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Braz Dent Sci 2019 Apr/Jun;22(2)
252
ORIGINAL ARTICLE
Effects of Bacillus subtilis on Candida albicans: biofilm formation,
filamentation and gene expression
Efeitos de
Bacillus
subtilis sobre Candida albicans: Formação de biofilme, filamentação e expressão gênica
Michelle Peneluppi SILVA1, Patrícia Pimentel de BARROS1, Adeline Lacerda JORJÃO1, Rodnei Dennis ROSSONI1, Juliana Campos
JUNQUEIRA1, Antonio Olavo Cardoso JORGE1.
1 – Univ Estadual Paulista (Unesp) - Institute of Science and Technology, São José dos Campos - Department of Biosciences and Oral Diagnosis
– São José dos Campos – SP - Brazil.
doi: 10.14295/bds.2019.v22i2.1692
UNIVERSIDADE ESTADUAL PAULISTA
“JÚLIO DE MESQUITA FILHO”
Instituto de Ciência e Tecnologia
Campus de São José dos Campos
Ciência
Odontológica
Brasileira
RESUMO
Objetivo: O objetivo deste estudo foi avaliar o efeito
de Bacillus subtilis sobre a formação de biofilme e
filamentação de Candida albicans através da avaliação
da expressão dos genes ALS3, HWP1, BCR1, EFG1 and
TEC1. Material e métodos: Biofilmes monotípicos
e mistos (C. albicans / B.subtilis) foram cultivados em
placas a 37°C por 48 h sob agitação, para a contagem
de células viáveis (UFC/mL) e para a análise da
expressão gênica por PCR em tempo real. O ensaio
de filamentação de C. albicans foi realizado em meio
contendo 10% de soro fetal bovino a 37°C por 6 h. Os
dados obtidos foram analisados por testes t-Student
e Mann–Whitney. Resultados: B.subtilis reduziu em
1 log a formação de biofilme por C. albicans quando
cultivados no mesmo ambiente (p<0.0001). Além disso,
reduziu significantemente a transição de levedura para
hifa, afetando assim, a morfologia de C. albicans. Em
relação aos genes analisados, os genes ALS3 e HWP1
foram os mais regulados negativamente, com uma
diminuição de 111,1 e 333,3 vezes, respectivamente, na
sua expressão em biofilmes de C. albicans associados a B.
subtilis. Conclusão: B. subtilis reduziu a filamentação e a
formação de biofilme de C. albicans através da regulação
negativa dos genes ALS3, HWP1, BCR1, EFG1 e TEC1,
que são essenciais na produção de hifas e de biofilme.
PALAVRAS-CHAVE
Bacillus subtilis; Candida albicans; biofi lme; fi
lamentação; expressão gênica.
ABSTRACT
Objective: The aim of this study was evaluate
the effect of Bacillus subtilis on Candida albicans
biofilm formation and filamentation by evaluating
the gene expression of ALS3, HWP1, BCR1, EFG1
and TEC1. Material and Methods: Mixed (C.
albicans / B.subtilis) and monotypic biofilms were
cultured in plates at 37°C for 48 h under shaking
for counting viable cells (CFU / mL) and analysis of
gene expression by real-time PCR. The C. albicans
filamentation assay was performed in medium
containing 10% fetal bovine serum at 37°C for 6
hours. Data was analysed by t-Student and Mann–
Whitney tests. Results: B. subtilis reduced the
biofilm formation of C. albicans in 1 log when
cultured in the same environment (p<0.0001). In
addition, it significantly reduced the yeast -
hypha transition
affecting the morphology of C. albicans. Among all
of the analyzed genes, the ALS3 and HWP1 genes
were the most affected, achieving 111.1- and 333.3-
fold decreases in the C. albicans biofilms associated
with B. subtilis, respectively. Conclusion: B. subtilis
reduced the biofilm formation and filamentation of
C. albicans by negatively regulating the ALS3, HWP1,
BCR1, EFG1 and TEC1 genes that are essential for
the production of biofilm and hyphae.
