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Verhoeven, P. O.

Publications and source records attributed to Verhoeven, P. O..

2 recordsLinked to original sources

Evaluation of in vitro activity of copper gluconate against SARS-CoV-2 using confocal microscopy-based high content screening

ContextSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged late in 2019 is the etiologic agent of coronavirus disease 2019 (Covid-19). There is an urgent need to develop curative and preventive therapeutics to limit the current pandemic and to prevent the re-emergence of Covid-19. This study aimed to assess the in vitro activity of copper gluconate against SRAS-CoV-2. MethodsVero E6 cells were treated with copper gluconate 18 hours before infection. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Infected cells were maintained in fresh medium containing copper gluconate for an additional 48-hour period. The infection level was measured by the confocal microscopy-based high content screening method. The cell viability in presence of copper gluconate was assessed by XTT assay. ResultsThe viability of Vero E6 cells treated with copper gluconate up to 200 M was found to be similar to that of untreated cells, but it dropped below 40% with 400 M of this agent. The infection rate was 23.8%, 18.9%, 20.6%, 6.9%, 5.3%,5.2% in cells treated with 0, 2, 10, 25, 50 and 100 M of copper gluconate respectively. As compared to untreated cells, the number of infected cells was reduced by 71%, 77%, and 78% with 25, 50, and 100 M of copper gluconate respectively (p < 0.05). ConclusionCopper gluconate was found to mitigate SARS-CoV-2 infection in Vero E6 cells. Furthers studies are needed to determine whether copper homeostasis could play a role in SARS-CoV-2 infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/422548v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f99949org.highwire.dtl.DTLVardef@1bebae6org.highwire.dtl.DTLVardef@e05e37org.highwire.dtl.DTLVardef@49a3b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

Construction and validation of EGFP-expressing Staphylococcus aureus clinical strains for adhesion and internalization assays on epithelial cells

BackgroundStaphylococcus aureus is both a major pathogen and a commensal bacterium in humans. It is able to adhere at the surface of epithelial cells of the anterior nares and can trigger its internalization inside these non-professional phagocytic cells. To better understand the interactions of clinical isolates with keratinocytes in the anterior nares, we developed and validated a one-step protocol expressing enhanced green fluorescent protein (EGFP) in S. aureus clinical strains with the aim to study adhesion to and internalization into mammalian cells.\n\nMethodsTwenty S. aureus clinical isolates belonging to clonal complexes 5, 8, 30, 45, 398 were selected for one-step transformation protocol with the EGFP-encoding plasmid pBSU101. EGFP expression was analysed by flow cytometry and confocal microscopy. Wild type and isogenic EGFP-expressing strains were compared for adhesion and internalization levels by using the HaCaT cell model.\n\nResultsTransformation was achieved in all the S. aureus strains regardless of their genetic background. The flow cytometry analysis showed that the mean proportion of EGFP-expressing bacteria was 97.2% ({+/-} 2.1) after 4h of incubation. Adhesion and internalization levels were similar in wild-type and isogenic EGFP-expressing S. aureus strains. Confocal laser scanning microscopy confirmed that EGFP-expressing S. aureus bacteria could be easily identified inside HaCaT keratinocytes.\n\nConclusionThis study reports an efficient protocol for expressing EGFP in S. aureus clinical strains and demonstrates that these EGFP-expressing strains are suitable for adhesion and internalization assays using HaCaT cells, which allows to perform static and dynamic in vitro studies of S. aureus colonization.

microbiology↗