Engineering a defined culture medium to grow Piscirickettsia salmonis for its use in vaccine formulations


Piscirickettsia salmonis is a facultative Gram-negative intracellular bacterium that produces piscirickettsiosis, disease that causes a high negative impact in salmonid cultures. The so-far-unidentified nutritional requirements have hindered its axenic culture at laboratory and industrial scales for the formulation of vaccines. The present study describes the development of a defined culture medium for P. salmonis. The culture medium was formulated through rational design involving auxotrophy test and statistical designs of experiments, considering the genome-scale metabolic reconstruction of P. salmonis reported by our group. The whole optimization process allowed for a twofold increase in biomass and a reduction of about 50% of the amino acids added to the culture medium. The final culture medium contains twelve amino acids, where glutamic acid, threonine and arginine were the main carbon and energy sources, supporting 1.65 g/L of biomass using 6.5 g/L of amino acids in the formulation. These results will contribute significantly to the development of new operational strategies to culture this bacterium for the production of vaccines.

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  1. 1.

    Albornoz R, Valenzuela K, Pontigo JP et al (2017) Identification of chemotaxis operon cheYZA and cheA gene expression under stressful conditions in Piscirickettsia salmonis. Microb Pathog 107:436–441

    CAS  Article  Google Scholar 

  2. 2.

    Arkush KD, McBride AM, Mendonca HL et al (2005) Genetic characterization and experimental pathogenesis of Piscirickettsia salmonis isolated from white seabass Atractoscion nobilis. Dis Aquat Organ 63:139–149

    CAS  Article  Google Scholar 

  3. 3.

    Athanassopoulou F, Groman D, Prapas T, Sabatakou O (2004) Pathological and epidemiological observations on rickettsiosis in cultured sea bass (Dicentrarchus labrax L.) from Greece. J Appl Ichthyol 20:525–529

    Article  Google Scholar 

  4. 4.

    Bartolucci AA, Singh KP, Bae S (2015) Robustness and Ruggedness. In: Bartolucci AA, Singh KP, Bae S (eds) Introduction to statistical analysis of laboratory data. Wiley, New York, pp 213–234

    Google Scholar 

  5. 5.

    Bhatnagar RK, Berry A, Hendry AT, Jensen RA (1989) The biochemical basis for growth inhibition by l-phenylalanine in Neisseria gonorrhoeae. Mol Microbiol 3:429–435

    CAS  Article  Google Scholar 

  6. 6.

    Birkbeck TH, Griffen AA, Reid HI et al (2004) Growth of Piscirickettsia salmonis to high titers in insect tissue culture cells. Infect Immun 72:3693–3694.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Braun PR, Al-Younes H, Gussmann J et al (2008) Competitive inhibition of amino acid uptake suppresses chlamydial growth: involvement of the chlamydial amino acid transporter BrnQ. J Bacteriol 190:1822–1830

    CAS  Article  Google Scholar 

  8. 8.

    Bravo S, Campos M (1989) Coho salmon syndrome in Chile. FHS/AFS Newsl 17:3

    Google Scholar 

  9. 9.

    Christgen SL, Becker DF (2019) Role of proline in pathogen and host interactions. Antioxid Redox Signal 30:683–709.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Contreras-Lynch S, Olmos P, Vargas A et al (2015) Identification and genetic characterization of Piscirickettsia salmonis in native fish from southern Chile. Dis Aquat Organ 115:233–244

    CAS  Article  Google Scholar 

  11. 11.

    Cortés MP, Mendoza SN, Travisany D et al (2017) Analysis of Piscirickettsia salmonis metabolism using genome-scale reconstruction, modeling, and testing. Front Microbiol 8:2462

    Article  Google Scholar 

  12. 12.

    Das M, Grover A (2018) Fermentation optimization and mathematical modeling of glycerol-based microbial poly(3-hydroxybutyrate) production. Process Biochem 71:1–11.

    CAS  Article  Google Scholar 

  13. 13.

    De Felice M, Levinthal M, Iaccarino M, Guardiola J (1979) Growth inhibition as a consequence of antagonism between related amino acids: effect of valine in Escherichia coli K-12. Microbiol Rev 43:42

    Article  Google Scholar 

  14. 14.

    Eliassen TM, Solbakk IT, Haugseth KT et al (2007) Process for culturing bacteria of the Piscirickettsia genus. Patent Application WO 2008/002152 A8.

  15. 15.

    Englesberg E, Bass R, Heiser W (1976) Inhibition of the growth of mammalian cells in culture by amino acids and the isolation and characterization of l-phenylalanine-resistant mutants modifying l-phenylalanine transport. Somat Cell Genet 2:411–428

    CAS  Article  Google Scholar 

  16. 16.

    Ferber DM, Ely B (1982) Resistance to amino acid inhibition in Caulobacter crescentus. Mol Gen Genet MGG 187:446–452

    CAS  Article  Google Scholar 

  17. 17.

