The role of bacterial extracellular vesicles in chronic wound infections: Current knowledge and future challenges

Chronic wounds are a significant global problem with an increasing economic and patient welfare impact. How wounds move from an acute to chronic, non‐healing, state is not well understood although it is likely that it is driven by a poorly regulated local inflammatory state. Opportunistic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa are well known to stimulate a pro‐inflammatory response and so their presence may further drive chronicity. Studies have demonstrated that host cell extracellular vesicles (hEVs), in particular exosomes, have multiple roles in both increasing and decreasing chronicity within wounds; however, the role of bacterial extracellular vesicles (bEVs) is still poorly understood. The aim of this review is to evaluate bEV biogenesis and function within chronic wound relevant bacterial species to determine what, if any, role bEVs may have in driving wound chronicity. We determine that bEVs drive chronicity by both increasing persistence of key pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa and stimulating a pro‐inflammatory response by the host. Data also suggest that both bEVs and hEVs show therapeutic promise, providing vaccine candidates, decoy targets for bacterial toxins or modulating the bacterial species within chronic wound biofilms. Caution should, however, be used when interpreting findings to date as the bEV field is still in its infancy and as such lacks consistency in bEV isolation and characterization. It is of primary importance that this is addressed, allowing meaningful conclusions to be drawn and increasing reproducibility within the field.


| INTRODUCTION
Wound healing is a complex and highly regulated process, typically divided into four stages: haemostasis, inflammation, proliferation and remodelling. 1 A proportion of wounds are slow to progress beyond the inflammation stage and as such are classified as chronic. An absolute definition of a chronic wound has yet to be agreed although generally wounds are typically classed as chronic if they have failed to show significant closure within a "reasonable" time frame, typically 12 weeks. [2][3][4] By their very definition, chronic wounds are long-term health problems. Guest et al 5 reported that 39% of the wounds within their study had not fully healed within the 12 month study period, similarly Posnett and Franks 6 reported that one-third of wounds are unresolved within 6 months, with a fifth of wounds taking 12 months or more. Within the United Kingdom, chronic wounds cost the national health service GBP £5.3 billion per annum, equivalent to 4% of the total National Health Service (NHS) annual budget. 5 In the United States, chronic wounds are estimated to cost US $ 9.7 billion, 7 with a combined (direct and societal) cost per individual of US $ 13,334, raising to US $ 33,499 per individual if amputation is required. 8 Infection within these chronic wounds is a complicating factor, leading not only to increased patient distress but also prolonged treatment and increased financial burden. 9,10 As the global population ages, becomes increasing sedentary and co-morbidities such as obesity and type two diabetes rise, so too do chronic wounds. 11 As such it is imperative that we gain a better understanding of not only the chronic wound itself but also how to successfully treat them. In this review, we have evaluated the evidence that extracellular vesicles (EVs), from both the host and bacterial species (hEVs and bEVs respectively), are able to contribute towards chronic wound pathogenesis and evaluated potential avenues for EV-based interventions. In particular, we focus on bEVs within the chronic wound as significantly less investigation of bEVs has been carried out to date.

| INFECTION WITHIN CHRONIC WOUNDS
Because of the presence of a complex microbial community upon the skin, 12 keeping chronic wounds sterile is an unrealistic goal, and many wound infections originate from the patient's own microbiome. 13 There is a continuous spectrum from microbial contamination of a wound, through colonization and finally infection, where colonization has become persistent and clinical signs of infection are present. 14 It is likely that the majority of chronic wounds are colonized; 15 however, diagnosis of infection is based on recognition of clinical infection indicators. 16 Prompers et al 17 reported that in their European study, 50% of diabetic foot ulcers were infected upon first presentation, whereas a US study reported a more conservative figure of 15%. 18 Metagenomic studies have shown that decreased microbial diversity and increased temporal stability of the microbial wound community are linked to delayed healing and poorer outcome. 19 Frequently microbes associated with chronic wounds originate from the patient or their environment.
Such species are referred to as "opportunistic pathogens" because they are able to colonize the body as a commensal but can take advantage of wound sites to establish an infection and proliferate uncontrollably. Opportunistic species commonly associated with chronic wounds include Staphylococcus epidermidis, a skin commensal, Staphylococcus aureus, a colonizer of the nasal passages, Pseudomonas aeruginosa, an environmental species found in soil and water, and Escherichia coli, a member of the gut community.
