With the rise of Klebsiella pneumoniae, a widely distributed pathogen, critical diseases such as endophthalmitis and bloodstream infections have become common (Lee et al., 2006, 2015; Yang et al., 2021). Carbapenems are often relied on for treating multidrug-resistant (MDR) infections; however, their overuse has resulted in the development of carbapenem-resistant Klebsiella pneumoniae (CRKP). Treatments available are increasingly limited, with only last-resort antibiotics like colistin and tigecycline remaining (Sheu et al., 2019). As the first glycylcycline antibiotic, it is a rare effective treatment for difficult-to-treat conditions, particularly those caused by CRKP (Seifert et al., 2018). Like tetracyclines, tigecycline binds reversibly to the 30S ribosomal subunit, disrupting aminoacyl-tRNA function and inhibiting bacterial translation (Pournaras et al., 2016). Tigecycline-resistant strains of K. pneumoniae have emerged rapidly since the clinical use of tigecycline, a problem likely exacerbated by the overuse of antibiotics, which has the potential to complicate treatment and pose a significant public health risk.

The current resistance mechanism of tigecycline is mainly related to the overexpression of RND efflux pumps, including AcrAB, OqxAB, MexAB-OprM, and Tmexd-toprJ (Pournaras et al., 2016; Chen et al., 2017; Lv et al., 2020; Avakh et al., 2023). The AcrAB-TolC pump is driven by the global transcriptional activator RamA and local inhibitory factor AcrR (Villa et al., 2014). The presence of a mutant in ramR, a local inhibitor of ramA, leads to elevated ramA expression and dysregulation of AcrAB expression, which ultimately leads to tigecycline resistance (Xu et al., 2021). Similarly, inactivation of OqxR enhances OqxAB transcription (Wan Nur Ismah et al., 2018). Furthermore, it has been demonstrated that mutations such as V57L in the rpsj gene can contribute to resistance even in the absence of ramR mutations (Herrera et al., 2021). The tet protein also has several known tigecycline resistance mechanisms, including tet(A), tet(L), tet(X), and tet(M) (Fiedler et al., 2016; Fan et al., 2024; Zou et al., 2024). Among them, mutations in the ribosome protection protein tet(M) can modify drug resistance by changing the binding site. Tetracycline mobile inactivating enzyme tet(X) and its variants can exist on a variety of mobile genetic elements, mediate the rapid spread of tigecycline resistance genes through horizontal transfer, and exist stably in drug-resistant strains at a very low adaptive cost, and significantly increase the level of resistance to tigecycline (Song et al., 2020; Hsieh et al., 2021).

The growing tigecycline resistance has significantly limited clinical treatment options for multidrug-resistant K. pneumoniae. Hence, it is crucial to examine TRKP isolates, particularly in regions like Southwest China, where data is lacking. This study aimed to evaluate the phenotypic characteristics, molecular prevalence and tigecycline resistance mechanism of TRKP isolates from southwest China. In brief, a drug susceptibility test was performed on clinically isolated K. pneumoniae, and the genetic relationship of TRKP isolates was studied using multi-site sequence typing (MLST) technique. At the same time, efflux pump inhibition assay was performed to verify efflux pump activity. Using PCR, DNA sequencing technology and reverse transcription PCR (RT-PCR), the determinants of drug resistance including tigecycline resistance genes, pump genes and their regulatory factors were studied.

The majority of TRKP strains in our hospital come from older adults and those in the ICU with catheters. Severe pneumonia caused by TRKP often results in extended hospital stays (3-370 days) and poor outcomes, with a 36.7% mortality rate. K. pneumoniae comes from a variety of specimens and can cause infections of the lungs, urinary tract, tissues, bile, and ducts. Antimicrobial susceptibility tests show that TRKP isolates exhibit high resistance to most antibiotics. The increased MIC of tigecycline may be linked to the use of other antibiotics, which are also expelled via the AcrAB-TolC pump (Roy et al., 2013). In this study, 80% of patients had no prior exposure to tigecycline, confirming that resistance can occur without direct exposure. Efflux pump inhibitors (EPIs) down-regulate their expression by interfering with the combination of efflux pump-related proteins, blocking the energy supply effect, and inhibiting the substrate’s passage through efflux pump channels [16]. To detect the presence of overexpression of efflux pumps in TRKP strains, we used efflux pump inhibitor (EPI) PAβN to evaluate efflux pump activity. In our study, 96.6% of TRKP strains showed a fourfold or greater reduction when PAβN was present. The MIC of tigecycline remained unchanged when PAβN was present in TR23. We believe that the following factors may be involved: First, changes in membrane permeability, such as mutations of ompK35 and ompK36 genes, may lead to decreased permeability of the outer membrane to tigecycline; Second, in the presence of PAβN, other efflux pumps may still expel tigecycline. Finally, the metabolic status of bacteria may also affect their susceptibility to drugs. We plan to further explore these resistance mechanisms through whole genome sequencing. However, most of the existing efflux pump inhibitors have obvious toxicity, and how to develop clinical drugs with high specificity, low toxicity, and high safety remains to be further explored.

