Multi-species biofilm of Candida albicans and non-Candida albicansCandida species on acrylic substrate
Identifieur interne : 001D56 ( Pmc/Corpus ); précédent : 001D55; suivant : 001D57Multi-species biofilm of Candida albicans and non-Candida albicansCandida species on acrylic substrate
Auteurs : Apurva K Pathak ; Sanjay Sharma ; Pallavi ShrivastvaSource :
- Journal of Applied Oral Science [ 1678-7757 ] ; 2012.
Abstract
In polymicrobial biofilms bacteria extensively interact with
The species of
In the present study, differences between the mean values of the biofilm-forming
ability of individual species (
The findings of this study showed that biofilm-forming ability was found greater
for non-
Url:
DOI: 10.1590/S1678-77572012000100013
PubMed: 22437681
PubMed Central: 3928775
Links to Exploration step
PMC:3928775Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Multi-species biofilm of <italic>Candida albicans</italic> and
non-<italic>Candida albicans</italic><italic>Candida</italic> species on acrylic substrate</title><author><name sortKey="K Pathak, Apurva" sort="K Pathak, Apurva" uniqKey="K Pathak A" first="Apurva" last="K Pathak">Apurva K Pathak</name><affiliation><nlm:aff id="aff01"> MSc. PhD, Sr. Lecturer, Department of Microbiology, Modern Dental College & Research Centre, Indore (M.P.), India.</nlm:aff></affiliation></author><author><name sortKey="Sharma, Sanjay" sort="Sharma, Sanjay" uniqKey="Sharma S" first="Sanjay" last="Sharma">Sanjay Sharma</name><affiliation><nlm:aff id="aff02"> BDS, MDS, Department of Prosthodontics with Crown, Bridge & Implantology, Modern Dental College & Research Centre, Indore (M.P.), India.</nlm:aff></affiliation></author><author><name sortKey="Shrivastva, Pallavi" sort="Shrivastva, Pallavi" uniqKey="Shrivastva P" first="Pallavi" last="Shrivastva">Pallavi Shrivastva</name><affiliation><nlm:aff id="aff03"> BDS, MDS, Department of Oral Medicine, Diagnosis & Radiology, Modern Dental College & Research Centre, Indore (M.P.), India.</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22437681</idno><idno type="pmc">3928775</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928775</idno><idno type="RBID">PMC:3928775</idno><idno type="doi">10.1590/S1678-77572012000100013</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">001D56</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001D56</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Multi-species biofilm of <italic>Candida albicans</italic> and
non-<italic>Candida albicans</italic><italic>Candida</italic> species on acrylic substrate</title><author><name sortKey="K Pathak, Apurva" sort="K Pathak, Apurva" uniqKey="K Pathak A" first="Apurva" last="K Pathak">Apurva K Pathak</name><affiliation><nlm:aff id="aff01"> MSc. PhD, Sr. Lecturer, Department of Microbiology, Modern Dental College & Research Centre, Indore (M.P.), India.</nlm:aff></affiliation></author><author><name sortKey="Sharma, Sanjay" sort="Sharma, Sanjay" uniqKey="Sharma S" first="Sanjay" last="Sharma">Sanjay Sharma</name><affiliation><nlm:aff id="aff02"> BDS, MDS, Department of Prosthodontics with Crown, Bridge & Implantology, Modern Dental College & Research Centre, Indore (M.P.), India.</nlm:aff></affiliation></author><author><name sortKey="Shrivastva, Pallavi" sort="Shrivastva, Pallavi" uniqKey="Shrivastva P" first="Pallavi" last="Shrivastva">Pallavi Shrivastva</name><affiliation><nlm:aff id="aff03"> BDS, MDS, Department of Oral Medicine, Diagnosis & Radiology, Modern Dental College & Research Centre, Indore (M.P.), India.</nlm:aff></affiliation></author></analytic><series><title level="j">Journal of Applied Oral Science</title><idno type="ISSN">1678-7757</idno><idno type="eISSN">1678-7765</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Objective</title><p>In polymicrobial biofilms bacteria extensively interact with
<italic>Candida</italic> species, but the interaction among the different
species of the <italic>Candida</italic> is yet to be completely evaluated. In the
present study, the difference in biofilm formation ability of clinical isolates of
four species of <italic>Candida</italic> in both single-species and multi-species
combinations on the surface of dental acrylic resin strips was evaluated.</p></sec><sec><title>Material and Methods</title><p>The species of <italic>Candida</italic>, isolated from multiple species oral
candidiasis of the neutropenic patients, were used for the experiment. Organisms
were cultured on Sabouraud dextrose broth with 8% glucose (SDB). Biofilm
production on the acrylic resins strips was determined by crystal violet assay.