KEYWORDS
Bacillus subtilis; Candida albicans; biofi lm; fi
lamentation; gene expression.
open access scientific journal volume 22 n002 - 2019
Campus de São José dos Campos
Partnership between
Institute of Science and Technology / Unesp, Brazil
and Ain Shams University, Egypt
Photo: Sérgio Eduardo de Paiva Gonçalves
Effects of
Bacillus subtilis
on
Bacillus subtilis
on
Bacillus subtilis
Candida albicans
:
Candida albicans
:
Candida albicans
Biofilm formation, filamentation and gene expression
Silva MP et al.
Braz Dent Sci 2019 Apr/Jun;22(2)
253
INTRODUCTION
C
andida albicans is a commensal fungus
found on mucosal surfaces of the oral
cavity, gastrointestinal and genitourinary
tracts. This yeast causes severe and recurrent
mucosal infections as well as fatal invasive
infections in both immunocompromised and
immunocompetent individuals [1].
Some virulence factors such as
dimorphism and the ability to adhere and
form biofilm on medical devices and/or
the host mucosal epithelium, enhance the
pathogenicity of C. albicans. The cells in
biofilms exhibit distinct phenotypes that
are similar to their free-living counterparts,
including extreme resistance to many
antimicrobial agents. Advances in genetic
manipulation and expression profiling have
helped define the regulatory pathways and
mechanisms that govern C. albicans biofilm
development [1,2,3]. Several classes of genes,
such as specify transcription factors (EFG1,
BCR1, TEC1) and adhesion (HWP1, ALS3),
control biofilm development on abiotic and
biotic surfaces [4,5].
In the recent past, microbial secondary
metabolites have engrossed great attention of
researchers as antifungal compounds. Most of
the antifungal compounds currently in use are
synthetic derivatives with known serious side
effects and toxicity [6]. Emergence of drug
resistant Candida has augmented the desire
for new biocompatible antifungal drugs [7,8].
The genus Bacillus includes a great
diversity of industrially important strains,
including Bacillus subtilis. This spore-forming
bacterium has been established as industrial
bacteria in the production of biological
indicators for sterilization, in studies of
biodefense and astrobiology methods as
well as disinfection agents, in treatment
evaluation and as potential adjuvants
or vehicles for vaccines, among other
applications. The metabolic diversity and
lack of reported incidenceof pathogenicity
makes this bacterium the most suitable for the
development of marketable products [9].
Bacillus strains could produce some
metabolites that inhibit pathogens, since
some Bacillus species used in commercially
available products have the ability to produce
antimicrobials, such as aminocumaim A [10]
and bacteriocin [11,12]. Zheng and Slavik
[11] isolated and characterized a bacteriocin
(molecular weight of approximately 34 kDa
and a pI value of approximately 47) of B.
subtilis 22. Firstly, the precipitated supernatant
from the B. subtilis 22 culture had inhibitory
activity in nine strains of Gram-negative and
Gram-positive bacteria by agar diffusion assay.
After partial purification of supernatant, the
bacteriocin was named bacillocin 22 making
this peptide potentially useful against the
food-borne pathogens. In a recent study,
Mayer and Kronstad [13] have identified the
soil bacterium, Bacillus safensis, as a potent
inhibitor of virulence factor production by
two major fungal pathogens of humans,
Cryptococcus neoformans, and C. albicans.
They determined that the anti-virulence factor
mechanism was, at least in part, baseded on
production of bacterial chitinases that target
and destabilize the fungal cell surface.
In this context, in an attempt to investigate
new compounds and/or metabolites with
antifungal properties produced by a sporulated
bacillus, the current study was evaluate the
effect of B. subtilis on C. albicans biofilm
formation and filamentation by evaluating the
gene expression of ALS3, HWP1, BCR1, EFG1
and TEC1.
MATERIAL AND METHODS
Microorganisms and growth
conditions
In this study, we used the reference
strains of B. subtilis (ATCC 19659) and C.
albicans (ATCC 18804); these strains were
plated on Brain Heart Infusion Agar (Difco,
Detroit, USA) and Sabouraud Dextrose Agar
(Difco, Detroit, USA), respectively, and were
Effects of
Bacillus subtilis
on
Bacillus subtilis
on
Bacillus subtilis
Candida albicans
:
Candida albicans
:
Candida albicans
Biofilm formation, filamentation and gene expression
Silva MP et al.
