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Evaluation of growth and gene expression of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis in defined medium

  • Laura Scherer Beier
  • Franciele Maboni Siqueira
  • Irene Silveira Schrank
Original Article

Abstract

Mycoplasmas belong to the Mollicutes class and possess low GC content and lack a cell wall, and also simplified metabolic pathways. Due to its reduced metabolic ability mycoplasmas are fastidious organisms growing with difficult under laboratory conditions. Its complex nutritional requirements render mycoplasmas to depend on external supplies of biosynthetic precursors. Aiming to develop and test defined media that could be used as a tool for Mycoplasma research, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis were cultivated in a complex medium supplemented with serum (Friis broth) and in four different defined media (YUS, YUSm, CMRL and CMRL+, that was developed in the present study). The cell concentration of both Mycoplasma species was assessed, by flow cytometry. Cellular viability was also analyzed in all defined media, indicating the presence of viable mycoplasma cells. All the defined media tested were able to maintain cell concentrations and viability and, amongst them, CMRL+ was the most suitable. For both Mycoplasma species, only the CMRL+ media showed similar cell density when compared to the complex medium. The transcriptional response of M. hyopneumoniae in CMRL+ broth was assessed by RT-qPCR, and the transcriptional profile of 18 genes in three cultures conditions (standard, heat shock and oxidative stress) was analyzed demonstrating gene expression regulation in response to the medium composition and to the culture conditions tested. The medium developed enables the definition of mycoplasmal nutritional requirements and metabolic pathways as well as genetic analysis.

Keywords

Mycoplasma Growth measurement Defined medium Gene regulation Flow cytometry 

Notes

Acknowledgements

This work was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES. CAPES—Biologia Computacional (Process Number: 23038.010043/2013-02) and Ministério da Ciência, Tecnologia e Inovação/Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCTI/CNPq) Universal (Process Number: 445228/2014-8).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

No human nor animal subjects were involved in this study.

Supplementary material

11033_2018_4413_MOESM1_ESM.doc (262 kb)
Supplementary material 1—Information of the composition of the defined media tested (DOC 262 KB)
11033_2018_4413_MOESM2_ESM.pdf (616 kb)
Supplementary material 2—Schematic representation of sample cultivation and processing. a: workflow of procedures to assess the growth rate in all the defined media to compare with complex medium. b: assessment of growth at different times of cultivation of M. hyopneumoniae and M. hyorhinis in Friis and CMRL+ broth. MHP: M. hyopneumoniae; MHR: M. hyorhinis (PDF 616 KB)
11033_2018_4413_MOESM3_ESM.pdf (150 kb)
Supplementary material 3—Schematic representation of the viability test protocol. Indicating the species and media utilized, pH alteration was seen through a color shift of the media. MHP: M. hyopneumoniae; MHR: M. hyorhinis (PDF 149 KB)
11033_2018_4413_MOESM4_ESM.pdf (236 kb)
Supplementary material 4—Schematic representation of procedures to assess gene regulation in CMRL+ and Friis broth. MHP: M. hyopneumoniae; MHR: M. hyorhinis (PDF 236 KB)
11033_2018_4413_MOESM5_ESM.doc (253 kb)
Supplementary material 5—Target genes and the oligonucleotide sequences and features used to assess transcriptional regulation (DOC 253 KB)
11033_2018_4413_MOESM6_ESM.pdf (5 mb)
Supplementary material 6—Viability test by color shift of the media. a: expected color change of media. In an earlier inoculated medium, the pH is alkaline (around 8.2), and the medium shows a red color, implying that there is no mycoplasmal growth (I). Once mycoplasma start to duplicate, growth metabolites cause medium acidification (II), decreasing the pH to about 6.6 after 48 h of cultivation (III). This pH alteration, seen as a color shift from red to yellow, denotes bacterial growth. b: viability test result. By the end of 48 h of cultivation, the alteration in color was only visualized in CMRL and CMRL+ media. After re-inoculation in Friis broth, neither culture presented the expected color shift (PDF 5095 KB)

