Wax esters protect tears from evaporative stress and help in preventing drying of the ocular surface. Being present in the outermost layer of the tear film, they serve as a mechanical barrier to microbial invasion. Wax esters are characteristically present in tear lipids produced by meibomian glands, and in skin lipids produced by sebaceous glands (Robosky et al., 2008; Smith and Thiboutot, 2008). Their presence on the outer surface of the body, such as skin, has a barrier function that provides protection from the outside environment and desiccation. Wax esters such as behenyl oleate and behenyl palmitoleate have been shown to have antifungal properties (Frank et al., 2018). The antibacterial properties of wax esters are not widely documented in literature, possibly due the fact that their extreme hydrophobicity makes it difficult to conduct conventional in vitro antimicrobial assays where bacterial pathogens need to be grown and tested in aqueous media.

Cholesterol ester inhibit growth of ocular pathogenic bacteria S. aureus and P. aeruginosa but do not kill these bacteria as revealed in an in vitro study using physiological conditions of tears (daSilva-Antunes et al., 2016). Cholesterol esters contribute to the inherent antibacterial activity of human nasal mucosa and Do et al. (2008) have demonstrated that cholesteryl linoleate and cholesteryl arachidonate exhibit direct antibacterial activity against P. aeruginosa and S. epidermidis in vitro, with cholesteryl linoleate being a more potent antibacterial lipid with a broader spectrum. Elevated levels of cholesteryl esters observed in sinus secretions of chronic rhinosinusitis patients (Lee et al, 2010) and bronchoalveolar lavage fluid of cystic fibrosis patients (Ma et al., 2015) indicate contribution of cholesterol esters to the innate host defense of the respiratory tract. In addition, cholesteryl linoleate in a liposome carrier has been shown to exhibit antibacterial activity against S. epidermidis and P. aeruginosa and lowering the minimum inhibitory concentration of vancomycin for vancomycin resistant Enterococcus faecalis (Cheung Lam et al., 2016).

Glycerides have antibacterial and antiviral properties. Monoglycerides containing linoleic acid show antiviral effects against enveloped vesicular stomatitis virus and cause reduction in virus titre in the antiviral activity assays in vitro (Thormar et al., 1987). Monocaprin, a monoglyceride containing capric acid, has been shown to be effective in killing three Gram-positive bacteria: Group A Streptococcus (GAS), Group B Streptococcus (GBS) and S. aureus. The study of the mode of action of monocaprin against GBS showed that the mechanism of killing was disruption of cell membrane because electron micrographs of bacteria treated with the lipid showed disintegrated cell membrane with cell wall left intact. It was suggested that the highly lethal effect of this monoglyceride could be utilized for treating infections caused by GBS (Bergsson et al., 2001). In an earlier work from the same group, monocaprin was shown to inactivate Chlamydia trachomatis by disrupting the membrane of the elementary bodies of the bacteria suggesting use of this lipid as a microbicidal agent (Bergsson et al., 1998). Monocaprin are also virucidal against herpex simplex virus, respiratory syncytial virus and parainfluenza virus (Hilmarsson et al., 2005; Hilmarsson et al., 2007). Another monoglyceride, monolaurin, shows a high antibacterial potency against S. aureus that is even greater than the antimicrobial effect of the fatty acid, lauric acid, derived from it (Kabara et al., 1972). Synergistic activities of monoglycerides have also been observed against Gram-positive bacteria, Streptococcus pyogenes and S. aureus (Batovska et al., 2009). Antimicrobial properties of monoglycerides and their fatty acids against various bacterial species suggest their possible therapeutic applications as alternative to antibiotics for combating infections (Churchward et al., 2018; Yoon et al., 2018).

Triglycerides are abundantly present in the human milk fat globules. Triglycerides upon hydrolysis by gastric lipases in the stomach of newborns produce free fatty acids and monoglycerides that can lyse the enveloped viruses, bacteria, and protozoa (Hamosh et al., 1999). Monoglycerides act additively with fatty acids and their combined concentration determines the antimicrobial lipid activity of human milk in which microbial inactivation happens by membrane destabilization (Isaacs, 2001). Similarly, triglycerides in skin lipids serve as source of fatty acids that act as potent antimicrobials at the skin surface (Drake et al., 2008; Fischer et al., 2014)

