ABSTRACT Major health challenges as the increasing number of cases of infections by antibiotic multiresistant microorganisms and cases of Alzheimer's disease have led to searching new control drugs. The present study aims to verify a new way of obtaining bioactive extracts from filamentous fungi with potential antimicrobial and acetylcholinesterase inhibitory activities, using epigenetic modulation to promote the expression of genes commonly silenced. For such finality, five filamentous fungal species (Talaromyces funiculosus, Talaromyces islandicus, Talaromyces minioluteus, Talaromyces pinophilus, Penicillium janthinellum) were grown or not with DNA methyltransferases inhibitors (procainamide or hydralazine) and/or a histone deacetylase inhibitor (suberohydroxamic acid). Extracts from T. islandicus cultured or not with hydralazine inhibited Listeria monocytogenes growth in 57.66 ± 5.98% and 15.38 ± 1.99%, respectively. Increment in inhibition of acetylcholinesterase activity was observed for the extract from P. janthinellum grown with procainamide (100%), when compared to the control extract (39.62 ± 3.76%). Similarly, inhibition of acetylcholinesterase activity increased from 20.91 ± 3.90% (control) to 92.20 ± 3.72% when the tested extract was obtained from T. pinophilus under a combination of suberohydroxamic acid and procainamide. Concluding, increases in antimicrobial activity and acetylcholinesterase inhibition were observed when fungal extracts in the presence of DNA methyltransferases and/or histone deacetylase modulators were tested.

Neste estudo, verificou-se a utilização de um filme de quitosana para o controle do crescimento e produção de aflatoxinas por A. parasiticus em amendoim. Os filmes foram aplicados sobre os grãos por meio de duas metodologias (aspersão e imersão). Os grãos recobertos foram inoculados com 2,5 ml de uma suspensão contendo 1,0×106 esporos/ml e incubados a 25 oC por 7 dias. A concentração de aflatoxinas foi determinada por cromatografia em camada delgada, utilizando a técnica de densitometria. A verificação da inibição do crescimento fúngico foi realizada por meio da inoculação do patógeno em placas de Petri contendo GYEP suplementado com quitosana a 2% e incubadas por 7 dias a 25 ºC. Após incubação, o diâmetro da colônia do patógeno foi estimado e comparado com o controle. Todos os testes foram realizados com cinco repetições. Para a verificação de alterações morfológicas, uma suspensão de esporos ou hifas incubados na presença de quitosana foi submetida à microscopia eletrônica de varredura. Foi observado que tanto o método de imersão quanto o de aspersão reduziram a produção de aflatoxina significativamente, 84,3% e 86,7% respectivamente. A presença de quitosana no meio de cultura reduziu o diâmetro das colônias e promoveu modificações morfológicas nos esporos de A. parasiticus. Com esse procedimento foi verificado que a utilização de quitosana a 2% na forma de filme, principalmente por aspersão, pode reduzir a concentração de esporos e aflatoxinas de A. parasiticus em amendoim, o que confere a essa substância uma grande perspectiva de utilização no controle de fungos aflatoxigênicos.

In this study, we evaluate the utilization on chitosan film to control the growth and aflatoxin production by A. parasiticus in peanut. The films were inoculated over the grains by aspersion or immersion methods. The chitosan coated grains were inoculated with 2.5 ml solution with 1.0×106spores/ml and incubated at 25 °C for 7 days. The aflatoxin concentration was estimated by thin layer chromatography and densitometry. The fungus growth inhibition was observed by pathogen inoculation in Petri dishes with GYEP media plus 2% chitosan and incubated by 7 days at 25 °C. After incubation the colony diameter was measured and compared with the control. All assays were repeated five times. To verify morphological alterations, a spore/hypha suspension was incubated in media with chitosan and observed in scanning electron microscopy.Both methods (immersion and aspersion) were able to reduce the aflatoxin production significantly, 84.3% and 86.7%, respectively. The chitosan reduced the colonies diameters and promoted morphological alterations in A. parasiticus spores. With this, was observed that a 2% chitosan film, specially inoculated by aspersion, can to reduce A. parasiticus spores and aflatoxins concentrations in peanuts. This characteristic gives to this substance an excellent perspective to be used in aflatoxigenic fungi control.

The Gram-negative intestinal microbiota of Hypostomus auroguttatus and Pimelodus maculatus, a detritivorous and an omnivorous fish species, respectively, were compared between fishes from the reservoir and the stretch of the river below the dam of the Funil hydroelectric plant, Rio de Janeiro, Brazil. Four selective culture media were used under aerobic and two under anaerobic conditions. The omnivorous species had microbiota with higher population levels compared to the detritivorous species. The number of morphotypes and population levels of total bacteria, vibrio and Bacteroides tended to be higher in summer and autumn in the reservoir, and not different in the river. The number of morphotypes of enterobacteria and total bacteria were higher in the lotic environment compared with the lentic one. The bacteria Aeromonas hydrophila and Plesiomonas shigelloides and the obligate anaerobic Fusobacterium mortiferum were the most frequently identified microorganisms in the intestine of both H. auroguttatus and P. maculatus. Both season and habitat influenced the Gram-negative intestinal microbiota of H. auroguttatus and P. maculatus. Environmental factors influenced the Gram-negative intestinal microbiota of both species with possible impact on the interrelationship between the fishes and their digestive ecosystem, although the gut microbiota composition of fishes may result from host-specific selective pressures within the gut.

