Genomic comparison of novel Staphylococcus aureus bacteriophage and their anti-biofilm properties against MRSA sequence type 22 and 36

Whittard, Elliot (2020) Genomic comparison of novel Staphylococcus aureus bacteriophage and their anti-biofilm properties against MRSA sequence type 22 and 36. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Staphylococcus aureus (including methicillin-resistant S. aureus - MRSA) remains a leading cause of both nosocomial and community acquired infections globally and despite constant improvement efforts to patient safety within healthcare, it still remains associated with considerable rates of morbidity and mortality. S. aureus is a common cause of biofilm-associated infections observed in chronic wounds, exhibiting a reduced susceptibility to the action of conventional antimicrobial agents and are often difficult to eradicate. The acquisition of resistance to almost any antibiotic with reference to MRSA has greatly reduced the number of alternative antimicrobial agents effective in the treatment of infections. Current development pipeline for new classes of antibiotics are greatly limited, requiring new, alternative approaches for therapeutic and prophylactic intervention in attempt to effectively control and overcome this current global health threat. Bacteriophage therapy exploits the natural killing ability of lytic bacteriophage (phage) as a means of controlling multidrug-resistant pathogenic bacteria. The utility of phage and their derivatives has been shown to effectively reduced the biofilms of major MRSA clones in vitro and in vivo. Global MRSA infections are caused by highly-successful isolates from a small number of epidemic lineages (clones). ST22 and ST36 are two of the most prevalent clones with global impact and largely responsible for the national epidemic of MRSA infections within UK healthcare system throughout the mid-1990s up until the mid-2000s. Understanding the phenotypic and genotypic characteristics of these clones in relation to the ability of bacteriophage to infect and disrupt established biofilms has yet to be explored. In this study, a total of 46 novel obligately lytic phage were isolated from wastewater samples by utilising a modified Staphylococcus carnosus TM300 isolate with expressed S. aureus wall teichoic acids to aid in phage adsorption. The addition of 32 more phage from our current laboratory stocks helped to establish a collection of 78 phage that were screened against a panel of 185 genetically diverse S. aureus, consisting of major clonal groups with high prevalence within the UK and United States, including 43 ST22 and 24 ST36 strains. The majority of the members displayed a wide host range against our panel. Based on this, the four most effective (wide host-range) phage were assessed for their anti-biofilm properties in polystyrene plates biofilm assays produced using four ST22 and four ST36 isolates. Treatment of mature biofilms was shown to significantly reduce biofilm biomass and viable cell counts. However these assays selected for the emergence of phage resistant mutants. Whole genome sequencing was performed on 22 phage isolates and these were found to share a high degree of similarity to genomes of 38 previously classified Twortvirinae phages represented in GenBank. Comparisons of these 60 phage genomes found a surprisingly high level of genetic diversity. Pairwise distances resolved groups of phage in distinct clusters representing individual genera within the Twortvirinae subfamily. Pan-genome analysis identified no single gene present amongst all phage genomes, however phage displayed a core genome amongst other members of the cluster. The structural homology tool HHpred was used to predict the protein structure of genes encoding for lytic enzymes among our phage genomes. We found that all phage encode a protein that shares high structural similarity to the same CHAP domain protein, a catalytic domain of endolysins employed by phage to degrade the bacterial host cell wall in order thus, mediate cell lysis. Suggesting that the all phage most likely share the same catalytic N-terminal endopeptidase domain of endolysins which have a modular domain structure. Interestingly, endolysins have been proposed as possible candidates for the control of antibiotic resistant S. aureus infections.