Healthy hosts rule within: ecological forces shaping the gut microbiota

A balanced gut microbiota is important for human health, but the mechanisms that maintain homeostasis are incompletely understood. Recent insights suggest the host plays a key role in shaping its gut microbiota to be beneficial. While host control in the small intestine curbs bacterial numbers to avoid competition for simple sugars and amino acids, the host limits oxygen availability in the large intestine to obtain microbial fermentation products from fiber. Epithelial cells are major players in imposing ecological control mechanisms, which involves the release of antimicrobial peptides by small-intestinal Paneth cells and maintenance of luminal anaerobiosis by epithelial hypoxia in the colon. Harnessing these epithelial control mechanisms for therapeutic means could provide a novel lynchpin for strategies to remediate dysbiosis.     

Changes in gut bacterial communities in canaries infected by Macrorhabdus ornithogaster

Macrorhabdus ornithogaster is an opportunistic yeast that colonizes the gastric mucosa of many avian species. Until now, no studies have focused on the influence of a gastric infection on the balance of the intestinal microbiota of birds. In this study, 44 faecal samples from individual canaries, with and without M. ornithogaster infection, were analysed. The detection of the yeast was evaluated by 18S rRNA PCR. In order to evaluate the impact of the Macrorhabdus infection on the bacterial communities, culture-independent methods, by the use of amplicon-based sequencing as well as 16S rRNA-DGGE, were adopted. The different health status of animals affected the relative abundance of the main OTUs, with a greater diversification of the gut microbiota in healthy animals compared to the infected. In particular, Lactococcus, Pseudomonas, Acinetobacter, Lachnospiraceae, Propionibacterium and Weissella were found to be characteristic of uninfected animals (FDR?<?0.05), while Lactobacillus and Candidatus Arthromitus were characteristic of infected animals (FDR?<?0.05). Both these taxa have been reported as immunostimulatory, involved in immunological disorders. In infected animals the inferred metagenome assessed by PICRUST clearly showed a positive correlation between the presence of M. ornithogaster and KEGG genes related to ether lipid metabolism, already reported to be immunostimulatory by activation of macrophages and to play a pathophysiological role in several immunological disorders. Finally, our results show an interaction between infection of the digestive tract and intestinal microbiota of pet birds and provide insight into the changing of the complex enteric bacterial community.

• HIGHLIGHTS

• Macrorabdus ornithogaster is a gastric yeast that colonizes a wide range of birds.

• Differences were found between infected and healthy animals in gut microbiota.

• Candidatus Arthromitus was closely associated with infected birds.

• M. ornithogaster can affect intestinal microbiota composition of canaries.

     

Impact of gut fungal and bacterial communities on the outcome of allogeneic hematopoietic cell transplantation

Patients receiving allogeneic hematopoietic cell transplantation (alloHCT) were previously shown to display a bacterial gut dysbiosis; however, limited data are available regarding the role of fungal microbiota in these patients. We evaluated the bacterial and fungal composition of the fecal microbiota at day 0 of alloHCT. Higher bacterial diversity was associated with an improved overall survival (OS) and disease-free survival (DFS). While fungal diversity had no impact on patient outcomes, we observed that high versus low relative abundance of Candida albicans in alloHCT patients at day 0 was associated with a significantly lower OS, DFS and graft-versus-host-free, relapse-free survival (GRFS) (p = 0.0008, p = 0.0064 and p = 0.026, respectively). While these results are limited by low patient numbers and low fungal read counts in some samples, they suggest a potentially important role for C albicans in alloHCT.     

