scholarly journals Screening of anti-mycobacterial compounds in a naturally infected zebrafish embryo model

2016 ◽  
Author(s):  
James P Dalton ◽  
Benedict Uy ◽  
Kazuhide Okuda ◽  
Christopher J Hall ◽  
William A Denny ◽  
...  
Keyword(s):  
Zebrafish Embryo ◽  
Natural Infection ◽  
Rapid Screening ◽  
Infection Model ◽  
Zebrafish Embryos ◽  
Drug Trials ◽  
Bioluminescent Signal ◽  
High Level ◽  
Selection Of

Mycobacterium tuberculosisis a deadly human pathogen that latently infects a third of the worlds population, resulting in approximately 1.5 million deaths per year. Due to the difficulties and expense of carrying out animal drug trials using M. tuberculosis and rodents, infections of the zebrafishDanio reriowithM. marinumhave been used as a surrogate. However the methods so far described require specialised equipment and a high level of operator expertise. We investigated a natural infection model where zebrafish embryos are infected through incubation in media containingM. marinum. Using bioluminescently labelledM. marinum, we have characterised the nature of infection and established a model for interventional drug therapy. We have used a selection of traditional and experimental compounds to validate this model for anti-mycobacterial drug discovery. We observed that only three of the six treatments tested (Delamonid, SN30527 and rifampicin) retarded the growth ofM. marinumin vitro. In contrast, five of the six treatments (Pretomanid, Delamanid, SN30488, SN30527 and rifampicin) retarded the growth ofM. tuberculosisin vitro. Importantly, these same five treatments significantly reduced the bioluminescent signal from naturally infected zebrafish embryos. Overall this study has demonstrated that zebrafish embryos naturally infected with bioluminescentM. marinumM can be used for the rapid screening of anti-mycobacterial compounds with readily available equipment and limited expertise. The result is an assay that can be carried out by a wide variety of laboratories for minimal cost and without high levels of zebrafish expertise.

scholarly journals Photodynamic Therapy Combined with Antibiotics or Antifungals against Microorganisms That Cause Skin and Soft Tissue Infections: A Planktonic and Biofilm Approach to Overcome Resistances

2021 ◽  
Vol 14 (7) ◽  
pp. 603
Author(s):  
Vanesa Pérez-Laguna ◽  
Isabel García-Luque ◽  
Sofía Ballesta ◽  
Antonio Rezusta ◽  
Yolanda Gilaberte
Keyword(s):  
Photodynamic Therapy ◽  
Soft Tissues ◽  
Combined Treatment ◽  
Resistance Pattern ◽  
Antimicrobial Photodynamic Therapy ◽  
Antimicrobial Drugs ◽  
Bacteria And Fungi ◽  
High Level ◽  
Selection Of

The present review covers combination approaches of antimicrobial photodynamic therapy (aPDT) plus antibiotics or antifungals to attack bacteria and fungi in vitro (both planktonic and biofilm forms) focused on those microorganisms that cause infections in skin and soft tissues. The combination can prevent failure in the fight against these microorganisms: antimicrobial drugs can increase the susceptibility of microorganisms to aPDT and prevent the possibility of regrowth of those that were not inactivated during the irradiation; meanwhile, aPDT is effective regardless of the resistance pattern of the strain and their use does not contribute to the selection of antimicrobial resistance. Additive or synergistic antimicrobial effects in vitro are evaluated and the best combinations are presented. The use of combined treatment of aPDT with antimicrobials could help overcome the difficulty of fighting high level of resistance microorganisms and, as it is a multi-target approach, it could make the selection of resistant microorganisms more difficult.

scholarly journals Zebrafish Embryo Model for Assessment of Drug Efficacy on Mycobacterial Persisters

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Susanna Commandeur ◽  
Nino Iakobachvili ◽  
Marion Sparrius ◽  
Mariam Mohamed Nur ◽  
Galina V. Mukamolova ◽  
...  
Keyword(s):  
Drug Screening ◽  
Zebrafish Embryo ◽  
Neutral Lipids ◽  
Drug Efficacy ◽  
Infection Model ◽  
Duration Of Treatment ◽  
Content Type ◽  
Set Up

