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Chlorous acid is a more potent antibacterial agent than sodium hypochlorite against Campylobacter
Journal Pre-proof Chlorous acid is a more potent antibacterial agent than sodium hypochlorite against Campylobacter Noritoshi Hatanaka, Sharda Prasad Awasthi, Hisataka Goda, Hiroyuki Kawata, Yuzuru Uchino, Takahiro Kubo, Shigeru Aoki, Atsushi Hinenoya, Shinji Yamasaki PII:
S0956-7135(19)30635-8
DOI:
https://doi.org/10.1016/j.foodcont.2019.107046
Reference:
JFCO 107046
To appear in:
Food Control
Received Date: 13 July 2019 Revised Date:
4 December 2019
Accepted Date: 8 December 2019
Please cite this article as: Hatanaka N., Awasthi S.P., Goda H., Kawata H., Uchino Y., Kubo T., Aoki S., Hinenoya A. & Yamasaki S., Chlorous acid is a more potent antibacterial agent than sodium hypochlorite against Campylobacter, Food Control (2020), doi: https://doi.org/10.1016/j.foodcont.2019.107046. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Author contribution section: N.H., H.G. and S.Y. designed research; N.H., S.P.A., and H.K. performed research; N.H., S.P.A., H.G., H.K., Y.U., T.K, S.A., A.H., and S.Y. analyzed data; N.H., H.G. and S.Y. wrote the paper.
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Chlorous acid is a more potent antibacterial agent than sodium
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hypochlorite against Campylobacter
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Noritoshi Hatanaka1, Sharda Prasad Awasthi1, Hisataka Goda2, Hiroyuki Kawata2, Yuzuru
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Uchino3, Takahiro Kubo1, 3, Shigeru Aoki2*, Atsushi Hinenoya1 and Shinji Yamasaki1, #
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1: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka,
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Japan
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2: Honbu Sankei Co. Ltd., Osaka, Japan
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3: Development Section, Honbusankei Co., Ltd., Hyogo, Japan
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*S.A. is deceased.
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Keywords: Campylobacter, chlorous acid (HClO2), sodium hypochlorite (NaClO), food
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protection, bactericidal．
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#
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Mailing address: Graduate School of Life and Environmental Sciences, Osaka Prefecture
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University, 1-58, Rinkuourai-kita, Izumisano, Osaka 598-8531, Japan
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Tel & Fax: +81-72-463-5653
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E-mail address: [email protected]
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Corresponding author: Shinji Yamasaki, Ph.D.
FC-rev191204
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Abstract
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Foodborne disease caused by campylobacters is one of the major global problems for
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food safety. Infection source of Campylobacter to human is mainly through contaminated
28
meat particularly chicken. Contamination of meat with Campylobacter usually occurs during
29
processing at slaughterhouse and to prevent such contaminations, sodium hypochlorite is
30
commonly used. However, it is well known that bactericidal activity of sodium hypochlorite
31
becomes weak under organic matter rich conditions. In this study, we compared the strength
32
of bactericidal activity of chlorous acid and sodium hypochlorite against Campylobacter
33
jejuni and Campylobacter coli strains under organic matter rich conditions. Bactericidal
34
activity against 5 representative C. jejuni and C. coli strains in chicken juice (an organic
35
matter rich condition) showed that minimum concentration of chlorous acid required for
36
complete killing of C. jejuni and C. coli cells is 200 to 400 ppm while that of sodium
37
hypochlorite is 2,000 to 4,000 ppm. Similar results were obtained by using Bolton broth.
38
Furthermore, it was observed that 400 ppm of chlorous acid but not 400 ppm of sodium
39
hypochlorite is highly effective in killing of 25 different Campylobacter strains (12 C. jejuni
40
and 13 C. coli strains) under the same conditions. To determine whether 400 ppm of chlorous
41
acid treatment had killed bacterial cells or induced them into viable but non-culturable
42
(VBNC) state, live and dead cell assay using DAPI and propidium iodide florescent dyes was
43
done. Such assay clearly indicated that Campylobacter cells were indeed killed and not
44
induced to VBNC state. Moreover, SDS-PAGE analysis of whole-cell lysates of
45
campylobacters indicated distinct effects in protein profiles of chlorous acid but not sodium
46
hypochlorite treated cells. The results strongly suggest that chlorous acid could efficiently kill
47
C. jejuni and C. coli cells with much lower concentration than sodium hypochlorite and the
2
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bactericidal mechanisms of chlorous acid may be due to damages of bacterial proteins. Thus,
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chlorous acid could be a better disinfectant in chicken slaughtering and processing to kill
50
campylobacters and prevent contamination.
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51
1. Introduction
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Campylobacter is one of the major causative agents of gastroenteritis worldwide.
53
Among 26 Campylobacter species so far reported, C.jejuni and C. coli are most frequently
54
isolated from human diarrheal patients (Man 2011). Furthermore, it is well known that C.
55
jejuni may cause autoimmune diseases such as Guillan-Barré syndrome and Miller Fisher
56
syndrome (Man 2011). Domestic animals are known to be primary reservoir of
57
Campylobacter. Poultry has particularly been recognized as a major infection source of C.
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jejuni and C. coli, which cause campylobacteriosis in human (Kaakoush et al., 2015).
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Campylobacter infects human mainly through consumption of undercooked meats, in
60
particular chicken and beef (Moore et al., 2005). Furthermore, in Japan, there is a food culture
61
that some people eat raw (Torisashi) or lightly roasted chicken (Tataki). This traditional food
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habit may have a role in higher incidence of food-borne disease caused by C. jejuni and C.
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coli in Japan. In fact, out of 1,014 incidences of food poisoning in Japan in 2017, 320 (31.6%)
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were caused by C. jejuni and C. coli (Ministry of Health, Labour and Welfare of Japan). It
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was speculated that as low as 360 most probable number (MPN) of C. jejuni might cause food
66
poisoning (Hara-Kudo et al., 2011). Therefore, it is important to reduce contamination of
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Campylobacter to foods for decreasing food poisoning caused by C. jejuni and C. coli.
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Sodium hypochlorite is commonly used for sanitization of food material such as meats,
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vegetables, cooking equipment etc. (Fukuzaki 2006). At chicken slaughterhouse, sodium
70
hypochlorite is also used in many steps (Cadena et al., 2019). During processing, feces
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containing Campylobacter probably contaminate carcasses and it might result in cross
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contamination of Campylobacter on chicken meat even though sodium hypochlorite is used
73
for sanitization. Furthermore, Misawa et al. (2012) reported that the number of 4
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Campylobacter on chicken meat was increased during processing at slaughterhouse. It is well
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known that sodium hypochlorite cannot demonstrate efficient bactericidal activity when used
76
in the presence of organic matter (Yamaoka et al., 2016).
77
Alternatively, chlorous acid (HClO2) is also used for sterilization of meats as a food
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additive not only in Japan, but also in the USA, Canada, Australia and New Zealand. Several
79
studies have been reported that chlorous acid showed bactericidal activity against pathogenic
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bacteria including Staphylococcus aureus, Escherichia coli, Candida albicans etc. (Horiuchi
81
et al. 2015). Furthermore, even in the presence of organic matter such as bovine serum
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albumin, it retained its bactericidal activity against MRSA and Clostridium difficile (Yamaoka
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et al. 2016, Goda et al. 2017), indicating that chlorous acid might be useful for sanitization of
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food-poisoning causing bacteria from meats such as chicken.
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The major aim of this study is to kill efficiently C. jejuni and C. coli cells or to reduce
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their number from chicken carcasses by a food additive to prevent food borne diseases in
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human. In this study, as a first step, we wanted to know whether chlorous acid could be more
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effective than sodium hypochlorite to decrease the number of C. jejuni and C. coli under
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organic matter rich conditions. Furthermore, we examined the mechanisms about how
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chlorous acid could kill C. jejuni and C. coli. Here, we show that chlorous acid had more
91
efficient bactericidal activity than sodium hypochlorite in the presence of organic matter and
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its bactericidal mechanism was most likely due to damage or denature of bacterial proteins of
93
C. jejuni and C. coli cells.
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2. Materials and Methods
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2. 1.Bacterial strains and growth media
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A total of 30 Campylobacter strains including 15 C. jejuni (6 clinical, 5 bovine and 4 5
97
chicken) and 15 C. coli (8 clinical and 7 swine) strains were used in this study (Table 1). Each
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strain was isolated from different host and individual, and identified the species by cdt
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gene-based species-specific multiplex PCR (Asakura et al. 2008). Campylobacters were
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grown on blood agar [blood agar base No. 2 (Oxoid Ltd., Basingstoke, United Kingdom)
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supplemented with 5% (v/v) defibrinated horse blood (Nippon Bio Supp. Center, Tokyo,
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Japan)] at 37°C for 48 h or more under anaerobic condition (10% CO2, 10% H2, 80% N2)
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(Hatanaka et al. 2017).
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2. 2. Bactericidal activity of chlorous acid and sodium hypochlorite in Bolton broth
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C. jejuni strains 81-176, ATCC 33560 and Chi2, and C. coli strains ATCC 33559 and
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Cc4 were cultured on blood agar as described above. Bacterial colonies were suspended in
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PBS(-) at pH 7.4 and bacterial concentration was adjusted to approximately 104 CFU/mL.
108
Then, 100 µL of bacterial suspension was inoculated into 5 mL of Bolton broth (Oxoid Ltd.)
