Surface-Active Compounds Produced by Microorganisms: Promising Molecules for the Development of Antimicrobial, Anti-Inflammatory, and Healing Agents

Surface-active compounds (SACs), biomolecules produced by bacteria, yeasts, and filamentous fungi, have interesting properties, such as the ability to interact with surfaces as well as hydrophobic or hydrophilic interfaces. Because of their advantages over other compounds, such as biodegradability, low toxicity, antimicrobial, and healing properties, SACs are attractive targets for research in various applications in medicine. As a result, a growing number of properties related to SAC production have been the subject of scientific research during the past decade, searching for potential future applications in biomedical, pharmaceutical, and therapeutic fields. This review aims to provide a comprehensive understanding of the potential of biosurfactants and emulsifiers as antimicrobials, modulators of virulence factors, anticancer agents, and wound healing agents in the field of biotechnology and biomedicine, to meet the increasing demand for safer medical and pharmacological therapies.


Introduction
Microorganisms can produce several surface-active compounds (SACs) with hydrophilic and hydrophobic moieties. These structural features allow them to interact with the surface and interfacial tensions, form micelles, and emulsify immiscible substances [1,2].
Biosurfactants (BSs) and bioemulsifiers (BEs) are considered SACs because of their ability to interfere and with modifying surfaces. Because these biomolecules are amphiphilic and are produced by different microorganisms, they have different physicochemical properties and physiological roles, which contribute to their specific functions in nature and biotechnological applications [3].
The present use of these biomolecules has aroused interest from several sectors because of their numerous functions and sustainable properties, allowing various applications in

Microorganisms Producing SACS
For many years, researchers have tirelessly searched for microorganisms that have the potential to produce secondary metabolites with surfactant or emulsifying properties. The amount of BS or BE produced depends on the type of microorganisms and their sources ( Table 1).

