scholarly journals Functional characterization of Ralstonia insidiosa, a bona fide resident at the maternal-fetal interface

2019 ◽  
Author(s):  
Lindsay A. Parnell ◽  
Graham G. Willsey ◽  
Chetanchandra S. Joshi ◽  
Yin Yin ◽  
Matthew J. Wargo ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Adverse Outcomes ◽  
Basal Plate ◽  
Functional Importance ◽  
16S Sequencing ◽  
Bona Fide ◽  
Ralstonia Insidiosa

AbstractControversy about whether there are microbes in the placenta and if they have any functional importance during pregnancy and for neonatal health is ongoing. Previous work has demonstrated that the basal plate (BP), comprising maternal and fetal derived cells harbors intracellular bacteria. 16S sequencing and bacterial species-specific analysis of term placentas revealed that the gram-negative bacillus Ralstonia insidiosa, native to aqueous environments and an effective biofilm promoter, comprises the most abundant species in the BP. Here, we demonstrate whether R. insidiosa cells home to a particular niche in the BP, how they may arrive there, and whether they are associated with adverse outcomes. We developed methods to detect and study cell-specific localization of R. insidiosa using ex vivo and in vitro models. Additionally, we studied potential routes of R. insidiosa entry into the placenta. We show that R. insidiosa is a bona fide resident in human placental BP. It can access trophoblast cells in culture and within basal plate tissues where it localizes to intracellular single-membrane vacuoles and can replicate. However, the presence of R. insidiosa does not cause cell death and does not induce a pro-inflammatory immune response suggesting that it is not harmful in and of itself. Finally, we show that in a pregnant mouse model, R. insidiosa traffics to the placenta via the intrauterine route but does not induce preterm labor or preterm birth. Together, our findings provide a foundation for understanding non-pathogenic placental cell-microbe interactions and the functional importance of R. insidiosa in placental health and physiology.

scholarly journals Function-adaptive clustered nanoparticles reverse Streptococcus mutans dental biofilm and maintain microbiota balance

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Fatemeh Ostadhossein ◽  
Parikshit Moitra ◽  
Esra Altun ◽  
Debapriya Dutta ◽  
Dinabandhu Sar ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Ex Vivo ◽  
Bacterial Species ◽  
Reactive Oxygen Species Generation ◽  
Biofilm Inhibition ◽  
16S Rrna Analysis ◽  
Dental Biofilm ◽  
Dental Plaques

AbstractDental plaques are biofilms that cause dental caries by demineralization with acidogenic bacteria. These bacteria reside inside a protective sheath which makes any curative treatment challenging. We propose an antibiotic-free strategy to disrupt the biofilm by engineered clustered carbon dot nanoparticles that function in the acidic environment of the biofilms. In vitro and ex vivo studies on the mature biofilms of Streptococcus mutans revealed >90% biofilm inhibition associated with the contact-mediated interaction of nanoparticles with the bacterial membrane, excessive reactive oxygen species generation, and DNA fragmentation. An in vivo examination showed that these nanoparticles could effectively suppress the growth of S. mutans. Importantly, 16S rRNA analysis of the dental microbiota showed that the diversity and richness of bacterial species did not substantially change with nanoparticle treatment. Overall, this study presents a safe and effective approach to decrease the dental biofilm formation without disrupting the ecological balance of the oral cavity.

scholarly journals Corneal Infection Models: Tools to Investigate the Role of Biofilms in Bacterial Keratitis

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2450
Author(s):  
Lucy Urwin ◽  
Katarzyna Okurowska ◽  
Grace Crowther ◽  
Sanhita Roy ◽  
Prashant Garg ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Bacterial Species ◽  
Resistance Mechanisms ◽  
Bacterial Keratitis ◽  
Corneal Surface ◽  
Corneal Infection ◽  
Infection Models

Bacterial keratitis is a corneal infection which may cause visual impairment or even loss of the infected eye. It remains a major cause of blindness in the developing world. Staphylococcus aureus and Pseudomonas aeruginosa are common causative agents and these bacterial species are known to colonise the corneal surface as biofilm populations. Biofilms are complex bacterial communities encased in an extracellular polymeric matrix and are notoriously difficult to eradicate once established. Biofilm bacteria exhibit different phenotypic characteristics from their planktonic counterparts, including an increased resistance to antibiotics and the host immune response. Therefore, understanding the role of biofilms will be essential in the development of new ophthalmic antimicrobials. A brief overview of biofilm-specific resistance mechanisms is provided, but this is a highly multifactorial and rapidly expanding field that warrants further research. Progression in this field is dependent on the development of suitable biofilm models that acknowledge the complexity of the ocular environment. Abiotic models of biofilm formation (where biofilms are studied on non-living surfaces) currently dominate the literature, but co-culture infection models are beginning to emerge. In vitro, ex vivo and in vivo corneal infection models have now been reported which use a variety of different experimental techniques and animal models. In this review, we will discuss existing corneal infection models and their application in the study of biofilms and host-pathogen interactions at the corneal surface.

