February 2016
Volume 57, Issue 2
Open Access
Cornea  |   February 2016
Glycerol Monolaurate Inhibits Lipase Production by Clinical Ocular Isolates Without Affecting Bacterial Cell Viability
Author Affiliations & Notes
  • Judith Louise Flanagan
    Brien Holden Vision Institute Sydney, Australia
    School of Optometry and Vision Science, University of New South Wales, Sydney, Australia
  • Neeta Khandekar
    Brien Holden Vision Institute Sydney, Australia
  • Hua Zhu
    Brien Holden Vision Institute Sydney, Australia
    School of Optometry and Vision Science, University of New South Wales, Sydney, Australia
    Vision Cooperative Research Centre, Sydney, Australia
  • Keizo Watanabe
    Kinki University School of Medicine, Osaka, Japan
  • Maria Markoulli
    School of Optometry and Vision Science, University of New South Wales, Sydney, Australia
  • John Terence Flanagan
    Sydney Business School, University of Wollongong, Wollongong, Australia
  • Eric Papas
    Brien Holden Vision Institute Sydney, Australia
    School of Optometry and Vision Science, University of New South Wales, Sydney, Australia
    Vision Cooperative Research Centre, Sydney, Australia
  • Correspondence: Judith Louise Flanagan, Level 3 Rupert Myers Building, University of New South Wales, Sydney, NSW 2052, Australia; j.flanagan@brienholdenvision.org
Investigative Ophthalmology & Visual Science February 2016, Vol.57, 544-550. doi:10.1167/iovs.15-17180
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      Judith Louise Flanagan, Neeta Khandekar, Hua Zhu, Keizo Watanabe, Maria Markoulli, John Terence Flanagan, Eric Papas; Glycerol Monolaurate Inhibits Lipase Production by Clinical Ocular Isolates Without Affecting Bacterial Cell Viability. Invest. Ophthalmol. Vis. Sci. 2016;57(2):544-550. doi: 10.1167/iovs.15-17180.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: We sought to determine the relative lipase production of a range of ocular bacterial isolates and to assess the efficacy of glycerol monolaurate (GML) in inhibiting this lipase production in high lipase–producing bacteria without affecting bacterial cell growth.

Methods: Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes, and Corynebacterium spp. were inoculated at a density of 106/mL in varying concentrations of GML up to 25 μg/mL for 24 hours at 37°C with constant shaking. Bacterial suspensions were centrifuged, bacterial cell density was determined, and production of bacterial lipase was quantified using a commercial lipase assay kit.

Results: Staphylococcus spp. produced high levels of lipase activity compared with P. acnes and Corynebacterium spp. GML inhibited lipase production by Staphylococcal spp. in a dose-dependent manner, with S. epidermidis lipase production consistently more sensitive to GML than S. aureus. Glycerol monolaurate showed significant (P < 0.05) lipase inhibition above concentrations of 15 μg/mL in S. aureus and was not cytotoxic up to 25 μg/mL. For S. epidermidis, GML showed significant (P < 0.05) lipase inhibition above 7.5 μg/mL.

Conclusions: Lipase activity varied between species and between strains. Staphylococcal spp. produced higher lipase activity compared with P. acnes and Corynebacterium spp. Glycerol monolaurate inhibited lipase production by S. aureus and S. epidermidis at concentrations that did not adversely affect bacterial cell growth. GML can be used to inhibit ocular bacterial lipase production without proving detrimental to commensal bacteria viability.

