Reginal dissemination of transferable repB(1701) plasmid carrying blaNDM-4 in Asia: emergence of tandem repeats with blaNDM-4 mediated by IS26 in Klebsiella pneumoniae

Reginal dissemination of transferable repB(1701) plasmid carrying blaNDM-4 in Asia: emergence of tandem repeats with blaNDM-4 mediated by IS26 in Klebsiella pneumoniae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Reginal dissemination of transferable repB(1701) plasmid carrying blaNDM-4 in Asia: emergence of tandem repeats with blaNDM-4 mediated by IS26 in Klebsiella pneumoniae Huanran Zhang, Hao Xu, Ruishan Liu, Xiaojing Liu, Yu Yang, Kexin Guo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4904070/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The worldwide spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) has led to a major challenge to human health. The emergence and prevalence of transferable plasmid carrying carbapenemase encoding gene is undoubtedly a potential factor in increasing the burden of carbapenem resistance. In this study, we described the reginal dissemination of repB(1701) plasmid carrying bla NDM-4 in China, with the coexistence of bla NDM-4 and bla OXA-9 in two independent K. pneumoniae isolates for the first time. Whole-genome sequencing and plasmid characterization of isolates L2388 and L3835 showed both bla NDM-4 and bla OXA-9 genes are situated on the transferable plasmid, designated as pL2388-NDM-OXA [120,100 bp, IncFII(K)-repB(R1701)] and pL3835-NDM-OXA [127,486 bp, repB(R1701)]. Notably, two copies of bla NDM-4 were identified in different orientations within pL3835-NDM-OXA. The bla NDM-4 sequence is relatively conserved ( bla NDM - 4 -ble-trpF-dsbD-cutA-groES-groL ), with various mobile genetic elements (MEGs) surrounding it, particularly IS 26 , which may play a pivotal role in gene-level dissemination. The previously unrecognized distribution of these plasmids across select regions of Southern China and Vietnam suggests a potentially wider epidemiological range than initially presumed in the context of microbiology and epidemiology. This is the first report of transferable IncFII(K)-repB(R1701)/repB(R1701) plasmids carrying the bla NDM-4 gene, relevant detection and investigation measures should be taken to control the prevalence. repB(R1701) NDM-4 mobile genetic elements tandem repeats Klebsiella pneumoniae Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Klebsiella pneumoniae is a Gram-negative bacterium known for its encapsulation and lack of motility. It can lead to a range of human infections. Similar to numerous Gram-negative bacteria, it is increasingly developing resistance to prominent antibiotics including carbapenems and cephalosporins. Notably, the rise and proliferation of transferable plasmids with carbapenemase-encoding genes are clearly major contributors to the growing challenge of carbapenem resistance. NDM-4, a class B carbapenemase, was first detected in ST648 Escherichia coli from an Indian patient which displayed significant hydrolytic activity against carbapenems and multiple cephalosporins [ 1 ]. Single-copy instance of bla NDM−4 is more prevalent. OXA-9, a constituent of the OXA-type β-lactamase family [ 2 ], was first detected by M.E. Tolmasky [ 3 ], exhibits heightened resistance to ceftazidime and various β-lactam antibiotics. The simultaneous presence of NDM-4 and OXA-9 is still rare on a global scale. Mobile genetic elements (MGEs; e.g., plasmid, insertion sequence (IS), transposons, and integron) play a key role in facilitating the acquisition and spread of antibiotic resistance genes (ARGs) [ 4 ]. The most common replicon types identified among NDM-4 are insertion sequence (IS) Aba12 5, IS 5 , IS 26 and IS 91 [ 5 – 8 ], Alterations in mobile genetic elements (MGEs) may facilitate the dissemination of NDM-4. The most common replicon types identified among bla NDM−4 harbouring plasmids in Enterobacteriaceae are IncX3 [ 5 , 9 , 10 ] or IncF [ 1 , 11 , 12 ]. It is noteworthy that NDM-4 genes are always associated with several ISs. The bla NDM−4 gene is located on a plasmid, enabling it to be horizontally transferred between bacteria, which allows its resistance to spread rapidly. Tracking and controlling the spread of this gene is a significant challenge. Additionally, IncF-repB(R1701) plasmids have recently been reported as a substantial threat to public health due to their mobile resistance to both tigecycline and carbapenem [ 13 ]. However, the link between bla NDM−4 gene and conjugative IncF-repB(R1701) plasmid has never been described. Here, we report the co-presence of OXA-9 and NDM-4 on a single plasmid in two strains of K. pneumoniae , detailing their genomic features through ARG profiling and whole-genome sequencing (WGS). Using a global dataset compiled from GenBank, we analyzed the worldwide epidemiological characteristics of plasmids harboring the bla NDM−4 gene. Materials and Methods Strains and antimicrobial susceptibility testing Strains L2388 and L3835 of K. pneumoniae were isolated and characterized from stool samples obtained from two tertiary hospitals in Hangzhou, Zhejiang Province, China. L2388 was isolated from a 35-year-old female patient after kidney transplantation in Shulan (Hang Zhou) Hospital. L3835 was isolated from a 45-year-old male patient presenting with non-Hodgkin lymphoma (NHL) in The First Affiliated Hospital of Zhejiang University. Subsequently, species were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Bruker, Bremen, Germany) The MICs of antibiotics were determined using the agar dilution method, and the results were interpreted following Clinical and Laboratory Standards Institution (CLSI) guidelines (M100-S25) except polymixin B and tigecycline, which were assessed using the broth microdilution method [ 14 ] . Pseudomonas aeruginosa ATCC27853 and E. coli ATCC25922 were used as the quality control. The tested antibiotics included: imipenem, meropenem, ceftriaxone, cefotaxime, ceftazidime, cefepime, levofloxacin, ciprofloxacin, amikacin, gentamicin, piperacillin/tazobactam, ceftazidime/avibactam, amoxicillin/clavulanic, tigecycline, fosfomycin, chloramphenicol, polymixin B, and aztreonam. S1-Pulsed-Field Gel Electrophoresis, Southern blotting and conjugational transfer We detected the numbers and sizes of plasmids in L2388 and L3835 using S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE). The positions of plasmids containing bla NDM-4 and bla OXA-9 were identified through Southern blotting and hybridization with specific probes labeled with digoxigenin. Moreover, we employed rifampin-resistant E. coli 600, rifampin-resistant P. aeruginosa PAO1R(PARI) and polymixin-resistant P. aeruginosa (PACO) as the recipient in conjugation experiments to explore the potential transfer of plasmids. The transconjugants were selected with three Mueller-Hinton agar medium (200 mg /L rifampicin and 2 mg /L meropenem, 200 mg /L polymixin and 2 mg /L meropenem, 400 mg /L rifampicin and 2 mg /L meropenem) and subsequently identified using MALDI-TOF/MS. We detected the presence of bla NDM-4 genes through PCR, along with conducting antimicrobial susceptibility testing to confirm the successful transfer of plasmids carrying the target genes. Whole-genome sequencing and bioinformatics analysis Whole-genome sequencing (WGS) was conducted for isolates L2388 and L3835 using both the Illumina NovaSeq 6000 (Illumina, San Diego, CA, United States) and Oxford Nanopore (Oxford Nanopore Technologies, Oxford, United Kingdom) platforms. Unicycler was used for data assembly, and sequence alignment and similarity searches were carried out with the BLASTn software. Prokka was used for annotatted. ARGs were identified using ResFinder 4.1, virulence genes by VirulenceFinder and the plasmid types were determined using PlasmidFinder 2.0 [15]. The multilocus sequence typing (MLST) was determined using the MLST service provided by CGE (http://www.genomicepidemiology.org/). The genetic context of the antimicrobial ARGs was visualized using Easyfig [16]. Lastly, the circular comparative images of multiplex plasmids were generated using the BLASTn Ring Image Generator (BRIG) [17]. Results Characterization of the strains L2388 was obtained from a stool sample of a kidney transplant patient undergoing immunosuppressive therapy. L3835 was obtained from a stool sample of a patient who was hospitalized for non-Hodgkinlymphoma and subsequently underwent systemic chemotherapy(Table 1 ). Table 1 Genome and plasmid analysis of K. pneumoniae strains isolated L2388 and L3835 Strains Isolation details Genome size MLST Chromosome/ plasmid Length Plasmid type Resistance genes Virulence genes L2388 Shulan (Hang Zhou) Hospital, Hangzhou, China, 2020 5,614,837 bp 726 Chromosome 5,293,684 / bla SHV−81 , bla SHV−110 , oqxA , oqxB fimH, iutA, mrkA pL2388_2 139,247 IncFIB(K) sul1 , sul2 , dfrA27 clpK1 pL2388-NDM-OXA 120,100 IncFII(K)-repB(R1701) bla NDM−4 , bla OXA−9 , bla CTX−M−14 , bla LAP−2 , traT pL2388_4 41,846 IncR bla TEM−1B , bla CTX−M−55 / pL2388_5 15,054 IncFIA(HI1) / / pL2388_6 4,906 / / / L3835 The First Affiliated Hospital of Zhejiang University Hangzhou, China, 2020 5,761,930 bp 15 Chromosome 5,390,661 / bla SHV−28 , bla SHV−106 , oqxA , oqxB fimH, fyuA, irp2, iutA, mrkA pL3835_2 216,715 IncQ1-IncFII(K)-IncFIB(K) bla SHV−106 , bla CTX−M−15 , dfrA27 , sul1 , sul2 traT, clpK1 pL3835-NDM-OXA 127,486 repB(R1701) bla NDM−4 , bla OXA−9 , bla TEM−1C , bla CTX−M−14 , bla LAP−2 traT pL3835_4 10,046 ColRNAI / ccl pL3835_5 4,935 / / / pL3835_6 4,232 / / / pL3835_7 3,559 / / / pL3835_8 2,238 / / / pL3835_9 2,058 ColpVC / / S1-PFGE showed there were three plasmids in L2388 and two in L3835. Southern blotting indicated that the bla NDM−4 were located on the third plasmid (Fig. 1 ). The antimicrobial susceptibility profiles of target strains and transconjugants were demonstrated in Table 2 . Both L2388 and L3835 were resistant to the carbapenem antibiotics, imipenem and meropenem, the cephalosporin antibiotics, cefaclor and ceftriaxone, amoxicillin/clavulanic, piperacillin/tazobactam and the penicillin antibiotics, susceptible to tigecycline and polymixin B. Moreover, the transconjugants L2388-PAO1Ri and L3835-PAO1Ri showed the similarity antibiotic resistance profile to L2388 and L3835. Table 2 Antimicrobial resistance profile of K. pneumoniae isolates L2388 and L3835 and transconjugants Antibiotic MIC values (µg/ml) L2388 L3835 L2388-PAO1R L3538-PAO1R Imipenem 4 (R) > 32 (R) 4 (R) 4 (R) Meropenem 8 (R) > 32 (R) 8 (R) 8 (R) Ceftriaxone > 128 (R) > 128 (R) > 128 (R) > 128 (R) Cefotaxime > 128 (R) > 128 (R) > 128 (R) > 128 (R) Ceftazidime > 128 (R) > 128 (R) > 128 (R) > 128 (R) Cefepime > 128 (R) > 128 (R) > 128 (R) > 128 (R) Ceftazidime/avibactam > 64 (R) > 64 (R) > 128 (R) > 128 (R) Levofloxacin > 64 (R) > 64 (R) 4 (S) 4 (S) Ciprofloxacin > 64 (R) > 64 (R) 2 (S) 2 (S) Amikacin 32 (R) 16 (R) 32 (R) 32 (R) Gentamicin > 128 (R) > 128(R) > 128 (R) > 128 (R) Piperacillin-tazobactam > 128 (R) > 128(R) > 128 (R) > 128 (R) Amoxicillin-clavulanic 32 (R) > 128(R) > 128 (R) > 128 (R) Tigecycline 1 (S) 2 (S) 0.06 (R) 0.6 (R) Fosfomycin 32 (R) 128 (R) 0.5 (S) 0.5 (S) Chloramphenicol > 128 (R) > 128 (R) 8 (R) 8 (R) Polymixin B 0.25 (S) 0.06 (S) 1 (S) 0.5 (S) Aztreonam ≤ 0.03 (S) > 128 (R) 16 (R) 8 (R) Genomics features The genome of L2388/L3835 consists a circular chromosome with a length of 5,614,837 and 5,761,930bp, respectively. In addition to the chromosome, L2388 carries six plasmids, while L3835 carries nine plasmids. Discrepancies from the S1-PFGE results may be attributed to the potential loss of smaller plasmids (< 10 kb) during bacterial processing, rendering them undetectable by S1-PFGE (Fig. 1 ). Therefore, WGS results were used as the criterion. WGS analysis confirmed that L2388/L3835 belongs to K. pneumoniae with MLST type 726/15, carried the antibiotic resistance and virulence genes were showed in Table 1 . Characterization of NDM-4 and OXA-9 co-producing plasmids ResFinder results reveal that both bla NDM-4 and bla OXA-9 within isolate L2388 are located on a 120,100 bp plasmid designated as pL2388-NDM-OXA. The PlasmidFinder analysis revealed that pL2388-NDM-OXA is a hybrid plasmid, containing both IncF and repB(1701) gene replication regions. Further analysis via BLASTn on GenBank indicated that shared the 99% similarity with pKP27-NDM4 ( K. pneumoniae , accession no: CP041642), pKP14ND1-1 ( K. pneumoniae , accession no: CP098376), pKP15ND44-1 ( K. pneumoniae , accession no: CP098382) and pKP17ND19-1 ( K. pneumoniae , accession no: CP098385). All these plasmids are classified as IncFII(K)-repB(R1701), as depicted in Fig. 2A and summarized in Table 3 . In contrast, both bla NDM-4 and bla OXA-9 genes within L3835 are situated on a 127,486 bp plasmid designated as pL3835-NDM-OXA. This plasmid classified to the repB(R1701) group and possesses a GC content of 54.1%. According to the result of the BLASTn search, plasmid pL3835-NDM-OXA shared the 99%-100% similarity with pKP15ND22-1 ( K. pneumoniae , accession no: CP098379), pKP14ND2-1 ( K. pneumoniae , accession no: CP098377), pCTXM15_020097 ( K. pneumoniae , accession no: CP043351) and pCTXM15_020035 ( K. pneumoniae , accession no: CP045989). The similar plasmids mentioned above are categorized within the IncFII(K)-repB(R1701) group, as depicted in Fig. 2B and summarized in Table 3 . Additionally, it is noteworthy that pL2388-NDM-OXA and pL3835-NDM-OXA also exhibit a high similarity of 99.9%. The gene environment analysis reveals a diverse array of sequences surrounding the bla NDM−4 , with the conserved structure sequence being bla NDM−4 - ble - trpF-dsbD-cutA-GroES-groL , present in eight strains. The predominant mobile genetic elements (MGEs) surrounding the bla NDM−4 gene include members of the IS 6 family (such as IS 15 and IS 26 ) and Tn 3 within our plasmid. Additionally, IS 91 , IS 30 and IS 26 were identified in the similar plasmids (Fig. 3). Within the pL3835-NDM-OXA plasmid, two copies of bla NDM−4 were identified in different orientations, located in two tandemly connected regions spanning 27,142 bp (Fig. 2, 3). Further analysis of the insertion sequences around bla NDM−4 revealed the presence of multiple copies of Tn 3 -hypothetical protein-IS 26 around each bla NDM−4 , two inverted repeats harboring NDM-4 are flanked by IS 15 . This observation may suggest potentially silent yet influential activity of MGEs. Table 3 Summary and genome characteristics of NDM-4/OXA9-positive plasmids downloaded from the NCBI database. Accession number Plasmid Collection date Isolation source Plasmid type Resistance genes Isolated strain Geographic location NZ_JASTSR010000003.1 pL2388-NDM-OXA 2022 stool IncFII(K)-repB(R1701) NDM-4, OXA-9 K. pneumoniae Hangzhou, China NZ_JASTSQ010000003.1 pL3835-NDM-OXA 2022 stool repB(R1701) NDM-4, OXA-9 K. pneumoniae Hangzhou,China CP041642 pKP27-NDM4 2015 sputum IncFII(K)-repB(R1701) NDM-4, OXA-9 K. pneumoniae Ho Chi Minh City, Viet Nam CP098376 pKP14ND1-1 2014 sputum IncFII(K)-repB(R1701) NDM-4 K. pneumoniae Ho Chi Minh City, Viet Nam CP098382 pKP15ND44-1 2015 blood IncFII(K)-repB(R1701) NDM-4 K. pneumoniae Ho Chi Minh City, Viet Nam CP098385 pKP17ND19-1 2017 sputum IncFII(K)-repB(R1701) NDM-4, OXA-9 K. pneumoniae Ho Chi Minh City, Viet Nam CP098377 pKP14ND2-1 2014 sputum IncFII(K)-repB(R1701) NDM-4 K. pneumoniae Ho Chi Minh City, Viet Nam CP098379 pKP15ND22-1 2015 pus IncFII(K)-repB(R1701) NDM-4 K. pneumoniae Ho Chi Minh City, Viet Nam AP018584 MH16-335M 2016 blood IncFII(K)-repB(R1701) NDM-4, OXA-9 K. pneumoniae Hanoi,Viet Nam CP098381 pKP15ND26-1 2015 endotracheal tube IncFIA(HI1)-IncR-repB(R1701) NDM-4 K. pneumoniae Ho Chi Minh City, Viet Nam CP032878 pNDM4_000837 2016 unspecified human sample IncFIA-repB(R1701) NDM-4 Escherichia coli Chengdu, China CP098383 pESC17ND15-1 2017 sputum IncFIA-repB(R1701) NDM-4 Escherichia coli Hanoi, Viet Nam Discussion The study provides a novel insight into the prevalence of bla NDM−4 gene, which was mediated by rare plasmid types, classified as IncFII(K)-repB(R1701). Additionally, it highlights the coexistence of OXA-9 and NDM-4 on individual plasmids, specifically pL2388-NDM-OXA and pL3835-NDM-OXA in K.pneumoniae . Furthermore, we report the first instance where a single plasmid harbors two inverted tandem repeats of the bla NDM−4 gene. These findings underscore the crucial need for robust surveillance methods to hinder the proliferation of antibiotic-resistant bacterial strains. Furthermore, they raise concerns regarding the potential escalation of resistance and dissemination of antibiotic-resistant bacteria. NDM-4 was first detected in 2012, Indian [ 1 ] and 2014 in China [ 18 ].Possessing a high ability to hydrolyze carbapenems and several bulky cephalosporins [ 1 ]. OXA-9, exhibits heightened resistance to ceftazidime and various β-lactam antibiotics, first reported in 1993 [ 19 ], with a relatively low incidence rate observed in China. In this study, we present the genome sequences of two carbapenem-resistant K. pneumoniae strains, L2388 and L3835, which were isolated from distinct hospitals in Hangzhou, China. These strains exhibited resistance to a broad spectrum of tested antibiotics (Table 2 ). Analysis of the genetic context of NDM-4 revealed that the predominant sequence was a 5,380 bp fragment designated as bla NDM−4 - ble-trpF-dsbD-cutA-GroES-groL , consistent with previous findings and exhibiting relative conservation. Although the surrounding MGEs varied, most plasmids from GenBank were flanked by IS 91 and a combination of IS 30 and IS 26 , aligning with prior reports of IS Aba125 , IS 5 , IS 26 , or IS 91 associated with bla NDM−4 [ 8 , 11 , 20 ]. Notably, in two K. pneumoniae isolates from Hangzhou, within plasmid pL2388-NDM-OXA, the NDM-4 gene was bordered by IS 15 at both ends. Of particular interest, in pL3835-NDM-OXA, we identified dual copies of bla NDM−4 arranged in inverted tandem, embedded within a Tn 3 -hypothetical protein-IS 26 - bla NDM - 4 -ble-trpF-dsbD-cutA-GroES-groL -hypothetical protein structure (the red box region in Fig. 2B, Fig. 3). Each upstream region of bla NDM - 4 featured a Tn 3 -hypothetical protein-IS 26 , also linked to IS 15 . This architecture suggests heightened mobility of these regions, implying that MGEs may exert profound effects on genomic dynamics, a phenomenon warranting further attention. IS 26 was found to be closely associated with many other MGEs, such as Tn 3 and class 1 integron, and with numerous different ARGs [ 21 ], such as deletions, inversions, and duplications through homologous recombination or non-homologous end joining mechanisms [ 22 ]. It plays a pivotal role in disseminating ARGs. While Tn 3 , located at the termini, does not directly participate in the inverse repetition of NDM-4, may presence as a major transposable element can augment genomic instability [ 23 ]. After separately delete the ‘red box’ region, BLASTn showed with 99.5% identity and 97% coverage to each of following plasmids: pKP14ND1-1(CP098376), pKP14ND2-1(CP098377), pKP15ND22-1(CP098379), with