Biomechanical forces shape the tumor microenvironment

The importance of the tumor microenvironment in cancer progression is indisputable, yet a key component of the microenvironment—biomechanical forces—remains poorly understood. Tumor growth and progression is paralleled by a host of physical changes in the tumor microenvironment, such as growth-induced solid stresses, increased matrix stiffness, high fluid pressure, and increased interstitial flow. These changes to the biomechanical microenvironment promote tumorigenesis and tumor cell invasion and induce stromal cells—such as fibroblasts, immune cells, and endothelial cells—to change behavior and support cancer progression. This review highlights what we currently know about the biomechanical forces generated in the tumor microenvironment, how they arise, and how these forces can dramatically influence cell behavior, drawing not only upon studies directly related to cancer and tumor cells, but also work in other fields that have shown the effects of these types of mechanical forces vis-à-vis cell behaviors relevant to the tumor microenvironment. By understanding how all of these biomechanical forces can affect tumor cells, stromal cells, and tumor–stromal crosstalk, as well as alter how tumor and stromal cells perceive other extracellular signals in the tumor microenvironment, we can develop new approaches for diagnosis, prognosis, and ultimately treatment of cancer.     

Beyond the gut bacterial microbiota: The gut virome

The gastrointestinal tract is colonized with a highly different population of bacterial, viral, ad fungal species; viruses are reported to be dominant. The composition of gut virome is closely related to dietary habits and surrounding environment. Host and their intestinal microbes live in a dynamic equilibrium and viruses stimulate a low degree of immune responses without causing symptoms (host tolerance). However, intestinal phages could lead to a rupture of eubiosis and may contribute to the shift from health to disease in humans and animals. Viral nucleic acids and other products of lysis of bacteria serve as pathogen‐associated molecular patterns (PAMPs) and could trigger specific inflammatory modulations. At the same time, phages could elicit innate antiviral immune responses. Toll‐like receptors (TLRs) operated as innate antiviral immune sensors and their activation triggers signaling cascades that lead to inflammatory response. J. Med. Virol. 88:1467–1472, 2016 . © 2016 Wiley Periodicals, Inc.

     

What immunologists should know about bacterial communities of the human bowel

The human bowel is home to a bacterial community of much complexity. This article summarizes current bacteriological knowledge of the community and highlights topics of potential interest to innovative immunologists. The role of the bacterial community in the development and regulation of the immune system of neonates seems likely to be a particularly important area of future research.     

Intestinal IgA synthesis: a primitive form of adaptive immunity that regulates microbial communities in the gut

Our intestine is colonized by an impressive community of bacteria, that has profound effects on the immune functions. The relationship between gut microbiota and the immune system is one of reciprocity: bacteria have important contribution in nutrient processing and education of the immune system and conversely, the immune system, particularly gut-associated lymphoid tissues (GALT) plays a key role in shaping the repertoire of gut microbiota. In this review we discuss new insights into the role of IgA in the maintenance of immune homeostasis and the reciprocal interactions between gut B cells and intestinal bacteria.     

Mechanisms of inflammation-driven bacterial dysbiosis in the gut

The gut microbiota has diverse and essential roles in host metabolism, development of the immune system and as resistance to pathogen colonization. Perturbations of the gut microbiota, termed gut dysbiosis, are commonly observed in diseases involving inflammation in the gut, including inflammatory bowel disease, infection, colorectal cancer and food allergies. Importantly, the inflamed microenvironment in the gut is particularly conducive to blooms of Enterobacteriaceae, which acquire fitness benefits while other families of symbiotic bacteria succumb to environmental changes inflicted by inflammation. Here we summarize studies that examined factors in the inflamed gut that contribute to blooms of Enterobacterieaceae, and highlight potential approaches to restrict Enterobacterial blooms in treating diseases that are otherwise complicated by overgrowth of virulent Enterobacterial species in the gut.     

Longitudinal linked-read sequencing reveals ecological and evolutionary responses of a human gut microbiome during antibiotic treatment

Gut microbial communities can respond to antibiotic perturbations by rapidly altering their taxonomic and functional composition. However, little is known about the strain-level processes that drive this collective response. Here, we characterize the gut microbiome of a single individual at high temporal and genetic resolution through a period of health, disease, antibiotic treatment, and recovery. We used deep, linked-read metagenomic sequencing to track the longitudinal trajectories of thousands of single nucleotide variants within 36 species, which allowed us to contrast these genetic dynamics with the ecological fluctuations at the species level. We found that antibiotics can drive rapid shifts in the genetic composition of individual species, often involving incomplete genome-wide sweeps of pre-existing variants. These genetic changes were frequently observed in species without obvious changes in species abundance, emphasizing the importance of monitoring diversity below the species level. We also found that many sweeping variants quickly reverted to their baseline levels once antibiotic treatment had concluded, demonstrating that the ecological resilience of the microbiota can sometimes extend all the way down to the genetic level. Our results provide new insights into the population genetic forces that shape individual microbiomes on therapeutically relevant timescales, with potential implications for personalized health and disease.

