Anticandida and antibiofilm activities of extract from Schinopsis brasiliensis Engl. against Candida spp

Abstract The pathogenic nature of infections caused by Candida spp. underscores the necessity for novel therapeutic agents. Extracts of Schinopsis brasilienses Engl are / a promising source of agents with antifungal effects. This study aimed to assess the antifungal potential of the leaf extract of S. brasilienses. The antifungal activity was evaluated by determining the minimum inhibitory concentrations and fungicide concentrations (MIC and MFC). The antibiofilm potential was assessed by counting colony-forming units/mL. The study examined the inhibition kinetics of fungal growth and potential synergism between gallic acid or the extract and nystatin using the Checkerboard method. Cytotoxicity was evaluated through the MTT assay. The extract exhibited antifungal effect against all tested strains, with MIC and MFC ranging from 31.25-250 μg/mL. Gallic acid, the main isolated compound, displayed a MIC of 2000 μg/mL. The extract of S. brasilienses at 31.25 μg/mL inhibited the formation of biofilm by C. albicans and significantly reduced the mass of mature biofilm after 24 and 48 h (p < 0. 05). At a concentration of 125 μg/mL, the extract demonstrated significant inhibition of fungal growth after 6 hours. The combination of gallic acid or extract with nystatin did not exhibit synergistic or antagonistic effect. Furthermore, the extract did not induce cytotoxicity to a human cell line. The extract of S. brasiliensis demonstrates antifungal activity against Candida, generally exhibiting fungicidal action and capacity to inhibit biofilm formation as well as reduce mature biofilms. Additionally, the extract showed low cytotoxicity to human cells.

Nanotopography and oral bacterial adhesion on titanium surfaces: in vitro and in vivo studies

Abstract The present study aimed to evaluate the influence of titanium surface nanotopography on the initial bacterial adhesion process by in vivo and in vitro study models. Titanium disks were produced and characterized according to their surface topography: machined (Ti-M), microtopography (Ti-Micro), and nanotopography (Ti-Nano). For the in vivo study, 18 subjects wore oral acrylic splints containing 2 disks from each group for 24 h (n = 36). After this period, the disks were removed from the splints and evaluated by microbial culture method, scanning electron microscopy (SEM), and qPCR for quantification of Streptococcus oralis, Actinomyces naeslundii, Fusobacterium nucleatum, as well as total bacteria. For the in vitro study, adhesion tests were performed with the species S. oralis and A. naeslundii for 24 h. Data were compared by ANOVA, with Tukey's post-test. Regarding the in vivo study, both the total aerobic and total anaerobic bacteria counts were similar among groups (p > 0.05). In qPCR, there was no difference among groups of bacteria adhered to the disks (p > 0.05), except for A. naeslundii, which was found in lower proportions in the Ti-Nano group (p < 0.05). In the SEM analysis, the groups had a similar bacterial distribution, with a predominance of cocci and few bacilli. In the in vitro study, there was no difference in the adhesion profile for S. oralis and A. naeslundii after 24 h of biofilm formation (p > 0.05). Thus, we conclude that micro- and nanotopography do not affect bacterial adhesion, considering an initial period of biofilm formation.

Anti-biofilm effectiveness of protocols for cleaning complete dentures in hospitalized patients: a randomized controlled trial

Abstract Denture biofilm acts as a potential reservoir for respiratory pathogens, considerably increasing the risk of lung infections, specifically aspiration pneumonia, mainly 48h after hospital admission. The establishment of a straightforward, affordable, and applicable hygiene protocol in a hospital environment for the effective control of denture biofilm can be particularly useful to prevent respiratory infections or reduce the course of established lung disease. Objectives To evaluate the anti-biofilm effectiveness of denture cleaning protocols in hospitalized patients. Methodology The maxillary complete dentures (MCDs) of 340 hospitalized participants were randomly cleaned once using one of the following 17 protocols (n=20): brushing with distilled water, toothpaste, or neutral liquid soap (controls); immersion in chemical solutions (1% sodium hypochlorite, alkaline peroxide, 0.12% or 2% chlorhexidine digluconate), or microwave irradiation (650 W for 3 min) combined or not with brushing. Before and after the application of the protocols, the biofilm of the intaglio surface of the MCDs was evaluated using two methods: denture biofilm coverage area (%) and microbiological quantitative cultures on blood agar and Sabouraud Dextrose Agar (CFU/mL). Data were subjected to the Wilcoxon and Kruskal-Wallis tests (α=0.05). Results All 17 protocols significantly reduced the percentage area of denture biofilm and microbial and fungal load (P<0.05). The highest percentage reductions in the area of denture biofilm were observed for 1% hypochlorite solution with or without brushing and for 2% chlorhexidine solution and microwave irradiation only in association with brushing (P<0.05). The greatest reductions in microbial and fungal load were found for the groups that used solutions of 2% chlorhexidine and 1% hypochlorite and microwave irradiation, regardless of the association with brushing (P<0.05). Conclusions A single immersion for 10 min in 1% sodium hypochlorite, even in the absence of brushing, proved to be a straightforward, rapid, low-cost, and effective protocol for cleaning the dentures of hospitalized patients.

Effect of N-acetyl cysteine, rifampicin, and ozone on biofilm formation in pan-resistant Klebsiella pneumoniae: an experimental study

ABSTRACT BACKGROUND: To the best of our knowledge, this is the first study to evaluate the effectiveness of specific concentrations of antibiofilm agents, such as N-acetyl cysteine (NAC), rifampicin, and ozone, for the treatment of pan-resistant Klebsiella pneumoniae (PRKp). OBJECTIVES: We evaluated the effectiveness of antibiofilm agents, such as NAC, rifampicin, and ozone, on biofilm formation in PRKp at 2, 6, 24, and 72 h. DESIGN AND SETTING: This single-center experimental study was conducted on June 15, 2017, and July 15, 2018, at Istanbul Faculty of Medicine, Istanbul University, Turkey. METHODS: Biofilm formation and the efficacy of these agents on the biofilm layer were demonstrated using colony counting and laser-screened confocal microscopy. RESULTS: NAC at a final concentration of 2 μg/mL was administered to bacteria that formed biofilms (24 h), and no significant decrease was detected in the bacterial counts of all isolates (all P > 0.05). Rifampicin with a final concentration of 0.1 μg/mL was administered to bacteria that formed biofilm (24 h), and no significant decrease was detected in bacterial count (all P > 0.05). Notably, ozonated water of even 4.78 mg/L concentration for 72 h decreased the bacterial count by ≥ 2 log10. CONCLUSION: Different approaches are needed for treating PRKp isolates. We demonstrate that PRKp isolates can be successfully treated with higher concentrations of ozone.

