Is there a placental microbiota? A critical review and re-analysis of published placental microbiota datasets.

The existence of a placental microbiota is debated. The human placenta has historically been considered sterile and microbial colonization was associated with adverse pregnancy outcomes. Yet, recent DNA sequencing investigations reported a microbiota in typical human term placentas. However, this detected microbiota could represent background DNA or delivery-associated contamination. Using fifteen publicly available 16S rRNA gene datasets, existing data were uniformly re-analyzed with DADA2 to maximize comparability. While Amplicon Sequence Variants (ASVs) identified as Lactobacillus, a typical vaginal bacterium, were highly abundant and prevalent across studies, this prevalence disappeared after applying likely  DNA contaminant removal to placentas from term cesarean deliveries. A six-study sub-analysis targeting the 16S rRNA gene V4 hypervariable region demonstrated that bacterial profiles of placental samples and technical controls share principal bacterial ASVs and that placental samples clustered primarily by study origin and mode of delivery. Contemporary DNA-based evidence does not support the existence of a placental microbiota.ImportanceEarly-gestational microbial influences on human development are unclear. By applying DNA sequencing technologies to placental tissue, bacterial DNA signals were observed, leading some to conclude that a live bacterial placental microbiome exists in typical term pregnancy. However, the low-biomass nature of the proposed microbiome and high sensitivity of current DNA sequencing technologies indicate that the signal may alternatively derive from environmental or delivery-associated bacterial DNA contamination. Here we address these alternatives with a re-analysis of 16S rRNA gene sequencing data from 15 publicly available placental datasets. After identical DADA2 pipeline processing of the raw data, subanalyses were performed to control for mode of delivery and environmental DNA contamination. Both environment and mode of delivery profoundly influenced the bacterial DNA signal from term-delivered placentas. Aside from these contamination-associated signals, consistency was lacking across studies. Thus, placentas delivered at term are unlikely to be the original source of observed bacterial DNA signals.

The Vibrio cholerae Type Six Secretion System Is Dispensable for Colonization but Affects Pathogenesis and the Structure of Zebrafish Intestinal Microbiome.

Zebrafish (Danio rerio) are an attractive model organism for a variety of scientific studies, including host-microbe interactions. The organism is particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae, an intestinal pathogen. V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. While numerous studies have explored various aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms, few, if any, have examined how a V. cholerae infection alters the resident intestinal microbiome and the role of the type six secretion system (T6SS) in that process. In this study, 16S rRNA gene sequencing was utilized to investigate how strains of V. cholerae both with and without the T6SS alter the aforementioned microbial profiles following an infection. V. cholerae infection induced significant changes in the zebrafish intestinal microbiome, and while not necessary for colonization, the T6SS was important for inducing mucin secretion, a marker for diarrhea. Additional salient differences to the microbiome were observed based on the presence or absence of the T6SS in the V. cholerae utilized for challenging the zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiome to enable colonization and that the T6SS is important for pathogenesis induced by the examined V. cholerae strains. Furthermore, the presence or absence of T6SS differentially and significantly affected the composition and structure of the intestinal microbiome, with an increased abundance of other Vibrio bacteria observed in the absence of V. cholerae T6SS.

Vibrio cholerae Infection Induces Strain-Specific Modulation of the Zebrafish Intestinal Microbiome.

Zebrafish (Danio rerio) is an attractive model organism to use for an array of scientific studies, including host-microbe interactions. Zebrafish contain a core (i.e., consistently detected) intestinal microbiome consisting primarily of Proteobacteria. Furthermore, this core intestinal microbiome is plastic and can be significantly altered due to external factors. Zebrafish are particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae. As an intestinal pathogen, V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. Members of the resident intestinal microbial community likely must be reduced or eliminated by V. cholerae for colonization, and subsequent disease, to occur. Many studies have explored a variety of aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms but few have researched how a V. cholerae infection changes the resident intestinal microbiome. In this study, 16S rRNA gene sequencing was used to examine how five genetically diverse V. cholerae strains alter the intestinal microbiome following an infection. We found that V. cholerae colonization induced significant changes in the zebrafish intestinal microbiome. Notably, changes in the microbial profile were significantly different from each other, based on the particular strain of V. cholerae used to infect zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiota to enable colonization and that specific microbes that are targeted depend on the V. cholerae genotype.

Sneathia: an emerging pathogen in female reproductive disease and adverse perinatal outcomes.

