Impact of enzymatic digestion on bacterial community composition in CF airway samples

BackgroundPrevious studies have demonstrated the importance of DNA extraction methods for molecular detection ofStaphylococcus,an important bacterial group in cystic fibrosis (CF). We sought to evaluate the effect of enzymatic digestion (EnzD) prior to DNA extraction on bacterial communities identified in sputum and oropharyngeal swab (OP) samples from patients with CF.MethodsDNA from 81 samples (39 sputum and 42 OP) collected from 63 patients with CF was extracted in duplicate with and without EnzD. Bacterial communities were determined by rRNA gene sequencing, and measures of alpha and beta diversity were calculated. Principal Coordinate Analysis (PCoA) was used to assess differences at the community level and Wilcoxon Signed Rank tests were used to compare relative abundance (RA) of individual genera for paired samples with and without EnzD.ResultsShannon Diversity Index (alpha-diversity) decreased in sputum and OP samples with the use of EnzD. Larger shifts in community composition were observed for OP samples (beta-diversity, measured by Morisita-Horn), whereas less change in communities was observed for sputum samples. The use of EnzD with OP swabs resulted in significant increase in RA for the generaGemella(p < 0.01),Streptococcus(p < 0.01), andRothia(p < 0.01).Staphylococcus(p < 0.01) was the only genus with a significant increase in RA from sputum, whereas the following genera decreased in RA with EnzD:Veillonella(p < 0.01),Granulicatella(p < 0.01),Prevotella(p < 0.01), andGemella(p = 0.02). In OP samples, higher RA of Gram-positive taxa was associated with larger changes in microbial community composition.DiscussionWe show that the application of EnzD to CF airway samples, particularly OP swabs, results in differences in microbial communities detected by sequencing. Use of EnzD can result in large changes in bacterial community composition, and is particularly useful for detection ofStaphylococcusin CF OP samples. The enhanced identification ofStaphylococcus aureusis a strong indication to utilize EnzD in studies that use OP swabs to monitor CF airway communities.

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Vaginal bacterial community composition and concentrations of estradiol at the time of artificial insemination in Brangus heifers

Journal of Animal Science ◽

10.1093/jas/skaa178 ◽

2020 ◽

Vol 98 (6) ◽

Cited By ~ 1

Author(s):

Riley D Messman ◽

Zully E Contreras-Correa ◽

Henry A Paz ◽

George Perry ◽

Caleb O Lemley

Keyword(s):

Bacterial Community ◽

Community Composition ◽

Artificial Insemination ◽

Beta Diversity ◽

Bacterial Community Composition ◽

Alpha Diversity ◽

Species Level ◽

Rrna Gene ◽

Microbial Composition ◽

Vaginal Tract

Abstract The knowledge surrounding the bovine vaginal microbiota and its implications on fertility and reproductive traits remains incomplete. The objective of the current study was to characterize the bovine vaginal bacterial community and estradiol concentrations at the time of artificial insemination (AI). Brangus heifers (n = 78) underwent a 7-d Co-Synch + controlled internal drug release estrus synchronization protocol. At AI, a double-guarded uterine culture swab was used to sample the anterior vaginal tract. Immediately after swabbing the vaginal tract, blood samples were collected by coccygeal venipuncture to determine concentrations of estradiol. Heifers were retrospectively classified as pregnant (n = 29) vs. nonpregnant (n = 49) between 41 and 57 d post-AI. Additionally, heifers were classified into low (1.1 to 2.5 pg/mL; n = 21), medium (2.6 to 6.7 pg/mL; n = 30), and high (7.2 to 17.6 pg/mL; n = 27) concentration of estradiol. The vaginal bacterial community composition was determined through sequencing of the V4 region from the 16S rRNA gene using the Illumina Miseq platform. Alpha diversity was compared via ANOVA and beta diversity was compared via PERMANOVA. There were no differences in the Shannon diversity index (alpha diversity; P = 0.336) or Bray–Curtis dissimilarity (beta diversity; P = 0.744) of pregnant vs. nonpregnant heifers. Overall, bacterial community composition in heifers with high, medium, or low concentrations of estradiol did not differ (P = 0.512). While no overall compositional differences were observed, species-level differences were present within pregnancy status and estradiol concentration groups. The implications of these species-level differences are unknown, but these differences could alter the vaginal environment thereby influencing fertility and vaginal health. Therefore, species-level changes could provide better insight rather than overall microbial composition in relation to an animal’s reproductive health.