Cholesterol being the main sterol in tear lipids was tested in an in vitro study and it inhibited the growth of clinical strains of eye pathogens, S. aureus, P. aeruginosa and S. marcescens, at low concentrations but it showed a little or no inhibitory effect at high concentrations (daSilva-Antunes, 2013). In this study, cells treated with cholesterol showed abnormal phenotype and loss of cellular content in scanning electron micrographs (daSilva-Antunes, 2013). In a previous study, Marquart et al. (2007) reported 1% cholesterol to be bactericidal against Streptococcus pneumonia in vitro and lower concentration of cholesterol being partially inhibitory in a concentration dependent manner. They proposed that topical application of cholesterol might be useful for the treatment of S. penumonae keratitis because cholesterol can inhibit pneumolysin and kill bacteria. Another study shows that targeted regulation of membrane cholesterol content is used as a host defense strategy to evade bacterial toxins that damage the animal cells by pore formation in the cell membrane (Zhou et al., 2020).

Oxysterols, the oxidation derivatives of cholesterol, have broad antiviral activity against enveloped and non-enveloped human viral pathogens (Lembo et al., 2016). Two oxysterols, namely 25-hydroxycholesterol and 27-hydroxycholesterol, possess broad antiviral activity and are involved in innate antiviral defense. 25-hydroxycholesterol has antiviral activity against Zika virus (Tricarico et al., 2019). 27-hydroxycholesterol present in colostrum is effective against paediatric viral pathogens, rotavirus and rhinovirus, suggesting that breastfeeding helps in transfer of protective factors to infants in the initial days of lactation (Civra et al., 2019). Cholesterol and its oxysterols also modulate the hepatic innate immune response against Hepatitis C virus (HCV) infection. It is proposed that cholesterol modifications can be used for adjuvant therapy and clinical management of patients with HCV infection (González-Aldaco et al., 2018).

Tears contain about 2% of free fatty acids and out of these oleic acid is the main fatty acid reported, although the amounts reported in literature vary a lot and are dependent on the analytical techniques used (Chen et al., 2010). In our previous study, oleic acid inhibited growth of clinical strains of eye pathogens, S. aureus, P. aeruginosa and S. marcescens in a concentration dependent manner with 1% concentration showing complete growth inhibition (Mudgil et al., 2014). Cells treated with oleic acid showed cellular distortions and cell lysis in scanning electron micrographs. Given its antimicrobial activity, oleic acid can be used to develop lipid-based treatment for eye infections helping in reducing antibiotics usage. Antibacterial activity associated with tear lipids identified in these bacteria may be relevant to other Gram-positive and Gram-negative bacteria with applications in treating a range of bacterial ocular infections. The mechanism by which oleic acid or other unsaturated fatty acids such as linoleic acid or palmitoleic acid may exhibit antibacterial action is by inhibition of bacterial fatty acid synthesis. These unsaturated fatty acids inhibit bacterial enoyl-acyl enoyl-acyl carrier protein reductase (FabI) which is an essential component of bacterial fatty acid synthesis (Zheng et al., 2005). FabI is responsible for catalysis of the final and rate-limiting step of the fatty acid chain elongation in bacteria. Inhibition of FabI by oleic acid and palmitoleic acid is noted against S. aureus and S. pyogenes but these lipids are not effective against Escherichia coli or P. aeruginosa (Zheng et al., 2005). Oleic acid and linoleic acid, also induce cell and protoplast lysis of Streptococcus faecalis by acting as membrane destabiliser (Carson and Daneo-Moore, 1980).

