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Clinical Infection & Immunity

Short Communication

Volume 7, Number 1, March 2022, pages 17-21

Antimicrobial Activity of Monolaurin Against Borrelia burgdorferi

Lyme disease (LD), also known as Lyme borreliosis, is a multisystem disorder characterized by host-mediated inflammation [1] that may involve musculoskeletal (Lyme arthritis), nervous (Lyme neuroborreliosis), or cardiovascular systems (carditis) [2]. Borrelia burgdorferi (Bb) is the causative agent and it is transmitted by ticks of the genus Ixodes.

While approximately 90% of patients respond to initial antibiotic therapy, some patients develop persistent mild symptoms of fatigue, arthralgia, myalgia, and mild cognitive complaints despite longer antibiotic treatment [3-5]. Randomized, double-blind, placebo-controlled clinical trials have shown that long-term therapy with approved antibiotic therapy provides no symptomatic relief in these patients [6, 7]. The occurrence of persistence of inflammatory symptoms have been attributed mainly to inadequate and insufficient therapy and increasing bacterial drug-resistance [8]. Therefore, exploration of novel treatment modalities would supplement existing therapies for LD and improve clinical outcomes.

Several naturally occurring compounds have been shown to provide antimicrobial activity against Borrelia species prevalent in Europe and North America [8]. Amongst these is monolaurin or glycerol monolaurate, an ester of glycerol and dodecanoic acid [8]. It is recognized as a safe emulsifying agent by the US Food and Drug Administration. Monolaurin has also shown antimicrobial action in combination with other compounds against gram-positive bacteria in vitro, including Staphylococcus aureus and Listeria monocytogenes [9, 10]. The anti-spirochetal activity of monolaurin might be due to spirochetal plasma membrane destabilization, disruption of biofilms, and interference with cell replication, without providing severe toxicity to human cells [8, 11-14].

In this study, we investigated the antimicrobial effect of monolaurin on Bb viability in vitro.

Bb strain B31, clone 5A2 (Bei Resources) was cultured in Barbour-Stoenner-Kelly (BSK) medium (Sigma-Aldrich). Spirochetes were temperature shifted [15].

A resazurin-based microdilution method [16] was used to calculate the minimum inhibitory concentration (MIC) of monolaurin against Bb. A liquid culture of Bb was transferred to a 96-well plate [17], and then exposed to serial dilutions of monolaurin (Sigma), 300 µg/mL, 150 µg/mL, 75 µg/mL, 37.5 µg/mL, 18.75 µg/mL, 9.4 µg/mL, and 4.7 µg/mL. Tetracycline (10 µg/mL) was used as a positive control. The plates were incubated for 24 h at 37 °C, followed by addition of 10 µL of resazurin and incubated for an additional 24 h. Absorbance was then measured at both 570 and 600 nm. An increased absorbance difference (570 - 600 nm) indicates higher metabolic activity.

The viability of Bb, treated with monolaurin, was assessed by LIVE/DEAD bacterial viability assay (ThermoFisher). Briefly, upon bacterial incubation with serial concentrations of monolaurin, propidium iodide and SYTO 13 were added, and slides were examined under a fluorescence microscope (IX81 Olympus). The percentage of live (green) and dead (red) spirochetes were quantified. Cultures treated with 70% ethanol (v/v) or no treatment, were used as negative and positive control, respectively.

Grouped analysis of untreated vs. treated cells using a two-way repeated measure analysis of variance (ANOVA) was performed using GraphPad Prism 9. A P value < 0.05 was considered statistically significant.

Institutional Review Board approval was not applicable. The study was conducted in compliance with the ethical standards of the Helsinki Declaration.

The effect of serially diluted concentration of monolaurin on the growth of Bb was examined. The results show that monolaurin exerted an antimicrobial effect on Bb with a MIC value of 75 - 150 µg/mL (Fig. 1a). Lower absorbance difference (570 - 600 nm) is suggestive of a decreased Bb viability.

Click for large image

Figure 1. Monolaurin has antimicrobial activity against Bb. Minimum inhibitory concentration (MIC) of monolaurin against Bb was determined using resazurin reduction assay. (a) Increasing concentration of monolaurin caused a dose-dependent reduction in Bb viability; monolaurin concentration between 75 and 150 µg/mL showed significant reduction in Bb viability. (b) Reduction in Bb viability by monolaurin. Data are reported as an average of triplicate runs (n = 3). *P < 0.05, paired t-test.

