Dissertations / Theses on the topic 'Prevotella intermedia'

Prevotella intermedia e Prevotella nigrescens são espécies comumente associadas Porphyromonas gingivalis. os objetivos foram verificar a co-agregação entre cepas de P. intermedia, P. nigrescens e P. gingivalis; quantificar a biomassa, avaliar a proporção dos microrganismos nos biofilmes mistos; verificar a interferência do co-cultivo pela técnica de dois compartimentos, avaliar os biofilmes em ensaios de Hibridização In Situ (FISH) e verificar o papel dos genes PINA0102 e PIN0398 de P. intermedia no biofilme. Assim, 9 isolados clínicos de P. intermedia e 5 de P. nigrescens, as cepas padrão P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277, os mutantes Pi17D0398 e Pi17D0102 foram avaliados em variados consórcios. Os resultados mostraram que as interações investigadas são cepa específicas.
Prevotella intermedia and Prevotella nigrescens are commonly associated with periodontal diseases. The goals of this study were to determine the co-aggregation of strains of P. intermedia, P. nigrescens and P. gingivalis; to quantify the biomass of these associations, to evaluate the ratio of these microorganisms in heterotypic biofilms, to verify the modulation of biofilms in co-culture using a trans-well system; to evaluate the structure of the biofilms by Fluorescent Hybridization In Situ (FISH) and to determine the role of genes PINA0102 and PIN0398 of P. intermedia in the modulation of its biofilm. Therefore, 9 clinical isolates of P. intermedia, 5 of P. nigrescens, type strains P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277 and mutant strains Pi17D0398 and Pi17D0102 were grown in consortia of two strains. Our data demonstrate that the associations of P. intermedia, P. nigrescens and P. gingivalis are strain-specific.

The present study evaluates the susceptibility to antibiotics of three anaerobic bacteria; Prevotella intermedia, Prevotella nigrescens and Parvimonas micra, bacterial species commonly found in root canal infections. Strains from two different time periods were tested against benzylpenicillin, amoxicillin and metronidazole. There are two collections of each bacterial species in this study. One of the collections is from 1976. Other collection includes the same bacterial species collected in recent years, (2009 to 2014). To identify all three bacteria species, the PCR (Polymerase Chain Reaction) method with species specific primers was used. To identify the minimal inhibitory concentrations (MIC) of selected antibiotics Etest strips were used according to Eucasts recommendations. The MIC values differ for P. micra, P. intermedia and P. nigrescens when exposed to the selected antibiotics. The new collection of P. micra showed higher MIC-values for amoxicillin and metronidazole than the old collection. The recent collection of P. intermedia showed higher MIC values for metronidazole compared to the old collection. On the other hand P. nigrescens showed no difference in MIC values between the two collections, although one recently isolated strain of P. nigrescens showed noticeable difference in MIC-values for benzylpenicillin and amoxicillin antibiotics compared to the old collection. The results indicate a susceptibility change for the tested antibiotics. Antibiotics susceptibility has changed during a 40 -year period for the selected bacterial species and it also shows that different strains of bacteria, although belonging to the same species, can have different susceptibility to antibiotics.

Orientador: Reginaldo Bruno Gonçalves
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O objetivo deste trabalho foi avaliar a diversidade genética da espécie Prevotella intermedia em indivíduos com doença periodontal, por meio de duas técnicas, a reação em cadeia da polimerase com primers arbitrários (AP-PCR) e análise heteroduplex de produtos amplificados pela reação em cadeia da polimerase (PCR). Amostras subgengivais foram obtidas de sítios com e sem destruição periodontal de 36 voluntários. Após o cultivo e identificação bacteriana, os isolados de P. intermedia foram submetidos à técnica de genotipagem utilizando AP-PCR. Adicionalmente, as amostras clínicas subgengivais, com resultado positivo para o cultivo de P. intermedia, foram submetidas à reação em cadeia da polimerase (PCR) com primer espécie específico e em seguida, os produtos amplificados foram submetidos à análise heteroduplex. Os resultados revelaram que os anaeróbios pigmentados negros foram mais freqüentes em sítios doentes, sendo a P. intermedia a mais prevalente dentre as espécies isoladas. A maioria dos indivíduos foi colonizada por um a dois tipos de genótipos, com relação positiva entre o número de clones e a profundidade de sondagem. Não houve detecção de genótipos idênticos entre os sítios de isolamento, em um mesmo ambiente bucal. Na análise interindivíduos, nenhum voluntário compartilhou o mesmo perfil eletroforético, indicando ampla heterogeneidade genética da espécie. Coincidindo com a técnica de AP -PCR, a análise heteroduplex detectou a presença de uma a dois clones distintos de P. intermedia nas amostras clínicas. Em conclusão, os indivíduos com doença periodontal abrigaram poucos tipos clonais e a coincidência de perfil genético entre sítios de um mesmo indivíduo e entre indivíduos não foi observada
Abstract: The aim of this study was to assess the genotypic diversity of P. intermedia on subjects with periodontal disease using two techniques, the arbitrarily primed polymerase chain reaction (AP-PCR) method and heteroduplex analysis from products amplified by polymerase chain reaction (PCR). Subgingival plaque samples were taken from the sites with and without periodontal destruction in 36 volunteers. After the culturing and identification procedures, the AP-PCR method was used for the genotypic characterization of P. intermedia strains. In addition, the clinical samples with a positive result for culture of the P. intermedia were amplified by polymerase chain reaction (PCR) method with a specie-specific primer and, after that, the PCR products were compared by the heteroduplex analysis. The results indicated that black-pigmented anaerobes were frequently cultured from periodontal diseased sites and P. intermedia were the most prevalent among the species recovered. Most of the subjects harbored between one to two distinct genotypes of P. intermedia, with a positive relation between numbers of genotypes and pocket probing depth. No matching of P. intermedia genotypes was observed between periodontal sites of the same individual. Interindividual genetic diversity has demonstrated absence of identical clones, indicating a high level of genetic diversity. The heteroduplex analyses of PCR products revealed that the majority of clinical samples harbored one to two clonal types, just like the AP-PCR technique. In conclusion, the subjects with periodontal disease harbored few clonal types and no identical genotypes between sites in the same individual and between individuals were found
Doutorado
Microbiologia e Imunologia
Doutor em Biologia Buco-Dental

P. gingivalis é um dos principais patógenos da doença periodontal, é encontrado em biofilmes orais com S. gordonii e P. intermedia e em células endoteliais da artéria coronária in vivo. P. gingivalis necessita de ferro em seu metabolismo e pode usar certas proteínas do hospedeiro como fontes deste íon em ambientes limitantes. Assim, este estudo investigou o papel dos genes PGN0741/PG0637 (receptor dependente de TonB) e PGN0531/PG1380 (fvW) de P. gingivalis na formação de biofilme em diferentes concentrações de ferro, em biofilmes mistos com S. gordonii e P. intermedia, e na adesão e invasão de células endoteliais da artéria coronária. Os resultados mostraram divergências no papel dos genes TonB e fvW na formação dos monobiofilmes e mistos e em diferentes concentrações de ferro, demonstrando uma relação cepa-dependente. Na adesão, fvW se mostrou importante para ambas cepas, mas na persistência apenas para P. gingivalis W83. Este trabalho enfatiza, assim, a necessidade do uso de mais de uma cepa de P. gingivalis no estudo do papel de genes em ensaios experimentais.
