Assessing and Reducing Sources of Gene Expression Variability in Staphylococcus Epidermidis Biofilms

Abstract

Gene expression quantification can be a useful tool in studying the properties of bacterial biofilms. Unfortunately, techniques such as RNA extraction, cDNA synthesis, and quantitative PCR (qPCR) can introduce variability into mRNA transcript measurements, obscuring biologically relevant results. Here we sought to identify the steps that impair accurate gene expression quantification from Staphylococcus epidermidis biofilm samples. We devised an experimental setup that could be used to determine the contribution of each experimental step to the variability of mRNA transcript measurement. Among factors tested, biofilm growth contributed the most bias to gene expression quantification. Additional experiments demonstrated that pooling biofilms together reduced this variability, resulting in more accurate gene expression analysis results. We therefore recommend pooling in order to reduce the variability associated with gene expression quantification from biofilm samples.

Characterization of an in vitro fed-batch model to obtain cells released from S. epidermidis biofilms

“…Quantitative PCR (qPCR) analysis was performed, as described elsewhere (Franca et al 2012 ), in order to determine the levels of transcription of genes involved in quorum sensing ( agrB ), biofilm accumulation ( icaA ) and dispersion ( atlE , psmβ ). In brief, after pooling together the bulk fluid of 10 biofilms, the 10 originating biofilms were washed twice and pooled together as well, in order to reduce the characteristic heterogeneity of this population (Sousa et al 2014 ). Thereafter, 2 mL of planktonic (24 h stationary phase), biofilm bulk fluid or biofilm cells were collected by centrifugation and total RNA immediately isolated using FastRNA ® Pro blue Kit (MP Biomedicals, Santa Ana, CA, USA).…”

Staphylococcus epidermidis Biofilm-Released Cells Induce a Prompt and More Marked In vivo Inflammatory-Type Response than Planktonic or Biofilm Cells

“…Biofilms were grown in 24-well plates made of polystyrene plastic (Orange Scientific, Braine-l' Alleud, Belgium) by inoculating 15 μL of the 1 × 10 8 CFU/mL bacterial suspension into 1 mL of TSB 0.65%G , then incubating at 37°C with agitation at 120 rpm. After 24 h of growth, biofilms were washed twice with apyrogenic Phosphate Buffered Saline (PBS, Gibco, MD, USA), 1 mL of fresh TSB 0.65%G was carefully added and biofilms allowed to grow, under the same temperature and agitation conditions, for additional 24 h. Biofilm-released cells, (i.e., the cells in the biofilm bulk-fluid), were collected as described before (Franca et al, 2016 ) from 12 originating biofilms and pooled together to decrease variability inherent to biofilm growth (Sousa et al, 2014 ). Four biofilms were washed twice with apyrogenic PBS, disrupted and also pooled together to reduce variability.…”

Unveiling the fate of adhering bacteria to antimicrobial surfaces: expression of resistance-associated genes and macrophage-mediated phagocytosis

“…Cells recovered from the control surfaces (PDMS and pDA) were grown in TSA whereas cells retrieved from the surfaces functionalized, PALM or VANC were grown in TSA supplemented with a sub-inhibitory concentration of Palm and vancomycin, respectively, to keep the stress conditions. The colonies recovered from two replicated surfaces were pooled together in RNase free water in order to reduce the variability associated with gene expression quantification from biofilm samples [27], and centrifuged to pellet before RNA extraction. Three independent experiments in duplicate were performed.…”