In silico characterization of IncX3 plasmids carrying bla OXA-181 in Enterobacterales.

Carbapenem-resistant Enterobacterales poses a global urgent antibiotic resistance threat because of its ability to transfer carbapenemase genes to other bacteria via horizontal gene transfer mediated by mobile genetic elements such as plasmids. Oxacillinase-181 (OXA-181) is one of the most common OXA-48-like carbapenemases, and OXA-181-producing Enterobacterales has been reported in many countries worldwide. However, systematic research concerning the overall picture of plasmids harboring bla OXA-181 in Enterobacterales is currently scarce. In this study, we aimed to determine the phylogeny and evolution of bla OXA-181-positive (gene encoding OXA-181) plasmids. To characterize the plasmids harboring bla OXA-181 in Enterobacterales, we identified 81 bla OXA-181-positive plasmids from 35,150 bacterial plasmids downloaded from the NCBI RefSeq database. Our results indicated that diverse plasmid types harbored bla OXA-181 but was predominantly carried by IncX3-type plasmids. We systematically compared the host strains, plasmid types, conjugative transfer regions, and genetic contexts of bla OXA-181 among the 66 bla OXA-181-positive IncX3 plasmids. We found that IncX3 plasmids harboring bla OXA-181 were mostly ColKP3-IncX3 hybrid plasmids with a length of 51 kb each and were mainly distributed in Escherichia coli and Klebsiella pneumoniae. Most of the IncX3 plasmids harboring bla OXA-181 were human origin. Almost all the bla OXA-181-positive IncX3 plasmids were found to carry genes coding for relaxases of the MOBP family and VirB-like type IV secretion system (T4SS) gene clusters, and all the 66 IncX3 plasmids were found to carry the genes encoding type IV coupling proteins (T4CPs) of the VirD4/TraG subfamily. Most IncX3 plasmids harbored both bla OXA-181 and qnrS1 in their genomes, and the two antibiotic resistance genes were found to a composite transposon bracketed by two copies of insertion sequence IS26 in the same orientation. Our findings provide important insights into the phylogeny and evolution of bla OXA-181-positive IncX3 plasmids and further address their role in acquiring and spreading bla OXA-181 genes in Enterobacterales.

Introduction

The rapid increase in carbapenemase-producing Enterobacterales has become a public-health threat (Kim et al., 2021). Surveillance studies have shown that OXA-48-like carbapenemases are the most common carbapenemases in Enterobacterales in certain regions of the world (Pitout et al., 2019). Oxacillinase-181 (OXA-181) is a carbapenem-hydrolyzing class D β-lactamase, a variant of OXA-48 differing by four amino acid substitutions, possessing a higher ability to hydrolyze carbapenems (Potron et al., 2011; Oueslati et al., 2015). The OXA-181 was first reported in clinical carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) and Enterobacter cloacae strains in Indian hospitals in 2007 (Castanheira et al., 2011). Since then, OXA-181-producing Enterobacterales, mainly K. pneumoniae and Escherichia coli (E. coli), have been reported in several countries worldwide (Balm et al., 2013; Liu et al., 2015; Rojas et al., 2017; Piazza et al., 2018; Mouftah et al., 2019), indicating a trend of increasing prevalence.

Enterobacterales cause both hospital- and community-acquired infections (Rood and Li, 2017). Carbapenem-resistant Enterobacterales (CRE) has now emerged worldwide as an urgent antibiotic resistance threat because these bacteria can transfer carbapenemase genes to other bacteria via horizontal gene transfer mediated by mobile genetic elements such as plasmids (Nordmann et al., 2011). Antimicrobial resistance (AMR) in CRE isolates is frequently encoded by plasmid-borne genes, and can disseminate clonally or horizontally (Rozwandowicz et al., 2018). Four different plasmid types belonging to the ColKP3, IncX3, IncN1, and IncT replicon types have been reported to harbor OXA-181 gene (bla OXA-181) (Pitout et al., 2019).

Conjugative plasmids are important vehicles for the dissemination of antibiotic resistant genes (ARGs) (Smillie et al., 2010; Ravi et al., 2018). These plasmids typically have the conserved backbone regions and the variable accessory regions (Brown et al., 2013; Sitter et al., 2021). The former contains genes encoding plasmid-related traits, such as replication control and conjugation functions, while the latter have accessory genes, such as genes encoding antibiotic resistance, which are usually located on the transposons or integrons (Norman et al., 2009; Norberg et al., 2011). However, systematic research on the backbone and accessory regions of plasmids harboring bla OXA-181 in Enterobacterales is currently scarce.

