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.

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.

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.

Examining the healthy human microbiome concept.

Human microbiomes are essential to health throughout the lifespan and are increasingly recognized and studied for their roles in metabolic, immunological and neurological processes. Although the full complexity of these microbial communities is not fully understood, their clinical and industrial exploitation is well advanced and expanding, needing greater oversight guided by a consensus from the research community. One of the most controversial issues in microbiome research is the definition of a 'healthy' human microbiome. This concept is complicated by the microbial variability over different spatial and temporal scales along with the challenge of applying a unified definition to the spectrum of healthy microbiome configurations. In this Perspective, we examine the progress made and the key gaps that remain to be addressed to fully harness the benefits of the human microbiome. We propose a road map to expand our knowledge of the microbiome-health relationship, incorporating epidemiological approaches informed by the unique ecological characteristics of these communities.

Tetracycline resistance genes acquired at birth.

Newborns acquire their first microbiota at birth. Maternal vaginal or skin bacteria colonize newborns delivered vaginally or by C-section, respectively (Dominguez-Bello et al. 2010 #884). We aimed to determine differences in the presence of four tetracycline (tet) resistance genes, in the microbes of ten newborns and in the mouth and vagina of their mothers, at the time of birth. DNA was amplified by PCR with primers specific for [tet(M), tet(O), tet(Q), and tet(W)]. Maternal vaginas harbored all four tet resistance genes, but most commonly tet(M) and tet(O) (63 and 38 %, respectively). Genes coding for tet resistance differed by birth mode, with 50 % of vaginally delivered babies had tet(M) and tet(O) and 16 and 13 % of infants born by C-section had tet(O) and tet(W), respectively. Newborns acquire antibiotic resistance genes at birth, and the resistance gene profile varies by mode of delivery.

Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns.

Upon delivery, the neonate is exposed for the first time to a wide array of microbes from a variety of sources, including maternal bacteria. Although prior studies have suggested that delivery mode shapes the microbiota's establishment and, subsequently, its role in child health, most researchers have focused on specific bacterial taxa or on a single body habitat, the gut. Thus, the initiation stage of human microbiome development remains obscure. The goal of the present study was to obtain a community-wide perspective on the influence of delivery mode and body habitat on the neonate's first microbiota. We used multiplexed 16S rRNA gene pyrosequencing to characterize bacterial communities from mothers and their newborn babies, four born vaginally and six born via Cesarean section. Mothers' skin, oral mucosa, and vagina were sampled 1 h before delivery, and neonates' skin, oral mucosa, and nasopharyngeal aspirate were sampled <5 min, and meconium <24 h, after delivery. We found that in direct contrast to the highly differentiated communities of their mothers, neonates harbored bacterial communities that were undifferentiated across multiple body habitats, regardless of delivery mode. Our results also show that vaginally delivered infants acquired bacterial communities resembling their own mother's vaginal microbiota, dominated by Lactobacillus, Prevotella, or Sneathia spp., and C-section infants harbored bacterial communities similar to those found on the skin surface, dominated by Staphylococcus, Corynebacterium, and Propionibacterium spp. These findings establish an important baseline for studies tracking the human microbiome's successional development in different body habitats following different delivery modes, and their associated effects on infant health.

Bacterial diversity in the cecum of the world's largest living rodent (Hydrochoerus hydrochaeris).

The capybara (Hydrochoerus hydrochaeris) is the world's largest living rodent. Native to South America, this hindgut fermenter is herbivorous and coprophagous and uses its enlarged cecum to digest dietary plant material. The microbiota of specialized hindgut fermenters has remained largely unexplored. The aim of this work was to describe the composition of the bacterial community in the fermenting cecum of wild capybaras. The analysis of bacterial communities in the capybara cecum is a first step towards the functional characterization of microbial fermentation in this model of hindgut fermentation. We sampled cecal contents from five wild adult capybaras (three males and two females) in the Venezuelan plains. DNA from cecal contents was extracted, the 16S rDNA was amplified, and the amplicons were hybridized onto a DNA microarray (G2 PhyloChip). We found 933 bacterial operational taxonomic units (OTUs) from 182 families in 21 bacterial phyla in the capybara cecum. The core bacterial microbiota (present in at least four animals) was represented by 575 OTUs. About 86% of the cecal bacterial OTUs belong to only five phyla, namely, Firmicutes (322 OTUs), Proteobacteria (301 OTUs), Bacteroidetes (76 OTUs), Actinobacteria (69 OTUs), and Sphirochaetes (37 OTUs). The capybara harbors a diverse bacterial community that includes lineages involved in fiber degradation and nitrogen fixation in other herbivorous animals.

The bacterial microbiota in the oral mucosa of rural Amerindians.