Antimicrobial Activities
The discovery of antibiotics in the last century can be considered a major advancement in medicine because the use of these antimicrobial agents significantly reduced morbidity and mortality associated with microbial infections. Antibacterial and antifungal factors reduce and eliminate the viability and growth of microbial populations through several mechanisms: (i) disruption of extracellular membranes and/or their cell wall, (ii) inhibition of gene expression, (iii) DNA damage, or (iv) manipulation of important metabolic pathways [74].
Bacteria become resistant to antimicrobial agents in several ways: through horizontal gene transfer between genetic elements of different strains and the environment that confer resistance and through mutations that interfere with basic cell functions in addition to conferring resistance to microorganisms [75,76].
The most resistant bacteria associated with serious hospital infections include Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, P. aeruginosa, and Enterobacter sp., which often result in high mortality rates [77]. Furthermore, other microorganisms such as Candida spp. can also be considered a global health threat because of their resistance to antimicrobial agents [78][79][80].
The increasing rates of antimicrobial resistance and the emergence of new microbial pathogens reinforce the need to find new antimicrobial compounds to fight microbial infections. Among these new strategies, SACs have promising antibiotic and disinfectant potential, as well as antibiotic delivery properties due to their physicochemical properties. Most of these biomolecules can break the outer and inner membranes of pathogens, thereby exploiting their charge and hydrophobicity. The advantages of using SACs as antimicrobials include their broad-spectrum bactericidal action and the absence of pathogen resistance mechanisms [81].
Cationic surfactants comprise the largest class of synthetic surfactants with antimicrobial properties because of their broad spectrum of biostatic and biocidal activities against planktonic pathogens. The hydrophobic chain of cationic surfactants penetrates the microbial cell membrane and interferes with membrane continuity and metabolic processes, leading to cell death [82]. Despite exhibiting antimicrobial efficiency mainly against Grampositive bacteria (29-32 mm), such as S. aureus and Bacillus subtilis, these compounds are less biodegradable than natural surfactants [83].
Previous studies reported the antimicrobial efficacy of glycolipid SACs produced by microorganisms. For example, RLs produced by P. aeruginosa significantly inhibited the growth of S. mutans UA159 and S. sanguinis ATCC10556. Furthermore, they completely inhibited the growth of Aggregatibacter actinomycetemcomitans Y4 at high concentrations [7].
Similarly, the synergistic action of two RL BSs produced by P. aeruginosa C2 and Bacillus stratosphericus A15 demonstrated bactericidal activity by rupturing the membrane of grampositive and gram-negative bacteria, such as S. aureus ATCC 25923 and Escherichia coli K8813 [84]. Because of these actions, the membrane disintegrates, leading to penetration into the cell wall and plasma membrane through the formation of pores, followed by leakage of internal cytoplasmic materials, leading to cell death [85].
A previous study demonstrated that the synergism between essential oils of oregano, cinnamon tree, and lavender with RLs produced by P. aeruginosa increased the antimicrobial effect against Candida albicans and S. aureus which are resistant to methicillin [86], revealing that SAC activity can be enhanced when they establish a synergistic relationship with other compounds. In addition to RLs, SLs are also easily extracted and are usually produced by Candida spp. yeast [87] either in the lactone form or the acid form or as a mixture of both forms [88,89].
A previous study showed that SL produced by C. albicans SC5314 and Candida glabrata CBS138 showed antibacterial properties against pathogenic bacteria Bacillus subtilis and E. coli [10]. Besides its antibacterial activity against both Gram-positive and Gram-negative bacteria, this class of BS also exhibited promising antifungal activity against pathogenic fungi including Colletotrichum gloeosporioides, Fusarium verticillioides, Fusarium oxysporum, Corynespora cassiicola, and Trichophyton rubrum [90].
The antimicrobial activity of SACs glycolipids was found to be dependent on the type of glycolipid and the interaction with the cell membrane. Diaz de Renzo et al. [63] demonstrated that RLs inhibit bacterial growth in the exponential phase while SLs inhibit growth between the exponential and stationary phases.
The antimicrobial potential of lipopeptide SACs has also been recognized; these biomolecules correspond to the most important components of metabolites that are synthesized by many species of the genus Bacillus spp., which characterize the strains of this genus as important parts of plant disease control and food safety [91][92][93].
Antimicrobial lipopeptides, such as iturin, fengycin, and surfactin, have been identified in Bacillus velezensis HC6. Surfactin exhibited strong antibacterial effects against Listeria monocytogenes and Bacillus cereus, while fengycin and iturin inhibited the growth of pathogenic fungi Aspergillus flavus, Aspergillus parasiticus, Aspergillus sulphureus, Fusarium graminearum, and Fusarium oxysporum [94]. Researchers also found that when B. velezensis HC6 is applied to corn, it reduced the levels of aflatoxin and ochratoxin produced by fungi.
Ohadi et al. [95] demonstrated that lipopeptides produced by Acinetobacter junii displayed nonselective activity against Gram-positive and Gram-negative bacterial strains. The data showed that this bioproduct had effective antibacterial activity at concentrations almost below the CMC and that the minimal inhibitory concentration (MIC) values were lower than the standard antifungal activity, exhibiting almost 100% inhibition against Candida utilis.
Other broad classes of bacterial metabolites with surface-active potential and antimicrobial effects include glycoproteins, peptides, and fatty acids. Lactobacillus spp. produced a bioactive glycolipoprotein surfactant with antimicrobial activity against C. albicans using sugarcane molasses as substrate, and some pathogenic gram-positive and gram-negative bacteria [96]. A cyclic heptapeptide containing a fatty acid moiety produced by Bacillus subtilis, called bacaucin 1, exhibited specific antibacterial activity against methicillin-resistant S. aureus (MRSA) by disrupting the membrane without detectable toxicity to mammalian cells or induction of bacterial resistance. In addition, this peptide was found to be efficient in preventing infections in both in vitro and in vivo models [97].
Finally, some microorganisms excrete mixtures of bioactive compounds with surfacereducing ability and emulsifying potential. For example, the actinomycete strains of Strep-tomyces griseoplanus NRRL-ISP5009 produced a BS component that is a complex mixture of proteins, carbohydrates, and lipids that have antimicrobial activity against gram-positive bacteria (Bacillus subtilis, S. aureus) and pathogenic fungi (C. albicans and Aspergillus fumigatus). However, it is only moderately active against Gram-negative bacteria E. coli and Salmonella typhimurium [37].

Antiviral Activity
Viruses represent a serious threat to human health at a global level. Previous studies have described secondary metabolites with surface-active properties for their antiviral properties against a variety of viruses. Antiviral activity by SACs was shown to be effective against various viruses, enveloped and nonenveloped ( Table 2). Viral infections represent one of the main causes of human and animal morbidity and mortality that lead to significant healthcare costs [107]. Therefore, secondary metabolites with surface-active properties should be considered promising substances for the development of antiviral compounds.

