scholarly journals Bacterial Microbiota in Unfed Ticks (Dermacentor nuttalli) From Xinjiang Detected Through 16S rDNA Amplicon Sequencing and Culturomics

Zoonoses ◽  
2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Kai Song ◽  
Yuxin Ji ◽  
Surong Sun ◽  
Xihong Yue ◽  
Cheng Wang ◽  
...  
Keyword(s):  
16S Rdna ◽  
Bacterial Species ◽  
Amplicon Sequencing ◽  
Zoonotic Diseases ◽  
Comprehensive Understanding ◽  
Bacterial Microbiota ◽  
Western Tianshan ◽  
Rdna Sequencing ◽  
16S Rdna Amplicon Sequencing ◽  
Acinetobacter Pittii

Background: Ticks are a major arthropod vector of zoonotic diseases affecting both humans and domestic animals worldwide. Thus, studying tick microbiota would aid in understanding of the potential threats posed by ticks. Methods: Approximately 8,000 unfed ticks, identified as Dermacentor nuttalli, were collected from the sylvosteppe in the western Tianshan mountains. To investigate their potential pathogens, we divided the ticks into 36 groups of 200–300 individuals each for examination with culturomics and 16S rDNA amplicon sequencing. Results: A total of 237 bacterial genera were identified with the two methods. Culturomics identified 46 bacterial species from 23 genera, predominantly Pseudomonas, Pantoea, and Bacillus, whereas 16S rDNA sequencing identified 461 OTUs from 233 genera, predominantly Pseudomonas (53.8%), Coxiella (17.2%), and Pantoea (6.4%). Coxiella, Rickettsia, and ten other genera were discovered only by sequencing, because optimal cultivating conditions were not used for their isolation, whereas Arthrobacter and three other genera were discovered only through culturomics. Conclusions: Several of the identified bacteria, such as line-related sepsis-causing Delftia acidovorans and the pneumonia agent Acinetobacter pittii, can cause human diseases. Thus, both sequencing and culturomics methods are crucial for comprehensive understanding of the microbiota of D. nuttalli.

scholarly journals Cave Drip Water-Related Samples as a Natural Environment for Aromatic Hydrocarbon-Degrading Bacteria

2019 ◽  
Vol 7 (2) ◽  
pp. 33 ◽  
Author(s):  
Eric Marques ◽  
Gislaine Silva ◽  
João Dias ◽  
Eduardo Gross ◽  
Moara Costa ◽  
...  
Keyword(s):  
Aromatic Hydrocarbon ◽  
Microbial Communities ◽  
16S Rdna ◽  
Aromatic Compounds ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Amplicon Sequencing ◽  
Drip Water ◽  
Rdna Sequencing ◽  
Degrading Bacteria ◽  
Cave Drip Water

Restricted contact with the external environment has allowed the development of microbial communities adapted to the oligotrophy of caves. However, nutrients can be transported to caves by drip water and affect the microbial communities inside the cave. To evaluate the influence of aromatic compounds carried by drip water on the microbial community, two limestone caves were selected in Brazil. Drip-water-saturated and unsaturated sediment, and dripping water itself, were collected from each cave and bacterial 16S rDNA amplicon sequencing and denaturing gradient gel electrophoresis (DGGE) of naphthalene dioxygenase (ndo) genes were performed. Energy-dispersive X-ray spectroscopy (EDX) and atomic absorption spectroscopy (AAS) were performed to evaluate inorganic nutrients, and GC was performed to estimate aromatic compounds in the samples. The high frequency of Sphingomonadaceae in drip water samples indicates the presence of aromatic hydrocarbon-degrading bacteria. This finding was consistent with the detection of naphthalene and acenaphthene and the presence of ndo genes in drip-water-related samples. The aromatic compounds, aromatic hydrocarbon-degrading bacteria and 16S rDNA sequencing indicate that aromatic compounds may be one of the sources of energy and carbon to the system and the drip-water-associated bacterial community contains several potentially aromatic hydrocarbon-degrading bacteria. To the best of our knowledge, this is the first work to present compelling evidence for the presence of aromatic hydrocarbon-degrading bacteria in cave drip water.

