PseudomonasDiversity in Crude-Oil-Contaminated Intertidal Sand Samples Obtained after thePrestigeOil Spill

ABSTRACTThe Galicia seashore, in northwestern Spain, was one of the shorelines affected by thePrestigeoil spill in November 2002. The diversity of autochthonousPseudomonaspopulations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of anrpoDgene library. The second involved the isolation of 94Pseudomonasstrains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifyingPseudomonasstrains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index forPseudomonasspecies was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the speciesP. stutzeri,P.putida,P. anguilliseptica, andP. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novelPseudomonasspecies. One isolate was considered representative of a novelP. stutzerigenomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by therpoDPCR method was a useful tool for evaluatingPseudomonascommunities and also for microdiversity studies ofPseudomonaspopulations.

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A culture independent method for the detection of Aeromonas sp. from water samples

Italian Journal of Food Safety ◽

10.4081/ijfs.2016.5489 ◽

2016 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Fadua Latif-Eugenín ◽

Roxana Beaz-Hidalgo ◽

María José Figueras

Keyword(s):

Water Samples ◽

Detection Method ◽

Cholesterol Acyltransferase ◽

Water Volume ◽

Molecular Method ◽

Fast Detection ◽

Culture Independent ◽

Pcr Method ◽

Culture Dependent ◽

Enrichment Step

The genus Aeromonas is present in a wide variety of water environments and is recognized as potentially pathogenic to humans and animals. Members of this genus are often confused with Vibrio when using automated, commercial identification systems that are culture-dependent. This study describes a PCR detection method for Aeromonas that is cultureindependent and that targets the gcat (Glycerophospholopid-Cholesterol Acyltransferase) gene, which is specific for this genus. The GCAT-PCR was 100% specific in artificially inoculated water samples, with a detection limit that ranged from 2.5 to 25 cfu/mL. The success at detecting this pathogen in 86 water samples using the GCAT-PCR method was identical to the conventional culturing method when a pre-enrichment step was carried out, yielding 83.7% positive samples. On the other hand, without a pre-enrichment step, only 77.9% of the samples were positive by culturing and only 15.1% with the GCAT-PCR. However, 83.7% positive samples were obtained for the GCAT-PCR when the water volume for the DNA extraction was increased from 400 μL to 4 mL. The proposed molecular method is much faster (5 or 29 h) than the culturing method (24 or 48 h) whether performed directly or after a pre-enrichment step and it will enable the fast detection of Aeromonas in water samples helping to prevent a possible transmission to humans.

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DNA enrichment and tagmentation method for species-level identification and strain-level differentiation using ON-rep-seq

Communications Biology ◽

10.1038/s42003-019-0617-x ◽

2019 ◽

Vol 2 (1) ◽

Author(s):

Łukasz Krych ◽

Josué L. Castro-Mejía ◽

Laura M. Forero-Junco ◽

Daniel N. Moesby ◽

Morten B. Mikkelsen ◽

...

Keyword(s):

Cost Effective ◽

Species Level ◽

Strain Level ◽

Read Length ◽

High Quality ◽

Bacterial Dna ◽

Sequencing Platform ◽

Dna Library ◽

Culture Independent ◽

Level Identification

Abstract Despite the massive developments within culture-independent methods for detection of microorganisms during the last decade, culture-based methods remain a cornerstone in microbiology. Yet, the problem of rapid, accurate and inexpensive identification of bacterial isolates down to species/strain level remains unresolved. We have developed a new method for bacterial DNA enrichment and tagmentation allowing fast (<24 h) and cost-effective species level identification and strain level differentiation using the MinION portable sequencing platform (ON-rep-seq). DNA library preparation for 96 isolates takes less than 5 h and ensures highly reproducible distribution of reads that can be used to generate strain level specific read length counts profiles (LCp). We have developed a pipeline that by correcting reads error within peaks of LCp generates a set of high quality (>99%) consensus reads. Whereas, the information from high quality reads is used to retrieve species level taxonomy, comparison of LCp allows for strain level differentiation.

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Microbiota identified from preserved Anopheles

Malaria Journal ◽

10.1186/s12936-021-03754-7 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Bianca E Silva ◽

Zvifadzo Matsena Zingoni ◽

Lizette L. Koekemoer ◽

Yael L. Dahan-Moss

Keyword(s):

