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Cited by 198Antimicrobial agents and applicationsEssential Oils and Antimicrobial ActivityBacterial biofilms and quorum sensing

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Glycerol monolaurate antibacterial activity in broth and biofilm cultures.

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Schlievert, Patrick M., Peterson, Marnie L.·2012·PLoS ONE
DOI 10.1371/journal.pone.0040350PMC PMC3394780
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Abstract

BACKGROUND: Glycerol monolaurate (GML) is an antimicrobial agent that has potent activity against gram-positive bacteria. This study examines GML antibacterial activity in comparison to lauric acid, in broth cultures compared to biofilm cultures, and against a wide range of gram-positive, gram-negative, and non-gram staining bacteria. METHODOLOGY/PRINCIPAL FINDINGS: GML is ≥ 200 times more effective than lauric acid in bactericidal activity, defined as a ≥ 3 log reduction in colony-forming units (CFU)/ml, against Staphylococcus aureus and Streptococcus pyogenes in broth cultures. Both molecules inhibit superantigen production by these organisms at concentrations that are not bactericidal. GML prevents biofilm formation by Staphylococcus aureus and Haemophilus influenzae, as representative gram-positive and gram-negative organisms, tested in 96 well microtiter plates, and simultaneously is bactericidal for both organisms in mature biofilms. GML is bactericidal for a wide range of potential bacterial pathogens, except for Pseudomonas aeruginosa and Enterobacteriaceae. In the presence of acidic pH and the cation chelator ethylene diamine tetraacetic acid, GML has greatly enhanced bactericidal activity for Pseudomonas aeruginosa and Enterobacteriaceae. Solubilization of GML in a nonaqueous delivery vehicle (related to K-Y Warming®) enhances its bactericidal activity against S. aureus. Both R and S, and 1 and 2 position lauric acid derivatives of GML exhibit bactericidal activity. Despite year-long passage of Staphylococcus aureus on sub-growth inhibitory concentrations of GML (0.5 x minimum bactericidal concentration), resistance to GML did not develop. CONCLUSIONS/SIGNIFICANCE: GML may be useful as a broad-spectrum human or animal topical microbicide and may be useful as an environmental surface microbicide for management of bacterial infections and contamination.

