Chapter 5

Aeromicrobiology
Ian L. Pepper and Charles P. Gerba

5.1 Introduction 5.6 Extramural Aeromicrobiology 5.8 Bioaerosol Control

5.2 Aerosols 5.6.1 Aerosolization of Indigenous 5.8.1 Ventilation
5.3 Nature of Bioaerosols Soil Pathogens 5.8.2 Filtration
5.4 Aeromicrobiological Pathway 5.6.2 Influenza Pandemics 5.8.3 Biocidal Control
5.4.1 Launching 5.6.3 Microbiology in the 5.8.4 Isolation
5.4.2 Transport Clouds 5.9 Biosafety in the Laboratory
5.4.3 Deposition 5.6.4 Agriculture 5.9.1 Biological Safety Cabinets
5.5 Microbial Survival in the Air 5.6.5 Waste Disposal 5.9.2 Biosafety Laboratories
5.5.1 Relative Humidity 5.6.6 Important Airborne Toxins 5.9.3 Biological Agent Classification
5.5.2 Temperature 5.7 Intramural Aeromicrobiology Questions and Problems
5.5.3 Radiation 5.7.1 Buildings References and Recommended Reading
5.5.4 Oxygen, OAF and Ions 5.7.2 Hospitals and Laboratories

5.1 INTRODUCTION intramural (indoor) and extramural (outdoor) aerobiology,

as they relate to the airborne transmission of environmen-
In the 1930s, F.C. Meier coined the term aerobiology to tally relevant microorganisms, including viruses, bacteria,
describe a project that involved the study of life in the air fungi, yeasts and protozoans.
(Boehm and Leuschner, 1986). Since then, aerobiology has
been defined by many as the study of the aerosolization,
aerial transmission and deposition of biological materials. 5.2 AEROSOLS
Others have defined it more specifically as the study of dis-
eases that may be transmitted via the respiratory route Particles suspended in air are called aerosols. These pose a
(Dimmic and Akers, 1969). Despite the variations in defi- threat to human health mainly through respiratory intake
nition, this evolving area is becoming increasingly impor- and deposition in nasal and bronchial airways. In addition,
tant in many aspects of diverse fields including public soil or dust particles can act as a “raft” for biological enti-
health, environmental science, industrial and agricultural ties known as bioaerosols (Brooks et al., 2004). Smaller
engineering, biological warfare and space exploration. aerosols travel further into the respiratory system and gen-
This chapter introduces the basics of aerobiology, erally cause more health problems than larger particles. For
including the nature of aerosols and the fundamentals of this reason, the United States Environmental Protection
the aeromicrobiological (AMB) pathway. The remainder of Agency (USEPA) has divided airborne particulates into
the chapter focuses on a subset of the science that we shall two size categories: PM10, which refers to particles with
term aeromicrobiology. Aeromicrobiology, as defined for diameters less than or equal to 10 µm (10,000 nm), and
the purpose of this text, involves various aspects of PM2.5, which are particles less than or equal to 2.5 µm

I.L. Pepper, C.P. Gerba, T.J. Gentry: Environmental Microbiology, Third edition. DOI: http://dx.doi.org/10.1016/B978-0-12-394626-3.00005-3
© 2015 Elsevier Inc. All rights reserved. 89
90 PART | II Microbial Environments

Information Box 5.1 Influence of Particle Size on

Velocity of Deposition of Particles in Air, Calculated
Using Stokes’ Law

Particle Diameter (mm) Particle Type Rate of Fall in

Air (cm s21)
1 Sand 7880
0.1 Silt 79
0.001 Clay 7.9 3 1025
0.002 Clostridial spore 0.016
From Pepper et al., 2006.

FIGURE 5.1 Mongolian dust over the Sea of Japan. Image provided where:
by NASA.
V 5 velocity of fall (cm/s21)
g 5 acceleration of gravity (980 cm/s22)
(2500 nm) in diameter. For this classification, the diameter D 5 diameter of particle (cm)
of aerosols is defined as the aerodynamic diameter: ρp 5 density of particle (density of quartz particles is
2.65 g/cm23)
1 ρ1 5 density of dispersion medium (air has a density
dpa 5 dps ðρp =ρw Þ /2 (Eq. 5.1)
of about 0.001213 g/cm23; water has a density of
where: about 1 g/cm23)
ρ 5 viscosity of the dispersion medium (about
dpa 5 aerodynamic particle diameter (µm) 1.83 3 1024 poise or g cm21s21 for air;
dps 5 Stokes’ diameter (µm) 1.002 3 1022 poise for water)
ρp 5 particle density (g/cm23)
ρw 5 density of water (g/cm23) Using Stokes’ law, we can calculate the rate of fall of
particles in air (Information Box 5.1). Small particles are
Atmospheric particulate concentration is expressed in thus a greater concern than larger particles for several rea-
micrograms of particles per cubic meter of air (µg/m3). The sons. Small particles stay suspended longer and so they
USEPA established a National Ambient Air Quality travel further and stay suspended longer. This results in an
Standard (NAAQS) for PM10 of 150 µg/m23 averaged over increased risk of exposure. Small particles also tend to
a 24-hour period, and 50 µg/m23 averaged annually. More move further into the respiratory system, exacerbating their
recently, separate standards for PM2.5 of 65 µg/m23 for effects on health. Stokes’ law explains why we can expect
24 hours and 15 µg/m23 annually have been introduced. viruses to persist as a bioaerosol longer than bacteria, which
Symptoms of particulate matter inhalation include: are much larger.
decreased pulmonary function; chronic coughs; bronchitis;
and asthmatic attacks. The specific causal mechanisms are
poorly understood. One well-documented episode 5.3 NATURE OF BIOAEROSOLS
occurred in London in 1952, when levels of smoke and
sulfur dioxide aerosols, largely associated with coal com- Biological contaminants include whole entities such as
bustion, reached elevated levels due to local weather con- bacterial and viral human pathogens. They also include
ditions. Over a 10-day period, approximately 4000 deaths airborne toxins, which can be parts or components of
were attributed to cardiovascular and lung disorders whole cells. In either case, biological airborne contami-
brought on or aggravated by these aerosols. nants are known as bioaerosols, which can be ingested or
Airborne particles can travel great distances. Intense inhaled by humans.
dust storms during 1998 and 2001 in the Gobi desert of Bioaerosols vary considerably in size, and composition
western China and Mongolia (Figure 5.1) elevated aerosol depends on a variety of factors including the type of micro-
levels to concentrations near the health standard in western organism or toxin, the types of particles they are associated
North America several thousand miles away. with such as mist or dust, and the gases in which the
Smaller particles tend to travel greater distances than large bioaerosol is suspended. Bioaerosols in general range from
particles. Stokes’ law (Eq. 5.2) is used to describe the fall of 0.02 to 100 µm in diameter and are classified on the basis
particles through a dispersion medium, such as air or water: of their size. The smaller particles (,0.1 µm in diameter)
are considered to be in the nuclei mode, those ranging from
V 5 ½D2 3 ðρp 2 ρ1 Þ 3 g=18ρ (Eq. 5.2) 0.1 to 2 µm are in the accumulation mode and larger
Chapter | 5 Aeromicrobiology 91

The composition of bioaerosols can be liquid or solid, or

Information Box 5.2 Possible Modes of Respiratory a mixture of the two, and should be thought of as microor-
Transmission of Influenza A ganisms associated with airborne particles, or as airborne
particles containing microorganisms. This is because it is
Direct Contact rare to have microorganisms (or toxins) that are not associ-
Transmission occurs when the transfer of microorganism results ated with other airborne particles such as dust or water. This
from direct physical contact between an infected or colonized
information is derived from particle size analysis experi-
individual and a susceptible host.
ments, which indicate that the average diameter of airborne
Indirect Contact bacterial particles is greater than 5 µm (Fengxiang et al.,
Transmission occurs by the passive transfer of microorganisms to 1992). By comparison, the average size of a soil-borne bacte-
a susceptible host via inanimate contaminated object or fomite. rium, 0.3 to 1 µm, is less than one-fifth this size. Similar par-
ticle size analysis experiments show the same to be true for
Droplet aerosolized microorganisms other than bacteria, including
Transmission occurs via large droplets ($5 µm diameter) gener- viruses.
ated from the respiratory tract of the infected individual during
coughing or sneezing, talking or during procedures such as suc-
tioning or bronchoscopy. These droplets are propelled a distance
of less than 1 m through the air, and are deposited on the nasal or
5.4 AEROMICROBIOLOGICAL PATHWAY
oral mucosa of the new host or in their immediate environment.
These large droplets do not remain suspended in the air and true The aeromicrobiological pathway describes: (1) the launch-
aerosolization does not occur. ing of bioaerosols into the air; (2) the subsequent transport
via diffusion and dispersion of these particles; and finally
Airborne (3) their deposition. An example of this pathway is that of
Transmission occurs via the dissemination of microorganisms by liquid aerosols containing the influenza virus launched into
aerosolization. Organisms are contained in droplet nuclei (air- the air through a cough, sneeze or even through talking.
borne particles less than 5 µm that result from the evaporation These virus-associated aerosols are dispersed by a cough or
of large droplets), or in dust particles containing skin cells and sneeze, transported through the air, inhaled, and deposited
other debris that remain suspended in the air for long periods of in the lungs of a nearby person, where they can initiate a
time. Microorganisms are widely dispersed by air currents and
new infection (Figure 5.3). Traditionally, the deposition of
inhaled by susceptible hosts.
viable microorganisms and the resultant infection are given
See also Section 5.6.2 and Case Study 5.2.
the most attention, but all three processes (launching, trans-
port and deposition) are of equal importance in understand-
ing the aerobiological pathway.
Protozoa