ABSTRACT Tuberculosis continues to kill millions of people each year. The main difficulty in eradication of the disease is the prolonged duration of treatment, which takes at least 6 months. Persister cells have long been associated with failed treatment and disease relapse because of their phenotypical, though transient, tolerance to drugs. By targeting these persisters, the duration of treatment could be shortened, leading to improved tuberculosis treatment and a reduction in transmission. The unique in vivo environment drives the generation of persisters; however, appropriate in vivo mycobacterial persister models enabling optimized drug screening are lacking. To set up a persister infection model that is suitable for this, we infected zebrafish embryos with in vitro-starved Mycobacterium marinum. In vitro starvation resulted in a persister-like phenotype with the accumulation of stored neutral lipids and concomitant increased tolerance to ethambutol. However, these starved wild-type M. marinum organisms rapidly lost their persister phenotype in vivo. To prolong the persister phenotype in vivo, we subsequently generated and analyzed mutants lacking functional resuscitation-promoting factors (Rpfs). Interestingly, the ΔrpfAB mutant, lacking two Rpfs, established an infection in vivo, whereas a nutrient-starved ΔrpfAB mutant did maintain its persister phenotype in vivo. This mutant was, after nutrient starvation, also tolerant to ethambutol treatment in vivo, as would be expected for persisters. We propose that this zebrafish embryo model with ΔrpfAB mutant bacteria is a valuable addition for drug screening purposes and specifically screens to target mycobacterial persisters.

scholarly journals In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa

2020 ◽  
Vol 75 (9) ◽  
pp. 2508-2515 ◽  
Author(s):  
María A Gomis-Font ◽  
Gabriel Cabot ◽  
Irina Sánchez-Diener ◽  
Pablo A Fraile-Ribot ◽  
Carlos Juan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Infection Model ◽  
Mechanisms Of Resistance ◽  
Resistance Development ◽  
C Elegans ◽  
Imipenem Resistance ◽  
High Level

Abstract Objectives We analysed the dynamics and mechanisms of resistance development to imipenem alone or combined with relebactam in Pseudomonas aeruginosa WT (PAO1) and mutator (PAOMS; ΔmutS) strains. Methods PAO1 or PAOMS strains were incubated for 24 h in Mueller–Hinton Broth with 0.125–64 mg/L of imipenem ± relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 mg/L of imipenem ± relebactam for 7 days. Two colonies per strain, replicate experiment and antibiotic from early (Day 1) and late (Day 7) cultures were characterized by determining the susceptibility profiles, WGS and determination of the expression of ampC and efflux-pump-coding genes. Virulence was studied in a Caenorhabditis elegans infection model. Results Relebactam reduced imipenem resistance development for both strains, although resistance emerged much faster for PAOMS. WGS indicated that imipenem resistance was associated with mutations in the porin OprD and regulators of ampC, while the mutations in imipenem/relebactam-resistant mutants were located in oprD and regulatoras of MexAB-OprM. High-level imipenem/relebactam resistance was only documented in the PAOMS strain and was associated with an additional specific (T680A) mutation located in the catalytic pocket of ponA (PBP1a) and with reduced virulence in the C. elegans model. Conclusions Imipenem/relebactam could be a useful alternative for the treatment of MDR P. aeruginosa infections, potentially reducing resistance development during treatment. Moreover, this work deciphers the potential resistance mechanisms that may emerge upon the introduction of this novel combination into clinical practice.

scholarly journals Selection of a Moxifloxacin Dose That Suppresses Drug Resistance inMycobacterium tuberculosis,by Use of an In Vitro Pharmacodynamic Infection Model and Mathematical Modeling

2004 ◽  
Vol 190 (9) ◽  
pp. 1642-1651 ◽  
Author(s):  
Tawanda Gumbo ◽  
Arnold Louie ◽  
Mark R. Deziel ◽  
Linda M. Parsons ◽  
Max Salfinger ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Drug Resistance ◽  
Infection Model ◽  
Selection Of

scholarly journals Cross-Competition in Transgenic Chloroplasts Expressing Single Editing Sites Reveals Shared cis Elements

