Carbapenemase OXA-58-like positive plasmid and host bacterium transmission risk assessment method thereof

Abstract

The invention discloses a carbapenemase OXA-58-like positive plasmid and a host bacterium propagation risk assessment method thereof, which are used for carrying out integrated assessment on the plasmid and the host bacterium in three dimensions of drug resistance, pathogenic potential and propagation potential, and carrying out comprehensive risk grading and classification by taking the OXA-58-like positive plasmid and the host bacterium thereof as a plasmid-host complex. The invention develops a set of OXA-58-like gene screening and systematic analysis process based on a public plasmid database aiming at the defects that the existing research of the OXA-58-like gene is mostly limited to a single species or a local sample and a standardized automatic analysis method is lacked. According to the method, all OXA-58-like gene positive plasmids in a database can be subjected to unified and efficient drug-resistant gene mining, the association rule between the drug-resistant genes and plasmid pedigree, host strains and geographical distribution is systematically disclosed, and important technical support is provided for drug resistance propagation monitoring and risk assessment.

Description

Technical Field

[0001] This invention relates to the fields of bioinformatics and microbial genomics, and in particular to a method for assessing the risk of transmission of carbapenemase OXA-58-like positive plasmids and their host bacteria. Background Technology

[0002] Antibiotic resistance (AMR) is spreading rapidly worldwide, posing a significant threat to human health. The widespread prevalence of AMR is primarily due to horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs), typically mediated by plasmids. Many plasmid-mediated resistance genes in pathogens originate from the environment, animal habitats, or human habitats. The ability of plasmids to acquire new genes through mobile genetic elements such as transposons or insertion sequences, and their capacity to replicate across a wide range of hosts, makes them ideal vectors for AMR transmission. With the long-term use of antimicrobial drugs in clinical and agricultural settings, plasmid-mediated resistance has become a major challenge to global public health security.

[0003] Chinese patent application CN102482712B discloses a primer set, kit, and method for detecting carbapenemase genes. The core technology lies in designing specific primers and utilizing multiplex real-time fluorescent polymerase chain reaction (PCR) combined with melting curve analysis to simultaneously detect multiple carbapenemase genes, including OXA-23, OXA-24, and OXA-58, from clinical samples. This method represents the mainstream research paradigm in this field for such drug resistance genes. Its key feature is its focus on laboratory genotyping of specific clinical isolates. The technology relies on specific primers and biochemical reactions targeting a limited set of targets, ultimately producing qualitative or limited genotyping results for individual samples.

[0004] The analytical perspective is limited to microscopic samples: Existing methods (such as multiplex PCR) are designed to detect specific samples from a single individual or a limited batch. This results in the analysis of isolated, localized data points, failing to systematically reveal the overall distribution frequency, transmission dynamics, and evolution over time of drug resistance genes in global or regional databases.

[0005] The technical approach relies on experimental procedures and cannot achieve high-throughput automated analysis: the core of this approach depends on specific physical reagents (such as primers and probes) and manual or semi-automated laboratory operations (such as nucleic acid extraction, PCR amplification, and curve analysis). This process is costly, has limited throughput, and is heavily dependent on the operator's experience and experimental conditions, making it difficult to directly apply to the rapid, batch, standardized, and in-depth screening and analysis of tens of thousands of genomic sequences in public databases.

[0006] The analysis is limited to a single dimension and cannot resolve multidimensional networks: the final output of existing technologies is usually only the "detected" or "not detected" status of the target gene, at most providing limited gene subtype information. It cannot automatically and systematically associate and reveal the plasmid background carried by the gene, the strain lineage of the host bacteria, and related geographical, temporal, and other multidimensional information, thus making it difficult to draw a complete transmission path and risk assessment map of drug resistance genes. Summary of the Invention

[0007] Purpose of the invention: The present invention aims to provide a standardized and automated method for assessing the risk of transmission of carbapenemase OXA-58-like positive plasmids and their host bacteria.

[0008] Technical solution: This invention provides a method for assessing the risk of transmission of carbapenemase OXA-58-like positive plasmids and their host bacteria, comprising the following steps:

[0009] The confirmed carbapenemase OXA-58-like positive plasmid sequences and their metadata information were obtained from publicly available drug resistance databases and used as reference sequences. The bacterial plasmid gene sets to be evaluated were compared and identified with the reference sequences. All positive plasmids carrying carbapenemase resistance genes were extracted from the bacterial plasmid gene sets to be evaluated, and a database of positive plasmid genome sequences to be evaluated was constructed.

[0010] Collinear resistance genes, mobile genetic elements, and virulence factors were annotated for the genomic sequences of the positive plasmids mentioned above. The various resistance genes, mobile genetic elements, and virulence factors carried by the positive plasmids were identified by comparison with public databases. Plasmid typing based on replication initiation proteins was performed on the genomic sequences of the positive plasmids mentioned above, and a maximum likelihood phylogenetic tree was constructed. Plasmid conjugation and transfer-related genes were identified for the genomic sequences of the positive plasmids mentioned above, and the horizontal transfer potential of the plasmids was assessed. Based on the above annotation and identification results, the positive plasmids to be evaluated were assessed to determine whether they possess multidrug resistance and / or strong transfer ability.

