Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).

Ocean acidification shows negligible impacts on high-latitude bacterial community structure in coastal pelagic mesocosms

The impact of ocean acidification and carbonation on microbial community structure was assessed during a large-scale in situ costal pelagic mesocosm study, included as part of the EPOCA 2010 Arctic campaign. The mesocosm experiment included ambient conditions (fjord) and nine mesocosms with pCO(2) levels ranging from similar to 145 to similar to 1420 mu atm. Samples for the present study were collected at ten time points (t-1, t1, t5, t7, t12, t14, t18, t22, t26 to t28) in seven treatments (ambient fjord (similar to 145), 2x similar to 185, similar to 270, similar to 685, similar to 820, similar to 1050 mu atm) and were analysed for "small" and "large" size fraction microbial community composition using 16S rRNA (ribosomal ribonucleic acid) amplicon sequencing. This high-throughput sequencing analysis produced similar to 20 000 000 16S rRNA V4 reads, which comprised 7000OTUs. The main variables structuring these communities were sample origins (fjord or mesocosms) and the community size fraction (small or large size fraction). The community was significantly different between the unenclosed fjord water and enclosed mesocosms (both control and elevated CO2 treatments) after nutrients were added to the mesocosms, suggesting that the addition of nutrients is the primary driver of the change in mesocosm community structure. The relative importance of each structuring variable depended greatly on the time at which the community was sampled in relation to the phytoplankton bloom. The sampling strategy of separating the small and large size fraction was the second most important factor for community structure. When the small and large size fraction bacteria were analysed separately at different time points, the only taxon pCO(2) was found to significantly affect were the Gammaproteobacteria after nutrient addition. Finally, pCO(2) treatment was found to be significantly correlated (non-linear) with 15 rare taxa, most of which increased in abundance with higher CO2.

Diverse profiles of N‐acyl‐homoserine lactone molecules found in cnidarians

Many marine habitats, such as the surface and tissues of marine invertebrates, including corals, harbour diverse populations of microorganisms, which are thought to play a role in the health of their hosts and influence mutualistic and competitive interactions. Investigating the presence and stability of quorum sensing (QS) in these ecosystems may shed light on the roles and control of these bacterial communities. Samples of 13 cnidarian species were screened for the presence and diversity of N-acyl-homoserine lactones (AHLs; a prevalent type of QS molecule) using thin-layer chromatography and an Agrobacterium tumefaciens NTL4 biosensor. Ten of 13 were found to harbour species-specific, conserved AHL profiles. AHLs were confirmed in Anemonia viridis using liquid chromatography tandem mass spectrometry. To assess temporal role and stability, AHLs were investigated in A. viridis from intertidal pools over 16 h. Patterns of AHLs showed conserved profiles except for two mid-chain length AHLs, which increased significantly over the day, peaking at 20:00, but had no correlation with pool chemistry. Denaturing gel electrophoresis of RT-PCR-amplified bacterial 16S rRNA showed the presence of an active bacterial community that changed in composition alongside AHL profiles and contained a number of bands that affiliate with known AHL-producing bacteria. Investigations into the quorum sensing-controlled, species-specific roles of these bacterial communities and how these regulatory circuits are influenced by the coral host and members of the bacterial community are imperative to expand our knowledge of these interactions with respect to the maintenance of coral health.

Rapid response of the active microbial community to CO2 exposure from a controlled sub-seabed CO2 leak in Ardmucknish Bay (Oban, Scotland)

The response of the benthic microbial community to a controlled sub-seabed CO2 leak was assessed using quantitative PCR measurements of benthic bacterial, archaeal and cyanobacteria/chloroplast 16S rRNA genes. Samples were taken from four zones (epicentre; 25 m distant, 75 m distant and 450 m distant) during 6 time points (7 days before CO2 exposure, after 14 and 36 days of CO2 release, and 6, 20 and 90 days after the CO2 release had ended). Changes to the active community of microphytobenthos and bacteria were also assessed before, during and after CO2 release. Increases in the abundance of microbial 16S rRNA were detected after 14 days of CO2 release and at a distance of 25 m from the epicentre. CO2 related changes to the relative abundance of both major and minor bacterial taxa were detected: most notably an increase in the relative abundance of the Planctomycetacia after 14 days of CO2 release. Also evident was a decrease in the abundance of microbial 16S rRNA genes at the leak epicentre during the initial recovery phase: this coincided with the highest measurements of DIC within the sediment, but may be related to the release of potentially toxic metals at this time point.

