Abstract
The determination of bacteriophage growth parameters, such as adsorption constant, latent period, and burst size, is essential for the proper design of bacteriophage production and the estimation of the efficacy of bacteriophage therapy. As they are dependent on the physiological state and cultivation conditions bacteria, they should be preferably determined in a non-invasive way. We propose a method that allows their determination under cultivation conditions. It is based on the cultivation of bacteria in a chemostat, the injection of bacteriophages, and monitoring of their concentration over a certain period. Phage growth parameters are determined by fitting a mathematical model to experimental data. E. coli–T4 system was investigated for various dilution rates covering a broad range of bacteria physiological states. Results were used for a prediction of bacteriophages and bacteria steady-state concentrations in a cellstat. A close match was found when adsorption of bacteriophages to the lysed cells was considered in the cellstat, while this mechanism can be neglected in the chemostat. Trends and values for burst size and latent period were consistent with literature data, demonstrating an increase in the burst size and decrease of the latent period with an increase of bacteria-specific growth rate (from 19 to 81 bacteriophage particles per cell and 89 to 29.8 min for a specific growth rate between 0.072 and 0.96 h−1, respectively). Adsorption constant also showed an increase with a specific growth rate increase (from 2.8E-10 to 4.0E-09 mL min−1), in contrast to chemostat literature data, probably due to its determination within the bioreactor. The proposed method also allowed estimation of latent period distribution. While its value for high-specific growth rates was determined to be constant of around 6 min, an increase of over an order of magnitude was found for the lowest specific growth rate, probably as a consequence of higher variability within bacteria population.
Key Points
• A method for determination of phage growth parameters under cultivating conditions was developed.
• The method was successfully tested on E. coli and T4 bacteriophage system comparing chemostat and cellstat values.
• Adsorption to lysed cells was found to be important for cellstat experiment but can be neglected in the chemostat.
• The determined burst size and latent period dependence on the bacterial physiological state was consistent with literature data, while differences were found for adsorption constant.
• Latent period distribution significantly increases for low bacteria–specific growth rates.
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Funding
This work was co-funded by the European Regional Development Fund and Slovenian Ministry of Education, Science and Sport (project BioPharm.Si) and project IMBI co-financed by European Regional Development Fund, Interreg V-A Italy-Slovenia programme 2014-2020.
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KŠ and AP contributed to the design of the research. KŠ carried out the experiments and performed the numerical calculations. Both KŠ and AP authors discussed the results and contributed to the manuscript. AP prepared final version of the manuscript.
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Šivec, K., Podgornik, A. Determination of bacteriophage growth parameters under cultivating conditions. Appl Microbiol Biotechnol 104, 8949–8960 (2020). https://doi.org/10.1007/s00253-020-10866-8
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DOI: https://doi.org/10.1007/s00253-020-10866-8