Biofilm formation and genetic variability of BCR1 gene in the Candida parapsilosis complex

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M: molecular weight marker (100-pb DNA ladder, Fermentas, Madrid, Spain). (I): C. albicans strains. (II): C. glabrata strains; S1’: C. albicans ATCC 90028; S2: C. glabrata ATCC 90030; SC: C. albicans SC5314; 22 and 17: vaginal C. albicans and C. glabrata strains, respectively." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Emira Noumi, Mejdi Snoussi, Inès Noumi, Fatma Saghrouni, Mahjoub Aouni, Eulogio Valentin" "autores" => array:6 [ 0 => array:2 [ "nombre" => "Emira" "apellidos" => "Noumi" ] 1 => array:2 [ "nombre" => "Mejdi" "apellidos" => "Snoussi" ] 2 => array:2 [ "nombre" => "Inès" "apellidos" => "Noumi" ] 3 => array:2 [ "nombre" => "Fatma" "apellidos" => "Saghrouni" ] 4 => array:2 [ "nombre" => "Mahjoub" "apellidos" => "Aouni" ] 5 => array:2 [ "nombre" => "Eulogio" "apellidos" => "Valentin" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1130140614000813?idApp=UINPBA00004N" "url" => "/11301406/0000003200000003/v1_201507300100/S1130140614000813/v1_201507300100/en/main.assets" ] "en" => array:21 [ "idiomaDefecto" => true "cabecera" => "Original article" "titulo" => "Biofilm formation and genetic variability of BCR1 gene in the Candida parapsilosis complex" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "180" "paginaFinal" => "184" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Rogelio de J. Treviño-Rangel, Irám P. Rodríguez-Sánchez, Adrián G. Rosas-Taraco, Romel Hernández-Bello, José G. González, Gloria M. González" "autores" => array:6 [ 0 => array:3 [ "nombre" => "Rogelio de J." "apellidos" => "Treviño-Rangel" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 1 => array:3 [ "nombre" => "Irám P." "apellidos" => "Rodríguez-Sánchez" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 2 => array:3 [ "nombre" => "Adrián G." "apellidos" => "Rosas-Taraco" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "c" "identificador" => "aff0015" ] ] ] 3 => array:3 [ "nombre" => "Romel" "apellidos" => "Hernández-Bello" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 4 => array:3 [ "nombre" => "José G." 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"apellidos" => "González" "email" => array:1 [ 0 => "gmglez@yahoo.com.mx" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "*" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:4 [ 0 => array:3 [ "entidad" => "Departamento de Microbiología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Departamento de Genética, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Departamento de Inmunología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico" "etiqueta" => "c" "identificador" => "aff0015" ] 3 => array:3 [ "entidad" => "Hospital Universitario, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico" "etiqueta" => "d" "identificador" => "aff0020" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Formación de biopelícula y variabilidad genética del gen BCR1 en el complejo Candida parapsilosis" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 998 "Ancho" => 2488 "Tamanyo" => 173456 ] ] "descripcion" => array:1 [ "en" => "Biofilm quantification in clinical isolates of C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis by crystal violet staining (a) and metabolic activity (b). The bars represent the optical density mean value of each group. The standard deviations are shown." ] ] ] "textoCompleto" => "The Candida parapsilosis complex (C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis) currently stands out as the second or third most common yeast species isolated from blood cultures in Latin America, Canada, Europe and Asia,<a class="elsevierStyleCrossRefs" href="#bib0160">1,9,15</a> becoming an important focus of attention, particularly among neonates and immunocompromised patients.<a class="elsevierStyleCrossRef" href="#bib0310">31</a> These opportunistic pathogens have been recovered from the hands of health-care workers,<a class="elsevierStyleCrossRef" href="#bib0160">1</a> and are well known as a cause of bloodstream infections associated with parenteral hyperalimentation and intravascular devices due to their capability to form biofilms.<a class="elsevierStyleCrossRef" href="#bib0260">21</a>Biofilms are complex surface-associated microbial communities embedded within an extracellular matrix. They are considered the most prevalent growth form of microorganisms.<a class="elsevierStyleCrossRef" href="#bib0275">24</a> Biofilm formation is an important virulence factor for a variety of Candida species, including C. parapsilosis, as it confers significant tolerance to antifungals and protects yeasts cells from host immune responses,<a class="elsevierStyleCrossRef" href="#bib0280">25</a> whereby biofilms themselves are a reservoir for persistent infections.<a class="elsevierStyleCrossRef" href="#bib0165">2</a>BCR1 (Biofilm and Cell wall Regulator 1) is a conserved fungal transcription factor required for biofilm formation in both Candida albicans and C. parapsilosis.<a class="elsevierStyleCrossRefs" href="#bib0175">4,16,18</a> Some major targets of Bcr1 in C. albicans include genes coding for adhesins and cell-wall proteins, suggesting that Bcr1 is involved in the early adhesion stage of biofilm development.<a class="elsevierStyleCrossRefs" href="#bib0180">5,16,18</a> However, it was demonstrated that the biofilm formation process in C. parapsilosis could be dependent and independent of BCR1.<a class="elsevierStyleCrossRef" href="#bib0255">20</a>The aim of this study was to quantify biofilm formation of a subset of 65 clinical isolates of the C. parapsilosis complex by two different methodologies: crystal violet staining and tetrazolium (XTT) reduction assay, as well as to analyze the nucleotide sequence of a fragment of the BCR1 gene looking for a possible association between the biofilm forming phenotype and genetic variants of the gene segment analyzed.Materials and methodsIdentification of isolatesA total of 65 clinical isolates of C. parapsilosis from a previous work<a class="elsevierStyleCrossRef" href="#bib0305">30</a> were included in this study. These were grown at 37°C for 24h on Potato-dextrose agar (PDA) (Difco, USA). The isolates were initially identified as C. parapsilosis by API 20C AUX strips (bioMérieux, Mexico) and standard morphological methods. Re-identification of the isolates was then performed using RFLP-BanI.<a class="elsevierStyleCrossRef" href="#bib0295">28</a> The isolate study collection consisted of 29 isolates each of C. parapsilosis sensu stricto and C. orthopsilosis, and 4 isolates of C. metapsilosis. A blood origin accounted for 48% of the isolates, while the remaining 52% were as follows: nail (9 isolates), skin (8 isolates), peritoneal fluid (7 isolates), urine (3 isolates), bronchial fluid (2 isolates), and 1 isolate each from spinal fluid, bile and otic secretion. Type strains C. parapsilosis sensu stricto ATCC 22019, C. orthopsilosis ATCC 96139, and C. metapsilosis ATCC 96144 were used as quality controls. All isolates were stored as suspensions in sterile distilled water at room temperature until testing.Biofilm induction and quantitationThe growth conditions and biofilm formation evaluated by crystal violet staining and XTT reduction assays were done according to the methodology described by Melo et al.<a class="elsevierStyleCrossRef" href="#bib0225">14</a>. Strains were initially grown on Sabouraud-dextrose agar (SDA) (Difco, Detroit, MI, USA) at 37°C for 24h, and one colony of each strain was further subcultured in RPMI 1640 broth medium with l-glutamine (Hardy Diagnostics, Santa Maria, CA, USA) at 37°C for 18h at 120–200rpm. The cells were harvested, washed twice with PBS, and adjusted to a concentration of 1×107cells/ml in RPMI 1640 medium. An aliquot of 100μl of the cell suspensions were transferred into each well of a sterile flat-bottomed 96-well polystyrene Corning Costar 9018 plate (Costar, Corning Incorporated, NY, USA). The plates were incubated at 37°C for 1.5h at 75rpm, washed with 150μl of PBS and then 100μl of fresh RPMI 1640 medium was added to each well. The plates were finally incubated at 37°C for 72h at 75rpm to allow biofilm growth. Test medium without cells was added to the final well of each plate as the negative control.To determine bulk biofilm formation, the crystal violet staining assay was performed. Briefly, after biofilm formation, each well was washed twice with 200μl of PBS and the plates were dried at 37°C for 20min. The washed biofilms were stained with 110μl of 0.4% aqueous crystal violet solution for 45min. The wells were then washed three times with 200μl of sterile distilled water and then faded with 200μl of 95% ethanol. After 45min, 100μl of detaining solution from each sample was transferred to a new plate and measured with a spectrophotometer plate reader at 595nm. The optical density (OD) values of the negative controls were subtracted from the values of the test wells in order to eliminate background interference. On the other hand, biofilm metabolic activity was determined by the XTT (tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino)-carbonyl-2H-tetrazoliumhydroxide) (Sigma, USA) reduction assay. After biofilm formation, the plate wells were washed twice with 200μl of PBS, then 200μl of PBS and 12μl of XTT-menadione solution were added to each well. The XTT-menadione solution was prepared on each day of testing by adding 1.5ml of XTT (1mg/ml in sterile saline) to 300μl of menadione solution (0.4mM in acetone; Sigma, St Louis, MO, USA). Plates were incubated at 37°C in darkness for 5h. Afterwards, 100μl of the reaction solution was transferred to a new plate and the concentration of the formazan product was spectrophotometrically determined at 490nm. The OD values of the negative control wells were subtracted from the values of the test wells. The amount of biofilm formed by an isolate was categorized as high (OD490≥0.1), low (0.025≤OD490<0.1) and no biofilm formation (OD490<0.025), according to the criteria previously established by Pannanusorn et al.