Rapid microbial identification and colistin resistance detection via MALDI-TOF MS using a novel on-target extraction of membrane lipids

Abstract

Rapid infection diagnosis is critical to improving patient treatment and outcome. Recent studies have shown microbial lipids to be sensitive and selective biomarkers for identifying bacterial and fungal species and antimicrobial resistance. Practical procedures for microbial lipid biomarker analysis will therefore improve patient outcomes and antimicrobial stewardship. However, current lipid extraction methods require significant hands-on time and are thus not suited for direct adoption as a clinical assay for microbial identification. Here, we have developed a method for lipid extraction directly on the surface of stainless-steel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) plates, termed fast lipid analysis technique or FLAT, which facilitates the identification of bacterial and fungal species using a sub-60-minute workflow. Additionally, our method detects lipid A modifications in Gram-negative bacteria that are associated with antimicrobial resistance, including to colistin.

Analyses of Lipid A Diversity in Gram-Negative Intestinal Bacteria Using Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

“…These results suggest that lipid A molecular profiles can be used as a taxonomic index and detection marker for bacteria. In fact, research on the detection of drug-resistant and pathogenic bacteria focusing on lipid A has recently emerged [ 31 , 32 , 33 ], and it is important to determine the structural diversity of lipid A for each bacterial strain and related phenotypes. The present study will contribute to deciphering the complex and heterogeneous landscape of lipid A in bacteria.…”

Colistin Heteroresistance Is Largely Undetected among Carbapenem-Resistant Enterobacterales in the United States

“…Previous studies have shown that colistin heteroresistance can be mediated by PhoPQ/PmrAB-dependent cationic sugar modifications to the lipid A component of lipopolysaccharide (LPS) (12)(13)(14)(15), resulting in an increased charge of the Gram-negative bacterial outer membrane and reduced susceptibility to colistin, a cationic antimicrobial peptide. Lipid A was analyzed using a new extraction method termed fast lipid analysis technique (FLAT) (16), as well as the previously used El Hamidi et al method (17), on a representative sample of 12 colistin-heteroresistant isolates from this study using matrixassisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry in the negative-ion mode (Table S5). After culture in the presence of colistin, which enriches for the resistant subpopulation, we observed an aminoarabinose (Dm/z 131 mass shift) modification to the terminal phosphate in all heteroresistant isolates evaluated, as well as an additional phosphoethanolamine (Dm/z 123 mass shift) modification in the Escherichia coli isolate analyzed (Fig.…”

Rapid identification of mcr-1-positive Escherichia coli from patient urine using a novel lipid-based MALDI-TOF-MS assay

“…The patient’s urine sample was collected for further analysis using a novel rapid lipid extraction protocol that allows for diagnosis directly from clinical samples, such as blood and urine cultures, without the need for growth by MALDI-TOF MS. This method is termed fast lipid analysis technology (FLAT) [ 12 ]. Briefly, 1 µl of urine, without concentrating, was spotted on a MALDI plate and overlaid with 1 µl of buffer, consisting of 0.2 M anhydrous citric acid and 0.1 M trisodium citrate dihydrate.…”