Topical Antimicrobial Treatments Can Elicit Shifts to Resident Skin Bacterial Communities and Reduce Colonization by Staphylococcus aureus Competitors

Abstract

The skin microbiome is a complex ecosystem with important implications for cutaneous health and disease. Topical antibiotics and antiseptics are often employed to preserve the balance of this population and inhibit colonization by more pathogenic bacteria. However, despite their widespread use, the impact of these interventions on broader microbial communities remains poorly understood. Here, we report the longitudinal effects of topical antibiotics and antiseptics on skin bacterial communities and their role in Staphylococcus aureus colonization resistance. In response to antibiotics, cutaneous populations exhibited an immediate shift in bacterial residents, an effect that persisted for multiple days posttreatment. By contrast, antiseptics elicited only minor changes to skin bacterial populations, with few changes to the underlying microbiota. While variable in scope, both antibiotics and antiseptics were found to decrease colonization by commensal Staphylococcus spp. by sequencing- and culture-based methods, an effect which was highly dependent on baseline levels of Staphylococcus Because Staphylococcus residents have been shown to compete with the skin pathogen S. aureus, we also tested whether treatment could influence S. aureus levels at the skin surface. We found that treated mice were more susceptible to exogenous association with S. aureus and that precolonization with the same Staphylococcus residents that were previously disrupted by treatment reduced S. aureus levels by over 100-fold. In all, the results of this study indicate that antimicrobial drugs can alter skin bacterial residents and that these alterations can have critical implications for cutaneous host defense.

Keywords: Staphylococcus aureus; antimicrobial agents; microbiome; skin.

FIG 1

Topical antibiotics induce long-term shifts to skin microbial residents. (a) Heat map of rarified abundances for the 30 most common phylotypes on murine skin in response to treatment with polyethylene glycol (PEG), mupirocin, petrolatum, or triple antibiotic ointment (TAO). Dendrograms represent hierarchical clustering of Euclidean distances using complete agglomeration. Horizontal bars above the map designate treatment and time point features for individual mice. (b to d) Breakdown and longitudinal analysis of rarified abundances for Enterobacteriaceae (b), Clostridiales (c), and Porphyromonadaceae (d). Data are presented as individual mice (a) or means ± standard errors of the means (SEMs) (b to d). Statistical significance was determined at each time point by the Wilcoxon rank sum test (Mann Whitney U test). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

Triple antibiotic ointment alters skin bacterial diversity. (a) Shannon diversity measurements of murine bacterial communities following treatment with antibiotics and vehicles over time. (b) Weighted UniFrac distances comparing longitudinal time points to baseline communities of bacterial residents in treated and untreated mice. (c) Principal coordinate analyses of weighted UniFrac distances for murine bacterial communities over time. Data are presented as means ± SEMs (a, b) or individual mice (c). Statistical significance was determined at each time point by the Kruskal-Wallis rank sum test (a) or Wilcoxon rank sum test (Mann Whitney U test) (b). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Antiseptic treatment does not significantly alter skin bacterial community structure. (a) Heat map of rarified abundances for the 30 most common phylotypes on murine skin following treatment with water, alcohol, or povidone-iodine at d1 posttreatment. Dendrograms represent hierarchical clustering of Euclidean distances using complete agglomeration. Horizontal bar above the map designates treatments for individual mice. (b) Shannon diversity of murine bacterial communities in response to treatment. (c) Weighted UniFrac principal-coordinate analysis representing differences in murine bacterial populations following treatment. (d) Bacterial load comparison of treated and untreated mice calculated by 16S rRNA gene content at the skin surface. U, untreated; W, water; A, alcohol; P-I, povidone-iodine. Treatments were compared by Kruskal-Wallis rank sum test (b, d) or the adonis statistical test for community similarity (c).

FIG 4

Antimicrobial treatment alters resident Staphylococcus colonization in a baseline-dependent manner. (a) Murine resident Staphylococcus CFUs in response to cage change frequency over time. Group 1 mice were changed every other day and group 2 mice were changed once per week at the start. Groups were switched to the alternate regimen at d28. Data are presented as individual mice with median bars. (b and c) Murine resident Staphylococcus CFUs in response to antibiotic treatment starting at low (b) or high (c) baseline levels. Statistical comparisons were made between polyethylene glycol (PEG) and mupirocin (*) or petrolatum and triple antibiotic ointment (TAO) (†). Data are presented as means ± SEMs. (d and e) Murine resident Staphylococcus CFUs in response to antiseptic treatment starting at low (d) or high (e) baseline levels. Data are presented as individual mice at baseline and at d1 posttreatment. U, untreated; W, water; A, alcohol; P-I, povidone-iodine. Statistical significance was determined by Wilcoxon rank sum test (Mann Whitney U test). *, P < 0.05; **, P < 0.01; ††, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 5

Resident Staphylococcus can reduce colonization by Staphylococcus aureus. (a) Staphylococcus aureus CFUs following exogenous administration in mice pretreated with alcohol or in untreated controls. (b) Phylogenetic tree of 16S rRNA gene diversity using approximate-maximum-likelihood to compare murine Staphylococcus residents (red) to known Staphylococcus isolates from the RDP database (black). (c) Growth curve analysis of resident Staphylococcus isolates at an optical density of 600 nm (OD₆₀₀). (d) Enumeration of Staphylococcus isolate CFUs following exogenous administration to mouse dorsa. (e) S. aureus CFU levels following precolonization of mouse dorsa with resident Staphylococcus isolates. Data are presented as means ± SEMs (a) or with median bars (d, e). Statistical significance was determined by Wilcoxon rank sum test (Mann Whitney U test). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.