Dry eye is one of the least well-defined disorders of the ocular surface, with characterization, prevalence, and etiology constantly reassessed in the literature. In 2007, the International Workshop on Dry Eye adopted the concept of dry eye disease as a multifactorial disease of the tears and ocular surface accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.1 Meibomian gland dysfunction (MGD) is believed to play a leading role in the development of dry eye and has been suggested as the leading cause of this disorder.2 The consensus definition of MGD encompasses terminal gland obstruction and alterations of the tear film, clinically apparent inflammation, and ocular surface disease.2 
The pathophysiology of MGD is complex, and the role of bacteria or parasites in the etiology of MGD remains controversial. It is well known that commensal bacteria such as Staphylococcus epidermidis, Staphylococcus aureus, Corynebacterium spp., and Propionibacterium acnes are associated with, and contribute to, the pathologic course of chronic blepharitis, but to date no reports implicate bacteria in the primary pathologic process of MGD.3 Paralleling the rising interest in the human microbiome in health and disease,4 the role of microbial flora in ocular health and disease is beginning to be appreciated.5 Quorum sensing has been implicated in perturbation of the ocular microbiome (as cause or effect of an altered eye lid microbial environment), leading to a pathologic imbalance of commensal bacteria.6 This imbalance can trigger release of exotoxins, lipopolysaccharides, and lipase activation, causing eyelid inflammation and lipidic changes (including the release of free fatty acids), leading to reduced tear film stability.6 
Bacterial lipases can alter tear lipids, leading to increased cytokine expression and inflammation associated with dry eye disease. Numerous studies710 have reported the benefit of tetracycline and its analogues in treating meibomianitis and blepharitis, demonstrating a persistent reduction in S. aureus and S. epidermidis colonization after 3 months. Tetracyclines are primarily used in MGD treatment for their anti-inflammatory and lipid-regulating effects.11 However, long-term use of antibiotics has deleterious side effects including emergence of resistance and elimination of commensal bacteria that can lead to undesirable side effects resulting from the collapse of the eye's own immunodefense system.12 The present study explores the potential of a compound that, in the absence of biocidal activity, inhibits bacterial lipases that can alter tear lipids. 
Generally recognized as safe by the US Food and Drug Administration for topical use at doses up to 100 mg/mL,13 glycerol monolaurate (GML) is a fatty acid monoester that has long been used in the food and cosmetics industries as an emulsifier and preservative.14 At concentrations lower than those required for biocidal action, GML blocks bacterial release of virulence factors including bacterial lipase.15 
We sought to determine lipase production in a range of Staphylococcus spp., P. acnes, and Corynebacterium spp. ocular isolates and to characterize the inhibitory actions of GML on bacterial lipase production in the absence of biocidal activity.16 We found that lipase production varies in a species- and strain-specific manner and that GML can inhibit lipase production without affecting bacterial cell viability. 
Materials and Methods
Chemicals
Glycerol monolaurate was purchased from (Corden Pharma Pty Ltd., Liestal, Switzerland) and dissolved in 100% ethanol to a final stock concentration of 200 mg/mL and stored at −20°C. 
Growth of Bacterial Strains
In total, clinical isolates primarily comprising ocular commensal species were examined. These included 37 isolates of Staphylococcus aureus, 31 of Staphylococcus epidermidis, 25 isolates of Propionibacteria acnes, and 8 of Corynebacterium spp. regrown from frozen stocks on chocolate agar for 24 hours in an ambient air incubator or, for P. acnes, an anaerobic jar at 37°C. 
Production of Bacterial Lipases
Single colonies were inoculated into 10 mL tryptone soy broth (TSB; Oxiod, Thermo Fisher Scientific Australia Pty Ltd., Adelaide SA, Australia) and incubated under the appropriate conditions. After 24 hours, OD660nm was measured to assess bacterial growth. Extracellular lipase enzymes in the supernatant were separated from bacterial cells by centrifugation (956g, 10 minutes). Supernatants were collected and filtered through a 0.22-μm filter to remove any residual bacterial cells and stored at −20°C till further use. 
Analyzing Lipase Activity
Lipase activity in culture supernatant (as a measure of lipase production) was quantified using a commercially available kit (QuantiChrom Lipase Assay Kit; BioAssay Systems, Hayward, CA, USA). The colorimetric assay was performed as per manufacturer's instructions. Lipase activity was measured in units per liter. 
Lipase Enzymic Activity in the Presence of GML
Staphylococcus aureus 020 was streaked on chocolate agar plates and incubated at 37°C for 24 hours. Resulting colonies were inoculated into 3 mL TSB and incubated at 37°C for 24 hours. Resulting bacterial suspensions were centrifuged to collect supernatant (containing lipases). These fractions were then filtered through a 0.22-μm filter to remove contaminating cell debris. To this “conditioned” supernatant was added GML up to 500 μg/mL. These supernatant + GML fractions were incubated 24 hours at 37°C. Lipase assay detection was performed as described above. Control comprised filtered bacterial supernatant from S. aureus 020 was grown overnight in the presence of GML (20 μg/mL) and similarly assayed for lipase activity. 
GML Inhibition of Bacterial Lipase Production
Two S. aureus and two S. epidermidis isolates with the highest lipase activities were selected and grown for 24 hours at 37°C in the presence or absence of GML (0–25 μg/mL) diluted in TSB. Lipase production (measured as lipase activity) was quantitated as above. 
Statistical Analysis
The Kolmogorov-Smirnov test and the Shapiro-Wilk test were used to test for normality of lipase production between isolates of S. aureus, S. epidermidis, P. acnes, and Corynebacterium spp. To overcome limitations due to determined nonnormality or small sample size, bootstrapping (Statistical Package for Social Sciences; http://www-01.ibm.com/software/analytics/spss/products/statistics/) was used to obtain unbiased estimates of 95% confidence intervals for the population median. Significant differences in lipase production of each of the four groups was determined using the nonparametric test of Oyeka and Okeh.17 This test is based on the statistic Q calculated from the ranks of items in the combined sample of size n = n1 + n2 (where n1 and n2 are ≥5)  where Ri is the sum of ranks in the combined sample of items from sample i, and Q is asymptotically χ2 distributed with one degree of freedom.  
Lipase inhibition results are presented as mean ± SD. The Student's t-test was performed for comparison of data comprising two groups at the P < 0.05 level of significance. 
Results
Bacterial Lipase Production
Levels of bacterial cell lipase production varied between species and within strains (Fig. 1). Because the distribution of lipase activities is positively skewed (Fig. 1) as determined by the Kolmogorov-Smirnov and Shapiro-Wilk tests, to determine significant differences in lipase production, we compared median lipase production (lower and upper 95% confidence limits, respectively; Fig. 2). Potential outliers as identified in the boxplot were included in our analysis as a more conservative test for significant differences because, as discussed by Hubert and Vandervieren,18 the SPSS Boxplot procedure is based on the tacit assumption that the data are drawn from a normal distribution and, in the case of positively skewed distributions (as our data are), typically misclassifies approximately 8% of the higher end values as possible outliers. 
Figure 1
 