[0011] Obtain the corresponding host genomes for each of the above positive plasmid genome sequences, thus obtaining the positive plasmid-host bacterial genome sequences with confirmed correspondences; identify the sequence type of each host genome for each positive plasmid genome sequence, and identify international clones based on the Pasteur MLST typing scheme; perform host bacterial phylogenetic analysis, collinear resistance gene annotation, collinear resistance gene classification, virulence factor system annotation, and virulence factor system identification on the above plasmid-host genome sequences; based on the above annotation and identification results, assess whether the host corresponding to each positive plasmid to be evaluated is a highly virulent host;

[0012] The risk level of transmission is assessed based on whether the positive plasmid to be evaluated has multidrug resistance, strong transmissibility, and a highly virulent host.

[0013] Preferably, the publicly available drug resistance databases include the CARD database, the Resifinder database, and the BLDB database.

[0014] Preferably, OXA-58-like includes OXA-58, OXA-97, OXA-164, OXA-96, OXA-420, OXA-512, OXA-397, OXA-1178, OXA-1376.

[0015] Preferably, Prodigal is used to predict genes in positive plasmid genomes, a metagenomic model is adopted to adapt to plasmid sequence characteristics, a closed-end model is used to prevent genes from predicting across boundaries, Prokka is used to perform automated functional annotation of predicted genes, and standardized format annotation files are generated for the annotation and identification of mobile genetic elements and virulence factors.

[0016] Preferably, a public database is used to genotype replication initiation proteins, extracting the replication initiation protein sequences corresponding to the same replication initiation protein family. Muscle is used for multiple sequence alignment, and IQ-TREE is used to construct a maximum likelihood phylogenetic tree to reveal the evolutionary relationships of replication initiation proteins.

[0017] Preferably, PlasmidFinder and the AcinetobacterPlasmidTyping database are used for replication initiation protein typing.

[0018] Preferably, a systematic analysis of the conjugation transfer core elements of the positive plasmid genome is performed to identify its transfer initiation site, relaxase, type IV coupling protein, and type IV secretion system-related genes, and to assess the horizontal transfer potential of the plasmid.

[0019] Preferably, the OriTfinder2 tool is used to perform a systematic analysis of the conjugation transfer core elements of the positive plasmid genome.

[0020] Preferably, the genome of the positive plasmid-host bacteria is annotated with comprehensive antibiotic resistance genes, bactericide and metal resistance genes; by comparing with comprehensive antibiotic resistance databases CARD and ResFinder and antimicrobial and metal resistance gene database BacMet, the various resistance genes carried by the host bacteria of the positive plasmid are systematically identified.

[0021] Preferably, in assessing the transmission risk level based on whether the positive plasmid to be evaluated has multidrug resistance, strong transmissibility, and a highly virulent host, the transmission risk levels are classified as follows: High risk: possesses multidrug resistance, strong transmissibility, and a highly virulent host; High risk: possesses multidrug resistance, but mainly relies on host clonal spread; Medium risk: only one of drug resistance, transmissibility, or pathogenicity is prominent; Low risk: drug resistance, transmissibility, and pathogenicity are all weak.

[0022] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: Addressing the issue that carbapenemase OXA-58-like genes are often horizontally propagated via plasmids, but lack systematic assessment of their transmission risk, this invention, for the first time, comprehensively assesses the potential risk of positive plasmids from multiple dimensions. By integrating drug resistance, virulence factor carriage status, and conjugation transfer capabilities, a plasmid transmission risk grading system is constructed, enabling the classification of risk levels for OXA-58-like positive plasmids and host bacteria, providing a theoretical basis and methodological support for clinical prevention and control and transmission early warning. Attached Figure Description

[0023] Figure 1 This is a flowchart of the method for assessing the transmission risk of positive plasmids and their host bacteria according to the present invention.

[0024] Figure 2 The characteristics and prevalence distribution of blaOXA-58-like gene variants and positive plasmids are shown below. Here, a) shows the sequence identity of eight blaOXA-58-like gene variants; b) is a histogram of the number of blaOXA-58-like gene variants in positive plasmids; c) is a box plot of the long distribution of blaOXA-58-like positive plasmids; d) shows the GC content distribution of blaOXA-58-like positive plasmids; e) shows the host classification information of blaOXA-58-like positive plasmids; f) shows the sample source of blaOXA-58-like positive plasmids; g) shows the year of sample collection for blaOXA-58-like positive plasmids; and h) shows the geographical information of blaOXA-58-like positive plasmids.

[0025] Figure 3 For the evolutionary analysis of resistance genes and plasmids based on blaOXA-58-like positive plasmids, a is the UpSet diagram of the coexistence relationship of the top 20 high-frequency resistance genes in the sample; b is the chord diagram of the direct relationship between the top 20 inserted sequences and different variants; c is the maximum likelihood tree of Rep3 superfamily plasmids based on rep protein.

