Taxonomic Structure and Stability of the Bacterial Community in Belgian Sourdough Ecosystems as Assessed by Culture and Population Fingerprinting

ABSTRACT A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.

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The Existence of Endophytic Actinobacteria from Rhododendron zoelerri Revealed by Culture-Dependent and Culture-Independent Approaches

HAYATI Journal of Biosciences ◽

10.4308/hjb.25.2.54 ◽

2018 ◽

Vol 25 (2) ◽

pp. 54

Author(s):

Yulin Lestari ◽

Lia Aseptin Murdini ◽

Dedy Duryadi Solihin

Keyword(s):

Community Structure ◽

16S Rrna ◽

16S Rrna Gene ◽

Denaturing Gradient Gel Electrophoresis ◽

Morphological Characteristics ◽

Rrna Gene ◽

Culture Independent ◽

Reference Strains ◽

Culture Dependent ◽

Endophytic Actinobacteria

Endophytic actinobacteria from medicinal plant may play a significant role in producing bioactive compounds. The information regarding their diversity is an important. Rhododendron are traditionally used for treating human disorders. One of the selected Rhododendron used in this study was R. zoelleri from Papua origin, which has been conserved and grown in Cibodas Botanical Garden, West Java, Indonesia. The aim of this study was to assess the existence of endophytic actinobacteria from R. zoelleri based on a culture-dependent and their community structure based on a culture-independent approach. Culturable actinobacteria were isolated and cultured on HV medium. Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) targeting the metagenomic 16S rRNA was used to analyse the structure of the actinobacterial community. Six culturable endophytic actinobacteria (200 cfu/g fresh weight) from R. zoelleri were successfully isolated, three isolates from leaf, and the other isolates were obtained from stem. The six culturable isolates were RZP 1.3, RZP 1.1, RZP 2.2, RZPB 1.1, RZPB 7.1, RZPB 4.1. Based on their morphological characteristics, the endophytes have Streptomyces characters. The existence of Streptomyces spp. were also confirmed with molecular analysis based on 16S rRNA gene. The phylogenetic analysis based on 16S rRNA gene to the reference strains available in EzTaxon-e database showed that six isolates were closely related to S. djakartensis strains of NBRC 15409ᵀ(99.19%), S. tritolerans strains of DAS 165T(99.90%), S. coelicoflavus strains of NBRC 15399T(99.59). However, they showed differences in morphological characteristics as compared with the reference strains. The metagenomic analysis of the DGGE profile based on 16S rRNA gene showed the community structure of endophytic actinobacteria from R. zoelleri which was represented by 13 DGGE bands. The bands were closely related to Agromyces, Gordonia, Microbacterium, Micromonospora, Propionibacterium, Saccharomonospora, Streptomyces which have 93.18%-100% similarity. Based on the data, it showed diversity of endophytic actinobacteria from R. zoelleri which may be further assess for their novelty and bioprospecting.

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PENDEKATAN CULTURE INDEPENDENT UNTUK ANALISIS KOMUNITAS BAKTERI

OSEANA ◽

10.14203/oseana.2017.vol.42no.1.34 ◽

2019 ◽

Vol 42 (1) ◽

pp. 9-17

Author(s):

Nur Fitriah Afianti ◽

Yeti Darmayati

Keyword(s):

Bacterial Community ◽

16S Rrna ◽

Target Genes ◽

Denaturing Gradient Gel Electrophoresis ◽

Single Strand Conformation Polymorphism ◽

Molecular Techniques ◽

Rrna Gene ◽

Culturable Bacteria ◽

Culture Independent ◽

Culture Dependent

CULTURE INDEPENDENT APPROACH FOR BACTERIAL COMMUNITY ANALYSIS. Analysis of bacterial community can be through two approaches, through cultivation (culture dependent) and without cultivation (culture independent). Culture dependent approach is conventional method which only covered few bacteria because not all bacteria could be cultured. Culture independent approach with molecular techniques based on DNA communities can provide more information about the structure and diversity of bacteria in nature, both culturable bacteria and unculturable bacteria. 16S rRNA gene is commonly target gene used in bacterial communities analysis. Other specific target genes also being developed for specific groups of bacteria. Several methods are developed for the analysis of molecular markers 16S rRNA or other specific genes, including Denaturing Gradient Gel Electrophoresis (DGGE), Terminal Restriction Fragment Length Polymorphism (TRFLP), and Single Strand Conformation Polymorphism (SSCP).

