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Growth Inhibitory Effects of Chlorine Dioxide on Bacteria

Authors:
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Biomedical Science Letters 2018, 24(3): 270~274
https://doi.org/10.15616/BSL.2018.24.3.270
eISSN : 2288-7415
Growth Inhibitory Effects of Chlorine Dioxide on Bacteria
Kyoung-Ju Song1,* and Suk-Yul Jung2,,**
1Purgofarm Co. Ltd, Hwasung-city, Gyeonggi 18627, Korea
2Department of Biomedical Laboratory Science, Molecular Diagnosis Research Institute,
Namseoul University, Chungnam 31020, Korea
Chlorine dioxide (ClO2) gas is a neutral chlorine compound. ClO2 gas was proven to effectively decontaminate different
environments, such as hospital rooms, ambulances, biosafety level 3 laboratories, and cafeterias. In this study, to evaluate
the effects of ClO2 gas, bacteria of clinical importance were applied. Staphylococci, Streptococci and Bacillus strains were
applied and Klebsiella, and others e.g., Escherichia coli, Shigella, Salmonella, Serratia were also done for the inhibitory
analysis. Bacteria plates were applied with a hygiene stick, namely, "FarmeTok (Medistick/Puristic)" to produce ClO2.
ClO2-releasing hygiene stick showed the very strong inhibition of bacterial growth but had different inhibitions to the
bacteria above 96.7% except for MRSA of 90% inhibition. It is difficult to explain why the MRSA were not inhibited
less than others at this point. It can be only suggested that more releasing ClO2 should be essential to kill or inhibit the
MRSA. B. subtilis, S. agalactiae, S. pyogenes, E. coli O157:H7, S. typhi (S. enterica serotype typhi) and S. marcesence
were inhibited over 99%. This study will provide fundamental data to research growth inhibition by ClO2 gas with
bacteria of clinical importance value.
Key Words: Chlorine dioxide, Bacteria, Inhibition, FarmeTok (Medistick/Puristic)
Chlorine dioxide (ClO2) gas is a neutral chlorine com-
pound. It is very different from elementary chlorine, both
in its chemical structure and in its behavior (Vogt et al.,
2010; Song and Jung, 2017).
ClO2 gas is an effective disinfectant agent with strong
oxidization ability and a broad biocidal spectrum (Gómez-
López et al., 2009; Wang et al., 2016). The antimicrobial
efficacy of ClO2 gas has been evaluated in previous studies,
and ClO2 gas was proven to effectively decontaminate dif-
ferent environments, such as hospital rooms (Luftman et al.,
2006; Lowe et al., 2013), ambulances (Lowe et al., 2013),
biosafety level 3 laboratories (Lowe et al., 2012), and cafe-
terias (Hsu et al., 2014).
It has been reported that chlorine dioxide, a strong oxidant,
can inhibit or destroy microorganisms (Ogata et al., 2008;
Morino et al., 2009; Sanekata et al., 2010; Ma et al., 2017;
Ofori et al., 2017). Sanekata et al., (2010) reported that
chlorine dioxide at concentrations ranging from 1 to 100
ppm produced potent antiviral activity, inactivating >or=
99.9% of the viruses with a 15 sec treatment for sensitization.
Our group has reported that in the clinics 11 micro-
organisms were isolated, and ClO2-releasing hygiene stick
showed the very strong inhibition of bacterial growth with
about 99.9% after 24 hr incubation (Song and Jung, 2017).
Brief Communication
Received: August 7, 2018 / Revised: August 17, 2018 / Accepted: August 23, 2018
*Chief Technology Officer, **Professor.
Corresponding author: Suk-Yul Jung. Department of Biomedical Laboratory Science, Molecular Diagnosis Research Institute, Namseoul University, 91
Daehak-ro, Seonghwan-eup, Seobuk-gu, Cheonan-city, Choongnam 31020, Korea.
Tel: +82-41-580-2723, Fax: +82-41-580-2932, e-mail: syjung@nsu.ac.kr
CThe Korean Society for Biomedical Laboratory Sciences. All rights reserved.
CC This is an Open Access article distributed under the terms of the Creativ e Commons Attribution Non-Commercial License (http://cr eativecommons.org/licenses/by-nc/3.0/) whic
h
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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ClO2 however was found to increase the permeability of the
outer and cytoplasmic membranes leading to the leakage of
membrane components such as 260 nm absorbing materials
and inhibiting the activity of the intracellular enzyme β-D-
galactosidase (Ofori et al., 2017).