P. aeruginosa and S. aureus are frequently identified as dominant microbial wound community members 20 and their presence linked to poor outcome. 21 Both species are important opportunistic pathogens, identified by the World Health Organization as priority pathogens, for which novel therapeutics are urgently required. 22 Microbial communities within chronic wounds exist as mixed species biofilm communities. 15,23 Biofilms are defined as communities of microbes that are surrounded by and embedded within a selfproduced extra-polysaccharide matrix (EPS). 24 Their presence within wounds is a significant complication and debridement has little longterm effect on the microbial community. 25 The biofilm's lifestyle limits bacterial clearance by phagocytosis, 26 drives the production of proinflammatory cytokines 27,28 and reduces bacterial susceptibility to antibiotics. 29 EVs are hypothesized to play a significant role in not only interactions between bacterial species within the biofilm, but also between bacteria and the host.

| BACTERIAL AND HOST EXTRACELLULAR VESICLES
EVs are particularly difficult to define definitively, but in general terms are small (<500 nm) membrane bounded vesicles, produced by conserved pathways. 30 Multiple EV types have been identified within both mammalian and bacterial populations. Morphological heterogeneity is commonly observed within EV populations, even when considering a single well-defined and homogeneous parent cell population. 31 Vesicles also harbour complex sets of molecular constituents, which are heterogeneously distributed amongst the EV population.
Normally investigators working with mammalian cells describe their EV preparations as being "enriched" for specific EV types, rather than claiming pure populations, although isolation strategies exhibiting higher resolutions are beginning to be increasingly utilized for more selective sub-population isolations. 32 Diverse mammalian vesicles have been extensively investigated in terms of their biophysical and molecular nature and their varied contributions to pathogenic processes.
Bacterial EVs are much less well studied, although interest within the field is increasing. Markers are yet to be determined for bEVs, with identification currently relying on microscopic observations and size measurements, but some protein profiling studies have also been undertaken. [33][34][35] Within bacterial cultures, the term outer membrane vesicles (OMV) is used to describe vesicles produced by Gramnegative bacteria and membrane vesicle (MV) for Gram-positive vesicles. Herein, the term bacterial EV (bEV) will be used to refer collectively to bacterial OMVs and MVs, while the term hEV refers to host EV. This review focuses predominantly on the role of bEVs produced by S. aureus and P. aeruginosa, respectively, Gram-positive and negative bacteria, because these are not only commonly wound pathogens but are also linked to adverse tissue healing outcomes.

| bEV CONCENTRATION AND COMPOSITION VARIES IN RESPONSE TO GROWTH CONDITIONS AND GENETIC BACKGROUND
4.1 | Isolation and characterization techniques used within bEV studies would benefit from standardization, identification of markers and minimum essential guidance A brief analysis of the bEV-focused manuscripts referenced within this work provided basic information about the methodologies used across the studies (Figure 1 and Table S1). Many of the studies referenced used ultracentrifugation, either alone or in combination with density gradients, for the purification of EVs. In contrast to hEV analysis, production and characterization of the bEV isolates across studies shows little consistency in both techniques presented and the data reported, with minimal vesicle-defining analyses taking place. For the majority of research papers analysed within this study, no more than two characterization assays were performed to determine the quality, concentration and composition of the bEV preparations obtained ( Figure 1(A)). We hypothesis that this is due to a lack of bEV standardization and minimal information guidelines, such as have been published for hEV studies in 2014 and 2018. 36,37 Following purification, many groups measured protein concentration, typically via Bradford Assay, and considered this as an indicator of the concentration of their EV preparations ( Figure 1(B)). Many studies also supported their characterization by carrying out several techniques, most commonly transmission electron microscopy (TEM) and particle analysis, to give further information about the size, density and purity of the preps (Figure 1(B)). Nanoparticle tracking analysis (NTA) is a method of interrogating the particle sizes and particle concentration of EV preparations. Commonly, NTA is carried out using NanoSight instruments, which allow visualization of EVs in suspension as they travel along a capillary tube using a microscope and camera.
