Dataset Information

Adaptive Laboratory Evolution of Staphylococcus aureus Resistance to Vancomycin and Daptomycin: Mutation Patterns and Cross-Resistance.

ABSTRACT: Vancomycin and daptomycin are first-line drugs for the treatment of complicated methicillin-resistant Staphylococcus aureus (MRSA) infections, including bacteremia. However, their effectiveness is limited not only by their resistance to each antibiotic but also by their associated resistance to both drugs. It is unknown whether novel lipoglycopeptides can overcome this associated resistance. Resistant derivatives from five S. aureus strains were obtained during adaptive laboratory evolution with vancomycin and daptomycin. Both parental and derivative strains were subjected to susceptibility testing, population analysis profiles, measurements of growth rate and autolytic activity, and whole-genome sequencing. Regardless of whether vancomycin or daptomycin was selected, most of the derivatives were characterized by a reduced susceptibility to daptomycin, vancomycin, telavancin, dalbavancin, and oritavancin. Resistance to induced autolysis was observed in all derivatives. Daptomycin resistance was associated with a significant reduction in growth rate. Resistance to vancomycin was mainly associated with mutations in the genes responsible for cell wall biosynthesis, and resistance to daptomycin was associated with mutations in the genes responsible for phospholipid biosynthesis and glycerol metabolism. However, mutations in walK and mprF were detected in derivatives selected for both antibiotics.

SUBMITTER: Gostev V

PROVIDER: S-EPMC10215302 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Adaptive Laboratory Evolution of Staphylococcus aureus Resistance to Vancomycin and Daptomycin: Mutation Patterns and Cross-Resistance.

Gostev Vladimir V Kalinogorskaya Olga O Sopova Julia J Sulian Ofelia O Chulkova Polina P Velizhanina Maria M Tsvetkova Irina I Ageevets Irina I Ageevets Vladimir V Sidorenko Sergey S

Antibiotics (Basel, Switzerland) 20230518 5

Vancomycin and daptomycin are first-line drugs for the treatment of complicated methicillin-resistant Staphylococcus aureus (MRSA) infections, including bacteremia. However, their effectiveness is limited not only by their resistance to each antibiotic but also by their associated resistance to both drugs. It is unknown whether novel lipoglycopeptides can overcome this associated resistance. Resistant derivatives from five S. aureus strains were obtained during adaptive laboratory ...[more]

PMID: 37237831

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Project description:It has been shown recently in a number of in vitro laboratory evolution experiments that under repetitive antibiotic exposure, bacterial populations can adapt quickly to the treatment condition by becoming tolerant and/or resistant to the drug. The repeated killing and regrowth cycles hasten the selection for tolerant/resistant mutants with survival advantages. Due to the random nature of mutagenesis and the large target size of tolerance mutations, this dynamic evolutionary process appears to be highly unpredictable, generating distinct mutants even under identical, well-controlled laboratory conditions. Here, we utilized an adaptive laboratory evolution (ALE) experiment to hunt for novel tolerance and resistance mutations by subjecting multiple lineages of methicillin-resistant Staphylococcus aureus (MRSA) to repetitive daptomycin treatment. By sequencing multiple isolates along the course of evolution, we obtained three tolerant mutants that have different tolerance levels and identified novel daptomycin resistance mutations in the mprF gene. In addition, we found that tolerance/resistance development is more rapid if the population is treated in the exponential phase than if it is treated in the stationary phase, which is likely attributable to the more effective killing of growing cells by the antibiotic. Through competition assays, we found that whether or not the resistant mutants can take over the population heavily depends on the relative survival advantages conferred by the tolerance and resistance mutations. This study reports novel daptomycin resistance and tolerance mutations and offers new insights into the dynamics of the development of tolerance and resistance in bacterial populations under antibiotic exposure. IMPORTANCE Although the phenotype of increased tolerance and/or resistance was commonly observed in evolved populations from typical adaptive laboratory evolution (ALE) experiments, a wide variety of mutations that underlie those phenotypes have been discovered. Therefore, performing ALE experiments in multiple populations in parallel would serve the purpose of mining for different tolerant/resistant mutants and would be useful to explore the diverse population dynamics of evolution. In this study, we performed in vitro evolution in a clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using a lethal concentration of a drug that is frequently used in the clinic, daptomycin. Using this strategy, we obtained three distinct daptomycin-tolerant mutants and identified six daptomycin resistance mutations in different locations on the mprF gene, collectively adding to our current knowledge of this important pathogen. In addition, we found out that in most cases, the daptomycin-resistant mutant outcompetes other susceptible and tolerant mutants and becomes established in the final population. Follow-up competition experiments offered an explanation; the resistant mutant cannot invade populations of tolerant mutants that confer higher survival advantages than itself. In summary, we demonstrated an experimental strategy to explore the landscape and dynamics of the evolution of tolerance and resistance in MRSA toward daptomycin and made observations that will guide future ALE experiments.

