Analysis of Indoor Fungal Contaminants Using Internal Transcribed Spacer Sequence Variability
M. Leo Antony,
D. Mubarak Ali,
R. Praveen Kumar
Fungi present in indoor environment always create serious health risks for
the individual dwellers. The present study was focused on the enumeration of
fungal contaminants in the newly constructed lab. Totally 8 different fungi
were isolated from the indoor environment by using sedimentation culture plate
method. The isolated species were identified conventionally, as well as using
the sequence homology of internal transcribed spacer regions. This ribosomal
non-coding unit sequence based analysis is the most popular locus for species
identification and to study the phylogenetic variation. The fungal species isolated
and described are already found to be reported as pathogens present in indoor
air. Based on the sequences obtained, phylogenetic tree was constructed using
both maximum likelihood and distance matrix depicts that the distance method
using ClustalW2 offers better resolution and relates the related genera. The
study reveals the methodology in fungal taxonomy and also for environmental
monitoring of fungi.
Lee et al., 2006; Wu
et al., 2003; Fischer and Dott, 2003). Nearly
10% of world populations are allergic to fungal spores and their concentration
in the environment depends on several factors like weather, vegetation, air
quality and various anthropological effects (Burge, 1986,
2001; Oliveira et al., 2010).
In general population, the incidence of mold allergy ranges from 6-24%, reaches
up to 44% among atopic individuals and 80% in asthmatic patients; it was found
more severe in immune-compromised individuals (Simon-Nobbe
et al., 2008; Oliveira et al., 2010).
The increased presence of fungal contamination in the air causes vigorous health
effects (Chakraborty et al., 2000) especially
the burden of illness from fungi present in homes and public buildings are controversial
because of limited study in this sector (Dales et al.,
1997). The majority of the fungi detected in the indoor air are the most
commonly existing in outdoor environment. Ventilation and water incursions in
buildings, paws of pets, shoes and clothing of human harbor these fungal particles
into the indoor environment (Gots et al., 2003).
Cladosporium, Aspergillus, Penicillium, Alternaria and Fusarium
are the most common indoor fungi followed by genera like Paecilomyces,
Mucor, Rhizopus, Trichoderma, Ulocladium, Stachybotrys,
Walneria and Curvularia were also reported (Fischer
and Dott, 2003; Wu et al., 2003).
Uninvited entry of biological hazards in a specific environment can be listed
out as contamination (Gorny, 2004). Monitoring the microorganisms
and quality assessment of air is essential and the simplest method for estimating
is that sedimentation culture plate method was adopted (Chakraborty
et al., 2000). In this case, identifying the fungus is more important
that describing them without knowing the taxon. This gives a clear indication
on fungal contamination and disease possibilities. Conventional and molecular
techniques were adopted for the identification of clinical and environmental
samples (Henry et al., 2000). The molecular analysis
of DNA helps in better understanding of fungal biology and the related aspects
(Crespo et al., 1997). The molecular analysis
of DNA helps in better understanding of fungal biology and the related aspects
(Crespo et al., 1997) 18s RNA, mitochondrial
DNA and the intergenic spacer and the Internal Transcribed Spacer (ITS) region
offers targets for the genus level identification. Varying sequence pattern
of ITS region can be used in the phylogenetics analysis of different organisms
(Henry et al., 2000; Guarro
et al., 1999) and also offers a higher sensitivity than other molecular
targets being existing in hundreds of copies per genome approximately. Nearly,
51,000 fungal ITS sequences where available so far and is the most popular locus
for the species identification and sub generic phylogenetic reference in sequence
based mycological research. These spacers occupy the small subunit coding sequence
(SSU) and the large subunit coding sequence (LSU) of the ribosomal operon (Martin
and Rygiewicz, 2005; Burge, 1986). Both 12 spacer
regions are useful in species level identification and also in phylogenetic
analysis among related species (Subramanian et al.,
2003; Henry et al., 2000; Guarro
et al., 1999). In the present work the newly constructed lab were
found to be contaminated with the fungal propagules. The prepared solid media
was frequently encountered fungal contamination and it creates an alarm of fungal
occurrences in the lab environment. The goal of the study was to isolate and
identify the fungal contaminants by both conventional and molecular methods
and to analyze the relatedness among the isolates.
