Aerosolization Methods and UV Inactivation of Bacterial Cells in Air

by

Wei Yao

A Thesis Presented in Partial Fulfillment

of the Requirements for the Degree

Master of Science

Approved November 2015 by the

Graduate Supervisory Committee:

Morteza Abbaszadegan, Chair

Absar Alum

Peter Fox

ARIZONA STATE UNIVERSITY

December 2015

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ABSTRACT

Since its first report in 1976, many outbreaks linked to Legionella have been

reported in the world. These outbreaks are a public health concern because of

legionellosis, which is found in two forms, Pontiac fever and Legionnaires disease.

Legionnaires disease is a type of pneumonia responsible for the majority of the illness in

the reported outbreaks of legionellosis. This study consists of an extensive literature

review and experimental work on the aerosolization and UV inactivation of E.coli and

Legionella under laboratory conditions. The literature review summarizes Legionella

general information, occurrence, environmental conditions for its survival, transmission

to human, collection and detection methodologies and Legionella disinfection in air and

during water treatment processes.

E. coli was used as an surrogate for Legionella in experimentation due to their

similar bacterial properties such as size, gram-negative rod-shaped, un-encapsulated and

non-spore-forming bacterial cells. The accessibility and non-pathogenicity of E. coli also

served as factors for the substitution.

Three methods of bacterial aerosolization were examined, these included an

electric spray gun, an air spray gun and a hand-held spray bottle. A set of experiments

were performed to examine E. coli aerosolization and transport in the aerosolization

chamber (an air tight box) placed in a Biological Safety Cabinet. Spiked sample was

sprayed through the opening from one side of the aerosolization chamber using the

selected aerosolization methods. The air sampler was placed at the other side to collect

100 L air sample from the aerosolization chamber. A Tryptic Soy Agar plate was placed

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inside the air sampler to collect and subsequently culture E. coli cells from air. Results

showed that the air spray gun has the best capability of aerosolizing bacteria cells under

all the conditions examined in this study compared to the other two spray methods. In this

study, we provide a practical and efficient method of bacterial aerosolization technique

for microbial dispersion in air. The suggested method can be used in future research for

microbial dispersion and transmission studies.

A set of experiments were performed to examine UV inactivation of E. coli and

Legionella cells in air. Spiked samples were sprayed through the opening from one side

of the aerosolization chamber using the air spray gun. A UV-C germicidal lamp inside

the Biological Safety Cabinet was turned on after each spray. The air samples were

collected as previously described. The application of UV-C for the inactivation of

bacterial cells resulted in removing aerosolized E. coli and Legionella cells in air. A 1 log

reduction was achieved with 5 seconds UV exposure time while 10 seconds UV exposure

resulted in a 2 log bacterial reduction for both bacteria. This study shows the applicability

of UV inactivation of pathogenic bacterial cells in air by short UV exposure time. This

method may be applicable for the inactivation of Legionella in air ducts by installing

germicidal UV lamps for protecting susceptible populations in certain indoor settings

such as nursing homes or other community rooms.

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TABLE OF CONTENTS

Page

LIST OF TABLES...........................................................................................................iii

CHAPTER

1 LITERATURE REVIEW ....................................................................................... 1

General Information of Legionella ............................................................ 1

Legionella Occurrence ............................................................................... 3

Environmental Conditions for Legionella Survival .................................. 7

Legionella Transmission to Human .......................................................... 9

Collection and Detection Methodologies of Legionella ......................... 11

Legionella Disinfection in Water Treatment ........................................... 13

Ultraviolet Inactivation and Application in Air Disinfection ................. 15

2 MATERIALS AND METHODS ......................................................................... 19

Aerosolization Methods for Dispersion of Bacterial Cells in Air .......... 21

UV Inactivation on Bacterial Cells in Air ............................................... 23

3 RESULTS AND DISCUSSION .......................................................................... 25

Aerosolization Methods for Dispersion of Bacterial Cells in Air .......... 25

UV Inactivation on Bacterial Cells in Air ............................................... 32

4 CONCLUSION ......................................................................................................... 37

REFERENCES ....................................................................................................................... 39

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LIST OF TABLES

Table Page

1. E. coli Aerosolization via Electric Spray Gun ................................................... 25

2. E. coli Aerosolization via Air Spray Gun ........................................................ 26

3. E. coli Aerosolization via Handheld Spray Bottle ........................................... 27

4. Summary of E. coli Aerosolization via Different Spray Methods .................. 28

5. Bacteria (E. coli) Aerosolizing Efficiency of Different Spray Methods under

Different Circumstances .................................................................................... 30

6. E. coli Aerosolization via Ultrasonic Humidifier ............................................ 32

7. UV Inactivation on E. coli Cells in Air with Different Exposure Times ........ 33

8. UV Inactivation on E. coli Cells in Air Simulating Real World Air Duct

Disinfection ...................................................................................................... 34

9. UV Inactivation of Legionella Cells in Air at Different Exposure Times ....... 35

10. UV Inactivation on Legionella Cells in Air Simulating Real World Air Duct

Disinfection........................................................................................................ 36

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CHAPTER 1

LITERATURE REVIEW

General Information of Legionella

History. An unexplained pneumonia outbreak occurred at the 1976 American

Legion Convention in Philadelphia, 221 of the attendees became ill with pneumonia, and

34 of those affected died (EPA 1999). In January 1977, Joseph McDade of the U.S.

Centers for Disease Control (CDC) identified a previously discovered bacteria while

investigating the outbreak (Brenner 1987). The aerobic gram-negative bacteria isolated

from infected lung tissue was identified as the causative agent of this pneumonia

outbreak and named Legionella pneumophila (Fang et al. 1989).

The symptoms associated from the 1976 Legionella outbreak were termed

Legionnaire’s disease. Legionella can affect humans in two ways: a potentially fatal

pneumonia which occurs in about 95% of infections (Legionnaire’s disease) and a non-

infectious influenza-like infection (Pontiac fever) (Hoge and Brieman 1991).

After identifying L. pneumophila, numerous species of Legionella have been discovered

due to the advances in growth and enrichment media (Brenner 1987).

Microbiology, Morphology, and Ecology. Legionella are aerobic gram-negative

rod-shaped bacteria. They are un-encapsulated, non-spore-forming, with physical

dimensions from 0.3 to 0.9 micrometers (μm) in width and from 2 to 20 μm in length

(Winn 1988). Most exhibit motility through one or more polar or lateral flagella (EPA

2001). Legionella have a non-fermentative respirative metabolism which is based on the

catabolism of amino acids for energy and carbon sources (Brenner et al. 1984).

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There are 53 known species of Legionella, and eighteen of them have been linked to

patients with pneumonia (Lück et al. 2010).