A dose-dependent Bb’s growth percentage inhibition by monolaurin was observed (Fig. 1b). The untreated condition (no monolaurin treatment) is used for calculation of Bb growth inhibition.

We then chose to assess Bb viability using a fluorescence assay (Fig. 2). A dose-dependent effect was observed with higher concentrations of monolaurin showing higher bactericidal activity (i.e., lower live/dead ratios) (Fig. 2a). Trend analysis was statistically significant (P < 0.0001). A plateau of the highest proportion of dead bacterial cells was observed with a monolaurin concentration between 125 and 150 µg/mL.

Click for large image

Figure 2. Bb viability determined using a LIVE/DEAD assay. (a) Serially diluted monolaurin concentrations displayed dose-dependent effect on Bb’s viability. Test conditions were compared to 70% ethanol and to untreated one. Data were presented as mean ± SD (n = 6) (two-way ANOVA) *P < 0.0001. (b) Representative images of Bb viability at the different concentrations. SD: standard deviation; ANOVA: analysis of variance.

Our study demonstrates the in vitro susceptibility of Bb upon exposure to monolaurin. Monolaurin is a medium-chain fatty acid containing an ester of glycerol and dodecanoic acid that is used as a surfactant in various products and fresh foods, but is not currently approved in the treatment of any disease [8, 12]. Monolaurin, also known as glycerol monolaurate, is a naturally occurring compound that has been demonstrated to have antimicrobial activity against a variety of bacteria in biofilms as well as the fungus Candida albicans [12, 18-20].

Bb can produce biofilm-like colonies that contain extracellular polymeric substances, extracellular DNA, and calcium to withstand adverse environmental conditions [21]. The formation of adherent polysaccharide-based matrices or biofilms may provide protection against antimicrobial agents and the host immune system. This protective defense may result in antibiotic efficacy against only free-floating bacterial forms and subsequent failure to completely eradicate the infection [22-24]. Nevertheless, there is evidence of antimicrobial activity of monolaurin against all morphological forms of Borrelia species as well as biofilms in vitro, with only mild or moderate toxicity against human cells at the minimum bactericidal concentration [8, 25]. We found a similar MIC in our study. The lipophilic nature of monolaurin may destabilize the biofilm matrix via cell membrane disruption, exposing bacteria to antimicrobial agents and the host immune system [11].

Several mechanisms of action have been suggested for the antimicrobial activity provided by monolaurin. In contrast to most antibiotics with specific bacterial targets, monolaurin is a fatty acid that targets many signal transduction molecules nonspecifically through self-insertion into plasma membranes [12-14].

Our study further supports the use of monolaurin as an antimicrobial agent against Bb. Additional treatment modalities are sought to address the deficiencies of current treatment regimens to prevent long-term complications of LD. Further in vivo studies are needed to determine the utility of monolaurin as an adjunct antimicrobial therapy for LD.

Acknowledgments

None to declare.

Financial Disclosure

None to declare.

Conflict of Interest

None to declare.

Informed Consent

Not applicable.

Author Contributions

LK and JC conceptualized the idea. SB, JB, LK, JK, BT performed the experiments. SB, AK, KA and JC wrote the manuscript.