Biological control of foodborne pathogens by lacticacid bacteria: A focus on juice processing industries

Abstract The use of lactic acid bacteria (LAB) in foods as biocontrol agents against foodborne pathogens has become increasingly known. Under the premise that controlling the adhesion of microorganisms to food contact surfaces is an essential step for meeting the goals of food processing, the aim of this work was to investigate the inhibitory and anti-biofilm effectiveness of Lactobacillus rhamnosus GG (ATCC 53103) and Lactobacillus casei (ATCC 393) against Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes. Lactobacillus strains (108UFCCFU/ml) and pathogens (104UFCCFU/ml) were evaluated to monitor LAB anti-adhesive and antibiofilm effect, in two main scenarios: (i) co-adhesion and (ii) pathogen incorporation to stainless steel surfaces with a protective biofilm of Lactobacillus cells. In (i) the predominant effect was observed in L. rhamnosus against S. enterica and L. monocytogenes, whereas in (ii) both LAB significantly reduced the number of pathogenic adherent cells. The effect of pre-established LAB biofilms was more successful in displacing the three pathogens than when they were evaluated under co-adhesion. These findings show that both LAB can be considered good candidates to prevent or inhibit the adhesion and colonization of L. monocytogenes, S. enterica and E. coli O157:H7 on surfaces and conditions of relevance for juice processing industries, offering alternatives for improving the safety and quality of fruit-based products.

Resumen Existe un creciente interés en el uso de bacterias ácido lácticas (BAL) como agentes de biocontrol frente a patógenos de transmisión alimentaria. Bajo la premisa de que el control de la adhesión de microorganismos a superficies de contacto con alimentos es el paso esencial para evitar su contaminación, el objetivo de este trabajo fue investigar la efectividad inhibitoria y antibiofilm de Lactobacillus rhamnosus GG (ATCC 53103) y Lactobacillus casei (ATCC 393) frente a Escherichia coli O157:H7, Salmonella enterica y Listeria monocytogenes. A fin de cumplir con el objetivo propuesto, las cepas de Lactobacillus (108UFCUFC/ml) y los patógenos (104UFCUFC/ml) se ensayaron en 2 escenarios: (1) coadhesión, y (2) incorporación de los patógenos a las superficies de acero inoxidable con un biofilm preformado de Lactobacillus. En (1), el efecto predominante se observó con L. rhamnosus frente a S. enterica y L. monocytogenes, mientras que en (2), ambas BAL redujeron significativamente el número de células patógenas adheridas. En función de estos resultados, concluimos que el efecto de un biofilm preformado de ambas BAL fue más exitoso en el desplazamiento de los 3 patógenos que en coadhesión. Ambas BAL pueden considerarse buenas candidatas para mitigar la adhesión y colonización de L. monocytogenes, S. enterica y E. coli O157:H7 en superficies en condiciones de relevancia para la industria procesadora de jugos, y, de esta manera, ofrecer alternativas para mejorar la seguridad y calidad de los alimentos a base de frutas.

Las biopelículas y su interrelación con la patología crónica del oído medio / Biofilms and their relationship with chronic middle ear pathology

El concepto de biopelículas ha surgido de forma paulatina durante un largo período; se presentan como estructuras tridimensionales compuestas por células sésiles de microorganismos que crecen y se adhieren irreversiblemente a superficies, tanto vivas como inertes. Su capacidad de desarrollarse, tanto en superficies bióticas como abióticas, es una característica que los relaciona directamente con la salud humana. Distintas infecciones óticas se han inculpado a la presencia de biopelículas en las mucosas como en la otitis media con efusión, de igual forma se manifiestan en la aparición y persistencia de la otitis media crónica. Las biopelículas afines con otitis media, generalmente, contienen uno o múltiples especies de bacterias otopatógenas primarias. La comprensión de la biopelicula auxiliará el progreso de nuevas terapias y estrategias de control, al evitar enfermedades infecciosas ya que las bacterias formadoras de biopelículas son una seria amenaza para la salud pública debido a su alta resistencia a los antimicrobianos.

The concept of biofilms has emerged gradually over a long period; they appear as three-dimensional structures composed of sessile cells of microorganisms that grow and adhere irreversibly to surfaces, both living and inert. Their ability to develop, both on biotic and abiotic surfaces, is a characteristic that directly relates them to human health. Different ear infections have been blamed on the presence of biofilms on the mucous membranes, such as otitis media with effusion, in the same way they manifest themselves in the appearance and persistence of chronic otitis media. Otitis media-related biofilms generally contain one or multiple species of primary otopathogenic bacteria. The understanding of the biofilm will help us refine new therapies and control strategies, by avoiding infectious diseases since biofilm-forming bacteria are a serious threat to public health due to their high resistance to antimicrobials.

Influencia de la higiene bucal en el estado de salud de los tejidos periimplantarios / Influence of oral hygiene on peri-implant tissue health status

Introducción: el estado de salud de los tejidos periimplantarios es de vital importancia en el éxito de la rehabilitación implantosoportada, por esta razón, es necesario observar todos aquellos factores que contribuyen a mantener este estado y dentro de ellos, principalmente: la higiene bucal. Objetivo: determinar la influencia de la higiene bucal en el estado de salud de los tejidos periimplantarios. Métodos: se realizó un estudio descriptivo, observacional y transversal en el servicio de Prótesis de la Facultad de Estomatología de Villa Clara, en el período comprendido entre los años 2017 y 2019. El universo de estudio estuvo constituido por 45 pacientes portadores de rehabilitaciones implantosoportadas; las unidades de análisis fueron los implantes y los tejidos que rodean a las 85 prótesis fijas realizadas a dichos pacientes que cumplieron con los criterios de inclusión. Se emplearon la observación clínica y radiográfica, y se elaboró un formulario como instrumento. Se evaluó la higiene bucal y el estado de los tejidos periimplantarios como principales variables. La información obtenida se recopiló en una base de datos, se procesó y se sometió a pruebas de independencia (el estadígrafo Ji cuadrado y su posibilidad asociada) para mostrar la relación entre las variables. Resultados: las variables analizadas evidenciaron una relación significativa de la higiene bucal con el estado de salud de los tejidos periimplantarios a favor de la buena higiene y los tejidos sanos. Conclusiones: la buena higiene bucal evidenciada contribuyó a que los tejidos periimplantarios se mantuvieran sanos.