Legionella mostly exist in aquatic environments while some have been isolated from

potting soils and moist soil samples (Fields 1996). They can survive in waters with

temperatures between 0-63 C, a pH range of 5.0-8.5, and 0.2-15 ppm dissolved oxygen

concentration (Nguyen et al. 1991).

Even though Legionella are ubiquitous in nature, they have specific culture

requirements. A typical media to sustain Legionella bacterial growth is charcoal yeast

extract (BCYE) agar, buffered to pH 6.9 (EPA 1985). The BCYE agar can be

supplemented with -ketoglutarate, L-cysteine, iron salts, and further with antibacterial

agents to suppress microflora (cefamandole and vancomycin to inhibit gram-positive

bacteria and polymyxin B to inhibit gram-negative bacteria), antifungal agents

(anisomycin for yeast), and inhibitors (glycine) (Nguyen et al. 1991). Optimal

temperatures for bacterial growth are 35-37C (EPA 1985). It can also be enhanced with a

CO2 concentration between 2.5- 5 percent, but not exceeding 8-10 percent, which will be

inhibitory (EPA 1985, Winn 1993).

Symbiosis in Microorganisms: Legionella proliferation is dependent on their

relationships with other microorganisms. Experiments have shown that Legionella in

sterile tap water possess long-term survival but without multiplication, while Legionella

in non-sterile tap water survive and multiply (Surman et al. 1994). Furthermore,

Legionella viability is maintained when they are combined with algae in culture but

decreases once the algae are removed (Winn 1988).

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Legionella’s symbiotic relationship with other microorganisms was first revealed

with the discovery of L. pneumophila’s co-existence in an algal mat from a thermally

polluted lake (EPA 1985). In contrast, Legionella environmental replication

predominantly occurs via parasitization of single-celled protozoa. In 1980, Rowbotham

demonstrated L. pneumophila’s ability to infect two types of amoeba, Acanthamoeba and

Naegleria (EPA 1985). It is now known that Legionella can infect 2 species of protozoa

and 13 species of amoebae (Fields 1996).

Legionella also can multiply within protozoan hosts (Vandenesch et al. 1990). Those

hosts provide Legionella with protection from harmful environmental conditions and,

thus, the bacteria are more resistant to water treatment with disinfectants, can survive in

habitats with a greater temperature range, and in dry conditions if encapsulated in cysts

(EPA 1999).

Legionella also grow symbiotically with aquatic bacteria attached to the surface of

biofilms (Kramer and Ford 1994). Biofilms provides them nutrients for growth and

protection from adverse environmental conditions (EPA 1999). The concentration of

Legionella in biofilms is affected by water temperature; they can effectively out compete

other bacteria at higher temperature (EPA 1999). Legionella have been found in biofilms

in the absence of amoeba (Kramer and Ford 1994). Because biofilms colonize drinking

water distribution systems, they provide a habitat suitable for Legionella growth in

potable water, which can lead to human exposure (EPA 1999).

Legionella Occurrence

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Worldwide Distribution. Legionellosis cases have been reported in Asia, Europe,

North and South America, Africa, Australia, New Zealand (Edelstein 1988). The true

incidence of legionellosis is difficult to determine because identification requires

adequate surveillance. Research suggests that Legionnaires’ disease is under reported to

national surveillance systems (Marston et al. 1994; Edelstein 1988). Its recognition

depends on physician awareness of the disease and resources available to diagnose it.

(EPA 2001).

Although legionellosis is widely distributed throughout the world, most cases have

been reported from the industrialized countries. The ecological niches that support

Legionella (complex recirculating water systems and hot water maintained at 35-55°C)

are not as common in developing countries, so the incidence of legionellosis may be

comparatively low in these countries (Bhopal 1993).However, most geographical

variation in the incidence of legionellosis is probably artefactual due to differences in

definitions, diagnostic methods, surveillance systems, or data presentation (Bhopal 1993).

Occurrence in Water. Legionella are widely distributed in the aqueous

environment in the United States and wherever they are sought (EPA 1985). Research has

indicated that Legionella thrive in biofilms, and their interaction with other organisms in

biofilms is essential for their survival and multiplication in aquatic environments (Kramer

and Ford 1994, Yu 1997, Lin et al. 1998a). The survival of Legionella is greater when the

bacteria form symbiotic relationships with other microorganisms. Sediment within

biofilms stimulates the growth of these commensal microflora, which stimulate the

growth of Legionella (EPA 1999). Legionella occurs in natural bodies of water, such as

5

surface water and groundwater, and man-made waters, such as potable water, cooling

towers, whirlpools, etc.

Natural Surface Waters. Legionella are ubiquitous in aquatic environments.

Several studies clearly demonstrate the widespread occurrence of Legionella from natural

surface freshwater sources (i.e. lakes and streams) in the United States (CDC 2005).

More recent studies indicate that Legionella are also common in marine waters (Ortiz-

Roque and Hazen 1987, Palmer et al. 1993). Their presence in an ocean shore swimming

area was due to surface runoff from a flood control channel and river. The channel and

river were tested and it was determined that the water was contaminated with Legionella

(Palmer et al. 1993).

Groundwater. Prior to 1985, there were no studies documenting the presence of

Legionella in groundwater (EPA 1999). More recently, studies have shown positive

samples in water supply system wells for the presence of L. pneumophila; however, other

studies have shown no positive samples (EPA 1999).

Man-Made Waters. Legionella bacteria thrive in drinking water biofilms due to

increase to standard water disinfection procedures, allowing Legionella to enter and

colonize potable water supplies (Kramer and Ford 1994, Lin et al. 1998a).

In the potable water supply, Legionella bacteria occupy niches suitable for their

survival and growth (e.g., components of water distribution systems, cooling towers, and

whirlpools), which function as amplifiers or disseminators of these bacteria (EPA 1985).

In 1980, British investigators first demonstrated that plumbing fixtures in potable

water systems contained Legionella (EPA 1985). Water distribution systems of hospitals,

6

hotels, clubs, public buildings, homes, and factories continue to be a major source of

Legionella exposure (EPA 1998, 1985).

Numerous outbreaks of legionellosis have been linked to heat-exchange units (e.g.,

cooling towers and evaporative condensers) in hospitals, hotels, and public buildings,

clearly establishing these reservoirs as habitats for Legionella (EPA 1998, 1985).

However, as knowledge and awareness of the ecology and epidemiology of Legionella

have increased, attention has shifted from heat-exchange units to potable water

distribution systems as the most important sources of human exposure and infection (Lin

et al. 1998b, Yu 1997).