[0026] Figure 4This study analyzes the spread and genomic background of the blaOXA-58-like gene variant in Acinetobacter baumannii hosts. Among them, a) shows the distribution of OXA-58-like variants in bacteria of different ST types; b) shows the phylogenetic consensus tree of species based on the core orthologous gene set; c) shows the minimum spanning tree of Acinetobacter baumannii isolates based on MLST; and d) shows the heatmap of the distribution patterns of collinear resistance genes and virulence factors. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] like Figure 1 As shown, this embodiment of the invention provides a method for assessing the transmission risk of carbapenemase OXA-58-like positive plasmids and their host bacteria, including the following steps:

[0029] The confirmed carbapenemase OXA-58-like positive plasmid sequences and their metadata information were obtained from publicly available drug resistance databases and used as reference sequences. The bacterial plasmid gene sets to be evaluated were compared and identified with the reference sequences. All positive plasmids carrying carbapenemase resistance genes were extracted from the bacterial plasmid gene sets to be evaluated, and a database of positive plasmid genome sequences to be evaluated was constructed.

[0030] Collinear resistance genes, mobile genetic elements, and virulence factors were annotated for the genomic sequences of the positive plasmids mentioned above. The various resistance genes, mobile genetic elements, and virulence factors carried by the positive plasmids were identified by comparison with public databases. Plasmid typing based on replication initiation proteins was performed on the genomic sequences of the positive plasmids mentioned above, and a maximum likelihood phylogenetic tree was constructed. Plasmid conjugation and transfer-related genes were identified for the genomic sequences of the positive plasmids mentioned above, and the horizontal transfer potential of the plasmids was assessed. Based on the above annotation and identification results, the positive plasmids to be evaluated were assessed to determine whether they possess multidrug resistance and / or strong transfer ability.

[0031] Obtain the corresponding host genomes for each of the above positive plasmid genome sequences, thus obtaining the positive plasmid-host bacterial genome sequences with confirmed correspondences; identify the sequence type of each host genome for each positive plasmid genome sequence, and identify international clones based on the Pasteur MLST typing scheme; perform host bacterial phylogenetic analysis, collinear resistance gene annotation, collinear resistance gene classification, virulence factor system annotation, and virulence factor system identification on the above plasmid-host genome sequences; based on the above annotation and identification results, assess whether the host corresponding to each positive plasmid to be evaluated is a highly virulent host;

[0032] The risk level of transmission is assessed based on whether the positive plasmid to be evaluated has multidrug resistance, strong transmissibility, and a highly virulent host.

[0033] Example 1: Identification and genomic characterization of OXA-58-like positive plasmids

[0034] This invention provides a method for identifying and analyzing the genomic characteristics of OXA-58-like positive plasmids, specifically including the following steps:

[0035] Step 1: Consult and collect reference sequences for all OXA-58-like variants from existing public resistance databases, including:

[0036] The databases include the CARD database (The Comprehensive Antibiotic Resistance Database), the Resifinder database (https: / / cge.cbs.dtu.dk / services / ResFinder), and the BLDB database (Beta-Lactamase Database, http: / / bldb.eu / ). OXA-58-like variants include: OXA-58, OXA-97, OXA-164, OXA-96, OXA-420, OXA-512, OXA-397, OXA-1178, and OXA-1376.

[0037] Step 2: Obtain bacterial plasmid sequences and their metadata (species, origin region, and sequencing information, etc.) from public databases, including:

[0038] Download all publicly available bacterial plasmid genome sequences from the PlasmidScope database (https: / / plasmid.deepomics.org / ) and the NCBI Genbank database (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0039] Step 3: Screening for OXA-58-like drug resistance genes and identification of positive plasmids based on nucleic acid alignment;

[0040] Using BLASTn v2.15.0, the reference sequence obtained in step one was compared with the gene sets of all target bacterial plasmids for nucleic acid sequence identification. The alignment parameters were set to coverage ≥90% and sequence identity ≥90%. The output was performed using the parameter -outfmt "6 qseqid qlen qstart qend sseqid stitle pident lengthevalue staxid", limiting each query sequence to a maximum of 5 alignment results (-num_alignments 5). All positive plasmids carrying the target drug resistance gene were extracted and a database was constructed.

[0041] Step 4: Sequence Feature Analysis and Metadata Integration of Positive Plasmids

[0042] A Perl script was used to batch process the positive plasmids obtained from screening, automatically calculating two key sequence features for each plasmid: length (bp) and GC content (%). Simultaneously, metadata such as host and country information was automatically extracted from annotation information in source files such as GenBank. For entries with incomplete information extracted automatically, manual completion was performed by tracing the NCBI BioSample / SRA database or consulting original literature. Finally, a comprehensive table of positive plasmid information containing complete biological characteristics and epidemiological metadata was constructed for subsequent statistical analysis and research on transmission patterns.