The composition of the gut microbiome varies among human populations and individuals, and it is thought to play a key role in maintaining health and reducing susceptibility to different diseases (Gill et al. 2006; Feng et al. 2015; Sharon et al. 2016; Halfvarson et al. 2017). Understanding how this microbial ecosystem changes from week to week—through periods of health, disease, and treatment—is important for personalized health management and design of microbiome-aware therapies (Spanogiannopoulos et al. 2016).Many studies have investigated intra-host dynamics at the species or pathway level (Jernberg et al. 2010; Dethlefsen and Relman 2011; Keeney et al. 2014; Buffie et al. 2015; Yin et al. 2015; Zaura et al. 2015; Raymond et al. 2016; Yassour et al. 2016; Lloyd-Price et al. 2017; Palleja et al. 2018; Ng et al. 2019). Among other findings, these studies have shown that oral antibiotics can strongly influence the composition of the gut microbiome over a period of days, whereas the community often regains much of its initial composition in the weeks or months after antibiotics are removed (Dethlefsen and Relman 2011; Buffie et al. 2015; Ng et al. 2019). This suggests an intriguing hypothesis, in which the long-term composition of a healthy gut community is buffered against brief environmental perturbations.However, the mechanisms that enable this ecological resilience are still poorly understood. Does species composition recover because external strains are able to recolonize the host? Or do resident strains persist in refugia and expand again once antibiotics are removed? In the latter case, do resident populations also acquire genetic differences during this time, either due to population bottlenecks or to new selection pressures that are revealed during treatment? These questions can be addressed by quantifying fine-scale microbiome genetic variation below the species or pathway level and tracking how it changes during periods of health, disease, and treatment.Recent advances in strain-resolved metagenomics and isolate sequencing (Scholz et al. 2016; Ward et al. 2016; Truong et al. 2017) have made it possible to detect DNA sequence variants within species and to track how they change within and between hosts. These studies have shown that gut bacteria can acquire genetic differences over time even in healthy human hosts and that these differences arise from a mixture of external replacement events (Schloissnig et al. 2013; Truong et al. 2017; Garud et al. 2019) and the evolution of resident strains (Ghalayini et al. 2018; Garud et al. 2019; Zhao et al. 2019). However, because these previous studies have included relatively few time points per host, or relatively shallow sampling of their microbiota, the population genetic processes that drive these strain-level dynamics remain poorly characterized. Understanding how the forces of mutation, recombination, selection, and genetic drift operate within hosts is critical for efforts to forecast personalized responses to drugs or other therapies.To bridge this gap, we used deep metagenomic sequencing to follow the genetic diversity within a single host microbiome at approximately weekly intervals over a period of 5 mo, which included periods of infectious disease and the oral administration of broad-spectrum antibiotics. We used a linked-read sequencing technique to generate each of our metagenomic samples: large molecules of bacterial DNA were isolated in millions of emulsified droplets, digested into shorter fragments, and labeled with a corresponding DNA barcode to follow linked reads from the same droplet. Previous work has shown that the linkage information encoded in these barcoded “read clouds” can improve genome assembly (Bishara et al. 2018) and taxonomic assignment (Danko et al. 2019) in human gut metagenomes. Here, we took a different approach and developed new statistical methods that leverage longitudinal linked-read sequencing to detect and interpret fine-scale genetic changes that take place within the resident populations of individual bacterial species over time. This reference-based strategy simultaneously captures the ecological and evolutionary dynamics of multiple strains in many resident species, without requiring assembly of complete genomes.Here, we sought to use this approach to characterize the population genetic forces that shape native gut microbiota through periods of health, disease, antibiotic treatment, and recovery. By analyzing the temporal dynamics of thousands of single nucleotide polymorphisms in 36 abundant species, we obtain new insights into the strain-level mechanisms that govern the ecological resiliency of this community, which have important potential implications for personalized health and disease.     

