KAJIAN MOLEKULER PADA PROBIOTIK ASAL AIR SUSU IBU DALAM SINTESIS EKSOPOLISAKARIDA (EPS)

Molecular Studies on Probiotic of Human Breast Milk in the Synthesis of Exopolysaccharide (EPS) The glucosyltransferase (gtf) gene has an important role in exopolysaccharide (EPS) synthesis in probiotic bacteria. The EPS produced is associated with the adhesion ability of bacteria to the intestinal mucosa. Therefore, the gtf gene can be used as a parameter in the selection of potential probiotic through a molecular approach. This study was conducted to determine the presence of the gtf gene in probiotic from human breast milk using PCR technique. The methods in this study include the following: reculture of probiotic isolates, DNA isolation, amplification of the 16S rRNA gene using universal primers (pA and pB), amplification of specific LAB primers (LABfw and LABrv), specific primary design for the gtf gene, and the amplification of the gtf gene. The results of 16S rRNA gene amplification using universal primers obtained the amplicons of 500-1,000 bp in size. The results of amplification using specific LAB primers obtained an amplicon of about 700 bp in all isolates. The results of amplification of the gtf gene using a specific primer produced an amplicon of 325 bp in all isolates. Based on this study, it was concluded that 16 probiotic isolates from human breast milk were proven to have the gtf gene. Gen glukosiltransferase (gtf) memiliki peran penting dalam sintesis eksopolisakarida (EPS) pada bakteri probiotik. EPS yang diproduksi berhubungan dengan kemampuan adhesi bakteri pada mukosa usus. Oleh karena itu, gen gtf dapat dijadikan sebagai salah satu parameter dalam seleksi probiotik potensial melalui pendekatan molekuler. Penelitian ini dilakukan untuk mengetahui adanya gen gtf pada probiotik asal air susu ibu (ASI) menggunakan teknik PCR. Metode pada penelitian ini meliputi: reculture isolat probiotik, isolasi DNA, amplifikasi gen 16S rRNA menggunakan primer universal (pA dan pB), amplifikasi primer spesifik BAL (LABfw dan LABrv), desain primer spesifik untuk gen gtf dan amplifikasi gen gtf. Hasil amplifikasi gen 16S rRNA menggunakan primer universal diperoleh amplikon berukuran antara 500-1.000 bp. Adapun hasil amplifikasi menggunakan primer spesifik BAL diperoleh amplikon berukuran sekitar 700 bp pada seluruh isolat. Hasil amplifikasi gen gtf menggunakan primer spesifik menghasilkan amplikon berukuran sekitar 325 bp pada seluruh isolat. Berdasarkan penelitian ini dapat disimpulkan bahwa 16 isolat probiotik asal ASI terbukti memiliki gen gtf.

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Identification and Assessment of Genetic Similarity of Soil Bacterial Isolates of Pseudomonas spp. Using Molecular Techniques

Polish Journal of Microbiology ◽

10.33073/pjm-2014-039 ◽

2014 ◽

Vol 63 (3) ◽

pp. 291-298

Author(s):

ANNA LISEK ◽

LIDIA SAS PASZ ◽

PAWEŁ TRZCIŃSKI

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Genetic Similarity ◽

Sour Cherry ◽

23S Rrna ◽

Rrna Gene ◽

Bacterial Isolates ◽

Bacterial Strains ◽

The 16S Rrna Gene ◽

Selection Of

Bacteria of the genus Pseudomonas are often components of bioproducts designed to enhance the condition of the soil and plants. The use of Pseudomonas bacteria in bioproducts must be preceded by the acquisition, characterization and selection of beneficial strains living in the soil. A prerequisite for the selection of bacterial strains for use in bioproducts is to be able to identify the isolates rapidly and accurately. To identify and differentiate 15 bacterial isolates obtained from the soil surrounding the roots of sour cherry trees and to assess their genetic similarity, the rep-PCR technique and restriction analysis of the 16S rRNA gene and the 16S-ITS-23S rRNA operon were used. In addition, a sequence analysis of the 16S rRNA gene was performed. The analyses made it possible to divide the isolates into four clusters and to confirm their affiliation with the Pseudomonas species. RFLP analysis of the 16S-ITS-23S rRNA operon enabled greater differentiation of the isolates than RFLP of the 16S rRNA gene. The greatest differentiation of isolates within the clusters was obtained after using the rep-PCR technique. However, none of the techniques was able to discriminate all the isolates, which indicates very high genetic similarity of the Pseudomonas isolates found in the same sample of soil from around the roots of sour cherry trees. The tests performed will find application for distinguishing and identifying Pseudomonas strains collected from the soil in order to select the most valuable bacterial strains that produce beneficial effects on plants.