P. gingivalis is one of the major pathogens of periodontal diseases. It is found in oral biofilms associated with S. gordonii and P. intermedia, and inside of coronary artery endothelial cells in vivo. P. gingivalis requires iron for growth and can exploit iron-carrying proteins of the host as sources in limiting environments. Thus, this work aimed to study the role of genes PGN0741/PG0637 (TonB-dependent receptor) and PGN0531/PG1380 (fvW) of P. gingivalis in biofilm formation under different iron concentrations, in mixed biofilms with S. gordonii and P. intermedia, and in the adhesion and invasion of coronary artery endothelial cells. Our data showed discordance for the role of TonB and fvW in homo- and heterotypic biofilm formation and in different iron concentrations. The relevance of both genes was strain-dependent. Gene fvW was relevant for adhesion to endothelial cells, but only for strain W83 during persistence. Therefore, our study emphasizes the importance of using different strains for a better understanding of the role of genes in experimental assays.

Periodontitis describes a set of related inflammatory diseases that damage the periodontal tissues that support the teeth. It is caused by bacterial infections that, if left untreated, can lead to progressive loss of alveolar bone and subsequent loosening and loss of teeth. Periodontitis is the primary cause of tooth loss in adults and treatment of more severe forms of the disease can be both painful and costly. Black-pigmenting bacterial species, such as Porphyromonas gingivalis and Prevotella intermedia, are among the major periodontal pathogens associated with severe periodontitis. These bacteria form black pigments consisting of iron protoporphyrin IX (haem; Fe(III)PPIX) when grown on blood-containing media. Development of the haem- containing black pigments by these organisms serves an important defensive role as the intrinsic catalase activity of ferrihaem destroys H202 (Smalley et al., 2000). Furthermore, formation of the pigment by strictly anaerobic P. gingivalis is a process which consumes oxygen and promotes anaerobiosis (Smalley et al., 1998; 2002). This study sought to further characterise some of the facets of the mechanisms of haem acquisition by these two bacteria, with particular emphasis on the role of specific virulence factors (InpA of P. intermedia, HmuY and the gingipains of P. gingivalis, and LPS from both species). This resulted in identification of Interpain A (InpA), a 90 kDa cysteine protease, and homologue of Streptococcus pyogenes streptopain (SpeB), which is expressed by P. intermedia as a potential major virulence factor in haem acquisition through its ability to degrade haemoglobin, haemalbumin and haern-haemopexin. InpA-mediated proteolysis of these haemoproteins was highly dependent on experimental factors such as pH and redox potential, which profoundly affected the protease sensitivity of the haem -containing protein substrates. This suggested that InpA activity would be hugely influenced by the changing conditions of the periodontal pocket during progressive periodontitis. A unique syntrophic relationship of haem acquisition from haemoglobin was also identified between the HmuY haemophore and the K- and R- gingipains of P. gingivalis. Furthermore, InpA facilitated greater haem extraction by HmuY by promoting haemoglobin oxidation to the methaemoglobin form. This suggested a degree of mutualism and possible cross-reactivity between the haem acqui sition systems of these two bacteria which has possibly arisen on account of their close proximity within the periodontal pocket biofilm. This study also identified a possible central role for LPS in binding and storage of haem at the cell surface of these black-pigmenting bacteria and presents evidence suggesting that the lipophilic nature of this molecule, in addition to the respective pericellular pHs of P intermedia and P gingivalis cells, may affect the species of haem present in the pigments.

