In silico characterization of blaNDM-harboring plasmids in Klebsiella pneumoniae

Klebsiella pneumoniae is a primary culprit of antibiotic-resistant nosocomial infections worldwide, and infections caused by NDM-producing strains are a major threat due to limited therapeutic options. The majority of bla_NDM cases occur on plasmids; therefore, we explored the relationships between plasmids and bla_NDM genes in K. pneumoniae by analyzing the variants of bla_NDM, replicon types, conjugative transfer regions of 171 bla_NDM-harboring plasmids from 4,451 K. pneumoniae plasmids. Of the nine identified bla_NDM variants, bla_NDM-1 (73.68%) and bla_NDM-5 (16.37%) were the most dominant. Over half of the bla_NDM-harboring plasmids of K. pneumoniae were classified into IncF plasmids. IncX3 single-replicon plasmids (46–57 kb) carried genes encoding relaxases of the MOB_P family, T4CP genes of the VirD4/TraG subfamily, and VirB-like T4SS gene clusters, which were mainly geographically distributed in China. We found 10 bla_NDM-harboring IncN plasmids (38.38–63.05 kb) carrying the NW-type origin of transfer (oriT) regions, genes coding for relaxases of MOB_F family, genes encoding T4CPs of the TrwB/TraD subfamily, and Trw-like T4SS gene clusters, which were also mainly geographically distributed in China. Moreover, we identified 21 IncC plasmids carrying bla_NDM-1 (140.1–329.2 kb), containing the A/C-type oriTs, genes encoding relaxases of MOB_H family, genes encoding T4CPs belonging to TrwB/TraD subfamily, and Tra_F-like T4SS gene clusters. The bla_NDM-harboring IncC plasmids were widely geographically distributed all over the world, mainly in the United States, China and Viet Nam. These findings enhance our understanding of the diversity of bla_NDM-harboring plasmids in K. pneumoniae.

Introduction

Klebsiella pneumoniae is a significant cause of nosocomial infections such as pneumonia, bloodstream infections, urinary tract infections, and septicemias (Pitout et al., 2015; Bengoechea and Sa Pessoa, 2019). Klebsiella pneumoniae represents one of the most concerning pathogens known for its high frequency and diversity of antimicrobial resistance (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). The emergence and spread of carbapenem-resistant K. pneumoniae have become severe medical problems worldwide (Navon-Venezia et al., 2017). Resistance to carbapenems in K. pneumoniae involves diverse mechanisms, e.g., production of carbapenemases (e.g., KPC, NDM, and OXA-48-like), alterations in outer membrane permeability and the upregulation of efflux systems (Pitout et al., 2015).

New Delhi metallo-β-lactamase (NDM), belonging to Ambler class B β-lactamase, has the ability to hydrolyze all β-lactam antibiotics (including carbapenems) except the monobactam aztreonam (Nordmann et al., 2011). NDM-1 was first reported in a K. pneumoniae isolate recovered in a Swedish patient who traveled to New Delhi in 2008 (Yong et al., 2009). According to the records of the Beta-lactamase database (BLDB; Naas et al., 2017) on September 8th, 2022, more than 40 variants of NDM have been identified so far. A variety of infections caused by NDM-producing Enterobacterales strains are associated with inferior prognosis and high mortality, especially in high-risk immunocompromised patients (Guducuoglu et al., 2018). NDM-producing Enterobacterales clinical isolates, mainly K. pneumoniae and Escherichia coli, have been found worldwide, with a higher prevalence in the Indian subcontinent, the Balkans, and the Middle East (Albiger et al., 2015; Wu and Feng, 2019).

Antimicrobial resistance (AMR) in carbapenem-resistant Enterobacterales (CRE) strains is often encoded by the plasmid-borne genes (Rozwandowicz et al., 2018). Plasmids, especially conjugative plasmids, play an essential role in mediating horizontal gene transfer (HGT) and dissemination of AMR (Jiang et al., 2020). The conjugative transfer regions of conjugative plasmids typically comprise four key modules, including origin of transfer (oriT) region, gene encoding relaxase, gene encoding type IV coupling protein (T4CP), and gene cluster for the bacterial type IV secretion system (T4SS) apparatus (de la Cruz et al., 2010). The relaxase initiates the bacterial conjugation by recognizing and cleaving the oriT of the plasmid in a site-specific manner, forming a relaxosome (Llosa et al., 2002; Carballeira et al., 2014). Currently, nine types of plasmid-borne oriT¹ and eight main relaxase families² have been identified (Li et al., 2018). Conjugation requires a pilus, which is assembled by T4SS, to connect the donor and the recipient strains (de la Cruz et al., 2010). Currently, five main types of T4SS gene clusters are defined, including 18 different kinds of systems³ (Bi et al., 2013). The T4CP connects the relaxosome to T4SS, which is required for conjugation, and currently, two main subfamilies of T4CPs⁴ exist (Li et al., 2018).