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Quantitative and qualitative evaluation of the impact of the G2 enhancer, bead sizes and lysing tubes on the bacterial community composition during DNA extraction from recalcitrant soil core samples based on community sequencing and qPCR

10.1101/365395 ◽

2018 ◽

Cited By ~ 1

Author(s):

Alex Gobbi ◽

Rui G. Santini ◽

Elisa Filippi ◽

Lea Ellegaard- Jensen ◽

Carsten S. Jacobsen ◽

...

Keyword(s):

Bacterial Community ◽

Dna Extraction ◽

Community Composition ◽

Bacterial Community Composition ◽

Microbial Community Composition ◽

Soil Core ◽

Soil Matrix ◽

Soil Dna ◽

Dna Recovery ◽

The Impact

AbstractSoil DNA extraction encounters numerous challenges that can affect both yield and purity of the recovered DNA. Clay particles lead to reduced DNA extraction efficiency, and PCR inhibitors from the soil matrix can negatively affect downstream analyses when applying DNA sequencing. Further, these effects impede molecular analysis of bacterial community compositions in lower biomass samples, as often observed in deeper soil layers. Many studies avoid these complications by using indirect DNA extraction with prior separation of the cells from the matrix, but such methods introduce other biases that influence the resulting microbial community composition.To address these issues, a direct DNA extraction method was applied in combination with the use of a commercial product, the G2 DNA/RNA Enhancer®, marketed as being capable of improving the amount of DNA recovered after the lysis step. The results showed that application of G2 increased DNA yields from the studied clayey soils from layers between 1.00 and 2.20 m below ground level.Importantly, the use of G2 did not introduce bias, as it did not result in any significant differences in the biodiversity of the bacterial community measured in terms of alpha and beta diversity and taxonomical composition.Finally, this study considered a set of customised lysing tubes for evaluating possible influences on the DNA yield. Tubes customization included different bead sizes and amounts, along with lysing tubes coming from two suppliers. Results showed that the lysing tubes with mixed beads allowed greater DNA recovery compared to the use of either 0.1 or 1.4 mm beads, irrespective of the tube supplier.These outcomes may help to improve commercial products in DNA/RNA extraction kits, besides raising awareness about the optimal choice of additives, offering opportunities for acquiring a better understanding of topics such as vertical microbial characterisation and environmental DNA recovery in low biomass samples.

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Effects of geographic location and water quality on bacterial communities in full-scale biofilters across North America

FEMS Microbiology Ecology ◽

10.1093/femsec/fiz210 ◽

2020 ◽

Vol 96 (2) ◽

Cited By ~ 1

Author(s):

Ben Ma ◽

Timothy M LaPara ◽

Ashley N. Evans ◽

Raymond M Hozalski

Keyword(s):

Water Quality ◽

North America ◽

Bacterial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Community Composition ◽

Bacterial Communities ◽

Bacterial Community Composition ◽

Geographic Location ◽

Rrna Gene

ABSTRACT Spatial patterns of bacterial community composition often follow a distance–decay relationship in which community dissimilarity increases with geographic distance. Such a relationship has been commonly observed in natural environments, but less so in engineered environments. In this study, bacterial abundance and community composition in filter media samples (n = 57) from full-scale rapid biofilters at 14 water treatment facilities across North America were determined using quantitative polymerase chain reaction and Illumina HiSeq high-throughput sequencing targeting the 16S rRNA gene, respectively. Bacteria were abundant on the filter media (108.8±0.3 to 1010.7±0.2 16S rRNA gene copies/cm3 bed volume) and the bacterial communities were highly diverse (Shannon index: 5.3 ± 0.1 to 8.4 ± 0.0). Significant inter-filter variations in bacterial community composition were observed, with weighted UniFrac dissimilarity values following a weak but highly significant distance–decay relationship (z = 0.0057 ± 0.0006; P = 1.8 × 10−22). Approximately 50% of the variance in bacterial community composition was explained by the water quality parameters measured at the time of media sample collection (i.e. pH, temperature and dissolved organic carbon concentration). Overall, this study suggested that the microbiomes of biofilters are primarily shaped by geographic location and local water quality conditions but the influence of these factors on the microbiomes is tempered by filter design and operating conditions.