Orientador: Rafael Nobrega Stipp
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O biofilme é uma população biológica com um elevado grau de organização, onde os microrganismos presentes formam uma comunidade estruturada, coordenada e funcional. O estudo do comportamento dos biofilmes é fundamental para melhorar as formas de controle, especialmente durante infecções, tais como as doenças periodontais. No primeiro capítulo, foram avaliados os efeitos da aplicação tópica e frequente do digluconato de clorexidina (CLX) em diferentes concentrações na periodontite induzida por ligadura nos primeiros molares de ratos. As ligaduras receberam 10 µl de soluções de CLX à 0,2%, 2%, 10%, 20% ou diluente, de quatro em quatro dias, em um total de quatro aplicações. Após eutanásia, a quantidade de células bacterianas no biofilme formado sobre a ligadura foi estimada por cultura e por PCR quantitativo. A reabsorção óssea foi mensurada em altura e área por fotografia digital e em volume por microtomógrafo. Depois de quatro dias a partir da última aplicação da CLX, as reduções bacterianas mantidas pelos tratamentos com CLX atingiram até 10-6. O grupo que recebeu CLX a 20% teve, em média, logs bacterianos 3,3× menor (p<0.01, Kruskal-Wallis). Não houve diferença estatística entre os grupos em relação à reabsorção óssea por ambos os métodos testados (p>0.05, Kruskal-Wallis), embora 55% dos sítios apresentaram menor reabsorção óssea. No segundo capítulo, foi avaliada a influência de diversas substâncias na formação de biofilme por Prevotella intermedia. Os biofilmes foram formados em placas de 48 poços contendo tratamento de superfície prévio com DNA purificado, hemina, CaCO3, Ca(OH)2, CaCl2, soro, albumina, dextrana, metionina, glicose, glutamina, KCl, complexo vitamínico, cistina ou mucina. O biofilme formado foi corado e quantificado por espectrofotometria. A arquitetura do biofilme foi visualizada por microscopia confocal de fluorescência por varredura laser. O tratamento da superfície com CaCl2 a 1 mg/cm2 permitiu a formação do biofilme em quantidade de 0,3 OD490nm (p<0.01, ANOVA Dunnet), sendo esse valor 10× superior quando a superfície foi tratada com 2,5 mg/cm2 (p<0.01, ANOVA Dunnet). As demais substâncias tiveram pouco ou nenhum impacto sobre a formação do biofilme. A visualização por microscopia confocal revelou uma comunidade estruturada e com vitalidade em toda sua espessura. Conclusões: os dados indicam que a CLX concentrada diminui a carga bacteriana na região da periodontite induzida, que reflete em menor reabsorção óssea apenas em parte das amostras. O pré-revestimento da superfície de crescimento com cálcio promove a formação de biofilme por P. intermedia
Abstract: Biofilms are biological communities with a high degree of organization, in which micro-organisms form structured, coordinated and functional communities. These biological communities are embedded in a self-created extracellular matrix. Biofilm is also associated with a high level of antimicrobial tolerance of the associated organisms. Understanding biofilm behavior is crucial to develop ways for its control during infections, such as periodontal disease. In the first chapter, topical and frequent application of various concentrations of chlorhexidine digluconate (CLX) where evaluated. Periodontitis were induced by ligature on first molars. Then, ligatures were treated with 10 µl of chlorhexidine solutions at 0.2%, 2%, 10%, 20% or diluent, every four days in a total of four applications periods. After euthanasia, bacterial loads on ligatures were estimated by both culture and qPCR. Bone resorption height and area were measured by digital photography and its volume by microtomography. Treated sites had bacterial reductions up to 10-6 cells. Treatment with 20% CLX showed mean of 3.3× lower bacterial levels (p<0.01, Kruskal-Wallis). There was no statistical difference between groups regarding bone resorption (p>0.05, Kruskal-Wallis), although 55% of the treated sites had some lower bone resorption. In the second chapter, substances that may enhance biofilm formation by Prevotella intermedia were investigated. Wells of 48-well plates were coated with DNA, hemin, CaCO3, Ca(OH)2, CaCl2, serum, albumin, dextran, methionine, glucose, glutamine, KCl, vitamin complex, cystine or mucin. Biofilms were grown for 24 h, washed, stained and quantified by spectrophotometry. Biofilm architecture and its viability were visualized by Confocal Laser Scanning Microscopy. Surfaces treated with 1 mg/cm2 of CaCl2 enhanced biofilm amount by 0.3 OD490nm (p<0.01, ANOVA Dunnet), while 2.5 mg/cm2 yielded 10-fold more biofilm mass (p<0.01, ANOVA Dunnet). Other substances had modest or no impact in biofilm mass. Confocal microscopy images showed structured and alive biofilms with no dead areas. Conclusions: concentrated CLX reduces bacterial load, which reflects in lower bone resorption in few sites. Surfaces pre-coated with calcium chloride enhance P. intermedia biofilm formation
Doutorado
Microbiologia e Imunologia
Doutor em Biologia Buco-Dental

Objectives: This investigation adapted the LIVE/DEAD� Baclight[TM] bacterial viability stain for the quantitative determination of bacterial cell viability of the aerotolerant anaerobes Porphyromonas gingivalis ATCC 33277 and Prevotella intermedia ATCC 25611. The Live/Dead stain was used to determine the influence of haem availability on the resistance of P. gingivalis and P. intermedia to the reactive oxygen species (ROS) superoxide anion and hydrogen peroxide and compare the sensitivities between the haem-requiring periodontal bacteria to ROS. Neutrophils use oxidative and non-oxidative killing mechanisms. During phagocytosis, neutrophils kill bacteria via a respiratory burst, producing ROS. P. gingivalis and P. intermedia are oxygen-tolerant gram-negative bacteria found in the gingival crevice. These bacteria express superoxide dismutase (SOD) activity, which extends some protection against superoxide radicals. Methods: Initially, experiments were performed to validate the reliability and accuracy of the fluorogenic Live/Dead stain using Escherichia coli ATCC 10798 (K-12), followed by experiments using P. gingivalis. The Live/Dead stain distinguishes viable:non-viable proportions of bacteria using mixtures of green (SYTO 9) and red (propidium iodide) fluorescent nucleic acid stains respectively. Bacterial cell viability was assessed