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Biofilm formation by zygomycetes: quantification,
structure and matrix composition
Rachna Singh, M. R. Shivaprakash and Arunaloke Chakrabarti
Correspondence
Arunaloke Chakrabarti
arunaloke@hotmail.com
Received 24 January 2011
Revised 12 May 2011
Accepted 26 May 2011
Division of Mycology, Department of Medical Microbiology, Postgraduate Institute of Medical
Education and Research (PGIMER), Chandigarh-160012, India
Most studies on fungal biofilms have focused on Candida in yeasts and Aspergillus in mycelial
fungi. To the authors’ knowledge, biofilm formation by zygomycetes has not been reported
previously. In this study, the biofilm-forming capacity of Rhizopus oryzae,Lichtheimia corymbifera,
Rhizomucor pusillus and Apophysomyces elegans was evaluated.At appropriate seeding spore
densities, Rhp. oryzae (10
5
c.f.u. ml
”1
), L. corymbifera (10
4
c.f.u. ml
”1
) and Rhm. pusillus
(10
4
c.f.u. ml
”1
) produced highly intertwined, adherent structures on flat-bottomed polystyrene
microtitre plates after 24 h at 37 6C. The adhered fungal hyphae were encased in an extracellular
matrix, as confirmed by phase-contrast and confocal microscopy. The thickness of Rhp. oryzae,
L. corymbifera and Rhm. pusillus biofilms was 109.67±10.02, 242±23.07 and 197±9.0 mm
(mean±SD), respectively. Biochemical characterization of the biofilm matrix indicated the
presence of glucosamine, constituting 74.54–82.22 % of its dry weight, N-acetylglucosamine,
glucose and proteins. Adherence and biofilm formation were not observed in A. elegans. Although
A. elegans spores germinated at all three seeding densities tested (1¾10
7
,1¾10
6
and 1¾10
5
c.f.u. ml
”1
), no significant difference was observed (P.0.05) between the A
490
of wells
inoculated with A. elegans and the cut-off A
490
for biofilm detection. This study highlights the
potential for biofilm formation by at least three medically important species of zygomycetes.
INTRODUCTION
Biofilms are dense, highly hydrated cell clusters that are
irreversibly attached to a substratum, to an interface or to
each other, and are embedded in a self-produced gelatinous
matrix composed of extracellular polymeric substances
(EPS) (Harding et al., 2009). The micro-organisms in these
biofilms exhibit an altered phenotype with respect to
growth rate, gene transcription, and resistance to physical,
chemical and biological stresses (Chandrasekar &
Manavathu, 2008; Harding et al., 2009; Mowat et al.,
2009). Biofilm formation has been widely implicated in the
pathogenesis of implant-associated and chronic infections
(Hall-Stoodley et al., 2004).
Most of the work describing biofilm genesis, architecture,
chemical composition, genetic regulation and antimicrobial
drug resistance has focused on bacteria and yeasts
(Chandrasekar & Manavathu, 2008; Harding et al., 2009;
Mowat et al., 2009). Occasionally, filamentous fungi have
also been reported to form biofilms (Chandrasekar &
Manavathu, 2008; Harding et al., 2009; Mowat et al., 2009).
Although filamentous fungi often penetrate the substrates
that they grow on, and this invasive growth may not
accurately represent the term biofilm (Harding et al., 2009),
the yeasts Candida albicans and Trichosporon asahii have
been shown to require differentiation to hyphal forms
during biofilm formation (Harding et al., 2009; Ramage
et al., 2009). Filamentation in fungi may therefore be a
prerequisite for robust biofilm development and virulence,
and fungal biofilms perhaps represent much more than a
mere biological coating (Harding et al., 2009). Biofilm
formation is claimed to be involved in the pathogenesis of
localized as well as invasive diseases caused by Aspergillus
fumigatus (Beauvais et al., 2007; Chandrasekar &
Manavathu, 2008; Harding et al., 2009; Loussert et al.,
2010; Mowat et al., 2007, 20\08, 2009). Aspergilloma, a
localized infection, has been shown to consist of highly
agglutinated hyphae encased in an extracellular matrix
(Loussert et al., 2010; Mowat et al., 2009). A similar
exopolysaccharide matrix is also produced at the surface of
fungal hyphae during invasive pulmonary aspergillosis
(Loussert et al., 2010).
