Resinous adhesive systems differentially affect the expression of cytokines by human monocytes stimulated or not with Streptococcus mutans in vitro.

OBJECTIVES: The polymerization of adhesive systems is incomplete and the residual monomers that have been released have a cytotoxic capacity. Some teeth develop into pulp necrosis after composite resin restorations. Considering frequent pulpal inflammation in response to cariogenic bacteria, substances released from the patches could affect the cells of the inflammatory infiltrate and interfere with the mechanisms of defense against microorganisms and protection of pulpal tissue. The aim of this study was to evaluate the effect of substances released by different resinous adhesive systems on cell viability and cytokine expression by human monocytes stimulated in vitro with Streptococcus mutans. DESIGN: Peripheral blood mononuclear cells from 10 healthy subjects were stimulated with S. mutans and then incubated with supernatants obtained from the Single Bond Universal (SBU) or Clearfil SE Bond (CSEB) adhesive systems for eight hours. Staining with Annexin V and 7AAD for analysis of apoptosis were performed and detection of monocytes expressing cytokines IL-1α, IL-6, IL-8, IL-10, IL-12 and TNF-α were performed by flow cytometry. RESULTS: No treatment significantly affected apoptosis in monocytes. SBU supernatant increased the frequency of monocytes expressing IL-8 and decreased the monocytes expressing IL-10. Considering S. mutans-stimulated cells, while SBU increased the frequency of IL-8+ monocytes, CSEB reduced the frequency of IL-6 and TNF-α positive monocytes. CONCLUSIONS: Products released from SBU seem to induce proinflammatory effects on monocytes while those from CSEB show an anti-inflammatory outcome. These effects may interfere in the control of cytokine-mediated immunoinflammatory pulp reactions, both in the presence and absence of stimulation by cariogenic bacteria.

Investigating cross-contamination by yeast strains from dental solid waste to waste-handling workers by DNA sequencing.

Trying to widen the discussion on the risks associated with dental waste, this study proposed to investigate and genetically compare yeast isolates recovered from dental solid waste and waste workers. Three samples were collected from workers' hands, nasal mucosa, and professional clothing (days 0, 30, and 180), and two from dental waste (days 0 and 180). Slide culture, microscopy, antifungal drug susceptibility, intersimple sequence repeat analysis, and amplification and sequencing of internal transcribed spacer regions were performed. Yeast strains were recovered from all waste workers' sites, including professional clothes, and from waste. Antifungal susceptibility testing demonstrated that some yeast recovered from employees and waste exhibited nonsusceptible profiles. The dendrogram demonstrated the presence of three major clusters based on similarity matrix and UPGMA grouping method. Two branches displayed 100% similarity: three strains of Candida guilliermondii isolated from different employees, working in opposite work shifts, and from diverse sites grouped in one part of branch 1 and cluster 3 that included two samples of Candida albicans recovered from waste and the hand of one waste worker. The results suggested the possibility of cross-contamination from dental waste to waste workers and reinforce the need of training programs focused on better waste management routines.

Actinomyces israelii in radicular cysts: a molecular study.

OBJECTIVE: To investigate whether the microscopic filamentous aggregates observed in radicular cysts are associated with the molecular identification of Actinomyces israelii. Moreover, to verify whether this bacterium can be detected in radicular cyst specimens not presenting aggregates. STUDY DESIGN: Microscopic colonies suggestive of Actinomyces were found in 8 out of 279 radicular cyst samples (case group). The case and control groups (n = 12; samples without filamentous colonies) were submitted to the semi-nested polymerase chain reaction to test the presence of A israelii. DNA sequencing was performed to validate polymerase chain reaction results. RESULTS: Two and 3 samples in the case and control groups, respectively, did not present a functional genomic DNA template and were excluded from the study. A israelii was identified in all samples of the case group and in 3 out of 9 samples of the control group. CONCLUSIONS: Although A israelii is more commonly identified in radicular cysts presenting filamentous aggregates, it also appears to be detected in radicular cysts without this microscopic finding.

Sub-Inhibitory Concentration of Piperacillin-Tazobactam May be Related to Virulence Properties of Filamentous Escherichia coli.

