Журнал микробиологии, эпидемиологии и иммунобиологии. 2020; 97: 401-412
Особенности изменений спектров жирных кислот бактерий семейства Enterobacteriaceae в процессе формирования устойчивых (дормантных) клеточных форм
https://doi.org/10.36233/0372-9311-2020-97-5-2Аннотация
Введение. С появлением парадигмы гетерогенности популяции бактерий возросло внимание к фенотипу дормантных (дремлющих) клеток, активная генерация которых происходит при неблагоприятных условиях среды обитания микроорганизмов. Эти клетки характеризуются метаболическим и репродуктивным покоем, а также резистентностью к антибиотикам. Однако при наступлении благоприятных для них условий обитания они способны вновь прорастать и вызывать обострение инфекционных заболеваний. С этими фенотипами патогенных бактерий связывают угрожающее снижение эффективности антимикробной терапии, рост уровня заболеваемости персистирующими, хроническими и госпитальными инфекциями. С учетом ключевой роли в адаптации бактерий жирных кислот (ЖК) целью исследования было выявление специфических особенностей изменений ЖК-состава грамотрицательных бактерий семейства Enterobacteriaceae в процессе их многолетнего хранения в экстремальных условиях и формирования дормантных (некультивируемых) субпопуляций клеточных форм.
Материалы и методы. Для исследования использовали статические культуры эталонных штаммов: Yersinia pseudotuberculosis, Salmonella enterica Typhimurium и Escherichia coli, хранившиеся под вазелиновым маслом при 4-8оС в течение 5-10 лет. Дормантные клеточные формы получали путем удаления масляного слоя и сбора микробной массы. Ультраструктурные признаки дормантных клеточных форм были подтверждены трансмиссионной электронной микроскопией. Жизнеспособность дормантных клеток оценивали молекулярно-генетическим методом. Отсутствие репродуктивной активности дормантных форм проверяли путем многократных посевов на LB-бульон, среды Эндо и Серова и инкубации при 4-6, 22-24 и 37оС. Получение метиловых эфиров общих ЖК проводили по методике, утвержденной Европейским комитетом по стандартизации и рекомендованной Sherlock MIS-протоколом. Анализ метиловых эфиров ЖК осуществляли методом газовой хроматографии в сочетании с масс-спектрометрией. После предварительной гомогенизации микробных масс бактерий липиды экстрагировали, спектры ЖК получали методом электронного удара при 70 эВ.
Результаты. Доказано, что в экстремальных условиях (низкая температура, недостаток питательных веществ, гипоксия) в популяции E. coli, Y. pseudotuberculosis и S. Typhimurium формируется фенотипическая некультивируемая генерация дормантных клеток. Сравнительный анализ изменений ЖК-спектра в дормантном фенотипе выявил определенные особенности по сравнению с вегетативными клетками, связанные со снижением индекса ненасыщенности и доминированием длинноцепочечных насыщенных ЖК (С14-С18).
Выводы. Биологическое значение выявленных трансформаций связано, по-видимому, с особой ролью этих фракций ЖК в обратимом формировании дремлющего (некультивируемого) клеточного фенотипа и как альтернативного источника углеводов в метаболически неактивном состоянии, а также в их последующей реверсии в вегетативные клетки при наступлении благоприятных условий существования.
