Resistome and Genome Analysis of an Extensively Drug-Resistant Klebsiella michiganensis KMIB106: Characterization of a Novel KPC Plasmid pB106-1 and a Novel Cointegrate Plasmid pB106-IMP Harboring bla_IMP-4 and bla_SHV-12

Abstract

Klebsiella michiganensis is a recently emerging human pathogen causing nosocomial infections. This study aimed to characterize the complete genome sequence of a clinical Klebsiella michiganensis strain KMIB106 which exhibited extensive drug-resistance. The whole genome of the strain was sequenced using PacBio RS III systems and Illumina Nextseq 500. Annotation, transposable elements and resistance gene identification were analyzed by RAST, prokka and Plasmid Finder, respectively. According to the results, KMIB106 was resistant to multiple antimicrobials, including carbapenems, but it remained susceptible to aztreonam. The genome of KMIB106 consisted of a single chromosome and three predicted plasmids. Importantly, a novel KPC plasmid pB106-1 was found to carry the array of resistance genes in a highly different order in its variable regions, including mphA, msrE, mphE, ARR-3, addA16, sul1, dfrA27, tetD and fosA3. Plasmid pB106-2 is a typical IncFII plasmid with no resistant gene. Plasmid pB106-IMP consists of the IncN and IncX3 backbones, and two resistance genes, bla_IMP-4 and bla_SHV-12, were identified. Our study for the first time reported an extensively drug-resistant Klebsiella michiganensis strain recovered from a child with a respiratory infection in Southern China, which carries three mega plasmids, with pB106-1 firstly identified to carry an array of resistance genes in a distinctive order, and pB106-IMP identified as a novel IncN-IncX3 cointegrate plasmid harboring two resistance genes bla_IMP-4 and bla_SHV-12.

1. Introduction

Klebsiella michiganensis was first identified in Michigan from a toothbrush holder in 2012 and was initially identified as Klebsiella oxytopca due to their highly similar 16S rRNA sequences (approximately 99% identity) until it was then recognized as a new species by DNA-DNA hybridization. Since then, K. michiganensis has been recognized as a potential emerging human pathogen responsible for nosocomial infections. Commonly isolated from blood and rectal swabs, this bacterium often infects immune compromised patients, with associated infection cases being increasingly reported in recent years. In addition, the antimicrobial resistance in K. michiganensis has become a major concern in the therapeutic treatment of relevant infections, especially for K. michiganensis exhibiting multidrug resistance (MDR) [1,2,3]. Also, the carriage of various antibiotic resistance genes has been found to be associated with the multidrug resistance phenotype [1,2,3]. In particular, over the past decades, carbapenemase-producing Klebsiella spp. and their carriage of various carbapenem resistance genes have been frequently identified, which has become a leading challenge to clinicians [2,4]. For K. michiganensis, strains harboring two to three carbapenem resistance genes have been previously reported in South Africa and China [5,6].

Carbapenemase is able to hydrolyze most of the β-lactam antibiotics [7]. As an Ambler class B metallo-β-lactamase, imipenemase (IMP) confers resistance to all the β-lactam antibiotics except for the monocyclic β-lactam antibiotic and is not susceptible to β-lactamases inhibitors [8]. Up to now, a total of 62 IMP variants have been identified (https://bitbucket.org/genomicepidemiology/resfinder_db/src/master/beta-lactam.fsa; accessed on 20 October 2020). Among them, IMP-4 is the prevalent imipenemase in China [9], which was first detected in Acinetobacter spp. in Hong Kong in 2001 and ever since has been spread worldwide [10]. The carriage of the bla_IMP-4 gene provides the host strain with high minimum inhibitory concentration (MIC) values for almost all β-lactams. Wide dissemination of IMP is often due to various transferable plasmids [11,12,13,14], and IMP genes are frequently reported to be carried in combination with other resistance genes. As a consequence, the coexistence of IMP and other resistance genes in a plasmid confers higher resistance to antibiotics, raising a significant challenge for clinical treatment [15].

Plasmids are DNA genetic elements in bacteria that can replicate themselves. Typical plasmids are composed of a backbone and several variable regions, which usually carries different species and different numbers of mobilizable elements including transposons, integrons and insertion sequences (ISs). Moreover, these mobilizable elements usually carry antibiotic resistance genes; therefore, horizontal gene transfer mediated by plasmids makes a significant contribution to the dissemination of antibiotic resistance genes between different bacterial species [16].

