Bactericidal Mechanism of Chlorous Acid Water in the Inactivation of Non-Tuberculous Mycobacteria

1. Introduction

In recent years, while the incidence of tuberculosis has been decreasing [1], the incidence of non-tuberculous mycobacteria (NTM) infections has been increasing [2]. Among the NTM, pulmonary infections by Mycobacterium avium complex (MAC) bacteria show the highest incidence and are becoming a serious concern for public health [3,4,5,6]. The main causative agents of pulmonary NTM infection are M. avium and M. intracellulare [7]. The prevalence of MAC isolated from pulmonary NTM infections shows a unique geographical distribution in Japan. M. avium is primarily isolated from pulmonary NTM infections in eastern Japan, whereas M. intracellulare is found in western Japan [7,8]. Pulmonary infections caused by M. intracellulare have a higher clinical morbidity than those caused by M. avium [9,10]. Pulmonary MAC infection is difficult to treat with antibiotics, and it requires long-term therapy (at least 1 year to achieve sputum culture negativity) with combinations of antibiotics, which include antituberculosis agents and macrolides [11]. Microbiological recurrence due to MAC reinfection is often observed even after successful clinical treatment [12,13].

NTM including MAC are widely distributed in the natural environment [14], and they are particularly abundant in moist or wet environments such as tap water and bathrooms [15]. Therefore, hygienic management of moist or wet environments is required to reduce exposure to NTM. Microbiological surveillance in the households of 49 pulmonary MAC disease outpatients and 43 healthy volunteers in Japan reported that MAC was isolated mainly from bathrooms and not from kitchen tap water, wash basins, and other sites [16]. In addition, the incidence of MAC in the bathrooms of patients was significantly higher than that of healthy volunteers [14].

The rapid detection and eradication of pathogenic microorganisms from infection sources is essential to prevent infectious diseases. However, effective methods for eradicating NTM remain to be established. NTM shows relatively high resistance to chlorine-based disinfectants [17], and of the NTM species, MAC is the most tolerant to disinfectant [18]. Effective sanitizing methods for bathrooms, which are rich in biological substances of human origin and biofilm materials, are needed.

Recently, a chlorite (ClO₂^-)-based disinfectant, chlorous acid water (CAW), has been approved as a Category II disinfectant in Japan. CAW has greater bactericidal effects under organic-matter-rich conditions when compared to sodium hypochlorite (NaClO), which is a representative chlorine-based disinfectant [19,20,21]. Therefore, CAW is a suitable disinfectant for environments contaminated by biological substances, which includes food processing equipment and hospital environments that are contaminated by human excretions.

The aims of this study were to examine whether CAW has a sufficient bactericidal effect against M. intracellulare and to elucidate the bactericidal mechanism of this disinfectant against mycobacteria.

2. Results

2.1. Bactericidal Effects of CAW and NaClO on NTM

We examined the bactericidal effects of chlorous acid water (CAW), CAW preparations, and sodium hypochlorite (NaClO) on Mycobacterium intracellulare NBRC 112750. CAW preparations (pH 7-8) are produced by adjusting the pH of CAW (pH 5-6) with phosphate buffer (pH 7.4). As shown in Figure 1A, CAW and a CAW preparation containing >5 mg/L free available chlorine (FAC) efficiently inactivated M. intracellulare within 1 min of treatment (>4.0 log₁₀ reduction in CFU/ml) in the absence of organic-matter load. The log₁₀ reduction in viable M. intracellulare by NaClO was <1.0 even with 200 mg/L FAC. When bovine serum albumin (BSA) was added to the reaction (0.5% final concentration), the sanitizing effect of all the test reagents was reduced (Figure 1B). To achieve effective killing (defined as a >4.0 log₁₀ reduction in CFU/ml) within 1 min, >100 mg/L FAC was required for both CAW and CAW preparations. On the other hand, NaClO reduced the concentration of viable cells only by around 1.0 log₁₀ CFU/ml even at 1,000 mg/L FAC in the presence of 0.5% BSA.

