Biofilmes multiespécies de Candida Albicans associados com streptococcus mitis e streptococcus sanguinis: estudo in vitro e in vivo

Pós-graduação em Odontologia Restauradora - ICT

Salivary IgA antibody responses to Streptococcus mitis and Streptococcus mutans in preterm and fullterm newborn children

Objectives: The intensities and specificities of salivary IgA antibody responses to antigens of Streptococcus mutans, the main pathogen of dental caries, may influence colonization by these organisms during the first 1.5 year of life. Thus, the ontogeny of salivary IgA responses to oral colonizers continues to warrant investigation, especially with regard to the influence of birth conditions, e.g. prematurity, on the ability of children to efficiently respond to oral microorganisms. In this study, we characterised the salivary antibody responses to two bacterial species which are prototypes of pioneer and pathogenic microorganisms of the oral cavity (Streptococcus mitis and Streptococcus mutans, respectively) in fullterm (FT) and preterm (PT) newborn children. Methods: Salivas from 123 infants (70 FT and 53 PT) were collected during the first 10 h after birth and levels of IgA and IgM antibodies and the presence of S. mutans and S. mitis were analysed respectively by ELISA and by chequerboard DNA-DNA hybridization. Two subgroups of 24 FT and 24 PT children were compared with respect to patterns of antibody specificities against S. mutans and S. mitis antigens, using Western blot assays. Cross-adsorption of 10 infant's saliva was tested to S. mitis, S. mutans and Enterococcus faecalis antigens. Results: Salivary levels of IgA at birth were 2.5-fold higher in FT than in PT children (Mann-Whitney; P < 0.05). Salivary IgA antibodies reactive with several antigens of S. mitis and S. mutans were detected at birth in children with undetectable levels of those bacteria. Adsorption of infant saliva with cells of S. mutans produced a reduction of antibodies recognizing S. mitis antigens in half of the neonates. The diversity and intensity of IgA responses were lower in PT compared to FT children, although those differences were not significant. Conclusion: These data provide evidence that children have salivary IgA antibodies shortly after birth, which might influence the establishment of the oral microbiota, and that the levels of salivary antibody might be related to prematurity. (C) 2011 Elsevier Ltd. All rights reserved.

A homologue of aliB is found in the capsule region of nonencapsulated Streptococcus pneumoniae

The epidemiology, phylogeny, and biology of nonencapsulated Streptococcus pneumoniae are largely unknown. Increased colonization capacity and transformability are, however, intriguing features of these pneumococci and play an important role. Twenty-seven nonencapsulated pneumococci were identified in a nationwide collection of 1,980 nasopharyngeal samples and 215 blood samples obtained between 1998 and 2002. On the basis of multilocus sequence typing and capsule region analysis we divided the nonencapsulated pneumococci into two groups. Group I was closely related to encapsulated strains. Group II had a clonal population structure, including two geographically widespread clones able to cause epidemic conjunctivitis and invasive diseases. Group II strains also carried a 1,959-bp homologue of aliB (aliB-like ORF 2) in the capsule region, which was highly homologous to a sequence in the capsule region of Streptococcus mitis. In addition, strains of the two major clones in group II had an additional sequence, aliB-like ORF 1 (1,968 to 2,004 bp), upstream of aliB-like ORF 2. Expression of aliB-like ORF 1 was detected by reverse transcription-PCR, and the corresponding RNA was visualized by Northern blotting. A gene fragment homologous to capN of serotypes 33 and 37 suggests that group II strains were derived from encapsulated pneumococci some time ago. Therefore, loss of capsule expression in vivo was found to be associated with the importation of one or two aliB homologues in some nonencapsulated pneumococci.

CXCL14 Displays Antimicrobial Activity against Respiratory Tract Bacteria and Contributes to Clearance of Streptococcus pneumoniae Pulmonary Infection

CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141-13. Interestingly, the central part of the molecule representing the β-sheet also maintained ∼62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia.

Homeologous recombination and mismatch repair during transformation in Streptococcus pneumoniae: saturation of the Hex mismatch repair system.

