Comparison of freeze-thaw and sonication cycle-based methods for extracting AMR-associated metabolites from Staphylococcus aureus

Abstract

Emerging antimicrobial resistance (AMR) among Gram-positive pathogens, specifically in Staphylococcus aureus (S. aureus), is becoming a leading public health concern demanding effective therapeutics. Metabolite modulation can improve the efficacy of existing antibiotics and facilitate the development of effective therapeutics. However, it remained unexplored for drug-resistant S. aureus (gentamicin and methicillin-resistant), primarily due to the dearth of optimal metabolite extraction protocols including a protocol for AMR-associated metabolites. Therefore, in this investigation, we have compared the performance of the two most widely used methods, i.e., freeze-thaw cycle (FTC) and sonication cycle (SC), alone and in combination (FTC + SC), and identified the optimal method for this purpose. A total of 116, 119, and 99 metabolites were identified using the FTC, SC, and FTC + SC methods, respectively, leading to the identification of 163 metabolites cumulatively. Out of 163, 69 metabolites were found to be associated with AMR in published literature consisting of the highest number of metabolites identified by FTC (57) followed by SC (54) and FTC + SC (40). Thus, the performances of FTC and SC methods were comparable with no additional benefits of combining both. Moreover, each method showed biasness toward specific metabolite(s) or class of metabolites, suggesting that the choice of metabolite extraction method shall be decided based on the metabolites of interest in the investigation.

Advancements in CHO metabolomics: techniques, current state and evolving methodologies

“…For example, in LC-MS of Klebsiella pneumoniae , 151 metabolites were identified in sample with metabolite extracted using Freeze thaw cycle (FTC), 103 by sonication cycle (SC) method and 132 metabolites by both (FTC + SC), enabling cumulative identification of 199 unique metabolites; more than any of the single method identified ( Kumar et al, 2022 ). Similarly, in LC-MS of Staphylococcus aureus , a total of 116, 119, and 99 metabolites were identified in samples with metabolite extracted using the FTC, SC, and FTC + SC methods, respectively, leading to the identification of 163 metabolites cumulatively ( Singh et al, 2023 ). This indicates that each method of metabolite extraction also has its own biasness ( Sowa et al, 2020 ; Singh et al, 2023 ).…”

“…Similarly, in LC-MS of Staphylococcus aureus , a total of 116, 119, and 99 metabolites were identified in samples with metabolite extracted using the FTC, SC, and FTC + SC methods, respectively, leading to the identification of 163 metabolites cumulatively ( Singh et al, 2023 ). This indicates that each method of metabolite extraction also has its own biasness ( Sowa et al, 2020 ; Singh et al, 2023 ). Therefore, utilizing multiple methods of metabolite extraction with multiple analytical methods (LC-MS/GC-MS and NMR) will present a clearer and more accurate picture of the metabolic profile due to increased metabolomic coverage and help to overcome the limitations of individual techniques/methods.…”

“…Different metabolite quenching and extraction methods have biased towards different classes of compounds (amino acids, carbohydrates, nucleotides, sugars, etc.). Hence, cataloguing methods with respect to their biasness towards metabolite extraction for general use can facilitate the detection of a larger range of metabolites or help in the quantification of a specific/targeted metabolite(s) ( Kumar et al, 2022 ; Singh et al, 2023 ). For example, the extraction of metabolites using 100% methanol extraction followed by water was found to be most effective for the recovery of the largest range of metabolites in CHO cells by GC-MS ( Sellick et al, 2010 ).…”

“…, erythropoietin and clotting factors, etc.) due to their ability to grow fast to achieve high-density in protein-free and chemically defined culture media, perform human-like post-translational modifications, appropriate protein folding, high productivity and low susceptibility to human viral infections ( Wurm, 2004 ; Lalonde and Durocher, 2017 ; Singh et al, 2023 ). However, the cost of such products is still high and requires significant improvements in the overall yield.…”