Bacterial diversity in rhizosphere soil from Antarctic vascular plants of Admiralty Bay, maritime Antarctica

The Antarctic is a pristine environment that contributes to the maintenance of the global climate equilibrium. The harsh conditions of this habitat are fundamental to selecting those organisms able to survive in such an extreme habitat and able to support the relatively simple ecosystems. The DNA of the microbial community associated with the rhizospheres of Deschampsia antarctica Desv (Poaceae) and Colobanthus quitensis (Kunth) BartI (Caryophyllaceae), the only two native vascular plants that are found in Antarctic ecosystems, was evaluated using a 16S rRNA multiplex 454 pyrosequencing approach. This analysis revealed similar patterns of bacterial diversity between the two plant species from different locations, arguing against the hypothesis that there would be differences between the rhizosphere communities of different plants. Furthermore, the phylum distribution presented a peculiar pattern, with a bacterial community structure different from those reported of many other soils. Firmicutes was the most abundant phylum in almost all the analyzed samples, and there were high levels of anaerobic representatives. Also, some phyla that are dominant in most temperate and tropical soils, such as Acidobacteria, were rarely found in the analyzed samples. Analyzing all the sample libraries together, the predominant genera found were Bifidobacterium (phylum Actinobacteria), Arcobacter (phylum Proteobacteria) and Faecalibacterium (phylum Firmicutes). To the best of our knowledge, this is the first major bacterial sequencing effort of this kind of soil, and it revealed more than expected diversity within these rhizospheres of both maritime Antarctica vascular plants in Admiralty Bay, King George Island, which is part of the South Shetlands archipelago. The ISME Journal (2010) 4, 989-1001; doi:10.1038/ismej.2010.35; published online 1 April 2010

Taxonomic and Functional Microbial Signatures of the Endemic Marine Sponge Arenosclera brasiliensis

The endemic marine sponge Arenosclera brasiliensis (Porifera, Demospongiae, Haplosclerida) is a known source of secondary metabolites such as arenosclerins A-C. In the present study, we established the composition of the A. brasiliensis microbiome and the metabolic pathways associated with this community. We used 454 shotgun pyrosequencing to generate approximately 640,000 high-quality sponge-derived sequences (similar to 150 Mb). Clustering analysis including sponge, seawater and twenty-three other metagenomes derived from marine animal microbiomes shows that A. brasiliensis contains a specific microbiome. Fourteen bacterial phyla (including Proteobacteria, Cyanobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Cloroflexi) were consistently found in the A. brasiliensis metagenomes. The A. brasiliensis microbiome is enriched for Betaproteobacteria (e.g., Burkholderia) and Gammaproteobacteria (e.g., Pseudomonas and Alteromonas) compared with the surrounding planktonic microbial communities. Functional analysis based on Rapid Annotation using Subsystem Technology (RAST) indicated that the A. brasiliensis microbiome is enriched for sequences associated with membrane transport and one-carbon metabolism. In addition, there was an overrepresentation of sequences associated with aerobic and anaerobic metabolism as well as the synthesis and degradation of secondary metabolites. This study represents the first analysis of sponge-associated microbial communities via shotgun pyrosequencing, a strategy commonly applied in similar analyses in other marine invertebrate hosts, such as corals and algae. We demonstrate that A. brasiliensis has a unique microbiome that is distinct from that of the surrounding planktonic microbes and from other marine organisms, indicating a species-specific microbiome.