MATERIALS AND METHODS
Sampling site: The indoor environment of a newly constructed lab was selected
as the sampling site. The lab was encountered with 20-30 people daily. Total
area of the lab was 22x15x12 sq. ft. The average temperature during the sampling
period was 28-35°C. The humidity was found to be very low in that season.
The external environment temperature was around 35-45°C as it was a period
of hot summer i.e., April-June 2010. These days are the initial phase of commencing
work in the lab and were examined for fungal exposure. Initially, the fungi
were found to contaminate the plates that were kept in the open environment
which were prepared for other cultivation purposes. The grown fungi were found
to be different from the already existed isolates.
Morphological characterization: The fungi grown as contaminants and
by using sedimentation culture method, the molds present in the lab were examined
using Saborauds Dextrose Agar
(SDA) plate. The plates were incubated at 28-30°C for 3-7 days and the grown
fungi were isolated in a fresh SDA plate. Stock of the isolate was prepared
and stored in SDA slant at 4°C for future use. The fungi grew for 4-5 days
were examined for both naked and microscopic morphology. The fungal filaments
were studied using light microscope (Optix, Italy) after staining with lactophenol
Total DNA extraction: Total cellular DNA was extracted from the fungal
mycelium grown on SDA plate. Spores were collected by pouring 50 μL of
triton-X100 over the grown propagules. This was done several times over the
same spot to collect enough material. The spore/triton-X 100 mixture was transferred
to a 1.5 mL microcentrifuge tube containing 500 μL of CTAB buffer [Stock
(pH 8): 100 mL 1 M Tris; 280 mL 5 M NaCl; 40 mL 0.5 M EDTA; 20 g Cetyl Trimethyl
Ammonium Bromide (CTAB) store at room temperature] (CTAB buffer: 5 mL of stock;
0.2 g polyvinylpyrrolidone; 25 μL of β-mercaptoethanol prepared right
before extraction) and a scoop of sterile glass powder. The mixture was agitated
using vortex mixer for 2 min following incubation in water bath at 65°C
for 30 min. Five hundred microliter of chloroform-isoamyl alcohol (24:1) was
added and mix well by inversion. Cellular debris was removed by centrifugation
at 12000 rpm for 5 min. The aqueous phase was removed and transferred to a new
1.5 mL microcentrifuge tube and the above steps were repeated one more time.
The DNA was precipitated by the addition of 233 μL of isopropanol along
with 32 μL of 4 M ammonium acetate to the aqueous phase. After a gentle
mix, the content was centrifuged at 12000 rpm for 10 min. The pellet was rinsed
with 70% ethanol and dried. The dried pellet was dissolved in 100 μL TE
buffer and stored at -20°C. The
isolated DNA was verified using 0.8% agarose gel electrophoresis.
DNA amplification: The internal transcribed spacer region of the rRNA
gene cluster was amplified from genomic DNA obtained from fungal mycelia by
PCR with the primers ITS1-F (5-CTTGGTCATTTAGAGGAAGTAA-3) (Gardes
and Bruns, 1993) and ITS4-R (5-TCCTCCGCT TATTGATATGC-3) (White
et al., 1990). The reaction mixture contained 20 μL 2X PCR premix
(GENET BIO, Korea), 1 μL of DNA (25-50 ng), 1 μL forward and reverses
primer each. PCR conditions consisted of an initial denaturation at 94°C
for 4 min; 35 cycles at 94°C for 60 s, 50°C for 45 s and 72°C for
60 s and a final 7 min extension at 72°C. The amplified products were examined
by agarose gel electrophoresis and the amplified DNA was sequenced.
