1H NMR-based metabolomics approach to understanding the temperature-dependent pathogenicity of Lactococcus garvieae

Nasim Safari Alighialo, Ruhollah Rahimi, Saeed Hajirezaee, Farzaneh Nikookhah


Lactococcus garvieae is known as main agent of the bacterial diseases, Lactococcosis in trout farms. The present study was aimed to study the metabolic bases of the temperature-dependent pathogenicity of the L. garvieae using 1H NMR spectroscopy. The bacteria were grown at different temperatures, including 10, 14, 18 and 22˚C and then the metabolites extracted, identified and quantified. The results of PLS-DA analysis clearly separated the experimental treatments. The main metabolites responsible for this separation were acetate, acetoacetate, creatine phosphate, succinylacetone and trehalose. Furthermore, the result of the analysis of variance indicated also significant differences in metabolome content between temperature treatments. The bacteria exposed to higher temperatures showed more concentration of acetate and acetoacetate compared to those grown at 10°C. The concentrations of trehalose were higher in the bacteria grown at 14 and 18°C compared to other temperature treatments. The higher levels of succinylacetone were found in the bacteria exposed to the temperature less than 14°C compared to those grown at 18 and 22°C. The creatine phosphate concentrations increased with temperature, however, a significant decline occurred at 22°C. The levels of isoeugenol, methionine and betaine significantly declined with increase of temperature from 10 to 22°C. Also, the concentration of N-Acetylglutamine significantly raised as the temperature increased from 10 to 22°C. In conclusion, the temperature altered the metabolome of L. garvie, which this may be linked to the pathogenicity. The temperature probably affects fermentation, homeostasis, energetic condition and metabolism of amino acids in L. garvieae.


Metabolomics, Pathogenicity, Lactococcus garvieae, Fish.

Full Text:



Akhtar Y., Pages E., Stevens A., Bradbury R., da Camara C.A., Isman M.B. (2012). Effect of chemical complexity of essential oils on feeding deterrence in larvae of the cabbage looper. Physiological Entomology, 37: 81-91.

Ankley G.T., Daston G.P., Degitz S.J., Denslow N.D., Hoke R.A., Kennedy S.W., Miracle A.L., Perkins E.J., Snape J., Tillitt D.E. (2006). Toxicogenomics in regulatory ecotoxicology. In. ACS Publications.

Argüelles J.C. (2000). Physiological roles of trehalose in bacteria and yeasts: a comparative analysis. Archives of Microbiology, 174: 217-224.

Åslund M.W., Celejewski M., Lankadurai B.P., Simpson A.J., Simpson M.J. (2011). Natural variability and correlations in the metabolic profile of healthy Eisenia fetida earthworms observed using 1H NMR metabolomics. Chemosphere, 83: 1096-1101.

Baeck G.W., Kim J.H., Gomez D.K., Park S.C. (2006). Isolation and characterization of Streptococcus sp. from diseased flounder (Paralichthys olivaceus) in Jeju Island. Journal of Veterinary Science 7: 53-58.

Bloem A., Bertrand A., Lonvaud‐Funel A., De Revel G. (2007). Vanillin production from simple phenols by wine‐associated lactic acid bacteria. Letters in Applied Microbiology, 44: 62-67.

Boroujerdi A.F., Vizcaino M.I., Meyers A., Pollock E.C., Huynh S.L., Schock T.B., Morris P.J., Bearden D.W. (2009). NMR-based microbial metabolomics and the temperature-dependent coral pathogen Vibrio coralliilyticus. Environmental Science and Technology, 43: 7658-7664.

Bundy J.G., Davey M.P., Viant M.R. (2009). Environmental metabolomics: a critical review and future perspectives. Metabolomics, 5: 3.

Caldas T., Demont-Caulet N., Ghazi A., Richarme G. (1999). Thermoprotection by glycine betaine and choline. Microbiology, 145: 2543-2548

Courtenay E., Capp M., Anderson C., Record M. (2000). Vapor pressure osmometry studies of osmolyte− protein interactions: implications for the action of osmoprotectants in vivo and for the interpretation of “osmotic stress” experiments in vitro. Biochemistry, 39: 4455-4471.

Csonka L.N. (1989). Physiological and genetic responses of bacteria to osmotic stress. Microbiological Reviews 53: 121-147.

Csonka L.N., Hanson A.D. (1991). Prokaryotic osmoregulation: genetics and physiology. Annual Review of Microbiology 45: 569-606.

Culham D.E., Lasby B., Marangoni A.G., Milner J.L., Steer B.A., van Nues R.W., Wood J.M. (1993). Isolation and sequencing of Escherichia coli gene proP reveals unusual structural features of the osmoregulatory proline/betaine transporter, ProP. Journal of Molecular Biology 229: 268-276.

Dal Pozzo M., Silva Loreto É., Flores Santurio D., Hartz Alves S., Rossatto L., Castagna de Vargas A., Viegas J., Matiuzzi da Costa M. (2012). Antibacterial activity of essential oil of cinnamon and trans-cinnamaldehyde against Staphylococcus spp. isolated from clinical mastitis of cattle and goats. Acta Scientiae Veterinariae 40: 1-5.