In this study, to evaluate the effects of ClO2 gas, bacteria
of clinical importance were applied.
Six gram positive bacteria and five gram negative bacteria
were applied. Bacteria were mentioned in results with growth
inhibition data. Briefly, 2 Staphylococci, 2 Streptococci and
1 Bacillus strains were applied and 2 Klebsiella, and others
e.g., Escherichia coli, Shigella, Salmonella, Serratia were
also done for the inhibitory analysis. In this study, the bacteria
were not divided by characteristics of diseases but simply
described with human infections above. Single colonies were
subcultured into other tryptic soy agar (TSA, MB cell, Korea)
plate at 37, and were double checked by Gram-staining
procedures (Lim et al., 1988).
To culture accurate colonies, obtained single colonies were
diluted with 0.85% NaCl and were adjusted into 0.5 of
McFaland turbidity, which could produce about 1.5 × 10
3
to 1.5 × 10
6 colony forming units (CFU)/mL (Song and
Jung, 2017). The adjusted bacteria grown in TSA plates were
applied for all subsequent experiments.
Bacteria plates were applied with a hygiene stick, namely,
"FarmeTok (Medistick/Puristic) kindly provided by Purgo-
farm, co, Ltd. (Hwasung, Gyeonggido, Korea)" to produce
ClO2 (Song and Jung, 2017). To efficiently observe and cul-
ture bacteria, bacterial plates were added into a plastic clear
chamber (250W × 350D × 200H) at a 37 incubator.
Table 1. CFU of bacteria by the hygiene stick of ClO2 gas. Bacteria were streaked onto the plate and the hygiene stick was located nea
r
the plate followed by counting of bacterial colonies
Gram
staining
Bacteria
(No. at KCTC) Groups Initial numbers
(CFU/mL)
Numbers after 24 hr
(CFU/mL)
*Growth inhibition rate
(%)
+
S. aureus (1621) Control 1.5 × 10
4 - -
ClO2 1.5 × 10
4 < 250 98.3
Methicillin-resistant
S. aureus (MRSA)
Control 1.5 × 10
3 - -
ClO2 1.5 × 10
3 < 150 90.0
B. subtilis (3613) Control 1.5 × 10
6 - -
ClO2 1.5 × 10
6 < 50 99.9
S. agalactiae Control 1.5 × 10
5 - -
ClO2 1.5 × 10
5 < 150 99.0
S. pyogenes Control 1.5 × 10
4 - -
ClO2 1.5 × 10
4 < 50 99.7
-
E. coli O157:H7 Control 1.5 × 10
4 - -
ClO2 1.5 × 104 < 50 99.7
K. oxytoca (1686) Control 1.5 × 10
4 - -
ClO2 1.5 × 10
4 < 300 98.0
K. pnuemoniae Control 1.5 × 10
3 - -
ClO2 1.5 × 10
3 < 50 96.7
S. typhi
(S. enterica serotype typhi)
Control 1.5 × 10
4 - -
ClO2 1.5 × 10
4 < 100 99.3
S. marcesence Control 1.5 × 10
6 - -
ClO2 1.5 × 10
6 < 100 99.9
S. sonnei Control 1.5 × 10
4 - -
ClO2 1.5 × 10
4 < 300 98.0
*100 - (Numbers after 24 hr / Initial numbers) × 100
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Bacterial growth was periodically observed until 24 hr and
was compared with ClO2 gas-untreated groups as a control.
All bacterial strains were below: S. aureus, Methicillin-
resistant S. aureus (MRSA), B. subtilis, S. agalactiae, S.
pyogenes, E. coli O157:H7, K. oxytoca, K. pnuemoniae, S.
typhi (S. enterica serotype typhi), S. marcesence, S. sonnei.
To analyze whether chlorine dioxide can inhibit the bacteria,
hygiene stick, namely, "FarmeTok (Medistick/Puristic)" which
produced the chlorine dioxide gas was co-incubated with the
bacteria. To avoid the release of the gas out, the hygiene stick
was put into a plastic chamber and was incubated at 37.
When the ClO2-releasing hygiene stick is ready for activation,
it is changed into yellow and release ClO2 (Song and Jung,
2017).
