Carbapenem-resistant Enterobacteriaceae (CRE) infections are characterized by increased prevalence, severe disease manifestations, longer hospital stays, higher healthcare costs, and increased mortality rates compared to susceptible carbapenem infections, thereby exacerbating a considerable global burden (Perez and Bonomo, 2019). In the 2024 updated WHO Bacterial Priority Pathogens List, CRE is classified as a critical group due to their ability to transfer resistance genes, which have been an urgent and continuous threat to global public health. An important mechanism for carbapenem resistance in CRE is the production of carbapenemases such as K. pneumoniae carbapenemase (KPC). KPC is a class A serine β-lactamase that belongs to group 2f in the Bush classification. KPC-2 is the most commonly identified variant that can efficiently hydrolyze all β-lactam antimicrobials, including carbapenems (Ding et al., 2023). The drastic increase in antibiotic-resistant strains exacerbates the failure to cure, posing a significant challenge to clinical anti-infective treatments.

Klebsiella michiganensis is an emerging species of pathogenic Klebsiella, one of the nine species of Klebsiella oxytoca complex (Yang et al., 2022). K. michiganensis has continued to spread in clinical settings, colonizing patients and causing nosocomial infections since it was first discovered in a toothbrush holder in 2012 (Saha et al., 2013). The clonal background of clinical isolates of K. michiganensis remains poorly understood and accurate species identification relies on genome sequencing and analysis. The chromosome-encoded OXY-type β-lactamase in K. michiganensis confers resistance to amino- and carboxy-penicillins. This species also acquires extended-spectrum β-lactamases (ESBLs) and carbapenemases through horizontal gene transfer (HGT), similar to other Enterobacteriaceae (Campos-Madueno et al., 2021). They also cause infections with severe clinical outcomes in hospitalized patients and can even lead to the outbreaks of clonal isolates (Chapman et al., 2020).

Carbapenemase genes can be horizontally transferred between resistant and susceptible strains via transposons and conjugative plasmids, enabling the rapid spread of resistance genes among Enterobacteriaceae and contributing to increasing CRE infections. Tn4401 is a removable element that commonly carries the bla _KPC-2 gene in Europe, Brazil, and the United States (Furlan et al., 2023). In Asia, bla _KPC-2 is mainly located on different variants of Tn1721-like transposons (Tang et al., 2017). Typical transposons Tn4401 and Tn1721, both members of the Tn3 family, have been demonstrated to mobilize the bla _KPC-2 at high transposition frequencies (Tang et al., 2022). Here, we characterized a bla _KPC-2–producing IncM2 plasmid harboring transposon ΔTn6296 in K. michiganensis LS81 recovered from a patient at a university-affiliated hospital in China. The ability of this strain to transfer the bla _KPC-2 genes was assessed by conjugation experiments. The genetic structure and location of the resistance genes on the IncM2 plasmid were analyzed through S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and whole-genome sequencing (WGS). Our comparison with the whole-genome sequence available from NCBI of K. michiganensis strains carrying bla _KPC-2 aimed to discern the driving force behind the bla _KPC-2 IncM2 plasmids.

The increasing prevalence and spread of the carbapenem resistance gene bla _KPC-2 poses a serious threat to public health, the bla _KPC-2 gene has been widely detected in different hosts. In recent years, other Enterobacteriaceae strains producing KPC-2 have been identified, including Enterobacter cloacae, Citrobacter freundii, Serrella marcescens, and Protesus mirabilis (Stewardson et al., 2019). K. michiganensis can colonize in the skin, mouth, intestines, and respiratory tract of healthy individuals and patients, and it is considered an opportunistic pathogen causing clinical infections. The extant studies highlight its critical clinical importance as a nosocomial pathogen, particularly in patients with underlying conditions causing multiple infections (Meng et al., 2022). Research has reported a propensity for K. michiganensis to cause bloodstream infections in immunocompromised individuals and severe, potentially fatal gastrointestinal damage in premature infants (Chen et al., 2020).