[0043] Based on the above method, a detailed practical exercise will be conducted. The specific steps and results are as follows:

[0044] Step 1: Consult and collect reference sequences for all OXA-58-like variants from existing public drug resistance databases.

[0045] Consulting the CARD, Resifinder, and BLDB databases, the currently identified OXA-58-like variants include: OXA-58, OXA-97, OXA-164, OXA-96, OXA-420, OXA-512, OXA-397, OXA-1178, and OXA-1376. Using these nine known OXA-58-like gene variants as reference sequences, a gene variant dataset with high similarity (>99%) was constructed.

[0046] Step 2: Obtain bacterial plasmid sequences from public databases

[0047] A total of 108,315 bacterial plasmid genome data were downloaded by combining the public databases GenBank and PlasmidScope (as of September 27, 2025).

[0048] Step 3: Identification of OXA-58-like positive plasmids

[0049] A systematic screening of plasmid genomes using the CARD and ResFinder databases identified 135 plasmids carrying the blaOXA-58-like gene, covering five variants (OXA-58, OXA-96, OXA-97, OXA-420, and OXA-164), with OXA-58 being the dominant variant. Figure 2 a. Sequence identity of eight blaOXA-58-like gene variants; Figure 2 b, Histogram of the number of blaOXA-58-like gene variants in blaOXA-58-like positive plasmids.

[0050] Step 4: Sequence Feature Analysis and Metadata Integration of Positive Plasmids

[0051] Of the 152 plasmids carrying blaOXA-58-like structures, the vast majority were single copies, with a few being double copies. The plasmid lengths varied widely, ranging from a minimum of 0.869 kb to a maximum of 339.8 kb. Different variants showed significant length differences, and GC content exhibited significant stratification (ANOVA p < 0.001). Figure 2 c. Box plot of the long distribution of blaOXA-58-like positive plasmid; Figure 2 d. GC content distribution of blaOXA-58-like positive plasmids. The blaOXA-58 plasmid has the widest GC content distribution, with some plasmids having high GC content (up to 0.586), suggesting that its genome has strong plasticity and can adapt to diverse host environments.

[0052] The blaOXA-58-like positive plasmid carries hosts spanning 2 orders, 3 families, and 3 genera, involving 21 bacterial species. Figure 2 e. Host classification information of blaOXA-58-like positive plasmids): Acinetobacter baumannii had the highest proportion, indicating extensive cross-species transmission capabilities. The temporal distribution spanned from 1997 to 2022, with a significant increase in sample size after 2010. Geographically, the distribution covered 22 countries worldwide, with China and its surrounding areas being the main concentration areas. Figure 2 f. Sample source of blaOXA-58-like positive plasmid; Figure 2 g, the year in which the blaOXA-58-like positive plasmid sample was collected; Figure 2 h, geographic information of blaOXA-58-like positive plasmid samples.

[0053] Example 2: Genomic functional annotation and conjugation transfer module analysis of OXA-58-like positive plasmids

[0054] This invention provides a method for genomic functional annotation and conjugation transfer module analysis of OXA-58-like positive plasmids, the steps of which are as follows:

[0055] Step 1: Gene prediction and annotation based on positive plasmid genomes

[0056] Prodigal was used to predict genes in positive plasmid genomes, employing a metagenomic mode (-p meta) to adapt to plasmid sequence characteristics and a closed-end mode (-c) to prevent gene prediction across boundaries. The results were output in GFF format (-f gff). Subsequently, Prokka was used for automated functional annotation of the predicted genes, integrating information from multiple databases to finally generate standardized annotation files in FAA, GBK, and other formats, providing a structured data foundation for subsequent functional analysis.

[0057] Step 2: Annotation and Classification of Collinear Drug Resistance Genes

[0058] Based on the genomic sequences of the positive plasmids selected using Scheme 1, comprehensive annotation of antibiotic resistance genes, bactericide and metal resistance genes was performed using uniform and stringent parameters (DIAMOND BLASTx, E-value ≤ 1e-5, coverage and consistency ≥ 80%). The various resistance genes carried by the positive plasmids were systematically identified by comparing them with the comprehensive antibiotic resistance databases CARD (Comprehensive Antibiotic Resistance Database) and ResFinder, as well as the antimicrobial, bactericide and metal resistance gene database BacMet.

[0059] Step 3: Systematic annotation and identification of mobile genetic elements and virulence factors

[0060] Based on the gene prediction files from step 1, nucleic acid sequences were batch-aligned and annotated using the Virulence Factor Database (VFDB) (using BLASTx, with E-value ≤ 1e-5, sequence identity ≥ 80%, and coverage ≥ 80% as screening thresholds) to identify potential virulence factors and their functional categories. Simultaneously, the bacterial mobile genetic element database (MGE) and the bacterial insertion sequence database (ISfinder) were used to further identify MGEs, including plasmid replicons, integrons, transposons, phage sequences, and insertion sequences (IS).

