Translational horizons in bacterial membrane vesicles: Hotspots and frontiers from basic medicine to clinical application by bibliometric analysis

ABSTRACT

Bacterial membrane vesicles (BMVs) represent a class of nanoscale lipid particles released by both Gram-positive and Gram-negative bacteria, serving as versatile mediators of intercellular communication and host–pathogen interactions. Their unique biogenesis pathways and functional properties have positioned them as promising targets for therapeutic and biotechnological applications. To comprehensively assess the research trends of this dynamic research field, we conducted a bibliometric analysis of 6,352 studies on BMVs published between 1 January 2014 and 19 November 2024 using VOSviewer, CiteSpace, and the R package “bibliometrix.” Our analysis revealed that the Chinese Academy of Sciences led institutional contributions, while Frontiers in Microbiology emerged as the most active journal. Kim, Yoon-Keun was the most prolific author, reflecting his significant influence in the field. Key research hotspots were categorized into four frontiers: biogenesis mechanisms, pathogenesis and immune regulation, clinical applications, and methodological and engineering innovations.

Introduction

Bacterial membrane vesicles (BMVs) are small extracellular vesicles released by the membrane of bacteria, typically ranging in size from 20 to 300 nm. According to the properties of bacterial cells, the vesicles produced specifically by Gram-negative bacteria are referred to as outer membrane vesicles (OMVs) and those released by Gram-positive bacteria are referred to as membrane vesicles (MVs).^1,2 While both types participate in intercellular communication and host–pathogen interactions, current research has predominantly focused on OMVs due to their broader therapeutic applications and better characterization.³ BMVs play multifaceted roles in various diseases, where they exhibit both therapeutic and diagnostic potential.⁴ In the realm of oncology, OMVs can be utilized in cancer immunotherapy as vaccines to activate the immune system against cancer cells.^5,6 Additionally, they can be engineered to act as drug carriers, enhancing targeted delivery and reducing side effects. OMVs derived from intratumoral microbiota also hold promise as biomarkers for noninvasive cancer detection and surveillance. Beside oncology, OMVs are also involved in the complex pathogenic mechanism of autoimmune diseases (AID) such as inflammatory bowel disease (IBD),⁷ where they have the capacity to both promote and inhibit disease progression through their interactions with the immune system.

Bibliometrics is a literature analysis method that explores literature system and bibliometric characteristics of publications by mathematical, statistical, and other econometric methods.^8,9 It can provide bibliometric relationship of authors, organizations, countries, and references in relevant research field. Common bibliometric tools including VOSviewer, CiteSpace, and R package “bibliometrix” are widely applied in medical fields. In 2020, Min Li et al. comprehensively demonstrated a recent update on the biomedical applications of bacterial OMVs.⁴ In 2023, Gisseth Magaña et al. comprehensively reviewed the role of OMVs in pathogenesis and host–cell interactions.¹⁰ However, even though the last 10 y have seen substantial growth in foundational and applied review on BMVs, systematic bibliometric analysis in this field remains sparse. In this research, we employed bibliometric analysis to methodically examine the trends and advancements in studies on BMVs from 1 January 2014 to 19 November 2024. Our goal is to unearth a quantitative and qualitative representation of the acadehmic progress in this field, thus shedding light on the emerging research trends and forecasting future developments.

Methods

Search strategy

We conducted a literature search on the Web of Science Core Collection (WoSCC) database. The time spans were limited from 1 January 2014 to 19 November 2024. The search strategy was TS = (“outer membrane vesicles” OR “bacterial outer membrane vesicles” OR “outer membrane derived vesicles” OR “membrane vesicles” OR “bacterial membrane vesicles”). Only documents with “article” and “review” type and written by English were included. Publications with other types and languages were excluded.

Data analysis

Microsoft Office Excel 2019 was used to conduct data analysis and to visualize the annual number of publications. The software VOSviewer (version 1.6.20) was harnessed to extract insights from the literature and investigate connections across various academic disciplines. In our research, VOSviewer facilitated the exploration of networks pertaining to countries, institutions, journals, authors, and keywords. Meanwhile, CiteSpace (version 6.3) enabled the creation of a dual-map overlay of journals and the analysis of references with citation bursts. The R package “bibliometrix” (version 4.0.0) available at (https://www.bibliometrix.org) was instrumental in mapping the global distribution of publications and analyzing trending topics. Information on the journal’s quartile and impact factor was sourced from the Journal Citation Reports 2023.

Results

Quantitative analysis of publication

Based on search terms, a total of 6,661 studies were identified from WoSCC, and meeting abstracts, editorial materials, corrections, letters, proceeding papers, new items and biographical-items were excluded, shown in Figure 1. Excluding 32 non-English studies, a total of 6,352 studies on BMVs were collected. The exact quantity of publications of each year was provided in Figure 2. During the last decade, the number of publications related to BMVs has kept high, showing long-term attention on this topic. There was a noticeable upward trend in the percentage, with gradual increases over the years from 2014 to 2024. Despite minor fluctuations, such as a slight decline in 2018, the overall trend indicated a significant growth and stayed at relatively high levels in recent years, having reached 10.0% from 2020 to 2024.

