Role of Proteus mirabilis flagella in biofilm formation

array:23 ["pii" => "S0325754123000202" "issn" => "03257541" "doi" => "10.1016/j.ram.2023.01.005" "estado" => "S300" "fechaPublicacion" => "2023-07-01" "aid" => "534" "copyright" => "Asociación Argentina de Microbiología" "copyrightAnyo" => "2023" "documento" => "article" "crossmark" => 1 "subdocumento" => "fla" "cita" => "Rev Argent Microbiol. 2023;55:226-34" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:1 [ "total" => 0 ] "itemSiguiente" => array:18 [ "pii" => "S0325754122001080" "issn" => "03257541" "doi" => "10.1016/j.ram.2022.07.003" "estado" => "S300" "fechaPublicacion" => "2023-07-01" "aid" => "528" "copyright" => "Asociación Argentina de Microbiología" "documento" => "article" "crossmark" => 1 "subdocumento" => "fla" "cita" => "Rev Argent Microbiol. 2023;55:235-9" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:1 [ "total" => 0 ] "es" => array:13 [ "idiomaDefecto" => true "cabecera" => "INFORME BREVE" "titulo" => "Actinomyces europaeus (Gleimia europaea) asociado con absceso cerebral: comunicación de tres casos" "tienePdf" => "es" "tieneTextoCompleto" => "es" "tieneResumen" => array:3 [ 0 => "es" 1 => "es" 2 => "en" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "235" "paginaFinal" => "239" ] ] "titulosAlternativos" => array:1 [ "en" => array:1 [ "titulo" => "Actinomyces europaeus (Gleimia europaea) associated with brain abscess: A report of three cases" ] ] "contieneResumen" => array:2 [ "es" => true "en" => true ] "contieneTextoCompleto" => array:1 [ "es" => true ] "contienePdf" => array:1 [ "es" => true ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Carla Álvarez, Marisa Almuzara, Claudia Tosello, Daniel Stecher, Carlos Vay, Claudia Barberis" "autores" => array:6 [ 0 => array:2 [ "nombre" => "Carla" "apellidos" => "Álvarez" ] 1 => array:2 [ "nombre" => "Marisa" "apellidos" => "Almuzara" ] 2 => array:2 [ "nombre" => "Claudia" "apellidos" => "Tosello" ] 3 => array:2 [ "nombre" => "Daniel" "apellidos" => "Stecher" ] 4 => array:2 [ "nombre" => "Carlos" "apellidos" => "Vay" ] 5 => array:2 [ "nombre" => "Claudia" "apellidos" => "Barberis" ] ] ] ] "resumen" => array:1 [ 0 => array:3 [ "titulo" => "Highlights" "clase" => "author-highlights" "resumen" => "<ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005">•Los abscesos cerebrales pueden estar relacionados con otitis media crónica.</li><li class="elsevierStyleListItem" id="lsti0010">•La espectrometría de masas es útil para identificar A. europaeus.</li><li class="elsevierStyleListItem" id="lsti0015">•Se destaca la relevancia de A. euroapeus y su asociación a la localización cerebral.</li></ul>" ] ] ] "idiomaDefecto" => "es" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754122001080?idApp=UINPBA00004N" "url" => "/03257541/0000005500000003/v1_202309280755/S0325754122001080/v1_202309280755/es/main.assets" ] "itemAnterior" => array:18 [ "pii" => "S0325754123000238" "issn" => "03257541" "doi" => "10.1016/j.ram.2023.01.008" "estado" => "S300" "fechaPublicacion" => "2023-07-01" "aid" => "537" "copyright" => "Asociación Argentina de Microbiología" "documento" => "article" "crossmark" => 1 "subdocumento" => "fla" "cita" => "Rev Argent Microbiol. 2023;55:214-25" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:1 [ "total" => 0 ] "es" => array:14 [ "idiomaDefecto" => true "cabecera" => "Original" "titulo" => "Primera evidencia de actividad enzimática nitrilasa en Xylaria sp., y su relación con la biosíntesis de ácido indol-3-acético" "tienePdf" => "es" "tieneTextoCompleto" => "es" "tieneResumen" => array:3 [ 0 => "es" 1 => "es" 2 => "en" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "214" "paginaFinal" => "225" ] ] "titulosAlternativos" => array:1 [ "en" => array:1 [ "titulo" => "First evidence of nitrilase enzymatic activity of Xylaria sp. and its relationship with the biosynthesis of indole-3-acetic acid" ] ] "contieneResumen" => array:2 [ "es" => true "en" => true ] "contieneTextoCompleto" => array:1 [ "es" => true ] "contienePdf" => array:1 [ "es" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figura 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 2098 "Ancho" => 3341 "Tamanyo" => 281148 ] ] "descripcion" => array:1 [ "es" => "Niveles de expresión génica relativa de una nitrilasa putativa de Xylaria sp. Los valores se expresan como log10 de 2-ΔΔCT. Se utilizó el gen de la β-tubulina como referencia endógena. Las líneas superiores de cada barra representan las desviaciones estándar de los valores de la media de las réplicas biológicas. Los niveles de expresión génica de cada ensayo (1-12) se muestran de manera individual y se componen del resultado de 4, 8 y 12h de fermentación." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Jorge Ricaño-Rodríguez, Celeste Ricaño-Rodríguez, Daniela Luis-Yong, Oswaldo Guzmán-López" "autores" => array:4 [ 0 => array:2 [ "nombre" => "Jorge" "apellidos" => "Ricaño-Rodríguez" ] 1 => array:2 [ "nombre" => "Celeste" "apellidos" => "Ricaño-Rodríguez" ] 2 => array:2 [ "nombre" => "Daniela" "apellidos" => "Luis-Yong" ] 3 => array:2 [ "nombre" => "Oswaldo" "apellidos" => "Guzmán-López" ] ] ] ] "resumen" => array:1 [ 0 => array:3 [ "titulo" => "Highlights" "clase" => "author-highlights" "resumen" => "<ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005">•Xylaria sp. es capaz de llevar a cabo actividad enzimática nitrilasa.</li><li class="elsevierStyleListItem" id="lsti0010">•Una secuencia homóloga de ADN transcribiría enzimas nitrilasas.</li><li class="elsevierStyleListItem" id="lsti0015">•La expresión genética es inducible con diversas fuentes de carbono y nitrógeno.</li><li class="elsevierStyleListItem" id="lsti0020">•El hongo produce AIA y promueve el desarrollo radicular en Arabidopsis thaliana.</li><li class="elsevierStyleListItem" id="lsti0025">•Las enzimas nitrilasas son intermediarias metabólicas de AIA.</li></ul>" ] ] ] "idiomaDefecto" => "es" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754123000238?idApp=UINPBA00004N" "url" => "/03257541/0000005500000003/v1_202309280755/S0325754123000238/v1_202309280755/es/main.assets" ] "en" => array:22 [ "idiomaDefecto" => true "cabecera" => "Original article" "titulo" => "Role of Proteus mirabilis flagella in biofilm formation" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "226" "paginaFinal" => "234" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Paola Scavone, Victoria Iribarnegaray, María José González, Nicolás Navarro, Nicole Caneles-Huerta, Jorge Jara-Wilde, Steffen Härtel, Pablo Zunino" "autores" => array:8 [ 0 => array:3 [ "nombre" => "Paola" "apellidos" => "Scavone" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 1 => array:3 [ "nombre" => "Victoria" "apellidos" => "Iribarnegaray" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "c" "identificador" => "aff0015" ] ] ] 2 => array:3 [ "nombre" => "María José" "apellidos" => "González" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 3 => array:3 [ "nombre" => "Nicolás" "apellidos" => "Navarro" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 4 => array:3 [ "nombre" => "Nicole" "apellidos" => "Caneles-Huerta" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 5 => array:3 [ "nombre" => "Jorge" "apellidos" => "Jara-Wilde" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 6 => array:3 [ "nombre" => "Steffen" "apellidos" => "Härtel" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 7 => array:4 [ "nombre" => "Pablo" "apellidos" => "Zunino" "email" => array:2 [ 0 => "pzunino@iibce.edu.uy" 1 => "pmzunino@gmail.com" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "*" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:3 [ 0 => array:3 [ "entidad" => "Department of Microbiology, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Laboratory for Scientific Image Processing (SCIAN-Lab), Biomedical Neuroscience Institute (BNI), Institute of Biomedical Sciences (ICBM), Faculty of Medicine, University of Chile, Santiago, Chile" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Department of Pathobiology, Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay" "etiqueta" => "c" "identificador" => "aff0015" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Papel de los flagelos de Proteus mirabilis en la formación de biofilms" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0030" "etiqueta" => "Figure 6" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr6.jpeg" "Alto" => 2348 "Ancho" => 3258 "Tamanyo" => 1230545 ] ] "descripcion" => array:1 [ "en" => "Time-lapse biofilms. (A) The dynamic flow system that consists of a medium reservoir (a), a peristaltic pump (b), the 3-way stop cock (c), the biofilm chamber (d) and the disposal flask (e). (B) Representative 3D stacks of each time point (24, 48 and 72h) of Pr2921 (left) and AF (right). The scale bar represents 10μm, and applies to all images. (C) Aspect ratio, defined as the length of the major axis divided into the minor axis of the ellipsoid that is fitted to each particle. Significant differences were calculated using the *** P<0.05." ] ] ] "textoCompleto" => "IntroductionProteus mirabilis(P. mirabilis) causes urinary tract infections (UTI), although it does not often colonize the normal unobstructed urinary tract<a class="elsevierStyleCrossRef" href="#bib0200">1</a>. It is more common in complicated UTI, particularly in catheter-associated urinary tract infections (CAUTIs)<a class="elsevierStyleCrossRef" href="#bib0365">34</a>.P. mirabilis can cause urinary stones and crystalline biofilm formation associated with the increase of urine pH, due to urease production. Environmental changes caused by hydrolyzation of the urine urea allow the precipitation of minerals, promoting the formation of typical mineral-encrusted biofilms<a class="elsevierStyleCrossRef" href="#bib0310">23</a>. Due to encrustation, these crystalline biofilms may block the catheters in patients with CAUTIs. Sixty-two percent (62%) of patients with recurrent P. mirabilis catheter encrustation developed bladder stones because of catheter colonization<a class="elsevierStyleCrossRef" href="#bib0320">25</a>. Other possible consequences associated with crystalline biofilms and blockages of the urinary catheter are urine retention, along with bladder and urethra mucosal trauma<a class="elsevierStyleCrossRef" href="#bib0280">17</a>.Different factors participate in biofilm development, including motility, which has often been considered a putative factor in this process<a class="elsevierStyleCrossRef" href="#bib0300">21</a>.P. mirabilis typical swarming motility is produced when vegetative swimmer cells differentiate into elongated, multinucleated and highly flagellated swarmer cells<a class="elsevierStyleCrossRef" href="#bib0200">1</a>. This process, mediated by flagella, is initiated by bacterial contact with a solid surface and probably contributes to biofilm formation and catheter colonization. The role of flagella and swarming in P. mirabilis UTI is still under debate<a class="elsevierStyleCrossRefs" href="#bib0210">3,24</a>. Different studies have suggested that flagella significantly contribute to P. mirabilis urovirulence<a