Marlise Klein | Universidade Estadual Paulista "Júlio de Mesquita Filho" (original) (raw)

Papers by Marlise Klein

Research paper thumbnail of pH-Activated Nanoparticles for Controlled Topical Delivery of Farnesol To Disrupt Oral Biofilm Virulence

ACS nano, Jan 13, 2015

Development of effective therapies to control oral biofilms is challenging, as topically introduc... more Development of effective therapies to control oral biofilms is challenging, as topically introduced agents must avoid rapid clearance from biofilm-tooth interfaces while targeting biofilm microenvironments. Additionally, exopolysaccharides-matrix and acidification of biofilm microenvironments are associated with cariogenic (caries-producing) biofilm virulence. Thus, nanoparticle carriers capable of binding to hydroxyapatite (HA), saliva-coated HA (sHA), and exopolysaccharides with enhanced drug release at acidic pH were developed. Nanoparticles are formed from diblock copolymers composed of 2-(dimethylamino)ethyl methacrylate (DMAEMA), butyl methacrylate (BMA), and 2-propylacrylic acid (PAA) (p(DMAEMA)-b-p(DMAEMA-co-BMA-co-PAA)) that self-assemble into ∼21 nm cationic nanoparticles. Nanoparticles exhibit outstanding adsorption affinities (∼244 L-mmol(-1)) to negatively charged HA, sHA, and exopolysaccharide-coated sHA due to strong electrostatic interactions via multivalent tertiary...

Research paper thumbnail of Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms

Frontiers in Cellular and Infection Microbiology, 2015

Biofilms are highly structured microbial communities that are enmeshed in a self-produced extrace... more Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS), eDNA, and lipoteichoic acid (LTA). EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan) synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases.

Research paper thumbnail of Genetic diversity of competence gene loci in clinical genotypes of Streptococcus mutans

Journal of clinical microbiology, 2006

The frequencies of 21 competence genes were analyzed in 94 genotypes of Streptococcus mutans. The... more The frequencies of 21 competence genes were analyzed in 94 genotypes of Streptococcus mutans. These include those of a main regulatory system (comCDE), structural, and other regulatory orthologues identified in the genome of strain UA159. PCR and Southern blot analysis revealed that all genes are widespread within the species.

Research paper thumbnail of Elevated incidence of dental caries in a mouse model of cystic fibrosis

PLoS ONE, 2011

Dental caries is the single most prevalent and costly infectious disease worldwide, affecting mor... more Dental caries is the single most prevalent and costly infectious disease worldwide, affecting more than 90% of the population in the U.S. The development of dental cavities requires the colonization of the tooth surface by acid-producing bacteria, such as Streptococcus mutans. Saliva bicarbonate constitutes the main buffering system which neutralizes the pH fall generated by the plaque bacteria during sugar metabolism. We found that the saliva pH is severely decreased in a mouse model of cystic fibrosis disease (CF). Given the close relationship between pH and caries development, we hypothesized that caries incidence might be elevated in the mouse CF model. We induced carious lesions in CF and wildtype mice by infecting their oral cavity with S. mutans, a well-studied cariogenic bacterium. After infection, the mice were fed a high-sucrose diet for 5 weeks (diet 2000). The mice were then euthanized and their jaws removed for caries scoring and bacterial counting. A dramatic increase in caries and severity of lesions scores were apparent in CF mice compared to their wildtype littermates. The elevated incidence of carious lesions correlated with a striking increase in the S. mutans viable population in dental plaque (20-fold increase in CF vs. wildtype mice; p value < 0.003; t test). We also found that the pilocarpine-stimulated saliva bicarbonate concentration was significantly reduced in CF mice (16 ± 2 mM vs. 31 ± 2 mM, CF and wildtype mice, respectively; p value < 0.01; t test). Considering that bicarbonate is the most important pH buffering system in saliva, and the adherence and survival of aciduric bacteria such as S. mutans are enhanced at low pH values, we speculate that the decrease in the bicarbonate content and pH buffering of the saliva is at least partially responsible for the increased severity of lesions observed in the CF mouse.

