Insights into incompatible plasmids in multidrug-resistant Raoultella superbugs

Abstract

The emergence of multidrug-resistant (MDR) Raoultella isolates is linked to the acquisition of antibiotic resistance genes (ARGs) with plasmids playing a pivotal role in this process. While plasmid-mediated transmission of ARGs in Raoultella has been extensively reported, limited attention has been given to genetically dissecting the modular structures of plasmids. This study aims to elucidate the genomic features of novel incompatible plasmids in MDR Raoultella by presenting 13 complete plasmid sequences from four isolates, along with an analysis of 16 related plasmids from GenBank. These 29 plasmids were classified into five distinct groups: IncFII single-replicon plasmids, dual-replicon plasmids containing the IncFII replicon, IncHI plasmids, IncR plasmids, and IncX8 plasmids. A new incompatible group, IncFII_{p23141−CTXM}, was identified, alongside five newly designated Inc groups based on previously determined sequences, namely IncFII_{pKPC2_EC14653}, IncFII_pCP020359, IncFII_pCP024509, IncFII_pKOX−137, and IncFII_pKDO1. Furthermore, this research marks the first report of four Inc groups of plasmids within Raoultella, namely IncFII_{p23141−CTXM} plasmid, IncFII_{pKPC2_EC14653} plasmid, IncX8 plasmid, and IncFII_pCP020359: IncFIB-7.1 dual-replicon plasmid. Moreover, novel mobile genetic elements, including two unit transposons (Tn6806 and Tn6891), one IS-based transposition unit (Tn6561), and four insertion sequences (ISRor6, ISRor7, ISRor8, and ISRor9) were discovered. Notably, this is the first report of mcr-9 in clinical Raoultella strains. At least 49 ARGs conferring resistance against 11 different categories of antimicrobials were identified on these 13 plasmids. Overall, this research deepens the understanding of incompatible plasmids in Raoultella, serving as a reference for exploring antibiotic resistance profiles and plasmid diversity in MDR Raoultella.

Introduction

Raoultella constitutes a Gram-negative, non-motile, encapsulated, facultatively anaerobic, rod-shaped bacterial genus within the Enterobacteriaceae family [1]. Previously, bacteria belonging to the Raoultella genus were classified under the Klebsiella genus. However, in 2001, molecular analysis of 16S ribosomal RNA (rRNA) and the gene encoding RNA polymerase β subunit (rpoB) led to the reclassification of K. planticola, K. ornithinolytica, and K. terrigena into the newly established genus Raoultella [2]. Raoultella is predominantly sourced from diverse natural environments, including plants, water, and soil [3]. Notably, in recent years, Raoultella has emerged as a significant opportunistic pathogen capable of inducing various infections in immunocompromised patients, such as bacteraemia, biliary tract infections, urinary tract infections, and sepsis [4,5,6,7]. Presently, the Raoultella genus encompasses four distinct species: R. planticola, R. terrigena, R. ornithinolytica, and R. electrica [3, 8, 9]. Among the Raoultella species frequently observed in clinical reports, R. planticola and R. ornithinolytica predominate [3].

Raoultella inherently exhibits resistance to penicillins due to the expression of chromosomal broad-spectrum β-lactamase genes [10]. In clinical practice, infections caused by Raoultella are commonly addressed with third- to fourth-generation cephalosporins, fluoroquinolones, and aminoglycosides [3]. However, there has been a growing body of evidence in recent years reporting that Raoultella strains confer resistance to these antibiotics, acquiring and carrying associated antimicrobial resistance genes (ARGs), including those encoding carbapenemases [11,12,13,14]. Plasmids, transposons, integrons, and other mobile genetic elements (MGEs) constitute DNA fragments that frequently harbour multiple ARGs, playing a crucial role in the acquisition, carriage, and dissemination of ARGs [15, 16]. Within Raoultella, ARGs are commonly captured and horizontally transferred by plasmids [14, 17]. Despite the widespread reporting of plasmid-mediated transmission of ARGs among Raoultella [18,19,20], there has been limited focus on detailed dissection of modular structures and the resistance gene environment of plasmids in Raoultella.

This study presents the complete sequences of 13 plasmids from four newly sequenced multidrug-resistant (MDR) Raoultella isolates in China. Detailed genetic dissection and genomic analyses were conducted. Novel incompatible groups of plasmids in Raoultella are identified. We anticipate that this research will contribute to a more profound genomics and bioinformatics understanding of the mechanisms underlying resistance adaption, evolution, and diversification of plasmids in Raoultella.

Methods

Sample collection and bacterial isolation

This study involves four clinical isolates of Raoultella, namely R. ornithinolytica strains 24150, 23141, and 602815, alongside R. planticola strain 208355. Strains 24150 and 208355 were isolated from a public hospital in Beijing, while strain 23141 originated from an affiliated hospital in Wenzhou, and strain 602815 was obtained from a public hospital in Nanjing. Strain 24150 was isolated in 2014 from a sputum sample of a 30-year-old male patient exhibiting fever and convulsions. Strain 23141 was isolated in 2016 from a 74-year-old patient, and strain 208355, collected in the same year, derived from a urine sample of a 44-year-old female patient diagnosed with vulvar melanoma. Lastly, strain 602815 was isolated in 2017 from a blood sample of a 55-year-old male patient with severe pancreatitis. Bacterial species identification was conducted through genome sequence-based average nucleotide identity (ANI) analysis (http://www.ezbiocloud.net/tools/ani), respectively [21].

