Around the world, Klebsiella pneumoniae is a pathogen of great clinical importance as it results in severe community-acquired and hospital-acquired infections [1,2]. Numerous diseases, including liver abscesses, bloodstream infections, pneumonia, and urinary tract infections, can result from it [3]. Carbapenem-resistant K. pneumoniae (CRKp) has become a major risk to human health [4]. Furthermore, the hypervirulent K. pneumoniae (hvKp) has also been spreading at a fast pace [5]. In recent years, the KPC-2-producing ST11-KL64 hypervirulent CRKp strain (CR-hvKp) has frequently emerged in China, which exhibits high transmissibility, hyper-resistance, and hypervirulence, leaving limited therapeutic options for severe infections [6].

A new cephalosporin named cefiderocol, which is conjugated with a siderophore, gets into bacteria through an active uptake mechanism by means of the iron transport system [7]. The new antibiotic agent contains a chlorocatechol sidechain that imitates catecholate-type siderophores (e.g., enterobactin) that chelate iron, allowing it to get into bacterial cells via TonB-dependent iron transporter channels [8]. The compound shows remarkable in vitro activity against a wide range of carbapenem-resistant bacteria, including CR-hvKp [7]. However, when its entry pathway into bacteria is obstructed, resistance can develop. EnvZ and OmpR are the typical two-component systems (TCSs) in K. pneumoniae, where EnvZ acts as a sensor kinase and OmpR acts as a response regulator [9]. Microarray data from the ΔompRΔenvZ mutant showed a marked upregulation of several Fur-regulated genes, especially those involved in enterobactin synthesis and transport [10]. Cefiderocol resistance may also arise as a result of TCS alterations. Yamano et al. [11] found that the mutations in envZ mutations were present in strains with an elevated MIC value for cefiderocol. Another study demonstrated that combined mutations in the KPC gene and envZ conferred a ≥256-fold increase in cefiderocol MIC [12]. Cefiderocol mimics the structure of the enterobactin siderophore, and the two often compete with each other during bacterial uptake. Yang et al. revealed that deletion of entB can reduce the cefiderocol MIC by more than eightfold [13]. Additionally, the function of the iron transporter depends on three inner membrane proteins, TonB–ExbB–ExbD, which transfer energy to the outer membrane for iron transport [8]. The mutations or downregulation in these proteins also caused resistance to cefiderocol [14]. However, investigations on these proteins are limited, further stuides is needed to elucidate the potential mechanisms of cefiderocol resistance.

A monitoring study named SIDERO-WT-2014 indicated that cefiderocol had 100% efficacy against Enterobacteriaceae that produce KPC, yet it was only 58% effective when it came to Enterobacteriaceae that produce NDM [15]. For most K. pneumoniae, the resistance to cefiderocol is caused by the combined influence of NDM expression and decreased expression or mutations in the siderophore receptor genes (e.g., cirA) [[16], [17], [18]]. Nevertheless, our previous research has found that KPC-producing CR-hvKp, a type of prevalent strain in China, can also develop resistance to cefiderocol [19]. Another study revealed that cefiderocol resistance in CR-hvKP can arise from cirA mutation combined with the co-production of KPC-2 and SHV-12 [20]. The KPC-31 variant, along with a conjugative low-copy-number vector containing the fec gene cluster, is another mechanism for strain resistance [21]. However, the cefiderocol resistance mechanisms in KPC-producing CR-hvKP are not fully understood.

This study, therefore, aimed to investigate novel cefiderocol resistance mechanisms in a KPC-producing CR-hvKP strain through in vitro laboratory evolution and multi-omics analysis.