Charles Bodet

Efficiency of water disinfectants against Legionella pneumophila and Acanthamoeba

Free-living amoebae might be pathogenic by themselves and be a reservoir for bacterial pathogens, such as Legionella pneumophila. Not only could amoebae protect intra-cellular Legionella but Legionella grown within amoebae could undergo physiological modifications and become more resistant and more virulent. Therefore, it is important to study the efficiency of treatments on amoebae and Legionella grown within these amoebae to improve their application and to limit their impact on the environment. With this aim, we compared various water disinfectants against trophozoites of three Acanthamoeba strains and L. pneumophila alone or in co-culture. Three oxidizing disinfectants (chlorine, monochloramine, and chlorine dioxide) were assessed. All the samples were treated with disinfectants for 1 h and the disinfectant concentration was followed to calculate disinfectant exposure (Ct). We noticed that there were significant differences of susceptibility among the Acanthamoeba strains. However no difference was observed between infected and non-infected amoebae. Also, the comparison between the three disinfectants indicates that monochloramine was efficient at the same level towards free or co-cultured L. pneumophila while chlorine and chlorine dioxide were less efficient on co-cultured L. pneumophila. It suggests that these disinfectants should have different modes of action. Finally, our results provide for the first time disinfectant exposure values for Acanthamoeba treatments that might be used as references for disinfection of water systems.

Acanthamoeba castellanii: cellular changes induced by chlorination.

Chlorination is a well-known disinfection method, used in water treatment to inactivate various microorganisms, it induces numerous cellular changes. Even though Acanthamoebae are frequently found in water, the cellular changes induced in Acanthamoebae have not been described in the literature. Acanthamoebae are pathogenic amoebae and may provide a reservoir for pathogenic bacteria such as Legionellapneumophila; it is consequently important to understand the response of this amoeba to chlorination, and our study was indeed aimed at examining cellular changes in Acanthamoebae following chlorination. Acanthamoeba trophozoites were treated at various chlorine concentrations (1-5mg/L). A 3-log reduction in Acanthamoebae population was achieved with 5mg/L of free chlorine. Confocal microscopy and flow cytometry experiments indicated that chlorination induced cell permeabilization, size reduction and likely intracellular thiol concentration. Our data show that among the non-cultivable cells some remained impermeabilized (negative staining with propidium iodide), thereby suggesting that these cells might remained viable. A similar state is described in other microorganisms as a VBNC (viable but not cultivable) state. Electron microscopy observations illustrate drastic morphological changes: the pseudopods disappeared and subcellular components, such as mitochondrion, were pronouncedly affected. In conclusion, depending on the concentration used, chlorination leads to many cellular effects on Acanthamoeba that could well arise in cell inactivation.

Cellular Response of the Amoeba Acanthamoeba castellanii to Chlorine, Chlorine Dioxide, and Monochloramine Treatments

ABSTRACT Acanthamoeba castellanii is a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested on A. castellanii trophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action on A. castellanii.

Chemokines and Antimicrobial Peptides Have a cag-Dependent Early Response to Helicobacter pylori Infection in Primary Human Gastric Epithelial Cells

ABSTRACT Helicobacter pylori infection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. The cag pathogenicity island (cag PAI) of H. pylori allows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response to H. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells with H. pylori B128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models with H. pylori B128ΔcagM, a cag PAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells with H. pylori, inflammatory-mediator production was largely due to cag PAI substrate-independent virulence factors. Thus, H. pylori cag PAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation during H. pylori infection.

Inflammatory signaling pathways induced by Helicobacter pylori in primary human gastric epithelial cells

Inflammatory signaling pathways induced by Helicobacter pylori remain unclear, having been studied mostly on cell-line models derived from gastric adenocarcinoma with potentially altered signaling pathways and nonfunctional receptors. Here, H. pylori-induced signaling pathways were investigated in primary human gastric epithelial cells. Inflammatory response was analyzed on chemokine mRNA expression and production after infection of gastric epithelial cells by H. pylori strains, B128 and B128ΔcagM, a cag type IV secretion system defective strain. Signaling pathway involvement was investigated using inhibitors of epidermal growth factor receptor (EGFR), MAPK, JAK and blocking Abs against TLR2 and TLR4. Inhibitors of EGFR, MAPK and JAK significantly reduced the chemokine mRNA expression and production induced by both H. pylori strains at 3 h and 24 h post-infection. JNK inhibitor reduced chemokine production at 24 h post-infection. Blocking Abs against TLR2 but not TLR4 showed significant reduction of chemokine secretion. Using primary culture of human gastric epithelial cells, our data suggest that H. pylori can be recognized by TLR2, leading to chemokine induction, and that EGFR, MAPK and the JAK/STAT signaling pathways play a key role in the H. pylori-induced CXCL1, CXCL5 and CXCL8 response in a cag pathogenicity island-independent manner.