class="elsevierStyleCrossRefs" href="#bib0305">22,24</a>. However, several works indicate that flagella are not critical for establishing UTI by P. mirabilis and other uropathogens<a class="elsevierStyleCrossRefs" href="#bib0285">18,19,35</a>.The role of flagella in P. mirabilis biofilm formation remains unclear and is seldomly addressed in the literature. A few studies have proposed that flagella and motility may play a role in the formation of biofilms<a class="elsevierStyleCrossRef" href="#bib0240">9</a>, while others consider flagella not to be critical in this process<a class="elsevierStyleCrossRef" href="#bib0275">16</a>.In this study, we assess the input of P. mirabilis flagella in biofilm formation using a flagellate mutant generated by allelic replacement, tested in different in vitro assays.Materials and methodsBacterial strainsThe clinical wild-type P. mirabilis Pr2921 strain was extensively characterized<a class="elsevierStyleCrossRef" href="#bib0375">36</a>. An isogenic non-flagellate allelic replacement mutant (AF strain) was used to evaluate the role of flagella in biofilm formation. This mutant has both flaA and flaB structural genes partially deleted and interrupted by a Kanamycin cassette<a class="elsevierStyleCrossRef" href="#bib0325">26</a>, following the procedure used to generate other flagellate mutants in our laboratory<a class="elsevierStyleCrossRef" href="#bib0290">19</a>. Absence of swimming and swarming motility was evaluated, and the lack of flagella was evidenced by Western blot<a class="elsevierStyleCrossRef" href="#bib0335">28</a>. Bacteria were cultured in Luria-Bertani (LB) broth or LB agar (1.5%). Artificial urine (AU) was prepared according to Scavone et al.<a class="elsevierStyleCrossRef" href="#bib0330">27</a> Growth curves were first assessed in LB and AU to check that the absence of flagella did not affect normal growth.Bacterial suspensions were prepared in phosphate-buffered saline (PBS) from fresh LB agar plates at an OD 600nm to inoculate LB or AU cultures in 96-multiwell plates<a class="elsevierStyleCrossRef" href="#bib0330">27</a>. pH of AU cultures of both strains was controlled serially.Hydrophobicity of planktonic cellsTen microliters of bacterial suspensions in PBS (OD540=0.2–0.3) were inoculated in 5ml of AU or LB, and incubated at 37°C for 48h. Then, bacterial suspensions (OD600=0.8) and 200μl of added xylene were mixed, shaken for 2min and left static for 20min. Subsequently, the aqueous phase (OD600nm) was measured and hydrophobicity was assessed according to Bibiloni et al.<a class="elsevierStyleCrossRef" href="#bib0205">2</a>. The assay was done in triplicate. Hydrophobicity of planktonic cells of the different strains and under different conditions was assessed using the Tukey's multiple comparison test.Swimming and swarming motilitySwimming motility was evaluated as described by Hola et al.<a class="elsevierStyleCrossRef" href="#bib0250">11</a>, and swarming motility was assessed as described by Jones et al.<a class="elsevierStyleCrossRef" href="#bib0270">15</a>. Both assays were performed in triplicate.Inhibition of swarming was evaluated using a specific P. mirabilis flagella rabbit anti-serum. For this purpose, a rabbit was immunized with four doses of 25μg of purified Pr2921 flagellin administered subcutaneously, using complete Freund's adjuvant (Sigma) for the first dose and incomplete Freund's adjuvant for the following ones, according to previous studies<a class="elsevierStyleCrossRef" href="#bib0290">19</a>. These procedures were approved by the Institutional Animal Care Committee and adequate measures were taken to minimize discomfort or stress of the animal. The antibody response was determined ten days after the last dose (day 55) and serum anti-flagella IgG titer measured by ELISA reached a value of 1:100000.For the swarming inhibition assay, the WT strain was grown on 50mm diameter plates containing 5ml of modified LB agar and LB agar supplemented with the specific rabbit anti-serum, using a 1:10 dilution.Bacterial migration across urethral cathetersThe ability to migrate across urinary catheters sections was assessed following the procedure proposed by Stickler and Huges<a class="elsevierStyleCrossRef" href="#bib0355">32</a>. The catheter materials were silicone and latex (Teleflex Medical, Kernen, Germany). Migration was tested 15 times for both catheter types.Quantification of biofilm formation on polystyrene multiwell platesThe effect of the flagellar mutation in biofilm formation was evaluated using the semi-quantification technique based on the adsorption of crystal violet (CV)<a class="elsevierStyleCrossRef" href="#bib0225">6</a>.A competition assay was also performed to assess the ability of both strains to remain attached to the polystyrene surface, forming a biofilm when cultured simultaneously following the procedure described above. However, in this case, the biofilms were scrapped and bacteria were cultured in nutrient agar and nutrient agar supplemented with kanamycin since AF carries a kanamycin-resistance gene incorporated into the mutagenic process<a class="elsevierStyleCrossRef" href="#bib0325">26</a>. Biofilm formation of the different strains and under different conditions was assessed using the Tukey's multiple comparison test.Biofilm formation over time by confocal laser scanning microscopyBiofilm formation of Pr2921 and AF was evaluated in 2, 5 and 7 day-LB and AU cultures under static conditions at 37°C<a class="elsevierStyleCrossRef" href="#bib0330">27</a>. This method allows biofilm formation on a coverslip placed into a tube subjected to fluorescent staining after different incubation periods. Staining techniques and microscopy analyses were performed according to Schlapp et al.<a class="elsevierStyleCrossRef" href="#bib0350">31</a>. Bacteria were stained with Syto 9 (Molecular Probes) and the extracellular matrix with FilmTracer™ SYPRO® Ruby Biofilm matrix stain. 3D images were acquired using a Leica TCS LSI super-zoom confocal fluorescent microscope with an iXon Ultra 897 back-illuminated imaging EMCCD camera, LAS Software, a 100× oil immersion objective (NA=1.35) and 488/520, 450/610nm excitation/emission wavelength.Three z-stacks were randomly chosen in each sample, using an acquisition step of 0.3μm in the z-axis and 1024×1024 pixels in the xy-plane with a pixel size of 170nm. After deconvolution using Huygens Software, segmentation, visualization, 3D reconstruction and biofilm parameter descriptors were calculated using ScianSoft<a class="elsevierStyleCrossRef" href="#bib0350">31</a>.Bacterial number, bacterial and matrix volumes were calculated to compare biofilm evolution. Results were compared using one-way ANOVA p≤0.05.Biofilm grown under a flow systemIn order to evaluate biofilm formation under a flow system, we have developed a system that consists of a medium reservoir, a peristaltic pump, a chamber where the biofilm was grown and a disposal flask. The chamber had two slides (bottom and top) in order to follow the biofilm formation by confocal microscopy. The system was set up with a LB flow at 0.5ml/min. First, the system was assembled and after removing air bubbles, the bacterial suspension was introduced into the chambers through a three-way stop cock. The biofilm was allowed to form for 1h, after that the flow was started for 24, 48 and 72h. Acridine orange (0.01mg/ml) was used to stain and visualize bacteria in the biofilm. At different time points, the system was stopped and the dye was added to the system through the three-way stop cock. Visualization was performed in a LSM Zeiss 800, using a 63× oil immersion objective (NA 1.4). Live images were taken every 127s for 3min. After acquisition, the system was loaded again until the next time point.Image processing of time-lapse biofilmImage processing and analysis were performed using FIJI/ImageJ software<a class="elsevierStyleCrossRef" href="#bib0345">30</a>. Morphological analysis was performed using MicrobeJ plugin<a class="elsevierStyleCrossRef" href="#bib0230">7</a>. Morphological parameters were defined according to the different morphotypes of P. mirabilis described by Jansen et al.<a class="elsevierStyleCrossRef" href="#bib0265">14</a> Swarmer cells with a length greater than 10μm and a maximum width of 3μm were excluded from the analysis. Thresholds were used for binarization and length/width exclusion criteria prior to analysis. Contour detection was performed using Fit-Shape mode. The shape descriptors calculated from the data were length, mean width and aspect ratio, defined as the length of the major axis divided by the minor axis of the ellipsoid fitted to each particle. Statistical analysis was performed by RStudio software (RStudio Team, 2020). The differences between each time-lapse were analyzed with the Wilcoxon test. Motility analyses were performed with TrackMate plugin<a class="elsevierStyleCrossRefs" href="#bib0235">8,33</a>. Motility analysis was performed using previously segmented time-lapse image sequences by Ilastik software. TrackMate auto intensity threshold tool was used to select all particles for track analysis. The trajectories of each individual bacterium were determined by LAP Tracker. It was set allowing a frame-to-frame linking and gap closing of 6μm, splitting and merging was set at 0.6μm, and ellipse long axis, ellipse short axis and max intensity were used as penalties for linking cost calculation. Mean square displacements (MSD) were calculated with R Studio software by Eq. <a class="elsevierStyleCrossRef" href="#eq0005">(1)</a> (RStudio Team, 2020). RStudio: Integrated Development for R. RStudio, PBC, Boston, MA (URL <a href="http://www.rstudio.com/">http://www.rstudio.com</a>).<elsevierMultimedia ident="eq0005"></elsevierMultimedia>ResultsLack of flagella did not affect bacterial features, except flagellin expressionGrowth curves of Pr2921 and AF cultured in LB or AU were similar, showing that the general biology of the mutant was not affected, and both strains grew better in LB compared with AU (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>).<elsevierMultimedia ident="fig0005"></elsevierMultimedia>pH of Pr2921 and AF cultures in AU reached a value of 9 after 6h of incubation in both cases, remaining constant for at least 24h, demonstrating that urease production was similar in both strains. Expression of MR/P fimbriae assessed by Western blotting (<a class="elsevierStyleCrossRef" href="#sec0110">Suppl. Fig. S1</a>), hemagglutination based on MR/P fimbrial expression, and hemolysis observed on blood agar plates were similar in both strains (data not shown).Genetic rearrangements due to allelic replacement mutagenesis were as expected and the mutant was unable to express flagellin<a class="elsevierStyleCrossRef" href="#bib0335">28</a>.Hydrophobicity index is increased in AUWhen bacteria were grown in LB, no significant differences were observed among the hydrophobicity indexes between both strains. However, when bacteria were grown in AU, the AF mutant exhibited a significant hydrophobicity increase compared with the wild-type Pr2921, in AU (p<0.001) (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>). In addition, hydrophobicity of planktonic bacteria grown in AU was significantly higher than bacteria grown in LB, in the case of both strains (p<0.05, <a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>).