Nonetheless, research into the full pathological implications of K. michiganensis remains limited. Owing to the capacity of MALDI-TOF/MS to rapidly identify the species level of pathogenic microorganisms, its application in clinical microbiology laboratories has proliferated in recent years. However, the reference spectra for these novel bacterial species may be absent from most laboratories’ MALDI-TOF/MS databases, complicating the differentiation of each species within the K. oxytoca complex. In some cases, distinguishing K. michiganensis from the K. oxytoca complex requires reliance on genomic analysis techniques such as WGS, which are not readily available in standard clinical laboratories, posing a significant challenge to accurate Klebsiella species identification. As K. michiganensis is often mistakenly identified as K. oxytoca, it is conceivable that some clinical conditions ostensibly due to K. oxytoca may actually be caused by K. michiganensis.

The bla _KPC-2 gene can be transferred by plasmids, transposons, integrons and so on, and the critical step is HGT of mobile genetic elements (MGEs) around this determinant. Plasmids are MGEs that can replicate independently of the chromosome, and many carry genes encoding beneficial phenotypes for the survival of host strains, such as antimicrobial resistance (AMR) and virulence (Grodner et al., 2024). The IncM2 plasmid carrying bla _KPC-2 in K. michiganensis LS81 could be autonomously transferred into E. coli EC600, the resistance phenotype of the conjugate was obtained by the pLS81-KPC plasmid. It is noteworthy that K. michiganensis LS81 and the transconjugant H-LS81 have similar drug resistance, and the transconjugant strain once cloned will pose significant clinical challenges.

Conjugative plasmids are considered to be the primary vectors for the dissemination of AMR because of their ability to transfer horizontally between bacteria, ensure their stability through toxin-antitoxin and distribution systems, and offset their own fitness costs through mutation (Partridge et al., 2018). Among the 46 bla _KPC-2–producing K. michiganensis genomes included in our study, 35 different plasmid subtypes were detected, with the most common Inc types being IncFII, IncFIB, and IncN. Similar to the report on the K. oxytoca complex, bla _KPC-2 is mainly found on IncN or IncF plasmids (Yang et al., 2022). The diversity of plasmid backgrounds in these isolates may provide opportunities for bla _KPC-2 exchange between different plasmid families. IncL/M plasmids were previously classified in the same incompatibility group, but, after verification that they were compatible with each other, IncL and IncM plasmids were separated into two different incompatibility groups with an overall nucleotide identity of about 94% (Getino et al., 2022). IncM plasmid poses a threat due to its broad host range and increasing prevalence in clinical, animal, and environmental isolate (Rozwandowicz et al., 2018). In our study, K. michiganensis pLS81-KPC carrying bla _CTX-M3 had almost the same stable region and conjugal transfer region as the IncM2 reference plasmid pCTX-M3, derived from a Citrobacter freundii isolate from Poland (Gołebiewski et al., 2007). As reported, the IncM pCTX-M-3 plasmid can even combine with other plasmid systems to exhibit more complex replication and transfer behavior, while the range of recipients of its conjugation system is broad, including α-, β-, and γ-proteobacteria and also Firmicutes (Kern-Zdanowicz, 2021).

In investigating neonatal sepsis in several low- and middle-income countries within Africa and South Asia, it has been found that diverse strains shared identical plasmid types and exhibited the same carbapenemase gene variants, indicating the potential occurrence of successful dissemination and acquisition across multiple species (Sands et al., 2021). Moreover, the discovery of identical carbapenemase ARGs across diverse plasmids substantiates the extensive dissemination of AMR (Brandt et al., 2019). In Germany, researchers observed a widespread distribution of the bla _KPC-2 gene across various species, a process enabled by both intra- and inter-species HGT involving a unique IncN plasmid; this transfer also resulted in the rise of highly antibiotic-resistant bacteria that possess up to 14 additional ARGs encoded on the same plasmid (Yao et al., 2024). These findings underscore the pivotal role of MGEs in enabling the worldwide dissemination of resistance via HGT among a diverse array of bacterial species.