References

  1. 1.
    Citti C, Blanchard A (2013) Mycoplasmas and their host: emerging and re-emerging minimal pathogens. Trends Microbiol 21(4):196–203CrossRefGoogle Scholar
  2. 2.
    Sirand-Pugnet P, Citti C, Barre A, Blanchard A (2007) Evolution of Mollicutes: down a bumpy road with twists and turns. Res Microbiol 158(10):754–766CrossRefGoogle Scholar
  3. 3.
    Thacker EL (2004) Diagnosis of Mycoplasma hyopneumoniae. J Swine Health Prod 12(5):252–254Google Scholar
  4. 4.
    Kobisch M, Friis NF (1996) Swine mycoplasmoses. Rev Sci Tech 15(4):6CrossRefGoogle Scholar
  5. 5.
    Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW (2012) Diseases of Swine. John Wiley & Sons, Inc., OxfordGoogle Scholar
  6. 6.
    Vasconcelos ATR, Ferreira HB, Bizarro CV, Bonatto SL, Carvalho MO, Pinto PM, Almeida DF, Almeida LGP, Almeida R, Alves L et al.(2005) Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae. J Bacteriol 187(16):5568–5577CrossRefGoogle Scholar
  7. 7.
    Siqueira FM, Gerber AL, Guedes RLM, Almeida LG, Schrank IS, Vasconcelos ATR, Zaha A (2014) Unravelling the transcriptome profile of the swine respiratory tract mycoplasmas. PLoS ONE 9(10):12CrossRefGoogle Scholar
  8. 8.
    Ferrarini MG, Siqueira FM, Mucha SG, Palama TL, Jobard E, Elena-Herrmann B, Vasconcelos ATR, Tardy F, Schrank IS, Zaha A et al.(2016) Insights on the virulence of swine respiratory tract Mycoplasmas through genome-scale metabolic modeling. BMC Genom 17:20CrossRefGoogle Scholar
  9. 9.
    Baseman JB, Tully JG (1997) Mycoplasmas: sophisticated, reemerging, and burdened by their notoriety. Emerg Infect Dis 3(1):21–32CrossRefGoogle Scholar
  10. 10.
    Greenbergofrath N, Terespolosky Y, Kahane I, Bar R (1993) Cyclodextrins as carriers of cholesterol and fatty-acids in cultivation of mycoplasmas. Appl Environ Microbiol 59(2):547–551Google Scholar
  11. 11.
    Minion FC, Lefkowitz EJ, Madsen ML, Cleary BJ, Swartzell SM, Mahairas GG (2004) The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. J Bacteriol 186(21):7123–7133CrossRefGoogle Scholar
  12. 12.
    Stemke GW, Robertson JA (1990) The growth-response of Mycoplasma hyopneumoniae and Mycoplasma flocculare based upon ATP-dependent luminometry. Vet Microbiol 24(2):135–142CrossRefGoogle Scholar
  13. 13.
    Razin S, Tully JG (1995) Molecular and diagnostic procedures in mycoplasmology. Academic Press, INC, CaliforniaGoogle Scholar
  14. 14.
    Gardner SW, Minion FC (2010) Detection and quantification of intergenic transcription in Mycoplasma hyopneumoniae. Microbiology 156:2305–2315CrossRefGoogle Scholar
  15. 15.
    van Belkum A, Scherer S, van Alphen L, Verbrugh H (1998) Short-sequence DNA repeats in prokaryotic genomes. Microbiol Mol Biol Rev 62(2):275–293PubMedPubMedCentralGoogle Scholar
  16. 16.
    Cattani AM, Siqueira FM, Guedes RLM, Schrank IS (2016) Repetitive elements in Mycoplasma hyopneumoniae transcriptional regulation. PLoS ONE 11(12):e0168626CrossRefGoogle Scholar
  17. 17.
    Madsen ML, Nettleton D, Thacker EL, Edwards R, Minion FC (2006) Transcriptional profiling of Mycoplasma hyopneumoniae during heat shock using microarrays. Infect Immun 74(1):160–166CrossRefGoogle Scholar
  18. 18.
    Madsen ML, Nettleton D, Thacker EL, Minion FC (2006) Transcriptional profiling of Mycoplasma hyopneumoniae during iron depletion using microarrays. Microbiology 152:937–944CrossRefGoogle Scholar
  19. 19.
    Oneal MJ, Schafer ER, Madsen ML, Minion FC (2008) Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to norepinephrine. Microbiology 154:2581–2588CrossRefGoogle Scholar
  20. 20.
    Schafer ER, Oneal MJ, Madsen ML, Minion FC (2007) Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to hydrogen peroxide. Microbiology 153:3785–3790CrossRefGoogle Scholar
  21. 21.
    Siqueira FM, de Morais GL, Higashi S, Beier LS, Breyer GM, de Sá Godinho CP, Sagot M-F, Schrank IS, Zaha A, de Vasconcelos ATR (2016) Mycoplasma non-coding RNA: identification of small RNAs and targets. BMC Genom 17(8):743CrossRefGoogle Scholar
  22. 22.
    Friis NF (1975) Some recommendations concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare—survey. Nordisk Veterinaer Med 27(6):337–339Google Scholar
  23. 23.
