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1.  In Vitro Communities Derived from Oral and Gut Microbial Floras Inhibit the Growth of Bacteria of Foreign Origins 
Microbial Ecology  2010;60(3):665-676.
The gastrointestinal (GI) tract is home to trillions of microbes. Within the same GI tract, substantial differences in the bacterial species that inhabit the oral cavity and intestinal tract have been noted. While the influence of host environments and nutritional availability in shaping different microbial communities is widely accepted, we hypothesize that the existing microbial flora also plays a role in selecting the bacterial species that are being integrated into the community. In this study, we used cultivable microbial communities isolated from different parts of the GI tract of mice (oral cavity and intestines) as a model system to examine this hypothesis. Microbes from these two areas were harvested and cultured using the same nutritional conditions, which led to two distinct microbial communities, each with about 20 different species as revealed by PCR-based denaturing gradient gel electrophoresis analysis. In vitro community competition assays showed that the two microbial floras exhibited antagonistic interactions toward each other. More interestingly, all the original isolates tested and their closely related species displayed striking community preferences: They persisted when introduced into the bacterial community of the same origin, while their viable count declined more than three orders of magnitude after 4 days of coincubation with the microbial flora of foreign origin. These results suggest that an existing microbial community might impose a selective pressure on incoming foreign bacterial species independent of host selection. The observed inter-flora interactions could contribute to the protective effect of established microbial communities against the integration of foreign bacteria to maintain the stability of the existing communities.
Electronic supplementary material
The online version of this article (doi:10.1007/s00248-010-9711-9) contains supplementary material, which is available to authorized users.
PMCID: PMC2954289  PMID: 20625712
2.  Oral-derived bacterial flora defends its domain by recognizing and killing intruders---- a molecular analysis using Escherichia coli as a model intestinal bacterium 
Microbial ecology  2010;60(3):655-664.
Within the same human gastrointestinal (GI) tract, substantial differences in the bacterial species that inhabit oral cavity and intestinal tract have been noted. Previous research primarily attributed the differences to the influences of host environments and nutritional availabilities (“host habitat” effect). Our recent study indicated that, other than the host habitat effect, an existing microbial community could impose a selective pressure on incoming foreign bacterial species independent of host-mediated selection (“community selection” effect). In this study, we employed in vitro microbial floras representing microorganisms that inhabit the oral cavities and intestinal tract of mice in combination with Escherichia coli as a model intestinal bacterium and demonstrated that E. coli displays a striking community preference. It thrived when introduced into the intestinal microbial community, and survived poorly in the microbial flora of foreign origin (oral community). A more detailed examination of this phenomenon showed that the oral community produced oxygen free radicals in the presence of wild type E. coli while mutants deficient in lipopolysaccharides (LPS) did not trigger significant production of these cell damaging agents. Furthermore, mutants of E. coli defective in the oxidative stress response experienced a more drastic reduction in viability when co-cultivated with the oral flora, while the exogenous addition of the anti-oxidant vitamin C was able to rescue it. We concluded that the oral-derived microbial community senses the E. coli LPS and kills the bacterium with oxygen free radicals. This study reveals a new mechanism of community invasion resistance employed by established microflora to defend their domains.
PMCID: PMC2954290  PMID: 20625713
3.  Oral-Derived Bacterial Flora Defends Its Domain by Recognizing and Killing Intruders—A Molecular Analysis Using Escherichia coli as a Model Intestinal Bacterium 
Microbial Ecology  2010;60(3):655-664.
Within the same human gastrointestinal tract, substantial differences in the bacterial species that inhabit oral cavity and intestinal tract have been noted. Previous research primarily attributed the differences to the influences of host environments and nutritional availabilities (“host habitat” effect). Our recent study indicated that, other than the host habitat effect, an existing microbial community could impose a selective pressure on incoming foreign bacterial species independent of host-mediated selection (“community selection” effect). In this study, we employed in vitro microbial floras representing microorganisms that inhabit the oral cavities and intestinal tract of mice in combination with Escherichia coli as a model intestinal bacterium and demonstrated that E. coli displays a striking community preference. It thrived when introduced into the intestinal microbial community and survived poorly in the microbial flora of foreign origin (oral community). A more detailed examination of this phenomenon showed that the oral community produced oxygen-free radicals in the presence of wild-type E. coli while mutants deficient in lipopolysaccharides (LPS) did not trigger significant production of these cell-damaging agents. Furthermore, mutants of E. coli defective in the oxidative stress response experienced a more drastic reduction in viability when cocultivated with the oral flora, while the exogenous addition of the antioxidant vitamin C was able to rescue it. We concluded that the oral-derived microbial community senses the E. coli LPS and kills the bacterium with oxygen-free radicals. This study reveals a new mechanism of community invasion resistance employed by established microflora to defend their domains.
Electronic supplementary material
The online version of this article (doi:10.1007/s00248-010-9708-4) contains supplementary material, which is available to authorized users.
PMCID: PMC2954290  PMID: 20625713
4.  Community-based interference against integration of Pseudomonas aeruginosa into human salivary microbial biofilm 
Molecular Oral Microbiology  2011;26(6):337-352.
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.
PMCID: PMC3327514  PMID: 22053962
bacterial interference; microbial flora; oral cavity; Pseudomonas aeruginosa; salivary biofilm
5.  Development of the Human Infant Intestinal Microbiota 
PLoS Biology  2007;5(7):e177.
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.
Author Summary
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.
PMCID: PMC1896187  PMID: 17594176
6.  The social structure of microbial community involved in colonization resistance 
The ISME Journal  2013;8(3):564-574.
