Tracking seasonal changes in diversity of pollen allergen exposure: Targeted metabarcoding of a subtropical aerobiome.

The importance of grass pollen to the global burden of allergic respiratory disease is well established but exposure to subtropical and temperate pollens is difficult to discern. Current monitoring of airborne pollen relies on light microscopy, limiting identification of taxa to family level. This informs seasonal fluctuations in pollen aerobiology but restricts analysis of aerobiological composition. We aimed to test the utility of DNA metabarcoding to identify specific taxa contributing to the aerobiome of environmental air samples, using routine pollen and spore monitoring equipment, as well as assess temporal variation of Poaceae pollen across an entire season. Airborne pollen concentrations were determined by light microscopy over two pollen seasons in the subtropical city of Brisbane (27°32'S, 153°00E), Australia. Thirty daily pollen samples were subjected to high throughput sequencing of the plastid rbcL amplicon. Amplicons corresponded to plants observed in the local biogeographical region with up to 3238 different operational taxonomic units (OTU) detected. The aerobiome sequencing data frequently identified pollen to genus levels with significant quantitative differences in aerobiome diversity between the months and seasons detected. Moreover, multiple peaks of Chloridoideae and Panicoideae pollen were evident over the collection period confirming these grasses as the dominant Poaceae pollen source across the season. Targeted high throughput sequencing of routinely collected airborne pollen samples appears to offer utility to track temporal changes in the aerobiome and shifts in pollen exposure. Precise identification of the composition and temporal distributions of airborne pollen is important for tracking biodiversity and for management of allergic respiratory disease.

Fluconazole resistance in Candida albicans is induced by Pseudomonas aeruginosa quorum sensing.

Microorganisms employ quorum sensing (QS) mechanisms to communicate with each other within microbial ecosystems. Emerging evidence suggests that intraspecies and interspecies QS plays an important role in antimicrobial resistance in microbial communities. However, the relationship between interkingdom QS and antimicrobial resistance is largely unknown. Here, we demonstrate that interkingdom QS interactions between a bacterium, Pseudomonas aeruginosa and a yeast, Candida albicans, induce the resistance of the latter to a widely used antifungal fluconazole. Phenotypic, transcriptomic, and proteomic analyses reveal that P. aeruginosa's main QS molecule, N-(3-Oxododecanoyl)-L-homoserine lactone, induces candidal resistance to fluconazole by reversing the antifungal's effect on the ergosterol biosynthesis pathway. Accessory resistance mechanisms including upregulation of C. albicans drug-efflux, regulation of oxidative stress response, and maintenance of cell membrane integrity, further confirm this phenomenon. These findings demonstrate that P. aeruginosa QS molecules may confer protection to neighboring yeasts against azoles, in turn strengthening their co-existence in hostile polymicrobial infection sites.

Beneficial changes in rumen bacterial community profile in sheep and dairy calves as a result of feeding the probiotic Bacillus amyloliquefaciens H57.

AIMS: The probiotic Bacillus amyloliquefaciens H57 increased weight gain, increased nitrogen retention and increased feed intake in ruminants when administered to the diet. This study aims to develop a better understanding of this probiotic effect by analysing changes in the rumen prokaryotic community. METHODS AND RESULTS: Sequencing the 16S rRNA gene PCR amplicons of the rumen microbiome, revealed that ewes fed H57 had a significantly different rumen microbial community structure to Control sheep. In contrast, dairy calves showed no significant differences in rumen community structure between treatment groups. In both instances, H57 was below detection in the rumen community profile and was only present at low relative abundance as determined by qPCR. CONCLUSIONS: The altered rumen microbial community in sheep likely contributes to increased weight gain through more efficient digestion of plant material. As no change occurred in the rumen community of dairy calves it is suggested that increased weight gain may be due to changes in community function rather than structure. The low relative abundance of H57 as determined by qPCR, suggests that weight gain was not directly mediated by the probiotic, but rather by influencing animal behaviour (feed consumption) and/or altering the native rumen community structure or function. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a novel look at the rumen prokaryotic community in both sheep and dairy calves when fed H57. These findings improve our understanding for the potential rumen community involvement in H57-enabled weight gain. The study reveals that the probiotic B. amyloliquefaciens H57 is capable of benefiting ruminants without colonizing the rumen, suggesting an indirect mechanism of action.

Isolation of Succinivibrionaceae implicated in low methane emissions from Tammar wallabies.