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Figures

Figure 1 GML and lauric acid inhibition of Staphylococcus aureus MN8 growth. GML and lauric acid were incubated with approximately 5×106 CFUs/ml of Staphylococcus aureus for 24 hours with shaking (200 revolutions/min) at 37°C in triplicate. Plate counts were used to determine CFUs/ml. Bars show standard deviations. Bactericidal activity was defined as the minimum concentration of agent required to reduce CFUs by ≥3 logs. * indicates GML mean significantly different from lauric acid mean with p<0.001. Dashed line indicates starting inoculum size.
Figure 1 GML and lauric acid inhibition of Staphylococcus aureus MN8 growth. GML and lauric acid were incubated with approximately 5×106 CFUs/ml of Staphylococcus aureus for 24 hours with shaking (200 revolutions/min) at 37°C in triplicate. Plate counts were used to determine CFUs/ml. Bars show standard deviations. Bactericidal activity was defined as the minimum concentration of agent required to reduce CFUs by ≥3 logs. * indicates GML mean significantly different from lauric acid mean with p<0.001. Dashed line indicates starting inoculum size.CC BY · PubMed Central
Figure 2 GML and lauric acid inhibition of Streptococcus pyogenes growth. GML and lauric acid were incubated with approximately 5×106 CFU/ml of Streptococcus pyogenes for 24 hours stationary in the presence of 7% CO2 at 37°C in triplicate. Plate counts were used to determine CFUs/ml. Bars show standard deviations. Bactericidal activity was defined as the minimum concentration of agent required to reduce CFUs by ≥3 logs. * indicates GML mean significantly different from lauric acid mean with p<0.001. Dashed line indicates starting inoculum size.
Figure 2 GML and lauric acid inhibition of Streptococcus pyogenes growth. GML and lauric acid were incubated with approximately 5×106 CFU/ml of Streptococcus pyogenes for 24 hours stationary in the presence of 7% CO2 at 37°C in triplicate. Plate counts were used to determine CFUs/ml. Bars show standard deviations. Bactericidal activity was defined as the minimum concentration of agent required to reduce CFUs by ≥3 logs. * indicates GML mean significantly different from lauric acid mean with p<0.001. Dashed line indicates starting inoculum size.CC BY · PubMed Central
Figure 3 Ability of pretreatment of GML with Staphylococcus aureus MN8 spent culture fluids to reduce antimicrobial activity. Staphylococcus aureus MN8 or Streptococcus pyogenes T253curedT12 sterile stationary phase culture fluids were incubated with GML (1000 μg) for 24 hours. Subsequently, treated GML was evaluated for antibacterial activity against Staphylococcus aureus MN8 in a 24 hour assay, in comparison to untreated GML. * indicates mean significantly reduced from starting inoculum mean at p<0.001. Dashed line indicates starting inoculum size.
Figure 3 Ability of pretreatment of GML with Staphylococcus aureus MN8 spent culture fluids to reduce antimicrobial activity. Staphylococcus aureus MN8 or Streptococcus pyogenes T253curedT12 sterile stationary phase culture fluids were incubated with GML (1000 μg) for 24 hours. Subsequently, treated GML was evaluated for antibacterial activity against Staphylococcus aureus MN8 in a 24 hour assay, in comparison to untreated GML. * indicates mean significantly reduced from starting inoculum mean at p<0.001. Dashed line indicates starting inoculum size.CC BY · PubMed Central
Figure 4 Comparison of GML and lauric acid for capacity to inhibit superantigen production. Staphylococcus aureus MN8 and Streptococcus pyogenes were cultured for 8 hours in the presence of GML or lauric acid. Toxic shock syndrome toxin-1 (TSST-1) production by Staphylococcus aureus MN8 and streptococcal pyrogenic exotoxin A (SPE A) production by Streptococcus pyogenes were quantified by Western immunoblot analysis. * indicates mean significantly reduced from the no GML mean at p<0.01.
Figure 4 Comparison of GML and lauric acid for capacity to inhibit superantigen production. Staphylococcus aureus MN8 and Streptococcus pyogenes were cultured for 8 hours in the presence of GML or lauric acid. Toxic shock syndrome toxin-1 (TSST-1) production by Staphylococcus aureus MN8 and streptococcal pyrogenic exotoxin A (SPE A) production by Streptococcus pyogenes were quantified by Western immunoblot analysis. * indicates mean significantly reduced from the no GML mean at p<0.01.CC BY · PubMed Central
Figure 5 Community-associated Staphylococcus aureus 128 growing as biofilms on tampon fibers and cellulose acetate dialysis tubing. Staphylococcus aureus 128, a TSST-1+ organism at 107/0.1 ml, was inoculated onto the inside of pre-wetted dialysis tubing that had been tied off on one end. A tampon was then inserted into the dialysis tubing and the tubing immersed under Todd Hewitt broth containing 0.8% agar. The open end of the dialysis tubing remained above the agar surface such that the only source of nutrients for the growing microbes was media absorbed across the dialysis tubing. Magnification of A was 6000x and B was 9000x. Both frames show organisms embedded in a complex matrix of extracellular material.
Figure 5 Community-associated Staphylococcus aureus 128 growing as biofilms on tampon fibers and cellulose acetate dialysis tubing. Staphylococcus aureus 128, a TSST-1+ organism at 107/0.1 ml, was inoculated onto the inside of pre-wetted dialysis tubing that had been tied off on one end. A tampon was then inserted into the dialysis tubing and the tubing immersed under Todd Hewitt broth containing 0.8% agar. The open end of the dialysis tubing remained above the agar surface such that the only source of nutrients for the growing microbes was media absorbed across the dialysis tubing. Magnification of A was 6000x and B was 9000x. Both frames show organisms embedded in a complex matrix of extracellular material.CC BY · PubMed Central