Student's t-test and ANOVA were used to compare <italic>in vitro</italic> biofilm
formation for the individual species of <italic>Candida</italic> and its different
multi-species combinations.</p></sec><sec><title>Results</title><p>In the present study, differences between the mean values of the biofilm-forming
ability of individual species (<italic>C. glabrata>C. krusei>C.
tropicalis>C. albicans</italic>) and in its multi-species' combinations (the
highest for <italic>C. albicans</italic> with <italic>C. glabrata</italic> and the
lowest for all the four species combination) were reported.</p></sec><sec><title>Conclusions</title><p>The findings of this study showed that biofilm-forming ability was found greater
for non-<italic>Candida albicans Candida</italic> species (NCAC) than for
<italic>C. albicans</italic> species with intra-species variation. Presence of
<italic>C. albicans</italic> in multi-species biofilms increased, whereas;
<italic>C. tropicalis</italic> decreased the biofilm production with all other
NCAC species.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Adam, B" uniqKey="Adam B">B Adam</name></author><author><name sortKey="Baillie, Gs" uniqKey="Baillie G">GS Baillie</name></author><author><name sortKey="Douglas, Lj" uniqKey="Douglas L">LJ Douglas</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Alberth, M" uniqKey="Alberth M">M Alberth</name></author><author><name sortKey="Majoros, L" uniqKey="Majoros L">L Majoros</name></author><author><name sortKey="Kovalecz, G" uniqKey="Kovalecz G">G Kovalecz</name></author><author><name sortKey="Borbas, E" uniqKey="Borbas E">E Borbás</name></author><author><name sortKey="Szegedi, I" uniqKey="Szegedi I">I Szegedi</name></author><author><name sortKey="Marton, Ij" uniqKey="Marton I">IJ Márton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baillie, Gs" uniqKey="Baillie G">GS 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uniqKey="Samaranayake Y">YH Samaranayake</name></author><author><name sortKey="Samaranayake, Lp" uniqKey="Samaranayake L">LP Samaranayake</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Watts, Rg" uniqKey="Watts R">RG Watts</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="case-report"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Appl Oral Sci</journal-id><journal-id journal-id-type="iso-abbrev">J Appl Oral Sci</journal-id><journal-id journal-id-type="publisher-id">J. Appl. Oral. Sci.</journal-id><journal-title-group><journal-title>Journal of Applied Oral Science</journal-title></journal-title-group><issn pub-type="ppub">1678-7757</issn><issn pub-type="epub">1678-7765</issn><publisher><publisher-name>Faculdade de Odontologia de Bauru da Universidade de São
Paulo</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22437681</article-id><article-id pub-id-type="pmc">3928775</article-id><article-id pub-id-type="doi">10.1590/S1678-77572012000100013</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>Multi-species biofilm of <italic>Candida albicans</italic> and
non-<italic>Candida albicans</italic><italic>Candida</italic> species on acrylic substrate</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>K PATHAK</surname><given-names>Apurva</given-names></name><xref ref-type="aff" rid="aff01">1</xref><xref ref-type="corresp" rid="c01"></xref></contrib><contrib contrib-type="author"><name><surname>SHARMA</surname><given-names>Sanjay</given-names></name><xref ref-type="aff" rid="aff02">2</xref></contrib><contrib contrib-type="author"><name><surname>SHRIVASTVA</surname><given-names>Pallavi</given-names></name><xref ref-type="aff" rid="aff03">3</xref></contrib></contrib-group><aff id="aff01"><label>1</label> MSc. PhD, Sr. Lecturer, Department of Microbiology, Modern Dental College & Research Centre, Indore (M.P.), India.</aff><aff id="aff02"><label>2</label> BDS, MDS, Department of Prosthodontics with Crown, Bridge & Implantology, Modern Dental College & Research Centre, Indore (M.P.), India.</aff><aff id="aff03"><label>3</label> BDS, MDS, Department of Oral Medicine, Diagnosis & Radiology, Modern Dental College & Research Centre, Indore (M.P.), India.</aff><author-notes><corresp id="c01"><bold>Corresponding address:</bold> Dr. Apurva K Pathak - Department of Microbiology,
Modern Dental College & Research Centre - Gandhinagar - Indore (M.P.) - India -
453112 - Phone: +91 731 2882695 - Ext. 341 - Fax: +91 731 2882697 - e-mail:
<email>pathak.apurva@gmail.com</email></corresp></author-notes><pub-date pub-type="ppub"><season>Jan-Feb</season><year>2012</year></pub-date><volume>20</volume><issue>1</issue><fpage>70</fpage><lpage>75</lpage><history><date date-type="received"><day>17</day><month>11</month><year>2009</year></date><date date-type="rev-recd"><day>16</day><month>2</month><year>2010</year></date><date date-type="accepted"><day>19</day><month>3</month><year>2010</year></date></history><permissions><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc/3.0/"><license-p>This is an Open Access article distributed under the terms of the Creative
Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the
original work is properly cited. </license-p></license></permissions><abstract><sec><title>Objective</title><p>In polymicrobial biofilms bacteria extensively interact with
<italic>Candida</italic> species, but the interaction among the different
species of the <italic>Candida</italic> is yet to be completely evaluated. In the
present study, the difference in biofilm formation ability of clinical isolates of
four species of <italic>Candida</italic> in both single-species and multi-species
combinations on the surface of dental acrylic resin strips was evaluated.</p></sec><sec><title>Material and Methods</title><p>The species of <italic>Candida</italic>, isolated from multiple species oral
candidiasis of the neutropenic patients, were used for the experiment. Organisms
were cultured on Sabouraud dextrose broth with 8% glucose (SDB). Biofilm
production on the acrylic resins strips was determined by crystal violet assay.
