RESUMO
OBJECTIVES: Putative new dioxygenases were identified in a metagenomic ß-lactam-resistance screening and, given their key role on aromatic metabolism, we raise the hypothesis that these enzymes maybe concomitantly related to antibiotic resistance and aromatic degradation. RESULTS: ORFs of three putative dioxygenases were isolated from resistant metagenomic clones. One of them, CRB2(1), was subcloned into pET24a expression vector and subjected to downstream phenotypic and bioinformatics analyses that demonstrated the "dual effect" of our metagenomic dioxygenase, on antibiotic and aromatic resistance. Furthermore, initial characterization assays strongly suggests that CRB2(1) open-reading frame is an extradiol-dioxygenase, most probably a bicupin domain gentisate 1,2-dioxygenase. This observation is, to our knowledge, the first description of a metagenomic dioxygenase and its action on ß-lactam resistance. CONCLUSION: Unraveling the diversity of antibiotic resistance elements on the environment could not only identify new genes and mechanisms in which bacteria can resist to antibiotics, but also contribute to biotechnology processes, such as in bioremediation.
Assuntos
Dioxigenases/genética , Dioxigenases/metabolismo , Resistência beta-Lactâmica , Biodegradação Ambiental , Brasil , Clonagem Molecular , Biblioteca Gênica , Genes Bacterianos , Metagenoma/efeitos dos fármacos , Fases de Leitura Aberta , Filogenia , Microbiologia do SoloRESUMO
Physalin B is a natural secosteroidal, extracted from the Solanaceae plant, Physalis angulata, and it presents immune-modulator effects on the bloodsucking bug, Rhodnius prolixus. In this work, R. prolixus was treated with physalin B at a concentration of 1 mg/ml of blood meal (oral application), or 20 ng/insect (applied topically) or 57 ng/cm(2) of filter paper (contact treatment), and infected with Trypanosoma cruzi Dm28c clone (2×10(6) epimastigotes/insect). The three types of applications significantly decreased the number of T. cruzi Dm28c in the gut comparing with the non-treated infected insects (controls). All groups of infected insects treated with physalin B had higher numbers of bacterial microbiota in the gut than the non-treated controls infected with T. cruzi. We observed that the infected physalin B insects with topical and contact treatments had a lower antibacterial activity in the gut when compared with control infected insects. Furthermore, infected insects with the physalin B oral treatment produced higher levels of nitrite and nitrate in the gut than control infected insects. These results demonstrate that physalin B decreases the T. cruzi transmission by inhibiting the parasite development in the insect vector R. prolixus. Herein the importance of physalin B modulation on the immune system and microbiota population in terms of parasite development and transmission are discussed.
Assuntos
Interações Hospedeiro-Parasita/efeitos dos fármacos , Rhodnius/efeitos dos fármacos , Secoesteroides/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Metagenoma/efeitos dos fármacos , Muda/efeitos dos fármacos , Nitratos/metabolismo , Nitritos/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Rhodnius/imunologia , Rhodnius/metabolismo , Rhodnius/microbiologia , Rhodnius/parasitologiaRESUMO
The gorgonian Pseudopterogorgia elisabethae collected at Providencia Island (Colombia) has an unfouled surface, free of obvious algal and invertebrate growth. This gorgonian produces significant amounts of the glycosilated diterpenes pseudopterosins and seco-pseudopterosins (Ps and seco-Ps). Our previous experiments have shown activity of these compounds against eukaryotic (human cancer cell lines and Candida albicans) and prokaryotic cells (Staphylococcus aureus and Enterococcus faecalis). However, the potential role of pseudopterosins on the regulation of the fouling process is still under study. We evaluated the activity of these compounds against bacteria isolated from heavily fouled marine surfaces as an indicator of antifouling activity. Additionally, we assessed their activity against bacteria isolated from P. elisabethae to determine whether potentially they play a role in preventing surface bacterial colonization, thus impairing presumptively the establishment of further successional stages of fouling communities. Results showed that Ps and seco-Ps seem to modulate bacterial growth (controlling Gram-positive bacterial growth and inducing Gram-negative bacterial associations). We thus hypothesized that Ps and seco-Ps may play a role in controlling microbial fouling communities on the surface of this gorgonian. By using bTEFAP and FISH we showed that the most abundant bacteria present in the microbial communities associated with P. elisabethae are Gram-negative bacteria, with Proteobacteria and Gammaproteobacteria the most representative. To evaluate whether Ps and seco-Ps have a direct effect on the structure of the bacterial community associated with P. elisabethae, we tested these compounds against culturable bacteria associated with the surface of P. elisabethae, finding remarkable selectivity against Gram-positive bacteria. The evidence presented here suggests that Ps and seco-Ps might have a role in the selection of organisms associated with the gorgonian surface and in the regulation of the associated bacterial community composition.