Studies on the comprehensive analysis of bla_NDM-harboring plasmids and their conjugative transfer regions in K. pneumoniae are scarce. In this work, we executed in silico typing and comparative analysis of bla_NDM-harboring plasmids of K. pneumoniae using the bacterial plasmids available in the NCBI GenBank database. We systematically analyzed the variants of bla_NDM, replicon types, phylogenetic patterns, and conjugative transfer regions of the bla_NDM-positive plasmids of K. pneumoniae. This study provides deep insights into the characteristics and diversity of bla_NDM-harboring plasmids in K. pneumoniae and further emphasizes their role in dissemination of resistance genes.

Materials and methods

Plasmid sequences from the NCBI database

The GenBank Genome database (Benson et al., 2018) collect all the plasmids belonging to K. pneumoniae.⁵ A total of 4,451 plasmids (without duplicates) of K. pneumoniae (Supplementary Table S1) were downloaded on April 26th, 2022. Files in FASTA DNA format of the 4,451 plasmids were downloaded in batches into our Linux-based server.

Identification of the bla_NDM-harboring plasmids of Klebsiella pneumoniae

The β-lactamase genes of the plasmids of K. pneumoniae were identified applying the ResFinder software, standalone version 4.1 (Bortolaia et al., 2020), with the minimum coverage of 60%, minimum identity of 90%, and species of “Klebsiella.” The term “bla_NDM” was used to search in the “Resistance gene” list of the ResFinder results in order to judge the bla_NDM-harboring plasmids of K. pneumoniae and identify the variants of the bla_NDM genes. For some bla_NDM-harboring plasmids, the variants of bla_NDM were not determined by the ResFinder software; instead, they were submitted to the CARD database⁶ (Alcock et al., 2020) and the Beta-lactamase database (BLDB; Naas et al., 2017) for further analysis.

Replicon typing of the bla_NDM-harboring plasmids of Klebsiella pneumoniae

Replicon typing of the bla_NDM-harboring plasmids was executed via the PlasmidFinder software (Carattoli and Hasman, 2020). Then, selecting the database “Enterobacteriales,” the FASTA-formatted DNA files were analyzed and classified in batches by using the PlasmidFinder tool version 2.0.1, with a minimum coverage cut-off of 60% and minimum identity cut-off of 95%. The database version was updated on November 29th, 2021.

Phylogenetic cladogram of the bla_NDM-harboring plasmids of Klebsiella pneumoniae

The files of the bla_NDM-harboring plasmids of K. pneumoniae, in GenBank format, were downloaded in batches using two Bioperl modules (Bio::SeqIO and Bio::DB::GenBank). Plasmid files containing protein sequences were compiled from the plasmid files in GenBank format through the Bioperl/Bio::SeqIO module. Phylogenetic cladogram based on the presence/absence of orthologous gene families of all the bla_NDM-harboring plasmids of K. pneumoniae were constructed. First, a binary gene presence/absence matrix was built using OrthoFinder software (Emms and Kelly, 2019), and subsequently a hierarchical cluster result was generated by PAST3 (Hammer et al., 2001) and eventually displayed by iTOL (Letunic and Bork, 2016).

Geographic location and host ST types of the bla_NDM-harboring plasmids in Klebsiella pneumoniae strains

Information about geographic location of bla_NDM-harboring plasmids and its host strains were extracted from the files of the bla_NDM-harboring plasmids in GenBank format. Table containing the correspondence between strains and plasmids of K. pneumoniae were downloaded from the GenBank.⁷ The bla_NDM-harboring plasmid-matched host K. pneumoniae strains were collected, and their DNA FASTA sequences were downloaded in batch using the Bioperl. The MLST software (Larsen et al., 2012) version 2.0.9 was downloaded from the website⁸ and installed on the Linux platform. The genomes of K. pneumoniae strains were analyzed in batch using MLST software.

