Chromosomal integration and plasmid fusion occurring in ST20 carbapenem-resistant Klebsiella pneumoniae isolates coharboring bla_NDM-1 and bla_IMP-4 induce resistance transmission and fitness variation

ABSTRACT

To investigate the epidemiology of ST20 carbapenem-resistant Klebsiella pneumoniae (CRKP) in China, and further explore the genomic characteristics of bla_IMP-4 and bla_NDM-1 coharboring isolates and plasmid contributions to resistance and fitness. Seven ST20 CRKP isolates were collected nationwide, and antimicrobial susceptibility testing was performed. Antimicrobial resistance genes, virulence genes, and plasmid replicons were identified via whole-genome sequencing, and clonality assessed via core-genome multilocus sequence typing. Furthermore, we found four dual-metallo-β-lactamases (MBL)-harbouring isolates, the gene location was detected by Southern blotting, and plasmid location analysis showed that bla_IMP-4 was located on a separate plasmid, a self-conjugative fusion plasmid, or the bacterial chromosome. These isolates were subjected to long-read sequencing, the presence of bla_IMP-4 in different locations was identified by genomic comparison, and transposon units were detected via inverse PCR. We subsequently found that bla_IMP-4 on the fusion plasmid and bacterial chromosome was formed via intact plasmid recombination by the IS26 and ltrA, respectively, and the circular transposon unit was related to cointegration, however, bla_IMP-4 in different locations did not affect the gene stability. The bla_NDM-1-harbouring plasmid contributed to the increased resistance to β-lactams and shortened survival lag time which was revealed in plasmid cured isolates. In summary, the K. pneumoniae ST20 clone is a high-risk resistant clone. With the use of ceftazidime/avibactam, MBL-positive isolates, especially dual-MBL-harbouring isolates, should be given additional attention.

KEYWORDS:

• Carbapenem-resistant Klebsiella pneumoniae
• ST20
• NDM
• chromosomal integrated IMP
• plasmid recombination

Introduction

Klebsiella pneumoniae is a nosocomial opportunistic pathogen that induces abdominal infections, bloodstream infections, and urinary tract infections, resulting in severe mortality [1]. Carbapenem is commonly used for treating Gram-negative bacterial infections; however, with the abuse of this kind of antibiotic, the prevalence of carbapenem-resistant K. pneumoniae (CRKP) is increasing. According to national surveillance data, the prevalence of CRKP in China is more than 25% [2], and the rapid increase in CRKP threatens public health, as it can lead to longer hospitalization stays, increased frequency of hospital-acquired infections, and higher medical costs [3].

The main mechanism of carbapenem resistance is the production of carbapenemase, which include class A, B, and D enzymes. Class A and D enzymes have a serine-based hydrolytic mechanism, and class B carbapenemases use one or two zinc ions in their active sites to catalyze the hydrolysis of β-lactam antibiotics. Thus, class B carbapenemases are called metallo-β-lactamases (MBLs). The most prevalent carbapenemase in CRKP in China is Klebsiella pneumoniae carbapenemase (KPC), followed by MBL, accounting for approximately 10% [4]. Inhibitors of class A, class C, and some class D enzymes, such as avibactam, have been approved; however, the development of MBL inhibitors is still an ongoing challenge, which had led to the lack of effective MBL-mediated CRKP treatments.

In addition to the predominant CRKP clone ST11 in China, several antimicrobial-resistant high-risk K. pneumoniae clones have emerged in recent years, which can evolve into dominant clones and cause local outbreaks. For instance, ST20 was reported as a multidrug resistant (MDR) clone, which has been identified in several countries including South Korea, Spain, Canada, New Zealand, Greece, Brazil, and China [5], and has been reported causing strain transmission in neonatal units within Greece and China [6,7]. Pei et al. indicated that this clone showed an apparent horizontal gene transfer (HGT) competency [8], and it caused sporadic outbreaks associated with pediatric infections in Wuhan and Shandong, China, by acquisition of bla_NDM-1 [9].