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and cultured at 37°C for 48 h under anaerobic condition as described above. Two hundred
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fifty microliters of the bacterial culture were transferred to a 1.5 mL tube and mixed with
111
equal volume of either PBS(-) at pH 7.4, 400, 2,000 or 4,000 parts per million (ppm) of
112
sodium hypochlorite (Wako Pure ChemicalIndustries, Ltd., Osaka, Japan) in which
113
concentration of available chlorine was measured by an iodometric method (Ref). Similar
114
assay was done with 400 ppm of chlorous acid (Care for Noro barrier plus; Honbu Sankei Co.
115
Ltd., Osaka, Japan) in which concentration of available chlorine was measured by DPD
116
method (Ref). Then, the mixture was kept at room temperature for 3 min. Thereafter, 250 µL
117
of 50 mM sodium thiosulfate (NacalaiTesque, Inc., Kyoto, Japan) was added to inactivate the
118
chemicals. The number of live bacteria remained was examined by colony counting using
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blood agar plate. 6
120
Similarly, all Campylobacter strains listed in Table 1 were treated with either PBS(-) at
121
pH 7.4, 400 ppm of sodium hypochlorite or 400 ppm of chlorous acid. Next, 100 µL of each
122
of these treated bacterial cultures was inoculated into 5 mL of Bolton broth, and cells were
123
allowed to grow at 37°C for 48 h under anaerobic condition. Then, a loopful of the culture
124
was streaked on blood agar plate and the plates were incubated as described previously (Ref).
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2. 3. Preparation of chicken juice
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One hundred grams of chicken meat were homogenized with 400 mL of sterilized
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distilled water and the homogenate was further passed through sterilized gauze in order to
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remove the debris. Then, protein concentration was determined and adjusted to approximately
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100 mg/mL by Bradford assay using bovine serum albumin (NalacaiTesque, Inc.) as a
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standard protein. Absence of campylobacters in chicken juice was confirmed by both culture
131
method and C16S PCR as described previously (Hatanaka et al. 2017).
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2. 4. Bactericidal activity of chlorous acid and sodium hypochlorite in chicken juice
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C. jejuni and C. coli strains were cultured in 5 mL of Bolton broth as described above,
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250 µL of the culture were centrifuged at 12,000 g for 5 min and supernatants were removed.
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The bacterial pellet was suspended with equal volume of chicken juice and 250 µL of PBS(-)
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(as a control) or 400/2,000/4,000 ppm each of sodium hypochlorite or 100/200/400 ppm each
137
of chlorous acid was added. The mixture was kept at room temperature for 3 min and 250 µL
138
of 50 mM sodium thiosulfate was added to neutralize the remaining activity of each chemical.
139
Thereafter, bacterial survival assay was done by colony counting method by plating each
140
dilution on mCCDA agar (Oxoid Ltd.).
141
Similarly, all Campylobacter strains listed in Table 1 were treated with 400 ppm of
142
chlorous acid in chicken juice. One hundred microliter of each treated bacterial culture was 7
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inoculated into 5 mL of Bolton broth supplemented with 5% (v/v) defibrinated horse blood
144
followed by incubation at 37°C for 48 h under anaerobic condition. Then, a loopful of the
145
culture was streaked on mCCDA agar plate and plate was as described previously (Ref).
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2. 5. Fluorescence microscopy using DAPI or propidium iodide
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C. jejuni strain 81-176 and C. coli strain ATCC 33559 were selected as a
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representative strain for each species and cultured in 5 mL of Bolton broth, respectively, and
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treated with PBS(-) (as a control) or400/2,000/4,000 ppm each of sodium hypochlorite or 400
150
ppm of chlorous acid as described above. Bacterial cell suspension was centrifuged at 6,000 g
151
for 5 min, supernatant was discarded and pellet was suspended with 500 µL of PBS(-).
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Then,the suspension was centrifuged under the same condition and pellet was re-suspended
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with 50 µL of fresh Bolton broth. Five microliters of each bacterial suspension were
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transferred into a 1.5 mL-tube and mixed with 20 µL of fluorescent dye mix [0.5 mg/mL of
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propidium iodide (PI) (Sigma-Aldrich Co. LLC, St. Louis, Missouri, USA) and 0.5 mg/mL of
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DAPI (Merck KGaA, Darmstadt, Germany) in PBS(-)]. After staining at room temperature for
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15 min under dark condition, the mixture was centrifuged at 6,000 g for 5 min, supernatant
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was discarded and pellet was fixed with 100 µL of 3.7% paraformaldehyde at room
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temperature for 30 min under dark condition. Then, sample was centrifuged as described
160
above, supernatant was discarded, pellet was re-suspendedwith 20 µL of PBS (-)and
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bacterialcells were observed by microscopy (Leica DM 2500: Leica Microsystems GmbH.).
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2. 6. Effect of chlorous acid on bacterial DNA
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C. jejuni strain 81-176 and C. coli strain ATCC 33559 were cultured on blood agar
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plates as described above. Genomic DNA of these strains were prepared as described
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previously (Wilson, 1987). Concentration of genomic DNA was adjusted to 500 ng/5 µL in 8
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Bolton broth and 5 µL of purified DNA was mixed with the same volume of PBS, 400, 2,000,
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4,000 ppm each of sodium hypochlorite or 400 ppm of chlorous acid solution. The mixture
168
was kept at room temperature for 3 min and 5 µL of 50 mM sodium thiosulfate was added to
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neutralize the remaining activity of each chemical. Samples were analyzed by electrophoresis
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using a 0.8% Agarose, LE, Analytical Grade (Promega Corporation, Madison, WI, USA) and
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DNAs were visualized with ultraviolet (UV) light after staining with ethidium bromide (1
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µg/mL). Images were captured on a ChemiDoc system (Bio-Rad Laboratories, Inc., Hercules,
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CA, USA).
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2. 7. Effect of chlorous acid on bacterial proteins
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C. jejuni strains81-176, ATCC 33560 and Chi2, and C. coli strains ATCC 33559 and
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Cc4were treated with PBS(-) (as a control) or 400 ppm of chlorous acid or 400/2,000/4,000
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ppmeach ofsodium hypochlorite as described above. Then, each suspension was centrifuged
178
at 12,000 g for 5 min, supernatant was discarded and bacterial pellet was re-suspended in
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PBS(-) at OD600=5.0 followed by sonication 3 times (each of 1 min duration) using a
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sonicator (Q500: Qsonica, LLC., Newtown, CT, USA) by keeping the culture tube on ice.
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Thereafter, proteins present in bacterial cell lysates were analyzed by SDS-polyacrylamide gel
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electrophoresis (SDS-PAGE) using 10% acrylamide gel. After separation by electrophoresis,
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proteins were visualized by staining with 0.25% Coomassie Brilliant Blue R-250
184
(NacalaiTesque, Inc.).
185
2. 8. Statistical analysis
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Statistical analyses were performed using Microsoft Excel 2016 (Microsoft, Redmond,
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WA, USA). Error bars show standard deviations. p value was calculated with Student’s t test
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using paired, two-tailed distribution. When a p value showed less than 0.05, it was concluded 9
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that there was statistically significant difference.
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3. Results
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3. 1. Bactericidal activity of chlorous acid and sodium hypochlorite in Bolton broth
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To examine the bactericidal activity of chlorous acid and sodium hypochlorite against
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C. jejuni and C. coli strains, the organisms were treated with 400 ppm of each chemical
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solution. Initially, bactericidal activity of sodium hypochlorite or chlorous acid was examined
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with five representative strains of C. jejuni and C. coli. When C. jejuni strains 81-176, ATCC
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33560 and Chi2, and C. coli strains ATCC 33559 and Cc4 were treated with PBS(-), viable
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cell counts were approximately 8.6 to 9.3 log CFU/mL per sample. When treated with 400
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ppm of sodium hypochlorite, number of cells of C. jejuni strains 81-176, ATCC 33560 and
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Chi2,and C. coli strains ATCC 33559 and Cc4 were reduced to be 5.4, 4.6, 4.8,4.2 and 5.4 log
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CFU/mL, respectively. On the other hand, when the same set of Campylobacter strains were
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treated with 400 ppm of chlorous acid, all of them failed to grow on blood agar, indicating
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that number of viable cells was reduced to less than detection limit (1 log CFU/mL) as shown
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in Fig. 1. In contrast, the number of viable cells was found to be significantly different in the
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case of bacterial cells treated with 400 ppm of sodium hypochlorite (Fig. 1).
205
Furthermore, to see whether bactericidal activity of chlorous acid is not limited to
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particular strains, various Campylobacter strains as shown in Table 1 were treated essentially
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as described above and followed by culturing in Bolton broth and plating of cultures on blood
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agar plates. Campylobacter strains were able to grow on blood agar plates when treated with
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400 ppm of sodium hypochlorite, however, none of the campylobacters grew on blood agar
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plates (less than 1 log CFU/mL) when treated with 400 ppm of chlorous acid. These data
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clearly indicate that the chlorous acid is more effective in terms of bactericidal activity against 10
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C. jejuni and C. coli strains than sodium hypochlorite in the presence of organic matter (Table
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1).
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Since 400 ppm of sodium hypochlorite was not able to completely kill the C. jejuni
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and C. coli strains used, the minimum concentration of sodium hypochlorite needed for
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complete killing of these cells was determined to compare bactericidal activities between
217
chlorous acid and sodium hypochlorite. When cells were treated with 2,000 ppm of sodium
218
hypochlorite, only C. jejuni strain ATCC 33560 and C. coli strain Cc4 failed to grow on blood
219
agar and the remaining 3 Campylobacter strains were able to grow. However, when all these
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strains were treated with 4,000 ppm of sodium hypochlorite, they failed to grow on blood agar
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as shown in Fig. 1.