<a class="elsevierStyleCrossRef" href="#bib0250">19</a>BCR1 gene amplification and sequencingYeast genomic DNA of each clinical isolate was extracted from a homogenized pellet using the phenol-chloroform method,<a class="elsevierStyleCrossRef" href="#bib0270">23</a> and then resuspended in nuclease-free water. Total genomic DNA was treated with RNase I (Invitrogen, USA) for 30min at 37°C to remove RNA traces, and the quality and integrity were assessed by standard spectrophotometric and elecrophoretic methods, respectively. Due to the fact that BCR1 gene sequence or trace archives of C. metapsilosis were not available at sequence data bases at the moment the experiments were done, the design of a consensus primer set to amplify BCR1 in C. parapsilosis complex (Sense 5′-AAA ACA CAC YGG TGA ACG AC-3′ and Anti-sense 5′-TCA CTC ATK GCT GAT CTT GG-3′) was based on the available highly conserved sequences previously reported for C. parapsilosis strain CDC317 (accession number: <a href="ncbi-n:HE605206">HE605206</a>) and C. orthopsilosis strain Co 90-125 (accession number: <a href="ncbi-n:HE681722">HE681722</a>) at GenBank (<a href="http://www.ncbi.nlm.nih.gov/">www.ncbi.nlm.nih.gov</a>) using the online primer-3 tool (<a href="http://biotools.umassmed.edu/bioapps/primer3_www.cgi">http://biotools.umassmed.edu/bioapps/primer3_www.cgi</a>). This primer set was used at 5μM with the commercial kit PCR master mix of Promega (USA). The amplification reactions were designed in a final volume of 25μl and carried out in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA). The standardized cycling parameters were as follows: 1 initial denaturation step of 4min at 94°C, 42 cycles of 1min at 94°C, 30s at 57°C, 90s at 72°C, and finally an elongation step of 6min at 72°C. The PCR products were visualized on 2% agarose gels stained with ethidium bromide and visualized under UV light (UVP BioImaging Systems, EpiChemi 3 Darkroom UVP Inc., Upland, CA). Amplicons were sequenced using Big Dye terminator cycle sequencing kit v3.1 using the same PCR primers. The reactions were analyzed in the ABI PRISM 3100 Genetic Analyzer using the Sequencing Analysis Software v5.3 of Applied Biosystems. The obtained sequences of each isolate were aligned using the CLUSTAL-W program and GeneStudio Pro software (GeneStudio, Inc., Suwanee, GA, USA), followed by manual corrections if needed. Sequences were then annotated using Sequin software from NCBI and the nucleotidic sequences were finally deposited in GenBank under the following accession numbers: <a href="ccdc:KJ610843">KJ610843</a> to <a href="ccdc:KJ610856">KJ610856</a>.StatisticsThe biofilm quantifications were carried out twice at different times, with two technical replicates each time. The OD values were expressed as means±standard deviation (SD) for each set of data determined and were compared using Student's t test (SPSS v17.0 for Windows; SPSS Inc., Chicago, IL). The biofilm categorization as high, low and negative biofilm production was compared among the C. parapsilosis species complex, clinical origin and genetic variants of BCR1 (looking for a possible association) by Pearson Chi-squared test; a P-value ≤0.05 was considered significant. The graphics were performed on GraphPad Prism v5.03 for Windows (GraphPad Software, San Diego, CA).ResultsBiofilm quantification by crystal violet staining is showed in <a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>(a). In this assay, the mean OD595 value and range for each species were as follows: C. parapsilosis sensu stricto, 0.121 (SD+0.124) with a range of 0.014–0.571; C. orthopsilosis, 0.082 (SD+0.076) with a range of 0.006–0.293; C. metapsilosis, 0.077 (SD+0.068) with a range of 0.013–0.175. C. parapsilosis sensu stricto showed the highest biofilm production as determined through crystal violet staining.<elsevierMultimedia ident="fig0005"></elsevierMultimedia><a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>(b) depicts the biofilm metabolic activity of the tested isolates. In this assay, the mean OD490 value and range for each species were as follows: C. parapsilosis sensu stricto, 0.075 (SD+0.045) with a range of 0.028–0.247; C. orthopsilosis, 0.092 (SD+0.054) with a range of 0.014–0.235; C. metapsilosis, 0.076 (SD+0.035) with a range of 0.049–0.135. C. orthopsilosis showed the highest biofilm production as determined through XTT reduction assay. Additionally, according to Pannanusorn et al. criteria,<a class="elsevierStyleCrossRef" href="#bib0250">19</a> 62 isolates (95.4%) produced biofilm, 26.2% were high biofilm producers, while 69.2% were categorized as low biofilm producers. Biofilms were predominantly highly produced by C. orthopsilosis (40%), whilst C. parapsilosis sensu stricto was mostly a low biofilm producer (86.7%). The non-biofilm producer phenotype was detected in just 3 isolates of C. orthopsilosis (4.6%) (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). C. parapsilosis sensu stricto was statistically associated with low biofilm production (P=0.0108), while C. orthopsilosis was significantly associated with both high and low biofilm production (P=0.0386 and P=0.0045, respectively).<elsevierMultimedia ident="tbl0005"></elsevierMultimedia>All the isolated DNAs were under the quality and quantity standards for PCR reaction. The designed consensus primer set amplified a fragment of BCR1 gene. These amplicons included a segment of exon and intron. From all the isolates a PCR product was generated. The nucleotide sequence analysis of amplicons corresponded to a fragment of the BCR1 gene previously reported in the BLAST analysis. These sequences showed genetic variability among the analyzed species. A total of eight genetic variants (designed as: Co-1 to 8) ranging from 913 to 1025bp, were detected in C. orthopsilosis, while C. metapsilosis presented two distinct genotypes of 872bp (Cm-1) and 984bp (Cm-2); moreover, genetic variability was not detected in C. parapsilosis sensu stricto, which amplified an approximate 872 bp-product (Cp-1).In the obtained sequences, we located the section corresponding to the exon, which was aligned. The open reading frame (ORF) was deducted from it, and also this sequence (supplementary material, Fig. S1) was aligned. According to the fragment of BCR1 exon analysis, the sequence of C. parapsilosis ATCC 22019 was identical to all C. parapsilosis sensu stricto strains (Cp-1). Moreover, the sequence of C. metapsilosis ATCC 96144 was the same to Cm-2, and 68.14% similar with respect to Cm-1, which is equal to the C. parapsilosis sensu stricto strains. On the other hand, the fragment of BCR1 exon of C. orthopsilosis ATCC 96139 was identical to those of C. parapsilosis sensu stricto strains, and conserved a homology ranging from 60.14% to 79.45% with respect to the eight genetic variants of C. orthopsilosis (Co-1 to 8). Finally, the most frequent variant of the analyzed fragment of BCR1 exon in C. parapsilosis complex, with 49.2% (32 isolates) was shared by C. parapsilosis ATCC 22019, Cp-1, C. orthopsilosis ATCC 96139, and Cm-1, which at the same time is identical to the deposited sequence of C. parapsilosis strain CDC317 (GenBank accession number: <a href="ncbi-n:HE605206">HE605206</a>). Finally, statistical associations between variants of the analyzed segment of BCR1 gene exon and the biofilm forming phenotypes of the isolates were analyzed by Pearson Chi-squared test, but were not found. Different genetic variants of BCR1 were classified within more than one biofilm forming phenotype, without any evident pattern of association with statistical support.DiscussionThe capability of Candida spp. to form biofilms may confer an ecological advantage to yeasts, aiding their survival as human commensals and pathogens. This may also be responsible for making them particularly well adapted to colonization of host tissues and indwelling devices.<a class="elsevierStyleCrossRef" href="#bib0275">24</a> To date, several methods for assessing Candida biofilm production have been reported.<a class="elsevierStyleCrossRef" href="#bib0170">3</a> In the present study, we evaluated the biofilm forming capability of a subset of 65 clinical isolates of the C. parapsilosis complex by two different techniques, crystal violet staining and XTT reduction assay. Crystal violet staining is probably the most reliable assay for determining bulk biofilm formation because it considers both metabolically-active and inactive cells of biofilms.<a class="elsevierStyleCrossRef" href="#bib0265">22</a> Another method that has been widely used to quantify Candida biofilms in vitro is XTT reduction assay,<a class="elsevierStyleCrossRef" href="#bib0210">11</a> which specifically considers the biofilm metabolically-active cells through XTT reduction by mitochondrial dehydrogenases.The study of biofilm forming capability among the C. parapsilosis species complex have not yet been clearly understood due to apparently contradictory findings. Song et al.<a class="elsevierStyleCrossRef" href="#bib0285">26</a> studied the capability of 159 isolates of the C. parapsilosis complex to produce biofilm through a spectrophotometric approach in which cultivated biofilms were incubated without agitation, and transmittance values were measured without previous staining at 405nm. They reported that C. parapsilosis sensu stricto (formerly C. parapsilosis group I) was the only species of the complex able to form biofilms. Later, Tavanti et al.<a class="elsevierStyleCrossRef" href="#bib0300">29</a> using the same methodology previously mentioned with few modifications, reported that none of the 33 C. orthopsilosis clinical isolates they studied could form biofilms in vitro. In contrast, Lattif et al.<a class="elsevierStyleCrossRef" href="#bib0220">13</a> based on XTT reduction assay, dry weight measurement, scanning electron microscopy and confocal laser scanning microscopy of 10 clinical isolates each of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis, established that all three species were able to form biofilms, which were similar in surface topography and architecture. Finally, Melo et al.<a class="elsevierStyleCrossRef" href="#bib0225">14</a> confirmed the biofilm forming capability of the C. parapsilosis species complex through the analysis of 20 isolates (7 C. parapsilosis sensu stricto, 8 C. orthopsilosis and 5 C. metapsilosis) by crystal violet staining, XTT reduction assay and scanning electron microscopy.The results we obtained about the biofilm forming capability of all three C. parapsilosis species complex on polystyrene microtiter plates under our experimental conditions are in contrast to those reported by Song et al.<a class="elsevierStyleCrossRef" href="#bib0285">26</a> and Tavanti et al.<a class="elsevierStyleCrossRef" href="#bib0300">29</a>, and in agreement with those reported earlier by Lattif et al.<a class="elsevierStyleCrossRef" href="#bib0220">13</a> and Melo et al.<a class="elsevierStyleCrossRef" href="#bib0225">14</a>. Although P values were not significant, according to crystal violet staining, the rank scale for biofilm production was C. parapsilosis sensu stricto>C. orthopsilosis>C. metapsilosis, while based on XTT reduction assay the rank was C. orthopsilosis>C. metapsilosis≥C. parapsilosis sensu stricto. Otherwise, the origin and selection of isolates can be one factor that influences biofilm formation in Candida spp.<a class="elsevierStyleCrossRefs" href="#bib0195">8,10</a> Recent reports have demonstrated that blood isolates produce greater quantities of biofilm compared with other clinical origins.<a class="elsevierStyleCrossRef" href="#bib0215">12</a> However, we have not found a significant statistical difference between the biofilm forming capability and the origin of the tested isolates, agreeing with Silva et al.<a class="elsevierStyleCrossRef" href="#bib0275">24</a>The microbial biofilm formation is a complex biological process finely regulated by inherent genetic mechanisms of the participating organisms. One of the master regulators of this phenomenon is the zinc finger transcription factor Bcr1.<a class="elsevierStyleCrossRef" href="#bib0185">6</a> It was identified in screens for mutant strains defective in biofilm formation on abiotic surfaces<a class="elsevierStyleCrossRefs" href="#bib0235">16,17</a> and in adherence to a silicone substrate.<a class="elsevierStyleCrossRef" href="#bib0190">7</a> Furthermore, Bcr1 is required for expression of several cell surface protein genes, such as ALS1, ALS3, and HWP1, which are the major functional Bcr1 targets.<a class="elsevierStyleCrossRefs" href="#bib0235">16,17</a> Recently, Srikantha et al.