The Tammar wallaby (Macropus eugenii) harbors unique gut bacteria and produces only one-fifth the amount of methane produced by ruminants per unit of digestible energy intake. We have isolated a dominant bacterial species (WG-1) from the wallaby microbiota affiliated with the family Succinivibrionaceae and implicated in lower methane emissions from starch-containing diets. This was achieved by using a partial reconstruction of the bacterium's metabolism from binned metagenomic data (nitrogen and carbohydrate utilization pathways and antibiotic resistance) to devise cultivation-based strategies that produced axenic WG-1 cultures. Pure-culture studies confirm that the bacterium is capnophilic and produces succinate, further explaining a microbiological basis for lower methane emissions from macropodids. This knowledge also provides new strategic targets for redirecting fermentation and reducing methane production in livestock.

Adaptation to herbivory by the Tammar wallaby includes bacterial and glycoside hydrolase profiles different from other herbivores.

Metagenomic and bioinformatic approaches were used to characterize plant biomass conversion within the foregut microbiome of Australia's "model" marsupial, the Tammar wallaby (Macropus eugenii). Like the termite hindgut and bovine rumen, key enzymes and modular structures characteristic of the "free enzyme" and "cellulosome" paradigms of cellulose solubilization remain either poorly represented or elusive to capture by shotgun sequencing methods. Instead, multigene polysaccharide utilization loci-like systems coupled with genes encoding beta-1,4-endoglucanases and beta-1,4-endoxylanases--which have not been previously encountered in metagenomic datasets--were identified, as were a diverse set of glycoside hydrolases targeting noncellulosic polysaccharides. Furthermore, both rrs gene and other phylogenetic analyses confirmed that unique clades of the Lachnospiraceae, Bacteroidales, and Gammaproteobacteria are predominant in the Tammar foregut microbiome. Nucleotide composition-based sequence binning facilitated the assemblage of more than two megabase pairs of genomic sequence for one of the novel Lachnospiraceae clades (WG-2). These analyses show that WG-2 possesses numerous glycoside hydrolases targeting noncellulosic polysaccharides. These collective data demonstrate that Australian macropods not only harbor unique bacterial lineages underpinning plant biomass conversion, but their repertoire of glycoside hydrolases is distinct from those of the microbiomes of higher termites and the bovine rumen.

Genomic analysis of "Elusimicrobium minutum," the first cultivated representative of the phylum "Elusimicrobia" (formerly termite group 1).

Organisms of the candidate phylum termite group 1 (TG1) are regularly encountered in termite hindguts but are present also in many other habitats. Here, we report the complete genome sequence (1.64 Mbp) of "Elusimicrobium minutum" strain Pei191(T), the first cultured representative of the TG1 phylum. We reconstructed the metabolism of this strictly anaerobic bacterium isolated from a beetle larva gut, and we discuss the findings in light of physiological data. E. minutum has all genes required for uptake and fermentation of sugars via the Embden-Meyerhof pathway, including several hydrogenases, and an unusual peptide degradation pathway comprising transamination reactions and leading to the formation of alanine, which is excreted in substantial amounts. The presence of genes encoding lipopolysaccharide biosynthesis and the presence of a pathway for peptidoglycan formation are consistent with ultrastructural evidence of a gram-negative cell envelope. Even though electron micrographs showed no cell appendages, the genome encodes many genes putatively involved in pilus assembly. We assigned some to a type II secretion system, but the function of 60 pilE-like genes remains unknown. Numerous genes with hypothetical functions, e.g., polyketide synthesis, nonribosomal peptide synthesis, antibiotic transport, and oxygen stress protection, indicate the presence of hitherto undiscovered physiological traits. Comparative analysis of 22 concatenated single-copy marker genes corroborated the status of "Elusimicrobia" (formerly TG1) as a separate phylum in the bacterial domain, which was so far based only on 16S rRNA sequence analysis.

Loss of bacterial diversity during antibiotic treatment of intubated patients colonized with Pseudomonas aeruginosa.

