Bacterial extracellular vesicles: A position paper by the microbial vesicles task force of the Chinese society for extracellular vesicles

Recently, the interest in extracellular vesicles released by bacteria has rapidly increased. Bacterial extracellular vesicles (BEVs) have been involved in bacteria‐bacteria and bacteria‐host interactions, which strengthen health or bring about various pathologies. However, BEV separation, characterization, and functional studies require the establishment of guidelines and further optimization in order to stimulate the development of science in BEV research and a following successful transformation into clinical applications. This position paper is authored by the Microbial Vesicles Task Force of the Chinese Society for Extracellular Vesicles (CSEV) composed of experienced medical laboratory specialists, microbiologists, virologists, biologists and material biologists who are actively engaged in BEV research. Herein, we present a concise description of BEV research and discover challenges and critical gaps in current BEV‐based analyses for clinical applications. Finally, we also offer suggestions and considerations to improve experimental reproducibility and interoperability in BEV research to promote progress in the field.


| TYPES OF BEVS
To better probe the biological functions of BEVs, it is currently divided into several categories based on different origins, where BEVs are released via "blebbing" mechanisms or via cell lysis processes that are lethal to the bacterial cell ( Figure 1).

| BEVs originated from gramnegative bacteria
BEVs released by gram-negative bacteria can be outer membrane vesicles (OMVs) as they have been thought to derive the outer membrane structures of parental bacteria. 16 The outer membrane (OM) of gram-negative bacteria is located outside the peptidoglycan layer. The OM of gram-negative bacteria consists of two leaflets, with the inner leaflet composed of phospholipids and the outer leaflet composed of lipopolysaccharides (LPS), which is composed of lipid A and the O-antigen. The OM also contains a variety of proteins that can function as receptors, adhesins, and transporters. Gram-negative bacteria have the ability to produce OMVs, small spherical structures that are released from the bacterial surface and can contain a variety of components, including LPS, phospholipids, and proteins. 17 However, according to the specific vesicle release mechanism, there are several types of BEVs released by gram-negative bacteria. 18 Outer membrane vesicles (OMVs) OMVs are classical BEVs. Based on this structure, the OMVs formation depends on the "blebbing" of OM, which is double-coated with lipids to form nanovesicles and secrete out of bacteria. Thus, OMVs are considered to have specific bacterial secretion pathways. 19 All extracellular vesicles secreted by gram-negative bacteria can be referred to as OMVs.
Outer inner membrane vesicles (OIMVs) The discovery of OIMVs has challenged the traditional view of bacterial secretion pathways. OIMVs are produced by the explosive of bacteria, which is triggered by phage-derived endolysin. The endolysin degrades the peptidoglycan cell wall, causing the inner membrane to protrude into the periplasm and resulting in the formation of OIMVs. 20 The existence of OIMVs suggests that bacterial secretion pathways are more complex than previously thought. The phage-derived endolysin triggers explosive cell lysis by degrading the peptidoglycan cell wall, which enables intercellular materials to enter the MVs. The discovery of OIMV was proposed as a novel mechanism for explaining how DNA passes through the inner membrane and enters OMVs.
Explosive outer-membrane vesicles (EOMVs) The production of EOMVs is also triggered by explosive bacterial lysis mentioned above. 15 After the degradation of the peptidoglycan, the cell undergoes rounding and lysis, causing membrane fragments to break apart and selfassemble into EOMVs. However, unlike MVs formed by bacteria "blebbing", EOMVs are composed at random of cytoplasmic components like chromosomal DNA and endolysins, which can kill bacteria. 15

| BEVs originated from grampositive bacteria
Gram-positive bacteria secrete extracellular vesicles known as cytoplasmic membrane vesicles (CMVs). Recent research has shown that CMVs can also be produced by dying cells, 21,22 particularly during "bubbling cell death" and through the activity of bacterial autolysins. It is possible that there are additional, non-lethal related mechanisms for CMV production. 15 The classification of and a greater understanding of the biogenesis of different types may provide further insight into their F I G U R E 1 Schematic illustration of BEVs biogenesis.Various factors influence the production and composition of BEVs. In gramnegative bacteria, BEVs can be generated through outer membrane blebbing or explosive cell lysis. Outer membrane vesicles (OMVs) result from outer membrane blebbing, while explosive outer-inner membrane vesicles (EOMVs/OIMVs) occur when the inner membrane protrudes due to weaknesses in the peptidoglycan layer. In gram-positive bacteria, BEVs from through bubbling cell death caused by endolysin-mediated degradation of the peptidoglycan layer, releasing cytoplasmic membrane vesicles (CMVs). Reproduced with permission. 15 Copyright 2023, Springer Nature. WEN ET AL.
-3 of 33 potential as indicators of pathological microbial activity both in gram-positive and gram-negative bacteria. By conducting research on BEVs, scientists may gain valuable insights into their underlying pathogenesis and potential beneficial effects on humans.

| COMPONENTS AND FUNCTIONS OF BEVS
BEVs perform various functions depending on their components, including lipids, proteins, nucleic acids and metabolites. This section outlines some of the significant and noteworthy components that have been reliably identified. Generally speaking, the special bilayer lipid structure of BEVs produced by parental bacteria can effectively protect the molecular cargo within them from degradation, enabling BEVs highly biocompatible and to deliver cargo with biological activity. 15,22 While these functional components enable them to regulate the physiological activities of the host cells. 19 The participation of BEVs (biological extracellular vesicles) in various biological processes is complex due to their multicomponent cargo of active molecules, which can undergo compositional changes in response to different physiological and pathological conditions. Therefore, clarifying the composition of BEVs is important for understanding their functions ( Figure 2).

| Lipid
Membrane-derived lipids are a major component of BEVs. 24 Electron micrographs showed that BEVs are spherical lipid bilayers, which are likely to be formed by budding from the bacterial OM. The phospholipid layer assures good membrane fluidity and permeability of BEVs to maintain membrane metabolism and normal physiological functions. Earlier studies found that OMVs of E. coli have a phospholipid profile similar to that of bacterial OM. 24 Meanwhile, BEVs also contain other types of lipids to perform different functions, as well as changes in lipid components affect their stability. 25 OMVs from Pseudomonas aeruginosa contain an abundant of phosphatidylglycerol, which is absent in bacterial OM. 14 In contrast, OMVs from Helicobacter pylori primarily use cardiolipin as the main lipid component. 26 Moreover, BEVs have a higher proportion of stearic acid compared to OM, which increases their membrane rigidity. The number of saturated fatty acyl chains is also relatively higher in BEVs than in bacterial OM. 13 In addition, Pseudomonas aeruginosa OMVs contain an abundance of both phosphatidylglycerol and stearic acid, which demonstrate greater rigidity of MVs. 27 The presence of different lipids from parental bacterial inner membranes was also detected in OMVs, 28 suggesting that they may have specific functions. Overall, this passage highlights the diverse lipid compositions of OMVs and their potential functional implications. The lipid composition of CMVs can vary depending on the bacterial species, and this variability may be related to bacterial adaptation and survival in different ecological niches. For example, CMVs released by Listeria monocytogenes are enriched in phosphatidylethanolamine, sphingolipids, and triacylglycerols, while glycoglycerolipids are underrepresented. 29 Additionally, lipids containing unsaturated fatty acids are more abundant in Listeria monocytogenes-derived vesicles than in parental bacteria. These findings highlight the diverse lipid compositions of OMVs and CMVs and their potential biological functions, prompting further investigation into the lipid compositions of different BEVs.

| Protein
BEVs play an important role in bacteria-host communication, and their protein components are essential for their functions. BEV proteins can be classified into six groups based on their functions: structural proteins, porins, ion channels, transporters, enzymes, and proteins related to stress response. 30 Structural proteins in BEVs maintain the stability of the vesicle structure and protect the cargo inside. For example, Jang et al. identified 134 vesicular proteins from OMVs of Campylobacter jejuni NCTC11168, categorized by their biological function and predicted cellular location. The most enriched terms were related to energy generation, such as aerobic respiration and the tricarboxylic acid cycle, and the most abundant enzymes belonged to the oxidoreductase family.
Flagellum assembly proteins were also highly represented. 31 Enzymes, virulence factors, and toxins in BEVs contribute to the disease-causing process. For example, murein hydrolases are responsible for the hydrolysis of certain cell wall glycopeptides, particularly peptidoglycans. 32 Proteomics provides technical support for the protein composition of BEVs. Proteomic analysis of BEVs from gram-positive bacteria, such as Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, and Bacillus anthracis, identified both membrane and cytoplasmic proteins. [33][34][35][36] A comprehensive study of BEV proteins can reveal their roles in bacterial life activities, such as disease occurrence and progression, and can help identify disease-related proteins as biomarkers for early diagnosis and prognosis evaluation of diseases. Here, we summarize the research on BEV proteomics and point out their scientific implications (Table 1).
Therefore, a thorough understanding of the BEV protein composition can aid in the discovery of more potential clinical applications for BEVs.

