Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen and one of the major causes of nosocomial infections, particularly affecting intensive care units (ICU) and immunocompromised patients, with a very high morbidity and mortality rate (Botelho et al., 2019; Thornton and Parkins, 2023). In addition, P. aeruginosa is the most frequent chronic respiratory infection contributing to the pathogenesis of cystic fibrosis (CF) and other chronic underlying diseases (Kawalek et al., 2020). The innate and acquired antibiotics resistance mechanism of P. aeruginosa includes reduced permeability, antibiotic efflux, expression change, antibiotic modification/degradation, target protection and target modification (including the acquisition of insensitive functional target) (Pang et al., 2019).

Infections caused by multidrug-resistant (MDR) or extensively drug-resistant (XDR) P. aeruginosa are extremely difficult to treat (Kawalek et al., 2020). In Asia, the MDR and XDR rates of P. aeruginosa nosocomial pneumonia were reported to 42.8 and 4.9%, respectively (Pang et al., 2019). Over the last few decades, the occurrence of epidemic outbreaks caused by antibiotic resistance strains within hospital environments has been given more attention (Talebi Bezmin Abadi et al., 2019). Recent works have provided further evidence that a small group of MDR/XDR global clones are disseminated in many hospitals worldwide (Telling et al., 2018; Hu et al., 2021; Gravningen et al., 2022). The most prevalent of these globally disseminated clones include ST235, ST111, ST233, ST244, ST357, ST308, ST175, ST277, ST654 and ST298, and they have been denominated high-risk clones (Oliver et al., 2015; del Barrio-Tofiño et al., 2020). Furthermore, some types of P. aeruginosa strains have developed resistance to carbapenems, called Carbapenem-Resistant P. aeruginosa (CRPA). The production of carbapenemases, especially Ambler class B Metallo-β-lactamases (MBLs), is an important mechanism of carbapenem resistance in P. aeruginosa (Yoon and Jeong, 2021). Moreover, in the absence of Carbapenemase-mediated resistance, the loss of outer membrane porin OprD, together with the overexpression of efflux pumps or ampC have been shown to be the main mechanisms leading to carbapenem resistance (Botelho et al., 2019). In China, only a few reports are available on the molecular epidemiology and genome information of P. aeruginosa high-risk clones. At the same time, research on Carbapenem-Resistant P. aeruginosa (CRPA) together with high-risk clones is even more limited.

Guangdong is the most populous province with the highest domestic migration in China (Xu et al., 2022). This study aims to characterize clinical P. aeruginosa strains isolated from two hospitals in Guangdong, in terms of clone diversity, antibiotics resistance profiles, and virulence determinants. Furthermore, the relationship between the carbapenem genes and P. aeruginosa high-risk clones will be explored. The outcome of this study will fill the gap related to cultures of P. aeruginosa and their associated genome sequences in China. The genetic and phenotypic characteristics of P. aeruginosa high-risk clones would provide novel insights into the epidemic and genomic mechanism background of this pathogen in China.

The emergence and increase of high-risk isolates, including their antibiotic resistance, has been recognized as a threat for human health (Oliver et al., 2015; del Barrio-Tofiño et al., 2020). However, our understanding of these clones’ global distribution and evolution is still incomplete due to uneven sampling in different regions of the world. Here, we investigated clinical P. aeruginosa clones isolated in Guangdong, China. The MLST analysis showed high genetic diversity among collected isolates in agreement with prior studies in China (Fan et al., 2016). Importantly, some international high-risk clones as well as new widely distributed clones (regarded as potential risk clones), were identified in our study. The observations of potential high-risk and locally distributed clones underscore the relevance of continued inspection of the population structure in clinical settings in different parts of the world.