<elsevierMultimedia ident="fig0010"></elsevierMultimedia>Swimming and swarming motility and migration across latex and silicone catheter bridgesThe flagellate mutant was unable to swim or swarm (<a class="elsevierStyleCrossRef" href="#sec0110">Suppl. Fig. S2</a>). In both assays, the mutant grew to form a defined colony without spreading beyond the colony limits.Swarming was completely inhibited when the WT strain (Pr2921) grew on LB agar supplemented with specific anti-flagella rabbit serum (1:10). Lower serum concentrations obtained by serial dilutions inhibited swarming in a dose-dependent relationship (data not shown).The influence of flagella on P. mirabilis migration across catheter sections was assessed. The WT strain crossed latex and silicone catheter bridges in every case (15/15), while the AF mutant was unable to cross any catheter sections (0/15) (<a class="elsevierStyleCrossRef" href="#sec0110">Suppl. Fig. S2</a>).Biofilm formation on polystyreneWhen biofilm formation was evaluated using the method based on associated CV adsorption, the AF mutant showed a significantly impaired ability to form biofilms compared to WT Pr2921 in LB (p<0.0001).When the assay was performed using AU, the level of biofilm formation of the mutant strain was lower than that formed by the WT strain but the difference was not significant (p>0.05) (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>). A significantly reduced biofilm was also observed when WT grew in AU compared with that observed in LB (p<0.0001).<elsevierMultimedia ident="fig0015"></elsevierMultimedia>A competition assay was also performed to evaluate if the mutant was outcompeted by the WT strain in the biofilm. Results indicated that the wild-type strain outcompeted the mutant taking into account viable bacterial counts of Pr2921/AF from co-cultured biofilms. At 24h, wild type and mutant counts (log) were 7.63±0.01 and 2.28±0.2, respectively (p<0.05). After 48h, wild type and mutant counts remained at similar levels (7.28±0.07 and 2.64±0.09, respectively, p<0.05).Biofilm formation dynamicsBiofilm formation was evaluated in LB and AU under static conditions at 37°C, at 2, 5 and 7 days of incubation, using CLSM and image analysis (<a class="elsevierStyleCrossRefs" href="#fig0020">Figs. 4 and 5</a>).<elsevierMultimedia ident="fig0020"></elsevierMultimedia><elsevierMultimedia ident="fig0025"></elsevierMultimedia>When biofilm formation was assessed after 2 days in LB or AU, the bacterial volume and the extracellular matrix of WT and the flagellate mutant AF were similar, showing in general low values with no significant differences among strains or media conditions (p>0.05) (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>). However, after 5 days, significant differences were observed among WT and the mutant AF strains grown in LB. The WT bacterial and matrix volumes significantly increased compared to the AF mutant (p<0.0001 in both cases). When both strains were cultured in AU, no AF bacteria remained attached to the coverslip, while WT Pr2921 exhibited a structured biofilm. However, the statistical difference was not significant since the WT biofilm was small. Furthermore, the AF matrix in AU was almost inexistent.After 7 days of incubation in LB, the bacterial and matrix volume of the biofilm formed by WT 2921 decreased and differences were not significant when compared to the biofilm formed by AF (p>0.05). When bacteria were cultured in AU, no statistical differences between strains were identified; however, a small WT 2921 biofilm (both bacteria and matrix) was still observed, but not with the AF mutant (<a class="elsevierStyleCrossRefs" href="#fig0020">Figs. 4 and 5</a>).The dynamic system allowed in vivo observation of the biofilm every 24h (<a class="elsevierStyleCrossRef" href="#fig0030">Fig. 6</a>A). Both strains formed biofilm, but differences were observed between WT and the AF mutant. At 24h, biofilms were flat as observed by the height in the 3D stacks (<a class="elsevierStyleCrossRef" href="#fig0030">Fig. 6</a>B upper panels). WT 2921 biofilm was around 1.25μm in height while the AF biofilm was 0.75μm in height. Moreover, significant differences were observed in the size and shape of the bacteria (<a class="elsevierStyleCrossRef" href="#fig0030">Fig. 6</a>B). WT 2921 was more elongated than AF as represented by the mean width (the length of the major axis divided by the minor axis of the ellipsoid fitted to each bacteria) (<a class="elsevierStyleCrossRef" href="#fig0030">Figs. 6B and C</a>). At 48h, WT 2921 increased the 3D biofilm structure reaching 2μm in height and forming channels while the AF biofilm remained flat. At 72h, AF showed a compacted and thin biofilm with shortened bacteria and WT 2921 had a heterogeneous bacterial morphology within a 3D biofilm with the presence of channels (<a class="elsevierStyleCrossRef" href="#fig0030">Fig. 6</a>B, <a class="elsevierStyleCrossRef" href="#sec0110">Suppl. MSD</a>). While WT 2921 formed a mature biofilm with different bacterial morphologies, the AF mutant strain showed a flat biofilm and the morphology of the bacteria was reduced over time. With regard to the length of the bacteria, we observed that 2921 acquired a more elongated morphology while AF was shortened (<a class="elsevierStyleCrossRef" href="#fig0030">Figs. 6B and C</a>).<elsevierMultimedia ident="fig0030"></elsevierMultimedia>DiscussionP. mirabilis is among the most frequent causes of complicated UTI<a class="elsevierStyleCrossRef" href="#bib0335">28</a>, and displays a broad and complex range of virulence factors that may participate in the colonization of different surfaces<a class="elsevierStyleCrossRefs" href="#bib0255">12,27</a>.Flagella have been associated with the formation of biofilms in different pathogens, including Bacillus cereus, Yersinia enterocolitica and Helicobacterpylori<a class="elsevierStyleCrossRefs" href="#bib0245">10,20</a>. However, the role of flagella and swarming motility in P. mirabilis UTI and biofilm formation has been thoroughly debated<a class="elsevierStyleCrossRefs" href="#bib0290">19,22,29</a>.In this study, we used a flagellate mutant to assess the role of flagella in P. mirabilis biofilm formation, through different experimental in vitro approaches, which are discussed below.Growth patterns were similar when WT Pr2921 and the AF mutant were grown in LB broth or AU, indicating that the mutation did not alter the general bacterial biology. In addition, a series of phenotypic pathogenic features were not altered by the mutagenesis process, including urease production, fimbriae expression, hemagglutination and hemolysis production.The influence of flagella on P. mirabilis surface-related features was assessed by measuring bacterial hydrophobicity. According to different authors, bacterial surface hydrophobicity is important for adherence to different interfaces and biofilm formation<a class="elsevierStyleCrossRef" href="#bib0215">4</a>. When the flagellate mutant was grown in LB broth, bacterial hydrophobicity did not vary. However, when grown in AU, hydrophobicity significantly increased, probably associated with an increased exposure of the lipid components of the bacterial surface. In other studies, no clear relationships between bacterial hydrophobicity and enhancement of binding to different substrates were observed<a class="elsevierStyleCrossRef" href="#bib0220">5</a>.In this study, we also assessed the ability of the isogenic flagellate mutant to swim, swarm and migrate over catheter sections of different materials. The mutant was unable to swim or swarm on LB agar or to migrate along sections of latex and silicone catheters, strongly confirming the role of flagella in the colonization of these clinical devices. These results are in accordance with previous studies<a class="elsevierStyleCrossRef" href="#bib0270">15</a>.The inclusion of specific P. mirabilis antiflagella antibodies in the culture medium also resulted in the abolition of motility, reinforcing the clear and specific role of flagellar function in motility and spreading over different surfaces through an indirect approach.When biofilm formation was assessed on an abiotic surface like polystyrene, the flagellate mutant grown in LB showed a significant smaller biofilm compared with the wild type Pr2921. When bacteria grew in AU, the mutant also formed a smaller biofilm although differences were not significant. In this case, it must be taken into account that P. mirabilis growth in AU was significantly less abundant than in LB broth, influencing the magnitude of growth differences in both strains.A competition assay based on the wild type and the mutant co-cultivation was performed to refine the assessment of flagella contribution in biofilm formation. We had used this kind of approach before to detect subtle effects of different P. mirabilis virulence factors in ascending UTI in mice<a class="elsevierStyleCrossRef" href="#bib0260">13</a>. Results of this assay revealed that although the mutant was outcompeted by the WT strain, a biofilm was formed that even persisted for at least 48h, indicating that biofilm formation was not completely abolished by the lack of flagella.When CLSM was used to assess biofilm formation in LB broth and AU, significant differences were observed among biofilm parameters at different stages. One of the most notorious differences was the impaired capacity of the AF mutant to grow and produce extracellular matrix at the biofilm maturation stage in the static model (5 days). At this stage, the AF mutant biofilm had almost disappeared in AU while the WT biofilm, although small, remained attached and showing a typical mature structure, remaining attached even after 7 days. Our results confirm that flagella are important for biofilm formation, under different conditions. Furthermore, differences in bacterial morphology were observed under dynamic conditions, but the AF mutant was able to form a biofilm.Different authors have reported that mutations of P. mirabilis virulence genes have different effects on biofilm formation that can induce a decrease or even an increase in matrix or bacterial biovolumes<a class="elsevierStyleCrossRefs" href="#bib0255">12,36</a>.Several studies have reported the role of bacterial flagella in adhesion to biotic and abiotic surfaces, including P. mirabilis, which can contribute to the initial contact with different surfaces and even among cells<a class="elsevierStyleCrossRefs" href="#bib0270">15,35</a>.As mentioned above, Jones et al.<a class="elsevierStyleCrossRef" href="#bib0275">16</a> suggested that neither swarming nor swimming motility are required for the attachment of P. mirabilis to catheters and that mutants unable to swarm and swim formed crystalline biofilms and blocked catheters even more rapidly than the wild-type strain. The authors hypothesize that on contact with a surface, the non-swarmer cells are more likely to remain at the site of attachment, divide or produce microcolonies that initiate biofilm formation. On the other hand, Fusco et al.