Daily topical application of chlorhexidine gluconate to the skin in dogs and its impact on skin barriers and cytotoxicity

Abstract

In recent years, the importance of using local disinfectants instead of systemic antibiotics for the treatment of infectious skin diseases to prevent the emergence of resistant bacteria has become widely recognized. Chlorhexidine gluconate (CHG) is commonly used in veterinary antibacterial shampoos; however, the daily topical application of diluted CHG solutions has also been adopted. Despite its widespread use, few studies have investigated the effects of CHG on the canine skin barrier. This study examined the skin barrier-damaging effects of CHG in dogs by applying diluted CHG (0.05%, 0.5%, and 4%) daily to six healthy dogs for 2 weeks. At the end of the trial, transepidermal water loss (TEWL), skin surface hydration (SSH), and number of skin bacteria were evaluated. Additionally, the cytotoxicity of CHG to the canine progenitor epidermal keratinocytes (CPEK) was examined using the water-soluble tetrazolium salt (WST-8) assay. Continuous application of 0.5% and 4% CHG to the skin led to a significant reduction in the number of skin bacteria. Additionally, at 4% CHG, a notable increase in TEWL and a decrease in SSH was observed. The WST assay revealed cytotoxicity of CHG at concentrations of 0.05%. In conclusion, although daily topical application of 4% CHG demonstrated the potential to disrupt the skin barrier, 0.5% CHG demonstrated sufficient antimicrobial activity without skin barrier disruption. Additionally, when treating dogs’ skin with a compromised stratum corneum, use of lower concentration of CHG is suitable.

INTRODUCTION

Oral and topical antibiotics are commonly used to treat bacterial skin infections in dogs. However, in recent years, the importance of topical therapy using disinfectants to prevent the emergence of antibiotic-resistant bacteria has been widely recognized [2, 12, 15]. Chlorhexidine preparations are a type of disinfectant used in dogs and are often included as ingredients in veterinary shampoos [6, 12]. However, for localized lesions, localized topical application is more convenient than shampoo application and is frequently used in a clinical setting, although few reports exists on its effectiveness [3, 4]. Recently, the efficacy of 3% chlorhexidine-containing wipes in dogs with atopic dermatitis was verified and showed a reduction in clinical scores and cytological bacterial counts when used once daily for 14 days [3]. Additionally, all dogs with superficial pyoderma show resolution of clinical symptoms when treated with 4% chlorhexidine shampoo twice a week and 4% chlorhexidine solution on non-shampoo days [4].

Chlorhexidine preparations are reportedly useful for skin disinfection; however, their repeated use in humans may lead to contact dermatitis, contact urticaria, photosensitivity, and fixed drug eruptions [10]. In dogs, there are reports indicating that the use of 3% chlorhexidine shampoo one to three times per week for up to 6 weeks showed effectiveness of >50% in dogs with Malassezia overgrowth. However, side effects such as erythema and itching have also been reported [11]. Additionally, a noticeable increase in transepidermal water loss (TEWL) after 4 weeks of using a shampoo containing 2% miconazole and 2% chlorhexidine twice a week in dogs with atopic dermatitis has been reported [8]. The occurrence of such side effects of rinsed-off shampoos raises concerns about the potential side effects of daily use without rinsing. However, detailed studies investigating the side effects of daily topical application of chlorhexidine preparations have not yet been conducted. Therefore, this study aimed to investigate how skin symptoms and barrier indices change after the daily application of a chlorhexidine solution daily for 2 weeks. Additionally, the study examined the cytotoxicity of the chlorhexidine solution on canine-derived cultured keratinocytes.

MATERIALS AND METHODS

Dogs

Six healthy Beagles (one male and five females) were included in this study. The dogs were 3 years old and their body weights ranged from 9.08 to 11.24 kg. None of the dogs exhibited any obvious signs of systemic and/or skin diseases. None of the dogs were administered any systemic or topical medications and were not bathed with shampoo for 2 weeks before and during the experiment. This study was approved by the Animal Care and Use Committee of Okayama University of Science (approval number: 2021-074).

Testing products

For daily topical application on the dogs, chlorhexidine gluconate (CHG; HIBITEN solution, Sumitomo Pharma Co., Ltd., Osaka, Japan) was diluted with sterile water to concentrations of 0.05%, 0.5%, and 4%. Concentrations of 0.05% and 0.5% are recommended for disinfecting human mucous membranes and skin, respectively, as stated in the accompanying document. A concentration of 4% was used as describes in a previous study [4]. Sterile water was used as a negative control. For the cell toxicity assay, CHG was diluted to 0.00005, 0.0005, 0.005, 0.05, and 0.5% in the culture medium. At concentrations greater than 0.5%, precipitation occurred upon dissolution into the culture medium, rendering the experiment unfeasible. All the test products were prepared immediately prior to use.

