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

doi:10.21203/rs.3.rs-4904070/v1

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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

https://doi.org/10.21203/rs.3.rs-4904070/v1

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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-4and 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-4sequence is relatively conserved (bla_NDM-₄-ble-trpF-dsbD-cutA-groES-groL), with various mobile genetic elements (MEGs) surrounding it, particularly IS26, 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

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) Aba125, IS5, IS26 and IS91 [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.

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].

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)
Antibiotic	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 IS6 family (such as IS15 and IS26) and Tn3 within our plasmid. Additionally, IS91, IS30 and IS26 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 Tn3-hypothetical protein-IS26 around each bla_NDM−4, two inverted repeats harboring NDM-4 are flanked by IS15. 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

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 IS91 and a combination of IS30 and IS26, aligning with prior reports of ISAba125, IS5, IS26, or IS91 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 IS15 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 Tn3-hypothetical protein-IS26-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 Tn3-hypothetical protein-IS26, also linked to IS15. This architecture suggests heightened mobility of these regions, implying that MGEs may exert profound effects on genomic dynamics, a phenomenon warranting further attention.

IS26 was found to be closely associated with many other MGEs, such as Tn3 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 Tn3, 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 IS26-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 Tn3-hypothetical protein-IS26 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 IS26 moiety involves recognition and binding to another IS26 located on a separate exogenous DNA sequences. IS26 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.

This study provides the first description of the concurrent presence of OXA-9 and NDM-4 in single plasmid of K. pneumoniae. 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.

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.

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No competing interests reported.

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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

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Abstract

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Introduction

Materials and Methods

Results

Characterization of the strains

Genomics features

Characterization of NDM-4 and OXA-9 co-producing plasmids

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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Version 1