Figure 1.

Publication screening flowchart.

Figure 2.

Annual number of publications on BMVs.

Country and institutional analysis

These publications spanned from 101 countries and 5,106 institutions, with the top ten countries primarily located in Europe (n = 6) (Table 1). The two leading nations, the United States (n = 1,680, 26.4%) from North America and China (n = 1,502, 23.6%) from Asia, collectively contributed to half of the total publications (n = 3,182, 50.0%). Countries with five or more publications were selected for visualization using VOSviewer, and their collaborative networks were illustrated in Figure 3, highlighting both the quantity and connections of their publications. Notably, the United States formed extensive collaborations with numerous countries, including China, the United Kingdom, Germany, South Korea, Japan, the Netherlands, and Spain.

Table 1.

Top ten countries and institutions on research of BMVs.

Rank	Country (Continent)	Publication Count	Institution (Country)	Publication Count
1	United States (North America)	1680 (26.4%)	Chinese Academy of Sciences (China)	106 (1.7%)
2	China (Asia)	1502 (23.6%)	University of Oxford (United Kingdom)	73 (1.1%)
3	Germany (Europe)	533 (8.4%)	Shanghai Jiao Tong University (China)	69 (1.1%)
4	United Kingdom (Europe)	441 (6.9%)	University of Chinese Academy of Sciences (China)	68 (1.1%)
5	Japan (Asia)	418 (6.6%)	Russian Academy of Sciences (Russia)	67 (1.1%)
6	Italy (Europe)	358 (5.6%)	Sichuan University (China)	59 (0.9%)
7	Spain (Europe)	283 (4.5%)	Umeå University (Sweden)	59 (0.9%)
8	France (Europe)	278 (4.4%)	Zhejiang University (China)	56 (0.9%)
9	South Kores (Asia)	257 (4.0%)	Chinese Academy of Agricultural Sciences (China)	54 (0.9%)
10	Netherlands (Europe)	231 (3.6%)	Cornell University (United States)	53 (0.8%)

Figure 3.

The visualization of countries on research of BMVs.

Each colored node represents a country, with node size corresponding to its publication count and color indicating its cluster affiliation based on collaborative patterns. Connecting lines between nodes demonstrate research partnerships, where thicker lines denote stronger co-authorship frequencies.

Three-fifths of the top ten research institutions are headquartered in China. The leading institutions included the Chinese Academy of Sciences (n = 106, 1.7%), University of Oxford (n = 73, 1.1%), Shanghai Jiao Tong University (n = 69, 1.1%), University of Chinese Academy of Sciences (n = 68, 1.1%), and the Russian Academy of Sciences (n = 67, 1.1%). Figure 4 illustrated the network of collaborations among 344 institutions that boasted over ten publications each. A notable close collaboration was observed between the Chinese Academy of Sciences and the University of Chinese Academy of Sciences. Within China, there was robust cooperation among local institutions, though these connections were less prevalent with international entities. The University of Oxford, however, demonstrated a diverse range of collaborative ties with institutions across different clusters. Conversely, the Russian Academy of Sciences showed limited interaction with other institutions.

Figure 4.

The visualization of institutions on research of BMVs.

Each node represents a research institution, where the node size corresponds to its publication count and the color indicates its cluster affiliation based on collaborative relationships. Connecting lines between nodes demonstrate research partnerships, where thicker lines denote stronger co-authorship frequencies.

Journals and co-cited journals

A total of 1,489 journals were analyzed for research on BMVs, as detailed in Table 2. Leading the publication count was Frontiers in Microbiology (n = 207, 3.3%), followed by the International Journal of Molecular Sciences (n = 176, 2.8%), and Scientific Reports (n = 144, 2.3%). Journal of Extracellular Vesicles (n = 65, 1.0%) boasts the highest impact factor (IF = 15.5, Q1) and is the only journal with an IF higher than 10. A filtered subset of 288 journals, each with over five relevant papers, was chosen for the bibliometric analysis depicted in Figure 5a. Notably, there was a close relationship between Frontiers in Microbiology and International Journal of Molecular Sciences.

Table 2.

Top 15 journals and co-cited journals for research of BMVs.