Daily topical application of CHG to the skin of dogs

The dog’s dorsal thorax was shaved to create four sections of 3 cm² (two sections on the left and two sections on the right) the day before the experiment began. The next day, the testing products were sprayed to allow the application of 0.2 mL/cm² [2]. The allocation of the test products to each shaved area was randomized for each dog. The application was repeated every 24 hr for 2 weeks. Before the initial application and 24 hr after the final application, clinical scoring of the rash and measurement of TEWL and skin surface hydration (SSH) were performed by a different person who sprayed the test products. These evaluations were conducted without information regarding the solutions applied to each area.

Clinical scores for rash

The shaved skin areas were observed before and after the trial, and clinical rash scores, including erythema, papules, and scales, were recorded as follows: 0=none; 1 mild; 2=moderate; and 3=severe [12].

Transepidermal water loss and skin surface hydration

The TEWL and SSH of the areas to which the test products were applied were measured before the initial application and 24 hr after the final application. The TEWL was measured after keeping the dogs on a platform in the measurement room for 20 min using a VAPO SCAN AS-VT100RS (Asch Japan Co., Ltd., Tokyo, Japan). The temperature in the measurement room was maintained at 25 ± 2°C, with a humidity level of 55 ± 5%. The SSH measurements were conducted immediately after the TEWL measurements using a Moisture Meter SC (Delfin Technologies, Kuopio, Finland). The TEWL and SSH values were calculated as the average of three measurements.

Bacterial count

The number of skin bacteria was measured before the initial application and 24 hr after the final application, following the measurement of TEWL and SSH. A dry sterile cotton swab was moistened with Dey-Engley (D/E) Neutralizing Broth (BD, Sparks Glencoe, MD, USA), swabbed vertically and horizontally five times on the shaved area, placed in a sterile tube containing 10 mL of D/E Neutralizing Broth, and mixed. The suspensions were kept on ice and transported to the laboratory. Thereafter, 100 μL of each suspension were placed in the center of the agar plates made with D/E Neutralizing Agar (BD). The cell suspensions were spread on a cell spreader (Fisher Scientific, Waltham, MA, USA) and the plates were incubated at 35°C for 48 hr and bacterial colonies were counted. Considering the possibility of skin barrier damage when swabbing the skin, the number of skin bacteria was not measured before the trial.

Cytotoxicity assay

The canine progenitor epidermal keratinocytes (CPEK), a canine epidermal keratinocyte progenitor cell line, was purchased from CELLnTEC Advanced Cell Systems (Bern, Switzerland) and used for the cytotoxicity assays. A 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (water-soluble tetrazolium salt-8 [WST-8]) assay was performed using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) with some modifications. The CEPK cells were cultured at 37°C and 5% CO₂ using CnT-09 (CELLnTEC advanced cell systems) until they reached 80% confluence. After washing the cells with phosphate-buffered saline (PBS) and detaching them from the flasks by incubation with 2.5 g/L of trypsin and 1 mM of ethylenediaminetetraacetic acid (Nakarai Tesque, Kyoto, Japan), 4 × 10⁴ cells in 100 µL of CnT-09 per well were plated in 96-well plates and incubated at 37°C and 5% CO₂ for 24 hr. The cells were washed with PBS, and incubated in CnT-09 with various concentrations of CHG at 37°C and 5% CO₂ for 5 min. The cells were washed with PBS twice and then incubated in CnT-09 containing WST-8 at 37°C and 5% CO₂ for 30 min. The absorbance was measured at 450 nm using a microplate reader (SH-1300; Corona Electric, Hitachinaka, Japan). Cell viability was calculated as follows: Cell viability (%)=(OD450 at each concentration of CHG / OD450 in the medium alone) × 100.

Statistical analysis

Statistical analyses were performed using EZR, a statistical software package in R version 4.3.1 [9]. The Kruskal-Wallis test followed by the Steel-Dwass test was used for bacterial count, the paired t-test for TEWL, the Wilcoxon signed-rank test for SSH, and analysis of variance (ANOVA) followed by Tukey’s test for cell viability. Normality was assessed using the Kolmogorov–Smirnov test, and the homogeneity of variances was verified using the F test or Bartlett test. Significance was defined as P values of less than 0.05.

RESULTS

The antibacterial effect of daily topical application of CHG

After 2 weeks of daily topical application of CHG to the skin of dogs, the bacterial count in the skin area decreased in a concentration-dependent manner (Fig. 1). The application of 0.5% and 4% CHG significantly decreased the number of skin bacteria compared to the application of water, whereas the application of 0.05% CHG showed a decreasing trend, but this was not significant.

Fig. 1.