Sub-inhibitory concentrations of antibiotics are always generated as a consequence of antimicrobial therapy and the effects of such residual products in bacterial morphology are well documented, especially the filamentation generated by beta-lactams. The aim of this study was to investigate some morphological and pathological aspects (virulence factors) of Escherichia coli cultivated under half-minimum inhibitory concentration (1.0 µg/mL) of piperacillin-tazobactam (PTZ sub-MIC). PTZ sub-MIC promoted noticeable changes in the bacterial cells which reach the peak of morphological alterations (filamentation) and complexity at 16 h of antimicrobial exposure. Thereafter the filamentous cells and a control one, not treated with PTZ, were comparatively tested for growth curve; biochemical profile; oxidative stress tolerance; biofilm production and cell hydrophobicity; motility and pathogenicity in vivo. PTZ sub-MIC attenuated the E. coli growth rate, but without changes in carbohydrate fermentation or in traditional biochemical tests. Overall, the treatment of E. coli with sub-MIC of PTZ generated filamentous forms which were accompanied by the inhibition of virulence factors such as the oxidative stress response, biofilm formation, cell surface hydrophobicity, and motility. These results are consistent with the reduced pathogenicity observed for the filamentous E. coli in the murine model of intra-abdominal infection. In other words, the treatment of E. coli with sub-MIC of PTZ suggests a decrease in their virulence.

Mimivirus Fibrils Are Important for Viral Attachment to the Microbial World by a Diverse Glycoside Interaction Repertoire.

UNLABELLED: Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the ∼ 1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCE: APMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion to other microorganisms could facilitate viral ingestion by amoebae, a mechanism never before described in the virosphere.

Synergistic growth effect among bacteria recovered from root canal infections

The objective of this study was to determine the ecological relationships between bacterial species that colonize infected root canals. Root canal bacteria recovered from one patient with pulp canal necrosis were evaluated in vitro for synergistic and antagonistic activities determined by mono and co-culture growth kinetics and the production of bacteriocin-like substances using the double layer diffusion method. Peptostreptococcus prevotii triggered a significant increase of Fusobacterium nucleatum growth, while the former bacteria did not affect the growth of P. prevotii. The bacterial species did not produce antagonism activity against itself or against any of the other two species. Despite many studies have demonstrated the capability of root canal microorganisms to produce antagonistic substances, these in vitro experimental tests show the synergistic effect of P. prevotii on the growth of F. nucleatum.

Effectiveness of chemomechanical preparation with alternating use of sodium hypochlorite and EDTA in eliminating intracanal Enterococcus faecalis biofilm.

INTRODUCTION: The elimination of microorganisms from root canals is a critical step in endodontic treatment. We aimed to evaluate the antimicrobial effectiveness of an alternating irrigation regimen during chemomechanical preparation (CMP). METHODS: During 21 days, root canals of extracted human teeth were infected with Enterococcus faecalis, and colonization was confirmed by scanning electron microscopy (SEM). Canals were irrigated with saline solution (control group), with 5.25% NaOCl followed by a final rinse with 17% EDTA (conventional irrigation group), or with the alternating use of NaOCl and EDTA (alternating irrigation [AI] group). Samples were taken before treatment (S1), after CMP (S2), and during the following 14 days. Two specimens/group were analyzed by SEM. RESULTS: The AI group yielded negative agar and liquid cultures from immediately after CMP and from the 5th day on, respectively. SEM confirmed several bacterium-free sites in the AI group. CONCLUSION: The irrigation regimen based on the alternating use of NaOCl and EDTA seems to be a promising endodontic tool because it promoted the elimination of root canal E. faecalis biofilms throughout the experimental period.

