Comparative proteomic investigation of multiple methicillin-resistant Staphylococcus aureus strains generated through adaptive laboratory evolution

Abstract

Summary Recent discoveries indicate that tolerance and resistance could rapidly evolve in bacterial populations under intermittent antibiotic treatment. In the present study, we applied antibiotic combinations in laboratory experiments to generate novel methicillin-resistant Staphylococcus aureus strains with distinct phenotypes (tolerance, resistance, and suppressed tolerance), and compared their proteome profiles to uncover the adaptation mechanisms. While the tolerant strains have very different proteomes than the susceptible ancestral strain, the resistant strain largely resembles the ancestral in terms of their proteomes. Our proteomics data and other assays support the connection between the detected mutations to the observed phenotypes, confirming the general understanding of tolerance and resistance mechanisms. While resistance directly counteracts the action mechanism of the antibiotic, tolerance involves complex substantial changes in the cells’ biological process to achieve survival advantages. Overall, this study provides insights into the existence of diverse evolutionary pathways for tolerance and resistance development under different treatment scenarios.

Novel Daptomycin Tolerance and Resistance Mutations in Methicillin-Resistant Staphylococcus aureus from Adaptive Laboratory Evolution

“…Because a larger set of possible mutations are associated with tolerance compared to that associated with resistance, tolerance mutations should appear more frequently and thus earlier in the evolutionary process ( 1 , 2 , 57 ). Mutations leading to daptomycin resistance are restricted to several genes, such as mprF (the most prevalent), cls , walKR , and the dlt operon, which all caused the repulsion of daptomycin from binding to the cell membrane ( 58 ), whereas daptomycin-tolerance mutations were observed in a wide range of genes not necessarily directly related to the action mechanism of the antibiotic (those found in this study, such as prkC , prs , addB , rpsR , and proP , and also those identified in other studies, such as pgsA [ 14 ], pitA [ 17 ], rpoC and purR [ 59 ], snoF , hmp1 , sspA , and many more [ 60 ]). Therefore, in our evolution experiments performed on exponential-phase cultures that eventually led to resistance, we should also expect tolerance mutations to have emerged earlier in the populations.…”

“…Although in most cases we observed that the resistant mutant eventually took over the population (lineages 1 to 3, and lineages 7 to 9) and could invade the tolerant mutant (lineage 2), this evolutionary pathway is not always taken. In a previous study, we also observed a situation where a daptomycin tolerance mutation (several base pairs upstream of the pgsA gene) that appeared after a week of cyclic daptomycin treatment was retained in the population even after the evolution experiment was extended for 2 weeks, without being replaced by a resistance mutation ( 14 ). We rationalized that this is because the survival advantage conferred by the mprF resistance mutation is lower than that of the pgsA tolerance mutation (the mprF mutation led to an ∼40-fold increase in survival, whereas the mutation upstream of pgsA led to an over 100-fold increase in survival upon 3 h of 10 mg/liter daptomycin treatment, which was the condition used during the evolution experiment).…”

“…To verify our hypothesis, we performed competition experiments where a small number of daptomycin-resistant strain (RES2) organisms were mixed with a larger number of susceptible ancestral wild-type or tolerant strains that confer different survival advantages (TOL2, TOL5, TOL6) and then treated with daptomycin ( Fig. 2d ) following similar protocols in the literature ( 14 , 15 , 59 ). This experiment mimics a scenario where a daptomycin-resistant strain emerged during our evolution experiment and would tell us whether the resistant mutant can invade the population or not.…”

“…The survival of the daptomycin-resistant mutant was evaluated by plating on MH agar containing daptomycin (1 μg/ml), and the survival of the nonresistant strains (WT and tolerant strains) was evaluated by plating on MH agar without the antibiotic, subtracted by the survival of the resistant mutants. This competition experiment has been used by other groups, and the experimental protocol used here is similar to those from the previous studies ( 14 , 15 , 59 ).…”

Proteomics and Transcriptomics Uncover Key Processes for Elasnin Tolerance in Methicillin-Resistant Staphylococcus aureus

“…We adopted a previously described sample preparation workflow that maximize protein identification on S. aureus samples, including the extraction of surface-associated proteins ( 54 ). The cell pellet was suspended in 350 μL lysis buffer (8 M urea, 50 mM Tris-HCl [pH 8.0]), frozen in liquid nitrogen, and sonicated for 15 min.…”