Research paper thumbnail of The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm

PLoS Pathogens, 2012

Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilm... more Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPSenmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (,5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPSmicrocolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidicmicroenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenicbiofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.

Research paper thumbnail of Influences of starch and sucrose on Streptococcus mutans biofilms

Oral Microbiology and Immunology, 2008

Research paper thumbnail of Transmission, diversity and virulence factors of Streptococcus mutans genotypes

Journal of Oral Science, 2005

Dental caries is an infectious and transmissible disease, in which many genetic, environmental an... more Dental caries is an infectious and transmissible disease, in which many genetic, environmental and behavioral risk factors interact. The mutans streptococci (MS), mainly Streptococcus mutans and Streptococcus sobrinus are the microorganisms most strongly associated with this disease. The main virulence factors associated with MS cariogenicity include adhesion, acidogenicity and acid tolerance. These properties work together to modify the physico-chemical properties of the biofilm, resulting in ecological changes in the form of increased proportions of S. mutans and other acidogenic and aciduric species. In addition, reports of higher numbers of S. mutans genotypes with increased virulence in caries-active subjects suggest the importance of microenvironmental factors in increasing the risk of caries. This review focuses on the transmission and establishment of different genotypes of S. mutans and the role they play in the development of dental caries. (J. Oral Sci. 47, 59-64, 2005)

Research paper thumbnail of Longitudinal Study of Transmission, Diversity, and Stability of Streptococcus mutans and Streptococcus sobrinus Genotypes in Brazilian Nursery Children

Journal of Clinical Microbiology, 2004

The aim of this study was to perform a follow-up evaluation of the Streptococcus mutans and Strep... more The aim of this study was to perform a follow-up evaluation of the Streptococcus mutans and Streptococcus sobrinus colonization profile of children's oral cavities, which included the pattern of vertical transmission from mother to child, genotypic diversity, and stability of the strains. The subjects were 16 mother-child pairs, who were monitored for 20 months. Samples of saliva, tongue dorsum, alveolar ridge mucosa, and dental plaque from the children were collected bimonthly. Saliva samples from the mothers were also collected. After isolation and identification, the arbitrarily primed PCR method was performed for the genotypic characterization of S. mutans (968 isolates) and S. sobrinus (111 isolates). At the time the strains were acquired, the children harbored one to four distinct genotypes of S. mutans and only one genotype of S. sobrinus. Although S. mutans prevalence and genotypic diversity were greater than those of S. sobrinus, the presence of matching genotypes of S. mutans and S. sobrinus was similar (in 81.25 and 83.33% of mother-child pairs, respectively), suggesting vertical transmission for both species. This longitudinal study showed an increase in genotypic diversity of S. mutans in the oral cavity during the follow-up period: most of the initially acquired genotypes persisted, normally those genotypes transmitted by the mother, and some were lost during follow-up; new strains were also acquired. In conclusion, S. mutans and S. sobrinus genotypes acquired from maternal or alternative sources may show effective persistence in the oral cavity and/or transitory detection in the children's mouths, reflecting the continuous development of oral microbiota in children.

Research paper thumbnail of Influences of trans-trans farnesol, a membrane-targeting sesquiterpenoid, on Streptococcus mutans physiology and survival within mixed-species oral biofilms

