Impact of ozonated water on perianal infectious diseases: A retrospective study

Abstract

BACKGROUND

Perianal abscess is an abscess caused by acute or chronic infection in the space surrounding the rectum and anal canal. If untreated or improperly managed, it may progress to a chronic infection and develop into an anal fistula, severely threatening patient health.

AIM

To investigate the therapeutic benefits of perianal infectious diseases by evaluating its bactericidal effects on pathogenic bacteria, its influence on tissue growth factor expression, and its macroscopic effects on tissue repair.

METHODS

Medical records of 65 patients with perianal abscess or anal fistula admitted to the Guihang Guiyang Hospital between January 2022 and May 2022 were retrospectively reviewed. The distribution of bacterial strains isolated from pus and secretion cultures collected pre-operatively and intra-operatively was analyzed, and the five most common pathogenic bacteria were identified. Ozonated water at varying concentrations was then applied to the cultured bacteria to evaluate bactericidal effects within the same exposure time and determine the minimum effective concentration (Cmin) capable of eliminating the major pathogenic bacteria associated with common perianal infectious diseases. Additionally, another patient cohort of 143 patients with perineal abscess or anal fistula treated between June 2022 and April 2024 was enrolled, including 79 patients in the observation group receiving ozone treatment and 64 patients in the control group receiving routine therapy. Both groups underwent surgical intervention. The control group additionally received routine anti-inflammatory therapy, sitz baths, and postoperative wound dressing changes. In addition to these treatments, the observation group received postoperative irrigation of the abscess cavity wound with ozonated water at concentration Cmin (100 mL, rinsed for 1 minute, once in the morning and once in the evening). After intervention, the expression levels of vascular endothelial growth factor, transforming growth factor-β, and platelet-derived growth factor in perianal wound tissue were assessed, and the wound healing outcomes were compared between groups.

RESULTS

Among the 65 patients, 56 showed positive bacterial cultures from pyogenic fluids. The identified bacteria were categorized as follows: (1) Pathogenic bacteria: Escherichia coli (34/56), Klebsiella pneumoniae (11/56), Staphylococcus aureus (4/56), Enterococcus faecium (3/56), and Proteus mirabilis (2/56); and (2) Nonpathogenic bacteria: Miscellaneous bacteria, including normal skin flora (2/56). Under the same exposure time, ozonated water exerted stronger bactericidal effects with increasing concentration and achieved complete elimination of the above pathogenic bacteria within 1 minute at a Cmin of 2.5 mg/L. The observation group showed significantly higher expression levels of tissue growth factors than the controls after treatment (vascular endothelial growth factor: 36.42 ± 1.60 vs 25.85 ± 1.18; transforming growth factor-β: 15.81 ± 2.04 vs 10.63 ± 0.80; platelet-derived growth factor: 33.32 ± 1.48 vs 24.75 ± 1.75; P < 0.001). At one week after treatment, the observation group also demonstrated significantly reduced wound secretion (0.92 ± 0.47 vs 1.27 ± 0.54; P < 0.001), tissue edema (0.54 ± 0.50 vs 0.94 ± 0.77; P < 0.05), and postoperative pain (1.77 ± 0.80 vs 2.94 ± 1.10; P < 0.001), along with significantly faster granulation tissue growth (0.67 ± 0.65 vs 1.19 ± 0.69; P < 0.001) compared with the control group.

CONCLUSION

Ozonated water irrigation is an effective approach for managing infected wounds in patients with perianal abscess or anal fistula and demonstrates promising clinical application value.

Key Words: Perianal abscess; Ozonated water; Growth factor; Wound healing; Retrospective study

Core Tip: If a perianal abscess is not treated promptly or is improperly managed, it may progress into a chronic infection and form an anal fistula, seriously threatening patient health. Surgical intervention remains the primary treatment approach. Although surgery provides reliable short-term efficacy, it is highly invasive and involves prolonged wound healing. Ozone, owing to its strong oxidizing properties, has been increasingly used in clinical practice and has demonstrated beneficial effects in promoting wound healing. However, few studies have investigated the use of ozonated water for treating infected wounds in the perianal region, which presents unique management challenges. Therefore, this study primarily evaluated the therapeutic efficacy of ozonated water for infected perianal wounds.

