COMMENTARY

Does a prenatal bacterial exist?

M Hornef1 and J Penders2

THE CONCEPT OF A PRENATAL the establishment of the neonate’s own meconium samples of 21 healthy human microbiota.4 Recently, maternal-fetal neonates born by either vaginal delivery The recent technical progress and enor- transmission of commensal or caesarean section and cultured mous efforts to unravel the manifold and the existence of a placental micro- bacteria of the genera Staphylococcus, interactions of the microbiota with the biome have been suggested.5–10 Coloni- Enterococcus, Streptococcus, Leuconos- host’s organism have provided striking zation of the healthy placental and/or toc, Bifidobacterium, Rothia, and unforeseen insights. This work fetal tissue with a diverse group of but also of the Klebsiella, assigns the microbiota a central role metabolically active bacteria would; Enterobacter and coli.6 in human health and has identified novel however, fundamentally challenge our Again, oral administration of the labeled strategies to prevent and fight diseases in current thinking of the development of E. faecium strain to pregnant mice led to the future. One particular aspect of this the fetus within a sterile, protected the detection in meconium samples.6 work has been the early colonization of environment. It would require new They concluded the presence of the newborn and a strong influence of concepts to explain how bacteria can ‘‘mother-to-child transmission’’ before maternal sources on the developing persist within host tissue but remain birth. Three other groups described the microbiota of the neonate.1,2 Birth, or anatomically restricted to prevent sys- PCR-based detection of bacteria in more accurately rupture of the amniotic temic spread within the fetal organism placental tissue. Rautava et al. detected membranes that surround the embryo and how preterm birth, a condition bacterial DNA mainly from the genus and separate it physically from the lumen causally linked to bacterial infection of Lactobacillus as well as the mostly of the uterus first exposes the neonate to the amniotic tissue, is prevented. It obligate anaerobic growing genera Bifi- the environment and is generally con- would also raise important questions dobacterium, Bacteroides and Clostri- sidered the start of the microbiota on the origin, composition and stability dium in 29 of 29 placental samples establishment. More recently, the exis- of the placental microbiome and its after elective cesarean section.7 The tence of a placental microbiome, and influence on the developing host and group of Versalovic performed a thus maternal-fetal transmission of postnatal microbiome. metagenomic approach on placental and microbial coloni- In support of the concept of a prenatal specimen collected under sterile condi- zation of the fetal organism before birth microbiome, Jime´nez et al. reported on tions from 320 individuals after vaginal has been suggested. This commentary the cultural detection of low numbers of delivery or cesarean section and critically discusses the available data. Enterococcus faecium, Staphylococcus described a low-abundance microbiome It is generally believed that the fetus in epidermidis and Propionibacterium including the phyla , Tener- utero develops in the absence of viable acnes from human cord blood samples icutes, Proteobacteria, Bacteroides and microorganisms. This is consistent with after elective cesarean section.5 This Fusobacteria.8 A recent study by Bassols the observation that cesarean section- analysis was complemented by a mouse et al., examined placental tissue of 22 born rodents can serve to generate germ- study in which they administered a vaginally delivered neonates from free animals.3 Only with rupture of genetically labeled human E. faecium mothers with or without gestational membranes and passage through the isolate orally to pregnant mice and diabetes aseptically collected and pre- birth canal, the newborn becomes reported detection of this strain from pared after childbirth in the delivery or exposed to colonized maternal body cultures of amniotic fluid.5 A subsequent operating room.9 PCR and 16S rDNA surfaces and the environment initiating study from the same group analyzed sequencing revealed the presence of

1Institute of Medical , RWTH University Hospital, Aachen, Germany and 2Department of Medical Microbiology, NUTRIM School of Nutrition and Translational Research in Metabolism & Caphri School for Public Health and Primary Care, Maastricht University Medical Centre, Maastricht, The Netherlands. Correspondence: MW Hornef ([email protected]) Published online 25 January 2017. doi:10.1038/mi.2016.141

