Influence of critical micelle concentration of
choline-based long chain fatty acid soaps on their
antibacterial activity against Methicillin resistant
Staphylococcus aureus

Helena Arellano, Tomasz Swebocki, Clémence Le Coeur, Sylvain Prévost, Marwan Abdallah, Véronique Nardello-Rataj, Anne-Laure Fameau

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To cite this version:

Helena Arellano, Tomasz Swebocki, Clémence Le Coeur, Sylvain Prévost, Marwan Abdallah, et al. Influence of critical micelle concentration of choline-based long chain fatty acid soaps on their antibacterial activity against Methicillin resistant Staphylococcus aureus. Journal of Colloid and Interface Science, 2024, 677, pp.314-323. 10.1016/j.jcis.2024.07.218. hal-04672571

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Regular Article

# Influence of critical micelle concentration of choline-based long chain fatty acid soaps on their antibacterial activity against *Methicillin resistant Staphylococcus aureus*

Helena Arellano<sup>a,1</sup>, Tomasz Swebocki<sup>b,1</sup>, Clémence Le Coeur<sup>c,d</sup>, Sylvain Prevost<sup>e</sup>, Marwan Abdallah<sup>f</sup>, Veronique Nardello-Rataj<sup>g</sup>, Anne-Laure Fameau<sup>a,*</sup>

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GRAPHICAL ABSTRACT

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ARTICLE INFO

Keywords:

Antibacterial lipids

Chaotropic counter-ions

Long chain saturated fatty acids

Self-assembly

Micelles

ABSTRACT

**Hypothesis:** Antimicrobial resistance (AMR) is a pressing global health concern. ESKAPEE pathogens, such as *Methicillin-resistant Staphylococcus aureus* (MRSA) are notable of concern in healthcare settings due to their resistance to critical antibiotics. To combat AMR, the development of alternatives such as bacterial membrane-active agents is crucial. Fatty acids (FAs) have emerged as a sustainable, antibiotic-free solution with inherent antibacterial activity. However, long chain saturated fatty acids (LCFAs) sodium soaps exhibit poorly antibacterial properties in comparison to short chain FAs, believed to be linked to limited solubility in aqueous media.

**Experiments:** We employed choline as a chaotropic organic counter-ion to enhance the solubility of LCFAs and investigated their antibacterial effects against MRSA. The optimal medium conditions for micelle formation for

<footnote><sup>a</sup> Univ. Lille, CNRS, INRAE, Centrale Lille, UMR 8207 – UMET – Unité Matériaux et Transformations, 59000 Lille, France</footnote>

<footnote><sup>b</sup> Univ. Lille, CNRS, Univ. Polytechnique Hauts-de-France, UMR 8520 – IEMN – Institut d'Electronique de Microélectronique et de Nanotechnologie, 59000 Lille, France</footnote>

<footnote><sup>c</sup> Laboratoire Léon Brillouin, CEA, Saclay, France</footnote>

<footnote><sup>d</sup> CNRS, ICMPE, UMR 7182, 2 rue Henri Dunant, Université Paris Est Creteil, 94320 Thiais, France</footnote>

<footnote><sup>e</sup> Institut Laue-Langevin, 71 Avenue des Martyrs, CS 20156, Cedex 9, 38042 Grenoble, France</footnote>

<footnote><sup>f</sup> Laboratoire Départemental Public du Nord, 59370 Villeneuve d'Ascq, France</footnote>

<footnote><sup>g</sup> Univ. Lille, CNRS, Centrale Lille, Univ. Artois, UMR 8181 – UCCS – Unité de Catalyse et Chimie du Solide, 59000 Lille, France</footnote>

<footnote>* Corresponding author.
E-mail address: anne-laure.fameau@inrae.fr (A.-L. Fameau).</footnote>

<footnote n="1">¹ Shared contribution.</footnote>
LCFAs was first investigated. Then, we determined the critical micelle concentration (CMC), micellar morphology, and aggregation number through surface tension measurements and small angle neutron scattering experiments. Antimicrobial activity was assessed using minimum bactericidal concentration (MBC) assays and time-kill experiments.

*Findings:* We have identified conditions where LCFAs are effective against MRSA for the first time, providing valuable insights for developing new antibacterial agents to fight AMR. LCFAs need to be used above their Krafft temperatures and CMC to exhibit antibacterial efficacy.

# 1. Introduction

The peril to human health posed by antimicrobial resistance (AMR) is widely recognized within the scientific community [1]. AMR is observed in a spectrum of bacteria, encompassing both Gram-positive and Gram-negative strains. The World Health Organization has identified 12 bacterial species, that pose a particular threat in hospitals, nursing homes, and among patients whose care requires devices such as ventilators and blood catheters [2]. This group of pathogens manifests resistance to a multitude of antibiotics and are stratified into three levels. The “critical group” encompasses Gram-negative ESKAPEE pathogens such as *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, *Klebsiella pneumoniae*, *Escherichia coli* and *Enterobacter spp.* Gram-positive strains are notably *Enterococcus faecium* and *Staphylococcus aureus* which are classified as “priority high” [3]. The acronym ESKAPEE is a concatenation of the initials of these bacterial strains, aptly alluding to their ability to evade contemporary treatment regimens and antibiotics [4].