<a class="elsevierStyleCrossRef" href="#bib0290">27</a> demonstrated that BCR1 gene conferred impermeability, impenetrability, and drug resistance to a/α biofilms of C. albicans. Particularly in C. parapsilosis, biofilm formation is both dependent and independent of BCR1, but even in strains which showed a BCR1 independent biofilm phenotype BCR1 has alternative physiological functions.<a class="elsevierStyleCrossRef" href="#bib0255">20</a> We did not find any association between a particular genetic variant of the analyzed BCR1 fragment and a biofilm forming phenotype of the C. parapsilosis complex isolates studied.Conflict of interestThe authors have no conflicts of interest to declare" "textoCompletoSecciones" => array:1 [ "secciones" => array:10 [ 0 => array:3 [ "identificador" => "xres536892" "titulo" => "Abstract" "secciones" => array:5 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Background" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Aims" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Methods" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Results" ] 4 => array:2 [ "identificador" => "abst0025" "titulo" => "Conclusions" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec557033" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres536893" "titulo" => "Resumen" "secciones" => array:5 [ 0 => array:2 [ "identificador" => "abst0030" "titulo" => "Antecedentes" ] 1 => array:2 [ "identificador" => "abst0035" "titulo" => "Objetivos" ] 2 => array:2 [ "identificador" => "abst0040" "titulo" => "Métodos" ] 3 => array:2 [ "identificador" => "abst0045" "titulo" => "Resultados" ] 4 => array:2 [ "identificador" => "abst0050" "titulo" => "Conclusiones" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec557034" "titulo" => "Palabras clave" ] 4 => array:3 [ "identificador" => "sec0005" "titulo" => "Materials and methods" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "sec0010" "titulo" => "Identification of isolates" ] 1 => array:2 [ "identificador" => "sec0015" "titulo" => "Biofilm induction and quantitation" ] 2 => array:2 [ "identificador" => "sec0020" "titulo" => "BCR1 gene amplification and sequencing" ] 3 => array:2 [ "identificador" => "sec0025" "titulo" => "Statistics" ] ] ] 5 => array:2 [ "identificador" => "sec0030" "titulo" => "Results" ] 6 => array:2 [ "identificador" => "sec0035" "titulo" => "Discussion" ] 7 => array:2 [ "identificador" => "sec0040" "titulo" => "Conflict of interest" ] 8 => array:2 [ "identificador" => "xack182066" "titulo" => "Acknowledgements" ] 9 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2014-06-08" "fechaAceptado" => "2014-11-11" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec557033" "palabras" => array:5 [ 0 => "Candida parapsilosis complex" 1 => "Biofilm" 2 => "Crystal violet staining" 3 => "XTT" 4 => "BCR1" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec557034" "palabras" => array:5 [ 0 => "Complejo Candida parapsilosis" 1 => "Biopelícula" 2 => "Tinción con cristal violeta" 3 => "XTT" 4 => "BCR1" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:3 [ "titulo" => "Abstract" "resumen" => "BackgroundCandida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis are cryptic species that belong to the C. parapsilosis complex, which has been increasingly associated to fungemia in various geographic regions, principally due to the capability of these yeasts to form biofilms on indwelling medical devices. BCR1 is one of the most studied genes related to Candida spp. biofilms. AimsTo evaluate the biofilm forming capability of a subset of 65 clinical isolates of the C. parapsilosis complex using two conventional approaches, and to look for an association between the biofilm forming phenotype and genetic variants of a fragment of BCR1. MethodsThe biofilm determination was carried out by crystal violet staining and tetrazolium reduction assay. On the other hand, a segment of BCR1 gene was sequenced by Sanger methodology. ResultsC. parapsilosis sensu stricto was statistically associated with a low biofilm production phenotype, while C. orthopsilosis was significantly associated with both phenotypes (high and low biofilm producers). According to the BCR1 sequence analysis, genetic variability was detected in C. orthopsilosis and C. metapsilosis without a particular biofilm formation phenotype association. ConclusionsUnder the adopted experimental design, C. parapsilosis sensu stricto was associated with the low biofilm phenotype and C. orthopsilosis with both phenotypes (high and low biofilm producers). On the other hand, an association between a biofilm forming phenotype and a particular genetic variant of the analyzed BCR1 fragment was not found." "secciones" => array:5 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Background" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Aims" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Methods" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Results" ] 4 => array:2 [ "identificador" => "abst0025" "titulo" => "Conclusions" ] ] ] "es" => array:3 [ "titulo" => "Resumen" "resumen" => "AntecedentesCandida parapsilosis sensu stricto, Candida orthopsilosis y Candida metapsilosis son especies crípticas que integran el complejo C. parapsilosis, asociado de forma creciente a fungemia en diversas regiones geográficas. Dicho crecimiento se debe principalmente a la capacidad de estas levaduras de crear biopelículas en los dispositivos médicos. El gen BCR1 es uno de los más estudiados en las biopelículas de Candida spp. ObjetivosEvaluar la capacidad de formación de biopelícula de un conjunto de 65 aislamientos clínicos del complejo C. parapsilosis mediante dos metodologías convencionales, así como establecer una posible asociación entre el fenotipo productor de la biopelícula y las variantes genéticas de un fragmento de BCR1. MétodosLa determinación de la presencia de biopelícula se llevó a cabo mediante tinción con cristal violeta y el análisis de reducción de la sal de tetrazolio. Además, se secuenció un segmento del gen BCR1 mediante el método Sanger. ResultadosC. parapsilosis sensu stricto presentó una asociación estadísticamente significativa con un fenotipo de baja producción de biopelícula, mientras que C. orthopsilosis tuvo una asociación estadísticamente significativa con ambos fenotipos (alta y baja producción de biopelícula). Según el análisis de la secuencia de BCR1, existe variabilidad genética en C. orthopsilosis y C. metapsilosis sin ninguna asociación particular a los fenotipos relacionados con la formación de biopelícula. ConclusionesBajo el diseño experimental adoptado, C. parapsilosis sensu stricto se asoció con un fenotipo de baja producción de biopelícula y C. orthopsilosis con ambos fenotipos (alta y baja producción de biopelícula). Por otra parte, no se encontró ninguna asociación estadísticamente significativa entre los fenotipos de formación de biopelícula y variantes genéticas particulares en el fragmento analizado de BCR1." "secciones" => array:5 [ 0 => array:2 [ "identificador" => "abst0030" "titulo" => "Antecedentes" ] 1 => array:2 [ "identificador" => "abst0035" "titulo" => "Objetivos" ] 2 => array:2 [ "identificador" => "abst0040" "titulo" => "Métodos" ] 3 => array:2 [ "identificador" => "abst0045" "titulo" => "Resultados" ] 4 => array:2 [ "identificador" => "abst0050" "titulo" => "Conclusiones" ] ] ] ] "apendice" => array:1 [ 0 => array:1 [ "seccion" => array:1 [ 0 => array:4 [ "apendice" => "The following are the supplementary data to this article:<elsevierMultimedia ident="upi0005"></elsevierMultimedia>" "etiqueta" => "Appendix A" "titulo" => "Supplementary data" "identificador" => "sec0050" ] ] ] ] "multimedia" => array:3 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 998 "Ancho" => 2488 "Tamanyo" => 173456 ] ] "descripcion" => array:1 [ "en" => "Biofilm quantification in clinical isolates of C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis by crystal violet staining (a) and metabolic activity (b). The bars represent the optical density mean value of each group. The standard deviations are shown." ] ] 1 => array:7 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:2 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="left" valign="top" scope="col">Species (No. of isolates) \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " colspan="6" align="center" valign="top" scope="col" style="border-bottom: 2px solid black">No. of isolates (%)</th></tr><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " colspan="2" align="left" valign="top" scope="col" style="border-bottom: 2px solid black">High biofilm production</th><th class="td" title="table-head " colspan="2" align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Low biofilm production</th><th class="td" title="table-head " colspan="2" align="left" valign="top" scope="col" style="border-bottom: 2px solid black">No presence of biofilm</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="table-entry " align="left" valign="top">C. parapsilosis sensu stricto (30) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">4 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(13.3) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">26 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(86.7)<a class="elsevierStyleCrossRef" href="#tblfn0005">*</a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(0) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " align="left" valign="top">C. orthopsilosis (30) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">12 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(40)<a class="elsevierStyleCrossRef" href="#tblfn0005">*</a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">15 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(50)<a class="elsevierStyleCrossRef" href="#tblfn0005">*</a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">3 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(10) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " align="left" valign="top">C. metapsilosis (5) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(20) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">4 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(80) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(0) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " colspan="7" align="left" valign="top"></td></tr><tr title="table-row"><td class="td" title="table-entry " align="left" valign="top">Total (65) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">17 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(26.2) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">45 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(69.2) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">3 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">(4.6) \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab863052.png" ] ] ] "notaPie" => array:1 [ 0 => array:3 [ "identificador" => "tblfn0005" "etiqueta" => "*" "nota" => "Statistical association (P≤0.05)." ] ] ] "descripcion" => array:1 [ "en" => "Categorization of biofilm formation of clinical isolates pertaining to C. parapsilosis complex, assessed by metabolic activity determination." ] ] 2 => array:5 [ "identificador" => "upi0005" "tipo" => "MULTIMEDIAECOMPONENTE" "mostrarFloat" => false "mostrarDisplay" => true "Ecomponente" => array:2 [ "fichero" => 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Original article

