Molecular characterization of hybrid virulence plasmids in ST11-KL64 KPC-2-producing multidrug-resistant hypervirulent Klebsiella pneumoniae from China

Introduction Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains combining virulence and multidrug resistance (MDR) features pose a great public health concern. The aim of this study is to explore the evolutionary characteristics of virulence in CR-HvKP by investigating the genetic features of resistance and virulence hybrid plasmids. Methods The resistance and virulence phenotypes were determined by using antimicrobial susceptibility testing and the mouse bacteremia infection model, respectively. Plasmid profiles were investigated by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting, conjugation assay, and whole genome sequencing (WGS). Bioinformatics tools were used to uncover the genetic features of the resistance and virulence hybrid plasmids. Results Two ST11-KL64 CRKP clinical isolates (KP18-3-8 and KP18-2079), which exhibited enhanced virulence compared with the classic CRKP, were detected positive for blaKPC−2 and rmpA2. The virulence level of the hypermucoviscous strain KP18-3-8 was higher than that of KP18-2079. S1-PFGE, Southern hybridization and WGS analysis identified two novel hybrid virulence plasmids in KP18-3-8 (pKP1838-KPC-vir, 228,158 bp) and KP18-2079 (pKP1838-KPC-vir, 182,326 bp), respectively. The IncHI1B/repB-type plasmid pKP1838-KPC-vir co-harboring blaKPC−2 and virulence genes (rmpA2, iucABCD and iutA) but lacking type IV secretion system could transfer into non-hypervirulent ST11 K. pneumoniae with the assistance of a helper plasmid in conjugation. The IncFII/IncR-type virulence plasmid pKP18-2079-vir may have been generated as a result of recombination between a typical pLVPK-like virulence plasmid and an MDR plasmid. Conclusion Our studies further highlight co-evolution of the virulence and resistance plasmids in ST11-CRKP isolates. Close surveillance of such hybrid virulence plasmids in clinical K. pneumoniae should be performed.


Introduction
As the most predominant and prevalent species within carbapenem-resistant Enterobacterales (CRE), carbapenemresistant Klebsiella pneumoniae (CRKP) is notable for its considerable resistance and the treatment challenges.Current studies revealed that multidrug-resistant (MDR) or extensive drug-resistant (XDR) CRKP isolates were only susceptible to tigecycline, polymyxin, and new β-lactamase inhibitor compound preparations such as ceftazidime/avibactam (Yang et al., 2022), and these agents were also prescribed for the treatment of CRKP infections in China (Yahav et al., 2020;Han et al., 2022).
Recently, the emergence and increasing reports of carbapenemresistant hypervirulent K. pneumoniae (CR-HvKP), which are simultaneously hypervirulent and multidrug resistant, represent a new serious threat in China for clinical treatment and therefore be recognized as a real superbug (Gu et al., 2018).The occurrence of CR-HvKP could be through either horizontal transfer of resistance plasmids into HvKP strains or through acquisition of the virulence plasmids in CRKP strains (Yang et al., 2021;Han et al., 2022), and the latter pathway was more frequently reported to be responsible for the emergence of CR-HvKP isolates since the MDR K. pneumoniae isolates have demonstrated a greater tendency to acquire virulence genes than hypervirulent clones to acquire resistance genes (Wyres et al., 2019).A recent national surveillance concerning CRKP from China identified a high proportion of CRKP isolates (34.2%) co-harboring bla KPC−2 and virulence plasmids in China (Wang et al., 2018;Zhang et al., 2020).The co-existence of virulence and MDR plasmids in the same CR-HvKP strain provide the opportunity for recombination between the two types of plasmids.Our previous report described the fusion of a virulence plasmid p17-16-vir (MK191024) and an MDR plasmid p17-16-CTX (MK192097) during conjugation (Li et al., 2020), and recent studies also reported the fusion of the non-conjugative virulence plasmid with the bla KPC -positive plasmid through homologous recombination (Xie et al., 2021;Zhao et al., 2022).In addition, recent research studies revealed that the pLVPK-like virulence plasmid can be transferred from HvKP strains to ST11 CRKP and Escherichia coli strains via a selftransferable IncF plasmid in a variety of modes (Li et al., 2020;Xu et al., 2021).Furthermore, IS element-mediated insertion of bla KPC−2 into the virulence plasmid is another way to generate hybrid plasmids that simultaneously expressed the carbapenem resistance-and hypervirulence-associated phenotypes (Dong et al., 2018;Jin et al., 2021).The emergence of such plasmids, coupled with the co-transfer of resistance and virulence determinants, may directly facilitate the transformation of classical K. pneumoniae into CR-HvKP.This phenomenon further exacerbates the spread of CR-HvKP strains, posing a severe threat to public health and requiring continuous monitoring.In this study, we have reported two novel hybrid plasmids encoding MDR/carbapenem resistance and virulence phenotypes in KPC-2 producing K. pneumoniae belonging to ST11-KL64, which is regarded as the most commonly detected type responsible for the majority of CRKP infections in China (Zhang et al., 2020;Wang et al., 2022), indicating further evolution of the pLVPK-like virulence plasmids in CRKP.