The skin bacterial count after daily chlorhexidine gluconate (CHG) topical application. Sterile water and various concentrations of CHG were applied every 24 hr for 2 weeks, and the applied area was swabbed using sterile cotton swabs 24 hr after the final application to collect skin bacteria. The collected bacteria were cultured on agar plates using disinfectant-neutralizing medium, and the number of colonies was counted to calculate the bacterial count on the skin. Box plots depict the distribution of bacterial count. The central line within each box represents the median, while the box itself spans the interquartile range. Whiskers extend to 1.5 times the interquartile range from the edges of the box.

The effect of daily topical application of CHG on rash clinical scores

Daily topical application of water, 0.05% CHG, or 0.5% CHG did not alter the rash clinical scores, including erythema, scales, and papules (Table 1). The application of 4% CHG tended to increase erythema and papules. All the dogs showed clinical scores of 2 or higher only after the application of 4% CHG.

Table 1. Clinical scores of rash before and after daily chlorhexidine gluconate (CHG) topical application.

Group	Dog number	Erythema		Scales		Papules
		Pre	Post	Pre	Post	Pre	Post
Water	1	0	0	0	0	0	0
	2	1	0	0	0	0	0
	3	0	0	0	0	1	0
	4	0	0	0	0	0	0
	5	0	0	0	0	0	0
	6	0	0	0	0	1	0
0.05% CHG	1	0	0	0	0	1	0
	2	0	0	0	0	0	0
	3	0	0	0	0	0	0
	4	0	0	0	0	0	0
	5	0	0	0	0	0	0
	6	0	0	0	0	0	0
0.5% CHG	1	0	0	0	0	1	0
	2	0	0	0	0	0	0
	3	0	0	0	0	0	0
	4	0	0	0	0	0	1
	5	0	1	0	0	0	1
	6	0	0	0	0	1	0
4% CHG	1	0	0	0	0	1	1
	2	0	2	0	0	0	1
	3	0	1	0	0	0	2
	4	0	1	0	0	0	2
	5	1	2	0	1	0	0
	6	0	0	0	0	0	1

The effect of daily topical application of CHG on the skin barrier

The TEWL values showed no significant change after the 2-week daily topical application of water, 0.05% CHG, or 0.5% CHG (Fig. 2A). In contrast, application of 4% CHG significantly elevated these values (Fig. 2A). Although not significant, the SSH values showed an upward trend after 2 weeks of daily water application (Fig. 3). This upward trend decreased as the CHG concentration increased, and a significant decrease was observed with the application of 4% CHG (Fig. 2B).

Fig. 2.

The skin barrier indices before and after daily chlorhexidine gluconate (CHG) topical application. The transepidermal water loss (TEWL) (A) and skin surface hydration (SSH) (B) were measured before the trial and 24 hr after the final application. The TEWL measurement was conducted after keeping the dogs on a platform in the measurement room for 20 min. The SSH measurement was conducted immediately after the TEWL measurement. Both the values of TEWL and SSH were calculated as the average of three measurements. The measurers were blind to the test product applied to the different sections. Box plots depict the distribution of TEWL or SSH. The central line within each box represents the median, while the box itself spans the interquartile range. Whiskers extend to 1.5 times the interquartile range from the edges of the box.

Fig. 3.

Cytotoxicity of chlorhexidine gluconate (CHG) on canine cultured keratinocytes. The canine epidermal keratinocyte (CPEK) cells (4 × 10⁴ cells in 100 µL/well) were plated in 96-well plates and incubated at 37°C, 5% CO₂ for 24 hr. The cells were washed with PBS, and incubated in culture medium with various concentrations of CHG at 37°C, 5% CO₂ for 5 min. The cells were washed with PBS twice and incubated in culture medium containing water-soluble tetrazolium salt (WST-8) at 37°C, 5% CO₂ for 30 min. Absorption at 450 nm was measured and the cell viability was calculated. Box plots depict the distribution of cell viability. The central line within each box represents the median, while the box itself spans the interquartile range.

Cytotoxicity of CHG on canine keratinocytes

Incubation for 5 min with CHG at concentrations ranging from 0.00005% to 0.005% resulted in no significant cytotoxicity to the CPEK (Fig. 3). However, incubation with 0.05% and 0.5% CHG clearly decreased the viability of the CPEK, and this difference was significant compared to no CHG (Fig. 3).

