Microbiology (2014), 160, 742–751
DOI 10.1099/mic.0.072520-0
Genetic and virulence variation in an environmental
population of the opportunistic pathogen
Aspergillus fumigatus
Fadwa Alshareef and Geoffrey D. Robson
Correspondence
Geoffrey D. Robson
[email protected]
Received 19 August 2013
Accepted 22 January 2014
Faculty of Life Sciences, Michael Smith Building, University of Manchester,
Manchester M16 8QW, UK
Environmental populations of the opportunistic pathogen Aspergillus fumigatus have been shown
to be genotypically diverse and to contain a range of isolates with varying pathogenic potential. In
this study, we combined two RAPD primers to investigate the genetic diversity of environmental
isolates from Manchester collected monthly over 1 year alongside Dublin environmental isolates
and clinical isolates from patients. RAPD analysis revealed a diverse genotype, but with three
major clinical isolate clusters. When the pathogenicity of clinical and Dublin isolates was
compared with a random selection of Manchester isolates in a Galleria mellonella larvae model, as
a group, clinical isolates were significantly more pathogenic than environmental isolates.
Moreover, when relative pathogenicity of individual isolates was compared, clinical isolates were
the most pathogenic, Dublin isolates were the least pathogenic and Manchester isolates showed
a range in pathogenicity. Overall, this suggests that the environmental population is genetically
diverse, displaying a range in pathogenicity, and that the most pathogenic strains from the
environment are selected during patient infection.
INTRODUCTION
Aspergillus fumigatus is an opportunistic fungal pathogen that can cause aspergillosis in immunocompromised
patients, such as those with neutropenia or on immunosuppressive therapy following solid organ transplantation
(Abad et al., 2010; Ben-Ami et al., 2010; Dagenais & Keller,
2009; Latgé, 1999; Rementeria et al., 2005). The principal
route of infection is through the inhalation of airborne
spores, which due to their small spore size are able to reach
the alveoli (Dagenais & Keller, 2009; Ibrahim-Granet et al.,
2003; Latgé, 1999; O’Gorman, 2011; Philippe et al., 2003).
While other aspergilli can cause opportunistic infections
(such as Aspergillus terreus and Aspergillus flavus), A.
fumigatus accounts for ~90 % of all cases despite airborne
spore numbers only accounting for ,1 % of all Aspergillus
spores (Rementeria et al., 2005). A number of studies
have shown a high degree of genetic variation within the
A. fumigatus population, and whilst some studies have
indicated potential geographical subgroups, comparisons
between clinical isolates and environmental isolates have
largely been unable to distinguish these populations
(Araujo et al., 2010; Aufauvre-Brown et al., 1992; Balajee
et al., 2008; Chazalet et al., 1998; Denning et al., 1990;
Duarte-Escalante et al., 2009; Leenders et al., 1999; Menotti
et al., 2005). Moreover, epidemiological studies have
largely failed to match genotypes in infected patients with
genotypes found in hospitals, in part due to the high level
of genetic diversity in the environmental populations
742
(Araujo et al., 2010; Bart-Delabesse et al., 1999; Chazalet
et al., 1998; Guinea et al., 2011; Leenders et al., 1999; de
Valk et al., 2007), and similar findings have been reported
in avian populations (Arné et al., 2011; Lair-Fulleringer
et al., 2003; Olias et al., 2011). Whilst genotyping of isolates
from some individual patients has revealed infection with
multiple isolates, most patients appear to be colonized by
only one genotype despite exposure to a genetically diverse
population (Alvarez-Perez et al., 2010; Bart-Delabesse et al.,
1999; Chazalet et al., 1998; Guinea et al., 2011; Menotti
et al., 2005; Tang et al., 1994; de Valk et al., 2007). This has
led to the suggestion that the lung environment selects for a
strain or strains from the environment that are the most
adapted and therefore pathogenic (Cimon et al., 2001;
Guinea et al., 2011; de Valk et al., 2007).
Animal models are used widely to determine microbial
pathogenicity, but their use is constrained by high costs
and ethical considerations. As a result, a number of insect
models have been developed as potential alternatives for
studying the pathogenicity of microbes and the efficacy of
antimicrobial agents (Desbois & Coote, 2012; Mak et al.,
2010; Papaioannou et al., 2013; Thomas et al., 2013) as they
contain functional and structural features similar to the
innate immune system of mammals (Arvanitis et al., 2013;
Browne et al., 2013). The greater wax moth larva (Galleria
mellonella), in particular, has been used successfully to
study pathogenicity for a range of microbial pathogens,
including Acinetobacter baumannii, Enterococcus faecium,
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Printed in Great Britain
Variation in an environmental population of A. fumigatus
Escherichia coli, Legionella pneumophila, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus,
Streptococcus pneumoniae, Candida albicans, Candida
tropicalis, Cryptococcus neoformans, A. flavus, A. fumigatus
and Fusarium spp. (Andrejko et al., 2009; Coleman et al.,
2011; Desbois & Coote, 2012; Dunphy et al., 1986; Evans &
Rozen, 2012; Fallon et al., 2011; Fuchs et al., 2010; Harding
et al., 2012; Harrison et al., 2006; Joyce & Gahan, 2010;
Lebreton et al., 2011; Leuko & Raivio, 2012; Mesa-Arango
et al., 2013; Miyata et al., 2003; Mylonakis et al.,
2005; Mukherjee et al., 2010; Peleg et al., 2009; Scully &
Bidochka, 2009; Soukup et al., 2012). Moreover, virulence
in these larvae has in many instances been shown to be
similar to that in mammals, and to involve similar
pathogenicity and virulence determinants (Brennan et al.,
2002; Cook & McArthur, 2013; Dunphy et al., 2003; Fuchs
& Mylonakis, 2006; Jander et al., 2000; Mukherjee et al.,
2010; Wand et al., 2011), including for A. fumigatus (Amich
et al., 2013; Jackson et al., 2009; Li et al., 2011; O’Hanlon
et al., 2011; Slater et al., 2011; Soukup et al., 2012).
broth in 250 ml conical flasks for 48 h at 37 uC in an orbital shaker
(200 r.p.m.).
In this study, we investigated the genetic variation in
airborne environmental A. fumigatus strains isolated over a
period of 12 months from the outdoor environment in
Manchester, Dublin environmental strains isolated from
the outdoor environment at one sample time and clinical
isolates using two RAPD primers. In addition, we also
compared the relative pathogenicity of environmental and
clinical isolates in a Galleria larva model, and report
that clinical isolates were at the most pathogenic end of
the spectrum whilst environmental isolates were found
throughout the spectrum from high to low, suggesting
clinical isolates represent the most pathogenic strains
present in the environment.
