Identification of a Novel Hybrid Plasmid Encoding KPC-2 and Virulence Factors in Klebsiella pneumoniae Sequence Type 11

ABSTRACT

Recent emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) coharboring bla_KPC-2 and pLVPK-like virulence plasmids represented a novel clinical challenge. In the present study, we characterized a bla_KPC-2 and virulence hybrid plasmid, designated pCRHV-C2244, from a clinical ST11-K64 CRKP strain. pCRHV-C2244 was non-self-transmissible due to incomplete conjugative elements but mobilizable together with a conjugative helper. Enhanced virulence and stable maintenance without significant fitness loss in its original host were confirmed in vitro and in vivo.

INTRODUCTION

Carbapenem-resistant Klebsiella pneumoniae (CRKP), primarily belonging to the dominant clonal lineage ST11 in China, is known to cause untreatable nosocomial infections largely due to the lack of therapeutic options, which creates a challenge for the clinician (1). Plasmids carrying bla_KPC-2 are the main genetic determinants for this phenotypic antibiotic resistance in ST11 strains (1, 2). In recent years, ST11 CRKP isolates have become more notorious due to the acquisition of a pLVPK-like virulence plasmid, which enables them to simultaneously express the carbapenem resistance- and hypervirulence-associated phenotypes (1). Typically, these CRKP isolates carry bla_KPC-2 and virulence genes on separate plasmids (3). To date, the insertion of bla_KPC-2 into virulence plasmid backbone has been described only in an ST23 CRKP isolate (4).

Recent studies identified a high proportion of CRKP isolates (34.2%) coharboring bla_KPC-2 and virulence plasmids (5, 6). Multiplasmid coexistence within the same host may bring out multiple forms of genetic translocations, transpositions, and homologous recombinations, which are likely mediated by insertion sequence (IS) elements among these plasmids (7, 8). IS26 was common in the ST11 CRKP genome and had been recognized to be involved in numerous plasmid fusion and recombination events with a distinctive translocatable mechanism (9, 10). In the present study, we identified a large hybrid plasmid, designated pCRHV-C2244, generated by movement of IS26-associated translocatable unit and encoding KPC-2 and virulence factors in a high-risk ST11-K64 CRKP host.

Clinical strain C2244 was recovered from the sputum culture of an 84-year-old male patient with chronic obstructive pulmonary disease in 2017 (Table 1). To resolve the complete plasmid sequence for strain C2244, the genomic DNA was extracted and subjected to whole-genome sequencing using a combination of Illumina NovaSeq 6000 and PacBio Sequel systems (11). BLASTN comparison with other plasmids in GenBank identified the majority (Fig. 1, 106,809 to 39,280 bp, regions 2 and 3) of pCRHV-C2244 was nearly identical to the known IncHIB/IncFIB(K) virulence plasmid, such as the prototypical virulence plasmid pLVPK, whereas the region covering 39,281 to 106,808 bp (Fig. 1, region 1) had a similar organization to the epidemic bla_KPC-2-bearing IncFII/IncR-type plasmid and was flanked by two IS26 elements. To find an appropriate reference plasmid sequence with high molecular homology to pCRHV-C2244, a maximum-likelihood phylogenetic tree was constructed based on the recombination-free single-nucleotide polymorphisms (12), and, accordingly, K. pneumoniae 9949 plasmids p9949_1 and p9949_2 were used to perform genomic linear comparison with pCRHV-C2244 (see Fig. S1 and S2 in the supplemental material).

TABLE 1.

Antimicrobial susceptibility of clinical K. pneumoniae C2244

Strain	MIC (μg/ml)
	Meropenem	Imipenem	Ertapenem	Colistin	Tigecycline	Aztreonam	Ceftazidime	Ceftazidime/avibactam	Cefepime	Amikacin	Ciprofloxacin
C2244a	>32	32	>32	0.25	1	>64	>64	4	128	≤0.5	>64
C2244-PC	≤0.5	≤0.5	≤0.5	0.25	1	>64	4	2	16	≤0.5	>64
ATCC 13883	≤0.5	≤0.5	≤0.5	0.125	0.25	≤0.5	≤0.5	≤0.5	≤0.5	≤0.5	≤0.25
ATCC 13883/pCRHV-C2244	>32	>32	>32	0.125	0.125	>64	>64	2	128	≤0.5	≤0.25

^aC2244 genotyping: sequence type ST11/serotype K64:O2/wzi 64. Antimicrobial resistance genes: bla_KPC-2, bla_CTX-M-65, bla_SHV-12, bla_SHV-11, oqxA, oqxB, fosA6. Virulence factors: yersiniabactin, aerobactin, salmochelin, RmpA2.

FIG 1.

Comparative plasmid maps of 12 virulence and bla_KPC-2 plasmids aligned with pCRHV-C2244. Sequence comparison revealed that region 1 of pCRHV-C2244 was very similar to two bla_KPC-2 plasmids, i.e., K. pneumoniae 9949 plasmid p9949_2 and XJ-K1 plasmid pXJ-K1_3, whereas regions 2 and 3 were highly similar to a multiple virulence plasmid. Virulence factors, such as iroBCDN, iucABCDiutA, and rmpA2, and β-lactamase genes, such as bla_KPC-2, bla_SHV-12, and bla_CTX-M-65, were located in regions 2 and 3, respectively. The circular map was generated using the BLAST Ring Image Generator (BRIG).

