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

Abstract

Bloodstream infections caused by Staphylococcus epidermidis are often misdiagnosed since no diagnostic marker found so far can unequivocally discriminate "true" infection from sample contamination. While attempts have been made to find genomic and/or phenotypic differences between invasive and commensal isolates, possible changes in the transcriptome of these isolates under in vivo-mimicking conditions have not been investigated. Herein, we characterized the transcriptome, by RNA sequencing, of three clinical and three commensal isolates after 2 h of exposure to whole human blood. Bioinformatics analysis was used to rank the genes with the highest potential to distinguish invasive from commensal isolates and among the ten genes identified as candidates, the gene SERP2441 showed the highest potential. A collection of 56 clinical and commensal isolates was then used to validate, by quantitative PCR, the discriminative power of the selected genes. A significant variation was observed among isolates, and the discriminative power of the selected genes was lost, undermining their potential use as markers. Nevertheless, future studies should include an RNA sequencing characterization of a larger collection of isolates, as well as a wider range of conditions to increase the chances of finding further candidate markers for the diagnosis of bloodstream infections caused by S. epidermidis.

Keywords: RNA sequencing; bloodstream infection diagnosis; clinical isolates; commensal isolates; ex vivo human blood model; molecular diagnosis markers.

Figure 1

Validation of the results obtained by RNA-seq. Data are presented in fold-change expression calculated using commensal isolates as control. (a) Technical validation (Tv) was performed using the same RNA utilized for library construction. The bars represent the mean and the standard error of the mean of two to three technical replicates. (b) Biological validation (Bv) was accomplished using RNA obtained from independent experiments. These experiments were performed with the same three clinical and three commensal isolates used for RNA-seq. The bars represent the mean and the standard error of the mean of three independent experiments performed using the blood of three different donors (n = 3). Statistical differences between technical and biological validations (b) were analyzed with the unpaired t-test. ** p < 0.001. RNA-seq, RNA sequencing, qPCR, quantitative PCR.

Figure 2

Evaluation of the discriminative potential of the gene SERP2441. Analysis of the gene SERP2441 expression stability (a) over time (n = 3, using the blood of the same donor) and (b) 4 h upon incubation with the blood of three different donors (n = 2 to 3 technical qPCR replicates). These experiments were performed in strains IE214 (clinical isolate) and SECOM0020A.1 (commensal isolate). Statistical differences among groups were analyzed with one-way ANOVA and Tukey’s multiple comparison test. * p < 0.05.

Figure 3

Evaluation of the expression of the gene SERP2441 in human and defibrinated horse blood over time. The bars represent the mean and the standard error of the mean of two independent assays (n = 2). These experiments were performed in strains IE214 (clinical isolate) and SECOM0020A.1 (commensal isolate). Statistical differences between groups in each time point were analyzed with the unpaired t-test. * p < 0.05.

Figure 4

Evaluation of the discriminative potential of the gene SERP2441 in a wide collection of isolates. This evaluation was performed 2 h after incubation with horse blood in a collection of 56 isolates. Of note, the isolates belonging to the “contamination” group are isolates collected from either contaminated samples or the skin of patients or staff. The horizontal line represents the grand mean of the transcription level of all isolates (n = 2 to 3 technical qPCR replicates). Statistical differences among groups were analyzed with one-way ANOVA and Tukey´s multiple comparison test.