Whirlpools and spas also serve as ideal habitats for Legionella because they are

maintained at temperatures ideal for their growth and organic nutrients suitable for

bacterial growth often accumulate in these waters (Hedges and Roser 1991). In addition,

whirlpools and spas can produce water droplets of respirable size that have the potential

to transmit Legionella to humans (Jernigan 1996). Other related sources where Legionella

have been identified include spring water spas and saunas (Bornstein et al. 1989a, 1989b,

Den Boer et al. 1998).

Legionella also have been detected in all phases of the sewage treatment process,

including treated effluent (Palmer et al. 1993, 1995).

Occurrence in Soil. Although water is the most documented source of Legionella in

the environment, these bacteria have been isolated from mud, moist soil, and potting soil

(EPA 1985, Steele et al. 1990). One species in particular, L. longbeachae, has been

shown to inhabit and thrive in potting soil.

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L. longbeachae was able to persist for seven months in two potting mixes stored at

room temperature (Steele et al. 1990).

Occurrence in Air. Legionella can be found in air as part of aerosols.

Aerosolization is an important component of Legionella transmission from the aquatic

environment to the human respiratory system (EPA 1985). The aerosolization process can

be accomplished by aerosol-generating systems such as cooling towers, evaporative

condensers, plumbing equipment (e.g., faucets, showerheads, hot water tanks),

humidifiers, respiratory therapy equipment (e.g., nebulizers), and whirlpool baths (Bollin

et al. 1985, EPA 1985, Seidel et al. 1987). Inhalation of Legionella-contaminated

aerosols is an important source of human exposure and infection (EPA 1985).

Environmental Conditions for Legionella Survival

Water Temperature. Legionella exhibit the ability to survive in wide range of

temperatures. As a lower limit, Bentham (1993) observed growth at a water temperature

of 16.5°C. The highest water temperature of a sample cultivated by Botzenhart et al.

(1986) was 64°C. Henke and Seidel (1986) claimed Legionella to be a “thermoresistant”

organism that exhibits survival in natural warm waters of up to 60°C and artificially

heated waters of 66.3°C. Optimum temperatures for Legionella reproduction range from

32 to 45°C (Vickers 1987, Kramer and Ford 1994).

Temperature has a pronounced effect on the persistence and dissemination of

Legionella in aquatic habitats. While Legionella populations seem to be controlled by

extremely low temperatures, they are enhanced by heat and elevated temperatures found

8

in areas like whirlpools and hot springs (Henke and Seidel 1986, Lee and West 1991,

Verissimo et al. 1991).

Colbourne and Dennis (1989) stated that although Legionella are not thermophilic, they

exhibit thermo-tolerance to temperatures between 40 and 60°C, which gives them a

survival advantage over other organisms competing in man-made warm water systems.

Although temperatures between 45 and 55°C are not optimal for Legionella, these

temperatures enable them to reach higher concentrations than other bacteria commonly

found in drinking water, thus providing Legionella with a selective advantage over other

microbes (Kramer and Ford 1994).

Microbial Interactions. The growth and survival of Legionella in the environment

is enhanced by their ability to form symbiotic relationships with larger microorganisms.

Legionella have been found to infect and incorporate themselves into at least 13 species

of amoebae and two strains of ciliates (Lee and West 1991, Paszko-Kolva et al. 1993,

States et al. 1989, Kramer and Ford 1994, Henke and Seidel 1986, Fields 1996,

Vandenesch et al. 1990).

Because Legionella replicate rapidly intracellularly within protozoan hosts for

prolonged periods of time, amoebic vesicles can contain hundreds of Legionella cells

(Berk et al. 1998, Lee and West 1991).

In addition, replication within protozoa may contribute to enhanced virulence of

Legionella (Kramer and Ford 1994).

The ability of Legionella to thrive within protozoa also allows them to survive over

a wider range of environmental conditions and to resist the effects of chlorine, biocides,

9

and other disinfectants (Fields 1996, Kramer and Ford 1994, Paszko-Kolva et al. 1993,

States et al. 1989).

Relationships with certain algae and bacteria in biofilms also foster the growth of

Legionella, presumably due to the increased availability of nutrients and protection from

disinfection (Kramer and Ford 1994).

Other Factors. Other factors influencing the survival of Legionella in the

environment include sediment accumulation and metal content (Kusnetsov, 1993, States

et al. 1987, Stout et al. 1992b, Stout et al. 1985, Vickers et al. 1987). These factors are

usually amplified by ideal water temperature or coexisting environmental factors (EPA

1999). The increase of sediment acts as a major source of nutrients for Legionella, which

increases the growth and survival of the bacteria (Stout et al. 1985). Sediment is

important to Legionella growth because it provides essential nutrients, aids in the growth

of other coexisting microflora, and shelters the organism as well (Vickers et al. 1987).

Additionally, total organic carbon and turbidity are also factors that affect the growth and

survival of Legionella because these factors are found in water zones rich in sediment

(EPA 1999). Changes in water pressure and flow rates of water distribution systems may

cause disruption of the biofilm, resulting in increased concentrations of Legionella in

water supplies (Kramer and Ford 1994).

Water hardness is determined by the amount of calcium and magnesium in scale

deposits. Legionella have been found to flourish in areas where these metallic cations are

present (Vickers et al. 1987). Low levels of iron, zinc, and vanadium also may stimulate

the growth of Legionella (Kusnetsov 1993, States et al. 1987, Stout et al. 1992), while

10

higher concentrations of metals like copper, iron, manganese, and zinc may actually be

toxic (Kusnetsov 1993).

Legionella Transmission to Human

Legionella are considered as opportunistic pathogens because although they are

highly prevalent in the environment, relatively few people develop a clinical infection

(EPA 2001). Yu and colleagues (1993) characterized the infection rate for Legionella as

"strikingly low."

The typical progression of a Legionella infection can be characterized by the

following steps (Cianciotto et al. 1989). First, Legionella is inhaled or instilled into the

lower airways of the lungs. Second, alveolar macrophages phagocytize the bacteria by

either a conventional or coiling mechanism. Intracellular survival of the bacteria may be

attributed to one or more of the following factors: reduced oxidative burst, failure of

phagosome to acidify, failure of phagosome to fuse with lysosome, and/or bacterial

resistance to lysosomal contents. Third, the bacteria undergo rapid intracellular growth.

The bacterial growth within infected macrophages has been estimated at 100- to 1000-

fold within 48 to 72 hours of infection, which is considered remarkable compared to that

of other intracellular opportunistic bacteria (e.g., Salmonella, Mycobacterium, Listeria)

(Friedman et al. 1998). Finally, the host cells lyse and releases the bacteria, which

escalates the bacterial infection.

