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Reply Re: "Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community".

ABSTRACT: How and when a newborn is first colonized by microbes continues to be of great interest due to its broad implications on human health and disease. Payne et al. express their opinion about our recent study in which we characterized the virome and bacterial microbiota of amniotic fluid from 24 uncomplicated term pregnancies. We conducted additional validation studies and respond to their comments. We conclude that in amniotic fluid from healthy term pregnancies, the bacterial microbiota is indistinguishable from contamination controls, and there is no evidence of a core virome.

SUBMITTER: Lim ES

PROVIDER: S-EPMC6373012 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Reply Re: "Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community".

Lim Efrem S ES Rodriguez Cynthia C Holtz Lori R LR

Microbiome 20190212 1

How and when a newborn is first colonized by microbes continues to be of great interest due to its broad implications on human health and disease. Payne et al. express their opinion about our recent study in which we characterized the virome and bacterial microbiota of amniotic fluid from 24 uncomplicated term pregnancies. We conducted additional validation studies and respond to their comments. We conclude that in amniotic fluid from healthy term pregnancies, the bacterial microbiota is indisti ...[more]

PMID: 30755269

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Project description:Cerebrospinal fluid (CSF) circulating in the human central nervous system has long been considered aseptic in healthy individuals, because normally, the blood-brain barrier can protect against microbial invasions. However, this dogma has been called into question by several reports that microbes were identified in human brains, raising the question of whether there is a microbial community in the CSF of healthy individuals without neurological diseases. Here, we collected CSF samples and other samples, including one-to-one matched oral and skin swab samples (positive controls), from 23 pregnant women aged between 23 and 40 years. Normal saline samples (negative controls), sterile swabs, and extraction buffer samples (contamination controls) were also collected. Twelve of the CSF specimens were also used to evaluate the physiological activities of detected microbes. Metagenomic and metatranscriptomic sequencing was performed in these 116 specimens. A total of 620 nonredundant microbes were detected, which were dominated by bacteria (74.6%) and viruses (24.2%), while in CSF samples, metagenomic sequencing found only 26 nonredundant microbes, including one eukaryote, four bacteria, and 21 viruses (mostly bacteriophages). The beta diversity of microbes compared between CSF metagenomic samples and other types of samples (except negative controls) was significantly different from that of the CSF self-comparison. In addition, there was no active or viable microbe in the matched metagenomic and metatranscriptomic sequencing of CSF specimens after subtracting those also found in normal saline, DNA extraction buffer, and skin swab specimens. In conclusion, our results showed no strong evidence of a colonized microbial community present in the CSF of healthy individuals. IMPORTANCE The microbiome is prevalent throughout human bodies, with profound health implications. However, it remains unclear whether it is present and active in human CSF, which has been long considered aseptic due to the blood-brain barrier. Here, we applied unbiased metagenomic and metatranscriptomic sequencing to detect the presence of a microbiome in CSF collected from 23 pregnant women with matched controls. Analysis of 116 specimens found no strong evidence to support the presence of a colonized microbiome in CSF. Our findings will strengthen our understanding of the internal environment of the CSF in healthy people, which has strong implications for human health, especially for neurological infections and disorders, and will help further disease diagnostics, prevention, and therapeutics in clinical settings.

Project description:PROBLEM:Neutrophils are capable of performing phagocytosis, a primary mechanism for microbial killing. Intra-amniotic infection is characterized by an influx of neutrophils into the amniotic cavity. Herein, we investigated whether amniotic fluid neutrophils could phagocytize bacteria found in the amniotic cavity of women with intra-amniotic infection. METHODS:Amniotic fluid neutrophils from women with intra-amniotic infection were visualized by transmission electron microscopy (n=6). The phagocytic activity of amniotic fluid neutrophils from women with intra-amniotic infection and/or inflammation (n=10) or peripheral neutrophils from healthy individuals (controls, n=3) was tested using ex vivo phagocytosis assays coupled with live imaging. Phagocytosis by amniotic fluid neutrophils was also visualized by confocal microscopy (n=10) as well as scanning and transmission electron microscopy (n=5). RESULTS:(i) Intra-amniotic infection-related bacteria including cocci (eg Streptococcus agalactiae), bacilli (eg Bacteriodes fragilis and Prevotella spp.), and small bacteria without a cell wall (eg Ureaplasma urealyticum) were found inside of amniotic fluid neutrophils; (ii) peripheral neutrophils (controls) rapidly phagocytized S. agalactiae, U. urealyticum, Gardnerella vaginalis, and Escherichia coli; (iii) amniotic fluid neutrophils rapidly phagocytized S. agalactiae and G. vaginalis; and (iv) amniotic fluid neutrophils slowly phagocytized U. urealyticum and E. coli; yet, the process of phagocytosis of the genital mycoplasma was lengthier. CONCLUSION:Amniotic fluid neutrophils can phagocytize bacteria found in the amniotic cavity of women with intra-amniotic infection, namely S. agalactiae, U. urealyticum, G. vaginalis, and E. coli. Yet, differences in the rapidity of phagocytosis were observed among the studied microorganisms. These findings provide a host defense mechanism whereby amniotic fluid neutrophils can kill microbes invading the amniotic cavity.

Project description:BACKGROUND: Preterm prelabor rupture of membranes (PPROM) complicated by microbial invasion of the amniotic cavity (MIAC) leading to histological chorioamnionitis (HCA) significantly impacts perinatal morbidity. Unfortunately, no well-established tool for identifying PPROM patients threatened by these disorders is available. METHODOLOGY/PRINCIPAL FINDINGS: We performed an unbiased exploratory analysis of amniotic fluid proteome changes due to MIAC and HCA. From among the top five proteins that showed the most profound and significant change, we sought to confirm results concerning cathelicidin (P49913, CAMP_HUMAN), since an ELISA kit was readily available for this protein. In our exploratory proteomic study, cathelicidin showed a ?6-fold higher concentration in PPROM patients with confirmed MIAC and HCA. We verified significantly higher levels of cathelicidin in exploratory samples (women without both MIAC and HCA: median 1.4 ng/ml; women with both conditions confirmed: median 3.6 ng/ml; p?=?0.0003). A prospective replication cohort was used for independent validation and for assessment of cathelicidin potential to stratify women with MIAC leading to HCA from women in whom at least one of these conditions was ruled out. We confirmed the association of higher amniotic fluid cathelicidin levels with MIAC leading to HCA (the presence of both MIAC and HCA: median 3.1 ng/ml; other women: median 1.4 ng/ml; p<0.0001). A cathelicidin concentration of 4.0 ng/ml was found to be the best cut-off point for identifying PPROM women with both MIAC and HCA. When tested on the validation cohort, a sensitivity of 48%, a specificity of 90%, a likelihood ratio of 5.0, and an area under receiver-operating characteristic curve of 71% were achieved for identification of women with MIAC leading to HCA. CONCLUSIONS: Our multi-stage study suggests cathelicidin as a candidate marker that should be considered for a panel of amniotic fluid proteins permitting identification of PPROM women with MIAC leading to HCA.

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Reply Re: "Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community".

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Reply Re: "Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community".

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