<a class="elsevierStyleCrossRef" href="#bib0240">9</a> compared different P. mirabilis strains and reported that motility ability was related to biofilm formation; however, they did not use isogenic mutants.ConclusionOverall, our results show that flagella critically contribute to the formation of P. mirabilis biofilms. However, the lack of this organelle could not completely prevent biofilm formation, suggesting that different elements drive biofilm formation and persistence. Like other factors (e.g., ATF fimbria)<a class="elsevierStyleCrossRef" href="#bib0375">36</a>, flagella could play a significant role in biofilm formation on abiotic surfaces rather than in urinary tract colonization and infection.This knowledge could contribute to the design of strategies for P. mirabilis biofilm prevention targeting the flagellar function, possibly in combination with other specific mechanisms.Conflict of interestThe authors declare that they have no conflict of interest." "textoCompletoSecciones" => array:1 [ "secciones" => array:13 [ 0 => array:3 [ "identificador" => "xres1976930" "titulo" => "Highlights" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0005" ] ] ] 1 => array:3 [ "identificador" => "xres1976931" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0010" ] ] ] 2 => array:2 [ "identificador" => "xpalclavsec1700507" "titulo" => "Keywords" ] 3 => array:3 [ "identificador" => "xres1976929" "titulo" => "Resumen" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0015" ] ] ] 4 => array:2 [ "identificador" => "xpalclavsec1700508" "titulo" => "Palabras clave" ] 5 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 6 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:8 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Bacterial strains" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Hydrophobicity of planktonic cells" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Swimming and swarming motility" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Bacterial migration across urethral catheters" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "Quantification of biofilm formation on polystyrene multiwell plates" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "Biofilm formation over time by confocal laser scanning microscopy" ] 6 => array:2 [ "identificador" => "sec0045" "titulo" => "Biofilm grown under a flow system" ] 7 => array:2 [ "identificador" => "sec0050" "titulo" => "Image processing of time-lapse biofilm" ] ] ] 7 => array:3 [ "identificador" => "sec0055" "titulo" => "Results" "secciones" => array:5 [ 0 => array:2 [ "identificador" => "sec0060" "titulo" => "Lack of flagella did not affect bacterial features, except flagellin expression" ] 1 => array:2 [ "identificador" => "sec0065" "titulo" => "Hydrophobicity index is increased in AU" ] 2 => array:2 [ "identificador" => "sec0070" "titulo" => "Swimming and swarming motility and migration across latex and silicone catheter bridges" ] 3 => array:2 [ "identificador" => "sec0075" "titulo" => "Biofilm formation on polystyrene" ] 4 => array:2 [ "identificador" => "sec0080" "titulo" => "Biofilm formation dynamics" ] ] ] 8 => array:2 [ "identificador" => "sec0085" "titulo" => "Discussion" ] 9 => array:2 [ "identificador" => "sec0090" "titulo" => "Conclusion" ] 10 => array:2 [ "identificador" => "sec0095" "titulo" => "Conflict of interest" ] 11 => array:2 [ "identificador" => "xack692868" "titulo" => "Acknowledgments" ] 12 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2022-09-05" "fechaAceptado" => "2023-01-25" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec1700507" "palabras" => array:6 [ 0 => "Urinary tract infection" 1 => "Proteus mirabilis" 2 => "Flagella" 3 => "Biofilm" 4 => "Catheter" 5 => "Prevention" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec1700508" "palabras" => array:6 [ 0 => "Infección del tracto urinario" 1 => "Proteus mirabilis" 2 => "Flagelos" 3 => "Biofilm" 4 => "Catéter" 5 => "Prevención" ] ] ] ] "tieneResumen" => true "highlights" => array:2 [ "titulo" => "Highlights" "resumen" => "<ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005">•P. mirabilis flagella have a medium-dependent role in cellular hydrophobicity.</li><li class="elsevierStyleListItem" id="lsti0010">•All motility and catheter migration models showed a significant role of flagella.</li><li class="elsevierStyleListItem" id="lsti0015">•A flagellate P. mirabilis mutant was outcompeted by the wild-type in biofilms.</li><li class="elsevierStyleListItem" id="lsti0020">•Biofilm formation of the mutant was impaired as seen in different in vitro assays.</li><li class="elsevierStyleListItem" id="lsti0025">•Flagellar function could be targeted to prevent biofilm formation.</li></ul>" ] "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "Proteus mirabilis(P. mirabilis) is a common etiological agent of urinary tract infections, particularly those associated with catheterization. P. mirabilis efficiently forms biofilms on different surfaces and shows a multicellular behavior called ‘swarming’, mediated by flagella. To date, the role of flagella in P. mirabilis biofilm formation has been under debate. In this study, we assessed the role of P. mirabilis flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin. Different approaches were used, such as the evaluation of cell surface hydrophobicity, bacterial motility and migration across catheter sections, measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models. Our findings indicate that P. mirabilis flagella play a role in biofilm formation, although their lack does not completely avoid biofilm generation. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets." ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "Proteus mirabilis(P. mirabilis) es un agente etiológico común de infecciones del tracto urinario, en particular de aquellas asociadas con cateterización. P. mirabilis forma biofilms eficientemente en diferentes superficies y muestra un comportamiento multicelular llamado swarming, mediado por flagelos. Hasta el momento, el papel de los flagelos en la formación de biofilms de P. mirabilis ha estado en discusión. En este estudio, se evaluó el papel de los flagelos de P. mirabilis en la formación de biofilms, utilizando una mutante isogénica generada por reemplazo alélico, incapaz de expresar flagelina. Se utilizaron diferentes enfoques, como la evaluación de la hidrofobicidad de la superficie celular, de la movilidad y la migración bacteriana sobre secciones de catéteres y medidas de biomasa y de la dinámica del biofilm mediante inmunofluorescencia y microscopia confocal, tanto en modelos estáticos como de flujo. Nuestros hallazgos indican que los flagelos de P. mirabilis desempeñan un papel en la formación de biofilms, aunque su falta no suprime por completo su generación. Asimismo, evidencian que la interferencia de la función flagelar puede contribuir a evitar la formación de biofilms en el contexto de estrategias centradas en blancos bacterianos particulares." ] ] "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" => "sec0110" ] ] ] ] "multimedia" => array:8 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 863 "Ancho" => 1591 "Tamanyo" => 62558 ] ] "descripcion" => array:1 [ "en" => "Growth curves of Pr2921 P. mirabilis wild-type strain and flagellate mutant AF in LB broth (LB) and artificial urine (AU). Growth curves were generated by serial OD measures of grown bacteria." ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1243 "Ancho" => 1425 "Tamanyo" => 179557 ] ] "descripcion" => array:1 [ "en" => "Hydrophobicity of Pr2921 P. mirabilis wild-type strain and flagellate mutant AF grown in LB broth (LB) and artificial urine (AU). When bacteria were grown in AU, the AF mutant exhibited a significant hydrophobicity increase compared with the wild-type Pr2921 strain in AU (p<0.001). Hydrophobicity was significantly higher in AU than in LB in the case of both strains (p<0.05). Statistical differences were calculated using the Tukey's multiple comparison test, **p<0.005, ***p<0.0005, ****p<0.0001." ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1640 "Ancho" => 1333 "Tamanyo" => 81061 ] ] "descripcion" => array:1 [ "en" => "Biofilm formation on polystyrene surfaces assessed by the semi-quantitative method based on crystal violet staining. Pr2921 P. mirabilis wild-type strain and flagellate mutant AF were grown in LB broth (LB) and in artificial urine (AU) and incubated at 37°C for 48h. Box plot of the OD values obtained in each condition are shown; asterisks show significant differences among different conditions. Statistical differences were calculated using the Tukey's multiple comparison test, **p<0.005, ****p<0.0001." ] ] 3 => array:7 [ "identificador" => "fig0020" "etiqueta" => "Figure 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 673 "Ancho" => 2925 "Tamanyo" => 101835 ] ] "descripcion" => array:1 [ "en" => "Bacteria and matrix volumes in biofilms formed by Pr2921 P. mirabilis wild-type strain and flagellate mutant AF in LB broth (LB) and artificial urine (AU) over time. Total bacteria and matrix volumes in biofilms formed in LB broth (LB) and artificial urine (AU) are shown in panels (A) and (B), respectively. *p≤0.05. The symbols represent the means of 3–5 random fields and their corresponding standard errors." ] ] 4 => array:7 [ "identificador" => "fig0025" "etiqueta" => "Figure 5" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr5.jpeg" "Alto" => 2668 "Ancho" => 3258 "Tamanyo" => 784589 ] ] "descripcion" => array:1 [ "en" => "3D reconstruction of biofilms formed by Pr2921 P. mirabilis wild-type strain and flagellate mutant AF in 7 day-cultures in LB broth (LB) and artificial urine (AU). All images were taken using the same magnification (63× oil immersion objective). Bacteria are represented in green and the matrix in red. D2: two day-cultures; D5: five day-cultures; D7: seven day-cultures. The scale bar represents 10μm." ] ] 5 => array:7 [ "identificador" => "fig0030" "etiqueta" => "Figure 6" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr6.jpeg" "Alto" => 2348 "Ancho" => 3258 "Tamanyo" => 1230545 ] ] "descripcion" => array:1 [ "en" => "Time-lapse biofilms. (A) The dynamic flow system that consists of a medium reservoir (a), a peristaltic pump (b), the 3-way stop cock (c), the biofilm chamber (d) and the disposal flask (e). (B) Representative 3D stacks of each time point (24, 48 and 72h) of Pr2921 (left) and AF (right). The scale bar represents 10μm, and applies to all images. (C) Aspect ratio, defined as the length of the major axis divided into the minor axis of the ellipsoid that is fitted to each particle. 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Original article

Papel de los flagelos de Proteus mirabilis en la formación de biofilms

Paola Scavonea, Victoria Iribarnegaraya,c, María José Gonzáleza, Nicolás Navarroa, Nicole Caneles-Huertab, Jorge Jara-Wildeb, Steffen Härtelb, Pablo Zuninoa,

Corresponding author

pzunino@iibce.edu.uy
pmzunino@gmail.com

Corresponding author.