It is well established that host-associated microbial communities can interfere with the colonization and establishment of microbes of foreign origins, a phenomenon often referred to as bacterial interference or colonization resistance. However, due to the complexity of the indigenous microbiota, it has been extremely difficult to elucidate the community colonization resistance mechanisms and identify the bacterial species involved. In a recent study, we have established an in vitro mice oral microbial community (O-mix) and demonstrated its colonization resistance against an Escherichia coli strain of mice gut origin. In this study, we further analyzed the community structure of the O-mix by using a dilution/regrowth approach and identified the bacterial species involved in colonization resistance against E. coli. Our results revealed that, within the O-mix there were three different types of bacterial species forming unique social structure. They act as ‘Sensor', ‘Mediator' and ‘Killer', respectively, and have coordinated roles in initiating the antagonistic action and preventing the integration of E. coli. The functional role of each identified bacterial species was further confirmed by E. coli-specific responsiveness of the synthetic communities composed of different combination of the identified players. The study reveals for the first time the sophisticated structural and functional organization of a colonization resistance pathway within a microbial community. Furthermore, our results emphasize the importance of ‘Facilitation' or positive interactions in the development of community-level functions, such as colonization resistance.
PMCID: PMC3930314  PMID: 24088624
colonization resistance; microbial community; social structure; host-associated microbiota; facilitation
7.  Adherence to Streptococci facilitates Fusobacterium nucleatum integration into an oral microbial community 
Microbial Ecology  2011;63(3):532-542.
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.
PMCID: PMC3313671  PMID: 22202886
coaggregation; Fusobacterium nucleatum; microbial flora; oral cavity; community resistance
8.  Diverse CRISPRs Evolving in Human Microbiomes 
PLoS Genetics  2012;8(6):e1002441.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with cas (CRISPR–associated) genes, form the CRISPR/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human Microbiome Project (HMP) enables us to explore the distribution and diversity of known CRISPRs in human-associated microbial communities and to discover new CRISPRs. We propose a targeted assembly strategy to reconstruct CRISPR arrays, which whole-metagenome assemblies fail to identify. For each known CRISPR type (identified from reference genomes), we use its direct repeat consensus sequence to recruit reads from each HMP dataset and then assemble the recruited reads into CRISPR loci; the unique spacer sequences can then be extracted for analysis. We also identified novel CRISPRs or new CRISPR variants in contigs from whole-metagenome assemblies and used targeted assembly to more comprehensively identify these CRISPRs across samples. We observed that the distributions of CRISPRs (including 64 known and 86 novel ones) are largely body-site specific. We provide detailed analysis of several CRISPR loci, including novel CRISPRs. For example, known streptococcal CRISPRs were identified in most oral microbiomes, totaling ∼8,000 unique spacers: samples resampled from the same individual and oral site shared the most spacers; different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences on the evolution of CRISPR defenses. We further demonstrate potential applications of CRISPRs to the tracing of rare species and the virus exposure of individuals. This work indicates the importance of effective identification and characterization of CRISPR loci to the study of the dynamic ecology of microbiomes.
Author Summary
Human bodies are complex ecological systems in which various microbial organisms and viruses interact with each other and with the human host. The Human Microbiome Project (HMP) has resulted in >700 datasets of shotgun metagenomic sequences, from which we can learn about the compositions and functions of human-associated microbial communities. CRISPR/Cas systems are a widespread class of adaptive immune systems in bacteria and archaea, providing acquired immunity against foreign nucleic acids: CRISPR/Cas defense pathways involve integration of viral- or plasmid-derived DNA segments into CRISPR arrays (forming spacers between repeated structural sequences), and expression of short crRNAs from these single repeat-spacer units, to generate interference to future invading foreign genomes. Powered by an effective computational approach (the targeted assembly approach for CRISPR), our analysis of CRISPR arrays in the HMP datasets provides the very first global view of bacterial immunity systems in human-associated microbial communities. The great diversity of CRISPR spacers we observed among different body sites, in different individuals, and in single individuals over time, indicates the impact of subtle niche differences on the evolution of CRISPR defenses and indicates the key role of bacteriophage (and plasmids) in shaping human microbial communities.
PMCID: PMC3374615  PMID: 22719260
9.  Using DGGE profiling to develop a novel culture medium suitable for oral microbial communities 
Molecular oral microbiology  2010;25(5):357-367.
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.
PMCID: PMC2951289  PMID: 20883224
oral microbial community; growth medium; 454 pyrosequencing
10.  Bacterial Communities of Diverse Drosophila Species: Ecological Context of a Host–Microbe Model System 
PLoS Genetics  2011;7(9):e1002272.
Drosophila melanogaster is emerging as an important model of non-pathogenic host–microbe interactions. The genetic and experimental tractability of Drosophila has led to significant gains in our understanding of animal–microbial symbiosis. However, the full implications of these results cannot be appreciated without the knowledge of the microbial communities associated with natural Drosophila populations. In particular, it is not clear whether laboratory cultures can serve as an accurate model of host–microbe interactions that occur in the wild, or those that have occurred over evolutionary time. To fill this gap, we characterized natural bacterial communities associated with 14 species of Drosophila and related genera collected from distant geographic locations. To represent the ecological diversity of Drosophilids, examined species included fruit-, flower-, mushroom-, and cactus-feeders. In parallel, wild host populations were compared to laboratory strains, and controlled experiments were performed to assess the importance of host species and diet in shaping bacterial microbiome composition. We find that Drosophilid flies have taxonomically restricted bacterial communities, with 85% of the natural bacterial microbiome composed of only four bacterial families. The dominant bacterial taxa are widespread and found in many different host species despite the taxonomic, ecological, and geographic diversity of their hosts. Both natural surveys and laboratory experiments indicate that host diet plays a major role in shaping the Drosophila bacterial microbiome. Despite this, the internal bacterial microbiome represents only a highly reduced subset of the external bacterial communities, suggesting that the host exercises some level of control over the bacteria that inhabit its digestive tract. Finally, we show that laboratory strains provide only a limited model of natural host–microbe interactions. Bacterial taxa used in experimental studies are rare or absent in wild Drosophila populations, while the most abundant associates of natural Drosophila populations are rare in the lab.