Fungi
5.4.1 Launching
Bacteria

Viruses
The process whereby particles become suspended within
Earth’s atmosphere is termed launching. Because
0.01 µm 0.1 µm 1.0 µm 10 µm 100 µm

Nuclei mode Accumulation Coarse mode

mode

Fine particles Coarse particles

FIGURE 5.2 Diagrammatic representation of the relative sizes of
bioaerosols. The depictions of the various kinds of organisms are indica-
tive of their potential sizes when associated with airborne particles
(rafts). The terminologies used to describe the various sizes of the
bioaerosols are also indicated.

particles are considered to be in the coarse mode

(Committee on Particulate Control Technology, 1980). As
shown in Figure 5.2, particles in nuclei or accumulation FIGURE 5.3 A cough or sneeze launches infectious microbes into the
mode are considered to be fine particles and those in coarse air. Anyone in the vicinity may inhale the microbes, resulting in a poten-
mode are considered coarse particles. tial infection.
92 PART | II Microbial Environments

Z-direction
n
tio
direc
Y-

X-d
irec
tion

FIGURE 5.4 Schematic representation of the type of bioaerosol distri-

bution expected from a point source, given three planes of diffusion:
(1) the X-direction is the mean direction in which the wind is blowing;
(2) the Y-direction is the lateral diffusion; and (3) the Z-direction is the
vertical diffusion.
FIGURE 5.5 A linear bioaerosol source using the example of the
release of biological warfare agents. This is an illustration of an instanta-
bioaerosols must be launched into the atmosphere to be neous linear bioaerosol release.
transported, it is important to understand this process. The
launching of bioaerosols is mainly from terrestrial and
aquatic sources, with greater airborne concentrations or Linear and area sources can also be divided into instanta-
atmospheric loading being associated with terrestrial neous and continuous launching points of origin. For exam-
sources than with aquatic sources. A recent model esti- ple, an instantaneous linear source might be a passing
mated that the total global emission of bacteria containing aircraft releasing a biological warfare agent (Figure 5.5).
particles to the atmosphere to be 7.6 3 1023 to 3.5 3 1024 A continuous area source might be exemplified by release
(Burrow et al., 2009). Some researchers speculate that there of bioaerosols from a large field that has received an appli-
may even be atmospheric sources of bioaerosols in addition cation of biosolids or animal manures.
to terrestrial and aquatic ones. This phenomenon is related
to the limited potential for microorganisms to reproduce 5.4.2 Transport
while airborne. This, however, is an area of aeromicrobiol-
ogy for which there is little available information. Transport or dispersion is the process by which kinetic
Launching into the surface boundary layers can energy provided by the movement of air is transferred to
include, but is certainly not limited to, diverse mechan- airborne particles, with resultant movement from one point
isms such as: air turbulence created by the movement of to another. This “energy of motion” gained by airborne
humans, animals and machines; the generation, storage, particles is considerable, and can result in dissemination of
treatment and disposal of waste material; natural mechan- airborne microorganisms over long distances. Transport of
ical processes such as the action of water and wind on bioaerosols can be defined in terms of time and distance.
contaminated solid or liquid surfaces; and the release of Submicroscale transport involves short periods of time,
fungal spores as a result of natural fungal life cycles. under 10 minutes, as well as relatively short distances,
Airborne particles can be launched from either point, under 100 m. This type of transport is common within
linear or area sources. A point source is an isolated and buildings or other confined spaces. Microscale transport
well-defined site of launching such as a pile of biosolid ranges from 10 minutes to 1 hour, and from 100 m to
material, before it is applied over a field. Point sources tend 1 km, and is the most common type of transport phenome-
to display a general conical-type dispersion (Figure 5.4). non. Mesoscale transport refers to transport in terms of
Point sources can be further defined on the basis of the type days and distances up to 100 km, and in macroscale trans-
of launching phenomenon: (1) instantaneous point sources, port, the time and distances are extended even further.
for example, a single event such as a sneeze; or (2) continu- Because most microorganisms have limited ability to sur-
ous point sources, from which launching occurs over vive when suspended in the atmosphere, the most common
extended periods of time, such as a biosolid pile. scales considered are the submicroscale and microscale. It
In contrast to point sources, linear sources and area should be noted, however, that some viruses, spores and
sources involve larger, less well-defined areas. When con- spore-forming bacteria have been shown to enter into
sidered on the same size scale, linear and area sources dis- mesoscale and even macroscale transport.
play more particulate wave dispersion as opposed to the As bioaerosols travel through time and space, different
conical type of dispersion displayed by point sources. forces act upon them such as diffusion, inactivation and
Chapter | 5 Aeromicrobiology 93

ultimately deposition. Diffusion is the scattering and/or where someone is holding a smoking candle. If there is
dissipation of bioaerosols in response to a concentration no air current in the room the smoke will still eventually
gradient as well as gravity, and is generally aided by air- reach you at the door, but it will be very diffuse as it is also
flow and atmospheric turbulence. The amount of turbu- spreading in every other direction. However, if there is a
lence associated with airflow, and thus the relative fan behind the person holding the smoking candle and this
amount of diffusion that may occur in association with fan is pointed at the door, then the smoke from the candle
particulates such as bioaerosols, can be estimated using will be carried by this air current. It will travel the same
the method of Osbert Reynolds. Reynolds found that fac- distance as it did before, but it will travel faster, undergo
tors associated with mean wind velocity, the kinetic vis- less diffusion and as a result be more concentrated when it
cosity of the air and the relative dimension of the reaches you. This is the principle of time-dependent diffu-
interfering structures could provide an indication of the sion as indicated by Tayler’s theory.
amount of turbulence associated with linear airflow.
Without turbulence, airborne particles from a point source
would travel in a concentrated stream directly downwind. 5.4.3 Deposition
The Reynolds equation is written as follows:
The last step in the aeromicrobiology pathway is deposi-
velocity 3 dimension tion. An airborne bioaerosol will eventually leave the turbu-
Reynolds number 5 (Eq. 5.3)
viscosity lence of the suspending gas and will ultimately be
deposited on a surface by one or a combination of interre-
Consider, for instance, a situation in which there are lated mechanisms. These mechanisms are discussed in the
relatively high winds (500 cm/sec) that are passing over a following sections and include: gravitational settling; down-
small bush (24 cm). Because the occurrence of frictional ward molecular diffusion; surface impaction; rain deposi-
turbulence associated with an object depends on the wind tion; and electrostatic deposition. These processes are
velocity being high enough, and the object it is flowing linked in many ways, and even though viewed separately,
over being large enough, we find that at normal air vis- they all combine to create a constant, if not steady, deposi-
cosity (0.14 cm2/sec) the Reynolds number (Re) becomes: tion of particles.

500 cm=sec 3 24 cm
Re 5 5 85; 700 (Eq. 5.4) 5.4.3.1 Gravitational Settling
0:14 cm2=sec
The main mechanism associated with deposition is the
The limiting value for the Reynolds equation is usually action of gravity on particles. The force of gravity acts
considered to be 2000, with values above this number indi- upon all particles heavier than air, pulling them down and
cating turbulent conditions. The higher this value, the high- essentially providing spatial and temporal limitations to
er the relative turbulence of the airflow, and the greater the the spread of airborne particles. Steady-state gravitational
microorganism-associated particle diffusion that occurs per deposition (Figure 5.6) in the absence of air movement
unit time. In the preceding example, one would expect a can be described in very simplistic terms by Stokes’ law,
great deal of turbulence around items such as a bush, which which takes into account gravitational pull, particle den-
would increase the diffusion rates of passing bioaerosols. sity, particle diameter and air viscosity (Section 5.2).
When dealing with particulate transport over time and
distance, Tayler (1915) indicated that diffusion during
horizontal transport could be viewed as an increase in the
5.4.3.2 Downward Molecular Diffusion
standard spatial deviation of particles from the source Downward molecular diffusion, as indicated by the name,
over time. What does this mean? For an instantaneous can be described as a randomly occurring process caused
point source under the influence of a mean wind direc- by natural air currents and eddies that promote and
tion, spread would be a standard spatial deviation from a enhance the downward movement of airborne particulates
linear axis (x) extending from the source (origin) in the (Figure 5.7). These random movements exist even in rela-
mean direction of wind flow, with diffusion caused by tively still air and tend to be in the downward direction
turbulence occurring in the lateral (y) and vertical (z) axes because of gravitational effects. As a result, measured
(Figure 5.4). The standard deviation of particulate diffu- rates of gravitational deposition tend to be greater than
sion cannot be considered constant over a particular spa- those predicted by the Stokes equation. The increase in
tial orientation, but is instead dependent on the time taken the rate of deposition is due to the added effects of down-
to reach the particular distance. Mathematical models that ward molecular diffusion. Molecular diffusion is also
attempt to estimate the transport of airborne particles use influenced by the force of the wind. Molecular diffusion-
this basic premise as a foundation for predictions. To pic- enhanced deposition rates tend to increase with increasing
ture this concept, imagine standing at the door of a room, wind speed and turbulence.
94 PART | II Microbial Environments

