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

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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.

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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).

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  1. Centro de Biotecnologia, Programa de Pós-Graduação em Biologia Celular e Molecular, Universidade Federal do Rio Grande do Sul (UFRGS), Avenida Bento Gonçalves, 9500, Porto Alegre, Rio Grande Do Sul, 91501-970, Brazil

    Laura Scherer Beier, Franciele Maboni Siqueira & Irene Silveira Schrank

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  1. Laura Scherer Beier
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  2. Franciele Maboni Siqueira
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Correspondence to Irene Silveira Schrank.

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Supplementary material 1—Information of the composition of the defined media tested (DOC 262 KB)

11033_2018_4413_MOESM2_ESM.pdf

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

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

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

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

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)

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Beier, L.S., Siqueira, F.M. & Schrank, I.S. Evaluation of growth and gene expression of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis in defined medium. Mol Biol Rep 45, 2469–2479 (2018). https://doi.org/10.1007/s11033-018-4413-3

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