In this study, we performed in silico typing and comparative analysis of bla OXA-181-positive plasmids from 35,150 bacterial plasmids downloaded from the NCBI RefSeq database. We analyzed and compared the host strains, plasmid replicon types, conjugative transfer regions, and genetic contexts of the bla OXA-181 gene among the bla OXA-181-positive plasmids. This study provides important insights into the phylogeny and evolution of bla OXA-181-positive plasmids and further addresses their role in the acquisition and spread of ARGs.

Materials and methods

Plasmid genomic sequences

RefSeq database at NCBI (O’Leary et al., 2016) is a comprehensive, integrated, non-redundant, well-annotated set of reference sequences. Via the FTP release directory “Plasmid (https://ftp.ncbi.nih.gov/refseq/release/plasmid/),” we accessed and downloaded all the plasmids available in the RefSeq database. A total of 35,150 bacterial plasmid genomic sequences were downloaded on July 14, 2021 from the NCBI RefSeq database ( ). The genome data (FASTA DNA format) were downloaded in batches using two Bioperl modules including Bio::DB::GenBank and Bio::SeqIO.

Identification of bacterial plasmids harboring bla OXA-181

The β-lactamase genes within the genomes of plasmids were identified in the 35,150 plasmids using the ResFinder software version 4.1 (https://cge.cbs.dtu.dk/services/ResFinder/) (Bortolaia et al., 2020), with a minimum coverage of 60% and minimum identity of 90%. The term “bla OXA-181” was used to search in the “Resistance gene” list within the ResFinder results to determine bla OXA-181-positive plasmids.

Plasmid replicon typing of the bla OXA-181-positive plasmids

Plasmid replicon typing was performed using the PlasmidFinder software (https://cge.cbs.dtu.dk/services/PlasmidFinder/) (Carattoli and Hasman, 2020). Selecting the database “Enterobacterales”, the DNA files in FASTA format were analyzed in batches using the PlasmidFinder software, with minimum coverage of 60% and minimum identity of 95%.

Phylogenetic analyses of the bla OXA-181-positive plasmids

The files of the bla OXA-181-positive plasmids identified by ResFinder in GenBank format were downloaded in batches using the Bio::DB::GenBank and Bio::SeqIO modules. Files containing protein sequences were extracted from the files in GenBank format using the Bioperl/Bio::SeqIO module. For all bla OXA-181-positive plasmids, phylogenetic patterns based on the presence/absence of orthologous gene families were analyzed. A binary protein presence/absence matrix was created using OrthoFinder (http://www.stevekellylab.com/software/orthofinder) (Emms and Kelly, 2019) with DIAMOND for sequence similarity searches, and then a hierarchical cluster result was generated by PAST3 (Hammer et al., 2001) and eventually displayed by iTOL (https://itol.embl.de/) (Letunic and Bork, 2016).

Characterization of the conjugative regions of bla OXA-181-positive plasmids

To determine the presence/absence of oriTs, relaxase genes, T4CP genes, and gene cluster for T4SS, the files in GenBank format of the bla OXA-181-positive plasmids were analyzed in batches using the software oriTfinder (https://bioinfo-mml.sjtu.edu.cn/oriTfinder/) (Li et al., 2018) (local version). Furthermore, the types of oriTs, relaxase genes, and T4CP genes were identified based on the exhibition of oriTDB database (https://bioinfo-mml.sjtu.edu.cn/oriTDB/index.php) (Li et al., 2018). In addition, the types of gene clusters for T4SS were classified based on the SecReT4 database (https://bioinfo-mml.sjtu.edu.cn/SecReT4/) (Bi et al., 2013).

Genetic context analysis towards the bla OXA-181 and other ARGs

The bacterial insertion sequences within the bla OXA-181-positive plasmids in Enterobacterales were explored using ISfinder software (Siguier et al., 2006). Comparisons among the genetic contexts of bla OXA-181 were performed using BLAST Ring Image Generator (BRIG) (Alikhan et al., 2011).