Lipase activity histograms showing distribution of lipase production in clinical isolates (A) S. aureus, (B) S. epidermidis, (C) P. acnes, and (D) Corynebacterium spp.
Figure 1
 
Lipase activity histograms showing distribution of lipase production in clinical isolates (A) S. aureus, (B) S. epidermidis, (C) P. acnes, and (D) Corynebacterium spp.
Figure 2
 
Lipase activity of clinical isolates. Box plot showing relative lipase activities of S. aureus, S. epidermidis, P. acnes, and Corynebacterium spp. Lipase activity was determined as in Materials and Methods. * and # indicate significant difference P < 0.05 from each of the other groups.
Figure 2
 
Lipase activity of clinical isolates. Box plot showing relative lipase activities of S. aureus, S. epidermidis, P. acnes, and Corynebacterium spp. Lipase activity was determined as in Materials and Methods. * and # indicate significant difference P < 0.05 from each of the other groups.
Median lipase production for S. aureus was significantly different to medians of S. epidermidis, P. acnes, and Corynebacterium spp. Median lipase production for S. epidermidis was similarly significantly different to median production for each of the other bacterial groups tested (Fig. 2). The difference in median lipase production for P. acnes and Corynebacterium spp. did not reach significance. Similarly, median lipase production was highest in S. aureus, followed by S. epidermidis isolates, P. acnes, and Corynebacterium spp., respectively. Lipase production by Staphylococcus spp. was generally much higher than that of P. acnes and Corynebacterium spp. isolates. Because lipase production by P. acnes and Cornyebacterium spp. was minimal, none of these isolates were tested further. 
Determination of Lipase Enzyme Stability in the Presence of GML
We sought to determine the mode of action of GML: whether inhibition of lipase production or inhibition of lipase activity (Fig. 3). In the presence of conditioned supernatant containing bacterially expressed lipase from overnight cultures (S. aureus 020), increasing concentrations of GML had no inhibitory effect on lipase activity. This is in stark contrast to the inhibition of lipase production evident in the bacteria control in which S. aureus was grown overnight in the presence of GML (20 μg/mL). That there was little lipase activity detected when S. aureus was grown in the presence of GML compared with the lipase activity remaining in the supernatants containing expressed lipase incubated with GML illustrates the mechanism of action of GML resides in inhibition of production rather than inhibition of lipase activity per se. 
Figure 3
 
