scholarly journals Quantification of Endospore-Forming Firmicutes by Quantitative PCR with the Functional Genespo0A

2013 ◽  
Vol 79 (17) ◽  
pp. 5302-5312 ◽  
Author(s):  
Matthieu Bueche ◽  
Tina Wunderlin ◽  
Ludovic Roussel-Delif ◽  
Thomas Junier ◽  
Loic Sauvain ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
High Sensitivity ◽  
Primer Design ◽  
Rrna Gene ◽  
Natural Environments ◽  
Natural Habitats ◽  
Content Type ◽  
Wide Range ◽  
Primer Sets ◽  
Reliable Quantification

ABSTRACTBacterial endospores are highly specialized cellular forms that allow endospore-formingFirmicutes(EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular, those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments, such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high-sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR (qPCR). For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered, but onlyspo0Awas retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures, including representatives of the generaBacillus,Paenibacillus,Brevibacillus,Geobacillus,Alicyclobacillus,Sulfobacillus,Clostridium, andDesulfotomaculum, as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception ofSulfobacillusandDesulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 104cells (or spores) per gram of initial material was calculated, indicating this method has a promising potential for the detection of EFF over a wide range of applications.

Usitatibacter rugosus gen. nov., sp. nov. and Usitatibacter palustris sp. nov., novel members of Usitatibacteraceae fam. nov. within the order Nitrosomonadales isolated from soil

2021 ◽  
Author(s):  
Selma Vieira ◽  
Katharina J. Huber ◽  
Meina Neumann-Schaal ◽  
Alicia Geppert ◽  
Manja Luckner ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Strain ◽  
Type Species ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
The Novel ◽  
Content Type ◽  
Link Type ◽  
Wide Range

Members of the metabolically diverse order Nitrosomonadales inhabit a wide range of environments. Two strains affiliated with this order were isolated from soils in Germany and characterized by a polyphasic approach. Cells of strains 0125_3T and Swamp67T are Gram-negative rods, non-motile, non-spore-forming, non-capsulated and divide by binary fission. They tested catalase-negative, but positive for cytochrome c-oxidase. Both strains form small white colonies on agar plates and grow aerobically and chemoorganotrophically on SSE/HD 1 : 10 medium, preferably utilizing organic acids and proteinaceous substrates. Strains 0125_3T and Swamp67T are mesophilic and grow optimally without NaCl addition at slightly alkaline conditions. Major fatty acids are C16 : 1  ω7c, C16 : 0 and C14 : 0. The major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine and phosphatidyglycerol. The predominant respiratory quinone is Q-8. The G+C content for 0125_3T and Swamp67T was 67 and 66.1 %, respectively. The 16S rRNA gene analysis indicated that the closest relatives (<91 % sequence similarity) of strain 0125_3T were Nitrosospira multiformis ATCC 25196T, Methyloversatilis universalis FAM5T and Denitratisoma oestradiolicum AcBE2-1T, while Nitrosospira multiformis ATCC 25196T, Nitrosospira tenuis Nv1T and Nitrosospira lacus APG3T were closest to strain Swamp67T. The two novel strains shared 97.4 % 16S rRNA gene sequence similarity with one another and show low average nucleotide identity of their genomes (83.8 %). Based on the phenotypic, chemotaxonomic, genomic and phylogenetic analysis, we propose the two novel species Usitatibacter rugosus sp. nov (type strain 0125_3T=DSM 104443T=LMG 29998T=CECT 9241T) and Usitatibacter palustris sp. nov. (type strain Swamp67T=DSM 104440T=LMG 29997T=CECT 9242T) of the novel genus Usitatibacter gen. nov., within the novel family Usitatibacteraceae fam. nov.

