Prophage-Free Enterococcus faecalis Strain for Monoclonal Phage Production
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Solution Overview
Problem
The production of therapeutic bacteriophage preparations against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium strains is hindered by contamination with temperate phages, which can propagate and spread bacterial virulence genes, necessitating the use of prophage-free bacterial strains for safe and effective phage therapy.
Innovation Solution
The development of a prophage-free Enterococcus faecalis strain, specifically the 1854wph strain, which is devoid of active prophages, allowing for the efficient multiplication and isolation of monoclonal therapeutic bacteriophages, ensuring high-titer preparations free from contaminating phages.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional Enterococcus faecalis strains are used for bacteriophage multiplication, then phage production is efficient, but contamination with temperate phages occurs which can spread bacterial virulence genes
Solution Approach 1:
The patent extracts and removes the harmful temperate prophages from the bacterial genome through site-specific recombination using Cre recombinase and loxP sites. This eliminates the source of contamination while preserving the bacterial strain's ability to support lytic phage multiplication, thus resolving the contradiction between productivity and safety.
Solution Approach 2:
The patent introduces an intermediary genetic system consisting of Cre recombinase and loxP sites that mediates the removal of temperate prophages. This intermediary mechanism allows controlled elimination of harmful elements without affecting the host bacterium's core functions or the lytic phage multiplication process.
2Adaptability or versatility
If temperate bacteriophages are present in bacterial cultures, then they can integrate into bacterial chromosomes and provide adaptive benefits, but they can also be spontaneously induced and spread virulence genes
Solution Approach 1:
The patent extracts temperate prophage elements from the bacterial genome using site-specific recombination systems. By removing these prophages entirely, the system eliminates the dual nature of temperate phages that provides both adaptive benefits and harmful spontaneous induction risks, choosing safety over adaptability in the context of therapeutic phage production.
3Reliability
If prophage-free bacterial strains are used for therapeutic bacteriophage production, then safety is improved by preventing virulence gene spread, but strain selection and development become more complex
Solution Approach 1:
The patent applies preliminary action by pre-modifying the bacterial strain to eliminate temperate prophages before phage therapy production. The Cre-loxP recombination system is预先 installed in the genome to enable complete and permanent removal of prophage elements, ensuring safety is established beforehand rather than requiring complex screening and validation during each production cycle.
Data Source
AI summary
Disclosed are Enterococcus faecalis strains for obtaining monoclonal preparations of lytic bacteriophages with high titer, particularly devoid of contamination with undesired bacteriophages, the strains guaranteeing the production of safe to use bacteriophage preparations, not being a source of dissemination of genes associated with bacterial virulence.