Microbicidal effects of slightly acidic hypochlorous acid water and weakly acidified chlorous acid water on foulbrood pathogens

Abstract

Paenibacillus larvae and Melissococcus plutonius are bacterial pathogens of honey bee brood. As decontamination of beekeeping equipment, including combs, is essential to control these pathogens, we evaluated the disinfecting effects of slightly acidic hypochlorous acid water (SAHAW) and weakly acidified chlorous acid water (WACAW) on the pathogens. Both disinfectants exhibited strong disinfecting effects in suspension tests under no organic matter conditions and reduced both pathogens by >5 log₁₀ CFU/ml. Although the microbicidal activity of SAHAW with an available chlorine concentration (ACC) of 10–30 ppm was decreased by organic matter, it reduced viable P. larvae spores in combs more efficiently than H₂O when the comb was not as dirty. However, its efficacy on combs decreased at 4°C and when overused or highly contaminated combs were tested. WACAW with an ACC of ≥600 ppm had a higher disinfecting capacity than SAHAW, and efficiently removed P. larvae spores from combs even under organic matter-rich and low-temperature conditions. However, even by WACAW, the amount of viable spores in combs was not markedly reduced depending on contamination levels and P. larvae genotypes. These results suggest the usefulness of both disinfectants for decontaminating beekeeping equipment depending on the situations expected.

American foulbrood (AFB) caused by Paenibacillus larvae and European foulbrood (EFB) caused by Melissococcus plutonius are two major bacterial infectious diseases of honey bee brood. Both diseases are globally distributed including in Japan [6], and may collapse many colonies when outbreaks occur. Thus, these diseases are recognized as economically important for the apiculture industry. In both diseases, strains of the causative pathogens are classified into different genotypes. P. larvae strains have been classified into four ERIC types (ERIC I, II, III and IV) by repetitive-element PCR [11] and 24 sequence types (STs) by multilocus sequence typing (MLST) [16, 20, 26] (https://pubmlst.org/plarvae/), whereas M. plutonius strains have been classified into more than 30 STs by MLST, and the STs have been further grouped into three clonal complexes (CC3, CC12 and CC13) [2, 5, 15, 24] https://pubmlst.org/mplutonius/). In both pathogens, strains with different genotypes have different phenotypes, including virulence [2, 11, 21]. In Japan, isolation of P. larvae ERIC II strains from AFB cases has been increasing recently [26], and M. plutonius CC12 strains have been frequently isolated from EFB cases [24].

P. larvae is a spore-forming bacterium and can produce over one billion spores in each infected larva. The spores are highly resistant to heat and chemical agents and can survive for many years in dried larval scales, hive products and equipment (http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.02.02_AMERICAN_FOULBROOD.pdf). Not only in diseased colonies, P. larvae spores may also exist in clinically healthy colonies. Indeed, analysis of honey samples that were harvested some years before the outbreak of AFB revealed that colonies were already contaminated with spores several years before the detection of clinical symptoms [27]. It is common in beekeeping to exchange hive material like honey or brood combs between colonies in order to reuse hive material when setting up a new colony and to unite weak colonies to build a strong colony [10]. If the exchanged material is contaminated with P. larvae spores, the practice causes the spread of AFB from colony to colony. Although M. plutonius is not a spore-forming bacterium, it is also a durable organism. In nature, honey bee larvae become infected with M. plutonius through the ingestion of brood food contaminated with the pathogen, and the ingested M. plutonius multiplies within the larval gut; however, the infection is not always lethal. Infected larvae may survive and deposit M. plutonius along with their feces into the brood comb when they pupate [9]. Hence, like AFB, the combs contaminated with M. plutonius cause transmission of EFB to other colonies. Therefore, decontamination of beekeeping equipment, including combs, is important for the control of both foulbroods.

In Japan, several disinfection methods, such as gamma irradiation and ethylene oxide sterilization, have been used to decontaminate combs. These methods have been demonstrated to be effective for sterilization of P. larvae spore-contaminated materials [4, 23]; however, as dedicated facilities are required for these methods, other disinfection methods that are effective and easily performed on site have been desired. For this purpose, slightly acidic hypochlorous acid water (SAHAW), also referred to as slightly acidic electrolyzed water, has recently been introduced for the decontamination of beekeeping equipment, including combs, in Japan. SAHAW is a chlorine-based disinfectant produced by the electrolysis of dilute hydrochloric acid solution in the electrolytic cell without a separating membrane and is permitted as a food additive by the Ministry of Health, Labour and Welfare (MHLW) of Japan. The principal ingredient of SAHAW with pH values of 5.0–6.5 is hypochlorous acid (HClO), which exhibits a wide spectrum of antimicrobial activity [19, 22, 25, 28]. As SAHAW has strong antimicrobial activity with a relatively low available chlorine concentration (ACC), it has the advantage of being less corrosive for equipment, less irritating for the human body [13, 14, 19] and having less of an impact on the environment. However, in the presence of organic matter, its antimicrobial activity is known to decrease [22].

Chlorous acid (HClO₂) water is another chlorine-based disinfectant approved as a food additive by the MHLW of Japan. Weakly acidified chlorous acid water (WACAW) is one of the chlorous acid-based disinfectants. WACAW mainly contains three kinds of oxygenated chlorines (HClO₂, ClO₂⁻ and ClO₂). Under weakly acidified conditions (pH 5.0–6.0), HClO₂ plays a major role in microbial killing as a predominant species. In previous studies, WACAW was reported to inactivate a wide variety of microorganisms, including endospores of Clostridium, Bacillus and Paenibacillus species [12, 17]. In addition, WACAW is relatively stable under organic matter-rich conditions and retains its microbicidal activity [12, 17]. Therefore, WACAW may also be used as an effective alternative for reducing pathogen contamination of beekeeping equipment.

