Complex Microbial Ecosystem Research Articles

Preservation and detection of microstructural and taxonomic correlations in the carbon isotopic compositions of individual Precambrian microfossils

Here we present techniques for, and new data from, in situ carbon isotope (δ13C) analysis of Precambrian permineralized microscopic fossils with a reproducibility of 1–2‰ using secondary ion mass spectrometry (SIMS). Individual microfossils, selected for their excellent preservation, were analyzed in petrographic thin sections of stromatolitic cherts from the Proterozoic Gunflint (∼1880Ma), Bitter Springs (∼830Ma), Min’yar (∼740Ma), and Chichkan (∼775Ma) Formations. The range of δ13C values (−34.6‰ to −22.1‰ VPDB) among the 46 individuals analyzed falls within that expected for photoautotrophic carbon fixation by ribulose bisphosphate carboxylase (RuBisCO), consistent with morphology-based taxonomic assignments for these specimens. Microfossils classified as cyanobacteria from the Gunflint, Bitter Springs, and Min’yar Formations (for which published carbonate carbon isotope data can be used to estimate the δ13C of the original dissolved inorganic carbon substrate) exhibit a consistent ∼19‰ total fractionation (δ13C of dissolved inorganic carbon−δ13C of biomass) similar to that observed in living cyanobacteria, over a wide range of δ13Ccarb values (−2.9‰ to 3.4‰). In stromatolitic chert of the Min’yar Formation, morphologically diverse microfossils preserved in a ∼1mm2 part of a microbial mat exhibit systematic isotopic differences among and within taxa that correlate with their morphologically inferred biological affinities and suggest that isotopic signatures of their original biosynthetic processes (e.g., lipid and peptidoglycan synthesis) are preserved. Isotopic offsets consistent with the different RuBisCO-based fractionations typical of cyanobacteria and photosynthetic eukaryotes are documented by the differing δ13C values of a colonial cyanobacterium (−22.6±0.5‰) and a phytoplanktonic protistan acritarch (−28.9±1.0‰) situated <1cm apart in the stromatolitic Chichkan chert. These findings show for the first time the possibility of using in situ isotopic microanalysis of fossil microbial mats and ancient sediments in order to distinguish metabolic fingerprints within complex microbial ecosystems and consortia.

Yeast populations associated with grapes during withering and their fate during alcoholic fermentation of high-sugar must

Background and Aims Grape mycobiota may be a determining factor for the population dynamics that develop during alcoholic fermentation for the production of wine. For sweet wine fermentations, high-sugar content grape musts are employed that represent complex microbial ecosystems. The focus of this study, the Passito di Caluso, is a sweet wine produced in the North of Italy from grapes harvested in the fall and subjected to a withering process. Methods and Results The withering process was studied by sampling and microbiological analysis, while the alcoholic fermentation was followed by both culture-dependent and culture-independent approaches. During the withering process we observed a succession of three yeast populations associated with grapes. A high degree of species biodiversity was detected the last day of the monitoring period. The dominance of Saccharomyces cerevisiae in the inoculated fermentation was confirmed. Conclusions A succession of yeast populations was observed during grape withering; species such as Candida zemplinina, Metschnikowia fructicola and Hanseniaspora uvarum were also detected during alcoholic fermentation. Autochthonous C. zemplinina populations could play an important technological role in sweet wine production. Significance of the Study The grape mycobiota during withering was described and its fate during alcoholic fermentation determined by molecular identification methods.

An Antiatherosclerotic Signaling Cascade Involving Intestinal Microbiota, MicroRNA-10b, and ABCA1/ABCG1-Mediated Reverse Cholesterol Transport