Список литературы
1. Miyaue S., Suzuki E., Komiyama Y, Kondo Y, Morikawa M., Maeda S. Bacterial memory of persisters: bacterial persister cells can retain their phenotype for days or weeks after withdrawal from colony-biofilm culture. Front. Microbiol. 2018; 9: 1396. https://doi.org/10.3389/fmicb.2018.01396
2. Hice S.A., Santoscoy M.C., Soupir M.L., Cademartiri R. Dis-tinguishing between metabolically active and dormant bacteria on paper. Appl. Microbiol. Biotechnol. 2018; 102(1): 367-75. https://doi.org/10.1007/s00253-017-8604-y
3. Bublitz D.C., Wright P.C., Wright P.C., Bodager J.R., Rasambainarivo F.T., Bliska J.B., et al. Epidemiology of pathogenic En-terobacteria in humans, livestock, and peridomestic rodents in rural Madagascar. PLoS One. 2014; 9(7): e101456. https://doi.org/10.1371/journal.pone.0101456
4. WHO. Antimicrobial resistance; 2015. Available at: https://www.who.int/antimicrobial-resistance/publications/global-action-plan/
5. Barak I., Muchova K. The role of lipid domains in bacterial cell processes. Int. J. Mol. Sci. 2013; 14(2): 4050-65. https://doi.org/10.3390/ijms14024050
6. Schennink A., Heck J.M., Bovenhuis H., Visker M.H., van Valenberg H.J., van Arendonk J.A. Milk fatty acid unsatura¬tion: genetic parameters and effects of stearoyl-CoA desaturase (SCD1) and acyl CoA: diacylglycerol acyltransferase 1 (DGAT1). J. Dairy Sci. 2008; 91(5): 2135-43. https://doi.org/10.3168/jds.2007-0825
7. Yao J., Rock C.O. Exogenous fatty acid metabolism in bacteria. Biochimie. 2017; 141: 30-9. https://doi.org/10.1016/j.biochi.2017.06.015
8. Андрюков Б.Г., Сомова Л.М., Матосова Е.В., Ляпун И.Н. фенотипическая пластичность бактерий как стратегия резистентности и объект современных антимикробных техно-логий (обзор). Современные технологии в медицине. 2019; 11(2): 164-82. https://doi.org/10.17691/stm2019.1L2.22
9. Ichihara K., Fukubayashi Y Preparation of fatty acid methyl es¬ters for gas-liquid chromatography. J. Lipid Res. 2010; 51(3): 635-40. https://doi.org/10.1194/jlr.D001065
10. Sherlock. Microbial Identification System. V 6.2. MIS Operat¬ing Manual. Newark: Sandy Dr.; 2012.
11. Cangelosi G.A., Meschke J.S. Dead or alive: molecular assess-ment of microbial viability. Appl. Environ. Microbiol. 2014; 80(19): 5884-91. https://doi.org/10.1128/aem.01763-14
12. Soejima T., Iida K., Qin T., Taniai H., Seki M., Takade A., et al. Photoactivated ethidium monoazide directly cleaves bacterial DNA and is applied to PCR for discrimination of live and dead bacteria. Microbiol. Immunol. 2007; 51(8): 763-75. https://doi.org/10.1111/j.1348-0421.2007.tb03966.x
13. Bligh E.G., Dyer W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959; 37(8): 911¬
14. https://doi.org/10.1139/o59-099
15. Li Y, Wu S., Wang L., Li Y, Shi F., Wang X. Differentiation of bacteria using fatty acid profiles from gas chromatography-tan-dem mass spectrometry. J. Sci. Food Agric. 2010; 90(8): 1380-3. https://doi.org/10.1002/jsfa.3931
16. Tan Y, Wu M., Liu H., Dong X., Guo Z., Song Z., et al. Cellular fatty acids as chemical markers for differentiation of Yersinia pestis and Yersinia pseudotuberculosis. Lett. Appl. Microbiol. 2010; 50(1): 104-11. https://doi.org/10.1111/j.1472-765X.2009.02762.x
17. Viarengo G., Sciara M.I., Salazar M.O., Kieffer P.M., Furlan R.L., Garda Vescovi E. Unsaturated long chain free fatty acids are input signals of the Salmonella enterica PhoP/PhoQ regulatory system. J. Biol. Chem. 2013; 288(31): 22346-58. https://doi.org/10.1074/jbc.M113.472829