Introduction: peri-implant tissue health state is of vital importance in the success of implant-supported rehabilitation; for this reason, it is necessary to observe all those factors that contribute to maintaining this state, mainly oral hygiene. Objective: to determine the influence of oral hygiene on peri-implant tissue health status. Methods: a descriptive, observational and cross-sectional study was carried out in the Prosthesis service at the Dental Faculty of Villa Clara between 2017 and 2019. The universe of study consisted of 45 patients with implant-supported rehabilitations; the units of analysis were the implants and the tissues surrounding the 85 fixed prostheses performed on those patients who met the inclusion criteria. Clinical and radiographic observations were used, and a form was developed as an instrument. Oral hygiene and peri-implant tissue state were evaluated as the main variables. The information obtained was compiled in a database as well as processed and subjected to independence tests (the Chi-square statistic and its associated possibility) to show the relationship among the variables. Results: the analyzed variables showed a significant relationship between oral hygiene and the peri-implant tissue health status in favour of good hygiene and healthy tissues. Conclusions: the evidenced good oral hygiene contributed to the maintenance of healthy peri-implant tissues.

Tipificación bioquímica y evaluación de la patogenicidad de aislamientos vulvovaginales del complejo Candida albicans

Introducción. Candida albicans, C. dubliniensis y C. africana forman el complejo Candida albicans. Objetivo. Identificar las características fenotípicas y patogénicas de aislamientos del complejo C. albicans conservados en una colección. Materiales y métodos. Se evaluaron 300 aislamientos identificados presuntivamente como del complejo C. albicans, utilizando CHROMagarTM Candida. Se determinó la producción del tubo germinal mediante tres métodos, se evaluó la producción de clamidosporas, se caracterizaron las colonias en agares artesanales (Rosmarinus officinalis y Nicotiana tabacum) y se utilizó MALDI-TOF como prueba de referencia para la identificación. Para detectar factores de patogenicidad, se evaluó la actividad hemolítica de los aislamientos independientes y en cocultivo con Staphylococcus aureus, la producción de enzima coagulasa y la formación de biopelículas. Resultados. El 43,7 % de los aislamientos produjo tubo germinal en caldo de medio infusión de cerebro-corazón y el 47 % generó clamidosporas. En los medios artesanales, en el 6 % de los aislamientos se obtuvieron colonias de color café en agar romero y, en el 5 %, en agar tabaco. Ninguna de las cepas hemolizó el agar sangre comercial (ni en presencia o ausencia de S. aureus), mientras que el 50 % hemolizó el agar papa dextrosa suplementado con sangre. Todos los aislamientos produjeron enzima coagulasa y la producción de biopelículas fue variable. Para la producción de tubo germinal, el método de suero humano mostró igual positividad que el de caldo de leche. Todos los aislamientos fueron identificados como C. albicans por MALDITOF. Conclusiones. Se requieren herramientas de proteómica y pruebas moleculares, o la combinación de métodos, para poder discriminar entre especies.

Introduction. Candida albicans, C. dubliniensis, and C. africana form the Candida albicans complex. Objective. To identify the phenotypic and pathogenic characteristics of isolates of the C. albicans complex preserved in a collection. Materials and methods. Three hundred presumptive strains of the C. albicans complex were evaluated using CHROMagarTM Candida. Germ tube production was determined by three methods, chlamydospores formation was assessed and colonies were characterized in artisanal agars (Rosmarinus officinalis and Nicotiana tabacum). MALDI-TOF was used as the gold standard identification test. To detect pathogenicity factors, we evaluated the hemolytic activity of each isolate and cocultured with Staphylococcus aureus, coagulase enzyme production, and biofilm formation. Results. Out of the 300 isolates, 43.7% produced germ tube in the heart-brain infusion broth and 47% of the isolates produced chlamydospores. In the artisan media, 6% of the isolates produced brown colonies on rosemary agar and 5% did so on tobacco agar. None of the strains hemolyzed the blood agar alone or cocultured with S. aureus. However, 50% of the isolates hemolyzed the potato dextrose agar supplemented with blood. All strains were coagulase producers, and biofilm production was variable. For germ tube production, the human serum method showed the same positivity as the milk broth method. All isolates were identified as C. albicans by MALDI-TOF. Conclusions. The use of proteomics, molecular tests or a combination of methods is required for species identification.

Evaluación de la formación de biopelículas en aislamientos bacterianos y fúngicos por el método semicuantitativo de microtitulación con cristal violeta y el cualitativo de agar con rojo Congo / Assessment of biofilms formation of bacterial and fungal isolates using qualitative Congo red agar and semiquantitative crystal violet microtiter methods

Introducción. El 65 % de las infecciones humanas son producidas por bacterias o levaduras, cuya capacidad de formar biopelículas las hace más resistentes a los antimicrobianos y antifúngicos. Objetivo. Determinar la capacidad de formación de biopelículas en aislamientos bacterianos y fúngicos por medio de los métodos cuantitativo de microtitulación con cristal violeta y cualitativo de cultivo en agar con rojo Congo. Materiales y métodos. Con el método cuantitativo, se utilizaron los medios de cultivo infusión cerebro-corazón, tripticasa de soya y Müeller-Hinton para aislamientos bacterianos; para levaduras, se usaron caldo infusión cerebro-corazón y Sabouraud dextrosa. Para el método cualitativo de cultivo en agar, se utilizaron los mismos medios de cultivo más una solución con 3 % de rojo Congo y 10 % de dextrosa. Cómo método de referencia, se utilizó la propuesta de Stepanovic et al. Resultados. Se evaluaron 103 aislamientos bacterianos y 108 de levaduras. No es recomendable sustituir el caldo infusión cerebro-corazón por los caldos tripticasa de soya y Müeller-Hinton en el método cuantitativo, para evaluar la formación de biopelículas en los aislamientos bacterianos. El medio Sabouraud dextrosa, en caldo y agar, puede sustituir al de infusión de cerebro-corazón para evaluar la formación de biopelículas en levaduras, tanto por el método cuantitativo como por el cualitativo. Conclusión. El estudio de las biopelículas en el laboratorio de microbiología, a partir del método cualitativo de cultivo en agar con rojo Congo, es un procedimiento sencillo, rápido y de bajo costo, que proporciona información útil para el diagnóstico y la terapéutica de infecciones persistentes causadas por bacterias y levaduras.

