Age and sex-associated variation in the multi-site microbiome of an entire social group of free-ranging rhesus macaques.

BACKGROUND: An individual's microbiome changes over the course of its lifetime, especially during infancy, and again in old age. Confounding factors such as diet and healthcare make it difficult to disentangle the interactions between age, health, and microbial changes in humans. Animal models present an excellent opportunity to study age- and sex-linked variation in the microbiome, but captivity is known to influence animal microbial abundance and composition, while studies of free-ranging animals are typically limited to studies of the fecal microbiome using samples collected non-invasively. Here, we analyze a large dataset of oral, rectal, and genital swabs collected from 105 free-ranging rhesus macaques (Macaca mulatta, aged 1 month-26 years), comprising one entire social group, from the island of Cayo Santiago, Puerto Rico. We sequenced 16S V4 rRNA amplicons for all samples. RESULTS: Infant gut microbial communities had significantly higher relative abundances of Bifidobacterium and Bacteroides and lower abundances of Ruminococcus, Fibrobacter, and Treponema compared to older age groups, consistent with a diet high in milk rather than solid foods. The genital microbiome varied widely between males and females in beta-diversity, taxonomic composition, and predicted functional profiles. Interestingly, only penile, but not vaginal, microbiomes exhibited distinct age-related changes in microbial beta-diversity, taxonomic composition, and predicted functions. Oral microbiome composition was associated with age, and was most distinctive between infants and other age classes. CONCLUSIONS: Across all three body regions, with notable exceptions in the penile microbiome, while infants were distinctly different from other age groups, microbiomes of adults were relatively invariant, even in advanced age. While vaginal microbiomes were exceptionally stable, penile microbiomes were quite variable, especially at the onset of reproductive age. Relative invariance among adults, including elderly individuals, is contrary to findings in humans and mice. We discuss potential explanations for this observation, including that age-related microbiome variation seen in humans may be related to changes in diet and lifestyle. Video abstract.

Changes in the vaginal microbiota across a gradient of urbanization.

The vaginal microbiota of healthy women typically has low diversity, which increases after perturbations. Among these, lifestyle associated with certain sexual and antimicrobial practices may be associated with higher diversity. To test this hypothesis, we characterized the vaginal microbiota in the cervicovaginal and introital sites in sexually active Amerindians (N = 82) spanning urbanization, and in urban mestizos (N = 29), in the Venezuelan Amazonas. HPV status was also considered. Sampling was performed in an urban gradient from remote villages to a town, and women were individually classified by the degree of urbanization (low, medium, and high). Amerindian cervicovaginal and introital microbiota diversity were not associated with major changes in urbanization or ethnicity. There was a non-significant trend of increased diversity with urbanization, with a few taxa found overrepresented in urban Amerindians (Brevibacterium linens and Peptoniphilus lacrimalis) or mestizos (Mobiluncus mulieris and Prevotella sp.). Among all women, cervicovaginal and introital samples clustered, respectively, in four and two community state types (CSTs), where most profiles were dominated by Lactobacillus iners, Gardnerella vaginalis or were highly diverse profiles. HPV status did not associate with microbial diversity. In conclusion, no association was found between urban level and the vaginal microbiome in Amerindian women, and little difference was found between ethnicities. L. iners and high diversity profiles, associated with vaginal health outcomes, prevail in these populations.

Maternal H. pylori is associated with differential fecal microbiota in infants born by vaginal delivery.

Helicobacter pylori colonization may affect the mucosal immune system through modification of microbiota composition and their interactions with the host. We hypothesized that maternal H. pylori status affects the maternal intestinal microbiota of both mother and newborn. In this study, we determine the structure of the fecal microbiota in mothers and neonates according to maternal H. pylori status and delivery mode. We included 22 mothers and H. pylori infection was determined by fecal antigen test. Eleven mothers (50%) were H. pylori-positive (7 delivering vaginally and 4 by C-section), and 11 were negative (6 delivering vaginally and 5 by C-section). Stool samples were obtained from mothers and infants and the fecal DNA was sequenced. The fecal microbiota from mothers and their babies differed by the maternal H. pylori status, only in vaginal birth, not in C-section delivery. All 22 infants tested negative for fecal H. pylori at 15 days of age, but those born vaginally -and not those by C-section- showed differences in the infant microbiota by maternal H. pylori status (PERMANOVA, p = 0.01), with higher abundance of Enterobacteriaceae and Veillonella, in those born to H. pylori-positive mothers. In conclusion, the structure of the infant fecal microbiota is affected by the maternal H. pylori status only in infants born vaginally, suggesting that the effect could be mediated by labor and birth exposures.