The ability of the Hex generalized mismatch repair system to prevent recombination between partially divergent (also called homeologous) sequences during transformation in Streptococcus pneumoniae was investigated. By using as donor in transformation cloned fragments 1.7-17.5% divergent in DNA sequence from the recipient, it was observed that the Hex system prevents chromosomal integration of the least and the most divergent fragments but frequently fails to do so for other fragments. In the latter case, the Hex system becomes saturated (inhibited) due to an excess of mismatches: it is unable to repair a single mismatch located elsewhere on the chromosome. Further investigation with chromosomal donor DNA, carrying only one genetically marked divergent region, revealed that a single divergent fragment can lead to saturation of the Hex system. Increase in cellular concentration of either HexA, the MutS homologue that binds mismatches, or HexB, the MutL homologue for which the essential role in repair as yet remains obscure, was shown to restore repair ability in previously saturating conditions. Investigation of heterospecific transformation by chromosomal DNA from two related streptococcal species, Streptococcus oralis and Streptococcus mitis, also revealed complete saturation of the Hex system. Therefore the Hex system is not a barrier to interspecies recombination in S. pneumoniae. These results are discussed in light of those described for the Mut system of Escherichia coli.

Impact of the Human Bacterial Environment on Mycobacteriosis and Allergy

My work describes two sectors of the human bacterial environment: 1. The sources of exposure to infectious non-tuberculous mycobacteria. 2. Bacteria in dust, reflecting the airborne bacterial exposure in environments protecting from or predisposing to allergic disorders. Non-tuberculous mycobacteria (NTM) transmit to humans and animals from the environment. Infection by NTM in Finland has increased during the past decade beyond that by Mycobacterium tuberculosis. Among the farm animals, porcine mycobacteriosis is the predominant NTM disease in Finland. Symptoms of mycobacteriosis are found in 0.34 % of slaughtered pigs. Soil and drinking water are suspected as sources for humans and bedding materials for pigs. To achieve quantitative data on the sources of human and porcine NTM exposure, methods for quantitation of environmental NTM are needed. We developed a quantitative real-time PCR method, utilizing primers targeted at the 16S rRNA gene of the genus of Mycobacterium. With this method, I found in Finnish sphagnum peat, sandy soils and mud high contents of mycobacterial DNA, 106 to 107 genome equivalents per gram. A similar result was obtained by a method based on the Mycobacterium-specific hybridization of 16S rRNA. Since rRNA is found mainly in live cells, this result shows that the DNA detected by qPCR mainly represented live mycobacteria. Next, I investigated the occurrence of environmental mycobacteria in the bedding materials obtained from 5 pig farms with high prevalence (>4 %) of mycobacteriosis. When I used for quantification the same qPCR methods as for the soils, I found that piggery samples contained non-mycobacterial DNA that was amplified in spite of several mismatches with the primers. I therefore improved the qPCR assay by designing Mycobacterium-specific detection probes. Using the probe qPCR assay, I found 105 to 107 genome equivalents of mycobacterial DNA in unused bedding materials and up to 1000 fold more in the bedding collected after use in the piggery. This result shows that there was a source of mycobacteria in the bedding materials purchased by the piggery and that mycobacteria increased in the bedding materials during use in the piggery. Allergic diseases have reached epidemic proportions in urbanized countries. At the same time, childhood in rural environment or simple living conditions appears to protect against allergic disorders. Exposure to immunoreactive microbial components in rural environments seems to prevent allergies. I searched for differences in the bacterial communities of two indoor dusts, an urban house dust shown to possess immunoreactivity of the TH2-type and a farm barn dust with TH1-activity. The immunoreactivities of the dusts were revealed by my collaborators, in vitro in human dendritic cells and in vivo in mouse. The dusts accumulated >10 years in the respiratory zone (>1.5 m above floor), thus reflecting the long-term content of airborne bacteria at the two sites. I investigated these dusts by cloning and sequencing of bacterial 16S rRNA genes from dust contained DNA. From the TH2-active urban house dust, I isolated 139 16S rRNA gene clones. The most prevalent genera among the clones were Corynebacterium (5 species, 34 clones), Streptococcus (8 species, 33 clones), Staphylococcus (5 species, 9 clones) and Finegoldia (1 species, 9 clones). Almost all of these species are known as colonizers of the human skin and oral cavity. Species of Corynebacterium and Streptococcus have been reported to contain anti-inflammatory lipoarabinomannans and immunmoreactive beta-glucans respectively. Streptococcus mitis, found in the urban house dust is known as an inducer of TH2 polarized immunity, characteristic of allergic disorders. I isolated 152 DNA clones from the TH1-active farm barn dust and found species quite different from those found from the urban house dust. Among others, I found DNA clones representing Bacillus licheniformis, Acinetobacter lwoffii and Lactobacillus each of which was recently reported to possess anti-allergy immunoreactivity. Moreover, the farm barn dust contained dramatically higher bacterial diversity than the urban house dust. Exposure to this dust thus stimulated the human dendritic cells by multiple microbial components. Such stimulation was reported to promote TH1 immunity. The biodiversity in dust may thus be connected to its immunoreactivity. Furthermore, the bacterial biomass in the farm barn dust consisted of live intact bacteria mainly. In the urban house dust only ~1 % of the biomass appeared as intact bacteria, as judged by microscoping. Fragmented microbes may possess bioactivity different from that of intact cells. This was recently shown for moulds. If this is also valid for bacteria, the different immunoreactivities of the two dusts may be explained by the intactness of dustborne bacteria. Based on these results, we offer three factors potentially contributing to the polarized immunoreactivities of the two dusts: (i) the species-composition, (ii) the biodiversity and (iii) the intactness of the dustborne bacterial biomass. The risk of childhood atopic diseases is 4-fold lower in the Russian compared with the Finnish Karelia. This difference across the country border is not explainable by different geo-climatic factors or genetic susceptibilities of the two populations. Instead, the explanation must be lifestyle-related. It has already been reported that the microbiological quality of drinking water differs on the two sides of the borders. In collaboration with allergists, I investigated dusts collected from homes in the Russian Karelia and in the Finnish Karelia. I found that bacterial 16S rRNA genes cloned from the Russian Karelian dusts (10 homes, 234 clones) predominantly represented Gram-positive taxa (the phyla Actinobacteria and Firmicutes, 67%). The Russian Karelian dusts contained nine-fold more of muramic acid (60 to 70 ng mg-1) than the Finnish Karelian dusts (3 to 11 ng mg-1). Among the DNA clones isolated from the Finnish side (n=231), Gram-negative taxa (40%) outnumbered the Gram-positives (34%). Out of the 465 DNA clones isolated from the Karelian dusts, 242 were assigned to cultured validly described bacterial species. In Russian Karelia, animal-associated species e.g. Staphylococcus and Macrococcus were numerous (27 clones, 14 unique species). This finding may connect to the difference in the prevalence of allergy, as childhood contacts with pets and farm animals have been connected with low allergy risk. Plant-associated bacteria and plant-borne 16S rRNA genes (chloroplast) were frequent among the DNA clones isolated from the Finnish Karelia, indicating components originating from plants. In conclusion, my work revealed three major differences between the bacterial communtites in the Russian and in the Finnish Karelian homes: (i) the high prevalence of Gram-positive bacteria on the Russian side and of Gram-negative bacteria on the Finnish side and (ii) the rich presence of animal-associated bacteria on the Russian side whereas (iii) plant-associated bacteria prevailed on the Finnish side. One or several of these factors may connect to the differences in the prevalence of allergy.

