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EFFICACY OF HYDROXYL RADICAL AEROSOLIZATION FOR INDUSTRIAL
SURFACE SANITATION AND HIGH-RISK AREA FUMIGATION
WANTHANAPORN BOONCHAN
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE MASTER DEGREE OF ENGINEERING IN
ENVIRONMENTAL ENGINEERING
FACULTY OF ENGINEERING
BURAPHA UNIVERSITY
AUGUST 2018
COPYRIGHT OF BURAPHA UNIVERSITY
ACKNOWLEDGEMENT
This thesis could not have been a success without help from many people. I
would like to express my appreciation and gratitude for the assistance given by those
who contributed to fulfilling this special research project. First of all, my advisor,
Asst. Prof. Dr. Aluck Thipayarat and my Co-advisor, Dr. Nuttinee Teerakulkittipong
for giving an opportunity to research this thesis project and giving a recommendation
all along. My appreciation goes to all project committee members including, Asst.
Prof. Dr. Chanan Phonprapai, Asst. Prof. Dr. Wirogana Ruengphrathuengsuka and Dr.
Emma Asanachinda for giving a valuable suggestion and knowledge during
presentation and project.
I am very grateful to have the strong industrial partnership with Rano Tech
Co., Ltd. for their funding to support equipment and chemical agents in my
laboratory. Special thanks are extended to the National Science and Technology
Development Agency for master degree student (Grant No. SCA-CO-2560-3504-TH).
Moreover, my special gratitude is extended to professors and all of the staff
members of Environmental Engineering Division, Burapha University who time-
honored an outstanding on a program to give me this great opportunity to progress my
knowledge and skills. Furthermore, I would like to thank all the Aluck Research
Team including, Wipavadee Sangadkit, Pattarin Supanichwatin, Adjima
Chayasitthisophon, Jirawan Supaproob, and Prof. Kenneth W. Foster at Syracuse
University, NY. and Dr. Nopphon Weeranoppanant at Burapha University for not
only their helpful suggestions but also how to work with other people as a team. Last,
of all, I also want to grateful thanks to all my friends/seniors at environmental
engineering division and Ph.D. student from the faculty of science, Romsan
Madmanang, for their support, helping and encouragement to reach the end.
Finally, great respect to my beloved parents and my brother for cheerfulness,
support throughout my study and always give encouragement and more have the
motivation to finish the study.
Wanthanaporn Boonchan
58910261: MAJOR: ENVIRONMENTAL ENGINEERING; M. Eng.
(ENVIRONMENTAL ENGINEERING)
KEYWORDS: AEROSOLIZATION/ SANITATION/ ADVANCED OXIDATION
PROCESS/ FUMIGATION/ HYDROGEN PEROXIDE
WANTHANAPORN BOONCHAN: EFFICACY OF HYDROXYL
RADICAL AEROSOLIZATION FOR INDUSTRIAL SURFACE SANITATION
AND HIGH-RISK AREA FUMIGATION. ADVISORY COMMITTEE: ALUCK
THIPAYARAT, Ph.D., NUTTINEE TEERAKULKITTIPONG, Ph.D. 85 P. 2018.
The use of combined UV-C and ozonation was applied to enhance the
production of hydroxyl radical (•OH) using hydrogen peroxide (H2O2) as a substrate.
The use of H2O2 as high as 5% alone was not effective for surface and hand
disinfectant when applying ultrasonic fumigation. The aerosols generated from this
technique were too small and well mist; hence, more effective oxidizing agents must
be developed. There were 3 other conventional disinfectants, including sodium
hypochlorite (NaOCl), chlorine dioxide (ClO2) and benzalkonium chloride (BKC) in
the experiments and their effectiveness as disinfectant fumes were compared to H2O2
and •OH fumes. Escherichia coli and Staphylococcus aureus were selected to
represent Gram-negative and positive bacteria. While 0.1% ClO2 fume was able to
disinfect contaminated surfaces of both bacteria, to reduce the pungent odors, and to
prevent usage difficulty of this chemical for practical applications of surface and hand
sanitizations. The development of •OH fume, on the other hand, was very effective to
inactivate bacteria contamination on both surface and hand applications. Due to its
short life and auto-oxidation to water and oxygen, this •OH fumigation technique was
an effective means for surface disinfection and hand sanitization that leaves no
footprints or toxic residue after usage making it as a useful alternative for such
applications.
CONTENTS
Page
ABSTRACT ........................................................................................................
CONTENTS..........................................................................................................
LIST OF TABLES................................................................................................
LIST OF FIGURES..............................................................................................
CHAPTER
1 INTRODUCTION......................................................................................
Objectives.............................................................................................
Work Scopes.........................................................................................
Benefitial outcomes..............................................................................
2 LITERATURE REVIEWS..........................................................................
Microbiological concerns in the industrial processing and the high
risk areas...............................................................................................
Microbial...............................................................................................
Escherichia coli...........................................................................
Staphylococcus aureus.................................................................
Conventional disinfection techniques...................................................
Chlorine dioxide (ClO2)...............................................................
Sodium hypochlorite (NaOCl).....................................................
Quaternary ammonium compounds (QACs)...............................
Hydrogen peroxide (H2O2)..........................................................
Advanced Oxidation Processes (AOPs)................................................
Hydrogen peroxide (H2O2)..........................................................
Ozone (O3)...................................................................................
Ultraviolet (UV)...........................................................................
Hydroxyl free radical (•OH)........................................................
Fogging Spray.......................................................................................
Mist blower..................................................................................
Thermal fog..................................................................................
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x
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CONTENTS (Cont.)
CHAPTER
Ultrasonic aerosolization.............................................................
Application Hand sanitation.................................................................
Alcohol-based hand rubs (ABHRs)......................................................
3 MATERIALS AND METHODS................................................................
Equipment and instruments...................................................................
The prototype of AOPs aerosolization..................................................
Production of hydroxyl radical aerosol.................................................
Bacterial strains and experimenting conditions....................................
Preparation of contaminated surfaces...................................................
Disinfectant aerosolization....................................................................
The effectiveness of hand fumigation for sanitation.............................
Preparation of hand contamination..............................................
Application of hand sanitization..................................................
Glove juice testing.......................................................................
Palm area testing..........................................................................
Finger area testing........................................................................
Data analysis.........................................................................................
4 RESULTS AND DISCUSSIONS...............................................................
Hydrogen peroxide fumigation.............................................................
Improvement of H2O2 treatment by AOPs............................................
Comparison to other alternative disinfectants.......................................
Application of •OH fume for hand sanitation.......................................
H2O2 /Ozonation/UV-C fume combined with 0.01% BKC..................
Comparison chart of disinfectants........................................................
5 CONCLUSION...........................................................................................
Recommendation..................................................................................
REFERENCES................................................................................................
Page
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CONTENTS (Cont.)
CHAPTER
APPENDIX...................................................................................................
APPENDIX A.......................................................................................
APPENDIX B.......................................................................................
BIOGRAPHY..................................................................................................
Page
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LIST OF TABLES
Tables Pages
2-1
4-1
4-2
4-3
4-4
4-5
Relative oxidation power of some oxidizing species....................................
Effect of H2O2 treatment on the initial cell loadings of 3, 5 and 7 log10
CFU/cm2. The colony growth results of the E. coli and S. aureus
inoculated surfaces were compared between the control treatment and the
treatment after 20 min of H2O2 fumigation...................................................
Visualization of finger area testing for finger print by 2 mL of E. coli
(5 log10 CFU/cm2) were treated with 0.5%, 1% and 3% H2O2 respectively,
for 30 seconds................................................................................................
Qualitative visualizations of E. coli hand contamination by palm area
testing procedures. 2 mL of E. coli (3 and 5 log10 CFU/cm2) were treated
3% H2O2. Imprints of contaminated palms were made onto PCA plates
and visualized after overnight incubation.....................................................
Qualitative visualizations of E. coli hand contamination by 4.5 ml of
E.coli was treated with 3% H2O2 and 0.01% BKC for 30 sec. Imprints of
contaminated palms were made onto PCA plates and visualized after
overnight incubation......................................................................................
Comparison characteristics, efficacies between H2O2/O3/UV fume with
conventional disinfectants.............................................................................
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LIST OF FIGURES
Figures Pages
2-1
2-2
3-1
3-2
4-1
4-2
4-3
4-4
4-5
Cells structure and colonial of E. coli...........................................................
Cells structure and colonial of S. aureus.......................................................
The schematic diagram of AOPs aerosolization...........................................
Diagram of the testing chamber made of transparent walls. Disinfectant
fume is introduced from the bottom and exited at the outlet on top.Testing
Petri dishes are installed near the outlet as indicated....................................
Viable cell counts of S. aureus and E. coli were treated by the vaporized
hydrogen peroxide at different concentrations.............................................
Effect of H2O2 concentrations combined with UV-C photocatalysis and
ozonation. H2O2 treatment on the initial cell 3, 5 and 7 log10 CFU/cm2 of
E. coli and S. aureus......................................................................................
Effect of UV-C, Ozone and UV-C/Ozonation treatments on the initial cell
3, 5 and 7 log10 CFU/cm2 of E. coli and S. aureus .......................................
Comparison of microbial disinfecting efficacies on contaminated agar
plates at the initial cell loading of 3, 5, and 7 log10 CFU/cm2.......................
Effect of E. coli treated by 0.5%, 1% and 3% were combined with ozone
and UV for handwashing...............................................................................
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CHAPTER 1
INTRODUCTION
Every year make a lot of people in the world sick globally diseases and
pathogenic which infections caused by poor hygiene and sanitation. Hutton and Haller
(2004) reported children worldwide lose 272 million school days. Not only do these
illnesses bar children from school attendance and achievement, poor hygiene and
sanitation can also inflict lots of negative impacts on their own development as well
as deepen the cycle of poverty to many underprivileged families in developing the
world. Healthcare-associated infections (HAIs) are also another good illustration of
how poor hygiene and sanitation can produce significant complications linking to
high morbidity and mortality due to additional infections occurred in healthcare
facilities (Magill et al., 2014). Recent statistics suggested that about 1 in 25 U.S.
hospital patients was diagnosed with at least one infection related to hospital care
alone. Normally, the health-care facility environment is rarely involved in the disease
transmission in patients, the immune-compromised patients (Sehulster, 2003).
Nonetheless, inadvertent exposures to the environmental pathogens (e.g., Aspergillus
spp. and Legionella spp.) or airborne pathogens (e.g., Mycobacterium tuberculosis
and varicella-zoster virus) can result in adverse patient outcomes and cause illness
among health-care workers. Environmental infection-control strategies and
engineering controls can effectively prevent these infections.
Several literatures suggested that bacteria have the ability to multiply and
attach to both engineered plastic and metal surfaces (e.g., polystyrene, polypropylene,
and stainless steel) (Barnes, Lo, Adams & Chamberlain, 1999; Suarez, Ferreiros &
Criado, 1992; Giaouris et al., 2014). Therefore, an effective method for reducing
bacterial contamination is important in achieving basic food safety standards. The
development of the less toxic and fast-acting sanitizing means or protocols still
proceed to control bacterial contamination, improve food sanitation practice and curve
the food outbreaks at the origins of the critical control points (Nitschke et al., 2009).
Many studies have been found the alternative aqueous sanitizers (e.g., organic acids,
chlorine dioxide, hydrogen peroxide, and ozonated water) which exhibit the highly
2
effective antimicrobial activity with the minimal toxicity of the residual chemical
(Yuk et al., 2006; Keskinen et al., 2009; Huang & Chen, 2011; Kingsley et al., 2014).
