Angela França

Optimizing a qPCR Gene Expression Quantification Assay for S. epidermidis Biofilms: A Comparison between Commercial Kits and a Customized Protocol

Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psmβ1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used.

Comparison of RNA extraction methods from biofilm samples of Staphylococcus epidermidis

BackgroundMicrobial biofilms are communities of bacteria adhered to a surface and surrounded by an extracellular polymeric matrix. Biofilms have been associated with increased antibiotic resistance and tolerance to the immune system. Staphylococcus epidermidis is the major bacterial species found in biofilm-related infections on indwelling medical devices. Obtaining high quality mRNA from biofilms is crucial to validate the transcriptional measurements associated with the switching to the biofilm mode of growth. Therefore, we selected three commercially available RNA extraction kits with distinct characteristics, including those using silica membrane or organic extraction methods, and enzymatic or mechanical cell lysis, and evaluated the RNA quality obtained from two distinct S. epidermidis bacterial biofilms.ResultsRNA extracted using the different kits was evaluated for quantity, purity, integrity, and functionally. All kits were able to extract intact and functional total RNA from the biofilms generated from each S. epidermidis strain. The results demonstrated that the kit based on mechanical lysis and organic extraction (FastRNA® Pro Blue) was the only one that was able to isolate pure and large quantities of RNA. Normalized expression of the icaA virulence gene showed that RNA extracted with PureLink™ had a significant lower concentration of icaA mRNA transcripts than the other kits tested.ConclusionsWhen working with complex samples, such as biofilms, that contain a high content extracellular polysaccharide and proteins, special care should be taken when selecting the appropriate RNA extraction system, in order to obtain accurate, reproducible, and biologically significant results. Among the RNA extraction kits tested, FastRNA® Pro Blue was the best option for both S. epidermidis biofilms used.

Dormancy within Staphylococcus epidermidis biofilms: a transcriptomic analysis by RNA-seq

The proportion of dormant bacteria within Staphylococcus epidermidis biofilms may determine its inflammatory profile. Previously, we have shown that S. epidermidis biofilms with higher proportions of dormant bacteria have reduced activation of murine macrophages. RNA-sequencing was used to identify the major transcriptomic differences between S. epidermidis biofilms with different proportions of dormant bacteria. To accomplish this goal, we used an in vitro model where magnesium allowed modulation of the proportion of dormant bacteria within S. epidermidis biofilms. Significant differences were found in the expression of 147 genes. A detailed analysis of the results was performed based on direct and functional gene interactions. Biological processes among the differentially expressed genes were mainly related to oxidation-reduction processes and acetyl-CoA metabolic processes. Gene set enrichment revealed that the translation process is related to the proportion of dormant bacteria. Transcription of mRNAs involved in oxidation-reduction processes was associated with higher proportions of dormant bacteria within S. epidermidis biofilm. Moreover, the pH of the culture medium did not change after the addition of magnesium, and genes related to magnesium transport did not seem to impact entrance of bacterial cells into dormancy.

Alterations in the Staphylococcus epidermidis biofilm transcriptome following interaction with whole human blood

Staphylococcus epidermidis biofilm formation on the surface of intravenous catheters is responsible for 22% of the cases of bloodstream infections, in patients in intensive care units in the USA. The ability of S. epidermidis to withstand the high bactericidal activity of human blood is therefore crucial for systemic dissemination. To identify the genes involved in the bacteriums survival, the transcriptome of S. epidermidis biofilms, upon contact with human blood, was assessed using an ex vivo model. Our results showed an increased transcription of genes involved in biosynthesis and metabolism of amino acids, small molecules, carboxylic and organic acids, and cellular ketones. One of the striking changes observed 4 h of S. epidermidis exposure to human blood was an increased expression of genes involved in iron utilization. This finding suggests that iron acquisition is an important event for S. epidermidis survival in human blood.

