Doxycycline post-exposure prophylaxis for sexually transmitted infections impacts the gut antimicrobial resistome

Abstract

Doxycycline post-exposure prophylaxis (doxy-PEP) reduces bacterial sexually transmitted infections (STIs) among men who have sex with men and transgender women. While poised for widespread clinical implementation, the impact of doxy-PEP on antimicrobial resistance remains a primary concern as its effects on the gut microbiome and resistome, or the antimicrobial resistance genes (ARGs) present in the gut microbiome, are unknown. To investigate these effects, we studied participants from a randomized clinical trial who either received doxy-PEP as a one-time doxycycline 200 mg taken after condomless sex (DP arm, n = 100) or standard of care treatment (SOC arm, n = 50). From self-collected rectal swabs at enrollment (day-0) and after 6 months (month-6), we performed metagenomic DNA sequencing (DNA-seq) or metatranscriptomic RNA sequencing (RNA-seq). DNA-seq data was analyzable from 127 samples derived from 89 participants, and RNA-seq data from 86 samples derived from 70 participants. We compared the bacterial microbiome and resistome between the two study arms and over time. Tetracycline ARGs were detected in all day-0 DNA-seq samples and 85% of day-0 RNA-seq samples. The proportional mass of tetracycline ARGs in the resistome increased between day-0 and month-6 in DP participants from 46-51% in the metagenome (p = 0.02) and 4-15% in the metatranscriptome (p < 0.01), but no changes in other ARG classes were observed. Exposure to a higher number of doxycycline doses correlated with proportional enrichment of tetracycline ARGs in the metagenome (Spearman's ρ = 0.23, p < 0.01) and metatranscriptome (Spearman's ρ = 0.55, p < 0.01). Bacterial microbiome alpha diversity, beta diversity, and total bacterial mass did not differ between day-0 and month-6 samples from DP participants when assessed by either DNA-seq or RNA-seq. In an abundance-based correlation analysis, we observed an increase over time in the strength of the correlation between tetracycline ARGs and specific bacterial taxa, including some common human pathogens. In sum, doxy-PEP use over a 6-month period was associated with an increase in the proportion of tetracycline ARGs comprising the gut resistome, and an increase in the expression of tetracycline ARGs. Notably, doxy-PEP did not significantly alter alpha diversity or taxonomic composition of the gut microbiome, and did not demonstrate significant increases in non-tetracycline ARG classes. Further studies and population level surveillance are needed to understand the implications of these findings as doxy-PEP is implemented as a public health strategy.

Keywords: antibiotic resistance; antimicrobial resistance; doxy-PEP; doxycycline; men who have sex with men; resistome; sexually transmitted infection.

Figure 1. Impact of Doxy-PEP use on the gut resistome for DNA-seq samples (A, C, E) and RNA-seq samples (B, D, F).

(A, B) Normalized resistome mass for doxy-PEP versus SOC participants (DNA-seq: n = 127, RNA-seq: n = 86). (C, D) Resistome alpha diversity (Shannon Diversity Index) in doxy-PEP versus SOC participants (DNA-seq: n = 127, RNA-seq: n = 86). (E, F) Resistome beta diversity (Bray-Curtis Index) in doxy-PEP versus SOC participants at six months (DNA-seq: n = 69, RNA-seq: n = 60). One outlier from the Doxy-PEP arm was omitted from the RNA-seq beta diversity plot for graphical purposes with coordinates of (NMDS1: 9.9, NMDS2: −0.3), but was included in the calculations. P-values were calculated using the Wilcoxon rank-sum test and adjusted for multiple comparisons (A-D). P-values for beta diversity was calculated using the PERMANOVA test and adjusted for multiple comparisons (E, F). Significant p-values (<0.05) are bolded. Abbreviations: ARG, antimicrobial resistance gene; p.adj, adjusted p-value; NMDS, non-metric multidimensional scaling.