Sneathia is an emerging pathogen implicated in adverse reproductive and perinatal outcomes. Although scarce, recent data suggest that vaginally residing Sneathia becomes pathogenic following its ascension into the upper urogenital tract, amniotic fluid, placenta, and foetal membranes. The role of Sneathia in women's health and disease is generally underappreciated because the cultivation of these bacteria is limited by their complex nutritional requirements, slow growth patterns, and anaerobic nature. For this reason, molecular methods are typically required for the detection and differential diagnosis of Sneathia infections. Here, we review the laboratory methods used for the diagnosis of Sneathia infections, the molecular mechanisms underlying its virulence, and its sensitivity to antibiotics. We further review the evidence of Sneathia's contributions to the pathogenesis of bacterial vaginosis, chorioamnionitis, preterm prelabour rupture of membranes, spontaneous preterm labour, stillbirth, maternal and neonatal sepsis, HIV infection, and cervical cancer. Collectively, growing evidence indicates that Sneathia represents an important yet underappreciated pathogen affecting the development and progression of several adverse clinical conditions diagnosed in pregnant women and their neonates, as well as in non-pregnant women.

Bacteria in the amniotic fluid without inflammation: early colonization vs. contamination.

OBJECTIVES: Intra-amniotic infection, defined by the presence of microorganisms in the amniotic cavity, is often accompanied by intra-amniotic inflammation. Occasionally, laboratories report the growth of bacteria or the presence of microbial nucleic acids in amniotic fluid in the absence of intra-amniotic inflammation. This study was conducted to determine the clinical significance of the presence of bacteria in amniotic fluid samples in the absence of intra-amniotic inflammation. METHODS: A retrospective cross-sectional study included 360 patients with preterm labor and intact membranes who underwent transabdominal amniocentesis for evaluation of the microbial state of the amniotic cavity as well as intra-amniotic inflammation. Cultivation techniques were used to isolate microorganisms, and broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was utilized to detect the nucleic acids of bacteria, viruses, and fungi. RESULTS: Patients whose amniotic fluid samples evinced microorganisms but did not indicate inflammation had a similar perinatal outcome to those without microorganisms or inflammation [amniocentesis-to-delivery interval (p=0.31), spontaneous preterm birth before 34 weeks (p=0.83), acute placental inflammatory lesions (p=1), and composite neonatal morbidity (p=0.8)]. CONCLUSIONS: The isolation of microorganisms from a sample of amniotic fluid in the absence of intra-amniotic inflammation is indicative of a benign condition, which most likely represents contamination of the specimen during the collection procedure or laboratory processing rather than early colonization or infection.

Microbial burden and inflammasome activation in amniotic fluid of patients with preterm prelabor rupture of membranes.

Background Intra-amniotic inflammation, which is associated with adverse pregnancy outcomes, can occur in the presence or absence of detectable microorganisms, and involves activation of the inflammasome. Intra-amniotic inflammasome activation has been reported in clinical chorioamnionitis at term and preterm labor with intact membranes, but it has not yet been investigated in women with preterm prelabor rupture of membranes (preterm PROM) in the presence/absence of detectable microorganisms. The aim of this study was to determine whether, among women with preterm PROM, there is an association between detectable microorganisms in amniotic fluid and intra-amniotic inflammation, and whether intra-amniotic inflammasome activation correlates with microbial burden. Methods Amniotic fluids from 59 cases of preterm PROM were examined for the presence/absence of microorganisms through culture and 16S ribosomal RNA (rRNA) gene quantitative real-time polymerase chain reaction (qPCR), and concentrations of interleukin-6 (IL-6) and ASC [apoptosis-associated spec-like protein containing a caspase recruitment domain (CARD)], an indicator of inflammasome activation, were determined. Results qPCR identified more microbe-positive amniotic fluids than culture. Greater than 50% of patients with a negative culture and high IL-6 concentration in amniotic fluid yielded a positive qPCR signal. ASC concentrations were greatest in patients with high qPCR signals and elevated IL-6 concentrations in amniotic fluid (i.e. intra-amniotic infection). ASC concentrations tended to increase in patients without detectable microorganisms but yet with elevated IL-6 concentrations (i.e. sterile intra-amniotic inflammation) compared to those without intra-amniotic inflammation. Conclusion qPCR is a valuable complement to microbiological culture for the detection of microorganisms in the amniotic cavity in women with preterm PROM, and microbial burden is associated with the severity of intra-amniotic inflammatory response, including inflammasome activation.

