Limited information is available about the effects of HIV and subsequent antiretroviral treatment on host-microbe interactions. This study aimed to determine the salivary microbial composition for 10 HIV-seropositive subjects, before and 6 months after highly active antiretroviral therapy (HAART), compared with that for 10 HIV-seronegative subjects. A conventional culture and two culture-independent analyses were used and consistently demonstrated differences in microbial composition among the three sets of samples. HIV-positive subjects had higher levels of total cultivable microbes, including oral streptococci, lactobacilli, Streptococcus mutans, and Candida, in saliva than did HIV-negative subjects. The total cultivable microbial levels were significantly correlated with CD4+ T cell counts. Denaturing gradient gel electrophoresis (DGGE), which compared the overall microbial profiles, showed distinct fingerprinting profiles for each group. The human oral microbe identification microarray (HOMIM) assay, which compared the 16S rRNA genes, showed clear separation among the three sample groups. Veillonella, Synergistetes, and Streptococcus were present in all 30 saliva samples. Only minor changes or no changes in the prevalence of Neisseria, Haemophilus, Gemella, Leptotrichia, Solobacterium, Parvimonas, and Rothia were observed. Seven genera, Capnocytophaga, Slackia, Porphyromonas, Kingella, Peptostreptococcaceae, Lactobacillus, and Atopobium, were detected only in HIV-negative samples. The prevalences of Fusobacterium, Campylobacter, Prevotella, Capnocytophaga, Selenomonas, Actinomyces, Granulicatella, and Atopobium were increased after HAART. In contrast, the prevalence of Aggregatibacter was significantly decreased after HAART. The findings of this study suggest that HIV infection and HAART can have significant effects on salivary microbial colonization and composition.
In an effort to suppress microbial outgrowth, the host sequesters essential nutrients in a process termed nutritional immunity. However, inflammatory responses to bacterial insult can restore nutritional resources. Given that nutrient availability modulates virulence factor production and biofilm formation by other bacterial species, we hypothesized that fluctuations in heme-iron availability, particularly at privileged sites, would similarly influence Haemophilus biofilm formation and pathogenesis. Thus, we cultured Haemophilus through sequential heme-iron deplete and heme-iron replete media to determine the effect of transient depletion of internal stores of heme-iron on multiple pathogenic phenotypes. We observed that prior heme-iron restriction potentiates biofilm changes for at least 72 hours that include increased peak height and architectural complexity as compared to biofilms initiated from heme-iron replete bacteria, suggesting a mechanism for epigenetic responses that participate in the changes observed. Additionally, in a co-infection model for human otitis media, heme-iron restricted Haemophilus, although accounting for only 10% of the inoculum (90% heme-iron replete), represented up to 99% of the organisms recovered at 4 days. These data indicate that fluctuations in heme-iron availability promote a survival advantage during disease. Filamentation mediated by a SulA-related ortholog was required for optimal biofilm peak height and persistence during experimental otitis media. Moreover, severity of disease in response to heme-iron restricted Haemophilus was reduced as evidenced by lack of mucosal destruction, decreased erythema, hemorrhagic foci and vasodilatation. Transient restriction of heme-iron also promoted productive invasion events leading to the development of intracellular bacterial communities. Taken together, these data suggest that nutritional immunity, may, in fact, foster long-term phenotypic changes that better equip bacteria for survival at infectious sites.
Clinical management of upper and lower respiratory tract diseases caused by nontypeable Haemophilus influenzae (NTHI) is a significant socioeconomic burden. Therapies targeting the pathogenic lifestyle of NTHI remain non-existent due to a lack of understanding of host microenvironmental cues and bacterial responses that dictate NTHI persistence. Iron availability influences bacterial virulence traits and biofilm formation; yet, host sequestration of iron serves to restrict bacterial growth. We predicted that fluctuations in availability of iron-containing compounds, typically associated with infection, would impact NTHI pathogenesis. We demonstrated that transient restriction of heme-iron triggered an epigenetic developmental program that enhanced NTHI biofilm architecture, directly influenced by induced morphological changes in bacterial length. Heme-iron restricted bacteria were primed for survival in the mammalian middle ear, due in part to an observed reduction in host inflammation coinciding with a striking reduction in host mucosal epithelial damage, compared to that observed in response to heme-iron replete NTHI. Moreover, transiently restricted NTHI were more invasive of epithelial cells resulting in formation of intracellular bacterial communities. Our findings significantly advance our understanding of how host immune pressure and nutrient availability influence pathogenic behaviors that impact disease severity.
Oral bacterial communities contain species that promote health and others that have been implicated in oral and/or systemic diseases. Culture-independent approaches provide the best means to assess the diversity of oral bacteria because most of them remain uncultivable.