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Proteobacteria, , Firmi- pipet tips and (even commercial) by PCR and culture and significant cutes, and .9 Placental reagents such as spin columns or differences in the main taxa of the tissue of women with gestational diabetes enzymes representing a source for false placental microbiome were described exhibited a distinct microbiota profile positive results. This problem is well in three other reports.8–10,13 which was amongst others characterized known to medical microbiologists or Additionally, transient bacteremia, by a lower relative abundance in the forensic scientists who are frequently i.e., the presence of viable bacteria in Pseudomonadales order and Acineto- confronted with the situation to judge on blood is occasionally observed in the bacter genus and an increased the value and meaning of a positive PCR healthy host. For example, bacteria can abundance in the Lachnospiraceae, signal. Of note, this problem can almost be cultured from blood samples after Coriobacteriaceae and Bradyrizobiaceae be neglected when studying densely dental hygiene.14 Similarly, mechanical families and the genera Escherichia and populated ecological niches such as manipulation of heavily colonized geni- Parabacteroides.9 Both PCR- and cul- the enteric microbiota but becomes a tal body surfaces as it occurs during ture-based methods were used by the critical issue when studying samples with vaginal delivery or the abdominal skin group of Salminen to examine placental low bacterial density. Therefore, exten- incision during cesarean section is tissue and amniotic fluid of 15 electively sive technical precautions during sample expected to facilitate bacterial entry into cesarean section delivered human new- acquisition and processing, the imple- the maternal bloodstream.15 Although borns. 16S rDNA sequencing detected a mentation of adequate internal controls bacteria are rapidly eliminated by serum- "low-richness, low-diversity" bacterial at all steps during sample preparation derived or cellular host defense mechan- composition with a predominance of and analysis and a critical interpretation isms in the healthy host, this can explain Proteobacteria.10 Culture mainly of the results are required. Previous the occasional detection of viable bac- revealed bacteria of the genera Staphy- studies did not incorporate internal teria at low-abundance in blood and lococcus and Propionibacterium (but controls or only at some stages during tissue samples including the heavily notably not the known human pathogen the experimental procedures (e.g., at perfused placental tissue. It can also S. aureus or any Proteobacteria). They DNA isolation and sequencing) but explain the detection of bacterial DNA at concluded the existence of ‘‘microbial not at sample collection.8,9 A bacterial sterile body sites. Although sensitive to transfer at the foeto-maternal interface’’ composition similar to that found in the nucleases, DNA remains detectable in and the presence of a ‘‘fetal micro- environment or the human oral or skin blood for a certain time and could reach biota’’.10 A recent PCR-based study on microbiota should raise doubts on the the placental tissue with the bloodstream early bacterial airway colonization origin. One study detected a similar or intracellularly in maternal phagocytic demonstrated the presence of a diverse microbial density and bacterial commu- migratory cells. and distinct lung microbiome at birth nity structure in placental specimen and Moreover, the detection of DNA by and also hypothesized fetal microbiota air swabs from the processing room.12 definition is unable to prove the exis- acquisition to explain their findings.11 Two other studies highlighted the simi- tence of a bona fide microbiota. The word Although maternal-fetal transfer of larity of the bacterial spectrum detected microbiota composed of the two greek commensal bacteria would reduce the in placental tissue with the human oral words mikros (small) and bios (life) environmental influence and could microbiome or the skin microbiota.7,8 describes a community of microorgan- thereby help to explain stable transmis- Another way to reveal a contamination isms that colonize an ecological niche sion of the microbiota over many gen- bias is to use different purification and refers to the presence of viable, erations, the currently available data methods with the same samples. A proliferating and metabolically active supporting the concept of a placental defined bacterial community is expected microorganisms. The detection of bac- or fetal microbiota at this point are highly to display a similar composition irre- terial DNA is not necessarily equivalent questionable for the following reasons. spective of the method and reagents to the presence of viable bacteria but used. A recent comparative 16S rDNA might be derived from microbial rem- DISCUSSION OF THE AVAILABLE based analysis of placental samples with nants at other body sites or the environ- EVIDENCE contamination controls employed two ment.7–10 Interestingly, one report The detection of DNA fragments by PCR different purification methods and failed described the detection of bacteria by using 25 or more cycles is extremely to provide evidence for a defined pla- light microscopy following Giemsa, sensitive. Minute amounts of even heav- cental spectrum of bacterial DNA.12 Gram, and Brown-Hopps staining of ily damaged DNA can already be Finally, a consistent detection of bacter- paraffin tissue sections of the basal plate detected. Deep sequencing with short ial DNA and a certain degree of similarity of the in 27% of pregnant reads as typically employed for NGS then in the bacterial composition between women without a significant link to reveals also very rare DNA molecules. different studies, i.e., a "core" micro- or preterm birth.16 The problem is that DNA of microbial biome would be postulated. However, Strikingly, the authors highlighted the origin is ubiquitous in our environment one study reported that all 16 amniotic intracellular localization of the detected and contaminates our laboratory equip- fluid samples obtained from healthy bacteria reminiscent of invasive infection ment including plastic tubes, forceps or pregnant females remained negative rather than colonization.16 Similar to the