Diler O., Altun S., Adiloglu A., Kubilay A., Istklt B. (2002). First occurrence of streptococcosis affecting farmed rainbow trout (Oncorhynchus mykiss) in Turkey. Bulletin-European Association of Fish Pathologists, 22: 21-26.

Dindo M., Conter C., Oppici E., Ceccarelli V., Marinucci L., Cellini B. (2018). Molecular basis of primary hyperoxaluria: clues to innovative treatments. Urolithiasis, 1-12.

Eldar A.A., Ghittino C. (1999). Lactococcus garvieae and Streptococcus iniae infections in rainbow trout Oncorhynchus mykiss: similar, but different diseases. Diseases of Aquatic Organisms, 36: 227-231.

Eyngor M., Zlotkin A., Ghittino C., Prearo M., Douet D-G., Chilmonczyk S., Eldar A. (2004). Clonality and diversity of the fish pathogen Lactococcus garvieae in Mediterranean countries. Applied and Environmental Microbiology, 70: 5132-5137.

Filippov A.E., Gorb S.N., Popov V.L. (2015). What can we learn from the “water bears” for the adhesion systems using in space applications? Facta Universitatis, Series: Mechanical Engineering, 13: 241-247.

Foster J., Ganatra M., Kamal I., Ware J., Makarova K., Ivanova N., Bhattacharyya A., Kapatral V., Kumar S., Posfai J. (2005). The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode. PLoS Biology, 3: e121.

Fujita Y., Matsuoka H., Hirooka K. (2007). Regulation of fatty acid metabolism in bacteria. Molecular Microbiology 66: 829-839.

Gomez Zavaglia A., Tymczyszyn E., De Antoni G., Anibal Disalvo E. (2003). Action of trehalose on the preservation of Lactobacillus delbrueckii ssp. bulgaricus by heat and osmotic dehydration. Journal of Applied Microbiology, 95: 1315-1320.

Gottschalk G. (2012) Bacterial metabolism. Springer Science & Business Media.

Haardt M., Kempf B., Faatz E., Bremer E. (1995). The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12. Molecular and General Genetics MGG, 246: 783-796.

Haran N., Kahana Z.E., Lapidot A. (1983). In vivo 15N NMR studies of regulation of nitrogen assimilation and amino acid production by Brevibacterium lactofermentum. Journal of Biological Chemistry, 258: 12929-12933.

Janssens J., Laekeman G.M., Pieters L.A., Totte J., Herman A.G., Vlietinck A.J. (1990). Nutmeg oil: identification and quantitation of its most active constituents as inhibitors of platelet aggregation. Journal of Ethnopharmacology, 29: 179-188.

Keun H.C., Ebbels T.M., Antti H., Bollard M.E., Beckonert O., Holmes E., Lindon J.C., Nicholson J.K. (2003). Improved analysis of multivariate data by variable stability scaling: application to NMR-based metabolic profiling. Analytica Chimica Acta, 490: 265-276.

Kinoshita S., Tanaka K. (1972). Glutamic acid. The microbial production of amino acids John Wiley, New York, N.Y. 263-324.

Lankadurai B.P., Nagato E.G., Simpson M.J. (2013). Environmental metabolomics: an emerging approach to study organism responses to environmental stressors. Environmental Reviews, 21: 180-205

Lerner A.B. (2009). Metabolism of phenylalanine and tyrosine. Advances in Enzymology 14:73-128.

Leslie S.B., Israeli E., Lighthart B., Crowe J.H., Crowe L.M. (1995). Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Applied and Environmental Microbiology, 61: 3592-3597.

Ljungdhal L. (1986). The autotrophic pathway of acetate synthesis in acetogenic bacteria. Annual Reviews in Microbiology, 40: 415-450.

López-Pedrosa J.M., Manzano M., Baxter J.H., Rueda R. (2007). N-Acetyl-L-Glutamine, a liquid-stable source of glutamine, partially prevents changes in body weight and on intestinal immunity induced by protein energy malnutrition in pigs. Digestive Diseases and Sciences, 52: 650.

Mah R., Hungate R., Ohwaki K. (1977). Acetate, a key intermediate in methanogenesis. In: Microbial Energy Conversion. Elsevier. pp: 97-106.

McCutcheon J.P., Moran N.A. (2012). Extreme genome reduction in symbiotic bacteria. Nature Reviews Microbiology, 10: 13.

Molenaar D., Hagting A., Alkema H., Driessen A., Konings W.N. (1993). Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis. Journal of Bacteriology, 175: 5438-5444.

Nakanishi T. (1978). Roles of ammonium and chloride ions in the conversion of L-glutamic acid fermentation to L-glutamine and N-acetyl-L-glutamine fermentation by Corynebacterium glutamicum. Journal of Fermentation Technology, 56: 179-188.

Newman J.C., Verdin E. (2014). Ketone bodies as signaling metabolites. Trends in Endocrinology and Metabolism, 25: 42-52.