Monoclonal Antibody Raised against PNAG Has Variable Effects on Static S. epidermidis Biofilm Accumulation In Vitro

Staphylococcus epidermidis causes infections commonly associated with patients with indwelling medical devices due to its ability to form biofilms, bacterial structures attached to a surface and embedded in a protective matrix. Cells within biofilms are known to be more resistant than free-floating planktonic cells to the host immune response and also to antibiotic therapy often leading to relapsing infections 1. Frequently, surgical removal of an infected device is required to resolve these infections, resulting in significant effects in a patients quality of life. Hence, preventative approaches are clearly needed to overcome this challenge. Antibodies have been shown to be one promising alternative to target surface-attached molecules and inhibit biofilm formation 2. We have previously shown that human monoclonal antibodies (mAb) specific for poly-β-1,6-N-acetylglucosamine (PNAG) were effective in killing S. epidermidis in opsonophagocytic in vitro assays, and in protecting the murine host against infection by PNAG-producing pathogens 3,4. Since S. epidermidis biofilm accumulation is mainly mediated by PNAG, it was hypothesized that the binding of this molecule by a specific mAb could impact biofilm accumulation, a process that has not previously been investigated. Here we tested the previously characterized mAb F598 3, for inhibition of S. epidermidis biofilm accumulation in vitro. Several concentrations of mAb F598 and the isotype control human mAb F429 (specific for the Pseudomonas aeruginosa alginate capsule) 5, were co-cultured with bacteria in static conditions for 1h at 37oC to allow antibody binding. Thereafter, cultures were incubated for 24h at 37oC with shaking at 250 rpm. Biofilm accumulation was then quantified by the standard crystal violet staining 6. Depending on the S. epidermidis strain used, the presence of mAb F598 had a differential effect on biofilm accumulation. In the case of the ATCC strain RP62A we observed a 42% reduction in biofilm accumulation at the highest mAb concentration tested, while the clinical strains 1457 and M184 grown in the presence of mAb F598 had a dose-dependent increase of the biofilm accumulation. In the case of the PNAG-deficient, ica-mutant strain 1457-M10, as expected, no significant effect was found on the biofilm biomass as no PNAG is produced. Furthermore, control mAb F429 had no significant effect on S. epidermidis biofilm accumulation. The results with the ica-mutant and mAb controls suggest altogether that the inhibitory or enhancing effect of the mAb is PNAG-dependent. As observed in other studies that have used antibodies specific for S. epidermidis surface molecules, the observed enhancement of biofilm formation could be a result of increased PNAG expression caused by the early blockage of the synthesis of this molecule 7. On the other hand, the specificity of mAb F598 for epitopes on PNAG that do not require the N-acetyl groups on the glucosamine monomers may have contributed to the differential effects in biofilm accumulation. These would thus depend on the level of PNAG acetylation of individual strains, ultimately, controlled by the IcaB extracellular deacetylase. Therefore, mAbs directed to other epitopes might be better suited for inhibition of in vitro biofilm accumulation. Additionally, the results presented here suggest that a difference between the effect of mAb F598 against PNAG-producing bacteria in animal models 3,4 and it efficiency at inhibiting in vitro static biofilm accumulation among different S. epidermidis strains. Notably, many biofilms are formed under flow conditions and it is not clear to what extent shear stress from flow over in vivo biofilms contributes to biofilm formation, and whether under those conditions the effect of mAb F598 might be different. While the stimulation of biofilm formation by S. epidermidis grown in vitro may raise questions regarding the usage of mAb F598 in vivo, the results do not necessarily exclude that mAb F598 could be effective in vivo against biofilm infections. The majority of studies that reported strong biofilm inhibition by monoclonal or polyclonal antibodies used only a few strains in the assays 2,8, which could result in misleading interpretations. The findings presented here further stress the necessity to use more than a few strains when testing the efficacy of new biofilm-inhibition strategies in order to ensure that the desired effect is observed in a representative number of clones of the species under study.