Comparative analysis of microbiome between accurately identified 16S rDNA and quantified bacteria in simulated samples

2014 ◽  
Vol 63 (3) ◽  
pp. 433-440 ◽  
Author(s):  
Haiyin Wang ◽  
Pengcheng Du ◽  
Juan Li ◽  
Yuanyuan Zhang ◽  
Wen Zhang ◽  
...  
Keyword(s):  
Microbial Communities ◽  
16S Rdna ◽  
Gold Standard ◽  
Clinical Utility ◽  
Bacterial Species ◽  
Relative Proportion ◽  
Rrna Gene ◽  
Low Concentrations ◽  
Rdna Sequencing ◽  
V3 Region

Although 16S rRNA gene (rDNA) sequencing is the gold standard for categorizing bacteria or characterizing microbial communities its clinical utility is limited by bias in metagenomic studies, in either the experiments or the data analyses. To evaluate the efficiency of current metagenomic methods, we sequenced seven simulated samples of ten bacterial species mixed at different concentrations. The V3 region of 16S rDNA was targeted and used to determine the distribution of bacterial species. The number of target sequences in individual simulated samples was in the range 1–1000 to provide a better reflection of natural microbial communities. However, for a given bacterial species present in the same proportion but at different concentrations, the observed percentage of 16S rDNAs was similar, except at very low concentrations that cannot be detected by real-time PCR. These results confirmed that the comparative microbiome in a sample characterized by 16S rDNA sequencing is sufficient to detect not only potential infectious pathogens, but also the relative proportion of 16S rDNA in the sample.

scholarly journals Composition and Diversity of the Ocular Surface Microbiota in Patients With Blepharitis in Northwestern China

2021 ◽  
Vol 8 ◽  
Author(s):  
Changhao Wang ◽  
Xiuhong Dou ◽  
Jian Li ◽  
Jie Wu ◽  
Yan Cheng ◽  
...  
Keyword(s):  
16S Rdna ◽  
Relative Abundance ◽  
Ocular Surface ◽  
Amplicon Sequencing ◽  
Northwestern China ◽  
Healthy Control Group ◽  
Control Group ◽  
Eyelid Margin ◽  
16S Rdna Amplicon Sequencing ◽  
Healthy Control

Purpose: To investigate the composition and diversity of the microbiota on the ocular surface of patients with blepharitis in northwestern China via 16S rDNA amplicon sequencing.Methods: Thirty-seven patients with blepharitis divided into groups of anterior, posterior and mixed blepharitis and twenty healthy controls from northwestern China were enrolled in the study. Samples were collected from the eyelid margin and conjunctival sac of each participant. The V3–V4 region of bacterial 16S rDNA in each sample was amplified and sequenced on the Illumina HiSeq 2500 sequencing platform, and the differences in taxonomy and diversity among different groups were compared.Results: The composition of the ocular surface microbiota of patients with blepharitis was similar to that of healthy subjects, but there were differences in the relative abundance of each bacterium. At the phylum level, the abundances of Actinobacteria, Cyanobacteria, Verrucomicrobia, Acidobacteria, Chloroflexi, and Atribacteria were significantly higher in the blepharitis group than in the healthy control group, while the relative abundance of Firmicutes was significantly lower (p < 0.05, Mann-Whitney U). At the genus level, the abundances of Lactobacillus, Ralstonia, Bacteroides, Akkermansia, Bifidobacterium, Escherichia-Shigella, Faecalibacterium, and Brevibacterium were significantly higher in the blepharitis group than in the healthy control group, while the relative abundances of Bacillus, Staphylococcus, Streptococcus, and Acinetobacter were significantly lower in the blepharitis group (p < 0.05, Mann-Whitney U). The microbiota of anterior blepharitis was similar to that of mixed blepharitis but different from that of posterior blepharitis. Lactobacillus and Bifidobacterium are biomarkers of posterior blepharitis, and Ralstonia is a biomarker of mixed blepharitis. There was no significant difference in the ocular surface microbiota between the eyelid margin and conjunctival sac with or without blepharitis.Conclusion: The ocular surface microbiota of patients with blepharitis varied among different study groups, according to 16S rDNA amplicon sequencing analysis. The reason might be due to the participants being from different environments and having different lifestyles. Lactobacillus, Bifidobacterium, Akkermansia, Ralstonia, and Bacteroides may play important roles in the pathogenesis of blepharitis.

