Gram-negative and Gram-positive bacteria differ in their biological origins and the substances they carry due to differences in their surface structure. Gram-negative bacteria produce extracellular vesicles by membrane vesiculation and cell blast lysis. Bacterial outer membrane vesicles (OMVs) derived from outer membrane vesicles consist of outer membrane and periplasmic components such as LPS. Similarly, outer-inner membrane vesicles (OIMVs) are formed by an autolysin-mediated blistering mechanism with a weakened peptidoglycan layer and contain the outer and inner membranes of the mother cell as well as cytoplasmic components. Degradation of the bacterial peptidoglycan layer by phage-derived endolysins leads to explosive cell lysis and random encapsulation of cytoplasmic contents in explosive outer membrane vesicles (EOMVs) and explosive outer-inner membrane vesicles (EOIMVs). These subtypes are often collectively referred to as OMVs because of the difficulty in accurately differentiating them. Gram-positive bacteria produce cytoplasmic membrane vesicles (CMVs) that lack an outer membrane because of the absence of an outer membrane structure. Thus, more cytoplasmic membrane vesicles are produced, and the production mechanism is similar to that observed in Gram-negative bacteria as part of explosive cell death. They can be categorized into cytoplasmic membrane vesicles (CMVs), explosive cytoplasmic membrane vesicles (ECMVs), and CMVs produced by a vesicular mechanism (Toyofuku et al., 2019).

Bacterial extracellular vesicles (BEVs) carry a variety of microbe-associated molecular patterns (MAMPs) and play a key role in host immune recognition. However, no standardized biomarkers can be used to differentiate BEVs specifically from Gram-negative or Gram-positive bacteria. Notably, outer membrane vesicles (OMVs) released by Gram-negative bacteria are often enriched in LPS, in which the lipid A structure can be specifically recognized by Toll-like receptor 4 (TLR4) (Kaparakis-Liaskos and Ferrero, 2015). In contrast, cytoplasmic membrane vesicles of Gram-positive bacterial origin express lipoteichoic acid on the surface of the vesicle, which can activate the TLR2 pathway (Brown et al., 2015; Kaparakis-Liaskos and Ferrero, 2015). In addition, BEVs produced by some pathogenic bacteria are enriched with specific virulence factors. For example, vesicles released by Porphyromonas gingivalis contain high levels of LPS, which induces M1-type polarization in host macrophages and enhances pro-inflammatory responses. In contrast, vesicles of Pseudomonas aeruginosa origin have been found to induce insulin resistance and impair glucose metabolism functions in skeletal muscle (Choi et al., 2015; Fleetwood et al., 2017). These BEVs play an important role in promoting inflammatory responses and disease processes by delivering virulence molecules to host cells.

It should be noted that the BEVs population itself is highly complex and heterogeneous. Their molecular composition and particle size distribution are not only regulated by the strain’s genetic background and biosynthetic mechanisms but also significantly influenced by a variety of environmental factors, including pH, temperature, nutritional status, and antibiotic exposure, which together regulate the efficiency of vesicle generation, the selective enrichment of bioactive components, and their biological effects on the host (Müller et al., 2021; Ye et al., 2021). Therefore, systematically resolving the dynamic composition of BEVs in different physiological or pathological states is of great significance to promote the standardization of functional studies of BEVs and an in-depth understanding of their mechanisms of action in the host. Figure 1 briefly summarizes the biogenesis of BEVs of different bacterial origins.