Fungi belonging to the class Zygomycetes and the order
Mucorales, including Rhizopus,Rhizomucor,Mucor,
Lichtheimia,Apophysomyces,Cunninghamella and Saksenaea,
often cause opportunistic infections which are similar to those
caused by Aspergillus and are characterized by angioinvasion
Abbreviations: CLSM, confocal laser scanning microscopy; Con-A,
concanavalin A conjugated to Alexa Fluor; ELLA, enzyme-linked
lectinsorbent assay; EPS, extracellular polymeric substance; G6PDH,
glucose-6-phosphate dehydrogenase; GlcN, glucosamine; GlcNAc,
N-acetylglucosamine; OG, Oregon green; ROC, rhino-orbito-cerebral;
WGA, wheatgerm agglutinin.
Microbiology (2011), 157, 2611–2618 DOI 10.1099/mic.0.048504-0
048504 G2011 SGM Printed in Great Britain 2611
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and fungal-ball formation (Chakrabarti et al., 2001, 2006,
2009; Goodnight et al., 1993; Kirkpatrick et al., 1979; Lahiri
et al., 2001; Robey et al., 2009). Mucormycosis is categorized
into rhino-orbito-cerebral (ROC), pulmonary, gastro-
intestinal, cutaneous and disseminated types depending upon
the clinical presentation and the anatomical sites involved
(Chakrabarti et al., 2001, 2006, 2009). While the ROC type is
often associated with individuals having uncontrolled
diabetes and diabetic ketoacidosis, the pulmonary, gastro-
intestinal and cutaneous types occur in patients with
haematological malignancies or neutropenia, severe mal-
nutrition, and trauma or burns, respectively (Chakrabarti
et al., 2001, 2006, 2009). The incidence of mucormycosis has
increased globally over the last two decades, with a
phenomenal rise in the number of cases reported from
India (Chakrabarti et al., 2001, 2006, 2009). Three case series
on mucormycosis have been reported from our tertiary-care
centre: 129 cases over 10 years (1990–1999), 178 cases during
the subsequent five years (2000–2004) and then 75 cases in an
18 month period during 2006–2007 (Chakrabarti et al., 2001,
2006, 2009). Rhizopus oryzae and Apophysomyces elegans
(sensu lato) were the predominant isolates in these series
(Chakrabarti et al., 2001, 2006, 2009).
Since more than 99 % of microbes have been reported to
grow as biofilms, and these surface-attached communities
have also been implicated in the pathogenesis of both
localized as well as invasive diseases caused by Aspergillus,
the present study was planned to elucidate the biofilm-
forming potential of four common pathogenic zygomy-
cetes: Rhizopus oryzae,Lichtheimia corymbifera,Rhizomucor
pusillus and Apophysomyces elegans.
METHODS
Fungal strains. Rhp. oryzae NCCPF 710004, L. corymbifera NCCPF
700002, Rhm. pusillus NCCPF 720004 and A. elegans NCCPF 102033
were used in this study. The strains were obtained from the National
Culture Collection of Pathogenic Fungi (NCCPF), Department of
Medical Microbiology, Postgraduate Institute of Medical Education
and Research (PGIMER), Chandigarh, India, and were preserved in
20 % glycerol at 270 uC until use.
Isolation of sporangiospores. Rhp. oryzae,L. corymbifera and Rhm.
pusillus were grown on Sabouraud dextrose agar (SDA) at 37 uC for
4–5 days. A. elegans was cultured on water agar at 37 uC for 7 days.
The plates were flooded with 10 ml PBS (0.1 M, pH 7.2) and
sporangiospores were harvested by repeated washing of the mycelia
with the added buffer. The resulting suspensions were centrifuged at
6000 r.p.m. at 4 uC for 10 min; the pellets were washed twice with
PBS and finally suspended in 1 ml PBS. Spores were counted using a
Neubauer haemocytometer and counts expressed as c.f.u. ml
21
.