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Rapid, inexpensive measurement of synthetic bacterial community composition by Sanger sequencing

10.1101/313932 ◽

2018 ◽

Author(s):

Nathan Cermak ◽

Manoshi Sen Datta ◽

Arolyn Conwill

Keyword(s):

Bacterial Community ◽

Microbial Ecology ◽

Community Composition ◽

Bacterial Communities ◽

Sanger Sequencing ◽

Bacterial Community Composition ◽

Marker Gene ◽

Compositional Analysis ◽

Time Variability ◽

Rrna Gene

AbstractSimple synthetic bacterial communities are powerful tools for studying microbial ecology and evolution, as they enable rapid iteration between controlled laboratory experiments and theoretical modeling. However, their utility is hampered by the lack of fast, inexpensive, and accurate methods for quantifying bacterial community composition. For instance, while next-generation amplicon sequencing can be very accurate, high costs (>$30 per sample) and turnaround times (>1 month) limit the nature and pace of experiments. Here, we introduce a new approach for quantifying composition in synthetic bacterial communities based on Sanger sequencing. First, for a given community, we PCR-amplify a universal marker gene (here, the 16S rRNA gene), which yields a mixture of amplicons. Second, we sequence this amplicon mixture in a single Sanger sequencing reaction, which produces a “mixed” electropherogram with contributions from each community member. We also sequence each community member’s marker gene individually to generate “individual” electropherograms. Third, we fit the mixed electropherogram as a linear combination of time-warped individual electropherograms, thereby allowing us to estimate the fractional amplicon abundance of each strain within the community. Importantly, our approach accounts for retention-time variability in electrophoretic signals, which is crucial for accurate compositional estimates. Using synthetic communities of marine bacterial isolates, we show that this approach yields accurate and reproducible abundance estimates for two-, four-, and seven-strain bacterial communities. Furthermore, this approach can provide results within one day and costs ~$5 USD per sample. We envision this approach will enable new insights in microbial ecology by increasing the number of samples that can be analyzed and enabling faster iteration between experiments and theory. We have implemented our method in a free and open-source R package called CASEU (“Compositional Analysis by Sanger Electropherogram Unmixing”), available at https://bitbucket.org/DattaManoshi/caseu.

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Bacterial community composition and diversity of two different forms of an organic residue of bioenergy crop

PeerJ ◽

10.7717/peerj.6768 ◽

2019 ◽

Vol 7 ◽

pp. e6768

Author(s):

Matheus A.P. Cipriano ◽

Afnan K.A. Suleiman ◽

Adriana P.D. da Silveira ◽

Janaína B. do Carmo ◽

Eiko E. Kuramae

Keyword(s):

Microbial Community ◽

Bacterial Community ◽

16S Rrna ◽

Community Composition ◽

Bacterial Community Composition ◽

Microbial Community Composition ◽

Nitrous Oxide Emissions ◽

Rrna Gene ◽

Bacterial Strains ◽

Microbial Composition

The use of residue of sugarcane ethanol industry named vinasse in fertirrigation is an established and widespread practice in Brazil. Both non-concentrated vinasse (NCV) and concentrated vinasse (CV) are used in fertirrigation, particularly to replace the potassium fertilizer. Although studies on the chemical and organic composition of vinasse and their impact on nitrous oxide emissions when applied in soil have been carried out, no studies have evaluated the microbial community composition and diversity in different forms of vinasse. We assessed the bacterial community composition of NCV and CV by non-culturable and culturable approaches. The non-culturable bacterial community was assessed by next generation sequencing of the 16S rRNA gene and culturable community by isolation of bacterial strains and molecular and biochemical characterization. Additionally, we assessed in the bacterial strains the presence of genes of nitrogen cycle nitrification and denitrification pathways. The microbial community based on16S rRNAsequences of NCV was overrepresented by Bacilli and Negativicutes while CV was mainly represented by Bacilli class. The isolated strains from the two types of vinasse belong to class Bacilli, similar toLysinibacillus, encode fornirKgene related to denitrification pathway. This study highlights the bacterial microbial composition particularly in CV what residue is currently recycled and recommended as a sustainable practice in sugarcane cultivation in the tropics.