Cellulolytic bacteria from soils in harsh environments

It is believed that the exposure of organisms to harsh climate conditions may select for differential enzymatic activities, making the surviving organisms a very promising source for bioprospecting. Soil bacteria play an important role in degradation of organic matter, which is mostly due to their ability to decompose cellulose-based materials. This work focuses on the isolation and identification of cellulolytic bacteria from soil found in two environments with stressful climate conditions (Antarctica and the Brazilian semi-arid caatinga). Cellulolytic bacteria were selected using enrichments at high and low temperatures (4 or 60A degrees C) in liquid media (trypic soy broth-TSB and minimum salt medium-MM) supplemented with cellulose (1%). Many of the isolates (119 out of 254-46.9%) displayed the ability to degrade carboxymethyl-cellulose, indicating the presence of endoglucolytic activity, while only a minority of these isolates (23 out of 254-9.1%) showed exoglucolytic activity (degradation of avicel). The obtained isolates revealed a preferential endoglucolytic activity according to the temperature of enrichments. Also, the identification of some isolates by partial sequencing of the 16S rRNA gene indicated that the Bacteroidetes (e.g., Pedobacter, Chryseobacterium and Flavobacterium) were the main phylum of cellulolytic bacteria isolated from soil in Antarctica; the Firmicutes (e.g., Bacillus) were more commonly isolated from samples from the caatinga; and Actinobacteria were found in both types of soil (e.g., Microbacterium and Arthrobacter). In conclusion, this work reports the isolation of bacteria able to degrade cellulose-based material from soil at very low or very high temperatures, a finding that should be further explored in the search for cellulolytic enzymes to be used in the bioenergy industry.

The gastric caeca of pentatomids as a house for actinomycetes

Abstract Background Microbes are extensively associated with insects, playing key roles in insect defense, nutrition and reproduction. Most of the associations reported involve Proteobacteria. Despite the fact that Actinobacteria associated with insects were shown to produce antibiotic barriers against pathogens to the hosts or to their food and nutrients, there are few studies focusing on their association with insects. Thus, we surveyed the Actinobacteria diversity on a specific region of the midgut of seven species of stinkbugs (Hemiptera: Pentatomidae) known to carry a diversity of symbiotically-associated Proteobacteria. Results A total of 34 phylotypes were placed in 11 different Actinobacteria families. Dichelops melacanthus held the highest diversity with six actinobacteria families represented by nine phylotypes. Thyanta perditor (n = 7), Edessa meditabunda (n = 5), Loxa deducta (n = 4) and Pellaea stictica (n = 3) were all associated with three families. Piezodorus guildini (n = 3) and Nezara viridula (n = 3) had the lowest diversity, being associated with two (Propionibacteriaceae and Mycobacteriaceae) and one (Streptomyceataceae) families, respectively. Corynebacteriaceae and Mycobacteriaceae were the most common families with phylotypes from three different insect species each one. Conclusions Many phylotypes shared a low 16S rRNA gene similarity with their closest type strains and formed new phyletic lines on the periphery of several genera. This is a strong indicative that stinkbug caeca can harbor new species of actinobacteria, which might be derived from specific associations with the species of stinkbugs studied. Although the well-known role of actinobacteria as a source of biomolecules, the ecological features of these symbionts on the stinkbugs biology remain unknown.

Phylogenetic identification of marine bacteria isolated from deep-sea sediments of the eastern South Atlantic Ocean

The deep-sea environments of the South Atlantic Ocean are less studied in comparison to the North Atlantic and Pacific Oceans. With the aim of identifying the deep-sea bacteria in this less known ocean, 70 strains were isolated from eight sediment samples (depth range between 1905 to 5560 m) collected in the eastern part of the South Atlantic, from the equatorial region to the Cape Abyssal Plain, using three different culture media. The strains were classified into three phylogenetic groups, Gammaproteobacteria, Firmicutes and Actinobacteria, by the analysis of 16s rRNA gene sequences. Gammaproteobacteria and Firmicutes were the most frequently identified groups, with Halomonas the most frequent genus among the strains. Microorganisms belonging to Firmicutes were the only ones observed in all samples. Sixteen of the 41 identified operational taxonomic units probably represent new species. The presence of potentially new species reinforces the need for new studies in the deep-sea environments of the South Atlantic.