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3. 2. Bactericidal activity of chlorous acid and sodium hypochlorite in chicken juice
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To understand whether chlorous acid could kill campylobacters under organic matter
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rich condition, the bactericidal activity of chlorous acid was examined and compared with that
225
of sodium hypochlorite in chicken juice.While protein concentration of Bolton broth was 10
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mg/mL, that of chicken juice prepared in this study was 100 mg/mL. When C. jejuni and C.
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coli strains were treated with PBS (-), about 8.6 to 8.9 log CFU/mL of campylobacters were
228
detected (Fig. 2). However, when treated with chlorous acid, the number of C. jejuni and C.
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coli was reduced in a dose-dependent manner. At 400 ppm concentration of chlorous acid,
230
both C. jejuni and C. coli strains failed to grow on mCCDA agar and C. coli strain ATCC
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33559 was not able to grow on mCCDA agar even in the presence of 200 ppm of chlorous
232
acid (Fig. 2).
233
To compare bactericidal activity between chlorous acid and sodium hypochlorite under
234
organic matter rich condition, we attempted to determine the minimum concentration of 11
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sodium hypochlorite needed for complete killing C. jejuni and C. coli in chicken juice. As
236
shown in Fig. 3, when treated with PBS(-), each of the 5 Campylobacter strains showed
237
growth of about 9 log CFU/mL. However, cells treated with sodium hypochlorite, the number
238
of campylobacters was reduced in a dose-dependent manner. When treated with 400 ppm of
239
sodium hypochlorite, all 5 campylobacters grew on mCCDA agar. However, when treated
240
with 2,000 ppm of sodium hypochlorite, C. coli strain ATCC 33559 was not able to grow. On
241
the other hand, all 5 Campylobacter strains failed to grow when treated with 4,000 ppm of
242
sodium hypochlorite.
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3. 3. Bactericidal effect of chlorous acid on Campylobacter
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SinceCampylobacter spp. failed to grow on agar plates after treatment with 400 ppm
245
of chlorous acid, bacterial cells treated with different concentrations of sodium hypochlorite
246
or 400 ppm of chlorous acid were stained with DAPI and PI to verify their viability. It is
247
known that while DAPI can stain DNA of live and dead bacteria (Chaveerach et. al. 2003) but
248
PI stains DNA of dead bacteria only (Ramamurthy et. al. 2014). In addition, DNA of bacteria
249
which entered into VBNC stage would be stained with only DAPI (Chaveerach et. al. 2003),
250
but not with PI (Chaisowwong et. al. 2012). While C. jejuni strains treated with PBS(-), 400
251
or 2,000 ppm of sodium hypochlorite showed distinct staining of their nucleoid structure with
252
DAPI (Fig. 4; A, C and E) but most of them failed to be stained with PI (Fig. 4; B, D and F).
253
In sharp contrast, C. jejuni cells treated with 4,000 ppm of sodium hypochlorite or 400 ppm of
254
chlorous acid failed to take both DAPI and PI (Fig. 4; G, H, I and J). It may be mentioned
255
here that treatment of C. coli strains showed similar results like that of C. jejuni (data not
256
shown).
257
3. 4. Effect of chlorous acid on bacterial DNA 12
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To examine whether chlorous acid damage bacterial DNA, purified DNA were treated
259
with chlorous acid in Bolton broth and analyzed by electrophoresis on a 0.8% agarose gel.
260
The results were also compared with those treated with sodium hypochlorite. When purified
261
DNA was treated with 400, 2,000 and 4,000 ppm of sodium hypochlorite or 400 ppm of
262
chlorous acid (Fig. 5A, B; lane 2-5), DNA profile of each sample was not significantly
263
different from those treated with PBS (-) as a negative control (Fig. 5A, B; lane 1).
264
3. 5. Effect of chlorous acid on proteins inCampylobacter
265
To understand how chlorous acid could kill Campylobacter cells, protein profile of
266
chlorous acid or sodium hypochlorite treated bacterial cells were analyzed by 10%
267
SDS-PAGE. When bacterial cells were treated with 400 or 2,000 ppm of sodium
268
hypochlorite, protein profiles of campylobacters (C. jejuni strains 81-176, ATCC 33560 and
269
Chi2, and C. coli strains ATCC 33559 and Cc4) were not significantly different from cells
270
which were treated with PBS(-) as a negative control (Fig. 6A-6E; lanes 1-3). However, cells
271
treated with 4,000 ppm of sodium hypochlorite, the protein bands became slightly lighter
272
compared to cells treated with PBS(-) as a control (Fig. 6A-6E; lanes1 and 4). In sharp
273
contrast, bacterial cells treated with 400 ppm of chlorous acid, most of the protein bands
274
disappeared from the resolving gel while proteins from PBS(-) treated cells were clearly
275
visible as shown in Fig. 6.
276
4. Discussion
277
Food borne disease caused by Campylobacter is one of the most serious public health
278
problems not only in Japan, but also in other parts of the world (Kaakoush et al., 2015).
279
Chicken meat is considered as one of themajor sources of Campylobacter food poisoning. C.
280
jejuni and C. coli have been known to colonize intestine in chicken without showing any 13
281
clinical sign and symptom (Moore et al., 2005). During meat processing, Campylobacter
282
contamination might occur but it is difficult to prevent Campylobacter contamination from
283
fecal matters to carcasses and meat. Therefore, to reduce the food poisoning caused by
284
Campylobacter, effective measure for sterilization of chicken meat during processing is
285
required. The Ministry of Health, Labour and Welfare of Japan has approved the use of
286
chlorous acid up to 400 ppm for sterilization of food materials. It has been reported that 300,
287
400 or 500 ppm of chlorous acid could sterilize Staphylococcus aureus, Salmonella Enteritidis
288
and C. jejuni from chicken meat without affecting quality of the meat (Ministry of health,
289
Labour and welfare of Japan, 2012). In this study, bactericidal activity of chlorous acid
290
against various C. jejuni and C. colistrains was evaluated in comparison to that of sodium
291
hypochlorite in the presence of organic matter (Table 1, and Figs. 1-3).
292
Although 400 ppm of sodium hypochlorite reduced the number of campylobacters but
293
this concentration was not enough to kill them completely (Figs. 1 and 3). In contrast, 400
294
ppm of chlorous acid completely killed Campylobacter strains regardless of the presence of
295
organic matters such as Bolton broth and chicken juice (Table 1, and Figs. 1-3). To kill all
296
campylobacters in Bolton broth or chicken juice by sodium hypochlorite, 4,000 ppm was
297
required (Figs. 1 and 3). These results indicate that chlorous acid is 10 times more effective
298
than sodium hypochlorite in killing C. jejuni and C. coli. Previously it has been demonstrated
299
that the chlorous acid is more effective than sodium hypochlorite in the presence of organic
300
matter (Yamaoka et al., 2015; Godaet al., 2017). In this study, we also demonstrated that 400
301
ppm of chlorous acid have a significant bactericidal activity on Campylobacter in both Bolton
302
broth and chicken juice where 100 mg/mL of protein was present. Therefore, if sodium
303
hypochlorite is replaced with chlorous acid in chiller pool etc. at chicken slaughterhouse, 14
304
chlorous acid may reduce more number of C. jejuni and C. coli at chicken slaughterhouse and
305
may be more effectiveto reduce cross contamination of campylobacters in chicken carcasses
306
during meat processing at slaughterhouse. However, further evaluation of bactericidal activity
307
of chlorous acid during chicken meat processing at slaughterhouse is required.
308
Since Campylobacter colonies were not obtained by culture method after treatment
309
with 400 ppm of chlorous acid, the treated cells were stained with DAPI and PI to see whether
310
bacteria were killed by chlorous acid. As expected, both the dyes were able to stain DNAs of
311
400 ppm of chlorous acid treated bacterial cells. Similar result was obtained when cells were
312
treated with 4,000 ppm of sodium hypochlorite. In contrast, when C. jejuni and C. coli cells
313
were treated with 400 or 2,000 ppm of sodium hypochlorite, most of them were stained with
314
DAPI and only a few with PI (Fig. 4). These results indicated that bacteria treated with 400 or
315
2,000 ppm of sodium hypochlorite were not killed while those treated with 400 ppm of
316
chlorous acid or 4,000 ppm of sodium hypochlorite were killed and they not entered into
317
VBNC stage.
318
Mechanisms of bactericidal activity of sodium hypochlorite are considered to be due
319
to inhibition of enzyme activities, damages to DNA and membrane proteins through HClO or
320
ClO- radicals (Ohnishi 2002, Fukuzaki 2006). On the other hand, mechanism of bactericidal
321
activity of chlorous acid remains unclear. To understand how chlorous acid killed
322
campylobacters, bacterial DNA and proteins were analyzed by agarose gel electrophoresis and
323
SDS-PAGE after treating the cells with each disinfectant, respectively (Figs. 5 and 6). In the
324
case of bacterial DNA, although purified DNA was directly treated with 400 ppm of chlorous
325
acid in Bolton broth, DNA damage was not observed on agarose gel regardless of chemicals
326
and their concentrations. On the other hand, when C. jejuni and C. coli themselves were 15
327
treated with 400 ppm of chlorous acid, proteins were aggregated and accumulated in stacking
328
gel. The data indicates that proteins of C. jejuni and C. coli were damaged or denatured by
329
400 ppm of chlorous acid. Most probably, this could be the reason for killing of C. jejuni and
330
C. coli by 400 ppm of chlorous acid.
331
5. Conclusion
332
This study shows that chlorous acid has more effective bactericidal activity than
333
sodium hypochlorite against C. jejuni and C. coli in the presence of organic matter.
