Antibiotics and probiotics impact gut antimicrobial resistance gene reservoir in COVID-19 patients

Abstract

Dysbiosis of gut microbiota is well-described in patients with coronavirus 2019 (COVID-19), but the dynamics of antimicrobial resistance genes (ARGs) reservoir, known as resistome, is less known. Here, we performed longitudinal fecal metagenomic profiling of 142 patients with COVID-19, characterized the dynamics of resistome from diagnosis to 6 months after viral clearance, and reported the impact of antibiotics or probiotics on the ARGs reservoir. Antibiotic-naive patients with COVID-19 showed increased abundance and types, and higher prevalence of ARGs compared with non-COVID-19 controls at baseline. Expansion in resistome was mainly driven by tetracycline, vancomycin, and multidrug-resistant genes and persisted for at least 6 months after clearance of SARS-CoV-2. Patients with expanded resistome exhibited increased prevalence of Klebsiella sp. and post-acute COVID-19 syndrome. Antibiotic treatment resulted in further increased abundance of ARGs whilst oral probiotics (synbiotic formula, SIM01) significantly reduced the ARGs reservoir in the gut microbiota of COVID-19 patients during the acute infection and recovery phase. Collectively, these findings shed new insights on the dynamic of ARGs reservoir in COVID-19 patients and the potential role of microbiota-directed therapies in reducing the burden of accumulated ARGs.

Keywords: COVID-19; SIM01; antimicrobial resistance gene; gut microbiome; synbiotic formula.

Figure 1.

Antibiotic-naive COVID-19 patients harbor more ARGs than non-COVID-19 controls. (a) Schematic overview of the study design, depicting the total number of samples and participants from whom data were available. The horizontal bars represent the sample collected at specific time point from antibiotic-naive COVID-19 patients. (b) Bray–Curtis-based beta diversity of baseline stool samples from COVID-19 patients and non-COVID-19 subjects based on ARG subtypes. The alpha diversity (Shannon, C), observed ARG types (d), observed ARG subtypes (e), and the normalized abundance of ARGs (f) were all significantly higher in stool samples from COVID-19 patients than that of non-COVID-19 subjects. (g) The prevalence of ARG types in COVID-19 patients and non-COVID-19 subjects.

Figure 2.

Resistome expansion persists for 6 months in antibiotic-naive COVID-19 patients. Dynamics of the observed ARG types (a), subtypes (b) and normalized abundance (c) in antibiotic-naive COVID-19 patients from baseline to 6-month follow-up. (d) Bray–Curtis dissimilarity of ARGs (‘types’) in stool samples from baseline to 6-month follow-up. (e) Abundance of antibiotic resistance ‘types’ in stool samples from baseline to 6-month follow-up. (f) The normalized abundance of ARGs in subjects with PACS is significantly higher than that of subjects without PACS at 6-month follow-up.

Figure 3.

Expanded resistome predicts risk of post-acute COVID-19 syndrome. (a) Bray–Curtis dissimilarity of ARGs (‘subtypes’) in stool samples from cluster 1 and cluster 2 at viral clearance. The observed ARG types (b), subtypes (c), and the normalized abundance of ARGs (d) were all significantly higher in stool samples from COVID-19 patients in cluster 1 than that of cluster 2 at viral clearance. (e) The prevalence of Klebsiella in COVID-19 patients of cluster 1 and cluster 2 at viral clearance, 3-month and 6-month  follow-up. (f) The prevalence of PACS in COVID-19 patients of cluster 1 and cluster 2 at 6-month  follow-up. Eigengap method was used to analyze the similarity of resistome of the 66 antibiotic-naive patients and divided them into two clusters based on the overall composition of resistome.

Figure 4.

Antibiotics further expand the resistome in COVID-19 patients. The increment of the observed ARG types (a), subtypes (b), and abundance (c) in antibiotic-naive COVID-19 patients is significantly higher than that of antibiotic-experienced COVID-19 patients. Increment refers to the expansion of resistome from baseline to viral clearance. (d) The increment of ARGs abundance in antibiotic-experienced COVID-19 patients is not associated with the length of antibiotic use time. (e) The increment of ARGs abundance in subjects who received two antibiotics is significantly higher than that of subjects who received one antibiotic. (f) Dynamics of ARGs abundance in antibiotic-naive COVID-19 patients and antibiotic-experienced COVID-19 patients. *P < .05, **P < .01, ***P < .001, ****P < .0001, Kruskal–Wallis and Dunn’s tests (all panels).

Figure 5.

Probiotics reduce ARGs in COVID-19 patients during virus-positive period. (a) Schematic overview of the study design, depicting the total number of samples and participants from whom data were available. The horizontal bars represent the sample collected at specific time point. (b) Probiotics were associated with an increased dissimilarity of resistome configuration compared with pre-treatment ARGs. The observed ARG types (c), subtypes (d), and abundance (e) in COVID-19 patients exhibited significant decrease after taking probiotics, but no significant trend was found in control group (Pearson Correlation).

Figure 6.

Probiotics reduce ARGs in COVID-19 patients after viral clearance. (a) Schematic overview of the study design, depicting the total number of samples and participants from whom data were available. The horizontal bars represent the sample collected at specific time point. (b) Bray–Curtis dissimilarities clearly separated the resistome at baseline and that after probiotics supplementation in subjects taking SIM01. The dynamics of the observed ARG types (c), subtypes (d), and abundance (e) in subjects taking SIM01 from baseline to week 12. *P < .05, **P < .01, ***P < .001, ****P < .0001, Kruskal–Wallis and Dunn’s tests (all panels).