PURPOSE: To determine the effect of probiotics on the development of chemically induced (1, 2-dimethylhydrazine) colonic preneoplastic lesions, in mice. METHODS: The animals were divided into five groups. The control group was injected with carcinogen alone and the other groups also received probiotics (1- Lactobacillus delbrueckii UFV-H2b20; 2- Bifidobacterium animalis var. lactis Bb12; 3- L. delbrueckii UFV-H2b20 plus B. animalis var. lactis Bb12; and 4- Saccharomyces boulardii) administered orally in drinking water throughout fourteen weeks. RESULTS: Consumption of lactobacilli and bifidobacteria alone resulted in a significant reduction of the total number of aberrant crypt foci (55.7% and 45.1%, respectively). Significant reduction in the number of these small foci (<3 aberrant crypts) was only observed in the group treated with lactobacilli (52.2%) in comparison to control group. The number of larger foci (>3 aberrant crypts) crypts had no significant reduction. CONCLUSION: L. delbrueckii UFV-H2b20 and B. animalis var. lactis Bb12 administered alone protect colonic preneoplastic lesions in mice, while the combined treatment of these bacteria and the administration of S.boulardii were not effective in reducing such colonic lesions.

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.

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.

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.

No presente estudo, a produção de substâncias antagonistas por Lactobacillus murinus contra bactérias enteropatogênicas foi avaliada in vivo assim como um possível efeito protetor contra um desafio oral com Salmonella Typhimurium utilizando um modelo animal gnotobiótico. Um maior tempo médio de sobrevida (P < 0,05) foi observado nos animais associados com L. murinus (7,89 ± 3,83 dias) quando comparado com os controles (4,44 ± 0,73 dias). Lactobacillus murinus exerceu um potente efeito antagonista in vivo contra S. sonnei e com menos intensidade contra S. Typhimurium como revelado pelos halos de inibição ao redor das fezes dos animais associados com L. murinus. Os diâmetros dos halos de inibição ao redor dos conteúdos intestinais aumentaram ao longo do trato digestivo, seguindo proporcionalmente os níveis populacionais de L. murinus nas respectivas porções intestinais. Concluindo, o presente estudo mostra que a associação com L. murinus em camundongos gnotobióticos retarda a morte após um desafio oral com S. Typhimurium e que compostos inibitórios difusíveis obtidos em ensaios in vitro foram também produzidos in vivo e podem ser responsáveis por este efeito.

In the present study, production of antagonistic substances by Lactobacillus murinus against enteropathogenic bacteria was evaluated in vivo as well as a possible protective effect against an oral challenge with Salmonella Typhimurium using a gnotobiotic animal model. A higher mean survival time (P < 0.05) was observed in L. murinus-associated animals (7.89 ± 3.83 days) when compared with the control ones (4.44 ± 0.73 days). Lactobacillus murinus exerted a potent antagonistic effect in vivo against S. sonnei and to a lesser extent against S. Typhimurium as revealed by inhibitory halos around the feces of Lactobacillus-associated animals. Diameters of inhibitory halos around intestinal contents increased along the intestine tract, following proportionally the L. murinus population levels in the respective intestinal portions. Concluding, the present study showed that L. murinus association in gnotobiotic mice delayed the death after an oral challenge with S. Typhimurium and that inhibitory diffusible compounds obtained in in vitro assays were also produced in vivo and may be responsible for this effect.

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.

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.

Neste trabalho, estudamos a infecção experimental de camundongos Suíços/NIH com Leishmania major em comparação com camundongos isogênicos C57BL/6 e BALB/c que são resistentes e suceptíveis a esta infecção, respectivamente. Camundongos Suiços mostraram lesões que se curaram espontaneamente e se restringiram ao local de inoculação. Células linfóides derivadas destes animais desenvolveram uma resposta do tipo Th1, caracterizada pela produção de níveis altos de IFN-<FONT FACE="Symbol">g</font> e níveis baixos de IL-4. Com o objetivo de estudar a importância da microbiota no desenvolvimento da leishmaniose cutânea neste modelo, camundongos Suiços sem germes foram infectados na pata com promastigotas de L. major, convencionalizados após 3 semanas de infecção e as lesões comparadas com as observadas em camundongos convencionais. Os camundongos convencionalizados apresentaram lesões significativamente maiores do que as observadas nos camundongos convencionais. A análise histopatológica das lesões de todos os animais convencionalizados mostrou abcessos de tamanhos e formas variáveis e a presença de numerosos macrófagos parasitados. Nas lesões dos camundongos convencionais não houve a formação de abscessos e foram observados poucos macrófagos parasitados. Entretanto, células de ambos os grupos experimentais desenvolveram uma resposta similar do tipo Th1, caracterizada pela produção de altos níveis de IFN-<FONT FACE="Symbol">g</font> e baixos níveis de IL-4. Neste trabalho, demonstramos que os camundongos Suíços/NIH são resistentes à infecção por L. major, com o desenvolvimento de um fenótipo do tipo Th1 e que a ausência da microbiota no início da infecção influencia significativamente o tamanho da lesão e no processo inflamatório nestes animais.

