The investigation of molecular epidemiological characteristics and resistance mechanism of tigecycline resistant Klebsiella pneumoniae from a large teaching hospital in southwest China, Chongqing

Abstract

Background: Klebsiella pneumoniae is one of the main pathogens of nosocomial infection, among which carbapenems can be used for multidrug-resistant Klebsiella pneumoniae. However, in the past decade, the resistance rate of carbapenem-resistant Klebsiella pneumoniae has increased yearly. Tigecycline has good antibacterial activity in treating severe bacterial infections, but the reports of tigecycline resistance are increasing. This study aimed to investigate the mechanism of drug resistance and epidemiological characteristics of tigecycline-resistant Klebsiella pneumoniae (TRKP) in a large teaching hospital in southwest China, Chongqing.

Methods: We isolated 30 TRKP strains from this hospital between August 2021 and December 2023. By PCR and sequencing, we examined the presence and mutation rates of genes associated with tigecycline resistance, including acrR, oqxR, ramR, tmexC, tet(x), tet(A), tet(L), and rpsj, and performed efflux pump inhibition experiments to verify efflux pump activity. At the same time, real-time RT-PCR was used to detect the expression levels of efflux pump genes (acrB and oqxB) and ramA. To investigate the prevalence trend of TRKP in our hospital, we performed multi-site sequence typing (MLST) analysis.

Results: The mutation rates of ramR (73.3%) and tet(A) (63.3%) were significant. In efflux pump inhibition experiments, PaβN could reverse the resistance of 29 TRKP strains (96.7%) to tigecycline. Real-time RT-PCR results showed that acrB and ramA genes were up-regulated in 22 strains, while oqxB genes were overexpressed in only 4 strains. MLST analysis showed that these strains could be divided into 25 different ST subtypes, indicating that no outbreak of TRKP occurred in our hospital. In addition, two tmexCD-torpj positive strains, ST661 and ST1561, were identified for the first time.

Conclusion: The efflux pump acrB and tet(A) mutations are the primary mechanisms of resistance to tigecycline-resistant Klebsiella pneumoniae at our hospital. The ramR mutation can mediate efflux pump activity of acrB by up-regulating ramA overexpression.

Keywords: Klebsiella pneumoniae; RND efflux pump; resistance mechanism; tet(A); tigecycline-resistance.

Figure 1

RamR protein mutations in TRKP clinical isolates. The open boxes indicate the nine α-helices of RamR (α1–α9). The reference sequence of K. pneumoniae MGH78578 (CP000647) was used to identify RamR mutations. Solid lines indicate efficiently transcribed RamR proteins and dashed lines indicate inefficiently transcribed RamR proteins.

Figure 2

The expression levels of resistance-nodulation-cell division (RND) efflux pump gene and ramA gene in Klebsiella pneumonia. The expression levels of resistance-nodulation-cell division (RND) efflux pump gene and ramA gene in K. pneumoniae were determined by qPCR and the target gene expression levels were divided into three groups according to the MIC value of tigecycline (=8 mg/L, = 16mg/L, = 32mg/L) for comparison, with tigecycline-susceptible Escherichia coli ATCC 25922 as control (expression = 1). (A) Relative expression (RE) level of acrB in TRKP strains; (B) RE level of oqxB in TRKP strains; (C) RE level of ramR in TRKP strains; (D)Average RE of acrB, oqxB, and ramA in TRKP isolates treated with different tigecycline MIC values. The bars represent the average value and the error bars represent the standard error of the mean value. Data were analyzed by Mann–Whitney U test (*P <0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).