Within the Gram-positive ESKAPEE group, methicillin-resistant *S. aureus* (MRSA) poses big challenges in healthcare settings due to its resistance to a broad range of antibiotics, including beta-lactams [5]. In 2021, findings revealed that 25 % of the 44 surveyed countries reported invasive MRSA isolates < 5 %, while a staggering 30 % of the included nations recorded isolate percentages surpassing 25 % [2]. MRSA is responsible for skin and soft tissue infections, but also for more serious diseases such as bacterial endocarditis, pleuropulmonary, septic arthritis and osteomyelitis [6,7]. Internationally, MRSA has exhibited an alarming rise in prevalence. At present, distinctive approaches are under development for the management of MRSA infections. While certain antibiotics remain in use, such as vancomycin, tedizolid or fluoroquinolones, the urgency of non-antibiotic treatments is clearly stated and different routes are exploited [2,6,8,9].

Antimicrobial peptides have been widely investigated, with their mode of action based on disrupting the bacterial cellular membranes. The high production costs and chemical instability highlight the need for other approaches [10–12]. Fatty acids (FAs), which are a sustainable, membrane-active agents have been recognized for their antibacterial properties for centuries, particularly in their salt form, known as soaps, leveraging their inherent surfactant properties [13–15] The hallmark characteristic of FAs lies in their ability to disrupt the cytoplasmic membrane [15]. FAs reported action are on the inhibition of DNA/RNA replication, suppression of cell wall biosynthesis in Gram-positive bacteria, and interference with protein synthesis, mechanisms described broadly in the work by Casillas-Vargas et al. [14].

Among the spectrum of FAs, some long chain saturated fatty acids (LCFAs) have demonstrated growth inhibition against different MRSA species [16]. Kitahara et al. reported that by increasing the alkyl chain lengths from 12 to 18 carbons (from lauric acid to stearic acid), the minimal inhibitory concentration (MIC) values increased drastically from 400 µg mL⁻¹ for lauric acid to ≥ 1600µg mL⁻¹ for stearic acid. The increase of the chain length of LCFAs, leads to a decrease of solubility in aqueous media, which could explain the drastic decrease of antibacterial effect [13,17]. For example, the solubility of LCFAs in water at 20 °C (pH around 7) was determined at 0.055 g/L and 0.0029 g/L for C12 and C18, respectively [18]. The solubility of FAs in water is intricately tied to the pH of the solution. FAs, as weakly ionizable surfactants, require the

solution to attain a pH surpassing their apparent pKₐ for them to adopt a soluble state, akin to the soap form (COO⁻) [19]. The apparent pKₐ is much higher than the pKₐ of a carboxylic acid due to the aggregation of FAs, and it could attain around 10 for saturated long chain FAs such as stearic acid [20,21]. This pH criterion introduces a limitation wherein achieving an effective interaction with the bacterial cytoplasmic membrane requires surpassing this pKₐ limit to ensure sufficient molecules are adequately solubilized [18]. In addition, the enhancement in LCFA solubility is also linked to the Krafft temperature, with the Krafft temperature increasing with the alkyl chain length of FAs. Below the Krafft temperature, LCFA soaps form crystals and are not efficient as antibacterial agent. Finally, the critical micellar concentration (CMC) decreases also by increasing the alkyl chain length of LCFAs, which results in a decrease of free monomers by increasing the alkyl chain length at a fixed FA concentration [22].

One strategy to enhance the FAs soap solubility is the substitution of commonly used alkali counter-ions by chaotropic organic ones such as choline [17,23–25]. Counter-ions play furthermore a pivotal role in decreasing the Krafft temperature [24,26]. Surprisingly, there is no detailed study in the literature on the use of organic counter-ion to increase the solubility of LCFAs, with the aim of improving the antibacterial effect of LCFAs. In this work the possible links between alkyl chain length, the critical micelle concentration, the aggregation number in the micelles and the antibacterial effect against MRSA were determined. LCFA soaps from lauric acid (C12) to stearic acid (C18) were produced by using choline hydroxide (ChOH) to take advantages of the presence of the chaotropic counter-ion – choline [27]. The conditions under which LCFAs form micelles in the presence of ChOH was determined by combining macroscopic observations and polarized optical microscopy (POM) with small angle neutron scattering (SANS) experiments. Additionally, the CMC was determined by tensiometry experiments for all formulations. The efficiency of synthesized LCFA soaps were furthermore tested against MRSA and *E. coli* strains using minimum bactericidal concentration (MBC) as well as time-kill kinetics assays (TKA) to investigate their time-of-action. Choosing judiciously the medium conditions and the LCFAs concentration, LCFAs from C12 to C18 became all effective against MRSA, a big step further in the development of new antibacterial mimetics.