Legionella are transmitted directly from the environment to humans. There is no

evidence of human-to-human transmission, or of any animal reservoirs with public health

relevance for this organism. One route of infection is the inhalation of an aerosol

11

containing respirable droplets of water (or other liquids) contaminated with Legionella.

Alternatively, Legionella may be deposited in upper airways and subsequently aspirated

into the deeper portions of the lung (EPA 1999).

It has been reported that the most common reservoirs of transmission for

community-acquired Legionella infection are aerosols from: heat-rejection equipment

(cooling towers, evaporative condensers, steam turbine cleaning), components of

plumbing systems (showers, faucets, hot water tanks), nebulizers, humidifiers, whirlpool

spas, or public fountains. Less common sources include: ingestion of potable water,

immersion in raw water, inhalation of contaminated oil/water mixtures, and excavations

(dust or soil) (EPA 1985). Additional types of aerosol generators (e.g., grocery store mist

machines) have been linked to outbreaks of Legionnaires ‘disease (Mahoney et al. 1992).

Most of these sources involve the aerosolization of water contaminated with

Legionella and subsequent inhalation or aspiration. Potable water, especially in hospitals

and other buildings with complex hot water systems, is considered to be the most

important source of Legionella transmission (Blatt et al. 1994, Stout and Yu 1997, Woo

et al. 1992, Yu 1993).

Collection and Detection Methodologies of Legionella

Legionella Sampling in Water and Air. Test water samples for Legionella

typically come from sources such as faucets, sink outlets, taps, filters, and showerheads,

which are usually sampled by disassembling, swabbing, and scraping to obtain

Legionella-bearing debris or scale (Stout et al. 1992, Helms et al. 1988, Stout and Yu

1997, Barbaree et al. 1987). The most effective manner of obtaining the sample is by

12

insertion of sterile cotton swabs into the interior surface of the water source (CDC 2005).

Swab sampling is the preferred sampling method because the swab is easier to transport

and requires less processing time than straight water samples (Ta et al. 1995). Filtration

and centrifugation are used to concentrate Legionella, which greatly improves the ability

to detect the bacteria in samples (Ta et al. 1995).

Additionally, heat and acid wash treatment are recommended to isolate Legionella

from environmental samples (EPA 1985). Since Legionella bacteria can survive at high

temperatures, heating at 60 °C for 1-2 minutes can reduce the numbers of other bacteria

in sample while leaving Legionella unaffected (EPA 1985). Acid wash treatment is also

used to isolate Legionella strains due to their acid resistance (Nguyen et al. 1991). Water

samples are pretreated with a buffer mixture at a pH of 2.2 for 3 minutes to reduce

development of other bacteria (EPA 1985).

Buffered Charcoal Yeast Agar. After the collection and pretreatment steps, the

samples are plated onto appropriate media. Legionella do not grow on standard culture

media because they have complex nutritional requirements, such as unusually high

amount of iron. The most common media used for culturing and isolating Legionella is

ACES BCYE agar medium supplemented with α-ketoglutarate, a Krebs-cycle

intermediate that is readily catabolized by these bacteria (Edelstein 1987). An incubation

period of two to six days is required for culturing Legionella on this medium (Grimont

1986). The buffer maintains the pH within a range that is critical for Legionella (around

pH 6.9) while the α-ketoglutarate stimulates growth. Growth is further enhanced by the

addition of L-cysteine, keto acids, and ferric ions. Antimicrobials such as glycine

13

(inhibitor), cefamandole, polymyxin B, vancomycin (antibacterials), and anisomycin and

cyclohexamide (antifungals) are added to inhibit or prevent the overgrowth of

contaminants (Nguyen et al. 1991). Selective media containing dyes, glycine,

vancomysin, and polymyxin (DGVP) is used for environmental sampling (Lin et al.

1998b).

Detection of Legionella in Environmental and Biological Samples. An array of

serological tests has been used for detecting Legionella in water, sputum, blood, serum,

and urine samples (EPA 1999). The two main serologic tests performed on bacteria are

direct and indirect fluorescent assays, which are applicable to both environmental and

clinical specimens (EPA 1999). Fluorescent organic compounds are attached to antibody

molecules that are bound to a cell or tissue’s surface antigens, and then a fluorescent

microscope detects these tags (EPA 1999). In the direct method, the antibody against the

organism is fluorescent, while the indirect method has the fluorescent antibody detected

against a non-fluorescent antibody on the surface of the cell. Other serologic tests include

enzyme-linked immunosorbent assays, monoclonal antibodies, and radioimmunoassay.

The serologic tests differ primarily in sensitivity, specificity, predictive value, and

complexity.

Molecular techniques used include Polymerase Chain Reaction (PCR), DNA

hybridization, genomic, oligonucleotide cataloguing of 16s rRNA, and plasmid analysis

(EPA 1985). Comparison of bacterial DNA and the use of antigenic analysis of proteins

and peptides are the best current methods to classify Legionella species, although some

phenotypic characteristics (i.e. gram reactivity, cell membrane fatty acid and ubiquinone

14

content, morphology, and growth on specific media) can be used to recognize bacteria at

the genus level (Bangsborg 1997, Fang et al. 1989, Winn 1988).

Legionella Disinfection in Water Treatment

There are several control methods available for disinfection of water distribution

systems. These include thermal (super heat and flush), hyperchlorination, copper-silver

ionization, ultraviolet light sterilization, ozonation, and instantaneous steam heating

systems. Because one methodology may not be sufficient, a combination of these

techniques may be the most effective way of managing water systems and preventing

future outbreaks (Yu et al. 1993).

Thermal Disinfection. Thermal disinfection is a common practice for water

distribution systems in hospitals, hotels, and other institutional buildings. The hot water

temperature is elevated to above 70°C (158° F), and distal sites, such as faucets and

showerheads, are flushed for thirty minutes (Nguyen et al. 1991, Miuetzner et al. 1997,

Stout and Yu 1997).

Hyperchlorination. Hyperchlorination of water distribution systems requires the

installation of a chlorinator. Shock hyperchlorination involves the addition of chlorine to

a water system, raising chlorine levels throughout the system for one to two hours (Lin et

al. 1998a). Continuous hyperchlorination entails the addition of chlorinated salts to the

water (Stout and Yu 1997, Muraca et al. 1990).

Copper-Silver Ionization. Copper-silver ionization distorts the permeability of the

Legionella cell, denatures proteins, and leads to lysis and cell death (Nguyen et al. 1991,

Miuetzner et al. 1997, Muraca et al. 1990).