Validation of the detection of Alexandrium species using specific RNA probes tested in a microarray format: Calibration of signal using variability of RNA content with environmental conditions.

The dinoflagellate genus Alexandrium contains several toxin producing species and strains, which can cause major economic losses to the shell fish industry. It is therefore important to be able to detect these toxin producers and also distinguish toxic strains from some of the morphologically identical non-toxic strains. To facilitate this DNA probes to be used in a microarray format were designed in silico or developed from existing published probes. These probes targeted either the 18S or 28S ribosomal ribonucleic acid (rRNA) gene in Alexandrium tamarense Group I, Group III and Group IV, Alexandrium ostenfeldii and Alexandrium minutum. Three strains of A. tamarense Group I, A. tamarense Group III, A. minutum and two strains of A. ostenfeldii were grown at optimal conditions and transferred into new environmental conditions changing either the light intensity, salinity, temperature or nutrient concentrations, to check if any of these environmental conditions induced changes in the cellular ribonucleic acid (RNA) concentration or growth rate. The aim of this experiment was the calibration of several species-specific probes for the quantification of the toxic Alexandrium strains. Growth rates were highly variable but only elevated or lowered salinity significantly lowered growth rate for A. tamarense Group I and Group III; differences in RNA content were not significant for the majority of the treatments. Only light intensity seemed to affect significantly the RNA content in A. tamarense Group I and Group III, but this was still within the same range as for the other treatments meaning that a back calibration from RNA to cell numbers was possible. The designed probes allow the production of quantitative information for Alexandrium species for the microarray chip.

Dynamic responses of the benthic bacterial community at the Western English Channel observatory site L4 are driven by deposition of fresh phytodetritus

The impact of the seasonal deposition of phytoplankton and phytodetritus on surface sediment bacterial abundance and community composition was investigated at the Western English Channel site L4. Sediment and water samples were collected from January to September in 2012, increasing in frequency during periods of high water column phytoplankton abundance. Compared to the past two decades, the spring bloom in 2012 was both unusually long in duration and contained higher than average biomass. Within spring months, the phytoplankton bloom was well mixed through the water column and showed accumulations near the sea bed, as evidenced by flow cytometry measurements of nanoeukaryotes, water column chlorophyll a and the appearance of pelagic phytoplankton at the sediment. Measurements of chlorophyll and chlorophyll degradation products indicated phytoplankton material was heavily degraded after it reached the sediment surface: the nature of the chlorophyll degradation products (predominantly pheophorbide, pyropheophorbide and hydroxychlorophyllone) was indicative of grazing activity. The abundance of bacterial 16S rRNA genes g−1 sediment (used as a proxy for bacterial biomass) increased markedly with the onset of the phytoplankton bloom, and correlated with measurements of chlorophyll at the surface sediment. Together, this suggests that bacteria may have responded to nutrients released via grazing activity. In depth sequencing of the 16S rRNA genes indicated that the composition of the bacterial community shifted rapidly through-out the prolonged spring bloom period. This was primarily due to an increase in the relative sequence abundance of Flavobacteria.

