Background Staphylococcus aureus is a major trigger of atopic dermatitis (AD) and causes the majority of skin and soft tissue infections. The precise staphylococcal factor that promotes type 2 inflammation in the skin, and the reason why recurrent skin infections are common despite seemingly adequate antibiotic treatment are currently unclear. Aims The aims of this study were to: 1. purify and identify S. aureus bioactive factor(s) that drive type 2 inflammation by primary human keratinocytes (NHEK). 2. to determine the ability of antibiotics to eliminate S. aureus internalised by NHEK. Methods Release of IL-33 and TSLP from NHEK by S. aureus and its filtered supernatant (FSA) were assessed by ELISA. Purification of the active factor was achieved by FPLC and confirmed by mass spectroscopy. Internalisation of S. aureus and S. epidermidis by NHEK were assessed by flow cytometry and confocal microscopy. Viability of intracellular S. aureus was determined by growth on nutrient agar plates after lysis of keratinocytes by physical disruption. Results S. aureus Second IgG binding protein (Sbi) was identified as the unique type 2 promoting biofactor. Confocal microscopy and flow cytometry showed that S. aureus, but not other staphylococci, was rapidly internalised into NHEK and could survive for 24 hours without inducing cytotoxicity or release of alarmins. The antimicrobials flucloxacillin, clindamycin, linezolid, and teicoplanin failed to eliminate intracellular S. aureus even at concentrations 20X MIC. Only rifampicin was effective at eradicating intracellular S. aureus. Conclusions Sbi was identified as the unique factor that induced type 2 promoting cytokine release by NHEK. The exact mechanism by which Sbi mediates inflammation still need to be determined. Many anti-staphylococcal antibiotics used to treat S. aureus infections have little effect on internalised microbes, possibly explaining recurrent reinfection. Rifampicin is the only antibiotic to effectively kill internalised S. aureus in our in vitro experiments.
|Date of Award
|1 Aug 2020
- The University of Manchester
|Peter Arkwright (Supervisor), Catherine O'Neill (Supervisor) & Joanne Pennock (Supervisor)