with fluorescence microscopy and subsequently quantitative measurement using a fluorescence microplate reader (BMG Fluorostar plus Optima). P. gingivalis and P. intermedia colonies were subcultured from frozen cultures, in Tryptic soy broth (TSB) (Difco) and incubated anaerobically for approximately five days. They were further subcultured in pre-reduced TSB, supplemented with menadione 0.5[mu]g/ml (TSB-M) and either 5 [mu]g/ml haemin (Haem 5), 50 [mu]g/ml haemin (Haem 50) or without supplemental haemin (Haem 0). Cultures were grown anaerobically at 37�C to early stationary phase (approximately 48 hours). For experimental purposes, bacteria were harvested, washed and resuspended in 10 mM Tris-buffered saline (pH 7.5) containing peptone (TBS-P) (0.1 mg/ml), with a final adjustment to OD₅₄₀ [approximately equals] 2.0 (which corresponds to 1 x 10⁹ bacteria/ml). Bacterial suspensions were diluted ([approximately equals] 10⁸/ml) into TBS-P containing the fluorogenic viability stain (BacLight, Molecular Probes). Either pyrogallol (0.02 - 2 mM) or hydrogen peroxide (0.01 - 100 mM) was added (except to control tubes); tubes were vortexed for ten seconds and incubated at 37�C. Viability was monitored fluorimetrically for three hours. Results: For both P. gingivalis and P. intermedia, a pyrogallol concentration of 0.2 mM resulted in 80 to 90% cell death; and a hydrogen peroxide concentration of 10 mM killed approximately 80 to 90% of cells. Irrespective of the haem status, no significant difference was determined between the overall maximum rate of killing of P. gingivalis and P. intermedia, in their response to either superoxide or hydrogen peroxide; with the exception that the P. intermedia Haem 0 group was significantly less susceptible to hydrogen peroxide than the P. gingivalis Haem 0 group. For the majority of the experiments, there was no significant difference between final bacterial cell viability in the Haem 0 and Haem 5 cells for both species, after 3 hours exposure to various concentrations of ROS. However, the Haem 50 cells showed a significant increased susceptibility (albeit, a small difference) to both hydrogen peroxide and superoxide. Conclusions: The Live/Dead bacterial viability stain provided a valuable method to monitor "real-time" killing, avoiding the difficulties associated with culture-based methods for assessing viability. Haem availability had no clear influence on the resistance to ROS of either P. gingivalis or P. intermedia Haem 0 and Haem 5 cells. The Haem 50 cells showed a very slight increase in susceptibility to hydrogen peroxide and superoxide. Although P. intermedia may be isolated in significant numbers from healthy gingivae, as well as from periodontally diseased sites, it was no more resistant to ROS than was P. gingivalis, which is associated with periodontal lesions and difficult to cultivate from relatively healthy (more oxygenated) sites. This suggests that resistance to ROS does not contribute to the ecological distinction between these two species. The finding that haem availability did not influence sensitivity implies that these bacteria do not accumulate haem for the purpose of protection from ROS.

Lors d'infections de l'endodonte des bacteries vont coloniser les canaux et adherer aux parois canalaires. Afin de mieux apprehender ce phenomene, et ainsi pouvoir tester de nouvelles therapeutiques, un modele d'etude original a ete developpe. Des bacteries sont mises en contact avec des blocs de racine d'incisives de bovin sur lesquels des cavites de faible profondeur sont taillees au depend de la dentine canalaire. Trois souches ont ete retenues: streptococcus sanguis (nctc 7863) et prevotella intermedia (nctc 93336 et sauvage). Apres trois heures d'incubation dans un milieu liquide a une concentration de 10. 9 cellules/ml, la surface canalaire est observee en microscopie electronique a balayage et les cellules y adherant sont comptabilisees. Deux series d'experimentations ont permis d'evaluer la fiabilite de ce modele: variations du ph du milieu (960 observations), a ph6, 7 et 8 il n'y a pas de difference concernant l'adherence de s. Sanguis. Traitement de l'etat de surface dentinaire (6060 observations) par des solutions d'hypochlorite de sodium a 6,25% et d'acide critique a 6% utilisees seules ou de facon combinee pendant 2 a 10 minutes. : 1) pour p. Intermedia, la souche sauvage a un potentiel de colonisation superieur a la souche de collection (f = 35,82) ; 2) c'est quand les parois canalaires sont tapissees de smear layer que les bacteries adherent le plus ; 3) l'adherence des cellules est independante de la nature de la dentine ; 4) c'est avec les chelateurs (acide citrique) que les meilleurs resultats sont obtenus. Ce modele permet donc de tester les solutions d'irrigation dans des conditions proches de la realite clinique.

Oral Biology
M.S.
Objectives: Prevotella intermedia and Prevotella nigrescens are two genetically distinct, gram-negative, anaerobic rods associated with the subgingival microbiome of human periodontitis. The two species are frequently isolated from subgingival dental plaque biofilms in chronic periodontitis patients clinically experiencing progressive destructive disease activity. In anaerobically-incubated liquid or solid culture media, P. intermedia and P. nigrescens exhibit nearly identical phenotypic properties, with regard to their colony morphology features and biochemical properties, which differ from other subgingival Prevotella and non-Prevotella microbial species. As a result, rapid differentiation and identification of P. intermedia/nigrescens group organisms from other bacterial species in anaerobically-cultivated subgingival dental plaque biofilms has been based upon examination of culture isolates for a dark-pigmented colony appearance, presence of brick-red autofluorescence of colonies to long-wave ultraviolet light exposure, and biochemical testing demonstrating a lack of colony lactose fermentation. However, the accuracy of this phenotypic-based identification scheme for periodontal P. intermedia/nigrescens group species has yet to be validated with a broad-based reference method that encompasses testing for a wide array of microbial species. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and associated analytic software, is recently approved for clinical microbiology diagnostic use in the United States by the Food and Drug Administration, and is capable of definitively identifying 4,613 different microbial species based on mass spectra of their bacterial proteins. To date, no performance evaluation has been carried out comparing the phenotypic-based identification scheme for periodontal P. intermedia/ nigrescens group species with definitive MALDI-TOF mass spectrometry identification of the organisms. As a result, the purpose of this study was to assess with MALDI-TOF mass spectrometry the accuracy of the rapid phenotypic-based periodontal P. intermedia/ nigrescens group species identification scheme widely utilized since 1986 by clinical periodontal microbiology laboratories and periodontal microbiology culture-based research studies. Methods: 84 fresh subgingival cultivable isolates from 23 chronic periodontitis patients were presumptively identified on anaerobically-incubated enriched Brucella blood agar primary isolation plates as P. intermedia/nigrescens group species based on their dark-pigmented colony morphology, presence of brick-red autofluorescence under long-wave ultraviolet light, and a negative MUG fluorescence test for lactose fermentation activity. Each of the putative P. intermedia/nigrescens clinical isolates were subjected to MALDI-TOF mass spectrometry analysis using a bench top mass spectrometer, Bruker FlexControl 3.0 software, and MALDI Biotyper 3.1 software (Bruker Daltonics, Billerica, MA, USA), which contains mass spectra for P. intermedia and P. nigrescens in its reference library of bacterial protein profiles. A MALDI Biotyper log score of equal to or larger than 1.7 was required for reliable taxonomic classification of the clinical isolates, with scores of equal to or larger than 2.0 representing more definitive species identification. Results: A total of 60 (71.4%) of the putative P. intermedia/nigrescens clinical isolates were reliably identified with MALDI-TOF mass spectrometry as either P. intermedia (25 isolates, with eight isolates exhibiting MALDI Biotyper log scores of equal to or larger than 2.0), or P. nigrescens (35 isolates, with nine isolates exhibiting MALDI Biotyper log scores of equal to or larger than 2.0). Among the 24 putative P. intermedia/nigrescens clinical isolates generating MALDI Biotyper log scores < 1.7, indicating a less reliable species identification, only P. intermedia (14 isolates) or P. nigrescens (10 isolates) were listed by the analytic software as the first choice among the most likely bacterial species. No other bacterial species other than P. intermedia or P. nigrescens were identified by MALDI-TOF mass spectrometry for any of the 84 tested putative P. intermedia/ nigrescens clinical isolates. Conclusions: These findings document, for the first time with MALDI-TOF mass spectrometry, the relative accuracy of a rapid phenotypic-based periodontal P. intermedia/nigrescens identification scheme based on dark-pigmented colony morphology, presence of brick-red long-wave ultraviolet light autofluorescence, and a negative MUG test for lactose fermentation activity. 100% of the 84 presumptive P. intermedia/nigrescens clinical isolates tested were identified with MALDI-TOF mass spectrometry, with varying levels of reliability, as being only either P. intermedia or P. nigrescens. These findings provide validation for the continued use of this rapid phenotypic identification scheme for periodontal P. intermedia/nigrescens.
Temple University--Theses

La salud gingival de la gestante sigue siendo un tema de estudio para los especialistas. Esta salud se relaciona muy íntimamente con la presencia de la placa bacteriana que la gestante pueda tener. Pero también existe el aumento de una bacteria en el surco gingival de la gestante, la Prevotella intermedia, que influye en el desarrollo de alteraciones gingivales en la gestante. Estudios desde el año 1981 (Jensen) y particularmente Tsai CC y Chen KS (China) en 1995, que realizan UN ESTUDIO DE HORMONAS SEXUALES EN EL FLUIDO CREVICULAR GINGIVAL Y LAS BACTERIAS PIGMENTADAS DE NEGRO EN LA PLACA SUBGINGIVAL DE MUJERES EMBARAZADAS, llegan a concluir que la Prevotella intermedia utiliza las hormonas esteroideas en la gestación para verse incrementada en su crecimiento. Esta relación ya se estableció y es reconocida. Sin embargo, el presente estudio busca encontrar otra relación: La higiene bucal y la presencia de la Prevotella intermedia. El trabajo se llevó a cabo en el Hospital Nacional Docente Madre-Niño San Bartolomé, se tomaron muestras del fluido crevicular del surco gingival de 138 mujeres gestantes de diferentes trimestres de gestación atendidas en el Departamento de Odontoestomatología. Se registraron datos clínicos en una ficha elaborada ad-hoc. Las muestras se tomaron con conos de papel estériles y se inocularon en tubos con un caldo estéril de tioglicolato, como medio de transporte. Los tubos se trasladaron a la Unidad de Microbiología del Servicio de Patología Clínica del Centro Médico Naval “Cirujano Mayor Santiago Tavara”, donde se procesaron con protocolos de aislamiento para bacterias anaerobias. Los datos se procesaron con tablas dinámicas para el cruce de variables, y se aplicaron pruebas estadísticas “t” y “z” para proporciones y hallar la independencia de los grupos; así como, la prueba del chi cuadrado para asociar los grupos o categorías y establecer la significancia. Se encontró que existe una relación entre las denominadas higiene bucal buena, regular y mala según el índice de placa de Greene y vermillión, y la presencia de la bacteria Prevotella intermedia: Mientras exista una higiene bucal BUENA, habrá predominantemente un crecimiento ESCASO (en el 80% de las gestantes) de esta bacteria en el surco gingival. Pero ante una higiene bucal REGULAR O MALA, predominará un crecimiento bacteriano MODERADO. Además, el crecimiento bacteriano de la Prevotella intermedia ante una higiene bucal BUENA en las gestantes, puede llegar a lo mucho en un porcentaje no mayor al 20% a un nivel de MODERADO, no así a niveles abundantes. Sin embargo, en casos de higiene regulares o malas el crecimiento si llega a niveles ABUNDANTE (que es en menor porcentaje en la higiene regular). De este modo, se puede concluir que a mejor control de la cantidad de placa bacteriana en las gestantes, menor crecimiento y presencia de la Prevotella intermedia se encontrará, y por ende, una mayor condición de salud gingival.
Tesis

Oral Biology
M.S.