However, little or no information is available on the microbicidal efficacy of SAHAW and WACAW on honey bee pathogens. Therefore, we evaluated the microbicidal efficacy of the two disinfectants on representative strains of ERIC I and II P. larvae and CC3, CC12 and CC13 M. plutonius in vitro. We also used honey bee combs that were used in apiaries for 1–5 years and contained different levels of organic matter, and evaluated the efficacy of SAHAW and WACAW in killing or removing P. larvae spores on the combs.

MATERIALS AND METHODS

Bacterial strains and culture conditions

M. plutonius DAT606 (genotype: ST3, CC3), DAT561 (ST12, CC12) and DAT585 (ST26, CC13) and P. larvae DTK386 (ERIC I, ST15) and DTK384 (ERIC II, ST10) isolated from diseased European honey bee larvae were used as representative strains from each genetic group in the present study. The genotypes of the strains were obtained from previous studies [16, 24]. M. plutonius strains were cultured on KSBHI agar [1] at 35°C under anaerobic conditions using the AnaeroPack System (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan). P. larvae strains were cultured on MYPGP agar [3] or Columbia agar (BBL; Becton, Dickinson and Co., Franklin Lakes, NJ, USA) supplemented with 5% defibrinated sheep blood at 35–37°C under air plus 5% CO₂ conditions.

Preparation of M. plutonius inoculum

M. plutonius strains cultured for two days were collected by sterile cotton swabs, suspended in sterile H₂O and adjusted to the optical density at 600 nm of approximately 1.98. In order to remove medium components, we collected bacterial cells from 1 ml of the suspension by centrifugation (15,000 rpm, 5 min) and removed the supernatants. The M. plutonius pellet was then suspended in sterile H₂O and adjusted to approximately 1 × 10⁸ CFU/ml.

Preparation of P. larvae spores

P. larvae spores were prepared according to standard methods for American foulbrood research [3] with some modifications. P. larvae strains grown on agar plates were suspended in MYPGP broth, and appropriately diluted bacterial suspensions were spread onto MYPGP agar plates. The plates were incubated at 35–37°C for 12–20 days under air plus 5% CO₂ conditions. After sporulation of more than 70–90% cells was confirmed by staining with malachite green, spores were collected from 10 or more agar plates, suspended in cold sterile H₂O and washed four times with cold sterile H₂O by collecting spores via centrifugation (12,000 × g, 15 min, 4°C), discarding the supernatant and suspending the spore pellet in 30 ml of cold sterile H₂O. Washed spores were suspended in 5–10 ml of cold sterile H₂O and stored at 4°C. One week or more later, the spores were washed several more times with 30 ml of cold sterile H₂O to remove organic matter from lysed vegetative cells. The re-washed spores were suspended in 5–10 ml of cold sterile H₂O and stored at 4°C until use. In some cases, amphotericin B was added to H₂O for the first washing at a final concentration of 16.8 µg/ml to avoid fungal contamination. Heat resistant and germinable spore concentrations were measured by heating a portion of the spore suspension at 65°C/80°C for 15 min, plating serial dilutions of the spore suspension on MYPGP agar plates and counting colonies on the plates after incubation at 35–37°C for seven days under air plus 5% CO₂ conditions.

Disinfectants

SAHAW (ACC, 10–30 ppm) was purchased from HOCL Inc. (Fujisawa, Japan). WACAW (ACC, 2,400 ppm) was kindly supplied by HONBUSANKEI Co., Ltd. (Osaka, Japan). WACAW was diluted in sterile H₂O as necessary. The disinfectants were protected from light and stored at 4°C until use.

Suspension tests

Approximately 1 × 10⁸ CFU/ml of M. plutonius inoculum was prepared as described above. Approximately 1 × 10⁷ CFU/ml of P. larvae spore inoculum was prepared by diluting the spore suspension in sterile H₂O and heating it at 80°C for 15 min. The final bacterial concentration in each inoculum was measured in each test by culturing serial dilutions of the inoculum at 35°C on KSBHI agar for four days under anaerobic conditions (for M. plutonius) or MYPGP agar for seven days under air plus 5% CO₂ conditions (for P. larvae). The results are shown in Supplementary Tables 1 and 2. As interfering conditions, 5% honey, 1% yeast extract plus 1% bovine serum albumin (BSA) (simulated high-level soiling conditions in the veterinary area [7]), 0.3% BSA (simulated low-level soiling conditions in the veterinary area/dirty conditions in food, industrial, domestic and institutional areas [7, 8]) and 0.03% BSA (simulated clean conditions in food, industrial, domestic and institutional areas [8]) were tested. Yeast extract and BSA were purchased from Becton, Dickinson and Co. and Sigma-Aldrich (St. Louis, MO, USA), respectively. The interfering substances were prepared as 10 × concentrated solutions and sterilized by filtration through a membrane with a pore size of 0.2 or 0.22 µm.

For evaluation of the microbicidal effects of SAHAW and WACAW on the foulbrood pathogens, the bacterial inoculums, 10 × concentrated organic matter solutions/sterile H₂O (for no organic matter conditions) and the disinfectants were mixed in 1.5-ml tubes at a ratio of 1:1:8, and the mixtures were incubated at 20°C. As controls, sterile H₂O was used instead of the disinfectants. At the end of each treatment time (5 min, 1 hr, 6 hr and 24 hr), a portion of the mixture was transferred to a sterile tube containing the same volume of neutralizing solution (3% yeast extract plus 3% BSA for SAHAW and 0.1 M Na₂SO₃ for WACAW) to halt disinfection. After neutralization, the number of viable M. plutonius cells and P. larvae spores in the treated samples was investigated by plating serial dilutions of the neutralized samples onto KSBHI and MYPGP agar plates, respectively, and counting colonies on the plates after incubation under the conditions described above. Tests were independently repeated three times. The efficacy of disinfectants was expressed as log₁₀ CFU/ml reduction, which was calculated by the following formula: log₁₀ CFU/ml reduction=log₁₀ (the viable cell concentration before the treatment/the viable cell concentration after the treatment).

