Enterococcus Species in the Oral Cavity: Prevalence, Virulence Factors and Antimicrobial Susceptibility.

Enterococci are considered as transient constituent components of the oral microbiome that may cause a variety of oral and systemic infections. As there is sparse data on the oral enterococcal prevalence, we evaluated the Enterococcus spp. and their virulence attributes including antimicrobial resistance in a healthy Brazilian cohort. A total of 240 individuals in different age groups were studied (children 4-11 yrs, adolescents 12-17 yrs, young adults 18-29 yrs, adults 30-59 yrs, elderly over 60 yrs). Oral rinses were collected and isolates were identified by API 20 Strep and confirmed by 16S rDNA sequencing. E. faecalis isolates, in particular, were evaluated for virulence attributes such as their biofilm formation potential, and susceptibility to antimicrobials and an antiseptic, chlorhexidine gluconate. A total of 40 individuals (16.6%) and 10% children, 4% adolescents, 14% young adults, 30% adults, and 25% elderly carried oral enterococci. The oral enterococcal burden in adolescents was significantly lower than in the adults (p = 0.000) and elderly (p = 0.004). The proportion of carriers was higher among females (p = 0.001). E. faecalis was the most frequent isolate in all the age groups (p = 0.000), followed by E. durans and E. faecium. Whilst all the clinical isolates were able to form biofilms, only a proportion of them were able to produce lipase (92%), hemolysin (38%), and gelatinase (39%). Of all the isolates 53.8% were resistant to tetracycline, 12.3% to amoxicillin, 16.0% to ampicillin, 20.8% to chloramphenicol and 43.4% to erythromycin. None of the isolates were resistant to vancomycin. Our data suggest that in this Brazilian cohort the oral cavity may act as a significant reservoir of rather virulent and antibiotic resistant enterococci, with an increasing degree of carriage in the adults and elderly. Hence clinicians should be cognizant of this silent reservoir of virulent enterococci that may pose a particular threat of nosocomial infection.

In vitro adherence of Candida albicans isolated from patients with chronic periodontitis

Adherence is considered an extremely important virulence factor in yeast. OBJECTIVE: The aim of this study was to analyze the adherence to epithelial cells of C. albicans isolated from patients with chronic periodontitis in comparison to healthy patients. MATERIAL AND METHODS: Candida albicans cells isolated from individuals with chronic periodontitis (n=25) and healthy controls (n=25) were included in this study. Suspensions of C. albicans (10(6) cells/mL) and epithelial cells (10(5) cells/mL) were mixed and incubated at 37ºC for 1 h. The number of yeasts adhered to 25 epithelial cells was counted. RESULTS: The number of C. albicans cells adhered to epithelial cells was statistically higher in the chronic periodontitis group than in the control group (Student's t-test, p=0.000). CONCLUSION:The results of the present study suggest a higher Candida adherence of samples isolated from patients with chronic periodontitis.

In vitro adherence of Candida albicans isolated from patients with chronic periodontitis.

UNLABELLED: Adherence is considered an extremely important virulence factor in yeast. OBJECTIVE: The aim of this study was to analyze the adherence to epithelial cells of C. albicans isolated from patients with chronic periodontitis in comparison to healthy patients. MATERIAL AND METHODS: Candida albicans cells isolated from individuals with chronic periodontitis (n=25) and healthy controls (n=25) were included in this study. Suspensions of C. albicans (10(6) cells/mL) and epithelial cells (10(5) cells/mL) were mixed and incubated at 37ºC for 1 h. The number of yeasts adhered to 25 epithelial cells was counted. RESULTS: The number of C. albicans cells adhered to epithelial cells was statistically higher in the chronic periodontitis group than in the control group (Student's t-test, p=0.000). CONCLUSION: The results of the present study suggest a higher Candida adherence of samples isolated from patients with chronic periodontitis.

Experimental systemic virulence of oral Candida dubliniensis isolates in comparison with Candida albicans, Candida tropicalis and Candida krusei.

