Monolaurin inhibits Antibiotic-resistant Staphylococcus aureus in Patients with Atopic Dermatitis

doi:10.21203/rs.3.rs-4883681/v1

Frequent use of antibiotics increases the incidence of antimicrobial-resistant Staphylococcus aureus in atopic dermatitis (AD), which prompts the search for new treatments. Monolaurin is a chemical byproduct found in coconut oil and has anti-bacterial properties. This study aimed to investigate the inhibitory effect of monolaurin on antimicrobial-resistant S. aureus. Thirty children and thirty adults diagnosed with AD were recruited and swabbed at three different sites: lesion, non-lesion, and nasal mucosa. Methicillin resistance and high-level mupirocin resistance in S. aureus were identified using mecA and mupA PCR, respectively, whilst fusidic acid resistance were detected by fusA gene sequencing. The broth microdilution method and tetrazolium bromide assays were used for monolaurin susceptibility and cellular cytotoxicity, respectively. We show that S. aureus was frequently isolated from lesions of both children and adults with AD. One isolate of methicillin-resistant S. aureus (MRSA) harboring mecA, one isolate of mupirocin-resistant S. aureus harboring mupA, and four isolates of fusidic acid-resistant S. aureus with novel point mutations of fusA were found in the children group. In silico molecular docking showed that these mutants interacted weakly with fusidic acid, explaining the mechanism of drug resistance. Monolaurin inhibited these antimicrobial-resistant S. aureus isolates with a minimal inhibitory concentration of 2 µg/mL without cytotoxicity to cultured epidermal and dermal cells. These data show that monolaurin could potentially be used to inhibit antimicrobial-resistant S. aureus in AD patients.

Biological sciences/Microbiology/Clinical microbiology

Health sciences/Diseases/Skin diseases

Atopic dermatitis (AD) is a chronic and recurrent eczematous skin disease commonly found in children with typical morphology and age-specific patterns ¹. There are several factors involved in the pathogenesis of AD, such as genetic predispositions, immunological disturbance, and certain environmental exposures such as irritants, allergens and pathogens ².

Staphylococcus aureus is a major pathogen associated with disease flare and severity of AD ³. It has been reported that S. aureus is found in up to 90% of AD patients, especially in children with worsened symptoms ⁴. In clinical practice, topical and systemic antibiotics are administered to eliminate S. aureus in patients with AD. Although frequently-used topical antibiotics (e.g., mupirocin and fusidic acid) and/or systemic antibiotics (e.g., cloxacillin and cephalexin) usually effectively eliminate this pathogen, the incidence of antimicrobial-resistant S. aureus has been rising and this has now become a challenging problem in AD treatment ^5,6.

A common antimicrobial-resistant S. aureus in patients with AD is methicillin-resistant S. aureus (MRSA). The main resistance mechanism of MRSA is production of penicillin-binding protein 2a (PBP2a, encoded by the mecA gene) that has low affinity to β-lactams ⁷. In addition, emerging evidence shows that mupirocin- and fusidic acid-resistant S. aureus are increasing in AD patients ^8–10. Mupirocin inactivates a bacterial enzyme, isoleucyl-t-RNA synthetase, which is essential for protein synthesis of S. aureus. However, high-level mupirocin-resistant S. aureus commonly carry a mupA gene encoding an alternative isoleucyl-t-RNA synthetase, which is not inhibited by mupirocin. In contrast, fusidic acid inhibits protein synthesis of S. aureus by binding with elongation factor G (EF-G, encoded by the fusA gene). Mutations of fusA gene are common in S. aureus during the development of fusidic acid resistance ^8–10.