Äidinmaito - terveysjuomaa ja normaalibakteereita

The chemical composition of breast milk has been studied in detail in the past decades. Hundreds of new antibacterial and antiviral components have been found. Several molecules have been found to promote the proper function of neonatal intestine. However, microbiological studies of breast milk have been, until recently, focused mainly on detecting harmful and pathogenic bacteria and viruses. Natural microbial diversity of human milk has not been widely studied before the work reported in this thesis. This is mainly because breast milk has traditionally been thought to be sterile - even if a certain amount of commensal bacteria have usually been detected in milk samples. The first part of this licentiate thesis contains a short literature review about the anatomy and physiology of breast feeding, human milk chemical and microbiological composition, mastitis, intestinal flora and bacteriocins. The second part reports on the experiments of the licentiate work, concentrating on the microbial diversity in the milk of healthy breast-feeding mothers, and the ability of these bacteria to produce antibacterial substances against pathogenic bacteria. The results indicate that human milk is a source of commensal bacteria for infant intestine. 509 random isolates from 40 breast milk samples were isolated and identified by 16S rRNA sequencing. Median bacterial count was about 600 colony forming units per milliliter. Over half of the isolates were staphylococci, and almost one third streptococci. The most common species were skin bacteria Staphylococcus epidermidis and oral bacteria Streptococcus salivarius and Streptococcus mitis. Lactic acid bacteria, identified as members of Lactobacillus-, Lactococcus- and Leuconostoc -genera, were found in five milk samples. Enterococci were found in three samples. A novel finding in this study is the capability of these commensal bacteria to inhibit the growth of pathogens. In 90 precent of the milk samples commensal bacteria inhibiting the growth of Staphylococcus aureus were found. In 40 precent of samples the colonies could block the growth completely. One fifth of the isolated Staph. epidermidis strains, half of Str. salivarius strains, and all lactic acid bacteria and enterococci could inhibit or block the growth of Staph. aureus. In further study also Listeria innocua- and Micrococcus luteus active isolates were found in 33 and 11 precent of milk samples (out of 140). Furthermore, two Lactococcus lactis isolates from the breast milk were shown to produce bacteriocin nisin, which is an antimicrobial molecule used as a food preservative. The importance of these human milk commensal bacteria in the development of newborn intestinal flora and immune system, as well as in preventing maternal breast infections, should be further explored.