Rank	Journal	Publication Count	IF	Q	Co-cited Journal	Co-citation Count	IF	Q
1	Frontiers in Microbiology	207 (3.3%)	4.0	Q2	Proceedings of the National Academy of Sciences of the United States of America (PNAS)	12549	9.4	Q1
2	International Journal of Molecular Sciences	176 (2.8%)	4.9	Q1	PLOS ONE	11239	2.9	Q1
3	Scientific Reports	144 (2.3%)	3.8	Q1	Journal of Biological Chemistry	10829	4.0	Q2
4	PLOS ONE	125 (2.0%)	2.9	Q1	Infection and Immunity	8632	2.9	Q3
5	Frontiers in Immunology	107 (1.7%)	5.7	Q1	Journal of Bacteriology	8471	2.7	Q3
6	Frontiers in Cellular and Infection Microbiology	70 (1.1%)	4.6	Q2	Nature	7601	50.5	Q1
7	Journal of Extracellular Vesicles	65 (1.0%)	15.5	Q1	Scientific Reports	6770	3.8	Q1
8	Mbio	56 (0.9%)	5.1	Q1	Nature Communications	6172	14.7	Q1
9	Applied and Environmental Microbiology	55 (0.9%)	3.9	Q2	Journal of Extracellular Vesicles	6155	15.5	Q1
10	Microorganisms	53 (0.8%)	4.1	Q2	Science	5932	44.7	Q1
11	Nature Communications	53 (0.8%)	14.7	Q1	Frontiers in Microbiology	5376	4.0	Q2
12	Vaccine	53 (0.8%)	4.5	Q2	Cell	5043	45.5	Q1
13	Cells	52 (0.8%)	5.1	Q2	Vaccine	4826	4.5	Q2
14	Journal of Biological Chemistry	51 (0.8%)	4.0	Q2	Journal of Immunology	4808	3.6	Q2
15	Proceedings of the National Academy of Sciences of the United States of America (PNAS)	50 (0.8%)	9.4	Q1	Applied and Environmental Microbiology	4431	3.9	Q2

Note: IF: Impact factor; Q: Quartile ranking (Q1–Q4) according to the Journal Citation Reports 2023.

Figure 5.

The visualization of journals (a) and co-cited journals (b) on research of BMVs.

Each colored node represents a journal, with node size corresponding to its publication count and color indicating its cluster affiliation based on collaborative patterns. Connecting lines between nodes demonstrate citation relationship, where thicker lines denote stronger journal citation frequencies.

Table 2 also revealed the top co-cited journals according to VOSviewer, all of which have been cited upward of 6,000 times. Proceedings of the National Academy of Sciences of the United States of America (PNAS) occupied the first place with 12,549 co-citations, followed by PLOS ONE (co-citation = 11,239) and Journal of Biological Chemistry (co-citation = 10,829). Among these, Nature stood out with the highest impact factor (IF = 50.5), though ranking sixth in the top ten of co-cited journals. Figure 5b illustrates a co-citation network that included 1,134 journals, each having received at least 50 co-citations. Within this network, PNAS showed robust co-citation links with other prestigious publications such as the Journal of Biological Chemistry, Frontiers in Microbiology, and Nature Communications.

In the dual-map overlay (Figure 6) presented by CiteSpace, citation dynamics were illustrated between journals. The diagram segregated citing journals on the left and cited journals on the right. Central to each cluster, labels identified the academic domains of the citing articles. Connecting these clusters, spline curves originated from the citing journals on the left side of the base map and targeted the cited journals on the right side. As shown in Figure 6, publications from journals in the Molecular/Biology/Genetics domain predominantly received citations from works within the Molecular/Biology/Immunology and the Physics/Materials/Chemistry fields.

Figure 6.

The dual-map overlay of journals on research of BMVs.

Authors and co-cited authors

There were 30,768 authors contributing to the research on BMVs. Notably, each of the top ten contributors has published no fewer than 20 papers, with Kim, Yoon-Keun leading by having authored 35 papers, as shown in the left section of Table 3. To explore the collaborative dynamics among these scholars, a network was constructed using VOSviewer for 605 authors who each had a minimum of five publications on BMVs (Figure 7a). This visualization omitted articles authored by more than 25 authors. Within the network, distinct clusters were apparent, yet inter-cluster connections were minimal, showing a tendency toward independence within each cluster.

Table 3.

Top ten authors and co-cited authors on research of BMVs.

Rank	Author	Publication Count	Co-cited Author	Co-citation Count
1	Kim, Yoon-Keun	35	Théry, Clotilde	1628
2	Gho, Yong Song	30	Schwechheimer, Carmen	1188
3	Micoli, Francesca	26	Toyofuku, Masanori	1059
4	Siadat, Seyed Davar	26	Raposo, Graça	857
5	Fuhrmann, Gregor	24	Lee, Eun Young	689
6	Nomura, Nobuhiko	24	Kulp, Adam	680
7	Toyofuku, Masanori	24	Valadi, Hadi	582
8	Bravo, Alejandra	21	György, Bence	580
9	Soberon, Mario	21	Kadurugamuwa, Jagath L	572
10	Wai, Sun Nyunt	21	Ellis, Terri N	533

Figure 7.

The visualization of authors (a) and co-cited authors (b) on research of BMVs.

In (a), nodes represent authors, with size indicating their publication volume and connecting lines showing co-authorship relationships. In (b), nodes represent cited authors, with size reflecting citation frequency and links indicating co-citation strength. Colors denote thematic clusters or research communities. Thicker lines represent stronger collaborative or co-citation ties.

Regarding co-cited authors, the most frequently co-cited author was Théry, Clotilde (n = 1,628), followed by Schwechheimer, Carmen (n = 1,188) and Toyofuku, Masanori (n = 1,059). The co-citation network, illustrated in Figure 7b, included 967 authors who each have been cited at least 50 times. Théry, Clotilde had active connection with several authors, including Schwechheimer, Carmen, Toyofuku, Masanori, and Van Niel, G.

