scholarly journals PENDEKATAN CULTURE INDEPENDENT UNTUK ANALISIS KOMUNITAS BAKTERI

OSEANA ◽  
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).

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

2008 ◽  
Vol 74 (8) ◽  
pp. 2414-2423 ◽  
Author(s):  
Ilse Scheirlinck ◽  
Roel Van der Meulen ◽  
Ann Van Schoor ◽  
Marc Vancanneyt ◽  
Luc De Vuyst ◽  
...  
Keyword(s):  
16S Rrna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Type I ◽  
Technological Parameters ◽  
Structure And Stability ◽  
Two Samples ◽  
Culture Independent ◽  
Culture Dependent

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.

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

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.

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 Effects of Antibiotics on the Bacterial Community, Metabolic Functions and Antibiotic Resistance Genes in Mariculture Sediments during Enrichment Culturing

2020 ◽  
Vol 8 (8) ◽  
pp. 604
Author(s):  
Meng-Qi Ye ◽  
Guan-Jun Chen ◽  
Zong-Jun Du
Keyword(s):  
Antibiotic Resistance ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Antibiotic Resistance Genes ◽  
Control Group ◽  
Rrna Gene ◽  
Metabolic Functions ◽  
Culture Dependent

The effect of antibiotics on the diversity and functioning of indigenous microorganisms in the environment has attracted much attention. In this study, effects of exposure to six different antibiotics on the bacterial community, metabolic functions and antibiotic resistance genes (ARGs) in marine sediments during enrichment culturing were investigated. Classical culture-dependent method and high-throughput 16S rRNA gene sequencing method were both applied. In the culture-dependent analysis, the obtained 1549 isolates belonged to four phyla (Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria) and 155 genera. Proteobacteria and Firmicutes were the dominant phyla. The diversity and abundance of obtained bacteria after antibiotic processing exhibited different degrees of decrease. Enrichment culturing for different time could also affect the bacterial community composition. Some genera of bacteria were not isolated in the control group, but they could be isolated in the antibiotic-treated groups. In high-throughput 16S rRNA gene amplicon sequencing analyses, all the effective reads were clustered into 2822 OTUs at 97% similarity cutoff; they were annotated to 49 phyla, 103 class, 220 orders, 347 families, 624 genera and 1122 species. An alpha diversity analysis indicated that the community diversity and richness decreased under antibiotic exposure. The changes at the genus level were much more obvious. Only 48 genera of 129 genera were shared by all the samples. A total of 29 genera which were not detected in the initial control sample could be detected in at least one antibiotic-treated group. SIMPER analysis showed that OTU2543 and OTU1450 were the most common taxa to the dissimilarity of bacterial community between antibiotic-treated groups and the control group. OTU2034 and OUT2543 were the most contributive taxa to dissimilarity of groups incubating for different time. Metabolism was the predominant bacterial function. A total of 30 ARGs were detected in the samples. This study mainly focused on the changes of microbiota under the selective pressure of antibiotics for different time and the results demonstrated that the antibiotic could affect the bacterial diversity and richness in the marine ecosystem.

scholarly journals Impact of Biochar Application to Soil on the Root-Associated Bacterial Community Structure of Fully Developed Greenhouse Pepper Plants

2011 ◽  
Vol 77 (14) ◽  
pp. 4924-4930 ◽  
Author(s):  
Max Kolton ◽  
Yael Meller Harel ◽  
Zohar Pasternak ◽  
Ellen R. Graber ◽  
Yigal Elad ◽  
...  
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
Relative Abundance ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Community Composition ◽  
Systemic Resistance ◽  
Rrna Gene ◽  
Molecular Fingerprinting ◽  
Content Type ◽  
Biochar Amendment

ABSTRACTAdding biochar to soil has environmental and agricultural potential due to its long-term carbon sequestration capacity and its ability to improve crop productivity. Recent studies have demonstrated that soil-applied biochar promotes the systemic resistance of plants to several prominent foliar pathogens. One potential mechanism for this phenomenon is root-associated microbial elicitors whose presence is somehow augmented in the biochar-amended soils. The objective of this study was to assess the effect of biochar amendment on the root-associated bacterial community composition of mature sweet pepper (Capsicum annuumL.) plants. Molecular fingerprinting (denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism) of 16S rRNA gene fragments showed a clear differentiation between the root-associated bacterial community structures of biochar-amended and control plants. The pyrosequencing of 16S rRNA amplicons from the rhizoplane of both treatments generated a total of 20,142 sequences, 92 to 95% of which were affiliated with theProteobacteria,Bacteroidetes,Actinobacteria, andFirmicutesphyla. The relative abundance of members of theBacteroidetesphylum increased from 12 to 30% as a result of biochar amendment, while that of theProteobacteriadecreased from 71 to 47%. TheBacteroidetes-affiliatedFlavobacteriumwas the strongest biochar-induced genus. The relative abundance of this group increased from 4.2% of total root-associated operational taxonomic units (OTUs) in control samples to 19.6% in biochar-amended samples. Additional biochar-induced genera included chitin and cellulose degraders (ChitinophagaandCellvibrio, respectively) and aromatic compound degraders (HydrogenophagaandDechloromonas). We hypothesize that these biochar-augmented genera may be at least partially responsible for the beneficial effect of biochar amendment on plant growth and viability.