An in-depth survey of the microbial landscape of the walls of a neonatal operating room.

Bacteria found in operating rooms (ORs) might be clinically relevant since they could pose a threat to patients. In addition, C-sections operations are performed in ORs that provide the first environment and bacterial exposure to the sterile newborns that are extracted directly from the uterus to the OR air. Considering that at least one third of neonates in the US are born via C-section delivery (and more than 50% of all deliveries in some countries), understanding the distribution of bacterial diversity in ORs is critical to better understanding the contribution of the OR microbiota to C-section- associated inflammatory diseases. Here, we mapped the bacteria contained in an OR after a procedure was performed; we sampled grids of 60x60 cm across walls and wall-adjacent floors and sequenced the V4 region of 16S rRNA gene from 260 samples. The results indicate that bacterial communities changed significantly (ANOSIM, p-value < 0.001) with wall height, with an associated reduction of alpha diversity (t-test, p-value <0.05). OR walls contained high proportions of Proteobacteria, Firmicutes, and Actinobacteria, with Proteobacteria and Bacteroidetes being the highest in floors and lowest in the highest wall sites. Members of Firmicutes, Deinococcus-thermus, and Actinobacteria increased with wall height. Source-track analysis estimate that human skin is the major source contributing to bacterial composition in the OR walls, with an increase of bacteria related to human feces in the lowest walls and airborne bacteria in the highest wall sites. The results show that bacterial exposure in ORs varies spatially, and evidence exposure of C-section born neonates to human bacteria that remain on the floors and walls, possibly accumulated from patients, health, and cleaning staff.

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.

The Skin Microbiota of Eleutherodactylus Frogs: Effects of Host Ecology, Phylogeny, and Local Environment.

Amphibian skin microbiota has a potential protective role against diseases. However, the effects of environmental and host factors on symbiotic bacterial communities are not well understood. Caribbean frogs in the genus Eleutherodactylus represent a case of congeneric species that differ in ecological specialization by the process of adaptive radiation. For a small clade of Eleutherodactylus from Puerto Rico, we investigated the role of local environments, host species, and microhabitat in the composition of their skin microbiome. The potential congruence between microbial communities in hosts that are most closely related phylogenetically was also addressed. We hypothesized that the skin microbiota of Eleutherodactylus frogs would be mostly associated to microhabitat use, but also differ according to locality, and to a lesser extent to host species. To test this hypothesis, we swabbed the skin of a total of 98 adult individuals of seven Eleutherodactylus species distributed in two nearby localities in Puerto Rico, and sequenced the V4 region of the 16S rRNA gene. Results showed that locality had the greatest effect on determining skin bacterial communities of amphibian hosts, but this effect was stronger on the composition (based on presence/absence) than on its structure (based on sequence abundance). The most ecologically distinct host, E. cooki, and the generalist E. coqui presented, respectively, the most dissimilar and similar microbiota compared to other hosts. Host phylogeny showed a weak influence on skin microbiota. Results suggest that both local environment and ecological specialization are structuring the skin bacterial community in these Eleutherodactylus species, but that characteristics intrinsic to species may also render unique hosts the ability to maintain distinct microbiotas.

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.

Comparative prevalence of Oxalobacter formigenes in three human populations.

There has been increasing interest in the human anaerobic colonic bacterium Oxalobacter formigenes because of its ability to metabolize oxalate, and its potential contribution to protection from calcium oxalate kidney stones. Prior studies examining the prevalence of this organism have focused on subjects in developed countries and on adults. Now using O. formigenes-specific PCR, we have compared the prevalence of these organisms among subjects in two remote areas in which modern medical practices have hardly been present with a USA group of mothers and their infants for the first three years of life. Among the Amerindians of the Yanomami-Sanema and Yekwana ethnic groups in Venezuela and the Hadza in Tanzania, O. formigenes was detected in 60-80% of the adult subjects, higher than found in adults from USA in this and prior studies. In young children, the prevalence was much lower in USA than in either tribal village. These data extend our understanding of the epidemiology of O. formigenes carriage, and are consistent with the hypothesis that the rising incidence of kidney stones is associated with the progressive loss of O. formigenes colonization in populations that have been highly impacted by modern medical practices.