Biofilm formation and genetic variability of BCR1 gene in the Candida parapsilosis complex

Formación de biopelícula y variabilidad genética del gen BCR1 en el complejo Candida parapsilosis

Rogelio de J. Treviño-Rangela, Irám P. Rodríguez-Sánchezb, Adrián G. Rosas-Taracoc, Romel Hernández-Belloa, José G. Gonzálezd, Gloria M. Gonzáleza,

Corresponding author

gmglez@yahoo.com.mx

Corresponding author.

a Departamento de Microbiología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico

b Departamento de Genética, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico

c Departamento de Inmunología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico

d Hospital Universitario, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico

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They are considered the most prevalent growth form of microorganisms&#46;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">24</span></a> Biofilm formation is an important virulence factor for a variety of <span class="elsevierStyleItalic">Candida</span> species&#44; including <span class="elsevierStyleItalic">C&#46; parapsilosis</span>&#44; as it confers significant tolerance to antifungals and protects yeasts cells from host immune responses&#44;<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">25</span></a> whereby biofilms themselves are a reservoir for persistent infections&#46;<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">2</span></a></p><p id="par0015" class="elsevierStylePara elsevierViewall">BCR1 &#40;Biofilm and Cell wall Regulator 1&#41; is a conserved fungal transcription factor required for biofilm formation in both <span class="elsevierStyleItalic">Candida albicans</span> and <span class="elsevierStyleItalic">C&#46; parapsilosis</span>&#46;<a class="elsevierStyleCrossRefs" href="#bib0175"><span class="elsevierStyleSup">4&#44;16&#44;18</span></a> Some major targets of Bcr1 in <span class="elsevierStyleItalic">C&#46; albicans</span> include genes coding for adhesins and cell-wall proteins&#44; suggesting that Bcr1 is involved in the early adhesion stage of biofilm development&#46;<a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">5&#44;16&#44;18</span></a> However&#44; it was demonstrated that the biofilm formation process in <span class="elsevierStyleItalic">C&#46; parapsilosis</span> could be dependent and independent of <span class="elsevierStyleItalic">BCR1</span>&#46;<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">20</span></a></p><p id="par0020" class="elsevierStylePara elsevierViewall">The aim of this study was to quantify biofilm formation of a subset of 65 clinical isolates of the <span class="elsevierStyleItalic">C&#46; parapsilosis</span> complex by two different methodologies&#58; crystal violet staining and tetrazolium &#40;XTT&#41; reduction assay&#44; as well as to analyze the nucleotide sequence of a fragment of the <span class="elsevierStyleItalic">BCR1</span> gene looking for a possible association between the biofilm forming phenotype and genetic variants of the gene segment analyzed&#46;</p><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Materials and methods</span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Identification of isolates</span><p id="par0025" class="elsevierStylePara elsevierViewall">A total of 65 clinical isolates of <span class="elsevierStyleItalic">C&#46; parapsilosis</span> from a previous work<a class="elsevierStyleCrossRef" href="#bib0305"><span class="elsevierStyleSup">30</span></a> were included in this study&#46; These were grown at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 24<span class="elsevierStyleHsp" style=""></span>h on Potato-dextrose agar &#40;PDA&#41; &#40;Difco&#44; USA&#41;&#46; The isolates were initially identified as <span class="elsevierStyleItalic">C&#46; parapsilosis</span> by API 20C AUX strips &#40;bioM&#233;rieux&#44; Mexico&#41; and standard morphological methods&#46; Re-identification of the isolates was then performed using RFLP-BanI&#46;<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">28</span></a> The isolate study collection consisted of 29 isolates each of <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> and <span class="elsevierStyleItalic">C&#46; orthopsilosis</span>&#44; and 4 isolates of <span class="elsevierStyleItalic">C&#46; metapsilosis</span>&#46; A blood origin accounted for 48&#37; of the isolates&#44; while the remaining 52&#37; were as follows&#58; nail &#40;9 isolates&#41;&#44; skin &#40;8 isolates&#41;&#44; peritoneal fluid &#40;7 isolates&#41;&#44; urine &#40;3 isolates&#41;&#44; bronchial fluid &#40;2 isolates&#41;&#44; and 1 isolate each from spinal fluid&#44; bile and otic secretion&#46; Type strains <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> ATCC 22019&#44; <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> ATCC 96139&#44; and <span class="elsevierStyleItalic">C&#46; metapsilosis</span> ATCC 96144 were used as quality controls&#46; All isolates were stored as suspensions in sterile distilled water at room temperature until testing&#46;</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Biofilm induction and quantitation</span><p id="par0030" class="elsevierStylePara elsevierViewall">The growth conditions and biofilm formation evaluated by crystal violet staining and XTT reduction assays were done according to the methodology described by Melo et al&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">14</span></a>&#46; Strains were initially grown on Sabouraud-dextrose agar &#40;SDA&#41; &#40;Difco&#44; Detroit&#44; MI&#44; USA&#41; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 24<span class="elsevierStyleHsp" style=""></span>h&#44; and one colony of each strain was further subcultured in RPMI 1640 broth medium with <span class="elsevierStyleSmallCaps">l</span>-glutamine &#40;Hardy Diagnostics&#44; Santa Maria&#44; CA&#44; USA&#41; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 18<span class="elsevierStyleHsp" style=""></span>h at 120&#8211;200<span class="elsevierStyleHsp" style=""></span>rpm&#46; The cells were harvested&#44; washed twice with PBS&#44; and adjusted to a concentration of 1<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">7</span><span class="elsevierStyleHsp" style=""></span>cells&#47;ml in RPMI 1640 medium&#46; An aliquot of 100<span class="elsevierStyleHsp" style=""></span>&#956;l of the cell suspensions were transferred into each well of a sterile flat-bottomed 96-well polystyrene Corning Costar 9018 plate &#40;Costar&#44; Corning Incorporated&#44; NY&#44; USA&#41;&#46; The plates were incubated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 1&#46;5<span class="elsevierStyleHsp" style=""></span>h at 75<span class="elsevierStyleHsp" style=""></span>rpm&#44; washed with 150<span class="elsevierStyleHsp" style=""></span>&#956;l of PBS and then 100<span class="elsevierStyleHsp" style=""></span>&#956;l of fresh RPMI 1640 medium was added to each well&#46; The plates were finally incubated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 72<span class="elsevierStyleHsp" style=""></span>h at 75<span class="elsevierStyleHsp" style=""></span>rpm to allow biofilm growth&#46; Test medium without cells was added to the final well of each plate as the negative control&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall">To determine bulk biofilm formation&#44; the crystal violet staining assay was performed&#46; Briefly&#44; after biofilm formation&#44; each well was washed twice with 200<span class="elsevierStyleHsp" style=""></span>&#956;l of PBS and the plates were dried at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 20<span class="elsevierStyleHsp" style=""></span>min&#46; The washed biofilms were stained with 110<span class="elsevierStyleHsp" style=""></span>&#956;l of 0&#46;4&#37; aqueous crystal violet solution for 45<span class="elsevierStyleHsp" style=""></span>min&#46; The wells were then washed three times with 200<span class="elsevierStyleHsp" style=""></span>&#956;l of sterile distilled water and then faded with 200<span class="elsevierStyleHsp" style=""></span>&#956;l of 95&#37; ethanol&#46; After 45<span class="elsevierStyleHsp" style=""></span>min&#44; 100<span class="elsevierStyleHsp" style=""></span>&#956;l of detaining solution from each sample was transferred to a new plate and measured with a spectrophotometer plate reader at 595<span class="elsevierStyleHsp" style=""></span>nm&#46; The optical density &#40;OD&#41; values of the negative controls were subtracted from the values of the test wells in order to eliminate background interference&#46; On the other hand&#44; biofilm metabolic activity was determined by the XTT &#40;tetrazolium salt 2&#44;3-bis&#40;2-methoxy-4-nitro-5-sulfophenyl&#41;-5-&#40;phenylamino&#41;-carbonyl-2H-tetrazoliumhydroxide&#41; &#40;Sigma&#44; USA&#41; reduction assay&#46; After biofilm formation&#44; the plate wells were washed twice with 200<span class="elsevierStyleHsp" style=""></span>&#956;l of PBS&#44; then 200<span class="elsevierStyleHsp" style=""></span>&#956;l of PBS and 12<span class="elsevierStyleHsp" style=""></span>&#956;l of XTT-menadione solution were added to each well&#46; The XTT-menadione solution was prepared on each day of testing by adding 1&#46;5<span class="elsevierStyleHsp" style=""></span>ml of XTT &#40;1<span class="elsevierStyleHsp" style=""></span>mg&#47;ml in sterile saline&#41; to 300<span class="elsevierStyleHsp" style=""></span>&#956;l of menadione solution &#40;0&#46;4<span class="elsevierStyleHsp" style=""></span>mM in acetone&#59; Sigma&#44; St Louis&#44; MO&#44; USA&#41;&#46; Plates were incubated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C in darkness for 5<span class="elsevierStyleHsp" style=""></span>h&#46; Afterwards&#44; 100<span class="elsevierStyleHsp" style=""></span>&#956;l of the reaction solution was transferred to a new plate and the concentration of the formazan product was spectrophotometrically determined at 490<span class="elsevierStyleHsp" style=""></span>nm&#46; The OD values of the negative control wells were subtracted from the values of the test wells&#46; The amount of biofilm formed by an isolate was categorized as high &#40;OD<span class="elsevierStyleInf">490</span><span class="elsevierStyleHsp" style=""></span>&#8805;<span class="elsevierStyleHsp" style=""></span>0&#46;1&#41;&#44; low &#40;0&#46;025<span class="elsevierStyleHsp" style=""></span>&#8804;<span class="elsevierStyleHsp" style=""></span>OD<span class="elsevierStyleInf">490</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;1&#41; and no biofilm formation &#40;OD<span class="elsevierStyleInf">490</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;025&#41;&#44; according to the criteria previously established by Pannanusorn et al&#46;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">19</span></a></p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090"><span class="elsevierStyleItalic">BCR1</span> gene amplification and sequencing</span><p id="par0040" class="elsevierStylePara elsevierViewall">Yeast genomic DNA of each clinical isolate was extracted from a homogenized pellet using the phenol-chloroform method&#44;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">23</span></a> and then resuspended in nuclease-free water&#46; Total genomic DNA was treated with RNase I &#40;Invitrogen&#44; USA&#41; for 30<span class="elsevierStyleHsp" style=""></span>min at 37<span class="elsevierStyleHsp" style=""></span>&#176;C to remove RNA traces&#44; and the quality and integrity were assessed by standard spectrophotometric and elecrophoretic methods&#44; respectively&#46; Due to the fact that <span class="elsevierStyleItalic">BCR1</span> gene sequence or trace archives of <span class="elsevierStyleItalic">C&#46; metapsilosis</span> were not available at sequence data bases at the moment the experiments were done&#44; the design of a consensus primer set to amplify <span class="elsevierStyleItalic">BCR1</span> in <span class="elsevierStyleItalic">C&#46; parapsilosis</span> complex &#40;<span class="elsevierStyleItalic">Sense</span> 5&#8242;-AAA ACA CAC YGG TGA ACG AC-3&#8242; and <span class="elsevierStyleItalic">Anti-sense</span> 5&#8242;-TCA CTC ATK GCT GAT CTT GG-3&#8242;&#41; was based on the available highly conserved sequences previously reported for <span class="elsevierStyleItalic">C&#46; parapsilosis</span> strain CDC317 &#40;accession number&#58; <a href="ncbi-n:HE605206">HE605206</a>&#41; and <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> strain Co 90-125 &#40;accession number&#58; <a href="ncbi-n:HE681722">HE681722</a>&#41; at GenBank &#40;<a href="http://www.ncbi.nlm.nih.gov/">www&#46;ncbi&#46;nlm&#46;nih&#46;gov</a>&#41; using the online primer-3 tool &#40;<a href="http://biotools.umassmed.edu/bioapps/primer3_www.cgi">http&#58;&#47;&#47;biotools&#46;umassmed&#46;edu&#47;bioapps&#47;primer3&#95;www&#46;cgi</a>&#41;&#46; This primer set was used at 5<span class="elsevierStyleHsp" style=""></span>&#956;M with the commercial kit PCR master mix of Promega &#40;USA&#41;&#46; The amplification reactions were designed in a final volume of 25<span class="elsevierStyleHsp" style=""></span>&#956;l and carried out in a Veriti 96-Well Thermal Cycler &#40;Applied Biosystems&#44; Thermo Fisher Scientific Inc&#46;&#44; Waltham&#44; MA&#41;&#46; The standardized cycling parameters were as follows&#58; 1 initial denaturation step of 4<span class="elsevierStyleHsp" style=""></span>min at 94<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; 42 cycles of 1<span class="elsevierStyleHsp" style=""></span>min at 94<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; 30<span class="elsevierStyleHsp" style=""></span>s at 57<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; 90<span class="elsevierStyleHsp" style=""></span>s at 72<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; and finally an elongation step of 6<span class="elsevierStyleHsp" style=""></span>min at 72<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The PCR products were visualized on 2&#37; agarose gels stained with ethidium bromide and visualized under UV light &#40;UVP BioImaging Systems&#44; EpiChemi 3 Darkroom UVP Inc&#46;&#44; Upland&#44; CA&#41;&#46; Amplicons were sequenced using Big Dye terminator cycle sequencing kit v3&#46;1 using the same PCR primers&#46; The reactions were analyzed in the ABI PRISM 3100 Genetic Analyzer using the Sequencing Analysis Software v5&#46;3 of Applied Biosystems&#46; The obtained sequences of each isolate were aligned using the CLUSTAL-W program and GeneStudio Pro software &#40;GeneStudio&#44; Inc&#46;&#44; Suwanee&#44; GA&#44; USA&#41;&#44; followed by manual corrections if needed&#46; Sequences were then annotated using Sequin software from NCBI and the nucleotidic sequences were finally deposited in GenBank under the following accession numbers&#58; <a href="ccdc:KJ610843">KJ610843</a> to <a href="ccdc:KJ610856">KJ610856</a>&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Statistics</span><p id="par0045" class="elsevierStylePara elsevierViewall">The biofilm quantifications were carried out twice at different times&#44; with two technical replicates each time&#46; The OD values were expressed as means<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation &#40;SD&#41; for each set of data determined and were compared using Student&#39;s <span class="elsevierStyleItalic">t</span> test &#40;SPSS v17&#46;0 for Windows&#59; SPSS Inc&#46;&#44; Chicago&#44; IL&#41;&#46; The biofilm categorization as high&#44; low and negative biofilm production was compared among the <span class="elsevierStyleItalic">C&#46; parapsilosis</span> species complex&#44; clinical origin and genetic variants of <span class="elsevierStyleItalic">BCR1</span> &#40;looking for a possible association&#41; by Pearson Chi-squared test&#59; a <span class="elsevierStyleItalic">P</span>-value &#8804;0&#46;05 was considered significant&#46; The graphics were performed on GraphPad Prism v5&#46;03 for Windows &#40;GraphPad Software&#44; San Diego&#44; CA&#41;&#46;</p></span></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Results</span><p id="par0050" class="elsevierStylePara elsevierViewall">Biofilm quantification by crystal violet staining is showed in <a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#40;a&#41;&#46; In this assay&#44; the mean OD<span class="elsevierStyleInf">595</span> value and range for each species were as follows&#58; <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span>&#44; 0&#46;121 &#40;SD<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>0&#46;124&#41; with a range of 0&#46;014&#8211;0&#46;571&#59; <span class="elsevierStyleItalic">C&#46; orthopsilosis</span>&#44; 0&#46;082 &#40;SD<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>0&#46;076&#41; with a range of 0&#46;006&#8211;0&#46;293&#59; <span class="elsevierStyleItalic">C&#46; metapsilosis</span>&#44; 0&#46;077 &#40;SD<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>0&#46;068&#41; with a range of 0&#46;013&#8211;0&#46;175&#46; <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> showed the highest biofilm production as determined through crystal violet staining&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0055" class="elsevierStylePara elsevierViewall"><a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#40;b&#41; depicts the biofilm metabolic activity of the tested isolates&#46; In this assay&#44; the mean OD<span class="elsevierStyleInf">490</span> value and range for each species were as follows&#58; <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span>&#44; 0&#46;075 &#40;SD<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>0&#46;045&#41; with a range of 0&#46;028&#8211;0&#46;247&#59; <span class="elsevierStyleItalic">C&#46; orthopsilosis</span>&#44; 0&#46;092 &#40;SD<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>0&#46;054&#41; with a range of 0&#46;014&#8211;0&#46;235&#59; <span class="elsevierStyleItalic">C&#46; metapsilosis</span>&#44; 0&#46;076 &#40;SD<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>0&#46;035&#41; with a range of 0&#46;049&#8211;0&#46;135&#46; <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> showed the highest biofilm production as determined through XTT reduction assay&#46; Additionally&#44; according to Pannanusorn et al&#46; criteria&#44;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">19</span></a> 62 isolates &#40;95&#46;4&#37;&#41; produced biofilm&#44; 26&#46;2&#37; were high biofilm producers&#44; while 69&#46;2&#37; were categorized as low biofilm producers&#46; Biofilms were predominantly highly produced by <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> &#40;40&#37;&#41;&#44; whilst <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> was mostly a low biofilm producer &#40;86&#46;7&#37;&#41;&#46; The non-biofilm producer phenotype was detected in just 3 isolates of <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> &#40;4&#46;6&#37;&#41; &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> was statistically associated with low biofilm production &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;0108&#41;&#44; while <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> was significantly associated with both high and low biofilm production &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;0386 and <span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;0045&#44; respectively&#41;&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0060" class="elsevierStylePara elsevierViewall">All the isolated DNAs were under the quality and quantity standards for PCR reaction&#46; The designed consensus primer set amplified a fragment of <span class="elsevierStyleItalic">BCR1</span> gene&#46; These amplicons included a segment of exon and intron&#46; From all the isolates a PCR product was generated&#46; The nucleotide sequence analysis of amplicons corresponded to a fragment of the <span class="elsevierStyleItalic">BCR1</span> gene previously reported in the BLAST analysis&#46; These sequences showed genetic variability among the analyzed species&#46; A total of eight genetic variants &#40;designed as&#58; Co-1 to 8&#41; ranging from 913 to 1025<span class="elsevierStyleHsp" style=""></span>bp&#44; were detected in <span class="elsevierStyleItalic">C&#46; orthopsilosis</span>&#44; while <span class="elsevierStyleItalic">C&#46; metapsilosis</span> presented two distinct genotypes of 872<span class="elsevierStyleHsp" style=""></span>bp &#40;Cm-1&#41; and 984<span class="elsevierStyleHsp" style=""></span>bp &#40;Cm-2&#41;&#59; moreover&#44; genetic variability was not detected in <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span>&#44; which amplified