Microsatellite genotyping of
environmental Aspergillus
fumigatus isolates
Nicole Abel
The University of
Tulsa
Introduction
• J&M FARMS
– Miami, OK (Ottawa Co.)
– Large outdoor compost
facility
– Commercial mushroom
farm
– Complaints about smell
and perceived health
effects
Concerning Statistics
• 13.4% of Ottawa County residents have
asthma
• Average asthma rate for rest of OK is
7.2%
Compost & Bioaerosols
• Several microorganisms break down the organic
matter in compost to create a usable product
• During composting process, compost is churned
mechanically
• Mechanical agitation can cause these biological
agents to become aerosolized
– “bioaerosol”
• Increasing concern about the potential impact of
composting on human health
Aspergillus fumigatus
• Fungus of the genus Aspergillus
• Dominant bioaerosol from compost
• Most common species in this genus to
cause disease in individuals with a
compromised immune system
Aspergillus fumigatus
Aspergillus
fumigatus
Aspergillus fumigatus
• Bioaerosol health concerns are focused on
Aspergillus fumigatus for several reasons:
– Grows abundantly in compost
– Can survive at peak compost temperatures
– Can survive temperature of the human body
– Conidia are easily airborne
– When inhaled, small enough to reach lung
– Several well-known health effects
Health effects of AF
• AF is known to cause a wide span of
invasive diseases collectively called
“Aspergillosis”
• Traditionally viewed as weak pathogen,
causing problems with asthma, allergies,
and other diseases:
– “Farmer’s lung”
– Aspergilloma
– Fungal sinusitis
Health effects of AF
– “Farmer’s lung”
• Inflammation of the lungs due to
repeated exposure to the spores
• Symptoms of acute farmer’s lung:
– Chills, Cough, Fever, Shortness of
breath
• Symptoms of chronic farmers lung:
– Breathlessness, Cough, Loss of
appetite, weight loss
• Often reverseable by avoiding additional
exposure
• Chronic form may lead to scarring of
lung tissue
Health effects of AF
– Aspergilloma
• an overgrowth of the fungus on
the surface of preexisting
cavities in the lungs
• This often occurs in patients
treated successfully for
tuberculosis
• Often no symptoms, but most
common symptom is coughing
up blood (hemoptysis)
• Most cases do not require
treatment
• With severe hemoptysis,
surgery may be required to
remove the aspergilloma and
stop the bleeding
Health effects of AF
– Fungal sinusitis
• Inflammation of sinuses
• Patients often have allergies or
asthma
• Thick fungal debris and sticky mucus
that must be surgically removed in
order to keep the inflammatory
condition under control
• At times this massive nasal polyposis
and fungal debris can expand and
erode towards the eyes or brain.
Health effects of AF
• The situation has become much more serious in
recent years
– increase in the number of immunosuppressed
patients
– degree of severity of modern
immunosuppressive therapies,
• Now most common airborne pathogenic fungus
• Causes wide span of severe and usually fatal
invasive infections in the immunocompromised
Aspergillosis
• Extrinsic asthma- most common, occurs in allergic
individuals, causes cough, wheezing, chills, aches, and
pains
• Extrinsic allergic alveolitis- can occur in those without
allergies, but with repeated exposure to AF spores and
many of the spores reach lung tissue, causes coughing,
difficulty in breathing, and fever
•
Allergic bronchiopulminary aspergillosis- more
serious form of “Farmer’s lung”, may follow entrinsic
asthma, but symptoms are chronic and more severe,
treatment is required or this disease can be fatal
Aspergillosis
• Invasive Aspergillosis
–
–
–
–
–
–
Most dangerous and rare
Occurs in the immunocompromised
AF grows from the lung into other organs
Often fatal
Causes 30% of fungal infections in cancer patients
Occurs in 10-25% of patients being treated for leukemia.
Even treated, the mortality rate for these patients is 8090%
– Major cause of death at transplant and leukemia centers
Peoria Tribe/University of Tulsa
Air Quality Studies in
Miami, OK
Phase 1
• Previous study recorded elevated airborne A.
fumigatus within 400 m of compost facility
• A. fumigatus spores were significantly higher
downwind of compost facility than control sites
• Problem: Air samples not done in populated
areas so not pertinant to public health
Present Study
2009-2010
• Objectives:
– Determine AF conc. in facility’s compost
– Determine airborne AF conc. in Ottawa
Co. vs. Tulsa Co.