The oral microbiota plays an important role in buccal health and in diseases such as periodontitis and meningitis. The study of the human oral bacteria has so far focused on subjects from Western societies, while little is known about subjects from isolated communities. This work determined the composition of the oral mucosa microbiota from six Amazon Amerindians, and tested a sample preservation alternative to freezing. Paired oral swabs were taken from six adults of Guahibo ethnicity living in the community of Platanillal, Amazonas State, Venezuela. Replicate swabs were preserved in liquid nitrogen and in Aware Messenger fluid (Calypte). Buccal DNA was extracted, and the V2 region of the 16S rRNA gene was amplified and pyrosequenced. A total of 17 214 oral bacterial sequences were obtained from the six subjects; these were binned into 1034 OTUs from 10 phyla, 30 families and 51 genera. The oral mucosa was highly dominated by four phyla: Firmicutes (mostly the genera Streptococcus and Veillonella), Proteobacteria (mostly Neisseria), Bacterioidetes (Prevotella) and Actinobacteria (Micrococcineae). Although the microbiota were similar at the phylum level, the Amerindians shared only 62 % of the families and 23 % of the genera with non-Amerindians from previous studies, and had a lower richness of genera (51 vs 177 reported in non-Amerindians). The Amerindians carried unidentified members of the phyla Bacteroidetes, Firmicutes and Proteobacteria and their microbiota included soil bacteria Gp1 (Acidobacteriaceae) and Xylanibacter (Prevotellaceae), and the rare genus Phocoenobacter (Pasteurellaceae). Preserving buccal swabs in the Aware Messenger oral fluid collection device substantially altered the bacterial composition in comparison to freezing, and therefore this method cannot be used to preserve samples for the study of microbial communities.

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.

Assessment of nutritional status in the Amazigh children of Amizmiz (Azgour Valley, High Atlas and Morocco).

The Berbers of the High Atlas (Amazigh) live in very severe socio-economic and climatic conditions, which expose children to the risk of malnutrition. In this study we used anthropometry and bioelectrical impedance analysis for the assessment of nutritional status. Height, weight and bioelectrical parameters were taken on 71 children (28 boys and 43 girls). Height and BMI were standardized using the 2007 WHO reference. The results show that 36.6% of the children were classified as stunted and 8.5% as wasted. Based on the Bioelectrical Impedance Vector Analysis, children from the High Atlas had an adequate body cell mass, but a high risk of dehydration (42.3%).

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.

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.

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.].

Do you have a probiotic in your future?

The possibility of using microbes to maintain health, and to prevent or treat disease is a topic as old as microbiology. However, one factor impeding the introduction of effective probiotics has been our very limited understanding of the composition of the human microbiome, as well as the biological requirements for these organisms. With advances in understanding the microbiome and its metagenome in humans and other mammals, we now can build a more robust scientific basis to develop probiotic strategies. Increasing knowledge of intramicrobial competition and cooperation, as well as host-microbe cross-signaling, will facilitate design of new probiotics and the modeling of their deployment, leading to eventual clinical trials.

Bacterial community in the crop of the hoatzin, a neotropical folivorous flying bird.

The hoatzin is unique among known avian species because of the fermentative function of its enlarged crop. A small-bodied flying foregut fermenter is a paradox, and this bird provides an interesting model to examine how diet selection and the gut microbiota contribute to maximizing digestive efficiency. Therefore, we characterized the bacterial population in the crop of six adult hoatzins captured from the wild. A total of 1,235 16S rRNA gene sequences were grouped into 580 phylotypes (67% of the pooled species richness sampled, based on Good's coverage estimator, with C(ACE) and Chao1 estimates of 1,709 and 1,795 species-level [99% identity] operational taxonomic units, respectively). Members of 9 of the approximately 75 known phyla in Bacteria were identified in this gut habitat; the Firmicutes were dominant (67% of sequences, belonging to the classes Clostridia, Mollicutes, and Bacilli), followed by the Bacteroidetes (30%, mostly in the order Bacteroidales), Proteobacteria (1.8%), and Lentisphaerae, Verrucomicrobia, TM7, Spirochaetes, Actinobacteria, and Aminanaerobia (all <0.1%). The novelty in this ecosystem is great; 94% of the phylotypes were unclassified at the "species" level and thus likely include novel cellulolytic lineages.

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.

Helicobacter pylori and intestinal parasites are not detrimental to the nutritional status of Amerindians.

Gastrointestinal parasites have evolved with humans and colonize many asymptomatic subjects. We investigated the influence of microbial gastrointestinal colonization on the nutritional status of rural Amerindians (40 males and 61 females). Helicobacter pylori was detected by 13C-breath test, and intestinal parasites were detected in fecal specimens. Body morphometry and bioelectrical impedance measurements were measured. Although Amerindians showed low height and weight for age, they had an adequate body mass index, morphometric parameters, and cell mass. Intestinal parasites were detected in 99% of the subjects, with no detrimental effect on nutritional parameters. Helicobacter pylori was present in 82% of adults and half the children, and was positively correlated with improved nutritional status. Despite the high prevalence of gastrointestinal microbes often associated with disease, the studied population of Amerindians had a body morphometry and composition indicative of good nutritional status, and improved in children positive for gastric H. pylori.

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.

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.

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.