Forschungsseminar: Vortrag von Prof. Dr. Philippe Corvini

Sulfonamides are the second most used antibiotics worldwide with a release of approximately 20,000 tons per year. Sulfamethoxazole (SMX) is often detected in environmental compartments. The spreading of antibiotic resistant bacteria and antibiotic resistant genes is of concern and shall be integrated in One Health approaches.

Microbacterium BR1 was isolated from a membrane bioreactor. Mutants defective for sul1 and/or sadA were obtained by sub-culturing Microbacterium BR1. Degradation experiments were carried out using growing and resting cells with SMX and 14C-labelled SMX. Metabolites were analyzed using LC-MS. Genomic data was acquired by Oxford nanopore and Illumina sequencing. The proteome was analyzed using 2D-gel electrophoresis and MS. The number of copies of sadA was determined using qPCR. Transcriptomic analyses were performed.

Microbacterium BR1 can feed on SMX as sole carbon and energy source. The degradation of SMX starts with an ipso-substitution, catalyzed by a flavin-dependent monooxygenase (SadA) acting with a FMN reductase (SadC). The resulting p-aminophenol enters the central metabolism through a second monooxygenase activity (SadB), which leads to products amenable to ring opening. The cluster of degradation genes was identified and each of these three enzymes (SadA, SadB and SadC) was expressed in E. coli. The resistance gene sul1 was also detected in Microbacterium BR1.

We discuss the relevance of these findings by addressing questions arising from this research. Finally, we isolated mutants defective for sul1, sadA and the double mutant lacking both genes. We demonstrated that the sadA gene is a novel sulfonamide resistance gene. We conclude that the sul1 mutant is even more resistant to SMX than the wild type. We demonstrate that the gene sadA of the cluster responsible for sulfonamide degradation confers sulfonamide antibiotic resistance to Microbacteriaceae.

This fact and another evidence, i.e. gene cluster flanked by relaxase encoding sequences point out to possible mobility of these genes and necessitate further works to elucidate whether sadA can be subject to horizontal gene transfer.