Acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans.

OBJECTIVE: The aim of this study was to evaluate the acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans. MATERIALS AND METHODS: The following strains were tested: Bifidobacterium dentium DSM20436, Parascardovia denticolens DSM10105, and Scardovia inopinata DSM10107. Streptococcus mutans UA159 and Lactobacillus acidophilus ATCC4356 were used as control. Bifidobacteria were studied planktonically as they were not able to form monospecies biofilm, they were grown in biofilms associated with S. mutans. Endogenous polysaccharide reserves of cultures at log phase were depleted. Standardized suspensions of the microorganisms were incubated in growth media supplemented with 10 mM glucose, lactose, raffinose, glucose, or xylitol. S. mutans biofilms were grown on glass cover slips for 24 h to which bifidobacteria were added. After 24 h, the dual-species biofilms were exposed to the same carbon sources, and after 3 h, the pH of spent culture media and concentrations of organic acids were measured. Statistical analyses were carried out using ANOVA and Tukey's test (α = 0.05). RESULTS: A higher pH drop was observed when S. mutans was associated with P. denticolens or S. inopinata, in either planktonic or biofilm cultures, than with S. mutans alone. Bifidobacteria showed a higher pH drop in the presence of raffinose than S. mutans or L. acidophilus. CONCLUSIONS: Dual-species biofilms of bifidobacteria and S. mutans produced more acid and greater pH drops than biofilms of S. mutans alone. CLINICAL RELEVANCE: New insights on the complex process of caries pathogenicity contribute to the establishment of preventive and therapeutic measures, in particular in specific cases, such as in early childhood caries.

Evaluation of caries-associated virulence of biofilms from Candida albicans isolated from saliva of pediatric patients with sickle-cell anemia

A previous study demonstrated that the amount of Candida spp. in saliva is higher in children with sickle-cell disease. The results from a recent study demonstrate its participation in the etiology of dental caries. Objective This study assessed caries-associated virulence (production of acid, extracellular polysaccharides, proteins and metabolic activity) of biofilms from Candida albicans isolated from saliva of patients with sickle-cell anemia in comparison to isolates obtained from matched healthy children. Material and Methods The isolates were previously obtained from 25 children (4-6 years) and their matched controls (healthy children). One isolate of C. albicans per children was used, totaling 25 isolates per group. The C. albicans biofilms were grown for five days and analyzed regarding the production of lactic acid, extracellular polysaccharides, proteins and metabolic activity. The production of lactic acid was determined by the enzymatic method. The concentration of extracellular polysaccharides was determined by the phenol-sulphuric acid method, and the concentration of the protein was analyzed using the QuantiPro BCA kit. The XTT reduction was used to verify the metabolic activity. The data were analyzed with GraphPad Prism at 5%. Results The Mean±standard deviation for acid production, extracellular polysaccharides, proteins and metabolic activity of isolates from sickle-cell group was, respectively: 7.1±5.0 mmol/L; 15.6±2.5 μg glucose/mg biofilm; 7,503±3,097 μg/mL; A490 3.5±0.7. For isolates from control group the values obtained were: 3.5±3.3 mmol/L; 12.8±3.4 μg glucose/mg biofilm; 4,995±682 μg/mL; A490 3.4±0.5. The C. albicans isolates from patients with sickle-cell anemia produced a significantly greater quantity of acids (p=0.025), polysaccharides (p=0.025) and proteins (p=0.047) compared with the isolates from control group. However, there was ...

Oral microbial colonization in children with sickle cell anaemia under long-term prophylaxis with penicillin.