    Fryer JL, Lannan CN, Garcés LH et al (1990) Isolation of a rickettsiales-like organism from diseased coho salmon (Oncorhynchus kisutch) in Chile. Fish Pathol 25:107–114

    Article  Google Scholar 

  18. 18.

    Fryer JL, Lannan CN, Giovannoni SJ, Wood ND (1992) Piscirickettsia salmonis gen. nov., sp. nov., the causative agent of an epizootic disease in salmonid fishes. Int J Syst Evol Microbiol 42:120–126

    CAS  Google Scholar 

  19. 19.

    Fu XT, Lin H, Kim SM (2009) Optimization of medium composition and culture conditions for agarase production by Agarivorans albus YKW-34. Process Biochem 44:1158–1163.

    CAS  Article  Google Scholar 

  20. 20.

    Fuentealba P, Aros C, Latorre Y et al (2017) Genome-scale metabolic reconstruction for the insidious bacterium in aquaculture Piscirickettsia salmonis. Bioresour Technol 223:105–114

    CAS  Article  Google Scholar 

  21. 21.

    Gesbert G, Ramond E, Tros F et al (2015) Importance of branched-chain amino acid utilization in Francisella intracellular adaptation. Infect Immun 83:173–183

    Article  Google Scholar 

  22. 22.

    Gómez F, Henríquez V, Marshall S (2009) Additional evidence of the facultative intracellular nature of the fish bacterial pathogen Piscirickettsia salmonis. Arch Med Vet 41:261–267

    Article  Google Scholar 

  23. 23.

    Henríquez M, González E, Marshall SH et al (2013) A novel liquid medium for the efficient growth of the salmonid pathogen Piscirickettsia salmonis and optimization of culture conditions. PLoS One 8:e71830.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Hobley L, Harkins C, MacPhee CE, Stanley-Wall NR (2015) Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes. FEMS Microbiol Rev 39:649–669

    CAS  Article  Google Scholar 

  25. 25.

    Jain SP, Singh PP, Javeer S, Amin PD (2010) Use of Placket–Burman statistical design to study effect of formulation variables on the release of drug from hot melt sustained release extrudates. AAPS PharmSciTech 11:936–944.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Janakiraman A, Lesser CF (2017) How to manage stress: lessons from an intracellular pathogen. Virulence 8:359–361.

    Article  PubMed  Google Scholar 

  27. 27.

    Johnson CL, Vishniac W (1970) Growth inhibition in Thiobacillus neapolitanus by histidine, methionine, phenylalanine, and threonine. J Bacteriol 104:1145–1150

    CAS  Article  Google Scholar 

  28. 28.

    Jolivet-Gougeon A, Bonnaure-Mallet M (2014) Biofilms as a mechanism of bacterial resistance. Drug Discov Today Technol 11:49–56

    Article  Google Scholar 

  29. 29.

    Koch AL (2007) Growth Measurement. In: Reddy CA, Beveridge TJ, Breznak JA et al (eds) Methods for general and molecular microbiology, 3rd edn. ASM Press, Washington D.C., pp 172–199

    Google Scholar 

  30. 30.

    Lawson J, Erjavec J (2017) Screening designs. In: Lawson J, Erjavec J (eds) Basic experimental strategies and data analysis for science and engineering. CRC Press, Boca Raton, pp 163–201

    Google Scholar 

  31. 31.

    Link H, Weuster-Botz D (2011) 2.11—medium formulation and development. In: Moo-Young M (ed) Comprehensive biotechnology, 2nd edn. Academic, Burlington, pp 119–134

    Google Scholar 

  32. 32.

    Maisey K, Montero R, Christodoulides M (2017) Vaccines for piscirickettsiosis (salmonid rickettsial septicaemia, SRS): the Chile perspective. Expert Rev Vaccines 16:215–228.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Manske C, Hilbi H (2014) Metabolism of the vacuolar pathogen Legionella and implications for virulence. Front Cell Infect Microbiol 4:125

    Article  Google Scholar 

  34. 34.

    Marshall SH, Gómez FA, Ramírez R et al (2012) Biofilm generation by Piscirickettsia salmonis under growth stress conditions: a putative in vivo survival/persistence strategy in marine environments. Res Microbiol 163:557–566

    CAS  Article  Google Scholar 

  35. 35.

    Mauel MJ, Ware C, Smith P (2008) Culture of Piscirickettsia salmonis on enriched blood agar. J Vet Diagn Investig 20:213–214.

    Article  Google Scholar 

  36. 36.

    Meibom KL, Charbit A (2010) Francisella tularensis metabolism and its relation to virulence. Front Microbiol.

    Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Mikalsen J, Skjaervik O, Wiik-Nielsen J et al (2008) Agar culture of Piscirickettsia salmonis, a serious pathogen of farmed salmonid and marine fish. FEMS Microbiol Lett 278:43–47.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Monds RD, O’Toole GA (2009) The developmental model of microbial biofilms: ten years of a paradigm up for review. Trends Microbiol 17:73–87.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Nagata Y, Chu KH (2003) Optimization of a fermentation medium using neural networks and genetic algorithms. Biotechnol Lett 25:1837–1842.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Navarrete-Bolaños JL, Téllez-Martínez MG, Miranda-López R, Jiménez-Islas H (2017) An experimental strategy validated to design cost-effective culture media based on response surface methodology. Prep Biochem Biotechnol 47:578–588.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Omsland A, Sixt BS, Horn M, Hackstadt T (2014) Chlamydial metabolism revisited: interspecies metabolic variability and developmental stage-specific physiologic activities. FEMS Microbiol Rev 38:779–801

    CAS  Article  Google Scholar 

  42. 42.

    Patil S, Nikam M, Patil H et al (2017) Bioactive pigment production by Pseudomonas spp. MCC 3145: statistical media optimization, biochemical characterization, fungicidal and DNA intercalation-based cytostatic activity. Process Biochem 58:298–305.

    CAS  Article  Google Scholar 

  43. 43.

    Pitol LO, Finkler ATJ, Dias GS et al (2017) Optimization studies to develop a low-cost medium for production of the lipases of Rhizopus microsporus by solid-state fermentation and scale-up of the process to a pilot packed-bed bioreactor. Process Biochem 62:37–47.

    CAS  Article  Google Scholar 

  44. 44.

    Plackett RL, Burman JP (1946) The design of optimum multifactorial experiments. Biometrika 33:305–325

    Article  Google Scholar 

  45. 45.

    Price CTD, Richards AM, Abu Kwaik Y (2014) Nutrient generation and retrieval from the host cell cytosol by intra-vacuolar Legionella pneumophila. Front Cell Infect Microbiol 4:111

    Article  Google Scholar 

  46. 46.

    Rinaudi LV, Giordano W (2010) An integrated view of biofilm formation in rhizobia. FEMS Microbiol Lett 304:1–11

    CAS  Article  Google Scholar 

  47. 47.

    Rozas M, Enríquez R (2014) Piscirickettsiosis and Piscirickettsia salmonis in fish: a review. J Fish Dis 37:163–188.

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Sargent B (2011) The need for defined, animal-free, components in vaccine production media.

  49. 49.

    Sernapesca (2018) Informe Sanitario de Salmonicultura en Centros Marinos—Año 2017.

  50. 50.

    Seth AK, Geringer MR, Hong SJ et al (2012) In vivo modeling of biofilm-infected wounds: a review. J Surg Res 178:330–338

    Article  Google Scholar 

  51. 51.

    Spaargaren DH (1996) The design of culture media based on the elemental composition of biological material. J Biotechnol 45:97–102.

    CAS  Article  Google Scholar 

  52. 52.

    Stellwagen E, Prantner JD, Stellwagen NC (2008) Do zwitterions contribute to the ionic strength of a solution? Anal Biochem 373:407–409.

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Tandberg JI, Lagos LX, Langlete P et al (2016) Comparative analysis of membrane vesicles from three Piscirickettsia salmonis isolates reveals differences in vesicle characteristics. PLoS One 11:e0165099.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Yañez AJ, Valenzuela K, Silva H et al (2012) Broth medium for the successful culture of the fish pathogen Piscirickettsia salmonis. Dis Aquat Organ 97:197–205.

    Article  PubMed  Google Scholar 

  55. 55.

    Yañez AJ, Silva H, Valenzuela K et al (2013) Two novel blood-free solid media for the culture of the salmonid pathogen Piscirickettsia salmonis. J Fish Dis 36:587–591

    Article  Google Scholar 

  56. 56.

    Zhang J, Greasham R (1999) Chemically defined media for commercial fermentations. Appl Microbiol Biotechnol 51:407–421.

    CAS  Article  Google Scholar 

  57. 57.

    Zuo S, Xiao J, Zhang Y et al (2018) Rational design and medium optimization for shikimate production in recombinant Bacillus licheniformis strains. Process Biochem 66:19–27.

    CAS  Article  Google Scholar 

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This work was supported by the FONDEF project [Grant Number D10I1185] and PhD scholarship CONICYT of Ministry of Education, Chile government.

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Correspondence to Claudia Altamirano.

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Fuentealba, P., Latorre, Y., González, E. et al. Engineering a defined culture medium to grow Piscirickettsia salmonis for its use in vaccine formulations. J Ind Microbiol Biotechnol 47, 299–309 (2020).

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  • Piscirickettsia salmonis
  • Nutritional requirement
  • Amino acid
  • Defined medium
  • Vaccines