an approximate 872 bp-product &#40;Cp-1&#41;&#46;</p><p id="par0065" class="elsevierStylePara elsevierViewall">In the obtained sequences&#44; we located the section corresponding to the exon&#44; which was aligned&#46; The open reading frame &#40;ORF&#41; was deducted from it&#44; and also this sequence &#40;supplementary material&#44; Fig&#46; S1&#41; was aligned&#46; According to the fragment of <span class="elsevierStyleItalic">BCR1</span> exon analysis&#44; the sequence of <span class="elsevierStyleItalic">C&#46; parapsilosis</span> ATCC 22019 was identical to all <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> strains &#40;Cp-1&#41;&#46; Moreover&#44; the sequence of <span class="elsevierStyleItalic">C&#46; metapsilosis</span> ATCC 96144 was the same to Cm-2&#44; and 68&#46;14&#37; similar with respect to Cm-1&#44; which is equal to the <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> strains&#46; On the other hand&#44; the fragment of <span class="elsevierStyleItalic">BCR1</span> exon of <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> ATCC 96139 was identical to those of <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> strains&#44; and conserved a homology ranging from 60&#46;14&#37; to 79&#46;45&#37; with respect to the eight genetic variants of <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> &#40;Co-1 to 8&#41;&#46; Finally&#44; the most frequent variant of the analyzed fragment of <span class="elsevierStyleItalic">BCR1</span> exon in <span class="elsevierStyleItalic">C&#46; parapsilosis</span> complex&#44; with 49&#46;2&#37; &#40;32 isolates&#41; was shared by <span class="elsevierStyleItalic">C&#46; parapsilosis</span> ATCC 22019&#44; Cp-1&#44; <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> ATCC 96139&#44; and Cm-1&#44; which at the same time is identical to the deposited sequence of <span class="elsevierStyleItalic">C&#46; parapsilosis</span> strain CDC317 &#40;GenBank accession number&#58; <a href="ncbi-n:HE605206">HE605206</a>&#41;&#46; Finally&#44; statistical associations between variants of the analyzed segment of <span class="elsevierStyleItalic">BCR1</span> gene exon and the biofilm forming phenotypes of the isolates were analyzed by Pearson Chi-squared test&#44; but were not found&#46; Different genetic variants of <span class="elsevierStyleItalic">BCR1</span> were classified within more than one biofilm forming phenotype&#44; without any evident pattern of association with statistical support&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Discussion</span><p id="par0070" class="elsevierStylePara elsevierViewall">The capability of <span class="elsevierStyleItalic">Candida</span> spp&#46; to form biofilms may confer an ecological advantage to yeasts&#44; aiding their survival as human commensals and pathogens&#46; This may also be responsible for making them particularly well adapted to colonization of host tissues and indwelling devices&#46;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">24</span></a> To date&#44; several methods for assessing <span class="elsevierStyleItalic">Candida</span> biofilm production have been reported&#46;<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">3</span></a> In the present study&#44; we evaluated the biofilm forming capability of a subset of 65 clinical isolates of the <span class="elsevierStyleItalic">C&#46; parapsilosis</span> complex by two different techniques&#44; crystal violet staining and XTT reduction assay&#46; Crystal violet staining is probably the most reliable assay for determining bulk biofilm formation because it considers both metabolically-active and inactive cells of biofilms&#46;<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">22</span></a> Another method that has been widely used to quantify <span class="elsevierStyleItalic">Candida</span> biofilms in vitro is XTT reduction assay&#44;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">11</span></a> which specifically considers the biofilm metabolically-active cells through XTT reduction by mitochondrial dehydrogenases&#46;</p><p id="par0075" class="elsevierStylePara elsevierViewall">The study of biofilm forming capability among the <span class="elsevierStyleItalic">C&#46; parapsilosis</span> species complex have not yet been clearly understood due to apparently contradictory findings&#46; Song et al&#46;<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">26</span></a> studied the capability of 159 isolates of the <span class="elsevierStyleItalic">C&#46; parapsilosis</span> complex to produce biofilm through a spectrophotometric approach in which cultivated biofilms were incubated without agitation&#44; and transmittance values were measured without previous staining at 405<span class="elsevierStyleHsp" style=""></span>nm&#46; They reported that <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> &#40;formerly <span class="elsevierStyleItalic">C&#46; parapsilosis</span> group I&#41; was the only species of the complex able to form biofilms&#46; Later&#44; Tavanti et al&#46;<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">29</span></a> using the same methodology previously mentioned with few modifications&#44; reported that none of the 33 <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> clinical isolates they studied could form biofilms in vitro&#46; In contrast&#44; Lattif et al&#46;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">13</span></a> based on XTT reduction assay&#44; dry weight measurement&#44; scanning electron microscopy and confocal laser scanning microscopy of 10 clinical isolates each of <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span>&#44; <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> and <span class="elsevierStyleItalic">C&#46; metapsilosis</span>&#44; established that all three species were able to form biofilms&#44; which were similar in surface topography and architecture&#46; Finally&#44; Melo et al&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">14</span></a> confirmed the biofilm forming capability of the <span class="elsevierStyleItalic">C&#46; parapsilosis</span> species complex through the analysis of 20 isolates &#40;7 <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span>&#44; 8 <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> and 5 <span class="elsevierStyleItalic">C&#46; metapsilosis</span>&#41; by crystal violet staining&#44; XTT reduction assay and scanning electron microscopy&#46;</p><p id="par0080" class="elsevierStylePara elsevierViewall">The results we obtained about the biofilm forming capability of all three <span class="elsevierStyleItalic">C&#46; parapsilosis</span> species complex on polystyrene microtiter plates under our experimental conditions are in contrast to those reported by Song et al&#46;<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">26</span></a> and Tavanti et al&#46;<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">29</span></a>&#44; and in agreement with those reported earlier by Lattif et al&#46;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">13</span></a> and Melo et al&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">14</span></a>&#46; Although <span class="elsevierStyleItalic">P</span> values were not significant&#44; according to crystal violet staining&#44; the rank scale for biofilm production was <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">C&#46; orthopsilosis</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">C&#46; metapsilosis</span>&#44; while based on XTT reduction assay the rank was <span class="elsevierStyleItalic">C&#46; orthopsilosis</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">C&#46; metapsilosis</span><span class="elsevierStyleHsp" style=""></span>&#8805;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span>&#46; Otherwise&#44; the origin and selection of isolates can be one factor that influences biofilm formation in <span class="elsevierStyleItalic">Candida</span> spp&#46;<a class="elsevierStyleCrossRefs" href="#bib0195"><span class="elsevierStyleSup">8&#44;10</span></a> Recent reports have demonstrated that blood isolates produce greater quantities of biofilm compared with other clinical origins&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">12</span></a> However&#44; we have not found a significant statistical difference between the biofilm forming capability and the origin of the tested isolates&#44; agreeing with Silva et al&#46;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">24</span></a></p><p id="par0085" class="elsevierStylePara elsevierViewall">The microbial biofilm formation is a complex biological process finely regulated by inherent genetic mechanisms of the participating organisms&#46; One of the master regulators of this phenomenon is the zinc finger transcription factor Bcr1&#46;<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">6</span></a> It was identified in screens for mutant strains defective in biofilm formation on abiotic surfaces<a class="elsevierStyleCrossRefs" href="#bib0235"><span class="elsevierStyleSup">16&#44;17</span></a> and in adherence to a silicone substrate&#46;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">7</span></a> Furthermore&#44; Bcr1 is required for expression of several cell surface protein genes&#44; such as <span class="elsevierStyleItalic">ALS1</span>&#44; <span class="elsevierStyleItalic">ALS3</span>&#44; and <span class="elsevierStyleItalic">HWP1</span>&#44; which are the major functional Bcr1 targets&#46;<a class="elsevierStyleCrossRefs" href="#bib0235"><span class="elsevierStyleSup">16&#44;17</span></a> Recently&#44; Srikantha et al&#46;<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">27</span></a> demonstrated that <span class="elsevierStyleItalic">BCR1</span> gene conferred impermeability&#44; impenetrability&#44; and drug resistance to a&#47;&#945; biofilms of <span class="elsevierStyleItalic">C&#46; albicans</span>&#46; Particularly in <span class="elsevierStyleItalic">C&#46; parapsilosis</span>&#44; biofilm formation is both dependent and independent of <span class="elsevierStyleItalic">BCR1</span>&#44; but even in strains which showed a <span class="elsevierStyleItalic">BCR1</span> independent biofilm phenotype <span class="elsevierStyleItalic">BCR1</span> has alternative physiological functions&#46;<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">20</span></a> We did not find any association between a particular genetic variant of the analyzed <span class="elsevierStyleItalic">BCR1</span> fragment and a biofilm forming phenotype of the <span class="elsevierStyleItalic">C&#46; parapsilosis</span> complex isolates studied&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Conflict of interest</span><p id="par0090" class="elsevierStylePara elsevierViewall">The authors have no conflicts of interest to declare</p></span></span>"
