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. 2025 May 25;15:18267. doi: 10.1038/s41598-025-01852-z

Antimicrobial properties and toxicity challenges of chlorine dioxide used in alternative medicine

Ruth Dudek-Wicher 1,✉, Malwina Brożyna 2, Justyna Paleczny 1, Beata Mączyńska 1, Bartłomiej Dudek 2, Paweł Migdał 3, Arleta Dołowacka-Jóźwiak 4, Jędrzej Fischer 5, Adam Junka 2
PMCID: PMC12104323  PMID: 40414916

Abstract

This study evaluates the in vitro antimicrobial efficacy and cytotoxicity of acidified sodium chlorite (ASC), a source of chlorine dioxide. Despite its controversial promotion in alternative medicine as a cure-all solution, known as "Miracle Mineral Solution" (MMS), the data on its factual medicinal activity is very limited. Therefore, we aimed to elucidate the activity of ASC against biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus mutans, Pseudomonas aeruginosa, Escherichia coli, and Lactobacillus sp. or an organic acid (ASC1, ASC2, respectively). The lowest antimicrobial concentration of ASC registered was 0.002992% (29.92 ppm) but did not exhibit stronger antimicrobial activity than polyhexamethylene biguanide. Biofilms of S. mutans and E. coli were the most susceptible to tested formulations. Biofilm formed by L. rhamnosus displayed susceptibility to concentrations lower than the minimum biofilm eradication concentration (0.09575%, 957.5 ppm). In the in vitro cytotoxic assay towards eukaryotic fibroblasts and in vivo model of Galleria mellonella larvae concentration-related increase of cytotoxic effects was observed. Our findings demonstrate that these concentrations of ASC which can effectively eradicate biofilms, also pose potential health risks due to their in vitro and in vivo cytotoxicity. It implies that ASC applied in humans can lead to damage to the mucous membrane of the gastrointestinal tract. This research contributes to the ongoing debate on the safety and efficacy of chlorine dioxide in clinical applications, highlighting the need for precise dosing to avoid mucosal damage in therapeutic contexts.

Keywords: Acidified sodium chlorite, Chlorine dioxide, Biofilm, Probiotics, Antibiofilm activity

Subject terms: Microbiology, Antimicrobials, Biofilms, Infectious diseases

Introduction

Sodium chlorite is an inorganic compound utilized in various applications, including the purification of drinking water. Neutral solutions of sodium chlorite demonstrate stability when stored in conditions devoid of light and heat exposure1. Acidification of these solutions with either organic or inorganic acids results in the formation of acidified sodium chlorite (ASC) and the stabilization of the pH value. This process is followed by a hydrolysis reaction, during which a significant quantity of chlorine dioxide (ClO2) is liberated:

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Sodium chlorite solutions are frequently marketed in alternative medicine circles as ‘Miracle Mineral Solution’ (MMS), a panacea for a wide array of illnesses. Distributors of MMS advocate that when mixed with citric acid, it exhibits efficacy against autism and cancer, as well as against various microbial pathogens including SARS-CoV-2-192–4.

ClO2 is a potent bleaching agent that is not intended for ingestion. ClO2 exerts its effect by interacting with biomolecules, leading to alterations in the electrochemical equilibria within mitochondrial and cellular membranes5. Such interactions have the potential to cause significant cellular and tissue damage. Although a tolerable daily intake of chlorite is 0.03 mg/kg/day, in 2019, the Food and Drug Administration (FDA) issued a stark warning regarding the potential adverse reactions associated with the consumption of MMS. These include severe vomiting, intense diarrhea, life-threatening hypotension due to dehydration, and acute liver failure5–7. Authorities in Switzerland, the United Kingdom, Australia, and Canada have also issued comparable alerts regarding the risks associated with MMS8–11. Despite these warnings, the MMS product continues to have a significant number of proponents and remains available for purchase through various online retailers12–16. Despite its irritant properties, aqueous solutions of ASC may be utilized under strictly regulated concentrations and conditions, as evidenced by numerous clinical studies and approvals from regulatory bodies. In the early 1980s, the relative safety of ingesting chlorine dioxide at a concentration of 5 mg/l was established over twelve consecutive weeks in clinical trials17. In 2013, the European Medicines Agency (EMA) approved a chlorite-based drug (known as NP001) in the dose of 2 mg/kg as an intravenous orphan drug for the treatment of amyotrophic lateral sclerosis18,19. Furthermore, ASC formulations (concentration range: ~ 0.01% wt/vol–0.006% wt/vol) have been developed into nasal aerosols, which were employed as an antiviral prophylactic measure among healthcare workers during the COVID-19 pandemic20. To maximize the utility of ClO2, Palcsó et al.created poly(ethylene oxide)-based nanofibrous mat loaded with sodium chlorite producing ClO2 under acidic conditions, that could be used as filters in face masks as protection against pathogens21. Additionally, Yates et al. demonstrated that the antiplaque activity of mouthwashes containing aqueous solutions of ASC is comparable to those containing 0.12% chlorhexidine, however neither the concentration of ASC nor malic acid was mentioned in the study22. In a clinical study focusing on geriatric patients with chronic oral candidiasis, the application of a ClO2-containing solution resulted not only in a reduction in the quantity of Candida spp., but also in an improvement in the appearance of soft tissues, attributed to a decrease in local inflammation23. Mouth rinse studies proved the antibacterial and antifungal properties of ASC for solutions ranging from 0.003% to 0.016% (wt/vol %) ClO224,25.

