Tigecycline

Transporters belonging to the chromosomally encoded resistance-nodulation-division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria. However, the cotransfer of large gene clusters encoding RND-type pumps from the chromosome to a plasmid appears infrequent, and no plasmid-mediated RND efflux pump gene cluster has yet been found to confer resistance to tigecycline. Here, we identified a novel RND efflux pump gene cluster, designated tmexCD1-toprJ1, on plasmids from five pandrug-resistant Klebsiella pneumoniae isolates of animal origin. TMexCD1-TOprJ1 increased (by 4- to 32-fold) the MICs of tetracyclines (including tigecycline and eravacycline), quinolones, cephalosporins, and aminoglycosides for K.pneumoniae, Escherichia coli, and Salmonella TMexCD1-TOprJ1 is closely related (64.5% to 77.8% amino acid identity) to the MexCD-OprJ efflux pump encoded on the chromosome of Pseudomonas aeruginosa In an IncFIA plasmid, pHNAH8I, the tmexCD1-toprJ1 gene cluster lies adjacent to two genes encoding site-specific integrases, which may have been responsible for its acquisition. Expression of TMexCD1-TOprJ1 in E. coli resulted in increased tigecycline efflux and in K. pneumoniae negated the efficacy of tigecycline in an in vivo infection model. Expression of TMexCD1-TOprJ1 reduced the growth of E. coli and Salmonella but not K. pneumoniaetmexCD1-toprJ1-positive Enterobacteriaceae isolates were rare in humans (0.08%) but more common in chicken fecal (14.3%) and retail meat (3.4%) samples. Plasmid-borne tmexCD1-toprJ1-like gene clusters were identified in sequences in GenBank from Enterobacteriaceae and Pseudomonas strains from multiple continents. The possibility of further global dissemination of the tmexCD1-toprJ1 gene cluster and its analogues in Enterobacteriaceae via plasmids may be an important consideration for public health planning.IMPORTANCE In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae, the most problematic pathogens in human clinical settings-especially carbapenem-resistant K.pneumoniae Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a "One Health" perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this.

Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has recently aroused increasing attention, especially ST11, the predominant CRKP clone in China. Here, we report a case of hv-CRKP-associated infection and reveal the in-host evolution of its mechanism of resistance to tigecycline and polymyxin under clinical therapy. A total of 11 K. pneumoniae carbapenemase (KPC)-producing CRKP strains were consecutively isolated from a male patient who suffered from continuous and multisite infections. String and antimicrobial susceptibility tests identified seven hypermucoviscous strains and three tigecycline-resistant and four colistin-resistant strains. Galleria mellonella larvae infection model confirmed the hypervirulence. Pulsed-field gel electrophoresis (PFGE) separated three PFGE clusters among all strains, and further Southern blotting detected that blaKPC-2 was located on the same-sized plasmid. Whole-genome sequencing showed that all strains belonged to the hv-CRKP ST11-KL64 clone. Diverse hypervirulence factors and resistance genes were identified. Further sequencing with the Nanopore platform was performed on the CRKP-Urine1 strain, which contained one virulence plasmid (pVi-CRKP-Urine1) and two resistance plasmids (pKPC-CRKP-Urine1 and pqnrS1-CRKP-Urine1). The gene mutations responsible for tigecycline or colistin resistance were then amplified with PCR followed by sequencing, which indicated that mutations of ramR and lon were the potential loci for tigecycline resistance and that the pmrB, phoQ and mgrB genes for colistin resistance. A novel frameshift mutation of lon was identified in the high-level tigecycline-resistant strain (MIC, 128 mg/L). The results indicate that the hypervirulent ST11-KL64 clone is a potential threat to antiinfection treatment and is capable of rapid and diverse evolution of resistance during tigecycline and polymyxin treatment.