In this study, an extensively drug-resistant K. michiganensis strain recovered from a child with a respiratory infection from a teaching hospital in Southern China was subjected to genomic sequencing and further bioinformative analysis. For the first time, we have identified three mega plasmids, with pB106-1 first identified to carry an array of resistance genes in a distinctive order which provides important evidence on the evolution of antimicrobial resistance in K. michiganensis

2. Materials and Methods

2.1. Isolation of the K. michiganensis KMIB106 Strain

The K. michiganensis strain KMIB106 was isolated from a 7-year-old girl in the First Affiliated Hospital of Guangzhou Medical University (FAHGMU) in Southern China. The child was hospitalized with a diagnosis of severe pneumonia in April 2019, who then suffered from fever with a maximum temperature of 41 °C and paroxysmal continuous cough. During the treatment, 3 different types of bacterial strains were isolated from independent sputum samples, including Acinetobacter baumannii, K. michiganensis and Elizabethkingia meningoseptica.

2.2. Bacterial Identification and Antimicrobial Susceptibility Testing

For all bacterial strains, biochemical identification to species level and the susceptibility test of 20 common antibiotics were performed using the Vitek2^TM Automated Susceptibility System [17], including ceftazidime, cefepime, cefuroxime sodium, ceftriaxone, cefuroxime axetil, cefoxitin, piperacillin/tazobactam, amoxicillin/clavulanic acid, ticacillin/clavulanic acid, compound sulfamethoxazole, minocycline, doxycycline, imipenem, ertapenem, colistin, aztreonam, amikacin, tobramycin, ciprofloxacin and levofloxacin. The MICs were interpreted according to the guidelines recommended by the Clinical and Laboratory Standards Institute (CLSI).

2.3. Genome Sequencing and De Novo Assembly and Correction

Bacterial genome sequencing was performed by PacBio RS III systems and Illumina Nextseq 500. For sequencing by PacBio RS III, genomic DNA was extracted using the CTAB method, followed by a concentration and quality test. Then, genomic DNA was fragmented with G-tubes and end-repaired to prepare SMRTbell DNA template libraries (with fragment size of >10 Kb selected by the Blue Pippin system). After the average fragment size was estimated on a Bioanalyzer 2100, SMRT sequencing was performed on the Pacific Biosciences Sequel. For sequencing by Illumina Nextseq 500, qualified genomic DNA was sonicated randomly, then end-repaired, A-tailed, and adaptor-ligated using NEBNext^® ΜLtra™ DNA Library Prep Kit for Illumina (NEB, TX, USA). DNA fragments with a length of 300–400 bp were enriched by PCR. PCR products were purified using the AMPure XP system (Beckman Coulter, Brea, CA, USA). The libraries were analyzed for size distribution by a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and quantified using real-time PCR. Genome sequencing was performed on the Illumina Novaseq 6000 sequencer using the pair-end technology. Continuous long reads obtained from SMRT sequencing runs were used for further de novo assembly using Falcon (version 0.3.0) [18]. Continuous long reads obtained from ONT sequencing were used for de novo assembly using Flye (version 2.8.1-b1676) [19]. Raw data from the Illumina platform were filtered using FASTP (version 0.20.0) [20] by the following standards: (1) removing reads with ≥10% unidentified nucleotides (N); (2) removing reads with ≥50% bases having Phred quality scores ≤ 20; (3) removing reads aligned to the barcode adapter. After filtering, the clean reads were used to correct the genome sequences to improve the quality of the assembly and determine the final genome sequences using Pilon (version 1.23) [21].

2.4. Genome Annotation and Analysis

The ORFs (open reading frames) were predicted using the NCBI prokaryotic genome annotation pipeline. For noncoding RNAs, rRNAs, tRNAs and sRNAs, predictions were carried out using rRNAmmer (version 1.2), tRNAscan (version 1.3.1) and cmscan (version 1.1.2), respectively. Gene islands and transposons were predicted using IslandPath-DIMOB (version 1.0.0) and Transposon PSI (version 20100822). Interspersed repeat elements were identified by Repeat Masker (version 4.0.5). The acquired genome sequences were further annotated with RAST and prokka tools [22,23]. The replicon types of plasmids were identified with Plasmid Finder-1.3 [24] and the resistance genes of plasmids were also predicted with ResFinder [25]. The genome of KMIB106 was searched for in CRISPR regions over the public databases using CRISPRfinder [26].

