Survey of Microbial Diversity in Flood Areas during Thailand 2011 Flood Crisis Using High-Throughput Tagged Amplicon Pyrosequencing

The Thailand flood crisis in 2011 was one of the largest recorded floods in modern history, causing enormous damage to the economy and ecological habitats of the country. In this study, bacterial and fungal diversity in sediments and waters collected from ten flood areas in Bangkok and its suburbs, covering residential and agricultural areas, were analyzed using high-throughput 454 pyrosequencing of 16S rRNA gene and internal transcribed spacer sequences. Analysis of microbial community showed differences in taxa distribution in water and sediment with variations in the diversity of saprophytic microbes and sulfate/nitrate reducers among sampling locations, suggesting differences in microbial activity in the habitats. Overall, Proteobacteria represented a major bacterial group in waters, while this group co-existed with Firmicutes, Bacteroidetes, and Actinobacteria in sediments. Anaeromyxobacter, Steroidobacter, and Geobacter were the dominant bacterial genera in sediments, while Sulfuricurvum, Thiovirga, and Hydrogenophaga predominated in waters. For fungi in sediments, Ascomycota, Glomeromycota, and Basidiomycota, particularly in genera Philipsia, Rozella, and Acaulospora, were most frequently detected. Chytridiomycota and Ascomycota were the major fungal phyla, and Rhizophlyctis and Mortierella were the most frequently detected fungal genera in water. Diversity of sulfate-reducing bacteria, related to odor problems, was further investigated using analysis of the dsrB gene which indicated the presence of sulfate-reducing bacteria of families Desulfobacteraceae, Desulfobulbaceae, Syntrobacteraceae, and Desulfoarculaceae in the flood sediments. The work provides an insight into the diversity and function of microbes related to biological processes in flood areas.     

Using Amplicon Sequencing To Characterize and Monitor Bacterial Diversity in Drinking Water Distribution Systems

Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic amplicon sequencing. Here, amplicon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user samples were more similar to the source water than to the postdisinfection water. Three sample locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira, were detected. Burkholderiales were abundant in samples containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic amplicon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs.     

Using Bloodmeal Analysis to Assess Disease Risk to Wildlife at the New Northern Limit of a Mosquito Species

The historically southeastern mosquito species Culex erraticus has over the last 30 years undergone a marked expansion north. We evaluated this species’ potential to participate in local disease cycles in the northeastern USA by identifying the vertebrate sources of blood in Cx. erraticus specimens from New Jersey. We found that the majority of bloodmeals (92.6%) were derived from birds, followed by 6.8% from mammals (of which half were human), and a single amphibian bloodmeal from a spring peeper (0.56%). Medium- and large-sized water birds from the order Pelecaniformes made up 60.4% of the bird species and 55.9% of all identified hosts. This group of birds is known enzootic hosts of arboviruses such as eastern equine encephalitis virus, for which Cx. erraticus is a competent vector. Additionally, we screened blooded mosquitoes for avian malaria parasites and identified three different lineages of Plasmodium, including what may represent a new Plasmodium species (likely a wetland bird specialist) in bloodmeals from Green Herons, a Great Egret, and a Double-Crested Cormorant. Our results support the utility of mosquito bloodmeals as sources of information about circulating wildlife pathogens and reveal the potential of range-expanding species to intensify local zoonoses and bridge enzootic pathogens to humans.     

Diversity of Microbial Communities in Production and Injection Waters of Algerian Oilfields Revealed by 16S rRNA Gene Amplicon 454 Pyrosequencing

The microorganisms inhabiting many petroleum reservoirs are multi-extremophiles capable of surviving in environments with high temperature, pressure and salinity. Their activity influences oil quality and they are an important reservoir of enzymes of industrial interest. To study these microbial assemblages and to assess any modifications that may be caused by industrial practices, the bacterial and archaeal communities in waters from four Algerian oilfields were described and compared. Three different types of samples were analyzed: production waters from flooded wells, production waters from non-flooded wells and injection waters used for flooding (water-bearing formations). Microbial communities of production and injection waters appeared to be significantly different. From a quantitative point of view, injection waters harbored roughly ten times more microbial cells than production waters. Bacteria dominated in injection waters, while Archaea dominated in production waters. Statistical analysis based on the relative abundance and bacterial community composition (BCC) revealed significant differences between production and injection waters at both OTUs_0.03 and phylum level. However, no significant difference was found between production waters from flooded and non-flooded wells, suggesting that most of the microorganisms introduced by the injection waters were unable to survive in the production waters. Furthermore, a Venn diagram generated to compare the BCC of production and injection waters of one flooded well revealed only 4% of shared bacterial OTUs. Phylogenetic analysis of bacterial sequences indicated that Alpha-, Beta- and Gammaproteobacteria were the main classes in most of the water samples. Archaeal sequences were only obtained from production wells and each well had a unique archaeal community composition, mainly belonging to Methanobacteria, Methanomicrobia, Thermoprotei and Halobacteria classes. Many of the bacterial genera retrieved had already been reported as degraders of complex organic molecules and pollutants. Nevertheless, a large number of unclassified bacterial and archaeal sequences were found in the analyzed samples, indicating that subsurface waters in oilfields could harbor new and still-non-described microbial species.     