Bactericidal tests for other mycobacterial species (Table S1) were conducted under the same conditions employed for M. intracellulare. Although species-dependent differences in the sensitivity to test sanitizers were observed, CAW and CAW preparations showed bactericidal effects on all the tested mycobacteria similar to the effect on M. intracellulare. A 1-min treatment with CAW or CAW preparations containing >50 mg/L and >100 mg/L FAC achieved efficient killing under organic-matter-free and 0.5% BSA-loading conditions, respectively (Figure 2A,B). M. intracellulare and M. avium were more resistant to NaClO than the other mycobacteria tested (Figure 1A and Figure 2A).

2.2. Effects of CAW and NaClO on the Morphology of Mycobacteria

To elucidate the bactericidal mechanisms of CAW and NaClO, M. intracellulare NBRC112750 was treated with each agent at a FAC concentration of 200 mg/L for 30 min, and the subsequent bacterial surface structure was observed by scanning electron microscopy (SEM). After this treatment, the bacterial count of M. intracellulare was below the detection limit(1.08), indicating that it was completely inactivated by CAW, whereas no significant decrease in viability was observed in the sterile water and NaClO-treated groups. As shown in Figure 3, an extracellular matrix, which is a structure contributing to biofilm formation, was clearly observed around the M. intracellulare cells in the sterile water group. Although NaClO treatment eliminated the extracellular matrix, no change in cell morphology was observed in this group. Likewise, CAW treatment did not alter the M. intracellulare cell morphology, but the extracellular matrix shrank and agglutinated around the cells. These results suggest that the bactericidal effect of CAW may not be due to direct damage to the bacterial surface.

2.3. Effects of CAW, CAW Preparations and NaClO on Mycobacterial Genomic DNA

We evaluated the degradation of mycobacterial DNA after treatment with CAW or NaClO. M. intracellulare NBRC112750 was exposed to each reagent at a FAC concentration of 200 mg/L for 30 min, followed by neutralization with 1 M sodium thiosulfate. As shown in Figure 4A, pulsed-field gel electrophoresis (PFGE) revealed that CAW treatment induced genomic DNA degradation in mycobacterial DNA, whereas NaClO treatment did not. To examine the direct action of CAW on DNA, we treated the purified genomic DNA from M. intracellulare NBRC112750 with CAW, a CAW preparation or NaClO under the same conditions. The results were contradictory to the PFGE analysis, in which CAW and CAW preparations did not break down the DNA, whereas NaClO did cause DNA degradation (Figure 4B). These results indicated that M. intracellulare DNA degradation did not result from the direct action of CAW, but from stress responses induced by the reagent.

Notably, the migration of the genomic DNA in the agarose gel was delayed by CAW treatment (200 mg/L, 30 min), while the CAW preparation (buffered around neutral pH with phosphate buffer) showed no effect (Figure 4B). S1 nuclease, which digests single-stranded DNA, largely degraded the genomic DNA after exposure to CAW (pH 5-6, unbuffered), which was not the case with the CAW preparation (Figure S1), indicating that CAW affects the hydrogen bonding pattern within double-strand DNA.

To determine whether DNA degradation is responsible for the bacterial killing by CAW, we extracted DNA from M. intracellulare NBRC112750 and treated the DNA with each disinfectant at the FAC levels used in the bactericidal tests (5, 10, 25, 50, 100, and 200 mg/L) for 1 min. None of the disinfectants affected the genomic DNA (Figure S2). These results indicate that DNA degradation is not the direct bactericidal mechanism for CAW and CAW preparations.

2.4. Effect of CAW, CAW Preparations and NaClO on Membrane Potential

We measured the membrane potential of M. intracellulare NBRC112750 treated with each disinfectant by using the BacLight™ Bacterial Membrane Potential kit. The kit employs the fluorescent dye DiOC₂(3), which permeates cells and normally exhibits green fluorescence. In cells with intact membrane potential, the dye polymerizes within the membrane and exhibits red fluorescence. By calculating the ratio of these fluorescence intensities (red/green ratio), it is possible to evaluate changes in membrane potential. A decoupling reagent, carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and sterilized distilled water were used as positive and negative controls, respectively.