Monolaurin or glycerol monolaurate is a monoglyceride found in coconut oil that is an ingredient in some natural skincare products ¹¹. Monolaurin inhibits the growth of fungi, viruses and bacteria ¹² and interferes with cell wall synthesis of Gram-positive bacteria ¹³. However, the effects of monolaurin have never been investigated in S. aureus in AD patients, in particular the strains of this pathogen that have developed antimicrobial resistance. In this study, we identified antimicrobial-resistant S. aureus isolated from AD patients and detected resistance genes of mecA, mupA, and de novo gene mutations in fusA, that were characterised by in silico molecular docking. Monolaurin was synthesized and purified in our laboratory, and we showed that it could inhibit these antimicrobial-resistant S. aureus.

Demographic data

The demographic characteristics of thirty children and thirty adults diagnosed with AD are shown in Table 1. The disease severity using EASI and SCORAD showed that children with AD had greater clinical severity than adults (p < 0.001) (Supplementary Table S3 online). The correlation between the severity scores and a presence of S. aureus on the lesions is shown in Table 2. The result demonstrated that both severity scores were significantly correlated with the presence of S. aureus (p < 0.001), particularly in patients with moderate to severe AD (EASI > 7 or SCORAD > 20) who showed higher incidences of S. aureus isolated from the lesion when compared to patients with mild symptoms.

Table 1. Demographic characteristics of AD patients

Demographic characteristics		Children (n=30)		Adults (n=30)
Demographic characteristics		Number	Percent	Number	Percent
Sex
	Male	17	56.6	9	30
	Female	13	43.3	21	70
BMI (Mean ± SD)		18.39 ± 4.41		23.57± 3.82
Allergic rhinitis/conjunctivitis		17	56.6	25	83.3
Allergic asthma		3	10	3	10
Drug/food allergy		10	33.3	8	26.7
Family history of allergic diseases
	allergic rhinitis/conjunctivitis	20	66.6	12	41.4
	allergic asthma	7	24.1	5	17.2
	atopic dermatitis	12	41.4	5	17.2
Smoking history/FH of smoking		4	13.3	12	40

Table 2. Disease severity with a presence of S. aureus isolated from the lesions in a combination of children (n=16) and adults (n=8) with AD

Score		S. *aureus*	p-value
Score		N (%)	p-value
EASI			0.001*
	mild (1-7)	5(20)
	moderate (7-20)	11(45.8)
	severe (>20)	8(33.3)
SCORAD
	mild (1-20)	0(0.0)	0.002*
	moderate (20-40)	8(33.3)
	severe (>40)	16(66.7)

* p<0.05

Treatments in patients with AD are shown in Supplementary Table S4 online and it was found that 90% of both groups were treated with topical corticosteroids, while 53.3% of children with AD received topical antibiotics whilst only 33.3% of adult patients used these medications. Systemic antibiotics were significantly used in 36.7% of children with AD (p < 0.05) whereas only 13.3% of adult patients were treated with systemic agents.

Bacterial identification in patients with AD

Clinical samples were swabbed from lesions, non-lesions and nasal mucosa of children (n = 30) and adults (n = 30) with AD. Figure 2 shows that S. aureus was found in 16 (53%) and 8 (27%) clinical isolates from lesions of children and adults with AD, respectively. Only 5 children (16%) and 2 adults (7%) with AD were colonized with S. aureus on non-lesions. In addition, coagulase-negative staphylococci (CoNS) were found on all sites, particularly in nasal cavities of adult patients, as shown in Fig. 2 and Supplementary Fig.S1 online.

Antibiotic susceptibility testing of S. aureus clinical isolates

The susceptibility of S. aureus isolated from lesions of children (n = 16) and adults (n = 8) with AD for different antibiotics was tested using the disk diffusion method. The MIC results of cefoxitin, mupirocin, and fusidic acid (Table 4), identified one MRSA isolate with a value of 8 µg/mL in the children group. One isolate of mupirocin-resistant S. aureus had a MIC of ≥ 256 µg/mL, while four isolates of fusidic acid-resistant S. aureus had MIC’s of 2, 2, 2, and 4 µg/mL, respectively (Table 4).