To investigate the epidemiology of ST20 CRKP in China, and further explore the genomic characteristics of isolates, we collected seven ST20 isolates across the country. Among these, four isolates coharbored bla_IMP-4 and bla_NDM-1, and we explored the location of carbapenem resistance gene, the transferability, and the plasmids cointegration mode. Furthermore, we evaluated the resistance and fitness contributions of the different plasmids.

Materials and methods

Collection of ST20 isolates, antimicrobial susceptibility testing (AST), and extended-spectrum beta-lactamases (ESBL) test

Seven ST20 isolates were collected in a national observational, multicenter CRKP epidemiological study in China from June 2018 to June 2019, which was approved by the local ethics committee of Sir Run Run Shaw Hospital (20180528-18). This epidemiological study collected 708 CRKP isolates from 28 hospitals throughout China in total [4].

The AST of these isolates was determined by agar dilution (for fosfomycin) or microbroth dilution (for meropenem, imipenem, ertapenem, cefoxitin, ceftazidime, cefepime, piperacillin/tazobactam, cefoperazone/sulbactam, ceftazidime/avibactam, aztreonam, amikacin, ciprofloxacin, levofloxacin, tigecycline, colistin, and cefiderocol) as previously described [10]. Escherichia coli ATCC 25922 was used as a quality control. The breakpoint of tigecycline was interpreted by the FDA standards, and the breakpoint of the remaining antimicrobial agents was interpreted by the Clinical and Laboratory Standards Institute (CLSI) guidelines (M100, 32nd edition) [11].

Isolates were characterized phenotypically for ESBL production using disk diffusion test as recommended by CLSI [11]. The test was done using both ceftazidime (30 μg) and cefotaxime (30 μg) alone and in combination with clavulanate (10 μg). A ≥ 5mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanate versus its zone diameter when tested alone was taken as positive result for ESBL production.

Genomic characteristics analysis

The antimicrobial resistance genes (ARGs) were determined by ABRicate (https://github.com/tseemann/abricate), based on the National Centre for Biotechnology Information (NCBI) AMRFinderPlus database [12]. Virulence associated genes were determined by Kleborate [13].

Clonality analysis by core-genome multilocus sequence typing (cgMLST)

The cgMLST of these isolates was performed by SeqSphere + 6.0.0 (Ridom GmbH, Münster, Germany) using the standard set of core genes for K. pneumoniae [14]. Then, the neighbor-joining (N-J) tree was exported and illustrated by iTol (https://itol.embl.de).

S1 pulsed-field gel electrophoresis (S1-PFGE) and southern blotting

S1-PFGE was performed using the contour-clamped homogeneous electric field (CHEF) technique as previously described [10]. Briefly, DNA was digested by S1 nuclease and electrophoresis was performed at 14°C and 6 V/cm with a 2.16–63.8 s pulse time gradient and an alternating pulse at 120° for 20 h. Southern blotting was performed with digoxigenin-labeled bla_NDM, bla_IMP, and bla_KPC probes (Roche Diagnostics, Basel, Switzerland), and the probe primers are listed in Table S1.

Long-read sequencing and plasmid comparison

The representative isolates CHN03027, CHN13011 and CHN13105 were selected for long-read sequencing by a MinION sequencer device (Nanopore Technologies, Oxford, UK) with a 1D flow cell as previously described [15]. Then, the long reads were assembled with Raven (https://github.com/lbcb-sci/raven) and further polished using the Illumina short reads by Polypolish [16].

The replicon types of the plasmids were detected by PlasmidFinder [17], and plasmid annotation was performed by Prokka [18]. Genomic comparison was subsequently performed by Easyfig (v2.2.5). Furthermore, the insert sequence (IS) and conjugal transfer capability were analyzed by ISfinder (https://isfinder.biotoul.fr) and oriTfinder [19].

Gene cloning and transposon unit (TU) detection

The bla_SHV genes were cloned and inserted into pCR2.1K, which was derived from pCR2.1 by knocking out the ampicillin resistance gene, as previously described [10]. Briefly, the identical predictive promoter along with bla_SHV-145 or bla_SHV-145-like was introduced into pCR2.1K between the HindIII and XhoI restriction enzyme sites.

