Intracellular polyphosphate levels in gluconacetobacter diazotrophicus affect tolerance to abiotic stressors and biofilm formation

Gluconacetobacter diazotrophicus is a plant growth-promoting bacterium, used as a bioinoculant. Phosphate (Pi) modulates polyphosphate (polyP) levels in Escherichia coli, affecting cellular fitness and biofilm formation capacity. Can environmental Pi modulate polyP levels in G. diazotrophicus to enhance fitness in view of its technological applications? In high Pi media, cells were able to accumulate polyP and to degrade it, improving survival, tolerance to stressors, biofilm formation capacity on abiotic and biotic surfaces and competence as a growth promoter of strawberry plant. Findings contribute to the importance of Pi and intracellular polyP as signals involved in the survival of G. diazotrophicus.Fil: Grillo Puertas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Delaporte Quintana, Paola Adriana Georgina. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pedraza, Raúl Osvaldo. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia; ArgentinaFil: Rapisarda, Viviana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

Environmental phosphate differentially affects virulence phenotypes of uropathogenic Escherichia coli isolates causative of prostatitis

K-12 Escherichia coli cells grown in static media containing a critical phosphate (Pi) concentration 25 mM maintained a high polyphosphate (polyP) level in stationary phase, impairing biofilm formation, a phenomenon that is triggered by polyP degradation. Pi concentration in human urine fluctuates according to health state. Here, the influence of environmental Pi concentration on the occurrence of virulence traits in uropathogenic E. coli (UPEC) isolated from acute prostatitis patients was evaluated. After a first screening, 3 isolates were selected according to differential biofilm formation profiles depending on media Pi concentration. For each isolate, biofilm positive and negative conditions were established. Regardless of the isolate, biofilm formation capacity was accompanied with curli and cellulose production and expression of some key virulence factors associated with adhesion. When the selected isolates were grown in their non-biofilm-forming condition, low concentrations of nalidixic acid and ciprofloxacin induced biofilm formation. Interestingly, similar to laboratory strains, polyP degradation induced biofilm formation in the selected isolates. Data demonstrated the complexity of UPEC responses to environmental Pi and the importance of polyP metabolism in the virulence of clinical isolates.Fil: Grillo Puertas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Martinez Zamora, Martin Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Rintoul, Maria Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Soto, Sara M.. Universidad de Barcelona; EspañaFil: Rapisarda, Viviana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentin

Environmental phosphate differentially affects virulence phenotypes of uropathogenic Escherichia coli isolates causative of prostatitis

K-12 Escherichia coli cells grown in static media containing a critical phosphate (Pi) concentration 25 mM maintained a high polyphosphate (polyP) level in stationary phase, impairing biofilm formation, a phenomenon that is triggered by polyP degradation. Pi concentration in human urine fluctuates according to health state. Here, the influence of environmental Pi concentration on the occurrence of virulence traits in uropathogenic E. coli (UPEC) isolated from acute prostatitis patients was evaluated. After a first screening, 3 isolates were selected according to differential biofilm formation profiles depending on media Pi concentration. For each isolate, biofilm positive and negative conditions were established. Regardless of the isolate, biofilm formation capacity was accompanied with curli and cellulose production and expression of some key virulence factors associated with adhesion. When the selected isolates were grown in their non-biofilm-forming condition, low concentrations of nalidixic acid and ciprofloxacin induced biofilm formation. Interestingly, similar to laboratory strains, polyP degradation induced biofilm formation in the selected isolates. Data demonstrated the complexity of UPEC responses to environmental Pi and the importance of polyP metabolism in the virulence of clinical isolates.Fil: Grillo Puertas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Martinez Zamora, Martin Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Rintoul, Maria Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Soto, Sara M.. Universidad de Barcelona; EspañaFil: Rapisarda, Viviana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentin

Bioremediation of soil and water contaminated with copper. "Escherichia coli mutant strains have different capacity to detoxify metal"

