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Outer membrane vesicles as molecular biomarkers for Gram-negative sepsis: Taking advantage of nature’s perfect packages

Open AccessPublished:September 12, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102483
      Sepsis is an often life-threatening response to infection, occurring when host proinflammatory immune responses become abnormally elevated and dysregulated. To diagnose sepsis, the patient must have a confirmed or predicted infection, as well as other symptoms associated with the pathophysiology of sepsis. However, a recent study found that a specific causal organism could not be determined in the majority (70.1%) of sepsis cases, likely due to aggressive antibiotics or localized infections. The timing of a patient’s sepsis diagnosis is often predictive of their clinical outcome, underlining the need for a more definitive molecular diagnostic test. Here, we outline the advantages and challenges to using bacterial outer membrane vesicles (OMVs), nanoscale spherical buds derived from the outer membrane of Gram-negative bacteria, as a diagnostic biomarker for Gram-negative sepsis. Advantages include OMV abundance, their robustness in the presence of antibiotics, and their unique features derived from their parent cell that could allow for differentiation between bacterial species. Challenges include the rigorous purification methods required to isolate OMVs from complex biofluids and the additional need to separate OMVs from similarly sized extracellular vesicles, which can share physical properties with OMVs.

      Keywords

      Abbreviations:

      EV (extracellular vesicle), IEC (ion exchange chromatography), LPS (lipopolysaccharide), OMV (outer membrane vesicle), SEC (size-exclusion chromatography), UC (ultracentrifugation)
      According to recent retrospective studies, sepsis is not only the most expensive condition treated in US hospitals (
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      ). Many of the altered physiological parameters measured by these scoring systems are not necessarily specific to sepsis, which makes it difficult to diagnose sepsis in early stages. The timing of a patient’s sepsis diagnosis is often predictive of their clinical outcome, thus underlining the need for a more definitive molecular diagnostic test (
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      A recent (2018) retrospective observational study looked at 2,566,689 sepsis cases from the Premier Healthcare Database, which included data from ∼20% of US inpatient discharges among private and academic hospitals (
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      ). Among the causal organisms identified, the primary included Escherichia coli, other Gram-negative bacteria, and Streptococcus (
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      ). In a separate smaller study of neonatal sepsis patient samples (n = 70), only 41% of blood cultures were positive for bacteria, but that number rose to 91% when the blood was tested using a more sensitive 16S rDNA quantitative PCR assay, suggesting that even in neonates, blood cultures, especially those procured after antibiotic treatment is initiated, are not a reliable determinant of bacterial infections (
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      The most recent Surviving Sepsis Campaign article, written as part of an international collaboration to provide evidence-based treatments and best practices to reduce mortality related to sepsis, recommends the initiation of antimicrobial treatment within 1 or 3 h of disease recognition for patients with and without possible septic shock, respectively (
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      ).” This recommendation, combined with the common occurrence of falsely negative blood cultures, underlines the need for a fast and reliable method for detecting and identifying bacteria in sepsis patient biofluid.
      Later, we introduce the concept of extracellular vesicles (EVs) as uniquely qualified to serve as molecular biomarkers for the diagnosis of bacterial sepsis due to their conserved, native-like content, their association with the host inflammatory response, and their robustness (and potential enhancement) in the presence of antibiotics. Due to their size, EVs are known to widely circulate in the body and in some cases readily cross tissue barriers, enabling potential diagnosis from easily accessed biofluids such as blood or urine.

      EVs as molecular biomarkers for sepsis

      EVs are nanoscale, lipid-bound species released from both prokaryotic and eukaryotic cells, which contain a multitude of cellular components, including intracellular soluble and membrane-associated proteins and nucleic acids, all originating from the parent cell from which they derive (Fig. 1). There are many proposed functions of EVs, including those related to intercellular communication and quorum sensing, pathogenesis, disease state regulation, and cellular survival (
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      Figure thumbnail gr1
      Figure 1Types of extracellular vesicles (EVs). A, bacterial OMVs and eukaryotic microvesicles are formed when the surface membrane is curved and then pinched off, releasing the spherical vesicle. These EVs, therefore, have similar surface properties to their parent cells. B, eukaryotic exosomes are intraluminal vesicles that are released upon fusion to the plasma membrane, allowing for the transport of cell-specific cargo to neighboring or distant cells. C, apoptotic bodies are a byproduct of programmed cell death, ranging from 50 to 5000 nm in diameter and containing nuclear fragments, cellular molecules, and organelles. D, Gram-positive EVs are produced from the pinching-off of the inner membrane, carrying a diverse array of cellular cargo to the extracellular space but not before transversing through the thick cell wall. E, fungus can release EVs in the form of smaller exosomes and larger microvesicles (up to 2000 nm in diameter). Like their counterparts, fungal EVs contain a variety of cellular contents, useful for intercellular communication.