scholarly journals Seasonal changes in bacterial communities associated with healthy and diseasedPoritescoral in southern Taiwan

2016 ◽  
Vol 62 (12) ◽  
pp. 1021-1033 ◽  
Author(s):  
Chorng-Horng Lin ◽  
Chih-Hsiang Chuang ◽  
Wen-Hung Twan ◽  
Shu-Fen Chiou ◽  
Tit-Yee Wong ◽  
...  
Keyword(s):  
Health Status ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequences ◽  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Southern Taiwan ◽  
The 16S Rrna Gene

We compared the bacterial communities associated with healthy scleractinian coral Porites sp. with those associated with coral infected with pink spot syndrome harvested during summer and winter from waters off the coast of southern Taiwan. Members of the bacterial community associated with the coral were characterized by means of denaturing gradient gel electrophoresis (DGGE) of a short region of the 16S rRNA gene and clone library analysis. Of 5 different areas of the 16S rRNA gene, we demonstrated that the V3 hypervariable region is most suited to represent the coral-associated bacterial community. The DNA sequences of 26 distinct bands extracted from DGGE gels and 269 sequences of the 16S rRNA gene from clone libraries were determined. We found that the communities present in diseased coral were more heterogeneous than the bacterial communities of uninfected coral. In addition, bacterial communities associated with coral harvested in the summer were more diverse than those associated with coral collected in winter, regardless of the health status of the coral. Our study suggested that the compositions of coral-associated bacteria communities are complex, and the population of bacteria varies greatly between seasons and in coral of differing health status.

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

2012 ◽  
Vol 535-537 ◽  
pp. 1046-1053 ◽  
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).

scholarly journals Monitoring Bacterial Communities in Raw Milk and Cheese by Culture-Dependent and -Independent 16S rRNA Gene-Based Analyses

2007 ◽  
Vol 73 (6) ◽  
pp. 1882-1891 ◽  
Author(s):  
Céline Delbès ◽  
Leila Ali-Mandjee ◽  
Marie-Christine Montel
Keyword(s):  
16S Rrna ◽  
Culture Media ◽  
Raw Milk ◽  
Single Strand Conformation Polymorphism ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Plate Count ◽  
Rrna Gene ◽  
Dual Culture ◽  
Culture Dependent

ABSTRACT The diversity and dynamics of bacterial populations in Saint-Nectaire, a raw-milk, semihard cheese, were investigated using a dual culture-dependent and direct molecular approach combining single-strand conformation polymorphism (SSCP) fingerprinting and sequencing of 16S rRNA genes. The dominant clones, among 125 16S rRNA genes isolated from milk, belonged to members of the Firmicutes (58% of the total clones) affiliated mainly with the orders Clostridiales and the Lactobacillales, followed by the phyla Proteobacteria (21.6%), Actinobacteria (16.8%), and Bacteroidetes (4%). Sequencing the 16S rRNA genes of 126 milk isolates collected from four culture media revealed the presence of 36 different species showing a wider diversity in the Gammaproteobacteria phylum and Staphylococcus genus than that found among clones. In cheese, a total of 21 species were obtained from 170 isolates, with dominant species belonging to the Lactobacillales and subdominant species affiliated with the Actinobacteria, Bacteroidetes (Chryseobacterium sp.), or Gammaproteobacteria (Stenotrophomonas sp.). Fingerprinting DNA isolated from milk by SSCP analysis yielded complex patterns, whereas analyzing DNA isolated from cheese resulted in patterns composed of a single peak which corresponded to that of lactic acid bacteria. SSCP fingerprinting of mixtures of all colonies harvested from plate count agar supplemented with crystal violet and vancomycin showed good potential for monitoring the subdominant Proteobacteria and Bacteroidetes (Flavobacteria) organisms in milk and cheese. Likewise, analyzing culturable subcommunities from cheese-ripening bacterial medium permitted assessment of the diversity of halotolerant Actinobacteria and Staphylococcus organisms. Direct and culture-dependent approaches produced complementary information, thus generating a more accurate view of milk and cheese microbial ecology.

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