DNA extraction. Mycelium was harvested on muslin cloth, washed
METHODS
Strains and maintenance. Clinical isolates were obtained from the
A. fumigatus culture collection, Wythenshaw Hospital, Manchester,
UK. Manchester environmental isolates were collected monthly over a
period of 12 months from the air at the University of Manchester
campus, between March 2009 and February 2010 (Alshareef &
Robson, 2014), and 60 strains were selected randomly for use in this
study. Dublin environmental isolates were kindly supplied by H.
Fuller (University of Dublin, Dublin, Ireland) and were isolated on a
single day from a single site at the Belfield Campus, University College
Dublin, Ireland. Isolates were grown on potato dextrose agar (PDA;
ForMedium) in tissue culture flasks and incubated at 37 uC until
confluent growth was obtained. Spores were harvested by gentle
agitation of the mycelial surface with a sterile loop with 20 ml 0.05 %
(v/v) Tween 20, filtered through four layers of lens tissue (Whatman)
to remove phialides and spore aggregates, and washed two times in
sterile water by centrifugation at 1500 g and resuspending the spores
in 20 ml sterile water. Spores were enumerated using a modified
Neubauer haemocytometer, and viability determined by serial
dilution and c.f.u. determination on PDA plates after 48 h incubation
at 37 uC. All strains had a spore viability .95 %. Spores were kept for
up to 1 week at 4 uC; for long-term storage, spore suspensions were
mixed with an equal volume of sterile glycerol and stored at 280 uC.
For DNA extraction, mycelium was grown in 50 ml potato dextrose
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Virulence determination in Galleria larvae. Galleria larvae (Live
Food Direct) were stored in wood shavings in the dark at 4 uC for
up to 10 days and transferred to room temperature for 2 h before
use. Galleria larvae in groups of 30 were inoculated with 10 ml A.
fumigatus spore suspension into the haemocoel through the last
right or left proleg, using a Hamilton 1 ml gas-tight syringe (Fisher
Scientific) with a 10 ml repeating dispenser attachment (Jaytee
Biosciences). Spore concentrations of 16108 to 16103 ml21
were used to infect larvae, which were incubated at 37 uC in the
dark in Petri dishes (10 larvae per plate) and the mortality
monitored daily for 7 days. Mortality was assessed by lack of
movement in response to stimulation and by discoloration
(melanization) of the cuticle.
The survival of the Galleria larvae was analysed using the Mann–
Whitney U test (Stats Direct) with P,0.05 considered significant, and
differences in the survival rates of larvae inoculated with different
strains were compared. A non-parametric estimate of the mean
survival time for Galleria larvae was obtained as the area under the
Kaplan–Meier estimate of the survival curve.
briefly with sterile water, and ground to a fine powder in liquid
nitrogen using a mortar and pestle. Ground mycelium was added to
50 ml Falcon tubes (on ice) to the 10 ml mark and an equal volume
of pre-heated DNA extraction buffer [0.7 M NaCl, 1 M Na2SO3,
0.1 M Tris/HCl (pH 7.5), 0.05 M EDTA, 1 % (w/v) SDS, 65 uC] was
added and mixed thoroughly with a pipette tip, and incubated
at 65 uC for 30 min to terminate nuclease activity and induce
protein denaturation. A NanoDrop 1000 spectrophotometer
(Thermo Scientific) was used to measure DNA concentrations in
samples.
RAPD. In total, 106 isolates were analysed using RAPD. The primers
used for RAPD were R108 (59-AGTGCACACC-39), UBC90 (59-GGGGGTTAGG-39), R151 (59-GCTGTAGTGT-39) and RC08 (59-AGGATGTCGAA-39) (Eurofins) as these have been used to successfully
fingerprint A. fumigatus populations in the past (O’Gorman et al.,
2009). The samples were run on 2.5 % (w/v) agarose gels in 40 mM
Tris/acetate EDTA buffer (pH 8.0). Only primers R108 and C08 were
used to compare all isolates, and RAPD bands included in the analysis
were scored as either absent (0) or present (1). The data from all
primers were pooled for each isolate and a pair’s similarity matrix was
calculated with Jaccard’s coefficient with the program FREETREE v.
0.9.1.50. A bootstrapped dendrogram with 1000 resamplings was
produced with NJPlot.
RESULTS
RAPD fingerprinting revealed genetic diversity in
the environmental population and clustering of
clinical isolates
Four primers were used initially for RAPD fingerprinting
(R108, RC08, R151 and UBC90) with seven A. fumigatus
isolates, and fingerprinting was repeated at least two times
to evaluate reproducibility. The four primers produced a
range of RAPD bands, from 0.4 to 6 kb. All four primers
gave several strong bands that were reproducible, along
with many fainter bands (data not shown). Of the four
primers tested, R108 and RC08 gave the most reproducible
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F. Alshareef and G. D. Robson
44
6
24
13
44
11
30
43
2
44
6
100
11
35
50
6
5
7
24
9
22
42
1
5
21
3
24
21
43
4
6
22
2
29
2
32
62
4
7
1
15
6
20
27
16
16
5
22
35
1
4
25
34
27
3
52
5
12
3
29
9
4
3
4
0.1
3
14
2
18
9
744
28
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Dec5
Dec9
Mar32
May13
Jan12
RB17
Aug14
Aug1
Aug13
22020
Jun11
22773
21932
22216
21636
22769
22577
22178
22636
22236
22821
21407
21522
Sep5
Sep6
Jan11
Jan9
Nov1
Dec11
Dec17
Dec3
Dec19
Dec26
AF1163
Dec20
Apr15
Dec4
Sep10
Sep1
Sep3
RB13
RB16
AF293
RB20
RB14
RB11
RB12
RB18
RB19
Nov2
RB15
17318
Oct9
Oct6
17768
Oct3
Jan7
Nov3
Aug2
17796
Mar15
Aug4
Aug3
Oct4
Jul9
Mar16
Jul2
Jun6
May1
May9
Jun9
Sep9
15991
17406
15562
17835
16975
Jun4
Jan1
Oct7
Oct8
Jun5
Mar2
Apr12
Mar7
Apr13
Apr7
Jul1
Mar12
Mar1
Mar17
17871
16258
17796
15819
Mar20
Mar13
Microbiology 160
Variation in an environmental population of A. fumigatus
Fig. 1. Dendrogram analysis of RAPD fingerprinting of A. fumigatus isolates (March 2009–February 2010) using combined
RAPD primers R108 and RC08. The strains are Manchester airborne isolates (month of isolation+code number) (indicated by
the absence of grey/black boxes on the right), clinical isolates (code number) (indicated by black boxes on the right), Dublin
airborne isolates (RB+code number) (indicated by grey boxes on the right), and sequenced strains AF1163 and AF293
(indicated by circles on the right). Bar, 0.1 (10 % genetic difference). Values at nodes represent the percentage bootstrap
support (based on 1000 resamplings).
results and had the greatest discriminatory power (0.992
and 0.868, respectively) (Bikandi et al., 2004) in a pilot
study, and were selected for fingerprinting the remaining
isolates. Only the strongest and reproducible bands were
included in the analysis, and fainter and variable bands
were excluded. To increase discrimination, RAPD data
from both primers were pooled prior to analysis. As the
number of isolates exceeded the maximum number of wells
on a gel, strain AF293 was run on every gel as an internal
control.