Nicholson J.K., Lindon J.C., Holmes E. (1999). 'Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica, 29:1181-1189

Pan Z., Raftery D. (2007). Comparing and combining NMR spectroscopy and mass spectrometry in metabolomics. Analytical and Bioanalytical Chemistry 387: 525-527.

Pastoris O., Dossena M., Foppa P., Catapano M., Arbustini E., Bellini O., Dal Bello B., Minzioni G., Ceriana P., Barzaghi N. (1998). Effect ofl-carnitine on myocardial metabolism: results of a balanced, placebo-controlled, double-blind study in patients undergoing open heart surgery. Pharmacological Research, 37:115-122.

Pereira F., Ravelo C., Toranzo A., Romalde J. (2004). Lactococcus garvieae, an emerging pathogen for the Portuguese trout culture. Bulletin of the European Association of Fish Pathologists, 24: 274-279.

Qu J., Chen W., Luo G., Wang Y., Xiao S., Ling Z., Chen G. (2002). Rapid determination of underivatized pyroglutamic acid, glutamic acid, glutamine and other relevant amino acids in fermentation media by LC-MS-MS. Analyst, 127: 66-69.

Racher K., Voegele R., Marshall E., Culham D., Wood J., Jung H., Bacon M., Cairns M., Ferguson S., Liang W-J. (1999). Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts. Biochemistry, 38: 1676-1684.

Ritota M., Casciani L., Han B-Z., Cozzolino S., Leita L., Sequi P., Valentini M. (2012). Traceability of Italian garlic (Allium sativum L.) by means of HRMAS-NMR spectroscopy and multivariate data analysis. Food Chemistry 135: 684-693.

Robertson D.G. (2005). Metabonomics in toxicology: a review. Toxicological Sciences 85: 809-822.

Roeßler M., Müller V. (2001). Osmoadaptation in bacteria and archaea: common principles and differences. Environmental Microbiology 3: 743-754.

Simpson M.J., McKelvie J.R. (2009). Environmental metabolomics: new insights into earthworm ecotoxicity and contaminant bioavailability in soil. Analytical and Bioanalytical Chemistry, 394: 137-149.

Snowden M., Baxter J., Mamula Bergana M., Reyzer I., Pound V. (2002). Stability of N‐Acetylglutamine and Glutamine in Aqueous Solution and in a Liquid Nutritional Product by an Improved HPLC Method. Journal of Food Science, 67: 384-389.

Soltani M., Nikbakht G., Ebrahimzadeh Moussavi H., Ahmadzadeh N. (2008). Epizootic outbreak of lactococcosis caused by Lactococcus garvieae in farmed rainbow trout (Oncorhynchus mykiss) in Iran. Bulletin of the European Association of Fish Pathologists, 28: 95-106.

Tian N., Wang J., Wang P., Song X., Yang M., Kong L. (2013). NMR-based metabonomic study of Chinese medicine Gegen Qinlian Decoction as an effective treatment for type 2 diabetes in rats. Metabolomics, 9: 1228-1242.

van de Guchte M., Serror P., Chervaux C., Smokvina T., Ehrlich S.D., Maguin E. (2002). Stress responses in lactic acid bacteria. In: Lactic Acid Bacteria: Genetics, Metabolism and Applications. Springer, pp: 187-216.

Van Ravenzwaay B., Herold M., Kamp H., Kapp M., Fabian E., Looser R., Krennrich G., Mellert W., Prokoudine A., Strauss V. (2012). Metabolomics: a tool for early detection of toxicological effects and an opportunity for biology based grouping of chemicals—from QSAR to QBAR. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 746: 144-150.

Vendrell D., Balcázar J.L., Ruiz-Zarzuela I., De Blas I., Gironés O., Múzquiz J.L. (2006). Lactococcus garvieae in fish: a review. Comparative Immunology, Microbiology and Infectious Diseases, 29: 177-198.

Verpoorte R., Choi Y., Mustafa N., Kim H. (2008). Metabolomics: back to basics. Phytochemistry Reviews, 7: 525-537.

Viant M.R., Bearden D.W., Bundy J.G., Burton I.W., Collette T.W., Ekman D.R., Ezernieks V., Karakach T.K., Lin C.Y., Rochfort S. (2008). International NMR-based environmental metabolomics intercomparison exercise. Environmental Science and Technology, 43: 219-225.

Xia J., Wishart D.S. (2011). Metabolomic data processing, analysis, and interpretation using MetaboAnalyst. Current Protocols in Bioinformatics 34: 14.10. 11-14.10. 48.

Xu L., Shao X. (2004). Methods of chemometrics. Academic Press: Beijing.

Zemek J., Košíková B., Augustin J., Joniak D. (1979). Antibiotic properties of lignin components. Folia Microbiologica, 24: 483-486.

Zemek J., Valent M., Pódová M., Košíková B., Joniak D. (1987). Antimicrobiai properties of aromatic compounds of plant origin. Folia Microbiologica, 32: 421-425.


  • There are currently no refbacks.