Oleic acid is also found in skin lipids and is known to be antibacterial against skin pathogens including methicillin-resistant S. aureus (MRSA) and group A streptococci (Speert et al., 1979; Chen et al., 2011). It kills S. aureus bacteria by breaking down the cell walls and is effective against many S. aureus strains including the multi-antibiotic resistant community associated MRSA USA 300 (Chen et al., 2011). Palmitoleic acid is another antimicrobial fatty acid in mammalian skin that protects against S. aureus and Gram-positive bacterial infections (Wille and Kydonieus, 2003; Parsons et al., 2012). It permeabilizes the cell membrane causing leakage of solutes and low-molecular-weight proteins into the medium (Parsons et al., 2012). Free fatty acids provide defense against S. aureus in healthy skin and their deficiency increases vulnerability of atopic dermatitis patients to colonization by S. aureus (Takigawa et al., 2005). They also create unfavorable growth conditions for bacteria by making the skin surface acidic (Fluhr et al., 2001). Sapienic acid is the major antimicrobial fatty acid uniquely present in human skin that arrests growth of S. aureus by countering the bacterial defense mechanisms (Subramanian et al., 2019). Sapienic acid has been shown to be antibacterial against Streptococcus sanguinis, Streptococcus mitis, and Fusobacterium nucleatum but not against E. coli, S. aureus, S. marcescens, and P. aeruginosa (Fischer et al., 2012). Lauric acid found in skin is a very potent antimicrobial and shows antibacterial activity against Gram-positive bacteria including S. aureus, S. mitis, S. sanguinis, Corynebacterium striatum, and Corynebacterium jeikeium, but it is not active against Gram-negative bacteria E. coli, S. marcescens, or P. aeruginosa, although it is active against F. nucleatum (Fischer et al., 2012). Lauric acid was found as the most potent fatty acid among a number of saturated fatty acids investigated against methicillin sensitive and resistant S. aureus (Kitahara et al., 2004). The strong antimicrobial effects of lauric acid against Propionibacterium acnes in vitro and in vivo show that it can be used as an alternative treatment for antibiotic therapy of acne vulgaris (Nakatsuji et al., 2009). A number of fatty acids including lauric acid and capric acid are antibacterial against GAS, GBS, S. aureus, and C. trachomatis (Bergsson et al., 1998; Bergsson et al., 2001), Fatty acids are also virucidal against herpes simplex virus and their activity increases even more in the acidic environment (Hilmarsson et al., 2005).

Host-derived fatty acids found in human milk play an important role in providing innate defense to newborns and infants. Antiviral activity in the human milk appears after storage at 4°C for 2 days. It reduces viral titre by as much as 10,000-fold and is due to antiviral fatty acids in the milk (Isaacs et al., 1986). The medium-chain saturated and long-chain unsaturated fatty acids in human milk are active against enveloped viruses vesicular stomatitis virus, herpes simplex virus, and visna virus in vitro. The antiviral activity results in the leakage of the viral envelope, and at higher concentration of fatty acids there is a complete disintegration of the envelope and the viral particles (Thormar et al., 1987). Fatty acids may exert a detergent-like effect on lipid-coated microbes. They can incorporate into the lipid membrane causing instability, which in turn results in the rupture of the membrane and death of the organism (Isaacs et al., 1986; Thormar et al., 1987).

Fatty acids typically have broad-spectrum antimicrobial effects. Unsaturated fatty acids generally have more antimicrobial effects than saturated fatty acids (Kabara et al., 1972; Zheng et al., 2005). Investigating antimicrobial effects of a variety of fatty acids, Kabara (1984) concluded that saturated fatty acids have highest activity when the chain length is C12 (lauric acid), monounsaturated fatty acids have highest activity in parmitoleic acid and the most active polyunsaturated fatty acid is linoleic acid. Another study with different Gram-positive bacteria indicated that unsaturated fatty acids having C18 chains such as oleic acid, linoleic acid and linolenic acid have potent antimicrobial activities (Galbraith et al., 1971). It is known that medium- and long-chain unsaturated fatty acids generally have more antimicrobial effects against Gram-positive bacteria in comparison to Gram-negative bacteria (Galbraith et al., 1971; Chen et al., 2011). The outer membrane of Gram-negative bacteria protects them from the destructive action of fatty acids (Speert et al., 1979). Fatty acids packaged in liposomes can be promising lipophilic antimicrobial agents. Palmitic acid and steric acid in liposome preparations have shown antibacterial activity against multidrug resistant S. epidermidis and vancomycin resistant E. faecalis (Cheung Lam et al., 2016). Lauric acid incorporated in a liposome kills P. acnes. The liposome fuses with the membrane of bacteria and releases the fatty acid directly on the bacterial membranes to kill the bacteria efficiently (Yang et al., 2009).