| Nucleic acids
Recent studies showed that nucleic acids, including DNAs, RNAs, and small regulatory RNAs, can be packaged into BEVs. 43 These nucleic acids are shielded from extracellular nucleases and can be internalized by neighboring bacteria or host cells, thereby modulating gene expression and contributing to bacterial virulence and pathogenesis. Understanding the role of nucleic acids T A B L E 1 The scientific implications of BEVs protein identification. in bacterial EVs is therefore critical for unraveling the mechanisms of bacterial communication and pathogenesis, and may ultimately lead to the development of new therapeutics for combating bacterial infections. The structure of the BEV bilayer lipid membrane protects nucleic acids from nuclease hydrolysis and transports them to target cells to regulate their activities. 44,45 Most DNA in Pseudomonas aeruginosa OMVs is associated with specific functions such as antibiotic resistance, virulence, and stress responses. 46 In some pathogens, small RNAs secreted by BEVs mediate disruption of the host immune response. Furthermore, many of the transferred small noncoding RNA sequences found in OMVs align with regions of the human genome that are implicated in epigenetic mechanisms, such as chromatin remodeling and histone modifications, or tissue-specific transcriptional control. 47 BEVs also encompass diverse genetic information. OMVs are potential vectors for the spread of the antibiotic resistance gene bla CTX-M- 15 in Enterobacteriaceae 48 Han et al. 49 demonstrated a novel mechanism for host gene regulation by miRNAs originating from OMVs of the periodontal pathogen Aggregatibacter actinomycetemcomitans. However, DNA and RNA sorting mechanisms are complex and have not yet been elucidated. Most current studies focus on the overall rather than the single particle level, which may miss some information. Thus, to better understand the functions of BEVs, a multifaceted investigation is required.

| Bacterial metabolites and other molecules
In addition to the lipids, proteins, and nucleic acids mentioned above, BEVs are also capable of transporting bacterial metabolites and other molecules. Metabonomics showed that BEVs contain a variety of parental bacterial metabolites. Differences in metabolite content between pathogenic and common strains of Bacteroides thetaiotaomicron are consistent with their ability to colonize and survive in the gut. 46 Metabolites packaged in OMVs can act as adaptation factors, facilitating bacterial survival in specific ecological niches. For instance, the antimicrobial peptide BSAP found in OMVs from Bacteroides fragilis has inhibitory activity against other Bacteroides in the human gut. 45 Recent studies have shown that Pseudomonas quinolone signal (PQS) is closely related to OMVs. 50 PQS also induced substantial release of MVs in all strains. 51 These OMVs can interact with other cells and release PQS into the surrounding environment. In this way, PQS can influence the behavior of other bacteria and encourage them to form complex structures such as biofilms. BEVs are known to transport a range of virulence factors, including toxins, adhesins, and immunomodulatory molecules. The transfer of these virulence factors through BEVs has been shown to significantly impact their interactions with host cells as evidenced by numerous studies. 52 OMVs secreted by enterohemorrhagic E. coli (EHEC) are believed to be a sophisticated secretion mechanism for simultaneous, coordinated, and direct delivery of bacterial virulence factors into host cells, making them potent virulence tools for these pathogens. 53 Studies have identified OMVs released by Shiga toxin-producing E. coli (STEC) as novel and effective virulence factors for these pathogens. 54,55 In gram-positive bacteria, Clostridium perfringens CMV transport genes encoding bacterial toxins, including αtoxin (plc) and perfringolysin O (pfoA). 56 Recent studies have shown that the gut microbiota have an impact on the gut-brain axis, 57 and BEVs may play a crucial role in the process of bacterial penetration across the bloodbrain barrier. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology, Wei et al. 58 discovered significant differences between the OMVs extracted from feces of Alzheimer's Disease (AD) patients and those of healthy controls. They found that fecal OMVs from AD patients exhibited significant enrichment of aspartate, L-aspartate, imidazole-4-acetic acid and Lglutamate. This finding suggests that BEVs may play an important role in the pathogenesis and development of AD and may be related to the pathological mechanism of the disease.
Hence, exploration and differentiation of the multiple components of BEVs will help to block the pathogenic bacterial infection, which is critical for understanding the disease pathogenesis and carrying out further targeted interventions.

CLINICAL APPLICATION
Bacteria are distributed in the human body, mainly at the skin barrier, the intestinal screen as well as in the respiratory tract, and under normal conditions, these probiotic and nonprobiotic bacteria mutualistically co-exist to achieve a balance of the microbiota.
Abnormal states or when the above barriers are damaged, the balance of flora is disrupted, and bacteria may affect our immune system and vasculature, leading to the occurrence and development of diseases.
As materials released by bacteria, BEVs play an important role in inter-and intra-bacterial communication with the body. Consequently, BEVs hold significant potential in disease diagnosis and treatment.

| BEVs serve as disease diagnostic markers
Bacterial infection remains a significant public health concern worldwide, and early detection and diagnosis are crucial for effective treatment and prevention of disease spread. Currently, the methods for diagnosing bacterial infections are highly diverse. The traditional "gold standard" in microbiological testing involves bacterial culture and drug sensitivity analysis, while the application of nucleic acid technology has also improved the accuracy and speed of bacterial detection. 59 However, both the "gold standard" and bacterial nucleic acid testing have certain limitations, such as longer processing times and a higher risk of contamination. To overcome these challenges, it is necessary to seek more refined diagnostic strategies. Increasing evidence suggests that BEVs have significant potential as diagnostic biomarkers for various bacterial infections. These tiny vesicles contain biomolecules that can provide valuable information about the bacteria and their host environment, enabling researchers to detect early signs of infection and identify the specific type of bacteria responsible for the illness. 60,61 Furthermore, BEVs not only contain bacterial DNA and RNA but also carry a variety of biomolecules such as lipids, proteins, and metabolites. In contrast, nucleic acid detection primarily focuses on the detection of DNA and RNA. The diverse analytical objectives present in BEVs expand their potential applications in the diagnosis of bacterial infection. In addition to bacterial infections, BEVs may hold diagnostic value for other conditions, such as cancer, neurodegenerative disorders, and autoimmune diseases. This groundbreaking research has the potential to transform the field of disease diagnosis and treatment, offering new tools to improve patient outcomes and enhance public health.
(1) Research basis The structure of lipid bilayer vesicles facilitates BEVs to transport substances across cell membranes and even biological barriers. 18 In the early stages of the disease (even before the patients develop clinical symptoms), BEVs are enabled to spread into the circulation with cargoes derived from parental bacteria. 55 BEVs can provide insights into the virulence and pathogenicity of bacteria. They can carry virulence factors, toxins, and other molecules that may contribute to novel markers, which can aid in the development of new diagnostic and therapeutic strategies. A study has used the Cre/LoxP system to modify E. coli, which can produce BEVs with red fluorescence. Their results showed that BEVs secreted by the modified E. coli were absorbed and ingested by single cells in the intestinal tract, liver, spleen, heart and kidney as well as immune cells after the modified E. coli was colonized in the intestinal tract of mice. Even individual nerve cells in the brain emit a red light signal. 62 These observations inspire us to explore BEVs to develop novel biomarkers for disease diagnosis.
(2) Novel techniques The investigation of BEVs has the potential to offer valuable insights into the pathogenesis of bacterial infection and facilitate the development of novel diagnostic and therapeutic approaches. Common techniques for detecting BEVs include transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting (WB). However, these technologies are difficult to achieve precise analysis of BEV, which is not conducive to clinical testing. In recent years, there has been a growing interest in developing rapid and sensitive methods for the detection of BEVs, which hold great promise for improving the diagnosis and treatment of bacterial infections. A recent study reported the development of BEV bioimaging with superior performance. The authors utilized aggregation-induced emission (AIE) to selectively label BEVs with enhanced fluorescence, resulting in a high signal-to-background ratio. This innovative approach provides a valuable tool for investigating the functions of BEVs in both health and disease contexts. 63 The promotion of BEV detection technology makes it possible to develop BEV biomarkers. Tony Hu and colleagues at Tulane University developed a nanoparticle surface plasmon resonance-enhanced immunoassay to detect LAM and LprG, two virulence factors that coexist on extracellular vesicles (EVs) secreted by Mtb-infected macrophages. This new diagnostic approach offers insights into the diagnosis of BEVs. 64 In recent years, new technologies, such as EV-FISHER, 65 aptamer-based methods, 66 and droplet-based single-exosome-counting, 67 have enabled the development of diagnostic BEVs.