International journal of oral science, 2011

Trans-trans farnesol (tt-farnesol) is a bioactive sesquiterpene alcohol commonly found in propoli... more Trans-trans farnesol (tt-farnesol) is a bioactive sesquiterpene alcohol commonly found in propolis (a beehive product) and citrus fruits, which disrupts the ability of Streptococcus mutans (S. mutans) to form virulent biofilms. In this study, we investigated whether tt-farnesol affects cell-membrane function, acid production and/or acid tolerance by planktonic cells and biofilms of S. mutans UA159. Furthermore, the influence of the agent on S. mutans gene expression and ability to form biofilms in the presence of other oral bacteria (Streptococcus oralis (S. oralis) 35037 and Actinomyces naeslundii (A. naeslundii) 12104) was also examined. In general, tt-farnesol (1 mmol x L(-1)) significantly increased the membrane proton permeability and reduced glycolytic activity of S. mutans in the planktonic state and in biofilms (P < 0.05). Moreover, topical applications of 1 mmol x L(-1) tt-farnesol twice daily (1 min exposure/treatment) reduced biomass accumulation and prevented ecologic...

Research paper thumbnail of Effect of Starch and Sucrose on Dental Biofilm Formation and on Root Dentine Demineralization

Caries Research, 2008

than in those formed in the presence of sucrose or sucrose/ starch combinations; however, no sign... more than in those formed in the presence of sucrose or sucrose/ starch combinations; however, no significant difference was observed among the groups containing sucrose (p 1 0.05). RNA was successfully isolated and purified from in situ biofilms and only biofilms formed in response to sucrose and starch/sucrose combinations showed detectable levels of gtf B and gtf C mRNA. The findings suggest that the combination of starch with sucrose may not be more cariogenic to dentine than sucrose alone.

Research paper thumbnail of Biotyping and genotypic diversity among oral Candida albicans strains from caries-free and caries-active healthy children

Brazilian Journal of Microbiology, 2006

Candida albicans oral strains collected from caries-free and caries-active healthy children rangi... more Candida albicans oral strains collected from caries-free and caries-active healthy children ranging from 24 to 36 months old, were studied. The aim of the study was to determine proteinase and phospholipase activities produced by Candida albicans in the two groups and to determine the phenotypic diversity of these enzymes based on genetic polymorphism using the AP-PCR method. Strains identified by morphological and fermentation tests as C. albicans were grown in proteinase and phospholipase agar media at 37ºC for 7 and 4 days, respectively. After the incubation period, the enzyme activity of the proteinase and phospholipase positive strains was measured. All strains were subjected to AP-PCR, using the arbitrary primer AP-3. The enzymatic analysis showed no differences between the two groups. The AP-PCR method was effective in demonstrating intra-individual genetic polymorphism in C. albicans, showing a greater clonal diversity in caries-active versus caries-free children. Dendograms of similarity showed only intra-individual clonal lineage. The results suggest that the enzymatic profile does not depend on the genotypic characteristics of the strains.

Research paper thumbnail of Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms

BMC Microbiology, 2009

The association of specific bioactive flavonoids and terpenoids with fluoride can modulate the de... more The association of specific bioactive flavonoids and terpenoids with fluoride can modulate the development of cariogenic biofilms by simultaneously affecting the synthesis of exopolysaccharides (EPS) and acid production by Streptococcus mutans, which enhanced the cariostatic effectiveness of fluoride in vivo. In the present study, we further investigated whether the biological actions of combinations of myricetin (flavonoid), tt-farnesol (terpenoid) and fluoride can influence the expression of specific genes of S. mutans within biofilms and their structural organization using real-time PCR and confocal fluorescence microscopy.

Research paper thumbnail of The specific degree-of-polymerization of A-type proanthocyanidin oligomers impacts Streptococcus mutans glucan-mediated adhesion and transcriptome responses within biofilms