Materials and methods
Bacterial isolates, antimicrobial susceptibility testing, and PCR detection Clinical K. pneumoniae strain 18-3-8 (KP18-3-8) was obtained from the urine culture of a 75-year-old man hospitalized in a respiratory unit of a local hospital in Pingdingshan city of Henan province on 14 July 2018, and KP18-2079 was recovered from the blood culture of a 42-year-old man, who was diagnosed with epilepsy and hospitalized at a neurosurgery unit of a teaching hospital of Zhengzhou University on 22 November 2018.The minimum inhibitory concentrations (MICs) of antimicrobial agents commonly used for the treatment of Enterobacterales infections were determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2018).The MIC results of the tested antibiotics were interpreted using the CLSI breakpoints, except tigecycline, which was interpreted in accordance with the EUCAST breakpoints (http://www.eucast.org/clinical_breakpoints/).An E. coli strain ATCC 25922 was used as the quality control.PCR and sequencing methods were employed to detect the presence of carbapenemaseencoding genes (bla KPC , bla NDM , bla IMP , and bla OXA−48−like ) and virulence factors rmpA/A2 in CRKP isolates (Qin et al., 2014;Yu et al., 2018).

S -PFGE and Southern blotting
S1-PFGE and Southern blotting were performed according to the method described in our previous study (Li et al., 2020).In brief, the agarose gel plugs containing whole-cell DNA of the strain were treated with S1 nuclease (TaKaRa, Dalian, China) and then PFGE was performed under the following conditions: 1% agarose solution for 18 h at 6 V/cm and 14 • C, with a pulse angle of 120 • and the pulse time linearly ramped from 2.16 s to 63.8 s.Digoxigenin-labeled bla KPC−2 and rmpA2-specific probes were hybridized with linear plasmids separated by S1-PFGE on nylon membranes (Millipore, USA) and then detected using an NBT/BCIP color detection kit (Roche, Germany).

Evaluation of hypermucoviscous and virulence phenotypes
The hypermucoviscous phenotype was determined using the string test (Russo et al., 2018).The mouse bacteremia infection model was used to assess the virulence of various K. pneumoniae strains as described previously (Yang et al., 2019).The nonhypermucoviscous ST1-K19 K. pneumoniae strain, KP18-208, was used as the control for low virulence (Qin et al., 2020), while an ST268-K20 K. penumoniae strain, KP19-2065, served as the control for hypervirulence.Mice in each group were infected intravenously with 5.0 × 10 5 CFU and 5.0 × 10 6 CFU of each K. pneumoniae strain tested.The survival rate of the mice was recorded daily for 5 days.The survival curves were plotted using the Kaplan-Meier method, and the statistical analysis was performed using the Log-rank test in GraphPad 9.5.1.

Results and discussion
Resistance phenotype and genotype of KPC--producing ST -KL K. pneumoniae isolates Both isolates KP18-3-8 and KP18-2079 were resistant to carbapenem and cephalosporins but were susceptible to colistin (Table 1).PCR-based screening for carbapenemase and virulenceencoding genes identified both bla KPC−2 and rmpA2 genes in each isolate.Further analysis using S1-PFGE and Southern blotting techniques with bla KPC−2 and rmpA2-specific probes indicated that these two genes were localized on plasmids of the same size in both KP18-3-8 (approximately 220 kb) and KP18-2079 (approximately 180 kb; Figure 1) strains, and therefore, we speculated that the two genes were located on the same plasmid.