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              "titulo" => "Identification of isolates"
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              "titulo" => "Biofilm induction and quantitation"
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        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Background</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">Candida parapsilosis sensu stricto</span>&#44; <span class="elsevierStyleItalic">Candida orthopsilosis</span>&#44; and <span class="elsevierStyleItalic">Candida metapsilosis</span> are cryptic species that belong to the <span class="elsevierStyleItalic">C&#46; parapsilosis</span> complex&#44; which has been increasingly associated to fungemia in various geographic regions&#44; principally due to the capability of these yeasts to form biofilms on indwelling medical devices&#46; <span class="elsevierStyleItalic">BCR1</span> is one of the most studied genes related to <span class="elsevierStyleItalic">Candida</span> spp&#46; biofilms&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Aims</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">To evaluate the biofilm forming capability of a subset of 65 clinical isolates of the <span class="elsevierStyleItalic">C&#46; parapsilosis</span> complex using two conventional approaches&#44; and to look for an association between the biofilm forming phenotype and genetic variants of a fragment of <span class="elsevierStyleItalic">BCR1</span>&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Methods</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">The biofilm determination was carried out by crystal violet staining and tetrazolium reduction assay&#46; On the other hand&#44; a segment of <span class="elsevierStyleItalic">BCR1</span> gene was sequenced by Sanger methodology&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Results</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> was statistically associated with a low biofilm production phenotype&#44; while <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> was significantly associated with both phenotypes &#40;high and low biofilm producers&#41;&#46; According to the <span class="elsevierStyleItalic">BCR1</span> sequence analysis&#44; genetic variability was detected in <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> and <span class="elsevierStyleItalic">C&#46; metapsilosis</span> without a particular biofilm formation phenotype association&#46;</p></span> <span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Conclusions</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Under the adopted experimental design&#44; <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> was associated with the low biofilm phenotype and <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> with both phenotypes &#40;high and low biofilm producers&#41;&#46; On the other hand&#44; an association between a biofilm forming phenotype and a particular genetic variant of the analyzed <span class="elsevierStyleItalic">BCR1</span> fragment was not found&#46;</p></span>"
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        "resumen" => "<span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Antecedentes</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">Candida parapsilosis sensu stricto</span>&#44; <span class="elsevierStyleItalic">Candida orthopsilosis</span> y <span class="elsevierStyleItalic">Candida metapsilosis</span> son especies cr&#237;pticas que integran el complejo <span class="elsevierStyleItalic">C&#46; parapsilosis</span>&#44; asociado de forma creciente a fungemia en diversas regiones geogr&#225;ficas&#46; Dicho crecimiento se debe principalmente a la capacidad de estas levaduras de crear biopel&#237;culas en los dispositivos m&#233;dicos&#46; El gen <span class="elsevierStyleItalic">BCR1</span> es uno de los m&#225;s estudiados en las biopel&#237;culas de <span class="elsevierStyleItalic">Candida</span> spp&#46;</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Objetivos</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Evaluar la capacidad de formaci&#243;n de biopel&#237;cula de un conjunto de 65 aislamientos cl&#237;nicos del complejo <span class="elsevierStyleItalic">C&#46; parapsilosis</span> mediante dos metodolog&#237;as convencionales&#44; as&#237; como establecer una posible asociaci&#243;n entre el fenotipo productor de la biopel&#237;cula y las variantes gen&#233;ticas de un fragmento de <span class="elsevierStyleItalic">BCR1</span>&#46;</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">M&#233;todos</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">La determinaci&#243;n de la presencia de biopel&#237;cula se llev&#243; a cabo mediante tinci&#243;n con cristal violeta y el an&#225;lisis de reducci&#243;n de la sal de tetrazolio&#46; Adem&#225;s&#44; se secuenci&#243; un segmento del gen <span class="elsevierStyleItalic">BCR1</span> mediante el m&#233;todo Sanger&#46;</p></span> <span id="abst0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Resultados</span><p id="spar0045" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> present&#243; una asociaci&#243;n estad&#237;sticamente significativa con un fenotipo de baja producci&#243;n de biopel&#237;cula&#44; mientras que <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> tuvo una asociaci&#243;n estad&#237;sticamente significativa con ambos fenotipos &#40;alta y baja producci&#243;n de biopel&#237;cula&#41;&#46; Seg&#250;n el an&#225;lisis de la secuencia de <span class="elsevierStyleItalic">BCR1</span>&#44; existe variabilidad gen&#233;tica en <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> y <span class="elsevierStyleItalic">C&#46; metapsilosis</span> sin ninguna asociaci&#243;n particular a los fenotipos relacionados con la formaci&#243;n de biopel&#237;cula&#46;</p></span> <span id="abst0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Conclusiones</span><p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Bajo el dise&#241;o experimental adoptado&#44; <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> se asoci&#243; con un fenotipo de baja producci&#243;n de biopel&#237;cula y <span class="elsevierStyleItalic">C&#46; orthopsilosis</span> con ambos fenotipos &#40;alta y baja producci&#243;n de biopel&#237;cula&#41;&#46; Por otra parte&#44; no se encontr&#243; ninguna asociaci&#243;n estad&#237;sticamente significativa entre los fenotipos de formaci&#243;n de biopel&#237;cula y variantes gen&#233;ticas particulares en el fragmento analizado de <span class="elsevierStyleItalic">BCR1</span>&#46;</p></span>"
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            "apendice" => "<p id="par0105" class="elsevierStylePara elsevierViewall">The following are the supplementary data to this article&#58;<elsevierMultimedia ident="upi0005"></elsevierMultimedia></p>"
            "etiqueta" => "Appendix A"
            "titulo" => "Supplementary data"
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          "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Biofilm quantification in clinical isolates of <span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span>&#44; <span class="elsevierStyleItalic">C&#46; orthopsilosis</span>&#44; and <span class="elsevierStyleItalic">C&#46; metapsilosis</span> by crystal violet staining &#40;a&#41; and metabolic activity &#40;b&#41;&#46; The bars represent the optical density mean value of each group&#46; The standard deviations are shown&#46;</p>"
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                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head  " align="left" valign="top" scope="col">Species &#40;No&#46; of isolates&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " colspan="6" align="center" valign="top" scope="col" style="border-bottom: 2px solid black">No&#46; of isolates &#40;&#37;&#41;</th></tr><tr title="table-row"><th class="td" title="table-head  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " colspan="2" align="left" valign="top" scope="col" style="border-bottom: 2px solid black">High biofilm production</th><th class="td" title="table-head  " colspan="2" align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Low biofilm production</th><th class="td" title="table-head  " colspan="2" align="left" valign="top" scope="col" style="border-bottom: 2px solid black">No presence of biofilm</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="table-entry  " align="left" valign="top"><span class="elsevierStyleItalic">C&#46; parapsilosis sensu stricto</span> &#40;30&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#40;13&#46;3&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">26&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#40;86&#46;7&#41;<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">&#42;</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#40;0&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry  " align="left" valign="top"><span class="elsevierStyleItalic">C&#46; orthopsilosis</span> &#40;30&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">12&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#40;10&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">17&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">45&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#40;69&#46;2&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">3&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#40;4&#46;6&#41;&nbsp;\t\t\t\t\t\t\n
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        "titulo" => "Acknowledgements"
        "texto" => "<p id="par0095" class="elsevierStylePara elsevierViewall">We thank Sergio Lozano-Rodriguez&#44; M&#46;D&#46; for the English review of the manuscript prior to submission&#46;</p>"
        "vista" => "all"
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]