Author Summary
All animals are associated with large consortia of non-pathogenic microbes. Most of these “microbiomes” are not well characterized despite their importance for many aspects of host biology including human and animal health and the agricultural impact of pest species. The fruit fly Drosophila melanogaster provides a powerful experimental model for investigating the dynamics and consequences of animal–microbial interactions. However, it is not clear whether the model bacteria studied in the lab are representative of natural microbial consortia. To establish an ecological and comparative background for experimental studies, we have conducted a global survey of bacterial communities associated with natural populations of 14 species of Drosophila and related genera. Despite the taxonomic and ecological diversity of these species, we find that they are associated with the same dominant bacterial groups. Based on our results, we propose a model of microbiome assembly where its composition is circumscribed by host diet and physiology but, within those limits, is highly dependent on chance environmental encounters. Consistent with this model, the microbiomes of wild flies differ significantly from those of laboratory strains, suggesting that experimental studies should be extended to include the bacteria that are most prevalent in natural communities.
PMCID: PMC3178584  PMID: 21966276
11.  Microbial Community Dynamics during Production of the Mexican Fermented Maize Dough Pozol 
The dynamics of the microbial community responsible for the traditional fermentation of maize in the production of Mexican pozol was investigated by using a polyphasic approach combining (i) microbial enumerations with culture media, (ii) denaturing gradient gel electrophoresis (DGGE) fingerprinting of total community DNA with bacterial and eukaryotic primers and sequencing of partial 16S ribosomal DNA (rDNA) genes, (iii) quantification of rRNAs from dominant microbial taxa by using phylogenetic oligonucleotide probes, and (iv) analysis of sugars and fermentation products. A Streptococcus species dominated the fermentation and accounted for between 25 and 75% of the total flora throughout the process. Results also showed that the initial epiphytic aerobic microflora was replaced in the first 2 days by heterofermentative lactic acid bacteria (LAB), including a close relative of Lactobacillus fermentum, producing lactic acid and ethanol; this heterolactic flora was then progressively replaced by homofermentative LAB (mainly close relatives of L. plantarum, L. casei, and L. delbrueckii) which continued acidification of the maize dough. At the same time, a very diverse community of yeasts and fungi developed, mainly at the periphery of the dough. The analysis of the DGGE patterns obtained with bacterial and eukaryotic primers targeting the 16S and 18S rDNA genes clearly demonstrated that there was a major shift in the community structure after 24 h and that high biodiversity—according to the Shannon-Weaver index—was maintained throughout the process. These results proved that a relatively high number of species, at least six to eight, are needed to perform this traditional lactic acid fermentation. The presence of Bifidobacterium, Enterococcus, and enterobacteria suggests a fecal origin of some important pozol microorganisms. Overall, the results obtained with different culture-dependent or -independent techniques clearly confirmed the importance of developing a polyphasic approach to study the ecology of fermented foods.
PMCID: PMC92204  PMID: 10966374
12.  Application of a Neutral Community Model To Assess Structuring of the Human Lung Microbiome 
mBio  2015;6(1):e02284-14.
DNA from phylogenetically diverse microbes is routinely recovered from healthy human lungs and used to define the lung microbiome. The proportion of this DNA originating from microbes adapted to the lungs, as opposed to microbes dispersing to the lungs from other body sites and the atmosphere, is not known. We use a neutral model of community ecology to distinguish members of the lung microbiome whose presence is consistent with dispersal from other body sites and those that deviate from the model, suggesting a competitive advantage to these microbes in the lungs. We find that the composition of the healthy lung microbiome is consistent with predictions of the neutral model, reflecting the overriding role of dispersal of microbes from the oral cavity in shaping the microbial community in healthy lungs. In contrast, the microbiome of diseased lungs was readily distinguished as being under active selection. We also assessed the viability of microbes from lung samples by cultivation with a variety of media and incubation conditions. Bacteria recovered by cultivation from healthy lungs represented species that comprised 61% of the 16S rRNA-encoding gene sequences derived from bronchoalveolar lavage samples.
Importance  Neutral distribution of microbes is a distinguishing feature of the microbiome in healthy lungs, wherein constant dispersal of bacteria from the oral cavity overrides differential growth of bacteria. No bacterial species consistently deviated from the model predictions in healthy lungs, although representatives of many of the dispersed species were readily cultivated. In contrast, bacterial populations in diseased lungs were identified as being under active selection. Quantification of the relative importance of selection and neutral processes such as dispersal in shaping the healthy lung microbiome is a first step toward understanding its impacts on host health.
Neutral distribution of microbes is a distinguishing feature of the microbiome in healthy lungs, wherein constant dispersal of bacteria from the oral cavity overrides differential growth of bacteria. No bacterial species consistently deviated from the model predictions in healthy lungs, although representatives of many of the dispersed species were readily cultivated. In contrast, bacterial populations in diseased lungs were identified as being under active selection. Quantification of the relative importance of selection and neutral processes such as dispersal in shaping the healthy lung microbiome is a first step toward understanding its impacts on host health.
PMCID: PMC4324308  PMID: 25604788
13.  The Role of Innate Immune Responses in the Outcome of Interspecies Competition for Colonization of Mucosal Surfaces 
PLoS Pathogens  2005;1(1):e1.
Since mucosal surfaces may be simultaneously colonized by multiple species, the success of an organism may be determined by its ability to compete with co-inhabitants of its niche. To explore the contribution of host factors to polymicrobial competition, a murine model was used to study the initiation of colonization by Haemophilus influenzae and Streptococcus pneumoniae. Both bacterial species, which occupy a similar microenvironment within the nasopharynx, persisted during colonization when given individually. Co-colonization, however, resulted in rapid clearance of S. pneumoniae from the upper respiratory tract, associated with increased recruitment of neutrophils into paranasal spaces. Systemic depletion of either neutrophil-like cells or complement was sufficient to eliminate this competitive effect, indicating that clearance was likely due to enhanced opsonophagocytic killing. The hypothesis that modulation of opsonophagocytic activity was responsible for host-mediated competition was tested using in vitro killing assays with elicited neutrophil-like cells. Components of H. influenzae (but not S. pneumoniae) stimulated complement-dependent phagocytic killing of S. pneumoniae. Thus, the recruitment and activation of neutrophils through selective microbial pattern recognition may underlie the H. influenzae–induced clearance of S. pneumoniae. This study demonstrates how innate immune responses may mediate competitive interactions between species and dictate the composition of the colonizing flora.