2.5. GenBank Accession Number

The whole genome sequences of KMIB106, including the chromosome and 3 plasmids, pB106-1, pB106-2 and pB106-IMP, were submitted to GenBank nucleotide sequence database under accession numbers CP067093.1, CP067094.1, CP067095.1 and CP067096.1, respectively.

3. Results

3.1. Antibiotic Susceptibilities

The antimicrobial susceptibility testing of the K. michiganensis strain KMIB106 was performed on 20 antibiotics, with MICs interpretation shown in

Table 1. Antimicrobial susceptibility profiles for KMIB106.

Group	Antibiotics	Breakpoints (μg/mL)		MIC (μg/mL)
		S	R
Fluoroquinolones	Ciprofloxacin	≤0.25	≥1	≤0.25
	Levofloxacin	≤0.5	≥2	≤0.12
Beta-lactams	Piperacillin/tazobactam	≤16/4	≥128/4	≥128/4
	Amoxicillin/clavulanic acid	≤8/4	≥32/16	≥32/16
	Ticacillin/clavulanic acid	≤16/2	≥128/2	≥128/2
	Ceftriaxone	≤1	≥4	≥64
	Ceftazidime	≤4	≥16	≥64
	Cefoxitin	≤8	≥32	≥64
	Cefuroxime axetil	≤8	≥32	≥64
	Cefuroxime sodium	≤8	≥32	≥64
	Aztreonam	≤4	≥16	≤1
	Cefepime	≤2	≥16	≥32
	Imipenem	≤1	≥4	≥16
	Ertapenem	≤0.5	≥2	≥8
Tetracyclines	Tigecycline	≤2	≥8	≤0.5
	Minocyline	≤4	≥16	≥16
	Doxycycline	≤4	≥16	≥16
Aminoglycosides	Amikacin	≤16	≥64	≤2
	Tobramycin	≤4	≥16	≤1
Folate pathway inhibitors	Compound sulfamethoxazole	≤2/38	≥4/76	≥320/80
Lipopeptides	Colistin	/	≥4	≤0.5

“/” indicates that the interpretive standard does not exist in Clinical and Laboratory Standards Institute. R, resistant; S, susceptible; MIC, minimum inhibitory concentration.

. According to the results, the K. michiganensis strain KMIB106 exhibited an extensive drug-resistance, with resistance to 14 different compounds, including ceftazidime (MIC ≥ 64), cefepime (MIC ≥ 32), cefuroxime sodium (MIC ≥ 64), ceftriaxone (MIC ≥ 64), cefuroxime axetil (MIC ≥ 64), cefoxitin (MIC ≥ 64), piperacillin/tazobactam (MIC ≥ 128), amoxicillin/clavulanic acid (MIC ≥ 32), ticacillin/clavulanic acid (MIC ≥ 128), compound sulfamethoxazole (MIC ≥ 320/80), minocycline (MIC ≥ 16), doxycycline (MIC ≥ 16), imipenem (MIC ≥ 16) and ertapenem (MIC ≥ 8). In contrast, the K. michiganensis strain KMIB106 was susceptible to tigecycline (MIC ≤ 0.5), colistin (MIC ≤ 0.5), aztreonam (MIC ≤ 1), amikacin (MIC ≤ 2), tobramycin (MIC ≤ 1), ciprofloxacin (MIC ≤ 0.25) and levofloxacin (MIC ≤ 0.12).

3.2. Genomic Analysis of Bacterial Chromosome

The average genome sequencing depth was more than 100X in coverage, including 1.2 G and 1.7 G data acquired from PacBio and Illumina platforms, respectively. The genome of the K. michiganensis strain KMIB106 was assembled into a single linear chromosome and three circular plasmids (Figure S1). The chromosome consisted of 5,907,184 bp with an overall G+C content of 55.48%. Among the 5667 predicted CDs from the chromosome, a total of 86 tRNA and 25 rRNA were identified, with 2 CRISPR-cas sequences further identified by the prokka and the CRISPRfinder server.

3.3. Profile of Resistance Gene

Analyses of antimicrobial resistance genes were performed on the genome of the K. michiganensis strain KMIB106 via ResFinder. A fluoroquinolone resistance gene oqxB [27] and a beta-lactam resistance gene bla_OXY-4-1 were found in the chromosome (

Table 2. Resistant determinants identified by genomic for KMIB106.

