scholarly journals Molecular Characterization of Subject-Specific Oral Microflora during Initial Colonization of Enamel

2006 ◽  
Vol 72 (4) ◽  
pp. 2837-2848 ◽  
Author(s):  
Patricia I. Diaz ◽  
Natalia I. Chalmers ◽  
Alexander H. Rickard ◽  
Colin Kong ◽  
Craig L. Milburn ◽  
...  
Keyword(s):  
Community Composition ◽  
Bacterial Species ◽  
Microbial Community Composition ◽  
Microbial Colonization ◽  
Rrna Gene ◽  
Dependent Manner ◽  
Clone Libraries ◽  
Tooth Surfaces ◽  
Biofilm Development

ABSTRACT The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.

scholarly journals Benchmarking laboratory processes to characterise low-biomass respiratory microbiota

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raiza Hasrat ◽  
Jolanda Kool ◽  
Wouter A. A. de Steenhuijsen Piters ◽  
Mei Ling J. N. Chu ◽  
Sjoerd Kuiling ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Microbial Community Composition ◽  
Positive Control ◽  
Rrna Gene ◽  
Elution Buffer ◽  
Community Profile ◽  
Microbial Community Profile ◽  
Microbial Dna ◽  
Pcr Conditions

AbstractThe low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available laboratory protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community profile, we amplified the 16S rRNA gene of respiratory samples using various bacterial loads and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 or V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community profile of low biomass samples. Purification methods and MiSeq reagent kits provided nearly similar microbiota profiles (paired Bray–Curtis dissimilarity median: 0.03 and 0.05, respectively). While profiles of positive controls were significantly influenced by the type of dilution solvent, the theoretical profile of the Zymo mock was most accurately analysed when the Zymo mock was diluted in elution buffer (difference compared to the theoretical Zymo mock: 21.6% for elution buffer, 29.2% for Milli-Q, and 79.6% for DNA/RNA shield). Microbiota profiles of DNA blanks formed a distinct cluster compared to low biomass samples, demonstrating that low biomass samples can accurately be distinguished from DNA blanks. In summary, to accurately characterise the microbial community composition we recommend 1. amplification of the obtained microbial DNA with 30 PCR cycles, 2. purifying amplicon pools by two consecutive AMPure XP steps and 3. sequence the pooled amplicons by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.

scholarly journals Nanopore-Sequencing Characterization of the Gut Microbiota of Melolontha melolontha Larvae: Contribution to Protection against Entomopathogenic Nematodes?

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 396
Author(s):  
Ewa Sajnaga ◽  
Marcin Skowronek ◽  
Agnieszka Kalwasińska ◽  
Waldemar Kazimierczak ◽  
Karolina Ferenc ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Entomopathogenic Nematodes ◽  
Bacterial Species ◽  
Antagonistic Activity ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Bacterial Phyla ◽  
Melolontha Melolontha

This study focused on the potential relationships between midgut microbiota of the common cockchafer Melolontha melolontha larvae and their resistance to entomopathogenic nematodes (EPN) infection. We investigated the bacterial community associated with control and unsusceptible EPN-exposed insects through nanopore sequencing of the 16S rRNA gene. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the most abundant bacterial phyla within the complex and variable midgut microbiota of the wild M. melolontha larvae. The core microbiota was found to include 82 genera, which accounted for 3.4% of the total number of identified genera. The EPN-resistant larvae differed significantly from the control ones in the abundance of many genera belonging to the Actinomycetales, Rhizobiales, and Clostridiales orders. Additionally, the analysis of the microbiome networks revealed different sets of keystone midgut bacterial genera between these two groups of insects, indicating differences in the mutual interactions between bacteria. Finally, we detected Xenorhabdus and Photorhabdus as gut residents and various bacterial species exhibiting antagonistic activity against these entomopathogens. This study paves the way to further research aimed at unravelling the role of the host gut microbiota on the output of EPN infection, which may contribute to enhancement of the efficiency of nematodes used in eco-friendly pest management.

scholarly journals Do initial concentration and activated sludge seasonality affect pharmaceutical biotransformation rate constants?

