Analysis of nitrifying bacterial communities in aerobic biofilm reactors with different DO conditions using molecular techniques

2008 ◽  
Vol 57 (12) ◽  
pp. 1889-1899 ◽  
Author(s):  
J. J. Park ◽  
I. G. Byun ◽  
J. C. Yu ◽  
S. R. Park ◽  
D. J. Ju ◽  
...  
Keyword(s):  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
In Situ Hybridisation ◽  
Amoa Gene ◽  
Biofilm Reactor ◽  
Molecular Techniques ◽  
Fluorescence In Situ Hybridisation ◽  
Do Concentration ◽  
Pcr Targeting ◽  
The Relationship

In order to assess the relationship between the dissolved oxygen (DO) concentration and the characteristics of nitrifying bacterial communities in an aerobic biofilm reactor, molecular techniques including denaturing gradient gel electrophoresis (DGGE)/cloning based on PCR targeting 16S rRNA and the amoA gene and fluorescence in situ hybridisation (FISH) were conducted. The D-1, D-2, D-3 and D-4 reactors with different DO concentrations (1, 3, 5 and 7 mg/L, respectively) were set up in the thermostat and acclimated. The optimal DO concentration with stable nitrification efficiency was above 5.0 mg/L. As was shown by the results of DGGE and cloning, the community of ammonia-oxidising bacteria (AOB) and the ratio of Nitrosomonas sp. changed only slightly despite their differing nitrification efficiencies. The results of FISH indicated that higher DO concentrations resulted in an increase in AOB and nitrite-oxidising bacteria (NOB), and a reduction in heterotrophic microorganisms. The INT-dehydrogenase activity (DHA) test demonstrated that the activity of AOB decreased with reductions in the DO concentration. This means that the DO concentration does not influence the community of AOB, but rather the activity of AOB. In the relationship between the attached biomass and the nitrification efficiency, only the active biomass affected the nitrification efficiencies.

scholarly journals HER-2 Evaluation in a Specific Gastric Cancer Population with the Highest Rate of Mortality in Spain

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
E. U. Cidon ◽  
R. G. Centeno ◽  
E. G. Lagarto ◽  
J. I. Peral
Keyword(s):  
Gastric Cancer ◽  
In Situ Hybridisation ◽  
Daily Practice ◽  
Radical Resection ◽  
Biological Factor ◽  
Fluorescence In Situ Hybridisation ◽  
Advanced Stages ◽  
Her 2 ◽  
The Relationship

Gastric cancer (GC) still represents the second cause of cancer-related death worldwide. Radical resection is the mainstay of early stages treatment with little impact on overall survival (OS) in the advanced ones. HER-2 is the most relevant biological factor involved.Purpose. This study aims to show the relationship between HER-2 positivity and survival in patients with completely resected GC.Methods. Retrospective study of GC patients diagnosed in 2003–2005 at our institution. Surgical specimens underwent immunohistochemistry (IHC), and in cases +/++/+++ samples underwent also fluorescence in situ hybridisation (FISH) analyses of HER-2 and graduated according to experts' consensus.Results. 120 cases included. Overall expression detected in 7.5%. Correlation between HER-2 positive and female sex, advanced stages or histological grades, or intestinal type was detected. Early recurrences higher in HER-2 positive (66.6% versus 35.4%, ). The median DFS for c-erbB-2 positive was 15 months (range 2–67 months), and OS was 25 months (range 10–67 months). In the case of patients with c-erbB-2, negative median DFS was 27 months (range 5–67 months) and OS for this sample is 47 months (range 29–67 months).Conclusions. These results emphasize the relevance of HER-2 positivity in GC as independent prognostic factor and support its current analyses in daily practice.

scholarly journals Hyperspectral microscope imaging methods for multiplex detection of Campylobacter

2019 ◽  
Author(s):  
Bosoon Park ◽  
Matthew Eady ◽  
Brian Oakley ◽  
Seung-Chul Yoon ◽  
Kurt Lawrence ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Pathogen Detection ◽  
Shigella Flexneri ◽  
In Situ Hybridisation ◽  
Foodborne Pathogen ◽  
Latex Agglutination ◽  
Molecular Techniques ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fluorescence In Situ Hybridisation ◽  
Microscope Imaging

Campylobacter is an emerging zoonotic bacterial threat in the poultry industry. The current methods for the isolation and detection of Campylobacter are culture-based techniques with several selective agars designed to isolate Campylobacter colonies, which is time-consuming, labour intensive and has low sensitivity. Several immunological and molecular techniques such as enzyme-linked immunosorbent assay (ELISA) and Latex agglutination are commercially available for the detection and identification of Campylobacter. However, these methods demand more advanced instruments as well as specially trained experts. A hyperspectral microscope imaging (HMI) technique with the fluorescence in situ hybridisation (FISH) technique has the potential for multiplex foodborne pathogen detection. Using Alexa488 and Cy3 fluorophores, the HMI (450–800 nm) technique was able to identify Campylobacter jejuni stains with high sensitivity and specificity. In addition, HMI was able to classify six bacteria using scattering intensity from their spectra without a FISH fluorophore. Overall classification accuracy of quadratic discriminant analysis (QDA) method for six bacteria including Bifidobacter longum, Campylobacter jejuni, Clostridium perfringens, Enterobacter cloacae, Lactobacillus salivarius and Shigella flexneri using the HMI technique without fluorescent markers was approximately 88.6 % with pixel-wise classification.