However, prior studies have not adequately addressed the variability in the responses of pathogenic species responsible for other local infections to aqueous solutions of chlorine dioxide (ASC), nor have they examined the effects of ASC on biofilms—complex microbial communities encased in extracellular polymeric substances and responsible for the persistence of infections. Biofilms represent a predominant form of microbial life with a multi-step formation process. Biofilms exhibit increased resistance to classical antibiotics and cause disease through device-related and non-device-associated infections, posing a severe threat to global health issues26.

Consequently, our study aims to identify the antibiofilm concentrations of ASC and evaluate its impact on biofilms formed by seven distinct bacterial species. This includes Staphylococcus aureus: MSSA (methicillin-sensitive S. aureus) and MRSA (methicillin-resistant S. aureus), Enterococcus faecalis, Streptococcus mutans, Pseudomonas aeruginosa, Escherichia coli, Lactobacillus rhamnosus and Lactobacillus casei. S. aureus is an opportunistic pathogen that colonizes the nose and skin and is the leading cause of healthcare-associated infections such as skin and surgical site infections. E. faecalis a gastrointestinal commensal capable of causing invasive diseases, particularly in hospitalized and immune-compromised patients. A major pathogen of dental caries—S. mutans, is regarded as a causative agent of infective endocarditis. P. aeruginosa is the most common Gram-negative organism identified in nosocomial pneumonia while E. coli, known to be a part of normal intestinal microbiota may be the cause of intestinal and extraintestinal illness, catheter-associated UTIs, and ventilator-associated pneumonia (VAP). Besides wide-spread pathogens we have tested probiotic strains representatives L. rhamnosus and L. casei, which are known constituents of the healthy microbiota in various body niches27.

To assess the potential cytotoxic effect of effective antimicrobial/antibiofilm concentrations of ASC, in vitro cell lines/in vivo model will be used.

The superior aim of this research is to provide empirical, scientific data on the efficacy and safety of ASC. This is crucial in either validating or refuting the claims made in alternative medicine circles. If the research finds specific concentrations and conditions under which ASC is effective and safe, it could lead to its more controlled and informed use in medical settings and managing infections. Conversely, if the results show limited efficacy or significant toxicity, it could help debunk unwarranted claims and protect patients and consumers from potentially harmful health advice.

Results

Minimal inhibitory concentration evaluation (MIC)

The MICs of ASC1 and ASC2 towards P. aeruginosa were lower than the MIC of control antiseptic agent—PHMB. For E. coli, the opposite results were obtained. The lowest MIC of ASC1 was observed towards MSSA while the lowest MIC of ASC2 was revealed for MSSA but also for E. coli. S. mutans, L. rhamnosus and L. casei were the most tolerant to ASC1 than to ASC2. ASC2 has shown greater antimicrobial activity against E. coli and L. casei than ASC1. The antimicrobial activity of ASC2 against L. casei was like the activity of PHMB. In the case of S. mutans, E. faecalis and L. casei, antimicrobial activity of ASC1 and ASC2 did not exceed the activity of SC (Table 1).

Table 1.

The MIC (%; ppm) of tested solutions.

Tested strain/tested solution MSSA MRSA E. faecalis S. mutans
% ppm % ppm % ppm % ppm
ASC1 (%ClO2) 0.002992 29.92 0.005984 59.84 0.005984 59.84 0.023938 239.38
ASC2 (%ClO2) 0.002992 29.92 0.005984 59.84 0.005984 59.84 0.023938 239.38
HA 0.08 800 0.08 800 0.08 800 0.08 800
GA 0.8 8000 0.8 8000 0.8 8000 0.8 8000
SC 0.0083 83 0.0083 83 0.0041 41 0.0165 165
PHMB (C +) 0.000098 0.98 0.000098 0.98 0.00039 3.9 0.000098 0.98
Tested strain/tested solution P. aeruginosa E. coli L. rhamnosus L. casei
% ppm % ppm % ppm % ppm
ASC1 (%ClO2) 0.005984 59.84 0.005984 59.84 0.023938 239.38 0.023938 239.38
ASC2 (%ClO2) 0.005984 59.84 0.002992 29.92 0.011969 119.69 0.005984 59.84
HA 0.08 800 0.08 800 0.08 800 0.08 800
GA 0.8 8000 0.8 8000 0.8 8000 0.8 8000
SC 0.0083 83 0.0041 41 0.0165 165 0.0165 165
PHMB (C +) 0.00625 62.5 0.000195 1.95 0.00625 62.5 0.00625 62.5
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Visualization of cells treated with MIC concentration with TEM

The TEM analysis performed on Gram-positive S. aureus and Gram-negative P. aeruginosa confirmed that ASC1 applied in MIC concentration mechanism of action relies on the destruction of cell walls/membranes leading to the cells’ deformation, cytoplasm leakage, and to death of cells (Fig. 1).

