Antimicrobial efficiency of chlorine dioxide and its potential use as anti-SARS-CoV-2 agent: mechanisms of action and interactions with gut microbiota (2024)

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Abstract

Introduction

Chemically active antimicrobials and disinfectants are used to control the spread of diseases caused mainly by the presence of pathogenic microorganisms and viruses. The effectiveness of disinfectants varies according to their chemical nature, which makes them effective in inactivating all kinds of viruses in addition to vegetative bacteria, mycobacteria, and bacterial spores. On the contrary, other disinfectants such as sodium hypochlorite and peroximonosulfate are only capable of eliminating vegetative bacteria and most enveloped viruses but are ineffective against nonenveloped viruses, mycobacteria, some fungi, and most bacteria spores (Campagna et al. 2016). Similarly, biofilm-forming bacterial cells have been reported to be between 10 and 100 times more resistant to chemical sanitizers than their planktonic cells, a fact that has represented an enormous challenge for the food industry due to the wide variety of food pathogenic bacteria that have the ability to generate biofilms (Yuan et al. 2021). In this sense, chlorine dioxide (ClO₂) is a strong oxidant agent recognized as an efficient disinfectant at low concentrations (0.1mg mL⁻¹) and a wide pH range (3–8). The applications of ClO₂ as a biocide include mainly the disinfection of drinking water, although its use in ensuring the microbiological safety of agricultural products has gained importance since these products are highly perishable due to the poor pre-harvest management and subsequent contamination by phytopathogenic and human pathogenic microorganisms, conditions that allow them to be identified as potential vehicles for disease outbreaks (Ran et al. 2019, Sun et al. 2019). The ClO₂ is used in different steps of food production, such as processing, transportation, and packaging, reducing the incidence of cholera, dysentery, and typhoid fever, being also used as an effective antiparasitic and antiviral reagent (Ofori et al. 2018, Ran et al. 2019). In post-harvest preservation of foods, the ClO₂ is used to control the presence of undesirable bacteria such as Escherichia coli and Listeria monocytogenes, spore-forming organisms, and toxigenic molds such as Aspergillus flavus and Nosema bombycis (Wang et al. 2010). These effects delay the senescence of strawberries, tomatoes, longan fruit, and other harvested fruits and vegetables (Liu et al. 2020). For instance, it has been demonstrated an efficient reduction in green mold disease produced by Botrytis cinerea in green pepper and winter jujube, positively correlated with the ClO₂ concentration, finding that 150 μmol L⁻¹ of ClO₂ inhibited the spore germination and changed the mycelial morphology (Fu et al. 2019). Similarly, these effects have been also reported in bacterial cultures of E. coli, where the mechanisms encompass inhibition of protein synthesis and oxidation of amino acids (Park et al. 2018).

Historically, ClO₂ achieved more popularity in the 1970s and 1980s; however, during the recent pandemic generated by Coronavirus Disease 2019 (COVID-19), its use has regained importance since it has been considered an effective option for the prevention of severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2) transmission, once it is applied as an oral treatment to reduce infections in humans (Arellano-Gutiérrez et al. 2021, Insignares-Carrione et al. 2021, Medina-Avitia et al. 2021). In March 2020, the World Health Organization (WHO 2023) declared COVID-19 disease a pandemic with 118000 confirmed cases, with 4291 deaths in 114 counties. Currently, the number of confirmed cases has reached 767364883, with 6938353 deaths worldwide until 5 June, 2023 (Burela et al. 2020, Jafari et al. 2022, WHO 2023). Due to the urgent need for COVID-19 treatment, humanity has turned to the development of new treatments to prevent the SARS-CoV-2 infection, including antiviral agents, repurposed drugs, and protease inhibitors to convalescent plasma, and monoclonal antibodies studied in clinical trials. Nevertheless, no licensed but potentially effective disinfectants, such as ClO₂, are used as a drug or medication able to avoid SARS-CoV-2 infection, even when there is no convincing scientific support about its mechanism of action, dose, administration protocol, and possible side effects (Burela et al. 2020, Jafari et al. 2022). The development of dysbiosis (an imbalance in the intestinal microbiota communities) has been one of the side effects reported as a consequence of the oral administration of ClO₂ in animal models and humans (Sultan et al. 2014, Lardieri et al. 2021, Sciurba et al. 2021). In this sense, it is important to consider that the uncontrolled intake of ClO₂ as an anti-SARS-CoV-2 agent can also trigger a malfunction of the gastrointestinal system (characterized by the presence of diarrhea), a decrease in the immune response, and, therefore, a greater severity of the symptoms of COVID-19 disease and a longer recovery time (Zuo et al. 2020). Hence, the present work aims to offer an extensive review of the mechanisms involved in the antifungal, antibacterial, and antiviral activities of ClO₂, as well as discuss the possible effects on host generated by its interactions with the gut microbiota after its oral consumption as a potential anti-SARS-CoV-2 agent.

Chemical and disinfectant properties of chlorine dioxide

ClO₂ is a water-soluble yellow gas with a short life in the air, but is stable as a free radical. ClO₂ is recognized as an attractive biocidal due to its strong oxidizing activity 2.5 times higher than that of diatomic chlorine, over a wide range of pH 3–8 and temperature, against proteins from viruses and bacteria (Kingsley and Annous 2019, Sun et al. 2019, Rubio-Casillas and Campra-Madrid 2021). The use of ClO₂ in the gas form is more effective for the disinfection of fruits and vegetables since it has the potential to reach more areas than liquid disinfectants due to its hydrophobic fruit coatings and possible surface air pockets (Kingsley and Annous 2019).

