The intestinal microbiota has become a relevant aspect of human health. Microbial colonization runs in parallel with immune system maturation and plays a role in intestinal physiology and regulation. Increasing evidence on early microbial contact suggest that human intestinal microbiota is seeded before birth. Maternal microbiota forms the first microbial inoculum, and from birth, the microbial diversity increases and converges toward an adult-like microbiota by the end of the first 3–5 years of life. Perinatal factors such as mode of delivery, diet, genetics, and intestinal mucin glycosylation all contribute to influence microbial colonization. Once established, the composition of the gut microbiota is relatively stable throughout adult life, but can be altered as a result of bacterial infections, antibiotic treatment, lifestyle, surgical, and a long-term change in diet. Shifts in this complex microbial system have been reported to increase the risk of disease. Therefore, an adequate establishment of microbiota and its maintenance throughout life would reduce the risk of disease in early and late life. This review discusses recent studies on the early colonization and factors influencing this process which impact on health.

The Oscillibacter type strain has valeric acid as its main metabolic end product, a homolog of neurotransmitter GABA, while Alistipes has previously been shown to be associated with induced stress in mice. In conclusion, the taxonomic correlations detected here may therefore correspond to mechanistic models.

Gut microbiota alterations are closely associated with immune dysfunction in HIV-1 patients, and these changes persist during short-term ART. Our data implicate that re-shaping the microbiota may be an adjuvant therapy in patients commencing successful ART.

The distinction between viable and dead cells is a major issue in many aspects of biological research. We have developed a novel concept for quantification of viable and dead cells in complex samples. The viable/dead stain ethidium monoazide (EMA) is used in combination with realtime PCR to inhibit amplification of DNA from dead cells that have taken up EMA (Fig. 1). Viable/dead determinations are key issues in many aspects of biological research. The current technologies addressing this important issue have severely limited application ranges (4,5,14,18,19). There are for instance no approaches enabling accurate viable/dead quantifications in mixed cell populations (2, 13).Real-time PCR is the most widely applied technology for direct quantification of cells in mixed samples. Real-time PCR is increasingly being used for direct detection and quantification of pathogens in foods and environmental or clinical samples. Still, a major obstacle with PCR diagnostics is how to distinguish between DNA from viable and dead cells. Intact DNA can be present although the organisms are dead. This is particularly relevant for pathogens subjected to killing treatments such as disinfections or antibiotics. Even greater challenges are encountered with organisms such as Campylobacter jejuni that have specific growth requirements and may enter a state where it is viable and infectious but not cultivable. The lack of viable/dead differentiation has been a serious limitation for the implementation of DNA diagnostics in routine applications (15,19,22).We have recently used ethidium monoazide (EMA)-PCR for qualitative DNA-based viable/dead differentiation of bacteria in pure monoculture models (21). Viable/dead analyses of pure monocultures are not new or novel. A wide range of different approaches exist (2,5,10,12,25,26). Methods for direct quantitative analyses of complex samples, however, are still lacking. Solving these analytical problems would be a major technological breakthrough. We discovered during the work with the monoculture models that EMA-PCR has this potential. Thus, the aim of the present work was to use EMA-PCR to show that it is possible to develop quantitative assays for specific viable and dead bacteria in complex samples with mixed bacterial populations. We developed the assay for the major food-borne pathogenic bacterium C. jejuni due to the apparent need for new viable/dead diagnostics of this bacterium. The conditions analyzed include detection in mixed and natural samples, survival in foods, and after disinfection and antibiotic treatments. This knowledge is crucial both for diagnostics and in the control of C. jejuni.A dynamic range of more than 4 log 10 was obtained for the EMA-PCR viable/dead assay. We were able to reliably quantify the fraction of viable C. jejuni under all conditions tested, including complex samples with mixed populations. This is to our knowledge the first time that quantitative viable/dead information has been obtained from specific bacteria in mixed populations. C. jejuni was used as...

SummaryBackgroundDysbiosis is associated with many diseases, including irritable bowel syndrome (IBS), inflammatory bowel diseases (IBD), obesity and diabetes. Potential clinical impact of imbalance in the intestinal microbiota suggests need for new standardised diagnostic methods to facilitate microbiome profiling.AimTo develop and validate a novel diagnostic test using faecal samples to profile the intestinal microbiota and identify and characterise dysbiosis.MethodsFifty‐four DNA probes targeting ≥300 bacteria on different taxonomic levels were selected based on ability to distinguish between healthy controls and IBS patients in faecal samples. Overall, 165 healthy controls (normobiotic reference collection) were used to develop a dysbiosis model with a bacterial profile and Dysbiosis Index score output. The model algorithmically assesses faecal bacterial abundance and profile, and potential clinically relevant deviation in the microbiome from normobiosis. This model was tested in different samples from healthy volunteers and IBS and IBD patients (n = 330) to determine the ability to detect dysbiosis.ResultsValidation confirms dysbiosis was detected in 73% of IBS patients, 70% of treatment‐naïve IBD patients and 80% of IBD patients in remission, vs. 16% of healthy individuals. Comparison of deep sequencing and the GA‐map Dysbiosis Test, (Genetic Analysis AS, Oslo, Norway) illustrated good agreement in bacterial capture; the latter showing higher resolution by targeting pre‐determined highly relevant bacteria.ConclusionsThe GA‐map Dysbiosis Test identifies and characterises dysbiosis in IBS and IBD patients, and provides insight into a patient's intestinal microbiota. Evaluating microbiota as a diagnostic strategy may allow monitoring of prescribed treatment regimens and improvement in new therapeutic approaches.

Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.Cyanobacteria are phototrophic organisms that often form water blooms in eutrophic or estuarine waters. These water blooms undergo fluctuations and may exhibit toxic states. One common genus in such water blooms, Microcystis, produces the hepatotoxin microcystin (6). There are approximately 65 known isoforms of microcystin, representing a family of cyclic heptapeptides having the common structure cyclo(D-Ala-L-X-D-MeAsp-L-Z-Adda-D-Glu-Mdha), where L-X and L-Z are variable L amino acids, Adda is 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyl-4,6-decandienoic acid, D-MeAsp is 3-methylaspartic acid, and Mdha is N-methyl-dehydroalanine (Fig.

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PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately 4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLightTM staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70C. However, for bacteria exposed to 100C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.