Summary1. The nuclear ribosomal internal transcribed spacer (ITS) region is the primary choice for molecular identification of fungi. Its two highly variable spacers (ITS1 and ITS2) are usually species specific, whereas the intercalary 5.8S gene is highly conserved. For sequence clustering and BLAST searches, it is often advantageous to rely on either one of the variable spacers but not the conserved 5.8S gene. To identify and extract ITS1 and ITS2 from large taxonomic and environmental data sets is, however, often difficult, and many ITS sequences are incorrectly delimited in the public sequence databases. 2. We introduce ITSx, a Perl-based software tool to extract ITS1, 5.8S and ITS2 -as well as full-length ITS sequences -from both Sanger and high-throughput sequencing data sets. ITSx uses hidden Markov models computed from large alignments of a total of 20 groups of eukaryotes, including fungi, metazoans and plants, and the sequence extraction is based on the predicted positions of the ribosomal genes in the sequences. 3. ITSx has a very high proportion of true-positive extractions and a low proportion of false-positive extractions. Additionally, process parallelization permits expedient analyses of very large data sets, such as a one million sequence amplicon pyrosequencing data set. ITSx is rich in features and written to be easily incorporated into automated sequence analysis pipelines. 4. ITSx paves the way for more sensitive BLAST searches and sequence clustering operations for the ITS region in eukaryotes. The software also permits elimination of non-ITS sequences from any data set. This is particularly useful for amplicon-based next-generation sequencing data sets, where insidious non-target sequences are often found among the target sequences. Such non-target sequences are difficult to find by other means and would contribute noise to diversity estimates if left in the data set.

The internal transcribed spacer ( ITS) region of the nuclear ribosomal repeat unit is the most popular locus for species identification and subgeneric phylogenetic inference in sequence-based mycological research. The region is known to show certain variability even within species, although its intraspecific variability is often held to be limited and clearly separated from interspecific variability. The existence of such a divide between intra- and interspecific variability is implicitly assumed by automated approaches to species identification, but whether intraspecific variability indeed is negligible within the fungal kingdom remains contentious. The present study estimates the intraspecific ITS variability in all fungi presently available to the mycological community through the international sequence databases. Substantial differences were found within the kingdom, and the results are not easily correlated to the taxonomic affiliation or nutritional mode of the taxa considered. No single unifying yet stringent upper limit for intraspecific variability, such as the canonical 3% threshold, appears to be applicable with the desired outcome throughout the fungi. Our results caution against simplified approaches to automated ITS-based species delimitation and reiterate the need for taxonomic expertise in the translation of sequence data into species names.

Antibiotic resistance and its wider implications present us with a growing healthcare crisis. Recent research points to the environment as an important component for the transmission of resistant bacteria and in the emergence of resistant pathogens. However, a deeper understanding of the evolutionary and ecological processes that lead to clinical appearance of resistance genes is still lacking, as is knowledge of environmental dispersal barriers. This calls for better models of how resistance genes evolve, are mobilized, transferred and disseminated in the environment. Here, we attempt to define the ecological and evolutionary environmental factors that contribute to resistance development and transmission. Although mobilization of resistance genes likely occurs continuously, the great majority of such genetic events do not lead to the establishment of novel resistance factors in bacterial populations, unless there is a selection pressure for maintaining them or their fitness costs are negligible. To enable preventative measures it is therefore critical to investigate under what conditions and to what extent environmental selection for resistance takes place. In addition, understanding dispersal barriers is not only key to evaluate risks, but also to prevent resistant pathogens, as well as novel resistance genes, from reaching humans.

BackgroundDNA sequences are increasingly seen as one of the primary information sources for species identification in many organism groups. Such approaches, popularly known as barcoding, are underpinned by the assumption that the reference databases used for comparison are sufficiently complete and feature correctly and informatively annotated entries.Methodology/Principal FindingsThe present study uses a large set of fungal DNA sequences from the inclusive International Nucleotide Sequence Database to show that the taxon sampling of fungi is far from complete, that about 20% of the entries may be incorrectly identified to species level, and that the majority of entries lack descriptive and up-to-date annotations.ConclusionsThe problems with taxonomic reliability and insufficient annotations in public DNA repositories form a tangible obstacle to sequence-based species identification, and it is manifest that the greatest challenges to biological barcoding will be of taxonomical, rather than technical, nature.