Data Availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

1. Brouwer MAE, van de Schoor FR, Vrijmoeth HD, Netea MG, Joosten LAB. A joint effort: The interplay between the innate and the adaptive immune system in Lyme arthritis. Immunol Rev. 2020;294(1):63-79.
   doi pubmed
2. Steere AC, Strle F, Wormser GP, Hu LT, Branda JA, Hovius JW, Li X, et al. Lyme borreliosis. Nat Rev Dis Primers. 2016;2:16090.
   doi pubmed
3. Kullberg BJ, Vrijmoeth HD, van de Schoor F, Hovius JW. Lyme borreliosis: diagnosis and management. BMJ. 2020;369:m1041.
   doi pubmed
4. Feder HM, Jr., Johnson BJ, O'Connell S, Shapiro ED, Steere AC, Wormser GP, Ad Hoc International Lyme Disease G, et al. A critical appraisal of "chronic Lyme disease". N Engl J Med. 2007;357(14):1422-1430.
   doi pubmed
5. Sanchez JL. Clinical manifestations and treatment of lyme disease. Clin Lab Med. 2015;35(4):765-778.
   doi pubmed
6. Berende A, ter Hofstede HJ, Vos FJ, van Middendorp H, Vogelaar ML, Tromp M, van den Hoogen FH, et al. Randomized Trial of longer-term therapy for symptoms attributed to lyme disease. N Engl J Med. 2016;374(13):1209-1220.
   doi pubmed
7. Klempner MS, Hu LT, Evans J, Schmid CH, Johnson GM, Trevino RP, Norton D, et al. Two controlled trials of antibiotic treatment in patients with persistent symptoms and a history of Lyme disease. N Engl J Med. 2001;345(2):85-92.
   doi pubmed
8. Goc A, Niedzwiecki A, Rath M. In vitro evaluation of antibacterial activity of phytochemicals and micronutrients against Borrelia burgdorferi and Borrelia garinii. J Appl Microbiol. 2015;119(6):1561-1572.
   doi pubmed
9. Umerska A, Cassisa V, Bastiat G, Matougui N, Nehme H, Manero F, Eveillard M, et al. Synergistic interactions between antimicrobial peptides derived from plectasin and lipid nanocapsules containing monolaurin as a cosurfactant against Staphylococcus aureus. Int J Nanomedicine. 2017;12:5687-5699.
   doi pubmed
10. Tokarskyy O, Marshall DL. Mechanism of synergistic inhibition of Listeria monocytogenes growth by lactic acid, monolaurin, and nisin. Appl Environ Microbiol. 2008;74(23):7126-7129.
    doi pubmed
11. Kabara JJ, Conley AJ, Swieczkowski DM. Antimicrobial action of isomeric fatty acids on group A Streptococcus. J Med Chem. 1973;16(9):1060-1063.
    doi pubmed
12. Schlievert PM, Deringer JR, Kim MH, Projan SJ, Novick RP. Effect of glycerol monolaurate on bacterial growth and toxin production. Antimicrob Agents Chemother. 1992;36(3):626-631.
    doi pubmed
13. Vetter SM, Schlievert PM. Glycerol monolaurate inhibits virulence factor production in Bacillus anthracis. Antimicrob Agents Chemother. 2005;49(4):1302-1305.
    doi pubmed
14. Projan SJ, Brown-Skrobot S, Schlievert PM, Vandenesch F, Novick RP. Glycerol monolaurate inhibits the production of beta-lactamase, toxic shock toxin-1, and other staphylococcal exoproteins by interfering with signal transduction. J Bacteriol. 1994;176(14):4204-4209.
    doi pubmed
15. Tokarz R, Anderton JM, Katona LI, Benach JL. Combined effects of blood and temperature shift on Borrelia burgdorferi gene expression as determined by whole genome DNA array. Infect Immun. 2004;72(9):5419-5432.
    doi pubmed
16. Elshikh M, Ahmed S, Funston S, Dunlop P, McGaw M, Marchant R, Banat IM. Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants. Biotechnol Lett. 2016;38(6):1015-1019.
    doi pubmed
17. Bioarray C. Resazurin cell viability assay. https://www.creative-bioarray.com/support/resazurin-cell-viability-assay.htm.
18. Seleem D, Freitas-Blanco VS, Noguti J, Zancope BR, Pardi V, Murata RM. In vivo antifungal activity of monolaurin against candida albicans biofilms. Biol Pharm Bull. 2018;41(8):1299-1302.
    doi pubmed
19. Krislee A, Fadly C, Nugrahaningsih DAA, Nuryastuti T, Nitbani FO, Jumina, Sholikhah EN. The 1-monolaurin inhibit growth and eradicate the biofilm formed by clinical isolates of Staphylococcus epidermidis. BMC Proc. 2019;13(Suppl 11):19.
    doi pubmed
20. Oh DH, Marshall DL. Destruction of Listeria monocytogenes Biofilms on Stainless Steel Using Monolaurin and Heat (1). J Food Prot. 1995;58(3):251-255.
    doi pubmed
21. Sapi E, Kaur N, Anyanwu S, Luecke DF, Datar A, Patel S, Rossi M, et al. Evaluation of in-vitro antibiotic susceptibility of different morphological forms of Borrelia burgdorferi. Infect Drug Resist. 2011;4:97-113.
    doi pubmed
22. Jefferson KK. What drives bacteria to produce a biofilm? FEMS Microbiol Lett. 2004;236(2):163-173.
    doi pubmed
23. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet. 2001;358(9276):135-138.
    doi
24. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999;284(5418):1318-1322.
    doi pubmed
25. Goc A, Niedzwiecki A, Rath M. Reciprocal cooperation of phytochemicals and micronutrients against typical and atypical forms of Borrelia sp. J Appl Microbiol. 2017;123(3):637-650.
    doi pubmed

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