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22 Comparison of vaginal microbiome and concentrations of estradiol at artificial insemination in Brangus heifers

Journal of Animal Science ◽

10.1093/jas/skz397.095 ◽

2020 ◽

Vol 98 (Supplement_2) ◽

pp. 41-42

Author(s):

Riley D Messman ◽

Zully Contreras-Correa ◽

Henry A Paz ◽

George Perry ◽

Caleb O Lemley

Keyword(s):

Bacterial Community ◽

Community Composition ◽

Artificial Insemination ◽

Diversity Index ◽

Bacterial Community Composition ◽

Principal Component ◽

Alpha Diversity ◽

Illumina Miseq ◽

Rrna Gene ◽

Vaginal Microbiome

Abstract The knowledge surrounding the bovine vaginal microbiome and its implications on fertility and reproductive traits remains incomplete. The objective of the current study was to characterize the bovine vaginal microbiome and estradiol concentrations at time of artificial insemination (AI). Brangus heifers (n = 78) underwent a 7-d Co-Synch + CIDR estrus synchronization protocol. At AI, a double guarded uterine culture swab was used to sample the anterior vaginal tract. Blood samples were collected by coccygeal venipuncture to determine concentrations of estradiol. Heifers were retrospectively classified as pregnant (n = 29) versus nonpregnant (n = 49) on day 35. Lastly, heifers were classified into low (1.1 - 2.5 pg/ml; n = 21), medium (2.6 - 6.7 pg/ml; n = 30), and high (7.2 - 17.6 pg/ml; n = 27) concentrations of estradiol. The vaginal bacterial community composition was determined through sequencing of the V4-V5 region from the 16S rRNA gene using the Illumina Miseq platform. ANOVA was used to compare the diversity metrics between treatment groups. PERMANOVA was utilized to determine variation in community structure. There were no statistical differences in the Shannon diversity index (alpha diversity; P = 0.336) or principal component analysis (beta diversity; P = 0.744) of pregnant versus nonpregnant animals. The vaginal microbiome of pregnant and nonpregnant animals was similar with the four most abundant phyla being Tenericutes, Proteobacteria, Fusobacteria, and Firmicutes. Overall bacterial community composition in animals with high, medium, or low concentrations of estradiol did not differ (P = 0.512). These results indicate that concentration of estradiol does not impact vaginal microbiome composition. In conclusion, the composition of the bovine vaginal microbiome, although dynamic, may not be directly linked to an animal’s reproductive ability.

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23 Comparison of vaginal microbiome and concentrations of estradiol at artificial insemination in Brangus heifers

Journal of Animal Science ◽

10.1093/jas/skz397.032 ◽

2020 ◽

Vol 98 (Supplement_2) ◽

pp. 14-15

Author(s):

Riley D Messman ◽

Zully Contreras-Correa ◽

Henry A Paz ◽

George Perry ◽

Caleb O Lemley

Keyword(s):