      EVs originating from eukaryotic and particularly human cells have been long studied with significant growing interest in recent years, but the term EV has been loosely used in the literature. In 2014, the International Society of Extracellular Vesicles (ISEV) established guidelines titled Minimal Information for Studies of Extracellular Vesicles to standardize protocols, nomenclature, and reporting, updating these guidelines again in 2018 (
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      ). The major classes of EVs originating from eukaryotic cells are defined by their biogenesis. Intraluminal vesicles originating in multivesicular bodies that are ultimately released upon fusion of these bodies with the plasma membrane are called exosomes (Fig. 1B). Microvesicles (Fig. 1A) are EVs that are regularly shed directly from the cell membrane upon outward budding. Due to their biogenesis, the lipid and membrane composition of exosomes and microvesicles differ as well as their luminal contents. Because a microvesicle results from the budding of the plasma membrane, its lipid composition, membrane-bound proteins, and surface markers closely mimic the surface of the parent cell. The third major class of EVs, apoptotic bodies (Fig. 1C), are released in the final stages of apoptosis through blebbing of the plasma membrane and have similar membrane composition to microvesicles. While exosomes are often the smallest class of EVs at just 20 to 200 nm in size, they do overlap in size with microvesicles, commonly 100 to 400 nm, as well as larger apoptotic bodies (50–5000 nm) (
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      Bacterial EVs

      Bacterial cells are known to release EVs, and their biogenesis is similar to EVs from human cells but with distinct differences, particularly between Gram-positive and Gram-negative cells (
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      OMVs are generally similar in size to eukaryotic exosomes and smaller microvesicles, typically between 20 to 250 nm in diameter. They function to secrete cellular components as a way of promoting pathogenesis, surviving stress conditions, or regulating microbial interactions within bacterial communities (
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      Advantages to using OMVs as biomarkers for Gram-negative sepsis

      Here, we describe three major advantages to using OMVs as biomarkers for Gram-negative sepsis: their ability to induce host inflammation and their probable role in bacterial sepsis (Fig. 2B), their parent-derived antigenic content (Fig. 2A) (although as discussed later, specific antigenic content can change with environmental conditions), and their robustness in the presence of antibiotics (Fig. 2C). While this review focuses on Gram-negative OMVs, we propose that EVs from Gram-positive bacteria may be similarly effective biomarkers for Gram-positive sepsis, allowing for possible differentiation between bacterial species and identification of the causal organism(s) for all cases of bacterial sepsis.
      Figure thumbnail gr2
      Figure 2Advantages to using OMVs as biomarkers for Gram-negative sepsis. A, OMVs contain biomolecules, including outer membrane and periplasmic proteins, nucleic acids, LPS, and other lipids, that are similar to its bacterial cell parent, allowing for the possible identification of the bacterial source of infection. B, OMVs can activate host immune cells, triggering the release of proinflammatory and anti-inflammatory cytokines and are capable of self-entry deep into host tissues, resulting in longer term, chronic inflammatory pathologies. C, the broad spectrum use of antibiotics to treat sepsis can lead to negative bacteria cultures. However, OMVs, which are continually released by bacteria and often enhanced in the presence of antibiotics, may offer an alternative method for identifying the causal organism of infection. OMV, outer membrane vesicle.
      Scientists have demonstrated that OMVs are capable of initiating the inflammatory response seen in the transition of an infection to sepsis, play a complex role in endothelial activation, and can induce cardiac injury, a sepsis complication that can worsen patient outcomes (
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      ). The delivery of toxins inside OMVs has several advantages. First, OMVs are capable of self-entry deep into host tissues, engaging both the innate and adaptive immune systems and resulting in longer term, chronic responses and inflammatory pathologies (
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      ). And finally, OMVs have been shown, in mice, to induce disseminated intravascular coagulation, a severe complication of sepsis that significantly increases the probability of mortality in septic patients (
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      LPS, also known as bacterial endotoxin, is the major lipid component in the outer leaflet of Gram-negative OMVs and is thought to be a major player in the induction of Gram-negative sepsis-related inflammation (
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      Treatment of gram-negative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin. A randomized, double-blind, placebo-controlled trial. The HA-1A Sepsis Study Group.
      ,
      • Hellman J.
      • Zanzot E.M.
      • Loiselle P.M.
      • Amato S.F.
      • Black K.M.
      • Ge Y.
      • et al.
      Antiserum against Escherichia coli J5 contains antibodies reactive with outer membrane proteins of heterologous gram-negative bacteria.
      ,
      • Hellman J.
      • Loiselle P.M.
      • Tehan M.M.
      • Allaire J.E.
      • Boyle L.A.
      • Kurnick J.T.
      • et al.
      Outer membrane protein A, peptidoglycan-associated lipoprotein, and murein lipoprotein are released by Escherichia coli bacteria into serum.
      ). E. coli OMVs contain LPS, OmpA, Lpp, and Pal, all of which have been shown to be released from E. coli as a complex in the presence of human sera and antibiotics, as well as in several animal models of sepsis (
      • Hellman J.
      • Zanzot E.M.
      • Loiselle P.M.
      • Amato S.F.
      • Black K.M.
      • Ge Y.
      • et al.
      Antiserum against Escherichia coli J5 contains antibodies reactive with outer membrane proteins of heterologous gram-negative bacteria.
      ,
      • Hellman J.
      • Loiselle P.M.
      • Tehan M.M.
      • Allaire J.E.
      • Boyle L.A.
      • Kurnick J.T.
      • et al.
      Outer membrane protein A, peptidoglycan-associated lipoprotein, and murein lipoprotein are released by Escherichia coli bacteria into serum.
      ,
      • Hellman J.
      • Loiselle P.M.
      • Zanzot E.M.
      • Allaire J.E.
      • Tehan M.M.
      • Boyle L.A.
      • et al.
