scholarly journals Interindividual Variation in Dietary Carbohydrate Metabolism by Gut Bacteria Revealed with Droplet Microfluidic Culture

mSystems ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Max M. Villa ◽  
Rachael J. Bloom ◽  
Justin D. Silverman ◽  
Heather K. Durand ◽  
Sharon Jiang ◽  
...  
Keyword(s):  
Bacterial Culture ◽  
Therapeutic Potential ◽  
Bacterial Species ◽  
Experimental Studies ◽  
Gut Bacteria ◽  
Droplet Microfluidics ◽  
Community Diversity ◽  
Interindividual Variation ◽  
Culture Methods ◽  
Carbohydrate Utilization

ABSTRACT Culture and screening of gut bacteria enable testing of microbial function and therapeutic potential. However, the diversity of human gut microbial communities (microbiota) impedes comprehensive experimental studies of individual bacterial taxa. Here, we combine advances in droplet microfluidics and high-throughput DNA sequencing to develop a platform for separating and assaying growth of microbiota members in picoliter droplets (MicDrop). MicDrop enabled us to cultivate 2.8 times more bacterial taxa than typical batch culture methods. We then used MicDrop to test whether individuals possess similar abundances of carbohydrate-degrading gut bacteria, using an approach which had previously not been possible due to throughput limitations of traditional bacterial culture techniques. Single MicDrop experiments allowed us to characterize carbohydrate utilization among dozens of gut bacterial taxa from distinct human stool samples. Our aggregate data across nine healthy stool donors revealed that all of the individuals harbored gut bacterial species capable of degrading common dietary polysaccharides. However, the levels of richness and abundance of polysaccharide-degrading species relative to monosaccharide-consuming taxa differed by up to 2.6-fold and 24.7-fold, respectively. Additionally, our unique dataset suggested that gut bacterial taxa may be broadly categorized by whether they can grow on single or multiple polysaccharides, and we found that this lifestyle trait is correlated with how broadly bacterial taxa can be found across individuals. This demonstration shows that it is feasible to measure the function of hundreds of bacterial taxa across multiple fecal samples from different people, which should in turn enable future efforts to design microbiota-directed therapies and yield new insights into microbiota ecology and evolution. IMPORTANCE Bacterial culture and assay are components of basic microbiological research, drug development, and diagnostic screening. However, community diversity can make it challenging to comprehensively perform experiments involving individual microbiota members. Here, we present a new microfluidic culture platform that makes it feasible to measure the growth and function of microbiota constituents in a single set of experiments. As a proof of concept, we demonstrate how the platform can be used to measure how hundreds of gut bacterial taxa drawn from different people metabolize dietary carbohydrates. Going forward, we expect this microfluidic technique to be adaptable to a range of other microbial assay needs.

scholarly journals Harvesting of Prebiotic Fructooligosaccharides by Nonbeneficial Human Gut Bacteria

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Zhi Wang ◽  
Alexandra S. Tauzin ◽  
Elisabeth Laville ◽  
Pietro Tedesco ◽  
Fabien Létisse ◽  
...  
Keyword(s):  
Genetic Locus ◽  
Bacterial Species ◽  
Strain Engineering ◽  
Gut Bacteria ◽  
Carbohydrate Binding ◽  
Phosphotransferase System ◽  
Human Gut ◽  
Carbohydrate Utilization ◽  
New Findings

