Characterization of a salmonella phage using omic tools and mathematical models to predict host-phage interaction

The increasing resistance of pathogenic bacteria to antibiotics in recent years has become a major problem in controlling infections in animals and humans. Salmonella enterica, which causes human food poisoning worldwide, is one of the most problematic bacteria with significant incidence in poultry. The development of alternatives to antibiotics has led to the resurgence of interest in (bacterio)phages, discovered before the antibiotics but which have succumbed to the efficiency of the later. The goal of the work described in this thesis was the biological, genomic and proteomic characterization of a broad host range Salmonella bacteriophage (PVPSE1) with high potential for therapy. It was also aimed at characterizing the population dynamics of the phage‐bacteria system by developing mathematic models that describe host‐phage interaction intended to predict the outcome of a therapeutical use of this phage and also the optimization of phage production. This phage has been proven to be efficient against Salmonella isolated from different sources and also shown the ability to lyse non‐pathogenic E. coli strains. Prior to the characterization steps a new method of phage detection based on the plaque assay was developed in order to enlarge the minimal size plaques formed by PVP‐SE1 which was rendering phage detection and enumeration very difficult. The method was based on the addition of glycerol and antibiotics at sub‐inhibitory concentrations which enabled the improvement of phage plaques size and contrast without changing its efficiency of plating. The phage genome sequencing was determined and a bioinformatic analysis was accomplished. This genomic characterization did not reveal any factor or gene responsible for lysogeny, pathogenesis or other which could exclude the use of this myovirus in vivo. Some of the phage proteins identified during this characterization represent an added value for biotechnological applications. From these, the PVP‐SE1 lysozyme is particularly interesting, not only for its lytic ability against pathogenic bacteria but also for the presence of a peptidoglycan binding domain, an unusual feature among phage lysozymes infecting Gram‐negative bacteria. The identification of the phage tail fibers, which are responsible for bacteria recognition, in such a broad host range Salmonella phage will lead to further investigation in their use to construct a diagnostic tool. Moreover, PVPSE1 was found to be phylogenetically unique, likely leading to the creation of a new phage genus. The mathematical model developed was able to explain the complicate hostphage interaction and allowed a good agreement between the predictions and the experimental data. The model has shown the importance of using a distribution of the latent period in simulations but more importantly it has shown that the bacterial physiological state and bacterial growth rate exerts a major influence in phage production. Due to the ability of the phage to lyse a non‐pathogenic host the possibility of producing the phage in the non‐pathogenic E. coli BL21 was studied. When produced in this alternative host the phage did not modify, maintaining its lytic ability and spectrum among the Salmonella strains. This new approach enables the production of a safer phage product by avoiding the risk of introducing phage resistant pathogenic bacteria in the final product thus diminishing costs of necessary purification techniques. In conclusion, it is presented here the biological, genomic, proteomic and population dynamics characterization of phage PVP‐SE1 which showed to be an added value as a biocontrol agent and as a diagnostic tool for the problematic pathogenic Salmonella. This characterization will be necessary and valuable in the development of a commercial product (therapeutic, diagnostic or other) based on this phage.

Name thesis:degree_name

Doutoramento em Engenharia Química e Biológica

Year dc:date.issued

2010

Author dc:creator

• Santos, Sílvio Roberto Branco

Advisors dc:contributor.advisor

• Ferreira, Eugénio C.
• Azeredo, Joana

Statement dc:rights

• restrictedAccess

Language dc:language.iso

por

Handle dc:identifier.uri

https://hdl.handle.net/1822/12276

OAI record oai:identifier

http://repositorium.sdum.uminho.pt/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arepositorium.uminho.pt%3A1822%2F12276