Use of AFLP Analyses to Assess Genetic Variation in Morone and Thunnus Species

The amplified fragment length polymorphism (AFLP) technique was used to assess genetic variation across the genera of 2 Morone and 3 Thunnus species. The AFLP profiles from 23 primers revealed higher levels of polymorphism in each of 3 Thunnus species than in either of the 2 Morone species. However, extensive variation (20 of 23 primers) was observed between the 2 Morone species, but much less variation was observed among the 3 Thunnus species. In addition, comparisons of banding patterns indicated that albacore is divergent from both Atlantic northern bluefin tuna and yellowfin tuna. This result is consistent with the findings of several previous studies employing either allozymes or mitochondrial DNA. Overall, these results demonstrate that AFLP is a useful technique for the assessment of both intraspecies and interspecies variation of fish. Furthermore, the species-specific patterns produced by AFLP can be used for the identification of closely related species.     

Diversity of Phytophthora Species from Declining Mediterranean Maquis Vegetation,including Two New Species,Phytophthora crassamura and P. ornamentata sp. nov.

The Mediterranean basin is recognized as a global biodiversity hotspot accounting for more than 25,000 plant species that represent almost 10% of the world’s vascular flora. In particular, the maquis vegetation on Mediterranean islands and archipelagos constitutes an important resource of the Mediterranean plant diversity due to its high rate of endemism. Since 2009, a severe and widespread dieback and mortality of Quercus ilex trees and several other plant species of the Mediterranean maquis has been observed in the National Park of La Maddalena archipelago (northeast Sardinia, Italy). Infected plants showed severe decline symptoms and a significant reduction of natural regeneration. First studies revealed the involvement of the highly invasive wide-host range pathogen Phytophthora cinnamomi and several fungal pathogens. Subsequent detailed research led to a better understanding of these epidemics showing that multiple Phytophthora spp. were involved, some of them unknown to science. In total, nine Phytophthora species were isolated from rhizosphere soil samples collected from around symptomatic trees and shrubs including Asparagus albus, Cistus sp., Juniperus phoenicea, J. oxycedrus, Pistacia lentiscus and Rhamnus alaternus. Based on morphological characters, growth-temperature relations and sequence analysis of the ITS and cox1 gene regions, the isolates were identified as Phytophthora asparagi, P. bilorbang, P. cinnamomi, P. cryptogea, P. gonapodyides, P. melonis, P. syringae and two new Clade 6 taxa which are here described as P. crassamura sp. nov. and P. ornamentata sp. nov. Pathogenicity tests supported their possible involvement in the severe decline that is currently threatening the Mediterranean maquis vegetation in the La Maddalena archipelago.     

rpoD Gene Pyrosequencing for the Assessment of Pseudomonas Diversity in a Water Sample from the Woluwe River

A water sample from a noncontaminated site at the source of the Woluwe River (Belgium) was analyzed by culture-dependent and -independent methods. Pseudomonas isolates were identified by sequencing and analysis of the rpoD gene. Culture-independent methods consisted of cloning and pyrosequencing of a Pseudomonas rpoD amplicon from total DNA extracted from the same sample and amplified with selective rpoD gene primers. Among a total of 14,540 reads, 6,228 corresponded to Pseudomonas rpoD gene sequences by a BLAST analysis in the NCBI database. The selection criteria for the reads were sequences longer than 400 bp, an average Q₄₀ value greater than 25, and >85% identity with a Pseudomonas species. Of the 6,228 Pseudomonas rpoD sequences, 5,345 sequences met the established criteria for selection. Sequences were clustered by phylogenetic analysis and by use of the QIIME software package. Representative sequences of each cluster were assigned by BLAST analysis to a known Pseudomonas species when the identity with the type strain was greater than or equal to 96%. Twenty-six species distributed among 12 phylogenetic groups or subgroups within the genus were detected by pyrosequencing. Pseudomonas stutzeri, P. moraviensis, and P. simiae were the only cultured species not detected by pyrosequencing. The predominant phylogenetic group within the Pseudomonas genus was the P. fluorescens group, as determined by culture-dependent and -independent analyses. In all analyses, a high number of putative novel phylospecies was found: 10 were identified in the cultured strains and 246 were detected by pyrosequencing, indicating that the diversity of Pseudomonas species has not been fully described.     