# 2. Materials and methods

## 2.1. Materials

Lauric acid (C12) (>98 %, Sigma Aldrich, United States), myristic acid (C14) (>99 %, TCI, Japan), palmitic acid (C16) (99 %, Sigma Aldrich, United States) and stearic acid (C18) (98 %, Alfa Aesar, United States). Sodium hydroxide (NaOH) (99 %, Verbiëse, France), choline hydroxide (47 wt% in aqueous solution, TCI, Tokyo) and ultrapure water type III (Sartorius, France) were used throughout all the experiments carried. Reactants were used without any further purification.

## 2.2. Protocol for the dispersion of fatty acids in aqueous solution

FAs soaps were obtained by mixing saturated fatty acids with a 1 M aqueous solution of ChOH or NaOH at different molar ratios, following the methodology outlined by Klein et al. [24]. Initially, precise amounts
of LCFAs powders were weighed, and ultrapure water was added to achieve a concentration of 100 mM. Subsequently, the appropriate volume of a 1 M aqueous solution of ChOH or NaOH solution was added to achieve the desired molar ratio as defined by Eq. (1). Homogenization of the mixtures was achieved through a series of freezing cycles: the mixtures were heated to 70 °C for 30 min., followed by a 30 s vortex mixing, and then immediately cooled down to -20 °C in a freezer for additional 30 min. The dispersed samples were left to settle down at room temperature before use. The molar ratio was defined as:

$$R = \frac{n(\text{counter-ion})}{n(\text{fattyacid})}(1).$$

## 2.3. Polarized optical microscopy

Micrographs were obtained on a scale of 10 to 100 µm utilizing polarized light at room temperature, using a VHX-700 digital microscope (Keyence, with VH-Z100UR zoom lens, France). 20 µL of each fatty acid soap were placed in a slide and observed immediately avoiding dryness.

## 2.4. Small angle neutron scattering

SANS patterns were recorded at D22 beamline at the Institut Laue-Langevin - The European Neutron Source (ILL, Grenoble, France), with a wavelength λ of 0.60 nm (relative full width at half maximum 10 %) and two ³He multi-PSD tube detectors, one at 1.4 m sample-to-detector distance and the second at 17.6 m, covering a total q-range of 0.023 to 6.4 nm⁻¹, where q is the magnitude of the wavevector (q = 4πsin(θ/2)/λ, with θ the scattering angle). For this experiment the samples were prepared in D₂O instead of ultrapure water to increase contrast and reduce background. Solutions were kept in quartz cuvettes of 1 mm pathway (type 110-QS, Hellma GmbH, Müllheim, Germany), placed on a sample-changer thermalized at 20 °C. Data reduction was performed with the program GRASP V.10.17i (https://doi.org/10.1107/S1600576723007379) to correct for flat field, parallax, transmission, scattering by the empty cell, detector background as measured with an absorber (sintered ¹⁰B₄C) at the sample position, using the monitor to normalize data, and with a measure of the direct beam attenuated with a calibrated chopper leading to corrected data in absolute units (cm⁻¹), which were radially averaged and binned according to resolution. Data are available at DOI: 10.5291/ILL-DATA.9-10-1747. Contributions from solvent and incoherent scattering were then subtracted. All spectra were fitted with a bi-axial ellipsoid model by using SasView 5.0.4 (https://www.sasview.org/). The form factor of an ellipsoid object is given by Eq. (2).

$$P(q, \alpha) = \frac{\text{scale}}{V} F^2(q, \alpha) + \text{background} \quad (2)$$

where:

$$F(q, \alpha) = \Delta\rho \cdot V \frac{3(\sin(qr) - q \cdot r \cos(qr))}{(qr)^3}$$

for α the angle between the axis of the ellipsoid (Eq. (3)).

$$\text{And } r = [R_e^2 \sin^2 \alpha + R_p^2 \cos^2 \alpha]^{1/2} \quad (3)$$

R_e and R_p are the equatorial and polar radius of the ellipsoid, respectively. The structure factor, S(q), which quantifies the inter-micellar interactions, was fitted with an “Hayter_MSA” model (Hayter and Penfold model) also displayed in the SASview software. It takes into account the repulsive screened coulombic intermicellar interaction potential. The scattering length density of the solvent and the fatty acids, the volume fraction, the salt concentration, the dielectric constant and the temperature were fixed (see details in Supporting Information). Only the polar radius, equatorial radius and the charge were fitted.

## 2.5. Critical micelle concentration (CMC)

CMC values were obtained by using the automatized surface tension plate reader Kibron Delta-8 (Kibron, Finland). A volume of 50 µL of aqueous dispersion was placed on the 96-hole platform. Measurements were performed in triplicates at 20 ± 1 °C after a waiting time of 10 min to ensure equilibrium at the air-water interface. A calibration was performed by using ultrapure water at 20 °C.

## 2.6. pH measurements

The pH apparent values were determined by using a high precision pH meter (Fisher Scientific, accumet® AE150, France). Measurements were performed at 20 °C. The pH values of each sample were determined by means of averaging measurements in triplicate.

