Plant-expressed bacteriophage lysins control pathogenic strains of Clostridium perfringens

Abstract

The anaerobic spore-forming bacterium Clostridium perfringens is a source of one of the most common food-borne illnesses in the United States and Europe. The costs associated with disease management are high and interventions are limited; therefore, effective and safe antimicrobials are needed to control food contamination by C. perfringens. A viable solution to this problem could be bacteriophage lysins used as food additives or food processing aids. Such antimicrobials could be produced cost-effectively and in ample supply in green plants. By using edible plant species as production hosts the need for expensive product purification can be reduced or obviated. We describe the first successful expression in plants of C. perfringens-specific bacteriophage lysins. We demonstrate that six lysins belonging to two different families (N-acetylmuramoyl-L-alanine amidase and glycosyl hydrolase 25) are active against a panel of enteropathogenic C. perfringens strains under salinity and acidity conditions relevant to food preparation environments. We also demonstrate that plant-expressed lysins prevent multiplication of C. perfringens on cooked meat matrices far better than nisin, the only currently approved bacteriocin food preservative to control this pathogen.

Techno‐economic analysis of a plant‐based platform for manufacturing antimicrobial proteins for food safety

“…11,14,22 Several salmocins and lysins can be expressed at high levels in spinach, which is comparable to expression levels in N. benthamiana. The alternative scenario models were designed in alignment with base case scenario inputs unless otherwise noted; each alternative scenario was chosen to isolate the impact of a key facility design assumption.…”

“…For example, recent literature highlights the challenge of the "viable but nonculturable" (VBNC) state of microorganisms in these foodsanitizing treatments. 11 In fact, various preparations of bacteriophages, such as the Salmonella-specific bacteriophage cocktail SalmoFresh™, 12 endolysins, 13,14 and bacteriocins, such as colicins 15,16 and nisins, 17 have already been granted Generally Recognized as Safe (GRAS) status as food antimicrobials by the US Food and Drug Administration (FDA). 10 Biotic approaches to food sanitization have high potential as supplementary treatments to de-risk the supply chain by employing efficacious and orthogonal protection against high-risk pathogens.…”

“…11,14,15,[22][23][24] In this study, we address cost sensitivity with a comprehensive techno-economic analysis of plant-based production of AMP for food safety applications. Plant-based platforms have the potential for producing market-relevant volumes of AMPs at competitive costs, because they do not require expensive bioreactors and culture media.…”

“…Therefore, there is a strong and intuitive case for acceptance of certain bacteriophage-and bacteriocin-derived antimicrobial treatments for food safety applications. 11 In fact, various preparations of bacteriophages, such as the Salmonella-specific bacteriophage cocktail SalmoFresh™, 12 endolysins, 13,14 and bacteriocins, such as colicins 15,16 and nisins, 17 have already been granted Generally Recognized as Safe (GRAS) status as food antimicrobials by the US Food and Drug Administration (FDA). It is anticipated that similar antimicrobial preparations will be granted GRAS status by FDA in the future, as the popularity of these technologies grows and additional regulatory notices are filed.…”

“…We have previously reported expression levels as high as 3 g/kg plant fresh weight (FW). 11,14,15,[22][23][24] In this study, we address cost sensitivity with a comprehensive techno-economic analysis of plant-based production of AMP for food safety applications. We used laboratoryscale results and working process knowledge from pilot and commercial processes to develop a process simulation model using SuperPro Designer ® to assess the commercial viability of the production platform and to identify economic "hotspots" to help guide future research and development.…”