Simply, the lid of bacterial plates was open to be released
to air and ClO2. Bacteria were streaked onto the plate and the
hygiene stick was located near the plate followed by counting
of bacterial colonies (Table 1). Bacterial numbers were dif-
ferent dud to the use of general growth media of TSA. ClO2-
releasing hygiene stick showed the very strong inhibition of
bacterial growth but had different inhibitions to the bac-
teria above 96.7% except for MRSA of 90% inhibition. It
Fig. 1. Bacterial plates by the co-incubation of the hygiene stick of ClO2 gas. Bacteria plates were applied with a hygiene stick to produc
e
ClO2. The bacterial plates were added into a plastic clear chamber at a 37 incubator. Bacterial growth was periodically observed unti
l
24 hr and was compared with ClO2 gas-untreated groups as a control.
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is difficult to explain why the MRSA were not inhibited less
than others at this point. It can be only suggested that more
releasing ClO2 should be essential to kill or inhibit the MRSA.
B. subtilis, S. agalactiae, S. pyogenes, E. coli O157:H7, S.
typhi (S. enterica serotype typhi) and S. marcesence were
inhibited over 99%. It can also suggest that the inhibition may
not be affected by the Gram positivity and Gram negativity.
Fig. 1. represented bacterial plates from the counting of
CFU. All bacteria could be easily counted post 24 hr co-
incubation with ClO2, but S. sonnei plate showed dispersed
patterns due to moisturized surface of the plate. Very inter-
estingly, the areas of growth inhibited plates were peripheral
but not the central, implied that diffusion of ClO2 gas affect
the margin and periphery at first and then go to the central
region.
ClO2 gas is required to sanitize a lot of areas and an equip-
ment to release the ClO2 gas may be necessary in hospitals.
The hygiene stick, namely, "FarmeTok (Medistick/Puristic)"
kindly provided by Purgofarm would be useful to release
ClO2 gas and were sufficient to inhibit bacterial growth for
24 hr release. In our previous study, 11 microorganisms in-
cluding Micrococcus luteus, Granulicatella adiacens, Sta-
phylococcus caprae, Sphingomonas paucimobilis, Kocuria
kristinae, etc which were isolated from the clinic were com-
pletely inhibited by the hygiene stick of ClO2 gas (Song and
Jung, 2017). Incomplete growth inhibition may be resulted
from different pathogenicity of those bacteria and this applied
bacteria.
All 11 bacterial strains in this study possess different patho-
genicity and require different growth media. TSA medium
was only used to check the bacterial growth, even if the
bacteria grew faster or slower. Interestingly, MRSA was not
completely inhibited by the hygiene stick of ClO2 gas, in
view of the 90% inhibition. The difference of its pathogeni-
city might be definitely described, but MRSA was antibiotics-
resistant bacterium of interests. Other 10 bacteria are killed
by broad antibiotics, but MRSA is characterized by resis-
tance. Even though only one antibiotics-resistant bacterium
was applied here, it implied that antibiotics-resistant bac-
teria require more dose of ClO2 gas to be killed or growth-
inhibited.
Some bacteria can be applied in specific condition and
environments. No detectable levels of E. coli (limit of detec-
tion 5 log) were determined in the water within 1 min after
E. coli was added to the ClO2 containing wash water (Banach
et al., 2018). And Five mg/L of ClO2, E. coli was reduced
>5 orders of magnitude after 3 min (COD 1,130 mg O2/L)
(Haute et al., 2017). Concentrations of ClO2 up to 385 ppm
were safely maintained in a hospital room with enhanced
environmental controls (Lowe et al., 2013). In this study,
the released ClO2 gas concentration was 13 ppmv/hr (data
not shown), so we suggest that this 'ready-to-use- ClO2 stick'
maybe useful tool for inhibition of nosocomial infection.
This study will provide fundamental data to research
growth inhibition by ClO2 gas with bacteria of clinical impor-
tance value.
ACKNOWLEDGMENTS
Funding for this paper was provided by Namseoul Uni-
versity.
CONFLICT OF INTEREST
The authors have no conflicts of interest to disclose.
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https://doi.org/10.15616/BSL.2018.24.3.270
Cite this article as: Song KJ, Jung SY. Growth Inhibitory
Effects of Chlorine Dioxide on Bacteria. Biomedical
Science Letters. 2018. 24: 270-274.
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