– Determine genetic relation between
airborne AF in Ottawa Co., AF in the
facilty’s compost, and if distinct from
Tulsa AF controls
Hypothesis
• AF concentrations in facility’s compost will be
high
• Airborne AF concentration will be greater in
Ottawa Co. vs. Tulsa Co.
• AF from compost & Ottawa Co. air will be
genetically identical
• AF from Tulsa air will be genetically different
from AF from Ottawa Co. air and compost
isolates
Compost
Compost Windrows
Miami, OK
Compost
• Purchased 6 truckloads of spent
compost from facility
• 1 gallon bag from each truckload
was brought to our lab at TU
• 10 random samples obtained from
each bag of compost
• Samples dilution plated onto MEA
containing Streptomycin in dilutions
of 10-1, 10-2, and 10-3
• Plated AF concentrations in the
compost ranged from 0-100,000
CFU/g
Compost 1 Sample 2
10-1
10-2
840 CFU/g
10-3
Compost 1 Sample 1
10-1
10-2
100,000 CFU/g
10-3
Average AF Concentrations in Facility's
Compost
Compost Bag #
6
660
5
1632
4
1938
3
154
2
11904
1
13904
0
5000
10000
AF CFU/g
15000
Air Samples
• Ottawa Co.- samples collected from 5 locations within 5
miles of facility (selected by the EPA)
• Tulsa Co.- samples collected from 5 random locations in
Tulsa (144 km upwind from compost site) to serve as
control
• Verified control sites do not use compost from this facility
• Sampling done once a week with
Anderson Single Stage Samplers
onto MEA plus streptomycin plates
for a total of 39 weeks
Ottawa Co. Air Sample Locations
Commerce
Picher
North Miami
Quapaw
Miami
Compost
Facility/
Mushroom farm
Tulsa Air Sample Locations
KendallWhittier
Mohawk
Park
Woodward
Park
University
of Tulsa
campus
Lakewood
Gardens
Average AF for Each Sample Location
20
15
10
5
wo
od
w
lak ard
ew
o
mo od
ha
w
ke k
nd
all
tu
ap
a
pit w
co che
mm r
e
n. r ce
mi
am
mi i
am
i
0
qu
Average AF CFU/m3
25
Sample Location
-------Ottawa Co.-------
-------Tulsa Co.-------
Average Airborne AF each Month
Ottawa Co. vs. Tulsa Co.
40
Average CFU/m 3
35
30
25
20
Ottawa
15
Tulsa
10
5
0
Apr May Jun Jul Aug Sep Oct Nov Dec Jan
Month
Number of times AF collected from air in 39 weeks:
Ottawa Co.- 17 Tulsa Co.-8
Overall Average Airborne AF CFU/m3
Ottawa Co. vs. Tulsa Co.
8
Average CFU/m 3
7
6
5
4
3
2
1
0
Ottawa
Tulsa
P=0.0238
(Statistically Significant)
Mushroom Side Project
• Several samples of the compost had
extremely high levels of AF
• Commercial mushrooms are grown in the
compost
• Could their mushrooms have elevated
AF on their surface when sold in grocery
store?
Mushrooms
• Purchased 7 brands of
mushrooms (including J&M)
• Compost from each brand was
removed from surface of
mushrooms
• Samples diluted to 10-1, 10-2,
and 10-3 onto MEA plates with
Streptomycin and incubated
• AF colonies were identified and
counted
J&M Grocery Mushrooms
AF on Surface of 7 Brands of Grocery Store Mushrooms
18000
16933
16000
12000
10000
8000
8000
6000
4000
2400
2000
613
20
0
40
G
re
en
Gi
an
t
ar
th
M
ot
he
rE
Ph
i ll i
ps
Jo
To
es
h
Fr
Tx
M
on
te
re
y
0
J&
M
AF CFU/g
14000
Mushroom Side project #2
• J&M mushrooms have excessive AF on
their surface
• AF is hydrophobic and becomes airborne
easily
• If these mushrooms are washed in a sink,
will they become airborne in the kitchen?