5.4.3.3 Surface Impaction

Surface impaction is the process by which particles make
contact with surfaces, such as leaves, trees, walls and com-
puters. With impaction there is an associated loss of kinetic
energy. In nature, it is rare to find flat, smooth surfaces on
which wind currents are unobstructed. Thus, surface impac-
tion is a very critical factor influencing transport and depo-
sition, especially for bioaerosols.
Impaction potential is the relative likelihood that an
airborne object will collide with another object in its
path. Impaction does not necessarily result in permanent
deposition, however. Once a particle collides with an
object, it has the potential to bounce. Bouncing off a sur-
face causes the particle to reenter the air current at a
lower rate, which can have one of two effects: (1) it can
allow subsequent downward molecular diffusion and
gravitational settling to occur, resulting in deposition on
another nearby surface; or (2) it can allow the particle to
escape the surface and once again reenter the air current.
Studies have shown that impaction is influenced by the
FIGURE 5.6 Schematic representation of gravitational settling, which
velocity and size of the particle, as well as the size and
is a function of Earth’s gravitational pull, particle density, particle diam-
eter and the viscosity of air. This figure does not take into account ran- shape of the surface it is approaching.
dom air movement. Stokes’ equation was developed to give an estimate
of the terminal velocity achieved by particles as a function of gravita-
tional settling. 5.4.3.4 Rain and Electrostatic Deposition
Rainfall and electrostatic charge also can affect deposition.
Rainfall deposition occurs as a condensation reaction
between two particles (raindrop and bioaerosol), which
combine and create a bioaerosol with a greater mass,
which settles faster. This can be described mathematically
using the Stokes equation. In the example presented in
Information Box 5.1, a clostridial spore alone has a calcu-
lated terminal velocity of 0.016 cm/sec. The same spore
(bioaerosol), if it condensed with another particle such as a
water droplet, has a greater mass and thus a greater termi-
nal velocity. For instance, if the clostridial spore were to
condense with a water droplet that doubled the bioaerosol
density from 1.3 to 2.6 g/cm3, the terminal velocity would
be increased from 0.016 to 0.032 cm/sec. The overall effi-
ciency of rain deposition also depends on the spread area
of the particle plume. Larger, more diffuse plumes undergo
stronger impaction than smaller, more concentrated
plumes. Rain deposition is also affected by the intensity of
the rainfall. The heavier the rainfall, the greater the overall
rates and numbers of the condensation reactions, and the
greater the subsequent increase in rain deposition.
Electrostatic deposition also condenses bioaerosols, but
is based on electrovalent particle attraction. All particles
tend to have some type of associated charge.
FIGURE 5.7 Schematic representation of downward molecular diffu- Microorganisms typically have an overall negative charge
sion, a naturally occurring process caused by the air currents and eddies
associated with their surfaces at neutral pH. These nega-
that promotes and enhance gravitational settling of airborne particles.
Although molecular diffusion can occur in any direction, due to the tively charged particles can associate with other positively
effects of gravity the overall trend of the process results in net down- charged airborne particles, resulting in electrostatic con-
ward movement and deposition. densation. The major phenomenon occurring may be a
Chapter | 5 Aeromicrobiology 95

coagulation effect between particles (much like the con- were among the first to show this phenomenon, indicating
densation of the clostridial spore with the water droplet), that as the relative humidity approaches 100%, the death
which would increase the bioaerosol mass and enhance rate of Escherichia coli increases. In general, it has been
deposition. It might also be assumed that as an electromag- reported that most Gram-negative bacteria associated with
netically charged bioaerosol comes into close proximity aerosols tend to survive for longer periods at low to mid
with an electromagnetically charged surface, electroattrac- levels of relative humidities, with enhanced decay at rela-
tive or electrorepulsive influences may be present. tive humidities above 80% (Brooks et al., 2004). The
opposite tends to be true for Gram-positive bacteria, which
tend to remain viable longer in association with high rela-
5.5 MICROBIAL SURVIVAL IN THE AIR tive humidities (Theunissen et al., 1993). Thus, the ability
of a microorganism to remain viable in a bioaerosol is
The atmosphere is an inhospitable climate for microorgan- related to the organism’s surface biochemistry. One mech-
isms mainly because of desiccation stress. This results in a anism that explains loss of viability in association with
limited time frame in which microbes can remain biologi- very low relative humidity is a structural change in the
cally active. Many microorganisms, however, have specific lipid bilayers of the cell membrane. As water is lost from
mechanisms that allow them to be somewhat resistant to the cell, the cell membrane bilayer changes from the typi-
the various environmental factors that promote loss of bio- cal crystalline structure to a gel phase. This structural
logical activity. Spore-forming bacteria, molds, fungi and phase transition affects cell surface protein configurations
cyst-forming protozoa all have specific mechanisms that and ultimately results in inactivation of the cell (Hurst
protect them from harsh gaseous environments, increasing et al., 1997). In general, Gram-negative bacteria react
their ability to survive aerosolization. For organisms that unfavorably to desiccation, whereas Gram-positive cells
have no such specific mechanisms, the survival in aerosols are more tolerant of desiccation stress (Mohr, 2001).
can often be measured in seconds. In contrast, organisms Early studies by Loosli et al. (1943) showed that the
with these mechanisms can survive indefinitely. influenza virus was also adversely affected by an increase
As a result, viability is highly dependent on the environ- in relative humidity. More recent work suggests that
ment, the amount of time the organism spends in the envi- viruses possessing enveloped nucleocapsids (such as the
ronment and the type of microorganism. In addition, influenza virus) have longer airborne survival when the
microbes may be viable but nonculturable (Chapter 3), but relative humidity is below 50%, whereas viruses with
for simplicity in this chapter we will use the term viable naked nucleocapsids (such as the enteric viruses) are
rather than the term culturable. Many environmental factors more stable at a relative humidity above 50% (Mohr,
have been shown to influence the ability of microorganisms 2001). It should be noted that viruses with enveloped
to survive. The most important of these are relative humid- nucleocapsids tend to have better survival in aerosols
ity and temperature. Oxygen content, specific ions, UV than those without. Some viruses are also stable in the
radiation, various pollutants and AOFs (air-associated fac- AMB pathway over large ranges of relative humidity,
tors) are also factors in the loss of biological activity. Each which makes them very successful airborne pathogens.
of these factors is discussed in the following sections.
The loss of biological activity can be termed inactiva-
tion and can generally be described using the following 5.5.2 Temperature
equation:
Temperature is a major factor in the inactivation of micro-
Xt 5 X0 e2kt (Eq. 5.5) organisms. In general, high temperatures promote inacti-
where: vation, mainly associated with desiccation and protein
denaturation, and lower temperatures promote longer sur-
Xt represents the viable organisms at time t vival times (Mohr, 2001). When temperatures approach
X0 is the starting concentration freezing, however, some organisms lose viability because
k is the inactivation constant, which is dependent on of the formation of ice crystals on their surfaces. The
the particular species of microorganisms as well as effects of temperature are closely linked with many other
a variety of environmental conditions environmental factors, including relative humidity.

5.5.1 Relative Humidity 5.5.3 Radiation

The relative humidity or the relative water content of the The main sources of radiation damage to microorganisms
air has been shown to be of major importance in the sur- including bacteria, viruses, fungi and protozoa are the
vival of airborne microorganisms. Wells and Riley (1937) shorter UV wavelengths and ionizing radiation such as
96 PART | II Microbial Environments