Anti-Inflammatory Activity
Inflammatory responses represent a crucial aspect of a tissue's response to certain injuries, chemical irritation, or microbial infections. This complex response involves leukocyte cells, macrophages, neutrophils, and lymphocytes. In response to inflammation, these cells release specialized substances, including amines and vasoactive peptides, eicosanoids, pro-inflammatory cytokines, and acute-phase proteins, which mediate the inflammatory process and prevent additional tissue damage [108].
Other studies showed that surfactin systematically induced CD4 + CD25 + FoxP3 + Tregs in the spleen of mice, which inhibit T cells from producing pro-inflammatory cytokines such as TNF-α and interferon (IFN)-γ. Moreover, surfactin attenuation of chronic inflammation increased IL-10 expression in atherosclerotic lesions of the aorta of mice, demonstrating that BSs can restore the balance in the Th1/Th2 response in mice [110], as well as induce the maturation of dendritic cells (DCs) and increase the expression of MHC-II molecules and other costimulatory factors [111].
In previous in vivo models, SLs reduced sepsis-related mortality and exhibited antiinflammatory effects in mice by inhibiting nitric oxide and inflammatory cytokine production [114,115]. On the other hand, the glycolipid complex had no significant effect on the proliferative effect of peripheral blood leukocytes because it activated the production of pro-inflammatory cytokines (IL-1β and TNF-α) without affecting the IL-6 production in vitro in the monocyte fraction [116].

Anticancer Activity
Cancer is considered a multistage disease, the etiology of which is associated with high incidence and mortality rates globally. Chemotherapy drugs, surgery, and radiation remain the most common treatments to fight the disease in humans. However, they are all associated with serious adverse effects, indicating the lack of specificity and the need to discover new antitumor agents to improve the effectiveness of conventional chemotherapy drugs while reducing the adverse effects [74].
For these purposes, several studies have demonstrated the antitumor potential of several SACs (Table 3). Biosurfactants have been proposed as suitable drug candidates for many diseases including cancer [117]. Given their wide applications, the interest in exploring their role in promoting human health continues to grow.

Antibiofilm Activity
Biofilms comprise microbial communities attached to the surface and embedded in an extracellular matrix composed of extracellular polymeric substances (EPS) secreted by cells that reside within them. In general, EPS is a mixture of polysaccharides, proteins, extracellular DNA (eDNA), and other smaller components. The biofilm matrix constituents' physical and chemical properties enable the matrix to protect resident cells from desiccation, chemical disturbance, invasion by other bacteria, and death from predators. However, biofilms often cause major medical problems and are the cause of chronic infections because biofilm communities can house bacteria that are tolerant and persistent against antibiotic treatment and are more resistant to antibiotics compared with planktonic bacteria [9,122].
Because of their composition, biofilms cause a wide range of chronic diseases due to the emergence of antibiotic-resistant bacteria that have become difficult to treat effectively. To date, available antibiotics are ineffective in treating these biofilm-related infections because of their higher MIC and minimal bactericidal concentration values, which may lead to in vivo toxicity. Therefore, designing or tracking antibiofilm molecules that can effectively minimize and eradicate biofilm-related infections is important [123].
Because of their antimicrobial, antiadhesive, and antibiofilm properties, SACs can be used to neutralize biofilm formation and the emergence of drug-resistant microorganisms [14]. These biomolecules tend to interact with antimicrobials [124,125], which are usually less effective against biofilms in general and multispecies biofilms associated with extremely complicated polymicrobial infections.
A mixture of lipopeptides (surfactin, iturin, and fengycin), which are synthesized by B. subtilis, prevented biofilm formation by inhibiting cell adhesion of Trichosporon spp. by up to 96.89% and dispersed mature biofilms (up to 99.2%), reducing their thickness and cell viability. This mixture reduced cell ergosterol content and altered the membrane permeability and surface hydrophobicity of planktonic cells [126].
Meanwhile, surfactin-type BS produced by B. subtilis reduced adhesion and stopped the formation of S. aureus biofilm on glass, polystyrene, and stainless-steel surfaces. Surfactin significantly decreased biofilm percentage and reduced icaA and icaD expressions, which are important for staphylococcal biofilm structure. Furthermore, lipopeptides have been shown to affect the quorum sensing (QS) system in S. aureus by regulating the autoinducer 2 activity [94].
In terms of the antibiofilm activity of glycolipids, Allegrone et al. [128] reported the effects of different types of RLs. They demonstrated that RL produced by P. aeruginosa 89 (R89BS) was 91.4% pure and comprised 70.6% of monorhamnolipids and 20.8% of dirhamnolipids. The pure extract R89BS inhibited S. aureus adhesion (97%) and biofilm formation (85%). Furthermore, purified monorhamnolipids (mR89BS) have been observed to induce dispersion of preformed biofilms at all concentrations (0.06-2 mg/mL) by 80-99%, unlike the pure extract R89BS and purified dirhamnolipids (dR89BS), which depended on the tested concentration.
Ceresa et al. [5] demonstrated that R89BS-coated silicone elastomeric disks significantly neutralized Staphylococcus spp. biofilm formation in terms of accumulated biomass (up to 60% inhibition in 72 h) and cellular metabolic activity (up to 68% inhibition in 72 h). The results suggested that RL coatings may be an effective antibiofilm treatment procedure and represent a promising strategy for preventing infections associated with implantable medical devices.
Shen et al. [129] demonstrated that besides inhibiting the formation of new biofilms, RLs were superior in eradicating mature biofilms formed by Helicobacter pylori, E. coli, and Streptococcus mutans in a dose-dependent manner, compared with other antibacterial agents even at concentrations below minimum inhibitory concentrations (MICs). They can enhance the effect of antimicrobial agents. Sidrim et al. [130] observed that these molecules significantly increased the activity of meropenem and amoxicillin-clavulanate against mature Burkholderia pseudomallei biofilms.
Rhamnolipids produced by P. aeruginosa SS14 also inhibited planktonic cells of filamentous fungi of Trichophyton rubrum and Trichophyton mentagrophytes. The formation and rupture of mature biofilms were dose-dependent, with the highest activity observed at concentrations of 2 × MIC against both pathogens [131].
Like RLs, SLs exhibited an effective inhibitory activity against biofilm formation. Ceresa et al. [132] obtained three different SL products: SLA (acid congeners), SL18 (lactonic congeners), and SLV (mixture of acid and lactone congeners), which all showed an inhibitory effect of 70%, 75%, and 80% for S. aureus, P. aeruginosa, and C. albicans, respectively. Using 0.8% w/v SLA on pre-coated medical silicone disks reduced S. aureus biofilm formation by 75%. In co-incubation experiments, 0.05% w/v SLA significantly inhibited S. aureus and C. albicans from forming biofilms and adhering to surfaces by 90-95% at concentrations between 0.025 and 0.1% w/v.
Antibiofilm activities were also demonstrated for BSs produced by probiotics of the genus Bacillus sp. that were isolated from cervicovaginal samples. This bioproduct, called BioSa3, was highly effective in the formation of biofilms of different pathogenic and multidrug-resistant strains, such as S. aureus and Staphylococcus haemolyticus. The anti-biofilm effect may be related to the ability of BioSa3 to alter the membrane physiology of the tested pathogens to cause a significant decrease in surface hydrophobicity [133].
Thus, SACs are good candidates for the emergence of new therapies to better control multidrug-resistant microorganisms and inhibit infections associated with biofilms, protecting surfaces from microbial contamination.

