Produção, purificação e caracterização de um peptídeo antimicrobiano produzido por uma linhagem de Bacillus sp.P34

Uma bactéria identificada como Bacillus sp. P34 isolada de intestino de peixe (Leporinus sp.) da Bacia Amazônica foi estudada quanto a sua capacidade de produzir substâncias do tipo-bacteriocina. As condições ótimas para produção da substância antimicrobiana foram determinadas. A produção da atividade antimicrobiana foi observada começando na fase exponencial de crescimento, sendo a atividade máxima observada no início da fase estacionária. Os resultados da Análise de Superfície de Resposta mostraram que a máxima produção da atividade antimicrobiana ocorreu a pH inicial entre 6.0 e 8.0 e temperaturas entre 25 e 37°C. A substância inibiu bactérias patogênicas e deteriorantes importantes em alimentos como Listeria monocytogenes, Bacillus cereus, Aeromonas hydrophila, Erwinia carotovora e Pasteurella haemolytica. O teste de termoestabilidade mostrou a perda de atividade quando a temperatura alcançou 100°C por 15 minutos. Foi sensível à ação das enzimas proteolíticas tripsina, papaína e pronase E. A substância antimicrobiana apresentou efeito bactericida e bacteriolítico sobre L. monocytogenes e B. cereus a 160 UA ml-1. O crescimento de Escherichia coli and Salmonella Enteritidis foi inibido somente quando o agente quelante EDTA foi adicionado juntamente. A atividade esporocida não foi observada. A análise da cultura de L. monocytogenes depois do tratamento com o composto antimicrobiano, usando espectroscopia de infravermelho com transformada de Fourier mostrou alterações no perfil de ácidos graxos e fosfolipídios da membrana celular bacteriana. Há evidências de que seu modo de ação interfira na membrana e na parede celular. A substância foi purificada pelo seguinte protocolo: precipitação com sulfato de amônio, cromatografias de gel filtração e de troca iônica. O peso molecular da substância foi determinado por espectroscopia de massas sendo 1498.68 Da. A substância antimicrobiana purificada apresentou sensibilidade ao tratamento com proteases e manutenção da atividade foi observada após congelamento e à incubação de 70°C por 30 minutos

Caracterização tecnológica de bactérias ácido lácticas isoladas de leite de ovelha para uso potencial como culturas não-iniciadoras

Sheep milk has different physicochemical properties, which means that its derived products have a high added value. Some of these properties are conferred by lactic bacteria which play important roles in the raw milk. This study aimed to analyze five lactic bacteria that were previously identified and evaluated for parameters such as their acidification, proteolysis, diacetyl and exopolysaccharides production, potential antimicrobial activity and safety parameters. The 16S rDNA gene identification via sequencing showed an agreement with the data obtained by MALDI-TOF/MS. The bacteria were identified as Lactococcus lactis MRS1, Lactococcus lactis MRS2, Lactococcus lactis MRS5, Lactococcus lactis MRS6, and Enterococcus faecalis M173. All isolates showed the same acidification profile, maintaining a pH of 4.5 from 6 h of incubation, under the conditions employed. Proteolytic activity, coexistence capacity, and production of exopolysaccharides were observed in all the isolates tested. Diacetyl production was only evident in the isolates Lactococcus lactis MRS1 and Lactococcus lactis MRS2. Regarding the presence of antimicrobial activity, Lactococcus lactis MRS6 and Enterococcus faecalis M173 isolates inhibited all tested cultures. In the evaluation of the safety parameters, none of the isolates presented high-level resistance to clinically important antibiotics and did not present gelatinase production and hemolytic activity. These results provide important information on the potential bacteria that could become valuable additives in sheep milk-derived products, in a condition of starters or adjunct cultures.O leite de ovelha possui diferentes propriedades físico-químicas, o que significa que seus derivados têm alto valor agregado. Parte dessas propriedades é conferida por bactérias lácticas que desempenham importantes atividades no leite cru. Esta pesquisa teve como objetivo analisar cinco bactérias lácticas previamente identificadas e avaliadas quanto a seus parâmetros de acidificação, proteólise, produção de diacetil e exopolissacarídeos, potencial atividade antimicrobiana e parâmetros de segurança. A identificação do gene 16S rDNA por sequenciamento mostrou concordância com os dados obtidos por MALDI-TOF/MS. As bactérias foram identificadas como Lactococcus lactis MRS1, Lactococcus lactis MRS2, Lactococcus lactis MRS5, Lactococcus lactis MRS6 e Enterococcus faecalis M173. Todos os isolados apresentaram o mesmo perfil de acidificação, mantendo o pH em 4,5 a partir de 6 horas de incubação, nas condições empregadas. Atividade proteolítica, capacidade de coexistência e produção de exopolissacarídeos foram observadas em todos os isolados testados. A produção de diacetil foi evidente nos isolados Lactococcus lactis MRS1 e Lactococcus lactis MRS2. Em relação à presença de atividade antimicrobiana, os isolados de Lactococcus lactis MRS6 e Enterococcus faecalis M173 inibiram todas as culturas testadas. Na avaliação dos parâmetros de segurança, nenhum dos isolados apresentou resistência de alto nível a antibióticos clinicamente importantes e não apresentaram produção de gelatinase e atividade hemolítica. Estes resultados fornecem uma informação importante sobre as potenciais bactérias a serem exploradas para a aplicação em produtos derivados de leite de ovelha, em condições de cultura starter ou adjuntas