A sobering fact is that human DNA represents <10% of the total DNA within each of us. Every mucosal surface harbors a diverse and distinct microbial composition, with the human intestines containing by far the largest and most complex microbial ecosystem (intestinal microbiota). Serving as the filter for our largest environmental exposure—what we eat—the human intestinal microbiota is enormous—at least 3.3 million nonredundant microbial genes, making it ≈150× larger than the human gene complement.1 Analyses of fecal metagenomes (genomic analysis of a community of microorganisms) from individuals, both with distinct long-term dietary patterns2 and from around the world,3 demonstrate the existence of a more limited number of stable clusters of microbial compositions, termed enterotypes, which represent relatively durable host-microbial symbiotic states. Metabolomics analyses reveal that intestinal microflora exert a significant effect on mammalian blood metabolites,4 raising the possibility that distinct enterotypes may respond differently to environmental (dietary or drug) exposures. Article, see p 967 Consistent with this notion, significant mounting evidence indicates that numerous cardiometabolic phenotypes within the human superorganism are influenced by a complex interplay among environmental (typically dietary nutrient) exposures, intestinal microbiota composition, and host factors. One of the earlier examples demonstrating the importance of intestinal microflora in a complex cardiometabolic phenotype was first reported by Turnbaugh et al,5 who showed that differences in the efficiency of energy harvest from food of distinct microflora compositions within lean versus obese inbred strains of mice could contribute to the development of obesity. Importantly, the obese phenotype was shown to be a transmissible trait, with obese versus lean cecal microbiota transplant into germ-free mice resulting in significantly greater body fat accumulation, despite equivalent caloric intake.5 Metabolomics studies by Nicholson et al6 suggested that intestinal microbiota may similarly play an active role in the development …

The human gastrointestinal microbiota—An unexplored frontier for pharmaceutical discovery

The mammalian gastrointestinal tract (GIT) harbors microorganisms (the microbiota) of vast phylogentic, genomic, and metabolic diversity, and recent years have seen a rapid development in the techniques for studying these complex microbial ecosystems. It is increasingly apparent that the GIT microbiota plays an intricate role in host health and disease. Targeted strategies for modulating human health through the modification of the GIT microbiota, however, are developing and in their infancy. This perspective article discusses the rationale, benefits and limitations of using the GIT microbiota as a pharmacological and nutritional target in the treatment of various diseases and disorders linked to imbalances in our microbiota.

Analysis of lactic acid bacteria communities and their seasonal variations in a spontaneously fermented dairy product (Malian fènè) by applying a cultivation/genotype-based binary model

Seasonal fluctuations of lactic acid bacteria (LAB) communities were analysed in spontaneous fermentation processes of the Malian sour milk fènè through the design of a novel flow-chart protocol for rapid typing of putative LAB. The protocol is based on cultivation on semi-selective complex agar media, followed by a binary scheme for genus- and species-specific genotypic identification. Presumptive LAB (n = 1583) were isolated and typed from 98 samples. Fènè samples displayed 108 cfu mL−1 in all seasons. Seasonal fluctuations indicated a higher prevalence of Streptococcus spp. and Enterococcus spp. in the cold and hot season, respectively. Analysis of enterococci (n = 596) and streptococci (n = 454) revealed the predominance of Streptococcus infantarius subsp. infantarius over Streptococcus thermophilus. The binary culture-based protocol is a rapid tool to follow LAB species diversity and seasonal variations in any complex dairy microbial ecosystem including the cultivation of strains for subsequent characterisation.

Inferring Dynamic Signatures of Microbes in Complex Host Ecosystems

The human gut microbiota comprise a complex and dynamic ecosystem that profoundly affects host development and physiology. Standard approaches for analyzing time-series data of the microbiota involve computation of measures of ecological community diversity at each time-point, or measures of dissimilarity between pairs of time-points. Although these approaches, which treat data as static snapshots of microbial communities, can identify shifts in overall community structure, they fail to capture the dynamic properties of individual members of the microbiota and their contributions to the underlying time-varying behavior of host ecosystems. To address the limitations of current methods, we present a computational framework that uses continuous-time dynamical models coupled with Bayesian dimensionality adaptation methods to identify time-dependent signatures of individual microbial taxa within a host as well as across multiple hosts. We apply our framework to a publicly available dataset of 16S rRNA gene sequences from stool samples collected over ten months from multiple human subjects, each of whom received repeated courses of oral antibiotics. Using new diversity measures enabled by our framework, we discover groups of both phylogenetically close and distant bacterial taxa that exhibit consensus responses to antibiotic exposure across multiple human subjects. These consensus responses reveal a timeline for equilibration of sub-communities of micro-organisms with distinct physiologies, yielding insights into the successive changes that occur in microbial populations in the human gut after antibiotic treatments. Additionally, our framework leverages microbial signatures shared among human subjects to automatically design optimal experiments to interrogate dynamic properties of the microbiota in new studies. Overall, our approach provides a powerful, general-purpose framework for understanding the dynamic behaviors of complex microbial ecosystems, which we believe will prove instrumental for future studies in this field.