Standardization of biofilm formation. Biofilm formation was
determined according to the method of Pierce et al. (2008). The
spores were adjusted to the required density (Rhp. oryzae,L.
corymbifera and Rhm. pusillus –1610
7
,1610
6
,1610
5
,1610
4
and
1610
3
c.f.u. ml
21
;A. elegans – 1610
7
,1610
6
,1610
5
c.f.u. ml
21
)in
RPMI 1640 (pH 7.2) buffered with MOPS (165 mM). Two hundred
microlitres of this suspension was inoculated per well in 96-well, flat-
bottomed polystyrene microtitre plates. Media-only blanks were also
set up. The plates were incubated at 37 uC for 24 h (up to 48 h for A.
elegans) and the resulting biofilms were washed twice with PBS, fixed
with 200 ml 95 % ethanol at 37 uC for 15 min and stained with 200 ml
0.1 % safranin for 5 min. Biofilm formation was observed under an
inverted microscope (Olympus CKX 41) with a 640 objective lens
and was quantified by measuring the absorbance of the bound
safranin, eluted with 200 ml 30 % glacial acetic acid, at 490 nm.
Experiments were performed in quadruplicate. The cut-off absor-
bance was calculated as the mean absorbance of the media-only
blanks plus three times their SD (A
490
50.075).
Adhesion kinetics. Two hundred microlitres of the standardized
spore suspension was inoculated per well in 96-well, flat-bottomed
polystyrene microtitre plates and incubated at 37 uC for 4, 8, 12 and
24 h. The experiments were performed in quadruplicate and media-
only blanks were set up in parallel. At each time point, the biofilms
were washed twice with PBS and were quantified as described
previously.
Confocal laser scanning microscopy (CLSM) of biofilms. Three
millilitres of the standardized spore suspension was inoculated in
35 mm Petri dishes and incubated at 37 uC for 24 h. The biofilms
formed were washed twice with PBS, and then stained with
wheatgerm agglutinin conjugated to Oregon green (WGA-OG,
0.1 mg ml
21
; Invitrogen BioServices India) for 15 min (Singh et al.,
2010) or with concanavalin A conjugated to Alexa Fluor 488 (Con-A,
50 mgml
21
) and 10 mM FUN-1 (Chandra et al., 2008) (Invitrogen
BioServices India) at 37 uC for 30 min in the dark. The biofilms were
then washed twice with PBS. CLSM was performed with an LSM 510
Meta (Carl Zeiss MicroImaging) attached to an Axioplan II
microscope using a 610/0.3 objective lens.
During CLSM using WGA-OG, HFT 488 was selected as the
excitation laser and WGA-OG was detected by fluorescence in the
green spectrum using BP505–530. Fungal hyphae were detected by
refraction of light in the red spectrum using LP560. NFT 545 was used
as the beam splitter and the images were acquired in multitrack mode.
For CLSM using dual staining with Con-A/FUN-1, the excitation
wavelengths were set to 488 nm and 543 nm for Con-A and FUN-1,
respectively. NFT 490 (Con-A) and NFT 545 (FUN-1) were used as
the beam splitters, and LP 505 (Con-A) and LP 560 (FUN-1) were
used as the emission filters. Image analysis was done using z-series
image stacks from four randomly chosen spots of each biofilm and
the biofilm architecture and mean thickness were elucidated using
LSM image browser version 4.2.0.121 and ZEN 2009.
Extraction of biofilm matrix. Ten millilitres of the standardized
spore suspension was inoculated per flask in five 50 ml polystyrene
tissue-culture flasks and incubated at 37 uC for 24 h. The biofilms
formed were washed twice with PBS, flooded with 10 ml sterile
MilliQ water per flask and vortexed mildly for 30 s. The suspensions
were then pooled and centrifuged at 6000 r.p.m. at 4 uC for 20 min.
The supernatant was collected, filter-sterilized and treated with 2
volumes of chilled 95 % ethanol overnight at 4 uC. The resulting
precipitates were collected by centrifugation at 6000 r.p.m. at 4 uC for
1 h, washed with chilled 95 % ethanol and dried. The EPS matrix was
dissolved in water and centrifuged at 6000 r.p.m. at 4 uC for 10 min.
