Optimizing a reliable ex vivo human blood model to analyze expression of Staphylococcus epidermidis genes

Abstract

Human blood is often used as an ex vivo model to mimic the environment encountered by pathogens inside the host. A significant variety of experimental conditions has been reported. However, optimization strategies are often not described. This study aimed to evaluate key parameters that are expected to influence Staphylococcus epidermidis gene expression when using human blood ex vivo models. Our data confirmed that blood antimicrobial activity was dependent on initial bacterial concentration. Furthermore, blood degradation over time resulted in lower antimicrobial activity, with a 2% loss of leukocytes viability correlating with a 5-fold loss of antimicrobial activity against S. epidermidis. We further demonstrated that the volume of human blood could be reduced to as little as 0.18 mL without affecting the stability of gene expression of the tested genes. Overall, the data described herein highlight experimental parameters that should be considered when using a human blood ex vivo model for S. epidermidis gene expression analysis.

Transcriptome Mining to Identify Molecular Markers for the Diagnosis of Staphylococcus epidermidis Bloodstream Infections

“…To reduce the use of human blood, for all validation studies, the co-incubation assays were performed in a smaller volume than the one used for RNA-seq analysis [ 37 ]. Defibrinated horse blood (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze gene expression in a larger collection of isolates.…”

“…To validate the transcription levels of the selected genes, qPCR was performed as optimized before [ 34 , 37 ]. For biological validation of the data obtained by RNA-seq, total RNA was extracted using the E.Z.N.A.…”

mazEF Homologue Has a Minor Role in Staphylococcus epidermidis 1457 Virulence Potential

“…Plasma was separated by centrifugation of whole blood for 20 min at 1,440 g and 4°C. The co-incubation of bacteria with either whole blood, plasma or TSB supplemented with heparin (TSB + heparin, control) was performed as described before ( França et al., 2016 ; Brás et al., 2020 ). Briefly, 50 µl of bacterial suspensions with 2 × 10 5 CFU/ml were mixed with 450 µl of whole blood, plasma or TSB + heparin and incubated at 37°C and 80 rpm for up to 4 h. Cells culturability was assessed at the beginning of the assay (T = 0 h) and 1, 2, and 4 h after incubation.…”