Functional metagenomics reveals novel salt tolerance loci from the human gut microbiome

Metagenomics is a powerful tool that allows for the culture-independent analysis of complex microbial communities. One of the most complex and dense microbial ecosystems known is that of the human distal colon, with cell densities reaching up to 10(12) per gram of faeces. With the majority of species as yet uncultured, there are an enormous number of novel genes awaiting discovery. In the current study, we conducted a functional screen of a metagenomic library of the human gut microbiota for potential salt-tolerant clones. Using transposon mutagenesis, three genes were identified from a single clone exhibiting high levels of identity to a species from the genus Collinsella (closest relative being Collinsella aerofaciens) (COLAER_01955, COLAER_01957 and COLAER_01981), a high G+C, Gram-positive member of the Actinobacteria commonly found in the human gut. The encoded proteins exhibit a strong similarity to GalE, MurB and MazG. Furthermore, pyrosequencing and bioinformatic analysis of two additional fosmid clones revealed the presence of an additional galE and mazG gene, with the highest level of genetic identity to Akkermansia muciniphila and Eggerthella sp. YY7918, respectively. Cloning and heterologous expression of the genes in the osmosensitive strain, Escherichia coli MKH13, resulted in increased salt tolerance of the transformed cells. It is hoped that the identification of atypical salt tolerance genes will help to further elucidate novel salt tolerance mechanisms, and will assist our increased understanding how resident bacteria cope with the osmolarity of the gastrointestinal tract.

Of Microbes and Meals

The human intestinal tract comprises a rich and complex microbial ecosystem. This intestinal microbota provides a large reservoir of potentially toxic molecules, including bacterial endotoxin (ie, lipopolysaccharide [LPS]). This potent inflammatory molecule is detectable in the circulation of healthy individuals, and levels transiently increase following ingestion of energy-rich meals. Chronic exposure to circulating endotoxin has been associated with obesity, diabetes, and cardiovascular disease. Western-style meals augment LPS translocation and by this mechanism may contribute to the pathogenesis of these diseases. By contrast, the gut and other organs have evolved mechanisms to detoxify endotoxin and neutralize the potentially inflammatory qualities of circulating endotoxin. Of specific interest to clinicians is evidence that acute postprandial elevation of circulating endotoxin is dependent on meal composition. In this review, the authors present an overview of the biochemical and cellular mechanisms that lead to endotoxemia, with emphasis on the interplay between microbial and nutrition determinants of this condition. The link between endotoxemia, diet, and changes in the intestinal microbiota raise the possibility that dietary interventions can, at least in part, ameliorate the detrimental outcomes of endotoxemia.

Importance of Bacteria as Trigger in Inflammatory Bowel Disease

Inflammatory bowel diseases (IBD) provide a complex model of host-microbe interactions underpinning disease pathogenesis. Although there is no widespread agreement on the aetiology of IBD, there is evidence that microorganisms lead to the often severe inflammatory response characteristic of the disease. IBD is thought to result from an inappropriate and continuing inflammatory response to pathobionts microbes in a genetically susceptible host. In this review, we discuss the complex microbial ecosystem of the mammalian gut, the underlying genetic factors that predispose to IBD, and how these gene variants may alter host-microbe interactions and propagate inflammation. Incentive should be given to research that will promote a better understanding of host-microbial interactions in the intestine and lay the foundations for new therapeutic approaches to both treat and prevent onset and relapse of intestinal inflammation in genetically susceptible hosts.

Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing

Periodontitis has a polymicrobial etiology within the framework of a complex microbial ecosystem. With advances in sequencing technologies, comprehensive studies to elucidate bacterial community differences have recently become possible. We used 454 sequencing of 16S rRNA genes to compare subgingival bacterial communities from 29 periodontally healthy controls and 29 subjects with chronic periodontitis. Amplicons from both the V1-2 and V4 regions of the 16S gene were sequenced, yielding 1,393,579 sequences. They were identified by BLAST against a curated oral 16S database, and mapped to 16 phyla, 106 genera, and 596 species. 81% of sequences could be mapped to cultivated species. Differences between health- and periodontitis-associated bacterial communities were observed at all phylogenetic levels, and UniFrac and principal coordinates analysis showed distinct community profiles in health and disease. Community diversity was higher in disease, and 123 species were identified that were significantly more abundant in disease, and 53 in health. Spirochaetes, Synergistetes and Bacteroidetes were more abundant in disease, whereas the Proteobacteria were found at higher levels in healthy controls. Within the phylum Firmicutes, the class Bacilli was health-associated, whereas the Clostridia, Negativicutes and Erysipelotrichia were associated with disease. These results implicate a number of taxa that will be targets for future research. Some, such as Filifactor alocis and many Spirochetes were represented by a large fraction of sequences as compared with previously identified targets. Elucidation of these differences in community composition provides a basis for further understanding the pathogenesis of periodontitis.