Wound Healing
Wound healing is an important but complicated process of tissue repair in humans or animals, comprising a multifaceted process organized by sequential and overlapping phases, including hemostasis, inflammation phase, proliferation phase, and remodeling phase [134,135]. Failure of one of these phases caused by a deregulated immune response or insufficient oxygenation impairs the healing process, leading to ulcerative skin defect (chronic wound) or excessive scar tissue formation (hypertrophic or keloid scarring) [136,137].
Treating wounds of different etiologies constitutes an important part of the total health budget, mostly affected by three important cost drivers: curing time, frequency of dressing change, and complications. Moreover, chronic wound infection, one of the leading causes of nonhealing, contributes significantly to rising healthcare costs. Although the treatment of an uncomplicated surgical incision is relatively inexpensive, the costs can increase significantly when infections occur [138].
Biofilms, commonly found in chronic wounds, contribute to infections, causing slower healing. Infections in chronic wounds are usually caused by multiple species [139], with P. aeruginosa and S. aureus being the most common. Although most microbial communities usually form on the wound's outer layer, some biofilms are also embedded in deeper layers, such as P. aeruginosa biofilms, which are difficult to diagnose via traditional wound smear culture [140,141]. Moreover, antibiotic resistance of bacteria in biofilms is a crucial problem in the management and treatment of chronic wounds [139].
For these reasons, physicians and the scientific community consider the management and treatment of wounds, as well as biofilm prevention, a top priority. In this context, SACs recently emerged as promising agents that promote wound healing with low irritation and high compatibility with human skin [14]. Furthermore, these bioproducts promote fibroblast and epithelial cell proliferation, faster re-epithelialization, and collagen deposition, leading to a faster healing process [142,143].
Surfactin A from B. subtilis promotes wound healing and scar inhibition. During the healing process, it up-regulates the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor, accelerates keratinocyte migration via mitogenactivated protein kinase (MAPK), and factor nuclear-κB (NF-κB) signaling pathways and also regulates pro-inflammatory cytokine secretion and macrophage phenotypic exchange. Furthermore, surfactin A inhibits scar tissue formation by influencing α-smooth muscle actin (α-SMA) and transforming growth factor (TGF-β) expression [144]. Therefore, the healing potency of the lipopeptides B. subtilis SPB1 is due to their antioxidant activity potential revealed in vitro [143].
A previously unknown lipopeptide 78 (LP78) from S. epidermidis inhibited TLR3mediated skin inflammation and promoted wound healing. The skin lesion activated TLR3/NF-κB, promoting p65 and PPARγ interaction in the nuclei and initiating the inflammatory response in keratinocytes. Next, LP78 activated the TLR2-SRC, inducing β-catenin phosphorylation in Tyr. Phospho-β-catenin is translocated into the nuclei to bind to PPARγ, thereby interrupting the p65 and PPARγ interaction. Dissociation between p65 and PPARγ reduced TLR3-induced inflammatory cytokine expression in skin wounds of normal and diabetic mice, which correlated with faster wound healing [145].
As an alternative to improve this healing process, the formulation of nanolipopeptide biosurfactant (NLPB) from the lipopeptide biosurfactant (LPB) produced by Acinetobacter junii was reported as promising for performing healing activity. The percentage of wound closure of mice treated with NLPB hydrogels at 2 mg/mL was approximately 80% on day 7 and 100% on day 15. The NLPB hydrogel formulation showed better efficacy in wound closure and healing when compared to the control [146].
A BS of glycolipid nature, which was synthesized by Bacillus licheniformis SV1, showed good cytocompatibility and increased 3T3/NIH fibroblasts proliferation in vitro. A previous study showed that the application of BS ointment in a skin excision wound in rats promoted re-epithelialization, fibroblast cell proliferation, and faster collagen deposition, demonstrating its potential transdermal properties to improve skin wound healing [147].
A previous study administered an RL-containing ointment (5 g/L) on the back of Wistar mice after creating an excision wound. Histopathological results revealed a significant healing effect of RL, demonstrating increased wound closure, improved collagenases, and reduced inflammation (decreasing the level of TNF-α) without inducing skin irritation [84]. Dirhamnolipid treatment has been suggested for cutaneous scar therapy, demonstrating an antifibrotic function in rabbit ear hypertrophic scar models with a significant reduction in the scar elevation index, type I collagen fibers, and α-SMA expression [148].
A cell culture model has demonstrated the wound healing capacity of SLs by using an in vitro human dermal fibroblast model as a substitute for human skin, revealing that SLs affected the ability of human skin fibroblasts to express collagen I mRNA (Col-I) and elastase inhibition (IC 50 = 38.5 µg/mL) [112]. In addition, Kwak et al. (2021), using an in vitro wound healing assay in human colorectal adenocarcinoma (HT-29) cell line, showed a significantly increased collagenase-1 expression (p < 0.05) 48 h after SL treatment. Moreover, all SL dosages significantly increased occludin and matrilysin-1 (MMP-7) expression [149].