Antibiotic susceptibility test

The bacterial antimicrobial susceptibility of these four Raoultella isolates was assessed using the BioMérieux VITEK 2 system and interpreted according to the 2020 Clinical and Laboratory Standards Institute (CLSI) guidelines. The corresponding antimicrobial drug susceptibility profiles are presented in Table S1.

Sequencing and sequence assembly

Whole-genome sequencing of strains 24150, 602815, and 208355 was performed using a sheared DNA library with an average size of 15 kb (ranging from 10 kb to 20 kb) on a PacBio RSII sequencer (Pacific Biosciences, CA, USA). In the case of strain 23141, genomic DNA was sequenced from a mate-pair library with an average insert size of 5 kb (ranging from 2 kb to 10 kb) using a MiSeq sequencer (Illumina).

Sequence annotation and comparison

Identification, thorough dissection, manual annotation, and additional sequence data mining of related plasmids were executed following the methodologies outlined in prior studies [22,23,24,25].

Plasmid conjugal transfer

The conjugal transfer of p23141-CTXM from the wild-type 23141 isolate into the rifampin-resistant recipient strain Escherichia coli EC600 was conducted following previously established protocols [26].

Phylogenetic reconstruction and analysis

A total of 116 sequences of Raoultella strains (last accessed 25 November, 2023, all sequenced Raoultella strains in the Genome database at the scaffold level or above and devoid of contamination) were retrieved from the RefSeq in NCBI. Strain HS11286 (accession number CP003200), the reference strain of K. pneumoniae, was selected as an outgroup. All sequences were compared to the reference genome, the chromosome sequence of R. planticola A2-F21 (accession number CP093334) using MUMmer [27]. Core single nucleotide polymorphisms (SNPs) in the backbone regions, shared by at least 95% of the strains, were identified and extracted. A maximum-likelihood phylogenetic tree was constructed following the methodologies outlined in prior studies [28]. Additionally, the replicon sequences of the indicated plasmids were aligned using Clustal Omega 1.2.2 [29], and then maximum-likelihood phylogenetic tree was constructed from aligned sequences using MEGA X 10.1.8 with a bootstrap iteration of 1000 [30].

Nucleotide sequence accession numbers

The complete chromosome and plasmid sequences of the four fully sequenced Raoultella isolates have been deposited in GenBank, with the exception of some gaps in the chromosome sequence of R. ornithinolytica 23141. The chromosome sequences for each of the four isolates (R. ornithinolytica 24150, 23141, 602815, and R. planticola 208355) are accessible through the following accession numbers: CP138912, CP138913, CP138839, and CP138840, respectively. The accession numbers for all 13 plasmids from these four isolates are provided in Table 1.

Results

Phylogenetic analysis of the four clinical Raoultella isolates

In this study, the complete genome sequences of the four Raoultella isolates were determined. Utilizing the 95% ANI threshold for genospecies identification [21], strains 23141, 24150, and 602815 were categorised as R. ornithinolytica, while strain 208355 was identified as R. planticola.

Phylogenetic analysis was undertaken to explore the diversity and evolutionary relationships among the four Raoultella strains. All Raoultella strains available in the RefSeq database (116 sequences, last accessed on November 25, 2023), with assembly levels at scaffold or above, were included in the phylogenetic analysis. After conducting multiple sequence alignments, a total of 397,640 SNPs within the core genome regions were identified and extracted to construct a maximum likelihood (ML) phylogenetic tree (Fig. 1). Further details regarding the strains included in the analysis can be found in Table S2.

The phylogenetic tree revealed distinct clusters for each species, with clear differences from other species. The four newly sequenced Raoultella strains in this study occupied different branches on the tree according to their species, consistent with previous species identification results. c24150 and GCF_018966505.1 were closely positioned on the phylogenetic tree, displaying a relatively short genetic distance between them. GCF_018966505.1 corresponds to R. ornithinolytica XF201, previously reported to be isolated from the Dongxiang wild rice rhizosphere [31]. In contrast, strain 24,150 was collected from a clinical environment. This observation suggests a possible connection between clinical and environmental strains.

Fig. 1

Population distribution of the four Raoultella isolates with 116 Raoultella genomes. The phylogenetic tree was constructed using the maximum-likelihood method, and bootstrap values were depicted using a bar featuring circles of varying sizes along each branch. The scale bar on the tree represented the degree of sequence divergence. Different background colors were used to denote the species. The four Raoultella isolates sequenced in this study were highlighted with red stars

Five groups of 13 plasmids

Each of the four Raoultella isolates harboured two to four plasmids, resulting in the identification of a total of 13 plasmids (Table 1 and Fig. S1): (i) p24150-KPC, p24150-arr, p24150-tet, and p24150-NR from strain 24150; (ii) p23141-KPC, p23141-CTXM, and p23141-mcr from strain 23141; (iii) p602815-KPC, p602815-tet, p602815-CTXM, and p602815-NR from strain 602815; (iv) p208355-IMP and p208355-NR from strain 208355. These 13 plasmids have circularly closed complete sequences, ranging from 41.9 kb to 333.5 kb in length, with mean G + C contents of 45.7–54.9%, and contain 58 to 362 predicted open reading frames (ORFs).