Fig. 1.

Fig. 1

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Visualization of S. aureus (A, B) and P. aeruginosa (C, D) treated (B, D) or non-treated (A, C) with MIC solution of tested solution. The deformation of cellular shapes and cell wall as well as loss of density of microbial cytoplasm (areas of lower electron density visualized by light grey color) are explicitly visible in the images of treated cells (C, D).

Minimal biofilm eradication concentration evaluation (MBEC)

None of the tested solutions were more effective against tested biofilms than PHMB. SC has shown greater antibiofilm activity against all species than ASC1 and ASC2. In turn, ASC2 displayed greater antibiofilm potential than ASC1. Biofilms formed by P. aeruginosa and L. casei were the most resistant to both ASC1 and ASC2 (Table 2).

Table 2.

The MBEC of tested compounds (%; ppm).

Tested strain/Tested solution MSSA MRSA E. faecalis S. mutans
% ppm % ppm % ppm % ppm
ASC1 (%ClO2) 0.1915 1915 0.1915 1915 0.1915 1915 0.09575 957.5
ASC2 (%ClO2) 0.047875 478.75 0.09575 957.5 0.09575 957.5 0.047875 478.75
HA 0.16 1600 0.16 1600 0.16 1600 0.08 800
GA 1.6 16,000 1.6 16,000 1.6 16,000 0.4 4000
SC 0.033125 331.25 0.06625 662.5 0.0165 165 0.0083 83
PHMB (C +) 0.0125 125 0.00625 62.5 0.003125 31.25 0.001563 15.63
Tested strain/tested solution P. aeruginosa E. coli L. rhamnosus L. casei
% ppm % ppm % ppm % ppm
ASC1 (%ClO2) 0.383 3830 0.09575 957.5 0.1915 1915 0.383 3830
ASC2 (%ClO2) 0.383 3830 0.09575 957.5 0.09575 957.5 0.383 3830
HA 0.16 1600 0.16 1600 0.16 1600 0.08 800
GA 1.6 16,000 1.6 16,000 1.6 16,000 0.8 8000
SC 0.033125 331.25 0.033125 331.25 0.0165 165 0.033125 331.25
PHMB (C +) 0.00625 62.5 0.00625 62.5 0.003125 31.25 0.00625 62.5
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Visualization of biofilm with fluorescence microscopy (FM)

The fluorescence microscopy images depict biofilms formed by S. aureus, P. aeruginosa, L. rhamnosus, and S. mutans after treatment with ASC1 and ASC2 at concentrations above, equal to, and below the minimum biofilm eradication concentration (MBEC) (Fig. 2). The biofilms were stained using the LIVE/DEAD Biofilm Tracer™ Viability Kit, with live cells fluorescing green and dead cells fluorescing red. The yellowish/orange color is found in partially damaged cells or area of high density of both live and dead cells. In the control (C +) samples, where no ASC treatment was applied, biofilms of all four bacterial species exhibit predominantly green fluorescence, indicating a high proportion of live cells. The [%] share of signal from SYTO-9 was 88%, 71%, 94%, 75% for S. aureus, P. aeruginosa, L. rhamnosus, S. mutans, respectively. The biofilm structure appears dense, with minimal disruption or dead cell presence, confirming the viability of untreated biofilms across all species. At concentrations above the MBEC, significant disruption of the biofilms is observed. For S. aureus, the biofilm was nearly entirely removed (fluorescence intensity measured from SYTO-9 constituted 2% of C + value), indicating a high level of cell death and effective biofilm eradication. Similarly, in P. aeruginosa and L. rhamnosus, a substantial decrease of green fluorescence is detected, with a large proportion of dead cells, especially in L. rhamnosus, where the biofilm structure seems fragmented. In contrast, S. mutans biofilms show less increase of red fluorescence (% Live:Dead is 53:47), suggesting some resistance to the treatment or incomplete eradication at this concentration. Biofilms treated with concentrations equal to MBEC exhibit intermediate effects. In S. aureus, almost the entire biofilm fluoresces red (% Live:Dead is 2:98), indicating that MBEC is sufficient for effective eradication. P. aeruginosa biofilms show a mixture of live (green) and dead (red) cells (% Live:Dead is 61:39), suggesting partial biofilm disruption. L. rhamnosus shows a similar pattern, with areas of red interspersed with green, indicating incomplete killing. In S. mutans, the biofilm still maintains a predominantly green fluorescence, indicating survival of a significant proportion of the cells, which might suggest that MBEC is less effective against this species compared to others. At concentrations below MBEC, the biofilms remain largely intact. S. aureus, P. aeruginosa, and L. rhamnosus biofilms predominantly fluoresce green, indicating that the treatment at this concentration is insufficient to cause significant cell death. Some localized areas of red fluorescence are visible, particularly in P. aeruginosa, but the biofilms largely remain viable. For S. mutans, the green fluorescence dominates across the biofilm, suggesting low effect of the ASCs at this sub-MBEC concentration.

