A Rapid Method for Performing a Multivariate Optimization of Phage Production Using the RCCD Approach

Abstract

Bacteriophages can be used in various applications, from the classical approach as substitutes for antibiotics (phage therapy) to new biotechnological uses, i.e., as a protein delivery vehicle, a diagnostic tool for specific strains of bacteria (phage typing), or environmental bioremediation. The demand for bacteriophage production increases daily, and studies that improve these production processes are necessary. This study evaluated the production of a T4-like bacteriophage vB_EcoM-UFV09 (an E. coli-infecting phage with high potential for reducing environmental biofilms) in seven types of culture media (Luria–Bertani broth and the M9 minimal medium with six different carbon sources) employing four cultivation variables (temperature, incubation time, agitation, and multiplicity of infection). For this purpose, the rotatable central composite design (RCCD) methodology was used, combining and comparing all parameters to determine the ideal conditions for starting to scale up the production process. We used the RCCD to set up the experimental design by combining the cultivation parameters in a specific and systematic way. Despite the high number of conditions evaluated, the results showed that when specific conditions were utilized, viral production was effective even when using a minimal medium, such as M9/glucose, which is less expensive and can significantly reduce costs during large-scale phage production.

Optimization of Escherichia coli Bacteriophages Production

“…There is very little information published on the optimization of key process variables for phage production. The rotatable central composite design (RCCD) methodology was used, combining and comparing all parameters to determine the ideal conditions for the production process of bacteriophages [37]. Directly influenced by environmental conditions like medium composition, temperature, pH, and agitation [38,39,40].…”

Genetically distant bacteriophages select for unique genomic changes in Enterococcus faecalis

“…Phage infection and development are determined by bacterial host factors such as nutritional state (Li et al, 2003 ). A recent study showed that carbon source is an important factor in phage production (Silva et al, 2021 ). A previous study using Bacillus subtilis showed that phage 29 infection downregulated CcpA‐dependent genes.…”

Investigation into scalable and efficient enterotoxigenic Escherichia coli bacteriophage production

“…Due to the prevalence of antibiotic resistance, studies have been carried out using both standalone phage therapy and combinations of phages and antibiotics to treat bacterial infections in humans and other animals 1 – 8 . However, for phage therapy to become more realistic on a global scale, efficient strategies to mass produce phages need to be established 9 , 10 .…”

“…Considering infection conditions, infection load (the amount of bacterial culture added in percent volume/volume) and MOI (ratio of infectious phages to viable bacterial cells) influence phage-host dynamics and the number of infection cycles that take place over the course of production 9 , 20 , 21 . An intermediate MOI may delay lysis, thereby allowing bacterial hosts to continue producing phages; in contrast an overly high or low MOI, results in rapid lysis or unimpeded bacterial growth respectively, sacrificing phage titer 14 , 15 , 21 .…”

“…An intermediate MOI may delay lysis, thereby allowing bacterial hosts to continue producing phages; in contrast an overly high or low MOI, results in rapid lysis or unimpeded bacterial growth respectively, sacrificing phage titer 14 , 15 , 21 . Growth conditions such as media composition 9 , 14 , 22 , supplementation of ions 14 , 23 , and temperature 16 , 24 can impact phage production 20 . Media formulations require careful consideration in large-scale propagation as defined media may alleviate issues relating to high costs, animal-sourced components, batch-to-batch variability, and more complex downstream processing associated with complex media 9 , 25 – 27 .…”