Stability of bacterial lipase enzyme activity in the presence of GML. Filtered supernatant from overnight cultures of S. aureus 020 grown in the absence of GML was incubated with increasing concentrations of GML, and lipase activity was assessed. Control culture S. aureus 020 was grown in the presence of GML (20 μg/mL), and lipase activity was similarly assessed.
Figure 3
 
Stability of bacterial lipase enzyme activity in the presence of GML. Filtered supernatant from overnight cultures of S. aureus 020 grown in the absence of GML was incubated with increasing concentrations of GML, and lipase activity was assessed. Control culture S. aureus 020 was grown in the presence of GML (20 μg/mL), and lipase activity was similarly assessed.
GML Inhibition of Bacterial Growth
Bacterial toxicity of GML was titrated to determine the bacteriocidal threshold. We chose to monitor the bacteriocidal activity of increasing concentrations of GML on the two highest lipase producing isolates from S. aureus and S. epidermidis, respectively: S. aureus 020 and 134, and S. epidermidis 001 and 024. We observed that growth of both clinical isolates of S. aureus remained unaffected in the presence of GML up to 25 μg/mL (Fig. 4A). Staphylococcus epidermidis exhibited a strain-dependent sensitivity to the growth inhibition of GML, with S. epidermidis 024 exhibiting a slight decline in growth when incubated in concentrations of GML greater than 15 μg/mL (Fig. 4B). 
Figure 4
 
Effect of increasing concentrations of GML on bacterial cell viability and lipase production. (A) Glycerol monolaurate proved nontoxic to S. aureus 020 and S. aureus 134 up to 25 μg/mL GML and inhibited the bacterial lipases in a dose-dependent manner. * and # indicate significant differences (P < 0.05) in the lipase production in the presence of GML by S. aureus 020 and S. aureus 134, respectively, compared with their lipase production in the absence of GML. ▓ S. aureus 020 ░ S. aureus 134 Image not availableS. aureus 020 growth × S. aureus 134 growth. (B) Glycerol monolaurate was nontoxic to S. epidermidis 001 and 024 up to a concentration of 15 μg/mL. Glycerol monolaurate inhibited the bacterial lipases in a dose-dependent manner. * and # show significant differences (P < 0.05) in the lipase production in the presence of GML by S. epidermidis 001 and S. epidermidis 024, respectively, compared with their lipase production in the absence of GML. ▓ S. epidermidis 001 ░ S. epidermidis 024 Image not availableS. epidermidis 001 growth × S. epidermidis 024 growth.
Figure 4
 
