scholarly journals Variability of bacterial behavior in the mammalian gut captured using a growth-linked single-cell synthetic gene oscillator

2018 ◽  
Author(s):  
David T Riglar ◽  
David L Richmond ◽  
Laurent Potvin-Trottier ◽  
Andrew A Verdegaal ◽  
Alexander D Naydich ◽  
...  
Keyword(s):  
Single Cell ◽  
Bacterial Growth ◽  
Bacterial Population ◽  
Bacterial Colonization ◽  
Growth Conditions ◽  
Inflammatory Conditions ◽  
Genetic Oscillator ◽  
Constant Period

AbstractThe dynamics of the bacterial population that comprises the gut microbiota plays key roles in overall mammalian health. However, a detailed understanding of bacterial growth within the gut is limited by the inherent complexity and inaccessibility of the gut environment. Here, we deploy an improved synthetic genetic oscillator to investigate dynamics of bacterial colonization and growth in the mammalian gut under both healthy and disease conditions. The synthetic oscillator, when introduced into both Escherichia coli and Salmonella Typhimurium maintains regular oscillations with a constant period in generations across growth conditions. We determine the phase of oscillation from individual bacteria using image analysis of resultant colonies and thereby infer the number of cell divisions elapsed. In doing so, we demonstrate robust functionality and controllability of the oscillator circuit’s activity during bacterial growth in vitro, in a simulated murine gut microfluidic environment, and in vivo within the mouse gut. We determine different dynamics of bacterial colonization and growth in the gut under normal and inflammatory conditions. Our results show that a precise genetic oscillator can function in a complex environment and reveal single cell behavior under diverse conditions where disease may create otherwise impossible-to-quantify variability in growth across the population.

scholarly journals Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
David T. Riglar ◽  
David L. Richmond ◽  
Laurent Potvin-Trottier ◽  
Andrew A. Verdegaal ◽  
Alexander D. Naydich ◽  
...  
Keyword(s):  
Single Cell ◽  
Bacterial Growth ◽  
Real Life ◽  
Cell Level ◽  
Bacterial Dynamics ◽  
Genetic Oscillator ◽  
Periodic Gene ◽  
Periodic Gene Expression

Abstract Synthetic gene oscillators have the potential to control timed functions and periodic gene expression in engineered cells. Such oscillators have been refined in bacteria in vitro, however, these systems have lacked the robustness and precision necessary for applications in complex in vivo environments, such as the mammalian gut. Here, we demonstrate the implementation of a synthetic oscillator capable of keeping robust time in the mouse gut over periods of days. The oscillations provide a marker of bacterial growth at a single-cell level enabling quantification of bacterial dynamics in response to inflammation and underlying variations in the gut microbiota. Our work directly detects increased bacterial growth heterogeneity during disease and differences between spatial niches in the gut, demonstrating the deployment of a precise engineered genetic oscillator in real-life settings.

scholarly journals Comparative Activity of Ceftriaxone, Ciprofloxacin, and Gentamicin as a Function of Bacterial Growth Rate Probed by Escherichia coli Chromosome Replication in the Mouse Peritonitis Model

2018 ◽  
Vol 63 (2) ◽  
pp. e02133-18 ◽  
Author(s):  
Maria Schei Haugan ◽  
Anders Løbner-Olesen ◽  
Niels Frimodt-Møller
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Bacterial Growth ◽  
Bacterial Population ◽  
Chromosome Replication ◽  
Bacterial Growth Rate ◽  
Peritonitis Model

