Coexistence of blaNDM-1 and blaIMP-4 in One Novel Hybrid Plasmid Confers Transferable Carbapenem Resistance in an ST20-K28 Klebsiella pneumoniae

Objectives We identified a novel hybrid plasmid simultaneously carrying blaNDM-1 and blaIMP-4 in an ST20-K28 carbapenem-resistant Klebsiella pneumoniae (CRKP) strain AZS099 and reported its detailed genetic and phenotypic characterization. Methods Antimicrobial susceptibility was characterized using broth microdilution method. Complete genome characteristics and plasmid detailed analysis were carried out by PacBio Sequel and Illumina sequencing and further bioinformatics analysis. Conjugation assay, S1-PFGE, Southern blot, plasmid stability, and fitness cost were conducted to the phenotypic characterization of this novel hybrid plasmid. Results AZS099 was isolated from a blood specimen obtained from a 3-month baby who presented with biliary tract infection. Susceptibility testing showed that AZS099 was resistant to almost all β-lactams examined, including cephalosporins, combinations of β-lactams and β-lactamase inhibitors, carbapenems, and aztreonam. PacBio and Illumina sequencing together with S1-PFGE and Southern blot showed that blaNDM-1 and blaIMP-4 were simultaneously located on a 296 kb IncFIB(K)/IncHI1B/IncX3 plasmid (pAZS099-NDM-IMP), which consists of four main parts that came from four different types of plasmids. The region harboring blaIMP-4 is located in a class 1 integron designated as In0, which is located in an IS6100-IS26 transposon-like structure with a total length of ~5 kb. The region harboring blaNDM-1 is located in the Tn125 transposon remnant. Conjugation and transformation assay confirmed that the plasmid pAZS099-NDM-IMP has the potential for horizontal transfer and displayed high stability (retention rate > 95%). Furthermore, growth curve assessment confirmed that the presence of pAZS099-NDM-IMP exhibits no growth pressure on bacteria. Conclusion Our research reported a hybrid plasmid coharboring blaNDM-1 and blaIMP-4 in an ST20-K28 CRKP strain. The emergence of novel hybrid plasmid could threaten the control of antimicrobial resistance and should be closely supervised.

In recent years, due to the widespread use of broad-spectrum antimicrobial drugs, K. pneumoniae has also shown multiple drug resistance, especially carbapenem resistance, representing a serious threat to human health and a serious challenge for clinicians (Meatherall et al., 2009). Moreover, carbapenem-resistant K. pneumoniae (CRKP) has led to severe infections in weak and immunocompromised patients around the world, resulting in prolonged length of hospital stays and increased mortality (Tzouvelekis et al., 2012). The primary type of CRKP is the high-risk clonal complex (CC) 258, including sequence type (ST) 258, ST11, and some closely related STs. Additionally, other clones may also give rise to the international spread of CRKP, and ST20 CRKP was firstly reported in 2020 (Qiao et al., 2020).
The main mechanism of carbapenem resistance in K. pneumoniae is the acquisition of carbapenemases (carbapenem hydrolyzing enzymes). The predominant carbapenemase in K. pneumoniae is KPC (class A serine β-lactamase), followed by class B metallo β-lactamase NDM and IMP . Both NDM and IMP can hydrolyze a broad spectrum of β-lactams covered penicillins, cephalosporins, even carbapenems. Not only that, strains containing NDM and IMP are also resistant to ceftazidime/avibactam (Bush, 2013;Bush and Bradford, 2019). Although IMP-producing CRKP and NDM-producing CRKP have been widely identified, CRKP with the coexistence of IMP-4 and NDM-1 has been rarely reported. To our knowledge, only three IMP-4 and NDM-1 co-producing CRKP isolates have been identified (Chen et al., 2015;Liu et al., 2018). The strains belong to ST1043, ST571, and ST273, respectively. In the ST273 strain, the structure of the plasmid was further characterized, and two carbapenemase genes are located on two different plasmids (bla IMP-4 carried by a self-transmissible IncHI5 plasmid and bla NDM-1 located on an IncN self-transmissible plasmid). Plasmid-encoded NDM/ IMP poses a critical clinical threat because they are carried by mobile genetic elements which can harbor and spread resistance genes to other antimicrobial classes (Bush and Bradford, 2019). However, so far, the detailed genomes of CRKP strains with two enzymes (NDM-1 and IMP-4) simultaneously located on the same plasmid have not been reported. In this study, we identified an ST20 CRKP clinical strain AZS099 simultaneously carrying bla NDM-1 and bla IMP-4 genes on one novel hybrid plasmid and firstly report its detailed structure characterization here.

