scholarly journals Applications of Blocker Nucleic Acids and Non-Metazoan PCR Improves the Discovery of the Eukaryotic Microbiome in Ticks

2021 ◽  
Vol 9 (5) ◽  
pp. 1051
Author(s):  
Yurie Taya ◽  
Gohta Kinoshita ◽  
Wessam Mohamed Ahmed Mohamed ◽  
Mohamed Abdallah Mohamed Moustafa ◽  
Shohei Ogata ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Miseq Platform ◽  
Pcr Method ◽  
Almost All ◽  
Tick Microbiome ◽  
Generation Sequencing

Ticks serve as important vectors of a variety of pathogens. Recently, the viral and prokaryotic microbiomes in ticks have been explored using next-generation sequencing to understand the physiology of ticks and their interactions with pathogens. However, analyses of eukaryotic communities in ticks are limited, owing to the lack of suitable methods. In this study, we developed new methods to selectively amplify microeukaryote genes in tick-derived DNA by blocking the amplification of the 18S rRNA gene of ticks using artificial nucleic acids: peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). In addition, another PCR using non-metazoan primers, referred to as UNonMet-PCR, was performed for comparison. We performed each PCR using tick-derived DNA and sequenced the amplicons using the Illumina MiSeq platform. Almost all sequences obtained by conventional PCR were derived from ticks, whereas the proportion of microeukaryotic reads and alpha diversity increased upon using the newly developed method. Additionally, the PNA- or LNA-based methods were suitable for paneukaryotic analyses, whereas the UNonMet-PCR method was particularly sensitive to fungi. The newly described methods enable analyses of the eukaryotic microbiome in ticks. We expect the application of these methods to improve our understanding of the tick microbiome.

scholarly journals 97 An insight into the piglet’s microbial colonization evolution: From birth towards weaning

2020 ◽  
Vol 98 (Supplement_3) ◽  
pp. 28-29
Author(s):  
Mireia Saladrigas García ◽  
Mario Durán ◽  
Jaume Coma ◽  
José Francisco Pérez ◽  
Susana María Martín-Orúe
Keyword(s):  
Alpha Diversity ◽  
Illumina Miseq ◽  
Microbial Colonization ◽  
Rrna Gene ◽  
Miseq Platform ◽  
Microbial Groups ◽  
Taxonomic Groups ◽  
The Impact ◽  
Biostatistical Analysis ◽  
Age Process

Abstract The aim of the present study was to explore the evolution of piglet gut microbiota from birth to weaning. Moreover, it was hypothesized that different farm environments could condition this process. Two farms, distinct in their use of antibiotics, and 10 litters per farm were selected. A total of 100 fecal samples were obtained from the same pig of each litter on d2, d7, d14 and d21 of lactation and d14 after weaning. The DNA was extracted by using the PSP® Spin Stool DNA Kit and sequencing of the 16S rRNA gene (V3-V4 region) performed by Illumina MiSeq Platform. Bioinformatics and biostatistical analysis were performed with QIIME and the open-source software R v3.5.3. (phyloseq package). Alpha diversity was strongly affected by age (P< 0.001), with an increasing richness of species through time. Beta diversity decreased after weaning (P< 0.001), suggesting a convergent evolution among individuals. Regarding the structure of the microbiota, a clear clustering of the samples according to age was observed (P< 0.001). A progressive decrease was observed as the piglets aged for Clostridiaceae, Enterobacteriaceae, Fusobacteriaceae, Pasteurellaceae and Streptococcaceae (P< 0.001). In contrast, Lachnospiraceae (P=0.003), Lactobacillaceae (P=0.003) and Veillonellaceae (P=0.025) increased along the d7–d14 period, but decreased afterwards. Campylobacteraceae, Erysipelotrichaceae, Ruminococcaceae (P< 0.001) and Prevotellaceae (P=0.005) gradually increased with age reflecting the change from a milk-oriented microbiome towards a butyrate-producing one. Regarding the impact of the farm, differences in species richness were found and also a distinct microbial structure (ANOSIM: P=0.025) associated to changes in some particular taxonomic groups. In conclusion, during the transition from birth to weaning, the pig microbiota showed a relevant succession of microbial groups towards a more stable ecosystem better adapted to the dry feed. In this relevant early-age process differences between farms seems to have a limited impact.

scholarly journals Changes in the Fecal Microbiota Associated with a Broad-Spectrum Antimicrobial Administration in Hospitalized Neonatal Foals with Probiotics Supplementation