ABSTRACT Commonly used antibiotics exert their effects predominantly on rapidly growing bacterial cells; yet, the growth dynamics taking place during infection in a complex host environment remain largely unknown. Hence, a means to measure in situ bacterial growth rate is essential to predict the outcome of antibacterial treatment. We have recently validated chromosome replication as a readout of in situ bacterial growth rate during Escherichia coli infection in the mouse peritonitis model. By the use of two complementary methods (quantitative PCR and fluorescence microscopy) for differential genome origin and terminus copy number quantification, we demonstrated the ability to track bacterial growth rate, both on a population average level and on a single-cell level, from one single biological specimen. Here, we asked whether the in situ growth rate predicts antibiotic treatment effect during infection in the same model. Parallel in vitro growth experiments were conducted as a proof of concept. Our data demonstrate that the activities of the commonly used antibiotics ceftriaxone and gentamicin correlated with pretreatment bacterial growth rate; both drugs performed better during rapid growth than during slow growth. Conversely, ciprofloxacin was less sensitive to bacterial growth rate, both in a homogenous in vitro bacterial population and in a more heterogeneous in vivo bacterial population. The method serves as a platform to test any antibiotic’s dependency on active in situ bacterial growth. Improved insight into this relationship in vivo could ultimately prove helpful in evaluating future antibacterial strategies.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals Zn-Containing Membranes for Guided Bone Regeneration in Dentistry

Polymers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1797
Author(s):  
Manuel Toledano ◽  
Marta Vallecillo-Rivas ◽  
María T. Osorio ◽  
Esther Muñoz-Soto ◽  
Manuel Toledano-Osorio ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Bone Regeneration ◽  
Bone Healing ◽  
Bone Repair ◽  
Complex Modulus ◽  
Bacterial Colonization ◽  
Guided Bone Regeneration ◽  
M2 Macrophage

Barrier membranes are employed in guided bone regeneration (GBR) to facilitate bone in-growth. A bioactive and biomimetic Zn-doped membrane with the ability to participate in bone healing and regeneration is necessary. The aim of the present study is to state the effect of doping the membranes for GBR with zinc compounds in the improvement of bone regeneration. A literature search was conducted using electronic databases, such as PubMed, MEDLINE, DIMDI, Embase, Scopus and Web of Science. A narrative exploratory review was undertaken, focusing on the antibacterial effects, physicochemical and biological properties of Zn-loaded membranes. Bioactivity, bone formation and cytotoxicity were analyzed. Microstructure and mechanical properties of these membranes were also determined. Zn-doped membranes have inhibited in vivo and in vitro bacterial colonization. Zn-alloy and Zn-doped membranes attained good biocompatibility and were found to be non-toxic to cells. The Zn-doped matrices showed feasible mechanical properties, such as flexibility, strength, complex modulus and tan delta. Zn incorporation in polymeric membranes provided the highest regenerative efficiency for bone healing in experimental animals, potentiating osteogenesis, angiogenesis, biological activity and a balanced remodeling. Zn-loaded membranes doped with SiO2 nanoparticles have performed as bioactive modulators provoking an M2 macrophage increase and are a potential biomaterial for promoting bone repair. Zn-doped membranes have promoted pro-healing phenotypes.

scholarly journals Single Cell Analysis Reveals That IL-4 Receptor/Stat6 Signaling Is Not Required for the In Vivo or In Vitro Development of CD4+Lymphocytes with a Th2 Cytokine Profile

2000 ◽  
Vol 164 (6) ◽  
pp. 3047-3055 ◽  
Author(s):  
Dragana Jankovic ◽  
Marika C. Kullberg ◽  
Nancy Noben-Trauth ◽  
Patricia Caspar ◽  
William E. Paul ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Cytokine Profile ◽  
Cell Analysis ◽  
In Vitro Development ◽  
Th2 Cytokine ◽  
Vitro Development ◽  
Cd4 Lymphocytes

scholarly journals Is There Potential for Repurposing Statins as Novel Antimicrobials?