      Release of gram-negative outer-membrane proteins into human serum and septic rat blood and their interactions with immunoglobulin in antiserum to Escherichia coli J5.
      ,
      • Hellman J.
      • Roberts J.D.
      • Tehan M.M.
      • Allaire J.E.
      • Warren H.S.
      Bacterial peptidoglycan-associated lipoprotein is released into the bloodstream in gram-negative sepsis and causes inflammation and death in mice.
      ,
      • Hellman J.
      • Tehan M.M.
      • Warren H.S.
      Murein lipoprotein, peptidoglycan-associated lipoprotein, and outer membrane protein A are present in purified rough and smooth lipopolysaccharides.
      ,
      • Hellman J.
      • Warren H.S.
      Outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and murein lipoprotein (MLP) are released in experimental Gram-negative sepsis.
      ,
      • Michel L.V.
      • Gallardo L.
      • Konovalova A.
      • Bauer M.
      • Jackson N.
      • Zavorin M.
      • et al.
      Ampicillin triggers the release of Pal in toxic vesicles from Escherichia coli.
      ). Additionally, Pal and Lpp have been shown to be inflammatory and to contribute to virulence on their own and in combination with LPS (
      • Hellman J.
      • Roberts J.D.
      • Tehan M.M.
      • Allaire J.E.
      • Warren H.S.
      Bacterial peptidoglycan-associated lipoprotein is released into the bloodstream in gram-negative sepsis and causes inflammation and death in mice.
      ,
      • Liang M.D.
      • Bagchi A.
      • Warren H.S.
      • Tehan M.M.
      • Trigilio J.A.
      • Beasley-Topliffe L.K.
      • et al.
      Bacterial peptidoglycan-associated lipoprotein: a naturally occurring toll-like receptor 2 agonist that is shed into serum and has synergy with lipopolysaccharide.
      ,
      • Hauschildt S.
      • Hoffmann P.
      • Beuscher H.U.
      • Dufhues G.
      • Heinrich P.
      • Wiesmüller K.H.
      • et al.
      Activation of bone marrow-derived mouse macrophages by bacterial lipopeptide: cytokine production, phagocytosis and Ia expression.
      ,
      • Bessler W.G.
      • Cox M.
      • Lex A.
      • Suhr B.
      • Wiesmüller K.H.
      • Jung G.
      Synthetic lipopeptide analogs of bacterial lipoprotein are potent polyclonal activators for murine B lymphocytes.
      ,
      • Bessler W.
      • Resch K.
      • Hancock E.
      • Hantke K.
      Induction of lymphocyte proliferation and membrane changes by lipopeptide derivatives of the lipoprotein from the outer membrane of Escherichia coli.
      ,
      • Hoffmann P.
      • Heinle S.
      • Schade U.F.
      • Loppnow H.
      • Ulmer A.J.
      • Flad H.D.
      • et al.
      Stimulation of human and murine adherent cells by bacterial lipoprotein and synthetic lipopeptide analogues.
      ,
      • Zhang H.
      • Peterson J.W.
      • Niesel D.W.
      • Klimpel G.R.
      Bacterial lipoprotein and lipopolysaccharide act synergistically to induce lethal shock and proinflammatory cytokine production.
      ). These results and the studies described previously all point to OMVs and their contents as significant contributors to the pathophysiology of Gram-negative sepsis.
      The diversity in functional roles of OMVs suggests that the inclusion of specific OMV cargo would be a highly controlled and orchestrated event. However, the mechanism behind the incorporation of specific molecules into OMVs has yet to be elucidated. OMVs typically contain a variety of outer membrane and periplasmic components, and several studies have demonstrated both the enrichment and exclusion of specific protein cargo (compared to concentrations in whole bacteria), suggesting there may be a cargo selection process or that the mechanism of OMV formation results in the enrichment and exclusion of certain molecules (
      • McBroom A.J.
      • Johnson A.P.
      • Vemulapalli S.
      • Kuehn M.J.
      Outer membrane vesicle production by Escherichia coli is independent of membrane instability.
      ,
      • Bonnington K.E.
      • Kuehn M.J.
      Protein selection and export via outer membrane vesicles.
      ). For example, the oral pathogen Porphyromonas gingivalis is thought to selectively sort outer membrane proteins into OMVs, enriching them with virulence factors, by accumulating the molecules into microdomains of the outer membrane that are primed for vesiculation (
      • Haurat M.F.
      • Aduse-Opoku J.
      • Rangarajan M.
      • Dorobantu L.
      • Gray M.R.
      • Curtis M.A.
      • et al.
      Selective sorting of cargo proteins into bacterial membrane vesicles.
      ). Similar microdomains have been proposed by others, formed by the accumulation of misfolded proteins or by their lack of linkages (via Lpp or Pal, for example) between the outer membrane and the peptidoglycan layer (
      • Schwechheimer C.
      • Kuehn M.J.
      Outer-membrane vesicles from gram-negative bacteria: biogenesis and functions.
      • Bonnington K.E.
      • Kuehn M.J.
      Protein selection and export via outer membrane vesicles.
      ,
      • Schwechheimer C.
      • Kulp A.
      • Kuehn M.J.
      Modulation of bacterial outer membrane vesicle production by envelope structure and content.
      ).
      While the mechanism of such cargo selection is still unknown, it is commonly accepted that OMVs contain components of their parent bacterium, which vary depending on growth conditions, including environmental stressors, and growth stage, which can affect the size and composition of the vesicles, as well as the expression and availability of proteins (
      • Bonnington K.E.
