Méthodes moléculaires de détection d'animaux porteurs d'actinobacillus pleuropneumoniae : comparaison et validation d'épreuves PCR

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Evaluación antimicrobiana de rifampicina nanoencapsulada contra aggregatibacter actinomycetemcomitans presente en la periodontitis

Introducción. A pesar de los esfuerzos tanto de la medicina como de la industria farmacéutica, el incremento en la prevalencia de resistencia en bacterias patógenas frente a antibióticos se ha vuelto uno de los mayores problemas en la medicina moderna. El área odontológica tampoco se encuentra exenta, siendo común el uso excesivo de antibióticos lo que contribuye al desarrollo de resistencia antimicrobiana. La primera etapa para el desarrollo de la enfermedad periodontal es la formación de un biofilm de bacterias periodontopatógenas, siendo el Aggregaribacter actinomycetemcomitans (A.a) uno de los más asociados a dicha enfermedad. El tratamiento de esta patología se basa en remover mecánicamente la placa dentobacteriana y, en segunda instancia, en el apoyo de terapia antimicrobiana para coadyuvar la eliminación de las bacterias periodontopatógenas, cuales tienen gran similitud con Mycobacterium tuberculosis. La rifampicina es uno de los antibióticos efectivos contra bacterias multi-resistentes y la primera elección en el tratamiento de tuberculosis activa. Con el fin de mejorar la terapia farmacológica y evadir la resistencia del agente infectivo, se han propuesto nuevas estrategias basadas en sistemas de liberación controlada. Entre los más estudiados en los últimos 10 años se encuentran las nanopartículas poliméricas. El objetivo del presente estudio fue evaluar la actividad antimicrobiana de la rifampicina nanoencapsulada contra el A.a presente en la periodontitis. Materiales y Métodos. Para el estudio, Se tomaron muestras de fluido crevicular en pacientes con bolsas periodontales de 5-10 mm de profundidad. Se inoculo caldo de tripticaseina de soya (TCS) con las muestras tomadas y se incubaron a 37 ° C en condiciones aeróbicas por 7 días. La presencia de Aggregatibacter actinomycetemcomitans (A.a) fue determinado mediante PCR en tiempo real. La Concentración Mínima Inhibitoria (MIC) de rifampicina para interferir con el crecimiento de bacterias orales fue determinada mediante la técnica de dilución de tubos. Posteriormente se prepararon mediante la técnica de nanoprecipitación NP de Eudragit® EPO, L100-55 y PLA entre 100 y 200 nm y su IP con distribución de tamaño homogéneo. Resultados. A.a fue detectado en muestras de fluido crevicular en pacientes con periodontitis, corroborando su asociación con dicha patología. La efectividad de la rifampicina libre contra bacterias orales fue confirmada, obteniéndose una CMI de 1 µg/ml. Las NP con Rifampicina se ajustaron a la misma CMI que la Rif libre. Las NP de Eudragit® EPO cargadas con Rif mostraron que la liberación de la Rif de la NP fue inmediata, mientras que el Eudragit® L100-55 y PLA con Rif no mostró inhibición durante los 5 días de incubación. Esto hace suponer que el fármaco no fue liberado o solo se liberó en una baja proporción que no permitió llegar a la CMI. Conclusión. La rifampicina es una excelente alternativa terapéutica para el tratamiento de la enfermedad periodontal, promoviendo resultados favorables en la evaluación clínica de pacientes. Sería interesante continuar con estudios utilizando otro polímero o mezcla de ellos para favorecer la liberación del fármaco en la NP.

Phylogenetic relationships of unclassified, satellitic Pasteurellaceae obtained from different species of birds as demonstrated by 16S rRNA gene sequence comparison

