Extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) is a great threat to public health due to its rising incidence, limited treatment options, high mortality rate and scarce data on optimal therapy (Horcajada et al., 2019; Del Barrio-Tofiño et al., 2020). Indeed, the incidence rate of XDR-PA ranging from 2.6 to 30.0% had been reported (Walkty et al., 2017; Sader et al., 2018; Horcajada et al., 2019; Pérez et al., 2019). Even though PA is a common cause of respiratory tract infection, urinary tract infection, bloodstream infection and other nosocomial infections, XDR-PA infections are associated with higher mortality (Horcajada et al., 2019). A meta-analysis reported a mortality rate of 62.5% among bacteremia patients infected with XDR-PA versus 30% in those with susceptible PA infections (Recio et al., 2018). Indeed, PA is highly adaptable and can survive in various ecological settings. Its genetic plasticity grants access to many metabolic pathways and a broad range of virulence and resistance factors, making it one of the most successful bacteria in clinical microorganism (Behzadi et al., 2022). The underlying mechanism of XDR-phenotype is heterogeneous and can be mediated through the chromosomal mutations or the horizontal transfer of resistance factors, particularly those encoding carbapenemases often co-transferred with aminoglycoside-modifying enzyme (Del Barrio-Tofiño et al., 2019). These resistance components are closely related to the global high-risk clones, such as ST111, ST235 and ST175 (Oliver et al., 2015). It is known that acquiring antimicrobial resistance often comes with a fitness cost that reduces virulence and disease severity (Gómez-Zorrilla et al., 2016). However, some XDR-PA strains have been linked to the specific virulence factors such as the T3SS exoU (Peña et al., 2015; Horna and Ruiz, 2021a). Indeed, PA possesses a virulence mechanism known as the T3SS. The T3SS injects cytotoxins, including exoU, exoS, exoT and exoY, into target eukaryotic cells, and each of these distinct virulence factors leads to a specific type of host tissue injury, with exoU being characterized as having a greater impact on bacterial virulence (Peña et al., 2015; Horna and Ruiz, 2021a).

The XDR-phenotype significantly limits treatment options, although the combination of CZA and ATM has demonstrated promising activity against Enterobacterales producing serine-β-lactamase and MBL, it remains uncertain whether this combination is still effective against XDR-PA due to the complex mechanisms underlying the XDR-phenotype (Biagi et al., 2019). Therefore, our study characterized the resistance mechanisms, virulence factors, and molecular epidemiology of XDR-PA, and further evaluated the synergistic antibacterial activity of CZA and ATM combination against XDR-PA, aimed at providing clues for the prevention, control and treatment strategies of XDR-PA infections.

Infections caused by XDR-PA have been increasingly reported worldwide, posing a significant threat to public health due to limited treatment options and high mortality rates (Montero et al., 2020). The resistance mechanisms of XDR-PA are multifactorial, involving a combination of intrinsic, acquired, and adaptive factors (Chevalier et al., 2017). In our study, the expressions of mexA were statistically different between the XDR-PA group and S-PA group, indicating that the overexpression of efflux pump may play an important role in XDR-PA phenotype. The efflux pump systems, which are widely present in microorganisms, can actively expel both metabolic wastes and antibiotics that inhibit bacterial growth (Lorusso et al., 2022). These systems exhibit a broad substrate spectrum and mediate resistance to most antibiotics, including aminoglycosides, fluoroquinolones, β-lactamase inhibitors, macrolides, and sulfonamides, thus their overexpression may contribute to extensive-drug resistance (Castanheira et al., 2019; Helmann et al., 2019). In this study, carbapenemase resistance genes were detected, and the results showed that the main resistance genes carried by XDR-PA strains were NDM-1 and VIM-2, indicating that MBL may play an important role in XDR phenotype. Previous studies have shown that NDM-1 was carried by various plasmids that co-contain multiple resistance genes, including carbapenemase, β-lactamase and fluoroquinolone resistance genes (Poirel et al., 2011; Jaidane et al., 2018; Politi et al., 2019; Shahin and Ahmadi, 2021), making NDM-1-carrying PA show resistance to most antibiotics, while remaining susceptible to only a few antibiotics such as polymyxin. Even though polymyxin demonstrates susceptibility in vitro and is regarded as an alternative for XDR-PA infections, its clinical use is restricted due to its narrow therapeutic window and toxicity (Motsch et al., 2020; Satlin, 2021). Thus, the optimal treatment for MBL-producing XDR-PA remains unclear. Novel antibiotic CZA has been approved for hospital-acquired pneumonia/ventilator-associated pneumonia, complex urinary tract infections and intra-abdominal infections (Carmeli et al., 2016). Avibactam (AVI) is a novel β-lactamase inhibitor, which can inhibit class A, class C and some class D (i.e., OXA-10 and OXA-48) β-lactamase, while it is not active against MBL-producing XDR-PA (Zhen et al., 2022). Therefore, it is urgent to find an effective therapy strategy for these XDR-PA strains. Although ATM remains stable against MBL-producing strains, it is usually inhibited by the frequent co-production of class A β-lactamases or ampC-type enzyme (Tan et al., 2021). Thus, the combination of ATM and CZA is considered as a potential treatment option for MBL-producing pathogens, as the combination will restore the activity of ATM against these strains attribute to the inhibition of the co-producing non-MBL β-lactamases by AVI (Mikhail et al., 2019; Cornely et al., 2020; Lee et al., 2021). However, clinical data on this combination is still scarce. Therefore, we performed the combined antimicrobial susceptibility tests of ATM and CZA on XDR-PA strains to provide some clues. The results revealed that the combination of ATM and CZA showed a synergistic effect on most MBL-producing XDR-PA strains, suggesting that the combination of ATM and CZA may be a potential treatment option for MBL-producing XDR-PA infections, which was consistent with some previous reports (Emeraud et al., 2019; Lee et al., 2021). Meanwhile, it is necessary to be vigilant against extensively drug-resistant mediated by KPC enzyme mutants or some D-class enzymes (i.e., OXA-14 and OXA-23), in which case the combination of ATM and CZA is usually ineffective.