For drinking water disinfection, a ClO₂ solution is used since its application is more tolerable because it does not generate taste and odors (Medina-Avitia et al. 2021). In drinking water, safe and permissible concentrations of ClO₂ and its by-product, the chlorite ion (ClO₂⁻), have been regulated by different agencies. For example, in the United States of America (USA), the EPA (Environmental Protection Agency) has established maximum concentrations of 0.8 and 1.0mg L⁻¹ for ClO₂ and ClO₂⁻ ions, respectively (Peltzer et al. 2022). Nevertheless, the FDA (Food and Drug Agency) in the USA, and the COFEPRIS (Federal Commission for Protection against Sanitary Risks) in Mexico, have pointed out that the prolongated administration (>90 days) of ClO₂ oral solution at concentrations >40ppm (∼40mg L⁻¹), can cause kidney and liver failure as well as red blood cells disruption (Rubio-Casillas and Campra-Madrid 2021, Cao et al. 2022). In addition to drinking water, the ClO₂ solutions have been successfully used as a disinfectant in fruits and vegetables since the gaseous ClO₂, dispersed in the aqueous solution, can exert higher disinfectant properties due to its ease of penetrating bacterial cells in comparison to sodium hypochlorite (Lee et al. 2018, Hatanaka et al. 2021). Lately, ClO₂ had been recommended for SARS-CoV-2 inactivation when it is used at concentrations significantly higher than those used in drinking water disinfection. For example, disinfection of domestic areas can be achieved applying a ClO₂ concentration of 20mg L⁻¹, whereas solutions over 500mg L⁻¹ are useful for disinfection of hospital and health care units (Malka and Park 2022, Peltzer et al. 2022).

The limitations for the use of ClO₂ as a disinfectant in food products and domestic or clinical surfaces are mainly based on its reactive nature related to its oxidizing capacity, which can make it toxic for human health at higher concentrations (Malka and Park 2022). Conversely, in Latin American countries the use and commercialization of ClO₂ solutions as a treatment and preventing therapy against SARS-CoV-2 infection increased in the last three years. This context represents an opportunity to venture into a new research field related to the emerging anti-SARS-CoV-2 agents, which must be studied and evaluated according to their antiviral effectiveness, as well as their impact host health (Peltzer et al. 2022). Therefore, it is necessary to describe the antimicrobial mechanisms of ClO₂ (specifically those involved in the SARS-CoV-2 inactivation), as well as its interactions with the gastrointestinal tract and gut microbiota (recognized as a modulator of the host immune response) once it is orally consumed as a therapeutic agent against SARS-CoV-2. This information may contribute to understand the effectiveness of ClO₂ as an antiviral agent and identify its safety under specific physiological conditions of host. Latest information related to this topic is given below.

Antimicrobial mechanisms of chlorine dioxide

Chemical disinfectants can be categorized into three classes according to their mechanisms of action, either as denaturants, reactants, and oxidants. Denaturing disinfectants include those substances that disrupt mainly protein and lipid structures from lipophilic enveloped viruses, such as, for example, quaternary ammonium salt compounds, phenolics, and alcohols. On the other hand, reactants (including aldehydes and ethylene dioxide) form and break covalent bonds, altering nucleic acids (DNA and RNA), and protein structure and synthesis. Finally, oxidants are the largest group used for disinfection and include peroxides, peroxymonosulfates, and halogens such as hypochlorite, iodine, and ClO₂. These disinfectants destabilize cell membranes, react with amino acids, interrupt protein synthesis, and oxidize proteins, enzymes, and possible DNA/RNA/proteins, making them useful for the elimination of a broad spectrum of pathogenic microorganisms (Campagna et al. 2016, Sun et al. 2019). The antimicrobial mechanisms of ClO₂ have not been deeply studied, but most of them are based on the generation of oxidative stress and the subsequent excessive production of reactive oxygen species (ROS), which finally cause cell injury (Medina-Avitia et al. 2021).

As mentioned, the mechanism by which ClO₂ inactivates microbial species encompasses selective oxidation, resulting in the formation of ClO₂⁻ and chlorate (ClO₃⁻) ions as the main by-products, with chlorite constituting ∼70% of the total by-products of the reaction (Ofori et al. 2018). Moreover, ClO₂ releases oxygen when it encounters acidic environments (generated by lactic acid or acidity of pathogens), becoming alkaline due to the elimination by oxidation of acidifying unicellular organisms, which are unable to tolerate and dissipate the electromagnetic overload (Insignares-Carrione et al. 2020). One of the main advantages of using ClO₂ as a possible therapeutic agent against different kinds of infections is the impossibility of resistance by bacteria and viruses, due to the oxidative stress it generates, which selectively affects compounds such as phenols and thiols, the latter present in amino acids such as cysteine, tryptophan, tyrosine, proline, and hydroxyproline, essential for viability and activity of the undesirable microorganisms (Loginova et al. 2008, Stewart et al. 2008, Insignares-Carrione et al. 2020). Also, in comparison to chlorine, ClO₂ does not chlorinate organic compounds to produce carcinogenic trihalomethanes and does not react with ammonia to form chloramines. Both advantages make ClO₂ very attractive for its use as an antimicrobial agent in foods (Kingsley and Annous 2019).

The mechanisms by which oxidation of vital molecules occurs are not fully understood and are dependent on the species of microorganism and its biochemical characteristics, as well as its composition (Fig.1). Generally, gaseous ClO₂ is less effective against Gram-positive than Gram-negative bacteria, while commonly molds and yeasts have an intermediated tolerance (Sun et al. 2019). Therefore, in the following lines, the antiviral, antifungal, and antibacterial activities of ClO₂ are listed, including their mechanisms of interaction and/or action.

Antibacterial properties

As reported by Singh et al. (2021), the gaseous form of ClO₂ has offered greater effectiveness for disinfection than the liquid form of ClO₂, since gaseous ClO₂ reaches and inactivates pathogenic bacterial cells because of its high diffusivity and penetrability, especially when it is used to obtain a safer food packaging. The antibacterial efficacy of ClO₂ is related to the destabilization of cell membranes, mainly by metabolism disruption after reactions between oxygenated compounds with ClO₂. Once ClO₂ penetrates the bacterial cell membranes, it can cause respiration inhibition and deterioration of the trans-membrane ionic gradient. In addition, gaseous ClO₂ can increase the permeability of the cell membrane by changing the spatial formation of lipids and proteins in the intermembrane space. Once inside the bacterial cell, ClO₂ oxidizes organic compounds, initiating cell metabolism interruptions, mainly bacterial protein synthesis derived from the oxidation of amino acids. Subsequently, in oxidated amino acids (cysteine, tyrosine, tryptophan, histidine, and proline), the ClO₂ forms disulfide analogs after oxidation of mercapto groups of enzymes, and finally a malfunction is observed in the physiological functions in those enzymes (Sun et al. 2019, Al-Sa'ady et al. 2020, Singh et al. 2021). For sporulated bacteria, such as Bacillus subtilis, the exposition to ClO₂ resulted in either the inactivation of spores or spore formation inhibition, both related to membrane damage (Sun et al. 2019).

