Screening of wild type Streptomyces isolates able to overproduce clavulanic acid

The selection of new microorganisms able to produce antimicrobial compounds is hoped for to reduce their production costs and the side effects caused by synthetic drugs. Clavulanic acid is a β-lactam antibiotic produced by submerged culture, which is widely used in medicine as a powerful inhibitor of β-lactamases, enzymes produced by bacteria resistant to antibiotics such penicillin and cephalosporin. The purpose of this work was to select the best clavulanic acid producer among strains of Streptomyces belonging to the Microorganism Collection of the Department of Antibiotics of the Federal University of Pernambuco (DAUFPE). Initially, the strains were studied for their capacity to inhibit the action of β-lactamases produced by Klebsiella aerogenes ATCC 15380. From these results, five strains were selected to investigate the batch kinetics of growth and clavulanic acid production in submerged culture carried out in flasks. The results were compared with the ones obtained by Streptomyces clavuligerus ATCC 27064 selected as a control strain. The best clavulanic acid producer was Streptomyces DAUFPE 3060, molecularly identified as Streptomyces variabilis, which increased the clavulanic acid production by 28% compared to the control strain. This work contributes to the enlargement of knowledge on new Streptomyces wild strains able to produce clavulanic acid by submerged culture.

Endophytic microorganisms from leaves of Spermacoce verticillata (L.): Diversity and antimicrobial activity.

Endophytic microorganisms from the base and top leaves of Spermacoce verticillata were isolated and the antimicrobial potential was evaluated. A total of 56 strains were isolated in pure culture, 44 fungi and 12 actinobacteria. The isolation frequency was higher in the base leaves (12.5%), when compared to the top leaves (3.05%). Among all fungi and actionobacteria identified, the majority belonged to the genus Guignardia (25%) and Microbispora (41.66%), respectively. The antimicrobial screening was firstly evaluated by agar plug assay and showed that 28.57% of the isolates presented activity mainly against gram positive bacteria Staphylococus aureus (ATCC-6538) and Bacillus subtilis (UFPEDA-16). The microorganisms that presented the best activities were then selected, evaluated in different culture media broth, and tested by disk diffusion assay using their fermented broth. The microorganisms selected for this assay exhibited antimicrobial activity mainly for Bacillus subtilis (UFPEDA-16). Since many isolates showed inhibitory activity against pathogenic microorganisms, it is suggestive that endophytic microorganisms from S. verticillata could be an interesting source to explore for bioactive metabolites and new tools need to be employed to explore the real potential of these microorganisms.

Resistência antimicrobiana e perfil plasmidial de Escherichia coli isolada de frangos de corte e poedeiras comerciais no Estado de Pernambuco / Antimicrobial resistance and plasmidial profile of Escherichia coli strain isolated from broilers and commercial layers in the state of Pernambuco, Brazil

Embora existam linhagens de Escherichia coli não patogênicas para aves, muitas outras possuem a capacidade de causar sérios danos à saúde das mesmas, sendo capazes de ocasionar diferentes tipos de processos infecciosos. As linhagens patogênicas são denominadas Avian Pathogenic Escherichia coli (APEC), possuindo genes relacionados ao processo de patogênese em epissomos (plasmídios) ou no cromossomo. A presença de plasmídios, contendo genes de resistência a antibióticos em linhagens aviárias, patogênicas ou não, indicam a possibilidade de transferência gênica lateral entre diferentes tipos de linhagens facilitando também a transferência de genes de patogenicidade ou virulência. Objetivou-se com este estudo avaliar o perfil de sensibilidade a antibióticos (13) de diferentes amostras (35) de E. coli isoladas de aves comerciais do Estado de Pernambuco apresentando, ou não, sinais clínicos de processos infecciosos e correlacionar esta resistência com a presença de plasmídios. Os testes utilizados demonstraram que 94,28% dos isolados foram resistentes a três ou mais antibióticos, com a lincomicina apresentando o maior percentual de resistência (100%). Na Concentração Inibitória Mínima (CIM) observou-se multirresistência a vários antimicrobianos. A presença de plasmídios foi detecada em 80,0% (28/35) dos isolados, com 16 isolados apresentando plasmídios com peso molecular aproximado de 88 MDa. Também foi verificada a presença de linhagens apresentando plasmídios de vários tamanhos. Concluiu-se que isolados de E. coli resistentes a antimicrobianos utilizados na avicultura estão presentes no Estado de Pernambuco, tanto em frangos de corte quanto em poedeiras comerciais. A presença de plasmídios detectados na maioria dos isolados pode estar associada à resistência aos antimicrobianos e sugere a presença de possíveis genes relacionados à patogenicidade. Monitorar a resistência a antibióticos em bactérias isoladas de animais torna-se um fator determinante para eleição e êxito do tratamento, bem como a possibilidade de eliminação daquelas que possuem plasmídios para se evitar a transferência de genes relacionados à patogenicidade.

