Prenatal versus Postnatal Initial Colonization of Healthy Neonates’ Colon Ecosystem by the Enterobacterium Escherichia coli

ABSTRACT The human colon is a microbial ecosystem whose initial bacterial colonization in neonates is an important step in establishing a beneficial microbiota for the body’s health. This study investigated the occurrence of viable culturable Escherichia coli in first-day meconium versus subsequent days’ stool to explore the prenatal versus postnatal initial colonization of the colon by E. coli in healthy neonates. E. coli occurrence was investigated on eosin-methylene blue (EMB) agar, followed by morphological and biochemical characterizations and phylogenetic analysis of 16S rRNA-encoding gene sequences. Viable culturable E. coli was not detected in meconium of healthy male or female neonates delivered either vaginally or by cesarean section. Neonates delivered surgically also showed no E. coli colonization on the second and third days, confirming postnatal colonization of the colon by this enterobacterium. E. coli’s initial colonization in the colon of neonates delivered vaginally occurred on the second day, which can be attributed to inoculation from the vaginal canal during delivery and, in comparison to the colonization in neonates delivered surgically, leads to the inference that the bacterium is not originally found in meconium. This study suggests no viability of the meconium microbiome in healthy neonates, possibly due to antimicrobial action in the prenatal colon’s meconium protecting babies’ gut from infection during delivery. IMPORTANCE The results of this study suggest that the initial postnatal colonization of neonates’ colon by beneficial bacteria is a naturally controlled process in which the prenatal colon’s meconium might play a role in protecting against infection of the babies’ gut during delivery.

The study reported the presence of viable culturable Escherichia coli in meconium and later fecal samples. Traditional culture methods and specific sanger sequencing were used to identify E.coli strains in healthy neonatal gut. Results showed that E.coli was not presence on meconium samples and C-section infant had a delay in E.coli detection compared to vaginal neonates. major comments: -Meconium sample is a sample that is collected <48h after birth, however, in terms of microbiota exposition, this matters. After 24h, the neonate is exposed to maternal skin, milk and oral microbes and also, family and environmental bacteria. Please, more detailed information is needed on sampling time.
-Did the authors check other culture media as LB and/or MacConkey? -Please, could you clarify if your method had a specific detection limit? there is a big difference between non detected and detected samples. The range is Non-detected and when the bacteria is present, the amount is aprox 1000000 CFU/gr. Could you detail the process and the technique? Does 10^6 the detection threshold? if yes, it is too high considering that meconium is a low biomass sample. -Please, include details and discussion on the problems related to the 16S fragment sequencing and the differentiation between E. coli/Salmonella/Shigella and also, the potential use of other genes as gyrB, atpD, infB, rpoB among others. - Figure 1 is not clear. Microbes had exponential growth and if data would be reported in log manner, the samples are more or less in same logaritmic unit (log 6) -please include a paragraph on limitations of the study.
-conclusion needs to be focused to the data obtained in the study. minor comments: -please, change "microflora" by "microbiota" -please, include the exact days where fecal samples were analyzed. The "subsequent days" is not sufficient information.
Reviewer #2 (Comments for the Author): This manuscript reports culture and 16S sequencing data for stool samples collected from human infants. The bacterial characterization is limited to E. Coli. The sample size is small. The work is of interest.
However, the final two bulleted "Highlights" in the supplemental file are not conclusions that can be drawn on the basis of the data presented in this manuscript.
It is not entirely clear, but it seems that the study design is cross-sectional. Is it correct that different infants are sampled at each time point presented.
More information about the demographics, diet (formula versus human milk), and antibiotic use for the participating infants needs to be provided to give the appropriate context for interpretation of the research results.
What is the standard of care for c-section deliveries at the hospital? Which antibiotics are used? How long could they be expected to be in the bloodstream of the mother and infant? Could that regimen affect the viability of microbes in the infant?
Detailed information about sample collection is required in the methods section. Were the samples collected from diapers? Or, was sampling done using swabs? By whom and when were the samples collected? Were samples stored at all before processing? If so, how?
Was regular Taq (line 135) not high fidelity polymerase used for preparation of the 16S genes for sequencing? If so, this could introduce errors in the sequences and should be listed as a limitation. This is especially true in light of the length of PCR product generated.
With respect to the collection of colonies for determination of E. Coli strains, please clarify if a single colony per infant was selected or how many colonies were sampled per infant for the sequencing.
The discussion section (and results section) could be reduced in length by removing the repeated statement of the same results that can found in many, many locations throughout the manuscript.
If you have not cited the work of Andreas Baumler in your discussion of the de-oxygenation of the gut and the role that plays in establishment of the gut microbiota, then please do.
In several places, it is suggested that the vagina might be the source of the E. Coli. However, it is more likely that fecal material would be the source of E. Coli.
Consider using the word "vaginal" instead of "normal" with reference to birth. Consider using the word, "sex" instead of the word, "gender." Staff Comments:

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Point-to-Point Response to Reviewers
Manuscript ID: Spectrum00379-21 The authors are grateful to the precious suggestions, constructive comments and careful corrections made by the two anonymous reviewers for further improvements of this paper. The manuscript has been revised according to the reviewers' comments.