The salivary microbiota from five adults was analyzed at three time-points by means of the 454 pyrosequencing technology. The V1-V3 region of the bacterial 16S rRNA genes was amplified by PCR using saliva lysates and broad-range primers. The bar-coded PCR products were pooled and sequenced unidirectionally to cover the V3 hypervariable region. Of 50,708 obtained sequences, 31,860 passed the quality control. Non-bacterial sequences (2.2%) were removed leaving 31,170 reads. Samples were dominated by seven major phyla: members of Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes and candidate division TM7 were identified in all samples; Fusobacteria and Spirochaetes were identified in all individuals, but not at all time-points. The dataset was represented by 3,011 distinct sequences (100%-ID phylotypes) of ~215 nucleotides and 583 phylotypes defined at ≥97% identity (97%-ID phylotypes). We compared saliva samples from different individuals in terms of the phylogeny of their microbial communities. Based on the presence and absence of phylotypes defined at 100% or 97% identity thresholds, samples from each subject formed separate clusters. Among individual taxa, phylum Bacteroidetes and order Clostridiales (Firmicutes) were the best indicators of intraindividual similarity of the salivary flora over time. Fifteen out of 81 genera constituted 73 to 94% of the total sequences present in different samples. Of these, 8 were shared by all time points of all individuals, while 15-25 genera were present in all three time-points of different individuals. Representatives of the class Sphingobacteria, order Sphingobacteriales and family Clostridiaceae were found only in one subject.
The salivary microbial community appeared to be stable over at least 5 days, allowing for subject-specific grouping using UniFrac. Inclusion of all available samples from more distant time points (up to 29 days) confirmed this observation. Samples taken at closer time intervals were not necessarily more similar than samples obtained across longer sampling times. These results point to the persistence of subject-specific taxa whose frequency fluctuates between the time points. Genus Gemella, identified in all time-points of all individuals, was not defined as a core-microbiome genus in previous studies of salivary bacterial communities. Human oral microbiome studies are still in their infancy and larger-scale projects are required to better define individual and universal oral microbiome core.
The salivary microbiota has been linked to both oral and non-oral diseases. Scant knowledge is available on the effect of environmental factors such as long-term dietary choices on the salivary microbiota and metabolome. This study analyzed the microbial diversity and metabolomic profiles of the saliva of 161 healthy individuals who followed an omnivore or ovo-lacto-vegetarian or vegan diet. A large core microbiota was identified, including 12 bacterial genera, found in >98% of the individuals. The subjects could be stratified into three “salivary types” that differed on the basis of the relative abundance of the core genera Prevotella, Streptococcus/Gemella and Fusobacterium/Neisseria. Statistical analysis indicated no effect of dietary habit on the salivary microbiota. Phylogenetic beta-diversity analysis consistently showed no differences between omnivore, ovo-lacto-vegetarian and vegan individuals. Metabolomic profiling of saliva using 1H-NMR and GC-MS/SPME identified diet-related biomarkers that enabled a significant discrimination between the 3 groups of individuals on the basis of their diet. Formate, urea, uridine and 5-methyl-3-hexanone could discriminate samples from omnivores, whereas 1-propanol, hexanoic acid and proline were characteristic of non-omnivore diets. Although the salivary metabolome can be discriminating for diet, the microbiota has a remarkable inter-individual stability and did not vary with dietary habits. Microbial homeostasis might be perturbed with sub-standard oral hygiene or other environmental factors, but there is no current indication that a choice of an omnivore, ovo-lacto-vegetarian or vegan diet can lead to a specific composition of the oral microbiota with consequences on the oral homeostasis.
The associations between oral diseases and increased risk of pancreatic cancer have been reported in several prospective cohort studies. In this study, we measured variations of salivary microbiota and evaluated their potential associations with pancreatic cancer and chronic pancreatitis.
This study was divided into three phases: (1) microbial profiling using the Human Oral Microbe Identification Microarray to investigate salivary microbiota variation between 10 resectable patients with pancreatic cancer and 10 matched healthy controls, (2) identification and verification of bacterial candidates by real-time quantitative PCR (qPCR) and (3) validation of bacterial candidates by qPCR on an independent cohort of 28 resectable pancreatic cancer, 28 matched healthy control and 27 chronic pancreatitis samples.
Comprehensive comparison of the salivary microbiota between patients with pancreatic cancer and healthy control subjects revealed a significant variation of salivary microflora. Thirty-one bacterial species/clusters were increased in the saliva of patients with pancreatic cancer (n=10) in comparison to those of the healthy controls (n=10), whereas 25 bacterial species/clusters were decreased. Two out of six bacterial candidates (Neisseria elongata and Streptococcus mitis) were validated using the independent samples, showing significant variation (p<0.05, qPCR) between patients with pancreatic cancer and controls (n=56). Additionally, two bacteria (Granulicatella adiacens and S mitis) showed significant variation (p<0.05, qPCR) between chronic pancreatitis samples and controls (n=55). The combination of two bacterial biomarkers (N elongata and S mitis) yielded a receiver operating characteristic plot area under the curve value of 0.90 (95% CI 0.78 to 0.96, p<0.0001) with a 96.4% sensitivity and 82.1% specificity in distinguishing patients with pancreatic cancer from healthy subjects.
The authors observed associations between variations of patients’ salivary microbiota with pancreatic cancer and chronic pancreatitis. This report also provides proof of salivary microbiota as an informative source for discovering non-invasive biomarkers of systemic diseases.