Studies have shown that BEVs can break through several biological barriers, including the intestinal epithelium, vascular endothelium, and blood-brain barrier, and eventually accumulate in organs such as the liver, lungs, and brain (Jones et al., 2024). For example, H. pylori-derived BEVs can co-localize near tight junctions (TJs) and carry the CagA protein, which alters the distribution of closure protein-1 (ZO-1) in the intestinal epithelium and increases the permeability of the intestinal barrier (Turkina et al., 2015). Similarly, BEVs of Campylobacter jejuni contain HtrA protease, which cleaves ZO-1 and E-cadherin in intestinal epithelial cells, weakening the integrity of the intestinal barrier (Elmi et al., 2016). These studies have shown that BEVs can increase the permeability of the intestinal barrier, making it easier for bacteria and their metabolites to enter the circulation, affecting distant organs. To more intuitively understand the translocation effect of BEVs, researchers used Cre-recombinase-labeled E. coli BEVs and through fluorescent tracer technology, observed that they could be expressed in mouse intestinal epithelial cells (including intestinal stem cells and mucosal immune cells) and fluorescent signals could be detected in organs such as the liver, spleen, kidneys, and the brain, which further confirms that BEVs can be transmitted across tissues in the host body (Jang et al., 2015). In addition, BEVs were similarly observed to enter the blood circulation through dynamin-dependent endothelial cell transport and ultimately accumulate in the liver by oral administration of BEVs to mice (Jones et al., 2020; Schaack et al., 2024).

It is worth noting that the transport of BEVs is not limited to when the intestinal barrier is compromised. Recent studies have shown that BEVs from commensal bacteria are equally capable of crossing the intestinal epithelium into the circulation via the paracellular pathway, even in a healthy state with an intact barrier. BEVs can cross the intestinal barrier relying on kinesin-mediated endocytosis, as well as the paracellular secretory pathway, without the need for disrupting tight junctions (Rubio et al., 2020; Juodeikis et al., 2022; Mottawea et al., 2025). This suggests that BEVs have a selective and controllable ability to cross the barrier. This unique mode of transport provides a new perspective for BEVs to achieve signaling molecules in the presence of a functionally intact intestinal barrier and lays the foundation for their potential role in regulating the intestinal-hepatic axis and remote inter-organ communication.

When BEVs cross the biological barrier, they interact with host cells through a variety of mechanisms, including direct activation of surface receptors through ligand binding, direct membrane fusion to deliver effector molecules to the cytoplasm or receptor cells through pathways such as giant cytotoxicity, phagocytosis, and endocytosis (Mulcahy et al., 2014; O’Donoghue et al., 2017). Existing studies have shown that BEVs carrying virulence factors can modulate cellular immunity and mediate apoptosis by affecting mitochondrial membrane potential, glucose metabolism, and so on when recognized by host cell receptors (Vanaja et al., 2016; Vitse and Devreese, 2020; Bitto et al., 2021; Balhuizen et al., 2022; Jain et al., 2024). These studies further emphasize that BEVs are not only important signaling mediators for bacteria at the site of colonization but also exert multiple biological effects in distant tissues. Figure 2  demonstrates a brief overview of BEVs crossing the intestinal barrier.

Metabolism-associated fatty liver disease (MAFLD), formerly known as nonalcoholic fatty liver disease (NAFLD), occurs mainly in genetically susceptible individuals due to overnutrition and insulin resistance. With the change in lifestyle, the prevalence of obesity and diabetes mellitus continues to rise globally, and MAFLD has become one of the most common chronic liver diseases in the world, with a prevalence of up to 25% (Younossi et al., 2019). Without timely intervention, MAFLD may progress to metabolism-associated steatohepatitis (MASH), liver fibrosis, cirrhosis, and even liver cancer (Younossi et al., 2019).

If the MAFLD/MASH stage is not effectively managed, patients may go on to develop liver fibrosis. This stage is characterized by the accumulation of collagen in the liver, forming a fibrotic network that limits normal liver function. As hepatic fibrosis progresses, cirrhosis may develop, which manifests as complete structural changes in the liver, including nodule formation and vascular remodeling. Cirrhosis is a critical stage in the loss of liver function and liver failure and a risk factor for hepatocarcinogenesis.

Hepatocellular carcinoma usually occurs in the setting of cirrhosis. As liver cells continue to be damaged, repair, and regenerate, abnormal cell proliferation may lead to the development of hepatocellular carcinoma (HCC). Hepatocellular carcinoma occurs as the final stage of liver disease and is usually accompanied by dramatic clinical symptoms and a poor prognosis. Therefore, early detection of hepatocellular carcinoma and intervention for early cirrhosis are key to improving patient prognosis.