Figure 6 TSST-1 production in biofilms in tampon sacs. Staphylococcus aureus MN8 (107/0.1 ml) were placed inside dialysis tubes that had been tied off on one end, and then tampons were inserted. The tampon sacs were immersed beneath Todd Hewitt 0.8% agar such that the open end of the dialysis tubing remained above the surface. The agar was allowed to solidify, and the tampons sacs were incubated for indicated times. TSST-1 was measured within the dialysis tube by knowing the pre- and post-weight of the tampons, eluting TSST-1 with distilled water, and quantifying TSST-1 by Western immunoblot analysis.
Figure 6 TSST-1 production in biofilms in tampon sacs. Staphylococcus aureus MN8 (107/0.1 ml) were placed inside dialysis tubes that had been tied off on one end, and then tampons were inserted. The tampon sacs were immersed beneath Todd Hewitt 0.8% agar such that the open end of the dialysis tubing remained above the surface. The agar was allowed to solidify, and the tampons sacs were incubated for indicated times. TSST-1 was measured within the dialysis tube by knowing the pre- and post-weight of the tampons, eluting TSST-1 with distilled water, and quantifying TSST-1 by Western immunoblot analysis.CC BY · PubMed Central
Figure 7 Effect of GML on three Staphylococcus aureus strains cultured in biofilms. Staphylococcus aureus strains methicillin-sensitive MN8, community-associated methicillin-resistant MNWH, and community-associated methicillin-resistant MW2 were cultured for 24 and 48 hours in 96 well microtiter plates. In one set of three wells for each microbe, the wells were agitated 3 times by pipetting up and down. CFU/ml were determined on supernates then removed from the wells. * indicates significant mean reduction compared to starting inoculum mean at p<0.001. Dashed line indicates starting inoculum size. Subsequent to removing unbound cells and then washing wells three times with phosphate-buffered saline, the wells were treated with crystal violet for 30 min. The wells were next washed three times with phosphate-buffered saline to remove unbound crystal violet. Finally, wells were treated with ethanol to solubilize biofilm-associated crystal violet. Absorbances at 595 nm were determined by an ELISA reader. # indicates significant mean reduction in absorbance at 595 nm wavelength between no GML and GML-treated wells at p<0.01.
Figure 7 Effect of GML on three Staphylococcus aureus strains cultured in biofilms. Staphylococcus aureus strains methicillin-sensitive MN8, community-associated methicillin-resistant MNWH, and community-associated methicillin-resistant MW2 were cultured for 24 and 48 hours in 96 well microtiter plates. In one set of three wells for each microbe, the wells were agitated 3 times by pipetting up and down. CFU/ml were determined on supernates then removed from the wells. * indicates significant mean reduction compared to starting inoculum mean at p<0.001. Dashed line indicates starting inoculum size. Subsequent to removing unbound cells and then washing wells three times with phosphate-buffered saline, the wells were treated with crystal violet for 30 min. The wells were next washed three times with phosphate-buffered saline to remove unbound crystal violet. Finally, wells were treated with ethanol to solubilize biofilm-associated crystal violet. Absorbances at 595 nm were determined by an ELISA reader. # indicates significant mean reduction in absorbance at 595 nm wavelength between no GML and GML-treated wells at p<0.01.CC BY · PubMed Central
Figure 8 Effect of GML on non-typable Haemophilus influenzae cultured in biofilms. Haemophilus influenzae was cultured for 24 and 48 hours in a 96 well microtiter plate. In one set of three wells for the microbe, the wells were agitated 3 times by pipetting up and down and then supernates removed for plate counting to determine CFUs/ml. * indicates significant mean reduction compared to starting inoculum mean at p<0.001. Dashed line indicates starting inoculum size. Subsequent to removing bacterial cells and washing three times with phosphate-buffered saline, the wells were treated with crystal violet for 30 min. The wells were then washed three times with phosphate-buffered saline to remove unbound crystal violet. Finally, the wells were treated with ethanol to solubilize biofilm-associated crystal violet. Absorbances at 595 nm were determined by an ELISA reader. # indicates significant mean reduction in absorbance at 595 nm wavelength between no GML and GML-treated wells at p<0.01.
Figure 8 Effect of GML on non-typable Haemophilus influenzae cultured in biofilms. Haemophilus influenzae was cultured for 24 and 48 hours in a 96 well microtiter plate. In one set of three wells for the microbe, the wells were agitated 3 times by pipetting up and down and then supernates removed for plate counting to determine CFUs/ml. * indicates significant mean reduction compared to starting inoculum mean at p<0.001. Dashed line indicates starting inoculum size. Subsequent to removing bacterial cells and washing three times with phosphate-buffered saline, the wells were treated with crystal violet for 30 min. The wells were then washed three times with phosphate-buffered saline to remove unbound crystal violet. Finally, the wells were treated with ethanol to solubilize biofilm-associated crystal violet. Absorbances at 595 nm were determined by an ELISA reader. # indicates significant mean reduction in absorbance at 595 nm wavelength between no GML and GML-treated wells at p<0.01.CC BY · PubMed Central