The potter’s wheel: the host’s role in sculpting its microbiota

Animals, ranging from basal metazoans to primates, are host to complex microbial ecosystems; engaged in a symbiotic relationship that is essential for host physiology and homeostasis. Epithelial surfaces vary in the composition of colonizing microbiota as one compares anatomic sites, developmental stages and species origin. Alterations of microbial composition likely contribute to susceptibility to several distinct diseases. The forces that shape the colonizing microbial composition are the focus of much current investigation, and it is evident that there are pressures exerted both by the host and the external environment to mold these ecosystems. The focus of this review is to discuss recent studies that demonstrate the critical importance of host factors in selecting for its microbiome. Greater insight into host-microbiome interactions will be essential for understanding homeostasis at mucosal surfaces, and developing useful interventions when homeostasis is disrupted.

High Genetic Diversity and Different Distributions of Glycosyl Hydrolase Family 10 and 11 Xylanases in the Goat Rumen

BackgroundThe rumen harbors a complex microbial ecosystem for efficient hydrolysis of plant polysaccharides which are the main constituent of the diet. Xylanase is crucial for hemicellulose hydrolysis and plays an important role in the plant cell wall degradation. Xylanases of ruminal strains were widely studied, but few studies have focused on their diversity in rumen microenvironment.Methodology/Principal FindingsWe explored the genetic diversity of xylanases belonging to two major glycosyl hydrolase families (GH 10 and 11) in goat rumen contents by analyzing the amplicons generated with two degenerate primer sets. Fifty-two distinct GH 10 and 35 GH 11 xylanase gene fragments (similarity <95%) were retrieved, and most had low identities with known sequences. Based on phylogenetic analysis, all GH 10 xylanase sequences fell into seven clusters, and 88.5% of them were related to xylanases from Bacteroidetes. Five clusters of GH 11 xylanase sequences were identified. Of these, 85.7% were related to xylanases from Firmicutes, and 14.3% were related to those of rumen fungi. Two full-length xylanase genes (one for each family) were directly cloned and expressed in Escherichia coli. Both the recombinant enzymes showed substantial xylanase activity, and were purified and characterized. Combined with the results of sheep rumen, Bacteroidetes and Firmicutes are the two major phyla of xylan-degrading microorganisms in rumen, which is distinct from the representatives of other environments such as soil and termite hindgut, suggesting that xylan-degrading microorganisms are environment specific.Conclusion/SignificanceThe numerous new xylanase genes suggested the functional diversity of xylanase in the rumen microenvironment which may have great potential applications in industry and agriculture. The phylogenetic diversity and different distributions of xylanase genes will help us understand their roles in plant cell wall degradation in the rumen microenvironment.

Recent Methodological Approaches to Human Microbiome

Human body is one of the most complex and diverse microbial ecosystem in which various microbes are living together with their hosts. Starting with Louis Pasteur's postulation that human health is dependent on gut-resident microbiota, microbes in the gastrointestinal tract have been studied using culture-based techniques. Cultivation has the great advantage that isolates can be recovered and used to further studies for their ability to utilize different substrates and other physiological properties. However, cultivation method is very labor-intensive and can not reveal representative microbial diversity of human intestinal tract. Only small fraction of the microbes residing in human intestine can be cultured and majority of intestinal microbes (approximately 60~70% of intestinal microbes) can not be come into view with currently available cultivation techniques. To avoid reliance on cultivation, many culture-independent molecular methods have been developed to analysis environmental microbes and our understanding of complex microbial communities has been greatly increased by molecular methods in recent decades. These culture-independent methods are mainly based on the use of microbial DNA sequences. Among prokaryotic DNAs targeted by molecular analysis, approximately 1.5 kb long 16S ribosomal DNA gene that encodes part of the small subunit (SSU) of ribosome is often used for analysis of microbial diversity. Molecular techniques introduced in microbial ecology have made it possible to study the composition of intestinal flora in a culture-independent way based on the detection of SSU rDNA.