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PCR results, this finding awaits con- local inflammatory response in placental steps during the experimental procedure firmation and the demonstration of tissue. Such an inflammatory response and the addition of prespecified and bacterial viability and diversity within has not been described and would be quantified bacterial mock communities individual samples. hard to reconcile with an ongoing to the examined samples will help to Many bacteria can multiply very . Chorioamnionitis, i.e., an reveal biases and identify batch rapidly. For example, E. coli can divide inflammation of the amniotic sac and effects.12,19 Subsequently, the demon- approximately every 20 min when sup- placental tissue is the most common stration of metabolically active and plied with sufficient nutrients. The con- known cause of preterm delivery.17 The proliferating diverse bacteria within the tact of the neonate’s nose, mouth, and bacteria associated with chorioamnioni- placental or fetal tissue will be required to anus with densely colonized maternal tis and preterm birth by cultural methods prove the existence of a viable, diverse body surfaces during passage through belong to the genera Ureaplasma, Myco- and unique bacterial community that the birth canal thus rapidly leads to plasma, Fusobacterium, Gardnerella, merits the term microbiota. Although colonized mucosal surfaces. Sample and Bacteroides.13 This spectrum of microbiota research has allowed unex- material acquired through the mouth cultured bacteria differs from what has pected and exciting insights during the or nose and meconium released through been detected by PCR-based methods.13 last years that undoubtedly will change the anus therefore does not represent a The reason is unclear but may again be our understanding of the host-microbial suitable sample material to proof the the fastidious growth or non-culturable relationship, we should keep a healthy presence of a fetal microbiota.6 Any state of individual bacterial species or dose of scepticism and critically view the contact of sample material with maternal DNA contamination. Some specialized current evidence on the existence of a body surfaces will inevitably contami- bacteria as well as endosym- prenatal bacterial microbiota. nate the material. bionts have adopted lifestyles that facil- Importantly, there is no need to The placenta attaches intimately to the itate long-term persistence within host postulate a placental or fetal microbiota uterus surface to facilitate efficient tissue with only minor or no inflamma- to explain the influence of microorgan- exchange of oxygen and nutrients tory response. Other bacterial species can isms or microbial components on cel- between the maternal and fetal circula- enter a stage of reduced metabolic and lular functions of the fetal tissue. Several tion. It does not exhibit a luminal space proliferative activity referred to as dor- reports have demonstrated systemic or internal mucosal surface and thus mant or "viable but not culturable", effects of the microbiota presumably colonizing microorganisms would have which might facilitate their prolonged through the dissemination of microbial to persist within host tissue and survive presence in tissue. The detection of constituents. The group of Jeff Weiser exposure to oxygen and humoral as well individual bacteria, however, would was the first to report an influence of gut as cellular host defense mechanisms. The not be referred to as a microbiota but microbiota-derived on bacteria suggested to contribute to a endosymbiosis or chronic infection neutrophil function in the bone mar- placental microbiota such as members of although the meaning of these terms row.20 Exposure of liver tissue to bacter- the genera Propionibacterium, Entero- may at some point overlap. ial DNA and lipopolysaccharide derived , Lactobacillus, Bacteroides, from the enteric microbiota and trans- Clostridium and coagulase-negative Sta- CONCLUSIONS AND FUTURE ported through the portal vein was phylococci (i.e., staphylococcal species RECOMMENDATIONS shown to promote non-alcoholic fatty other than S. aureus) are typical com- Altogether, there is no compelling evi- liver disease by the Flavell group.21 mensal bacteria and colonize the adult dence for the existence of a universal Pulendran and coworkers highlighted skin or intestinal tract at high density. mammalian placental or fetal microbiota the systemic effect of bacterial flagellin Infections with these bacteria are rarely to date. The available results, however, from as a natural adju- observed in the healthy host but warrant further analysis. Larger sample vant during intramuscular vaccine associated with foreign bodies (i.e., sizes, the simultaneous use of different administration.22 More recently, the intravenous catheters) or strong detection methods, the elimination of group of Andrew MacPherson demon- immunosuppression (i.e., after bone extracellular DNA prior to molecular strated antibody-dependent spread of marrow transplantation). It is question- microbial profiling and rigorous controls microbial constituents to the fetal organ- able whether these bacteria are able to for reagents and equipment at all steps ism during pregnancy.23 resist the cellular and serum-derived during sample processing and analysis antibacterial defense mechanisms and are required to establish a consistent ACKNOWLEDGMENTS 18 survive for prolonged time in healthy bacterial profile. The determination of This work has been supported by the Deutsche tissue. Other reported microorganisms the relative abundance of bacterial Forschungsgemeinschaft (Ho2236/8-1 and such as Staphylococcus aureus and groups should be preceded by an abso- Priority Program 1656 to M.W.H). are known to resist lute quantification of the bacterial load in DISCLOSURE the innate host defense. Yet they typically samples and controls (e.g., using a The authors declare no conflict of interest. cause pyogenic infections and would quantitative eubacterial PCR). Negative therefore be expected to induce a strong controls should be implemented at all & 2017 Society for Mucosal Immunology

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