VESPA: an optimized protocol for accurate metabarcoding-based characterization of vertebrate eukaryotic endosymbiont and parasite assemblages.

Protocols for characterizing taxonomic assemblages by deep sequencing of short DNA barcode regions (metabarcoding) have revolutionized our understanding of microbial communities and are standardized for bacteria, archaea, and fungi. Unfortunately, comparable methods for host-associated eukaryotes have lagged due to technical challenges. Despite 54 published studies, issues remain with primer complementarity, off-target amplification, and lack of external validation. Here, we present VESPA (Vertebrate Eukaryotic endoSymbiont and Parasite Analysis) primers and optimized metabarcoding protocol for host-associated eukaryotic community analysis. Using in silico prediction, panel PCR, engineered mock community standards, and clinical samples, we demonstrate VESPA to be more effective at resolving host-associated eukaryotic assemblages than previously published methods and to minimize off-target amplification. When applied to human and non-human primate samples, VESPA enables reconstruction of host-associated eukaryotic endosymbiont communities more accurately and at finer taxonomic resolution than microscopy. VESPA has the potential to advance basic and translational science on vertebrate eukaryotic endosymbiont communities, similar to achievements made for bacterial, archaeal, and fungal microbiomes.

Environmental pollutant exposure associated with altered early-life gut microbiome: Results from a birth cohort study.

Emerging evidence shows that the gut microbiota interacts with environmental pollutants, but the effect of early exposure on the neonatal microbiome remains unknown. We investigated the association between maternal exposure to environmental pollutants and changes in early-life gut microbiome development. We surveyed 16S rRNA gene on meconium and fecal samples (at 1, 3, and 6 months) from the Brazilian birth cohort, and associated with levels of metals, perfluoroalkyl chemicals (PFAS), and pesticides in maternal and umbilical cord blood. The results indicate that the magnitude of the microbiome changes associated with increasing pollutant exposure was bigger in cesarean-section (CS) born and CS-born-preterm babies, in relation to vaginally (VG) delivered infants. Breastfeeding was associated with a stronger pollutant-associated effect on the infant feces, suggesting that the exposure source could be maternal milk. Differences in microbiome effects associated with maternal or cord blood pollutant concentrations suggest that fetal exposure time - intrauterine or perinatal - may matter. Finally, despite the high developmental microbiota variability, specific microbionts were consistently affected across all pollutants, with taxa clusters found in samples from infants exposed to the highest toxicant exposure. The results evidence that perinatal exposure to environmental pollutants is associated with alterations in gut microbiome development which may have health significance.

Home chemical and microbial transitions across urbanization.

Urbanization represents a profound shift in human behaviour, and has considerable cultural and health-associated consequences1,2. Here, we investigate chemical and microbial characteristics of houses and their human occupants across an urbanization gradient in the Amazon rainforest, from a remote Peruvian Amerindian village to the Brazilian city of Manaus. Urbanization was found to be associated with reduced microbial outdoor exposure, increased contact with housing materials, antimicrobials and cleaning products, and increased exposure to chemical diversity. The degree of urbanization correlated with changes in the composition of house bacterial and microeukaryotic communities, increased house and skin fungal diversity, and an increase in the relative abundance of human skin-associated fungi and bacteria in houses. Overall, our results indicate that urbanization has large-scale effects on chemical and microbial exposures and on the human microbiota.

Correction: The fecal, oral, and skin microbiota of children with Chagas disease treated with benznidazole.

[This corrects the article DOI: 10.1371/journal.pone.0212593.].

The fecal, oral, and skin microbiota of children with Chagas disease treated with benznidazole.