(3) Previous study on diagnostic BEVs
BEVs are expected to be new targets for disease diagnosis. Early detection using biomarkers of salivary BEVs may facilitate timely prevention. Research found that LPS + BEVs, global 5 mC methylation and BEVs secreted by four periodontal pathogens (Tannerella denticola, Eikenella corrodens, Porphyromonas gingivalis, and Fusobacterium nucleatum) were significantly increased in the saliva of periodontitis patients compared with healthy individuals. 68 Another study developed a rapid and costeffective protocol for isolating BEV-associated DNA, which was then used for 16S rRNA gene sequencing to identify bacterial origins of the blood microbiome in both healthy individuals and patients with inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis. This approach provides a basis for conducting larger studies to determine the potential use of blood microbiota profiling as a diagnostic tool for IBD. 69 Furthermore, BEVs can also be applied in tumor potentially. Kim et al. isolated EVs derived from microbes present in serum samples and analyzed the metagenomic profiles of 166 ovarian cancers and 76 benign ovarian tumors. The study also investigated the relative abundance of specific microbiomes at the genus level in combination with patient age and serum CA-125 levels. A new diagnostic model to differentiate between benign and malignant ovarian cancer was successfully developed, which exhibited better diagnostic performance than models without microbe biomarkers. 70 Poore et al. 71 used artificial intelligence to analyze the gene fragments from microorganisms in The Cancer Genome Atlas (TCGA) and discovered a unique microbial signal which was displayed in the blood and tissue samples of most cancer patients. They focused on comparing bloodderived microbial DNA (mbDNA) with circulating tumor DNA (ctDNA) that is being used in the clinic and found that even low-grade tumors that cannot be detected by ctDNA or tumors without any genomic changes can be well identified by mbDNA. Notably, it can also distinguish different types of cancer. 71 These findings indicate that the aforementioned DNA were largely identified to originate from BEVs, and they can serve as new tools for cancer diagnosis, improving cancer detection, and achieving a major breakthrough in cancer liquid biopsy. Here we list some examples about BEVs as disease biomarkers ( Table 2).

| BEVs serve as therapeutic agents
BEVs have nanosized structures, low toxicity and good biocompatibility and always act with target cells in a variety of ways to produce therapeutic effects. BEVs have been shown to be critically involved in the treatment of several diseases. In this position paper, we focused on the use of BEVs in the aspects of natural and engineered BEVs.

| Natural BEVs
Natural bacterial extracellular vesicles (nBEVs) are small, spherical structures that are surrounded by a lipid bilayer and contain a variety of biomolecules, including proteins, lipids, and nucleic acids. They are produced by various bacteria and play a crucial role in intercellular communication as well as the delivery of virulence factors.
nBEVs play a role in bacterial communication and can transport cargo between bacterial cells as well as between bacteria and their host organisms. They have been found to be involved in a variety of processes, including biofilm formation, pathogenesis, and virulence. In addition to being closely related to bacteria, the role of BEVs in human diseases is gaining attention. BEVs help combat virus infection by activating the host immune system. OMVs derived from E. coli can activate TLR responses in macrophages, resulting in the production of IFN-α, IFN-β, IL-1β, and MCP-1. Furthermore, these OMVs can trigger antiviral responses through a type I IFN-dependent mechanism. After being given E. coli OMVs for 3 days, the mice were resistant to multi-strain viruses, such as pandemic H1N1, PR8, H5N2, and highly pathogenic H5N1 viruses. 76 Furthermore, OMVs derived from Lactobacillus species such as Lactobacillus crispatus or Lactobacillus gasseri have been shown to restrict HIV-1 replication in human cervicovaginal cells, indicating their potential as a means of modulating antiviral immunity. 77 In addition, the regulation of the immune system by nBEVs can also provide new strategies for disease treatment. Fusobacterium nucleatum OMVs contain the virulence determinant FadA that translocates into the joints, T A B L E 2 BEVs as novel disease biomarkers.

Classification Disease Source for BEVs Analysis Reference
Oral cavity Periodontitis Saliva DNA methylation 68 Reproductive system Ovarian cancer Serum Metabolites 68 Digestive system Intestinal barrier dysfunction Plasma, feces LPS 72 Colorectal cancer Feces Metabolites 73 Nervous system Alzheimers disease Feces, urine Metabolites 58 Immune system Atopic dermatitis Serum Metagenomic alternations 74 Respiratory system Lung diseases Serum Detection of OMV antibodies 75

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triggering local inflammatory responses. The results indicate that Fusobacterium nucleatum OMVs play a causal role in exacerbating rheumatoid arthritis (RA) and offer potential therapeutic targets for improving the clinical outcomes of RA. 10 Another study found that Akkermansia muciniphila-derived extracellular vesicles (AmEVs) were transported from the gastrointestinal (GI) tract to the placenta and alleviated pre-eclamptic symptoms in mice with Preeclampsia (PE). These findings unveiled the potential therapeutic benefits of utilizing AmEVs for treating PE and emphasized the significant interactions between the host and the microbiota. 78 Here we summarize the potential applications of nBEVs (Table 3). However, the efficacy, stability and bio-safety of nBEVs should not be ignored. For instance, nBEVs may cause inflammation and immune responses, which may lead to cell death, tissue damage and the occurrence of diseases. Moverover, nBEVs are known to transfer antibiotic resistance genes, 85 which may lead to bacterial resistance to antibiotics and thus make infections more difficult to treat. Therefore, the use of nBEVs in disease treatment still needs further investigation.

| Engineered BEVs
Due to their complicated components and high heterogeneity, nBEVs may have limited therapeutic efficacy and pose safety concerns when used for delivering therapeutic cargos. Additionally, challenges, such as inefficient isolation and scale-up production of EVs as well as accurate monitoring of the dosage of therapeutic cargos in EVs, may hinder their clinical translation. 86 To address these issues, there has been increasing interest in modifying, engineering, or designing BEVs to enhance their efficiency, specificity, and safety. 87 Engineered BEVs have emerged as a promising alternative approach for EV-based therapy.
(1) Approaches for loading cargos into BEVs A primary issue for engineered BEVs is to improve the biosafety. nBEVs can contain adjuvants or antigens that may lead to serious side effects such as septic shock in some cases, which could potentially reduce the therapeutic efficacy for bacterial infections. 88 For these reasons, some modifications are required for improving the biosafety of BEVs. Furthermore, engineered BEVs may be an effective strategy to improve efficacy and reduce sideeffects. For example, BEVs contain the bacteriotoxin-like LPS, which can provoke uncontrolled adverse responses like inflammation in the body. 89 Although nBEVs can cause adverse effects like septic shock due to their adjuvants or antigens, engineered BEVs obtained after specific modifications no longer exhibit these toxicities. This offers the potential for safe delivery of therapeutic agents in vivo while also maintaining the protein activity and enhancing the target ability and therapeutic effect of the BEVs. Furthermore, engineering techniques can be used to amplify and control the bactericidal ability of BEVs, and the underlying mechanisms will be further understood in future studies. 79,90 We summarize the approches for uploading engineered BEVs in Table 4.
(2) Engineered BEVs for therapeutic application Due to their nanosized structures, low toxicity, drug loading capacity, and good biocompatibility, BEVs are a promising platform for the treatment of various diseases (Table 5). BEVs drive an immunostimulatory state in tumor microenvironment (TME). OMVs naturally T A B L E 3 The applications of different types of nBEVs. released by gram-negative bacteria from Klebsiella pneumoniae are used to prepare doxorubicin-loaded OMVs (DOX-OMVs) due to their proper immunogenicity. Affected by DOX-OMVs, macrophages are recruited in the tumor microenvironment and perform a synergistic antitumor effect with the OMVs. 98 The biofilm composite nano-drug delivery system (OMVs-MSN-5-FU) has also been confirmed to have good anti-tumor effect in lymph node metastasis from oral squamous cell carcinoma. 99 In a recent study, BEVs were used to co-load paclitaxel (PTX) and regulate the expression of DNA damage response 1 (Redd1)-siRNA, thereby suppressing tumor growth and modulating the tumor metabolism microenvironment. The siRNA@M-/PTX-CA-OMVs developed in this study were able to release PTX in response to the acidic tumor microenvironment (pH 6.8) in a triplenegative breast cancer model. 100 Scientists used OMVs as immunostimulants to reprogram TME by coating the surface with pH-sensitive CaP shells that integrate with functional compounds such as folic acid or photosensitizers for synergistic therapeutic effects in combination therapy. 97 BEVs can also improve the effective response rate of immune checkpoint inhibitors (ICI) therapy. Immunosuppressive TMEs take responsibility for the failure of ICI treatment. BEVs as TME activated participants can effectively improve the effect of ICI treatment.