A new type of Brazilian propolis: Prenylated benzophenones in propolis from Amazon and effects against cariogenic bacteria

Ethanol extracts of four propolis samples (E1-E4) from Manaus (Brazilian Amazon) were analysed by HPLC/DAD/ESI-MS/MS and GC/EIMS. The major constituents of E2 and E4 were analysed by NMR ((1)H and (13)C) and ESI/MS/MS. The main constituents of E2 and E4 are polyprenylated benzophenones: 7-epi-nemorosone, 7-epi-clusianone (major E4 constituents), xanthochymol and gambogenone (major E2 constituents), making up a chemical profile so far unreported for Brazilian propolis. Aristhophenone, methyl insigninone, 18-ethyloxy-17-hydroxy-17,18-dihydroscrobiculatone B, and derivatives of dimethyl weddellianone A and B, propolones, and a scrobiculatone derivative, were detected as minor constituents. Triterpenoids (beta-amyrins, beta-amyrenone, lupeol and lupenone) were ubiquitous and predominant in El and E3. The extracts E2 and E4 were highly active against the cariogenic bacteria Streptococcus mitis, Streptococcus mutans and Streptococcus salivarius. E2 was more active than E4, probably due to a higher content of 2-epi-nemorosone, while the latter was richer in di-hydroxylated compounds. (C) 2010 Elsevier Ltd. All rights reserved.

Preparation and antimicrobial activity of gelatin microparticles containing propolis against oral pathogens

Gelatin microparticles containing propolis ethanolic extractive solution were prepared by spray-drying technique. Particle,, with regular morphology, mean diameter ranging of 2.27 mu m to 2.48 mu m, and good entrapment efficiency for propolis were obtained. The in vitro antimicrobial activity of microparticles was evaluated against microorganisms of oral importance (Enterococcus faecalis, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus mitis, Streptococcus mutans, Streptococcus sobrinus, Candida albicans, and Lactobacillus casei). The utilized techniques were diffusion in agar and determination of minimum inhibitory concentration. The choice of the method to evaluate the antimicrobial activity of microparticles showed be very important. The microparticles displayed activity against all tested strains of similar way to the propolis, showing greater activity against the strains of E. salivarius, S. sanguinis, S. mitis, and C albicans.

Detecção da microbiota endodôntica de dentes sem vitalidade pulpar com ou sem lesão periapical radiográfica, por meio das técnicas de checkerboard DNA-DNA hybridization e cultura microbiológica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Investigação das propriedades antimicrobianas de frações obtidas a partir dos extratos hidroalcoólico e etanólico de Croton doctoris S. Moore

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Investigação das propriedades anticariogênicas de óleos essenciais: atividade antimicrobiana e caracterização química

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação in vitro da atividade antimicrobiana de extratos vegetais sobre microrganismos relacionados à lesão de mucosite oral