Characterization of the conjugative transfer regions of bla_NDM-harboring plasmids

Files in GenBank format of the bla_NDM-harboring plasmids in K. pneumoniae were analyzed in batches using oriTfinder software (local version; Li et al., 2018) to identify the presence/absence of oriTs, relaxase-coding genes, T4CP-coding genes, and gene clusters for T4SS. Furthermore, the types of oriTs, relaxases, T4CPs, and T4SSs toward the plasmids were determined based on the exhibition of the oriTDB database⁹ (Li et al., 2018). In addition, the types of gene clusters for T4SS were classified based on the SecReT4 database¹⁰ (Bi et al., 2013).

Bipartite network construction, clustering and visualization of the bla_NDM-harboring plasmids of Klebsiella pneumoniae

The bipartite network was constructed based on all the bla_NDM-harboring plasmids of K. pneumoniae using the AccNet software using default parameters (Lanza et al., 2017). The obtained network files including nodes, edges and clusters were then imported into the Cytoscape software (Shannon et al., 2003) for visualization. We displayed the relative genomic content of each plasmid by making the diameter of each node proportional to its degree.

Results

Variants of bla_NDM genes in the bla_NDM-harboring plasmids of Klebsiella pneumoniae

Based on the results analyzed by ResFinder, 171 (3.84%) bla_NDM-harboring plasmids (Supplementary Table S2) were identified from 4,451 plasmids of K. pneumoniae, which were downloaded from the GenBank Genome database. Among the 171 bla_NDM-harboring plasmids of K. pneumoniae, nine different variants of bla_NDM were identified (Figure 1A). Among the nine variants of bla_NDM, bla_NDM-1 was found to be the predominant variant, accounting for 73.68% (126 bla_NDM-1-harboring plasmids), followed by bla_NDM-5, accounting for 16.37% (28 bla_NDM-5-harboring plasmids) (Figure 1A).

FIGURE 1

Figure 1. Characteristics of the 171 bla_NDM-harboring plasmids of Klebsiella pneumoniae. (A) Histogram of number of variants of bla_NDM genes among the 171 bla_NDM-harboring plasmids of K. pneumoniae. bla_NDM* representing an unknown variant of bla_NDM. (B) An overview of the plasmid replicon types among the 171 bla_NDM-harboring plasmids of K. pneumoniae analyzed using software PlasmidFinder.

Replicon types of bla_NDM-harboring plasmids of Klebsiella pneumoniae

Replicon typing of the 171 bla_NDM-harboring plasmids of K. pneumoniae was executed using PlasmidFinder. Of the 171 plasmids, 154 were successfully identified with their replicon types, including 103 single-replicon plasmids and 51 multi-replicon plasmids (44 plasmids with two replicons, three plasmids with three replicons, and four plasmids with four replicons; Figure 1B; Supplementary Figure S1). For the 103 single-replicon plasmids harboring bla_NDM in K. pneumoniae, the TOP5 prevalent replicons (in descending order) were IncX3 (29 plasmids), IncC (15 plasmids), IncFIB(pQil) (11 plasmids), IncFII (11 plasmids), and IncFII(Yp) (11 plasmids). Of the 44 bla_NDM-harboring plasmids with two replicons, 25 contained replicons IncFIB(pNDM-Mar) and IncHI1B(pNDM-MAR), which were the most prevalent two-replicon plasmids harboring bla_NDM in K. pneumoniae (Figure 1B; Supplementary Figure S1).

In summary, 21 of the 171 bla_NDM-harboring plasmids of K. pneumoniae were found to carry the replicon of IncC, accounting for 12.28% of all the bla_NDM-harboring plasmids of K. pneumoniae in this study (Figure 1B; Supplementary Figure S1). Notably, 91 of the 171 bla_NDM-harboring plasmids in our study were found to be the IncF plasmids, including IncFI and IncFII plasmids, accounting for 53.22% of all the bla_NDM-harboring plasmids of K. pneumoniae in this study (Figure 1B; Supplementary Figure S1).

Diversity of the bla_NDM-harboring plasmids in Klebsiella pneumoniae

To get the comprehensive overview of bla_NDM-harboring plasmids in K. pneumoniae, we created a phylogenetic cladogram of the 171 bla_NDM-harboring plasmids (Figure 2). Based on phylogenetic patterns of the 171 plasmids, combined with the replicon types, conjugative transfer regions, and genome sizes of the bla_NDM-harboring plasmids, most of the 171 bla_NDM-harboring plasmids were clustered into 10 main clades (clades I–X), representing 10 plasmid patterns carrying bla_NDM genes in K. pneumoniae (Table 1).