INTRODUCTION

Perianal abscess is an abscess caused by acute and chronic infection in the perianal and anorectal space and commonly occurs in individuals aged 20-40 age years[1]. Affected patients are often immunocompromised, receive long-term oral hormone therapy or chemoradiotherapy, or have comorbidities such as diabetes, acquired immunodeficiency syndrome, or hematological diseases, including leukemia and lymphoma[2]. Lifestyle factors such as prolonged sitting or standing, sleep deprivation, and constipation may also contribute to disease onset. Without timely intervention, the disease may progress to septic shock, which can be life-threatening in severe cases. Additionally, untimely or improper management of perianal abscess may result in chronic infection and anal fistula formation, leading to a prolonged disease course characterized by recurrent painful perianal masses, ulceration, and purulent discharge. Perianal abscess is also usually accompanied by anal fistula, seriously threatening patient well-being[3]. Surgery remains the primary treatment for this disease. Although the short-term therapeutic effect is well established, the procedure is invasive and requires postoperative wound healing spanning weeks to months. This not only causes substantial pain and economic burden for patients but is also associated with a relatively high postoperative recurrence rate, thereby negatively affecting quality of life. Ozone, because of its strong oxidative properties, has been increasingly applied in clinical practice in recent years. It can eliminate pathogenic bacteria, induce fibroblast proliferation, and modulate endotheliocyte and macrophage function, thereby promoting wound repair[4,5]. As a carrier of ozone, water can deliver ozone to human tissues and organs. Through the slow release of ozone, ozonated water may reduce the adverse effects associated with pure ozone gas and facilitate daily clinical management. However, evidence regarding the effectiveness of ozonated water for treating infected perineal wounds remains limited, and the clinical value of this approach remains unclear. Therefore, this study aimed to further investigate this issue through a stepwise analysis.

MATERIALS AND METHODS

Case data

This study was designed as a retrospective study. A total of 65 patients and 143 patients with perianal abscess or anal fistula admitted to the Department of Gastrointestinal-Anorectal Surgery at Guihang Guiyang Hospital between January 2022 and May 2022 and between June 2022 to June 2024, respectively, were consecutively enrolled.

Eligibility criteria were as follows: (1) Diagnosis of perianal abscess according to the China Expert Consensus on Clinical Diagnosis and Treatment of Perianal Abscess, further confirmed by digital rectal examination, perianal B-ultrasound, and anoscopy; (2) Age ≥ 14 years old; and (3) Eligibility for surgical treatment without absolute contraindications to surgery.

Ineligibility criteria were as follows: (1) Concurrent malignancies, dysfunction of vital organs, mental illness, perianal skin diseases, or venereal diseases; and (2) Trauma, pregnancy or lactation, allergic constitution, and poor treatment compliance.

The eligible patients were assigned to either a control group receiving conventional treatment (surgery + antibiotics + sitz bath + dressing changes) or an observation group additionally treated with ozonated water therapy. The control group received wound irrigation with sterile normal saline (0.9% NaCl) following the same protocol as the observation group. No statistically significant differences were observed between groups regarding sex, age, abscess site, comorbid anal fistula status, or complications (P > 0.05), indicating good baseline comparability. This retrospective study was approved by the Ethics Committee of Guihang Guiyang Hospital (Approval No. GHGYYYLL-KY-202501A). Figure 1 shows the study flowchart.

Figure 1 Research flowchart. VEGF: Vascular endothelial growth factor; TGF: Transforming growth factor; PDGF: Platelet-derived growth factor.

Main reagents and instruments

Enzyme-linked immunosorbent assay kits for detecting human vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β, and platelet-derived growth factor (PDGF) were all purchased from Beijing Solarbio Science & Technology Co., Ltd. The medical ozonated water therapeutic instrument was supplied by Shenzhen Ozone Spring Technology Co., Ltd.

Methods

We retrospectively reviewed 65 cases of perianal abscess treated in our department between January 2022 and May 2022. Pus samples were collected preoperatively and intraoperatively for bacterial culture, and the types and frequencies of common pathogenic bacteria were analyzed.