Effect of increasing concentrations of GML on bacterial cell viability and lipase production. (A) Glycerol monolaurate proved nontoxic to S. aureus 020 and S. aureus 134 up to 25 μg/mL GML and inhibited the bacterial lipases in a dose-dependent manner. * and # indicate significant differences (P < 0.05) in the lipase production in the presence of GML by S. aureus 020 and S. aureus 134, respectively, compared with their lipase production in the absence of GML. ▓ S. aureus 020 ░ S. aureus 134 Image not availableS. aureus 020 growth × S. aureus 134 growth. (B) Glycerol monolaurate was nontoxic to S. epidermidis 001 and 024 up to a concentration of 15 μg/mL. Glycerol monolaurate inhibited the bacterial lipases in a dose-dependent manner. * and # show significant differences (P < 0.05) in the lipase production in the presence of GML by S. epidermidis 001 and S. epidermidis 024, respectively, compared with their lipase production in the absence of GML. ▓ S. epidermidis 001 ░ S. epidermidis 024 Image not availableS. epidermidis 001 growth × S. epidermidis 024 growth.
Effect of Varying Concentrations of GML on Bacterial Lipase Production
Glycerol monolaurate exerted a dose-dependent inhibition of bacterial lipase production by S. aureus 020 and 134 (Fig. 4A) and S. epidermidis 001 and 024 (Fig. 4B). Glycerol monolaurate concentrations of 15 μg/mL and above induced significant (P < 0.05) inhibition of lipase production by both S. aureus strains; lipases were reduced by approximately 60% in the presence of GML at 15 μg/mL and almost 90% at 25 μg/mL GML. 
Staphylococcus epidermidis lipase production was consistently more sensitive to GML than were S. aureus isolates. Glycerol monolaurate at 10 μg/mL for S. epidermidis 001 and as low as 5 μg/mL for S. epidermidis 024 significantly (P < 0.05) reduced bacterial lipase production relative to no GML. The lipase inhibition was concentration dependent for both strains. 
Discussion
Lipase production activities were determined for S. aureus, coagulase-negative staphylococci, P. acnes, and Corynebacterium spp. The level of bacterial lipase activity varied widely between the ocular species and strains examined: P. acnes and Corynebacterium spp. produced relatively low lipase activity, whereas the Staphylococcus spp. produced relatively high lipase activities. 
The finding of low lipase production by P. acnes and Corynebacterium spp. is interesting in light of evidence from Lee et al.19 that, in blepharitis subjects, the relative abundance of P. acnes in the ocular microbiome was decreased compared with healthy subjects, whereas that of Staphylococcus spp. was increased. In an earlier study,20 Corynebacterium spp. were more likely to be detected in patients suffering from blepharitis who presented without dry eye than those blepharitis patients with dry eye. The species we examined in the present study are the most frequently isolated from blepharitis patients.21 
Our observations are consistent with the idea that a higher proportion of low lipase producing bacteria might be protective against tear film lipid degradation. Conversely, an overrepresentation of high lipase–producing bacteria might destabilize the tear film. In the present study, S. aureus and S. epidermidis showed a divergent range of lipase activities, with S. aureus generally exhibiting higher lipase activities than the S. epidermidis isolates. It has been proposed that S. epidermidis might have evolved not to cause disease but to maintain a benign relationship with the host22 and further, might play a probiotic role in preventing colonization by more severe pathogens, such as S. aureus.23 
Culture independent analyses of the ocular microbiome—in contrast to the relatively low culturable bioburden on the eye—are reporting a robust community of complex dynamics.19 As with other regions of the body, microbial dysbiosis characterized by shifts in populations and a loss of species diversity—a feature of chronic disease24—can occur in the ocular microbiome. How these communities shift in chronic dry eye is a matter of great interest. Zegans and Van Gelder25 recently suggested that many idiopathic ocular disorders, including dry eye disease, that are characterized by an inflammatory component might be in part due to dysregulation of the ocular microbiota and the concomitant release of specific toxins or the triggering of a large immune response. This is consistent with our hypothesis of the contribution of dysregulation of lipase-producing commensal bacteria to the etiology of dry eye disease. Staphylococcus epidermidis (a ubiquitous constituent of commensal microflora26), along with the more virulent S. aureus,22 can become opportunistic under the appropriate conditions. Exploring dry eye disease in terms of a tipping point in the ecologic balance of the ocular microbiota might shed new light on this disorder. We previously reported (Zhu H, et al. IOVS 2013;54:ARVO E-Abstract 926) that in normal subjects (without dry eye or other ocular disease), the average number of bacteria recovered from eyes of younger females was lower than that of older females or males and that this reduced bacterial burden correlated with clinical measures of increased meibum quality and function. 
Lipase inhibitors exert a specific bacteriostatic action on Gram-positive bacteria through a delay in growth initiation and a decrease in maximum growth achieved.27,28 Concentrations at which GML was observed to inhibit growth are in keeping with previous reports of minimal inhibitory concentrations (between 10 and 63 μg/mL) of GML against S. aureus strains; the magnitude of the range was ascribed to inherent strain variation, inoculum size, and culture conditions.13,16,28,29 