As part of the human gastrointestinal tract, the oral cavity represents a complex biological system and harbors diverse bacterial species. Unlike the gut microbiota which is often considered a health asset, studies of the oral commensal microbial flora have been largely limited to their implication in oral diseases such as dental caries and periodontal diseases; Little emphasis has been given to their potential beneficial roles, especially the protective effects against oral colonization by foreign/pathogenic bacteria. In this study, we used the salivary microbiota derived from healthy human subjects to investigate protective effects against the colonization and integration of Pseudomonas aeruginosa, an opportunistic bacterial pathogen, into developing and pre-formed salivary biofilms. When co-cultivated in saliva medium, P. aeruginosa persisted in the planktonic phase, but failed to integrate into salivary microbial community during biofilm formation. Furthermore, in the saliva medium supplemented with 0.05% (w/v) sucrose, the oral flora inhibited the growth of P. aeruginosa by producing lactic acid. More interestingly, while pre-formed salivary biofilms were able to prevent P. aeruginosa colonization, the same biofilms recovered from mild chlorhexidine gluconate treatment displayed a shift in microbial composition and showed a drastic reduction in protection. Our study indicates that normal oral communities with balanced microbial compositions could be important in effectively preventing the integration of foreign/pathogenic bacterial species, such as P. aeruginosa.
bacterial interference; microbial flora; oral cavity; Pseudomonas aeruginosa; salivary biofilm
Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms.
Fungal infections contribute substantially to human morbidity and mortality. During infectious processes, fungi have evolved mechanisms to obtain iron from high-affinity iron-binding proteins. In the current study, we demonstrated that hemoglobin is the preferential host iron source for the thermodimorphic fungus Paracoccidioides spp. To acquire hemoglobin, the fungus presents hemolytic activity and the ability to internalize protoporphyrin rings. A putative hemoglobin receptor, Rbt5, was demonstrated to be GPI-anchored at the yeast cell surface. Rbt5 was able to bind to hemin, protoporphyrin and hemoglobin in vitro. When rbt5 expression was inhibited, the survival of Paracoccidioides sp. inside macrophages and the fungal burden in mouse spleen diminished, which indicated that Rbt5 could participate in the establishment of the fungus inside the host. Drugs or vaccines could be developed against Paracoccidioides spp. Rbt5 to disturb iron uptake of this micronutrient and, thus, the proliferation of the fungus. Moreover, this protein could be used in routes to introduce antifungal agents into fungal cells.
Although bacteria are continually acquired over the lifetime of an individual, the phylogenetic relationships of great ape species is mirrored in the compositions of their gut microbial communities.
Multiple factors over the lifetime of an individual, including diet, geography, and physiologic state, will influence the microbial communities within the primate gut. To determine the source of variation in the composition of the microbiota within and among species, we investigated the distal gut microbial communities harbored by great apes, as present in fecal samples recovered within their native ranges. We found that the branching order of host-species phylogenies based on the composition of these microbial communities is completely congruent with the known relationships of the hosts. Although the gut is initially and continuously seeded by bacteria that are acquired from external sources, we establish that over evolutionary timescales, the composition of the gut microbiota among great ape species is phylogenetically conserved and has diverged in a manner consistent with vertical inheritance.
The microbial communities that inhabit the gastrointestinal tract of humans and other mammals are complex, dynamic, and critical to both health and disease. The composition and constituents of these communities are influenced by multiple factors such as host diet, geography, physiology, and disease state. Given the central role of the gut microbiota in the physiology of the host, it is important to determine whether it is predictable and substantially determined by the host, or variable and largely determined by the external environment (including diet) experienced by the host. A valuable way of determining the relative contributions of such factors is by comparing gut microbial communities in closely related host species. Applying a high-throughput sequencing approach, we profiled the distal gut microbiotae of great ape species sampled in their native ranges and then employed a parsimony-based analysis of phylogenetically informative phylotypes (i.e., bacterial taxa residing in multiple individuals) to determine the relationships among the diverse microbial communities. Our analyses revealed a clear species-specific signature of microbial community structure. Moreover, the pattern of relationships among the five great ape species (Homo sapiens, Pan troglodytes, P. paniscus, Gorilla gorilla, and G. beringei) inferred from their fecal microbial communities was identical to that inferred from host mitochondrial DNA, indicating that host phylogeny shapes the gut microbiota over evolutionary timescales. It seems after all that you are not what you eat.
The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.
Artificial human gut microbial communities implanted into germ-free mice provide insights into how species-level responses to changes in diet give rise to community-level structural and functional reconfiguration and how types of bacteria prioritize use of available nutrients in vivo.
The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many of these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs) in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising >90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism's 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These results offer insight into how gut microbes adapt to dietary perturbations at both a community level and from the perspective of a well-adapted symbiont with exceptional saccharolytic capabilities, and illustrate the value of artificial communities.