Transposon unit detection was performed using the genomic DNA of CHN13011 and CHN13105 as templates, and it was detected by inverse PCR using paired outward-facing primers. The primers used here are listed in Table S1.

Conjugation experiment

E. coli EC600 served as the recipient, and K. pneumoniae strains (CHN03027, CHN13011, and CHN13105) served as the donors. Rifampin (500 mg/L) and ertapenem (1 mg/L) were simultaneously used for transconjugant selection. The presence of conjugates was confirmed by MALDI-TOF and S1-PFGE for the species and plasmid profiles, respectively, and the presence of the ARGs bla_NDM-1 and bla_IMP-4 was confirmed by PCR.

Stability of bla_IMP-4 based on experimental passage

Three independent colonies were incubated in LB broth with shaking (200 rpm) at 37°C overnight. Then, 40μL of the overnight culture was passaged in 4mL of fresh medium (1:100 dilution ratio) every day. The cultures at days 0 and 7 were spread on LB plates without antibiotic, and 40 colonies per isolate were picked for bla_IMP-4 amplification.

The remaining cultures at days 0, 1, 2, 3, and 7 were used for genomic DNA extraction. The copy numbers of bla_IMP-4 were determined by quantitative PCR with a LightCycler 480 II (Roche, Basel, Switzerland), and the housekeeping gene pgi was used as the internal reference. The relative copy number was determined using TB Green Master Mix (Takara, Dalian, China) and calculated by the 2^−ΔΔCt method; each experiment was repeated three times. The primers used are listed in Table S1.

Plasmid curing

The CRISPR-Cas9 plasmid curing system was established in this study. Briefly, the specific spacer sequences of the plasmid replicons (IncX3, IncFIB, and IncHI1B) were inserted into pSGKP-hyg at the BsaI restriction enzyme site, and the spacer primers used in this study are listed in Table S1. Then, the isolates harbouring pCasKP was induced by L-arabinose, and the pSGKP-hyg-spacer was electroporated into them. The plasmid curing system was removed by overnight culture on sucrose agar at 37°C. The plasmid cured isolates were screened by PCR, and confirmed by S1-PFGE.

Growth curve measurements

An overnight culture of each isolate was diluted 1:1000 into 200 μL of fresh MH medium without antibiotics or with 2 mg/L meropenem, after which the mixture was transferred to microwell plates (four wells per isolate). The plates were incubated at 37°C in a Bioscreen C (Oy Growth Curves Ab Ltd., Turku, Finland) with continuous shaking, and the optical density (OD) at 600 nm was measured every 5 min for 18 h. All the experiments were performed in triplicate. The growth rates and lag times were calculated with an R script as previously described [20]. Multiple comparisons of the relative growth rate and lag time were performed by Dunn's multiple comparisons test, and an adjusted p value less than 0.05 was considered to indicate statistical significance.

Nucleotide sequence accession numbers

The Illumina raw reads of seven K. pneumoniae isolates were deposited in the NCBI SRA database (accession number: SRR23784455 to SRR23784461), and the nanopore raw reads of three K. pneumoniae isolates were deposited in the NCBI SRA database (accession number: SRR23750731 to SRR23750733).

Results

Clonality, genetic characteristics and antimicrobial susceptibility of ST20 isolates in China

A total of seven ST20 isolates were collected, and the prevalence of ST20 was 0.99% (7/708), which took the fourth place in prevalence of CRKP in nationwide surveillance (Table S2). To determine the clonality of ST20 in China, the N-J tree based on cgMLST was generated. The results showed that CHN03027, CHN03034, CHN13011, CHN13052, and CHN13105 were closely related (cluster A), with average differences of 3.2 ± 2.9 core genes among these isolates. The isolates in cluster A all harboured bla_IMP-4, and 80% (4/5) of them were bla_NDM-1 positive. However, the outgroup isolates, CHN03052 and CHN06080, were bla_NDM-5 and bla_KPC-2 positive, respectively (Figure 1A).

