Characterization of a Conjugative Hybrid Plasmid Coharboring bla_KPC-2 and bla_IMP-4 in a Klebsiella quasipneumoniae Clinical Isolate

ABSTRACT

Generation of hybrid MDR plasmids accelerated the evolution and transmission of resistance genes. In this study, we characterized a bla_KPC-2- and bla_IMP-4-coharboring conjugative hybrid plasmid constituted of an IncHI5 plasmid-like region, an IncFII(Yp)/IncFIA plasmid-like region, and a KPN1344 chromosome-like region from a clinical ST852-KL18 Klebsiella quasipneumoniae strain. The bla_IMP-4 gene was captured by a novel integron In1965, and the bla_KPC-2 gene was located on a new non-Tn4401 group I NTE_KPC element. Both bla_KPC-2- and bla_IMP-4-containing genetic architectures were distinguished from classical structures, highlighting the constant evolution of these genetic elements.

IMPORTANCE The emergence of carbapenem-resistant Enterobacterales (CRE) that coexpress serine- and metallo-carbapenemases is a severe threat to the efficacy of ceftazidime-avibactam (CZA), which has been proven to be extremely effective against KPC-producing Enterobacterales strains. Our study described the cooccurrence of KPC-2, a serine β-lactamase, and IMP-4, a metallo-β-lactamase (MBL), on a conjugative hybrid plasmid from a clinical carbapenem-resistant K. quasipneumoniae strain, and it revealed an alternative route for IncHI5 plasmid to evolve by recombining with other plasmids to form a hybrid plasmid. Moreover, this hybrid plasmid can be transferred into other Klebsiella species and stably persist during passage. The propagation of two important carbapenemase genes with a new genetic background using well-evolved plasmids in the clinical setting promotes the emergence of superbugs that require careful monitoring.

OBSERVATION

The prevalence of carbapenem-resistant Enterobacterales (CRE) poses a significant threat to public health. Within CRE, carbapenem-resistant Klebsiella pneumoniae (CRKP) is the predominant bacterial species (1). Besides the most commonly detected class A serine-β-lactamase KPC-2/KPC-3, metallo-β-lactamases such as NDM, VIM, and IMP were also described in CRKP conferring carbapenem resistance. Recently, the emergence of KPC-2- and NDM-1-coproducing CPKP brings challenges to the clinical treatment of CRKP; the synergistic effect of serine-β-lactamase and metallo-β-lactamase in a single isolate severely compromised the utility of ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam, which are effective means to treat infections caused by KPC-producing CRKP (2). Similar to the KPC-2- and NDM-1-coproducing CPKP, current reports of bla_KPC- and bla_IMP-coharboring strains, including K. pneumoniae (3), Klebsiella oxytoca (4), Serratia marcescens (5), and Pseudomonas aeruginosa (6), revealed that the two different types of carbapenemase genes were carried by distinct plasmids. To date, only a nonconjugative plasmid coharboring bla_KPC and bla_IMP but without genetic feature description was identified in Raoultella ornithinolytica (7). Here, we characterized a novel conjugative hybrid plasmid named pKP18-31-IMP coharboring bla_KPC-2 and bla_IMP-4 in a K. quasipneumoniae strain of clinical origin.

The clinical strain KP18-31 was isolated from the blood culture of a 12-year-old female patient with severe aplastic anemia in 2018 (Table 1). By using BIGSdb (https://pubmlst.org/) and Kleborate software (8, 9), the in silico species identification results showed that KP18-31 belonged to K. quasipneumoniae, which has been reported as an emerging pathogen in 2004 that can cause bloodstream infections in healthy individuals (10). For measuring the antimicrobial susceptibility profiles, agar dilution (for fosfomycin) and broth microdilution methods according to the Clinical and Laboratory Standards Institute (CLSI) guidelines were recruited (11). KP18-31 was resistant to a variety of antibiotics, including meropenem and aztreonam (Table 1). Whole-genome sequencing (WGS) results indicated that KP18-31 carried both bla_KPC-2 and bla_IMP-4. In order to investigate the transferability of bla_KPC-2 and bla_IMP-4, KP18-31 was used as the donor strain, and ST11 K. pneumoniae HS11286YZ6 was used as the recipient strain for conjugation assay (12). According to conjugation assay, both bla_KPC-2 and bla_IMP-4 were successfully transferred into the recipient strain HS11286YZ6. S1 pulsed-field gel electrophoresis (PFGE) and Southern hybridization revealed that the bla_KPC-2 and bla_IMP-4 genes were colocated on an ~370-kb plasmid (Fig. 1A) in KP18-31 and the corresponding transconjugant, suggesting that the plasmid simultaneously encoding two important carbapenemases was able to spread horizontally. Furthermore, the conjugation frequency (the number of transconjugants/total number of recipients) of this plasmid was 2.0 × 10⁻⁵, and stability tests demonstrated that it could be stably maintained in the transconjugant at 150th generations (66.7%) without antibiotic selection, indicating it was well adapted to the host (13).