Bacterial infection commonly begins with organisms that colonize and proliferate on mucosal surfaces. These microenvironments may be occupied by multiple microbial species, suggesting that successful colonizers are distinguished by their capacity to prevail over their competitors. This study examines interactions between two bacterial species that both colonize and infect the human upper respiratory tract. In a mouse model, strains of both Haemophilus influenzae and Streptococcus pneumoniae efficiently colonize the nasal mucosa when tested individually. In contrast, following co-inoculation, H. influenzae rapidly and completely outcompetes S. pneumoniae. This competitive effect is dependent on the local responses from the host in the form of a specific type of white blood cell (neutrophil) that acts to engulf and kill microorganisms that have been labeled by proteins that bind to microbial surfaces (complement). The results of this study show that recognition of microbial products from one species may activate inflammatory responses that promote the clearance of another competing species. This study also demonstrates how manipulations such as antibiotics or vaccines, which are meant to diminish the presence of a single pathogen, may inadvertently alter the competitive interactions of complex microbial communities.
PMCID: PMC1238736  PMID: 16201010
14.  Dismicrobism in inflammatory bowel disease and colorectal cancer: Changes in response of colocytes 
World Journal of Gastroenterology : WJG  2014;20(48):18121-18130.
Patients with inflammatory bowel disease (IBD) have an increased risk of 10%-15% developing colorectal cancer (CRC) that is a common disease of high economic costs in developed countries. The CRC has been increasing in recent years and its mortality rates are very high. Multiple biological and biochemical factors are responsible for the onset and progression of this pathology. Moreover, it appears absolutely necessary to investigate the environmental factors favoring the onset of CRC and the promotion of colonic health. The gut microflora, or microbiota, has an extensive diversity both quantitatively and qualitatively. In utero, the intestine of the mammalian fetus is sterile. At birth, the intestinal microbiota is acquired by ingesting maternal anal or vaginal organisms, ultimately developing into a stable community, with marked variations in microbial composition between individuals. The development of IBD is often associated with qualitative and quantitative disorders of the intestinal microbial flora (dysbiosis). The healthy human gut harbours about 10 different bacterial species distributed in colony forming units which colonize the gastrointestinal tract. The intestinal microbiota plays a fundamental role in health and in the progression of diseases such as IBD and CRC. In healthy subjects, the main control of intestinal bacterial colonization occurs through gastric acidity but other factors such as endoluminal temperature, competition between different bacterial strains, peristalsis and drugs can influence the intestinal microenvironment. The microbiota exerts diverse physiological functions to include: growth inhibition of pathogenic microorganisms, synthesis of compounds useful for the trophism of colonic mucosa, regulation of intestinal lymphoid tissue and synthesis of amino acids. Furthermore, mucus seems to play an important role in protecting the intestinal mucosa and maintaining its integrity. Changes in the microbiota composition are mainly influenced by diet and age, as well as genetic factors. Increasing evidence indicates that dysbiosis favors the production of genotoxins and metabolites associated with carcinogenesis and induces dysregulation of the immune response which promotes and sustains inflammation in IBD leading to carcinogenesis. A disequilibrium in gut microflora composition leads to the specific activation of gut associated lymphoid tissue. The associated chronic inflammatory process associated increases the risk of developing CRC. Ulcerative colitis and Crohn’s disease are the two major IBDs characterized by an early onset and extraintestinal manifestations, such as rheumatoid arthritis. The pathogenesis of both diseases is complex and not yet fully known. However, it is widely accepted that an inappropriate immune response to microbial flora can play a pivotal role in IBD pathogenesis.
PMCID: PMC4277951  PMID: 25561781
Dismicrobism; Inflammatory bowel disease; Colorectal Cancer; Dysbiosis; Eubiosis; Heat shock proteins
15.  The outer mucus layer hosts a distinct intestinal microbial niche 
Nature Communications  2015;6:8292.
The overall composition of the mammalian intestinal microbiota varies between individuals: within each individual there are differences along the length of the intestinal tract related to host nutrition, intestinal motility and secretions. Mucus is a highly regenerative protective lubricant glycoprotein sheet secreted by host intestinal goblet cells; the inner mucus layer is nearly sterile. Here we show that the outer mucus of the large intestine forms a unique microbial niche with distinct communities, including bacteria without specialized mucolytic capability. Bacterial species present in the mucus show differential proliferation and resource utilization compared with the same species in the intestinal lumen, with high recovery of bioavailable iron and consumption of epithelial-derived carbon sources according to their genome-encoded metabolic repertoire. Functional competition for existence in this intimate layer is likely to be a major determinant of microbiota composition and microbial molecular exchange with the host.
The inner layer of the mucus that covers our intestine is nearly sterile. Here, the authors show in mice that the outer mucus layer constitutes a unique microbial niche hosting bacterial communities with distinct proliferation rates and resource utilization activities.