Fig. 2.

Fig. 2

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Fluorescent visualization of 4 tested biofilms after application MBEC, < MBEC and > MBEC concentrations of ASC1. Green staining represents live cells, and red/yellow staining represents dead cells.

Biofilm eradication from HAPd

Gram-negative strains displayed higher sensitivity to ASC1 than Gram-positive strains. In all cases, the eradication level exceeded 50% (Fig. 3A). Simultaneously, various patterns of susceptibility to ASC1 were observed among tested P. aeruginosa strains (Fig. 3B). Statistical significance was observed in the level of biofilm reduction between species from the same genera such as MRSA vs. S. aureus or L. rhamnosus vs. L. casei (Fig. 3C).

Fig. 3.

Fig. 3

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Eradication of biofilms formed by tested strains on HAPd. ns—no statistical significance, **- small statistical significance, ***-moderate statistical significance, ****-strong statistical significance.

Visualization of biofilm with SEM

Figure 4 presents the effect of applying MBECs on selected species. Full biofilm eradication has not been demonstrated.

Fig. 4.

Fig. 4

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SEM visualization of biofilm formed on HAP surface before (C +) and after MBEC application. Magnification: S. aureus (× 20,000), P. aeruginosa (× 20,000), L. rhamnosus (× 10,000), S. mutans (× 10,000).

Evaluation of antibiofilm activity of ASC after different activation times

Obtained results (Table 3) revealed that activation time has an impact on the antibiofilm effectiveness of tested mixtures. Both ASC1 and ASC2 were characterized by greater antibiofilm activity after applying more than 60 s of acidification time. However, extending acidification time over 180 s did not translate into an increase in the antibiofilm activity of the tested solutions.

Table 3.

MBEC values obtained after applying different times of activation of tested ASC.

Acidification time [s] MBEC
MSSA 		MBEC
P. aeruginosa
%ClO2 ppm %ClO2 ppm
ASC 1 60 0.1915 1915 0.383 3830
180 0.047875 478.75 0.047875 478.75
360 0.047875 478.75 0.047875 478.75
ASC2 60 0.047875 478.75 0.383 3590
180 0.023938 239.38 0.047875 478.75
360 0.023938 239.38 0.047875 478.75
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Cytotoxicity of tested solutions

In vitro

The cytotoxicity study showed that the solutions displayed similar cytotoxicity, regardless of whether SC was activated with an organic acid (GA) or an inorganic acid (HA). The observed differences were statistically insignificant between concentrations of ASC1 vs ASC2 (Mann–Whitney test, α = 0.05), with exception of 59.84 ppm and 478.75 ppm where assessed p-values were, 0.012 and 0.016, respectively. For both tested combinations, the lowest cytotoxic concentration was observed for ASC1 and ASC2 at 0.002992%, which demonstrated antimicrobial effectiveness against S. aureus and E. coli in MIC evaluation. In Table 4, the results of in vitro cytotoxicity tests are presented.

Table 4.

Cytotoxicity of selected concentrations of ASC1 and ASC2.

Concentrations tested Cytotoxicity [%]
ClO2% ppm
ASC1 0.002992 29.92 26.13 (± 11.98)
0.005984 59.84 75.39 (± 12.43)
0.011969 119.69 96.96 (± 2.37)
0.023938 239.38 97.12 (± 0.89)
0.047875 478.75 98.15 (± 0.91)
ASC2 0.002992 29.92 27.52 (± 8.60)
0.005984 59.84 86.70 (± 2.69)
0.011969 119.69 96.4 (± 1.58)
0.023938 239.38 97.62 (± 1.12)
0.047875 478.75 96.90 (± 1.17)
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In vivo

The data on ASC1 cytotoxicity obtained in a two-dimensional model of fibroblast culture was developed using larvae in vivo model. Concentration A (0.383% ClO2) displayed rapid cytotoxic activity (exceeding the activity of highly concentrated ethanol), while concentration B (0.002992% ClO2) did not lead to a significant drop in viability of larvae between hours 0 and 72 from the injection. After that time, c.a. 20% decrease in larvae viability was observed (Fig. 5).

Fig. 5.

Fig. 5

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(A) Average survival of larvae treated with concentration A (0.383% ClO2) or B (0.002992% ClO2) of ASC1, PHMB, PBS, and 70% ethanol (B) larvae treated with concentration B of ASC1 display full turgor, mobility and viability. (C) Larvae treated with concentration A of ASC1 after two hours stopped moving and started to vomit red-colored liquid. Within the next hours these larvae became melanized and did not react to physical stimuli. Average [%] survival of larvae treated using concentration A or ethanol already after 1 h post-exposure was 0%; [%] survival of larvae treated using concentration B after 120 h was 75%]; [%] survival of larvae treated using 0.1% PHMB + betaine or PBS was 100%.