Sudden Increase and Regrowth of Fecal Coliforms and Escherichia Coli in Wastewater Biosolids After High Solids Centrifugation

Treatment plants that operate either thermophilic or mesophilic anaerobic digesters with centrifugal dewatering processes have consistently observed densities of fecal coliform and Escherichia coli, both indicator bacteria, that decrease during digestion but then increase after dewatering and storage. The increases have been characterized as two separate phenomena to explain this observation: 1) “Sudden Increase,” or SI, which is defined as the increase that occurs immediately after dewatering and 2) “regrowth,” which is defined as an increase during storage of cake samples over a period of hours or days. The SI observation appears to be more prevalent with biosolids that are generated with thermophilic processes and dewatered by centrifugation. Both thermophilic and mesophilic digesters with centrifuge dewatering processes have observed the regrowth phenomena. This research hypothesizes that the SI phenomenon is due to the presence of viable nonculturable (VNC) bacteria that are reactivated during dewatering. In other words, the bacteria were always present but were not enumerated by standard culturing methods (SCM). Analysis of the E. coli density in thermally treated solids by SCMs and quantitative real-time polymerase chain reaction (qPCR) indicated that E. coli densities are often underestimated by SCM. When analyzed with qPCR, the E. coli density after digestion can be 4-5 orders of magnitude greater than the non-detect levels identified by SCMs, which supports the non-culturable hypothesis. The VNC state describes a condition where bacteria are alive but unable to sustain the metabolic process needed for cellular division. Supplements added to culturing media were investigated to determine if the resuscitation of VNC bacteria could be enhanced. The autoinducer molecules Nhexanoyl- L-Homoserine lactone (C6-HSL), 3-oxo-N-octanoyl-L-Homoserine lactone (3-oxo- C8-HSL), and norepinephrine were unable to induce the resuscitation of VNC E. coli. Additional sampling was performed to determine if autoinducer molecules, peroxides, or other as of yet unknown inhibitory agents and toxins could be removed from biosolids during SCM. Culture media supplemented with the peroxide degrading compounds catalase, α-ketoglutaric acid, and sodium pyruvate was unable to resuscitate non-culturable E. coli. The additions of bentonite and exponential growth phase E. coli cell-free supernatant to culturing media were also unable to increase the culturability of E. coli. To remove inhibitory agents and toxins, a cell washing technique was employed prior to performing SCM; however, this cell washing technique may have increased cellular stresses that inhibited resuscitation since cell densities decreased. A novel laboratory-scale dewatering process was also investigated to determine if the SI and regrowth phenomena observed in full-scale centrifugal dewatering could be mimicked in the laboratory using a lab shearing device. Fecal coliform and E. coli densities in laboratory prepared cake samples were observed to be an order of magnitude higher than full-scale dewatered cakes. Additionally, the laboratory-scale dewatering process was able to resuscitate fecal coliforms and E. coli in stored sludge such that the density increased by 4-5 orders of magnitude from nondetect values. Lastly, the addition of aluminum sulfate during centrifuge dewatering at a full-scale utility produced an increased regrowth of fecal coliforms and E. coli that was sustained for 5 days.

Identification of the Causative Bacteria in Musculoskeletal Infections Using 16s rDNA - Denaturing Gradient Gel Electrophoresis Analysis