Each of these chemicals has its own unique characteristic and drawback. Hydrogen
peroxide (H2O2) solution, for instance, has been proven to be effective in controlling
the spread of hospital pathogens and used as the decontamination procedure for
clinical surfaces in hospital settings (Ali et al., 2016). As a highly active biocide,
H2O2 exhibits antimicrobial activity through the generation of hydroxyl free radicals
that penetrate the cell wall to attack lipids, proteins, and DNA (McDonnell &
Russell., 1999). Owing to its non-selective biocidal property, it can inhibit viruses,
spores, and fungi as well as bacteria (Block, 1991) and does not react with the organic
matter to form the toxic residues (Herdt & Feng., 2009; Jiang et al., 2017). The
reaction is terminated by forming benign by-products (e.g., oxygen and water)
(Luukkonen et al., 2014).
This research has combined the use of H2O2 and ultrasonic fogging systems
to create and disperse a disinfectant aerosol to disinfect the food processing surfaces
and also at the difficult-to-reach areas, especially the overhead surfaces, cracks and
the crevasses of food equipment and so on. For surface disinfection, fogging can be
effective only if a sufficient amount of disinfectant is deposited onto the surface with
the ample dose to inactivate microbial survival (Kakurinov, 2014). Other advanced
oxidation processes (AOPs), like O3 and UV, were installed to enhance the formation
of hydroxyl radicals (Kommineni, 2000). H2O2/O3 systems appear to be the most
tested and applied AOP protocol in much antimicrobial disinfection relative to the
other AOPs. The knowledge of these hydroxyl radicals enhancement to improve the
efficacy of H2O2 fumigation was limited. This study aims to investigate the
application of O3 and UV to compromise the strength of H2O2 applied to achieve the
highly biocidal effect on E. coli. The use of AOPs to decrease the toxicity of residual
H2O2 can facilitate the application of a highly efficient disinfecting protocol for food
processing area and surfaces with the minimal toxicity of chemical residue.
This research aims to investigate an affordable and more environmental-
friendly application with safer, less irritating and requires shorter exposure times for
fast-acting sanitizing with hydroxyl radical fume for surface and hand sanitation.
Together with the advanced oxidation processes (AOPs), this H2O2 fumigation have
3
constituted a promising technology since many industries incorporated H2O2 solution
into their routine applications in controlling the spread of hospital pathogens and as a
common decontamination procedure for clinical surfaces in food industries (Ali et al.,
2016) especially hard-to-reach cracks and cleavages. Nevertheless, using the high
concentration of H2O2 has a critical impact for material and environmental samples.
Therefore, the application of H2O2 on surface sanitization and its application on hand
disinfection were utilized at a low concentration of H2O2 to create dry fog and carry
•OH with the water mist in micron size.
Objectives
1. To compare the efficacy of hydroxyl radical fumigation generated from
advanced oxidation processes (AOPs) against other industrial means of surface
sanitization.
2. To create new applications of hydroxyl radical fumigation for surface
sanitization
3. To determine the critical factors affecting the production of effective
hydroxyl radical surface sanitization for new applications.
4. To develop the concept of hydroxyl radical aerosolization for hand
disinfection fumigation to improve the processing sanitation and hygiene.
5. To evaluate the efficacy of processing hand sanitization using hydroxyl
radical aerosolization technique.
Work Scopes
1. Gather fundamental knowledge and mechanisms of how the advanced
oxidation processes destroy E. coli and S. aureus.
2. Evaluate the industrial use of other surface sanitation techniques to
eliminate E. coli and S. aureus contamination on surfaces.
3. Compare the conventional techniques to hydroxyl radical aerosolization
using advanced oxidation processes for microbial decontamination.
4. Determine the optimal condition of hydroxyl radical aerosolization for
high-risk area fumigation.
4
5. Fabricate a semi-industrial prototype of hydroxyl radical fumigator and
evaluate its performance on microbial surface decontamination using bacteria.
Benefitial outcomes
1. Improve sanitation quality and hygienicity of industrial processes and
high-risk areas.
2. Develop a less toxic and environmentally friendly alternative approach to
enhance good sanitation.
3. Demonstrate the effectiveness of hydroxyl radical disinfection using our
constructed semi-industrial prototype in eliminating bacterial contaminations.
CHAPTER 2
LITERATURE REVIEWS
This chapter presents the theory and literature review related to this study
including the important for control the microbial contamination, disinfectant used,
foodborne pathogenic bacteria, healthcare-associated infections (HAIs) and Advance
oxidation process (AOPs) in this research.
Microbiological concerns in the industrial processing and the high-
risk areas
People who work in food factories as well as cleanrooms, the hand
sanitation is a vital importance in relation to personnel working. Because their hands
carry many types of microorganisms and these cells can be promptly transferred from
person to person, from person to equipment or onto the critical surfaces.
Staphylococcus, Micrococcus, and Propionibacterium are the group of bacteria which
presented on the skin and are unable to multiply microorganisms released from the
shed skin cells (Larson, 1988). Fresh produce can be contaminated by the water, air,
soil, insect vectors, an equipment or the improper handling by the workers
(Martinez et al., 2014; Meireles et al., 2016). For example, microbial adhesion on
food-contact surfaces including the conveyor belts, the containers used along the food
cycle in harvesting, post-harvesting, and packaging (Food and Drug Administration,
1998), they can easily lead to the formation of biofilms (Yaron & Romling, 2014),
following the produce contamination. In each year, the diseases and the pathogenic
infections caused by poor hygiene and sanitation make a lot of people sick worldwide.
For the hospital, a number of measures concerning the patient and the operating
environment can be taken to reduce the risks of surgical site infections
(Mangram et al., 1999; Chauveaux, 2015; Tammelin & Blomfeldt, 2016) including
keeping the number of bacteria in the operating room air as low as possible. This can
be achieved by a ventilation system that dilutes and/or sweeps away the bacteria
carrying particles in the air (Chauveaux, 2015).
6
Not only the equipment in the process showed a high-risk area for
pathogenic infection, but also the air in the environment can carry the microorganism.
Air is the main component in the atmosphere which can cause the bacterial infections.
Because it is the source of all living and nonliving forms (Sattar et al., 2016) and can
be contributed into the surrounding. For a human, it affected the profound health in all
indoor environments, where we spend most of our time (Kowalski, 2012;
Fernstrom & Goldblatt, 2013; Traistaru, 2013). The quality of indoor air is easy
changing by there are many controllable and uncontrollable factors, which are
virtually everywhere. Particularly indoor air can expose to noxious chemicals,
particulates, pollen, allergens, and a variety of infectious agents.
(Mandal & Brandl, 2011; Mandin et al., 2012). There are many groups of
emerging pathogens, such as Acinetobacter baumannii (Munoz et al., 2013;
Spellberg & Bonomo, 2013), noroviruses (Nenonen et al., 2014), and Clostridium
difficile (Best et al., 2010). They have also been detected in indoor air, with a strong
potential for airborne dissemination. Hence, there is a renewed emphasis on the
potential of indoor air for transmitting many types of infectious agents by direct
inhalation (Gralton et al., 2011; Sattar et al., 2016). Moreover, the airborne pathogens
also may settle on the environmental surfaces which could then become secondary
vehicles indoors (Muzslay et al., 2013). Airborne isolation is created by placing
infectious patients into rooms which having inward air flow and sustained negative air
pressure to prevent the spread of pathogens (Mousavi et al., 2015). This system
function is required for patients diagnosed with varicella, rubella, and tuberculosis as
well as an increasing number of new and emerging diseases. It is suspected of being
transmitted through the airborne route such as severe acute respiratory.
Microbial
Pathogens can survive on the fomites such as glass, steel, laminate, clothes
and upholstery, plastics, and carpets (Chambers, 2001; Kramer et al., 2006;
Coughenour et al., 2010; Desai et al., 2011). Especially they were founded higher
levels of pathogenic bacteria on vinyl surfaces than carpets, but generally lower
numbers of bacterial genera on vinyl surfaces compared with carpets
7
(Harris et al., 2010; Gupta et al., 2017). Moreover, nontraditional surfaces including
floors, have been studied sparingly (Edmonds, 2009). Microbiological quality is the
key attributes to indicate the integrity of food products and hygiene of food
processing. Coliforms and E. coli detection are the main criteria in the food industry
to determine the microbiological quality of food products. (Feng & Hartman, 1982;
Bredie & Boer, 1992; Sangadkit et al., 2012). The contamination of E. coli was
founded from direct or indirect fecal origins of humans and warm-blooded animals
(Kaspar et al., 1987).
Pathogen bacteria such as Salmonella spp., E. coli and S. aureus can cause
foodborne illnesses. They have presented a continuous challenge problem for food
safety and are considered as a common, costly, global public health concern
(McLinden et al., 2014; Havelaar et al., 2015; Wu et al., 2016; Liua et al., 2017).
Centers for Disease Control and Prevention (CDC) in the US estimates that
approximately 1 in 6 Americans get sick from contaminated food or beverages and
3000 die each year. They lost costing $15.6 billion in the US each year
(Israelsen et al., 2016).
Bacteria employ to form biofilm show up to 1000 times greater tolerance to
antibiotics and biocides resistant than their planktonic counterparts (Ceri et al., 1999).
Biofilms are difficult to eliminate once they are formed on the contact surface and are
therefore a persistent source of contamination in the food industry. Biofilms
can develop directly on food or on food-contact surfaces whereby they cause
contamination of the food (Shi & Zhu, 2009) leading to food spoilage or to the spread
of foodborne illnesses. Non-typhoidal Salmonella, Listeria monocytogenes, and
Campylobacter spp. are amongst the top five of pathogens causing death from
foodborne illnesses in the USA (Scallan et al., 2011). All of these bacteria readily
form to biofilms. Pseudomonas and Lactic acid bacteria species are common causes
of food spoilage (Gram et al., 2002) and are also able to form biofilms. The report
from the Centers for Disease Control and Prevention revealed that 48 million
citizens in the USA become ill from ingesting foodborne pathogens every year
(Scallan et al., 2011). One-third of foods globally go to waste due to spoilage
(Food and Agricultural Organization United Nations, 2011). These numbers indicated
that how serious problems to improve food preservation. For instance, both E. coli
8
and Salmonella Typhimurium are able to penetrate the leaves of iceberg lettuce
(Golberg et al., 2011; Meireles et al., 2016), while Seo and Frank (1999)
demonstrated that E. coli O157:H7 can penetrate 20 –100 μm below the surface of
lettuce leaves. Biofilms are sessile communities of microorganisms that initially
attach to the wet solid surface, and subsequently grow to produce extracellular
polymeric substances (EPS). Its keep the cells strongly together and also protect them
from external stress conditions (Kumar & Anand, 1998).
Escherichia coli
Escherichia coli (E. coli) is a Gram-negative, A cell structure of E. coli like
rod-shaped (see Figure 2-1a), and E. coli colonies are off-white and growth with a
steady pattern, colonies have no pigment (Figure 2-1b), but its change color when
transformed by a plasmid. The strain ATCC 25922 is commonly used to control the
strained quality, particularly in antibody sensitivity assays. Originally the strain is
isolated from a human clinical sample. E. coli, a large group of bacteria,
usually inhabit in the intestinal tract of humans and other warm-blooded animals
(i.e., mammals, birds). Most E. coli strains harmlessly colonize the gastrointestinal
tract of humans within two days and animals as a normal flora.
However, there are some strains that have evolved into pathogenic E. coli by
acquiring virulence factors through plasmids, transposons, bacteriophages, and/or
pathogenicity islands. These pathogenic E. coli can be categorized based on antigenic
differences and mechanism of pathogenicity. E. coli causing diarrhea can be classified
into 6 groups: Enteroaggregative Escherichia coli (EAEC), Enterohemorrhagic
Escherichia coli (EHEC), Enteroinvasive Escherichia coli (EIEC), Enteropathogenic
Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), and Diffusely
adherent Escherichia coli (DAEC) (Kaper et al., 2004; Amani et al., 2015;
Esfandiari et al., 2017). Among them, a prevalence of diarrhea caused by ETEC tend
to be higher, especially in the deprived areas, and people who travel to such areas that
also called traveler's diarrhea (Velarde et al., 2007). In many developing countries,
Diarrheal diseases caused by Escherichia coli strains are one of the most important
health problems in different human communities (Zhang & Sack, 2015;
Hayat et al., 2016; Mirhoseini et al., 2018) especially in areas with a lack of taking the
health (Nazarian et al., 2014; Madhavan & Sakellaris, 2015). There are many factors
9
that causing diarrhea diseases such as bacteria and viruses. They can lead to the
deaths of hundreds of thousands of people including children (Amani et al., 2015;
Bourgeois et al., 2016). Enterohemorrhagic E. coli (EHEC) is defined as pathogenic.