Figure 2. Impact of Doxy-PEP use on tetracycline and non-tetracycline ARGs for DNA-seq samples (A, C, E) and RNA-seq samples (B, D, F).

(A, B) Tetracycline ARG richness and (C, D) tetracycline ARG proportion of resistome mass were compared between standard of care and doxy-PEP arms at each visit and over time (DNA-seq: n = 127, RNA-seq: n = 86). (E, F) The proportion of the resistome mass by ARG classes over time within the doxy-PEP arm (DNA-seq: n = 80, RNA-seq: n = 55). P-values were calculated using the Wilcoxon rank-sum test and adjusted for multiple comparisons. Significant p-values (<0.05) are bolded. Abbreviations: ARG, antimicrobial resistance gene; p.adj, adjusted p-value; MLS, macrolide-lincosamide-streptogramin.

Figure 3. Impact of Doxy-PEP use on tetracycline ARGs by number of doxycycline doses received for DNA-seq samples (A, B) and RNA-seq samples (C, D).

A test of trend was used to compare (A, C) tetracycline ARG richness and (B, D) proportion of tetracycline ARG mass to resistome mass by number of doxycycline doses received (DNA-seq: n = 127, RNA-seq: n = 86). P-values were calculated using the Wilcoxon rank-sum test and adjusted for multiple comparisons. The Spearman’s rank correlation test was used to calculate Spearman’s ρ and p-value. Significant p-values (<0.05) are bolded. Abbreviations: ARG, antimicrobial resistance gene; p.adj, adjusted p-value.

Figure 4. Impact of Doxy-PEP use on tetracycline ARGs in sets of paired DNA-seq samples (A, C, E) and paired RNA-seq samples (B, D).

(A, B) Tetracycline ARG richness and (C, D) the proportion of tetracycline ARG mass to resistome mass were compared between standard of care and doxy-PEP arms at each visit and over time (DNA-seq: n = 38 sets of paired samples, RNA-seq: n = 16 sets of paired samples). (E) Heatmap of the tetracycline ARGs detected in DNA-seq data of paired samples (day-0 and month-6 samples) for the standard of care and Doxy-PEP arms (n = 38 sets of paired samples). P-values were calculated using the Wilcoxon signed-rank test for paired samples and adjusted for multiple comparisons. Significant p-values (<0.05) are bolded. Abbreviations: ARG, antimicrobial resistance gene; p.adj, adjusted p-value; D0, day-0; M6, month-6.

Figure 5. Bacterial microbiome metrics and correlation with tetracycline ARGs for DNA-seq samples.

(A) Normalized mass of the bacterial microbiome and (B) alpha diversity measured by Shannon Diversity Index were compared between standard of care and doxy-PEP arms at each visit and over time (n = 127). (C) Beta diversity was compared between the SOC and doxy-PEP arms at the month-6 time point (n = 69). (D) A differential abundance analysis of bacterial genera was performed using DeSeq2 comparing doxy-PEP samples at day-0 and month-6 with a significance level of 0.05, adjusted for multiple comparisons (n = 80). (E) A plot demonstrating the change in Pearson’s correlation coefficient between tetracycline ARG abundance and bacterial genera abundance from day-0 to month-6 doxy-PEP samples for tetracycline ARGs and bacterial genera that were found to be significantly (p.adj <0.05, adjusted for multiple comparisons) correlated at month-6 in the doxy-PEP arm (n = 80). Color and size of the square filling represent the degree of change in the correlation coefficient. Blank squares represent cases where there was not a significantly correlation between the tetracycline ARG and the bacterial taxa at month 6 or there was not enough data to evaluate for correlation. P-values for mass, alpha diversity, and species of interest were calculated using the Wilcoxon signed-rank test and adjusted for multiple comparisons. The p-value for beta diversity was calculated using the PERMANOVA test and adjusted for multiple comparisons. Abbreviations: ARG, antimicrobial resistance gene; p.adj, adjusted p-value.