Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

Abstract

Genetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a unique Staphylococcus aureus strain via a specific S. aureus bacteriophage, ⌽187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar ⌽187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinical Staphylococcus epidermidis isolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion. C oagulase-negative staphylococci (CoNS), such as human skin-colonizing Staphylococcus epidermidis or Staphylococcus lugdunensis, are frequently isolated from hospital-associated infections and represent emerging bacterial pathogens (1, 2). Many CoNS, in particular hospital-associated S. epidermidis, are resistant to available antibiotics, such as methicillin (Ն75% of S. epidermidis isolates), clindamycin, tetracycline, trimethoprim, macrolides, and aminoglycosides (3). Such strains typically cause a variety of complicated infections, particularly infections associated with indwelling medical devices in combination with strong biofilm formation or, even more alarming, life-threatening systemic disease, such as endocarditis or sepsis (2, 4-6).Genetic engineering of the well-studied pathogen Staphylococcus aureus has revolutionized research activities in the field of staphylococci in the past. However, current knowledge of the physiology and pathogenicity of CoNS is very limited because most available techniques used for genetic manipulation (e.g., electroporation of shuttle plasmids) often fail, most likely because of strong genetic barrier mechanisms, such as restriction-modification (R-M) systems or clustered regularly interspaced short palindromic repeats (CRISPR), previously shown to impede horizontal gene transfer (HGT) events between bacteria (7, 8). However, specifically engineered Escherichia coli strains lacking the dcm gene required for cytosine methylation of DNA have been developed to produce plasmid DNA that is not degraded upon electroporation of S. aureus or of specific S. lugdunensis strains, or even of the clinical S. epidermidis isolate RP62A (9, 10). This approach is promising bu...

Molecular characterization of a new efficiently transducing bacteriophage identified in meticillin-resistant Staphylococcus aureus

“…However, recently it has been documented by the detection and quantification of bacterial DNA encapsidated in phage particles that w81 of serological group A, which was considered to be nontransducing, also contains bacterial DNA, although in significantly lower amounts than the transducing bacteriophages (Mašlaň ová et al, 2013). Furthermore, serogroup L bacteriophage w187 was used for successful interspecies transfer of plasmid DNA from S. aureus to coagulasenegative staphylococci (Winstel et al, 2015). These findings indicate that packaging followed by transfer of mobile genetic elements with antibiotic resistance genes and virulence factors is widespread in S. aureus bacteriophages.…”

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis

“…However, this tool does not account for the type I RM barriers; as a result, its application to S. epidermidis proved limited. Other genetic tools, like the recently reported S. aureus phage 187 adapted to coagulase-negative staphylococci (CoNS) (25), have augmented the number of transformable S. epidermidis strains to include strains that are recalcitrant to electroporation. However, 187 transduction is dependent on the presence of the cognate phage receptor on the recipient strains; plasmid DNA, once inside the clinical isolate, is still subjected to type I restriction.…”

The metalloprotease SepA governs processing of accumulation‐associated protein and shapes intercellular adhesive surface properties in Staphylococcus epidermidis

“…Electroporation of S. epidermidis was performed using plasmids isolated from E. coli strain DC10B (Monk et al, 2012;Maliszewski and Nuxoll, 2014). Transduction by phage 187 was performed according to Winstel et al, with S. aureus PS187DhsdRDSauUS1 carrying the respective plasmid as the donor strain (Winstel et al, 2015(Winstel et al, , 2016. S. epidermidis double and triple mutants were created by phage crosses with phage 71 (Dean et al, 1973;Olson and Horswill, 2014) or phage A6C (Rohde et al, 2005).…”