Biofilm dormancy enhances antimicrobial tolerance in S. epidermidis

Our well-being and our microbes Annual Meeting of the New Zealand Microbiological SocietyIndwelling medical devices have been increasingly used in modern medicine and have saved millions of lives worldwide. However, they can also be an important source of infections, most commonly caused by coagulase negative-staphylococci, particularly by biofilm forming Staphylococcus epidermidis. A key feature of biofilms is its enhanced tolerance to antibiotics. Several mechanisms have been proposed to contribute to this phenomenon. We recently developed an in vitro model able to stimulate the induction or prevention of biofilm dormancy. Herein, we used that model to determine if biofilms with induced dormancy presented a distinct antimicrobial tolerance profile than biofilms with prevented dormancy. Both clinical or commensal isolates where included and a total of 43 unique isolates, from different parts of the world were tested. Biofilms were exposed to tetracycline, vancomycin and rifampicin and where analysed by flow citometry, CFU counts and CLSM. Three unique observations were obtained. First, biofilm dormancy was found as a widespread condition in both clinical and commensal isolates, suggesting this is a fundamental process not only related to the infectious process. Second, while vancomycin did not presented any significant effect on the tested biofilms, tetracycline and rifampicin significantly reduced the number of CFUs in biofilms with prevented dormancy tested (up to 4 log killing under 8 h), but were significantly less effective in biofilms with induced dormancy. The third and more curious observation was that the very high reduction in cultivable bacteria was not correlated with the reduction of total and viable cells. Overall, our data suggests in one hand that biofilms with induced dormancy are more tolerant to tetracycline and rifampicin and that those antibiotics further induce dormancy in biofilms, instead of effective eliminating the biofilm bacteria.info:eu-repo/semantics/publishedVersio

The pathogenesis of Staphylococcus epidermidis biofilm-associated infections: the host and the pathogen perspective

Book of Abstracts of CEB Annual Meeting 2017info:eu-repo/semantics/publishedVersio

Efficacy studies of phage phiIBB-Sep1 against S. epidermidis cells under different metabolic states

Staphylococcus epidermidis has recently become known as a usual cause of nosocomial infections, predominantly in patients with indwelling medical devices. Although, S. epidermidis infections only rarely develop into life-threatening diseases, they are very frequent and difficult to treat due to the ability of this bacterium to adhere to the surfaces of indwelling medical devices and form biofilms. When S. epidermidis cells are in a biofilm they are more resistant to antibiotics and to the immune system. The importance of biofilms in the pathogenesis of the S. epidermidis infections is becoming more understandable, consequently several studies are needed, in order to develop effective methods for biofilm control. The use of bacteriophages (phages) to eradicate biofilms can be seen as a potentially valuable approach. Phages are virus that infect bacteria and are the most abundant organisms on Earth. They are generally very efficient antibacterial agents and possess many advantages over antibiotics. Our aim is to search for virulent phages with broad host range for S. epidermidis biofilm therapy. Using wastewater treatment plants raw effluents we were able to isolate 5 phages. Their lytic activity was screened against 40 clinical S. epidermidis isolates with different genetic profiles and it was found to be different ranging from 46% to 95% of positive results. Further morphologic and genetic characterization of these isolated phages is now being performed. Efficacy studies results show that phage phiIBB-Sep1 is able to cause a 6 Log CFU/ml reduction of the cell titre in <2h for some of the clinical strains at exponential phase and in <4h for stationary phase cells (using a MOI of 1). This phage has also the capacity of reducing by up to 2 Log CFU/ml 24h biofilm cells and in some strains it was observed 50% cell reduction on biofilms. Besides CFU counting, all the cell counts were confirmed by flow cytometry assays. Additionally, flow cytometry allow the observation that this phage kill cells under different metabolic states from the biofilm. Work developed with non biofilm forming strains showed that possibly PNAG might be the cell receptor of the phage. The high amounts of PNAG on biofilms, might the lower activity of this phage on biofilms. These are promising results, since phage phiIBB-Se1 presents a broad host range and ability to control S. epidermidis under different metabolic states. Ongoing studies are being performed with 4 other phages, with the purpose of developing a phage cocktail to be used against S. epidermidis biofilm infections

A first new look into the interaction of Staphylococcus epidermidis biofilm-released cells with the host immune system