Microbial contamination and antimicrobial resistance profiles indicate potential risks of infection at the veterinary medical teaching hospital - UFRGS, Porto Alegre, Brazil

Background: This study aimed to assess the level of bacterial contamination in the Small Animals Sector of the Veterinary Medical Teaching Hospital (HCV) of the Universidade Federal do Rio Grande do Sul (UFRGS). Firstly, a committee was invited to complete a questionnaire and to list critical sample sites for collection. With the identification of the places to be sampled, collections were made with sterile swabs on different surfaces of environments of the Veterinary Hospital. The identification of important bacteria in the veterinary area, in the different sampled environments, raises the concern for hygiene procedures in the veterinary hospital environment. Materials, Methods & Results: Sixteen samples were collected from these different areas, and microbiological analyses were performed. Standard counts of viable and strictly aerobic mesophilic microorganisms were realized. Collections were made to assess ambient air quality. With the microbiological analysis performed, bacteria of clinical importance were identified. To assess the resistance profile of the bacteria, the susceptibility test to antimicrobials was performed. MALDI-TOF/MS measurement identified 29 bacteria at the genus level and 10 bacteria at the species level and the antimicrobial susceptibility test was realized. Most of the isolates identified (60%) were bacteria of the genus Staphylococcus spp. Regarding antimicrobial susceptibility analysis the 10 bacteria identified at the species level were assessed. Test results showed that the isolates S. aureus, S. epidermidis and S. haemolyticus - collected from treatment room 2 - and S. haemolyticus, which had been isolated from samples from treatment room 2 of the cattery, presented multiresistance. Pantoea ananatis isolates from room 5 also showed a multiresistant profile for erythromycin, cephalothin, vancomycin and ampicillin. Micrococcus luteus isolates from the x-ray room and the kennel showed resistance to ceftazidime. Staphylococcus equorum isolates from room 4 were sensitive to all tested antimicrobials. Discussion: In Brazilian legislation there are no official microbiological parameters for surfaces in a veterinary hospital environment. The microorganisms present in the air are transient and variable, and the number and types of airborne agents is determined by the various sources of contamination in the environment. These microorganisms can be found in suspension, particulate matter and water droplets. Veterinary medical care tables are potentially contaminated by the animals handling, including those that sometimes defecate or urinate during their medical visit. Frequent handwashing is also known to be an important means of personal protection and disease prevention, although it is estimated that only 40% of practitioners do so routinely. Based on these results, we recommend a plan of bacterial control and disinfection that should be implemented to ensure more effective sanitary conditions. Microorganism counts were high in some of the veterinary hospital environments tested, indicating that current disinfection and hygiene practices are not sufficient to control the establishment of these microorganisms at the study sites. In view of this, it is reasonable to conclude that permanent monitoring and assessment of the effectiveness of hygiene protocols is needed in different sectors of the hospital. This may be an essential tool in a preventive approach to stop the spread of selectively resistant microorganisms, as well as cases of hospital infections. In addition, continuous staff training and awareness of the importance of personal and environmental hygiene is vital for minimizing the presence of these microorganisms in hospitals and avoid their transmission to patients. Finally, a more systematic hygiene guideline should be implemented in areas that showed higher counts