Article information

ISSN: 11301406
Original language: English

DOI:10.1016/j.riam.2014.11.001

The statistics are updated each day

Year/Month	Html	Pdf	Total

2024 November	4	0	4
2024 October	16	10	26
2024 September	26	3	29
2024 August	22	4	26
2024 July	20	3	23
2024 June	18	5	23
2024 May	25	6	31
2024 April	28	8	36
2024 March	49	5	54
2024 February	24	5	29
2024 January	25	7	32
2023 December	33	9	42
2023 November	39	4	43
2023 October	40	9	49
2023 September	18	3	21
2023 August	23	4	27
2023 July	16	5	21
2023 June	40	9	49
2023 May	74	5	79
2023 April	83	6	89
2023 March	101	12	113
2023 February	49	7	56
2023 January	28	9	37
2022 December	21	9	30
2022 November	21	16	37
2022 October	21	8	29
2022 September	21	17	38
2022 August	27	6	33
2022 July	26	15	41
2022 June	24	9	33
2022 May	22	16	38
2022 April	37	16	53
2022 March	51	10	61
2022 February	28	19	47
2022 January	53	27	80
2021 December	68	19	87
2021 November	37	16	53
2021 October	35	23	58
2021 September	31	27	58
2021 August	35	12	47
2021 July	15	14	29
2021 June	12	12	24
2021 May	28	8	36
2021 April	85	36	121
2021 March	21	17	38
2021 February	16	12	28
2021 January	26	14	40
2020 December	21	14	35
2020 November	16	6	22
2020 October	28	12	40
2020 September	25	20	45
2020 August	19	6	25
2020 July	28	6	34
2020 June	25	14	39
2020 May	23	10	33
2020 April	41	8	49
2020 March	34	6	40
2020 February	42	10	52
2020 January	22	11	33
2019 December	32	16	48
2019 November	15	4	19
2019 October	22	5	27
2019 September	23	4	27
2019 August	27	11	38
2019 July	28	13	41
2019 June	53	33	86
2019 May	111	19	130
2019 April	65	11	76
2019 March	13	1	14
2019 February	14	9	23
2019 January	10	3	13
2018 December	10	2	12
2018 November	9	3	12
2018 October	20	16	36
2018 September	27	1	28
2018 August	2	3	5
2018 July	10	3	13
2018 June	9	2	11
2018 May	4	7	11
2018 April	7	0	7
2018 March	7	2	9
2018 February	15	3	18
2018 January	9	0	9
2017 December	11	5	16
2017 November	20	5	25
2017 October	13	2	15
2017 September	5	6	11
2017 August	20	7	27
2017 July	15	7	22
2017 June	47	10	57
2017 May	19	17	36
2017 April	19	12	31
2017 March	16	28	44
2017 February	17	2	19
2017 January	16	2	18
2016 December	31	8	39
2016 November	19	3	22
2016 October	37	13	50
2016 September	29	9	38
2016 August	28	6	34
2016 July	28	2	30
2016 February	1	0	1
2015 October	4	1	5
2015 September	0	2	2
2015 August	1	4	5

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