Bacteria-host interactions play an important role in tumorigenesis and development, and BEVs, as a key mediator between bacteria and host cannot be ignored (Liang et al., 2024). The mechanism of BEVs’ action in tumorigenesis is mainly twofold: firstly, they induce genetic instability in the organism by carrying biologically active substances, such as proteins and nucleic acids; secondly, they stimulate pre-cancerous lesions by activating inflammatory response to stimulate precancerous lesions (Li et al., 2023). Recent studies have shown that the abundance and diversity of BEVs and microbiota in the feces of tumor patients are significantly reduced, but the proteasome carried by BEVs in tumor patients is more diverse and enriched with proteins related to amino acid and carbohydrate metabolism, nucleotide binding, and oxidoreductase activity compared to normal subjects (Mishra et al., 2025). Studies have shown that BEVs are more likely to accumulate in solid tumor tissues such as non-small cell lung cancer, colon adenocarcinoma, melanoma, and breast cancer compared to normal organs (Gujrati et al., 2019; Eyvazi et al., 2020; Kuerban et al., 2020; Basu et al., 2022). These studies reveal the potential role of BEVs in tumorigenesis and development.

Hepatocellular carcinoma, as a solid tumor with a high degree of malignancy, usually involves multiple molecular mechanisms in its development and progression (Yeo et al., 2025). The TGF-β signaling pathway plays an important role in the tumor microenvironment, invasion and metastasis of hepatocellular carcinoma, and blocking the TGF-β signaling pathway can effectively alleviate the development of hepatocellular carcinoma (Xin et al., 2024). In contrast, BEVs can activate the cellular TGF-β signaling pathway through the bioactive substances they carry, activate HSCs, and promote the progression of hepatic fibrosis, and the persistent hepatic fibrous state is a major risk factor for the development of hepatocellular carcinoma (Dhar et al., 2020; Dorner et al., 2024). For example, H. pylori-derived BEVs significantly upregulate gene expression in the TGF-β signaling pathway and enhance the invasive ability of HepG2 cells (Mohammadi Azad et al., 2024). In addition, C. difficile-derived BEVs can decrease mitochondrial membrane potential and increase intracellular ROS accumulation, which mediates mitochondrial dysfunction, and induce enhanced cellular glycolysis to promote hepatocellular carcinoma progression by up-regulating the expression of the key enzymes of glycolysis, HK2, PDK1, LDHA and PKM2 (Caballano-Infantes et al., 2023).

In the diagnosis of liver diseases (e.g., MAFLD, liver fibrosis, hepatocellular carcinoma, etc.), traditional methods usually require invasive histologic analyses, whereas BEVs, as bacterial-derived extracellular vesicles, not only have the advantages of being easily accessible and non-invasive but also have stronger immunogenicity than host cell-derived vesicles, which provides better specificity in diagnosis (Kaparakis-Liaskos and Ferrero, 2015; Loomba et al., 2021). With the development of multi-omics technologies, BEVs have demonstrated diagnostic potential in the diagnosis of infectious diseases, neurodegenerative diseases, respiratory diseases, and tumors (Dey et al., 2019; Wei et al., 2019; Yang et al., 2020). For example, An Hendrix’s team successfully isolated LPS-positive BEVs from the plasma of patients with intestinal barrier dysfunction and analyzed them quantitatively and qualitatively, and found that the levels of LPS-positive BEVs in the blood of patients with intestinal barrier dysfunction were significantly elevated and positively correlated with the levels of plasma ZO-1 proteins, which implies that the intestinal mucosal integrity of such patients is reduced and permeability is increased, suggesting that they could be used as a potential biomarker of intestinal barrier damage (Jones et al., 2021). In the context of liver disease diagnosis, researchers’ macrogenomic analysis of circulating BEVs from HCC and healthy controls showed an AUC of up to 0.879 based on a model of five genera of BEVs (Pseudomonas, Streptococcus, Staphylococcus, Bifidobacterium and Trabulsiella) (Cho et al., 2019). Therefore, by effectively isolating BEVs from patients’ body fluids and combining them with multi-omics analysis, it is expected to provide new tools for diagnosis and prognosis prediction of liver diseases in the future.