Copper is an essential nutrient for organisms but when in excess, it becomes potentially toxic. The copper contamination of soils, rivers and deep water has increased dangerously. Bacteria, algae and fungi are receiving increased attention for removing and/or recovery heavy metals due to its effectiveness, low cost and easy availability. In our laboratory it was demonstrated that certain strains of Escherichia coli might detoxify liquid media with high concentrations of copper. The objectives of this study were: a) adequately process the soil samples, artificially contaminated with copper to release the metal of the liquid and then be captured by bacteria, b) using different mutants of E. coli to remediate the waste water from one of the steps in an sequential oxidative treatment (SOT) for controlling green mold in citrus, which contains in its composition a copper salt. a) Different soil samples artificially contaminated with copper were processed and the acid was obtained in better extraction was HCl. Three sequential extractions of 30 min each released about 90% of the metal. Strains of E. coli showed a higher capture of copper when the supernatants had a pH above 5. A single bacterial extraction recovered 90% of the metal. b) In solutions of SOT containing CuSO4, to recover 90% of the metal from it, were required perform three consecutive bacterial extractions. Some mutants in respiratory chain components were more effective than wild type in this process. This study confirms that some strains of E. coli, in an ex situ process could be efficiently used to detoxify soil or water contaminated with copper.El cobre es un nutriente esencial para los organismos sin embargo cuando está en exceso, se vuelve potencialmente tóxico. La contaminación con cobre de suelos, ríos y aguas profundas se ha incrementado peligrosamente. Las bacterias, algas y hongos están recibiendo una atención creciente para la eliminación-recuperación de metales pesados debido a su efectividad, bajo costo y fácil disponibilidad. En nuestro laboratorio se demostró que ciertas cepas de Escherichia coli podían detoxificar medios líquidos con altas concentraciones de cobre. Los objetivos del presente trabajo fueron: a) procesar adecuadamente muestras de suelos, contaminados artificialmente con cobre, para liberar el metal al medio líquido y a partir de allí ser capturado por las bacterias; b) utilizar diferentes mutantes de E. coli para remediar el agua de desecho de uno de los pasos del tratamiento secuencial oxidativo (TSO) para el control de moho verde en citrus, que contiene en su composición una sal de cobre. a) Diferentes muestras de tierra contaminadas artificialmente con cobre fueron procesadas y el ácido con el que se obtuvo una mejor extracción fue el HCl. Tres extracciones secuenciales de 30 min cada una solubilizaban alrededor del 90% del metal. Las cepas de E. coli presentaron una mayor captura del cobre cuando los sobrenadantes tenían un pH superior a 5. Con una sola extracción bacteriana se recuperaba el 90% del metal. b) En soluciones del TSO conteniendo CuSO4, para recuperar el 90% del metal de la solución, se necesitaron realizar tres extracciones bacterianas consecutivas. Algunas mutantes en componentes de la cadena respiratoria fueron más efectivas que la cepa salvaje en este proceso. Este estudio confirma que algunas cepas de E. coli, en un proceso ex situ, podrían ser eficientemente utilizadas para detoxificar suelos o aguas contaminados con cobre.Asociación de Universidades Grupo Montevide

Transcriptional Responses of Herbaspirillum seropedicae to Environmental Phosphate Concentration

Herbaspirillum seropedicae is a nitrogen-fixing endophytic bacterium associated with important cereal crops, which promotes plant growth, increasing their productivity. The understanding of the physiological responses of this bacterium to different concentrations of prevailing nutrients as phosphate (Pi) is scarce. In some bacteria, culture media Pi concentration modulates the levels of intracellular polyphosphate (polyP), modifying their cellular fitness. Here, global changes of H. seropedicae SmR1 were evaluated in response to environmental Pi concentrations, based on differential intracellular polyP levels. Cells grown in high-Pi medium (50 mM) maintained high polyP levels in stationary phase, while those grown in sufficient Pi medium (5 mM) degraded it. Through a RNA-seq approach, comparison of transcriptional profiles of H. seropedicae cultures revealed that 670 genes were differentially expressed between both Pi growth conditions, with 57% repressed and 43% induced in the high Pi condition. Molecular and physiological analyses revealed that aspects related to Pi metabolism, biosynthesis of flagella and chemotaxis, energy production, and polyhydroxybutyrate metabolism were induced in the high-Pi condition, while those involved in adhesion and stress response were repressed. The present study demonstrated that variations in environmental Pi concentration affect H. seropedicae traits related to survival and other important physiological characteristics. Since environmental conditions can influence the effectiveness of the plant growth-promoting bacteria, enhancement of bacterial robustness to withstand different stressful situations is an interesting challenge. The obtained data could serve not only to understand the bacterial behavior in respect to changes in rhizospheric Pi gradients but also as a base to design strategies to improve different bacterial features focusing on biotechnological and/or agricultural purposes

PhoB activation in non-limiting phosphate condition by the maintenance of high polyphosphate levels in the stationary phase inhibits biofilm formation in Escherichia coli

Polyphosphate (polyP) degradation in Escherichia coli stationary phase triggers biofilm formation via the LuxS quorum sensing system. In media containing excess of phosphate (Pi), high polyP levels are maintained in the stationary phase with the consequent inhibition of biofilm formation. The transcriptional-response regulator PhoB, which is activated under Pi limitation, is involved in the inhibition of biofilm formation in several bacterial species. In the current study, we report, for the first time, we believe that E. coli PhoB can be activated in non-limiting Pi conditions, leading to inhibition of biofilm formation. In fact, PhoB was activated when high polyP levels were maintained in the stationary phase, whereas it remained inactive when the polymer was degraded or absent. PhoB activation was mediated by acetyl phosphate with the consequent repression of biofilm formation owing to the downregulation of c-di-GMP synthesis and the inhibition of autoinducer-2 production. These results allowed us to propose a model showing that PhoB is a component in the signal cascade regulating biofilm formation triggered by fluctuations of polyP levels in E. coli cells during stationary phase.Fil: Grillo Puertas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Rintoul, Maria Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Rapisarda, Viviana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentin

Removal of pathogenic bacterial biofilms by combinations of oxidizing compounds

Bacterial biofilms are commonly formed on medical devices and food processing surfaces. The antimicrobials used have limited efficacy against the biofilms; therefore, new strategies to prevent and remove these structures are needed. Here, the effectiveness of brief oxidative treatments, based on the combination of sodium hypochlorite (NaClO) and hydrogen peroxide (H2O2) in the presence of copper sulfate (CuSO4), were evaluated against bacterial laboratory strains and clinical isolates, both in planktonic and biofilm states. Simultaneous application of oxidants synergistically inactivated planktonic cells and prevented biofilm formation of laboratory Escherichia coli, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Staphylococcus aureus strains, as well as clinical isolates of Salmonella enterica subsp. enterica, Klebsiella oxytoca, and uropathogenic E. coli. In addition, preformed biofilms of E. coli C, Salmonella Typhimurium, K. pneumoniae, and Salmonella enterica exposed to treatments were removed by applying 12 mg/L NaClO, 0.1 mmol/L CuSO4, and 350 mmol/L H2O2 for 5 min. Klebsiella oxytoca and Staphylococcus aureus required a 5-fold increase in NaClO concentration, and the E. coli clinical isolate remained unremovable unless treatments were applied on biofilms formed within 24 h instead of 48 h. The application of treatments that last a few minutes using oxidizing compounds at low concentrations represents an interesting disinfection strategy against pathogens associated with medical and industrial settings.Des biofilms bactériens se forment souvent sur la surface de dispositifs médicaux et d’équipements de traitement des aliments. Puisque les antibiotiques actuels n’ont que peu d’emprise sur eux, il est nécessaire d’élaborer de nouvelles stratégies aptes a` prévenir et éliminer ces structures. Dans la présente étude, on a évalué l’efficacité de brefs traitements d’oxydants, basés sur une combinaison de NaClO et de H2O2 en présence de CuSO4, pour contrer des souches bactériennes de laboratoire et des isolats cliniques en phase planctonique ou sous forme de biofilm. L’application simultanée d’oxydants a inactivé les cellules planctoniques de manière synergique tout en prévenant la formation de biofilm formé par des souches de laboratoire d’Escherichia coli, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae et Staphylococcus aureus ainsi que des isolats cliniques de Salmonella enterica subsp. enterica, Klebsiella oxytoca et E. coli uropathogène. En outre, des biofilms préformés d’E. coli C, Salmonella Typhimurium, K. pneumoniae et Salmonella enterica exposés aux traitements ont été éliminés en appliquant 12 mg/L de NaClO, 0,1 mmol/L de CuSO4 et 350 mmol/L de H2O2 pendant 5 min. On a eu besoin de 5 fois plus de NaClO pour venir a` bout de K. oxytoca et Staphylococcus aureus et l’isolat clinique d’E. coli était impossible a` éliminer a` moins de raccourcir la durée de formation de ses biofilms (24 h au lieu de 48 h). L’application d’une durée de quelques minutes de traitements a` base de substances oxydantes a` faibles concentrations représente une stratégie attrayante pour lutter contre les pathogènes associés aux installations médicales et industrielles.Fil: Olmedo, Gabriela María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Grillo Puertas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Cerioni, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Rapisarda, Viviana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Volentini, Sabrina Inès. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentin

Copper tolerance mediated by polyphosphate degradation and low-affinity inorganic phosphate transport system in Escherichia coli

Metal tolerance in bacteria has been related to polyP in a model in which heavy metals stimulate the polymer hydrolysis, forming metal-phosphate complexes that are exported. As previously described in our laboratory, Escherichia coli cells grown in media containing a phosphate concentration >37 mM maintained an unusually high polyphosphate (polyP) level in stationary phase. The aim of the present work was to evaluate the influence of polyP levels as the involvement of low-affinity inorganic phosphate transport (Pit) system in E. coli copper tolerance.Fil: Grillo Puertas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Schurig Briccio, Lici Ariane. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; Argentina. University of Illinois. Urbana - Champaign; Estados Unidos. Illinois; Estados UnidosFil: Rodríguez Montelongo, Luisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Rintoul, Maria Regina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; ArgentinaFil: Rapisarda, Viviana Andrea. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; Argentin

Polyphosphate Degradation in Stationary Phase Triggers Biofilm Formation via LuxS Quorum Sensing System in <em>Escherichia coli</em>

<div><p>In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that <em>Escherichia coli</em> cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in <em>E. coli</em>. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in <em>ppk⁻ppx⁻</em> or <em>ppk⁻ppx⁻</em>/<em>ppk⁺</em> strains, deficient in polyP synthesis and hydrolysis, respectively. <em>luxS</em> and <em>lsrK</em> mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.</p> </div

Biofilm formation after reinoculation in CM.

*<p>Biofilm formation was determined 24 h after the cells transference to CM. 48 h MT cultures were used as control. Different letters indicate significant differences according to Tukey's test with a <i>p</i>-value of 0.05.</p