Fatty acid potassium had beneficial bactericidal effects and removed Staphylococcus aureus biofilms while exhibiting reduced cytotoxicity towards mouse fibroblasts and human keratinocytes

Article abstract of DOI:10.3390/ijms20020312

Wounds frequently become infected or contaminated with bacteria. Potassium oleate (C18:1K), a type of fatty acid potassium, caused >4 log colony-forming unit (CFU)/mL reductions in the numbers of Staphylococcus aureus and Escherichia coli within 10 min and a >2 log CFU/mL reduction in the number of Clostridium difficile within 1 min. C18:1K (proportion removed: 90.3%) was significantly more effective at removing Staphylococcus aureus biofilms than the synthetic surfactant detergents sodium lauryl ether sulfate (SLES) (74.8%, p < 0.01) and sodium lauryl sulfate (SLS) (78.0%, p < 0.05). In the WST (water-soluble tetrazolium) assay, mouse fibroblasts (BALB/3T3 clone A31) in C18:1K (relative viability vs. control: 102.8%) demonstrated a significantly higher viability than those in SLES (30.1%) or SLS (18.1%, p < 0.05). In a lactate dehydrogenase (LDH) leakage assay, C18:1K (relative leakage vs. control: 108.9%) was found to be associated with a significantly lower LDH leakage from mouse fibroblasts than SLES or SLS (720.6% and 523.4%, respectively; p < 0.05). Potassium oleate demonstrated bactericidal effects against various species including Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Clostridium difficile; removed significantly greater amounts of Staphylococcus aureus biofilm material than SLES and SLS; and maintained fibroblast viability; therefore, it might be useful for wound cleaning and peri-wound skin.

Full text of DOI:10.3390/ijms20020312

International Journal of
Molecular Sciences
Article
Fatty Acid Potassium Had Beneficial Bactericidal
Effects and Removed Staphylococcus aureus Biofilms
while Exhibiting Reduced Cytotoxicity towards
Mouse Fibroblasts and Human Keratinocytes
Takayoshi Kawahara 1 , Miki Takita , Akihiro Masunaga , Hayato Morita ,
,2
1
1
1
3
4
4
5,
Tadayuki Tsukatani , Kohji Nakazawa , Daisuke Go and Sadanori Akita *
1
Shabondama Soap Co., Ltd., Kitakyushu 808-0195, Japan; kawahara@shabon.com (T.K.);
miki-takita@shabon.com (M.T.); akihiro-masunaga@shabon.com (A.M.); h-morita@shabon.com (H.M.)
Department of Microbiology, University of Occupational and Environmental Health,
2
Kitakyushu 807-0804, Japan
Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Kurume 839-0861, Japan;
3
tukatani@fitc.pref.fukuoka.jp
Department of Life and Environment Engineering, The University of Kitakyushu, Kitakyushu 808-0135,
4
Japan; nakazawa@kitakyu-u.ac.jp (K.N.); y7mab007@eng.kitakyu-u.ac.jp (D.G.)
Department of Plastic Surgery, Wound Repair and Regeneration, Fukuoka University,
5
Fukuoka 814-0180, Japan
*
Correspondence: akitas@hf.rim.or.jp; Tel.: +81-92-866-8250
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 13 December 2018; Accepted: 10 January 2019; Published: 14 January 2019
Abstract: Wounds frequently become infected or contaminated with bacteria. Potassium oleate
C18:1K), a type of fatty acid potassium, caused >4 log colony-forming unit (CFU)/mL reductions
(
in the numbers of Staphylococcus aureus and Escherichia coli within 10 min and a >2 log CFU/mL
reduction in the number of Clostridium difficile within 1 min. C18:1K (proportion removed: 90.3%) was
significantly more effective at removing Staphylococcus aureus biofilms than the synthetic surfactant
detergents sodium lauryl ether sulfate (SLES) (74.8%, p < 0.01) and sodium lauryl sulfate (SLS) (78.0%,
p < 0.05). In the WST (water-soluble tetrazolium) assay, mouse fibroblasts (BALB/3T3 clone A31)
in C18:1K (relative viability vs. control: 102.8%) demonstrated a significantly higher viability than
those in SLES (30.1%) or SLS (18.1%, p < 0.05). In a lactate dehydrogenase (LDH) leakage assay,
C18:1K (relative leakage vs. control: 108.9%) was found to be associated with a significantly lower
LDH leakage from mouse fibroblasts than SLES or SLS (720.6% and 523.4%, respectively; p < 0.05).
Potassium oleate demonstrated bactericidal effects against various species including Staphylococcus
aureus, Escherichia coli, Bacillus cereus, and Clostridium difficile; removed significantly greater amounts
of Staphylococcus aureus biofilm material than SLES and SLS; and maintained fibroblast viability;
therefore, it might be useful for wound cleaning and peri-wound skin.