Differential Secretion of Inflammatory Cytokines by Human Trophoblasts in the Presence of Escherichia coli, Lactobacillus crispatus, and Lactobacillus jensenii.

INTRODUCTION: The existence of a placental microbiome would require a non-antagonistic relationship between potentially colonizing bacteria and trophoblasts. OBJECTIVE: The immunologic response of trophoblasts to specific potentially invading bacteria needs further analysis. METHODOLOGY: Immortalized first trimester human trophoblasts Swan 71 (Sw.71) were coincubated with Escherichia coli, Lactobacillus jensenii, Lactobacillus crispatus, and incubated alone (i.e., control group; 4 conditions with n = 6 for each condition). Chemokines and cytokines were measured. ANOVA with post hoc pairwise analysis was used to compare cytokines/chemokines concentrations in the 4 culture media. RESULTS: Sw.71 co-incubated with E. coli, L. jensenii or L. crispatus resulted in differential secretion of 11 of the 26 assayed cytokines/chemokines. Sw.71 co-incubated with any of the 3 bacteria responded with significant increased secretion of interleukin (IL)-8 and granulocyte macrophage colony-stimulating factor. All bacteria elicited the secretion of IL-6 and interferon (IFN) α2, 2 proinflammatory cytokines. In addition, Lactobacillus species resulted in increased secretion of IL-12p40 and IFNγ. While E. coli did not modify secretion of anti-inflammatory cytokines, Sw.71 cells responded to co-incubation with Lactobacillus species by secreting increased levels of IL-10 and IL-1ra. Both Lactobacillus species led to a decreased secretion of IL-4. CONCLUSION: All 3 bacterial species triggered significant release of chemokines and inflammatory cytokines, suggesting that a commensal relationship with trophoblasts may not be feasible.

Experimental evidence that symbiotic bacteria produce chemical cues in a songbird.

Symbiotic microbes that inhabit animal scent glands can produce volatile compounds used as chemical signals by the host animal. Though several studies have demonstrated correlations between scent gland bacterial community structure and host animal odour profiles, none have systematically demonstrated a causal relationship. In birds, volatile compounds in preen oil secreted by the uropygial gland serve as chemical cues and signals. Here, we tested whether manipulating the uropygial gland microbial community affects chemical profiles in the dark-eyed junco (Junco hyemalis). We found an effect of antibiotic treatment targeting the uropygial gland on both bacterial and volatile profiles. In a second experiment, we cultured bacteria from junco preen oil, and found that all of the cultivars produced at least one volatile compound common in junco preen oil, and that most cultivars produced multiple preen oil volatiles. In both experiments, we identified experimentally generated patterns in specific volatile compounds previously shown to predict junco reproductive success. Together, our data provide experimental support for the hypothesis that symbiotic bacteria produce behaviourally relevant volatile compounds within avian chemical cues and signals.

Does the human placenta delivered at term have a microbiota? Results of cultivation, quantitative real-time PCR, 16S rRNA gene sequencing, and metagenomics.

BACKGROUND: The human placenta has been traditionally viewed as sterile, and microbial invasion of this organ has been associated with adverse pregnancy outcomes. Yet, recent studies that utilized sequencing techniques reported that the human placenta at term contains a unique microbiota. These conclusions are largely based on the results derived from the sequencing of placental samples. However, such an approach carries the risk of capturing background-contaminating DNA (from DNA extraction kits, polymerase chain reaction reagents, and laboratory environments) when low microbial biomass samples are studied. OBJECTIVE: To determine whether the human placenta delivered at term in patients without labor who undergo cesarean delivery harbors a resident microbiota ("the assemblage of microorganisms present in a defined niche or environment"). STUDY DESIGN: This cross-sectional study included placentas from 29 women who had a cesarean delivery without labor at term. The study also included technical controls to account for potential background-contaminating DNA, inclusive in DNA extraction kits, polymerase chain reaction reagents, and laboratory environments. Bacterial profiles of placental tissues and background technical controls were characterized and compared with the use of bacterial culture, quantitative real-time polymerase chain reaction, 16S ribosomal RNA gene sequencing, and metagenomic surveys. RESULTS: (1) Twenty-eight of 29 placental tissues had a negative culture for microorganisms. The microorganisms retrieved by culture from the remaining sample were likely contaminants because corresponding 16S ribosomal RNA genes were not detected in the same sample. (2) Quantitative real-time polymerase chain reaction did not indicate greater abundances of bacterial 16S ribosomal RNA genes in placental tissues than in technical controls. Therefore, there was no evidence of the presence of microorganisms above background contamination from reagents in the placentas. (3) 16S ribosomal RNA gene sequencing did not reveal consistent differences in the composition or structure of bacterial profiles between placental samples and background technical controls. (4) Most of the bacterial sequences obtained from metagenomic surveys of placental tissues were from cyanobacteria, aquatic bacteria, or plant pathogens, which are microbes unlikely to populate the human placenta. Coprobacillus, which constituted 30.5% of the bacterial sequences obtained through metagenomic sequencing of placental samples, was not identified in any of the 16S ribosomal RNA gene surveys of these samples. These observations cast doubt as to whether this organism is really present in the placenta of patients at term not in labor. CONCLUSION: With the use of multiple modes of microbiologic inquiry, a resident microbiota could not be identified in human placentas delivered at term from women without labor. A consistently significant difference in the abundance and/or presence of a microbiota between placental tissue and background technical controls could not be found. All cultures of placental tissue, except 1, did not yield bacteria. Incorporating technical controls for potential sources of background-contaminating DNA for studies of low microbial biomass samples, such as the placenta, is necessary to derive reliable conclusions.