Although commonly used in EV studies, it was not frequently used in the bEV studies referenced in this review. NTA systems measure whole particle numbers, with no distinction able to be made between EV particles and other debris in the suspensions. As such care must be taken to measure the particle sizes and concentration of the vehicle used to both prepare EVs and for dilution prior to NTA. This non-specificity is particularly problematic when carrying out NTA using complex and undefined samples, for example, from EV preparations originating from body fluids or cultures growing in rich broths or serum. Recent work indicated that repeated measures using a single machine and operator on one day were reproducible but results varied when machines in two separate geographical locations were used, even when used by the same operator and matched software settings. 38 Similarly, Bachurski et al 39 demonstrated that both NanoSight and ZetaVeiw, both NTA tools, showed poor accuracy in estimating particle concentration and size, respectively.
These studies highlight that although NTA can be a rapid and useful tool to assess quality of EV preparations, the data generated by NTA should be interpreted with care. Bachurski et al 39 concluded that TEM, a tool used in many of the studies described here, was superior to NTA in accurately determining EV particle size and preparation purity.
The majority of the data discussed within this manuscript focuses on the activity of P. aeruginosa and S. aureus bEVs within chronic wounds, and as such the majority of the studies referenced focus on these two species. It is, however, interesting that that majority of studies to date have focused on using lab strains of bacteria, rather than clinical isolates, the latter being more representative of chronic wound isolates. The bEV analysis of P. aeruginosa was dominated by two well commonly used laboratory strains: PA01 and PA14, with greater variation in isolate choice shown within the S. aureus bEV studies. The chosen isolates are genetically tractable, well adapted to laboratory growth and genetically and phenotypically well characterized, making their selection understandable; however, caution must be used when comparing studies and extrapolating clinical significance from the obtained data. Prolonged growth of bacterial isolates, including P. aeruginosa, within the laboratory is known to lead to genetic and phenotypic variations, 40,41 with differences in antimicrobial susceptibility, biofilm formation and virulence between different clinical and laboratory isolates also widely reported. [42][43][44] As such, within this manuscript, caution has been used to interpret how findings obtained from bEV cultures derived from in vitro, single species and planktonic bacterial cultures may translate to bEV activity within the complex, multi-species environment of the chronic wound. Details of each study using bEVs along with bacterial isolates, growth conditions, bEV isolation and characterization techniques are shown in Table S1.

| Culture conditions and genetic background of bacterial isolates are important considerations
Originally bEVs were thought to be a mechanism of clearing misfolded and damaged components from bacterial cells but there is now significant evidence that clearance is not their only role. Conditions in which fundamental bacterial process are inhibited have been shown to increase bEV biogenesis in both P. aeruginosa and S. aureus. For example, oxidative stress but not temperature stress was shown to increase bEV production in P. aeruginosa 45 and the presence of ciprofloxacin, an antibiotic that inhibits DNA replication, increases S. aureus bEV biogenesis. 46 The production of bEVs by bacteria is a dynamic process influenced by several factors. A comparison of two P. aeruginosa strains, PA01 and PA14, reported that PA14 produced significantly more bEVs than PA01 under the same growth conditions, indicating that the genetic background is relevant. 47 However, the bEV production rate of PA01 could be increased by culturing in nutrient-rich medium, 47 highlighting an additional role for environmental factors.  Table S1 Gentamicin also appears to increase bEV release from P. aeruginosa. 48 Although Gentamicin's activity is primarily via inhibition of protein synthesis, it is able to destabilize the cells wall of P. aeruginosa, likely contributing to elevated vesicle output. 49 Both S. aureus and P. aeruginosa have been reported to alter bEV production during different growth phases, with peak bEV production reported during the stationary phase. 50,51 Much of the bEV field to date has focused on collecting vesicles were highly abundant. 53 Taken together, these findings suggest that P. aeruginosa can not only vary bEV-associated proteins depending on its lifestyle but may produce bEVs that are focused less on virulence and more on persistence when in a biofilm community.