BackgroundThe recent emergence and dissemination of high-level mobile tigecycline resistance Tet(X) challenge the clinical effectiveness of tigecycline, one of the last-resort therapeutic options for complicated infections caused by multidrug-resistant Gram-negative and Gram-positive pathogens. Although tet(X) has been found in various bacterial species, less is known about phylogeographic distribution and phenotypic variance of different genetic variants.MethodsHerein, we conducted a multiregional whole-genome sequencing study of tet(X)-positive Acinetobacter isolates from human, animal, and their surrounding environmental sources in China. The molecular and enzymatic features of tet(X) variants were characterized by clonal expression, microbial degradation, reverse transcription, and gene transfer experiments, while the tet(X) genetic diversity and molecular evolution were explored by comparative genomic and Bayesian evolutionary analyses.ResultsWe identified 193 tet(X)-positive isolates from 3846 samples, with the prevalence ranging from 2.3 to 25.3% in nine provinces in China. The tet(X) was broadly distributed in 12 Acinetobacter species, including six novel species firstly described here. Besides tet(X3) (n = 188) and tet(X4) (n = 5), two tet(X5) variants, tet(X5.2) (n = 36) and tet(X5.3) (n = 4), were also found together with tet(X3) or tet(X4) but without additive effects on tetracyclines. These tet(X)-positive Acinetobacter spp. isolates exhibited 100% resistance rates to tigecycline and tetracycline, as well as high minimum inhibitory concentrations to eravacycline (2-8 μg/mL) and omadacycline (8-16 μg/mL). Genetic analysis revealed that different tet(X) variants shared an analogous ISCR2-mediated transposon structure. The molecular evolutionary analysis indicated that Tet(X) variants likely shared the same common ancestor with the chromosomal monooxygenases that are found in environmental Flavobacteriaceae bacteria, but sequence divergence suggested separation ~ 9900 years ago (7887 BC), presumably associated with the mobilization of tet(X)-like genes through horizontal transfer.ConclusionsFour tet(X) variants were identified in this study, and they were widely distributed in multiple Acinetobacter spp. strains from various ecological niches across China. Our research also highlighted the crucial role of ISCR2 in mobilizing tet(X)-like genes between different Acinetobacter species and explored the evolutionary history of Tet(X)-like monooxygenases. Further studies are needed to evaluate the clinical impact of these mobile tigecycline resistance genes.

The presence of tigecycline heteroresistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) can compromise the efficacy of antimicrobial therapy. This study aimed to investigate the phenotypic and genomic characteristics of Tgc-HR in CRKP clinical isolates. A total of 52 non-repetitive CRKP isolates, all tigecycline-susceptible, were collected from January 2021 to December 2023. Tigecycline heteroresistance was confirmed by K-B disk diffusion, E-test screening, growth curves, population analysis profiling (colony pattern analysis), and time-kill curve in this study. Protein fingerprinting clustering, WGS analysis, and bioinformatics were used to investigate the phenotypic characteristics and resistance genes, virulence factors, and plasmid profiles of the isolates. The heteroresistant subclones could proliferate at tigecycline levels as high as 16 mg/L, and their resistant phenotype stayed stable even after repeated passages without antibiotic exposure. Analysis of protein fingerprint clustering suggested that the heteroresistant subclones were derived directly from the original strain, not from contamination by an external strain. All isolates were identified as ST-11 through multilocus sequence typing. The genomic analysis showed that the isolates contained numerous resistance genes, especially the tetracycline efflux pump gene tet(A), β-lactamase genes (blaTEM and blaCTX-M), the aminoglycoside-modifying enzyme gene (aadA), and the efflux pump regulators ramR and acrR. These resistance genes were found to co-occur with virulence factors, such as the type 3 fimbriae (mrk-associated loci), based on genomic co-occurrence analysis (which involves assessing the presence of genes in close proximity or co-localization on the genome), suggesting that resistance and virulence together could heighten pathogenicity and make treatment outcomes more challenging in clinical scenarios. The plasmid typing results indicated that the isolates contained a multireplicon plasmid capable of carrying several resistance genes and closely resembling known high-risk resistant plasmids, aiding in the dissemination of resistance. In this study, we systematically investigated the phenotypic and genomic features of tigecycline heteroresistance among clinical CRKP isolates in China and demonstrated its stability and plausibly genetic basis and hinted at treatment implications. The findings are predictions based off of genomic data estimations and have not been confirmed by transcriptomic nor functional research. More work is required to validate these mechanisms.ImportanceThis study provides crucial insights into the phenomenon of tigecycline heteroresistance (Tgc-HR) in carbapenem-resistant Klebsiella pneumoniae. It highlights the stability of heteroresistant subpopulations and their ability to persist without selective antibiotic pressure, complicating treatment outcomes. The research shows that Tgc-HR is linked to multiple resistance and virulence factors, suggesting a co-evolution of resistance and pathogenicity. This finding underscores the importance of monitoring for heteroresistance in clinical settings, as it may lead to undetected therapeutic failures. Additionally, the study contributes to understanding the role of plasmids in the spread of resistance, emphasizing the need for comprehensive genomic surveillance to inform better antimicrobial strategies.