Sympatric chimpanzees and gorillas harbor convergent gut microbial communities

The gut microbial communities within great apes have been shown to reflect the phylogenetic history of their hosts, indicating codiversification between great apes and their gut microbiota over evolutionary timescales. But because the great apes examined to date represent geographically isolated populations whose diets derive from different sources, it is unclear whether this pattern of codiversification has resulted from a long history of coadaptation between microbes and hosts (heritable factors) or from the ecological and geographic separation among host species (environmental factors). To evaluate the relative influences of heritable and environmental factors on the evolution of the great ape gut microbiota, we assayed the gut communities of sympatric and allopatric populations of chimpanzees, bonobos, and gorillas residing throughout equatorial Africa. Comparisons of these populations revealed that the gut communities of different host species can always be distinguished from one another but that the gut communities of sympatric chimpanzees and gorillas have converged in terms of community composition, sharing on average 53% more bacterial phylotypes than the gut communities of allopatric hosts. Host environment, independent of host genetics and evolutionary history, shaped the distribution of bacterial phylotypes across the Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria, the four most common phyla of gut bacteria. Moreover, the specific patterns of phylotype sharing among hosts suggest that chimpanzees living in sympatry with gorillas have acquired bacteria from gorillas. These results indicate that geographic isolation between host species has promoted the evolutionary differentiation of great ape gut bacterial communities.The compositions of the gut microbial communities harbored by great apes reflect the phylogeny of their hosts in a manner suggesting that host species and their gut microbiota have codiversified over evolutionary timescales (Ochman et al. 2010; Degnan et al. 2012). This pattern of codiversification between hosts and their gut microbiota could stem from both heritable factors, such as host genetics and the vertical, generation-to-generation transmission of gut microbes (Vaishampayan et al. 2010), and environmental factors, such as host diet and geography (Ley et al. 2008a,b; Turnbaugh et al. 2008, 2009; De Filippo et al. 2010; La Serre et al. 2010; Muegge et al. 2011; Claesson et al. 2012; Yatsunenko et al. 2012). However, because the great ape species sampled to date represent populations that are at once phylogenetically, ecologically, and geographically distinct, it has not been possible to separate the relative influences of heritable and environmental factors on the evolution of the great ape gut microbiota.One approach to parsing the effects of environmental factors on the gut microbiota from those of heritable factors is to compare sympatric (i.e., co-occurring) and allopatric (i.e., geographically separated) host populations. Gorillas diverged from the lineage leading to humans and chimpanzees/bonobos at least 6 million years ago (Glazko and Nei 2003; Langergraber et al. 2012), but since that time, the two groups have come into secondary contact throughout equatorial Africa. When living in sympatry, great ape species experience dietary convergence in addition to shared geography (Williamson et al. 1990; Tutin and Fernandez 1993; Shannon et al. 2006; Yamagiwa and Basabose 2006), but they do not mingle or interbreed, and their phylogenetic distinctiveness is maintained. Therefore, the effects on the gut microbiota of the environmental factors shared exclusively by sympatric chimpanzees and gorillas can be measured quantitatively as the degree of compositional convergence between the gut microbiota of sympatric populations relative to those of allopatric populations.To quantify the effects of shared environmental factors on the gut microbiota of sympatric great apes, we have investigated the gut microbiota of sympatric and allopatric populations of chimpanzees, bonobos, and gorillas from Tanzania, Cameroon, the Central African Republic (CAR), and the Democratic Republic of the Congo (DRC). We show that, while hosts of different species always maintain distinct gut microbiota (even when living in sympatry), the gut microbiota of sympatric Pan and Gorilla share significantly more bacterial phylotypes than do those of allopatric Pan and Gorilla. Moreover, the specific patterns of phylotype sharing indicate a history of transfer of gut bacteria between the two host species, with chimpanzees acquiring bacteria from sympatric gorillas. Recent analyses of human populations have shown how geographic factors can shape intraspecific variation in gut microbiota composition (De Filippo et al. 2010; Yatsunenko et al. 2012); our results broaden this principle to include a role for geographic isolation in maintaining differences in gut microbiota composition among closely related heterospecific hosts.     

