Fast identification of mycobacterium species by GC analysis with trimethylsulfonium hydroxide (TMSH) for transesterification

Abstract

SummaryTrimethylsulfonium hydroxide (TMSH) reproducibly converts fatty acids bound in, e-g., biomolecules such as phospholipids and/or glycerides, into the corresponding fatty acid methyl esters (FAMEs). The transesterification can be performed at room temperature in a fast single step reaction. Surprisingly, secondary alcohols and mycolic acid cleavage products (MACPs) are also released from mycobacteria under these conditions.The complex reaction mixtures containing FAMEs, MACPs, and secondary alcohols can easily be separated by high resolution temperature-programmed capillary GC. Different species of mycobacteria give rise to characteristic chromatographic patterns and the amount of lipids from a single colony of mycobacteria is sufficient for reliable identification of the bacteria. The profiles of the chromatograms match well those obtained from other sample preparation techniques.The TMSH method of identification of mycobacteria from the patterns of the gas chromatograms is faster and more sensitive than conventional methods, which also involve transesterifkation. The identification of mycobacterial species by microbiological culture techniques is difficult to perform and requires several weeks.

A high‐throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis

“…For high‐throughput GC analyses, about 30 seeds harvested from each mature T 2 transgenic line were aliquoted to custom 96‐well stainless steel plates, and were cracked using a custom 96‐tip hand‐held ball‐crushing device. Fatty acyl methyl esters (FAMEs) were derivatized for 15 min in 100 μ l trimethylsulfonium hydroxide solution (Muller et al. , 1993) and diluted with 100 μ l methanol; both were delivered into the 96‐well plates using a LEAP Twin‐PAL dual‐rail robot (LEAP Technologies, Carrboro, NC, USA) fitted to an Agilent 6890 dual‐FID GC (Palo Alto, CA, USA).…”

Fatty Acid Methyl Ester (FAME) Profiling Identifies Carbapenemase-Producing Klebsiella pneumoniae Belonging to Clonal Complex 258

“…Approximately 15 mg of bacterial cells were harvested from the surface of the agar plates and transferred to 1.5 mL Eppendorf tubes. Cells were suspended in 10 µL of distilled water, and then 30 µL of methanolic trimethylsulfonium hydroxide solution (0.25 M) was added to simultaneously lyse cells and transesterify the lipid fraction, according to the procedure reported by Müller and colleagues [13]. The reaction mixture was dried under a nitrogen stream, dissolved in 200 µL of a tert-butyl-methyl ether/methanol (MeOH) mixture (10:1 v/v), and directly injected into the GC-MS system.…”

“…Specifically, we evaluated the FAME profiles of six KPC-producing isolates belonging to the CC258 lineage (KPC + /258 + ), six KPC-producing isolates belonging to non-CC258 lineages (KPC + /258 − ), and six non-KPC-producing isolates belonging to non-CC258 lineages (KPC − /258 − ). We utilized a single-step sample preparation method to simultaneously lyse bacterial cells and transesterify the lipid fraction, according to a procedure previously reported by Müller and colleagues [13]. The FAME profiles were then analyzed using gas chromatography-mass spectrometry (GC-MS) and discriminatory FAMEs were identified using the machine learning algorithm Random Forest (RF) [14].…”

Fatty acid profiling of lipid classes by silica rod TLC-thermally assisted hydrolysis and methylation–GC/MS

“…In the past, attempts were made by scrapping off separated lipid bands from TLC plates followed by FA analysis using GC [1][2][3][4][5][6]. These strategies gave FA information on small amounts of sample ($20 mg) [17,18] but care had to be taken to control the weight ratio of silica gel versus ''scrapped-off'' lipid, where significant loss of polyunsaturated fatty acids (PUFAs) was observed [3].…”

“…Thermally assisted hydrolysis and methylation (THM) techniques [12] have been applied in recent years for profiling fatty acids in bacteria [13][14][15][16][17][18][19][20], zooplankton [20][21][22], microalgae [23], and polyunsaturated fatty acid (PUFA) rich oil [24,25]. THM procedures show a series of advantages over conventional fatty acid analysis protocols that include: smaller sample size, minimum sample treatment and handling, and faster analysis time.…”