RamR T116I mutation confers tigecycline resistance in ST23 hypervirulent Klebsiella pneumoniae

To the Editor: Klebsiella pneumoniae (K. pneumoniae) is a common opportunistic pathogen with rising prevalence in clinical settings. A multicenter study reported its detection rate among hospitalized adults with community-acquired pneumonia (CAP) has increased annually, making it the leading cause of severe CAP (SCAP), surpassing Streptococcus pneumoniae.[1] Hypervirulent K. pneumoniae (hvKP), especially sequence type (ST) 23,[2] exhibits enhanced virulence and colonization, driven by multiple siderophore systems and virulence plasmids, and is linked to high mortality.[3] Alarmingly, tigecycline resistance in ST23 strains is increasing. While tigecycline is crucial for treating multidrug-resistant and carbapenem-resistant K. pneumoniae, resistance—mainly due to efflux pump overexpression, enzymatic inactivation, and target site mutations—has been widely reported.[4] Mutations in regulators such as RamR (a TetR-family transcriptional repressor regulating ramA) are involved,[5] but their roles in hvKP remain unclear. In this study, a novel RamR point mutation was identified and validated via whole-genome sequencing, gene editing, quantitative real-time polymerase chain reaction (qRT-PCR), proteomics, and electrophoretic mobility shift assay (EMSA). This mutation reduced ramA repression, resulting in efflux pump overexpression, and conferred tigecycline resistance. The Medical Ethics Committee of Qingdao Municipal Hospital approved this study (No. XM: 2023-030). All statistical analyses were conducted using GraphPad Prism 9.3.0 (GraphPad Software, San Diego, CA, USA). For proteomics data processing and quantification, SpectronautTM 19 (Biognosys AG, Zurich, Switzerland) was used. For qRT-PCR analysis, each sample was analyzed with three biological and three technical replicates. Gene expression was normalized to the endogenous control gene rpoB using the 2–ΔΔCt method. For statistical comparisons, two-tailed Student’s t-test was performed for normally distributed continuous data, while Mann-Whitney U test was used for non-normally distributed continuous data. All data are presented as the mean ± standard deviation (SD). For proteomics analysis (n = 3 per group), data quantification and initial identification of differentially expressed proteins were performed using SpectronautTM 19. Proteins with differential expression were identified based on a two-tailed t-test (P <0.05) and a fold change (FC) >1.2. Two ST23 hvKP strains were isolated from different sites in the same patient: KP124 from the bloodstream and KP126 from liver abscess. Whole-genome sequencing was performed with a P2solo system (Oxford Nanopore Technologies, Oxford, UK; GenBank accession number: PRJNA1206319). Although they shared identical key genetic determinants [Supplementary Table 1, https://links.lww.com/CM9/C637], they exhibited markedly different susceptibility to tigecycline. KP124 was susceptible to tigecycline (minimum inhibitory concentration [MIC] = 0.5 μg/mL), whereas KP126 displayed an MIC of 4 μg/mL, exceeding the resistance threshold defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (MIC >0.5 μg/mL). Further antimicrobial susceptibility testing confirmed that KP126 was also resistant to other tetracycline-class antibiotics, including eravacycline (MIC = 16 μg/mL) and tetracycline (MIC = 8 μg/mL). Detailed susceptibility data are provided in Supplementary Table 2, https://links.lww.com/CM9/C637. To elucidate the mechanism underlying the differential tigecycline resistance between the two strains, we conducted further investigations. To identify the genetic determinants of tigecycline resistance, comparative genomic analysis was performed, revealing an extremely high level of genomic similarity between the KP124 and KP126 strains, with an average nucleotide identity (ANI) of 99.996% and the presence of only 165 single-nucleotide polymorphisms (SNPs). Notably, comparative analysis revealed a nonsynonymous point mutation in the tigecycline resistance-associated transcriptional regulator RamR in KP126, leading to a threonine-to-isoleucine