X-rays. The main target of UV irradiation damage is the acids (Donaldson and Ferris, 1975). Therefore, OAFs
nucleotides that make up DNA. Ionizing radiation or have been strongly linked to microbial survival in the air.
X-rays cause several types of DNA damage, including The formation of other ions, such as those containing
single strand breaks, double strand breaks and alterations chlorine, nitrogen or sulfur, occurs naturally as the result
in the structure of nucleic acid bases. UV radiation causes of many processes. These include the action of lightning,
damage mainly in the form of intrastrand dimerization, shearing of water and the action of various forms of radi-
with the DNA helix becoming distorted as thymidines are ation that displace electrons from gas molecules, creating
pulled toward one another (Freifelder, 1987). This in turn a wide variety of anions and cations not related to the
causes inhibition of biological activity such as replication oxygen radicals. These ions have a wide range of biologi-
of the genome, transcription and translation. cal activity. Positive ions cause only physical decay of
Several mechanisms have been shown to protect microorganisms, e.g., inactivation of cell surface proteins,
organisms from radiation damage. These include associa- whereas negative ions exhibit both physical and biologi-
tion of microbes with larger airborne particles, possession cal effects such as internal damage to DNA.
of pigments or carotenoids, high relative humidity and
cloud cover, all of which tend to absorb or shield bioaero-
sols from radiation. Many types of organisms also have 5.6 EXTRAMURAL AEROMICROBIOLOGY
mechanisms for repair of the DNA damage caused by
UV radiation. An example of an organism that has a radia- Extramural aeromicrobiology is the study of microorgan-
tion resistance mechanism is Dienococcus radiodurans. isms associated with outdoor environments. In the extra-
D. radiodurans is a soil bacterium that is considered the mural environment, the expanse of space and the presence
most highly radiation-resistant organism that has yet been of air turbulence are two controlling factors in the move-
isolated. An important component of its radiation resis- ment of bioaerosols. Environmental factors such as UV
tance is the ability to enzymatically repair damage to chro- radiation, temperature and relative humidity modify the
mosomal DNA. The repair mechanism used by these effects of bioaerosols by limiting the amount of time that
bacteria is so highly efficient that much of the metabolic aerosolized microorganisms will remain viable. This sec-
energy of the cell is dedicated exclusively to this function. tion provides an overview of extramural aeromicrobiology
that includes: aerosolization of indigenous soil pathogens;
influenza pandemics; the spread of agricultural pathogens;
5.5.4 Oxygen, OAF and Ions the spread of airborne pathogens associated with waste
environments; and important airborne toxins.
Oxygen, open air factors (OAFs) and ions are environmen-
tal components of the atmosphere that are difficult to study
at best. In general, it has been shown that these three fac- 5.6.1 Aerosolization of Indigenous Soil
tors combine to inactivate many species of airborne Pathogens
microbes. Oxygen toxicity is not related to the dimolecular
form of oxygen (O2), but is instead important in the inacti- Geo-indigenous pathogens are those found in soils that are
vation of microorganisms when O2 is converted to more capable of metabolism, growth and reproduction (Pepper
reactive forms (Cox and Heckley, 1973). These include et al., 2009). These are found in all soils and include both
superoxide radicals, hydrogen peroxide and hydroxide prokaryotic and eukaryotic organisms, many of which are
radicals. These radicals arise naturally in the environment spore formers. Such spores can potentially be aerosolized
from the action of lightning, UV radiation, pollution, etc. and cause human infections. Bacillus anthracis is a bacte-
Such reactive forms of oxygen cause damage to DNA by rial geo-indigenous pathogen that causes lethal disease in
producing mutations, which can accumulate over time. humans via pulmonary, gastrointestinal or cutaneous
The repair mechanisms described in the previous section modes of infection (Gentry and Pepper, 2002). The organ-
are responsible for control of the damaging effects of reac- ism is found worldwide and, because it is a spore former,
tive forms of oxygen. can remain viable in soil for years.
Similarly, the open air factor (OAF) is a term coined Studies have shown the potential for anthrax to be dis-
to describe an environmental effect that cannot be repli- seminated by aerosols. Turnbull et al. (1998) found air-
cated in laboratory experimental settings. It is closely borne concentrations of anthrax spores as high as
linked to oxygen toxicity, and has come to be defined as 2.1 3 1022 CFU L21 of air, and airborne movement as far
a mixture of factors produced when ozone and hydrocar- as 18 m from a contaminated carcass in Etosha National
bons (generally related to ethylene) react. For example, Park, Namibia. However, the majority of samples taken
high levels of hydrocarbons and ozone can cause were negative, and the number of spores collected in pos-
increased inactivation rates for many organisms, probably itive samples was very low, making airborne contraction
because of damaging effects on enzymes and nucleic of disease at a distance from the carcass unlikely. A more
Chapter | 5 Aeromicrobiology 97

serious outbreak in humans resulting from a B. anthracis grows in the soils of semiarid regions of the Southwest
aerosol is described in Case Study 5.1. United States, including California, Arizona, New Mexico
Important fungal geo-indigenous pathogens include and Texas (Baptista-Rosas et al., 2007). Symptoms can
Coccidioides immitis and Histoplasma capsulatum. be mild to fatal. Histoplasma capsulatum, another fungus
Coccidiodes immitis is a soil-borne fungi that causes a causing respiratory infections, is found worldwide in
respiratory illness known as Valley Fever. It preferentially soils, but, in the United States, it is endemic to southeast-
ern and midwestern states (Deepe and Gibbons, 2008).
Histoplasmosis can be asymptomatic or mild, but the
infections can be very serious or even fatal for immuno-
Case Study 5.1 Anthrax compromised individuals.

In 1979, an anthrax outbreak occurred in Sverdlovsk, in the

then U.S.S.R., due to the accidental release of a bioaerosol
from a military microbiological facility (Meselson et al., 1994). 5.6.2 Influenza Pandemics
At least 66 people died as a result of the release. Human
anthrax cases extended 4 km along an axis to the south of the Influenza pandemic is the term given to an epidemic of
military facility and livestock cases extended up to 50 km in an influenza virus that occurs on a worldwide scale with
the same direction. The geographic distribution of human and a resultant infection of a large proportion of the human
animal cases was consistent with meteorological patterns exist- population. Known colloquially as the “flu,” influenza is
ing when the accidental release was believed to have occurred. an infectious disease of birds and mammals caused by an
There has been no indication that human anthrax cases have
RNA virus of the family Orthomyxoviridae. Influenza can
occurred in Sverdlovsk since 1979.
cause the common flu symptoms of muscle ache, head-
ache, coughing, weakness and fatigue, or pneumonia
which can be fatal.
Avian influenza refers to a large group of influenza
viruses that primarily affect birds, but have the potential
Case Study 5.2 The Spanish Influenza Pandemic of to adapt and infect humans. An influenza pandemic
1918 occurs when an avian influenza virus adapts into a strain
that is contagious among humans and that has not previ-
This pandemic affected approximately one-third of the world ously circulated within humans. Such adaptations can be
population at that time, with 36% dying (Barry, 2004). The devastating, as illustrated in Table 5.1.
pandemic lasted from January 1918 to December 1920, the Influenza virus transmission among humans can occur
responsible virus being H1N1. This was the first outbreak via four mechanisms: by direct contact with infected indi-
resulting from H1N1, with the second epidemic occurring in viduals; by indirect contact with contaminated objects of
2009. Although the pandemic did not originate in Spain, the fomites; by inhalation of droplets that contain the virus;
term “Spanish flu” was coined due to the severity of the infec- or by inhalation of aerosolized virus. Interestingly, despite
tions in Spain. It is believed that the pandemic began in
70 years of research since the influenza A virus was dis-
Haskell County, Kansas, before spreading rapidly to Europe.
covered, there is still debate about the modes of influenza
Estimates of the total number of deaths range from 50 to 100
million worldwide with 500,000 to 675,000 deaths in the U.S.
transmission, specifically whether influenza is mainly
A. (Barry, 2004). transmitted via true bioaerosols, or by droplets, or by
direct or indirect contact (Brankston et al., 2007).

TABLE 5.1 History of Major Influenza Pandemics

Name of Pandemic Period Deaths Influenza Subtype

Asiatic (Russian) flu 18891890 1 million Unknown
Spanish flu 19181920 Up to 50 million H1N1
Asian flu 19571958 2 million H2N2
Hong Kong flu 19681969 1 million H3N2
Swine flu 20092010 D18,000 H1N1
98 PART | II Microbial Environments

FIGURE 5.8 Cloud environmental factors that can

adversely influence microbes.

Acidic
Sunlight environment
Low temperature Osmolarity
Lack of
nutrients
Desiccation Oxidants

Deposition
Aerosolization

Bubbling
Mechanical aerosolization

Vegetation
Water Soil

5.6.3 Microbiology in the Clouds staple crops that are paramount to world food security.
Major pathogens of such crops are the wheat rust fungi.
Recent studies have suggested that microbes can poten- These spore-forming fungi cause some of the most devas-
tially affect meteorological processes. In particular, some tating diseases of wheat and other grains. In 1993, one type
microorganisms, called ice nucleators, efficiently catalyze of wheat rust (leaf rust) was responsible for the loss of
ice formation and may play a role in the formation and over 40 million bushels of wheat in Kansas and Nebraska
precipitation within clouds (Chistner, 2012). Based on alone. Even with selective breeding for resistance in wheat
recent studies, 95% of ice nuclei are biological particles plants, leaf rust continues to have major economic impacts.
and at least 40% originate from bacteria. Microorganisms The high concentration of wheat in areas ranging from
are present in both clouds and fog. The abundance of cul- northern Texas to Minnesota and up into the Dakotas
turable bacteria and fungi in clouds varies with the season, makes this whole region highly susceptible to rust
with greater numbers occurring in the summer and fall. epidemics.
While only 1% of the bacteria and 50% of the fungi in Spores of wheat rust are capable of spreading hundreds
clouds are culturable, studies suggest that the majority are if not thousands of kilometers through the atmosphere
metabolically active (Delort et al., 2010). Bacterial num- (Ingold, 1971). The airborne spread of rust disease has
bers range from 103 to 104/ml compared to fungal num- been shown to follow a predictable trend, which starts dur-
bers of 102 to 104/ml. The cloud environment is a harsh ing the fall with the planting of winter wheat in the south-
environment with UV light irradiation, desiccation, low ern plains. Any rust-infected plant produces thousands of
temperatures and other factors potentially adversely affect- spores, which are released into the air (Figure 5.9) by
ing microbes (Figure 5.8). Microorganisms may modify either natural atmospheric disturbance or mechanical dis-
this environment by metabolizing organic compounds, and turbance during the harvesting process. Once airborne,
also by playing a role in cloud chemistry and physics, but these spores are capable of long-distance dispersal, which
much additional research is needed because the cloud can cause downwind deposition onto other susceptible
environment is difficult to study. wheat plants. The generation time of new spores is mea-
sured in weeks, after which new spores are again released
from vegetative fungi into the AMB pathway. For exam-
5.6.4 Agriculture ple, during the harvest of winter wheat in Texas, the pre-
vailing wind currents are from south to north, which can
Numerous plant pathogens are spread by the aeromicrobio- allow rust epidemics to spread into the maturing crops far-
logical pathway (Information Box 5.3). Contamination of ther north in Kansas and up into the young crops in the
crops and animals via bioaerosols has a large worldwide Dakotas (Figure 5.10). This epidemic spread of wheat rust
economic impact. Rice and wheat are two of the major and the resulting economic destruction produced are
Chapter | 5 Aeromicrobiology 99