Keywords: fatty acid potassium; biofilm; less cytotoxicity; bactericide; wound
1
. Introduction
Wounds increase healthcare costs, and a pivotal report has estimated that the US healthcare system
spends an estimated 25 billion dollars annually treating chronic wounds alone [1]. Chronic wounds
persist for an average of one year, frequently recur in up to 70% of subjects, and can lead to functional
impairment and decreased quality of life. In addition, lower extremity wounds are a significant cause
of morbidity [2], and diabetic foot ulcers are becoming more prevalent and more difficult to treat as well
as being associated with high treatment costs [3]. Wounds frequently become infected or contaminated
Int. J. Mol. Sci. 2019, 20, 312; doi:10.3390/ijms20020312
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Int. J. Mol. Sci. 2019, 20, 312
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with bacteria. Staphylococcus aureus is known to form biofilms and to colonize the skin of caregivers as
well as wounds, making it a leading cause of healthcare-associated infections, whereas Clostridium
difficile forms spores that make it more resistant to conventional antimicrobial therapy.
In contrast, surgical site infections (SSIs) are a major complication of surgery and are related to
longer hospital stays and higher hospital costs. Antiseptic solutions are widely used for preoperative
bathing or showering in the belief that they help prevent SSIs from developing.
In eyelid preoperative cleansing, the use of baby shampoo was as good as povidone-iodine or
isopropyl alcohol in a prospective, randomized, and comparative study [4] and skin cleansing with
the topical use of chlorhexidine gluconate (CHG) was able to reduce the risk of central-line-associated
bloodstream infection, SSIs, and the colonization of vancomycin-resistant entecocci (VRE) or
methicillin-resistant Staphylococcus aureus (MRSA) in a systematic review [5]; however, the use, safety,
and effectiveness of natural soap has not been reported in the literature.
In a previous study, the effects of 4% CHG, an antiseptic alone; CHG and bathing; or other
hygiene products such as soap on SSIs were examined; however, CHG did not demonstrate superior
efficacy [
6]. In a surgical intensive care unit, daily skin cleansing with a bar of soap was found to
exhibit comparable efficacy to CHG against bloodstream infections via central lines [
7
], which might
be attributed to the fact that MRSA isolates display a reduced susceptibility to CHG [8].
A Markov model in chronic venous leg ulcers (VLUs) was constructed to evaluate the cost and
clinical benefits (healing and decreased infection rates) of the two treatment modalities over a 1-year
period using data from four randomized, controlled clinical studies (RCTs) included in a late Cochrane
review and the cost data from a recently published economic evaluation of VLUs. Costs were calculated
using 2014 United States dollars; wound outcomes included complete healing in 212 patients reported
in the Cochrane meta-analysis and quality-adjusted life years (QALYs) with utility values obtained
from 200 patients with VLUs calculated using standard gamble. Treatment with CI, Cadexomer Iodine,
over one year was $7259 compared to $7901 for SC. This resulted in a cost saving of $643/patient
in favor of CI when compared with SC, Standard Care. More patients treated with CI (61%) had
their wounds healed when compared to the 54% treated with SC. Furthermore, patients treated with
CI + SC experienced six additional ulcer-free weeks compared to patients treated with SC alone (i.e.,
2
5 ulcer- free weeks compared to 19 ulcer-free weeks, respectively). Overall, CI resulted in 0.03 more
QALYs (i.e., 0.86 QALYs compared to 0.83 for SC). The use of CI in addition to SC compared to SC
alone over 52 weeks resulted in more wounds being healed and more QALYs along with a decrease of
overall costs Therefore, the results of this study suggest that CI is cost effective when compared to SC
alone in the management of patients with chronic VLUs [9].
Disinfection and the effective removal of pathogenic bacteria such as Staphylococcus aureus,
its resistant form MRSA, and biofilm-forming MRSA, are important to prevent infections and provide
wound care. We sought to investigate whether natural soap, which is devoid of any preservatives,
additives, or synthetic materials, or its core ingredients such as fatty acid potassium could be used for
such purposes. Thus, the cytotoxicity, bactericidal activity, and ability to remove MRSA of various
types of fatty acid potassium were investigated.
2
. Results
2
.1. Structures of Fatty Acid Potassium and Synthetic Surfactant Detergents
Various different types of fatty acid potassium were examined in this study, and the synthetic
surfactant detergents sodium lauryl ether sulfate (SLES) and sodium lauryl sulfate (SLS) were used as
controls (Figure 1).

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Figure 1. Structures of the fatty acid potassium molecules and synthetic surfactant detergents.
(
(
(
a
d
g
) Potassium caprylate (C8K); (
) potassium myristate (C14K); (
) potassium oleate (C18:1K); ( ) potassium linoleate (C18:2K); (
b
e
) potassium caprate (C10K); (
) potassium palmitate (C16K); (
) SLES; and (j
c
f
) potassium laurate (C12K);
) potassium stearate (C18K);
) SLS. Carbon: gray,
h
i
hydrogen: white, oxygen: red, sulfur: yellow, potassium ions: purple, sodium ions: green
2
.2. Cytotoxic Assays
To assess cytotoxicity, lactate dehydrogenase (LDH) leakage was measured and cell viability was
determined using the WST (water-soluble tetrazolium) assay.
