C. difficile may be overdiagnosed in adults and is a prevalent commensal in infants

Reviewer #1 (Public Review):

Summary:

The authors present a comprehensive meta-analysis of Clostridioides difficile (CD) occurrence across 42,900 metagenomes from 253 public studies, largely representing stool samples from human adults, infants, and with a smaller fraction of samples from non-gut body sites and from environmental samples (e.g., non-human animals, wastewater, soil, etc.). In particular, the authors looked at adults who were healthy, diseased (but not with C. diff), and with diagnosed C. diff infection (CDI) and found that CD occurrence was fairly low: ~30% in adult CDI samples, ~2% in adult diseased samples, and ~1% in healthy samples. CD was much more prevalent in infants (15 and 40% in healthy and diseased infants, respectively). These findings, if they hold true, would be significant because they would suggest an over-diagnosis of CDI and an under-diagnosis of other putative enteric pathogens (also enriched in CDI samples) across the population. Furthermore, these results suggest that the asymptomatic carriage of CD in adults (~1-5%, depending on demographics) may be much lower than some prior estimates (some as high as ~30-40%).

Strengths:

The authors have done an admirable job pulling down an enormous data set for this CD-focused meta-analysis, which is a valuable service to the field. The results push against some common wisdom in the field, in terms of the prevalence of CD in CDI patients, which will be impactful if they hold up to further scrutiny. Furthermore, the identification of commensal bacteria that are positively or negatively associated with CD presence in both healthy and diseased people at different periods of the lifespan (infant, child, and adult), is a valuable synthesis with potential translational value. The manuscript is clearly written and the figures are presented well. The methodology is robust, although I have a few suggestions for improvement.

Weaknesses:

My main critique relates to detection limitations, both in terms of sequencing depth and read-mapping. Given that CD detection is the root of the main conclusions reported here, this deserves some additional care. The authors have already done some work to address this by including sequencing depth in their linear mixed effects model, which is great. Furthermore, they were conservative with how they labeled CD positive/negative individuals with multiple time points (i.e., if you had CD detected at any point, this sample was selected for the cross-sectional analysis, and that individual was labeled as CD positive). I have a few additional suggestions to explore this issue, which I outline in the recommendations for authors.

Reviewer #2 (Public Review):

Summary:

C. difficile infection (CDI) is clinically important as a hospital-acquired infection and a frequent cause of antibiotic-associated diarrhea, which is associated with high morbidity and mortality and increases in prevalence. It is also the prime example of a disease that is associated with gut microbiome dysbiosis and successfully treated with fecal microbiota transfer, highlighting the important but unclear functional or structural role of this bacterial pathogen and the condition of CDI for the gut microbiome, which is the focus of this study.

Ferretti et al. assembled an impressive gut metagenome dataset from previous and ongoing microbiome studies, which involves a large number of samples from patients with CDI or other diarrheal and non-diarrheal diseases and from healthy individuals, as well as from infants, adolescents, and adults. The authors analyze the prevalence and relative abundance of C. difficile in this dataset in relation to CDI diagnosis, host age and disease background, and the composition of the remaining microbiota. They detect C. difficile only in a minority of samples labelled as originating from CDI patients but frequently identify other pathogens and their toxin genes in the same samples. In infants, they detect C. difficile at high frequency and relative abundance in samples without clinical symptoms. They associate C. difficile presence in infant samples with "multiple indicators of healthy gut microbiome maturation' and suggest 'distinct biotic and physiological contexts in infants and adults' for C. difficile.

Strengths:

The manuscript provides an important overview of the complex relationship of C. difficile with the gut microbiome of healthy and diseased infants and adults, mostly due to the large studied dataset and convincing applied analysis that underlies the presented findings. This includes a number of interesting findings including, for example, that CDI can be reliably predicted based on taxonomic microbiota compositions, without including C. difficile itself or that C. difficile in infants appears not to originate from maternal sources.

Weaknesses:

Inconsistent associations of C. difficile with what is clinically labeled CDI, as well as the frequent detection of C. difficile in healthy infants, have been reported before and the manuscript does not reveal to what extent this bacterium reflects or even directly influences the gut microbiome of infants and adults. Whether the increased microbiota diversity, richness, and compositional similarity of C. difficile-positive infants to their mothers is sufficient to associate this bacterium with "healthy gut microbiome maturation" seems questionable, since C. difficile was also found to be more prevalent in preterm infants, formula-fed or antibiotically treated infants, and infants born by C-section, all of which are typically considered detrimental influences on microbiota development. The conclusion that "C. difficile may be a transient hallmark of healthy gut microbiome maturation" therefore appears too strong.

In addition, the statement that "its asymptomatic carriage in adults depends on microbial context" is not sufficiently supported by the presented data. Apparently, the authors are unable to define or measure "asymptomatic carriage", as they convincingly show that many patients diagnosed with "CDI" appear not to carry C. difficile, suggesting that neither asymptomatic nor symptomatic "CDI" conditions are necessarily linked to C. difficile.

The manuscript includes a large number of samples from poorly defined, but diverse patient backgrounds. It might be helpful to better define these samples (e.g. fecal samples vs. other gut samples) and to specify subcategories for samples from "diseased control subjects without CDI". Maybe this information could help validate the interesting suggestion from the manuscript that C. difficile may be (one of several) dysbiosis marker rather than the cause of (CDI) dysbiosis.

The phylogenetic analysis of C. difficile from metagenomic sequence data seems to suggest that there is a large mostly toxin gene-free cluster that is only identified in infants (Supplementary Figure 13). Could this indicate that there are, in fact, less pathogenic C. difficile lineages that are more prevalent in infants?

The authors argue in the Discussion that "Differential diagnosis against multiple enteropathogens may therefore stratify patients with CDI-like symptoms, towards adapted therapeutic interventions." It might be helpful to expand this discussion of different clinical options that could be adapted to highlight the clinical applicability of the presented findings.

Author Response:

We thank the reviewers and editors for their constructive and encouraging feedback on our manuscript. We have carefully studied the reviewer comments and found that we agree with almost all of them; we will implement these suggestions and prepare a revised submission. In particular, we will aim to address the reviewers’ valid concerns regarding metagenomic detection limits via a high-sensitivity re-analysis of the data based on metagenomic read mapping, orthogonal to our current analyses based on read mapping to mOTU single copy marker genes. Moreover, we will revise the manuscript text for clarity and streamline the phrasing on some observations and claims. We are confident that our work will improve as a result and look forward to future feedback and interactions.

Sincerely, for the authors,

Sebastian Schmidt & Peer Bork