Distribution, detection of enterotoxigenic strains and antimicrobial drug susceptibility patterns of Bacteroides fragilis group in diarrheic and non-diarrheic feces from Brazilian infants

Despite the importance of gastrointestinal diseases and their global distribution, affecting millions of individuals around the world, the role and antimicrobial susceptibility patterns of anaerobic bacteria such as those in the Bacteroides fragilis group (BFG) are still unclear in young children. This study investigated the occurrence and distribution of species in the BFG and enterotoxigenic strains in the fecal microbiota of children and their antimicrobial susceptibility patterns. Diarrheic (n=110) and non-diarrheic (n=65) fecal samples from children aged 0-5 years old were evaluated. BFG strains were isolated and identified by conventional biochemical, physiological and molecular approaches. Alternatively, bacteria and enterotoxigenic strains were detected directly from feces by molecular biology. Antimicrobial drug susceptibility patterns were determined by the agar dilution method according to the guidelines for isolated bacteria. BFG was detected in 64.3% of the fecal samples (55% diarrheic and 80.4% non-diarrheic), and 4.6% were enterotoxigenic. Antimicrobial resistance was observed against ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, meropenem, ceftriaxone, clindamycin and chloramphenicol. The data show that these bacteria are prevalent in fecal microbiota at higher levels in healthy children. The molecular methodology was more effective in identifying the B. fragilis group when compared to the biochemical and physiological techniques. The observation of high resistance levels stimulates thoughts about the indiscriminate use of antimicrobial drugs in early infancy. Further quantitative studies are needed to gain a better understanding of the role of these bacteria in acute diarrhea in children.

Composition analysis of dental solid waste in Brazil.

When developing proper waste management strategies, it is essential to characterize the volume and composition of solid waste. The aim of this work was to evaluate the composition of dental waste produced by three dental health services in Belo Horizonte, Minas Gerais State, Brazil. Two universities, one public and one private, and one public dental health service were selected. Waste collection took place from March to November 2007. During this period, three samples were collected from each dental health service. The total amount of dental waste produced in one day of dental work was manually separated into three categories: infectious and potentially infectious waste, accounting for 24.3% of the total waste; non-infectious waste, accounting for 48.1%; and domestic-type waste, accounting for 27.6% (percentages are for mean weights of solid waste). Our results showed that most of the waste considered as biomedical may be misclassified, consequently making the infectious waste amount appear much larger. In addition, our results suggest that the best waste minimization method is recycling, and they help to define an appropriate waste management system in all three of the dental health services involved in this study.

Partial characterization of antagonistic substance produced by a Clostridium butyricum strain

The production of antagonistic substance by bacterium present in the infected root canal system (RCS) probably is an important ecological factor for its successful colonization of the local. The objective of this study was to partially characterize an antagonistic substance produced by a Clostridium butyricum isolated from infected RCS.Production of inhibitory compound was evaluated by the agar double layer diffusion technique using Fusobacterium nucleatum and Bifidobacterium adolescentis as indicator bacteria. The physicochemical and biochemical factors tested for the partial characterization were influence of pH and temperature and susceptibility to the action of some proteolytic enzymes. An inhibition zone was observed against the two indicator strains and acidity and bacteriophage were rejected as responsible for this phenomenon. The inhibitory activity showed to be decreasing in a pH range from 3.5 to 6.5 and being stable at temperatures of 60º, 70º and 100ºC, but completely inactivated when exposed at 121ºC. The antagonistic activity was resistant to the proteolytic action of trypsin, a-chymotrypsin and papain. An antagonistic substance was produced by C. butyricum, which was thermo-resistant and probably of non-protein nature.

A produção de substâncias antagonistas por espécies bacterianas presentes em sistema de canais radiculares (SCR) infectados, tem um papel importante na colonização deste sítio. O objetivo deste estudo foi caracterizar parcialmente a substância antagonista produzida por amostra de Clostridium butyricum isolado de SCR infectados.A produção de substância antagonista foi avaliada pela técnica de difusão em ágar utilizando como bactérias indicadoras Fusobacterium nucleatum e Bifidobacterium adolescentis. Os parâmetros físico-químicos utilizados durante a caracterização parcial foram: pH, estabilidade térmica, susceptibilidade à ação das enzimas tripsina, a-quimiotripsina e papaína. Foi observada zona de inibição contra as duas amostras indicadoras e ainda foi demonstrado que ácidos e bacteriófagos não eram responsáveis por este fenômeno. A atividade inibitória mostrou-se diminuída em uma faixa de pH de 3.5 a 6.5 e estável em temperaturas de 60º, 70º e 100ºC, sendo completamente inativada quando exposta a 121ºC. A atividade antagonista foi resistente à ação das enzimas proteolíticas: tripsina, a-quimiotripsina e papaína. A substância antagonista produzida por C. butyricum é termoresistente e provavelmente de natureza não protéica.