PMCID: PMC4595636  PMID: 26392213
16.  Noma Affected Children from Niger Have Distinct Oral Microbial Communities Based on High-Throughput Sequencing of 16S rRNA Gene Fragments 
We aim to understand the microbial ecology of noma (cancrum oris), a devastating ancient illness which causes severe facial disfigurement in>140,000 malnourished children every year. The cause of noma is still elusive. A chaotic mix of microbial infection, oral hygiene and weakened immune system likely contribute to the development of oral lesions. These lesions are a plausible entry point for unidentified microorganisms that trigger gangrenous facial infections. To catalog bacteria present in noma lesions and identify candidate noma-triggering organisms, we performed a cross-sectional sequencing study of 16S rRNA gene amplicons from sixty samples of gingival fluid from twelve healthy children, twelve children suffering from noma (lesion and healthy sites), and twelve children suffering from Acute Necrotizing Gingivitis (ANG) (lesion and healthy sites). Relative to healthy individuals, samples taken from lesions in diseased mouths were enriched with Spirochaetes and depleted for Proteobacteria. Samples taken from healthy sites of diseased mouths had proportions of Spirochaetes and Proteobacteria that were similar to healthy control individuals. Samples from noma mouths did not have a higher abundance of Fusobacterium, casting doubt on its role as a causative agent of noma. Microbial communities sampled from noma and ANG lesions were dominated by the same Prevotella intermedia OTU, which was much less abundant in healthy sites sampled from the same mouths. Multivariate analysis confirmed that bacterial communities in healthy and lesion sites were significantly different. Several OTUs in the Orders Erysipelotrichales, Clostridiales, Bacteroidales, and Spirochaetales were identified as indicators of noma, suggesting that one or more microbes within these Orders is associated with the development of noma lesions. Future studies should include longitudinal sampling of viral and microbial components of this community, before and early in noma lesion development.
Author Summary
Noma is a traumatic disease characterized by oral-facial lesions that often lead to severe disfigurement and ultimately shame and isolation from the community. Because the causes of noma are likely to be numerous, and reaching those who suffer from this illness is challenging, the etiology of noma remains ill-defined. Although it is known that oral hygiene and nutrition influence the development of noma, evidence suggests that one or more microbes play a crucial role in development of noma lesions. Previous studies have examined the DNA of microbes in lesions to determine which species are present and how their abundances differ between healthy mouth sites and noma lesions. These studies used techniques that were state-of-the-art at the time, though we know they likely only scratched the surface of the resident microbial diversity. Here we extend these studies by digging deeper to characterize a larger diversity of microbial species in noma and control samples, with the goal of better identifying which microbes are uniquely present or have altered abundances in noma lesions.
PMCID: PMC4256271  PMID: 25474262
17.  A short-oligonucleotide microarray that allows improved detection of gastrointestinal tract microbial communities 
BMC Microbiology  2008;8:195.
The human gastrointestinal (GI) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. Increasingly molecular profiling techniques are being employed to examine this complex microbial community. The purpose of this study was to develop a microarray technique based on 16S ribosomal gene sequences for rapidly monitoring the microbial population of the GI tract.
We have developed a culture-independent, semi-quantitative, rapid method for detection of gut bacterial populations based on 16S rDNA probes using a DNA microarray. We compared the performance of microarrays based on long (40- and 50-mer) and short (16–21-mer) oligonucleotides. Short oligonucleotides consistently gave higher specificity. Optimal DNA amplification and labelling, hybridisation and washing conditions were determined using a probe with an increasing number of nucleotide mismatches, identifying the minimum number of nucleotides needed to distinguish between perfect and mismatch probes. An independent PCR-based control was used to normalise different hybridisation results, and to make comparisons between different samples, greatly improving the detection of changes in the gut bacterial population. The sensitivity of the microarray was determined to be 8.8 × 104 bacterial cells g-1 faecal sample, which is more sensitive than a number of existing profiling methods. The short oligonucleotide microarray was used to compare the faecal flora from healthy individuals and a patient suffering from Ulcerative Colitis (UC) during the active and remission states. Differences were identified in the bacterial profiles between healthy individuals and a UC patient. These variations were verified by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing.
In this study we demonstrate the design, testing and application of a highly sensitive, short oligonucleotide community microarray. Our approach allows the rapid discrimination of bacteria inhabiting the human GI tract, at taxonomic levels ranging from species to the superkingdom bacteria. The optimised protocol is available at: . It offers a high throughput method for studying the dynamics of the bacterial population over time and between individuals.
PMCID: PMC2628385  PMID: 19014434
18.  Metabolic Reconstruction for Metagenomic Data and Its Application to the Human Microbiome 
PLoS Computational Biology  2012;8(6):e1002358.
Microbial communities carry out the majority of the biochemical activity on the planet, and they play integral roles in processes including metabolism and immune homeostasis in the human microbiome. Shotgun sequencing of such communities' metagenomes provides information complementary to organismal abundances from taxonomic markers, but the resulting data typically comprise short reads from hundreds of different organisms and are at best challenging to assemble comparably to single-organism genomes. Here, we describe an alternative approach to infer the functional and metabolic potential of a microbial community metagenome. We determined the gene families and pathways present or absent within a community, as well as their relative abundances, directly from short sequence reads. We validated this methodology using a collection of synthetic metagenomes, recovering the presence and abundance both of large pathways and of small functional modules with high accuracy. We subsequently applied this method, HUMAnN, to the microbial communities of 649 metagenomes drawn from seven primary body sites on 102 individuals as part of the Human Microbiome Project (HMP). This provided a means to compare functional diversity and organismal ecology in the human microbiome, and we determined a core of 24 ubiquitously present modules. Core pathways were often implemented by different enzyme families within different body sites, and 168 functional modules and 196 metabolic pathways varied in metagenomic abundance specifically to one or more niches within the microbiome. These included glycosaminoglycan degradation in the gut, as well as phosphate and amino acid transport linked to host phenotype (vaginal pH) in the posterior fornix. An implementation of our methodology is available at This provides a means to accurately and efficiently characterize microbial metabolic pathways and functional modules directly from high-throughput sequencing reads, enabling the determination of community roles in the HMP cohort and in future metagenomic studies.