[0061] Step 4: Plasmid typing and phylogenetic analysis based on the rep protein

[0062] Based on the genomic sequences of positive plasmids, replication initiation proteins (rep) were genotyped using PlasmidFinder and the AcinetobacterPlasmidTyping database. Rep protein sequences corresponding to the same rep family were extracted, and multiple sequence alignment was performed using Muscle. A maximum likelihood phylogenetic tree was constructed using IQ-TREE (parameters: -m MFP model selection, -bb1000 for ULTRA rapid bootstrapping, -alrt 1000 for SH-aLRT support test) to reveal the evolutionary relationships of the rep proteins.

[0063] Step 5: Identification of plasmid conjugation transfer-related genes

[0064] The OriTfinder2 tool was used to perform a systematic analysis of the core elements of conjugation transfer in positive plasmid genomes, identifying related genes such as the transfer initiation site (oriT), relaxase, type IV conjugate protein (T4CP), and type IV secretion system (T4SS), and assessing the horizontal transfer potential of the plasmids.

[0065] Based on the above method, a detailed practical exercise will be conducted. The specific steps and results are as follows:

[0066] Step 1: Annotation and Classification of Collinear Drug Resistance Genes

[0067] Based on gene prediction of positive plasmid genomes using prodigal, resistance gene annotation was performed using the CARD and Resfinder databases. A total of 66 ARGs were detected, covering eight classes of antibiotics: aminoglycosides, macrolides, quinolones, sulfonamides, tetracyclines, trimethoprim, β-lactams, and chloramphenicol. The blaOXA-58-like positive plasmid exhibited broad multidrug resistance characteristics, particularly high detection rates in aminoglycosides and macrolides, demonstrating its important role in the spread of clinical drug resistance. Most positive plasmids showed multidrug resistance characteristics, and the resistance genes they carried could mediate resistance to multiple antibiotic classes. Among them, 14 drug resistance genes (including oxa-58, NDM-1, mph(E), msr(E), tet(X3), aac(6')-Ib9, aph(3'')-Ib, bpr(MBL), sul1, sul2, aph(3')-Via, aph(6)-Id, and arr-3) were detected simultaneously in 7 plasmids. These genes can mediate resistance to carbapenems, tetracyclines, macrolides, aminoglycosides, sulfonamides, and rifampin antibiotics. Figure 3 a, UpSet plot showing the coexistence of the top 20 high-frequency resistance genes in the sample.

[0068] Step 2: Systematic annotation and identification of mobile genetic elements

[0069] Using ISfinder, in the analysis of the correspondence between the inserted sequences in the 5 kb regions upstream and downstream of the constructed blaOXA-58-like gene and the blaOXA-58-like gene variants, the distribution of multiple IS elements on the flanks of different variants was identified. Figure 3 b. Upset plot of coexistence relationships of the top 20 insertion sequences in the sample. The results showed that ISAba3 was the IS most closely associated with blaOXA-58-like. ISAba3 can not only enhance blaOXA-58-like gene expression by forming heterozygous promoters itself or with other IS (such as IS1006), but it also frequently forms complex transposons with copies at both ends, serving as an insertion target for other IS (such as ISAba825), collectively creating a dynamic and complex genetic environment. This multiple mechanism of action may explain the dominant role of ISAba3 in driving the spread of blaOXA-58-like genes.

[0070] Steps 3-5: Plasmid typing and phylogenetic analysis based on the rep protein

[0071] Plasmid replicon typing was performed using Acinetobacter Plasmid Typing and Plasmidfinder. Analysis showed that 62.5% (95 / 152) of the positive OXA-58-like plasmids carried the replicase gene, and all belonged to the Rep 3 superfamily (pfam01051). Furthermore, 22 replicon types were successfully identified among the OXA-58-like positive Rep 3 superfamily plasmids.

[0072] A maximum likelihood phylogenetic tree of Rep 3 superfamily blaOXA-58-like positive plasmids was constructed based on plasmid replication initiation proteins. Figure 3 c. Maximum likelihood tree of Rep 3 superfamily plasmids based on the rep protein. Plasmids were classified according to the PlasmidFinder and Acinetobacter PlasmidTyping database typing schemes (nucleotide coverage of at least 80% and nucleotide identity of at least 75% for the same group of replicase genes). The coupling transfer modules of each positive plasmid, including the types of oriTs, relaxase genes, T4CP genes, and T4SS genes, were determined using the oriTfinder network tool.

[0073] Using VFDB annotation of virulence factors, it was found that all positive plasmids carried only a single virulence factor, and no coexistence of multiple virulence factors was observed. A total of 15 different virulence factors were identified, and their distribution showed a clear preference: Type 3 fimbriae and TraJ (conjugation transfer regulatory protein) were the most dominant types, followed by T3SS (type III secretion system), ToxB (toxin B), and Hemolysin (hemolysin).

