To culture or not to culture: a snapshot of culture-dependent and culture-independent bacterial diversity from peanut rhizosphere

Background Sequencing driven metagenomics studies have been instrumental in various aspects of microbiology including identification of newer taxa. While this culture-independent approach has its own merits and demerits, several studies have focussed on comparing it with traditional culture-dependent (CD) approach. However, most of these comparative studies rely on Sanger sequencing of complete 16S rRNA gene from pure culture colonies to determine the culturable bacterial diversity. This approach undercounts culturable diversity as only fewer isolates are selected, sequenced, and identified. Methods In this study, we have used an Illumina based partial 16S sequencing to identify all the microbes growing on the media and directly comparing with its culture-independent (CI) counterpart. Eight different media were used to target different organisms from soil. Diversity on these media were compared with their CI counterpart. The NGS data was analysed using DADA2 to provide more resolution to the data. Results In line with studies of similar nature, current study presented higher bacterial diversity in CI approach. However, the current study reflected that a greater number of sequence variants were missed out in CI approach as compared to number of sequence variants shared with CD approach. We observed around 322 (5.98%) ASVs (Amplicon Sequence Variants) exclusively present in CD samples while, 234 (4.35%) ASVs were shared between both approaches. Most of these 322 CD exclusive ASVs were classified as Enterobacteriaceae family and Bacillus genus, with several ASVs annotated at the species level as well, and these organisms are more commonly observed in soil and were also detected in CI approach. Furthermore, 22 genera were exclusively detected in CD samples, most of which were reported from soil and water.

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

Parasites & Vectors ◽

10.1186/s13071-020-04550-z ◽

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.

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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

Journal of Dairy Research ◽

10.1017/s0022029921000765 ◽

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.

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Molecular bacterial diversity and bioburden of commercial airliner cabin air

Canadian Journal of Microbiology ◽

10.1139/w07-093 ◽

2007 ◽

Vol 53 (11) ◽

pp. 1259-1271 ◽

Cited By ~ 14

Author(s):

Myron T. La Duc ◽

Tara Stuecker ◽

Kasthuri Venkateswaran

Keyword(s):

Bacterial Diversity ◽

Rrna Gene ◽

Human Respiratory Tract ◽

Atp Assay ◽

Gram Positive Bacteria ◽

Air Impingement ◽

Culture Independent ◽

Bacterial Enumeration ◽

Gradual Accumulation ◽

Opportunistic Pathogenic

Culture-independent, biomarker-targeted bacterial enumeration and identification strategies were employed to estimate total bacterial burden and diversity within the cabin air of commercial airliners. Samples from each of 4 flights on 2 commercial carriers were collected via air-impingement. The total viable microbial population ranged from below detection limits to 4.1 × 106cells/m3of air, as assessed by the ATP assay. A gradual accumulation of microbes was observed from the time of passenger boarding through mid-flight, followed by a sharp decline in bacterial abundance and viability from the initiation of descent through landing. Representatives of the α-, β-, and γ-Proteobacteria, as well as Gram-positive bacteria, were isolated in varying abundance. Neisseria meningitidis rRNA gene sequences were retrieved in great abundance from Airline A followed by Streptococcus oralis/mitis sequences. Pseudomonas synxantha sequences dominated Airline B clone libraries, followed by those of N. meningitidis and S. oralis/mitis. The cabin air samples examined herein housed low bacterial diversity and were often dominated by a particular subset of bacteria: opportunistic pathogenic inhabitants of the human respiratory tract and oral cavity.

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The culture-dependent and culture-independent analysis for determination of bacterial diversity within Limnatis nilotica (Clitellata: Hirudinea)

Biologia ◽

10.2478/s11756-019-00194-2 ◽

2019 ◽

Vol 74 (6) ◽

pp. 639-648

Author(s):

Ali Sevim ◽

Elif Sevim

Keyword(s):

Bacterial Diversity ◽

Independent Analysis ◽

Culture Independent ◽

Culture Dependent

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

Biogeosciences ◽

10.5194/bg-8-301-2011 ◽

2011 ◽

Vol 8 (2) ◽

pp. 301-309 ◽

Cited By ~ 44

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.

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