Author Summary
The human body is inhabited by trillions of bacteria and other microbes, which have recently been studied in many different habitats (including gut, mouth, skin, and urogenital) by the Human Microbiome Project (HMP). These microbial communities were assayed using high-throughput DNA sequencing, but it can be challenging to determine their biological functions based solely on the resulting short sequences. To reconstruct the metabolic activities of such communities, we have developed HUMAnN, a method to accurately infer community function directly from short DNA reads. The method's accuracy was validated using a collection of synthetic microbial communities. Applying HUMAnN to data from the HMP, we showed that, unlike individual microbial species, many metabolic processes were present among all body habitats. However, the frequencies of these processes varied dramatically, and some were highly enriched within individual habitats to provide niche specialization (e.g. in the gut, which is abundant in food matter but low in oxygen). Other community functions were linked specifically to properties of the human host, such as biochemical processes only present in vaginal habitats with particularly high or low pH. Studying additional environmental or disease-associated communities using HUMAnN will further improve our understanding of how the microbial organisms in a community are linked to the biological processes they carry out.
PMCID: PMC3374609  PMID: 22719234
Colonization of the gastrointestinal tract by bacteria of the normal flora was followed by bacteriological and special histological techniques in mice from several colonies. These histological techniques were designed to preserve the intimate associations that become established between particular strains of microorganisms and the epithelium of the mucosa of certain areas of the gut. The findings were as follows: 1. The various strains of bacteria of the normal flora became established in the different areas of the guts of infant mice according to a definite time sequence. 2. The first types of bacteria that could be cultured from the gut were lactobacilli and Group N streptococci. Within the first day after birth, these bacteria colonized the entire digestive tract and formed layers on the stratified squamous epithelium of the nonsecreting portion of the stomach and of the distal esophagus. 3. The bacterial types that appeared next were coliforms and enterococci. From about the 9th to the 18th day after birth, these bacteria could be cultured in extremely high numbers from the cecum and the colon. Histological sections of those organs taken during the first 2 or 3 days of that interval revealed microcolonies of Gram-positive cocci in pairs and tiny Gram-negative rods embedded in the mucous layer of the epithelium. The microcolonies were well separated from the mixture of digesta and bacteria that occupied the center of the lumen; they may have consisted of the coliforms and enterococci mentioned above; but this possibility remains to be proved. 4. Histological sections also revealed that, at about the 12th day after birth, long, thin Gram-variable rods with tapering ends were present, side by side, with the small Gram-negative rods and Gram-positive cocci in the mucous layer. By the 15th day after birth, the fusiform bacteria formed thick layers in the mucus, and seemed to be the only bacteria remaining in that location. It has not yet been possible to enumerate these tapered rods by culture methods, but as judged by visual appearances in the histological sections, they seemed to outnumber all other bacteria in the cecum and the colon by a factor of as much as 1000. It must be stressed that these bacterial layers are readily disrupted and even washed away by conventional histological techniques; their discovery was largely due to the use of the special histological techniques described in the text. The bacteriological and histological findings described here constitute further evidence for the hypothesis that symbiotic associations exist between microorganisms and animals, and that a very large percentage of the bacteria in the gastrointestinal tract constitutes a true autochthonous flora. The constant occurrence of several distinct associations of bacteria with the special histological structures of the animal host renders obsolete the notion that the intestine constitutes a chemostat in which the bacterial populations are randomly mixed. For a full understanding of the ecology of the normal microflora, it is necessary to think of body surfaces as distinct microenvironments in which virtually pure cultures of a few species of microorganisms interact with their host and the adjacent microbial populations. Experiments based on this hypothesis are admittedly difficult to design, but on the other hand studies based on the assumption that microorganisms exist as mixtures in the gastrointestinal tract will be only of limited value and may often be misleading.
PMCID: PMC2138434  PMID: 4169441
20.  Metagenomic chromosome conformation capture (meta3C) unveils the diversity of chromosome organization in microorganisms 
eLife  null;3:e03318.
Genomic analyses of microbial populations in their natural environment remain limited by the difficulty to assemble full genomes of individual species. Consequently, the chromosome organization of microorganisms has been investigated in a few model species, but the extent to which the features described can be generalized to other taxa remains unknown. Using controlled mixes of bacterial and yeast species, we developed meta3C, a metagenomic chromosome conformation capture approach that allows characterizing individual genomes and their average organization within a mix of organisms. Not only can meta3C be applied to species already sequenced, but a single meta3C library can be used for assembling, scaffolding and characterizing the tridimensional organization of unknown genomes. By applying meta3C to a semi-complex environmental sample, we confirmed its promising potential. Overall, this first meta3C study highlights the remarkable diversity of microorganisms chromosome organization, while providing an elegant and integrated approach to metagenomic analysis.
eLife digest
Microbial communities play vital roles in the environment and sustain animal and plant life. Marine microbes are part of the ocean's food chain; soil microbes support the turnover of major nutrients and facilitate plant growth; and the microbial communities residing in the human gut support digestion and the immune system, among other roles. These communities are very complex systems, often containing 1000s of different species engaged in co-dependent relationships, and are therefore very difficult to study.
The entire DNA sequence of an organism constitutes its genome, and much of this genetic information is stored in large structures called chromosomes. Examining the genome of a species can provide important clues about its lifestyle and how it evolved. To do this, DNA is extracted from cells and is then usually cut into smaller fragments, amplified, and sequenced. The small stretches of sequence obtained, called reads, are finally assembled, yielding ideally the complete genome of the organism under study.
Metagenomics attempts to interpret the combined genome of all the different species in a microbial community and has been instrumental in deciphering how the different species interact with each other. Metagenomics involves sequencing stretches of the community's DNA and matching these pieces to individual species to ultimately assemble whole genomes. While this may be a relatively straightforward task for communities that contain only a handful of members, the metagenomes derived from complex microbial communities are huge, fragmented, and incomplete. This often makes it very difficult or even nearly impossible to match the inferred DNA stretches to individual species.
A method called chromosome conformation capture (or ‘3C’ for short) can reveal the physical contacts between different regions of a chromosome and between the different chromosomes of a cell. How often each of these chromosomal contacts occurs provides a kind of physical signature to each genome and each individual chromosome within it.
