General considerations, future perspectives, and conclusion

4.4.1 Endolysins for control of foodborne pathogens

For the application of endolysin in foods, several other aspects next to its antimicrobial activity need to be considered. For example, endolysin production costs are expected to be relatively high with current technologies (Oliveira et al., 2012). Though, this is argued to be an acceptable challenge for the application of endolysin, since many different enzymes used in the food industry are produced cost-effectively on large scale. However, technological developments for more efficient endolysin production would be required to make its use as food control agent financially appealing.

Another aspect which needs to be considered is consumer safety and acceptance. Although endolysins have already been successfully applied in several animal infection models of human disease (as reviewed by Nelson et al., 2012), more comprehensive and systematic investigations into the safety of these enzymes for human consumption will be required for its approval. To this respect, legislative approval for the use of endolysin on foods might pose one of the biggest challenges. Whereas phages are considered a natural product and starting to find acceptance either as additive (USA) or food processing aid (EU; Perera et al., 2015, Whitworth, 2016), endolysins are purified from genetically modified organisms, which increases the hurdles in their approval process. Despite considerable progress in recent years, the use of genetically modified organism for the production of enzymes that are added to food either as food additive or processing aid, are only slowly finding legislative approval (EFSA, 2014). Similarly, the classification of endolysin as either food processing aid or additive could greatly influence consumer acceptance. Since the production of microbiologically safe and clean label food products is one of the main advantages of HHP processing, classification of endolysin as food additive would also limit interest for their combined application. Although the concern about genetically modified food is slowly declining (Deloitte, 2010), only about one decade ago consumers were still most concerned about genetic modification among several other food processing techniques (Wright et al., 2007).

DISCUSSION 97

A final but very important consideration for the use of phages or their lysins as food antimicrobials is resistance development. Fister et al., 2015 isolated multiple phage P100 resistance strains from an Austrian dairy plant where the phage was experimentally used. This illustrates the importance for the intelligent use of phages as biocontrol agents of food pathogens, for example by the use of phage cocktails and rotation schemes of cocktails with different phage compositions. For endolysins, the development of bacterial resistance is, compared to antibiotics or phages, much rarer. Despite extensive efforts of several groups, a less susceptible mutant strain has only been isolated once until now (1.4.2.4). Nonetheless, to ensure long-term efficacy beyond what can be tested on a laboratory scale, similar strategies as mentioned above for the application of phages will be required. The combination of multiple endolysins with different cleavage specificities is usually put forward as strategy to prevent resistance development. Although this might reduce the risk on resistance development, the only endolysin resistant mutant strain ever isolated directly demonstrated an increased resistance towards a whole range of peptidoglycan hydrolases with different enzymatic targets (Schmelcher et al., 2015), which indicates that the use of endolysin cocktail does not eliminate the risk of resistance development. Nonetheless, the use of a combination of different endolysins is recommended because it might provide additional other benefits as well. For example, several groups have shown a synergistic inactivation of target bacteria by the combination of different peptidoglycan hydrolases (Loeffler and Fischetti, 2003, Becker et al., 2008). In the current work, the combination of different Listeria phage endolysin merely resulted in an additive but not synergistic effect (3.4.1.2).

Although PlyP40, Ply511, and PlyP825 cleave different bonds, their CBD all specifically recognize the peptidoglycan backbone structure of Listeria, which might have restricted any synergistic effects.

Although the combination of endolysins with different binding targets might thus be desired to achieve maximum lytic activity, highly variable structures such as the peptidoglycan’s interlinking peptide sequence should be excluded as possible binding target because of the increased risk on resistance development, which limits the binding targets of interest to the highly conserved polysaccharide backbone and N-acetylmuramoyl-L-alanine amide bond. In conclusion, to reduce the risk on resistance development, cocktails containing endolysins with both different enzymatic as well as conserved binding targets are recommended.

4.4.2 Endolysin as biological tool to study the role of the cell wall

The present work investigated the direct application of endolysin to food products. The antimicrobial properties of endolysin have also been investigated as enzybiotic or in food processing environments against biofilms, while the high affinity CBD is being exploited for the rapid detection of pathogens. In several experiments throughout this work, the combined application of endolysin and HHP caused a major reduction in cell count, whereas individual treatments hardly had any effect. This synergism was discussed before to be the result of a partial breakdown of the peptidoglycan layer, which destabilizes the cytoplasmic membrane and makes the cell more susceptible to pressure (4.2.1.). This work thereby indicates that the peptidoglycan layer plays a major role in pressure resistance. Although the mechanistic background of bacterial high pressure inactivation has been extensively studied in the last century, the role of this layer in pressure resistance has been mostly neglected, which is somewhat surprising while the peptidoglycan layer is commonly put forward as the main cellular component responsible for the difference in pressure resistance between Gram-positive and -negative bacteria (Hogan et al., 2005). Considering the above, endolysin might find another interesting application as biotechnological tool to study the mostly unexplored role of the peptidoglycan layer in bacterial

98 DISCUSSION

pressure resistance. Modular endolysins, with their highly diverse cleavage target sites, binding properties, and the ability to swap domains creating enzymes with desired characteristics, would provide a perfect toolbox to examine the role of the murein layer in bacterial pressure resistance.

SUMMARY 99