scholarly journals Elucidation of the microbial community structure within a laboratory scale activated sludge process using molecular techniques

2006 ◽  
Author(s):  
◽  
Pamela Padayachee
Keyword(s):  
Microbial Community ◽  
Activated Sludge ◽  
Denaturing Gradient Gel Electrophoresis ◽  
In Situ Hybridisation ◽  
Molecular Techniques ◽  
Laboratory Scale ◽  
Oligonucleotide Probes ◽  
Mass Balances ◽  
Nitrogen Levels ◽  
Gradient Gel Electrophoresis

The microbial community present in a laboratory-scale modified Ludzack-Ettinger activated sludge system was investigated using a combination of novel molecular techniques. The parent system was investigated for a duration of one year and samples were taken at regular intervals to determine the profile and structure of the microbial community present within the anoxic and aerobic zones of the MLE system. The combination of molecular techniques included fluorescent in situ hybridisation (FISH) and denaturing gradient gel electrophoresis (DGGE). FISH was performed using oligonucleotide probes, which were complementary to conserved regions of the rRNA for the alpha, beta and gamma subclasses of the gram negative family Proteobacteria as well as a group-specific HGC oligonucleotide probe as a representative of the gram positive actinomycetes branch. The total eubacteria present was determined using the EUB oligonucleotide probes, EUB388, EUB388-II and EUB388-III. The DGGE analysis of PCR-amplified 16S rDNA gene segments was used to examine the microbial community profile in the anoxic and aerobic zones. The profile for each of the zones revealed a number of consistent bands throughout the duration of the laboratory-scale process. However, the profiles obtained suggested that a diverse microbial community existed within the aerobic and anoxic zones. The bands also indicated the presence of dominant and less dominant species of bacteria. Hybridisations obtained from the FISH analyses indicated that the alpha and gamma subclasses were predominant within the anoxic zone and the aerobic zone showed a dominance of the beta subclass of Proteobacteria. The steady state behaviour of the MLE system was confirmed with the results obtained from COD, TKN, nitrates and OUR analytical tests. COD and nitrogen mass balances were conducted to confirm the acceptance of the results obtained for each batch as an indication of the system performance for the MLE model. Nitrogen mass balances indicated an upset in the nitrogen levels for batches two and seven.

Impact of oxygen on the coexistence of nitrification, denitrification, and sulfate reduction in oxygen-based membrane aerated biofilm

2015 ◽  
Vol 61 (3) ◽  
pp. 237-242 ◽  
Author(s):  
Hong Liu ◽  
Shuying Tan ◽  
Zhiya Sheng ◽  
Tong Yu ◽  
Yang Liu
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Aerobic Oxidation ◽  
Bulk Water ◽  
Biofilm Reactor ◽  
Molecular Techniques ◽  
Bulk Liquid ◽  
Rrna Gene ◽  
Ammonia Oxidizing Bacteria ◽  
Microbial Composition ◽  
Gradient Gel Electrophoresis

Membrane aerated biofilms (MABs) are subject to “counter diffusion” of oxygen and substrates. In a membrane aerated biofilm reactor, gases (e.g., oxygen) diffuse through the membrane into the MAB, and liquid substrates pass from the bulk liquid into the MAB. This behavior can result in a unique biofilm structure in terms of microbial composition, distribution, and community activity in the MAB. Previous studies have shown simultaneous aerobic oxidation, nitrification, and denitrification within a single MAB. Using molecular techniques, we investigated the growth of sulfate-reducing bacteria (SRB) in the oxygen-based MAB attached to a flat sheet membrane. Denaturing gradient gel electrophoresis of the amplified 16S rRNA gene fragments and functional gene fragments specific for ammonia-oxidizing bacteria (amoA), denitrifying bacteria (nirK), and SRB (dsrB) demonstrated the coexistence of nitrifiers, denitrifiers, and SRB communities within a single MAB. The functional diversities of SRB and denitrifiers decreased with an increase in the oxygen concentration in the bulk water of the reactor.

scholarly journals Changes in Bacterial Communities of the Marine Sponge Mycale laxissima on Transfer into Aquaculture†

2007 ◽  
Vol 74 (4) ◽  
pp. 1209-1222 ◽  
Author(s):  
Naglaa M. Mohamed ◽  
Julie J. Enticknap ◽  
Jayme E. Lohr ◽  
Scott M. McIntosh ◽  
Russell T. Hill
Keyword(s):  
16S Rrna ◽  
Marine Sponge ◽  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Surrounding Water ◽  
Clone Libraries ◽  
Recirculating Aquaculture Systems ◽  
Mycale Laxissima