FIGURE 2

Figure 2. Details of variants of bla_NDM genes, replicon types, conjugative transfer regions, geographical distributions, host STs, and length distribution of the 171 bla_NDM-harboring plasmids of K. pneumoniae. The four categories of information present in this figure include the phylogenetic cladogram, variants of bla_NDM, replicon types, conjugative transfer regions (oriT, relaxase, T4CP, and T4SS), geographical distributions, ST types of host strains, and length distribution of the 171 bla_NDM-harboring plasmids of K. pneumoniae. bla_NDM* representing an unknown variant of bla_NDM.

TABLE 1

Table 1. Summary of the 171 bla_NDM-harboring plasmids of Klebsiella pneumoniae.

Clade I: A total of 29 IncX3 plasmids were found in the clade I cluster, mainly bla_NDM-1 and bla_NDM-5 (Figure 2). For the 29 IncX3 plasmids harboring bla_NDM, their genome sizes varied from 45.1 to 159.3 kb (25th percentile = 46.2 kb; 75th percentile = 57.3 kb), with a median size of 53.1 kb (Supplementary Figure S2). For the conjugative transfer regions, all the plasmids belonging to clade I were found to carry genes encoding relaxases of the MOB_P family characterized by the domain “Relaxase (Pfam: PF03432),” T4CPs of the VirD4/TraG subfamily characterized by the domain “T4SS-DNA_transf (Pfam: PF02534),” and VirB-like T4SS gene clusters (Figure 2; Supplementary Figure S3). Members of clade I were mainly geographically distributed in China (Figure 3; Table 1; Supplementary Table S3). No predominant ST types of isolates were found in the plasmids harboring bla_NDM in K. pneumoniae (Table 1; Supplementary Table S3).

FIGURE 3

Figure 3. Worldwide distribution of bla_NDM-harboring plasmids of K. pneumoniae. The geographical distribution of the 10 clades (Clade I–Clade X) from the bla_NDM-positive plasmids of K. pneumoniae was calculated and displayed by pie chart. For the plasmids isolated in European countries, only those from the United Kingdom, Italy, and Switzerland were displayed by pie chart, others were displayed in the tabular form. For the plasmids isolated in Southeast Asia, only those from Viet Nam were displayed by pie chart, others were displayed in the tabular form. For the plasmids isolated in South Asia, only those from India were displayed by pie chart, others were displayed in the tabular form.

Clade II: Ten bla_NDM-positive IncN plasmids were clustered into clade II, mainly carrying bla_NDM-1 (Figure 2). The genome sizes of the 10 bla_NDM-harboring IncN plasmids varied from 38.4 to 63.1 kb (25th percentile = 47.2 kb; 75th percentile = 59.8 kb), with a median size of 52.0 kb (Supplementary Figure S1). Almost all the IncN plasmids carried the NW-type oriTs and genes encoding relaxases of MOB_F family characterized by the domain “TrwC (PF08751).” All the 10 bla_NDM-positive IncN plasmids carried the genes encoding T4CPs of the TrwB/TraD subfamily characterized by the domain “TrwB_AAD_bind (PF10412)” and Trw-like T4SS gene clusters (Figure 2; Supplementary Figure S4). The members of clade II were mainly geographically distributed in China (Figure 3; Table 1; Supplementary Table S3). The 10 bla_NDM-positive IncN plasmids were distributed in seven ST types of K. pneumoniae strains, and four plasmids were distributed in K. pneumoniae ST15 (Table 1; Supplementary Table S3).

Clade III: Eleven bla_NDM-1-positive IncF plasmids with the IncFIB(pQil) replicon were grouped into clade III, and most were 54-kb plasmids (Figure 2). Moreover, no conjugative transfer regions were identified in the 11 plasmids of clade III, indicating that the 11 plasmids should be non-transferable. Plasmids belonging to clade III were mainly geographically distributed in Italy and United States (Figure 3; Table 1; Supplementary Table S3). All the members of clade III were harbored by the strains of K. pneumoniae ST147 (Table 1; Supplementary Table S3).

Clade IV: Five bla_NDM-positive untypeable plasmids were clustered into clade IV, involving three bla_NDM-1-positive plasmids and two bla_NDM-4-positive plasmids (Figure 2). These five untyped plasmids, with lengths ranging from 75.3 to 86.0 kb, all carried the genes encoding relaxases of MOB_F family, genes encoding T4CPs of TrwB/TraD subfamily, and Tra_F-like T4SS gene clusters (Figure 2; Supplementary Figure S5). For the five plasmids, three were found in Viet Nam, one was found in China, and one was found in South Korea (Figure 3; Table 1; Supplementary Table S3). The STs of K. pneumoniae host strains containing the clade IV plasmids were distributed into ST395 and ST16 (Table 1; Supplementary Table S3).