BACKGROUND: Chagas disease is still prevalent in rural areas of South America. In endemic areas of Bolivia, school children are screened for the program of Chagas disease eradication of the Ministry of Health, and positive children are treated. Here, we compared the fecal, oral and skin microbiomes of children with or without Chagas disease, and before and after benznidazol treatment of infected children. METHODS: A total of 543 Bolivian children (5-14 years old) were tested for Chagas disease, and 20 positive children were treated with Benznidazole. Fecal samples and oral and skin swabs were obtained before and after treatment, together with samples from a group of 35 uninfected controls. The 16S rRNA genes were sequenced and analyzed using QIIME to determine Alpha diversity differences and community distances, and linear discriminant analyses to determine marker taxa by infection status or treatment. RESULTS: Twenty out of 543 children screened were seropositive for Chagas disease (3.7%) and were included in the study, together with 35 control children that were seronegative for the disease. Fecal samples, oral and skin swabs were taken at the beginning of the study and after the anti-protozoa therapy with Benznidazole to the chagasic children. Infected children had higher fecal Firmicutes (Streptococcus, Roseburia, Butyrivibrio, and Blautia), and lower Bacteroides and also showed some skin -but not oral- microbiota differences. Treatment eliminated the fecal microbiota differences from control children, increasing Dialister (class Clostridia) and members of the Enterobacteriaceae, and decreasing Prevotella and Coprococcus, with minor effects on the oral and skin bacterial diversity. CONCLUSIONS: The results of this study show differences in the fecal microbiota associated with Chagas disease in children, and also evidence that treatment normalizes fecal microbiota (makes it more similar to that in controls), but is associated with oral and skin microbiota differences from control children. Since microbiota impacts in children, it is important to determine the effect of drugs on the children microbiota, since dysbiosis could lead to physiological effects which might be avoidable with microbiota restoration interventions.

Characterization of the intestinal microbiota of the sea cucumber Holothuria glaberrima.

High-throughput 16S rRNA gene sequencing has been used to identify the intestinal microbiota of many animal species, but that of marine invertebrate organisms remains largely unknown. There are only a few high-throughput sequencing studies on the intestinal microbiota of echinoderms (non-vertebrate Deuterostomes). Here we describe the intestinal microbiota of the sea cucumber Holothuria glaberrima, an echinoderm, well-known for its remarkable power of regeneration. We characterized the microbiota from the anterior descending intestine, the medial intestine (these two comprise the small intestine) and the posterior descending intestine (or large intestine), using pyrosequencing to sequence the V4 region of the 16S rRNA gene. We compared animals in their natural marine environment and in sea-water aquaria. A total of 8,172 OTU's were grouped in 10 bacterial phyla, 23 classes, 44 orders, 83 families, 127 genera and 1 group of unknown bacteria, present across the digestive tract of 10 specimens. The results showed that the anterior intestine is dominated by Proteobacteria (61%) and Bacteroidetes (22%), the medium intestine is similar but with lower Bacteroidetes (4%), and the posterior intestine was remarkably different, dominated by Firmicutes (48%) and Bacteroidetes (35%). The structure of the community changed in animals kept in aquaria, which had a general dominance of Firmicutes and Bacteroidetes, regardless the intestinal segment. Our results evidence that in the natural sea environment, there is intestinal segment differentiation in the microbiota of H. glaberrima, which is lost in artificial conditions. This is relevant for physiological studies, such as mechanisms of digestive regeneration, which might be affected by the microbiota.

Changes in the Gut Microbiota of Urban Subjects during an Immersion in the Traditional Diet and Lifestyle of a Rainforest Village.

People living traditional lifestyles have higher gut microbiota diversity than urban subjects. We hypothesized that shifting lifestyles from an urban environment to a traditional rainforest village would lead to changes in the microbiota of visitors, which would become more similar to the microbiota of villagers. Here, we characterized at different time points the microbiota of 7 urban visitors (5 adults and 2 children) staying in a rainforest Amerindian village for 16 days and compared them with a reference collection of samples from age-matched local villagers. We performed a 16S rRNA gene survey of samples from multiple body sites (including fecal, oral, nasal, and skin samples) using Illumina MiSeq sequencing. The main factor segregating the microbiotas of each body site was the human group (i.e., visitors versus villagers), with the visitor microbiota tending to have lower alpha diversity; the lowered alpha diversity was statistically significant in the microbiota of skin and in the children's fecal and oral microbiota. During the rainforest period, all visitors experienced microbiota changes within their personal cloud of variation. For all body sites, the microbiota conformations in the visitor children better matched the microbiota conformations in villagers of the same age than did those of the visitor adults, which showed a lower "microbiota age" than the microbiota of the villagers. The results suggest higher stability in the adult microbiota, with the less resilient children's microbiota responding more to dietary changes.IMPORTANCE Despite the limitations of a small study, our results evidence higher resilience of the gut microbiota with respect to dietary manipulation in adults than in children and urge further studies to understand the extent of microbiota plasticity in response to dietary changes and the mechanisms underlying microbiota resilience. These studies are relevant to the potential of future human pre- and probiotics in preventing or curing microbiota-associated diseases.