substitution at position 116 (T116I). We highly suspect that this mutation confers resistance to tigecycline. Two complementary experiments were performed to validate our hypothesis. Firstly, the transformation of KP126 with wild-type ramR (cloned and inserted into pACYC184-spc) successfully restored tigecycline susceptibility, reducing the MIC fourfold to 1 μg/mL. Secondly, because genetic modification of KP124 and KP126 was difficult, we instead employed the tigecycline-susceptible K. pneumoniae strain KP437 (MIC = 0.5 μg/mL) as an alternative model. This strain has a clean genetic background and is susceptible to all tested antibiotics, and it had previously been used successfully for gene knockout and complementation in our studies. In this model, ramR was deleted using the clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) system, followed by chromosomal replacement with the T116I variant, which increased tigecycline resistance to an MIC of 2 μg/mL. Detailed antimicrobial susceptibility results are provided in Supplementary Table 3, https://links.lww.com/CM9/C637, and all primers, plasmids, and spacers used are provided in Supplementary Table 4, https://links.lww.com/CM9/C637. qRT-PCR was performed for ramA, acrB, and ramR. This was aimed at investigating whether the T116I mutation in RamR leads to derepression of ramA expression, which in turn causes overexpression of the AcrAB-TolC multidrug efflux pump, a major tripartite efflux pump in K. pneumoniae that pumps various substrates, including tigecycline, out of the cell. All primers used are listed in Supplementary Table 4, https://links.lww.com/CM9/C637. Raw cycle threshold (Ct) values and calculated relative gene expression levels (2–ΔΔCt) are provided in Supplementary Tables 5 and 6, https://links.lww.com/CM9/C637. Compared with sensitive strains, the tigecycline-resistant strains presented statistically significantly upregulated expression of ramA, acrB, and ramR (all P <0.001) [Figure 1A–H], which is consistent with the hypothesis that the T116I RamR mutation leads to tigecycline resistance by derepressing RamA, leading to elevated expression of AcrAB-TolC. However, the observed increase in ramR expression does not rule out a functional defect in the RamR protein. It is plausible that the T116I mutation leads to either post-translational degradation or a change in the protein’s conformation, which in turn reduces its ability to repress ramA expression. To investigate these possibilities, we performed a proteomic analysis.Figure 1: Globally present RamR T116I mutation led to tigecycline resistance by derepressing the AcrAB-TolC efflux pump. (A–H) Gene expression levels of acrB, ramA, and ramR in Klebsiella pneumoniae cultured in LB broth without antibiotics. (I) Volcano plot of differentially expressed proteins in K. pneumoniae KP126 compared to KP124. (J) The structure predicts by alphafold3. Overall structure of RamR protein, showing its DNA-binding domain (orange) and dimerization domain (cyan). Hydrogen bond network (yellow dashed lines) of T116 (red) with surrounding residues in wild-type RamR, forming six hydrogen bonds. Hydrogen bond network of I116 (red) with the same residues in the T116I mutant, forming only three hydrogen bonds, indicating weakened interaction. (K) Electrophoretic mobility shift assay of the binding of RamR with the ramA promoter. (L) Phylogenetic tree of K. pneumoniae strains carrying the RamR T116I mutation. ADH: Alcohol dehydrogenase; DegP: Serine endoprotease; LivG: High-affinity branched-chain amino acid ABC transporter ATP-binding protein; Lpp: Outer membrane lipoprotein; MlaE: Lipid asymmetry maintenance ABC transporter permease subunit; OmpA lipoprotein: Outer membrane protein A; K. pneumoniae: Klebsiella pneumoniae.Label-free data-independent acquisition (DIA) proteomics was performed on K. pneumoniae KP124 and KP126 using a VanquishNeo LC-timsTOF HT system (Thermo Fisher Scientific Inc., Waltham, USA; iProX project ID: IPX0012731001). Volcano plots were generated using the ggplot2 package (v3.4.4) in R (v4.2.1, R Development Core Team, Vienna, Austria), visualizing proteins with a |log2 fold change| >0.58 and