Braz Dent Sci 2019 Apr/Jun;22(2)
254
incubated at 37 °C for 24 h. After growth,
bacterial colonies were transferred and
cultured in Brain Heart Infusion Broth (BHI)
(Difco Laboratories Inc., Detroit, MI, USA)
and the yeast in Yeast Nitrogen Base - YNB
(Difco Laboratories Inc., Detroit, MI, USA) at
37 °C for 24 h.
Biofi lm formation
Suspensions of each microorganism
sample were adjusted to 107 cells/mL using
a spectrophotometer (B582, Micronal, São
Paulo, Brazil). The formation of monotypic and
mixed biofilms was based on the methodology
described by Seneviratne et al. [14] and Bridier
et al. [15] with modifications. Biofilms were
formed in the bottom of a 96-well microtiter
plate (TPP®, Trasadingen, Switzerland), by
pipetting 100 µL of a standardized suspension
containing 107 cells/mL of each microorganism,
C. albicans and B. subtilis, associated in each
well to the formation of a mixed biofilm. For
monotypic biofilm formation, 100 µL of only
one microorganism and 100 µL BHI broth
were pipetted in each well. The plates were
incubated under shaking at 75 rpm rotation
(Quimis, Diadema, Brazil) for 90 min at 37
°C with only the microorganisms to facilitate
initial adhesion.
After adhesion phase, the suspension of
non-adhered microorganisms was aspirated,
and each well was washed with 200 µL of
0.85% NaCl to remove the non-adhered cells;
200 µL of BHI broth was added in all formed
biofilms, both monotypic and mixed groups. All
plates were incubated at 37°C for 48 h under
shaking at 75 rpm rotation. After 24 h, the
broth was changed and after 48 h, the biofilms
were aspirated and washed with 0.85% NaCl
to remove the non-adhered cells. Biofilms
were evaluated in relation to cell viability
(quantification of the colony forming units -
CFU/mL) and gene expression in interspecies
interaction. For each of these evaluations
the experiment was performed in triplicate
at two different times with 10 trials for each
experimental group, resulting in 180 trials.
Quantifi cation of viable cells (CFU/
mL)
After washing the biofilm, 200 µL
of 0.85% NaCl was added in each well for
homogenization during 30 s in an ultrasonic
homogenizer (Vibra Cell-Sonics & Materials,
Inc. Newtown, Connecticut, USA), with 25%
amplification to detach the biofilms. Serial
dilutions of suspensions were performed, and
100 µL aliquots of each dilution were plated
in the culture medium, according to the
microorganism: BHI Agar supplemented with
fluconazole for B. subtilis and HiCrome Candida
Differential Agar (Himedia, Mumbai, India) for
C. albicans. The plates were incubated at 37 °C
for 48 h to count the CFU/mL and converted
into logarithm to base 10 (Log).
C. albicans fi lamentation production
The filamentation assay was performed in
24-well microtiter plates (TPP®, Trasadingen,
Switzerland) following a methodology
described previously with modifications [16].
The experimental groups formed were: C.
albicans +PBS (Phosphate Buffered Saline)
(control group) and C. albicans + B. subtilis
(interaction group). After 18 hours of growth
C. albicans and B. subtilis were centrifuged to
prepare standard suspensions corresponding
to 107 cells/mL in a spectrophotometer as
previously described. In 24-well cell culture
plates, 1 mL distilled water supplemented
with 10% bovine fetal serum was added to a
100 µL C. albicans suspension. Then, a 100 µL
B. subtilis suspension was added to each well.
The control group used 100 µL PBS.
The plate was incubated at 37°C for 6 h
and 50 µL of the inoculum contained in each
well was placed on microscopic glass slides
with 10 fields previously marked on the back
and observed under an optical microscope
with a magnification of 400 x. The images
were analyzed in relation to C. albicans
morphology and hyphae quantification in 10
microscopic fields per slide and classified as
scores from 0 to 4 attributed to each field
according to the number of hyphae present:
Effects of
Bacillus subtilis
on
Bacillus subtilis
on
Bacillus subtilis
Candida albicans
:
Candida albicans
:
Candida albicans
Biofilm formation, filamentation and gene expression
Silva MP et al.
Braz Dent Sci 2019 Apr/Jun;22(2)
255
score 0: no hyphae; score 1: 1 to 20 hyphae;
score 2: 21 to 40 hyphae; score 3: 41 to 50
hyphae; and score 4: more than 50 hyphae.
