Cross-Genus "Boot-Up" of Synthetic Bacteriophage in Staphylococcus aureus by Using a New and Efficient DNA Transformation Method.

Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections and food poisoning in humans with antibiotic resistance, specifically to methicillin, compounding the problem. Bacteriophages (phages) provide an alternative treatment strategy, but these only infect a limited number of circulating strains and may quickly become ineffective due to bacterial resistance. To overcome these obstacles, engineered phages have been proposed, but new methods are needed for the efficient transformation of large DNA molecules into S. aureus to "boot-up" (i.e., rescue) infectious phages. We presented a new, efficient, and reproducible DNA transformation method, NEST (non-electroporation Staphylococcus transformation), for S. aureus to boot-up purified phage genomic DNA (at least 150 kb in length) and whole yeast-assembled synthetic phage genomes. This method was a powerful new tool for the transformation of DNA in S. aureus and will enable the rapid development of engineered therapeutic phages and phage cocktails against Gram-positive pathogens. IMPORTANCE The continued emergence of antibiotic-resistant bacterial pathogens has heightened the urgency for alternative antibacterial strategies. Phages provide an alternative treatment strategy but are difficult to optimize. Synthetic biology approaches have been successfully used to construct and rescue genomes of model phages but only in a limited number of highly transformable host species. In this study, we used a new, reproducible, and efficient transformation method to reconstitute a functional nonmodel Siphophage from a constructed synthetic genome. This method will facilitate the engineering of Staphylococcus and Enterococcus phages for therapeutic applications and the engineering of Staphylococcus strains by enabling transformation of higher molecular weight DNA to introduce more complex modifications.

Complete Genome Sequence of Staphylococcus aureus Phage SA75, Isolated from Goat Feces.

Antibiotic-resistant Staphylococcus aureus is an opportunistic pathogen causing serious human infections worldwide. Here, we report the complete annotated genome of bacteriophage SA75, a member of the Siphoviridae family which could be an alternative to traditional antibiotics for treating Staphylococcus infections. We used a hybrid approach combining MinION and Illumina MiSeq sequencing, which yielded a 43,134-bp genome and 65 open reading frames.