Marbouty et al. took advantage of these interactions to develop a technique that combines metagenomics and chromosome conformation capture—called meta3C—that can analyze the DNA of many different species mixed together. Testing meta3C on artificial mixtures of a few species of yeast or bacteria showed that meta3C can separate the genomes of the different species without any prior knowledge of the composition of the mix. In a single experiment, meta3C can identify individual chromosomes, match each of them to its species of origin, and reveal the three-dimensional structure of each genome in the mix. Further tests showed that meta3C can also interpret more complex communities where the number and types of the species present are not known.
Meta3C holds great promise for understanding how microbial communities work and how the genomes of the species within a community are organized. However, further developments of the technique will be required to investigate communities as diverse as those present in most natural environments.
PMCID: PMC4381813  PMID: 25517076
Hi-C; meta3C; metagenomics; plasmid F; meta Hi-C; genome assembly; B. subtilis; E. coli; S. cerevisiae
21.  Nod1 Signaling Overcomes Resistance of S. pneumoniae to Opsonophagocytic Killing 
PLoS Pathogens  2007;3(8):e118.
Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp) leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi), provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1), which is involved in recognition of γ-D-glutamyl-meso-diaminopimelic acid (meso-DAP) contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1−/− mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo.
Author Summary
Pathogens are generally studied in the laboratory one species at a time. Most exist, however, in complex environments where they must adapt not only to their host but also to other members of the microbial flora. Using a mouse model of co-colonization, we have shown that one bacterial species (Haemophilus influenzae) can take advantage of the innate immune response of its host to outcompete and eliminate another species (Streptococcus pneumoniae) that resides in the same microenvironment of the upper respiratory tract. The molecular mechanism for this effect involves recognition of a cell wall fragment found on H. influenzae, but not on S. pneumoniae. The response to this immunostimulatory fragment requires Nod1, a host molecule that transmits inflammatory signals in response to specific peptides of the bacterial cell wall. This Nod1-mediated inflammatory stimulation triggers an increase in the ability of a type of white blood cell (neutrophil) to engulf and then kill S. pneumoniae, effectively removing it from its niche on the mucosal surface of the host airway. Our study, therefore, provides a demonstration of the importance of Nod1 in neutrophil-mediated clearance of bacterial infection. In addition, we have described a mechanism for interspecies competition between microbes that occurs through selective stimulation of host innate immune responses.
PMCID: PMC1950946  PMID: 17722978
22.  Microbial community structure and function on sinking particles in the North Pacific Subtropical Gyre 
Sinking particles mediate the transport of carbon and energy to the deep-sea, yet the specific microbes associated with sedimenting particles in the ocean's interior remain largely uncharacterized. In this study, we used particle interceptor traps (PITs) to assess the nature of particle-associated microbial communities collected at a variety of depths in the North Pacific Subtropical Gyre. Comparative metagenomics was used to assess differences in microbial taxa and functional gene repertoires in PITs containing a preservative (poisoned traps) compared to preservative-free traps where growth was allowed to continue in situ (live traps). Live trap microbial communities shared taxonomic and functional similarities with bacteria previously reported to be enriched in dissolved organic matter (DOM) microcosms (e.g., Alteromonas and Methylophaga), in addition to other particle and eukaryote-associated bacteria (e.g., Flavobacteriales and Pseudoalteromonas). Poisoned trap microbial assemblages were enriched in Vibrio and Campylobacterales likely associated with eukaryotic surfaces and intestinal tracts as symbionts, pathogens, or saprophytes. The functional gene content of microbial assemblages in poisoned traps included a variety of genes involved in virulence, anaerobic metabolism, attachment to chitinaceaous surfaces, and chitin degradation. The presence of chitinaceaous surfaces was also accompanied by the co-existence of bacteria which encoded the capacity to attach to, transport and metabolize chitin and its derivatives. Distinctly different microbial assemblages predominated in live traps, which were largely represented by copiotrophs and eukaryote-associated bacterial communities. Predominant sediment trap-assocaited eukaryotic phyla included Dinoflagellata, Metazoa (mostly copepods), Protalveolata, Retaria, and Stramenopiles. These data indicate the central role of eukaryotic taxa in structuring sinking particle microbial assemblages, as well as the rapid responses of indigenous microbial species in the degradation of marine particulate organic matter (POM) in situ in the ocean's interior.
PMCID: PMC4436931  PMID: 26042105
metagenomics; marine particles; sediment trap; biological pump; microbiology
23.  Analysis of the Upper Respiratory Tract Microbiotas as the Source of the Lung and Gastric Microbiotas in Healthy Individuals 
mBio  2015;6(2):e00037-15.
No studies have examined the relationships between bacterial communities along sites of the upper aerodigestive tract of an individual subject. Our objective was to perform an intrasubject and intersite analysis to determine the contributions of two upper mucosal sites (mouth and nose) as source communities for the bacterial microbiome of lower sites (lungs and stomach). Oral wash, bronchoalveolar lavage (BAL) fluid, nasal swab, and gastric aspirate samples were collected from 28 healthy subjects. Extensive analysis of controls and serial intrasubject BAL fluid samples demonstrated that sampling of the lungs by bronchoscopy was not confounded by oral microbiome contamination. By quantitative PCR, the oral cavity and stomach contained the highest bacterial signal levels and the nasal cavity and lungs contained much lower levels. Pyrosequencing of 16S rRNA gene amplicon libraries generated from these samples showed that the oral and gastric compartments had the greatest species richness, which was significantly greater in both than the richness measured in the lungs and nasal cavity. The bacterial communities of the lungs were significantly different from those of the mouth, nose, and stomach, while the greatest similarity was between the oral and gastric communities. However, the bacterial communities of healthy lungs shared significant membership with the mouth, but not the nose, and marked subject-subject variation was noted. In summary, microbial immigration from the oral cavity appears to be the significant source of the lung microbiome during health, but unlike the stomach, the lungs exhibit evidence of selective elimination of Prevotella bacteria derived from the upper airways.