Figure 1. (A) Phylogenetic tree of seven ST20 isolates. The red stars represent carbapenem resistance genes, the red circles represent other ARGs, and the blue circles represent virulence genes. (B) S1 nuclease-digested plasmid pattern and Southern blotting using IMP, NDM, and KPC probes.

The isolates were isolated from abdominal drainage, throat swabs, urine, or blood. All the isolates were resistant to carbapenems, including meropenem, imipenem, and ertapenem. These isolates were all resistant to cefoxitin, ceftazidime, cefepime, piperacillin/tazobactam, and cefoperazone/sulbactam (Table 1). One isolate (CHN06080) producing KPC-2 carbapenemase was susceptible to ceftazidime/avibactam, and the other isolates producing MBLs were resistant to ceftazidime/avibactam. These isolates were all resistant to aztreonam except CHN13052. The ARG analysis indicated that the remaining isolates all harboured ESBLs, including bla_CTX-M-14, bla_CTX-M-15, bla_CTX-M-3, and bla_SHV-12. CHN13052 harboured bla_SHV-145-like, which was similar to bla_SHV-145 with an identity of 99.88%. The gene cloning experiment showed that both SHV-145 and SHV-145_L118R were susceptible to aztreonam, and the ESBL confirmation experiment showed that neither was ESBL positive (Table S3). The other antimicrobial agents, such as fosfomycin, amikacin, tigecycline, colistin, and cefiderocol were effective against all the ST20 isolates in vitro.

Table 1. The specimen types and the antimicrobial susceptibility testing results of ST20 isolates.

Isolate ID	Time	City	Specimen type	Antimicrobial MIC (mg/L)*
				MEM	IPM	ETP	FOX	CAZ	FEP	PTZ	F/S	AZT	FOS	AK	CIP	LEV	TGC	COL	CZA	FDC
CHN03027	2018.6	Tianjin	Abdominal drainage	64	32	64	>256	>128	32	512/4	>128/64	>128	16	0.5	0.06	0.06	0.125	1	>128/4	2
CHN03034	2018.6	Tianjin	Abdominal drainage	64	32	>64	>256	>128	32	512/4	>128/64	>128	8	0.5	0.06	0.06	0.125	1	>128/4	2
CHN03052	2018.6	Tianjin	Throat swab	>128	64	>64	>256	>128	128	>512/4	>128/64	128	16	0.5	4	1	0.5	0.5	>128/4	2
CHN06080	2018.8	Shenyang	Urine	16	16	32	32	32	16	512/4	128/64	128	4	0.5	32	4	0.25	1	0.5/4	1
CHN13011	2018.6	Guangzhou	Blood	128	32	>64	>256	>128	64	512/4	>128/64	>128	4	0.25	0.06	0.06	0.25	1	>128/4	1
CHN13052	2018.9	Guangzhou	Blood	32	8	32	>256	>128	64	32/4	>128/64	0.25	4	0.25	0.06	0.06	0.25	0.5	>128/4	1
CHN13105	2018.8	Guangzhou	Blood	128	32	>64	>256	>128	32	512/4	>128/64	>128	8	0.5	0.06	0.06	0.125	1	>128/4	1

*MEM: meropenem, IPM: imipenem, ETP: ertapenem, FOX: cefoxitin, CAZ: ceftazidime, FEP: cefepime, PTZ: piperacillin/tazobactam, F/S: cefoperazone/sulbactam (2:1), AZT: aztreonam, FOS: fosfomycin, AK: amikacin, CIP: ciprofloxacin, LEV: levofloxacin, TGC: tigecycline, COL: colistin, CZA: ceftazidime/avibactam, FDC: cefiderocol

**The bold numbers were interpreted as resistant (R).

Different locations of bla_NDM-1 and bla_IMP-4 among the ST20 isolates

There were three isolates harbouring a single carbapenemase, and S1-PFGE showed that these carbapenemases were all located on plasmids; specifically, CHN03052 was positive for bla_NDM-5 on an ∼33-55 kb plasmid, CHN13052 was positive for bla_IMP-4 on an ∼250 kb plasmid, and CHN06080 was positive for bla_KPC-2 on an ∼33-55 kb plasmid (Figure 1B).