Bacterial Strains
The strain was isolated on August 21, 2018, from a blood specimen obtained from a 3-month baby who presented with biliary tract infection in Guangzhou, Guangdong, China, and was identified by Vitek MS MALDI-TOF (BioMérieux) system in Peking Union Medical College Hospital. The hemogram indexes are the results of the first examination after admission within 1-3 days of the occurrence of a bloodstream infection. Escherichia coli rifampicin-resistant strain EC600 were used in the construction of transconjugants experiments.

Antimicrobial Susceptibility Testing
We used the broth microdilution method for antimicrobial susceptibility testing as per the Clinical and Laboratory Standards Institute (CLSI) recommendations (CLSI, 2020). MICs were interpreted according to the CLSI M100-S30 guidelines (CLSI, 2020). Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and K. pneumoniae ATCC700603 were used as quality controls.
sequencing was implemented using the PacBio Sequel platform. A 10 kb SMRTbell library was prepared from sheared genomic DNA (≥5 g) with an additional bead clean-up step before primer annealing (Zhu et al., 2016).
We also re-sequenced this isolate using Illumina sequencing platform in order to correct the polymer errors generated during PacBio sequencing. Paired-end libraries were constructed from 5 μg of isolated genomic DNA using a TruSeq DNA sample prep kit (Illumina Inc., San Diego, California, United States), and sequenced using Illumina with a read length of 2 × 150 bp. A threshold of 0.01 (phred score of 20) was used for raw reads filtration. Genome assembly was conducted using Unicycler (Wick et al., 2017) from short and long sequencing reads.
Genome sequences were initially annotated with the rapid prokaryotic genome annotation software Prokka (Seemann, 2014) and further annotated by BLAST searches against the RefSeq and UniProtKB/Swiss-Prot databases. Pairwise sequence comparisons were also performed using BLAST. All mobile elements were identified using ExPASy (Artimo et al., 2012), ISFinder (Siguier et al., 2006), the Transposon Registry (Tansirichaiya et al., 2019), INTEGRALL (Moura et al., 2009), and Integron Finder. 1 Comparisons of plasmid structures were conducted to analyze the sequence homology. BRIG software was used for the generation of plasmid circular structure maps.
Sequence Type, Serotype, Virulence, and Antimicrobial Resistance Genes Analysis STs were determined using SRST2 (Inouye et al., 2014) based on the Illumina reads. Serotype was analyzed using SerotypeFinder 2.0 (Joensen et al., 2015) based on the assembled contigs. Virulence genes were downloaded from Virulence Factor Database (VFDB). FASTA sequences of the downloaded virulence genes were used for searching corresponding genes by BLAST with coverage of 50% and identity of 90%. Antimicrobial resistance genes were analyzed through ResFinder 4.0 based on the whole genomic sequences.