Although its main application consists of drinking water disinfection, gaseous ClO₂ dissolved in water, has been successfully used against pathogenic bacteria of clinical origin. According to this, the use of ClO₂ has shown a higher antibacterial activity against Streptococcus pneumoniae and Klebsiella pneumoniae (isolated from sputum), Enterobacter aerogenes (isolated from urine), and methicillin-resistant Staphylococcus aureus (isolated from wound infection), in comparison with pure chlorine (Cl₂), which was 4–7 times less effective than ClO₂, against these same bacterial species (Al-Sa’ady et al. 2020).

The low resistance of Gram-negative bacteria to gaseous ClO₂ is boosted by the presence of a thin layer of peptidoglycan that is more easily penetrated by gaseous ClO₂ (Sun et al. 2019). In the food industry, the use of ClO₂ has evolved beyond its application as a sanitizing gas. For example, a custom-made antimicrobial film coated with sustained-release ClO₂ microcapsules in fresh mangos packaging (Zhang et al. 2019). The authors of the previous report observed that control mangos (without antibacterial ClO₂ film), showed a loss of edible and commercial value after 21 days of storage, while the mangos packaged with the antimicrobial ClO₂ film still retained edible and commercial value at the same storage time. These findings exhibit a potential use of solid ClO₂ for fresh food (mainly fruits and vegetables) preservation, based on its antimicrobial characteristics. In the same way, it has been reported that ClO₂ gas can reduce the bacterial load of L. monocytogenes (ATCC 19111, ATCC 19115, and ATCC 15313), E. coli O157:H7 (ATCC 35150, ATCC 43889, and ATCC 43890), and Salmonella Typhimurium (ATCC 19585, ATCC 43971, and DT 104), inoculated on fresh tomatoes surface, observing that when the ClO₂ gas concentration, relative humidity (RH), and treatment time increase, inactivation levels also reduced the bacterial load below the detection limit (0.48 log CFU per cm²), either within 15min of exposition to 30 ppmv of ClO₂ gas at 70% RH and within 10min at 90% RH at the same ClO₂ gas concentration (Park et al. 2018). In this sense, it is possible to alter the effectiveness of ClO₂ by modifying intrinsic factors such as sample surface characteristics or food product characteristics, and extrinsic factors including gas concentration, RH, and treatment time (Park and Kang 2018).

Secondly, the ClO₂ gas has been also used to sanitize food contact surfaces by the inactivation of the biofilm cells of foodborne pathogens. For example, an exposure time of 20min to 50 ppmv of ClO₂ gas at 60% of RH, resulted in log reductions from 2.08 to 4.62 and 2.08 to 4.41 of the biofilm cells of three pathogenic bacterial strains (E. coli O157:H7, S. Typhimurium and L. monocytogenes), on stainless steel and high-density polyethylene surfaces. While, with 50 ppmv of ClO₂ gas at 90% of RH, the levels of biofilm cells of all mentioned pathogens were reduced to below the detection limit [0.48 log CFU (cm²)⁻¹], after 20min exposure (Kim and Park 2022). Therefore, and based on the previous results, to establish an optimum ClO₂ gas treatment for the disinfection of both fresh fruits and vegetables as well as food processing facilities, environmental conditions such as RH, must be considered to maximize the antibacterial efficacy of ClO₂ (Kim and Park 2022).

Contrary to what was expected, there are currently no reliable reports about the impact of the use of ClO₂, either as an aqueous solution or as a gas, on those desirable bacteria in food, such as lactic acid bacteria and/or probiotics (e.g. Lactobacillus sakei subsp. carnosus and Leuconostoc mesenteroides subsp. mesenteroides), which, according to the constitution of their cell wall, they could suffer less damage than Gram-negative bacteria once they interact with this disinfectant. Differences in tolerance between Gram-positive and Gram-negative bacteria to ClO₂, in a gaseous form, are based on the structural constitution of their cell wall. While Gram-negative bacteria have a thinner peptidoglycan layer, easily penetrated by gaseous ClO₂, Gram-positive bacteria show greater resistance since their peptidoglycan layer is thicker and more difficult to penetrate by the same gaseous ClO₂ (Vandekinderen et al. 2009, Sun et al. 2019). It should be noted that in the last two years, the intake of ClO₂ has become popular due to its potential activity as an anti-SARS-CoV-2 agent at a concentration of 2.19mg L⁻¹ (Peltzer et al. 2022). This activity and the recurrent consumption of ClO₂ suggest the presence of interactions of the sanitizing agent with the intestinal microbiota, including probiotics, potentially probiotic lactic acid bacteria, and other beneficial Gram-positive bacteria. However, the outcome of these interactions and their possible health effects are not fully understood or are still under analysis.