These 13 plasmids can be categorised into five distinct groups based on their replicon types: (i) IncFII single-replicon plasmids; (ii) dual-replicon plasmids containing the IncFII replicon; (iii) IncHI plasmids; (iv) IncR plasmids; and (v) IncX8 plasmids. The modular structure of each plasmid was divided into backbone regions (responsible for plasmid replication, maintenance, and/or conjugal transfer) and accessory modules, defined as acquired DNA segments flanked by or associated with MGEs. Each of the 13 plasmids contained a variable number of accessory modules, ranging from one to nine, encompassing both resistance and non-resistance modules (Table 1). With the exception of plasmids p24150-NR, p602815-NR, and p208355-NR, which do not contain any ARGs, the remaining 10 plasmids harboured varying numbers (ranging from 1 to 16) of ARGs, all of which were located in the accessory resistance modules (Table S3). Subsequent analysis was concentrated on these 13 plasmids, with the reference plasmid and plasmids of the same type from Raoultella strains in GenBank selected for further investigation.

Table 1 Major features of sequenced plasmids

IncFII single-replicon plasmids

Backbone regions of IncFII single-replicon plasmids

In this study, three newly sequenced IncFII single-replicon plasmids, namely p23141-KPC, p23141-CTXM, and p24150-NR, were identified (Table 1 and Fig. S1). Each of these plasmids harboured a novel replicon belonging to the IncFII family, however, they could not be categorised into any known subgroup of IncFII [32].

Through BLAST verification, the replicon of p23141-KPC was found to be identical to the replicon present in pKPC2-EC14653 from Enterobacter cloacae (accession number KP868646). Since pKPC2-EC14653 represents the first sequenced plasmid in this subgroup, it has been designated as the reference plasmid for this subgroup, denoted as IncFII_{pKPC2_EC14653}. Meanwhile, p23141-KPC is the first sequenced IncFII_{pKPC2_EC14653} plasmid from Raoultella. p23141-KPC lacks approximately 40-kb backbone region found in pKPC2-EC14653, which comprises a 15.5-kb plasmid maintenance region and a 24.5-kb conjugal transfer region (Fig. 2A). The plasmid p23141-KPC carries repA3-2-1-4, responsible for plasmid replication. Additionally, its maintenance region includes serC (serine recombinase), ecoRII (restriction enzyme), pinE (site-specific DNA recombinase), parA (partitioning ATPases), umuCD (translesion error-prone DNA polymerase V subunit), pld (phospholipase D), etc. The conjugal transfer region encompasses Δcpl (coupling protein), rlx (relaxase), tivF16 (F-type type IV secretion system, pilin acetylase) and finO (IncF plasmid conjugative transfer fertility inhibition protein) (Fig. S1).

p23141-CTXM harboured a distinctive IncFII-type replicon and was classified into a novel IncFII subgroup designated as IncFII_{p23141−CTXM}. It represents the initial sequenced IncFII_{p23141−CTXM} plasmid. The backbone of p23141-CTXM demonstrated minimal sequence homology with all publicly available plasmid sequences. Key backbone genes or loci of p23141-CTXM include the four-gene array repA3-2-1-4, responsible for replication initiation, parDE (type II toxin-antitoxin) and parABC (type Ib partitioning) for plasmid maintenance, along with residual conjugal transfer genes cpl, tivF16, and finO (Fig. 2B and S1).

The replicon of p24150-NR exhibited 100% BLAST coverage and ≥ 99% nucleotide identity to the counterparts of pCP024509 (accession number CP024509) and pCP041249 (accession number CP041249), thereby constituting a novel subgroup designated as IncFII_pCP024509. pCP024509 was the first sequenced plasmid in this subgroup. In Raoulella, two sequenced IncFII_pCP024509 plasmids exist: p24150-NR (this study) and pCP041249. Despite their membership in the same subgroup, there are notable differences in their backbone regions. p24150-NR displayed a distinct partition system compared to the other two plasmids, and certain genes in the plasmid maintenance region were absent in p24150-NR, including psiA, gvpC, and some ORFs encoding proteins of unknown function. Both pCP041249 and pCP024509 possess intact F-type type IV secretion system genes involved in conjugal transfer, while the deletion of this region (including tivF1-11, tivF16, tivF18, dtr1/2, eex, and cpl) occurred in p23141-NR (Fig. 2C and S1).

It can be inferred that p23141-KPC, p23141-CTXM, and p24150-NR are incapable of being transferred through conjugation, attributable to the absence of crucial genes in their conjugal transfer region. In order to validate the conjugative transfer capability of the newly identified plasmid Inc group IncFII_{p23141−CTXM}, conjugation experiments were conducted. Despite multiple attempts, the IncFII_{p23141−CTXM} group plasmid p23141-CTXM could not be transferred from the wild-type strain 23141 into E. coli EC600 through conjugation. This observation aligns with the genomic structure of p23141-CTXM, which lacks crucial genes in its conjugal transfer region. However, it is important to note that this result does not conclusively suggest that IncFII_{p23141−CTXM} plasmids are incapable of conjugative transfer, as there is currently limited information on the prevalence of these plasmids. Further investigation is warranted to ascertain the conjugative transfer ability of IncFII_{p23141−CTXM} plasmids.