no reported 100% coverage plasmid. Consequently, we hypothesized the precursor of pL3835-NDM-OXA, proposed two models to elucidate the novel mechanism behind IS 26 -mediated tandem duplication of bla NDM−4 : (1) Homologous recombination model: the plasmid initially harbors a single ‘red box’ region. Introduction of the second ‘red box’ region, either through replication errors or acquisition of external DNA, brings about a stretch of sufficient homology between the two Tn 3 -hypothetical protein-IS 26 regions. This homologous segment triggers a homologous recombination event, which, under imprecise action of recombinases, integrating in reverse orientation to form the inverted tandem repeat [ 24 ]; (2) Post-acquisition bla NDM−4 insertion model: we posit a plasmid initially lacking the bla NDM−4 gene, with both ‘red box’ region acquired externally. The transposition of one IS 26 moiety involves recognition and binding to another IS 26 located on a separate exogenous DNA sequences. IS 26 facilitates the formation of a DNA ring, which then inserts back into the genome, resulting in the generation of an inverted tandem duplication [ 22 , 24 ] (Fig. 2C-2D). Studies have indicated that the multiplication of ARGs can augment resistance levels [ 25 ]. However, there is currently a paucity of direct evidence specifically addressing how an increased copy number of bla NDM−4 impacts carbapenem resistance. Further research is warranted to elucidate this relationship comprehensively. The integration of novel MGEs in proximity to NDM-4 enriches its genetic context, emphasizing the critical function of MGEs in the lateral dissemination of ARGs. Successful conjugation assays have illustrated the plasmid-mediated transfer of bla NDM−4 to recipient bacteria. Our hypothesis posits that the emergence of novel MGEs in conjunction with NDM-4 expedite the dissemination of NDM-4 resistance, consequently exacerbating concerns over the escalating trend of antimicrobial resistance. Plasmid analysis showed pL2388-NDM-OXA and similar plasmid types, except for pL3835-NDM-OXA, were all IncFII(K)-repB(R1701) (Table 3 ). Previous studies have reported that NDM-4 plasmids were mainly IncX3 [ 5 , 9 , 10 ] or IncF [ 1 , 11 , 12 ]. To our knowledge, this study is the first to report the NDM-4 plasmid type as IncFII(K)-repB(R1701)/repB(R1701). This suggests that these plasmids share a common replication initiation protein, repB(R1701), and fall within the IncF family, which is typically associated with efficient conjugative transfer capabilities and broad host range among diverse bacterial species. The plasmid harboring dual NDM-4 segments is labeled only as repB(R1701), without clear assignment to a specific Inc group, possibly indicating less clarity in its classification or highlighting the unique feature of its repB(R1701) protein. To our knowledge, this study represents the first report characterizing the plasmid types carrying bla NDM−4 . Further analysis of NDM-4-positive plasmids (Table 3 ) reveals a novel conjugative plasmid type, IncF and repB(R1701), mostly IncFII(K)-repB(R1701), which is geographically concentrated in Vietnam and China, predominantly in Vietnam and southern China. Isolates originate from various human clinical samples including stool, sputum, blood et al., collected between 2014 and 2020, but primarily deposited in the NCBI database in 2022, hence the recent availability of data for analysis. The outcomes of this study suggest that both L2388 and L3835 isolates, sourced from patients without foreign residency history and originating from separate hospitals, indicate a broader distribution of these strains than previously anticipated. This delay potentially signals a belated public health alert regarding the severity of NDM-4 resistance and its potentially underestimated transmission dynamics. It is imperative for public health authorities to promptly review and analyze these data to implement measures aimed at curtailing the further spread of resistance. Conclusion This study provides the first description of the concurrent presence of OXA-9 and NDM-4 in single plasmid of K. pneum oniae. The identification of IncFII(K)-repB(R1701)/repB(R1701) plasmids carrying the bla NDM−4 gene, previously unrecognized, indicates clandestine transmission across select regions of Viet Nam and China. This suggests a potentially broader epidemiological reach than initially presumed in the realms of microbiology and epidemiology. Continuous observation and exploration are essential to control its spread. Declarations Data availability statement The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material. This Whole Genome Shotgun project has been deposited at NCBI/ GenBank(https://submit.ncbi.nlm.nih.gov/subs/genbank/) with the sequence data for K. pneumoniae L2388/L3835 under the BioProject number PRJNA390758. The 2 whole-genome sequenced data have been deposited in the NCBI database BioProject:PRJNA390758. Author contributions Huanran Zhang and Beiwen Zheng conceived and designed the experiments. Hao Xu and Ruishan Liu collected samples and performed the experiments. Xiaojing Liu，Yu Yang and Kexin Guo collected the data and prepared the article. Huanran Zhang wrote the manuscript. Maotao Chen, Yuanqiang Lu and Beiwen Zheng critically revised the manuscript. Beiwen Zheng reviewed and finalized the manuscript. All authors contributed to the article and approved the submitted version. Ethical approval In this study，informed consent was obtained from all participants. The study was approved by the clinical research ethics committee of the First Affiliated Hospital, Zhejiang University School of Medicine [number 2021-IIT-631]. All methods were performed following the relevant guidelines and regulations. Funding This work was supported by the National Key Research and Development Program of China (2020YFE0204300); National Natural Science Foundation of China (82072314); Zhejiang Provincial Natural Science Foundation of China (LHDMZ22H190002); the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-045); and the Fundamental Research Funds for the Central Universities (2022ZFJH003). Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Disclosure The authors report no conflicts of interest in this work. Consent for publication Not applicable. References Nordmann, P., A.E. Boulanger, and L. Poirel, NDM-4 metallo-beta-lactamase with increased carbapenemase activity from Escherichia coli. Antimicrob Agents Chemother, 2012. 56 (4): p. 2184-2186. Evans, B.A. and S.G. Amyes, OXA beta-lactamases. Clin Microbiol Rev, 2014. 27 (2): p. 241-263. Dortet, L., et al., Intercontinental travels of patients and dissemination of plasmid-mediated carbapenemase KPC-3 associated with OXA-9 and TEM-1. J Antimicrob Chemother, 2008. 61 (2): p. 455-457. Partridge, S.R., et al., Mobile Genetic Elements Associated with Antimicrobial Resistance. Clin Microbiol Rev, 2018. 31 (4). Choudhury, N.A., et al., IncX3 plasmid mediated occurrence of bla(NDM-4) within Escherichia coli ST448 from India. J Infect Public Health, 2018. 11 (1): p. 111-114. Zhang, F., et al., Molecular Characteristics of an NDM-4 and OXA-181 Co-Producing K51-ST16 Carbapenem-Resistant Klebsiella pneumoniae: Study of Its Potential Dissemination Mediated by Conjugative Plasmids and Insertion Sequences. Antimicrob Agents Chemother, 2023. 67 (1): p. e0135422. Kim, J.S., et al., Emergence of NDM-4 and OXA-181 carbapenemase-producing Klebsiella pneumoniae. J Glob Antimicrob Resist, 2020. 20 : p. 332-333. Adler, A., et al., Clinical and molecular features of NDM-producing Acinetobacter baumannii in a multicenter study in Israel. Ann Clin Microbiol Antimicrob, 2023. 22 (1): p. 52. Espedido, B.A., et al., The use of whole-genome sequencing for molecular epidemiology and antimicrobial surveillance: identifying the role of IncX3 plasmids and the spread of blaNDM-4-like genes in the Enterobacteriaceae. J Clin Pathol, 2015. 68 (10): p. 835-838. Hirabayashi, A., et al., A Transferable IncC-IncX3 Hybrid Plasmid Cocarrying bla(NDM-4), tet(X), and tmexCD3-toprJ3 Confers Resistance to Carbapenem and Tigecycline. mSphere, 2021. 6 (4): p. e0059221. Zhang, X., et al., Cryptic transmission of ST405 Escherichia coli carrying bla (NDM-4) in hospital. Sci Rep, 2018. 8 (1): p. 390. Ahmad, N., S.M. Ali, and A.U. Khan, Molecular characterization of novel sequence type of carbapenem-resistant New Delhi metallo-beta-lactamase-1-producing Klebsiella pneumoniae in the neonatal intensive care unit of an Indian hospital. Int J Antimicrob Agents, 2019. 53 (4): p. 525-529. Yao, H., et al., Conjugative plasmids facilitate the transmission of tmexCD2-toprJ2 among carbapenem-resistant Klebsiella pneumoniae. Sci Total Environ, 2024. 906 : p. 167373. Performance Standards for Antimicrobial Susceptibility Testing , in M100 . 2023, Clinical and Laboratory Standards Institute: Wayne, PA. Carattoli, A., et al., In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother, 2014. 58 (7): p. 3895-3903. Sullivan, M.J., N.K. Petty, and S.A. Beatson, Easyfig: a genome comparison visualizer. Bioinformatics, 2011. 27 (7): p. 1009-1010. Alikhan, N.F., et al., BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics, 2011. 12 : p. 402. Qin, S., et al., First identification of NDM-4-producing Escherichia coli ST410 in China. Emerg Microbes Infect, 2016. 5 (11): p. e118. Tolmasky, M.E. and J.H. Crosa, Genetic organization of antibiotic resistance genes (aac(6')-Ib, aadA, and oxa9) in the multiresistance transposon Tn1331. Plasmid, 1993. 