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2283
Author(s):  
Francesca Freccero ◽  
Aliai Lanci ◽  
Jole Mariella ◽  
Elisa Viciani ◽  
Sara Quercia ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Fecal Microbiota ◽  
Antimicrobial Treatment ◽  
Rrna Gene ◽  
The Mean ◽  
Mean Time ◽  
The 16S Rrna Gene ◽  
Generation Sequencing

There is a wide array of evidence across species that exposure to antibiotics is associated with dysbiosis, and due to their widespread use, this also raises concerns also in medicine. The study aimed to determine the changes on the fecal microbiota in hospitalized neonatal foals administered with broad-spectrum antimicrobials and supplemented probiotics. Fecal samples were collected at hospital admission (Ta), at the end of the antimicrobial treatment (Te) and at discharge (Td). Feces were analysed by next-generation sequencing of the 16S rRNA gene on Illumina MiSeq. Seven foals treated with IV ampicillin and amikacin/gentamicin were included. The mean age at Ta was 19 h, the mean treatment length was 7 days and the mean time between Te and Td was 4.3 days. Seven phyla were identified: Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, TM7 and Verrucomicrobia. At Ta, Firmicutes (48.19%) and Proteobacteria (31.56%) were dominant. The alpha diversity decreased from Ta to Te, but it was the highest at Td. The beta diversity was higher at Ta than at Te and higher at Td than at Te. An increase in Akkermansia over time was detected. The results suggest that the intestinal microbiota of neonatal foals rapidly returns to a high diversity after treatment. It is possible that in foals, the effect of antimicrobials is strongly influenced or overshadowed by the time-dependent changes in the developing gut microbiota.

scholarly journals PSIX-6 Gastrointestinal microbiome of calves fed solid diets containing different levels and sources of NDF

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 418-419
Author(s):  
Gercino F Virgínio Júnior ◽  
Milaine Poczynek ◽  
Ana Paula Silva ◽  
Ariany Toledo ◽  
Amanda Cezar ◽  
...  
Keyword(s):  
Illumina Miseq ◽  
Diversity Indices ◽  
Dairy Calves ◽  
Rrna Gene ◽  
Gastrointestinal Microbiome ◽  
Fecal Microbiome ◽  
Miseq Platform ◽  
Holstein Calves ◽  
Ad Libitum ◽  
Different Levels

Abstract Different levels and sources of NDF can modify the gastrointestinal microbiome. This study evaluated 18 Holstein calves housed in not-bedded suspended individual cages and fed one of three treatments: 22NDF - conventional starter containing 22% NDF (n = 7); 31NDF - starter with 31% NDF, replacing part of the corn by soybean hull (n = 6); and 22NDF+H - conventional starter with 22% NDF plus coast-cross hay ad libitum (n = 5). All animals received 4 L of milk replacer daily (24% CP; 18.5% fat; diluted to 12.5% solids), divided into two meals, being weaned at 8th week of age. After weaning, animals were housed in tropical shelters, fed with the respective solid diet and coast-cross hay ad libitum for all treatments. To evaluate the microbiome, ruminal fluid samples were collected using a modified Geishauser oral probe at weeks 2, 4, 6, 8 and 10, two hours after the morning feeding, and fecal samples were collected at birth (0) and at weeks 1, 2, 4, 8 and 10. The microbial community was determined by sequencing V3 and V4 region amplicons of the 16S rRNA gene that was amplified by PCR and sequenced by the Illumina MiSeq platform. Ruminal microbiome had no differences in diversity for the effects of weeks, treatments or interaction of both factors (Table 1). In feces, the diversity indices and evenness were higher for 22NDF+H when compared to 22NDF, with no difference for 31NDF. All indices were significantly affected by calves age. At birth, calves had the greatest diversity and richness. Week 1 and 2 had less evenness and diversity. Bacteroidota, Firmicutes_A and Firmicutes_C were the most abundant phylum in rumen and feces. The supply of hay was only effective in modifying the fecal microbiome of dairy calves, suggesting a resilience in the ruminal microbiome.

scholarly journals Comparative analysis of bacterioplankton assemblages from two subtropical karst reservoirs of southwestern China with contrasting trophic status

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Qiang Li ◽  
Yadan Huang ◽  
Shenglin Xin ◽  
Zhongyi Li
Keyword(s):  
Trophic Status ◽  
Alpha Diversity ◽  
Decisive Role ◽  
Sensu Stricto ◽  
Karst Area ◽  
Southwestern China ◽  
Rrna Gene ◽  
Karst Water ◽  
Bacterioplankton Community ◽  
Generation Sequencing