The bla_KPC-2 gene region from pCRHV-C2244 (region 1) was compared with K. pneumoniae 9949 plasmid p9949_2, and this 67.5-kb homologous part was found to consist of multiple β-lactamase genes and IS26 elements, as well as conjugative transfer genes (traM and traA) (Fig. 2A). However, a traA/Y/J/M operon, encoding the plasmid conjugation machinery, was not cointegrated into the hybrid plasmid. The downstream sequence of bla_KPC-2 underwent an inversion of a 45.9-kb fragment bordered by IS26 sequences. Furthermore, the entire region 1 sequence was bordered by two copies of inverted IS26, but only one IS26 was located upstream of the corresponding part in plasmid p9949_2.

FIG 2.

Linear comparisons of pCRHV-C2244 with other plasmids with homologous structures and proposed formation model. (A) pCRHV-C2244 sequence was aligned with virulence and bla_KPC-2 plasmids coharbored by K. pneumoniae isolate 9949 or XJ-K1. The identical regions are shaded in gray, or pink for inverted regions. (B) Proposed model of virulence-resistance plasmid generation that involved IS26-mediated recombination between a bla_KPC-2 segment and a virulence plasmid backbone. Large-fragment inversion events were detected in both virulence plasmid and bla_KPC-2 plasmid.

Compared with K. pneumoniae 9949 plasmid p9949_1, the pLVPK-like backbone from pCRHV-C2244 (regions 2 and 3) showed high gene synteny with 92% query coverage and 99% nucleotide identity (Fig. 2A). This fragment can be divided into two regions according to the sequence direction with the corresponding plasmid. Region 2 can be aligned reversely with plasmid p9949_1 with two opposite IS26 elements at the boundaries. Region 3 exhibited the same orientation and significant nucleotide similarity with the corresponding region in plasmid p9949_1. However, a 16-kb segment comprised of the salmochelin synthesis operon and an intact ISKpn26 element were lost in plasmid p9949_1.

Considering the distinctive mechanism of IS26-mediated transposition, we proposed a potential pathway for the generation of pCRHV-C2244. First, a circular translocatable unit excised from a p9949_2-like plasmid was integrated into a p9949_1-like plasmid, resulting in formation of a 293-kb cointegrate. Then, region 2 of the cointegrate underwent a complete inversion mediated by IS26-associated intramolecular replicative transposition and finally generated the pCRHV-C2244-like plasmid. Also, IS26 was involved in the 45.9-kb fragment inversion within region 1 of the p9949_2-like plasmid (Fig. 2B).

Additionally, pCRHV-C2244 could be transferred into strain ATCC 13883 using the triparental mating method with the helper plasmid pRK2013 (Escherichia coli HB101). The conjugation frequency was much lower, at 1.1 × 10⁻¹⁰ per recipient cell. Incomplete conjugal transfer loci were a probable reason for the inability of self-transferability of pCRHV-C2244. Plasmid stability tests were performed with a wild-type C2244 strain and its transconjugant (see Fig. S3A in the supplemental material). After passaging approximating 140 generations, pCRHV-C2244 was stably maintained in original and conjugated K. pneumoniae isolates (retaining a frequency of 98% and 96%, respectively). The growth curve showed no significant fitness difference between the original C2244 strain and the plasmid-cured (PC) C2244 strain (Fig. S3B).

To determine whether the acquisition of pCRHV-C2244 could cause a significant increase in the production of capsular polysaccharide and the elevated virulence level in vivo, uronic acid quantification and mouse lethality assays were performed (13, 14). Although the pCRHV-C2244 transconjugant produced a comparable level of capsular polysaccharide to that of the wild-type recipient, the PC C2244 strain exhibited decreased capsule production compared with the wild-type C2244 strain (Fig. S3C). These findings were consistent with those in the mouse systemic infection model. Mice infected with the PC C2244 strain showed a higher survival rate than those infected with the original C2244 strain. However, the pCRHV-C2244 transconjugant strain did not display a significant increase in virulence in vivo despite the 12.5% mortality caused by this strain (Fig. S3D).

Taken together, the present study results characterized a novel hybrid plasmid coharboring bla_KPC-2 and virulence factors in an ST11 K. pneumoniae isolate that may have been generated as a consequence of transposition events mediated by IS26. This virulence-resistance-encoding plasmid may simultaneously confer carbapenem resistance and enhanced virulence in the clinical CRKP strain and maintain stability without fitness cost. This finding is of grave concern and merits further studies on the epidemiology of such a hybrid plasmid and the interaction between the vehicles expressing virulence and resistance.

Data availability.

The complete nucleotide sequences of the C2244 chromosome and pCRHV-C2244 were deposited in GenBank under accession numbers CP071464 and MT644086, respectively.