We have demonstrated that the bacterial communities of the healthy lung overlapped those found in the mouth but were found at lower concentrations, with lower membership and a different community composition. The nasal microbiome, which was distinct from the oral microbiome, appeared to contribute little to the composition of the lung microbiome in healthy subjects. Our studies of the nasal, oral, lung, and stomach microbiomes within an individual illustrate the microbiological continuity of the aerodigestive tract in healthy adults and provide culture-independent microbiological support for the concept that microaspiration is common in healthy individuals.
PMCID: PMC4358017  PMID: 25736890
24.  Implication of the Mosquito Midgut Microbiota in the Defense against Malaria Parasites 
PLoS Pathogens  2009;5(5):e1000423.
Malaria-transmitting mosquitoes are continuously exposed to microbes, including their midgut microbiota. This naturally acquired microbial flora can modulate the mosquito's vectorial capacity by inhibiting the development of Plasmodium and other human pathogens through an unknown mechanism. We have undertaken a comprehensive functional genomic approach to elucidate the molecular interplay between the bacterial co-infection and the development of the human malaria parasite Plasmodium falciparum in its natural vector Anopheles gambiae. Global transcription profiling of septic and aseptic mosquitoes identified a significant subset of immune genes that were mostly up-regulated by the mosquito's microbial flora, including several anti-Plasmodium factors. Microbe-free aseptic mosquitoes displayed an increased susceptibility to Plasmodium infection while co-feeding mosquitoes with bacteria and P. falciparum gametocytes resulted in lower than normal infection levels. Infection analyses suggest the bacteria-mediated anti-Plasmodium effect is mediated by the mosquitoes' antimicrobial immune responses, plausibly through activation of basal immunity. We show that the microbiota can modulate the anti-Plasmodium effects of some immune genes. In sum, the microbiota plays an essential role in modulating the mosquito's capacity to sustain Plasmodium infection.
Author Summary
The Anopheles gambiae mosquito that transmits the malaria-causing parasite Plasmodium has an intestinal bacterial flora, or microbiota, which comprises a variety of species. Elimination of this microbiota with antibiotic treatment will render the Anopheles mosquito more susceptible to Plasmodium infection. In this study we show that these bacteria can inhibit the infection of the mosquito with the human malaria parasite Plasmodium falciparum through a mechanism that involves the mosquito's immune system. Our study suggests that the microbial flora of mosquitoes is stimulating a basal immune activity, which comprises several factors with known anti-Plasmodium activity. The same immune factors that are needed to control the mosquito's microbiota are also defending against the malaria parasite Plasmodium. This complex interplay among the mosquito's microbiota, the innate immune system, and the Plasmodium parasite may have significant implications for the transmission of malaria in the field where the bacterial exposure of mosquitoes may differ greatly between ecological niches.
PMCID: PMC2673032  PMID: 19424427
25.  Viral dark matter and virus–host interactions resolved from publicly available microbial genomes 
eLife  null;4:e08490.
The ecological importance of viruses is now widely recognized, yet our limited knowledge of viral sequence space and virus–host interactions precludes accurate prediction of their roles and impacts. In this study, we mined publicly available bacterial and archaeal genomic data sets to identify 12,498 high-confidence viral genomes linked to their microbial hosts. These data augment public data sets 10-fold, provide first viral sequences for 13 new bacterial phyla including ecologically abundant phyla, and help taxonomically identify 7–38% of ‘unknown’ sequence space in viromes. Genome- and network-based classification was largely consistent with accepted viral taxonomy and suggested that (i) 264 new viral genera were identified (doubling known genera) and (ii) cross-taxon genomic recombination is limited. Further analyses provided empirical data on extrachromosomal prophages and coinfection prevalences, as well as evaluation of in silico virus–host linkage predictions. Together these findings illustrate the value of mining viral signal from microbial genomes.
eLife digest
Viruses are infectious particles that can only multiply inside the cells of microbes and other organisms. Little is known about the genetic differences between virus particles (so-called ‘genetic diversity’), especially compared to what we know about the diversity of bacteria, archaea, and other single-celled microbes. This lack of knowledge hampers our understanding of the role viruses play in the evolution of microbial communities and their associated ecosystems.
Studying the genetics of the viruses in these communities is challenging. There is no single ‘marker’ gene that can be used to identify all viruses in environmental samples. Also, many of the fragments of viral genomes that have been identified have not yet been linked to their host microbes. Many viruses integrate their genome into the DNA of their host cell, and there are computational tools available that exploit this ability to identify viruses and link them to their host. However, other viruses can live and multiply inside cells without integrating their genome into the host's DNA.
Earlier in 2015, researchers developed a new computational tool called VirSorter that can predict virus genome sequences within the DNA extracted from microbes. VirSorter identifies viral genome sequences based on the presence of ‘hallmark’ genes that encode for components found in many virus particles, together with a reference database of genomes from many viruses.
Now, Roux et al.—including some of the researchers from the earlier work—use VirSorter to predict viral DNA from publicly available bacteria and archaea genome data. The study identifies over 12,000 viral genomes and links them to their microbial hosts. These data increase the number of viral genome sequences that are publically available by a factor of ten and identify the first viruses associated with 13 new types of bacteria, which include species that are abundant in particular environments.
It is possible for several different viruses to infect a single cell at the same time. Some viruses are known to be able to exchange DNA, and if this happens frequently in other viruses, it could have a big impact on how viruses evolve. Roux et al.'s findings suggest that although it is common for several different viruses to infect the same cell, it is relatively rare for these viruses to exchange genetic material.
Roux et al.'s findings demonstrate the value of searching publicly available microbial genome data for fragments of viral genomes. These new viral genomes will serve as a useful resource for researchers as they explore the communities of viruses and microbes in natural environments, the human body and in industrial processes.
PMCID: PMC4533152  PMID: 26200428
virus; phage; prophage; virus-host adaptation; none

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