Antifungal properties

As part of its antifungal activity, the ClO₂ can inhibit the growth of Penicillium digitatum, using as a mechanism of action the destruction of membrane integrity of fungal cells (Liu et al. 2020). However, other alterations such as the promotion of apoptosis and changes in the membrane potential were observed as a response to the diffusive transport of ClO₂ into the cell. Once inside the fungal cell, the ClO₂ promotes significant levels of protein release, lipid peroxidation, changes in cell permeability, and hence the exudation of cell components. Also, in this same study, ClO₂ was related to different negative effects on mitochondrial functions, including abnormal membrane potential, compromising microbial respiration, and promoting the loss of cell viability. Treatments consisting of short exposure times and high concentrations of ClO₂ have been investigated to determine the optimum conditions for fungal inhibition through in vivo assays. An example of this is the complete inhibition of mycelial growth of Alternaria alternata and Stemphylium vesicarium after 3min of treatment with 10mg mL⁻¹ of gaseous ClO₂, observed on the fresh tomato peel surface (Trinetta et al. 2013). Meanwhile, this same ClO₂ concentration was used in an in vivo study in which complete inhibition of A. alternata and S. vesicarium, previously inoculated on the fresh Roma tomatoes peel surface, was achieved after 7 and 5min, respectively. As can be seen, the efficacy of ClO₂ can be reduced in in vivo studies since, in some cases, its application is carried out in low RH environments (RH≤70%), which decreases its diffusion and subsequent penetration into the microbial cell (Park and Kang 2018, Park et al. 2018). In comparison, in vivo reduction of fungal populations by ClO₂ can be more efficient at RH of 90% than 70%, because at high humidity spore swelling occurs and, thus, the size of openings available to ClO₂ molecules increases, resulting in a greater antimicrobial efficacy (Sun et al. 2019). Further, the use of ClO₂ is not limited to fresh fruits, but also it has been used as an antifungal agent for fruit and vegetable packing house surfaces, being effective in the reduction of Botrytis cinerea, Penicillium expansum, Mucor piriformis, and Cryptospotiopsis perennas by spore inactivation after 1min exposure to 3–5μg L⁻¹ ClO₂ aqueous solution, while effective control of Stachybotrys chartatum, Chaetomium globosum, Penicillium chrysogenum, and Cladosporium cladosporioides was observed at 1.4–1.8μg L⁻¹ ClO₂ solution after 24h treatment (Trinetta et al. 2013).

As for the yeasts, the mechanism of action of ClO₂ is like that exerted in bacteria cells. An alteration in cell structure, as well as an inhibition of key enzyme activities of metabolic pathways, was observed in Saccharomyces cerevisiae strain 13482 after exposure to 100mg L⁻¹ of ClO₂. Moreover, ion leakages of K⁺, Ca⁺, and Mg⁺ were detected and related to the inactivation rate. Nonetheless, protein and DNA leakage were not observed during and after exposure to ClO₂ (Zhu et al. 2013).

Antiviral properties

The antiviral activity of ClO₂ has been well documented, specifically against echoviruses, enteroviruses, polioviruses, noroviruses, caliciviruses, mumps virus, and coronaviruses (in which SARS-CoV-2 is included) (Wang et al. 2005, Jin et al. 2013, Montazeri et al. 2017, Zhong et al. 2017). The antiviral activity of ClO₂ has been tested in vitro against human and animal viruses; for example, human influenza was inactivated by 99.99% at 15s using 1ppm, while with 10ppm, the inactivation of Measles and Herpes viruses was achieved at 30 and 15s, respectively (Sanekata et al. 2010). Similarly, ClO₂ concentrations from 0.2 to 10ppm were used to inactivate or destroy poliomyelitis and hepatitis A viruses, rotavirus, human immunodeficiency virus type 1 (HIV-1), and coronavirus that causes SARS (Rubio-Casillas and Campra-Madrid 2021). In foods, it has been demonstrated that constant low levels of ClO₂ (0.63ppm-h g⁻¹) can inactivate foodborne viruses such as Tulane virus, a human norovirus surrogate, on the surface of different berries (blueberries, raspberries, blackberries, and strawberries). Interestingly, the use of a direct constant level of ClO₂ allows equal treatments regardless of the size of the berry, since all the samples treated with ClO₂ showed the same reduction of 2.9 PFU (plaque-forming units) g⁻¹ of virus load in all the samples (Kingsley and Annous 2019).

In addition to the doses used for virus inactivation, other factors such as temperature and pH have an important role in the viricidal activity of ClO₂. For example, Jin et al. (2013), reported the inactivation of enterovirus 71 (EV71) in drinking water samples, using a 4.24–6.62mg L⁻¹ min⁻¹ of ClO₂. However, the inactivation of EV71 was temperature- and pH-dependent, with a maximum EV71 reduction of 4-log when the water samples were treated with the mentioned ClO₂ concentrations at 36°C and the pH adjusted to 8.2. Furthermore, damage in the EV71 genome was observed because of ClO₂ exposure. Through an RT-PCR analysis, it was possible to identify damage on the 5′ non-coding region (1–118nt length), which is closely related to the viral infectivity. EV71 is an enteric human virus that spreads via the fecal–oral route and can be excreted from an infected individual to aquatic environments and spread to drinking water. In infected individuals, the EV71 is associated with hand, foot, and mouth disease, aseptic meningitis, encephalitis, and poliomyelitis-like paralysis (Solomon et al. 2010, Plevka et al. 2012). Thus, the EV71 inactivation in drinking water is an important feature to consider in the development of new antiviral agents.

ClO₂ inactivates virus by the denaturation of envelope proteins responsible for cell entry, a mechanism based on the oxidation of amino acids, mainly thiol (-SH) residues of tyrosine, threonine, and tryptophan. So, these covalent oxidative modifications trigger the denaturation of proteins from the viral envelope decreasing the viral infectiveness (Al-Sa'ady et al. 2020, Rubio-Casillas and Campra-Madrid 2021). An example of this is the inactivation of influenza virus by ClO₂ through oxidation of tryptophan residue (W153) in hemagglutinin (a spike protein of the viruses), thereby suppressing its receptor-binding capacity (Káli-Kullai et al. 2020). In addition to the antiviral effect previously described, ClO₂ has also demonstrated control and modulation over the symptomatic response generated by a viral infection. For example, an in vitro study carried out by Zhu et al. (2019), showed that ClO₂ can inhibit the synthesis of pro-inflammatory molecules (IL-1, IL-6, and TNF-α), derived from the exposition of pig alveolar macrophages and MARC-145 cells to porcine reproductive and respiratory syndrome virus (PRRSV1). In this study, the authors concluded that ClO₂ can reduce the viral replication of PRRSV1 by interrupting the synthesis of RNA and proteins, and thus, the pathogenesis of this disease is diminished. These findings suggest that the ClO₂ can not only be used as a useful agent to prevent viral infections, but also as an adjuvant in the control of the disease generated once the infection is present in the host.