2
.2.1. LDH Leakage
After 5 min of treatment, the BALB/3T3 cells treated with the test solutions demonstrated
significantly less LDH leakage than the SLES- or SLS-treated BALB/3T3 cells (percentage relative
to the untreated control: potassium caprylate [C8K]: 104.1 3.3%; potassium caprate [C10K]:
00.2 3.3%; potassium laurate [C12K]: 103.9 3.5%; potassium myristate [C14K]: 104.9 3.6%;
potassium palmitate [C16K]: 105.8 3.2%; potassium stearate [C18K]: 111.0 3.3%; potassium oleate
C18:1K]: 108.9 10.2%; potassium linoleate [C18:2K]: 107.0 3.2%; SLES: 720.6 45.8%; and SLS:
23.4 ± 61.4%; p < 0.05 for all test substances vs. SLES and SLS) (Figure 2).
±
1
±
±
±
±
±
[
5
±
±
±
Figure 2. LDH leakage of BALB/3T3 cells treated with 0.5 mM fatty acid potassium, SLES, or SLS for
min. Untreated cells were used as the control. The percentage LDH leakage was calculated relative to
the control value. Results are expressed as mean standard deviation (SD) values (n = 8). ** p < 0.01;
# p < 0.01 compared to the control.
5
±
#

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After 5 min of treatment, NHEK-Ad cells treated with the test solutions demonstrated significantly
less LDH leakage than the SLES-treated NHEK-Ad cells (percentage relative to the untreated control:
potassium caprylate [C8K]: 49.2
C12K]: 75.3 8.4%; potassium oleate [C18:1K]: 116.6
8.5 8.6%; SLES: 442.3 15.5%; and SLS: 96.5 9.5%; p < 0.01 for all test substances vs. SLES)
Figure 3).
±
7.4%; potassium caprate [C10K]: 44.5
±
9.9%; potassium laurate
[
5
(
±
±
23.4%; potassium linoleate [C18:2K]:
±
±
±
Figure 3. LDH leakage of NHEK-Ad cells treated with 0.5 mM fatty acid potassium, SLES, or SLS for
min. Untreated cells were used as the control. The percentage LDH leakage was calculated relative to
the control value. Results are expressed as mean standard deviation (SD) values (n = 8). ** p < 0.01;
# p < 0.01 compared to the control.
5
±
#
2
.2.2. Cell Viability
BALB/3T3 cells were cultured for 24 h, then treated with one of the test substances for 5 min.
The WST assay indicated that the cells treated with fatty acid potassium exhibited significantly
greater viability than those treated with SLES or SLS (percentage viability relative to untreated
control: untreated control: potassium caprylate [C8K]: 114.4
09.9 12.7%; potassium laurate [C12K]: 111.5 10.7%; potassium myristate [C14K]: 111.5
potassium palmitate [C16K]: 104.8 15.4%; potassium stearate [C18K]: 102.0 10.7%; potassium oleate
C18:1K]: 102.8 13.4%; potassium linoleate [C18:2K]: 108.0 10.6%; SLES: 30.1 4.8%; and SLS:
8.1 ± 0.6%; p < 0.05 for all test substances vs. SLES and SLS) (Figure 4).
NHEK-Ad cells were cultured for 48 h, then treated with one of the test substances for 5 min.
±
8.7%; potassium caprate [C10K]:
1
±
±
±
12.0%;
±
±
[
1
±
±
±
The WST assay indicated that the cells treated with fatty acid potassium exhibited significantly
greater viability than those treated with SLES or SLS (percentage viability relative to untreated
control: potassium caprylate [C8K]: 92.8
laurate [C12K]: 95.9 7.1%; potassium oleate [C18:1K]: 110.7
2.9 11.3%; SLES: 31.5 1.3%; and SLS: 56.0 6.4%; p < 0.05 for all test substances vs. SLES )
Figure 5).
±
13.7%; potassium caprate [C10K]: 60.9
±
9.9%; potassium
±
±
8.1%; potassium linoleate [C18:2K]:
9
(
±
±
±

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Figure 4. Viability of BALB/3T3 cells that were cultured in Dulbecco’s modified Eagle’s medium
DMEM) for 24 h after they had been treated with 0.5 mM fatty acid potassium, SLES, or SLS for
min. Untreated cells were used as the control. Percentage cell viability was calculated relative to the
(
5
control value. Results are expressed as mean
compared to the control.
±
SD values (n = 8). ** p < 0.01, # p < 0.05 and ## p < 0.01
Figure 5. Viability of NHEK-Ad cells treated with 0.5 mM fatty acid potassium, SLES, or SLS for
min. Untreated cells were used as the control. Percentage cell viability was calculated relative to the
5
control value. Results are expressed as mean
compared to the control.