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Methanotroph Diversity in Landfill Soil: Isolation of Novel Type I and Type II Methanotrophs Whose Presence Was Suggested by Culture-Independent 16S Ribosomal DNA Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.65.11.4887-4897.1999 ◽

1999 ◽

Vol 65 (11) ◽

pp. 4887-4897 ◽

Cited By ~ 136

Author(s):

Mark G. Wise ◽

J Vaun McArthur ◽

Lawrence J. Shimkets

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Ribosomal Dna ◽

Rrna Gene ◽

Type I ◽

Type Ii ◽

Partial Sequencing ◽

16S Ribosomal Dna ◽

Culture Independent ◽

The 16S Rrna Gene

ABSTRACT The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4.8 versus 6.8), air/CH4/CO2 headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1× nitrate minimal salts [NMS] versus 0.2× NMS). Screening of the isolates showed that the nutrient-rich 1× NMS selected for type I methanotrophs, while the nutrient-poor 0.2× NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobiumgroup, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly publishedMethylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.

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Comparative Analysis of Bacterial Diversity for Cutlassfish (Trichiurus haumela) during Cold Storage by the Culture-Dependent and Culture-Independent Methods

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.535-537.1046 ◽

2012 ◽

Vol 535-537 ◽

pp. 1046-1053 ◽

Cited By ~ 1

Author(s):

Wei Qing Lan ◽

Jing Xie

Keyword(s):

16S Rrna ◽

Pseudomonas Fluorescens ◽

Cold Storage ◽

Denaturing Gradient Gel Electrophoresis ◽

Rrna Gene ◽

Region Gene ◽

Gradient Gel Electrophoresis ◽

Dominant Bacteria ◽

V3 Region ◽

Culture Dependent

The methods of culture-dependent and denaturing gradient gel electrophoresis (DGGE) based on the sequence of 16S rRNA V3 region gene were described to comparatively characterize the microbial population and community structure of cutlassfish (Trichiurus haumela) under the cold storage. The results showed that 13 kinds of bacteria were identified by the traditional culture-dependent methods, the dominant bacteria belonged to Shewanella putrefaciens and Pseudomonas fluorescens. To determine the community profiles of the samples on variable V3 region, the bacteria of 16S rRNA gene were amplified by PCR and 11 distinct PCR products were separated by DGGE fingerprinting technology. From the sequence analysis, Psychrobacter sp. was found to be the predominant bacteria in the initial stage of the storage. The proportion of Shewanella sp., Pseudomonas sp. increased gradually with the extension of storage time, and they took the place of Psychrobacter sp. to be the dominant bacteria. Thereinto, both Pseudomonas fluorescens and Vibrio sp. took high proportions in the process of storage due to the deterioration of cutlassfish (Trichiurus haumela).

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Culture-dependent and -independent approaches establish the complexity of a PAH-degrading microbial consortium

Canadian Journal of Microbiology ◽

10.1139/w05-090 ◽

2005 ◽

Vol 51 (11) ◽

pp. 897-909 ◽

Cited By ~ 36

Author(s):

Marc Viñas ◽

Jordi Sabaté ◽

Caterina Guasp ◽

Jorge Lalucat ◽

Anna M Solanas

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Consortium ◽

Denaturing Gradient Gel Electrophoresis ◽

Sole Source ◽

Rrna Gene ◽

Dominant Group ◽

Clone Libraries ◽

Variable Regions ◽

Culture Dependent

A microbial consortium (AM) obtained by sequential enrichment in liquid culture with a polycyclic aromatic hydrocarbon (PAH) mixture of three- and four-ringed PAHs as a sole source of carbon and energy was examined using a triple-approach method based on various cultivation strategies, denaturing gradient gel electrophoresis (DGGE), and the screening of 16S and 18S rRNA gene clone libraries. Eleven different sequences by culture-dependent techniques and seven by both DGGE and clone libraries were obtained. The comparison of three variable regions (V3–V5) of the 16S rRNA gene between the sequences obtained yielded 19 different microbial components. Proteobacteria were the dominant group, representing 83% of the total, while the Cytophaga–Flexibacter–Bacteroides group (CFB) was 11% and the Ascomycota fungi 6%. β-Proteobacteria were predominant in the DGGE and clone library methods, whereas they were a minority in culturable strains. The highest diversity and number of noncoincident sequences were achieved by the cultivation method that showed members of the α-, β-, and γ-Proteobacteria; CFB bacterial group; and Asco mycota fungi. Only six of the 11 strains isolated showed PAH-degrading capability. The bacterial strain (AMS7) and the fungal strain (AMF1), which were similar to Sphingomonas sp. and Fusarium sp., respectively, achieved the greatest PAH depletion. The results indicate that polyphasic assessment is necessary for a proper understanding of the composition of a microbial consortium.Key words: microbial consortium, microbial diversity, PAHs, DGGE, 16S rRNA gene.