BackgroundAntibacterial biocides and metals can co-select for antibiotic resistance when bacteria harbour resistance or tolerance genes towards both types of compounds. Despite numerous case studies, systematic and quantitative data on co-occurrence of such genes on plasmids and chromosomes is lacking, as is knowledge on environments and bacterial taxa that tend to carry resistance genes to such compounds. This effectively prevents identification of risk scenarios. Therefore, we aimed to identify general patterns for which biocide/metal resistance genes (BMRGs) and antibiotic resistance genes (ARGs) that tend to occur together. We also aimed to quantify co-occurrence of resistance genes in different environments and taxa, and investigate to what extent plasmids carrying both types of genes are conjugative and/or are carrying toxin-antitoxin systems.ResultsCo-occurrence patterns of resistance genes were derived from publicly available, fully sequenced bacterial genomes (n = 2522) and plasmids (n = 4582). The only BMRGs commonly co-occurring with ARGs on plasmids were mercury resistance genes and the qacE∆1 gene that provides low-level resistance to quaternary ammonium compounds. Novel connections between cadmium/zinc and macrolide/aminoglycoside resistance genes were also uncovered. Several clinically important bacterial taxa were particularly prone to carry both BMRGs and ARGs. Bacteria carrying BMRGs more often carried ARGs compared to bacteria without (p < 0.0001). BMRGs were found in 86 % of bacterial genomes, and co-occurred with ARGs in 17 % of the cases. In contrast, co-occurrences of BMRGs and ARGs were rare on plasmids from all external environments (<0.7 %) but more common on those of human and domestic animal origin (5 % and 7 %, respectively). Finally, plasmids with both BMRGs and ARGs were more likely to be conjugative (p < 0.0001) and carry toxin-antitoxin systems (p < 0.0001) than plasmids without resistance genes.ConclusionsThis is the first large-scale identification of compounds, taxa and environments of particular concern for co-selection of resistance against antibiotics, biocides and metals. Genetic co-occurrences suggest that plasmids provide limited opportunities for biocides and metals to promote horizontal transfer of antibiotic resistance through co-selection, whereas ample possibilities exist for indirect selection via chromosomal BMRGs. Taken together, the derived patterns improve our understanding of co-selection potential between biocides, metals and antibiotics, and thereby provide guidance for risk-reducing actions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2153-5) contains supplementary material, which is available to authorized users.

The high and sometimes inappropriate use of antibiotics has accelerated the development of antibiotic resistance, creating a major challenge for the sustainable treatment of infections world-wide. Bacterial communities often respond to antibiotic selection pressure by acquiring resistance genes, i.e. mobile genetic elements that can be shared horizontally between species. Environmental microbial communities maintain diverse collections of resistance genes, which can be mobilized into pathogenic bacteria. Recently, exceptional environmental releases of antibiotics have been documented, but the effects on the promotion of resistance genes and the potential for horizontal gene transfer have yet received limited attention. In this study, we have used culture-independent shotgun metagenomics to investigate microbial communities in river sediments exposed to waste water from the production of antibiotics in India. Our analysis identified very high levels of several classes of resistance genes as well as elements for horizontal gene transfer, including integrons, transposons and plasmids. In addition, two abundant previously uncharacterized resistance plasmids were identified. The results suggest that antibiotic contamination plays a role in the promotion of resistance genes and their mobilization from environmental microbes to other species and eventually to human pathogens. The entire life-cycle of antibiotic substances, both before, under and after usage, should therefore be considered to fully evaluate their role in the promotion of resistance.

Pharmaceuticals are typically found in very low concentrations in the aquatic environment. Accordingly, environmental effects clearly assigned to residual drugs are consistent with high affinity interactions with conserved targets in affected wildlife species rather than with a general toxic effect. Thus, evolutionarily well-conserved targets in a given species are associated with an increased risk. In this study orthologs for 1318 human drug targets were predicted in 16 species of which several are relevant for ecotoxicity testing. The conservation of different functional categories of targets was also analyzed. Zebrafish had orthologs to 86% of the drug targets while only 61% were conserved in Daphnia and 35% in green alga. The predicted presence and absence of orthologs agrees well with published experimental data on the potential for specific drug target interaction in various species. Based on the conservation of targets we propose that aquatic environmental risk assessments for human drugs should always include comprehensive studies on aquatic vertebrates. Furthermore, individual targets, especially enzymes, are well conserved suggesting that tests on evolutionarily distant organisms would be highly relevant for certain drugs. We propose that the results can guide environmental risk assessments by improving the possibilities to identify species sensitive to certain types of pharmaceuticals or to other contaminants that act through well defined mechanisms of action. Moreover, we suggest that the results can be used to interpret the relevance of existing ecotoxicity data.

Antibiotic resistance has become a major human health concern due to widespread use, misuse and overuse of antibiotics. In addition to antibiotics, antibacterial biocides and metals can contribute to the development and maintenance of antibiotic resistance in bacterial communities through co-selection. Information on metal and biocide resistance genes, including their sequences and molecular functions, is, however, scattered. Here, we introduce BacMet (http://bacmet.biomedicine.gu.se)—a manually curated database of antibacterial biocide- and metal-resistance genes based on an in-depth review of the scientific literature. The BacMet database contains 470 experimentally verified resistance genes. In addition, the database also contains 25 477 potential resistance genes collected from public sequence repositories. All resistance genes in the BacMet database have been organized according to their molecular function and induced resistance phenotype.