TABLE 1.

Antimicrobial susceptibility profiles of K. quasipneumoniae KP18-31

Antimicrobial category	Antimicrobial agent	MIC (μg/mL) of:a
		KP18-31b	C18-31-YZ6c	YZ6d
Penicillins	Ampicillin	>256	>256	>256
Antipseudomonal penicillins with β-lactamase inhibitors	Piperacillin-tazobactam	128	>512	512
Nonextended spectrum cephalosporins	Cefazolin	>256	>256	>256
Extended-spectrum cephalosporins	Ceftazidime	>64	>64	8
	Cefepime	>256	>256	128
Carbapenems	Imipenem	32	32	0.5
	Meropenem	32	16	0.06
Monobactams	Aztreonam	64	>64	64
Fluoroquinolones	Ciprofloxacin	<0.015	4	8
Aminoglycosides	Gentamicin	<0.25	1	0.5
	Amikacin	0.5	1	1
Phenicols	Chloramphenicol	16	4	4
Tetracyclines	Doxycycline	>128	64	4
Phosphonic acid	Fosfomycin	<1	256	256
Glycylcyclines	Tigecycline	0.25	0.25	1
Polymyxin C	Colistin	<0.125	<0.125	1
Ceftazidime-avibactam	Ceftazidime-avibactam	128/4	0.5/4	0.5/4
Aztreonam-avibactam	Aztreonam-avibactam	0.5/4	0.5/4	0.5/4

^aMICs were determined by broth or agar dilution as proposed by CLSI and interpreted using its guidelines.

^bDonor strain.

^cCorresponding transconjugant.

^dRecipient strain.

FIG 1.

(A) PFGE of S1-digested plasmid DNA and Southern blot hybridization for bla_IMP-4 (red) and bla_KPC-2 (green). The black bands showed positive signals by Southern blot hybridization using the bla_IMP probe or bla_KPC probe. Lane M, Salmonella strain H9812 as a molecular marker. (B) Circular comparison between the hybrid plasmid pKP18-31-IMP and other reported similar plasmids or chromosomes. The plasmid pKP18-31-IMP at the outermost circle was used as the reference plasmid. Three circles with different sizes marked in blue, green, and gray dash lines represent the different genetic modules of pKP18-31-IMP. The circular map was generated using the BLAST Ring Image Generator (BRIG). (C) Linear alignment of plasmid pKP18-31-IMP with plasmid pRo24724 and plasmid pK516_KPC. The linear map was generated using EasyFig.

To resolve the complete plasmid sequence of KP18-31, the whole-genome DNA of strain KP18-31 was extracted (Wizard genomic DNA purification kit) and used for WGS by Illumina HiSeq2500 platform (San Diego, CA, United States) and PacBio RS platform (Pacific Biosciences, Menlo Park, CA, USA). Multilocus sequence typing (MLST), the distribution of plasmid replicons, antimicrobial resistance (AMR) genes, and insertion sequences were confirmed by online tools (https://cge.food.dtu.dk/services/) and Kleborate software (8). Strain KP18-31, which was assigned to ST852 and KL18 based on Kleborate analysis, was found to harbor one chromosome of 5,157,396 bp (GenBank accession no. CP045641) and three plasmids named pKP18-31-IMP (GenBank accession no. MN661402), pKP18-31-2 (GenBank accession no. MN661403), and pKP18-31-3 (GenBank accession no. MN661404) of 377,346 bp, 119,801 bp, and 107,000 bp, respectively. Resistome analysis revealed four AMR genes, including oqxA, oqxB, bla_OKP-B-2, and fosA, were detected on the chromosome, while the remaining eight AMR genes, including bla_KPC-2, bla_IMP-4, msr(E), mph(E), arr-3, sul1, tet(D), and tet(R) were identified on plasmid pKP18-31-IMP, and no AMR gene was carried by the other two smaller plasmids, pKP-18-31-2 and pKP-31-3, which belonged to IncFII(K) and untypeable plasmids, respectively.

