Gut bacteria are rarely shared by co-hospitalized premature infants, regardless of necrotizing enterocolitis development

Thank you for sending your work entitled “Gut bacteria are rarely shared by co-hospitalized premature infants, regardless of necrotizing enterocolitis development” for consideration at eLife. Your article has been favorably evaluated by Detlef Weigel (Senior editor), Roberto Kolter (Reviewing editor), and two reviewers.

The Reviewing editor and the two reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

You present your work detailing the microbiota of the feces in premature infants with and without necrotizing enterocolitis (NEC) using metagenomic methods. Overall we found that the findings presented are a very important contribution to the field. While the fact that the authors did not find of any commonality of microorganisms that contribute to NEC is somewhat disappointing, the finding of the unique microbiota at a strain level for most infants in this study will shape the way scientists and physicians will think about disease pathogenesis and gut colonization in these infants.

The manuscript presents a large amount of important work, but they are currently presented as a loose compilation of findings. As it stands, one comes away feeling that less has been accomplished than is actually the case. Thus, the authors should make every effort to revise the written presentation so as to generate a text that flows better and makes a compelling case for the importance of the findings, despite the current lack of a common thread leading to NEC. In revising the text, the authors should pay particular attention to responding the following major concerns, some of which ask for analyses that may indeed provide interesting insights:

1) What were the strain level differences between closely related taxa? The suggested reasons for the high level of unique diversity between infants are compelling, but we would like to understand how different these strains are? Are we talking major rearrangements, or SNP variance? Even with partial to near-complete fragments the authors should be able to start to think about this. This would help to narrow down which of the several possibilities provided by the team are likely. Do any of the differences seem likely to have occurred due to local niche differentiation as a result of host genetics? Might it be possible to generate a figure that shows, perhaps by alignment, the degree of similarity between the different strains of the same species between different infant guts?

2) While there is no particular suite of taxa that are always present with NEC, is it possible that Random Forest could be used to determine the predictive potential of a subset of genomic strains for NEC, or other health statistics that were also collected?

3) Figure 1 is not well explained in the manuscript. While it is useful to see an actual timeline with the dates of the samples, the authors don't really make a correlation between bacteria per gram of feces and anything else in the manuscript. Also, there should be some text in the manuscript detailing the time points sampled for these infants.

4) The authors’ method is presented as a two-week protocol and authors claim that this is 'clinically relevant time scale.' However, in the case of NEC, 12-14 days is in fact not clinically relevant. Neither the time frame nor the amount of labor involved in producing these types of results can be considered clinically relevant. There is the possibility of the current analyses to be eventually modified to become useful in clinical practice, but that would require considerable modifications. The authors do not provide any description as to how the protocols would have to be modified to actually make them clinically relevant, as two weeks for a diagnostic test for NEC would be of no utility in the diagnosis or treatment of these patients. Thus, the authors should revise the manuscript to indicate that their current protocols are in fact not really clinically relevant and that modifications would have to be developed to be useful in diagnostics.

1) What were the strain level differences between closely related taxa? The suggested reasons for the high level of unique diversity between infants are compelling, but we would like to understand how different these strains are? Are we talking major rearrangements, or SNP variance? Even with partial to near-complete fragments the authors should be able to start to think about this. This would help to narrow down which of the several possibilities provided by the team are likely. Do any of the differences seem likely to have occurred due to local niche differentiation as a result of host genetics? Might it be possible to generate a figure that shows, perhaps by alignment, the degree of similarity between the different strains of the same species between different infant guts?

We thank the reviewers for this important comment. We have added a new figure and figure supplement (Figure 6, Figure 6–figure supplement 1) that provide a visualization of differences between strains for Enterococcus faecalis (a highly prevalent species) and Clostridium paraputrificum (one of the rare species in which one genotype was shared among some of the infants). Differences were visualized by mapping reads from various samples from which different strains were recovered to a specific scaffold from one of these assemblies, and constructing a multiple alignment of the consensus sequences derived from each mapping. In the case of E. faecalis we selected a 1 Mbp scaffold recovered from infant 9 and constituting a third of the genome. This view shows that most differences can be attributed to SNP and small indels, but that those are present throughout the sequence (∼90% identity for most strain pairs in the multiple alignment), suggesting that most sequence pairs did not diverge very recently. A few larger indel regions (20-30 Kbp) were detected and contained among other things a sucrose metabolism operon, mobile elements and genes related to Fe-S protein biogenesis. However, some strains (specifically two pairs of strains, the one in infants #3 and early samples of infant #5, and the one in infants #2 and #7) were much more similar to each other, with very few detected differences across the compared sequence. Judging by the small extent of differences and their type (e.g., a prophage insertion in infants #2 and #7), it is possible that differentiation between those closely related strains occurred very recently, perhaps even within the host, though not necessarily due to host genetics.

2) While there is no particular suite of taxa that are always present with NEC, is it possible that Random Forest could be used to determine the predictive potential of a subset of genomic strains for NEC, or other health statistics that were also collected?

While a predictive model is definitely a holy grail in the study of NEC, given the small number of studied cases so far and the large number of potentially contributing factors, a reliable model that predicts NEC development without over-fitting cannot be constructed at this point. However, accumulation of similar data may ultimately enable the construction of such models. We are in the process of acquiring additional data, and hope that we will be able to present such models in the future. Presently however, we would like to note that current data does not show any strong correlation between NEC and any of the health statistics collected. We now discuss this issue in the main text.

3) Figure 1 is not well explained in the manuscript. While it is useful to see an actual timeline with the dates of the samples, the authors don't really make a correlation between bacteria per gram of feces and anything else in the manuscript. Also, there should be some text in the manuscript detailing the time points sampled for these infants.

We thank the reviewers for drawing our attention to this issue. Discussion of these results was inadvertently left out. We now discuss the strategy for the sampling schedule shown in this figure. In addition, we note that quantification of the bacterial load in each sample was in general agreement with previous measurements in full-term infants of similar postnatal ages (De Leoz et al. 2014), and that the measured variation in the number of microbes per gram feces did not exceed a 100 fold across all samples. Finally, we state that we did not observe a consistent trend of change in bacterial load prior to or following diagnosis of NEC.

4) The authors’ method is presented as a two-week protocol and authors claim that this is 'clinically relevant time scale.' However, in the case of NEC, 12-14 days is in fact not clinically relevant. Neither the time frame nor the amount of labor involved in producing these types of results can be considered clinically relevant. There is the possibility of the current analyses to be eventually modified to become useful in clinical practice, but that would require considerable modifications. The authors do not provide any description as to how the protocols would have to be modified to actually make them clinically relevant, as two weeks for a diagnostic test for NEC would be of no utility in the diagnosis or treatment of these patients. Thus, the authors should revise the manuscript to indicate that their current protocols are in fact not really clinically relevant and that modifications would have to be developed to be useful in diagnostics.

The reviewers are correct by saying that the current timescale of our analyses is not relevant for diagnosis and treatment of NEC cases. When we referred to clinical relevance, we meant to suggest that this timescale could be relevant in other, less acute, clinical situations where long-term health problems are involved. In the future, as technology and algorithm improvement significantly reduce these timescales, this type of analyses could be made possible also for real-time management of acute illnesses like NEC. We have now clarified this in the text, and we hope that the reviewers will find our re-phrasing of this section satisfactory.