Enviphage recommendation guide

enviphage EVALUATE THE EFFECT OF PHAGES USE IN ENVIRONMENTAL BACTERIAL ECOLOGY EVALUAR EL USO DE FAGOS EN LA ECOLOGÍA BACTERIANA AMBIENTAL AVALIAR O EFEITO DE FAGOS NA ECOLOGIA BACTERIANA AMBIENTAL

Recommendation guide

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The aim of this document is to provide a concise and helpful guide to regulatory bodies in order to take in consideration during phage safety evaluation The base of this guide is the results obtained along the LIFE Enviphage project and from different points of views. This topic has been considered by other researchers, and we encourage to the interested persons to consult the papers annotated in the Bibliography section.

Can bacteriophages affect eukaryotic cells?

No. All bacteriophages require strictly prokaryotes (bacteria) cells to multiply. Due to their metabolic and biochemical characteristics, phages are unable to affect eukaryotic cells, and, hence, can´t replicate at expenses of animal, human, vegetal or fungi cells.

How can bacteriophages affect human/animal/vegetal?

As we have demonstrated, bacteriophages would affect the bacteria community of each ecosystem only if the phage-target specie is present. Bacterial balance is important in all biological fields, from our skin, the intestinal tracks, the foliar area or the geoclycles, like the nitrogen cycle. In some cases, phages would eliminate some target species from the system (i.e. a pathogenic host from the fish-farm water), which could beneficiate other no-target bacteria of the microbiota. But, in other cases, no-target species would be beneficiate by the new ecological niche.

The main advantage of bacteriophages is that they would not modify the ecosystem were the target bacteria is not present (see the reports of action B2, C1 and C2 of this project).

Why are bacteriophages a good alternative to antibiotics?

Bacteriophages present some characteristics those made them an interesting alternative to antibiotics, including: 1) Phages are highly specific. Some phages are specific for a to a single bacteria genus, more often for a specific specie or even for few strains of bacteria. Therefore, as we have demonstrated, phages cause much less damage to the normal intestinal fish flora and to naturally present non-target bacteria. This effect was predicted

by Pereira et al., 2011.

2) Limited resistance development. Bacteria will certainly develop resistance to phages, but since phages have a higher mutation and replication rate, they can get round the adaptation of the bacteria and development of resistance is therefore limited. Moreover, it is comparably easier to find new phages than new antibiotics because phages co-evolving with their host bacteria, outnumbering bacteria in the environment by tenfold, make possible the rapid isolation of new lytic phages from the environment for phage-resistant bacteria mutants. So, even if the bac-

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teria acquire phage resistance new mutant phage that acts lytically against these bacteria can be used against the targeted bacteria (Matsuzaki et al., 2003). Smith et al. (1983) demonstrated that infections produced by phage-resistant mutants of an enteropathogenic strain of E. coli and their parents could be successfully controlled with mutant phage derived from phage that had been active against the parent bacteria (DĂşran et al.,2002; Lucena et al., 2004). It is already possible to prepare a mixture of different strains of phages that would prevent the emergence of resistant strains during phage treatment. Therefore, the solution to the problem of phage-resistant bacteria can be found.

3) Limited impact, unlike antibiotics, phages are self-replicating as well as self-limiting.

They replicate exponentially as bacteria do and decline when bacteria number decreases. Depending of the form of application, a single dose may be sufficient. This project demonstrated that low or no phages are recovered/remnant after a single use when the host is not present in the ecosystem. Reports have revealed that a single treatment with phage leads to recovery in mice with infections caused by E. coli (Smith et al., 1983), P. aeruginosa (Hagens et al., 2004), methicillin-resistant S. aureus (Matsuzaki et al, 2003), and vancomycin-resistant E. faecium (Biswas et al., 2002) and in human skin by P. aeruginosa (Vieira et al., 2012).

4) Regulatory approval, since phages are naturally occurring and very abundant, there may be substantially fewer problems involved in obtained regulatory approval for their use. LIFE Envipahge project has developed new methodologies in order to support this approvals.

5) High resistance to environmental conditions, phages are found within the same environment as their bacterial hosts, indicating the ability of these phages to survive in the same surrounding as their host bacteria. It is well known that phages are more resistant to environmental conditions than bacteria (DĂşran et al.,2002; Lucena et al., 2004).

6) Technology for phage production is flexible, faster and cheaper than for antibiotics. Action A1 in LIFE Enviphage project has demonstrated that phage isolation, characterization and production is cheap, fast and effective.

Which aspects should be taken in consideration prior bacteriophages use?