Bacterial lipases in growing culture can hydrolyze GML to lauric acid.14 Lauric acid is more stable in the presence of bacterial lipases and similarly inhibits staphylococcal exoprotein production.14 In the skin, lauric acid is released from sebaceous triglycerides through bacterial lipase action,30 acting to protect against pathogens and drying. We speculate that a similar action might occur in the tear film and meibomian triglycerides once the meibum has been exposed to the ocular microbiota and that a balanced presence of lipase producing S. epidermidis is part of a healthy ocular environment. Bacteriostasis could check overgrowth of opportunistic pathogens and help rebalance the commensal microflora, producing sufficient lipase to effect a steady concentration of lauric acid (as a product of hydrolysis of meibum) while limiting deleterious lipase action on tear film stability. In support of this, in 1982, Doughtery and McCulley31 reported significantly reduced amounts of C12:0 fatty acids in mixed seborrheic/staphylococcal blepharitis and meibomian seborrheic blepharitis compared with normals, whereas recently, Arita et al.32 reported the presence of C12:0 fatty acids in meibum of dry eye patients and indicated a continual decrease of this fatty acid species as meibum moved from clear to cloudy to yellow, suggesting that healthier meibum is associated with increased concentration of C12:0 fatty acids. 
Tetracycline analogues have been used to treat dry eye symptoms.8 The mechanism of action of these analogues in treating meibomianitis is not yet fully elucidated; however, tetracycline analogues are known to inhibit bacterial lipase activity, inducing a concomitant reduction in free fatty acids generated from hydrolysis of wax and sterol esters.10,33,34 As it is well known that long-term use of antibiotics has deleterious consequences including increased resistance and a potentially catastrophic shift in the commensal flora, lipase inhibition in the absence of antimicrobial action is preferable. 
Use of GML to reduce virulence factors in toxic shock syndrome has shown GML does not alter mucosal or microflora integrity and stabilizes mammalian cell membranes leading to reduced production of proinflammatory cytokines from epithelial cells.35,36 As GML is safe for use on skin and mucosal surfaces13 and is considered to be an ocular nonirritant,37 it could potentially play a role in protecting against bacterially mediated tear film instability. 
The role of lipases in bacterial pathogenesis is incompletely understood; however, these enzymes are thought to contribute to overall fitness of the pathogens by providing a nutrition source and releasing fatty acids to which the bacteria can adhere.26,38 Additionally, S. epidermidis lipases appear to play a role in limiting colonization by S. aureus, and S. aureus lipases interfere with phagocytosis of S. aureus by granulocytes.39 Controlling lipase production that results from overgrowth of opportunistic pathogens could reduce overall fitness of these bacteria and assist in rebalancing the ocular microbiome. 
Interestingly, Dave et al.40 provide evidence that efficacy of azithromycin administration for blepharitis might not be due to its antibacterial properties but more from the outcompeting of S. aureus by S. epidermidis, suggesting that azithromycin renders S. aureus “less fit” relative to S. epidermidis. Application of GML to the ocular surface might similarly affect the fitness of the higher lipase producer (S. aureus) relative to that of S. epidermidis
As eloquently expressed by Andersen et al.: “To study bacterial cells, we must remove them from the host environment…which may release them from selective pressures that we wish to understand.”41 Bacterial virulence factors (including S. aureus lipases) are regulated largely through quorum sensing.42 As such, expression of virulence determinants in isolated strains could differ substantially compared with behaviors within the context of community. 
Hence, a limitation of the present work is the determination of bacterial lipase production by isolated strains. Because more than 600 genera43 have been identified from conjunctiva of healthy subjects, in vitro modeling of community interactions would be extremely complex. Whole community shotgun genomics or proteomics with in silico analysis of potential community interactions could offer a powerful alternative.44,45 In addition, studying lipase production and the influence of GML in the context of the dynamics of the in vivo bacterial community is a natural extension of these investigations. 
The significant finding in our study is that GML inhibits lipase production by S. aureus and S. epidermidis at low concentrations without adversely affecting bacterial cell growth and thus potentially could be used in vivo to inhibit lipases produced by ocular isolates without proving detrimental to commensal bacteria, leading to a more stable tear film. Judicious choice of a bacterial lipase inhibitor that protects the commensal ocular flora could potentially reduce the progression of dry eye disease. Improved understanding of the dynamics of the ocular microbiome in health and disease, including the role of bacterial lipases and the action of GML in vivo, might add considerably to our understanding of dry eye disease. 
Acknowledgments
The authors thank Rani Bandara and Amali Ariyavidana for excellent technical assistance. 
Supported by the Brien Holden Vision Institute. 
Disclosure: J.L. Flanagan, P; N. Khandekar, None; H. Zhu, P; K. Watanabe, None; M. Markoulli, None; J.T. Flanagan, None; E. Papas, None 
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Figure 1
 