Nowadays, the humanity is adapting to the “new normality” once cases of SARS-CoV-2 infections worldwide have been brought under control; however, during the period of confinement, alternative treatments using emergent anti-infective agents, including ClO₂, were explored. Specifically, ClO₂ gained popularity by being considered an anti-SARS-CoV-2 agent administered orally as an aqueous solution, even though its effectiveness and its possible side effects were not fully identified or verified. In this context, in the following lines, current information about the anti-SARS-CoV-2 efficiency of ClO₂, as well as its possible side effects derived from its interaction with the gut microbiota, once it is orally consumed, are described.

Effect of chlorine dioxide on SARS-CoV-2

As mentioned above, the main mechanism by which ClO₂ exerts its antiviral properties is related to its ability to denature the viral envelop proteins responsible for the cell attachment, preventing the subsequent replication, and host infection. In relation to SARS-CoV-2, previous computational studies using the Chemistry at HARvard Macromolecular Mechanics (CHARMM) (C36) force field (FF), have demonstrated, through molecular optimization and dynamics, a potentially anti-SARS-CoV-2 effect of ClO₂ based on its denaturing effect specifically on the disulfide bonds, formed between the spike protein of SARS-CoV-2 and ACE-2 (Angiotensin-converting enzyme type 2) receptors, located in the human alveolar cells (Hati and Bhattacharyya 2020, WHO 2020a). Such simulations, based on molecular structure, carried out to study protein structure–dynamics–function relationships, allowed to predict possible interactions of ClO₂ with the amino acids responsible for the formation of disulfide bonds. Based on the 54 tyrosine, 12 tryptophan, and 40 cysteine residues present in the spike protein of the SARS-CoV-2. Computational 3D simulations based on the data obtained from electron cryomicroscopy and manipulated with the ChimeraX (UCSF) augmented reality software, allowed to identify to the cysteine residues (Cys480–Cys488) as the target residues to be oxidize by ClO₂ to prevent the binding between SARS-CoV-2 spike protein and the ACE-2 receptor (Insignares-Carrione et al. 2020). Thus, the oxidative behavior of ClO₂ emerges as a therapeutic mechanism by which the formation of disulfide bonds and binding between the ACE-2 and spike protein of SARS-CoV-2 could be prevented, attenuating the incidence of COVID-19 (Insignares-Carione et al. 2021). In this regard, it was confirmed that the binding capacity of purified spike proteins to ACE-2 human receptors was inhibited by 50% in a concentration-dependent manner with concentrations of 7.6 and 5.8 μmol L⁻¹ of ClO₂ for the SARS-CoV-2 type variants from England (variant B.1.1.7) and South Africa (variant B.1.351), respectively, indicating that ClO₂ can be a useful strategy in blocking the transmission of not just a specific SARS-CoV-2 variant (Ogata and Miura 2021). Commonly, aqueous solutions of sodium hypochlorite are used as an anti-SARS-CoV-2 agent; however, the use of hypochlorite for human administration is not approved and is very controversial. So, it has been reported that ClO₂ gas dissolved in water can be a up to 10 times stronger antiviral agent against SARS-CoV-2 than sodium hypochlorite. This fact was observed by Hatanaka et al. (2021), who used both disinfectant agents in the treatment of VeroE6/TMPRSS2 cells infected with SARS-CoV-2, observing inhibition of 99.99% of the virus within 10s of exposure to a solution of 24ppm of ClO₂. Conversely, a solution of 24ppm of sodium chlorite only inactivated from 90 to 99% of the virus after 3min of exposure to ClO₂ under similar conditions. Additionally, the antiviral activity of ClO₂ for the treatment of COVID-19 disease has been already evaluated in a clinical study carried out by Insignares-Carrione et al. (2021). In this research, the antiviral activity of ClO₂ was evaluated in 20 patients with active infection of SARS-CoV-2, who received a daily dose of a ClO₂ solution at 30mg L¹ for 21 days. The results obtained were compared to those of a control group (20 patients who no received the oral ClO₂ solution). At the end of the 14th day of the study, symptoms in infected patients such as fever and chills decreased in their entirety, while cough and dyspnea decreased by 60 and 75%, respectively, in comparison with the control group. In summary, the studies previously discussed highlight the use of aqueous solutions of ClO₂, at controlled concentrations, as an alternative for the treatment of COVID-19; although, to offer more information about its effectiveness and safety, deeper clinical studies are still necessary.

Responses of gut microbiota to SARS-CoV-2 infection and chlorine dioxide oral administration

Concerning the foregoing, the following lines discuss the most recent findings related to the gut microbiota responses during SARS-CoV-2 infection and after oral administration of ClO₂.

Gut microbiota response to SARS-CoV-2 infection

Gut microbiota can indirectly mediate both the colonization of non-indigenous microorganisms and viral infections by stimulating host responses such as mucosal immune defenses (Xiang et al. 2021). Therefore, the gut microbiota is closely related to the proper functioning of immune system and can be altered by many immunologic diseases, including COVID-19. However, it is important to mention that gut microbiota composition is highly heterogeneous between individuals of different ages, eating habits, and geographies, thus the following reported findings may not entirely consistent across the different in vitro and clinical studies (Patel and Roper 2021).

Gastrointestinal tract is considered an extrapulmonary site for SARS-CoV-2 infection since receptor ACE-2 is highly expressed in the intestinal enterocyte. So, once the SARS-CoV-2 infects and actively replicates in the intestinal enterocytes, symptomatic gastrointestinal COVID-19 is triggered in the host (Zuo et al. 2021). These facts suggest the possibility that SARS-CoV-2 interacts (directly or indirectly) with the gut microbiota adhered to the intestinal walls, resulting in dysbiotic communities that may persist even after recovery from COVID-19 (Kaźmierczak-Siedlecka et al. 2020, Patel and Roper 2021, Xiang et al. 2021). Thus, SARS-CoV-2 infection modifies the composition of the gut microbiota, which could predispose patients not only to greater severity and mortality of COVID-19, but also to prolonging such symptoms weeks or months after SARS-CoV-2 infection, a phenomenon known as long COVID or post-COVID syndrome (Raveendran et al. 2021). Similarly, the microbial imbalance of gut microbiota can cause that COVID-19 patients have a greater probability of suffering co-infections and super-infections with other respiratory or systemic viruses (Fig.2; Cox et al. 2020).