Source for BEVs Indications Reference
Researchers created a LyP1 polypeptide-modified outer membrane vesicle (LOMV) that carried a PD-1 plasmid. Through OMV's targeting capability, this nano-carrier accumulated in tumor tissue. The outer membrane protein of LOMV recruited cytotoxic lymph cells and natural killer cells to the tumor tissue, stimulating them to secrete IFN-γ, thus enhancing the antitumor activity of PD-1/PD-L1 self-blocking therapy. 101 Bacterial infection is a common clinical disease that can cause severe symptoms. Antibiotics have an important position in clinical antibacterial therapy. However, the misuse of antibiotics has led to serious drug resistance. Also, due to the low drug permeability of the host cell membrane, it is difficult for many antibiotics to treat intracellular infection. BEVs carry a variety of antimicrobial substances and work in intracellular and extracellular infections. 11 BEVs as antibiotic delivery may provide new strategies for dealing with antibioticresistant organisms (Figure 3). Some studies have designed novel antibiotic-loaded OMVs by exploiting a resistance mechanism mediated and demonstrated that they could efficiently enter and kill pathogenic bacteria in vitro. Low-dose oral administration of OMVs containing antibiotics were given to the mice of intestinal bacterial infection, but no systemic drug spread was observed after administration in 12 h, which hints at the great potential of OMVs in the use of antibiotics in infectious diseases. 11 In addition, engineered BEVs are also used in the development of disease vaccines, which will be detailed in section 4.3. (

3) Challenges
Despite the promising therapeutic applications of engineered BEVs, several barriers remain before their clinical translation. Safety is the foremost challenge to overcome before BEVs can be used in humans, given that their effectiveness and potential side effects are still unclear in human studies. Additionally, the absence of standardized production and isolation methods presents another obstacle to the clinical development of BEVs. Different production and isolation techniques can cause significant heterogeneity, which hamper the reproducibility and repeatability of results. 87 Despite the ongoing challenges, continued research on engineered BEVs will eventually yield innovative solutions for clinical translation.

| BEVs serve as preventive care
Prevention plays a crucial role in healthcare, alongside disease diagnosis and treatment. Vaccination is an important part of preventive care as it is a cost-effective and efficient way to prevent diseases. However, developing safe and effective vaccine is a challenging task. It is found that bacterial substances present in BEVs can effectively regulate the activities of different immune cells, highlighting their potential in disease prevention. BEVs derived from E. coli Nissle 1917 (EcN) and commensal E. coli strains activate DCs to promote strainspecific differentiation of T cell subsets, driving complex Th responses including Th1, Th2, Th17, Th22, and Treg, which provide immunity to extracellular parasites, protective tissue responses, protective immunity against pathogens, antimicrobial responses, and immune F I G U R E 3 Design of antibiotic-loaded BEVs for intestinal bacterial infection. A novel group of antibiotic-loaded bacterial outer membrane vesicles, known as antibiotic-OMVs, have been discovered to provide protection against intestinal bacterial infections. Reproduced with permission. 11 Copyright 2023, Elsevier. tolerance, respectively. Furthermore, probiotics from the genus Bifidobacterium can reduce allergic reactions in addition to their protective effects against colitis. Specifically, the probiotic B. bifidum LMG13195 elicits tolerance responses. 102 Thus, BEV is a novel immunization strategy that has great potential for the development of vaccines.
(1) Vaccines for cancer BEVs are candidates for vaccine development because they carry a large number of bacterial virulence-related proteins, but without the ability to replicate. Tumor vaccine is required to awaken the host immune system and produce specific immune protection. Notably, BEVs just meet all requirements above by containing a large number of pathogen-associated molecular patterns (PAMPs), 101,103 which can be recognized and phagocytosed by immune cells to promote the activation and maturation of other cells. 104 Figure 4 illustrates the immune response involving BEVs. BEVs can be fused with tumor cell membranes (TCMs) to wrap polymer PLGA to develop a vesicle cancer nanovaccine named BTs129. With bacterial-derived PAMPs, BTs129 can be used as adjuvants to enhance the targeting of DCs. Meanwhile, TCMs in BTs collect a variety of tumorspecific antigens from the tumor cell membrane to create a diverse T-cell repertoire. This is achieved by amplifying the processing and presentation of these antigens, which are necessary for inducing the proliferation and activation of antigen-specific CD8 + T cells. [105][106][107] Another advantage of the BEV vaccine is that it penetrates the cell membrane smoothly without vesicle structure damage. Vipul Gujrati and his colleagues 108 isolated OMVs derived from genetically ΔmsbB mutant E. coli (strain K-12W3110) with low endotoxicity and immunogenicity. They selected human epidermal growth factor receptor 2 (HER2) as the receptor for tumor cells and loaded siRNA to target gene silencing of kinesin spindle protein (KSP), which regulates mitosis, to synthesize Affi HER2 OMVsiRNA vaccine to target tumor tissue apoptosis. The Affi HER2 OMVsiRNA was taken up by HER2-overexpressing tumor cells through receptormediated endocytosis, and then slowly degraded in a specific acidic environment to release free siRNA, thereby reducing the non-specific killing effect of siRNA on the normal tissue cells.
Individualized BEV tumor vaccines are on the horizon. The tumor antigen is presented on the outer surface of OMVs by fusing it with the ClyA protein. A Plug-and-Display system, consisting of a tag/capture protein pair, simplifies the antigen presentation process. OMVs can simultaneously display multiple tumor antigens decorated with different protein traps to provoke a synergistic antitumor immune response. Furthermore, bioengineered OMVs carrying various tumor antigens have demonstrated the ability to eliminate lung melanoma metastasis and inhibit subcutaneous colorectal cancer growth. The rapid and simultaneous antigen display capabilities of bioengineered BEV-based platforms may help develop personalized tumor vaccines. 110 Moreover, BEVs serve as a delivery platform for mRNA through genetic engineering using surface decoration with RNA-binding protein L7Ae and lysosomal escape protein listeriolysin O (OMV-LL). This approach provides an alternative mRNA delivery technology to lipid nanoparticles (LNPs) for personalized tumor vaccination. The versatile "Plug-and-Display" strategy enables this platform's application in various mRNA vaccines. 111 (2) Vaccines for infectious diseases presented. 113 Another study showed that immunization with V. cholerae or ETEC OMVs induced a speciesspecific immune response. However, combining both OMV species resulted in a high-titer protective immune response against both pathogens. In another study, OMVs from Salmonella with high levels of antigens displayed on their surface were administered intranasally as vaccine, effectively inducing strong protection against pneumococcal colonization in a murine model without requiring a mucosal adjuvant. 114,115 A recent study introduced a novel SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium, decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD), showcasing the value and versatility of OMV-based vaccines. 116 BEVs are a promising vaccine delivery system due to their selfadjuvant properties and ability to be decorated with antigens. The study also demonstrated an effective method for displaying SARS-CoV-2 antigens both in the lumen and on the surface of the same OMV, highlighting the potential of OMVs as general multi-antigen carriers. 117 However, the presence of OMV-specific IgG antibodies may induce antibody-dependent immune clearance, thereby reducing the antigen uptake of APCs and weakening the subsequent immune response. On the other hand, the existence of highly reactive components of OMVs, such as LPS, may weaken the effectiveness of the specific immune response to the target antigen. In addition, the selection of tumor antigens is also a complicated process, which requires more research to verify. In the future, tumor vaccines based on BEVs have a broad application prospect in becoming a safe and effective option for disease prevention.