The widespread application of indwelling medical devices in the clinical setting, together with the remarkable ability of the commensal Staphylococcus epidermidis to adhere to these surfaces and form biofilms, has given to this bacterium the recognition of being a leading causative agent of nosocomial infections. Biofilms lifecycle is currently divided into 4 main steps: initial adhesion, accumulation, maturation and, disassembly. Biofilm disassembly, the release of the cells within the biofilm into the involving environment, is the less understood of all steps despite its involvement in the development of several serious acute infections such as endocarditis, bacteremia and pneumonia. Hence, due to its important consequences in human health and disease, the study of the cells released from S. epidermidis biofilms is crucial to create effective therapeutic strategies against these serious infections. For that reason, in order to better characterize S. epidermidis biofilm-released cells, we assessed their cell properties by determining 1) the expression of key genes involved in initial adhesion, biofilm regulation and disassembly, 2) the total protein profile, 3) the susceptibility to routinely used antibiotics for the treatment of staphylococcal infection, and 4) the adhesion ability to coated and uncoated surfaces. Additionally, 5) the interaction of these cells with the host immune system was also assessed using an intravenous mouse infection model. Planktonic and biofilm cells were also used for comparison purposes. Our results revealed that S. epidermidis biofilm-released cells share some particular features with planktonic cells, such as expression of psmβ, but at the same time share some features similar to biofilms, such as high antibiotic tolerance. Moreover, although these shared features, the cells released from the S. epidermidis biofilms produced a unique protein that is not detected in the other assessed phenotypes. Additionally, S. epidermidis biofilm-released cells elicit a different in vivo response than their planktonic counterparts, by stimulating a lower production of inflammatory chemokines KC and MCP-1, and interleukine-6 as well. Altogether, these results indicate that this particular phenotype may present some advantageous features allowing a more effective host colonization and infection spreading. Thus, the targeting of the particular properties of the biofilm-released cells could present new opportunities to more effectively prevent the pathologic events associated with dissemination of cells from a biofilm to more distant sites.This work was funded by Fundação para a Ciência e a Tecnologia (The Foundation for Science and Technology: FCT) and COMPETE (Programa Operacional Factores de Competitividade) grants PTDC/BIA-MIC/113450/2009 and FCOMP-01-0124-FEDER-014309. AF and VC were funded by FCT fellowship SFRH/BD/62359/2009 and SFRH/BD/78235/2011, respectively

Medium supplementation with magnesium prevented the induction of dormancy in biofilms of clinical and commensal isolates of Staphylococcus epidermidis

Staphylococcus epidermidis is a commensal bacterium that colonizes the skin and mucous membranes, being the most prevalent staphylococcal species found in humans. However, S. epidermidis has the ability of colonize indwelling medical devices establishing biofilms, which makes this bacteria a common cause of bacteraemia particularly in immunocompromised individuals and neonates [1][2]. The presence of high amounts of dormant bacteria (viable but non-culturable cells, VBNC) is a hallmark of biofilms, making them more tolerant to antimicrobials and elusive to the host immune response [3]. Glucose (G) is commonly used in vitro to induce a biofilm mode of growth in S. epidermidis cultures [4] due to the medium acidification derived from carbon metabolism [5]. Previous studies in our laboratory demonstrated that the accumulation of VBNC bacteria inside S. epidermidis biofilms could be prevented by supplementation of the medium with magnesium (Mg) (unpublished data). In the present work, a flow cytometric live/dead staining [6], in combination with the spreadplate method, were used to evaluate the effect of magnesium in preventing dormancy on glucose excess-grown biofilms in 53 strains of international clinical, Portuguese clinical, and Portuguese commensal isolates of S. epidermidis. Culture supplementation with Mg2+ prevented the accumulation of death bacteria inside the biofilms in all the groups of strains tested, being the proportion of live bacteria (SYBR+PI-) significantly increased by Portuguese clinical isolates. The quantification of colony forming units (CFUs) revealed that Mg hampered the induction of dormancy in all the groups of strains studied (International clinical G: 3,60,8×107 vs. G+Mg: 2,60,5×108, Portuguese clinical 6,82,0×107 vs. 4,31,4×108, Portuguese commensal 7,21,5×107 vs. 2,60,4×108 CFUs/mL). These results confirmed that dormancy modulation is widespread in different S. epidermidis isolates obtained from clinical and commensal sources, suggesting that this is indeed an important mechanism of S. epidermidis biofilm physiology

Staphylococcus epidermidis biofilms are resistant to phage infection due to the protective effect of the biofilm matrix