Management of airway infections caused by Pseudomonas aeruginosa is a serious clinical challenge, but little is known about the microbial ecology of airway infections in intubated patients. We analyzed bacterial diversity in endotracheal aspirates obtained from intubated patients colonized by P. aeruginosa by using 16S rRNA clone libraries and microarrays (PhyloChip) to determine changes in bacterial community compositions during antibiotic treatment. Bacterial 16S rRNA genes were absent from aspirates obtained from patients briefly intubated for elective surgery but were detected by PCR in samples from all patients intubated for longer periods. Sequencing of 16S rRNA clone libraries demonstrated the presence of many orally, nasally, and gastrointestinally associated bacteria, including known pathogens, in the lungs of patients colonized with P. aeruginosa. PhyloChip analysis detected the same organisms and many additional bacterial groups present at low abundance that were not detected in clone libraries. For each patient, both culture-independent methods showed that bacterial diversity decreased following the administration of antibiotics, and communities became dominated by a pulmonary pathogen. P. aeruginosa became the dominant species in six of seven patients studied, despite treatment of five of these six with antibiotics to which it was sensitive in vitro. Our data demonstrate that the loss of bacterial diversity under antibiotic selection is highly associated with the development of pneumonia in ventilated patients colonized with P. aeruginosa. Interestingly, PhyloChip analysis demonstrated reciprocal changes in abundance between P. aeruginosa and the class Bacilli, suggesting that these groups may compete for a similar ecological niche and suggesting possible mechanisms through which the loss of microbial diversity may directly contribute to pathogen selection and persistence.

Quantitative phylogenetic assessment of microbial communities in diverse environments.

The taxonomic composition of environmental communities is an important indicator of their ecology and function. We used a set of protein-coding marker genes, extracted from large-scale environmental shotgun sequencing data, to provide a more direct, quantitative, and accurate picture of community composition than that provided by traditional ribosomal RNA-based approaches depending on the polymerase chain reaction. Mapping marker genes from four diverse environmental data sets onto a reference species phylogeny shows that certain communities evolve faster than others. The method also enables determination of preferred habitats for entire microbial clades and provides evidence that such habitat preferences are often remarkably stable over time.

NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes.

Microbiologists conducting surveys of bacterial and archaeal diversity often require comparative alignments of thousands of 16S rRNA genes collected from a sample. The computational resources and bioinformatics expertise required to construct such an alignment has inhibited high-throughput analysis. It was hypothesized that an online tool could be developed to efficiently align thousands of 16S rRNA genes via the NAST (Nearest Alignment Space Termination) algorithm for creating multiple sequence alignments (MSA). The tool was implemented with a web-interface at http://greengenes.lbl.gov/NAST. Each user-submitted sequence is compared with Greengenes' 'Core Set', comprising approximately 10,000 aligned non-chimeric sequences representative of the currently recognized diversity among bacteria and archaea. User sequences are oriented and paired with their closest match in the Core Set to serve as a template for inserting gap characters. Non-16S data (sequence from vector or surrounding genomic regions) are conveniently removed in the returned alignment. From the resulting MSA, distance matrices can be calculated for diversity estimates and organisms can be classified by taxonomy. The ability to align and categorize large sequence sets using a simple interface has enabled researchers with various experience levels to obtain bacterial and archaeal community profiles.

Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB.

A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages in the Archaea and Bacteria.

PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed, paraffin-embedded tissues.

The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.

The development and use of real-time PCR for the quantification of nitrifiers in activated sludge.

Chemical analytical data has long been used to monitor the performance of activated sludge plants even though the process relies on the performance of microorganisms. It is now evident that a rapid and reliable quantitative method is required, to be able to monitor the organisms responsible for nutrient transformation and their activities, allowing avenues for more efficient nutrient removal. The development of real-time or quantitative polymerase chain reaction (PCR) also known as TaqMan or 5'-nuclease assay has allowed the rapid, quantitative analysis of DNA templates, eliminating some of the variability traditionally associated with other quantitative techniques. In this study analysis of Nitrospira spp., one of the key organisms in nitrite oxidation in wastewater treatment, was used to validate real-time PCR for the their quantification in activated sludge. A probe and primer set, targeting the 16S rRNA gene of Nitrospira spp. was designed according to the constraints of the TaqMan specifications. Samples used to evaluate the method included DNA from the sludge from full-scale wastewater treatment plants and laboratory scale systems. The reproducibility, quantitative efficiency and specificity were assessed in the evaluation. It was concluded that the method is sensitive and reproducible but has some constraints on the quantitative efficiency. A survey of full-scale systems for Nitrospira spp. was carried out and the results are presented here.

Intellectual property. Europe's database experiment.