Evidence that intra-amniotic infections are often the result of an ascending invasion - a molecular microbiological study.

Background Microbial invasion of the amniotic cavity resulting in intra-amniotic infection is associated with obstetrical complications such as preterm labor with intact or ruptured membranes, cervical insufficiency, as well as clinical and histological chorioamnionitis. The most widely accepted pathway for intra-amniotic infection is the ascension of microorganisms from the lower genital tract. However, hematogenous dissemination of microorganisms from the oral cavity or intestine, retrograde seeding from the peritoneal cavity through the fallopian tubes, and introduction through invasive medical procedures have also been suggested as potential pathways for intra-amniotic infection. The primary reason that an ascending pathway is viewed as most common is that the microorganisms most often detected in the amniotic fluid are those that are typical inhabitants of the vagina. However, thus far, no studies have shown that microorganisms in the amniotic cavity are simultaneously present in the vagina of the woman from which they were isolated. The objective of the study was to determine the frequency with which microorganisms isolated from women with intra-amniotic infection are also present in the lower genital tract. Methods This was a cross-sectional study of women with intra-amniotic infection with intact membranes. Intra-amniotic infection was defined as a positive culture and elevated concentrations of interleukin-6 (IL-6) (>2.6 ng/mL) in amniotic fluid and/or acute histologic chorioamnionitis and funisitis. Microorganisms isolated from bacterial cultures of amniotic fluid were taxonomically identified through matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and 16S ribosomal RNA (rRNA) gene sequencing. Vaginal swabs were obtained at the time of amniocentesis for the identification of microorganisms in the lower genital tract. The overall bacterial profiles of amniotic fluids and vaginal swabs were characterized through 16S rRNA gene sequencing. The bacterial profiles of vaginal swabs were interrogated for the presence of bacteria cultured from amniotic fluid and for the presence of prominent (>1% average relative abundance) operational taxonomic units (OTUs) within the overall 16S rRNA gene bacterial profiles of amniotic fluid. Results (1) A total of 75% (6/8) of women had bacteria cultured from their amniotic fluid that are typical residents of the vaginal ecosystem. (2) A total of 62.5% (5/8) of women with bacteria cultured from their amniotic fluid also had these bacteria present in their vagina. (3) The microorganisms cultured from amniotic fluid and also detected in the vagina were Ureaplasma urealyticum, Escherichia coli, and Streptococcus agalactiae. (4) 16S rRNA gene sequencing revealed that the amniotic fluid of women with intra-amniotic infection had bacterial profiles dominated by Sneathia, Ureaplasma, Prevotella, Lactobacillus, Escherichia, Gardnerella, Peptostreptococcus, Peptoniphilus, and Streptococcus, many of which had not been cultured from the amniotic fluid samples. (5) Seventy percent (7/10) of the prominent (>1% average relative abundance) OTUs found in amniotic fluid were also prominent in the vagina. Conclusion The majority of women with intra-amniotic infection had bacteria cultured from their amniotic fluid that were typical vaginal commensals, and these bacteria were detected within the vagina at the time of amniocentesis. Molecular microbiological interrogation of amniotic fluid from women with intra-amniotic infection revealed that the bacterial profiles of amniotic fluid were largely consistent with those of the vagina. These findings indicate that ascension from the lower genital tract is the primary pathway for intra-amniotic infection.

