scholarly journals Single-Cell Analyses Revealed Transfer Ranges of IncP-1, IncP-7, and IncP-9 Plasmids in a Soil Bacterial Community

2013 ◽  
Vol 80 (1) ◽  
pp. 138-145 ◽  
Author(s):  
Masaki Shintani ◽  
Kazuhiro Matsui ◽  
Jun-ichi Inoue ◽  
Akira Hosoyama ◽  
Shoko Ohji ◽  
...  
Keyword(s):  
Single Cell ◽  
Soil Bacteria ◽  
Fluorescent Protein ◽  
Independent Method ◽  
Rrna Gene ◽  
Reporter Protein ◽  
Content Type ◽  
Culture Independent ◽  
Soil Bacterial ◽  
Culture Dependent

ABSTRACTThe conjugative transfer ranges of three different plasmids of the incompatibility groups IncP-1 (pBP136), IncP-7 (pCAR1), and IncP-9 (NAH7) were investigated in soil bacterial communities by culture-dependent and culture-independent methods.Pseudomonas putida, a donor of each plasmid, was mated with soil bacteria, and green fluorescent protein (GFP), encoded on the plasmid, was used as a reporter protein for successful transfer. GFP-expressing transconjugants were detected and separated at the single-cell level by flow cytometry. Each cell was then analyzed by PCR and sequencing of its 16S rRNA gene following either whole-genome amplification or cultivation. A large number of bacteria within the phylumProteobacteriawas identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phylaActinobacteria,Bacteroidetes, andFirmicuteswere detected only by the culture-independent method. Members of the genusPseudomonas(classGammaproteobacteria) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereasDelftiaspecies (classBetaproteobacteria) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCAR1-containingDelftiastrains could not be cultivated after a one-to-one filter mating assay between the donor and cultivableDelftiastrains as recipients, fluorescencein situhybridization detected pCAR1-containingDelftiacells, suggesting thatDelftiawas a “transient” host of pCAR1.

Comparison of culture-dependent and culture-independent techniques in the detection of lactic acid bacteria biodiversity and dynamics throughout the ripening process: The case of Turkish artisanal Tulum cheese produced in the Anamur region

2021 ◽  
pp. 1-7
Author(s):  
Talha Demirci ◽  
Aysun Oraç ◽  
Kübra Aktaş ◽  
Enes Dertli ◽  
Ismail Akyol ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Dna Isolation ◽  
Independent Method ◽  
Rrna Gene ◽  
Ripening Process ◽  
Culture Independent ◽  
Tulum Cheese ◽  
V3 Region ◽  
Culture Dependent ◽  
Bacterial 16S Rrna Gene

Abstract Our objective was to analyze the diversity of the microbiota over 180 d of ripening of eight batches of artisanal goatskin Tulum cheeses by culture-dependent and culture-independent (PCR-DGGE) methods. V3 region of the bacterial 16S rRNA gene was amplified with the PCR after direct DNA isolation from the cheese samples. Nine different species and five genera were determined by culturing, while 11 species were identified in the PCR-DGGE technique. This diversity revealed the uniqueness of artisanal cheese varieties. The dominant genera in all the cheese samples were composed of Enterococcus species. The culture-dependent method revealed five genera (Enterococcus,Bacillus,Lactococcus,Lactobacillus, Sphingomonas) while three genera (Enterococcus, Streptococcus, Lactococcus) were detected in the culture-independent method. It was concluded that combining the two methods is important for characterizing the whole microbiota of the Tulum cheese varieties produced in the Anamur region.

scholarly journals Biodiversity in Oscypek, a Traditional Polish Cheese, Determined by Culture-Dependent and -Independent Approaches

2012 ◽  
Vol 78 (6) ◽  
pp. 1890-1898 ◽  
Author(s):  
Ángel Alegría ◽  
Pawel Szczesny ◽  
Baltasar Mayo ◽  
Jacek Bardowski ◽  
Magdalena Kowalczyk
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Starter Cultures ◽  
Rrna Gene ◽  
Content Type ◽  
Protected Designation Of Origin ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis ◽  
Microbial Groups ◽  
The Tatra Mountains ◽  
Culture Dependent

ABSTRACTOscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g.,Lactococcus,Lactobacillus,Leuconostoc,Streptococcus, andEnterococcus, identified by all three methods, other, subdominant bacteria belonging to the familiesBifidobacteriaceaeandMoraxellaceae(mostlyEnhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures.