Our intestines are populated by an almost unimaginably large number of microbial cells, most of which are bacteria. This species assemblage operates as a microbial metabolic organ, performing myriad tasks that contribute to our well-being, including processing components of our diet. The way this incredible machine assembles itself and operates remains mysterious. One approach to understanding its properties is to create artificial communities composed of a limited number of sequenced human gut bacterial species and to install them in the guts of germ-free mice that are then fed different diets. In this report, we adopt this approach. We describe the genome sequence of a new gut bacterial isolate, Bacteroides cellulosilyticus WH2, which is equipped with an unprecedented number of carbohydrate active enzymes. Deploying four different “omics” technologies, we characterize the response to diet, the relative stability, and the temporal dynamics of a 12-species artificial bacterial assemblage (including B. cellulosilyticus WH2) implanted in germ-free mouse guts. We also combine high-throughput substrate utilization screens and RNA-Seq to generate reference data analogous to a “Rosetta stone” in order to decipher what types of carbohydrates B. cellulosilyticus encounters and uses within the gut, and how it interacts with other organisms that have similar and/or distinct “professions.” This work sets the stage for future ecological and metabolic studies of more complex assemblages that more fully emulate the properties of our native gut communities.
Root exudate composition and quantity vary in relation to plant nutritional status, but the impact of the differences on rhizosphere microbial communities is not known. To examine this question, we performed an experiment with barley (Hordeum vulgare) plants under iron-limiting and iron-sufficient growth conditions. Plants were grown in an iron-limiting soil in root box microcosms. One-half of the plants were treated with foliar iron every day to inhibit phytosiderophore production and to alter root exudate composition. After 30 days, the bacterial communities associated with different root zones, including the primary root tips, nonelongating secondary root tips, sites of lateral root emergence, and older roots distal from the tip, were characterized by using 16S ribosomal DNA (rDNA) fingerprints generated by PCR-denaturing gradient gel electrophoresis (DGGE). Our results showed that the microbial communities associated with the different root locations produced many common 16S rDNA bands but that the communities could be distinguished by using correspondence analysis. Approximately 40% of the variation between communities could be attributed to plant iron nutritional status. A sequence analysis of clones generated from a single 16S rDNA band obtained at all of the root locations revealed that there were taxonomically different species in the same band, suggesting that the resolving power of DGGE for characterization of community structure at the species level is limited. Our results suggest that the bacterial communities in the rhizosphere are substantially different in different root zones and that a rhizosphere community may be altered by changes in root exudate composition caused by changes in plant iron nutritional status.
Almost immediately after a human being is born, so too is a new microbial ecosystem, one that resides in that person's gastrointestinal tract. Although it is a universal and integral part of human biology, the temporal progression of this process, the sources of the microbes that make up the ecosystem, how and why it varies from one infant to another, and how the composition of this ecosystem influences human physiology, development, and disease are still poorly understood. As a step toward systematically investigating these questions, we designed a microarray to detect and quantitate the small subunit ribosomal RNA (SSU rRNA) gene sequences of most currently recognized species and taxonomic groups of bacteria. We used this microarray, along with sequencing of cloned libraries of PCR-amplified SSU rDNA, to profile the microbial communities in an average of 26 stool samples each from 14 healthy, full-term human infants, including a pair of dizygotic twins, beginning with the first stool after birth and continuing at defined intervals throughout the first year of life. To investigate possible origins of the infant microbiota, we also profiled vaginal and milk samples from most of the mothers, and stool samples from all of the mothers, most of the fathers, and two siblings. The composition and temporal patterns of the microbial communities varied widely from baby to baby. Despite considerable temporal variation, the distinct features of each baby's microbial community were recognizable for intervals of weeks to months. The strikingly parallel temporal patterns of the twins suggested that incidental environmental exposures play a major role in determining the distinctive characteristics of the microbial community in each baby. By the end of the first year of life, the idiosyncratic microbial ecosystems in each baby, although still distinct, had converged toward a profile characteristic of the adult gastrointestinal tract.
It has been recognized for nearly a century that human beings are inhabited by a remarkably dense and diverse microbial ecosystem, yet we are only just beginning to understand and appreciate the many roles that these microbes play in human health and development. Knowing the composition of this ecosystem is a crucial step toward understanding its roles. In this study, we designed and applied a ribosomal DNA microarray-based approach to trace the development of the intestinal flora in 14 healthy, full-term infants over the first year of life. We found that the composition and temporal patterns of the microbial communities varied widely from baby to baby, supporting a broader definition of healthy colonization than previously recognized. By one year of age, the babies retained their uniqueness but had converged toward a profile characteristic of the adult gastrointestinal tract. The composition and temporal patterns of development of the intestinal microbiota in a pair of fraternal twins were strikingly similar, suggesting that genetic and environmental factors shape our gut microbiota in a reproducible way.
Microarray profiling of the microbial communities of infant guts throughout the first year shows initial variation then convergence on the adult flora, providing new insight into this human ecosystem.