1) Only virulent (strictly lityc) phages are suitable for biocontrol purposes. Lityc phages cannot integrate their genome in bacterial chromosome and will always kill infected target cells.

2) Safety of the phage genome should be checked. The absence of virulence genes, confirmed by complete genome sequentiation and annotation

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3) The low proportion of gene transfer due the bacteriophages, in order to prevent a dispensation of infective genes, antibiotic resistance genes, toxic production genes.

4) The phage:bacteria ratio for a good activity, in order to allow a correct sanitation with a single application.

5) The bacterial specificity, in order to avoid not-desirable effect, both in the intestine or environmental bacteria.

6) The immune response of the host.

7) The effect on bacterial ecology in real situation. This project trends to be a good example about how to board this a aspect.

8) The stability at different storage and application conditions: Test phages for durability within the intended-use environment.

9) The ability to propagate in surrogate non-pathogenic hosts for large-scale commercial production is also important from an economical point of view How to apply phages in aquaculture facilities?

Phages can be applied in aquaculture facility directly in the tanks, at different single or multiple points, as a liquid to a large volume of water.

Can phages be use during food processing?

Yes. Different application strategies can be used, depending on the objectives, the target bacteria and the matrix to be treated. Phages can be use to sanitize working surfaces, processing instrumental, food surface,‌..

Do bacteriophages modify the environmental bacterial ecology?

This project, LIFE+ Enviphage project, has demonstrated that it’s possible to select phages that do not modify the environmental bacterial ecology.

The results presented in this project are valid for every phages?

Not, effects on environmental and intestinal bacterial population should be stated phageby-phage.

Can bacteriophages be used as antibiotic substitutes in agricultural or veterinary applications?

Most authors, including the ones those have worked in this project, would say yes. Significant efforts are being made to boost this alternative, and next years we will see the real potential of this application.

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Are bacteriophages an industrial-viable alternative?

Yes, but the economical viability depends, among others, on the phages, the selected host, the production conditions and the effective concentration. Also, the regulatory frame would determine the real application of bacteriophages as antibacterial agents.

REFERENCES Biswas B, Adhya S, Washart P, Paul B, Trostel AN, Powell B, Carlton R, Merril CR. (2002). Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infect Immun. 2002 Jan;70(1):204-10.

Durán AE, Muniesa M, Méndez X, Valero F, Lucena F, Jofre J. (2002). Removal and inactivation of indicator bacteriophages in fresh waters. J Appl Microbiol. 2002;92(2):338-47.

Hagens S, Habel A, von Ahsen U, von Gabain A, Bläsi U. (2004). Therapy of experimental pseudomonas infections with a nonreplicating genetically modified phage. Antimicrob Agents Chemother. 2004 Oct;48(10):3817-22.

Lucena F, Duran AE, Morón A, Calderón E, Campos C, Gantzer C, Skraber S, Jofre J. (2004). Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments. J Appl Microbiol.

2004;97(5):1069-76.

Matsuzaki S1, Yasuda M, Nishikawa H, Kuroda M, Ujihara T, Shuin T, Shen Y, Jin Z, Fujimoto S, Nasimuzzaman MD, Wakiguchi H, Sugihara S, Sugiura T, Koda S, Muraoka A, Imai S.(2003). Experimental protection of mice against lethal Staphylococcus aureus infection by novel bacteriophage phi MR11. J Infect Dis. 2003 Feb 15;187(4):613-24.

Pereira C, Silva YJ, Santos AL, Cunha A, Gomes NC, Almeida A. (2011). Bacteriophages with potential for inactivation of fish pathogenic bacteria: survival, host specificity and effect on bacterial community structure. Mar Drugs. 2011;9(11):2236-55. doi: 10.3390/md9112236.

Smith HW, Huggins MB. (1983). Effectiveness of phages in treating experimental Escherichia coli diarrhoea in calves, piglets and lambs. J Gen Microbiol. 1983 Aug;129(8):2659-75.

Vieira A, Silva YJ, Cunha A, Gomes NC, Ackermann HW, Almeida A. (2012). Phage therapy to control multidrug-resistant Pseudomonas aeruginosa skin infections: in vitro and ex vivo experiments. Eur J Clin Microbiol Infect Dis. 2012 Nov;31(11):3241-9. doi: 10.1007/s10096-012-1691-x. Epub 2012 Jul 10.

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enviphage EVALUATE THE EFFECT OF PHAGES USE IN ENVIRONMENTAL BACTERIAL ECOLOGY EVALUAR EL USO DE FAGOS EN LA ECOLOGÍA BACTERIANA AMBIENTAL AVALIAR O EFEITO DE FAGOS NA ECOLOGIA BACTERIANA AMBIENTAL

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