Fig. 2

Linear comparison of IncFII single-replicon plasmids. Genes are denoted by arrows. Genes and other features are colored based on their functional classification. Shade regions denotes regions of homology (light blue: nucleotide identity ≥ 95%; light red: very low nucleotide identity with conserved gene functions)

Accessory resistance modules of IncFII single-replicon plasmids

Three accessory resistance modules were identified in p23141-KPC, p23141-CTXM, and p24150-NR, encompassing the MDR region from p23141-KPC and Tn6377 from both p23141-KPC and p23141-CTXM (Fig. 2 and S1). The bla_KPC region from pKPC2-EC14653 was included in the comparative analysis due to its similarity to Tn6296, a Tn1722-based transposon commonly found carrying the bla_KPC platform in China [26, 33].

The MDR region of p23141-KPC carried two resistance elements: ΔTn6296_{p23141 − KPC} carrying bla_KPC−2, and Tn6561 carrying addA2 and sul1. Similar to p23141-KPC, within the bla_KPC region of pKPC2-EC14653, bla_KPC was located in ΔTn6296_{pKPC2 − EC14653}. However, these two Tn6296 derivatives had undergone different evolutionary events. Compared to the prototype of Tn6296, ΔTn6296_{pKPC2 − EC14653} underwent deletion of Tn1722-3’ and insertion of bla_TEM−1 downstream of ISKpn27. ΔTn6296_{p23141 − KPC} was further truncated from ΔTn6296_{pKPC2 − EC14653} by deletion of ΔTn1722-5’, truncation of Tn6376, and loss of Δbla_TEM−1. Tn6561 was a newly identified composite transposon harboring In127, an In4-like integron containing a single aadA2 cassette. IS26 was found on both sides of In127, forming an IS26-flanked composite transposon Tn6561 with direct repeats (DRs) at its ends (Fig. 3).

Fig. 3

Organization of MDR region from p23141-KPC, and comparison to related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity ≥ 95%). Numbers in brackets indicate nucleotide positions within corresponding plasmids. Accession numbers of Tn6296 and Tn5563 used as reference are FJ628167 and U88088, respectively

Tn6377 is a Tn1722-based unit transposon generated by integrating Tn6503a, an IS-based transposition unit serving as the prototype bla_CTX−M−14 genetic platform, into the mcp gene [25, 34]. The complete structure of Tn6377 was identified in both p23141-KPC and p23141-CTXM in strain 23141. Various plasmids, including pPM64421a (accession number MF150118), pX39-8 (accession number CP023985), p11219-CTXM (accession number MF133442), and p397108-Ct2 (accession number MH917284) were found to contain different derivatives of Tn6377 with distinct deletions or insertions (Fig. 4) [35].

Fig. 4

Organization of Tn6377 from p23141-KPC and p23141-CTXM, and comparison to related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity ≥ 95%). Numbers in brackets indicate nucleotide positions within corresponding plasmids. Accession number of Tn1722 used as reference is X61367

Dual-replicon plasmids containing IncFII replicon

Backbone regions of dual-replicon plasmids containing IncFII replicon

IncFII replicons often coexist with other replicons, such as IncFIA, IncFIB, IncN, and IncR, to form multi-replicon plasmids capable of overcoming the IncFII incompatibility barrier [32]. The plasmids identified in this study, namely p24150-KPC, p602815-CTXM, and p602815-NR, were dual-replicon plasmids, each carrying an IncFII family replicon (Table 1 and Fig. S1).

p24150-KPC contains two distinct plasmid replication regions: one hosting the classical IncFII structure, designated as the IncFII_pCP020359 replicon, and the other carrying repFIB and its iterons, known as the IncFIB-7.1 replicon [36]. IncFII_pCP020359 was newly designated in this study due to its divergence from existing IncFII subgroups, with plasmid pCP020359 (accession number CP020359) being the first sequenced single-replicon plasmid in the IncFII_pCP020359 group. pKpQIL-LS6 (accession number JX442975), the first sequenced single-replicon plasmid in the IncFIB-7.1 group, was also included in the comparative analysis.

The backbone region of p24150-KPC exhibited a mosaic structure, comprising a 27.8-kb region derived from pCP020359, a 31.5-kb region derived from pKpQIL-LS6, and a 6.9-kb region not present in either pCP020359 or pKpQIL-LS6 (Fig. 5A). The pCP020359-derived regions of p24150-KPC included a set of F-type type IV secretion system genes involved in conjugal transfer, along with a 3.6-kb plasmid maintenance region carrying dsbA. Compared to pCP020359, the pCP020359-derived regions of p24150-KPC displayed two notable modular deletions: (i) the removal of the region between traQ and cpl, leading to the truncation of cpl; (ii) the deletion of the region between rlx and finO, resulting in the truncation of both rlx and finO. The pKpQIL-LS6-derived regions of p24150-KPC, containing parABC, umuCD, stbAB, and psiAB, were almost identical to the counterparts in pKpQIL-LS6.

