Alterations of human skin microbiome and expansion of antimicrobial resistance after systemic antibiotics

Abstract

Although systemic antibiotics are critical in controlling infections and reducing morbidity and mortality, overuse of antibiotics is presumed to contribute to negative repercussions such as selection of antimicrobial-resistant organisms and collateral damage to commensal microbes. In a prospective, randomized study of four clinically relevant antibiotic regimens [doxycycline (20 mg or 100 mg), cephalexin, or trimethoprim/sulfamethoxazole], we investigated microbial alterations on skin after administration of systemic antibiotics to healthy human volunteers. Samples from different skin and oral sites, as well as stool, were collected before, during, and up to 1 year after antibiotic use, and shotgun metagenomic sequencing was performed. Taxonomic analysis showed that subjects receiving doxycycline 100 mg and trimethoprim/sulfamethoxazole (TMP/SMX) exhibited greater changes to their skin microbial communities, as compared to those receiving other regimens or untreated controls. Oral and stool microbiota also demonstrated fluctuations after antibiotics. Bacterial culturing in combination with whole-genome sequencing revealed specific emergence, expansion, and persistence of antibiotic-resistant staphylococci harboring tetK or tetL and dfrC or dfrG genes in all subjects who received doxycycline 100 mg or TMP/SMX, respectively. Last, analysis of metagenomic data revealed an increase of genes involved in gene mobilization, indicating stress responses of microbes to antibiotics. Collectively, these findings demonstrate direct, long-lasting effects of antibiotics on skin microbial communities, highlighting the skin microbiome as a site for the development and persistence of antibiotic resistance and the risks of overprescribing.

Fig. 1.. Changes and resilience of skin microbiome after systemic antibiotics.

(A) Study design. Healthy subjects were randomized to 4 different antibiotic regimens. Skin swabs for metagenomic sequencing and in vitro culturing were collected at indicated time points (red arrows), skin biopsies for mass-spectrometry were collected at D0 and D14 (blue arrows) and oral/fecal samples were collected at D0, D14, D56 and D112 for doxycycline treated subjects (brown arrows). BID = twice daily; TID = three times daily. See Methods for details. *Fecal samples from Ceph and TMP/SMX were not sequenced. (B) Changes in microbiome similarity of Vf for each subject. Points indicate Bray-Curtis distance at each time point compared to D0 for each subject. Lines connect points from the same individual. Colored points/lines are data from subjects who received antibiotics, and gray points/lines indicate untreated healthy subjects (n=10, 5 samples excluded). Light gray shaded area marks the time period when subjects received antibiotics. (C) Bray-Curtis distances in (B) are summarized by time point and statistical significance determined using Wilcoxon rank-sum test (*P<0.05, **P<0.01, ***P<0.005); including Ac (n=10, 2 samples excluded) and Ra (n=10). (D) and (E) Average Bray-Curtis distance of Doxy100 subjects (D) and TMP/SMX subjects (E) to all of untreated healthy subjects (Vf). Statistical significance determined using Wilcoxon rank-sum test (*P<0.05, **P<0.01)

Fig. 2.. Expansion and emergence of antibiotic-resistant S. epidermidis within skin-associated microbial communities.

(A) Experimental design for in vitro culturing of doxycycline-resistant (DoxyR) or TMP-resistant (TMPR)skin bacteria. Skin swabs from D0, D14, D56, D112, and D336 were plated on TSB and RCA agar plate (with or without Doxy 2ug/ml or 100ug/ml), and incubated under aerobic and anaerobic conditions, respectively. After 72 hours (up to 120 hours to allow for minor and slow growing microbes to make visible colony), colonies were counted, grown independently, DNA extracted, and sequenced. (B) to (D) Summary tables for in vitro culture results of (B) Doxy100 subjects, (C) Doxy20 subjects and (D) TMP/SMX subjects. (+: 1–10 colonies, ++: 10–100 colonies, +++: >100 colonies, pink boxes highlight presence of DoxyR colonies). Cells with read shading indicate where resistant colonies were found. Species of isolated resistant bactera are listed at the bottom of each table.

Fig. 3.. Phylogenetic analyses and dynamics of S. epidermidis skin isolates in Doxy100 subjects.

(A) and (B) Phylogeny of S. epidermidis isolates from Subject 5 (A), and 8 (B). Dashed lines connect with doxycycline-resistant genes profiles (heatmap), with red denoting presence of specific resistant genes. Minimum inhibitory concentration (MIC) for doxycyline is summarized on the middle column. Time point (days) of isolation for each isolates are indicated on the right panel. STNR*: allele combination not reported in MLST allele database (MLST.net). (C) to (F) Changes of relative abundances of S. epidermidis STs from metagenomic data of Doxy100 subjects. Subject 5 (C), 6 (D), 7 (E) and 8 (F). Relative abundances of each ST were estimated by BIB (see Methods for detail). (G) and (H) Changes of relative abundances of tetK/L from metagenomic data of Doxy100 (G) and Doxy20 (H) groups. FPKM (fragments per kilobase per million reads) was calculated based on the number of reads mapped at high confidence to either tetK or tetL to the total of non-human reads (see Methods).

Fig. 4.. Dynamics of S. epidermidis strains in TMP/SMX group.

(A) to (C) Phylogeny of S. epidermidis isolates from Subject 12 (A), 13 (B) and 14 (C). Dashed lines connect with TMP-resistant genes profiles (heatmap), with red denoting presence of specific resistant genes. Minimum inhibitory concentration (MIC) for TMP is summarized on the middle column. Time point (days) of isolation for each isolates are indicated on the right pane l. (D) to (F) Changes of relative abundances of S. epidermidis STs from metagenomic data of TMP/SMX subjects. Subject 12 (D), 13 (E) and 14 (F). Relative abundances of each ST were estimated by BIB (see Methods for detail).

Fig. 5.. Increased gene mobilization of skin microbiomes with antibiotic use.

(A) and (C) Comparison of MGC relative abundances between D0 and D56 in Doxy100 subjects (A) and D0 and D42 in TMP/SMX subjects (C). The x-axis indicates log-transformed mean MGC abundances (MGC abundance per million reads (MPM), see Methods for detail) at D0 and D14 and the y-axis indicates log-transformed mean MPM at D56 (A) and D42 (C). Hexbin (blue to dark blue colors) indicate the number of MGCs in each hexagon and red points indicate significantly increased or decreased MGCs (adjusted P<0.05). (B) and (D) Analysis for Gene Ontology (GO, biological process) enrichment of increased MGCs in Doxy100 subjects (B) and in TMP/SMX subjects (D). GO enrichment was calculated by dividing of MGC with indicated GO term to the all differentially abundant MGC (x-axis). Red color indicates P<0.05 (Fisher’s exact test, see Methods).