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

Applied and Environmental Microbiology ◽

10.1128/aem.06081-11 ◽

2012 ◽

Vol 78 (6) ◽

pp. 1890-1898 ◽

Cited By ~ 84

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.

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Analysis of Vaginal Lactobacilli from Healthy and Infected Brazilian Women

Applied and Environmental Microbiology ◽

10.1128/aem.00284-08 ◽

2008 ◽

Vol 74 (14) ◽

pp. 4539-4542 ◽

Cited By ~ 32

Author(s):

Rafael C. R. Martinez ◽

Sílvio A. Franceschini ◽

Maristela C. Patta ◽

Silvana M. Quintana ◽

Álvaro C. Nunes ◽

...

Keyword(s):

Bacterial Vaginosis ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Restriction Analysis ◽

Rrna Gene ◽

Vaginal Lactobacilli ◽

Culture Independent ◽

Gradient Gel Electrophoresis ◽

Gene Restriction ◽

Culture Dependent

ABSTRACT Culture-dependent PCR-amplified rRNA gene restriction analysis and culture-independent (PCR-denaturing gradient gel electrophoresis) methodologies were used to examine vaginal lactobacilli from Brazilian women who were healthy or had been diagnosed with vulvovaginal candidiasis (VVC) or bacterial vaginosis. Only Lactobacillus crispatus was detected accordingly by both methods, and H2O2-producing lactobacilli were not associated with protection against VVC.

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Culture-Independent Analysis of Bacterial Fuel Contamination Provides Insight into the Level of Concordance with the Standard Industry Practice of Aerobic Cultivation

Applied and Environmental Microbiology ◽

10.1128/aem.02317-10 ◽

2011 ◽

Vol 77 (13) ◽

pp. 4527-4538 ◽

Cited By ~ 31

Author(s):

Judith White ◽

Jack Gilbert ◽

Graham Hill ◽

Edward Hill ◽

Susan M. Huse ◽

...

Keyword(s):

16S Rrna ◽

Denaturing Gradient Gel Electrophoresis ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Content Type ◽

Aerobic Culture ◽

Aerobic Cultivation ◽

Culture Independent ◽

Gradient Gel Electrophoresis ◽

Industry Practice

ABSTRACTBacterial diversity in contaminated fuels has not been systematically investigated using cultivation-independent methods. The fuel industry relies on phenotypic cultivation-based contaminant identification, which may lack accuracy and neglect difficult-to-culture taxa. By the use of industry practice aerobic cultivation, 16S rRNA gene sequencing, and strain genotyping, a collection of 152 unique contaminant isolates from 54 fuel samples was assembled, and a dominance ofPseudomonas(21%),Burkholderia(7%), andBacillus(7%) was demonstrated. Denaturing gradient gel electrophoresis (DGGE) of 15 samples revealedProteobacteriaandFirmicutesto be the most abundant phyla. When 16S rRNA V6 gene pyrosequencing of four selected fuel samples (indicated by “JW”) was performed,Betaproteobacteria(42.8%) andGammaproteobacteria(30.6%) formed the largest proportion of reads; the most abundant genera wereMarinobacter(15.4%; JW57),Achromobacter(41.6%; JW63),Burkholderia(80.7%; JW76), andHalomonas(66.2%; JW78), all of which were also observed by DGGE. However, theClostridia(38.5%) andDeltaproteobacteria(11.1%) identified by pyrosequencing in sample JW57 were not observed by DGGE or aerobic culture. Genotyping revealed three instances where identical strains were found: (i) aPseudomonassp. strain recovered from 2 different diesel fuel tanks at a single industrial site; (ii) aMangroveibactersp. strain isolated from 3 biodiesel tanks at a single refinery site; and (iii) aBurkholderiavietnamiensisstrain present in two unrelated automotive diesel samples. Overall, aerobic cultivation of fuel contaminants recovered isolates broadly representative of the phyla and classes present but lacked accuracy by overrepresenting members of certain groups such asPseudomonas.

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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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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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