ABSTRACT Prebiotic oligosaccharides, such as fructooligosaccharides, are increasingly being used to modulate the composition and activity of the gut microbiota. However, carbohydrate utilization analyses and metagenomic studies recently revealed the ability of deleterious and uncultured human gut bacterial species to metabolize these functional foods. Moreover, because of the difficulties of functionally profiling transmembrane proteins, only a few prebiotic transporters have been biochemically characterized to date, while carbohydrate binding and transport are the first and thus crucial steps in their metabolization. Here, we describe the molecular mechanism of a phosphotransferase system, highlighted as a dietary and pathology biomarker in the human gut microbiome. This transporter is encoded by a metagenomic locus that is highly conserved in several human gut Firmicutes, including Dorea species. We developed a generic strategy to deeply analyze, in vitro and in cellulo, the specificity and functionality of recombinant transporters in Escherichia coli, combining carbohydrate utilization locus and host genome engineering and quantification of the binding, transport, and growth rates with analysis of phosphorylated carbohydrates by mass spectrometry. We demonstrated that the Dorea fructooligosaccharide transporter is specific for kestose, whether for binding, transport, or phosphorylation. This constitutes the biochemical proof of effective phosphorylation of glycosides with a degree of polymerization of more than 2, extending the known functional diversity of phosphotransferase systems. Based on these new findings, we revisited the classification of these carbohydrate transporters. IMPORTANCE Prebiotics are increasingly used as food supplements, especially in infant formulas, to modify the functioning and composition of the microbiota. However, little is currently known about the mechanisms of prebiotic recognition and transport by gut bacteria, while these steps are crucial in their metabolism. In this study, we established a new strategy to profile the specificity of oligosaccharide transporters, combining microbiomics, genetic locus and strain engineering, and state-of-the art metabolomics. We revisited the transporter classification database and proposed a new way to classify these membrane proteins based on their structural and mechanistic similarities. Based on these developments, we identified and characterized, at the molecular level, a fructooligosaccharide transporting phosphotransferase system, which constitutes a biomarker of diet and gut pathology. The deciphering of this prebiotic metabolization mechanism by a nonbeneficial bacterium highlights the controversial use of prebiotics, especially in the context of chronic gut diseases.

scholarly journals High-throughput isolation and culture of human gut bacteria with droplet microfluidics

2019 ◽  
Author(s):  
Max M Villa ◽  
Rachael J Bloom ◽  
Justin D Silverman ◽  
Heather K Durand ◽  
Sharon Jiang ◽  
...  
Keyword(s):  
High Throughput ◽  
Community Dynamics ◽  
Experimental Studies ◽  
Gut Bacteria ◽  
Droplet Microfluidics ◽  
Human Gut ◽  
Single Experiment ◽  
High Throughput Dna Sequencing ◽  
Metabolic Variation ◽  
Kinetics Of

AbstractIsolation and culture of gut bacteria enable testing for microbial roles in disease and may also lead to novel therapeutics. However, the diversity of human gut microbial communities (microbiota) impedes comprehensive experimental studies of individual bacterial taxa. Here, we combine advances in droplet microfluidics and high-throughput DNA sequencing to develop a platform for isolating and assaying microbiota members in picoliter droplets (MicDrop). MicDrop can be used to create millions of distinct bacterial colonies in a single experiment while using off-the-shelf parts compact enough to fit in an anaerobic chamber. In proof-of-concept experiments, we used the platform to characterize inter-individual metabolic variation among hundreds of polysaccharide-degrading gut bacteria from nine stool donors. We also used MicDrop to test the hypothesis that growth kinetics of individual gut bacterial taxa are associated with longterm community dynamics in an artificial gut. These demonstrations suggest the MicDrop platform could support future diagnostic efforts to personalize microbiota-directed therapies, as well as to provide comprehensive new insights into the ecology of human gut microbiota.

scholarly journals Phenotypic and phylogenetic characterization of Lactobacillus species isolated from traditional Lighvan cheese

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Haleh Forouhandeh ◽  
Sepideh Zununi Vahed ◽  
Hossein Ahangari ◽  
Vahideh Tarhriz ◽  
Mohammad Saeid Hejazi
Keyword(s):  
16S Rrna ◽  
Random Amplified Polymorphic Dna ◽  
Bacterial Species ◽  
16S Rrna Sequencing ◽  
Lactobacillus Species ◽  
Culture Methods ◽  
Phylogenetic Characterization ◽  
Rrna Sequencing ◽  
Specific Culture ◽  
Lighvan Cheese