a Department of Microbiology, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay

b Laboratory for Scientific Image Processing (SCIAN-Lab), Biomedical Neuroscience Institute (BNI), Institute of Biomedical Sciences (ICBM), Faculty of Medicine, University of Chile, Santiago, Chile

c Department of Pathobiology, Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay

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        "titulo" => "Papel de los flagelos de <span class="elsevierStyleItalic">Proteus mirabilis</span> en la formaci&#243;n de biofilms"
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          "en" => "<p id="spar0075" class="elsevierStyleSimplePara elsevierViewall">Time-lapse biofilms&#46; &#40;A&#41; The dynamic flow system that consists of a medium reservoir &#40;a&#41;&#44; a peristaltic pump &#40;b&#41;&#44; the 3-way stop cock &#40;c&#41;&#44; the biofilm chamber &#40;d&#41; and the disposal flask &#40;e&#41;&#46; &#40;B&#41; Representative 3D stacks of each time point &#40;24&#44; 48 and 72<span class="elsevierStyleHsp" style=""></span>h&#41; of Pr2921 &#40;left&#41; and AF &#40;right&#41;&#46; The scale bar represents 10<span class="elsevierStyleHsp" style=""></span>&#956;m&#44; and applies to all images&#46; &#40;C&#41; Aspect ratio&#44; defined as the length of the major axis divided into the minor axis of the ellipsoid that is fitted to each particle&#46; Significant differences were calculated using the &#42;&#42;&#42; P&#60;0&#46;05&#46;</p>"
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Introduction</span><p id="par0030" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Proteus mirabilis</span><span class="elsevierStyleItalic">&#40;P&#46; mirabilis&#41;</span> causes urinary tract infections &#40;UTI&#41;&#44; although it does not often colonize the normal unobstructed urinary tract<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">1</span></a>&#46; It is more common in complicated UTI&#44; particularly in catheter-associated urinary tract infections &#40;CAUTIs&#41;<a class="elsevierStyleCrossRef" href="#bib0365"><span class="elsevierStyleSup">34</span></a>&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">P&#46; mirabilis</span> can cause urinary stones and crystalline biofilm formation associated with the increase of urine pH&#44; due to urease production&#46; Environmental changes caused by hydrolyzation of the urine urea allow the precipitation of minerals&#44; promoting the formation of typical mineral-encrusted biofilms<a class="elsevierStyleCrossRef" href="#bib0310"><span class="elsevierStyleSup">23</span></a>&#46; Due to encrustation&#44; these crystalline biofilms may block the catheters in patients with CAUTIs&#46; Sixty-two percent &#40;62&#37;&#41; of patients with recurrent <span class="elsevierStyleItalic">P&#46; mirabilis</span> catheter encrustation developed bladder stones because of catheter colonization<a class="elsevierStyleCrossRef" href="#bib0320"><span class="elsevierStyleSup">25</span></a>&#46; Other possible consequences associated with crystalline biofilms and blockages of the urinary catheter are urine retention&#44; along with bladder and urethra mucosal trauma<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">17</span></a>&#46;</p><p id="par0040" class="elsevierStylePara elsevierViewall">Different factors participate in biofilm development&#44; including motility&#44; which has often been considered a putative factor in this process<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">21</span></a>&#46;</p><p id="par0045" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">P&#46; mirabilis</span> typical swarming motility is produced when vegetative swimmer cells differentiate into elongated&#44; multinucleated and highly flagellated swarmer cells<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">1</span></a>&#46; This process&#44; mediated by flagella&#44; is initiated by bacterial contact with a solid surface and probably contributes to biofilm formation and catheter colonization&#46; The role of flagella and swarming in <span class="elsevierStyleItalic">P&#46; mirabilis</span> UTI is still under debate<a class="elsevierStyleCrossRefs" href="#bib0210"><span class="elsevierStyleSup">3&#44;24</span></a>&#46; Different studies have suggested that flagella significantly contribute to <span class="elsevierStyleItalic">P&#46; mirabilis</span> urovirulence<a class="elsevierStyleCrossRefs" href="#bib0305"><span class="elsevierStyleSup">22&#44;24</span></a>&#46; However&#44; several works indicate that flagella are not critical for establishing UTI by <span class="elsevierStyleItalic">P&#46; mirabilis</span> and other uropathogens<a class="elsevierStyleCrossRefs" href="#bib0285"><span class="elsevierStyleSup">18&#44;19&#44;35</span></a>&#46;</p><p id="par0050" class="elsevierStylePara elsevierViewall">The role of flagella in <span class="elsevierStyleItalic">P&#46; mirabilis</span> biofilm formation remains unclear and is seldomly addressed in the literature&#46; A few studies have proposed that flagella and motility may play a role in the formation of biofilms<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">9</span></a>&#44; while others consider flagella not to be critical in this process<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">16</span></a>&#46;</p><p id="par0055" class="elsevierStylePara elsevierViewall">In this study&#44; we assess the input of <span class="elsevierStyleItalic">P&#46; mirabilis</span> flagella in biofilm formation using a flagellate mutant generated by allelic replacement&#44; tested in different <span class="elsevierStyleItalic">in vitro</span> assays&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Bacterial strains</span><p id="par0060" class="elsevierStylePara elsevierViewall">The clinical wild-type <span class="elsevierStyleItalic">P&#46; mirabilis</span> Pr2921 strain was extensively characterized<a class="elsevierStyleCrossRef" href="#bib0375"><span class="elsevierStyleSup">36</span></a>&#46; An isogenic non-flagellate allelic replacement mutant &#40;AF strain&#41; was used to evaluate the role of flagella in biofilm formation&#46; This mutant has both <span class="elsevierStyleItalic">flaA</span> and <span class="elsevierStyleItalic">flaB</span> structural genes partially deleted and interrupted by a Kanamycin cassette<a class="elsevierStyleCrossRef" href="#bib0325"><span class="elsevierStyleSup">26</span></a>&#44; following the procedure used to generate other flagellate mutants in our laboratory<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">19</span></a>&#46; Absence of swimming and swarming motility was evaluated&#44; and the lack of flagella was evidenced by Western blot<a class="elsevierStyleCrossRef" href="#bib0335"><span class="elsevierStyleSup">28</span></a>&#46; Bacteria were cultured in Luria-Bertani &#40;LB&#41; broth or LB agar &#40;1&#46;5&#37;&#41;&#46; Artificial urine &#40;AU&#41; was prepared according to Scavone et al&#46;<a class="elsevierStyleCrossRef" href="#bib0330"><span class="elsevierStyleSup">27</span></a> Growth curves were first assessed in LB and AU to check that the absence of flagella did not affect normal growth&#46;</p><p id="par0065" class="elsevierStylePara elsevierViewall">Bacterial suspensions were prepared in phosphate-buffered saline &#40;PBS&#41; from fresh LB agar plates at an OD 600<span class="elsevierStyleHsp" style=""></span>nm to inoculate LB or AU cultures in 96-multiwell plates<a class="elsevierStyleCrossRef" href="#bib0330"><span class="elsevierStyleSup">27</span></a>&#46; pH of AU cultures of both strains was controlled serially&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Hydrophobicity of planktonic cells</span><p id="par0070" class="elsevierStylePara elsevierViewall">Ten microliters of bacterial suspensions in PBS &#40;OD<span class="elsevierStyleInf">540</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;2&#8211;0&#46;3&#41; were inoculated in 5<span class="elsevierStyleHsp" style=""></span>ml of AU or LB&#44; and incubated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 48<span class="elsevierStyleHsp" style=""></span>h&#46; Then&#44; bacterial suspensions &#40;OD<span class="elsevierStyleInf">600</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;8&#41; and 200<span class="elsevierStyleHsp" style=""></span>&#956;l of added xylene were mixed&#44; shaken for 2<span class="elsevierStyleHsp" style=""></span>min and left static for 20<span class="elsevierStyleHsp" style=""></span>min&#46; Subsequently&#44; the aqueous phase &#40;OD<span class="elsevierStyleHsp" style=""></span>600<span class="elsevierStyleHsp" style=""></span>nm&#41; was measured and hydrophobicity was assessed according to Bibiloni et al&#46;<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">2</span></a>&#46; The assay was done in triplicate&#46; Hydrophobicity of planktonic cells of the different strains and under different conditions was assessed using the Tukey&#39;s multiple comparison test&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Swimming and swarming motility</span><p id="par0075" class="elsevierStylePara elsevierViewall">Swimming motility was evaluated as described by Hola et al&#46;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">11</span></a>&#44; and swarming motility was assessed as described by Jones et al&#46;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">15</span></a>&#46; Both assays were performed in triplicate&#46;</p><p id="par0080" class="elsevierStylePara elsevierViewall">Inhibition of swarming was evaluated using a specific <span class="elsevierStyleItalic">P&#46; mirabilis</span> flagella rabbit anti-serum&#46; For this purpose&#44; a rabbit was immunized with four doses of 25<span class="elsevierStyleHsp" style=""></span>&#956;g of purified Pr2921 flagellin administered subcutaneously&#44; using complete Freund&#39;s adjuvant &#40;Sigma&#41; for the first dose and incomplete Freund&#39;s adjuvant for the following ones&#44; according to previous studies<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">19</span></a>&#46; These procedures were approved by the Institutional Animal Care Committee and adequate measures were taken to minimize discomfort or stress of the animal&#46; The antibody response was determined ten days after the last dose &#40;day 55&#41; and serum anti-flagella IgG titer measured by ELISA reached a value of 1&#58;100<span class="elsevierStyleHsp" style=""></span>000&#46;</p><p id="par0085" class="elsevierStylePara elsevierViewall">For the swarming inhibition assay&#44; the WT strain was grown on 50<span class="elsevierStyleHsp" style=""></span>mm diameter plates containing 5<span class="elsevierStyleHsp" style=""></span>ml of modified LB agar and LB agar supplemented with the specific rabbit anti-serum&#44; using a 1&#58;10 dilution&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Bacterial migration across urethral catheters</span><p id="par0090" class="elsevierStylePara elsevierViewall">The ability to migrate across urinary catheters sections was assessed following the procedure proposed by Stickler and Huges<a class="elsevierStyleCrossRef" href="#bib0355"><span class="elsevierStyleSup">32</span></a>&#46; The catheter materials were silicone and latex &#40;Teleflex Medical&#44; Kernen&#44; Germany&#41;&#46; Migration was tested 15 times for both catheter types&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Quantification of biofilm formation on polystyrene multiwell plates</span><p id="par0095" class="elsevierStylePara elsevierViewall">The effect of the flagellar mutation in biofilm formation was evaluated using the semi-quantification technique based on the adsorption of crystal violet &#40;CV&#41;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">6</span></a>&#46;</p><p id="par0100" class="elsevierStylePara elsevierViewall">A competition assay was also performed to assess the ability of both strains to remain attached to the polystyrene surface&#44; forming a biofilm when cultured simultaneously following the procedure described above&#46; However&#44; in this case&#44; the biofilms were scrapped and bacteria were cultured in nutrient agar and nutrient agar supplemented with kanamycin since AF carries a kanamycin-resistance gene incorporated into the mutagenic process<a class="elsevierStyleCrossRef" href="#bib0325"><span class="elsevierStyleSup">26</span></a>&#46; Biofilm formation of the different strains and under different conditions was assessed using the Tukey&#39;s multiple comparison test&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Biofilm formation over time by confocal laser scanning microscopy</span><p id="par0105" class="elsevierStylePara elsevierViewall">Biofilm formation of Pr2921 and AF was evaluated in 2&#44; 5 and 7 day-LB and AU cultures under static conditions at 37<span class="elsevierStyleHsp" style=""></span>&#176;C<a class="elsevierStyleCrossRef" href="#bib0330"><span class="elsevierStyleSup">27</span></a>&#46; This method allows biofilm formation on a coverslip placed into a tube subjected to fluorescent staining after different incubation periods&#46; Staining techniques and microscopy analyses were performed according to Schlapp et al&#46;<a class="elsevierStyleCrossRef" href="#bib0350"><span class="elsevierStyleSup">31</span></a>&#46; Bacteria were stained with Syto 9 &#40;Molecular Probes&#41; and the extracellular matrix with FilmTracer&#8482; SYPRO&#174; Ruby Biofilm matrix stain&#46; 3D images were acquired using a Leica TCS LSI super-zoom confocal fluorescent microscope with an iXon Ultra 897 back-illuminated imaging EMCCD camera&#44; LAS Software&#44; a 100&#215; oil immersion objective &#40;NA<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>1&#46;35&#41; and 488&#47;520&#44; 