Lipase activity histograms showing distribution of lipase production in clinical isolates (A) S. aureus, (B) S. epidermidis, (C) P. acnes, and (D) Corynebacterium spp.
Figure 1
 
Lipase activity histograms showing distribution of lipase production in clinical isolates (A) S. aureus, (B) S. epidermidis, (C) P. acnes, and (D) Corynebacterium spp.
Figure 2
 
Lipase activity of clinical isolates. Box plot showing relative lipase activities of S. aureus, S. epidermidis, P. acnes, and Corynebacterium spp. Lipase activity was determined as in Materials and Methods. * and # indicate significant difference P < 0.05 from each of the other groups.
Figure 2
 
Lipase activity of clinical isolates. Box plot showing relative lipase activities of S. aureus, S. epidermidis, P. acnes, and Corynebacterium spp. Lipase activity was determined as in Materials and Methods. * and # indicate significant difference P < 0.05 from each of the other groups.
Figure 3
 
Stability of bacterial lipase enzyme activity in the presence of GML. Filtered supernatant from overnight cultures of S. aureus 020 grown in the absence of GML was incubated with increasing concentrations of GML, and lipase activity was assessed. Control culture S. aureus 020 was grown in the presence of GML (20 μg/mL), and lipase activity was similarly assessed.
Figure 3
 
Stability of bacterial lipase enzyme activity in the presence of GML. Filtered supernatant from overnight cultures of S. aureus 020 grown in the absence of GML was incubated with increasing concentrations of GML, and lipase activity was assessed. Control culture S. aureus 020 was grown in the presence of GML (20 μg/mL), and lipase activity was similarly assessed.
Figure 4
 
Effect of increasing concentrations of GML on bacterial cell viability and lipase production. (A) Glycerol monolaurate proved nontoxic to S. aureus 020 and S. aureus 134 up to 25 μg/mL GML and inhibited the bacterial lipases in a dose-dependent manner. * and # indicate significant differences (P < 0.05) in the lipase production in the presence of GML by S. aureus 020 and S. aureus 134, respectively, compared with their lipase production in the absence of GML. ▓ S. aureus 020 ░ S. aureus 134 Image not availableS. aureus 020 growth × S. aureus 134 growth. (B) Glycerol monolaurate was nontoxic to S. epidermidis 001 and 024 up to a concentration of 15 μg/mL. Glycerol monolaurate inhibited the bacterial lipases in a dose-dependent manner. * and # show significant differences (P < 0.05) in the lipase production in the presence of GML by S. epidermidis 001 and S. epidermidis 024, respectively, compared with their lipase production in the absence of GML. ▓ S. epidermidis 001 ░ S. epidermidis 024 Image not availableS. epidermidis 001 growth × S. epidermidis 024 growth.
Figure 4
 
Effect of increasing concentrations of GML on bacterial cell viability and lipase production. (A) Glycerol monolaurate proved nontoxic to S. aureus 020 and S. aureus 134 up to 25 μg/mL GML and inhibited the bacterial lipases in a dose-dependent manner. * and # indicate significant differences (P < 0.05) in the lipase production in the presence of GML by S. aureus 020 and S. aureus 134, respectively, compared with their lipase production in the absence of GML. ▓ S. aureus 020 ░ S. aureus 134 Image not availableS. aureus 020 growth × S. aureus 134 growth. (B) Glycerol monolaurate was nontoxic to S. epidermidis 001 and 024 up to a concentration of 15 μg/mL. Glycerol monolaurate inhibited the bacterial lipases in a dose-dependent manner. * and # show significant differences (P < 0.05) in the lipase production in the presence of GML by S. epidermidis 001 and S. epidermidis 024, respectively, compared with their lipase production in the absence of GML. ▓ S. epidermidis 001 ░ S. epidermidis 024 Image not availableS. epidermidis 001 growth × S. epidermidis 024 growth.
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