BACKGROUND AND OBJECTIVE: Sickle cell anaemia (SCA) is the most frequent haematological hereditary disease. Children with SCA are submitted to long-term prophylactic therapy with penicillin, but little is known about its impact on oral microflora. The aim of this study was to evaluate the oral microbial colonization of paediatric patients with SCA. DESIGN: Forty children (4-11yrs old) with SCA (genotype SS) under long-term prophylactic treatment with penicillin were included in the study. Age/gender-matched control group of healthy children was also included. Scores of dmft/DMFT (number of decayed (D), missing (M), or filled (F) teeth; dmft, for primary dentition; DMFT, for permanent dentition) were obtained and stimulated saliva was sampled. Salivary flow rate and buffering capacity were evaluated. Counts of microorganisms (mutans streptococci, lactobacilli and yeasts) were determined by plating method. Yeasts were identified by API 20C AUX and PCR. RESULTS: Mean dmft/DMFT values were similar in the studied groups (SCA 2.13/1.60 and control 2.38/1.3). Although no significant differences between cariogenic microorganism counts were observed, significantly higher yeasts oral levels were observed in SCA group. Controls showed lower salivary buffering capacity. Candida albicans was the most frequently isolated species in both groups. Candida famata, Candida parapsilosis and Candida tropicalis were also isolated from controls. Candida dubliniensis, Candida rugosa and Candida sphaerica were found only in SCA group. CONCLUSIONS: Based on the results, it could be concluded that paediatric patients with SCA showed significantly higher oral level of yeasts. Uncommon fungal species were found in SCA group. Similar caries prevalence and counts of lactobacilli and streptococci in relation to controls were observed.

Evaluation of caries-associated virulence of biofilms from Candida albicans isolated from saliva of pediatric patients with sickle-cell anemia.

A previous study demonstrated that the amount of Candida spp. in saliva is higher in children with sickle-cell disease. The results from a recent study demonstrate its participation in the etiology of dental caries. Objective This study assessed caries-associated virulence (production of acid, extracellular polysaccharides, proteins and metabolic activity) of biofilms from Candida albicans isolated from saliva of patients with sickle-cell anemia in comparison to isolates obtained from matched healthy children. Material and Methods The isolates were previously obtained from 25 children (4-6 years) and their matched controls (healthy children). One isolate of C. albicans per children was used, totaling 25 isolates per group. The C. albicans biofilms were grown for five days and analyzed regarding the production of lactic acid, extracellular polysaccharides, proteins and metabolic activity. The production of lactic acid was determined by the enzymatic method. The concentration of extracellular polysaccharides was determined by the phenol-sulphuric acid method, and the concentration of the protein was analyzed using the QuantiPro BCA kit. The XTT reduction was used to verify the metabolic activity. The data were analyzed with GraphPad Prism at 5%. Results The Mean±standard deviation for acid production, extracellular polysaccharides, proteins and metabolic activity of isolates from sickle-cell group was, respectively: 7.1±5.0 mmol/L; 15.6±2.5 µg glucose/mg biofilm; 7,503±3,097 µg/mL; A490 3.5±0.7. For isolates from control group the values obtained were: 3.5±3.3 mmol/L; 12.8±3.4 µg glucose/mg biofilm; 4,995±682 µg/mL; A490 3.4±0.5. The C. albicans isolates from patients with sickle-cell anemia produced a significantly greater quantity of acids (p=0.025), polysaccharides (p=0.025) and proteins (p=0.047) compared with the isolates from control group. However, there was no difference in metabolic activity (XTT) between groups (p=0.750). Conclusion The C. albicans biofilms from patients with sickle-cell anemia presented a greater caries-associated virulence than isolates from healthy children.

Atividade antifúngica de uma pomada à base de chamomilla recutita sobre candida albicans / Antifungal activity of chamomilla recutita based pomade on candida albicans

O objetivo do estudo foi avaliar a atividade antifúngica de uma pomada à base de [Chamomilla recutita (L) Rauschert] (camomila) sobre espécies do gênero Cândida. Vinte e seis isolados clínicos Cân- dida (e. a/bicans, e. dub/iniensis, e. krusei, e. parapsi/osis) e três cepas de referência foram semeadas em ágar Sabouraud dextrose a 3rC/24h antes da realização do experimento. Foi realizada a diluição seriada do produto nas concentrações de 500f0, 250f0, 12,5%, 6,25%, 3,12%, 1,56% e 0,78% em ágar RPMI tamponado com MOPS. Foram obtidas suspensões padronizadas na escala 5 de McFarland em solução fisiológica esterilizada (NaCI 0,9%) de cada isolado a ser avaliado. Posteriormente, as suspensões dos microrganismos foram semeadas em placas de Petri contendo o produto teste + ágar RPMI através de replicador de Steers e incubadas em estufa bacteriológica a 3rC/24h. Um controle de crescimento foi incluído no estudo e os testes foram realizados em duplicata. O resultado foi avaliado através da observação da presença ou ausência de crescimento de colônias no ágar. Os testes mostraram que das 26 amostras avaliadas, 14 amostras (53,9%) foram inibidas na concentração de 500f0, 11 amostras (42,3%) foram inibidas na concentração de 25%, uma amostra (3,9%) foi inibida na concentração de 12,5%. A pomada não teve efeitos inibitórios sobre a cepa de referência e. krusei. A partir dos resultados obtidos, concluiu-se que o produto testado apresentou atividade antifúngica in vitro sobre a maioria dos isolados de Cândida avaliados.