In genetic variation and evolution of bacteria, transposable elements play an important role. The bla _KPC-2 gene is mainly located in the Tn4401 and Tn1721 transposable elements. Deletions or insertions at different sites in Tn4401 divide the gene into subtypes; there are at least nine subtypes of Tn4401a–Tn4401I, of which Tn4401a and Tn4401b are the most common (Araújo et al., 2018). The Tn1721 variant has been reported to be the dominant structure in Asia, particularly in China, and the structure of the Tn1721 variant in the bla _KPC-2 epidemic is complex and diverse. In 2016, a scientific research team in China studied the genetic environment of the bla _KPC-2 gene in Klebsiella pneumoniae isolates and found that bla _KPC-2 is located in two types of genetic structures, Tn1721–bla _KPC-2–Tn3 and Tn1721– bla _KPC-2–ΔTn3-IS26 (Shen et al., 2016). In 2022, this team again reported that transposition of the Tn1721 variant promoted the evolution and spread of the KPC-2 plasmid in Enterobacteriaceae, bla _KPC-2 can be located on three different variants of Tn1721, A1-type (Tn1721-bla _KPC-2), A2-type (Tn1721–bla _KPC-2–IS26), and B-type (Tn1721–bla _KPC-2–IRL2) (Tang et al., 2022).

Tn6296, a transposon of the Tn3 family originally identified in the Klebsiella pneumoniae plasmid pKP048 (Jiang et al., 2010). The core structure of Tn6376–bla _KPC-2–ΔISKpn6-korC-klcA-orf279-orf396-ΔrepB was inserted into Tn1722, truncated and split into ΔTn1722-5′ and ΔTn1722-3′. Unlike the most common bla _KPC-2–producing mobile element transposon, the Tn6296/Tn6376 chimera may have evolved by recombination of the Tn4401. Insertions and deletions in the exogenous insertion region bla _KPC-2 lead to different ΔTn6296 variant structures, and different lengths of truncated Tn6296 can be observed on different plasmids. Tn6296-derived complex transposons are present in common species of Enterobacteriaceae, such as Klebsiella pneumoniae pHS062105-3 (Shanghai, China, KF623109), Klebsiella pneumoniae pVA833-92 (Santiago de Chile, Chile, CP093457), and many other bacterial species such as Serratia marcescens pKPC-2-HENAN1602 (Zhengzhou, China, CP047392) (Wang et al., 2022b), Citrobacter freundii PP10159-3 (Chongqing, China, MF072963) (Ouyang et al., 2018), Aeromonas hydrophila PK522-A-KPC and pK522-B-KPC (Taizhou, China, CP118700, CP118703) (Jing et al., 2024), Citrobacter koseri pCK1008-KPC-123 (Hangzhou, China, ON209376) (Wang et al., 2022a), Aeromonas media pE31A (Jinan, China, CP067418), and Klebsiella aerogenes pC212158-KPC_75k (Hangzhou, China, CP139398) (Li et al., 2024). Tn6296-derived complex transposons show diversity in bacterial clones carrying the bla _KPC-2 gene. Notably, they share a common core structure ISKpn27–bla _KPC-2–ISKpn6, we speculate that ISKpn27–bla _KPC-2–ISKpn6 may be the main genetic structure in the gene microenvironment carrying bla _KPC-2. These transposons not only carry the bla _KPC-2 gene but can also contain other ARGs, forming complex chimeric structures for rapid adaptation and evolution under antibiotic selection pressure.

In conclusion, this study is the first to characterized a bla _KPC-2–producing IncM2 plasmid pLS81-KPC harboring transposon ΔTn6296 in K. michiganensis strain in China, suggesting that it may play an important role in the dissemination of bla _KPC-2 in K. michiganensis. In addition, the presence and significance of K. michiganensis in clinical pathology may be underestimated because most strains designated as K. oxytoca in research have not been subjected to accurate species identification. Consequently, this species might be implicated in diseases previously attributed to the K. oxytoca complex and other members of the genus, with the epidemiology of colonization and infection of each species in humans remaining largely uncharted. Although China is considered an endemic area for CRE, large-scale molecular epidemiological studies of the carbapenem-resistant K. oxytoca complex have been rare in China. As an emerging pathogen, K. michiganensis has relatively few reported clinical infections and its genomic characteristics and multidrug resistance profile need further investigation.