| Nomenclature
BEVs are commonly used to name the bilayer lipid vesicles released by bacteria, which have no proliferative ability but high bioactivity similar to parental bacteria. 3 However, there is no nomenclature rule for BEVs yet. Currently, BEVs are mainly named after their parental bacteria 21 : (1) BEVs from gram-negative bacteria are named outer membrane vesicles (OMVs); (2) BEVs from gram-positive bacteria are named cytoplasm membrane vesicles (CMVs). But more accurate nomenclature of BEVs is urgently needed, which will be beneficial to facilitate further research on BEVs detection and its application in disease diagnosis and treatment. With the growing sophistication of techniques like TEM, the ways that BEVs release from gram-negative bacteria have been captured, and according to the different ways, these BEVs can be divided into OMVs, explosive outer membrane vesicles (EOMVs), and outer inner membrane vesicles (OIMVs). 15 In addition to the study of nBEVs, there is growing interest in engineered BEVs. Engineered BEVs are usually modified with certain molecules to enhance their effects. 118 Here, we recommend that the name of such BEVs should reflect two points: (1) the source of parental bacteria and (2) the engineered substances. Here we give some naming examples (Table 5). If the use of newly coined terms is considered inevitable, the definition should be highlighted at the beginning of the article.

| Bacterial culture medium
The bacteria culture conditions are varied for different bacteria, but are generally as follows. According to the bacterial characteristics, the medium conducive to bacterial growth was selected, and at the suitable growth temperature of bacteria, the bacteria were cultured into the logarithmic growth phase, the OD600 value was detected, and the bacterial pretreatment experiment was carried out when it was located at 0.6-0.8. The bacterial supernatant will be isolated by a series of centrifugation for BEVs. In short, cell-free culture supernatant was collected and followed by sequential centrifugation at 5000 g (15 min, 4°C) and 10,000 g (30 min, 4°C). To ensure the removal of the remaining bacteria, the supernatant was afterward filtered through a 0.45 μm membrane filter and transferred to a new bottle. 120,121 However, it should be pointed out that, according to previous studies, there may be differences in the cargo and concentration of particles in the BEVs produced by repeatedly cultured strains and the original strains, leading to variations in the biological functions they exert. Therefore, when culturing strains in expanded cultures, attention should be paid to contamination, the conditions under which the strains are cultured and the number of generations of the strains. It is essential to determine the appropriate time to harvest based on how the crop grows and how vital the crop appears. BEVs should be isolated during the logarithmic growth phase of the strain and the first generation of strains should be used. 120 But, BEVs purified from Pseudomonas aeruginosa from the stationary phase had a higher particle concentration than those purified from the exponential growth phase, and the same phenomenon was observed with Francisella novicida. 122,123 The dynamic levels of BEVs indicate that their biological functions and active levels may change with their growth status. Prior to the comparison of the protein composition of BEVs from different culture conditions or different BEV strains, the growth curve should be determined. BEVs, even if they require different incubation times to reach optimum optical density, should then be harvested at the same growth stage. Protein or lipid concentrations should be quantified and normalised to the bacterial count in order to compare BEV production at different harvest times. Also, during the initial isolation of supernatant and strain by differential centrifugation, the centrifugation speed should be chosen to adapt to the bacteria since excessively high speed may cause the bacterium to rupture, releasing the contents and resulting in impure isolation of EVs. 23 Another continuing concern is how to yield BEVs. Previous studies have shown that under pressure selection, bacteria increase BEV production in response to environmental changes. [124][125][126][127] Specific pressure choices are as follows: (1) Iron depletion: Iron deficiency in the medium can lead to increased production of BEVs in some strains, accompanied by increased levels of BEV proteins, such as increased expression of OMV-associated virulence factors and changes in the composition of the LPS and BEV envelope in Helicobacter pylori. Under low iron/hemoglobin conditions, high levels of proteins involved in hemoglobin acquisition and biofilm formation were observed in BEVs of Porphyromonas gingivalis. 128,129 (2) Oxidative stress: Increased secretion of BEVs has been observed when Pseudomonas aeruginosa is exposed to hydrogen peroxide. Similarly, the intracellular signal for increased vesicle formation in Neisseria meningitidis can be mimicked by cysteine depletion. Large numbers of BEVs are currently being produced to guide vaccine development using this approach. 7,130 (3) Temperature stress: Pseudomonas putida increased BEV production after being challenged by heat shock at 55°C. In Serratia marcescens, the biological process of BEVs was found to be a thermoregulatory process, but with an opposite trend, that is, increased BEV production at low temperatures (22°C and 30°C), but least production at high temperatures (37°C). 131,132 (4) Other methods of vesicle induction: The use of Hypertonic Shock (2 M NaCl), Ionic chelators (ethylenediamine-tetraacetic acid, EDTA) and Organically Solvents (1-octanol) can enhance the yield of BEVs from Pseudomonas putida. The cell wall biosynthesis blocker d-cycloserine and the OM-targeted antimicrobial peptide polymyxin B resulted in increased secretion of BEVs by Pseudomonas aeruginosa. However, when interfering with BEV production, it should be noted that these compounds may lead to the destruction of BEVs. 7,131 Additionally, the incremental BEV production can be achieved by genetic manipulation of the parent strain, where the strain's BEV production can be regulated by modifying the parental strain's genes to make gene deletions or mutations. 9,132-134

| Biological fluids
BEVs are present in biological fluids including "healthy" blood traditionally considered as a "germ-free" niche. 135,136 Each biological fluid presents specific biophysical and chemical characteristics that contribute to the complexity of BEV separation. Methods used for the collection and pre-processing of body fluids (blood, urine, saliva, nasal secretions, breast milk, bronchoalveolar lavage fluid, bile, etc.) may largely impact bacterial richness, and the recovery of BEVs, for subsequent omics and functional analyses. It is promising to standardize if the whole process could be managed and unified. As specific guidelines covering all BEV-dedicated research have not been established, it is recommended to follow the general recommendations related to the collection and pre-processing of the biological fluids by the authorities including the Clinical Laboratories and Standard Institute (CLSI) (H21-A5; H03-A6; GP16-A2). In addition, the position papers by the International Society of Extracellular Vesicles (ISEV) for eukaryotic cell-derived extracellular vesicles (EEVs) 121 could be consulted due to the similarity in structure, the proximity in size and density between EEVs and BEVs. Furthermore, the key point to note is full aseptic operation, ensuring the subject researched is BEVs rather than bacteria. 137,138 In the pre-processing phase, 139 viscosity, volume, 140 and pattern optimization [141][142][143][144][145][146] are critical factors needed to pay attention to, each of which determines the dilution multiple of the sample, preference to appropriate centrifugal rotor or other enrichment equipment, and removal efficiency of contaminating components, respectively, and further affect purity and recovery of BEVs after isolating. Standardized and optimized preprocessing methods for BEV isolation should therefore be carefully determined for each of the biological fluids separately. Moreover, filtering through 0.22 μm membrane is recommended to remove residual bacteria. However, it can be challenging to eliminate EEVs during pre-processing, which underscores the need for better isolation strategies for BEVs.
The proposed checklists for sample collection and pre-processing of the specific specimens are provided in Tables 6 and 7, respectively. 14 of 33 -WEN ET AL.

