scholarly journals No Consistent Evidence for Microbiota in Murine Placental and Fetal Tissues

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Kevin R. Theis ◽  
Roberto Romero ◽  
Jonathan M. Greenberg ◽  
Andrew D. Winters ◽  
Valeria Garcia-Flores ◽  
...  
Keyword(s):  
Bacterial Isolate ◽  
Fetal Brain ◽  
16S Rrna Gene Sequencing ◽  
Fetal Tissue ◽  
In Utero ◽  
Rrna Gene ◽  
Tissue Samples ◽  
Fetal Tissues ◽  
Rrna Gene Sequencing ◽  
Colonization Hypothesis

ABSTRACT The existence of a placental microbiota and in utero colonization of the fetus have been the subjects of recent debate. The objective of this study was to determine whether the placental and fetal tissues of mice harbor bacterial communities. Bacterial profiles of the placenta and fetal brain, lung, liver, and intestine samples were characterized through culture, quantitative real-time PCR (qPCR), and 16S rRNA gene sequencing. These profiles were compared to those of the maternal mouth, lung, liver, uterus, cervix, vagina, and intestine, as well as to background technical controls. Positive bacterial cultures from placental and fetal tissue samples were rare; of the 165 total bacterial cultures of placental tissue samples from the 11 mice included in this study, only nine yielded at least a single colony, and five of those nine positive cultures came from a single mouse. Cultures of fetal intestinal tissue samples yielded just a single bacterial isolate, Staphylococcus hominis, a common skin bacterium. Bacterial loads of placental and fetal brain, lung, liver, and intestinal tissues were not higher than those of DNA contamination controls and did not yield substantive 16S rRNA gene sequencing libraries. From all placental or fetal tissue samples (n = 51), there was only a single bacterial isolate that came from a fetal brain sample having a bacterial load higher than that of contamination controls and that was identified in sequence-based surveys of at least one of its corresponding maternal samples. Therefore, using multiple modes of microbiological inquiry, there was not consistent evidence of bacterial communities in the placental and fetal tissues of mice. IMPORTANCE The prevailing paradigm in obstetrics has been the sterile womb hypothesis, which posits that fetuses are first colonized by microorganisms during the delivery process. However, some are now suggesting that fetuses are consistently colonized in utero by microorganisms from microbial communities that inhabit the placenta and intra-amniotic environment. Given the established causal role of microbial invasion of the amniotic cavity (i.e., intra-amniotic infection) in pregnancy complications, especially preterm birth, if the in utero colonization hypothesis were true, there are several aspects of current understanding that will need to be reconsidered; these aspects include the magnitude of intra-amniotic microbial load required to cause disease and its potential influence on the ontogeny of the immune system. However, acceptance of the in utero colonization hypothesis is premature. Herein, we do not find consistent evidence for placental and fetal microbiota in mice using culture, qPCR, and DNA sequencing.

scholarly journals No consistent evidence for microbiota in murine placental and fetal tissues

2019 ◽  
Author(s):  
Kevin R. Theis ◽  
Roberto Romero ◽  
Jonathan M. Greenberg ◽  
Andrew D. Winters ◽  
Valeria Garcia-Flores ◽  
...  
Keyword(s):  
Bacterial Communities ◽  
Bacterial Isolate ◽  
Fetal Brain ◽  
16S Rrna Gene Sequencing ◽  
In Utero ◽  
Rrna Gene ◽  
Bacterial Cultures ◽  
Fetal Tissues ◽  
Rrna Gene Sequencing ◽  
Colonization Hypothesis