Treatment with CAW and a CAW preparation at >10 mg/L FAC significantly reduced the membrane potential (Figure 5A) and the survival rate (Figure 5B) of M. intracellulare cells, while significant reductions in the membrane potential and survival rate were only observed at >50 mg/L FAC after NaClO treatment. No significant reduction relative to the negative control was observed when M. intracellulare cells were inoculated with pre-neutralized reagents. These results indicate that CAW and CAW preparations disrupt the membrane potential in a similar manner, but at lower FAC, as NaClO (Figure 6).

2.5. Alterations in Intracellular ATP Levels After Treatment with CAW, CAW Preparation and NaClO

To verify the membrane potential disruption by CAW or CAW preparation, we periodically measured ATP production using the BacTiter-Glo reagent and the GloMax Navigator system for a period of seven days after the treatment (Figure 7). After 24 h of treatment, ATP synthesis did not recover in any of the treated M. intracellulare cultures. However, after 7 days, ATP levels in the culture that was treated with NaClO at 5 to 50 mg/L FAC recovered ATP production at levels equivalent to, or higher than, those of the control (25 and 50 mg/L). On the other hand, in the M. intracellulare cultures treated with CAW or a CAW preparation, ATP production did not recover until 4 days after treatment. Recovery of ATP production was observed in the cells treated with CAW or a CAW preparation at low FAC (5, 10, or 25 mg/L), but the level was lower than that of the control. The cells treated with CAW and the CAW preparation at 50 mg/L did not recover ATP production at all. These results indicate that CAW and the CAW preparation cause irreversible damage to the respiratory chain.

2.6. Reactive Oxygen Species (ROS) Generation After Treatment with Each Sanitizer

Based on the results described above, we speculated that CAW and the CAW preparation caused severe damage to M. intracellulare membrane proteins, leading to dysfunction of the respiratory chain, loss of membrane potential and impairment of ATP synthesis. NADH dehydrogenase, which is a well-known constituent of the respiratory chain, is a major contributor to intracellular ROS production during redox reactions. Therefore, we measured intracellular ROS production in M. intracellulare after contact with the test reagents. We used hydrogen peroxide (H₂O₂) as a positive control to induce intracellular ROS production. H₂O₂ induced intracellular ROS production in a concentration-dependent manner (Figure 8A). In contrast, 25 mg/L and 50 mg/L CAW or CAW preparation decreased the intracellular ROS level (Figure 8B,C). On the other hand, 25 mg/L and 50 mg/L NaClO did not alter the intracellular ROS level (Figure 8D). At low FAC (5 mg/L and 10 mg/L), all reagents increased the intracellular ROS level, but a steeper curve was observed for NaClO than for CAW or the CAW preparation: the ratios of the last/initial fluorescence values were 6.4, 4.0, 1.4, and 1.4 for H₂O₂ (1 mM), NaClO (5 mg/L), CAW (5 mg/L) and CAW preparation (5 mg/L), respectively.

3. Discussion

Chlorite (ClO₂^-)-based sanitizers, which include acidified sodium chlorite (ASC) and CAW preparations, are used for the sanitation of food and food processing facilities [22,23]. CAW has recently been approved as a category 2 drug in Japan. The microbicidal activity of CAW preparations against a wide spectrum of microorganisms, including Campylobacter jejuni [24,25], Clostridioides difficile [19], M. tuberculosis [26], human norovirus [21] and SARS-CoV-2 [20], has been reported. These studies commonly reported that CAW showed superior microbicidal effects to NaClO under organic matter-rich conditions. The incidence of pulmonary disease caused by MAC has been increasing in several countries where the incidence of tuberculosis is decreasing [1,2]. Moist and wet environments in households such as bathroom and kitchen sinks and water supply systems are reservoirs for NTM [15]. These environments are rich in organic matter derived from humans and from food ingredients, leading to biofilm and/or slime formation. Under these conditions, it is likely to be difficult to sanitize NTM using simple sanitation approaches. Therefore, we aimed to examine the microbicidal effect of CAW on NTM.