Musculoskeletal infections are infections of the bone and surrounding tissues. They are currently diagnosed based on culture analysis, which is the gold standard for pathogen identification. However, these clinical laboratory methods are frequently inadequate for the identification of the causative agents, because a large percentage (25-50%) of confirmed musculoskeletal infections are false negatives in which no pathogen is identified in culture. My data supports these results. The goal of this project was to use PCR amplification of a portion of the 16S rRNA gene to test an alternative approach for the identification of these pathogens and to assess the diversity of the bacteria involved. The advantages of this alternative method are that it should increase sample sensitivity and the speed of detection. In addition, bacteria that are non-culturable or in low abundance can be detected using this molecular technique. However, a complication of this approach is that the majority of musculoskeletal infections are polymicrobial, which prohibits direct identification from the infected tissue by DNA sequencing of the initial 16S rDNA amplification products. One way to solve this problem is to use denaturing gradient gel electrophoresis (DGGE) to separate the PCR products before DNA sequencing. Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on their melting point, which is determined by their DNA sequence. This analytical technique allows a mixture of PCR products of the same length that electrophoreses through agarose gels as one band, to be separated into different bands and then used for DNA sequence analysis. In this way, the DGGE allows for the identification of individual bacterial species in polymicrobial-infected tissue, which is critical for improving clinical outcomes. By combining the 16S rDNA amplification and the DGGE techniques together, an alternative approach for identification has been used. The 16S rRNA gene PCR-DGGE method includes several critical steps: DNA extraction from tissue biopsies, amplification of the bacterial DNA, PCR product separation by DGGE, amplification of the gel-extracted DNA, and DNA sequencing and analysis. Each step of the method was optimized to increase its sensitivity and for rapid detection of the bacteria present in human tissue samples. The limit of detection for the DNA extraction from tissue was at least 20 Staphylococcus aureus cells and the limit of detection for PCR was at least 0.05 pg of template DNA. The conditions for DGGE electrophoreses were optimized by using a double gradient of acrylamide (6 – 10%) and denaturant (30-70%), which increased the separation between distinct PCR products. The use of GelRed (Biotium) improved the DNA visualization in the DGGE gel. To recover the DNA from the DGGE gels the gel slices were excised, shredded in a bead beater, and the DNA was allowed to diffuse into sterile water overnight. The use of primers containing specific linkers allowed the entire amplified PCR product to be sequenced and then analyzed. The optimized 16S rRNA gene PCR-DGGE method was used to analyze 50 tissue biopsy samples chosen randomly from our collection. The results were compared to those of the Memorial Hermann Hospital Clinical Microbiology Laboratory for the same samples. The molecular method was congruent for 10 of the 17 (59%) culture negative tissue samples. In 7 of the 17 (41%) culture negative the molecular method identified a bacterium. The molecular method was congruent with the culture identification for 7 of the 33 (21%) positive cultured tissue samples. However, in 8 of the 33 (24%) the molecular method identified more organisms. In 13 of the 15 (87%) polymicrobial cultured tissue samples the molecular method identified at least one organism that was also identified by culture techniques. Overall, the DGGE analysis of 16S rDNA is an effective method to identify bacteria not identified by culture analysis.

Exposure to the Asp f 1 allergen in nonindustrial occupational environments

Sick Building Syndrome is a prevalent problem with patient complaints similar to typical allergy symptoms. Unlike most household allergens, the Asp f 1 allergen is conceivably ubiquitous in the work environment. This project examined levels of the Asp f 1 allergen in office and non-industrial occupational environments, and studied the bioaerosol and dust reservoirs of Aspergillus fumigatus responsible for those levels. ^ Culturable bioaerosols of total mesophilic fungi were sampled with Andersen N6 impactors. Aggressive airborne and bulk dust samples were concurrently collected and assayed for Asp f 1. Bulk dusts were selectively cultured for A. fumigatus. Samples were collected during both wet and dry climatological conditions to examine the possibility of Asp f 1 increases due to fungal growth blooms. ^ Only very low levels of Asp f 1 were detected in relatively few samples. Analysis of wet versus dry period samples showed no differences in Asp f 1 levels, although A. fumigatus counts from dusts did fluctuate significantly with exterior moisture events as did indoor prevalence of total colony forming units. These results indicate that even in the presence of elevated fungal concentrations, levels of Asp f 1 are extremely low. These levels do not correlate with climatological moisture events, despite distinct fungal blooms in the days immediately following those events. Non-industrial office buildings devoid of indoor air quality issues did not demonstrate significant levels or occurrence of Asp f 1 contamination in the geographical region of this study. ^

Comprehensive environmental study in high risk areas for cancer patients comparing air and water Aspergillus species samples