scholarly journals Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections

2016 ◽  
Vol 54 (5) ◽  
pp. 1295-1303 ◽  
Author(s):  
Hana Obručová ◽  
Radka Tihelková ◽  
Iva Kotásková ◽  
Filip Růžička ◽  
Veronika Holá ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Fungal Infections ◽  
Rapid Identification ◽  
Candida Guilliermondii ◽  
Rrna Gene ◽  
Content Type ◽  
Culture Independent ◽  
Panfungal Pcr ◽  
Reference Strains ◽  
Culture Dependent

Early diagnosis of fungal infection is critical for initiating antifungal therapy and reducing the high mortality rate in immunocompromised patients. In this study, we focused on rapid and sensitive identification of clinically importantCandidaspecies, utilizing the variability in the length of theITS2rRNA gene and fluorescent capillary electrophoresis (f-ITS2-PCR-CE). The method was developed and optimized on 29 variousCandidareference strains from which 26Candidaspecies were clearly identified, whileCandida guilliermondii,C. fermentati, andC. carpophila, which are closely related, could not be distinguished. The method was subsequently validated on 143 blinded monofungal clinical isolates (comprising 26 species) and was able to identify 88% of species unambiguously. This indicated a higher resolution power than the classical phenotypic approach which correctly identified 73%. Finally, the culture-independent potential of this technique was addressed by the analysis of 55 retrospective DNA samples extracted directly from clinical material. The method showed 100% sensitivity and specificity compared to those of the combined results of cultivation and panfungal PCR followed by sequencing used as a gold standard. In conclusion, this newly developed f-ITS2-PCR-CE analytical approach was shown to be a fast, sensitive, and highly reproducible tool for both culture-dependent and culture-independent identification of clinically importantCandidastrains, including species of the “psilosis” complex.

scholarly journals Culture-Independent and Culture-Dependent Characterization of the Black Soldier Fly Gut Microbiome Reveals a Large Proportion of Culturable Bacteria with Potential for Industrial Applications

2021 ◽  
Vol 9 (8) ◽  
pp. 1642
Author(s):  
Dorothee Tegtmeier ◽  
Sabine Hurka ◽  
Sanja Mihajlovic ◽  
Maren Bodenschatz ◽  
Stephanie Schlimbach ◽  
...  
Keyword(s):  
Gut Microbiome ◽  
Amplicon Sequencing ◽  
Industrial Applications ◽  
Culture Collection ◽  
Rrna Gene ◽  
Organic Substrates ◽  
Culturable Bacteria ◽  
Black Soldier Fly ◽  
Culture Independent ◽  
Culture Dependent

Black soldier fly larvae (BSFL) are fast-growing, resilient insects that can break down a variety of organic substrates and convert them into valuable proteins and lipids for applications in the feed industry. Decomposition is mediated by an abundant and versatile gut microbiome, which has been studied for more than a decade. However, little is known about the phylogeny, properties and functions of bacterial isolates from the BSFL gut. We therefore characterized the BSFL gut microbiome in detail, evaluating bacterial diversity by culture-dependent methods and amplicon sequencing of the 16S rRNA gene. Redundant strains were identified by genomic fingerprinting and 105 non-redundant isolates were then tested for their ability to inhibit pathogens. We cultivated representatives of 26 genera, covering 47% of the families and 33% of the genera detected by amplicon sequencing. Among these isolates, we found several representatives of the most abundant genera: Morganella, Enterococcus, Proteus and Providencia. We also isolated diverse members of the less-abundant phylum Actinobacteria, and a novel genus of the order Clostridiales. We found that 15 of the isolates inhibited at least one of the tested pathogens, suggesting a role in helping to prevent colonization by pathogens in the gut. The resulting culture collection of unique BSFL gut bacteria provides a promising resource for multiple industrial applications.

scholarly journals The bacterial community of the lone star tick (Amblyomma americanum)

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
L. Paulina Maldonado-Ruiz ◽  
Saraswoti Neupane ◽  
Yoonseong Park ◽  
Ludek Zurek
Keyword(s):  
Bacterial Community ◽  
Amblyomma Americanum ◽  
Rrna Gene ◽  
Culturable Bacteria ◽  
Core Microbiome ◽  
Lone Star Tick ◽  
Animal Pathogens ◽  
Wide Range ◽  
Culture Independent ◽  
Culture Dependent