±
SD values (n = 6). ** p < 0.01, # p < 0.05 and ## p < 0.01
2
.3. Anti-Bacterial Test
The bactericidal effects of fatty acid potassium (potassium oleate [C18:1K]) and an alcohol-based
disinfectant on various bacterial suspensions were compared over 10 min. Regarding Staphylococcus
aureus, potassium oleate exhibited significantly weaker bactericidal activity than the alcohol-based
disinfectant at 1 min (reduction in bacterial number: 1.7
CFU)/mL, p < 0.05), but both treatments displayed similar levels of bactericidal activity from 5 to
0 min.
Both treatments caused consistent reductions (of
cereus from 1 to 10 min.
Potassium oleate demonstrated consistently greater bactericidal activity against Clostridium difficile
than the alcohol-based disinfectant from 1 to 10 min (bacterial number reductions: 1 min: >2.4 0.5 vs.
.4 0.2, p < 0.05; 5 min: >2.4 0.5 vs. 0.5 0.3, p < 0.05; 10 min: >2.4 0.5 vs. 0.5 0.2, p < 0.01;
potassium oleate vs. the alcohol-based disinfectant, respectively) (Table 1).
±
0.9 vs. >4.5
±
0.3 log colony-forming units
(
1
≤
0.3 log CFU/mL) in the numbers of Bacillus
±
0
±
±
±
±
±

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Table 1. Bactericidal activity of C18:1K and an alcohol-based disinfectant.
C18:1K (log CFU/mL) Alcohol-Based Disinfectant (log CFU/mL)
1
min
5 min
10 min
1 min
5 min
10 min
Escherichia coli
Staphylococcus aureus
Bacillus cereus
3.3 (0.4)
1.7 (0.9) *
0.2 (0.2)
>4.3 (0.2)
>4.4 (0.1)
0.2 (0.3)
>4.3 (0.2)
>4.4 (0.2)
0.3 (0.3)
>4.3 (0.2)
>4.5 (0.3)
0.1 (0.2)
0.4 (0.2)
>4.3 (0.2)
>4.4 (0.3)
0.2 (0.3)
0.5 (0.3)
>4.3 (0.2)
>4.4 (0.2)
0.2 (0.4)
0.5 (0.2)
Clostridium difficile
>2.4 (0.5) *
>2.4 (0.5) *
>2.4 (0.5) **
Anti-bacterial testing was performed by mixing 0.2 mL of each bacterial suspension with 20 mL of the relevant test
substance. Results are expressed as mean ± SD values (n = 3). * p < 0.05, ** p < 0.01.
2
.4. Effects on Biofilm-Forming MRSA
2
.4.1. Crystal Violet Assay
By examining the amount of 0.1% crystal violet left after biofilms were placed in contact with one
of five different fatty acids (potassium caprylate [C8K], potassium caprate [C10K], potassium laurate
C12K], potassium oleate [C18:1K], and potassium linoleate [C18:2K]), SLES, or SLS for 1 min,
it was demonstrated that C18:1K (percentage biofilm clearance relative to control: 90.3 2.9%)
was significantly more effective against biofilm-forming MRSA than SLES (74.8 9.7%, p < 0.01) and
SLS (78.0 7.7%, p < 0.05). In addition, potassium caprylate (C8K) demonstrated a significantly greater
ability to remove the MRSA biofilm than SLES and SLS (89.3 3.3%, p < 0.01 and 0.05, respectively)
[
±
±
±
±
(Figure 6).
Figure 6. Percentage MRSA OJ-1 biofilm removal values of various types of fatty acid potassium, SLES,
and SLS. The formed biofilm was placed in contact with the test substance for 1 min. The biofilm
remaining in the well was stained with 0.1% crystal violet and quantified based on the absorbance at
5
70 nm using a microplate reader. The results are expressed as mean
±
SD values (n = 6). * p < 0.05,
*
* p < 0.01.
2
.4.2. Ultra-Microstructural Appearance after Treatment with Potassium Oleate (C18:1K)
Scanning electron microscopy demonstrated that the MRSA biofilm that formed after 24 h
incubation had almost completely disappeared after 1 min of treatment with potassium oleate
Figure 7).
(

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Figure 7. Scanning electron microscopy. Scanning electron microscopy micrographs of the untreated
(
a
) and C18:1K-treated (
b
) biofilms that formed on the test pieces (2000
×
). MRSA biofilms formed on
◦
the test pieces after they had been incubated for 24 h at 37 C. The MRSA biofilms that formed on the
test pieces were removed by C18:1K. (a) An untreated biofilm that formed on a test piece; (b) A biofilm
that had been treated with C18:1K for 1 min.
3
. Discussion
As the use of antiseptics or antibiotics can lead to secondary resistance, new methods are required
to prevent and effectively treat wound infections. A biocompatible, non-ionic, completely water-soluble
surfactant polymer dressing was found to improve wound healing via the direct effects on
Gram-negative bacteria such as Pseudomonas aeruginosa. Specifically, it was shown to affect the
lipid-based outer membranes of Pseudomonas aeruginosa and Staphylococcus aureus by disrupting
their biofilm matrices and membrane glycoproteins, which decreased their metabolic activity [10].
