Evolution of XDR Pseudomonas aeruginosa ST463 strains with two plasmids harboring multiple antimicrobial resistance genes

ABSTRACT

The extensively drug-resistant (XDR) Pseudomonas aeruginosa ST463 strains, which co-harbor plasmid-associated metallo-β-lactamase (MBL) and bla_KPC-2 genes, exhibit significant resistance and virulence, posing great clinical treatment challenges. Here, we report on three XDR P. aeruginosa ST463 strains, PA64, PA3117, and PA30, all carrying two plasmid types. One plasmid was a ~450 kb IncP-2-type megaplasmid named pPA64_1, pPA3117_1, and pPA30_1 in strains PA64, PA3117, and PA30, respectively. The other plasmid was a type I plasmid named pPA64_2, pPA3117_2, and pPA30_2 in strains PA64, PA3117, and PA30, respectively, harboring the bla_KPC-2 gene in the core genetic platform ISKpn27- bla_KPC-2-ISKpn6. The bla_KPC-2 gene copies were associated with IS26-mediated inversion or duplication events. Notably, the IncP-2 megaplasmids pPA64_1, pPA3117_1, and pPA30_1 were associated with a variable ~57.3 kb Tn1403-like transposon named Tn6485g, Tn6485h, and Tn6485f, respectively. Tn6485g carried the MBL gene bla_IMP-45, which was located in the class 1 integron In786, followed by an ISCR1-associated armA module and the IS26-composite transposon Tn6309. On this basis, other ISCR1-associated modules (ISCR1-qnrVC6, ISCR1-bla_PER-1, and ISCR1-bla_AFM-1) were inserted between In786 derivatives and ISCR1-armA, resulting in a novel transposon, Tn6485h, carrying two MBL genes, bla_IMP-45 and bla_AFM-1. In contrast to Tn6485h, Tn6485f had another inserted copy of ISCR1-qnrVC6. We inferred that the evolution of the Tn1403-like transposon might be driven by the recruitment of ISCR1-associated antimicrobial resistance (AMR) modules under antibiotic pressure in a clinical setting.

INTRODUCTION

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of hospital-acquired infections. This pathogen is often associated with conditions, such as cystic fibrosis infections, ventilator-associated pneumonia, urinary tract infections, otitis externa, osteoarthritic infections, and bloodstream infections (1). Carbapenem antibiotics are typically the first-line treatment for multidrug-resistant P. aeruginosa infections. However, the rate of carbapenem-resistant P. aeruginosa (CRPA) infections has rapidly increased in recent years due to the excessive use of carbapenem antibiotics (2). A global multicenter study published in The Lancet demonstrated that there were approximately 38,100 deaths worldwide as a result of CRPA infections in 2019 (3). The rapid spread of CRPA has emerged as a significant public health concern, posing a major threat to public safety.

There are several reasons for carbapenem resistance in P. aeruginosa, with the acquisition of the carbapenemase gene being the most significant mechanism (4). Among the carbapenemase genes, metallo-β-lactamase (MBL) genes, in particular bla_IMP and bla_VIM, are the most common type in P. aeruginosa worldwide (5, 6). In 2018, a novel MBL gene, bla_AFM-1, was first discovered in Alcaligenes faecalis in China (7) and subsequently identified in P. aeruginosa strains (8). ST463, initially identified as a predominant clone in east China, has emerged as a new potential high-risk clone due to its high virulence (9). Notably, ST463 is the only clone positive for both the exoU and exoS genes in P. aeruginosa (10). Moreover, the mortality rate associated with bloodstream infections caused by ST463 CRPA is higher than that associated with other clones (11). A significant proportion of ST463 strains carrying the carbapenemase gene bla_KPC-2 is resistant to all β-lactam antibiotics, except ceftazidime/avibactam (12).

In this study, we isolated three strains of ST463 CRPA harboring both the bla_KPC-2 and MBL genes. Of these strains, two carried bla_KPC-2, bla_IMP-45, and bla_AFM-1 (PA30 and PA3117), whereas the remaining strain carried bla_KPC-2 and bla_IMP-45 (PA64). Notably, all carbapenemase genes were located on plasmids. We conducted a thorough analysis of these strains’ genetic characteristics and assessed the plasmids’ evolutionary patterns.

MATERIALS AND METHODS

Bacterial strain and antimicrobial susceptibility testing

Three P. aeruginosa strains were isolated from samples from inpatients at the Affiliated Jinhua Hospital, Zhejiang University School of Medicine in Zhejiang Province, China. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify the strains. The antimicrobial susceptibility of the strains was determined via the broth microdilution method, and the results were interpreted according to the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) of 2024.