The current lack of biofilm bEV-focused studies is a significant limitation in fully understanding how bEVs might contribute to pathogenicity within chronic wounds. As described above, several researchers have reported fundamental differences in the characteristics of bEVs from biofilm or planktonic populations, leading to the assumption that their activity could also differ fundamentally. These studies, combined with the report by Florez et al 47 that nutrient availability can also distinctly alter the number of EVs produced by genetically identical bacterial populations, suggest that only when bEVs are collected in chronic wound relevant conditions can their exact function within the chronic wound be accurately determined. Techniques and growth mediums have now been developed to allow investigation of chronic wound relevant biofilms, [54][55][56][57] raising the exciting possibility that chronic wound relevant bEVs will soon be able to be investigated in greater detail. 5 | BIOGENESIS OF P. aeruginosa AND S. aureus bEV TAKES PLACE VIA BOTH ACTIVE AND PASSIVE MECHANISMS 5.1 | P. aeruginosa bEV biogenesis occurs via binding and deformation of the outer membrane Gram-negative bEV and outer membrane preparations, including those produced by P. aeruginosa, have similar protein profiles, 35,58 suggesting bEV originate from the outer membrane. Several P. aeruginosa bEV biogenesis mechanisms have been proposed, the best described of which utilizes the Pseudomonas quinolone signal (PQS) pathway. PQS plays an important role in population density sensing and is particularly important for stationary phase transition, biofilm formation and maintenance. 59 P. aeruginosa bEV production peaks during stationary phase growth 51 and several groups have confirmed that PQS plays a role in P. aeruginosa bEV biogenesis. [60][61][62][63] Inactivation of genes involved in PQS, such as pqsH and pqsA, significantly reduce P. aeruginosa bEV production 51,64,65 while supplementation with exogenous PQS restores bEV production. 64,65 Interestingly, this was still observed in isolates with defective PQS downstream signalling due to deletion of the transcriptional regulator mvfR, 64 hinting at the possibility that the action of PQS is not due to regulation, but acts as a mechanical factor for biogenesis ( Figure 2, upper panel). As such, P. aeruginosa bEV production is related to the exogenous, not internal, PQS molecule concentration. 47 Interestingly, this effect is not species specific as exogenous P. aeruginosa PQS molecules were reported to increase bEV production in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. 65

| Passive mechanisms of bEV biogenesis may occur in both Gram-negative and positive populations
It is important to note that small amounts of bEVs are produced by pqsA inactivated mutants, suggesting that PQS is not solely responsible for P. aeruginosa bEV biogenesis. Recently, the P. aeruginosa explosive cell lysis required to release eDNA within biofilms was demonstrated to also form membrane vesicles between 110 and 800 nm in diameter, independent of PQS ( Figure 2, lower panel). Vesicle formation was due to recircularization of membrane fragments, trapping local cell cytoplasm components within them. 66 As described for P. aeruginosa, sub-populations of S. aureus lyse during the early stages of biofilm formation, also providing a source of eDNA within the biofilm EPS. 67,68 Although no link has yet been made between S. aureus lysis during biofilm formation and bEV genesis, it is interesting to speculate that this may also contribute to bEV populations within biofilms. Certainly, cell wall disruption within planktonic cultures contributes to S. aureus bEV production. The presence of the β lactam antibiotics flucloxacillin and ceftaroline, which perturb Gram-positive cell walls causing lysis, increased S. aureus bEV formation, as did the presence of lysogenic phages. 46

| Biogenesis of bEVs by S. aureus requires modification and loosening of the bacterial cell wall