Confocal laser-scanning microscopy confirmed that the same surfactant-based wound dressing
caused the effective detachment and dispersion of bacterial biofilms including those produced by
Staphylococcus aureus and MRSA in 48-h in vitro in the Center for Disease Control and Prevention
biofilm reactor, filter biofilm, and chamber slide biofilm models, even in the absence of 1% silver
sulfadiazine, an antimicrobial agent [11]. In our study, Escherichia coli, Staphylococcus aureus,
and Bacillus cereus were incubated for 48 h before being treated with potassium oleate or the control
(an alcohol-based detergent) for 10 min. Both of these treatments led to similar reductions in the
numbers of these bacteria. The numbers of both Escherichia coli and Staphylococcus aureus were reduced
by 4-fold, but Bacillus cereus was resistant to both substances. Bacillus cereus can produce protective
endospores, and thus, is even resistant to novel antimicrobial agents such as platelet (PLT)-poor plasma,
PLT-rich plasma, PLT gel, and solvent/detergent-treated PLT lysate biomaterials [12].
In the current study, potassium oleate caused a 2.4-fold reduction in the number of Clostridium
difficile CFU within 1 min, whereas the alcohol-based disinfectant caused a 0.4-fold reduction
in the number of Clostridium difficile CFU during this period. In children with severe burns,
Clostridium difficile-associated diarrhea resulted in a 5-fold greater in-hospital mortality rate,
longer hospital stays relative to the percentage total burnt body surface area, and prolonged acidosis
due to diarrhea [13]. Thus, the use of potassium oleate rather than alcohol-based disinfectants is
recommended in cases of Clostridium difficile-related infections.
In a study involving the in vitro decellularization of mouse skin, a detergent-free method was
compared with non-ionic and anionic detergent methods, and sulfated glycosaminoglycan content
was only significantly reduced (p < 0.05) in the ionic detergent treatment group. In contrast to the
detergent-free method, all of the detergent-based methods caused significant reductions in mechanical
scaffold strength and elastin content (p < 0.05) [14]. In the latter experiment, although fibroblasts
were removed by both the detergent-free method and the non-ionic, synthetic, and anionic detergents,
the detergents caused reductions in scaffold strength and the amount of extracellular matrix tissue,
which is harmful and can adversely affect the condition of the remnant tissue. In our experiment,

Int. J. Mol. Sci. 2019, 20, 312
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fatty acid potassium, which is found in anionic detergents, was demonstrated to be significantly less
cytotoxic to mouse fibroblasts and human keratinocytes than SLES after treatment for 24 and 48 h.
In this experiment, the viability of the mouse fibroblasts and human primary keratinocytes were
assessed using the WST assay and LDH leakage assay, which were shown to be useful for assessing
the cytotoxicity of substances towards human glioma stem cells (U87) and endothelial cells (HUVEC)
in a microfluidic system [15]. In mouse fibroblasts, there were significantly less cytotoxic and more
viability in all fatty acids than SLS, whereas in human primary keratinocytes, there was significantly
greater cell viability among the selective fatty acids such as C8K, C12K, C18:1K, and C18:2K than
SLS, but no LDH leakage. This may be due to the interaction between SLS and human keratinocytes
(NCTC2455 keratinocyte cell line) in the photoallergy test [16].
In a case-control study of trauma in the US military, the occurrence of a polymicrobial infection
was reported to be a significant risk factor for persistent infections. Therefore, biofilm production
by multiple clinically important bacterial strains is significantly associated with persistent wound
infections [17]. A type of MRSA (ATCC, BAA-2856; formerly named OJ-1) that we isolated from a
clinical sample exhibited increased resistance to vancomycin in a mouse dermal chip model [18,19].
In addition, it was highly capable of producing highly virulent biofilms, and was able to survive
in mouse Kupffer cells [20]. In the biofilm removal rate experiment conducted in the current study,
which involved the above-mentioned biofilm-forming MRSA, C18:1K was significantly more effective
at biofilm removal than SLES (by 15%, p < 0.01) and SLS (by 12%, p < 0.05). Another fatty acid
potassium, C8K, was also significantly more effective at biofilm removal than SLES and SLS. Thus,
other types of fatty acid potassium apart from potassium oleate are more effective at removing
biofilms than SLES and SLS. In a previous study, SLS demonstrated activity against Staphylococcus
epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa, as did oleic acid and benzalkonium
chloride, in musculocutaneous wounds [21]. In contrast, SLES is an ingredient in cosmetics and
has been shown to have cytotoxic effects [22], even though a shower gel containing SLES was
demonstrated to transiently dampen inflammation and reduce bacterial growth in suction-blistered
wounds [23]. Below the critical micelle concentration (0.1 mg/mL), SLS has minimal influence on
the enzymatic activity of collagenase [24] and is also known to penetrate skin and cause cutaneous
irritation. Cumulative SLS treatment significantly increased the concentration of this compound in the
underlying epidermis, at least in vivo [25]. In contrast to these synthetic anionic detergents, the toxicity
of fatty acid potassium and sodium oleate can change in systems that mimic natural rivers; i.e., it is
higher upstream, but lower downstream, due to cation-dependent detoxification (forming metallic
soaps) caused by the precipitation of sodium oleate in hard water. The toxic levels of oleate and
palmitate salts were found to be 10-fold lower than those of laurate and myristate salts. When river
water and local tap water were used for culturing, the toxic levels of all fatty acid salts were markedly
higher (by 30- to 100-fold); i.e., the substances were less toxic than when ultra-pure water was used [26].