The distribution of Fasciola hepatica and Fasciola gigantica within southern Tanzania - constraints associated with the intermediate host

In East Africa, Fasciola gigantica is generally the causative agent of fasciolosis but there have been reports of F. hepatica in cattle from highland regions of Kenya, Ethiopia, Uganda and Zaire. The topography of the Southern Highlands of Tanzania provides an environment where the climatic conditions exist for the sustenance of lymnaeid species capable of Supporting both Fasciola hepatica and F. gigantica. Theoretically this would allow interaction between fasciolid species and the possible creation of hybrids. In this report we present molecular data confirming the existence of the snail, Lymnaea truncatula, at high altitude on the Kitulo Plateau of the Southern Highlands, Tanzania, along with morphometric and molecular data confirming the presence of F. hepatica in the corresponding area. At lower altitudes, where climatic conditions were unfavourable for the existence of L. truncatula, the presence of its sister species L. natalensis was confirmed by molecular data along with its preferred fasciolid parasite, F. gigantica. Analysis based on a 618 bp sequence of the 28S rRNA gene did not reveal the presence of hybrid fasciolids in our fluke samples.

The invasive genus Asparagopsis (Bonnemaisoniaceae, Rhodophyta): Molecular systematics, morphology, and ecophysiology of Falkenbergia isolates

The genus Asparagopsis was studied using 25 Falkenbergia tetrasporophyte strains collected worldwide. Plastid (cp) DNA RFLP revealed three groups of isolates, which differed in their small subunit rRNA gene sequences, temperature responses, and tetrasporophytic morphology (cell sizes). Strains from Australia, Chile, San Diego, and Atlantic and Mediterranean Europe were identifiable as A. armata Harvey, the gametophyte of which has distinctive barbed spines. This species is believed to be endemic to cold-temperate waters of Australia and New Zealand and was introduced into Europe in the 1920s. All isolates showed identical cpDNA RFLPs, consistent with a recent introduction from Australia. Asparagopsis taxiformis (Delile) Trevisan, the type and only other recognized species, which lacks spines, is cosmopolitan in warm-temperate to tropical waters. Two clades differed morphologically and ecophysiologically and in the future could be recognized as sibling species or subspecies. A Pacific/Italian clade had 4-8degrees C lower survival minima and included a genetically distinct apomictic isolate from Western Australia that corresponded to the form of A. taxiformis originally described as A. sanfordiana Harvey. The second clade, from the Caribbean and the Canaries, is stenothermal (subtropical to tropical) with some ecotypic variation. The genus Asparagopsis consists of two or possibly three species, but a definitive taxonomic treatment of the two A. taxiformis clades requires study of field-collected gametophytes.

FACT facilitates chromatin transcription by RNA polymerases I and III

Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III-transcribed genes.

Frequency and distribution of group I intron genotypes of Candida albicans colonising critically ill patients

A study is performed to examine the distribution and frequency of 25S rRNA intron genotypes of Candida albicans isolated from different anatomical sites of patients in an intensive care unit (ICU) setting. Germ-tube positive Candida isolates (n=65) from 65 patients are included and isolates are characterised by 25S intron genotyping, whereby all can be subdivided into four genotypes (A-D). Results demonstrated that there were no significant differences between the frequency and genotype distribution of the Candida isolates and the anatomical site of colonisation. Furthermore, analysis of the transposable intron region in the 25S rRNA gene demonstrated equal distribution, regardless of age and anatomical site of isolation (groin, throat, etc.). Therefore, there does not appear to be any selective pressure associated with any anatomical site, resulting in an ecological shift in the frequency of genotypes present. This suggests that C. albicans intron genotypes equally colonise those sites of the body examined in this study. Although such an ecological finding as this is interesting, it perpetuates the continued need to find a genotypic typing scheme that helps to identify the source (nosocomial or endogenous) and mode of entry of C. albicans into patients in the ICU setting, resulting in C. albicans bloodstream infection.

