Characterization of microorganisms present in a slaughterhouse and beef processing/chilling environment

The ingestion of contaminated foods of animal origin has considerably increased the occurrence of food-borne diseases. Several bacterium species are deemed responsible for outbreaks of those diseases and the presence of such species in food processing plants depends on the proper hygiene routines there adopted. The aims of this study were: (i) to isolate and characterize the microorganisms present in a bovine slaughterhouse and processing/chilling environment, and (ii) to identify enterotoxigenic Escherihcia coli (ETEC) among isolates using the PCR amplification protocol. Out of the total 580 bacteria isolated and identified by biochemical assays, 168 were Staphylococcus aureus, 123 Escherichia coli, and 79 Corynebacterium vitarumem. The remaining belonged to the Bacillus spp. and Corynebacterium spp. genera, and to the Enterobacteriaceae family. The 123 bacteria identified as E. coli underwent a plasmid DNA extraction routine for the PCR assays. The results revealed 37 E. coli isolates to be positive for the ST gene, out of which 20 were correspondingly positive for the LT gene and 2 were only LT positive. The data evidenced the predominance of bacteria indicating faecal contamination and of bacteria found in bovine hides. The sampling sites of higher contamination were desks, in both the slaughterhouse and in processing/chilling plants inspected

Avaliação in vitro da atividade antagônica de fungos isolados de sementes e folhas de trigo contra o fitopatógeno Bipolaris sorokiniana

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Evaluation of the antifungal activity of Streptomyces sp. on Bipolaris sorokiniana and the growth promotion of wheat plants

Streptomyces sp. R18(6) and Streptomyces sp. 6(4) strains were evaluated for their ability to control brown spot and common root rot caused by Bipolaris sorokiniana in wheat crops. The antifungal activity of these isolates was tested using a double-layer assay and culture pairing at 28 °C. Physiological and enzymatic activity performed through siderophore, indole-3-acetic acid, nitrogen fixation and phosphate solubilization tests. The biocontrol of the disease and growth-promoting efficiency of wheat seedlings were assessed using in vivo assays in a greenhouse. In the culture pairing assays, both strains inhibited B. sorokiniana mycelial growth, while in the double-layer only Streptomyces sp. R18(6). Streptomyces sp. 6(4) produced auxin, siderophores, fixed nitrogen and solubilized phosphate, whereas R18(6) did not produce siderophores. In the greenhouse assays, strain R18(6) showed statistical differences in shoot dry mass and root dry mass compared with those of strain 6(4) in the presence of the phytopathogen (P ≤ 0.05), and these results were more evident when the environmental temperature was higher. In the absence of the phytopathogen, Streptomyces sp. 6(4) strain increased the root dry mass compared with that of the control during the same period. Therefore, these isolates can potentially control root rot and brown spotting and may promote the growth of wheat plants

Temporal variation of bacterial population and response to physical and chemical parameters along a petrochemical industry wastewater treatment plant

The petrochemical industry has played a considerable role in generation and release of waste in the environment. Activated sludge and facultative lagoons are commonly used for domestic and industrial wastewater treatment due to their low-cost and minimal need for operational requirements. Microorganisms present in wastewater treatment plant (WWTP) are responsible for most nutrient removal. In this study, microbiological and physicochemical parameters were used to estimate changes in bacterial community in a petrochemical industrial WWTP. The activated sludge was the place with higher heterotrophic bacterial quantification. Denitrifying bacteria was reduced at least 5.3 times throughout all collections samples. We observe a decrease in the total Kjeldahl nitrogen, oxygen demand and phosphate throughout the WWTP. In this work, we also use Matrix-Assisted Laser Desorption Ionisation-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for bacteria isolates identification comparing with 16S rDNA sequencing. The MALDITOF MS allowed the identification of 93% of the isolates and only 5% show different results from 16S rDNA sequencing showing that the MALDI-TOF MS can be a tool for identifying environmental bacteria. The observation of microbial community dynamics in the WWTP is important in order to understand the functioning of the ecological structure formed in a specific environment

O uso de PCR na detecção de Escherichia coli enterotoxigênica em amostras de água de esgoto