The cell walls of Gram-negative bacteria are composed of two thin layers separated by a periplasmic space, whereas the Gram-positive cell well comprises only a single thick peptidoglycan layer. 69 This difference means the cell wall is more rigid and inflexible and therefore resistant to vesicle formation by budding. No definitive method of bEV release has yet been demonstrated for Gram-positive bacteria; F I G U R E 2 Both active and passive bEV biogenesis mechanisms are utilised by the Gram-negative chronic wound pathogen P. aeruginosa. Within P. aeruginosa two mechanisms of biogenesis are shown: genesis via membrane deformation and budding following PQS molecule binding (upper panel) and biogenesis via explosive cell lysis and recircularization of membrane fragments (lower panel). The former mechanism has been demonstrated to be selective, with currently undescribed mechanisms sorting and transporting cargo to the budding membrane, which is deformed, circularised, and eventually separated into a vesicle via binding of PQS molecules to the membrane. In contrast, the latter mechanism of explosive cell lysis is a random process where cargo is packaged only due to its close proximity to the vesicle at the time of recircularization. During this process the membrane ruptures, releasing cell membrane fragments and the cell contents. Membrane recircularization occurs passively and randomly, creating vesicles of varying size and contents. The blue, green and red triangles represent the range of bacterial components which are able to be packaged, both actively or passively, into the Gram-negative bEVs (i.e. siderophores, enzymes, virulence factors, etc.) F I G U R E 3 The S. aureus bEV biogenesis mechanism is fundamentally different to that of P. aeruginosa. Only one mechanism of S. aureus biogenesis has been described in detail to date and is presented here. As with P. aeruginosa PQS dependent biogenesis, S. aureus bEV biogenesis is known to involve selective packaging of components, via a currently unidentified transport mechanism. The process appears to be controlled by the accessory gene regulator (agr) which has a role in controlling many nongrowth functions of S. aureus biology. Vesicle formation occurs via enzymatic loosening of the cell wall to allow membrane blebbing and budding through the newly created cell wall gaps. The blue, green and red triangles represent the range of bacterial components which are able to be packaged into the Gram-positive bEV (i.e. siderophores, enzymes, virulence factors, etc.) nevertheless, it is indisputable that bEV production occurs. 70 Thus far, three models of release have been proposed, including (i) forcing of bEV through the cell wall by turgor pressure, (ii) enzymatic loosening of the cell wall to allow bEV passage and (iii) transport of EVs through channels in the cell wall 71 (Figure 3).
S. aureus bEV proteomic analysis identified enzymes with cell wall components activity, including N-acetylmuramoyl-L-alanine amindase, lipoteichoic acid synthase and penicillin binding proteins 34 ; suggesting that loosening of cell wall bonds could assist bEV release. Phenol soluble modulins (PSM) that have surfactant properties and are able to disrupt membrane integrity [72][73][74] are also important in S. aureus bEV release. 75 Inactivation of single or multiple PSM genes reduced bEV production, whereas overexpression increased bEV numbers. 75 Similarly, inactivation of other peptidoglycan degrading enzymes, Sle1 and Atl, decreased S. aureus bEV production. 75 Taken together, these findings indicate that S. aureus bEV biogenesis occurs when membrane, containing EV components, is forced through enzymatically created cell wall breaks, forming membrane blebs that bud off and form vesicles ( Figure 3).
S. aureus bEV biogenesis has been proposed to be controlled by the accessory gene regulator, agr. As is the case with PQS within P. aureginosa, agr is responsible for regulating multiple S. aureus functions including virulence factor production and biofilm formation. 76 Deletion of argA decreased bEVs to a undetectable level although some bEV related (antibiofilm) activity was still present, suggesting that perhaps bEV production, although undetectable, was not completely lost. 77 In contrast, Wang et al 78 did not see different bEV production in their inactivated agr strain, although the bEVs produced by the mutant showed significantly reduced cytotoxicity to, and ability to, stimulate a pro-inflammatory response in a macrophage cell line (THP1).