Delivery Mode and the Transition of Pioneering Gut-Microbiota Structure, Composition and Predicted Metabolic Function.

Cesarean (C-section) delivery, recently shown to cause excess weight gain in mice, perturbs human neonatal gut microbiota development due to the lack of natural mother-to-newborn transfer of microbes. Neonates excrete first the in-utero intestinal content (referred to as meconium) hours after birth, followed by intestinal contents reflective of extra-uterine exposure (referred to as transition stool) 2 to 3 days after birth. It is not clear when the effect of C-section on the neonatal gut microbiota emerges. We examined bacterial DNA in carefully-collected meconium, and the subsequent transitional stool, from 59 neonates [13 born by scheduled C-section and 46 born by vaginal delivery] in a private hospital in Brazil. Bacterial DNA was extracted, and the V4 region of the 16S rRNA gene was sequenced using the Illumina MiSeq (San Diego, CA, USA) platform. We found evidence of bacterial DNA in the majority of meconium samples in our study. The bacterial DNA structure (i.e., beta diversity) of meconium differed significantly from that of the transitional stool microbiota. There was a significant reduction in bacterial alpha diversity (e.g., number of observed bacterial species) and change in bacterial composition (e.g., reduced Proteobacteria) in the transition from meconium to stool. However, changes in predicted microbiota metabolic function from meconium to transitional stool were only observed in vaginally-delivered neonates. Within sample comparisons showed that delivery mode was significantly associated with bacterial structure, composition and predicted microbiota metabolic function in transitional-stool samples, but not in meconium samples. Specifically, compared to vaginally delivered neonates, the transitional stool of C-section delivered neonates had lower proportions of the genera Bacteroides, Parabacteroides and Clostridium. These differences led to C-section neonates having lower predicted abundance of microbial genes related to metabolism of amino and nucleotide sugars, and higher abundance of genes related to fatty-acid metabolism, amino-acid degradation and xenobiotics biodegradation. In summary, microbiota diversity was reduced in the transition from meconium to stool, and the association of delivery mode with microbiota structure, composition and predicted metabolic function was not observed until the passing of the transitional stool after meconium.

Increased weight gain by C-section: Functional significance of the primordial microbiome.

Epidemiological evidence supports a direct association between early microbiota impact-including C-section-and obesity. We performed antibiotic-free, fostered C-sections and determined the impact on the early microbiota and body weight during development. Mice in the C-section group gained more body mass after weaning, with a stronger phenotype in females. C-section-born mice lacked the dynamic developmental gut microbiota changes observed in control mice. The results demonstrate a causal relationship between C-section and increased body weight, supporting the involvement of maternal vaginal bacteria in normal metabolic development.

The Human Microbiome before Birth.

The conservation of the microbiota within humans and other hominids suggests an ancient assembly that has been selected to optimize host fitness. Pregnancy induces changes in the maternal microbiome just before the intergenerational hand-off of the microbiota. Interventions, including peri-partum antibiotics and Cesarean sections, may have unintended effects on babies.

The Gestational Vaginal Microbiome and Spontaneous Preterm Birth among Nulliparous African American Women.