29 (1): p. 31-40. Wu, J.W., et al., Investigation of carbapenem-resistant Klebsiella pneumoniae in Taiwan revealed strains co-harbouring bla(NDM) and bla(OXA-48-like) and a novel plasmid co-carrying bla(NDM-1) and bla(OXA-181). Int J Antimicrob Agents, 2023. 62 (5): p. 106964. Khalifa, H.O., et al., NDM-4- and NDM-5-Producing Klebsiella pneumoniae Coinfection in a 6-Month-Old Infant. Antimicrob Agents Chemother, 2016. 60 (7): p. 4416-4417. Harmer, C.J., C.H. Pong, and R.M. Hall, Structures bounded by directly-oriented members of the IS26 family are pseudo-compound transposons. Plasmid, 2020. 111 : p. 102530. Nicolas, E., et al., The Tn3-family of Replicative Transposons. Microbiol Spectr, 2015. 3 (4). Harmer, C.J. and R.M. Hall, IS26 and the IS26 family: versatile resistance gene movers and genome reorganizers. Microbiol Mol Biol Rev, 2024: p. e0011922. Zhang, P., et al., Emergence of ceftazidime/avibactam resistance in carbapenem-resistant Klebsiella pneumoniae in China. Clin Microbiol Infect, 2020. 26 (1): p. 124 e121-124 e124. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4904070","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":359083710,"identity":"dfdc8c1d-9e37-4da0-ad5e-897152d8d270","order_by":0,"name":"Huanran Zhang","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Huanran","middleName":"","lastName":"Zhang","suffix":""},{"id":359083711,"identity":"979c6050-8640-4845-bd1b-5adee3768ae7","order_by":1,"name":"Hao Xu","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Xu","suffix":""},{"id":359083712,"identity":"a712ac8a-7658-4939-b0dd-5eafbe820920","order_by":2,"name":"Ruishan Liu","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ruishan","middleName":"","lastName":"Liu","suffix":""},{"id":359083713,"identity":"8bfecccc-06b7-444c-98b9-a2811a2169fa","order_by":3,"name":"Xiaojing Liu","email":"","orcid":"","institution":"National Medical Center for Infectious Diseases, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaojing","middleName":"","lastName":"Liu","suffix":""},{"id":359083715,"identity":"bce5d47f-4446-46f6-8396-c760c554c359","order_by":4,"name":"Yu Yang","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Yang","suffix":""},{"id":359083716,"identity":"4397dc1a-1df6-49f3-b27f-1424e251a665","order_by":5,"name":"Kexin Guo","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kexin","middleName":"","lastName":"Guo","suffix":""},{"id":359083718,"identity":"8567aba8-5c52-4abb-b429-231a3f4ba820","order_by":6,"name":"Mantao Chen","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Mantao","middleName":"","lastName":"Chen","suffix":""},{"id":359083720,"identity":"9673f9ce-9b72-4ec4-89f5-00f816489876","order_by":7,"name":"Yuanqiang Lu","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Yuanqiang","middleName":"","lastName":"Lu","suffix":""},{"id":359083722,"identity":"0a33e3bc-872c-4986-b7f3-9f65390ea8cb","order_by":8,"name":"Beiwen Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYDACCSBmbGCQY2A4AGSxkaDFmHQtiQ1gHjFa5Gc3H3v4dcfh9PmNZwwYPpQdZuCf3YBfC+OcY+nGsmcO5zY2nDFgnHHuMIPEnQP4tTBL5JhJS7bdzm1mOGPAzNt2mMFAIgG/FjaJ/G8gLelsIC1/idHCI5HDJvmx7XYCD0gLIzFaJCTSzKQZz/w3nMFwrOBgz7l0HokbBLTIz0h+JvlzR5q8/IzDGx/8KLOW459BQAsIMPOA7TsAjkwewuqBgPEHiORvIErxKBgFo2AUjEAAALM5Q62B/praAAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"Beiwen","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2024-08-13 04:51:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4904070/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4904070/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65691108,"identity":"71a3b165-b818-4a39-a50a-1f27a923f291","added_by":"auto","created_at":"2024-10-01 10:13:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":265743,"visible":true,"origin":"","legend":"\u003cp\u003ePlasmid profiles and Southern blot-hybridization of L2388 and L3835. Southern blot-hybridization of S1-nuclease digested DNA using a specific probe (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4\u003c/sub\u003e). M: XbaI digested total DNA of salmonella enterica serotype Braenderup H9812 as a size marker.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4904070/v1/2cde05ee0f11c0cd7c35451d.jpg"},{"id":65691109,"identity":"a7ff56d4-0b82-4d02-aadf-6ca8255f2351","added_by":"auto","created_at":"2024-10-01 10:13:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7345195,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4904070/v1/8e5c5c9dba87e71ec1309532.jpg"},{"id":65691106,"identity":"dcef728d-2062-421f-8f28-c67eef512e3f","added_by":"auto","created_at":"2024-10-01 10:13:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3387147,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4904070/v1/e14ad7d8f4f1ab2145ee1c83.jpg"},{"id":65691465,"identity":"a0f5efb9-6450-46b5-a773-e371b7b5bfc7","added_by":"auto","created_at":"2024-10-01 10:21:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1124541,"visible":true,"origin":"","legend":"\u003cp\u003eReginal dissemination of transferable repB(1701) plasmid carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4\u003c/sub\u003e in Asia.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4904070/v1/1d9f56f24ff1169942dee7ec.jpg"},{"id":86351212,"identity":"93a5cc9f-bbe1-43bd-9fe7-0f3368535761","added_by":"auto","created_at":"2025-07-09 16:01:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13131011,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4904070/v1/e65ecb9f-f5bb-4990-bd43-276c1662626e.pdf"},{"id":65691111,"identity":"0f77ac16-7ada-4399-ba62-10e48f33a645","added_by":"auto","created_at":"2024-10-01 10:13:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13905416,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4904070/v1/33aed7338c28b279a42dd954.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reginal dissemination of transferable repB(1701) plasmid carrying blaNDM-4 in Asia: emergence of tandem repeats with blaNDM-4 mediated by IS26 in Klebsiella pneumoniae","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e is a Gram-negative bacterium known for its encapsulation and lack of motility. It can lead to a range of human infections. Similar to numerous Gram-negative bacteria, it is increasingly developing resistance to prominent antibiotics including carbapenems and cephalosporins. Notably, the rise and proliferation of transferable plasmids with carbapenemase-encoding genes are clearly major contributors to the growing challenge of carbapenem resistance.\u003c/p\u003e \u003cp\u003eNDM-4, a class B carbapenemase, was first detected in ST648 \u003cem\u003eEscherichia coli\u003c/em\u003e from an Indian patient which displayed significant hydrolytic activity against carbapenems and multiple cephalosporins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Single-copy instance of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e is more prevalent. OXA-9, a constituent of the OXA-type β-lactamase family [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], was first detected by M.E. Tolmasky [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], exhibits heightened resistance to ceftazidime and various β-lactam antibiotics. The simultaneous presence of NDM-4 and OXA-9 is still rare on a global scale.\u003c/p\u003e \u003cp\u003eMobile genetic elements (MGEs; e.g., plasmid, insertion sequence (IS), transposons, and integron) play a key role in facilitating the acquisition and spread of antibiotic resistance genes (ARGs) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The most common replicon types identified among NDM-4 are insertion sequence (IS) \u003cem\u003eAba12\u003c/em\u003e5, IS\u003cem\u003e5\u003c/em\u003e, IS\u003cem\u003e26\u003c/em\u003e and IS\u003cem\u003e91\u003c/em\u003e [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], Alterations in mobile genetic elements (MGEs) may facilitate the dissemination of NDM-4. The most common replicon types identified among \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e harbouring plasmids in Enterobacteriaceae are IncX3 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] or IncF [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It is noteworthy that NDM-4 genes are always associated with several ISs. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e gene is located on a plasmid, enabling it to be horizontally transferred between bacteria, which allows its resistance to spread rapidly. Tracking and controlling the spread of this gene is a significant challenge. Additionally, IncF-repB(R1701) plasmids have recently been reported as a substantial threat to public health due to their mobile resistance to both tigecycline and carbapenem [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the link between \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e gene and conjugative IncF-repB(R1701) plasmid has never been described.\u003c/p\u003e \u003cp\u003eHere, we report the co-presence of OXA-9 and NDM-4 on a single plasmid in two strains of \u003cem\u003eK. pneumoniae\u003c/em\u003e, detailing their genomic features through ARG profiling and whole-genome sequencing (WGS). Using a global dataset compiled from GenBank, we analyzed the worldwide epidemiological characteristics of plasmids harboring the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e gene.