| The bEV genesis mechanism may influence both composition and role within the chronic wound
The extent of the differences in the composition and properties of the bEVs formed by different biogenesis mechanisms is still largely unexplored, yet is essential to understanding bEV roles during wound colonization. Preliminary data suggest that inclusion of proteins and genetic material within passively created P. aeruginosa bEVs was random, with no enrichment of specific molecules. 66 DNA sequencing of bEVs created by explosive lysis showed they contained DNA representative of the whole genome. 66 In contrast, bEVs produced by active mechanisms appear to be more selective in specific components, with multiple reports showing that both the protein and genetic profiles of P. aeruginosa and S. aureus bEVs differ from both whole cell and cell membrane profiles. 48 34,70,79,84 Investigation of the "core proteome" of several S. aureus isolate bEVs showed 36% of bEV related proteins were virulence-associated. 84 This is proportionally much higher than would be expected if bEV packaging had simply been random, strongly suggesting the existence of processes for virulence protein selection, inclusion and hence function in the context of an expelled EV. As a comparator, S. aureus protein isolates showed that 102 out of a total of 1135 non-redundant peptides were virulence-associated (comprising only 11% of the total whole cell lysate). 85 5.5 | The role of bEVs, and their components, in driving chronicity of wounds 5.5.1 | bEVs in biofilm formation and maintenance bEVs appear to have multiple functions throughout the biofilm life cycle (see Table 1 for a summary). The bEVs of the oral pathogen Porphyromonas gingivalis stimulate aggregation of S. aureus with Streptococcus spp. and the yeast pathogen Candida albicans, all three of which are also found within chronic wound biofilm communities. 93 P. gingivalis is a poor biofilm former, typically integrating into pre-existing oral biofilms rather than creating de novo biofilms. 97 As such it is interesting to speculate that this pro-aggregation property of P. gingivalis bEVs develops a favourable environment for the organism, increasing its survival. In contrast, the bEVs of S. aureus decreases the attachment of competitor species, in particular Acinetobacter baumannii, to abiotic surfaces. 77 Biofilm EPS typically comprises LPS, eDNA and protein 98 and bEVs have a role in providing EPS materials. Transmission electron microscopy showed that P. aeruginosa bEVs were present in in vitro and in vivo biofilms and a major source of EPS associated LPS. 52 Between 20% and 30% of the total S. aureus and P. aeruginosa biofilm EPS protein content originates from their bEVs. 53,80 Both P. aeruginosa and S. aureus bEV also provide a structural role.
P. aeruginosa bEVs bind biofilm eDNA, 95 and S. aureus bEVs are incorporated into S. aureus biofilms. In the case of one methicillin resistant S. aureus isolate, the presence of the antibiotic ceftazidime decreased EPS production; as such bEVs became the primary EPS component and provided structural support. 96 These studies highlight that bEVs therefore contribute to EPS, and are likely to form important structural elements, which are highly dynamic in responses to therapeutic agents.

| Proteins associated with bEVs protect biofilm communities and aid nutrient acquisition
Iron is an important nutrient for bacteria, and microbial chronic wound communities must be able to obtain free iron from the host environment or nearby microbes. In turn, the host restricts iron availability, reducing colonization. 99 P. aeruginosa and S. aureus bEV each contain several proteins with iron sequestering function. 84 PQS, which is so important in the production of P. aeruginosa bEV, remains associated with bEV after thier formation and has iron chelating activity. 100 In turn it is able to interact with vesicle-associated TseF, a type VI secretion system effector, to deliver iron to P. aeruginosa cells via the outer membrane proteins OprF and FptA. 91 Siderophore associated domains were identified within the bEV of S. aureus isolated from human, bovine and ovine hosts. These domains formed part of the "core" proteome identified in bEVs from all the isolates tested. 84 The identification, and even enrichment, of siderophores within the bEVs of diverse bacterial species, with both pathogenic and commensal functions, highlights the importance of bEVs as an iron sequestering tool. [88][89][90] The proximity of biofilm inhabitants drives exchange of antimicrobial resistance (AMR) genes and molecules. Multiple groups showed the inclusion of β lactamases, conferring penicillin resistance, within bEV. 34,70,83,92 Inclusion appears to be specific to the enzyme with bEV-associated β lactamase reported in both Gram-positive 34 and -negative 92 species. Tetracycline and chloramphenicol resistance cannot be conferred via bEVs, further demonstrating that bEV inclusion is β lactamase specific. 83 Transfer of the β lactamase gene from S. aureus to a recipient E. coli strain led to the expression of functioning β lactamase enzyme within the E. coli bEV. 83 Interestingly, however, the β lactamase was shown to be packed into the lumen of EVs, as treatment of EVs with Protease K did not result in attenuation of β lactamases activity. 83 This raises the interesting question of how are the β lactamases released from the EVs to provide protection? To the best of the authors' knowledge, no mechanism is currently proposed for liberation from EV association and it is not known if this involves bEVs merging with bacterial cells or simply lysing within the extracellular milieu. Despite this, bEV-associated β lactamases are demonstrated to be active and able to protect any microorganisms from penicillin treatment, regardless of species. 83

| bEVs as a source of mobile genetic elements within chronic wound biofilms
Alongside AMR enzymes, the presence of AMR genes within bEVs is of concern as they could lead to the spread of AMR genes throughout biofilm populations. Bacterial genetic competence is the ability of bacterial species to bind and uptake DNA from donors or the environment and incorporate it into their genetic repertoire. Competence can either be naturally present (referred to as naturally competence) or induced in vitro by chemical or electrical means. Once the DNA is T A B L E 1 Summary of the roles bEV play in biofilm formation, maintenance and supporting the biofilm inhabitants Benefactors are dependent on the molecules associated with bEV. To the best of the authors' knowledge, to date only PQS has been shown to be bEV associated. However, if universal quorum sensing molecules (i.e. Autoinducer-2) are also present then multiple species may be able to sense and respond to bEV associated signals. b Bacterial species other than the originating species may also benefit from either increased or decreased attachment and biofilm formation; however, to date no evidence exists that this effect is intended to benefit organisms other than the originating species.
