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 Microbiological Study of Lactic Acid Fermentation of Caper Berries by Molecular and Culture-Dependent Methods

2005 ◽  
Vol 71 (12) ◽  
pp. 7872-7879 ◽  
Author(s):  
Rubén Pérez Pulido ◽  
Nabil Ben Omar ◽  
Hikmate Abriouel ◽  
Rosario Lucas López ◽  
Magdalena Martínez Cañamero ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Community Dynamics ◽  
Accurate Determination ◽  
Lactic Acid Fermentation ◽  
Rrna Gene ◽  
Acid Fermentation ◽  
Rapd Pcr ◽  
Lactobacillus Paraplantarum ◽  
Culture Independent ◽  
Culture Dependent

ABSTRACT Fermentation of capers (the fruits of Capparis sp.) was studied by molecular and culture-independent methods. A lactic acid fermentation occurred following immersion of caper berries in water, resulting in fast acidification and development of the organoleptic properties typical of this fermented food. A collection of 133 isolates obtained at different times of fermentation was reduced to 75 after randomly amplified polymorphic DNA (RAPD)-PCR analysis. Isolates were identified by PCR or 16S rRNA gene sequencing as Lactobacillus plantarum (37 isolates), Lactobacillus paraplantarum (1 isolate), Lactobacillus pentosus (5 isolates), Lactobacillus brevis (9 isolates), Lactobacillus fermentum (6 isolates), Pediococcus pentosaceus (14 isolates), Pediococcus acidilactici (1 isolate), and Enterococcus faecium (2 isolates). Cluster analysis of RAPD-PCR patterns revealed a high degree of diversity among lactobacilli (with four major groups and five subgroups), while pediococci clustered in two closely related groups. A culture-independent analysis of fermentation samples by temporal temperature gradient electrophoresis (TTGE) also indicated that L. plantarum is the predominant species in this fermentation, in agreement with culture-dependent results. The distribution of L. brevis and L. fermentum in samples was also determined by TTGE, but identification of Pediococcus at the species level was not possible. TTGE also allowed a more precise estimation of the distribution of E. faecium, and the detection of Enterococcus casseliflavus (which was not revealed by the culture-dependent analysis). Results from this study indicate that complementary data from molecular and culture-dependent analysis provide a more accurate determination of the microbial community dynamics during caper fermentation.

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.

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 DNA isolation of potentially probiotic lactic acid bacteria from durian arilus fermentation (jruek drien)

2019 ◽  
Vol 1 (1) ◽  
pp. 34-38
Author(s):  
Yulia Sari Ismail ◽  
◽  
Cut Yulvizar ◽  
Risa Riani Ramlan ◽  
Mahyuddin Mahyuddin ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Dna Isolation ◽  
Incubation Temperature ◽  
Fermented Food ◽  
Subtilis Strain ◽  
Rrna Gene ◽  
Dna Concentration ◽  
Purity Level

Jruek drien is an Acehnese traditional fermented food containing lactic acid bacteria (LAB) from durian arilus fermentation. Lactic acid bacteria from jruek drien were considered as potential probiotics. The LAB from jruek drien were first identified by phenotyping and genotyping analysis. The aim of the research is to isolate DNA from lactic acid bacteria (JD-2 and JD-3) from jruek drien for identification based on 16S rDNA gene sequence analysis. The research steps consist of samples regeneration in MRSA medium with temperature of incubation at 37°C, samples culturing in TSB medium, DNA isolation, and measurement of the DNA concentration and DNA purity using nanophotometer. The result showed that the JD-2 and the JD-3 isolates grow well on selective media MRSA by incubation temperature at 37°C. JD-2 has a DNA concentration of 40.0 ng/µl with a purity level of 2.0, and JD-3 has a DNA concentration of 32.5 ng/µl with a purity level of 1.8 at the A260/A280 ratio. The 16S rRNA gene of JD-2 isolate was successfully amplified at 1426 bp and JD-3 isolate at 1435 bp. The JD-2 isolate was identified as Bacillus subtilis because it has the highest similarity with Bacillus subtilis strain WA3-4. The JD-3 isolate was identified as Lactobacillus plantarum because it has the highest similarity with L. plantarum strain CSI9 and strain CSI3.

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 Influence of Turning and Environmental Contamination on the Dynamics of Populations of Lactic Acid and Acetic Acid Bacteria Involved in Spontaneous Cocoa Bean Heap Fermentation in Ghana

2007 ◽  
Vol 74 (1) ◽  
pp. 86-98 ◽  
Author(s):  
Nicholas Camu ◽  
Ángel González ◽  
Tom De Winter ◽  
Ann Van Schoor ◽  
Katrien De Bruyne ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Lactic Acid ◽  
Environmental Contamination ◽  
Acetic Acid Bacteria ◽  
Cocoa Bean ◽  
Relative Population ◽  
Acetobacter Pasteurianus ◽  
Culture Independent ◽  
Dynamics Of Populations ◽  
Culture Dependent

ABSTRACT The influence of turning and environmental contamination on six spontaneous cocoa bean heap fermentations performed in Ghana was studied through a multiphasic approach, encompassing both microbiological (culture-dependent and culture-independent techniques) and metabolite target analyses. A sensory analysis of chocolate made from the fermented, dried beans was performed as well. Only four clusters were found among the isolates of acetic acid bacteria (AAB) identified: Acetobacter pasteurianus, Acetobacter ghanensis, Acetobacter senegalensis, and a potential new Acetobacter lovaniensis-like species. Two main clusters were identified among the lactic acid bacteria (LAB) isolated, namely, Lactobacillus plantarum and Lactobacillus fermentum. No differences in biodiversity of LAB and AAB were seen for fermentations carried out at the farm and factory sites, indicating the cocoa pod surfaces and not the general environment as the main inoculum for spontaneous cocoa bean heap fermentation. Turning of the heaps enhanced aeration and increased the relative population size of AAB and the production of acetic acid. This in turn gave a more sour taste to chocolate made from these beans. Bitterness was reduced through losses of polyphenols and alkaloids upon fermentation and cocoa bean processing.

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

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12035
Author(s):  
Ankit Hinsu ◽  
Ashvin Dumadiya ◽  
Anjali Joshi ◽  
Rohitkumar Kotadiya ◽  
Kavan Andharia ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Rrna Gene ◽  
Sequence Variants ◽  
Similar Nature ◽  
Soil Diversity ◽  
16S Sequencing ◽  
Culture Independent ◽  
The Media ◽  
Ngs Data ◽  
Culture Dependent

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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