A number of mechanisms have been proposed for the antimicrobial action of fatty acids. They mainly target the bacterial cell membrane and affect cellular protection and functions (Desbois and Smith, 2010; Yoon et al., 2018). The proposed mechanisms include (1) exhibiting deleterious detergent effects on the cell membrane causing pore formation, leakage, and cell lysis, (2) interfering with the cellular energy production by disrupting the electron transport chain and uncoupling oxidative phosphorylation, (3) inhibiting membrane enzymes activity and nutrient uptake, and (4) formation of hydroperoxides causing oxidative stress. Fatty acids can insert into the bacterial cell membrane and increase its permeability. This membrane-lytic action causes destabilization, pore formation, leakage of contents and cell lysis (Greenway and Dyke, 1979; Speert et al., 1979; Carson and Daneo-Moore, 1980; Chamberlain et al., 1991). The electron transport chain in the cell membrane of bacteria is the site for energy production. Medium- and long-chain saturated and unsaturated fatty acids can disrupt the electron transport chain by binding to the electron carriers resulting in reduced energy production (Galbraith and Miller, 1973; Kenny et al., 2009). Fatty acids can further reduce energy production through uncoupling of oxidative phosphorylation by decreasing the membrane potential and proton gradient, or by directly binding with the ATP synthase (Sheu and Freese, 1972; Galbraith and Miller, 1973). Fatty acids can directly inhibit membrane enzymes and target membrane-associated proteins in bacteria, for example, inhibiting glucosyl transferase affecting glucan production (Won et al., 2007), and inhibiting enoyl-acyl enoyl-acyl carrier protein reductase (FabI) affecting fatty acid synthesis (Zheng et al., 2005). These effects are generally greater for unsaturated fatty acids than saturated fatty acids (Zheng et al., 2005). Fatty acid can starve bacteria by inhibiting their ability to uptake nutrients such as amino acids (Galbraith and Miller, 1973). Formation of hydroperoxides causing oxidative stress is another mechanism suggested for the bactericidal effects of polyunsaturated fatty acids (Knapp and Melly, 1986).

Squalene shows antimicrobial activities against bacteria, Sarcina lutea and E. coli, and fungi causing aspergillosis (Biswas and Chakraborty, 2013). Squalene also has anti-oxidative properties and used as adjuvant in vaccines and cosmetics (Fox, 2009). Squalene in microemulsions has been shown to be antimicrobial against MRSA (Fang et al., 2019), and effective in the treatment of COVID-19 patients (Ebrahimi et al., 2022).

The in vitro studies with the ocular pathogenic bacteria S. aureus and P. aeruginosa show that phosphatidylcholine (PC) inhibits bacterial growth slightly but time kill assays indicate that it does not kill these bacteria (daSilva-Antunes et al., 2016). Lysophosphatidic acid (LPA), a polar lipid involved in cell proliferation and wound healing, has a protective role in the activation of innate immune response and it enhances antimycobacterial activity both in vitro and ex vivo (Garg et al., 2006). LPA is present in solution form bound to albumin in many extracellular fluids including aqueous humor in the eye and is released in vitro. It is termed as a ‘bioactive’ phospholipid whose receptors and metabolic enzymes can be promising pharmacological targets in finding relevance of bioactivity of LPA in vivo (Saulnier-Blache, 2004).

Phospholipids can themselves be inhibitory to bacterial growth or enhance activity of antibiotics against antibiotic-resistant bacteria by increasing permeability of outer membrane to antibiotics through their ability to chelate divalent cations. P. aeruginosa is an opportunistic pathogen that causes chronic lung infection in patients with cystic fibrosis. Biofilm formation by this bacteria enhances development of cystic fibrosis. Monopalmitoylphosphatidic acid (MPPA), a host-derived lysophospholipid that accumulates in inflammation, has been shown to slow the growth of antibiotic-resistant strains of P. aeruginosa isolated from sputum of cystic fibrosis patients (Krogfelt et al., 2000). It hinders pathogenesis of P. aeruginosa PAO1 by inhibiting bacterial virulence factors such as extracellular accumulation of alginate, elastase, LasA protease, and siderophore pyoverdin, as well as biofilm formation (Laux et al., 2002). The inhibitory effect of MPPA is partly attributed to its ability to bind divalent cations and to physically disrupt the bacterial membrane structure. Phospholipids can also enhance the activity of β-lactam antibiotics against P. aeruginosa strains (Krogfelt et al., 2000). MPPA enhances activity of ampicillin in vitro against P. aeruginosa PAO1 by chelating divalent cations, and enhances the activity of pireracillin and ceftazidime against P. aeruginosa strains isolated from cystic fibrosis patients (Krogfelt et al., 2000). Liposomes containing bioactive lipids such as phosphatidic acid and phosphatidylinositol stimulate pulmonary cells to kill drug-resistant bacterial pathogens by augmenting immune response showing that liposomes delivered bioactive lipids enhance antimicrobial response and can be used as an additional host-directed strategy for the control of chronic drug-resistant infections (Poerio et al., 2017).