Co-cited references

In the last decade, research on BMVs has referenced a total of 267,301 co-citations. Shown in Table 4, two references were co-cited more than 100 times. Seven hundred and ninety-one references with at least 40 co-citations were filtered to the construction of the co-cited network map (Figure 8). “Théry C, 2009, Nat Rev Immunol, V9, P5813¹³” displayed active co-cited relationships with “Valadi H, 2007, Nat Cell Biol, V9, P6544¹⁴” and “Schwechheimer C, 2015, Nat Rev Microbiol, V13, P6051¹¹,” etc. Notably, several references displayed no connection with others at all.

Table 4.

Top ten co-cited references on BMVs.

Rank	Co-cited Reference	Main Research Content	Citation Count
1	Schwechheimer C, 2015, Nat Rev Microbiol, V13, P60511	A review on recent advances in the field regarding OMV biogenesis and cargo selection, as well as the functions of OMVs during nutrient and iron acquisition, interbacterial communication, stress relief and pathogenesis.	862
2	Kulp A, 2010, Annu Rev Microbiol, V64, P16312	A review on the function and biogenesis of gram-negative bacterial OMVs.	679
3	Théry C, 2009, Nat Rev Immunol, V9, P58113	A review on the role of membrane vesicles, in particular exosomes, in the communication between immune cells, and between tumor and immune cells.	644
4	Valadi H, 2007, Nat Cell Biol, V9, P65414	Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells	581
5	Raposo G, 2013, J Cell Biol, V200, P37315	A review on the characterization of EVs and currently proposed mechanisms for their formation, targeting, and function.	515
6	Toyofuku M, 2019, Nat Rev Microbiol, V17, P1316	A review on the structures and compositions of the various vesicle types and their novel formation routes.	498
7	Kaparakis-Liaskos M, 2015, Nat Rev Immunol, V1517	A review on the mechanisms through which OMVs induce host pathology or immune tolerance and the development of OMVs as innovative nanotechnologies.	454
8	György B, 2011, Cell Mol Life Sci, V68, P266718	A review on extracellular vesicles from a systems biology perspective and recent developments and some burning questions in the field.	412
9	Ellis TN, 2010, Microbiol Mol Biol R, V74, P8119	A review on the role of bacterial outer membrane vesicles in the virulence of gram-negative bacterial pathogens.	395
10	Théry C, 2018, J Extracell Vesicles, V720	A guideline named minimal information for studies of extracellular vesicles 2018 (MISEV2018) with protocols to document specific EV-associated functional activities.	368

Figure 8.

The visualization of co-cited references on BMVs.

Each colored node represents a reference with node size corresponding to its citation count and color indicating its cluster affiliation based on collaborative patterns. Connecting lines between nodes demonstrate co-citation relationship, where thicker lines denote stronger relationship.

References with citation bursts

Reference with citation burst refers to papers with a surge in citation frequency after publication, indicating a high attention to relevant topics. Figure 9 provides the top 25 references experiencing the most significant citation bursts determined by CiteSpace. A red bar highlighted the period of intense citation activity from 2014 to 2024. These bursts started appearing as early as 2014 and as recently as 2022. The reference experiencing the most significant citation burst (strength = 95.18) ranked 15 in Table 5 and was titled “Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions,” authored by Schwechheimer C and Kuehn MJ, showing heightened citation activity from 2017 to 2020. Coming in second (strength = 70.33) was titled “Extracellular vesicles: Exosomes, microvesicles, and friends.” It was published in Journal of Cell Biology by Raposo G and Stoorvogel W with 5-y citation bursts (2014–2018). Detailed information on these top 25 references was listed in Table 5 and was arranged according to the order in Figure 9, with burst strengths ranging from 33.33 to 95.18 and durations of increased citation frequency lasting between 2 and 5 y.

Figure 9.

Top 25 references with strong citation bursts. A red bar indicates high citations in that year.

Table 5.

The main research contents of the 25 references with strong citations bursts.

