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.

中文翻译：

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在培养条件下确定噬菌体生长参数。

摘要

确定噬菌体生长参数，例如吸附常数，潜伏期和爆发大小，对于正确设计噬菌体生产和评估噬菌体疗法的有效性至关重要。由于它们取决于细菌的生理状态和培养条件，因此应优选以非侵入性方式确定它们。我们提出了一种在培养条件下可以测定它们的方法。它基于在恒化器中培养细菌，注入噬菌体并在一定时期内监测其浓度。通过将数学模型拟合到实验数据来确定噬菌体的生长参数。大肠杆菌研究了–T4系统的各种稀释率，涵盖了广泛的细菌生理状态。结果用于预测细胞稳定器中的噬菌体和细菌稳态浓度。当在cellstat中考虑噬菌体吸附到裂解的细胞上时，发现紧密匹配，而在化学恒温器中可以忽略这种机制。爆发大小和潜伏期的趋势和值与文献数据一致，表明爆发大小的增加和潜伏期的减少随着细菌特异性生长速率的增加（每细胞噬菌体颗粒从19个增加到81个，噬菌体从89个增加到29.8个）在0.072至0.96 h ^-1之间的特定生长速率下为min， 分别）。与恒化器文献数据相比，吸附常数还显示出随着特定生长速率增加（从2.8E-10到4.0E-09 mL min ^-1）的增加，这可能是由于其在生物反应器中的测定。所提出的方法还允许估计潜伏期分布。尽管其高比增长率的值确定为恒定在6分钟左右，但发现最低比增长率增加了一个数量级，这可能是细菌种群内部变异性更高的结果。

关键点

•开发了一种在培养条件下测定噬菌体生长参数的方法。

•该方法已在大肠杆菌和T4噬菌体系统上成功测试，比较了恒化器和细胞稳定剂的值。

•发现对裂解细胞的吸附对于cellstat实验很重要，但在恒化器中可以忽略不计。

•确定的猝发大小和潜伏期对细菌生理状态的依赖性与文献数据一致，但发现吸附常数存在差异。

•对于低细菌特异性生长速率，潜伏期分布显着增加。