DNA sequencing and analysis: Nucleotide sequences were analyzed and
edited by using BioEdit software (Hall, 1999). The sequences
of spacer region obtained were used to search the GenBank database with BLASTN
algorithm to determine the phylogenetic positions of the isolates. The aligned
sequences were incorporated to construct phylogenetic tree using maximum-likelihood
method (Saitou and Nei, 1987). MEGA 4 software (Tamura
et al., 2007). To determine the variation among the isolated strains;
the tree was also constructed using online multiple alignment tool ClustalW2.Manipulation
and tree editing was done by using TreeView. The sequences obtained were deposited
in GenBank for accession.
RESULTS AND DISCUSSION
Isolation and morphological characterization: To study the molds present
in the newly constructed lab, fungi grown as contaminants and the fungal growth
obtained from the open plate method were purified and identified. Both morphological
and sequence based methods were used in identification. Morphological characterization
was carried out based on the description given in the standard manual for soil
fungi (Gilman, 1959). Since there is no standard manual
for air borne fungi, the manual for soil fungi helps in partial identification
of the fungus. Colour, nature of mycelium in both obverse and reverse position,
mycelial and spore morphology observed under microscope were used for morphological
||Overview of the fungi isolated from the study, f: Front view,
b: Back view, 1-NTLF01 T. longibrachiatum, 2: NTLF02 A. terreus,
3: NTLF03 E. nidulans, 4: NTLF04 Nodulisporium sp. 5: NTLF05
P. citrinum, 6: NTLF06 A. fumigatus, 7: NTLF07 Fusarium
species, 8: NTLF08 Fusarium species
|| Microscopic view o f the fungi stained with lactophenol cotton
blue, Magnification: x400 a: NTLF01 T. longibrachiatum, b: NTLF02
A. terreus, c: NTLF03 E. nidulans, d: NTLF04, Nodulisporium
sp. e: NTLF05 P. citrinum, f: NTLF06 A. fumigatus, g:
NTLF07 Fusarium sp., h: NTLF08 Fusarium species
Morphological characteristics of the fungi were detected and documented by
plating and microscopic analysis (Fig. 1, 2),
respectively. Six genera were noted from the eight isolated species from the
Trichoderma: Trichoderma longibrachiatum isolated from
the study, are the common species abundant in nature, saprophytes and also reported
as indoor air pollutant (Druzhinina and Kubicek, 2005;
Thrane et al., 2001). The mycelium of the isolate
was initially dark green and upon maturation became yellow to white color, forming
a flat, firm turf. Conidiophores were erect, rising from short, branched sides,
branching usually opposite, apex were not swollen with bore terminal conidial
heads. Conidia are small, mostly globose, oblong to ellipsoidal with bright
colored hyphae (Tang et al., 2003). Kredics
et al. (2003) reported that Trichoderma longibrachiatum can
act as an opportunistic pathogen for mammals as well as humans especially severe
under immunocompromised state. They are known to be major causative agent of
Trichoderma associated mycoses and Allergic Fungal Sinusitis (AFS). Tang
et al. (2003), later Druzhinina et al.
(2008) from his studies emphasized that the relationship between the clinical
and wild T. longibrachiatum strains was unclear.
Aspergillus: Four different Aspergillus species were detected
and were found to be dominated in the sampled site. Aspergillus has already
been reported several times as a part of indoor air fungal communities. It has
been proved that the major causative agent of many diseases like fungal allergy,
aspergillosis, allergic bronchopulmonary disease, mycotic keratitis, otomycosis,
nasal sinusitis and invasive infection in many individuals (Hyvarinen
et al., 2010).
The species identified from the study Aspergillus terreus, E. nidulans (anamorph
A. nidulans) and A. fumigatus are the one reported several times
as opportunistic pathogens (Simon-Nobbe et al., 2008).
