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Resistance

First we must define phage resistance. In the scientific literature any strain that a particular phage is unable to form plaques on (replicate in) will be defined as resistant. However phage bio-control in foods does not rely on phages being able to replicate. Hence, for the purpose of phage bio-control resistance must be redefined as the inability of a phage to kill a particular strain when 1 phage infects it.

Why is this distinction important? Because bacteria can have inherent phage resistance mechanisms: Abortive infection mechanisms (ABIs), restriction methylation mechanisms (RM) and CRISPR-Cas systems. All of these, if successful, will not let phages replicate and phages that cannot replicate within a population that does grow will lose out. Ecologically these approaches are useful to the bacteria. But with virulent (as opposed to temperate) phages all these resistance systems are likely to result in abortive infection and death of the individual cells that are infected. In the case of ABIs there can be no other outcome as the infected cell actively commits suicide. But even RM and CRISPRs will result in abortive infection. This is because they act after the phage has infected the cell and early genes have been expressed. Most virulent phage early genes will have one particular effect on the cell. These genes ensure that cell division (not growth) is halted. This causes the cell to elongate – a process that in itself eventually leads to cell death (Wagemans et al 2014.) It should also be mentioned that such mechanisms cannot be introduced by phage pressure. They are either present in a population or not – they cannot spontaneously come into existance.

Petri dish PhageGuardChallenging a bacterial population with phages holds the risk of resistance development through mutation of the phage receptor. The bacteria become “invisible” to the phage. In such a population such mutants have an advantage. On foods themselves one cannot speak of a population. Some bacteria inadvertently manage to contaminate the food from one source or another. In treating the food there is no pressure put on the original population. This would be different if phages were applied in the environment, but even there measures can be taken to reduce this risk to the point where benefits of phage application outweigh any risks.

The key lies in not applying phage pressure continuously on a given population. Mutations are generally not advantageous to bacteria and can result in a decrease of fitness on other levels (growth speed, virulence etc).

A key component in this issue is the removal of pressure for certain periods. Food processing facilities are cleaned regularly and intensely. Sanitizing agents tend to be both anti-bacterial and antiviral. Since phages, unlike bacteria, lack repair mechanisms any damage sustained is permanent. Therefore, most sanitizing agents will affect phages more heavily than bacteria and normal sanitation procedures are likely to completely remove phage presence after being applied.

Applying phage occasionally and ensuring treated areas are cleaned afterwards holds a minimal risk of resistance development. This would be especially true for food contact surfaces which usually undergo daily rigorous sanitation procedures.

Lastly, it should be mentioned that mutations are generally detrimental. In the absence of phage pressure such mutations are likely to disappear because carrying the mutation is only an advantage in the presence of phages. Mutant are likely to be less fir that wild-type cells which may be reflected in decreased growth speeds or loss of pathogenicity.

 

References:

Wagemans J, Blasdel BG, Van den Bossche A, Uytterhoeven B, De Smet J, Paeshuyse J, Cenens W, Aertsen A, Uetz P, Delattre AS, Ceyssens PJ, Lavigne R. (2014) Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa. Cell Microbiol. 6(12):1822-35

Eugster MR, Morax LS, Hüls VJ, Huwiler SG, Leclercq A, Lecuit M, Loessner MJ. (2015) Bacteriophage predation promotes serovar diversification in Listeria monocytogenes. Mol Microbiol. 97(1):33-46.