A Whole-Cell Assay for Detection of Antibacterial Activity in Actinomycete Culture Supernatants

Abstract

Whole-cell antibacterial assays are particularly suitable for fast detection and semi-quantification of bioactivities in extracts or other solutions such as microbial culture supernatants. As Actinomycetales, including the members of the genus Streptomyces, are one of the most potent “suppliers” of antibiotics and other bioactive compounds, there is a strong interest in the development of useful assays enabling early identification of such valuable producers. Furthermore, such assays facilitate the screening of a large collection of clones for the detection of engineered “super-producers” that are essential for industrial manufacturing of the respective product. In this protocol, we describe a whole-cell assay for a fast detection of antimicrobial agents in culture supernatants. As model, Streptomyces fradiae DSM 41546, the producer of the antibiotic tylosin, and the test strain Bacillus subtilis ATCC 6051 are used. Culture supernatants of S. fradiae DSM 41546 and controls are diluted, and their antibiotic activity is tested against B. subtilis ATCC 6051. For the dilutions of tylosin-containing culture supernatant, a clear concentration-dependent growth inhibition effect on B. subtilis ATCC 6051 is demonstrated. In contrast, dilutions of the culture supernatant lacking tylosin do not inhibit the growth of B. subtilis. Moreover, including defined concentrations of tylosin facilitates the semi-quantification of tylosin in the culture supernatants. Our results confirm the applicability of the assay in fast screenings for antimicrobial products in culture supernatants. The protocol can be used as positive control within screening campaigns in the drug discovery field.