Interactions between human behaviour and the built environment in terms of facility management

Facilities ◽  
2018 ◽  
Vol 36 (1/2) ◽  
pp. 2-12 ◽  
Author(s):  
Darja Kobal Grum
Keyword(s):  
Built Environment ◽  
Low Income ◽  
Well Being ◽  
Human Behaviour ◽  
Natural Environments ◽  
Psychological Well Being ◽  
Content Type ◽  
Sustainable Solutions ◽  
Wide Range ◽  
The Impact

Purpose In comparison with the relations between the human and natural environments that have been the central focus of environmental psychology for many years, the interactions between the psychological processes underlying human behaviour and the built environment have only recently regained the interest of researchers. In this paper, the author first discusses the reasons for the slower development of human – built environment relations. Afterwards, the author systematically examines the impact that the research of environmental stress, namely, poor housing and poor neighbourhood quality, had on the contemporary understanding of human – built environment relations. Design/methodology/approach The author focuses on social, biophilic and evidence-based design. The author proposes deeper psychological engagement in correlation with human behaviour, psychological well-being and society. The author highlights the inclusion of psychologists in interdisciplinary research teams addressing the development of sustainable solutions to the issues of residential environments. Findings It has been shown that substandard house quality, high noise, lack of natural light in houses, poorer physical quality of urban neighbourhoods, living in a low-income neighbourhood, etc. are linked to elevated physiological and psychological stress. Despite this evidence, there is still a gap between building designers and building users in modern industrialised societies, which could deepen tenants’ dissatisfaction due to specific behavioural needs and consequently lower their psychological well-being and health risk behaviour. Research limitations/implications These are potential risks of error arising from the use of assumptions, limited samples size and data from the secondary resources. Originality/value The major contributions of this paper are as follows. If the environment is understood as a dynamic, constantly changing and complex system of a wide range of players, the author can discern in this environment a dynamic that is otherwise characteristic of emotional dynamics. Expressed participants’ high satisfaction with residential status does not necessarily generate high expectations regarding real estate factors.

scholarly journals Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa

2014 ◽  
Vol 80 (17) ◽  
pp. 5515-5521 ◽  
Author(s):  
Suzanne L. Ishaq ◽  
André-Denis G. Wright
Keyword(s):  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Hypervariable Regions ◽  
Blast Database ◽  
Primer Sets ◽  
18S Rrna Genes ◽  
Ciliate Protozoa

ABSTRACTFour new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplifiedEntodinium simplexandOstracodiniumspp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the generaBandia,Blepharocorys,Polycosta, andTetratoxumand betweenHemiprorodon gymnoprosthiumandProrodonopsiscoli, none of which are normally found in the rumen.

scholarly journals Establishment of an Analytical System for the Human Fecal Microbiota, Based on Reverse Transcription-Quantitative PCR Targeting of Multicopy rRNA Molecules

2009 ◽  
Vol 75 (7) ◽  
pp. 1961-1969 ◽  
Author(s):  
Kazunori Matsuda ◽  
Hirokazu Tsuji ◽  
Takashi Asahara ◽  
Kazumasa Matsumoto ◽  
Toshihiko Takada ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Intestinal Microbiota ◽  
Reverse Transcription ◽  
Rrna Gene ◽  
Specific Primer ◽  
Human Intestinal Microbiota ◽  
Reverse Transcription Quantitative Pcr ◽  
Primer Sets ◽  
Analytical System ◽  
Pcr Targeting

ABSTRACT An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) was established for the precise evaluation of human intestinal microbiota. Group- and species-specific primer sets for Clostridium perfringens, Lactobacillus spp. (six subgroups and three species), Enterococcus spp., and Staphylococcus spp. targeting 16S rRNA gene sequences were newly developed for the quantitative analysis of such subdominant populations in human intestines. They were used together with previously reported group-specific primer sets for Enterobacteriaceae, Pseudomonas spp., and six predominant bacterial groups (the Clostridium coccoides group, the Clostridium leptum subgroup, the Bacteroides fragilis group, Bifidobacterium spp., the Atopobium cluster, and Prevotella spp.) for the examination of fecal samples from 40 healthy adults by RT-qPCR with lower detection limits of 102 to 104 cells per g of feces. The RT-qPCR method gave data equivalent to those yielded by qPCR for predominant populations of more than 108 cells per g of feces and could quantify bacterial populations that were not detectable (Staphylococcus and Pseudomonas) or those only detected at lower incidences (Prevotella, C. perfringens, Lactobacillus, and Enterococcus) by qPCR or the culture method. The RT-qPCR analysis of Lactobacillus spp. at the subgroup level revealed that a subject has a mean of 4.6 subgroups, with an average count of log10(6.3 ± 1.5) cells per g of feces. These results suggest that RT-qPCR is effective for the accurate enumeration of human intestinal microbiota, especially the entire analysis of both predominant and subdominant populations.