The water-soluble fraction (supernatant) was collected and the water-
insoluble material (pellet) was then dissolved in trichloroacetic acid
(TCA). The experiment was performed three times and the matrix
was characterized biochemically each time.
Characterization of biofilm matrix. The intracellular enzyme
glucose-6-phosphate dehydrogenase (G6PDH) was used as a marker
for detecting cell lysis and thereby a contamination of the harvested
matrix with cellular components (Flemming & Wingender, 2010).
Ten microlitres of the water-soluble fraction was added to 290 mlof
R. Singh, M. R. Shivaprakash and A. Chakrabarti
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the assay buffer containing 250 mM glycine buffer (pH 7.4), 60 mM
glucose 6-phosphate, 20 mM NADP and 300 mM MgCl
2
, and
incubated at 25 uC for 5 min. The production of NADPH was
elucidated by measuring the change in absorbance at 340 nm. A
molar absorption coefficient of 6.22 mM
21
cm
21
was used and 1 U
G6PDH was defined as the enzyme activity catalysing the formation
of 1 mmol NADPH min
21
. Protein concentration was determined in
the water-soluble fraction by the bicinchoninic acid method (Smith
et al., 1985). Total carbohydrates in the water-soluble fraction were
determined by the phenol/sulphuric acid method (Dubois et al.,
1951). Glucose was quantified in the water-soluble fraction after acid
hydrolysis by using a glucose oxidase kit (Sigma-Aldrich) according
to the manufacturer’s instructions. The presence of amino sugars in
the water-soluble fraction was detected by UV spectroscopy
(Kumirska et al., 2010). Briefly, the UV absorption of the fractions
was scanned over 190–350 nm in 1 nm step increments. The first-
derivative spectra of the samples were elucidated and compared with
those of standard solutions containing N-acetylglucosamine
(GlcNAc) and glucosamine (GlcN): GlcNAc only, GlcN only, and
GlcNAc and GlcN mixed in different ratios (4 : 1, 3 : 2, 1 : 1, 2 : 3 and
1 : 4). The percentage of acetylated and non-acetylated hexosamine in
the samples was determined from standard curves of l
min
vs
percentage GlcNAc as well as calibrated peak height (H) vs percentage
GlcNAc. GlcNAc was quantified in the water-soluble and TCA-
soluble fractions by enzyme-linked lectinsorbent assay (ELLA)
according to the method of Singh et al. (2010), using a standard
curve of GlcNAc (mg) vs A
490
. GlcN in the water-soluble fraction was
determined by its N-acetylation using acetic anhydride in methanol
(Levvy & McAllan, 1959), followed by quantification of the GlcNAc
formed by ELLA. All experiments were performed in quadruplicate.
Statistical analysis. The two-tailed, unpaired t-test was used for
comparing the adhesion capacity of test strains versus the media-only
blanks.
RESULTS AND DISCUSSION
Mucorales usually cause acute angioinvasive ROC, pulmo-
nary, gastrointestinal or cutaneous infections in immuno-
compromised patients (Chakrabarti et al., 2001, 2006,
2009). In addition, these fungi have been implicated in
central-venous-catheter-associated fungaemia (Chan-Tack
et al., 2005), peritonitis in patients undergoing continuous
ambulatory peritoneal dialysis (Branton et al., 1990; Fergie
et al., 1992; Nakamura et al., 1989; Nannini et al., 2003;
Nayak et al., 2007; Polo et al., 1989; Serna et al., 2003),
pleuritis following implantation of drainage catheters
(Kimura et al., 2009), endocarditis on native (Mehta
et al., 2004; Mitchell et al., 2010) and artificial heart valves
(Gubarev et al., 2007; Sanchez-Recalde et al., 1999),
osteomyelitis (Chaudhuri et al., 1992; Eaton et al., 1994;
Lopes et al., 1995; Meis et al., 1994; Wilkins et al., 2009),
and paranasal fungal balls (Goodnight et al., 1993;
Kirkpatrick et al., 1979; Lahiri et al., 2001; Robey et al.,
2009). Many of these infections are associated with biofilm
formation (Costerton et al., 1999; Hall-Stoodley et al.,
2004; Loussert et al., 2010) and were reported to exhibit a
chronic course (Eaton et al., 1994; Lahiri et al., 2001; Lopes
et al., 1995). Furthermore, surface-attached growth of
zygomycetes has often been used for industrial purposes
(Cao et al., 1997). However, biofilm formation by
zygomycetes has not yet been demonstrated. This is
believed to be the first study to elucidate the biofilm-
forming capacity of Rhp. oryzae,L. corymbifera,Rhm.
pusillus and A. elegans, and study their adhesion kinetics,
biofilm morphology and matrix composition.