All S. aureus isolated from lesions of adult patients (n = 8) were susceptible to cefoxitin, mupirocin, and fusidic acid (Table 3). Furthermore, S. aureus found in lesions of children with AD were resistant to tetracycline (n = 4, 25%), ciprofloxacin (n = 1, 6%), clindamycin (n = 2, 13%), and erythromycin (n = 3, 19%) whilst there were only 2 clinical isolates (25%) from adult patients were resistant to tetracycline.

Table 3. Antibiotic susceptibilities of S. aureus isolated from lesions of children and adults with AD (S=sensitive, R=resistant)

Antibiotic susceptibility testing of S. aureus		Children (n=16)		Adults (n=8)		P-value
Antibiotic susceptibility testing of S. aureus		Number	Percent	Number	Percent	P-value
Cefoxitin
	S	15	93.75	8	100	0.497
	R	1	6.25	0	0
Sulfamethoxazole/trimethoprim
	S	16	100	8	100	-
	R	0	0	0	0
Fusidic acid
	S	12	75	8	100	0.037*
	R	4	25	0	0
Mupirocin
	S	15	93.75	8	100	0.126
	R	1	6.25	0	0
Tetracycline
	S	11	68.75	6	75	0.883
	R	5	31.25	2	25
Ciprofloxacin
	S	15	93.75	8	100	0.497
	R	1	6.25	0	0
Clindamycin
	S	14	87.5	8	100	0.326
	R	2	12.5	0	0
Erythromycin
	S	13	81.25	8	100	0.22
	R	3	18.75	0	0

* p<0.05

The resistance mechanism of antimicrobial-resistant S. aureus

The clinical isolate of MRSA contained the mecA gene, while the mupirocin-resistant S. aureus clinical isolate contained the mupA gene (ileS2) as detected by PCR (Supplementary Fig.S2 online). As fusidic acid-resistant S. aureus mostly result from mutations of the fusA gene encoding EF-G, the mutations of the four fusidic acid-resistant S. aureus clinical isolates were identified by nucleotide sequencing and compared with wild-type S. aureus using the MultAlin program. The results showed that the predicted amino acid sequences of EF-G from fusidic acid-resistant S. aureus differed from the wild-type sequence. Moreover, novel EF-G mutations were observed in this study in which two isolates possessed P404Q (404 proline/glutamine) and Q505H (505 glutamine/histidine) substitutions, one isolate possessed S238A (238 serine/alanine) and C258W (258 cysteine/tryptophan) substitutions, and one isolate possessed a H457Y (457 histidine/tyrosine) substitution (Supplementary Table S5 online).

To understand and compare the binding properties of fusidic acid with wild-type and mutant EF-G molecules (P404Q, Q505H, S238A, C258W and H457Y) of S. aureus (EF-G vs mEF-G), post-docking analysis was performed using AutoDock v4.2. The results showed that fusidic acid (binding energy: -6.84 kcl/mol, Ki: 9.64 µM) formed two hydrogen bonds with two different amino acids in wild-type EF-G (Fig. 3b). The OH group of the 2-hydroxy toluene ring formed a hydrogen bond with OH of Thr24 present in the H5 Helices ¹⁴ with a distance of 2.029 Å. The OH of Thr68 formed a hydrogen bond with OH of the pentenoic acid moiety in the fusidic acid at a distance of 1.973 Å, presented the interfaces of domains I, II, and III in the C-terminal ¹⁵ and completely occupied the Hydrophobic pocket in wild-type EF-G (Fig. 3c). In the case of mEF-G (P404Q, Q505H, S238A, C258W and H457Y), the binding energy of fusidic acid was found to be -5.79 kcl/mol with a Ki value of 56.75 µM, which was much lower than the wild type EF-G. Fusidic acid formed only one hydrogen bond with the mutant protein (Fig. 3c). The NH group of Lys23 formed a hydrogen bond with the carboxylic acid group of fusidic acid (NH - - -OH) at a distance of 2.053 Å (Fig. 3c). Due to the mutations of residues 404 and 457, the ligand was unable to form any interaction in the hydrophobic pocket.