Biofouling, 2013

Cranberry A-type proanthocyanidins (PACs) have been recognized for their inhibitory activity agai... more Cranberry A-type proanthocyanidins (PACs) have been recognized for their inhibitory activity against bacterial adhesion and biofilm-derived infections. However, the precise identification of the specific classes of degree-of-polymerization (DP) conferring PACs bioactivity remains a major challenge owing to the complex chemistry of these flavonoids. In this study, chemically characterized cranberries were used in a multistep separation and structure-determination technique to isolate A-type PAC oligomers of defined DP. The influences of PACs on the 3D architecture of biofilms and Streptococcus mutans-transcriptome responses within biofilms were investigated. Treatment regimens that simulated topical exposures experienced clinically (twice-daily, 60 s each) were used over a saliva-coated hydroxyapatite biofilm model. Biofilm accumulation was impaired, while specific genes involved in the adhesion of bacteria, acid stress tolerance, and glycolysis were affected by the topical treatments (vs the vehicle-control). Genes (rmpC, mepA, sdcBB, and gbpC) associated with sucrose-dependent binding of bacteria were repressed by PACs. PACs of DP 4 and particularly DP 8 to 13 were the most effective in disrupting bacterial adhesion to glucan-coated apatitic surface (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;85% inhibition vs vehicle control), and gene expression (eg rmpC). This study identified putative molecular targets of A-type cranberry PACs in S. mutans while demonstrating that PAC oligomers with a specific DP may be effective in disrupting the assembly of cariogenic biofilms.

Research paper thumbnail of Genetic diversity and exoenzyme activities of Candida albicans and Candida dubliniensis isolated from the oral cavity of Brazilian periodontal patients

Archives of Oral Biology, 2008

J.F. Hö fling). a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o... more J.F. Hö fling). a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . i n t l . e l s e v i e r h e a l t h . c o m / j o u r n a l s / a r o b 0003-9969/$ -see front matter #

Research paper thumbnail of Structural and Molecular Basis of the Role of Starch and Sucrose in Streptococcus mutans Biofilm Development

Applied and Environmental Microbiology, 2009

The interaction of sucrose and starch with bacterial glucosyltransferases and human salivary amyl... more The interaction of sucrose and starch with bacterial glucosyltransferases and human salivary amylase may enhance the pathogenic potential of Streptococcus mutans within biofilms by influencing the structural organization of the extracellular matrix and modulating the expression of genes involved in exopolysaccharide synthesis and specific sugar transport and two-component systems.

Research paper thumbnail of Periodontal and microbiologic evaluation of 2 methods of archwire ligation: Ligature wires and elastomeric rings

American Journal of Orthodontics and Dentofacial Orthopedics, 2008

Introduction: Prophylactic programs to prevent dental biofilm accumulation must be implemented to... more Introduction: Prophylactic programs to prevent dental biofilm accumulation must be implemented to minimize the risk for periodontal diseases in orthodontic patients. Therefore, we assessed the possible periodontal and microbiologic changes resulting from the use of 2 methods of orthodontic archwire ligation: elastomeric rings and steel ligatures. Methods: The following parameters were measured: plaque index, gingival bleeding index, probing depth, and biofilm samples from the maxillary second premolars and the mandibular lateral incisors were evaluated in 14 subjects without clinical signs of gingival inflammation before orthodontic appliance placement and after 6 months of treatment. Each orthodontic arch was fixed with elastomeric rings on 1 side of the midline, and steel ligatures were used on the opposite side. Polymerase chain reaction analysis was used to detect Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and P nigrescens. Results: The elastomeric rings were associated with a higher score for plaque index and bleeding than steel ligatures, as well as many positive sites of T forsythia and P nigrescens (P Ͻ0.05). Conclusions: Elastomeric rings favored these 2 periodontopathogens and harmed gingival conditions. (Am J Orthod Dentofacial Orthop 2008;134:506-12)

Research paper thumbnail of Increase in Probing Depth Is Correlated With a Higher Number of Prevotella intermedia Genotypes

Journal of Periodontology, 2006

Background: The aims of this study were to determine the genotypic diversity of Prevotella interm... more Background: The aims of this study were to determine the genotypic diversity of Prevotella intermedia in subgingival plaque samples by using two techniques, arbitrarily primed polymerase chain reaction (AP-PCR) and heteroduplex analysis, and to assess the relationship of this diversity with increase in probing depth.