Avian haemophili demonstrating in vitro satellitic growth, also referred to as the V-factor or NAD requirement, have mainly been classified with Avibacterium paragallinarum (Haemophilus paragallinarum), Avibacterium avium (Pasteurella avium), Avibacterium volantium (Pasteurella volantium) and Avibacterium sp. A (Pasteurella species A). The aim of the present study was to assess the taxonomic position of 18 V-factor-requiring isolates of unclassified Haemophilus-like organisms isolated from galliforme, anseriforme, columbiforme and gruiforme birds as well as kestrels and psittacine birds including budgerigars by conventional phenotypic tests and 16S rRNA gene sequencing. All isolates shared phenotypical characteristics which allowed classification with Pasteurellaceae. Haemolysis of bovine red blood cells was negative. Haemin (X-factor) was not required for growth. Maximum-likelihood phylogenetic analysis including bootstrap analysis showed that six isolates were related to the avian 16S rRNA group and were classified as Avibacterium according to 16S rRNA sequence analysis. Surprisingly, the other 12 isolates were unrelated to Avibacterium. Two isolates were unrelated to any of the known 16S rRNA groups of Pasteurellaceae. Two isolates were related to Volucribacter of the avian 16S rRNA group. Seven isolates belonged to the Testudinis 16S rRNA group and out of these, two isolates were closely related to taxa 14 and 32 of Bisgaard, whereas four other isolates were found to form a genus-like group distantly related to taxon 40 and one isolated remained distantly related to other members of the Testudinis group. One isolate was closely related to taxon 26 (a member of Actinobacillus sensu stricto). The study documented major genetic diversity among V-factor-requiring avian isolates beyond the traditional interpretation that they only belong to Avibacterium, underlining the limited value of satellitic growth for identification of avian members of Pasteurellaceae. Our study also emphasized that these organisms will never be isolated without the use of special media satisfying the V-factor requirement.

Probiotic activity of Lactobacillus delbrueckii subsp. bulgaricus in the oral cavity

Yogurt consumption has been related to longevity of some populations living on the Balkans. Yogurt starter L. delbrueckii subsp. bulgaricus and Str. thermophilus have been recognized as probiotics with verified beneficial health effects. The oral cavity emerges as a arget for probiotic applications. Probiotics have demonstrated promising results in controlling dental diseases and oral yeast infections. However, L. bulgaricus despite its broad availability in dairy products has not been evaluated for probiotic activity in the mouth. These series of studies investigated in vitro properties of L. bulgaricus to outline its potential as an oral probiotic. Prerequisite probiotic properties in the mouth are resistance to oral defense mechanisms, adherence to saliva-coated surfaces, and inhibition of oral pathogens. L. bulgaricus strains showed a strain-dependent inhibition of oral streptococci and Aggregatibacter actinomycetemcomitans, whereas none of the dairy starter strains could affect growth of Porphyromonas gingivalis and Fusobacterium nucleatum. Adhesion is a factor contributing to colonization of the species at the target site. Radiolabeled L. bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated surfaces. The effects of lysozyme on adhesion and adhesion of Streptococcus sanguinis after lactobacilli pretreatment were also assessed. Adhesion of L. bulgaricus remained lower in comparison to L. rhamnosus GG. One L. bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment significantly increased Lactobacillus adhesion. Low gelatinolytic activity was observed for all strains and no conversion of proMMP-9 to its active form was induced by L. bulgaricus. Safety assessment ruled out deleterious effects of L. bulgaricus on extracellular matrix structures. Cytokine response of oral epithelial cells was assessed by measuring IL-8 and TNF-α in cell culture supernatants. The effect of P. gingivalis on cytokine secretion after lactobacilli pretreatment was also assessed. A strain- and time-dependent induction of IL-8 was observed with live bacteria inducing the highest levels of cytokine secretion. Levels of TNF-α were low and only one of ten L. bulgaricus strains stimulated TNF-α secretion similar to positive control. The addition of P. gingivalis produced immediate reduction of cytokine levels within the first hours of incubation irrespective of lactobacilli strains co-cultured with epithelial cells. According to these studies strains among the L. delbrueckii subsp. bulgaricus species may have beneficial probiotic properties in the mouth. Their potential in prevention and management of common oral infectious diseases needs to be further studied.

Inflammation-induced atherogenesis, liver alterations, and cardiovascular outcome