Information Box 5.3 Examples of Airborne Plant

Pathogens

Fungal Plant Disease Pathogen

Dutch Elm disease Ceratocystis ulmi
Potato late blight Phytophthora infestans
Leaf rust Puccinia recondite
Loose smut of wheat Ustilago tritici
Downy mildew Pseudoperonospora humuli
Maize rust Puccinia sorghi
Powdery mildew of barley Erysiphe graminis
Southern corn leaf blight Helminthosporium maydis

The figure shows the airborne spread of late blight of potato

that caused the 1845 epidemic known as the Irish potato famine.
Phytophthora infestans spread from Belgium (mid-June) through-
out Europe by mid-October. Famine related deaths are estimated
from 750,000 to 1,000,000. Economic devastation from this fam- FIGURE 5.9 Field of wheat highly infected by phytopathogenic wheat
ine caused the population of Ireland to decrease from approxi- rust. The field is being harvested by a hay machine, which is releasing a
mately 8 million to 4 million from 1840 to 1911. cloud of rust spores into the aeromicrobiological pathway. These spores
can spread thousands of miles and infect other crops downwind, causing
catastrophic losses to wheat crops.

Norway
Sweden

Ireland
UK
Poland
Belgium

France Germany
FIGURE 5.10 The arrow indicates the northern path of wheat rust
Italy infections as spread by the aeromicrobiological pathway. The wheat rust
infection begins in the winter harvest in Texas and spreads northward
Spain
with the prevailing wind currents. The epidemic spread of these phyto-
pathogens infects maturing crops in Kansas and then moves up into the
young crops in the Dakotas.

fungicide application is timed to coincide with spore

release. This approach minimizes use of harmful chemi-
indicative of the impact that airborne microbial pathogens cals. Thus, efficient aeromicrobiology pathway sampling,
can have on agriculture. monitoring, detection and modeling have the ability to
A factor that complicates the control of such diseases aid in the control of airborne pathogens.
is that chemical treatment for the control of pathogens is The airborne spread of pathogenic microorganisms is
viewed as undesirable. This is because many pesticides also highly important in the animal husbandry industry
have long half-lives and their residence in an ecosystem (Information Box 5.4). The occurrence of foot-and-mouth
can be extremely harmful. Therefore, instead of using disease is an example of the importance of bioaerosols in
wheat rust fungicides, attempts are being made to breed the spread of airborne disease (Case Study 5.3). It has
strains of wheat that are more resistant to the fungi. long been thought that bioaerosol spread is linked primar-
Another method used for controlling phytopathogenic ily to respiratory pathogens, but there is growing evidence
(plant pathogenic) fungi is spore monitoring as a disease that gastrointestinal pathogens are also important in air-
control strategy. In this approach, the life cycle of the borne transmission of disease among animals. One exam-
fungi, especially the release of spores, is monitored, and ple of bioaerosol spread of a gastrointestinal pathogen is
100 PART | II Microbial Environments

Information Box 5.4 Examples of Airborne Animal

Pathogens

Animal Disease Pathogen

Bacterial diseases
Tuberculosis Mycobacterium bovis
Brucellosis Brucella spp.
Fungal diseases
Aspergillosis Aspergillus spp.
Coccidioidomycosis Coccidioides immitis
Viral diseases
Influenza Influenza virus
Rabies Rhabdoviridae
Foot-and-mouth disease Aphthovirus

FIGURE 5.11 Application of secondary treated wastewater onto agri-

cultural lands. This method is highly efficient at conserving water and
has been shown to improve the fertility of soils. Due to the presence of
pathogens in wastewater, and the nature of these land application sys-
Case Study 5.3 The United Kingdom Foot and Mouth tems, there are high concentrations of bioaerosols generated. Currently,
Crisis 2001 however, there is little epidemiological and microbial risk assessment
information available to determine if there may be health concerns for
Outbreaks of foot and mouth disease have occurred worldwide populations living in the vicinity of such operations, though there is a
including multiple occurrences within the U.S.A. However, growing base of information on the concentration and types of pathogens
found in these bioaerosols.
one of the more devastating outbreaks occurred in the U.K. in
the spring and summer of 2001. Foot and mouth viruses are
from the Aphthovirus genus of the family Picornaviridae, and within a few days. Thus, the aeromicrobiology pathway
are single-stranded RNA viruses. The 2001 U.K. outbreak was can be important even in the spread of diseases for which
due to a type O pan-Asia strain that was thought to have arisen
pathogens are not normally considered airborne.
from infected meat illegally imported into the U.K.
Transmission of the virus can occur via direct contact or via
bioaerosols (Grubman and Baxt, 2004). Overall, 2000 cases
were reported throughout Britain, resulting in the culling of 7 5.6.5 Waste Disposal
million sheep and cattle, costing the U.K.D$16 million.
Waste disposal is a multibillion dollar industry in the
United States. However, there are many hazards inherent
in the treatment and disposal of wastewater (Figure 5.11),
animal manures and biosolid material. Figures 5.125.14
transmission of Salmonella typhimurium among calves illustrate the potential for bioaerosol production via vari-
that are housed individually in small pens (Hinton et al., ous methods of land application of biosolids and also load-
1983). The potential for bioaerosol spread of this patho- ing operations. Major hazards associated with waste
gen was recognized because the initial symptoms resem- effluents are pathogenic microorganisms including bacte-
bled those of pneumonia and appeared randomly within ria, viruses, protozoa and helminths. Wastewater treatment
these animals, two factors that are not characteristic of plants utilize activated sludge and trickling filter systems,
oral transmission. Oral transmission generally occurs and all of these treatment processes potentially create rela-
sequentially from one pen to the next, whereas aerial tively large amounts of aerosols, which have been shown
transmission can carry organisms past nearby pens, infect- to include pathogenic microorganisms. Other aspects of
ing calves randomly. Furthermore, Wathes et al. (1988) the treatment process such as composting and land dis-
showed that S. typhimurium could survive for long peri- posal are also associated with the generation of aerosols
ods in an airborne state, and calves and mice exposed to containing pathogenic microorganisms.
aerosolized S. typhimurium developed symptoms, proving One of the primary methods for the disposal of biosolids
that gastrointestinal pathogens could be spread by aero- and manure is agricultural land application. The major con-
solization. Finally, Baskerville et al. (1992) showed that cern associated with the aerosolization process in relation to
aerosolized Salmonella enteritidis could infect laying waste disposal operations is the exposure of waste disposal
hens. These hens showed clinical symptoms and were workers to pathogenic microorganisms (occupational risk).
shedding the test strain of salmonellae in their feces In addition, nearby population centers are also potential
Chapter | 5 Aeromicrobiology 101

exposure risks (community risk). The potential for aerosoli-

zation of pathogens from land application of biosolids has
become a nationally debated issue. A major national study
on aerosolization from land application in the United States
was conducted by Brooks et al. (2005a,b). This study
showed that occupational risks of infection from bioaerosols
was greater than for offsite communities, where risks were
minimal (Brooks et al., 2012) (Case Study 5.4). Baertsch
et al. (2007) used DNA-based microbial source tracking to
measure aerosolization during land application.

