Collection of XDR-PA strains and screening of XDR-hvPA

A total of 77 non-repetitive P. aeruginosa strains were isolated from various types of clinical specimens taken from 77 patients; five of these patients were emergency patients, and the remainder were hospitalized patients. In terms of the distribution of patients in departments, most of the patients were in the intensive care unit (ICU, 40.3%, 31/77), followed by integrated Chinese and Western medicine departments (ICWM, 22.0%, 17/77), rehabilitation departments [11.7% (9/77)], emergency departments [6.5% (5/77)], respiratory departments [3.9% (3/77)], and other departments [15.6% (12/77)]. Among the sample sources, 85.7% (66/77) were respiratory specimens, followed by urine specimens [5.2% (4/77)], fecal specimens [3.9% (3/77)], wound secretion specimens [3.9% (3/77)], and tissue specimen [1.3% (1/77)].

According to the results of antimicrobial susceptibility tests, all 77 strains were susceptible only to polymyxin B, indicating XDR-PA. Notably, the drug sensitivity results of 14 ST235 strains carrying the blaGES gene to CZA showed that the MIC value was 2/4∼8/4 mg/L, and five strains (P54, P62, P68, P71, and P88) showed the MIC value of CZA at 8/4 mg/L, which was close to the resistance cutoff point (MIC break point for sensitivity: ≤8/4 mg/L).

In our study, the G. mellonella infection model and the important virulence-related genes (exoU and exoS) were used to evaluate the virulence of all collected strains, and the results suggested that 47 strains were XDR-hvPA. (Fig. 1).

Fig. 1

The virulence of 77 XDR-PA isolates. The virulence characterization in the G. mellonella infection model. PBS was used as the negative group. After infection with XDR-PA isolates carrying exoE/exoS virulence-related genes, the mortality rate of the G. mellonella was significantly higher

Clinical characterizations of XDR-hvPA and XDR-non-hvPA

To clarify the clinical characteristics of XDR-hvPA and XDR-non-hvPA, a comparative analysis was conducted on these indicators, including demographic information, ICU admission, underlying diseases, invasive procedures, and patients’ history of antibiotic exposure. Patients were divided into three age groups: ≤18 years, 18–59 years, and ≥ 60 years. In patients aged ≥ 60 years, the detection rate of XDR-hvPA was significantly higher than that of XDR-non-hvPA (72.3% vs. 40.0%, P = 0.005); however, the rate did not differ significantly in the other two age groups. There was no significant difference in sex, length of hospitalization < 30 days, ICU admission, underlying diseases, invasive procedures, and previous antibiotic exposure. It was suggested that elderly patients were more likely to contract XDR-hvPA infection (Table 1).

Table 1 Clinical characteristics of infection patients caused by XDR-hvPA and XDR-non-hvPAResistance and virulence genes of XDR-hvPA strains

Among 47 XDR-hvPA strains, 51.1% (24/47) strains carried the carbapenemase gene, suggesting that carbapenem-producing played a major role in drug resistance of P. aeruginosa. Six types of carbapenem-resistant genes were detected, namely blaGES-1, blaVIM-2, blaGES-14, blaIMP-45, blaKPC-2, and blaNDM-14, with positivity rates of 21.3% (10/47), 12.8% (6/47), 8.5% (4/47), 4.3% (2/47), 2.1% (1/47), and 2.1% (1/47), respectively. Notably, some resistance genes are rare types of genes in XDR-hvPA, such as blaIMP-45, blaKPC-2, and blaNDM-14. (Fig. 2). In addition, these strains also co-carried other drug resistance-related genes, as shown in Figure S1.

Fig. 2

Molecular characteristics of 47 XDR-hvPA strains. PAO1 (GenBank accession: NC_002516.2) was used as the reference strain

The detection rates of the virulence-associated genes exoT, exoY, exoU, and exoS, which are the important effectors of the type III secretion system of P. aeruginosa, were 100.0% (47/47), 97.9% (46/47), 66.0% (31/47), and 38.3% (18/47), respectively. Interestingly, two strains (P59 and P69) co-carried exoU and exoS simultaneously (Fig. 2). Genes encoding flagella, type IV pili and non-pilus adhesins, and extracellular virulence factors were also detected (Figure S2).

Homology analysis of XDR-hvPA

Notably, 47 XDR-hvPA strains belonged to 14 ST types, with ST235 (n = 14, 29.8%) being the most prevalent, followed by ST1158 (n = 13, 27.7%) and ST1800 (n = 7, 14.9%). All strains of ST235 carried the blaGES gene; ST1158 did not carry the carbapenemase gene, and three strains of ST1800 carried the blaVIM-2 gene. The strain carrying the blaNDM-14 gene belonged to ST4; the strain carrying blaIMP-45 belonged to ST2068 and ST3755, and the strain carrying the blaKPC-2 gene belonged to ST463. The 47 XDR-hvPA strains were divided into 17 clusters. Interestingly, the isolate carrying the blaGES gene (ST235) was identified to belong to the same cluster with the difference in SNP from 2 to 51 (mainly from 2021 to 2022), indicating the existence of clonal transmission (Fig. 2).

We downloaded the genetic data of global epidemic ST235 P. aeruginosa from the Pseudomonas genome database (https://www.pseudomonas.com/) and analyzed the evolutionary relationship between ST235 P. aeruginosa globally and XDR-hvPA strains in this study. Thus far, 206 strains of ST235 P. aeruginosa have been uploaded to the system globally. In addition, we conducted homology analysis on these 206 strains of ST235 P. aeruginosa, 15 strains of ST235 P. aeruginosa from Hangzhou, Zhejiang, which were previously reported by Li et al. [14], and 14 strains of XDR-hvPA reported in this study. The results showed that these 235 strains could be divided into 11 clusters, with clusters A, B, C, and D being the most prevalent, accounting for 98.3% of all strains, indicating clonal transmission. Fourteen XDR-hvPA strains were classified into A clone cluster. These had the closest similarity to JAPZLY01, which were obtained from Hangzhou, Zhejiang, in a previous report by Li et al. [14], with an SNP difference of 1–5 (except for P51 and P87), indicating that ST235 P. aeruginosa may have clonal transmission in China and should be highly valued. (Fig. 3).

Fig. 3

The phylogenetic analyses of ST235 PA from the global region. The first circle represents the cluster; the second circle shows the countries or regions where the strains were distributed; the third circle indicates the O serotype. P54 in our study was used as the reference strain

Evolution relationship of serotype in the past six years

This study analyzed the change of O serotype during the evolution of the strain. A total of six O serotypes of 47 XDR-hvPA strains were detected, namely O1, O4, O5, O6, O7, and O11. Among them, O11 (40.4%, 19/47), O7 (27.6%, 13/47), and 04 (19.1%, 9/47) accounted for the highest proportion. From 2018 to 2020, O4 and O7 were the prevalent serotypes, whereas in the last three years, O11 was the most prevalent serotype, accounting for 61.5% of all serotypes (16/26). (Fig. 2). Notably, 235 strains of ST235 P. aeruginosa isolated globally were O11, except one strain (SAMN12127367) was O10. (Fig. 3).