Birgitte Moen

Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

ABSTRACT The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log10 was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.

Development of gut microbiota in infants not exposed to medical interventions.

Eggesbø M, Moen B, Peddada S, Baird D, Rugtveit J, Midtvedt T, Bushel PR, Sekelja M, Rudi K. Development of gut microbiota in infants not exposed to medical interventions. APMIS 2010.

Explorative Multifactor Approach for Investigating Global Survival Mechanisms of Campylobacter jejuni under Environmental Conditions

ABSTRACT Explorative approaches such as DNA microarray experiments are becoming increasingly important in microbial research. Despite these major technical advancements, approaches to study multifactor experiments are still lacking. We have addressed this problem by using rotation testing and a novel multivariate analysis of variance (MANOVA) approach (50-50 MANOVA) to investigate interacting experimental factors in a complex experimental design. Furthermore, a new rotation testing based method was introduced to calculate false-discovery rates for each response. This novel analytical concept was used to investigate global survival mechanisms in the environment of the major food-borne pathogen C. jejuni. We simulated nongrowth environmental conditions by investigating combinations of the factors temperature (5 and 25°C) and oxygen tension (anaerobic, microaerobic, and aerobic). Data were generated with DNA microarrays for information about gene expression patterns and Fourier transform infrared (FT-IR) spectroscopy to study global macromolecular changes in the cell. Microarray analyses showed that most genes were either unchanged or down regulated compared to the reference (day 0) for the conditions tested and that the 25°C anaerobic condition gave the most distinct expression pattern with the fewest genes expressed. The few up-regulated genes were generally stress related and/or related to the cell envelope. We found, using FT-IR spectroscopy, that the amount of polysaccharides and oligosaccharides increased under the nongrowth survival conditions. Potential mechanisms for survival could be to down regulate most functions to save energy and to produce polysaccharides and oligosaccharides for protection against harsh environments. Basic knowledge about the survival mechanisms is of fundamental importance in preventing transmission of this bacterium through the food chain.

Development and application of new nucleic acid-based technologies for microbial community analyses in foods.

Several challenges still persist in the analysis of microorganisms in foods, particularly in studies of complex communities. Nucleic acid-based methods are promising tools in addressing new questions concerning microbial communities. We have developed several new methods in the field of nucleic acid-based microbial community analyses. These methods cover both sample preparation and detection approaches. The sample preparation method involves simplified DNA purification using paramagnetic beads. As an extension of this method, the same paramagnetic beads are used for both cell separation and DNA purification. This enables full automation. The separate detection of viable and dead bacteria is a major issue in nucleic acid-based diagnostics. We have applied a living/dead dye that binds covalently to DNA and inhibits the PCR from dead cells. In addition, a DNA array-based detection assay has been developed. The assay combines the specificity obtained by enzymatic labeling of DNA probes with the possibility of detecting several targets simultaneously by DNA array hybridization. In combination with 16S rDNA amplification, this is a promising tool for community analyses. Also, we have developed a novel approach for multiplex quantitative PCR. The multiplex PCR has been combined with our DNA array-based detection method. Finally, we are now in the process of adapting a system for monitoring microbial growth and death in real-time through the tagging of bacteria with green fluorescent protein (GFP) combined with fluorescence detection using a high-resolution confocal laser scanner.

Novel Developmental Analyses Identify Longitudinal Patterns of Early Gut Microbiota that Affect Infant Growth

It is acknowledged that some obesity trajectories are set early in life, and that rapid weight gain in infancy is a risk factor for later development of obesity. Identifying modifiable factors associated with early rapid weight gain is a prerequisite for curtailing the growing worldwide obesity epidemic. Recently, much attention has been given to findings indicating that gut microbiota may play a role in obesity development. We aim at identifying how the development of early gut microbiota is associated with expected infant growth. We developed a novel procedure that allows for the identification of longitudinal gut microbiota patterns (corresponding to the gut ecosystem developing), which are associated with an outcome of interest, while appropriately controlling for the false discovery rate. Our method identified developmental pathways of Staphylococcus species and Escherichia coli that were associated with expected growth, and traditional methods indicated that the detection of Bacteroides species at day 30 was associated with growth. Our method should have wide future applicability for studying gut microbiota, and is particularly important for translational considerations, as it is critical to understand the timing of microbiome transitions prior to attempting to manipulate gut microbiota in early life.