2002 ◽  
Vol 22 (24) ◽  
pp. 8448-8456 ◽  
Author(s):  
Anne-Laure Chateigner-Boutin ◽  
Maureen R. Hanson
Keyword(s):  
Editing Site ◽  
Competition Effect ◽  
High Level Expression ◽  
Consensus Sequences ◽  
Endogenous Genes ◽  
High Level ◽  
Cross Competition ◽  
Selection Of

ABSTRACT RNA editing in organelles of angiosperm plants results in alteration of Cs to Us in transcripts. In most editing sites analyzed in vitro or in vivo, sequences within approximately 30 nucleotides (nt) 5′ and 10 nt 3′ of the edited C have been found to be required for selection of the correct C editing target and for editing efficiency, but no consensus sequences have been identified. The effect of high-level expression of two different minigenes carrying either the rpoB-2 or the ndhF-2 editing site on editing was determined for all 31 known edited Cs in two transgenic tobacco lines. The editing efficiencies of both the corresponding rpoB and ndhF editing sites in the endogenous genes' transcripts and in several other genes' transcripts were reduced in the chloroplast transgenic plants. Conserved nucleotides could be identified in the sequences immediately 5′ of each overexpressed editing site and in the sites in the additional genes that experienced a competition effect, though the conserved nucleotides differ 5′ of rpoB-2 and 5′ of ndhF-2. Inspection of sequences surrounding chloroplast and mitochondrial editing sites reveals that they can be grouped into clusters which carry conserved nucleotides within 30 nt 5′ of the C target of editing. The data are consistent with a model in which the same trans factor recognizes several chloroplast or mitochondrial editing sites, depending on the particular sequence 5′ of the edited C.

scholarly journals In Vitro Development of Resistance to Five Quinolones and Amoxicillin-Clavulanate in Streptococcus pneumoniae

1999 ◽  
Vol 43 (5) ◽  
pp. 1177-1182 ◽  
Author(s):  
Todd A. Davies ◽  
Glenn A. Pankuch ◽  
Bonifacio E. Dewasse ◽  
Michael R. Jacobs ◽  
Peter C. Appelbaum
Keyword(s):  
Streptococcus Pneumoniae ◽  
Quinolone Resistance ◽  
In Vitro Development ◽  
Development Of Resistance ◽  
Amoxicillin Clavulanate ◽  
Efflux Mechanism ◽  
High Level ◽  
Vitro Development ◽  
Selection Of

ABSTRACT The ability of 50 sequential subcultures in subinhibitory concentrations of ciprofloxacin, levofloxacin, grepafloxacin, sparfloxacin, trovafloxacin, and amoxicillin-clavulanate to select for resistance was studied for six penicillin-susceptible and four penicillin-intermediate pneumococci. Subculturing in ciprofloxacin, grepafloxacin, levofloxacin, and sparfloxacin led to selection of mutants requiring increased MICs for all 10 strains, with MICs rising from (i) 0.5 to 4.0 to (ii) 4.0 to 32.0 μg/ml after 7 to 12 passages for ciprofloxacin, from (i) 0.06 to 0.25 to (ii) 0.5 to 8.0 μg/ml after 5 to 23 passages for grepafloxacin, from (i) 0.5 to 1.0 to (ii) 4.0 to 64 μg/ml after 14 to 49 passages for levofloxacin, and from (i) 0.125 to 0.25 to (ii) 1.0 to 16.0 μg/ml after 8 to 26 passages for sparfloxacin. Subculturing in trovafloxacin led to increased MICs for eight strains, with MICs rising from (i) 0.06 to 0.125 to (ii) 0.5 to 8.0 μg/ml after 6 to 28 passages. Subculturing in amoxicillin-clavulanate led to raised MICs for only one strain, with the MIC rising from 0.015 to 0.125 μg/ml after 24 passages. Double mutations in both ParC and GyrA led to high-level quinolone resistance when ParC mutations were at S79. Trovafloxacin MICs were 1 to 2 μg/ml in double mutants with ParC mutations at positions other than S79 (e.g., D83). Mutations in ParE (at D435, R447, and E474) and GyrB (at S405, D406, and D435) were found in four and six mutants, respectively. In the presence of reserpine, 29 mutants had lower ciprofloxacin MICs (2 to 16 times lower), 8 mutants had lower levofloxacin MICs (2 times), and one mutant had a lower trovafloxacin MIC (2 times), suggesting the involvement of an efflux mechanism. In contrast to the case for quinolones, subculturing in the presence of amoxicillin-clavulanate did not select for resistance to this drug.

