Plasma levels of trimethylamine-N-oxide can be increased with 'healthy' and 'unhealthy' diets and do not correlate with the extent of atherosclerosis but with plaque instability.

AIMS: The microbiome-derived metabolite trimethylamine-N-oxide (TMAO) has attracted major interest and controversy both as a diagnostic biomarker and therapeutic target in atherothrombosis. METHODS AND RESULTS: Plasma TMAO increased in mice on 'unhealthy' high-choline diets and notably also on 'healthy' high-fibre diets. Interestingly, TMAO was found to be generated by direct oxidation in the gut in addition to oxidation by hepatic flavin-monooxygenases. Unexpectedly, two well-accepted mouse models of atherosclerosis, ApoE-/- and Ldlr-/- mice, which reflect the development of stable atherosclerosis, showed no association of TMAO with the extent of atherosclerosis. This finding was validated in the Framingham Heart Study showing no correlation between plasma TMAO and coronary artery calcium score or carotid intima-media thickness (IMT), as measures of atherosclerosis in human subjects. However, in the tandem-stenosis mouse model, which reflects plaque instability as typically seen in patients, TMAO levels correlated with several characteristics of plaque instability, such as markers of inflammation, platelet activation, and intraplaque haemorrhage. CONCLUSIONS: Dietary-induced changes in the microbiome, of both 'healthy' and 'unhealthy' diets, can cause an increase in the plasma level of TMAO. The gut itself is a site of significant oxidative production of TMAO. Most importantly, our findings reconcile contradictory data on TMAO. There was no direct association of plasma TMAO and the extent of atherosclerosis, both in mice and humans. However, using a mouse model of plaque instability we demonstrated an association of TMAO plasma levels with atherosclerotic plaque instability. The latter confirms TMAO as being a marker of cardiovascular risk.

Applying microbial biogeography in soil forensics.

The ubiquity, heterogeneity and transferability of soil makes it useful as evidence in criminal investigations, especially using new methods that survey the microbial DNA it contains. However, to be used effectively and reliably, more needs to be learned about the natural distribution patterns of microbial communities in soil. In this study we examine these patterns in detail, at local to regional scales (2 m-260 km), across an environmental gradient in three different soil types. Geographic location was found to be more important than soil type in determining the microbial community composition: communities from the same site but different soil types, although significantly different from each other, were still much more similar to each other than were communities from the same soil type but from different sites. At a local scale (25-1000 m), distance-decay relationships were observed in all soil types: the farther apart two soil communities were located, even in the same soil type, the more they differed. At regional-scale distances (1-260 km), differences between communities did not increase with increased geographic distance between them, and the dominant factor determining the community profile was the physico-chemical environment, most notably annual precipitation (R2 = 0.69), soil sodium (R2 = 0.49) and soil ammonium (R2 = 0.47) levels. We introduce a likelihood-ratio framework for quantitative evaluation of soil microbial DNA profile evidence in casework. In conclusion, these profiles, along with detailed knowledge of natural soil microbial biogeography, provide valuable forensic information on soil sample comparison and allow the determination of approximate source location on large (hundreds of km) spatial scales. Moreover, at small spatial scales it may enable pinpointing the source location of a sample to within at least 25 m, regardless of soil type and environmental conditions.

Soil characterisation by bacterial community analysis for forensic applications: A quantitative comparison of environmental technologies.

The ubiquity and transferability of soil makes it a resource for the forensic investigator, as it can provide a link between agents and scenes. However, the information contained in soils, such as chemical compounds, physical particles or biological entities, is seldom used in forensic investigations; due mainly to the associated costs, lack of available expertise, and the lack of soil databases. The microbial DNA in soil is relatively easy to access and analyse, having thus the potential to provide a powerful means for discriminating soil samples or linking them to a common origin. We compared the effectiveness and reliability of multiple methods and genes for bacterial characterisation in the differentiation of soil samples: ribosomal intergenic spacer analysis (RISA), terminal restriction fragment length polymorphism (TRFLP) of the rpoB gene, and five methods using the 16S rRNA gene: phylogenetic microarrays, TRFLP, and high throughput sequencing with Roche 454, Illumina MiSeq and IonTorrent PGM platforms. All these methods were also compared to long-chain hydrocarbons (n-alkanes) and fatty alcohol profiling of the same soil samples. RISA, 16S TRFLP and MiSeq performed best, reliably and significantly discriminating between adjacent, similar soil types. As TRFLP employs the same capillary electrophoresis equipment and procedures used to analyse human DNA, it is readily available for use in most forensic laboratories. TRFLP was optimized for forensic usage in five parameters: choice of primer pair, fluorescent tagging, concentrating DNA after digestion, number of PCR amplifications per sample and number of capillary electrophoresis runs per PCR amplification. This study shows that molecular microbial ecology methodologies are robust in discriminating between soil samples, illustrating their potential usage as an evaluative forensic tool.