## 2.7. Krafft temperature determination

To determine the Krafft temperatures, direct visual observations were performed. All the samples were cooled down from 70 °C (temperature at which all samples were completely clear and isotropic) to 5 °C. For the samples, for which turbidity appeared at 5 °C, the Krafft temperatures were determined by re-heating them slowly until they turned into clear and isotropic samples.

## 2.8. Study of the antibacterial effect of fatty acid soaps

### 2.8.1. Bacterial strains and reagents

Gram-positive MRSA (ATCC 43300) and E. coli (K-12) were grown on sterile culture solid medium and broths which were previously prepared as advised by the commercial supplier. Tryptic Soy Agar (TSA, Sigma Aldrich, France) and Mueller-Hinton Broth (MHB, Sigma Aldrich, France) were accurately weighted and dissolved in ultrapure water and further sterilized by autoclave.

### 2.8.2. Minimal Inhibitory/Bactericidal concentration

Initially a single colony of MRSA or E. coli was cultured during 3–5 h in 3 mL of Mueller Hinton Broth (MHB) at 35 °C. After incubation, the standardized inoculum was prepared by adjusting the optical density (OD₆₀₀) of the bacterial suspension to 0.1, which corresponds to ≈ 1.5 × 10⁸ CFU (colony forming units) mL⁻¹ using an UV-Vis spectrophotometer (GeneQuant Pro, AMERSHAM Biosciences, France). The inoculum was used in a time range of less than 15 min. to avoid further bacterial growth. MIC assays was carried out by microdilution growth inhibition assay. The experiment followed the protocol outlined by Swebocki and colleagues [28], which is detailed as follows: samples were prepared in flat-bottomed 96-well microplate (Nunc®, Thermo Scientific, France) by two-fold serial dilution, using 50 µL of tested sample diluted in 50 µL of MHB. MRSA or E. coli without investigated substance and sterile MHB were used as positive and negative controls respectively. Finally, 50 µL of the bacterial suspension diluted to 10⁶ CFU/mL were deposited in the wells and incubated at 35 °C overnight. This test was performed for each of the fatty acid soaps, as well as the counter-ions alone in the form of hydroxides – NaOH and ChOH. Due to the high turbidity caused by mixing bacterial dilution and fatty acid soaps, conducting a standard MIC assessment using a plate reader or digital imaging (naked eye) was not feasible, as it could compromise the reliability of the data. Therefore, MBC assessment, where the bacterial concentration in each well an over of incubation was determined by plating serial dilutions onto Tryptone Soy Agar (TSA) Petri dishes. Subsequently, colony counting were performed after an overnight incubation at 35 °C. This approach enabled the evaluation MBC and introducing a parameter that we called the Starting to Kill Concentration (SKC). The SKC represents the concentration at which a notable reduction in the number of bacteria occurs compared to the initial concentration of the inoculum. For NaOH and ChOH aqueous solution (limpid), the standard procedure was followed,
with OD measurements using plate reader (Nivo®, PerkinElmer, France). The obtained data was plotted and modelled using Gompertz model.

### 2.8.3. Time-Kill kinetics assay

Time-kill kinetics assay (TKA) was conducted in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines outlined in M26-A as also described by Swebocki and colleagues [9]. For this test, all FAs soaps were tested in a range of concentrations equal to the SKC, 2 × SKC, 4 × SKC, and 8 × SKC, in a total volume of 10 mL of MHB. Subsequently, 33 µL of bacterial culture, previously adjusted to OD₆₀₀ = 0.01, were introduced into each tube. The tubes were subjected to vortex-shaking and promptly transferred to an incubator set to a temperature of 35 °C.

At designated time intervals (0.5, 1, 2, 4, 8, 24 h), 200 µL aliquots were withdrawn from each tube to subsequently dilute them with sodium chloride solution (9 g/L) and further plated onto TSA plates, followed by incubation of the plates for 24 h. A three-fold reduction in colony count, corresponding to 0.01 % of the initial starting colony count (equivalent to 500 colony-forming units per milliliter, CFU/mL), was considered as the threshold.

## 3. Results and discussion

### 3.1. Effect of fatty acids' alkyl chain length and molar ratio on Krafft temperature and CMC

In this study, four LCFAs were chosen differing in alkyl chain lengths: lauric acid (C12), myristic acid (C14), palmitic acid (C16) and stearic acid (C18) (Fig. 1). These LCFAs were mixed with either choline hydroxide (ChOH) or sodium hydroxide (NaOH), used as control here. Initially, the four LCFA soaps were formulated with ChOH at a molar ratio of 1:1 maintaining a fixed concentration of 100 mM (Figure S1).