334
Experimental evidence indicates that chlorous acid killed C. jejuni and C. coli cells most
335
probably through damaging bacterial proteins. Chlorous acid may be more useful disinfectant
336
than sodium hypochlorite to sterilize C. jejuni and C. coli contaminated on chicken carcasses
337
during meat processing at slaughterhouse. Further studies are required to evaluate the effect of
338
chlorous acid by using contaminated chicken.
339 340 341 342
ACKNOWLEDGMENTS We thank Dr. Rupak K. Bhadra, CSIR-Indian Institute of Chemical Biology, Kolkata, India for critically reading the manuscript.
343 344 345 346
CONFLICT OF INTEREST This study was performed as a collaborative research of Honbu Sankei Co., Ltd., and financially supported by this company.
347 348
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Chaveerach, P., ter, Huurne, A.A., Lipman, L.J., van, Knapen, F. (2003) Survuval and
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Fukuzaki, S. (2006) Mechanisms od actions of sodium hypochlorite in cleaning and
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Goda, H., Yamaoka, H., Nakayama-Imaohji, H., Kawata, H., Horiuchi, I., Fujita, Y., Nagao, T.,
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Tada, A., Terada, A. and Kuwahara, T. (2017) Microbicidal effects of weakly acidified
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Hatanaka, N., Shimizu, A., Somroop, A., Li, Y., Asakura, M., Nagita, A., Prasad Awashi S.,
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Hinenoya A. and Yamasaki S. (2017) High prevalence of Campylobacter ureolyticusinstool
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Horiuchi, I., Kawata, H., Nagao, T.,Imaohji, H., Murakami, K., Kino, Y., Yamasaki, H.,
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Koyama, A.H., Fujita, Y., Goda, H. and Kuwahara, T. (2015) Antimicrobial activity and
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Kaakoush, N.O., Castaǹo-Rodriguez, N., Mitchell, H.M. and Man, S.M. (2015) Global
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(in Japanese)
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Moore, J.E., Corcoran, D., Dooley, J.S., Fanning, S., Lucey, B., Matsuda, M., McDowell,
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D.A., Mègraud, F., Millar, B.C., ÓMahony, R., ÓRiordan, L., ÓRourke, M., Rao, J.R.,
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Rooney, P.J., Sails, A. and Whyte, P. (2005) Campylobacter. Vet Res.36, 351-382.
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Ohnishi, S., Murata, M., Kawanishi, S. (2002) DNA damage induced by hypochlorite and
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Ramamurthy, T., Ghosh, A., Pazhani, G.P., Shinoda, S. (2014) Current perspectives on viable
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Japan.
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H., Goda, H. and Kuwahara, T. (2016) Tetramethylbenzidine method for monitoring the free
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available
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Wilson, K. (1987). Preparation of genomic DNA from bacteria. In: Ausubel, F.M., Brent, R.,
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Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. (Eds.), Current Protocols
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in Molecular Biology. John Wiley & Sons, New York, NY, pp. 2.4.1–2.4.5.
chlorine
and
microbicidal
activity
of
chlorite-based
sanitizers
under
403
19
404
Figure legends
405
Fig. 1. Comparison of bactericidal activity against C. jejuni and C. coli between chlorous
406
acid and sodium hypochlorite in Bolton broth.
407
C. jejuni strains 81-176, ATCC 33560 and Chi2, and C. coli strains ATCC 33559 and Cc4
408
were treated with PBS (-), 400, 2,000, 4,000 ppm of sodium hypochlorite or 400 ppm of
409
chlorous acid in Bolton broth. Viable bacterial number was determined by colony counting
410
using blood agar. Dotted line indicates detection limit by colony counting.* indicates that
411
there are significant differences between PBS and 400 ppm sodium hypochlorite.
412
that there are significant differences between 400 ppm sodium hypochlorite and 2,000 ppm,
413
4,000 ppm sodium hypochlorite or 400 ppm chlorous acid.
414
Fig. 2. Bactericidal activity against C. jejuni and C. coli bychlorous acid in chicken juice.
415
C. jejuni strains 81-176, ATCC 33560 and Chi2, and C. coli strains ATCC 33559 and Cc4
416
were treated with PBS (-), 100, 200 or 400 ppm of chlorous acid in chicken juice. Viable
417
bacterial number was determined by colony counting using mCCDA agar. Dotted line
418
indicates detection limit by colony counting.* indicates that there were significant differences
419
between PBS and 100 ppm, 200 ppm or 400 ppm chlorous acid.
420
Fig.3. Comparative analysis of bactericidal activity against C. jejuni and C. colibetween
421
chlorous acid and sodium hypochlorite in chicken juice.
422
C. jejuni strains 81-176, ATCC 33560 and Chi2, and C. coli strains ATCC 33559 and Cc4
423
were treated with PBS (-), 400, 2,000, 4,000 ppm of sodium hypochlorite or 400 ppm of
424
chlorous acid in chicken juice. Viable bacterial number was determined by colony counting
425
using mCCDA agar. Dotted line indicates detection limit by colony counting.* indicates that
426
there are significant differences between PBS and 400 ppm sodium hypochlorite.’ and
#
indicates
#
20
427
indicates that there are significant differences between 400 ppm sodium hypochlorite and
428
2,000 ppm, 4,000 ppm sodium hypochlorite or 400 ppm chlorous acid.
429
Fig. 4. Staining of C. jejuniby DAPI and propidium iodide after treated with either PBS,
430
sodium hypochlorite or chlorous acid.
431
C. jejuni strain 81-176 was treated with PBS(-) (A, B), 400 (C, D), 2,000 (E, F) or 4,000 (G,
432
H) ppm of sodium hypochlorite, or 400 ppm of chlorous acid (I. J), and then stained with
433
DAPI (A, C, E, G, I) and PI (B, D, F, H. J). Bacterial DNA was observed by fluorescence
434
microscopy. Scale bars correspond to 20 µm in all images.
435
Fig. 5. Effect of chlorous acid on bacterial DNA in C. jejuniand C. coli.
436
Genomic DNA were purified from C. jejuni strain 81-176 (A) and C. coli strain ATCC 33559
437
(B) and treated with PBS (-) (lane 1), 4,000, 2,000, 400 ppm of sodium hypochlorite (lanes 2,
438
3 and 4) or 400 ppm of chlorous acid (lane 5). DNA profile was analyzed by electrophoresis
439
on a 0.8% agarose gel.
440
Fig. 6. Effect of chlorous acid on bacterial proteins in C. jejuniand C. coli.
441
C. jejuni strains 81-176 (A), ATCC 33560 (B) and Chi2 (C) and C. coli strains ATCC 33559
442
(D) and Cc4 (E) were treated with PBS (-)(lane 1), 400, 2,000, 4,000 ppm of sodium
443
hypochlorite (lane 2, 3, 4) or 400 ppm of chlorous acid (lane 5). Protein profiles of C. jejuni
444
and C. coli treated with the disinfectantwere analyzed by SDS-PAGE and stained with CBB.
445
M, SDS-protein marker. Arrowhead indicates that proteins of C. jejuni and C. colitreated with
446
400 ppm of chlorous acid were stacked in staking gel.
21
1 2
Table 1. Bactericidal activity of sodium hypochlorite and chlorous acid against various Campylobacter strains Bactericidal activity
3 4
Species
Source
C. jejuni (n=15)
Clinical
Strain
81-176 JCM2013 Co2-037 Co3-007 P8219F-1 P8224F-1 Bovine ATCC33560 8214c 8215a B01 B86 Chicken Chi2 Chi7 Chi17 Chi34 C. coli Clinical Cc4 (n=15) CO1-017 CO1-106 CO1-124 CO1-130 CO1-179 P8015C-1 P8216F-1 Swine ATCC33559 WLD4-1 D3-2 LW9-9 W8-7 L8-1 WLD6♀ ‘-’ indicates no bactericidal activity used to treat campylobacters.
Sodium hypochlorite Chlorous acid (400 ppm) in (400 ppm) in Bolton Bolton broth chicken juice broth + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + and ‘+' indicates bactericidal activity of the disinfectant
1
(log CFU/mL) 10.00 10.0 9.00 9.0 8.00 8.0
C. jejuni 81-176 C. jejuni ATCC33560
7.00 7.0
C. jejuni Chi2
6.00 6.0
*
5.00 5.0
C. coli ATCC33559
* *
C. coli Cc4
* *
4.00 4.0
# 3.00 3.0
#
#
2.00 2.0
#
#
#
#
#
#
#
#
#
#
#
1.00 1.0 0.00 0
PBS
400 ppm
2,000 ppm
Sodium hypochlorite
Fig. 1
4,000 ppm
400 ppm Chlorous acid
#
(log CFU/mL) 10.00 10.0
C. jejuni 81-176
9.00 9.0
C. jejuni ATCC33560
8.00 8.0
C. jejuni Chi2
*
7.00
7.0
*
C. coli ATCC33559
*
C. coli Cc4
*
*
6.00 6.0
*
5.00 5.0
*
* *
4.00 4.0 3.00 3.0 2.00 2.0
*
1.00 1.0
*
*
*
*
0.00 0
PBS
100 ppm
200 ppm Chlorous acid
Fig. 2
400 ppm
*
(log CFU/mL) 10.00 10.0 9.00 9.0 8.00 8.0
C. jejuni 81-176 C. jejuni ATCC33560
7.00 7.0
C. jejuni Chi2 C. coli ATCC33559
6.00 6.0
*
*
5.00 5.0
*
C. coli Cc4 #
* 4.00 4.0
#
*
3.00 3.0
#
2.00 2.0
# #
#
#
#
#
#
#
#
#
#
#
1.00 1.0 0.00 0
PBS
Fig. 3
400 ppm
2,000 ppm
Sodium hypochlorite
4,000 ppm
400 ppm Chlorous acid
DAPI
Propidium iodide (A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
(I)
(J)
PBS
Sodium hypochlorite
400 ppm
2,000 ppm
4,000 ppm
400 ppm Chlorous acid
Fig. 4
(A)
1
Fig. 5
2
3
4
5
(B)
1
2
3
4
5
(A)
(B)
M 1 2
3
4 5
(C)
M 1 2
3
4 5
(kDa)
116 66 45
31
(D) M 1 (kDa)
116 66 45
31
Fig. 6
(E) 2 3 4 5
M 1
2 3 4 5
M 1 2
3
4 5
Highlights Chrolous acid possess stronger bactericidal activity than sodium hypochlorite. Chrolous acid showed bactericidal activity even under high protein concentration. Chrolous acid killed Campylobacter by affecting their proteins rather DNA. Chrolous acid might be useful disinfectant in chicken processing steps.