More than 700 bacterial species have been detected in human oral cavity. They form highly organized microbial communities and are responsible for many oral infectious diseases, such as dental caries and periodontal disease. The prevention and treatment of these diseases require a comprehensive knowledge of oral microbial communities, which largely relies on culture-dependent methods to have detailed phenotypic and physiological analysis of these communities. However, most of the currently available lab media can only selectively support the growth of a limited number of bacterial species within these communities, and fail to sustain the original oral microbial diversity. In this study, using denaturing gradient gel electrophoresis (DGGE) as an index to systematically survey and analyze the selectivity of commonly used lab media, we developed a new medium (SHI medium) by combining the ingredients of several selected media which can support different sub-populations within the original oral microbial community derived from pooled saliva. DGGE and 454 pyrosequencing analysis showed that SHI medium was capable of supporting a more diversified community with a microbial profile closest to that of the original oral microbiota. Furthermore, 454 pyrosequencing revealed that SHI medium supported the growth of many oral species that have not been cultured so far. Crystal violet assay and the CLSM (confocal laser scanning microscope) analysis indicated that, compared with other media, SHI medium is able to support more complex saliva-derived biofilm with higher biomass yield and more diversified species. This DGGE-guided method could also be used to develop novel media for other complex microbial communities.
oral microbial community; growth medium; 454 pyrosequencing
Bioavailable levels of trace metals, such as iron and zinc, for bacterial growth in nature are sufficiently low that most microbes have evolved high-affinity binding and transport systems. The microbe Campylobacter jejuni lives in the gastrointestinal tract of chickens, the principal source of human infection. A high-affinity ABC transporter for zinc uptake is required for Campylobacter survival in chicken intestines in the presence of a normal microbiota but not when chickens are raised with a limited microbiota. Mass spectrometric analysis of cecal contents revealed the presence of numerous zinc-binding proteins in conventional chicks compared to the number in limited-microbiota chicks. The presence of a microbiota results in the production of host zinc-binding enzymes, causing a growth restriction for bacteria that lack the high-affinity zinc transporter. Such transporters in a wide range of pathogenic bacteria make them good targets for the development of broad-spectrum antimicrobials.
Importance Zinc is an essential trace element for the growth of most organisms. Quantities of zinc inside cells are highly regulated, as too little zinc does not support growth, while too much zinc is toxic. Numerous bacterial cells require zinc uptake systems for growth and virulence. The work presented here demonstrates that the microbiota in the gastrointestinal tract reduces the quantity of zinc. Without a high-affinity zinc transporter, Campylobacter jejuni, a commensal organism of chickens, is unable to replicate or colonize the gastrointestinal tract. This is the first demonstration of zinc competition between microbiota in the gastrointestinal tract of a host. These results could have profound implications in the field of microbial pathogenesis and in our understanding of host metabolism and the microbiota.
Zinc is an essential trace element for the growth of most organisms. Quantities of zinc inside cells are highly regulated, as too little zinc does not support growth, while too much zinc is toxic. Numerous bacterial cells require zinc uptake systems for growth and virulence. The work presented here demonstrates that the microbiota in the gastrointestinal tract reduces the quantity of zinc. Without a high-affinity zinc transporter, Campylobacter jejuni, a commensal organism of chickens, is unable to replicate or colonize the gastrointestinal tract. This is the first demonstration of zinc competition between microbiota in the gastrointestinal tract of a host. These results could have profound implications in the field of microbial pathogenesis and in our understanding of host metabolism and the microbiota.
The development of multispecies oral microbial communities involves complex intra- and interspecies interactions at various levels. The ability to adhere to the resident bacteria or the biofilm matrix and overcome community resistance are among the key factors that determine whether a bacterium can integrate into a community. In this study, we focus on community integration of Fusobacterium nucleatum, a prevalent Gram-negative oral bacterial species that is considered an important member of the oral community due to its ability to adhere to Gram-positive as well as Gram-negative species. This interaction with a variety of different species is thought to facilitate the establishment of multispecies oral microbial community. However, the majority of experiments thus far has focused on the physical adherence between two species as measured by in vitro co-aggregation assays, while the community-based effects on the integration of F. nucleatum into multispecies microbial community remains to be investigated. In this study, we demonstrated using an established in vitro mice oral microbiota (O-mix) that the viability of F. nucleatum was significantly reduced upon addition to the O-mix due to cell contact-dependent induction of hydrogen peroxide (H2O2) production by oral community. Interestingly, this inhibitory effect was significantly alleviated when F. nucleatum was allowed to adhere to its known interacting partner species (such as Streptococcus sanguinis) prior to addition. Furthermore, this aggregate formation-dependent protection was absent in the F. nucleatum mutant strain ΔFn1526 that is unable to bind to a number of Gram-positive species. More importantly, this protective effect was also observed during integration of F. nucleatum into a human salivary microbial community (S-mix). These results support the idea that by adhering to other oral microbes, such as streptococci, F. nucleatum is able to mask the surface components that are recognized by H2O2 producing oral community members. This evasion strategy prevents detection by antagonistic oral bacteria and allows integration into the developing oral microbial community.
coaggregation; Fusobacterium nucleatum; microbial flora; oral cavity; community resistance
The variation of microbial communities associated with the human body can be the cause of many factors, including the human genetic makeup, diet, age, surroundings, and sexual behavior. In this study, we investigated the effects of intimate kissing on the oral microbiota of 21 couples by self-administered questionnaires about their past kissing behavior and by the evaluation of tongue and salivary microbiota samples in a controlled kissing experiment. In addition, we quantified the number of bacteria exchanged during intimate kissing by the use of marker bacteria introduced through the intake of a probiotic yoghurt drink by one of the partners prior to a second intimate kiss.