Prevention of intestinal Campylobacter jejuni colonization in broilers by combinations of in‐feed organic acids

Aim:  We have tested the effect of various combinations of formic acid and sorbate on Campylobacter jejuni colonization in broiler chickens to reduce the colonization of this zoonotic pathogen in broiler chicken flocks.

An eight-year investigation of bovine livestock fecal microbiota

Cattle represent a major source of fecal contamination worldwide. Understanding the natural variation of the bovine livestock fecal microbiota is therefore important. For this reason we addressed the yearly differences of the fecal microbiota for bovine livestock reared in the same geographical region from 1999 to 2007 - analyzing a total of 300 samples representing a range of experimental regimes. The aim of our work was to determine the effect of year visa experimental regime of the bovine livestock fecal microbiota. We used a newly developed high-throughput 16S rRNA sequencing approach (a single mixed Sanger sequence was generated per sample) in combination with deep pyrosequencing. We found that similar feeding and treatment regimes for different years showed major differences in the fecal microbiota, suggesting other factors important for shaping the fecal microbiota than those experimentally controlled. Ruminococcaeae, Peptostreptococcaceae, Acinetobacter, Escherichia/Shigella, Lachnospiraceae and Lactobacillales were the main taxa associated with the yearly fluctuations. Furthermore, we found that fecal samples with high levels of Lactobacillales, Ruminococcaea and Lachnospiraceae had the most even species distributions. The Peptostreptococcaceae and Acinetobacter dominated samples, on the other hand, showed a few highly dominant taxa. Testing of neutrality showed that the evenly distributed samples were explained by a neutral mode (that the assembly of the microbiota was random), while for the other samples there were overrepresentation of the dominant species (indicating bacterial-bacterial nice competition). We therefore propose that there are natural yearly fluctuations of the bovine livestock microbiota - both with respect to ecology and composition. This knowledge will have impact on our management of fecal bacteria in the environment, since it is very difficult to predict risk based on historical data.

Effect of Dietary Fibers on Cecal Microbiota and Intestinal Tumorigenesis in Azoxymethane Treated A/J Min/+ Mice

Foods naturally high in dietary fiber are generally considered to protect against development of colorectal cancer (CRC). However, the intrinsic effect of dietary fiber on intestinal carcinogenesis is unclear. We used azoxymethane (AOM) treated A/J Min/+ mice, which developed a significantly higher tumor load in the colon than in the small intestine, to compare the effects of dietary inulin (IN), cellulose (CE) or brewers spent grain (BSG) on intestinal tumorigenesis and cecal microbiota. Each fiber was tested at two dose levels, 5% and 15% (w/w) content of the AIN-93M diet. The microbiota was investigated by next-generation sequencing of the 16S rRNA gene (V4). We found that mice fed IN had approximately 50% lower colonic tumor load than mice fed CE or BSG (p<0.001). Surprisingly, all three types of fiber caused a dose dependent increase of colonic tumor load (p<0.001). The small intestinal tumor load was not affected by the dietary fiber interventions. Mice fed IN had a lower bacterial diversity than mice fed CE or BSG. The Bacteroidetes/Firmicutes ratio was significantly (p = 0.003) different between the three fiber diets with a higher mean value in IN fed mice compared with BSG and CE. We also found a relation between microbiota and the colonic tumor load, where many of the operational taxonomic units (OTUs) related to low tumor load were significantly enriched in mice fed IN. Among the OTUs related to low tumor load were bacteria affiliated with the Bacteroides genus. These results suggest that type of dietary fiber may play a role in the development of CRC, and that the suppressive effect of IN on colonic tumorigenesis is associated with profound changes in the cecal microbiota profile.

Microbial dynamics in mixed culture biofilms of bacteria surviving sanitation of conveyor belts in salmon-processing plants.

The microbiota surviving sanitation of salmon‐processing conveyor belts was identified and its growth dynamics further investigated in a model mimicking processing surfaces in such plants.

Reduced spread of Campylobacter jejuni in broiler chickens by stimulating the bird's natural barriers

We have tested the effect of feed structure and feeding regime to prevent the spread of the zoonotic pathogen Campylobacter jejuni in broiler chicken flocks.