scholarly journals Metagenomic Analysis of Slovak Bryndza Cheese Using Next-Generation 16S rDNA Amplicon Sequencing

2016 ◽  
Vol 15 (1) ◽  
pp. 23-34 ◽  
Author(s):  
Matej Planý ◽  
Tomáš Kuchta ◽  
Katarína Šoltýs ◽  
Tomáš Szemes ◽  
Domenico Pangallo ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rdna ◽  
Lactococcus Lactis ◽  
Microbial Population ◽  
Starter Culture ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Next Generation ◽  
16S Rdna Amplicon Sequencing

Abstract Knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. Aim of this study was to investigate microbial consortia composition in Slovak bryndza cheese. For this purpose, we used culture-independent approach based on 16S rDNA amplicon sequencing using next generation sequencing platform. Results obtained by the analysis of three commercial (produced on industrial scale in winter season) and one traditional (artisanal, most valued, produced in May) Slovak bryndza cheese sample were compared. A diverse prokaryotic microflora composed mostly of the genera Lactococcus, Streptococcus, Lactobacillus, and Enterococcus was identified. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. Second most abundant species, detected in all bryndza cheeses, were Lactococcus fujiensis and Lactococcus taiwanensis, independently by two different approaches, using different reference 16S rRNA genes databases (Greengenes and NCBI respectively). They have been detected in bryndza cheese samples in substantial amount for the first time. The narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. Metagenomic analysis by high-throughput sequencing using 16S rRNA genes seems to be a powerful tool for studying the structure of the microbial population in cheeses.

scholarly journals Technical note: overcoming host contamination in bovine vaginal metagenomic samples with Nanopore adaptive sequencing

2021 ◽  
Author(s):  
Chian Teng Ong ◽  
Elizabeth M Ross ◽  
Gry B Boe-Hansen ◽  
Conny Turni ◽  
Ben J Hayes ◽  
...  
Keyword(s):  
16S Rdna ◽  
Sequence Data ◽  
Amplicon Sequencing ◽  
Technical Note ◽  
Metagenomic Data ◽  
High Coverage ◽  
Long Reads ◽  
Oxford Nanopore ◽  
16S Rdna Amplicon Sequencing ◽  
Functional Profiles

Abstract Animal metagenomic studies, in which host-associated microbiomes are profiled, are an increasingly important contribution to our understanding of the physiological functions, health and susceptibility to diseases of livestock. One of the major challenges in these studies is host DNA contamination, which limits the sequencing capacity for metagenomic content and reduces the accuracy of metagenomic profiling. This is the first study comparing the effectiveness of different sequencing methods for profiling bovine vaginal metagenomic samples. We compared the new method of Oxford Nanopore Technologies (ONT) adaptive sequencing, which can be used to target or eliminate defined genetic sequences, to standard ONT sequencing, Illumina 16S rDNA amplicon sequencing, and Illumina shotgun sequencing. The efficiency of each method in recovering the metagenomic data and recalling the metagenomic profiles was assessed. ONT adaptive sequencing yielded a higher amount of metagenomic data than the other methods per 1 Gb of sequence data. The increased sequencing efficiency of ONT adaptive sequencing consequently reduced the amount of raw data needed to provide sufficient coverage for the metagenomic samples with high host-to-microbe DNA ratio. Additionally, the long reads generated by ONT adaptive sequencing retained the continuity of read information, which benefited the in-depth annotations for both taxonomical and functional profiles of the metagenome. The different methods resulted in the identification of different taxa. Genera Clostridium, which was identified at low abundances and categorised under Order “Unclassified Clostridiales” when using the 16S rDNA amplicon sequencing method, was identified to be the dominant genera in the sample when sequenced with the three other methods. Additionally, higher numbers of annotated genes were identified with ONT adaptive sequencing, which also produced high coverage on most of the commonly annotated genes. This study illustrates the advantages of ONT adaptive sequencing in improving the amount of metagenomic data derived from microbiome samples with high host-to-microbe DNA ratio and the advantage of long reads in preserving intact information for accurate annotations.

