Streptomyces from rhizospheres and compost
Description
This information was obtained in order to demonstrate the presence of actinobacteria in different rhizospheres and in a compost system, which would inhibit the growth of bacteria resistant to some antibiotic. In addition, the aim of this study was to identify by molecular methods and phylogenetic analysis the species of native bacteria with antimicrobial activity, mainly of the Streptomyces genus, recovered from these environments. In these data it is possible to see the number of isolates obtained from different rhizospheres and from a composting system, the culture media used for their isolation and the antimicrobial activity against antibiotic resistant and non-resistant bacteria; this activity was initially measured by direct antagonism by perpendicular streak technique. In the photo of the preliminary test, some isolates with inhibitory potential can be identified according to the space without growth between the streak of the native organism and the streak of the control bacteria. An initial enzyme profile of all the isolates obtained is also presented, which allowed the physiological characterization of native Streptomyces with antimicrobial activity. A biochemical analysis was carried out through conventional identification tests and the microscopic and macroscopic morphological characteristics are reported, the latter analyzed through a comparison of the color of the colonies obtained in different culture media. The data also shows the percentages of inhibition obtained with the active isolates against bacteria resistant to antibiotics. These calculations were made through the perpendicular streak method comparing the area that presented growth of the control microorganism and the areas where it was inhibited. The native isolates that showed activity were identified through molecular tests and phylogenetic analysis, for which sequences reported in GenBank for the 16S ribosomal gene were organized and shown in the respective sequence bank file.
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Soils of rhizospheres were collected from a private land in Medellín, Colombia. Compost samples were taken from a one-week-old artisanal organic solid waste treatment system. The collected samples were taken at a depth of 10-20 cm. Actinobacteria were isolated using GYM medium and starch casein nitrate medium, both culture media were supplemented with 2.5 µg/ml rifampicin and pH was adjusted in 7.2. The plates were incubated at 28 °C for seven days. The isolated colonies corresponding to Gram-positive filamentous bacteria were subcultured on GYM agar and stored in Brain Heart Infusion broth with 15 % glycerol at -20 °C. To select the native isolates with antibacterial activity, a cross-streak method was performed. Plates of Mueller-Hinton agar were inoculated by a single streak in the center of the Petri dish with actinobacterial suspensions; these plates were incubated at 30 ºC for 10 days. The test microorganisms were incubated on LB (Miller) agar at 37 °C for 24 h, and bacterial suspensions equivalent to 0.5 McFarland standard were seeded by streaking perpendicular to the line of actinobacterial growth, and reincubated at 37 °C for 24 h. Antagonism was observed based on the inhibitory interaction between the actinobacteria and test strains. The isolates that showed promising antibacterial activity against resistant bacteria were morphologically and physiologically characterized according to International Streptomyces Project (ISP) and Bergey's Manual of Systematic Bacteriology. Growth at 20-40 ºC and NaCl concentrations 0.2-10 % were examined by growing the isolates on starch casein nitrate medium. Nitrate reduction were stablished by growing in nitrate containing nutrient broth for 72 h and addition of Griess-Illosvay’s reagent. Urea hydrolysis was checked in basal medium with 2 % urea. Catalase activity was evaluated by hydrogen peroxide effervescence and oxidase activity using Bactident® diagnostic test. Utilization of different carbon sources were studied by adding 1 % filter-sterilized sugars to the basal medium ISP-9. The production of hydrolytic enzymes was evaluated in different culture media including soluble starch for amylolytic activity, carboxymethylcellulose for cellulolytic activity, gelatin for proteolytic activity, olive oil for lipolytic activity. For verification and comparison with available sequences in GenBank, all congener sequences having above 96 % of identity were downloaded and included in the final dataset. Kitasatospora aureofaciens and Micromonospora echinospora species from GenBank were used as outgroup. Isolates and GenBank sequences were then aligned and manually edited and trimmed to generate a final dataset for further phylogenetical delimitation analysis.