Although previous studies had showed that high-risk clones were predominant among multidrug-resistance strains (del Barrio-Tofiño et al., 2020; Hu et al., 2021; Teo et al., 2021; Aguilar-Rodea et al., 2022), we found that 50/212 isolates (23.58%) belong to the 18 previously reported high-risk clones, and that the remaining isolates showed high diversity together with other reported STs in all strains.

Among the high-risk clones found in our study, we observed that ST111 included two MDR/non-MDR strains from different hospitals. ST235 is the most widely spread P. aeruginosa high-risk clone associated MDR/XDR isolates and carries various resistance genes (Treepong et al., 2018; Tahmasebi et al., 2022). Three ST235 MDR strains were isolated from the neurology ward, of which two were carrying the bla_VIM-2 gene. The most prevalent high-risk clone in all isolated strains was ST244 (n = 9); six multidrug resistance strains were collected from the ICU, of which one harbored the bla_VIM-2 gene. ST277 was reported to be highly prevalent in Brazil, and commonly associated with MBL SPM-1 (Galetti et al., 2019; Silveira et al., 2020). We found ST277 isolates composed of 4 MDR/XDR strains, but the specific MBLs genes were not identified. ST357 with bla_IMP and bla_VIM type MBLs was previously reported in Turkey (Çekin et al., 2021) and Czech (Papagiannitsis et al., 2017), three ST357 stains identified in our study showed MDR/XDR phenotype, and one of them harbored bla_CARB-3 gene. ST298 is the subclade of the CC446 clonal complex (Pincus et al., 2019). One ST298 isolate and one ST446 isolate, both with MDR/XDR phenotype, were identified in different hospitals. In some regional research projects, the clonal diversity of P. aeruginosa is much lower among multidrug-resistant isolates (del Barrio-Tofiño et al., 2020; Hu et al., 2021; Teo et al., 2021; Aguilar-Rodea et al., 2022). This is not entirely the case among our MDR subsets from 2018–2020. We have observed 84 (39.62%) different STs in the 130 MDR/XDR strains. However, only 12.26% (n = 26) of MDR/XDR isolates are actually related to the reported international high-risk clones.

We note that our study is focused on the Guangdong Province. This region is the most populous province with the largest population inflow in China. We speculate that trade and mobility in this region have contributed to the high level of diversity observed among the isolated strains. Overall, our survey of regional P. aeruginosa high-risk clones in China may assist in a better understanding of the evolution of these important clone types.

Besides the high-risk clones, eight founder STs of clonal groups/subgroups have been identified (Figure 2), and these founder STs have been reported to be distributed in at least three different countries (Table 2). Furthermore, one special disseminated and multidrug-resistant clone, ST1971 (n = 7, 3.30%), has only been identified in China (Wang et al., 2017). This clone can be defined as a regional epidemic risk type for nosocomial healthcare. Some of the STs identified in this study have been previously described as new potential high-risk clones, including ST108 (Telling et al., 2018), ST179 (Moloney et al., 2020), ST253 (Fischer et al., 2020), ST260 (Martak et al., 2022), ST274 (Sewunet et al., 2022), ST348 (Dix et al., 2022), ST395 (Petitjean et al., 2017), ST446 (Pincus et al., 2019), ST463 (Hu et al., 2021), ST532 (Founou et al., 2020), ST699 (Rada et al., 2021) and ST773 (Singh et al., 2021).

The antibiotic profiles of all isolated P. aeruginosa strains were quantified by MIC assays. 196 strains (92.45%) were found to be CRPA, and most of them represented resistance to multiple antibiotics (126/196, 64.29%). It was previously reported that meropenem and imipenem resistance rates for P. aeruginosa range from 21.6 to 25.6% in China (Wang and Wang, 2020), and the detection rate of CRPA in Guangdong was 41.9, 17.9 and 19.2% from 2018 to 2020, respectively. The CRPA rates determined in this study were higher than the previous studies. Previous studies reported that the CRPA isolating rate of inpatients and those admitted to an ICU is higher than outpatients and those in non-ICU wards (Gill, Nicolau, and on behalf of the ERACE-PA Global Study Group, 2022). Here, 35.85% of strains were isolated from ICU patients, which might explain the high prevalence of CRPA in the collection. In future studies, collecting strains from more diverse wards and improving the randomness of the evaluation would be beneficial.