Bacterial Community ◽

Community Composition ◽

Artificial Insemination ◽

Diversity Index ◽

Bacterial Community Composition ◽

Principal Component ◽

Alpha Diversity ◽

Illumina Miseq ◽

Rrna Gene ◽

Vaginal Microbiome

Abstract The knowledge surrounding the bovine vaginal microbiome and its implications on fertility and reproductive traits remains incomplete. The objective of the current study was to characterize the bovine vaginal microbiome and estradiol concentrations at time of artificial insemination (AI). Brangus heifers (n = 78) underwent a 7-day Co-Synch + CIDR estrus synchronization protocol. At AI, a double guarded uterine culture swab was used to sample the anterior vaginal tract. Blood samples were collected by coccygeal venipuncture to determine concentrations of estradiol. Heifers were retrospectively classified as pregnant (n = 29) versus nonpregnant (n = 49) on day 35. Lastly, heifers were classified into low (1.1 - 2.5 pg/ml; n = 21), medium (2.6 - 6.7 pg/ml; n = 30), and high (7.2 - 17.6 pg/ml; n = 27) concentrations of estradiol. The vaginal bacterial community composition was determined through sequencing of the V4-V5 region from the 16S rRNA gene using the Illumina Miseq platform. ANOVA was used to compare the diversity metrics between treatment groups. PERMANOVA was utilized to determine variation in community structure. There were no statistical differences in the Shannon diversity index (alpha diversity; P = 0.336) or principal component analysis (beta diversity; P = 0.744) of pregnant versus nonpregnant animals. The vaginal microbiome of pregnant and nonpregnant animals was similar with the four most abundant phyla being Tenericutes, Proteobacteria, Fusobacteria, and Firmicutes. Overall bacterial community composition in animals with high, medium, or low concentrations of estradiol did not differ (P = 0.512). These results indicate that concentration of estradiol does not impact vaginal microbiome composition. In conclusion, the composition of the bovine vaginal microbiome, although dynamic, may not be directly linked to an animal’s reproductive ability.

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Mercury and Other Heavy Metals Influence Bacterial Community Structure in Contaminated Tennessee Streams

Applied and Environmental Microbiology ◽

10.1128/aem.01715-10 ◽

2010 ◽

Vol 77 (1) ◽

pp. 302-311 ◽

Cited By ~ 93

Author(s):

Tatiana A. Vishnivetskaya ◽

Jennifer J. Mosher ◽

Anthony V. Palumbo ◽

Zamin K. Yang ◽

Mircea Podar ◽

...

Keyword(s):

Community Structure ◽

Microbial Community ◽

Bacterial Community ◽

Community Composition ◽

Bacterial Community Structure ◽

Bacterial Community Composition ◽

Microbial Community Composition ◽

Inorganic Mercury ◽

Rrna Gene ◽

Oak Ridge

ABSTRACTHigh concentrations of uranium, inorganic mercury [Hg(II)], and methylmercury (MeHg) have been detected in streams located in the Department of Energy reservation in Oak Ridge, TN. To determine the potential effects of the surface water contamination on the microbial community composition, surface stream sediments were collected 7 times during the year, from 5 contaminated locations and 1 control stream. Fifty-nine samples were analyzed for bacterial community composition and geochemistry. Community characterization was based on GS 454 FLX pyrosequencing with 235 Mb of 16S rRNA gene sequence targeting the V4 region. Sorting and filtering of the raw reads resulted in 588,699 high-quality sequences with lengths of >200 bp. The bacterial community consisted of 23 phyla, includingProteobacteria(ranging from 22.9 to 58.5% per sample),Cyanobacteria(0.2 to 32.0%),Acidobacteria(1.6 to 30.6%),Verrucomicrobia(3.4 to 31.0%), and unclassified bacteria. Redundancy analysis indicated no significant differences in the bacterial community structure between midchannel and near-bank samples. Significant correlations were found between the bacterial community and seasonal as well as geochemical factors. Furthermore, several community members within theProteobacteriagroup that includes sulfate-reducing bacteria and within theVerrucomicrobiagroup appeared to be associated positively with Hg and MeHg. This study is the first to indicate an influence of MeHg on thein situmicrobial community and suggests possible roles of these bacteria in the Hg/MeHg cycle.

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Mucus composition and bacterial communities associated with the tissue and skeleton of three scleractinian corals maintained under culture conditions

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s002531541000130x ◽

2010 ◽

Vol 91 (3) ◽

pp. 649-657 ◽

Cited By ~ 25

Author(s):

Pascale Tremblay ◽

Markus G. Weinbauer ◽

Cécile Rottier ◽

Yann Guérardel ◽

Christian Nozais ◽

...