Associations of birth mode with cord blood cytokines, white blood cells, and newborn intestinal bifidobacteria.

The associations of Cesarean delivery with offspring metabolic and immune-mediated diseases are believed to derive from lack of mother-to-newborn transmission of specific microbes at birth. Bifidobacterium spp., in particular, has been hypothesized to play a health-promoting role, yet little is known about how delivery mode modifies colonization of the newborn by this group of microbes. The aim of this research was to examine the presence of Bifidobacterium in meconium and in the transitional stool, and to assess cytokine levels and hematological parameters in the venous cord blood of infants born by elective, pre-labor Cesarean section vs. vaginal delivery in Southern Brazil. We recruited 89 mother-newborn pairs (23 vaginal delivery and 66 elective cesarean delivery), obtained demographic information from a structured questionnaire and clinical information from medical records. We obtained umbilical cord venous blood and meconium samples following delivery and the transitional stool (the first defecation after meconium) before discharge. We determined plasma levels of IL-1ß, IL-10, IL-6, GM-CSF, IL-5, IFN-γ, TNF-α, IL-2, IL-4 and IL-8 in the cord blood, and presence of stool Bifidobacterium by real time PCR. Compared to vaginally-delivered neonates, Cesarean-delivered neonates had a lower leukocyte count (p = 0.037), lower hemoglobin (p = 0.04), and lower levels of the cytokine GM-CSF (p = 0.009) in the cord blood. Moreover, Bifidobacterium was detected less often in the transitional stool of Cesarean-delivered neonates compared to vaginally-delivered neonates (p = 0.001). The results indicate that pre-labor Cesarean birth may be associated with microbial and hematological alterations in the neonate. The clinical significance of these findings remains to be determined in larger prospective birth cohort studies.

Complex virome in feces from Amerindian children in isolated Amazonian villages.

The number of viruses circulating in small isolated human populations may be reduced by viral extinctions and rare introductions. Here we used viral metagenomics to characterize the eukaryotic virome in feces from healthy children from a large urban center and from three Amerindian villages with minimal outside contact. Numerous human enteric viruses, mainly from the Picornaviridae and Caliciviridae families, were sequenced from each of the sites. Multiple children from the same villages shed closely related viruses reflecting frequent transmission clusters. Feces of isolated villagers also contained multiple viral genomes of unknown cellular origin from the Picornavirales order and CRESS-DNA group and higher levels of nematode and protozoan DNA. Despite cultural and geographic isolation, the diversity of enteric human viruses was therefore not reduced in these Amazonian villages. Frequent viral introductions and/or increased susceptibility to enteric infections may account for the complex fecal virome of Amerindian children in isolated villages.

High Rate of Infection by Only Oncogenic Human Papillomavirus in Amerindians.

Human papillomavirus (HPV), an etiological agent of cervical cancer (CC), has infected humans since ancient times. Amerindians are the furthest migrants out of Africa, and they reached the Americas more than 14,000 years ago. Some groups still remain isolated, and some migrate to towns, forming a gradient spanning urbanization. We hypothesized that, by virtue of their history, lifestyle, and isolation from the global society, remote Amerindian women have lower HPV diversity than do urban women (Amerindian or mestizo). Here we determined the diversity of the 25 most relevant cervical HPV types in 82 Amerindians spanning urbanization (low, medium, and high, consistent with the exposure to urban lifestyles of the town of Puerto Ayacucho in the Venezuelan Amazonas State), and in 29 urban mestizos from the town. Cervical, anal, oral, and introitus samples were taken, and HPVs were typed using reverse DNA hybridization. A total of 23 HPV types were detected, including 11 oncogenic or high-risk types, most associated with CC. Cervical HPV prevalence was 75%, with no differences by group, but Amerindians from low and medium urbanization level had significantly lower HPV diversity than mestizos did. In Amerindians, but not in mestizos, infections by only high-risk HPVs were higher than coinfections or by exclusively low-risk HPVs. Cervical abnormalities only were observed in Amerindians (9/82), consistent with their high HPV infection. The lower cervical HPV diversity in more isolated Amerindians is consistent with their lower exposure to the global pool, and transculturation to urban lifestyles could have implications on HPV ecology, infection, and virulence.IMPORTANCE The role of HPV type distribution on the disparity of cervical cancer (CC) incidence between human populations remains unknown. The incidence of CC in the Amazonas State of Venezuela is higher than the national average. In this study, we determined the diversity of known HPV types (the viral agent of CC) in Amerindian and mestizo women living in the Venezuelan Amazonas State. Understanding the ecological diversity of HPV in populations undergoing lifestyle transformations has important implication on public health measures for CC prevention.