However, it is worth noting that BEVs from different body fluid sources have their own advantages and disadvantages in terms of their potential for disease diagnosis. Fecal-sourced BEVs can effectively reflect the compositional characteristics of the gut microbiota due to their high yield and diversity, have a strong discriminatory ability in distinguishing disease states from healthy controls, and may even be used to identify different stages or subtypes of diseases, such as the staging of colorectal cancer or the subtyping of inflammatory bowel disease (Park et al., 2021). However, the collection and processing of fecal samples is susceptible to factors such as environmental contamination, and fecal-derived BEVs may be difficult to accurately reflect systemic disease processes or extraintestinal manifestations (Jones et al., 2021; Liu et al., 2024). In contrast, BEVs in peripheral blood have more potential as markers reflecting systemic status, but their biomass is low, the risk of contamination is high, their isolation and characterization still face major technical challenges, and their yield is usually lower than that of fecal samples (Jones et al., 2021; Ou et al., 2023; Liu et al., 2024). Therefore, future studies need to optimize the methods for the detection and analysis of body fluid BEVs further and incorporate longitudinal clinical data with larger sample sizes to comprehensively assess the validity and clinical utility of BEVs as novel disease biomarkers.

Probiotic therapy plays a key role in liver diseases, and the BEVs secreted by probiotics are inanimate substances that are beneficial to host health, not only avoiding the potential harm caused by live bacteria to the host but also preserving the beneficial and immunogenic properties of the parental bacteria. Therefore, BEVs from probiotics are expected to be novel nanomaterials for the prevention and treatment of corresponding diseases. Table 2 summarizes the results of studies on the beneficial effects of intestinal symbiosis and probiotic-derived extracellular vesicles on the host, which are mainly related to the regulation of metabolism, the improvement of apoptosis, and the improvement of intestinal barrier function.

Much of the current work on the application of engineered BEVs in liver diseases focuses on their role in liver tumors. Disturbances in intestinal flora can alter the immune microenvironment of liver cancer, such as inducing T-cell depletion and immunosuppression, thus increasing tumor susceptibility, and remodeling TME has become an important direction in tumor therapy (Levy et al., 2017; Behary et al., 2021). Some studies have shown that BEVs exhibit great potential in improving the tumor microenvironment. Animal experiments have shown that intravenous injection of genetically engineered modified E. coli BEVs can achieve long-term resistance to the immune response of a variety of tumors by relying on interferon-γ, for example, liver tumors, renal tumors, and colorectal tumors, etc., and can completely eradicate established tumors without showing significant side effects. In addition, BEVs are also targeted; when systematically administered, BEVs can be observed to specifically target and accumulate in tumor tissues, subsequently inducing the production of anti-tumor cytokines CXCL10 and interferon-γ in the tumor microenvironment and inducing the aggregation of immune cells, such as NK cells and T cells, so as to achieve the therapeutic purpose (Kim et al., 2017). Surface engineering technology reduces the immunogenicity of BEVs by attaching CD47 to their membranes, remodels the TME by inducing macrophage M1 polarization and blocking immunosuppressive pathways, and the effect also exhibits long-term immune memory in the event of recurrent tumors (Feng et al., 2022). In addition, through drug encapsulation, BEVs are equally effective in inducing phenotypic conversion of macrophages in the TME from M2 to M1, thereby suppressing tumor development (Jiang et al., 2023). Immune checkpoint therapy also plays an important role in treating tumors; however, this approach is often challenged by immune and drug resistance. Studies have shown that BEVs can serve as essential immune adjuvants to overcome this dilemma. By inserting the OM structural domain of PD1 altered on BEVs, they can be made to bind specifically to PD-L1 on tumor cells and protect T cells from PD1/PD-L1 immunosuppression (Li et al., 2020). Currently, more and more studies have shown that BEVs combined with other treatments can further enhance tumor immunotherapy, completely eradicate tumors, and even prevent tumor recurrence and metastasis (Kuerban et al., 2020; Liu et al., 2023). However, it is also important to note that the existing studies still lack clinical trial support, while the natural immunogenicity of BEVs may have an impact on the human body. The rational use of the immunogenicity of BEVs combined with engineered modifications may be a new strategy for tumor therapy.