Rank	Strength*	Main Research Content
1	70.33	A review on the characterization of EVs and currently proposed mechanisms for their formation, targeting, and function.15
2	59.92	A review on the function and biogenesis of gram-negative bacterial OMVs.12
3	59.19	A review on extracellular vesicles from a systems biology perspective and recent developments and some burning questions in the field.18
4	46.48	An article on the function of melanoma-derived exosomes in the formation of primary tumors and metastases.21
5	38.20	Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.22
6	35.74	Comparison of vesicle release and the resulting functional impact across bacteria, eukaryotes and archaea.23
7	35.53	An innovative approach for delivering siRNA to the brain in mice using targeted exosomes.24
8	35.17	A review on recent progress in the role of extracellular vesicles in intercellular communication and their therapeutic opportunities.25
9	33.77	A review on the role of bacterial outer membrane vesicles in the virulence of gram-negative bacterial pathogens.19
10	56.64	A comprehensive overview of the current understanding of the physiological roles of EVs.26
11	52.42	A review on biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.27
12	49.93	A review on the mechanisms through which OMVs induce host pathology or immune tolerance and the development of OMVs as innovative nanotechnologies.17
13	37.76	An article of the role of tumor-derived exosomes integrins in the determination of organotropic metastasis.28
14	33.33	A review on the current knowledge of EV uptake mechanisms.29
15	95.18	A review on recent advances in the field regarding OMV biogenesis and cargo selection, as well as the functions of OMVs during nutrient and iron acquisition, interbacterial communication, stress relief and pathogenesis.11
16	45.77	A review on the current status of vesiculogenesis research in thick-walled microorganisms and discuss the cargo and functions associated with EVs in these species.30
17	33.33	An article that found explosive cell lysis act as a mechanism for the production of bacterial MVs.31
18	43.90	A review on OMVs of gram-negative bacteria.32
19	54.24	A guideline named minimal information for studies of extracellular vesicles 2018 (MISEV2018) with protocols to document specific EV-associated functional activities.20
20	36.37	An article on the potential of bacterial outer membrane vesicles as therapeutic agents to treat cancer via immunotherapy.33
21	35.87	A review on the cellular processes that govern extracellular vesicle biology.34
22	65.13	A review on the structures and compositions of the various vesicle types and their novel formation routes.16
23	40.69	An article on Bioengineered bacteria-derived outer membrane vesicles as a versatile antigen display platform for tumor vaccination via Plug-and-Display technology.35
24	35.18	A review on the biogenesis, the function, and the potential clinical applications of exosomes in disease.36
25	34.40	A review on the multiple roles of OMVs, the current knowledge of OMV biogenesis, and the biotechnological applications of OMV.2

Note: *Strength: It quantifies the frequency surge of a reference over time. Higher values denote greater attention and potential emergence as a research frontier or hotspot in the field.

Hotspots and frontiers

The co-occurrence analysis of keywords helps to capture the hotspots in the field of BMVs, and the top 30 high-frequency keywords calculated by VOSviewer were summarized in Table 6. Excluding keywords like extracellular vesicles (count = 1,025), outer membrane vesicles (count = 826), exosomes (count = 718), membrane vesicles (count = 328), and microvesicles (count = 149), important keywords, such as vaccines (count = 249), biomarkers (count = 157), cancer (count = 132), drug delivery (count = 132), immunotherapy (count = 78), and antibiotic resistance (count = 88), indicate the medical and therapeutic applications of BMVs and are very likely to represent the research hotspots of BMVs.

Table 6.

Top 30 keywords on research of BMVs.

Rank	Keyword	Publication Count	Rank	Keyword	Publication Count
1	extracellular vesicles	1025	16	antibiotic resistance	88
2	outer membrane vesicles	826	17	immunotherapy	78
3	exosomes	718	18	Bacillus thuringiensis	77
4	membrane vesicles	328	19	microbiota	74
5	vaccines	249	20	Pseudomonas aeruginosa	73
6	biomarkers	157	21	Porphyromonas gingivalis	65
7	microvesicles	149	22	apoptosis	60
8	cancer	132	23	gut microbiota	59
9	drug delivery	132	24	mesenchymal stem cells	59
10	microRNA	132	25	immune response	58
11	autophagy	131	26	COVID-19	56
12	inflammation	126	27	Helicobacter pylori	56
13	proteomics	111	28	virulence	56
14	biofilm	103	29	tumor microenvironment	53
15	bacteria	91	30	pathogenesis	50

Author keywords appearing more than ten times were subjected to a co-occurrence and cluster analysis, generating a bibliometric map (Figure 10a). Different colors represented different research directions and there were five major clusters performed in Figure 10a, respectively, colored mainly in light blue, dark blue, green, red, and yellow.

Figure 10.

Keyword cluster analysis (a) and trend topic analysis (b).

In (a), each node represents a keyword, with size proportional to its frequency in the literature and color indicating its thematic cluster. Connecting lines between keywords denote co-occurrence in the same publications, where thicker lines reflect stronger associations.

The trend topic analysis of the keywords presented in Figure 10b reveals intriguing shifts in research focus over the years. From 2014 to 2018, the research landscape was primarily dominated by an exploration of mechanisms related to flow cytometry, ABC transporter, protease, vacuole, P-glycoprotein, invasion, endothelium, and atherosclerosis. These keywords collectively highlighted a deep dive into the underlying mechanisms of BMVs, suggesting a period of intense investigation into their structure, function, and interaction with host cells. Shifting gears, from 2018 to 2023, the research focus transitioned toward leveraging BMVs for practical applications. Notably, there was a surge in research on the utilization of BMVs in the context of cancer, biomarkers, COVID-19, vaccines, drug delivery, and immunotherapy. This shift reflected a growing interest in harnessing the unique properties of BMVs for therapeutic and prophylactic purposes, particularly in response to emerging health challenges such as the COVID-19 pandemic. By recent years, emerging terms such as ferroptosis, genetic engineering, and the gut-brain axis indicated new avenues of exploration that were gaining attention.

Discussion

General information

Over the past decade, the volume of published research has remained high, indicating a sustained interest in BMVs. The United States led in this research area, followed by China and Germany, suggesting strong scientific and economic foundations in these countries contribute significantly to advancements in BMVs studies. Notably, six of the top ten institutions engaged in BMVs researches are located in China, while the remaining four are based in the United Kingdom, Russia, Sweden, and the United States. This distribution highlighted the global effort in understanding and utilizing BMVs although with particular emphasis in countries with robust healthcare technologies and research capabilities. The significant representation of China indicated a concentrated effort and governmental support in the field of medical research. High population metrics and increasing healthcare demands likely drove the focused growth in BMVs research.