Rapid differentiation between fluconazole-sensitive and -resistant species of Candida directly from positive blood-culture bottles by real-time PCR

In view of both the delay in obtaining identification by conventional methods following blood-culture positivity in patients with candidaemia and the close relationship between species and fluconazole (FLC) susceptibility, early speciation of positive blood cultures has the potential to influence therapeutic decisions. The aim was to develop a rapid test to differentiate FLC-resistant from FLC-sensitive Candida species. Three TaqMan-based real-time PCR assays were developed to identify up to six Candida species directly from BacT/Alert blood-culture bottles that showed yeast cells on Gram staining at the time of initial positivity. Target sequences in the rRNA gene complex were amplified, using a consensus two-step PCR protocol, to identify Candida albicans, Candida parapsilosis, Candida tropicalis, Candida dubliniensis, Candida glabrata and Candida krusei; these are the most commonly encountered Candida species in blood cultures. The first four of these (the characteristically FLC-sensitive group) were identified in a single reaction tube using one fluorescent TaqMan probe targeting 1 8S rRNA sequences conserved in the four species. The FLC-resistant species C. krusei and C. glabrata were detected in two further reactions, each with species-specific probes. This method was validated with clinical specimens (blood cultures) positive for yeast (n=33 sets) and the results were 100% concordant with those of phenotypic identification carried out concomitantly. The reported assay significantly reduces the time required to identify the presence of C. glabrata and C. krusei in comparison with a conventional phenotypic method, from ~72 to

Microbial Communities Associated with Healthy and White Syndrome-Affected Echinopora lamellosa in Aquaria and Experimental Treatment with the Antibiotic Ampicillin

Prokaryotic and ciliate communities of healthy and aquarium White Syndrome (WS)-affected coral fragments were screened using denaturing gradient gel electrophoresis (DGGE). A significant difference (R = 0.907, p < 0.001) in 16S rRNA prokaryotic diversity was found between healthy (H), sloughed tissue (ST), WS-affected (WSU) and antibiotic treated (WST) samples. Although 3 Vibrio spp were found inWS-affected samples, two of these species were eliminated following ampicillin treatment, yet lesions continued to advance, suggesting they play a minor or secondary role in the pathogenesis. The third Vibrio sp increased slightly in relative abundance in diseased samples and was abundant in non-diseased samples. Interestingly, a Tenacibaculum sp showed the greatest increase in relative abundance between healthy and WS-affected samples, demonstrating consistently high abundance across all WS-affected and treated samples, suggesting Tenacibaculum sp could be a more likely candidate for pathogenesis in this instance. In contrast to previous studies bacterial abundance did not vary significantly (ANOVA, F2, 6 = 1.000, p = 0.422) between H, ST, WSU or WST. Antimicrobial activity (assessed on Vibrio harveyi cultures) was limited in both H and WSU samples (8.1% ±8.2 and 8.0% ±2.5, respectively) and did not differ significantly (Kruskal-Wallis, χ2 (2) = 3.842, p = 0.146). A Philaster sp, a Cohnilembus sp and a Pseudokeronopsis sp. were present in all WS-affected samples, but not in healthy samples. The exact role of ciliates in WS is yet to be determined, but it is proposed that they are at least responsible for the neat lesion boundary observed in the disease.

Performance of a Novel Molecular Method in the Diagnosis of Late-Onset Sepsis in Very Low Birth Weight Infants

OBJECTIVE: To compare the use of a generic molecular assay to 'standard' investigations used to assist the diagnosis of late onset bacterial sepsis in very low birth weight infants (VLBW, <1500g).

METHODS: VLBW infants, greater than 48 hours of age, who were clinically suspected to have sepsis were investigated using standard tests (full blood count, C-reactive protein (at presentation) and blood culture), in addition, blood was taken for a universal molecular assay (16S rRNA reverse transcriptase PCR) for comparison. Clinical data were recorded during the suspected infection episode. A validated sepsis score (NEO-KISS) was used to retrospectively determine the presence of sepsis (independent of blood culture). The performance of each of the tests were compared by sensitivity, specificity, positive/negative likihood ratios (+/-LR) and postive/negative predictive values (PPV/NPV).

