Evaluation of in vitro and in vivo antibiotic efficacy against a novel bioluminescent Shigella flexneri

Abstract Shigella spp., the bacteria responsible for shigellosis, are one of the leading causes of diarrheal morbidity and mortality amongst children. There is a pressing need for the development of novel therapeutics, as resistance of Shigella to many currently used antibiotics is rapidly emerging. This paper describes the development of robust in vitro and in vivo tools to study antibiotic efficacy against Shigella flexneri. A novel bioluminescent S. flexneri strain (S. flexneri lux1) was generated, which can be used in a mammalian epithelial cell co-culture assay to evaluate antibiotic intracellular and extracellular efficacy. In addition, the S. flexneri lux1 strain was used with an intraperitoneal (IP) murine model of shigellosis to test the efficacy of ciprofloxacin and ampicillin. Both antibiotics significantly reduced the observed radiance from the gastrointestinal tissue of infected mice compared to vehicle control. Furthermore, plated gastrointestinal tissue homogenate confirmed antibiotic treatment significantly reduced the S. flexneri infection. However, in contrast to the results generated with tissue homogenate, the radiance data was not able to distinguish between the efficacy of ampicillin and ciprofloxacin. Compared to traditional methods, these models can be utilized for efficient screening of novel antibiotics aiding in the discovery of new treatments against shigellosis.

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Author Correction: Evaluation of in vitro and in vivo antibiotic efficacy against a novel bioluminescent Shigella flexneri

Scientific Reports ◽

10.1038/s41598-020-59766-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Molly C. McCloskey ◽

Shareef Shaheen ◽

Lesley Rabago ◽

Matthew A. Hulverson ◽

Ryan Choi ◽

...

Keyword(s):

Shigella Flexneri ◽

Antibiotic Efficacy

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Villin Severing Activity Enhances Actin-based Motility In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0423 ◽

2007 ◽

Vol 18 (3) ◽

pp. 827-838 ◽

Cited By ~ 22

Author(s):

Céline Revenu ◽

Matthieu Courtois ◽

Alphée Michelot ◽

Cécile Sykes ◽

Daniel Louvard ◽

...

Keyword(s):

Shigella Flexneri ◽

Actin Dynamics ◽

Actin Binding ◽

Loss Of Function ◽

Mesenchymal Transition ◽

Capping Protein ◽

Function Strategy ◽

In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

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Host oyster tissue extracts modulate in vitro protease expression and cellular differentiation in the protozoan parasite, Perkinsus marinus

Parasitology ◽

10.1017/s003118200200286x ◽

2003 ◽

Vol 126 (4) ◽

pp. 293-302 ◽

Cited By ~ 15

Author(s):

E. A. MACINTYRE ◽

C. G. EARNHART ◽

S. L. KAATTARI

Keyword(s):

Serine Proteases ◽

Protozoan Parasite ◽

Eastern Oyster ◽

Defined Medium ◽

Tissue Homogenate ◽

Oyster Tissue ◽

Perkinsus Marinus ◽

Tissue Extracts

Perkinsus marinus is responsible for a chronic disease (Dermo) of the Eastern oyster, Crassostrea virginica. In order to simulate the in vivo environment more closely, a chemically defined medium (JL-ODRP-3) was supplemented with tissue homogenate extracts or plasma from oysters possessing varying degrees of susceptibility to P. marinus infection. In media supplemented with extracts from highly susceptible oysters (C. virginica), P. marinus cells secreted elevated amounts of a set of low molecular weight serine proteases (LMP: 30–45 kDa) as assessed by enhanced digestion within gelatin-substrate SDS–PAGE gels. Oyster species of low susceptibility (C. gigas and C. ariakensis) did not exhibit this ability to upregulate P. marinus LMP expression. Oyster extract supplementation also led to pronounced changes in P. marinus cellular morphology, such that the cells were comparable to those observed within naturally infected oysters.

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

Journal of Biomaterials Applications ◽

10.1177/08853282211051183 ◽

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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The Use of Lectins as Tools to Combat SARS-CoV-2

Current Pharmaceutical Design ◽

10.2174/1381612827666210830094743 ◽

2021 ◽

Vol 27 ◽

Author(s):

Daniela Martinez ◽

Diego Amaral ◽

David Markovitz ◽

Luciano Pinto

Keyword(s):

Viral Infection ◽

Host Cells ◽

Viral Fusion ◽

Cell Receptors ◽

First Case ◽

Mannose Binding ◽

New Treatments ◽

Viral Surface

Background: in december 2019, china announced the first case of an infection caused by an, until then, unknown virus: sars-cov-2. since then, researchers have been looking for viable alternatives for the treatment and/or cure of viral infection. among the possible complementary solutions are lectins, and proteins that are reversibly bound to different carbohydrates. the spike protein, present on the viral surface, can interact with different cell receptors: ace2, cd147, and dc-signr. since lectins have an affinity for different carbohydrates, the binding with the glycosylated cell receptors represents a possibility of preventing the virus from binding to the receptors of host cells. Objective: in this review we discuss the main lectins that are possible candidates for use in the treatment of covid-19, highlighting those that have already demonstrated antiviral activity in vivo and in vitro, including mannose-binding lectin, griffithsin, banlec, and others. we also aim to discuss the possible mechanism of action of lectins, which appears to occur through the mediation of viral fusion in host cells, by binding of lectins to glycosylated receptors found in human cells and/or binding of these proteins with the spike glycoprotein, present in virus surface.moreover, we also discuss the use of lectins in clinical practice. Conclusion: Even with the development of effective vaccines, new cases of viral infection with the same virus, or new outbreaks with different viruses can occur; so, the development of new treatments should not be discarded. moreover, the discussions made in this work are relevant regarding the anti-viral properties of lectins.