Plasmid pKP18-31-IMP in this study simultaneously possessed four plasmid replicons, including IncFII(Yp), IncFIA, IncFIB-like, and IncHI5B. Previously, bla_IMP-positive plasmids carrying multiple plasmid replicons have also been identified in species other than K. quasipneumoniae, such as an IncFII/IncU/IncFIB/IncHI5B-type plasmid pRo24724 (GenBank accession no. CP021328) found in Raoultella ornithinolytica and an IncHIB/IncFIA/IncR-type plasmid pKOX75251 (GenBank accession no. CP065475) found in Klebsiella michiganensis (7, 14). Given that IncFIB-like and IncHI5B replication genes are used to distinguish IncHI5 plasmids (15), the presence of two extra replication genes suggested that the formation of plasmid pKP18-31-IMP might have been driven by the recombination of an IncHI5 plasmid with another plasmid. A BLASTN search against the NCBI nucleotide database revealed that pKP18-31-IMP displayed 80% query coverage and 99.97% nucleotide identity to plasmid pRo24724 (GenBank accession no. CP021328) from the R. ornithinolytica strain, 59% query coverage and 99.95% nucleotide identity to IncHI5 plasmid pSIM-1-BJ01 (GenBank accession no. MH681289) from the K. pneumoniae strain, and 31% query coverage and 99.81% nucleotide identity to IncFII(Yp)/IncFIA plasmid pK516_KPC (GenBank accession no. CP022349) from the K. michiganensis strain. Detailed sequence analysis of pKP18-31-IMP showed it was a cointegrate plasmid consisting of an ~226-kb pRo24724-derived module (IncHI5 plasmid-like region, region 3), an ~110-kb pK516_KPC-derived module (IncFII(Yp)/IncFIA plasmid-like region, region 1), and an ~28-kb segment contained open reading frames (ORFs) coding for products related to a bifunctional protein (zinc-containing alcohol dehydrogenase and quinone oxidoreductase), beta-galactosidase LacZ, lactose permease LacY, and maltoporin; it shares high identity (88% query coverage and 100% nucleotide identity) with the corresponding fragment in the chromosome of K. pneumoniae strain KPN1344 (GenBank accession no. CP033901) (KPN1344 chromosome-like region, region 2) (Fig. 1B and C). Notably, linear comparison revealed that a 34,216-bp region carrying genes related to plasmid replication and transfer (within the red box) organized as repFII-finO-traXIDGH-trbB-traQF-trbE-traNCUW-trbI-traCVPAKELAYW in pKP18-31-IMP was shared by pRo24724 and pK516_KPC, suggesting that homologous recombination within this region might have led to the formation of a pRo24724:pK516_KPC hybrid (Fig. 1C). Moreover, inversion of a 30,235-bp region in pK516_KPC-derived region 1, integration of an ~28-kb segment highly homologous to the KPN1344 chromosome (region 2), and an ~5-kb IS26-flanked segment carrying tet(R) and tet(D) genes between regions 1 and 2, and rearrangement of a 12,539-bp (within the red box) segment in pRo24724-derived region 3, further suggested that the formation of pKP18-31-IMP has undergone complex and multiple recombination processes based on pRo24724-like and pK516_KPC-like structures. Recently, we have identified another IncHI5-like plasmid, pC39-334kb, and inferred that the plasmid was evolved from the pNDM-1-EC12-like IncHI5 plasmids (16). These findings jointly highlight the promising evolutionary potential of IncHI5 plasmids, as they may lead to the formation of novel multidrug resistance plasmids and further expand their host range (17).

