A fast and reliable method for monitoring of prophage‐activating chemicals

Abstract

SummaryBacteriophages, that is viruses that infect bacteria, either lyse bacteria directly or integrate their genome into the bacterial genome as so‐called prophages, where they remain at a silent state. Both phages and bacteria are able to survive in this state. However, prophages can be reactivated with the introduction of chemicals, followed by the release of a high number of phage particles, which could infect other bacteria, thus harming ecosystems by a viral bloom. The basics for a fast, automatable analytical method for the detection of prophage‐activating chemicals are developed and successfully tested here. The method exploits the differences in metabolic heat produced by Escherichia coli with (λ+) and without the lambda prophages (λ−). Since the metabolic heat primarily reflects opposing effects (i.e. the reduction of heat‐producing cells by lysis and enhanced heat production to deliver the energetic costs for the synthesis of phages), a systematic analysis of the influence of the different conditions (experimentally and in silico) was performed and revealed anoxic conditions to be best suited. The main advantages of the suggested monitoring method are not only the possibility of obtaining fast results (after only few hours), but also the option for automation, the low workload (requires only few minutes) and the suitability of using commercially available instruments. The future challenge following this proof of principle is the development of thermal transducers which allow for the electronic subtraction of the λ+ from the λ‐ signal.

An enhanced bioindicator for calorimetric monitoring of prophage‐activating chemicals in the trace concentration range

“…The anaerobic growth conditions allow for providing the quickest results and responses to still traces of the test chemical. These findings are in accord with previous reports dealing with a simpler λ+ bioindicator [27]. Practical advantages of the proposed method are the potential for automation, prevention of sampling errors, ease of data evaluation, and consumption of still fewer and less expensive chemicals for the media.…”

“…The prophage‐free E. coli DSM4230 (λ−) host strain was obtained from the DSMZ (Braunschweig, Germany). The construction and characterization of the prophage wearing strain as well as the applied media are described in . All induction experiments were conducted at 37°C.…”

“…Two types of calorimetric experiments were carried out (A: an anaerobic in liquid culture; and B: an aerobic on solid agar). The first experimental set up was chosen because under such conditions the highest heat effect per mole of generated ATP is to be expected (for the calculation see ), whereas the second experimental set up was selected because under its conditions any oxygen diffusion effects on the measurement can mainly be excluded . In the anaerobic experiments (A), 1.9 mL LB media, 0.1 mL bacterial suspension (containing 0.09 mL E. coli (λ−), and 0.01 mL E. coli (λ+)) in LB medium (both with OD600 = 0.1) and different concentrations of the test chemicals were transferred to the calorimetric vessel.…”

“…For these reasons, we successfully developed and tested a simple and reliable method to assess the influence of chemicals on prophage activation that is based on microcalorimetry. This method was not only shown to be feasible with the established commercially available instruments, but also suitable for automation and high‐throughput measurements .…”

Rapid Calorimetric Detection of Bacterial Contamination: Influence of the Cultivation Technique

“…The method is also applicable to strict anaerobic microorganisms if oxygen is excluded during both preparation and the conduction of the experiments. This can be achieved by purging with an inert gas (e.g., N 2 ) (Maskow and Babel, 2003) or by overcoating of the culture with paraffin oil as described by Xu et al (2018).…”