Laboratory investigation into the role of largemouth bass virus (Ranavirus, Iridoviridae) in smallmouth bass mortality events in Pennsylvania rivers.

BACKGROUND: Mortality episodes have affected young-of-year smallmouth bass (Micropterus dolomieu) in several river systems in Pennsylvania since 2005. A series of laboratory experiments were performed to determine the potential role of largemouth bass virus (Ranavirus, Iridoviridae) in causing these events. RESULTS: Juvenile smallmouth bass experimentally infected with the largemouth bass virus exhibited internal and external clinical signs and mortality consistent with those observed during die-offs. Microscopically, infected fish developed multifocal necrosis in the mesenteric fat, liver, spleen and kidneys. Fish challenged by immersion also developed severe ulcerative dermatitis and necrotizing myositis and rarely panuveitis and keratitis. Largemouth bass virus-challenged smallmouth bass experienced greater mortality at 28 °C than at 23 or 11 °C. Co-infection with Flavobacterium columnare at 28 °C resulted in significant increase in mortality of smallmouth bass previously infected with largemouth bass virus. Aeromonas salmonicida seems to be very pathogenic to fish at water temperatures < 23 °C. While co-infection of smallmouth bass by both A. salmonicida and largemouth bass virus can be devastating to juvenile smallmouth bass, the optimal temperatures of each pathogen are 7-10 °C apart, making their synergistic effects highly unlikely under field conditions. CONCLUSIONS: The sum of our data generated in this study suggests that largemouth bass virus can be the causative agent of young-of-year smallmouth bass mortality episodes observed at relatively high water temperature.

Ultrastructure and molecular phylogeny of Pleistophora hyphessobryconis (Microsporidia) infecting hybrid jundiara (Leiarius marmoratus × Pseudoplatystoma reticulatum) in a Brazilian aquaculture facility.

A microsporidian infecting the skeletal muscle of hybrid jundiara (Leiarius marmoratus × Pseudoplatystoma reticulatum) in a commercial aquaculture facility in Brazil is described. Affected fish exhibited massive infections in the skeletal muscle that were characterized by large opaque foci throughout the affected fillets. Histologically, skeletal muscle was replaced by inflammatory cells and masses of microsporidial developmental stages. Generally pyriform spores had a wrinkled bi-layer spore wall and measured 4·0 × 6·0 µm. Multinucleate meronts surrounded by a simple plasma membrane were observed. The polar filament had an external membrane and a central electron dense mass. The development of sporoblasts within a sporophorous vesicle appeared synchronized. Ultrastructural observations and molecular analysis of 16S rDNA sequences revealed that the microsporidian was Pleistophora hyphessobryconis. This study is the first report of a P. hyphessobryconis infection in a non-ornamental fish.

Isolation of the Fathead Minnow Nidovirus from Muskellunge Experiencing Lingering Mortality.

In 2011, the Fathead Minnow nidovirus (FHMNV; Genus Bafinivirus, Family Coronaviridae, Order Nidovirales) was isolated from pond-raised juvenile Muskellunge Esox masquinongy suffering from lingering mortality at the Wild Rose Hatchery in Wild Rose, Wisconsin. Moribund Muskellunge exhibited tubular necrosis in the kidneys as well as multifocal coalescing necrotizing hepatitis. The FHMNV was also isolated from apparently healthy juvenile Muskellunge at the Wolf Lake State Fish Hatchery in Mattawan, Michigan. The identity of the two syncytia-forming viruses (designated MUS-WR and MUS-WL from Wild Rose Hatchery and Wolf Lake State Fish Hatchery, respectively) as strains of FHMNV was determined based on multiple-gene sequencing and phylogenetic analyses. The pathogenicity of the MUS-WL FHMNV strain was determined by experimentally infecting naive juvenile Muskellunge through intraperitoneal injection with two viral concentrations (63 and 6.3 × 10(3) TCID50/fish). Both doses resulted in 100% mortality in experimentally infected fish, which exhibited severely pale gills and petechial hemorrhaging in eyes, fins, and skin. Histopathological alterations in experimentally infected fish were observed mainly in the hematopoietic tissues in the form of focal areas of necrosis. Phylogenetic analysis of concatenated partial spike glycoprotein and helicase gene sequences revealed differences between the MUS-WL FHMNV, MUS-WR FHMNV, and two other FHMNV originally isolated from moribund Fathead Minnows Pimephales promelas including the index FHMNV strain (GU002364). Based on a partial helicase gene sequence, a reverse transcriptase PCR assay was developed that is specific to FHMNV. These results give evidence that the risks posed to Muskellunge by FHMNV should be taken seriously. Received May 1, 2015; accepted February 8, 2016.