Standardization of biofilm formation
The spore density of Rhp. oryzae,L. corymbifera and Rhm.
pusillus in the initial inoculum was found to be important for
germination of the adhered spores as well as structural
integrity of the biofilms formed (Fig. 1). Whilst very high
seeding density (Rhp. oryzae,1610
7
and 1610
6
c.f.u. ml
21
;
L. corymbifera and Rhm. pusillus,1610
7
,1610
6
and 1610
5
c.f.u. ml
21
) resulted in poor germination of the adhered
spores, low inoculum concentration (Rhp. oryzae,1610
4
and 1610
3
c.f.u. ml
21
;L. corymbifera and Rhm. pusillus,
1610
3
c.f.u. ml
21
) reduced the hyphal density. Similar
results have been previously reported for Aspergillus
fumigatus (Mowat et al.,2007),Trichosporon asahii
(Di Bonaventura et al., 2006) and various Candida species
including C. albicans (Ramage et al., 2009), and may be
associated with the production of quorum-sensing molecules
(Ramage et al., 2009). Initial seeding densities of 1610
5
c.f.u.
ml
21
(Rhp. oryzae)and1610
4
c.f.u. ml
21
(L. corymbifera
and Rhm. pusillus) produced robust, filamentous adherent
structures and were selected for further experiments. At these
inoculum concentrations, the mean adherence of Rhp.
oryzae,L. corymbifera and Rhm. pusillus on polystyrene
plates after 24 h of incubation was 2.06±0.02, 2.74±0.29
and 1.44±0.05 (A
490
, mean±SD), respectively.
Apophysomyces elegans spores germinated at all the three
seeding densities tested but adherence and biofilm
formation were not detected. No significant difference
(P.0.05) was observed between the A
490
of wells
inoculated with A. elegans compared to the cut-off A
490
for biofilm detection [1610
7
c.f.u. ml
21
, 0.092±0.0007
(24 h) and 0.091±0.0 (48 h); 1610
6
c.f.u. ml
21
,
0.078±0.0 (24 h) and 0.083±0.0007 (48 h); 1610
5
c.f.u.
ml
21
, 0.073±0.018 (24 h) and 0.097±0.013 (48 h)].A.
elegans is usually associated with superficial infections in
immunocompetent hosts, although infections in deep
tissue have also been described (Chakrabarti et al., 2003,
2010). We reported an upsurge of such A. elegans
infections over the past decade in India (Chakrabarti
et al., 2003, 2010). These findings indicate that biofilm
formation may not play an important role in the
pathogenesis of A. elegans infections. Species-specific
differences in biofilm-forming capacity have also been
reported in Candida, and have been correlated with the
pathogenesis and the associated risk factors (Hawser &
Douglas, 1994; Kuhn et al., 2002; Li et al., 2003; Ramage
et al., 2009).
Adhesion kinetics
A direct correlation was observed between biomass,
quantified by measuring the absorbance of the bound
Biofilm formation by Mucorales
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safranin, and hyphal development (Figs 2 and 3).
Following initial seeding, the spores adhered to the
polystyrene surface and began to swell and germinate in
4–6 h. Hyphae were observed within 8–10 h of incuba-
tion. These hyphae formed monolayers by 10–12 h, and
then further intertwined and increased in density over the
next 12 h. This pattern resembles the kinetics of biofilm
formation in Aspergillus fumigatus (Mowat et al.,2007).
The intertwining of the mycelial mass provides stability
and integrity to the biofilms and is achieved by extensive
branching, followed by elongation of the hyphal branches
by apical growth (Chandrasekar & Manavathu, 2008;
Mowat et al., 2009).