According to the manufacturer’s instructions, the ozonated water generator was used to prepare ozonated water at concentrations of 0.5 mg/L, 1.0 mg/L, 1.5 mg/L, 2.0 mg/L, 2.5 mg/L, and 3.0 mg/L. To maintain ozone stability, ozonated water was prepared extemporaneously using the medical ozonated water treatment device and used within 10 minutes of production. Before each treatment batch, dissolved ozone concentrations were calibrated using a portable ozone ultraviolet spectrophotometer to ensure that the initial concentration reached the target range.

The five most frequently detected pathogens in bacterial cultures of pyogenic fluids from perianal abscesses were selected and cultured separately on agar slants for 18-24 hours. Fresh bacterial cultures were aseptically suspended in sterile saline, and bacterial concentrations were adjusted using McFarland turbidimetry. To minimize the effects of nutrient deprivation on bacterial sensitivity to ozone, ozonated water treatment was initiated immediately (within 5 minutes) after the suspension reached the target concentration (1 × 10⁸ CFU/mL). Subsequently, 1.0 mL bacterial suspension was mixed with 4.0 mL ozonated water stock solution. Sterile normal saline served as the negative control, whereas a mixture of 1.0 mL bacterial suspension and 4.0 mL normal saline served as the positive control. The mixtures were shaken thoroughly and allowed to react for 1 minute. Then, 0.5 mL of the mixed solution was added to a test tube containing 4.5 mL neutralizing solution consisting of sodium thiosulfate (10 g/L)-supplemented phosphate buffer solution. To ensure complete termination of the ozone reaction, sodium thiosulfate neutralizer was added at a volume ratio of 1:1 to the sample. The mixture was vortexed for 30 seconds to ensure homogeneity. Neutralization efficacy was preverified according to standardized disinfection technical codes, confirming that residual ozone became undetectable immediately after mixing. After 10 minutes, 1.0 mL of the sample was aspirated, evenly inoculated onto blood agar plates, and incubated at 37 °C. Colony growth was assessed after 24 hours, with the presence or absence of bacterial growth recorded as (+) and (-), respectively. The mean values from three repeated measurements were used for analysis to determine the minimum effective concentration (Cmin) of ozonated water capable of eliminating all pathogenic bacteria in this study.

According to the eligibility and ineligibility criteria, another 143 patients admitted between June 2022 and June 2024 were enrolled and allocated to either the observation group (conventional treatment + ozone treatment) and control group (conventional treatment alone). All the patients underwent surgery and received routine postoperative sitz baths, wound dressing changes beginning on postoperative day 1, supplemented with broad-spectrum antibiotic treatment. In addition to conventional treatment, the observation group received ozonated water irrigation. Specifically, 100 mL ozonated water at concentration Cmin was slowly and continuously used to irrigate the perianal wound through a 50 mL syringe over a 2-3 minutes interval, twice daily (morning and evening). After irrigation, the wound was wiped clean with no need to re-rinse it with clear water. A 7-day treatment course was conducted.

On postoperative day 1 and day 7, wound tissue samples measuring 0.2 cm × 0.2 cm × 0.1 cm were collected from each patient, supplemented with phosphate-buffered saline (pH = 7.4), and stored at -80 °C for subsequent analysis. After thawing to 2 °C-8 °C, additional phosphate-buffered saline (pH = 7.4) was added, and the specimens were manually homogenized to ensure thorough mixing. Following centrifugation at 2000 rpm for approximately 20 minutes, the supernatant was carefully collected for enzyme-linked immunosorbent assay-based quantification of VEGF, TGF-β, and PDGF levels according to the kit instructions.

Endpoints

Common pathogenic bacteria and ozonated water bactericidal tests: Colony growth on blood agar plates was recorded as “+++++”, “++++”, “++++”, “++”, or “+”, whereas colony-free growth was denoted as “-“.

Tissue growth factor expression: Expression levels of VEGF, TGF-β, and PDGF in local wound tissues before and after treatment were statistically analyzed.