Skin innate immune response to flaviviral infection

Skin is a complex organ and the largest interface of the human body exposed to numerous stress and pathogens. Skin is composed of different cell types that together perform essential functions such as pathogen sensing, barrier maintenance and immunity, at once providing the first line of defense against microbial infections and ensuring skin homeostasis. Being inoculated directly through the epidermis and the dermis during a vector blood meal, emerging Dengue, Zika andWest Nile mosquito-borne viruses lead to the initiation of the innate immune response in resident skin cells and to the activation of dendritic cells, which migrate to the draining lymph node to elicit an adaptive response. This literature review aims to describe the inflammatory response and the innate immune signalization pathways involved in human skin cells during Dengue, Zika and West Nile virus infections.

Antibacterial Polyketide Heterodimers from Pyrenacantha kaurabassana Tubers

Two heterodimers comprising anthraquinone and methylbenzoisocoumarin moieties (1 and 2) were isolated, together with emodin and physcion from the tubers of Pyrenacantha kaurabassana. The structures of 1 and 2 were established by NMR spectroscopy, including the analysis of a 2D INADEQUATE spectrum. On the basis of the data obtained, the structures that were previously proposed in the literature for these compounds were revised. Compounds 1 and 2 showed antibacterial activity against three different strains of Staphylococcus aureus. Compound 2 also showed bactericidal activity against Helicobacter pylori.

Th-17 response and antimicrobial peptide expression are uniformly expressed in gastric mucosa of Helicobacter pylori -infected patients independently of their clinical outcomes

The pathological determinism of H. pylori infection is explained by complex interplay between bacterial virulence and host inflammatory response. In a large prospective multicenter clinical study, Th17 response, expression of antimicrobial peptides (AMPs), cagA and vacA status, and bacterial density were investigated in the gastric mucosa of H. pylori ‐infected patients.

Interleukin-17A-induced production of acute serum amyloid A by keratinocytes contributes to psoriasis pathogenesis

Background Acute-serum Amyloid A (A-SAA), one of the major acute-phase proteins, is mainly produced in the liver but extra-hepatic synthesis involving the skin has been reported. Its expression is regulated by the transcription factors NF-κB, C/EBPβ, STAT3 activated by proinflammatory cytokines. Objectives We investigated A-SAA synthesis by resting and cytokine-activated Normal Human Epidermal Keratinocytes (NHEK), and their inflammatory response to A-SAA stimulation. A-SAA expression was also studied in mouse skin and liver in a model mimicking psoriasis and in the skin and sera of psoriatic and atopic dermatitis (AD) patients. Methods NHEK were stimulated by A-SAA or the cytokines IL-1α, IL-17A, IL-22, OSM, TNF-α alone or in combination, previously reported to reproduce features of psoriasis. Murine skins were treated by imiquimod cream. Human skins and sera were obtained from patients with psoriasis and AD. A-SAA mRNA was quantified by RT qPCR. A-SAA proteins were dosed by ELISA or immunonephelemetry assay. Results IL-1α, TNF-α and mainly IL-17A induced A-SAA expression by NHEK. A-SAA induced its own production and the synthesis of hBD2 and CCL20, both ligands for CCR6, a chemokine receptor involved in the trafficking of Th17 lymphocytes. A-SAA expression was increased in skins and livers from imiquimod-treated mice and in patient skins with psoriasis, but not significantly in those with AD. Correlations between A-SAA and psoriasis severity and duration were observed. Conclusion Keratinocytes could contribute to psoriasis pathogenesis via A-SAA production, maintaining a cutaneous inflammatory environment, activating innate immunity and Th17 lymphocyte recruitment.

Anti-inflammatory activity of Crateva adansonii DC on keratinocytes infected by Staphylococcus aureus: From traditional practice to scientific approach using HPTLC-densitometry

ETHNOPHARMACOLIGICAL RELEVANCE Leaves of Crateva adansonii DC (Capparidaceae), a small bush found in Togo, are widely used in traditional medicine to cure infectious abscesses. Traditional healers of Lomé harvest only budding leaves early in the morning, in specific area in order to prepare their drugs. AIM OF THE STUDY The main goal was to validate the ancestral picking practices, and to assess the activity of C. adansonii medicine towards infectious abscesses. MATERIALS AND METHODS A phytochemical screening of various C. adansonii leaf samples was performed using an original HPTLC-densitometry protocol and major flavonoids were identified and quantified. C. adansonii samples were collected in different neighborhoods of Lomé, at different harvesting-times and at different ages. Radical scavenging capacity, using DPPH assay, was used to quickly screen all extracts. Extracts were tested for anti-Staphylococcus aureus activity and anti-inflammatory effect on human primary keratinocytes infected by S. aureus. IL6, IL8 and TNFα expression and production were assessed by RT-PCR and ELISA assays. RESULTS Using antioxidant activity as selection criteria, optimal extracts were obtained with budding leaves, collected at 5:00am in Djidjolé neighborhood. This extract showed the strongest anti-inflammatory effect on S. aureus-infected keratinocytes by reducing IL6, IL8 and TNFα expression and production. None of the extracts inhibited the growth of S. aureus. CONCLUSIONS Those results validate the traditional practices and the potential of C. adansonii as anti-inflammatory drug. Our findings suggest that traditional healers should add to C. adansonii leaves an antibacterial plant of Togo Pharmacopeia, in order to improve abscess healing.