The impact of different antimicrobial exposures on the gut microbiome in the ARMORD observational study

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors provide a study among healthy individuals, general medical patients and patients receiving haematopoietic cell transplants (HCT) to study the gut microbiome through shotgun metagenomic sequencing of stool samples. The first two groups were sampled once, while the patients receiving HCT were sampled longitudinally. A range of metadata (including current and previous (up to 1 year before sampling) antibiotic use) was recorded for all sampled individuals. The authors then performed shotgun metagenomic sequencing (using the Illumina platform) and performed bioinformatic analyses on these data to determine the composition and diversity of the gut microbiota and the antibiotic resistance genes therein. The authors conclude, on the basis of these analyses, that some antibiotics had a large impact on gut microbiota diversity, and could select opportunistic pathogens and/or antibiotic resistance genes in the gut microbiota.

Strengths:

The major strength of this study is the considerable achievement of performing this observational study in a large cohort of individuals. Studies into the impact of antibiotic therapy on the gut microbiota are difficult to organise, perform and interpret, and this work follows state-of-the-art methodologies to achieve its goals. The authors have achieved their objectives and the conclusion they draw on the impact of different antibiotics and their impact on the gut microbiota and its antibiotic resistance genes (the 'resistome', in short), are supported by the data presented in this work.

Weaknesses:

The weaknesses are the lack of information on the different resistance genes that have been identified and which could have been supplied as Supplementary Data. In addition, no attempt is made to assess whether the identified resistance genes are associated with mobile genetic elements and/or (opportunistic) pathogens in the gut. While this is challenging with short-read data, alternative approaches like long-read metagenomics, Hi-C and/or culture-based profiling of bacterial communities could have been employed to further strengthen this work. Unfortunately, the authors have not attempted to perform corrections for multiple testing because many antibiotic exposures were correlated.

Impact:

The work may impact policies on the use of antibiotics, as those drugs that have major impacts on the diversity of the gut microbiota and select for antibiotic resistance genes in the gut are better avoided. However, the primary rationale for antibiotic therapy will remain the clinical effectiveness of antimicrobial drugs, and the impact on the gut microbiota and resistome will be secondary to these considerations.

Reviewer #2 (Public Review):

Summary:

In this manuscript by Peto et al., the authors describe the impact of different antimicrobials on gut microbiota in a prospective observational study of 225 participants (healthy volunteers, inpatients and outpatients). Both cross-sectional data (all participants) and longitudinal data (a subset of 79 haematopoietic cell transplant patients) were used. Using metagenomic sequencing, they estimated the impact of antibiotic exposure on gut microbiota composition and resistance genes. In their models, the authors aim to correct for potential confounders (e.g. demographics, non-antimicrobial exposures and physiological abnormalities), and for differences in the recency and total duration of antibiotic exposure. I consider these comprehensive models an important strength of this observational study. Yet, the underlying assumptions of such models may have impacted the study findings (detailed below). Other strengths include the presence of both cross-sectional and longitudinal exposure data and the presence of both healthy volunteers and patients. Together, these observational findings expand on previous studies (both observational and RCTs) describing the impact of antimicrobials on gut microbiota.

Weaknesses:

(1) The main weaknesses result from the observational design. This hampers causal interpretation and corrects for potential confounding necessary. The authors have used comprehensive models to correct for potential confounders and for differences between participants in duration of antibiotic exposure and time between exposure and sample collection. I wonder if some of the choices made by the authors did affect these findings. For example, the authors did not include travel in the final model, but travel (most importantly, south Asia) may result in the acquisition of AMR genes [Worby et al., Lancet Microbe 2023; PMID 37716364). Moreover, non-antimicrobial drugs (such as proton pump inhibitors) were not included but these have a well-known impact on gut microbiota and might be linked with exposure to antimicrobial drugs. Residual confounding may underlie some of the unexplained discrepancies between the cross-sectional and longitudinal data (e.g. for vancomycin).

In addition, the authors found a disruption half-life of 6 days to be the best fit based on Shannon diversity. If I'm understanding correctly, this results in a near-zero modelled exposure of a 14-day-course after 70 days (purple line; Supplementary Figure 2). However, it has been described that microbiota composition and resistome (not Shannon diversity!) remain altered for longer periods of time after (certain) antibiotic exposures (e.g. Anthony et al., Cell Reports 2022; PMID 35417701). The authors did not assess whether extending the disruption half-life would alter their conclusions.

(2) Another consequence of the observational design of this study is the relatively small number of participants available for some comparisons (e.g. oral clindamycin was only used by 6 participants). Care should be taken when drawing any conclusions from such small numbers.

(3) The authors assessed log-transformed relative abundances of specific bacteria after subsampling to 3.5 million reads. While I agree that some kind of data transformation is probably preferable, these methods do not address the compositional data of microbiome data and using a pseudocount (10-6) is necessary for absent (i.e. undetected) taxa [Gloor et al., Front Microbiol 2017; PMID 29187837]. Given the centrality of these relative abundances to their conclusions, a sensitivity analysis using compositionally-aware methods (such as a centred log-ratio (clr) transformation) would have added robustness to their findings.

(4) An overall description of gut microbiota composition and resistome of the included participants is missing. This makes it difficult to compare the current study population to other studies. In addition, for correct interpretation of the findings, it would have been helpful if the reasons for hospital visits of the general medical patients were provided.