[0074] Example 3: Genome analysis of host bacteria of positive plasmids

[0075] This invention provides a method for analyzing the genome of a host bacterium with a positive plasmid, comprising the following steps:

[0076] Step 1: Obtain the corresponding host genome of the OXA positive plasmid.

[0077] Based on the PubMed number in the GenBank file of each positive plasmid, the GenBank ID of the genome containing the positive plasmid was located, and the genomes were collected and downloaded in batches. Prokka v1.14.6 software was used to perform uniform functional annotation on all genomes, and the protein amino acid sequence files (.faa format) of each genome were output.

[0078] Step 2: Identify the host bacterium ST type and international clone type

[0079] Using mlst v2.23.0 (https: / / github.com / tseemann / mlst), the sequence type (ST) of each host genome was identified based on seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA). International clones (ICs) were identified using the goeBURST algorithm based on the Pasteur MLST typing scheme.

[0080] Step 3: Host bacterial phylogenetic analysis

[0081] All .faa files obtained in step one were input into OrthoFinder v2.5.4 for ortholog analysis of the entire proteome. This analysis successfully clustered >99% of the genes into orthologous groups, and from these, single-copy orthologous gene sets present in all species were selected, constructing a consensus phylogenetic tree of species evolutionary relationships. Using the iTOL (https: / / itol.embl.de / ) online tool, based on the strain's host origin, IC, and the bla carried by it... OXA Variant types, tree nodes are visually annotated.

[0082] Step 4: Annotation and Classification of Collinear Drug Resistance Genes

[0083] Gene prediction was performed on the host bacterial genomes of positive plasmids using Prodigal. A metagenomic mode (-pmeta) was employed to accommodate plasmid sequence characteristics, while a closed-end mode (-c) prevented cross-boundary gene prediction. Results were output in GFF format (-fgff). Based on the host bacterial genome sequences selected in step 1, comprehensive annotation of antibiotic resistance genes, bactericide and metal resistance genes was performed using uniform and stringent parameters (DIAMOND BLASTx, E-value ≤ 1e-5, coverage and consistency ≥ 80%). Various antibiotic resistance genes carried by the host bacteria of positive plasmids were systematically identified by comparing against comprehensive antibiotic resistance databases CARD and ResFinder, and the antimicrobial, bactericide and metal resistance gene database BacMet.

[0084] Step 5: Systematic annotation and identification of virulence factors

[0085] Based on the gene prediction results from step 2, nucleic acid sequences were batch aligned and annotated using the VFDB virulence factor database (BLASTx, with E-value ≤ 1e-5, sequence identity ≥ 80%, and coverage ≥ 80% as screening thresholds) to identify potential virulence factors and their functional categories. Combining the annotation results from step 2, the presence / deletion information of ARGs and VFs from all strains was integrated to generate a binary matrix. Using the R language's pheatmap package, a heatmap of the co-distribution of resistance genes and virulence factors was plotted, displaying the resistance and virulence characteristic spectra of different ST serotypes.

[0086] Based on the above method, a detailed practical exercise will be conducted. The specific steps and results are as follows:

[0087] Step 1: Acquisition and Standardized Annotation of Host Bacterial Genome Sequence

[0088] A total of 75.6% (115 / 152) of the available host bacterial genomes with positive plasmids were collected, all from the genus Acinetobacter. Prokka was used to perform uniform functional annotation on all genomes, and the protein amino acid sequence files (.faa format) for each genome were output.

[0089] Step 2: Identify the host bacterium ST type and international clone type

[0090] The corresponding host genomes for each blaOXA-positive plasmid were collected and downloaded in batches. The ST type of each host genome was identified using mlst v2.23.0 based on seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA). Of the 135 plasmid host strains, 106 were assigned to 66 known ST types (…). Figure 4 a. Distribution of OXA-58-like variants in different ST-type host bacteria. Due to allelic differences or incomplete database information, the ST-type host strains of the other 46 plasmids are unknown. Acinetobacter baumannii had a high detection rate in this study (42.5%, 45 / 106). A minimum spanning tree was constructed based on the MLST of this bacterium, revealing that the ST of this bacterium has high diversity and type specificity, and that there is an association between different ST types and specific blaOXA-58-like variants. Figure 4 c, Minimum spanning tree of Acinetobacter baumannii isolates based on MLST.

[0091] Step 3: Host bacterial phylogenetic analysis

[0092] Using OrthoFinder, >99% of the genes were clustered into orthologous groups, and single-copy orthologous gene sets present in all species were selected to construct a consensus phylogenetic tree of species evolutionary relationships. Figure 4 b, Species phylogenetic consensus tree based on core orthologous gene sets.

