肠杆菌科细菌在形成稳定(休眠)细胞形态过程中脂肪酸光谱的变化特征

B. Andryukov, L. Somova, I. Lyapun, M. Bynina, E. Matosova
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引用次数: 0

摘要

介绍。随着细菌群体异质性范式的出现,休眠细胞的表型引起了人们的关注,休眠细胞的活性产生发生在不利的微生物环境条件下。这些细胞的特点是代谢和生殖休眠,以及抗生素耐药性。然而,一旦出现有利的生存条件,它们就能再次发芽,造成传染病的加剧。近年来,抗菌素治疗有效性的威胁下降以及持续、慢性和医院感染发生率的增加与这些病原菌的表型有关。鉴于脂肪酸(FA)在细菌适应中的关键作用,本研究的目的是确定革兰氏阴性肠杆菌科细菌在极端条件下的长期储存和休眠(未培养)细胞亚群形成过程中脂肪酸组成变化的具体特征。材料和方法。研究中使用了以下参考菌株的静态培养:假结核耶尔森菌、肠炎鼠伤寒沙门氏菌和大肠杆菌,在4-8°С的凡士林油中保存5-10年。通过去除油层和收集微生物团块获得休眠细胞形态。透射电镜证实了休眠细胞形态的超微结构特征。采用分子遗传学方法评价休眠细胞的活力。通过在LB肉汤、Endo和Serov培养基上反复接种,并在4-6°C、22-24°C和37°С孵育,检查休眠形式的繁殖活性。根据欧洲标准化委员会批准的程序和Sherlock MIS协议推荐的程序获得总FAs的甲酯。采用气相色谱-质谱联用技术对脂肪酸甲酯进行分析。对菌团进行初步匀浆后,提取脂质,并在70 ev下通过电子冲击获得FA谱。结果表明,在极端条件下(低温、营养缺乏、缺氧),大肠杆菌、假结核杆菌和鼠伤寒沙门氏菌可形成表型未培养的休眠细胞代。通过对休眠表型脂肪酸谱变化的比较分析,揭示了与营养细胞相比,休眠表型脂肪酸谱的某些特征与不饱和指数的降低和长链饱和脂肪酸的优势(C14-C18)有关。所观察到的转化的生物学意义显然与这些FA组分在休眠(未培养)细胞表型可逆形成中的特殊作用有关,并且在代谢不活跃状态下作为碳水化合物的替代来源,以及它们随后在有利的生活条件下逆转为营养细胞。
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Features of changes in spectra of fatty acids of the bacteria of the Enterobacteriaceae family in the process of forming stable (dormant) cell forms
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.
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来源期刊
Zhurnal mikrobiologii, epidemiologii, i immunobiologii
Zhurnal mikrobiologii, epidemiologii, i immunobiologii Immunology and Microbiology-Immunology and Microbiology (miscellaneous)
CiteScore
1.40
自引率
0.00%
发文量
51
审稿时长
8 weeks
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