ABSTRACTThe existence of a placental microbiota and in utero colonization of the fetus has been the subject of recent debate. The objective of this study was to determine whether the placental and fetal tissues of mice harbor bacterial communities. Bacterial profiles of the placenta and fetal brain, lung, liver, and intestine were characterized through culture, qPCR, and 16S rRNA gene sequencing. These profiles were compared to those of the maternal mouth, lung, liver, uterus, cervix, vagina, and intestine, as well as to background technical controls. Positive bacterial cultures from placental and fetal tissues were rare; of the 165 total bacterial cultures of placental tissues from the 11 mice included in this study, only nine yielded at least a single colony, and five of those nine positive cultures came from a single mouse. Cultures of fetal intestinal tissues yielded just a single bacterial isolate: Staphylococcus hominis, a common skin bacterium. Bacterial loads of placental and fetal brain, lung, liver, and intestinal tissues were not higher than those of DNA contamination controls and did not yield substantive 16S rRNA gene sequencing libraries. From all placental or fetal tissues (N = 49), there was only a single bacterial isolate that came from a fetal brain sample having a bacterial load higher than that of contamination controls and that was identified in sequence-based surveys of at least one of its corresponding maternal samples. Therefore, using multiple modes of microbiologic inquiry, there was not consistent evidence of bacterial communities in the placental and fetal tissues of mice.IMPORTANCEThe prevailing paradigm in obstetrics has been the sterile womb hypothesis, which posits that fetuses are first colonized by microorganisms during the delivery process. However, some are now suggesting that fetuses are consistently colonized by microorganisms in utero by microbial communities that inhabit the placenta and intra-amniotic environment. Given the established causal role of microbial invasion of the amniotic cavity (i.e. intra-amniotic infection) in pregnancy complications, especially preterm birth, if the in utero colonization hypothesis were true, there are several aspects of current understanding that will need to be reconsidered including the magnitude of intra-amniotic microbial load required to cause disease and their potential influence on the ontogeny of the immune system. However, acceptance of the in utero colonization hypothesis is premature. Herein, we do not find consistent evidence for placental and fetal microbiota in mice using culture, qPCR, and DNA sequencing.

scholarly journals Streptococcus australis and Ralstonia pickettii as Major Microbiota in Mesotheliomas

2021 ◽  
Vol 11 (4) ◽  
pp. 297
Author(s):  
Rumi Higuchi ◽  
Taichiro Goto ◽  
Yosuke Hirotsu ◽  
Sotaro Otake ◽  
Toshio Oyama ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Pcr Amplification ◽  
16S Rrna Gene Sequencing ◽  
Abundant Species ◽  
Rrna Gene ◽  
Tissue Samples ◽  
Ralstonia Pickettii ◽  
Operational Taxonomic Units ◽  
Rrna Gene Sequencing ◽  
Almost All

The microbiota has been reported to be correlated with carcinogenesis and cancer progression. However, its involvement in the pathology of mesothelioma remains unknown. In this study, we aimed to identify mesothelioma-specific microbiota using resected or biopsied mesothelioma samples. Eight mesothelioma tissue samples were analyzed via polymerase chain reaction (PCR) amplification and 16S rRNA gene sequencing. The operational taxonomic units (OTUs) of the effective tags were analyzed in order to determine the taxon composition of each sample. For the three patients who underwent extra pleural pneumonectomy, normal peripheral lung tissues adjacent to the tumor were also included, and the same analysis was performed. In total, 61 OTUs were identified in the tumor and lung tissues, which were classified into 36 species. Streptococcus australis and Ralstonia pickettii were identified as abundant species in almost all tumor and lung samples. Streptococcus australis and Ralstonia pickettii were found to comprise mesothelioma-specific microbiota involved in tumor progression; thus, they could serve as targets for the prevention of mesothelioma.

scholarly journals Microbiome in lung explants of idiopathic pulmonary fibrosis: a case–control study in patients with end-stage fibrosis

Thorax ◽  
2017 ◽  
Vol 73 (5) ◽  
pp. 481-484 ◽  
Author(s):  
Georgios D Kitsios ◽  
Mauricio Rojas ◽  
Daniel J Kass ◽  
Adam Fitch ◽  
John C Sembrat ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Case Control Study ◽  
16S Rrna Gene Sequencing ◽  
Case Control ◽  
Rrna Gene ◽  
Tissue Samples ◽  
Rrna Gene Sequencing ◽  
End Stage ◽  
Control Study

The microbiome has been proposed to play a role in the progression of idiopathic pulmonary fibrosis (IPF) based on bronchoalveolar lavage analyses, but the microbiome of lung tissue in IPF has not been explored. In a case–control study of lung explants analysed by 16S rRNA gene sequencing, we could not reliably detect bacterial DNA in basilar tissue samples from patients with either chronic or acute exacerbations of IPF, in contrast to control candidate-donor lungs or cystic fibrosis explants. Thus, our data do not indicate microbiome alterations in regions of IPF lung with advanced fibrosis.

scholarly journals Fetal Tissues Tested for Microbial Sterility by Culture- and PCR-Based Methods Can be Safely Used in Clinics