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStrains and antimicrobial susceptibility testing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrains L2388 and L3835 of \u003cem\u003eK. pneumoniae\u003c/em\u003e were isolated and characterized from stool samples obtained from two tertiary hospitals in Hangzhou, Zhejiang Province, China. L2388 was isolated from a 35-year-old female patient after kidney transplantation in Shulan (Hang Zhou) Hospital. L3835 was isolated from a 45-year-old male patient presenting with non-Hodgkin lymphoma (NHL) in\u0026nbsp;The First Affiliated Hospital of Zhejiang University.\u0026nbsp;Subsequently, species were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Bruker, Bremen, Germany)\u003c/p\u003e\n\u003cp\u003eThe MICs of antibiotics were determined using the agar dilution method, and the results were interpreted following Clinical and Laboratory Standards Institution (CLSI) guidelines (M100-S25) except polymixin B and tigecycline, which were assessed using the broth microdilution method\u003cu\u003e\u0026nbsp;[\u003c/u\u003e\u003cu\u003e14\u003c/u\u003e\u003cu\u003e]\u003c/u\u003e.\u003cem\u003e\u0026nbsp;Pseudomonas aeruginosa\u003c/em\u003e ATCC27853 and \u003cem\u003eE. coli\u003c/em\u003e ATCC25922 were used as the quality control.\u0026nbsp;The tested antibiotics included: imipenem, meropenem, ceftriaxone, cefotaxime, ceftazidime, cefepime, levofloxacin, ciprofloxacin, amikacin, gentamicin, piperacillin/tazobactam, ceftazidime/avibactam, amoxicillin/clavulanic, tigecycline, fosfomycin, chloramphenicol, polymixin B, and aztreonam.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eS1-Pulsed-Field Gel Electrophoresis, Southern blotting and conjugational transfer\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe detected the numbers and sizes of plasmids in L2388 and L3835 using S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE). The positions of plasmids containing \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-9\u003c/sub\u003e were identified through Southern blotting and hybridization with specific probes labeled with digoxigenin. Moreover, we employed rifampin-resistant \u003cem\u003eE. coli\u003c/em\u003e 600, rifampin-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1R(PARI) and polymixin-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e (PACO) as the recipient in conjugation experiments to explore the potential transfer of plasmids.\u0026nbsp;The transconjugants were selected with three Mueller-Hinton agar medium (200 mg /L rifampicin and 2 mg /L meropenem, 200 mg /L polymixin and 2 mg /L meropenem, 400 mg /L rifampicin and 2 mg /L meropenem) and subsequently identified using MALDI-TOF/MS.\u0026nbsp;We detected the presence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4\u003c/sub\u003e genes through PCR, along with conducting antimicrobial susceptibility testing to confirm the successful transfer of plasmids carrying the target genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWhole-genome sequencing and bioinformatics analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-genome sequencing (WGS) was conducted for isolates L2388 and L3835 using both the Illumina NovaSeq 6000 (Illumina, San Diego, CA, United States) and Oxford Nanopore (Oxford Nanopore Technologies, Oxford, United Kingdom) platforms. Unicycler was used for data assembly, and sequence alignment and similarity searches were carried out with the BLASTn software. Prokka was used for annotatted. ARGs were identified using ResFinder 4.1, virulence genes by VirulenceFinder and the plasmid types were determined using PlasmidFinder 2.0 [15]. The multilocus sequence typing (MLST) was determined using the MLST service provided by CGE (http://www.genomicepidemiology.org/). The genetic context of the antimicrobial ARGs was visualized using Easyfig [16]. Lastly, the circular comparative images of multiplex plasmids were generated using the BLASTn Ring Image Generator (BRIG) [17].\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of the strains\u003c/h2\u003e \u003cp\u003eL2388 was obtained from a stool sample of a kidney transplant patient undergoing immunosuppressive therapy. L3835 was obtained from a stool sample of a patient who was hospitalized for non-Hodgkinlymphoma and subsequently underwent systemic chemotherapy(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenome and plasmid analysis of \u003cem\u003eK. pneumoniae\u003c/em\u003e strains isolated L2388 and L3835\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsolation details\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGenome size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMLST\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChromosome/\u003c/p\u003e \u003cp\u003eplasmid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLength\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmid type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eResistance genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eVirulence genes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eL2388\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eShulan (Hang Zhou) Hospital, Hangzhou, China, 2020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e5,614,837 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e726\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5,293,684\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;81\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;110\u003c/sub\u003e, \u003cem\u003eoqxA\u003c/em\u003e, \u003cem\u003eoqxB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003efimH, iutA, mrkA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL2388_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e139,247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIncFIB(K)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003esul1\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e, \u003cem\u003edfrA27\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eclpK1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL2388-NDM-OXA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e120,100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIncFII(K)-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;9\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;14\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eLAP\u0026minus;2\u003c/sub\u003e,\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003etraT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL2388_4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e41,846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIncR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u0026minus;1B\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;55\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003e/\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL2388_5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15,054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIncFIA(HI1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL2388_6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4,906\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eL3835\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eThe First Affiliated Hospital of Zhejiang University\u003c/p\u003e \u003cp\u003eHangzhou, China, 2020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e5,761,930 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5,390,661\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;28\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;106\u003c/sub\u003e, \u003cem\u003eoqxA\u003c/em\u003e, \u003cem\u003eoqxB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003efimH, fyuA, irp2, iutA, mrkA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL3835_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e216,715\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIncQ1-IncFII(K)-IncFIB(K)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;106\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;15\u003c/sub\u003e, \u003cem\u003edfrA27\u003c/em\u003e, \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003etraT, clpK1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL3835-NDM-OXA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e127,486\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003erepB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;9\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u0026minus;1C\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;14\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eLAP\u0026minus;2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003etraT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL3835_4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10,046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eColRNAI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eccl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL3835_5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4,935\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL3835_6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4,232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL3835_7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3,559\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL3835_8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2,238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epL3835_9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2,058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eColpVC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eS1-PFGE showed there were three plasmids in L2388 and two in L3835. Southern blotting indicated that the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e were located on the third plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The antimicrobial susceptibility profiles of target strains and transconjugants were demonstrated in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Both L2388 and L3835 were resistant to the carbapenem antibiotics, imipenem and meropenem, the cephalosporin antibiotics, cefaclor and ceftriaxone, amoxicillin/clavulanic, piperacillin/tazobactam and the penicillin antibiotics, susceptible to tigecycline and polymixin B. Moreover, the transconjugants L2388-PAO1Ri and L3835-PAO1Ri showed the similarity antibiotic resistance profile to L2388 and L3835.