Resistance Gene	Location	Identity	Query/Template Length	Position	Predicted Phenotype	Accession Number
oqxB	chromosome	99.10	3131/3153	280,484..283,614	FluoroquinoloneResistance	EU370913
blaOXY-4-1	chromosome	99.88	869/873	5,016,109..5,016,977	Beta-lactam resistance	AY077481
tetD	pB106-1	100.0	1185/1185	213,924..215,108	TetracyclineResistance	AF467077
mphA	pB106-1	100.0	906/906	82,715..83,620	Macrolide resistance	D16251
msrE	pB106-1	100.0	1476/1476	188,852..190,327	Macrolide resistance	FR751518
mphE	pB106-1	100.0	885/885	190,383..191,267	Macrolide resistance	DQ839391
ARR-3	pB106-1	100.0	453/453	90,760..91,212	Rifamycin resistance	JF806499
addA16	pB106-1	99.65	846/846	89,128..89,973	Aminoglycosides Resistance	EU675686
fosA3	pB106-1	100.0	417/417	81,087..81,503	Fosfomycin resistance	AB522970
sul1	pB106-1	100.0	840/840	87,831..88,670	Folate pathway antagonist	U12338
dfrA27	pB106-1	100.0	474/474	90,154..90,627	Folate pathway antagonist	FJ459817
blaSHV-12	pB106-IMP	98.88	861/861	42,832..43,692	Beta-lactam resistance	KF976405
blaIMP-4	pB106-IMP	100.0	741/741	48,488..49,228	Beta-lactam resistance	AF244145

). Nine resistance genes were found in the novel KPC plasmid pB106-1, including genes conferring resistance to macrolide (mphA, msrE, mphE), rifamycin (ARR-3), aminoglycosides (addA16), folate pathway antagonist (sul1, dfrA27), fosfomycin (fosA3) and tetracycline (tetD). In addition, 2 β-lactamase genes bla_SHV-12 and bla_IMP-4 were found in plasmid pB106-IMP.

3.4. Correlation between Antimicrobial Resistance Phenotype and Genes Profile

The correlation between antimicrobial resistance phenotype and resistance genes profile was further analyzed in the K. michiganensis strain KMIB106. As shown in Table 2, the antimicrobial resistance phenotype was in accordance with the carriage of relevant resistance gene. For each antimicrobial agent group to which the K. michiganensis strain KMIB106 was resistant, the strain carried the relevant resistance gene. Remarkably, high MIC values were obtained for Beta-lactams and compound sulfamethoxazole, for which KMIB106 harbors 3 bla genes and sul1 with dfrA27, respectively.

3.5. Genomic Analysis of the Novel KPC Plasmid pB106-1

The size of plasmid pB106-1 was 284,262 bp, with the average G+C content at 47.04% and the total number of predicted CDs at 382. The sequence of plasmid pB106-1 showed high similarity (91% coverage, 99% identity) with plasmid pKP18-31-IMP-KPC (accession number: MN661402.1) (Figure 1). Both plasmid pB106-1 and pKP18-31-IMP-KPC contained a highly similar 230 kb backbone with a slight difference in the variable regions. In plasmid pB106-1, this backbone shared 80% identity with 41 plasmids in GenBank, with more than 95% query coverage. Among such 41 plasmids, 20, 7 and 4 were from K. pneumoniae, K. michiganensis and Raoultella planticola, suggesting the plasmid pB106-1 was likely derived from K. pneumoniae. This backbone of plasmid pB106-1 contained sequences responsible for plasmid maintenance (stbE and stbD) and conjugative transfer (traD, trbB, traE, trbN and trhU). Also, the replication sequences could be incorporated into two replicons (repB and repHI5B). Analyzed by ResFinder, plasmid pB106-1 did not belong to any incompatibility groups of Enterobacteriaceae.

Importantly, in plasmid pB106-1, the 18,748 bp variable region VR1 (nt 78,572 to 97,319) contained three fragments, including a composite transposon organized as IS26-acrR-hp-hp-fosA3-IS26-mphA-mrxA-acrR-IS600, a class 1 integron and a unique transposon Tn3 (lack of resolvase tnpR). Most of the resistance genes in plasmid pB106-1 were located in the VR1 to form a related subsidiary drug-resistant area. The VR1 was inserted in the same position in plasmid pKP18-31-IMP, KPC. However, the array of genes was arranged in a different order from pB106-1 and pKP18-31-IMP, KPC. These two variable regions both contained IS26 and IS5075 as boundaries and their class 1 integron had the same 5′-conserved segment (5′-CS), 3′-CS and ARR-3, whereas the variable region of plasmid pKP18-31-IMP, KPC contained three different fragments, a unit transposon Tn1696, a class 1 integron and a composite transposon organized as ISCR1-msrE-mphE-hp-repA-ISKpn26-IS26 (Figure 1).