RAPD analysis of the Manchester environmental isolates
showed a high genetic diversity in the population (Fig. 1).
With few exceptions, isolates collected at the same time
points could be distinguished from each other, indicating
multiple sources of airborne spores. In addition, comparison of Manchester isolates collected monthly for 12
months indicated that the source of airborne spores varied
at each time point. When environmental isolates were
analysed along with clinical isolates, clinical isolates
displayed a degree of clustering with 21 of the 25 clinical
isolates forming three clusters accounting for 12, 5 and 4
isolates, although, with the exception of isolates 22178 and
22636 and isolates 17871 and 16258, they could be
distinguished from each other.
Virulence testing in Galleria larvae demonstrated
a broad range in pathogenicity in the
environmental population
The virulence of ten randomly selected Manchester
environmental isolates, ten Dublin environmental isolates,
ten clinical isolates, and sequenced strains AF293 and
AF1163 was determined in Galleria larvae inoculated with
16103 to 16106 spores per larva by monitoring survival
over 7 days. Initial spore inoculum size had a large effect on
Galleria larvae survival in all strains, with all strains
showing the greatest survival after 7 days with 16103
spores and lowest survival with 16106 spores (data not
shown). The greatest discrimination in survival between
the different strains was seen at an inoculum level of
16105 spores per larva (Fig. 2). In order to compare the
relative pathogenicity of the Manchester, Dublin and
sequenced strains, survival over 7 days for each spore
inoculum for each group was combined and subjected to
Mann–Whitney and Kaplan–Meier analyses (Fig. 3). The
clinical isolates were significantly more pathogenic than the
Dublin and Manchester isolates (P,0.0001) at the four
different doses. Moreover, the Manchester isolates were
significantly more pathogenic than the Dublin isolates (106
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and 105, P,0.0001; 104, P50.0135; 103, P50.0043). This
was also apparent when the survival of each group 4 days
after inoculation was compared at initial spore concentrations from 16103 to 16106 spores per larva, confirming
that the greater pathogenicity of the clinical isolates was
across the spectrum of spore inoculum concentrations (Fig.
4). When the percentage survival values after 4 days for
each individual strain at an inoculum of 16105 spores per
larva were compared, the clinical isolates clustered together
as the most pathogenic, the Dublin isolates the least
pathogenic and the Manchester isolates showed a broad
spectrum of pathogenicity (Fig. 5).
DISCUSSION
A variety of molecular typing methods have been
developed over the past 20 years to investigate genetic
diversity in the population of a wide variety of fungal
species. Many of these techniques have also been used to
study genetic diversity in the A. fumigatus population. The
methods include RAPD (Anderson et al., 1996; AufauvreBrown et al., 1992; Mondon et al., 1995; O’Gorman et al.,
2009), RFLP (Spreadbury et al., 1990), restriction enzyme
analysis (Denning et al., 1990), amplified fragment length
polymorphism (Vos et al., 1995) and sequence-specific
DNA primer analysis (Mondon et al., 1997), and more
recently microsatellite length polymorphism (Balajee et al.,
2008; Bart-Delabesse et al., 1998; de Valk et al., 2007),
MLST (Bain et al., 2007) and variable number of short
tandem repeat typing (Vanhee et al., 2009). These methods
have also demonstrated that there is a large genetic
diversity within the A. fumigatus population (Balajee
et al., 2008; Chazalet et al., 1998; Leenders et al., 1999; de
Valk et al., 2009; Vanhee et al., 2009; O’Gorman et al.,
2009) and have largely been used to investigate the
epidemiology of isolates following clinical outbreaks of
disease in hospitals (Alvarez-Perez et al., 2010; BartDelabesse et al., 1999; Vanhee et al., 2010; Guinea et al.,
2011), as well as to study genotypic diversity of strains in
avian disease (Arné et al., 2011; Lair-Fulleringer et al., 2003;
Olias et al., 2011). The recent discovery of a functional
sexual cycle in A. fumigatus probably accounts for the
genetic diversity observed in the population (O’Gorman
et al., 2009), and much of this diversity appears to be
concentrated in discrete genomic islands within the DNA
that encode for proteins involved in heterokaryon incompatibility and cell-wall-associated proteins that may be
involved in virulence (Fedorova et al., 2008). In addition,
whilst the macromorphology of A. fumigatus strains
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F. Alshareef and G. D. Robson
Mean survival time (days)
(a) 100
90
80
70
60
50
40
30
20
8
7
6
5
4
3
2
1
0
103
10
104
105
106
Spore inoculum concentration (c.f.u. per larva)
0
(b) 100
Fig. 3. Mann–Whitney analysis of the mean survival of G.
mellonella inoculated with Manchester environmental (black bars),
Dublin environmental (white bars) and clinical (grey bars) strains of
A. fumigatus at spore concentrations of 1¾103, 1¾104, 1¾105 and
1¾106 spores per larva after 7 days. Controls were inoculated with
sterile 0.05 % (v/v) Tween 20 and retained 100 % viability over 7
days. Error bars represent SEM.
90
Survival (%)
80
70
60
50
40
30
20
10
appears to be largely similar within the population, the
micromorphology (such as conidia and vesicle size and
shape), pathogenicity and enzyme secretion have been
found to be variable between isolates (Alp & Arikan,
2008; Aufauvre-Brown et al., 1998; Ben-Ami et al., 2010;
Birch et al., 2004; Chamilos et al., 2007, 2010; Mondon
et al., 1995, 1996; Olias et al., 2011; Rinyu et al., 1995).
Limited studies on enzyme secretion have demonstrated a
range of expression levels and isoenzymes from different
isolates (Alp & Arikan, 2008; Birch et al., 2004; Blanco
et al., 2002; Lin et al., 1995; Rinyu et al., 1995; Rodriguez
et al., 1996).