Analysis of bacterial communities associated with the benthic amphipod Diporeia in the Laurentian Great Lakes Basin.

Bacterial communities play important roles in the biological functioning of crustaceans, yet little is known about their diversity, structure, and dynamics. This study was conducted to investigate the bacterial communities associated with the benthic amphipod Diporeia, an important component in the Great Lakes foodweb that has been declining over the past 3 decades. In this study, the combination of 16S rRNA gene sequencing and terminal restriction fragment length polymorphism revealed a total of 175 and 138 terminal restriction fragments (T-RFs) in Diporeia samples following treatment with the endonucleases HhaI and MspI, respectively. Relatively abundant and prevalent T-RFs were affiliated with the genera Flavobacterium and Pseudomonas and the class Betaproteobacteria. T-RFs affiliated with the order Rickettsiales were also detected. A significant difference in T-RF presence and abundance (P = 0.035) was detected among profiles generated for Diporeia collected from 4 sites in Lake Michigan. Comparison of profiles generated for Diporeia samples collected in 2 years from lakes Superior and Michigan showed a significant change in diversity for Lake Superior Diporeia but not Lake Michigan Diporeia. Profiles from one Lake Michigan site contained multiple unique T-RFs compared with other Lake Michigan Diporeia profiles, most notably one that represents the genus Methylotenera. This study generated the most extensive list of bacteria associated with Diporeia and sheds useful insights on the microbiome of Great Lakes Diporeia that may help to reveal potential causes of the decline of Diporeia populations.

Spatio-temporal dynamics of parasites infecting Diporeia spp. (Amphipoda, Gammaridae) in southern Lake Michigan (USA).

Since the 1990s, populations of the benthic amphipod Diporeia spp. (Diporeia) have sharply declined across much of the Laurentian Great Lakes. This study was undertaken to identify contemporary and historical community composition, structure, and dynamics of parasites infecting Diporeia collected from nine sites in the southern basin of Lake Michigan, where declines of the amphipod have been well documented over the past 20years. An additional aim of this study was to assess whether infection dynamics and dreissenid densities could explain the declines in Diporeia densities that have occurred. We found that Diporeia were host to eight groups of uni- and multicellular pathogens. Of the 3082 amphipods analyzed, 1624 individuals (52.7%) were infected with at least one type of parasite. Ciliophora was the most prevalent parasite (50.08% prevalence of infection), followed by Gregarinasina (2.79%), Microsporidia (0.68%), Cestoda (0.45%), Acanthocephala (0.36%), Haplosporidia (0.23%), Yeast (0.32%), and filamentous Fungi (0.10%). Considerable spatial and temporal variability were observed in parasite prevalences, with prevalences frequently appearing to cycle between low and high values. Parasite species belonging to Microsporidia and Haplosporidia were associated with tissue alteration and host inflammatory response; however, parasite prevalences explained very little in terms of Diporeia density declines at assessed sites. Despite these findings, we do not discount the possibility that parasitic infections may have played a role in declining Diporeia densities in the Great Lakes, as the cyclical prevalences that were observed are possibly suggestive of parasitic outbreaks that are followed by die-offs at affected sites. This study suggests that if parasites have affected Diporeia densities in the Laurentian Great Lakes, then the relationship may be a complicated one.

Long access heroin self-administration significantly alters gut microbiome composition and structure.

