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Cornea  |   November 2014
Antimicrobial Role of Human Meibomian Lipids at the Ocular Surface
Author Notes
  • School of Medicine, University of Western Sydney, Penrith, New South Wales, Australia 
  • Correspondence: Poonam Mudgil, School of Medicine, Campbelltown Campus, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia; p.mudgil@uws.edu.au
Investigative Ophthalmology & Visual Science November 2014, Vol.55, 7272-7277. doi:10.1167/iovs.14-15512
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      Poonam Mudgil; Antimicrobial Role of Human Meibomian Lipids at the Ocular Surface. Invest. Ophthalmol. Vis. Sci. 2014;55(11):7272-7277. doi: 10.1167/iovs.14-15512.

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Abstract

Purpose.: Human meibomian lipids form the outermost lipid layer of the tear film and serve many important functions to maintain its integrity. Although not investigated earlier, these lipids may have antimicrobial properties that help in strengthening the innate host defense of tears at the ocular surface. The aim of this study was to investigate the antimicrobial role of human meibomian lipids.

Methods.: Ocular pathogenic bacteria, Staphylococcus aureus 31, Pseudomonas aeruginosa 19, Pseudomonas aeruginosa 20, and Serratia marcescens 35, were grown in the presence and absence of human meibomian lipids in an artificial tear solution at the physiological temperature. Viable counts were obtained to note the number of bacteria surviving the treatment with meibomian lipids. Bacterial cells were imaged using scanning electron microscopy to observe the damages caused by meibomian lipids.

Results.: Viable count results showed that in the presence of meibomian lipids, growth of all bacteria was considerably lower. Scanning electron microscopy showed that meibomian lipids caused extensive cellular damage to bacteria as manifested in smaller size, loss of aggregation, abnormal phenotype, cellular distortion, damaged cell wall, and cell lysis.

Conclusions.: This is the first-ever report of the antimicrobial role of human meibomian lipids. These lipids possess antimicrobial properties against both Gram-positive and Gram-negative bacteria and are involved in the innate host defense of tears in protecting the ocular surface against microbial pathogens.

Introduction
Human meibomian lipids secreted by meibomian glands form the outermost lipid layer of the tear film at the ocular surface. These lipids serve multiple physiological functions to maintain integrity of the tear film, acting, for example, as a barrier to prevent skin lipids from entering into the tear film and to avoid spillover of tears, as a lubricant to facilitate smooth movement of eyelids during blinking, as a blanket to retard evaporation of aqueous tears, and as surfactants to maintain a low surface tension of the tear film. Research shows that meibomian lipids form highly compressible, noncollapsible, multilayered liquid films—characteristics particularly suited for maintaining stability and functioning of the tear film.1 
Meibomian lipids have been extensively studied for their composition2,3 and various functions,1,48 but a completely unexplored area of investigation is the antimicrobial role these lipids might play in providing the innate host defense to tears at the ocular surface. This role is likely because lipids found in other body secretions have been shown to possess antimicrobial properties that help in the intrinsic host defense. 
One of the prime examples is skin lipids, a complex mixture of lipids, some of which are carried to the surface from epidermis and others of which are secreted onto the surface by sebaceous glands. Skin lipids contain fatty acids, cholesterol, ceramides, sphingosine bases (from stratum corneum)911 and squalene, wax monoesters, and triglycerides (from sebum).12,13 The antimicrobial property of skin lipids, particularly fatty acids present therein, against Staphylococcus aureus is known.14,15 Fatty acids also prevent bacterial adherence by making the skin surface acidic.16 Sphingosines in skin lipids are known to be potent antibacterials.17 Lipids in vernix caseosa, a white substance covering the skin of the fetus and newborns, protect neonates from infections.18 
Besides those in skin, many other body lipids with antimicrobial properties have been shown to provide innate host defense in humans. Oral mucosal and salivary lipids show antimicrobial activity against bacteria, Prophyromonas gingivalis, which colonize the oral cavity and cause periodontitis.19 Sinus secretions contain elevated levels of antimicrobial lipids in rhinosinusitis that contribute to the innate host defense of the respiratory tract.20 Similarly, human milk lipids defend infants against microbial infections. Breastfeeding decreases the amount of bacteria in the gastrointestinal tract, and it is suggested that lipids play a role in the antimicrobial defense in addition to proteins (lysozyme and lactoferrin) in the milk.21,22 
Meibomian lipids, like skin lipids, are a complex mixture of nonpolar and polar lipids. These include wax esters; cholesteryl esters; diesters; mono-, di-, and triglycerides; cholesterol; and fatty acids.2,3 The diversity in the species of lipids means each type can have its own effect adding to the overall property of lipids. For example, it is known that fatty acids are broad-spectrum antimicrobials with potency comparable to that of antimicrobial peptides14,23,24; cholesterol and cholesteryl esters show antimicrobial activity against Streptococcus pneumonia25 and Pseudomonas aeruginosa,26 respectively; and mono- and triglycerides are effective antimicrobials.27 Since these lipid species and many more are found in meibomian lipids, they are likely to affect a variety of pathogens encountered by the tear film. 
The aim of this research was to study the antibacterial property of human meibomian lipids against known ocular pathogens in an artificial tear fluid at the physiological temperature. The findings of this study help in elaborating the functions of human meibomian lipids and advancing understanding of the innate defense mechanism of tears to combat corneal ocular infections, which in acute cases can lead to impaired visual function. 
Materials and Methods
Bacterial media and components were obtained from Oxoid (Thermofisher Scientific, Adelaide, SA, Australia). Glutaraldehyde was obtained from ProSci Tech (Kirwan, QLD, Australia). All other chemicals were purchased from Sigma (Castle Hill, NSW, Australia), and HPLC-grade chloroform from Sigma was used as a solvent for meibomian lipids. 
Meibomian Lipids
Collection of human meibomian lipids from healthy volunteers was done as described by Mudgil et al.6 in accordance with the Declaration of Helsinki. Eyelids were pressed between sterile cotton tips to express meibum. Lipids were collected with a sterile spatula in a glass tube containing chloroform. Samples were pooled, vacuum evaporated, and reconstituted at 1 mg/mL in chloroform. 
Bacterial Strains and Culture Conditions
Bacterial strains S. aureus 31, P. aeruginosa 19, P. aeruginosa 20, and Serratia marcescens 35 were obtained from Mark Willcox, University of New South Wales, Australia. These strains have been used in earlier studies,2830 and characteristics of these bacterial strains are given in the Table. Bacterial cultures were always freshly streaked on LB (Luria-Bertani) plates and allowed to grow at 37°C overnight before use. For long-term storage, glycerol cultures were maintained at −80°C. 
Table.
 