scholarly journals Using a Novel Lysin To Help Control Clostridium difficile Infections

2015 ◽  
Vol 59 (12) ◽  
pp. 7447-7457 ◽  
Author(s):  
Qiong Wang ◽  
Chad W. Euler ◽  
Aurelia Delaune ◽  
Vincent A. Fischetti
Keyword(s):  
Clostridium Difficile ◽  
Catalytic Domain ◽  
Ex Vivo ◽  
Bacterial Species ◽  
Lytic Activity ◽  
Content Type ◽  
Intestinal Pathogen ◽  
Drug Treatments ◽  
Hospital Acquired

ABSTRACTAs a consequence of excessive antibiotic therapies in hospitalized patients,Clostridium difficile, a Gram-positive anaerobic spore-forming intestinal pathogen, is the leading cause of hospital-acquired diarrhea and colitis. Drug treatments for these diseases are often complicated by antibiotic-resistant strains and a high frequency of treatment failures and relapse; therefore, novel nonantibiotic approaches may prove to be more effective. In this study, we recombinantly expressed a prophage lysin identified from aC. difficilestrain, CD630, which we named PlyCD. PlyCD was found to have lytic activity against specificC. difficilestrains. However, the recombinantly expressed catalytic domain of this protein, PlyCD1–174, displayed significantly greater lytic activity (>4-log kill) and a broader lytic spectrum againstC. difficilestrains while still retaining a high degree of specificity towardC. difficileversus commensal clostridia and other bacterial species. Our data also indicated that noneffective doses of vancomycin and PlyCD1–174when combinedin vitrocould be significantly more bactericidal againstC. difficile. In anex vivotreatment model of mouse colon infection, we found that PlyCD1–174functioned in the presence of intestinal contents, significantly decreasing colonizingC. difficilecompared to controls. Together, these data suggest that PlyCD1–174has potential as a novel therapeutic for clinical application againstC. difficileinfection, either alone or in combination with other preexisting treatments to improve their efficacy.

[14] IN VITRO AND EX VIVO FUNCTIONAL CHARACTERIZATION ON NEWLY IDENTIFIED ATP BINDING CASSETTE A1 MUTATIONS

2009 ◽  
Vol 19 ◽  
pp. S4
Author(s):  
C. Candini ◽  
R. Franssen ◽  
A.W. Schimmel ◽  
J. Peter ◽  
G.M. Dallinga-Thie ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Functional Characterization ◽  
Atp Binding ◽  
Atp Binding Cassette

scholarly journals Arginine Improves pH Homeostasis via Metabolism and Microbiome Modulation

2017 ◽  
Vol 96 (8) ◽  
pp. 924-930 ◽  
Author(s):  
M. Agnello ◽  
L. Cen ◽  
N.C. Tran ◽  
W. Shi ◽  
J.S. McLean ◽  
...  
Keyword(s):  
Microbial Community ◽  
Bacterial Species ◽  
Oral Microbiome ◽  
Ecological Effect ◽  
Sequencing Analysis ◽  
Ph Homeostasis ◽  
16S Sequencing ◽  
Plaque Ph

Dental caries can be described as a dysbiosis of the oral microbial community, in which acidogenic, aciduric, and acid-adapted bacterial species promote a pathogenic environment, leading to demineralization. Alkali generation by oral microbes, specifically via arginine catabolic pathways, is an essential factor in maintaining plaque pH homeostasis. There is evidence that the use of arginine in dentifrices helps protect against caries. The aim of the current study was to investigate the mechanistic and ecological effect of arginine treatment on the oral microbiome and its regulation of pH dynamics, using an in vitro multispecies oral biofilm model that was previously shown to be highly reflective of the in vivo oral microbiome. Pooled saliva from 6 healthy subjects was used to generate overnight biofilms, reflecting early stages of biofilm maturation. First, we investigated the uptake of arginine by the cells of the biofilm as well as the metabolites generated. We next explored the effect of arginine on pH dynamics by pretreating biofilms with 75 mM arginine, followed by the addition of sucrose (15 mM) after 0, 6, 20, or 48 h. pH was measured at each time point and biofilms were collected for 16S sequencing and targeted arginine quantification, and supernatants were prepared for metabolomic analysis. Treatment with only sucrose led to a sustained pH drop from 7 to 4.5, while biofilms treated with sucrose after 6, 20, or 48 h of preincubation with arginine exhibited a recovery to higher pH. Arginine was detected within the cells of the biofilms, indicating active uptake, and arginine catabolites citrulline, ornithine, and putrescine were detected in supernatants, indicating active metabolism. Sequencing analysis revealed a shift in the microbial community structure in arginine-treated biofilms as well as increased species diversity. Overall, we show that arginine improved pH homeostasis through a remodeling of the oral microbial community.

