Insights Into the Evolution of Staphylococcus aureus Daptomycin Resistance From an in vitro Bioreactor Model

Abstract

The extensive use of daptomycin for treating complex methicillin-resistant Staphylococcus aureus infections has led to the emergence of daptomycin-resistant strains. Although genomic studies have identified mutations associated with daptomycin resistance, they have not necessarily provided insight into the evolution and hierarchy of genetic changes that confer resistance, particularly as antibiotic concentrations are increased. Additionally, plate-dependent in vitro analyses that passage bacteria in the presence of antibiotics can induce selective pressures unrelated to antibiotic exposure. We established a continuous culture bioreactor model that exposes S. aureus strain N315 to increasing concentrations of daptomycin without the confounding effects of nutritional depletion to further understand the evolution of drug resistance and validate the bioreactor as a method that produces clinically relevant results. Samples were collected every 24 h for a period of 14 days and minimum inhibitory concentrations were determined to monitor the acquisition of daptomycin resistance. The collected samples were then subjected to whole genome sequencing. The development of daptomycin resistance in N315 was associated with previously identified mutations in genes coding for proteins that alter cell membrane charge and composition. Although genes involved in metabolic functions were also targets of mutation, the common route to resistance relied on a combination of mutations at a few key loci. Tracking the frequency of each mutation throughout the experiment revealed that mutations need not arise progressively in response to increasing antibiotic concentrations and that most mutations were present at low levels within populations earlier than would be recorded based on single-nucleotide polymorphism (SNP) filtering criteria. In contrast, a serial-passaged population showed only one mutation in a gene associated with resistance and provided limited detail on the changes that occur upon exposure to higher drug dosages. To conclude, this study demonstrates the successful in vitro modeling of antibiotic resistance in a bioreactor and highlights the evolutionary paths associated with the acquisition of daptomycin non-susceptibility.

Genome-Wide Association Studies for the Detection of Genetic Variants Associated With Daptomycin and Ceftaroline Resistance in Staphylococcus aureus

“…For Cls2 we detected the AAS I298T (15-00711) and S466P (15-01065) that are located in close proximity to the putative cadiolipin synthase domains at residues 229–256 and 407–434 ( Figure 3 ; Peleg et al, 2012 ). AAS in Cls2 and varying levels of cls2 transcription have already been suggested to be involved in the development of DAP resistance in S. aureus , either alone or in combination with mprF mutations ( Camargo et al, 2008 ; Peleg et al, 2012 ; Jiang et al, 2019 ; Lasek-Nesselquist et al, 2019 ). Interestingly, within our strain collection we found a DAP-R isolate (MIC = 8 mg/L; median of all DAP-R isolates = 4 mg/L) harboring MprF S337L, Cls2 S466P, and RpoB Q468K (15-01065; Figure 3 ), indicating that a combination of mutations in different loci might lead to synergistic effects.…”

Insights into collateral susceptibility and collateral resistance in Acinetobacter baumannii during antimicrobial adaptation

“…Moreover, the acquisition of antibiotic resistance is associated with the homeostasis of cell wall and cell membrane (Lasek‐Nesselquist et al . 2019).…”

“…Unlike mutation, adaptation occurs bacteria are exposed to increasing concentrations of antibiotics (Lasek‐Nesselquist et al . 2019).…”

Addition of daptomycin to levofloxacin increased the efficacy of levofloxacin monotherapy against a methicillin-susceptible Staphylococcus aureus strain in experimental meningitis and prevented development of resistance in vitro

“…An additional amino acid substitution was detected in GyrB (W592F) and its role in increased MIC of levofloxacin is not known. The mutant selected under daptomycin contained four different mutations, including amino acid substitutions in MprF (P314L) and Cls2 (A23V), which correspond to known mutations associated with resistance to daptomycin resistance in clinical S. aureus isolates and in in vitro generated mutants [9,10,28]. The two other mutations were detected in the shikimate kinase AroK and the immunoglobulin G-binding protein A Spa, whose associations with decreased susceptibility to daptomycin are not known.…”