Research paper thumbnail of pH-Activated Nanoparticles for Controlled Topical Delivery of Farnesol To Disrupt Oral Biofilm Virulence

ACS nano, Jan 13, 2015

Development of effective therapies to control oral biofilms is challenging, as topically introduc... more Development of effective therapies to control oral biofilms is challenging, as topically introduced agents must avoid rapid clearance from biofilm-tooth interfaces while targeting biofilm microenvironments. Additionally, exopolysaccharides-matrix and acidification of biofilm microenvironments are associated with cariogenic (caries-producing) biofilm virulence. Thus, nanoparticle carriers capable of binding to hydroxyapatite (HA), saliva-coated HA (sHA), and exopolysaccharides with enhanced drug release at acidic pH were developed. Nanoparticles are formed from diblock copolymers composed of 2-(dimethylamino)ethyl methacrylate (DMAEMA), butyl methacrylate (BMA), and 2-propylacrylic acid (PAA) (p(DMAEMA)-b-p(DMAEMA-co-BMA-co-PAA)) that self-assemble into ∼21 nm cationic nanoparticles. Nanoparticles exhibit outstanding adsorption affinities (∼244 L-mmol(-1)) to negatively charged HA, sHA, and exopolysaccharide-coated sHA due to strong electrostatic interactions via multivalent tertiary...

Research paper thumbnail of Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms

Frontiers in Cellular and Infection Microbiology, 2015

Biofilms are highly structured microbial communities that are enmeshed in a self-produced extrace... more Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS), eDNA, and lipoteichoic acid (LTA). EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan) synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases.

Research paper thumbnail of Genetic diversity of competence gene loci in clinical genotypes of Streptococcus mutans

Journal of clinical microbiology, 2006

The frequencies of 21 competence genes were analyzed in 94 genotypes of Streptococcus mutans. The... more The frequencies of 21 competence genes were analyzed in 94 genotypes of Streptococcus mutans. These include those of a main regulatory system (comCDE), structural, and other regulatory orthologues identified in the genome of strain UA159. PCR and Southern blot analysis revealed that all genes are widespread within the species.

Research paper thumbnail of Elevated incidence of dental caries in a mouse model of cystic fibrosis

PLoS ONE, 2011

Dental caries is the single most prevalent and costly infectious disease worldwide, affecting mor... more Dental caries is the single most prevalent and costly infectious disease worldwide, affecting more than 90% of the population in the U.S. The development of dental cavities requires the colonization of the tooth surface by acid-producing bacteria, such as Streptococcus mutans. Saliva bicarbonate constitutes the main buffering system which neutralizes the pH fall generated by the plaque bacteria during sugar metabolism. We found that the saliva pH is severely decreased in a mouse model of cystic fibrosis disease (CF). Given the close relationship between pH and caries development, we hypothesized that caries incidence might be elevated in the mouse CF model. We induced carious lesions in CF and wildtype mice by infecting their oral cavity with S. mutans, a well-studied cariogenic bacterium. After infection, the mice were fed a high-sucrose diet for 5 weeks (diet 2000). The mice were then euthanized and their jaws removed for caries scoring and bacterial counting. A dramatic increase in caries and severity of lesions scores were apparent in CF mice compared to their wildtype littermates. The elevated incidence of carious lesions correlated with a striking increase in the S. mutans viable population in dental plaque (20-fold increase in CF vs. wildtype mice; p value &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.003; t test). We also found that the pilocarpine-stimulated saliva bicarbonate concentration was significantly reduced in CF mice (16 ± 2 mM vs. 31 ± 2 mM, CF and wildtype mice, respectively; p value &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01; t test). Considering that bicarbonate is the most important pH buffering system in saliva, and the adherence and survival of aciduric bacteria such as S. mutans are enhanced at low pH values, we speculate that the decrease in the bicarbonate content and pH buffering of the saliva is at least partially responsible for the increased severity of lesions observed in the CF mouse.