LCFA soaps formed using C12 to C16 are all optically clear at room temperature. Trough polarized optical microscopy experiments; no micron-sized self-assemblies were observed (Figure S1). The Krafft temperatures were ≤ 11 °C for C12-C16, while for C18 it was 42 °C. The C18 sample exhibited turbidity, and microscopy and SANS measurements revealed the presence of micron-sized crystals at room temperature (Fig. S1-2). Micelles were only obtained above the Krafft

temperature (Figure S2). The molar ratio was increased to 2:1, which enhanced the solubility of LCFA soaps formed and lowered their Krafft temperatures. Upon increasing to a molar ratio of 2:1, all samples from C12 to C18 appeared optically clear, with no observable crystals at room temperature under polarized optical microscopy (Fig. 2). An excess of ChOH is thus required to lower the Krafft temperature below 5 °C in all cases, also for LCFA soaps such as C18 (Table 1) [24]. The molar ratio of 2:1 was maintained throughout our study to ensure that all the LCFAs from C12 to C18 remained soluble with a Krafft temperature significantly lower than room temperature. Therefore, for the microbiology experiments, all the LCFAs were in the same soluble state.

All formed LCFA soaps exhibited pH values between 12.5 and 13.1, significantly exceeding the pKₐ values of the respective FAs species and indicating that all FA molecules were predominantly in their ionized carboxylate state (Table 1). The CMC of each LCFA soap at 20 °C was determined through surface tension measurements (Figure S3). Notably, the CMC for C12 was approximately 12.7 mM, while for C18, it was notably lower at only 0.5 mM. Decreasing CMC with increasing alkyl chain length, has been reported before in the presence of alternative organic chaotropic counter-ions, such as tetrabutylammonium hydroxide [29]. This decrease in CMC with increasing alkyl chain length reflects an augmentation in the hydrophobicity of the soap molecules, promoting their aggregation at lower concentrations (Figure S4).

### 3.2. Effect of fatty acids' alkyl chain length on the self-assembly of the fatty acid soaps

To elucidate the self-assembled structures, present in all previously described LCFA soaps at a molar ratio of 2:1, SANS was employed (Fig. 3). In all the scattering spectra, the intensity increase observed at low Q, signifies the presence of few remaining crystals within the system. At medium and high Q values, the scattering spectra exhibit characteristic features indicative of small micelles interacting via repulsive forces. Notably, a Porod-like decay proportional to Q⁻⁴ and distinct oscillations around 0.15 Å⁻¹ characteristic of small micelles, were observed. Additionally, a correlation peak at approximately 8.10⁻² Å⁻¹ was identified. This peak arises from electrostatic repulsions between micelles formed by negatively charged LCFA soaps, resulting in a broad correlation peak that corresponds, in real space, to the average distance between spherical micelles. As the alkyl chain length of FA

<figure><img src="image_7.png" /><figcaption>Fig. 1. Chemical structures and nomenclature of the compounds used for the production of LCFA soaps.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Fig. 2. POM micrographs of the FAs soaps at 100 mM with choline hydroxide at a molar ratio 2:1 at room temperature.</figcaption></figure>

**Table 1**

Values of pH, Krafft temperature, CMC and surface tension at the CMC for LCFAs soaps with ChOH at molar ratio 2:1, and for LCFAs soaps with NaOH at a molar ratio 1:1. The estimated errors for CMC are less than 5 %.

<table><thead><tr><th>LCFA Soap</th><th>pH</th><th>Krafft temperature</th><th>CMC (mM)</th><th>σ<sub>CMC</sub> (mN/m)</th></tr></thead><tbody><tr><td>C12 (ChOH)</td><td>13.1 ± 0.05</td><td>&lt;5 °C</td><td>12.7</td><td>46.5</td></tr><tr><td>C14 (ChOH)</td><td>12.8 ± 0.03</td><td>&lt;5 °C</td><td>2.8</td><td>45.1</td></tr><tr><td>C16 (ChOH)</td><td>12.9 ± 0.02</td><td>&lt;5 °C</td><td>0.7</td><td>43.5</td></tr><tr><td>C18 (ChOH)</td><td>12.5 ± 0.02</td><td>~5 °C</td><td>0.5</td><td>42.4</td></tr><tr><td>C12 (NaOH)</td><td>n/a</td><td>26 °C [30]</td><td>24.4 [30]</td><td>n/a</td></tr><tr><td>C14 (NaOH)</td><td>n/a</td><td>46 °C [30]</td><td>6.9 [30]</td><td>n/a</td></tr></tbody></table>

soaps increases, the position of the correlation peak shifts slightly to lower Q values, indicating an increase in the distance between micelles. This qualitative observation can be attributed to an increase in the size and aggregation number of each micelle as the length of the fatty acid alkyl chain increases. To quantitatively confirm these findings, all scattering spectra were fitted using classical models.

Initially, the data were fitted by a classical spherical model, which failed to adequately capture both the large Q oscillation and the Q⁻⁴ slope. Moreover, literature reports suggest that C18 tends to form ellipsoidal micelles rather than spherical ones in the presence of chaotropiс counter-ions [31]. Consequently, the data were fitted using an “elliptical micelles” model to reflect this shape for all LCFA soaps (Table S1). From the fit, both the polar and equatorial radii were determined (Table 2). The ellipticity (e), defined as the ratio of the polar radius to the equatorial radius, exhibited a linear decrease with increasing chain length from 2.21 for C12 to 0.77 for C18 (Fig. 4). For C18, the ellipticity indicates an oblate micellar shape consistent with observations made with ethanolamine as a chaotropiс counter-ion [31]. C12, C14, and C16 led to prolate micelles. Therefore, by increasing the FAs chain length, we observed an increase of the equatorial radius and a concomitant decrease of the polar radius leading to a change in micellar shape from prolate to oblate.