Results

General characteristics of bla OXA-181-positive plasmids

Using ResFinder, 81 plasmids bearing bla OXA-181 ( ) were identified from 35150 bacterial plasmids downloaded from the NCBI RefSeq database. By analyzing the taxonomy of the bacterial strains containing the bla OXA-181-positive plasmids, we found that the 81 plasmids were distributed in seven different species ( ). The predominant species carrying bla OXA-181-positive plasmids was E. coli, accounting for 59.26% (48 bla OXA-181-positive plasmids), followed by K. pneumoniae, accounting for 30.10% (26 bla OXA-181-positive plasmids). Other species with bla OXA-181-positive plasmids were Enterobacter hormaechei (two plasmids), Morganella morganii (two plasmids), Citrobacter freundii (one plasmid), Klebsiella variicola (one plasmid), and Providencia rettgeri (one plasmid). Overall, all the strains harboring bla OXA-181-positive plasmids belonged to the order Enterobacterales, including family Enterobacteriaceae and family Morganellaceae.

Figure 1

Characteristics of 81 bla OXA-181-positive plasmids in Enterobacterales. (A) Histogram about number of plasmids distributed in different species for the 81 bla OXA-181-positive in Enterobacterales. (B) Length distribution of the bla OXA-181-positive plasmids in different species. (C) GC content distribution of the bla OXA-181-positive plasmids in different species. (D) Histogram of number of replicons per plasmid for the 81 bla OXA-181-positive plasmids. (E) Histogram of number of combination modes of different replicons among the 81 bla OXA-181-positive plasmids. *representing ColKP3+IncFIB(pB171)+IncFII; **representing ColKP3+IncFIB(AP001918)+IncFIC(FII); ***representing ColKP3+IncFIA+IncFIB(AP001918)+IncFII(pRSB107).

We analyzed and compared the genome sizes of the bla OXA-181-positive plasmids, and compared the sizes of the bla OXA-181-positive plasmids distributed in different species. The genome sizes of the 81 bla OXA-181-positive plasmids varied from 6.103 kb to 155.5 kb, with the 25th percentile, median, and 75th percentile being 51.47 kb, 51.48 kb, and 51.48 kb, respectively ( ). The sizes of the 48 bla OXA-181-positive plasmids in E. coli varied from 50.14 kb to 155.5 kb (25th percentile = 51.48 kb; 75th percentile = 51.48 kb), with a median size of 51.48 kb. The sizes of the 26 bla OXA-181-positive plasmids in K. pneumoniae varied from 6.103 kb to 123.3 kb (25th percentile = 49.50 kb; 75th percentile = 51.48 kb), with a median size of 51.48 kb.

We calculated the GC contents of the 81 bla OXA-181-positive plasmids. The GC contents of the 81 bla OXA-181-positive plasmids ranged from 36.51% to 53.20% (25th percentile = 46.37%; 75th percentile = 46.38%), with a median GC content of 46.37% ( ). The GC contents of the 48 bla OXA-181-positive plasmids of E. coli varied from 46.22% to 52.19% (25th percentile = 46.37%; 75th percentile = 46.38%), with a median GC content of 46.37%. The GC contents of the 26 bla OXA-181-positive plasmids of K. pneumoniae varied from 45.88% to 53.20% (25th percentile = 46.37%; 75th percentile = 49.87%), with a median GC content of 46.37%.

Replicon types in plasmids harboring bla OXA-181

Among the 81 bla OXA-181-bearing plasmids, 80 were successfully identified their replicon types, including 10 single-replicon plasmids and 70 multi-replicon plasmids (67 plasmids with two replicons, two plasmids with three replicons, and one plasmid with four replicons) ( ). Of the 67 plasmids with two replicons, 64 plasmids were ColKP3-IncX3 hybrid plasmids ( ). Of the 10 single-replicon plasmids, five plasmids had ColKP3 replicon ( ). Notably, 75 of the 81 bla OXA-181-positive plasmids in our study carried ColKP3 replicon.

Identification of the bla OXA-181-positive conjugative IncX3 plasmids

To obtain a comprehensive overview of bla OXA-181-positive plasmids, we constructed phylogenetic trees ( ). Based on the phylogenetic patterns, replicon types, and conjugative transfer regions, the 81 bla OXA-181-positive plasmids were classified into two main clades. One clade contained 66 bla OXA-181-positive plasmids (81.48%) and represented the most common conjugative plasmid pattern carrying bla OXA-181 gene. Of the 66 bla OXA-181-positive plasmids, 65 carried genes encoding relaxases of the MOBP family. All the 66 bla OXA-181-positive plasmids harbored genes encoding type IV coupling proteins (T4CPs) of the VirD4/TraG subfamily. In addition, 65 of the 66 bla OXA-181-positive plasmids contained VirB-like type IV secretion system (T4SS) gene clusters ( , ). Most of the VirB-like T4SS gene clusters were composed of 11 core genes ( ). The current version of oriTfinder could not identify the oriT sites of the clade; however, 354-bp intergenic sequences flanking the relaxase genes were identified as oriT–like regions and had the inverted repeat (IR) sequence “TAACTA..TAGTTA” ( ). Of the 66 bla OXA-181-positive conjugative IncX3 plasmids, 64 plasmids were identified with two replicons: ColKP3 and IncX3, and the other two plasmids were single-replicon IncX3 plasmids.

