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Antimicrobial resistance is a major threat to human health as resistant pathogens spread globally, and the development of new antimicrobials is slow. Since many antimicrobials function by targeting cell wall and membrane components, high-throughput lipidomics for bacterial phenotyping is of high interest for researchers to unveil lipid-mediated pathways when dealing with a large number of lab-selected or clinical strains. However, current practice for lipidomic analysis requires the cultivation of bacteria on a large scale, which does not replicate the growth conditions for high-throughput bioassays that are normally carried out in 96-well plates, such as susceptibility tests, growth curve measurements, and biofilm quantitation. Analysis of bacteria grown under the same condition as other bioassays would better inform the differences in susceptibility and other biological metrics. In this work, a high-throughput method for cultivation and lipidomic analysis of antimicrobial-resistant bacteria was developed for standard 96-well plates exemplified by methicillin-resistant Staphylococcus aureus (MRSA). By combining a 30-mm liquid chromatography (LC) column with ion mobility (IM) separation, elution time could be dramatically shortened to 3.6 min for a single LC run without losing major lipid features. Peak capacity was largely rescued by the addition of the IM dimension. Through multi-linear calibration, the deviation of retention time can be limited to within 5%, making database-based automatic lipid identification feasible. This high-throughput method was further validated by characterizing the lipidomic phenotypes of antimicrobial-resistant mutants derived from the MRSA strain, W308, grown in a 96-well plate. [ABSTRACT FROM AUTHOR]

Dalbavancin is a lipoglycopeptide active against methicillin-resistant Staphylococcus aureus (MRSA). Its long half-life (8.5–16 days) allows for once-weekly or single-dose treatments but could prolong the mutant selection window, promoting resistance and cross-resistance to related antimicrobials such as vancomycin. The objective of this study was to evaluate the capacity of post-distributional pharmacokinetic exposures of dalbavancin to select for resistance and cross-resistance in MRSA. We simulated average, post-distributional exposures of single-dose (1500 mg) dalbavancin (f Cmax 9.9 μg/mL, β-elimination t 1/2 204 h) in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model for 28 days (672 h) against five MRSA strains and one methicillin-susceptible strain (MSSA). Samples were collected at least daily, and surviving colonies were enumerated and screened for resistance on drug-free and dalbavancin-supplemented medium respectively. Isolates from resistance screening plates were subjected to whole-genome sequencing (WGS) and susceptibly testing against dalbavancin, vancomycin, daptomycin, and six β-lactams with varying penicillin-binding protein (PBP) affinities. Dalbavancin was bactericidal against most strains for days 1–4 before regrowth of less susceptible subpopulations occurred. Isolates with eight-fold increases in dalbavancin MIC were detected as early as day 4 but increased 64–128-fold in all models by day 28. Vancomycin and daptomycin MICs increased 4–16-fold, exceeding the susceptibly breakpoints for both antibiotics; β-lactam MICs generally decreased by two-to eight-fold, suggesting a dalbavancin–β-lactam seesaw effect, but increased by eight-fold or more in certain isolates. Resistant isolates carried mutations in a variety of genes, most commonly walKR , apt , stp1 , and atl. In our in vitro system, post-distributional dalbavancin exposures selected for stable mutants with reduced susceptibility to dalbavancin, vancomycin, and daptomycin, and generally increased susceptibility to β-lactams in all strains of MRSA tested. The clinical significance of these findings remains unclear, but created an opportunity to genotype a unique collection of dalbavancin-resistant strains for the first time. Mutations involved genes previously associated with vancomycin intermediate susceptibility and daptomycin non-susceptibility, most commonly walKR -associated genes. [ABSTRACT FROM AUTHOR]