DISCUSSION

In the present study, the daily topical application of 0.5% and 4% CHG significantly reduced the bacterial count on the skin of dogs, whereas 0.05% CHG did not show a significant reduction. Previous reports have indicated that 0.05% CHG reduced bacterial counts in experimentally created wounds on the backs of beagles after 48 hr. It has been confirmed that CHG exhibits antibacterial activity against Staphylococcus pseudintermedius in vitro at concentrations ranging from 0.00006247% to 0.0007% [1, 15]. The reason for the lack of a significant reduction in the bacterial count in this study is unclear, but may be attributed to the sampling of bacteria 24 hr after the final application. Although 0.05% CHG exhibited antibacterial activity, it may have had a limited residual efficacy. CHG is known to precipitate in the presence of organic matter or ions, and its solid residues may persist on the skin, providing sustained antibacterial activity compared to disinfectants such as alcohol [1, 6]. In the present study, the near absence of detectable bacteria 24 hr after the final application of 0.5% and 4% CHG to the skin was likely due to sustained antibacterial activity. Therefore, when sustained antibacterial effects on the skin are expected, such as during the treatment of pyoderma, CHG at concentrations of 0.5% or higher may be effective. Concentrations of 0.1%, which were not evaluated in this study, require further investigation.

In previous reports, daily, 4% CHG showed therapeutic effects and did not cause worsening clinical skin symptoms. However, these studies also included CHG-containing shampoos, twice-weekly; therefore, the effects of daily topical CHG application have not been clearly elucidated [4]. Given that this study did not find significant differences in the antibacterial effects of 4% and 0.5% CHG on canine skin, similar results may be achieved with 0.5% CHG. Furthermore, after 14 days of daily topical application of 4% CHG, an increase in erythema and papules, significantly increased TEWL values, and significantly decreased SSH values were observed. Therefore, the daily use of 4% CHG may potentially impair the skin barrier, which is not recommended. In contrast, 0.5% CHG showed sufficient and sustained antibacterial effects without worsening clinical scores or skin barrier function indices. Thus, a concentration of 0.5% CHG is recommended for daily application to canine skin. However, as this study used healthy dogs without skin diseases, prospective studies evaluating the efficacy of daily topical application of 0.5% CHG for the treatment of canine pyoderma are needed. In addition, using 3% chlorhexidine-impregnated wipes containing moisturizers for 2 weeks improved the clinical symptoms of dogs with atopic dermatitis and localized bacterial infections, without adverse effects on the skin [3]. This highlights the importance of exploring the concentrations not examined in this study as well as the potential skin barrier protective effects of combining chlorhexidine with moisturizing agents in future research.

A previous study reported that the application of disinfectants might alter the resident microbiota in humans [14]. However, it has also been demonstrated that changes in the resident microbiota are limited to a short period of 6 hr post-application. Additionally, some reports have indicated that the use of alcohol-based solutions or 2% chlorhexidine does not affect the diversity of the resident microbiota in humans, and that the long-term adverse impact of disinfectants on the microbiota remains unclear [7, 16]. At present, it is unclear how disinfectants affect the resident microbiota of canine skin; however, in this study, healthy dogs were used, suggesting that the antibacterial effects of CHG influenced the resident microbiota in the patients’ skin. Considering the potential risks of adverse effects of CHG, the application of CHG to the skin in the absence of bacterial overgrowth should be avoided.

In vitro cytotoxicity experiments showed that CHG significantly reduced cell viability, even at low concentrations (0.05%). The results of the present study indicated that 0.05% CHG was cytotoxic to canine keratinocytes, whereas at 0.005%, its cytotoxicity was less pronounced, which was consistent with previous reports where the survival threshold for canine fibroblasts was at concentrations of approximately 0.013% acetic acid chlorhexidine [13]. It has been reported that the IC₅₀ of ethanol for cultured human keratinocytes ranges from 2% to 5% [5]. However, 70–80% ethanol formulations are widely used for hand disinfection, suggesting that the safe concentration in vitro differs from that in vivo. This discrepancy is likely attributable to the barrier function of the stratum corneum. Therefore, in situations where the stratum corneum is compromised and keratinocytes are directly exposed to the disinfectant, CHG cytotoxicity may occur within a short period. In cases of erosion or similar conditions, it is advisable to use CHG at lower concentrations, such as 0.005% or less. However, reports indicate that concentrations lower than 0.05% may not provide sufficient antibacterial effects because significant survival of Staphylococcus aureus has been observed in vitro [13]. Therefore, prospective studies are necessary to determine whether lower concentrations of CHG are effective for treating canine pyoderma.

The limitations of this study include the use of healthy dogs, which may not accurately reflect the effectiveness of CHG on the skin of dogs with bacterial infections of the skin. Dogs with conditions such as atopic dermatitis, which involves skin barrier abnormalities, could potentially exhibit more severe manifestations of rash and impaired skin barrier function as observed in this study. In addition, because rinsing or wiping was not performed after application in this study, the effects of such practices were not evaluated. It is thus possible that rinsing off CHG after a certain period, particularly at higher concentrations, minimizes its effects on the skin barrier.

In conclusion, the daily topical application of 0.5% CHG effectively controlled microbes without disrupting the skin barrier. The use of lower concentrations should be considered in dogs with suspected compromised skin-barrier function.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.