AbstractAlthough bacterioplankton play an important role in aquatic ecosystems, less is known about bacterioplankton assemblages from subtropical karst reservoirs of southwestern China with contrasting trophic status. Here, 16S rRNA gene next-generation sequencing coupled with water chemistry analysis was applied to compare the bacterioplankton communities from a light eutrophic reservoir, DL Reservoir, and a mesotrophic reservoir, WL Reservoir, in subtropical karst area of southwestern China. Our findings indicated that Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Cyanobacteria and Verrucomicrobia dominated bacterioplankton community with contrasting relative frequency in the two subtropical karst reservoirs. Proteobacteria and Bacteroidetes were the core communities, which played important roles in karst biogeochemical cycles. Though WT, TN and DOC play the decisive role in assembling karst aquatic bacterioplankton, trophic status exerted significantly negative direct effects on bacterioplankton community composition and alpha diversity. Due to contrasting trophic status in the two reservoirs, the dominant taxa such as Enterobacter, Clostridium sensu stricto, Candidatus Methylacidiphilum and Flavobacteriia, that harbor potential functions as valuable and natural indicators of karst water health status, differed in DL Reservoir and WL Reservoir.

scholarly journals Effect of Bovine MFGM and LF in Infant Formula on Gut Microbiome and Metabolome at Four Months of Age

2021 ◽  
Author(s):  
Maciej Chichlowski ◽  
Nicholas Bokulich ◽  
Cheryl L Harris ◽  
Jennifer L Wampler ◽  
Fei Li ◽  
...  
Keyword(s):  
Infant Formula ◽  
Beta Diversity ◽  
Milk Fat ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Term Infants ◽  
Linear Discriminant ◽  
Alpha And Beta Diversity ◽  
Metabolite Profiles

Abstract Background Milk fat globule membrane (MFGM) and lactoferrin (LF) are human milk bioactive components demonstrated to support gastrointestinal (GI) and immune development. Significantly fewer diarrhea and respiratory-associated adverse events through 18 months of age were previously reported in healthy term infants fed a cow's milk-based infant formula with added source of bovine MFGM and bovine LF through 12 months of age. Objectives To compare microbiota and metabolite profiles in a subset of study participants. Methods Stool samples were collected at Baseline (10–14 days of age) and Day 120 (MFGM + LF: 26, Control: 33). Bacterial community profiling was performed via16S rRNA gene sequencing (Illumina MiSeq) and alpha and beta diversity were analyzed (QIIME 2). Differentially abundant taxa were determined using Linear discriminant analysis effect size (LefSE) and visualized (Metacoder). Untargeted stool metabolites were analyzed (HPLC/mass spectroscopy) and expressed as the fold-change between group means (Control: MFGM + LF ratio). Results Alpha diversity increased significantly in both groups from baseline to 4 months. Subtle group differences in beta diversity were demonstrated at 4 months (Jaccard distance; R2 = 0.01, P = 0.042). Specifically, Bacteroides uniformis and Bacteroides plebeius were more abundant in the MFGM + LF group at 4 months. Metabolite profile differences for MFGM + LF vs Control included: lower fecal medium chain fatty acids, deoxycarnitine, and glycochenodeoxycholate, and some higher fecal carbohydrates and steroids (P < 0.05). After applying multiple test correction, the differences in stool metabolomics were not significant. Conclusions Addition of bovine MFGM and LF in infant formula was associated with subtle differences in stool microbiome and metabolome by four months of age, including increased prevalence of Bacteroides species. Stool metabolite profiles may be consistent with altered microbial metabolism. Trial registration:  https://clinicaltrials.gov/ct2/show/NCT02274883).

Prevalence and Antimicrobial Resistance of Campylobacter spp. Isolated from Poultry Carcasses in Poland

2013 ◽  
Vol 76 (8) ◽  
pp. 1451-1455 ◽  
Author(s):  
KINGA WIECZOREK ◽  
IWONA KANIA ◽  
JACEK OSEK
Keyword(s):  
Campylobacter Jejuni ◽  
23S Rrna ◽  
Rrna Gene ◽  
Campylobacter Coli ◽  
Genetic Determinants ◽  
Gyra Gene ◽  
23S Rrna Gene ◽  
Pcr Method ◽  
Almost All ◽  
Campylobacter Spp