incorporated into a bacterium's genetic repertoire, the cell is referred to as "transformed". 101 P. aeruginosa has recently been shown to be naturally competent within biofilms, 102

| The role of bEVs in promoting competition between bacterial species
The majority of biofilm inhabitants participate in synergistic and/or antagonistic interactions with other community members, creating a high degree of spatial organization. 112 The interactions between P. aeruginosa and S. aureus are usually reported to be antagonistic, with P. aeruginosa dominating cystic fibrosis lung communities. [113][114][115] This paradigm has recently been shown to be more nuanced (for a review see Reference 116), particularly within chronic biofilm infections, such as those found in wounds. In the chronic wound, S. aureus and P. aeruginosa have been shown to co-exist in vivo, although they are spatially separated within the wound. 117 The presence of S. aureus biofilm may increase the biofilm biomass of P. aeruginosa 118 and it has been shown that P. aeruginosa presence can promote S. aureus colonization within an in vivo mouse lung infection model, even when in vitro testing suggested an antagonistic relationship. 119 The role of bEVs within these biofilm interactions is still largely unknown; however, evidence from planktonic studies strongly suggests a role in promoting competition and co-operation.
P. aeruginosa bEVs appear to have significant bactericidal activity, with greater protease, phospholipase C and alkaline phosphatase activity than whole cell lysates. In total, 50% of exogenous alkaline phosphatase activity was linked to P. aeruginosa bEVs. 48 This activity contributes to the ability of P. aeruginosa bEVs to lyse multiple Gramnegative species, although lysis activity was less evident against Gram-positive organisms, with no ability to lyse Staphylococcus sp. observed. 87 In contrast, Mashburn and Whiteley 64 reported the inhibitory activity of P. aeruginosa bEVs against S. epidermidis, an organism closely related to S. aureus and also commonly identified within chronic wounds. 120  Similarly, in murine studies, S. aureus bEVs stimulated a pro-inflammatory response when either inhaled into the lung or applied directly to tape stripped skin. 126,127 It must also be noted that evidence is emerging to indicate that the host is also able to degrade bEVs and package components into hEVs (for an overview, see Schorey et al 128 ). This is important for the immunosurveillance of intracellular pathogens, a lifestyle demonstrated by both S. aureus and P. aeruginosa, [129][130][131][132][133] although to the best of the authors' knowledge the presence of bEV components from these bacterial species within hEVs has not yet been determined (Figure 4 upper panel).
The bEVs from some S. aureus isolates also have a cytotoxic effect on host cells (Figure 4 lower panel). This is not unexpected as many S. aureus bEV factors, including α-, δand γ-haemolysin, S. aureus M060 contained exfoliative toxin A, 79 suggesting that perhaps this compound was responsible for the observed cytotoxicity. In contrast to S. aureus, there are few reports of P. aeruginosa bEV cytotoxicity, probably due to a lack of relevant studies rather than activity.