Assuntos
Antozoários/química , Antozoários/microbiologia , Bactérias/efeitos dos fármacos , Diterpenos/isolamento & purificação , Diterpenos/farmacologia , Fenômenos Ecológicos e Ambientais , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Metagenoma/efeitos dos fármacos , Animais , Antibacterianos/análise , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bactérias/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Incrustação Biológica/prevenção & controle , Diterpenos/análise , Glicosídeos/análise , Índias OcidentaisRESUMO
AIMS/HYPOTHESIS: A high-fat dietary intake induces obesity and subclinical inflammation, which play important roles in insulin resistance. Recent studies have suggested that increased concentrations of circulating lipopolysaccharide (LPS), promoted by changes in intestinal permeability, may have a pivotal role in insulin resistance. Thus, we investigated the effect of gut microbiota modulation on insulin resistance and macrophage infiltration. METHODS: Swiss mice were submitted to a high-fat diet with antibiotics or pair-feeding for 8 weeks. Metagenome analyses were performed on DNA samples from mouse faeces. Blood was collected to determine levels of glucose, insulin, LPS, cytokines and acetate. Liver, muscle and adipose tissue proteins were analysed by western blotting. In addition, liver and adipose tissue were analysed, blinded, using histology and immunohistochemistry. RESULTS: Antibiotic treatment greatly modified the gut microbiota, reducing levels of Bacteroidetes and Firmicutes, overall bacterial count and circulating LPS levels. This modulation reduced levels of fasting glucose, insulin, TNF-α and IL-6; reduced activation of toll-like receptor 4 (TLR4), c-Jun N-terminal kinase (JNK), inhibitor of κ light polypeptide gene enhancer in B cells, kinase ß (IKKß) and phosphorylated IRS-1 Ser307; and consequently improved glucose tolerance and insulin tolerance and action in metabolically active tissues. In addition, there was an increase in portal levels of circulating acetate, which probably contributed to an increase in 5'-AMP-activated protein kinase (AMPK) phosphorylation in mice. We observed a striking reduction in crown-like structures (CLS) and F4/80(+) macrophage cells in the adipose tissue of antibiotic-treated mice. CONCLUSIONS/INTERPRETATION: These results suggest that modulation of gut microbiota in obesity can improve insulin signalling and glucose tolerance by reducing circulating LPS levels and inflammatory signalling. Modulation also appears to increase levels of circulating acetate, which activates AMPK and finally leads to reduced macrophage infiltration.
Assuntos
Antibacterianos/farmacologia , Dieta Hiperlipídica/efeitos adversos , Trato Gastrointestinal/microbiologia , Insulina/fisiologia , Metagenoma/efeitos dos fármacos , Obesidade/fisiopatologia , Transdução de Sinais/fisiologia , Quinases Proteína-Quinases Ativadas por AMP , Acetatos/sangue , Animais , Bacteroides/isolamento & purificação , Movimento Celular/fisiologia , Citocinas/sangue , Modelos Animais de Doenças , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/patologia , Resistência à Insulina/fisiologia , Lipopolissacarídeos/sangue , Macrófagos/patologia , Masculino , Camundongos , Obesidade/etiologia , Obesidade/patologia , Proteínas Quinases/fisiologiaRESUMO
Trypanosoma cruzi in order to complete its development in the digestive tract of Rhodnius prolixus needs to overcome the immune reactions and microbiota trypanolytic activity of the gut. We demonstrate that in R. prolixus following infection with epimastigotes of Trypanosoma cruzi clone Dm28c and, in comparison with uninfected control insects, the midgut contained (i) fewer bacteria, (ii) higher parasite numbers, and (iii) reduced nitrite and nitrate production and increased phenoloxidase and antibacterial activities. In addition, in insects pre-treated with antibiotic and then infected with Dm28c, there were also reduced bacteria numbers and a higher parasite load compared with insects solely infected with parasites. Furthermore, and in contrast to insects infected with Dm28c, infection with T. cruzi Y strain resulted in a slight decreased numbers of gut bacteria but not sufficient to mediate a successful parasite infection. We conclude that infection of R. prolixus with the T. cruzi Dm28c clone modifies the host gut immune responses to decrease the microbiota population and these changes are crucial for the parasite development in the insect gut.
