Horizontal gene transfer via interspecies recombination confers virulence and fitness advantages compared to de novo mutation in Beta-lactam resistant Streptococcus pneumoniae

Streptococcus pneumoniae is an opportunistic Gram-positive pathogen and leading cause of hospitalization and mortality in young children and the elderly. Beta-lactams are frequently used as first-line treatments of pneumococcal infection, though global resistance to Beta-lactams have complicated pneumococcal infection treatment. As opposed to de novo mutation, S. pneumoniae Beta-lactam resistance is often conferred by mosaic variants of the target penicillin-binding proteins (PBPs). These variants result from homologous recombination during horizontal transfer within species and/or from related oral streptococcal species. We hypothesized that interspecies recombination may confer fitness advantages over de novo PBP mutation and facilitate the spread of Beta-lactam resistance. The wild-type TIGR4 pneumococcal strain was repeatedly transformed up to three times (first-, second-, and third-round transformations) and screened on successively increasing concentrations of penicillin to generate Beta-lactam-resistant pneumococci with recombinant genomic DNA sourced from resistant oral streptococcal species. Early-round recombinant strains retained virulence in vivo comparable to the wild type parental strain, whereas subsequent (primarily third-round) recombinants and experimentally-evolved strains encoding de novo point mutations were attenuated. Tolerance to penicillin was also observed among third-round transformants. In vitro characterization of virulence phenotypes did not identify differences consistent with virulence disparities observed in vivo. However, genome sequencing revealed large-scale changes in PBP2X and PBP2B in early-round and late-round recombinants, respectively as well as non-PBP-related gene changes in several recombinant isolates. Our results indicate that horizontal transfer of Beta-lactam resistance engenders initial favorable or minimal cost changes in vivo compared to de novo mutation(s).