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Characterisation of a novel Staphylococcus aureus lineage : Clonal Complex 75 (CC75), Staphylococcus argenteus

Ng, Jacklyn Wei Sze (2014). Characterisation of a novel Staphylococcus aureus lineage : Clonal Complex 75 (CC75), Staphylococcus argenteus. PhD Thesis, Charles Darwin University.

Document type: Thesis
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Author Ng, Jacklyn Wei Sze
Title Characterisation of a novel Staphylococcus aureus lineage : Clonal Complex 75 (CC75), Staphylococcus argenteus
Institution Charles Darwin University
Publication Date 2014-04
Thesis Type PhD
Supervisor Giffard, Philip Morrison
Subjects MEDICAL AND HEALTH SCIENCES
Abstract Staphylococcus aureus is a major opportunistic human pathogen, commonly found in the nose and on the skin of healthy individuals. S. aureus can cause a broad spectrum of diseases, ranging from minor skin infections to life threatening sepsis and necrotising pneumonia. First reported in Australia in 2004, Clonal complex 75 (CC75), defined by multilocus sequence typing (MLST), is highly divergent from S. aureus at each MLST locus, and is believed to be a novel lineage of S. aureus. CC75 is common in tropical northern Australia where it is associated with skin lesions in the Indigenous population, although CC75 has also been reported elsewhere in the Asia Pacific region and South America. This thesis presents the characterisation of CC75 with the following aims: (i) determining the evolutionary position of CC75 within the genus Staphylococcus; (ii) investigating the resolving power of high-resolution melt (HRM) analysis for rapid screening to identify new CC75 MLST alleles; and (iii) investigating whether saturating dyes such as LCGreen+ are superior to non-saturating dyes such as SYBR Green for the discrimination of sequence variants utilising HRM analysis. Major findings from this thesis are: (i) the striking divergence in the multilocus sequence analysis (MLSA) and MLST housekeeping genes within the core genome between CC75 and S. aureus and the phenotypic characteristic lacking staphyloxanthin, responsible for the golden colour pigment in S. aureus, support CC75 to be a different species from S. aureus (CHAPTER 2); (ii) HRM using SYBR Green in full length MLST amplicons reliably discriminates CC75 MLST alleles at only two (tpi and gmk) out of seven loci (CHAPTER 3); (iii) the robustness of PCR (as measured by CT values) correlates inversely with the reproducibility of HRM curves (as measured by the variation of Tm values) (CHAPTER 3); (iv) HRM using SYBR Green on full length MLST amplicons lacks the resolving power to detect new CC75 MLST alleles (CHAPTER 4); and (v) saturating dyes such as LCGreen+ are not necessarily superior to non-saturating dyes such as SYBR Green for the discrimination of sequence variants (CHAPTER 5). In the presence of LCGreen+ at low MgCl2 concentration (2mM), HRM is confounded by an inverse correlation between DNA concentration and Tm. This effect is eliminated when the MgCl2 concentration is increased to 6mM (CHAPTER 5).
Keyword Clonal Complex 75
CC75
genotyping
high-resolution melt
HRM
Tm
magnesium chloride
MgCl2
multilocus sequence analysis
MLSA
multilocus sequence typing
MLST
mutation scanning
neighbour-joining trees
population structure
phylogeny
single nucleotide polymorphism
SNP
Staphylococcus argenteus
S. argenteus
Staphylococcus aureus
S. aureus
SYBR Green
real-time PCR


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