Introduction Early markers to identify pregnant women at high risk for spontaneous preterm birth (SPTB) have not been established and preventive options are limited. Recent attention has focused on examining the importance of characterizing the vaginal microbiome to predict SPTB. Results We examined the diversity and structure of the vaginal microbiome in nulliparous African American women during early pregnancy and compared 13 women who delivered preterm and 27 women who delivered at term. Samples were taken at one of two points in gestation, before 16 weeks or between 20 and 24 weeks. Among women who delivered preterm, we found lower bacterial diversity with lower abundance of Coriobacteriaceae, Sneathia, Prevotella, and Aerococcus compared with women delivering at term (linear discriminant analysis score > 3.0). The Shannon diversity index was not significantly different between the groups (p-value = 0.239). Phylogenetic diversity and Chao1 suggested a lower diversity in the vaginal microbiota of women who delivered preterm compared with term, but these findings were not significantly different (p = 0.077 and p = 0.066, respectively). Conclusion These data suggest that the vaginal microbiome of women delivering preterm had lower diversity than women delivering after 37 weeks, although these findings need to be explored in a larger sample of nulliparous African American women.

Walls talk: Microbial biogeography of homes spanning urbanization.

Westernization has propelled changes in urbanization and architecture, altering our exposure to the outdoor environment from that experienced during most of human evolution. These changes might affect the developmental exposure of infants to bacteria, immune development, and human microbiome diversity. Contemporary urban humans spend most of their time indoors, and little is known about the microbes associated with different designs of the built environment and their interaction with the human immune system. This study addresses the associations between architectural design and the microbial biogeography of households across a gradient of urbanization in South America. Urbanization was associated with households' increased isolation from outdoor environments, with additional indoor space isolation by walls. Microbes from house walls and floors segregate by location, and urban indoor walls contain human bacterial markers of space use. Urbanized spaces uniquely increase the content of human-associated microbes-which could increase transmission of potential pathogens-and decrease exposure to the environmental microbes with which humans have coevolved.

Partial restoration of the microbiota of cesarean-born infants via vaginal microbial transfer.

Exposure of newborns to the maternal vaginal microbiota is interrupted with cesarean birthing. Babies delivered by cesarean section (C-section) acquire a microbiota that differs from that of vaginally delivered infants, and C-section delivery has been associated with increased risk for immune and metabolic disorders. Here we conducted a pilot study in which infants delivered by C-section were exposed to maternal vaginal fluids at birth. Similarly to vaginally delivered babies, the gut, oral and skin bacterial communities of these newborns during the first 30 d of life was enriched in vaginal bacteria--which were underrepresented in unexposed C-section-delivered infants--and the microbiome similarity to those of vaginally delivered infants was greater in oral and skin samples than in anal samples. Although the long-term health consequences of restoring the microbiota of C-section-delivered infants remain unclear, our results demonstrate that vaginal microbes can be partially restored at birth in C-section-delivered babies.

Human Milk Bacterial and Glycosylation Patterns Differ by Delivery Mode.

Mammals have evolved to nourish their offspring exclusively with maternal milk for around half of the lactation period, a crucial developmental window. In view of oral-breast contact during lactation and the differences in oral microbiota between cesarean section (C-section) and vaginally delivered infants, we expected differences in milk composition by delivery mode. We performed a cross-sectional study of banked human milk and found changes related to time since delivery in bacterial abundance and glycosylation patterns only in milk from women who delivered vaginally. The results warrant further research into the effects of delivery mode on milk microbes, milk glycosylation, and postpartum infant development.

The first microbial environment of infants born by C-section: the operating room microbes.

BACKGROUND: Newborns delivered by C-section acquire human skin microbes just after birth, but the sources remain unknown. We hypothesized that the operating room (OR) environment contains human skin bacteria that could be seeding C-section born infants. RESULTS: To test this hypothesis, we sampled 11 sites in four operating rooms from three hospitals in two cities. Following a C-section procedure, we swabbed OR floors, walls, ventilation grids, armrests, and lamps. We sequenced the V4 region of the 16S rRNA gene of 44 samples using Illumina MiSeq platform. Sequences were analyzed using the QIIME pipeline. Only 68 % of the samples (30/44, >1000 sequences per site) yielded sufficient DNA reads to be analyzed. The bacterial content of OR dust corresponded to human skin bacteria, with dominance of Staphylococcus and Corynebacterium. Diversity of bacteria was the highest in the ventilation grids and walls but was also present on top of the surgery lamps. Beta diversity analyses showed OR dust bacterial content clustering first by city and then by hospital (t test using unweighted UniFrac distances, p < 0.05). CONCLUSIONS: We conclude that the dust from ORs, collected right after a C-section procedure, contains deposits of human skin bacteria. The OR microbiota is the first environment for C-section newborns, and OR microbes might be seeding the microbiome in these babies. Further studies are required to identify how this OR microbiome exposure contributes to the seeding of the neonatal microbiome. The results might be relevant to infant health, if the current increase in risk of immune and metabolic diseases in industrialized societies is related to lack of natural exposure to the vaginal microbiome during labor and birth.

