Identification of metabolite extraction method for targeted exploration of antimicrobial resistance associated metabolites of Klebsiella pneumoniae

Abstract

Antimicrobial resistant Klebsiellapneumoniae (K. pneumoniae), as being a pathogen of critical clinical concern, urgently demands effective therapeutic options. However, the discovery of novel antibiotics over the last three decades has declined drastically and necessitates exploring novel strategies. Metabolomic modulation has been the promising approach for the development of effective therapeutics to deal with AMR; however, only limited efforts have been made to-date, possibly due to the unavailability of suitable metabolites extraction protocols. Therefore, in order to establish a detailed metabolome of K. pneumoniae and identify a method for targeted exploration of metabolites that are involved in the regulation of AMR associated processes, metabolites were extracted using multiple methods of metabolites extraction (freeze–thaw cycle (FTC) and sonication cycle (SC) method alone or in combination (FTC followed by SC; FTC + SC)) from K. pneumoniae cells and then identified using an orbitrap mass analyzer (ESI-LC–MS/MS). A total of 151 metabolites were identified by using FTC, 132 metabolites by using FTC+SC, 103 metabolites by using SC and 69 metabolites common among all the methods used which altogether enabled the identification of 199 unique metabolites. Of these 199, 70 metabolites were known to have an association with AMR phenotype and among these, the FTC + SC method yielded better (identified 55 metabolites), quantitatively and qualitatively compared to FTC and SC alone (identified 51 and 41 metabolites respectively). Each method of metabolite extraction showed a definite degree of biasness and specificity towards chemical classes of metabolites and jointly contributed to the development of a detailed metabolome of the pathogen. FTC method was observed to give higher metabolomic coverage as compared to SC alone and FTC + SC. However, FTC + SC resulted in the identification of a higher number of AMR associated metabolites of K. pneumoniae compared to FTC and SC alone.

Improved workflow for untargeted metabolomics and NMR analysis of intracellular and extracellular metabolites isolated from Gram positive and Gram negative bacteria

“…In this study, they detected 1,320 metabolites, which is considerably higher than the capabilities of NMR metabolomics and Chenomx software. However, these data are not comparable with our workflow as the sensitivity of NMR is known to be notably lower than MS/MS methods (Kumar et al, 2022) and the Chenomx library (version 9.0) only contains information on 336 metabolites. However, despite the reduced sensitivity of NMR metabolomics, this technology is considered to be simpler, faster, more cost-effective and more reproducible than MS/MS methods (Zhong et al, 2022), making NMR highly desirable for metabolomics research.…”

“…A number of drawbacks in the field of NMR metabolomics has resulted in the limited use of these techniques in microbiology research. While studies have been performed using untargeted metabolomics approaches for bacteria, they are often limited to individual species or they solely focus on isolating IC or EC compounds and not both simultaneously (Aries and Cloninger, 2020; Kumar et al, 2022; Ye et al, 2021). When both groups of compounds have been extracted in a singular protocol, it has commonly been performed using a singular species (Foschi et al, 2018; Hoerr et al, 2016).…”

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

“…In this investigation, the maximum number of metabolites from S. aureus were identified using SC (119 metabolites) and subsequently by FTC (116 metabolites) and FTC + SC (99 metabolites). Whereas for K. pneumoniae , the maximum number of metabolites were identified by the FTC (151) method followed by FTC + SC (132 metabolites) and SC (103 metabolites; Supplementary Table 2 ) ( Kumar et al, 2022 ). Notably, the number of metabolites identified in K. pneumoniae was more when compared to S. aureus and the potential reason remains unknown.…”

“…For metabolite extraction, the stored cell pellet was thawed for 10 min on ice, followed by resuspension in 500 μl of mass spectrometry (MS) grade chilled methanol (Sigma). To evaluate the metabolites extraction efficiency of three extraction methods, i.e., FTC, SC, FTC + SC, 13C-labeled L-valine (40 ng/ml), an internal standard, was added to each sample before metabolite extraction ( Kumar et al, 2022 ). For the freeze-thaw cycle-based metabolite extraction method, the resuspended cells were subjected to repeated freeze-thaw cycles (10 min at −80°C followed by 10 min on ice) three times.…”

“…A decrease in intracellular ATP levels has been linked to the development of the S. aureus persister phenotype making them resistant to antibiotics ( Conlon et al, 2016 ). However, existing knowledge of target(s) for regulating metabolomic modulation to treat resistant infections is still limited, owing to limited attempts to investigate the comprehensive metabolome of S. aureus ( Kumar et al, 2022 ). This may potentially be due to the unavailability of the appropriate protocols and pipeline for investigating the metabolome of the Gram-positive bacteria, S. aureus (specifically drug-resistant strains), including the very first step of extracting AMR-associated metabolites.…”