scholarly journals Native Valve Endocarditis Caused by Corynebacterium striatum with Heterogeneous High-Level Daptomycin Resistance: Collateral Damage from Daptomycin Therapy?

2012 ◽  
Vol 56 (6) ◽  
pp. 3461-3464 ◽  
Author(s):  
Truc T. Tran ◽  
Siraya Jaijakul ◽  
Cole T. Lewis ◽  
Lorena Diaz ◽  
Diana Panesso ◽  
...  
Keyword(s):  
Invasive Disease ◽  
Native Valve ◽  
Positive Skin ◽  
Content Type ◽  
Native Valve Endocarditis ◽  
Corynebacterium Striatum ◽  
Skin Flora ◽  
High Level ◽  
Selection Of

ABSTRACTWe describe a patient who developedCorynebacterium striatumnative valve endocarditis after receiving two 6-week courses of daptomycin for the treatment of methicillin-resistantStaphylococcus aureusbacteremia and osteomyelitis. The organism exhibitedin vitroheteroresistance to daptomycin, with two subpopulations showing daptomycin susceptibility (MIC of ≤0.094 μg/ml) and high-level resistance to daptomycin (MIC of ≥256 μg/ml). The selection of daptomycin-resistant Gram-positive skin flora with the potential of causing invasive disease may be a concern during prolonged courses of daptomycin.

scholarly journals Innate secretory antibodies protect against natural Salmonella typhimurium infection

2006 ◽  
Vol 203 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Odilia L.C. Wijburg ◽  
Tania K. Uren ◽  
Kim Simpfendorfer ◽  
Finn-Eirik Johansen ◽  
Per Brandtzaeg ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
Natural Infection ◽  
Oral Route ◽  
Microbial Pathogens ◽  
Infection Model ◽  
Knock Out ◽  
Immunoglobulin Receptor ◽  
Secretory Antibodies

The production of IgA is induced in an antigen-unspecific manner by commensal flora. These secretory antibodies (SAbs) may bind multiple antigens and are thought to eliminate commensal bacteria and self-antigens to avoid systemic recognition. In this study, we addressed the role of “innate” SAbs, i.e., those that are continuously produced in normal individuals, in protection against infection of the gastrointestinal tract. We used polymeric immunoglobulin receptor (pIgR−/−) knock-out mice, which are unable to bind and actively transport dimeric IgA and pentameric IgM to the mucosae, and examined the role of innate SAbs in protection against the invasive pathogen Salmonella typhimurium. In vitro experiments suggested that innate IgA in pIgR−/− serum bound S. typhimurium in a cross-reactive manner which inhibited epithelial cell invasion. Using a “natural” infection model, we demonstrated that pIgR−/− mice are profoundly sensitive to infection with S. typhimurium via the fecal-oral route and, moreover, shed more bacteria that readily infected other animals. These results imply an important evolutionary role for innate SAbs in protecting both the individual and the herd against infections, and suggest that the major role of SAbs may be to prevent the spread of microbial pathogens throughout the population, rather than protection of local mucosal surfaces.