The purpose of the present study was to determine the prevalence of Campylobacter in poultry carcasses at slaughter in Poland. For the isolated strains, resistance to selected antibiotics and the associated genetic determinants were identified. A total of 498 Campylobacter isolates were obtained from 802 poultry samples during the 2-year study period. Strains were identified to species with the PCR method; 53.6% of the strains were Campylobacter jejuni and 46.4% were Campylobacter coli. A high percentage of the tested Campylobacter strains were resistant to ciprofloxacin and nalidixic acid (74.1 and 73.5%, respectively) followed by tetracycline (47.4%) and streptomycin (20.5%). Only one C. jejuni and two C. coli isolates were resistant to gentamicin. Seventy-nine (15.9%) of the 498 strains were resistant to three or more classes of antibiotics examined. Higher levels of resistance, irrespective of the antimicrobial agent tested, were found within the C. coli group. Almost all strains resistant to quinolones (99.5%) and to tetracycline (99.6%) carried the Thr-86-to-Ile mutation in the gyrA gene and possessed the tet(O) marker, respectively. All isolates resistant to erythromycin had the A2075G mutation in the 23S rRNA gene. These results reveal that poultry carcasses in Poland are a reservoir of potentially pathogenic and antimicrobial-resistant Campylobacter strains for humans, which may pose a public health risk.

scholarly journals Forfeiting the founder effect: turnover defines biofilm community succession

2018 ◽  
Author(s):  
Colin J. Brislawn ◽  
Emily B. Graham ◽  
Karl Dana ◽  
Peter Ihardt ◽  
Sarah J. Fansler ◽  
...  
Keyword(s):  
Bacterial Colonization ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Community Succession ◽  
Ecological Processes ◽  
Functional Capabilities ◽  
Resolution Imaging ◽  
Successional Stages ◽  
Almost All

ABSTRACTMicrobial community succession is a fundamental process that effects underlying functions of almost all ecosystems; yet the roles and fates of the most abundant colonizers are poorly understood. Does early abundance spur long term persistence? How do deterministic and stochastic processes influence the roles of founder species? We performed a succession experiment within a hypersaline microbial mat ecosystem to investigate how ecological processes contributed to the turnover of founder species. Bacterial and micro-eukaryotic founder species were identified from primary succession and tracked through a defined maturation period using 16S and 18S rRNA gene amplicon sequencing in combination with high resolution imaging that utilized stable isotope tracers to evaluate basic functional capabilities. The majority of the founder species did not maintain high relative abundances in later stages of succession. Turnover (versus nestedness) was the dominant process shaping the final community structure. We also asked if different ecological processes acted on bacteria versus eukaryotes during successional stages and found that deterministic and stochastic forces corresponded more with eukaryote and bacterial colonization, respectively. Our results show that taxa from different kingdoms, that share habitat in the tight spatial confines of a biofilm, were influenced by different ecological forces and time scales of succession.

scholarly journals Faecal Microbiota of Dogs Offered a Vegetarian Diet with or without the Supplementation of Feather Meal and either Cornmeal, Rye or Fermented Rye: A Preliminary Study

2020 ◽  
Vol 8 (9) ◽  
pp. 1363
Author(s):  
Julia Hankel ◽  
Amr Abd El-Wahab ◽  
Richard Grone ◽  
Birgit Keller ◽  
Eric Galvez ◽  
...  
Keyword(s):  
Intestinal Microbiota ◽  
Hypervariable Region ◽  
Illumina Miseq ◽  
Vegetarian Diet ◽  
Rrna Gene ◽  
Feather Meal ◽  
Illumina Miseq Platform ◽  
Faecal Samples ◽  
Miseq Platform ◽  
Preliminary Study

Anthropomorphism of dogs has affected feeding and the choice of components present in diets for dogs. Conflicting trends are present: raw or vegetarian appear more prevalent. Animal-derived proteins seem to have unfavourable impacts on intestinal microflora by decreasing the presence of Bacteroidetes. This preliminary study evaluates whether effects of diets with animal proteins on intestinal microbiota can be compensated by the addition of certain carbohydrates to dog diet. Eight female beagles were included in a cross-over study and fed a vegetarian diet or the same diet supplemented with feather meal (2.7%) and either 20% of cornmeal, fermented or non-fermented rye (moisture content of the diets about 42%). A 16S rRNA gene amplification was performed within the hypervariable region V4 on faecal samples and sequenced with the Illumina MiSeq platform. The Firmicutes/Bacteroidetes ratio tended to shift to the advantage of Firmicutes when feather meal and cornmeal were added (Firmicutes/Bacteroidetes ratio of 5.12 compared to 2.47 when offered the vegetarian diet) and tended to switch back to the advantage of Bacteroidetes if rye: fermented (2.17) or not (1.03) was added. The addition of rye might have the potential to compensate possible unfavourable effects of diets with animal proteins on intestinal microbiota of dogs.