One preliminary study has reported cytotoxicity to airway epithelial cells, 122 suggesting that further studies are likely to confirm that P. aeruginosa has similar cytotoxicity described for S. aureus. Both Broadly the activity of bEV within chronic wounds can be divided into two distinct areas: 1) driving wound chronicity by stimulating a host inflammatory response (upper panel) and 2) association of bEVs with components improving bacterial survival (lower panel). These components have multiple roles ranging from causing cell death and lysis thereby releasing nutrients to sequestering of freed nutrients for use by the originating bacterial cells. Small yellow circles denote bEV whole orange circles represent hEVs. Blue, orange and green triangles represent the cell components (i.e., proteins, metal compounds, etc.) which are able to able to be sequestered by local bacteria or their bEV bacterial cultures, highlighting their potency. 144 The pro-wound healing mechanism of activity was suggested to be based on the photosynthetic ability of the bacterium and the improved oxygenation this brought to damaged tissue. 145 However, in vivo healing was also reported in mice kept in dark conditions, suggesting that the mechanism is likely to be multi-factorial. 144 There has also been significant interest in the potential of hEVs, Although MSC hEV studies to date have shown significant promise in stimulating chronic wound healing both in vitro and in vivo, several limitations need to be overcome before therapeutics can be developed efficiently. Increasing the scale of hEV production and improving product purity and constancy must be addressed (for an overview of the challenges see 157,158 ). The use of 3D tissue culture, rather than monolayer culture, has been shown to improve hEV yield 159,160 as has exposing cell lines to low intensity ultrasound radiation. 161 Alterations in EV generation must be carried out with great care, however, as alterations in the conditions originating cells are exposed to are reported to alter the composition of both bEV (discussed above) and hEV. 162 As such, care must be taken to show that changes to EV generation and purification processes do not impact the specific components of interest.
The wound environment itself also provides challenges for thera-  163 Similarly, a chitosan hydrogel incorporating endometrial stem cell hEVs improved closure of mouse full thickness wounds and enhanced fibroblast migration in vitro. 164 As well as improving in vivo wound closure, in vitro antimicrobial activity has been reported for hEV impregnated hydrogels, 165 although not all the hydrogel types tested were antibacterial, suggesting that both the hEV and gel type may be important in determining the final activity of the product.

| Bacterial EVs as a potential vaccine candidate
Identifying suitable vaccine candidates for S. aureus and P. aeruginosa Much however, still remains to be discovered; fundamental mechanisms of bEV biogenesis such as the targeting and packaging of bEV contents is still poorly understood, yet as described above the con- Finally, there is a lack of bEV markers. Although we recognize that the intra-and inter-species diversity of prokaryotes will likely make identification of "universal" bacterial markers impossible, it would be helpful for classification purposes to identify molecular factors to help define bEV subsets. The standardization and classification of human vesicle types, as described in the MISEV2018 guidelines, 37 have been essential to increase the robustness of the Eukaryotic EV field, and the bEV field would benefit greatly from a similar approach. Markers of bEV allowing differentiation between bacterial genus and/or species would be particularly useful, especially when attempting to investigate the complex hEV and bEV populations that are found within human infections, including chronic wounds. Currently, although it is possible to isolate whole EV populations from chronic wounds, it is not possible to separate bEV and hEV. Markers specific for bEV might make this possible in the future. An approach similar to that applied to shotgun metagenomics might prove useful in this instance. Shotgun metagenomics harvests and sequences the total DNA from any community, regardless of its source. Computational analysis of the sequencing reads then allows the community to be interrogated and separated into host and DNA from various microbial sources.
This allows identification of bacterial, fungal/yeast and viral isolates to the species level and highlights the presence of AMR and virulence genes within the community. 170 Multi-omics methods, combining analysis of DNA, gene and protein expression are now being used to investigate biofilm communities 171 and such approaches could be applied to EV populations in the future. Unfortunately, all 'omics tools' are heavily reliant on the availability of well curated databases and knowledge of markers that allow components to be linked to likely originating species or genus. Since databases are not yet widely available for EVs, the focus must currently be on not only identifying markers and components, but creating freely available and curated databases, such as are now available for DNA and protein analysis. Biofilm communities are very rarely composed of a single species, and the interactions within these communities are important for persistence. As we demonstrate in this review, it is abundantly clear that bEVs have a critical role in these interactions, but a better understanding of bEV biology and type is required before these findings can be confidently tested.

ACKNOWLEDGEMENT
Funding for HLB is provided by the Dunhill Medical Trust (Grant ID RPGF1902\133).