Each experimental group had 5 trials at
two different times, totaling 20 tests. The
percentage of germination was calculated
considering the mixed group formed by C.
albicans- B. subtilis in relation to monotypic
group of C. albicans.
Evaluation of ALS3, HWP1, BCR1,
EFG1 and TEC1 gene expression with qPCR
The evaluation of C. albicans virulence
genes was based on the methodology described
by Barros et al. [17] with modifications.
Biofilms were formed in 24-well microtiter
plates (TPP®, Trasadingen, Switzerland)
with the same concentration and conditions
as in the “Biofilm formation” section. The
expression of ALS3, HWP1, BCR1, EFG1
and TEC1 genes was evaluated in relation
to four reference genes, ACT1 (β-Actin),
RIP1 (Ubiquinol Cytochrome-c Reductase
Complex Component), PMA1 (Adenosine
Triphosphatase) and LSC2 (Succinyl-CoA
Synthetase β-Subunit Fragment), described
in the literature [18], in all experimental
groups. The results were analyzed at http://
www.leonxie.com/referencegene.phpe, and
the reference gene RIP1 was selected for this
experiment.
Total RNA was extracted using
the Trizol reagent (Ambion®, Carlsbad,
USA), according to the manufacturer’s
recommendations. The purity, quality and
concentration of the RNA were analyzed
by the NanoDrop 2000 spectrophotometer
(Thermo Fisher Scientific Inc., Wilmington,
USA). To remove contaminating DNA, RNA
(1µg) extracted was treated with DNase
I (Turbo DNase Treatment and Removal
Reagents - InvitrogenTM, Carlsbad, USA) and
transcribed into complementary DNA (cDNA)
using the SuperScript™ III First-Strand
Synthesis Super Mix for qRT-PCR Kit with
SYBR®Green (InvitrogenTM, Carlsbad, USA),
according to the protocol recommended by
the manufacturer.
Transcribed cDNA was amplified for
the relative quantification of ALS3, HWP1,
BCR1, EFG1 and TEC1 gene expression
levels in relation to the concentration of the
selected reference gene, RIP1. Quantitative
real-time PCR was conducted using the
Platinum® SYBR® Green qPCR SuperMix-
UDG Kit (Applied Biosystems, Framingham,
MA, USA) in the StepOnePlus™ apparatus
(Applied Biosystems, Framingham, MA, USA).
The 2-DDCT method was used to analyze the
relative changes in gene expression from the
quantitative RT-qPCR experiment [19].
Statistical analysis
Student’s t-test was used to compare
the CFU/mL results from the in vitro biofilm
formation assay and the relative quantification
of gene expression. The scores obtained from
the filamentation analysis were compared
using the Mann–Whitney test. All analyses
were performed using the GraphPad Prism 5
Program (GraphPad Software, Inc., USA) and
5% level of signifcance was adopted.
RESULTS
The CFU/mL count of C. albicans in
monotypic biofilms showed higher values
compared to mixed biofilms formed by
associations between C. albicans and B. subtilis
(Figure 1). Comparing the results obtained
in monotypic biofilms with mixed biofilms,
there was a reduction of 1.0 log of C. albicans
with a statistically significant difference (p
= 0.001). B. subtilis was not affected in the
mixed biofilm compared to monotypic (p =
0.7002). The results, expressed in mean and
standard deviation, for CFU/mL counts of C.
albicans in monotypic biofilms were 5.38 ±
0.149 log and 4.37 ± 0.183 in mixed biofilms.
For B. subtilis, the means were 5.33 ± 0.202
log in monotypic biofilms and 5.11 ± 0.179 in
mixed biofilms.
In relation to the filamentation test, the
results showed reduction of morphological
transition (yeast/hyphae) for C. albicans in
Effects of
Bacillus subtilis
on
Bacillus subtilis
on
Bacillus subtilis
Candida albicans
:
Candida albicans
:
Candida albicans
Biofilm formation, filamentation and gene expression
Silva MP et al.
Braz Dent Sci 2019 Apr/Jun;22(2)
256
the presence of B. subtilis (C. albicans and
B. subtilis interaction), with a statistically
significant reduction (p=0.0001) compared
to the control group with PBS (Figure 2).
