Features of changes in spectra of fatty acids of the bacteria of the Enterobacteriaceae family in the process of forming stable (dormant) cell forms

Abstract

Introduction. With the advent of the paradigm of heterogeneity of the bacterial population, attention has been drawn to the phenotype of dormant cells, the active generation of which occurs when adverse environmental conditions of microorganisms appear. These cells are characterized by metabolic and reproductive dormancy, as well as antibiotic resistance. However, upon the occurrence of favorable living conditions, they are able to germinate again and cause an exacerbation of infectious diseases. In recent years, a threatening decrease in the effectiveness of antimicrobial therapy and an increase in the incidence of persistent, chronic and hospital infections have been associated with these phenotypes of pathogenic bacteria. Given the key role of fatty acid (FA) in the adaptation of bacteria, the aim of this study was to identify the specific features of changes in the fatty acid composition of gram-negative bacteria from the Enterobacteriaceae family during their long-term storage under extreme conditions and the formation of dormant (uncultured) subpopulations of cell forms.Materials and methods. Static cultures of following reference strains were used in the study: Yersinia pseudotuberculosis, Salmonella enterica Typhimurium, and Escherichia coli, stored under vaseline oil at 4-8°С for 5-10 years. Dormant cell forms were obtained by removing the oil layer and collecting the microbial mass. The ultrastructural features of the dormant cell forms were confirmed by transmission electron microscopy. The viability of dormant cells was assessed by a molecular genetic method. The lack of reproductive activity of dormant forms was checked by repeated inoculations on LB broth, Endo and Serov media and incubation at 4-6°C, 22-24°C, and 37°С. Methyl esters of total FAs were obtained according to the procedure approved by the European Committee for Standardization and recommended by the Sherlock MIS protocol. Analysis of fatty acid methyl esters was carried out by gas chromatography in combination with mass spectrometry. After preliminary homogenization of the bacterial masses, lipids were extracted, and FA spectra were obtained by electron impact at 70 eVResults. It was demonstrated that phenotypic uncultured generation of dormant cells is formed under extreme conditions (low temperature, nutrient deficiency, hypoxia) in populations of E. coli, Y. pseudotuberculosis and S. Typhimurium. A comparative analysis of changes in the fatty acid spectrum in the dormant phenotype revealed certain features compared to vegetative cells associated with a decrease in the unsaturation index and the dominance of long-chain saturated FAs (C14-C18).Conclusion. The biological significance of the observed transformations is apparently associated with the special role of these FA fractions in the reversible formation of dormant (uncultivated) cell phenotype and as an alternative source of carbohydrates in a metabolically inactive state, as well as their subsequent reversal to vegetative cells upon favorable living conditions.