Change in the Gut Microbiome and Immunity by Lacticaseibacillus rhamnosus Probio-M9

ABSTRACT With the exploding growth of the global market for probiotics and the rapid awakening of public awareness to manage health by probiotic intervention, there is still an active debate about whether the consumption of probiotics is beneficial for nonpatients, which is due to the lack of systematic analysis based on time series multiomics data sets. In this study, we recruited 100 adults from a college in China and performed a random case-control study by using a probiotic (Lacticaseibacillus rhamnosus Probio-M9) as an intervention for 6 weeks, aiming to achieve a comprehensive evaluation and understanding of the beneficial effect of Probio-M9 consumption. By testing advanced blood immunity indicators, sequencing the gut microbiome, and profiling the gut metabolome at baseline and the end of the study, we found that although the probiotic intervention has a limited impact on the human immunity and the gut microbiome and metabolome, the associations between the immunity indicators and multiomics data were strengthened, and further analysis of the gut microbiome’s genetic variations revealed inhibited generation of single nucleotide variants (SNVs) by probiotic consumption. Taken together, our findings indicated an underestimated influence of the probiotic, not on altering the microbial composition but on strengthening the association between human immunity and commensal microbes and stabilizing the genetic variations of the gut microbiome. IMPORTANCE Although the global market for probiotics is growing explosively, there is still an active debate about whether the consumption of probiotics is beneficial for nonpatients. In this study, we recruited 100 adults from a college in China and performed 6 weeks of intervention for half of the volunteers. By analyzing the time series multiomics data in this study, we found that the probiotic intervention (i) has a limited effect on human immunity or the global structure of the gut microbiome and metabolome, (ii) can largely influence the correlation of the development between multiomics data and immunity, which was not able to be discovered by conventional differential abundance analysis, and (iii) can inhibit the generation of SNVs in the gut microbiome instead of promoting it.

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The manuscript titled 'Stabilization of the gut microbiome by Lacticaseibacillus rhamnosus Probio-M9' has explored whether probiotic ingestion is beneficial for healthy individuals as a prophylaxis, by conducting a systemic analysis with large datasets. These kind of studies should be encouraged, especially it shows both positive and negative results. However, there are some concerns.
1. It seems that there are two arms of this study: increasing the correlation between immune parameters and microbiota changes; decreasing the genetic variation of the gut microbiome. However, the title only highlights the latter.
2. One main cocern of this manuscript is its content of introduction and discussion. For introduction, there are two paragraphs, one about the importance and the application of the probiotics. One was a summary of this study (almost like the abstract). It is not sufficient to explain why it is necessary to test the effects of probiotics on host immunity and metabolism, for instance. Same problem with the discussion. One paragraph of findings summary. One paragraph about the study limitation. No specific discussion about results. Although there are no profound changes of immunity, there are some difference. For example, the results in Fig. 1c. Perhaps there are very few studies of probiotics in healthy humans, but the microbiome, immune parameters, their meaning have had been intenstively studied.
5. Does this manuscript contain a statistical analysis section (could not find one)? If not, it should.
The authors found that the probiotic intervention: has a limited effect on human immunity or the global structure of the gut microbiome and metabolome; can largely influence the correlation of the development between multi-omics data and immunity, which was not able to be discovered by conventional differential abundance analysis; and inhibits the generation of adaptive SNVs in the gut microbiome instead of promoting it. The authors indicated an underestimated influence of probiotics, not on altering the microbial composition but on strengthening the association between human immunity and commensal microbes and stabilizing the genetic variations of the gut microbiome.
The data analysis methods are correct. The English of the text is well written and well readable. The uniqueness of the text is more than 90% by AntiPlagiarism.NET.

One main concern of this manuscript is its content of introduction and discussion. For the introduction, there are two paragraphs, one about the importance and the application of probiotics. One was a summary of this study (almost like the abstract). It is not sufficient to explain why it is
necessary to test the effects of probiotics on host immunity and metabolism, for instance. Same problem with the discussion. One paragraph of findings summary. One paragraph about the study limitation. No specific discussion about the results. Although there are no profound changes of immunity, there are some differences. For example, the results in Fig. 1c. Perhaps there are very few studies of probiotics in healthy humans, but the microbiome, immune parameters, their meaning have had been intensively studied.