15

Ultraviolet Light Sterilization. Ultraviolet light kills Legionella by disrupting

cellular DNA synthesis (Muraca et al. 1990). An ultraviolet light sterilization system can

be installed easily. It can be positioned to disinfect the incoming water, or it can be

installed at a specific place in the pipe system that services a designated area. No

chemical by-products are produced, and the taste and odor of water from a water

distribution system containing a UV sterilizer are not affected (Muraca et al. 1990). The

UV sterilization system requires continuous maintenance in order to prevent scale from

coating the UV lamps. The system does not provide residual protection, so distal areas

must be disinfected (Nguyen et al. 1991, Muraca et al. 1990). Operational problems, such

as electrical malfunction and water leaks, are possible, in which case experienced

technicians are needed (Muraca et al. 1990).

Ozonation. Ozone can be used to kill L. pneumophila. It can be created using

ozonators, which electrically excite oxygen (O2) to ozone (O3).

Instantaneous Steam Heating. Instantaneous steam heating systems entail flash

heating water to temperatures greater than 88°C (190°F) and then blending the hot water

with cold water to attain a designated water temperature (Nguyen et al. 1991, Muraca et

al. 1990).

Ultraviolet Inactivation and Application in Air Disinfection

Ultraviolet germicidal irradiation (UVGI) is an established means of disinfection

and can be used to prevent the spread of certain infectious diseases. UV-C radiation kills

or inactivates microbes by damaging their deoxyribonucleic acid (DNA). The principal

mode of inactivation occurs when the absorption of a photon forms pyrimidine dimers

16

between adjacent thymine bases and renders the microbe incapable of replicating (Reed

2010).

UVGI can be used to disinfect water, air, and surfaces although surface disinfection

is limited by micro shadows and absorptive protective layers (Reed 2010). UV irradiation

has been widely implemented for drinking and waste water disinfection during the last 20

years in Europe and the US due to the high quality assurance of the UV disinfection

plants (Hijnen et al., 2006; Sommer et al., 2008). Cervero-Arago et al. applied UV

irradiation on 5 strains of Legionella spp. and 2 different co-cultures of Legionella

pneumophila with free-living amoeba strains in suspension. No significant differences in

the UV inactivation behavior were observed among Legionella strains tested which were

3 logs reduced for fluences around 45 J/m2 (Cervero-Arago et al., 2014). In contrast, the

results showed that the association of L. pneumophila with free-living amoebae decreases

the effectiveness of UV irradiation against the bacteria in a range of 1.5 - 2 fold (Cervero-

Arago et al., 2014).

UVGI air disinfection is accomplished via several methods: irradiating the upper-

room air only, irradiating the full room (when the room is not occupied or protective

clothing is worn), and irradiating air as it passes through enclosed air-circulation and

heating ventilation, and air-conditioning(HVAC) systems (Reed 2010). UVGI is also

used in self-contained room air disinfection units (Reed, 2010).

Upper-room UVGI is one of two primary applications of UVGI air disinfection.

Designed for use in occupied rooms without using protective clothing, upper-room UVGI

uses wall-mounted and ceiling-suspended louvered/shielded UVGI fixtures to confine the

17

germicidal radiation to the entire room area above people's heads and greatly minimizes

exposure to occupants in the lower room (Reed 2010).

In-duct UVGI is the other primary application of UVGI air disinfection. Designed to

disinfect air as it passes through the HVAC system and before it is recirculated or

exhausted, in-duct UVGI irradiates the entire cross-section of a duct at high intensities

not accessible to room occupants, and may include the use of highly UV-reflective

materials to further increase irradiance levels (Reed 2010).

Inactivation of microorganisms by UV irradiation in air may differ from that in

water because water can influence microbial sensitivity to UV radiation. In air, the

effectiveness of UV radiation as a disinfectant is governed by various factors, including

UV intensity, UV exposure time, microbial susceptibility to UV, and relative humidity

(RH) of UV-irradiance environments (Peccia et al., 2001). To date, many UVGI studies

on infectious bioaerosols are mainly focused on preventing the transmission of

tuberculosis using Mycobacterium as the test organism (Ko et al., 2000; Peccia and

Hernandez, 2004; Xu et al., 2005). Chang et al. tested UVGI on Legionella pneumophila

aerosols with a bioaerosol generation and UVGI test system built in the laboratory and

achieved a 2.2 – 4.3 log reduction (Chang et al., 2012).

UVGI is most effective in preventing infections spread chiefly by droplet nuclei, not

by direct contact or larger respiratory droplets, although some surface decontamination

likely occurs (Reed 2010). Also, the location(s) where UVGI is employed must also be

the primary location(s) of disease transmission (i .e. there cannot be a high risk of

acquiring the same infection outside the location where UVGI is used) (Reed 2010).

18

Although it is clear that UVGI can be effective in test chambers, engineering

specifications for a given room application remain elusive and are currently based more

on common sense and historical practice than on actual evidence (Reed 2010).

19

CHAPTER 2

MATERIALS AND METHODS

Experiments were conducted under laboratory conditions to aerosolize bacterial

cells and measure the dispersion of bacterial cells in air. To contain and prevent

spreading of aerosolized bacterial cells, all spray trials were tested in an airtight container

(Sterilite® ClearView LatchTM; Townsend, MA, USA) within a Class II A/B3

Biological Safety Cabinet (Forma Scientific, Inc.; Marietta, OH, USA). All air samples

were taken using a PBI SAS-Super ISO Air Sampler (VWR International PBI S.r.L.; Via

San Guisto, Milano, Italy). Three aerosolization methods tested in this study include an

electric paint spray gun (Krause & BeckerTM, China), a HVLP gravity feed spray gun

(Central Pneumatic®, China) connecting to an air valve in the lab, and a hand-held spray

bottle (up&upTM teardrop spray bottle, Target Corporation; Minneapolis, MN, USA). A

Cool Mist Ultrasonic Humidifier (The Sharper ImageTM, USA) was also tested. A stock

culture of E. coli (ATCC strain 25922) was used as subject bacteria. Tryptic Soy Broth

(TSB) and Tryptic Soy Agar (TSA) were used to culture E. coli.

The dimensions of the Sterilite® ClearViewTM Latch container were measured to

be 87.9 cm in length, 47.6 cm in width and 32.1 cm in height. The volume was 104 L. On

one side of the box, a 3 cm diameter opening was made for the spray input and on the

opposite side, a 12.5 cm diameter opening was made for the air sampler intake.

Preparation of E. coli stock culture. Pure culture of E. coli, strain 25922, was

obtained from the American Type Culture Collection (ATCC) (Manassas, VA). An

overnight culture was prepared by adding 1 mL of pure culture of E. coli to 9 mL of

20

Tryptic Soy Broth (TSB). The test tube was placed in an incubator at 37 °C for 16-18

hours. The concentration of this overnight culture was determined using a DR/4000U

Spectrophotometer (HACH Company; Loveland, CO). The spectrophotometer estimated

1 optical density unit (OD) at 600 nanometers (nM) of E. coli to reflect approximately

3x108 CFU/mL. The spectrophotometer was first zeroed with a cuvette filled with TSB.