Project description:The genomic evolution in vivo in persistent infection was critical information for understanding how methicillin-resistant Staphylococcus aureus (MRSA) was adapted to host environments with high antibiotic selective pressure. Thirty-two successive MRSA blood isolates with incremental non-susceptibility to vancomycin (VISA), daptomycin (DRSA), and/or linezolid (LRSA) were isolated from a patient failing multiple courses of antimicrobial therapy during 1,356 days of bacteremia. Whole genome sequencing (WGS) for all consecutive isolates were conducted to characterize the evolutionary pathways, resistance-associated mutations and their temporal relationship with antimicrobial treatment. The WGS-based phylogeny categorized the isogenic strains into three major clades, I (22 isolates), II (7 isolates), and III (3 isolates), respectively, harboring a median (range) of 7 (1-30), 62 (53-65), and 118 (100-130) non-synonymous mutations when compared to the very first isolate. Clade I strains were further grouped into early and late subclades, which, respectively, shared the most recent common ancestor with Clade III strains at day 393.7 and Clade II strain at day 662.5. Clade I and Clade III strains were characterized, respectively, with high rates of VISA (9/22, 40.9%) and VISA-and-DRSA phenotype (2/3, 66.7%). Linezolid-resistance including VISA-DRSA-and-LRSA phenotype was exclusively identified in Clade II strains after eight courses of linezolid treatment. The LRSA displayed a small colony variant phenotype and were associated with G2576T mutations in domain V region of 23S rRNA. Substantial loss of mobile elements or alleles mediating resistance or virulence were identified during the evolution of multi-resistance. However, the gene loss might not be correlated to the development of VISA, DRSA, or LRSA phenotype. In conclusion, MRSA in persistent bacteremia was adapted to harsh host environment through multiple pathways involving both resistance-associated mutations and extensive gene loss.

Project description:IntroductionVancomycin-intermediate Staphylococcus aureus (VISA) is typically associated with a decline in virulence. We previously reported a WalK(S221P) mutation that plays an important role in mediating vancomycin resistance in VISA XN108. Whether this mutation is implicated in bacterial virulence remains unknown.ObjectivesThis study aimed to investigate the effect of WalK(S221P) mutation on the virulence of VISA and the underlying mechanism of this effect.MethodsThe influence of WalK(S221P) mutation on VISA virulence and its underlying mechanism were explored using animal models, RNA-seq analysis, RT-qPCR, hemolytic assay, slide coagulase test, Western blot, β-galactosidase assay, and electrophoresis mobility shift assay (EMSA).ResultsCompared with XN108, WalK(S221P)-reverted strain XN108-R exacerbated cutaneous infections with increased lesion size and extensive inflammatory infiltration in mouse models. The bacterial loads of S. aureus XN108-R in murine kidney increased compared with those of XN108. RNA-seq analysis showed upregulation of a set of virulence genes in XN108-R, which exhibited greater hemolytic and stronger coagulase activities compared with XN108. Introduction of WalK(S221P) to methicillin-resistant S. aureus USA300 and methicillin-susceptible strain Newman increased the vancomycin resistance of the mutants, which exhibited reduced hemolytic activities and decreased expression levels of many virulence factors compared with their progenitors. WalK(S221P) mutation weakened agr promoter-controlled β-galactosidase activity. EMSA results showed that WalK-phosphorylated WalR could directly bind to the agr promoter region, whereas WalK(S221P)-activated WalR reduced binding to the target promoter. Inactivation of agr in S. aureus did not affect their vancomycin susceptibility but mitigated the virulence alterations caused by WalK(S221P) mutation.ConclusionThe results of our study indicate that WalK(S221P) mutation can enhance vancomycin resistance in S. aureus of diverse genetic backgrounds. WalK(S221P)- bearing S. aureus strains exhibit reduced virulence. WalK(S221P) mutation may directly impair the activation of the agr system by WalR, thereby decreasing the expression of virulence factors in VISA.

Dataset Information

Adaptive Laboratory Evolution of Staphylococcus aureus Resistance to Vancomycin and Daptomycin: Mutation Patterns and Cross-Resistance.

Publications

Adaptive Laboratory Evolution of <i>Staphylococcus aureus</i> Resistance to Vancomycin and Daptomycin: Mutation Patterns and Cross-Resistance.

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