5.6.6 Important Airborne Toxins

Microbial toxins can also be airborne. For example, a toxin
FIGURE 5.12 Land application of liquid biosolids via a spray applica- from Clostridium botulinum (botulinum A toxin) is a poten-
tion, and collection of air samples via biosamplers. tial biological warfare agent (Amon et al., 2001). Botulinum
toxin is a neurotoxin that is normally associated with inges-
tion of contaminated food. However, the lethal dose is so
small that aerosolization can also be a means of dissemina-
tion. The lethal dose for botulinum toxin by inhalation is
0.3 µg, with death occurring 12 hours after exposure. Death
is due to asphyxiation caused by the paralysis of respiratory
muscles. Another toxin produced by bacteria is staphylococ-
cal enterotoxin. On occasion, this toxin can be fatal with the
lethal dose estimated to be 25 µg by inhalation. The symp-
toms include cramping, vomiting and diarrhea, which occur
within 1 hour of exposure by aerosolization.
An important airborne toxin is lipopolysaccharide
(LPS) (Hurst et al., 1997). Lipopolysaccharide is derived
from the outer membrane of Gram-negative bacteria. It is
also referred to as endotoxin and is a highly antigenic bio-
logical agent that, when associated with airborne particles
FIGURE 5.13 Land application of liquid biosolids via a sprinkler such as dust, is often associated with acute respiratory
system. symptoms such as chest tightness, coughing, shortness of
breath, fever and wheezing. Due to the ubiquity of Gram-
negative bacteria, especially in soil, LPS is considered by
some to be the most important aerobiological allergen.
LPS (Figure 5.15) has three major components: a lipid A
moiety, which is a disaccharide of phosphorylated gluco-
samines with associated fatty acids; a core polysaccharide;
and an O-side chain. The lipid A moiety and the core poly-
saccharide are similar among Gram-negative bacteria, but
the O-side chain varies among species and even strains. It
is the O-side chain that is responsible for the hyperaller-
genic reaction. There are many sources associated with the
production of high levels of LPS, such as cotton mills,
haystacks, sewage treatment plants, solid waste handling
facilities, swine confinement buildings, poultry houses,
and even homes and office buildings. LPS is liberated
when Gram-negative bacteria in these environments are
lysed but can also be released when they are actively
growing.
FIGURE 5.14 Land application of cake biosolids via a slinger in In soils, bacterial concentrations routinely exceed 108
Solano County, California. per gram and soil particles containing sorbed microbes can
102 PART | II Microbial Environments

Case Study 5.4 Occupational and Community Risks of Infection from Bioaerosols Generated During Land Application
of Biosolids

In 2002, the National Research Council (NRC) issued a report N 5 number of pathogens inhaled per day
titled, Biosolids Applied to Land: Advancing Standards and Practices.
The annual risk of infection can be calculated from the daily
One of the recommendations of this report was the need to docu-
risk using
ment and evaluate the risk of infections from bioaerosols gener-
ated during land application. In response to this the University of Pyear 5 1 2 ð12Pi Þd
Arizona conducted a national study to evaluate occupational and
where d 5 number of days exposed per year.
community risks from such bioaerosols. Overall, more than 1000
aerosol samples were collected and analyzed for bacterial and
viral pathogens (Tanner et al., 2005; Brooks et al., 2005b).
Occupational Risk of Infection
Annual risk of infection for Coxsackie virus A21 during loading
The study was undertaken in two parts. First, the emission
operations was 2.1 3 1022.
rate of pathogens generated during loading of biosolids from
This risk suggests that approximately 1 worker per 50 is likely
trucks into spreaders, and also during land application of bioso-
to be infected with Coxsackie virus A11 working on-site over the
lids, was evaluated. This was assumed to be direct exposure to
course of 1 year.
biosolid workers on-site, that is, occupational risk, since there
was no pathogen transport required to for exposure. For commu-
nity risk, fate and transport of pathogens was taken into account,
Community Risk of Infection
Risk was calculated for a distance of 30 m from a land application
since residents live off-site, allowing for natural attenuation of
site assuming 6 days of land application annually, and 8 h expo-
pathogens due to environmental factors such as desiccation and
sure duration.
ultraviolet light.
Annual risk of infection for Coxsackie virus A21 during load-
Based on exposure data gathered on-site during land applica-
ing was 3.8 3 1025.
tion, occupational risk of infection from Coxsackie virus A21 was
Annual risk of infection for Coxsackie virus A21 during land
determined using a one-hit exponential model: Pi 5 1 2 exp
application was 2.1 3 1025.
(2rN), where:
These data imply that community risks of infection are mini-
Pi 5 the probability of infection per work day,
mal. As a comparison, for drinking water a 1:10,000 risk of infec-
r 5 parameter defining the probability of an organism initiating
tion per hear is considered acceptable (Haas et al., 2014).
infection 5 0.0253 for Coxsackie A21, and

be aerosolized, and hence act as a source of endotoxin. In comparison with the extramural environment, intramu-
Farming operations such as driving a tractor across a field ral environments have limited circulation of external air
have been shown to result in endotoxin levels of 469 endo- and much less UV radiation exposure. Indoor environ-
toxin units (EU) per cubic meter as measured by the ments also have controlled temperature and relative
Limulus amebocyte assay. These values are comparable to humidity, which are generally in the ranges that allow
those found during land application of biosolids operations extended microbial survival. Thus, these conditions are
(Table 5.2) (Brooks et al., 2006). Daily exposures of as lit- suitable for the accumulation and survival of microorgan-
tle as 10 EU/m23 from cotton dust can cause asthma and isms within many enclosed environments, including office
chronic bronchitis. However, dose response is dependent buildings, hospitals, laboratories and even spacecraft. In
on the source of the material, the duration of exposure and this section, we will consider these three diverse areas as
the frequency of exposures (Brooks et al., 2004). The data examples of current topics related to intramural aeromicro-
in Table 5.2 illustrate that endotoxin aerosolization can biology. Again, it should be noted that this section does
occur during both wastewater treatment and land applica- not cover all aspects of intramural aeromicrobiology, but
tion of biosolids. However, the data also show that endo- instead attempts to show the wide diversity of the science.
toxin of soil origin resulting from dust generated during
tractor operations results in similar amounts of aerosolized
endotoxin (see also Section 26.3.2).
5.7.1 Buildings
Many factors can influence bioaerosols and therefore how
5.7 INTRAMURAL AEROMICROBIOLOGY “healthy” or how “sick” a building is. These include: the
presence and/or efficiency of air filtering devices, the design
The home and workplace are environments in which air- and operation of the air circulation systems, the health and
borne microorganisms create major public health concerns. hygiene of the occupants, the amount of clean outdoor air
Chapter | 5 Aeromicrobiology 103

legionellae can find a niche. These include cooling

Mannose----Abequose towers, evaporative condensers, plumbing systems, whirl-
|
O-side chain Rhamnose pools, shower heads and hot-water faucets (Bollin et al.,
|
Galactose
n
1985). In the case of the American Legion convention,
|
Glucose----GlcN the reservoir for the organism that caused the outbreak
|
Galactose was a poorly maintained cooling tower, which provided
|
Glucose----Galactose optimal conditions for Legionella proliferation. Because
|
Heptose O O of the poor design of the air circulation system at the con-
Core polysaccharide |
+ vention, this proliferation led to the subsequent aerosoli-
Heptose P O P O CH2 CH2 NH3
| zation and spread of the organisms throughout the
| O O
2-Keto-3-deoxyoctanoate (KDO) building.
| O
|
+
What conditions promote the proliferation of
2-KD O P O CH2 CH2 NH3
| Legionella spp.? Stagnant water and temperatures in the
range of 3546 C are factors that can lead to the rapid
| O
O | O
|
–
O P O GlcN---------GlcN O P O–
multiplication of background levels of Legionella spp.
O
Another interesting aspect of the ecology of Legionella is
O
that they can grow intracellularly within cyanobacteria
Lipid A and protozoa. How can growth and spread of Legionella
spp. be avoided? Several strategies can be used. In the
maintenance of hot-water plumbing systems, operating
temperatures should be greater than 50 C. All potential
MAFA MA FA places where water can stagnate in water pipes should be
avoided. For cooling towers, the recommendations
FIGURE 5.15 Schematic structural representation of the lipopolysac- involve the installation of ozonization units, dry convec-
charide from Salmonella typhimurium. The number of repeating units
(n) in the side chain varies from 10 to 40. The sugars found in the side
tive heat exchange designs and the avoidance of any
chain vary among bacterial species, whereas the composition of the core design that could potentially mix the wet system with the
polysaccharide is usually the same. There is a molecule of beta- supply air. Biocidal agents such as chlorine or copper can
hydroxymyristic acid (MA), which is a14-carbon fatty acid attached to also be effective when used regularly at low levels.
each N-acetylglucosamine (GlcN) residue. Other fatty acids (FA) are
attached to these residues as well.

5.7.2 Hospitals and Laboratories

circulated through the building, the type of lighting used, Hospitals and microbiology laboratories are the two
the ambient temperature in the building, and the relative indoor environments with perhaps the greatest potential
humidity (Information Box 5.5). for the aerosolization of pathogenic microorganisms.
Some pathogens are uniquely adapted for survival and Hospitals, because they are centers for the treatment of
transmission in the intramural environment. One good patients with diseases, have a high percentage of indivi-
example of such an organism is Legionella pneumophilia, duals, including patients and staff, who are active carriers
the causative agent of both Legionnaires’ disease and of infectious, airborne pathogens. Of particular concern
Pontiac fever. Legionnaires’ disease or legionellosis is a are neonatal wards, surgical transplant wards and surgical
pneumonia that causes disease in up to 5% of those theaters, all critical areas where the control of nosocomial
exposed. Of those who contract the disease, up to 39% die infection is imperative. Illustrating this point is a study by
from the infection. Pontiac fever is associated with flu-like Portner et al. (1965) that evaluated airborne microbial
symptoms and affects up to 100% of those exposed, concentrations in surgical theaters, industrial clean rooms,
although it is generally not associated with mortality. The typical industrial manufacturing areas and a horizontal
causative agent of both diseases is a poorly staining, Gram- laminar flow clean room designed for the space industry.
negative bacillus called L. pneumophila. This organism is The surgical theater had by far the highest counts of path-
named in association with the first highly characterized ogenic airborne microbial contaminants, followed by the
outbreak of the disease, which occurred in 1976 at an industrial manufacturing area, the industrial clean room
American Legion convention in Philadelphia. and finally the laminar flow room, which had the lowest
Legionella spp. are ubiquitous in the environment. counts of airborne microbes.
They are found in association with lakes, ponds, compost Because microbiology laboratories often handle patho-
and streams, and have even been found in deep terrestrial gens, procedures have been developed and refined to pro-
subsurface environments. In addition to natural reservoirs, tect laboratory workers. However, even under the strictest
there are many human-made systems within which of conditions, aerosolization events may occur. In 1988, for
104 PART | II Microbial Environments