Assuntos
Intestinos/imunologia , Intestinos/parasitologia , Metagenoma/imunologia , Rhodnius/microbiologia , Rhodnius/parasitologia , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Antibacterianos/farmacologia , Doença de Chagas/imunologia , Doença de Chagas/metabolismo , Doença de Chagas/microbiologia , Imunidade Humoral/efeitos dos fármacos , Imunidade Humoral/imunologia , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Metagenoma/efeitos dos fármacos , Monofenol Mono-Oxigenase/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Rhodnius/efeitos dos fármacos , Rhodnius/metabolismo , Especificidade da Espécie , Análise de Sobrevida , Trypanosoma cruzi/patogenicidadeRESUMO
BACKGROUND: Previous work showed that daily ingestion of an aqueous soy extract fermented with Enterococcus faecium CRL 183 and Lactobacillus helveticus 416, supplemented or not with isoflavones, reduced the total cholesterol and non-HDL-cholesterol levels, increased the high-density lipoprotein (HDL) concentration and inhibited the raising of autoantibody against oxidized low-density lipoprotein (ox-LDL Ab) and the development of atherosclerotic lesions. OBJECTIVE: The aim of this study was to characterize the fecal microbiota in order to investigate the possible correlation between fecal microbiota, serum lipid parameters and atherosclerotic lesion development in rabbits with induced hypercholesterolemia, that ingested the aqueous soy extract fermented with Enterococcus faecium CRL 183 and Lactobacillus helveticus 416. METHODS: The rabbits were randomly allocated to five experimental groups (n = 6): control (C), hypercholesterolemic (H), hypercholesterolemic plus unfermented soy product (HUF), hypercholesterolemic plus fermented soy product (HF) and hypercholesterolemic plus isoflavone-supplemented fermented soy product (HIF). Lipid parameters and microbiota composition were analyzed on days 0 and 60 of the treatment and the atherosclerotic lesions were quantified at the end of the experiment. The fecal microbiota was characterized by enumerating the Lactobacillus spp., Bifidobacterium spp., Enterococcus spp., Enterobacteria and Clostridium spp. populations. RESULTS: After 60 days of the experiment, intake of the probiotic soy product was correlated with significant increases (P < 0.05) on Lactobacillus spp., Bifidobacterium spp. and Enterococcus spp. and a decrease in the Enterobacteria population. A strong correlation was observed between microbiota composition and lipid profile. Populations of Enterococcus spp., Lactobacillus spp. and Bifidobacterium spp. were negatively correlated with total cholesterol, non-HDL-cholesterol, autoantibodies against oxidized LDL (oxLDL Ab) and lesion size. HDL-C levels were positively correlated with Lactobacillus spp., Bifidobacterium spp., and Enterococcus spp. populations. CONCLUSION: In conclusion, daily ingestion of the probiotic soy product, supplemented or not with isoflavones, may contribute to a beneficial balance of the fecal microbiota and this modulation is associated with an improved cholesterol profile and inhibition of atherosclerotic lesion development.
Assuntos
Aterosclerose/prevenção & controle , Fezes/microbiologia , Glycine max , Metagenoma/efeitos dos fármacos , Extratos Vegetais/farmacologia , Probióticos/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/etiologia , Carga Bacteriana/efeitos dos fármacos , Colesterol/efeitos adversos , Avaliação Pré-Clínica de Medicamentos , Enterococcus faecium , Fermentação , Hipercolesterolemia/sangue , Hipercolesterolemia/induzido quimicamente , Hipercolesterolemia/complicações , Lipídeos/sangue , Masculino , CoelhosRESUMO
Little was known about the development of the gastrointestinal (GI) tract microbiota, until recently, because of difficulties in obtaining sufficient sequence information from enough people or time points. Now, with decreased costs of DNA sequencing and improved bioinformatic tools, we can compare GI tract bacterial communities among individuals, of all ages from infancy to adulthood. Some key recent findings are that the initial bacterial community, even in the GI tract, depends strongly on delivery mode; that the process of early development of the microbiota is highly unstable and idiosyncratic; that the microbiota differs considerably among children from different countries; and that older adults have substantially different GI tract communities than younger adults, indicating that the GI tract microbiota can change throughout life. We relate these observations to different models of evolution including the evolution of senescence and suggest that probiotics be selected based on patient age. Studies of the microbiota in older people might tell us which probiotics could increase longevity. Drug metabolism varies among individuals with different microbial communities, so age- and region-specific clinical trials are required to ensure safety and efficacy.