Figure 9 Ability of GML to remove pre-formed biofilms. Staphylococcus aureus MN8 and nontypable Haemophilus influenzae were grown for 48 hours in 96 well microtiter plates to allow biofilm formation. CFUs/ml were determined after suspension of organisms (A). Subsequently, the wells were treated or not treated with GML (500 μg/ml) for 60 min, and then CFUs/ml determined again (A). * indicates significant mean reduction compared to starting inoculum mean at p<0.001. Dashed line indicates starting inoculum size. Wells were stained with crystal violet after removal of non-adherent bacteria or after removal of non-adherent bacteria and treatment with GML, followed by removal of unbound crystal violet and determination of absorbance at 595 nm (B). # indicates significant mean reduction in absorbance at 595 nm wavelength between no GML and GML-treated wells at p<0.01.
Figure 9 Ability of GML to remove pre-formed biofilms. Staphylococcus aureus MN8 and nontypable Haemophilus influenzae were grown for 48 hours in 96 well microtiter plates to allow biofilm formation. CFUs/ml were determined after suspension of organisms (A). Subsequently, the wells were treated or not treated with GML (500 μg/ml) for 60 min, and then CFUs/ml determined again (A). * indicates significant mean reduction compared to starting inoculum mean at p<0.001. Dashed line indicates starting inoculum size. Wells were stained with crystal violet after removal of non-adherent bacteria or after removal of non-adherent bacteria and treatment with GML, followed by removal of unbound crystal violet and determination of absorbance at 595 nm (B). # indicates significant mean reduction in absorbance at 595 nm wavelength between no GML and GML-treated wells at p<0.01.CC BY · PubMed Central
Figure 10 Ability of R versus S and 1/3 versus 2 forms of GML to inhibit growth of Streptococcus pyogenes 594. R form and mixture of R and S forms of GML and 2 position lauric acid versus mixture of 1/3 and 2 position lauric of GML were incubated with Streptococcus pyogenes 594, a highly susceptible organism to GML, for 24 hours. Plate counts were used to determine GML killing. * indicates the mean for the 2 form of GML is significantly different from the mixture at p<0.001. Dashed line indicates starting inoculum size.
Figure 10 Ability of R versus S and 1/3 versus 2 forms of GML to inhibit growth of Streptococcus pyogenes 594. R form and mixture of R and S forms of GML and 2 position lauric acid versus mixture of 1/3 and 2 position lauric of GML were incubated with Streptococcus pyogenes 594, a highly susceptible organism to GML, for 24 hours. Plate counts were used to determine GML killing. * indicates the mean for the 2 form of GML is significantly different from the mixture at p<0.001. Dashed line indicates starting inoculum size.CC BY · PubMed Central
Figure 11 Ability of EDTA to synergize with GML in antibacterial activity against Escherichia coli. Escherichia coli Watson strain was incubated with EDTA or EDTA + GML (100 μg/ml) for 24 hours and then plate counts used to determine CFUs/ml. GML (5000 μg/ml) alone did not inhibit Escherichia coli growth. * indicates mean for EDTA + GML differs significantly from mean for EDTA alone at p<0.001. Dashed line indicates starting inoculums size.
Figure 11 Ability of EDTA to synergize with GML in antibacterial activity against Escherichia coli. Escherichia coli Watson strain was incubated with EDTA or EDTA + GML (100 μg/ml) for 24 hours and then plate counts used to determine CFUs/ml. GML (5000 μg/ml) alone did not inhibit Escherichia coli growth. * indicates mean for EDTA + GML differs significantly from mean for EDTA alone at p<0.001. Dashed line indicates starting inoculums size.CC BY · PubMed Central
Figure 12 Effect of pH on GML activity against Escherichia coli Watson strain. Todd Hewitt broth was buffered to pH 7.0, 6.0, and 5.0. Susceptibility of Escherichia coli to GML at the indicated pH was measured in 24 hour assays. CFUs/ml were determined by plate counts. * indicates means of No GML samples and GML samples differ significantly at p<0.001. Dashed line indicates the inoculum size.
Figure 12 Effect of pH on GML activity against Escherichia coli Watson strain. Todd Hewitt broth was buffered to pH 7.0, 6.0, and 5.0. Susceptibility of Escherichia coli to GML at the indicated pH was measured in 24 hour assays. CFUs/ml were determined by plate counts. * indicates means of No GML samples and GML samples differ significantly at p<0.001. Dashed line indicates the inoculum size.CC BY · PubMed Central
Figure 13 Effect of pH on GML activity against Pseudomonas aeruginosa. Todd Hewitt broth was buffered to pH 7.0, 6.0, and 5.0. Susceptibility of Pseudomonas aeruginosa PA01 to GML at the indicated pH was measured in 24 hour assays. CFUs/ml were determined by plate counts. * indicates sample means differ significantly from the no GML control at p<0.001. Dashed line indicates the inoculum size.
Figure 13 Effect of pH on GML activity against Pseudomonas aeruginosa. Todd Hewitt broth was buffered to pH 7.0, 6.0, and 5.0. Susceptibility of Pseudomonas aeruginosa PA01 to GML at the indicated pH was measured in 24 hour assays. CFUs/ml were determined by plate counts. * indicates sample means differ significantly from the no GML control at p<0.001. Dashed line indicates the inoculum size.CC BY · PubMed Central
Figure 14 Effect of a nonaqueous (NA) gel on GML antibacterial activity against Staphylococcus aureus MN8. Non-aqueous gel was diluted to 25% and 10% with Todd Hewitt broth. GML and Staphylococcus aureus MN8 were added, and cultures were incubated for 24 hours. CFU/ml were determined by plate counts. Dashed line indicates the inoculum size. * indicates mean differs significantly from the GML alone control at p<0.001.
Figure 14 Effect of a nonaqueous (NA) gel on GML antibacterial activity against Staphylococcus aureus MN8. Non-aqueous gel was diluted to 25% and 10% with Todd Hewitt broth. GML and Staphylococcus aureus MN8 were added, and cultures were incubated for 24 hours. CFU/ml were determined by plate counts. Dashed line indicates the inoculum size. * indicates mean differs significantly from the GML alone control at p<0.001.CC BY · PubMed Central

