Creation of a non-Western humanized gnotobiotic mouse model through the transplantation of rural African fecal microbiota

ABSTRACT Gut microbiota are increasingly being recognized as a contributing factor in the etiology of numerous diseases and as a potential determinant in the immune response to various treatments. Recent work has suggested that the suboptimal immunogenic response to vaccination in low- and middle-income countries may be associated with differences in the gut microbiome, which are known to be substantially different between Western and non-Western countries. However, insufficient consideration has been given to the characterization of non-Western microbiomes and their relationship with well-being and immunity. Humanized gnotobiotic mouse models have been used to better understand the causal associations between the gut microbiota and health outcomes but have largely been limited to the study of Western microbiota. Thus, we were interested in determining the applicability of gavage strategies used to humanize germ-free mice with Western microbiota to the humanization of germ-free mice with rural African fecal samples. Here, we assessed the impact of the number and frequency of gavages and the effect of a donor-matched diet on the colonization of Malian fecal microbiota in germ-free mice. One gavage was insufficient to provide a stable establishment of the Malian microbiome, whereas four weekly gavages resulted in a more consistent colonization of the human donor taxa. Interestingly, the donor-matched diet did not improve colonization over the fixed-formula, grain-based mouse chow. Subsequent phenotypic studies using African gut microbiota-humanized gnotobiotic mouse models will allow for a better understanding of the interaction between African gut microbiota and well-being and potentially aid in developing improved treatments for microbiota-dependent diseases in non-Western populations. IMPORTANCE There is increasing evidence that microbes residing within the intestines (gut microbiota) play important roles in the well-being of humans. Yet, there are considerable challenges in determining the specific role of gut microbiota in human diseases owing to the complexity of diverse internal and environmental factors that can contribute to diseases. Mice devoid of all microorganisms (germ-free mice) can be colonized with human stool samples to examine the specific contribution of the gut microbiota to a disease. These approaches have been primarily focused on stool samples obtained from individuals in Western countries. Thus, there is limited understanding as to whether the same methods used to colonize germ-free mice with stool from Western individuals would apply to the colonization of germ-free mice with stool from non-Western individuals. Here, we report the results from colonizing germ-free mice with stool samples of Malian children.

Introduction N/A Methods Place the Materials and Methods section after the Discussion per ASM guidelines.Line 116 -Is there a specific approval number/ID that can be cited here?Same with line 145.Line 121 -Should this be "scraped off"?Line 147 -Is there a reference available for these ingredients representing a Malian diet, even if general and/or secondary?This is relevant to line 194 as well.Line 175 -On what instrument was the sequence data generated?What specific protocols were used to process the sequence data?Minimally a reference which enumerates the process used.

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We would like to thank the reviewers for the me they have invested to carefully review our manuscript and the suppor ve comments they have provided.We would also like to thank the reviewers for the though ul and construc ve comments they have provided to us to improve the manuscript.Please find below our responses to each of these comments and how the manuscript has been revised.

Reviewer comments:
Reviewer #1 (Comments for the Author): I found the research interes ng and impac ul.

Comments:
Why the authors decided to mix the 5 fecal samples and did not used them as independent microbiome communi es?I think one combined sample is not representa ve of the varia on in the microbiome of the given popula on.What are the preexis ng condi ons of the donors?why the authors chose random pa ents?preexis ng condi ons have huge impact of diversity of gut microbes.

How mixing samples would interfere with interpreta on of the results?
The ideal condi ons for engra ment likely vary between fecal samples, even from those within the same community and those from the same donor over me.While it would be ideal to have a custom designed gavage procedure for each individual fecal sample, it would be unfeasible to do this with the number of samples required to obtain sta s cally significant results in human microbiome studies.Thus, we endeavored to design a generally applicable gavage procedure for non-Western fecal samples, or at the very least, for rural African samples, as this is our study popula on of interest.The use of a mixture of five samples was done with the hope of capturing a wider breath of the possible taxa present in this community than would be found within a single sample, while also crea ng a more representa ve sample that was less likely to be as influenced by deviancies present within the individual samples.While it is possible that the use of five individual samples (each given to five mice in each of the four separate groups) may have shown that some of the individual samples engra ed slightly be er with another gavage regimen, it is likely that the consensus regimen would have been the same.The choice of randomized pa ents was done as there is a lack of informa on regarding what cons tutes a "normal" microbiome in rural African popula on, thus choosing samples to capture the known variability based on the limited informa on that we currently possess likely would have inadvertently biased our fecal sample.
The fecal samples that were chosen came from children with no known pre-exis ng condi ons.The children that gave the fecal samples were enrolled into the larger study popula on in May 2011, therefore we have informa on obtained from their clinical visits for the three years prior to the start of our study arm of interest in 2014.The children previously went to the clinic for various injury-and illness-related issues (toe disloca on, heel wound, fever, chills, headache, diarrhea, vomi ng, conjunc vi s, tonsil infec on, ear inflamma on, pneumopathy, etc.).
However, none of the children were currently experiencing a symptoma c illness when they gave the samples that were used in our study.Two of the five samples came from children that were posi ve for Plasmodium falciparum by PCR and asymptoma c.Asymptoma c carriage of P. falciparum is very common in this popula on of children, and our current data shows that asymptoma c infec on with P. falciparum does not significantly change the gut microbiome in these children compared to the gut microbiome of uninfected children.
authors performed the microbiome recovery under anaerobic condi ons? why only saline was used? the authors could try to grow the collec on in vitro in a supplemented Gut microbiome growth media for 24-48 hrs before transferring to the mice to enhance the growth before these microbes got lost in the GIT of the recipient animal and give them be er chance of coloniza on.
The fecal gavage mixtures were not created under anaerobic condi ons.The samples were collected at a rural clinic without the capacity to manipulate the samples in anaerobic condi ons, thus the samples were previously exposed to aerobic condi ons before we received them.Furthermore, we observed excellent coloniza on of known strict anaerobes (such as Faecalibacterium prausnitzii), which suggests that the limited exposure of the samples to oxygen had minimal impact on the ability of these anaerobes to colonize the mice.The samples were made fresh at each me point immediately before being gavaged into the recipient mice.Since the microbes were spending a limited amount of me in the solu on before gavage, they did not need addi onal media supplementa on and we wanted to avoid modifying the original condi ons of the fecal sample as much as possible.For that reason, we also administered the fecal solu on directly, without an intermediate incuba on step.Growing the samples in vitro would likely enhance the prevalence of some of the microbes, but many microbes are unable to be cultured outside of the host and this culturing step would likely decrease the abundance of these microbes, if not completely eliminate their viable popula on from our sample, dras cally modifying the composi on of the gavage solu on administered to the mice.Addi onally, our lab, and many other labs, have observed that microbes ini ally present at very low amounts within the original sample can greatly expand once within a mouse and microbes present at rela vely elevated levels can be lost, thus the success of coloniza on is likely more dependent on the host condi ons than on their original abundances within the gavage sample.