Abstract Background The lone star tick (Amblyomma americanum), an important vector of a wide range of human and animal pathogens, is very common throughout the East and Midwest of the USA. Ticks are known to carry non-pathogenic bacteria that may play a role in their vector competence for pathogens. Several previous studies using the high throughput sequencing (HTS) technologies reported the commensal bacteria in a tick midgut as abundant and diverse. In contrast, in our preliminary survey of the field collected adult lone star ticks, we found the number of culturable/viable bacteria very low. Methods We aimed to analyze the bacterial community of A. americanum by a parallel culture-dependent and a culture-independent approach applied to individual ticks. Results We analyzed 94 adult females collected in eastern Kansas and found that 60.8% of ticks had no culturable bacteria and the remaining ticks carried only 67.7 ± 42.8 colony-forming units (CFUs)/tick representing 26 genera. HTS of the 16S rRNA gene resulted in a total of 32 operational taxonomic units (OTUs) with the dominant endosymbiotic genera Coxiella and Rickettsia (> 95%). Remaining OTUs with very low abundance were typical soil bacterial taxa indicating their environmental origin. Conclusions No correlation was found between the CFU abundance and the relative abundance from the culture-independent approach. This suggests that many culturable taxa detected by HTS but not by culture-dependent method were not viable or were not in their culturable state. Overall, our HTS results show that the midgut bacterial community of A. americanum is very poor without a core microbiome and the majority of bacteria are endosymbiotic.

scholarly journals Bacteria from the Genus Arcobacter Are Abundant in Effluent from Wastewater Treatment Plants

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Jannie Munk Kristensen ◽  
Marta Nierychlo ◽  
Mads Albertsen ◽  
Per Halkjær Nielsen
Keyword(s):  
Wastewater Treatment ◽  
Pathogenic Bacteria ◽  
Municipal Wastewater ◽  
Wastewater Treatment Plants ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Content Type ◽  
Effluent Quality ◽  
Culture Independent ◽  
Influent Wastewater

ABSTRACT Pathogenic bacteria in wastewater are generally considered to be efficiently removed in biological wastewater treatment plants. This understanding is almost solely based on culture-based control measures, and here we show, by applying culture-independent methods, that the removal of species in the genus Arcobacter was less effective than for many other abundant genera in the influent wastewater. Arcobacter was one of the most abundant genera in influent wastewater at 14 municipal wastewater treatment plants and was also abundant in the “clean” effluent from all the plants, reaching up to 30% of all bacteria as analyzed by 16S rRNA gene amplicon sequencing. Metagenomic analyses, culturing, genome sequencing of Arcobacter isolates, and visualization by fluorescent in situ hybridization (FISH) confirmed the presence of the human-pathogenic Arcobacter cryaerophilus and A. butzleri in both influent and effluent. The main reason for the high relative abundance in the effluent was probably that Arcobacter cells, compared to those of other abundant genera in the influent, did not flocculate and attach well to the activated sludge flocs, leaving a relatively large fraction dispersed in the water phase. The study shows there is an urgent need for new standardized culture-independent measurements of pathogens in effluent wastewaters, e.g., amplicon sequencing, and an investigation of the problem on a global scale to quantify the risk for humans and livestock. IMPORTANCE The genus Arcobacter was unexpectedly abundant in the effluent from 14 Danish wastewater treatment plants treating municipal wastewater, and the species included the human-pathogenic A. cryaerophilus and A. butzleri. Recent studies have shown that Arcobacter is common in wastewater worldwide, so the study indicates that discharge of members of the genus Arcobacter may be a global problem, and further studies are needed to quantify the risk and potentially minimize the discharge. The study also shows that culture-based analyses are insufficient for proper effluent quality control, and new standardized culture-independent measurements of effluent quality encompassing most pathogens should be considered.

scholarly journals Advancing Weed Management Strategies Using Metagenomic Techniques

Weed Science ◽  
2013 ◽  
Vol 61 (2) ◽  
pp. 171-184 ◽  
Author(s):  
Jenny Kao-Kniffin ◽  
Sarah M. Carver ◽  
Antonio DiTommaso
Keyword(s):  
Weed Management ◽  
Soil Bacteria ◽  
Control Strategies ◽  
Management Strategies ◽  
Past Research ◽  
Management Research ◽  
Herbicide Resistant ◽  
Culture Independent ◽  
Living Organisms ◽  
Culture Dependent

Global occurrences of herbicide resistant weed populations have increased the demand for development of new herbicides targeting novel mechanisms of action. Metagenomic approaches to natural drug discovery offer potential for isolating weed suppressive compounds from microorganisms. In past research, traditional techniques entailed isolating compounds from living organisms, whereas metagenomic approaches involve extracting fragments of DNA from soil and exploring for compounds of interest produced by the transformed hosts. Several herbicidal compounds have been isolated from soil bacteria through culturing methods and have led to the development of popular herbicides, such as glufosinate. In this review, we discuss the emergence of metagenomic approaches for weed management in the context of natural product discovery using traditional culture-dependent isolation and the more recent culture-independent methods. The same techniques can be used to isolate herbicide resistance genes. Adoption of metagenomic approaches in pest management research can lead to novel control strategies in cropping and landscape systems.