Therefore, potassium oleate, which is a type of fatty acid potassium, could be a useful wound-cleansing
agent as it exhibits bactericidal activity, is effective at removing MRSA, is less toxic against host cells,
and displays a beneficial eco-toxicity profile.
4
. Materials and Methods
4
.1. Reagents
Lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and glutaraldehyde
were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Caprylic acid,
capric acid, linoleic acid, SLS, sodium chloride (NaCl), disodium hydrogen phosphate
1
2-water (Na HPO4 12H O), potassium dihydrogen phosphate (KH PO ), sodium chloride (KCl),
•
2
2
2
4
potassium hydroxide (KOH), hydrochloric acid (HCl), manganese (II) chloride tetrahydrate
(
(
MnCl2
FeSO4
•
4H O), magnesium sulfate heptahydrate (MgSO 7H O), iron (II) sulfate heptahydrate
•
2
4 2
•
7H O), calcium chloride dihydrate (CaCl2 2H O), agar, crystal violet, Hank’s balanced
•
2
2

Int. J. Mol. Sci. 2019, 20, 312
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salt solution (HBSS)(+) and 99.5% ethanol (EtOH) were purchased from FUJIFILM Wako Pure
Chemical Corporation (Osaka, Japan). SLES was purchased from the NOF Corporation (Tokyo,
Japan). The Dulbecco’s phosphate-buffered saline (D-PBS)(+) preparation reagent (with Ca and Mg)
(
100
×
) and fetal bovine serum (FBS) were purchased from Nacalai Tesque (Kyoto, Japan) and Thermo
®
Fisher Scientific K.K. (Tokyo, Japan), respectively. WELPAS antiseptic solution for hands (0.2%),
an alcohol-based disinfectant, was purchased from Maruishi Pharmaceutical Co. Ltd. (Osaka, Japan).
4
.2. Preparation of Surfactant Solutions for the Cytotoxicity Assay
A total of eight different fatty acids, potassium caprylate (C8K), potassium caprate (C10K),
potassium laurate (C12K), potassium myristate (C14K), potassium palmitate (C16K), potassium stearate
C18K), potassium oleate (C18:1K), and potassium linoleate (C18:2K), and the synthetic surfactants
(
SLES and SLS were used in the cytotoxicity assays. D-PBS(+) or HBSS(+) buffer, containing Ca and
Mg, was used to produce the fatty acid solutions. All fatty acid salts (C8K, C10K, C12K, C14K, C16K,
C18K, C18:1K, and C18:2K; all 0.5 mM) were prepared by mixing the relevant fatty acid (C8, C10, C12,
C14, C16, C18, C18:1, and C18:2, respectively) with KOH that had been solubilized with D-PBS(+) or
◦
HBSS(+) at 80 C. Then, the pH of each fatty acid salt was adjusted to 10.4 by adding KOH. SLES and
SLS (0.5 mM) were prepared by diluting them with D-PBS(+) or HBSS(+). Both of these solutions had
final pH values of 7.7.
4
.3. Preparation of Surfactant Solutions for the Antibacterial Test
C18:1K and an alcohol-based disinfectant were used in the antibacterial test. Oleic acid (315 mmol)
and purified water were stirred while being heated. An equimolar amount of KOH was added and
◦
heated at 80 C for one hour to convert the oleic acid to potassium oleate (C18:1K). Finally, the pH
of the aqueous 315 mM C18:1K solution was adjusted to 10.4 by adding KOH. The alcohol-based
disinfectant was used as is.
4
.4. Preparation of Surfactant Solutions for the Biofilm Removal Test
A total of five different fatty acids, C8K, C10K, C12K, C18:1K, and C18:2K, and the synthetic
surfactants SLES and SLS were used in the biofilm removal test. All fatty acid salts (C8K, C10K, C12K,
C18:1K, and C18:2K; all 31.2 mM) were prepared by mixing the relevant fatty acid (C8, C10, C12,
◦
C18:1, and C18:2, respectively) with KOH that had been solubilized with purified water at 80 C. Then,
the pH of each fatty acid salt was adjusted to 10.4 by adding KOH. SLES and SLS (31.2 mM) were
prepared by diluting them with purified water. The final pH values of the SLES and SLS solutions
were 4.2 and 7.0, respectively.