18. JanBen H.J., Steinbuchel A. Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnol. Biofuels. 2014; 7(1): 7. https://doi.org/10.1186/1754-6834-7-7
19. Huang C.B., Alimova Y, Myers T.M., Ebersole J.L. Shortand medium-chain fatty acids exhibit antimicrobial activity for oral microorganisms. Arch. Oral. Biol. 2011; 56(7): 650-4. https://doi.org/10.1016/j.archoralbio.2011
20. Matsumoto K., Kusaka J., Nishibori A., Hara H. Lipid domains in bacterial membranes. Mol. Microbiol. 2006; 61(5): 1110-7. https://doi.org/10.1111/j.1365-2958.2006.05317.x
21. Bousfield I.J., Smith G.L., Dando T.R., Hobbs G. Numerical analysis of total fatty acid profiles in the identification of coryneform, nocardioform and some other bacteria. Microbiology. 1983; 129(2): 375-94. https://doi.org/10.1099/00221287-129-2-375
22. Van Teeseling M.C.F., de Pedro M.A., Cava F. Determinants of bacterial morphology: from fundamentals to possibilities for antimicrobial targeting. Front. Microbiol. 2017; 8: 1264. https://doi.org/10.3389/fmicb.2017.01264
23. Paget M.S. Bacterial sigma factors and anti-sigma factors: structure, function and distribution. Biomolecules. 2015; 5(3): 1245-65. https://doi.org/10.3390/biom5031245
24. Shen G., Li X. The multifaceted role of hypoxia-inducible fac¬tor 1 (HIF1) in lipid metabolism, hypoxia and human diseases. https://doi.org/10.5772/65340 Available at: https://www.inte-chopen.com/books/hypoxia-and-human-diseases/the-multifac-eted-role-of-hypoxia-inducible-factor-1 -hif1-in-lipid-metabo-lism
25. Del Portillo P., Garda-Morales L., Menendez M.C., Anzola J.M., Rodriguez J.G., Helguera-Repetto A.C., et al. Hypoxia is not a main stress when Mycobacterium tuberculosis is in a dormancy-like long-chain fatty acid environment. Front. Cell Infect. Microbiol. 2019; 8: 449. https://doi.org/10.3389/fcimb.2018.00449
26. Mocali S., Chiellini C., Fabiani A., Decuzzi S., de Pascale D., Parrilli E., et al. Ecology of cold environments: new insights of bacterial metabolic adaptation through an integrated genomicphenomic approach. Sci. Rep. 2017; 7(1): 839. https://doi.org/10.1038/s41598-017-00876-4
27. Hassan N., Anesio A.M., Rafiq M., Holtvoeth J., Bull I., Haleem A., et al. Temperature driven membrane lipid adaptation in glacial psychrophilic bacteria. Front. Microbiol. 2020; 11: 824. https://doi.org/10.3389/fmicb.2020.00824
28. Battesti A., Bouveret E. Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism. Mol. Microbiol. 2006; 62(4): 1048-63. https://doi.org/10.1111/j.1365-2958.2006.05442.x
29. Cronan J.E., Thomas J. Bacterial fatty acid synthesis and its re-lationships with polyketide synthetic pathways. Methods Enzymol. 2009; 459: 395-33. https://doi.org/10.1016/S0076-6879(09)04617-5
30. Joers A., Vind K., Hernandez S.B., Maruste R., Pereira M., Brauer A., et al. Muropeptides stimulate growth resumption from stationary phase in Escherichia coli. Sci. Rep. 2019; 9(1): 18043. https://doi.org/10.1038/s41598-019-54646-5
31. Burkert A., Douglas T.A., Waldrop M.P., Mackelprang R. Changes in the active, dead, and dormant microbial commu¬nity structure across a Pleistocene permafrost chronosequence. Appl. Environ. Microbiol. 2019; 85(7): e02646-18. https://doi.org/10.1128/AEM.02646-18
Journal of microbiology, epidemiology and immunobiology. 2020; 97: 401-412
Features of changes in spectra of fatty acids of the bacteria of the Enterobacteriaceae family in the process of forming stable (dormant) cell forms
https://doi.org/10.36233/0372-9311-2020-97-5-2Abstract
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 eV
Results. 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.