| Feces
Like other biofluids mentioned above, standardized operating procedures for stool collection, transport, preprocessing and storage for BEV isolation should be developed as the gut is the main habitat for the microbial community. 161 Depending on the time (within 4 h or 24 h) the feces can be handled in the biology laboratory, we should follow the different International Human Microbiome Standards (IHMS) Consortium-quality procedures for sample collection (http://www.microbiomestandards.org/). Sampling kits could be evaluated by combining the subject preference and previous or pre-experimental BEV analysis results. 162 After transportation to the laboratory, the stool can be aliquoted or dissolved in solution, and then frozen at −80°C. 163 If you do the latter, selecting the sterile buffer (e.g., phosphate buffer saline) to dissolve the stool is key in preprocessing. The dilution ratio is at least w/v (g/ mL) = 1/10 to prevent oversaturation. 164,165 Furthermore, shaking for a few hours or soaking at 4°C for longer until the feces dissolve completely is both feasible, but it needs to be documented in detail. The appropriate shaking frequency and intensity should be assessed to prevent bacteria from breaking, and interfere with BEV analysis. Carefully recording the centrifugation time, force and temperature is truly essential to provide the comparability of studies. The parameters may be adjusted according to the needs of the experiment, 73,163-167 but it is worth exploring the effect of different parameters on the recovery and purity of BEVs isolation in processing. During the whole process, we should pay attention to personal protection (biosafety cabinet, protective clothing, mask, and gloves) and keep the fully dissolved sample on ice. We really look forward to establish a standardized protocol for increased accuracy, repeatability and stability.

| Tissues
Tissue isolation of BEVs has rarely been studied, with most studies focusing on isolating total tissue EVs, but there is a need for isolation of BEVs in infected traumatic T A B L E 6 Considerations in sample collection.

Parameters What to report Recommendations References
Personal information -Age; -Gender; -Race; -Physical examination (e.g., height, weight, blood pressure); -Clinical data, including laboratory indicators and imaging parameters; -Lifestyle habits (e.g., smoking, drinking, diet, physical activity and rest pattern) -The project conducted in accordance with international principles and regulations on ethics in biomedical research should be respected the personal privacy. -Record clinical or laboratory information for subsequent normalization of dynamic results such as BEV yield, and exploration of spatial and temporal heterogeneity in organism. 147 Sample collection -Container (e.g., type of blood collection tube, material and sealing degree); -Time (e.g., morning, random, spot, 24 h for urine); -Type (e.g., venous, fingertip for blood); -Specimen status (e.g., viscosity, hemolysis, lipid turbidity, microbial contamination); -Volume; -Chemical substances (e.g., protease inhibitors or preservatives for urine, anticoagulant for plasma) -Perform in accordance with the standard operating procedures (SOPs), and sterility must be maximized. -Biosafety measures need to strictly enforce on the basis of recommendations by U. S. Department of health and human services (HHS). -Sample properties, such as viscosity, are extremely critical, influencing bacteria richness and BEV recovery. 148 Sample transport -Manner (e.g., insulated medical shipping box, upright or lying flat on ice, protecting from light); -Temperature (e.g., room temperature, 2-10°C); -Time from sample collection -Keep the samples at 4°C immediately after collection and transport on ice for stability of constituents. -Excessive agitation of the specimens, such as blood, must be avoided to minimize the changes in composition (hemolysis). 138 wounds and in tumor tissue where microorganisms are present. Briefly, after isolating the tissue pieces, they were cut into 2-mm pieces and then added to PBS containing 1% collagenase and digested overnight at 37°C. The next day, after the tissue had dissolved into a thin paste, it was ground and filtered, and a single cell suspension was obtained from tissue fragmentation using a gentle tissue fragmentation separator. Differential centrifugation (300 � g for 10 min, 5000 � g for 15 min, 10,000 � g for 30 min) removed cell debris to obtain an extracellular matrix suspension of the tissue, which was stored at −80°C or directly subjected to ultracentrifugation to separate the EVs. 60,[168][169][170] After the total EVs have been obtained, the BEVs should be further separated and purified using a specific marker or a specific method, which can be characterized in section 4.3.2.

| Storage
Upon arrival at the laboratory, it is preferable to carry out immediate isolation of BEVs as fresh specimens are best for BEV analysis. In cases where prompt processing is not possible, aliquoting samples after essential preprocessing and then freezing at −80°C for several weeks or months is recommended. It is important to assess the effect of different storage temperatures on the physicochemical properties of BEVs and minimize the number of freeze-thaw cycles. For isolated BEVs, storage in PBS at −80°C is recommended. Additionally, emerging techniques such as freeze-drying, spray-drying, and cryopreservation could be valid alternatives for improved structural integrity, stability and immunogenicity. 79,167 Furthermore, the T A B L E 7 Pre-processing of BEVs for biological fluid samples.

Sample type
Pre-processing Other biofluids -The samples not handled in time store at −80°C. -Frozen samples are thawed on ice, centrifuged at 200 � g for 10 min, 2, 000-3, 000 � g for 15 min, 10,000 � g for 30 min.
-Carefully evaluate the sample's properties, select the appropriate pre-processing methods (e.g., reductants are used to break down the mucus). The most commonly used approaches for the isolation of BEV populations include Differential centrifugation (Ultracentrifugation), Density gradient centrifugation (Ultracentrifugation), Ultrafiltration, Molecular-exclusion chromatography, Microfluidic technology, PEG precipitation and Immunomagnetic bead separation, Size exclusion chromatography (SEC) and Tangential flow filtration (TFF). Their specific operating procedures, strengths and precautions are shown in Table 8.

| Steps of BEV characterization
We mainly identify BEVs by size, concentration, morphology, density and composition.
(1) Concentration and Size The diameter of BEVs from gram-negative bacteria OMVs is about 20-250 nm, while BEVs from grampositive bacteria CMVs are about 20-400 nm. 8,15,181 The NTA and Dynamic Light Scattering technique (DLS) have been widely used to characterize the size and distribution of BEVs. NTA can quickly and accurately analyze particles at lower concentration levels and combine with the graph to draw intuitive conclusions. However, its specificity is poor and it is difficult to distinguish between BEVs, cell debris, and possible contaminants. 15,29,40,[182][183][184] DLS is easy to operate and prepare samples and can quickly measure particles with a lower limit of 1 nm but it can only measure relatively high concentrations of nanoparticles and has low specificity. In addition, there are many optical and non-optical methods available to analyze the size and concentration of nanoparticles. [182][183][184] Tunable resistive pulse sensing (TRPS) can calculate particle size and concentration by monitoring the current and velocity flowing through the adjustable nanopore. For larger BEVs (>150 nm), TRPS detected more BEVs than NTA, but the accuracy of the TRPS is not high enough and the specificity is low. 182 Raman scattering (RS) can analyze the molecular structure and chemical composition of particles by the scattered light generated when light strikes the material. 183,184 Flow cytometry (FCM) can use antibodies corresponding to specific markers on the particle surface to quantitatively analyze and classify particles. FCM has the characteristics of fast speed, high precision, high flux, low required sample concentration, good repeatability and good quantitative measurement ability. 183,184 However, when used to detect BEVs with small diameters and low refractive indexes, their accuracy and resolution are poor. BEVs can also be identified by electrophoretic mobility, fluorescence, membrane stiffness, and refractive index. Each of the above methods has its own advantages and disadvantages, so in fact, in the study of BEVs, it is often necessary to combine different methods for detection.
(2) Density The density of BEVs is greater than the EVs, and the density of EVs is generally at 1.083 g/mL~1.111 g/mL, while the density of BEVs is generally greater than 1.111 g/mL.
(3) Morphology Conventional light microscopes have difficulty resolving structures less than 200 nm in size. Therefore, various microscope techniques are used to observe the morphology and structure of BEVs. TEM is the most commonly used method to visualize BEVs with a resolution up to 0.1-0.2 nm. The internal structure and morphology of vesicles can be observed, and it can also be combined with immunocolloidal gold technology to obtain further molecular biological characteristics. The TEM images of BEVs we captured reveal spherical vesicles with clear membrane structures, exhibiting the classic saucer-like morphology ( Figure 5). These images provide visual evidence of the morphological characteristics of BEVs, confirming their presence and distinct shape.
However, the sample preparation of TEM is complex, the field of view is limited, the detection flux is low, and some chemical stains or coatings used in sample preparation may damage the vesicle structure. 184,185 Scanning electron microscopy (SEM) has simple sample preparation, a wide range of image magnification, high resolution, a rich three-dimensional sense, and can perform microcomponent analysis. However, the resolution of SEM may be lower than that of TEM. The heterogeneous EV population cannot be analyzed, the imaging conditions of polydispersed samples are harsh, the throughput is low, and cannot reflect the EVs morphology and biological components in living cells. 184,186 Cyro-electron WEN ET AL.