Introduction. Sixty-five percent of human infections are caused by bacteria or yeasts able to form biofilms. This feature makes them more resistant to antimicrobials and antifungals. Objective. To determine biofilm formation capacity of bacterial and fungal isolates by quantitative crystal violet microtiter and qualitative Congo red agar methods. Materials and methods. Brain-heart infusion, trypticase soy broth and Müeller-Hinton culture media were used in bacterial isolates for the quantitative method; brain-heart infusion broth and Sabouraud dextrose were used for yeasts. The same culture media plus 3% Congo red and 10% dextrose were used to apply the qualitative method in agar. The proposal by Stepanovic, et al. was used as a reference method. Results. We evaluated 103 bacterial isolates and 108 yeasts isolates. We did not recommend substitute brain-heart infusion broth for trypticase soy and Müeller-Hinton broths for biofilm formation assessment in bacterial isolates using the quantitative method. Sabouraud dextrose medium, both broth and agar, can replace brain-heart infusion to assess biofilm formation in yeasts, quantitatively and qualitatively. Conclusion. The study of biofilms in the microbiology laboratory, using Congo red agar qualitative method, is a simple, fast, and inexpensive procedure that provides precise information for the diagnosis and treatment of persistent infections caused by bacteria and yeasts.

Evaluation of the effects of Albizia lebbeck bark extracts on the cariogenic properties of Streptococcus mutans

Introduction: Oral diseases continue to be a major and common health problem worldwide and affect the quality of life by causing considerable pain and discomfort. While the mechanistic methods of caries prevention and treatment are in vogue, the concern about adverse effects of the constituents of these methods persists. Thus, there has always existed a need to develop effective and safe strategies to combat oral pathogens such as Streptococcus mutans (S. mutans). Aim: To examine the action of Albizia lebbeck (A. lebbeck) bark extracts on the growth and virulence factors of S. mutans such as- biofilm formation, surface adherence, cell surface hydrophobicity and acid production. Materials and Methods: Ethyl acetate, hexane, and chloroform extracts of the bark of A. lebbeck was prepared and tested for their activity against the ATCC 25175 S. mutans. The phytochemical constituents of the extracts were determined by biochemical assays and the MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) were obtained using the well diffusion assay. The effect of the sub-MIC concentrations of extracts on biofilm formation & eradication, microbial growth, adherence of the strain to glass surfaces, cell surface hydrophobicity and acid production were also determined. Results: All extracts inhibited the organism’s growth, and MIC was determined as 25 mg/ml. the sub-MIC concentrations of the extracts were found to- inhibit the formation of biofilms, eradicate formed biofilms and interfere with the adherence and acid production of S. mutans. Conclusion: The ethyl acetate and ethanolic extracts were found to be active at low concentrations (0.02mg/ml) and demonstrate a potential to be used in the formulation of anti-caries preparations.

Epithelial responses and Candida albicans pathogenicity are enhanced in the presence of oral streptococci

Abstract Experimental models that consider host-pathogen interactions are relevant for improving knowledge about oral candidiasis. The aim of this study was to assess the epithelial immune responses, Candida penetration of cell monolayers, and virulence during mixed species culture infections. Single species cultures of Candida albicans and mixed cultures (C. albicans, Streptococcus mutans, and Streptococcus sanguinis) were used to infect monolayers of HaCaT and FaDu ATCC HTB-43 cells for 12 h. After infection, IL-18 and IL-34 gene expression was measured to assess epithelial cell immune responses, and lactate dehydrogenase (LDH) activity was measured as an indicator of cell damage. Microscopy determined C. albicans morphology and penetration of fungal cells through the keratinocyte monolayer. Monolayers devoid of infection served as controls. Data were analyzed by an ANOVA one-way test followed by Tukey's post-hoc test (α = 0.05). The results found that IL-18 and IL-34 gene expression and LDH activity were significantly (p < 0.05) upregulated for both cell lines exposed to mixed species cultures compared with C. albicans alone. Candida albicans yeast and hyphae were evident in C. albicans only infections. In contrast, monolayers infected by C. albicans, S. mutans, and S. sanguinis exhibited higher microbial invasion with several hyphal aggregates detected. The presence of streptococci in C. albicans infection enhances the virulence and pathogenicity of the fungus with associated increased immune responses and tissue damage. Extrapolation of these findings to oral infection would indicate the added potential benefit of managing bacterial components of biofilms during treatment.

Resumo O objetivo deste estudo foi avaliar a resposta epithelial imune, a colonização da Candida albicans em monocamadas celulares e sua virulência em resposta a infecções de culturas de biofilme multiespécie. Culturas de biofilme monoespécie de C. albicans e culturas mistas (C. albicans, Streptococcus mutans e Streptococcus sanguinis) foram utilizadas para infectar monocamadas de células HaCaT e FaDu por 12 h. Após a infecção, a expressão dos genes IL-18 e IL-34 foi medida para avaliar as respostas imunes das células epiteliais. A atividade da lactato desidrogenase (LDH) foi medida como um indicador de dano celular. A microscopia determinou a morfologia de C. albicans e a penetração das células fúngicas através da monocamada de queratinócitos. Monocamadas em que não houve infecção serviram como controles. Os dados foram analisados por um teste ANOVA one-way seguido pelo teste post-hoc de Tukey (α = 0,05). Os resultados demonstraram que a expressão gênica de IL-18 e IL-34 e a atividade de LDH foram (p < 0,05) reguladas positivamente para ambas as linhagens de células expostas a culturas de espécies mistas em comparação com C. albicans isoladamente. Leveduras de C.albicans e hifas foram evidentes em infecções apenas por C. albicans. Entretanto, monocamadas infectadas por C. albicans, S. mutans e S. sanguinis exibiram maior invasão microbiana com vários agregados de hifas detectados. Dessa maneira, a presença de estreptococos na infecção por C. albicans aumentou a virulência e a patogenicidade do fungo com respostas imunes aumentadas associadas a danos nos tecidos. A extrapolação desses achados para a infecção oral indicaria o potencial benéfico do controle dos componentes bacterianos em biofilmes durante a terapia da candidíase

Effect of different disinfection protocols in bacterial viability of an intraradicular biofilm formed in situ

Abstract The present study aimed to evaluate bacterial viability after the use of different disinfection protocols in root canals infected with a multispecies biofilm (MB) formed in situ. Palatal roots with a single canal were obtained from extracted maxillary molars and sterilized before being inserted into the mouth. The roots were contaminated with a MB in an intraoral appliance worn by ten volunteers. All volunteers wore six roots simultaneously in two intraoral devices for 21 days. One root from each volunteer was assigned to each group (n=10): PUI - passive ultrasonic irrigation; EC - Easy Clean; XPF - XP-endo Finisher; aPDT - antimicrobial photodynamic therapy; CI - conventional irrigation; and NC - negative control. The samples were evaluated under confocal laser scanning microscopy. The percentage of viable cells (VC) was calculated over the total percentage of MB biovolume. Data were statistically analyzed (α=5%). The cell viability in the entire root canal or for each third was compared between groups (Kruskal-Wallis test, Dunn post-hoc test) and for the same group (Friedman test, Dunn post-hoc test). Disinfection protocols were not significantly different from each other (P>.05). Samples in EC, PUI, and aPDT had lower cell viability than in NC (P<.05). In the coronal third of samples in the EC, XPF, PUI and aPDT, the percentage of VC biovolume was lower than in the NC (P<.05). The percentage of VC in EC samples was lower in the coronal and middle thirds than in the apical third (P<.05). EC, PUI and aPDT had significant effects on cell viability in intraradicular multispecies biofilm formed in situ when compared with untreated samples.