2016 ◽  
Vol 60 (9) ◽  
pp. 5111-5121 ◽  
Author(s):  
Emma Hennessy ◽  
Claire Adams ◽  
F. Jerry Reen ◽  
Fergal O'Gara
Keyword(s):  
Serum Cholesterol ◽  
Bacterial Growth ◽  
Bacterial Infections ◽  
Statin Treatment ◽  
Therapeutic Agents ◽  
Pleiotropic Effects ◽  
Current Information ◽  
Potential Use

ABSTRACTStatins are members of a class of pharmaceutical widely used to reduce high levels of serum cholesterol. In addition, statins have so-called “pleiotropic effects,” which include inflammation reduction, immunomodulation, and antimicrobial effects. An increasing number of studies are emerging which detail the attenuation of bacterial growth andin vitroandin vivovirulence by statin treatment. In this review, we describe the current information available concerning the effects of statins on bacterial infections and provide insight regarding the potential use of these compounds as antimicrobial therapeutic agents.

scholarly journals Anaerobic Conditions Induce Expression of Polysaccharide Intercellular Adhesin in Staphylococcus aureus and Staphylococcus epidermidis

2001 ◽  
Vol 69 (6) ◽  
pp. 4079-4085 ◽  
Author(s):  
Sarah E. Cramton ◽  
Martina Ulrich ◽  
Friedrich Götz ◽  
Gerd Döring
Keyword(s):  
Staphylococcus Aureus ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Extracellular Polysaccharide ◽  
Growth Conditions ◽  
Anaerobic Conditions ◽  
Anaerobic Environment ◽  
Specific Mrna

ABSTRACT Products of the intercellular adhesion (ica) operon in Staphylococcus aureus and Staphylococcus epidermidis synthesize a linear β-1,6-linked glucosaminylglycan. This extracellular polysaccharide mediates bacterial cell-cell adhesion and is required for biofilm formation, which is thought to increase the virulence of both pathogens in association with prosthetic biomedical implants. The environmental signal(s) that triggers ica gene product and polysaccharide expression is unknown. Here we demonstrate that anaerobic in vitro growth conditions lead to increased polysaccharide expression in both S. aureus and S. epidermidis, although the regulation is less stringent inS. epidermidis. Anaerobiosis also dramatically stimulates ica-specific mRNA expression inica- and polysaccharide-positive strains of both S. aureus and S. epidermidis.These data suggest a mechanism whereby ica gene expression and polysaccharide production may act as a virulence factor in an anaerobic environment in vivo.

scholarly journals Glycoside Hydrolases Degrade Polymicrobial Bacterial Biofilms in Wounds

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Derek Fleming ◽  
Laura Chahin ◽  
Kendra Rumbaugh
Keyword(s):  
Glycoside Hydrolase ◽  
Bacterial Population ◽  
Chronic Wounds ◽  
Glycoside Hydrolases ◽  
Bacterial Biofilms ◽  
Planktonic Cells ◽  
Content Type ◽  
Biomass Dissolution

ABSTRACT The persistent nature of chronic wounds leaves them highly susceptible to invasion by a variety of pathogens that have the ability to construct an extracellular polymeric substance (EPS). This EPS makes the bacterial population, or biofilm, up to 1,000-fold more antibiotic tolerant than planktonic cells and makes wound healing extremely difficult. Thus, compounds which have the ability to degrade biofilms, but not host tissue components, are highly sought after for clinical applications. In this study, we examined the efficacy of two glycoside hydrolases, α-amylase and cellulase, which break down complex polysaccharides, to effectively disrupt Staphylococcus aureus and Pseudomonas aeruginosa monoculture and coculture biofilms. We hypothesized that glycoside hydrolase therapy would significantly reduce EPS biomass and convert bacteria to their planktonic state, leaving them more susceptible to conventional antimicrobials. Treatment of S. aureus and P. aeruginosa biofilms, grown in vitro and in vivo, with solutions of α-amylase and cellulase resulted in significant reductions in biomass, dissolution of the biofilm, and an increase in the effectiveness of subsequent antibiotic treatments. These data suggest that glycoside hydrolase therapy represents a potential safe, effective, and new avenue of treatment for biofilm-related infections.

