The acquisition of clinically relevant amoxicillin resistance in Streptococcus pneumoniae requires ordered horizontal gene transfer of four loci

Antimicrobial resistance in the opportunistic human pathogen Streptococcus pneumoniae is on the rise. Understanding how resistance spreads is critical for optimal application of new treatments in the clinic. The pneumococcus is naturally competent, and commonly takes up DNA from closely related strains and species in its environment. In S. pneumoniae, genetic resistance to beta-lactam antibiotics is mediated by recombination events in genes encoding the target proteins, resulting in reduced drug binding affinity. However, for amoxicillin, one of the front-line antibiotics for the treatment of pneumococcal infections, the exact mechanism of resistance still needs to be elucidated. Through successive rounds of transformation with genomic DNA from a clinically resistant isolate, we followed amoxicillin resistance development. We show that the final minimum inhibitory concentration differs depending on the recipient genome, and the fitness landscape that is being sampled. Using whole genome sequencing, we showed that multiple recombination events occurred at different loci during one round of transformation. We also found examples of non-contiguous recombination, and demonstrate that this can occur through simultaneous D-loop formation from one donor DNA molecule or by the uptake of multiple DNA fragments. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we show that pbp1a, pbp2b, pbp2x, murM are the main resistance determinants for amoxicillin resistance, and that the order of allele uptake matters. We conclude that recombination events are complex, and that this complexity contributes to the highly diverse genotypes of amoxicillin resistant pneumococcal isolates.