References
Immunomagnetic bead separation Due to their unique topology, BEVs are positively selected using special markers and negatively selected using nanospike depletion to remove environmental contaminants (protein precipitates, cellular debris). Size exclusion chromatography (SEC) 1. Choose an appropriate SEC column with the desired molecular weight resolution. Calibrate the column using a series of known molecular weight standards.
2. Collect the BEVs from the culture media or biological fluids and process the sample to remove cellular debris and other impurities.
3. Inject the prepared BEVs sample into the SEC column using an autosampler or manual loading.
4. Allow the buffer to flow through the column. Larger vesicles and aggregates will elute first as they pass through the column more quickly, while smaller vesicles will be retained in the column due to their size. This separates the vesicles based on their sizes.
5. Start collecting eluted solutions from the bottom of the column, which will contain the separated BEVs.

High integrity and biological activity of
EVs.
1. The process requires specialized equipment and packing materials.
2. There is a possibility of contamination by non-EVs particles. 179 Tangential flow filtration (TFF) 1. Assemble the TFF system, including the filtration membrane, pump, pressure sensors, and appropriate tubing. microscopy (cryo-EM) is also used to visualize the morphology and structure of BEVs, which has high resolution, fast detection speed, and no dehydration or chemical fixation damage in sample preparation. However, it has the disadvantages of low signal-to-noise ratio, high cost and low flux, and it is difficult to determine whether EVs are endocytosis or exocytosis. 184,187 Atomic force microscopy (AFM) samples require no special treatment, have no damage, high spatial resolution, and wide applicability. It can be used to characterize the dynamics of individual EVs and their interactions with biomolecules, such as stiffness and adhesion. But it also has the shortcomings of small imaging range, large influence of probe, high sample quality requirements, a complex detection process, and a slow detection speed. 141,184,186 In addition to characterization requirements, we need to conduct tracer labeling of BEVs when studying their role in physiological and pathological processes. Depending on the medium, BEV tracer labeling methods include fluorescent dyes, fluorescent proteins, luciferase and physical labeling methods, which can selectively label the outer membrane, inner membrane or internal components of exosomes. Among them, fluorescent dye labeling is a more commonly used method, 188 such as carbonyl cyanine dye, PKH dye and permeable dye.

(4) Biomarker
There are no universal biomarkers for microbial vesicles. EVs secreted by microorganisms contain proteins, nucleic acids, metabolites, LPS, and peptidoglycan. SDS-PAGE is often used to identify the expression  72 In addition, the content of LPS in BEVs can be calculated by colorimetry. 191 Combined with the above methods, the BEVs were analyzed comprehensively.

| Downstream analysis of BEV
EVs encapsulate virtually any biomolecular type found in the respective donor cells, namely: DNA, RNA, protein, lipid, or metabolite. 4 Variations in EV metabolites, such as sugars, amino acids, lipids, and nucleotides, may reflect the status of the bacteria, and thus analysis of the metabolic cargo may provide insight into bacterial interhost processes. Monitoring methods such as micronuclear magnetic resonance, small-angle X-ray scattering and anomalous SAXS can also be used to determine EVs based on study needs. Immunoblotting is a convenient way to identify EV proteins by evaluating the characteristic protein markers of EVs. In addition, measuring the amount of total proteins present in the EVs by the Bradford assay gives a rough idea of the number of EVs secreted by the bacteria. Monitoring methods such as atomic force microscopy, micronuclear magnetic resonance, small-angle X-ray scattering, anomalous SAXS, and resistance pulse sensing can also be used to determine EV based on the study needs.

| Nucleic acid
BEVs have several forms of nucleic acids, and RNA and DNA are contained in them.The most important nucleic acid component of BEVs is RNA. Since the first discovery of RNA in animal EVs, researchers have begun to delve into the RNAs of BEVs. The RNAs of BEVs have a variety of biological functions, particularly in regulating the host immune system. 192 For the RNAs of BEVs, the current common technical method is to perform high-resolution flow cytometric analysis of the RNA content of individual EVs using fluorescent RNA tracing dyes (e.g., acridine orange, etc.). In addition, RNAs in EVs can also be detected indirectly by microscopy, among which RNAbound fluorescent protein probes are the most widely used. Similar to RNA detection and analysis, nextgeneration sequencing (NGS), PCR and other technologies can also be used to analyze or verify EV-DNA content. 68 Typically, NGS and microarrays are primarily used for the analysis of nucleic acids in whole EVs. PCR, RT-qPCR, Digital PCR, Droplet PCR and RNA-binding fluorescent protein probes are commonly used for the detection of the specific DNAs or RNAs. Compared with PCR technology, isothermal amplification of nucleic acids, such as LAMP, RPA, and RCA, greatly simplifies the requirements of the instrument, greatly reduces the reaction time and can better meet the needs of rapid and simple. A study has reported a system for the amplification and detection of exosome miRNA based on the isothermal amplification technology. 193 In related studies, it was found that after adding DNase, about 80% of the DNA carried by the vesicles remained intact, indicating that the DNA eliminated by DNase is likely to be adsorbed on the OM of the vesicles from pathogens. The exclusion of nucleic acid interference from sources other than BEVs is a key point. If it is determined to be derived from plasmids, agarose electrophoresis can be attempted and the fragment of interest can be recovered by cutting the gel. However, how to exclude interference from host-derived nucleic acids is still difficult and requires further research. The common techniques for nucleic acid detection are listed in Table 9.

| Analysis of the BEV lipidome
According to previous reports, the lipid composition of BEVs is rich and diverse, and different lipids play different roles in life activities. In recent years, the main method of BEV lipid composition analysis is still mass spectrometry, including liquid chromatography-tandem mass spectrometry (MS/MS). However, problems such as degradation and destruction of lipids exist in the process of extraction, so the extraction of total lipids needs to follow standardized procedures. Due to the high diversity of lipid molecules, it is easy to produce the overlap of mass spectrum ions. In order to improve the resolution efficiency of lipids, a highresolution LC-MS/MS method is recommended. In the process of using the LC-MS/MS method, it is also necessary to pay attention to the internal recalibration of the scanning mass spectrum using known standards. 198

| Analysis of the BEV proteome
It has been proved that BEVs carry various kinds of proteins, which may be associated with bacterial markers, virulence, etc. As previously reported, the BEVs derived from Candida auris present immunogenic/drug resistance-implicated proteins, including alcohol dehydrogenase 1 as well as Candida albicans Mp65-like and Xog1-like proteins in high quantities. 199 Therefore, the identification of proteins carried in BEVs is particularly important for the study of pathogenic, genetic and other mechanisms of bacterial life activities as well as the early diagnosis and treatment of diseases. Although BEVs can be separated from other substances such as bacteria and proteins by using related separation techniques, a small number of extracapsular proteins and soluble proteins remain unavoidable. Even though technology is available to remove soluble precipitates, it is still not available for the analysis of the BEV proteome. There are many proteomic analysis techniques, but there is no clear recommendation on which technique is most suitable for BEV proteomic analysis. Based on literature research, we found that mass spectrometry is currently the most commonly used method for proteomic analysis of BEVs, including pathogenicity and immune response of pathogens, 200 antibiotic resistance of pathogens, 201 etc. In addition, other techniques are available for proteomic analysis of tumor-derived EVs, 202 and circular EVs, 203 such as Aptamer, Proximity extension assays (PEA), Low density array (LDA) analysis, and Immunoaffinity assays. These methods may also be applicable to BEVs proteomic analysis. Therefore, we have compiled the advantages and limitations of these methods in Table 10 as reference for readers.

| Metabolites and other molecules
BEVs contain metabolites and effector molecules that regulate target cell function.The specific metabolite type is related to the type of microorganism producing vesicles. BEVs play a pivotal role in the process of interaction between bacteria and host cells. In pathogens, BEVs carrie virulence factors and related toxins that assist bacteria in attacking host cells. 208 Metabolomics analysis of BEVs is often performed using liquid chromatographymass spectrometry/mass spectrometry (LC-MS/MS). The glycans on the surface of BEVs are diverse.We can analyze the glycomics using qualitative and quantitative methods of N-glycans.However, many metabolites of BEVs have different chemical properties, which means that no single method can simultaneously analyze all metabolites in BEVs.