Biological activity of bacteria isolated from wetland sediments collected from a conservation unit in the southern region of Brazil

Wetlands are ecosystems rich in biodiversity and their ecological importance is recognized worldwide. Sediment samples were subjected to physical-chemical analysis and organic carbon content varied from 3.0% to 4.8%, the clay between 32 and 40%, silt with 41% and 43%, sand coarse varied between 6 and 11% and fi ne sand between 7 and 16%. The nitrogen values varied from 0.25% to 0.48%, the pH from 5.4 to 7.5 and the humidity varied from 44 to 56%. The selected isolates were evaluated for enzymatic properties and 64% showed positive results for amylase, 16% for gelatinase, 37% for lipase, 91% for protease and 2.7% for inulinase. Six bacterial isolates were selected for the overlapping assay and Bacillus sp. sed 2.2 showed inhibitory activity against Corynebacterium fi mi NCTC 7547, and the antimicrobial substance was partially purifi ed. The characterization of the substance was carried and the substance was stable at 100° C for up to 10 minutes and sensitive to the enzymes papain and trypsin. This substance was active against some species of Listeria, including Listeria monocytogenes ATCC 7644. The microorganims obtained from sediment samples were important sources of bioactive compounds, including enzymes and peptides, being a source of bioactive compounds to be studied

Elaboração de iogurte probiótico a base de leite orgânico

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The probiotic properties of Enterococcus faecium strains isolated from buffalo milk : food matrix studies

Buffalo milk has been increasingly explored in the production of dairy foods. Given the diversity of lactic acid bacteria present in this raw material, the functional potential of this type of milk should be studied. This study aims to assess the probiotic potential of two strains of Enterococcus faecium isolated from buffalo milk. We evaluated E. faecium M7AN7-1 and E. faecium M7AN10 for sustained cell viability under different conditions of conservation. We also assessed adhesion and cell viability in a simulated gastrointestinal transit test. E. faecium M7AN10 was selected for microencapsulation in sodium alginate and testing in the food matrix. The isolates maintained cell viability after refrigeration, freezing, and freeze-drying. E. faecium M7AN10 showed higher concentrations of viable cells than E. faecium M7AN7-1 after 180 min of contact with simulated gastric fluid, reaching 7.19 ± 0.59 Log10 CFU mL-1. We also observed sustained cell viability after exposure to simulated intestinal fluid. After encapsulation, E. faecium M7AN10 was tested in the food matrix of UHT milk. Cell count viability was maintained and its release was sustained in this medium for 30 days. The results of the in vitro assessment demonstrated that E. faecium M7AN10 remained functionally active under these experimental conditions. Similarly, they showed that it is a bacterium capable of sustaining viability in this type of food and after transit in a simulated gastrointestinal tract. Based on the data, we suggest this isolate may be a probiotic bacterial candidate for in vivo behavioral assessment

Characterization of the antimicrobial activity produced by Bacillus sp. isolated from wetland sediment

