부산대학교 도서관

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 that this could occur either through multiple D-loop formation from one donor DNA molecule, or by the integration of multiple DNA fragments. We also show that the final minimum inhibitory concentration (MIC) differs depending on recipient genome, explained by differences in the extent of recombination at key loci. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we confirm that pbp1a, pbp2b, pbp2x, and murM are the main resistance determinants for amoxicillin resistance, and that the order of allele uptake is important for successful resistance evolution. We conclude that recombination events are complex, and that this complexity contributes to the highly diverse genotypes of amoxicillin-resistant pneumococcal isolates. Author summary: Streptococcus pneumoniae (the pneumococcus) is an asymptomatic coloniser of the human nasopharynx and a common cause of middle ear infections and pneumonia. The frontline treatment for bacterial respiratory tract infections is amoxicillin, a β-lactam antibiotic that inhibits cell wall synthesis. The pneumococcus is naturally competent and β-lactam resistance in this species is acquired by recombination of large stretches of foreign DNA into genes encoding the target penicillin binding proteins, resulting in reduced drug binding affinity. For amoxicillin, the exact mechanism of resistance development is not well understood. Here, we sequentially transformed genomic DNA from a resistant clinical isolate into two different susceptible strains and selected on amoxicillin. This resulted in a large collection of strains with varying amoxicillin resistances and a known pedigree. Using whole genome sequencing and fluorescently labelled penicillin binding assays, we were able to confirm that alterations to penicillin binding proteins Pbp2x, Pbp2b, and Pbp1a, as well as another cell wall synthesis protein MurM are essential for amoxicillin resistance. We also show that the resistant alleles must be acquired in a specific order for optimal resistance evolution, and that the inherent complexity of recombination itself may contribute to the enormous diversity of resistant pneumococcal isolates. [ABSTRACT FROM AUTHOR]