a P value <0.05 [Figure 1I]. The proteomic analysis results were consistent with the qRT-PCR results, confirming the high expression of the AcrAB-TolC efflux pump and RamA in strain KP126. The upregulation of RamR confirmed that ramA upregulation was not caused by the repression of RamR. We performed protein structure prediction using AlphaFold3 (https://alphafoldserver.com/). RamR consists of a DNA-binding domain (helices α1–α3) and a dimerization domain (helices α4–α11). The T116 residue is in a critical loop connecting helices α6 and α7, adjacent to the DNA-binding domain. Although the T116I substitution did not cause major changes in the overall protein structure, in wild-type RamR, T116 forms six hydrogen bonds with N56 (α4), T119, and R120 (α7), contributing to structural stability. In contrast, the T116I mutant forms only three hydrogen bonds with the same residues, weakening this interaction network. This disruption likely reduces the stability of the dimerization domain, increases the flexibility of the DNA-binding domain, and consequently alters the DNA-binding activity. The predicted structures of wild-type and T116I mutant RamR are illustrated in Figure 1J. Next, we purified the wild-type and mutant RamR proteins using the BL21-pET15b expression system [Supplementary Figure 1, https://links.lww.com/CM9/C637] and performed electrophoretic mobility shift assays with DNA fragments containing the ramA promoter. All primers and plasmids used are listed in Supplementary Table 4, https://links.lww.com/CM9/C637. As expected, the T116I mutation reduced the binding affinity of RamR for the ramA promoter: the KP124 RamR protein-initiated probe bound at a 1:20 DNA:protein ratio, whereas the KP126 RamR protein began binding at a 1:40 ratio [Figure 1K]. To assess the prevalence of the T116I mutation, we performed a BLAST search of the National Center for Biotechnology Information Non-redundant protein sequence database (NCBI NR) for RamR carrying the T116I mutation. This search identified nine additional strains with the mutation, which were isolated between 2015 and 2023, from six countries, including China, the United States, Germany, Austrialia, Japan, and Thailand. We constructed a phylogenetic tree using these eleven genomes [Figure 1L] with Snippy (v4.6.0) and Gubbins (v3.3.1) and visualized it using Interactive Tree Of Life (https://itol.embl.de/). Most of these strains presented diverse sequence types and serotypes, suggesting independent evolutionary origins rather than clonal dissemination. We identified a novel RamR T116I mutation in an ST23 hvKP strain that confers tigecycline resistance by impairing RamR-mediated repression of ramR, leading to efflux pump overexpression. Our findings demonstrate that hvKP can acquire resistance under selective pressure, even within a single host, highlighting the importance of early detection and continuous genomic surveillance. Whole-genome sequencing enabled precise identification of this mutation and discrimination between otherwise near-identical isolates, while functional assays confirmed its role in resistance. Given the hypervirulent background and increasing clinical use of tigecycline, rapid genomic diagnostics and prudent antimicrobial stewardship are critical to prevent dissemination. Acknowledgments We would like to thank Dr. Jingyao Qu, Dr. Jing Zhu, Dr. Zhifeng Li, and Mr. Guannan Lin of Core Facilities for Life and Environmental Sciences, State Key Laboratory of Microbial Technology of Shandong University for protein analysis. Funding This work was supported by grants from the National Key Research and Development Program of China (No. 2022YFE0199800), Qingdao Science and Technology Wellness Promotion Demonstration Program (No. 24-1-8-smjk-13-nsh), the National Natural Science Foundation of China (No. 82271658), the SKLMT Frontiers and Challenges Project (No. SKLMTFCP-2023-01), the Shandong Provincial Natural Science Foundation (Nos. ZR2024QD228 and ZR2024QC311), the Qingdao Natural Science Foundation (No. 24-4-4-zrjj-40-jch), and the Opening Project of the Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP) (No. FDLAP24008). Conflcts of Interest None.