Close relationships were observed among the United States, China, the United Kingdom, and Germany. Chinese institutions like the Chinese Academy of Sciences dominated research publication but mostly collaborated within the country, indicating a more insular network. The University of Oxford stood out with its extensive international collaborations, enhancing global knowledge exchange. Conversely, the Russian Academy of Sciences exhibited limited collaborations outside Russia, which could reflect geopolitical or funding constraints affecting its global research connectivity.

About 3.3% articles were published in Frontiers in Microbiology (IF = 4.0, Q2) and 2.8% articles were published in International Journal of Molecular Sciences (IF = 4.9, Q1). PNAS (IF = 9.4, Q1) occupied the 15th spot in the journal list. Meanwhile, this journal ranked first in the list of co-cited journals. Such a high co-citation frequency implied that while PNAS might not have the highest number of BMVs publications, the research it published significantly influenced the field. This illustrated the journal’s scope and its role in disseminating influential foundational studies that shaped ongoing research trends and methodologies related to BMVs. Besides, the dual-map overlay of journals indicated that research on BMVs published in Molecular/Biology/Genetics journals had interdisciplinary relevance and impact, influencing studies not only in closely related fields like molecular biology and immunology but also extending its influence to seemingly distant fields such as physics, materials science, and chemistry. This suggested the fundamental nature of BMVs and their potential applications across a broad spectrum of scientific disciplines.

Regarding author contributions, Kim, Yoon-Keun published the most articles with the number of 35. His research primarily focused on the mechanism and application of microbial EVs therapy as diagnostic biomarkers and therapeutic agents for various diseases, including allergic asthma,³⁷ gastric cancer,³⁸ colorectal cancer,³⁹ and brain tumor.⁴⁰ Following him, Gho, Yong Song, who is affiliated with the same institution as Kim, Yoon-Keun, ranked as the second most published author. They have also coauthored several papers together, indicating a robust collaborative relationship in the field of BMVs.

With regard to the co-cited authors, Théry, Clotilde was the most frequent co-cited author (citation = 1,628), followed by Schwechheimer, Carmen (citation = 1,188) and Toyofuku, Masanori (citation = 1,059). Théry, Clotilde is a leading figure in exosome research, having established the gold standard for the extraction, isolation, and identification of exosomes.⁴¹ In 2016, Théry, Clotilde published a review in Cell, which focused on the latest findings regarding the role of EVs in cancer metastasis.⁴² In 2018, she and her coworkers published Minimal information for studies of extracellular vesicles 2018 (MISEV2018),²⁰ which is also the 19th reference with citation burst, setting up standardized protocols for studies in this field that are crucial for verifying research findings and facilitating comparisons of results across different studies.

Knowledge base

A co-cited reference is defined as one research that is cited together by multiple publications. Top 10 co-cited references were selected to clarify the research basis of BMVs. Among them, eight references are reviews and two are articles. These references cover topics ranging from the biogenesis and functions of BMVs, as highlighted in references ranked 1, 2, 5, 6, and 8,^{11,12,15,16,18} to the mechanisms of genetic material transfer facilitated by vesicles, as discussed in references ranked 3 and 4.^13,14 Additionally, references ranked 7 and 9^, examine how BMVs contribute to bacterial virulence and immune responses. Technical insights and methodological guidelines are provided by previously discussed MISEV2018.^17,19,20 Overall, the biogenesis, function, pathogenesis, and protocol of vesicles have provided the foundational work of studies on BMVs.

Hotspots and frontiers

References with citation bursts reflect on the emerging topics in a particular field. According to the burst period (Figure 9) and main contents of publications (Table 5), relevant researches primarily focused on the underlying mechanisms of BMVs, suggesting a period of intense investigation into their structure, function, and interaction with host cells before 2018. Since 2018, the disease associations and potential clinical applications of BMVs have been the hotspots in the research field of BMVs, aligning with the current emphasis on their significance in medical sciences.

Additionally, the top 30 keywords (Table 6) support this conclusion. The keywords predominantly encompass three categories. The first is types and general aspects of vesicles, with keywords including extracellular vesicles, outer membrane vesicles, exosomes, membrane vesicles, and microvesicles. These keywords indicate a foundational interest in various types of vesicles, their biogenesis, properties, and their distinct functions. The second is disease associations and pathology, including keywords such as cancer, inflammation, COVID-19, Helicobacter pylori, Pseudomonas aeruginosa, Porphyromonas gingivalis, apoptosis, tumor microenvironment, and virulence. The third is medical and therapeutic applications, mainly involving keywords including vaccines, biomarkers, drug delivery, and immunotherapy. BMVs are being explored for vaccines, targeted drug delivery systems and immunotherapeutic agents, aligning with diagnostic approaches. The broad spectrum spanning from fundamental science to clinical applications highlights their significant potential. The trend of topic terms shown in Figure 10b also provides some new aspects of future research. The interest in “ferroptosis” within the context of BMVs probably lies in their potential implications in bacterial pathogenicity and host–pathogen interactions. “Genetic engineering” is a power technology which can realize targeted therapy and reduce vesicle toxicity. The term “gut-brain axis” indicates that BMVs in the gastrointestinal tract might influence neurological health, thus opening up avenues for novel therapeutic strategies. Combining the abovementioned information, we derived four hotspots and frontiers for research on BMVs, especially OMVs.