Riscos à saúde, associados à transmissão de doenças através do meio aquático, tornam importante a detecção de microrganismos patogênicos nesse ambiente. Portanto, o objetivo deste trabalho foi utilizar a técnica de PCR para uma detecção rápida de Escherichia coli enterotoxigênica (ETEC) em água de esgoto. Com o propósito de isolar esta bactéria, as amostras de esgoto foram inoculadas no meio seletivo ágar eosina azul de metileno (EMB). As colônias isoladas foram identificadas através de testes bioquímicos e as E. coli encontradas foram submetidas à extração de DNA plasmideal por lise alcalina e posteriormente à reação de PCR. Desses isolados, 5 apresentaram resultado positivo: 3 isolados foram positivos para o gene da toxina LT e 2 isolados para os genes de ambas as toxinas. As condições utilizadas na reação de PCR não detectaram a presença de E. coli, quando aplicada diretamente nas amostras de esgoto. Os produtos de amplificação dos 5 isolados de E. coli foram digeridos com enzimas de restrição e os fragmentos resultantes confirmaram a especificidade da amplificação. Os resultados sugerem que a técnica de PCR reduz o tempo de detecção do microrganismo, porém a amostra necessita de um pré-enriquecimento para aumentar a sensibilidade da técnica

Evaluation of the activity of disinfectants against coliform bacteria group strains isolated from a sewage treatment plant (ETE-Ipanema)

Municipal wastewater usually presents pathogenic enteric bacteria, viruses and intestinal parasites. Primary, secondary and tertiary wastewater treatment eliminates 90-99.9% of these microorganisms and if a more efficient reduction of microorganisms is needed, disinfection with chemical compounds must be done. The extraordinary genetic variability of bacteria gives them adaptation ability against theses chemical agents, which indiscriminate use brings about serious health problems. In this study, chemical agents commonly used in disinfection procedures like chlorhexidine gluconate, sodium hypochlorite and quaternary ammonium compounds were investigated for their activity against bacteria from the coliform group, isolated from a sewage treatment plant, using the macrodilution method. It was observed that the addition of organic matter interferes in sensitivity levels and also that the lower the concentration and the exposure time, the higher the resistance. Sodium hypochlorite was the most effective against the coliforms, once inhibited their growth in every exposure time and at every disinfectant concentration used . Quaternary ammonium compounds and chlorhexidine gluconate were also effective, although a higher percentage of resistance was observed for both

Occurrence and susceptibility of yeasts present in the polluted waters of Dilúvio Stream, Porto Alegre, Brazil

Water is one of the naturally occurring essential requirements of all life functions.  Today water pollution has become one of the major problems for all life forms. Dilúvio Stream is one of the main watercourses in Porto Alegre, Brazil and receives domestic and hospital sewage and rain waters, which favors the growth of microorganisms. This study investigates the presence of yeasts in water samples collected from Dilúvio Stream and assesses virulence factors and the susceptibility of antifungal agents commonly prescribed for the treatment of mycoses. Samples were collected at three sites, in two seasons of the year, seeded and isolated in different culture media supplemented with chloramphenicol. Biochemical assays, thermotolerance at 37ºC, proteinase, and phospholipase activities were carried out to identify isolates. Tests to determine resistance to antifungal included a susceptibility test and minimum inhibitory concentration (MIC) evaluation using different antifungal agents. Results showed that Ascomycota prevailed among the isolates and that 25% of which were potentially opportunistic yeasts. The susceptibility test revealed that the isolates were resistant to fluconazole and voriconazole, while the minimum inhibitory concentration test showed resistance to fluconazole and to itraconazole

Purification and characterization of a thermostable alkaline cellulase produced by Bacillus licheniformis 380 isolated from compost

During composting processes, the degradation of organic waste is accomplished and driven by a succession of microbial populations exhibiting a broad range of functional competencies. A total of 183 bacteria, isolated from a composting process, were evaluated for cellulase activity at different temperatures (37, 50, 60, and 70°C) and pH values. Out of the 22 isolates that showed activity, isolate 380 showed the highest cellulase activity. Its ability to produce cellulase was evaluated in culture medium supplemented with carboxymethyl cellulose, microcrystalline cellulose, wheat straw, and rice husk. The culture medium supplemented with carboxymethyl cellulose induced higher enzyme activity after 6 hours of incubation (0.12 UEA mL-1 min-1). For wheat straw and rice husk, the results were 0.08 UEA mL-1 min-1 for both, while for microcrystalline cellulose, 0.04 UEA mL-1 min-1 were observed. The highest carboxymethyl cellulase activity was observed at 60°C (0.14 UEA mL-1 min-1) for both crude and partially purified enzyme after 30 and 120 min of incubation, respectively. Alkalinization of the medium was observed during cultivation in all substrates. The cellulase had a molecular mass of 20 kDa determined by SDS-Page. Isolate 380 was identified as Bacillus licheniformis. This work provides a basis for further studies on composting optimization