      • Kuehn M.J.
      Protein selection and export via outer membrane vesicles.
      ,
      • Bomberger J.M.
      • Maceachran D.P.
      • Coutermarsh B.A.
      • Ye S.
      • O’Toole G.A.
      • Stanton B.A.
      Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles.
      ,
      • Bhar S.
      • Edelmann M.J.
      • Jones M.K.
      Characterization and proteomic analysis of outer membrane vesicles from a commensal microbe, Enterobacter cloacae.
      ,
      • Bai J.
      • Kim S.I.
      • Ryu S.
      • Yoon H.
      Identification and characterization of outer membrane vesicle-associated proteins in Salmonella enterica serovar Typhimurium.
      ,
      • Lee E.-Y.
      • Bang J.Y.
      • Park G.W.
      • Choi D.-S.
      • Kang J.S.
      • Kim H.-J.
      • et al.
      Global proteomic profiling of native outer membrane vesicles derived from Escherichia coli.
      ,
      • Lee E.-Y.
      • Choi D.-S.
      • Kim K.-P.
      • Gho Y.S.
      Proteomics in gram-negative bacterial outer membrane vesicles.
      ,
      • Zavan L.
      • Bitto N.J.
      • Johnston E.L.
      • Greening D.W.
      • Kaparakis-Liaskos M.
      Helicobacter pylori growth stage determines the size, protein composition, and preferential cargo packaging of outer membrane vesicles.
      ,
      • Valguarnera E.
      • Scott N.E.
      • Azimzadeh P.
      • Feldman M.F.
      Surface exposure and packing of lipoproteins into outer membrane vesicles are coupled processes in Bacteroides.
      ,
      • Orench-Rivera N.
      • Kuehn M.J.
      Differential packaging into outer membrane vesicles upon oxidative stress reveals a general mechanism for cargo selectivity.
      ). Cargo that are conserved among a bacterium’s OMVs, independent of growth conditions and stage, would serve as ideal biomarkers, as well as cargo specific to a given bacterium that would allow for rapid identification and differentiation between bacterial species. Therefore, in order to utilize OMVs as effective biomarkers for sepsis, cargo that is conserved among the bacterial OMVs of interest must first be identified. At a minimum, LPS should be able to serve as a detectable biomarker for Gram-negative bacteria due to its abundance in the outer leaflet of the OMV outer membrane. And although outside the scope of this review, in the case that few or no proteins are conserved across species-specific OMVs, the amplification and detection of nucleic acids may be an alternative method for identifying the parent bacterial source of infection (
      • Omar S.
      • Murphy S.
      • Gheevarghese R.
      • Poppleton N.
      A retrospective evaluation of a multiplex polymerase chain reaction test directly applied to blood for the management of sepsis in the critically ill.
      ), especially considering the unique protection afforded to nucleic acids contained within membrane-bound OMVs.
      As described previously, one of the biggest challenges in diagnosing sepsis and identifying the causal organism is the common occurrence of falsely negative blood cultures, due in part to the presence of antibiotics (wherein the bacterial infection source is unable to grow due to bactericidal or bacteriostatic levels of antibiotics in the host) and the inherent challenges in culturing bacteria in vitro (
      • Ter S.K.
      • Rattanavong S.
      • Roberts T.
      • Sengduangphachanh A.
      • Sihalath S.
      • Panapruksachat S.
      • et al.
      Molecular detection of pathogens in negative blood cultures in the Lao people’s democratic republic.
      ,
      • Fischer G.W.
      • Longfield R.
      • Hemming V.G.
      • Valdes-Dapena A.
      • Smith L.P.
      Pneumococcal sepsis with false-negative blood cultures.
      ,
      • Papafilippou L.
      • Claxton A.
      • Dark P.
      • Kostarelos K.
      • Hadjidemetriou M.
      Nanotools for sepsis diagnosis and treatment.
      ). Despite these challenges and the additional hurdle of bacterial cultures taking up to 24 h for results, lab culture testing remains the most common method for identifying the causal organism of infection (
      • Evans L.
      • Rhodes A.
      • Alhazzani W.
      • Antonelli M.
      • Coopersmith C.M.
      • French C.
      • et al.
      Surviving sepsis Campaign: international guidelines for management of sepsis and septic shock 2021.
      ,
      • Thompson K.
      • Venkatesh B.
      • Finfer S.
      Sepsis and septic shock: current approaches to management.
      ,
      • Tabak Y.P.
      • Vankeepuram L.
      • Ye G.
      • Jeffers K.
      • Gupta V.
      • Murray P.R.
      Blood culture turnaround time in U.S. Acute care hospitals and implications for laboratory process optimization.
      ). As an alternative, OMVs and their parent-like features could be used to identify the bacterium. Unlike their bacterial parent, OMVs can withstand the inundation of most antimicrobials. Since bacteria release OMVs as part of their stress response, many bacteria have been shown to enhance OMV production and release in the presence of antimicrobials, such as gentamicin (
      • Kadurugamuwa J.L.
      • Mayer A.
      • Messner P.
      • Sára M.
      • Sleytr U.B.
      • Beveridge T.J.
      S-layered aneurinibacillus and Bacillus spp. are susceptible to the lytic action of Pseudomonas aeruginosa membrane vesicles.