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial resistance profile of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates L2388 and L3835 and transconjugants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntibiotic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eMIC values (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL2388\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL3835\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eL2388-PAO1R\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eL3538-PAO1R\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImipenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMeropenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCeftriaxone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCefotaxime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCeftazidime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCefepime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCeftazidime/avibactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLevofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmikacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGentamicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePiperacillin-tazobactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmoxicillin-clavulanic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTigecycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.06 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.6 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFosfomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloramphenicol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolymixin B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.06 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAztreonam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.03 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGenomics features\u003c/h2\u003e \u003cp\u003eThe genome of L2388/L3835 consists a circular chromosome with a length of 5,614,837 and 5,761,930bp, respectively. In addition to the chromosome, L2388 carries six plasmids, while L3835 carries nine plasmids. Discrepancies from the S1-PFGE results may be attributed to the potential loss of smaller plasmids (\u0026lt;\u0026thinsp;10 kb) during bacterial processing, rendering them undetectable by S1-PFGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, WGS results were used as the criterion. WGS analysis confirmed that L2388/L3835 belongs to \u003cem\u003eK. pneumoniae\u003c/em\u003e with MLST type 726/15, carried the antibiotic resistance and virulence genes were showed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of NDM-4 and OXA-9 co-producing plasmids\u003c/h2\u003e \u003cp\u003eResFinder results reveal that both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-9\u003c/sub\u003e within isolate L2388 are located on a 120,100 bp plasmid designated as pL2388-NDM-OXA. The PlasmidFinder analysis revealed that pL2388-NDM-OXA is a hybrid plasmid, containing both IncF and repB(1701) gene replication regions. Further analysis via BLASTn on GenBank indicated that shared the 99% similarity with pKP27-NDM4 (\u003cem\u003eK. pneumoniae\u003c/em\u003e, accession no: CP041642), pKP14ND1-1 (\u003cem\u003eK. pneumoniae\u003c/em\u003e, accession no: CP098376), pKP15ND44-1 (\u003cem\u003eK. pneumoniae\u003c/em\u003e, accession no: CP098382) and pKP17ND19-1 (\u003cem\u003eK. pneumoniae\u003c/em\u003e, accession no: CP098385). All these plasmids are classified as IncFII(K)-repB(R1701), as depicted in Fig.\u0026nbsp;2A and summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In contrast, both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-9\u003c/sub\u003e genes within L3835 are situated on a 127,486 bp plasmid designated as pL3835-NDM-OXA. This plasmid classified to the repB(R1701) group and possesses a GC content of 54.1%. According to the result of the BLASTn search, plasmid pL3835-NDM-OXA shared the 99%-100% similarity with pKP15ND22-1 (\u003cem\u003eK. pneumoniae\u003c/em\u003e, accession no: CP098379), pKP14ND2-1 (\u003cem\u003eK. pneumoniae\u003c/em\u003e, accession no: CP098377), pCTXM15_020097 (\u003cem\u003eK. pneumoniae\u003c/em\u003e, accession no: CP043351) and pCTXM15_020035 (\u003cem\u003eK. pneumoniae\u003c/em\u003e, accession no: CP045989). The similar plasmids mentioned above are categorized within the IncFII(K)-repB(R1701) group, as depicted in Fig.\u0026nbsp;2B and summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Additionally, it is noteworthy that pL2388-NDM-OXA and pL3835-NDM-OXA also exhibit a high similarity of 99.9%.\u003c/p\u003e \u003cp\u003eThe gene environment analysis reveals a diverse array of sequences surrounding the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e, with the conserved structure sequence being \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e-\u003cem\u003eble\u003c/em\u003e-\u003cem\u003etrpF-dsbD-cutA-GroES-groL\u003c/em\u003e, present in eight strains. The predominant mobile genetic elements (MGEs) surrounding the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e gene include members of the IS\u003cem\u003e6\u003c/em\u003e family (such as IS\u003cem\u003e15\u003c/em\u003e and IS\u003cem\u003e26\u003c/em\u003e) and Tn\u003cem\u003e3\u003c/em\u003e within our plasmid. Additionally, IS\u003cem\u003e91\u003c/em\u003e, IS\u003cem\u003e30\u003c/em\u003e and IS\u003cem\u003e26\u003c/em\u003e were identified in the similar plasmids (Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eWithin the pL3835-NDM-OXA plasmid, two copies of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e were identified in different orientations, located in two tandemly connected regions spanning 27,142 bp (Fig.\u0026nbsp;2, 3). Further analysis of the insertion sequences around \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e revealed the presence of multiple copies of Tn\u003cem\u003e3\u003c/em\u003e-hypothetical protein-IS\u003cem\u003e26\u003c/em\u003e around each \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e, two inverted repeats harboring NDM-4 are flanked by IS\u003cem\u003e15\u003c/em\u003e. This observation may suggest potentially silent yet influential activity of MGEs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary and genome characteristics of NDM-4/OXA9-positive plasmids downloaded from the NCBI database.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAccession number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCollection date\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIsolation source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlasmid type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eResistance genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIsolated strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGeographic location\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNZ_JASTSR010000003.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epL2388-NDM-OXA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003estool\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(K)-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4, OXA-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHangzhou, China\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNZ_JASTSQ010000003.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epL3835-NDM-OXA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003estool\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003erepB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4, OXA-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHangzhou,China\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP041642\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKP27-NDM4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003esputum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(K)-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4, OXA-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHo Chi Minh City, Viet Nam\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP098376\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKP14ND1-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003esputum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(K)-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHo Chi Minh City, Viet Nam\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP098382\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKP15ND44-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eblood\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(K)-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHo Chi Minh City, Viet Nam\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP098385\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKP17ND19-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003esputum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(K)-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4, OXA-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHo Chi Minh City, Viet Nam\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP098377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKP14ND2-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003esputum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(K)-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHo Chi Minh City, Viet Nam\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP098379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKP15ND22-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(K)-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHo Chi Minh City, Viet Nam\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAP018584\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMH16-335M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eblood\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(K)-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4, OXA-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHanoi,Viet Nam\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP098381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKP15ND26-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eendotracheal tube\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFIA(HI1)-IncR-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHo Chi Minh City, Viet Nam\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP032878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epNDM4_000837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eunspecified human sample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFIA-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChengdu, China\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP098383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epESC17ND15-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003esputum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFIA-repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDM-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHanoi, Viet Nam\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe study provides a novel insight into the prevalence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e gene, which was mediated by rare plasmid types, classified as IncFII(K)-repB(R1701). Additionally, it highlights the coexistence of OXA-9 and NDM-4 on individual plasmids, specifically pL2388-NDM-OXA and pL3835-NDM-OXA in \u003cem\u003eK.pneumoniae\u003c/em\u003e. Furthermore, we report the first instance where a single plasmid harbors two inverted tandem repeats of the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e gene. These findings underscore the crucial need for robust surveillance methods to hinder the proliferation of antibiotic-resistant bacterial strains. Furthermore, they raise concerns regarding the potential escalation of resistance and dissemination of antibiotic-resistant bacteria.\u003c/p\u003e \u003cp\u003eNDM-4 was first detected in 2012, Indian [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and 2014 in China [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].Possessing a high ability to hydrolyze carbapenems and several bulky cephalosporins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. OXA-9, exhibits heightened resistance to ceftazidime and various β-lactam antibiotics, first reported in 1993 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], with a relatively low incidence rate observed in China. In this study, we present the genome sequences of two carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e strains, L2388 and L3835, which were isolated from distinct hospitals in Hangzhou, China. These strains exhibited resistance to a broad spectrum of tested antibiotics (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnalysis of the genetic context of NDM-4 revealed that the predominant sequence was a 5,380 bp fragment designated as \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e-\u003cem\u003eble-trpF-dsbD-cutA-GroES-groL\u003c/em\u003e, consistent with previous findings and exhibiting relative conservation. Although the surrounding MGEs varied, most plasmids from GenBank were flanked by IS\u003cem\u003e91\u003c/em\u003e and a combination of IS\u003cem\u003e30\u003c/em\u003e and IS\u003cem\u003e26\u003c/em\u003e, aligning with prior reports of IS\u003cem\u003eAba125\u003c/em\u003e, IS\u003cem\u003e5\u003c/em\u003e, IS\u003cem\u003e26\u003c/em\u003e, or IS\u003cem\u003e91\u003c/em\u003e associated with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Notably, in two \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from Hangzhou, within plasmid pL2388-NDM-OXA, the NDM-4 gene was bordered by IS\u003cem\u003e15\u003c/em\u003e at both ends. Of particular interest, in pL3835-NDM-OXA, we identified dual copies of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e arranged in inverted tandem, embedded within a Tn\u003cem\u003e3\u003c/em\u003e-hypothetical protein-IS\u003cem\u003e26\u003c/em\u003e-\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e-\u003csub\u003e4\u003c/sub\u003e\u003cem\u003e-ble-trpF-dsbD-cutA-GroES-groL\u003c/em\u003e-hypothetical protein structure (the red box region in Fig.\u0026nbsp;2B, Fig.\u0026nbsp;3). Each upstream region of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e-\u003csub\u003e4\u003c/sub\u003e featured a Tn\u003cem\u003e3\u003c/em\u003e-hypothetical protein-IS\u003cem\u003e26\u003c/em\u003e, also linked to IS\u003cem\u003e15\u003c/em\u003e. This architecture suggests heightened mobility of these regions, implying that MGEs may exert profound effects on genomic dynamics, a phenomenon warranting further attention.\u003c/p\u003e \u003cp\u003eIS\u003cem\u003e26\u003c/em\u003e was found to be closely associated with many other MGEs, such as Tn\u003cem\u003e3\u003c/em\u003e and class 1 integron, and with numerous different ARGs [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], such as deletions, inversions, and duplications through homologous recombination or non-homologous end joining mechanisms [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It plays a pivotal role in disseminating ARGs. While Tn\u003cem\u003e3\u003c/em\u003e, located at the termini, does not directly participate in the inverse repetition of NDM-4, may presence as a major transposable element can augment genomic instability [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter separately delete the \u0026lsquo;red box\u0026rsquo; region, BLASTn showed with 99.5% identity and 97% coverage to each of following plasmids: pKP14ND1-1(CP098376), pKP14ND2-1(CP098377), pKP15ND22-1(CP098379), with no reported 100% coverage plasmid. Consequently, we hypothesized the precursor of pL3835-NDM-OXA, proposed two models to elucidate the novel mechanism behind IS\u003cem\u003e26\u003c/em\u003e-mediated tandem duplication of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e: (1) Homologous recombination model: the plasmid initially harbors a single \u0026lsquo;red box\u0026rsquo; region. Introduction of the second \u0026lsquo;red box\u0026rsquo; region, either through replication errors or acquisition of external DNA, brings about a stretch of sufficient homology between the two Tn\u003cem\u003e3\u003c/em\u003e-hypothetical protein-IS\u003cem\u003e26\u003c/em\u003e regions. This homologous segment triggers a homologous recombination event, which, under imprecise action of recombinases, integrating in reverse orientation to form the inverted tandem repeat [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]; (2) Post-acquisition \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e insertion model: we posit a plasmid initially lacking the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e gene, with both \u0026lsquo;red box\u0026rsquo; region acquired externally. The transposition of one IS\u003cem\u003e26\u003c/em\u003e moiety involves recognition and binding to another IS\u003cem\u003e26\u003c/em\u003e located on a separate exogenous DNA sequences. IS\u003cem\u003e26\u003c/em\u003e facilitates the formation of a DNA ring, which then inserts back into the genome, resulting in the generation of an inverted tandem duplication [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] (Fig.\u0026nbsp;2C-2D).\u003c/p\u003e \u003cp\u003eStudies have indicated that the multiplication of ARGs can augment resistance levels [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, there is currently a paucity of direct evidence specifically addressing how an increased copy number of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e impacts carbapenem resistance. Further research is warranted to elucidate this relationship comprehensively. The integration of novel MGEs in proximity to NDM-4 enriches its genetic context, emphasizing the critical function of MGEs in the lateral dissemination of ARGs. Successful conjugation assays have illustrated the plasmid-mediated transfer of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e to recipient bacteria. Our hypothesis posits that the emergence of novel MGEs in conjunction with NDM-4 expedite the dissemination of NDM-4 resistance, consequently exacerbating concerns over the escalating trend of antimicrobial resistance.