Evaluatie van bacteriële virussen en afgeleide producten voor de controle van Staphylococcus aureus

摘要

Staphylococcus aureus is a major problem in health care settings and livestock breeding across the world. This opportunistic bacterium is responsible for various infections of the skin, systemic infections and sepsis. Moreover, its increasing antibiotic resistance and ability to form biofilms, sessile communities of bacterial cells embedded in an extracellular matrix, necessitates alternatives for classical treatment and therefore drives the renewed interest in (bacterio)phages. However, the therapeutic use of these bacterial viruses (phage therapy) remains underdeveloped. Two phages infecting S. aureus, Romulus and Remus, were isolated from sewage of the UZ Leuven Campus Gasthuisberg (Belgium). Phage ISP, obtained from the Eliava Institute of Bacteriophage, Microbiology and Virology in Tbilisi (Georgia), was already available. The newly isolated phages as well as phage ISP are myoviruses, known by their long contractile tail, and taxonomic members of the genus Twortlikevirus. Romulus and Remus are very closely related to each other and to S. aureus phage SA11, but display no more than 60% DNA homology with other staphylococcal myoviruses. Therefore, three species within the genus Twortlikevirus, represented by phages K, Remus and Twort, were proposed based on phenotypical properties and genome organization.A thorough microbiological, genomic and proteomic characterization was carried out for the three phages. Host range screening of a large batch of S. aureus isolates revealed activity of ISP, Romulus and Remus against 87%, 69% and 68% of the isolates, including relevant MRSA isolates. Furthermore, where all human S. aureus isolates were sensitive to ISP infection, Romulus and Remus infected 28 and 30 out of 36 S. aureus isolates of patients, respectively. In contrast to the 22 phage propagation strains infected by ISP, only nineteen and eighteen of the 31 phage propagation strains were sensitive to Romulus and Remus, respectively. Hence, the host range of Romulus and Remus is relatively narrow compared to phage ISP. Considering their close relatedness, the differences in host range and efficiency of plating between Romulus and Remus are remarkable. Furthermore, S. haemolyticus and S. epidermidis isolates appeared to be insensitive to the three phages. The adsorption to and subsequent infection of S. aureus in liquid culture was studied. The adsorption experiments revealed similar adsorption characteristics for ISP, Romulus and Remus, whereas the in vitro infection patterns of Romulus and Remus showed a faster killing of liquid S. aureus cultures compared to ISP. In a proof-of-concept experiment, phage Remus seemed to be most promising in controlling a S. aureus PS47 biofilm. To determine optimal storage and application conditions, the survival of ISP, Romulus and Remus at different temperatures and pH values was tested. Phages ISP, Romulus as well as Remus were stable within a pH range of 5 to 9. Consequently, oral administration of the phages would require protection from the acidic environment of the stomach. The three phages appeared to be stable at 4°C and 16°C. Whereas ISP was stable at 37°C and 42°C as well, the Romulus and Remus titers drop with approximately one logarithmic unit at 37°C and at 42°C the logarithmic decrease was considerable. This should be taken into account in case of systemic use. Specifically for phage ISP, the frequency of bacterial mutations conferring resistance was determined and an alternative purification method using CIM® Disk Monolithic Columns was optimized. Furthermore, subcutaneous as well as nasal and oral ISP administration to rabbits was demonstrated to cause no adverse effects.ISP, Romulus and Remus encode 215, 180 and 189 gene products on their 138,339 bp, 131,333 bp and 134,643 bp genomes, respectively. Promoters, terminators, tRNAs and protein functions were predicted. No toxic, lysogeny- or virulence-associated proteins were observed. Structural proteome analysis identified 22 and nineteen virion proteins for ISP and Remus, respectively. Exploration of phage-encoded proteins represented an intriguing sidestep of this research. Although several candidates were indicated, no ISP-encoded exopolysaccharide depolymerase was detected. On the other hand, a receptor-binding protein of phage ISP, possibly targeting the cell wall teichoic acids in the S. aureus envelope, was identified. Moreover, this receptor-binding protein, Gp40, appeared to be the short tail fiber of phage ISP. Another ISP-encoded structural protein, Gp38, was localized near the baseplate, but no function could be suggested. The microbiological and molecular characterization of ISP, Romulus and Remus supports their application for therapeutic purposes, although some of their characteristics are not ideal for specific situations in phage therapy. In addition, fundamental insight in taxonomy, genome organization and the distribution of group I introns in twortlikeviruses was obtained. Based on bioinformatic analysis and immunogold localization experiments, four components of the ISP virion (Gp18, Gp19, Gp33 and Gp40) were indicated in a preliminary Twortlikevirus tail model.