Research paper thumbnail of The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm

PLoS Pathogens, 2012

Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilm... more Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPSenmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (,5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPSmicrocolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidicmicroenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenicbiofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.

Research paper thumbnail of Influences of starch and sucrose on Streptococcus mutans biofilms

Oral Microbiology and Immunology, 2008

Research paper thumbnail of Transmission, diversity and virulence factors of Streptococcus mutans genotypes

Journal of Oral Science, 2005

Dental caries is an infectious and transmissible disease, in which many genetic, environmental an... more Dental caries is an infectious and transmissible disease, in which many genetic, environmental and behavioral risk factors interact. The mutans streptococci (MS), mainly Streptococcus mutans and Streptococcus sobrinus are the microorganisms most strongly associated with this disease. The main virulence factors associated with MS cariogenicity include adhesion, acidogenicity and acid tolerance. These properties work together to modify the physico-chemical properties of the biofilm, resulting in ecological changes in the form of increased proportions of S. mutans and other acidogenic and aciduric species. In addition, reports of higher numbers of S. mutans genotypes with increased virulence in caries-active subjects suggest the importance of microenvironmental factors in increasing the risk of caries. This review focuses on the transmission and establishment of different genotypes of S. mutans and the role they play in the development of dental caries. (J. Oral Sci. 47, 59-64, 2005)

Research paper thumbnail of Longitudinal Study of Transmission, Diversity, and Stability of Streptococcus mutans and Streptococcus sobrinus Genotypes in Brazilian Nursery Children

Journal of Clinical Microbiology, 2004

The aim of this study was to perform a follow-up evaluation of the Streptococcus mutans and Strep... more The aim of this study was to perform a follow-up evaluation of the Streptococcus mutans and Streptococcus sobrinus colonization profile of children's oral cavities, which included the pattern of vertical transmission from mother to child, genotypic diversity, and stability of the strains. The subjects were 16 mother-child pairs, who were monitored for 20 months. Samples of saliva, tongue dorsum, alveolar ridge mucosa, and dental plaque from the children were collected bimonthly. Saliva samples from the mothers were also collected. After isolation and identification, the arbitrarily primed PCR method was performed for the genotypic characterization of S. mutans (968 isolates) and S. sobrinus (111 isolates). At the time the strains were acquired, the children harbored one to four distinct genotypes of S. mutans and only one genotype of S. sobrinus. Although S. mutans prevalence and genotypic diversity were greater than those of S. sobrinus, the presence of matching genotypes of S. mutans and S. sobrinus was similar (in 81.25 and 83.33% of mother-child pairs, respectively), suggesting vertical transmission for both species. This longitudinal study showed an increase in genotypic diversity of S. mutans in the oral cavity during the follow-up period: most of the initially acquired genotypes persisted, normally those genotypes transmitted by the mother, and some were lost during follow-up; new strains were also acquired. In conclusion, S. mutans and S. sobrinus genotypes acquired from maternal or alternative sources may show effective persistence in the oral cavity and/or transitory detection in the children's mouths, reflecting the continuous development of oral microbiota in children.

Research paper thumbnail of Influences of trans-trans farnesol, a membrane-targeting sesquiterpenoid, on Streptococcus mutans physiology and survival within mixed-species oral biofilms

