Viable bacterial colonization is highly limited in the human intestine in utero
Authors: V F Mendoza
T D Burt
J Halkias
E M Fukui
K E Fujimura
E Rackaityte
C Hayzelden
S V Lynch
E D Crawford
Journal: Nature Medicine (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Mucosal immunity; fetal intestine; microbes; meconium; scanning electron microscopy
Affiliations: Division of Gastroenterology, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA., Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA., Division of Neonatology, Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA., Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA., College of Science and Engineering, San Francisco State University, San Francisco, CA, USA., Chan Zuckerberg Biohub, San Francisco, CA, USA., Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA., Genentech, South San Francisco, CA, USA., Duke University School of Medicine, Durham, NC, USA., Division of Gastroenterology, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA. .

Abstract:  Mucosal immunity develops in the human fetal intestine by 11-14 weeks of gestation, yet whether viable microbes exist in utero and interact with the intestinal immune system is unknown. Bacteria-like morphology was identified in pockets of human fetal meconium at mid-gestation by scanning electron microscopy (n = 4), and a sparse bacterial signal was detected by 16S rRNA sequencing (n = 40 of 50) compared to environmental controls (n = 87). Eighteen taxa were enriched in fetal meconium, with Micrococcaceae (n = 9) and Lactobacillus (n = 6) the most abundant. Fetal intestines dominated by Micrococcaceae exhibited distinct patterns of T cell composition and epithelial transcription. Fetal Micrococcus luteus, isolated only in the presence of monocytes, grew on placental hormones, remained viable within antigen presenting cells, limited inflammation ex vivo and possessed genomic features linked with survival in the fetus. Thus, viable bacteria are highly limited in the fetal intestine at mid-gestation, although strains with immunomodulatory capacity are detected in subsets of specimens. 

Retracted: No

PubPeer comments:
 Neither *Lactobacillus* nor *Micrococcus* could be cultured from *Micrococcus*-enriched meconium using traditional selective medium. Using cRPMI medium with THP1 monocytes, suddenly *Micrococcus* and the following bacteria were cultured: *Pseudomonas*, *Stenotrophomonas*, *Microbacterium* (Supplementary Table 5). All four genera are known members of the kitome : https: bmcbiol.biomedcentral.com articles 10.1186 s12915-014-0087-z tables 1 , 
 Thank you for reading and bringing this comment to our attention. In our view, the two statements that you highlight are not in contradiction. We were unable to isolate Micrococcus and Lactobacillus using traditional selective media for these taxa, only when we mimicked the fetal environment by the addition of monocytes was Micrococcus culturable. We isolated a few other species under these conditions that you also mention, which we list in Supplementary Table 5. We thank you for citing Salter et al, it is a study we considered when designing ours. For one, it prompted us to avoid using commercial kits for our extractions (see methods). We additionally isolated from samples that were in parallel preserved and never encountered lab buffers for extraction (Supplementary Figure 1). Finally, in our article we state:  Whether bacteria are present in utero is contentious because of the inherent limitations of molecular methods that are commonly used to identify bacteria in low-burden environments. [Background noise and false positives in technical negative controls are common when bacterial burden is extremely low] , therefore simple removal of all taxa detected in these controls is not deemed appropriate. In our study, despite improving the current molecular methods to boost bacterial signal (Methods), 16S rRNA sequencing data remained noisy and identified a sparse bacterial signal that was indistinguishable from procedural or fetal kidney controls in the majority of samples (70%). Using a taxon-filtering approach that focused on the signal detected in the majority of negative controls led to the identification of a Micrococcus taxon as one of a small number of discriminant taxa enriched in meconium samples. With these molecular data as a guide for fetal bacterial species isolation, we subsequently cultured M. luteus from parallel preserved samples that never encountered extraction buffers during the course of sample processing. The molecular bacterial signal was proportionately more sparse than the signal identified by microscopy, which indicates that our removal of contaminant taxa may have suppressed a broader bacterial signal and supports previous reports of false-positive classification of contaminant taxa in buffer controls. Our molecular data identified only 17 additional taxa not attributable to contamination in our cohort of 50 fetal intestinal specimens, suggesting that conditions in the fetal gut highly limit bacteria, the mechanisms of which warrant further investigation. While there is debate regarding the best methods to address contamination in low-burden bacterial environments, our data suggest that current molecular methods alone are insufficient to support or reject the in utero sterility hypothesis. By combining molecular bacterial detection, immune correlates, microscopy, strain isolation and ex vivo experiments, our study provides direct and indirect evidence for the presence of sparse but viable bacteria in the human fetal intestine at mid-gestation with the capacity to limit inflammatory potential by fetal immune T cell populations. It is possible that the bacterial signal identified may arise from contamination from a source not investigated in this study, yet in our judgment, the corroborating evidence suggests that restriction of bacterial entry into the human fetal intestine is not absolute.  We propose to our readers to consider the data presented as a whole and we welcome alternative interpretations. There is much more work to be done to fully resolve this question and we are eager to read the work of others in this field. , 
 I believe that the authors of this paper did **not** do enough to show that they didn t write this entire paper about kit contaminants. I would have done the following experiments to help support the claims of this paper, **especially since these claims are highly controversial and would be expected to be met with criticism by others in the field and they are using so many precious human tissue samples**: 1. Grow several isolates of M. luteus (which is easily grown on rich media) from supposed meconium-originating cells and also from the samplers, lab reagents, etc. and compare their genomes by simply aligning reads to a reference genome and comparing SNVs. If they are contaminants, you would not expect them to be related to each other by fewer than a hand-full of SNVs by chance. 2. Explain why every other EM image of M. luteus online shows structures several time smaller than those in your EM( 1um as opposed to several um in this paper). You should mount and image a dilute culture of pure M.luteus cells to show that you end up with similarly sized structures, because all the EM in thisa paper is doing is showing that there are bacteria-shaped things, but not even that they are the right size. one could also imagine using FISH or immunogold, but at least showing some support for the EM would be useful. 3. a. I think the growth curves in Fig. 3 do little support that their isolate is  adapted to the fetal environment if they don t even use a reference strain as a control.... all you are doing is showing that a very common human commensal is able to grow in some given condition, not that it is specially  adapted . b. Adapted is a wholly irresponsible word to use in the title of figure 3 if you aren t actually going to demonstrate evidence for adaptation in any of the panels except C (which still only uses lab-adapted reference strains from ATCC, which is almost certainly less able to live APCs by chance and the human isolated controls i mentioned would have been the appropriate choice). While these are my main gripes from my first pass through the paper and it isn t exhaustive, it shows my train of thinking. I would like to see a followup with this lab using appropriate controls and where the experiments which take into account the within-species diversity of M. luteus wherein you can t expect an ATCC strain serve as a null model in all your experiments. , 
 Were sample IDs assigned sequentially? From Supplementary Table 2 it seems as though procedural kidney swab controls were performed only from sample ID 1633 onwards and that after these controls began to be collected OTU10 (Micrococcus) was only detected in 1 sample. Were any other collection procedures changed when collection of these negative controls was initiated? Also, I would like to know whether the samples shown in SEM images correspond to any other data-were they sequenced? cultured? ,