After this, a cuvette filled with E. coli overnight culture in TSB was sent loaded the

spectrophotometer to determine the optical density.

Preparation of TSA Media for E. coli. Tryptic Soy Agar (Becton Dickinson

211043) was used for detection and enumeration of E. coli growth, visible by off-white

growths on the surface of the agar. First, 40 grams of TSA medium was added to 1 L of

DI water. Then the solution was heated to boiling on a hot plate (Thermo Scientific

Cimarec™ Digital Stirring Hotplates; USA or VWR® Hot Plate/Stirrer; Radnor, PA).

The solution was continuously mixed with a magnetic bar making sure all of the media

was completely dissolved. The TSA was then autoclaved for 15 minutes at 121 °C with

liquid setting. After the media was autoclaved, 15 mL of it was dispensed to individual

petri dishes using aseptic technique. The dishes were allowed to solidify and then were

placed in a sealed bag and stored at 4 °C in between experimental trials.

Preparation of Phosphate-Buffered Saline (PBS). Phosphate Buffered Saline was

utilized to contain E. coli cells in the aerosolization devices and more importantly,

maintain a favorable pH and osmotic pressure for the bacterial cells for aerosolization.

To prepare 1 L of 0.5 M PBS solution, 4 g of sodium chloride (NaCl), 0.1 g potassium

chloride (KCl), 0.72 g disodium phosphate (Na2HPO4) and 0.12 g monopotassium

21

phosphate (KH2PO4) were added to 800 mL of distilled water. The pH of this solution

was then adjusted to be approximately 7.4 and the volume was brought to 1 L. The

solution was then sterilized by autoclaving for 15 minutes at 121 °C.

Preparation of Spiked Samples for Aerosolization of E. coli. The concentration

of bacteria used in each spray method was held constant at 104 CFU/ml. 300 mL of 0.5 M

PBS was loaded into each spray device containing E. coli. A series of dilutions from the

E. coli working stock were conducted in order to achieve the concentration levels

necessary to perform aerosolization. A 10-fold dilution series was performed by

transferring 1 mL from an E. coli overnight culture into 9 mL of PBS buffer. Then the 10

mL sample was vortexed to ensure complete mixing and the 10-fold dilution was

continued to reach target concentration.

Additionally, spread plates were performed to confirm the concentration of the

spiked sample. Spread plates were conducted by transferring 100 μL (0.1 mL) of E. coli

sample from 102 CFU/mL and 103 CFU/mL dilution tubes onto TSA media. The number

of bacterial cells expected to grow on the plates were 10 and 100, respectively.

Aerosolization Methods for Dispersion of Bacterial Cells in Air

A set of experiments were performed to examine E. coli aerosolization and transport

in the aerosolization chamber within the Biological Safety Cabinet. Spiked sample was

sprayed through the opening from one side of the aerosolization chamber using the three

aerosolization methods: the electric spray gun, the air spray gun, and the handheld spray

bottle. The air sampler was placed at the other side of the chamber, lengthwise, to collect

22

100 L air sample from the aerosolization chamber. A TSA plate was placed inside the air

sampler to culture E. coli cells from the air sample.

To test the bacterial cells dispersion with different levels of aerosolization, spray

time was incorporated as a variable: each spray method was tested with 1 second and 5

second spray times. Additionally, to test the bacterial cells dispersion with different levels

of bacterial aerosol settlement, elapsed time was incorporated as another variable: for

each spray method, after an initial spray into the box, a specific amount of time (0 sec or

5 mins) was allowed to pass for aerosolized particles to settle before sampling.

Testing was conducted by spraying spiked sample (0.5 M PBS containing 104

CFU/ml E. coli) for a certain amount of time (1 sec, 5secs) into the aerosolization

chamber via a selected spray method (electric spray gun, air spray gun, handheld spray

bottle). After a specific elapsed time (0 sec, 5 mins), the air sampler was turned on and

100 L of air from the box was collected. After each sample, the TSA plate was removed

from the sampler and placed in the incubator at 37oC for 16-18 hours.

The ability of a humidifier to aerosolize bacteria was also of interest to this

experiment. Because most humidifiers are filled with non-sterile tap water that might

contains bacteria cells, these bacteria cells may be aerosolized as the humidifier runs,

thereby enabling human infection. This experiment was carried out in the Bio-Safety

cabinet because the humidifier could not be placed in the box due to size limitations. E.

coli spiked sample was placed in the chamber of a humidifier and the air sampler was

placed 87.9 cm (the length of the box) from the humidifier, inside of the Bio-Safety

cabinet. The humidifier was run for a certain amount of time (1 sec, 5secs) and a 100 L

23

air sample was taken after a specific elapsed time (0 sec, 5 mins). After each sample, the

TSA plate was removed from the sampler and placed in the incubator at 37oC for 16-18

hours.

UV Inactivation on Bacterial Cells in Air

A set of experiments were performed to examine UV inactivation on E. coli cells in

air in the Biological Safety Cabinet. Spiked sample was sprayed through the opening

from one side of the aerosolization chamber using the air spray gun. A UV-C germicidal

lamp inside the Biological Safety Cabinet was turned on after each spray. The air sampler

was placed at the other side to collect 100 L air sample. To avoid false positive results,

the intake of the air sampler was covered with parafilm during spraying. A TSA plate was

placed inside the air sampler to culture E. coli cells from the air sample.

To test for UV inactivation on E. coli aerosols with different level of UV dose,

exposure time was incorporated as a variable: after each spray, a specific amount of time

(5 secs, 10 secs, 20secs) was allowed to pass for aerosolized particles to be exposed to

UV-C rays.

Testing was conducted by spraying spiked sample (0.5 M PBS containing 104

CFU/ml E. coli) for 5secs into the aerosolization chamber with lid off via air spray gun.

The UV-C germicidal lamp inside the Biological Safety Cabinet was turned on for a

certain amount of time (5 sec, 10 secs, 20 secs) after each spray. Then the parafilm

covering the air sampler intake was removed and the air sampler was turned on to collect

100 L of air sample. After each sample, the TSA plate was removed from the sampler and

placed in the incubator at 37oC for 16-18 hours.

24

In order to calculate UV inactivation rate, controls were tested in the same procedure

with no UV inactivation.

Additionally, to simulate a real world scenario of air duct disinfection with UV

germicidal lamp, a UV inactivation test was conducted by spraying spiked sample (0.5 M

PBS containing 104 CFU/ml E. coli) for 5secs into the aerosolization chamber with the

lid off via an air spray gun. The UV-C germicidal lamp inside the Biological Safety

Cabinet was turned on as the spray started and turned off as the spray ended. The air

sampler was turned on the same time as the spray occurred and collected 100 L of air

sample without using parafilm to cover the intake during spraying.