In plasmid pB106-1, another 23,276 bp variable region VR2 (nt 175,964 to 199,240) was similar to that of plasmid pKP18-31-IMP, KPC (94% coverage, 99% identity). Resistance genes msrE and mphE were located on an ISCR1-IS26-mediated composite transposon, organized as ISCR1-msrE-mphE-hp-repB-IS26. In addition, the third variable region VR3 (nt 212,456 to 217,506, 5051 bp) contained the tetD resistance gene organized as IS26-tetR-tetD-frmA-frmB-IS26.

3.6. Genomic Analysis of Plasmid pB106-2

Belonging to IncFII incompatibility groups, the size of plasmid pB106-2 was 189,346 bp, with the average G+C content at 51.74% and the total number of predicted CDs at 228. The plasmid pB106-2 exhibited similarity (94% coverage, 99% identity) with an unnamed plasmid (unnamed1, accession number: CP033823.1) from the Klebsiella sp. FDAARGOS_511 strain. The plasmid pB106-2 contained two replicons, IncFII and IncFIB, but with none of the antimicrobial resistance gene.

3.7. Genomic Analysis of Plasmid pB106-IMP

The size of plasmid pB106-IMP was 85,326 bp, with the average G+C content at 48.80% and the total number of predicted CDs at 132. The backbone of plasmid pB106-IMP included sequences responsible for plasmid maintenance (stbC and stbA genes), antirestriction function (adrA, adrR and adrK genes), UV protection (mucA and mucB genes) and conjugative transfer (traM, trbA and trbK genes).

It is noteworthy that two different replication genes were found in the backbone regions of plasmid pB106-IMP, including the IncN-type replication gene repE and the IncX3-type replication gene repB. Remarkably, approximately 43.37% of the sequences were similar to a series of IncX3-type plasmids (identity > 99%, coverage 100%) and 51.92% of the sequences were similar to a series of IncN-type plasmids (identity 100%, coverage > 99%), with both regions accounting for more than 95% of the whole sequences of pB106-IMP. In addition, these two regions were linked by a composite transposon, and this composite transposon was bracketed by two identical IS26, with a ESBLs gene bla_SHV-12 inserted. Another resistance gene bla_IMP-4 was located on the IS-mediated transposition unit, organized as IS26-ΔIntI1-bla_IMP-4-ItrA-mobC-IS6100. A 54-bp segment of the integrase gene was deleted and the IS26 was located at the 5’-end of In823 integrons. A similar structure was reported in the bla_IMP-4-carrying plasmid pIMP-HZ1 (the GenBank accession number: KU886034.1) originating from the K. pneumoniae strain in China [28,29]. The sequence of bla_IMP-4 was 100% identical to those frequently detected in the broad host range of IncN-type plasmids [28]. The most common genetic environments of bla_IMP-4 were complex class 1 integrons (In809 and In823). In809 is a genetic structure with bla_IMP-4 as the core (bla_IMP-4-qacG2-aacA4-catB3-ΔqacE) and IS26 nearby. In823 was first reported in plasmid pIMP-HZ1 from the K. pneumoniae strain [28,29], in which bla_IMP-4 was captured as a cassette by the class 1 integrons. A type II intron, Kl.pn.I3, was located downstream of bla_IMP-4 and a qnrS1 (quinolone resistance gene) region was located upstream of In823. However, in our study, in In823, the upstream of truncated IntI1 was IS26 and a genetic structure of bla_SHV-12 (IS26-bla_SHV-12-ygbI-IS26) was found at the same variable region in plasmid pB106-IMP [30]. As a consequence, we hypothesized that the formation of the variable region in plasmid pB106-IMP occurs due to the recombination event mediated by the transposition formed by two copies of IS26 in the transposon.

