The relationship between gut and nasopharyngeal microbiome composition can predict the severity of COVID-19

Reviewer #1 (Public Review):

Summary:

The research study under review investigated the relationship between the gut and identified potential biomarkers derived from the nasopharyngeal and gut microbiota-based that could aid in predicting COVID-19 severity. The study reported significant changes in the richness and Shannon diversity index in nasopharyngeal microbiome associated with severe symptoms. The study showed a high abundance of Bacillota and Pesudomonadota in patients exhibiting severe symptomatology. Positive correlations were also found between Corynebacterium, Acinetobacter, Staphylococcus, and Veillonella, with the severity of SARS-CoV-2 infection.

Strengths:

The study successfully identified differences in the microbiome diversity that could indicate or predict disease severity. Furthermore, the authors demonstrated a link between individual nasopharyngeal organisms and the severity of SARS-CoV-2 infection. The density of the nasopharyngeal organism was shown to be a potential predictor of the severity of COVID-19.

Weaknesses:

The authors claimed an association between nasopharyngeal organisms and severity of SARS-CoV-2 infection but omitted essential data on the statistical significance of these associations between groups. The authors frequently referred to a p-value < 0.05 without presenting the actual p-values and percentages to show the significance of their results. The discussion is hard to understand (lacked clarity), as it contained an extensive literature review without discussing the study findings. A more focused discussion and results section on the main findings could have improved the overall readability of the paper. The role of potential confounders, such as HIV infection, and ethnicity which impacts the nasopharyngeal microbiome composition, was not included in the paper. Addressing the potential confounders would contribute to a more comprehensive understanding of the study's implications, specifically the role of the nasopharyngeal microbiome as a predictor of COVID-19 severity.

Reviewer #2 (Public Review):

The study conducted by Benita et al studied the gut and nasopharyngeal microbiome in covid-19 severity. There are a lot of studies on this topic, and this study therefore cannot stand out from a pool of such similar studies. Beyond that, I have a number of major concerns:

(1) The sample size is limited. There were 3 cohorts, but only ~100 subjects in total. This indicates that there were only a small number of subjects in each cohort (the authors did not list this information), and beyond that, there was a lack of healthy individuals as controls. A cohort-specific effect should usually exist, I believe with such a small number of patients (they were further divided into 3 groups), the authors cannot find reproducible data between cohorts.

(2) The study did not meet the study goal. The authors say "Many factors have been described to be correlated with its severity but no specific determinants of infection outcome have been identified yet". However, numerous studies have shown the relationship between microbiome and covid. The present study only again showed a correlation between microbiome and covid severity and did not provide further insights, nor did they find specific determinants.

(3) This study only studied 16s-seq for microbiome profiling, which made this study lack depth and resolution. Many peer papers have used metagenomics sequencing for in-depth interrogation.

(4) Since there are fecal and nasopharyngeal microbiome data, the authors only listed their respective associations with covid severity yet did not provide further insights into whether and how these two microbiome types are linked to covid, or into whether there is a microbiome priority, resistance or transmission.

(5) The abstract is amiss where each sentence lacks a key message - I don't understand each of the sentences or the underlying meanings. One example of an unclear expression is "this ratio" - what ratio?

(6) The figures are all unclear and need significant improvement

Reviewer #3 (Public Review):

Summary:

How the microbial composition of the human body is influenced by and influences disease progression is an important topic. For people with COVID-19, symptomatic progression and deterioration can be difficult to predict. This manuscript attempts to associate the nasal and fecal microbiomes of COVID-19 patients with the severity of disease symptoms, with the goal of identifying microbial markers that can predict disease outcomes. However, the value of this work is held back by unclear methods and data presentation.

Strengths:

Analysis of microbiomes from two distinct anatomical locations and across three distinct patient groups is a substantial undertaking. How these microbiomes influence and are influenced by COVID-19 disease progression is an important question. In particular, the putative biomarker identified here could be of clinical value with additional research.

Weaknesses:

The methods and statistics used for several figures and comparisons are unclear or used in non-standard ways. For instance: the description of the Bray-Curtis test for Figure 1 is inaccurate and conflicts between the text and figure legend; the method used to compare the relative abundance of genera in Figure 2 is not clear; and it is not stated how the "total amount" of detected bacteria is inferred from the data presented in Figures 2C and 2D.

The description of results for Figure 1 is overstated or unclear for both the alpha diversity among disease groups and the overlap for nasal samples.

The most abundant phyla from nasal samples cumulatively account for less than 1% of abundance and it is unclear why this would be expected or how it compares to other work. Relatedly, the potential biological relevance of the very small proportional changes among phyla in the nasal samples is also not clear.

There is no real discussion of how the identified biomarkers might work in practice. While some microbes are detected in one condition but not others, it is unclear whether these organisms are expected to already exist below the detection threshold and then increase in abundance along with disease severity, or if they are picked up from the environment. For instance, would the presence of these 'severe' - associated microbes in patients with mild or moderate disease justify additional treatment to prevent disease progression?

