Analysis of hand environment factors contributing to the hand surface infection barrier imparted by lactic acid

Abstract Background Organic acids on the surface of human hands contribute to the barrier against transient pathogens. This is the first study to explore the synergistic contribution of lactic acid and other hand environment‐related features on the antibacterial properties of the hand surface. Materials and Methods We estimated the contribution of fingerprint depth, skin pH, stratum corneum water content, skin temperature, and sweat rate of the hands to the infection barrier using an observational survey of 105 subjects. The relationship between each factor and the antibacterial activity of the hands was analyzed using Pearson's correlation coefficient. We performed molecular dynamics simulations to study the interaction between lactic acid and bacterial membranes. Results The amount of lactic acid on the hands and skin temperature contributed positively to the antimicrobial activity (r = 0.437 and P = 3.18 × 10−6, r = 0.500 and P = 5.66 × 10−8, respectively), while the skin pH contributed negatively (r = −0.471, P = 3.99 × 10−7). The predicted value of the combined antimicrobial effect of these parameters was [antimicrobial activity] = 0.21 × [lactic acid] − 0.25 × [skin pH] + 0.26 × [skin temperature] + 0.98. The coefficient of determination (R2) was 0.50. Conclusion The increase in the amount of non‐ionic lactic acid due to lower pH and improvement in the fluidity of the cell membrane due to higher temperatures enable the efficient transport of lactic acid into cells and subsequent antimicrobial activity. The proposed mechanism could help to develop an effective hand infection barrier technology.


| INTRODUC TI ON
Contact infection through the "hands" is an important route in the transmission of pathogens by direct physical contact with infected individuals or by indirect contact through contaminated surfaces. 1,2 A study involving the surfaces of both dominant and non-dominant hands revealed that more than 150 distinct species-level bacterial phylotypes reside on the average palmer surface of human hands. 3 Some of these bacteria are not part of the natural microbial microflora of the skin, but are transmitted pathogens that can cause various infectious diseases including gastrointestinal and respiratory illnesses. 4,5 These transmittable pathogens on hand surfaces are challenged by factors such as skin dryness, antimicrobial host defense, and exposures to soaps, detergents and other antimicrobial compounds, and UV radiation. From a public health perspective, the preventive effect of hand washing on infectious diseases has been strongly demonstrated epidemiologically. [5][6][7] In addition, the perceptions of hand washing are closely associated with health behavior. 8 However, existing hand hygiene practices are unable to fully prevent contact infections due to both the high frequency of self-inoculation 9 and the high stability of pathogens in the environment. 10 A recent study revealed the prevalence of the collective habit of frequent face touching, even in a pandemic situation such as COVID-19. 11 As the outcome of the existing hand hygiene practices focuses on active interventions to remove and inactivate the pathogens attached to the hands, it is considered as difficult to manage the risk of contact infection in the context of habitual and social behaviors in daily life. In addition, intensive hand washing regimens can be practical challenging, and the frequent use of alcohol-based hand sanitizers and gloves can lead to various adverse skin conditions. 12,13 Therefore, we have been focusing on the natural ability of humans against pathogens.
Previous studies have shown that the surface of human hands naturally has some levels of antimicrobial activity. 10,14,15 In addition, we reported that this antibacterial activity of the hands is suggested to be associated with the susceptibility to infections. 16 Lactic acid has prominent antimicrobial properties. 17,18 Furthermore, our yet to be published data that include a comprehensive analysis of the components on the hand surface shows that not only the amount of lactic acid is abundant on the surface of the hands, but it also has a high positive correlation with the antimicrobial activity of the hands and that applying different amounts of lactic acid on the skin could improve the hand surface infection barrier. 16 Given these, it is expected that a new hand hygiene technology could be developed by leveraging the hands' innate antimicrobial mechanism in the form of a leave-on lactic acid-based formulation.
Recent studies have highlighted the importance of the surface structure in the stability of pathogens. 19,20 Therefore, we assumed that the antimicrobial effects of hand surface components can differ at the individual level and hypothesized that additional hand environmental factors such as fingerprint depth, skin pH, stratum corneum water content, skin temperature, and sweat rate could have synergistic effects for improving the hand surface infection barrier. In this study, we investigated which hand environment factors are involved in the antibacterial activity of lactic acid and elucidated the mechanism of the hand surface infection barrier with the aim of creating a hand barrier technology.