CONFLICT OF INTEREST This study was performed as a collaborative research of Honbu Sankei Co., Ltd., and financially supported by this company.
S0956-7135(19)30635-8
DOI:
https://doi.org/10.1016/j.foodcont.2019.107046
Reference:
JFCO 107046
To appear in:
Food Control
Received Date: 13 July 2019 Revised Date:
4 December 2019
Accepted Date: 8 December 2019
Please cite this article as: Hatanaka N., Awasthi S.P., Goda H., Kawata H., Uchino Y., Kubo T., Aoki S., Hinenoya A. & Yamasaki S., Chlorous acid is a more potent antibacterial agent than sodium hypochlorite against Campylobacter, Food Control (2020), doi: https://doi.org/10.1016/j.foodcont.2019.107046. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Author contribution section: N.H., H.G. and S.Y. designed research; N.H., S.P.A., and H.K. performed research; N.H., S.P.A., H.G., H.K., Y.U., T.K, S.A., A.H., and S.Y. analyzed data; N.H., H.G. and S.Y. wrote the paper.
1
Chlorous acid is a more potent antibacterial agent than sodium
2
hypochlorite against Campylobacter
3 4
Noritoshi Hatanaka1, Sharda Prasad Awasthi1, Hisataka Goda2, Hiroyuki Kawata2, Yuzuru
5
Uchino3, Takahiro Kubo1, 3, Shigeru Aoki2*, Atsushi Hinenoya1 and Shinji Yamasaki1, #
6 7
1: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka,
8
Japan
9
2: Honbu Sankei Co. Ltd., Osaka, Japan
10
3: Development Section, Honbusankei Co., Ltd., Hyogo, Japan
11
*S.A. is deceased.
12 13 14
Keywords: Campylobacter, chlorous acid (HClO2), sodium hypochlorite (NaClO), food
15
protection, bactericidal．
16 17 18
#
19
Mailing address: Graduate School of Life and Environmental Sciences, Osaka Prefecture
20
University, 1-58, Rinkuourai-kita, Izumisano, Osaka 598-8531, Japan
21
Tel & Fax: +81-72-463-5653
22
E-mail address: [email protected]
23
Corresponding author: Shinji Yamasaki, Ph.D.
FC-rev191204
24 1
25
Abstract
26
Foodborne disease caused by campylobacters is one of the major global problems for
27
food safety. Infection source of Campylobacter to human is mainly through contaminated
28
meat particularly chicken. Contamination of meat with Campylobacter usually occurs during
29
processing at slaughterhouse and to prevent such contaminations, sodium hypochlorite is
30
commonly used. However, it is well known that bactericidal activity of sodium hypochlorite
31
becomes weak under organic matter rich conditions. In this study, we compared the strength
32
of bactericidal activity of chlorous acid and sodium hypochlorite against Campylobacter
33
jejuni and Campylobacter coli strains under organic matter rich conditions. Bactericidal
34
activity against 5 representative C. jejuni and C. coli strains in chicken juice (an organic
35
matter rich condition) showed that minimum concentration of chlorous acid required for
36
complete killing of C. jejuni and C. coli cells is 200 to 400 ppm while that of sodium
37
hypochlorite is 2,000 to 4,000 ppm. Similar results were obtained by using Bolton broth.
38
Furthermore, it was observed that 400 ppm of chlorous acid but not 400 ppm of sodium
39
hypochlorite is highly effective in killing of 25 different Campylobacter strains (12 C. jejuni
40
and 13 C. coli strains) under the same conditions. To determine whether 400 ppm of chlorous
41
acid treatment had killed bacterial cells or induced them into viable but non-culturable
42
(VBNC) state, live and dead cell assay using DAPI and propidium iodide florescent dyes was
43
done. Such assay clearly indicated that Campylobacter cells were indeed killed and not
44
induced to VBNC state. Moreover, SDS-PAGE analysis of whole-cell lysates of
45
campylobacters indicated distinct effects in protein profiles of chlorous acid but not sodium
46
hypochlorite treated cells. The results strongly suggest that chlorous acid could efficiently kill
47
C. jejuni and C. coli cells with much lower concentration than sodium hypochlorite and the
2
48
bactericidal mechanisms of chlorous acid may be due to damages of bacterial proteins. Thus,
49
chlorous acid could be a better disinfectant in chicken slaughtering and processing to kill
50
campylobacters and prevent contamination.
3
51
1. Introduction
52
Campylobacter is one of the major causative agents of gastroenteritis worldwide.
53
Among 26 Campylobacter species so far reported, C.jejuni and C. coli are most frequently
54
isolated from human diarrheal patients (Man 2011). Furthermore, it is well known that C.
55
jejuni may cause autoimmune diseases such as Guillan-Barré syndrome and Miller Fisher
56
syndrome (Man 2011). Domestic animals are known to be primary reservoir of
57
Campylobacter. Poultry has particularly been recognized as a major infection source of C.
58
jejuni and C. coli, which cause campylobacteriosis in human (Kaakoush et al., 2015).
59
Campylobacter infects human mainly through consumption of undercooked meats, in
60
particular chicken and beef (Moore et al., 2005). Furthermore, in Japan, there is a food culture
61
that some people eat raw (Torisashi) or lightly roasted chicken (Tataki). This traditional food
62
habit may have a role in higher incidence of food-borne disease caused by C. jejuni and C.
63
coli in Japan. In fact, out of 1,014 incidences of food poisoning in Japan in 2017, 320 (31.6%)
64
were caused by C. jejuni and C. coli (Ministry of Health, Labour and Welfare of Japan). It
65
was speculated that as low as 360 most probable number (MPN) of C. jejuni might cause food
66
poisoning (Hara-Kudo et al., 2011). Therefore, it is important to reduce contamination of
67
Campylobacter to foods for decreasing food poisoning caused by C. jejuni and C. coli.
68
Sodium hypochlorite is commonly used for sanitization of food material such as meats,
69
vegetables, cooking equipment etc. (Fukuzaki 2006). At chicken slaughterhouse, sodium
70
hypochlorite is also used in many steps (Cadena et al., 2019). During processing, feces
71
containing Campylobacter probably contaminate carcasses and it might result in cross
72
contamination of Campylobacter on chicken meat even though sodium hypochlorite is used
73
for sanitization. Furthermore, Misawa et al. (2012) reported that the number of 4
74
Campylobacter on chicken meat was increased during processing at slaughterhouse. It is well
75
known that sodium hypochlorite cannot demonstrate efficient bactericidal activity when used
76
in the presence of organic matter (Yamaoka et al., 2016).
77
Alternatively, chlorous acid (HClO2) is also used for sterilization of meats as a food
78
additive not only in Japan, but also in the USA, Canada, Australia and New Zealand. Several
79
studies have been reported that chlorous acid showed bactericidal activity against pathogenic
80
bacteria including Staphylococcus aureus, Escherichia coli, Candida albicans etc. (Horiuchi
81
et al. 2015). Furthermore, even in the presence of organic matter such as bovine serum
82
albumin, it retained its bactericidal activity against MRSA and Clostridium difficile (Yamaoka
83
et al. 2016, Goda et al. 2017), indicating that chlorous acid might be useful for sanitization of
84
food-poisoning causing bacteria from meats such as chicken.
85
The major aim of this study is to kill efficiently C. jejuni and C. coli cells or to reduce
86
their number from chicken carcasses by a food additive to prevent food borne diseases in
87
human. In this study, as a first step, we wanted to know whether chlorous acid could be more
88
effective than sodium hypochlorite to decrease the number of C. jejuni and C. coli under
89
organic matter rich conditions. Furthermore, we examined the mechanisms about how
90
chlorous acid could kill C. jejuni and C. coli. Here, we show that chlorous acid had more
91
efficient bactericidal activity than sodium hypochlorite in the presence of organic matter and
92
its bactericidal mechanism was most likely due to damage or denature of bacterial proteins of
93
C. jejuni and C. coli cells.
94
2. Materials and Methods
95
2. 1.Bacterial strains and growth media
96
A total of 30 Campylobacter strains including 15 C. jejuni (6 clinical, 5 bovine and 4 5
97
chicken) and 15 C. coli (8 clinical and 7 swine) strains were used in this study (Table 1). Each
98
strain was isolated from different host and individual, and identified the species by cdt
99
gene-based species-specific multiplex PCR (Asakura et al. 2008). Campylobacters were
100
grown on blood agar [blood agar base No. 2 (Oxoid Ltd., Basingstoke, United Kingdom)
101
supplemented with 5% (v/v) defibrinated horse blood (Nippon Bio Supp. Center, Tokyo,
102
Japan)] at 37°C for 48 h or more under anaerobic condition (10% CO2, 10% H2, 80% N2)
103
(Hatanaka et al. 2017).