Discussion

ClO2 is known for its antimicrobial, antiviral, and antifungal activity when applied in controlled range of concentrations between 5 and 8000 ppm1,23,24,28. Numerous researchers explored its potential in in vitro, in vivo studies and clinical trials17,24–35. Also advocates of MMS assert its capability to treat a range of illnesses. However, such claims lack empirical support regarding both its safety and therapeutic effectiveness.

Herein, we aimed to investigate antimicrobial/antibiofilm activity and cytotoxicity of ASC, a source of ClO2 (Tables 1, 2, 3 and 4, Figs. 1, 2, 3 and 4). In our study, ClO2 was obtained by acidifying SC using either an inorganic or an organic acid (ASC1, ASC2, respectively).

The lowest MIC of 0.002992% (29.92 ppm) was observed against MSSA and E. coli when ASC2 was applied. For ASC1, the same MIC value of 0.002992% (29.92 ppm) was observed only against MSSA (Table 1). Both formulations at this concentration showed acceptable levels of cytotoxicity but did not exhibit stronger antimicrobial activity than PHMB (the clinically used antiseptic agent) or the 5 and 20 ppm ASC solutions used by Ma et al.28 (Table 4). The discrepancies observed may be related to either the different strains tested in this work or the ClO2 production method, as the electrolytic method reduces impurities in the solution, potentially affecting its antimicrobial efficacy28,34.

Conversely, MIC of PHMB was higher than ASC2 towards P. aeruginosa and L. casei and of ASC1 towards P. aeruginosa. Our results are consistent with results of Morino et. al., which have shown the effectiveness of extremely low concentration (0.01 ppm, 0.028 mg/m3) ClO2 against P. aeruginosa and E. coli36. In the case of E. coli and L. casei, ASC2 displayed a higher antimicrobial effect than ASC1. This may indicate that gluconic acid may have an additional role in the antibacterial effect.

Björnsdóttir et al. have used gluconic acid as a non-inhibitory low-pH buffer while investigating the acid-resistance of E. coli, however Eisenberg et al. revealed that gluconate induces the Entner-Doudoroff pathway activity and catabolism of glucose in E. coli which could additionally disturb the functioning of bacterial cell and reduce its viability37,38. Gluconate-induced pathway of glucose catabolism in L. casei is unconfirmed as far. The observed reduction in L. casei number may indicate that when drinking, ASC solution may disturb the gut microbiome and speculatively vaginal microbiota as well. All biofilms were more tolerant to applied compounds than their planktonic counterparts (Tables 1 and 2)39,40. S. mutans and E. coli biofilms were the most susceptible to ClO2. Accordingly, Herczegh et al. showed that 0.0015% and 0.003% solutions of hyper-pure ClO2, obtained with dedicated methods, are promising preventive and therapeutic adjuvants in dental practice as they reduced the number of S. mutans in saliva samples collected from patients41. The susceptibility of E. coli biofilm to ASC2 may be related to the activation of the Entner-Doudoroff pathway as mentioned above38.

SC was more effective against the biofilm of S. mutans than ASC1 and ASC2 (Table 2, Fig. 2), which stays in line with reports showing that biofilm formation and matrix gene transcription can be stimulated by sublethal (4 μg/ml; 150 ppm) doses of ClO242,43.

It was shown that L. rhamnosus biofilm displayed high susceptibility to sub-MBEC (0.09575%, 957.5 ppm) which may suggest that consuming even low amount of ClO2 may interfere negatively with microbiota (Fig. 2). However, our results encroach on Ahmed et al. observations indicating that ClO2 in concentrations 0.05%; 500 ppm and 0.1%; 1000 ppm did not affect the Lactobacillus spp.44.

The different species within Lactobacillus genera display various susceptibility to 75 ppm and 125 ppm ASC which have been explained by various adaptations to fermentative process conditions and so, different resistance to antimicrobial agents45.

The certain discrepancy observed at MBEC (Fig. 2), where green-dyed cells remain in the fluorescence microscopy data despite complete eradication of biofilms observed in the 96-well microplate model (Table 3), may be partially attributed to the differences in the experimental setup. The microplate model utilizes a 96-well plate format, while the fluorescence microscopy was conducted in a 24-well plate. Although the same concentrations of ASC1 and ASC2 were applied in both setups, scaling these concentrations 1:1 between the two models may only serve as an estimation rather than a precise match. Differences in the surface area-to-volume ratios, diffusion dynamics, and overall biofilm density between the 96-well and 24-well plates could influence how the antiseptics interact with the biofilms46. In particular, the larger well size in the 24-well plate may result in more uneven distribution of the antiseptic or altered biofilm growth patterns, allowing some cells to survive. Nonetheless, despite these differences, the results from both models were largely consistent, with the fluorescence microscopy data supporting the findings from the microplate model to a significant extent. This consistency reinforces the validity of the MBEC values derived from the microplate model, though it also highlights the need to account for such scaling factors when comparing data across different experimental platforms.