Response 4:
We thank Reviewer #4 for pointing out the issues. We have modified the introduction (the first paragraph below) and discussion sections (the second paragraph below) as below: (27-29). Although previous studies supported a beneficial effect of probiotic in enhance immunity against the common cold, which can reduce the incidence(30), duration(31), and symptoms(32) of the common cold (28, 33, 34), some reported that the effects of probiotic on the immune system and gastrointestinal symptoms in nonpatients are limited(29, 35). One approach by which probiotics impact the host immune system is through the microbial metabolism that arises from intestinal microbiota catabolism(36-39), e.g., functional metagenomic studies have identified the associations between host pro-inflammatory cytokines and microbial tryptophan and palmitoleic acid metabolic pathways (37, 38, 40-42). Moreover, previous studies have illustrated that the gut microbiota and metabolite activate the host immune system, thereby increasing the expression of endocrine peptides and promoting host metabolic homeostasis (37, 43, 44). In addition, the characterization of stable and changeable genetic components in the gut microbiome is crucial for further understanding the role of the gut microbiome in human health and phenotypic changes (45), which was rarely been investigated in probiotic studies. Therefore, a comprehensive evaluation of the role of gut microbiome, the change of microbial metabolites, and variations of their genetic structure in the beneficial effect of probiotic consumption is still urgently warranted due to the lack of systematic analysis of objective immunity indicators and time-series multi-omics data in previous studies." … "…Therefore, there is an urgent need in understanding the benefits of probiotics on nonpatients through immunity indicators, the gut microbiome, and microbial metabolism using time-series data in  (53,54). Although there is just slightly altered overall human immunity after the probiotic intervention compared to the placebo, six cytokines such as Eotaxin, IL-1α, IL-8, MCP-1, RANTES TNF-β, and one lymphocyte CD3+CD8+ T cells showed significant improvements within the normal range, which is in line with previous findings that the T lymphocytes and cytokines interleukin are activated after probiotic consumption (28,31,55,56). Complex ecological communities are generally thought to be more stable and resilient, and gut microbial diversity is often used as a proxy for human health (58-60). Even though six weeks of Lacticaseibacillus rhamnosus Probio-M9 consumption did not dramatically affect the overall gut microbiome and metabolites, it reduced the population heterogeneity of individuals in view of the multi-omics perspective. To better understand such impacts on the host, we innovatively evaluated the probiotic's effects by categorizing them into direct effects and indirect effects. As expected, we found a significantly underestimated indirect effect of probiotics on the other intestinal microorganisms, metabolites, and immune indicators, underling the intermediary effect of Probio-M9 that regulates intestinal microbes and microbial metabolites and eventually affect the host immune system."

line 60, please revise 'increasing immunity'
Response 5: We thank Reviewer #4 for this comment. We have revise 'increasing immunity' into 'enhanced immunity' 4. line193-194, it does not seem that the study involves immunity development investigation.

Response 6:
We thank Reviewer #4 for this comment and sorry for misleading. We have changed the subtitle to: "The underestimated influence of probiotic intake on the association between multi-omics data and immunity" to avoid the confusion of "immunity development" and "change of immunity related indexes".

Response 7:
We thank Reviewer #4 for this comment. We didn't provide an independent "Statistical analysis" section. Rather, we described those details in each section, e.g., the alpha diversity and beta diversity analysis methods in the "Alpha and beta diversity analysis" section; the differentially abundant species taxa identification in the "Differential abundance analysis" section. Now, we have added a "Statistical analysis" section in our revised manuscript, it reads: "The alpha diversity of bacterial composition, fungal composition, microbial function, and metabolites were measured by the Shannon index and calculated using the "Vegan" package in R 4.0.5. With the same package, Bray-Curtis dissimilarity was calculated to measure the beta diversity for the multi-omics data. The differences in BC dissimilarity were determined using a non-parametric multivariate analysis of variance (Permanova test) and visualized by the UMAP using R package "umap"(46). The convergence of microbial composition is measured by the within group BC