Bacterial communities in the gut of the freshwater copepod Eudiaptomus gracilis

Eudiaptomus gracilis is the most abundant member of the zooplankton, plays a key role in the food web of Lake Balaton (Hungary). In the present study the composition of bacterial communities of this copepod was investigated based on cultivation and molecular cloning. The cultivated bacterial strains from the gut homogenate samples of Eudiaptomus gracilis belonged to four different clades: Firmicutes, Actinobacteria, Bacteriodetes and Proteobacteria. Clone library showed high species diversity, Firmicutes, Actinobacteria, Proteobacteria, representatives of Deinococcus-Thermus lineage and Cyanobacteria were detected. The isolated strains were very effective in degradation of different biopolymers. Many of the detected bacteria are known as opportunistic human or fish pathogens (Pseudomonas spp., Aeromonas spp., Chryseobacterium sp. and Staphylococcus sp.).     

Helminth-induced alterations of the gut microbiota exacerbate bacterial colitis

Infection with the intestinal helminth parasite Heligmosomoides polygyrus exacerbates the colitis caused by the bacterial enteropathogen Citrobacter rodentium. To clarify the underlying mechanism, we analyzed fecal microbiota composition of control and helminth-infected mice and evaluated the functional role of compositional differences by microbiota transplantation experiments. Our results showed that infection of Balb/c mice with H. polygyrus resulted in significant changes in the composition of the gut microbiota, characterized by a marked increase in the abundance of Bacteroidetes and decreases in Firmicutes and Lactobacillales. Recipients of the gut microbiota from helminth-infected wide-type, but not STAT6-deficient, Balb/c donors had increased fecal pathogen shedding and significant worsening of Citrobacter-induced colitis compared to recipients of microbiota from control donors. Recipients of helminth-altered microbiota also displayed increased regulatory T cells and IL-10 expression. Depletion of CD4⁺CD25⁺ T cells and neutralization of IL-10 in recipients of helminth-altered microbiota led to reduced stool C. rodentium numbers and attenuated colitis. These results indicate that alteration of the gut microbiota is a significant contributor to the H. polygyrus-induced exacerbation of C. rodentium colitis. The helminth-induced alteration of the microbiota is Th2-dependent and acts by promoting regulatory T cells that suppress protective responses to bacterial enteropathogens.     

The gram negative bacterial flora of the avian gut

Earlier papers describing the bacterial flora of the avian digestive tract suggest that the finding of Escherichia coli in the intestines of some grain and fruit eating birds, notably the parrots and parakeets, is abnormal. It has been postulated that coliforms in these species are likely to be pathogenic and that antibiotics should be used on a prophylactic basis in such cases. The evidence presented to support these suppositions is not convincing. A recent analysis of the records of post-mortem examinations carried out at the Zoological Society of London's collection at Regent's Park revealed that E. coli was present in 180 of 271 apparently normal birds. This suggests that E. coli is regularly found in the digestive tracts of the species under question and that there is no evidence that the organism is necessarily pathogenic.     

Exploring the bacterial gut microbiota of supralittoral talitrid amphipods

Talitrid amphipods (sandhoppers and beach fleas) are typical of the supralittoral zone. They are known to thrive on stranded materials, including detrital marine angiosperms and macroalgae, as well as occasional dead animals. In this work, the gut microbiota of five species of talitrid amphipods (Talitrus saltator, Talorchestia ugolinii, Sardorchestia pelecaniformis, Orchestia montagui and Orchestia stephenseni) collected in Sardinia (Italy) has been investigated through: i) metabarcoding analysis of the amplified 16S rRNA V4 region; and ii) quantification of family 48 glycosyl hydrolase genes (GHF48), involved in cellulose degradation. Results indicate that, though talitrid gut biodiversity is not directly related to taxon or sampling locality, the animals' digestive tracts may host species-specific bacterial communities. In particular, gut microbiota of O. montagui, an inhabitant of Posidonia banquettes and macro-algae mat, showed the greatest differences in taxonomic composition and the highest proportion of GHF48 genes with respect to 16S rRNA genes. These results suggest that the different talitrid species may host species-specific bacterial communities whose function could partially reflect the different microhabitats and food preferences of their host.     

