Transcriptomic Analysis of Staphylococcus epidermidis Biofilm-Released Cells upon Interaction with Human Blood Circulating Immune Cells and Soluble Factors

摘要

Background The colonization of indwelling medical devices by biofilm-forming bacteria is one of the major causes of healthcare-associated infections (Percival et al., 2015 ). Staphylococcus epidermidis , a biofilm-forming commensal bacterium that inhabits human skin and mucosae, is considered one of most important causes of medical devices-related infections, being particularly associated with the use of intravascular catheters (Mack et al., 2013 ). Although S. epidermidis biofilms are classically associated with the development of chronic infections (Costerton et al., 1999 ), the release of cells from the biofilm has been associated with onset of acute infections such as embolic events of endocarditis (Pitz et al., 2011 ), bacteremia, or even septicemia (Cole et al., 2016 ). Bloodstream infections caused by S. epidermidis are typically indolent and difficult to eradicate significantly increasing patient's morbidity (Kleinschmidt et al., 2015 ) and mortality among immunocompromised (Khashu et al., 2006 ) and immunosuppressed patients (Bender and Hughes, 1980 ). In addition, the costs associated with the diagnosis and treatment of these secondary infections is estimated to be approximately $20,000 per occurrence (Kilgore and Brossette, 2008 ). Henceforth, it is imperative to redefine strategies for the management of the pathologic events associated with biofilm disassembly. Since bloodstream infections are one of the most frequent complications caused by S. epidermidis biofilm disassembly (Cole et al., 2016 ), a comprehensive analysis of the interplay between S. epidermidis biofilm-released cells (BRC) and hosts' blood components would be invaluable. Herein, as the first step toward the understanding of this interaction, we have characterized, using RNA sequencing (RNAseq) technology, the transcriptome of S. epidermidis BRC upon interaction with whole human blood, polymorphonuclear, or mononuclear leukocytes and plasma. Materials and methods Ethics statement Human blood was collected from healthy adult volunteers, under a human subject's protocol approved by the Institutional Review Board of the University of Minho (SECVS 002/2014). Furthermore, this procedure was performed in agreement with Helsinki declaration and Oviedo convention. All donors gave written consent before blood collection. Bacteria and growth conditions S. epidermidis strain 9142, isolated from a blood culture (Mack et al., 1992 ), was used for this study. BRC were obtained using a fed-batch system in the presence of Tryptic Soy Broth (TSB) supplemented with 0.65% glucose and under agitation conditions, as detailed elsewhere (Fran?a et al., 2016 ). BRC cells were collected from 12 different originating biofilms and pooled together to decrease the variability inherent to biofilm growth (Sousa et al., 2014 ). After 10 s sonication at 33% amplitude (Cole-Parmer 750-Watt Ultrasonic Homogenizer 230 VAC, IL, USA), the concentration of BRC was adjusted to 1 × 10~(9)total cells/mL, by flow cytometry (EC800, Sony Biotechnology Inc., CA, USA), using SYBR Green (Invitrogen, CA, USA) and propidium iodide (Sigma, MO, USA) staining as previously optimized (Cerca et al., 2011 ). Blood collection and fractioning Peripheral blood was collected into BD Vacutainer? tubes coated with lithium heparin (BD?, NJ, USA). Plasma was separated from the cellular fraction by centrifuging whole blood at 1440 g for 20 min at 4°C. Mononuclear (MN) leukocytes were purified from whole blood using Histopaque 1077 gradient (Sigma) as indicated by the manufacturer. Thereafter, the mononuclear cells-depleted pellet resultant from the Histopaque 1077 gradient was incubated with 1.5% (v/v) dextran solution during 35 min, at room temperature, in order to separate polymorphonuclear (PMN) cells from erythrocytes. PMN cells (present in the supernatant) were then transferred in to a new tube and harvested by centrifugation at 450 g for 15 min at 4°C. Both PMN and MN cells were incubated with water for 30 s to lyse the remaining erythrocytes and, after readjusting the isotonic conditions by adding 10 × PBS, leukocytes were collected by centrifugation at 200 g for 15 min at 4°C. Finally, leukocytes were suspended in 0.5 mL of donor's plasma and samples purity and viability determined by flow cytometry (EC800, Sony) using, respectively, CD15 (PMN) and CD3 (MN) (eBioscience, CA, USA) and propidium iodide staining (5 μg/mL, Sigma). Only samples with purity ≥90% and death ≤ 15% were used. The number of PMN and MN cells was determined also by flow cytometry and the concentration adjusted, in donor's plasma, to 1.0 × 10~(6)cells/mL. Co-incubation of bacteria with whole human blood and its circulating immune factors In 2 mL tubes, 100 μL of a suspension of 1 × 10~(9)total BRC/mL were mixed with 900 μL of whole human blood, PMN, or MN at 1.0 × 10~(6)cells/mL, plasma or TSB (containing the same concentration of heparin as blood and its components) and incubated at 80 rpm (in a 10 mm orbit incuba