Resumo O presente estudo teve como objetivo avaliar a viabilidade bacteriana após o uso de diferentes protocolos de desinfecção em canais radiculares infectados com um biofilme multiespécies (MB) formado in situ. Raízes palatinas com canal único foram obtidas de molares superiores extraídos e esterilizadas antes de serem inseridas na boca. As raízes foram contaminadas com MB em um aparelho intraoral usado por dez voluntários. Todos os voluntários usaram seis raízes simultaneamente em dois dispositivos intrabucais por 21 dias. Uma raiz de cada voluntário foi atribuída a cada grupo (n=10): PUI - irrigação ultrassônica passiva; EC - Easy clean; XPF - XP-endo Finisher; aPDT - terapia fotodinâmica antimicrobiana; IC - irrigação convencional; e, NC - controle negativo. As amostras foram avaliadas em microscopia confocal de varredura a laser. A porcentagem de células viáveis (VC) foi calculada sobre a porcentagem total do biovolume de MB. Os dados foram analisados estatisticamente (α=5%). A viabilidade celular em todo o canal radicular ou em cada terço foi comparada entre os grupos (teste de Kruskal-Wallis, teste post-hoc de Dunn) e no mesmo grupo (teste de Friedman, teste post-hoc de Dunn). Os protocolos de desinfecção não foram significativamente diferentes entre si (P>0,05). Amostras dos grupos EC, PUI e aPDT apresentaram menor viabilidade celular do as do NC (P<0,05). No terço cervical das amostras do EC, XPF, PUI e aPDT, a porcentagem de biovolume de VC foi menor do que no NC (P<0,05). A porcentagem de VC nas amostras do EC foi menor nos terços cervical e médio do que no terço apical (P<0,05). EC, PUI e aPDT tiveram efeitos significativos na viabilidade celular do biofilme multiespécies intrarradicular formado in situ quando comparado com amostras não tratadas. Estudos clínicos devem investigar o papel da redução de cargas bacterianas viáveis no sistema de canais radiculares para o sucesso do tratamento endodôntico.

Inhibitory Effect of Gallium Nitrate against Staphylococcus aureus and Its Action on the Formation of Biofilms on Titanium Surfaces

Aims: This study aimed to investigate the effect of gallium nitrate [Ga(NO3)3], an inorganic antimicrobial agent, against the growth and biofilm formation of Staphylococcus aureus on the titanium surface. Study Design: This study is a laboratory investigation involving the determination of the inhibitory concentration (IC) of Ga(NO3) 3 against the planktonic strain of S. aureus and biofilm formation on titanium coupons. Place and Duration of Study: Sample: Center of Science and Technology for Sustainability (CCTS), Federal University of São Carlos, SP, Brazil, between March 2020 and March 2022. Methodology: The inhibitory concentration of gallium nitrate was determined in a 96-well microtiter plate in Muller Hinton broth. The potential of the antimicrobial agent to inhibit biofilm formation by S. aureus on titanium surfaces was evaluated by Scanning Electron Microscopy (SEM). The cytotoxicity potential of Ga(NO3)3 was determined on V79 cells. Results: The results showed that the susceptibility of gallium nitrate against S. aureus was 1.40 µM, while SEM images revealed that concentrations of 90 µM inhibited biofilm formation by S. aureus. Conclusion: This research has shown promising results regarding gallium nitrate's potential of inhibiting the growth of both planktonic and sessile S. aureus cells. In addition, coating titanium surfaces with Ga(NO3)3 would be an extra alternative to prevent implant-associated infections due to its non-toxicity to cells.

Antibacterial Efficacy of Silver Diamine Fluoride Compared to Casein Phosphopeptide-Amorphous Calcium Phosphate Against Streptococcus mutans in a Biofilm Caries Model

ABSTRACT Objective: To compare the antibacterial efficacy of silver diamine fluoride (SDF) with a product containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) against Streptococcus mutans using a biofilm caries model. Material and Methods: Twenty-seven saliva-coated dentine blocks obtained from extracted human teeth were inoculated with Streptococcus mutans monospecies biofilm in this in vitro study. The biofilms were then exposed to 10% sucrose in brain heart infusion broth eight times daily for seven days. After the biofilm growth period, the dentine blocks (n=9 per group) were treated with one of the following substances: 1) sterile saline (control), 2) 38% SDF, and 3) a product containing CPP-ACP. Then, the samples were incubated at 37ºC for 48 hours, and the numbers of viable microorganisms in the biofilms were counted and compared. ANOVA and Tukey's HSD tests were used to analyze the data (p<0.05). Results: The number of viable bacteria, as determined by the number of colony-forming units (CFU mL-1) of Streptococcus mutans, was significantly reduced following treatment with SDF and the CPP-ACP product (p<0.05). However, SDF showed superior antibacterial activity compared to the CPP-ACP product (mean CFU mL-1 =zero compared to 96 x106) (p<0.05). Conclusion: SDF has higher antibacterial activity against cariogenic Streptococcus mutans biofilm than the CPP-ACP product. The CPP-ACP product showed antibacterial activity, but it was limited.