Reshaping the gut microbiome with bacterial transplantation and antibiotic intake

The intestinal microbiota consists of over 1000 species, which play key roles in gut physiology and homeostasis. Imbalances in the composition of this bacterial community can lead to transient intestinal dysfunctions and chronic disease states. Understanding how to manipulate this ecosystem is thus essential for treating many disorders. In this study, we took advantage of recently developed tools for deep sequencing and phylogenetic clustering to examine the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. In our rat model, deep sequencing revealed an intestinal bacterial diversity exceeding that of the human gut by a factor of two to three. The transplantation produced a marked increase in the microbial diversity of the recipients, which stemmed from both capture of new phylotypes and increase in abundance of others. However, when transplantation was performed after antibiotic intake, the resulting state simply combined the reshaping effects of the individual treatments (including the reduced diversity from antibiotic treatment alone). Therefore, lowering the recipient bacterial load by antibiotic intake prior to transplantation did not increase establishment of the donor phylotypes, although some dominant lineages still transferred successfully. Remarkably, all of these effects were observed after 1 mo of treatment and persisted after 3 mo. Overall, our results indicate that the indigenous gut microbial composition is more plastic that previously anticipated. However, since antibiotic pretreatment counterintuitively interferes with the establishment of an exogenous community, such plasticity is likely conditioned more by the altered microbiome gut homeostasis caused by antibiotics than by the primary bacterial loss.The human intestinal tract harbors the most abundant, and among the most diverse, microbial community of all body sites (Ley et al. 2008; Costello et al. 2009). As in most mammals, the gut microbiome is dominated by four bacterial phyla: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria (Ley et al. 2008), which represent more than 1000 different molecular species or phylotypes (Dethlefsen et al. 2008; Claesson et al. 2009). Remarkably, this phylotype composition can be specific and stable for each individual. Repeated sampling of the same individuals indicates that samples from the same subject are more similar than samples from different subjects (Costello et al. 2009; Turnbaugh et al. 2009), and in a 2-yr interval an individual conserves over 60% of phylotypes of the gut microbiome (Manichanh et al. 2008).The gut is considered the primary site for cross-talk between the host immune system and microorganisms, in part because of the size and complexity of its microbiota and the presence of specialized lymphoid structures in the mucosa (Guarner et al. 2006). This close relationship is important for maintaining an adequate homeostasis between the individual and the external environment (Backhed et al. 2005; Guarner et al. 2006). Imbalances of the intestinal microbial composition, named dysbiosis, may disturb homeostasis, and therefore lead to a dysfunction or disease state. For instance, specific changes of this microbial ecosystem were recently associated with two of the major inflammatory bowel diseases (IBD) (Ott et al. 2004; Manichanh et al. 2006; Frank et al. 2007; Dicksved et al. 2008). A large reduction of microbial diversity was found in patients with Crohn''s disease (Manichanh et al. 2006; Dicksved et al. 2008), and a selective reduction of Faecalibacterium prausnitzii, a member of the Firmicutes phylum, was reported in patients with ulcerative colitis (Sokol et al. 2009). Remarkably, in both inflammatory bowel diseases, most bacteria that decrease in abundance relative to healthy controls