Biological and molecular characterization of South African bacteriophages infective against Staphylococcus aureus subsp. aureus Rosenbach 1884, casual agent of bovine mastitis.

Abstract

Bacteriophage therapy has been exploited for the control of bacterial diseases in fauna, flora and humans. However, the advent of antibiotic therapy lead to a cessation of most phage research. Recently, the problem of antibiotic resistance has rendered many commonly used antibiotics ineffective, thereby renewing interest in phage therapy as an alternative source of control. This is particularly relevant in the case of bovine mastitis, an inflammatory disease of bovine mammary glands, caused by strains such as Staphylococcus aureus subsp. aureus Rosenbach 1884. Antibiotic resistance (primarily towards penicillin and methicillin) by staphylococcal strains causing mastitis is regularly reported. Phage therapy can provide a stable, effective and affordable system of mastitis control with little to no deleterious effect on the surrounding environment or the affected animal itself. Several studies have delved into the field of biocontrol of bovine mastitis using phages. Results are variable. While some phage-based products have been commercialized for the treatment of S. aureus-associated infections in humans, no products have yet been formulated specifically for the strains responsible for bovine mastitis. If the reliability of phage therapy can be resolved, then phages may become a primary form of control for bovine mastitis and other bacterial diseases. This study investigated the presence of S. aureus and its phages in a dairy environment, as well as the lytic ability of phage isolates against antibiotic-resistant strains of mastitic S. aureus. The primary goals of the thesis were to review the available literature on bovine mastitis and its associated control, and then to link this information to the use of phages as potential control agents for the disease, to conduct in vitro bioassays on the selected phages, to conduct phage sensitivity assays to assess phage activity against different chemical and environmental stresses, to morphologically classify the selected phages using transmission electron microscopy, to characterize the phage proteins using one-dimensional electrophoresis, and lastly, to characterize phage genomes, using both electrophoresis as well as full genome sequencing. Twenty-eight phages were isolated and screened against four strains of S. aureus. Only six phages showed potential for further testing, based on their wide host range, high titres and common growth requirements. Optimal growth conditions for the host S. aureus strain was 37°C for 12hr. This allowed for optimal phage replication. At an optimal titre of between 6.2x10⁷ to 2.9x10⁸ pfu.mlˉ¹(at 10ˉ⁵ dilution of phage stock), these phages were able to reduce live bacterial cell counts by 64-95%. In addition, all six phages showed pathogenicity towards another 18 S. aureus strains that were isolated from different milk-producing regions during a farm survey. These six phages were named Sabp-P1, Sabp-P2, Sabp-P3, Sabp-P4, Sabp-P5 and Sabp-P6. Sensitivity bioassays, towards simulated environmental and formulation stresses were conducted on six identified phages. Phages Sabp-P1, Sabp-P2 and Sabp-P3 showed the most stable replication rates at increasing temperatures (45-70°C), in comparison to phages Sabp-P4, Sabp-P5 and Sabp-P6. The effect of temperature on storage of phages showed that 4ºC was the minimum temperature at which phages could be stored without a significant reduction in their lytic and replication abilities. Furthermore, all phages showed varying levels of sensitivity to chloroform exposure, with Sabp-P5 exhibiting the highest level of reduction in activity (74.23%) in comparison to the other phages. All six phages showed optimal lytic ability at pH 6.0-7.0 and reduced activity at any pH above or below pH 6.0-7.0. Exposure of phages to varying glycerol concentrations (5-100%) produced variable results. All six phages were most stable at a glycerol concentration of 10-15%. Three of the six isolated phages, Sabp-P1, Sabp-P2 and Sabp-P3, performed optimally during the in vitro assays and were used for the remainder of the study. Morphological classification of phages Sabp-P1, Sabp-P2 and Sabp-P3 was carried out using transmission electron microscopy. All three phages appeared structurally similar. Each possessed an icosahedral head separated from a striated, contractile tail region by a constricted neck region. The head capsules ranged in diameter between 90-110nm with the tail length ranging from 150-200nm in the non-contractile state and 100-130nm in the contractile state. Rigid tail fibres were also visible below the striated tail. The major steps in the virus replicative cycle were also documented as electron micrographs. Ultra-thin sections through phage plaques were prepared through a modification of traditional methods to speed up the process, with no negative effects on sample integrity. The major steps that were captured in the phage replicative cycle were (1) attachment to host cells, (2) replication within host cells, and, (3) release from cells. Overall results suggested that all three phages are strains from the order Caudovirales and are part of the Myoviridae family. A wealth of information can be derived about an organism based on analysis of its proteomic data. In the current study, one-dimensional electrophoretic methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and ultra-thin layer isoelectric focusing (UTLIEF), were used to analyse the proteins of three phages, Sabp-P1, Sabp-P2 and Sabp-P3, in order to determine whether these strains differed from each other. SDS-PAGE analysis produced unique protein profiles for each phage, with band fragments ranging in size from 8.86-171.66kDa. Combined similarity matrices showed an 84.62% similarity between Sabp-P1 and Sabp-P2 and a 73.33% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 69.23% similarity to Sabp-P3. UTLIEF analysis showed protein isoelectric charges in the range of pI 4.21-8.13, for all three phages. The isoelectric profiles for each phage were distinct from each other. A combined similarity matrix of both SDS-PAGE and UTLIEF data showed an 80.00% similarity between phages Sabp-P1 and Sabp-P2, and a 68.29% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 70.59% similarity to Sabp-P3. Although the current results are based on putative protein fragments analysis, it can be confirmed that phages Sabp-P1, Sabp-P2 and Sabp-P3 are three distinct phages. This was further confirmed through genomic characterization of the three staphylococcal phages, Sabp-P1, Sabp-P2 and Sabp-P3, using restriction fragment length analysis and whole genome sequencing. Results showed that the genomes of phages Sabp-P1, Sabp-P2 and Sabp-P3 were all different from each other. Phages Sabp-P1 and Sabp-P3 showed sequence homology to a particular form of Pseudomonas phages, called "giant" phages. Phage Sabp-P3 showed sequence homology to a Clostridium perfringens phage. Major phage functional proteins (the tail tape measure protein, virion structural proteins, head morphogenesis proteins, and capsid proteins) were identified in all three phages. However, although the level of sequence similarity between the screened phages and those already found on the databases, enabled preliminary classification of the phages into the order Caudovirales, family Myoviridae, the level of homology was not sufficient enough to assign each phage to a particular type species. These results suggest that phage Sabp-P1 might be a new species of phage within the Myoviridae family. One longer-term objective of the study is to carry out complete assembly and annotation of all the contigs for each phage. This will provide definitive conclusions in terms of phage relatedness and classification.