Whole-genome sequencing and plasmid analysis

The genomic DNA of the three P. aeruginosa strains was extracted using the PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) and subsequently sequenced on the Illumina HiSeq X10 (San Diego, CA, USA) and Nanopore MinION (Oxford, UK) platforms. The Illumina and Nanopore reads were then hybrid-assembled via Unicycler v0.4.8 (13). The resulting contigs were annotated using RAST (14). Multilocus sequence typing (MLST) was performed using the PubMLST database for P. aeruginosa (https://pubmlst.org/organisms/pseudomonas-aeruginosa). Resistance and virulence genes were identified via ABRicate 1.0.0 (https://github.com/tseemann/abricate). Plasmid sequence alignment was conducted via BLAST Ring Image Generator (BRIG) (15). Transposon Registry (16) and ISfinder (17) were utilized for identifying the transposons and insertion sequences, respectively. The further confirmations of the construction of the transposons were manually refined by BLASTn/BLASTp (18). Equal comparisons of the plasmids and transposons were performed using Easyfig (19).

Conjugation experiments

Conjugation experiments were conducted using PA30, PA3117, and PA64 as the donor strains and a rifampin-resistant mutant of P. aeruginosa PAO1 (PAO1-RifR) as the recipient strain. Transconjugants were selected on Mueller-Hinton (MH) agar media supplemented with 800 µg/mL rifampin and 32 µg/mL ceftazidime. The conjugation experiments for each strain were repeated more than three times. The growing colonies on the selective plates were confirmed via polymerase chain reaction (PCR) amplification.

RESULTS

Clinical and microbiological characteristics

The PA30 strain was isolated from the catheter of a 67-year-old male patient who had undergone a clearance operation for an intracranial hematoma. The patient had a fever of 37.8°C after the operation and recovered after consecutive treatment with cephalosporins, linezolid, and meropenem. It was considered that the isolated PA30 strain was more likely related to colonization than to infection. The PA3117 strain was isolated from the bile drainage fluid of a 62-year-old female patient admitted to the Department of Hepatobiliary and Pancreatic Surgery. The patient was diagnosed with cholangitis after undergoing choledochotomy and T-tube drainage. A combination of vancomycin with levofloxacin and vancomycin with imipenem was applied successively, and the CRPA strain PA3117 was cultured after these treatments. The patient was cured after polymyxin treatment and T-tube removal. The remaining strain, PA64, was isolated from the sputum sample of a 73-year-old male patient who was treated for left pulmonary adenocarcinoma. After left upper lobectomy and mediastinal lymph node dissection, the patient received successive anti-infection treatments with cefazolin, cefoperazone/sulbactam, imipenem, levofloxacin, and vancomycin. The patient was discharged after treatment.

All three strains were resistant to piperacillin, cefepime, ceftazidime, imipenem, meropenem, aztreonam, amikacin, gentamicin, levofloxacin, ciprofloxacin, and ceftazidime-avibactam. However, they were susceptible to colistin. All the strains demonstrated an extensively drug-resistant (XDR) phenotype. Antimicrobial susceptibility testing showed that PA30 and PA64 were susceptible to cefiderocol, while PA3117 demonstrated intermediate susceptibility. This finding provided novel insights for guiding antimicrobial treatment strategies against these three strains.

The PA64, PA3117, and PA30 strains harbored two kinds of plasmids. One plasmid type was identified as an IncP-2-type megaplasmid carrying genes encoding MBLs (pPA64_1, pPA3117_1, and pPA30_1), whereas the other type carried one or two copies of the bla_KPC-2 gene (pPA64_2, pPA3117_2, and pPA30_2) (Table 1). The single-nucleotide polymorphism (SNP) difference between PA30 and PA3117 was 3 (SNP = 3), and that between PA30 and PA64 was 0 (SNP = 0). We constructed a phylogenetic tree based on the SNPs (Fig. S1). The phylogenetic tree clustered primarily into three major clades. PA30, PA64, and PA3117 clustered closely together within the predominant major clade.

TABLE 1.