Meta-analysis indicated that up to 20% of COVID-19 patients had gastrointestinal symptoms, including nausea, vomiting, abdominal pain, and diarrhea (Vodnar et al. 2020, Zuo et al. 2021). Additionally, fecal calprotectin was founded elevated in COVID-19 patients, indicating gut inflammation. These effects are related to gut microbiota modulation during and after SARS-CoV-2 infection; however, it is unknown about the activity of SARS-CoV-2 in the gut or gut microbiota of COVID-19 patients (Zuo et al. 2021). In the gut, the SARS-CoV-2 can reduce the expression of ACE-2 and the number of circulating angiogenic cells, thus endangering the gut endothelium, promoting a dysbiosis and effecting the hots immune response and metabolism (Romani et al. 2022). In this sense, in previous report, Yeoh et al. (2021), compared the microbiota composition of hospitalized COVID-19 and non-COVID-19 subjects, finding a more abundance of phylum Bacteroidetes (23.9%) in COVID-19 patients, while in non-COVID-19 individuals, Actinobacteria (26.1%) were more abundant phylum. It is important to note that these changes in the diversity of the intestinal microbiota can persist even when the symptomatic and hospitalization periods of COVID-19 patients has ended. However, this prolonged imbalance of microbial diversity is not entirely negative, since it has been observed that some species of Bacteroides (such as Bacteroides dorei, Bacteroides thetaiotaomicron, Bacteroides massiliensis, and Bacteroides ovatus), downregulated the ACE-2 expression in colon and thus, play a potential role by blocking host entry, preventing future reinfections by SARS-CoV-2 (Donati Zeppa et al. 2020, Zuo et al. 2020). Nevertheless, during this period of dysbiosis, concentrations of pro-inflammatory cytokines and blood biomarkers (C protein reactive, lactate dehydrogenase, aspartate aminotransferase, and γ-glutamyl transferase, TNF-α, CXCL10, CCL10, and IL10) were elevated, thereby increasing the COVID-19 severity (Vabret et al. 2020). In accordance with the above, significant alterations in fecal microbiomes of COVID-19 patients, in which the abundance of Coprobacillus, Clostridium ramosum, and Clostridium hathewayi have been correlated with COVID-19 severity (Zuo et al. 2020). Additionally, several gut commensal bacteria with known immunomodulatory potential, such as Eubacterium rectale, Faecalibacterium prausnitzii, and bifidobacteria, underrepresented in COVID-19 patients, remaining at low concentrations up to 30 days after disease clearance (Zuo et al. 2020). Therefore, gut microbiota can be involved in the magnitude of COVID-19 severity, either modulating the immune response or increasing the symptoms during and after disease resolution. Based on the previous studies, it is possible to establish that the gut microbiota plays an important role in the pathogenesis process of the SARS-CoV-2 and the eventual illness of COVID-19.

Currently, the existence of a “quiescent” intestinal infection of SARS-CoV-2 has been reported in patients with COVID-19, being characterized by a persisting viral infection and replication at intestinal level and by a higher abundance of opportunistic pathogens such as Collinsella aerofaciens, Collinsella tanakaei, Streptococcus infantis, and Morganella morganii in fecal samples. Based on these findings, it has been postulated that SARS-CoV-2 may be transmitted through the fecal-oral route, even from those individuals with asymptomatic COVID-19 or with a negative respiratory tract but positive fecal SARS-CoV-2 tests (Zuo et al. 2021). However, future research involved in the infectivity and pathogenesis of SARS-CoV-2 in the gastrointestinal tract, as well as measures gut infection level and interactions with gut microbiota, are needed.

Gut microbiota response to oral administration of dioxide during SARS-CoV-2 infection

Nutrition, diet, environmental factors, genetics, medication, and comorbidities play an important role in shaping the diversity of gut microbiota. The low microbial diversity of gut microbiota during dysbiosis or derived from uncontrolled medication are associated with higher rates of serious bacterial and viral infections, including influenza, pneumonia, and SARS-CoV-2. So, improving the profile of gut microbiota can be considered as a prophylactic measure to minimize the impact of COVID-19, mainly in elderly and immunodeficient patients (Vodnar et al. 2020). Mostly, COVID-19 clinical conditions and fatalities occur in elderly people, mainly due to aging-related factors that render the elderly more prone to this disease. Comorbidities such as hypertension, hyperlipidemias, cardiovascular diseases, diabetes, and cancer are commonly related to the consumption of more than five medicines per day, which not only implies an increase in drug side effects but also affects the gut microbiota and gradually decreases the antiviral capacity of the host (Donati Zeppa et al. 2020, Lithander et al. 2020). Research studies have demonstrated alterations in gut microbiota composition at increasing the number of co-administered drugs. For example, the maintenance of relative abundance of pathogenic Helicobacter and reduction of Lachnospiraceae and Succinivibrionaceae (aids in the maintenance of host cardiorespiratory health and regulation of inflammation), were observed in patients using additional drugs during viral infection. So, an increase in mortality in COVID-19 patients may be related to the lowest gut microbiota diversity index (Ticinesi and Milani 2017, Donati Zeppa et al. 2020). Accordingly, the uncontrolled use of anti-SARS-CoV-2 medications not yet authorized, such as ClO₂, can represent a health risk since this disinfectant, rather than eliminate the SARS-CoV-2, alter the diversity of the gut microbiota, ameliorating the immune response of the host, and generating a state of dysbiosis that can increase the severity of the disease (Xiang et al. 2021).