Other Considerations
We also consider that there are SACs molecules obtained by chemical synthesis processes, such as ultrashort synthetic surface active (USSA) [150,151]. Some of these can be synthesized as C-terminal amides on Rink amide (4-Methylbenzhydrylamine (MBHA) resin using 9-fluorenylmethoxycarbonyl/t-butylcarbamate [151]. The fundamental difference of the USSA, as lipopeptoids (modified SAC) in relation to the natural ones, is their immunomodulatory capacity. As seen in mouse infection models, they reduce the exacerbation of the disease, even if not presenting direct antibacterial activity [151]. This characteristic would be a limiting activity, since many natural ones lead to a disturbance of biological membranes, with antifungal and antibacterial actions [151].
New possibilities can be obtained for the SACs, as transformation systems applying recombinant plasmids have been employed to substantially increase the productivity of microbial biosurfactants, e.g., the engineered strain Pseudozyma sp. SY16, which increases the production of mannosylerythritol lipids (MELs) by up to 31.5%, suggesting that genetic engineering can improve the industrial application of yeast [152].

Conclusions
The BS and BE surface-active compounds have drawn the attention of the scientific community as a new generation of products with high potential in the biomedical and pharmaceutical fields. Their use, whether alone or in combination with other antimicrobial or chemotherapeutic agents, opens paths for new strategies to prevent and combat infections caused by bacteria, fungi, and viruses, as well as the formation and proliferation of biofilms. Furthermore, new anticancer treatments and wound healing applications can be explored in future studies.
These molecules affect various biological activities, making them suitable candidates for use in new generations of agents in the biotechnological, biomedical, and pharmaceutical fields. However, it is necessary to investigate their applications, cost-effectiveness, and availability further.