Similarity indices of microbial communities show that average partners have a more similar oral microbiota composition compared to unrelated individuals, with by far most pronounced similarity for communities associated with the tongue surface. An intimate kiss did not lead to a significant additional increase of the average similarity of the oral microbiota between partners. However, clear correlations were observed between the similarity indices of the salivary microbiota of couples and self-reported kiss frequencies, and the reported time passed after the latest kiss. In control experiments for bacterial transfer, we identified the probiotic Lactobacillus and Bifidobacterium marker bacteria in most kiss receivers, corresponding to an average total bacterial transfer of 80 million bacteria per intimate kiss of 10 s.
This study indicates that a shared salivary microbiota requires a frequent and recent bacterial exchange and is therefore most pronounced in couples with relatively high intimate kiss frequencies. The microbiota on the dorsal surface of the tongue is more similar among partners than unrelated individuals, but its similarity does not clearly correlate to kissing behavior, suggesting an important role for specific selection mechanisms resulting from a shared lifestyle, environment, or genetic factors from the host. Furthermore, our findings imply that some of the collective bacteria among partners are only transiently present, while others have found a true niche on the tongue’s surface allowing long-term colonization.
Intimate kiss; Oral microbiota; Tongue; Saliva; Next generation sequencing; Streptococcus; Lactobacillus
Staphylococcus aureus and other coagulase-positive staphylococci (CPS) colonize skin and mucous membrane sites and can cause skin and soft tissue infections (SSTIs) in humans and animals. Factors modulating methicillin-resistant S. aureus (MRSA) colonization and infection in humans remain unclear, including the role of the greater microbial community and environmental factors such as contact with companion animals. In the context of a parent study evaluating the households of outpatients with community MRSA SSTI, the objectives of this study were 1) to characterize the microbiota that colonizes typical coagulase-positive Staphylococcus spp. carriage sites in humans and their companion pets, 2) to analyze associations between Staphylococcus infection and carriage and the composition and diversity of microbial communities, and 3) to analyze factors that influence sharing of microbiota between pets and humans.
We enrolled 25 households containing 56 pets and 30 humans. Sampling locations were matched to anatomical sites cultured by the parent study for MRSA and other CPS. Bacterial microbiota were characterized by sequencing of 16S ribosomal RNA genes. Household membership was strongly associated with microbial communities, in both humans and pets. Pets were colonized with a greater relative abundance of Proteobacteria, whereas people were colonized with greater relative abundances of Firmicutes and Actinobacteria. We did not detect differences in microbiota associated with MRSA SSTI, or carriage of MRSA, S. aureus or CPS. Humans in households without pets were more similar to each other than humans in pet-owning households, suggesting that companion animals may play a role in microbial transfer. We examined changes in microbiota over a 3-month time period and found that pet staphylococcal carriage sites were more stable than human carriage sites.
We characterized and identified patterns of microbiota sharing and stability between humans and companion animals. While we did not detect associations with MRSA SSTI, or carriage of MRSA, S. aureus or CPS in this small sample size, larger studies are warranted to fully explore how microbial communities may be associated with and contribute to MRSA and/or CPS colonization, infection, and recurrence.
Electronic supplementary material
The online version of this article (doi:10.1186/s40168-014-0052-7) contains supplementary material, which is available to authorized users.
16S rRNA; Methicillin-resistant Staphylococcus aureus (MRSA); Microbiome; Pet; Staphylococcus; Skin and soft tissue infection (SSTI)
Iron (Fe) deficiency affects an estimated 2 billion people worldwide and Fe supplements are a common corrective strategy. The impact of Fe deficiency and Fe supplementation on the complex microbial community of the child gut was studied using in vitro colonic fermentation models inoculated with immobilized fecal microbiota. Chyme media (all Fe chelated by 2,2’-dipyridyl to 26.5 mg Fe L-1) mimicking Fe deficiency and supplementation were continuously fermented. Fermentation effluent samples were analyzed daily on the microbial composition and metabolites by qPCR, 16S rRNA gene 454-pyrosequencing and HPLC. Low Fe conditions (1.56 mg Fe L-1) significantly decreased acetate concentrations and subsequent Fe supplementation (26.5 mg Fe L-1) restored acetate production. High Fe following normal Fe conditions had no impact on the gut microbiota composition and metabolic activity. During very low Fe conditions (0 . 9 m g F e L-1 or Fe chelated b y 2,2’-dipyridyl), a decrease of Roseburia spp./Eubacterium rectale, Clostridium Cluster IV members and Bacteroides spp. was observed while Lactobacillus spp. and Enterobacteriaceae increased consistent with a decrease of butyrate (-84%) and propionate (-55%). The strong dysbiosis of the gut microbiota together with decrease of main gut microbiota metabolites observed with very low iron conditions could weaken the barrier effect of the microbiota and negatively impact gut health.