scholarly journals Downregulation of Rv0189c, encoding a dihydroxyacid dehydratase, affects growth of Mycobacterium tuberculosis in vitro and in mice

Microbiology ◽  
2011 ◽  
Vol 157 (1) ◽  
pp. 38-46 ◽  
Author(s):  
Vinayak Singh ◽  
Deepak Chandra ◽  
Brahm S. Srivastava ◽  
Ranjana Srivastava
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Acid Stress ◽  
Auxotrophic Mutant ◽  
Isoleucine Leucine ◽  
E Coli ◽  
Antisense Mrna

Dihydroxyacid dehydratase (DHAD), a key enzyme involved in branched-chain amino acid (BCAA) biosynthesis, catalyses the synthesis of 2-ketoacids from dihydroxyacids. In Mycobacterium tuberculosis, DHAD is encoded by gene Rv0189c, and it shares 40 % amino acid sequence identity and conserved motifs with DHAD of Escherichia coli encoded by ilvD. In this study, Rv0189c was overexpressed in E. coli and the resultant protein was characterized as a homodimer (∼155 kDa). Functional characterization of Rv0189c was established by biochemical testing and by genetic complementation of an intron-disrupted ilvD-auxotrophic mutant of E. coli to prototrophy. Growth of M. tuberculosis, E. coli BL21(DE3) and recombinant E. coli BL21(DE3) ΔilvD carrying Rv0189c was inhibited by transient nitric oxide (NO) exposure in minimal medium but growth was restored if the medium was supplemented with BCAA (isoleucine, leucine and valine). This suggested that inactivation of Rv0189c by NO probably inhibited bacterial growth. The role of Rv0189c in M. tuberculosis was elucidated by antisense and sense RNA constructs. Growth of M. tuberculosis transformed with a plasmid encoding antisense mRNA was markedly poor in the lungs of infected mice and in Middlebrook 7H9 broth compared to that of sense and vector-alone transformants, but growth was normal when the medium was supplemented with BCAA. Upregulation of Rv0189c was observed during the early exponential phase of growth, under acid stress and ex vivo, suggesting that Rv0189c has a role in the survival of M. tuberculosis during normal and stress conditions. It may be concluded that the DHAD encoded by Rv0189c is essential for the survival of M. tuberculosis and could be a potential drug/vaccine target, as it is absent in mammals.

scholarly journals Engineered phage endolysin eliminates Gardnerella in bacterial vaginosis without damaging the healthy vaginal microbiome

2020 ◽  
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Agnieszka Latka ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Ex Vivo ◽  
Bacterial Species ◽  
Drug Candidate ◽  
Vaginal Microbiome ◽  
Promising Alternative ◽  
Binding Domains ◽  
Cell Wall Binding ◽  
Homologous Genes

BackgroundBacterial vaginosis is characterized by an imbalance of the vaginal microbiome in which the normally predominant lactobacilli are replaced by other bacterial species. Initiated by strains of the bacterium Gardnerella, a characteristic biofilm forms on the vaginal epithelium, explaining the typical presence of clue cells. This biofilm contributes to the resilience of the bacteria to antibiotic treatment, which may explain the frequent recurrence of BV.ObjectiveIn this study, we investigate whether a therapy based on bacteriophage endolysins which specifically lyse Gardnerella, in particular the drug candidate PM-477, might be a promising alternative to broad-spectrum antibiotics and antiseptics.Study designTo identify Gardnerella specific endolysins, we searched for endolysin-encoding sequences in regions of Gardnerella genomes that are of prophage origin. This search identified fourteen homologous genes predicted to encode 1,4-beta-N-acetylmuramidase-type endolysins. When expressed in Escherichia coli and purified, the recombinant proteins demonstrated strong bactericidal activity against four different Gardnerella species. By shuffling the N-terminal catalytic domains and C-terminal cell wall-binding domains between the homologues, we produced 81 chimeric endolysins. These endolysins were tested for their activity and specificity in vitro and ex-vivo on vaginal samples from fifteen BV positive patients. Fluorescence in situ hybridization was used for visualization.ResultsSeveral engineered endolysins were 10-fold more active than the most active wild-type enzymes. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/ml. PM-477 had no effect on Lactobacillus strains or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested on vaginal samples from fifteen patients with either first time or recurring bacterial vaginosis. In fourteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome.ConclusionThe high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising drug candidate and an alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

scholarly journals Composition of the Gut Microbiome Influences Production of Sulforaphane-Nitrile and Iberin-Nitrile from Glucosinolates in Broccoli Sprouts