CLSM of biofilms
Microbial biofilms are characterized by the presence of
surface-attached cells enmeshed in a self-produced gelat-
inous matrix made primarily of polysaccharides (Costerton
et al., 1999; Hall-Stoodley et al., 2004). This matrix, also
termed slime or EPS, forms a scaffold for the three-
dimensional architecture of the biofilm (Flemming &
Wingender, 2010; Hall-Stoodley et al., 2004). It provides
stability to the biofilm structure, helps in adherence as well
as in trapping the nutrients, and also protects the biofilm
cells from desiccation, UV radiation, protozoan grazers,
antimicrobials and host immune defences (Flemming &
Wingender, 2010; Hall-Stoodley et al., 2004).
To detect the presence of polysaccharide matrix and
elucidate the biofilm architecture, confocal microscopy
was performed using the plant-based lectins WGA and
concanavalin A. These markers bind to GlcNAc (WGA)
(Singh et al., 2010), glucose and mannose (concanavalin A)
(Chandra et al., 2008), and therefore detect these specific
residues in the hyphae and the matrix, if any. Individual
fungal hyphae were also detected via the red light refracted
by the cells (WGA-OG staining) or FUN-1 (Con-A/FUN-1
staining), and the images obtained were compared with
WGA-OG or Con-A fluorescence for differentiation
between the binding of WGA or Con-A to the hyphae
and the matrix.
Phase-contrast images of the confocal micrographs
revealed a haze-like film, suggestive of a polysaccharide
matrix, covering the fungal hyphae in the biofilms (Fig. 4).
CLSM images also indicated the presence of highly
intertwined mycelia mass, with WGA-OG staining many
inter-hyphal regions as well (Fig. 4). These results
demonstrate the existence of an exopolysaccharide matrix
in Rhp. oryzae,L. corymbifera and Rhm. pusillus biofilms
and further satisfy the criteria proposed for detecting
biofilm formation in filamentous fungi (Harding et al.,
2009). Also, the staining of many inter-hyphal regions with
WGA-OG suggests the presence of GlcNAc in the matrix.
In contrast, Con-A detected the fungal hyphae but inter-
hyphal regions were not significantly stained with this
marker, indicating that glucans and mannans were not a
major component of the matrix. This was further
confirmed by biochemical characterization of EPS
extracted from the biofilms. The thickness of Rhp. oryzae,
L. corymbifera and Rhm. pusillus biofilms was 109.67±
10.02 mm, 242±23.07 mm and 197±9.0 mm (mean±SD),
respectively.
Fig. 1. Effect of seeding density on the development of Rhp. oryzae NCCPF 710004 biofilms after 24 h at 37 6C. Very high
(A, 1¾10
7
;B,1¾10
6
c.f.u. ml
”1
) or low spore concentrations (D, 1¾10
4
;E,1¾10
3
c.f.u. ml
”1
) resulted in poor germination and
reduced hyphal density, respectively. An initial seeding density of 1¾10
5
c.f.u. ml
”1
(C) produced robust, filamentous adherent
structures and was selected for further experiments. The images were taken at ¾40 using an inverted microscope. Scale bar,
10 mm.
R. Singh, M. R. Shivaprakash and A. Chakrabarti
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Characterization of the biofilm matrix
EPS has often been called the ‘dark matter’ of biofilms,
owing to the large range of biopolymers it may contain,
and the difficulty in analysing them (Flemming &
Wingender, 2010). It accounts for a major component of
the biofilm biomass and consists primarily of polysacchar-
ides, although proteins (including many enzymes), lipids
and extracellular DNA may also be present (Flemming &
Wingender, 2010).