4
.5. Cell Culture
A permanent mouse fibroblast cell line, BALB/3T3 clone A31 cells (JCRB Cell Bank, Tokyo,
Japan, JCRB9005). and a primary human keratinocyte cell line, NHEK-Ad cells (Lonza Japan Ltd.,
Tokyo, Japan) were cultured in DMEM (Thermo Fisher Scientific) supplemented with FBS (10%),
1
00 U/mL penicillin (Meiji Seika Pharma Co. Tokyo, Japan), and 100
µ
g/mL streptomycin (Meiji Seika
4
Pharma) [27
,
28] and in KBM-Gold medium (Lonza Japan), respectively. In total, 1
×
10 cells were
added to each well of a 96-well Nunc MicroWell microplate (Thermo Fisher Scientific) before being
cultured for 24 h for BALB/3T3 and 48 h for NHEK-Ad cells in a humidified atmosphere containing
◦
5
% CO at 37 C [29].
2
4
.6. Cytotoxicity Assay
LDH leakage and cell viability were evaluated as indices of cytotoxicity [15]. BALB/3T3 cells
were cultured for 24 h and treated with 100
µL/well of 0.5 mM C8K, C10K, C12K, C14K, C16K,
18K, 18:1K, C18:2K, SLES, or SLS for 5 min at room temperature. D-PBS(+)-treated cells were used

Int. J. Mol. Sci. 2019, 20, 312
10 of 14
as the control. Then, the solution was collected from each well and used for the LDH leakage
assay. BALB/3T3 cells were washed five times with the culture medium and then cultured for 24 h.
After being cultured, BALB/3T3 cells were washed five times with the culture medium, and then
their viabilities were evaluated (Figure 8). NHEK-Ad cells were cultured for 48 h and treated with
1
00 µL/well of 0.5 mM C8K, C10K, C12K, 18:1K, C18:2K, SLES, or SLS for 5 min at room temperature.
HBSS(+)-treated cells were used as the control and either the LDH assay or washed 3-times following
the viability assay (Figure 9). LDH leakages and cell viabilities were assessed in each well using the
cytotoxicity LDH assay kit-WST (Dojindo Laboratories, Kumamoto, Japan) and cell counting kit-8
(Dojindo Laboratories), respectively.
Figure 8. Cytotoxicity test procedure for the BALB/3T3 cells. BALB/3T3 cells that had been cultured
in DMEM for 24 h were treated with 100 mL/well of each sample for 5 min. The solution was collected
from each well and used for the LDH leakage assay. After the cells had been cultured for 24 h,
their viability was assessed.
Figure 9. Cytotoxicity test for the NHEK-Ad cells. NHEK-Ad cells that had been cultured in KBM-Gold
medium for 48 h were treated with 100 mL/well of each sample for 5 min. The solution was collected
from each well and used for the LDH leakage assay. After that, their viability was assessed.
4
.7. Preparation of the Bacterial Suspensions
Escherichia coli NBRC3972, Staphylococcus aureus subsp. aureus NBRC12732, and Bacillus cereus
NBRC15305 were purchased from the National Institute of Technology and Evaluation (Tokyo, Japan),
and Clostridium difficile ATCC9689 was purchased from Kanto Chemical Co. Inc. (Tokyo, Japan).
Escherichia coli and Staphylococcus aureus subsp. aureus were cultured on soybean casein digest
◦
agar (SCDA; Nissui Seiyaku Co., Tokyo, Japan) at 30–35 C for 18–24 h. Several colonies were then
added to 10 mL of Trypticase soy broth (TSB; bioMerieux Japan Ltd., Tokyo, Japan) and incubated with
◦
7
8
shaking at 35 C for 18–24 h. Each bacterial suspension was diluted to 10 –10 CFU/mL by adding
purified water.
The culture media for Bacillus cereus and Clostridium difficile were prepared by adding 784 mL
of ion exchanged water to 32 g of Trypticase soy agar (TSA; bioMerieux Japan Ltd.), which was
◦
sterilized by autoclaving and then maintained at around 50 C. Next, an 8 mL solution of 0.306 g
MnCl2
•
4H O, 2.5 g MgSO4
•
7H O, and 0.003 g FeSO4 7H O dissolved in 100 mL of 0.01 mol/L
•
2
2
2
HCl and an 8-mL solution of 1.5 g CaCl
and mixed. Bacillus cereus and Clostridium difficile were inoculated into the culture medium and
cultured at 30–35 C until sufficient spore formation could be confirmed. After the culturing procedure,
•
2H O dissolved in 100 mL of 0.01 mol/L HCl were added
2
2
◦
sterile purified water was added, and a suspension was created with a sterile bacterial spreader.

Int. J. Mol. Sci. 2019, 20, 312
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◦
The suspension liquid was collected and heat-treated for 15 min at 75 C, before being immediately
7
8
cooled and then diluted with purified water to about 10 –10 CFU/mL.