Objectives: Systemic antibiotics are generally recognized as providing a beneficial impact in treatment of both aggressive and chronic periodontitis. Since strains of periodontal pathogens among periodontitis patients may vary in their antibiotic drug resistance, the American Academy of Periodontology recommends antimicrobial susceptibility testing of suspected periodontal pathogens prior to administration of systemic periodontal antibiotic therapy, to reduce the risk of a treatment failure due to pathogen antibiotic resistance. E-test and MIC Test Strip assays are two in vitro antimicrobial susceptibility testing systems employing plastic- and paper-based, respectively, carriers loaded with predefined antibiotic gradients covering 15 two-fold dilutions. To date, no performance evaluations have been carried out comparing the Etest and MIC Test Strip assays in their ability to assess the in vitro antimicrobial susceptibility of periodontal bacterial pathogens. As a result, the purpose of this study was to compare the in vitro performance of E-test and MIC Test Strip assays in assessing minimal inhibitory concentration (MIC) values of four antibiotics frequently utilized in systemic periodontal antibiotic therapy against 11 fresh clinical subgingival isolates of the putative periodontal pathogen, Prevotella intermedia/ nigrescens, and to compare the distribution of P. intermedia/ nigrescens strains identified with interpretative criteria as "susceptible" and "resistant" to each of the four antibiotics using MIC values determined by the two antimicrobial susceptibility testing methods. Methods: Standardized cell suspensions, equivalent to a 2.0 McFarland turbidity standard, were prepared with 11 fresh clinical isolates of P. intermedia/nigrescens, each recovered from the subgingival microbiota of United States chronic periodontitis subjects, and plated onto to the surfaces of culture plates containing enriched Brucella blood agar. After drying, pairs of antibiotic-impregnated, quantitative, gradient diffusion strips from two manufacturers (E-test, bioMérieux, Durham, NC, USA, and MIC Test Strip, Liofilchem s.r.l., Roseto degli Abruzzi, Italy) for amoxicillin, clindamycin, metronidazole, and doxycycline were each placed apart from each other onto the inoculated enriched Brucella blood agar surfaces, so that an antibiotic test strip from each manufacturer was employed per plate against each P. intermedia/ nigrescens clinical isolate for antibiotic susceptibility testing. After 48-72 hours anaerobic jar incubation, individual MIC values for each antibiotic test strip against P. intermedia/nigrescens were read in μg/ml at the point where the edge of the bacterial inhibition ellipse intersected with the antibiotic test strip. MIC50, MIC90, and MIC range were calculated and compared for each of the test antibiotics, with essential agreement (EA) values determined per test antibiotic for the level of outcome agreement between two antimicrobial susceptibility testing methods. In addition, the identification of antibiotic "susceptible" and "resistant" strains among the P. intermedia/nigrescens clinical isolates was determined for each test antibiotic using MIC interpretative criteria from the MIC interpretative standards developed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for gram-negative anaerobic bacteria for amoxicillin, clindamycin, and metronidazole findings, and from the French Society of Microbiology breakpoint values for anaerobic disk diffusion testing for doxycycline data. Results: For amoxicillin, higher MIC50 and MIC90 values against the P. intermedia/ nigrescens strains were found with the MIC Test Strip assay than with E-test strips, resulting in a relatively low EA value of 45.5% between the two susceptibility testing methods. A higher percentage of amoxicillin "resistant" P. intermedia/nigrescens strains (72.7%) were identified by MIC Test Strips as compared to E-test strips (54.5%), although both methods found the same proportion of amoxicillin "susceptible" strains (27.3%). For clindamycin, both susceptibility testing methods provided identical MIC values (EA value = 100%), and exactly the same distributions of "susceptible" and "resistant" strains of P. intermedia/nigrescens. For metronidazole, only very poor agreement (EA value = 9.1%) was found between the two susceptibility testing methods, with MIC Test Strips exhibiting markedly higher MIC50 and MIC90 values against P. intermedia/nigrescens as compared to E-test strips. However, the distribution of "susceptible" and "resistant" P. intermedia/ nigrescens were identical between the two susceptibility testing methods. For doxycycline, relatively good agreement (EA value = 72.7%) was found in MIC concentrations between the two susceptibility testing methods, although generally lower MIC values were associated with MIC Test Strips. In addition, identical distributions of "susceptible" and "resistant" P. intermedia/nigrescens were provided by both susceptibility testing methods. Conclusions: Relative to MIC values measured against periodontal strains of P. intermedia/nigrescens, MIC Test Strips gave higher MIC values with amoxicillin and metronidazole, equal MIC values with clindamycin, and lower MIC values with doxycycline, as compared to MIC values measured with the E-test assay. Relative to the identification of antibiotic "susceptible" periodontal P. intermedia/ nigrescens strains, both susceptibility testing methods provided identical findings, suggesting that both methods appear to be interchangeable for clinical decision making in regard to identification of antibiotic-sensitive strains of periodontal P. intermedia/nigrescens. However, for epidemiologic surveillance of drug susceptibility trends, where exact MIC values are important to track over time, the relatively higher proportion of non-exact MIC differences between the two susceptibility testing methods argues against using them interchangeably. Instead, one or the other method should be used consistently for such studies. Further comparative studies of the E-test and MIC Test Strip assays are indicated using other periodontopathic bacterial species besides P. intermedia/ nigrescens, and to assess the reproducibility of MIC values provided by both in vitro susceptibility testing methods over time.