p602815-CTXM harboured the Inc_pA1763−KPC replicon (repA_{IncpA1763−KPC} and its iterons) and the IncFII_pKDO1 replicon (repB3-repB2-repB-repB1), with repB1 in the IncFII_pKDO1 replicon being disrupted by the insertion of unit transposon Tn6377. pKDO1 (accession number JX424423) from K. pneumoniae served as the reference plasmid since it was the first sequenced plasmid with these two replicons [37], and pCP026048 (accession number CP026048) was the first sequenced Inc_pA1763−KPC: IncFII_pKDO1 plasmid in Raoultella. These three plasmids shared core backbone regions (Fig. 5B), all of which contained essential genes or gene loci responsible for plasmid maintenance, including parA, resA, ccdAB, ardAB, stbAB, umuCD, and a set of F-type type IV secretion system genes involved in conjugal transfer.

p602815-NR contained two replicons, including IncFII_pKOX−137 replicon (repA2-6-1-4) and IncFIB-4.1 replicon (repB and its iterons) [36]. The backbone region of p602815-NR comprised regions derived from pKOX-137 (accession number CP008789) and IncFIB-4.1, as well as additional regions not present in either plasmid. Notably, the entire conserved backbone region of the IncFIB-4.1 plasmid is encompassed by p602815-NR. In comparison to pKOX-137, the regions of p602815-NR derived from pKOX-137 exhibited two distinct differences (Fig. 5C): (i) a deletion of 12-genes region between orf300 and tivF18 in the conjugative transfer region, potentially resulting in reduced or lost ability for conjugative transfer; (ii) an insertion of the ISEsa2-to-ISRor7 region into orf342, leading to its disruption. pWLK-107717 (accession number CP038276) is the first sequenced IncFII_pKOX−137: IncFIB-4.1 plasmid in Raoultella [38]. This plasmid shared the key backbone region with p602815-NR, but also contained an ars locus within its backbone region, which is absent in p602815-NR.

Fig. 5

Linear comparison of dual-replicon plasmids containing the IncFII replicon. Genes are denoted by arrows. Genes and other features are colored based on their functional classification. Shade regions denotes regions of homology (nucleotide identity ≥ 95%)

A phylogenetic tree (Fig. 6) was constructed from the IncFII replicon sequences of 19 representative plasmids, including five reference plasmids representing previously reported IncFII subgroups [24, 39] and the above 14 analyzed plasmids with IncFII as the sole or auxiliary replicon. Additionally, the IncFIA replicon sequence and the IncFIB replicon sequence of plasmid F were included. These 19 plasmids could be divided into 11 separately clustering subgroups, comprised of five previously reported IncFII subgroups and six newly designated IncFII subgroups in this study.

Fig. 6

A maximum-likelihood phylogenetic tree of IncFII replicon sequences. Bootstrap values are shown next to each branch. Bar denote scale of sequence divergence. Triangles indicate the plasmids harbouring IncFII replicon that were fully sequenced in this study

Accessory resistance modules of dual-replicon plasmids containing IncFII replicon

Each of these plasmids contained two to four accessory regions (Fig. 5 and S1): (i) p24150-KPC harboured the bla_KPC region and ISKpn25; (ii) p602815-CTXM contained Tn6377, tauBC region, ΔISKpn25, and S.ma.I1; (iii) p602815-NR carried LAR_p602815−NR, KI.pn.I5-like region, and the ISEsa2-to-ISRor7 region.

The bla_KPC region from p24150-KPC was organized sequentially as ISKpn19, ΔTn6296, and IS26 (Fig. 7). bla_KPC−2 was harboured within a Tn6296 derivative, similar to the genetic context of p23141-KPC. Compared to the prototype of Tn6296, ΔTn6296 in p24150-KPC underwent the deletion of ΔTn1722-5’ and ΔTn1722-3’, as well as the insertion of IS26 within tnpA of Tn6376. Subsequently, the 5’- and 3’-terminal of ΔTn6296 were respectively linked to IS26 and ISKpn19, thereby forming the bla_KPC region. Tn6377 from p602815-CTXM, a resistant module that carried bla_CTX−M−14, was identical to Tn6377 presented in both p23141-KPC and p23141-CTXM.

Fig. 7

Organization of bla_KPC region from p24150-KPC, and comparison to related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity ≥ 95%). Numbers in brackets indicate nucleotide positions within corresponding plasmids. Accession number of Tn6296 used as reference is FJ628167

IncHI plasmids

Backbone regions of IncHI plasmids

The IncHI group can be classified into five subgroups, namely IncHI1 to IncHI5, based on their nucleotide sequence homology [17]. In this study, we identified two IncHI2 plasmids, namely p23141-mcr and p208355-NR, as well as three IncHI5 plasmids, namely p24150-arr, p602815-tet, and p208355-IMP (Table 1 and Fig. S1).

Four IncHI2 plasmids, namely p23141-mcr, p208355-NR, R478 (the IncHI2 reference plasmid, accession number BX664015), and pHNTS48-1 (the first sequenced IncHI2 plasmid in Raoultella, accession number MF135534), were included for comparative genomics analysis (Fig. S2). Each of these four plasmids contained a conserved IncHI2 backbone, encompassing essential genes or gene loci such as repHI2A and repHI2C for replication, parAB for partition, and tra1 and tra2 for conjugal transfer.