The aim of this study was to evaluate the antifungal activity of a chamomile [Chamomilla recutita (L) Rauschert] based ointment. Twenty-six clinical isolates of Candida (e. a/bicans, e. dub/iniensis, e. krusei, e. parapsi/osis) and three reference strains were spread onto Sabouraud dextrose agar and incubated under 37°C/24 h before the experiment. The ointment was serially diluted (500f0, 25%, 12.5%, 6.25%,3.12%, 1.56% and 0.78%) in RMPI agar buffered with MOPS. Standardized suspensions of each strain were prepared in saline solution (NaCl 0.9%) based on scale 5 of McFarland. Next, the suspensions of each microorganism were plated on Petri using a Steers replicator and incubated at 37°C/24 h. A positive control was included and the tests were performed in duplicate. The result was evaluated by the observation the presence or absence the colonies on the agar. The tests showed that from the 26 isolates evaluated, 14 (53,9%) were inhibited by the concentration of 500/0, 11 strains (42,3%) were inhibited by the concentration of 25%, one strain (3.9%) were inhibited by the concentration of 12.5%. The ointment did not show inhibitory effects on e. krusei reference strain. Based on the results obtained, we concluded that the product evaluated showed antifungal activity in vitro on the majority of the tested isolates.

Atividade antifúngica do extrato alcoólico de Mentha piperita sobre Candida albicans e C. tropicalis / Antifungal activity of Mentha piperita alcoholic extract on Candida albicans and C. tropicalis

O objetivo do estudo foi avaliar a atividade antifúngica do extrato alcoólico de hortelã sobre Candida albicans e C. tropicalis. Incluíram-se no estudo C. albicans (ATCC 18804, ATCC 36802 e 20 isolados clínicos) e C. tropicalis (ATCC 13803 e 20 isolados clínicos) e foram realizados testes de screening com cepas padrão de C. dubliniensis (NCPF 3108), C. glabrata (ATCC 90030), C. krusei (ATCC 6258) e C. parapsilosis (ATCC 22019). O extrato de hortelã foi testado frente às amostras de Candida spp. pelo método de microdiluição. Inicialmente, realizou-se diluição seriada do extrato de hortelã em caldo RPMI, utilizando placas de microtitulação; nestas, inocularam-se suspensões padronizadas contendo 10 6 células. mL–1 de cada cepa a ser avaliada e foi incluído também um grupo controle do veículo (álcool). Os testes foram realizados em duplicata, as placas incubadas a 37 °C por 24 horas, e a leitura realizada em leitor de microplacas para determinação da concentração inibitória mínima (CIM). A seguir, determinou-se a concentração fungicida mínima (CFM) por meio da semeadura do conteúdo de cada poço da microplaca em ágar Sabouraud dextrose. Os resultados mostraram ação do extrato de hortelã (CIM e CFM) sobre C. albicans (p = 0,00001), mas não sobre C. tropicalis [p = 0,5296 (CIM) e p = 0,6504 (CFM)]. O extrato de hortelã apresentou ação superior sobre C. albicans quando comparada à C. tropicalis, p = 0,00001 (CIM e CFM). Concluiu-se que o extrato de hortelã apresentou atividade inibitória e fungicida sobre as amostras de C. albicans avaliadas.

