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Genetic engineering of untransformable coagulase-negative staphylococcal pathogens

Abstract

Coagulase-negative staphylococci (CoNS) are recognized as significant opportunistic pathogens. However, current knowledge of virulence mechanisms is very limited because a significant proportion of CoNS are refractory to available techniques for DNA transformation. We describe an efficient protocol for plasmid transfer using bacteriophage Φ187, which can transduce plasmid DNA to a wide range of CoNS from a unique, engineered Staphylococcus aureus strain. The use of a restriction-deficient, modification-proficient S. aureus PS187 mutant, which has a CoNS-type bacteriophage surface receptor, allows plasmid transfer to CoNS even when they are refractory to electroporation. Once the Φ187 titer reaches 109 plaque-forming units per milliliter, plasmid transfer can be accomplished within 1–2 d. Thus, our protocol is a major technical advance offering attractive opportunities for research on CoNS-mediated infections.

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Figure 1: Workflow of bacteriophage Φ187–mediated plasmid transfer to coagulase-negative staphylococcal (CoNS) pathogens.
Figure 2: Cyclic repetition of bacteriophage Φ187 propagation in liquid culture.
Figure 3: Ultracentrifugation of phage or plasmid-containing phage lysates.
Figure 4: Timeline of tasks required for bacteriophage Φ187–mediated plasmid transfer.

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Acknowledgements

This work was supported by German Research Council grants TRR34 and SFB766 to A.P. and Ro2413/4-1 to H.R., and by German Center for Infection Research (DZIF) grants to H.R. and A.P.

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V.W., H.R. and A.P. designed the study. V.W. and P.K. performed the experiments. V.W., P.K. and A.P. wrote the paper.

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Correspondence to Andreas Peschel.

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Winstel, V., Kühner, P., Rohde, H. et al. Genetic engineering of untransformable coagulase-negative staphylococcal pathogens. Nat Protoc 11, 949–959 (2016). https://doi.org/10.1038/nprot.2016.058

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