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Virulent Shigella flexneri subverts the host innate immune response through manipulation of antimicrobial peptide gene expression

Journal of Experimental Medicine ◽

10.1084/jem.20071698 ◽

2008 ◽

Vol 205 (5) ◽

pp. 1121-1132 ◽

Cited By ~ 105

Author(s):

Brice Sperandio ◽

Béatrice Regnault ◽

Jianhua Guo ◽

Zhi Zhang ◽

Samuel L. Stanley ◽

...

Keyword(s):

Innate Immunity ◽

Shigella Flexneri ◽

Host Cells ◽

Virulence Plasmid ◽

Cationic Peptides ◽

Immunity Genes ◽

Genes Encoding ◽

Peptide Gene

Antimicrobial factors are efficient defense components of the innate immunity, playing a crucial role in the intestinal homeostasis and protection against pathogens. In this study, we report that upon infection of polarized human intestinal cells in vitro, virulent Shigella flexneri suppress transcription of several genes encoding antimicrobial cationic peptides, particularly the human β-defensin hBD-3, which we show to be especially active against S. flexneri. This is an example of targeted survival strategy. We also identify the MxiE bacterial regulator, which controls a regulon encompassing a set of virulence plasmid-encoded effectors injected into host cells and regulating innate signaling, as being responsible for this dedicated regulatory process. In vivo, in a model of human intestinal xenotransplant, we confirm at the transcriptional and translational level, the presence of a dedicated MxiE-dependent system allowing S. flexneri to suppress expression of antimicrobial cationic peptides and promoting its deeper progression toward intestinal crypts. We demonstrate that this system is also able to down-regulate additional innate immunity genes, such as the chemokine CCL20 gene, leading to compromised recruitment of dendritic cells to the lamina propria of infected tissues. Thus, S. flexneri has developed a dedicated strategy to weaken the innate immunity to manage its survival and colonization ability in the intestine.

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Intracellular localisation of Mycobacterium tuberculosis affects antibiotic efficacy

10.1101/2020.11.25.398636 ◽

2020 ◽

Author(s):

Pierre Santucci ◽

Daniel J. Greenwood ◽

Antony Fearns ◽

Kai Chen ◽

Haibo Jiang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Host Cell ◽

Combination Chemotherapy ◽

Human Macrophage ◽

Ion Microscopy ◽

Antibiotic Efficacy ◽

Subcellular Level ◽

Intracellular Localisation

AbstractTo be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of Mycobacterium tuberculosis (Mtb). However, how host cell environments affect antibiotic accumulation and efficacy remains elusive. Pyrazinamide (PZA) is a key antibiotic against TB, yet its behaviour is not fully understood. Here, by using correlative light, electron, and ion microscopy to image PZA at the subcellular level, we investigated how human macrophage environments affect PZA activity. We discovered that PZA accumulates heterogeneously between individual bacteria in multiple host cell environments. Crucially, Mtb phagosomal localisation and acidification increase PZA accumulation and efficacy. By imaging two antibiotics commonly used in combined TB therapy, we showed that bedaquiline (BDQ) significantly enhances PZA accumulation by a host cell mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy; explaining the potent in vivo efficacy compared to its modest in vitro activity and the critical contribution to TB combination chemotherapy.

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Some biochemical effects of triamcinolone acetonide on rat liver and muscle

Biochemical Journal ◽

10.1042/bj1160349 ◽

1970 ◽

Vol 116 (3) ◽

pp. 349-355 ◽

Cited By ~ 22

Author(s):

R. F. Peters ◽

M. C. Richardson ◽

Margaret Small ◽

A. M. White

Keyword(s):

Amino Acids ◽

Triamcinolone Acetonide ◽

Time Course ◽

Muscle Protein ◽

Specific Radioactivity ◽

Synthetic Activity ◽

Tissue Homogenate ◽

Glycogen Concentration

1. The powerful anti-inflammatory glucocorticoid triamcinolone acetonide, administered to rats at 20 and 2.5mg/kg, leads to a decrease in the incorporation in vivo of [3H]uridine and [32P]orthophosphate into hind-limb skeletal muscle. 2. At the higher dose, this decrease in the rate of incorporation of precursors into RNA precedes a decrease in the incorporating ability of muscle ribosomes, which commences about 4–5h after drug administration, but is unaccompanied by any changes in the concentration of tissue ATP or free amino acids. 3. The ribosomal dysfunction extends to polyribosomes, which can only be successfully isolated from the muscle of triamcinolone-treated animals after the addition of α-amylase to the tissue homogenate to remove glycogen. 4. The specific radioactivity of muscle protein labelled in vivo with 14C-labelled amino acids does not decrease progressively after triamcinolone administration. After 2h there is an apparent stimulation of incorporation which leads to an overall discrepancy between measurements of protein-synthetic activity made in vivo and in vitro. 5. There is a significant increase in muscle-glycogen concentration between 8 and 12h after the administration of triamcinolone acetonide (20mg/kg), although a significant decrease occurs after 4h. The fall in glycogen concentration may be due to a decrease in the rate of synthesis of protein essential for glucose uptake into the tissues. 6. As judged by (a) incorporation of 14C-labelled amino acids into protein, (b) [3H]uridine and [32P]-orthophosphate incorporation into RNA, (c) the rate of induction of tryptophan pyrrolase and (d) changes in the pool sizes of taurine and tryptophan, the responses in liver followed the same time-course as those in muscle after administration of the drug.

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