As in the previous study, we observed that CAW showed superior killing of NTM compared with NaClO at the same FAC level. Notably, MAC including M. avium and M. intracellulare showed higher resistance to NaClO than other NTM such as M. fortuitum and M. abscessus (Figure 2A). This indicates that chlorine levels in tap water are insufficient to inactivate NTM in water supply systems.

Even under organic matter loading conditions, CAW at >100 mg/L achieved a >4 log₁₀ reduction in viable cells of all the tested NTM strains within 1 min of treatment, in contrast to NaClO, which did not achieve this level of reduction even at 1000 mg/L. The chloroperoxyl radical (ClOO●) is a main active component of CAW [27]. This novel radical species can be detected by electron spin resonance without spin trap reagents, indicating that it is a long-lived radical. In addition, Goda et al. reported that CAW showed selective reactivity toward amino acids, reacting only with cysteine and histidine among the 15 amino acids tested [21]. These findings indicate that the tolerance of CAW to organic matter can be attributed to the long-lived chloroperoxyl radical and its selective reactivity. However, the microbicidal mechanisms of CAW have not been fully elucidated.

We first investigated whether the bactericidal mechanism of CAW was due to oxidative DNA damage. DNA purified from M. intracellulare was exposed to each of the FAC reagents at a concentration of 200 mg/L for 30 minutes. Contrary to the PFGE results, NaClO degraded the DNA band, whereas CAW and the CAW formulation did not reduce its intensity. However, CAW treatment resulted in an upward shift of the band. The shift in the DNA band observed after CAW treatment may suggest dissociation of double-stranded DNA (Figure 4B).

This is likely due to disruption of electrostatic interactions between double-stranded DNA, as predicted by experiments using S1 nuclease, which degrades single-stranded DNA (Figure S2).

Furthermore, DNA was extracted after a 1-minute contact time, during which CAW and the CAW formulation exerted efficient bactericidal activity. Although 200 mg/L NaClO reduced the intensity of the DNA band, DNA extracted from M. intracellulare remained unchanged after treatment with either reagent at 200 mg/L for 1 min (Figure S1).

Since there was no correlation between the DNA degradation and bactericidal effect of CAW, we concluded that DNA damage is not a primary bactericidal mechanism of CAW. Gupta et al. reported that exonucleases are induced in Escherichia coli cells by oxidative stress [28]. In the case of M. intracellulare, the DNA degradation shown in the PFGE analysis seemed to be a secondary phenomenon due to lethal oxidative stress induced by CAW. Transcriptome analysis will be needed to fully explain how physiological changes in NTM are induced by CAW. On the other hand, NaClO degraded purified DNA, but not DNA inside M. intracellulare cells, indicating that it is difficult for NaClO to penetrate the lipid-rich cell walls of mycobacteria. It was noteworthy that CAW preparations, which consist of CAW in phosphate buffer around neutral pH, did not degrade the DNA, but rather seemed to stabilize the DNA. This idea is based on the agarose gel image in which the smearing of DNA decreased after treatment with the CAW preparation (Figure S1). The reason for this effect is unknown, but the negative charges of the phosphate ion electrostatically interact with divalent cations such as Mg²⁺, which is a cofactor enhancing DNase activity. The pH adjustment of CAW with phosphate buffer may decrease the genotoxic effects of CAW or other oxidative sanitizers.

Hatanaka et al. reported that CAW agglutinated membrane proteins without affecting the genomic DNA, indicating that protein degeneration is a major bactericidal mechanism [25]. Accordingly, we measured the membrane potential, and the results showed that the membrane potential declines in a manner that correlates well with the bactericidal effects of CAW and CAW preparations on NTM. The reduction of membrane potential by CAW was attributed to the deterioration of respiratory chain function. CAW and CAW preparations were considered to cause irreversible degeneration of membrane proteins since ATP synthesis did not recover after treatment with CAW or CAW preparations at 50 mg/L, even seven days after treatment. On the other hand, M. intracellulare treated with NaClO recovered ATP synthesis, including higher ATP production than the control when the cells were treated with NaClO at 25 mg/L and 50 mg/L, indicating a compensatory response to increased energy demand for recovery from cellular damage.