Isolamento e identificação da microbiota periodontal de cães da raça Pastor Alemão

The indigenous gingival microbiota of dogs is not totally described, although such identification is an important step to establish the etiopathogenesis and adequate therapy for the periodontal disease. The aims of this study were to isolate and identify the periodontal microbiota of German Shepherd dogs from healthy and with periodontal desease sites. Twenty nine German Shepherd dogs from three to six years of age were used in this study. Clinical specimens were analysed from healthy periodontal sites of 12 dogs and sites presenting gingivitis of 17 dogs. A total amount of 672 microbial samples, were isolated where the predominant genera were Pasteurella spp., Staphylococcus spp., Porphyromonas spp. and Fusobacterium spp. The microbiological population of the affected sites was similar to the healthy sites, consisting on an indigenous microbiota. The microbiota on the affecteded sites was higher in number than on the healthy sites, showing change in the environment of the periodontal sites.

A microbiota indígena gengival de cães não está totalmente descrita, sendo sua identificação uma etapa importante no estabelecimento da etiopatogenia e terapia da doença periodontal. O objetivo deste trabalho foi isolar e identificar a microbiota periodontal de cães da raça Pastor Alemão, considerando sítios saudáveis e com doença periodontal. Foram utilizados 29 cães, com idade variando de três a seis anos, sendo analisados espécimes clínicos de sítios periodontais saudáveis de 12 cães e sítios com periodontite de outros 17. Foram isoladas 672 amostras microbianas, com predomínio dos gêneros Pasteurella, Staphylococcus, Porphyromonas e Fusobacterium. A microbiota dos sítios saudáveis equiparou-se à dos sítios doentes, tratando-se de uma microbiota indígena. A microbiota dos sítios doentes apresentou-se aumentada em relação a dos sítios saudáveis, indicando mudança do ambiente do sítio periodontal.

Isolamento e identificação da microbiota periodontal de cães da raça Pastor Alemão

The indigenous gingival microbiota of dogs is not totally described, although such identification is an important step to establish the etiopathogenesis and adequate therapy for the periodontal disease. The aims of this study were to isolate and identify the periodontal microbiota of German Shepherd dogs from healthy and with periodontal desease sites. Twenty nine German Shepherd dogs from three to six years of age were used in this study. Clinical specimens were analysed from healthy periodontal sites of 12 dogs and sites presenting gingivitis of 17 dogs. A total amount of 672 microbial samples, were isolated where the predominant genera were Pasteurella spp., Staphylococcus spp., Porphyromonas spp. and Fusobacterium spp. The microbiological population of the affected sites was similar to the healthy sites, consisting on an indigenous microbiota. The microbiota on the affecteded sites was higher in number than on the healthy sites, showing change in the environment of the periodontal sites.

A microbiota indígena gengival de cães não está totalmente descrita, sendo sua identificação uma etapa importante no estabelecimento da etiopatogenia e terapia da doença periodontal. O objetivo deste trabalho foi isolar e identificar a microbiota periodontal de cães da raça Pastor Alemão, considerando sítios saudáveis e com doença periodontal. Foram utilizados 29 cães, com idade variando de três a seis anos, sendo analisados espécimes clínicos de sítios periodontais saudáveis de 12 cães e sítios com periodontite de outros 17. Foram isoladas 672 amostras microbianas, com predomínio dos gêneros Pasteurella, Staphylococcus, Porphyromonas e Fusobacterium. A microbiota dos sítios saudáveis equiparou-se à dos sítios doentes, tratando-se de uma microbiota indígena. A microbiota dos sítios doentes apresentou-se aumentada em relação a dos sítios saudáveis, indicando mudança do ambiente do sítio periodontal.

Antiproliferative activity of Actinobacillus (Haemophilus) actinomycetemcomitans and Fusobacterium nucleatum in peripheral blood mononuclear cells.