Carriers used for carrier tests

Four combs (nos. 1–4) used as carriers were kindly provided from two apiaries where no AFB outbreak has been recorded. The absence of P. larvae in the combs was confirmed by collecting swab samples from five cells of each comb and culturing the samples on Columbia agar supplemented with 5% defibrinated sheep blood and MYPGP agar at 37°C for five days under air plus 5% CO₂ conditions. As controls to evaluate the efficacy of disinfectants in killing or removing of P. larvae spores under no organic matter conditions, 96-well microplates were used as carriers.

ATP-bioluminescence assay of combs

The amount of organic matter that remained on the surface of the combs used in this study was indirectly estimated by measuring the amount of ATP and AMP originating from organisms and their residues. The amount of ATP and AMP was measured by the ATP-bioluminescence assay using Lumitester PD-30 (Kikkoman Biochemifa Co., Tokyo, Japan) according to the manufacturer’s instructions, and the results were expressed in relative light units (RLU). With the Lumitester PD-30 system, AMP can also be detected by converting it into ATP by pyruvate orthophosphate dikinase. Organic matter adhered to the internal wall and bottom of cells was collected by inserting the special cotton-tipped swab moistened with sterile H₂O into a cell, pressing the swab lightly on the bottom of the cell and rotating it three times. In total, 12 samples were collected from 12 cells for each comb. The cells used in this assay were not used for the evaluation of disinfection efficiency.

Carrier tests using combs

For the carrier tests, the combs were cut in multiple pieces, and each piece was fixed in a sterile petri dish. After heating a portion of the P. larvae spore suspension at 65°C for 15 min, approximately 1 × 10⁵ or 1 × 10⁷ CFU was inoculated in each cell of the combs or in each well of the 96-well microplates and dried in an incubator at 25°C for 17–25 hr. The number of inoculated spores was measured in each test by culturing serial dilutions of the spore suspension on MYPGP agar or Columbia agar supplemented with 5% defibrinated sheep blood at 37°C for seven days under air plus 5% CO₂ conditions, and the results are shown in Supplementary Table 3. The cells and wells were then filled with SAHAW (ACC, 10–30 ppm), WACAW (ACC, 2,400, 1,200, 600 and 300 ppm) or sterile H₂O (ACC, 0 ppm; for control) and incubated at 20°C or 4°C. In some tests, P. larvae spore-inoculated cells were prewashed by filling them with 40°C sterile H₂O, incubating at 25°C for 1 hr and removing the water from the cells. Prewashed cells were then dried, filled with disinfectants or H₂O and incubated at 20°C. After a 16-hr incubation, disinfectants and H₂O were removed, and the cells and wells were dried in an incubator at 25°C for approximately 16 hr. The number of viable spores that remained in the cells and wells was measured by adding 100 µl of sterile H₂O into the cells and wells, pipetting up and down 50 times, and culturing serial dilutions of the collected suspensions under the conditions described above. In carrier tests, neutralization of the disinfectants was not performed in order to remain as close as possible to actual practice. Tests were repeated at least three times using different cells. The efficacy of disinfectants was expressed as log₁₀ CFU/cell or well reduction, which was calculated by the following formula: log₁₀ CFU/cell or well reduction=log₁₀ (the number of viable spores in inoculum/the number of viable spores recovered after the treatment).

Statistical analysis

All the statistical analyses were performed in EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [18], which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). For each experiment, Kruskal-Wallis test followed by Steel-Dwass post-hoc test, Welch’s t-test or one-way ANOVA followed by Bonferroni post-hoc test was used depending on the type of variables and the number of levels in factors with a significance level of 0.05. In this study, the minimum detectable value of each test was used for the analyses of data below the detection limit.

RESULTS

Microbicidal activity of SAHAW and WACAW on M. plutonius in suspension tests

In the suspension tests, SAHAW with an ACC of 10–30 ppm and WACAW with that of 2,400 ppm were used, and the microbicidal activity of the disinfectants was evaluated on the representative M. plutonius strains of CC3, CC12 and CC13. In the absence of organic matter, both disinfectants inactivated all M. plutonius within 5 min (>5 log₁₀ CFU/ml reduction) (Fig. 1 and Supplementary Table 1). SAHAW also reduced all three M. plutonius strains by >5 log₁₀ CFU/ml within 5 min under 5% honey and 0.03% BSA conditions (Fig. 1A and Supplementary Table 1). Under these conditions, regardless of the M. plutonius genotypes, microbicidal activity of the disinfectants was significantly higher than that of H₂O (Welch’s t-test, P<0.01 [in all CCs]) (Fig. 1). However, microbicidal activity of SAHAW decreased with increasing concentrations of organic matter. As the viability of some M. plutonius strains decreases under hypotonic conditions, viable bacterial counts decreased with time under 0.3% BSA conditions even in the absence of disinfectants (0.02–3.15 log₁₀ CFU/ml reduction at 24 hr); however, the reduction was not promoted by the addition of SAHAW under these conditions (0.57–3.69 log₁₀ CFU/ml reduction at 24 hr) (Welch’s t-test at 24 hr, P>0.15 [in all CCs]) (Fig. 1A and Supplementary Table 1). Moreover, in the presence of 1% yeast extract plus 1% BSA, SAHAW had little microbicidal activity (0.63 log₁₀ CFU/ml or lower reduction at 24 hr), and the reduction was not significantly different from that of H₂O (0.55 or lower at 24 hr) (Welch’s t-test at 24 hr, P>0.44 [in all CCs]) (Fig. 1A and Supplementary Table 1).