0
(c) 100
90
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
Time since inoculation (days)
100
90
746
80
70
Survival (%)
Fig. 2. Percentage survival of wax moth larvae inoculated with
various strains of A. fumigatus over 7 days. Thirty Galleria larvae
were inoculated with isolates of A. fumigatus at 1¾105 c.f.u. per
larva and incubated at 37 6C for 7 days. The number of surviving
Galleria larvae was determined daily. Controls were inoculated
with sterile 0.05 % (v/v) Tween 20 (¾). (a) Manchester environmental strains: Mar13 (D), Mar15 (e), Mar20 ($), Mar32 (&),
Apr12 (m), Apr15 (¤), May1 (grey circle), Jun4 (grey square),
Jun5 (grey triangle) and Jul1 (grey diamond). (b) Dublin
environmental strains: RB11 (D), RB12 (e), RB13 ($), RB14
(&), RB15 (m), RB16 (¤), RB17 (grey circle), RB18 (grey
square), RB19 (grey triangle) and RB20 (grey diamond). (c)
Clinical strains 17871 (D), 15562 (e), 177796 ($), 16258 (&),
16795 (m), 15819 (¤), 17406 (grey circle), 17768 (grey square),
17882 (grey triangle) and 16916 (grey diamond). Sequenced
strains AF293 (#) and AF1163 (h) were included as internal
controls.
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
Time since inoculation (days)
Fig. 4. Kaplan–Meier approximation of the mean survival time of G.
mellonella inoculated with A. fumigatus clinical (&), Manchester
environmental (h), Dublin environmental ($) and AF293 and
AF1163 (#) strains over 7 days following inoculation with
1¾105 c.f.u. per larva. Controls were inoculated with sterile
0.05 % (v/v) Tween 20 (¾).
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Microbiology 160
Variation in an environmental population of A. fumigatus
100
90
80
Survival (%)
70
60
50
40
30
20
10
17768
15562
17882
16795
17406
16916
Mar1
17871
Apr15
15819
Mar20
AF1163
Jun5
Jul1
16258
Mar13
AF293
17796
RB14
RB18
RB20
RB16
RB19
Apr12
RB12
RB13
RB15
Mar15
Mar32
RB17
Jun4
RB11
0
Strain
Fig. 5. Percentage survival of Galleria larvae 4 days after inoculation with 1¾105 spores per larva of various strains of A.
fumigatus: clinical (black bars), Manchester environmental (white bars) and Dublin environmental (grey bars). Controls were
inoculated with sterile 0.05 % (v/v) Tween 20.
In our study, RAPD typing also demonstrated a large
degree of genetic variability within the airborne population
of A. fumigatus collected monthly over a period of 12
months from the same site in Manchester (Fig. 1).
Moreover, isolates typed from the same time point showed
several genotypes, indicating a mixture of sources contributed to the airborne population at any one time. This
contrasted with the small number of isolates from Dublin,
which were also collected at a single time point from a
single site (H. Fuller, personal communication), that with
one exception showed identical genotypes with both
primers (Fig. 1), indicating that the majority of airborne
spores at the time of sampling may have arisen from a single
source. However, other isolates collected from Dublin at
different time points also showed a range of phenotypes,
indicating variable sources over time (O’Gorman et al.,
2009). That a range of genotypes was found at each monthly
sampling point in this study is not unexpected as A.
fumigatus spores are dispersed readily in the atmosphere
due to their small size and samples were taken in the open
outdoor environment. Other studies that have typed A.
fumigatus spores collected from the environment have
also shown a high degree of genotypic diversity in the
population, including the enclosed environments of hospitals where it might have been expected that the number of
contributing sources would have been smaller (Araujo et al.,
2010; Debeaupuis et al., 1997; Guinea et al., 2011; Menotti
et al., 2005).
Previous studies attempting to identify environmental
sources of patient infection in hospital environments have
generally found only a low number of patient isolate
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genotypes and only a small number could be matched with
an environmental phenotype, probably due to insufficient
environmental samples being collected to capture the high
level of genotype diversity reported (Araujo et al., 2010;
Guinea et al., 2011). In addition, in those studies where
environmental samples were taken at different time periods
or locations within the same building, in only a few
instances was an identical genotype recovered, indicating a
high degree of variability occurs at the same location over
time and at any one time in different locations (Araujo
et al., 2010; Guinea et al., 2011). Many aspects of the
pathogenesis of aspergillosis remain to be clarified, including the contribution of the place of acquisition, incubation
period, virulence variation in different Aspergillus isolates
and the possible contribution of more than one clone in
human infections (Alvarez-Perez et al., 2010). Interestingly,
in our study there was some genetic clustering amongst the
clinical isolates where 21 of the 25 isolates clustered into
three groups that also contained some environmental
isolates, suggesting that some traits were detected that were
shared amongst some isolates that may be associated with a
higher pathogenicity (Fig. 1).
Whilst many studies have used mammalian models to
investigate the impact of specific gene function on
pathogenicity (Askew, 2008; Rementeria et al., 2005), there
have been few studies that have compared the pathogenicity of different isolates of A. fumigatus, and those that have
reported a range of pathogenicity between isolates (Jackson
et al., 2009; Lionakis et al., 2005; Reeves et al., 2004).
Invertebrate models of pathogenicity offer a cheaper and
quicker alternative to animal models, enabling large
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F. Alshareef and G. D. Robson
numbers of isolates to be compared (Chamilos et al., 2010;
Kavanagh & Reeves, 2004). In A. fumigatus, these models
have included Galleria larvae and Toll-deficient mutants of
Drosophila (Jackson et al., 2009; Lionakis et al., 2005;
Reeves et al., 2004), and relative pathogenicity has been
correlated with pathogenicity levels in mammalian models
(Slater et al., 2011; Chamilos et al., 2010). In this present
study, a range in pathogenicity against Galleria larvae was
apparent in both clinical isolates and environmental
isolates (Fig. 2). Moreover, as a group, the clinical isolates
were significantly (P,0.005) more pathogenic than the
environmental isolates (Fig. 3) with the exception of the
clinical isolates and the Manchester environmental isolates
at an inoculum level of 16106 spores per larva. However,
the environmental isolates showed an overlap in pathogenicity with the clinical strains (Fig. 5), suggesting that
clinical isolates may represent the most pathogenic of the
environmental isolates and that the infection process in
patients may select for the most pathogenic strains present
in the environment. Selection for the most pathogenic
strains from the environment has been suggested previously as an explanation for why patients are often
infected with only one strain and in those cases where coinfection is present, only two or three genotypes have been
observed despite a highly diverse environmental population (Alvarez-Perez et al., 2010; Debeaupuis et al., 1997;
Menotti et al., 2005; Guinea et al., 2011; de Valk et al.,
2007; Tang et al., 1994). Moreover, in one study, whilst
several genotypes were recovered from the respiratory tract
of a patient, only one was recovered from invasive disease
from solid organs, suggesting further selection for invasion
(de Valk et al., 2007). It is now clear that the pathogenicity
of this opportunistic pathogen is polygenetic rather than
being dependent on any one virulence factor (Askew, 2008;
Rhodes, 2006), with a broad range of factors being
implicated as playing a role in the pathogenicity, including
extracellular hydrolases, nutrient acquisition and metabolism, toxin production, melanin and transcription factors,
and secondary messenger pathways with broad genotypic
and phenotypic effects (Amich et al., 2013; Amich &
Krappmann, 2012; Jackson et al., 2009; Richie et al., 2009;
Kim et al., 2013; Krappmann et al., 2004; Li et al., 2011;
O’Hanlon et al., 2011; Schrettl & Haas, 2011; Soukup et al.,
2012). Differing levels of expression of these factors in the
population may therefore give rise to the range in
pathogenicity observed in the A. fumigatus population,
and the ability to adapt and infect the lung. In addition,
infection also depends on host factors that will vary in
differing individuals (Hartmann et al., 2011; Sales-Campos
et al., 2013). Thus, selection in the lung of A. fumigatus
from a diverse range of genotypes will be multifactorial
dependent on the interplay between A. fumigatus and host
genotype.