scholarly journals Relevance of Biofilm Models in Periodontal Research: From Static to Dynamic Systems

2021 ◽  
Vol 9 (2) ◽  
pp. 428
Author(s):  
María Carmen Sánchez ◽  
Andrea Alonso-Español ◽  
Honorato Ribeiro-Vidal ◽  
Bettina Alonso ◽  
David Herrera ◽  
...  
Keyword(s):  
Research Group ◽  
Surface Composition ◽  
Bacterial Colonization ◽  
Implant Surface ◽  
Biofilm Growth ◽  
Biofilm Model ◽  
Dental Implant Surface ◽  
The Impact

Microbial biofilm modeling has improved in sophistication and scope, although only a limited number of standardized protocols are available. This review presents an example of a biofilm model, along with its evolution and application in studying periodontal and peri-implant diseases. In 2011, the ETEP (Etiology and Therapy of Periodontal and Peri-Implant Diseases) research group at the University Complutense of Madrid developed an in vitro biofilm static model using representative bacteria from the subgingival microbiota, demonstrating a pattern of bacterial colonization and maturation similar to in vivo subgingival biofilms. When the model and its methodology were standardized, the ETEP research group employed the validated in vitro biofilm model for testing in different applications. The evolution of this model is described in this manuscript, from the mere observation of biofilm growth and maturation on static models on hydroxyapatite or titanium discs, to the evaluation of the impact of dental implant surface composition and micro-structure using the dynamic biofilm model. This evolution was based on reproducing the ideal microenvironmental conditions for bacterial growth within a bioreactor and reaching the target surfaces using the fluid dynamics mimicking the salivary flow. The development of this relevant biofilm model has become a powerful tool to study the essential processes that regulate the formation and maturation of these important microbial communities, as well as their behavior when exposed to different antimicrobial compounds.

Clindamycin-loaded titanium prevents implant-related infection through blocking biofilm formation

2021 ◽  
pp. 088532822110511
Author(s):  
Youbin Li ◽  
Shaochuan Wang ◽  
Shidan Li ◽  
Jun Fei
Keyword(s):  
Surface Modification ◽  
Rat Model ◽  
Biofilm Formation ◽  
High Performance ◽  
Bacterial Colonization ◽  
Tissue Homogenate ◽  
Tartrate Resistant Acid Phosphatase ◽  
Layer By Layer

Implant-related infection is a disastrous complication. Surface modification of titanium is considered as an important strategy to prevent implant-related infection. However, there is no recognized surface modification strategy that can be applied in clinic so far. We explored a new strategy of coating. The clindamycin-loaded titanium was constructed by layer-by-layer self-assembly. The release of clindamycin from titanium was detected through high performance liquid chromatography. Different titanium was co-cultured with Staphylococcus aureus for 24 h in vitro, then the effect of different titanium on bacterial colonization and biofilm formation was determined by spread plate method and scanning electron microscopy. Cytotoxicity and cytocompatibility of clindamycin-loaded titanium on MC3T3-E1 cells were measured by CCK8. The antibacterial ability of clindamycin-loaded titanium in vivo was also evaluated using a rat model of osteomyelitis. The number of osteoclasts in bone defect was observed by tartrate-resistant acid phosphatase staining. Bacterial burden of surrounding tissues around the site of infection was calculated by tissue homogenate and colony count. Clindamycin-loaded titanium could release clindamycin slowly within 160 h. It reduced bacterial colonization by three orders of magnitude compare to control ( p < .05) and inhibits biofilm formation in vitro. Cells proliferation and adhesion were similar on three titanium surfaces ( p > .05). In vivo, clindamycin-loaded titanium improved bone healing, reduced microbial burden, and decreased the number of osteoclasts compared control titanium in the rat model of osteomyelitis. This study demonstrated that clindamycin-loaded titanium exhibited good biocompatibility, and showed antibacterial activity both in vivo and in vitro. It is promising and might have potential for clinical application.

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