Cleansing effect of NO sustained-release silica nanoparticles on endoscopic biofilm and its clinical application / 中华消化内镜杂志

Objective:To explore the cleansing effect of Nitric Oxide (NO) sustained-release silica nanoparticles (short for NO sustained-release agent) on endoscopic biofilm and its clinical application.Methods:A total of 160 clinical endoscopes were randomly divided into two groups: the cleansing agent group (80 pieces, disinfected with cleansing agents), NO group (80 pieces, disinfected with NO sustained-release agent). A biofilm model of Pseudomonas aeruginosa was constructed and used as the control for phosphate buffered solution (PBS) treatment. A biofilm model of Pseudomonas aeruginosa on the surface of endoscopic lumen was built first in vitro. Scanning electron microscopy was then used to observe the microstructure of biofilm after treatment with NO sustained-release agent. Viable counting method was used to evaluate the cleansing effect of NO sustained-release agent on biofilm. Finally, at the clinical level, the actual disinfection effect of NO sustained-release agent on clinical endoscopy was evaluated by detecting the protein residues, viable counting and adenosine triphosphate (ATP) biofluorescence detection. Results:The scanning electron microscopy showed that the biofilm was intact in the model group, but scattered bacteria were observed on the biofilm surface in the NO group and the detergent group. Compared with the model group [(4.86±2.67)×10 6(colony-forming units, CFU)/mL], the standard CFUs of the NO group [(1.37±0.61)×10 4CFU/mL] and the detergent group [(1.31±0.21)×10 5CFU/mL] were significantly lower (detergent group VS model group, P=0.009; NO group VS model group, P=0.008), and there was significant difference between the detergent group and the model group ( t=9.53, P=0.000 6). The levels of residual proteins in the endoscopic lumens before and after the treatment were 8.03±1.47 mg/mL and 0.50±0.37 mg/mL in the NO group, 8.01±1.51 mg/mL and 0.91±0.52 mg/mL in the detergent group with significant difference ( P<0.01), and the reduction effect of the NO group was more significant. The disinfection of NO group and cleaning agent group was within the qualifying range, but the ATP bioluminescence value, protein residue and colony count of NO group (78.56±42.59 RLU, 0.50±0.37 mg/mL, 7.55±4.56 CFU) were significantly lower than those of detergent agent group (120.80±54.00 RLU,0.91±0.52 mg/mL,11.50±4.75 CFU, P<0.01). Conclusion:NO sustained-release agent can effectively clear endoscopic biofilm and further improve the disinfection effect on endoscopes, which may be of great significance for improving the effects on treatment and prognosis of patients.

Effect of S. mutans biofilm on the hybrid ceramic-resin cement bond strength assessed by different methods

Aim: The purpose of this study was to investigate the biofilm effect on the hybrid ceramic-resin cement bond strength (BS) by comparing two methods. Methods: Teeth were distributed into groups (n=5), according to the resin cement (Maxcem Elite-(MC) or NX3 Nexus-(NX)) and degradation method (24h or 7 days in distilled water; 7 or 30 days incubated with biofilm and 30 days in sterile media). Treated surfaces of Vita Enamic blocks (5x6x7mm) were luted to treated or no treated dentin surfaces and light-cured. After 24h, beams were obtained (1x1x10mm) and stored accordingly. The flexural bond strength (FBS) was assessed by four-point bending test. Additional beams were obtained from new teeth (n=5), stored for 24h or 7 days in distilled water, and submitted to a microtensile bond strength (µTBS) assay. Failure modes were determined by scanning electron microscopy (100X). The flexure strength of the cements (n=10) was assessed by a four-point bending test. Data were analyzed by 1 and 2-ways ANOVA, and Tukey's test (α=0.05). Results: There was no significant difference between the degradation methods for the FBS groups. For the µTBS, the significant difference was as follows: NX 7days > NX 24h > MC 7days = MC 24h. Failure mode was mainly adhesive and mixed, but with an increase of cohesive within cement and pre-failures for the MC groups assessed by µTBS. NX had better performance than MC, regardless of the method. Conclusions: The biofilm had no effect on the materials BS and FBS test was a useful method to evaluate BS of materials with poor performance

Fibra de carbono ativada: avaliação das interações biológicas in vitro / Activated carbon fiber: evaluation of biological interactions in vitro

Devido a constante necessidade de desenvolver materiais biocompatíveis com propriedades osteocondutores e osteoindutoras, a presente tese conta com o desenvolvimento de dois estudos in vitro com fibra de carbono obtida a partir de fibra PAN têxtil, incorporada com diferentes íons de metais, na osteogênese com vistas à compreensão das necessidades da engenharia tecidual no desenvolvimento desse biomaterial com adequadas propriedades biológicas. As células foram obtidas dos fêmures de 09 ratos machos adultos (Wistar) pesando 300g, com 90 dias.Estudo 1: A partir da preparação da fibras foram obtidos corpos de prova de 4 mm de diâmetro e 2 mm de altura, dos seguintes grupos: fibra de carbono não ativada (FCNA), fibra carbono ativada (FCA) e fibra carbono ativada com prata (FCAAg). Após plaqueamento (n=5) em meio suplementado (MTS) e meio suplementado osteogênico (MTSO) foram analisados: viabilidade celular, conteúdo de proteína total (PT), atividade de fosfatase alcalina (ALP), interaçãocelular e formação de nódulos de mineralização. Foi avaliada a formação de biofilme nos corpos de prova, utilizando cepas de S. aureus, P. aeruginosa e E. coli. Na viabilidade celular, houve diferença estatística entre grupo controle celular (C) e FCA-MTS, FCAAg-MTS e FCAAg-MTSO. Em PT, não houvediferença, na ALP houve diferença entre C-MTS e as fibras, C-MTSO se mostrou semelhante. Em nódulos, houve diferença entre C-MTS e C-MTSO e as fibras do MTSO. Houve redução de formação de biofilme do S. aureus na FCAAg.Estudo 2: Foram obtidos corpos de prova da mesma dimensão do estudo 1 (n=5) dos seguintes grupos: fibra carbono ativada com prata (FCAAg), fibra carbono ativada com ouro (FCAAu), fibra carbono ativada com cobre (FCACu), fibra carbono ativada com paládio (FCAPd) e fibra carbono ativada com platina (FCAPt). Foram quantificadas a proliferação celular, viabilidade celular, formação de nódulos de mineralização, conteúdo de PT e ALP. Todas as amostras mostraram-se semelhantes quanto a proliferação celular, com exceção do grupo FCAAg comparado ao grupo controle (C). Sobre viabilidade celular, C obteve maior viabilidade que os outros grupos, e FCA obteve maior taxa que os grupos FCAAg, FCACu, FCAPt, sendo semelhante aos grupos FCAAu e FCAPd. Já os grupos FCAAu e FCAPd apresentaram diferença aos grupos FCAAg e FCACu. Na análise de expressão de PT apenas houve diferença entre FCA e FCAAu, sendo FCAAu com menor expressão de produção de PT. Na avaliação da ALP os grupos FCAAg e FACu mostraram diferença estatística e inferior com os grupos C, FCAAu, FCAPd e FCAPt, além disso, o grupo FCA mostrou menor taxa que C.Conclusões: As fibras utilizadas de base para a incorporação dos íons demonstraram grande potencial para uso como scaffold para reparação óssea, isso porque em ambos os estudos, na forma ativada e não ativada, as fibras apresentaram viabilidade celular e quantificação de cálcio satisfatórias. Sendo a versão não ativada mais econômica no que diz respeito ao tempo e custo de preparação. Mais estudos devem ser empregados a fim de assegurar sua segurança clínica em relação à citotoxicidade da incorporação de íons de ouro e paládio.(AU)