Efficacy of single and multiple oral doses of fosfomycin against Pseudomonas aeruginosa urinary tract infections in a dynamic in vitro bladder infection model

2020 ◽  
Vol 75 (7) ◽  
pp. 1879-1888 ◽  
Author(s):  
Iain J Abbott ◽  
Elke van Gorp ◽  
Rixt A Wijma ◽  
Jordy Dekker ◽  
Peter D Croughs ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Single Dose ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Infection Model ◽  
Post Exposure ◽  
High Level ◽  
Tract Infections ◽  
Emergence Of Resistance

Abstract Objectives We used a dynamic bladder infection in vitro model with synthetic human urine (SHU) to examine fosfomycin exposures to effectively kill, or prevent emergence of resistance, among Pseudomonas aeruginosa isolates. Methods Dynamic urinary fosfomycin concentrations after 3 g oral fosfomycin were simulated, comparing single and multiple (daily for 7 days) doses. Pharmacodynamic response of 16 P. aeruginosa (MIC range 1 to >1024 mg/L) were examined. Baseline disc diffusion susceptibility, broth microdilution MIC and detection of heteroresistance were assessed. Pathogen kill and emergence of resistance over 72 h following a single dose, and over 216 h following daily dosing for 7 days, were investigated. The fAUC0–24/MIC associated with stasis and 1, 2 and 3 log10 kill were determined. Results Pre-exposure high-level resistant (HLR) subpopulations were detected in 11/16 isolates after drug-free incubation in the bladder infection model. Five of 16 isolates had >2 log10 kill after single dose, reducing to 2/16 after seven doses. Post-exposure HLR amplification occurred in 8/16 isolates following a single dose and in 11/16 isolates after seven doses. Baseline MIC ≥8 mg/L with an HLR subpopulation predicted post-exposure emergence of resistance following the multiple doses. A PK/PD target of fAUC0–24/MIC >5000 was associated with 3 log10 kill at 72 h and 7 day-stasis. Conclusions Simulated treatment of P. aeruginosa urinary tract infections with oral fosfomycin was ineffective, despite exposure to high urinary concentrations and repeated daily doses for 7 days. Emergence of resistance was observed in the majority of isolates and worsened following prolonged therapy. Detection of a baseline resistant subpopulation predicted treatment failure.

scholarly journals Quantifying endothelial cell proliferation in the zebrafish embryo

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 1032
Author(s):  
George Bowley ◽  
Timothy JA Chico ◽  
Jovana Serbanovic-Canic ◽  
Paul C Evans
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cell ◽  
Zebrafish Embryo ◽  
Time Lapse ◽  
Endothelial Cell Proliferation ◽  
Small Scale ◽  
Zebrafish Embryos ◽  
Time Lapse Imaging

Introduction: Endothelial cell (EC) proliferation is a fundamental determinant of vascular development and homeostasis, and contributes to cardiovascular disease by increasing vascular permeability to blood-borne lipoproteins. Rodents have been traditionally used to analyse EC proliferation mechanisms in vascular health and disease; however, alternative models such as the zebrafish embryo allow researchers to conduct small scale screening studies in a physiologically relevant vasculature whilst reducing the use of mammals in biomedical research. In vitro models of EC proliferation are valuable but do not fully recapitulate the complexity of the in vivo situation. Several groups have used zebrafish embryos for vascular biology research because they offer the advantages of an in vivo model in terms of complexity but are also genetically manipulable and optically transparent. Methods: Here we investigated whether zebrafish embryos can provide a suitable model for the study of EC proliferation. We explored the use of antibody, DNA labelling, and time-lapse imaging approaches. Results: Antibody and DNA labelling approaches were of limited use in zebrafish due to the low rate of EC proliferation combined with the relatively narrow window of time in which they can label proliferating nuclei. By contrast, time-lapse imaging of fluorescent proteins localised to endothelial nuclei was a sensitive method to quantify EC proliferation in zebrafish embryos. Discussion: We conclude that time-lapse imaging is suitable for analysis of endothelial cell proliferation in zebrafish, and that this method is capable of capturing more instances of EC proliferation than immunostaining or cell labelling alternatives. This approach is relevant to anyone studying endothelial cell proliferation for screening genes or small molecules involved in EC proliferation. It offers greater biological relevance than existing in vitro models such as HUVECs culture, whilst reducing the overall number of animals used for this type of research.

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