25

CHAPTER 3

RESULTS AND DISCUSSION

Aerosolization Methods for Dispersion of Bacterial Cells in Air

For each aerosolization method two experiments with two duplicate culture assays

were performed. The results were acquired as the number of E. coli colonies detected in

100 liters of air samples (CFU/100 L of air). The calculated average and each of the

duplicate data are presented the Tables below.

Table 1

E. coli Aerosolization via Electric Spray Gun

Spray Time (sec) Elapsed Time*

(min)

Number of E. coli Detected in Air

Samples (CFU/100 L air)

Duplicates Average

1

0

233

330 247

408

432

5

19

30 25

32

44

5

0

516

750 564

916

1004

5

49

65 61

73

87

26

*Elapsed Time: The time between completion of each spray and the start of air sample

collection.

Table 2

E. coli Aerosolization via Air Spray Gun

Spray Time (sec) Elapsed Time

(min)

Number of E. coli Detected in Air

Samples (CFU/100 L air)

Duplicates Average

1

0

538

675 562

750

850

5

100

175 120

150

330

5

0

856

1250 944

1600

1600

5

241

330 279

336

464

27

Table 3

E. coli Aerosolization via Handheld Spray Bottle

Spray Time* (sec) Elapsed Time

(min)

Number of E. coli Detected in Air

Samples (CFU/100 L air)

Duplicates Average

1

0

34

70 56

82

108

5

0

0 0

0

0

5

0

198

310 248

344

450

5

7

13 9

12

24

*One spray per second

28

Table 4

Summary of E. coli Aerosolization via the Three Spray Methods

Spray Method Spray Time (sec) Elapsed Time

(min)

Number of E. coli

Detected in Air

Samples (CFU/100 L

air)

Electric Spray Gun

1 0 330

5 30

5 0 750

5 65

Air Spray Gun

1 0 675

5 175

5 0 1250

5 330

Handheld Spray

Bottle

1 0 70

5 0

5 0 310

5 13

The results showed that for each of the spray methods, the number of E. coli

detected in air samples increased as the spray time increased from 1 to 5 seconds but

decreased significantly as the elapsed time increased from 0 to 5 minutes. These

relationships are understandable due to the fact that with a longer spray time, more E. coli

cells were aerosolized into the box and get detected in air samples. On the other hand,

more E. coli cells could settle from the air with a longer elapsed time due to the action of

gravity and, thus, were not detected in the air samples.

It was shown that within the three spray methods, the air spray gun aerosolized the

most E. coli cells under all the condition with a highest number of 1250 CFU/ 100 L air

29

at 5 seconds spray time and 0 minute elapsed time. It was also shown that compared to

other spray methods, the handheld spray bottle aerosolized the least E. coli cells under all

the condition with a lowest number of 0 CFU/ 100 L air at 1 second spray time and 5

minutes elapsed time.

Another finding was that the variables affected the E.coli aerosolization from

different spray methods differently. The aerosolization results of the handheld spray

method were largely affected by the elapsed time. For example, for 0 min and 5 min

elapsed time with 5 secs spray time, the number of E. coli in the air samples decreased

from 310 to 13 CFU/100 L air, indicating that only 4.2 % of E. coli cells remained

aerosolized after 5 minutes. In comparison, the results at the same conditions for the air

spray gun were 1,250 and 330 CFU/100 L air, meaning that 26.4% of E. coli remained in

the air after 5 minutes. An explanation for the findings could be due to the differences in

the sizes of aerosols emitted by different sprayers; larger droplets settled faster in the

same time period.

To better understand the aerosolization of E. coli cells via different spray methods,

bacteria aerosolizing efficiency of the three sprayers were defined and calculated. The

formula used for determining this is as follows:

Bacteria Aerosolizing Efficiency = {Number of Bacteria Detected in Air Samples /

Number of Bacteria Sprayed} * 100%, where Number of Bacteria Sprayed (CFU) =

Bacteria Concentration (CFU/ mL) * Volume of each spray (mL)

30

The volume sprayed for each spray time was measured and the efficiency of

bacterial aerosolization at different conditions for each spray method was calculated, as

shown in Table 5.

Table 5

The Efficiency of Bacterial Aerosolization by the Three Spraying Methods

Spray

Method

Spray Time

(sec)

Elapsed

Time (min)

Number of

E. coli

Detected in

Air

Samples

(CFU/100

L air)

Volume

Sprayed

(mL)

Bacteria (E.

coli)

Aerosolizing

Efficiency

Electric

Spray Gun

1

0 330 10 0.33%

5 30 10 0.03%

5

0 750 32 0.24%

5 65 32 0.02%

Air Spray

Gun

1

0 675 6 1.1%

5 175 6 0.3%

5

0 1250 20 0.63%

5 330 20 0.17%

Handheld

Spray

Bottle

1

0 70 1 0.7%

5 0 1 0

31

5

0 310 5 0.62%

5 13 5 0.03%

The efficiency results showed that the air spray gun has the best capability of

aerosolizing bacteria cells under all the conditions examined in this study compared to

the other two spray methods. The highest aerosolizing efficiency calculated was at 1.1%

with a spray time of 1 second and an elapsed time of 0 minute, which means there was

approximately 1 bacterial cell detected in air from 100 cells sprayed.

The electric spray gun showed the lowest capability of aerosolizing bacterial cells of

the three spray methods. One of the reason for having the lowest efficiency is because the

electric spray gun has a much higher spray volume than the handheld spray bottle,

resulting in lower calculated aerosolizing efficiency than that of the spray bottle method.

Another finding was that only the handheld spray bottle’s spray volume increases at

the same ratio as the spray time increases, which explains its significant rise in number of

E. coli detected in air samples as spray time increases.

The number of cells detected in air samples and the calculated aerosolization

efficiency are the two criteria for selecting the best spraying method. The cells in air

samples indicate the number of bacteria aerosolized by the sprayer while the calculated

efficiency describes the aerosolizing capability of the sprayer. The results for both criteria

suggest that the air spray gun is the best aerosolizing method.

32

Table 6

E. coli Aerosolization via Ultrasonic Humidifier

Operation Time

(sec)

Elapsed Time

(min)

Number of E. coli Detected in Air

Samples (CFU/100 L air)

Duplicates Average

1

0

2

3.75 4

4

5

5

0

0 0

0

0

5

0

2

6 2

8

12

5

0

0 0

0

0

Positive results were observed from some of the plates, proving the ultrasonic

humidifier is capable of aerosolizing bacteria cells. E. coli number detected in air sample

was very low compared to that of the electric spray gun, air sprayer and handheld spray

bottle. This is partly because only a certain portion of E. coli cells aerosolized by the

humidifier were sampled since the test was not conducted inside the aerosolization

33

chamber. The actual number of E. coli aerosolized, the aerosolizing efficiency of the

humidifier and whether its aerosolization process kills E. coli remain to be determined.