104
2. 2. Bactericidal activity of chlorous acid and sodium hypochlorite in Bolton broth
105
C. jejuni strains 81-176, ATCC 33560 and Chi2, and C. coli strains ATCC 33559 and
106
Cc4 were cultured on blood agar as described above. Bacterial colonies were suspended in
107
PBS(-) at pH 7.4 and bacterial concentration was adjusted to approximately 104 CFU/mL.
108
Then, 100 µL of bacterial suspension was inoculated into 5 mL of Bolton broth (Oxoid Ltd.)
109
and cultured at 37°C for 48 h under anaerobic condition as described above. Two hundred
110
fifty microliters of the bacterial culture were transferred to a 1.5 mL tube and mixed with
111
equal volume of either PBS(-) at pH 7.4, 400, 2,000 or 4,000 parts per million (ppm) of
112
sodium hypochlorite (Wako Pure ChemicalIndustries, Ltd., Osaka, Japan) in which
113
concentration of available chlorine was measured by an iodometric method (Ref). Similar
114
assay was done with 400 ppm of chlorous acid (Care for Noro barrier plus; Honbu Sankei Co.
115
Ltd., Osaka, Japan) in which concentration of available chlorine was measured by DPD
116
method (Ref). Then, the mixture was kept at room temperature for 3 min. Thereafter, 250 µL
117
of 50 mM sodium thiosulfate (NacalaiTesque, Inc., Kyoto, Japan) was added to inactivate the
118
chemicals. The number of live bacteria remained was examined by colony counting using
119
blood agar plate. 6
120
Similarly, all Campylobacter strains listed in Table 1 were treated with either PBS(-) at
121
pH 7.4, 400 ppm of sodium hypochlorite or 400 ppm of chlorous acid. Next, 100 µL of each
122
of these treated bacterial cultures was inoculated into 5 mL of Bolton broth, and cells were
123
allowed to grow at 37°C for 48 h under anaerobic condition. Then, a loopful of the culture
124
was streaked on blood agar plate and the plates were incubated as described previously (Ref).
125
2. 3. Preparation of chicken juice
126
One hundred grams of chicken meat were homogenized with 400 mL of sterilized
127
distilled water and the homogenate was further passed through sterilized gauze in order to
128
remove the debris. Then, protein concentration was determined and adjusted to approximately
129
100 mg/mL by Bradford assay using bovine serum albumin (NalacaiTesque, Inc.) as a
130
standard protein. Absence of campylobacters in chicken juice was confirmed by both culture
131
method and C16S PCR as described previously (Hatanaka et al. 2017).
132
2. 4. Bactericidal activity of chlorous acid and sodium hypochlorite in chicken juice
133
C. jejuni and C. coli strains were cultured in 5 mL of Bolton broth as described above,
134
250 µL of the culture were centrifuged at 12,000 g for 5 min and supernatants were removed.
135
The bacterial pellet was suspended with equal volume of chicken juice and 250 µL of PBS(-)
136
(as a control) or 400/2,000/4,000 ppm each of sodium hypochlorite or 100/200/400 ppm each
137
of chlorous acid was added. The mixture was kept at room temperature for 3 min and 250 µL
138
of 50 mM sodium thiosulfate was added to neutralize the remaining activity of each chemical.
139
Thereafter, bacterial survival assay was done by colony counting method by plating each
140
dilution on mCCDA agar (Oxoid Ltd.).
141
Similarly, all Campylobacter strains listed in Table 1 were treated with 400 ppm of
142
chlorous acid in chicken juice. One hundred microliter of each treated bacterial culture was 7
143
inoculated into 5 mL of Bolton broth supplemented with 5% (v/v) defibrinated horse blood
144
followed by incubation at 37°C for 48 h under anaerobic condition. Then, a loopful of the
145
culture was streaked on mCCDA agar plate and plate was as described previously (Ref).
146
2. 5. Fluorescence microscopy using DAPI or propidium iodide
147
C. jejuni strain 81-176 and C. coli strain ATCC 33559 were selected as a
148
representative strain for each species and cultured in 5 mL of Bolton broth, respectively, and
149
treated with PBS(-) (as a control) or400/2,000/4,000 ppm each of sodium hypochlorite or 400
150
ppm of chlorous acid as described above. Bacterial cell suspension was centrifuged at 6,000 g
151
for 5 min, supernatant was discarded and pellet was suspended with 500 µL of PBS(-).
152
Then,the suspension was centrifuged under the same condition and pellet was re-suspended
153
with 50 µL of fresh Bolton broth. Five microliters of each bacterial suspension were
154
transferred into a 1.5 mL-tube and mixed with 20 µL of fluorescent dye mix [0.5 mg/mL of
155
propidium iodide (PI) (Sigma-Aldrich Co. LLC, St. Louis, Missouri, USA) and 0.5 mg/mL of
156
DAPI (Merck KGaA, Darmstadt, Germany) in PBS(-)]. After staining at room temperature for
157
15 min under dark condition, the mixture was centrifuged at 6,000 g for 5 min, supernatant
158
was discarded and pellet was fixed with 100 µL of 3.7% paraformaldehyde at room
159
temperature for 30 min under dark condition. Then, sample was centrifuged as described
160
above, supernatant was discarded, pellet was re-suspendedwith 20 µL of PBS (-)and
161
bacterialcells were observed by microscopy (Leica DM 2500: Leica Microsystems GmbH.).
162
2. 6. Effect of chlorous acid on bacterial DNA
163
C. jejuni strain 81-176 and C. coli strain ATCC 33559 were cultured on blood agar
164
plates as described above. Genomic DNA of these strains were prepared as described
165
previously (Wilson, 1987). Concentration of genomic DNA was adjusted to 500 ng/5 µL in 8
166
Bolton broth and 5 µL of purified DNA was mixed with the same volume of PBS, 400, 2,000,
167
4,000 ppm each of sodium hypochlorite or 400 ppm of chlorous acid solution. The mixture
168
was kept at room temperature for 3 min and 5 µL of 50 mM sodium thiosulfate was added to
169
neutralize the remaining activity of each chemical. Samples were analyzed by electrophoresis
170
using a 0.8% Agarose, LE, Analytical Grade (Promega Corporation, Madison, WI, USA) and
171
DNAs were visualized with ultraviolet (UV) light after staining with ethidium bromide (1
172
µg/mL). Images were captured on a ChemiDoc system (Bio-Rad Laboratories, Inc., Hercules,
173
CA, USA).
174
2. 7. Effect of chlorous acid on bacterial proteins
175
C. jejuni strains81-176, ATCC 33560 and Chi2, and C. coli strains ATCC 33559 and
176
Cc4were treated with PBS(-) (as a control) or 400 ppm of chlorous acid or 400/2,000/4,000
177
ppmeach ofsodium hypochlorite as described above. Then, each suspension was centrifuged
178
at 12,000 g for 5 min, supernatant was discarded and bacterial pellet was re-suspended in
179
PBS(-) at OD600=5.0 followed by sonication 3 times (each of 1 min duration) using a
180
sonicator (Q500: Qsonica, LLC., Newtown, CT, USA) by keeping the culture tube on ice.
181
Thereafter, proteins present in bacterial cell lysates were analyzed by SDS-polyacrylamide gel
182
electrophoresis (SDS-PAGE) using 10% acrylamide gel. After separation by electrophoresis,
183
proteins were visualized by staining with 0.25% Coomassie Brilliant Blue R-250
184
(NacalaiTesque, Inc.).
185
2. 8. Statistical analysis
186
Statistical analyses were performed using Microsoft Excel 2016 (Microsoft, Redmond,
187
WA, USA). Error bars show standard deviations. p value was calculated with Student’s t test
188
using paired, two-tailed distribution. When a p value showed less than 0.05, it was concluded 9
189
that there was statistically significant difference.
190
3. Results
191
3. 1. Bactericidal activity of chlorous acid and sodium hypochlorite in Bolton broth
192
To examine the bactericidal activity of chlorous acid and sodium hypochlorite against
193
C. jejuni and C. coli strains, the organisms were treated with 400 ppm of each chemical
194
solution. Initially, bactericidal activity of sodium hypochlorite or chlorous acid was examined
195
with five representative strains of C. jejuni and C. coli. When C. jejuni strains 81-176, ATCC
196
33560 and Chi2, and C. coli strains ATCC 33559 and Cc4 were treated with PBS(-), viable
197
cell counts were approximately 8.6 to 9.3 log CFU/mL per sample. When treated with 400
198
ppm of sodium hypochlorite, number of cells of C. jejuni strains 81-176, ATCC 33560 and
199
Chi2,and C. coli strains ATCC 33559 and Cc4 were reduced to be 5.4, 4.6, 4.8,4.2 and 5.4 log
200
CFU/mL, respectively. On the other hand, when the same set of Campylobacter strains were
201
treated with 400 ppm of chlorous acid, all of them failed to grow on blood agar, indicating
202
that number of viable cells was reduced to less than detection limit (1 log CFU/mL) as shown
203
in Fig. 1. In contrast, the number of viable cells was found to be significantly different in the
204
case of bacterial cells treated with 400 ppm of sodium hypochlorite (Fig. 1).
205
Furthermore, to see whether bactericidal activity of chlorous acid is not limited to
206
particular strains, various Campylobacter strains as shown in Table 1 were treated essentially
207
as described above and followed by culturing in Bolton broth and plating of cultures on blood
208
agar plates. Campylobacter strains were able to grow on blood agar plates when treated with
209
400 ppm of sodium hypochlorite, however, none of the campylobacters grew on blood agar
210
plates (less than 1 log CFU/mL) when treated with 400 ppm of chlorous acid. These data
211
clearly indicate that the chlorous acid is more effective in terms of bactericidal activity against 10
212
C. jejuni and C. coli strains than sodium hypochlorite in the presence of organic matter (Table
213
1).