Iron deficiency; gut microbiota; butyrate; fermentation model
Many different species of acidophilic prokaryotes, widely distributed within the domains Bacteria and Archaea, can catalyze the dissimilatory oxidation of ferrous iron or reduction of ferric iron, or can do both. Microbially mediated cycling of iron in extremely acidic environments (pH < 3) is strongly influenced by the enhanced chemical stability of ferrous iron and far greater solubility of ferric iron under such conditions. Cycling of iron has been demonstrated in vitro using both pure and mixed cultures of acidophiles, and there is considerable evidence that active cycling of iron occurs in acid mine drainage streams, pit lakes, and iron-rich acidic rivers, such as the Rio Tinto. Measurements of specific rates of iron oxidation and reduction by acidophilic microorganisms show that different species vary in their capacities for iron oxido-reduction, and that this is influenced by the electron donor provided and growth conditions used. These measurements, and comparison with corresponding data for oxidation of reduced sulfur compounds, also help explain why ferrous iron is usually used preferentially as an electron donor by acidophiles that can oxidize both iron and sulfur, even though the energy yield from oxidizing iron is much smaller than that available from sulfur oxidation. Iron-oxidizing acidophiles have been used in biomining (a technology that harness their abilities to accelerate the oxidative dissolution of sulfidic minerals and thereby facilitate the extraction of precious and base metals) for several decades. More recently they have also been used to simultaneously remediate iron-contaminated surface and ground waters and produce a useful mineral by-product (schwertmannite). Bioprocessing of oxidized mineral ores using acidophiles that catalyze the reductive dissolution of ferric iron minerals such as goethite has also recently been demonstrated, and new biomining technologies based on this approach are being developed.
acidophiles; iron; oxidation; reduction
Supragingival plaque is permanently in contact with saliva. However, the extent to which the microbiota contributes to the salivary bacterial population remains unclear. We compared the compositional shift in the salivary bacterial population with that in supragingival plaque following periodontal therapy. Samples were collected from 19 patients with periodontitis before and after periodontal therapy (mean sample collection interval, 25.8±2.6 months), and their bacterial composition was investigated using barcoded pyrosequencing analysis of the 16S rRNA gene. Phylogenetic community analysis using the UniFrac distance metric revealed that the overall bacterial community composition of saliva is distinct from that of supragingival plaque, both pre- and post-therapy. Temporal variation following therapy in the salivary bacterial population was significantly smaller than in the plaque microbiota, and the post-therapy saliva sample was significantly more similar to that pre-therapy from the same individual than to those from other subjects. Following periodontal therapy, microbial richness and biodiversity were significantly decreased in the plaque microbiota, but not in the salivary bacterial population. The operational taxonomic units whose relative abundances changed significantly after therapy were not common to the two microbiotae. These results reveal the compositional stability of salivary bacterial populations against shifts in the supragingival microbiota, suggesting that the effect of the supragingival plaque microbiota on salivary bacterial population composition is limited.
This study was undertaken to characterize the vaginal microbiota throughout normal human pregnancy using sequence-based techniques. We compared the vaginal microbial composition of non-pregnant patients with a group of pregnant women who delivered at term.
A retrospective case–control longitudinal study was designed and included non-pregnant women (n = 32) and pregnant women who delivered at term (38 to 42 weeks) without complications (n = 22). Serial samples of vaginal fluid were collected from both non-pregnant and pregnant patients. A 16S rRNA gene sequence-based survey was conducted using pyrosequencing to characterize the structure and stability of the vaginal microbiota. Linear mixed effects models and generalized estimating equations were used to identify the phylotypes whose relative abundance was different between the two study groups. The vaginal microbiota of normal pregnant women was different from that of non-pregnant women (higher abundance of Lactobacillus vaginalis, L. crispatus, L. gasseri and L. jensenii and lower abundance of 22 other phylotypes in pregnant women). Bacterial community state type (CST) IV-B or CST IV-A characterized by high relative abundance of species of genus Atopobium as well as the presence of Prevotella, Sneathia, Gardnerella, Ruminococcaceae, Parvimonas, Mobiluncus and other taxa previously shown to be associated with bacterial vaginosis were less frequent in normal pregnancy. The stability of the vaginal microbiota of pregnant women was higher than that of non-pregnant women; however, during normal pregnancy, bacterial communities shift almost exclusively from one CST dominated by Lactobacillus spp. to another CST dominated by Lactobacillus spp.
We report the first longitudinal study of the vaginal microbiota in normal pregnancy. Differences in the composition and stability of the microbial community between pregnant and non-pregnant women were observed. Lactobacillus spp. were the predominant members of the microbial community in normal pregnancy. These results can serve as the basis to study the relationship between the vaginal microbiome and adverse pregnancy outcomes.