The Shiga toxins (Stxs) that produce from the E. coli strains can cause hemorrhagic
colitis (HC) and the life-threatening sequelae hemolytic uremic syndrome (HUS)
in humans. Several serotypes in EHEC are frequently associated with human
diseases such as O26:H11, O91:H21, O111:H8, O157:NM, and O157:H7
(Melton-Celsa et. al., 1996; Paton & Paton, 1999). E. coli O157:H7 is the most
frequently isolated serotype of EHEC from ill persons in the United States, Japan,
and the United Kingdom.
a) b)
Figure 2-1 a) Cells structure and b) colonial of E. coli (Zhang et al., 2016; The
Editors of Encyclopaedia Britannica, 2018)
The CDC has estimated that 85% of E. coli O157:H7 infections are
foodborne in origin (Mead et. al., 1999). Many food products have been associated
with E. coli O157:H7 outbreaks, such as ground beef, venison, sausages, dried (non-
cooked) salami, unpasteurized milk and cheese, unpasteurized apple juice and cider
(Cody et al., 1999), orange juice, alfalfa and radish sprouts (Breuer et al., 2001),
lettuce, spinach, and water (Friedman et al., 1999). In fact, consumption of this food
or beverage that becomes contaminated by an animal (especially cattle). The manure
can result in contracting the disease. In 1982, the first outbreak of E. coli O157:H7
occurred and was traced down to contaminated from hamburger meat
10
(Riley et al., 1983; Rahal et al., 2012). Not only the outbreak came from the beef
product particularly undercooked hamburgers but also unpasteurized milk was also
caused the outbreak (Griffin & Tauxe, 1991). Other food sources (vegetable and fruit)
are rising because marked changes in the epidemiology of human infections have
taken place.
Staphylococcus aureus
This microorganism was discovered and first described by the surgeon Sir
Alexander Ogston in 1880. He observed the cell structure like grape of bacteria
(Figure 2-2a) when examining a purulent discharge from patients with post-operative
wounds during microscopy and named them staphylé. (Taylor & Unakal, 2017). In
1884, Rosenbach could isolate yellow bacterial colonies from abscesses and named
them Staphylococcus aureus that “aureus” was come from the Latin word for gold.
Staphylococcus aureus is part of the genus Staphylococcus under the Micrococcaceae
family which contains more than 30 species such as S. epidermidis, S. saprophyticus,
and S. haemolyticus.
S. aureus is Gram-positive bacteria (stain purple by Gram stain) that are
cocci-shaped with diameters of 0.5–1.5 µm. It is characterized as coagulase- and
catalase positive, non-motile, non-spore-forming. S. aureus is a facultative anaerobe
so it can grow under both aerobic and anaerobic conditions. However, growth occurs
slower rate under anaerobic conditions (Stewart et al., 2003). It can grow in up to 10%
salt and colonies are often golden or yellow (aureus means golden or yellow) on
nutrient-rich media (Figure 2-2b). The temperature range for growth of S. aureus is
7–48°C, with an optimum of 37°C. For the pH range, S. aureus can grow well in the
pH range of 4.0–10.0, with an optimum of 6–7 (Stewart et al., 2003). S. aureus is
resistant in freezing condition and survives well in food stored below -20°C; however,
viability is reduced at temperatures of -10 to 0°C. S. aureus can be killed during
pasteurization or cooking.
S. aureus is the most virulent and pathogenic for humans. It is known as for
its capacity to cause a broad range of important infections in humans
(Costa et al., 2013). The capacity can be expressed in an array of factors which
participate in the pathogenesis of infection. It permits the bacterium to adhere to
surfaces/tissues, avoid or invade the immune system and cause harmful toxic effects
11
to the host (Lowy, 1998; Foster & Höök, 1998; Dinges et al., 2000). MRSA is the
multi-drug resistant strains for resisting Staphylococcus aureus and is essential to
hospital-acquired settings and treatment to manage the emergency. S. aureus can be
founded in the environment and normal human flora. It is usually located on the skin
and mucous membranes.
a) b)
Figure 2-2 a) Cells structure and b) colonial of S. aureus (Zhang et al., 2016;
Wikipedia, 2018)
Antibiotic resistance At first, penicillin was used to treat S. aureus
infections (Stark, 2013). Soon afterward, resistance emerged when strains acquired a
genetic element coding for β-lactamase production, and today over 80% of all S.
aureus strains are resistant to penicillins. The next drug to be introduced for treating
infections; Penicillinase-resistant penicillin named oxacillin or methicillin is
semi-synthetic and is introduced for treating infections with S. aureus. Despite
shortly after its introduction, the first isolate with resistance was detected
(Chambers & DeLeo, 2009).
Methicillin-resistant Staphylococcus aureus The massive consumption
of antibiotics over the past 50 years has led to the selection of drug-resistance among
S. aureus strains, and by far the most important is the resistance against methicillin. In
1961, methicillin (celbenin) became available for the treatment of penicillin-resistant
S. aureus strains. Only six months thereafter, the first methicillin-resistant S. aureus
was detected and nosocomial infections began to increase, and in Sweden efforts to
12
combat the spread was established. In the 1980s the detection of MRSA isolates
suddenly increased, and a few strains began to expand.
Conventional disinfection techniques
Disinfection is the process for destroying the pathogenic microorganisms to
control the bacterial growth or other undesired microorganisms in most food factories
or hospitals by using the disinfectant substances. Various types of disinfectants were
synthesis to inhibit the growth of pathogens bacteria. The mechanisms of disinfectants
upon the microbial physiology which are varied in their mode of action. The major
modes of action are cell wall synthesis inhibition, cell membrane inhibition, hindrance
of protein and DNA synthesis in the bacterial life cycle and competitive inhibition of
metabolic reactions (Todar, 2008). In this research, several types of disinfectants were
used in the commercial according to the historical reviewed literature. There are many
disinfectants in food microbiology which was used to focus on point of species.
Chlorine dioxide (ClO2)
Chlorine dioxide (ClO2) is a powerful sanitizer and has a broad
antimicrobial activity. It’s more stable and has a higher oxidizing capacity than
chlorine about 2.5 times (Chen and Zhu, 2011; Karabulut et al., 2009). ClO2 in the
form of unstable gas is generated by the addition of acid or chlorine to sodium
chloride (Scholz, 2015). When its react with the water leading to produce two
unstable acids products such as chlorous acid and chlorite acid which can act as
disinfectants. ClO2 does not react with nitrogen-containing compounds or ammonia to
form dangerous chloramine compounds (Chen et al., 2010). So it is considered as an
alternative to sanitizing fruit and vegetable processing for its effectiveness and safety
in water (US Food and Drug Administration, 2008). Moreover, ClO2 is a novel and
effective method for minimizing pathogens on fresh produce (Richardson et al., 1998;
Rodgers et al., 2004; Mahmoud et al., 2008). Du et al (2002). The processes have
been done in the research that described the antimicrobial efficacy of ClO2 on fruits.
A 5.5 log CFU reduction of L. monocytogenes on apple skin was achieved by
treatment with 4.0 mg/L ClO2 gas for 10 min. Additionally, more than a 5 log
reduction of E. coli O157:H7 on apple skin was achieved by treatment with 7.2 mg/L
13
ClO2 gas for 10 min (Du et al., 2003). Mahmoud et al. (2007) reported that
approximately a 4.3–4.7 log CFU reduction per strawberry of E. coli O157:H7,
L. monocytogenes, and Salmonella Enterica was achieved by treatment with 5 mg/L
ClO2 for 10 min.
Sodium hypochlorite (NaOCl)
Sodium hypochlorite is usually called household bleach (Rutala et al., 2008)
and is aqueous solutions of 5.25%–6.15% sodium hypochlorite. It is the most
prevalent chlorine products in the United States. They have a broad spectrum
of antimicrobial activity and have a low incidence of serious toxicity
(Jakobsson et al., 1991). Sodium hypochlorite is unaffected by water hardness.
They are inexpensive and fast acting (Rutala & Weber, 1997) by removing dried or
fixed organisms and biofilms from surfaces (Merritt et al., 2000). The concentration
of Sodium hypochlorite approximately 5.25 to 6.15 percent can produce ocular
irritation or oropharyngeal, esophageal, and gastric burns.
The data suggest that current disinfection and sterilization practices are
appropriate for managing patient-care equipment and environmental surfaces.
The contaminated patients are potentially evaluated and/or admitted in a
health-care facility after exposure to a bioterrorist agent. For example, sodium
hypochlorite can be used for surface disinfection. A 1:50 dilution of 5.25%–6.15%
sodium hypochlorite (household bleach) for 5 minutes should be effective
(Rutala et al., 1998). NaOCl has been used as an irritant agent in root canal treatments
for permanent teeth since the 1920s and has been shown to have good antimicrobial
effects without being a significant pulpal irritant (Tang et al., 2000; Orstavik, 2003).
Rosenfeld et al., 1978 showed that placement of 5% NaOCl on non-instrumented vital
pulp tissue acted only at the superficial surface with minimal effects on deeper pulpal
tissue. Hafez et al. (2000, 2002) showed normal soft tissue reorganization and
dentinal bridge formation after hemorrhage control was obtained with 3% NaOCl in
pulpotomized adult monkey teeth.
It is common for dentists to use commercial household bleach as the source
of NaOCl for root canal irrigation (Jungbluth et al., 2012). The recommended
concentration of NaOCl ranges from 0.5% to 6% in the past, with no consensus on the
ideal concentration. Previous studies showed negligible differences in antibacterial
14
activity among 5.25%, 2.5% and 1% NaOCl in infected root canals. When using 0.5%
NaOCl in larger volumes and longer irrigation times, it possesses good bactericidal
activity (Siqueira, et al., 2000). All concentrations of NaOCl were effective in
eliminating endodontically-relevant resistant microbes, including Candida albicans,
Pseudomonas aeruginosa, Enterococcus faecalis, Bacillus subtilis, Streptococcus
mutans and Staphylococcus aureus (Arcangelo et al., 1999; Arias-Moliz et al., 2009).
Conversely, the tissue dissolving effect of NaOCl is directly related to its
concentration. To maximize the tissue dissolving and antimicrobial effects, NaOCl is
frequently used in ‘‘full strength”, namely, at the highest end of the concentration
range (Cullen et al., 2015). The concentration approximately 8.25% NaOCl became
available in 2012. However, many dentists are not aware of this change. Although
more and more endodontists are using this concentrated NaOCl version. The available
information showed the data about the difference between various concentrations of
NaOCl at 2%, 4%, 6%, and 8%, affected their effects on bacterial cells and human
tissues.
Quaternary ammonium compounds (QACs)
Quaternary ammonium compounds (QACs) derived from substituted
ammonium salts with a chlorine or bromine anion (Holah, 2014) and were cationic
membrane active antibacterial agents. It is usually used in healthcare disinfectants,
agriculture, home, and the food industry (Gerba, 2015; Tezel & Pavlostathis, 2015).
QACs disinfection performance showed having more effective against
Gram-positive bacteria (e.g. staphylococci and streptococci) at lower concentrations
(< 50 ppm, 10°C) than Gram-negative bacteria like coliforms and psychotropic
organisms (Sandle, 2013). They are also considerably less effective against bacterial
spores. QACs are sometimes classified as surfactants (i.e., benzalkonium chloride).