The different surges of COVID-19 have triggered the use of drugs with not enough scientific support and lack of sanitary authorization as alternatives for the effective treatment of COVID-19 worldwide (Burela et al. 2020, WHO 2020b, Lardieri et al. 2021). In this sense, the use of ClO₂ has generated controversy due to its toxic reactions reported in humans exposed to different concentrations (Liester 2021). In animal studies, adverse reactions including oral and digestive irritation, anemia and methemoglobinemia, altered thyroid function, and neurotoxicity have been observed (Rubio-Casillas and Campra-Madrid 2021). Regarding this, the FDA has published a warning about the use of ClO₂ as an anti-SARS-CoV-2 agent, due to its oxidative behavior and the adverse effects after consumption, including vomiting, diarrhea, dehydration, hypotension, abdominal pain, methemoglobinemia, and systemic failures. Nevertheless, several studies have demonstrated that all these symptoms are completely reversible after ClO₂ consumption, highlighting that its toxicity is strongly dose-dependent (Bathina et al. 2013, Romanovsky et al. 2013, Loh and Shafi 2014, Ma et al. 2017, FDA 2020, Lardieri et al. 2021).

Nowadays, gastrointestinal damage symptoms have been reported because of an administration of a liquid solution ClO₂ (FDA 2020, Lardieri et al. 2021). It has been observed that drinking water with concentrations from 1 to 1000mg mL⁻¹ of ClO₂, decreased red blood cell counts, half-life erythrocytes hemoglobin (HEB) concentration, and packed cell volume in animal and in vitro models at 30–60 days of exposure. Likewise, the consumption of ClO₂ has been related to the presence of oxidative stress, since the ClO₂⁻ can oxidize HEB to methemoglobin (mHEB), which suppresses glutathione concentration (an antioxidant mechanism), increasing the levels of the oxidative hydrogen peroxide. At the gastrointestinal level, exposure to ClO₂ affects the gastric mucosa, promoting symptoms such as nausea, vomiting, and abdominal pain (Medina-Avitia et al. 2021).

As mentioned, the SARS-CoV-2 infection causes a prolongated dysbiosis in gut microbiota, which can be postponed until after COVID-19 recovery. This imbalance of the diversity of the gut microbiota can be increased with the consumption of uncontrolled medications, which can mean additional damage to the host’s health. An example of this is the indiscriminate consumption of ClO₂ as a possible anti-SARS-CoV-2 agent and the lack of information about its possible impacts on the intestinal microbiota and host health both during and after COVID-19. There are few studies related to the consumption of ClO₂ and its impact on the composition of the gut microbiota. For instance, a murine study showed that the daily administration of 100ppm ClO₂ tablet to C57BL/6 mice for 28 days generated a greater overall effect on gut microbiota than a 70% ethanol solution, but a lesser effect than the hydrogen peroxide and potassium peroxymonosulfate. In addition, the taxon-by-taxon analysis showed no differences in Firmicutes, Bacteroidetes, or any of the lower abundance phyla, between the cecum microbiota of mice treated with ClO₂ and that of the water control mice. On the contrary, in fecal microbiota, significant differences in lower abundance phyla (Verrucomicrobia, Actinobacteria, and Proteobacteria) were observed between ClO₂ and water-control mice at 14, 21, and 28 days (Sciurba et al. 2021). These results indicate that ClO₂ can be considered a potential anti-SARS-CoV-2 agent with minimal effects on the gut microbiota; however, it is necessary to clarify the maximum concentration allowed to avoid possible side effects that compromise the health of the host.

Toxic and safety aspects of oral administration of chlorine dioxide

Derived from the indiscriminate oral consumption of ClO₂ during COVID-19 pandemic, different regulatory agencies and scientific societies have postulated statements warning about the lack of solid evidence for the efficiency of ClO₂ in SARS-CoV-2 inactivation and modulation of COVID-19 disease symptoms (Andrés et al. 2022). Studies have reported toxic effects of ClO₂ exposition or consumption, either by inhalation, parenteral, or oral routes. Specifically, oral administration of ClO₂ facilitates its absorption at the gastrointestinal level, and once absorbed, maximum peaks levels in blood can be observed within one hour after a single dose is administered. Once in blood, most of the ClO₂ and its derivatives (chlorite, chlorate, and chloride) remain in plasma, followed by kidneys, lungs, stomach, intestines, liver, and spleen, to be finally excreted through urine and feces within 72h after oral consumption (Burela et al. 2020, Soriano-Moreno et al. 2021).

Separately to oral administration, some adverse reactions, such as acute liver failure, arterial hypotension, hydroelectrolytic imbalance, nausea, vomiting, diarrhea, digestive mucosa irritation, respiratory failure, arrythmias, headaches, dizziness, tremors, anxiety, drowsiness, and mainly methemoglobinemia or hemoglobinuria, have been identified as a response to direct exposition to ClO₂ or to its derivatives (Andrés et al. 2022). Methemoglobinemia is characterized by an oxidation of hemoglobin generated by ClO₂. This oxidation turns hemoglobin into methemoglobin, which is uncapable to bind oxygen molecules and thus hinders oxygen transport in the body, resulting in a hypoxia and gradual respiratory failure. Previous reports indicate that hypoxia and methemoglobinemia can be developed either by accidentally ingestion or low doses of ClO₂ (<100mL of 28% ClO₂ solution). However, in both cases, to save the lives of the patients, endotracheal intubation, and kidney transplant, were required, respectively. The endotracheal intubation was required to counteract a severe hypoxia with no response to oxygen therapy, while the kidney transplant, accompanied by blood transfusions, were necessary to maintain constant blood oxygen levels, which dropped due to the development of methemoglobinemia (Romanovsky et al. 2013, Hagiwara and Inoue 2015). Nevertheless, as mentioned previously, the toxic effects of ClO₂ are dependent of several factors, including the physiological state of host, the concentration of ClO₂, and the time of exposition to the agent. Therefore, the possibility of ClO₂ being cytotoxic to humans is low; however, this cytotoxicity can be increased either by mixing with other disinfectants, e.g. chlorine or derived from the presence of its released by-products, including chlorite (ClO₂⁻), chlorate (ClO₃⁻), and chloride (Cl⁻) (Jefri et al. 2022). In animal models, it has been shown that the cytotoxicity of ClO₂ is size- or weight-dependent. For example, a study reported by Ma et al. (2017), demonstrated that ClO₂ concentrations up to 40ppm in drinking water did not show toxicity in mouse lung fibroblasts L929 cells, but antimicrobial and antifungal effects were observed at ClO₂ concentrations of 5 and 20ppm, respectively. Meanwhile, a similar test carried out in African green monkeys (Cercopithecus aethiops) demonstrated that a ClO₂ concentration of 200ppm in drinking water caused erythema and ulceration of the oral mucosa after one week exposure (Bercz et al. 1982). These reports corroborate the fact that, based on dimensions of the subjects, the ClO₂ may not be considered a toxic agent; however, the concentration and exposure time are factors to consider for future controlled applications in humans, mainly as a potentially anti-SARS-CoV-2 agent.