The anionic surfactant lipids of the lung, phosphatidylglycerol (PG) and phosphatidylinositol (PI), exert potent antiviral activities in vitro and in vivo against respiratory viruses including respiratory syncytial virus and influenza A virus (Numata et al., 2010; Numata et al., 2012; Numata et al., 2015). PG and PI thus paly complementary role in innate immune antiviral defense in the lung. Surfactant preparations containing PG can be used for treating respiratory viral infections and potentially for improving lung function in COVID-19 patients (Bollag and Gonzales, 2020; Ji et al., 2021).

Oxidized phospholipids that are naturally released from dead cells block the replication of RNA viruses during the early stage of viral infection in human epithelial cells (Ernandes and Kagan, 2021). Slowing viral growth prior to infection by oxidized phospholipids allows time for other immune mechanisms to take over, hence, helping in innate host defense against RNA viral infections. Endogenously produced oxidized phospholipids inhibit inflammation and provide protection from lethal endotoxin shock in severe Gram-negative bacterial infections showing that oxidized phospholipids that have ability to inhibit endotoxins can be used for developing drugs for sepsis (Bochkov et al., 2002).

Sphingomyelin, the phospholipid or more specifically a sphingophospholipid, present in human milk provides protection to neonates from bacterial infections (Silva et al., 2021). Bactericidal activity of sphingolipids of milk lipids against pathogenic strains of E. coli, Salmonella enteritidis, Campylobacter jejuni, Listeria monocytogenes has been shown in vitro (Sprong et al., 2001).

Sphingosines, the base in the sphingophospholipid molecules, are potent broad acting antimicrobial present on the skin and constitute the innate immune defense of skin (Drake et al., 2008; Fischer et al., 2014). It is proposed that exogenous application of these lipids to skin can be a therapeutic option for people at risk of infection (Fischer et al., 2014). Reduced levels of sphinogosine is associated with vulnerability of atopic dermatitis patients to colonization by S. aureus (Arikawa et al., 2002). The antibacterial activity of sphingosine has been shown in vitro against many Gram-negative bacteria and Gram-positive bacteria including E. coli, S. mitis, S. aureus, S. sanguinis, Corynebacterium bovis, C. striatum, C. jeikeium, and F. nucleatum but not against S. marcescens and P. aeruginosa (Fischer et al., 2012). Sphingosines are also antibacterial against S. pyogenes, Micrococcus luteus, P. acnes, Brevibacterium epidermidis, and Candida albicans in vitro (Bibel et al., 1992). The bactericidal activity of sphingosine against S. aureus has been shown in vitro, and the in vivo infections in mice indicate that lack of sphingosine causes susceptibility to lung infection by Staphylococcus aureus in cystic fibrosis (Tavakoli Tabazavareh et al., 2016). Sphingosine is directly involved in pathogenic defense and provides protection from P. aeruginosa infections (Baker et al., 2018). In vitro studies indicate that sphingosines are antibacterial against P. aeruginosa, Acinetobacter baumannii, and Moraxella catarrhalis, and in vivo studies in mice indicate that sphingosine can prevent lung infection by P. aeruginosa in cystic fibrosis patients (Pewzner-Jung et al., 2014).

Ceramides are derived from sphingomyelins and they have been shown to regulate mammalian defense against P. aeruginosa and S. aureus pathogens that are commonly found in pneumonia (Baker et al., 2018). Ceramides also have potent bactericidal activity against pathogenic Neisseriae. Antibacterial activity of ceramides has been observed against Neisseria meningitidis and Neisseria gonorrhoeae in vitro with kinetic assays showing killing of N. meningitidis within 2 h (Becam et al., 2017).

OAHFAs found in tears are also found in equine amniotic fluid and semen (Wood, 2020), and vernix caseosa in newborns (Kalužíková et al., 2017). OAHFAs act as surfactants in these fluids. While antimicrobial action of OAHFAs is not well reported, antimicrobial properties of other hydroxyl fatty acids are known. Hydroxy polyunsaturated fatty acids exert antiviral activity against influenza virus by interfering with the binding of virus to host cell receptors and reducing viral titres (de Toledo-Piza et al., 2018). Hydroxy fatty acids also show antibacterial activities against Gram-positive bacteria, Bacillus subtilis, L. monocytogenes, S. aureus, and Gram-negative bacteria, P. aeruginosa (Shin et al., 2004; Desbois and Lawlor, 2013).