Detection of phage-associated virulence/resistance genes in induced prophages of Streptococcus pyogenes clinical isolates

Objectives: The genome of each sequenced Streptococcus pyogenes strain shows to contain many prophages encoding proven or putative extracellular virulence factors and antibiotic resistance determinants. In this work we determined the presence of prophage-associated virulence/resistance genes (F-vir) and the induction profile of prophages harbouring virulence/resistance genes in fifty-nine S. pyogenes pharyngeal isolates. Methods: PCR was used to assess the presence of those genes (i.e. speA, speC, speH, speI, speK, speL, speM, ssa, spd1, spd3, spd4, sdn, sda, sla, mefA and TetO) known to be prophage-associated in S. pyogenes. Each strain was treated with mitomycin C to induce release of functional phages, and the corresponding unrestricted total DNA was then analysed by pulsed field gel electrophoresis (PFGE). S. pyogenes SF370 and strain-6 (mefA/TetO) were used as control strains. Single or multiple bands, corresponding to phage DNA, were obtained only in mitomycin treated cells. They were excised and analysed by PCR to detect the specific F-vir. Results: Five percent of the strains did not contain any of the known F-vir, while 76% had at least two. The distribution of F-vir was greatly variable and the overall mean number of F-vir per isolate was 3.8 (± 2.3). The release of phage DNA was achieved in 17 strains. Among these, PCR analysis detected a single F-vir in the phage DNA released by 35.3% of the strains, and three F-vir in 17.6%. One strain released phage DNA containing two F-vir, whereas another strain released phage DNA positive to five F-vir. The F-vir most frequently associated with released phage DNAs were sdn, spd4, speC, spd1 and spd3. Conclusion: The pharynx is colonised by S. pyogenes harbouring a variable number and assortment of F-vir. A limited number of virulence genes are hosted by functional prophages and, therefore, have the potentiality to be horizontally transferred. Many strains possess different important F-vir that, at least in the conditions used, cannot be detected in released phage DNAs. These results suggest that the population of Fvir-lacking functional prophages is vast and that the polylysogeny would be the product of the accumulation of temperate phages that eventually become defective.