There are no previous studies on the comparative virulence of Candida dubliniensis with other non-albicans species. The aim of this study was to compare the virulence and infection kinetics of C. dubliniensis and other species. Candida albicans, C. dubliniensis, Candida tropicalis and Candida krusei (reference strains) were inoculated intravenously in mice. For infection kinetics evaluation, a group of five animals were sacrificed after 6 h, 3, 7, 14 and 21 days. Microbiological evaluations (liver, spleen, kidneys, lungs and brain) and histopathological examination of the kidney were performed. The results of virulence evaluation were analysed using Kaplan-Meier survival analysis (5%). Candida dubliniensis-inoculated mice survived for longer periods compared with those with C. albicans (P = 0.005). No differences were detected in relation to C. tropicalis (P = 0.326) and C. krusei (P = 0.317). Most of the organs were persistently colonised by C. albicans and C. dubliniensis even by day 21. Tendency of C. krusei clearance was observed in all organs. Fungal masses and renal lesions were observed after inoculation of C. albicans, C. dubliniensis and C. tropicalis. Within the limits of the study, data on survival rate and dissemination capacity suggest that C. dubliniensis is less virulent than C. albicans.

Experimental pathogenicity of Candida albicans and Candida dubliniensis with continuous and discontinuous fringes morphotypes.

A possible correlation between the presence of discontinuous fringes and high virulence has been previously suggested. The aim of this study was to compare the pathogenicity of Candida albicans and Candida dubliniensis with continuous and discontinuous fringes morphotypes on mice. For C. albicans, two discontinuous fringe morphotype isolates (PN 69, PN 74), two continuous fringe morphotype isolates (N 60, N 33) and one reference strain were used. For C. dubliniensis, three discontinuous fringe morphotype isolates (97487, 97464, 97519), two continuous fringe morphotype isolates (97040, 98026) and one reference strain were used. Swiss male mice were inoculated with a standardised suspension of the microorganisms and observed for 35 days. The pathogenicity of the isolates was analysed according to parameters proposed previously. Three isolates were considered pathogenic: PN 74, N 60 and 98026. Strain N 60 killed the highest amount of mice (80%). Animals inoculated with C. albicans did not show differences on survival estimate. Candida dubliniensis 98026 was more pathogenic than samples 97464 and 97519. On the other hand, the sample 97487 showed a higher pathogenicity when compared with 97040 (Kaplan-Meier test, P = 0.008). Strains with continuous fringe morphotypes were also associated with Candida sp. virulence in vivo.

Evaluation of alternative methods for the disinfection of toothbrushes.

The aim of this study was to evaluate alternative methods for the disinfection of toothbrushes considering that most of the previously proposed methods are expensive and cannot be easily implemented. Two-hundred toothbrushes with standardized dimensions and bristles were included in the study. The toothbrushes were divided into 20 experimental groups (n = 10), according to microorganism considered and chemical agent used. The toothbrushes were contaminated in vitro by standardized suspensions of Streptococcus mutans, Streptococcus pyogenes, Staphylococcus aureus or Candida albicans. The following disinfectants were tested: 0.12% chlorhexidine digluconate, 50% white vinegar, a triclosan-containing dentifrice solution, and a perborate-based tablet solution. The disinfection method was immersion in the disinfectant for 10 min. After the disinfection procedure, the number of remaining microbial cells was evaluated. The values of cfu/toothbrush of each group of microorganism after disinfection were compared by Kruskal-Wallis ANOVA and Dunn's test for multiple comparisons (5%). The chlorhexidine digluconate solution was the most effective disinfectant. The triclosan-based dentifrice solution promoted a significant reduction of all microorganisms' counts in relation to the control group. As to the disinfection with 50% vinegar, a significant reduction was observed for all the microorganisms, except for C. albicans. The sodium perborate solution was the less effective against the tested microorganisms. Solutions based on triclosan-containing dentifrice may be considered effective, nontoxic, cost-effective, and an easily applicable alternative for the disinfection of toothbrushes. The vinegar solution reduced the presence of S. aureus, S. mutans and S. pyogenes on toothbrushes.