Clinical characteristics and MICs for Pseudomonas aeruginosa strains^c

Strain	Date	Source	ST	Virulence gene	I/C	ICUa	MICs (μg/mL)													Plasmid characteristics
							PIP	FEP	CAZ	IMP	MEM	AZT	AK	GM	LEV	CIP	CZA	COL	FDC	Plasmid	Size(bp)	Carbapenemase genes
PA30	2021-08-28	Catheter	463	exoU+ /exoS+	C	Yesb	256	1,024	1,024	128	>1,024	64	>1,024	>1,024	128	16	>1,024/4	0.5	4	pPA30_1	453250	blaIMP-45, blaAFM-1
																				pPA30_2	49370	blaKPC-2, blaKPC-2
PA3117	2022-09-04	Bile	463	exoU+ /exoS+	I	Yes	>1,024	>1,024	>1,024	1,024	>1,024	>64	>1,024	>1,024	256	16	>1,024/4	0.5	8	pPA3117_1	449377	blaIMP-45, blaAFM-1
																				pPA3117_2	37887	blaKPC-2
PA64	2022-10-21	Sputum	463	exoU+ /exoS+	C	Yes	>1,024	>1,024	>1,024	1,024	>1,024	>64	>1,024	>1,024	256	16	>1,024/4	0.5	1	pPA64_1	426741	blaIMP-45
																				pPA64_2	37533	blaKPC-2
PA3117-PAO1-RifR	/	This study	/	/	/	/	512	>1,024	>1,024	64	>1,024	>64	>1,024	>1,024	2	1	>1,024/4	0.5	8	/	/	/
PAO1-RifR	/	This study	/	/	/	/	4	4	4	1	0.5	8	<0.5	2	<0.5	0.12	2/4	0.5	0.5	/	/	/
PAO1	/	This study	547	exoU− /exoS+	/	/	4	4	2	1	1	4	<0.5	2	<0.5	0.06	1/4	1	0.5	/	/	/

^a ICU, intensive care unit.

^b Yes, the isolation of strain was before admission to the ICU.

^c I, infection; C, colonization;PIP, piperacillin; FEP, cefepime; CAZ, ceftazidime; IMP, imipenem; MEM, meropenem; AZT, aztreonam; AK, amikacin; GM, gentamicin; LEV, levofloxacin; CIP, ciprofloxacin; CZA, ceftazidime-avibactam; COL, colistin; FDC, cefiderocol./:not applicable.

We performed conjugation assays to examine the transferability of the megaplasmids. A transconjugant was obtained from the PA3117-PAO1-RifR conjugation experiment with a very low conjugation frequency (2.78 × 10⁻¹⁴). We failed to obtain transconjugants from PA64-PAO1-RifR and PA30-PAO1-RifR in more than three independent conjugation experiments. To further characterize the transconjugant, we conducted antibiotic susceptibility assays of the transconjugant PA3117-PAO1-RifR (Table 1). The results revealed that the transconjugant PA3117-PAO1-RifR maintained resistance profiles comparable to the donor strain PA3117 across all tested antibiotic classes, except for quinolones, to which it showed susceptibility.

Genetic features of the plasmids

The length of the base pairs of megaplasmids pPA64_1, pPA3117_1, and pPA30_1 was greater than 450 kb, and the plasmid backbones were highly similar, with over 95% query coverage and 99% nucleotide similarity. All three megaplasmids encoded identical RepA replication and ParAB partition proteins with 100% amino acid sequence identities. The genetic elements of the backbones were conserved and included the conjugative module traGBV, which is responsible for horizontal plasmid transmission. The tellurite resistance operon terABCDEZ and the chemotaxis operon cheBARZWY, the intrinsic structures of IncP-2 megaplasmids, were also identified (Fig. 1).

Fig 1.

Genetic comparison of three IncP-2 megaplasmids. The sequence of pPA30_1 is taken as the reference. The innermost circle indicates the scale, and the second and third circles illustrate the GC content and skew, respectively. The colored circles from the inner to the outer represent each plasmid, as shown in the right column. The solid regions demonstrate a sequence similar to that of the reference, whereas the gaps represent regions lacking sequence similarity. The annotations on the outermost circle indicate the locations of the main features of pPA30_1.

The complete plasmid sequences of pPA64_2 and pPA3117_2 were 37,533 and 37,887 bp long, respectively. These two plasmids shared identical backbones and carried a single copy of bla_KPC-2 on the ISKpn27-ΔISKpn6 gene platform flanked by two IS26 insertion elements. The IS26-associated module was duplicated and inverted in pPA30_2, forming two symmetric IS26-bla_KPC-2-IS26 units and yielding two copies of the bla_KPC-2 genes and adjacent IS26 mosaic structures (Fig. 2).

Fig 2.