G6PDH activity was not detected in the matrices extracted
from Rhp. oryzae,L. corymbifera and Rhm. pusillus biofilms,
confirming an absence of contaminating cellular compo-
nents. Hexosamine and N-acetylhexosamine constituted
93.4–95.6 % and 4.4–6.6 %, respectively, of the amino sugars
in the water-soluble EPS fraction. GlcN was found to be the
primary component of Rhp. oryzae,L. corymbifera and Rhm.
pusillus biofilm matrix, constituting 745.38±115.25,
822.20±178.99 and 803.94±135.43 mg (mg EPS)
21
(mean±SD), respectively. In contrast, GlcNAc constituted
43.49±6.72, 47.97±10.44 and 46.91±7.90 mg (mg EPS)
21
(mean±SD)inRhp. oryzae,L. corymbifera and Rhm. pusillus
biofilms, respectively. These results suggest the presence of
the chitin-derivative chitosan, a partially acetylated b-1,4-
linked GlcN polymer, which is usually soluble in water at a
high degree of deacetylation (Kumirska et al., 2010). In
contrast to other fungi, chitin and chitosan constitute a
major component of the cell wall in zygomycetes (Bartnicki-
Garcia, 1968). Chitosan cements the cell wall, protects chitin
from enzymic attack and also adsorbs ionic material by salt
or complex formation (Bartnicki-Garcia, 1968). The content
of this aminopolysaccharide in zygomycetes’ cell wall may be
up to three times the amount of chitin (Bartnicki-Garcia,
1968). A similar partially deacetylated b-1,6-linked GlcNAc
polymer termed polysaccharide intercellular adhesin (PIA)
is the primary component of Staphylococcus aureus and
Staphylococcus epidermidis biofilm matrix and it mediates
cell-to-cell interaction in these bacteria (Singh et al., 2010).
Hexosamine has also been reported as an important
component of EPS in Candida tropicalis biofilms (Al-
Fattani & Douglas, 2006).
The high concentration of amino sugars interfered with the
determination of carbohydrates by the phenol/sulphuric
acid method. Upon reaction with this reagent, the samples
exhibited a peak absorbance at 405 nm, compared to the
usual 490 nm observed in sugars. Glucose content was
therefore determined in the water-soluble fraction using a
Fig. 2. Hyphal development during adhesion of
Rhp. oryzae NCCPF 710004 on 96-well, flat-
bottomed polystyrene microtitre plates. The
plates were incubated at 37 6C for 4 h (A), 8 h
(B), 12 h (C) and 24 h (D). The images were
taken at ¾40 using an inverted microscope.
Scale bar, 10 mm.
Fig. 3. Adhesion kinetics of Rhp. oryzae NCCPF 710004 (X–X),
L. corymbifera NCCPF 700002 (&---&) and Rhm. pusillus
NCCPF 720004 (m????m) on 96-well, flat-bottomed polystyrene
microtitre plates. The plates were incubated at 37 6C for 4, 8, 12
and 24 h (note the non-linear time scale). Data are represented as
A
490
. Experiments were performed in quadruplicate. Error bars
indicate standard deviation (not shown where smaller than symbols).
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glucose oxidase kit. Although glucose constitutes 32–74 % of
the biofilm matrix in Aspergillus fumigatus (Beauvais et al.,
2007) and Candida species (Al-Fattani & Douglas, 2006;
Silva et al., 2009), it accounted for only a small proportion of
the biofilm-matrix weight in the Mucorales that we studied.
Rhp. oryzae,L. corymbifera and Rhm. pusillus biofilm EPS
contained 7.42±0.92, 7.92±1.20 and 7.17±0.71 mg glucose
(mg EPS)
21
(mean±SD), respectively. Proteins were also
detected in the matrix preparations, constituting 2.23±0.10,
7.70±0.31 and 3.17±0.61 mg (mg EPS)
21
(mean±SD)in
Rhp. oryzae,L. corymbifera and Rhm. pusillus, respectively.
However, these results probably underestimate the protein
content of the slime. The solubilized matrix harvested from
the biofilms was concentrated using ethanol, an organic
solvent which preferentially precipitates polysaccharides
(Flemming & Wingender, 2010). The protein content of
the suspensions before ethanol precipitation was about
sevenfold higher than that of the precipitated EPS.
The TCA-soluble EPS fraction was processed for the
detection of chitin, a b-1,4-linked GlcNAc polymer insoluble
in water and many acids (Kumirska et al., 2010), and its
monomer GlcNAc was quantified by ELLA. The concentra-
tion of GlcNAc in the TCA-soluble fraction of Rhp. oryzae,
L. corymbifera and Rhm. pusillus biofilm matrix was
59.43±0.80, 74.28±8.09 and 56.89±4.40 mg (mg EPS)
21
(mean±SD), respectively.