Clade V: Thirteen plasmids with the IncFII replicon, mainly carrying bla_NDM-5, were classified into the clade V (Figure 2). For the 13 IncFII plasmids harboring bla_NDM, genome sizes varied from 75.3 to 140.6 kb (25th percentile = 88.8 kb; 75th percentile = 101.4 kb), with a median size of 96.2 kb (Supplementary Figure S1). They all carried the F-type oriTs and Tra_F-like T4SS gene clusters (Figure 2; Supplementary Figure S6). Most of the plasmids clustered into clade V were found to carry genes encoding relaxases of the MOB_F family and genes encoding T4CPs of the TrwB/TraD subfamily (Figure 2). The members of clade V were widely distributed in India, Southeast Asia, North America, East Asia, and Europe, with the highest prevalence in India (Figure 3; Table 1; Supplementary Table S3). The STs of K. pneumoniae host strains containing all Clade V plasmids were mainly distributed in ST16, ST147, and ST2096 (Table 1; Supplementary Table S3).

Clade VI: Four bla_NDM-1-positive plasmids, including one IncM2 plasmid and three untyped plasmids, were classified into a small cluster named clade VI in our study (Figure 2). These four plasmids, with lengths ranging from 75.6 to 100.2 kb, all carried the L/M-type oriTs, genes encoding relaxases of MOB_P family, genes encoding T4CPs of TrwB/TraD subfamily, and Tra_I-like T4SS gene clusters (Figure 2; Supplementary Figure S7). The four plasmids were sporadically discovered in Canada, Germany, United Kingdom, and China (Figure 3; Table 1; Supplementary Table S3). No prevalent STs of K. pneumoniae host strains containing all clade VI plasmids were found (Table 1; Supplementary Table S3).

Clade VII: Four bla_NDM-1-positive plasmids with the IncFII(pKPX1) replicon were classified into clade VII (Figure 2). The genome sizes of the four IncFII(pKPX1) plasmids varied from 96.8 to 250.4 kb. Similar to the conjugative transfer regions of plasmids belonging to clade VI, they all carried the L/M-type oriTs, genes encoding relaxases of the MOB_P family, genes encoding T4CPs of the TrwB/TraD subfamily, and Tra_I-like T4SS gene clusters (Figure 2; Supplementary Figure S8). The four plasmids were sporadically discovered in United States, India, Switzerland, and China (Figure 3; Table 1; Supplementary Table S3). No obvious common STs of strains were found (Table 1; Supplementary Table S3).

Clade VIII: Eighteen IncF plasmids, mainly carrying bla_NDM-1, were grouped into the clade VIII cluster (Figure 2). Most of the IncF plasmids contained the IncFII(Yp) or IncFII(K) replicon in their genomes. For the 18 bla_NDM-harboring plasmids of clade VIII, genome sizes varied from 94.4 to 316.2 kb (25th percentile = 106.8 kb, 75th percentile = 150.1 kb), with a median size of 110.6 kb (Supplementary Figure S1). Most of the plasmids of clade VIII were found to contain the F-type oriTs. They all carried the genes encoding relaxases of the MOB_F family, genes encoding T4CPs of the TrwB/TraD subfamily, and Tra_F-like T4SS gene clusters (Figure 2; Supplementary Figure S9). Notably, K. pneumoniae strain JNQH116 plasmid pJNQH116-2 (NZ_CP070900), belonging to the clade VIII cluster, was found to contain both Tra_F-like and Trw-like T4SS gene clusters in its genome. For clade VIII, its members were widely geographically distributed all over the world, including China, India, Southeast Asia, Middle East, North America (Canada and United States), South Africa, and some European countries (e.g., Germany, Romania, and the United Kingdom; Figure 3; Table 1; Supplementary Table S3). No prevalent STs of K. pneumoniae host strains containing all clade VIII plasmids were found (Table 1; Supplementary Table S3).