Furthermore, the aggregation number of the micelles, denoted as N, was determined using two distinct methods and subsequently compared. The first method, referred to as Nₚ, involved utilizing the position of the

correlation peak, denoted as Q*. Under the assumption of spatially homogeneous distribution of micelles in aqueous solution, the mean distance (dₘean) between homogeneous micelles can be calculated as depicted in Eq. (4):

$$d_{\text{mean}} = \frac{2\pi}{Q^*} \qquad (4)$$

The second method, termed Nₚvolume was determined by comparing the total volume of the ellipsoid to the volume of a single fatty acid molecule, expressed as in Eq. (5),

$$N_{\text{volume}} = \frac{V_{\text{ellipsoid}}}{V_{\text{fattyacid}}} \qquad (5)$$

To approximate the volume of fatty acid molecule, the Tanford’s formula was employed (Eq. (6)), which considers the alkyl chain volume, V:

$$V = 27.4 + 26.9n_c \qquad (6)$$

where n_c represents the number of alkyl chain carbons. Both methods demonstrated a consistent increase in the aggregation number with respect to the alkyl chain length of the FAs soaps (Fig. 4). Specifically, the aggregation number, Nₚ exhibited an almost linear increase from 52 to 92 as the alkyl chain progressed from C12 to C18, respectively. Similarly, the aggregation number, N_v also displayed a nearly linear rise, ranging from 72 to 170 for C12 to C18, with an approximate two-fold difference between the results obtained from the two methods. However, it’s crucial to acknowledge potential limitations in the quantitative assessment of N_v. Firstly, the empirical formula used to calculate the volume of fatty acids may not precisely reflect their volume within the micelle. Moreover, our fitting process did not explicitly consider the contribution to the scattered signal of the positively charged counter-ion, choline, which is relatively large. Consequently, the measured size likely encompasses contributions from both LCFAs and choline. Therefore, it may be more appropriate to interpret N_v qualitatively rather than quantitatively.

Nevertheless, the combined findings from both methods unequivocally demonstrate that the number of micelle aggregations increases with the increase of the alkyl chain length. Thus, since all the LCFAs
<figure><img src="image_9.png" /><figcaption>**Fig. 3.** SANS scattering spectra at 20 °C for LCFAs/ChOH soaps with a molar ratio of 2:1: C12 (blue), C14 (purple), C16 (green) and C18 (orange). The black line corresponds to the best fit according the model described in the main text. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<table><caption>**Table 2**
Values obtained by fitting the SANS data by using an elliptical sphere model.</caption><thead><tr><th>FA Soap</th><th>Polar radius (Å)</th><th>Equatorial Radius (Å)</th><th>Charge (e-)</th></tr></thead><tbody><tr><td>C12 (ChOH)</td><td>31</td><td>14</td><td>14</td></tr><tr><td>C14 (ChOH)</td><td>31</td><td>18</td><td>15</td></tr><tr><td>C16 (ChOH)</td><td>29</td><td>22</td><td>15</td></tr><tr><td>C18 (ChOH)</td><td>23</td><td>30</td><td>14</td></tr></tbody></table>

soaps were at the same concentration, there is a decrease in the number of micelles by increasing the LCFAs chain length.

### 3.3. Assessment of antibacterial properties

#### 3.3.1. Minimum inhibitory/bactericidal concentration

A series of antibacterial assessments were carried out. The minimum inhibitory concentration (MIC), that is the lowest concentration of an

antimicrobial agent that inhibits the visible growth of a microorganism, and the minimum bactericidal concentration (MBC), the lowest concentration of an antimicrobial agent that kills a specific bacterium, were determined. Unlike the MIC, which only indicates an inhibition of the growth of bacteria, the MBC reflects the concentration required to actually eradicate the population of bacteria and bring it to under the detection limit [32]. As stated in the Materials and Methods section, the high turbidity prevented us from determining the MIC values. Consequently, we established a parameter called the Starting to Kill Concentration (SKC), which indicates the concentration at which a significant reduction in the bacterial population occurs compared to the initial inoculum concentration.

Unlike MRSA, *E. coli* was not significantly inhibited by any of the investigated LCFA soaps. This result confirms that LCFA soaps exhibit a much higher affinity towards Gram-positive strains [16,33]. A clear correlation between the increase in alkyl chain length and the rise in SKC values is observed for MRSA (Fig. 5). Specifically, C12 shows the