NADH dehydrogenase, which is a large, multi-subunit enzyme forming part of the respiratory chain, is a major contributor to intracellular ROS [29]. We confirmed the reduction of ROS generation after treatment with CAW or CAW preparations, correlating with the reduction in membrane potential after treatment with these sanitizers (Figure 5). As mentioned above, CAW preferentially reacts with cysteine and histidine. These amino acid residues are essential for the structures of protein complexes responsible for electron transfer in the respiratory chain, which contain cofactors such as heme and iron-sulfur clusters [30,31,32], supporting our conclusion that the bactericidal mechanism of CAW and CAW preparations on NTM involves membrane protein degeneration, especially the components of the respiratory chain.

A limitation of this study is that the results are obtained from in vitro analysis. NTM form biofilms even in laboratory cultures. Therefore, bactericidal assays in practical settings will be essential to confirm the sanitizing effect of CAW and CAW preparations on NTM colonizing wet environments. It may be necessary to consider the combined use of CAW.

4. Materials and Methods

4.1. Bacterial Strains and Culture Conditions

The NTM strains used in this study are listed in Table S1. For all the strains, glycerol stocks stored at -80℃ were streaked onto Middlebrook 7H10 agar (Becton, Dickinson and Company, BD) and incubated at 37 ℃ for one week. The resulting colonies were picked and suspended in Middlebrook 7H9 liquid medium (Sigma-Aldrich), then cultured at 37 ℃ with shaking (130 rpm) for two weeks until sufficient growth was obtained.

4.2. Disinfectants

Chlorous acid water (CAW) and CAW preparations were obtained from Sankei Co., Ltd. Sodium hypochlorite (NaClO) was pur4.3.chased from Oyalox Co. Ltd. Free available chlorine (FAC) levels were measured by the method employing N,N-diethyl-p-phenylenediamine (DPD) according to a previous study [33]. FACs levels are expressed in mg/L in this study. DPD was obtained from FUJIFILM Wako Pure Chemical Co.

4.3. Bactericidal Assays

Mycobacterial cultures (5.0 mL) were centrifuged (14,000 g, 4 ℃, 5 min) and resuspended in 1.0 mL saline solution with 0.05% Tween 20. Optical densities at 600 nm (OD₆₀₀) of the individual cell suspensions were adjusted to 1.0 with saline/0.05% Tween 20. For the assays with organic-matter-free conditions, 0.9 mL of each of the disinfectants was mixed with 0.1 mL of bacterial suspension. In the case of organic-matter-load conditions, 0.1 mL of the bacterial inoculum, which was prepared by equally mixing 7.5% bovine serum albumin (BSA, Sigma-Aldrich) and the bacterial suspension (OD₆₀₀=1.0), was added to 0.9 ml of each of the disinfectants. The treatment time at 25 ℃ was 1 min for both conditions. After treatment, 0.2 mL of 1 M sodium thiosulfate solution (FUJIFILM Wako Pure Chemical Co.) was immediately added to neutralize residual chlorine. After neutralization, 10-fold serial dilutions were prepared with saline/0.05% Tween 20. Appropriate dilutions (0.1 ml) were spread on Middlebrook 7H10 agar medium, and the plates were incubated at 37 ℃ for 4 weeks for M. intracellulare and 1 week for other mycobacteria tested The number of surviving bacteria was measured by counting the colonies formed on the plates. As a control, the viability of the bacteria in pre-neutralized disinfectants was monitored using standard plating methods.