For IncHI5 plasmids, we conducted comparative genomics analysis on five selected plasmids (Fig. S3): p24150-arr, p602815-tet, p208355-IMP, pA324-IMP (the IncHI5 reference plasmid, accession number MF344566) [40], and pYNKP001-dfrA (the first sequenced IncHI5 plasmid in Raoultella, accession number KY270853) [17]. These plasmids all featured the presence of both IncHI5-type replication gene repHI5B and an additional IncFIB-6.2-type replication gene repB [36]. Plasmids p602815-tet, p208355-IMP, pA324-IMP, and pYNKP001-dfrA displayed highly conserved IncHI5 backbone regions, incorporating crucial gene loci such as parABC and tra1/tra2. However, the tra1 region in p24150-arr exhibited only 84% nucleotide identity with the sequences of the other four plasmids, while the tra2 region was fragmented into three segments, and Δtra2-3 region demonstrated a mere 76% nucleotide identity with the corresponding regions in the other four plasmids. Nevertheless, both the tra1 and Δtra2-3 regions in p24150-arr exhibited conserved gene functions as observed in their counterparts on other four plasmids.

Accessory resistance modules of IncHI plasmids

Each of the four IncHI2 plasmids harboured six to nine accessory modules distributed along the IncHI2 backbone at various sites (Fig. S1 and S2). Plasmids R478, pHNTS48-1, and p23141-mcr each contained an accessory module of 23–33 kb in length inserted between orf159-1 and orf819, indicating a potential hotspot for insertion. Of the two sequenced plasmids, p23141-mcr and p208355-NR, only p23141-mcr carried two distinct accessory resistance modules, namely the MDR-1 region and the tet region. The MDR-1 region harboured resistance genes addA2, sul1, strAB, mcr-9.1, while the tet region contained tetA(D) and tetR(D).

In the MDR-1 region of p23141-mcr, three drug resistance loci were identified, comprising In127, Tn5393c remnant, and mcr-9.1 region (Fig. 8). In127, resembling an In4-like integron, carried a single aadA2 cassette, with its intI1 truncated by IS26. Additionally, within the MDR-1 region, another In4-like integron lacking gene cassette was identified, specifically referred to as In0.

Fig. 8

Organization of MDR-1 region from p23141-mcr, and comparison to related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity ≥ 95%). Numbers in brackets indicate nucleotide positions within corresponding plasmids. Accession number of Tn5393c used as reference is AF262622

Tn5393c, a unit transposon carrying strAB, featured the structure IRL (inverted repeat left)–tnpA (transposase)–res–tnpR (resolvase)–strAB–IRR (inverted repeat right) [41]. In the Tn5393c remnant, the 5’ end was truncated while retaining the ΔtnpR–strAB–IRR structure. The mcr-9.1 region exhibited a structure of rcnR–rcnA–pcoE–pcoS–IS903B–mcr-9.1–wbuC, representing the core structure of all known mcr-9 cassettes [42]. The tet region of p23141-mcr was derived from the IS26–tetR(D)–tetA(D)–IS26 unit, in which the IS26 element downstream of tetR(D) was replaced by IS903B (Fig. S4).

Each of the five IncHI5 plasmids contained four to nine accessory modules (Fig. S1 and S3). Each plasmid harboured a single accessory resistance region. With the exception of p24150-arr, the remaining four plasmids positioned their accessory resistance regions between relB and orf342, identified as ARI-A islands. In p24150-arr, the accessory resistance region, named In469-related region, was inserted between ΔterZ and terY1.

The ARI-A islands of these four plasmids were identified as Tn1696 derivatives, including Tn6382 from pA324-IMP, Tn6338 from pYNKP001-dfrA, Tn6806 from p602815-tet, and Tn6891 from p208355-IMP. Tn1696, a unit transposon belonging to the Tn21 subfamily, originated from the integration of a class 1 integron In4 into the resolution (res) site within a primary backbone structure: IRL–tnpA–tnpR–res–mer (mercury resistance locus)–IRR [43].

The four ARI-A islands were derived from Tn1696 through the insertion of distinct integrons or integron-related regions instead of In4 in Tn1696, resulting in the formation of four intact transposons (Fig. 9). Among them, Tn6806 and Tn6891 were novel. In Tn6806, In609 replaced In4 in Tn1696, then IS26–tetA(D)–tetR(D)–IS26 unit was inserted between In609 and the mer locus, resulting in the truncation of the mer locus. In Tn6891, In4 in the prototype Tn1696 was replaced by other MGEs, arranged sequentially as follows: In37, IS26–mph(A)–IS6100 unit, ΔTn21, aacC2–tmrB region, and In809.