ABSTRACT The changes in bacterial communities associated with the marine sponge Mycale laxissima on transfer to aquaculture were studied using culture-based and molecular techniques. M. laxissima was maintained alive in flowthrough and closed recirculating aquaculture systems for 2 years and 1 year, respectively. The bacterial communities associated with wild and aquacultured sponges, as well as the surrounding water, were assessed using 16S rRNA gene clone library analysis and denaturing gradient gel electrophoresis (DGGE). Bacterial richness and diversity were measured using DOTUR computer software, and clone libraries were compared using S-LIBSHUFF. DGGE analysis revealed that the diversity of the bacterial community of M. laxissima increased when sponges were maintained in aquaculture and that bacterial communities associated with wild and aquacultured M. laxissima were markedly different than those of the corresponding surrounding water. Clone libraries of bacterial 16S rRNA from sponges confirmed that the bacterial communities changed during aquaculture. These communities were significantly different than those of seawater and aquarium water. The diversity of bacterial communities associated with M. laxissima increased significantly in aquaculture. Our work shows that it is important to monitor changes in bacterial communities when examining the feasibility of growing sponges in aquaculture systems because these communities may change. This could have implications for the health of sponges or for the production of bioactive compounds by sponges in cases where these compounds are produced by symbiotic bacteria rather than by the sponges themselves.

Survival of Campylobacter jejuni in potable water biofilms: a comparative study with different detection methods

2006 ◽  
Vol 54 (3) ◽  
pp. 57-61 ◽  
Author(s):  
M.J. Lehtola ◽  
T. Pitkänen ◽  
L. Miebach ◽  
I.T. Miettinen
Keyword(s):  
Drinking Water ◽  
Foodborne Pathogens ◽  
Potable Water ◽  
In Situ Hybridisation ◽  
Biofilm Reactor ◽  
Detection Methods ◽  
Fluorescence In Situ Hybridisation ◽  
Culture Methods ◽  
Number Of Bacteria

Campylobacteria are important foodborne pathogens. C. jejuni bacteria have caused several drinking water-related epidemics in Finland. Normally, C. jejuni is not able to multiply in drinking water or in biofilms although it may survive in biofilms. The survival of C. jejuni in biofilms was studied using the Propella™ biofilm reactor. The number of bacteria was analysed with traditional culture methods and with fluorescence in situ hybridisation (FISH). By culture methods C. jejuni was detectable for only 1 d after spiking whereas bacteria were found from biofilms for at least 1 week after spiking and from outlet water of the reactor for 3 weeks when using FISH. These results suggested that C. jejuni may survive in biofilms and culture methods probably seriously underestimate the real number in water and in biofilms.

scholarly journals Molecular detection of cattle Sarcocystis spp. in North-West Italy highlights their association with bovine eosinophilic myositis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Selene Rubiola ◽  
Tiziana Civera ◽  
Felice Panebianco ◽  
Davide Vercellino ◽  
Francesco Chiesa
Keyword(s):  
18S Rdna ◽  
Inflammatory Myopathy ◽  
Molecular Techniques ◽  
Diaphragm Muscle ◽  
Economic Losses ◽  
Intermediate Hosts ◽  
Slaughter Cattle ◽  
Significant Difference ◽  
Pcr Targeting

Abstract Background Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa because of the evidence supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based on either morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis. Methods To reach our aim, individual striated muscle samples from BEM condemned carcasses (N = 54) and diaphragm muscle samples from randomly sampled carcasses (N = 59) were obtained from Northwest Italy slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S rDNA and cox1 genes. PCR products amplified using the genus-specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification. Results Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species in slaughter cattle (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses (42.6% and 1.8%, respectively) and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in two carcasses. Conclusions Our study contributes to updating the data on the prevalence of the different Sarcocystis spp. in cattle in Italy, highlighting the presence of three Sarcocystis spp., S. cruzi, S. hominis and S. bovifelis, in BEM lesions and allowing us to speculate on the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved in bovine eosinophilic myositis. Graphic Abstract

scholarly journals Rapid Multi-Hybridisation FISH Screening for Balanced Porcine Reciprocal Translocations Suggests a Much Higher Abnormality Rate Than Previously Appreciated

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 250
Author(s):  
Rebecca E O’Connor ◽  
Lucas G Kiazim ◽  
Claudia C Rathje ◽  
Rebecca L Jennings ◽  
Darren K Griffin
Keyword(s):  
Semen Analysis ◽  
In Situ Hybridisation ◽  
Economic Loss ◽  
Environmental Damage ◽  
Fluorescence In Situ Hybridisation ◽  
Reciprocal Translocations ◽  
Chromosome Translocations ◽  
Farrowing Rate ◽  
Significant Economic Loss

With demand rising, pigs are the world’s leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.

scholarly journals Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Caitlin M. Singleton ◽  
Francesca Petriglieri ◽  
Jannie M. Kristensen ◽  
Rasmus H. Kirkegaard ◽  
Thomas Y. Michaelsen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Wastewater Treatment Plants ◽  
In Situ Hybridisation ◽  
Amplicon Sequencing ◽  
Rrna Genes ◽  
Fluorescence In Situ Hybridisation ◽  
Sequencing Data ◽  
High Quality ◽  
16S Rrna Amplicon Sequencing ◽  
Long Read

AbstractMicroorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

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