450&#47;610<span class="elsevierStyleHsp" style=""></span>nm excitation&#47;emission wavelength&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">Three z-stacks were randomly chosen in each sample&#44; using an acquisition step of 0&#46;3<span class="elsevierStyleHsp" style=""></span>&#956;m in the <span class="elsevierStyleItalic">z</span>-axis and 1024<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>1024 pixels in the <span class="elsevierStyleItalic">xy</span>-plane with a pixel size of 170<span class="elsevierStyleHsp" style=""></span>nm&#46; After deconvolution using Huygens Software&#44; segmentation&#44; visualization&#44; 3D reconstruction and biofilm parameter descriptors were calculated using ScianSoft<a class="elsevierStyleCrossRef" href="#bib0350"><span class="elsevierStyleSup">31</span></a>&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">Bacterial number&#44; bacterial and matrix volumes were calculated to compare biofilm evolution&#46; Results were compared using one-way ANOVA <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#8804;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Biofilm grown under a flow system</span><p id="par0120" class="elsevierStylePara elsevierViewall">In order to evaluate biofilm formation under a flow system&#44; we have developed a system that consists of a medium reservoir&#44; a peristaltic pump&#44; a chamber where the biofilm was grown and a disposal flask&#46; The chamber had two slides &#40;bottom and top&#41; in order to follow the biofilm formation by confocal microscopy&#46; The system was set up with a LB flow at 0&#46;5<span class="elsevierStyleHsp" style=""></span>ml&#47;min&#46; First&#44; the system was assembled and after removing air bubbles&#44; the bacterial suspension was introduced into the chambers through a three-way stop cock&#46; The biofilm was allowed to form for 1<span class="elsevierStyleHsp" style=""></span>h&#44; after that the flow was started for 24&#44; 48 and 72<span class="elsevierStyleHsp" style=""></span>h&#46; Acridine orange &#40;0&#46;01<span class="elsevierStyleHsp" style=""></span>mg&#47;ml&#41; was used to stain and visualize bacteria in the biofilm&#46; At different time points&#44; the system was stopped and the dye was added to the system through the three-way stop cock&#46; Visualization was performed in a LSM Zeiss 800&#44; using a 63&#215; oil immersion objective &#40;NA 1&#46;4&#41;&#46; Live images were taken every 127<span class="elsevierStyleHsp" style=""></span>s for 3<span class="elsevierStyleHsp" style=""></span>min&#46; After acquisition&#44; the system was loaded again until the next time point&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Image processing of time-lapse biofilm</span><p id="par0125" class="elsevierStylePara elsevierViewall">Image processing and analysis were performed using FIJI&#47;ImageJ software<a class="elsevierStyleCrossRef" href="#bib0345"><span class="elsevierStyleSup">30</span></a>&#46; Morphological analysis was performed using MicrobeJ plugin<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">7</span></a>&#46; Morphological parameters were defined according to the different morphotypes of <span class="elsevierStyleItalic">P&#46; mirabilis</span> described by Jansen et al&#46;<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">14</span></a> Swarmer cells with a length greater than 10<span class="elsevierStyleHsp" style=""></span>&#956;m and a maximum width of 3<span class="elsevierStyleHsp" style=""></span>&#956;m were excluded from the analysis&#46; Thresholds were used for binarization and length&#47;width exclusion criteria prior to analysis&#46; Contour detection was performed using Fit-Shape mode&#46; The shape descriptors calculated from the data were length&#44; mean width and aspect ratio&#44; defined as the length of the major axis divided by the minor axis of the ellipsoid fitted to each particle&#46; Statistical analysis was performed by RStudio software &#40;RStudio Team&#44; 2020&#41;&#46; The differences between each time-lapse were analyzed with the Wilcoxon test&#46; Motility analyses were performed with TrackMate plugin<a class="elsevierStyleCrossRefs" href="#bib0235"><span class="elsevierStyleSup">8&#44;33</span></a>&#46; Motility analysis was performed using previously segmented time-lapse image sequences by Ilastik software&#46; TrackMate auto intensity threshold tool was used to select all particles for track analysis&#46; The trajectories of each individual bacterium were determined by LAP Tracker&#46; It was set allowing a frame-to-frame linking and gap closing of 6<span class="elsevierStyleHsp" style=""></span>&#956;m&#44; splitting and merging was set at 0&#46;6<span class="elsevierStyleHsp" style=""></span>&#956;m&#44; and ellipse long axis&#44; ellipse short axis and max intensity were used as penalties for linking cost calculation&#46; Mean square displacements &#40;MSD&#41; were calculated with R Studio software by Eq&#46; <a class="elsevierStyleCrossRef" href="#eq0005">&#40;1&#41;</a> &#40;RStudio Team&#44; 2020&#41;&#46; RStudio&#58; Integrated Development for R&#46; RStudio&#44; PBC&#44; Boston&#44; MA &#40;URL <a href="http://www.rstudio.com/">http&#58;&#47;&#47;www&#46;rstudio&#46;com</a>&#41;&#46;<elsevierMultimedia ident="eq0005"></elsevierMultimedia></p></span></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Results</span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Lack of flagella did not affect bacterial features&#44; except flagellin expression</span><p id="par0130" class="elsevierStylePara elsevierViewall">Growth curves of Pr2921 and AF cultured in LB or AU were similar&#44; showing that the general biology of the mutant was not affected&#44; and both strains grew better in LB compared with AU &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0135" class="elsevierStylePara elsevierViewall">pH of Pr2921 and AF cultures in AU reached a value of 9 after 6<span class="elsevierStyleHsp" style=""></span>h of incubation in both cases&#44; remaining constant for at least 24<span class="elsevierStyleHsp" style=""></span>h&#44; demonstrating that urease production was similar in both strains&#46; Expression of MR&#47;P fimbriae assessed by Western blotting &#40;<a class="elsevierStyleCrossRef" href="#sec0110">Suppl&#46; Fig&#46; S1</a>&#41;&#44; hemagglutination based on MR&#47;P fimbrial expression&#44; and hemolysis observed on blood agar plates were similar in both strains &#40;data not shown&#41;&#46;</p><p id="par0140" class="elsevierStylePara elsevierViewall">Genetic rearrangements due to allelic replacement mutagenesis were as expected and the mutant was unable to express flagellin<a class="elsevierStyleCrossRef" href="#bib0335"><span class="elsevierStyleSup">28</span></a>&#46;</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Hydrophobicity index is increased in AU</span><p id="par0145" class="elsevierStylePara elsevierViewall">When bacteria were grown in LB&#44; no significant differences were observed among the hydrophobicity indexes between both strains&#46; However&#44; when bacteria were grown in AU&#44; the AF mutant exhibited a significant hydrophobicity increase compared with the wild-type Pr2921&#44; in AU &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46; In addition&#44; hydrophobicity of planktonic bacteria grown in AU was significantly higher than bacteria grown in LB&#44; in the case of both strains &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#44; <a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Swimming and swarming motility and migration across latex and silicone catheter bridges</span><p id="par0150" class="elsevierStylePara elsevierViewall">The flagellate mutant was unable to swim or swarm &#40;<a class="elsevierStyleCrossRef" href="#sec0110">Suppl&#46; Fig&#46; S2</a>&#41;&#46; In both assays&#44; the mutant grew to form a defined colony without spreading beyond the colony limits&#46;</p><p id="par0155" class="elsevierStylePara elsevierViewall">Swarming was completely inhibited when the WT strain &#40;Pr2921&#41; grew on LB agar supplemented with specific anti-flagella rabbit serum &#40;1&#58;10&#41;&#46; Lower serum concentrations obtained by serial dilutions inhibited swarming in a dose-dependent relationship &#40;data not shown&#41;&#46;</p><p id="par0160" class="elsevierStylePara elsevierViewall">The influence of flagella on <span class="elsevierStyleItalic">P&#46; mirabilis</span> migration across catheter sections was assessed&#46; The WT strain crossed latex and silicone catheter bridges in every case &#40;15&#47;15&#41;&#44; while the AF mutant was unable to cross any catheter sections &#40;0&#47;15&#41; &#40;<a class="elsevierStyleCrossRef" href="#sec0110">Suppl&#46; Fig&#46; S2</a>&#41;&#46;</p></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Biofilm formation on polystyrene</span><p id="par0165" class="elsevierStylePara elsevierViewall">When biofilm formation was evaluated using the method based on associated CV adsorption&#44; the AF mutant showed a significantly impaired ability to form biofilms compared to WT Pr2921 in LB &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;0001&#41;&#46;</p><p id="par0170" class="elsevierStylePara elsevierViewall">When the assay was performed using AU&#44; the level of biofilm formation of the mutant strain was lower than that formed by the WT strain but the difference was not significant &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#41;&#46; A significantly reduced biofilm was also observed when WT grew in AU compared with that observed in LB &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;0001&#41;&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0175" class="elsevierStylePara elsevierViewall">A competition assay was also performed to evaluate if the mutant was outcompeted by the WT strain in the biofilm&#46; Results indicated that the wild-type strain outcompeted the mutant taking into account viable bacterial counts of Pr2921&#47;AF from co-cultured biofilms&#46; At 24<span class="elsevierStyleHsp" style=""></span>h&#44; wild type and mutant counts &#40;log&#41; were 7&#46;63<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;01 and 2&#46;28<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;2&#44; respectively &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; After 48<span class="elsevierStyleHsp" style=""></span>h&#44; wild type and mutant counts remained at similar levels &#40;7&#46;28<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;07 and 2&#46;64<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;09&#44; respectively&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Biofilm formation dynamics</span><p id="par0180" class="elsevierStylePara elsevierViewall">Biofilm formation was evaluated in LB and AU under static conditions at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; at 2&#44; 5 and 7 days of incubation&#44; using CLSM and image analysis &#40;<a class="elsevierStyleCrossRefs" href="#fig0020">Figs&#46; 4 and 5</a>&#41;&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia><elsevierMultimedia ident="fig0025"></elsevierMultimedia><p id="par0185" class="elsevierStylePara elsevierViewall">When biofilm formation was assessed after 2 days in LB or AU&#44; the bacterial volume and the extracellular matrix of WT and the flagellate mutant AF were similar&#44; showing in general low values with no significant differences among strains or media conditions &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>&#41;&#46; However&#44; after 5 days&#44; significant differences were observed among WT and the mutant AF strains grown in LB&#46; The WT bacterial and matrix volumes significantly increased compared to the AF mutant &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;0001 in both cases&#41;&#46; When both strains were cultured in AU&#44; no AF bacteria remained attached to the coverslip&#44; while WT Pr2921 exhibited a structured biofilm&#46; However&#44; the statistical difference was not significant since the WT biofilm was small&#46; Furthermore&#44; the AF matrix in AU was almost inexistent&#46;</p><p id="par0195" class="elsevierStylePara elsevierViewall">After 7 days of incubation in LB&#44; the bacterial and matrix volume of the biofilm formed by WT 2921 decreased and differences were not significant when compared to the biofilm formed by AF &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; When bacteria were cultured in AU&#44; no statistical differences between strains were identified&#59; however&#44; a small WT 2921 biofilm &#40;both bacteria and matrix&#41; was still observed&#44; but not with the AF mutant &#40;<a class="elsevierStyleCrossRefs" href="#fig0020">Figs&#46; 4 and 5</a>&#41;&#46;</p><p id="par0200" class="elsevierStylePara elsevierViewall">The dynamic system allowed <span class="elsevierStyleItalic">in vivo</span> observation of the biofilm every 24<span class="elsevierStyleHsp" style=""></span>h &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>A&#41;&#46; Both strains formed biofilm&#44; but differences were observed between WT and the AF mutant&#46; At 24<span class="elsevierStyleHsp" style=""></span>h&#44; biofilms were flat as observed by the height in the 3D stacks &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>B upper panels&#41;&#46; WT 2921 biofilm was around 1&#46;25<span class="elsevierStyleHsp" style=""></span>&#956;m in height while the AF biofilm was 0&#46;75<span class="elsevierStyleHsp" style=""></span>&#956;m in height&#46; Moreover&#44; significant differences were observed in the size and shape of the bacteria &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>B&#41;&#46; WT 2921 was more elongated than AF as represented by the mean width &#40;the length of the major axis