The specific role of bioactive molecules carried by BEVs on host cells has not been clarified. Most of the current studies have focused on the overall effects of BEVs, and fewer studies have delved into which specific class of bioactive substances in BEVs plays a key role in cell damage. Only some studies have also elucidated the damaging effects of LPS and bacterial DNA carried by BEVs on host cells (Tulkens et al., 2020b). Currently, the multi-omics analysis of BEVs only stops at the transcriptome to find the source, and there is no multi-omics joint analysis to elaborate on the specific pathogenic factors of BEVs. Therefore, further study of the downstream components of BEVs through multi-omics, such as genome, proteome and metabolome, is more conducive to the understanding of bacteria-bacteria and bacteria-host interactions and lays the foundation for clarifying their pathogenic mechanisms and regulatory pathways.

How to efficiently separate BEVs from body fluids remains an open question worthy of further exploration. Numerous studies have demonstrated the presence of BEVs in human body fluids, yet no standardized method exists for their separation and detection. Effectively isolating BEVs from various body fluids is a critical step toward their clinical application. Common methods for isolating BEVs include ultracentrifugation, ultrafiltration, size-exclusion chromatography, and density gradient centrifugation (Bauman and Kuehn, 2006; Northrop-Albrecht et al., 2022). Among these, ultracentrifugation is the most commonly used technique, relying on centrifugal speed to separate vesicles from cellular debris, bacteria, and other components. However, the high centrifugal force can damage the structure and function of BEVs, leading to the loss of some bioactive substances. Ultrafiltration utilizes membranes with different molecular weight cutoffs to selectively separate and concentrate vesicles, offering the highest recovery efficiency but low separation purity. Size-exclusion chromatography separates particles of different sizes using a porous polymer gel matrix, achieving high purity but being limited to small sample volumes. Density gradient centrifugation separates BEVs into specific density layers via ultrahigh-speed centrifugation, providing the highest purity but being time-consuming and technically complex, which limits its clinical translation. Tulkens et al. (2020a) proposed an efficient separation method combining density gradient centrifugation with size-exclusion chromatography, which effectively separates bacterial outer membrane vesicles, host cell-derived vesicles, and protein components from human fluids. Although this approach shows clinical feasibility, it also has limitations: density gradient centrifugation may result in the loss of some low-density BEVs, and there is no standardized method for separating BEVs from different body fluids. Therefore, further exploration and optimization of BEVs separation methods—particularly density gradient centrifugation combined with size-exclusion chromatography—are essential for clinical translation. Developing more efficient, standardized, and automated BEVs separation strategies is a core prerequisite for advancing their clinical application.

Following the separation of BEVs, accurate characterization is equally crucial. Commonly used methods include nanoparticle tracking analysis (NTA) for measuring the particle size distribution and concentration of BEVs. However, NTA requires strict detection conditions (e.g., temperature, concentration, calibration) and has limited specificity, as it cannot distinguish BEVs from cellular debris or potential contaminants, let alone differentiate BEVs from various sources. Dynamic light scattering (DLS) can also be used to measure BEV particle size, but it is only suitable for samples with high concentrations and uniform particle sizes. Furthermore, DLS cannot provide information on particle concentration, limiting its specificity (Coelho et al., 2019; Mei et al., 2020). Morphological characterization of BEVs typically relies on transmission electron microscopy (TEM) (Jung and Mun, 2018; Shao et al., 2018). In addition, no universally accepted biomarkers specific to BEVs have been established. Preliminary identification of BEVs from different origins can only be achieved by detecting known lipid compositions, outer membrane proteins, specific genes, or functional proteins (Cao et al., 2020; Díaz-Garrido et al., 2021; Tiku et al., 2021). Therefore, in-depth characterization of BEV composition, as well as identifying their sources and functions, will provide systematic support for BEV research and applications.