Evaluation of alternative methods for the disinfection of toothbrushes

The aim of this study was to evaluate alternative methods for the disinfection of toothbrushes considering that most of the previously proposed methods are expensive and cannot be easily implemented. Two-hundred toothbrushes with standardized dimensions and bristles were included in the study. The toothbrushes were divided into 20 experimental groups (n = 10), according to microorganism considered and chemical agent used. The toothbrushes were contaminated in vitro by standardized suspensions of Streptococcus mutans, Streptococcus pyogenes, Staphylococcus aureus or Candida albicans. The following disinfectants were tested: 0.12 percent chlorhexidine digluconate, 50 percent white vinegar, a triclosan-containing dentifrice solution, and a perborate-based tablet solution. The disinfection method was immersion in the disinfectant for 10 min. After the disinfection procedure, the number of remaining microbial cells was evaluated. The values of cfu/toothbrush of each group of microorganism after disinfection were compared by Kruskal-Wallis ANOVA and Dunn's test for multiple comparisons (5 percent). The chlorhexidine digluconate solution was the most effective disinfectant. The triclosan-based dentifrice solution promoted a significant reduction of all microorganisms' counts in relation to the control group. As to the disinfection with 50 percent vinegar, a significant reduction was observed for all the microorganisms, except for C. albicans. The sodium perborate solution was the less effective against the tested microorganisms. Solutions based on triclosan-containing dentifrice may be considered effective, nontoxic, cost-effective, and an easily applicable alternative for the disinfection of toothbrushes. The vinegar solution reduced the presence of S. aureus, S. mutans and S. pyogenes on toothbrushes.

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.

Phenotypic characterization of Candida spp. isolates from chronic periodontitis patients

Aim: Several typing methods for Candida spp. have been suggested in the literature in order to distinguish isolates for studies about the virulence or infection routes of these microorganisms and, in particular, for epidemiological purposes. The aim of this study was to establish a comparison between the phenotypic profile of oral Candida isolates from periodontitis patients and control individuals. Methods: The morphotyping and biotyping of 35 C. albicans isolates obtained from chronic periodontitis patients and 48 isolates from control individuals were performed. For morphotyping, the isolates were plated on malt extract agar and incubated for 10 days. Sixteen different morphotypes were observed for C. albicans, the most frequently observed being 0000 and 0001. Results: Biotype 0000 (complete absence of fringe) was most prevalent among the isolates obtained from periodontitis patients compared to those from control individuals, with statistical significance. Biotyping revealed 5 different biotypes with higher prevalence of the biotype 357 among the isolates from control and periodontitis groups. Conclusions: The results obtained by biotyping of the isolates did not permit to differentiate a characteristic model related to periodontal disease, whilst the morphotype 0000 was most frequently isolated from periodontitis patients.

Efetividade de um novo produto a base de gel de triclosan e Pimpinella anisum na anti-sepsia de mãos em Odontologia / Effectiveness of a new product with triclosan and Pimpinella anisum for hand antisepsis in Odontology