Background. Nosocomial invasive aspergillosis (a highly fatal disease) is an increasing problem for immunocompromised patients. Aspergillus spp. can be transmitted via air (most commonly) and by water. ^ The hypothesis for this prospective study was that there is an association between patient occupancy, housekeeping practices, patients, visitors, and Aspergillus spp. loading. Rooms were sampled as not terminally cleaned (dirty) and terminally cleaned (clean). The secondary hypothesis was that Aspergillus spp. positive samples collected from more than one sampling location within the same patient room represent the same isolate. ^ Methods. Between April and October 2004, 2873 environmental samples (713 air, 607 water, 1256 surface and 297 spore traps) were collected in and around 209 “clean” and “dirty” patient rooms in a large cancer center hospital. Water sources included aerosolized water from patient room showerheads, sinks, drains, and toilets. Bioaerosol samples were from the patient room and from the running shower, flushing toilet, and outside the building. The surface samples included sink and shower drains, showerheads, and air grills. Aspergillus spp. positive samples were also sent for PCR, molecular typing (n = 89). ^ Results. All water samples were negative for Aspergillus spp. There were a total of 130 positive culturable samples (5.1%). The predominant species found was Aspergillus niger. Of the positive culturable samples, 106 (14.9%) were air and 24 (3.8%) were surface. There were 147 spore trap samples, and 49.5% were positive for Aspergillus/Penicillum spp. Of the culturable positive samples sent for PCR, 16 were indistinguishable matches. There was no significant relationship between air and water samples and positive samples from the same room. ^ Conclusion. Primarily patients, visitors and staff bring the Aspergillus spp. into the hospital. The high number of A. niger samples suggests the spores are entering the hospital from outdoors. Eliminating the materials brought to the patient floors from the outside, requiring employees, staff, and visitors to wear cover up over their street clothes, and improved cleaning procedures could further reduce positive samples. Mold strains change frequently; it is probably more significant to understand pathogenicity of viable spores than to commit resources on molecular strain testing on environmental samples alone. ^

Microbiota and Microsatellite genotypes of Pacific oysters stemming from three oyster bed in the Northern Wadden Sea

Background: Studies of oyster microbiomes have revealed that a limited number of microbes, including pathogens, can dominate microbial communities in host tissues such as gills and gut. Much of the bacterial diversity however remains underexplored and unexplained, although environmental conditions and host genetics have been implicated. We used 454 next generation 16S rRNA amplicon sequencing of individually tagged PCR reactions to explore the diversity of bacterial communities in gill tissue of the invasive Pacific oyster Crassostrea gigas stemming from genetically differentiated beds under ambient outdoor conditions and after a multifaceted disturbance treatment imposing stress on the host. Results: While the gill associated microbial communities in oysters were dominated by few abundant taxa (i.e. Sphingomonas, Mycoplasma) the distribution of rare bacterial groups correlated to relatedness between the hosts under ambient conditions. Exposing the host to disturbance broke apart this relationship by removing rare phylotypes thereby reducing overall microbial diversity. Shifts in the microbiome composition in response to stress did not result in a net increase in genera known to contain potentially pathogenic strains. Conclusion: The decrease in microbial diversity and the disassociation between population genetic structure of the hosts and their associated microbiome suggest that disturbance (i.e. stress) may play a significant role for the assembly of the natural microbiome. Such community shifts may in turn also feed back on the course of disease and the occurrence of mass mortality events in oyster populations.

Table 1 Microorganisms, identified with DOGE analysis. 16S rDNA sequences, obtained from sequencing of DOGE bands, were assigned to the corresponding microorganisms

Microorganisms play an important role in the transformation of material within the earth's crust. The storage of CO2 could affect the composition of inorganic and organic components in the reservoir, consequently influencing microbial activities. To study the microbial induced processes together with geochemical, petrophysical and mineralogical changes, occurring during CO2 storage, long-term laboratory experiments under simulated reservoir P-T conditions were carried out. Clean inner core sections, obtained from the reservoir region at the CO2 storage site in Ketzin (Germany) from a depth of about 650 m, were incubated in high pressure vessels together with sterile synthetic formation brine under in situ P-T conditions of 5.5 MPa and 40°C. A 16S rDNA based fingerprinting method was used to identify the dominant species in DNA extracts of pristine sandstone samples. Members of the alpha- and beta-subdivisions of Proteobacteria and the Actinobacteria were identified. So far sequences belonging to facultative anaerobic, chemoheterotrophic bacteria (Burkholderia fungorum, Agrobacterium tumefaciens) gaining their energy from the oxidation of organic molecules and a genus also capable of chemolithoautotrophic growth (Hydrogenophaga) was identified. During CO2 incubation minor changes in the microbial community composition were observed. The majority of microbes were able to adapt to the changed conditions. During CO2 exposure increased concentrations of Ca**2+, K**+, Mg**2+ and SO4**2- were observed. Partially, concentration rises are (i) due to equilibration between rock pore water and synthetic brine, and (ii) between rock and brine, and are thus independent on CO2 exposure. However, observed concentrations of Ca**2+, K**+, Mg**2+ are even higher than in the original reservoir fluid and therefore indicate mineral dissolution due to CO2 exposure.