REFERENCES
Abad, A., Fernández-Molina, J. V., Bikandi, J., Ramı́rez, A., Margareto,
J., Sendino, J., Hernando, F. L., Pontón, J., Garaizar, J. & Rementeria, A.
(2010). What makes Aspergillus fumigatus a successful pathogen? Genes
and molecules involved in invasive aspergillosis. Rev Iberoam Micol 27,
155–182.
Alp, S. & Arikan, S. (2008). Investigation of extracellular elastase, acid
proteinase and phospholipase activities as putative virulence factors in
clinical isolates of Aspergillus species. J Basic Microbiol 48, 331–337.
Alshareef, F. & Robson, G. D. (2014). Prevalence, persistence and
phenotypic variation of Aspergillus fumigatus in the outdoor environment in Manchester (UK) over a two year period. Med Mycol (in
press).
Alvarez-Perez, S., Blanco, J. L., Alba, P. & Garcia, M. E. (2010).
Mating type and invasiveness are significantly associated in Aspergillus
fumigatus. Med Mycol 48, 273–277.
Amich, J. & Krappmann, S. (2012). Deciphering metabolic traits of
the fungal pathogen Aspergillus fumigatus: redundancy vs. essentiality.
Front Microbiol 3, 414.
Amich, J., Schafferer, L., Haas, H. & Krappmann, S. (2013).
Regulation of sulphur assimilation is essential for virulence and
affects iron homeostasis of the human-pathogenic mould Aspergillus
fumigatus. PLoS Pathog 9, e1003573.
Anderson, M. J., Gull, K. & Denning, D. W. (1996). Molecular typing
by random amplification of polymorphic DNA and M13 southern
hybridization of related paired isolates of Aspergillus fumigatus. J Clin
Microbiol 34, 87–93.
Andrejko, M., Mizerska-Dudka, M. & Jakubowicz, T. (2009).
Antibacterial activity in vivo and in vitro in the hemolymph of
Galleria mellonella infected with Pseudomonas aeruginosa. Comp
Biochem Physiol B Biochem Mol Biol 152, 118–123.
Araujo, R., Amorim, A. & Gusmão, L. (2010). Genetic diversity of
Aspergillus fumigatus in indoor hospital environments. Med Mycol 48,
832–838.
Arné, P., Thierry, S., Wang, D., Deville, M., Le Loc’h, G., Desoutter, A.,
Féménia, F., Nieguitsila, A., Huang, W. & other authors (2011).
Aspergillus fumigatus in poultry. Int J Microbiol 2011, 746356.
Arvanitis, M., Glavis-Bloom, J. & Mylonakis, E. (2013). Invertebrate
models of fungal infection. Biochim Biophys Acta 1832, 1378–1383.
Askew, D. S. (2008). Aspergillus fumigatus: virulence genes in a street-
smart mold. Curr Opin Microbiol 11, 331–337.
Aufauvre-Brown, A., Cohen, J. & Holden, D. W. (1992). Use of
randomly amplified polymorphic DNA markers to distinguish
isolates of Aspergillus fumigatus. J Clin Microbiol 30, 2991–2993.
Aufauvre-Brown, A., Brown, J. S. & Holden, D. W. (1998).
Comparison of virulence between clinical and environmental isolates
of Aspergillus fumigatus. Eur J Clin Microbiol Infect Dis 17, 778–780.
Bain, J. M., Tavanti, A., Davidson, A. D., Jacobsen, M. D., Shaw, D., Gow,
N. A. R. & Odds, F. C. (2007). Multilocus sequence typing of the
pathogenic fungus Aspergillus fumigatus. J Clin Microbiol 45, 1469–1477.
Balajee, S. A., de Valk, H. A., Lasker, B. A., Meis, J. F. & Klaassen, C. H.
(2008). Utility of a microsatellite assay for identifying clonally related
outbreak isolates of Aspergillus fumigatus. J Microbiol Methods 73, 252–
256.
Bart-Delabesse, E., Humbert, J.-F., Delabesse, E. & Bretagne, S.
(1998). Microsatellite markers for typing Aspergillus fumigatus
isolates. J Clin Microbiol 36, 2413–2418.
ACKNOWLEDGEMENTS
Bart-Delabesse, E., Cordonnier, C. & Bretagne, S. (1999). Usefulness
The authors would like to thank the government of Saudi Arabia for a
scholarship to F. A.
748
of genotyping with microsatellite markers to investigate hospitalacquired invasive aspergillosis. J Hosp Infect 42, 321–327.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 14:56:53
Microbiology 160
Variation in an environmental population of A. fumigatus
Ben-Ami, R., Lamaris, G. A., Lewis, R. E. & Kontoyiannis, D. P. (2010).
Desbois, A. P. & Coote, P. J. (2012). Utility of greater wax moth larva
Interstrain variability in the virulence of Aspergillus fumigatus and
Aspergillus terreus in a Toll-deficient Drosophila fly model of invasive
aspergillosis. Med Mycol 48, 310–317.
(Galleria mellonella) for evaluating the toxicity and efficacy of new
antimicrobial agents. Adv Appl Microbiol 78, 25–53.
Bikandi, J., San Millán, R., Rementeria, A. & Garaizar, J. (2004). In
silico analysis of complete bacterial genomes: PCR, AFLP-PCR and
endonuclease restriction. Bioinformatics 20, 798–799.
Birch, M., Denning, D. W. & Robson, G. D. (2004). Comparison of
extracellular phospholipase activities in clinical and environmental
Aspergillus fumigatus isolates. Med Mycol 42, 81–86.
Blanco, J. L., Hontecillas, R., Bouza, E., Blanco, I., Pelaez, T., Muñoz,
P., Perez Molina, J. & Garcia, M. E. (2002). Correlation between the
elastase activity index and invasiveness of clinical isolates of
Aspergillus fumigatus. J Clin Microbiol 40, 1811–1813.