Characteristics of Bacterial Strains
Table.
 
Characteristics of Bacterial Strains
Bacteria Source Mechanism/Action Reference
S. aureus 31 Contact lens peripheral ulceration Causes ulceration 28
P. aeruginosa 16 (6294) Contact lens–associated microbial keratitis Invasive 29
P. aeruginosa 20 (6206) Cornea–keratitis Cytotoxic 29
S. marcescens 35 Microbial keratitis Causes microbial keratitis 30
Antimicrobial Testing
Bacterial cultures were grown in Tryptone Soy Broth overnight at 37°C. Cells were centrifuged (2,000g for 10 minutes), washed, suspended in Artificial Tear (AT) solution, and equilibrated to 106 CFU (colony-forming units)/mL. The AT solution was derived from the artificial tear solution of Mirejovsky et al.31 and emulated the salt composition and pH of tears (NaCl 6.6 g/L; KCl 1.7 g/L; NaHCO3 1.4 g/L; lactic acid 0.27 g/L; CaCl2·2H2O 0.15 g/L, NaH2PO4·H2O 0.1 g/L; glucose 0.8 g/L; MOPS 4.18 g/L; pH 7.4). Meibomian lipids solution (20 μL) was slowly added drop-wise using a microsyringe onto AT solution, and chloroform was allowed to evaporate for 30 minutes. Control contained just the AT solution without meibomian lipids. A chloroform control in which 20 μL solvent chloroform was added to AT was also included. Bacterial suspension was added (1 in 10) to the AT solution and incubated at 35°C on a shaking incubator for 24 hours. Cultures were serially diluted and plated on LB agar plates and incubated at 37°C for 24 hours, and CFU/mL was calculated. Experiments were repeated on three separate occasions with three replicates and gave similar results. 
Electron Microscopy
Bacterial cells after incubation with and without meibomian lipids were centrifuged and washed three times with 0.1 M sodium phosphate buffer (pH 7.4). Cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) overnight at 4°C. Cells were washed three times with sodium phosphate buffer. Dehydration was done in graded ethanol series (30%, 50%, 70%, 80%, 90%, and 100%) followed by incubation in 100% ethanol for 1 hour. The cells were then spread on silica wafers and examined using a JEOL JSM-7001F Field Emission Scanning Electron Microscope (JEOL Ltd., Akishima, Tokyo, Japan) at 5 kV. Cells were examined across multiple experiments and multiple fields to present the representative micrographs. 
Results
Growth of all four bacterial isolates in the presence of meibomian lipids was considerably lower in comparison to the bacteria grown without meibomian lipids (Fig. 1). Growth of bacteria in control (just AT) and chloroform control (AT and chloroform) was similar, showing that chloroform was completely evaporated in the experiment and did not have an effect on the bacterial growth. Viable counts in CFU/mL were converted into log10 values as presented in Figure 1
Figure 1
 