Phenotypic and Genetic Diversity of Aeromonas Species Isolated from Fresh Water Lakes in Malaysia

Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions—exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.     

Diversity and Significance of Mold Species in Norwegian Drinking Water

In order to determine the occurrence, distribution, and significance of mold species in groundwater- and surface water-derived drinking water in Norway, molds isolated from 273 water samples were identified. Samples of raw water, treated water, and water from private homes and hospital installations were analyzed by incubation of 100-ml membrane-filtered samples on dichloran-18% glycerol agar. The total count (number of CFU per 100 ml) of fungal species and the species diversity within each sample were determined. The identification of mold species was based on morphological and molecular methods. In total, 94 mold species belonging to 30 genera were identified. The mycobiota was dominated by species of Penicillium, Trichoderma, and Aspergillus, with some of them occurring throughout the drinking water system. Several of the same species as isolated from water may have the potential to cause allergic reactions or disease in humans. Other species are common contaminants of food and beverages, and some may cause unwanted changes in the taste or smell of water. The present results indicate that the mycobiota of water should be considered when the microbiological safety and quality of drinking water are assessed. In fact, molds in drinking water should possibly be included in the Norwegian water supply and drinking water regulations.     

Intra-Species Diversity and Panmictic Structure of Cryptosporidium parvum Populations in Cattle Farms in Northern Spain

The intra-herd and intra-host genetic variability of 123 Cryptosporidium parvum isolates was investigated using a multilocus fragment typing approach with eleven variable-number tandem-repeat (VNTR) loci and the GP60 gene. Isolates were collected from intensively farmed diarrheic pre-weaned calves originating from 31 dairy farms in three adjoining regions in northern Spain (País Vasco, Cantabria and Asturias). The multilocus tool demonstrated an acceptable typeability, with 104/123 samples amplifying at all twelve loci. The ML2, TP14, GP60 and the previously un-described minisatellite at locus cgd2_3850 were the most discriminatory markers, while others may be dismissed as monomorphic (MSB) or less informative (CP47, ML1 and the novel minisatellites at loci Cgd1_3670 and Cgd6_3940). The 12-satellite typing tool provided a Hunter-Gaston index (HGDI) of 0.987 (95% CI, 0.982–0.992), and differentiated a total of 70 multilocus subtypes (MLTs). The inclusion of only the four most discriminatory markers dramatically reduced the number of MLTs (n: 44) but hardly reduced the HGDI value. A total of 54 MLTs were distinctive for individual farms, indicating that cryptosporidiosis is an endemic condition on most cattle farms. However, a high rate of mixed infections was detected, suggesting frequent meiotic recombination. Namely, multiple MLTs were seen in most farms where several specimens were analyzed (90.5%), with up to 9 MLTs being found on one farm, and individual specimens with mixed populations being reported on 11/29 farms. Bayesian Structure analysis showed that over 35% of isolates had mixed ancestry and analysis of evolutionary descent using the eBURST algorithm detected a high rate (21.4%) of MLTs appearing as singletons, indicating a high degree of genetic divergence. Linkage analysis found evidence of linkage equilibrium and an overall panmictic structure within the C. parvum population in this discrete geographical area.     

