The risk of pathogenicity and antibiotic resistance in deep-sea cold seep microorganisms

Abstract

Deep-sea cold seeps host high microbial biomass and biodiversity that thrive on hydrocarbon and inorganic compound seepage, exhibiting diverse ecological functions and unique genetic resources. However, potential health risks from pathogenic or antibiotic-resistant microorganisms in these environments remain largely overlooked, especially during resource exploitation and laboratory research. Here, we analyzed 165 metagenomes and 33 metatranscriptomes from 16 global cold seep sites to investigate the diversity and distribution of virulence factors (VFs), antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs). A total of 2,353 VFs are retrieved in 689 metagenome-assembled genomes (MAGs), primarily associated with indirect pathogenesis like adherence. In addition, cold seeps harbor nearly 100,000 ARGs, as important reservoirs, with high-risk ARGs (11.22%) presenting at low abundance. Compared to other environments, microorganisms in cold seeps exhibit substantial differences in VF and ARG counts, with potential horizontal gene transfer facilitating their spread. These virulome and resistome profiles provide valuable insights into the evolutionary and ecological implications of pathogenicity and antibiotic resistance in extreme deep-sea ecosystems. Collectively, these results indicate that cold seep sediments pose minimal public health risks, shedding light on environmental safety in deep-sea resource exploitation and research.

Importance: In the "One Health" era, understanding pathogenicity and antibiotic resistance in vast and largely unexplored regions like deep-sea cold seeps is critical for assessing public health risks. These environments serve as critical reservoirs where resistant and virulent bacteria can persist, adapt, and undergo genetic evolution. The increasing scope of human activities, such as deep-sea mining, is disrupting these previously isolated ecosystems, heightening the potential for microbial exchange between deep-sea communities and human or animal populations. This interaction poses a significant risk for the dissemination of resistance and virulence genes, with potential consequences for global public health and ecosystem stability. This study offers the first comprehensive analysis of virulome, resistome, and mobilome profiles in cold seep microbial communities. While cold seeps act as reservoirs for diverse ARGs, high-risk ARGs are rare, and most VFs were low risk that contribute to ecological functions. These results provide a reference for monitoring the spread of pathogenicity and resistance in extreme ecosystems, informing environmental safety assessments during deep-sea resource exploitation.

Keywords: antibiotic resistance genes; cold seeps; microbiome; virulence factors.

Fig 1

VFs detected in the non-redundant gene catalog from cold seep sediments. (A) Relative proportions of different VF categories in the non-redundant gene catalog, with each category represented by a distinct color as shown in the legend. (B) Abundances of VF categories at different cold seep sites. Each point represents the abundance of a VF gene at a specific site, with vertical bars denoting the minimum and maximum values. Gene abundances are expressed in genes per million (GPM) and are plotted on a log10(x + 1) scale. Details are shown in Table S2. (C) Transcript abundance of different categories of VFs from 33 cold seep sediment samples. Each point represents the transcript abundance of a VF gene at a cold seep site. Vertical bars indicate the minimum and maximum VF transcript abundances. Transcript abundances are represented in units of transcripts per million (TPM), with values shown on the graph as log10(x + 1). Details are shown in Table S3. (D) Wind rose diagram showing the top 15 expression levels for different VFs in cold seeps. Transcript abundances are represented in units of transcripts per million (TPM), with values shown on the graph as log10(x + 1). Each italicized text indicates a VF gene, with the site abbreviation shown below each label.

Fig 2

Taxonomic distribution of VFs. (A) Bar chart showing the counts of different VF categories identified in archaeal and bacterial MAGs derived from 165 cold seep sediment samples. Additional details are provided in Table S4. (B) Relative proportions of different VF categories across bacterial phyla. (C) Relative proportions of archaeal phyla within immune modulation VFs. (D) Relative proportions of VF categories across the top 15 taxonomic phyla. The number of MAGs in each phylogenetic cluster is indicated in brackets. Different VF categories are colored as indicated in the legend. (E) Association of VF categories with different phyla. Asterisks indicate significant enrichment according to Fisher’s exact test (*/**/***, odds ratio > 1 and P-value < 0.05/0.01/0.001 after Benjamini-Hochberg correction). The heatmap colors exhibit the number of each VF category in each phylum. The bar chart at the top represents the total count of each VF category across all 15 phyla, while the bar chart on the right shows the total count of all VF categories for each phylum.