Several studies indicate Actinobacillus (Haemophilus) actinomycetemcomitans and Fusobacterium nucleatum as etiologic agents of periodontal disease. Immunosuppressive factors produced by microorganisms probably contribute to the initiation and evolution of this disease. This study evaluated the antiproliferative activity of ammonium precipitate fractions of A. (H.) actinomycetemcomitans and F. nucleatum isolates from humans and marmosets both with and without periodontal disease. All A. (H.) actinomycetemcomitans and most F. nucleatum strains inhibited PBMC proliferation in a dose-dependent manner. The degree of cell proliferative inhibition of each bacterial species differed among the strains and was independent of host clinical status. The in vitro inhibition of stimulated lymphocyte proliferation induced by different A. (H.) actinomycetemcomitans and F. nucleatum isolates demonstrated the importance of this phenomenon in bacterial virulence, playing a possible suppressor role in host defense mechanisms in vivo. Moreover, our findings pointed out a marked difference between A. (H.) actinomycetemcomitans and F. nucleatum cytoplasmic extracts in their antiproliferative activity, regarding the antigen concentration required for maximum inhibition and their vulnerability to heating and proteolytic treatment.

In vitro activation of the hemolysin in Prevotella nigrescens ATCC 33563 and Prevotella intermedia ATCC 25611.

Hemolytic activity was evaluated in the putative periodontopathogens Prevotella intermedia and Prevotella nigrescens. Whole cells of both species present weak hemolytic activity evidenced only by solid media assays after 48 h of bacterial growth or after 5 h of interaction with erythrocytes at 37 degrees C in liquid assays. In this work we show that the use of crude extract allowed the detection of a higher hemolytic activity for P. intermedia, but surprisingly not for P. nigrescens. Incubation at 37 degrees C for 9 h, or treatment with trypsin or proteinase K, increased or exposed the hemolytic activity of P. intermedia and P. nigrescens crude extract, respectively. The activation process was inhibited by TLCK and PMSF but not by EDTA, E-64 or pepstatin A, indicating the serino-protease nature of the factor involved in activation of P. intermedia and P. nigrescens hemolysins. Both the buffer and the pH employed for cell fractionation influenced the activation of hemolysin, and the best results were obtained with Universal buffer at pH 8.0. The activated hemolysins acted optimally at pH 6.5 at 37 degrees C and the maximum hemolytic activity was detected at the early log phase of growth. The results of this study show for the first time a strong hemolytic activity for P. nigrescens and evidence of proteolytic activation of hemolysins produced by periodontopathogens.

IMPORTÂNCIA DA AVALIAÇÃO CLÍNICA NO DIAGNÓSTICO DE DOENÇA PERIODONTAL EM CÃES DA RAÇA PASTOR ALEMÃO

As doenças periodontais resultam de resposta inflamatória progressiva do hospedeiro ao acúmulo de placa bacteriana, incluindo os metabólitos no dente e nos tecidos gengivais. Assim, avaliou-se, clinicamente, o periodonto de cães da raça Pastor Alemão, levando-se em consideração presença de tártaro, sangramento à sondagem e mobilidade dentária em animais com sítios saudáveis e com doença periodontal. Para isso, utilizaram-se 29 cães,da raça Pastor Alemão, com idade variando de três a seis anos. Clinicamente, 27 (93,10%) cães apresentavam vários sítios com sintomatologia clínica de doença periodontal e somente dois apresentavam sítios saudáveis. Os dentes mais acometidos foram os molares, os pré-molares e os caninos,com sinais clínicos mais intensos nas superfícies dentárias e maior acúmulo de tártaro e placa bacteriana. Os resultados demonstraram a necessidade de se proceder a uma limpeza preventiva dos dentes, para proporcionar maior saúde para os cães. PALAVRAS-CHAVE: Periodonto, cães, dente.

