scholarly journals Contamination Is Not Linked to the Gestational Microbiome

2019 ◽  
Vol 85 (19) ◽  
Author(s):  
Michelle D. Rodriguez ◽  
Kevin K. Yu ◽  
Zubin S. Paul ◽  
Maureen Keller-Wood ◽  
Charles E. Wood ◽  
...  
Keyword(s):  
16S Rrna ◽  
Fetal Liver ◽  
In Utero ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Tissue Samples ◽  
Bacterial Dna ◽  
Culture Independent ◽  
Active Transfer ◽  
Colonization Hypothesis

ABSTRACT Differentiating between contamination and the genuine presence of 16S rRNA genes in gestational tissue samples is the gold standard for supporting the in utero colonization hypothesis. During gestation, the fetus undergoes significant physiological changes that may be directly affected by maternal colonization of key bacterial genera. In this study, lab benches, necropsy tables, and air ducts were swabbed at the same time as clinical sampling. The relative and absolute abundance of bacteria present in sheep samples was determined by culture-independent and culture-dependent means. Of 14 healthy pregnant ewes, there was no evidence of any bacteria in the fetal liver, spleen, or brain cortex using culture-independent techniques despite evidence of the presence of bacteria in various locations of the necropsy room used for 11 of these 14 sheep. Of the 336 bacterial genera found in the room swabs, only 12 (5%) were also found in the saliva and vaginal swabs among the three ewes for which bacteria were detected. These 12 taxa represent 1.32% of the relative abundance and approximately 393 16S rRNA copies/swab in these three ewes. Using careful necropsy protocols, bacterial contamination of sheep tissues was avoided. Contamination of saliva and vaginal samples was limited to less than 2% of the bacterial population. IMPORTANCE Recent evidence for a gestational microbiome suggests that active transfer between mother and fetus in utero is possible, and, therefore, actions must be taken to clarify the presence versus absence of these organisms in their respected sources. The value of this study is the differentiation between bacterial DNA identified in the necropsy rooms of animals and bacterial DNA whose origin is purely clinical in nature. We do not know the extent to which microorganisms traverse maternal tissues and infiltrate fetal circulation, so measures taken to control for contamination during sample processing are vital for addressing these concerns.

scholarly journals Census of the Viral Metagenome within an Activated Sludge Microbial Assemblage

2010 ◽  
Vol 76 (8) ◽  
pp. 2673-2677 ◽  
Author(s):  
Larissa C. Parsley ◽  
Erin J. Consuegra ◽  
Stephen J. Thomas ◽  
Jaysheel Bhavsar ◽  
Andrew M. Land ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
Activated Sludge ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Isolates ◽  
Microbial Assemblage ◽  
Culture Independent ◽  
Culture Dependent

ABSTRACT The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.

scholarly journals Bacteria Associated with Mucus and Tissues of the Coral Oculina patagonica in Summer and Winter

2006 ◽  
Vol 72 (8) ◽  
pp. 5254-5259 ◽  
Author(s):  
Omry Koren ◽  
Eugene Rosenberg
Keyword(s):  
16S Rrna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Culturable Bacteria ◽  
Vibrio Splendidus ◽  
Marine Agar ◽  
Tissue Samples ◽  
Operational Taxonomic Units ◽  
Sybr Gold ◽  
16S Rrna Clone Library

ABSTRACT The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Oculina patagonica was determined by analyses of the 16S rRNA genes of isolated colonies and from a 16S rRNA clone library of extracted DNA. By SYBR gold staining, the numbers of bacteria in mucus and tissue samples were 6.2 × 107 and 8.3 × 108/cm2 of coral surface, respectively, 99.8% of which failed to produce colonies on Marine Agar. From analysis of mucus DNA, the most-abundant bacterium was Vibrio splendidus, representing 68% and 50% of the clones from the winter and summer, respectively. After removal of mucus from coral by centrifugation, analyses of DNA from the crushed tissue revealed a large diversity of bacteria, with Vibrio species representing less than 5% of the clones. The most-abundant culturable bacteria were a Pseudomonas sp. (8 to 14%) and two different α-proteobacteria (6 to 18%). Out of a total 1,088 16S rRNA genes sequenced, 400 different operational taxonomic units were identified (>99.5% identity). Of these, 295 were novel (<99% identical to any sequences in the GenBank database). This study provides a comprehensive database for future examinations of changes in the bacterial community during bleaching events.

scholarly journals Increase in Terminal Restriction Fragments of Bacteroidetes-Derived 16S rRNA Genes after Administration of Short-Chain Fructooligosaccharides

2006 ◽  
Vol 72 (9) ◽  
pp. 6271-6276 ◽  
Author(s):  
Yusuke Nakanishi ◽  
Koichiro Murashima ◽  
Hiroki Ohara ◽  
Takahisa Suzuki ◽  
Hidenori Hayashi ◽  
...  
Keyword(s):  
16S Rrna ◽  
Intestinal Microbiota ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Intestinal Bacteria ◽  
Short Chain ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Fragments ◽  
Culture Independent