The inhibitory effect of monolurin on antimicrobial-resistant S. aureus

Monolaurin inhibited S. aureus ATCC 29213 strain, MRSA, mupirocin-resistant S. aureus, and fusidic acid-resistant S. aureus at MICs of 1, 2, 2 and 2 µg/mL, respectively (Table 4 and Supplementary Fig.S3 online). Moreover, MBCs was determined by subculturing each isolate of antimicrobial-resistant S. aureus at the same concentrations determined in the MIC, and the results showed that the MBCs were 2 µg/mL for MRSA, mupirocin-resistant S. aureus, and fusidic acid-resistant S. aureus, as there was no growth detected of each isolate (Table 4).

Table 4. MICs and MBCs of antibiotics and monolaurin against antimicrobial-resistant S. aureus isolated from children with AD and a standard strain (ATCC 29213)

*S. aureus* isolates	MIC^a (µg/mL)		MBC^f
*S. aureus* isolates	Antibiotics	Monolaurin	Monolaurin
*S. aureus* ATCC 29213^b	1-4	1	NG
Cefoxitin	1-4	1	NG
Mupirocin	0.06-0.5	1	NG
Fusidic acid	0.06-0.25	1	NG
*S. aureus* MRSA^c	8 (R)	2	NG
*S. aureus* MupRSA^d	≥256 (R)	2	NG
*S. aureus* FusA_1^e	2 (R)	2	NG
*S. aureus* FusA_2^e	2 (R)	2	NG
*S. aureus* FusA_3^e	2 (R)	2	NG
*S. aureus* FusA_4^e	4 (R)	2	NG

^a MIC: lowest concentration at which drug can inhibit microorganism. Concentration for monolaurin is µg/ml.

^b S. aureus ATCC 29213: Control for broth microdilution method

^c MRSA: Methicillin-resistant S. aureus clinical isolate

^d MupRSA: Mupirocin-resistant S. aureus clinical isolate

^e FusA: Fusidic acid-resistant S. aureus 4 clinical isolates (FusA_1-4)

^f NG: no bacterial growth after 24-h incubation at MICs

R: resistant

The cytotoxicity of monolaurin on human keratinocytes and dermal fibroblasts

The potential cytotoxicity of monolaurin on HEKn and dermal fibroblasts was determined by MTT assay and it was found that monolaurin at concentrations of 2–4 µg/mL was not toxic to HEKn (Fig. 4). Similarly, monolaurin at concentrations of 2–8 µg/mL was not toxic to dermal fibroblasts. However, higher concentrations of monolaurin (16–32 µg/mL) showed significant decreases in cell viability of both HEKn and dermal fibroblast cells (p < 0.05).

S. aureus is a major aggravating factor of AD. This pathogen not only drives pathogenesis of the disease but also triggers disease recurrence and flare ¹⁶. In previous studies, it was demonstrated that S. aureus was found in up to 90% of the lesions ¹⁷, and their toxins can induce acute exacerbation of AD patients ^18,19. In this study, we first identified S. aureus in swabs from the lesions of AD patients using conventional methods (e.g., coagulase test, catalase, and biochemical tests) and rapid test (APISTAPH kit) and then confirmed identity by PCR (nuc gene expression) ²⁰. The results showed that S. aureus was more commonly found on lesions, particularly in children with AD, than other species of Staphylococcus (Fig. 2). Moreover, the lesions had more colonization by S. aureus in half of these children (53%) when compared with non-lesions and the nasal mucosa. Interestingly, patients with moderate to severe AD (EASI > 7 or SCORAD > 20) had a higher incidence of S. aureus isolated from the lesions (Table 2). These findings are consistent with previous studies and support a strong association between S. aureus colonization and disease severity of AD ^{17–19,21,22}. Notably, S. aureus was less-commonly isolated from adult patients than children (Fig. 2), and this is probably because adults had milder symptoms than children (Supplementary Table S3 online), again highlighting the link between colonization by this pathogen and disease severity.