Introduction: It is well known that chronic opioid use disorder is associated with alterations in gastrointestinal (GI) function that include constipation, reduced motility, and increased bacterial translocation due to compromised gut barrier function. These signs of disrupted GI function can be associated with alterations in the gut microbiome. However, it is not known if long-access opioid self-administration has effects on the gut microbiome. Methods: We used 16S rRNA gene sequencing to investigate the gut microbiome in three independent cohorts (N=40 for each) of NIH heterogeneous stock rats before onset of long-access heroin self-administration (i.e., naïve status), at the end of a 15-day period of self-administration, and after post-extinction reinstatement. Measures of microbial α- and ß-diversity were evaluated for all phases. High-dimensional class comparisons were carried out with MaAsLin2. PICRUSt2 was used for predicting functional pathways impacted by heroin based on marker gene sequences. Results: Community α-diversity was not altered by heroin at any of the three phases by comparison to saline-yoked controls. Analyses of ß-diversity showed that the heroin and saline-yoked groups clustered significantly apart from each other using the Bray-Curtis (community structure) index. Heroin caused significant alterations at the ASV level at the self-administration and extinction phases. At the phylum level, the relative abundance of Firmicutes was increased at the self-administration phase. Deferribacteres was decreased in heroin whereas Patescibacteria was increased in heroin at the extinction phase. Potential biomarkers for heroin emerged from the MaAsLin2 analysis. Bacterial metabolomic pathways relating to degradation of carboxylic acids, nucleotides, nucleosides, carbohydrates, and glycogen were increased by heroin while pathways relating to biosynthesis of vitamins, propionic acid, fatty acids, and lipids were decreased. Discussion: These findings support the view that long access heroin self-administration significantly alters the structure of the gut microbiome by comparison to saline-yoked controls. Inferred metabolic pathway alterations suggest the development of a microbial imbalance favoring gut inflammation and energy expenditure. Potential microbial biomarkers and related functional pathways likely invoked by heroin self-administration could be targets for therapeutic intervention.

The Vaginal Microbiota of Pregnant Women Varies with Gestational Age, Maternal Age, and Parity.

The composition of the vaginal microbiota is heavily influenced by pregnancy and may factor into pregnancy complications, including spontaneous preterm birth. However, results among studies have been inconsistent due, in part, to variation in sample sizes and ethnicity. Thus, an association between the vaginal microbiota and preterm labor continues to be debated. Yet, before assessing associations between the composition of the vaginal microbiota and preterm labor, a robust and in-depth characterization of the vaginal microbiota throughout pregnancy in the specific study population under investigation is required. Here, we report a large longitudinal study (n = 474 women, 1,862 vaginal samples) of a predominantly African-American cohort-a population that experiences a relatively high rate of pregnancy complications-evaluating associations between individual identity, gestational age, and other maternal characteristics with the composition of the vaginal microbiota throughout gestation resulting in term delivery. The principal factors influencing the composition of the vaginal microbiota in pregnancy are individual identity and gestational age at sampling. Other factors are maternal age, parity, obesity, and self-reported Cannabis use. The general pattern across gestation is for the vaginal microbiota to remain or transition to a state of Lactobacillus dominance. This pattern can be modified by maternal parity and obesity. Regardless, network analyses reveal dynamic associations among specific bacterial taxa within the vaginal ecosystem, which shift throughout the course of pregnancy. This study provides a robust foundational understanding of the vaginal microbiota in pregnancy and sets the stage for further investigation of this microbiota in obstetrical disease. IMPORTANCE There is debate regarding links between the vaginal microbiota and pregnancy complications, especially spontaneous preterm birth. Inconsistencies in results among studies are likely due to differences in sample sizes and cohort ethnicity. Ethnicity is a complicating factor because, although all bacterial taxa commonly inhabiting the vagina are present among all ethnicities, the frequencies of these taxa vary among ethnicities. Therefore, an in-depth characterization of the vaginal microbiota throughout pregnancy in the specific study population under investigation is required prior to evaluating associations between the vaginal microbiota and obstetrical disease. This initial investigation is a large longitudinal study of the vaginal microbiota throughout gestation resulting in a term delivery in a predominantly African-American cohort, a population that experiences disproportionally negative maternal-fetal health outcomes. It establishes the magnitude of associations between maternal characteristics, such as age, parity, body mass index, and self-reported Cannabis use, on the vaginal microbiota in pregnancy.

Blockade of IL-6R prevents preterm birth and adverse neonatal outcomes.