scholarly journals “Candidatus Finniella” (Rickettsiales, Alphaproteobacteria), Novel Endosymbionts of Viridiraptorid Amoeboflagellates (Cercozoa, Rhizaria)

2015 ◽  
Vol 82 (2) ◽  
pp. 659-670 ◽  
Author(s):  
Sebastian Hess ◽  
Andreas Suthaus ◽  
Michael Melkonian
Keyword(s):  
Rrna Gene ◽  
The Novel ◽  
Single Nucleotide ◽  
Content Type ◽  
Obligate Intracellular ◽  
Wide Range ◽  
Candidate Species ◽  
Novel Bacteria ◽  
Obligate Intracellular Bacteria

ABSTRACTTheRickettsiales(Alphaproteobacteria) are obligate intracellular bacteria that colonize a wide range of eukaryotic hosts, including diverse metazoa and protists. Here, we characterize rickettsial endosymbionts discovered in the cytoplasm of the algivorous amoeboflagellatesViridiraptor invadensandOrciraptor agilis(Viridiraptoridae, Cercozoa, Rhizaria), supplying evidence of free-living, phagotrophic members of the Cercozoa serving as hosts forRickettsiales. According to 16S rRNA gene phylogenies, the bacteria represent two closely related but distinct genotypes within a deep-branching rickettsial clade, which contains the genera “CandidatusOdyssella,” “CandidatusParacaedibacter,” and “CandidatusCaptivus.” Using the full-cycle rRNA approach, we detected the novel bacteria in four of nine viridiraptorid strains tested. Furthermore, two specific oligonucleotide probes with a single-nucleotide-difference discriminated both bacterial genotypes by fluorescencein situhybridization (FISH). We establish the candidate species “CandidatusFinniella inopinata” (found inViridiraptor invadens) and “CandidatusFinniella lucida” (found inOrciraptor agilis) for the novel bacteria and propose a new, provisional family ofRickettsiales, “CandidatusParacaedibacteraceae.”

scholarly journals DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

2015 ◽  
Vol 8 ◽  
pp. MBI.S29736 ◽  
Author(s):  
Kenjiro Nagamine ◽  
Guo-Chiuan Hung ◽  
Bingjie Li ◽  
Shyh-Ching Lo
Keyword(s):  
Safety Concern ◽  
Rapid Development ◽  
High Sensitivity ◽  
High Specificity ◽  
Clostridium Tetani ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Pcr Assays ◽  
Primer Sets ◽  
Species Specific

Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

scholarly journals Community Structure Analysis of Methanogens Associated with Rumen Protozoa Reveals Bias in Universal Archaeal Primers

2012 ◽  
Vol 78 (11) ◽  
pp. 4051-4056 ◽  
Author(s):  
Lisa D. Tymensen ◽  
Tim A. McAllister
Keyword(s):  
Community Structure ◽  
Species Composition ◽  
Structure Analysis ◽  
Small Subunit ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Content Type ◽  
Ssu Rrna Gene ◽  
Rumen Protozoa ◽  
Primer Sets

ABSTRACTThe diversity of protozoan-associated methanogens in cattle was investigated using five universal archaeal small-subunit (SSU) rRNA gene primer sets.Methanobrevibacterspp. and rumen cluster C (distantly related toThermoplasmaspp.) were predominant. Significant differences in species composition among libraries indicate that some primers used previously to characterize rumen methanogens exhibit biased amplification.

scholarly journals Host Selection of Symbiotic Cyanobacteria in 31 Species of the Australian Cycad Genus: Macrozamia (Zamiaceae)

2010 ◽  
Vol 23 (6) ◽  
pp. 811-822 ◽  
Author(s):  
Michelle M. Gehringer ◽  
Jasper J. L. Pengelly ◽  
William S. Cuddy ◽  
Claus Fieker ◽  
Paul I. Forster ◽  
...  
Keyword(s):  
Microscopic Analysis ◽  
Fungal Species ◽  
Host Specialization ◽  
Rrna Gene ◽  
Natural Habitats ◽  
Diverse Range ◽  
Cyanobacterial Species ◽  
Wide Range ◽  
In The Wild ◽  
Selection Of