Brennan, M., Thomas, D. Y., Whiteway, M. & Kavanagh, K. (2002).
Correlation between virulence of Candida albicans mutants in mice
and Galleria mellonella larvae. FEMS Immunol Med Microbiol 34, 153–
157.
Browne, N., Heelan, M. & Kavanagh, K. (2013). An analysis of the
structural and functional similarities of insect hemocytes and
mammalian phagocytes. Virulence 4, 597–603.
Chamilos, G., Lionakis, M. S., Lewis, R. E. & Kontoyiannis, D. P.
(2007). Role of mini-host models in the study of medically important
fungi. Lancet Infect Dis 7, 42–55.
Chamilos, G., Bignell, E. M., Schrettl, M., Lewis, R. E., Leventakos, K.,
May, G. S., Haas, H. & Kontoyiannis, D. P. (2010). Exploring the
concordance of Aspergillus fumigatus pathogenicity in mice and Tolldeficient flies. Med Mycol 48, 506–510.
Chazalet, V., Debeaupuis, J. P., Sarfati, J., Lortholary, J., Ribaud, P.,
Shah, P., Cornet, M., Vu Thien, H., Gluckman, E. & other authors
(1998). Molecular typing of environmental and patient isolates of
Aspergillus fumigatus from various hospital settings. J Clin Microbiol
36, 1494–1500.
Cimon, B., Symoens, F., Zouhair, R., Chabasse, D., Nolard, N.,
Defontaine, A. & Bouchara, J. P. (2001). Molecular epidemiology of
Duarte-Escalante, E., Zúñiga, G., Ramı́rez, O. N., Córdoba, S., Refojo,
N., Arenas, R., Delhaes, L. & Reyes-Montes, M. R. (2009). Population
structure and diversity of the pathogenic fungus Aspergillus fumigatus
isolated from different sources and geographic origins. Mem Inst
Oswaldo Cruz 104, 427–433.
Dunphy, G. B., Morton, D. B., Kropinski, A. & Chadwick, J. M. (1986).
Pathogenicity of lipopolysaccharide mutants of Pseudomonas aeruginosa for larvae of Galleria mellonella: bacterial properties associated
with virulence. J Invertebr Pathol 47, 48–55.
Dunphy, G. B., Oberholzer, U., Whiteway, M., Zakarian, R. J. &
Boomer, I. (2003). Virulence of Candida albicans mutants toward
larval Galleria mellonella (Insecta, Lepidoptera, Galleridae). Can
J Microbiol 49, 514–524.
Evans, B. A. & Rozen, D. E. (2012). A Streptococcus pneumoniae
infection model in larvae of the wax moth Galleria mellonella. Eur
J Clin Microbiol Infect Dis 31, 2653–2660.
Fallon, J. P., Troy, N. & Kavanagh, K. (2011). Pre-exposure of Galleria
mellonella larvae to different doses of Aspergillus fumigatus conidia
causes differential activation of cellular and humoral immune
responses. Virulence 2, 413–421.
Fedorova, N. D., Khaldi, N., Joardar, V. S., Maiti, R., Amedeo, P.,
Anderson, M. J., Crabtree, J., Silva, J. C., Badger, J. H. & other
authors (2008). Genomic islands in the pathogenic filamentous
fungus Aspergillus fumigatus. PLoS Genet 4, e1000046.
Fuchs, B. B. & Mylonakis, E. (2006). Using non-mammalian hosts to
study fungal virulence and host defense. Curr Opin Microbiol 9, 346–
351.
Fuchs, B. B., Eby, J., Nobile, C. J., El Khoury, J. B., Mitchell, A. P. &
Mylonakis, E. (2010). Role of filamentation in Galleria mellonella
killing by Candida albicans. Microbes Infect 12, 488–496.
Guinea, J., Garcı́a de Viedma, D., Peláez, T., Escribano, P., Muñoz,
P., Meis, J. F., Klaassen, C. H. & Bouza, E. (2011). Molecular
airway colonisation by Aspergillus fumigatus in cystic fibrosis patients.
J Med Microbiol 50, 367–374.
epidemiology of Aspergillus fumigatus: an in-depth genotypic analysis
of isolates involved in an outbreak of invasive aspergillosis. J Clin
Microbiol 49, 3498–3503.
Coleman, J. J., Muhammed, M., Kasperkovitz, P. V., Vyas, J. M. &
Mylonakis, E. (2011). Fusarium pathogenesis investigated using
Harding, C. R., Schroeder, G. N., Reynolds, S., Kosta, A., Collins, J. W.,
Mousnier, A. & Frankel, G. (2012). Legionella pneumophila patho-
Galleria mellonella as a heterologous host. Fungal Biol 115, 1279–
1289.
genesis in the Galleria mellonella infection model. Infect Immun 80,
2780–2790.
Cook, S. M. & McArthur, J. D. (2013). Developing Galleria mellonella
Harrison, F., Browning, L. E., Vos, M. & Buckling, A. (2006).
as a model host for human pathogens. Virulence 4, 350–353.
Dagenais, T. R. & Keller, N. P. (2009). Pathogenesis of Aspergillus
fumigatus in invasive aspergillosis. Clin Microbiol Rev 22, 447–465.
de Valk, H. A., Meis, J. F., de Pauw, B. E., Donnelly, P. J. & Klaassen,
C. H. (2007). Comparison of two highly discriminatory molecular
Cooperation and virulence in acute Pseudomonas aeruginosa infections. BMC Biol 4, 21.
Hartmann, T., Sasse, C., Schedler, A., Hasenberg, M., Gunzer, M. &
Krappmann, S. (2011). Shaping the fungal adaptome – stress
responses of Aspergillus fumigatus. Int J Med Microbiol 301, 408–
416.
fingerprinting assays for analysis of multiple Aspergillus fumigatus
isolates from patients with invasive aspergillosis. J Clin Microbiol 45,
1415–1419.
Ibrahim-Granet, O., Philippe, B., Boleti, H., Boisvieux-Ulrich, E.,
Grenet, D., Stern, M. & Latgé, J. P. (2003). Phagocytosis and
de Valk, H. A., Klaassen, C. H., Yntema, J. B., Hebestreit, A., Seidler,
M., Haase, G., Müller, F. M. & Meis, J. F. (2009). Molecular typing and
intracellular fate of Aspergillus fumigatus conidia in alveolar macrophages. Infect Immun 71, 891–903.
colonization patterns of Aspergillus fumigatus in patients with cystic
fibrosis. J Cyst Fibros 8, 110–114.
Jackson, J. C., Higgins, L. A. & Lin, X. (2009). Conidiation color
Debeaupuis, J. P., Sarfati, J., Chazalet, V. & Latgé, J. P. (1997).