The authors use the term "nasopharyngeal-faecal axis", but there is no substantial discussion of how these two microbiomes interact to influence disease progression, or how they are jointly affected to yield useful biomarkers. With one exception, correlation values between nasal and fecal microbes range from negligible to modest. It is unclear, then, how much parallel influence disease has on these microbiomes.

Author Response

eLife assessment

We appreciate the assessment carried out by the editorial team at eLife. Therefore, we plan to review the methods section in order to make the statistical analysis more comprehensible for each of the displayed figures.

Public reviews

Reviewer 1

We would like to express our gratitude to Reviewer 1 for providing a thorough summary of our work and highlighting its strengths. With regards to the weaknesses, we are committed to improve the manuscript by performing the necessary changes. First, we will specify the exact p-value in all cases.

Regarding the discussion section, we acknowledge the feedback regarding its potential confusion. In line with the reviewer's suggestion, we will reduce the literature review and highlight our findings.

Finally, for the preprint we did not include cofounders such as HIV infection and ethnicity as our study population did not exhibit viral infections and comprised only Hispanic individuals. We will make a more thorough description of the population of study and address these characteristics explicitly in both the methods section and the initial part of the results.

Reviewer 2

We appreciate and thank reviewer 2 for the commentaries. Although it is true that several papers have described the role of microbiome in COVID-19 severity, we firmly believe that our current work stands out.

There is not much information related to this association in mediterranean countries, especially in the south of Spain. In addition, most of the studies only describe microbiota composition in stool or nasopharyngeal samples separately, without investigating any potential relationships between them as we do.

(1) We agree with the reviewer idea of a limited sample size. We faced the challenge of collecting the samples during the peak of COVID-19 pandemia. Thus, doctors and nurses were overwhelmed and not always available for carrying out patient recruitment following the inclusion criteria. Despite these constraints, we ensured that all included samples met our specified inclusion criteria and were from subjects with confirmed symptomatology.

In addition, our main goal was to identify whether severity of the disease could be assessed through microbiota composition. Therefore we did not include a healthy group. Despite not having a large N, our results should be reproducible as they are supported by statistical analysis.

(2) We thank reviewer commentary, and since our original sentence may have lacked clarity, we intend to modify it to ensure it conveys the intended meaning more effectively.

Nonetheless, we remain confident in the significance of our findings. Not only have we found correlation between microbiota and COVID severity, but we have also described how specific bacteria from each condition is associated with key biochemical parameters of clinical COVID infection.

(3) We appreciate the feedback provided by the reviewer. In this case, we have performed 16S analysis due to its cost-effectiveness compared to metagenomic approaches. Furthermore, 16S analysis has undergone refinements that ensure comprehensive coverage and depth, along with standardized analysis protocols. Unlike 16S, metagenomic approaches lack software tools such as QIIME that facilitate standardization of analysis and, thus, reduce reproducibility of results.

(4) We sincerely appreciate this insightful suggestion. simply listing associations between both microbiomes and COVID-19 severity could not be enough, we intend to discuss how microbiota composition may be linked to the mechanisms underlying COVID-19 pathogenesis in our discussion.

(5) We are grateful for the constructive criticism and intend to rewrite our abstract to enhance clarity. Additionally, we will thoroughly review all figures and their descriptions to ensure accuracy and comprehensibility.

Reviewer 3

We acknowledge the annotations made by reviewer 3 and are committed to addressing all identified weaknesses to enhance the quality of our work. Our idea is to modify the methods section and figures to make them easier to understand.

Specifically, in the case of Figure 1, we recognize an error in the description of the Bray-Curtis test. We appreciate the commentary and we will make the necessary changes. Moreover, there is another observation related to Figure 1 description. We are going to modify it in order to gain accuracy.

For figure 2 we are planning to add a supplementary table showing the abundance of detected genus. Nevermind, we will also update the manuscript text to provide clarification on how we obtained this result.Regarding the clarification about "1% abundance," we want to emphasize that we are referring to relative abundance, where 1 represents 100%. To avoid confusion, we will explicitly state this in both the methods section and figure descriptions. Besides, it is true that the statistical test employed for the analysis is not mentioned in the figure description and we recognize that the image may be difficult to interpret. Therefore, we will modify the text and a supplementary table displaying the abundance and p values is going to be added.

Furthermore, we agree with the reviewer's suggestion to investigate whether the bacteria identified as potential biomarkers for each condition are specific to their respective severity index or if there is a threshold. Thus, we will reanalyze the data and include a supplementary table with the abundance of each biomarker for each condition. We will also place greater emphasis on these results in our discussion.

Finally, in response to the reviewer's suggestion, we are going to go through the nasopharyngeal-fecal axis part in the discussion. It is well described that COVID-19 induces a dysbiosis in both microbiomes.

Consequently, we understand that the ratio we have described could be an interesting tool for assessing COVID severity development as it considers alterations in both environments. However, we acknowledge that there may be room for improvement in clarifying the significance of this intriguing finding and its implications.