<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Evolution of the ellipticity as a function of the fatty acid carbon number inside the alkyl chain for FAs soaps (left) and aggregation number, N, as a function of the fatty acid carbon number inside the alkyl chain for FAs soaps, with N<sub>p</sub> corresponding to the aggregation number determined by the correlation peak position, and with N<sub>v</sub> corresponding to the aggregation number determined by using the volume of the ellipsoid as described in the main text (right).</figcaption></figure>
lowest SKC value, approximately 0.56 mM, followed by C14 with a value of 0.74 mM, C16 with 2.31 mM, and C18 with 10.95 mM. However, the trend is not mirrored in the MBC values. Among all LCFAs soaps, C14 exhibits the lowest MBC value of 3.1 mM, followed by C12 with an MBC of 6.2 mM, C18 with 12.5 mM, and C16 with 25 mM. A decrease in the inhibitory and killing efficiencies (SKC and MBC, respectively) by increasing the alkyl chain length of FAs soaps was also reported in the literature [34,35]. The critical concentration required for inhibition and killing is also related to CMC, which determines the availability of FA monomers that can act on the bacterial membrane (Table 1) [36]. Since the free LCFAs concentration is limited by CMC, bacteria are exposed to higher monomeric concentrations of the shorter LCFAs (C12 and C14) when compared with the longer LCFAs (C16 and C18) having lower CMC values.

Control experiments were performed by using NaOH to produce the soaps. For NaOH LCFAs soaps, turbidity increased with alkyl chain length in the samples due to crystallization observed in C12, and C14 retaining a semi-gelled form, while C16 and C18 were nearly solid. Based on these observations, we chose to measure only NaOH C12 and C14 soaps (Fig. 6). The SKC values were 0.28 and 9.51 mM for C12 and C14, with corresponding MBC values of 0.8 mM and 12.5 mM, respectively. The SKC values for C12 were similar between ChOH and NaOH, whereas the SKC values for C14 were much higher in the presence of NaOH than ChOH. This observation could be explained by the fact that for C12 NaOH, C12 was still in soluble micellar state at the temperature at which the experiments were performed (35 °C) since the Krafft temperature is around 26 °C. This is in contrast to C14 for which a Krafft temperature < 5 °C for ChOH and ≈ 46 °C for NaOH. In order to verify if the saponification of LCFAs had any effect on MIC values, both counterions were tested as well. Both ChOH and NaOH aqueous solution exhibited relatively high MIC values of around 49.55 mM and 46.75 mM, respectively (Figure S5). Thus, both NaOH and ChOH have very low and similar antibacterial properties coming from the hydroxide ions. This result indicates that the effect on LCFAs soaps did not come from an effect of the nature of the counter-ion but rather is linked to the Krafft temperatures and LCFAs soap solubility.

#### 3.3.1.1. *Time-Kill kinetics assay.*
Knowing SKC and MBC values of the FAs soaps, the time-kill kinetics assays were investigated. TKA is used to assess the antimicrobial activity of a substance over a specific period of time and provides information on how quickly and effectively an antimicrobial agent can reduce or kill microorganisms. This assay can provide valuable insights into the speed of action, potency, and potential for resistance development.

All LCFAs ChOH soaps underwent testing within a concentration range spanning from 1 × SKC to 8 × SKC over an antimicrobial action time of 24 h. A value of t99.9% was evaluated, which indicates a time after which a three log units' reduction was observed. As illustrated in Fig. 7 and detailed in Table 3, each investigated soap exhibited distinct kinetic profiles.

C18 demonstrated the swiftest time-to-action across all tested concentrations, followed by the C16 which also displayed efficacy, although it required much longer durations to surpass the t99.9% threshold at all concentrations. C12 and C14 did not completely eradicate the bacteria, at lower concentration ranges (1 × and 2 × SKC for both, and additionally 4 × SKC for C14), instead showing bacteriostatic or inhibitory effects. However, it is noteworthy that C12 effectively eliminated MRSA within 4 h at 4 × and 8 × SKC concentrations, whereas for C14, only 8 × SKC proves capable of achieving eradication.

To explain these results, it is important to compare the CMC and the SKC values used for the TKA measurements. For C18, the SKC value is almost 20 times above the CMC. At 1 × SKC already a high quantity of free monomers is present, which could interact with the bacterial membrane with a huge reservoir of possible monomers inside the micelles which could liberate new free monomers at the bacteria surface if needed. At 1 × SKC, all the bacteria were killed in less than 4 h. At 8 × SKC, the C18 concentration was so high that in less than 30 min all bacteria were killed. For C12, the SKC value is almost 10 times below the CMC, that is to say that the free monomers concentration was very low in comparison to C18 at the same concentration. Even after 24 h, for C12 at 1 × SKC, the free monomers concentration was not enough to kill all the bacteria. It is only from 4 × SKC, that all the bacteria were killed in less than 4 h even if the concentration was still below the corresponding

<figure><img src="image_11.png" /><figcaption>**Fig. 5.** SKC/MBC assays against MRSA for LCFAs ChOH soaps: C12, C14, C16 and C18. MBC values have been circled with short-dotted line. The SKC values are given in mM.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Fig. 6.** SKC/MBC assays against MRSA for NaOH LCFA soaps: C12 (left) and C14 (right). MBC values have been circled with short-dotted line. The SKC values are given in mM.</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Fig. 7.** TKA assays against MRSA showing the evolution of bacterial population with time for LCFAs ChOH soaps: C12, C14, C16 and C18. Red line indicates a three unit log reduction; any value below it means an eradication below 99.9 % (grey zone) (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<table><caption>**Table 3**