An
IncHI1B/repB-type plasmid pKP1838-KPC-vir (MT035874) with a length of 228,158 bp was found to be the largest plasmid in the CRKP strain, KP18-3-8.It shared 99% of its identity and 93% of its coverage with a typical non-conjugative pLVPK-like virulence plasmid pvir-CR-HvKP267 (MG053312), which was carried by a tigecycline resistant KPC-2 producing hypervirulent K. pneumoniae KP267 isolated in 2015 in our previous study (Yao et al., 2018).Notably, compared with pvir-CR-HvKP267, the plasmid pKP1838-KPC-vir carried an additional 19 kb segment containing the carbapenemase-encoding bla KPC−2 gene, indicating that pKP1838-KPC-vir might have undergone further evolution by integrating exogenous drug resistance genes on the backbone of traditional virulence plasmids.In the KP18-3-8 strain, two bla KPC−2 genes were identified, each borne on distinct plasmids, pKP1838-KPC-vir and pKP1838-KPC.Detailed comparative analyses of the gene surrounding bla KPC−2 revealed a 45-kb segment harboring the gene within a non-Tn4401 element (NTE KPC−1b ), along with gene clusters conferring resistance to heavy metals such as mercury, copper, and silver.Despite a high degree of sequence homology (99% query coverage and 100% nucleotide identity between the segments), notable variations were observed in their genomic arrangement, including an inversion of the entire NTE KPC−1b element associated with the IS26 element in pKP1838-KPC relative to pKP1838-KPC-vir (Figure 2).Based on the above observations, we supposed that the plasmid pKP1838-KPC-vir co-harboring bla KPC−2 and virulence factors possibly evolved from a typical pLVPK-like virulence plasmid, and the genetic environment surrounding bla KPC−2 on pKP1838-KPC-vir similar to that on the bla KPC−2 -bearing plasmid pKP1838-KPC indicates that pKP1838-KPC might have been the source of the bla KPC−2 -carrying fragment for the plasmid pKP1838-KPC-vir.
The conjugative assay revealed that, despite lacking a type IV secretion system (T4SS), pKP1838-KPC-vir was successfully transferred to K. pneumoniae HS11286YZ6 at a frequency of 6.37 × 10 −8 per donor cell.Our previous study and recent reports demonstrated that the non-conjugative virulence plasmids could be mobilized by the conjugative plasmids belonging to IncF (Li et al., 2020;Xu et al., 2021).Thus, we suggested that the plasmid pKP1838-IncFII, which contained four necessary components [oriT, relaxase gene, type IV coupling protein (T4CP) gene, and T4SS gene cluster] for self-transfer in the same strain, might have served as a helper plasmid by sharing its T4SS with pKP1838-KPCvir to transfer.
A recent nationwide surveillance study on CRKP in China revealed that over 90% CRKP isolates were KPC-type carbapenemase producers, especially KPC-2, and over 30% KPC-2 producing CRKP isolates were positive for one or more plasmidborne virulence genes such as rmpA, rmpA2, iucA, and iroN (Zhang et al., 2020).Typically, the bla KPC−2 and virulence genes were carried by distinct plasmids.However, two recent reports described the homologous recombination-mediated fusion of the KPC plasmid and the virulence plasmid during the conjugation process, resulting in the generation of conjugative hybrid plasmids (Xie et al., 2021;Zhao et al., 2022).In addition to this homologous recombination pathway to generate the hybrid bla KPC−2 -bearing virulence plasmids, bla KPC−2 was rarely reported inserted into the virulence plasmid backbone.To date, only two plasmids, namely pKP70-2 (MF398271) and pCRHV-C2244 (MT644086), which were presumed to be generated as a consequence of an IS26-mediated bla KPC -bearing fragment inserted into the virulence plasmid, were identified in the ST23-K1 HvKP strain KP70-2 isolated from China and the ST11-K64 CRKP strain C2244 isolated from China, respectively (Dong et al., 2018;Jin et al., 2021).However, there was no additional bla KPC−2 -bearing plasmid other than the hybrid virulence-and resistance-encoding plasmid in each strain.In this study, the co-occurrence of bla KPC−2 that was carried by the NTE KPC−1b element in both virulence and MDR plasmids in the same isolate provided a more direct evidence of recombination events that occurred between virulence and MDR plasmids.Characteristics of an IncFII/IncR type multi-drug resistant-virulence plasmid An 182,326 bp IncFII/IncR-type virulence plasmid pKP18-2079-vir (MT090958) containing 237 open reading frames, with GC contents of 50.60%, was identified in K. pneumoniae KP18-2079 (Table 2).This hybrid plasmid harbored both virulence genes (iutA-iucABCD, rmpA, rmpA2, and peg344) and multiple antimicrobial resistance determinants, including beta-lactamase genes bla TEM−1B and bla CTX−M−65 , aminoglycoside resistance gene rmtB1, and phosphonic acids resistance gene fosA3, simultaneously.The BLAST analysis showed that this type of plasmid has not ) was observed between KP --/ KP -and KP -in × CFU and × CFU.

FIGURE
A comparison of Klebsiella pneumoniae KP -and KP --with hvKp KP -and the non-mucoviscous KP -.The hypermucoviscosity phenotype of K. pneumoniae KP -and KP --evidenced by the string test.After the overnight incubation at • C on blood agar, a mucoviscous string of at least mm in length was observed when lifting the KP -and KP --colony using a disposable inoculation loop.The ST -K hvKP strain KP -was used as a positive control while the non-mucoviscous ST -K strain KP -was used as a negative control.