Keyword(s):

Bacterial Community ◽

Community Composition ◽

Bacterial Communities ◽

Coral Species ◽

Denaturing Gradient Gel Electrophoresis ◽

Close Association ◽

Bacterial Community Composition ◽

Microbial Community Composition ◽

Coral Tissue ◽

Species Specific

Corals live in close association with bacterial communities, but the nature of the relationship is still poorly understood. In this study, three scleractinian coral species,Galaxea fascicularis, Pavona cactusandTurbinaria reniformiswere incubated under different laboratory conditions, and the composition of the bacterial community associated with their tissue or skeleton was compared between species or between species and seawater using denaturing gradient gel electrophoresis (DGGE). The amount of dissolved organic carbon (DOC) excreted and the mucus glycoconjugate composition were also determined for each species. The aim of the study was to assess if the bacterial community composition was species-specific or linked either to the seawater composition, or to the quality and quantity of carbon released by each coral. Results obtained showed that DOC release was significantly different (P< 0.0001) for the three species, with the highest excretion rate forG. fascicularis. Also, the mucus ofG. fascicularisandP. cactusmainly contained galactose and glucose whereas the mucus ofT. reniformiscontained more glucose and xylose. Cluster analyses of microbial community composition showed that the bacterial community was species-specific in the coral tissue but not in the skeleton, in all conditions. It remained specific when corals were incubated in the same or in different aquaria, and under different seawater renewal rates. Since DOC release rates and bacterial composition were both different according to the coral species considered, a link might be suggested between the two parameters. Sequencing of DGGE bands indicated that some bacterial phylotypes were consistently retrieved in all samples of a given species.

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The bacterial community composition and its environmental drivers in the rivers around eutrophic Chaohu Lake, China

BMC Microbiology ◽

10.1186/s12866-021-02252-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Keqiang Shao ◽

Xin Yao ◽

Zhaoshi Wu ◽

Xingyu Jiang ◽

Yang Hu ◽

...

Keyword(s):

Bacterial Community ◽

Community Composition ◽

Bacterial Communities ◽

Bacterial Community Composition ◽

Cyanobacterial Blooms ◽

Environmental Drivers ◽

Chaohu Lake ◽

Rrna Gene ◽

Functional Profiles ◽

16 S Rrna

Abstract Background Bacterial community play a key role in environmental and ecological processes in river ecosystems. Rivers are used as receiving body for treated and untreated urban wastewaters that brings high loads of sewage and excrement bacteria. However, little is known about the bacterial community structure and functional files in the rivers around the eutrophic Chaohu Lake, the fifth largest freshwater lake in China, has been subjected to severe eutrophication and cyanobacterial blooms over the past few decades. Therefore, understanding the taxonomic and functional compositions of bacterial communities in the river will contribute to understanding aquatic microbial ecology. The main aims were to (1) examine the structure of bacterial communities and functional profiles in this system; (2) find the environmental factors of bacterial community variations. Results We studied 88 sites at rivers in the Chaohu Lake basin, and determined bacterial communities using Illumina Miseq sequencing of the 16 S rRNA gene, and predicted functional profiles using PICRUSt2. A total of 3,390,497 bacterial 16 S rRNA gene sequences were obtained, representing 17 phyla, and 424 genera; The dominant phyla present in all samples were Bacteroidetes (1.4-82.50 %), followed by Proteobacteria (12.6–97.30 %), Actinobacteria (0.1–17.20 %). Flavobacterium was the most numerous genera, and accounted for 0.12–80.34 % of assigned 16 S reads, followed by Acinetobacter (0.33–49.28 %). Other dominant bacterial genera including Massilia (0.06–25.40 %), Psychrobacter (0-36.23 %), Chryseobacterium (0.01–22.86 %), Brevundimonas (0.01–12.82 %), Pseudomonas (0-59.73 %), Duganella (0.08–23.37 %), Unidentified Micrococcaceae (0-8.49 %). The functional profiles of the bacterial populations indicated an relation with many human diseases, including infectious diseases. Overall results, using the β diversity measures, coupled with heatmap and RDA showed that there were spatial variations in the bacterial community composition at river sites, and Chemical oxygen demand (CODMn) and (NH4+ )were the dominant environmental drivers affecting the bacterial community variance. Conclusions The high proportion of the opportunistic pathogens (Acinetobacter, Massilia, Brevundimonas) indicated that the discharge of sewage without adequate treatment into the rivers around Chaohu Lake. We propose that these bacteria could be more effective bioindicators for long-term sewage monitoring in eutrophic lakes.

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