Comparison of gizzard and intestinal microbiota of wild neotropical birds.

Gut bacterial communities have been shown to be influenced by diet, host phylogeny and anatomy, but most of these studies have been done in captive animals. Here we compare the bacterial communities in the digestive tract of wild birds. We characterized the gizzard and intestinal microbiota among 8 wild Neotropical bird species, granivorous or frugivorous species of the orders Columbiformes and Passeriformes. We sequenced the V4 region of the 16S rRNA gene in 94 collected samples from 32 wild birds from 5 localities, and compared bacterial communities by foraging guild, organ, locality and bird taxonomy. 16S rRNA gene-based sequencing data were examined using QIIME with linear discriminant analysis effect size (LEfSe) and metabolic pathways were predicted using PICRUSt algorism. We identified 8 bacterial phyla, dominated by Firmicutes, Actinobacteria and Proteobacteria. Beta diversity analyses indicated significant separation of gut communities by bird orders (Columbiformes vs. Passerifomes) and between bird species (p<0.01). In lower intestine, PICRUSt shows a predominance of carbohydrate metabolism in granivorous birds and xenobiotics biodegradation pathways in frugivorous birds. Gizzard microbiota was significantly richer in granivorous, in relation to frugivorous birds (Chao 1; non-parametric t-test, p<0.05), suggesting a microbial gizzard function, beyond grinding food. The results suggest that the most important factor separating the bacterial community structure was bird taxonomy, followed by foraging guild. However, variation between localities is also likely to be important, but this could not been assessed with our study design.

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.

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.

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.

Fecal microbiota imbalance in Mexican children with type 1 diabetes.

Dysbiosis of the intestinal microbiota affecting the gut barrier could be triggering Type 1 Diabetes (T1D), the second most frequent autoimmune disease in childhood. This study compared the structure of the fecal microbiota in 29 mestizo children aged 7-18 years, including 8 T1D at onset, 13 T1D after 2 years treatment, and 8 healthy controls. Clinical information was collected, predisposing haplotypes were determined; the fecal DNA was extracted, the V4 region of the 16S rRNA gene amplified and 454-pyrosequenced. The newly diagnosed T1D cases had high levels of the genus Bacteroides (p < 0.004), whereas the control group had a gut microbiota dominated by Prevotella. Children with T1D treated for ≥2 years had levels of Bacteroides and Prevotella compared to those of the control group. The gut microbiota of newly diagnosed T1D cases is altered, but whether it is involved in disease causation or is a consequence of host selection remains unclear.

Biphasic assembly of the murine intestinal microbiota during early development.

The birth canal provides mammals with a primary maternal inoculum, which develops into distinctive body site-specific microbial communities post-natally. We characterized the distal gut microbiota from birth to weaning in mice. One-day-old mice had colonic microbiota that resembled maternal vaginal communities, but at days 3 and 9 of age there was a substantial loss of intestinal bacterial diversity and dominance of Lactobacillus. By weaning (21 days), diverse intestinal bacteria had established, including strict anaerobes. Our results are consistent with vertical transmission of maternal microbiota and demonstrate a nonlinear ecological succession involving an early drop in bacterial diversity and shift in dominance from Streptococcus to Lactobacillus, followed by an increase in diversity of anaerobes, after the introduction of solid food. Mammalian newborns are born highly susceptible to colonization, and lactation may control microbiome assembly during early development.