      ), antimicrobial quinolone PQS (
      • Mashburn-Warren L.
      • Howe J.
      • Garidel P.
      • Richter W.
      • Steiniger F.
      • Roessle M.
      • et al.
      Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation.
      ,
      • Macdonald I.A.
      • Kuehn M.J.
      Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa.
      ,
      • Tashiro Y.
      • Ichikawa S.
      • Nakajima-Kambe T.
      • Uchiyama H.
      • Nomura N.
      Pseudomonas quinolone signal affects membrane vesicle production in not only gram-negative but also gram-positive bacteria.
      ), polymixin B (
      • Macdonald I.A.
      • Kuehn M.J.
      Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa.
      ), ciprofloxacin (
      • Maredia R.
      • Devineni N.
      • Lentz P.
      • Dallo S.F.
      • Yu J.
      • Guentzel N.
      • et al.
      Vesiculation from Pseudomonas aeruginosa under SOS.
      ), mitomycin C (
      • Dutta S.
      • Iida K.
      • Takade A.
      • Meno Y.
      • Nair G.B.
      • Yoshida S.
      Release of Shiga toxin by membrane vesicles in Shigella dysenteriae serotype 1 strains and in vitro effects of antimicrobials on toxin production and release.
      ), and other antibiotics, especially those known to target the outer membrane, peptidoglycan, or LPS (
      • Michel L.V.
      • Gallardo L.
      • Konovalova A.
      • Bauer M.
      • Jackson N.
      • Zavorin M.
      • et al.
      Ampicillin triggers the release of Pal in toxic vesicles from Escherichia coli.
      ,
      • Kohanski M.A.
      • Dwyer D.J.
      • Collins J.J.
      How antibiotics kill bacteria: from targets to networks.
      ,
      • Volgers C.
      • Savelkoul P.H.M.
      • Stassen F.R.M.
      Gram-negative bacterial membrane vesicle release in response to the host-environment: different threats, same trick?.
      ). OMVs are also thought to be released by bacteria to act as decoys, absorbing antimicrobials and antibodies so that the bacteria itself can evade the host’s innate and adaptive immune responses (
      • Volgers C.
      • Savelkoul P.H.M.
      • Stassen F.R.M.
      Gram-negative bacterial membrane vesicle release in response to the host-environment: different threats, same trick?.
      ,
      • Manning A.J.
      • Kuehn M.J.
      Contribution of bacterial outer membrane vesicles to innate bacterial defense.
      ).
      Taken together, these advantages suggest that OMVs, which are continually released by Gram-negative bacteria and enhanced in the presence of environmental stressors, would allow for more efficient identification of the bacterial source of infection, even in the presence of antibiotics. However, before OMVs can become a reliable biomarker for Gram-negative sepsis, several significant challenges must be addressed, as described in the following section.

      Challenges to using OMVs as biomarkers and possible strategies to overcome them

      The short- and long-term prevalence of OMVs in biofluids postinfection is still relatively unknown and understudied. We can surmise that bacteria will continuously release OMVs during the infection process, but post-infection, while the dysregulated inflammatory response wreaks havoc in the patient, how long will OMVs remain in circulation before being filtered out by the body? And compared to the patient’s own EV population, which includes EVs released by organisms in the host micro/mycobiome, how many OMVs will circulate, and how quickly do those numbers change during disease progression? Further, can we count on a detectable level of OMVs to be released into the bloodstream, independent of bacterial source and/or level of infection? One thought is that even very low levels of OMVs may be detectable and allow for identification of the bacterial source, although more sensitive detection techniques may be required, such as PCR-based methods. While exogenous nucleic acids from the bacteria will be quickly degraded by host nucleases, DNA or RNA contained within OMVs may be protected from degradation indefinitely. Effective isolation and purification of EVs from complex biofluids remain a challenge, and solving this will enable a variety of diagnostic strategies that will improve our understanding of sepsis progression and treatment for patients.
      To detect OMVs in a complex biofluid such as human plasma, one must consider the OMV titer during infection and the sensitivity and selectivity of the detection device. Low titers of OMVs and/or low sensitivity of the detection device (e.g., weak antibody binding to the antigenic OMV target) could result in a weak positive signal, and high titers of extraneous EVs or poor selectivity of the detection device (e.g., nonselective antibody binding to non-OMV targets) could result in false-negative results. Therefore, an initial purification and/or concentration step of the OMVs could allow for improved sensitivity and selectivity.
      Purification of human and bacterial EVs from complex biofluids such as serum and urine is technically demanding. In addition to whole cells and cellular fragments, these fluids contain many types of proteins, lipid complexes, extracellular RNA and DNA, as well as other biological nanoparticles with overlapping density and size (
      • Brennan K.
      • Martin K.
      • FitzGerald S.P.
      • O’Sullivan J.
      • Wu Y.
      • Blanco A.
      • et al.
      A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum.
      ,
      • Lee Y.X.F.
      • Johansson H.
      • Wood M.J.A.
      • El Andaloussi S.
      Considerations and implications in the purification of extracellular vesicles – a cautionary tale.
      ). More specifically, in addition to human extracellular vesicles, there will likely be a background level of vesicles released by bacteria from the host microbiome and fungi from the host mycobiome (
      • Ghannoum M.A.
      • Jurevic R.J.
      • Mukherjee P.K.
      • Cui F.