Community stability; Longitudinal sampling; Pregnancy; Vaginal microbiome; Lactobacillus; Dynamics
Iron sequestration by host iron-binding proteins is an important mechanism of resistance to microbial infections. Inside oral epithelial cells, iron is stored within ferritin, and is therefore not usually accessible to pathogenic microbes. We observed that the ferritin concentration within oral epithelial cells was directly related to their susceptibility to damage by the human pathogenic fungus, Candida albicans. Thus, we hypothesized that host ferritin is used as an iron source by this organism. We found that C. albicans was able to grow on agar at physiological pH with ferritin as the sole source of iron, while the baker's yeast Saccharomyces cerevisiae could not. A screen of C. albicans mutants lacking components of each of the three known iron acquisition systems revealed that only the reductive pathway is involved in iron utilization from ferritin by this fungus. Additionally, C. albicans hyphae, but not yeast cells, bound ferritin, and this binding was crucial for iron acquisition from ferritin. Transcriptional profiling of wild-type and hyphal-defective C. albicans strains suggested that the C. albicans invasin-like protein Als3 is required for ferritin binding. Hyphae of an Δals3 null mutant had a strongly reduced ability to bind ferritin and these mutant cells grew poorly on agar plates with ferritin as the sole source of iron. Heterologous expression of Als3, but not Als1 or Als5, two closely related members of the Als protein family, allowed S. cerevisiae to bind ferritin. Immunocytochemical localization of ferritin in epithelial cells infected with C. albicans showed ferritin surrounding invading hyphae of the wild-type, but not the Δals3 mutant strain. This mutant was also unable to damage epithelial cells in vitro. Therefore, C. albicans can exploit iron from ferritin via morphology dependent binding through Als3, suggesting that this single protein has multiple virulence attributes.
Iron is an essential nutrient for all microbes. Many human pathogenic microbes have developed sophisticated strategies to acquire iron from the host as most compartments in the body contain little free iron. For example, in oral epithelial cells intracellular iron is bound to ferritin, a protein that is highly resistant to microbial attack. In fact, no microorganism has so far been shown to directly exploit ferritin as an iron source during interaction with host cells. This study demonstrates that the pathogenic fungus Candida albicans can use ferritin as the sole source of iron. Most intriguingly, C. albicans binds ferritin via a receptor that is only exposed on invasive hyphae. This receptor is Als3, which is a member of the Als-protein family. Als3 was previously demonstrated to be an adhesin with invasin-like properties. Mutants lacking Als3 failed to bind ferritin, grew poorly with ferritin as an iron source and were unable to damage epithelial cells. Strains of the baker's yeast expressing C. albicans Als3, but not two closely related proteins, Als1 or Als5, were able to bind ferritin. Therefore, C. albicans uses an additional morphology specific and unique iron uptake strategy based on ferritin while invading into host cells where ferritin is located.
Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.
In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.
Iron homeostasis is important for all living organisms; too much iron confers cell toxicity, and too little iron results in reduced cell fitness. While crucial for many cellular processes in both man and pathogens, a battle for this essential nutrient erupts during infection between the host and the invading bacteria. Iron is principally stored in ferritin, a large molecule able to bind several thousand iron ions. Although host ferritins represent a mine of iron for pathogens, studies of the mechanisms involved in its acquisition by bacteria are scarce. In the human opportunistic pathogen Bacillus cereus, the surface protein IlsA is able to bind several host iron sources in vitro. In this study, we show that IlsA acts as a ferritin receptor and enhances iron release from the ferritin through direct interaction with each ferritin subunit. Moreover, we demonstrate that the siderophore bacillibactin, a small secreted iron chelator, is essential for ferritin iron acquisition and takes part in B. cereus virulence. We propose a new iron acquisition model that provides new insights into bacterial host adaptation.
Epigeic earthworms are key organisms in organic matter decomposition because of the interactions they establish with microorganisms. The earthworm species and the quality and/or substrate availability are expected to be major factors influencing the outcome of these interactions. Here we tested whether and to what extent the epigeic earthworms Eisenia andrei, Eisenia fetida and Perionyx excavatus, widely used in vermicomposting, are capable of altering the microbiological properties of fresh organic matter in the short-term. We also questioned if the earthworm-induced modifications to the microbial communities are dependent on the type of substrate ingested.
To address these questions we determined the microbial community structure (phospholipid fatty acid profiles) and microbial activity (basal respiration and microbial growth rates) of three types of animal manure (cow, horse and rabbit) that differed in microbial composition, after being processed by each species of earthworm for one month. No differences were found between earthworm-worked samples with regards to microbial community structure, irrespective of type of manure, which suggests the existence of a bottleneck effect of worm digestion on microbial populations of the original material consumed. Moreover, in mesocosms containing cow manure the presence of E. andrei resulted not only in a decrease in bacterial and fungal biomass, but also in a reduced bacterial growth rate and total microbial activity, while no such reduction was found with E. fetida and P. excavatus.
Our results point to the species of earthworm with its associated gut microbiota as a strong determinant of the process shaping the structure of microbial communities in the short-term. This must nonetheless be weighed against the fact that further knowledge is necessary to evaluate whether the changes in the composition of microbiota in response to the earthworm species is accompanied by a change in the microbial community diversity and/or function.