Investigetion (detection) of environment microbial contamination by mass spectrometry. Part 2, 3-hydroxy fatty acids as a lipopolysaccharides (endotoxin) biomarkers of the gram-negative bacterial cell walls

Abstract

Microorganisms synthesize several monomeric chemical structures that are not found elsewhere in nature, e.g. muramic acid (an amino sugar) and D-amino acids (D-alanine and D-glutamic acid) are ubiquitous in bacterial peptidoglycan (PG). Characteristic sugars (e.g. heptoses) and 3-hydroxylated fatty acids are found in the endotoxin (lipopolysaccharide, LPS) of gram-negative bacteria [1]. The best way to protect against environment contamination is microbial control. Methods in current use for monitoring microorganisms mainly include culture and direct microscopy. However, several factors, including samples collection, growth conditions, incubation temperature and interaction between different organisms all affected the culture results. Additionally, culture based methods can detect only viable organisms and they are also time consuming, sometimes taking days or weeks. However, since both living and dead microorganisms express irritating and toxic structures, they should all be taken into consideration. Muramic acid has been suggested for use as a chemical marker in gas chromatography-mass spectrometry (GC-MS) determination of bacterial peptidoglycan [2]. While 3-hydroxylated fatty acids are the best proposition for use as a chemical markers in gas chromatography-mass spectrometry determination of bacterial lipopolysaccharide (endotoxin) of gram-negative bacteria in both clinical and environmental samples [38]. Two derivatives have been applied, including the trimethylsilyl (TMS) and pentafluorobenzoyl (PFBO) derivatives [80]. Both derivatives (TMS and PFBO) have been proven suitable for use with GC-ion-trap tandem MS [3]. The aim of our proposition is trial of application of gas chromatography-mass spectrometry (GC-MS) method as an alternative or complement to culturing, microscopy and other assays for detection, characterization and monitoring of microbial contamination of environment (e.g. water, air, air-conditioning systems), contamination of biochemical and food production chain processes, packaging for foodstuffs etc. by analysis of bacterial 3-hydroxylated fatty acids as a biochemical markers. A method is described for the quantitation of methyl esters of 3-hydroxyacids, markers of bacterial lipopolysaccharide (endotoxin), as trimethylsilyl or pentafluorobenzoyl derivatives using GC/MS method. The described methods are quick and simple, can be applied for monitoring microbial contamination directly, without prior culturing, in complex environmental samples. This method can be also applied for testing processes of cleaning and disinfections on packaging materials or on both packaging materials/foodstuffs in order to decrease their microbial load and thus to ensure better shelf-life. [1] Z. Mielniczuk, K. Bal, Spektrometria mas w badaniach skażeń mikrobiologicznych środowiska. Część I. Kwas muraminowy jako biomarker ścian komórkowych bakterii, Wiad. Chem., 2012, 66, 445. [2] K. Bal, L. Larsson, E. Mielniczuk, Z. Mielniczuk, Structure of muramic acid TMS derivative mass spectrum’s base ion (m/z=185) used for quantification of bacterial peptidoglycan, J. Microbiol. Meth., 2002, 48, 267. [3] A. Saraf, L. Larsson, Identification of microorganisms by mass spectrometry, Advances in Mass Spectrometry, 1998, 14, 449. [38] Z. Mielniczuk, E. Mielniczuk, L. Larsson, Gas chromatography-mass spectrometry methods for analysis of 2- and 3-hydroxylated fatty acids: Application for endotoxin measurement, J. Microbiol. Meth., 1993, 17, 91. [80] Z. Mielniczuk, S. Alugupalli, E. Mielniczuk, L. Larsson, Gas chromatography-mass spectrometry of lipopolysaccharide 3-hydroxy fatty acids: comparison of pentafluorobenzoyl and trimethylsilyl methyl ester derivatives, J. Chromatogr., 1992, 623, 115