S. mutans biofilm was also the most resistant to ASC1 in the investigation of biofilm eradication from hydroxyapatite surface investigation (Fig. 3A–C). It is known that S. mutans can grow in continuous cultures in pH values of 4.5–5.0 however, this microorganism can survive brief periods of extreme acidification (pH 2.5). Gram-positive microorganisms demonstrated higher resilience to ClO2 than Gram-negative ones (Fig. 3) which may be explained by the enhanced stability of the membrane structure and the mechanical stability of their cell walls47,48. A high resistance to ClO2 (0,03%) was also described in the case of other Gram-positive Bacillus subtilis vegetative forms isolated from a washer‐disinfector49.

The biofilm formed by E. coli and L. casei was the most susceptible to ASC1 (Fig. 3B, C). Although ASC1 is effective against biofilm formed by Gram-negative species, a higher eradication effect was noted against E. coli than P. aeruginosa biofilm, confirming the results obtained in previous methods and proving that bacterial species differ in susceptibility to antimicrobials even if they share similar cell structures that form cell wall (Table 2, Figs. 2 and 3B). However, a relatively high level of biofilm eradication was observed also in the case of P. aeruginosa biofilm which is opposite to MBEC results. This may be related to the structure of the biofilm formed by this species. P. aeruginosa biofilm consists of densely packed populations of cells embedded in tick, mucosal extracellular polymeric substances (EPS)50. This can affect the detachment of large cell aggregates from the structure of the biofilm during the procedure performance. Therefore, the results obtained in this step of investigation may be burdened with a bias arising from the methodology applied51. The fact that L. casei was eradicated more effectively from HAP surface than L. rhamnosus biofilm, confirms that the intrinsic susceptibilities of microorganisms to disinfectants vary widely [B]. Nevertheless, all tested microorganisms formed robust biofilm structures on HAP surfaces (Fig. 4) what may satisfactorily explain the high differences between recorded MIC and MBEC values.

It is believed that the duration of acidification of SC has an impact on its biological activity3. In this study, we have shown that after 180 s of acidification, effective concentrations of ASC were 2—8 times lower against MSSA and P. aeruginosa biofilms, respectively, compared to the situation when 60 s of acidification was applied. This result may influence the production of ASC solution for medical purposes20–24. To make ClO2, an acid is mixed with chlorite, which slowly releases the gas. The reaction normally requires high acidity (low pH) which is irritating for human tissues. Also in this study, we have observed a concentration-related increase of cytotoxic effects in both in vitro and in vivo analysis (Table 4, Fig. 5) after the use of antimicrobially-active ASC concentrations. While the G. mellonella model is a widely recognized model for toxicity evaluation, methodological parameters implemented into this research provide data on subchronic toxicity the limitation of this study is the absence of long-term toxicity assessments on mammalian cells52–54. Therefore, the use of obtained cytotoxicity data should be approached with caution due to physiological differences between insect and mammalian systems. A robust summary concerning the overall perspective on the toxicology of chlorine dioxide and chlorite is available on the Agency for Toxic Substances and Disease Registry website (Atlanta, USA)55. Nevertheless, based on the data obtained it may be hypothesized that choosing mild acid with a pH closer to neutral and prolonged acidification time may result in a product optimized for use on the body. Interestingly, the rapid death (within 2 h) of larvae after application of ASC (Fig. 5) was effect even harsher than level of cytotoxicity observed after exposure of fibroblast cell line to ASC.

Our results indicate that it is difficult to establish a precise antimicrobial dose of ASC while being effective against pathogens, safe for tissues and sparing probiotic species. We have shown that the dosage recommended by alternative medicine proponents cannot lead to the achievement of safe and therapeutic concentrations. At the same time, taking an ASC solution with a pH of 2.5 several times a day can led to damage to the mucous membrane of the gastrointestinal tract. Taking into consideration existing research results concerning ClO2 it can be noted that it has the potential to be used for medicinal purposes, but more investigation is needed to develop safe methods of its application and to evaluate a factual benefit/risk ratio.

Materials and methods

The following 7 solutions were applied:

  • A.

    Hydrochloric acid (HA), (5%, Chempur, Poland)

  • B.

    Gluconic acid (GA), (50%, Chempur, Poland)

  • C.

    Sodium chlorite (SC), (25%, Chempur, Poland)

  • D.

    Prontosan—0.1% polihexanidine (PHMB, B.Braun, Germany)

  • E.

    ASC1—acidified sodium chlorite solution acidified with HA

  • F.

    ASC2—acidified sodium chlorite solution acidified with GA

SC (25%) was diluted in a ratio of 1:1 with 5% HA (ASC1) or 50% GA (ASC2). After 60 s of activation, deionized water was added to interrupt the hydrolysis process. The final ASC1 working solution contained 0.53% SC and 0.16% HA (pH = 2.5). ASC2 contained 0.53% SC and 1.6% GA (pH = 3.12). The concentration of ClO2 in ASC1 and ASC2 was 0.383% (3830 ppm). Control solutions of 0.53% SC (pH = 7.86); 0.16% HA (pH = 1.63); 1.6% GA were prepared (pH = 2.86). pH measurement was performed with Pehametr Elmetron CPI-505 (Emetron, Poland).