In clinical practice, topical (e.g., mupirocin and fusidic acid) and systemic antibiotics (e.g., cloxacillin, first-generation cephalosporin: cephalexin) are usually prescribed in AD patients with severe symptoms and/or clinical signs of S. aureus infection. However, the prevalence of antimicrobial-resistant S. aureus is increasing, particularly MRSA, leading to decreased clinical responses to the treatment of AD ^23,24. Although mupirocin and fusidic acid are usually effective antibiotics against S. aureus, including MRSA, the prevalence of mupirocin and fusidic acid-resistant S. aureus has also been reported in AD patients ²⁴. In this study, antimicrobial-resistant S. aureus on the lesions of AD patients was found only in children with AD (n = 1 MRSA; n = 1 mupirocin-resistant S. aureus: n = 4 fusidic acid-resistant S. aureus). Unexpectedly, no antimicrobial-resistant S. aureus were isolated from adult patients (Table 3), and this is probably due to lower disease severity and infrequent/unnecessary use of topical antibiotics in adult patients (Supplementary Table S4 online). Nevertheless, the incidence of antimicrobial-resistant S. aureus in children with AD was lower than the incidence in previous studies ²⁵ probably because we recruited a limited number of patients (n = 30) in both groups, and sampling took place during the COVID-19 pandemic, when hand hygiene procedures were more commonplace.

The mechanisms responsible for antibiotic resistance were investigated in the S. aureus isolates from AD patients. We found that mecA and mupA genes were expressed in one MRSA and one mupirocin-resistant S. aureus clinical isolate, respectively. In contrast, four fusidic acid-resistant S. aureus with newly-identified fusA gene mutations were isolated (Supplementary Table S5 online). The results showed alleles encoding EF-G acquisition with the amino acid substitutions of P404Q (404 proline/glutamine), Q505H (505 glutamine/histidine), S238A (238 serine/alanine), C258W (258 cysteine/tryptophan) and H457Y (457 histidine/tyrosine) arising from point mutations in these clinical isolates: these mutations, except H457 ^10,26,27, have never been reported previously. Moreover, molecular docking studies further suggested that these de novo mutations resulted in weak binding of fusidic acid to the active site of the mutated EF-G (Fig. 3), which could explain fusidic acid resistance. Notably, the incidence of fusidic acid-resistant S. aureus was higher than other antimicrobial-resistant S. aureus. This finding may be because fusidic acid is more commonly prescribed in our patient population, particularly children with AD (Supplementary Table Table S4 online) and frequent use of this agent drives fusidic acid resistance of S. aureus. In addition, the higher incidence of fusidic acid–resistant S. aureus may be related to the methods used and breakpoint values applied for detection of fusidic acid resistance (MIC > 1 µg/mL) ^{28 29}. Fortunately, we did not find S. aureus clinical isolates with multidrug resistance (cefoxitin, mupirocin and fusidic acid) in our population.

Although the incidence of antimicrobial-resistant S. aureus is low in this study (Table 3), the incidence across the world has been increasing. We therefore evaluated a chemical product for its potential to address this challenging problem. Recently, monolaurin extracted from natural products (i.e. coconut oil) has shown antimicrobial activity against different organisms such as bacteria including S. aureus ^30,31. It has been found that monolaurin inhibits the synthesis of staphylococcal toxins and other exoproteins, and expression of virulence factors such as protein A and toxic shock syndrome toxin-1 (TSST-1). Moreover, it interferes with the regulation of bacterial signaling pathways that are critical for survival of gram-positive bacteria ³², and signal transduction in the synthesis of 𝜷-lactamase ^33,34.

In this study, monolaurin was synthesized in our laboratory and the chemical properties and purity of monolaurin were demonstrated using the Nuclear Magnetic Resonance (NMR) spectra of proton and carbon (Fig. 1), and the results were consistent to previous studies^35–37. The inhibitory effect of monolaurin against S. aureus including antimicrobial-resistant S. aureus was investigated. We showed that monolaurin significantly inhibited MRSA, mupirocin- and fusidic acid-resistant S. aureus at the MIC and MBC of 2 µg/mL (Table 4). As a previous study reported that monolaurin (25 or 50 µM; 6.8 or 13.72 µg/mL) was toxic to keratinocytes (OBA-9 cells) and fibroblasts (HGF-1 cells) ³⁸, we therefore further investigated the cytotoxic effect of monolaurin on primary human keratinocytes and dermal fibroblasts. We showed that there was no direct toxicity to these skin cell types at concentrations that completely inhibited S. aureus. (Fig. 4). Our preliminary findings suggest that monolaurin could potentially be used as an alternative topical treatment for antimicrobial-resistant S. aureus on skin lesions of AD patients. This would require clinical trials of this agent.

In conclusion, this study demonstrated that S. aureus was frequently found on the lesions of children with AD, and it was associated with disease severity. Both topical and systemic antibiotics are usually prescribed in children with AD which probably resulted in higher incidences of antimicrobial-resistant S. aureus, particularly fusidic acid-resistant S. aureus. We have characterised novel fusidic acid mutants and propose that monolaurin, a new chemical extract is a safe inhibitor of S. aureus, including antimicrobial-resistant strains without cytotoxicity to skin cells. Our preliminary data suggests that monolaurin could potentially be used as an alternative treatment for AD patients with antimicrobial-resistant S. aureus infections.

Data and biological sample collection

Children (3 months – 16 years old) and adults diagnosed with AD using Hannifin and Rajka clinical criteria ³⁹ who attended dermatology, pediatric dermatology, and pediatric allergy clinics at the King Chulalongkorn Memorial Hospital were recruited in this study during the COVID-19 pandemic. The demographic data, including clinical presentation, treatments, and severity scores using EASI and SCORAD ^40,41 were recorded. The exclusion criteria were patients with current bacterial skin infection, systemic infection, contact dermatitis, and recent (within the past 4 weeks) use of topical or systemic antibiotics, or systemic corticosteroids, or recent hospital admission. This study was approved by the Institutional Review Board (IRB No. 640/60), Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Written informed consent and/or assent forms were obtained from all participants. For human participants that are minors, the informed consent and/or assent forms were obtained from parents and/or legal guardians for both study participation and publication of identifying information in an online open-access publication. We confirm that all methods and experiments were performed in accordance with relevant guidelines and regulations. Biological samples from 3 different sites of the skin (lesion, non-lesion, and nasal mucosa) were swabbed using separate sterile cotton sticks, and samples were inoculated onto 5% sheep blood agar and mannitol salt agar (MSA; a selective media for S. aureus).

Bacterial identification and Antibiotic susceptibility testing

The inoculated plates were incubated at 35°C for 18–24 h in ambient air. The bacterial colonies from MSA and 5% sheep blood agar plates were identified by Gram-staining, catalase, coagulase, biochemical tests, API Staph kit (bioMérieux, France), and confirmed by polymerase chain reaction (PCR) using a specific gene (nuc) as shown in Supplementary Table S1-S2 online.

Antimicrobial resistance in S. aureus isolated was initially detected using the disk diffusion method (CLSI). Briefly, pure colonies of isolated S. aureus were suspended in normal saline solution and adjusted to be equivalent to 0.5 McFarland standard before spreading onto Muller-Hinton agar (BBL, USA). Disks of cefoxitin (30 µg), clindamycin (2 µg), erythromycin (15 µg), trimethoprim/sulfamethoxazole (5 µg), ciprofloxacin (5 µg), tetracycline (30 µg), fusidic acid (10 µg), and mupirocin (200 µg) were mounted on the agar surface and incubated at 35°C for 18–24 h. The result (susceptible; S, intermediate; I, and resistant; R) was interpreted using the Clinical and Laboratory Standards Institute (CLSI) ⁴² and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria ²⁸. S. aureus ATCC 25923 was used as a control strain (zone diameter ≥ 22 mm). Minimum inhibitory concentrations (MICs) of cefoxitin (0.03 to 32 µg/mL), mupirocin (> 256 µg/mL), and fusidic acid (0.03 to 32 µg/mL) were determined by broth microdilution assay for phenotypic detection of methicillin, mupirocin, and fusidic acid resistances.