Importância da avaliação clínica no diagnóstico de doença periodontal em cães da raça pastor alemão

As doenças periodontais resultam de resposta inflamatória progressiva do hospedeiro ao acúmulo de placa bacteriana, incluindo os metabólitos no dente e nos tecidos gengivais. Assim, avaliou-se, clinicamente, o periodonto de cães da raça Pastor Alemão, levando-se em consideração presença de tártaro, sangramento à sondagem e mobilidade dentária em animais com sítios saudáveis e com doença periodontal. Para isso, utilizaram-se 29 cães,da raça Pastor Alemão, com idade variando de três a seis anos. Clinicamente, 27 (93,10%) cães apresentavam vários sítios com sintomatologia clínica de doença periodontal e somente dois apresentavam sítios saudáveis. Os dentes mais acometidos foram os molares, os pré-molares e os caninos,com sinais clínicos mais intensos nas superfícies dentárias e maior acúmulo de tártaro e placa bacteriana. Os resultados demonstraram a necessidade de se proceder a uma limpeza preventiva dos dentes, para proporcionar maior saúde para os cães. PALAVRAS-CHAVE: Periodonto, cães, dente.

Bacterial concentrations determine the ability to implant in the root canal system and translocate to lymph nodes in germ-free mice.

The purpose of this study was to evaluate the influence of the infecting dose on bacterial colonization in root canal systems (RCS) and translocation to the submandibular lymph node (SML) of gnotobiotic (germ-free) mice. RCS were inoculated with 10(2) and 10(1) colony-forming units (CFU) of Enterococcus faecalis (ATCC 4083) or 10(5), 10(4), 10(3), and 10(2) CFU of Prevotella nigrescens (ATCC 33563). E. faecalis implanted in 83.3% of the cases, for both inocula. Translocation was detected in 58.3% of lymph nodes for the 10(2) CFU inoculum and in 33.3% of lymph nodes for the 10(1) CFU inoculum. P. nigrescens implanted in 25%, 16.7%, 8.3%, and 0%, for the 10(5), 10(4), 10(3), and 10(2) CFU inocula, respectively, and did not translocate at any of the concentrations used. These results indicate that E. faecalis was able to implant in the RCS and translocate to the SML, whereas P. nigrescens implanted in lower percentages in the RCS and did not translocate to the SML.

Dominant culturable bacterial microbiota in the digestive tract of the American black vulture (Coragyps atratus Bechstein 1793) and search for antagonistic substances

Strict and facultative culturable anaerobic bacteria from the digestive tract of six American black vultures (Coragyps atratus Bechstein 1793) were isolated and identified. After capture, the birds received a non-contaminated diet for one week to eliminate possible allochthonous microorganisms. Then, specimens collected from tongue, stomach and intestines were weighed, submitted to decimal dilution in an anaerobic chamber, inoculated into culture media and incubated aerobically and anaerobically at 37ºC for enumeration, isolation and identification. Isolated bacteria were submitted to tests to detect possible antagonisms between them. The total bacterial population along the digestive tract ranged from 3.46 ± 0.39 log CFU/g in the stomach to 10.75 ± 0.37 log CFU/g in the distal intestine. Some bacteria were isolated for the first time from the digestive tract of C. atratus: Actinomyces bovis, Lactobacillus cellobiosus, Micrococcus luteus, Neisseria sicca, Clostridium bifermentans, Enterobacter agglomerans, Peptostreptococcus sp., Sarcina sp., Serratia odorifera, and Shigella flexneri. Associations between microorganisms were observed during isolation on two occasions, one involving A. bovis and N. sicca, and the other involving A. bovis and a Gram-negative rod. Hetero-, iso- and autoantagonisms were observed, suggesting the ecological role of these indigenous microorganisms in terms of population auto-control and environmental barrier in the digestive tract of carrion-feeding birds.