BEVs can be used as biomarkers for the diagnosis and prediction of diseases, and many studies have described their potential as biomarkers for future diseases (Yang et al., 2020; Han et al., 2021; Yoon et al., 2023). However, BEVs are heterogeneous, and BEVs produced from different strain sources as well as different survival environments are usually very different; for example, H. pylori-derived BEVs induce hepatic fibrosis progression in liver fibrosis through activation of HSCs as well as modulation of autophagy, but in hepatocellular carcinoma, H. pylori-derived BEVs promote tumor progression through activation of the TGF-β signaling pathway (Zahmatkesh et al., 2022; Shegefti et al., 2023; Mohammadi Azad et al., 2024). In addition, while pathogenic E. coli-derived BEVs can promote liver fibrosis progression, E. coli BEVs derived from the Nissle 1917 strain can ameliorate liver disease by regulating intestinal flora species and elevating serum SCFA levels (Natsui et al., 2023; Shi et al., 2023). This shows that BEVs of the same bacterial origin have different roles in different diseases, and even BEVs of the same bacterial origin from different strains have different roles in liver diseases. Moreover, there is no effective method to identify specific BEVs from complex body fluids such as feces and blood (Ban et al., 2016; Younossi et al., 2018; Hong et al., 2019). Therefore, more sophisticated technologies such as proteomics, lipidomics or metabolomics are needed to screen for specific “molecular fingerprints” of BEVs for non-invasive early diagnosis (e.g., an alternative to liver puncture), accurate typing (to distinguish viral or alcoholic liver disease) and treatment monitoring.

Maintaining the activity of BEVs requires strict storage conditions, and preserving the activity of isolated BEVs is crucial for advancing subsequent research. Different storage temperatures and durations have varying impacts on BEVs activity (Hong et al., 2019; Noonin et al., 2022). Therefore, optimizing storage and transport conditions tailored to BEVs derived from different body fluid sources is essential for their clinical application. To ensure the functional integrity of BEVs as research materials or therapeutic agents, systematic studies on the optimal storage environment, anti-degradation mechanisms, and transportation methods are needed. Establishing a standardized, high-fidelity, and highly controllable storage protocol will provide a solid foundation for future research and clinical use.

The use of live probiotics may increase the risk of intestinal dysbiosis or sepsis, especially in immunocompromised populations (Doron and Snydman, 2015). In contrast, beneficial BEVs offer key advantages: they are non-replicative, can produce effects similar to those of their parental bacteria, and do not carry the risk of live bacteria entering the bloodstream. However, a potential limitation lies in the complexity of probiotic BEV composition. BEVs derived from Gram-negative bacteria retain active components but cannot completely avoid carrying LPS, so safety concerns remain. Therefore, systematic evaluation of bioactive molecules carried by different types of probiotic exosomes is essential to assess potential unintended effects. For example, glycine supplementation has been shown to greatly enhance exosome production from the probiotic E. coli strain Nissle 1917 while reducing the endotoxin activity of these exosomes (Hirayama and Nakao, 2020). Future BEV production processes should focus on optimizing in vitro culture conditions to maximize therapeutic efficacy for specific diseases while minimizing potential side effects. To ensure the effectiveness of BEVs, further studies on their pharmacokinetics and dosage regulation principles are necessary. Given the possible limitations of probiotic BEVs from a single strain in modulating host physiological functions—especially for complex diseases such as liver diseases, which involve multiple pathological processes—the combined application of BEVs from multiple sources may achieve synergistic effects by targeting multiple pathways. This approach may provide a new strategy for the comprehensive treatment of liver diseases, following thorough elucidation of the biological functions and immunomodulatory properties of different probiotic BEVs.

In summary, although the research and application of BEVs still face many challenges, including the lack of efficient isolation and purification techniques, the screening difficulties of specific molecular markers, the insufficient standardization of storage and transportation conditions, and the imperfection of the immunosafety assessment system, their natural immunomodulatory ability, trans-tissue barrier transport ability, and engineered design make them not only expected to become “humoral signal sensors” reflecting the state of liver disease but also likely to be developed into smart therapeutic carriers integrating diagnostic and targeted delivery functions. However, its natural immunomodulatory ability, cross-tissue transport ability and engineered design make it not only a “fluid signal sensor” reflecting the state of liver disease but also a smart therapeutic carrier integrating diagnosis, intervention and targeted delivery. With the deepening of mechanism research and continuous breakthroughs in key technologies, BEVs are expected to play a central role in the precise diagnosis, early intervention and individualized treatment of liver diseases and to drive the entero-liver axis into a new era of “intervention-based microecological medicine.”