scholarly journals Bacterial Contamination of Plant in vitro Cultures in Commercial Production Detected by High‑Throughput Amplicon Sequencing

2019 ◽  
Vol 67 (4) ◽  
pp. 1005-1014
Author(s):  
Dorota Tekielska ◽  
Eliška Peňázová ◽  
Tamás Kovács ◽  
Břetislav Křižan ◽  
Jana Čechová ◽  
...  
Keyword(s):  
High Throughput ◽  
Bacterial Contamination ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Commercial Laboratory ◽  
Three Samples ◽  
Plant Tissue Cultures ◽  
The 16S Rrna Gene

The study overviews results of bacterial incidence in in vitro plant tissue cultures obtained from commercial laboratory dealing with plants micropropagation. For the exploration, the 454 pyrosequencing of the 16S rRNA gene was used. Three samples of plant in vitro cultures with visual bacterial contamination were subjected for identification of present bacteria. Eleven genera as Acinetobacter, Lactobacillus, Methylobacterium, Roseomonas, Microbacterium, Mycobacterium, Curtobacterium, Acidovorax, Magnetospirillum, Chryseobacterium and Ralstonia were detected. Obtained results confirmed the advantages of high‑throughput amplicon sequencing analysis for identification of bacterial communities in contaminated plant in vitro cultures what provides an important information for applying appropriate measures to eliminate bacterial contamination.

scholarly journals Changes in the Bacterial Diversity of Human Milk during Late Lactation Period (Weeks 21 to 48)

Foods ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1184
Author(s):  
Wendy Marin-Gómez ◽  
Mᵃ José Grande ◽  
Rubén Pérez-Pulido ◽  
Antonio Galvez ◽  
Rosario Lucas
Keyword(s):  
Bacterial Diversity ◽  
Single Mother ◽  
Variable Region ◽  
Sampling Period ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Lactation Period ◽  
Relative Abundances ◽  
The 16S Rrna Gene

Breast milk from a single mother was collected during a 28-week lactation period. Bacterial diversity was studied by amplicon sequencing analysis of the V3-V4 variable region of the 16S rRNA gene. Firmicutes and Proteobacteria were the main phyla detected in the milk samples, followed by Actinobacteria and Bacteroidetes. The proportion of Firmicutes to Proteobacteria changed considerably depending on the sampling week. A total of 411 genera or higher taxons were detected in the set of samples. Genus Streptococcus was detected during the 28-week sampling period, at relative abundances between 2.0% and 68.8%, and it was the most abundant group in 14 of the samples. Carnobacterium and Lactobacillus had low relative abundances. At the genus level, bacterial diversity changed considerably at certain weeks within the studied period. The weeks or periods with lowest relative abundance of Streptococcus had more diverse bacterial compositions including genera belonging to Proteobacteria that were poorly represented in the rest of the samples.

scholarly journals rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

2019 ◽  
Author(s):  
Jean-Claude OGIER ◽  
Sylvie Pagès ◽  
Maxime Galan ◽  
Matthieu Barret ◽  
Sophie Gaudriault
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Taxonomic Resolution ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Specificity And Sensitivity ◽  
The 16S Rrna Gene ◽  
Mock Communities

Abstract Background Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene. Results We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii. Conclusions Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

scholarly journals rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

2019 ◽  
Author(s):  
Jean-Claude OGIER ◽  
Sylvie Pagès ◽  
Maxime Galan ◽  
Matthieu Barret ◽  
Sophie Gaudriault
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Taxonomic Resolution ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Specificity And Sensitivity ◽  
The 16S Rrna Gene ◽  
Mock Communities

Abstract Background Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene. Results We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii. Conclusions Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

scholarly journals rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

2019 ◽  
Author(s):  
Jean-Claude Ogier ◽  
Sylvie Pagès ◽  
Maxime Galan ◽  
Mathieu Barret ◽  
Sophie Gaudriault
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Taxonomic Resolution ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Specificity And Sensitivity ◽  
The 16S Rrna Gene ◽  
Mock Communities

AbstractBackgroundMicrobiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene.ResultsWe first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii.ConclusionsOur results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

scholarly journals The microbial community of the gut differs between piglets fed sow milk, milk replacer or bovine colostrum

2017 ◽  
Vol 117 (7) ◽  
pp. 964-978 ◽  
Author(s):  
Ann-Sofie R. Poulsen ◽  
Nadieh de Jonge ◽  
Sugiharto Sugiharto ◽  
Jeppe L. Nielsen ◽  
Charlotte Lauridsen ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Amplicon Sequencing ◽  
Bovine Colostrum ◽  
Milk Replacer ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Faecal Samples ◽  
Milk Replacers ◽  
The 16S Rrna Gene ◽  
Gut Microbiota Composition

AbstractThe aim of this study was to characterise the gut microbiota composition of piglets fed bovine colostrum (BC), milk replacer (MR) or sow milk (SM) in the post-weaning period. Piglets (n36), 23-d old, were randomly allocated to the three diets. Faecal samples were collected at 23, 25, 27 and 30 d of age. Digesta from the stomach, ileum, caecum and mid-colon was collected at 30 d of age. Bacterial DNA from all samples was subjected to amplicon sequencing of the 16S rRNA gene. Bacterial enumerations by culture and SCFA analysis were conducted as well. BC-piglets had the highest abundance ofLactococcusin the stomach (P<0·0001) and ileal (P<0·0001) digesta, whereas SM-piglets had the highest abundance ofLactobacillusin the stomach digesta (P<0·0001). MR-piglets had a high abundance of Enterobacteriaceae in the ileal digesta (P<0·0001) and a higher number of haemolytic bacteria in ileal (P=0·0002) and mid-colon (P=0·001) digesta than SM-piglets. BC-piglets showed the highest colonic concentration of iso-butyric and iso-valeric acid (P=0·02). Sequencing and culture showed that MR-piglets were colonised by a higher number of Enterobacteriaceae, whereas the gut microbiota of BC-piglets was characterised by a change in lactic acid bacteria genera when compared with SM-piglets. We conclude that especially the ileal microbiota of BC-piglets had a closer resemblance to that of SM-piglets in regard to the abundance of potential enteric pathogens than did MR-piglets. The results indicate that BC may be a useful substitute for regular milk replacers, and as a feeding supplement in the immediate post-weaning period.

