Integrity of prokaryotic mRNA isolated from complex samples for in vivo bacterial transcriptome analysis.

Even though several in vitro studies have focused on bacterial biology, the extent of such knowledge is not complete when considering an actual infection. As culture-independent microbiology methods such as high-throughput sequencing became available, important aspects of host-bacterium interactions will be elucidated. Based on microbiological relevance, we considered Bacteroides fragilis in a murine experimental infection as a model system to evaluate the in vivo bacterial transcriptome in host exudates. A disproportionate number of reads belonging to the host genome were retrieved in the first round of pyrosequencing, even after depletion of ribosomal RNA; the average number of reads related to the eukaryotic genome was 71.924-67.7%, whereas prokaryotic reads represented 34.338-32.3% in host exudates. Thus, different treatments were used to improve the prokaryotic RNA yield: i) centrifugation; ii) ultrasonic treatment; and iii) ultrasonic treatment followed by centrifugation. The latter treatment was found to be the most efficient in generating bacterial yields, as it resulted in a higher number of Bacteroides cells. However, the RNA extracted after this treatment was not of sufficient quality to be used in cDNA synthesis. Our results suggest that the methodology routinely used for RNA extraction in transcriptional analysis is not appropriate for in vivo studies in complex samples. Furthermore, the most efficient treatment for generating good bacterial cell yields was not suitable to retrieve high-quality RNA. Therefore, as an alternative methodological approach to enable in vivo studies on host-bacterium interactions, we advise increasing the sequencing depth despite the high costs.

Dynamics of antibiotic resistance genes and presence of putative pathogens during ambient temperature anaerobic digestion.

AIMS: This study was focused on evaluating the persistency of antimicrobial resistance (AR) genes and putative pathogenic bacteria in an anaerobic digesters operating at mesophilic ambient temperature, in two different year seasons: summer and winter. METHODS AND RESULTS: Abundance and dynamic of AR genes encoding resistance to macrolides (ermB), aminoglycosides (aphA2) and beta-lactams (blaTEM -1 ) and persistency of potentially pathogenic bacteria in pilot-scale anaerobic digesters were investigated. AR genes were determined in the influent and effluent in both conditions. Overall, after 60 days, reduction was observed for all evaluated genes. However, during the summer, anaerobic digestion was more related to the gene reduction as compared to winter. Persistency of potentially pathogenic bacteria was also evaluated by metagenomic analyses compared to an in-house created database. Clostridium, Acinetobacter and Stenotrophomonas were the most identified. CONCLUSIONS: Overall, considering the mesophilic ambient temperature during anaerobic digestion (summer and winter), a decrease in pathogenic bacteria detection through metagenomic analysis and AR genes is reported. Although the mesophilic anaerobic digestion has been efficient, the results may suggest medically important bacteria and AR genes persistency during the process. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to show AR gene dynamics and persistency of potentially pathogenic bacteria through metagenomic approach in cattle manure ambient temperature anaerobic digestion.

Determination of the relevant magnetic interactions in low-dimensional molecular materials: the fundamental role of single crystal high frequency EPR.

A new one-dimensional copper(II) complex with formula [Cu(hfac)(2)(N(3)TEMPO)](n) (hfac = hexafluoroacetylacetonate and N(3)TEMPO = 4-azido-2,2,6,6-tetramethylpiperidine-1-oxyl) has been synthesized and investigated by X-ray crystallography, magnetometry and multifrequency single crystal EPR. The system crystallizes in the P1 space group with two non equivalent copper(II) ions in the unit cell, the two nitroxide radicals being coordinated to Cu(1) in axial positions. The copper(II) ions are bridged by N(3)TEMPO radicals resulting in a zig-zag chain structure. The magnetic susceptibility data were at first satisfactorily modeled assuming an alternating spin chain along the monodimensional covalent skeleton, with a ferromagnetic interaction between Cu(1) and the nitroxide moieties and a weaker antiferromagnetic interaction between these and Cu(2) (J(1) = -13.8 cm(-1), J(2) = +2.4 cm(-1)). However, single crystal EPR studies performed at the X- and W-band clearly demonstrate that the observed magnetic monodimensional character of the complex is actually due to the intermolecular contacts involving N(3)TEMPO ligands. This prompted us to fit the magnetic data using a consistent model, pointing out the fundamental role of single crystal EPR data in defining a correct model to describe the magnetic properties of molecular low dimensional systems.