Infection of Swiss/NIH mice with Leishmania major was compared with infection in isogenic resistant C57BL/6 and susceptible BALB/c mice. Swiss/NIH mice showed self-controlled lesions in the injected foot pad. The production of high levels of interferon-<FONT FACE="Symbol">g</font> (IFN-<FONT FACE="Symbol">g</font>) and low levels of interleukin-4 (IL-4) by cells from these animals suggests that they mount a Th1-type immune response. The importance of the indigenous microbiota on the development of murine leishmaniasis was investigated by infecting germfree Swiss/NIH in the hind footpad with L. major and conventionalizing after 3 weeks of infection. Lesions from conventionalized Swiss/NIH mice were significantly larger than conventional mice. Histopathological analysis of lesions from conventionalized animals showed abscesses of variable shapes and sizes and high numbers of parasitized macrophages. In the lesions from conventional mice, besides the absence of abscess formation, parasites were rarely observed. On the other hand, cells from conventional and conventionalized mice produced similar Th1-type response characterized by high levels of IFN-<FONT FACE="Symbol">g</font> and low levels of IL-4. In this study, we demonstrated that Swiss/NIH mice are resistant to L. major infection and that the absence of the normal microbiota at the beginning of infection significantly influenced the lesion size and the inflammatory response at the site of infection.

O aparecimento de halo de inibição contra o Vibrio cholerae a partir das fezes de ratos Wistar e Fischer nas idades de 10 a 42 dias foi observado usando um teste ex vivo em placa. A frequência de ratos Wistar apresentando halo aumentou de 0% (10 dias) até um máximo de 80,0% (29 dias) antes de decair para 53,3% (42 dias). Um perfil similar foi obtido com os ratos Fischer mas com valores inferiores (frequência máxima de 50,0% no dia 36). Num experimento separado quando ratos Wistar foram alimentados com uma ração deficiente em proteína a freqüência de halo diminuiu drasticamente. Três morfologias de colonias aparentemente distintas foram isoladas da microbiota fecal dominante: uma bactéria anaeróbia facultativa (ANF) e duas anaeróbias estritas (ANS). O teste inibitório ex vivo mostrou halo ao redor das fezes de camundongos isentos de germes monoassociados com a bactéria ANF ou uma das bactérias ANS mas não para os animais isentos de germes. Após desafio oral de todos os grupos com o V. cholerae, barreiras permissiva e drástica foram observadas em camundongos associados com as bactérias ANF e ANS, respectivamente. As bactérias ANF e uma ANS usadas em monoassociação nos desafios in vivo foram identificadas como Escherichia coli e Streptococcus intermedius, respectivamente. O potente antagonismo demostrado pela microbiota intestinal de ratos contra V. cholerae parece devido, em parte, a compostos difusíveis e este fenômeno depende aparentemente da idade, de espécie e da nutrição do animal. Esses dados preliminares sugerem também que este antagonismo seja devido a mais de um componente bacteriano num instante dado.

In an ex vivo agar plate assay, we monitored the appearance of an inhibitory halo against Vibrio cholerae from the feces of Wistar and Fischer rats aged 10 to 42 days. The frequency of Wistar rats showing halo increased from 0% (10 days) to a maximum of 80.0% (29 days) and then decreased to 53.3% (42 days). A similar pattern was obtained with Fischer rats but with a lower intensity (maximum frequency of 50.0% by day 36). In a separate experiment, when Wistar rats were fed a low-protein diet for 7 days, the inhibitory halo decreased drastically. Three apparently different colony morphologies were isolated from the dominant fecal microbiota: a facultative anaerobe (FAN) and two strict anaerobes (SAN). The ex vivo inhibitory test showed a halo around the feces of germfree mice monoassociated with the FAN bacterium or one of the SAN bacterium but not of the germfree ones. After oral challenge of all groups with V. cholerae, a permissive and a drastic barrier effects were observed in mice with FAN and SAN associated bacteria, respectively. The FAN and one SAN bacteria used in the in vivo challenges were identified as Escherichia coli and Streptococcus intermedius, respectively. The potent antagonism developed by the rat intestinal microbiota against V. cholerae seems to be due, in part, to diffusible compounds and this phenomenon depends apparently on age, strain and nutrition of the animals. These preliminary results also suggest that this effect was due to more than one bacterial component at any given moment.

A leishmaniose tegumentar foi muito mais grave em camundongos convencionais e convencionalizados do que em seus pares isentos de germes ou gnotobióticos. Usou-se um inóculo de Leishmania mexicana amazonensis.

Cutaneous leishmaniasis was much more severe in conventional than in gnotobiotic mice as revealed by macro and microscopic examination. An inoculum of Leishmania mexicana amazonensis was used.