Mushroom Washing
• J&M mushrooms were purchased and washed
in a sink to mimic home use
• Air samples collected next to the sink using an
Anderson single stage sampler with MEA plates
before, during, and after washing
• This process was repeated with separate
packages of mushrooms for a total of three trials
Airborne AF During Mushroom Washing
70
70
AF (CFU/m 3)
60
50
40
35 35
35
trial 2
30
trial 3
20
10
0
trial 1
0
0
before
0
0
during
0
after
Subcultures
• All plates from air samples, compost, and
mushrooms were incubated at 45° C for
48 hours
• All AF isolates were sub-cultured & grown
in malt extract broth
Microsatellite
• A microsatellite is a specific sequence of
DNA bases which are repeated a certain # of
times. For example:
– GTGTGTGTGTGT---’GT’ X 6
– CTGCTGCTGCTG---’CTG’ X 4
– ACTCACTCACTCACTC---’ACTC’ X 4
• Microsastellites can differ in # of repeats & are
hereditary, so # of repeats can determine
relatedness
• AKA
– simple sequence repeats (SSR)
– short tandem repeats (STR)
– variable number tandem repeats (VNTR)
Microsatellite Applications
• Forensics
– Link a suspect with a sample of blood, semen
or hair taken from a crime
– Link a sample found on a suspect's clothing
with a victim
– Investigating paternity in order to establish
rape or incest
– Linking DNA samples with relatives of a
missing person
Microsatellite Applications
– Diagnosis & identification of human
diseases
• Change in length early in development of
some cancers
• Early cancer detection
• Polymorphic, so useful in locating genes
responsible for various genetic disorders
Microsatellite Applications
– Population studies
• Variation of microsatellites in populations
can help to make inferences about pop.
–Structures
–Differences
–Genetic drift
–Genetic bottlenecks
–Date of a last common ancestor
Microsatellite Applications
• Conservation biology
• Detect sudden change in population,
• Effects of population fragmentation
• Interaction of different populations
• Identification of new and incipient
populations.
DNA extraction
• The DNA was extracted from all
isolates using EZNA kit
• DNA concentrations extracted from
each isolate were documented using
the Nanodrop 8000
Identification and PCR
• Isolates positively identified as AF using:
– Microscopic analysis
– DNA analysis of ITS gene
• DNA extraction
• PCR
• Sequencing
Genotyping
• 9 microsatellites were used to discriminate
between the isolates of AF using the method
of de Valk et al. (2005)
– Genotyped using complete panel of 9
microsatellites (ABI 3130xl Capillary
Sequencer)
– Genotypes analyzed
– Strains and relatedness determined
(GeneMapper v 4.0)
AF STRAINS
Conclusions
Compost
• Aspergillus fumigatus concentration in
J&M’s compost was extremely variable (0100,000 CFU/g)
• Bags #1 & #2 contains extremely high
amounts of AF
• Some J&M compost contains very high
levels of AF and is mechanically agitated, it
is possible this could cause elevated
airborne AF in the surrounding area.
Conclusions
Air Samples
• Total airborne spore concentration significantly
higher in Ottawa Co. vs. Tulsa Co.
• P=0.0238
• The sampling location with highest airborne
conc. Is most often downwind from J&M
Conclusions
Genetic Analysis
• Genetic variation exists amongst the isolates;
however:
– 3 of 3 isolates from mushrooms match compost
– 2 of 2 Isolates from air sampling during
mushroom washing match compost
– 12 of 19 isolates from Ottawa Co. air match
compost
– 30 of 48 isolates from compost match Ottawa
Co. air samples
– Tulsa isolates distinct from all J&M isolates
Acknowledgments
•
•
•
•
•
•
Dr. Estelle Levetin
Dr. Mark Buchheim
Dr. Ron Bonett
Jacob Crowley
Justin Downs and Brandon Bartley
Funding for this study was provided by the
Peoria Tribe of Indians through an
• EPA grant
Thank you!
Questions?