International journal of oral science, 2011

Trans-trans farnesol (tt-farnesol) is a bioactive sesquiterpene alcohol commonly found in propoli... more Trans-trans farnesol (tt-farnesol) is a bioactive sesquiterpene alcohol commonly found in propolis (a beehive product) and citrus fruits, which disrupts the ability of Streptococcus mutans (S. mutans) to form virulent biofilms. In this study, we investigated whether tt-farnesol affects cell-membrane function, acid production and/or acid tolerance by planktonic cells and biofilms of S. mutans UA159. Furthermore, the influence of the agent on S. mutans gene expression and ability to form biofilms in the presence of other oral bacteria (Streptococcus oralis (S. oralis) 35037 and Actinomyces naeslundii (A. naeslundii) 12104) was also examined. In general, tt-farnesol (1 mmol x L(-1)) significantly increased the membrane proton permeability and reduced glycolytic activity of S. mutans in the planktonic state and in biofilms (P < 0.05). Moreover, topical applications of 1 mmol x L(-1) tt-farnesol twice daily (1 min exposure/treatment) reduced biomass accumulation and prevented ecologic...

Research paper thumbnail of Effect of Starch and Sucrose on Dental Biofilm Formation and on Root Dentine Demineralization

Caries Research, 2008

than in those formed in the presence of sucrose or sucrose/ starch combinations; however, no sign... more than in those formed in the presence of sucrose or sucrose/ starch combinations; however, no significant difference was observed among the groups containing sucrose (p 1 0.05). RNA was successfully isolated and purified from in situ biofilms and only biofilms formed in response to sucrose and starch/sucrose combinations showed detectable levels of gtf B and gtf C mRNA. The findings suggest that the combination of starch with sucrose may not be more cariogenic to dentine than sucrose alone.

Research paper thumbnail of Biotyping and genotypic diversity among oral Candida albicans strains from caries-free and caries-active healthy children

Brazilian Journal of Microbiology, 2006

Candida albicans oral strains collected from caries-free and caries-active healthy children rangi... more Candida albicans oral strains collected from caries-free and caries-active healthy children ranging from 24 to 36 months old, were studied. The aim of the study was to determine proteinase and phospholipase activities produced by Candida albicans in the two groups and to determine the phenotypic diversity of these enzymes based on genetic polymorphism using the AP-PCR method. Strains identified by morphological and fermentation tests as C. albicans were grown in proteinase and phospholipase agar media at 37ºC for 7 and 4 days, respectively. After the incubation period, the enzyme activity of the proteinase and phospholipase positive strains was measured. All strains were subjected to AP-PCR, using the arbitrary primer AP-3. The enzymatic analysis showed no differences between the two groups. The AP-PCR method was effective in demonstrating intra-individual genetic polymorphism in C. albicans, showing a greater clonal diversity in caries-active versus caries-free children. Dendograms of similarity showed only intra-individual clonal lineage. The results suggest that the enzymatic profile does not depend on the genotypic characteristics of the strains.

Research paper thumbnail of Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms

BMC Microbiology, 2009

The association of specific bioactive flavonoids and terpenoids with fluoride can modulate the de... more The association of specific bioactive flavonoids and terpenoids with fluoride can modulate the development of cariogenic biofilms by simultaneously affecting the synthesis of exopolysaccharides (EPS) and acid production by Streptococcus mutans, which enhanced the cariostatic effectiveness of fluoride in vivo. In the present study, we further investigated whether the biological actions of combinations of myricetin (flavonoid), tt-farnesol (terpenoid) and fluoride can influence the expression of specific genes of S. mutans within biofilms and their structural organization using real-time PCR and confocal fluorescence microscopy.

Research paper thumbnail of The specific degree-of-polymerization of A-type proanthocyanidin oligomers impacts Streptococcus mutans glucan-mediated adhesion and transcriptome responses within biofilms

Biofouling, 2013

Cranberry A-type proanthocyanidins (PACs) have been recognized for their inhibitory activity agai... more Cranberry A-type proanthocyanidins (PACs) have been recognized for their inhibitory activity against bacterial adhesion and biofilm-derived infections. However, the precise identification of the specific classes of degree-of-polymerization (DP) conferring PACs bioactivity remains a major challenge owing to the complex chemistry of these flavonoids. In this study, chemically characterized cranberries were used in a multistep separation and structure-determination technique to isolate A-type PAC oligomers of defined DP. The influences of PACs on the 3D architecture of biofilms and Streptococcus mutans-transcriptome responses within biofilms were investigated. Treatment regimens that simulated topical exposures experienced clinically (twice-daily, 60 s each) were used over a saliva-coated hydroxyapatite biofilm model. Biofilm accumulation was impaired, while specific genes involved in the adhesion of bacteria, acid stress tolerance, and glycolysis were affected by the topical treatments (vs the vehicle-control). Genes (rmpC, mepA, sdcBB, and gbpC) associated with sucrose-dependent binding of bacteria were repressed by PACs. PACs of DP 4 and particularly DP 8 to 13 were the most effective in disrupting bacterial adhesion to glucan-coated apatitic surface (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;85% inhibition vs vehicle control), and gene expression (eg rmpC). This study identified putative molecular targets of A-type cranberry PACs in S. mutans while demonstrating that PAC oligomers with a specific DP may be effective in disrupting the assembly of cariogenic biofilms.

Research paper thumbnail of Genetic diversity and exoenzyme activities of Candida albicans and Candida dubliniensis isolated from the oral cavity of Brazilian periodontal patients

Archives of Oral Biology, 2008

J.F. Hö fling). a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o... more J.F. Hö fling). a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . i n t l . e l s e v i e r h e a l t h . c o m / j o u r n a l s / a r o b 0003-9969/$ -see front matter #

Research paper thumbnail of Structural and Molecular Basis of the Role of Starch and Sucrose in Streptococcus mutans Biofilm Development

Applied and Environmental Microbiology, 2009

The interaction of sucrose and starch with bacterial glucosyltransferases and human salivary amyl... more The interaction of sucrose and starch with bacterial glucosyltransferases and human salivary amylase may enhance the pathogenic potential of Streptococcus mutans within biofilms by influencing the structural organization of the extracellular matrix and modulating the expression of genes involved in exopolysaccharide synthesis and specific sugar transport and two-component systems.

Research paper thumbnail of Periodontal and microbiologic evaluation of 2 methods of archwire ligation: Ligature wires and elastomeric rings

American Journal of Orthodontics and Dentofacial Orthopedics, 2008

Introduction: Prophylactic programs to prevent dental biofilm accumulation must be implemented to... more Introduction: Prophylactic programs to prevent dental biofilm accumulation must be implemented to minimize the risk for periodontal diseases in orthodontic patients. Therefore, we assessed the possible periodontal and microbiologic changes resulting from the use of 2 methods of orthodontic archwire ligation: elastomeric rings and steel ligatures. Methods: The following parameters were measured: plaque index, gingival bleeding index, probing depth, and biofilm samples from the maxillary second premolars and the mandibular lateral incisors were evaluated in 14 subjects without clinical signs of gingival inflammation before orthodontic appliance placement and after 6 months of treatment. Each orthodontic arch was fixed with elastomeric rings on 1 side of the midline, and steel ligatures were used on the opposite side. Polymerase chain reaction analysis was used to detect Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and P nigrescens. Results: The elastomeric rings were associated with a higher score for plaque index and bleeding than steel ligatures, as well as many positive sites of T forsythia and P nigrescens (P Ͻ0.05). Conclusions: Elastomeric rings favored these 2 periodontopathogens and harmed gingival conditions. (Am J Orthod Dentofacial Orthop 2008;134:506-12)

Research paper thumbnail of Increase in Probing Depth Is Correlated With a Higher Number of Prevotella intermedia Genotypes

Journal of Periodontology, 2006

Background: The aims of this study were to determine the genotypic diversity of Prevotella interm... more Background: The aims of this study were to determine the genotypic diversity of Prevotella intermedia in subgingival plaque samples by using two techniques, arbitrarily primed polymerase chain reaction (AP-PCR) and heteroduplex analysis, and to assess the relationship of this diversity with increase in probing depth.