2021 ◽  
Author(s):  
Tamara J. H. M. van Bergen ◽  
Ana B. Rios-Miguel ◽  
Tom M. Nolte ◽  
Ad M. J. Ragas ◽  
Rosalie van Zelm ◽  
...  
Keyword(s):  
Microbial Community ◽  
Activated Sludge ◽  
Community Composition ◽  
Rate Constants ◽  
Wastewater Treatment Plants ◽  
Microbial Community Composition ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Initial Concentration ◽  
Different Seasons

Abstract Pharmaceuticals find their way to the aquatic environment via wastewater treatment plants (WWTPs). Biotransformation plays an important role in mitigating environmental risks; however, a mechanistic understanding of involved processes is limited. The aim of this study was to evaluate potential relationships between first-order biotransformation rate constants (kb) of nine pharmaceuticals and initial concentration of the selected compounds, and sampling season of the used activated sludge inocula. Four-day bottle experiments were performed with activated sludge from WWTP Groesbeek (The Netherlands) of two different seasons, summer and winter, spiked with two environmentally relevant concentrations (3 and 30 nM) of pharmaceuticals. Concentrations of the compounds were measured by LC–MS/MS, microbial community composition was assessed by 16S rRNA gene amplicon sequencing, and kb values were calculated. The biodegradable pharmaceuticals were acetaminophen, metformin, metoprolol, terbutaline, and phenazone (ranked from high to low biotransformation rates). Carbamazepine, diatrizoic acid, diclofenac, and fluoxetine were not converted. Summer and winter inocula did not show significant differences in microbial community composition, but resulted in a slightly different kb for some pharmaceuticals. Likely microbial activity was responsible instead of community composition. In the same inoculum, different kb values were measured, depending on initial concentration. In general, biodegradable compounds had a higher kb when the initial concentration was higher. This demonstrates that Michealis-Menten kinetic theory has shortcomings for some pharmaceuticals at low, environmentally relevant concentrations and that the pharmaceutical concentration should be taken into account when measuring the kb in order to reliably predict the fate of pharmaceuticals in the WWTP. Key points • Biotransformation and sorption of pharmaceuticals were assessed in activated sludge. • Higher initial concentrations resulted in higher biotransformation rate constants for biodegradable pharmaceuticals. • Summer and winter inocula produced slightly different biotransformation rate constants although microbial community composition did not significantly change. Graphical abstract

scholarly journals Temporal Dynamics of Cloacal Microbiota in Adult Laying Chickens With and Without Access to an Outdoor Range

2021 ◽  
Vol 11 ◽  
Author(s):  
Janneke Schreuder ◽  
Francisca C. Velkers ◽  
Alex Bossers ◽  
Ruth J. Bouwstra ◽  
Willem F. de Boer ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Intestinal Microbiota ◽  
Environmental Changes ◽  
Temporal Dynamics ◽  
Microbial Community Composition ◽  
Sudden Change ◽  
Rrna Gene ◽  
Poultry House ◽  
Over Time

Associations between animal health and performance, and the host’s microbiota have been recently established. In poultry, changes in the intestinal microbiota have been linked to housing conditions and host development, but how the intestinal microbiota respond to environmental changes under farm conditions is less well understood. To gain insight into the microbial responses following a change in the host’s immediate environment, we monitored four indoor flocks of adult laying chickens three times over 16 weeks, during which two flocks were given access to an outdoor range, and two were kept indoors. To assess changes in the chickens’ microbiota over time, we collected cloacal swabs of 10 hens per flock and performed 16S rRNA gene amplicon sequencing. The poultry house (i.e., the stable in which flocks were housed) and sampling time explained 9.2 and 4.4% of the variation in the microbial community composition of the flocks, respectively. Remarkably, access to an outdoor range had no detectable effect on microbial community composition, the variability of microbiota among chickens of the same flock, or microbiota richness, but the microbiota of outdoor flocks became more even over time. Fluctuations in the composition of the microbiota over time within each poultry house were mainly driven by turnover in rare, rather than dominant, taxa and were unique for each flock. We identified 16 amplicon sequence variants that were differentially abundant over time between indoor and outdoor housed chickens, however none were consistently higher or lower across all chickens of one housing type over time. Our study shows that cloacal microbiota community composition in adult layers is stable following a sudden change in environment, and that temporal fluctuations are unique to each flock. By exploring microbiota of adult poultry flocks within commercial settings, our study sheds light on how the chickens’ immediate environment affects the microbiota composition.