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  1. JJ Kabara, 1980, GRAS antimicrobial agents for cosmetic products., J Soc Cosmet Chem, 31, 1

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Metadata

Authors
Schlievert, P. M., & Peterson, M. L.
Affiliations
  • University of Iowa (US)
  • University of Minnesota (US)
Corresponding author
Schlievert, Patrick M.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-07-11
Volume
7
Issue
7
Pages
e40350
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0040350
PMC
PMC3394780
OpenAlex
W2035186660
Cited by
198
References
33
Citations by year
2026: 6 · 2025: 13 · 2024: 32 · 2023: 15 · 2022: 15 · 2021: 23 · 2020: 18 · 2019: 20 · 2018: 19 · 2017: 10 · 2016: 11 · 2015: 7 · 2014: 3 · 2013: 5
FWCI
2.21
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institutes of Health
  • National Institute of Allergy and Infectious Diseases (U54 AI 57153, R01AI073366, R01 AI73366, U54AI057153, R01AI074283)
Topics
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Antimicrobial agents and applications
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Essential Oils and Antimicrobial Activity
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
Keywords
glycerol monolaurate, lauric acid, antibacterial activity, biofilm inhibition, Staphylococcus aureus, Streptococcus pyogenes, Haemophilus influenzae, Pseudomonas aeruginosa, Enterobacteriaceae, bactericidal activity, microbicides, topical antimicrobial agents, surface disinfectants, antimicrobial resistance
MeSH terms
Anti-Bacterial Agents, Bacteria, Glycerol, Humans, Hydrogen-Ion Concentration, Laurates, Lauric Acids, Microbial Sensitivity Tests, Community-Acquired Infections, Superantigens, Biofilms, Monoglycerides

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