To elucidate the inhibitory mechanisms
of B. subtilis on C. albicans biofilms, this study
was extended to analyze C. albicans gene
expression, concentrating on genes involved
in biofilm formation and filamentation. The
expression levels of the adhesion genes (ALS3
and HWP1) and transcriptional regulatory
genes (TEC1, BCR1 and EFG1) were quantifed
in C. albicans cells from monotypic and mixed
biofilms using qPCR. In C. albicans and B.
subtilis association, ALS3, HWP1, BCR1, TEC1
and EFG1 genes were downregulated when
compared to the C. albicans control group with
statistically significant differences (p=0.0001)
(Figure 3). Among all of the analyzed genes,
the ALS3 and HWP1 genes were the most
affected, achieving 111.1- and 333.3-fold
decreases in the C. albicans biofilms associated
with B. subtilis, respectively. These results
agreed with those obtained for the CFU/mL
counts and filamentation assay and suggest
that B. subtilis exerts inhibitory effects on C.
albicans by interfering with yeast adherence
during biofilm formation and reducing its
development during filamentation.
Figure 1 - Quantitative analysis of in vitro biofilm formation by
CFU/mL count: Means and standard deviations of C. albicans
ATCC18804 and B. subtilis ATCC19659 CFU/mL (Log) values in
the following groups: monotypic, formed by C. albicans (Control
group); mixed, formed by C. albicans + B. subtilis, monotypic,
formed by B.subtilis; mixed, formed by C. albicans + B. subtilis.
Student’s t-test, p ≤ 0.05.
Figure 2 - Bacillus subtilis reduce C. albicans filamentation.
(A): Median scores obtained by determining the number of
hyphae in the in vitro Candida, filamentation assay for the
following groups: C. albicans control group, interaction group
with B subtilis. A significant hyphal reduction was observed in
the interaction group compared to the control group (Mann-
Whitney test, p ≤ 0.05). (B) Percentage germination, expressed
as the mean values of hyphae, in the viability of C. albicans
when associated with B. subtilis.
Figure 3 - Bacillus subtilis decreased the expression of
C. albicans genes involved in biofilm formation. Relative
quantification (Log) of TEC1, BCR1, EFG1, ALS3 and HWP1
genes in monotypic and mixed biofilms formed by C. albicans.
The units in the Y-axis were calculated based on the 2-DDCT
method, and are expressed as the means and standard
deviations. Each gene was normalized and compared with
the expression of monotypic biofilms of C. albicans (control
groups) using the reference gene RIP1. Student’s t-test was
used to compare gene expression between the groups (p ≤
0.05). ***indicates p ≤ 0.0001.
Effects of
Bacillus subtilis
on
Bacillus subtilis
on
Bacillus subtilis
Candida albicans
:
Candida albicans
:
Candida albicans
Biofilm formation, filamentation and gene expression
Silva MP et al.
Braz Dent Sci 2019 Apr/Jun;22(2)
257
DISCUSSION
Biofilm growth is a main model
of microbial life and up to 80% of all
microorganisms are sessile attached to the
surface of biofilm communities. Within this
group of microorganisms, 65 to 80% can cause
microbial infections in humans from pathogenic
biofilm formation [20]. The capability of biofilm
formation and morphological transformation
from yeast to hyphae are important to C. albicans
pathogenicity [21]. Biofilms create an area of
higher antimicrobial resistance for the residing
microorganisms [20]. Thus, the interactions
between bacteria and fungi can influence survival
and proliferation, which can be beneficial for
microorganisms because it increases resistance
to antimicrobials or antagonistic that lead to
death [22].
In this study, the results demonstrated
that C. albicans was reduced in association
with B. subtilis during biofilm formation. The
quantification of CFU/mL showed a reduction
of 87.44% in C. albicans cells when compared
to the control group. Thus, it demonstrated that
in mixed biofilm the microorganisms interacted
competitively and antagonistic, suggesting an
inhibitory effect of B. subtilis on C. albicans. A
possible explanation for the reduction of biofilm
formation by C. albicans during the microbial
interaction is the fact that Bacillus spp. secrete
antimicrobial agents, such as bacteriocins,
which have effects for suppressing the growth of
competitive and opportunistic microorganisms
[8,9]. One example is Subtilisin A, which is a
bacteriocin isolated from B. subtilis [23]. B.
subtilis in mixed biofilms did not present a
significant reduction in relation to the monotypic
biofilm. According to López et al. [24] B. subtilis
uses a mechanism called cannibalism to slow its
sporulation, and these cannibal cells secrete two
toxins, skf and sdp, in which they can interfere
with the growth of other microorganisms,
constituting as a defense mechanism of B.
subtilis.