The other four isolates harboured dual MBLs. CHN03027 and CHN03034 had similar plasmid profiles; briefly, bla_IMP-4 was located on an ∼250 kb plasmid, and bla_NDM-1 was located on an ∼55 kb plasmid. However, bla_NDM-1 and bla_IMP-4 of CHN13105 were colocalized on an ∼310 kb plasmid. Furthermore, bla_NDM-1 was located on an ∼55 kb plasmid in CHN13011; however, CHN13011 was bla_IMP-4 negative according to Southern blotting, which indicated that bla_IMP-4 of CHN13011 might be located on the chromosome (Figure 1B).

Here, we found three different locations for bla_NDM-1 and bla_IMP-4. Then, we selected the representative isolates CHN03027, CHN13011, and CHN13105 for long-read sequencing. The results showed that in CHN03027, bla_NDM-1 was located on an IncX3-type plasmid, and bla_IMP-4 was located on an IncFIB(K)/HI1B-type plasmid. Like in CHN13011, bla_NDM-1 was located on the same IncX3 plasmid, while bla_IMP-4 was located on the chromosome where the IncFIB(K) and IncHI1B replicons could also be identified. In addition, in CHN13105, the fusion plasmid contained the three replicon types mentioned above, which carried both bla_IMP-4 and bla_NDM-1 (Table 2).

Table 2. The genomic characteristics and antimicrobial resistance genes among CHN03027, CHN13011, and CHN13105.

Isolates	size (bp)	ORFs	GC%	Replicon type	Antimicrobial resistance genes
CHN03027
03027-chromosome	5,184,567	4,780	57.6		oqxA, oqxB32, blaSHV-145, fosA
p03027-IMP	263,082	256	51.6	IncFIB(K), IncHI1B	blaIMP-4, aph(3'‘)-Ib, aph(6)-Id, sul2
p03027-NDM	53,983	63	49.0	IncX3	blaNDM-1, bleMBL, blaSHV-12
p03027-3	15,263	14	45.5		-
CHN13011
13011-chromosome	5,468,368	5,057	57.3	IncFIB(K), IncHI1B	oqxA, oqxB32, blaSHV-145, fosA, blaIMP-4, aph(3'‘)-Ib, aph(6)-Id, sul2
p13011-NDM	56,668	68	49.0	IncX3	blaNDM-1, bleMBL, blaSHV-12
p13011-3	15,234	13	45.4		-
CHN13105
13105-chromosome	5,187,434	4,777	57.6		oqxA, oqxB32, blaSHV-12, fosA
p13105-NDM-IMP	296,385	299	50.9	IncFIB(K), IncHI1B, IncX3	blaNDM-1, bleMBL, blaSHV-12, blaIMP-4, aph(3'‘)-Ib, aph(6)-Id, sul2
p13105-3	15,410	14	45.5		-

The conjugation experiment showed that the bla_NDM-1-harbouring plasmid in CHN03027 (p03027-NDM) could be transferred to the EC600 strain, but the bla_IMP-4 plasmid (p03027-IMP) could not be transmitted. Conjugal transfer capability analysis revealed that relaxase, type IV coupling protein (T4CP), and type IV secretion system (T4SS) were present on p03027-NDM, whereas they could not be found on p03027-IMP, which was consistent with the results of the conjugation experiment. Moreover, the conjugation results showed that the bla_NDM-1-harbouring plasmid in CHN13011 (p13011-NDM) could be transmitted, but the bla_IMP-4 gene on the chromosome could not be transferred. The bla_IMP-4 and bla_NDM-1 genes present on the same plasmid in CHN13105 (p13105-NDM-IMP) could be transferred simultaneously. p13011-NDM and p13105-NDM-IMP were positive for relaxase, T4CP, and T4SS.