Genetic diversity among clinical and environmental isolates of
Aspergillus fumigatus. Infect Immun 65, 3080–3085.
mutants of Aspergillus fumigatus are highly pathogenic to the
heterologous insect host Galleria mellonella. PLoS ONE 4, e4224.
Jander, G., Rahme, L. G. & Ausubel, F. M. (2000). Positive correlation
between virulence of Pseudomonas aeruginosa mutants in mice and
insects. J Bacteriol 182, 3843–3845.
Denning, D. W., Clemons, K. V., Hanson, L. H. & Stevens, D. A.
(1990). Restriction endonuclease analysis of total cellular DNA of
Joyce, S. A. & Gahan, C. G. (2010). Molecular pathogenesis of Listeria
Aspergillus fumigatus isolates of geographically and epidemiologically
diverse origin. J Infect Dis 162, 1151–1158.
monocytogenes in the alternative model host Galleria mellonella.
Microbiology 156, 3456–3468.
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 14:56:53
749
F. Alshareef and G. D. Robson
Kavanagh, K. & Reeves, E. P. (2004). Exploiting the potential of
insects for in vivo pathogenicity testing of microbial pathogens. FEMS
Microbiol Rev 28, 101–112.
Mondon, P., Thélu, J., Lebeau, B., Ambroise-Thomas, P. & Grillot, R.
(1995). Virulence of Aspergillus fumigatus strains investigated by
Kim, S. S., Kim, Y. H. & Shin, K. S. (2013). The developmental
random amplified polymorphic DNA analysis. J Med Microbiol 42,
299–303.
regulators, FlbB and FlbE, are involved in the virulence of Aspergillus
fumigatus. J Microbiol Biotechnol 23, 766–770.
Mondon, P., De Champs, C., Donadille, A., Ambroise-Thomas, P. &
Grillot, R. (1996). Variation in virulence of Aspergillus fumigatus
Krappmann, S., Bignell, E. M., Reichard, U., Rogers, T., Haynes, K. &
Braus, G. H. (2004). The Aspergillus fumigatus transcriptional
activator CpcA contributes significantly to the virulence of this
fungal pathogen. Mol Microbiol 52, 785–799.
Lair-Fulleringer, S., Guillot, J., Desterke, C., Seguin, D., Warin, S.,
Bezille, A., Chermette, R. & Bretagne, S. (2003). Differentiation
between isolates of Aspergillus fumigatus from breeding turkeys and
their environment by genotyping with microsatellite markers. J Clin
Microbiol 41, 1798–1800.
Latgé, J. P. (1999). Aspergillus fumigatus and aspergillosis. Clin
Microbiol Rev 12, 310–350.
Lebreton, F., Le Bras, F., Reffuveille, F., Ladjouzi, R., Giard, J. C.,
Leclercq, R. & Cattoir, V. (2011). Galleria mellonella as a model for
studying Enterococcus faecium host persistence. J Mol Microbiol
Biotechnol 21, 191–196.
Leenders, A. C., van Belkum, A., Behrendt, M., Luijendijk, A. &
Verbrugh, H. A. (1999). Density and molecular epidemiology of
strains in a murine model of invasive pulmonary aspergillosis. J Med
Microbiol 45, 186–191.
Mondon, P., Brenier, M. P., Symoens, F., Rodriguez, E., Coursange,
E., Chaib, F., Lebeau, B., Piens, M. A., Tortorano, A. M. & other
authors (1997). Molecular typing of Aspergillus fumigatus strains by
sequence-specific DNA primer (SSDP) analysis. FEMS Immunol Med
Microbiol 17, 95–102.
Mukherjee, K., Altincicek, B., Hain, T., Domann, E., Vilcinskas, A. &
Chakraborty, T. (2010). Galleria mellonella as a model system for
studying Listeria pathogenesis. Appl Environ Microbiol 76, 310–317.
Mylonakis, E., Moreno, R., El Khoury, J. B., Idnurm, A., Heitman, J.,
Calderwood, S. B., Ausubel, F. M. & Diener, A. (2005). Galleria
mellonella as a model system to study Cryptococcus neoformans
pathogenesis. Infect Immun 73, 3842–3850.
O’Gorman, C. M. (2011). Airborne Aspergillus fumigatus conidia: a
risk factor for aspergillosis. Fungal Biol Rev 25, 151–157.
O’Gorman, C. M., Fuller, H. & Dyer, P. S. (2009). Discovery of a sexual
Aspergillus in air and relationship to outbreaks of Aspergillus
infection. J Clin Microbiol 37, 1752–1757.
cycle in the opportunistic fungal pathogen Aspergillus fumigatus.
Nature 457, 471–474.
Leuko, S. & Raivio, T. L. (2012). Mutations that impact the
O’Hanlon, K. A., Cairns, T., Stack, D., Schrettl, M., Bignell, E. M.,
Kavanagh, K., Miggin, S. M., O’Keeffe, G., Larsen, T. O. & Doyle, S.
(2011). Targeted disruption of nonribosomal peptide synthetase pes3
enteropathogenic Escherichia coli Cpx envelope stress response
attenuate virulence in Galleria mellonella. Infect Immun 80, 3077–
3085.
Li, H., Barker, B. M., Grahl, N., Puttikamonkul, S., Bell, J. D., Craven,
K. D. & Cramer, R. A., Jr (2011). The small GTPase RacA mediates
intracellular reactive oxygen species production, polarized growth,
and virulence in the human fungal pathogen Aspergillus fumigatus.
Eukaryot Cell 10, 174–186.
Lin, D., Lehmann, P. F., Hamory, B. H., Padhye, A. A., Durry, E.,
Pinner, R. W. & Lasker, B. A. (1995). Comparison of three typing
methods for clinical and environmental isolates of Aspergillus
fumigatus. J Clin Microbiol 33, 1596–1601.
Lionakis, M. S., Lewis, R. E., Torres, H. A., Albert, N. D., Raad, I. I. &
Kontoyiannis, D. P. (2005). Increased frequency of non-fumigatus
augments the virulence of Aspergillus fumigatus. Infect Immun 79,
3978–3992.
Olias, P., Gruber, A. D., Hafez, H. M., Lierz, M., Slesiona, S., Brock, M.
& Jacobsen, I. D. (2011). Molecular epidemiology and virulence
assessment of Aspergillus fumigatus isolates from white stork chicks
and their environment. Vet Microbiol 148, 348–355.
Papaioannou, E., Utari, P. D. & Quax, W. J. (2013). Choosing an
appropriate infection model to study quorum sensing inhibition in
Pseudomonas infections. Int J Mol Sci 14, 19309–19340.