Figure 2

Details of replicon types, conjugative transfer regions, and host strains of the 81 bla OXA-181-positive plasmids in Enterobacterales. The six categories of information present in this figure include the phylogenetic tree, replicon types, conjugative transfer regions (oriT, relaxase, T4CP, and T4SS), origin, collection date, and host strains of 81 bla OXA-181-positive plasmids.

Figure 3

Details of the VirB-like T4SS gene clusters in the 66 bla OXA-181-positive IncX3 plasmids in Enterobacterales. Red/blue colors indicate gene presence/absence.

General characteristics of the bla OXA-181-positive conjugative IncX3 plasmids

In this study, the bla OXA-181-positive conjugative IncX3 plasmids were mainly distributed in the strains of E. coli (44 plasmids) and K. pneumoniae (18 plasmids) ( and ). Overall, all the bla OXA-181-positive conjugative IncX3 plasmids included 65 bla OXA-181-positive conjugative plasmids belonging to family Enterobacteriaceae and one plasmid belonging to family Morganellaceae ( and ).

Of the 66 bla OXA-181-harboring IncX3 plasmids, 54 were human origin, accounting for 81.82% ( and ). In addition, four bla OXA-181-harboring IncX3 plasmids were found to be animal origin, five IncX3 plasmids harboring bla OXA-181 were animal origin, and one bla OXA-181-bearing IncX3 plasmid was food origin ( and ).

We analyzed and compared the genome sizes of all the 66 bla OXA-181-harboring conjugative plasmids. The genome sizes of 66 bla OXA-181-positive IncX3 plasmids varied from 45.90 kb to 74.95 kb (25th percentile = 51.48 kb; 75th percentile = 51.48 kb), with the median size of 51.48 kb ( ). The genome sizes of the 44 bla OXA-181-positive conjugative IncX3 plasmids in E. coli ranged from 45.14 kb to 74.95 kb (25th percentile = 51.48 kb; 75th percentile = 51.48 kb), with a median size of 51.48 kb ( ). The genome sizes of the 18 bla OXA-181-positive conjugative IncX3 plasmids in K. pneumoniae ranged from 45.90 kb to 69.76 kb (25th percentile = 50.98 kb; 75th percentile = 51.48 kb), with a median of 51.48 kb ( ).

Figure 4

Distribution of lengths and GC contents of the 81 bla OXA-181-harboring plasmids in Enterobacterales. The 66 bla OXA-181-harboring IncX3 plasmids were highlighted in light green. The outermost grey bars denote the lengths of the bla OXA-181-harboring plasmids. The bars in light blue denote the GC content of the 81 bla OXA-181-harboring plasmids.

Co-existence of bla OXA-181 and qnrS1 within bla OXA-181-positive conjugative IncX3 plasmids

Among the 66 bla OXA-181-positive IncX3 plasmids, 63 were found to carry two acquired ARGs: bla OXA-181 and qnrS1 in their genomes; the former encoded OXA-181-type carbapenemase and the latter was responding for resistance to quinolone ( ). Two bla OXA-181-positive IncX3 plasmids from K. pneumoniae (K. pneumoniae strain BA39649 plasmid pColKP3_IncX3 with GenBank accession number of NZ_CP058945 and K. pneumoniae strain RIVM_C017275 plasmid pRIVM_C017275_2 with GenBank accession number of NZ_CP068868) only contained the bla OXA-181 gene ( ). In the E. coli strain EC2 IncX3 plasmid pEC2_1 (NZ_CP041956), in addition to the bla OXA-181 and qnrS1, six other acquired ARGs were also identified, including bla CTX-M-15, bla TEM-1B, bla OXA-1, aac(6’)-Ib-cr, catB3, and tet(A) ( ). In the K. pneumoniae strain RIVM_C018652 IncX3 plasmid pRIVM_C018652_2 (NZ_CP068835), eight acquired ARGs [mph(A), bla TEM-1B, rmtB, bla NDM-5, sul1, aadA2, dfrA12, and bla OXA-181] were found but no qnrS1 was present in its genome ( ).