Virulence of KPC--producing ST -KL K. pneumoniae isolates
The mouse infection model was employed to assess the virulence of KP18-3-8 and KP18-2079.As shown in Figure 4, infections of mice with 5 × 10 6 CFU of strains KP19-2065, KP18-3-8, and KP18-2079 led to a 100% mortality rate at 12 h, whereas the mortality rate was recorded as 0% for the strain KP18-208, a classic CRKP control strain (Figure 4A).Consistently, when infected at the lower dose of 5 × 10 5 CFU of KP19-2065, KP18-3-8, and KP18-2079, it resulted in a 100% mortality rate at 12, 36, and 60 h, respectively, whereas a 0% mortality rate was observed for KP18-208 (Figure 4B).These data confirmed that KP18-3-8 and KP18-2079 exhibited a virulence level slightly lower than that of KP19-2065 but much higher than that of KP18-208, a classic CRKP strain without a virulence plasmid.In addition, the virulence level of KP18-2079 without the hypermucoviscosity phenotype was lower than that of KP18-3-8, a hypermucoviscous strain which was positive for the string test (Figure 5).
ST11-KL64 CR-HvKP, which was found to be derived from an ST11-KL47-like ancestor through recombination, has demonstrated enhanced virulence and higher mortality rates than that of ST11-KL47 CRKP (Zhou et al., 2020).Moreover, a recent surveillance study on CR-HvKP revealed that ST11-KL64 CR-HvKP became the most common detected CR-HvKP type in different regions of China, especially in Henan province (Zhang et al., 2020).In our study, we isolated two ST11-KL64 CRKP strains that exhibited an XDR profile, with susceptibility only to colistin.Notably, these strains demonstrated heightened virulence compared to their non-virulent plasmid-bearing CRKP counterparts.The presence of composite plasmids encoding both virulence and resistance factors significantly contributed to their carbapenem resistance/MDR profiles and virulent phenotypes.

Conclusion
Overall, our study identified two novel hybrid virulence plasmids in ST11-KL64 CRKP strains.The IncHI1B/repB-type plasmid pKP1838-KPC-vir co-harboring bla KPC−2 and virulence factors may have been generated as a consequence of integration of bla KPC−2 -bearing fragment into a typical pLVPK-like virulence plasmid, and the IncFII/IncR-type virulence plasmid pKP18-2079-vir may have been generated as a result of recombination between a typical pLVPK-like virulence plasmid and an MDR plasmid.Our findings revealed further evolution of the pLVPKlike virulence plasmids which were found to be present in ST11 CRKP since 2015 (Gu et al., 2018).The acquisition of such a virulence-and resistance-encoding plasmid, especially the bla KPC−2 -bearing virulence plasmid simultaneously conferring carbapenem resistance and enhanced virulence in classical K. pneumoniae isolates, may enable them to become CR-HvKP.The emergence of such strains, which may cause severe infections in individuals difficult to treat with current antibiotics, warrants worldwide attention.Close surveillance on the epidemiology of such hybrid plasmids is urgently warranted to control their further dissemination.organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

FIGURES-
FIGURE S -PFGE and Southern hybridization analyses of KP -and KP --using the bla KPC− (I) and rmpA probes (II).Lane M, reference standard strain H restricted with XbaI (sizes are given in kilobases).

FIGURE
FIGUREThe rmpA and bla KPC− co-harboring plasmid pKP -KPC-vir and the bla KPC− -bearing plasmid pKP -KPC in Klebsiella pneumoniae strain KP --.Circular and linear comparisons of pKP -KPC-vir, pKP -KPC, p _L , and pLVPK plasmids.The pKP -KPC-vir and pKP -KPC plasmids located at the outermost circle was used as the reference plasmid to perform sequence alignment with BLASTn by BRIG software.

FIGURE
FIGURE The MDR virulence plasmid p --vir and the bla KPC− -bearing plasmid p --KPC in Klebsiella pneumoniae strain KP -. (A, B) Circular maps of plasmid p --KPC and p --vir recovered from K. pneumoniae KP -with plasmids recorded in the NCBI database by BLAST Ring Image Generator (BRIG).(C, D) Linear alignment of plasmid p --KPC and p --vir recovered from K. pneumoniae KP -using Easyfig.

FIGURE
FIGURE Virulence potential of Klebsiella pneumoniae KP --and KP -in a mouse infection model with an inoculum of × CFU and × CFU.Survival of mice (n = ) infected by the indicated inoculum of each K. pneumoniae strain at h is shown.The test strains included K. pneumoniae strain KP --, K. pneumoniae strain KP -, ST -K hypervirulent K. pneumoniae strain KP -(hypervirulence control), and ST -K classic K. pneumoniae strain KP -(low-virulence control).A log-rank (Mantel-Cox) test was performed for the indicated curves.A significant di erence (P < .)was observed between KP --/ KP -and KP -in × CFU and × CFU.
TABLE Molecular characteristics of XDR CRKP strains KP --, KP -and their plasmids.