EMBO Conference on Viruses of Microbes III: Structure and Function - from Molecules to Communities (Programme and Abstract Book)info:eu-repo/semantics/publishedVersio

Genomic characterization of the Staphylococcus epidermidis-specific bacteriophage SEP1 and evaluation of its lytic activity against bacterial under different metabolic states

Staphylococcus epidermidis is one of the most frequent causative agents of nosocomial infections, predominantly in patients with indwelling medical devices. This microrganism may form biofilms which are microbial structures very tolerant to the host immune defenses and to antibiotherapy. Therefore, studies are needed in order to develop effective methods for biofilm control. Currently, bacteriophages (phages) are seen as an important tool to combat pathogenic organisms. These bacteria-specific viruses are generally very efficient antibacterial agents and possess many advantages over antibiotics. The present study concerns the search for virulent phages with broad host range for S. epidermidis biofilm therapy. Using wastewater treatment plant raw effluents, a novel phage was isolated and characterized. This virus was named phiIBB-SEP1 and TEM micrographs suggested that it belonged to the Twortlikevirus genus. Phage phiIBB-SEP1 is able to infect 41 S. epidermidis clinical isolates used in this study, and contrarily to other polyvalent viruses of the Twortlikevirus genus, phiIBB-SEP1 is highly specific for S. epidermidis strains. The genome of this phage was fully sequenced and presents the typical structure of a member of the Twortlikevirus. However, when compared to other staphylococcal members of this genus, it showed DNA sequence identities no greater than 58.2%, suggesting that phiIBB-SEP1 is a new species within this subfamily. Efficacy studies results showed that phage phiIBB-SEP1 is able to cause a 6 Log CFU per ml reduction of the cell titre in less than 2h for some of the clinical strains in exponential phase; and, in less than 4h for stationary phase cells (using a multiplicity of infection of 1). This phage has also the capacity of reducing, by up to 2 Log CFU per ml, 24h scraped biofilm cells, and in some strains it was observed 50% cell reduction. Besides CFU counting, this cell reduction was confirmed by flow cytometry counting. Additionally, live/death flow cytometry staining allowed the observation that this phage kills biofilms bacteria in different metabolic states. These are promising results, since phage phiIBB-SEP1 presents a broad host strain range and the ability to control S. epidermidis bacteria in different metabolic states. Keywords: bacteriophage, Twortlikevirus, S. epidermidis, staphylococci, biofilm

Lipid Antigen Presentation by CD1b and CD1d in Lysosomal Storage Disease Patients

The lysosome has a key role in the presentation of lipid antigens by CD1 molecules. While defects in lipid antigen presentation and in invariant Natural Killer T (iNKT) cell response were detected in several mouse models of lysosomal storage diseases (LSD), the impact of lysosomal engorgement in human lipid antigen presentation is poorly characterized. Here, we analyzed the capacity of monocyte-derived dendritic cells (Mo-DCs) from Fabry, Gaucher, Niemann Pick type C and Mucopolysaccharidosis type VI disease patients to present exogenous antigens to lipid-specific T cells. The CD1b- and CD1d-restricted presentation of lipid antigens by Mo-DCs revealed an ability of LSD patients to induce CD1-restricted T cell responses within the control range. Similarly, freshly isolated monocytes from Fabry and Gaucher disease patients had a normal ability to present α-Galactosylceramide (α-GalCer) antigen by CD1d. Gaucher disease patients' monocytes had an increased capacity to present α-Gal-(1-2)-αGalCer, an antigen that needs internalization and processing to become antigenic. In summary, our results show that Fabry, Gaucher, Niemann Pick type C, and Mucopolysaccharidosis type VI disease patients do not present a decreased capacity to present CD1d-restricted lipid antigens. These observations are in contrast to what was observed in mouse models of LSD. The percentage of total iNKT cells in the peripheral blood of these patients is also similar to control individuals. In addition, we show that the presentation of exogenous lipids that directly bind CD1b, the human CD1 isoform with an intracellular trafficking to the lysosome, is normal in these patients.This work was financed by Gaucher Generation Program, supported by Sanofi-Genzyme and by Norte-01-0145-FEDER-000012—Structured program on bioengineered therapies for infectious diseases and tissue regeneration, supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). BP-C was temporally supported by a grant from the American Portuguese Biomedical Research Fund (APBRF).info:eu-repo/semantics/publishedVersio