scholarly journals A core microbiota dominates a rich microbial diversity in the bovine udder and may indicate presence of dysbiosis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Davide Porcellato ◽  
Roger Meisal ◽  
Alberto Bombelli ◽  
Judith A. Narvhus
Keyword(s):  
Microbial Diversity ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Lactation Period ◽  
Strong Negative Correlation ◽  
Udder Health ◽  
Milk Samples ◽  
Lactating Cows ◽  
Definition Of ◽  
The 16S Rrna Gene

AbstractThe importance of the microbiome for bovine udder health is not well explored and most of the knowledge originates from research on mastitis. Better understanding of the microbial diversity inside the healthy udder of lactating cows might help to reduce mastitis, use of antibiotics and improve animal welfare. In this study, we investigated the microbial diversity of over 400 quarter milk samples from 60 cows sampled from two farms and on two different occasions during the same lactation period. Microbiota analysis was performed using amplicon sequencing of the 16S rRNA gene and over 1000 isolates were identified using MALDI-TOF MS. We detected a high abundance of two bacterial families, Corynebacteriaceae and Staphylococcaceae, which accounted for almost 50% of the udder microbiota of healthy cows and were detected in all the cow udders and in more than 98% of quarter milk samples. A strong negative correlation between these bacterial families was detected indicating a possible competition. The overall composition of the udder microbiota was highly diverse and significantly different between cows and between quarter milk samples from the same cow. Furthermore, we introduced a novel definition of a dysbiotic quarter at individual cow level, by analyzing the milk microbiota, and a high frequency of dysbiotic quarter samples were detected distributed among the farms and the samples. These results emphasize the importance of deepening the studies of the bovine udder microbiome to elucidate its role in udder health.

scholarly journals High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

2019 ◽  
Vol 47 (18) ◽  
pp. e103-e103 ◽  
Author(s):  
Benjamin J Callahan ◽  
Joan Wong ◽  
Cheryl Heiner ◽  
Steve Oh ◽  
Casey M Theriot ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Full Length ◽  
Rrna Gene ◽  
Single Nucleotide ◽  
Full Complement ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

scholarly journals The Microbial Communities of Leaves and Roots Associated with Turtle Grass (Thalassia testudinum) and Manatee Grass (Syringodium filliforme) are Distinct from Seawater and Sediment Communities, but Are Similar between Species and Sampling Sites

2018 ◽  
Vol 7 (1) ◽  
pp. 4 ◽  
Author(s):  
Kelly Ugarelli ◽  
Peeter Laas ◽  
Ulrich Stingl
Keyword(s):  
Carbon Sink ◽  
Amplicon Sequencing ◽  
Thalassia Testudinum ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Carbon Cycles ◽  
Syringodium Filiforme ◽  
Turtle Grass ◽  
Shoreline Protection ◽  
The 16S Rrna Gene

Seagrasses are vital members of coastal systems, which provide several important ecosystem services such as improvement of water quality, shoreline protection, and serving as shelter, food, and nursery to many species, including economically important fish. They also act as a major carbon sink and supply copious amounts of oxygen to the ocean. A decline in seagrasses has been observed worldwide, partly due to climate change, direct and indirect human activities, diseases, and increased sulfide concentrations in the coastal porewaters. Several studies have shown a symbiotic relationship between seagrasses and their microbiome. For instance, the sulfur, nitrogen, and carbon cycles are important biochemical pathways that seem to be linked between the plant and its microbiome. The microbiome presumably also plays a key role in the health of the plant, for example in oxidizing phyto-toxic sulfide into non-toxic sulfate, or by providing protection for seagrasses from pathogens. Two of the most abundant seagrasses in Florida include Thalassia testudinum (turtle grass) and Syringodium filliforme (manatee grass), yet there is little data on the composition of the microbiome of these two genera. In this study, the microbial composition of the phyllosphere and rhizosphere of Thalassia testudinum and Syringodium filiforme were compared to water and sediment controls using amplicon sequencing of the V4 region of the 16S rRNA gene. The microbial composition of the leaves, roots, seawater, and sediment differ from one another, but are similar between the two species of seagrasses.

scholarly journals First Case Report of Fatal Sepsis Due to Campylobacter upsaliensis

2014 ◽  
Vol 53 (2) ◽  
pp. 713-715 ◽  
Author(s):  
Itaru Nakamura ◽  
Nami Omori ◽  
Ayaka Umeda ◽  
Kiyofumi Ohkusu ◽  
Tetsuya Matsumoto
Keyword(s):  
Case Report ◽  
Rare Case ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Content Type ◽  
First Case ◽  
Campylobacter Upsaliensis ◽  
Fatal Sepsis ◽  
The 16S Rrna Gene

We encountered a rare case of severe fatal infection in a 70-year-old woman due toCampylobacter upsaliensis, identified by PCR amplification and sequencing analysis of the 16S rRNA gene using DNA extracted from the isolates. To our knowledge, fatal sepsis due to this organism has never been described to date.

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