Plasmid Conjugation Assay
The transferability of pAZS099-NDM-IMP among isolates was determined using AZS099 as donor and E. coli EC600 as the recipient by conjugation assay. The procedure of conjugation was performed according to the protocol previously described (Yang, 2015). 100 μl overnight culture of clinical isolate (AZS099) in Luria-Bertani (LB) broth containing meropenem (2 μg/ml) and 200 μl overnight culture of recipient EC600 in LB broth containing rifampicin (100 μg/ml) were mixed into 5-ml antibiotic-free LB broth and were incubated at 37°C for 18 h. Then, the conjugant mixtures were placed on selective LB agar containing 100 μg/ml rifampin and 1 μg/ml meropenem. PCR analysis and agarose gel electrophoresis were performed to confirm the effects of transconjugation. 1 https://github.com/gem-pasteur/Integron_Finder S1-PFGE, Southern Blot, and PCR Amplification and Sanger Sequencing To further characterize the plasmid pAZS099-NDM-IMP in AZS099 and the transconjugant EC600-pAZS099-NDM-IMP, S1 nuclease digestion analyzed with PFGE was performed using a CHEF Mapper XA apparatus (Bio-Rad Laboratories, Hercules, CA). Briefly, the adjusted bacterial suspension mixed in a 1% Seakem Golden Agarose and 1% Sodium dodecyl sulfate (SDS) was digested with proteinase K for 2 h at 56°C and then further treated with S1 nuclease. XbaI-digested Salmonella enterica serovar Braenderup H9812 as a reference molecular weight marker. The electrophoresis conditions used in this study were as follows: initial switch time, 3 s; a final switch time, 36 s; gradient, 6 V/cm; angle, 120; temperature, 14°C; and time, 18 h. After the electrophoresis was complete, the gel was stained with Ultra GelRed and visualized with the ImageQuant LAS 500 (GE LifeScience, Pittsburgh, United States) or transferred to nylon membrane (GE LifeScience, Pittsburgh, United States), followed by hybridization with digoxigenin-labelled probes specific to NDM-1 or IMP-4. Probe labelling and signal detection were done by DIG DNA Labeling and Detection Kit (Roche Diagnostics, GmbH, Germany).

Plasmid Stability Testing
Plasmid stability was assessed upon several successive subculture of plasmid-carrier AZS099 strain for 10 days with 1:1000 dilution on antibiotic-free LB broth at 37°C in a shaking bath (180 rpm). The 10th day's culture was serially diluted and plated onto LB plates with or without meropenem (1 mg/L). The retention rate of plasmid was calculated by dividing the number of colonies on LB plates supplemented with meropenem by the number of colonies on LB plates (Gao et al., 2020). 50 colonies on the antibiotic-free LB plates were randomly selected and subjected to PCR validation of bla IMP (primer sequence 5′-3′: Forward TGGTTTGTGGAACGTGGCTA; Reverse GAGTGAT GCGTCTCCAGCTT) and bla NDM (primer sequence 5′-3′: Forward CTCGCATTTGCGGGGTTTTT; Reverse ACGCCTC TGTCACATCGAAA) genes.

Fitness Cost Assessment
Growth curve was used to assess the fitness impact of plasmid carriage under noncompetitive conditions as Wang et al. described previously . The recipients and transconjugants carrying pAZS099-NDM-IMP plasmid from AZS099 isolate were cultured overnight in LB broth without or with 2 mg/L meropenem at 37°C, respectively. Bacterial suspensions were diluted 1:1000 in LB medium (approximately Frontiers in Microbiology | www.frontiersin.org 10 6 CFU/ml) and grown at 37°C in triplicate for 36 h by Epoch™ 2 Microplate Spectrophotometer from BioTek Instruments. The OD600 of each culture was measured every 30 min, and the plates were shaken for 15 s before measuring. GraphPad Prism version 8 (GraphPad Software, Inc., United States) was used to estimate the growth curves. Statistical significance was determined for an overall error at 0.05 level (95% confidence interval) using one-way ANOVA, followed by Tukey tests.

Clinical Characteristics of Patients With AZS099
The Klebsiella pneumoniae strain AZS099 was isolated from a blood specimen obtained from a 3-month baby who presented with biliary tract infection. The patient was hospitalized with cholangitis after biliary atresia and had a fever >39°C lasting for more than 3 days. The total white blood cells (WBC), neutrophil percent (NEU), C-reactive protein (CRP), and procalcitonin (PCT) were 12.85*10 9 /L, 22.7%, 161.71 mg/L, and 1.52 ng/ml, respectively. In the duration of hospitalization, the patient was treated with meropenem (0.15 g, q 8 h, 5 days).