TABLE 5.2 Aerosolized Endotoxin Concentrations Detected Downwind of Biosolids Operations, a Wastewater
Treatment Plant Aeration Basin, and a Tractor Operation

Aerosolized Endotoxin (EU ma)

Sample Type # of Samples Distance From Avg Median Minimum Maximum
Collected Site (m)
Controls
Background 12 NA 2.6 2.49 2.33 3.84
Biosolids operations
Loading 39 250 343.7 91.5 5.6 1807.6
Slinging 24 10200 33.5 6.3 4.9 14.29
Biosolids pile 6 2 103 85.4 48.9 207.1
Total operation 33 10200 133.9 55.6 5.6 623.6
Wastewater treatment plant
Aeration basin 6 2 627.3 639 294.4 891.1
Nonbiosolids field
Tractor 6 2 469.8 490.9 284.4 659.1

EU m 5 Endotoxin units per m3.

a

5.8 BIOAEROSOL CONTROL

Information Box 5.5 Molds in Buildings
The control of airborne microorganisms can be handled in a
In moist environments within buildings mold and bacteria can variety of ways. Launching, transport and deposition are all
proliferate rapidly within days and become established as colo- points at which the airborne spread of pathogens can be
nies on solid surfaces, subsequently releasing toxins and/or aller- controlled. The mechanisms used to control bioaerosols
gens into the air. The most common indoor molds are
include ventilation, filtration, UV treatment, biocidal agents
Cladosporium, Penicillium, Aspergillus and Alternaria. Molds can
and physical isolation. These are discussed in the following
cause both allergic reactions and chemical toxigenic responses
from direct exposure to spores, cell wall components and myco- sections.
toxins. Molds and endotoxins can also be found within tobacco
smoke (Pauly and Paszkiewicz, 2011).
5.8.1 Ventilation
Ventilation is the method most commonly used to prevent
the accumulation of airborne particles. This mechanism
involves creating a flow of air through areas where air-
instance, eight employees in a clinical microbiological lab- borne contamination occurs. This can mean simply open-
oratory developed acute brucellosis (Staszkiewicz et al., ing a window and allowing outside air to circulate
1991). A survey of the laboratory and the personnel inward, or use of air-conditioning and heating units that
showed that a cryogenically stored clinical isolate of pump outside air into a room. Ventilation is considered
Brucella sp. had been thawed and subcultured without the one of the least effective methods for controlling airborne
use of a biosafety cabinet. Other than this, the laboratory pathogens, but is still very important. Ventilation relies
worker claimed to have used good technique. This example on mixing of intramural air with extramural air to reduce
demonstrates the ease with which a bioaerosol can spread the concentration of airborne particles. However, in some
within areas where pathogens are handled for research and cases the addition of extramural air can actually increase
clinical purposes, and indicates the importance of bioaero- airborne particles. For example, one study showed that
sol control methodologies. The following sections describe hospitals in Delhi, India, that relied on ventilation alone
how bioaerosol formation and spread is actually controlled contained airborne fungal loads that were higher inside
in the laboratory. the hospital than those outside. This indicates that
Chapter | 5 Aeromicrobiology 105

ventilation alone may not be sufficient to significantly air. The guinea pigs in the untreated-air compartment
reduce circulating bioaerosols. Thus, for most public developed TB, but none of the animals in the UV-treated
buildings, especially hospitals, other forms of bioaerosol compartment became infected. The American Hospital
control need to be implemented. Association (1974) indicated that, properly utilized, UV
radiation can kill nearly all infectious agents, although the
effect is highly dependent on the UV intensity and expo-
5.8.2 Filtration sure time. Thus, major factors that affect survival (tem-
perature, relative humidity, UV radiation, ozone) in the
Unidirectional airflow filtration is a relatively simple and extramural environment can be used to control the spread
yet effective method for control of airborne contamination. of contagion in the intramural environment.
Some filters, for example, high-efficiency particulate air
(HEPA) filters are reported to remove virtually all infec-
tious particles. These types of filters are commonly used in 5.8.4 Isolation
biological safety hoods. However, because of their high
cost, they are not often used in building filtration systems. Isolation is the enclosure of an environment through the
Instead, other filtration systems that rely on baghouse filtra- use of positive or negative pressurized air gradients and air-
tion (a baghouse works on the same principle as a vacuum tight seals. Negative pressure exists when cumulative air-
cleaner bag) are used. Typically, air filters (baghouse, flow travels into the isolated region. Examples of this are
HEPA, etc.) are rated using the dust-spot percentage, which the isolation chambers of the tuberculosis wards in hospi-
is an index of the size of the particles efficiently removed tals used to protect others outside the TB wards from the
by the filter, with higher percentages representing greater infectious agent generated within these negative-pressure
filtration efficiencies. The typical rating for the filters used areas. This type of system is designed to protect other peo-
in most buildings is 30 to 50%. Studies have shown that a ple in the hospital from the pathogens (Mycobacterium
97% dust-spot rating is required to effectively remove virus tuberculosis) present inside the isolation area. Air from
particles from the air. Other factors that influence filtration these rooms is exhausted into the atmosphere after passing
efficiency are related to the type of circulation system and through a HEPA filter and biocidal control chamber.
how well it mobilizes air within the building, the type of Positive-pressure isolation chambers work on the
baghouse system used and the filter material chosen (nylon opposite principle by forcing air out of the room, thus pro-
wound, spun fiberglass, etc.) as well as the filter’s nominal tecting the occupants of the room from outside contamina-
porosity (1 µm5 µm). All these factors combine to influ- tion. One can reason that the TB ward is a negative-
ence the efficiency of the air filtration and removal of par- pressure isolation room, while the rest of the hospital, or at
ticles including bioaerosols. In spite of the high level of least the nearby anterooms, are under positive-pressure
efficiency that can be achieved with filtration, many sys- isolation. Other examples are the hospitals critical care
tems still cannot stop the circulation of airborne microor- wards for immunosuppressed patients such as organ trans-
ganisms, especially viruses, and added treatments may be plant, human immunodeficiency virus (HIV)-infected and
required to ensure that air is safe to breathe. chemotherapy patients. These areas are protected from
exposure to any type of pathogen or opportunistic patho-
gens. The air circulating into these critical care wards is
5.8.3 Biocidal Control filtered using HEPA filters, generating purified air essen-
tially free of infectious agents.
Biocidal control represents an added treatment that can be
used to eradicate all airborne microorganisms, ensuring
they are no longer viable and capable of causing infection. 5.9 BIOSAFETY IN THE LABORATORY
Many eradication methods are available, for example,
superheating, superdehydration, ozonation and UV irradi- Many microbiological laboratories work specifically with
ation. The most commonly used of these methods is pathogenic microorganisms, some of which are highly
UVGI or ultraviolet germicidal radiation. UVGI has been dangerous, especially in association with the aeromicro-
shown to be able to control many types of pathogens, biology pathway. Also, many types of equipment, such as
although some microbes show various levels of resistance. centrifuges and vortexes (instruments for mixing of
The control of contagion using UV irradiation was tested microbial suspensions) that are commonly used in micro-
in a tuberculosis (TB) ward of a hospital. Contaminated biological laboratories can promote the aerosolization of
air was removed from the TB ward through a split ventila- microorganisms. Thus, laboratories and specialized equip-
tion duct and channeled into two animal holding pens that ment used in these laboratories (e.g., biosafety cabinets)
contained guinea pigs. One pen received air that had been are designed to control the spread of airborne microorgan-
treated with UV irradiation; the other received untreated isms. There are essentially four levels of control designed
106 PART | II Microbial Environments