Minor comments Wri ng; overall decent but some terms are not common such as "faithful coloniza on"
The two instances of "faithful coloniza on" was changed to more standard terms.

Reviewer #2 (Comments for the Author):
The manuscript aims to evaluate the op mal approach for establishing a humanized gnotobio c mouse model based on fecal samples from a non-Westernized, Malian popula on.The number and frequency of gavages, as well as the influence of mouse diet, were considered.The manuscript is informa ve, well wri en, and there has been clear a en on to detail in the study design and its communica on.

Abstract
Line 40 -This is a very general statement.For the abstract, consider being explicit regarding "mul ple" and "faithful".In the Introduc on, this wording is fine."mul ple" and "faithful" were changed to be more explicit.

Methods
Place the Materials and Methods sec on a er the Discussion per ASM guidelines.Materials and Methods were moved to a er the Discussion.
Line 116 -Is there a specific approval number/ID that can be cited here?Same with line 145.All relevant protocol and ID numbers for the clinical study can be found in the trial listed at ClinicalTrials.gov.This posi on of this reference in the relevant sec on has been moved slightly to make this clearer.The protocol numbers for the mouse work have been added.
Line 121 -Should this be "scraped off"?Scrapped was changed to be scraped.Line 147 -Is there a reference available for these ingredients represen ng a Malian diet, even if general and/or secondary?This is relevant to line 194 as well.The ingredients used were based on informal observa ons at the study site and the rela ve amounts were based on the average of nutri onal studies conducted in Mali, as indicated in the legend of Table S1.If needed, the emails from our collaborators regarding the ingredient composi on of the typical meals eaten at the study site can be included as text in the supplementary file.
Line 175 -On what instrument was the sequence data generated?What specific protocols were used to process the sequence data?Minimally a reference which enumerates the process used.All informa on regarding the instrument used to generate the sequence data and the protocols used to generate the OTU table can be found in the cita on listed for MVRSION.The cita on was added again in the Material and Methods sec on a er reference was made to the construc on of the OTU table by GTAC to clarify this point.Further clarifica on about the analysis of the data on our end has been included in the relevant sec on of the Materials and Methods.
Line 222, 270, 307, 457 -Use "mouse" or "murine" to be consistent?These words are synonyms in the instances in which they are used.
Line 235 -First appearance of "PCoA", thus write out here The defini on of PCoA (principal coordinate analysis) was added to the first appearance of PCoA.

Figures 4-6 -Consider merging some or all elements into a mul panel figure
This can be done if the journal would prefer this organiza on.Otherwise, we would prefer to keep the figures as they are.

Discussion
Line 407 -change "bacteria" to "bacterium" The word "bacteria" is in reference to F. prausnitzii as a taxa, not an individual bacterium.Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.
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Sincerely, Kevin Theis Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org Figure 1B-I, Figure 3 -It is not clear what the dotted lines in the figure panels represent Discussion Line 407 -change "bacteria" to "bacterium" Staff Comments: , legend of Figure S1, label of Figure S2, line 264)

Figure
Figure 1B-I, Figure 3 -It is not clear what the do ed lines in the figure panels represent The do ed line represents the level present in the input.This is indicated within the figure for Figure 1B-1 and Figure 3.
-23R1 (Creation of a non-Western humanized gnotobiotic mouse model through the transplantation of rural African fecal microbiota) Dear Dr. Nathan W. Schmidt: • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred