Effects of Mannosylerythritol-Lipids-B on Cutibacterium acnes ATCC 6919

Abstract

Mannosylerythritol-lipids-B (MEL-B) are microbial-produced glycolipids with skincare properties, notably moisturizing, antimelanogenic, antimicrobial, and antiaging. Thus, there is a potential use of MEL-B in a formulation for treating acne-prone skin. This study investigated the antimicrobial effect of MEL-B against the Gram-positive bacteria Cutibacterium acnes. The broth macro dilution method was used to evaluate the growth of C. acnes (3–4 CFU/mL), in the absence (positive control) or presence of MEL-B (128, 192, 256, and 512 μg/mL). Additionally, the leakage of genetic materials was used to determine the potential drug-induced membrane disruption of glycolipids. The amount of DNA and RNA release was quantified spectrophotometrically at 260 nm. Macro dilution technique and membrane integrity experiments showed that MEL-B does not have antimicrobial activity against C. acnes. Indeed, MEL-B assisted C. acnes growth. Ultimately, MEL-B has been reported as a remarkably active compound for skincare formulations; however, preliminarily, it should be avoided for acneic skin.

MEL-B is a glycolipid that has shown promising activity in several studies in the skincare field. Among the properties of MEL-B, it can be highlighted anti-melanogenic and lightening effects, antiaging by modulation of aquaporin-3 expression, moisturizing, and enhancer agent of delivery transdermal of water-soluble compounds [1–3]. Moreover, this glycolipid presented antitumoral activity [4], which can be promising for advanced skincare formulation. Regarding the action of MEL-B on bacterial strains, it can act as an antimicrobial [1] or prebiotic [5] compound.

Cutibacterium acnes is a pathogenic bacterium, classified as Gram-positive, fastidious, aerotolerant nevertheless anaerobiotic. This specie is the agent of acne vulgaris, a disease characterized by an imbalance in the skin microbiota that results in an inflammatory process. The symptoms include altered follicular keratinization, follicular colonization, and inflammation of the pilosebaceous unit [6, 7].

In this sense, the MEL-B properties cited above raise questions about its viability for acne-prone skin since this type of skin is more likely to favor the development of C. acnes. Thus, assuming MEL-B as an ingredient of the skincare formula, the current study aimed to evaluate the effect of this glycolipid against C. acnes.

Cutibacterium acnes ATCC 6919 was purchased from Fundação André Tosello (Brazil). MEL-B (95% purity degree, Japan) was produced from Pseudozyma tsukubaensis and purified using liquid–liquid extraction followed by silica chromatography. Other materials used were Reinforced Clostridium Medium (RCM, Merck), Agar (Merck), Sodium chloride (99% of purity, Dinamica, Brazil), Anaerobic chamber 2.5 L, Anaerobac® (PROBAC, Brazil), and polysulfone membrane 0.22 µm (Millipore).

The bacterial strain was activated by seeding it in 5 mL of RCM broth. The tube was sealed with 2 mL of liquid agar, as recommended by the manufacturer, and incubated in an anaerobic chamber with Anaerobac® at 37 °C for 24 h.

The inoculum was prepared with the bacterial liquid culture after 24 h of growth, followed by dilution in saline solution (0.85% NaCl) until factor 10^–6, reaching around 3–4 CFU/mL. Then, these standardized inoculants were used for all assays.

In growth kinetics experiments, 100 µL of inoculum was added to tubes containing 5 mL of RCM broth. Then, 100 µL of culture was sampled at different time intervals. The samples' optical density at 600 nm was measured in a spectrophotometer (Biospectro SP220). In parallel, the samples were serially diluted with saline solution (0.85%) and plated by the double-layer poured plate method for colony forming units (CFU) determination. These Petri dishes were incubated under anaerobic conditions at 37 °C (the optimal temperature for the growth of C. acnes) for 48 h. Then, the colonies were counted using the colony counter [8].

The Baranyi and Roberts [9] model was fitted to experimental growth data using the Combase web platform (www.combase.cc).

The antimicrobial activity of MEL-B was screened by the broth macro dilution method, according to the standards of CLSI (Clinical and Laboratory Standards Institute) [10] for anaerobic and fastidious bacteria.