Aspergillus terreus forms white, aerial mycelium. Sometimes it forms
brown mycelium whereas new mycelium always white in color. Reverse and agar
occurs bright yellow to deep brown. Conidia are elliptical to globose (Gilman,
1959). It is one of the causative agents of invasive aspergillosis and capable
of producing toxin called ochratoxin (Henry et al.,
2000; Simon-Nobbe et al., 2008).
Emericella nidulans (anamorph: Aspergillus nidulans) were typical
in morphology and a representative of perfect Aspergillus. They are homothallic
fungi, able to produce nest-like fruiting body called cleistothecium
(Han, 2009). In the initial culturing phase it looks
green which on sub culturing forms orange color hyphae. Whitish new mycelium
upon maturation forms pink colored mycelial mass. On the reverse it forms a
light yellow to white color, upon aging it forms a liquid over the mycelium
in the form of dew drops (Matsuzawa et al., 2010).
In case of A. fumigatus, mature hyphae produce aerial mycelium and the
new forms have flattened mycelium. It forms green to dark green colonies. Reverse
and substratum is colorless to yellow. Conidia are crowded. This fungus can
grow in mucous secretions in the lung and cause type I and III hypersensitivities
and severe bronchopulmonary infections and reported to be grown in indoor environment
(Srikanth et al., 2008). A. fumigatus
is one of the major species associated with invasive aspergillosis and Aspergillus
associated diseases (Zhao et al., 2001; Benneth
and Klich, 2003; Matsuzawa et al., 2010).
Penicillium: Both Aspergillus and Penicillium are
the most studied ascomycetes and are already described to be major contaminants
of indoor air (Burge, 1986). Spores of Aspergillus
and Penicillium are readily airborne when compared to many other
fungal spores and the main indoor fungal contaminants (Burge,
2001). These two are xerophilic molds, dominates in indoor air when compared
to outdoor air (Thrasher and Crawley, 2009). Penicillium
rarely causes diseases in human but excessive inhalation of the spores cause
atopic asthma in sensitive persons (Simon-Nobbe et al.,
2008). The only Penicillium species predominant in the lab was P.
citrinum which produces characteristic red pigment but the property was
lost on subsequent cultivation. It forms aerial mycelium, pale green hyphae,
on the reverse it forms wrinkled white which turns to red color upon maturation.
They produce a characteristic two series of elements called phialides and forms
thick sclerotium and can consistently grow at high temperature (Houbraken
et al., 2010). Penicillium citrinum is ubiquitous in the environment
rarely cause human infection (Mok et al., 1997).
According to the report of Wei et al. (1993),
P. citrinum is the most predominant Penicillium species detected
in the indoor environment. It also reported that they can produce toxin called
citrinin which acts as a nephrotoxin in test animals but its significance with
human disease was unconfirmed (Benneth and Klich, 2003).
Fusarium: Two different types of Fusarium were isolated
from the indoor environment. Conidiospores were sickle shaped in the isolate
Fusarium sp. NTLF07 and in NTLF09 no distinct spores was identified.
Fusarium forms aerial mycelium in which new mycelium forms white to pink
color and on maturation it turns to brown. Reverse of the plate is pale brown
to dark brown. In NTLF07 the conidia has a maximum of up to 7 septa with irregular
conidiophores. It has been reported that Fusarium is an important pathogen
producing different species specific toxins namely fumonisin, deoxynivalenol,
and zearalenone etc. (Benneth and Klich, 2003; Jarvis
and Miller, 2005). Fusarium species frequently encountered in localized
infections in immunocompetent and disseminated in severely immunocompromised
patients. Development of skin lesions is the most common infectious aspect of
this genus and often used in diagnosis of the infection (Nucci
and Anaissie, 2002).
Nodulisporium: Nodulisporium sp. forms characteristic brown color
with flattened mycelium. Hyphae are irregular and branched. They are the common
wood decaying fungi of ascomycetes group of Xylariaceae. They also implicated
in clinical reports. They were marked as LF4 Nodulisporium sp. in
Fig. 2 and identification up to species level is difficult.