Fig. 9

Organization of ARI-A from IncHI5 plasmids, and comparison to related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity ≥ 95%). Numbers in brackets indicate nucleotide positions within corresponding plasmids. Accession numbers of Tn1696, Tn6502a, and Tn21 used as reference are U12338, KF914891, and AF071413, respectively

IncR plasmids

We identified an IncR plasmid named p24150-tet and performed a comparative genomics analysis on three selected plasmids: p24150-tet, pHN84KPC (accession number KY296104), and pWP3-W18-ESBL-01_2DNA (accession number AP021971). pHN84KPC served as the reference IncR plasmid [44], while pWP3-W18-ESBL-01_2DNA was an IncR plasmid previously identified in Raoultella. Both pHN84KPC and pWP3-W18-ESBL-01_2DNA harboured the complete IncR backbone regions with key backbone genes including repB, parAB, umuCD, retA, vagCD, and resD. However, the absence of retA and umuD, as well as truncation of umuC, was observed in p24150-tet.

Each of these three plasmids possessed a distinct accessory module: an MDR region in p24150-tet; a bla_KPC−2 region in pHN84KPC, and a Tn1721-related region in pWP3-W18-ESBL-01_2DNA (Fig. S5). The MDR region of p24150-tet contained three resistant elements (Fig. 10): (i) ΔTn1721, resulting from the truncation of two terminal regions of Tn1721, forming the tetR(A)–tetA(A)–pecM–ΔtnpA structure; (ii) In363, a class 1 integron carrying gene cassettes dfrA1 and gcuC; (iii) a truncated IS26–bla_LAP−2–qnrS1–IS26 unit harboring qnrS1 and bla_LAP−2, with truncations at both ends compared to the prototype [45].

Fig. 10

Organization of MDR region from p24150-tet, and comparison to related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity ≥ 95%). Numbers in brackets indicate nucleotide positions within corresponding plasmids. Accession numbers of Tn1721 and IS26–bla_LAP−2–qnrS1–IS26 unit used as reference are X61367 and HF545433, respectively

IncX8 plasmids

The plasmid p602815-KPC (Table 1 and Fig. S1), categorised within the IncX8 group, displayed a conserved IncX8 backbone region, closely resembling that of the reference plasmid pCAV1043-58 (accession number CP011588) [46]. These two plasmids shared backbone genes or gene loci (Fig. S6), including plasmid replication genes repA (pir) and bis, plasmid maintenance genes parA, topB–hha–hns, relBE, kikA, mpr, and ardR, as well as conjugal transfer genes tivB, cpl, rlx, and dtr [46]. The only modular difference observed in their backbone regions was the deletion of resD in p602815-KPC.

Each of these two plasmids featured a single accessory module (Fig. 11): a bla_KPC−2 region carrying bla_KPC−2 and Δbla_TEM−1 in p602815-KPC, and a msrAB region with no resistance genes in pCAV1043-58. Consistent with the accessory modules observed in previously sequenced IncX8 plasmids [46], these two modules were found to be associated with a miniature inverted repeat transposable elements (MITEs) [47]. Both the bla_KPC−2 region in p602815-KPC and the msrAB region in pCAV1043-58 harboured a copy of MITE256, potentially facilitating the transposition and mobilization of resistance genes [14]. Similar to p24150-KPC and p23141-KPC, bla_KPC−2 in p602815-KPC was harboured within a distinct Tn6296 derivative exhibiting a klcA–korC–ΔISKpn6–bla_KPC−2–Δbla_TEM−1–ΔTn6376 structure.

Fig. 11

Organization of accessory modules from IncX8 Plasmids, and comparison with related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity ≥ 95%). Numbers in brackets indicate nucleotide positions within corresponding plasmids. Accession number of Tn6296 used as reference is FJ628167

Discussion

This study presents the complete sequences of 13 plasmids in Raoultella. Detailed genetic dissection and comparison were conducted on these 13 plasmids, together with additional 16 related plasmids from GenBank. Based on their replicon types, these plasmids could be classified into five distinct groups: IncFII single-replicon plasmids, dual-replicon plasmids containing the IncFII replicon, IncHI plasmids, IncR plasmids, and IncX8 plasmids. Among the 13 sequenced plasmids in this work, a novel Inc group named IncFII_{p23141−CTXM} was identified. Additionally, five newly designated Inc groups were identified (first designated in this study but with previously determined sequences), namely IncFII_{pKPC2_EC14653}, IncFII_pCP020359, IncFII_pCP024509, IncFII_pKOX−137, and IncFII_pKDO1. This study reports the first instances of IncFII_{p23141−CTXM} plasmid, IncFII_{pKPC2_EC14653} plasmid, IncX8 plasmid, and IncFII_pCP020359: IncFIB-7.1 dual-replicon plasmid in Raoultella. Furthermore, two unit transposons Tn6806 within p602815-tet and Tn6891 within p208355-IMP, one IS-based transposition unit Tn6561 within p23141-KPC, and four ISs (ISRor6 and ISRor9 within p24150-arr, ISRor7 and IS Ror8 within p602815-NR) were newly identified herein.