Abstract Lighvan cheese (Lighvan panir) is among the most famous traditional cheese in Iran for its desired aroma and flavor. Undoubtedly, the lactic acid bacteria especially the genus Lactobacillus are the critical factors in developing the aroma, flavor, and texture in Lighvan cheese. In this study, the Lactobacillus population of the main Lighvan cheese was investigated. The Lactobacillus of the main Lighvan cheese was isolated using specific culture methods according to previously published Guidelines. Then, the phylogenetic features were investigated and the phenotypic characteristics were examined using specific culture methods. Twenty-eight Gram-positive bacterial species were identified belonged to the genus Lactobacillus. According to the same sequences as each other, three groups (A, B, and C) of isolates were categorized with a high degree of similarity to L. fermentum (100%) and L. casei group (L. casei, L. paracasei, and L. rhamnosus) (99.0 to 100%). Random amplified polymorphic DNA (RAPD) fingerprint analysis manifested the presence of three clusters that were dominant in traditional Lighvan cheese. Cluster І was divided into 4 sub-clusters. By the result of carbohydrate fermentation pattern and 16S rRNA sequencing, isolates were identified as L. rhamnosus. The isolates in clusters II and III represented L. paracasei and L. fermentum, respectively as they were identified by 16S rRNA sequencing and fermented carbohydrate patterns. Our result indicated that the specific aroma and flavor of traditional Lighvan cheese can be related to its Lactobacillus population including L. fermentum, L. casei, L. paracasei, and L. rhamnosus. Graphical abstract

scholarly journals Antimicrobial Resistance among Neonates with Bacterial Sepsis and Their Clinical Outcomes in a Tertiary Hospital in Kathmandu Valley, Nepal

2021 ◽  
Vol 6 (2) ◽  
pp. 56
Author(s):  
Bijendra Raj Raghubanshi ◽  
Karuna D. Sagili ◽  
Wai Wai Han ◽  
Henish Shakya ◽  
Priyanka Shrestha ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Neonatal Sepsis ◽  
Bacterial Culture ◽  
Bacterial Species ◽  
Antibiotic Sensitivity ◽  
College Teaching ◽  
Hospital Treatment ◽  
Resistant Bacteria ◽  
Coagulase Negative Staphylococcus ◽  
Cross Sectional

Globally, antibiotic resistance in bacteria isolated from neonatal sepsis is increasing. In this cross-sectional study conducted at a medical college teaching hospital in Nepal, we assessed the antibiotic resistance levels in bacteria cultured from neonates with sepsis and their in-hospital treatment outcomes. We extracted data of neonates with sepsis admitted for in-patient care from June 2018 to December 2019 by reviewing hospital records of the neonatal intensive care unit and microbiology department. A total of 308 neonates with sepsis were admitted of which, blood bacterial culture antibiotic sensitivity reports were available for 298 neonates. Twenty neonates (7%) had bacteriologic culture-confirmed neonatal sepsis. The most common bacterial species isolated were Staphylococcus aureus (8), followed by coagulase-negative Staphylococcus (5). Most of these bacteria were resistant to at least one first-line antibiotic used to manage neonatal sepsis. Overall, there were 7 (2%) deaths among the 308 neonates (none of them from the bacterial culture-positive group), and 53 (17%) neonates had left the hospital against medical advice (LAMA). Improving hospital procedures to isolate bacteria in neonates with sepsis, undertaking measures to prevent the spread of antibiotic-resistant bacteria, and addressing LAMA’s reasons are urgently needed.

scholarly journals N-Acetylcysteine (NAC): Impacts on Human Health

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 967
Author(s):  
Micaely Cristina dos Santos Tenório ◽  
Nayara Gomes Graciliano ◽  
Fabiana Andréa Moura ◽  
Alane Cabral Menezes de Oliveira ◽  
Marília Oliveira Fonseca Goulart
Keyword(s):  
Human Health ◽  
Therapeutic Potential ◽  
Experimental Studies ◽  
Primary Role ◽  
Necrosis Factor Alpha ◽  
Biochemical Basis ◽  
Tnf Α ◽  
Factor Alpha ◽  
Anti Inflammatory