Cardiovascular diseases, which presently are considered inflammatory diseases, affect millions of people worldwide. Chronic infections may contribute to the systemic inflammation suggested to increase the risk for cardiovascular diseases. Such chronic infections are periodontitis and Chlamydia pneumoniae infection. They are highly prevalent as approximately 10% of adult population and 30% of people over 50 years old are affected by severe periodontitis and 70-80% of elderly people are seropositive for C. pneumoniae. Our general aim was to investigate the role of infection and inflammation in atherosclerosis both in animal and human studies. We aimed to determine how the two pathogens alter the atherosclerosis-associated parameters, and how they affect the liver inflammation and lipid composition. Furthermore, we evaluated the association between matrix metalloproteinase-8 (MMP-8), a proteinase playing a major role in inflammation, and the future cardiovascular diseases (CVD) events in a population-based cohort. For the animal experiments, we used atherosclerosis-susceptible apolipoprotein E deficient (apoE-/-) mice. They were kept in germ free conditions and fed with a normal chow diet. The bacteria were administered either intravenously (A. actinomycetemcomitans) or intranasally (C. pneumoniae). Several factors were determined from serum as well as from aortic and hepatic tissues. We also determined how cholesterol efflux, a major event in the removal of excess cholesterol from the tissues, and endothelial function were affected by these pathogens. In the human study, serum MMP-8 and its tissue inhibitor (TIMP-1) concentrations were measured and their associations during the follow-up time of 10 years with CVD events were determined. An infection with A. actinomycetemcomitans increased concentrations of inflammatory mediators, MMP production, and cholesterol deposit in macrophages, decreased lipoprotein particle size, and induced liver inflammation. C. pneumoniae infection also elicited an inflammatory response and endothelial dysfunction, as well as induced liver inflammation, microvesicular appearance and altered fatty acid profile. In the population-based cohort, men with increased serum MMP-8 concentration together with subclinical atherosclerosis (carotid artery intima media thickness > 1mm) had a three-fold increased risk for CVD death during the follow-up. The results show that infections with A. actinomycetemcomitans and C. pneumoniae induce proatherogenic changes, as well as affect the liver. These data therefore support the concept that common infections have systemic effects and could be considered as cardiovascular risk factors. Furthermore, our data indicate that, as an independent predictor of fatal CVD event, serum MMP-8 could have a clinical significance in diagnosing cardiovascular diseases.

Evaluation of a multiplex PCR to identify and serotypeActinobacillus pleuropneumoniaeserovars 1, 5, 7, 12 and 15

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species-specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar-specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations. Significance and Impact of the Study A multiplex PCR that can recognize serovars 1, 5, 7, 12 and 15 of A. pleuropneumoniae was developed and validated. This novel diagnostic tool will enable frontline laboratories to provide key information (the serovar) to guide targeted prevention and control programmes for porcine pleuropneumonia, a serious economic disease of pigs. The previous technology, traditional serotyping, is typically provided by specialized reference laboratories, limiting the capacity to respond to this key disease.

The association between subgingival periodontal pathogens and systemic inflammation

Aim To investigate associations between periodontal disease pathogens and levels of systemic inflammation measured by C-reactive protein (CRP). Methods A representative sample of dentate 60-70-year-old men in Northern Ireland had a comprehensive periodontal examination. Men taking statins were excluded. Subgingival plaque samples were analysed by quantitative real time PCR to identify the presence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia. High-sensitivity CRP (mg/l) was measured from fasting blood samples. Multiple linear regression analysis was performed using log-transformed CRP concentration as the dependent variable, with the presence of each periodontal pathogen as predictor variables, with adjustment for various potential confounders. Results A total of 518 men (mean age 63.6 SD 3.0 years) were included in the analysis. Multiple regression analysis showed that body mass index (p < 0.001), current smoking (p < 0.01), the detectable presence of P. gingivalis (p < 0.01) and hypertension (p = 0.01), were independently associated with an increased CRP. The detectable presence of P. gingivalis was associated with a 20% (95% confidence interval 4-35%) increase in CRP (mg/l) after adjustment for all other predictor variables. Conclusion In these 60-70-year-old dentate men, the presence of P. gingivalis in subgingival plaque was significantly associated with a raised level of C-reactive protein.

Microbiological Profile of Aggressive Periodontitis in a Sudanese Population

Background: Several bacterial species have been identified as being associated with aggressive periodontitis (AgP) notably Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg). There are limited data on bacterial associations with AgP in African populations. Objective: To investigate possible associations between specific bacteria and AgP in a Sudanese population. Methods: Subgingival plaque samples were collected from 93 (20 male, 73 female) Sudanese patients diagnosed with AgP and from 72 (23 male, 48 female) periodontally healthy Sudanese controls. Quantitative PCR was used to identify Aa, Pg, Treponema denticola (Td) and Fusobacterium nucleatum (Fn). The prevalence of these bacterial species was compared using Chi-square analysis. Odds ratios (OR) were calculated using standard methods. Results: The cases with AgP were well matched in age with the controls: 24.8 (SD 5.1) compared with 23.5 (SD 3.7) years, p=0.07. There was a significantly higher prevalence of Pg in AgP (73%) than in the controls (33%), p<0.0001. The OR for Pg to be associated with AgP was 5.44 (95% confidence intervals 2.78-10.64). In 26 (38%) of the AgP cases positive for Pg there were low levels of this bacterium (<100 copies). Both Td and Fn were identified in virtually all (>95%) the plaque samples studied from both AgP and controls. Aa was the least frequently identified species and was present in only 28% of AgP and 18% of controls, p=0.14. The OR for Aa to be associated with AgP was slightly increased at 1.76 (95% CI 0.83-3.74), however, this was not significant (p=0.14). Conclusion: In the Sudanese subjects studied Pg but not Aa was associated with AgP. There were very low levels of Pg in many of the plaque samples from AgP.