Microbial Diversity ◽

Vector Competence ◽

Mosquito Species ◽

Anopheles Funestus ◽

Cost Effective ◽

Diversity Indices ◽

Malaria Vectors ◽

Microbial Composition ◽

Culture Independent ◽

Culture Dependent

Abstract Background Mosquito species from the Anopheles gambiae complex and the Anopheles funestus group are dominant African malaria vectors. Mosquito microbiota play vital roles in physiology and vector competence. Recent research has focused on investigating the mosquito microbiota, especially in wild populations. Wild mosquitoes are preserved and transported to a laboratory for analyses. Thus far, microbial characterization post-preservation has been investigated in only Aedes vexans and Culex pipiens. Investigating the efficacy of cost-effective preservatives has also been limited to AllProtect reagent, ethanol and nucleic acid preservation buffer. This study characterized the microbiota of African Anopheles vectors: Anopheles arabiensis (member of the An. gambiae complex) and An. funestus (member of the An. funestus group), preserved on silica desiccant and RNAlater® solution. Methods Microbial composition and diversity were characterized using culture-dependent (midgut dissections, culturomics, MALDI-TOF MS) and culture-independent techniques (abdominal dissections, DNA extraction, next-generation sequencing) from laboratory (colonized) and field-collected mosquitoes. Colonized mosquitoes were either fresh (non-preserved) or preserved for 4 and 12 weeks on silica or in RNAlater®. Microbiota were also characterized from field-collected An. arabiensis preserved on silica for 8, 12 and 16 weeks. Results Elizabethkingia anophelis and Serratia oryzae were common between both vector species, while Enterobacter cloacae and Staphylococcus epidermidis were specific to females and males, respectively. Microbial diversity was not influenced by sex, condition (fresh or preserved), preservative, or preservation time-period; however, the type of bacterial identification technique affected all microbial diversity indices. Conclusions This study broadly characterized the microbiota of An. arabiensis and An. funestus. Silica- and RNAlater®-preservation were appropriate when paired with culture-dependent and culture-independent techniques, respectively. These results broaden the selection of cost-effective methods available for handling vector samples for downstream microbial analyses.

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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

Microorganisms ◽

10.3390/microorganisms9081642 ◽

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.

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Assessing Genotypic and Environmental Effects on Endophyte Communities of Fraxinus (Ash) Using Culture Dependent and Independent DNA Sequencing

Journal of Fungi ◽

10.3390/jof7070565 ◽

2021 ◽

Vol 7 (7) ◽

pp. 565

Author(s):

Anindita Lahiri ◽

Brian R. Murphy ◽

Trevor R. Hodkinson

Keyword(s):

Community Composition ◽

Environmental Effects ◽

High Throughput Sequencing ◽

Fungal Community Composition ◽

Species Identity ◽

Ash Dieback ◽

Hymenoscyphus Fraxineus ◽

Culture Independent ◽

The Impact ◽

Culture Dependent

Fraxinus excelsior populations are in decline due to the ash dieback disease Hymenoscyphus fraxineus. It is important to understand genotypic and environmental effects on its fungal microbiome to develop disease management strategies. To do this, we used culture dependent and culture independent approaches to characterize endophyte material from contrasting ash provenances, environments, and tissues (leaves, roots, seeds). Endophytes were isolated and identified using nrITS, LSU, or tef DNA loci in the culture dependent assessments, which were mostly Ascomycota and assigned to 37 families. Few taxa were shared between roots and leaves. The culture independent approach used high throughput sequencing (HTS) of nrITS amplicons directly from plant DNA and detected 35 families. Large differences were found in OTU diversity and community composition estimated by the contrasting approaches and these data need to be combined for estimations of the core endophyte communities. Species richness and Shannon index values were highest for the leaf material and the French population. Few species were shared between seed and leaf tissue. PCoA and NMDS of the HTS data showed that seed and leaf microbiome communities were highly distinct and that there was a strong influence of Fraxinus species identity on their fungal community composition. The results will facilitate a better understanding of ash fungal ecology and are a step toward identifying microbial biocontrol systems to minimize the impact of the disease.

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Integration of culture-dependent and independent methods provides a more coherent picture of the pig gut microbiome

FEMS Microbiology Ecology ◽

10.1093/femsec/fiaa022 ◽

2020 ◽

Vol 96 (3) ◽

Cited By ~ 2

Author(s):

Gavin J Fenske ◽

Sudeep Ghimire ◽

Linto Antony ◽

Jane Christopher-Hennings ◽

Joy Scaria

Keyword(s):

Bacterial Communities ◽

Bacterial Species ◽

Shotgun Metagenomics ◽

Culture Techniques ◽

Microbiome Composition ◽

Culture Independent ◽

Health And Disease ◽

The Media ◽

And Function ◽

Culture Dependent

ABSTRACT Bacterial communities resident in the hindgut of pigs, have profound impacts on health and disease. Investigations into the pig microbiome have utilized either culture-dependent, or far more commonly, culture-independent techniques using next generation sequencing. We contend that a combination of both approaches generates a more coherent view of microbiome composition. In this study, we surveyed the microbiome of Tamworth breed and feral pigs through the integration high throughput culturing and shotgun metagenomics. A single culture medium was used for culturing. Selective screens were added to the media to increase culture diversity. In total, 46 distinct bacterial species were isolated from the Tamworth and feral samples. Selective screens successfully shifted the diversity of bacteria on agar plates. Tamworth pigs are highly dominated by Bacteroidetes primarily composed of the genus Prevotella whereas feral samples were more diverse with almost equal proportions of Firmicutes and Bacteroidetes. The combination of metagenomics and culture techniques facilitated a greater retrieval of annotated genes than either method alone. The single medium based pig microbiota library we report is a resource to better understand pig gut microbial ecology and function. It allows for assemblage of defined bacterial communities for studies in bioreactors or germfree animal models.

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