Due to the constant need to develop biocompatible materials with osteoconductive and osteoinductive properties, this thesis involves the development of two in vitro studies with carbon fiber obtained from textile PAN fiber, incorporated with different metal ions, in osteogenesis with a view to understanding the needs of tissue engineering in the development of this biomaterial with adequate biological properties. The cells were obtained from the femurs of 9 adult male rats (Wistar) weighing 300g, aged 90 days. Study 1: From the fiber preparation, specimens measuring 4 mm in diameter and 2 mm in height were obtained from the following groups: non-activated carbon fiber (FCNA), activated carbon fiber (FCA) and silver-activated carbon fiber (FCAAg). After plating (n=5) in supplemented medium (MTS) and supplemented osteogenic medium (MTSO), cell viability, total protein content (PT), alkaline phosphatase (ALP) activity, cell interaction and formation of mineralization nodules were analyzed. . Biofilm formation was evaluated in the specimens, using strains of S. aureus, P. aeruginosa and E. coli. In cell viability, there was a statistical difference between the cell control group (C) and FCAMTS, FCAAg-MTS and FCAAg-MTSO. In PT, there was no difference, in ALP there was a difference between C-MTS and fibers, C-MTSO was similar. In nodules, there was a difference between C-MTS and C-MTSO and MTSO fibers. There was a reduction in S. aureus biofilm formation on FCAAg. Study 2: Specimens of the same size as in study 1 (n=5) were obtained from the following groups: carbon fiber activated with silver (FCAAg), carbon fiber activated with gold (FCAAu), carbon fiber activated with copper (FCACu), palladium-activated carbon fiber (FCAPd) and platinum-activated carbon fiber (FCAPt). Cell proliferation, cell viability, formation of mineralization nodules, PT and ALP content were quantified. All samples were similar in terms of cell proliferation, with the exception of the FCAAg group compared to the control group (C). Regarding cell viability, C obtained higher viability than the other groups, and FCA obtained a higher rate than the FCAAg, FCACu, FCAPt groups, being similar to the FCAAu and FCAPd groups. The FCAAu and FCAPd groups showed differences to the FCAAg and FCACu groups. In the analysis of PT expression, there was only a difference between FCA and FCAAu, with FCAAu having lower expression of PT production. In the ALP assessment, the FCAAg and FACu groups showed a lower statistical difference compared to the C, FCAAu, FCAPd and FCAPt groups, in addition, the FCA group showed a lower rate than C. Conclusions: The fibers used as the basis for the incorporation of ions demonstrated great potential for use as a scaffold for bone repair, because in both studies, in activated and non-activated form, the fibers showed satisfactory cell viability and calcium quantification. The non-activated version is moreeconomical in terms of preparation time and cost. More studies must be carried out to ensure its clinical safety in relation to the cytotoxicity of the incorporation of gold and palladium ions. (AU)

Effectiveness of disinfectant solutions associated or not with brushing on the biofilm control of a 3D printed-denture base resin

Abstract The formation of biofilm on denture bases is a recurrent clinical problem that favors the development of denture stomatitis. The effectiveness of a hygiene protocol in a 3D-printed denture base resin is still uncertain. Objective To evaluate of the effectiveness of immersion, associated or not with brushing in a soap solution, on the biofilm control of a 3D-printed denture base resin. Methodology Specimens of denture base resins [Cosmos Denture (COS) and Classico (CLA/control)] were contaminated in vitro with Candida albicans and immersed in sodium hypochlorite 0.25% (SH, alkaline peroxide) AP, chlorhexidine digluconate 2% (CD or PBS-Control), associated or not with brushing with 0.78% Lifebuoy soap. Roughness was evaluated before and after brushing and immersion. The effectiveness of the protocols was assessed by CFU/mL, cellular metabolism (XTT), scanning electron microscopy (SEM), and confocal scanning laser microscopy. Data were analyzed by T student, ANOVA/Welch, and Tukey/Gomes-Howell pos-hoc tests (α = 0.05). Results CLA showed greater roughness than COS. CFU/mL and XTT were higher in COS resin with a higher hyphae formation. Immersion in SH and CD eliminated CFU/mL and reduced XTT for both resins, associated or not with brushing. AP reduced CFU/mL only when associated with brushing. Conclusions The biofilm on the 3D-printed resin was thicker and presumably more pathogenic, regardless of its smoother surface. Immersions in SH 0.25% and CD 2% are effective hygiene protocols for both resins, associated or not with brushing. AP should be recommended when associated with brushing with a Lifebuoy 0.78% solution.

Biofilme em parafusos ortopédicos prontos para uso adquiridos por meio de sistema de consiganação/comodato / Biopelícula en tornillos ortopédicos listos para uso adquiridos por medio del sistema de consignación/comodato / Biofilm on patient-ready orthopaedic screws acquired through the loaner system

Resumo Objetivo Avaliar a integridade da superfície e as condições microbiológicas de parafusos prontos para uso em bandejas ortopédicas após múltiplos processamentos. Métodos Após o processamento completo, as bandejas utilizadas em cirurgias de pequenos fragmentos, fornecidas por meio de sistema de consignação/comodato em um hospital brasileiro, foram selecionadas aleatoriamente durante quatro meses. Os parafusos mais utilizados (números 14, 16 e 18 - Grupo 1) e menos utilizados (números 10 e 38 - Grupo 2), portanto, os mais e menos expostos a agentes biológicos, químicos e físicos, foram aleatoriamente removidos e submetidos a inspeção visual (n=126), seguido de cultura bacteriana (n=6 parafusos/bandeja, 9 bandejas), teste de proteínas (n=6 parafusos/bandeja, 9 bandejas) e Microscopia Eletrônica de Varredura (MEV) (n=2 parafusos/bandeja, 9 bandejas). As culturas positivas foram submetidas a métodos automatizados de identificação bacteriana e suscetibilidade antimicrobiana. Resultados Foram detectadas ranhuras em 8,7% dos parafusos, predominantemente no Grupo 2 (8/11). Proteína residual foi detectada em 96,3%, e não houve diferença estatisticamente significativa na quantidade de proteína entre os grupos (P=0,07). Crescimento bacteriano foi identificado em 3/54 parafusos. Danos na superfície e presença de sujidade foram visualizados em todos os parafusos submetidos a MEV. Formação de biofilmes extensos foi detectada em oito parafusos, três do Grupo 1 e cinco do Grupo 2. Conclusão Recuperação de bactérias viáveis, acúmulo de biofilme e danos na superfície foram detectados nos parafusos prontos para uso. Os parafusos costumam permanecer nas bandejas cirúrgicas e serem submetidos a múltiplos processamento, sendo expostos a contaminação e danos repetidas vezes. Esses achados apontam para a necessidade de discutir e repensar a forma como esses implantes de uso único são atualmente disponibilizados para cirurgias.