Log10 CFU/mL bacterial cells grown in the absence and presence of meibomian lipids in artificial tear solution. Viable counts (converted into log10 CFU/mL) of S. aureus 31, P. aeruginosa 19, P. aeruginosa 20, and S. marcescens 35, grown without and with meibomian lipids, show considerably lower number of viable cells in the presence of meibomian lipids, indicating the antimicrobial nature of these lipids. Results represent mean of three experiments with three replicates (bar: standard error) for each bacterial pathogen.
Figure 1
 
Log10 CFU/mL bacterial cells grown in the absence and presence of meibomian lipids in artificial tear solution. Viable counts (converted into log10 CFU/mL) of S. aureus 31, P. aeruginosa 19, P. aeruginosa 20, and S. marcescens 35, grown without and with meibomian lipids, show considerably lower number of viable cells in the presence of meibomian lipids, indicating the antimicrobial nature of these lipids. Results represent mean of three experiments with three replicates (bar: standard error) for each bacterial pathogen.
Scanning electron microscopy (SEM) of bacterial cells grown in the presence of meibomian lipids showed various forms of damage to the cell morphology, ranging from smaller size to cell lysis. 
Staphylococcus aureus 31 control cells grown without meibomian lipids exhibited typical staphylococcal morphology of round cells present in groups (Fig. 2A). Scanning electron microscopy of cells treated with meibomian lipids showed fewer numbers of cells, mostly separated from each other. Cells were smaller in size (Fig. 2B) and broken (Fig. 2C) and showed lysis (Figs. 2D, 2E). Parts of lysed cells were seen lying around (Fig. 2F). Very few healthy cells could be seen. 
Figure 2
 
Scanning electron microscopy of S. aureus 31 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were smaller in size (B), broken (C), and lysed (D, E), and parts of lysed cells were lying around (F).
Figure 2
 
Scanning electron microscopy of S. aureus 31 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were smaller in size (B), broken (C), and lysed (D, E), and parts of lysed cells were lying around (F).
Pseudomonas aeruginosa 19 control cells grown without meibomian lipids exhibited typical morphology of rod-shaped elongated cells present in groups (Fig. 3A). Scanning electron microscopy of cells treated with meibomian lipids showed very few cells, mostly scattered. Some cells were seen in groups, but very few groups were seen. Various forms of damage to cells were seen, such as smaller size, distorted cells (Fig. 3B), bleb formation (Fig. 3C), twisted and broken cells (Fig. 3D), lysed cells (Fig. 3E). Contents of some cells were seen leaking and spilling out (Fig. 3F). 
Figure 3
 
Scanning electron microscopy of P. aeruginosa 19 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were distorted (B), showed blebs (C), were twisted and broken (D), and were lysed (E), and leaky contents were spilling out (F).
Figure 3
 
Scanning electron microscopy of P. aeruginosa 19 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were distorted (B), showed blebs (C), were twisted and broken (D), and were lysed (E), and leaky contents were spilling out (F).
Pseudomonas aeruginosa 20 control cells grown without meibomian lipids exhibited typical morphology of rod-shaped elongated cells present in groups (Fig. 4A). Scanning electron microscopy of cells treated with meibomian lipids showed fewer numbers of cells, mostly single cells. Types of damage included smaller size, broken cells (Fig. 4B), distorted cells with blebs (Fig. 4C), lysed cells (Fig. 4D), and contents leaking out of the cell (Figs. 4E, 4F). 
Figure 4
 
Scanning electron microscopy of P. aeruginosa 20 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A), and cells grown with meibomian lipids were broken (B), showed blebs (C), and were lysed (D), and leaky contents were spilling out (E, F).
Figure 4
 