Temple University--Theses

L'empirisme lors de la prescription antibiotique en parodontologie persiste toujours. Dans la 1ère partie, nous avons procédé à une revue de la littérature concernant les bases fondamentales de l'antibiothérapie curative et préventive. La notion de variabilité de la sensibilité bactérienne a été introduite. Dans la 2è partie, nous avons étudié, à partir de 60 prélèvements de flore sous gingivale effectués chez 20 patients atteints de parodontites agressives, la variabilité de la sensibilité de 82 souches bactériennes. Dix antibiotiques ont été testés (2 méthodes d'antibiogramme). De fortes variabilités inter individuelles et intra individuelles ont été observées spécialement avec les antibiotiques dits de 1ère génération (pénicilline G, tétracycline et érythromycine) et la ciprofloxacine. P. Intermedia montra le coefficient de variation le plus élevé. La réalisation de plusieurs antibiogrammes à partir de CFU distinctes de P. Intermedia est recommandée dans les parodontites agressives
The frequent use of antibiotics in developed countries has led to the emergence of a wide-spread bacterial resistance. The first part of our thesis reviewed all the modalities of prescribing antibiotics in periodontology. In the second part, experimental, the inter-individual and intra-individual variabilities of the antibiotic susceptibility of 82 putative micro-organisms in aggressive periodontitis (20 patients, 60 samples) has been evaluated by means of VC (variation coefficient). Inter-individual antibiotics susceptibility of periopathogens is not homogenous and seems to vary according to bacterial species and antibiotic molecules. Intra-individual variability is heterogeneous too and very random. This inter- and intra-individual variability seems to be fairly more important with old molecules (PEN G, TET, ERY) than observed with more recent ones (AMC, AMX, AMP and DOX). P. Intermedia appeared to be the less sensitive bacteria and the one that showed the highest VC

A cavidade oral é um dos locais do organismo humano que revela a mais complexa e heterogénea população microbiana, da qual fazem parte mais de meio milhar de espécies bacterianas. A doença periodontal (DP) é uma patologia oral de origem bacteriana caracterizada por um estado inflamatório que começa por afetar o tecido gengival e cuja progressão leva frequentemente à perda de dentes na idade adulta. As gengivites e periodontites são das patologias periodontais que surgem com maior predomínio na rotina da clinica dentária. A identificação do microbioma responsável por este tipo de patologias é extremamente importante na adoção de medidas mais eficazes, quer de tratamento, quer de prevenção. Certas estirpes de bactérias de Gram negativo têm sido encontradas de forma consistente em lesões periodontais, nomeadamente as espécies Porphyromonas gingivalis e Prevotella intermedia. Estas bactérias são de Gram negativo, anaeróbias estritas, imóveis, não esporuladas e produtoras de pigmento negro. A identificação destes microrganismos pode ser realizada pelos métodos convencionais, sendo tecnicamente complexos, dispendiosos e morosos ou por diversas técnicas de biologia molecular, nomeadamente a Polymerase Chain Reaction (PCR). A prevenção e o controlo das doenças periodontais residem na eliminação do biofilme microbiano, através de hábitos de higiene oral e tratamento mecânico local. No entanto, em alguns tipos de DP torna-se necessário o uso de antisépticos e/ou antibióticos. A identificação das espécies bacterianas implicadas na DP permite a prescrição do antibiótico específico, uma vez que se tem verificado uma tendência para a abordagem terapêutica empírica o que tem conduzido para um aumento da resistência aos antibióticos, particularmente nas espécies do género Porphyromonas e Prevotella. Face ao exposto e à escassa informação acerca da identidade de estirpes bacterianas em infecções periodontais em Portugal e eventuais resistências desenvolvidas realizou-se um estudo preliminar de pesquisa das estirpes produtoras de pigmento negro, nomeadamente P.gingivalis e P. intermedia e da pesquisa da suscetibilidade antimicrobiana aos antibióticos β-lactâmicos, metronidazol, clindamicina e tetraciclina; assim como também, a pesquisa de enzimas β-lactamases. Neste trabalho foram analisadas 42 amostras de isolados subgengivais provenientes de pacientes com periodontites, da clínica dentária da Faculdade de Ciências da Saúde, da Universidade Fernando Pessoa, Porto. Das 42 amostras, apenas 14 tiveram crescimento de colónias com pigmento negro. Destas, 57,1% foram identificadas como P. intermedia, 28,6% eram P. gingivalis e 14,2% pertenciam a outras espécies pigmentadas. Relativamente à prova de sensibilidade antimicrobiana, verificou-se que todas as amostras de P. intermedia foram sensíveis aos antibióticos testados, embora 14,3% de Prevotella spp tenha-se mostrado resistente à penincilina, amoxicilina, clindamicina e tetraciclina. Nenhum dos isolados da espécie P. gingivalis mostrou resistência aos agentes antimicrobianos ensaiados. A pesquisa de β-lactamases revelou que apenas 2 amostras tinham capacidade para a produção destas enzimas. Os dois métodos utilizados neste trabalho mostraram-se eficazes no conhecimento das espécies bacterianas intervenientes na DP. Contudo, no método clássico várias amostras perderam viabilidade durante o processo de congelação, inviabilizando a sua cultura. Por outro lado, os métodos moleculares amplificaram todas as amostras tendo-se demonstrado como previsto uma técnica rápida e precisa. As estirpes isoladas de P. gingivalis e P. intermedia foram sensíveis a todos os antibióticos testados, ao contrário do referido na literatura. Este resultado pode dever-se à reduzida amostragem deste estudo. Da avaliação destes resultados preliminares é nossa proposta em estudos futuros alargar significativamente a amostragem, bem como a amplificação direta do ADN bacteriano colhido nas bolsas periodontais, de forma a não existir seleção cultural dos agentes microbianos. O estudo da suscetibilidade antimicrobiana com recurso a técnicas de biologia molecular é, também nossa proposta para a contribuição de uma maior sensibilidade e precisão de resultados.