Tópicos especiais em microbiologia.

Taxonomia bacteriana ? aspectos atuais e perspectivas. Aggregatibacter actinomycetemcomitans: fatores de virulência e modulação do sistema imune. Escherichia coli diarreiogênica em animais. Fatores de virulência em escherichia coli patogênica Extraintestinal. Enterococcus sp em alimentos: paradigmas. Resistência a carbapenêmicos em enterobactérias. Resistência em Staphylococcus aureus. Relação mútua entre Candida albicans e imunidade. Switching fenotípico em Candida spp Phytomonas spp.: modelo para estudo de processos biológicos da família Trypanosomatidae? Rotavirus. Antimicrobianos naturais produzidos por microrganismos: da busca à identificação. Antivirais naturais. Nanopartículas metálicas com atividade antimicrobiana. Plantas medicinais: A busca de novos fármacos no tratamento de doenças causadas por protozoários tripanossomatídeos. Introdução, estabelecimento e adaptação de Bradirrizóbios simbiontes da soja em solos brasileiros. Microrganismos e processos microbianos como bioindicadores de qualidade ambiental.

Zinc as an agent for the prevention of biofilm formation by pathogenic bacteria

Les biofilms sont des communautés structurées de micro-organismes enrobées dans une matrice extracellulaire. Les biofilms sont impliqués dans la persistance de plusieurs maladies infectieuses et la matrice extracellulaire du biofilm protège les bactéries contre les cellules du système immunitaire de l'hôte, les antibiotiques et les désinfectants. Récemment notre laboratoire a démontré que le zinc inhibe la formation de biofilm chez Actinobacillus pleuropneumoniae, une bactérie pathogène du porc. Le but de cette étude est d'évaluer l'effet du zinc sur la croissance et la formation du biofilm chez différentes bactéries pathogènes du porc, telles que Bordetella bronchiseptica, Escherichia coli, Haemophilus parasuis, Salmonella, Staphylococcus aureus et Streptococcus suis. Les bactéries ont été cultivées dans des plaques de 96 puits sous condition optimale de formation de biofilm et les biofilms ont été colorés au cristal violet. La présence du biofilm a été confirmée par microscopie confocale à balayage laser à l’aide du marqueur fluorescent FilmTracerTM FM ® 1-43. À des concentrations micromolaires, le zinc inhibe faiblement la croissance bactérienne et bloque d'une manière dose-dépendante la formation de biofilm d’A. pleuropneumoniae, Salmonella Typhimurium et H. parasuis. De plus, la formation de biofilm de E. coli, S. aureus et S. suis a été faiblement inhibée par le zinc. Nos résultats indiquent que le zinc a un effet inhibiteur sur la formation de biofilm de la plupart des pathogènes bactériens d'origine porcine. Cependant, le mécanisme sous-jacent de l'activité anti-biofilm du zinc reste à être caractérisé.

Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an epidemiological survey.

By the end of 2004, the Canadian swine population had experienced a severe 2 increase in the incidence of Porcine circovirus-associated disease (PCVAD), a problem that was 3 associated with the emergence of a new Porcine circovirus-2 genotype (PCV-2b), previously 4 unrecovered in North America. Thus it became important to develop a diagnostic tool that could 5 differentiate between the old and new circulating genotypes (PCV-2a and -2b, respectively). 6 Consequently, a multiplex real-time quantitative polymerase chain reaction (mrtqPCR) assay that 7 could sensitively and specifically identify and differentiate PCV-2 genotypes was developed. A 8 retrospective epidemiological survey that used the mrtqPCR assay was performed to determine if 9 cofactors could affect the risk of PCVAD. From 121 PCV-2–positive cases gathered for this 10 study, 4.13%, 92.56% and 3.31% were positive for PCV-2a, PCV-2b, and both genotypes, 11 respectively. In a data analysis using univariate logistic regressions, PCVAD compatible 12 (PCVAD/c) score was significantly associated with the presence of Porcine reproductive and 13 respiratory syndrome virus (PRRSV), PRRSV viral load, PCV-2 viral load, and PCV-2 14 immunohistochemistry (IHC) results. Polytomous logistic regression analysis revealed that 15 PCVAD/c score was affected by PCV-2 viral load (P = 0.0161) and IHC (P = 0.0128), but not by 16 the PRRSV variables (P > 0.9); suggesting that mrtqPCR in tissue is a reliable alternative to IHC. 17 Logistic regression analyses revealed that PCV-2 increased the odds ratio of isolating 2 major 18 swine pathogens of the respiratory tract, Actinobacillus pleuropneumoniae and Streptococcus 19 suis serotypes 1/2, 1, 2, 3, 4, and 7, which are serotypes commonly associated with clinical 20 diseases.