For antimicrobial assays, a volume of 100 µL of inoculum (around 3–4 CFU/mL) was added to each tube containing the MEL-B solutions (128, 192, 256, and 512 µg/mL), followed by incubation at 37 °C for 8 and 16 h in an anaerobic environment.

Then, for counting, samples were centrifuged at 3500 rpm for 15 min, the supernatant was discarded, and biomass was washed twice with saline solution (0.85% NaCl) to remove residual MEL-B. Subsequently, samples were suspended in an RCM medium and vortexed for 20 s. After that, 100 µL of bacteria suspension was collected, serially diluted into saline solution, and plated in solid RCM according to the double-layer poured plate method. The plates were incubated under anaerobic conditions at 37 °C for 48 h, and samples containing colonies between 30 and 300 were used to estimate log CFU/mL.

Bacterial suspension in RCM was used as the positive control, while tetracycline solution (4 µg/mL) was used as the negative control [11].

The release of nucleic acids was used to estimate cell membrane damage [12] caused by MEL-B. For this, the culture of C. acnes after 8 and 16 h was taken out, filtrated by a 0.22 µm filter membrane, and used to quantify double-stranded DNA and RNA release in the culture supernatants. The concentration of nucleic acids was determined by absorbance measurement using NanoVue Plus spectrophotometer. All experiments were conducted in duplicate. Two-way ANOVA (Analysis of variance) was used to analyze the data statistically (p < 0.05).

Figure 1 shows the C. acnes growth curve determined by plating count and spectrophotometry measurements. The Baranyi and Roberts model [8] was used to fit the CFU/mL growth data, obtaining a determination coefficient (R²) of 0.994, indicating the goodness of fit. The growth curves presented a sigmoidal format containing such lag, exponential and stationary phases. However, optical density data are not trustful out of the range 0.3–0.8 [13]; thus, no model adjustment was used to those values presented in Fig. 1. The lag phase duration and maximum specific growth rate were estimated by counting colonies on solid medium, with values of 6.8 ± 0.8 h and 0.77 h⁻¹ (R² 0.99), respectively. The stationary phase was observed after approximately 24 h of incubation.

Fig. 1.

Cutibacterium acnes growth curve in RCM medium determined by (□) plate count (CFU/mL) and (○) optical density at 600 nm. (-) Baranyi and Roberts model, and (---) optical density minimum and maximum confidence values

The treatment of planktonic cells with MEL-B showed that glycolipid enhances the strain growth, achieving a 2–3 log increase after 8 h (early exponential phase) or 16 h (middle exponential phase) of incubation.

In the early exponential phase, the increase in the bacterial population initially occurs gradually, and at the end, the cell divisions are more regulars (time). In the middle exponential phase, the cell division rate is maximum, in which the bacterial population is considered as uniform as possible, in all terms chemically, morphologically, and physiologically (metabolic activity) [14].

The statistical analysis reveals that MEL-B concentrations (Table 1) have a similar effect (p value = 0.15) on bacterial growth (log CFU/mL); F critical (6.38) was greater than F calculated (3.02). On the other hand, the strain development was influenced by incubation time (p value = 0.002; F_calculated 52.7 > F_critical 7.70). Thus, we speculated that glycolipid was in the intracellular space; nevertheless, it was hydrolyzed (especially the fatty acid chains) and then used as a nutrient source (the β-oxidation), assisting C. acnes growth. Therefore, MEL-B seems to be an unsuitable active compound for acne-prone skin products.

Table 1.

Cutibacterium acnes ATCC 6919 counts after exposure to MEL-B concentrations of 128, 192, 256, and 512 µg/mL under 8 and 16 h of incubation

MEL-B concentration (µg/mL)	Count after 8 h of incubation	Count after 16 h of incubation
	Log CFU/mL	Log CFU/mL
0 (positive control)	4.54	8.92
128	7.42	9.44
192	7.25	11.17
256	6.47	11.35
512	6.88	12.12

Some antimicrobial compounds can damage bacterial membranes [15], which leads to an increased genetic material release in the culture medium. Membrane integrity assays (Table 2) show that the DNA and RNA concentration for almost all study conditions slightly exceeds the control values. However, it does not indicate membrane damage; it seems that bacteria cell death accelerates. This behavior can be due to the decrease in the nutrient constituents due to the bacterial biomass increase caused by MEL-B (Table 1).