2017 ◽  
Vol 26 (2) ◽  
pp. 339-350 ◽  
Author(s):  
Yakov Vitrenko ◽  
Iryna Kostenko ◽  
Kateryna Kulebyakina ◽  
Alla Duda ◽  
Mariya Klunnyk ◽  
...  
Keyword(s):  
16S Rrna ◽  
Soft Tissues ◽  
Fetal Tissue ◽  
Rrna Gene ◽  
Tissue Samples ◽  
Fetal Tissues ◽  
Microbiological Control ◽  
Pcr Products ◽  
Human Fetal Tissues ◽  
Taxonomic Groups

Cell preparations to be used in clinical practice must be free of infectious agents. Safety concerns are especially elevated upon the use of human fetal tissues, which are otherwise highly advantageous in cell therapy. We demonstrate that treating fetal samples with antibiotic, extensive washing, and homogenization prior to cryoconservation efficiently removes microbes in general. Screening a large collection by an automatic culture system showed that 89.2% fetal tissue samples were sterile, while contamination was detected in 10.8% samples. Liver and chorion were contaminated more than the brain, kidney, lung, and soft tissues. Broad-range PCR from the bacterial 16s rRNA gene was adopted as a confirmatory assay; however, the concordance between the culture-based and PCR assays was weak. Taxonomic identification was done for contaminated samples by bacteriological methods and sequencing 16s rRNA PCR products. The two approaches revealed different spectra of taxonomic groups sharing only Lactobacillus, the most frequently found genus. In addition, other representatives of vaginal microbiota were detected by culture-based identification, while PCR product sequencing has also revealed a subset of nosocomial microorganisms. Importantly, species known to cause sepsis were identified by both techniques, arguing for their indispensability and mutual complementarity. We suggest that most contaminations are taken up during collection of fetal material rather than originating from an in utero infection. In conclusion, a rigorous microbiological control by culture and PCR is a prerequisite for safe clinical use of fetal tissue suspensions.

Screening and Molecular Characterization of Cellulase Producing Actinobacteria from Litchi Orchard

2019 ◽  
Vol 13 (1) ◽  
pp. 90-101
Author(s):  
Sanju Kumari ◽  
Utkarshini Sharma ◽  
Rohit Krishna ◽  
Kanak Sinha ◽  
Santosh Kumar
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Evolutionary Relationship ◽  
Gene Sequencing ◽  
Cellulase Activity ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Background: Cellulolysis is of considerable economic importance in laundry detergents, textile and pulp and paper industries and in fermentation of biomass into biofuels. Objective: The aim was to screen cellulase producing actinobacteria from the fruit orchard because of its requirement in several chemical reactions. Methods: Strains of actinobacteria were isolated on Sabouraud’s agar medium. Similarities in cultural and biochemical characterization by growing the strains on ISP medium and dissimilarities among them perpetuated to recognise nine groups of actinobacteria. Cellulase activity was measured by the diameter of clear zone around colonies on CMC agar and the amount of reducing sugar liberated from carboxymethyl cellulose in the supernatant of the CMC broth. Further, 16S rRNA gene sequencing and molecular characterization were placed before NCBI for obtaining recognition with accession numbers. Results: Prominent clear zones on spraying Congo Red were found around the cultures of strains of three groups SK703, SK706, SK708 on CMC agar plates. The enzyme assay for carboxymethylcellulase displayed extra cellulase activity in broth: 0.14, 0.82 and 0.66 µmol mL-1 min-1, respectively at optimum conditions of 35°C, pH 7.3 and 96 h of incubation. However, the specific cellulase activities per 1 mg of protein did not differ that way. It was 1.55, 1.71 and 1.83 μmol mL-1 min-1. The growing mycelia possessed short compact chains of 10-20 conidia on aerial branches. These morphological and biochemical characteristics, followed by their verification by Bergey’s Manual, categorically allowed the strains to be placed under actinobacteria. Further, 16S rRNA gene sequencing, molecular characterization and their evolutionary relationship through phylogenetics also confirmed the putative cellulase producing isolates of SK706 and SK708 subgroups to be the strains of Streptomyces. These strains on getting NCBI recognition were christened as Streptomyces glaucescens strain SK91L (KF527284) and Streptomyces rochei strain SK78L (KF515951), respectively. Conclusion: Conclusive evidence on the basis of different parameters established the presence of cellulase producing actinobacteria in the litchi orchard which can convert cellulose into fermentable sugar.

scholarly journals Diversity, Composition and Functional Inference of Gut Microbiota in Indian Cabbage white Pieris canidia (Lepidoptera: Pieridae)