A mucosite oral é a complicação oral mais freqüente nos pacientes sob quimioterapia e/ou radioterapia. Vários microrganismos podem estar presentes nesta lesão o que dificulta o seu tratamento. A propriedade antimicrobiana de plantas tem sido estudada com o intuito de confirmar cientificamente sua ação, e o possível potencial no controle de doenças infecciosas, principalmente devido ao aumento de microrganismos resistentes aos antimicrobianos conhecidos. O estudo teve por objetivo observar a ação inibidora de extratos das plantas Arrabidaea chica, Bryophyllum calycinum, Mansoa alliacea, Azadirachta indica, Senna alata, Vatairea guianensis, Vismia guianensis, Ananas erectifolius, Psidium guajava, Euterpe oleracea e Symphonia globulifera sobre cepas de microrganismos frequentemente envolvidos em lesões de mucosite oral, tais como, Streptococcus mitis (ATCC 903), Streptococcus sanguis (ATCC 10557), Streptococcus mutans (ATCC 25175), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), Candida albicans (ATCC 40175), Candida krusei (ATCC 40147) e Candida parapsilosis (ATCC 40038). A avaliação da atividade antimicrobiana e a determinação da Concentração Inibitória Mínima (CIM) foram realizadas através do método de disco-difusão em meio sólido. Os extratos brutos das plantas foram testados nas concentrações de 500, 250, 125, 62,50, 31,25 e 15,62 mg/ml utilizando como solvente o Dimetil-Sulfóxido (DMSO). Os extratos de anani e de pirarucu foram os que apresentaram maior espectro de ação, inibindo o crescimento de sete microrganismos dentre os oito testados. As menores CIM foram obtidas com os extratos de anani, lacre e mata pasto. O extrato de anani foi o mais ativo tendo demonstrado boa atividade antimicrobiana (CIM abaixo de 100 mg/mL) contra sete microrganismos (S. aureus, C. albicans, C. krusei, C. parapsilosis, S. mitis. S. sanguis e S. mutans, sendo inativo apenas para P. aeruginosa). O extrato de lacre demonstrou boa atividade frente a cinco microrganismos. Mata pasto teve boa atividade contra S. aureus, S. mitis e C. albicans. P. aeruginosa foi o microrganismo mais resistente sendo suscetível apenas para os extratos de pariri e pirarucu. Dentre os extratos avaliados, apenas o curauá não apresentou atividade sobre nenhum dos microrganismos testados. Os resultados obtidos demonstraram a capacidade antimicrobiana dos produtos vegetais testados. Entretanto, estudos futuros são necessários para esclarecer os seus mecanismos de ações e as possíveis interações com as drogas antimicrobianas, visando seu aproveitamento na terapêutica de doenças infecciosas.

Extratos de Pyrostegia venusta: caracterização físico-química, capacidade imunomoduladora e prevenção da formação do biofilme dental

A espécie Pyrostegia venusta é uma liana trepadeira popularmente conhecida como flor-de-São-João. É utilizada na medicina popular para o tratamento de diversas doenças. Na literatura científica, vários compostos bioativos já foram isolados e algumas atividades biológicas importantes, como a atividade antimicrobiana e imunomoduladora, já foram atribuídas a esta espécie. A cárie e a doença periodontalsão as doenças bucais mais prevalentes na população brasileira, sendo o biofilme dental o seu fator etiológico primário. A candidíase oral ocorre devido à infecção por espécies do gênero Candida que, em determinadas situações, podem se comportar como patógenos oportunistas. O objetivo deste trabalho foi realizar um estudo físico, químico e biológico da espécie vegetal Pyrostegia venusta e investigar seu potencial na prevenção das principais doenças bucais. Foram realizados ensaios de citotoxicidade do extrato bruto e frações em células mononucleares do sangue periférico e em macrófagos murinos. Para avaliar a atividade imunomoduladora do extrato bruto e frações, foi realizada dosagem de óxido nítrico (NO) produzido por macrófagos estimulados com lipopolissacarídeo (LPS). Foi realizado ensaio de microdiluição em caldo para determinar a concentração inibitória mínima (CIM), concentração bactericida mínima (CBM) e concentração fungicida mínima (CFM) do extrato bruto, frações e marcadores. Também foram avaliados o efeito na inibição da aderência de Streptococcus mutans em lamínula de vidro, o efeito na redução do pH e a capacidade de inibição do brotamento de Candida albicans do extrato bruto e frações. A espécie Pyrostegia venusta não foi citotóxica em baixas concentrações. A produção de NO foi inibida pelas frações acetato de etila e n-butanólica. O extrato bruto, frações e marcadores apresentaram atividade antimicrobiana contra Streptococcus mutans, Streptococcus mitis, Streptococcus oralis e Candida albicans, capacidade de inibição da aderência e redução do pH. O extrato bruto e frações hexânica, acetato de etila e n-butanólica apresentaram capacidade de inibição do brotamento. A espécie Pyrostegia venusta pode ser uma alternativa na prevenção e tratamento das principais doenças bucais. No entanto, mais estudos são necessários antes de considerá-la realmente promissora.

Systematic Screening of Plant Extracts from the Brazilian Pantanal with Antimicrobial Activity against Bacteria with Cariogenic Relevance

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)