Alignment of the genetic context of pPA64_2, pPA3117_2, and pPA30_2. The shaded regions denote nucleotide identity (100%). The antibiotic resistance genes bla_KPC-2 are denoted by red arrows. The IS elements IS26, ISKpn27, and ΔISKpn6 are denoted by yellow, blue, and orange arrows, respectively.

Tn1403-like transposon evolution in IncP-2 megaplasmids

The primary distinguishing regions among pPA64_1, pPA3117_1, and pPA30_1 were recognized as the Tn1403-like transposon regions carrying the antimicrobial resistance (AMR) genes encoding MBLs. A Tn1403-like transposon possessed tnpA and tnpR, demonstrating 99% amino acid sequence identity to those of Tn1403 (20). The transposon Tn1403 was an important vehicle for resistance gene dissemination, which featured a backbone structure organized as tnpAR-res site-sup-uspA-dksA-yjiK (7). Tn6485-like transposons, the derivatives of Tn1403, inserted bla_IMP-45-bearing In786 into the res site and retained the ancestral recombination module (tnpAR-res site) critical for transposition but lost most of the downstream Tn1403 backbone elements, resulting in a truncated architecture specialized for disseminating the acquired antibiotic resistance genes (21). The megaplasmid pPA64_1 carried the MBL gene bla_IMP-45 located in a novel Tn6485-like transposon named Tn6485g, which is closely related to Tn6485b from the plasmid pR31014-IMP (GenBank accession no. MF344571.1). The genetic architecture of the bla_IMP-45 gene was identical in both Tn6485-like transposons, with an arrangement of antimicrobial resistance cassettes aacA4-bla_IMP-45-gcu3-bla_OXA-1-catB3 in a class 1 integron In786. The 3′ conserved segment (3′CS) of the integron included the qacEΔ1 and sul1 genes, followed by an identical array of genes, including the composition of the ISCR1-armA module, except for the ISPpu29 insertion element, which was absent in the IS26-composite transposon [IS26-tetAR(C)-IS26] in Tn6485g (Fig. 2).

Compared to Tn6485g in pPA64_1, pPA3117_1 carried another novel Tn6485-like transposon named Tn6485h, which was very similar to Tn6485e in pHS17-127 (GenBank accession no. CP061377.1) and acquired an additional MBL gene, bla_AFM-1, and the bla_PER-1 and qnrVC6 genes via the insertion of ISCR modules. The genetic environment of bla_AFM-1 in pPA3117_1 was bracketed by two ISCR27-like elements (ISCR27n3 and ΔISCR27n1) in a conserved region of groEL/ΔgroEL-ΔfloR-bla_AFM-1-ble-ΔtrpF-ΔISCR27n2-ΔISPme1-msrB2-msrA-yghU-corA. The AMR genes bla_PER-1 and qnrVC6 are related to the ISCR1-qnrVC6 and the ISCR1-bla_PER-1 modules located between In786 and the ISCR27n3-bla_AFM-1 module. In PA30_1, the transposon Tn6485f also harbored two MBL genes, bla_IMP-45 and bla_AFM-1, and one more ISCR1-qnrVC6 module was identified upstream of the ISCR1-bla_PER-1 module that was not present in Tn6485h (Fig. 3).

Fig 3.

Alignment of the genetic context of Tn1403-like transposons from pPA64_1, pPA3117_1, and pPA30_1. The shaded regions denote nucleotide identity (95–100%). Red arrows denote the genes bla_IMP-45 and bla_AFM-1.

DISCUSSION

ST463 has recently emerged as a novel potential high-risk clone in China because of its notable virulence and resistance traits. Previous studies have shown that 40.4% of CRPA carrying bla_KPC-2 in China and ST463 represent the predominant clone in bla_KPC-2-positive CRPA populations (12). The bla_KPC-2 gene in the ST463 strains is carried mainly by type I plasmids. The core genetic platform harboring bla_KPC-2 is ISKpn27- bla_KPC-2 -ISKpn6, and the adjacent region varies by IS26-mediated inversion or duplication events, which amplify bla_KPC-2 gene copies and facilitate the transmission of bla_KPC-2 in ST463 strains (22).