Fig 3

Different ARGs across phyla and sites in cold seeps. (A) Bar chart showing the counts of ARGs conferring different antibiotic resistance identified in archaeal and bacterial MAGs derived from 165 cold seep sediment samples. Details are shown in Table S5. (B and C) Relative proportions of different ARG types across (B) archaeal and (C) bacterial phyla. (D) Relative abundance of ARG types across different microbial phyla (ARGs > 500). The number of MAGs in each phylogenetic cluster is indicated in brackets. Different ARG types are colored as indicated in the legend. (E) Association of ARG types with different phyla. Asterisks indicate significant enrichment according to one-sided Fisher’s exact test (*/**/***, odds ratio >1 and P-value < 0.05/0.01/0.001 after Benjamini-Hochberg correction). The heatmap colors exhibit the number of each ARG type in each phylum, log-transformed [log2(x  +  1)] for plotting. The bar chart at the top represents the total count of each ARG type across all phyla, while the bar chart on the right shows the total count of all ARG types for each phylum.

Fig 4

Deep-sea cold seep microbiomes harbor diverse efflux pumps conferring antibiotic resistance (A) The heatmap displaying gene abundances of the top 100 abundant ARG subtypes across different cold seep sites. Abundance was log-transformed [i.e., log10(GP16S  ×  10⁶  +  1)] for the plot. Each row indicates a sample (arranged by sample type). Each row represents a cold seep site, with sample abbreviations indicated on the left. Each column represents an ARG subtype, affiliated with an ARG type indicated by different colors at the top. (B) Sankey diagram illustrating the taxonomic distribution of MAGs containing the macAB-tolC pump. (C) Predicted structures of MacA, MacB, and TolC proteins, as well as their assembly into the complete efflux pump complex, predicted by AlphaFold3. The left side shows the individual protein structures, while the right side illustrates the predicted interactions, forming the trimeric TolC, hexameric MacA, and dimeric MacB complexes. The full efflux pump comprises 11 proteins, with outer membrane (OM) and inner membrane (IM) boundaries indicated by dashed lines.

Fig 5

Tree and alignment of MsbA recovered from 3,164 cold seep MAGs. (A) Structural tree of MsbA predicted by ESMFold (n = 260) and the reference structures (n = 202) downloaded from the AlphaFold Protein Structure Database (AlphaFoldDB) and the Protein Data Bank (PDB). Reference structures are colored in black (B). Predicted structure of MsbA homodimer. The top section shows the alignment of the predicted structure from FR_cobin_47 and the crystal structure of MsbA from Salmonella typhimurium (PDB ID: 3B5Z). The bottom section displays the predicted MsbA homodimer structure from FR_cobin_47, with inner membrane (IM) boundaries indicated by dashed lines.

Fig 6

Comparison of ARGs, VFs, and MGEs identified in cold seep sediments. (A) Relative proportion of different VF positions detected in 3,164 cold seep MAGs. The positions of VFs are categorized into different types, including ambiguous (phage/chromosome), ambiguous (plasmid/phage), chromosome, phage, plasmid, and unclassified. (B) A chord diagram illustrating the association between different microbial phyla and the types of VFs carried on MGEs. (C) Phylogenetic tree of 82 VF-ARG carrying MAGs identified in cold seep sediments. The outer rings display the types and quantities of VFs, ARGs, and MGEs present in these genomes. (D) Boxplots comparing the counts of ARGs, VFs, and MGEs per genome across different ecosystems, including cold seeps and other habitats.