4.4. Scanning Electron Microscopy

M. intracellulare NBRC 112750 cells, which were treated with distilled water and 200 mg/L each of CAW or NaClO, were fixed overnight with 2% glutaraldehyde in cacodylate buffer (pH 7.4) at 4°C. For scanning electron microscopy (SEM), each cell sample was dehydrated with a series of acetone solutions ranging in 10% increments from 50% (vol/vol) ethanol in distilled water to absolute acetone. All samples were dried to the critical point using a critical point dryer, coated with gold, and examined by SEM (Hitachi S-800; Hitachi, Tokyo, Japan).

4.5. Pulsed-Field Gel Electrophoresis (PFGE)

PFGE of M. intracellulare NBRC 112750 was conducted to assess the chromosomal degradation after treatment with CAW or NaClO according to the method described by Samir et al. [34]. In brief, M. intracellulare NBRC 112750 cells were collected by centrifugation (14,000 g, 5 min, 4 ℃) from the reaction mixture after 30 min of treatment with saline, CAW or NaClO following neutralization. The pellet was washed with 500 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and then resuspended in 200 μL of TE buffer. The sample plug molds, which were prepared with 200 μL of the cell suspension, were immersed in 4 mg/L lysozyme solution (Sigma Aldrich) and treated at 37 ℃ for 48 h (lysozyme solution was exchanged every 24 h). Subsequently, they were immersed in 0.5 M EDTA (DOJINDO) solution containing 2 mg/L proteinase K (FUJIFILM Wako Pure Chemical Corporation) and 1% lauroyl sarcosine sodium (Nacalai Tesque), and the samples were statically incubated at 55 ℃ for 7 days (the solution was exchanged on the 5th day). The plugs were washed four times with TE buffer (each wash lasting 1 h). The obtained sample plugs containing DNA fragments were analyzed by pulsed-field gel electrophoresis (PFGE) using a CHEF-DR2 (Bio-Rad Laboratories) under the following conditions: running buffer, 1X Tris-borate EDTA; running temperature, 14℃; running time, 48 h; voltage gradient, 6 V/cm; switching time: 5 s to 35 s for 48 h.

4.6. DNA Extraction

After M. intracellulare NBRC 112750 cells were collected from the cultures by centrifugation (14,000 g, 5 min, 4 ℃), pellets were suspended in 0.9 mL of TE buffer (pH 8.0) containing 4 μg/mL lysozyme and incubated at 37 ℃ for 1 h. Subsequently, 50 μL of 5.8 mg/mL achromopeptidase (FUJIFILM Wako Pure Chemical Co) was added, and the reaction mixture was incubated at 37 ℃ for 1 h. Then, 110 μL of 20 μg/mL proteinase K was added, and the mixture was further incubated at 55 ℃ for 1 to 1.5 h. Next, 120 μL of 10% sodium dodecyl sulfate (FUJIFILM Wako Pure Chemical Co) was added and incubated at 55 ℃ for 1 h. The DNA was extracted using phenol/chloroform/isoamyl alcohol (25:24:1, Nippon Gene) followed by ethanol precipitation at -80 ℃ overnight. The extracted DNA was dissolved in TE buffer (pH 8.0) and stored at -20 ℃ until use.

4.7. Digestion of Single Stranded DNA

To evaluate the amount of single-stranded DNA produced by treatment with CAW or NaClO, extracted bacterial DNAs were digested with S1 nuclease (Takara Bio). The digested DNAs were electrophoresed in 0.8% Agarose S (Nippon Gene) and DNA fragmentation was evaluated using ethidium bromide (Thermo Fisher) staining of the gels.

4.8. Measurement of Membrane Potential

After bactericidal assays were performed (treatment time 1 min) as described above, M. intracellulare NBRC 112750 cells were collected by centrifugation (14,000 g, 4 ℃, 5 min), suspended in 1 mL of Middlebrook 7H9 medium supplemented with ADC (0.2% (w/v) dextrose, 0.2% (v/v) glycerol and 0.5% bovine serum albumin) and incubated at 37 ℃ for 30 min. As a control, we heated a M. intracellulare NBRC 112750 cell suspension at 100 ℃ for 10 min and filtered the suspension through a 40 μm cell strainer (FALCON). We also prepared carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-treated M. intracellulare NBRC 112750 cells as a control. Membrane potentials of the M. intracellulare NBRC 112750 cells after each treatment were measured using BacLight™ Bacterial Membrane Potential Kit (Thermo Fisher) according to a previous report [35]. Green and red fluorescence were measured using a flow cytometer (Merck Millipore), and the changes in membrane potential were evaluated based on the ratio of red fluorescence to green fluorescence of the stained cells.