Wound-related scores: Wound-related scores were evaluated before treatment and at 1 week after treatment in both groups. These include: (1) Wound secretion score: 0 points: No secretion; 1 point: Minimal secretion not saturating a small gauze; 2 points: Moderate secretion soaking 1-3 pieces of small gauze; and 3 points: Large amounts of secretions soaking ≥ 3 pieces of small gauze[1]; (2) Periwound tissue edema score: 0 points: No edema around the wound; 1 point: Mild edema with visible dermatoglyphics; 2 points: Obvious edema with indistinct dermatoglyphics; 3 points: Severe edema without visible dermatoglyphics[6]; (3) Wound pain severity assessment: Pain severity was evaluated using the Visual Analog Scale. A score of 0 indicated no pain; 1-3 points indicated mild, tolerable pain not affecting sleep; 4-6 points indicated moderate pain affecting sleep; and 7-10 points indicated severe, intolerable pain affecting sleep[2]; and (4) Granulation tissue growth evaluation: 0 points: Ruddy granulation tissue; 1 point: Whitish granulation tissue with bleeding tendency on wiping; 2 points: Pale granulation tissue without bleeding tendency on wiping; 3 points: Dark, swollen granulation tissue without bleeding tendency on wiping[3].

Statistical analysis

Data analysis was performed using SPSS 23.0. Continuous variables are expressed as mean ± SD and compared between groups using t-tests. Counting data are expressed as rates or constituent ratios and compared using χ² tests. A P < 0.05 indicated statistical significance, whereas P < 0.001 indicated highly significant statistical differences.

RESULTS

Bactericidal effects of ozonated water

Among the 65 patients who underwent surgery for perianal abscess, 56 had positive bacterial cultures of pyogenic fluid.

The identified pathogens included Escherichia coli (34/56), Klebsiella pneumoniae (11/56), Staphylococcus aureus (4/56), Enterococcus faecium (3/56), and Proteus mirabilis (2/56). Before and after ozonated water intervention, colony growth on blood agar plates was graded as “+++++”, “++++”, “+++”, “++”, or “+”, whereas colony-free growth was recorded as “-“ (Table 1). Ozonated water exerted bactericidal effects against common perianal pathogenic bacteria, with higher concentrations showing stronger antibacterial activity. At a concentration of 2.5 mg/L, complete bacterial elimination was achieved within 1 minute.

Table 1 Bactericidal effects of ozonated water in 1 minute.

Residual bacteria	Ozonated water concentration (mg/L)
	0	0.5	1	1.5	2	2.5	3
Escherichia coli	+++++	++	+	-	-	-	-
Staphylococcus aureus	+++++	++++	+++	++	+	-	-
Klebsiella pneumoniae	+++++	++	+	-	-	-	-
Enterococcus faecium	+++++	+++	+	+	-	-	-
Proteus mirabilis	+++++	+++	++	+	-	-	-

Colony growth on blood agar plates was recorded as “+++++”, “++++”, “++++”, “++”, or “+”, whereas colony-free growth was denoted as “-“.

Expression of tissue growth factors before and after treatment

As shown in Table 2, expression levels of growth factors increased significantly in both groups after treatment compared with pretreatment levels, with the observation group demonstrating greater increases than the control group (P < 0.001).

Table 2 Expression of tissue growth factors across groups pre-treatment and post-treatment, mean ± SD.

Group	VEGF (pg/mL)		TGF-β (pg/mL)		PDGF (pg/mL)
	Before treatment	1-week post-treatment	Before treatment	1-week post-treatment	Before treatment	1-week post-treatment
Control group (n = 64)	19.75 ± 1.94	25.85 ± 1.18a	7.16 ± 1.66	10.63 ± 0.80a	15.75 ± 2.40	24.75 ± 1.75a
Observation group (n = 79)	19.71 ± 1.86	36.42 ± 1.60a,b	6.80 ± 1.50	15.81 ± 2.04a,b	15.35 ± 2.19	33.32 ± 1.48a,b

^aP < 0.001 vs before therapy within the groups.

^bP < 0.001 vs control group at one weeks after treatment.

VEGF: Vascular endothelial growth factor; TGF: Transforming growth factor; PDGF: Platelet-derived growth factor.