Fig. 1.

Microbicidal effects of slightly acidic hypochlorous acid water (SAHAW) (available chlorine concentration [ACC], 10–30 ppm) (A) and weakly acidified chlorous acid water (WACAW) (ACC, 2,400 ppm) (B) on Melissococcusplutonius in suspension tests. Approximately 1 × 10⁸ CFU/ml of M. plutonius cells, 10 × concentrated organic matter solutions/sterile H₂O and the disinfectants were mixed at a ratio of 1:1:8, and incubated at 20°C. As controls, sterile H₂O was used instead of the disinfectants. At the end of each treatment time, a portion of the mixture was mixed with the same amount of neutralizing solution. The number of remaining viable bacterial cells was investigated as described in MATERIALS AND METHODS. Data were collected from three independent tests, and the survival of M. plutonius strains was expressed as fold changes in viable cell concentrations. BDL represents “below the detection limit” of the test. Asterisks indicate statistically significant efficacy (P<0.05; disinfectants vs. H₂O) against clonal complex (CC) 3 (blue asterisks), CC12 (red asterisks) and CC13 (green asterisks) strains.

WACAW was more stable under organic matter-rich conditions than SAHAW. WACAW inactivated M. plutonius CC3 and CC13 strains within 5 min and the CC12 strain within 1 hr (>5 log₁₀ CFU/ml reduction) even in the presence of 0.3% BSA, and the reduction was significantly higher than that of H₂O (Welch’s t-test, P<0.01 [in all CCs]) (Fig. 1B and Supplementary Table 1). Under 1% yeast extract plus 1% BSA conditions, the microbicidal effects of WACAW varied among M. plutonius strains. WACAW reduced viable CC13 cells with time and achieved a reduction of >5 log₁₀ CFU/ml (i.e., below the detection limit) after 24-hr treatment. The log₁₀ CFU/ml reduction of CC13 cells was always significantly higher than that of H₂O (Welch’s t-test, P<0.05) (Fig. 1B and Supplementary Table 1). WACAW also reduced the number of viable CC3 cells to a certain extent (1.93–2.21 log₁₀ CFU/ml reduction at 24 hr), and significantly efficient microbicidal activity was observed at 6 and 24 hr (Welch’s t-test, P<0.05). However, viable CC3 cells were still detected even after 24-hr treatment (Fig. 1B and Supplementary Table 1). The CC12 strain was the most resistant to WACAW among the three strains. Although significant difference in the log₁₀ CFU/ml reduction between WACAW and H₂O was detected at 24 hr (Welch’s t-test, P=0.03), the reduction by WACAW was only 0.55 or lower (Fig. 1B and Supplementary Table 1). That is, the CC12 strain was hardly killed under the 1% yeast extract plus 1% BSA conditions.

Sporicidal activity of SAHAW and WACAW on P. larvae in suspension tests

SAHAW with an ACC of 10–30 ppm and WACAW with that of 2,400 ppm were also used for P. larvae spores, and their sporicidal activity was evaluated on the representative ERIC I and ERIC II strains. P. larvae spores were more resistant to the disinfectants than M. plutonius. Although SAHAW inactivated P. larvae spores (>5 log₁₀ CFU/ml reduction) in the absence of organic matter, it took 1 hr for the ERIC I strain and 6 hr for the ERIC II strain (Fig. 2A and Supplementary Table 2). In the presence of organic matter, even in small amounts, SAHAW exhibited almost no sporicidal effects and achieved a reduction of only a 0.64 log₁₀ CFU/ml at 24 hr (Fig. 2A and Supplementary Table 2).

Fig. 2.

Sporicidal effects of slightly acidic hypochlorous acid water (SAHAW) (available chlorine concentration [ACC], 10–30 ppm) (A) and weakly acidified chlorous acid water (WACAW) (ACC, 2,400 ppm) (B) on Paenibacillus larvae spores in suspension tests. Approximately 1 × 10⁷ CFU/ml of P. larvae spores, 10 × concentrated organic matter solutions/sterile H₂O and the disinfectants were mixed at a ratio of 1:1:8, and incubated at 20°C. As controls, sterile H₂O was used instead of the disinfectants. At the end of each treatment time, a portion of the mixture was mixed with the same amount of neutralizing solution. The number of remaining viable spores was investigated as described in MATERIALS AND METHODS. Data were collected from three independent tests, and the survival of spores was expressed as fold changes in viable spore concentrations. BDL represents “below the detection limit” of the test. Asterisks indicate statistically significant efficacy (P<0.05; disinfectants vs. H₂O) against ERIC I (blue asterisks) and ERIC II (red asterisks) spores.

Similar to the effects on M. plutonius, WACAW exhibited stronger sporicidal activity than SAHAW. In the presence of 5% honey and 0.03% BSA as well as under no organic matter conditions, WACAW exhibited significant sporicidal activity in 5 min (Welch’s t-test, P<0.001) and completely sterilized spores of both genotypes (>5 log₁₀ CFU/ml reduction) within 1 hr (Fig. 2B and Supplementary Table 2). However, even using WACAW with a high ACC (2,400 ppm), P. larvae spores were hardly inactivated under the 0.3% BSA and 1% yeast extract plus 1% BSA conditions (0.53 log₁₀ CFU/ml or lower reduction at 24 hr) (Fig. 2B and Supplementary Table 2).