| Bioinformatics and databases
With the explosive growth of BEV-related data, bioinformatics analysis of these multidimensional data has become critical. However, there is currently no dedicated BEV database.  121 When it comes to BEVs, it brings up a common question whether the activity is predominantly associated with BEVs or with co-isolated components. It can be settled by comparing the effects of the original microbial culture supernatant, BEVs isolated from the same volume of supernatant, and the same volume of supernatant removing BEVs on specific functions. At present, GW4869, cambinol and other drugs are often used to inhibit the production of neutral sphingomyelinase and ceramide, which reduce the secretion of mammalian extracellular vesicles. 138 However, their ability to inhibit BEVs is poor, and effective BEV inhibitors deserve further exploration. Here, we present more possible directions and suggestions that can be considered in functional studies of BEVs.

| BEVs and occurrence of diseases
BEVs play an important role in the process of various diseases because they carry proteins, nucleic acids, lipopolysaccharides and other substances of parental bacteria and have significant advantages of stable existence in the circulation, which can mediate the interaction between bacteria and bacteria, and can also participate in remote communication between bacteria and hosts. 213 In terms of bacteria-to-bacteria interaction, under antibiotic pressure, bacteria can exert a defensive function and protect themselves by secreting BEVs, binding antibiotics or hydrolyzing antibiotics. 214,215 In addition, BEVs can mediate the transmission of virulence factors and horizontal gene transfer between the same bacteria and different species of bacteria, transforming bacteria from low-virulence strains to high-virulence strains, and sensitive strains into drug-resistant strains, resulting in a rapid epidemic of carbapenem-resistant bacteria worldwide in a short period of time. 44,216 In recent years, the functions of BEVs in protecting bacterial survival, transmitting virulence factors, mediating horizontal gene transfer and cell-to-cell communication have been elucidated in detail, but how BEVs respond to antibiotics and the efficiency of BEVs-mediated horizontal gene transfer need to be further studied. In terms of bacteria-host interaction, BEVs can transport bacterial toxins and other virulence factors, such as adhesins and lipopolysaccharides to host cells, induce inflammatory responses, release pro-inflammatory cytokines and promote the transition from local infection to systemic infection. 217 In addition, BEVs carry microbial associated molecular patterns (MAMPs) that can be recognized by specific receptors of host immune cells, thereby modulating immune responses and mediating immunopathogenesis. 218 In the future, BEVs may provide new insights for elucidating the pathogenesis of infectious diseases and immune diseases.

| BEVs and treatment of diseases
Treatment strategies for diseases based on BEVs have received more and more attention in recent years. (1) Genetically engineered BEVs: BEVs have high physicochemical stability and biocompatibility, so they can be used as an excellent carrier for a variety of bioactive substances and used in the treatment of tumors and other diseases. Natural BEVs still have the disadvantage of insufficient targeting in vivo, and their complex structural components can reduce the therapeutic effect and even bring safety problems. Moreover, due to the lack of efficient separation and industrial production technology, the application of natural BEVs in clinical practice is still limited. Therefore, the use of bioengineering strategies to modify BEVs and target host cells for tumor and antibacterial treatment is expected to become a more valuable therapeutic strategy. 219 (2) Fluorescent probe-labeled BEVs: labeling BEVs with fluorescent probes can trace their uptake by organelles or tissues, providing support for visualizing the distribution of BEVs in vivo and providing a basis for more intuitive efficacy evaluation. 63 (3) Probiotic-derived BEVs: the way to improve intestinal health and increase immunity with probiotics has been favored by more and more people. Compared with direct supplementation with probiotics, probiotic-derived EVs, as a safer intervention, have the effect of regulating host immune response and alleviating inflammation, are not easy to cause secondary infection and have therapeutic effects on a variety of diseases represented by inflammatory bowel disease. 220 However, the mechanism of probiotic-derived EVs remains to be further explored. Therefore, BEVs have important potential in the treatment of diseases and are worth further exploiting.

| BEVs and diagnosis of diseases
With the development of multi-omics technology, the microbiome has received more and more attention in the prediction and diagnosis of diseases. Although BEVs have been observed to play a key role in the pathogenesis of the disease, research on BEVs as biomarkers for disease diagnosis is still lacking. In the future, the ability of BEVs as potential markers could be explored from the 24 of 33 following two directions. (1) BEVs: The differences in protein and lipid components of BEVs between disease and health states can be better understood through the multi-omics analysis of BEVs themselves, so as to predict or diagnose diseases. (2) EVs of host cells infected with BEVs: After the body is infected by pathogenic bacteria, BEVs are taken up by host cells, which may have an impact on the secretion of EVs and cell metabolism. For example, after host cells are infected, EVs can selectively enrich specific miRNAs to promote tumor metastasis. 221 Therefore, combining multi-omics analysis to elucidate the biological functions of various components of BEVs and the influence of BEVs on the secretion of EVs of host cells will provide new ideas for the diagnosis of diseases. In conclusion, the task force recognizes that functional research on BEVs is still in its early stages. However, it has great application prospects in the occurrence, diagnosis and treatment of a variety of diseases ( Figure 6). With the joint help of multiple techniques, more fundamental and clinical research majoring on BEVs will emerge that will benefit our understanding of the diseases.

| CONCLUSION AND PERSPECTIVE
BEVs are a specific type of lipid vesicle released by bacteria into the outside world. BEVs play a vital role in bacteria-host communication, with features of no proliferative ability but biological viability. Changes in BEVs are associated with a variety of diseases. With the establishment of evidence that BEVs are involved in the occurrence and development of diseases, such as cancer, 110 bacterial infection 64 and viral infection, 76,77 it is suggested that the mystery between BEVs and human diseases are worth discovering, which might open a series of exciting new ways for disease diagnosis and treatment, a realization of "bacteria-human" crossspecies communication.
However, there are two major challenges for researchers to tackle before BEVs can be used in clinical use. The first challenge is to establish an efficient method for BEV separation and extraction.
According to ISEV Worldwide survey, ultracentrifugation is the most commonly used technique for primary EV separation and enrichment. 121 At present, BEV separation is mainly dependent on ultracentrifugation, but it has a low BEV output, which is difficult to meet the huge demand for subsequent clinical demand. Moreover, it may also face problems such as contamination of other proteins or loss of BEVs by high centrifugal force. Therefore, we propose to establish a recognized method for the separation of BEVs to lay a technical foundation for its future clinical application (Figure 7). In addition to solving the separation strategy of different types of BEVs, we should F I G U R E 6 Function of BEVs in diseases. BEVs play a significant role in human diseases, primarily in three areas: disease occurrence, disease treatment, and disease diagnosis. Firstly, BEVs can contain genes, virulence factors, and active proteins that offer valuable insights into disease pathogenesis, Secondly, the transport properties of BEVs can be harnessed for targeted anti-bacterial or anti-tumor therapy. Lastly, precise analysis of BEV biomarkers holds promise as a diagnostic approach for various diseases.  provide solutions for BEV detection, follow-up research and clinical application. Here comes the second challenge that is to build a high-precision BEV characterisation platform. The growing maturity of big data analysis and its unique advantages make it a favorable tool for studying a variety of biological problems. BEVs from bacteria contain a variety of proteins, nucleic acids and other substances, making it more difficult to analyze BEV heterogeneity and function mining. Therefore, the key step in BEV identification and analysis is to aim at the carried characteristic substances. Sometimes, we may struggle to recognize BEVs as we cannot recall all their specific markers. In order to resolve this dilemma, we devote to build a BEV marker information database to include the protein and specific molecular information of different types of BEVs, so as to facilitate the early realization of quantitative analysis of BEV single particles. In addition, the database can also help the clinical treatment of BEVs. For example, the database can include indications, administration routes, preservation methods, biocompatibility and safety of therapeutic BEVs. The database can also record the specific preparation methods of engineering BEVs, such as the selection of model bacteria and engineering transformation/drug loading methods.
Currently, research related to BEVs is developing rapidly and is expected to elucidate the complex network of microbe-host communication, which is essential for maintaining human health and provide a solid foundation for driving new therapeutic strategies based on BEVs. Although facing many challenges, BEVs are certainly worth exploring because they may promote new changes the way of diagnosis and treatment in no longer future. 1