Isolation and characterisation of bacteriophages infecting Gram-positive bacteria

Details

Supervisors

Gillis, Annika

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences agronomiques, à finalité spécialisée

Abstract

Bacteriophages (a.k.a. phages) are natural bacteria-killer viruses that enjoy a renewed scientific interest in the context of the antimicrobial resistance landscape. Phages-based applications for food safety biocontrol or therapeutic purposes are increasingly studied and seem promising in the fight against pathogenic multi-drug resistant bacteria. The aim of this master’s dissertation is to isolate and characterise phages infecting species belonging to the following three genera: Bacillus, Listeria and Staphylococcus. Species belonging to these genera are commonly encountered in our daily life food or are part of our microbiome. Some can prove to be pathogenic or pathogenic opportunistic and are of a major food safety or medicine interest. To achieve this objective, a sampling campaign was performed to collect environmental samples among diverse structures and environments (i.e. agricultural soil, water treatment plant and hospital samples). The content of phage in those samples was assessed via laboratory procedures, including spot assays and enrichment techniques. Through these procedures, four putative lytic phages were successfully isolated: three phages infecting Bacillus thuringiensis and one infecting Staphylococcus aureus. It was decided to focus on the characterisation of the Staphylococcus phage, dubbed Tournay. The stability towards several physicochemical parameters was assessed to gain insight into further applications and the intrinsic resistance of the phage to the parameters used. Together with a host range, growth parameters were assessed. A preliminary bioinformatics analysis of the genome was also carried out. Leading outcomes of this work is that phage Tournay would belong to the genus Kayvirus. It is stable under the conditions that sustain the growth of S. aureus (thermal stability from 4 to 60 °C and pH stability from 4 to 10). Moreover, it is not a lipid-containing phage since it can stand chloroform concentrations up to 5 % V/V, at least for one month. The host range highlights a broad scope of action (20 strains infected out of the 26 strains used), although the appearance of turbid zones requires in-depth investigations. Phage Tournay seems to be a relevant candidate to study resistance systems and defence mechanisms among Staphylococcus due to the rapid appearance of resistance on the host isolation strain, exposed by the killing assays performed. Latent period (20 min) is consistent with other Staphylococcus phages, though the burst size (17 phage particles) is lower compared to other staphylococci-infecting phages. Overall, this master’s dissertation enabled isolating four phages infecting Gram-positive bacteria. The work raises numerous questions for research outlooks. The characterisation of the three Bacillus phages needs to be further performed to assess their potential application in bio-controlling schemes, especially towards biofilm degradation and eradication. Regarding phage Tournay, completing the bioinformatics analysis remains pivotal to better understand the interactions between the phage and the bacteria leading to potential applications in the future.