\u003c/p\u003e \u003cp\u003ePlasmid analysis showed pL2388-NDM-OXA and similar plasmid types, except for pL3835-NDM-OXA, were all IncFII(K)-repB(R1701) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Previous studies have reported that NDM-4 plasmids were mainly IncX3 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] or IncF [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To our knowledge, this study is the first to report the NDM-4 plasmid type as IncFII(K)-repB(R1701)/repB(R1701). This suggests that these plasmids share a common replication initiation protein, repB(R1701), and fall within the IncF family, which is typically associated with efficient conjugative transfer capabilities and broad host range among diverse bacterial species. The plasmid harboring dual NDM-4 segments is labeled only as repB(R1701), without clear assignment to a specific Inc group, possibly indicating less clarity in its classification or highlighting the unique feature of its repB(R1701) protein. To our knowledge, this study represents the first report characterizing the plasmid types carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eFurther analysis of NDM-4-positive plasmids (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) reveals a novel conjugative plasmid type, IncF and repB(R1701), mostly IncFII(K)-repB(R1701), which is geographically concentrated in Vietnam and China, predominantly in Vietnam and southern China. Isolates originate from various human clinical samples including stool, sputum, blood et al., collected between 2014 and 2020, but primarily deposited in the NCBI database in 2022, hence the recent availability of data for analysis. The outcomes of this study suggest that both L2388 and L3835 isolates, sourced from patients without foreign residency history and originating from separate hospitals, indicate a broader distribution of these strains than previously anticipated. This delay potentially signals a belated public health alert regarding the severity of NDM-4 resistance and its potentially underestimated transmission dynamics. It is imperative for public health authorities to promptly review and analyze these data to implement measures aimed at curtailing the further spread of resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides the first description of the concurrent presence of OXA-9 and NDM-4 in single plasmid of \u003cem\u003eK. pneum\u003c/em\u003eoniae. The identification of IncFII(K)-repB(R1701)/repB(R1701) plasmids carrying the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;4\u003c/sub\u003e gene, previously unrecognized, indicates clandestine transmission across select regions of Viet Nam and China. This suggests a potentially broader epidemiological reach than initially presumed in the realms of microbiology and epidemiology. Continuous observation and exploration are essential to control its spread.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability statement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets presented in this study can be\u0026nbsp;found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material. This Whole Genome Shotgun project has been deposited at NCBI/ GenBank(https://submit.ncbi.nlm.nih.gov/subs/genbank/) with the sequence data for\u003cem\u003e\u0026nbsp;K. pneumoniae\u003c/em\u003e L2388/L3835 under the BioProject number PRJNA390758.\u003c/p\u003e\n\u003cp\u003eThe 2 whole-genome sequenced data have been deposited in the NCBI database BioProject:PRJNA390758.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuanran Zhang and\u0026nbsp;Beiwen Zheng\u0026nbsp;conceived and designed the experiments. Hao Xu and Ruishan Liu collected samples and performed the experiments. Xiaojing Liu，Yu Yang and Kexin Guo collected the data and prepared the article. Huanran Zhang wrote the manuscript. Maotao Chen, Yuanqiang Lu and Beiwen Zheng critically revised the manuscript. Beiwen Zheng reviewed and finalized the manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study，informed consent was obtained from all participants. The study was approved by the clinical research ethics committee of the First Affiliated Hospital, Zhejiang University School of Medicine [number 2021-IIT-631]. All methods were performed following the relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2020YFE0204300); National Natural Science Foundation of China (82072314); Zhejiang Provincial Natural Science Foundation of China (LHDMZ22H190002); the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-045); and the Fundamental Research Funds for the Central Universities (2022ZFJH003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNordmann, P., A.E. 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Crosa, \u003cem\u003eGenetic organization of antibiotic resistance genes (aac(6\u0026apos;)-Ib, aadA, and oxa9) in the multiresistance transposon Tn1331.\u003c/em\u003e Plasmid, 1993. \u003cstrong\u003e29\u003c/strong\u003e(1): p. 31-40.\u003c/li\u003e\n\u003cli\u003eWu, J.W., et al., \u003cem\u003eInvestigation of carbapenem-resistant Klebsiella pneumoniae in Taiwan revealed strains co-harbouring bla(NDM) and bla(OXA-48-like) and a novel plasmid co-carrying bla(NDM-1) and bla(OXA-181).\u003c/em\u003e Int J Antimicrob Agents, 2023. \u003cstrong\u003e62\u003c/strong\u003e(5): p. 106964.\u003c/li\u003e\n\u003cli\u003eKhalifa, H.O., et al., \u003cem\u003eNDM-4- and NDM-5-Producing Klebsiella pneumoniae Coinfection in a 6-Month-Old Infant.\u003c/em\u003e Antimicrob Agents Chemother, 2016. \u003cstrong\u003e60\u003c/strong\u003e(7): p. 4416-4417.\u003c/li\u003e\n\u003cli\u003eHarmer, C.J., C.H. Pong, and R.M. Hall, \u003cem\u003eStructures bounded by directly-oriented members of the IS26 family are pseudo-compound transposons.\u003c/em\u003e Plasmid, 2020. \u003cstrong\u003e111\u003c/strong\u003e: p. 102530.\u003c/li\u003e\n\u003cli\u003eNicolas, E., et al., \u003cem\u003eThe Tn3-family of Replicative Transposons.\u003c/em\u003e Microbiol Spectr, 2015. \u003cstrong\u003e3\u003c/strong\u003e(4).\u003c/li\u003e\n\u003cli\u003eHarmer, C.J. and R.M. Hall, \u003cem\u003eIS26 and the IS26 family: versatile resistance gene movers and genome reorganizers.\u003c/em\u003e Microbiol Mol Biol Rev, 2024: p. e0011922.\u003c/li\u003e\n\u003cli\u003eZhang, P., et al., \u003cem\u003eEmergence of ceftazidime/avibactam resistance in carbapenem-resistant Klebsiella pneumoniae in China.\u003c/em\u003e Clin Microbiol Infect, 2020. \u003cstrong\u003e26\u003c/strong\u003e(1): p. 124 e121-124 e124.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"repB(R1701), NDM-4, mobile genetic elements, tandem repeats, Klebsiella pneumoniae","lastPublishedDoi":"10.21203/rs.3.rs-4904070/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4904070/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe worldwide spread of carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (CRKP) has led to a major challenge to human health. The emergence and prevalence of transferable plasmid carrying carbapenemase encoding gene is undoubtedly a potential factor in increasing the burden of carbapenem resistance. In this study, we described the reginal dissemination of repB(1701) plasmid carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4\u003c/sub\u003e in China, with the coexistence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4 \u003c/sub\u003eand\u003cem\u003e bla\u003c/em\u003e\u003csub\u003eOXA-9\u003c/sub\u003e in two independent \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates for the first time. Whole-genome sequencing and plasmid characterization of isolates L2388 and L3835 showed both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-9\u003c/sub\u003e genes are situated on the transferable plasmid, designated as pL2388-NDM-OXA\u003csup\u003e \u003c/sup\u003e[120,100 bp, IncFII(K)-repB(R1701)] and pL3835-NDM-OXA [127,486 bp, repB(R1701)]. Notably, two copies of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4\u003c/sub\u003e were identified in different orientations within pL3835-NDM-OXA. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4 \u003c/sub\u003esequence is relatively conserved (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e-\u003csub\u003e4\u003c/sub\u003e\u003cem\u003e-ble-trpF-dsbD-cutA-groES-groL\u003c/em\u003e), with various mobile genetic elements (MEGs) surrounding it, particularly IS\u003cem\u003e26\u003c/em\u003e, which may play a pivotal role in gene-level dissemination. The previously unrecognized distribution of these plasmids across select regions of Southern China and Vietnam suggests a potentially wider epidemiological range than initially presumed in the context of microbiology and epidemiology. This is the first report of transferable IncFII(K)-repB(R1701)/repB(R1701) plasmids carrying the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-4\u003c/sub\u003e gene, relevant detection and investigation measures should be taken to control the prevalence.\u003c/p\u003e","manuscriptTitle":"Reginal dissemination of transferable repB(1701) plasmid carrying blaNDM-4 in Asia: emergence of tandem repeats with blaNDM-4 mediated by IS26 in Klebsiella pneumoniae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-01 10:13:01","doi":"10.21203/rs.3.rs-4904070/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d0f40e74-45f7-4a28-8d43-b13dec9c92f3","owner":[],"postedDate":"October 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-09T15:53:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-01 10:13:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4904070","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4904070","identity":"rs-4904070","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}