References
1. Miyaue S., Suzuki E., Komiyama Y, Kondo Y, Morikawa M., Maeda S. Bacterial memory of persisters: bacterial persister cells can retain their phenotype for days or weeks after withdrawal from colony-biofilm culture. Front. Microbiol. 2018; 9: 1396. https://doi.org/10.3389/fmicb.2018.01396
2. Hice S.A., Santoscoy M.C., Soupir M.L., Cademartiri R. Dis-tinguishing between metabolically active and dormant bacteria on paper. Appl. Microbiol. Biotechnol. 2018; 102(1): 367-75. https://doi.org/10.1007/s00253-017-8604-y
3. Bublitz D.C., Wright P.C., Wright P.C., Bodager J.R., Rasambainarivo F.T., Bliska J.B., et al. Epidemiology of pathogenic En-terobacteria in humans, livestock, and peridomestic rodents in rural Madagascar. PLoS One. 2014; 9(7): e101456. https://doi.org/10.1371/journal.pone.0101456
4. WHO. Antimicrobial resistance; 2015. Available at: https://www.who.int/antimicrobial-resistance/publications/global-action-plan/
5. Barak I., Muchova K. The role of lipid domains in bacterial cell processes. Int. J. Mol. Sci. 2013; 14(2): 4050-65. https://doi.org/10.3390/ijms14024050
6. Schennink A., Heck J.M., Bovenhuis H., Visker M.H., van Valenberg H.J., van Arendonk J.A. Milk fatty acid unsatura¬tion: genetic parameters and effects of stearoyl-CoA desaturase (SCD1) and acyl CoA: diacylglycerol acyltransferase 1 (DGAT1). J. Dairy Sci. 2008; 91(5): 2135-43. https://doi.org/10.3168/jds.2007-0825
7. Yao J., Rock C.O. Exogenous fatty acid metabolism in bacteria. Biochimie. 2017; 141: 30-9. https://doi.org/10.1016/j.biochi.2017.06.015
8. Andryukov B.G., Somova L.M., Matosova E.V., Lyapun I.N. fenotipicheskaya plastichnost' bakterii kak strategiya rezistentnosti i ob\"ekt sovremennykh antimikrobnykh tekhno-logii (obzor). Sovremennye tekhnologii v meditsine. 2019; 11(2): 164-82. https://doi.org/10.17691/stm2019.1L2.22
9. Ichihara K., Fukubayashi Y Preparation of fatty acid methyl es¬ters for gas-liquid chromatography. J. Lipid Res. 2010; 51(3): 635-40. https://doi.org/10.1194/jlr.D001065
10. Sherlock. Microbial Identification System. V 6.2. MIS Operat¬ing Manual. Newark: Sandy Dr.; 2012.
11. Cangelosi G.A., Meschke J.S. Dead or alive: molecular assess-ment of microbial viability. Appl. Environ. Microbiol. 2014; 80(19): 5884-91. https://doi.org/10.1128/aem.01763-14
12. Soejima T., Iida K., Qin T., Taniai H., Seki M., Takade A., et al. Photoactivated ethidium monoazide directly cleaves bacterial DNA and is applied to PCR for discrimination of live and dead bacteria. Microbiol. Immunol. 2007; 51(8): 763-75. https://doi.org/10.1111/j.1348-0421.2007.tb03966.x
13. Bligh E.G., Dyer W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959; 37(8): 911¬
14. https://doi.org/10.1139/o59-099
15. Li Y, Wu S., Wang L., Li Y, Shi F., Wang X. Differentiation of bacteria using fatty acid profiles from gas chromatography-tan-dem mass spectrometry. J. Sci. Food Agric. 2010; 90(8): 1380-3. https://doi.org/10.1002/jsfa.3931
16. Tan Y, Wu M., Liu H., Dong X., Guo Z., Song Z., et al. Cellular fatty acids as chemical markers for differentiation of Yersinia pestis and Yersinia pseudotuberculosis. Lett. Appl. Microbiol. 2010; 50(1): 104-11. https://doi.org/10.1111/j.1472-765X.2009.02762.x
17. Viarengo G., Sciara M.I., Salazar M.O., Kieffer P.M., Furlan R.L., Garda Vescovi E. Unsaturated long chain free fatty acids are input signals of the Salmonella enterica PhoP/PhoQ regulatory system. J. Biol. Chem. 2013; 288(31): 22346-58. https://doi.org/10.1074/jbc.M113.472829
