Molecular characterization of endocarditis-associated Staphylococcus aureus

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted

Virulence characteristics and diagnostic implications of invasive Staphylococcus aureus disease

Staphylococcus aureus is a pathogenic bacterium of significant global concern. It is essential to understand the molecular characteristics associated with invasive S. aureus infections and develop tools for rapid and accurate diagnosis of infections cause by multidrug resistant strains. This thesis investigates molecular characteristics associated with infective endocarditis, characterises a leukotoxin that acts as a direct neutrophil chemoattractant and examines infrared spectroscopy as a potential diagnostic tool to identify antibiotic resistance. This work contributes to the knowledge of S. aureus virulence, host cell interactions and antimicrobial resistance, which can help monitor the spread of this pathogen and combat infections

Virulence attributes of successful methicillin-resistant Staphylococcus aureus lineages.

SUMMARYMethicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of severe and often fatal infections. MRSA epidemics have occurred in waves, whereby a previously successful lineage has been replaced by a more fit and better adapted lineage. Selection pressures in both hospital and community settings are not uniform across the globe, which has resulted in geographically distinct epidemiology. This review focuses on the mechanisms that trigger the establishment and maintenance of current, dominant MRSA lineages across the globe. While the important role of antibiotic resistance will be mentioned throughout, factors which influence the capacity of S. aureus to colonize and cause disease within a host will be the primary focus of this review. We show that while MRSA possesses a diverse arsenal of toxins including alpha-toxin, the success of a lineage involves more than just producing toxins that damage the host. Success is often attributed to the acquisition or loss of genetic elements involved in colonization and niche adaptation such as the arginine catabolic mobile element, as well as the activity of regulatory systems, and shift metabolism accordingly (e.g., the accessory genome regulator, agr). Understanding exactly how specific MRSA clones cause prolonged epidemics may reveal targets for therapies, whereby both core (e.g., the alpha toxin) and acquired virulence factors (e.g., the Panton-Valentine leukocidin) may be nullified using anti-virulence strategies

Vibrational spectroscopy as a sensitive probe for the chemistry of intra-phase bacterial growth

Bacterial growth in batch cultures occurs in four phases (lag, exponential/log, stationary and death phase) that differ distinctly in number of different bacteria, biochemistry and physiology. Knowledge regarding the growth phase and its kinetics is essential for bacterial research, especially in taxonomic identification and monitoring drug interactions. However, the conventional methods by which to assess microbial growth are based only on cell counting or optical density, without any insight into the biochemistry of cells or processes. Both Raman and Fourier transform infrared (FTIR) spectroscopy have shown potential to determine the chemical changes occurring between different bacterial growth phases. Here, we extend the application of spectroscopy and for the first time combine both Raman and FTIR microscopy in a multimodal approach to detect changes in the chemical compositions of bacteria within the same phase (intra-phase). We found a number of spectral markers associated with nucleic acids (IR: 964, 1082, 1215 cm−1; RS: 785, 1483 cm−1), carbohydrates (IR: 1035 cm−1; RS: 1047 cm−1) and proteins (1394 cm−1, amide II) reflecting not only inter-, but also intra-phase changes in bacterial chemistry. Principal component analysis performed simultaneously on FTIR and Raman spectra enabled a clear-cut, time-dependent discrimination between intra-lag phase bacteria probed every 30 min. This demonstrates the unique capability of multimodal vibrational spectroscopy to probe the chemistry of bacterial growth even at the intra-phase level, which is particularly important for the lag phase, where low bacterial numbers limit conventional analytical approaches

Targeting NLRP3 and staphylococcal pore-forming toxin receptors in human-induced pluripotent stem cell-derived macrophages

Staphylococcus aureus causes necrotizing pneumonia by secreting toxins such as leukocidins that target front-line immune cells. The mechanism by which leukocidins kill innate immune cells and trigger inflammation during S. aureus lung infection, however, remains unresolved. Here, we explored human-induced pluripotent stem cell-derived macrophages (hiPSC-dMs) to study the interaction of the leukocidins Panton-Valentine leukocidin (PVL) and LukAB with lung macrophages, which are the initial leukocidin targets during S. aureus lung invasion. hiPSC-dMs were susceptible to the leukocidins PVL and LukAB and both leukocidins triggered NLPR3 inflammasome activation resulting in IL-1β secretion. hiPSC-dM cell death after LukAB exposure, however, was only temporarily dependent of NLRP3, although NLRP3 triggered marked cell death after PVL treatment. CRISPR/Cas9-mediated deletion of the PVL receptor, C5aR1, protected hiPSC-dMs from PVL cytotoxicity, despite the expression of other leukocidin receptors, such as CD45. PVL-deficient S. aureus had reduced ability to induce lung IL-1β levels in human C5aR1 knock-in mice. Unexpectedly, inhibiting NLRP3 activity resulted in increased wild-type S. aureus lung burdens. Our findings suggest that NLRP3 induces macrophage death and IL-1β secretion after PVL exposure and controls S. aureus lung burdens