Student's t-test and ANOVA were used to compare <italic>in vitro</italic> biofilm
formation for the individual species of <italic>Candida</italic> and its different
multi-species combinations.</p></sec><sec><title>Results</title><p>In the present study, differences between the mean values of the biofilm-forming
ability of individual species (<italic>C. glabrata>C. krusei>C.
tropicalis>C. albicans</italic>) and in its multi-species' combinations (the
highest for <italic>C. albicans</italic> with <italic>C. glabrata</italic> and the
lowest for all the four species combination) were reported.</p></sec><sec><title>Conclusions</title><p>The findings of this study showed that biofilm-forming ability was found greater
for non-<italic>Candida albicans Candida</italic> species (NCAC) than for
<italic>C. albicans</italic> species with intra-species variation. Presence of
<italic>C. albicans</italic> in multi-species biofilms increased, whereas;
<italic>C. tropicalis</italic> decreased the biofilm production with all other
NCAC species.</p></sec></abstract><kwd-group><kwd>Oral candidiasis</kwd><kwd>Biofilms</kwd><kwd>Crystal violet</kwd><kwd>Assay</kwd></kwd-group></article-meta></front><body><sec><title>INTRODUCTION</title><p><italic>Candida</italic> is the most frequently isolated fungal pathogen in humans
causing a variety of afflictions ranging from superficial mucosal infections to systemic
mycoses. Oral fungal infections develop frequently in immunocompromised patients,
particularly in patients with prolonged, severe neutropenic episodes<sup><xref rid="r02" ref-type="bibr">2</xref></sup>. <italic>In vivo</italic> studies
indicate that microbial contamination of denture acrylic resin occurs quite rapidly and
implanted devices like denture prostheses provide refuge to candidal organisms as either
single-species or multi-species biofilms<sup><xref rid="r05" ref-type="bibr">5</xref>,<xref rid="r13" ref-type="bibr">13</xref></sup>. Consequently, the
immunocompromised denture wearers are more prone to the fungal infections.</p><p>One of the major factors contributing to the virulence of <italic>Candida</italic> is
its ability in acclimatize to a variety of different habitats for growth and formation
of surface-attached microbial communities known as "biofilms". Biofilms are defined as
microbial communities encased in a matrix of extracellular polymeric substance (EPS),
which display phenotypic features that differ from their planktonic or free-floating
counterparts<sup><xref rid="r07" ref-type="bibr">7</xref>,<xref rid="r16" ref-type="bibr">16</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup>.</p><p><italic>In vitro</italic> studies indicate that microbial contamination of denture
acrylic resin occurs quite rapidly and the yeast cells adhere strongly to denture
materials<sup><xref rid="r04" ref-type="bibr">4</xref>,<xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r18" ref-type="bibr">18</xref></sup>. As bacteria
and non-<italic>Candida albicans Candida</italic> species are often found with
<italic>Candida albicans</italic> in polymicrobial biofilms <italic>in vivo</italic>,
it is likely that extensive interspecies interactions take place in these adherent
populations<sup><xref rid="r16" ref-type="bibr">16</xref>,<xref rid="r17" ref-type="bibr">17</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup>.</p><p>A large number of <italic>in vitro</italic> model systems have been used to investigate
characteristics of single-species and mixed-species biofilms consisting of <italic>C.