Clade IX: A total of 21 IncC plasmids carrying bla_NDM-1 were grouped into the clade IX cluster of the phylogenetic cladogram (Figure 2). Their genome sizes varied from 140.1 to 329.2 kb, with the 25th percentile, median size, and 75th percentile being 144.3, 147.9, and 178.7 kb, respectively (Supplementary Figure S1). For the conjugative transfer modules, all the plasmids belonging to clade IX carried the A/C-type oriTs, genes encoding relaxases of the MOB_H family characterized by the domain “TraI_2 (Pfam: PF07514),” mostly genes encoding T4CPs of TrwB/TraD subfamily, and Tra_F-like T4SS gene clusters (Figure 2; Supplementary Figure S10). The IncC plasmids harboring bla_NDM-1 were widely geographically distributed all over the world, mainly in the United States, Viet Nam, and China (Figure 3; Table 1; Supplementary Table S3). ST11 and ST1967 were the common STs strains containing bla_NDM-1-harboring IncC plasmids (Table 1; Supplementary Table S3).

Clade X: A total of 40 mega plasmids, where the length range varied from 238.0 to 401.6 kb (25th percentile = 293.5 kb; median = 327.3 kb; 75th percentile = 355.1 kb), mainly carrying bla_NDM-1, were grouped into a large cluster, named clade X in our study (Figure 2). Of the plasmids belonging to clade X, 27 (67.5%) were found to contain both replicons IncFIB(pNDM-Mar) and IncHI1B(pNDM-MAR), seven (17.5%) were unable to be typed, and four (10.0%) were IncQ1 plasmids. Moreover, all the plasmids of clade X carried genes encoding T4CPs of the TrwB/TraD subfamily and Tra_F-like T4SS gene clusters (Figure 2; Supplementary Figure S11). Most of the plasmids belonging to clade X were found to have no oriT and harbored the genes encoding relaxases of the MOB_H family. For the clade with the largest number, clade X, its members were widely distributed all over the world, mainly in China and the United States (Figure 3; Table 1; Supplementary Table S3). ST14, ST11, and ST147 were the common STs strains containing the plasmids of clade X (Table 1; Supplementary Table S3).

We also perform a bipartite network analysis with the 171 bla_NDM-harboring plasmids of K. pneumoniae. The bipartite network consisted of two classes of nodes: 171 plasmid units (PUs) and 2,502 homologous protein clusters (HPCs, protein families according to amino acid sequence identity, coverage, and E-value; Figure 4). Edges connected every PU with the HPC that it contained. The PUs of the bipartite network clearly showed distinct clustering phenomena. Overall, one homologous PU clusters (PUCs) contained the almost the same members of Clade X in the analysis above (Figure 4), which was clearly distinct from other PUs. One large region including clades III, VI – IX was also identified, which were mostly IncF and IncC plasmids. In addition, clades I, II, IV, and V were also found their corresponding PUCs in the PU-HPC bipartite network.

FIGURE 4

Figure 4. The PU-HPC bipartite network visualization of the bla_NDM-harboring plasmids of K. pneumoniae. Plasmid units (PUs, 171) and homologous protein clusters (HPCs, 2,502) are represented as colored circles, with red for PUs and pink for HPCs. The size of a circle is ranked as the degree calculated with the Cytoscape’s built-in NetworkAnalyzer tool. The 10 clades (Clade I–Clade X) were labeled into the location of corresponding PUCs.

Discussion

Global spread of the NDM-type carbapenemases can be partly attributed to the dissemination of various bla_NDM-harboring plasmids (Lee et al., 2016; Dong et al., 2022). Therefore, to characterize plasmids harboring bla_NDM in K. pneumoniae, we systematically analyzed the variants of bla_NDM, replicon types, and conjugative transfer regions of 4,451 plasmids belonging to K. pneumoniae from the NCBI GenBank database. Overall, 171 bla_NDM-harboring plasmids of K. pneumoniae were identified.

In our study, nine different variants of bla_NDM were identified from the 171 bla_NDM-harboring plasmids in K. pneumoniae, with bla_NDM-1 and bla_NDM-5 being highly prevalent; bla_NDM-1-carrying plasmids were the most prevalent and accounted for 73.68% of the 171 bla_NDM-harboring plasmids. NDM-1 was first reported in 2008 on a 180-kb plasmid of K. pneumoniae strain isolated from a Swedish patient hospitalized in New Delhi, India (Yong et al., 2009). After the first report, NDM-1 was reported in many clinical isolates, mainly K. pneumoniae and E. coli (Kumarasamy et al., 2010). In addition, bla_NDM-5 was another common variant in our work, accounting for 16.37% of the 171 bla_NDM-harboring plasmids. The variant NDM-5 was first reported on an IncF plasmid of E. coli EC405, isolated from a 41-year-old British patient who had a travel history to India (Hornsey et al., 2011). Notably, bla_NDM-5 was reported to be the predominant variant in bla_NDM-harboring plasmids of E. coli (Zhang et al., 2021).