Intestinal microbiota and overweight

The microbes in our gut can influence our weight by providing us with energy through the degradation of nondigestable carbohydrates and by affecting the cellular energy status of liver and muscle cells and the accumulation of lipids in adipose tissue. Thus, it is not surprising that in several studies the gastrointestinal microbiota of overweight and obese subjects has been found to differ from that of lean subjects. The initial findings linked obesity with proportionally decreased levels of the phylum Bacteroidetes and increased levels of the phylum Firmicutes. Later, several studies have assessed the association between overweight or obesity and the gastrointestinal microbiota, applying an array of molecular methods targeting the microbiota as a whole or specific bacterial groups or species within. However, at present it is difficult to draw conclusions on which of the observed microbiota alterations are relevant; essentially all of the bacterial groups that have been studied in more than one trial have given contradictory results in regard to their association with weight. Some of these discrepancies can result from methodological issues and some from the nature of the gastrointestinal microbiota, which is an extremely complex and dynamic microbial ecosystem with high subject specificity. In addition, selecting subjects purely based on weight may result in a largely heterogeneous group with several potentially confounding factors. While it may be premature to conclude which specific groups of bacteria are prominent in the intestinal tract of overweight and obese subjects, it appears clear that microbes contribute to weight gain and related health issues, such as the metabolic syndrome and type II diabetes. Therefore, it is important to continue to search for common microbial markers and predictors of obesity, and to study how these may be modulated with probiotics and prebiotics to promote health.

Construction and characterization of Enterococcus faecalis CG110/gfp/pRE25*, a tool for monitoring horizontal gene transfer in complex microbial ecosystems

Enterococci are among the most notorious bacteria involved in the spread of antibiotic resistance (ABR) determinants via horizontal gene transfer, a process that leads to increased prevalence of antibiotic-resistant bacteria. In complex microbial communities with a high background of ABR genes, detection of gene transfer is possible only when the ABR determinant is marked. Therefore, the conjugative multiresistance plasmid pRE25, originating from a sausage-associated Enterococcus faecalis, was tagged with a 34-bp random sequence marker spliced by tet(M). The plasmid constructed, designated pRE25(*) , was introduced into E. faecalis CG110/gfp, a strain containing a gfp gene as chromosomal marker. The plasmid pRE25(*) is fully functional compared with its parental pRE25, occurs at one to two copies per chromosome, and can be transferred to Listeria monocytogenes and Listeria innocua at frequencies of 6 × 10(-6) to 8 × 10(-8) transconjugants per donor. The markers on the chromosome and the plasmid enable independent quantification of donor and plasmid, even if ABR genes occur at high numbers in the background ecosystem. Both markers were stable for at least 200 generations, permitting application of the strain in long-running experiments. Enterococcus faecalis CG110/gfp/pRE25(*) is a potent tool for the investigation of horizontal ABR gene transfer in complex environments such as food matrices, biofilms or colonic models.

Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis

The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in large cohort studies. In order to enhance the DNA extraction step we combined mechanical disruption of microbial cells by repeated bead-beating (RBB) with two automated DNA extraction methods, KingFisher with InviMag Stool DNA kit (KF) and NucliSENS easyMAG (NeM). The semi-automated DNA extraction methods, RBB combined with either KF or NeM, were compared to the manual extraction method currently considered the most suited method for fecal DNA extraction by assessing the yield of 16S rRNA gene copies by qPCR and total microbiota composition by the HITChip, a phylogenetic microarray. Parallel DNA extractions from infant fecal samples by using the three methods showed that the KF and manual methods gave comparable yields of 16S rRNA gene copies as assessed by qPCR, whereas NeM showed a significantly lower yield. All three methods showed highly similar microbiota profiles in HITChip. Both KF and NeM were found to be suitable methods for DNA extraction from fecal samples after the mechanical disruption of microbial cells by bead-beating. The semi-automated methods could be performed in half of the time required for the manual protocol, while being comparable to the manual method in terms of reagent costs.