Wound-related scores in the two groups

As shown in Table 3, no statistically significant differences were observed between groups in pretreatment scores for wound secretion, periwound tissue edema, wound pain intensity, or granulation tissue growth status (P > 0.05). However, at one week posttreatment, the observation group showed significantly lower scores than the control group for wound secretion, periwound tissue edema, wound pain intensity, and granulation tissue growth scores (P < 0.05).

Table 3 Comparison of wound-related scores, mean ± SD.

Groups	Wound secretion score		Periwound tissue edema score		Wound pain severity score		Granulation tissue growth score
	Pre-treatment	1-week post-treatment	Pre-treatment	1-week post-treatment	Pre-treatment	1-week post-treatment	Pre-treatment	1-week post-treatment
Observation group (n = 79)	2.51 ± 0.50	0.92 ± 0.47	2.54 ± 0.50	0.54 ± 0.50	7.95 ± 0.90	1.77 ± 0.80	2.58 ± 0.50	0.67 ± 0.65
Control group (n = 64)	2.52 ± 0.50	1.27 ± 0.54	2.69 ± 0.47	0.94 ± 0.77	8.11 ± 1.01	2.94 ± 1.10	2.64 ± 0.48	1.19 ± 0.69
t value	-0.110	-4.018	-1.764	-3.511	-0.998	-7.340	-0.707	-4.589
P value	0.913	< 0.001	0.08	0.001	0.320	< 0.001	0.481	< 0.001

DISCUSSION

Perianal abscess and anal fistula remain challenging clinical conditions that affect many patients. Their treatment is complicated by extensive postoperative wounds, prolonged healing time, easy formation of complex anal fistulas, high recurrence rates, and uncertain curative effects, profoundly compromising postoperative quality of life. Therefore, accelerating wound healing postoperatively for perianal abscess, alleviating patient suffering, reducing treatment costs, and lowering recurrence rates remain major focuses in disease diagnosis and treatment.

Ozone is an activated form of oxygen and a strong oxidant. Ozone has been shown to react with various biological components, inducing moderate oxidative stress that exerts antibacterial, antifungal, antiviral, and antiprotozoal effects. In liquid form, ozone may also promote tissue repair by enhancing tissue oxygen utilization, promoting tissue regeneration, and reducing platelet aggregation[7,8]. Ulger[9] considered ozone an important antimicrobial agent capable of effectively eliminating microorganisms without inducing drug resistance. To date, ozone therapy has been applied to more than 50 diseases, including abscesses, acne, eczema, oral mucosal diseases, cardiovascular diseases, and cancer, with favorable therapeutic effects reported[10]. Based on previous research, this study dissolved ozone in water to prepare ozonated water at different concentrations and applied it to common pathogenic bacteria associated with perianal abscess. Ozonated water demonstrated bactericidal effects against all identified pathogenic bacteria, and stronger bactericidal activity increased with higher concentrations. Therefore, ozone therapy is effective against common pathogenic bacteria involved in perianal infections, with its bactericidal effect further validated. However, higher ozone concentrations are not necessarily better because excessive doses may induce some adverse reactions, including respiratory irritation such as cough, dyspnea, and pulmonary edema, as well as skin desquamation, dryness, burning sensation, and pigmentation after contact with ozonated water[11]. Küçük et al[12] evaluated the toxicity of ozonated water at different concentrations on dental pulp cells and found that lower concentrations (2 mg/L and 4 mg/L) more readily promoted cell proliferation than higher concentrations (8 mg/L and 16 mg/L). Accordingly, this study was conducted in two steps. First, experiments were conducted to evaluate the bactericidal effects of ozonated water on pathogenic bacteria isolated from infected perianal wounds. Through repeated testing, the Cmin capable of eliminating all tested pathogens was identified. Second, this concentration was applied clinically. Specifically, when Cmin = 2.5 mg/L, ozonated water completely eliminated the common perianal pathogenic bacteria within 1 minute. This concentration achieved satisfactory bactericidal effects without requiring higher ozone levels, thereby reducing the risk of concentration-related discomfort. However, Bialoszewski et al[13] reported that organic matter may inhibit the antimicrobial activity of ozonated water, with high concentrations of bovine serum completely inhibiting ozone’s bactericidal effects. Perianal wounds contain large amounts of protein and blood clots that may rapidly consume dissolved ozone. Nevertheless, clinical irrigation is a dynamic cleansing process. Although organic matter consumes ozone, continuous irrigation can first remove the organic matter and subsequently exert bactericidal effects. Thus, despite interference from organic matter, the basic antibacterial threshold can still be maintained. Moreover, due to the low cytotoxicity of ozonated water, the selected concentration strikes a balance between safety and efficacy.

