scholarly journals Microbial diversity in freshwater samples and their contaminating human and bovine hosts

2017 ◽  
Author(s):  
Nancy C Stoppe ◽  
Tatiana T Torres ◽  
Fabricio C Leotti ◽  
Maria Inês Z Sato ◽  
Laura MM Ottoboni
Keyword(s):  
Water Samples ◽  
Interaction Network ◽  
Pollution Source ◽  
Rrna Gene ◽  
Operational Taxonomic Units ◽  
Chemical Factors ◽  
Stool Samples ◽  
The One ◽  
Human Pollution ◽  
V3 Region

DNA extracted from feces (human and bovine) and water samples was used for the massive pyrosequencing of the hypervariable V3 region of the 16S rRNA gene, revealing 4296 operational taxonomic units (OTUs). The greatest diversity was observed in samples of cattle feces, and the smallest diversity was found in a pristine water sample. Firmicutes was the predominant group in samples of human feces, while in bovine feces the dominant groups were Firmicutes and Bacteroidetes. The interaction network showed that the stool samples had the greatest diversity and, among the water samples, the one with human pollution source had the highest diversity. The LEfSe method was used to identify host biomarkers. Actinobacteria, Betaproteobacteria, and Firmicutes were identified as human biomarkers, while for cattle, the potential markers were Bacteroidetes, Tenericutes, and Spirochaetes. Host-specific markers were identified, but were not found in the water samples, suggesting either that the tools used did not have the resolution to identify markers in environmental samples, or that the contamination in the water bodies was mixed. Additionally, as the host-specific markers were isolated from non-autochthonous microorganisms, they could be affected by adverse environmental effects including physical-chemical factors and competition with native organisms.

scholarly journals Microbial diversity in freshwater samples and their contaminating human and bovine hosts

2017 ◽  
Author(s):  
Nancy C Stoppe ◽  
Tatiana T Torres ◽  
Fabricio C Leotti ◽  
Maria Inês Z Sato ◽  
Laura MM Ottoboni
Keyword(s):  
Water Samples ◽  
Interaction Network ◽  
Pollution Source ◽  
Rrna Gene ◽  
Operational Taxonomic Units ◽  
Chemical Factors ◽  
Stool Samples ◽  
The One ◽  
Human Pollution ◽  
V3 Region

DNA extracted from feces (human and bovine) and water samples was used for the massive pyrosequencing of the hypervariable V3 region of the 16S rRNA gene, revealing 4296 operational taxonomic units (OTUs). The greatest diversity was observed in samples of cattle feces, and the smallest diversity was found in a pristine water sample. Firmicutes was the predominant group in samples of human feces, while in bovine feces the dominant groups were Firmicutes and Bacteroidetes. The interaction network showed that the stool samples had the greatest diversity and, among the water samples, the one with human pollution source had the highest diversity. The LEfSe method was used to identify host biomarkers. Actinobacteria, Betaproteobacteria, and Firmicutes were identified as human biomarkers, while for cattle, the potential markers were Bacteroidetes, Tenericutes, and Spirochaetes. Host-specific markers were identified, but were not found in the water samples, suggesting either that the tools used did not have the resolution to identify markers in environmental samples, or that the contamination in the water bodies was mixed. Additionally, as the host-specific markers were isolated from non-autochthonous microorganisms, they could be affected by adverse environmental effects including physical-chemical factors and competition with native organisms.

Endophytic bacterial communities and spatiotemporal variations in cotton roots in Xinjiang, China

2020 ◽  
Author(s):  
Yingwu Shi ◽  
Hongmei Yang ◽  
Min Chu ◽  
Xinxiang Niu ◽  
Xiangdong Huo ◽  
...  
Keyword(s):  
Community Structure ◽  
Endophytic Bacteria ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Operational Taxonomic Units ◽  
Cotton Sample ◽  
Endophytic Bacterial Community ◽  
V3 Region ◽  
Illumina Amplicon Sequencing

Endophytic bacteria may be important for plant health and other ecologically relevant functions of cotton. However, the endophytic bacterial community structure and diversity in cotton is still poorly characterized. We investigated the community structure of endophytic bacteria in cotton roots growing in Xinjiang, China, using the Illumina amplicon sequencing. A total of 60.84 M effective sequences of 16S rRNA gene V3 region were obtained from cotton samples. These sequences revealed huge amount of operational taxonomic units (OTUs) in cotton, that is, 81-338 OTUs in a cotton sample, at 3% cutoff level and sequencing depth of 50000 sequences. We identified 23 classes from the resulting 2,723,384 sequences. Gammaproteobacteria were the dominant class in all cottons, followed by Alphaproteobacteria, Actinobacteria and Bacilli. A marked difference in the diversity of endophytic bacteria in cotton for different growth periods was evident. The greatest number of OTUs was detected during seedling (654 OTUs) and budding (381 OTUs). Endophytic bacteria diversity was reduced during flowering (350 OTUs) and boll-opening (351 OTUs). 217 OTUs were common to all four periods. There were more tags of Pantoea in Shihezi than other locations. While there were more tags of Erwinia in Hami than other locations. The dynamics of endophytic bacteria communities were influenced by plant growth stage. These results show the complexity of the bacterial populations present in inner tissues of cotton.