Detection of antimicrobial resistance genes by PCR

Genomic DNA of the S. aureus was extracted as a template of PCR. mecA, mupA, fusA genes were amplified by PCR using specific primers (Supplementary Fig.S2 online) ^7–9,43. The PCR conditions were 30 cycles of 94°C (mecA) or 95°C (mupA and fusA) for 30 sec; 55°C (mecA) or 57°C (mupA and fusA) for 30 sec and 72°C for 4 min. Genotypes of methicillin resistance and mupirocin resistance were detected by the presence of mecA and mupA amplicons using agarose gel electrophoresis. The PCR product of the fusA gene was purified using the QIAquick PCR purification kit for nucleotide sequencing at the 1st BASE Inc, Malaysia.

Analysis of nucleotide sequences

Nucleotide sequences and deduced amino acid sequences were analyzed using Online Software available at the National Center for Biotechnology Information (NCBI). (https://blast.ncbi.nlm.nih.gov), SnapGene (version 5.0.7) and ExPASy (www.expasy.org). Multiple sequence alignment was performed using Multilin (http://multalin.toulouse.inra.fr/multalin). The fusA sequences of fusidic acid-resistant S. aureus isolates were compared with the fusA sequence from S. aureus NCTC 8325 (GenBank accession no. NC_007795), using Mega 4 software (Biodesign Institute, Tempe, AZ, USA).

Molecular docking

The AutoDock v4.2 tool was used to perform a molecular docking simulation of fusidic acid with both wild-type EF-G and compared with mutated EF-G (mEF-G). The structure of wild type EF-G obtained from rcsb.org/ (PDB: 2XEX), and mutations were modeled by PyMOL [DeLano, 2002] (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.). Both molecules were analysed in the same manner by deleting the co-crystalized waters, adding polar hydrogen, and computing gasteiger charge. The ligand was modified in silico by adding polar hydrogen and computing gasteiger charge, before initiating the docking process. The grid file was generated with 60 × 60 × 60 as the number of points in x, y, and z directions, and the center spacing was 0.375Å. A docking output file was prepared with Lamarckian Genetic Algorithm using default settings. Finally, both grid and docking output files were performed using a script of autogrid4 and autodock4 (The Scripps Research Institute). The interactions were analyzed using MGL tools.

Synthesis of monolaurin

Figure 1 shows the chemical structure of monolaurin. 0.3 g (2.27 mmol) of 1,2-isopropylideneglycerol in 10 mL of CH₂Cl₂ under N₂ gas conditions was mixed with lauroyl chloride (2.25 mmol) and triethylamine (3.4 mmol) ⁴⁴. The reaction mixture was stirred for 30 min at room temperature and washed with water before evaporation using a rotatory evaporator ⁴⁵. The concentrated compound (0.5 mmol) was dissolved in 50 mL of acetone, then mixed with 10 mL of aqueous 3N HCl and stirred for 1.5 h at room temperature. The solution was re-evaporated, neutralized with NaHCO₃ and extracted with CH₂Cl₂. The residue was purified by silica gel column chromatography and the final product (monolaurin) was collected as a powder. The spectral signals of monolaurin as determined by Nuclear Magnetic Resonance (NMR) of proton (¹H NMR) were: ¹H-NMR (500 MHz, CDCl₃): δ 4.16 (dd, J = 13.6, 5.3 Hz, 2H), 3.91 (s, 1H), 3.67 (d, J = 4.0 Hz, 1H), 3.59 (d, J = 5.8 Hz, 1H), 2.42 (s, 2H), 2.33 (s, 2H), 1.61 (s, 2H), 1.24 (s, 16H), 0.86 (s, 3H); and carbon (¹³C-NMR):¹³C-NMR (126 MHz, CDCl₃): δ 174.51, 70.35, 65.23, 63.43, 34.25, 31.99, 29.69, 29.54, 29.42, 29.34, 29.21, 24.99, 22.77, 14.21 (as shown in Fig. 1).