      • Sikaroodi M.
      • Naqvi A.
      • et al.
      Characterization of the oral fungal microbiome (mycobiome) in healthy individuals.
      ,
      • Kalluri R.
      • LeBleu V.S.
      The biology, function, and biomedical applications of exosomes.
      ,
      • Rayner S.
      • Bruhn S.
      • Vallhov H.
      • Andersson A.
      • Billmyre R.B.
      • Scheynius A.
      Identification of small RNAs in extracellular vesicles from the commensal yeast Malassezia sympodialis.
      ,
      • Johansson H.J.
      • Vallhov H.
      • Holm T.
      • Gehrmann U.
      • Andersson A.
      • Johansson C.
      • et al.
      Extracellular nanovesicles released from the commensal yeast Malassezia sympodialis are enriched in allergens and interact with cells in human skin.
      ). While the remainder of this review will focus on separation techniques required to isolate EVs, we acknowledge the additional challenge that comes with differentiating vesicles from the infection source and those derived from the host, including those produced by the host’s own micro/mycobiome.
      A variety of approaches have been employed to purify and separate EVs, ranging from centrifugation to novel microfluidic technologies (Fig. 3). Comprehensive reviews cataloging and comparing separation approaches have been published elsewhere (
      • Brennan K.
      • Martin K.
      • FitzGerald S.P.
      • O’Sullivan J.
      • Wu Y.
      • Blanco A.
      • et al.
      A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum.
      ,
      • Lee Y.X.F.
      • Johansson H.
      • Wood M.J.A.
      • El Andaloussi S.
      Considerations and implications in the purification of extracellular vesicles – a cautionary tale.
      ,
      • Shirejini S.Z.
      • Inci F.
      The Yin and Yang of exosome isolation methods: conventional practice, microfluidics, and commercial kits.
      ,
      • Liangsupree T.
      • Multia E.
      • Riekkola M.-L.
      Modern isolation and separation techniques for extracellular vesicles.
      ,
      • Tulkens J.
      • De Wever O.
      • Hendrix A.
      Analyzing bacterial extracellular vesicles in human body fluids by orthogonal biophysical separation and biochemical characterization.
      ). While most of these reviews have focused on human EVs, the same principles generally apply to OMVs. A summary of the most common techniques for isolating EVs and OMVs from septic patient biofluids and from each other are outlined later.
      Figure thumbnail gr3
      Figure 3Isolation and purification approaches for OMVs. A, centrifugation is one of the most widely used techniques for isolating extracellular vesicles, including OMVs. The most common centrifugal approach is differential, which involves sequential centrifugation steps ending with ultracentrifugation. B, membrane filtration is also commonly used in the isolation of vesicles. A prefiltration step is used in several isolation approaches to rapidly remove large contaminants from small vesicles and proteins. Tangential flow filtration (TFF) is often used for high-volume isolation needs. Low molecular weight filters are sometimes used to concentrate vesicles after ultracentrifugation or chromatography. C, microfluidic devices incorporating novel isolation and separation technologies from acoustics to electrokinetics aim to improve recovery as well as separate vesicle subpopulations. OMV, outer membrane vesicle.
      Two techniques became relatively common early on for the purification of EVs, ultracentrifugation (UC) and polymer precipitation. Precipitation with PEG was one of the first commercially available kits for EV isolation and gained relatively quick adoption because of its simplicity. Since the beginning of its use, however, several publications have shown that precipitation methods result in significant coprecipitation of contaminating proteins, lipoprotein complexes, and extracellular RNA, resulting in the discontinuation of this technique by many researchers (
      • Taylor D.D.
      • Shah S.
      Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.
      ,
      • Rider M.A.
      • Hurwitz S.N.
      • Meckes D.G.
      ExtraPEG: a polyethylene glycol-based method for enrichment of extracellular vesicles.
      ). On the other hand, UC remains a gold standard for the purification of EVs (Fig. 3A), although newer techniques are proving to offer higher purity and recovery (
      • Liangsupree T.
      • Multia E.
      • Riekkola M.-L.
      Modern isolation and separation techniques for extracellular vesicles.
      ,
      • Théry C.
      • Amigorena S.
      • Raposo G.
      • Clayton A.
      Isolation and characterization of exosomes from cell culture supernatants and biological fluids.
      ). Differential or sequential UC relies upon differing sedimentation rates of the biofluid constituents, typically recovering the supernatant and discarding the pellet until a final high speed centrifugation where EVs are ideally pelleted, while most proteins remain in the supernatant (
      • Bobrie A.
      • Colombo M.
      • Krumeich S.
      • Raposo G.
      • Théry C.
      Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation.
      ). Fundamentally, this technique cannot provide an absolute high purity separation as proteins, lipid complexes, and other non-EV species can aggregate and copellet. At the same time, high speed centrifugation can lead to fusion of the target EVs (
      • Jeppesen D.K.
      • Fenix A.M.
      • Franklin J.L.
      • Higginbotham J.N.
      • Zhang Q.
      • Zimmerman L.J.
      • et al.
      Reassessment of exosome composition.
      ,
      • Nakai W.
      • Yoshida T.
      • Diez D.
      • Miyatake Y.
      • Nishibu T.
      • Imawaka N.
      • et al.
      A novel affinity-based method for the isolation of highly purified extracellular vesicles.
      ,
      • Kowal J.
      • Arras G.