scholarly journals Sediment-associated microbial community profiling: sample pre-processing through sequential membrane filtration for 16s rDNA amplicon sequencing

2020 ◽  
Author(s):  
Joeselle M. Serrana ◽  
Kozo Watanabe
Keyword(s):  
Microbial Community ◽  
16S Rdna ◽  
Community Composition ◽  
Membrane Filtration ◽  
Amplicon Sequencing ◽  
Sediment Samples ◽  
Community Profiling ◽  
Significant Difference ◽  
16S Rdna Amplicon Sequencing ◽  
Read Abundance

ABSTRACTSequential membrane filtration as a pre-processing step for the isolation of microorganisms could provide good quality and integrity DNA that can be preserved and kept at ambient temperatures before community profiling through culture-independent molecular techniques, e.g., 16s rDNA amplicon sequencing. Here, we assessed the impact of pre-processing sediment samples by sequential membrane filtration (from 10, 5 to 0.22 μm pore size membrane filters) for 16s rDNA-based community profiling of sediment-associated microorganisms. Specifically, we examined if there would be method-driven differences between non- and pre-processed sediment samples regarding the quality and quantity of extracted DNA, PCR amplicon, resulting high-throughput sequencing reads, microbial diversity, and community composition. We found no significant difference in the quality and quantity of extracted DNA and PCR amplicons between the two methods. Although we found a significant difference in raw and quality-filtered reads, read abundance after bioinformatics processing (i.e., denoising and the chimeric-read filtering steps) were not significantly different. These results suggest that read abundance after these read processing steps were not influenced by sediment processing or lack thereof. Although the non- and pre-processed sediment samples had more unique than shared amplicon sequence variants (ASVs), we report that their shared ASVs accounted for 74% of both methods’ absolute read abundance. More so at the genus level, the final collection filter identified most of the genera (95% of the reads) captured from the non-processed samples, with a total of 51 false-negative (2%) and 59 false-positive genera (3%). Accordingly, the diversity estimates and community composition were not significantly different between the non- and pre-processed samples. We demonstrate that while there were differences in shared and unique taxa, both methods revealed comparable microbial diversity and community composition. We also suggest the inclusion of sequential filters (i.e., pre- and mid-filters) in the community profiling, given the additional taxa not detected from the non-processed and the final collection filter. Our observations highlight the feasibility of pre-processing sediment samples for community analysis and the need to further assess sampling strategies to help conceptualize appropriate study designs for sediment-associated microbial community profiling.

scholarly journals Comparing 16S rDNA amplicon sequencing and hybridization capture for pea aphid microbiota diversity analysis

2018 ◽  
Vol 11 (1) ◽  
Author(s):  
Marie Cariou ◽  
Céline Ribière ◽  
Stéphanie Morlière ◽  
Jean-Pierre Gauthier ◽  
Jean-Christophe Simon ◽  
...  
Keyword(s):  
16S Rdna ◽  
Amplicon Sequencing ◽  
Pea Aphid ◽  
Diversity Analysis ◽  
Hybridization Capture ◽  
16S Rdna Amplicon Sequencing ◽  
Microbiota Diversity

scholarly journals Approaches for diversity analysis of cultivable and non-cultivable bacteriain real soil