Phylogenetic and metabolic diversity of bacteria associated with cystic fibrosis

In patients afflicted with cystic fibrosis (CF), morbidity and mortality are primarily associated with the adverse consequences of chronic microbial bronchial infections, which are thought to be caused by a few opportunistic pathogens. However, recent evidence suggests the presence of other microorganisms, which may significantly affect the course and outcome of the infection. Using a combination of 16S rRNA gene clone libraries, bacterial culturing and pyrosequencing of barcoded 16S rRNA amplicons, the microbial communities present in CF patient sputum samples were examined. In addition to previously recognized CF pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus, >60 phylogenetically diverse bacterial genera that are not typically associated with CF pathogenesis were also detected. A surprisingly large number of fermenting facultative and obligate anaerobes from multiple bacterial phyla was present in each sample. Many of the bacteria and sequences found were normal residents of the oropharyngeal microflora and with many containing opportunistic pathogens. Our data suggest that these undersampled organisms within the CF lung are part of a much more complex microbial ecosystem than is normally presumed. Characterization of these communities is the first step in elucidating potential roles of diverse bacteria in disease progression and to ultimately facilitate advances in CF therapy.

Detection and quantification of microbial activity in the subsurface

The subsurface harbors a large fraction of Earth’s living biomass, forming complex microbial ecosystems. Without a profound knowledge of the ongoing biologically mediated processes and their reaction to anthropogenic changes it is difficult to assess the long-term stability and feasibility of any type of geotechnical utilization, as these influence subsurface ecosystems. Despite recent advances in many areas of subsurface microbiology, the direct quantification of turnover processes is still in its infancy, mainly due to the extremely low cell abundances. We provide an overview of the currently available techniques for the quantification of microbial turnover processes and discuss their specific strengths and limitations. Most techniques employed so far have focused on specific processes, e.g. sulfate reduction or methanogenesis. Recent studies show that processes that were previously thought to exclude each other can occur simultaneously, albeit at very low rates. Without the identification of the respective processes it is impossible to quantify total microbial activity. Even in cases where all simultaneously occurring processes can be identified, the typically very low rates prevent quantification. In many cases a simple measure of total microbial activity would be a better and more robust measure than assays for several specific processes. Enzyme or molecular assays provide a more general approach as they target key metabolic compounds. Depending on the compound targeted a broader spectrum of microbial processes can be quantified. The two most promising compounds are ATP and hydrogenase, as both are ubiquitous in microbes. Technical constraints limit the applicability of currently available ATP-assays for subsurface samples. A recently developed hydrogenase radiotracer assay has the potential to become a key tool for the quantification of subsurface microbial activity.

Role of gastrointestinal microbiota in fish

The gastrointestinal (GI) tract of an animal consists of a very complex and dynamic microbial ecosystem that is very important from a nutritional, physiological and pathological point of view. A wide range of microbes derived from the surrounding aquatic environment, soil/sediment and feed are found to colonize in the GI tract of fish. Among the microbial groups, bacteria (aerobic, facultative anaerobic and obligate aneraobic forms) are the principal colonizers in the GI tract of fish, and in some fish, yeasts are also reported. The common bacterial colonizers in the GI tract of freshwater and marine fish include Vibrio, Aeromonas, Flavobacterium, Plesiomonas, Pseudomonas, Enterobacteriaceae, Micrococcus, Acinetobacter, Clostridium, Fusarium and Bacteroides, which may vary from species to species as well as environmental conditions. Besides, several unknown bacteria belonging to Mycoplasma, Arthrobacter, Brochothrix, Jeotgailbacillus, Ochrobactrum, Psychrobacter and Sejongia species in the GI tract of different fish species have now been identified successfully using culture-independent techniques. Gnotobiotic and conventional studies indicate the involvement of GI microbiota in fish nutrition, epithelial development, immunity as well as disease outbreak. This review also highlights the need for manipulating the gut microbiota with useful beneficial microbes through probiotic, prebiotic and synbiotic concepts for better fish health management.

Culture independent analyses and wine fermentation: an overview of achievements 10 years after first application

Wine fermentations are complex microbial ecosystems, with both yeasts and bacteria taking part in the transformation process with their metabolic activities. Traditional microbiological methods do not allow a complete understanding of the microbial ecology of complex systems. This is due mainly to the capacity of certain microorganisms to grow on microbiological media preferentially with respect to others. Moreover, with these methods, stressed or damaged cells cannot be detected on the plates. In the last 10 years new methods based on the analysis of nucleic acids (DNA and RNA) extracted directly from the sample without the need for microbial cultivation have been developed. A method often used in this type of study is the polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE). This paper aims to report the most important contributions of PCR-DGGE to the study of microbiological ecology during wine fermentation.