Life ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 254
Author(s):  
Ying Wang ◽  
Jianqing Zhu ◽  
Jie Fang ◽  
Li Shen ◽  
Shuojia Ma ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
16S Rrna Gene Sequencing ◽  
Adult Survival ◽  
Rrna Gene ◽  
Life Stages ◽  
Microbial Composition ◽  
Significant Difference ◽  
Functional Inference ◽  
Rrna Gene Sequencing ◽  
Insect Fitness

We characterized the gut microbial composition and relative abundance of gut bacteria in the larvae and adults of Pieris canidia by 16S rRNA gene sequencing. The gut microbiota structure was similar across the life stages and sexes. The comparative functional analysis on P. canidia bacterial communities with PICRUSt showed the enrichment of several pathways including those for energy metabolism, immune system, digestive system, xenobiotics biodegradation, transport, cell growth and death. The parameters often used as a proxy of insect fitness (development time, pupation rate, emergence rate, adult survival rate and weight of 5th instars larvae) showed a significant difference between treatment group and untreated group and point to potential fitness advantages with the gut microbiomes in P. canidia. These data provide an overall view of the bacterial community across the life stages and sexes in P. canidia.

scholarly journals Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson’s disease individuals

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Janis R. Bedarf ◽  
Naiara Beraza ◽  
Hassan Khazneh ◽  
Ezgi Özkurt ◽  
David Baker ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
False Positive ◽  
Gene Sequencing ◽  
Bacterial Biomass ◽  
16S Rrna Gene Sequencing ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Abstract Background Recent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson’s disease (PD) and Alzheimer’s disease (AD) or as a “brain microbiome” in the context of healthy patients’ brain samples. Methods Using 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR. Results Amplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination. We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue’s sterility and could also be found in independent brain 16S rRNA gene sequences. Conclusions Taxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains.

scholarly journals The growth response of rice (Oryza sativa L. var. FARO 44) in vitro after inoculation with bacterial isolates from a typical ferruginous ultisol

2021 ◽  
Vol 45 (1) ◽  
Author(s):  
Musa Saheed Ibrahim ◽  
Beckley Ikhajiagbe
Keyword(s):  
16S Rrna ◽  
Bacillus Cereus ◽  
Proteus Mirabilis ◽  
Growth Response ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rice Seedlings ◽  
Rrna Gene Sequencing

Abstract Background Rice forms a significant portion of food consumed in most household worldwide. Rice production has been hampered by soil factors such as ferruginousity which has limited phosphorus availability; an important mineral component for the growth and yield of rice. The presence of phosphate-solubilizing bacteria (PSB) in soils has been reported to enhance phosphate availability. In view of this, the present study employed three bacteria species (BCAC2, EMBF2 and BCAF1) that were previously isolated and proved P solubilization capacities as inocula to investigate the growth response of rice germinants in an in vitro setup. The bacteria isolates were first identified using 16S rRNA gene sequencing and then applied as inoculum. The inolula were prepared in three concentrations (10, 7.5 and 5.0 ml) following McFarland standard. Viable rice (var. FARO 44) seeds were sown in petri dishes and then inoculated with the three inocula at the different concentrations. The setup was studied for 28 days. Results 16S rRNA gene sequencing identified the isolates as: isolate BCAC2= Bacillus cereus strain GGBSU-1, isolate BCAF1= Proteus mirabilis strain TL14-1 and isolate EMBF2= Klebsiella variicola strain AUH-KAM-9. Significant improvement in rice germination, morphology, physiology and biomass parameters in the bacteria-inoculated setups was observed compared to the control. Germination percentage after 4 days was 100 % in the inoculated rice germinants compared to 65% in the control (NiS). Similarly, inoculation with the test isolates enhanced water-use efficiency by over 40%. The rice seedlings inoculated with Bacillus cereus strain GGBSU-1 (BiS) showed no signs of chlorosis and necrosis throughout the study period as against those inoculated with Proteus mirabilis strain TL14-1 (PiS) and Klebsiella variicola strain AUH-KAM-9 (KiS). Significant increase in chlorophyll-a, chlorophyll-b and alpha amylase was observed in the rice seedlings inoculated with BiS as against the NiS. Conclusion Inoculating rice seeds with Bacillus cereus strain GGBSU-1, Proteus mirabilis strain TL14-1 and Klebsiella variicola strain AUH-KAM-9 in an in vitro media significantly improved growth parameters of the test plant. Bacillus cereus strain GGBSU-1 showed higher efficiency due to a more improved growth properties observed.

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