ABSTRACT It is well known that short chain fructooligosaccharides (scFOS) modify intestinal microbiota in animals as well as in humans. Since most murine intestinal bacteria are still uncultured, it is difficult for a culturing method to detect changes in intestinal microbiota after scFOS administration in a mouse model. In this study, we sought markers of positive change in murine intestinal microbiota after scFOS administration using terminal restriction fragment length polymorphism (T-RFLP) analysis, which is a culture-independent method. The T-RFLP profiles showed that six terminal restriction fragments (T-RFs) were significantly increased after scFOS administration. Phylogenetic analysis of the 16S rRNA partial gene sequences of murine fecal bacteria suggested that four of six T-RFs that increased after scFOS administration were derived from the 16S rRNA genes of the class Bacteroidetes. Preliminary quantification of Bacteroidetes by real-time PCR suggests that the 16S rRNA genes derived from Bacteroidetes were increased by scFOS administration. Therefore, the T-RFs derived from Bacteroidetes are good markers of change of murine intestinal microbiota after scFOS administration.

scholarly journals Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinIONTMnanopore sequencer

2018 ◽  
Author(s):  
Shinichi Kai ◽  
Yoshiyuki Matsuo ◽  
So Nakagawa ◽  
Kirill Kryukov ◽  
Shino Matsukawa ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bacterial Identification ◽  
Direct Pcr ◽  
16S Rrna Gene Analysis ◽  
Bacterial Dna

AbstractRapid identification of bacterial pathogens is crucial for appropriate and adequate antibiotic treatment, which significantly improves patient outcomes. 16S ribosomal RNA (rRNA) gene amplicon sequencing has proven to be a powerful strategy for diagnosing bacterial infections. We have recently established a sequencing method and bioinformatics pipeline for 16S rRNA gene analysis utilizing the Oxford Nanopore Technologies MinION™ sequencer. In combination with our taxonomy annotation analysis pipeline, the system enabled the molecular detection of bacterial DNA in a reasonable timeframe for diagnostic purposes. However, purification of bacterial DNA from specimens remains a rate-limiting step in the workflow. To further accelerate the process of sample preparation, we adopted a direct PCR strategy that amplifies 16S rRNA genes from bacterial cell suspensions without DNA purification. Our results indicate that differences in cell wall morphology significantly affect direct PCR efficiency and sequencing data. Notably, mechanical cell disruption preceding direct PCR was indispensable for obtaining an accurate representation of the specimen bacterial composition. Furthermore, 16S rRNA gene analysis of mock polymicrobial samples indicated that primer sequence optimization is required to avoid preferential detection of particular taxa and to cover a broad range of bacterial species. This study establishes a relatively simple workflow for rapid bacterial identification via MinIONTMsequencing, which reduces the turnaround time from sample to result, and provides a reliable method that may be applicable to clinical settings.

scholarly journals Screening of a Fosmid Library of Marine Environmental Genomic DNA Fragments Reveals Four Clones Related to Members of the OrderPlanctomycetales

1998 ◽  
Vol 64 (8) ◽  
pp. 3075-3078 ◽  
Author(s):  
Kevin L. Vergin ◽  
Ena Urbach ◽  
Jeffery L. Stein ◽  
Edward F. DeLong ◽  
Brian D. Lanoil ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Genomic Dna ◽  
Pcr Amplification ◽  
Fosmid Library ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Dna ◽  
Marine Environmental

ABSTRACT A fosmid library with inserts containing approximately 40 kb of marine bacterial DNA (J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya, and E. F. DeLong, J. Bacteriol. 178:591–599, 1996) yielded four clones with 16S rRNA genes from the orderPlanctomycetales. Three of the clones belong to thePirellula group and one clone belongs to thePlanctomyces group, based on phylogenetic and signature nucleotide analyses of full-length 16S rRNA genes. Sequence analysis of the ends of the genes revealed a consistent mismatch in a widely used bacterium-specific 16S rRNA PCR amplification priming site (27F), which has also been reported in some thermophiles and spirochetes.

scholarly journals Flow-Cytometric Cell Sorting and Subsequent Molecular Analyses for Culture-Independent Identification of Bacterioplankton Involved in Dimethylsulfoniopropionate Transformations

2005 ◽  
Vol 71 (3) ◽  
pp. 1405-1416 ◽  
Author(s):  
Xiaozhen Mou ◽  
Mary Ann Moran ◽  
Ramunas Stepanauskas ◽  
José M. González ◽  
Robert E. Hodson
Keyword(s):  
16S Rrna ◽  
Cell Sorting ◽  
Marine Ecosystem ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Coastal Marine ◽  
Four Seasons ◽  
Culture Independent

ABSTRACT Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 μM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, α-Proteobacteria); Caulobacter (α-Proteobacteria); and Brachymonas and Xenophilus (β-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, α-Proteobacteria) and novel γ-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.