The certain alkaline compounds (anionic wetting agents) in QAC can reduce the
bactericidal action. However, the factors that can impair their bactericidal
effectiveness are the presence of organic matter, water hardness. They can reduce
their activity and the type of organism. QACs are stable in concentrated form and
have long shelf-life. In concentrated form, they are much safer to handle than
hypochlorite solutions and they are relatively non-corrosive to metals. Owing to their
15
high surface activity, an excessive foam can be produced during circulation through
the plant and hence QACs are sometimes difficult to rinse away.
Benzalkonium chloride (BKC) is a quaternary ammonium compound
that has been in clinically used since 1935. It is an antimicrobial additive
(Marple et al., 2004). It has been used to maintain the sterility of a variety of
prescription and over-the-counter products, such as cosmetics, infant care products,
pharmaceutical nasal sprays, ophthalmic solutions, and otic drops (Liebert, 1989).
As reported in the Journal of American College of Toxicology, the Cosmetic
Ingredient Review panel concluded that BKC can be safely used as an antimicrobial
agent at concentrations up to 0.1%. In addition to the intranasal products containing
BKC, there have been conflicting reports about the damage to human nasal epithelial
and/or exacerbation of rhinitis medicaments. Velázquez et al. (2009) showed that
benzalkonium chloride at 0.1 ppm damaged lettuce leaves with the appearance of
yellow spots on the produce after 7 days of storage.
Benzalkonium chloride (BKC) is a subgroup of quaternary ammonium
compounds (QACs) and contain benzyl dimethyl ammonium chlorides with an
attached alkyl chain of C8–C18 (Khan et al., 2017). The commercial use of
surfactants was 12 million tons in 2010, of which approximately 15% were QACs
(Brycki et al., 2014). Total BACs have been measured in environmental samples
worldwide. Concentrations of total BKC ranged from 0.05 g/L to 6.03 mg/L in
hospital effluents in several European countries (Kümmerer et al., 1997). In some
wastewater treatment plants (WWTP) in Austria, BKC was measured in the range of
0.02–0.31 mg/L in the influents (Clara et al., 2007). BKC in the range of 0.022–0.21
mg/kg were also found in river sediments close to WWTP and other urban areas in the
USA (Ferrer & Furlong, 2002), and from 0.05 to 1.1 mg/kg in China (Li et al., 2014).
Environmentally relevant concentrations of BKC are toxic, particularly to aquatic life
(Ferk et al., 2007; Pérez et al., 2009).
BKC is the active ingredient of many pharmaceutical formulations,
cosmetics, commercial disinfectants, industrial sanitizers and food preservatives
(Tezel & Pavlostathis, 2009). It is widely used as a clinical disinfectant and antiseptic
in health care and domestic facilities. There is an antimicrobial preservative in drugs,
an antiseptic for preoperative skin or for wounds, burns, etc. Moreover, it is a
16
disinfectant in processing lines and on surfaces in the food industry, and also as an
antimicrobial agent in the treatment of common infections of the mouth and throat
(Mangalappalli-Illathu & Korber, 2006).
Hydrogen peroxide (H2O2)
Hydrogen peroxide with the formula of H2O2 is a chemical substance. The
pure form compound is colorless liquid, slightly, and more viscous than water. H2O2
is the simplest peroxide (a compound with an oxygen-oxygen single bond). It is used
as an oxidizer, bleaching agent and disinfectant. For the biological function, it has
important roles as a signaling molecule in the regulation of a wide variety of
biological processes (Giorgio, 2007). The compound is a major factor implicated in
the free-radical. Based on the H2O2 can be decomposed into a hydroxyl radical and
produced superoxide radical byproducts. These hydroxyl radicals in turn readily react
with and damage vital cellular components (Veal, 2007; González, 2010), especially
those of the mitochondria.
Advanced Oxidation Processes (AOPs)
Advanced oxidation processes (AOPs) are defined as the oxidation
processes which generate very powerful, non-selective hydroxyl radicals that are
utilized in water treatment (Munter, 2001). The hydroxyl radicals (•OH) are the
principal reactive oxidizing agents in water (Table 2-1) and are highly active in the
inactivation of bacteria and virus (Selma et al., 2008). Many systems are qualified
under this broad definition of AOPs. The combination of strong oxidants (e.g., O3 and
H2O2, catalysts, transition metal ions or photocatalyst, irradiation, ultraviolet (UV),
ultrasound (US), or electron beam) were used in these systems. AOPs may be sorted
into three main groups, for example, photocatalysis and hydrogen peroxide
photolysis, the Fenton reaction based processes, and ozonation processes. The
advanced oxidation processes where two or more oxidants are used simultaneously.
The most common process used to generate •OH is through the use of combined
catalytic oxidants such as ozone-ultraviolet (O3/UV), hydrogen peroxide-ultraviolet
(H2O2/UV) and hydrogen peroxide-ozone (H2O2/O3). Although these processes can
produce •OH, the H2O2/UV combination provides the maximum yield of •OH per
oxidant. The key difference between the ozonation and AOP processes is the ozone
17
process relies mainly on the direct oxidation with aqueous ozone while AOPs rely
primarily on oxidation with hydroxyl radicals. As stated above the aim of AOPs is to
produce the hydroxyl radical in the aqueous medium (Jung et al., 2008).
For this reason, the use of H2O2 and ultrasonic fogging systems have been
attracting increasing research interest. However, little information is currently
available about the use of H2O2 and ultrasonic fogging treatment for sanitizing the
microbial in the food industry.
Table 2-1 Relative oxidation power of some oxidizing species (Goi, 2005)
Oxidation species Oxidation potential (eV)
Fluorine 3.06
Hydroxyl radical 2.80
Nascent oxygen 2.42
Ozone 2.07
Hydrogen peroxide 1.77
Perhydroxyl radical 1.70
Hypochlorous Acid 1.49
Chlorine 1.36
A common reaction is the abstraction of a hydrogen atom to initiate a
radical chain oxidation (Munter, 2001).
RH + OH• → H2O + R (2-1)
OH → H2O2 (2-2)
R + H2O2 → ROH + OH• (2-3)
R + O2 → ROO (2-4)
ROO + RH → ROOH + R (2-5)
Hydrogen peroxide (H2O2)
Hydrogen peroxide (H2O2) is an oxidizer that can form cytotoxic species.
The formation of these cytotoxic species is what assures its antimicrobial properties
18
(Ölmez & Kretzschmar, 2009; Rahman et al., 2010; Rico et al., 2007) which can be
either bactericidal or bacteriostatic (Brul & Coote, 1999; Ölmez & Kretzschmar,
2009). It depends on the concentration, pH, and temperature (Beuchat, 1998). This
disinfectant can be applied on food-contact surfaces (Rico et al., 2007). However, the
use of H2O2 cannot avoid the cross-contamination which can still occur in the
vegetables washing water (Haute et al., 2015), as a result, its decomposition is fast
and the disinfection kinetics is slow. Another disadvantage is the cause of the
browning effects of the vegetables, particularly to lettuce. It is an environmentally
friendly disinfectant and is quickly decomposes, despite the fact that the
concentrations used are very high.
Ozone (O3)
Ozone (O3) is generated as a gas that can be dissolved in water. When it is
used in a dissolved form, only a small concentration about 1–5 ppm is needed to exert
antimicrobial activity. The sufficient in destroying the pathogens appears the retention
time between 5 and 10 min. This method is effective in killing pathogens. In the water
treatment, the ozone is a strong oxidizing and showed the effectiveness in removing
taste, odor, iron, manganese, and color residual from waters. However at higher
concentrations are required when it is used as the gas since the humidity of the air
affects its penetration into the cells and the consequent disinfection process
(Chauret, 2014; Horvitz & Cantalejo, 2014). It is a very powerful oxidizer with
showed high antiseptic potential (Cunningham et al., 2012) and can spontaneously
decompose toward to a nontoxic product as O2. (Atungulu & Pan, 2012;
Kim et al., 2003). However ozone not only provides the effectiveness in disinfection
bacterial but also get some disadvantages, it is unstable and rapidly decomposes
(Chawla et al., 2012). Moreover, it can become very toxic (Chauret, 2014)
that can affect the respiratory tract and cause irritation to the eyes and throat
(Artés et al., 2009). The ozone is quite sensitive to the presence of organic matter, as a
result, the performance of ozone will reduce. It has to be generated on site
(Chauret, 2014) and is not suitable to be used on the produce. Since it can affect its
physicochemical properties (Cunningham et al., 2012) and is potentially corrosive to
the equipment (Sapers, 2009).
19
Ultraviolet (UV)
UV radiation has been used for the disinfection of surfaces, fluids and
drinking water because it is demolition to bacteria, yeasts viruses, algae and protozoa
(Koivunen & Tanski, 2005). When UV radiation is absorbed by the cell, the
antimicrobial effect of UV radiation occurs due to the photochemical changes that
take place in proteins and nucleic acids. Various foods and beverages have been
treated by UV radiation to decrease bacterial content and eliminate pathogens such as
L. monocytogenes (Bintsis et al., 2000). Many food researchers have applied ozone
and UV-C to various fruit juices during processing, for example, apple cider, orange
juice, strawberry juice, and apple juice (Kumar et al., 2016). Previous research has
shown ozone and UV treatment to be beneficial in reducing bacterial contaminated in
water when used individually and in combination.
Hydroxyl free radical (•OH)
The hydroxyl free radical is created from H2O2 that can destroy bacterial
cells (Ikai et al., 2010; Shirato et al., 2012; Toki et al., 2015; Nakamura et al., 2016).
Hydroxyl radical molecules are reactive oxygen sorts that supply other substances of
an electron donate (i.e. oxidizes them). When hydroxyl radicals interact with bacteria,
they cause lethal oxidative damage. However, it has a very short lifetime in liquid
only 10-9 s (Pryor, 1986; Sies et al., 1992) and it cannot be formulated as a ready-
made disinfectant. In the photolysis reaction, hydroxyl radicals are generated by the
light irradiation at the wavelength less than 405 nm and their yield depends on light
intensity, irradiation time and concentration of irradiated H2O2 (Ikai et al., 2010).
Furthermore, residual toxicity in the environment negligible because of the short
lifetime of hydroxyl radicals (Yamada et al., 2012; Kanno et al., 2012). Therefore,
this technique is expected to be applicable to prophylaxis and/or treatment of
superficial infections, including dental caries caused by acidogenic bacteria in dental
plaque. However, whether this technique can kill highly resistant bacteria embedded
in biofilms remains unclear.
Fogging Spray
The fogging spray is the disinfectant aerosol which is applied to disinfect
food processing surfaces. It can be created by the systems as follow;
20
Mist Blower
It consists of a gallon tank which contains the hydrogen peroxide solution
and the air blowers together with high power, low weight, and superior engine
efficiency. It provides a powerful air stream and the chemical from the gallon tank is
turned to a fine mist which disinfects the microbial on food processing surfaces.
Thermal fog
A thermal fogger is an equipment that normally used to terminate a pest
problem in an outdoor area. In this system, the fogging solution was heated to produce
a mist or fog that can easily penetrate to reach outdoor areas. (i.e., shrubbery, grass,
treetops) and many other difficult areas. Thermal foggers are often used for mosquito
control, microbial disinfection and so on. The insecticide or other fogging liquid is
filled into the container. The fogging solution in the tank is carried out by the pump
through the heating assembly located on the front of the fogger. The pumping is done
by a manual operating the fogger when a fogging trigger is pressed by the user.
Ultrasonic aerosolization
Ultrasonic aerosolization is the process to create and disperse a disinfectant
aerosol for disinfecting food processing surfaces. The disinfectant aerosol can be
conducted by an ultrasonic aerosol generator for delivering a liquid formulation
(i.e. H2O2) at a high output rate. This device contains at least a liquid reservoir/
aerosolization chamber, a piezoelectric engine, a relief aperture, and an aerosol
delivery element. This equipment releases cooler vapor than the steam-type and
releases the most of the dissolved and suspended components of the water, including
microorganisms and pathogens, into the air.