Outbreaks of CRPA producing IMP-45 occurred in Shanghai, China in 2015, with the study identifying bla_IMP-45 in 9.83% of CRPA strains (23). CRPA strains co-producing IMP-45 and AFM-1 have been documented in China (7). In these strains, both bla_IMP-45 and bla_AFM-1 genes reside on the same IncP-2-type plasmid within P. aeruginosa, and this co-harboring plasmid has been confirmed to be conjugative, facilitating horizontal gene transfer. Additionally, CRPA strains co-producing KPC-2 and AFM-1 (bla_KPC-2 and bla_AFM-1) have also been detected in China (24). Unlike the IMP-45/AFM-1 co-producers, in these isolates, bla_KPC-2 was plasmid-borne, whereas bla_AFM-1was chromosomally integrated. Epidemiological surveillance data highlighted the clinical significance: among 192 CRPA strains analyzed, eight (4.17%) co-harbored bla_KPC-2 and bla_AFM-1, and infections caused by these strains resulted in five patient fatalities (25).

In this study, we identified an ST463 P. aeruginosa strain (PA64) harboring both bla_KPC-2 and bla_IMP-45, the latter of which was carried by an IncP-2-type megaplasmid that is highly related to the spread of MBL genes in P. aeruginosa (23, 26). The IncP-2 megaplasmids share a common core genetic backbone that includes genes involved in replication, segregation, and conjugation, improving the efficiency of vertical and horizontal transmission of megaplasmids (27). These beneficial traits of megaplasmids provide adaptive advantages for acquiring and disseminating resistance genes. The extensively variable regions are associated with multiple cassette-borne AMR genes, such as bla_VIM-2 genes in In461 and bla_IMP-45 genes in the variable region of In786, encoding MBLs disseminated in nosocomial P. aeruginosa isolates (23, 26). The bla_IMP-45 gene in PA64 was also carried in In786 located in an ~57.3 kb Tn1403-like transposon newly named as Tn6485g. Tn1403 was recovered from a multiple antibiotic-resistant clinical strain of P. aeruginosa and acted as a vehicle for a series of resistance modules associated with MGEs (20). The Tn1403 backbone contains the tnpA and tnpR genes, which encode the TnpA transposase, TnpR resolvase, and most of the res site. The bla_IMP-45-bearing In786 derivatives were embedded within the res site but lost most of the Tn1403 backbone structure, followed by an ISCR1-associated armA module and an IS26-composite transposon Tn6309 (7).

On this basis, another novel Tn1403-like transposon, Tn6485h, which was inserted by various ISCR1-associated modules (ISCR1-qnrVC6, ISCR1-bla_PER-1, and ISCR1-bla_AFM-1), was identified from the IncP-2 megaplasmid of the ST463 P. aeruginosa strain PA3117. Additionally, one more copy of ISCR1-qnrVC6 was inserted, resulting in Tn6485f of pPA30_1. This finding suggests that the Tn1403-like transposons are highly variable in terms of the recruitment of ISCR1-associated modules carrying multiple AMR genes, which are captured by the IncP-2 megaplasmid and exhibit multiple drug resistance (MDR) properties in the host. The evolution of these MDR transposons may be driven by the pressure of antibiotic therapy in a specific clinical setting since all three patients with infections with novel Tn1403-like transposons in our study were admitted to the same ICU ward. The small SNP differences between these three isolates indicated that they were genetically indistinguishable at the core genome level and likely represented a recent clonal transmission event within the healthcare setting. This finding provided strong molecular evidence for nosocomial cross-transmission and underscored the need for targeted infection control interventions. However, the details of the evolutionary path are lacking, and the realities are likely more complex than anticipated.

In conclusion, we report on three clinical XDR P. aeruginosa ST463 strains, all containing two plasmids. One plasmid is an IncP-2 megaplasmid containing a variable Tn1403-like transposon that might be modified by recruiting ISCR1-associated AMR modules under antibiotic pressure in a clinical setting. The other plasmid, which is common in ST463 strains, harbors the bla_KPC-2 gene in the core genetic platform ISKpn27- bla_KPC-2- ISKpn6, and the bla_KPC-2 gene copies are associated with IS26-mediated inversion or duplication events. These transferable IncP-2 megaplasmids, which harbor XDR transposons and coexist with plasmids carrying bla_KPC-2 genes in P. aeruginosa ST463 strains, pose a substantial challenge to clinical treatment.

DATA AVAILABILITY

The data used in our research are available for access. The complete sequences of the chromosomes and plasmids of PA64 and PA3117 have been submitted to GenBank under the BioProject accession numbers PRJNA1123618 and PRJNA224116, respectively. The BioProject accession number of PA30 is PRJNA866310.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aac.01697-24.

Fig. S1. aac.01697-24-s0001.docx.

Phylogenetic tree of the ST463 strains isolated in China from the NCBI Pathogen Database and the three strains (PA30, PA64, and PA3117) from this study.

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