4
.8. Anti-Bacterial Test
The anti-bacterial test was performed by thoroughly mixing 0.2 mL of the bacterial suspension
with 20 mL of C18:1K or the alcohol-based disinfectant at room temperature [30]. Samples were
taken from the reaction mixture after 1, 5, and 10 min and were neutralized by diluting them 100
times with soya casein digest lecithin polysorbate (SCDLP) broth (Eiken Chemical Co. Ltd., Tokyo,
Japan). Ten-fold dilutions of neutralized samples of Escherichia coli, Staphylococcus aureus, and Bacillus
◦
cereus were cultured on SCDA at 30–35 C for 40–48 h. Ten-fold dilutions of neutralized samples
◦
of Clostridium difficile were cultured on Columbia agar plates at 30–35 C for 48–72 h. The number
of colonies was counted and expressed as CFU/mL. Bactericidal activity was expressed as the log
CFU reduction in the viable count compared with the log CFU viable count seen after the addition of
purified water.
4
.9. Biofilm Formation
The MRSA (OJ-1, ATCC No. BAA-2856™) was provided by Shiro Jimi (Central Laboratory for
Pathology and Morphology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan) and was
◦
cultured on a TSA slant for 24 h at 37 C. Several colonies were then diluted with TSB to an optical
density at 600 nm of 0.1, and 1 mL of the prepared suspension was then added to each well of a 12-well
◦
cell culture plate (Corning Inc., Corning, NY, USA). After the plates had been cultured for 24 h at 37 C
to encourage biofilm formation [31], the resultant biofilms were washed with saline to remove any
planktonic cells.
4
.10. Biofilm Removal Test
To measure the biofilm removal rate, 1 mL of the relevant sample was brought into contact with
the biofilm for 1 min at room temperature [32]. To remove any planktonic cells, the biofilm was
washed three times with saline. Subsequently, the biofilm remaining in the well was stained with
0.1% crystal violet for one hour [33] and then washed with saline twice. After being decolorized
with ethanol, the amount of biofilm remaining was quantified based on the absorbance at 570 nm,
which was evaluated with a Synergy H4 hybrid microplate reader (BioTek Instruments Inc., Winooski,
VT, USA). The biofilm removal rate was determined using the following equation:
(
Ac − A ) − (As − A )
b
b
Biofilm removal rate (%) =
× 100
(
Ac − A )
b
where Ac, A , and As are the absorbance of the control, blank, and sample, respectively
b
4
.11. Biofilm Structure According to Scanning Electron Microscopy (SEM)
The test pieces used measured 8 mm 8 mm and were obtained from a Petri dish for cells
Corning Inc.). The sterilized test pieces were placed in each well of a 12-well cell culture plate.
×
(
The prepared bacterial suspension (1.5 mL) was added to each well and then incubated for 24 h at
◦
3
7 C to allow biofilms to form on the test pieces. Subsequently, the biofilm removal test was performed
using these test pieces according to the above-mentioned procedure. The biofilm was rinsed with
saline to remove any loosely adherent cells, and then fixed in 2.0% glutaraldehyde solution for 2 h
◦
at 4 C. Subsequently, the biofilms were dehydrated through a graded series of ethanol solutions to
9
9.5%, before being air-dried overnight and coated with platinum/palladium ions for the SEM analysis.
The SEM was carried out with a Miniscope TM-1000 electron microscope (Hitachi, Tokyo, Japan).

Int. J. Mol. Sci. 2019, 20, 312
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4
.12. Statistical Analysis
Data were presented as mean
±
SD values. Statistical analyses were performed using
repeated-measures analysis of variance for the LDH leakage and WST assays, and the Student’s
t-test for the antibacterial and biofilm removal tests. p-Values of <0.05 were considered statistically
significant in each analysis.
5
. Conclusions
Potassium oleate, one of the types of fatty acid potassium found in natural soap, can kill various
bacteria including MRSA, and can also effectively remove biofilm-forming MRSA. As for its effects on
normal host cells, it caused significantly less LDH leakage and significantly lower reductions in cell
viability when compared with SLES and SLS, which are synthetic detergents.
Thus, potassium oleate might contribute to the provision of cost-effective wound care.
Author Contributions: T.K., H.M., T.T., K.N., and S.A. conceived and designed the experiments; M.T., A.M., T.T.,
and D.G. performed the experiments; T.K., M.T., T.T., and K.N. analyzed the data; and T.K., M.T., A.M., and S.A.
prepared the draft manuscript. All authors reviewed and approved the paper.
Funding: This research received no external funding.
Acknowledgments: The authors are grateful Shiro Jimi and Hiroyuki Ohjimi for providing OJ-1 cells.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
MRSA Methicillin-resistant Staphylococcus aureus
SLES
SLS
SSI
Sodium lauryl ether sulfate
Sodium lauryl sulfate
Surgical site infection
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).