The efficacy of SAHAW and WACAW for the decontamination of honey bee combs

As stated above, both SAHAW and WACAW had the ability to kill foulbrood pathogens in suspension tests; however, P. larvae spores were more resistant to the disinfectants than M. plutonius, and their microbicidal activity was greatly affected by organic matter. In order to investigate the potential of these disinfectants to reduce the contamination level of combs with P. larvae spores, we next evaluated the efficacy of SAHAW and WACAW in killing or removing P. larvae spores in cells of the combs. For this test, we used combs that have been used for one (comb nos. 1 and 2), three (comb no. 3) or five (comb no. 4) years in AFB-free apiaries in Japan. Although the amount of organic matter remaining in the cells of the combs varied according to the cells tested, the cells of the comb used for five years contained more organic matter (32,305.08 ± 6,709.82 RLU) than those of the combs used for one (5,186.08 ± 2,919.13 [no. 1] or 11,009.58 ± 1,247.41 [no. 2] RLU) or three (3,306.17 ± 714.64 RLU) years (Kruskal-Wallis test and Steel-Dwass post-hoc test, P<0.01) (Supplementary Fig. 1). Therefore, we regarded the combs used for one and three years and that used for five years as clean and dirty combs, respectively, for convenience. For no organic matter controls, wells of 96-well microplates were used as dummy cells. As it took a long time to inactivate all ERIC II P. larvae spores in suspension tests even under no organic matter conditions, we treated spore-inoculated carriers with disinfectants for 16 hr in this study.

In our carrier tests, after incubation of spore-inoculated cells/wells with disinfectants or sterile H₂O, the liquid was removed from the cells/wells and absorbed by paper towels by covering the cells/wells with paper towels and plastic lids, turning them over and shaking the liquid off from the cells/wells thoroughly. Therefore, even by H₂O, some of the inoculated spores were washed off, and the contamination levels with spores decreased to some extent. Indeed, when microplate wells inoculated with P. larvae spores at doses of 0.18–5.0 × 10⁵ CFU/well were treated at 20°C for 16 hr, a reduction of 1.62 log₁₀ CFU/well was observed even with H₂O (Fig. 3 and Supplementary Table 3). However, as both SAHAW (ACC, 10–30 ppm) and WACAW (ACC, 2,400 ppm) significantly more efficiently removed P. larvae spores from the wells of the microplates (>3.26 log₁₀ CFU/well reduction) than H₂O (one-way ANOVA and Bonferroni test, P<0.001 [in both ERIC I and II]) (Fig. 3 and Supplementary Table 3), these disinfectants were confirmed to have sporicidal activity even on solid substance surfaces under the no organic matter conditions.

Fig. 3.

Efficacy of slightly acidic hypochlorous acid water (SAHAW) (available chlorine concentration [ACC], 10–30 ppm) and weakly acidified chlorous acid water (WACAW) (ACC, 300–2,400 ppm) in killing or removing Paenibacillus larvae spores in cells of a comb used for one year (a clean comb). As controls, 96-well microplates were also used as carriers. Approximately 1 × 10⁵ CFU of P. larvae spores was inoculated in each cell of the comb or in each well of 96-well microplates. After drying the cells and wells, they were filled with disinfectants and incubated at 20°C for 16 hr. As controls, sterile H₂O was used instead of the disinfectants. The number of remaining viable spores was investigated as described in MATERIALS AND METHODS. Data were collected from four independent tests, and the survival of spores was expressed as fold changes in viable spore numbers. BDL represents “below the detection limit” of the test. Asterisks indicate statistically significant efficacy of disinfectants (P<0.05; disinfectants vs. H₂O) against ERIC I (blue asterisks) and ERIC II (red asterisks) spores.

When a clean comb inoculated with P. larvae spores at doses of 0.18–5.0 × 10⁵ CFU/cell was used, some of the spores were also removed from the cells physically by H₂O after incubation at 20°C for 16 hr; however, the log₁₀ CFU/cell reduction was only 1.63 or lower (Fig. 3 and Supplementary Table 3). Under the same conditions, SAHAW achieved a reduction of >3.89–4.08 log₁₀ CFU/cell (below the detection limit) in four of the eight cells used for the test, but the efficacy differed according to the cells, and SAHAW did not efficiently remove the spores from the other four cells (1.18–1.86 log₁₀ CFU/cell reduction) (Fig. 3 and Supplementary Table 3). The efficacy of SAHAW may also differ according to the P. larvae genotypes. When ERIC I spores were inoculated, viable spores were eliminated from three of the four cells by SAHAW, and the efficacy was significantly higher than that of H₂O (Welch’s t-test, P=0.03). On the other hand, although SAHAW always reduced ERIC II spores (1.18–>4.04 log₁₀ CFU/cell reduction) more efficiently than H₂O (−0.19–1.08 log₁₀ CFU/cell reduction) (Supplementary Table 3), statistically significant difference was not detected between SAHAW and H₂O (Welch’s t-test, P=0.098). In contrast, WACAW removed P. larvae spores significantly more efficiently than H₂O regardless of its ACC and ERIC types (one-way ANOVA and Bonferroni test, P<0.05), and viable spores were eliminated (i.e., >3.26–4.70 log₁₀ CFU/cell reduction) from most of the cells tested after treatment of the cells with WACAW at an ACC of 600–2,400 ppm (Fig. 3 and Supplementary Table 3).