Genomic Characteristics and Antimicrobial-Resistance Genotype Analysis
To reveal the genetic basis of the multidrug-resistant (MDR) phenotype, we obtained the complete genome of the clinical isolate AZS099 including a 5.2-Mb chromosome, a 296-kb plasmid (pAZS099-NDM-IMP), and a 15-kb plasmid (pAZS099-2; Table 2 and Figure 1). Bioinformatics analysis provided the general information of the chromosome genome, including GC% content (57.63%), predicted protein-coding genes (4,998), average gene length (922 bp), and coding region (88.82%). The two plasmids possess a shorter average gene length, lower ratio of coding regions, and lower GC% content. Sequence type (ST) and serotype analysis showed that AZS099 belongs to ST20 and K28.
We also performed a full-plasmid BLAST comparative analysis of pAZS099-2 in NCBI, and it exhibited 99% identity with a plasmid from K. pneumoniae strain FDAARGOS_630 (CP044037.1; Figure 2B). This plasmid (pAZS099-2) is IncFIA(HI1) type, 15,410 bp in length, constituting a circular DNA with an average G + C content of 45.55% and 17 open reading frames. No virulence and antimicrobial-resistance genes were found.

Transferability, Stability, and Fitness Cost of the Resistance Plasmid (pAZS099-NDM-IMP)
To evaluate the transferability of the resistance plasmid carrying bla NDM-1 and bla IMP-4 , conjugation assays were performed by co-culturing AZS099 with E. coli EC600. S1-PFGE confirmed the size of the plasmid, which was consistent with the donor AZS099, in the transconjugant (Supplementary Figure S1A). The following Southern blot and PCR results confirmed the coexistence of bla NDM-1 and bla IMP-4 on the plasmid pAZS099-NDM-IMP (Supplementary Figures S1B,C, S2, S3). These results indicated that the plasmid pAZS099-NDM-IMP harboring bla NDM-1 and bla IMP-4 in the clinical strain AZS099 has the potential for horizontal transfer. Moreover, the two carbapenem resistance genes bla NDM-1 and bla IMP-4 were also transferred along with the plasmid. As shown in Table 1, the susceptibility results of the transconjugant were consistent with the donor (clinical isolate AZS099): resistant to cephalosporins, carbapenems, combinations of β-lactams and β-lactamase inhibitors. And the MIC range of meropenem for NDM-IMP-bearing transconjugants was >16 μg/ml.
The stability of plasmid pAZS099-NDM-IMP was also evaluated during serial passage in the laboratory last for 10 days. It displayed high stability, as the retention rates were still over 95% at the end of the experiment for both bla NDM-1 and bla  , which were confirmed by PCR.
Further, the effect of acquiring bla NDM-1 and bla IMP-4 plasmid on biological fitness cost was evaluated. Of interest, no significant differences in the growth rate were observed among the recipient strain EC600 and the transconjugant harboring bla NDM-1 and bla IMP-4 plasmid pAZS099-NDM-IMP (p > 0.05; Figure 4). Therefore, the antibiotic resistance mediated by pAZS099-NDM-IMP did not increase the growth fitness cost of the resistant strains compared to their susceptible counterparts.