Urease and Dental Plaque Microbial Profiles in Children.

OBJECTIVE: Urease enzymes produced by oral bacteria generate ammonia, which can have a significant impact on the oral ecology and, consequently, on oral health. To evaluate the relationship of urease with dental plaque microbial profiles in children as it relates to dental caries, and to identify the main contributors to this activity. METHODS: 82 supragingival plaque samples were collected from 44 children at baseline and one year later, as part of a longitudinal study on urease and caries in children. DNA was extracted; the V3-V5 region of the 16S rRNA gene was amplified and sequenced using 454 pyrosequencing. Urease activity was measured using a spectrophotometric assay. Data were analyzed with Qiime. RESULTS: Plaque urease activity was significantly associated with the composition of the microbial communities of the dental plaque (Baseline P = 0.027, One Year P = 0.012). The bacterial taxa whose proportion in dental plaque exhibited significant variation by plaque urease levels in both visits were the family Pasteurellaceae (Baseline P<0.001; One Year P = 0.0148), especially Haemophilus parainfluenzae. No association was observed between these bacteria and dental caries. Bacteria in the genus Leptotrichia were negatively associated with urease and positively associated with dental caries (Bonferroni P<0.001). CONCLUSIONS: Alkali production by urease enzymes primarily from species in the family Pasteurellaceae can be an important ecological determinant in children's dental plaque. Further studies are needed to establish the role of urease-associated bacteria in the acid/base homeostasis of the dental plaque, and in the development and prediction of dental caries in children.

Effect of influenza-induced fever on human bioimpedance values.

BACKGROUND AND AIMS: Bioelectrical impedance analysis (BIA) is a widely used technique to assess body composition and nutritional status. While bioelectrical values are affected by diverse variables, there has been little research on validation of BIA in acute illness, especially to understand prognostic significance. Here we report the use of BIA in acute febrile states induced by influenza. METHODS: Bioimpedance studies were conducted during an H1N1 influenza A outbreak in Venezuelan Amerindian villages from the Amazonas. Measurements were performed on 52 subjects between 1 and 40 years of age, and 7 children were re-examined after starting Oseltamivir treatment. Bioelectrical Impedance Vector Analysis (BIVA) and permutation tests were applied. RESULTS: For the entire sample, febrile individuals showed a tendency toward greater reactance (p=0.058) and phase angle (p=0.037) than afebrile individuals, while resistance and impedance were similar in the two groups. Individuals with repeated measurements showed significant differences in bioimpedance values associated with fever, including increased reactance (p<0.001) and phase angle (p=0.007), and decreased resistance (p=0.007) and impedance (p<0.001). CONCLUSIONS: There are bioelectrical variations induced by influenza that can be related to dehydration, with lower extracellular to intracellular water ratio in febrile individuals, or a direct thermal effect. Caution is recommended when interpreting bioimpedance results in febrile states.

The infant microbiome development: mom matters.

The infant microbiome plays an essential role in human health and its assembly is determined by maternal-offspring exchanges of microbiota. This process is affected by several practices, including Cesarean section (C-section), perinatal antibiotics, and formula feeding, that have been linked to increased risks of metabolic and immune diseases. Here we review recent knowledge about the impacts on infant microbiome assembly, discuss preventive and restorative strategies to ameliorate the effects of these impacts, and highlight where research is needed to advance this field and improve the health of future generations.