Peleg, A. Y., Jara, S., Monga, D., Eliopoulos, G. M., Moellering, R. C.,
Jr & Mylonakis, E. (2009). Galleria mellonella as a model system to
study Acinetobacter baumannii pathogenesis and therapeutics.
Antimicrob Agents Chemother 53, 2605–2609.
Aspergillus species in amphotericin B- or triazole-pre-exposed cancer
patients with positive cultures for aspergilli. Diagn Microbiol Infect Dis
52, 15–20.
Philippe, B., Ibrahim-Granet, O., Prévost, M. C., Gougerot-Pocidalo,
M. A., Sanchez Perez, M., Van der Meeren, A. & Latgé, J. P. (2003).
Mak, P., Zdybicka-Barabas, A. & Cytryńska, M. (2010). A different
Killing of Aspergillus fumigatus by alveolar macrophages is mediated
by reactive oxidant intermediates. Infect Immun 71, 3034–3042.
repertoire of Galleria mellonella antimicrobial peptides in larvae
challenged with bacteria and fungi. Dev Comp Immunol 34, 1129–
1136.
Menotti, J., Waller, J., Meunier, O., Letscher-Bru, V., Herbrecht, R. &
Candolfi, E. (2005). Epidemiological study of invasive pulmonary
aspergillosis in a haematology unit by molecular typing of environmental and patient isolates of Aspergillus fumigatus. J Hosp Infect 60,
61–68.
Reeves, E. P., Messina, C. G., Doyle, S. & Kavanagh, K. (2004).
Correlation between gliotoxin production and virulence of Aspergillus
fumigatus in Galleria mellonella. Mycopathologia 158, 73–79.
Rementeria, A., López-Molina, N., Ludwig, A., Vivanco, A. B., Bikandi,
J., Pontón, J. & Garaizar, J. (2005). [Genes and molecules involved in
Aspergillus fumigatus virulence]. Rev Iberoam Micol 22, 1–23 (in
Spanish).
Mesa-Arango, A. C., Forastiero, A., Bernal-Martı́nez, L., CuencaEstrella, M., Mellado, E. & Zaragoza, O. (2013). The non-mammalian
Rhodes, J. C. (2006). Aspergillus fumigatus: growth and virulence.
host Galleria mellonella can be used to study the virulence of the
fungal pathogen Candida tropicalis and the efficacy of antifungal
drugs during infection by this pathogenic yeast. Med Mycol 51, 461–
472.
Richie, D. L., Hartl, L., Aimanianda, V., Winters, M. S., Fuller, K. K.,
Miley, M. D., White, S., McCarthy, J. W., Latgé, J. P. & other authors
(2009). A role for the unfolded protein response (UPR) in virulence
Miyata, S., Casey, M., Frank, D. W., Ausubel, F. M. & Drenkard, E.
(2003). Use of the Galleria mellonella caterpillar as a model host to
study the role of the type III secretion system in Pseudomonas
aeruginosa pathogenesis. Infect Immun 71, 2404–2413.
750
Med Mycol 44 (Suppl. 1), 77–81.
and antifungal susceptibility in Aspergillus fumigatus. PLoS Pathog 5,
e1000258.
Rinyu, E., Varga, J. & Ferenczy, L. (1995). Phenotypic and genotypic
analysis of variability in Aspergillus fumigatus. J Clin Microbiol 33,
2567–2575.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 14:56:53
Microbiology 160
Variation in an environmental population of A. fumigatus
Rodriguez, E., De Meeüs, T., Mallie, M., Renaud, F., Symoens, F.,
Mondon, P., Piens, M. A., Lebeau, B., Viviani, M. A. & other authors
(1996). Multicentric epidemiological study of Aspergillus fumigatus
RNA gene complex of Aspergillus nidulans. J Gen Microbiol 136, 1991–
1994.
isolates by multilocus enzyme electrophoresis. J Clin Microbiol 34,
2559–2568.
epidemiological study of invasive pulmonary aspergillosis in a renal
transplantation unit. Eur J Clin Microbiol Infect Dis 13, 318–321.
Sales-Campos, H., Tonani, L., Cardoso, C. R. & Kress, M. R. (2013).
The immune interplay between the host and the pathogen in
Aspergillus fumigatus lung infection. Biomed Res Int 2013, 693023.
Tang, C. M., Cohen, J., Rees, A. J. & Holden, D. W. (1994). Molecular
Thomas, R. J., Hamblin, K. A., Armstrong, S. J., Müller, C. M., BokoriBrown, M., Goldman, S., Atkins, H. S. & Titball, R. W. (2013). Galleria
Aspergillus fumigatus? Curr Opin Microbiol 14, 400–405.
mellonella as a model system to test the pharmacokinetics and efficacy
of antibiotics against Burkholderia pseudomallei. Int J Antimicrob
Agents 41, 330–336.
Scully, L. R. & Bidochka, M. J. (2009). An alternative insect
Vanhee, L. M., Nelis, H. J. & Coenye, T. (2009). Rapid detection and
pathogenic strategy in an Aspergillus flavus auxotroph. Mycol Res
113, 230–239.
quantification of Aspergillus fumigatus in environmental air samples
using solid-phase cytometry. Environ Sci Technol 43, 3233–3239.
Slater, J. L., Gregson, L., Denning, D. W. & Warn, P. A. (2011).
Vanhee, L. M., Nelis, H. J. & Coenye, T. (2010). What can be learned
Schrettl, M. & Haas, H. (2011). Iron homeostasis – Achilles’ heel of
Pathogenicity of Aspergillus fumigatus mutants assessed in Galleria
mellonella matches that in mice. Med Mycol 49 (Suppl. 1 ), S107–
S113.
Soukup, A. A., Farnoodian, M., Berthier, E. & Keller, N. P. (2012).
NosA, a transcription factor important in Aspergillus fumigatus stress
and developmental response, rescues the germination defect of a laeA
deletion. Fungal Genet Biol 49, 857–865.
from genotyping of fungi? Med Mycol 48 (Suppl. 1), S60–S69.
Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M.,
Frijters, A., Pot, J., Peleman, J. & other authors (1995). AFLP: a new
technique for DNA fingerprinting. Nucleic Acids Res 23, 4407–4414.
Wand, M. E., Müller, C. M., Titball, R. W. & Michell, S. L. (2011).
Spreadbury, C. L., Bainbridge, B. W. & Cohen, J. (1990). Restriction
Macrophage and Galleria mellonella infection models reflect the
virulence of naturally occurring isolates of B. pseudomallei, B.
thailandensis and B. oklahomensis. BMC Microbiol 11, 11.
fragment length polymorphisms in isolates of Aspergillus fumigatus
probed with part of the intergenic spacer region from the ribosomal
Edited by: K. Haynes
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 14:56:53
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