scholarly journals Census of the Bacterial Community of the Gypsy Moth Larval Midgut by Using Culturing and Culture-Independent Methods

2004 ◽  
Vol 70 (1) ◽  
pp. 293-300 ◽  
Author(s):  
Nichole A. Broderick ◽  
Kenneth F. Raffa ◽  
Robert M. Goodman ◽  
Jo Handelsman
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
Gypsy Moth ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Composition Analysis ◽  
White Oak ◽  
Microbial Composition ◽  
Larval Midgut ◽  
Culture Independent

ABSTRACT Little is known about bacteria associated with Lepidoptera, the large group of mostly phytophagous insects comprising the moths and butterflies. We inventoried the larval midgut bacteria of a polyphagous foliivore, the gypsy moth (Lymantria dispar L.), whose gut is highly alkaline, by using traditional culturing and culture-independent methods. We also examined the effects of diet on microbial composition. Analysis of individual third-instar larvae revealed a high degree of similarity of microbial composition among insects fed on the same diet. DNA sequence analysis indicated that most of the PCR-amplified 16S rRNA genes belong to the γ-Proteobacteria and low G+C gram-positive divisions and that the cultured members represented more than half of the phylotypes identified. Less frequently detected taxa included members of the α-Proteobacterium, Actinobacterium, and Cytophaga/Flexibacter/Bacteroides divisions. The 16S rRNA gene sequences from 7 of the 15 cultured organisms and 8 of the 9 sequences identified by PCR amplification diverged from previously reported bacterial sequences. The microbial composition of midguts differed substantially among larvae feeding on a sterilized artificial diet, aspen, larch, white oak, or willow. 16S rRNA analysis of cultured isolates indicated that an Enterococcus species and culture-independent analysis indicated that an Entbacter sp. were both present in all larvae, regardless of the feeding substrate; the sequences of these two phylotypes varied less than 1% among individual insects. These results provide the first comprehensive description of the microbial diversity of a lepidopteran midgut and demonstrate that the plant species in the diet influences the composition of the gut bacterial community.

scholarly journals Functional Gene Analysis of Freshwater Iron-Rich Flocs at Circumneutral pH and Isolation of a Stalk-Forming Microaerophilic Iron-Oxidizing Bacterium

2013 ◽  
Vol 79 (17) ◽  
pp. 5283-5290 ◽  
Author(s):  
Shingo Kato ◽  
Clara Chan ◽  
Takashi Itoh ◽  
Moriya Ohkuma
Keyword(s):  
16S Rrna ◽  
Carbon Fixation ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Content Type ◽  
Culture Independent ◽  
Iron Oxidizers ◽  
Circumneutral Ph

ABSTRACTIron-rich flocs often occur where anoxic water containing ferrous iron encounters oxygenated environments. Culture-independent molecular analyses have revealed the presence of 16S rRNA gene sequences related to diverse bacteria, including autotrophic iron oxidizers and methanotrophs in iron-rich flocs; however, the metabolic functions of the microbial communities remain poorly characterized, particularly regarding carbon cycling. In the present study, we cultivated iron-oxidizing bacteria (FeOB) and performed clone library analyses of functional genes related to carbon fixation and methane oxidization (cbbMandpmoA, respectively), in addition to bacterial and archaeal 16S rRNA genes, in freshwater iron-rich flocs at groundwater discharge points. The analyses of 16S rRNA,cbbM, andpmoAgenes strongly suggested the coexistence of autotrophic iron oxidizers and methanotrophs in the flocs. Furthermore, a novel stalk-forming microaerophilic FeOB, strain OYT1, was isolated and characterized phylogenetically and physiologically. The 16S rRNA andcbbMgene sequences of OYT1 are related to those of other microaerophilic FeOB in the familyGallionellaceae, of theBetaproteobacteria, isolated from freshwater environments at circumneutral pH. The physiological characteristics of OYT1 will help elucidate the ecophysiology of microaerophilic FeOB. Overall, this study demonstrates functional roles of microorganisms in iron flocs, suggesting several possible linkages between Fe and C cycling.

scholarly journals Elimination of Bacterial DNA from TaqDNA Polymerases by Restriction Endonuclease Digestion

1999 ◽  
Vol 37 (10) ◽  
pp. 3402-3404 ◽  
Author(s):  
Nora M. Carroll ◽  
Peter Adamson ◽  
Narciss Okhravi
Keyword(s):  
16S Rrna ◽  
Dna Polymerase ◽  
Restriction Enzyme ◽  
Clinical Applications ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Endonuclease Digestion ◽  
Bacterial Dna ◽  
Diagnosis Of Infection ◽  
Endonuclease Digestion

The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S rRNA genes. The method was used prior to a PCR that amplified 10 fg of bacterial DNA. This method can be readily adapted to suit other sensitive PCRs required for clinical applications.

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