In contrast to the high rate of carbapenem resistance, the MBLs genes carrying strains were with very low proportion (n = 6, 2.83%) in all collected isolates. Only 3 bla_IMP harbored strains and 3 bla_VIM-2 harbored strains were found. The oprD gene has been reported as the main determinant of imipenem resistance (Quinn et al., 2022). The MIC data show that the imipenem resistance rate is 87.26%. The wild-type oprD gene was not found in any newly sequenced strains, which implied that the mutational inactivation of the oprD gene may play a role in carbapenem resistance. However, the relationship between the mutations in the oprD gene and its function cannot be fully understood only by whole genome sequencing, and future investigations should focus on mechanistic studies of these mutations.

Some variants of PDC enzymes (class C β-lactamases) are responsible for the resistance to imipenem and newer β-lactam/β-lactamase inhibitor combinations such as ceftolozane-tazobactam (Papp-Wallace et al., 2020). Furthermore, the production of class D β-lactamases (oxacillinase; OXA) can lead to third-generation cephalosporins or multiple-drug resistance (Vrancianu et al., 2020). In this study, PDC β-lactamase and OXA β-lactamase genes were prevalent in collected strains, which can induce multidrug resistance to some extent by antibiotic inactivation. Previous studies showed that the presence of qnrVC genes plays important roles in quinolone resistance (Liu et al., 2018; Saki et al., 2022), and 2.3% of collected P. aeruginosa isolates from Guangdong carried qnrVC genes (Lin et al., 2020), which is consistent with this study.

The type III secretion system is an important virulence factor of P. aeruginosa, contributing to microbial cytotoxicity and invasion (Horna and Ruiz, 2021). The exoU and exoS genes encode type III secretion virulence effector proteins. Previous studies have shown the mutual exclusion of the exoU and exoS genes (Bradbury et al., 2010; Juan et al., 2017). Through the VFDB analysis, we found the co-presence of both genes in 5 clone types strains (ST273, ST274, ST463, and ST3658). The exoS⁺/exoU⁺ strains have no significant differences in the resistance phenotype, but the strains carrying two virulence genes were all classified into XDR (CRPA).

Pseudomonas aeruginosa isolates from hospitals in this study exhibited multidrug resistance to various drugs and a high rate of resistance against carbapenem. MLST analysis further revealed the genetic diversity of these clinical isolates of P. aeruginosa. The stLFR technology was first applied for the bacteria antibiotic gene profiling, and the findings indicate that the sequence clone types are independent of carbapenem resistance or MDR/XDR phenotype. The strains harboring bla_OXA and bla_PDC genes mainly circulate in the hospital environments studied. The spectrum of high-risk clones is also different from previous reports and has obvious regional characteristics. This means that the control measures for the circulation and success of high-risk clones and CRPA must have a unified global standard and custom local strategies.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number (s) can be found in the article/Supplementary material.

The study was approved by the Ethics Committee (full name: The Ethics Committee of the Zhujiang Hospital of Southern Medical University), the reference number 2021-KY-046-01. The samples were obtained with written informed consent and reviewed by the ethical board in accordance with the tenets of the Declaration of Helsinki.

YZ designed the experimental plan, analyzed WGS data, and finished the draft manuscript writing. DC collected strains and participated in project design. BJ prepared MLST distribution and digital chart. XZ contributed to manuscript revision. MA participated in project design. LJ contributed to conceptualization, project administration, and manuscript revision. All authors participated in the manuscript critical review and editing, and agreed with the above contribution details.

This work was supported by a research grant to LJ from the Independent Research Fund Denmark (9039-00350A).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.