T<sub>99.9%</sub> values in hours of LCFAs ChOH soaps: C12, C14, C16 and C18 against MRSA.</caption><thead><tr><th rowspan="2">LCFA Soap</th><th colspan="4">t<sub>99.9%</sub> (hours)</th></tr><tr><th>1 × SKC</th><th>2 × SKC</th><th>4 × SKC</th><th>8 × SKC</th></tr></thead><tbody><tr><td>C12 (ChOH)</td><td>n/a</td><td>n/a</td><td>&lt;4</td><td>≈2</td></tr><tr><td>C14 (ChOH)</td><td>n/a</td><td>n/a</td><td>n/a</td><td>&lt; 24</td></tr><tr><td>C16 (ChOH)</td><td>&lt; 24</td><td>≈8</td><td>&lt;4</td><td>&lt;4</td></tr><tr><td>C18 (ChOH)</td><td>&lt;4</td><td>≈4</td><td>&lt;2</td><td>&lt; 0.5</td></tr></tbody></table>

CMC. The same conclusions could be applied for C14, but in this case it is important to highlight the alkyl chain length effect. Indeed, even if the CMC of C14 is much lower than that of C12, t<sub>99.9%</sub> threshold at 4 and 8 × SKC were reached much faster for C12 than for C14, respectively. This result confirms findings in the literature showing that C12 exhibits better antibacterial properties than C14 due to the effect of the alkyl chain length on the bacteria membrane [34,35,37]. Since FAs disrupt bacterial membranes by insertion into the lipid bilayer, the length, and the rigidity of the alkyl chain of FAs play a key role in the membranes’ disruption. However, the LCFAs multiple mechanisms of antibacterial action remain poorly understood for the moment [15–36]. The most important processes occur within and at the membrane surface. These
mechanisms include inhibition of enzymatic activity, cytoplasmic
membrane disruption, formation of peroxidation or auto-oxidation
products, DNA/RNA replication inhibitors, as well as protein and cell
wall biosynthesis inhibitors [36].

4. Conclusions

In this study, we utilized the chaotropic choline counter-ion to enhance the solubility of long-chain fatty acid soaps, enabling a comprehensive investigation into the interplaying effects of alkyl chain length, Krafft temperatures, and critical micelle concentration on the antibacterial properties against the pathogen *Methicillin-resistant Staphylococcus aureus* (MRSA). Increasing the alkyl chain length of LCFAs resulted in a decrease in CMC, consistent with existing literature [29]. It also revealed a transition in micellar shape from prolate for C12 to oblate for C18 and an increase in aggregation number within the micelles of around two times from C12 to C18 [29]. Our study highlighted the effect of the alkyl chain length of LCFAs on the shape and number of monomers inside the micelles. For the first time, we determined the decrease in the start to kill concentration (SKC) and minimum bacterial concentration (MBC) with increasing chain length of FA soaps without solubility-related ambiguities in contrary to previous reports [16,37,38]. The critical concentration required for inhibition and killing was found to be influenced by CMC, impacting the availability of LCFAs at the bacterial membrane interface. Time-kill assay demonstrated that while all LCFAs exhibit strong antibacterial activity against MRSA, higher concentrations are required for longer LCFAs (C16 and C18) due to their lower CMC. Moreover, achieving an efficient antibacterial effect necessitates concentrations above the respective CMC and reducing Krafft temperatures to suitable levels; something which could only be achieved by using chaotropic counter-ions such as choline. Our findings suggest the potential use of LCFAs:ChOH as novel antibacterial agents against Gram-positive bacteria, although further research is needed to elucidate their mechanism of action, such as molecular dynamic simulations to assess the interaction of LCFA soaps with bacteria cell membrane [36], coupled with electron microscopy for study of any morphological changes of the bacterial cell [9]. These fatty acid soaps could be used for cleaning and disinfection procedures of medical devices.

CRediT authorship contribution statement

**Helena Arellano:** Writing – original draft, Validation, Methodology, Investigation. **Tomasz Swebocki:** Writing – original draft, Validation, Methodology, Investigation, Data curation. **Clémence Le Coeur:** Writing – review & editing, Formal analysis, Data curation. **Sylvain Prevost:** Writing – review & editing, Formal analysis, Data curation. **Marwan Abdallah:** Writing – review & editing, Validation, Methodology, Data curation. **Veronique Nardello-Rataj:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. **Anne-Laure Fameau:** Writing – original draft, Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgments

The doctorate of T. S. was financed by the former French Ministry of Higher Education, Research, and Innovation (MESRI). The authors thank Dr. R. Boukherroub for constructive comments on the manuscript. The Chevreul Institute is thanked for its help in the development of this work through the ARCHI-CM project supported by the MESRI, the region "Hauts-de-France", the ERDF program of the European Union and the "Métropole Européenne de Lille". The graphical abstract was exported under a paid subscription of Biorender and created with BioRender.com.

**Appendix A. Supplementary material**

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcis.2024.07.218.

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