From the report of Cox et al. (1994) and Tang
et al. (2003) also noted that it can cause allergic fungal sinusitis
like Trichoderma longibrachiatum which being a common allergic disease
caused by Aspergillus sp.
Molecular identification: The application of molecular tools helps in
identification and to study the relationship among the organisms. ITS variability
was examined for the 8 isolates based on the sequence homology of the amplified
regions. The sequences of ITS regions were deposited in the GenBank for the
accession numbers and was as follows: Aspergillus terreus NTLF02-HQ219673,
Emericella nidulans NTLF03-HQ141711, Nodulisporium sp. NTLF04-HQ219674,
Penicillium citrinum NTLF05-HQ245157, Aspergillus fumigatus NTLF06
HQ141712, Fusarium sp. NTLF07-HQ141713, Fusarium sp. NTLF08-HQ219675
and Trichoderma longibrachiatum NTLF01-HQ141714.
The ITS sequences obtained were analyzed to find identical species by using
BLAST search. Most of the species matched with the sequences of similar species.
The similarity search helps in identification up to species level. From the
report of Planning workshop on all fungi DNA barcoding, 2007, ITS
can be used as a marker for fungal DNA barcoding except Fusarium and
certain AM fungi, which show variation when compared to all other true fungi
(Rossman, 2007). With the help of ITS sequences and
the BLAST tool Nodulisporium sp. was identified and verified with the
report of Cox et al. (1994).
Phylogenetic analysis: From the report of Lee and
Taylor (1992), 0.3-14.6% of nucleotide difference and comparisons of variable
regions permitted the phylogenetic analysis and study of evolutionary relationships.
The evolutionary history was inferred using maximum-likelihood method based
on Kimura 2 and the phylogenetic tree was observed in Fig. 3.
The bootstrap consensus tree inferred from 500 replicates is taken to represent
the evolutionary history of the taxa analyzed.
||Phylogenetic tree was constructed using maximum likelihood
method from MEGA4 software
|| Phylogenetic tree was constructed using CLUSTALW2 a multiple
alignment online tool
All positions containing gaps and missing data were eliminated from the dataset.
This dataset shows the two Fusarium sp. in different clade reveals that
ITS region shows much variation to distinguish among species and also not suitable
for using as an identity marker for Fusarium as per the report of international
symposium on DNA barcoding (Rossman, 2007).
The tree was also constructed using ClustalW2 an online alignment tool which
depicts the closely related species into a single clade when compared to the
evolutionary relationship study using MEGA4. The phylogenetic tree was calculated
using distance matrix. Figure 4 clearly distinguishes the
Nodulisporium NTLF4 from other ascomycetes group. Trichoderma longibrachiatum
NTLF01 falls in a separate clade in both of the phylograms. It shows a close
relationship within the species, whereas Aspergillus and Penicillium
fall separately when compared to other four distinct species. Thus the
multiple alignments using ClustalW2 gives a better resolution and joins the
associated isolates in a single clade when compared to maximum likelihood method.
Of all genera mentioned above, Aspergillus was found to be the most
dominant ones found in the lab environment. The genera like Aspergillus,
Penicillium, Fusarium and Trichoderma were common in indoor environment
and were analyzed by internal transcribed spacer region and microscopic identification.
Partial sequences of ITS region were deposited in GenBank (NCBI) with accession
numbers mentioned above. Though the work creates only a rough idea about the
fungal contamination over the newly constructed buildings and their environment,
it prospect to put more effort to study indoor air contaminants and their relative
aspects to avoid risk of infection.
Department of Biotechnology (DBT) and Council for Scientific Industrial Research
(CSIR) Government of India for the grant for financial support and Senior Research
Fellowship are highly acknowledged, respectively." class="btn btn-success" target="_blank">View Fulltext