[0093] Steps 4-5: Annotation and identification of collinear drug resistance genes and virulence factors in the host bacterial genome

[0094] In addition to the core OXA-58 class of carbapenemase genes, the strains generally carry multiple other classes of resistance genes, forming a complex co-resistance background. Among them, resistance genes against aminoglycosides (such as aadA, APH(3')-Ia), sulfonamides (such as sul1, sul2), and tetracyclines (such as tet(39), tet(A)) are widely present. Particularly noteworthy is the presence of efflux pump systems mediating multidrug resistance (such as adeABC, adeFGH) genes in the vast majority of strains, providing a basis for their resistance to multiple antibiotics. Furthermore, some strains also showed the presence of armA (16S rRNA methyltransferase), qacEdelta1 (disinfectant resistance), and a few metallo-β-lactamase genes (…). Figure 4 d, Heatmap of distribution patterns of collinear resistance genes and virulence factors.

[0095] Virulence factors are highly conserved and abundant among different strains. The vast majority of strains (>90%) carry more than 80 core virulence factors, including bfmR, pilG, lpxC, and ompA, which are involved in several key pathogenic processes such as biofilm formation, pili synthesis, lipopolysaccharide metabolism, and outer membrane integrity.

[0096] Integrating the phylogenetic tree with the aforementioned phenotypic profiles revealed that closely related strains typically possess highly similar virulence and resistance gene profiles, further supporting the view that clonal dissemination is the primary driver of the diffusion of these traits. Conversely, strains with more distant phylogenetic relationships exhibited significant differences in their phenotypic profiles. Notably, despite the high conservation of core virulence factors, the distribution of some genes associated with specific virulence islands or motility elements (such as the csu, pga, and other fimbrial-related gene clusters, as well as TSS-type VI secretion system genes) varied among different clones, suggesting that horizontal gene transfer may also play a role in the acquisition of specific virulence traits.

[0097] Example 4: A method for assessing and classifying the risk level of OXA-58-like positive plasmids and host bacteria based on drug resistance, pathogenicity, and transmission potential.

[0098] This embodiment provides a method for assessing and classifying the risk level of OXA-58-like positive plasmids and host bacteria based on drug resistance, pathogenicity, and transmission potential. Based on the results of Examples 1-3, and through an integrated assessment of the plasmid and host bacteria across three dimensions—drug resistance, pathogenicity, and transmission potential—the OXA-58-like positive plasmid and its host bacteria are now considered as a "plasmid-host" complex, and a comprehensive risk classification and grading are performed as follows:

[0099] Category I (High Risk): Possesses multiple drug resistance, strong transmissibility, and highly virulent hosts, posing the highest risk of outbreaks and epidemics;

[0100] Category II (Higher Risk): Possesses multidrug resistance, but mainly relies on host clonal spread, which can cause persistent or endemic epidemics;

[0101] Category III (Medium Risk): Only one of the following is particularly prominent: drug resistance, transmissibility, or pathogenicity; overall risk is limited.

[0102] Category IV (Low Risk): Drug resistance, transmissibility, and pathogenicity are all relatively weak, with the lowest risk of transmission and pathogenicity.

[0103] Based on the above method, a detailed practical exercise will be conducted. The specific steps and results are as follows:

[0104] Based on Examples 1-3, a four-layer risk classification system was established by integrating the analysis of the plasmid-host complex in three dimensions: drug resistance, transmission potential, and host pathogenicity.

[0105] Among them, the Class I (high-risk) combination accounts for only 1.1%, and its typical characteristics are that the plasmid has both autonomous conjugation and transfer capabilities and a multidrug resistance gene spectrum, and the host is Acinetobacter baumannii of the international clone IC2 type with high epidemic potential, which poses a threat of potentially causing an outbreak.

[0106] Category II (higher risk) combinations represent the most significant clinical threat, accounting for 47.8% of cases. The core characteristic of this type of combination is that the plasmid carries a wide range of multidrug resistance genes, but its dissemination mainly relies on the clonal expansion of the host (primarily Acinetobacter baumannii) rather than on the plasmid's own conjugation transfer.

[0107] The combinations of Category III (medium risk) and Category IV (low risk) accounted for 44.4% and 6.7%, respectively. The former may be due to either a host (such as Acinetobacter baumannii) with high pathogenicity but a limited plasmid resistance spectrum, or a host (such as other Acinetobacter species) with high plasmid resistance but limited pathogenicity and transmissibility. The latter, however, exhibited low risk across all three dimensions. While these two types of strains individually pose a low risk of widespread transmission, their potential evolutionary risks as reservoirs of resistance genes cannot be ignored.

[0108] In summary, this classification system reveals that Acinetobacter baumannii clones carrying multidrug-resistant plasmids (especially highly prevalent clones such as IC2) are priority targets for infection control. Active monitoring and isolation measures for such strains are of great significance in blocking the spread of carbapenemase resistance such as OXA-58-like.