| Bacterial strains and culture medium
The Escherichia coli NBRC3301 strain (NBRC, National Institute of Technology and Evaluation Biological Resource Center) was used.
As a pre-culture, a single colony was grown on soybean casein digest (SCD) agar medium (Nihon Pharmaceutical. Co., Ltd) and then inoculated into 4 mL of Luria-Bertani (LB) liquid medium (Nihon Pharmaceutical. Co., Ltd.), and cultured overnight at 37°C and 180 rpm. Next, 1% of the obtained culture solution was inoculated into LB liquid medium, cultured for 15 hour, washed twice with sterile water, and stored on ice. 21

| Quantitative measurement of the bacterial count using the bioluminescence ATP assay
The bioluminescence ATP assay was used to count the number of viable bacteria by measuring the luminescence level of the ATPluciferase reaction, 22 since the presence of ATP can be considered as proof of the presence of a living organism. 23 The ATP luminescence intensity of the suspension was evaluated using a luciferin-luciferase ATP assay reagent kit (Lucifer HS Set, Kikkoman Biochemifa Co.,), according to the manufacturer's instructions. Fifty microliters of ATP scavenging reagent were added to 500 μL of the sample, and the mixture was allowed to react for 30 minutes. ATP extract solution (100 μL) was added to 100 μL of the reaction solution and mixed for 20 seconds using a vortex mixer. Immediately after the reaction, 100 μL of luminescent reagent was mixed, and the luminescence intensity was measured using a luminometer (Lumitester C-110, Kikkoman Biochemifa Co.,).

| Study design
To investigate the physiological factors of the hands that are involved in the antibacterial activity, we conducted an observational study.

| Volunteer recruitment
Based on the number of variables included in the multiple regression model in this study, we estimated that a sample size of 60 would be adequate. 24 Finally, a total of 106 healthy subjects, including 57 men and 49 women, were recruited randomly. The test was conducted from July 27 to August 28, 2018. Participants of Japanese and mongoloid races were recruited from the Kao Corporation with the following inclusion criteria: (1) healthy female or male aged between 20 and 60 years and (2) provided written informed consent to participate in the complete test process. Subjects were excluded if (1) they had skin symptoms such as atopic dermatitis and rosacea-like dermatitis; (2) they had allergic symptoms due to the use of external medicines, cosmetics, quasi-drugs, etc, in the past; (3) they had an external wound at the observation site; (4) they were taking antibiotics or antifungal agents and had taken it within the past month from the test date; and (5) those who were pregnant or may be pregnant and those who were less than 6 months after delivering the baby.

| Ethical approval
The study protocol, including sample collection, was reviewed and approved by the Ethical Committee of the Kao Corporation with approval number as S181-180613. Informed written consent was obtained from all participants after the procedures were explained with documentation. All experiments were conducted in accordance with the principles of the Declaration of Helsinki.

| Sample collection
Subjects performed standard hand washing with a commercially available test soap (Biore U Rg, Kao Corporation) formulated with alkyl ether sulfate, alkyl ether carboxylic acid, and alkyl glucoside without antimicrobial compounds, 25 rinsed with tap water for 30 seconds, and then washed using purified water for 10 seconds.
To avoid contact with the evaluation site, they wore polyethylene gloves (0950; SHOWA GLOVE Co.) for 2 hour of acclimation (20°C, 40% humidity). The measurement areas of antibacterial activity and physiological properties are shown in Figure S1(a-h). where the bacteria were not applied was swabbed using the same procedure ( Figure S1b) and used as a negative control. The amount of residual number of applied bacteria on the hand was calculated by subtracting the number of negative controls from the number on areas on which the sample was applied. Then, the antibacterial activity of the hands was evaluated relative to the initial viable bacterial number.

| Physiological measurements on hand
The amount of lactic acid, fingerprint depth, pH, and water content of the stratum corneum, temperature, and sweat rate were measured as follows. Regarding the measurement of lactic acid, an acrylic cylinder with an inner diameter of 1.5 cm was placed on the hand, 250 μL of pure water was added, and the mixture was pipetted. The concentration of L-lactic acid was measured using Lactate Pro 2 (Arkray Co., Ltd.) using the extracted solution. 26 The measurements were performed twice, and the values were averaged.
The fingerprint depth was obtained as an index related to the surface shape of the hand. To measure the fingerprint depth, a replica of the thumb was made using a dental silicone resin (GC Examix Fine, GC Corporation) for 5 minutes with the thumb pressed. The fingerprint depth of the replica was measured by 3D imaging using a microscope (Digital Microscope VHX-5000, KEYENCE Corp.). 27 The pH of the skin was measured using a multi-skin measuring instrument (MDD4) and skin pH meter (pH 905; Courage +Khazaka Electric GmbH). The water content of the stratum corneum was measured using a multi-skin measuring instrument, MDD4 and Corneometer CM825 (Courage +Khazaka Electric GmbH). The skin temperature was measured with a contact thermometer TM-300 (AS ONE Corp.). 28 The sweat rate was considered as an index related to the water evaporation rate near the hand surface. The sweat rate was measured using a micro sweat meter TPL series (Techno Science Co., Ltd.). 29 2.3.6 | Statistical analysis JMP14 (manufactured by SAS Institute) was used for the statistical analysis. Since the amount of lactate in one of the 106 subjects was below the lower limit of detection (0.5 mmol L −1 ), this subject was excluded from the analysis, with n = 105. To investigate the relationship between each factor and the antibacterial activity of the hands, Pearson's correlation coefficient was calculated. In the Pearson correlation coefficient test, the significance level was set at <1%. To investigate the contribution of each factor, a multiple regression model was calculated based on the least-squares method with in vivo antibacterial activity as the objective value. The Bayesian information criterion was adopted to optimize the factors. To compare the contributions of each factor, the explanatory variables were scaled (maximum value = 1, minimum value = −1, average value = 0) in the model.