The research was performed on reference strains (American Type Culture Collection (ATCC, Manassas, VA, USA; and Polish Collection of Microorganisms (PCM, Poland)):

  • Staphylococcus aureus ATCC 6538—(MSSA—Methicillin sensitive)

  • Staphylococcus aureus ATCC 33,591—(MRSA—Methicillin resistant)

  • Enterococcus faecalis ATCC 25,212

  • Lactobacillus rhamnosus PCM 489

  • Lactobacillus casei PCM 2639

  • Pseudomonas aeruginosa ATCC 15,442

  • Escherichia coli ATCC 25,922

  • Streptococcus mutans ATCC 25,175

Evaluation of the minimal inhibitory concentration (MIC)

The standard serial microdilution method, performed in a 96-well plate, was applied to compare the antimicrobial activity of analyzed solutions against tested reference strains. The solutions’ antimicrobial activity was compared to the activity of PHMB (positive control C +). The following microbial strains growing on agar plates were transferred to liquid Tryptone Soy Broth (TSB; BTL, Poland): S. aureus, E. faecalis, P. aeruginosa, E.coli, strains of L. rhamnosus and L. casei to Man—Rogosa—Sharp (MRS; Biomaxima, Poland) and strain of S. mutans to Brain–Heart Infusion (BHI; Franklin Lakes, NJ, USA) and incubated at 37 °C/5%CO2/24 h. Reduction of colorless TTC to red formazan confirmed the presence of metabolically active microorganisms in the plate’s well. The first well where formazan did not appear was determined as a MIC. Growth and sterility control and control tests with HA, GA, and SC were performed. The highest tested concentrations were HA: 0.08%; GA: 0.08%; SC: 0.27%; PHMB: 0.05%. All procedures were performed in triplicates. To additionally check the correctness of the TTC-based MIC assay, the microbial turbidity studies were performed using Thermo Scientific Multiskan Go spectrometer (Thermo-Fischer Scientific, Finland) with a wavelength of 600 nm. MIC values were expressed as the percentage (%ClO2) and part per million (ppm) concentration of ClO2 obtained in the reaction of SC and acid applied56–59.

Visualization with transmission electron microscopy (TEM)

The samples of MSSA and P. aeruginosa were fixed in glutaraldehyde (POCH, 2.5%) and subjected to centrifugation (5 min, 50 µf). The samples were then passed through an ascending alcohol series and embedded in a medium-hard epoxy resin. After polymerization, ultra-thin sections were prepared on an ultramicrotome (Leica). Sections of 60 nm were prepared from the resin blocks and placed on copper grids (400 Mesh) with formvar film and carbon coating. The contrasting was performed using 2% uranyl acetate (MicroShop, Poland) (10 min) and 2% osmium tetroxide (Agar Scientific, UK) (2 h) as described elsewhere60. Imaging was performed using a JEOL 1200, (JEOL, Japan) transmission microscope, operated at an accelerating voltage of 100 kV. Micrographs were captured at a magnification of 20,000 × to visualize cellular ultrastructure, with an exposure time of 1 s per image using a standard electron beam current optimized for contrast and resolution of bacterial cell walls and cytoplasm.

Evaluation of the minimal biofilm eradication concentration (MBEC)

Suspensions of density 105 of each species were prepared as described in Sect. “Minimal inhibitory concentration evaluation (MIC)”. 200 mL of each suspension was added to the wells of 96-well plate and incubated at 37 °C/5%CO2/24 h. Next, a series of tested compounds’ dilutions in appropriate liquid media were prepared. The highest tested ClO2 concentration was 0.383% (3830 ppm) obtained from both formulations: ASC1 and ASC2. The highest tested concentrations of control substances were HA: 0.16%; GA: 0.16%; SC: 0.53%; PHMB: 0.1%. The analyses were performed as we described earlier61. The first well where formazan did not appear was determined as a MBEC. The absorbance was measured with a wavelength of 490 nm. MBEC values were expressed as %ClO2 and ppm of ClO2. The tests were performed in triplicates.

Visualization of biofilm with fluorescence microscopy (FM) and extraction of quantitative data

Concentrations equal MBEC, < MBEC, and > MBEC of ASC1 were applied against tested biofilms preformed in the wells of 24-well plates. To visualize live cells, positive control of each chosen strain was prepared. Plates were incubated at 37 °C/5%CO2/24 h. Then biofilms were dyed with the mixture of SYTO-9 and propidium iodide (PI) dyes (FilmTracer™ LIVE/DEAD® Biofilm Viability kit, Invitrogen, Ltd., Paisley PA4 9RF, UK) to visualize live and dead cells, respectively62. The biofilm samples were visualized using a fluorescence microscope Lumascope 620 (Etaluma) equipped with a 20 µm magnification Olympus IPC phase objective produced by Shinjuku. The biofilm-forming cells were stained using the BiofilmTracer fluorescent dye mixture (propidium iodide/SYTO-9). Propidium iodide, which selectively stains dead cells with compromised membranes, was excited at 535–540 nm, and its fluorescence was captured at 615–620 nm (red channel). SYTO 9, which penetrates both live and dead cells, was excited with blue light at 480–500 nm, and its fluorescence was detected in the 500–540 nm range (green channel). The biofilm samples were prepared in triplicate wells to account for biological variability. To ensure representative visualization, approximately 12 fields of view per sample were manually surveyed at 20 × magnification across the central and peripheral regions of each well. The six biofilm images/well were processed using ImageJ v. 8 (National Institutes of Health, Bethesda, MD, USA) software. First, RGB images were split into R or G channel sub-images and transformed into 32-bite grey types. Next, the mean grey value (MGV) was obtained from these particular images (with the use of following commends: Analyze- > Set Measurements- > Mean Grey Value- > Measure). The recorded mean grey value was correlated with the value of fluorescence intensity and presented as the combined values of all pixels divided by the pixels’ number. The combined values of all pixels’ intensity were considered 100%, and based on that [%] share of green or red intensity was calculated.