Bacteria of the genus Bacillus sp. present the potential for inhibiting various pathogens, making them a promising starting point in the search for new antimicrobial substances. In this study, bacteria were isolated from sediment samples from humid areas of a Natural Conservation Unit in the state of Rio Grande do Sul, Brazil. The isolate Bacillus sp. sed 1.4 was selected for production of antimicrobial activity, and was characterized by MALDI-TOF and 16S rDNA sequencing. Phylogenetic analysis showed that Bacillus sed 1.4 was closely related to Bacillus altitudinis and Bacillus pumilus. The cell-free supernatant was partially purifi ed using ammonium sulfate precipitation, gel fi ltration chromatography (Sephadex G-200) and an ultrafi ltration membrane. Partial purifi cation resulted in specifi c activity of 769.23 AU/mg, with a molecular mass of approximately 148 kDa. This antimicrobial substance showed stability at 100°C for 5 min, and was inactivated by proteolytic enzymes. An antimicrobial effect against Listeria species was observed. Considering the importance of the Listeria genus in the area of food safety, this antimicrobial activity should be further explored, specifi cally in the fi eld of dairy products and with a focus on food biopreservation studies

Atividade antimicrobiana in vitro de óleos essenciais de Heterothalamus Less. (Asteraceae) contra espécies bacterianas e fúngicas clinicamente relevantes

Essential oils are natural, complex, and volatile compounds produced by the secondary metabolism of plants, which often serve as a protection against fungal or bacterial attack. Two species of the Asteraceae family, Heterothalamus alienus (Spreng.) O. Kuntze and Heterothalamus psiadioides Less., are used in folk medicine and recognised for their production of bioactive substances. However, little evidence of their antimicrobial properties currently exists. The aim of this study was to examine the in vitro antimicrobial activity of essential oils from H. alienus and H. psiadioides against clinically relevant bacterial and fungal species. The major chemical components of the essential oils used are terpene substances, of which β-pinene is the major component. Analysis of antibacterial activity was conducted using the disc diffusion method against 22 bacteria (18 gram-positive strains, including 13 enterococci strains resistant or multiresistant to antibiotics, and 4 Gram- -negative strains) and 9 fungi (6 yeasts and 3 filamentous fungi). The results indicated that essential oils from Heterothalamus species have a strong antimicrobial effect against gram-positive bacteria and yeasts, variable activity against filamentous fungi, and no activity against gram-negative bacteria. The most prominent results were observed in the sensitivity of Enterococcus faecalis (antibiotic- and multidrug-resistant strains) to the essential oils. In conclusion, this study demonstrates the potential for essential oils from Heterothalamus species for use as both an antiseptic and disinfectant in future approaches to control of clinically relevant gram-positive bacteria and fungi.Óleos essenciais são compostos naturais, complexos e voláteis produzidos no metabolismo secundário de plantas, os quais muitas vezes servem como proteção contra o ataque de fungos ou bactérias. Duas espécies da família Asteraceae, Heterothalamus alienus (Spreng.) O. Kuntze e Heterothalamus psiadioides Less., são utilizadas na medicina popular e reconhecidas por suas substâncias bioativas. Entretanto, existem poucas evidências sobre suas proprieda- -des antimicrobianas. O objetivo deste estudo foi avaliar a atividade antimicrobiana in vitro dos óleos essenciais de H. alienus e H. psiadioides contra cepas clinicamente relevantes de bactérias e fungos. Os principais componentes químicos dos óleos essenciais utilizados são as substâncias terpênicas, da qual o β-pineno é o componente majoritário. A análise da atividade antimicrobiana foi conduzida usando a técnica de difusão em disco contra 22 bactérias (18 gram-positivas, incluindo 13 enterococos resistentes ou multirresistentes a antibióticos, e 4 gram-negativas) e 9 fungos (6 leveduras e 3 fungos filamentosos). Nossos resultados indicaram que os óleos essenciais das espécies de Heterothalamus possuem forte atividade antimicrobiana contra bactérias gram-positivas e leveduras, atividade variável contra fungos filamentosos e nenhuma atividade contra bactérias gram-negativas. O resultado mais proeminentes observado foi a sensibilidade de Enterococcus faecalis resistentes e multirresistentes aos óleos essenciais. Em conclusão, este estudo demonstra o potencial dos óleos essenciais das espécies Heterothalamus para serem usados como antissépticos e desinfetantes, abordando o controle de bactérias gram-positivas e fungos de importância clínica