| Evaluation of the bactericidal activity through the in vitro experiments
L-Lactic acid is a natural enantiomer in humans and is present in human sweat in amounts higher than that of D-Lactic acid. 30,31 In this study, we focused on the L-form in the in vitro analysis. A lactic acid solution of 0.2 wt% was prepared using L-lactic acid (Tokyo Chemical Industries Co., Ltd.). Regarding pH adjustment, 48% so-

| Molecular Dynamics (MD) simulation conditions
Bacterial membranes are one of the targets of antimicrobial agents.
To enhance the efficacy of a lactic acid-based technology in maintaining skin hygiene, we aimed to understand the interaction of lac-  Figure 1 (A-F) shows a scatter plot of the amount of each factor, including lactic acid, and the in vivo antimicrobial activity of the hand. As a result of testing the null hypothesis using Pearson's correlation coefficient for six factors, a significant correlation (P-value < significance level α = 0.01) was found in the following four factors (Table 1) (Table 3). However, the correlation between the amount of lactate and skin pH was lower than that between each factor and the in vivo antimicrobial activity (r = 0.437, P = 3.18 × 10 −6 and r = −0.471, P = 3.99 × 10 −7 , respectively) ( Table 1). On the other hand, the sweat rate was significantly correlated with the amount of lactic acid (r = 0.397, P = 2.72 × 10 −5 ) (Table 3), and this correlation was higher than that between the sweat rate and in vivo antimicrobial activity (r = 0.290, P = 2.68 × 10 −3 ) ( Table 1).  Note: Correlation 1 and P-value 2 were calculated using the Pearson correlation coefficients. Bold numbers indicate statistically significant differences (P < 0.01).

| The effect of pH on the antimicrobial action of lactic acid
Statistical analysis showed that there was a negative correlation between skin pH and in vivo antimicrobial activity, indicating that the lower the skin pH, the higher the in vivo antimicrobial activity.
However, it is not clear whether this is due to the solitary effect of skin pH or the synergistic effect of the two factors, skin pH and lactic acid. Therefore, we compared the in vitro antimicrobial activities of the lactic acid solution with that of the HCl solution at the same pH ( Figure 3A). In this experiment, antimicrobial activity was  Note: F-value 1 and P-value 2 were calculated using a multiple regression model based on the least-squares method with in vivo antimicrobial activity as the objective variable. Bold numbers indicate statistically significant differences (P < 0.01). Note: Correlation coefficients and P-values were calculated using the Pearson correlation coefficients. Bold numbers indicate statistically significant differences (P < 0.01).

| D ISCUSS I ON
Bacteria grow and thrive on human skin, and many of them have adapted to survive in various regions of the skin surface. Human hands are one of the most dynamic regions for complex microbial habitats because of their continuous and varied exposure to different environmental surfaces. In this study, we investigated the contribution of specific hand surface characteristics to the infection barrier by conducting an observational study. As an earlier study suggested, between the two lactic acid isomers, L-lactic acid was significantly more effective than D-lactic acid for killing E coli, 30 disturbance of pH homeostasis in cells, 44,47 and the accumulation of toxic anions. 48 Usually, because of their membrane, gram-negative bacteria are typically less susceptible to weak acids. 49 Lactic acid is a weak acid with pKa =3.86, suggesting that non-ionic lactic acid under low pH conditions can inactivate bacteria through the same mechanism, leveraging its ability to permeabilize the membrane of the bacteria. In addition, the antimicrobial activity of lactic acid is reported to depend on the temperature. 50 The temperature dependence of the in vitro antibacterial activity of lactic acid ( Figure 3B) suggests that the improvement of the antibacterial activity is due to the synergistic effect of temperature and lactic acid. A previous report showed that temperature affects the fluidity of the phospholipid bilayer membrane of bacteria. 51 Thus, it is considered that the fluidity of the bacterial cell membrane improves as the temperature increases and the inflow of lactic acid inside the bacterium increases.

| CON CLUS ION
The proposed mechanism in this study would lead to the construction of an effective leave-on technology to improve the hand infection barrier. In addition, the statistical analysis provided a multiple regression model that predicted antibacterial activities based on the amount of lactic acid, pH, and temperature. Thus, this prediction might be used as a monitoring system for the hand surface infection barrier using a simple device without using bacteria. By combining leave-on technologies and the monitoring system, we can provide a new hand hygiene model to improve the hand surface infection barrier at the appropriate time for individuals.

CO N FLI C T S O F I NTE R E S T
All authors are employees of the study sponsor, Kao Corporation, Tokyo, Japan.