HEPA filtered air

Room air
Contaminated air
A
B

FIGURE 5.17 Schematic representation of the airflow paths within a

typical Class II biosafety cabinet. Room air is dawn in from the top and
from the front as indicated by the blue arrows. The nonpurified atmo-
spheric air (blue) entering from the top of the cabinet is drawn in by an
air pump (A) and then is purified by a HEPA filter (B) as it enters the
workspace. Nonpurified air from the room (blue) entering from the front
of the cabinet passes into the front grill and passes up through the top of
the cabinet where it also passes through a HEPA filter before entering
FIGURE 5.16 Biosafety cabinet Class II. From Telstar Life Science the workspace (C). This prevents the experiments in the workspace from
Solutions, photo courtesy J. Bliznick. being contaminated by airborne room contaminants. As the purified
room air is exposed to the work environment and becomes contaminated
(red) it is passed through yet another HEPA filter before being exhausted
to the atmosphere. This pattern of airflow and purification ensures that
into laboratories, depending on the type of research being the worker and the atmosphere are not exposed to the biohazards con-
conducted. These levels of control are termed biosafety tained within the biosafety cabinet.
levels 14, with 1 being the lowest level of control and 4
the highest level of control. Within these laboratories,
biosafety cabinets are essentially isolation chambers that negative-pressure airflow that provides protection from
provide safe environments for the manipulation of patho- infectious bioaerosols generated within the cabinet
genic microorganisms. In this section we will discuss bio- (Figures 5.16 and 5.17), and Class III biosafety cabinets
safety cabinets and biosafety suits, followed by a short are characterized by total containment (Figure 5.18). Class
discussion of the actual biosafety levels imposed to I cabinets are also in existence, but they are no longer pro-
achieve specific levels of control. duced and are being replaced by Class II cabinets for all
applications.
Class II biosafety cabinets, of which there are several
5.9.1 Biological Safety Cabinets types, are suitable for most work with moderate-risk
pathogens (Table 5.3). Class II biosafety cabinets operate
Biological safety cabinets (BSC) are among the most by drawing airflow past the worker and down through the
effective and commonly used biological containment front grill. This air is then passed upward through con-
devices in laboratories that work with infectious agents duits and downward to the work area after passing
(US Department of Health and Human Services: CDC- through a HEPA filter. Room air is also drawn into the
NIH, 1993). There are two basic types of biosafety cabi- cabinet through the top of the unit, where it joins the cir-
nets currently available (Class II and Class III), each of culating air and passes through the HEPA filter and into
which has specific characteristics and applications that dic- the work area. About 70% of the air circulating in the
tate the type of microorganism it is equipped to contain. work area is then removed by passing it through the rear
Properly maintained biosafety cabinets provide safe envir- grill of the cabinet, where it is discharged into the exhaust
onments for working with microorganisms. Class II bio- system. The remaining 30% is passed through the front
safety cabinets are characterized by having considerable grill, essentially recirculating in the cabinet (Figure 5.17).
Chapter | 5 Aeromicrobiology 107

Room air
Contaminated air TABLE 5.3 Examples of Classification of Biological
HEPA filtered air Agents According to Risk
B
Class Type of Agent Agent
Class I Bacterial All those which have been assessed
C Fungal for risk and do not belong in higher
Protozoal classes
Viral Influenza virus reference strains
Newcastle virus
Class II Bacterial Campylobacter spp.
Clostridium spp.
E. coli spp.
A D Klebsiella spp.
Mycobacteria spp.
Front view Side view Shigella spp.
Vibrio spp.
Salmonella spp.
Fungal Penicillium spp.
FIGURE 5.18 Schematic representation of a Class III biological safety Cryptococcus spp.
cabinet. This cabinet is completely sealed from the environment. Any Microsporum spp.
materials entering or leaving the cabinet are passed through a chemical Protozoal Cryptosporidium spp.
dunk tank or autoclave (A) in order to sterilize them and prevent envi- Giardia spp.
ronmental contamination. Air entering or leaving these cabinets is passed Encephalitozoon spp.
through HEPA filters (B). Access to the workspace is by means of rubber Enterocytozoon spp.
gloves (D) and the workspace is visualized through a sealed window (C). Babesia spp.
These biosafety cabinets are utilized when working with highly patho- Echinococcus spp.
genic microorganisms to protect workers and the environment. Class III Entamoeba spp.
cabinets can be used to work with all biohazardous agents except those Fasciola spp.
specifically designated for biosafety level 4 containment. Leishmania spp.
Plasmodium spp.
Schistosoma spp.
Trypanosoma spp.
Laboratory personnel require special training in order to Viral Adenoviruses
properly use Class II cabinets and to ensure proper con- Corona viruses
Cowpox virus
tainment of bioaerosols. One of the major hazards associ- Coxsackie A and B viruses
ated with Class II cabinets is the potential for the Echoviruses
disruption of the negative airflow. Many mechanical Hepatitis viruses A, B, C, D and E
actions can disrupt the protective airflow, such as EpsteinBarr virus
repeated insertion and withdrawal of arms, opening or Influenza viruses
Vaccinia virus
closing of doors in the laboratory, or even someone walk- Rhinoviruses
ing past the cabinet while it is in use. Any of these
actions can potentially allow the escape of bioaerosols Class III Bacterial Brucella spp.
Mycobacterium bovis
from the cabinet. Mycobacterium tuberculosis
The Class III biosafety cabinet (Figure 5.18) is a Rickettsia spp.
completely enclosed environment that offers the highest Yersinia pestis
degree of personnel and environmental protection from Fungal Coccidioides immitis
bioaerosols. Class III cabinets are used for high-risk Histoplasma capsulatum
Protozoal None
pathogens (Table 5.3). All operations in the work area of Viral Dengue virus
the cabinet are performed through attached rubber gloves. Monkey pox virus
Class III cabinets use complete isolation to protect work- Yellow fever virus
ers. All air entering the cabinet is filtered using a HEPA Bacterial None
filter, and the air leaving the cabinet is filtered by two Class IV Fungal None
HEPA filters in series. The exhaust may also include bio- Protozoal None
cidal treatment such as incineration following the HEPA Viral Hemorrhagic fever agents
Ebola fever virus
filtration to further ensure complete biological inactiva-
Marburg virus
tion. In addition to these safeguards, Class III cabinets are
connected with airtight seals to all other laboratory equip- Adapted from University of Pennsylvania Biological Safety Manual.
ment (such as incubators, refrigerators and centrifuges)
108 PART | II Microbial Environments

that is needed for working with the pathogens while using 5.9.2 Biosafety Laboratories
the cabinet. The Class III cabinet must also be connected
to autoclaves and chemical dunk tanks used to sterilize or Biosafety laboratories are carefully designed environments
disinfect all materials entering or exiting the cabinet. where infectious or potentially infectious agents are han-
Another type of containment that typically provides the dled and/or contained for research or educational pur-
same level of protection as a Class III biosafety hood is the poses. The purpose of a biosafety laboratory is to prevent
biological safety suit (Figure 5.19). The biological suit, the exposure of workers and the surrounding environment
unlike biosafety cabinets, operates under positive pressure to biohazards. There are four levels of biohazard control,
created by an external air supply, thus protecting the which are designated as biosafety levels 1 through 4.
wearer. Like the biosafety cabinets, the biosafety suit iso- Biosafety level 1, as defined by the Centers for
lates the laboratory worker wearing it from bioaerosols. Disease Control (US Department of Health and Human
Biosafety suits are typically used in airtight complete bio- Services: CDC-NIH, 1993), indicates laboratories where
containment areas, and are decontaminated by means of well-characterized agents that are not associated with dis-
chemical showers upon exiting the biohazard area. Some ease in healthy adult humans are handled. In general, no
biosafety suits are portable and can be used in environ- safety equipment is used other than sinks for hand wash-
ments outside the laboratory such as “hot zones” (epidemi- ing, and only general restrictions are placed on public
ological areas that are currently under the influence of access to these laboratories. Work with the microorgan-
epidemic cases of diseases caused by high-risk pathogens) isms can be done on bench tops using standard microbio-
so that microbiologists and physicians working in these logical techniques. A good example of a biosafety 1
areas can minimize their risk of exposure to pathogens. As laboratory is a teaching laboratory used for undergraduate
in biosafety cabinets, the air entering and leaving the bio- microbiology classes.
safety suit passes through two HEPA filters. Biosafety 2 indicates an area where work is performed
using agents that are of moderate hazard to humans and
the environment. These laboratories differ from biosafety
1 laboratories in that the personnel have specialized train-
ing in the handling of pathogens, and access to the work
areas is limited. Many procedures that may cause aerosol-
ization of pathogenic microorganisms are conducted in
biological safety level II cabinets or other physical con-
tainment equipment, to protect the laboratory workers.
Biosafety 3 indicates laboratories where agents that can
cause serious or fatal disease as a result of AMB exposure
are handled. As with biosafety 2, all personnel are specifi-
cally trained to handle pathogenic microorganisms. All pro-
cedures involving these infectious agents are conducted in
biological safety level II cabinets or other physical contain-
ment devices. These facilities also have permanent locks to
control access, negative airflow and filtered ventilation in
order to protect the public and the surrounding environ-
ments. With certain pathogens used in biosafety 3 laborato-
ries, Class III safety hoods may also be used, and clothes
must be changed before leaving the premises.
Biosafety 4 is the highest level of control and is indi-
cated for organisms that have high potential for life-
threatening disease in association with aerosolization. To
work in these facilities, personnel must have specialized
training beyond that required for biosafety levels 2 and 3.
Biosafety level 4 laboratories are 100% isolated from
other areas of a building and may even be separated from
other buildings altogether. Work in these areas is con-
fined exclusively to Class III biological safety cabinets
unless one-piece positive-pressure ventilation suits are
worn, in which case Class II biosafety cabinets may be
used. These laboratories are also specially designed to
FIGURE 5.19 Biosafety suit. Source: Centers for Disease Control. prevent microorganisms from being disseminated into the
Chapter | 5 Aeromicrobiology 109

environment. The laboratories have complete contain- l duration of exposure 5 8 hours

ment, and require personnel to wear specialized clothing, l infectivity constant r 5 0.0253
which is removed and sterilized before leaving the con- l breathing rate 5 0.83 m3 per hour
tainment areas. Personnel are also required to shower
before leaving the facility. In general, all air into and out
of these laboratories is sterilized by filtration and germi-
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