Claims

1. A method for assessing the risk of transmission of carbapenemase OXA-58-like positive plasmids and their host bacteria, characterized in that, Includes the following steps: The confirmed carbapenemase OXA-58-like positive plasmid sequences and their metadata information were obtained from publicly available drug resistance databases and used as reference sequences. The bacterial plasmid gene sets to be evaluated were compared and identified with the reference sequences. All positive plasmids carrying carbapenemase resistance genes were extracted from the bacterial plasmid gene sets to be evaluated, and a database of positive plasmid genome sequences to be evaluated was constructed. Collinear resistance genes, mobile genetic elements, and virulence factors were annotated for the genomic sequences of the positive plasmids mentioned above. The various resistance genes, mobile genetic elements, and virulence factors carried by the positive plasmids were identified by comparison with public databases. Plasmid typing based on replication initiation proteins was performed on the genomic sequences of the positive plasmids mentioned above, and a maximum likelihood phylogenetic tree was constructed. Plasmid conjugation and transfer-related genes were identified for the genomic sequences of the positive plasmids mentioned above, and the horizontal transfer potential of the plasmids was assessed. Based on the above annotation and identification results, the positive plasmids to be evaluated were assessed to determine whether they possess multidrug resistance and / or strong transfer ability. Obtain the corresponding host genomes for each of the above positive plasmid genome sequences, thus obtaining the positive plasmid-host bacterial genome sequences with confirmed correspondences; identify the sequence type of each host genome for each positive plasmid genome sequence, and identify international clones based on the Pasteur MLST typing scheme; perform host bacterial phylogenetic analysis, collinear resistance gene annotation, collinear resistance gene classification, virulence factor system annotation, and virulence factor system identification on the above plasmid-host genome sequences; based on the above annotation and identification results, assess whether the host corresponding to each positive plasmid to be evaluated is a highly virulent host; The risk level of transmission is assessed based on whether the positive plasmid to be evaluated has multidrug resistance, strong transmissibility, and a highly virulent host.

2. The method for assessing the transmission risk of carbapenemase OXA-58-like positive plasmids and their host bacteria according to claim 1, characterized in that, Publicly available drug resistance databases include the CARD database, the Resifinder database, and the BLDB database.

3. The method for assessing the transmission risk of carbapenemase OXA-58-like positive plasmids and their host bacteria according to claim 1, characterized in that, OXA-58-like includes OXA-58, OXA-97, OXA-164, OXA-96, OXA-420, OXA-512, OXA-397, OXA-1178, and OXA-1376.

4. The method for assessing the transmission risk of carbapenemase OXA-58-like positive plasmids and their host bacteria according to claim 1, characterized in that, Prodigal was used to predict genes in positive plasmid genomes. A metagenomic model was adopted to adapt to the characteristics of the plasmid sequence, and a closed-end model was used to prevent genes from predicting across boundaries. Prokka was used to perform automated functional annotation of the predicted genes, and standardized format annotation files were generated for the annotation and identification of mobile genetic elements and virulence factors.

5. The method for assessing the transmission risk of carbapenemase OXA-58-like positive plasmids and their host bacteria according to claim 1, characterized in that, Replication initiation proteins were genotyped using public databases, and protein sequences of replication initiation proteins corresponding to the same replication initiation protein family were extracted. Muscle was used for multiple sequence alignment, and IQ-TREE was used to construct a maximum likelihood phylogenetic tree to reveal the evolutionary relationships of replication initiation proteins.

6. The method for assessing the transmission risk of carbapenemase OXA-58-like positive plasmids and their host bacteria according to claim 5, characterized in that, Replication initiation protein typing was performed using PlasmidFinder and the AcinetobacterPlasmidTyping database.

7. The method for assessing the transmission risk of carbapenemase OXA-58-like positive plasmids and their host bacteria according to claim 1, characterized in that, A systematic analysis of the conjugation transfer core elements of positive plasmid genomes was performed to identify their transfer initiation sites, relaxases, type IV coupling proteins, and type IV secretion system-related genes, and to assess the horizontal transfer potential of the plasmids.

8. The method for assessing the transmission risk of carbapenemase OXA-58-like positive plasmids and their host bacteria according to claim 7, characterized in that, Systematic analysis of conjugation transfer core elements was performed on positive plasmid genomes using the OriTfinder2 tool.

9. The method for assessing the transmission risk of carbapenemase OXA-58-like positive plasmids and their host bacteria according to claim 1, characterized in that, The genomes of positive plasmids and host bacteria were annotated to include comprehensive antibiotic resistance genes, bactericide and metal resistance genes. By comparing the comprehensive antibiotic resistance databases CARD and ResFinder with the antimicrobial, bactericide and metal resistance gene database BacMet, the various resistance genes carried by the host bacteria of positive plasmids were systematically identified.

10. The method for assessing the transmission risk of carbapenemase OXA-58-like positive plasmids and their host bacteria according to claim 1, characterized in that, The risk level of transmission is assessed based on whether the positive plasmid to be evaluated possesses multidrug resistance, strong transmissibility, and a highly virulent host. The risk levels are classified as follows: High risk: Possesses multidrug resistance, strong transmissibility, and a highly virulent host; High risk: Possesses multidrug resistance, but mainly relies on host clonal spread; Medium risk: Only one of drug resistance, transmissibility, or pathogenicity is prominent; Low risk: Drug resistance, transmissibility, and pathogenicity are all weak.

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