Furthermore, we analyzed hyphae
formation in C. albicans, and the results showed
a significant reduction in the number of hyphae
when compared to the control group of C.
albicans, which confirms the results previously
obtained by the inhibitory effect exerted by B.
subtilis on C. albicans biofilm. Ability of Candida
to reversibly switch between budding yeast
and filamentous form is crucial for establishing
infection. This phenotypic plasticity is tightly
regulated by several environmental cues and
internal signaling pathways [25]. The hyphal
formation is related to gene expression that
encodes virulence factors, such as the hyphae
wall proteins HWP1 and ALS3 [25].
To confirm the results obtained in the CFU/
mL and filamentation assays, we analyzed the
expression of ALS3, HWP1, BCR1, EFG1 and TEC1,
which are C. albicans virulence genes responsible
for biofilm formation and morphogenesis
of yeast, by the relative quantification
method qPCR [21,25]. All of these genes
were significantly downregulated compared
to monotypic biofilms, demonstrating the
reduction effect of B. subtilis on morphogenesis
and biofilm formation in C. albicans. HWP1 and
ALS3 were the most downregulated genes in
the presence of B. subtilis, and this result can be
associated with the decrease in the CFU counts,
the hyphae formation interfering in biofilm
formation. Our findings are in agreement with
Rautela et al. [8] that evaluated the influence
of cyclic lipopeptides, fengycin and iturin,
produced by Bacillus amyloliquefaciens strain
AR2 in C. albicans biofilms. They showed
that the fungicidal activity was concentration
dependent and sub-minimal concentrations
of the lipopeptide showed a decrease in the
hydrophobicity of the cell surface, formation of
germinative tubes and reduced of the hyphae
specific genes, such as HWP1 and ALS3.
Our results show the potential antifungal
effect of B. subtilis, since this species interferes
in the transcriptional analysis of C. albicans.
Recently, Subramenium et al. [26] evaluated the
antibiofilm effect of B. subtilis, a marine bacterial
isolate from Palk Bay against C. albicans. This
bacterium was able to reduce the biofilm of C.
albicans by up to 90%, the filamentation and
Effects of
Bacillus subtilis
on
Bacillus subtilis
on
Bacillus subtilis
Candida albicans
:
Candida albicans
:
Candida albicans
Biofilm formation, filamentation and gene expression
Silva MP et al.
Braz Dent Sci 2019 Apr/Jun;22(2)
258
production of hydrolytic enzymes. In addition,
the genes ERG11, FLU1, CDR2 and CDR1
(involved in drug resistance mechanisms) were
downregulated when associated with B. subtilis.
The interactions between bacteria and
fungi are highly complex and many factors need
to be analyzed together, such as microorganism
virulence associated with environmental factors
and interactions between different species
of microorganisms present in certain niches,
requiring proper development of in vitro and in
vivo models to characterize these interactions.
The results from the experimental conditions
described in this study demonstrated that B.
subtilis inhibited C. albicans, suggesting that
this bacterium produced compounds and/or
metabolites with antifungal activity.
CONCLUSION
In conclusion, this study demonstrated
that B. subtilis reduced the biofilm formation
and filamentation of C. albicans by negatively
regulating the ALS3, HWP1, BCR1, EFG1 and
TEC1 genes that are essential for the production
of biofilm and hyphae.
ACKNOWLEDGEMENTS
This study was supported by the São
Paulo Council of Research - FAPESP, Brazil
(Grants 2012/15250-0, 2013/25181-8 and
2015/09770-9) for supporting this research.
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Patrícia Pimentel de Barros
(Corresponding address)
Department of Biosciences and Oral Diagnosis
Univ Estadual Paulista/UNESP
Av. Eng Francisco José Longo 777, São Dimas, São José dos Campos
CEP: 12245-000, SP, Brazil.
E-mail: barrosdnapp@yahoo.com.br
Date submitted: 2018 Nov 13
Accept submission: 2019 Feb 26