O objetivo deste estudo foi avaliar a efetividade antimicrobiana de um novo anti-séptico para mãos a base de triclosan e Pimpinella anisum. Foi realizado estudo in vitro incluindo cepas de Staphylococcus aureus (ATCC 6.538), Escherichia coli (ATCC 25.922), Proteus mirabilis, Citrobacter freundii (CCUT 22.001), Enterobacter aerogenes (IAL 2.482) e Candida albicans (20 isolados clínicos). A metodologia utilizada foi de diluição em ágar (Müeller Hinton ou RPMI/MOPS) e os resultados foram expressos em valores de concentração inibitória mínima (CIM). A CIM para Escherichia coli, Citrobacter freundii e Entterobacter aerogenes foi de 0,03%; para Staphylococcus aureus 6,25%; Proteus mirabilis 12,5% e Candida albicans 12,5%. Para avaliação in vivo foram incluídos 60 voluntários dividos em três grupos: Produto A (triclosan, álcool etílico e Pimpinella anisum - Erva doce); Produto B (irgasan e álcool etílico a 70%) e controle (clorexidina 2% gel). Para a realização da coleta, as mãos foram divididas em oito partes iguais sendo que a coleta inicial e a final foram realizadas em quatro partes alternadas diferentes das mãos. As amostras foram coletadas com auxílio de swab esterilizado, umedecido em solução salina esterilizada e semeadas em duplicata em ágar sangue. Após a incubação, por 48 horas/37ºC, foram obtidos valores de unidades formadoras de colônias. O estudo in vivo demonstrou redução significativa das contagens em todos os grupos estudados (p < 0,001), sendo de 92,5% para o produto A, 91,1% para o produto B e 98,4% para o controle. Não houve diferença estatisticamente significativa entre as reduções de contagens dos grupos testados (p = 0,30). Concluiu-se que o novo produto apresentou alta efetividade antimicrobiana similar a dos demais grupos testados.

Avaliação in vitro de selante protetor dentinário sobre aderência de Streptococcus mutans / Microleakage in nickel-chromium copings subited to the acction of S. mutans

O objetivo deste estudo foi avaliar in vitro o efeito do selante dessensibilizador e protetor para dentina exposta Seal & Protect® (Dents-ply) sobre a aderência de Streptococcus mutans ATCC 35688 à superfície e dentária. As vantagens finas de Strptococcus mutans aderidas obitidas para o grupo selante foram significativamente menores em relaçãos às do grupo controle (p = 0,008). O percentual de redução de unidades formadoras de colônia...

Desinfecção de superfície em odontologia: avaliação do álcool gel 70°INPM, lenços embebidos em solução de clorexidina e spray de cloreto benzalcônio / Surface disinfection in dentistry

Com a introdução no mercado de novos agentes desinfetantes tornou-se necessário o estudo destes para se estabelecer uma desinfecção segura. O objetivo deste trabalho foi analisar a efetividade e estabelecer comparação entre a efetividade do álcool gel 70°INPM, lenços umedecidos em solução de clorexidina e o cloreto de benzalcônio. Foram utilizadas bandejas clínicas nas quais foram depositados 0,5 ml de suspensão contendo 10 na sexta potência células das amostras dos seguintes microrganismos: Staphylococcus aureus, Candida albicans, Enterococcus faecalis, Escherichia coli e Streptococcus mutans. Foram coletadas amostras utilizando-se placas de superfície RODAC contendo ágar Mitis salivarius, ágar BHI ou ágar Sabouraud dextrose de acordo com o microrganismo a ser analisado, antes e depois da desinfecção utilizando a técnica spray-wipe-spray. Os resultados foram analisados estatisticamente utilizando-se o teste Kruskal-Wallis para comparação de proporção de inibição entre os desinfetantes testados, o qual mostrou que houve diferença estatisticamente significativa entre os grupos. Conclui-se que todos se mostraram efetivos agentes de desifecção promovendo uma diminuição expressiva do número de microrganismos. O cloreto de benzalcônico e o álcool gel foram significantemente mais efetivos em relação à solução de clorexidina (p=0,000 e p=0,004, respectivamente). Concluiu-se que o álcool gel 70°INPM e o cloreto de benzalcônico foram igualmente mais efetivos em relação aos lenços embebidos em solução de clorexidina

Prevalence of yeasts in the oral cavity of children treated with inhaled corticosteroids.