As bactérias anaeróbias estritas e facultativas cultiváveis do trato digestivo de seis urubus (Coragyps atratus Bechstein 1793) foram isoladas e identificadas. Após a captura, as aves receberam uma alimentação de baixa contaminação durante uma semana para eliminar possíveis microorganismos alóctonos. A seguir, amostras colhidas na língua, estomago e intestinos foram pesadas, submetidas a diluições decimais numa câmara anaeróbia, inoculadas em meios de cultura e incubadas em aerobiose e anaerobiose a 37ºC para enumeração, isolamento e identificação. As bactérias isoladas foram usadas posteriormente como produtoras e reveladoras para detectar possíveis fenômenos de antagonismo. A população bacteriana total ao longo do trato digestivo variou de 3,46 ± 0,39 log UFC/g no estômago até 10,75 ± 0,37 log UFC/g no intestino distal. Algumas bactérias foram isoladas pela primeira vez do trato digestivo de C. atratus: Actinomyces bovis, Lactobacillus cellobiosus, Micrococcus luteus, Neisseria sicca, Clostridium bifermentans, Enterobacter agglomerans, Peptostreptococcus sp., Sarcina sp., Serratia odorifera, and Shigella flexneri. Associações entre microorganismos foram observadas durante o isolamento em dois casos, um envolvendo A. bovis e N. sicca, e o outro envolvendo A. bovis e um bastonete Gram-negativo. Hetero-, iso- e autoantagonismos foram observados, sugerindo um papel ecológico para esses microorganismos em termos de autocontrole populacional e de barreira ambiental no trato digestivo dessas aves.

LEUKOTOXIC ACTIVITY OF ACTINOBACILLUS ACTINOMYCETEMCOMITANS ISOLATED FROM HUMAN AND NON-HUMAN PRIMATES

Actinobacillus actinomycetemcomitans is a clinically relevant periodontopathogenic Gram-negative coccobacillus that produces a leukotoxin of the RTX cytolysin family. In this study, we evaluated the leukotoxic activity of A. actinomycetemcomitans strains isolated from human and marmosets by Trypan blue exclusion and by the chemiluminescence assays. Among eight A. actinomycetemcomitans human strains studied, two (P2.17 and P8.12) were classified as high leukotoxin producers and among eight marmoset strains, one (M22.11) showed high leukotoxin production, as determined by Trypan blue exclusion assay. The reference strains ATCC 29523 and FDC Y4 respectively behaved like moderate and low producers. The chemiluminescence assay was used to evaluate the leukotoxic activity of M22.11 and P2.17 strains submitted to different growth conditions. Leukotoxic activity was detected on cells at the logarithmic phase and was similar under anaerobic and microaerophilic growth conditions. It was greatly reduced when cells were grown at glucose concentrations lower or higher than 0.75% (0.25% and 1.5%) in thioglycolate medium. Leukotoxin production mainly by the M22.11 strain was low in BHI broth, whereas production in TSB medium showed a similar level as in thioglycolate broth medium. Sodium bicarbonate at 10 mM did not affect leukotoxin production.

Actinobacillus actinomycetemcomitans é um cocobacilo Gram negativo, periodontopatógeno clinicamente importante, que produz uma leucotoxina pertencente à família das citolisinas RTX. Neste estudo, avaliou-se a atividade leucotóxica de amostras de A. actinomycetemcomitans isoladas de seres humanos e de calitriquídeos pelos métodos de exclusão de azul de Tripan e quimioluminescência. Duas (P2.17 e P8.12) entre oito amostras de A. actinomycetemcomitans isoladas de seres humanos, e uma (M22.11) entre 8 amostras isoladas de sagüis se apresentaram como altamente produtoras de leucotoxina, como determinado pelo teste de exclusão de azul de Tripan. As amostras de referências de A. actinomycetemcomitans ATCC 29523 e FDC Y4 se comportaram como média e baixa produtoras de leucotoxina, respectivamente. O teste de quimioluminescência foi usado para avaliar a atividade leucotóxica das amostras M22.11 e P2.17 submetidas a diferentes condições de crescimento. A atividade leucotóxica foi detectada em células durante a fase logarítmica e foi similar sob crescimento em anaerobiose e microaerofilia. A atividade leucotóxica foi muito reduzida quando as células foram crescidas em concentrações de glicose mais baixa e mais alta que 0,75% (0,25% e 1,5%), em meio tioglicolato. A produção de leucotoxina, especialmente pela amostra M22.11, foi mais baixa em caldo BHI, enquanto em meio TSB a produção foi em nível similar a aquele em meio tioglicolato. Bicarbonato de sódio 10mM não afetou a produção de leucotoxina.