxacin; GEN, gentamicin.