Response to Reviewer #1
Reviewer comments: Reviewer #1 (Comments for the Author): The study reported the presence of viable culturable Escherichia coli in meconium and later fecal samples. Traditional culture methods and specific sanger sequencing were used to identify E. coli strains in healthy neonatal gut. Results showed that E.
coli was not presence on meconium samples and C-section infant had a delay in E.
coli detection compared to vaginal neonates. major comments: -Meconium sample is a sample that is collected <48h after birth, however, in terms of microbiota exposition, this matters. After 24h, the neonate is exposed to maternal skin, milk and oral microbes and also, family and environmental bacteria. Please, more detailed information is needed on sampling time.
Response to the reviewer comment: Done; Once first-pass meconium collected in first day after birth or subsequent days stool samples were obtained normally in diapers, they were sent to the bacteriology laboratory and directly used for examining the occurrence of E. coli and determining its CFU (Colony Forming Units). This was outlined in the revised form of the manuscript.
-Did the authors check other culture media as LB and/or MacConkey?
Response to the reviewer comment: Done, No E. coli or other enteric bacteria were detected in first-pass meconium on EMB, LB agar or MacConkey agar. The first-pass meconium also showed no viable lactic acid bacteria (LAB) on MRS agar medium in this study or in a previous study (Al-Balawi and Morsy, 2020) where LAB were detected in 2 nd day stool and above. This was outlined in the revised form of the manuscript.
-Please, could you clarify if your method had a specific detection limit? there is a big difference between non detected and detected samples. The range is Non-detected and when the bacteria is present, the amount is aprox 1000000 CFU/gr. Could you detail the process and the technique? Does 10^6 the detection threshold? if yes, it is too high considering that meconium is a low biomass sample.
Response to the reviewer comment: Done; Non detected in this study means no bacterial colonies at all (Zero colonies detected). This was outlined under table 1 in the revised form of the manuscript.
-Please, include details and discussion on the problems related to the 16S fragment sequencing and the differentiation between E. coli/Salmonella/Shigella and also, the potential use of other genes as gyrB, atpD, infB, rpoB among others.
Response to the reviewer comment: Done; Because of its high degree of conservation in closely related species (Spröer et al., 1999;Paradis et al., 2005) and its several copies within the genome (Paradis et al., 2005;Kembel et al., 2012;Větrovský and Baldrian, 2013;Louca et al., 2018;Ibal et al., 2019), it has been shown that gyrB, atpD, infB, rpoB genes sequence analysis is a useful alternative to 16S rRNA analysis for the classification of the enterobacteria Salmonella, Shigella and E. coli (Mollet et al., 1997;Hedegaard et al., 1999;Fukushima et al., 2002;Paradis et al., 2005) for exploring the phylogenetic relationships at the species level in particular of closely related ones (Fukushima et al., 2002). This was outlined in the coli colonized no baby of cesarean section surgical delivery neither male nor female up to babies of 3 days old, the bacterium sharply colonized the babies at 4 th day after birth and the CFU was slightly lower than that of normal birth at 5 th and 6 th day ( Fig.1). These results indicate a sharper colonization after inoculation from the surrounding environment of newborn babies of cesarean section surgical delivery which might be attributed to a more space for colonization due to late inoculation and to possibly a more suitability of colonization condition at this age of 5 and 6 days old.) -please include a paragraph on limitations of the study. -conclusion needs to be focused to the data obtained in the study.
Response to the reviewer comment: Done, revised.

Response to the reviewer comment: Done
-please, include the exact days where fecal samples were analyzed. The "subsequent days" is not sufficient information.
Response to the reviewer comment: Done; As mentioned above; Once first-pass meconium collected in first day after birth or subsequent days stool samples were obtained normally in diapers, they were sent to the bacteriology laboratory and directly used for examining the occurrence of E. coli and determining its CFU. This was outlined in the revised form of the manuscript.

Response to Reviewer #2
Reviewer #2 (Comments for the Author): This manuscript reports culture and 16S sequencing data for stool samples collected from human infants. The bacterial characterization is limited to E. coli. The sample size is small. The work is of interest.
However, the final two bulleted "Highlights" in the supplemental file are not conclusions that can be drawn on the basis of the data presented in this manuscript.
Response to the reviewer comment: Done; Highlights were revised.
It is not entirely clear, but it seems that the study design is cross-sectional. Is it correct that different infants are sampled at each time point presented.
Response to the reviewer comment: Done; Different neonates were chosen to have wide view of the initial colonization where healthy different neonates were selected randomly of age one day for obtaining the first-pass meconium while subsequent days stool samples were obtained from babies of age 2 to 6 days old. This was outlined in the revised form of the manuscript.
More information about the demographics, diet (formula versus human milk), and antibiotic use for the participating infants needs to be provided to give the appropriate context for interpretation of the research results.
Response to the reviewer comment: Done; All newborn babies' subjects in this study were chosen with normal breast feeding. Further future studies for comparing In several places, it is suggested that the vagina might be the source of the E. coli.
However, it is more likely that fecal material would be the source of E. coli.
Response to the reviewer comment: Done; As E. coli appeared slightly earlier in vaginal birth neonates than C-section ones, the vagina might be the source of inoculation of the neonates' gut by this bacterium during vaginal delivery. This was outlined clear in the revised form of the manuscript Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed information on submitting your revised paper are below.

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Reviewer #2 (Comments for the Author): Thank you for addressing most of my comments. However, there are still issues with this manuscript.
For instance, in lines 203-205 and again in lines 275-280, you mention the vagina as the original source of the E. Coli. However, during vaginal birth infants are exposed to maternal fecal matter --babies mouths actually are oriented toward the maternal rectum. Most women will poop a little during vaginal birth. Thus, this fecal matter may be the source of the E. Coli.
The phrase "healthy babies" still appears in this manuscript, but it seems that some of the infants may not have been healthy if I am interpreting your methods section correctly.
Furthermore, even with edits, bullets 5&6 (the last two) in "highlights" are not appropriate given the results reported in the manuscript.
Staff Comments:

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