Nutrients ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 3013
Author(s):  
John A. Bouranis ◽  
Laura M. Beaver ◽  
Jaewoo Choi ◽  
Carmen P. Wong ◽  
Duo Jiang ◽  
...  
Keyword(s):  
Gut Microbiome ◽  
Ex Vivo ◽  
Human Subjects ◽  
Individual Variability ◽  
16S Sequencing ◽  
Microbiome Composition ◽  
Broccoli Sprouts ◽  
Broccoli Sprout

Isothiocyanates, such as sulforaphane and iberin, derived from glucosinolates (GLS) in cruciferous vegetables, are known to prevent and suppress cancer development. GLS can also be converted by bacteria to biologically inert nitriles, such as sulforaphane-nitrile (SFN-NIT) and iberin-nitrile (IBN-NIT), but the role of the gut microbiome in this process is relatively undescribed and SFN-NIT excretion in humans is unknown. An ex vivo fecal incubation model with in vitro digested broccoli sprouts and 16S sequencing was utilized to explore the role of the gut microbiome in SFN- and IBN-NIT production. SFN-NIT excretion was measured among human subjects following broccoli sprout consumption. The fecal culture model showed high inter-individual variability in nitrile production and identified two sub-populations of microbial communities among the fecal cultures, which coincided with a differing abundance of nitriles. The Clostridiaceae family was associated with high levels, while individuals with a low abundance of nitriles were more enriched with taxa from the Enterobacteriaceae family. High levels of inter-individual variation in urine SFN-NIT levels were also observed, with peak excretion of SFN-NIT at 24 h post broccoli sprout consumption. These results suggest that nitrile production from broccoli, as opposed to isothiocyanates, could be influenced by gut microbiome composition, potentially lowering efficacy of cruciferous vegetable interventions.

scholarly journals Adult human circulating CD34−Lin−CD45−CD133− cells can differentiate into hematopoietic and endothelial cells

Blood ◽  
2011 ◽  
Vol 118 (8) ◽  
pp. 2105-2115 ◽  
Author(s):  
Elisa Ciraci ◽  
Silvia Della Bella ◽  
Ombretta Salvucci ◽  
Cristina Rofani ◽  
Marta Segarra ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Adult Life ◽  
Hematopoietic Tissue ◽  
Developmental Potential ◽  
Adult Human

Abstract A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34−Lin−CD45−CD133− cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34−Lin−CD45−CD133− cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34−Lin−CD45−CD133− cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34−Lin−CD45−CD133− cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34−Lin−CD45−CD133− cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.

scholarly journals Isolation and functional characterization of two dioxygenasese putatively involved in bixin biosynthesis in annatto (Bixa orellana L.)

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7064 ◽  
Author(s):  
Victor Manuel Carballo-Uicab ◽  
Yair Cárdenas-Conejo ◽  
Alba Adriana Vallejo-Cardona ◽  
Margarita Aguilar-Espinosa ◽  
Jacobo Rodríguez-Campos ◽  
...  
Keyword(s):  
Food Industry ◽  
Developmental Stages ◽  
Functional Characterization ◽  
Economic Value ◽  
Bixa Orellana ◽  
Reverse Transcription Pcr ◽  
Bona Fide

Carotenoid cleavage dioxygenases (CCDs) are enzymes that have been implicated in the biosynthesis of a wide diversity of secondary metabolites with important economic value, including bixin. Bixin is the second most used pigment in the world’s food industry worldwide, and its main source is the aril of achiote (Bixa orellana L.) seeds. A recent transcriptome analysis of B. orellana identified a new set of eight CCD members (BoCCD4s and BoCCD1s) potentially involved in bixin synthesis. We used several approaches in order to discriminate the best candidates with CCDs genes. A reverse transcription-PCR (RT-qPCR) expression analysis was carried out in five developmental stages of two accessions of B. orellana seeds with different bixin contents: (P13W, low bixin producer and N4P, high bixin producer). The results showed that three BoCCDs (BoCCD4-1, BoCCD4-3, and BoCCD1-1) had an expression pattern consistent with bixin accumulation during seed development. Additionally, an alignment of the CCD enzyme family and homology models of proteins were generated to verify whether the newly proposed CCD enzymes were bona fide CCDs. The study confirmed that these three enzymes were well-preserved and belonged to the CCD family. In a second selection round, the three CCD genes were analyzed by in situ RT-qPCR in seed tissue. Results indicated that BoCCD4-3 and BoCCD1-1 exhibited tissue-specific expressions in the seed aril. To test whether the two selected CCDs had enzymatic activity, they were expressed in Escherichia coli; activity was determined by identifying their products in the crude extract using UHPLC-ESI-QTOF-MS/MS. The cleavage product (bixin aldehyde) was also analyzed by Fourier transform infrared. The results indicated that both BoCCD4-3 and BoCCD1-1 cleave lycopene in vitro at 5,6-5′,6′.

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