DISCUSSION
In this study, we identified an ST20-K28 bloodstream infection CRKP carrying a novel hybrid plasmid coharboring bla NDM-1 and bla IMP-4 and characterized the detailed structure of this hybrid plasmid. Moreover, we assessed the transferability, stability, and impact on the strain survival of the plasmid.
The continuous and often inappropriate use of β-lactam drugs has led to the emergence and global spread of extendedspectrum β-lactamases (ESBLs) and, more recently, carbapenemases, which limits the use of all β-lactam agents. According to previous epidemiological studies, the most commonly identified carbapenemases in K. pneumoniae is KPC-2 Yang et al., 2020). And K. pneumoniae strains with bla KPC-2 have been widely disseminated in several regions, including Europe, South and North America, and Asia (Nordmann et al., 2009). Additionally, the emergence of FIGURE 4 | Growth curve comparison. Growth curves of recipient strain E. coli EC600 and the transconjugants harboring bla NDM-1 and bla IMP-4 plasmid pAZS099-NDM-IMP with and without meropenem (value of p >0.05). EC600 means the recipient strain E. coli EC600. EC600 + pAZS099-NDM-IMP means the transconjugant harboring bla NDM-1 and bla IMP-4 plasmid pAZS099-NDM-IMP in antibiotic-free LB broth. EC600 + pAZS099-NDM-IMP+MEM means the transconjugant harboring bla NDM-1 and bla IMP-4 plasmid pAZS099-NDM-IMP in LB agar plates containing meropenem. metallo-β-lactamases (MBLs) NDM-1 and IMP-4 has also become an established major public health, posing new challenges to the treatment of infectious diseases. MBLs are of great concern due to their carbapenemase activity, their rapid spread in major human opportunistic pathogens, while no clinically useful inhibitor is available yet (Carcione et al., 2021;Legru et al., 2021). Avibactam is one of the most important drugs and inhibits β-lactamases of class A, including KPC, class C, and certain class D β-lactamases, mainly OXA-48. Unfortunately, it is not active against MBL enzymes (β-lactamases of class B) (Tooke et al., 2019;Carcione et al., 2021). Researchers to develop MBL inhibitors have been focused on compounds that bind and/or chelate zinc ions at the active site, but the high heterogeneity of these enzymes poses a serious problem. Currently, no drugs are close to the clinic (Tooke et al., 2019;Gonzalez-Bello et al., 2020;Carcione et al., 2021;Davies and Everett, 2021).
At present, IMP-producing CRKP and NDM-producing CRKP have been widely identified. However, CRKP with the coexistence of IMP-4 and NDM-1 has been rarely reported. To our knowledge, only three IMP-4 and NDM-1 co-producing CRKP isolates have been identified in Southeast Asia (Chen et al., 2015;Liu et al., 2018). In 2015, Chen et al. reported the coexistence of bla NDM-1 and bla IMP-4 in two K. pneumoniae isolates (ST1043 and ST571). However, the sequence features of plasmids carrying these two genes were not characterized (Chen et al., 2015). In 2018, Liu et al. reported a ST273 CRKP carrying bla NDM-1 and bla IMP-4 . The two carbapenemase genes are located on two different plasmids . Except for K. pneumoniae, Zhang et al. reported a KPC-2-, NDM-1-, and IMP-4-producing K. michiganensis isolate recently, and bla KPC-2 , bla NDM-1 , and bla IMP-4 were carried on different conjugative plasmids (Zhang et al., 2022). Moreover, Li et al. described a hybrid plasmid co-harboring bla NDM-1 and bla IMP-4 from K. michiganensis (Li et al., 2021). However, no similar plasmids were reported in K. pneumoniae. Our study, for the first time, reported a CRKP strain with the two enzymes (IMP-4 and NDM-1) simultaneously located on one hybrid plasmid and characterized the detailed structure of this hybrid plasmid (pAZS099-NDM-IMP). Additionally, the MIC of meropenem, imipenem, and ertapenem for AZS099 is >16 μg/ml, > 16 μg/ ml, and > 4 μg/ml, respectively. We noticed that the MIC of both meropenem and imipenem for most reported NDM-1 harboring K. pneumoniae strains is 8 μg/ml . And the MIC of ertapenem for IMP-4 harboring K. pneumoniae strains is >4 μg/ml, while it is <8 μg/ml of meropenem and imipenem (Limbago et al., 2011;Lai et al., 2017). This shows that there is an increase in MIC value of the strain AZS099 coharboring bla NDM-1 and bla IMP-4 . Although the patient was eventually cured and discharged, which might be due to timely surgical drainage, followed by appropriate ICU care and early goal directed therapy, this kind of strain should also be paid more attention.