scholarly journals  Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

2012 ◽  
Vol 57 (No. 5) ◽  
pp. 224-232 ◽  
Author(s):  
M. Adamska ◽  
A. Leonska-Duniec ◽  
M. Sawczuk ◽  
A. Maciejewska ◽  
B. Skotarczak
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
High Concentration ◽  
Intestinal Protozoan ◽  
Wide Range ◽  
Stool Samples

Cryptosporidium parvum is a common intestinal protozoan parasite infecting humans and a wide range of animals, whose diagnostics present considerable difficulties. These arise from the exceptionally robust nature of the oocyst’s walls, which necessitates more stringent treatments for disruption and recovery of DNA for analysis using molecular methods. In the case of water, which is the major source of Cryptosporidium oocysts, investigations concern the detection of the presence of the oocysts. Their concentration in water is very low, and moreover, many substances that may have significance as inhibitors of DNA amplification, are present in environmental water and stool. We have carried out trials in order to assess the effectiveness of recovery of C. parvum oocysts, from spiked environmental and distilled water samples, filtrated and concentrated with the use of special laboratory equipment. Inactivation of inhibitors was carried out with use of bovine serum albumin (BSA) in PCR mixes at ten different concentrations. DNA extraction was carried out from stool samples spiked with C. parvum oocysts, concentrated using two methods, and unconcentrated. Nested PCR and a TaqMan nested real time PCR assay, targeting the 18S rRNA gene, was used to detect C. parvum DNA in spiked water and additionally in spiked stool samples. The obtained results showed that losses of C. parvum oocysts occur during the filtration and concentration of spiked water samples. The addition of small amounts of BSA (5–20 ng/µl) to PCR and TaqMan PCR mixes increases the sensitivity of both methods, but a high concentration of BSA (100 ng/µl and above) has an inhibiting effect on the polymerase reaction. The extraction of DNA from C. parvum oocysts from spiked stool samples preceded by concentration with PBS, ether and Percoll resulted in a higher copy number of the 18S rRNA gene.  

scholarly journals Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Sandra Reitmeier ◽  
Thomas C. A. Hitch ◽  
Nicole Treichel ◽  
Nikolaos Fikas ◽  
Bela Hausmann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Diversity Analysis ◽  
Careful Attention ◽  
Operational Taxonomic Units ◽  
Basic Concepts ◽  
Gnotobiotic Mice ◽  
Mock Communities

Abstract16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

scholarly journals Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities

2009 ◽  
Vol 75 (23) ◽  
pp. 7537-7541 ◽  
Author(s):  
Patrick D. Schloss ◽  
Sarah L. Westcott ◽  
Thomas Ryabin ◽  
Justine R. Hall ◽  
Martin Hartmann ◽  
...  
Keyword(s):  
16S Rrna ◽  
Software Package ◽  
Sequence Data ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Laptop Computer ◽  
Operational Taxonomic Units ◽  
Β Diversity ◽  
Single Piece

ABSTRACT mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the α and β diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

scholarly journals The Effects of Genetic Relatedness on the Preterm Infant Gut Microbiota

2021 ◽  
Vol 9 (2) ◽  
pp. 278
Author(s):  
Shen Jean Lim ◽  
Miriam Aguilar-Lopez ◽  
Christine Wetzel ◽  
Samia V. O. Dutra ◽  
Vanessa Bray ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Preterm Infant ◽  
Neonatal Intensive Care ◽  
Genetic Relatedness ◽  
Bovine Milk ◽  
Alpha Diversity ◽  
Neonatal Intensive Care Units ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Operational Taxonomic Units

The preterm infant gut microbiota is influenced by environmental, endogenous, maternal, and genetic factors. Although siblings share similar gut microbial composition, it is not known how genetic relatedness affects alpha diversity and specific taxa abundances in preterm infants. We analyzed the 16S rRNA gene content of stool samples, ≤ and >3 weeks postnatal age, and clinical data from preterm multiplets and singletons at two Neonatal Intensive Care Units (NICUs), Tampa General Hospital (TGH; FL, USA) and Carle Hospital (IL, USA). Weeks on bovine milk-based fortifier (BMF) and weight gain velocity were significant predictors of alpha diversity. Alpha diversity between siblings were significantly correlated, particularly at ≤3 weeks postnatal age and in the TGH NICU, after controlling for clinical factors. Siblings shared higher gut microbial composition similarity compared to unrelated individuals. After residualizing against clinical covariates, 30 common operational taxonomic units were correlated between siblings across time points. These belonged to the bacterial classes Actinobacteria, Bacilli, Bacteroidia, Clostridia, Erysipelotrichia, and Negativicutes. Besides the influence of BMF and weight variables on the gut microbial diversity, our study identified gut microbial similarities between siblings that suggest genetic or shared maternal and environmental effects on the preterm infant gut microbiota.