divided by the minor axis of the ellipsoid fitted to each bacteria&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Figs&#46; 6B and C</a>&#41;&#46; At 48<span class="elsevierStyleHsp" style=""></span>h&#44; WT 2921 increased the 3D biofilm structure reaching 2<span class="elsevierStyleHsp" style=""></span>&#956;m in height and forming channels while the AF biofilm remained flat&#46; At 72<span class="elsevierStyleHsp" style=""></span>h&#44; AF showed a compacted and thin biofilm with shortened bacteria and WT 2921 had a heterogeneous bacterial morphology within a 3D biofilm with the presence of channels &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>B&#44; <a class="elsevierStyleCrossRef" href="#sec0110">Suppl&#46; MSD</a>&#41;&#46; While WT 2921 formed a mature biofilm with different bacterial morphologies&#44; the AF mutant strain showed a flat biofilm and the morphology of the bacteria was reduced over time&#46; With regard to the length of the bacteria&#44; we observed that 2921 acquired a more elongated morphology while AF was shortened &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Figs&#46; 6B and C</a>&#41;&#46;</p><elsevierMultimedia ident="fig0030"></elsevierMultimedia></span></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Discussion</span><p id="par0205" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">P&#46; mirabilis</span> is among the most frequent causes of complicated UTI<a class="elsevierStyleCrossRef" href="#bib0335"><span class="elsevierStyleSup">28</span></a>&#44; and displays a broad and complex range of virulence factors that may participate in the colonization of different surfaces<a class="elsevierStyleCrossRefs" href="#bib0255"><span class="elsevierStyleSup">12&#44;27</span></a>&#46;</p><p id="par0210" class="elsevierStylePara elsevierViewall">Flagella have been associated with the formation of biofilms in different pathogens&#44; including <span class="elsevierStyleItalic">Bacillus cereus</span>&#44; <span class="elsevierStyleItalic">Yersinia enterocolitica</span> and <span class="elsevierStyleItalic">Helicobacter</span><span class="elsevierStyleItalic">pylori</span><a class="elsevierStyleCrossRefs" href="#bib0245"><span class="elsevierStyleSup">10&#44;20</span></a>&#46; However&#44; the role of flagella and swarming motility in <span class="elsevierStyleItalic">P&#46; mirabilis</span> UTI and biofilm formation has been thoroughly debated<a class="elsevierStyleCrossRefs" href="#bib0290"><span class="elsevierStyleSup">19&#44;22&#44;29</span></a>&#46;</p><p id="par0215" class="elsevierStylePara elsevierViewall">In this study&#44; we used a flagellate mutant to assess the role of flagella in <span class="elsevierStyleItalic">P&#46; mirabilis</span> biofilm formation&#44; through different experimental <span class="elsevierStyleItalic">in vitro</span> approaches&#44; which are discussed below&#46;</p><p id="par0220" class="elsevierStylePara elsevierViewall">Growth patterns were similar when WT Pr2921 and the AF mutant were grown in LB broth or AU&#44; indicating that the mutation did not alter the general bacterial biology&#46; In addition&#44; a series of phenotypic pathogenic features were not altered by the mutagenesis process&#44; including urease production&#44; fimbriae expression&#44; hemagglutination and hemolysis production&#46;</p><p id="par0225" class="elsevierStylePara elsevierViewall">The influence of flagella on <span class="elsevierStyleItalic">P&#46; mirabilis</span> surface-related features was assessed by measuring bacterial hydrophobicity&#46; According to different authors&#44; bacterial surface hydrophobicity is important for adherence to different interfaces and biofilm formation<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">4</span></a>&#46; When the flagellate mutant was grown in LB broth&#44; bacterial hydrophobicity did not vary&#46; However&#44; when grown in AU&#44; hydrophobicity significantly increased&#44; probably associated with an increased exposure of the lipid components of the bacterial surface&#46; In other studies&#44; no clear relationships between bacterial hydrophobicity and enhancement of binding to different substrates were observed<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">5</span></a>&#46;</p><p id="par0230" class="elsevierStylePara elsevierViewall">In this study&#44; we also assessed the ability of the isogenic flagellate mutant to swim&#44; swarm and migrate over catheter sections of different materials&#46; The mutant was unable to swim or swarm on LB agar or to migrate along sections of latex and silicone catheters&#44; strongly confirming the role of flagella in the colonization of these clinical devices&#46; These results are in accordance with previous studies<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">15</span></a>&#46;</p><p id="par0235" class="elsevierStylePara elsevierViewall">The inclusion of specific <span class="elsevierStyleItalic">P&#46; mirabilis</span> antiflagella antibodies in the culture medium also resulted in the abolition of motility&#44; reinforcing the clear and specific role of flagellar function in motility and spreading over different surfaces through an indirect approach&#46;</p><p id="par0240" class="elsevierStylePara elsevierViewall">When biofilm formation was assessed on an abiotic surface like polystyrene&#44; the flagellate mutant grown in LB showed a significant smaller biofilm compared with the wild type Pr2921&#46; When bacteria grew in AU&#44; the mutant also formed a smaller biofilm although differences were not significant&#46; In this case&#44; it must be taken into account that <span class="elsevierStyleItalic">P&#46; mirabilis</span> growth in AU was significantly less abundant than in LB broth&#44; influencing the magnitude of growth differences in both strains&#46;</p><p id="par0245" class="elsevierStylePara elsevierViewall">A competition assay based on the wild type and the mutant co-cultivation was performed to refine the assessment of flagella contribution in biofilm formation&#46; We had used this kind of approach before to detect subtle effects of different <span class="elsevierStyleItalic">P&#46; mirabilis</span> virulence factors in ascending UTI in mice<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">13</span></a>&#46; Results of this assay revealed that although the mutant was outcompeted by the WT strain&#44; a biofilm was formed that even persisted for at least 48<span class="elsevierStyleHsp" style=""></span>h&#44; indicating that biofilm formation was not completely abolished by the lack of flagella&#46;</p><p id="par0250" class="elsevierStylePara elsevierViewall">When CLSM was used to assess biofilm formation in LB broth and AU&#44; significant differences were observed among biofilm parameters at different stages&#46; One of the most notorious differences was the impaired capacity of the AF mutant to grow and produce extracellular matrix at the biofilm maturation stage in the static model &#40;5 days&#41;&#46; At this stage&#44; the AF mutant biofilm had almost disappeared in AU while the WT biofilm&#44; although small&#44; remained attached and showing a typical mature structure&#44; remaining attached even after 7 days&#46; Our results confirm that flagella are important for biofilm formation&#44; under different conditions&#46; Furthermore&#44; differences in bacterial morphology were observed under dynamic conditions&#44; but the AF mutant was able to form a biofilm&#46;</p><p id="par0255" class="elsevierStylePara elsevierViewall">Different authors have reported that mutations of <span class="elsevierStyleItalic">P&#46; mirabilis</span> virulence genes have different effects on biofilm formation that can induce a decrease or even an increase in matrix or bacterial biovolumes<a class="elsevierStyleCrossRefs" href="#bib0255"><span class="elsevierStyleSup">12&#44;36</span></a>&#46;</p><p id="par0260" class="elsevierStylePara elsevierViewall">Several studies have reported the role of bacterial flagella in adhesion to biotic and abiotic surfaces&#44; including <span class="elsevierStyleItalic">P&#46; mirabilis</span>&#44; which can contribute to the initial contact with different surfaces and even among cells<a class="elsevierStyleCrossRefs" href="#bib0270"><span class="elsevierStyleSup">15&#44;35</span></a>&#46;</p><p id="par0265" class="elsevierStylePara elsevierViewall">As mentioned above&#44; Jones et al&#46;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">16</span></a> suggested that neither swarming nor swimming motility are required for the attachment of <span class="elsevierStyleItalic">P&#46; mirabilis</span> to catheters and that mutants unable to swarm and swim formed crystalline biofilms and blocked catheters even more rapidly than the wild-type strain&#46; The authors hypothesize that on contact with a surface&#44; the non-swarmer cells are more likely to remain at the site of attachment&#44; divide or produce microcolonies that initiate biofilm formation&#46; On the other hand&#44; Fusco et al&#46;<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">9</span></a> compared different <span class="elsevierStyleItalic">P&#46; mirabilis</span> strains and reported that motility ability was related to biofilm formation&#59; however&#44; they did not use isogenic mutants&#46;</p></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Conclusion</span><p id="par0270" class="elsevierStylePara elsevierViewall">Overall&#44; our results show that flagella critically contribute to the formation of <span class="elsevierStyleItalic">P&#46; mirabilis</span> biofilms&#46; However&#44; the lack of this organelle could not completely prevent biofilm formation&#44; suggesting that different elements drive biofilm formation and persistence&#46; Like other factors &#40;<span class="elsevierStyleItalic">e&#46;g&#46;</span>&#44; ATF fimbria&#41;<a class="elsevierStyleCrossRef" href="#bib0375"><span class="elsevierStyleSup">36</span></a>&#44; flagella could play a significant role in biofilm formation on abiotic surfaces rather than in urinary tract colonization and infection&#46;</p><p id="par0275" class="elsevierStylePara elsevierViewall">This knowledge could contribute to the design of strategies for <span class="elsevierStyleItalic">P&#46; mirabilis</span> biofilm prevention targeting the flagellar function&#44; possibly in combination with other specific mechanisms&#46;</p></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Conflict of interest</span><p id="par0280" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflict of interest&#46;</p></span></span>"
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          "titulo" => "Introduction"
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          "identificador" => "sec0010"
          "titulo" => "Materials and methods"
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              "identificador" => "sec0015"
              "titulo" => "Bacterial strains"
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              "titulo" => "Hydrophobicity of planktonic cells"
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              "identificador" => "sec0025"
              "titulo" => "Swimming and swarming motility"
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            3 => array:2 [
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              "titulo" => "Bacterial migration across urethral catheters"
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              "titulo" => "Quantification of biofilm formation on polystyrene multiwell plates"
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              "titulo" => "Biofilm formation over time by confocal laser scanning microscopy"
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              "identificador" => "sec0045"
              "titulo" => "Biofilm grown under a flow system"
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          "titulo" => "Results"
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              "titulo" => "Lack of flagella did not affect bacterial features&#44; except flagellin expression"
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            1 => array:2 [
              "identificador" => "sec0065"
              "titulo" => "Hydrophobicity index is increased in AU"
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            2 => array:2 [
              "identificador" => "sec0070"
              "titulo" => "Swimming and swarming motility and migration across latex and silicone catheter bridges"
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            3 => array:2 [
              "identificador" => "sec0075"
              "titulo" => "Biofilm formation on polystyrene"
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              "identificador" => "sec0080"
              "titulo" => "Biofilm formation dynamics"
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          "palabras" => array:6 [
            0 => "Urinary tract infection"
            1 => "<span class="elsevierStyleItalic">Proteus mirabilis</span>"
            2 => "Flagella"
            3 => "Biofilm"
            4 => "Catheter"
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            0 => "Infecci&#243;n del tracto urinario"
            1 => "<span class="elsevierStyleItalic">Proteus mirabilis</span>"
            2 => "Flagelos"
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            4 => "Cat&#233;ter"
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    "highlights" => array:2 [
      "titulo" => "Highlights"
