Dottorato in MALATTIE INFETTIVE, MICROBIOLOGIA E SANITA' PUBBLICA

Titolo della tesi: Genotypic and phenotypic features of Staphylococcus aureus strains isolated in subjects with atopic dermatitis

Background: The human skin hosts several microbial communities, called skin microbiota (SM). It is known that SM cooperates with the skin immune system and protects against pathogens, representing an important factor for skin health. In fact, the dysbiosis of the SM has been associated with the development of different skin disorders, like atopic dermatitis (AD). AD is a chronic inflammatory skin disease and its pathogenesis is not fully understood, but many data revealed an alteration of the bacterial diversity, with increased colonization by Staphylococcus aureus, on the inflamed skin of AD patients. Moreover, it is documented that some strains of S. aureus produce several virulence factors, colonize skin via biofilm and can invade keratinocytes, thus contributing to increase inflammation. However, the functional role of S. aureus in the AD is poorly understood. The aim of the study is to evaluate the genotypic and phenotypic differences among S. aureus strains from AD patients, during the different phases of AD. Methods: A total of 15 AD patients were enrolled. To evaluate AD status, the score index SCORAD was used. AD patients were sampled during the flare phase (t0) and the post flare phase (t1), representing the clinical remission phase, four weeks after the treatment with monoclonal antibody (Dupilumab), that acts blocking the Th2 immune response. For each patient, swabs from nose, lesional and non-lesional skin at the flare (t0) and from nose and skin at post flare (t1) were collected. Cultural analysis of all swabs was carried out to isolate, identify and collect S. aureus strains. All isolates were identified by MALDI-TOF and genetically characterized by RAPD-PCR. Then, the genome of not-clonal S. aureus strains from AD patients and the genome of S. aureus strains from healthy carriers were analyzed by WGS. Biofilm production of S. aureus strains was determined through crystal violet staining. Finally, to test the S. aureus invasion ability, in vitro gentamicin protection assays on human keratinocytes (HaCat cells) were also performed. Results: By cultural analysis, a total of 55 S. aureus isolates were collected from AD patients. At t0, S. aureus was recovered in 100% and 60% of lesional and non-lesional skin samples respectively, with a high degree of colonization (10^4-10^5 CFU/ml); at t1 S. aureus was recovered in 50% of skin samples with a lower degree of colonization (10^2-10^3 CFU/ml). Results obtained by molecular analysis revealed a total of 24 S. aureus strains. In almost all patients, at t0 S. aureus isolates from the nose and the skin of the same patient belonged to the same clonal group. Differently, at t1, in each patient, nasal isolates were clonal to those recovered at t0, while the skin isolates belonged to a different clonal group. Moreover, all S. aureus strains from AD patients were strong or moderate biofilm producers, while those from healthy carriers were moderate or weak biofilm producers. Regarding the invasive capacity, most of the strains from healthy carriers and only half of the strains from AD were invasive. Conclusions: These data confirm that S. aureus is the key pathogen in AD. The therapeutic treatment with Dupilumab reduces both the clinical signs of disease and the bacterial load of S. aureus, but it is not able to induce a complete S. aureus skin eradication. Overall, the ability to produce biofilm and to invade keratinocytes could be a functional tract for S. aureus persistence in this hostile environment. Currently, to deepen genomic and functional features associated with pathogenic mechanisms, the analysis of the complete genome sequence of S. aureus strains from AD patients and healthy carriers is in progress. Such information could evidence the bacterial virulence traits linked to S. aureus capability to survive and to induce inflammatory processes in human skin.