On the other hand, when a dirty comb inoculated with P. larvae spores at doses of 0.18–5.0 × 10⁵ CFU/cell was used, SAHAW did not exhibit strong spore-removal effects in all tests. The log₁₀ CFU/cell reduction by SAHAW (0.18–2.08) was similar to that by H₂O (−0.38–2.02) (Fig. 4 and Supplementary Table 3), and no significant difference was observed in the reduction between SAHAW and H₂O (Welch’s t-test, P=0.95 [in ERIC I] and 0.36 [in ERIC II]). In contrast, the log₁₀ CFU/cell reduction by WACAW at an ACC of 2,400, 1,200 or 600 ppm (2.97–>4.70, 3.36–>4.30 and 2.95–>4.30, respectively) was significantly higher than that of H₂O and SAHAW (one-way ANOVA and Bonferroni test, P<0.01 [in both ERIC I and II]). However, viable spores were not completely removed from approximately half of the cells tested. At an ACC of 300 ppm, viable spores were detected from all the treated cells (the log₁₀ CFU/cell reduction, 0.74–4.30), and no significant difference was observed in the reduction when compared with that of H₂O (one-way ANOVA and Bonferroni test, P=0.60 [in ERIC I] and 0.13 [in ERIC II]) (Fig. 4 and Supplementary Table 3).

Fig. 4.

Efficacy of slightly acidic hypochlorous acid water (SAHAW) (available chlorine concentration [ACC], 10–30 ppm) and weakly acidified chlorous acid water (WACAW) (ACC, 300–2,400 ppm) in killing or removing Paenibacillus larvae spores in cells of a comb used for five years (a dirty comb). As controls, 96-well microplates were also used as carriers. Approximately 1 × 10⁵ CFU of P. larvae spores was inoculated in each cell of the comb or in each well of 96-well microplates. After drying the cells and wells, they were filled with disinfectants and incubated at 20°C for 16 hr. As controls, sterile H₂O was used instead of the disinfectants. The number of remaining viable spores was investigated as described in MATERIALS AND METHODS. Data were collected from at least three independent tests, and the survival of spores was expressed as fold changes in viable spore numbers. BDL represents “below the detection limit” of the test. Asterisks indicate statistically significant efficacy of disinfectants (P<0.05; disinfectants vs. H₂O) against ERIC I (blue asterisks) and ERIC II (red asterisks) spores.

As combs may be maintained, including disinfection, during winter, we also evaluated the efficacy of SAHAW and WACAW at 4°C. For this test, a clean comb was used as a carrier. P. larvae spores were inoculated to wells/cells at doses of 1.2–3.6 × 10⁵ CFU/well or cell, and the carriers were treated with SAHAW or WACAW for 16 hr. In 96-well microplates, both disinfectants reduced viable spores (≥4.0 log₁₀ CFU/well reduction) more efficiently than H₂O (0.25–1.44 log₁₀ CFU/well reduction) (one-way ANOVA and Bonferroni test, P<0.001 [in both ERIC I and II]), and no viable spore was detected from most of wells after treatment with the disinfections (Fig. 5 and Supplementary Table 3). However, under the low temperature conditions, SAHAW did not decontaminate the comb efficiently, and the log₁₀ CFU/cell reduction (0.70–1.90) was similar to that by H₂O (0.37–1.31) (Fig. 5 and Supplementary Table 3) (Welch’s t-test, P=0.24 [in ERIC I] and 0.30 [in ERIC II]). In contrast, WACAW more efficiently removed P. larvae spores from the comb than SAHAW even at 4°C, and viable spores in inoculated cells became undetectable (>4.08 log₁₀ CFU/cell reduction) after treatment at an ACC of 1,200–2,400 ppm. At 600 ppm, WACAW reduced ERIC I and ERIC II P. larvae by 3.43–>4.30 and 1.94–>4.20 log₁₀ CFU/cell, respectively, and the efficacy was significantly higher than that of H₂O and SAHAW (one-way ANOVA and Bonferroni test, P<0.05 [in both ERIC I and II]) (Fig. 5 and Supplementary Table 3). Even at 300 ppm, ERIC I and ERIC II spores were reduced by 2.38–2.95 and 1.49–2.19 log₁₀ CFU/cell, respectively; however, when compared with H₂O, significant reduction was detected only in ERIC I spores (one-way ANOVA and Bonferroni test, P<0.001 [in ERIC I] and P=0.31 [in ERIC II]) (Fig. 5 and Supplementary Table 3).

Fig. 5.

Efficacy of slightly acidic hypochlorous acid water (SAHAW) (available chlorine concentration [ACC], 10–30 ppm) and weakly acidified chlorous acid water (WACAW) (ACC, 300–2,400 ppm) in killing or removing Paenibacillus larvae spores in cells under low temperature conditions (i.e., simulated winter conditions). A comb used for one year (a clean comb) and 96-well microplates were used as carriers. Approximately 1 × 10⁵ CFU of P. larvae spores was inoculated in each cell of the comb or in each well of 96-well microplates. After drying the cells and wells, they were filled with disinfectants and incubated at 4°C for 16 hr. As controls, sterile H₂O was used instead of the disinfectants. The number of remaining viable spores was investigated as described in MATERIALS AND METHODS. Data were collected from three independent tests, and the survival of spores was expressed as fold changes in viable spore numbers. BDL represents “below the detection limit” of the test. Asterisks indicate statistically significant efficacy of disinfectants (P<0.05; disinfectants vs. H₂O) against ERIC I (blue asterisks) and ERIC II (red asterisks) spores.