The aim of this study was to observe the prevalence of Candida spp. in the oral cavity of children undergoing treatment with inhaled corticosteroids. Thirty children treated with inhaled corticosteroids and thirty control children were studied. Saliva samples were collected through oral rinses with phosphate buffered saline (PBS). The samples were plated on Sabouraud's dextrose agar and incubated at 37 degrees C for 48 h. After this period, the number of colony-forming units per ml (cfu/ml) of saliva was calculated. The isolates were identified by phenotypic characterization. Candida spp. was isolated from 43.33% of the samples of children treated with corticosteroids, with a mean of 780 cfu/ml of saliva, and from 30% of the samples of the control group, with a mean of 560 cfu/ml of saliva. No significant statistical difference was observed between the groups. C. albicans was the prevalent species in both groups, followed by C. guilliermondii, C. parapsilosis and C. stellatoidea. Furthermore, Rhodotorula rubra and C. lusitaniae were also isolated from the treated group. We concluded that there was no significant increase in the prevalence and number of Candida spp. in the oral cavity of children treated with inhaled corticosteroids.

Prevalence of yeasts in the oral cavity of children treated with inhaled corticosteroids

O objetivo deste trabalho foi verificar a prevalência de Candida spp. na cavidade bucal de crianças tratadas com corticosteróides inalados. Foram estudadas 30 crianças usuárias de corticosteróides inalados e 30 crianças controle. A coleta de saliva foi realizada através de enxágües bucais com solução fisiológica tamponada (PBS). As amostras foram semeadas em ágar Sabouraud dextrose e incubadas a 37ºC por 48 horas. A seguir, foi realizada a contagem de unidades formadoras de colônias por ml (ufc/ml) de saliva, e as cepas isoladas foram identificadas por caracterização fenotípica. Foram encontradas leveduras do gênero Candida em 43,33% dos pacientes usuários de corticosteróides, com média de 780 ufc/ml de saliva, e em 30% do grupo controle, com média de 560 ufc/ml de saliva, não ocorrendo diferença estatisticamente significante entre os grupos. C. albicans foi a espécie prevalente em ambos os grupos, seguida por C. guilliermondii, C. parapsilosis e C. stellatoidea. No grupo usuário de corticosteróide, verificou-se também a presença de Rhodotorula rubra e C. lusitaniae. Concluiu-se que não houve aumento significante na presença e na quantidade de leveduras do gênero Candida na cavidade bucal de crianças tratadas com corticosteróides inalados.

Estudo in vitro de dentifrícios comercialmente disponíveis no Brasil: efeito inibitório sobre Steptococcus mutans / In vitro study of commercially available toothpastes in Brazil: inhibitory effect on Streptococcus mutans

Introdução - A importância do controle do biofilme dentário na manutenção da saúde bucal e na prevenção da cárie e doença periodontal tem sido relatada na literatura por diversos autores. A escovação utilizando dentifrícios é o meio mais comumente usado no controle do biofilme dentário. O objetivo deste estudo foi verificar in vitro o efeito inibitório de dentifrícios comercialmente disponíveis no Brasil sobre cepas de Streptococcus mutans. Métodos - Foram obtidas diluições dos componentes solúveis de 10 diferentes dentifrícios. Placas contendo Brain Heart Infusion Ágar (Difco) e concentrações seqüenciais dos componentes solúveis dos dentifrícios foram preparadas. Vinte e quatro amostras de S. mutans, isoladas há menos de seis meses e uma amostra padrão ATCC 35688 foram inoculadas sobre a superficie do meio de cultura e após a incubação por 48 h a 37°C e 5 po cento CO2, a concentração inibitória mínima (MIC) de cada dentifrício foi determinada. Resultados - Os dentifrícios Colgate Gel, Prevent Anti-placa, Sorriso Herbal com Própolis e Colgate Tripla Ação, apresentaram MIC90 = 7,5mg/mL. Os dentifrícios Close-up Triple, Sorriso Juá e Própolis, Gessy Cristal Juá, Canela e Menta, Sorriso Dentes Brancos, Sorriso Fresh Crystal Mint apresentaram MIC90 = 15mg/mL, e Colgate Máxima Proteção Anticáries apresentou MIC90 = 30mg/mL. Conclusão - Conclui-se que os dentifrícios Colgate Gel, Prevent Anti-placa, Sorriso Herbal com Própolis e Colgate Tripla Ação apresentaram atividade inibitória mais efetiva sobre S. mutans em relação aos demais testados