Detection of the hallmark of cointegrate formation

To investigate the formation of the fusion plasmid (p13105-NDM-IMP), cointegration sites were identified. The sequence analysis results showed that IS26 was related to the formation of the fusion plasmid. Specifically, the 8-bp left and right flanking sequences of IS26 (1) on p13105-NDM-IMP were CACATTGT and CAAAATCT, which were identical to the left flanking sequence of IS26 (3) on p03027-NDM and the right flanking sequence of IS26 (4) on p03027-IMP, respectively. The 8-bp left and right flanking sequences of IS26 (2) on p13105-NDM-IMP were CTCAGGCC and AACGCCGG, which were identical to the left flanking sequence of IS26 (4) on p03027-IMP and the right flanking sequence of IS26 (3) on p03027-NDM, respectively (Figure 2A).

Figure 2. (A) Genomic comparison of CHN03027, CHN13011, and CHN13105. The yellow arrows represent IMP and NDM, the dark green arrows represent IS26, and the light arrows represent ltrA. (B-C) Diagrams of circular transposon unit formation and DNA gel electrophoresis for CHN13011 and CHN13105.

To determine the location of bla_IMP-4 on the CHN13011 chromosome, we performed a comparison between the chromosomes of CHN13011 and CHN03027. The results showed that CHN13011 had an intact plasmid (p03027-IMP) that was integrated on the chromosome at the site of the group II intron-encoded protein (LtrA), which led to formation of the repeats of ltrA on the chromosome of CHN13011 (Figure 2A).

Furthermore, we determined whether bla_IMP-4 integration was related to the transposon unit (TU) on the chromosome or fusion plasmid. To detect the excised circular TU on CHN13011, primers facing outward from ltrA were used, and these primers did not generate the amplicon; however, electrophoresis revealed an ∼2,000 bp fragment, which was identical to the result when CHN03027 was used as the template. These results indicated that the circular TU was formed on CHN13011 (Figure 2B). Similarly, we detected circular TU formation in CHN13105. The primers designed for p13105-NDM-IMP were separated by approximately 55 kb and would not generate the amplicon; however, they would generate an amplicon if the circular TU was present, and the results showed an ∼750 bp fragment on the gel. These results indicated that IS26 was related to TU formation in the fusion plasmid (Figure 2C).

Stability of the bla_IMP-4 gene and the plasmids contribution to resistance and fitness

We performed an experimental passage to determine the stability of the bla_IMP-4 gene. All the clones picked on day 7 were positive for bla_IMP-4, and the relative copy number of bla_IMP-4 was stable during the experimental passage. A comparison between the first day and the last day of passage revealed no significant difference (p > 0.05), which indicated that the location of bla_IMP-4 was not related to its stability (Figure S1).

To determine the resistance and fitness contributions of the different plasmids, we utilized the CRISPR-Cas9 plasmid curing system to eliminate the bla_IMP-4- and bla_NDM-1-harbouring plasmids in CHN03027. We obtained CHN03027-ΔIMP, CHN03027-ΔNDM, and CHN03027-ΔIMP-ΔNDM from CHN03027, which was confirmed by S1-PFGE (Figure 3A). The AST results indicated that when both the p03027-IMP and p03027-NDM plasmids were cured, the isolates were susceptible to all the tested antimicrobial agents. When p03027-IMP was eliminated, the resistance level was equal to that of the parental strain (CHN03027); however, the resistance of the p03027-NDM cured isolates to carbapenems decreased at least 8-fold, and the resistance to cefepime, piperacillin/tazobactam, cefoperazone/sulbactam, and aztreonam was also affected (Table 3). The results of the growth curve analysis showed that when these isolates were incubated in MH broth without antibiotics, the growth curves were similar. However, when these isolates were incubated in MH supplemented with 2 mg/L meropenem, the lag time of CHN03027-ΔNDM was significantly longer than that of CHN03027 and CHN03027-ΔIMP (Figure 4).

Figure 3. (A) The S1 nuclease-digested plasmids of CHN03027, CHN13011, CHN13105 and the corresponding plasmid cured isolates. (B) Diagram of the differences in recombination between IS26 in CHN13105-ΔNDM.