Isolation and Characterization of Levoglucosan Metabolizing Bacteria

2021 ◽  
Author(s):  
Ajay S. Arya ◽  
Minh T. H. Hang ◽  
Mark A. Eiteman
Keyword(s):  
Recombinant Expression ◽  
Bacterial Species ◽  
Soluble Carbohydrate ◽  
Rrna Gene ◽  
Gene Products ◽  
16S Rrna Gene Sequences ◽  
E Coli ◽  
Isolation And Characterization ◽  
Charred Wood

Bacteria were isolated from wastewater and soil containing charred wood remnants based on their ability to use levoglucosan as a sole carbon source and on their levoglucosan dehydrogenase (LGDH) activity. On the basis of their 16S rRNA gene sequences, these bacteria represented diverse genera of Microbacterium, Paenibacillus , Shinella , and Klebsiella . Genomic sequencing of the isolates verified that two isolates represented novel species, Paenibacillus athensensis MEC069 T and Shinella sumterensis MEC087 T , while the remaining isolates were closely related to either Microbacterium lacusdiani or Klebsiella pneumoniae . The genetic sequence of LGDH, lgdA , was found in the genomes of these four isolates as well as Pseudarthrobacter phenanthrenivorans Sphe3. The identity of the P. phenanthrenivorans LGDH was experimentally verified following recombinant expression in E. coli . Comparison of the putative genes surrounding lgdA in the isolate genomes indicated that several other gene products facilitate the bacterial catabolism of levoglucosan, including a putative sugar isomerase and several transport proteins. Importance Levoglucosan is the most prevalent soluble carbohydrate remaining after high temperature pyrolysis of lignocellulosic biomass, but it is not fermented by typical production microbes such as Escherichia coli and Saccharomyces cerevisiae . A few fungi metabolize levoglucosan via the enzyme levoglucosan kinase, while several bacteria metabolize levoglucosan via levoglucosan dehydrogenase. This study describes the isolation and characterization of four bacterial species which degrade levoglucosan. Each isolate is shown to contain several genes within an operon involved in levoglucosan degradation, furthering our understanding of bacteria which metabolize levoglucosan.

scholarly journals Changes in the Active, Dead, and Dormant Microbial Community Structure across a Pleistocene Permafrost Chronosequence

2019 ◽  
Vol 85 (7) ◽  
Author(s):  
Alexander Burkert ◽  
Thomas A. Douglas ◽  
Mark P. Waldrop ◽  
Rachel Mackelprang
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Community Composition ◽  
Environmental Conditions ◽  
Microbial Community Composition ◽  
Rrna Gene ◽  
Subzero Temperatures ◽  
Dormant Cells

ABSTRACTPermafrost hosts a community of microorganisms that survive and reproduce for millennia despite extreme environmental conditions, such as water stress, subzero temperatures, high salinity, and low nutrient availability. Many studies focused on permafrost microbial community composition use DNA-based methods, such as metagenomics and 16S rRNA gene sequencing. However, these methods do not distinguish among active, dead, and dormant cells. This is of particular concern in ancient permafrost, where constant subzero temperatures preserve DNA from dead organisms and dormancy may be a common survival strategy. To circumvent this, we applied (i) LIVE/DEAD differential staining coupled with microscopy, (ii) endospore enrichment, and (iii) selective depletion of DNA from dead cells to permafrost microbial communities across a Pleistocene permafrost chronosequence (19,000, 27,000, and 33,000 years old). Cell counts and analysis of 16S rRNA gene amplicons from live, dead, and dormant cells revealed how communities differ between these pools, how they are influenced by soil physicochemical properties, and whether they change over geologic time. We found evidence that cells capable of forming endospores are not necessarily dormant and that members of the classBacilliwere more likely to form endospores in response to long-term stressors associated with permafrost environmental conditions than members of theClostridia, which were more likely to persist as vegetative cells in our older samples. We also found that removing exogenous “relic” DNA preserved within permafrost did not significantly alter microbial community composition. These results link the live, dead, and dormant microbial communities to physicochemical characteristics and provide insights into the survival of microbial communities in ancient permafrost.IMPORTANCEPermafrost soils store more than half of Earth’s soil carbon despite covering ∼15% of the land area (C. Tarnocai et al., Global Biogeochem Cycles 23:GB2023, 2009, https://doi.org/10.1029/2008GB003327). This permafrost carbon is rapidly degraded following a thaw (E. A. G. Schuur et al., Nature 520:171–179, 2015, https://doi.org/10.1038/nature14338). Understanding microbial communities in permafrost will contribute to the knowledge base necessary to understand the rates and forms of permafrost C and N cycling postthaw. Permafrost is also an analog for frozen extraterrestrial environments, and evidence of viable organisms in ancient permafrost is of interest to those searching for potential life on distant worlds. If we can identify strategies microbial communities utilize to survive in permafrost, it may yield insights into how life (if it exists) survives in frozen environments outside of Earth. Our work is significant because it contributes to an understanding of how microbial life adapts and survives in the extreme environmental conditions in permafrost terrains.