N-acetylcysteine (NAC) is a medicine widely used to treat paracetamol overdose and as a mucolytic compound. It has a well-established safety profile, and its toxicity is uncommon and dependent on the route of administration and high dosages. Its remarkable antioxidant and anti-inflammatory capacity is the biochemical basis used to treat several diseases related to oxidative stress and inflammation. The primary role of NAC as an antioxidant stems from its ability to increase the intracellular concentration of glutathione (GSH), which is the most crucial biothiol responsible for cellular redox imbalance. As an anti-inflammatory compound, NAC can reduce levels of tumor necrosis factor-alpha (TNF-α) and interleukins (IL-6 and IL-1β) by suppressing the activity of nuclear factor kappa B (NF-κB). Despite NAC’s relevant therapeutic potential, in several experimental studies, its effectiveness in clinical trials, addressing different pathological conditions, is still limited. Thus, the purpose of this chapter is to provide an overview of the medicinal effects and applications of NAC to human health based on current therapeutic evidence.

scholarly journals Biological activity and mechanisms of therapeutic action of rosmarinic acid, luteolin and its sulphated derivatives

2016 ◽  
Vol 62 (1) ◽  
pp. 22-30 ◽  
Author(s):  
A.M. Popov ◽  
O.N. Krivoshapko ◽  
A.A. Klimovich ◽  
A.A. Artyukov
Keyword(s):  
Type 2 Diabetes ◽  
Biological Activity ◽  
Lipid Metabolism ◽  
Phenolic Compounds ◽  
Rosmarinic Acid ◽  
Therapeutic Potential ◽  
Experimental Studies ◽  
Therapeutic Action ◽  
Carbohydrate And Lipid Metabolism

The review considers recent experimental studies of biological activity and mechanisms of therapeutic action of rosmarinic acid, luteolin and its sulfated derivatives in diseases associated with disorders of carbohydrate and lipid metabolism. Particular attention is focused on the results of studies showing a high therapeutic potential of these phenolic compounds in their prophylactic and therapeutic use at experimental modeling of type 2 diabetes and hyperlipidemia. Based on the analysis of our results and the literature data putative mechanisms of therapeutic action of rosmarinic acid, luteolin and its sulfated derivatives have been proposed.

scholarly journals The intersectional genetics landscape for human

2019 ◽  
Author(s):  
Andre Macedo ◽  
Alisson M. Gontijo
Keyword(s):  
Gene Expression ◽  
Therapeutic Potential ◽  
Regulatory Element ◽  
Logic Gate ◽  
Cell Types ◽  
Boolean Logic ◽  
Interindividual Variation ◽  
Model Organisms ◽  
Cell Type ◽  
Diagnostic Potential

The human body is made up of hundreds, perhaps thousands of cell types and states, most of which are currently inaccessible genetically. Genetic accessibility carries significant diagnostic and therapeutic potential by allowing the selective delivery of genetic messages or cures to cells. Research in model organisms has shown that single regulatory element (RE) activities are seldom cell type specific, limiting their usage in genetic systems designed to restrict gene expression posteriorly to their delivery to cells. Intersectional genetic approaches can increase the number of genetically accessible cells. A typical intersectional method acts like an AND logic gate by converting the input of two or more active REs into a single synthetic output, which becomes unique for that cell. Here, we systematically assessed the intersectional genetics landscape of human using a curated subset of cells from a large RE usage atlas obtained by Cap Analysis of Gene Expression Sequencing (CAGE-Seq) of thousands of primary and cancer cells (the FANTOM5 consortium atlas). We developed the heuristics and algorithms to retrieve and quality rank AND gate intersections intra- and inter-individually. We find that >90% of the 154 primary cell types surveyed can be distinguished from each other with as little as 3 to 4 active REs, with quantifiable safety and robustness. We call these minimal intersections of active REs with cell-type diagnostic potential “Versatile Entry Codes” (VEnCodes). We show that VEnCodes could be found for 100% of the 158 cancer cell types surveyed, and that most of these are highly robust to intra- and interindividual variation. Our tools for generating and quality-ranking VEnCodes can be adapted to other RE usage databases and to other intersectional methods using alternative Boolean logic operations. Our work demonstrate the potential of intersectional approaches for future gene delivery technologies in human.