Tet (A) belongs to the MFS efflux pump family with mutations that allow tigecycline to accumulate within bacterial cells leading to resistance (Linkevicius et al., 2016). In 2017, Chiu et al. first discovered that type 1 tet(A) raised the MIC of tigecycline by a factor of 8. It was also demonstrated that in the case of tet(A) mutation, loss of RamR protein has a synergistic effect on tigecycline resistance in K. pneumoniae (Chiu et al., 2017). Five years later, Peng et al. demonstrated through cloning experiments that type 3 tet (A) could raise the MIC of tigecycline fourfold (Peng et al., 2022a). In our experiments, we found that almost all isolates carried tet (A) mutations or ramR mutations. No ramR was detected in five of the isolates, suggesting that their ramR gene may have been truncated or deleted, similar to the fully ramR deletion mutant of Klebsiella pneumoniae strain KPBj1 M3 Lev (Bialek-Davenet et al., 2013). Furthermore, the deletion and insertion of various fragments among the seven isolates contributed to the premature appearance of the stop codon. This may result in the loss of the α8-α9 region, disrupt dimerization, and ultimately lead to a loss of function (Yamasaki et al., 2013). Fortunately, A large study has shown that although ramR mutants can enhance bacterial resistance, they also induce an enhanced immune response by modulating the structure of lipid A, thereby reducing the pathogen’s ability to kill in organs and blood (Yu et al., 2024).

Point mutations in the local suppressor oqxR have been shown to cause the OqxAB efflux pump to become overactive, giving the isolates increased virulence and multidrug-resistant characteristics (Bialek-Davenet et al., 2015). Previous studies have shown that amino acid substitution mutations such as V102G and V130A confer resistance to tigecycline (Bialek-Davenet et al., 2015; Chiu et al., 2017). This paper identifies for the first time two additional amino acid substitutions (E24R and C100Y) and frameshift deletions involving amino acids 226 to 227, which may negatively impact function. Therefore, the effect of these mutations on drug resistance requires further investigation. The recent discovery of the novel tetracycline-inactivating enzyme tet(X) homologs and the efflux pump gene clusters Tmexd-toprj has been linked to high levels of tigecycline resistance. These resistance mechanisms can be horizontally transferred via mobile elements like plasmids, spreading to humans, animals, and the environment, and posing a significant public health threat (Guo et al., 2024; Pan et al., 2024). A report identified 237 bacterial strains worldwide carrying the tmexCD-toprJ gene, with 92.83% originating from China (Dong et al., 2022). These strains represent 50 unique sequence types. Our study marks the first identification of two novel tmexCD-toprJ-positive strains, ST661 and ST1561, offering fresh insights into microbial research and underscoring the significance of bacterial diversity alongside its vast research potential. Tmexcd-positive strains were previously found mainly on chromosomes, but the discovery of plasmids in the past five years suggests a possible transmission mechanism. Despite attempts to perform conjugation experiments to determine whether tmexC is located on the plasmid and its ability to transfer, they were unsuccessful, suggesting that they may indeed be located on chromosomes. To further test this hypothesis, whole genome sequencing is planned to confirm the specific location and function of tmexCD-toprJ. In addition, patients infected with ST1561 were observed to survive, while those infected with ST661 died, a difference that may reflect significant differences in the pathogenicity and virulence of the strains. rpsj mutations linked to tigecycline resistance are generally found in the amino acids located between positions 53 and 60 of the S10 ribosomal protein (Beabout et al., 2015). Fortunately, we did not find any rpsj mutations related to tigecycline in the strains, nor did we detect any cases of tet(X) carrying.

Both AcrAB and OqxAB efflux pumps are common resistance mechanisms in enterobacteria (Liu et al., 2018). Research shows that OqxAB overexpression can lead to resistance to several antimicrobials, including chloramphenicol, quinolones, furantoin, and tigecycline (Li et al., 2019). In our study, acrB expression increased with varying MIC levels, while oqxB was overexpressed in only 4 bacterial strains. This aligns with findings by Perez et al., showing that while OqxAB is widely distributed in K. pneumoniae, it is not always overexpressed (Perez et al., 2013). It is important to note that the ramR gene was absent from the highly resistant strain TR4 (MIC 8µg/ml), and efflux pump inhibition tests demonstrated that the resistance mechanism was linked to the efflux pump even though acrB expression was only 2.32 times higher. Furthermore, no known resistance determinants were discovered, indicating that resistance may emerge through other pathways such as KpgABC or MacAB-TolC efflux pumps. Mutation of ramR, as an inhibitor of ramA, leads to up-regulation of ramA expression, which in turn increases the expression of acrAB or oqxAB efflux pump. In this study, three isolates (TR10, TR16, TR25) showed elevated ramA expression without any ramR mutations. This may be linked to mutations in the RamR recognition sites (PI and PII promoters) or Lon protease mutations (Rosenblum et al., 2011; Ricci et al., 2014), requiring further investigation to clarify this finding. Although it has been established that efflux mechanisms generally only lead to low levels of resistance to tigecycline, we speculate that multiple mechanisms may be at work at the same time, with possible synergistic effects. Therefore, more in-depth research is urgently needed to explore the interrelationships and effects of these mechanisms.

Additionally, 30 K. pneumoniae isolates were classified into 25 distinct ST types, reflecting the significant genetic diversity of TRKP. Previous research has indicated that most TRKP strains are resistant to multiple antibiotics and carry virulence factors, heightening the risk of resistance and virulence gene transfer. Monitoring the spread of these clones is essential to prevent their emergence as clinical pathogens (Peng et al., 2022a).

This study’s limitation is its single-center scope, which may limit representativeness. Additionally, only certain resistance genes were screened, leaving other mechanisms unexplored. Future efforts will expand sample size through multi-center studies and broaden genetic screening to better understand resistance mechanisms and enhance the study’s clinical relevance. In conclusion, acrB overexpression and tet(A) mutations are key contributors to tigecycline resistance in K. pneumoniae in southwest China.