Application Hand sanitation
Regarded as the simplest yet most cost-effective intervention in reducing
healthcare-associated infections (HAIs), hand hygiene is important in any health care
concern around the world (Cruz & Bashtawi, 2016). It is an essential component of
infection control which is critical to ensuring patients’ safety in hospitals
(Colet et al., 2015). Due to the increasing incidence rate of HAIs and the growing
burden accompanying them, the increasing complexity of illnesses and their
complications affected the soaring cost of hospitalization and the occurrence of
21
multiple-resistant pathogens causing new types of infections. Therefore the necessity
for strict and effective compliance with hand hygiene has been emphasized (Mathur,
2011). A wide array of studies supports indicated that the effectiveness of hand
hygiene can reduce cross-contamination and infection in a healthcare facility
(Boyce & Pittet, 2002; Kampf & Kramer, 2004; Mathur, 2011).
In March 2015, the World Health Organization (WHO) and the United
Nations Children’s Fund (UNICEF) released the report (World Health Organization,
& Unicef, 2015) on the status of water and sanitation in health-care facilities from 54
low- and middle-income countries (Bartram et al., 2015). Data representing 66,000
health facilities show that water was not readily available in about 40%
(Bain et al., 2014). The facilities lacked soap for hand washing and lacked toilets in
the third and fifth respectively. In many countries, there is no guarantee about the
safety for consumption in spite of the available water in facilities.
The total aerobic bacterial counts on the scalp are colonized ranging from
more than 1 x 106 CFU/cm2 on the scalp, 5 x 105 CFU/cm2 in the axilla, and 4 x 104
CFU/cm2 on the abdomen to 1 x 104 CFU/cm2 on the forearm (Selwyn, 1980).
Total bacterial counts on the hands of HCWs have ranged from 3.9 x 104 to 4.6 x 106
CFU/cm2 (Price, 1938; Larson, 1984; Larson et al., 1998; Maki, 1978). Fingertip
contamination ranged from 0 to 300 CFU when sampled by agar contact methods
(Pittet et al., 1999). These bacteria may be lead to many diseases. The gastrointestinal
infections, such as Salmonella, and respiratory infections, such as influenza. It can be
spread from one person to another.
Washing your hands properly can help prevent the spread of the germs (like
bacteria and viruses) that cause these diseases. Some forms of gastrointestinal and
respiratory infections can cause serious complications in the group of young children,
the elderly, or those with a weakened immune system.
Hand-washing products are not uniformly equal in their ability to prevent
the spread of infection (Messina et al., 2008). Mechanical hand-wash products most
often contain esterified fatty acids with sodium or potassium hydroxide and are used
for social hand washing. These detergent/surfactant products remove loosely adherent
microorganisms and viruses from the hands by mechanical means. They have no
effect on resident hand flora after 2 minutes of hand washing. (Boyce & Pittet, 2002;
22
Kampf & Kramer, 2004) In fact, contamination of the hands may occur in the process
of hand washing with non-medicated bar soaps by contacting the surrounding
environment, faucet, paper towel handle, or the sink edge (Boyce & Pittet, 2002).
Furthermore, bacteria adhere more readily to wet hands can cause increases the risk of
cross-contamination.
Alcohol-based hand rubs (ABHRs)
Alcohol-based hand rubs (ABHRs) have been used for the prevention of
transmission of infections for many years and having been shown to be safe and
highly effective (Pittet et al., 2000; Loveday et al., 2014). Alcohol consists of two
water-soluble chemical compounds such as ethyl alcohol and isopropyl alcohol in the
healthcare setting that have generally underrated germicidal characteristics
(Spaulding, 1964). FDA has not confirmed any liquid chemical sterilant or high-level
disinfectant with alcohol as the main active ingredient. These alcohols are destroying
bactericidal rapidly rather than bacteriostatic including vegetative forms of bacteria
and they also are tuberculocidal, fungicidal, and virucidal. However, these agents can
not destroy bacterial spores. The diluted solution below 50% concentration drops the
cidal activity sharply. The optimum bactericidal concentration is approximately 60%-
90% solutions in water (v/v) (Block, 1991)
ABHRs were introduced in the healthcare environment as an alternative to
hand washing for use when hands are physically clean (Cheeseman et al., 2009). Their
introduction resulted in much higher compliance rates and this has led to a reduction
in infection rates (Karabay et al., 2005). There are many AHRs commercially
available, each with a different formulation. Although these products all claim to kill
a number of pathogenic micro-organisms, different efficacy test protocols are being
used by manufacturers. These include methods such as EN 1500, which measure
efficacy against a small number of standard bacterial strains in suspension
(EN 1500, 1997). Many available agents for hand aseptic are alcohol-based and are
widely used with good results but may provoke skin reactions (Humes & Lobo, 2006;
Appelgrein et al., 2016). Alternatives substances for hand aseptic that do not cause
skin reactions but still provide effective hand asepsis would facilitate workplace
compliance.
CHAPTER 3
MATERIALS AND METHODS
Equipment and instruments
1. Hot air incubator, Memmert Model ULM500, Japan
2. Laminar flow cabinet, DWYER Series 0325, USA
3. 6-microwell plate, Costar, USA
4. Autoclave, BECTHAI and HIRAYAMA Model HA300D, Japan
5. Auto pipette volume 10 microliter, Autopipette, USA
6. Auto pipette volume 200 microliter, Autopipette, USA
7. Auto pipette volume 1000 microliter, Autopipette, USA
8. Balance accuracy 0.0001 grams, Metter Toledo Model AG204,
Switzerland
9. Balance accuracy 0.01 grams, Metter Toledo Model GG4002-4,
Switzerland
10. Digital camera, Olympus SP 570 UZ, Indonesia
11. Petri dish plastic, Citotest, China
The prototype of AOPs aerosolization
This equipment shows how to fumigate a model chamber with the
dimension of 34 x 34 x 34 centimeters cube.
1. Oxygen tank, Sangsap, Thailand
2. The ozone generator, Rano tech, Thailand
3. Venturi
4. UV-C lamp sterilizer
5. Flowmeter
6. Water Pump
7. Liquid tank 10 liters
8. Valve
9. Conduit
10. Chamber
24
11. Fumigator, Meiyan, China
11.1 Ultrasonic mist maker
11.2 Level switch
11.3 Electric fan
Production of hydroxyl radical aerosol
Strong oxidizing molecules including •OH are generated in liquid phase
with our patent-pending technology combining ozonation and UV-C photocatalysis
and aerosolized to micron-size particles. A prototype of •OH fumigator was
constructed as shown in Figure 3-1. The system which consists of an ozone generator,
UV-C unit, and ultrasonic fumigator. The reservoir contained 10 L liquid of each
disinfection reagents. An oxygen tank, flow rates 2 L/min, was connected to the ozone
generator. The ozone gas was subsequently forced into a venturi in which the ozone
gas was mixed with circulating water before transferring into a fumigator. The
fumigator (Meiyan, China) contained twelve ultrasonic mist maker produces aerosols.
The aerosols were further dispersed by a fan in the fumigator. The system also has
15W UV-C lamps installed in the circulation line to activate more •OH production.
The fumes, containing the radicals, were then sent to a testing chamber with bacterial
inoculated plates installed.
Figure 3-1 The schematic diagram of AOPs aerosolization.
25
Bacterial strains and experimenting conditions
Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923
stock cultures were received from the Department of Medical Sciences Thailand
(DMST, Bangkok, Thailand) and kept in the Trypticase Soy Broth (TSB, Himedia,
Mumbai, India) containing 20% glycerol and stored at –80°C. Prior to use, the
bacterial stocks were grown in TSB for 18–24 hr at 37°C (Keskinen et al., 2009). TSB
was prepared by mixing 3 g of TSB into 100 mL of distilled water (Supanivatin et al.,
2011). TSB was sterilized at 121°C for 15 min in an autoclave.
Preparation of contaminated surfaces
Artificially-contaminated agar plates of E. coli and S. aureus were fabricated
by serially diluted the culture stock and varying the cell concentration between 3 to 8
log10 CFU/mL using saline water. 100 µL of inoculum was introduced onto the 90
mm disposable Petri dish (Citotest, Haimen, China) containing Plate Count Agar
(PCA, Difco, USA). PCA was prepared by mixing 4.7 g of PCA powder into 200 mL
of distilled water. Then PCA was sterilized at 121°C for 15 min in an autoclave. The
final cell densities achieved in each inoculated plated were approximately varied from
1 to 7 log10 CFU/cm2. The plates were installed on the top side inside the fumigated
chamber as shown in figure 3-2 where the incoming fume of tested sanitizing agents
was entered from the bottom. The total volume of the cube is 0.04 m3 measured 0.34
m on each side.
26
Figure 3-2 Diagram of the testing chamber made of transparent walls. Disinfectant
fume is introduced from the bottom and exited at the outlet on top.
Testing petri dishes are installed near the outlet as indicated.
Disinfectant aerosolization
Four disinfectants (i.e., hydrogen peroxide (H2O2, Merck, Germany), sodium
hypochlorite (NaOCl, AGC chemical, Thailand), chlorine dioxide (ClO2, Hydro-bio,
Thailand) and benzalkonium chloride (BKC, Chemipan, Thailand) were selected to
compare their bactericidal effectiveness to aerosolized •OH. The H2O2 application
was adjusted to three concentrations (1%, 3% and 5%) where the concentrations of
the other disinfectants were selected from their lower and upper bounds used for
common disinfection applications found in literatures (i.e., 0.04% and 0.5% for
NaOCl, 0.0003% and 0.1% for ClO2, and 0.004% and 0.1% for BKC). The treatment
times were varied to 5, 10, 15, and 20 min in order to demonstrate any differences in
their ability to kill bacteria on the surface of the inoculated plates. After each
treatment, the disinfected plates were inoculated at 37°C for 18-24 hr and evaluated
the cell culture dishes on the top of the chamber were fumigated already and
evaluated visually by observing density of colonies compared to the control
treatments.
27
The effectiveness of hand fumigation for sanitation
Preparation of hand contamination
A 1.5 mL aliquot of E. coli suspension at two cell densities (i.e., 1 and 8
log10 CFU/mL) was applied onto each volunteer’s cupped hands. The aliquot was
rubbed thoroughly over the hand’s surfaces for 1 min and left to be air dried for 20
sec. This procedure (i.e., dispensing, rubbing, and drying steps) was repeated three
times to apply the total aliquot volume of 4.5 mL. After the third aliquot, the hand
contamination was complete and ready for hand sanitization experiment.
Application of hand sanitization
Using the same prototype of •OH fumigator, other tested disinfectants (i.e.,
H2O2, NaOCl, ClO2, and BKC) were aerosolized via ultrasonic fumigation while the
UV-C photocatalytic and ozonation units were inactivated. During hand sanitization,
the disinfectant fume was generated at all time and both of the bacterial applied hands
were rubbed against each other thoroughly and vigorously. Exposure times were
varied for every 5 sec up to 60 sec by using the procedure described in ASTM E-1174
(Standard Test Method for Evaluation of the Effectiveness of Health Care Personnel
Handwash Formulations) (Macinga et al., 2011).
Glove juice testing
The background colony count on the tested hands was first determined by
massaging the fingertips in the sterile plastic bag containing 100 mL of distilled water
for 1 min (Wilkinson et al., 2017). The contaminated sample was kept in an eppendorf
tube two times each of the sample and then 0.1 mL diluted samples were plated onto
PCA agar plates and incubated at 37°C for 24 hr. Total numbers of CFU were counted
for each plate.
Palm area testing
Palm area testing was prepared by dropped 2 mL of E. coli suspension on
each of the subject’s cupped hands. The aliquot was rubbed over all surfaces of palm
hands for 1 min. After applied fumigation for hand sanitization, the contaminated
hands (palm) were imprinted onto the surface of PCA agar plates two times each of
the sample. The plates were then incubated at 37°C for 24 hr before taking photos for
bacterial evaluation.