scholarly journals Virulent secondary metabolites of entomopathogenic bacteria genera, Xenorhabdus and Photorhabdus, inhibit phospholipase A2 to suppress host insect immunity

2020 ◽  
Author(s):  
Md. Mahi Imam Mollah ◽  
Yonggyun Kim
Keyword(s):  
Secondary Metabolites ◽  
Bacterial Culture ◽  
Bacterial Species ◽  
Culture Broth ◽  
Insect Immunity ◽  
Bacterial Isolates ◽  
Bacterial Pathogenicity ◽  
Entomopathogenic Bacteria ◽  
Organic Extracts ◽  
Insecticidal Activities

Abstract Background: Xenorhabdus and Photorhabdus are entomopathogenic bacteria that cause septicemia and toxemia in insects. They produce secondary metabolites to induce host immunosuppression. Their metabolite compositions vary among bacterial species. Little is known about the relationship between metabolite compositions and the bacterial pathogenicity. The objective of this study was to compare pathogenicity and production of secondary metabolites of 14 bacterial isolates (species or strains) of Xenorhabdus and Photorhabdus. Results: All bacterial isolates exhibited insecticidal activities after hemocoelic injection to Spodoptera exigua (a lepidopteran insect) larvae, with median lethal doses ranging from 168.8 to 641.3 CFU per larva. Bacterial infection also led to immunosuppression by inhibiting eicosanoid biosynthesis. Bacterial culture broth was fractionated into four different organic extracts. All four organic extracts of each bacterial species exhibited insecticidal activities and resulted in immunosuppression. These organic extracts were subjected to GC-MS analysis which predicted 182 compounds, showing differential compositions for 14 bacteria isolates. There were positive correlations between total number of secondary metabolites produced by each bacterial culture broth and its bacterial pathogenicity based on immunosuppression and insecticidal activity. From these correlation results, 70 virulent compounds were selected from secondary metabolites of high virulent bacterial isolates by deducting those of low virulent bacterial isolates. These selected virulent compounds exhibited significant immunosuppressive activities by inhibiting eicosanoid biosynthesis. They also exhibited relatively high insecticidal activities. Conclusion: Virulence variation between Xenorhabdus and Photorhabdus is determined by their different compositions of secondary metabolites, of which PLA2 inhibitors play a crucial role.

scholarly journals F-type Pyocins are Diverse Non-Contractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Senjuti Saha ◽  
Chidozie D. Ojobor ◽  
Erik Mackinnon ◽  
Olesia I. North ◽  
Joseph Bondy-Denomy ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bactericidal Activity ◽  
Pathogenic Bacteria ◽  
Therapeutic Potential ◽  
Experimental Studies ◽  
Opportunistic Pathogen ◽  
Multidrug Resistant ◽  
Antibiotic Resistant ◽  
Genes Encoding ◽  
Intraspecies Competition

ABSTRACTMost Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or non-contractile phage tails known as R-type and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3’-end of the F-type pyocin cluster are divergent in sequence, and likely determine bactericidal specificity. We use sequence similarities among these proteins to define 11 distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly re-assorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins emerged earlier or have been subject to distinct evolutionary pressures. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as anti-bacterial therapeutics.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes a broad spectrum of antibiotic resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great interest in the development of alternative therapeutics. One alternative is protein-based antimicrobials called bacteriocins, which are produced by one strain of bacteria to kill other strains. In this study, we investigate F-type pyocins, bacteriocins naturally produced by P. aeruginosa that resemble non-contractile phage tails. We show that they are potent killers of P. aeruginosa, and distinct pyocin groups display different killing specificities. We have identified the probable specificity determinants of F-type pyocins, which opens up the potential to engineer them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well characterized phage tails will greatly facilitate their development into effective antibacterials.

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