Resumen Objetivo Evaluar la integridad de la superficie y las condiciones microbiológicas de tornillos listos para uso en bandejas ortopédicas después de múltiples procesamientos. Métodos Después del procesamiento completo, fueron seleccionadas aleatoriamente durante cuatro meses las bandejas utilizadas en cirugías de pequeños fragmentos, proporcionadas mediante el sistema de consignación/comodato en un hospital brasileño. Los tornillos más utilizados (números 14, 16 y 18 - Grupo 1) y menos utilizados (números 10 y 38 - Grupo 2), por lo tanto, los más y menos expuestos a agentes biológicos, químicos y físicos, fueron quitados aleatoriamente y sometidos a inspección visual (n=126), seguido de cultivo bacteriano (n=6 tornillos/bandeja, 9 bandejas), prueba de proteínas (n=6 tornillos/bandeja, 9 bandejas) y microscopía electrónica de barrido (MEB) (n=2 tornillos/bandeja, 9 bandejas). Los cultivos positivos fueron sometidos a métodos automatizados de identificación bacteriana y susceptibilidad antimicrobiana. Resultados Se detectaron ranuras en el 8,7 % de los tornillos, predominantemente en el Grupo 2 (8/11). Se detectó proteína residual en el 96,3 % y no se encontró diferencia estadísticamente significativa en la cantidad de proteína entre los grupos (P=0,07). En 3/54 tornillos se identificó crecimiento bacteriano. Se visualizaron daños en la superficie y presencia de suciedad en todos los tornillos sometidos a MEB. En ocho tornillos se detectó la formación de biopelículas, tres del Grupo 1 y cinco del Grupo 2. Conclusión Se detectó recuperación de bacterias viables, acumulación de biopelícula y daños en la superficie en los tornillos listos para uso. Los tornillos suelen permanecer en las bandejas quirúrgicas y son sometidos a múltiples procesamientos, donde están expuestos a contaminación y daños repetidas veces. Estos descubrimientos señalan la necesidad de discutir y repensar la forma como estos implantes de uso único se ponen a disposición para cirugía actualmente.

Abstract Objective Assess the surface integrity and microbiological conditions of patient-ready screws in orthopaedic trays that had been multiply reprocessed. Methods After full reprocessing, clinical trays used for small fragment surgery provided through a loaner system to a Brazilian hospital were randomly selected during four months. The most (numbers 14, 16 and 18 - Group 1) and least (numbers 10 and 38 - Group 2) frequently implanted screws, therefore, the ones estimated to be the most and least exposed to biological, chemical and physical agents, were randomly removed and subjected to visual inspection (n=126), followed by bacterial culture (n=6 screws/tray, 9 trays), protein test (n=6 screws/tray, 9 trays) and Scanning Electron Microscopy (SEM) (n=2 screws/tray, 9 trays). Positive cultures were subjected to automated bacterial identification and antimicrobial susceptibility tests. Results Grooves were detected on 8.7% screws, predominantly in Group 2 (8/11). Residual protein was detected on 96,3%, and there was no statistically significant difference in the amount of protein between the groups (P=0.07). Bacterial growth was identified in 3/54 screws. Surface damage and soil were visualized on all screws subjected to SEM. Extensive biofilms were detected on eight screws, three from Group 1 and five from Group 2. Conclusion Recovery of bacteria, biofilm accumulation and surface damage were detected on patient-ready screws. Screws frequently remain in surgical trays for multiple reprocessing; thus they are repeatedly exposed to contamination and damage. These findings point to the need to discuss and review the way these single-use implants are currently made available for surgeries.

Effect of anaerobic or/and microaerophilic atmosphere on microcosm biofilm formation and tooth demineralization

Abstract Microcosm biofilms can reproduce the complexity of a dental biofilm. However, different forms of cultivation have been used. The impact of the culture atmosphere on the development of microcosm biofilms and their potential to cause tooth demineralization has not yet been deeply studied. Objective This study analyzes the effects of three experimental cultivation models (microaerophile vs. anaerobiosis vs. experimental mixed) on the colony-forming units (CFU) of the cariogenic microorganisms and tooth demineralization. Methodology 90 bovine enamel and 90 dentin specimens were distributed into different atmospheres: 1) microaerophilia (5 days, 5% CO2); 2) anaerobiosis (5 days, jar); 3) mixed (2 days microaerophilia and 3 days anaerobiosis), which were treated with 0.12% chlorhexidine (positive control - CHX) or Phosphate-Buffered Saline (negative control - PBS) (n=15). Human saliva and McBain's saliva containing 0.2% sucrose were used for microcosm biofilm formation, for 5 days. From the second day to the end of the experiment, the specimens were treated with CHX or PBS (1x1 min/day). Colony-forming units (CFU) were counted, and tooth demineralization was analyzed using transverse microradiography (TMR). Data were subjected to two-way ANOVA and Tukey's or Sidak's test (p<0.05). Results CHX was able to reduce total microorganism's CFU compared to PBS (differences of 0.3-1.48 log10 CFU/mL), except for anaerobiosis and microaerophilia in enamel and dentin biofilm, respectively. In the case of dentin, no effect of CHX on Lactobacillus spp. was observed. CHX significantly reduced enamel demineralization compared to PBS (78% and 22% reductions for enamel and dentin, respectively). Enamel mineral loss did not differ when compared with the other atmospheres; however, the enamel lesion depth was greater under anaerobiosis. Dentin mineral loss was lower under anaerobiosis when compared with the other atmospheres. Conclusion The type of atmosphere has, in general, little influence on the cariogenic ability of the microcosm biofilm.