Genetic contexts associated with bla OXA-181 and qnrS1 in the bla OXA-181-positive conjugative IncX3 plasmids

In the accessory regions of the 63 IncX3 plasmids harboring both bla OXA-181 and qnrS1 in their genomes, the bla OXA-181 and qnrS1 were located in a composite transposon, which was bracketed by two copies of insertion sequence IS26 in the same orientation ( and ). Of the 63 IncX3 plasmids with both bla OXA-181 and qnrS1, 56 were found to carry almost identical genetic contexts associated with bla OXA-181 and qnrS1, the intact IS26-flanked composite transposon ( ). Ten bla OXA-181-positive IncX3 plasmids were found to carry the truncated IS26-flanked composite transposon ( ).

Figure 5

Schematic map of bla OXA-181-associated genetic structures identified among the 66 bla OXA-181-positive IncX3 plasmids in Enterobacterales. The pEC21-OXA-181_like represent the 56 plasmids carrying the intact IS26-flanked composite transposon.

The immediate genetic environment of bla OXA-181 (IS26–ΔIS3000–ΔISEcp1–bla OXA-181–ΔlysR–Δere–ΔrepA–ISKpn19) were found to situated in all the 66 bla OXA-181-positive IncX3 plasmids. For the immediate genetic environment of qnrS1, the qnrS1 was flanked by a Tn3-like transposon (truncated by an ISKpn19) and an IS2-like insertion sequence (truncated by an IS26) ( ).

Discussion

OXA-181 is one of the most common OXA-48-like derivative, and the OXA-181-producing Enterobacterales has been reported in many countries worldwide (Pitout et al., 2019). To characterize the plasmids harboring bla OXA-181 in Enterobacterales, we identified and analyzed 81 bla OXA-181-positive plasmids, which were selected from 35,150 bacterial plasmids collected from all over the world. Our results show that diverse plasmid types harbor bla OXA-181 but IncX3 plasmids, especially the ColKP3-IncX3 hybrid plasmids, predominantly carry it, indicating the potential of IncX3 plasmids as vehicles in the global dissemination of OXA-181. IncX3 plasmids are narrow-host range plasmids in Enterobacterales (Johnson et al., 2012), which have been reported to carry various carbapenemase genes in CRE worldwide (Mouftah et al., 2019). To further characterize the IncX3 plasmids harboring bla OXA-181, we systematically compared the plasmid types, conjugative transfer regions, as well as the genetic features associated with bla OXA-181 in the 66 bla OXA-181-positive IncX3 plasmids.

The common species bearing the bla OXA-181-positive IncX3 plasmids were E. coli (44 plasmids) and K. pneumoniae (18 plasmids). OXA-181 was first described for K. pneumoniae (Castanheira et al., 2011; Potron et al., 2011). K. pneumoniae represents one of the most concerning pathogens known for its high frequency and diversity of AMR genes (Navon-Venezia et al., 2017; Wyres and Holt, 2018), and it has been classified as an ESKAPE organism (De Oliveira et al., 2020). Herein, we found that K. pneumoniae was the second most prevalent species harboring the bla OXA-181-positive IncX3 plasmids. In our study, E. coli was the predominant species carrying the bla OXA-181-positive IncX3 plasmids. AMR in E. coli has become an issue of concern in both human and veterinary health worldwide (Poirel et al., 2018).

Our work indicated that almost all the IncX3 plasmids harboring bla OXA-181 were ColKP3-IncX3 hybrid plasmids. ColKP3-type plasmid was also reported to harbor bla OXA-232, another gene encoding OXA-48-like carbapenemase (Shu et al., 2019). By comparing the sizes of IncX3 plasmids harboring bla OXA-181, we found that the OXA-181-encoding gene bla OXA-181 was mostly located in 51-kb IncX3-type plasmids. A previous study has demonstrated that bla OXA-181-positive IncX3 plasmid, 51-kb pOXA181_EC14828 in E. coli ST410 from China, was a self-transmissible plasmid, as it could be transferred to the recipient strain in the conjugation experiment (Liu et al., 2015).