Pyrosequencing Reveals Contrasting Soil Bacterial Diversity and Community Structure of Two Main Winter Wheat Cropping Systems in China

Microbes are key components of the soil environment, playing an important role in maintaining soil health, sustainability, and productivity. The composition and structure of soil bacterial communities were examined in winter wheat–rice (WR) and winter wheat–maize (WM) cropping systems derived from five locations in the Low-Middle Yangtze River plain and the Huang-Huai-Hai plain by pyrosequencing of the 16S ribosomal RNA gene amplicons. A total of 102,367 high quality sequences were used for multivariate statistical analysis and to test for correlation between community structure and environmental variables such as crop rotations, soil properties, and locations. The most abundant phyla across all soil samples were Proteobacteria, Acidobacteria, and Bacteroidetes. Similar patterns of bacterial diversity and community structure were observed within the same cropping systems, and a higher relative abundance of anaerobic bacteria was found in WR compared to WM cropping systems. Variance partitioning analysis revealed complex relationships between bacterial community and environmental variables. The effect of crop rotations was low but significant, and interactions among soil properties, locations, and crop rotations accounted for most of the explained variation in the structure of bacterial communities. Soil properties such as pH, available P, and available K showed higher correlations (positive or negative) with the majority of the abundant taxa. Bacterial diversity (the Shannon index) and richness (Chao1 and ACE) were higher under WR than WM cropping systems.     

Effects Of Vegetation and Soil on Species Diversity of Soil Dwelling Diptera in a Heathland Ecosystem

The role of vegetation and soil factors on the biodiversity of two soil dwelling, saprophagous, fly families (Sphaeroceridae and Lonchopteridae) in a heathland ecosystem were investigated. The fly community is primarily affected by soil humidity and the amount of organic matter while the vegetation structure and species composition only indirectly influence the fly communities. There was no correlation between plant species richness and the fly diversity indices. Based on our results and literature data, we hypothesise that the direct effects of the vegetation is more evident for herbivorous insects than for species that do not feed on plants. The investigated families show a clear response to microhabitat differences in the soil factors, which makes them promising indicators for soil health and as tool for monitoring environmental changes.     

The Use of Structured Imagery and Dispositional Measurement to Assess Situational Use of Mindfulness Skills

The recent proliferation of studies on mindfulness produced varying theoretical models, each based in part on how mindfulness is assessed. These models agree, however, that mindfulness encompasses moment-to-moment or situational experiences. Incongruence between dispositional and situational assessment would be problematic for theory and empirical research. In particular, it remains to be established whether situational measurement is an accurate method for mindfulness assessment and whether dispositional measures are able to accurately detect mindfulness skills in various situations. The association between dispositional and situational mindfulness processes (i.e., situational attention awareness and emotion acceptance) was examined in two studies. In Study 1 (N = 148), independent groups who reported high and low levels of dispositional mindfulness skills were compared on a continuous measure of situational mindfulness skills. In Study 2 (N = 317), dispositional mindfulness questionnaires were used to predict situational use of mindfulness skills. Results suggest not only that situational measures accurately detect use of mindfulness skills, but also that dispositional measures can predict one’s use of situational mindfulness skills. Findings from both studies were consistent across both positive and negative situations. Moreover, neither neuroticism nor extraversion was shown to have a moderating effect on the relationship between dispositional and situational use of mindfulness skills. The implications of these findings for clinical practice and future investigations pertaining to measurement validity in this area are discussed.     

佳西热带雨林土壤芽胞杆菌分离与多样性分析

采用热处理法从海南省佳西热带雨林土壤中分离到147株芽胞杆菌,并利用16S rDNA PCR-RFLP与序列分析技术对其遗传多样性进行了研究。16S rDNA PCR-RFLP酶切图谱UPGMA聚类分析结果表明,在100%的相似性水平上,这些芽胞杆菌分属13个遗传类群。不同遗传类型代表菌株的16S rRNA基因序列分析结果显示,它们分布在Bacillaceae、Planococcaceae和Paenibacillaceae科的Bacillus、Lysinibacillus、Paucisalibacillus、Bhargavaea和Paenibacillus五个属,其中Bacillus为优势属(占50%);有3株芽胞杆菌的16S rRNA基因序列与数据库中相应模式菌株的最大相似性在98.3%~98.9%之间。结果表明,佳西热带雨林土壤中芽胞杆菌有着较为丰富的遗传多样性。     

The Transport of Water from Soil to Shoot in Wheat Seedlings

A technique is described for precisely measuring the drop inwater potential, , between the soil and the leaf xylem of wheatseedlings. The technique was used to explore the relation betweentranspiration rate and at various times during the monotonicdrying of the soil in which the plants were growing. When thesoil was wet, the relation was linear, but, as the soil dried,nonlinearities appeared which were, in the main, explicablein terms of simple soil physical models describing the flowof water through the soil to the roots. There was no sign ofthe major hydraulic resistance at the root: soil interface thatother people have recently found.     

Optimization of Procedures for Isolation of Mycobacteria from Soil and Water Samples Obtained in Northern India

For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.