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

Abstract

Understanding how antimicrobial resistance spreads is critical for optimal application of new treatments. In the naturally competent human pathogen Streptococcus pneumoniae, resistance to β-lactam antibiotics is mediated by recombination events in genes encoding the target proteins, resulting in reduced drug binding affinity. However, for the front-line antibiotic amoxicillin, 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. Using whole genome sequencing, we showed that multiple recombination events occurred at different loci during one round of transformation. We found examples of non-contiguous recombination, and demonstrated for the first time that this can occur through multiple D-loop formation from one donor DNA molecule or by the uptake of multiple DNA fragments. We also show that the final minimum inhibitory concentration differs depending on recipient genome, and the sampled fitness landscape. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we show that pbp1a, pbp2b, pbp2x, and 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.

Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as indicated by sCRilecs-seq

“…In S. pneumoniae , resistance toward β-lactam antibiotics is mostly caused by mosaic PBPs. These resistance-conferring mosaic PBPs are formed by recombination events following horizontal gene transfer from β-lactam resistant donor strains ( Jensen et al, 2015 ; Hakenbeck et al, 2012 ; Gibson et al, 2021 ). It has been shown that many resistance-conferring PBP mutations are associated with considerable fitness costs that need to be compensated for ( Albarracín Orio et al, 2011 ; Zerfass et al, 2009 ; Smith and Klugman, 2001 ) and that mosaic PBPs significantly alter the cell wall structure, potentially by a decrease in affinity for their natural substrate ( Garcia-Bustos and Tomasz, 1990 ).…”