4.9. Measurement of ATP

ATP levels in M. intracellulare NBRC 112750 cultures were measured according to the method reported by Yuroff et al. [36]. Cell suspensions of M. intracellulare NBRC 112750 were adjusted to an OD₆₀₀ of 1.0 with saline/0.5% Tween 20. Next, 0.1 mL of the cell suspension was mixed with 0.9 mL of the test reagent (H₂O, CAW, CAW preparation or NaClO) and incubated at room temperature for 1 min. Subsequently, residual chlorine was neutralized with 0.2 mL of 1 M sodium thiosulfate. The treated cells were collected by centrifugation (14,000 g, 4 ℃, 5 min) and resuspended in 1 mL of Middlebrook 7H9 medium supplemented with ADC. The cell suspension was diluted 10-fold with the same medium and cultured at 37 ℃ for pre-scheduled intervals. The culture (0.5 ml) was periodically sampled and frozen at -80℃ for 30 min. The samples were then thawed at room temperature to lyse the cells and 50 μL of each cell lysate was dispensed into a 96-well plate. Sterilized distilled water (dH₂O) and 50 μL of BacTiter-Glo™ reagent (Promega) were added and statically reacted at room temperature for 5 min. The luminescence intensity was measured using a GloMax® Navigator System (Promega). The obtained luminescence values were evaluated as an indicator of surviving bacterial cells based on the amount of ATP.

4.10. ROS Measurement

The reactive oxygen species (ROS) which were generated in M. intracellulare NBRC 112750 after treatment with CAW or NaClO were measured using the OxiSelect™ Intracellular ROS Assay Kit (Cell Biolabs Inc.) according to the manufacturer’s instructions. In brief, M. intracellulare NBRC 112750 cell suspensions were adjusted to an OD₆₀₀ of 2.0 with Middlebrook 7H9 medium supplemented with ADC, then centrifuged (14,000 g at 4 ℃, 5 min) and resuspended in 200 μL of a 1X solution of dichlorofluorescein-diacetate (DCFH-DA) (Thermo Fisher). After the reaction mixture was incubated at 37 ℃ for 1 h in the dark, the cells were collected by centrifugation (14,000 g, 4 ℃, 5 min) and resuspended in 2 mL of 1X PBS (pH 7.4). Then, 100 μL of each test reagent (CAW or NaClO) was dispensed into a 96-well plate and mixed with the same volume of the bacterial suspension. Hydrogen peroxide (1 mM and 10 mM) was used as a positive control. Immediately after mixing, changes in fluorescence intensity due to oxidative stress were recorded at 1-min intervals for 1 h. Fluorescence was monitored at an excitation wavelength of 480 nm and an emission wavelength of 530 nm using a fluorometer (CORONA ELECTRIC Co., Ltd).

4.11. Statistical Analysis

All statistical analyses were performed using R software (version 4.4.2). A p-value of <0.05 was considered to be statistically significant unless otherwise noted. Primary statistical analyses were conducted using a one-way analysis of variance (ANOVA) followed by Tukey’s test or Dunnett’s test. For the membrane potential assays, comparisons of the fluorescence ratios were performed using two-sample t-tests.

5. Conclusions

We showed in this study that CAW and CAW preparations effectively inactivate NTM even under conditions that are rich in organic matter, possibly by disrupting NTM membrane integrity. CAW and CAW preparations lead to lower levels of direct DNA damage than NaClO, predicting the lower genotoxicity of CAW. CAW and CAW preparations are considered to be effective sanitizers that decrease the NTM load in wet environments, possibly reducing the risk of NTM exposure to susceptible individuals.