Figure 4. (A-B) Growth curves of CHN03027, CHN13011, CHN13105 and the corresponding plasmid cured isolates in MH broth or MH broth supplemented with 2 mg/L meropenem. (C) Comparison of lag times among the wide type and corresponding plasmid cured isolates.

Table 3. The susceptibility of parental dual carbapenemase harbouring isolate and its plasmid curing isolates.

Isolates	Antimicrobial MIC (mg/L)*
	MEM	IPM	ETP	FOX	CAZ	FEP	PTZ	F/S	AZT	CZA
CHN03027	64	32	64	>256	>128	32	512/4	>128/64	>128	>128/4
CHN03027-ΔIMP	128	32	64	>256	>128	64	512/4	>128/64	>128	>128/4
CHN03027-ΔNDM	4	4	8	>256	>128	8	8/4	64/32	0.25	>128/4
CHN03027-ΔIMP-ΔNDM	0.125	0.5	0.03	4	0.25	0.06	4/4	0.5/0.25	0.25	0.25/4
CHN13011	128	32	>64	>256	>128	64	512/4	>128/64	>128	>128/4
CHN13011-ΔNDM	8	4	8	>256	>128	8	8/4	64/32	0.25	>128/4
CHN13105	128	32	>64	>256	>128	32	512/4	>128/64	>128	>128/4
CHN13105-ΔNDM-8	4	4	8	>256	>128	8	8/4	64/32	0.25	>128/4

*MEM: meropenem, IPM: imipenem, ETP: ertapenem, FOX: cefoxitin, CAZ: ceftazidime, FEP: cefepime, PTZ: piperacillin/tazobactam, F/S: cefoperazone/sulbactam (2:1), AZT: aztreonam, CZA: ceftazidime/avibactam

**The bold numbers were interpreted as resistant (R).

Then, we obtained a plasmid cured isolate (CHN13011-ΔNDM) from CHN13011 that harboured only bla_IMP-4 on its chromosome; we used the same protocol to cure the fusion plasmid in CHN13105. However, we obtained bla_NDM-1-negative but bla_IMP-4-positive isolates, and CHN13015-ΔNDM-8 had a longer plasmid than CHN13015-ΔNDM-5 (Figure 3A). Next, we investigate the deletion region of p13105-NDM-IMP, and the results showed that recombination with IS26 resulted in different lengths of CHN13015-ΔNDM (Figure 3B). Similarly, bla_NDM-1 strongly contributed to the resistance to carbapenems and other antimicrobial agents. When bla_NDM-1 was knocked out, the MIC of carbapenems decreased at least 8-fold. Moreover, the bla_NDM-1 knockout isolates had a significantly longer lag time than their corresponding parental isolates (p = 0.0001 and p < 0.0001) when they were grown in 2 mg/L meropenem-containing MH broth (Figure 4).

For this reason, CHN03027, CHN13011 and CHN13105 had only 1-2 differences in core genes, and we tried to compare the differences in resistance and fitness among the isolates harbouring bla_IMP-4 at different location. The results showed that the antimicrobial susceptibility and growth curves of CHN03027-ΔNDM, CHN13011-ΔNDM and CHN13105-ΔNDM were similar, which indicated that the location of bla_IMP-4, whether on the chromosome or a plasmid, did not influence the resistance level or fitness.

Discussion

IMP was the first MBL detected in CRKP isolates in 1996, and IMP-4 is now the most common carbapenemase detected in Gram-negative bacteria in Austria [21]. NDM was initially detected in Sweden from a patient who previously travelled to New Delhi and constitutes a threat of major concern [22]. Nationwide surveillance in China showed that the most common carbapenemase in CRKP is KPC-2, accounting for approximately 80% of carbapenemases [4]. However, fewer MBL producers are found in China; for instance, the prevalence of NDM-1 is 1-4%, and that of IMP-4 is 1-2% [2,23,24]. Furthermore, bla_NDM and bla_IMP coharboring isolates have rarely been reported. However, with the use of innovative β-lactamase inhibitors, the epidemiological trends of carbapenemases might change, and the frequency of MBL producers has increased markedly [25], for instance, the frequency of MBLs increased from 2.0% in 2019 to 7.1% in 2020, and 12.2% in 2021 among CRKP. Considering that ceftazidime/avibactam was approved for use in 2019 in China, we believe that the number of MBL-producing isolates will increase rapidly in China; hence, these isolates should receive additional attention.