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Usefulness of rpoB Gene Sequencing for Identification of Afipia and Bosea Species, Including a Strategy for Choosing Discriminative Partial Sequences

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6740-6749.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6740-6749 ◽

Cited By ~ 27

Author(s):

Atieh Khamis ◽

Philippe Colson ◽

Didier Raoult ◽

Bernard La Scola

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Housekeeping Genes ◽

Rpob Gene ◽

Universal Primers ◽

Strain Identification ◽

Rrna Gene ◽

Dna Relatedness ◽

The 16S Rrna Gene

ABSTRACT Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability “hot spots” in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.

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Cultivable bacterial diversity of the canine dental plaque as a potential source of bacterial infections

Acta Veterinaria Brno ◽

10.2754/avb202190020171 ◽

2021 ◽

Vol 90 (2) ◽

pp. 171-178

Author(s):

Marián Maďar ◽

Jana Kačírová ◽

Aladár Maďari ◽

Rastislav Mucha ◽

Eva Styková ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dental Plaque ◽

Pathogenic Bacteria ◽

Companion Animals ◽

Oral Bacteria ◽

Universal Primers ◽

Rrna Gene ◽

Persistent Problem ◽

The 16S Rrna Gene

Dental diseases are a persistent problem, not only in humans, but very often in companion animals as well. Aetiological agents of these diseases are the dental plaque bacteria. In the present study, we focused on identifying cultivable bacteria living in the dental plaque of dogs, specifically dogs suffering from the early stages of periodontal disease. Canine oral bacteria pose a risk to humans that get bitten by the dog, but they also have a zoonotic potential. Dental plaque samples were taken from five dogs of small breeds. Samples were cultured under aerobic and anaerobic conditions on several types of microbiological agars. All obtained and selected bacterial colonies were identified by PCR with universal primers for the 16S rRNA gene and the sequences of the 16S rRNA gene were compared with the sequences available in the GenBank database using BLASTn analysis. A total of 75 bacteria belonging to five phyla, predominantly to Firmicutes and Proteobacteria, were identified. The most frequent species was Pasteurella canis which was detected in all samples. In addition, representatives of the genera Actinomyces, Bacillus, Bacteroides, Corynebacterium, Frederiksenia, Fusobacterium, Haemophilus, Lactobacillus, Leucobacter, Neisseria, Ottowia, Porphyromonas, Pseudomonas, Staphylococcus, Stenotrophomonas and Streptococcus were detected in the samples. In the present study, a broad spectrum of bacteria in dental plaque samples, including canine periodontal pathogens such as Porphyromonas gulae or Porphyromonas macacae were identified. In addition, highly pathogenic bacteria, specifically Actinomyces hordeovulneris, Bacillus circulans, and Bacteroides pyogenes, which pose a serious risk to human health, were detected in samples.

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Study of Microbiocenosis of Canine Dental Biofilms

10.21203/rs.3.rs-698153/v1 ◽

2021 ◽

Author(s):

Jana Kačírová ◽

Aladár Maďari ◽

Rastislav Mucha ◽

Lívia K. Fecskeová ◽

Izabela Mujakic ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Companion Animals ◽

Amplicon Sequencing ◽

Universal Primers ◽

Rrna Gene ◽

Bacterial Phyla ◽

Dental Biofilm ◽

Solid Media ◽

The 16S Rrna Gene

Abstract Dental biofilm is a complex microbial community influenced by many exogenous and endogenous factors. Despite long-term studies, its bacterial composition is still not clearly understood. While most of the research on dental biofilms was conducted in humans, much less information is available from companion animals. In this study, we analyzed the composition of canine dental biofilms using both standard cultivation on solid media and amplicon sequencing, and compared the two approaches. The 16S rRNA gene sequences were used to define the bacterial community of canine dental biofilm with both, culture-dependent and culture-independent methods. After DNA extraction from each sample, the V3-V4 region of the 16S rRNA gene was amplified and sequenced via Illumina MiSeq platform. Isolated bacteria were identified using universal primers and Sanger sequencing. Representatives of 18 bacterial genera belonging to 5 phyla were isolated from solid media. Amplicon sequencing largely expanded this information identifying in total 284 OTUs belonging to 10 bacterial phyla. Amplicon sequencing revealed much higher diversity of bacteria in the canine dental biofilms, when compared to standard cultivation approach. In contrast, cultured representatives of several bacterial families were not identified by amplicon sequencing.