BACKGROUND: Preterm birth preceded by spontaneous preterm labour often occurs in the clinical setting of sterile intra-amniotic inflammation (SIAI), a condition that currently lacks treatment. METHODS: Proteomic and scRNA-seq human data were analysed to evaluate the role of IL-6 and IL-1α in SIAI. A C57BL/6 murine model of SIAI-induced preterm birth was developed by the ultrasound-guided intra-amniotic injection of IL-1α. The blockade of IL-6R by using an aIL-6R was tested as prenatal treatment for preterm birth and adverse neonatal outcomes. QUEST-MRI evaluated brain oxidative stress in utero. Targeted transcriptomic profiling assessed maternal, foetal, and neonatal inflammation. Neonatal biometrics and neurodevelopment were tested. The neonatal gut immune-microbiome was evaluated using metagenomic sequencing and immunophenotyping. FINDINGS: IL-6 plays a critical role in the human intra-amniotic inflammatory response, which is associated with elevated concentrations of the alarmin IL-1α. Intra-amniotic injection of IL-1α resembles SIAI, inducing preterm birth (7% vs. 50%, p = 0.03, Fisher's exact test) and neonatal mortality (18% vs. 56%, p = 0.02, Mann-Whitney U-test). QUEST-MRI revealed no foetal brain oxidative stress upon in utero IL-1α exposure (p > 0.05, mixed linear model). Prenatal treatment with aIL-6R abrogated IL-1α-induced preterm birth (50% vs. 7%, p = 0.03, Fisher's exact test) by dampening inflammatory processes associated with the common pathway of labour. Importantly, aIL-6R reduces neonatal mortality (56% vs. 22%, p = 0.03, Mann-Whitney U-test) by crossing from the mother to the amniotic cavity, dampening foetal organ inflammation and improving growth. Beneficial effects of prenatal IL-6R blockade carried over to neonatal life, improving survival, growth, neurodevelopment, and gut immune homeostasis. INTERPRETATION: IL-6R blockade can serve as a strategy to treat SIAI, preventing preterm birth and adverse neonatal outcomes. FUNDING: NICHD/NIH/DHHS, Contract HHSN275201300006C. WSU Perinatal Initiative in Maternal, Perinatal and Child Health.

Optimization and validation of two multiplex qPCR assays for the rapid detection of microorganisms commonly invading the amniotic cavity.

Microbial invasion of the amniotic cavity (MIAC) leading to infection is strongly associated with adverse pregnancy and neonatal outcomes. Limitations of current diagnostic assays to detect MIAC rapidly and accurately have hindered the ability of obstetricians to identify and treat intra-amniotic infections. We developed, optimized, and validated two multiplex quantitative polymerase chain reaction (qPCR) assays for the simultaneous detection and quantification of microbial taxa commonly associated with MIAC. The first assay allows for the quantification of general bacterial and fungal loads in amniotic fluid and includes a human reference gene to allow for assessing the integrity of clinical samples and the DNA extraction process. The second assay allows for the detection and quantification of four specific bacterial taxa commonly associated with MIAC: Ureaplasma spp., Mycoplasma hominis, Streptococcus agalactiae, and Fusobacterium nucleatum. The qPCR assays were validated by using both microbial isolates and clinical amniotic fluid samples. The assays were further validated by comparing qPCR amplification results to those from the microbial culture and bacterial 16S rRNA gene sequencing of amniotic fluid. Both assays demonstrated high reproducibility and are sensitive and specific to their intended targets. Therefore, these assays represent promising molecular diagnostic tools for the detection of MIAC. Most importantly, these assays may allow for administration of timely and targeted antibiotic interventions to reduce adverse perinatal outcomes attributed to intra-amniotic infections.

Does the Amniotic Fluid of Mice Contain a Viable Microbiota?

The existence of an amniotic fluid microbiota (i.e., a viable microbial community) in mammals is controversial. Its existence would require a fundamental reconsideration of fetal in utero exposure to and colonization by microorganisms and the role of intra-amniotic microorganisms in fetal immune development as well as in pregnancy outcomes. In this study, we determined whether the amniotic fluid of mice harbors a microbiota in late gestation. The profiles of the amniotic fluids of pups located proximally or distally to the cervix were characterized through quantitative real-time PCR, 16S rRNA gene sequencing, and culture (N = 21 dams). These profiles were compared to those of technical controls for bacterial and DNA contamination. The load of 16S rRNA genes in the amniotic fluid exceeded that in controls. Additionally, the 16S rRNA gene profiles of the amniotic fluid differed from those of controls, with Corynebacterium tuberculostearicum being differentially more abundant in amniotic fluid profiles; however, this bacterium was not cultured from amniotic fluid. Of the 42 attempted bacterial cultures of amniotic fluids, only one yielded bacterial growth - Lactobacillus murinus. The 16S rRNA gene of this common murine-associated bacterium was not detected in any amniotic fluid sample, suggesting it did not originate from the amniotic fluid. No differences in the 16S rRNA gene load, 16S rRNA gene profile, or bacterial culture were observed between the amniotic fluids located Proximally and distally to the cervix. Collectively, these data indicate that, although there is a modest DNA signal of bacteria in murine amniotic fluid, there is no evidence that this signal represents a viable microbiota. While this means that amniotic fluid is not a source of microorganisms for in utero colonization in mice, it may nevertheless contribute to fetal exposure to microbial components. The developmental consequences of this observation warrant further investigation.