scholarly journals Metabarcoding Analyses of Gut Microbiota of Nile Tilapia (Oreochromis niloticus) from Lake Awassa and Lake Chamo, Ethiopia

2020 ◽  
Vol 8 (7) ◽  
pp. 1040
Author(s):  
Negash Kabtimer Bereded ◽  
Manuel Curto ◽  
Konrad J. Domig ◽  
Getachew Beneberu Abebe ◽  
Solomon Workneh Fanta ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Oreochromis Niloticus ◽  
Nile Tilapia ◽  
Large Scale ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Operational Taxonomic Units ◽  
Nile Tilapia Oreochromis Niloticus ◽  
Miseq Platform ◽  
Ethiopian Lakes

The Nile tilapia (Oreochromis niloticus) gut harbors a diverse microbial community; however, their variation across gut regions, lumen and mucosa is not fully elucidated. In this study, gut microbiota of all samples across gut regions and sample types (luminal content and mucosa) were analyzed and compared from two Ethiopian lakes. Microbiota were characterized using 16S rRNA Illumina MiSeq platform sequencing. A total of 2061 operational taxonomic units (OTUs) were obtained and the results indicated that Nile tilapia from Lake Chamo harbored a much more diversified gut microbiota than Lake Awassa. In addition, the gut microbiota diversity varied significantly across the gut region based on the Chao1, Shannon and Simpson index. The microbiome analyses of all samples in the midgut region showed significantly higher values for alpha diversity (Chao 1, Shannon and Simpson). Beta diversity analysis revealed a clear separation of samples according to sampling areas and gut regions. The most abundant genera were Clostridium_sensu_stricto and Clostridium_XI genera across all samples. Between the two sampling lakes, two phyla, Phylum Fusobacteria and Cyanobacteria, were found to be significantly different. On the other hand, six phyla (Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria and Cyanobacteria) were significantly different across gut regions. In this study, we found that all samples shared a large core microbiota, comprising a relatively large number of OTUs, which was dominated by Proteobacteria, Firmicutes, Cyanobacteria, Fusobacteria and Actinobacteria. This study has established the bases for future large-scale investigations of gut microbiota of fishes in Ethiopian lakes.

scholarly journals The cultivation of Pyropia haitanensis has important impacts on the seawater microbial community

2020 ◽  
Vol 32 (4) ◽  
pp. 2561-2573
Author(s):  
Wenlei Wang ◽  
Lei Wu ◽  
Kai Xu ◽  
Yan Xu ◽  
Dehua Ji ◽  
...  
Keyword(s):  
Microbial Community ◽  
Growth And Development ◽  
Alpha Diversity ◽  
18S Rrna Gene ◽  
Pyropia Haitanensis ◽  
Rrna Gene ◽  
Cultivation Period ◽  
Rrna Gene Sequencing ◽  
Bacteria And Fungi ◽  
Surrounding Seawater

Abstract Microorganisms play important roles in the growth and development of macroalgae. Still, the biodiversity of the epiphytic microbial community associated with the economically important red alga Pyropia haitanensis during the cultivation period remains uncharacterized, especially the effects of P. haitanensis cultivation on the microbial community of surrounding seawater. Here, we isolated epiphytic microbes from P. haitanensis during the thallus stage during oceanic cultivation and the conchocelis stage during industrial cultivation. The dynamic diversity patterns, as determined by 16S and 18S rRNA gene sequencing of the bacterial and fungal communities, respectively, associated with P. haitanensis and seawater in the presence and absence of algal cultivation were investigated. A notable distinction was observed between the microbial communities of seawater with and without P. haitanensis cultivation. Additionally, the alpha-diversity of seawater with P. haitanensis cultivation was significantly greater than without P. haitanensis cultivation. Cyanobacteria were the dominant species in the latter, while Rhodobacteraceae was enriched in the former. Furthermore, there were significant differences in the microbial community of P. haitanensis at the thallus and conchocelis stages. Seawater properties had significant direct effects on the microbial diversity of P. haitanensis and cultivation seawater, but not on non-cultivation seawater. The enriched microbial presence might promote thallus morphogenesis and be beneficial for the growth and development of both the thallus and conchocelis stages. These findings expand our knowledge of the bacteria and fungi that are beneficial for Pyropia nursery seeding and cultivation, as well as the effects of P. haitanensis cultivation on the seawater microbial community.

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