UV Inactivation on Bacterial Cells in Air

For each UV inactivation trial, two experiments with two duplicate culture assays

were performed. The results were acquired as the number of E. coli or Legionella

colonies detected in 100 liters of air samples (CFU/100 L air). Duplicate values for the

calculated mean aerosol concentrations and UV inactivation rate (as log reduction) are

presented in the Tables below.

Table 7

UV Inactivation of E.coli Cells in Air at Different Exposure Times

UV

exposure

Time (sec)

Control (CFU/100 L air)

Number of E. coli

Detected in Air Samples

(CFU/100 L air) Log

Reduction

Duplicates Average Duplicates Average

5

350

355

33

34 1.02

360 35

10

290

298

2

3.5 1.93

306 5

34

20

164

168

0

0 > 2

172 0

A significant reduction between control and experimental UV inactivation results

could be observed. The results proved that UV-C inactivation is capable of removing

aerosolized E. coli cells in air. A 1 log reduction was achieved with 5 seconds UV

exposure time while 10 seconds UV exposure caused a 2 log reduction. For the 20

seconds UV exposure time test, no E. coli was detected in air samples, thus achieving at

least a 2 log reduction, although the exact value is unknown.

Table 8

UV Inactivation on E. coli Cells in Air Simulating Real World Air Duct Disinfection

UV

exposure

Time (sec)

Control (CFU/100 L air)

Number of E. coli

Detected in Air Samples

(CFU/100 L air) Log

Reduction

Duplicates Average Duplicates Average

0 - 5

380

424

270

275 0.19

468 280

To simulate a real world air duct UV disinfection scenario, UV inactivation on E.

coli cells within constant air flow was examined. Unlike experiment trials in Table 7, this

test does not have a certain UV exposure time for all aerosolized particles because

35

spraying, UV inactivation and air sampling occurred at the same time. UV log reduction

in this test was calculated to be 0.19. This was less than the log reduction for the 5

seconds UV exposure test shown in Table 7 due to the UV exposure time being uncertain

and less than 5 seconds for a portion of aerosolized E. coli cells. In addition, false

positive results were generated in the control test as the intake of the air sampler couldn’t

be covered with parafilm during spraying for the air duct simulation scenario.

Table 9

UV Inactivation of Legionella Cells in Air at Different Exposure Times

UV exposure

Time (sec)

Control (CFU/100 L air)

Number of Legionella

Detected in Air Samples

(CFU/100 L air) Log Reduction

Duplicates Average Duplicates Average

5

267

384

4

9.25 1.62

293 9

472 11

504 13

10

178

233.5

0

0 > 2

196 0

256 0

304 0

20

138

190

0

0 > 2

142 0

36

232 0

248 0

A significant reduction between control and experimental UV inactivation results

could be observed. The results proved that UV-C inactivation is capable of removing

aerosolized Legionella cells in air. A 1.62 log reduction was achieved with 5 seconds UV

exposure time. For the 10 and 20 seconds UV exposure time test, no Legionella was

detected in air samples, thus achieving at least a 2 log reduction, although the exact value

is unknown. Compared to UV inactivation of E.coli cells (Table 7), Legionella shows

similar aerosolization results but a higher susceptibility to UV-C.

Table 10

UV Inactivation of Legionella Cells in Air Simulating Real World Air Duct Disinfection

UV

exposure

Time (sec)

Control (CFU/100 L air)

Number of Legionella

Detected in Air Samples

(CFU/100 L air) Log

Reduction

Duplicates Average Duplicates Average

0 - 5

578

761.25

400

541 0.15 667 444

880 640

920 680

37

CHAPTER 4

CONCLUSION

The results of the experiments indicate that for each of the spray methods, the

number of cells detected in air samples increased with the spray time from 1 to 5 seconds

but decreased substantially as the elapsed time increased from 0 to 5 minutes. For the

three spray methods tested, the air spray gun aerosolized the most E. coli cells under all

conditions, with a maximum of 1,250 CFU/ 100 L air at 5 seconds spray time and 0

minute elapsed time.

To better understand the aerosolization of E. coli cells via different spray methods,

bacteria aerosolizing efficiency of the three sprayers were defined and calculated. The

efficiency results showed that the air spray gun has the highest capability of aerosolizing

bacteria under the conditions examined in this study compared to the other two spray

methods. The highest aerosolizing efficiency calculated was at 1.1% with a spray time of

1 second and an elapsed time of 0 minute, meaning that approximately 1 bacterial cell

was detected in air from 100 cells sprayed. It is important to note that bacterial

cultivability can significantly decreased within aerosols due to rapid dehydration while

suspended in air. The number of cells detected in air samples and the calculated

aerosolization efficiency are the two criteria for selecting the best spraying method. The

cells in air samples indicate the number of bacteria aerosolized by the sprayer while the

calculated efficiency describes the aerosolizing capability of the sprayer. The results for

both criteria suggest that the air spray gun was the best aerosolizing method. This

experiment suggests a practical and efficient method of bacterial aerosolization technique

38

for microbial dispersion in air. The suggested method can be used in future research for

microbial dispersion and transmission studies.

In addition, air samples were taken from a humidifier filled with a solution of PBS

and E. coli bacteria, serving as a proof of concept for airborne pathogen transmission via

a humidifier. The presence of colony formation on sampling plates was evidence of the

humidifiers’ capability for bacterial aerosolization.

The application of UV-C for the inactivation of bacterial cells resulted in removing

aerosolized E. coli and Legionella cells in air. For E. coli cells, 1 log reduction was

achieved with 5 seconds UV exposure time while 10 seconds UV exposure resulted in a 2

log bacterial reduction, UV log reduction in the real world air duct UV disinfection

simulation test was calculated to be 0.19 log. For Legionella cells, 1.62 log reduction was

achieved with 5 seconds UV exposure time while 10 seconds UV exposure resulted in a >

2 log bacterial reduction, UV log reduction in the real world air duct UV disinfection

simulation test was calculated to be 0.15 log. These results demonstrate the applicability

of UV inactivation for pathogenic bacterial cells in air via short UV exposure time. This

method may be applicable for the inactivation of Legionella in air ducts by installing

germicidal UV lamps for protecting susceptible populations in certain indoor settings

such as nursing rooms or other large gathering rooms.

39

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