Database protection laws in Europe may have produced a modest, one-time growth spurt for commercial providers. Unfortunately, Europe's new database laws have also produced significant problems including overprotection of low-value data (e.g., telephone books), new barriers to database integration, and further erosion of the public domain. The authors of this Policy Forum caution that these problems could cripple commercial and academic science.

Multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes.

A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with delta-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the delta-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the delta-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.

Clinical and economic impact of diabetes mellitus on percutaneous and surgical treatment of multivessel coronary disease patients: insights from the Arterial Revascularization Therapy Study (ARTS) trial.

BACKGROUND: Our aims were to compare coronary artery bypass grafting (CABG) and stenting for the treatment of diabetic patients with multivessel coronary disease enrolled in the Arterial Revascularization Therapy Study (ARTS) trial and to determine the costs of these 2 treatment strategies. METHODS AND RESULTS: Patients (n=1205) were randomly assigned to stent implantation (n=600; diabetic, 112) or CABG (n=605; diabetic, 96). Costs per patient were calculated as the product of each patient's use of resources and the corresponding unit costs. Baseline characteristics were similar between the groups. At 1 year, diabetic patients treated with stenting had the lowest event-free survival rate (63.4%) because of a higher incidence of repeat revascularization compared with both diabetic patients treated with CABG (84.4%, P<0.001) and nondiabetic patients treated with stents (76.2%, P=0.04). Conversely, diabetic and nondiabetic patients experienced similar 1-year event-free survival rates when treated with CABG (84.4% and 88.4%). The total 1-year costs for stenting and CABG in diabetic patients were $12 855 and $16 585 (P<0.001) and in the nondiabetic groups, $10 164 for stenting and $13 082 for surgery. CONCLUSIONS: Multivessel diabetic patients treated with stenting had a worse 1-year outcome than patients assigned to CABG or nondiabetics treated with stenting. The strategy of stenting was less costly than CABG, however, regardless of diabetic status.

A multiple-outgroup approach to resolving division-level phylogenetic relationships using 16S rDNA data.

The 16S rRNA gene (16S rDNA) is currently the most widely used gene for estimating the evolutionary history of prokaryotes. To date, there are more than 30,000 16S rDNA sequences available from the core databases, GenBank, EMBL and DDBJ. This great number may cause a dilemma when composing datasets for phylogenetic analysis, since the choice and number of reference organisms are known to affect the resulting tree topology. A group of sequences appearing monophyletic in one dataset may not be so in another. This can be especially problematic when establishing the relationships of distantly related sequences at the division (phylum) level. In this study, a multiple-outgroup approach to resolving division-level phylogenetic relationships is suggested using 16S rDNA data. The approach is illustrated by two case studies concerning the monophyly of two recently proposed bacterial divisions, OP9 and OP10.

Investigation of candidate division TM7, a recently recognized major lineage of the domain Bacteria with no known pure-culture representatives.

A molecular approach was used to investigate a recently described candidate division of the domain Bacteria, TM7, currently known only from environmental 16S ribosomal DNA sequence data. A number of TM7-specific primers and probes were designed and evaluated. Fluorescence in situ hybridization (FISH) of a laboratory scale bioreactor using two independent TM7-specific probes revealed a conspicuous sheathed-filament morphotype, fortuitously enriched in the reactor. Morphologically, the filament matched the description of the Eikelboom morphotype 0041-0675 widely associated with bulking problems in activated-sludge wastewater treatment systems. Transmission electron microscopy of the bioreactor sludge demonstrated that the sheathed-filament morphotype had a typical gram-positive cell envelope ultrastructure. Therefore, TM7 is only the third bacterial lineage recognized to have gram-positive representatives. TM7-specific FISH analysis of two full-scale wastewater treatment plant sludges, including the one used to seed the laboratory scale reactor, indicated the presence of a number of morphotypes, including sheathed filaments. TM7-specific PCR clone libraries prepared from the two full-scale sludges yielded 23 novel TM7 sequences. Three subdivisions could be defined based on these data and publicly available sequences. Environmental sequence data and TM7-specific FISH analysis indicate that members of the TM7 division are present in a variety of terrestrial, aquatic, and clinical habitats. A highly atypical base substitution (Escherichia coli position 912; C to U) for bacterial 16S rRNAs was present in almost all TM7 sequences, suggesting that TM7 bacteria, like Archaea, may be streptomycin resistant at the ribosome level.