The bla_IMP-4 gene was identified within class 1 integron with the intI1-bla_IMP-4-K1.pn.I3-arr-3-qacEΔ1-sul1 genetic array, which was given a new integron number, In1965, by the INTEGRALL database. The variable region of In1965 was interrupted by the insertion of group II intron K1.pn.I3, downstream of the attC site (also named the 59-base element) of the bla_IMP-4 gene cassette. Compared to the In1589 reported previously (18), the two integrons were identical except for the loss of two genes, qacG2 and aacA4 (Fig. 2A). The bla_IMP-4 gene was usually captured by a class 1 integron, including In1589, In823, In1310, In809, etc. (19). In this study, we described a novel bla_IMP-4-K1.pn.I3-harboring class 1 integron. As previously reported, bla_IMP-4-K1.pn.I3 has been regarded as the most common structure in IMP-4-producing isolates in China (18). Previous studies showed that transposons such as Tn6017 and Tn1696 are important vectors mediating the spread of bla_IMP in Enterobacterales. Whether transposons can promote the spread of the bla_IMP-4-harboring integron-carrying In1965 requires further investigation.

FIG 2.

(A) Linear comparison of genetic environment of bla_IMP-4 between pKP18-31-IMP and pIMP-4-EC62. (B) Linear comparison of the genetic environment of bla_KPC-2 among pKPHS2, pKP048, pKP18-31-IMP, and pZZ40-KPC. The plasmid comparison figure was generated by EasyFig.

The bla_KPC-2 gene was located on a non-Tn4401 element with the structure of IS26-ISKpn8-bla_KPC-2-ΔISKpn6/traN-korC-klcA-ΔrepA. Compared to the classical NTEKPC-Ib structure in pKPHS2 (GenBank accession no. CP003224) (20), ΔtnpR upstream of bla_KPC-2 and Tn4653 tnpR and Tn1721 tnpR downstream of bla_KPC-2 were absent in the bla_KPC-2 genetic context of pKP18-31-IMP. Alternatively, this genetic array also shared high homology to the NTEKPC-Ia structure in pKP048 (GenBank accession no. FJ628167) (Fig. 2B). In addition, we found that the genetic environment of bla_KPC-2 in plasmid pZZ40-KPC (GenBank accession no. MN891679) had 91% query coverage compared with that in plasmid pKP18-31-IMP. However, the main difference between them is the upstream region of bla_KPC-2 with different genes (IS26 and ΔISSwi) (Fig. 2B). These findings provided substantial evidence that the bla_KPC-2 gene was located on a new non-Tn4401 element in this study. Since the different genetic organizations containing bla_KPC-2 were detected from the traditional Tn4401 in 2009 (21), various mutations around bla_KPC were reported (22). This novel structure could be assigned to the group I NTE element because no insertion sequence was observed upstream of bla_KPC-2 (20). However, unlike group I NTE elements that usually have a tnpR gene upstream of ISKpn8, the tnpR gene is missing in the new element, suggesting that the formation of the structure may undergo various recombination events.

In conclusion, we characterized a bla_IMP-4- and bla_KPC-2-coharboring conjugative hybrid plasmid pKP18-31-IMP from K. quasipneumoniae. Three regions of an IncHI5 plasmid-like region, an IncFII(Yp)/IncFIA plasmid-like region, and a KPN1344 chromosome-like region construct of the basic structure of the plasmid highlight the strong evolutionary ability of IncHI5 plasmids. Both bla_IMP-4 and bla_KPC-2 were surrounded by novel genetic contexts, which differ to some extent from that reported previously. The spread of two critical AMR genes with the novel genetic contexts using the hybrid plasmid as the vehicle in clinical settings will facilitate the emergence of superbugs, which requires stringent surveillance.

Data availability.

The complete sequences of KP18-31 were submitted to the NCBI GenBank database with the following accession numbers: chromosome, CP045641; pKP18-31-IMP, MN661402; pKP18-31-2, MN661403; and pKP18-31-3, MN661404.