Biogenesis mechanisms

First, several articles delve into the biogenesis mechanism of OMVs. Since the first observation of OMVs production in 1965,⁴³ despite extensive studies, a definitive or universal mechanism remains elusive due to the complexity and variability among different bacterial species. Here are some of the proposed mechanisms. One theory is the disruption or absence of cross-linking proteins, which connect the outer phospholipid bilayer to the peptidoglycan layer of the outer membrane of gram-negative bacteria, leads to the curving of the outer membrane, subsequently resulting in OMVs formation.⁶ In 2016, another proposed model is the explosive cell lysis model, where high-stress conditions trigger abrupt membrane disruption, leading to OMVs release.³¹ Further, OMVs originate when the outer membrane spontaneously curves due to disruptions in lipid asymmetry or from interactions between outer membrane proteins and lipopolysaccharides (LPS).⁴⁴ For example, the VacJ/Yrb ATP-binding cassette (ABC) Transporter System plays a critical role in maintaining lipid asymmetry, with disruptions potentially causing imbalances that result in OMVs formation.⁴⁵ Additionally, protein misfolding in the periplasm may accumulate toxic byproducts, pushing the membrane to bud off as OMVs as a means of quality control.⁴⁶

Due to the multiplicity of these mechanisms, it is likely that OMVs production is highly dependent on their contents and could involve different processes among different species or environmental conditions. Continued research is needed to understand the exact trajectories and their regulation.

Pathogenesis and immune responses

Second, OMVs are studied extensively for their roles in pathogenesis and immune responses. OMVs carry bacterial components such as LPS, proteins, and nucleic acids which can directly interact with host immune cells including macrophages and dendritic cells, modulating immune responses. Furthermore, OMVs can act as decoys, binding antibodies and diverting immune responses away from the actual pathogens, thus enhancing bacterial survival. They also facilitate horizontal gene transfer, spreading antibiotic resistance genes and virulence factors between bacteria,⁴⁷ which complicates treatment strategies. For example, OMVs from Pseudomonas aeruginosa, an opportunistic pathogen, contain enzymes, toxins, and other effector molecules that contribute to tissue damage and evasion of host immune responses.⁴⁸ By delivering these factors directly to host cells, they can cause severe infections, particularly in immunocompromised patients. Additionally, OMVs can induce inflammation through the activation of pattern recognition receptors, and by triggering inflammasomes.⁴⁹ The interplay of these factors demonstrates the complexity and significance of OMVs in bacterial pathogenesis and presents ongoing challenges and opportunities in therapeutic development against bacterial infections.

Furthermore, OMVs have attracted significant attention in cancers that are caused by infectious microorganisms, including oral cancer,⁵⁰ gastric cancer,⁵¹ and colorectal cancer.^50,52 In Table 6, keyword Porphyromonas gingivalis is linked to oral cancer, and keyword Helicobacter pylori is a major risk factor for gastric cancer. For instance, previous research has found that H. pylori outer membrane vesicles may affect carcinosis by inhibiting human T cell responses via induction of monocyte cyclo-oxygenase-2 (COX-2) expression.⁵³

Clinical applications

Third, OMVs are being explored for their potential in clinical applications. This includes their use in vaccine development, drug delivery system, cancer immunotherapy, and diagnostic application.

Given that OMVs inherently carry pathogen-associated molecular patterns (PAMPs), particularly when developing vaccines for bacterial pathogens, they provoke a potent immune response. This advantage is significant as it eliminates the requirement for live pathogens in vaccine formulation.⁵⁴ OMVs from bacteria like Neisseria meningitidis have already been used in licensed vaccines.⁵⁵

OMVs are explored as natural drug delivery vehicles because they can encapsulate various therapeutic agents, including small molecules, proteins, or nucleic acids.⁵⁶ The lipid bilayer and natural composition of OMVs allow them to merge with other cellular membranes, facilitating targeted delivery of their cargo directly into the cytoplasm of recipient cells. This can be particularly useful for the delivery of drugs that must enter cells to be effective or for therapies that target specific types of cells.