The plasmid p03027-IMP was an IncFIB/HI1B-type plasmid, that was generated from three different plasmids, namely, pNH25.1 (CP024875.1), pIMP-4_IncN (CP050160.1), and pKPN-65 (CP015026.1). Specifically, the main plasmid backbone originated from pNH25.1, and the IMP-4 originated from pIMP-4_IncN; among these, pIMP-4_IncN had conjugative gene elements; however, p03027-IMP lost these genetic segments. Additionally, p03027-IMP can integrate at different locations. We found two different locations for the bla_IMP-4 gene in this study: one was on the fusion plasmid (p13105-NDM-IMP), and the other was chromosomal integrated (CHN13011-IMP-chr).

A previous study reported a similar fusion plasmid AZS099-NDM-IMP (accession number: CP086762.1), and bla_IMP-4 and bla_NDM-1 were simultaneously located on this 296 kb fusion plasmid [26]. In this study, a conjugation experiment showed that p03027-NDM was a self-transferable plasmid and that p03027-IMP could not be transferred. The formation of fusion plasmids, such as in CHN13105, cause both ARGs to be transferred together, which increases the infection risk of these isolates. These phenomena were also reported for the transmission of the pLVPK-like virulence plasmid. Xie et al. reported that the fusion plasmid was generated through the interaction of p16ZR-187-Vir and p16ZR-187-Hp in a 241 bp homologous region, after which the fusion plasmid became conjugative [27].

The carbapenem resistance gene bla_IMP is typically carried by plasmids but is occasionally reported to be carried on the chromosome [28]; for instance, Abe et al. reported that bla_IMP-6 was located on the chromosome in E. coli [29], and Miyazato et al. reported that bla_IMP-60 was located on the chromosome in Enterobacter asburiae [30]. In this study, we found a chromosomally integrated bla_IMP-4 gene in K. pneumoniae. The results showed that recombination was mediated by a group II intron-encoded protein (LtrA), which was previously reported in the MDR virulence convergence strain KP17-15, and that p17-15-vir was derived from p17-16-vir and p17-16-CTX [31].

Li et al. reported a bla_IMP-4- and bla_NDM-1-positive K. michiganensis isolate, and experimental passaging indicated that isolates containing bla_IMP-4 but lacking bla_NDM-1 had decreased MICs of carbapenems [32]. Eliminating plasmids directly from bacterial hosts is the best way to determine the relationship between the presence of plasmids and fitness costs [33]. In this study, we eliminated the bla_IMP-4-and bla_NDM-1-harbouring plasmids in natural bacterial hosts and further clarified the contributions of different plasmids. We found that when the bla_NDM-1 gene was knocked out, carbapenem resistance decreased; however, the resistance level did not change when bla_IMP-4 was knocked out, and we further found that loss of bla_NDM-1 prolonged the lag time in these isolates. Sequence analysis revealed a weak Pc promoter within intI1 and a 14 bp spacer sequence between the −35 and −10 boxes of P2 in these isolates, which has been proven to not contribute to the transcription of the downstream bla_IMP gene (Figure S2) [34].

Conclusion

In this study, we investigated the epidemiology of ST20 CRKP in China, furthermore, we found three different integration locations of the bla_IMP-4 gene, namely, a separate plasmid, a self-conjugative fusion plasmid, and the bacterial chromosome. The bla_IMP-4 fusion plasmid and chromosomally integrated bla_IMP-4 were generated from p03027-IMP recombination, which was mediated by IS26 and ltrA, respectively. The bla_NDM-1 gene increases resistance to carbapenems and shortens the lag time more than bla_IMP-4.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by National Key Research and Development Program of China: [grant no 2023YFC2307100]; National Natural Science Foundation of China: [grant no 32141001].