    Yus E, Maier T, Michalodimitrakis K, van Noort V, Yamada T, Chen WH, Wodke JAH, Guell M, Martinez S, Bourgeois R et al.(2009) Impact of genome reduction on bacterial metabolism and its regulation. Science 326(5957):1263–1268CrossRefGoogle Scholar
  24. 24.
    Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, Moorman AFM (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37(6):12CrossRefGoogle Scholar
  25. 25.
    Madsen ML, Puttamreddy S, Thacker EL, Carruthers MD, Minion FC (2008) Transcriptome changes in Mycoplasma hyopneumoniae during infection. Infect Immun 76(2):658–663CrossRefGoogle Scholar
  26. 26.
    Cook BS, Beddow JG, Manso-Silvan L, Maglennon GA, Rycroft AN (2016) Selective medium for culture of Mycoplasma hyopneumoniae. Vet Microbiol 195:158–164CrossRefGoogle Scholar
  27. 27.
    Razin S (1969) Structure and function in Mycoplasma. Annu Rev Microbiol 23:317–356CrossRefGoogle Scholar
  28. 28.
    Razin S, Tully JG (1970) Cholesterol requirement of mycoplasmas. J Bacteriol 102(2):306–310PubMedPubMedCentralGoogle Scholar
  29. 29.
    Rodwell AW, Abbot A (1961) Function of glycerol, cholesterol and long-chain fatty acids in nutrition of Mycoplasma mycoides. J Gen Microbiol 25(2):201–214CrossRefGoogle Scholar
  30. 30.
    Miles RJ, Wadher BJ, Henderson CL, Mohan K (1998) Increased growth yields of Mycoplasma spp. in the presence of pyruvate. Lett Appl Microbiol 7(6):149–151CrossRefGoogle Scholar
  31. 31.
    Kamminga T, Slagman SJ, Bijlsma JJE, Martins dos Santos VAP, Suarez-Diez M, Schaap PJ (2017) Metabolic modeling of energy balances in Mycoplasma hyopneumoniae shows that pyruvate addition increases growth rate. Biotechnol Bioeng 114(10):2339–2347CrossRefGoogle Scholar
  32. 32.
    Constantopoulos G, McGarrity GJ (1987) Activities of oxidative enzymes in mycoplasmas. J Bacteriol 169(5):2012–2016CrossRefGoogle Scholar
  33. 33.
    Lin YC, Miles RJ, Nicholas RAJ, Kelly DP, Wood AP (2008) Isolation and immunological detection of Mycoplasma ovipneumoniae in sheep with atypical pneumonia, and lack of a role for Mycoplasma arginini. Res Vet Sci 84(3):367–373CrossRefGoogle Scholar
  34. 34.
    Bertin C, Pau-Roblot C, Courtois J, Manso-Silvan L, Thiaucourt F, Tardy F, Le Grand D, Poumarat F, Gaurivaud P (2013) Characterization of free exopolysaccharides secreted by Mycoplasma mycoides Subsp mycoides. PLoS ONE 8(7):9Google Scholar
  35. 35.
    Friis NF (1971) Selective medium for Mycoplasma suipneumoniae. Acta Vet Scand 12(3):454–456PubMedGoogle Scholar
  36. 36.
    Razin S (1994) DNA probes and PCR in diagnosis of mycoplasma-infections. Mol Cell Probes 8(6):497–511CrossRefGoogle Scholar
  37. 37.
    Jaffe JD, Stange-Thomann N, Smith C, DeCaprio D, Fisher S, Butler J, Calvo S, Elkins T, Fitzgerald MG, Hafez N et al.(2004) The complete genome and proteome of Mycoplasma mobile. Genome Res 14(8):1447–1461CrossRefGoogle Scholar
  38. 38.
    Gardella RS, Delgiudice RA (1995) Growth of Mycoplasma hyorhinis cultivar-alpha on semisynthetic medium. Appl Environ Microbiol 61(5):1976–1979PubMedPubMedCentralGoogle Scholar
  39. 39.
    Calus D, Maes D, Vranckx K, Villareal I, Pasmans F, Haesebrouck F (2010) Validation of ATP luminometry for rapid and accurate titration of Mycoplasma hyopneumoniae in Friis medium and a comparison with the color changing units assay. J Microbiol Methods 83(3):335–340CrossRefGoogle Scholar
  40. 40.
    Buysschaert B, Byloos B, Leys N, Van Houdt R, Boon N (2016) Reevaluating multicolor flow cytometry to assess microbial viability. Appl Microbiol Biotechnol 100(21):9037–9051CrossRefGoogle Scholar
  41. 41.
    Gusarov I, Nudler E (2005) NO-mediated cytoprotection: instant adaptation to oxidative stress in bacteria. Proc Natl Acad Sci USA 102(39):13855–13860CrossRefGoogle Scholar
  42. 42.
    Dascher CC, Poddar SK, Maniloff J (1990) Heat-shock response in mycoplasmas, genome-limited organisms. J Bacteriol 172(4):1823–1827CrossRefGoogle Scholar
  43. 43.
    Plesofsky N, Higgins L, Markowski T, Brambl R (2016) Glucose starvation alters heat shock response, leading to death of wild type cells and survival of MAP kinase signaling mutant. PLoS ONE 11(11):31CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Centro de Biotecnologia, Programa de Pós-Graduação em Biologia Celular e MolecularUniversidade Federal do Rio Grande do Sul (UFRGS)Porto AlegreBrazil

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