Scanning electron microscopy of P. aeruginosa 20 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A), and cells grown with meibomian lipids were broken (B), showed blebs (C), and were lysed (D), and leaky contents were spilling out (E, F).
Serratia marcescens 35 control cells grown without meibomian lipids exhibited typical morphology of rod-shaped cells present in groups (Fig. 5A). Scanning electron microscopy of cells treated with meibomian lipids showed overall fewer numbers of cells; some were in groups and others were scattered. Damages were seen as cellular distortions with concave and wrinkled cells (Figs. 5B, 5C), abnormal phenotype with flat cells and raised blebs (Fig. 5D), and cell lysis (Figs. 5E, 5F). 
Figure 5
 
Scanning electron microscopy of S. marcescens 35 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were concave and wrinkled (B, C), showed abnormal phenotype with flat cells and raised blebs (D), and were lysed (E, F).
Figure 5
 
Scanning electron microscopy of S. marcescens 35 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were concave and wrinkled (B, C), showed abnormal phenotype with flat cells and raised blebs (D), and were lysed (E, F).
Discussion
This study reports for the first time the evidence for the antimicrobial role of human meibomian lipids. The results show that these lipids possess antimicrobial properties against both Gram-positive and Gram-negative bacteria and thus contribute to the innate host defense mechanism of tears in protecting the ocular surface against microbial pathogens. The study has relevance to the physiological conditions of the eye because experiments were performed in a tear-like solution at physiological temperature and pH using clinical ocular surface pathogens. 
Tears act as the first line of defense, fighting against microbial pathogens by physical and chemical means.3234 Physical mechanisms are exhibited by reflex tearing and washing action of tears, which retard bacterial attachment to the ocular surface. Chemical mechanisms are exhibited by an assortment of tear proteins including bactericidal lysozyme, bacteriostatic lactoferrin and lipocalin, phagocytic secretory IgA, and so on. So far, knowledge of antimicrobial compounds in tears has been limited to the presence of proteins.35 This study indicates that meibomian lipids that are part of tears also play a role in the host defense. It appears that lipids and proteins both contribute to the innate host defense mechanism of tears. This interplay is relevant to other body secretions as well, such as those from skin, oral and nasal mucosa, and respiratory fluid. All of these contain proteins as well as lipids. Multiple antimicrobials with different mechanisms of action would help body fluids in getting rid of a broad spectrum of pathogens, and cooperative interactions between these molecules would mean that they can be effective at lower concentrations. Indeed, synergistic interactions between antimicrobial lipids and proteins for intrinsic host defense have been demonstrated in some reports.18,26,36 
A review of the normal ocular microbiota shows that numbers of microorganisms that can be isolated from tears (<100 CFU/μL) are strikingly less than those isolated from saliva (107–108 CFU/μL).37 Although these secretions have similar antimicrobial proteins for innate defense, the apparent difference in the microbial load may be attributed to the lipid quantities that the two secretions contain. While the amount of total lipids in human saliva is found to be 0.013 mg/mL,38 a quantitative estimation of tear lipids shows approximately 7 mg/mL in the normal eye,39 which is some 500 times higher. This adds to the results of this study and to the assertion that besides proteins, lipids too play a role in the innate antimicrobial defense and help in strengthening it. This is further supported by the association of decreased lipids levels with lower host defense. For example, decreased levels of fatty acids and sphingosines make atopic dermatitis patients vulnerable to colonization by S. aureus.40,41 Removal of lipids from the nasal fluid decreases its antimicrobial activity, and their resupplementation restores the activity.26 Lipids added to human milk and infant formulas provide increased protection from infection to infants,42 and children feeding on low-fat milk are five times more susceptible to gastrointestinal infection than those on whole milk.43 
Scanning electron microscopy of bacterial cells in this study helps in exploring the potential antimicrobial effects of meibomian lipids on ocular pathogens. These lipids caused various forms of cellular damage, including smaller size, loss of aggregation, abnormal phenotype, cellular distortion, damaged cell walls, and cell lysis. The extent of damage was wide because fewer numbers of cells were seen; most of these were damaged, with very few staying intact. Similar cellular damage, noticed using SEM, is done to P. gingivalis by oral mucosal lipids.19 Sphingoid bases found in skin also cause similar intracellular and extracellular damage to S. aureus, which has been revealed by SEM.44 
The mechanism of antimicrobial action of meibomian lipids would be difficult to predict due to its mixture nature. However, knowledge of the action of different lipid classes can help in understanding the likely effect of these lipids on bacteria. For example, fatty acids have been shown to exhibit deleterious detergent effects by interacting with the cell membrane, causing pore formation, leakage, and cell lysis.14 Some of these effects were seen in the SEM results of this study. Fatty acids can also affect bacterial energy production by disrupting the electron transport chain and oxidative phosphorylation.14 Other lipid classes such as cholesterol and phospholipids can exert their action by affecting virulence factors and direct killing and inhibition of biofilm formation.25,45 Given the SEM results of this study and the fact that meibomian lipids possess surfactant properties,1 membrane destabilization by surfactant/detergent activity14,19 may be a likely mechanism of their antimicrobial action. 
In summary, this study provides the evidence for the antimicrobial role of human meibomian lipids. This is the first report of the antimicrobial role of lipids in the intrinsic host defense in the ocular environment. This adds to the overall concept of lipids playing a role in the innate host defense in humans. Different lipid classes in meibomian lipids are expected to exhibit different antimicrobial mechanisms, and together they create effective and potent antimicrobial protection. Since these lipids are present in tears along with proteins, as in other body secretions, cooperative interactions between them provide a robust intrinsic host defense that is reflected in the self-sterilizing property and low microbial load of tears at the ocular surface. Future research will involve studying antimicrobial properties of distinct lipid classes of meibomian lipids to identify their therapeutic potential for treating ocular surface infections. 
Acknowledgments
The author thanks John Whitehall, FRACP, University of Western Sydney, for his helpful critique, and Richard Wuhrer, PhD, UWS Advanced Materials Characterisation Facility (AMCF), for training on scanning electron microscopy. 
Disclosure: P. Mudgil, None 
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Figure 1
 