In the past decades, ClO₂ was widely used as a drinking water sanitizing agent, which can produce a variety of oxidized by-products once it reacts with organic material. Even so, the EPA had listed the ClO₂ in group D (no classifiable for human carcinogenicity), due to the controversial data related to the genotoxicity in human and animal models (Buschini et al. 2004). In this sense, recent studies related to the genotoxicity of ClO₂ have reported that, before rapidly degrading into its by-products, ClO₂ can damage DNA and RNA structures by inducing DNA mutations and hindering its ability to act as a template for RNA replication (Zhang et al. 2023). However, these effects are dependent on the concentration of ClO₂ used and, in some cases, exceed the limits established by the normativity. For example, Buschini et al. (2004), reported that ClO₂ showed a weakly genotoxic damage on human leukocytes with a dose of 0.2ppm, while a genotoxic effect was only observed in cells of S. cerevisiae strain D7 exposed to doses higher (5- to 10-fold) than the concentration used for water disinfection. In this context, Monarca et al. (2000), observed, through an Ames test, that wastewater treated with 1.5ppm of ClO₂ exhibited mutagenicity in Vibrio fischeri, a marine bacterium commonly founded in wastewater effluents. Moreover, a genotoxicity test using human HepG2 cells revealed that the genotoxicity of ClO₂ is triggered by the presence of its derivative compounds, such as ClO₃⁻, which led to an increase of DNA damage in cells after exposure to 0.001ppm for 24h at 37°C with 5% CO₂ (Feretti et al. 2008).

Regarding its genotoxic effects on viral species, studies have revealed that ClO₂ decreased the viral infectivity of EV71 by altering the 1–118nt region located at 5′-non-coding region of the viral genome (Jin et al. 2013). Similarly, ClO₂ was able to produce RNA damage and decrease the complete infectivity of Poliovirus 1 after 3min of exposure to 5ppm of ClO₂ (Simonet and Gantzer 2006). Thus, the ClO₂ exerts genotoxic effects as antiviral mechanisms, either preventing the viral infection or replication inside the infected cell (Ge et al. 2021).

As mentioned above, during the disinfection process, the ClO₂ encounters naturally occurring organic or inorganic matter and then, rapidly degrades to its different by-products (ClO₂⁻, ClO₃⁻, and Cl⁻). There is evidence supporting that ClO₂ by-products, specifically chlorite, have genotoxic effects. For instance, a study conducted on E. coli strains deficient in oxygen-scavenging enzymes, demonstrated that chlorite was highly cytotoxic due to the formation of reactive oxygen species that promote oxidative damage in cells (Ueno et al. 2000, Richardson et al. 2007). On the contrary, there is information indicating relative lower genotoxic effects and non-carcinogenic risks of ClO₂ used for drinking water disinfection (Kimura et al. 2019, Du et al. 2021). Based on these findings, it is possible to infer that the genotoxic properties of the ClO₂ are more related to the release and presence of its by-products than to the original molecule. However, studies focused on clarifying the mechanisms by which the ClO₂ or its by-products (ClO₂⁻, ClO₃⁻, and Cl⁻) exert their genotoxic effects are still necessary.

Despite the above information, there are currently no published scientific evidence nor competent authorities that supports the positive benefits of ClO₂ on human health. Likewise, studies focused on the oral administration of ClO₂ as an anti-SARS-CoV-2 agent and its safety for human health are scarce, and in some of them, the results are controversial or not conclusive. So, more studies in the field are still required.

Conclusions and future perspectives

In the industry, ClO₂ is a well-researched and widely used disinfectant agent with strong antifungal, antibacterial, and antiviral activities reported both in in vitro and in vivo studies. As an antimicrobial agent, the ClO₂ gas is recognized for its greater penetration abilities and minimal impact on the operators. Conversely, aqueous solutions of ClO₂ present several undesirable effects, including low reactivity and penetration capacity, as well as bleaching effects in food products after their application.

Regarding its toxicity, it has been observed that controlled administration of ClO₂, at low concentrations (≤40ppm), does not represent a risk to human health, since the possible effects are latent while the exposure continues. On the contrary, high concentrations (>40ppm) of ClO₂ can cause adverse effects, such as hematologic and renal complications, which disappear once the exposure to ClO₂ is suspended and when the host health damage is not serious. In the same way, the ClO₂ cytotoxicity is low for humans; however, it can be increased either by mixing with other disinfectants or derived from the presence of its released by-products (e.g. ClO₂⁻, ClO₃⁻, and Cl⁻). This information potentializes the use of ClO₂ as a possible treatment for COVID-19. Nevertheless, randomized clinical controlled and double-blind studies related to the use of ClO₂ for prevention and treatment of COVID-19 are still needed to explore the efficiency in the treatment and the possible side effects, either in healthy individuals as well as in immunosuppressed patients or in those with comorbidities such as chronic degenerative diseases. Likewise, the potential use of ClO₂ as an anti-SARS-CoV-2 agent demands more in-depth studies related to the impact that this disinfectant can generate on the gut microbiota composition since an imbalance of microbial species or dysbiosis could increase the severity of symptoms in COVID-19 patients, such as inflammation or diarrhea.

Conflict of interest

None of the authors have conflict of interest to be declared.

Author contributions

Audry Peredo-Lovillo (Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing), Haydee Eliza Romero-Luna (Formal analysis, Investigation, Methodology, Supervision, Visualization, Writing – review & editing), Naida Juárez-Trujillo (Investigation, Methodology, Supervision, Writing – review & editing) and Maribel Jiménez-Fernández (Conceptualization, Formal analysis, Investigation, Project administration, Validation, Visualization, Writing – review & editing)

Data availability

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

References

© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.

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