214
Since 400 ppm of sodium hypochlorite was not able to completely kill the C. jejuni
215
and C. coli strains used, the minimum concentration of sodium hypochlorite needed for
216
complete killing of these cells was determined to compare bactericidal activities between
217
chlorous acid and sodium hypochlorite. When cells were treated with 2,000 ppm of sodium
218
hypochlorite, only C. jejuni strain ATCC 33560 and C. coli strain Cc4 failed to grow on blood
219
agar and the remaining 3 Campylobacter strains were able to grow. However, when all these
220
strains were treated with 4,000 ppm of sodium hypochlorite, they failed to grow on blood agar
221
as shown in Fig. 1.
222
3. 2. Bactericidal activity of chlorous acid and sodium hypochlorite in chicken juice
223
To understand whether chlorous acid could kill campylobacters under organic matter
224
rich condition, the bactericidal activity of chlorous acid was examined and compared with that
225
of sodium hypochlorite in chicken juice.While protein concentration of Bolton broth was 10
226
mg/mL, that of chicken juice prepared in this study was 100 mg/mL. When C. jejuni and C.
227
coli strains were treated with PBS (-), about 8.6 to 8.9 log CFU/mL of campylobacters were
228
detected (Fig. 2). However, when treated with chlorous acid, the number of C. jejuni and C.
229
coli was reduced in a dose-dependent manner. At 400 ppm concentration of chlorous acid,
230
both C. jejuni and C. coli strains failed to grow on mCCDA agar and C. coli strain ATCC
231
33559 was not able to grow on mCCDA agar even in the presence of 200 ppm of chlorous
232
acid (Fig. 2).
233
To compare bactericidal activity between chlorous acid and sodium hypochlorite under
234
organic matter rich condition, we attempted to determine the minimum concentration of 11
235
sodium hypochlorite needed for complete killing C. jejuni and C. coli in chicken juice. As
236
shown in Fig. 3, when treated with PBS(-), each of the 5 Campylobacter strains showed
237
growth of about 9 log CFU/mL. However, cells treated with sodium hypochlorite, the number
238
of campylobacters was reduced in a dose-dependent manner. When treated with 400 ppm of
239
sodium hypochlorite, all 5 campylobacters grew on mCCDA agar. However, when treated
240
with 2,000 ppm of sodium hypochlorite, C. coli strain ATCC 33559 was not able to grow. On
241
the other hand, all 5 Campylobacter strains failed to grow when treated with 4,000 ppm of
242
sodium hypochlorite.
243
3. 3. Bactericidal effect of chlorous acid on Campylobacter
244
SinceCampylobacter spp. failed to grow on agar plates after treatment with 400 ppm
245
of chlorous acid, bacterial cells treated with different concentrations of sodium hypochlorite
246
or 400 ppm of chlorous acid were stained with DAPI and PI to verify their viability. It is
247
known that while DAPI can stain DNA of live and dead bacteria (Chaveerach et. al. 2003) but
248
PI stains DNA of dead bacteria only (Ramamurthy et. al. 2014). In addition, DNA of bacteria
249
which entered into VBNC stage would be stained with only DAPI (Chaveerach et. al. 2003),
250
but not with PI (Chaisowwong et. al. 2012). While C. jejuni strains treated with PBS(-), 400
251
or 2,000 ppm of sodium hypochlorite showed distinct staining of their nucleoid structure with
252
DAPI (Fig. 4; A, C and E) but most of them failed to be stained with PI (Fig. 4; B, D and F).
253
In sharp contrast, C. jejuni cells treated with 4,000 ppm of sodium hypochlorite or 400 ppm of
254
chlorous acid failed to take both DAPI and PI (Fig. 4; G, H, I and J). It may be mentioned
255
here that treatment of C. coli strains showed similar results like that of C. jejuni (data not
256
shown).
257
3. 4. Effect of chlorous acid on bacterial DNA 12
258
To examine whether chlorous acid damage bacterial DNA, purified DNA were treated
259
with chlorous acid in Bolton broth and analyzed by electrophoresis on a 0.8% agarose gel.
260
The results were also compared with those treated with sodium hypochlorite. When purified
261
DNA was treated with 400, 2,000 and 4,000 ppm of sodium hypochlorite or 400 ppm of
262
chlorous acid (Fig. 5A, B; lane 2-5), DNA profile of each sample was not significantly
263
different from those treated with PBS (-) as a negative control (Fig. 5A, B; lane 1).
264
3. 5. Effect of chlorous acid on proteins inCampylobacter
265
To understand how chlorous acid could kill Campylobacter cells, protein profile of
266
chlorous acid or sodium hypochlorite treated bacterial cells were analyzed by 10%
267
SDS-PAGE. When bacterial cells were treated with 400 or 2,000 ppm of sodium
268
hypochlorite, protein profiles of campylobacters (C. jejuni strains 81-176, ATCC 33560 and
269
Chi2, and C. coli strains ATCC 33559 and Cc4) were not significantly different from cells
270
which were treated with PBS(-) as a negative control (Fig. 6A-6E; lanes 1-3). However, cells
271
treated with 4,000 ppm of sodium hypochlorite, the protein bands became slightly lighter
272
compared to cells treated with PBS(-) as a control (Fig. 6A-6E; lanes1 and 4). In sharp
273
contrast, bacterial cells treated with 400 ppm of chlorous acid, most of the protein bands
274
disappeared from the resolving gel while proteins from PBS(-) treated cells were clearly
275
visible as shown in Fig. 6.
276
4. Discussion
277
Food borne disease caused by Campylobacter is one of the most serious public health
278
problems not only in Japan, but also in other parts of the world (Kaakoush et al., 2015).
279
Chicken meat is considered as one of themajor sources of Campylobacter food poisoning. C.
280
jejuni and C. coli have been known to colonize intestine in chicken without showing any 13
281
clinical sign and symptom (Moore et al., 2005). During meat processing, Campylobacter
282
contamination might occur but it is difficult to prevent Campylobacter contamination from
283
fecal matters to carcasses and meat. Therefore, to reduce the food poisoning caused by
284
Campylobacter, effective measure for sterilization of chicken meat during processing is
285
required. The Ministry of Health, Labour and Welfare of Japan has approved the use of
286
chlorous acid up to 400 ppm for sterilization of food materials. It has been reported that 300,
287
400 or 500 ppm of chlorous acid could sterilize Staphylococcus aureus, Salmonella Enteritidis
288
and C. jejuni from chicken meat without affecting quality of the meat (Ministry of health,
289
Labour and welfare of Japan, 2012). In this study, bactericidal activity of chlorous acid
290
against various C. jejuni and C. colistrains was evaluated in comparison to that of sodium
291
hypochlorite in the presence of organic matter (Table 1, and Figs. 1-3).
292
Although 400 ppm of sodium hypochlorite reduced the number of campylobacters but
293
this concentration was not enough to kill them completely (Figs. 1 and 3). In contrast, 400
294
ppm of chlorous acid completely killed Campylobacter strains regardless of the presence of
295
organic matters such as Bolton broth and chicken juice (Table 1, and Figs. 1-3). To kill all
296
campylobacters in Bolton broth or chicken juice by sodium hypochlorite, 4,000 ppm was
297
required (Figs. 1 and 3). These results indicate that chlorous acid is 10 times more effective
298
than sodium hypochlorite in killing C. jejuni and C. coli. Previously it has been demonstrated
299
that the chlorous acid is more effective than sodium hypochlorite in the presence of organic
300
matter (Yamaoka et al., 2015; Godaet al., 2017). In this study, we also demonstrated that 400
301
ppm of chlorous acid have a significant bactericidal activity on Campylobacter in both Bolton
302
broth and chicken juice where 100 mg/mL of protein was present. Therefore, if sodium
303
hypochlorite is replaced with chlorous acid in chiller pool etc. at chicken slaughterhouse, 14
304
chlorous acid may reduce more number of C. jejuni and C. coli at chicken slaughterhouse and
305
may be more effectiveto reduce cross contamination of campylobacters in chicken carcasses
306
during meat processing at slaughterhouse. However, further evaluation of bactericidal activity
307
of chlorous acid during chicken meat processing at slaughterhouse is required.
308
Since Campylobacter colonies were not obtained by culture method after treatment
309
with 400 ppm of chlorous acid, the treated cells were stained with DAPI and PI to see whether
310
bacteria were killed by chlorous acid. As expected, both the dyes were able to stain DNAs of
311
400 ppm of chlorous acid treated bacterial cells. Similar result was obtained when cells were
312
treated with 4,000 ppm of sodium hypochlorite. In contrast, when C. jejuni and C. coli cells
313
were treated with 400 or 2,000 ppm of sodium hypochlorite, most of them were stained with
314
DAPI and only a few with PI (Fig. 4). These results indicated that bacteria treated with 400 or
315
2,000 ppm of sodium hypochlorite were not killed while those treated with 400 ppm of
316
chlorous acid or 4,000 ppm of sodium hypochlorite were killed and they not entered into
317
VBNC stage.