Log10 CFU/mL bacterial cells grown in the absence and presence of meibomian lipids in artificial tear solution. Viable counts (converted into log10 CFU/mL) of S. aureus 31, P. aeruginosa 19, P. aeruginosa 20, and S. marcescens 35, grown without and with meibomian lipids, show considerably lower number of viable cells in the presence of meibomian lipids, indicating the antimicrobial nature of these lipids. Results represent mean of three experiments with three replicates (bar: standard error) for each bacterial pathogen.
Figure 1
 
Log10 CFU/mL bacterial cells grown in the absence and presence of meibomian lipids in artificial tear solution. Viable counts (converted into log10 CFU/mL) of S. aureus 31, P. aeruginosa 19, P. aeruginosa 20, and S. marcescens 35, grown without and with meibomian lipids, show considerably lower number of viable cells in the presence of meibomian lipids, indicating the antimicrobial nature of these lipids. Results represent mean of three experiments with three replicates (bar: standard error) for each bacterial pathogen.
Figure 2
 
Scanning electron microscopy of S. aureus 31 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were smaller in size (B), broken (C), and lysed (D, E), and parts of lysed cells were lying around (F).
Figure 2
 
Scanning electron microscopy of S. aureus 31 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were smaller in size (B), broken (C), and lysed (D, E), and parts of lysed cells were lying around (F).
Figure 3
 
Scanning electron microscopy of P. aeruginosa 19 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were distorted (B), showed blebs (C), were twisted and broken (D), and were lysed (E), and leaky contents were spilling out (F).
Figure 3
 
Scanning electron microscopy of P. aeruginosa 19 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were distorted (B), showed blebs (C), were twisted and broken (D), and were lysed (E), and leaky contents were spilling out (F).
Figure 4
 
Scanning electron microscopy of P. aeruginosa 20 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A), and cells grown with meibomian lipids were broken (B), showed blebs (C), and were lysed (D), and leaky contents were spilling out (E, F).
Figure 4
 
Scanning electron microscopy of P. aeruginosa 20 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A), and cells grown with meibomian lipids were broken (B), showed blebs (C), and were lysed (D), and leaky contents were spilling out (E, F).
Figure 5
 
Scanning electron microscopy of S. marcescens 35 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were concave and wrinkled (B, C), showed abnormal phenotype with flat cells and raised blebs (D), and were lysed (E, F).
Figure 5
 
Scanning electron microscopy of S. marcescens 35 cells grown in the absence and presence of meibomian lipids. Control cells without meibomian lipids showed typical morphology (A). Cells grown with meibomian lipids were concave and wrinkled (B, C), showed abnormal phenotype with flat cells and raised blebs (D), and were lysed (E, F).
Table.
 
Characteristics of Bacterial Strains
Table.
 
Characteristics of Bacterial Strains
Bacteria Source Mechanism/Action Reference
S. aureus 31 Contact lens peripheral ulceration Causes ulceration 28
P. aeruginosa 16 (6294) Contact lens–associated microbial keratitis Invasive 29
P. aeruginosa 20 (6206) Cornea–keratitis Cytotoxic 29
S. marcescens 35 Microbial keratitis Causes microbial keratitis 30
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