The Diversity of Staphylococcus aureus Prophage Integrases and Their Association with Virulence and Antibiotic Resistance Factors

Staphylococcus aureus is a Gram-positive bacterium that causes a wide range of infectious diseases. An important source of virulence factors of Staphylococcus aureus are prophages - bacteriophages integrated into bacterial genomic DNA. Prophages are highly mosaic, making it difficult to establish links between genes. However, some virulence factors have previously been found to be linked to prophage integrases, the enzymes responsible for integrating prophages into the bacterial genome. The aim of this work was to study the diversity of prophage integrases in a global sample of S. aureus and to assess their association with virulence and antibiotic resistance factors. The genomes of 1300 S. aureus strains were downloaded from the NCBI database and the sequencing type was determined for all samples in silico. The search for prophage sequences was performed by the Phaster programme; subsequently, genomes for which prophage sequences were determined at the chromosome edges were excluded from the analysis. The annotation of prophages was performed using the Pharokka resource. Prophage integration sites and precise boundaries were determined using Vector NTI. Refined prophage sequences were compared with genomes from the International Committee on Taxonomy of Viruses (ICTV) database: trees based on pairwise distances between phage genomes were derived using the ViPTree programme. Virulence factors were searched using the VFDB database. A total of 857 S. aureus genomes containing prophage sequences not on chromosome edges were included in the study. Strains belonged to 143 sequencing types, a significant proportion of genomes belonged to epidemically significant ST: ST8 (n=161), ST5 (n=118), ST398 (n=50). In total, 3440 prophage sequences were identified for the strains in the collection, about half of which were ~40 thousand bp in length, which corresponds to the length of moderate Staphylococcus aureus phages. Comparison of annotated prophage sequences and known integrases revealed 1532 matches: the most abundant integrases were Sa3 (n=540), Sa2 (n=330), Sa1 (n=172), Sa6 (n=144), Sa7 (n=114) and Sa5 (n=103). The integrases Sa9, Sa8, Sa12 and Sa4 were encountered in 20, 7, 9 and 8 prophages, respectively. Both known and previously undescribed integration sites were identified for the resulting integrases. Eight new subtypes of classical types and three new types of integrases were also characterised during the study. Phylogenetic analysis of the prophage sequences confirmed the following genera in most cases: Peeveelvirus, Dubowvirus, Biseptimavirus, Phietavirus and Triavirus. Analysis of virulence factors revealed their highest representation among prophages containing Sa3 integrases; Sa3 were most frequently associated with the immune evasion gene cluster and the Chp protein. Sa5 carried the LukMF toxin. Sa1 prophages carried the eap cell attachment protein and the immune evasion gene cluster. In MDR genes, Sa3 prophages carried the aac(6')-Ie/aph(2'')-Ia aminoglcoside resistance gene and Sa2 prophages carried the dfrG gene, which is responsible for trimethoprim resistance.