28
Finger area testing
Finger area testing was prepared by dropped 2 mL of E. coli suspension on
fingers each of the hands. The aliquot was rubbed over all surfaces of fingers for 1
min. The contaminated hands (finger) were imprinted onto agar media two times each
of the sample. The plates were then incubated at 37°C for 24 hr prior to visualization.
Data analysis
For each treatment, the mean and standard deviation for the survivor ratios
of bacterial were calculated (Sauer & Moraru, 2009).
CHAPTER 4
RESULTS AND DISCUSSIONS
This research has combined the use of H2O2 as well as other common
disinfectant used commercially and ultrasonic fogging systems to create and disperse
disinfectant aerosols for hand sanitization. The same concept can be applied to
disinfect the food processing surfaces and also at the difficult-to-reach areas,
especially the ventilation and air conditioning systems, cracks and the crevasses of
food equipment and so on. For hand sanitization, airborne, micron-size fume can be
effectively and evenly dispersed on to total hand surface and with sufficient
bactericidal strength and mass deposited onto the surfaces this fumigation
methodology should readily inactivate any bacteria present on hands.
Hydrogen peroxide fumigation
Figure 4-1 shows the effectiveness of H2O2 fumigation generated from our
prototype equipment on contaminated surfaces with S. aureus and E. coli at the initial
cell loadings of 3, 5 and 7 log10 CFU/cm2 over the course of 20 min. The 5% H2O2
treatment produced slightly better bacterial kill comparing to the 3% H2O2 treatment.
At the same strength of H2O2, longer treatment reduced the bacteria present on the
contaminated agar surface and S. aureus in general was more vulnerable than E. coli.
At high initial cell contamination (i.e, 7 log10 CFU/cm2), only 5% H2O2 returned 1 log
reduction after 20 minutes treatment. At lower cell density (e.g., 3 log10 CFU/cm2), as
high as 3 log reduction can be observed on S. aureus contaminated plates.
In general, hydrogen peroxide has a greater antimicrobial activity against
both of gram-negative than gram-positive bacteria (Buck, 2001). Unlike like most
disinfectants, hydrogen peroxide is unaffected by the addition of organic matter and
salts and without chemical residue. Gram–negative bacteria are generally less
susceptible to biocides because of their complex cell wall (Sheldon, 2005). The outer
cell wall membrane composed of peptidoglycan, lipoproteins and the
lipopolysaccharides act as permeability barrier in limiting or prevention the entry of
many chemically unrelated types into the bacterial cell (Russell et al., 1998;
30
Sheldon, 2005; Al-Jubory et al., 2012). Generally, Gram–positive bacteria observed
and neither of these appears to act as effective barrier to the break-in of antiseptics
and disinfectants. Therefore high molecular weight substances can readily pass into
the S. aureus and vegetative cell of Bacillus spp.
a) 3% H2O2 b) 5% H2O2
Figure 4-1 Viable cell counts of S. aureus and E. coli were treated by the vaporized
hydrogen peroxide at different concentrations
The evaluation of bacterial kill on the inoculated surfaces was demonstrated
on Table 4-1. When the significant portion of bacterial colonies was destroyed
comparing to the control, the surface was qualitatively declared positive for
disinfection.
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10C
FU
/cm
2)
Time (min)
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10C
FU
/cm
2)
Time (min)
31
Table 4-1 Effect of H2O2 treatment on the initial cell loadings of 3, 5 and 7 log10
CFU/cm2. The colony growth results of the E. coli and S. aureus
inoculated surfaces were compared between the control treatment and the
treatment after 20 min of H2O2 fumigation.
Microorganisms Concentration
inoculated
Control
treatment
(0 min)
Fumigation results
(after 20 min)
Negative Positive
E. coli 7 log10
CFU/cm2
5 log10
CFU/cm2
3 log10
CFU/cm2
S. aureus 7 log10
CFU/cm2
5 log10
CFU/cm2
3 log10
CFU/cm2
.
Increasing the antimicrobial agent concentration and/or treatment time
generally improved the effectiveness of the treatment. Huang, Ye and Chen (2012)
32
used 3% of H2O2 to wash and decontaminate baby spinach leaves for 5 min and
achieved 1.6 log10 CFU/g reduction of E. coli O157:H7. Similar H2O2 washing on
spinach leaves was performed by Huang and Chen (2011) and achieved similar log
CFU reduction (1.5 log CFU/g) of E. coli O157:H7 using 2% H2O2.
Ukuku and Fett (2002) used 5% H2O2 solution to disinfect melon surface for
2 min and obtained 2.0–3.5 log CFU/cm2 reduction of L. monocytogenes. In the
aerosolized form, 10 min treatment of hydrogen peroxide vapor treatment at 1, 3, and
5% was able to provide significant bacterial decontamination of S. Typhimurium
(1.48, 2.09, and 2.63 log10 CFU/g reduction, respectively) and E. coli O157:H7 (1.62,
2.14, and 2.94 log10 CFU/g reduction) on lettuce leaves (Back et al., 2014). Similarly,
our experiments showed 2-3 log reduction at lower cell contamination on the agar
plates. But the H2O2 fumigation was less effective when initial contamination was
high (i.e., 7 log10 CFU/cm2)
Improvement of H2O2 treatment by AOPs
In this experiment, ozonation and UV-C photocatalysis are combined with
H2O2 fumigation to generate •OH fume for bacterial decontamination. In the AOPs
scheme, H2O2 serves as a substrate to excessively produce reactive oxygen derivatives
(e.g., hydroxyl radicals, superoxide anions), which are able to non-selectively attack
essential cell components such as DNA, lipids, and proteins (Kahnert et al., 2005).
Figure 4-2 demonstrates the significant improvement of our patent-pending
technology of •OH fumigator in comparing to the same concentration of H2O2
fumigation in Figure AOPs. At same 3% H2O2 as in Figure 4-1a, the conversion of
H2O2 to •OH in Figure 4-2a was able to produce substantial reduction of both
S. aureus and E. coli; although, E. coli was much more resilient than S. aureus.
All S. aureus contamination levels were brought down to complete sterility within 5
min of treatment time but the E. coli contamination higher than 5 log10 CFU/cm2
requires more than 20 min to total sterile condition. Nevertheless, the stronger H2O2
concentration (i.e., 5% H2O2 in Figure 4-2b) was able to inactive E. coli
contamination as high as of 7 log10 CFU/cm2 within 15 min.
33
a) 3% H2O2/Ozonation/UV-C b) 5% H2O2/Ozonation/UV-C
Figure 4-2 Effect of H2O2 concentrations combined with UV-C photocatalysis and
ozonation. H2O2 treatment on the initial cell 3, 5 and 7 log10 CFU/cm2 of
E. coli and S. aureus
The improvement of bacterial destruction by the combined UV-C
photocatalysis and ozonation can be depicted by their individual effect on pure water.
Figure 4-3a shows no remnant effect of UV-C in the water that fumed the E. coli and
S. aureus contaminated surfaces and there was no evidence of bacterial kills.
When the ozonation application without UV-C was applied, the aerosolized ozonated
water enabled the reduction of both E. coli and S. aureus (Figure 4-3b). Presumably
the synergy between the ozone gas and ozonated water produced significant bacterial
reduction via the generation of •OH and the effect of ozone itself. Even in pure water,
the combined effects of UV-C photocatalysis and ozonation were able to harness
enough •OH to slightly outperform the ozone experiment (Figure 4-3c). Then H2O2
at 0.5% was added to increase the substrate to generate •OH and a significant
improvement in efficacy of bacterial reduction was achieved, especially at the
treatments with low initial cell densities (Figure 4-3d).
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10
CF
U/c
m2)
Time (min)
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10
CF
U/c
m2)
Time (min)
34
a) UV-C b) Ozonated water
c) UV-C/Ozonation d) 0.5% H2O2/Ozonation/UV-C
Figure 4-3 Effect of UV-C, Ozone and UV-C/Ozonation treatments on the initial cell
3, 5 and 7 log10 CFU/cm2 of E. coli and S. aureus
Koller (1965) reported the production of •OH using UV-C treatment of
wavelength between 4 and 400 nm and concluded that UV-C photolysis applications
in water happened in the UV-C range (200-280 nm). In our experiment, none of the
bacterial killing using the fumigation of the UV water was observed. UV-C alone can
decrease some organic compounds but it is not efficient enough for bacteria removal
and •OH generated (Jing & Cao, 2012). However, several researchers found that
UV-C can increase the oxidation potential of the others oxidation processes.
H2O + hv → • H + • OH (4-1)
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10C
FU
/cm
2)
Time (min)
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10C
FU
/cm
2)
Time (min)
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li(l
og
10
CF
U/c
m2)
Time (min)
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10
CF
U/c
m2)
Time (min)
35
However, substantial reduction of both E. coli and S. aureus was achieved
and assumed to be derived from the oxidation with •OH. When ozone is dissolved in
water, the reaction between hydroxide ions and ozone generates •O2- anion and a
•HO2 radical (Gunten, 2003). This •HO2 radical has around 4.8 and readily forms an
•O2- radical at the pH higher than this pKa value.
O3 + OH− → • O2
− + • HO2 (4-2)
A series of radical chain reaction occurs as •OH are formed.
•HO2 → • O2− + H+ (4-3)
O3 + • O2− → • O3
− + O2 (4-4)
• O3− + H+ → • HO3 (4-5)
• HO3 → • OH + O2 (4-6)
• OH + O3 → • HO4 (4-7)
•HO4 → • HO2 + O2 (4-8)
At the end, radical scavengers neutralize the above chain reaction and inhibit
ozone decay.
• OH + • HO2 → O2 + H2O (4-9)
When UV-C and ozonation are combined, it was proposed that dissolved
ozone was able to absorb UV photocatalysis to produce H2O2. This H2O2 intermediate
was further photocatalyzed to the highly reactive •OH (Kommineni et al., 2000;
Munter, 2001; Krishnan et al., 2017). These intermediate radicals perhaps participated
in the destruction of organic substances, but •OH was believed to be the predominant
oxidizing agent.
O3 + H2O hv→ H2O2 + O2 (<300 nm) (4-10)
2O3 + H2O2hv→ 2OH •+3O2 (4-11)
36
H2O2hv→ 2 • OH (4-12)
H2O2 +• HO2hv→ • OH + H2O + O2 (4-13)
H2O2 +• OHhv→ • HO2 + H2O (4-14)
• 2HO2hv→ H2O2 + O2 (4-15)
An addition of H2O2 provide a surplus of substrate to move the reaction to
the right and enhance •OH production. Presumably, the synergy of H2O2/Ozonation
/UV-C water maintained high level of reactive •OH in the fume facilitating instant
oxidation and fast microbial inactivation at the point of contact similar to other
research works (Glaze et al., 1987; Andreozzi et al., 1999; Kommineni et al., 2000).
Ozonated water and H2O2 adsorbs UV-C light at a wavelength of 254 nm producing a
wide array of free radical intermediates, including H2O2 radicals (Munter, 2001).
Hence, the combination of UV-C photocatalysis and ozonation in H2O2 fumigation
provides highly oxidizing conditions (higher activation energy) and involves the
production of many free radical species, including, H2O2, hydroxyl radical (•OH)
Perhydroxyl radical (•HO2) and so on (Kommineni et al., 2000; Munter, 2001) as
summarized in Equation 1-15.
Comparison to other alternative disinfectants
To apply this fumigation concept for surface disinfection and hand
sanitization, other commercially-available disinfectants were explored. This study
included 3 other potential candidates (i.e., NaOCl, ClO2, and BKC) to create
disinfectant fume via ultrasonic fumigation by using their minimum and maximum
concentrations for such application. Figure 4-4 compares the effectiveness of each
chemical by comparing their ability to reduce bacterial counts on both E. coli- and S.
aureus-contaminated agar surfaces. Only 0.1% ClO2 and 5% H2O2/ozonation/UV-C
fume were able to inactivate both bacteria completely at any initial cell loadings.