RESULTS: Sixty-five babies with suspected clinical sepsis were prospectively included. The performance indicators are presented with 95% confidence limits. For the detection of bacteria, blood culture had sensitivity of 0.57 (0.34-0.78), specificity of 0.45 (0.30-0.61); +LR of 1.05 (0.66-1.66) and-LR of 0.94 (0.52-1.7); PPV of 33.3 (18.56-50.97) and NPV of 68.97 (49.17-87.72). Serum CRP had sensitivity of 0.92 (0.64-1) and specificity of 0.36 (0.17-0.59); +LR of 1.45 (1-2.1) and-LR of 0.21 (0.03-1.5); PPV of 44.46 (26.6-66.6) and NPV of 88.9 (51.8-99.7). The universal molecular assay had sensitivity of 0.76 (0.53-0.92), specificity of 0.95 (0.85-0.99); +LR of 16.8 (4.2-66.3) and-LR of 0.25 (0.1-0.5); PPV of 88.9 (65.3-98.6) and NPV of 89.4 (76.9-96.5).

CONCLUSIONS: In VLBW infants this universal molecular assay performed better in the diagnosis of late onset sepsis (LOS) than blood culture and CRP. Further development is required to explore and improve the performance of the assay in real-time diagnosis.

Metagenomic characterisation of the viral community of lough neagh, the largest freshwater lake in Ireland

Lough Neagh is the largest and the most economically important lake in Ireland. It is also one of the most nutrient rich amongst the world's major lakes. In this study, 16S rRNA analysis of total metagenomic DNA from the water column of Lough Neagh has revealed a high proportion of Cyanobacteria and low levels of Actinobacteria, Acidobacteria, Chloroflexi, and Firmicutes. The planktonic virome of Lough Neagh has been sequenced and 2,298,791 2×300 bp Illumina reads analysed. Comparison with previously characterised lakes demonstrates that the Lough Neagh viral community has the highest level of sequence diversity. Only about 15% of reads had homologs in the RefSeq database and tailed bacteriophages (Caudovirales) were identified as a major grouping. Within the Caudovirales, the Podoviridae and Siphoviridae were the two most dominant families (34.3% and 32.8% of the reads with sequence homology to the RefSeq database), while ssDNA bacteriophages constituted less than 1% of the virome. Putative cyanophages were found to be abundant. 66,450 viral contigs were assembled with the largest one being 58,805 bp; its existence, and that of another 34,467 bp contig, in the water column was confirmed. Analysis of the contigs confirmed the high abundance of cyanophages in the water column.

Yeast diversity in the Mediterranean strawberry tree (Arbutus unedo L.) fruits' fermentations

In the Mediterranean region the fruits of the strawberry tree (Arbutus unedo L.) may be fermented and distilled to produce a traditional beverage very much appreciated in Southern Europe. The aim of the present work was to study the diversity of the yeast population and the killer activity of the isolates identified as Saccharomyces cerevisiae, obtained during solid state industrial fermentations of the arbutus berries. The identification of the isolates was performed by the 5.8S rRNA-ITS region restriction analysis and by sequencing the D1/D2 region of the large subunit of the rRNA gene. At the start of the fermentations, various non-Saccharomyces species were detected including Aureobasidium pullulans, Dothichiza pithyophila, Dioszegia zsoltii, Hanseniaspora uvarum and yeasts belonging to the genera Metschnikowia, Cryptococcus and Rhodotorula. However, as the biological processes progressed the number of different species decreased with S. cerevisiae and Pichia membranaefaciens becoming dominant at advanced stages of the must fermentation that is characterized by high concentrations of ethanol. Forty three isolates identified as S. cerevisiae were tested for killer activity against two sensitive reference strains and Zygosaccharomyces bailii. Their killer sensitivity in relation to five killer referenced toxins (K2, K5, K8, K9 and K10) was also studied. Out of the isolates analyzed, 95.3% were sensitive and 4.7% were tolerant against the killer toxins tested. Only three isolates revealed killer activity against one sensitive strain and two of them against the spoiler yeast Z. bailii. The microbiota obtained revealed an interesting potential to be used as starter cultures to overcome unpredictable uncontrolled fermentations of the arbutus fruits as well as in other applications of biotechnological interest. (C) 2012 Elsevier Ltd. All rights reserved.