PREreview of Commensal acidification of specific gut regions produces a protective priority effect against enteropathogenic bacterial infection

This review resulted from the graduate-level course "How to Read and Evaluate Scientific Papers and Preprints" from the University of São Paulo, which aimed to provide students with the opportunity to review scientific articles, develop critical and constructive discussions on the endless frontiers of knowledge, and understand the peer review process.

In this manuscript, the group led by Yang sought to investigate the relationship between strains of Lactobacillus plantarum and Serratia marcescens isolated from a wild Drosophila melanogaster fly and their effects on the health of D. melanogaster in germ-free conditions. Through a series of experiments, it was possible to observe that colonization by L. plantarum had a protective effect against S. marcescens on D. melanogaster health, elongating the flies’ lifespan. The mechanisms behind this effect were investigated, leading the authors to propose that L. plantarum had a role in acidification of the pH in different digestive tract regions, causing a priority effect that hampered S. marcescens colonization. This study has a clearly stated objective that is investigated through meaningful experiments. The findings presented by the group have relevance in their field, given the importance of D. melanogaster as a model organism for studies in microbiomes and the absence of studies investigating the mechanism behind the protective effects between strains. Therefore, this study could be the basis for understanding these relationships in other more complex models.

Major comments:

1. The authors state that they disregarded isolated strains of L. plantarum due to the biofilm formation associated with these strains, which would render them unsuitable for the intended tests. Therefore, even when isolating wild-type-associated strains, it was found that there is variability between the two different L. plantarum isolates in a single fly. One must wonder whether this also applies to different flies under the same conditions, under different conditions or even originating from different environmental populations. This indicates that the observed results can only be correlated with the specific strains used in the study. Nevertheless, the conclusions are presented in a generalized manner, as if the experiments conducted proved that the results reflect the behaviour of the entire species.

2. Another characteristic of the study that diminishes its possibility of generalizing its results is the absence of a control group with the ‘normal’ microbiome of D. melanogaster. While the importance of isolating two bacteria to gain a better understanding of their relationship is clear, the intricacy of the relationships between the components of the microbiome must also be taken into consideration. Given the issues raised above, it is suggested to either limit the conclusions to the specific observed scenario and to include more speculative interpretations in the discussion as indicators of possible new hypotheses, or promote the inclusion of the additional control group, broadening the validity of the study and making it even more meaningful.

3. As for the methodology used, most of the experiments chosen provided insights into the investigated problem, however, there are questions to be raised about the sensitivity of some of the experiments. The first three experiments focused on describing the interactions between L. plantarum and S. marcescens and provided logical insights into the mechanisms that explained the observed outcome, however, the methods chosen for the fourth experiment are questionable due to the low sensitivity of the pH measuring method.

4. As was previously mentioned, despite the clear narrative, we noticed that some conclusions are somehow overstated to the scope of the experiments performed. In order to avoid overstatements, the authors could employ more cautious terminology, such as “indicate” or “suggest”, given the conditional nature of the experiments. This would provide a more accurate representation of the findings and their limitations.

Minor comments

1. It would be interesting if the authors provided an explanation as to why the fly group colonized only by L. plantarum in the first experiment survived less than the group kept germ free, since the literature states that L. plantarum is a probiotic agent for D. melanogaster.

2. There are instances where italics are missing in the nomenclature in the “L. plantarum colonization diminishes S. marcescens abundance in the Drosophila gut” section.

Competing interests

The authors declare that they have no competing interests.