      • Colombo M.
      • Jouve M.
      • Morath J.P.
      • Primdal-Bengtson B.
      • et al.
      Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.
      ). Gradient UC can be performed after an initial purification to separate species that differ slightly in their density, even when similar in size, but some lipoproteins can still coisolate (
      • Yuana Y.
      • Levels J.
      • Grootemaat A.
      • Sturk A.
      • Nieuwland R.
      Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation.
      ). Differences in biofluid viscosity, protein, and lipid concentration can dramatically alter UC results. For these reasons, EV researchers have sought solutions using a variety of other bioseparation approaches.
      Several types of chromatography have been used to purify or isolate EVs (
      • Tengattini S.
      Chromatographic approaches for purification and analytical characterization of extracellular vesicles: recent advancements.
      ,
      • Staubach S.
      • Bauer F.N.
      • Tertel T.
      • Börger V.
      • Stambouli O.
      • Salzig D.
      • et al.
      Scaled preparation of extracellular vesicles from conditioned media.
      ). Size-exclusion chromatography (SEC) has proven successful in separating smaller proteins from EVs, which are large enough to be excluded from the size-exclusion beads, passing more quickly into the collection fractions. Ion-exchange chromatography (IEC) has been used to target EVs that generally have a net negative charge. However, ion-exchange will also select for protein complexes with a similar charge. Vesicles that are not rich in negatively charged glycans, phosphoryl, and sulfo groups may not bind to the IEC matrix (
      • Kosanović M.
      • Milutinović B.
      • Goč S.
      • Mitić N.
      • Janković M.
      Ion-exchange chromatography purification of extracellular vesicles.
      ). Furthermore, the negative surface charge of most EVs can change as environmental conditions, such as pH, vary in the biofluids (
      • Midekessa G.
      • Godakumara K.
      • Ord J.
      • Viil J.
      • Lättekivi F.
      • Dissanayake K.
      • et al.
      Zeta potential of extracellular vesicles: toward understanding the attributes that determine colloidal stability.
      ). Affinity chromatography can be used to select for specific surface markers of a particular EV subpopulation through the use of antibodies, aptamers, or other ligands (
      • Staubach S.
      • Bauer F.N.
      • Tertel T.
      • Börger V.
      • Stambouli O.
      • Salzig D.
      • et al.
      Scaled preparation of extracellular vesicles from conditioned media.
      ). Unlike SEC, both IEC and affinity chromatography typically require elution buffers with significant changes in pH or ionic strength, which could affect the properties and functionality of the EVs. In all cases, chromatography solutions generally result in significant dilution, often requiring a final membrane concentration and buffer exchange step, which can lead to further loss.
      A classic approach to purifying biomolecules from various biofluids is membrane filtration (Fig. 3B). Filtration can be applied in several manners depending on user requirements. Membranes have been used in EV purification pipelines in several ways (
      • Liangsupree T.
      • Multia E.
      • Riekkola M.-L.
      Modern isolation and separation techniques for extracellular vesicles.
      ), from prefiltration of cellular debris to concentration following chromatography to the separation of EVs from other small biomolecules using tangential flow filtration (
      • Heinemann M.L.
      • Vykoukal J.
      Sequential filtration: a gentle method for the isolation of functional extracellular vesicles.
      ). Prefiltration often utilizes relatively large pores to allow EVs and proteins to pass while retaining cellular debris (
      • Van Deun J.
      • Mestdagh P.
      • Sormunen R.
      • Cocquyt V.
      • Vermaelen K.
      • Vandesompele J.
      • et al.
      The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.
      ). The use of vacuum and syringe filtration membranes operating in normal flow, also known as dead-end filtration, is common, but prone to cake formation (accumulation of matter at the membrane surface) and significant loss in cell and protein-rich biofluids (
      • Dehghani M.
      • Lucas K.
      • Flax J.
      • McGrath J.
      • Gaborski T.
      Tangential flow microfluidics for the capture and release of nanoparticles and extracellular vesicles on conventional and ultrathin membranes.
      ). Due to the substantial dilution that occurs during chromatography elution, EV-rich fractions are often combined and then concentrated with a low molecular weight cut-off membrane, where EVs are retained above the filter, while excess fluid passes through. EVs forced onto the membrane and into pores can be damaged and lost during this process (
      • Welton J.L.
      • Webber J.P.
      • Botos L.-A.
      • Jones M.
      • Clayton A.
      Ready-made chromatography columns for extracellular vesicle isolation from plasma.
      ). Finally, tangential flow filtration uses a sweeping process across the membrane to minimize cake formation and concentration polarization while passing smaller species, such as proteins, while retaining EVs (
      • Dehghani M.
      • Lucas K.
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      Tangential flow microfluidics for the capture and release of nanoparticles and extracellular vesicles on conventional and ultrathin membranes.
      ,
      • Kim K.
      • Park J.
      • Jung J.-H.
      • Lee R.
      • Park J.-H.
      • Yuk J.M.
      • et al.
      Cyclic tangential flow filtration system for isolation of extracellular vesicles.
      ,
      • Busatto S.
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      Tangential flow filtration for highly efficient concentration of extracellular vesicles from large volumes of fluid.
      ). This approach is common in large-scale purification solutions but is gaining popularity in smaller formats due to higher purity and less loss compared to other filtration methods.
      On the horizon are a number of promising technologies that rely on fluidic (asymmetric flow field-flow fractionation), electrokinetic, and acoustic focusing principles, often in combination with one or more traditional separation approaches (
      • Shirejini S.Z.
      • Inci F.
      The Yin and Yang of exosome isolation methods: conventional practice, microfluidics, and commercial kits.
      ). Many of these microfluidic technologies (Fig. 3C) have the potential to significantly improve purity with rapid processing times but are generally limited to relatively small sample volumes. Microfluidics can also enable the combination of size and affinity approaches in a single platform. While volume limitation may inhibit their widespread adoption for all EV isolation needs, improvements in purity and speed may be ideal for diagnostic purposes where sample volumes are relatively small.
      In addition to the more generic challenges to isolating EVs, as described previously, isolating EVs from human serum offers its own unique challenges. Protein concentration in serum is typically very high (60–80 mg/ml) and can lead to almost immediate membrane fouling in normal or dead-end filtration modes used in vacuum or syringe filters. In contrast, tangential flow filtration can minimize this effect to maintain throughput (
      • Lucas K.
      • Ahmad S.D.
      • Dehghani M.
      • Gaborski T.
      • McGrath J.
      Critical flux behavior of ultrathin membranes in protein-rich solutions.
      ). Serum also contains lipids such as high-density lipoprotein (7–13 nm) and low-density lipoprotein (18–23 nm) that are smaller than most EVs and can still be removed with size-based methods (
      • Brennan K.
      • Martin K.
      • FitzGerald S.P.
      • O’Sullivan J.
      • Wu Y.
      • Blanco A.
      • et al.
      A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum.
      ). High lipid concentrations, however, can foul some membranes and affect pellet formation during centrifugation. Larger lipids such as very low-density lipoprotein (30–80 nm) and chylomicrons (80–1200 nm) can overlap in size with EVs but have different densities. These nanoparticles can be separated from EVs using density gradient centrifugation (
      • Brennan K.
      • Martin K.
      • FitzGerald S.P.
      • O’Sullivan J.
      • Wu Y.
      • Blanco A.
      • et al.
      A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum.
      ,
      • Carter D.C.
      • Ho J.X.
      Structure of serum albumin.
      ). Additionally, lipids and lipoproteins can be removed using affinity and, in some cases, charge-based separation techniques as discussed earlier. Urine has lower protein concentration, few lipid complexes, and overall, much lower viscosity than serum but also likely far fewer OMVs originating from sepsis and may not be a desirable or reliable source for these important biomarkers.
      Separating EVs and bacterial OMVs from each other is yet another challenge, because they are similar in size. Physical separation approaches such as membrane filtration, SEC, and asymmetric flow field flow fractionation are not effective on their own. Separating vesicles based on surface antigens using affinity approaches is one possible strategy that could utilize chromatography, magnetic beads, or microfluidic capture on functionalized surfaces. Recently, some groups have successfully isolated human EVs by targeting specific phospholipids using Tim4 protein and annexin V (
      • Nakai W.
      • Yoshida T.
      • Diez D.
      • Miyatake Y.
      • Nishibu T.
      • Imawaka N.
      • et al.
      A novel affinity-based method for the isolation of highly purified extracellular vesicles.
      ,
      • Kang Y.-T.
      • Purcell E.
      • Palacios-Rolston C.
      • Lo T.-W.
      • Ramnath N.
      • Jolly S.
      • et al.
      Isolation and profiling of circulating tumor-associated exosomes using extracellular vesicular lipid-protein binding affinity based microfluidic device.
      ). Similarly, anti-LPS or LPS-binding effector TeoL could be used to isolate LPS-rich OMVs (
      • Li C.
      • Zhu L.
      • Wang D.
      • Wei Z.
      • Hao X.
      • Wang Z.
      • et al.
      T6SS secretes an LPS-binding effector to recruit OMVs for exploitative competition and horizontal gene transfer.
      ). Additionally, some are investigating whether proteins that sense and bind to highly curved phospholipid membranes and the peptides derived from them can be used to selectively capture EVs (
      • Gori A.
      • Romanato A.
      • Greta B.
      • Strada A.
      • Gagni P.
      • Frigerio R.
      • et al.
      Membrane-binding peptides for extracellular vesicles on-chip analysis.
      ,
      • Flynn A.D.
      • Yin H.
      Lipid-targeting peptide probes for extracellular vesicles.
      ). The net surface charge of OMVs likely differs from human EVs based on the variation in antigens, glycans, phospholipids, and the presence of LPS. The most successful strategy in isolating OMVs from human biofluids will likely use a combination of traditional bioseparation approaches, such as membrane filtration, in combination with vesicle-selective capture via affinity or charge interaction.
      While a combination of isolation and detection approaches will likely be required, OMVs remain a highly attractive biomarker for Gram-negative sepsis diagnosis. The common occurrence of falsely negative bacterial cultures in sepsis patient biofluids continues to be a mystery, perplexing doctors and leaving patients with more questions than answers. Where bacteria fail (thankfully, due to stalwart antimicrobial treatments), OMVs may persist and allow for a more definitive diagnosis and a more targeted approach to treatment.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Author contributions

      L. V. M. and T. G. conceptualization; L. V. M. and T. G. writing–original draft.

      Funding and additional information

      L. V. M. and T. G. are supported by NIAID of the National Institutes of Health under award number R21AI163782. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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