Moreover, the spread of plasmid-mediated carbapenemresistant clinical isolates is a severe threat to global health. The two enzymes (IMP-4 and NDM-1) are located on the same plasmid, which makes it easier for the two enzymes to spread at the same time. Our result also implied that the hybrid plasmid co-harboring bla NDM-1 and bla IMP-4 is stable and has the potential for horizontal transfer. In addition, this hybrid plasmid did not increase the growth fitness cost of the clinical strain, which highlights the importance of controlling the spread of NDM-1-IMP-4-CRKPs. Detailed plasmid structure analysis showed that the plasmid replicon type of pAZS099-NDM-IMP was IncFIB(K)/IncHI1B/IncX3. bla NDM-1 came from the IncX3-type plasmid similar to pA575-NDM and bla IMP-4 was integrated into this region. The IncX3-type plasmid has been found to be widely distributed in various species in different regions of China and the United Arab Emirates, and may contribute to the spread of bla NDM-1 (Qu et al., 2015). In our strain, bla NDM-1 is located in the Tn125 transposon remnant without ISAba125 fragment. We speculated that bla NDM-1 might become a relatively fixed component on the plasmid and disseminated together with IncX3 plasmid (Ho et al., 2012). In this hybrid plasmid, the bla IMP-4 gene in class 1 integron In0 flanked by IS26 and IS6100 on both sides. Class 1 integrons are responsible for the dissemination of the bla IMP gene (Huang et al., 2021). Besides, the insertion sequence also plays important roles in the transmission of resistant genes. Previous studies on bla IMP-4 -carrying plasmids showed that IS26 might play important roles in disseminating bla  in different plasmids (Liu et al., 2021). Moreover, the region of bla NDM-1 , bla SHV-12 and bla IMP-4 lay adjacent to one another and formed a continuous 20 kb multidrug-resistant region. We further compared pAZS099-NDM-IMP (296,148 bp) with the reported hybrid plasmid (pKOC7525_1, 397,447 bp) co-harboring bla NDM-1 and bla IMP-4 in K. michiganensis (Li et al., 2021). The result showed that the plasmid sequences and structure of the two strains were different from each other, and pKOX7525_1 only covered 25.67% of pAZS099-NDM-IMP (Supplementary Figures S4, S5), which indicated the different sources of these two bla NDM-1 and bla IMP-4 co-harboring plasmids. Moreover, bla NDM-1 and bla IMP-4 are ~100 kb apart from each other on the plasmid (pKOC7525_1) of K. michiganensis, while it is only ~20 kb on pAZS099-NDM-IMP, which might greatly increase the risk of simultaneous transfers of this multidrugresistant region in K. pneumoniae. We also compare pAZS099-NDM-IMP with the plasmids containing NDM-1 (pK210011_ NDM) and IMP-4 (pK210011_IMP) in K. michiganensis isolate K210011 reported by Zhang et al. recently (Zhang et al., 2022). In K. michiganensis isolate K210011, bla NDM-1 , and bla IMP-4 were carried by different conjugative plasmids, and pK210011_NDM covered 31.83%, pK210011_IMP covered 17.87% of pAZS099-NDM-IMP, respectively (Supplementary Figures S4, S5). These results further illustrate the novelty of the plasmid pAZS099-NDM-IMP.
Our research reported a hybrid plasmid coharboring bla NDM-1 and bla IMP-4 in an ST20-K28 CRKP strain. The emergence of novel hybrid plasmid may contribute to the further dissemination of carbapenem-resistant genes and pose new challenges to the treatment of infectious diseases. Surveillance of carbapenemases especially MBLs, in K. pneumoniae is urgently needed to control and prevent the spread of these resistance determinants in China.