Our results showed that IncX3 single-replicon plasmids were important carriers of bla_NDM in K. pneumoniae, mainly bla_NDM-1 and bla_NDM-5. IncX3 plasmid is narrow-host range plasmids in Enterobacterales (Johnson et al., 2012), which has been reported to harbor diverse carbapenemase genes in CRE worldwide (Mouftah et al., 2019). Of the 29 bla_NDM-harboring IncX3 plasmids grouped into clade I, most were relatively small with lengths of 46–57 kb (25th percentile = 46.2 kb; median size = 53.1 kb; 75th percentile = 57.3 kb). Based on the results analyzed by the oriTfinder software, all the 29 bla_NDM-harboring IncX3 plasmids of clade I contained genes encoding for relaxases belonging to the MOB_P family, with TraI protein encoded by the IncPα plasmid RP4 (Pansegrau et al., 1993) as a representative. T4CPs encoded by the 29 bla_NDM-harboring IncX3 plasmids of clade I belonged to the VirD4/TraG subfamily, with the TraG protein of plasmid RP4 and the VirD4 protein of Ti plasmids as representatives (Gomis-Rüth et al., 2004). The bla_NDM-harboring IncX3 plasmids classified into clade I contained VirB-like T4SS gene clusters, which are the best-characterized T4SS (Guglielmini et al., 2014). However, no known oriT site was found in most of the IncX3 plasmids harboring bla_NDM belonging to clade I of the phylogenetic cladogram, indicating a new type of oriT site, different from the nine oriT families collected in the oriTDB database (Li et al., 2018).

We found 10 bla_NDM-harboring IncN plasmids, with IncN or IncN2 replicons, clustered into clade II of the phylogenetic cladogram. They were also relatively small plasmids, with genome sizes varying from 38.38 to 63.05 kb. These bla_NDM-harboring IncN plasmids carried the NW-type oriTs, which were characterized by the conserved nick region KGTST|ATAGC (“|” refers to the nic site of oriT), with oriT sites of IncN plasmid R46 (Hall and Vockler, 1987) and IncW plasmid R388 (Revilla et al., 2008) as representatives. Almost all the plasmids of clade II contained genes coding for relaxases of the MOB_F family, which was characterized by the domain “TrwC (PF08751),” with R388 TrwC and F TraI as representatives (de la Cruz et al., 2010). The T4CPs encoded by the bla_NDM-positive IncN plasmids belonged to the TrwB/TraD subfamily, which was characterized by the domain “TrwB_AAD_bind (PF10412),” with the TrwB encoded by plasmid R388 from E. coli as a representative (Gomis-Rüth et al., 2004). In addition, the bla_NDM-positive IncN plasmids carried Trw-like T4SS gene clusters. The Trw T4SS clusters were regarded as the bacterial conjugation machines that mediate the spread of plasmids among bacterial populations (e.g., the trw locus of broad-host-range IncW plasmid R388; Seubert et al., 2003) while also mediating host-specific erythrocyte infection (e.g., the pathogenesis-related Trw system of Bartonella; Vayssier-Taussat et al., 2010).

Our work showed that 21 IncC plasmids carrying bla_NDM-1, with genome sizes from 140.1 kb to 329.2 kb, were clustered into the clade IX of the phylogenetic cladogram constructed by the 171 bla_NDM-harboring plasmids in K. pneumoniae. The broad-host-range IncC mega plasmids are essential contributors to the dissemination of antibiotic resistance genes, and more than 200 fully sequenced IncC plasmids have been reported (Ambrose et al., 2018). The bla_NDM-1-harboring IncC plasmids of clade IX contained the A/C-type oriTs, with the oriT site of IncA/C conjugative pVCR94ΔX from Vibrio cholera as the prototype (Carraro et al., 2014). Furthermore, these bla_NDM-1-harboring IncC plasmids carried genes encoding relaxases of the MOB_H family, characterized by the domain “TraI_2 (Pfam: PF07514),” with TraI encoded by IncHI plasmid R27, TraI encoded by IncA/C plasmid pIP1202, TraI encoded by IncJ plasmid R391, and TraI encoded by IncT plasmid Rts1 as representatives (de la Cruz et al., 2010). In addition, most of the IncC plasmids clustered into clade IX contained genes encoding T4CPs of the TrwB/TraD subfamily and Tra_F-like T4SS gene clusters.