scholarly journals Characterization of the SOS Regulon of Caulobacter crescentus

2007 ◽  
Vol 190 (4) ◽  
pp. 1209-1218 ◽  
Author(s):  
Raquel Paes da Rocha ◽  
Apuã César de Miranda Paquola ◽  
Marilis do Valle Marques ◽  
Carlos Frederico Martins Menck ◽  
Rodrigo S. Galhardo
Keyword(s):  
Dna Damage ◽  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Direct Repeat ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Wild Type ◽  
Promoter Regions ◽  
Potential Promoter

ABSTRACT The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN7GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.

scholarly journals Comparative Analysis of Eukaryotic Marine Microbial Assemblages from 18S rRNA Gene and Gene Transcript Clone Libraries by Using Different Methods of Extraction

2012 ◽  
Vol 78 (11) ◽  
pp. 3958-3965 ◽  
Author(s):  
Amy Koid ◽  
William C. Nelson ◽  
Amy Mraz ◽  
Karla B. Heidelberg
Keyword(s):  
Community Composition ◽  
18S Rrna ◽  
Northwest Pacific ◽  
Rrna Gene ◽  
Gene Transcript ◽  
Acid Extraction ◽  
Phylogenetic Groups ◽  
Northwest Pacific Ocean ◽  
Clone Libraries ◽  
Sequencing Platform

ABSTRACTEukaryotic marine microbes play pivotal roles in biogeochemical nutrient cycling and ecosystem function, but studies that focus on the protistan biogeography and genetic diversity lag-behind studies of other microbes. 18S rRNA PCR amplification and clone library sequencing are commonly used to assess diversity that is culture independent. However, molecular methods are not without potential biases and artifacts. In this study, we compare the community composition of clone libraries generated from the same water sample collected at the San Pedro Ocean Time Series (SPOTs) station in the northwest Pacific Ocean. Community composition was assessed using different cell lysis methods (chemical and mechanical) and the extraction of different nucleic acids (DNA and RNA reverse transcribed to cDNA) to build Sanger ABI clone libraries. We describe specific biases for ecologically important phylogenetic groups resulting from differences in nucleic acid extraction methods that will inform future designs of eukaryotic diversity studies, regardless of the target sequencing platform planned.

Current state of knowledge: the canine gastrointestinal microbiome

2012 ◽  
Vol 13 (1) ◽  
pp. 78-88 ◽  
Author(s):  
Seema Hooda ◽  
Yasushi Minamoto ◽  
Jan S. Suchodolski ◽  
Kelly S. Swanson
Keyword(s):  
Bacterial Species ◽  
Rrna Gene ◽  
Dietary Interventions ◽  
Health Indices ◽  
Recent Emergence ◽  
Spatial Differences ◽  
Bowel Diseases ◽  
Health And Disease ◽  
Cultivation Techniques

AbstractGastrointestinal (GI) microbes have important roles in the nutritional, immunological, and physiologic processes of the host. Traditional cultivation techniques have revealed bacterial density ranges from 104to 105colony forming units (CFU)/g in the stomach, from 105to 107CFU/g in the small intestine, and from 109to 1011CFU/g in the colon of healthy dogs. As a small number of bacterial species can be grown and studied in culture, however, progress was limited until the recent emergence of DNA-based techniques. In recent years, DNA sequencing technology and bioinformatics have allowed for better phylogenetic and functional/metabolic characterization of the canine gut microbiome. Predominant phyla include Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, and Actinobacteria. Studies using 16S ribosomal RNA (rRNA) gene pyrosequencing have demonstrated spatial differences along the GI tract and among microbes adhered to the GI mucosa compared to those in intestinal contents or feces. Similar to humans, GI microbiome dysbiosis is common in canine GI diseases such as chronic diarrhea and inflammatory bowel diseases. DNA-based assays have also identified key pathogens contributing to such conditions, including variousClostridium,Campylobacter,Salmonella, andEscherichiaspp. Moreover, nutritionists have applied DNA-based techniques to study the effects of dietary interventions such as dietary fiber, prebiotics, and probiotics on the canine GI microbiome and associated health indices. Despite recent advances in the field, the canine GI microbiome is far from being fully characterized and a deeper characterization of the phylogenetic and functional/metabolic capacity of the GI microbiome in health and disease is needed. This paper provides an overview of recent studies performed to characterize the canine GI microbiome.

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