Although exist poultry non-pathogenic Escherichia coli strains, many others have capacity to impose serious damages to this birds, being able to cause different infectious diseases. Pathogenic strains are termed Avian pathogenic Escherichia coli (APEC) strains. APEC strains harbor chromossomal and plasmid pathogenicity-related genes. The presence of resistance plasmids in avian E. coli strains could facilitate horizontal tranfer of virulence gene between pathogenic and non pathogenic strains. The aim of this paper was to determine the resistance level to 13 different antibacterial drugs of avian E. coli strains (35) isolated from commercial poultry of Pernambuco State, Brazil, and to correlate the detected resistance level to the presence of plasmids. The results show that 94.28% of strains were resistant to at least three different antibacterial drugs with the highest percentage to lincomycin. The Minimal Inibitory Concentration (MIC) showed that multi- resistance to various antibacterial drugs was present in these strains. Plasmids of several sizes, including plasmids of approximately 88Mda were detected in most of the studied strains. The results herein obtained suggest that the high resistance level observed could be due to the presence of plasmids, what could facilitate the transfer of pathogenicity related genes among pathogenic and non pathogenic strains; it is necessary to take a constant survey on the resistance level to antimicrobial drugs of avian E. coli strains to reach a better control of APEC strains and avoid transfer of pathogenicity related genes between strains.

Optimization of clavulanic acid production by Streptomyces daufpe 3060 by response surface methodology

Clavulanic acid is a β-lactam antibiotic which has a potent β-lactamase inhibiting activity. In order to optimize its production by the new isolate Streptomyces DAUFPE 3060, the influence of two independent variables, temperature and soybean flour concentration, on clavulanic acid and biomass concentrations was investigated in 250 mL-Erlenmeyers according to a 2² central composite design. To this purpose, temperature and soybean flour (SF) concentration were varied in the ranges 26-34°C and 10-50 g/L, respectively, and the results evaluated utilizing the Response Surface Methodology. The experimental maximum production of clavulanic acid (629 mg/L) was obtained at 32°C and 40 g/L SF after 48 h, while the maximum biomass concentration (3.9 g/L) at 30°C and 50 g/L soybean flour, respectively. These values are satisfactorily close to those (640 mg/L and 3.75 g/L, respectively) predicted by the model, thereby demonstrating the validity of the mathematical approach adopted in this study.

Isolation of endophytic actinomycetes from roots and leaves of maize (Zea mays L.)

Actinomycetes were isolated from surface sterilized leaves and roots of maize. A total of 53 isolates were obtained, 31 of them from leaves and 22 from roots. The genus Microbispora was the most frequently found followed by the genera Streptomyces and Streptosporangium. From the isolated actinomycetes, 43.4 percent showed antimicrobial activity against one or more tested bacteria and yeast.

Microrganismos endofíticos são frequentemente encontrados no interior de plantas cultivadas. Embora vários tipos de microrganismos endofíticos tenham sido isolados de milho (Zea mays) não existiam dados sobre certos grupos de actinomicetos obtidos a partir desta espécie de planta cultivada. No presente trabalho, actinomicetos endofíticos foram isolados de folhas e raízes de milho. Um total de 53 isolados foram obtidos sendo 31 deles a partir de folhas e 22 a partir de raízes. O gênero Microbispora foi o mais frequente (62 por cento), seguido dos gêneros Streptomyces e Streptosporangium. Dos actinomicetos isolados, 43,4 por cento apresentaram atividade antimicrobiana contra um ou mais microrganismos ensaiado.