As for cancer immunotherapy, owing to the bacterial components, they can interact with host cells to induce immune responses. In 2017, Kim, Oh Youn et al. presented the first report of utilizing OMVs as cancer immunotherapeutic agent. The results revealed trypsin-sensitive surface proteins of OMVs are the key inducers of IFN-γ production within the tumor microenvironment to activate antitumor responses.³³ Another strategy for cancer immunotherapy is to block immune checkpoints. For example, Li, Yao et al. engineered OMVs by inserting programmed death 1 (PD1), thus allowing OMV-PD1 to bind to PD-L1 on the cancer cell surface. In this way, T cells are being protected and can continue their immune functions.⁵⁷ Furthermore, combining the aforementioned vaccine and delivery vehicle applications of OMVs, OMVs can also be engineered to deliver immune-stimulating molecules directly into the tumor microenvironment (TME).⁵⁸

Additionally, the potential of using OMVs as biomarkers or for the detection of certain diseases is also an exciting area of research. OMVs play a vital role in the communication and pathogenic mechanisms of bacteria, with differences observed between healthy individuals and patients. These vesicles can be detected in blood, urine, and stool from patients. Previous studies have demonstrated the diagnostic function of OMVs in various diseases, including IBD, gastric cancer, hepatocellular carcinoma, and colorectal cancer.^39,59,60

While the potential of OMVs in these areas is substantial, there are technical and safety issues to resolve. Ensuring the stability of OMVs, the efficiency of the targeting mechanism, the controlled release of their cargo, and minimizing any potential side effects are crucial areas of ongoing research. Moreover, scaling up production and meeting rigorous safety standards for clinical use are challenges that need to be addressed in future studies.

Methodological and engineering innovations

Forth, the development of methodological and engineering innovations for OMVs applications should not be overlooked. From traditional methods of OMVs isolation and purification to advanced biotechnological approaches for the large-scale production and characterization of OMVs, technological advancements have diversified the options for utilizing OMVs. This provides a fertile ground for the potential future development of OMVs-based therapies and diagnostics.

Originally, the focus was on naturally secreted OMVs from gram-negative bacteria, which were utilized primarily in vaccine development and drug delivery due to their inherent adjuvant properties and biocompatibility. However, the need for more tailored and efficient delivery systems has driven research toward engineering OMVs. One prevalent engineering strategy is the incorporation of specific ligands or receptors to target specific cell types. For example, Sun, Jianan et al. conjugated an OMV-based nanosized immune cell engager (OMV-NICE) targeting CD47/SIRPα and PD-1/PD-L1 pathways to realize precise immunotherapy.⁶¹ Other engineering strategies include the encapsulation of novel therapeutic agents, such as oncolytic adenovirus⁶² and plasmid,⁶³ and the methods for loading agents into OMVs, such as incubation, electroporation, and extrusion.⁶ Moreover, the development of synthetic OMVs-like vesicles, which mimic the structure and function of natural OMVs but with enhanced properties, is also an active area of research.

Furthermore, the integration of emerging technologies, such as nanotechnology and artificial intelligence, with OMVs-based systems holds promise for even more innovative applications. For example, in 2024, Tang, Songsong et al. introduced OMVs-based nanorobots that were bioengineered with cell-penetrating peptide capable of tumor targeting and penetration, and proved their efficacy and safety in precision cancer therapy.⁶⁴ With ongoing advancements in our understanding of OMVs biology and the development of new technologies, it is anticipated that the applications of OMVs will continue to expand, offering new solutions for a wide range of medical challenges.

Limitations

Inevitably, there are some limitations in this study. First, the exclusive use of the WoSCC may lead to an incomplete perspective as important studies from other databases like PubMed, Scopus or Google Scholar are excluded. This limitation restricts the study to a subset of the available literature and may overlook significant contributions available elsewhere. Second, by only including studies published in English, the research introduces a linguistic bias. Important findings and discussions published in other languages are not considered, which could potentially miss out on pertinent data and perspectives from non-English-speaking regions. Finally, the use of specific data analysis software such as VOSviewer affects the analysis outcomes. For example, when constructing relationship visualization, the threshold for the number of documents might overlook some important literature and omit useful insights.

Conclusion

The consistent research activity over the recent years underscores the sustained interest and perceived importance of OMVs in global health contexts. OMVs are being viewed as both a pivotal component in understanding bacterial function and interactions, and as a versatile tool in biotechnological and medical applications, highlighting their dual roles in science as both fundamental biological structures and powerful applicational tools. Researchers are increasingly focused on the incorporation of bioengineering techniques and advanced technologies in OMVs to enhance their safety and efficacy. More preclinical and clinical trials are needed to guarantee the effective translation of OMVs basic research into practical medical applications.

Biographies

Shao-Gang Wang is a prominent urologist at Tongji Hospital, specializing in minimally invasive surgeries for prostate, kidney, bladder, and adrenal cancers, as well as urinary tract stones. His expertise in laparoscopic and robotic techniques has advanced urological surgery, improving patient outcomes. A pioneer in extracellular vesicle research, Professor Wang’s team investigate their role in urological cancers, focusing on tumor microenvironment regulation, immune modulation, and drug delivery system. His groundbreaking studies have identified EV-based biomarkers and therapeutic targets, enhancing early diagnosis and precision treatment.

Qi-Dong Xia is a postdoctoral fellow at Tongji Hospital. As a prolific researcher in urology and oncology, Dr. Xia has made substantial contributions to the field. His work has been presented at prestigious conferences, including the Chinese Urological Association (CUA), European Association of Urology (EAU), and American Association for Cancer Research (AACR). He also serves as a youth editorial board member for journals. His research contributions have garnered significant academic influence in the field.

Funding Statement

The author(s) reported that there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).