albicans</italic> and bacteria<sup><xref rid="r01" ref-type="bibr">1</xref>,<xref rid="r03" ref-type="bibr">3</xref>,<xref rid="r10" ref-type="bibr">10</xref>,<xref rid="r11" ref-type="bibr">11</xref>,<xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r17" ref-type="bibr">17</xref></sup>. Some studies have also
been done on the interaction between different species of <italic>Candida</italic> on
<italic>in vitro</italic> dual-species biofilm formation<sup><xref rid="r20" ref-type="bibr">20</xref>,<xref rid="r25" ref-type="bibr">25</xref></sup>. The study on
the interaction between different species of <italic>Candida</italic> on <italic>in
vitro</italic> multi-species biofilm formation is still scarce<sup><xref rid="r27" ref-type="bibr">27</xref></sup>.</p><p>In the present study, the differences in biofilm-forming ability of clinical isolates of
four species of <italic>Candida</italic> in single-species and multi-species combination
on the surface of dental acrylic resin strips, commonly used for dental appliances, was
evaluated.</p></sec><sec sec-type="materials|methods"><title>MATERIAL AND METHODS</title><sec><title>Organisms</title><p>The test organisms included 24 isolates of 4 different species of
<italic>Candida</italic> i.e. <italic>C. albicans, C. kruzi, C. glabrata and C.
tropicalis</italic> (six isolates of each), isolated from the oral lesions of
multiple species (≥2 <italic>Candida</italic> species) Oropharyngeal Candidiasis
(OPC) of the neutropenic patients (absolute neutrophil count <1.5×10<sup>9</sup>cells/L)<sup><xref rid="r28" ref-type="bibr">28</xref></sup>. Pure culturing
and identification was done at the Department of Microbiology, Modern Dental College
and Research Centre. The identification of <italic>Candida</italic> species was
conducted by culture characteristics on HiChrome <italic>Candida</italic> agar medium
(HiMedia, Mumbai, India), assessing germ tube, chlamydospore formation and sugar
assimilation patterns<sup><xref rid="r06" ref-type="bibr">6</xref>,<xref rid="r22" ref-type="bibr">22</xref></sup>.</p></sec><sec><title>Simulated acrylic resins plates fabrication</title><p>Thirty-six square acrylic resins (polymethylmethacrylate) strips of 10x10x3 mm were
fabricated. The acrylic resins strips were prepared as described by Samaranayake and
MacFarlane<sup><xref rid="r21" ref-type="bibr">21</xref></sup> (1980) with some
modifications. Wax patterns were invested in denture flasks, boiled out, packed with
the denture base resins, and heat polymerized according to manufacturer instructions
at a temperature 73ºC for 6 h. Strips were removed from flask after bench cooling.
One surface of strips was polished on buff wheel with pumice slurry. Other surface
was left untouched to simulate intaglio surface. The resultant acrylic resins strips
were immersed in distilled water for 1 week to leach excess monomer. Following this
strips were disinfected by dipping in 70 % alcohol for 1 min, washed with sterile
distilled water, dried and used for the experiment after checking their
sterility.</p></sec><sec><title>Determination of biofilm production</title><p>Sabouraud dextrose broth (SDB) prepared from powdered Sabouraud broth (HiMedia,
Mumbai, India) supplemented with 60 g of glucose <italic>per</italic> liter (final
glucose concentration, 80 g/liter or 8%) (Qualigens, Navi Mumbai, India), was
according to manufacturer's instructions. Fresh pure cultures of testing organism
were prepared on SDA medium by subculturing clinical isolate. A loop full of
organisms from each SDA plate was inoculated into modified Sabouraud dextrose broth
(8% of glucose concentration) for 24 h at 35±2ºC. The turbidity of each suspension
was adjusted to the equivalent of 1x10<sup>7</sup> CFU/mL with SDB as determined by
comparative plate counts.</p><p>Next, 1 mL of suspension of isolated species and testing combination of different
species was prepared by mixing equal volume of tested species, inoculated into a test
tube with a screw cap (HiMedia, Mumbai, India) containing 9 mL of SDB, to make the
final turbidity of each suspension to 1x10<sup>6</sup> CFU/mL. Strips were placed in
SDB, and then incubated at 35±2ºC for 24 h without agitation. After 24 h of
incubation, the culture broth in the tube was aspirated gently, and then acrylic
strips were taken out for further investigation.</p><p>The acrylic resins strips, on which biofilms developed, were washed once with
distilled water, and then incubated in a crystal violet (HiMedia, Mumbai, India)
staining solution (0.1% in distilled water) for 15 min. These were then washed three
times with distilled water. The stain was then dissolved in de-staining solution (95%
ethanol) and absorbance in terms of optical density (OD) was measured at 570 nm as
previously described<sup><xref rid="r15" ref-type="bibr">15</xref></sup>. Untreated
acrylic strips were used as a control for the amount of the crystal violet stain in
the de-staining solution. The absorbance values of controls were subtracted from the
test values to minimize background interference.</p></sec><sec><title>Statistical analysis</title><p>The ODs of the amount of the crystal violet in the de-staining solution, measured for