The aim of the study was evaluate the antifungal activity of the peppermint alcoholic extract on Candida albicans and C. tropicalis. It was included in the study C. albicans (ATCC 18804, ATCC 36802 and 20 clinical isolates) and C. tropicalis (ATCC 13803 and 20 clinical isolates), and performed screening tests with C. dubliniensis (NCPF 3108), C. glabrata (ATCC 90030), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019). The peppermint extract was evaluated on Candida spp. by microdilution method. Firstly, it was performed serial dilutions of the extract into RPMI broth in microdilution plates where was inoculated standardized suspensions with 106 cells/ml of each strain to be evaluated, and a control group (alcohol) was also included. The tests were made in duplicate, the plates were incubated at 37 °C/24 hours and the reading was performed in a microplates reader to determine the minimal inhibition concentration (MIC). Following, the minimal fungicide concentration (MFC) was determined by plating the content of each well of the microplate on Sabouraud dextrose agar. The results showed activity of the peppermint extract (MIC and MFC) on C. albicans (p = 0.00001), but not on C. tropicalis, p = 0.5296 (MIC) and p = 0.6504 (MFC). The peppermint extract showed higher activity on C. albicans when it was compared with C. tropicalis, p = 0.00001 (MIC and MFC). We concluded that the peppermint extract showed inhibitory and fungicide activity on the C. albicans strains tested.

Comparação da atividade antimicrobiana de soluções de peróxido de hidrogênio e malva sobre candida albicans / Comparison of hydrogen peroxide and malva mouthrinses antimicrobial activity on candida albicans

O estudo avaliou a atividade antimicrobiana de enxaguatórios bucais sobre Candida albicans. Vinte e uma amostras clínicas e uma padrão de C. albicans (ATCC 18804) foram testadas frente à enxaguatórios à base de peróxido de hidrogênio 1,5% e tintura de malva. Solução de gluconato de clorexidina 0,12% foi utilizada como controle positivo e foram obtidos valores para máxima diluição inibitória (MDI) e máxima diluição fungicida (MDF). Foram obtidas diluições seriadas dos produtos (50% a 0,02%), e adicionadas 100μL de suspensão de cada cepa e foram incubadas a 37°C/24h, e a MDI determinada. As amostras foram semeadas em ágar Sabouraud para determinação da MDF. Para a solução à base de peróxido de hidrogênio a MDI=0,78% para 86,35% das amostras e MDF=3,1% para 77,27% das amostras. Para a solução à base de gluconato de clorexidina, a MDI foi de 0,2% e 0,1% para 72,7% das amostras e MDF foi de 1,56% e 0,78% para 90,9% das amostras. Para a solução à base de tintura de malva a MDI foi de 1,56% e 0,78% para 72,72% das amostras e não houve atividade fungicida para 54,54% das amostras. A análise estatística (ANOVA Kruskal-Wallis, α=5%) mostrou diferenças significativas entre os valores médios de MDI (p=0,000) e de MDF (p=0,003). Concluiu-se que a solução à base de gluconato de clorexidina apresentou melhor atividade antimicrobiana sobre C. albicans, seguida pela solução à base de peróxido de hidrogênio. A solução à base de tintura de malva não apresentou atividade fungicida sobre a maioria das cepas de C. albicans.

The study evaluated the antimicrobial activity of mouthrinses on Candida albicans. Twenty-one clinical isolates and one reference strain of C. albicans (ATCC 18804) were tested with 1.5% hydrogen peroxide and malva tincture based mouthrinses. Chlorhexidine gluconate solution (0.12%) was included as positive control and values of maximum inhibitory dilution (MID) and maximum fungicide dilution (MFD) values were obtained. Serial dilutions of the products were obtained, from 50% to 0.02%, and added 100μL of suspension of each strain that were incubated at 37°C for 24h, and the MID was determinated. Samples were plated in Sabouraud agar for the determination of MFD. Results obtained for hydrogen peroxide-based solution was 0.78% for MID to most of the isolates (86.35%) and MFD was 3.1% for 77.27% of the samples. For chlorhexidine-based solution, MID was 0.2% and 0.1% for most of the isolates (72.7%) and MFD was 1.56% and 0.78% for 90.9% of the strains. For malva tincture-based solution, MID values were between 1.56% and 0.78% for 72.72% of the isolates and no fungicide activity for 54.54% of the isolates was observed. Statistical analyses of the results (ANOVA Kruskal-Wallis, α=5%) showed significant differences among the mean values of MID (p=0.000) and of MFD (p=0.003). It could be concluded that chlorhexidine gluconate-based solution showed the best antimicrobial activity on C. albicans, followed by hydrogen peroxide-based solution. Malva tincture-based solution did not show fungicide activity on most of the C. albicans isolates tested.