Other authors reported that ClO2 was more effective to Gram-negative than Gram-
positive or acid fast bacteria (Toda et al. 2006; Morino et al., 2011).
37
LeChevallier & Au, (2004) rationalized the survival of Gram- positive after chlorine
disinfection was possibly because Gram-positive bacteria have thicker cell walls than
Gram-negative bacteria. While both ClO2 and H2O2 treatments have their own
characteristic smells when they were in use, 0.1% ClO2 treatment had heavily pungent
fume and caused irritations of eyes and nose. Perhaps it may not be suitable for such
applications with direct human contacts.
Several studies have shown that Gram-positive bacteria were more
susceptible to BKC than Gram-negative (Marple et al., 2004; Khajavi et al., 2007;
Fazlara & Ekhtelat, 2012). For instance, 0.1% BKC was highly effective to disinfect
S. aureus (representing Gram-positive bacteria) in all levels of contaminations but less
effective to E. coli (representing Gram-negative bacteria). A few authors had
observed that the Gram-negative bacteria with an outer lipopolysaccharide membrane
modulating the accessibility of a cell had more intrinsic resistant to antiseptics and
disinfectants than nonsporulating Gram-positive bacteria (Helander et al., 1997;
Brula & Cooteb, 1999; Mcdonnell & Russell, 1999; Fazlara & Ekhtelat, 2012).
Morrissey et al. (2014) reported the minimum inhibitory concentration (MIC) for
BKC of most Gram-positive bacteria was 4-16 mg/L (as opposed to 32-128 mg/L in
Gram-negative bacteria).
The 0.5% NaOCl was more effective toward E. coli than S. aureus.
However, the use of NaOCl in high concentrations (more than 0.05%) is highly
corrosive to metals and its reaction products of chorine and hypochlorite with organic
pollution are potentially cancerogenic and mutagenic (Zajic, 1999). At lower
concentrations, the effectiveness of NaOCl was rather limited. It was more restricted
to apply as a general surface disinfectant since it released of toxic chlorine gas if
mixed with ammonia or acid (Mrvos et al., 1993; Reisz & Gammon, 1986;
Gapany et al., 1982).
In Figure 4-4, the H2O2/Ozonation/UV-C treatment showed non-selective
disinfecting characteristic against the types of bacteria. At 5% H2O2, the
H2O2/Ozonation/UV-C treatment was able to completely disinfect both E. coli- and
S. aureus-contamined agar plates within 20 min. Note that the H2O2/Ozonation/UV-C
treatment was to combine UV-C and ozonation to the H2O2 treatment and able to
improve the efficacy of bacterial disinfection tremendously in comparing to the H2O2
38
treatment alone.) The concentration of H2O2 affected the disinfecting efficacy,
especially at higher levels of initial microbial contamination. It was assumed that the
major oxidant in this H2O2/Ozonation/UV-C treatment is the hydroxyl radical that can
react with organic compounds non-selectively facilitating the microbial inactivation at
a reaction rate constant as high as 109 M-1 sec-1 through hydrogen atom abstraction or
by addition of the hydroxyl radical (Munter, 2001). H2O2/Ozonation/UV its high
potential for application in hand sanitation and less toxic to human and environment.
a) S. aureus
Figure 4-4 Comparison of microbial disinfecting efficacies on contaminated agar
plates at the initial cell loading of 3, 5, and 7 log10 CFU/cm2. The
disinfecting fumes were generated from H2O2 (hydrogen peroxide), H2O2
combined with ozonation and ultraviolet, ClO2 (Chlorine dioxide), BKC
(Benzalkonium chloride) and NaOCl (Sodium hypochlorite) and applied
in an enclosed chamber for 20 min.
0
10
20
30
40
50
60
70
80
90
100
3% H2O2 5% H2O2 3% H2O2
+ UV
+Ozone
5% H2O2
+ UV
+Ozone
0.0003%
ClO2
0.1%
ClO2
0.004%
BKC
0.1%
BKC
0.04%
NaOCl
0.5%
NaOCl
% R
edu
ctio
n
of
S.a
ure
us
Alternative method
39
b) E. coli
Figure 4-4 (Cont.)
Application of •OH fume for hand sanitation
Selwyn (1980) reported that the normal human skin usually has the total
aerobic bacterial counts ranging from more than 1 x 106 CFU/cm2 (e.g., scalp and
axilla) to 1 x 104 CFU/cm2 (e.g., forearm). Statistics showed that healthcare workers
can on average have the total bacterial counts from 3.9 x 104 to 4.6 x 106 CFU/cm2
(Price, 1938; Maki, 1978; Larson, 1984; Larson et al., 1998) and normal fingertip
areas can harbor as many as 300 CFU if counted by agar contact methods
(Pittet et al., 1999). Many infectious diseases can be spread from one person to
another by contacting to these contaminated body surfaces. The use of
H2O2/Ozonation/UV-C fume was proposed as an alternative to common hand-
washing products to effectively sanitize hands and contaminated surfaces.
Ethanol (ethyl alcohol, C2H5OH) and 2-propanol (isopropyl alcohol,
(CH3)2CHOH) have similar disinfectant properties. The feasible explanation for the
antimicrobial action of alcohol is denaturation of proteins. The absolute ethyl alcohol
is observed in the antimicrobial reaction. This dehydrating agent facilitate less
bactericidal than mixtures of alcohol and water because proteins are denatured more
0
10
20
30
40
50
60
70
80
90
100
3% H2O2 5% H2O2 3% H2O2
+ UV
+Ozone
5% H2O2
+ UV
+Ozone
0.0003%
ClO2
0.1%
ClO2
0.004%
BKC
0.1%
BKC
0.04%
NaOCl
0.5%
NaOCl
% R
edu
ctio
n o
f E
.co
li
Alternative method
E. coli, 3 log CFU/cm2 E. coli, 5 log CFU/cm2 E. coli, 7 log CFU/cm2
40
rapidly in the presence of water. The ethanol works by denaturing proteins and
dissolving lipids moreover it is effectively destroying many types of bacterial and
viral cells. Ethanol is normally used at 70% concentration because at higher
concentrations evaporate very quickly. It is ineffective against spores and may
not kill all types of non-lipid viruses (World Health Organisation Staff & World
Health Organization, 2004).
The broad spectrum efficacy of VHP has been shown against a wide range
of micro-organisms over the last decade including bacteria, viruses, fungi, and
bacterial spores (Heckert et al. 1997; Kahnert et al., 2005). From the six of seven
exposure trials were investigated of 3% concentration for 150 min. killed 106 spores
and hydrogen peroxide for reducing spacecraft bacterial populations, a complete
kill of 106 spores (i.e., Bacillus species) occurred 60-min contact time
(Rutala & Weber, 2008). Therefore, the effect of VHP on the sporicidal action
on the hand would be interestingly study in the future research.
Rubbing of both hands in the H2O2/Ozonation/UV-C fume using the
standard hand-washing protocol (Safety & World Health Organization, 2009) up to 60
sec produced various degrees of microbial disinfection depending on the levels of
initial E. coli contaminations and the concentrations of H2O2 used. At 1-2 log10
CFU/cm2, all E. coli cells can be removed from volunteer’s hands within 15 sec using
the H2O2 concentration as low as 0.5%. At the higher initial E. coli contaminations,
only 3% H2O2/Ozonation/UV-C fume was able to reduce the E. coli count to zero in
30 sec. The lower H2O2 concentrations were unable to produce complete disinfection
even after 60 sec of constant rubbing in the H2O2/Ozonation/UV-C fume.
Higher concentration of H2O2 may be needed to achieve total sterility using brief
rubbing treatment or else a different mechanism (e.g., spraying) may be required to
collect more mass of disinfecting solution containing •OH onto the hands. The
ultrasonic fume generate very fine airborne aerosols that only lightly wetted and
hardly accumulated on the skin surfaces.
41
a) 1-2 log10 CFU/cm2 b) 3-4 log10 CFU/cm2
c) 5-6 log10 CFU/cm2
Figure 4-5 Effect of a) E. coli population on hands treated by 0.5% were combined
with ozone and UV b) E. coli on hands treated by 1% were combined
with ozone and UV-C and c) 3% H2O2 respectively, used for
handwashing. The inoculate at the initial cell loading of 1, 3 and 5 log10
CFU/cm2
Also the finger area test was conducted to test the ability of the
H2O2/Ozonation/UV-C fume in disinfecting E. coli on the finger areas after rubbing
for 1 min. This test is to reflect the normal behavior of people spending time to wash
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60
E.c
oli
(lo
g C
FU
/cm
2)
Time (sec)
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60
E.c
oli
(lo
g1
0C
FU
/cm
2)
Time (sec)
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60
E. co
li (
Lo
g1
0C
FU
cm
2)
Time (sec)
42
their hands not exceed 30 sec (Wilkinson et al., 2017). This test usually utilizes higher
concentration of E. coli cell (e.g., 5 log10 CFU/cm2) and 2 mL of disinfectant solution
dropped and rubbed only on finger area for 30 sec. Our technique the amount of •OH
solution applied to the finger area was much less due to the nature of fuming but the
rubbing time was kept constant at 30 sec. Table 4 indicates that the use of
H2O2/Ozonation/UV-C fume is able to reduce E. coli counts on the finger area tested.
Higher H2O2 concentrations used were able to produce more bactericidal effect
showing less E. coli colonies grown on the agar medium. When the H2O2
concentration reached 3%, no growth of E. coli was shown on the finger prints and 30
sec rubbing in the H2O2/Ozonation/UV-C fume was suffice to generate total sterility
on the finger area.
Table 4-2 Visualization of finger area testing for finger print by 2 mL of E. coli (5
log10 CFU/cm2) were treated with 0.5%, 1% and 3% H2O2 respectively, for
30 sec.
Treatments Left & right hand finger prints
CONTROL
Applying 2 ml of E.
coli
(5 log10 CFU/cm2)
and rubbing for 1 min
Good E. coli growth on finger prints from both hands
Sanitizing with 0.5%
H2O2 fume for
30 sec
Fair E. coli growth with much less than the control
treatment
43
Table 4-2 (Cont.)
Treatments Left & right hand finger prints
Sanitizing with 1%
H2O2 fume for
30 sec
Sparsely populated E. coli growth on finger prints
Sanitizing with 3%
H2O2 fume for
30 sec
Only finger print marks without E. coli growth
The palm area test was also performed using 3% H2O2/Ozonation/UV-C
treatment and varying the initial E. coli contamination (i.e., 3 and 5 log10 CFU/cm2).
Table 4-3 shows that rubbing both hand in 3% H2O2/Ozonation/UV-C fume for 30 sec
can was effective to aseptically disinfect both hands if the initial E. coli contamination
is less than 3 log10 CFU/cm2. For the 5 log10 CFU/cm2 treatment, there were still
some sporadic growth of E. coli colonies but this palm print result were significantly
less than the control treatment. From these finger and palm test results, it is clearly
shown that the H2O2/Ozonation/UV-C fume using at least 3% H2O2 has the ability to
disinfect hands and skin surfaces in preventing the spread of bacterial infection and
diseases. Arguably this hand sanitization protocol using the H2O2/Ozonation/UV-C
fume is more effective than mechanical hand washing where hand-washing soaps
often containing esterified fatty acids with sodium or potassium hydroxide or other
detergent/surfactant products are served to remove loosely adherent microorganisms
and viruses from the hands by mechanical means (Messina et al., 2008).
44
Contamination can also occur in the process of hand washing with non-
bactericidal hand-washing products by contacting the contaminated surfaces after
washing (Boyce & Pittet, 2002). Also, the moist surrounding and surfaces generated
in the process of regular water-based hand washing can provide suitable environment
for bacteria growth and increase the risk of cross-contamination. Although many
alternative agents are available for hand sanitation such as alcohol-based hand
rubbing liquid and are tested with good results but may provoke skin allergy reactions
(Humes & Lobo, 2006; Appelgrein et al., 2016). The H2O2/Ozonation/UV-C fume is
present itself as an alternatives to alcohol for hand asepsis that do not cause skin
reactions but provide effective hand asepsis as shown in the palm and finger test
experiments.