are producers of butyrate, which has strong anti-inflammatory effects (Nancey et al. 2002; Hamer et al. 2009). Therefore, although the mechanisms underlying these disorders are yet unclear, it is now well accepted that intestinal microorganisms play a key role in the initiation and maintenance of IBD (Round and Mazmanian 2009).Experimental manipulation has great potential to go beyond observational studies and allow us to decode the physiological roles of the gut bacterial community, and also define new therapeutic strategies based on altering this microbiome. In principle, the stability of the gut microbiome could be disrupted by the use of prebiotics, probiotics, and antibiotics. Intake of prebiotics (i.e., specific nondigestible food ingredients) is expected to stimulate the growth and/or bacterial activity in the gut. So far, however, no prebiotic has been shown to have a persistent effect in modifying the gut microbial composition. Similarly, intake of probiotics (i.e., live microorganisms) confers only transient effects on digestive physiology, and long-term persistent alteration of the indigenous gut microbial composition remains controversial. Attempts to manipulate the composition of the intestinal microbiome by fecal bacteriotherapy have now become the focus of an extensive body of clinical case reports with promising results (Borody et al. 2003; You et al. 2008; Khoruts et al. 2009; Shanahan 2009). For instance, it has recently been shown that fecal transplantation from a healthy donor restored both gut microbiota composition and function in a human patient that suffered from recurrent Clostridium difficile–associated diarrhea (Khoruts et al. 2009). Finally, in contrast to prebiotic and probiotic intake, antibiotics have been shown to produce drastic short- and long-term alterations of the human indigenous microbiota. In these studies, microbial compositions were examined using DNA fingerprint techniques (Lofmark et al. 2006; Jernberg et al. 2007), microarrays (Palmer et al. 2007), and, more recently, by taking advantage of DNA pyrosequencing (Dethlefsen et al. 2008; Antonopoulos et al. 2009). All of the above studies indicated that after antibiotic intake there is a drastic disruption of the intestinal microbiota, resulting in a long-term decrease of its overall diversity.The above observations clearly anticipate that experimental manipulation of the gut bacterial community should be feasible to some extent, for example, in the well-established transplantation of exogenous microbiota into germ-free animals. The result of this procedure is a stable colonization by the transplanted community that keeps most of its original diversity (Rawls et al. 2006; Alpert et al. 2008). Therefore, although host factors probably have a major effect in broadly shaping the intestinal microbial ecosystem, long-term alterations of an indigenous consortium might also be induced, especially at the phylotype level. Such changes can now be uncovered due to the rapid development of genomic approaches and computational methods, which permit more detailed comparisons of the compositions of microbial ecosystems. In the present study, we used recently developed tools for deep sequencing and phylogenetic clustering to examine the degree to which the gut ecosystem could be intentionally manipulated. Using rats as a model system, we compared the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. We tested the hypothesis that antibiotics, by reducing bacterial load, would promote establishment of the transferred microbiota, this outcome would have important implications for clinical practice in situations where the goal is to colonize the gut with a new microbiota. The results were surprising, and indicated that the indigenous gut microbial composition could be reshaped to an extent not anticipated in previous studies.     