different <italic>Candida</italic> species, were compared by the paired Student's
t-test by using the SPSS Win 12.0 program (SPSS Inc, Chicago, IL, USA). Differences
between the isolated species and its multi-species combinations were considered to be
significant for <italic>P</italic> of 0.05. The null hypothesis (HO) rejected in
favor of the alternative hypothesis (H1) at significance level α (0.05) if;
T>t<sub>n-1</sub>, α/2 (value of the Student table with n -1 degrees of
freedom). The null hypothesis is H0: δ=0 (there is no difference in biofilm formation
ability among the tested species) and the alternative hypothesis is (was) H1: δ=0
(there is a difference in biofilm formation ability among the tested species).</p></sec></sec><sec sec-type="results"><title>RESULTS</title><p>Twenty-four <italic>Candida</italic> species isolated from clinical samples and were
used for this study. Amongst the 24 isolates, 6 each of <italic>C. albicans</italic>,
<italic>C. glabrata</italic>, <italic>C. krusei</italic>, and <italic>C. tropicalis
</italic> were used to test the biofilm formation ability both in single-species and in
multi-species combination. All the isolates produce moderate to high degree of biofilms
on the surface of acrylic material (<xref ref-type="fig" rid="f01">Figure 1</xref>). The
biofilm-forming ability of <italic>C. glabrata</italic> were reported to be the highest
on acrylic substances (OD - 0.3833±0.02066), whereas; of <italic>C. albicans</italic> to
be the lowest (OD - 0.1467±0.01366). In case of multiple species conditions, it was
observed that the <italic>C. albicans</italic> had a positive impact on biofilms
formation on acrylic substances as the highest degree of slim production occurred when
<italic>C. albicans</italic> were inoculated with <italic>C. glabrata</italic> (OD-
0.7850±0.03209); whereas <italic>C. krusei</italic> had a negative impact in
combination. Under multiple species condition when all the four species were in
inoculums, the biofilms forming activity were severely hampered (OD -
0.1133±0.01033).</p><fig id="f01" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Optical Density of de-staining solution <sup>#</sup></p></caption><p><sup>#</sup> OD., Optical Density; A., C. albicans; T., C. tropicalis; K., C. krusei;
G., C. glabrata; +, Combination of tested species</p><graphic xlink:href="jaos-20-01-0070-g01"></graphic></fig><p>To test the hypothesis of no difference or no relationship between biofilm-forming
ability of isolated species with that of in-combination of other species, paired t-test
was performed (<xref ref-type="table" rid="t01">Table 1</xref>). For all the four
species, 7 different multi-species combinations were prepared, which made a total of 39
pairs. Eighty-five percent of the pairs rejected null hypothesis in favor of alternate
hypothesis. Only 15% of pairs showed no difference between the mean values of the
biofilm-forming ability of single-species and in its multi-species combination.</p><table-wrap id="t01" orientation="portrait" position="float"><label>Table 1</label><caption><p>Paired samples t-test of <italic>Candida</italic> species combinations<xref ref-type="table-fn" rid="t01-fn01">$</xref></p></caption><table frame="hsides" rules="groups"><thead><tr style="background-color:#CCCCCC"><td colspan="2" align="center" valign="top" rowspan="1"><bold>Pairs</bold></td><td align="center" valign="top" rowspan="1" colspan="1"><bold>t-test</bold></td><td align="center" valign="top" rowspan="1" colspan="1"><bold>Significance level (2-tailed)</bold></td><td align="center" valign="top" rowspan="1" colspan="1"><bold>Decision (α=0.05)</bold></td></tr></thead><tbody><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 1</td><td align="center" valign="top" rowspan="1" colspan="1">A - A+T</td><td align="center" valign="top" rowspan="1" colspan="1">-5,452</td><td align="center" valign="top" rowspan="1" colspan="1">0,003</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 2</td><td align="center" valign="top" rowspan="1" colspan="1">A - A+K</td><td align="center" valign="top" rowspan="1" colspan="1">-6,928</td><td align="center" valign="top" rowspan="1" colspan="1">0,001</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 3</td><td align="center" valign="top" rowspan="1" colspan="1">A - A+G</td><td align="center" valign="top" rowspan="1" colspan="1">-65,11</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 4</td><td align="center" valign="top" rowspan="1" colspan="1">A - A+K+G</td><td align="center" valign="top" rowspan="1" colspan="1">-2,907</td><td align="center" valign="top" rowspan="1" colspan="1">0,034</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 5</td><td align="center" valign="top" rowspan="1" colspan="1">A - A+T+K</td><td align="center" valign="top" rowspan="1" colspan="1">-2,697</td><td align="center" valign="top" rowspan="1" colspan="1">0,043</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 6</td><td align="center" valign="top" rowspan="1" colspan="1">A - A+T+G</td><td align="center" valign="top" rowspan="1" colspan="1">-31,55</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 7</td><td align="center" valign="top" rowspan="1" colspan="1">A - A+T+K+G</td><td align="center" valign="top" rowspan="1" colspan="1">5,423</td><td align="center" valign="top" rowspan="1" colspan="1">0,003</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 8</td><td align="center" valign="top" rowspan="1" colspan="1">T - A+T</td><td align="center" valign="top" rowspan="1" colspan="1">-4,443</td><td align="center" valign="top" rowspan="1" colspan="1">0,007</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 9</td><td align="center" valign="top" rowspan="1" colspan="1">T - T+G</td><td align="center" valign="top" rowspan="1" colspan="1">5,477</td><td align="center" valign="top" rowspan="1" colspan="1">0,003</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 10</td><td align="center" valign="top" rowspan="1" colspan="1">T - T+K</td><td align="center" valign="top" rowspan="1" colspan="1">1</td><td align="center" valign="top" rowspan="1" colspan="1">0,363</td><td align="center" valign="top" rowspan="1" colspan="1">Non-significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 11</td><td align="center" valign="top" rowspan="1" colspan="1">T - A+T+K</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">1</td><td align="center" valign="top" rowspan="1" colspan="1">Non-significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 12</td><td align="center" valign="top" rowspan="1" colspan="1">T - A+T+G</td><td align="center" valign="top" rowspan="1" colspan="1">-29,39</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 13</td><td align="center" valign="top" rowspan="1" colspan="1">T - T+K+G</td><td align="center" valign="top" rowspan="1" colspan="1">0,889</td><td align="center" valign="top" rowspan="1" colspan="1">0,415</td><td align="center" valign="top" rowspan="1" colspan="1">Non-significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 14</td><td align="center" valign="top" rowspan="1" colspan="1">T - A+T+K+G</td><td align="center" valign="top" rowspan="1" colspan="1">8,367</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 15</td><td align="center" valign="top" rowspan="1" colspan="1">K - A+K</td><td align="center" valign="top" rowspan="1" colspan="1">-3,503</td><td align="center" valign="top" rowspan="1" colspan="1">0,017</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 16</td><td align="center" valign="top" rowspan="1" colspan="1">K - K+G</td><td align="center" valign="top" rowspan="1" colspan="1">8,73</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 17</td><td align="center" valign="top" rowspan="1" colspan="1">K - T+K</td><td align="center" valign="top" rowspan="1" colspan="1">4,392</td><td align="center" valign="top" rowspan="1" colspan="1">0,007</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 18</td><td align="center" valign="top" rowspan="1" colspan="1">K - A+K+G</td><td align="center" valign="top" rowspan="1" colspan="1">2,169</td><td align="center" valign="top" rowspan="1" colspan="1">0,082</td><td align="center" valign="top" rowspan="1" colspan="1">Non-significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 19</td><td align="center" valign="top" rowspan="1" colspan="1">K - A+T+K</td><td align="center" valign="top" rowspan="1" colspan="1">7</td><td align="center" valign="top" rowspan="1" colspan="1">0,001</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 20</td><td align="center" valign="top" rowspan="1" colspan="1">K - T+K+G</td><td align="center" valign="top" rowspan="1" colspan="1">5</td><td align="center" valign="top" rowspan="1" colspan="1">0,004</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 21</td><td align="center" valign="top" rowspan="1" colspan="1">K - A+T+K+G</td><td align="center" valign="top" rowspan="1" colspan="1">8,919</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 22</td><td align="center" valign="top" rowspan="1" colspan="1">G - A+G</td><td align="center" valign="top" rowspan="1" colspan="1">-32,85</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 23</td><td align="center" valign="top" rowspan="1" colspan="1">G - K+G</td><td align="center" valign="top" rowspan="1" colspan="1">27,568</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 24</td><td align="center" valign="top" rowspan="1" colspan="1">G - T+G</td><td align="center" valign="top" rowspan="1" colspan="1">21,422</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 25</td><td align="center" valign="top" rowspan="1" colspan="1">G - A+K+G</td><td align="center" valign="top" rowspan="1" colspan="1">20,684</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 26</td><td align="center" valign="top" rowspan="1" colspan="1">G - A+T+G</td><td align="center" valign="top" rowspan="1" colspan="1">-1,025</td><td align="center" valign="top" rowspan="1" colspan="1">0,352</td><td align="center" valign="top" rowspan="1" colspan="1">Non-significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 27</td><td align="center" valign="top" rowspan="1" colspan="1">G - T+K+G</td><td align="center" valign="top" rowspan="1" colspan="1">37,997</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 28</td><td align="center" valign="top" rowspan="1" colspan="1">G - A+T+K+G</td><td align="center" valign="top" rowspan="1" colspan="1">60,374</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 