Unlike ordinary infected wounds, postoperative wounds following perianal abscess and anal fistula surgery are continuously exposed to fecal contamination. The bactericidal effects observed in this study suggest that ozonated water may provide a relatively sterile environment conducive to perianal wound healing. Wound healing is a complex pathophysiological process involving local tissue regeneration, repair, and reconstruction after tissue injury, with granulation tissue proliferation serving as the basis of healing. Granulation tissue consists of abundant capillaries, microvessels, fibroblasts, and inflammatory cell infiltration. Cytokines, including VEGF, PDGF, and TGF-β, play critical roles in promoting angiogenesis and accelerating wound healing[6,14]. Accumulating evidence has demonstrated strong associations between wound healing and the expression levels of VEGF, TGF-β, and PDGF. Dhamnaskar et al[15] showed that ozone gas bath therapy combined with conventional treatment improved diabetic foot ulcer healing, possibly through the upregulation of VEGF expression. Karakaya et al[16] reported that ozone baths applied to rats with deep second-degree burns ameliorated local pathological injury and promoted the expression of wound healing-related cytokines, including TGF-β. Although the pathophysiological characteristics of diabetic foot ulcers and burn wounds differ from the acute contaminated environment of perianal wounds, these conditions share a common challenge: Impaired healing secondary to high bacterial load and local hypoxia. Therefore, while the underlying etiologies vary, the biological responses of granulation tissue to ozone-induced oxidative stimulation may involve conserved signaling pathways across different wound types. In a study investigated ozonated autohemotherapy on finger survival and vascular repair after the replantation of severed fingers, He et al[17] reported higher survival rates and increased VEGF, TGF-β, and PDGF expression levels in the observation group compared with the control group (all P < 0.05). However, evidence regarding the effects of ozone on growth factor expression and wound healing in fecally contaminated perianal wounds remain limited. In this study, VEGF, TGF-β, and PDGF expression levels were significantly higher in local wound tissues following ozone treatment than in controls, supporting the role of ozone therapy in promoting the expression of growth factors and facilitating tissue repair in perianal wounds. However, it should also be noted that growth factors such as VEGF and TGF-β exhibit complex dynamic expression patterns throughout the wound healing process. Although a single measurement on postoperative day 7 cannot fully capture the entire kinetic profile, this time point was selected because it represents a critical transition from the inflammatory phase to the proliferative phase in perianal wounds. The elevated growth factor levels observed at day 7 suggest that ozonated water may help accelerate this transition, potentially shortening the inflammatory phase. Meanwhile, ozonated water treatment (2.5 mg/L) was associated with greater reductions in wound secretion, faster resolution of periwound skin and tissue edema, improved postoperative pain relief, and faster granulation tissue growth, consistent with the findings reported[18]. These findings further indicate that ozonated water irrigation therapy can accelerate perianal wound healing and alleviate patient pain. Additionally, low-concentration ozonated water did not cause obvious discomfort.

By evaluating both the bactericidal effects of ozonated water and the expression of wound healing-related growth factors, this suggests that ozone exerts dual therapeutic effects. Specifically, it may provide a relatively sterile environment for wound healing while simultaneously promoting the expression of growth factors and the proliferation of granulation tissue. However, given the current clinical study design, it remains challenging to determine whether the observed pro-healing effects result directly from physiological stimulation by ozone or indirectly from reduced microbial interference. Nevertheless, this study’s results support the potential for broader clinical application of ozonated water therapy.

CONCLUSION

This study has several limitations, including a relatively small sample size, limited bacterial spectrum coverage, and short postoperative follow-up duration. Future studies should therefore expand patient recruitment, evaluate the bactericidal effects of ozonated water against additional bacterial species, and expand postoperative follow-up to assess the epithelialization of irrigated abscess cavity wounds and long-term recurrence rates.