Date Published: January 25, 2019
Publisher: Public Library of Science
Author(s): Maria Loza-Correa, Juan A. Ayala, Iris Perelman, Keith Hubbard, Miloslav Kalab, Qi-Long Yi, Mariam Taha, Miguel A. de Pedro, Sandra Ramirez-Arcos, Surajit Das.
Staphylococcus epidermidis is a bacterium frequently isolated from contaminated platelet concentrates (PCs), a blood product used to treat bleeding disorders in transfusion patients. PCs offer an accidental niche for colonization of S. epidermidis by forming biofilms and thus avoiding clearance by immune factors present in this milieu. Using biochemical and microscopy techniques, we investigated the structural changes of the peptidoglycan (PG) and the biofilm matrix of S. epidermidis biofilms formed in whole-blood derived PCs compared to biofilms grown in glucose-supplemented trypticase soy broth (TSBg). Both, the PG and the biofilm matrix are primary mechanisms of defense against environmental stress. Here we show that in PCs, the S. epidermidis biofilm matrix is mainly of a proteinaceous nature with extracellular DNA, in contrast to the predominant polysaccharide nature of the biofilm matrix formed in TSBg cultures. PG profile studies demonstrated that the PG of biofilm cells remodels during PC storage displaying fewer muropeptides variants than those observed in TSBg. The PG muropeptides contain two chemical modifications (amidation and O-acetylation) previously associated with resistance to antimicrobial agents by other staphylococci. Our study highlights two key structural features of S. epidermidis that are remodeled when exposed to human platelets and could be used as targets to reduce septic transfusions events.
Staphylococcus epidermidis is part of the normal human skin microbiome [1, 2]. It helps in the maintenance of a healthy skin, outcompeting harmful microorganisms such as Staphylococcus aureus [3, 4]. Although S. epidermidis does not produce virulence factor such as exotoxins, it has emerged as a significant opportunistic pathogen associated with healthcare-associated infections [3, 5]. Its ability to adhere to plastics for medical use and subsequent formation of surface-attached aggregates of bacteria known as biofilms is its most important virulence trait [3–5]. In transfusion medicine, S. epidermidis is the most frequent aerobic pathogen isolated from contaminated platelet concentrates (PCs), a blood product administered to patients with bleeding disorders . PCs can be manufactured by pooling buffy coat fractions obtained from whole blood donations of four to five donors. The pooled buffy coats are suspended in the plasma of one of the donors. Alternative, PCs can be collected from a single donor using an apheresis (centrifugation) device. Independently of the manufacturing method, the final PC product can be suspended in plasma or in a mix of plasma and a buffering additive solution . PCs are susceptible to bacterial contamination due to their storage conditions in gas-permeable plastic bags, containing high concentration of glucose, incubated at 20–24°C with agitation for up to 7 days, all of which are amenable for bacterial growth. A number of practices such as donor skin disinfection, diversion of the first aliquot of the donated blood, PC sterility testing using culture methods, and pathogen reduction technologies, have been implemented to minimize the risk of transfusing bacterially–contaminated blood products . Biofilm formation in PCs has been shown to increase missed detection during sterility screening [8, 9]. Proliferation of S. epidermidis in the PC storage environment may be linked to its ability to form biofilms, which confer protection from host defense molecules such as antimicrobial peptides (AMPs) derived from platelets [2, 10–14]. We have recently demonstrated that although AMPs could prevent biofilm formation by S. epidermidis, established mature staphylococcal biofilms are resistant to the bactericidal action of these immune factors . Interestingly, S. epidermidis isolates displaying a biofilm-negative phenotype convert to a biofilm-positive phenotype when grown in PCs, dependant on the presence of plasma factors [8, 16, 17]. Typical biofilm formation in S. epidermidis depends on the production of the exo-polysaccharide intracellular adhesin (PIA), encoded by the icaADBC operon. PIA mediates the accumulation stage of biofilm formation and is the main component of the biofilm matrix [18, 19]. Some PIA-negative strains are able to form biofilms in an ica-independent manner involving several cell wall anchored proteins [18, 20], while others display a biofilm-negative phenotype . Interestingly, certain strains are able to switch between PIA-dependent and protein-dependent biofilm formation with chemical changes in the structure of the biofilm matrix [22, 23]. Biofilm formation is a highly regulated process that involves a wide number of molecular factors including cell-surface proteins involved in adhesion to biotic or abiotic surfaces (Bhp, AltE, SSP-1 and SSP2) or the biofilm accumulation phase (Aap, Embp); extracellular DNA (eDNA); and teichoic acids (TA) [24, 25]. Importantly, relative amounts of the biofilm matrix components such as PIA and TA are dependent on growth conditions . The matrix is a mesh of molecules that interact with each other, for example, Aap can be covalently linked to peptidoglycan (PG) forming fibril-like structures [27, 28]. Similarly, TA increase bacterial adhesion to fibronectin-coated surfaces and contribute to biofilm formation .The study of the PG profile in S. epidermidis has not gained much attention in comparison to other staphylococci [29, 30]. Here we show that changes in the PG and biofilm matrix structure occur during biofilm formation of S. epidermidis in buffy coat PCs suspended in plasma. We explored the PG composition of S. epidermidis cells from mature biofilms formed in PCs compared to biofilm cells grown in laboratory media under optimal growth conditions. Furthermore, we explored the changes in the biofilm matrix composition and structure of S. epidermidis biofilms formed in PCs.
In the present work, we have shown that S. epidermidis biofilm cells undergo structural changes in their PG and biofilm matrix composition when grown in buffy coat PCs compared to optimal laboratory conditions. PCs are known to provide ideal conditions for bacteria to grow; however, the PC milieu also provides a stressful environment with bactericidal properties due to the presence of immune factors such as AMPs [10, 11]. Biofilm formation by S. epidermidis is likely a survival mechanism to resist such antimicrobial factors . The biofilm has an architecture including the extracellular matrix that provides resistance to antibiotics and other antimicrobial agents in different environments . Here we have shown that the biofilm matrix of different S. epidermidis strains in PCs is mainly composed of proteins and eDNA, which differs from the chemical structure when the bacteria are grown in regular media. It is important to recognize that proteins associated to the biofilm matrix could also be plasma proteins present in the PCs. These observations open new questions about alternative unexplored mechanisms for biofilm formation that are induced when S. epidermidis is grown in PCs. Understanding of the proteinaceous nature of the biofilm matrix of S. epidermidis formed in PCs would provide the identification of potential molecular targets to avoid biofilm formation and consequently improve PC transfusion safety. In S. aureus, formation of a proteinaceous matrix containing cytoplasmic proteins exported to the cell surface has been described to be linked to a decrease in pH in the growth medium . The molecular mechanisms that regulate this phenomenon remain unclear. The presence of glucose in the growth medium for bacteria leads to the accumulation of acidic by-products from fermentation that is critical for the decrease in pH and the induction of biofilms. In PCs, the high levels of glucose might be one of the factors that contributes to the induction of biofilm formation . It would be reasonable to hypothesize that the utilization of glucose by bacteria could create a microenvironment with a decreased pH that stimulates the exportation of cytoplasmic proteins to the cell surface to form a proteinaceous biofilm matrix. Indeed, a decrease in pH in contaminated PCs has been associated to high titres of bacterial contamination [48, 49].