Thus, the extracellular matrix in Rhp. oryzae,L. corymbifera
and Rhm. pusillus biofilms is primarily composed of amino
sugars (GlcN and GlcNAc), with glucose and proteins also
Fig. 4. Phase-contrast (Aa and Ba) and confocal laser scanning micrographs (Ab and Ac, Bb and Bc, Ca and Cb) of Rhm.
pusillus NCCPF 720004 biofilms. (A) WGA-OG staining. WGA-OG (Ab) was used to detect GlcNAc in the fungal hyphae and
the biofilm matrix. Individual fungal hyphae were also detected via the red light refracted by the cells (Ac) and the images
obtained were compared with WGA-OG fluorescence for differentiation between the binding of WGA to the hyphae and the
matrix. The arrow indicates a representative inter-hyphal region stained by WGA-OG. Scale bars, 100 mm; (B) Con-A and FUN-1
staining. Con-A (Bb) was used to detect glucose and mannose in the fungal hyphae as well as in the biofilm matrix, if any. Individual
fungal hyphae were also detected via FUN-1 (Bc) and the images obtained were compared with Con-A fluorescence for
differentiation between the binding of Con-A to the hyphae and the matrix, if any. Scale bars, 100 mm. (C) Three-dimensional
reconstruction of biofilms after staining with WGA-OG. (Ca) Detection of GlcNAc in the hyphae and matrix with WGA-OG;
(Cb) detection of hyphae by collecting the red-refracted light.
R. Singh, M. R. Shivaprakash and A. Chakrabarti
2616 Microbiology 157
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being present in small amounts. By contrast, glucose is the
major component of Candida and Aspergillus fumigatus
biofilms (Al-Fattani & Douglas, 2006; Baillie & Douglas,
2000; Beauvais et al., 2007; Lal et al., 2010; Silva et al.,
2009). In A. fumigatus biofilms, the EPS consists of glucose
(74 %), mannitol (18 %), trehalose (3 %), glycerol (5 %),
and melanin and proteins (2 %) (Beauvais et al., 2007).
Immunolabelling studies have further revealed the presence
of galactomannan, galactosaminogalactan and a-1,3 glu-
cans (Beauvais et al., 2007; Loussert et al., 2010). C.
albicans biofilms usually contain glucose (16–32.2 %),
hexosamine (3.3 %), proteins (5 %), phosphorus (0.5 %)
and uronic acid (0.1 %) as well as mannose, rhamnose and
galactose in the matrix (Al-Fattani & Douglas, 2006; Baillie
& Douglas, 2000; Lal et al., 2010). However, C. tropicalis
biofilm matrix comprises mainly hexosamine (27.4 %),
with smaller amounts of other carbohydrates (3.3 %,
including 0.5 % glucose), protein (3.3 %) and phosphorus
(0.2 %) (Al-Fattani & Douglas, 2006).
In conclusion, our results reveal the biofilm-forming
potential of Rhp. oryzae,L. corymbifera and Rhm. pusillus,
but not A. elegans. At appropriate seeding densities, these
fungi produced robust, highly intertwined, filamentous,
adherent structures that were encased in an extracellular
matrix composed primarily of GlcN and GlcNAc. Although
Mucorales are usually implicated in angio-invasive
infections, biofilm formation may be involved in the
pathogenesis of paranasal fungal balls, endocarditis,
osteomyelitis and catheter-based infections caused by these
pathogens. It may also be important for the survival of these
saprophytic fungi in the environment (Ramage et al., 2009).
ACKNOWLEDGEMENTS
This work was supported by funding from the Indian Council of
Medical Research (ICMR). We thank Dr Alok Mondal, Scientist Gr.
IV(5) and Mr Deepak Bhatt, Technical assistant Gr III(1), Institute of
Microbial Technology (IMTECH), Chandigarh, India, for help with
the CLSM.
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Edited by: I. J. Van der Klei
R. Singh, M. R. Shivaprakash and A. Chakrabarti
2618 Microbiology 157