Oral cavity is one of the places in the human body that reveals the most complex and heterogeneous microbial population, which are a part over half of a thousand bacterial species. Periodontal Disease (PD) is an oral pathology of bacterial origin characterized by an inflammatory state that stars affecting gingival tissue and progresses frequently to tooth lost in adult age. Gingivitis and periodontitis are the periodontal pathologies more frequently found in clinical dentistry routine. The identification of the microbiome responsible for theses pathologies is of extreme importance due to the adoption of more effective measures of treatment and prevention. Certain strains of Gram-negative bacteria have been consistently found in periodontal lesions namely Porphyromonas gingivalis and Prevotella intermedia. These are Gramnegative bacteria, anaerobic, non-sporulating and producers of black pigment. The identification of these strains can be performed by conventional methods, which are complex, expensive and time-consuming, or by molecular biology techniques, namely Polymerase Chain Reaction (PCR). The prevention and control of periodontal diseases is based on the microbial biofilm elimination, through oral hygiene or mechanical and local treatment. However in some types of PD its necessary the use of anti-septic and/or antibiotics. The identification of the bacterial species involved in PD allows the prescription of a specific antibiotic, which is another approach to the empirical therapeutic lately, leading to the increase of antibiotic resistance namely of the species Porphyromonas and Prevotella. Facing all the above and due to the lack of information regarding the identity of the bacterial strains in periodontal infections in Portugal and also due to antibiotic resistance, a preliminary study was developed based on the black pigment producers strains P. gingivalis and P. intermedia; search of antimicrobial susceptibility to β-lactam antibiotics, metronidazole, clindamycin and tetracycline, as well as the research of the β-lactamases enzymes. In this work it was analyzed 42 samples of subgingival isolates from patients with periodontitis from the dentistry clinic of Health Science faculty, Fernando Pessoa University of Oporto. Of the 42 samples, only 14 revealed growth of black pigment colonies. From this ones, 57,1% were identified as P. intermedia, 28,6% were P. gingivalis and 14,2% belong to other pigmented species. Concerning the antimicrobial sensitivity to the antibiotic tested, all the samples of P. intermedia were sensitive, although 14,3% of Prevotella spp. was resistant to penicillin, amoxicillin, clindamycin and tetracycline. None of the isolates of P. gingivalis showed resistance to the antimicrobial agents tested. The search for β-lactamases shows that only 2 strains had the ability to produce it. The two methods used in this work were effective in the identification of the bacterial strains involved in PD. However it was verified that using the classical method several samples loss viability during the freezing process, so they could not be cultured. On the other hand the molecular methods amplified all the samples as expected it was quick and precise. The isolated strains of P. gingivalis and P. intermedia were sensitive to the antibiotics tested, opposite to literature references. This result may be due to the reduced sample number. From this preliminary result evaluation is our proposal to engage future studies with a broader number of samples and using direct DNA amplification collected from periodontal pockets, a way from avoiding strain selection of the bacteria. The study of antimicrobial susceptibility based also in molecular biology techniques is, according to us, a better contribution to increase the sensibility and precision of the results.

Indiana University-Purdue University Indianapolis (IUPUI)
Periodontal disease is the leading cause of tooth loss in adults. Bacterial biofilm on tooth surfaces is the primary initiator of periodontal disease. Various factors contribute to the severity of periodontal disease including the different virulence factors of the bacteria within the biofilm. In the progression of periodontal disease, the microflora evolves from a predominantly Gram positive microbial population to a mainly Gram negative population. Specific gram negative bacteria with pronounced virulence factors have been implicated in the etiology and pathogenesis of periodontal disease, namely Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola which form the red complex of bacteria. The orange complex bacteria become more dominant in the maturation process of dental plaque and act to bridge the early colonizers of plaque with the later more dominant red complex bacterial and consists of such bacteria as Campylobacter showae, Campylobacter rectus, Fusobacterium nucleatum and Prevotella intermedia. Perhaps the most investigated contributing factor is the relationship between smoking and periodontal disease. When examining the association between cigarette smoking and interproximal bone loss, greater bone loss is associated with higher cigarette consumption, longer duration (i.e., pack year history) and higher lifetime exposure. The presence of various virulence factors such as the production of a capsular material, as well as the proteolytic activity of the various periopathodontic bacteria has been associated with the pathogenesis of periodontitis. Even though many different enzymes are produced in large quantities by these periodontal bacteria, trypsin-like enzymes, chymotrypsin-like enzymes and elastase-like enzymes, as well as dipeptidyl peptidase-like enzymes, have been thought to increase the destructive potential of the bacterium and mediate destruction of the periodontal apparatus. More specifically, it is hypothesized that the proteolytic activity of other clinically important periodontal pathogens, such as Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas assacharolyticus, is increased in the presence of nicotine. The purpose of this study was to determine the effects of nicotine on F. nucleatum, P. intermedia and P. assacharolyticus proteolytic activity. Cultures were maintained on anaerobic blood agar plates containing 3% sheep blood. Bacterial cells were harvested from the plates and washed. Washed F. nucleatum, P. intermedia and P. assacharolyticus cells were incubated with 1 mg/ml of nicotine. Bacterial cells not incubated with nicotine were used as positive controls. Secreted enzymatic activity was measured using the synthetic chromogenic substrates glycyl-L-proline-p-nitroanilide (GPPNA), N-succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide (SAAAPNA), N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (SAAPPPNA) and N-α-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) (Sigma-Aldrich Products, St. Louis, MO, USA). Appropriate means and standard deviations were determined for each of the enzymatic activities measured and analysis of variance (ANOVA) was used to compare the groups utilizing a 5% significance level for all comparisons. Results demonstrated that after 60 minutes of incubation of F. nucleatum, P. intermedia and P. assacharolyticus cells with 1 mg/ml of nicotine and the various synthetic substrates, had the following proteolytic activity for GPPNA: 0.83 ± 0.14, 0.72 ± 0.03 and 0.67 ± 0.10, respectively; SAAAPNA: 0.82 ± 0.06, 0.76 ± 0.05 and 0.68 ± 0.08, respectively; SAAPPPNA: 0.90 ± 0.13, 0.85 ± 0.17 and 0.72 ± 0.03, respectively; and BAPNA: 0.81 ± 0.15, 0.74 ± 0.13 and 0.74 ± 0.16, respectively. In conclusion, the results indicate that in the presence of 1 mg/ml of nicotine, the proteolytic activity of F. nucleatum and P. assacharolyticus was increased with all of the synthetic substrates (with statistical significance seen only in the increases with F. nucleatum and GPPNA, SAAAPNA and BAPNA). The proteolytic activity exhibited an increasing trend in activity for P. intermedia with SAAPPPNA and BAPNA but a decreasing trend in activity with GPPNA and SAAAPNA when incubated with 1 mg/ml of nicotine, once again demonstrating no statistical significance for any of the substrates. Therefore, it could be concluded that based on these results nicotine at a concentration of 1 mg/ml may increase the proteolytic activity of periodontal pathogens and thus may increase periodontal disease activity and subsequent periodontal breakdown. Further studies are needed to validate these results utilizing different concentrations of nicotine.