Conjugative plasmids play a central role in facilitating horizontal genetic exchange and therefore promote the acquisition and spread of AMR genes (Partridge et al., 2018; Jiang et al., 2020). The conjugative transfer regions of plasmids typically consist of four modules: oriT region, relaxase gene, T4CP gene, and gene cluster for T4SS apparatus (de la Cruz et al., 2010). Herein, we attempted to analyze and compare the conjugative transfer regions located in the bla OXA-181-positive IncX3 plasmids in Enterobacterales using the software oriTfinder (Li et al., 2018). Almost all the IncX3 plasmids harboring bla OXA-181 contained genes coding for relaxases belonging to the MOBP family characterized by the domain “Relaxase (Pfam: PF03432)”. The TraI encoded by the IncPα plasmid RP4 (Pansegrau et al., 1993) is regarded as a representative of MOBP family. T4CPs are essential elements in conjugative T4SSs and are also key elements in many pathogenic T4SSs (Álvarez-Rodríguez et al., 2020). In this study, all the 66 bla OXA-181-positive plasmids in Enterobacterales carried the genes encoding T4CPs of the VirD4/TraG subfamily characterized by the domain “T4SS-DNA_transf (Pfam: PF02534)”. Almost all bla OXA-181-positive IncX3 plasmids contained VirB-like T4SS gene clusters, which are by far the best characterized T4SS (Guglielmini et al., 2014). Notably, we cannot identify the oriT sites in bla OXA-181-positive IncX3 plasmids using oriTfinder, but the 354-bp intergenic sequences flanking the relaxase genes were oriT–like regions.

In our study, we found that 63 of the 66 IncX3 plasmids harbored both bla OXA-181 and qnrS1 in their genomes. The two acquired ARGs were found to be embedded in a composite transposon, which is bracketed by two copies of the insertion sequence IS26 oriented in the same direction. The IS26 has been shown to play a major role in the dissemination of ARGs in gram-negative bacteria (Harmer and Hall, 2021) because IS26 can recruit ARGs into the mobile gene pool by forming transposons carrying many different resistance genes (Harmer and Hall, 2016). The bla OXA-181 harbored by all the 66 IncX3 plasmids was adjacent to ISEcp1. The ISEcp1 is a member of the IS1380 family known to mobilize adjacent DNA sequences by a so-called one-ended transposition mechanism (Poirel et al., 2005), and it has been shown to be involved in mobilization of different antibiotic resistance genes such as bla CTX-M (Poirel et al., 2003). The truncation of the ISEcp1 adjacent to bla OXA-181 in the 66 bla OXA-181-positive IncX3 plasmids suggested that the IS26-flanked composite transposon has the potential to mobilize bla OXA-181 independent of the action of ISEcp1.

Conclusion

In this study, we identified 81 bla OXA-181-harboring plasmids from 35,150 bacterial plasmids downloaded from the NCBI RefSeq database. Diverse plasmid types harbored bla OXA-181 but was predominantly carried by IncX3-type plasmids. Our study mainly focused on in silico characterization of the 66 bla OXA-181-bearing IncX3 plasmids, including host strains, plasmid types, origin, conjugative transfer regions, and genetic contexts. We found that IncX3 plasmids harboring bla OXA-181 were mostly ColKP3-IncX3 hybrid plasmids with a length of 51 kb each and were mainly distributed in Escherichia coli and Klebsiella pneumoniae. Most of the IncX3 plasmids harboring bla OXA-181 were found to be human origin. For the conjugative transfer regions, almost all the bla OXA-181-positive IncX3 plasmids were found to carry genes coding for relaxases of the MOBP family and VirB-like type IV secretion system (T4SS) gene clusters, and all the 66 IncX3 plasmids were found to carry the genes encoding type IV coupling proteins (T4CPs) of the VirD4/TraG subfamily. Plasmid analysis revealed that bla OXA-181 together with the quinolone resistance gene qnrS1 were carried by an IS26-flanked composite transposon on the IncX3-type plasmids. Our findings enhance our understanding of the genetic diversity and characteristics of bla OXA-181-harboring IncX3 plasmids and further address their role in acquiring and spreading bla OXA-181 genes in Enterobacterales.

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/ .

Author contributions

XL, ML and XF designed the study. XL and ZY analysed all the data and drafted the manuscript. SH, TL, QL, and ZXZ contributed to the data acquisition and methodology. ZRZ and LS reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This work was supported financially by the National Natural Science Foundation of China (Grant No. 81902460 and 82002170), the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2019A1515011725), the Xiangshan Talent Project of Zhuhai People’s Hospital (Grant No. 2020XSYC-02), the Cultivation Project of Zhuhai People’s Hospital (2019PY-19 and 2019PY-22).

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.

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