The nitrogen-fixing cyanobacterium Nostoc is a commonly occurring terrestrial and aquatic cyanobacterium often found in symbiosis with a wide range of plant, algal, and fungal species. We investigated the diversity of cyanobacterial species occurring within the coralloid roots of different Macrozamia cycad species at diverse locations throughout Australia. In all, 74 coralloid root samples were processed and 56 endosymbiotic cyanobacteria were cultured. DNA was isolated from unialgal cultures and a segment of the 16S rRNA gene was amplified and sequenced. Microscopic analysis was performed on representative isolates. Twenty-two cyanobacterial species were identified, comprising mostly Nostoc spp. and a Calothrix sp. No correlation was observed between a cycad species and its resident cyanobiont species. The predominant cyanobacterium isolated from 18 root samples occurred over a diverse range of environmental conditions and within 14 different Macrozamia spp. Phylogenetic analysis indicated that endosymbionts were not restricted to previously described terrestrial species. An isolate clustering with Nostoc PCC7120, an aquatic strain, was identified. This is the first comprehensive study to identify the endosymbionts within a cycad genus using samples obtained from their natural habitats. These results indicate that there is negligible host specialization of cyanobacterial endosymbionts within the cycad genus Macrozamia in the wild.

scholarly journals Fusibacter bizertensis sp. nov., isolated from a corroded kerosene storage tank

2015 ◽  
Vol 65 (Pt_1) ◽  
pp. 117-121 ◽  
Author(s):  
Latifa Smii ◽  
Wajdi Ben Hania ◽  
Jean-Luc Cayol ◽  
Manon Joseph ◽  
Moktar Hamdi ◽  
...  
Keyword(s):  
Storage Tank ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
Positive Staining ◽  
Northern Tunisia ◽  
Content Type ◽  
Link Type ◽  
Lateral Flagellum ◽  
Wide Range

Strain LTF Kr01T, a novel mesophilic, anaerobic, halotolerant, rod-shaped bacterium, was isolated from a drain at the bottom of a corroded kerosene storage tank of the Société Tunisienne des Industries de Raffinage (STIR), Bizerte, northern Tunisia. Cells were Gram-positive-staining rods, occurred singly or in pairs, and were motile by one lateral flagellum. Strain LTF Kr01T grew at temperatures between 15 and 40 °C (optimum 30 °C), between pH 5.5 and 8.2 (optimum pH 7.2) and at NaCl concentrations between 0 and 50 g l−1 (optimum 5 g l−1). It reduced thiosulfate and elemental sulfur into sulfide, but did not reduce sulfate or sulfite. It utilized a wide range of carbohydrates (cellobiose, d-glucose, d-fructose, d-mannitol, d-ribose, sucrose, d-xylose, maltose, d-galactose, starch and trehalose) and produced acetate, CO2 and H2 as end products from glucose fermentation. The DNA G+C content was 37.4 mol%. The predominant cellular fatty acids were C14 : 0 and C16 : 0. Phylogenetic analysis of the 16S rRNA gene sequence suggested that Fusibacter tunisiensis was the closest relative of strain LTF Kr01T (gene sequence similarity of 94.6 %). Based on phenotypic, phylogenetic and genotypic taxonomic characteristics, strain LTF Kr01T is proposed to represent a novel species of the genus Fusibacter , order Clostridiales , for which the name Fusibacter bizertensis sp. nov. is proposed. The type strain is LTF Kr01T ( = DSM 28034T = JCM 19376T).

scholarly journals Distribution of Human-Specific Bacteroidales and Fecal Indicator Bacteria in an Urban Watershed Impacted by Sewage Pollution, Determined Using RNA- and DNA-Based Quantitative PCR Assays

2014 ◽  
Vol 81 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Vikram Kapoor ◽  
Tarja Pitkänen ◽  
Hodon Ryu ◽  
Michael Elk ◽  
David Wendell ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Fecal Pollution ◽  
Detection Sensitivity ◽  
Rrna Gene ◽  
Fecal Bacteria ◽  
Combined Sewer Overflows ◽  
Fecal Indicator ◽  
Content Type ◽  
Detection Frequency ◽  
Human Specific

ABSTRACTThe identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci,Enterococcus faecalis, andEnterococcus faeciummarkers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specificBacteroidalesmarkers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specificBacteroidalesmarkers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources.

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