Table 4-3 Qualitative visualizations of E. coli hand contamination by palm area
testing procedures. 2 mL of E. coli (3 and 5 log10 CFU/cm2) were treated
3% H2O2. Imprints of contaminated palms were made onto PCA
plates and visualized after overnight incubation.
Concentration of E. coli
Hand wash by H2O2/Ozonation/UV-C fume results
Control Treated with 3% H2O2
for 30 seconds
3 log10 CFU/cm2
Dropped 2 ml of E. coli
(3 log10 CFU/cm2) and
rubbing for 1 min
.
Only palm print without
E. coli colony
45
Table 4-3 (Cont.)
Concentration of E. coli
Hand wash by H2O2/Ozonation/UV-C fume results
Control Treated with 3% H2O2
for 30 seconds
5 log10 CFU/cm2
Dropped 2 ml of E. coli
(5 log10 CFU/cm2)
and rubbing for 1 min
Sporadic E. coli colonies
grew on the agar medium.
H2O2 /Ozonation/UV-C fume combined with 0.01% BKC
For the palm area testing, using 3% H2O2/Ozonation/UV-C can be effective
for hand wash and take a few minute to contact the contaminated surfaces. This
concentration is sufficient in killing bacteria as well as inactivate bacteria growth.
This results corresponding to the Journal of American College of Toxicology that the
Cosmetic Ingredient Review panel concluded that BKC can be safely used as an
antimicrobial agent at concentrations up to 0.1% (Liebert, 1989; Marple et al., 2004)
and use in nasal spray containing BKC 0.01% to preservative-free saline on nasal
mucociliary clearance (Rizzo et al., 2006). The results from the glove juice testing
was also performed using 3% H2O2/Ozonation/UV-C combined with 0.01% BKC and
varied the initial E. coli concentration (i.e., 3, 5 and 7 log10 CFU/cm2) which spiked
on the hand panel (Table 4-4). The results showed that rubbing both hands by a
disinfectant aerosol for 30 sec can reduce in a few colonies at high initial cells
(i.e., 7 log10 CFU/cm2). The survival E. coli cells after treatment (i.e., 3 log10
CFU/cm2) were significantly less than the control.
46
Table 4-4 Qualitative visualizations of E. coli hand contamination by 4.5 ml of E.
coli was treated with 3% H2O2 and 0.01% BKC for 30 sec. Imprints of
contaminated palms were made onto PCA plates and visualized after
overnight incubation.
Initial cells
of E. coli
Control
(No fume)
4.5 ml of E. coli was treated
with 3% H2O2 and 0.01%
BKC for 30 seconds
7 log10
CFU/mL
5 log10
CFU/mL
3 log10
CFU/mL
Comparison chart of disinfectants
To design an optimum disinfectant for sanitizing contaminated surfaces,
different techniques were tested against the same sample and disinfecting conditions.
Several practical aspects including, x, y, z and a, were included in our experiments
(Table 4-5). While BKC is the least expensive fuming technique in term of chemical
cost, the efficacy of bactericide was only observed at high concentration and gave
good result with Gram-positive bacteria. The experimental results shows great
potential as broad spectrum fuming for surface disinfection. Most chorine-based
chemical are soon to be obsolete because of their toxicity and toxic residues that
polute environment.
47
Table 4-5 Comparison of characteristics, efficacies between H2O2/O3/UV fume with
conventional disinfectants.
Chemical
Charasterics
Disinfectants
H2O2/O3/UV NaOCl BKC ClO2
Odors Weak odor Strong odor Weak odor Strong odor
Chlorinated
byproducts
leftover
No generated generated No generated generated
Decomposion
of trace
residue
Rapidly Very Slowly Moderate Slowly
Side effects No odor or
irritation
issues
Serious eye
irritant (safety
glasses)
Respiratory
irritant
Eye irritation
with contact
and
respiratory
irritant (safety
glasses and
safety mask)
Safety of
usage
No impact and
no residue
white smoke
residue white
smoke in
laboratory
room and
create chlorine
smell
(Ventilation
room)
No impact and
no residue
white smoke
residue white
smoke in
laboratory
room and
create
chlorine smell
(Ventilation
room)
Fuming cost
(baht/hr)
112.94 277.34 12.8 238.92
Note: H2O2/O3/UV cost can be divided into H2O2 103.6 baht/h, O3 4.67 baht/h and
UV 4.67 baht/h
CHAPTER 5
CONCLUSION
This research was to propose the new protocol for killing bacteria by using
advanced oxidation technology to reduce the risk for HAIs, including hand hygiene
practices, environmental cleaning, and disinfecting in the high-risk area (e.g., hospital,
airport, kindergarten and public places). The research has combined the use of H2O2
and ultrasonic fogging systems to create and disperse a disinfectant aerosol to
disinfect factory (food and pharmaceutical) processing surfaces as well as difficult-to-
reach areas, especially overhead surfaces, cracks, and crevasses of food equipment.
The use of ultrasonic fumigation to generate disinfectant fumes by different
bactericidal chemicals was explored. The use of advanced oxidation technology by
applying ozonation and UV-C photocatalysis was able to enhance the bactericidal
effectiveness of H2O2 fume. The generation of •OH in the H2O2/Ozonation/UV-C
fume was used to explain the significant improvement from the H2O2 fume alone.
Owing to the non-selectiveness oxidation towards any bacteria, the
H2O2/Ozonation/UV-C fume were demonstrated for surface and hand sanitization.
Depending on the degree of contamination, the concentration of H2O2 can be selected
to produce a substantial reduction of contamination or complete elimination on the
intended surfaces. This optimum concentration did not affect for material and
environmental samples. At approximately 3-4 log 10 CFU/cm2, it was compulsory to
use at least 3% H2O2/Ozonation/UV-C fume to achieve complete surface sanitation in
30 sec. The glove juice and palm tests also confirmed the effectiveness of the 3%
H2O2/Ozonation/UV-C fume for 30 sec hand sanitation.
Recommendations
The results from this research show a chance to reduce pathogen bacteria in a
few time and reduce survive rate of E.coli and S. aureus that caused diarrhea diseases.
The result showed great potential to reduce bacteria contamination. Using 3%
H2O2/Ozonation/UV-C fume for 30 sec can sanitize the bacteria and completely
reduce pathogens presented in several conditions. To implement these protocols, the
49
user or the factory can choose to apply in many industries. It depends on their
practicality, effectiveness, and feasibility.
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APPENDIX
76
APPENDIX A
CALCULATIONS
77
1. Colony forming unit (cfu/mL) = Total of colony (CFU)
Dilution factor × Volume of sample (mL)
2. % Reduction = C0 − Cn
C0 ×100 %
78
APPENDIX B
EXPERIMENTAL DATA
79
a) 6 log CFU/mL
b) 4 log CFU/mL
Figure Appendix B-1 Effect of 5 % H2O2 treatment on the initial cell 2, 4 and 6 log10
CFU/mL Figure a), b) and c), respectively, of S. aureus
compared with control (0 min) to 60 sec.
0
1
2
3
4
5
6
7
8
0 5 10 15 20 25 30 35 40 45 50 55 60
S.a
ure
us
(lo
g1
0C
FU
/ml.
)
Time (sec)
Control 5% H2O2 +O3+UV, S. aureus, 6 log 10 CFU/mL
0
1
2
3
4
5
6
7
8
0 5 10 15 20 25 30 35 40 45 50 55 60
S.a
ureu
s (l
og
10
CF
UI/
ml.
)
Time (sec)
Control 5% H2O2+O3+UV, S. aureus,4 log 10 CFU/mL
80
c) 2 log CFU/mL
Figure Appendix B-1 (Cont.)
a) 7 log10 CFU/mL
Figure Appendix B-2 Effect of 5 % H2O2 treatment on the initial cell 2, 4 and 6 log10
CFU/ mL of E. coli compared with control (0 min) to 60 sec.
0
1
2
3
4
5
6
7
8
0 5 10 15 20 25 30 35 40 45 50 55 60
S. a
ure
us
(lo
g1
0C
FU
/ml.
)
Time (sec)
Control 5% H2O2+O3+UV, S. aureus, 2 log 10 CFU/mL
0
1
2
3
4
5
6
7
8
9
0 10 20 30 40 50 60
E. co
li (
log
10
CF
U/m
l.)
Time (sec)
Control 5% H2O2+O3+UV, E. coli, 7 log 10 CFU/mL
81
b) 5 log10 CFU/mL
c) 3 log10 CFU/mL
Figure Appendix B-2 (Cont.)
0
1
2
3
4
5
6
7
8
9
0 10 20 30 40 50 60
E. co
li (
log
10
CF
U/m
l.)
Time (sec)
Control 5% H2O2+O3+UV, E. coli, 5 log 10 CFU/mL
0
1
2
3
4
5
6
7
8
9
0 10 20 30 40 50 60
E.
coli
(lo
g10
CF
U/m
l.)
Time (sec)
Control 5% H2O2+ O3 + UV, E. coli, 3 log 10 CFU/mL
82
a) 0.0003% ClO2
b) 0.1% ClO2
Figure Appendix B-3 Effect of chlorine dioxide fume on E. coli and S. aureus
contamination on surfaces, initial cells 3, 5 and 7 log10
CFU/cm2. Contact time 5, 10, 15 and 20 min.
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10
CF
U/c
m2)
Time (min)
S. aureus, 7 log CFU/cm2 E.coli, 7 log CFU/cm2 S. aureus, 5 log CFU/cm2
E. coli, 5 log CFU/cm2 S.aureus, 3 log10 CFU/cm2 E. coli, 3 log10 CFU/cm2
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10
CF
U/c
m2)
Time (min)
S. aureus, 7 log CFU/cm2 E.coli, 7 log CFU/cm2 S. aureus, 5 log CFU/cm2
E. coli, 5 log CFU/cm2 S.aureus, 3 log10 CFU/cm2 E. coli, 3 log10 CFU/cm2
83
a) 0.004% BKC
b) 0.1% BKC
Figure Appendix B-4 Effect of benzalkonium chloride fume to E. coli and S. aureus
contamination on surfaces, initial cells 3, 5 and 7 log10
CFU/cm2
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10
CF
U/c
m2
)
Time (min)
S. aureus, 7 log CFU/cm2 E.coli, 7 log CFU/cm2 S. aureus, 5 log CFU/cm2
E. coli, 5 log CFU/cm2 S.aureus, 3 log10 CFU/cm2 E. coli, 3 log10 CFU/cm2
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10
CF
U/c
m2)
Time (min)
S. aureus, 7 log CFU/cm2 E.coli, 7 log CFU/cm2 S. aureus, 5 log CFU/cm2
E. coli, 5 log CFU/cm2 S.aureus, 3 log10 CFU/cm2 E. coli, 3 log10 CFU/cm2
84
a) 0.04% NaOCl
b) 0.5% NaOCl
Figure Appendix B-5 Effect of Sodium hypochlorite fume to E. coli and S. aureus
contamination on surfaces, initial cells 3, 5 and 7 log10
CFU/cm2
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10
CF
U/c
m2)
Time (min)
S. aureus, 7 log CFU/cm2 E.coli, 7 log CFU/cm2 S. aureus, 5 log CFU/cm2
E. coli, 5 log CFU/cm2 S.aureus, 3 log10 CFU/cm2 E. coli, 3 log10 CFU/cm2
0
1
2
3
4
5
6
7
8
0 5 10 15 20
S.a
ure
us
& E
.co
li (
log
10
CF
U/c
m2)
Time (min)
S. aureus, 7 log CFU/cm2 E.coli, 7 log CFU/cm2 S. aureus, 5 log CFU/cm2
E. coli, 5 log CFU/cm2 S.aureus, 3 log10 CFU/cm2 E. coli, 3 log10 CFU/cm2