An interleukin-1 beta (IL-1 beta) single-nucleotide polymorphism at position 3954 and red complex periodontopathogens independently and additively modulate the levels of IL-1 beta in diseased periodontal tissues

Inflammatory cytokines such as interieukin-1 beta (IL-1 beta) are involved in the pathogenesis of periodontal diseases. A high individual variation in the levels of IL-10 mRNA has been verified, which is possibly determined by genetic polymorphisms and/or by the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans. In this study, we investigated the role of an IL-10 promoter single-nucleotide polymorphism at position 3954 [IL-1 beta(3954) SNP] and the presence of the periodontopathogens in the determination of the IL-1 beta levels in the periodontal tissues of nonsmoking chronic periodontitis (CP) patients (n = 117) and control (C) subjects in = 175) and the possible correlations with the clinical parameters of the disease. IL-1 beta(3954) SNP was investigated by restriction fragment length polymorphism, while the IL-1 beta levels and the presence of the periodontopathogens were determined by real-time PCR. Similar frequencies of IL-1 beta(3954) SNP were found in the C and CP groups, in spite of a trend toward a higher incidence of T alleles in the CP group. The IL-1 beta (3954) SNP CT and TT genotypes, as well as P. gingivalis, T. forsythia, and T. denticola, were associated with higher IL-1 beta levels and with higher values of the clinical parameters of disease severity. Concomitant analyses demonstrate that IL-1 beta(3954) and the red complex periodontopathogens were found to independently and additively modulate the levels of IL-1 beta in periodontal tissues. Similarly, the concurrent presence of both factors was associated with increased scores of disease severity. IL-1 beta(3954) genotypes and red complex periodontopathogens, individually and additively, modulate the levels of IL-1 beta in the diseased tissues of nonsmoking CP patients and, consequently, are potentially involved in the determination of the disease outcome.

Exogenous Administration of 15d-PGJ(2)-Loaded Nanocapsules Inhibits Bone Resorption in a Mouse Periodontitis Model

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

Detecção de bactérias periodontopatogênicas cultiváveis e não cultiváveis em placas ateromatosas

Periodontal infections consist of a group of inflammatory conditions caused by microorganisms that colonize the tooth surface through the formation of dental biofilm. Chronic infections such as periodontitis have been associated to the development and progression of atherosclerosis. AIM: Detect cultivatable and non-cultivatable periodontopathogenic bacteria in atheromatous plaques; search for factors associated to the presence of these bacteria in the atheromatous plaques and characterize the presence of cultivatable and non-cultivatable bacteria in these plaques. METHODOLOGY: A cross-sectional study was performed with a sample of 30 patients diagnosed with atherosclerosis in the carotid, coronary or femoral arteries and surgically treated with angioplasty and stent implant, bypass or endarterectomy. The plaques were collected during surgery and analyzed using the PCR molecular technique for the presence of the DNA of the cultivatable bacteria Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Treponema denticola and of the non-cultivatable Synergistes phylotypes. The patients were examined in the infirmary, after the surgery, where they also responded to a questionnaire aimed at determining factors associated to the presence of periodontopathogenic bacteria in the atheromatous plaques. RESULTS: All patients with tooth (66,7%) possessed disease periodontal, being 95% severe and 65% widespread. No periodontopathogenic bacteria were found in the atheromatous plaques. However, four samples (13.3%) were positive for the presence of bacteria. Of these, three participants were dentate, being two carriers of widespread severe chronic periodontite and one of located severe chronic periodontitis. None of them told the accomplishment of procedures associated to possible bacteremia episodes, as treatment endodontic, extraction the last six months or some procedure surgical dental. CONCLUSION: The periodontopathogenic bacteria studied were not found in the atheromatous plaques, making it impossible to establish the prevalence of these pathogens or the factors associated to their presence in plaques, the detection of positive samples for bacteria suggests that other periodontal and non-periodontal pathogens be studied in an attempt at discovering the association or not between periodontal disease and/or others infections and atherosclerosis, from the presence of these bacteria in atheromas