The Protective Effect of Microbiota on S The Protective Effect of Microbiota on S. aureus Skin Colonization Depends on the Integrity of the Epithelial Barrier  Marc Burian, Katharina Bitschar, Beatrice Dylus, Andreas Peschel, Birgit Schittek  Journal of Investigative Dermatology  Volume 137, Issue 4, Pages 976-979 (April 2017) DOI: 10.1016/j.jid.2016.11.024 Copyright © 2016 The Authors Terms and Conditions

Figure 1 S. epidermidis-secreted factors protect primary human keratinocytes against S. aureus infection. (a) Infection model and experimental time course. Treatment of differentiated primary human keratinocytes with live S. epidermidis bacterial cells or S. epidermidis-conditioned medium before S. aureus infection. At 1.5 hours after S. aureus infection, adherent and internalized staphylococci species were analyzed, and 3 hours after initial S. aureus infection, only internalized staphylococci species were determined. (b, c) Capability of S. aureus to adhere to and invade primary human keratinocytes. Differentiated primary human keratinocytes were pretreated with S. epidermidis 1457 (MOI = 30) for 2 hours or S. epidermidis-conditioned mediumfor 24 hours. Subsequently, cells were infected (MOI = 30) with S. aureus (b) USA300 or (c) SA113 for 1.5 hours, followed either by cell lysis (adhesion and invasion) or lysostaphin-treatment for 1.5 hours (invasion). Serial dilutions of the lysate were spotted on blood agar plates and CFUs were determined. Data represent mean values ± standard error of the mean of three independent experiments. Significant differences to the untreated control were analyzed by ordinary one-way analysis of variance followed by Dunnett posttest: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (d, e) Capability of CM of different S. epidermidis isolates or of other skin commensals to protect against S. aureus adhesion and invasion. Differentiated primary human keratinocytes were pretreated with indicated CM for 24 hours. Subsequently, cells were infected (MOI = 30) with S. aureus USA300 for 1.5 hours followed by cell lysis (adhesion and invasion). Serial dilutions of the lysate were spotted on blood agar plates, and CFUs were determined. Data represent mean values ± standard error of the mean of three independent experiments. Significant differences to the untreated control were analyzed by ordinary one-way analysis of variance, followed by Dunnett posttest: ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. CFU, colony-forming unit; CM, conditioned medium; CnT, epidermal keratinocyte medium; M, mol/L; MOI, multiplicity of infection; n.s., not significant; OD, optical density; TSB, tryptic soy broth. Journal of Investigative Dermatology 2017 137, 976-979DOI: (10.1016/j.jid.2016.11.024) Copyright © 2016 The Authors Terms and Conditions

Figure 2 S. epidermidis-induced skin protection against S. aureus infection depends on the integrity of the epithelial barrier. (a) Epicutaneous mouse skin infection model. 1 × 109 live S. epidermidis bacterial cells, S. epidermidis CM, or PBS as a control were epicutaneously applied 24 hours before S. aureus USA300 application on the back skin of shaved C57BL/6 mice. Skin of mice was either unaffected or superficially disrupted by multiple stripping with an adhesive tape. (b, c) Mouse infection of unaffected skin. Unaffected back skin of C57BL/6 mice was pretreated with either 1 × 109 live S. epidermidis bacterial cells, S. epidermidis CM, or PBS as a control 24 hours before S. aureus application. After 1 day the number of (b) S. aureus CFUs for surface-attached bacteria and (c) bacteria located in deeper skin tissue was determined. Horizontal lines represent the mean of each group ± standard error of the mean. Significant differences to the PBS control were analyzed by ordinary one-way analysis of variance followed by Dunnett posttest: ∗∗P < 0.01, ∗∗∗∗P < 0.0001. (d, e) Mouse infection of superficially disrupted skin. Tape-stripped back skin of C57BL/6 mice was pretreated with either 1 × 109 live S. epidermidis bacterial cells, S. epidermidis CM, or PBS as a control 24 hours before S. aureus application. After 1 day the number of S. aureus CFUs for (d) surface-attached bacteria as well as (e) bacteria located in deeper skin tissue was determined. Horizontal lines represent the mean of each group ± standard error of the mean. Significant differences to the PBS control were analyzed by ordinary one-way analysis of variance followed by Dunnett posttest: ∗P < 0.05. CFU, colony-forming unit; CM, conditioned medium; n.s., not significant; PBS, phosphate buffered saline. Journal of Investigative Dermatology 2017 137, 976-979DOI: (10.1016/j.jid.2016.11.024) Copyright © 2016 The Authors Terms and Conditions