Cytosine deamination and selection of CpG suppressed clones are the two major independent biological forces that shape codon usage bias in coronaviruses

Using the complete genome sequences of 19 coronavirus genomes, we analyzed the codon usage bias, dinucleotide relative abundance and cytosine deamination in coronavirus genomes. Of the eight codons that contain CpG, six were markedly suppressed. The mean NNU/NNC ratio of the six amino acids using either NNC or NNU as codon is 3.262, suggesting cytosine deamination. Among the 16 dinucleotides, CpG was most markedly suppressed (mean relative abundance 0.509). No correlation was observed between CpG abundance and mean NNU/NNC ratio. Among the 19 coronaviruses, CoV-HKU1 showed the most extreme codon usage bias and extremely high NNU/NNC ratio of 8.835. Cytosine deamination and selection of CpG suppressed clones by the immune system are the two major independent biochemical and biological selective forces that shape codon usage bias in coronavirus genomes. The underlying mechanism for the extreme codon usage bias, cytosine deamination and G+C content in CoV-HKU1 warrants further studies.     

Formation and structure of mixed bacterial communities.

Mixed bacterial communities are formed by unrelated bacteria on solid media. Mixed bacterial communities on solid media are similar to "classical" colonies and are formed after the growth of a large number of unrelated bacteria simultaneously plated onto a limited area of agar. The morphology of the mixed bacterial communities was similar for different combinations of bacteria and did not change when the bacteria were plated on different media. Different bacterial strains form zones of individual and mixed growth in the structure of mixed bacterial communities. The results of electron microscopic examination indicate that mixed bacterial communities are isolated from their external environment by a surface film. The basic part of this film is formed by an elementary membrane. The membrane of the surface film of mixed bacterial communities is a stable structure occupying a large surface area. The results of this investigation seem to indicate the existence of a special type of co-operation between different species of bacteria. This type of co-operation may be very important in the regulation of interactions between different bacteria and between bacteria and the environment.     

Modulation of genotoxic enzyme activities by non-digestible oligosaccharide metabolism in in-vitro human gut bacterial ecosystems

Supplementation of the human diet with prebiotic substances such as inulin and non-digestible oligosaccharides (NDO), e.g., galacto-oligosaccharides (GOS), has been associated with various health benefits. However, little information is available regarding the spatial location of their metabolism in human gut bacterial ecosystems. Therefore, the present study investigated the metabolism of inulin and GOS with respect to bacterial growth, bifidobacterial stimulatory properties and anti-mutagenicity potential, in a three-stage continuous culture model of the colon which reproduces the physicochemical characteristics of the proximal (V1) and distal (V2, V3) colons. Fermentation of both carbohydrates was rapid, and occurred primarily in V1, as evidenced by acid formation. Inulin metabolism was associated with 10-fold stimulation of lactobacillus populations, together with smaller increases in bifidobacterial cell counts in V1. However, peptostreptococci, enterococci and Clostridium perfringens also increased in this fermentation vessel. In contrast, GOS was only weakly bifidogenic in V1, although these bacteria did proliferate in V2. GOS also increased lactobacilli by an order of magnitude in V1. However, overall changes in microbial populations resulting from inulin or GOS addition were minimal in V2 and V3. Potential beneficial effects of inulin metabolism included minor reductions in beta-glucosidase and beta-glucuronidase, whereas GOS strongly suppressed these enzymes, together with arylsulphatase (AS). Growth of putatively health promoting micro-organisms was not only associated with reductions in enzymes linked to genotoxicity. For example, both carbohydrates stimulated synthesis of nitroreductase and azoreductase, throughout the fermentation system, while inulin increased AS. Colonic transit time is an important factor in bacterial metabolism in the large bowel, and these data suggest that, in some circumstances, NDO fermentation will occurprincipally in the proximal colon.     

Molecular ecological analysis of planktonic bacterial communities in constructed wetlands invaded by Culex (Diptera: Culicidae) mosquitoes

The succession of the planktonic bacterial community during the colonization by Culex (Diptera: Culicidae) mosquitoes of 0.1-ha treatment wetlands was studied using denaturing gradient gel electrophoresis (DGGE) methodology. Relationships between apparent bacterial diversity and ecological factors (water quality, total bacterial counts, and immature mosquito abundance) were determined during a 1-mo flooding period. Analysis of DGGE banding patterns indicated that days postflooding and temporal changes in water quality were the primary and secondary determinants, respectively, of diversity in bacterial communities. Lower levels of diversity were associated with later postflood stages and increases in ammoniacal nitrogen concentration and total bacterial counts. Diversity was therefore most similar for bacteria present on the same sampling date at wetland locations with similar flooding regimes and water quality, suggesting that wastewater input was the driving force shaping bacterial communities. Comparatively small changes in bacterial diversity were connected to natural processes as water flowed through the wetlands. Greater immature mosquito abundance coincided with less diverse communities composed of greater total numbers of bacteria. Five individual DGGE bands were directly associated with fluctuations in mosquito production, and an additional 16 bands were associated with hydrological aspects of the environment during the rise and fall of mosquito populations. A marked decline in mosquito numbers 21 d after inundation may have masked associations of bacterial communities and mosquito recruitment into the sparsely vegetated wetlands. DGGE was an effective tool for the characterization of bacteria in mosquito habitat in our study, and its potential application in mosquito ecology is discussed.