29</td><td align="center" valign="top" rowspan="1" colspan="1">A+T – (A,T)</td><td align="center" valign="top" rowspan="1" colspan="1">5,27</td><td align="center" valign="top" rowspan="1" colspan="1">0,003</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 30</td><td align="center" valign="top" rowspan="1" colspan="1">A+K – (A,K)</td><td align="center" valign="top" rowspan="1" colspan="1">5,966</td><td align="center" valign="top" rowspan="1" colspan="1">0,002</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 31</td><td align="center" valign="top" rowspan="1" colspan="1">A+G – (A,G)</td><td align="center" valign="top" rowspan="1" colspan="1">50,747</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 32</td><td align="center" valign="top" rowspan="1" colspan="1">K+G – (K,G)</td><td align="center" valign="top" rowspan="1" colspan="1">-23,95</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 33</td><td align="center" valign="top" rowspan="1" colspan="1">T+G – (T,G)</td><td align="center" valign="top" rowspan="1" colspan="1">-16,84</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 34</td><td align="center" valign="top" rowspan="1" colspan="1">T+K – (T,K)</td><td align="center" valign="top" rowspan="1" colspan="1">-3,841</td><td align="center" valign="top" rowspan="1" colspan="1">0,012</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 35</td><td align="center" valign="top" rowspan="1" colspan="1">A+K+G – (A,K,G)</td><td align="center" valign="top" rowspan="1" colspan="1">-11,87</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 36</td><td align="center" valign="top" rowspan="1" colspan="1">A+T+K – (A,T,K)</td><td align="center" valign="top" rowspan="1" colspan="1">0,183</td><td align="center" valign="top" rowspan="1" colspan="1">0,862</td><td align="center" valign="top" rowspan="1" colspan="1">Non-significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 37</td><td align="center" valign="top" rowspan="1" colspan="1">A+T+G – (A,T,G)</td><td align="center" valign="top" rowspan="1" colspan="1">16,021</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr style="background-color:#CCCCCC"><td align="center" valign="top" rowspan="1" colspan="1">Pair 38 </td><td align="center" valign="top" rowspan="1" colspan="1">T+K+G – (T,K,G)</td><td align="center" valign="top" rowspan="1" colspan="1">-30,62</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Pair 39</td><td align="center" valign="top" rowspan="1" colspan="1">A+T+K+G – (A,T,K,G)</td><td align="center" valign="top" rowspan="1" colspan="1">-25,85</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">Significant</td></tr></tbody></table><table-wrap-foot><fn id="t01-fn01"><label>$</label><p><italic>C. albicans</italic>; T., <italic>C. tropicalis</italic>; K.,
<italic>C. krusei</italic>; G., <italic>C. glabrata;</italic> +, Combination
of tested species; (,),Average of the biofilm-forming ability of tested species
independently.</p></fn></table-wrap-foot></table-wrap></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>In patients with advanced immunodeficiency, mucosal infections can lead to severe oral
and esophageal candidiasis, resulting in disseminated candidiasis and sometimes early
death. One of the most important virulence factors of <italic>Candida</italic> species
is its ability to form biofilms, which has an important clinical consequence, as it
confers resistance to antifungal therapy and capacity for yeast cells within the
biofilms to withstand host immune defenses<sup><xref rid="r03" ref-type="bibr">3</xref>,<xref rid="r05" ref-type="bibr">5</xref>,<xref rid="r06" ref-type="bibr">6</xref>,<xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup>. Changes in the oral environment effected by tooth loss
or denture wearing can cause changes in oral microflora. The carriage rates of single
and multiple <italic>Candida</italic> species were reported to be significantly higher
in denture wearers<sup><xref rid="r11" ref-type="bibr">11</xref></sup>. In the light of
above fact <italic>Candida,</italic> isolated from multiple species candidimia lesion,
were evaluated for their biofilms formation ability on acrylic surface <italic>in
vitro</italic>, in single-species and multi-species combination.</p><p><italic>Candida</italic> biofilms formation has been described on polymethylmethacrylate
strips which occur essentially in three overlapping phases: early (0-11 h), intermediate
(12-30 h), and maturation (38-72 h) phases. The early stage is characterized by
adherence and development of blastospores into distinct microcolonies. By 18 to 24 h,
the <italic>Candida</italic> biofilm community can be seen as a bilayered structure
comprising a mixture of yeasts, germ tubes, and young hyphae; this intermediate phase is
distinguished by the production of extracellular polymeric substance (EPS). During
maturation, the biofilms becomes a thick EPS layer in which a dense network of yeasts,
pseudohyphae, and hyphae are embedded<sup><xref rid="r04" ref-type="bibr">4</xref>,<xref rid="r18" ref-type="bibr">18</xref></sup>.</p><p>The biofilm-forming ability of clinical isolates of <italic>C. albicans, C. krusei, C.