P. larvae can produce over one billion spores in each infected larva (http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.02.02_AMERICAN_FOULBROOD.pdf); therefore, we inoculated high doses (0.78–4.0 × 10⁷ CFU) of P. larvae spores to each cell of clean combs assuming highly contaminated combs and investigated the efficacy of the disinfectants on decontamination of the cells. Under no organic matter conditions (i.e., in microplate wells), viable ERIC I spores became undetectable (>5.89 log₁₀ CFU/well reduction) following treatment with SAHAW (ACC, 10–30 ppm) or WACAW (ACC, 2,400 ppm), and the efficacy was significantly higher than that of H₂O (one-way ANOVA and Bonferroni test, P<0.001) (Fig. 6 and Supplementary Table 3). However, both disinfectants did not work well on ERIC II spores. The log₁₀ CFU/well reduction by SAHAW (1.81–2.17) was not significantly different from that by H₂O (0.70–2.16) (one-way ANOVA and Bonferroni test, P=1) (Fig. 6 and Supplementary Table 3). In addition, although WACAW reduced ERIC II spores more efficiently (2.51–4.90 log₁₀ CFU/well reduction) than H₂O (one-way ANOVA and Bonferroni test, P<0.05), it could not eliminate viable spores completely.

Fig. 6.

Efficacy of slightly acidic hypochlorous acid water (SAHAW) (available chlorine concentration [ACC], 10–30 ppm) and weakly acidified chlorous acid water (WACAW) (ACC, 300–2,400 ppm) in killing or removing a large quantity of P. larvae spores in cells. Combs used for 1–3 years (clean combs) and 96-well microplates were used as carriers. Approximately 1 × 10⁷ CFU of P. larvae spores was inoculated in each cell of the combs or in each well of 96-well microplates and dried. In some tests, cells inoculated with P. larvae spores were prewashed with 40°C sterile H₂O. The cells and wells were then filled with disinfectants and incubated at 20°C for 16 hr. As controls, sterile H₂O was used instead of the disinfectants. The number of remaining viable spores was investigated as described in MATERIALS AND METHODS. Data were collected from at least three independent tests, and the survival of spores was expressed as fold changes in viable spore numbers. BDL represents “below the detection limit” of the test. Asterisks indicate statistically significant efficacy of disinfectants (P<0.05; disinfectants vs. H₂O) against ERIC I (blue asterisks) and ERIC II (red asterisks) spores.

On the highly contaminated combs, SAHAW was ineffective against both ERIC I and ERIC II spores (1.91–3.28 and 2.01–2.97 log₁₀ CFU/cell reduction, respectively), and the degree of reduction was similar to that by H₂O (2.16–3.30 and 1.70–2.72 log₁₀ CFU/cell reduction for ERIC I and ERIC II, respectively) (one-way ANOVA, P>0.5 [in both ERIC I and II]) (Fig. 6 and Supplementary Table 3). Before SAHAW treatment, we added a prewashing step with 40°C sterile H₂O as described in MATERIALS AND METHODS; however, the prewashing of the combs did not improve the disinfecting effects by SAHAW (one-way ANOVA, P>0.5 [in both ERIC I and II]) (Fig. 6 and Supplementary Table 3). In contrast, WACAW at an ACC of 600–2,400 ppm sterilized or sanitized the highly contaminated combs more efficiently than H₂O, but only when they were contaminated with ERIC I spores (log₁₀ CFU/cell reduction, >5.89 at 2,400 and 1,200 ppm [below the detection limit] and ≥4.48 at 600 ppm) (one-way ANOVA and Bonferroni test, P<0.001). Consistent with the results in microplate wells, ERIC II spores were more resistant to WACAW than ERIC I spores under the highly contaminated conditions, and even at an ACC of 2,400 ppm, the degree of reduction by WACAW (2.21–3.15 log₁₀ CFU/cell reduction) was similar to that by H₂O (1.70–2.72 log₁₀ CFU/cell reduction) (one-way ANOVA and Bonferroni test, P=1) (Fig. 6 and Supplementary Table 3).

DISCUSSION

SAHAW is recognized as a safe disinfectant [13, 14, 19] and can be produced on-site using purchasable compact generators at a relatively low cost, thus it began to be used to decontaminate beekeeping equipment in Japan. When a questionnaire survey on disinfectants was carried out in a region in Japan, 30% of respondent beekeepers used SAHAW and 23% of respondents planned on using it (Ohashi, I., unpublished data). In our study, under no organic matter conditions, SAHAW sterilized M. plutonius quickly (Fig. 1A). This disinfectant was also effective for P. larvae spores if the spores were exposed to it for several hours (Fig. 2A). Even under 5% honey and 0.03% BSA conditions, SAHAW exhibited strong antimicrobial activity against M. plutonius (Fig. 1A). In addition, SAHAW reduced the contamination levels of P. larvae spore-inoculated cells more efficiently than H₂O when a relatively clean comb was used as a carrier (Fig. 3). These results suggest that SAHAW is worth considering as an option to reduce the number of foulbrood pathogens in combs when the combs are relatively clean and not old. However, the efficacy of SAHAW is not 100% even under such desirable conditions. Indeed, some cells in the relatively clean comb were not effectively decontaminated by SAHAW (Fig. 3). As the amount of remaining organic matter varied among cells in a comb, as shown in Supplementary Fig. 1, the cells that were not well decontaminated by SAHAW may contain more organic matter than well decontaminated cells. In addition, the efficacy of SAHAW markedly decreased when disinfection was performed at 4°C and when overused or highly contaminated combs were used as carriers (Figs. 4, 5, 6). Of note, comb no. 2 was used under low temperature conditions. Although it was only used for one year and categorized as a clean comb, its cells contained slightly more organic matter than those of the other clean combs (nos. 1 and 3) (Supplementary Fig. 1). In addition, even under the low temperature conditions, SAHAW removed P. larvae spores from 96-well microplates significantly more efficiently than H₂O (one-way ANOVA and Bonferroni test, P<0.001) (Fig. 5); therefore, we cannot rule out the possibility that the reduced effects of SAHAW at 4°C were partially due to the organic matter remaining in comb no. 2. Even if this is the case, when low temperature conditions are expected, the reduction of SAHAW efficacy needs to be taken into consideration to prevent AFB efficiently.