Diversity of halophilic bacteria isolated from Rambla Salada, Murcia (Spain)

2014 ◽  
Vol 60 (12) ◽  
pp. 839-846 ◽  
Author(s):  
Rocío Luque ◽  
Victoria Béjar ◽  
Emilia Quesada ◽  
Inmaculada Llamas
Keyword(s):  
Bacterial Community ◽  
Halophilic Bacteria ◽  
Operational Taxonomic Units ◽  
Sequence Identity ◽  
Abundant Genus ◽  
Phenotypic Tests ◽  
Sampling Season ◽  
V3 Region ◽  
Rrna Sequences ◽  
16S Rrna Sequences

In this study we analyzed the diversity of the halophilic bacteria community from Rambla Salada during the years 2006 and 2007. We collected a total of 364 strains, which were then identified by means of phenotypic tests and by the hypervariable V1–V3 region of the 16S rRNA sequences (around 500 bp). The ribosomal data showed that the isolates belonged to Proteobacteria (72.5%), Firmicutes (25.8%), Actinobacteria (1.4%), and Bacteroidetes (0.3%) phyla, with Gammaproteobacteria the predominant class. Halomonas was the most abundant genus (41.2% isolates) followed by Marinobacter (12.9% isolates) and Bacillus (12.6% isolates). In addition, 9 strains showed <97% sequence identity with validly described species and may well represent new taxa. The diversity of the bacterial community analyzed with the DOTUR package determined 139 operational taxonomic units at 3% genetic distance level. Rarefaction curves and diversity indexes demonstrated that our collection of isolates adequately represented all the bacterial community at Rambla Salada that can be grown under the conditions used in this work. We found that the sampling season influenced the composition of the bacterial community, and bacterial diversity was higher in 2007; this fact could be related to lower salinity at this sampling time.

scholarly journals Detection of Protozoan Hosts for Legionella pneumophila in Engineered Water Systems by Using a Biofilm Batch Test

2010 ◽  
Vol 76 (21) ◽  
pp. 7144-7153 ◽  
Author(s):  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Dick van der Kooij
Keyword(s):  
Legionella Pneumophila ◽  
Biofilm Formation ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Batch Test ◽  
Free Living ◽  
Water Types ◽  
Operational Taxonomic Units ◽  
Hartmannella Vermiformis

ABSTRACT Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C.

Comparison of culture-dependent and culture-independent techniques in the detection of lactic acid bacteria biodiversity and dynamics throughout the ripening process: The case of Turkish artisanal Tulum cheese produced in the Anamur region

2021 ◽  
pp. 1-7
Author(s):  
Talha Demirci ◽  
Aysun Oraç ◽  
Kübra Aktaş ◽  
Enes Dertli ◽  
Ismail Akyol ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Dna Isolation ◽  
Independent Method ◽  
Rrna Gene ◽  
Ripening Process ◽  
Culture Independent ◽  
Tulum Cheese ◽  
V3 Region ◽  
Culture Dependent ◽  
Bacterial 16S Rrna Gene

Abstract Our objective was to analyze the diversity of the microbiota over 180 d of ripening of eight batches of artisanal goatskin Tulum cheeses by culture-dependent and culture-independent (PCR-DGGE) methods. V3 region of the bacterial 16S rRNA gene was amplified with the PCR after direct DNA isolation from the cheese samples. Nine different species and five genera were determined by culturing, while 11 species were identified in the PCR-DGGE technique. This diversity revealed the uniqueness of artisanal cheese varieties. The dominant genera in all the cheese samples were composed of Enterococcus species. The culture-dependent method revealed five genera (Enterococcus,Bacillus,Lactococcus,Lactobacillus, Sphingomonas) while three genera (Enterococcus, Streptococcus, Lactococcus) were detected in the culture-independent method. It was concluded that combining the two methods is important for characterizing the whole microbiota of the Tulum cheese varieties produced in the Anamur region.

Evidence of bacterial DNA presence in chorionic villi and amniotic fluid in the first and second trimester of pregnancy

2021 ◽  
Author(s):  
Giuseppina Campisciano ◽  
Mariachiara Quadrifoglio ◽  
Manola Comar ◽  
Francesco De Seta ◽  
Nunzia Zanotta ◽  
...  
Keyword(s):  
Amniotic Fluid ◽  
Chorionic Villus ◽  
Chorionic Villus Sampling ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Chorionic Villi ◽  
Uncomplicated Pregnancy ◽  
Bacterial Microbiota ◽  
Sequencing Method ◽  
V3 Region

The sterile-womb dogma in uncomplicated pregnancy has been lively debated. Data regarding the in utero microbiome environment are based mainly on studies performed at the time of delivery. Aim: To determine whether human placenta and amniotic fluid are populated by a bacterial microbiota in the first and second trimesters of pregnancy. Materials & methods: We analyzed by next-generation sequencing method 24 and 29 samples from chorionic villus sampling (CVS) and amniocentesis (AC), respectively. The V3 region of the 16S rRNA gene was sequenced. Results: 37.5% of CVS and 14% of AC samples showed the presence of bacterial DNA. Conclusion: Our study suggests that bacterial DNA can be identified in the placenta and amniotic fluid during early prenatal life.

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