18. JanBen H.J., Steinbuchel A. Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnol. Biofuels. 2014; 7(1): 7. https://doi.org/10.1186/1754-6834-7-7
19. Huang C.B., Alimova Y, Myers T.M., Ebersole J.L. Shortand medium-chain fatty acids exhibit antimicrobial activity for oral microorganisms. Arch. Oral. Biol. 2011; 56(7): 650-4. https://doi.org/10.1016/j.archoralbio.2011
20. Matsumoto K., Kusaka J., Nishibori A., Hara H. Lipid domains in bacterial membranes. Mol. Microbiol. 2006; 61(5): 1110-7. https://doi.org/10.1111/j.1365-2958.2006.05317.x
21. Bousfield I.J., Smith G.L., Dando T.R., Hobbs G. Numerical analysis of total fatty acid profiles in the identification of coryneform, nocardioform and some other bacteria. Microbiology. 1983; 129(2): 375-94. https://doi.org/10.1099/00221287-129-2-375
22. Van Teeseling M.C.F., de Pedro M.A., Cava F. Determinants of bacterial morphology: from fundamentals to possibilities for antimicrobial targeting. Front. Microbiol. 2017; 8: 1264. https://doi.org/10.3389/fmicb.2017.01264
23. Paget M.S. Bacterial sigma factors and anti-sigma factors: structure, function and distribution. Biomolecules. 2015; 5(3): 1245-65. https://doi.org/10.3390/biom5031245
24. Shen G., Li X. The multifaceted role of hypoxia-inducible fac¬tor 1 (HIF1) in lipid metabolism, hypoxia and human diseases. https://doi.org/10.5772/65340 Available at: https://www.inte-chopen.com/books/hypoxia-and-human-diseases/the-multifac-eted-role-of-hypoxia-inducible-factor-1 -hif1-in-lipid-metabo-lism
25. Del Portillo P., Garda-Morales L., Menendez M.C., Anzola J.M., Rodriguez J.G., Helguera-Repetto A.C., et al. Hypoxia is not a main stress when Mycobacterium tuberculosis is in a dormancy-like long-chain fatty acid environment. Front. Cell Infect. Microbiol. 2019; 8: 449. https://doi.org/10.3389/fcimb.2018.00449
26. Mocali S., Chiellini C., Fabiani A., Decuzzi S., de Pascale D., Parrilli E., et al. Ecology of cold environments: new insights of bacterial metabolic adaptation through an integrated genomicphenomic approach. Sci. Rep. 2017; 7(1): 839. https://doi.org/10.1038/s41598-017-00876-4
27. Hassan N., Anesio A.M., Rafiq M., Holtvoeth J., Bull I., Haleem A., et al. Temperature driven membrane lipid adaptation in glacial psychrophilic bacteria. Front. Microbiol. 2020; 11: 824. https://doi.org/10.3389/fmicb.2020.00824
28. Battesti A., Bouveret E. Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism. Mol. Microbiol. 2006; 62(4): 1048-63. https://doi.org/10.1111/j.1365-2958.2006.05442.x
29. Cronan J.E., Thomas J. Bacterial fatty acid synthesis and its re-lationships with polyketide synthetic pathways. Methods Enzymol. 2009; 459: 395-33. https://doi.org/10.1016/S0076-6879(09)04617-5
30. Joers A., Vind K., Hernandez S.B., Maruste R., Pereira M., Brauer A., et al. Muropeptides stimulate growth resumption from stationary phase in Escherichia coli. Sci. Rep. 2019; 9(1): 18043. https://doi.org/10.1038/s41598-019-54646-5
31. Burkert A., Douglas T.A., Waldrop M.P., Mackelprang R. Changes in the active, dead, and dormant microbial commu¬nity structure across a Pleistocene permafrost chronosequence. Appl. Environ. Microbiol. 2019; 85(7): e02646-18. https://doi.org/10.1128/AEM.02646-18