WACAW had a higher disinfecting capacity than SAHAW, especially under the organic matter-rich conditions and at low temperatures (Figs. 4 and 5). The ACC of WACAW used in this study (300–2,400 ppm) was higher than that of SAHAW (10–30 ppm). As the bactericidal effects of chlorine-based disinfectants usually increase with a higher ACC, the high disinfecting capacity of WACAW may have simply been due to the high ACC. In addition, the stable microbicidal activity of WACAW under organic matter-rich conditions may have resulted from the large dissociation of total chlorine level and free available chlorine (FAC) level in the disinfectant [12]. WACAW mainly contains HClO₂, ClO₂⁻ and ClO₂ as oxygenated chlorines. ClO₂⁻ has very weak oxidative potential and makes only a minor contribution to the FAC level and antimicrobial killing. In WACAW, the ClO₂⁻ content is considered to be much higher than that of HClO₂ and ClO₂ [12]. This ClO₂⁻ may continuously supply HClO₂ even after FAC is consumed from contact with microorganisms or organic matter [12]. Although WACAW has not been used in apiculture, it was approved as a food additive in 2013 in Japan and has since been applied to food and environmental sanitation [12, 17]. The maximum HClO₂ concentration approved as a food additive is 400 ppm, which corresponds to an ACC of approximately 800 ppm according to the manufacturer (HONBUSANKEI Co., Ltd.). In the present study, WACAW with an ACC of 600 ppm worked well for removing viable P. larvae spores from combs, suggesting WACAW as an alternative safe and promising disinfectant for beekeeping equipment. However, for efficient decontamination of combs, all cells in the combs have to be filled with disinfectants, thus a relatively large amount of disinfectants is needed in apiaries. WACAW products are purchasable, but it cannot be produced on-site because its generator is not on the market; therefore, the use of WACAW is currently more expensive than SAHAW.

Of note, the efficacy of the disinfectants varied between different strains. In suspension tests using M. plutonius, the representative strain of CC12 was the most resistant to the disinfectants among the three strains tested and was hardly killed under the 1% yeast extract plus 1% BSA conditions even by WACAW with an ACC of 2,400 ppm (Fig. 1B). Microbicidal effects of the disinfectants tested are attributable to their oxidative potential. Interestingly, although M. plutonius CC3 and CC13 strains tested (DAT606 and DAT585, respectively) cannot grow under aerobic conditions, DAT561 of CC12 can grow aerobically [1], implying that DAT561 is more resistant to oxidative stress than DAT606 and DAT585. This different oxidative stress susceptibility might have resulted in different disinfectant susceptibility between the strains. In suspension tests with P. larvae, inactivation of the ERIC II spores by SAHAW took longer than inactivation of the ERIC I spores under no organic matter conditions (Fig. 2A). Moreover, it was more difficult to remove viable ERIC II spores from highly contaminated combs than ERIC I spores (Fig. 6). Of note, it was more difficult to produce spores of the representative ERIC II strain used in this study on MYPGP agar plates than the ERIC I strain. Although we devised methods for spore preparation to reduce remaining vegetative cells and their debris by extending the culture time of the ERIC II strain and washing spore solutions repeatedly at intervals of one or more weeks, we cannot rule out the possibility that ERIC II spore solutions contained more remaining impurities (vegetative cells and their debris) than ERIC I spore solutions, and the impurities may have reacted with available chlorine, resulting in the loss of sporicidal activity of the disinfectants. To show clear correlation between bacterial genotypes and disinfectant susceptibility, it is necessary to test at least several strains for each genotype. As we tested only a single strain from each genetic group, this result may have nothing to do with the genotypes. However, M. plutonius CC12 and P. larvae ERIC II strains have been involved in many foulbrood cases in Japan [24, 26], thus the lower disinfectant susceptibility of strains with these genotypes than the others may become a problem for efficient prevention of AFB in the field.

In conclusion, if combs to be disinfected are not old and relatively clean (e.g., when the average RLU of the cells by the Lumitester PD-30 system is approximately 10,000 or less), SAHAW, which is available at a relatively low cost, will be a useful option to reduce contamination levels of foulbrood pathogens. However, even under such favorable conditions, disinfection with SAHAW should be carried out on a warm day. In addition, although prewashing of combs did not improve the disinfecting effects by SAHAW on highly contaminated combs in our study (Fig. 6), we consider prewashing of combs to be prerequisite for removing visible content from the cells and maximizing the effects of SAHAW. If the combs are old or not clean, WACAW with an ACC of 600 ppm or higher will more effectively reduce contamination levels than SAHAW. Strong spore-removal effects of WACAW can also be expected at low temperatures. However, even if WACAW is used, it may not be sufficient for the prevention of foulbroods depending on contamination levels and genotypes of bacterial strains. Therefore, for effective control of foulbroods, it is important to use different disinfectants and other control methods (e.g., replacement of combs, gamma irradiation and ethylene oxide sterilization) properly according to the conditions and available budget. Honey bees are affected not only by foulbroods but also other infectious diseases, including chalkbrood by the fungus Ascosphaera apis, nosemosis by the microsporidian Nosema apis and N. ceranae and various virus infections. The two disinfectants used in this study may also be useful to control these diseases, although there is little or no information regarding the effects of SAHAW and WACAW on other honey bee pathogens. Further studies using the disinfectants will provide additional useful information for infectious disease control in apiculture.