IncFII plasmids are frequently isolated from various species in Enterobacteriaceae and play crucial roles in the dissemination of ARGs [32]. Previous research has indicated that the IncFII group can be further subdivided into different subgroups, generating numerous compatible variants [26]. This implies the possible existence of undiscovered subgroups within the IncFII group. In this study, three IncFII plasmids (p23141-KPC, p23141-CTXM, and p24150-NR) were identified from two Raoultella strains. Based on their respective replicon types and comparisons with other IncFII plasmid replicons in GenBank, these three plasmids could be classified into three distinct subgroups of IncFII: IncFII_{pKPC2_EC14653}, IncFII_{p23141−CTXM}, and IncFII_pCP024509. It is noteworthy that the IncFII_{pKPC2_EC14653} plasmid (p23141-KPC) and the IncFII_{p23141−CTXM} plasmid (p23141-CTXM) coexisted in the same bacterial strain (Raoultella 23141). This coexistence may suggest that these less-explored IncFII subgroups provide conditions for IncFII plasmids to overcome the incompatibility barrier.

Additionally, IncFII replicons frequently coexist with other replicons, such as IncFIA, IncFIB, IncN, and IncR, forming multi-replicon plasmids. In this study, three dual-replicon plasmids carrying the IncFII replicon were identified: p24150-KPC, which harbors both IncFII_pCP020359 and IncFIB-7.1 replicons; p602815-CTXM, containing a disrupted IncFII_pKDO1 replicon and an Inc_pA1763−KPC replicon; and p602815-NR, carrying an IncFII_pKOX−137 replicon and an IncFIB-4.1 replicon. Previous research has demonstrated that IncFII replicons are free to diverge when associated with FIA or FIB replicons, as FII replicons may not be involved in the initiation of plasmid replication [25, 32, 39]. In this study, p602815-NR simultaneously carries an IncFII replicon and an IncFIB-4.1 replicon, and it coexisted with p602815-CTXM, which harbours the IncFII replicon. This phenomenon may provide evidence supporting previous conclusions that the combination of IncFII multi-replicons associated with FIA or FIB can potentially generate new compatible variants, thereby overcoming the incompatibility barrier with incoming IncF plasmids. However, it is worth noting that the IncFII replicons carried by p602815-CTXM and p602815-NR do not belong to the same subgroup, which may also contribute to their coexistence. Similar scenarios are observed in the plasmids, p24150-KPC and p24150-NR, both identified in strain 24150. The coexistence of these plasmids may be attributed either to the divergence of their IncFII replicons into distinct subgroups or to the influence of the FIB replicon. In summary, whether carrying multiple replicons or forming new IncFII subgroups, IncFII plasmids demonstrate an expanded host range, thereby facilitating the extensive dissemination of antibiotic resistance genes. Therefore, IncFII plasmids warrant further attention in future studies.

IncHI plasmids, typically > 200 kb in size, exhibit a wide host range [48]. The IncHI group can be classified into five subgroups, IncHI1 to IncHI5, based on their nucleotide sequence homology [17]. However, the incompatibility interactions among these subgroups remain unclear. In this study, we identified five IncHI plasmids in Raoultella, ranging from 231 kb to 334 kb, including two IncHI2 plasmids and three IncHI5 plasmids. Similar to previously reported IncHI plasmids, each of these sequenced plasmids harboured numerous accessory modules integrated at various sites within the IncHI backbone, leading to considerable diversification of IncHI backbones. Furthermore, we identified strain 208355 harboring both the IncHI2 plasmid p208355-NR and the IncHI5 plasmid p208355-IMP, thereby providing evidence for compatibility between these subgroups.

IncR plasmids exhibit a distinctive characteristic: they possess small backbones capable of integrating various accessory modules [44]. The IncR plasmid p24150-tet, with a length of 102 kb, retained the conserved IncR backbone while integrating a 93-kb MDR region. The IncX8 subgroup, designated in 2018, is a newly identified subgroup within the IncX group [46]. Currently, there are a total of 53 IncX8 plasmids (including p602815-KPC sequenced in this study) available in GenBank (last accessed 7 November, 2023). All these plasmids originated from Enterobacteriaceae strains such as K. pneumoniae, E. coli, and Citrobacter freundii. Notably, p602815-KPC is the first and currently only identified IncX8 plasmid found in Raoultella.

A total of 49 accessory modules were identified in the 13 plasmids sequenced in this study. Among them, 14 accessory modules contained antimicrobial and heavy metal resistance genes, carrying at least 49 MGEs conferring resistance to β-lactam, aminoglycoside, macrolide, tetracycline, rifampicin, quinolone, trimethoprim, phenicol, sulphonamide, tunicamycin, and colistin. Notably, this is the first report of mcr-9 in clinical Raoultella strains. Intact or truncated ISs, unit transposons, composite transposons, and integrons were found in these accessory modules, indicating that the assembly of these accessory modules involves complex processes, such as transposition and homologous recombination, originating from diverse collections of MGEs and associated genetic elements.

Conclusions

In conclusion, this study conducted a comprehensive genetic dissection and comparison analysis of 13 plasmids (together with 16 plasmids from GenBank) from four Raoultella strains. A novel plasmid Inc group was newly identified, five newly Inc groups were newly designated, and four types of plasmids harbouring in Raoultella were first discovered, along with seven novel MGEs that were newly observed. Additionally, this study reports, for the first time in a clinical setting, the presence of Raoultella carrying mcr-9. The implication of these newly identified plasmids and other elements underscores their pivotal role in the emergence of MDR Raoultella isolates. We anticipate that this research will serve as a foundation for future investigations into the evolution and diversity of plasmids in Raoultella.