T3SS, mainly includes exoY, exoT, exoU and exoS, play an important role in PA infection. PA transports a series of self-secretion proteins to host cells via the T3SS and destroys the actin skeleton and cause host cells death (Horna and Ruiz, 2021b; Moir et al., 2021). In our study, the T3SS virulence genes carried by XDR-PA strains mainly were exoY+/exoT+/exoU+/exoS-. Previous studies have reported that PA with exoU+/exoS- had a higher antibiotic resistance rate than that with exoU-/exoS+, may be because that PA with exoU+/exoS- frequently exhibiting overexpression of efflux pump (Takata et al., 2018; Ali, 2022). In addition, the strains carrying exoU+/exoS- exhibit high virulence, making their infection involve a wide range of lesions and increases their exposure to antibiotics, thus resulting in higher resistance rate (Takata et al., 2018; Ali, 2022). However, the relationship between antibiotic resistance and virulence remains controversial. Some researchers believe that the acquisition of resistance mechanisms may have a negative impact on adaptability (namely adaptability cost), resulting in bacteria physiological damage and loss of virulence (Gómez-Zorrilla et al., 2016). On the contrary, it has been reported that some resistance mutations are not associated with adaptive costs (Subedi et al., 2018). XDR-PA strains may develop other compensatory or inhibitory mutations that allow them to regain virulence without affecting resistance (Subedi et al., 2018).

Global clones associated with MDR/XDR-PA phenotypes are known as high-risk clones and pose great threat in hospitals around the world (Del Barrio-Tofiño et al., 2020). In our study, the 32 XDR-PA strains belong to 8 different ST types, and the predominant ST type was ST1971, with carrying both NDM-1 and exoY+/exoT+/exoU+/exoS-, suggesting that NDM-1-producing ST1971 XDR-PA with exoY+/exoT+/exoU+/exoS- was the predominant strain in our study. ST1971, a high-risk clone predominantly reported in China, is commonly associated with high virulence, it was first isolated in Beijing in 2015 and later in Guangdong in 2023 (Zhao et al., 2023a; Zhao et al., 2023b), which alarmed the further surveillance of this highly virulence and resistant clone. In addition, we also identified other high-risk global clones, including ST308, ST244 and ST357, which were recognized as the top 10 high-risk global clones (Del Barrio-Tofiño et al., 2020). Therefore, it is essential to implement effective infection control measures to prevent the spread of these high-risk clones.

Our study revealed the resistance mechanisms, molecular epidemiology and virulence genes of XDR-PA strains, as well as the synergistic activity of ATM and CZA combination against XDR-PA. The main limitation is that no in vivo validation was included in our study. In addition, the genes detected are limited compared with whole-genome sequencing. More studies on the mechanisms of synergistic activity between ATM and CZA combination as well as in vivo validation will be performed in subsequent research.

In summary, the underlying resistance mechanisms of XDR-PA involved the overexpression of efflux pump mexA and the existence of MBL. In addition, ST1971 was the predominant ST type in our study, with carrying both NDM-1 and exoY+/exoT+/exoU+/exoS-. Furthermore, combined antimicrobial susceptibility testing suggested that CZA and ATM combination has potential against MBL-producing exoY+/exoT+/exoU+/exoS- XDR-PA. These findings may provide clues for the prevention, control and treatment strategies of XDR-PA infections.