Table 2.

Cutibacterium acnes ATCC 6919 DNA and RNA release after exposure to MEL-B concentrations of 128, 192, 256, and 512 µg/mL under 8 and 16 h of incubation

MEL-B concentration (µg/mL)	Samples after 8 h of incubation		Samples after 16 h of incubation
	DNA ± CV (ng/µL)	RNA ± CV (ng/µL)	DNA ± CV (ng/µL)	RNA ± CV (ng/µL)
Positive control	362.5 ± 0.080	196.8 ± 0.046	492.0 ± 0.032	267.6 ± 0.070
128	393.5 ± 0.032	191.2 ± 0.022	433.5 ± 0.093	221.6 ± 0.121
192	499.0 ± 0.053	272.8 ± 0.151	425.0 ± 0.178	215.2 ± 0.130
256	392.5 ± 0.069	208.4 ± 0.128	329.5 ± 0.130	174.8 ± 0.114
512	514.5 ± 0.142	281.6 ± 0.062	368.0 ± 0.002	196.8 ± 0.051
Negative control	467.5 ± 0.002	251.6 ± 0.033	470.1 ± 0.036	263.2 ± 0.116

CV coefficient of variation

Statistical analysis showed that MEL-B concentration (p value = 0.67; F_calculated 0.65 < F_critical 5.05) and incubation time (p value = 0.65; F_calculated 0.22 < F_critical 6.60) do not influence the leakage of DNA. Similar results were found for RNA leakage—the statistical parameters values were p value = 0.63; F_calculated 0.72 > F_critical 5.05, and p value = 0.67; F_calculated 0.20 > F_critical 6.60 for concentration and incubation time, respectively.

Further experiments can deeply elucidate the effect of MEL-B on seboregulation, an important feature for acne manifestation. It is worth mentioning that oily skin does not necessarily imply acneic skin [16]. Oily skin is a clinical condition that can be observed even in patients without acne and is related to larger facial pores [16, 17]. However, the stimulation of sebum secretion is a key factor in triggering C. acnes overgrowth and inflammation mechanisms [14]. In particular, the detection and quantification of porphyrins appear to be another interesting aspect since these compounds are produced by C. acnes and indicate pathogenicity severity [17, 18].

Thus, for studied conditions, it is evident that MEL-B has stimulated the growth of C. acnes. In this sense, the insertion of this glycolipid in skincare formulations must be evaluated with caution since it could be problematic for the health of the epidermis. In addition, understanding the effect of MEL-B on non-pathogenic strains of the epidermis is primordial to defining how harmful or beneficial this compound can be in the skin microenvironment.

Mannosylerythritol-lipids-B (MEL-B) is a microbial glycolipid with skincare properties—moisturizing, anti-melanogenic, antimicrobial, and antiaging. In this regard, there is a gap about the potential use of MEL-B in a formulation for treating acne-prone skin. This study investigated the antimicrobial effect of MEL-B against the Gram-positive bacteria Cutibacterium acnes. To this end, the traditional macro-dilution and plate counting methods were used. The trials were realized with different MEL-B concentrations (0, 128, 192, 256, and 512 µg/mL). Additionally, the leakage of intracellular nucleic acids was used to determine the potential drug-induced membrane disruption by glycolipids. The amount of DNA and RNA release was quantified spectrophotometrically at 260 nm. Macro dilution technique and membrane integrity experiments showed that MEL-B does not have antimicrobial activity against C. acnes. Indeed, MEL-B assisted C. acnes growth. Ultimately, MEL-B has been reported as a remarkably active compound for skincare formulations; however, preliminarily, it should be avoided for acneic skin.

Author contributions

All authors contributed to the manuscript’s conception and design. Material preparation and data collection were performed by ALSC and DAL. Data analysis was performed by all authors. The first draft of the manuscript was written by ALSC. All authors commented on previous versions, revised the language, and approved the final manuscript.

Data availability

Data available on request from the authors.

Declarations

Conflict interest

The authors declare that they have no conflict of interest.