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Study of microbiocenosis of canine dental biofilms

Scientific Reports ◽

10.1038/s41598-021-99342-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jana Kačírová ◽

Aladár Maďari ◽

Rastislav Mucha ◽

Lívia K. Fecskeová ◽

Izabela Mujakic ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Companion Animals ◽

Amplicon Sequencing ◽

Universal Primers ◽

Rrna Gene ◽

Bacterial Phyla ◽

Dental Biofilm ◽

Solid Media ◽

The 16S Rrna Gene

AbstractDental biofilm is a complex microbial community influenced by many exogenous and endogenous factors. Despite long-term studies, its bacterial composition is still not clearly understood. While most of the research on dental biofilms was conducted in humans, much less information is available from companion animals. In this study, we analyzed the composition of canine dental biofilms using both standard cultivation on solid media and amplicon sequencing, and compared the two approaches. The 16S rRNA gene sequences were used to define the bacterial community of canine dental biofilm with both, culture-dependent and culture-independent methods. After DNA extraction from each sample, the V3–V4 region of the 16S rRNA gene was amplified and sequenced via Illumina MiSeq platform. Isolated bacteria were identified using universal primers and Sanger sequencing. Representatives of 18 bacterial genera belonging to 5 phyla were isolated from solid media. Amplicon sequencing largely expanded this information identifying in total 284 operational taxonomic units belonging to 10 bacterial phyla. Amplicon sequencing revealed much higher diversity of bacteria in the canine dental biofilms, when compared to standard cultivation approach. In contrast, cultured representatives of several bacterial families were not identified by amplicon sequencing.

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How conserved are the conserved 16S-rRNA regions?

PeerJ ◽

10.7717/peerj.3036 ◽

2017 ◽

Vol 5 ◽

pp. e3036 ◽

Cited By ~ 21

Author(s):

Marcel Martinez-Porchas ◽

Enrique Villalpando-Canchola ◽

Luis Enrique Ortiz Suarez ◽

Francisco Vargas-Albores

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Frequency Analysis ◽

Universal Primers ◽

Rrna Gene ◽

Conserved Regions ◽

Hypervariable Regions ◽

Prokaryotic Diversity ◽

Gene Data ◽

The 16S Rrna Gene

The 16S rRNA gene has been used as master key for studying prokaryotic diversity in almost every environment. Despite the claim of several researchers to have the best universal primers, the reality is that no primer has been demonstrated to be truly universal. This suggests that conserved regions of the gene may not be as conserved as expected. The aim of this study was to evaluate the conservation degree of the so-called conserved regions flanking the hypervariable regions of the 16S rRNA gene. Data contained in SILVA database (release 123) were used for the study. Primers reported as matches of each conserved region were assembled to form contigs; sequences sizing 12 nucleotides (12-mers) were extracted from these contigs and searched into the entire set of SILVA sequences. Frequency analysis shown that extreme regions, 1 and 10, registered the lowest frequencies. 12-mer frequencies revealed segments of contigs that were not as conserved as expected (≤90%). Fragments corresponding to the primer contigs 3, 4, 5b and 6a were recovered from all sequences in SILVA database. Nucleotide frequency analysis in each consensus demonstrated that only a small fraction of these so-called conserved regions is truly conserved in non-redundant sequences. It could be concluded that conserved regions of the 16S rRNA gene exhibit considerable variation that has to be considered when using this gene as biomarker.

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Faculty Opinions recommendation of Low Maternal Microbiota Sharing across Gut, Breast Milk and Vagina, as Revealed by 16S rRNA Gene and Reduced Metagenomic Sequencing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.733207420.793548246 ◽

2018 ◽

Author(s):

Gregor Reid

Keyword(s):

Breast Milk ◽

16S Rrna ◽

16S Rrna Gene ◽

Rrna Gene ◽

Metagenomic Sequencing

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Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.052993-0 ◽

2014 ◽

Vol 64 (Pt_3) ◽

pp. 781-786 ◽

Cited By ~ 33

Author(s):

Maximo Sánchez ◽

Martha-Helena Ramírez-Bahena ◽

Alvaro Peix ◽

María J. Lorite ◽

Juan Sanjuán ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Sequence Similarity ◽

Carbon Sources ◽

Lotus Corniculatus ◽

Culture Conditions ◽

Rrna Gene ◽

Content Type ◽

Link Type ◽

The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

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