Klebsiella pneumoniae (Kp) is becoming an important clinical opportunist due to its highly varied antimicrobial resistance features caused by its relatively strong genome plasticity (Wyres and Holt, 2018). Sequence type (ST) 11, a member of the worldwide prevalent clonal group (CG) 258 and one of the dominant subtypes of Kp, was identified as a reservoir of various antimicrobial resistance genes and is highly prevalent in China (Wang et al., 2018). More importantly, the convergence of resistance to last-line antimicrobials, such as carbapenem resistance, and hypervirulence within ST11 Kp (CR-hvKp), associated with significant morbidity and mortality rates, is emerging and rapidly increasing, posing a great threat to public health (Gu et al., 2018, Liu et al., 2022).

In addition to the resistances that are typically displayed as stable traits, unstable resistances have also been observed. Seemingly identical bacterial cells in a population can exhibit resistant phenotypic heterogeneity, namely, heteroresistance, leading to difficulties in identifying the bacterial population as susceptible or resistant and very poor prognoses. Moreover, it has been observed that heteroresistance could greatly impact the antibacterial effectiveness of some novel antimicrobials, such as cefiderocol (FDC) (Choby et al., 2021). FDC is a novel siderophore-conjugated cephalosporin that displays great potential antibacterial activity in vitro (Candel et al., 2022, Shortridge et al., 2022, Zhanel et al., 2019). Previous study reported that FDC presented 98% activity among global carbapenem-resistant Enterobacterales (CRE) isolates (Kazmierczak et al., 2019). Similarly, extremely low FDC resistance among carbapenem-resistant Escherichia coli and CRKP strains has been reported in multicenter studies in China (Lan et al., 2022, Wang et al., 2022). However, FDC heteroresistance in Kp has been reported to be associated with mutations in the siderophore receptor cirA and β-lactamase activities. Increased blaNDM-5 copy numbers have been recently reported to contribute to FDC resistance in vivo (Simner et al., 2022). Furthermore, blaSHV-12, a common β-lactamase gene, was also found to be related to reduced FDC susceptibility in Enterobacteriaceae in vitro (Kohira et al., 2020, Poirel et al., 2022).

More importantly, resistance to novel antimicrobials could even emerge before the use of these drugs via a mechanism known as cross-resistance. Antimicrobials belonging to the same class are usually chemically similar; therefore, bacteria that are resistant to one member of the class might also be resistant to the other members. Pre-exposure to some cephalosporins could induce cross-resistance to FDC (Shields et al., 2020). Ceftazidime/avibactam (CZA)-resistant KPC variants have a possible impact on FDC by increasing the minimum inhibitory concentrations (MICs) of FDC, leading to an inoculum effect caused by using CZA before FDC, which could greatly impact the effectiveness of FDC (Hobson et al., 2021). The emergence of cross-resistance to cefepime, CAZ, and FDC in Enterobacter hormaechei resulting from the emergence of the AmpC variant was also reported (Shields et al., 2020). These cross-resistances have been observed both in vivo and in vitro; however, the impacts are still not well understood.

Here, during active surveillance, we identified two Kp infection cases in which the isolates displayed FDC heteroresistance both in vivo and in vitro, and more notably, the two patients had never been pre-exposed to FDC. Using nanopore sequencing technology, we first found that tandem amplification of blaSHV-12 occurred and that the copy number rapidly changed both in vivo and in vitro, subsequently contributing to FDC resistance without significant fitness costs.