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

Both dynamic and fed-batch systems have been used for the study of biofilms. Dynamic systems, whose hallmark is the presence of continuous flow, have been considered the most appropriate for the study of the last stage of the biofilm lifecycle: biofilm disassembly. However, fed-batch is still the most used system in the biofilm research field. Hence, we have used a fed-batch system to collect cells released from Staphylococcus epidermidis biofilms, one of the most important etiological agents of medical device-associated biofilm infections. Herein, we showed that using this model it was possible to collect cells released from biofilms formed by 12 different S. epidermidis clinical and commensal isolates. In addition, our data indicated that biofilm disassembly occurred by both passive and active mechanisms, although the last occurred to a lesser extent. Moreover, it was observed that S. epidermidis biofilm-released cells presented higher tolerance to vancomycin and tetracycline, as well as a particular gene expression phenotype when compared with either biofilm or planktonic cells. Using this model, biofilm-released cells phenotype and their interaction with the host immune system could be studied in more detail, which could help providing significant insights into the pathophysiology of biofilm-related infections.

Comparative proteomic and transcriptomic profile of Staphylococcus epidermidis biofilms grown in glucose-enriched medium

Staphylococcus epidermidis is an important nosocomial agent among carriers of indwelling medical devices, due to its strong ability to form biofilms on inert surfaces. Contrary to some advances made in the transcriptomic field, proteome characterization of S. epidermidis biofilms is less developed. To highlight the relation between transcripts and proteins of S. epidermidis biofilms, we analyzed the proteomic profile obtained by two mechanical lysis methods (sonication and bead beating), associated with two distinct detergent extraction buffers, namely SDS and CHAPS. Based on gel electrophoresis-LC-MS/MS, we identified a total of 453 proteins. While lysis with glass beads provided greater amounts of protein, CHAPS extraction buffer allowed identification of a higher number of proteins compared to SDS. Our data shows the impact of different protein isolation methods in the characterization of the S. epidermidis biofilm proteome. Furthermore, the correlation between proteomic and transcriptomic profiles was evaluated. The results confirmed that proteomic and transcriptomic data should be analyzed simultaneously in order to have a comprehensive understanding of a specific microbiological condition.

Assessing and reducing sources of gene expression variability in Staphylococcus epidermidis biofilms.

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.

Farnesol induces cell detachment from established S. epidermidis biofilms

Antibiotic resistance is a serious problem in Staphylococcus epidermidis infections as many clinical isolates of this organism are resistant to up to eight different antibiotics. The increased resistance to conventional antibiotic therapy has lead to the search for new antimicrobial therapeutic agents. Farnesol, an essential oil found in many plants, has been shown to be active against S. epidermidis. Using a type control strain we recently described that although farnesol was not efficient at killing biofilm bacteria, a strong reduction on biofilm biomass was detected, and we hypothesize that farnesol could, somehow, induce biofilm detachment. In this report, to test our hypothesis we used 36 representative clinical strains of S. epidermidis from different geographic locations and characterized them in terms of genetic variability by multilocus sequence typing and staphylococcal chromosome cassette mec. Strains were tested for biofilm formation, and the presence of ica, bhp and aap genes was determined. Stronger biofilms had always the presence of ica operon but often co-harbored bhp and aap genes. Farnesol was then used in biofilm-forming strains, and biofilm detachment was detected in half of the strains tested. Furthermore, we also showed that farnesol inability to kill biofilm bacteria was not the result of the biofilm structure but was related to high cell density. Our results demonstrate, for the first time, that the biomass reduction previously found by us, and many other groups, is the result not of cell killing but instead is the result of biofilm detachment.

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

Staphylococcus epidermidis biofilm formation on indwelling medical devices is frequently associated with the development of chronic infections. Nevertheless, it has been suggested that cells released from these biofilms may induce severe acute infections with bacteraemia as one of its major associated clinical manifestations. However, how biofilm-released cells interact with the host remains unclear. Here, using a murine model of hematogenously disseminated infection, we characterized the interaction of cells released from S. epidermidis biofilms with the immune system. Gene expression analysis of mouse splenocytes suggested that biofilm-released cells might be particularly effective at activating inflammatory and antigen presenting cells and inducing cellular apoptosis. Furthermore, biofilm-released cells induced a higher production of pro-inflammatory cytokines, in contrast to mice infected with planktonic cells, even though these had a similar bacterial load in livers and spleens. Overall, these results not only provide insights into the understanding of the role of biofilm-released cells in S. epidermidis biofilm-related infections and pathogenesis, but may also help explain the relapsing character of these infections.