scholarly journals Detection and phylogenetic analysis of coastal bioaerosols using culture dependent and independent techniques

2011 ◽  
Vol 8 (2) ◽  
pp. 301-309 ◽  
Author(s):  
R. Urbano ◽  
B. Palenik ◽  
C. J. Gaston ◽  
K. A. Prather
Keyword(s):  
Atmospheric Chemistry ◽  
Cloud Droplet ◽  
Independent Study ◽  
Rrna Gene ◽  
Monitoring Site ◽  
Microbial Composition ◽  
Atmospheric Processes ◽  
Erosion Processes ◽  
Culture Independent ◽  
Culture Dependent

Abstract. Bioaerosols are emerging as important yet poorly understood players in atmospheric processes. Microorganisms can impact atmospheric chemistry through metabolic reactions and can potentially influence physical processes by participating in ice nucleation and cloud droplet formation. Microbial roles in atmospheric processes are thought to be species-specific and potentially dependent on cell viability. Using a coastal pier monitoring site as a sampling platform, culture-dependent (i.e. agar plates) and culture-independent (i.e. DNA clone libraries from filters) approaches were combined with 18S rRNA and 16S rRNA gene targeting to obtain insight into the local atmospheric microbial composition. From 13 microbial isolates and 42 DNA library clones, a total of 55 sequences were obtained representing four independent sampling events. Sequence analysis revealed that in these coastal samples two fungal phyla, Ascomycota and Basidiomycota, predominate among eukaryotes while Firmicutes and Proteobacteria predominate among bacteria. Furthermore, our culture-dependent study verifies the viability of microbes from all four phyla detected through our culture-independent study. Contrary to our expectations and despite oceanic air mass sources, common marine planktonic bacteria and phytoplankton were not typically found. The abundance of terrestrial and marine sediment-associated microorganisms suggests a potential importance for bioaerosols derived from beaches and/or coastal erosion processes.

scholarly journals Fungal Endophytic Communities in Grapevines (Vitis vinifera L.) Respond to Crop Management

2012 ◽  
Vol 78 (12) ◽  
pp. 4308-4317 ◽  
Author(s):  
Michael Pancher ◽  
Marco Ceol ◽  
Paola Elisa Corneo ◽  
Claudia Maria Oliveira Longa ◽  
Sohail Yousaf ◽  
...  
Keyword(s):  
Vitis Vinifera ◽  
Fungal Endophytes ◽  
Intergenic Spacer ◽  
Fungal Communities ◽  
Vitis Vinifera L ◽  
Multivariate Statistical ◽  
Content Type ◽  
Colony Pcr ◽  
Culture Independent ◽  
Culture Dependent

ABSTRACTWe studied the distribution of fungal endophytes of grapevine (Vitis viniferaL.) plants in a subalpine area of northern Italy, where viticulture is of high economic relevance. We adopted both cultivation-based and cultivation-independent approaches to address how various anthropic and nonanthropic factors shape microbial communities. Grapevine stems were harvested from several locations considering organic and integrated pest management (IPM) and from the cultivars Merlot and Chardonnay. Cultivable fungi were isolated and identified by internal-transcribed-spacer sequence analysis, using a novel colony-PCR method, to amplify DNA from fungal specimens. The composition of fungal communities was assessed using a cultivation-independent approach, automated ribosomal intergenic spacer analysis (ARISA). Multivariate statistical analysis of both culture-dependent and culture-independent data sets was convergent and indicated that fungal endophytic communities in grapevines from organically managed farms were different from those from farms utilizing IPM. Fungal communities in plants of cv. Merlot and cv. Chardonnay overlapped when analyzed using culture-dependent approaches but could be partially resolved using ARISA fingerprinting.

scholarly journals Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes by Escherichia coli with a Novel Ligation-Independent Cloning Vector Based on the Autotransporter YfaL

2012 ◽  
Vol 78 (9) ◽  
pp. 3051-3058 ◽  
Author(s):  
Hyeok-Jin Ko ◽  
Eunhye Park ◽  
Joseph Song ◽  
Taek Ho Yang ◽  
Hee Jong Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Surface Display ◽  
Cell Surface Display ◽  
Heterologous Proteins ◽  
Display System ◽  
Reporter Protein ◽  
Content Type ◽  
Ligation Independent Cloning

ABSTRACTAutotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) ofEscherichia colifor the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes fromSaccharophagus degradans2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 104molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growingE. coli.

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