4. Discussion

In this study, the whole genomic sequences of the K. michiganensis strain KMIB106 were analyzed, including a chromosome and three mega plasmids. For the profile of resistance genes, only two resistance genes, oqxB and bla_OXY-4-1, were located on the chromosome. As previously found, bacteria carrying the resistance gene bla_OXY-4-1 showed natural resistance to amino and carboxy penicillins. In comparison with the chromosome, plasmid pB106-1 and pB106-IMP were found to carry a large number of resistance genes which are associated with resistance to cephalosporins, β-Lactamase inhibitors, carbapenems, tetracycline antibiotics and compound sulfamethoxazole. Coincidentally, the K. michiganensis strain KMIB106 exhibited antimicrobial resistance to such antibiotics. Thus, the large proportion of the resistance genes that are likely relevant to the resistance phenotypes in KMIB106 were located on plasmids, which strongly suggested such mega plasmids with various resistance genes are the crucial factor responsible for the multidrug resistance of the host strain. Thus, horizontal gene transfer mediated by mobile genetic elements, such as plasmids, transposons and integrons, plays a major role in the dissemination of antibiotic resistance genes, as well as the ultimate adaptation of the host strain to the antibiotic pressure.

In general, each plasmid has one specific replicon gene and a plasmid might be driven away by other plasmids if they carry an incompatible replicon. However, recently, a large number of mega plasmids containing two or even more functional replicons have been reported. Importantly, the carriage of multiple replicons in one single plasmid may allow these plasmids to contain more than one incompatible replicon, and thus expand the host range and further facilitate the dissemination of these plasmids. Concerning bla_IMP-4 and bla_shv-12, the plasmids containing bla_IMP-4 often belong to several replicon types, including IncN, IncHI2, IncF, IncL/M and IncI1 [31,32,33], and the plasmid replicon types associated with the widespread dissemination of bla_shv-12 include IncX3, IncA/C, IncF, IncHI2, IncI1α/γ, IncL/M, IncN, IncP, IncR, and IncK [34]. In our study, the plasmid pB106-IMP can be divided into three regions, including the IncN section, the IncX3 section and the 4029 bp segment carried bla_SHV-12. The IncN section was similar to an IncN plasmid pIMP-HZ1, containing several conjugative transfer genes which implies the potential capability of horizontal transmission by conjugation for plasmid pB106-IMP. The IncX3 section of plasmid pB106-IMP was identical to an IncX3 plasmid p128379-NDM (the GenBank accession number: MF344560.1) which carries both bla_NDM-1 and bla_SHV-12 genes. In comparison, the 4029 bp bla_SHV-12 segment in plasmid pB106-IMP linking the IncN and IncX3 section was likely derived from the plasmid p128379-NDM (100% coverage, 99.96% identity). Furthermore, there were three identical IS26 fragments in plasmid pB106-IMP which were located at the junction of the IncX3 section; the 4029 bp segment carried bla_SHV-12 and IncN section, respectively. As a consequence, it could be inferred that the cointegrate plasmid pB106-IMP was formed by fusion of an IncX3 plasmid and an IncN plasmid, with the IS26 structure located between these two sections (Figure 2, Figure 3). Notably, this is the first report of an IncN and IncX3 cointegrate plasmid. As a novel IncN-IncX3 cointegrate plasmid, pB106-IMP is potentially capable of facilitating the dissemination of bla_shv-12 and bla_IMP-4 among Klebsiella spp.

ISs are simple autonomous mobile elements that mediate recombination events within genomes and further cause structural rearrangements. For a genetic structure composed of terminal inverted repeats and one ORF-encoded transposase, the terminal inverted repeats function as the specific recognition sites for the transposase, and the transposase further conducts the strand cleavage and transfer reactions. Various ISs are reported to play critical roles in the formation of these cointegrate plasmids [35]. IS26, as an important member of the IS6 family, contains an 820 bp nucleotide sequence, with two inverted repeats and only one gene tnp26 encoding 234 amino acids. IS26 has been reported to play an important role in genetic mobility in plasmids and chromosomes among Enterobacteriaceae, which reorganize plasmids by replicative transposition or targeted conservative [36].

5. Conclusions

This study, for the first time, described a novel IncN-IncX3 cointegrate plasmid pB106-IMP. It contained two replicons and two carbapenem genes bla_IMP-4 and bla_SHV-12, giving its nature host KMIB106 carbapenem-resistance. Our work first expanded the diversity of the plasmids incompatibility group in Enterobacterales. Revealing the mechanisms underlying the formation of this novel cointegrate plasmid will help to find new ways to inhibit dissemination of resistance genes through this type of plasmid.