318
Mechanisms of bactericidal activity of sodium hypochlorite are considered to be due
319
to inhibition of enzyme activities, damages to DNA and membrane proteins through HClO or
320
ClO- radicals (Ohnishi 2002, Fukuzaki 2006). On the other hand, mechanism of bactericidal
321
activity of chlorous acid remains unclear. To understand how chlorous acid killed
322
campylobacters, bacterial DNA and proteins were analyzed by agarose gel electrophoresis and
323
SDS-PAGE after treating the cells with each disinfectant, respectively (Figs. 5 and 6). In the
324
case of bacterial DNA, although purified DNA was directly treated with 400 ppm of chlorous
325
acid in Bolton broth, DNA damage was not observed on agarose gel regardless of chemicals
326
and their concentrations. On the other hand, when C. jejuni and C. coli themselves were 15
327
treated with 400 ppm of chlorous acid, proteins were aggregated and accumulated in stacking
328
gel. The data indicates that proteins of C. jejuni and C. coli were damaged or denatured by
329
400 ppm of chlorous acid. Most probably, this could be the reason for killing of C. jejuni and
330
C. coli by 400 ppm of chlorous acid.
331
5. Conclusion
332
This study shows that chlorous acid has more effective bactericidal activity than
333
sodium hypochlorite against C. jejuni and C. coli in the presence of organic matter.
334
Experimental evidence indicates that chlorous acid killed C. jejuni and C. coli cells most
335
probably through damaging bacterial proteins. Chlorous acid may be more useful disinfectant
336
than sodium hypochlorite to sterilize C. jejuni and C. coli contaminated on chicken carcasses
337
during meat processing at slaughterhouse. Further studies are required to evaluate the effect of
338
chlorous acid by using contaminated chicken.
339 340 341 342
ACKNOWLEDGMENTS We thank Dr. Rupak K. Bhadra, CSIR-Indian Institute of Chemical Biology, Kolkata, India for critically reading the manuscript.
343 344 345 346
CONFLICT OF INTEREST This study was performed as a collaborative research of Honbu Sankei Co., Ltd., and financially supported by this company.
347 348
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chlorine
and
microbicidal
activity
of
chlorite-based
sanitizers
under
403
19
404
Figure legends
405
Fig. 1. Comparison of bactericidal activity against C. jejuni and C. coli between chlorous
406
acid and sodium hypochlorite in Bolton broth.
407
C. jejuni strains 81-176, ATCC 33560 and Chi2, and C. coli strains ATCC 33559 and Cc4
408
were treated with PBS (-), 400, 2,000, 4,000 ppm of sodium hypochlorite or 400 ppm of
409
chlorous acid in Bolton broth. Viable bacterial number was determined by colony counting
410
using blood agar. Dotted line indicates detection limit by colony counting.* indicates that
411
there are significant differences between PBS and 400 ppm sodium hypochlorite.
412
that there are significant differences between 400 ppm sodium hypochlorite and 2,000 ppm,
413
4,000 ppm sodium hypochlorite or 400 ppm chlorous acid.
414
Fig. 2. Bactericidal activity against C. jejuni and C. coli bychlorous acid in chicken juice.
415
C. jejuni strains 81-176, ATCC 33560 and Chi2, and C. coli strains ATCC 33559 and Cc4
416
were treated with PBS (-), 100, 200 or 400 ppm of chlorous acid in chicken juice. Viable
417
bacterial number was determined by colony counting using mCCDA agar. Dotted line
418
indicates detection limit by colony counting.* indicates that there were significant differences
419
between PBS and 100 ppm, 200 ppm or 400 ppm chlorous acid.
420
Fig.3. Comparative analysis of bactericidal activity against C. jejuni and C. colibetween
421
chlorous acid and sodium hypochlorite in chicken juice.
422
C. jejuni strains 81-176, ATCC 33560 and Chi2, and C. coli strains ATCC 33559 and Cc4
423
were treated with PBS (-), 400, 2,000, 4,000 ppm of sodium hypochlorite or 400 ppm of
424
chlorous acid in chicken juice. Viable bacterial number was determined by colony counting
425
using mCCDA agar. Dotted line indicates detection limit by colony counting.* indicates that
426
there are significant differences between PBS and 400 ppm sodium hypochlorite.’ and
#
indicates
#
20
427
indicates that there are significant differences between 400 ppm sodium hypochlorite and
428
2,000 ppm, 4,000 ppm sodium hypochlorite or 400 ppm chlorous acid.
429
Fig. 4. Staining of C. jejuniby DAPI and propidium iodide after treated with either PBS,
430
sodium hypochlorite or chlorous acid.
431
C. jejuni strain 81-176 was treated with PBS(-) (A, B), 400 (C, D), 2,000 (E, F) or 4,000 (G,
432
H) ppm of sodium hypochlorite, or 400 ppm of chlorous acid (I. J), and then stained with
433
DAPI (A, C, E, G, I) and PI (B, D, F, H. J). Bacterial DNA was observed by fluorescence
434
microscopy. Scale bars correspond to 20 µm in all images.
435
Fig. 5. Effect of chlorous acid on bacterial DNA in C. jejuniand C. coli.
436
Genomic DNA were purified from C. jejuni strain 81-176 (A) and C. coli strain ATCC 33559
437
(B) and treated with PBS (-) (lane 1), 4,000, 2,000, 400 ppm of sodium hypochlorite (lanes 2,
438
3 and 4) or 400 ppm of chlorous acid (lane 5). DNA profile was analyzed by electrophoresis
439
on a 0.8% agarose gel.
440
Fig. 6. Effect of chlorous acid on bacterial proteins in C. jejuniand C. coli.
441
C. jejuni strains 81-176 (A), ATCC 33560 (B) and Chi2 (C) and C. coli strains ATCC 33559
442
(D) and Cc4 (E) were treated with PBS (-)(lane 1), 400, 2,000, 4,000 ppm of sodium
443
hypochlorite (lane 2, 3, 4) or 400 ppm of chlorous acid (lane 5). Protein profiles of C. jejuni
444
and C. coli treated with the disinfectantwere analyzed by SDS-PAGE and stained with CBB.
445
M, SDS-protein marker. Arrowhead indicates that proteins of C. jejuni and C. colitreated with
446
400 ppm of chlorous acid were stacked in staking gel.
21
1 2
Table 1. Bactericidal activity of sodium hypochlorite and chlorous acid against various Campylobacter strains Bactericidal activity
3 4
Species
Source
C. jejuni (n=15)
Clinical
Strain
81-176 JCM2013 Co2-037 Co3-007 P8219F-1 P8224F-1 Bovine ATCC33560 8214c 8215a B01 B86 Chicken Chi2 Chi7 Chi17 Chi34 C. coli Clinical Cc4 (n=15) CO1-017 CO1-106 CO1-124 CO1-130 CO1-179 P8015C-1 P8216F-1 Swine ATCC33559 WLD4-1 D3-2 LW9-9 W8-7 L8-1 WLD6♀ ‘-’ indicates no bactericidal activity used to treat campylobacters.
Sodium hypochlorite Chlorous acid (400 ppm) in (400 ppm) in Bolton Bolton broth chicken juice broth + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + and ‘+' indicates bactericidal activity of the disinfectant
1
(log CFU/mL) 10.00 10.0 9.00 9.0 8.00 8.0
C. jejuni 81-176 C. jejuni ATCC33560
7.00 7.0
C. jejuni Chi2
6.00 6.0
*
5.00 5.0
C. coli ATCC33559
* *
C. coli Cc4
* *
4.00 4.0
# 3.00 3.0
#
#
2.00 2.0
#
#
#
#
#
#
#
#
#
#
#
1.00 1.0 0.00 0
PBS
400 ppm
2,000 ppm
Sodium hypochlorite
Fig. 1
4,000 ppm
400 ppm Chlorous acid
#
(log CFU/mL) 10.00 10.0
C. jejuni 81-176
9.00 9.0
C. jejuni ATCC33560
8.00 8.0
C. jejuni Chi2
*
7.00
7.0
*
C. coli ATCC33559
*
C. coli Cc4
*
*
6.00 6.0
*
5.00 5.0
*
* *
4.00 4.0 3.00 3.0 2.00 2.0
*
1.00 1.0
*
*
*
*
0.00 0
PBS
100 ppm
200 ppm Chlorous acid
Fig. 2
400 ppm
*
(log CFU/mL) 10.00 10.0 9.00 9.0 8.00 8.0
C. jejuni 81-176 C. jejuni ATCC33560
7.00 7.0
C. jejuni Chi2 C. coli ATCC33559
6.00 6.0
*
*
5.00 5.0
*
C. coli Cc4 #
* 4.00 4.0
#
*
3.00 3.0
#
2.00 2.0
# #
#
#
#
#
#
#
#
#
#
#
1.00 1.0 0.00 0
PBS
Fig. 3
400 ppm
2,000 ppm
Sodium hypochlorite
4,000 ppm
400 ppm Chlorous acid
DAPI
Propidium iodide (A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
(I)
(J)
PBS
Sodium hypochlorite
400 ppm
2,000 ppm
4,000 ppm
400 ppm Chlorous acid
Fig. 4
(A)
1
Fig. 5
2
3
4
5
(B)
1
2
3
4
5
(A)
(B)
M 1 2
3
4 5
(C)
M 1 2
3
4 5
(kDa)
116 66 45
31
(D) M 1 (kDa)
116 66 45
31
Fig. 6
(E) 2 3 4 5
M 1
2 3 4 5
M 1 2
3
4 5
Highlights Chrolous acid possess stronger bactericidal activity than sodium hypochlorite. Chrolous acid showed bactericidal activity even under high protein concentration. Chrolous acid killed Campylobacter by affecting their proteins rather DNA. Chrolous acid might be useful disinfectant in chicken processing steps.
CONFLICT OF INTEREST This study was performed as a collaborative research of Honbu Sankei Co., Ltd., and financially supported by this company.