Monolaurin susceptibility testing

The MIC and minimum bactericidal concentration (MBC) of monolaurin were determined using the broth microdilution method. Monolaurin powder was dissolved in 30% ethanol (w/v), and concentrations of 0.0625 to 32 µg/mL were adjusted using two-fold dilution with Muller Hinton II broth (cation-adjusted) (BBL, USA). Antimicrobial-resistant S. aureus (1.5x10⁸ CFU/mL) clinical isolates were incubated with different concentrations of monolaurin at 35°C for 24 h. The MIC was determined at the lowest concentration of monolaurin that resulted in no visible growth of isolates. To determine the MBC, each isolate (0.01 mL of bacterial suspension) of antimicrobial-resistant S. aureus at the same concentrations determined in the MIC was subcultured and inoculated onto blood agar and further incubated at 35°C for 24 h. The concentration of monolaurin with no growth of isolates was identified.

Cytotoxicity Assay

Primary epidermal keratinocytes (HEKn) and dermal fibroblasts were seeded (1 x 10⁵ cells/mL) into 96-well plates and incubated with Dermal Cell Basal Medium and Dulbecco’s Modified Eagle Medium (DMEM), respectively (Cytiva, USA) at 37°C in 5% CO₂ for 24 h. Different concentrations of monolaurin (0.0625-32 µg/mL) were added into each well and the cells were incubated at 37°C in 5% CO₂ for 24 h. The cell culture media was removed, and fresh medium with 10 µL of thiazolyl blue tetrazolium bromide method (MTT) (Sigma, USA) was added into each well, followed by incubation at 37°C in 5% CO₂ for 4 h. The solution was discarded and DMSO was added to dissolve the formazan crystals. The optical density (OD) was measured at 570 nm using a microplate reader.

Statistical analyses

The statistical analyses were performed using GraphPad Prism version 9.2.0 (GraphPad Software, USA), Pearson’s chi-squared test and Fisher exact test (for categorical data) and Paired t-test (for continuous data). The results are presented as the mean ± standard deviation (SD) and differences with a p-value < 0.05 were considered statistically-significant.

Acknowledgment (optional)

We would like to thank the Division of Dermatology, Skin, and Allergy Research Unit, Department of Medicine and Division of Dermatology, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand for helping with the biospecimen collection.

Statement of Ethics

This study was approved by the Institutional Review Board (IRB No. 640/60), Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Written informed consent and/or assent forms were obtained from all participants.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

Ratchadapiseksompotch Fund, Faculty of Medicine, Chulalongkorn University, grant number RA66/005

Author Contributions

M.L, S.S ; performed the experiments, D.L.W, V.N.B, and P.H; molecular docking analysis and co-wrote the manuscript, S.W.E, N.S, Pan.C, P.L, P.R, K.C, S.C, S.W, A.T, R.P and Pat.C; co-investigation and co-wrote the manuscript, M.L, S.S, A.T, and D.C; data curation and co-wrote the manuscript, Pat.C, W.C, T.C, and D.C; resources and validation; S.S, M.L, T.C, and D.C writing-review and editing; M.L and S.S visualization; T.C and D.C project administration

Data Availability Statement

All data underlying the results are available as part of the article and no additional source data are required.

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No competing interests reported.

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Monolaurin inhibits Antibiotic-resistant Staphylococcus aureus in Patients with Atopic Dermatitis

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