tropicalis</italic> and <italic>C. glabrata</italic> recovered from multiple species
candidimia lesions, was evaluated by measuring absorbance of the de-staining solution
containing crystal violet dye (crystal violet assay). The present showed <italic>C.
glabrata</italic> forming thickest slim layer on acrylic resins strip followed by
<italic>C. krusei,</italic><italic>C. tropicalis</italic> and C<italic>. albicans,</italic> contrasting with the
findings of previous researchers<sup><xref rid="r09" ref-type="bibr">9</xref>,<xref rid="r24" ref-type="bibr">24</xref></sup>. However Silva, et al.<sup><xref rid="r23" ref-type="bibr">23</xref></sup> (2009) reported that <italic>C.
glabrata</italic> biofilms matrix was high in both protein and carbohydrates, which
that probably enabled it to adsorbed more amount of crystal violet contents.</p><p>Shin, et al.<sup><xref rid="r22" ref-type="bibr">22</xref></sup> (2002) observed that
biofilm formation was most frequent for isolates of <italic>C. tropicalis </italic>(80%), followed by <italic>C. parapsilosis</italic> (73%), <italic>C. glabrata </italic>(28%), and <italic>C. albicans</italic> (8%). This finding contrasts with the present
adhesion studies, probably due to the sources of isolates. Different strains of the same
<italic>Candida</italic> species were found to be different in their ability to form
biofilms was also reported in this study, indicating "strong" and "weak"
biofilms-forming strains might exist within each <italic>Candida </italic>species<sup><xref rid="r26" ref-type="bibr">26</xref></sup>. Shin, et
al.<sup><xref rid="r22" ref-type="bibr">22</xref></sup> (2002) also reported that
biofilms-forming ability was greater for NCAC than for <italic>albicans</italic> species
using similar protocol.</p><p>Presence of one species of microorganism on a surface can promote the adhesion of
another<sup><xref rid="r14" ref-type="bibr">14</xref></sup>. Thein, et
al.<sup><xref rid="r25" ref-type="bibr">25</xref></sup> (2007) reported
competitive interaction in a dual-species biofilms of <italic>C. albicans</italic> and
<italic>C. krusei</italic>; whereas Pereira-Cenci, et al.<sup><xref rid="r20" ref-type="bibr">20</xref></sup> (2008) did not report competitive interaction in a
dual-species biofilms of <italic>C. albicans</italic> and <italic>C. glabrata</italic>.
In the present study, the highest degree of slim production was seen with the
combination of <italic>C. albicans</italic> and <italic>C. glabrata</italic>, followed
by the combination of <italic>C. albicans, C. tropicalis</italic> and <italic>C.
glabrata</italic>, and the lowest slim production was seen with the multi-species
biofilms of all the four species of <italic>Candida</italic>. Presence of <italic>C.
albicans</italic> in multi-species biofilms increased the production of slim when
inoculated with all other NCAC species; whereas <italic>C. tropicalis</italic> retarded
the production of slim under multi-species condition except with <italic>C.
albicans</italic>. In this way, C<italic>. albicans</italic> might be able to
successfully provide a substratum to the NCAC species on the acrylic prosthesis.</p><p>One of the most significant features of microbial biofilms is its resistance to a
variety of antimicrobial agents. Studies have demonstrated drug resistance when
<italic>Candida</italic> biofilms are even grown on surfaces like denture acrylic.
The possible mechanisms of biofilm resistance to antimicrobial agents are: restricted
penetration of drugs through the biofilms matrix; phenotypic switching, surface-induced
expression of resistance genes and a small number of "persistent" cells<sup><xref rid="r08" ref-type="bibr">8</xref></sup>. Presence of two or more species in a
biofilm could aggregate these factors. Synergistic effects of these factors can pose
major problems to the clinicians.</p></sec><sec sec-type="conclusions"><title>CONCLUSION</title><p>Based on the results of this study, biofilm-forming ability was found greater for NCAC
than for <italic>albicans</italic> species, isolated from multi-species oral candidiasis
of the neutropenic patients. Presence of <italic>C. albicans</italic> in multi-species
biofilms increased the slim production with all other NCAC species, whereas <italic>C.
tropicalis</italic> impeded the production of slim under multi-species condition
except for <italic>C. albicans.</italic></p></sec></body><back><ack><sec><title>ACKNOWLEDGEMENTS</title><p>Authors are greatly thankful to Dr. P.V. Wanjari, Honorable Dean, Modern Dental
College and Research Centre, Indore, for providing academic and materialistic
supports, without which it was hard to perform this work. We also acknowledge the
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