Estudo de Produção de Compostos com Atividade Antimicrobiana produzidos por Streptomyces sp . 1S

Em um estudo preliminar de avaliação da atividade antimicrobiana, um isolado de Streptomyces caracterizado como linhagem 1S foi selecionado em decorrência do seu alto potencial como produtor de metabólitos bioativos. Este isolado apresentou um amplo espectro de atividade inibindo 46 dos 54 microrganismos-alvos (bactérias Gram-positivas e Gram-negativas, leveduras e fungos filamentosos). No presente estudo, foi avaliada a influência das condições ambientais como meio de cultivo, temperatura de incubação, cultivo estático ou com agitação e tempo de crescimento para a produção de moléculas bioativas. Para tanto, foram utilizados sete meios de cultura: caldo amido caseína (AC); caldo Czapeck Dox modificado (CPD); meio proposto por Sahin (S); meio Bennett´s (B); caldo nutritivo (CN); meio extrato de malte e levedura (ISP2) e caldo tripticaseina de soja (TSB). As temperaturas avaliadas foram 28, 30, 35 e 40 °C. Para cada um dos ensaios foi determinada a atividade antimicrobiana, o pH e a biomassa celular. Das condições de cultivo avaliadas, a melhor atividade antimicrobiana foi observada nos extratos provenientes do crescimento de Streptomyces sp. 1S no meio de cultura ISP2, a uma temperatura de 28 °C e com 96 horas de cultivo. Nestas condições, o isolado inibiu 15, dos 17 microrganismos-alvo empregados neste estudo, incluindo microrganismos multirresistentes como Staphylococcus aureus MRSA, Enterococcus sp. e  Salmonella enterica subsp. enterica Enteretidis. Palavras-chave: Streptomyces. Atividade antimicrobiana. Resistência.  Compostos bioativos

Antifungal activity of actinobacteria against fungus isolates of clinical importance

The escalating use of antifungal agents and long-term treatment approaches has led to an increased prevalence of fungus species that are resistant to the most common antifungal drugs. Actinobacteria produce a wide variety of bioactive secondary metabolites. This study investigated actinomycete isolates with the potential to produce bioactive compounds against dermatophyte fungus species and Candida spp. of clinical importance. Antifungal activity of actinomycetes was assessed using the double-layer agar technique. Active isolates were cultivated in starch casein broth (SCB) at 30 ºC for 8 days. Aliquots were retrieved at 24-h intervals and centrifuged to obtain extracts. Extract activity was assessed using the well-diffusion method. No dermatophyte fungus isolate was inhibited in the double-layer assay, although isolates 1S, R18(6) and 6(2) were active against all Candida spp. used in the assay. The well-diffusion method revealed that isolate R18(6) inhibited the six Candida spp. in a 72-h growth period in SCB broth, showing good potential to yield a compound with antifungal activity.(Atividade antifúngica de actinobactérias contra fungos isolados de importância clínica).O uso crescente de antifúngicos e os tratamentos prolongados vêm aumentando a incidência de fungos resistentes às drogas antifúngicas comumente utilizadas. As actinobactérias são conhecidas por produzirem uma grande variedade de metabólitos secundários bioativos e este trabalho teve como objetivo selecionar isolados de actinomicetos com potencial para produção de compostos bioativos contra fungos dermatófitos e espécies de Candida de importância clínica. A atividade antifúngica dos actinomicetos foi avaliada pela técnica da dupla camada. Os isolados que apresentaram atividade foram cultivados em caldo amido caseína (AC) à temperatura de 30 ºC por oito dias e foram retiradas e centrifugadas alíquotas a cada 24h, para obtenção do extrato. A atividade dos extratos foi avaliada através da técnica de difusão em poço. Nenhum dos isolados de fungos dermatófitos foi inibido no ensaio de dupla camada e os isolados 1S, R18(6) e 6(2) mostraram atividade frente todas as espécies de Candida testadas. No ensaio de difusão em poço com os extratos, o isolado R18(6) inibiu as seis espécies de Candida em 72h de crescimento em caldo AC e mostrou grande potencial para obtenção de composto com atividade antifúngica