Biofilm eradication from hydroxyapatite (HAP) surface

MBEC concentrations of ASC1 were applied against 24-h old biofilms preformed on hydroxyapatite discs (HAPd) (manufactured as described by Junka et al.) in the wells of a 24-well plate63 in 37 °C/5%CO2/24 h conditions. Then, all samples were stained, and the analyses were performed as we described earlier59. The absorbance was measured with a wavelength of 490 nm.

Visualization of biofilm with scanning electron microscopy (SEM)

MSSA, P. aeruginosa, S. mutans and L. rhamnosus biofilms formed on HAPd were selected for this stage of research. Biofilms were fixed in 2.5% glutamate aldehyde (Carl Roth, Germany) for 24 h/4 °C. Then, the samples were rinsed two times with a 0.2 M cacodyl buffer to remove any residuals. The dehydration process was conducted with increasing concentrations of ethanol (30, 60, 80, 90, and 9.99%). The obtained samples were coated with a 15 nm layer of carbon using a high vacuum carbon coater (ACE 600, Germany) and imaged with the ZEISS Auriga 60 scanning electron microscope (Zeiss, Germany). Imaging was conducted at an accelerating voltage of 5 kV, with a working distance of 8 mm, to optimize surface detail and minimize charging of the biofilm samples. Magnifications were set at 20,000× for S. aureus and P. aeruginosa, and 10,000× for L. rhamnosus and S. mutans, with a dwell time of 50 µs per pixel to ensure high-resolution imaging of biofilm structures59,64.

Evaluation of antibiofilm activity of ASC after different activation time

SC (25%) was diluted in a ratio of 1:1 with 5% HA or 50% GA. At this stage different acidification times were applied: 60 s, 120 s, 360 s before distilled, deionized water was added to tested mixtures. Next, all steps of this stage of the study were performed as described in section "Evaluation of the minimal biofilm eradication concentration (MBEC)" 61.

Cytotoxicity of tested solutions

In vitro—towards L929 fibroblast cells

The standard neutral red cytotoxicity test was performed. A 100 mL of suspension of fibroblasts L929 (American Type Culture Collection, Rockville, MD, USA) of 105 cells/mL density was added to 96-well plate and incubated for 24 h/37 °C. Next, the medium was removed and MIC and MBEC concentrations of ASC1 and ASC2 were poured (100 µL/well)65.

In vivo—in model of Galleria mellonella larvae

The in vivo model of Galleria mellonella larvae was applied to perform analysis of cytotoxicity of ASC1 in A- 0.383% and B- 0.002992% concentrations. The control setting was the PHMB, 75% ethanol (ChemPur, Poland) and Dulbecco’s Phosphate Buffered Saline (PBS; Biowest, Riverside, MO, USA). The G. mellonella larvae in a stadium of sixth instar (weight of 0.21 g), were selected. 20 µL of compounds mentioned above were injected into the larvae. Each compound was injected totally into 20 larvae in two separate experiments (2 × 10 larvae). The larvae were then incubated, and their mortality was monitored after 2 h from injection and subsequently every 24 h up to 120th hour. Death was stated when the larvae were nonmobile, melanized, and did not react to physical stimuli66,67.

Statistical calculations

The statistical analyses were conducted using GraphPad Prism 8.0. (GraphPad Software, San Diego, CA, USA). The normality of distribution was checked with Shapiro–Wilk’s test, then he Mann–Whitney U test was performed with a significance level (α) set at 0.005. Results were considered statistically significant when p-values were less than or equal to 0.005.

Author contributions

R.D.W—conception, design, methodology, investigation, writing—original draft preparation, M.B.—investigation, data curation, J.P.—investigation, data curation, B.M.- resources, data curation, writing—original draft preparation, B.D—methodology, investigation, analysis, visualization; P.M.—investigation, formal analysis, visualization; A.D.J—resources, project administration, writing—original draft preparation; J.F.—validation, project administration, funding acquisition; A.J.—validation, writing—review and editing, supervision. All authors have read and approved the submitted version and agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally.

Funding

This research was supported by the subsidy funds no. SUBK.D230.22.074 and by Wroclaw Medical University statutory grant no. SUBZ.D230.24.001.

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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