2009 ◽  
Vol 55 (No. 9) ◽  
pp. 389-396 ◽  
Author(s):  
P. Štursa ◽  
O. Uhlík ◽  
V. Kurzawová ◽  
J. Koubek ◽  
M. Ionescu ◽  
...  
Keyword(s):  
16S Rdna ◽  
Molecular Phylogenetics ◽  
Bacterial Species ◽  
Pseudomonas Stutzeri ◽  
Population Based ◽  
Phylogenetic Relations ◽  
Advantages And Disadvantages ◽  
Active Population ◽  
Cultivable Bacteria ◽  
Rdna Sequencing

Until recently, investigators had no idea how accurately cultivated microorganisms represented the overall microbial diversity. The cultivation-dependent approach is limited by the fact that the overwhelming majority of microorganisms present in soil cannot be cultivated under laboratory conditions. The development of molecular phylogenetics has recently enabled characterization of naturally occurring microbial biota without cultivation. There is a vast amount of information held within the genomes of cultivable and non-cultivable microorganisms, and new methods based on analysis of DNA allow to investigate this potential. In this work we show some aspects, advantages and disadvantages of classical and new approaches in taxonomical and functional description of bacteria present in natural microbial assemblages on the example of cultivable bacteria isolated from rhizosphere of plants, tobacco and black nightshade, planted in PCB contaminated soil. Biochemical analysis of isolates showed 8 different bacterial species. This identification was compared by discrimination using MALDI-TOF mass spectrometry and identity evaluation after sequencing of 16S rDNA. Six strains from original number of 8 were positively identified after 16S rDNA sequencing and their phylogenetic relations were compared. These analyses confirmed closed relations of all species (two of isolates exhibited the same characteristics and were discriminated as the same species <I>Pseudomonas stutzeri</I>) and also of <I>Burkholderia xenovorans</I> LB 400, a well-known PCB degrader. Nevertheless, only two isolates gave a positive reaction after amplification of the biphenyl dioxygenase gene and exhibited potential to degrade PCB. These results indicate that only a subset of the recovered molecular information, derived from active population based on molecular and functional analysis is relevant to microbial ecology.

scholarly journals Infection of theBiomphalaria glabrata vector snailbySchistosoma mansoniparasites drives snail microbiota dysbiosis

2018 ◽  
Author(s):  
Anaïs Portet ◽  
Eve Toulza ◽  
Ana Lokmer ◽  
Camille Huot ◽  
David Duval ◽  
...  
Keyword(s):  
Control Strategies ◽  
Amplicon Sequencing ◽  
Biomphalaria Glabrata ◽  
Individual Level ◽  
Host Parasite Interactions ◽  
Bacterial Microbiota ◽  
Rnaseq Data ◽  
16S Rdna Amplicon Sequencing ◽  
The Individual ◽  
Host Parasite

SummaryHost-associated microbiota cari affect the fitness of its host i η a number of ways, including the modification of host-parasite interactions and thus the outcome of disease.Biomphalaria glabratais the vector snail of the trematodeSchistosoma mansoni,the agent of human schistosomiasis, causing hundreds of thousands of deaths every year. Here, we present the first study of the snail bacterial microbiota in response toSchistosomainfection. To examine the interplay betweenB. glabrata, S. mansoniand snail microbiota, snails were infected and the microbiota composition was analysed by massive 16S rDNA amplicon sequencing approach. We characterized theBiomphalariabacterial microbiota at the individual level in both naive and infected snails. Sympatric and allopatric strains of parasites were used for infections and re­infections to analyse the modification or dysbiosis of snail microbiota in different host-parasite co-evolutionary contexts. Concomitantly, using RNAseq data, we investigated the link between bacterial microbiota dysbiosis and snail anti-microbial peptide immune response. This work paves the way for a better understanding of snail/schistosome interaction, and would have critical consequences in terms of snail control strategies for fighting schistosomiasis disease in the field.

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