      "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall"><ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005"><span class="elsevierStyleLabel">&#8226;</span><p id="par0005" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">P&#46; mirabilis</span> flagella have a medium-dependent role in cellular hydrophobicity&#46;</p></li><li class="elsevierStyleListItem" id="lsti0010"><span class="elsevierStyleLabel">&#8226;</span><p id="par0010" class="elsevierStylePara elsevierViewall">All motility and catheter migration models showed a significant role of flagella&#46;</p></li><li class="elsevierStyleListItem" id="lsti0015"><span class="elsevierStyleLabel">&#8226;</span><p id="par0015" class="elsevierStylePara elsevierViewall">A flagellate <span class="elsevierStyleItalic">P&#46; mirabilis</span> mutant was outcompeted by the wild-type in biofilms&#46;</p></li><li class="elsevierStyleListItem" id="lsti0020"><span class="elsevierStyleLabel">&#8226;</span><p id="par0020" class="elsevierStylePara elsevierViewall">Biofilm formation of the mutant was impaired as seen in different <span class="elsevierStyleItalic">in vitro</span> assays&#46;</p></li><li class="elsevierStyleListItem" id="lsti0025"><span class="elsevierStyleLabel">&#8226;</span><p id="par0025" class="elsevierStylePara elsevierViewall">Flagellar function could be targeted to prevent biofilm formation&#46;</p></li></ul></p></span>"
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        "resumen" => "<span id="abst0010" class="elsevierStyleSection elsevierViewall"><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">Proteus mirabilis</span><span class="elsevierStyleItalic">&#40;P&#46; mirabilis&#41;</span> is a common etiological agent of urinary tract infections&#44; particularly those associated with catheterization&#46; <span class="elsevierStyleItalic">P&#46; mirabilis</span> efficiently forms biofilms on different surfaces and shows a multicellular behavior called &#8216;swarming&#8217;&#44; mediated by flagella&#46; To date&#44; the role of flagella in <span class="elsevierStyleItalic">P&#46; mirabilis</span> biofilm formation has been under debate&#46; In this study&#44; we assessed the role of <span class="elsevierStyleItalic">P&#46; mirabilis</span> flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin&#46; Different approaches were used&#44; such as the evaluation of cell surface hydrophobicity&#44; bacterial motility and migration across catheter sections&#44; measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models&#46; Our findings indicate that <span class="elsevierStyleItalic">P&#46; mirabilis</span> flagella play a role in biofilm formation&#44; although their lack does not completely avoid biofilm generation&#46; Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets&#46;</p></span>"
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        "resumen" => "<span id="abst0015" class="elsevierStyleSection elsevierViewall"><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">Proteus mirabilis</span><span class="elsevierStyleItalic">&#40;P&#46; mirabilis&#41;</span> es un agente etiol&#243;gico com&#250;n de infecciones del tracto urinario&#44; en particular de aquellas asociadas con cateterizaci&#243;n&#46; <span class="elsevierStyleItalic">P&#46; mirabilis</span> forma biofilms eficientemente en diferentes superficies y muestra un comportamiento multicelular llamado <span class="elsevierStyleItalic">swarming</span>&#44; mediado por flagelos&#46; Hasta el momento&#44; el papel de los flagelos en la formaci&#243;n de biofilms de <span class="elsevierStyleItalic">P&#46; mirabilis</span> ha estado en discusi&#243;n&#46; En este estudio&#44; se evalu&#243; el papel de los flagelos de <span class="elsevierStyleItalic">P&#46; mirabilis</span> en la formaci&#243;n de biofilms&#44; utilizando una mutante isog&#233;nica generada por reemplazo al&#233;lico&#44; incapaz de expresar flagelina&#46; Se utilizaron diferentes enfoques&#44; como la evaluaci&#243;n de la hidrofobicidad de la superficie celular&#44; de la movilidad y la migraci&#243;n bacteriana sobre secciones de cat&#233;teres y medidas de biomasa y de la din&#225;mica del biofilm mediante inmunofluorescencia y microscopia confocal&#44; tanto en modelos est&#225;ticos como de flujo&#46; Nuestros hallazgos indican que los flagelos de <span class="elsevierStyleItalic">P&#46; mirabilis</span> desempe&#241;an un papel en la formaci&#243;n de biofilms&#44; aunque su falta no suprime por completo su generaci&#243;n&#46; Asimismo&#44; evidencian que la interferencia de la funci&#243;n flagelar puede contribuir a evitar la formaci&#243;n de biofilms en el contexto de estrategias centradas en blancos bacterianos particulares&#46;</p></span>"
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            "etiqueta" => "Appendix A"
            "titulo" => "Supplementary data"
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          "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Growth curves of Pr2921 <span class="elsevierStyleItalic">P&#46; mirabilis</span> wild-type strain and flagellate mutant AF in LB broth &#40;LB&#41; and artificial urine &#40;AU&#41;&#46;</p> <p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Growth curves were generated by serial OD measures of grown bacteria&#46;</p>"
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          "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Hydrophobicity of Pr2921 <span class="elsevierStyleItalic">P&#46; mirabilis</span> wild-type strain and flagellate mutant AF grown in LB broth &#40;LB&#41; and artificial urine &#40;AU&#41;&#46;</p> <p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">When bacteria were grown in AU&#44; the AF mutant exhibited a significant hydrophobicity increase compared with the wild-type Pr2921 strain in AU &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46; Hydrophobicity was significantly higher in AU than in LB in the case of both strains &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; Statistical differences were calculated using the Tukey&#39;s multiple comparison test&#44; &#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;005&#44; &#42;&#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;0005&#44; &#42;&#42;&#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;0001&#46;</p>"
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          "en" => "<p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Biofilm formation on polystyrene surfaces assessed by the semi-quantitative method based on crystal violet staining&#46;</p> <p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Pr2921 <span class="elsevierStyleItalic">P&#46; mirabilis</span> wild-type strain and flagellate mutant AF were grown in LB broth &#40;LB&#41; and in artificial urine &#40;AU&#41; and incubated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 48<span class="elsevierStyleHsp" style=""></span>h&#46; Box plot of the OD values obtained in each condition are shown&#59; asterisks show significant differences among different conditions&#46; Statistical differences were calculated using the Tukey&#39;s multiple comparison test&#44; &#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;005&#44; &#42;&#42;&#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;0001&#46;</p>"
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          "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Bacteria and matrix volumes in biofilms formed by Pr2921 <span class="elsevierStyleItalic">P&#46; mirabilis</span> wild-type strain and flagellate mutant AF in LB broth &#40;LB&#41; and artificial urine &#40;AU&#41; over time&#46; Total bacteria and matrix volumes in biofilms formed in LB broth &#40;LB&#41; and artificial urine &#40;AU&#41; are shown in panels &#40;A&#41; and &#40;B&#41;&#44; respectively&#46; &#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#8804;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#46; The symbols represent the means of 3&#8211;5 random fields and their corresponding standard errors&#46;</p>"
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          "en" => "<p id="spar0075" class="elsevierStyleSimplePara elsevierViewall">Time-lapse biofilms&#46; &#40;A&#41; The dynamic flow system that consists of a medium reservoir &#40;a&#41;&#44; a peristaltic pump &#40;b&#41;&#44; the 3-way stop cock &#40;c&#41;&#44; the biofilm chamber &#40;d&#41; and the disposal flask &#40;e&#41;&#46; &#40;B&#41; Representative 3D stacks of each time point &#40;24&#44; 48 and 72<span class="elsevierStyleHsp" style=""></span>h&#41; of Pr2921 &#40;left&#41; and AF &#40;right&#41;&#46; The scale bar represents 10<span class="elsevierStyleHsp" style=""></span>&#956;m&#44; and applies to all images&#46; &#40;C&#41; Aspect ratio&#44; defined as the length of the major axis divided into the minor axis of the ellipsoid that is fitted to each particle&#46; Significant differences were calculated using the &#42;&#42;&#42; P&#60;0&#46;05&#46;</p>"
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                            5 => "&#321;&#46; Lechowicz"
                            6 => "D&#46; Gmiter"
                            7 => "P&#46; Kowalczyk"
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                    0 => array:2 [
                      "doi" => "10.1007/s00203-016-1249-x"
                      "Revista" => array:6 [
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                      "autores" => array:1 [
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                            0 => "R&#46; de los Santos"
                            1 => "M&#46; Fern&#225;ndez"
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                0 => array:2 [
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                    0 => array:2 [
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                      "autores" => array:1 [
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                          "etal" => false
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                      "doi" => "10.1038/nmicrobiol.2016.77"
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                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:15 [
                            0 => "D&#46; Ershov"
                            1 => "M&#46;-S&#46; Phan"
                            2 => "J&#46;W&#46; Pylv&#228;n&#228;inen"
                            3 => "S&#46;U&#46; Rigaud"
                            4 => "L&#46; Le Blanc"
                            5 => "A&#46; Charles-Orszag"
                            6 => "R&#46; James"
                            7 => "W&#46; Conway"
                            8 => "F&#46; Romain"
                            9 => "N&#46; Laine"
                            10 => "N&#46;H&#46;P&#46; Roy"
                            11 => "D&#46; Bonazzi"
                            12 => "G&#46; Dum&#233;nil"
                            13 => "G&#46; Jacquemet"
                            14 => "J&#46;Y&#46; Tinevez"
                          ]
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                    ]
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                        0 => array:2 [
                          "etal" => false
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                            0 => "A&#46; Fusco"
                            1 => "L&#46; Coretti"
                            2 => "V&#46; Savio"
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                          ]
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                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.3390/ijms18020414"
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                        "paginaInicial" => "414"
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                      "titulo" => "<span class="elsevierStyleItalic">Helicobacter pylori</span> biofilm involves a multigene stress-biased response&#44; including a structural role for flagella"
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                          "etal" => false
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                            0 => "S&#46; Hathroubi"
                            1 => "J&#46; Zerebinski"
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                          ]
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                  ]
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                        "fecha" => "2018"
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                            0 => "V&#46; Hola"
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                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
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                            0 => "N&#46; Holling"
                            1 => "D&#46; Lednor"
                            2 => "S&#46; Tsang"
                            3 => "A&#46; Bissell"
                            4 => "L&#46; Campbell"
                            5 => "J&#46; Nzakizwanayo"
                            6 => "C&#46; Dedi"
                            7 => "J&#46;A&#46; Hawthorne"
                            8 => "G&#46; Hanlon"
                            9 => "L&#46;A&#46; Ogilvie"
                            10 => "J&#46;P&#46; Salvage"
                            11 => "B&#46;A&#46; Patel"
                            12 => "L&#46;M&#46; Barnes"
                            13 => "B&#46;V&#46; Jones"
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                      ]
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                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1128/IAI.01652-13"
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Article information

ISSN: 03257541
Original language: English

DOI:10.1016/j.ram.2023.01.005

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