In our work, 53.22% (91 out of 171 plasmids) of the bla_NDM-harboring plasmids of K. pneumoniae were found to be IncF plasmids, and most were multi-replicon IncF plasmids, especially IncFI-type plasmids. IncF plasmids are commonly low-copy-number plasmids, >100 kb in size (Villa et al., 2010); however, in our study, the bla_NDM-harboring IncF plasmids in K. pneumoniae were heterogeneous in size. For example, the bla_NDM-1-positive IncF plasmids, with the IncFIB(pQil) replicon, clustered into clade III were mostly 54-kb plasmids; the genome sizes of the IncFII plasmids grouped into clade V varied from 75.31 to 140.6 kb (25th percentile = 88.81 kb; 75th percentile = 101.4 kb); and the 27 plasmids with replicon IncFIB(pNDM-Mar) belonging to clade X were > 250 kb in size. The IncF plasmids comprise a diverse set of conjugative plasmids frequently found in Enterobacterales, which contribute to spreading AMR genes (Villa et al., 2010; Carattoli, 2011). The bla_NDM-harboring IncF plasmids in K. pneumoniae were also heterogeneous in types of conjugative transfer regions. The IncFII-type plasmids, including clades V and VIII, carried F-type oriTs, genes encoding relaxases of the MOB_F family, genes encoding T4CPs of the TrwB/TraD subfamily, and Tra_F-like T4SS gene clusters belonging to the classical F-like conjugative system (de la Cruz et al., 2010). Mega plasmids with replicons IncFIB(pNDM-Mar):IncHI1B(pNDM-MAR) belonging to clade X mostly harbored the genes encoding relaxases of the MOB_H family. In our study, we found 11 bla_NDM-1-positive IncFIB(pQil) plasmids classified into clade III without any classical conjugative transfer regions, which were predicted as non-transferable plasmids.

Conclusion

In this study, we analyzed the variants of bla_NDM, replicon types, conjugative transfer regions, host STs, and geographical distributions of 171 bla_NDM-harboring plasmids from 4,451 K. pneumoniae plasmids, which were downloaded from the GenBank database. Nine variants of bla_NDM were found among the 171 bla_NDM-positive plasmids, with bla_NDM-1 (73.68%) and bla_NDM-5 (16.37%) as the most dominant. Over half of the bla_NDM-harboring plasmids of K. pneumoniae were classified into IncF plasmids. In addition, IncX3 single-replicon plasmids (46–57 kb), IncN plasmids (38.4–63.1 kb), IncC plasmids (140.1–329.2 kb) were also the common carriers of bla_NDM in K. pneumoniae. The bla_NDM-harboring IncX3 and IncN plasmids were mainly geographically distributed in China. The IncC plasmids harboring bla_NDM-1 were widely geographically distributed all over the world, mainly in the United States, China, and Viet Nam. This study provides important insights into the diversity of bla_NDM-harboring plasmids in K. pneumoniae and further addresses their role in the acquisition and spread of resistance genes. However, the genetic diversity and characteristics of bla_NDM-harboring plasmids in other Gram-negative species need further study in the future.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Author contributions

XL, LuL, and XD: conceptualization. ZhuZ: methodology. LeL and LiL: software. SH and JY: validation. WL, LZ, QL, and ZhiZ: formal analysis. ZhuZ and XL: writing—original draft preparation. XL and LuL: writing—review and editing. XL: supervision. All authors contributed to the article and approved the submitted version.

Funding

This work was supported financially by the grants from the National Natural Science Foundation of China (grant nos. 81902460 and 82002170), the Xiangshan Talent Project of Zhuhai People’s Hospital (grant no. 2020XSYC-02), and the Cultivation Project of Zhuhai People’s Hospital (2019PY-19).

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.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2022.1008905/full#supplementary-material

Footnotes

1. ^https://bioinfo-mml.sjtu.edu.cn/oriTDB/browse_oriT_type_p.php

2. ^https://bioinfo-mml.sjtu.edu.cn/oriTDB/browse_relaxase.php

3. ^https://bioinfo-mml.sjtu.edu.cn/SecReT4/browse_type.php

4. ^https://bioinfo-mml.sjtu.edu.cn/oriTDB/browse_t4cp.php

5. ^https://www.ncbi.nlm.nih.gov/genome/browse/#!/plasmids/815/

6. ^https://card.mcmaster.ca

7. ^https://www.ncbi.nlm.nih.gov/genome/browse/#!/prokaryotes/815/

8. ^https://cge.food.dtu.dk/services/MLST/

9. ^https://bioinfo-mml.sjtu.edu.cn/oriTDB/index.php

10. ^https://bioinfo-mml.sjtu.edu.cn/SecReT4/

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