Prokaryotic growth and presence of metabolic products and contamination tracers in rocks samples from ODP Leg 187 sites

The microbial population in samples of basalt drilled from the north of the Australian Antarctic Discordance (AAD) during Ocean Drilling Program Leg 187 were studied using deoxyribonucleic acid (DNA)-based methods and culturing techniques. The results showed the presence of a microbial population characteristic for the basalt environment. DNA sequence analysis revealed that microbes grouping within the Actinobacteria, green nonsulfur bacteria, the Cytophaga/Flavobacterium/Bacteroides (CFB) group, the Bacillus/Clostridium group, and the beta and gamma subclasses of the Proteobacteria were present in the basalt samples collected. The most dominant phylogenetic group, both in terms of the number of sequences retrieved and the intensities of the DNA bands obtained with the denaturing gradient gel electrophoresis analysis, was the gamma Proteobacteria. Enrichment cultures showed phylogenetic affiliation with the Actinobacteria, the CFB group, the Bacillus/Clostridium group, and the alpha, beta, gamma, and epsilon subclasses of the Proteobacteria. Comparison of native and enriched samples showed that few of the microbes found in native basalt samples grew in the enrichment cultures. Only seven clusters, two clusters within each of the CFB and Bacillus/Clostridium groups and five clusters within the gamma Proteobacteria, contained sequences from both native and enriched basalt samples with significant similarity. Results from cultivation experiments showed the presence of the physiological groups of iron reducers and methane producers. The presence of the iron/manganese-reducing bacterium Shewanella was confirmed with DNA analysis. The results indicate that iron reducers and lithotrophic methanogenic Archaea are indigenous to the ocean crust basalt and that the methanogenic Archaea may be important primary producers in this basaltic environment.

Comparison of fecal indicators with pathogenic bacteria and rotavirus in rural Bangladesh groundwater

Groundwater is routinely analyzed for fecal indicators but direct comparisons of fecal indicators to the presence of bacterial and viral pathogens are rare. This study was conducted in rural Bangladesh where the human population density is high, sanitation is poor, and groundwater pumped from shallow tubewells is often contaminated with fecal bacteria. Five indicator microorganisms (E. coli, total coliform, F+RNA coliphage, Bacteroides and human-associated Bacteroides (HuBacteroides)) and various environmental parameters were compared to the direct detection of waterborne pathogens by quantitative PCR in groundwater pumped from 50 tubewells. Rotavirus was detected in groundwater filtrate from the largest proportion of tubewells (40%), followed by Shigella (10%), Vibrio (10%), and pathogenic E. coli (8%). Spearman rank correlations and sensitivity-specificity calculations indicate that some, but not all, combinations of indicators and environmental parameters can predict the presence of pathogens. Culture-dependent fecal indicator bacteria measured on a single date did not predict bacterial pathogens, but annually averaged monthly measurements of culturable E. coli did improve prediction for total bacterial pathogens. F+RNA coliphage were neither correlated nor sufficiently sensitive towards rotavirus, but were predictive of bacterial pathogens. A qPCR-based E. coli assay was the best indicator for the bacterial pathogens, rotavirus and all pathogens combined. Since groundwater cannot be excluded as a significant source of diarrheal disease in Bangladesh and neighboring countries with similar characteristics, the need to develop more effective methods for screening tubewells with respect to microbial contamination is necessary.