Staphylococcus epidermidis biofilms: importance and implications.

The coagulase-negative staphylococci and, in particular, Staphylococcus epidermidis, have emerged as major nosocomial pathogens associated with infections of implanted medical devices. These organisms, which are among the most prevalent bacteria of the human skin and mucous membrane microflora, present unique problems in the diagnosis and treatment of infections involving biofilm formation on implanted biomaterials. Epidemiological data that address whether invasive S. epidermidis strains can be traced to commensal organisms or an endemic occurrence of distinct strains with enhanced virulence have important implications for the implementation of appropriate infection control measures. An extracellular polysaccharide adhesin represents a key virulence determinant in S. epidermidis and is required for biofilm formation. Production of this adhesin, which is encoded by the ica operon, is subject to phase variable regulation (ON <---> OFF switching). Recent advances in understanding the molecular events controlling polysaccharide adhesin synthesis and the potential clinical implications of its phase variable regulation are outlined. Further research in this area may contribute to the development of novel strategies for therapeutic intervention. Finally, in addition to antibiotic prophylaxis, preventive strategies to control S. epidermidis medical device-related infections are focusing on the development of improved biomaterials and physical electrical barriers to impede bacterial colonisation.     

芍药苷对白色念珠菌生物膜的作用

背景：研究证实中药赤芍有效成分对白色念珠菌有较好的抑制作用,但其单体芍药苷对白色念珠菌生物膜是否有抑制作用未见报道。  目的：观察芍药苷对体外白色念珠菌生物膜的影响。 方法：用RPMI-1640分别按2倍稀释法制备5个浓度梯度(4,2,1,0.5,0.25 g/L)的芍药苷溶液。用RPMI-1640稀释洗必泰为5个浓度梯度(2%,1%,0.5%,0.25%,0.125%)。采用琼脂扩散法检测不同浓度梯度芍药苷或洗必泰对白色念珠菌的抑菌直径。MTT法检测不同浓度洗必泰或芍药苷对白色念珠菌细胞黏附的作用,以及对白色念珠菌生物膜的抑制作用,并且利用激光共聚焦扫描显微镜和死菌活菌荧光染色技术相结合方法观察常态及药物作用下的白色念珠菌生物膜。 结果与结论：洗必泰与芍药苷均有抑菌能力,抑菌环直径与药物浓度呈正相关;除2 g/L芍药苷组与1%,2%洗必泰组抑菌环直径无差异外,其余组间两两比较差异均有显著性意义。不同质量浓度芍药苷对白色念珠菌的细胞黏附都具有抑制作用,对白色念珠菌生物膜也具有抑制作用,且抑制率与药物质量浓度呈正相关。观察48 h时常态生物膜结构中大部分是活菌,有少量死菌存在;随芍药苷质量浓度改变白色念珠菌生物膜中死菌比例不断增高,其抑菌活性相对弱于洗必泰。表明芍药苷对体外白色念珠菌生物膜有较明显的抑制作用。|中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Activity of coumarin against Candida albicans biofilms

Objective

The aim of this study was to investigate the antibiofilm activity of coumarin against Candida albicans.

Photodynamic action of merocyanine 540 on Staphylococcus epidermidis biofilms

Photodynamic treatment (PDT) has been proposed as a new approach for inactivation of biofilms associated with medical devices that are resistant to chemical additives or biocides. In this study, we evaluated the antimicrobial activity of merocyanine 540 (MC 540), a photosensitizing dye that is used for purging malignant cells from autologous bone marrow grafts, against Staphylococcus epidermidis biofilms. Effect of the combined photodynamic action of MC 540 and 532 nm laser was investigated on the viability and structure of biofilms of two Staphylococcus epidermidis strains, RP62A and 1457. Significant inactivation of cells was observed when biofilms were exposed to MC 540 and laser simultaneously. The effect was found to be light dose-dependent but S. epidermidis 1457 biofilm proved to be slightly more susceptible than S. epidermidis RP62A biofilm. Furthermore, significant killing of both types of cells was attained even when a fixed light dose was delivered to the biofilms. Confocal laser scanning microscope (CLSM) analysis indicated damage to bacterial cell membranes in photodynamically treated biofilms, while disruption of PDT-treated biofilm was confirmed by scanning electron microscopy (SEM).     

Microfluidic devices for studying growth and detachment of Staphylococcus epidermidis biofilms

Microfluidic devices were used to study the influences of hydrodynamics of local microenvironments on Staphylococcus epidermidis (S. epidermidis) biofilm formation and the effects of a poly(beta-1,6-N-acetyl glucosamine)-hydrolyzing enzyme (dispersin B) and/or an antibiotic (rifampicin) on the detachment of the biofilm. Elongated, monolayered biofilm morphologies were observed at high flow velocity and fluid shear locations whereas large clump-like, multilayered biofilm structures were produced at low flow velocity and fluid shear locations. Upon dispersin B treatment, most of the biofilm was detached from the microchannel surface. However, a trace amount of bacterial cells could not be removed from corner locations most likely due to the insufficient wall shear stress of the fluid at these locations. Dispersin B or rifampicin treatment was effective in delaying the dispersal behavior of bacterial cells, but could not completely remove the biofilm. Combined dynamic delivery of dispersin B and rifampicin was found to be effective for complete removal of the S. epidermidis biofilm.     

Ultrasonically enhanced vancomycin activity against Staphylococcus epidermidis biofilms in vivo

Infection of implanted medical devices by Gram-positive organisms such as Staphylococcus ssp. is a serious concern in the biomaterial community. In this research the application of low frequency ultrasound to enhance the activity of vancomycin against implanted Staphylococcus epidermidis biofilms was examined. Polyethylene disks covered with a biofilm of S. epidermidis were implanted subcutaneously in rabbits on both sides of their spine. The rabbits received systemic vancomycin for the duration of the experiment. Following 24 h of recovery, one disk was insonated for 24 or 48 h while the other was a control. Disks were removed and viable bacteria counted. At 24 h of insonation, there was no difference in viable counts between control and insonated biofilms, while at 48 h of insonation there were statistically fewer viable bacteria in the insonated biofilm. The S. epidermidis biofilms responded favorably to combinations of ultrasound and vancomycin, but longer treatment times are required for this Gram-positive organism than was observed previously for a Gram-negative species.     

Candida albicans interaction with Gram-positive bacteria within interkingdom biofilms

Candida albicans is a commensal of the human body and an opportunistic pathogen frequently responsible for nosocomial bloodstream infections. Most of these infections are linked to the development of a biofilm in or on implanted medical devices. C. albicans cells have the capacity to interact with bacteria within biofilms, especially by the way of chemical or metabolic indirect interactions and/or direct physical contacts involving specifically the yeast or hyphal form of the fungal cell, or more rarely involving both forms. According to the species, C. albicans-bacteria interactions can be antagonistic or synergistic, competitive or not. The polymicrobial nature of biofilms may deeply influence the physiopathology of infections as well as the efficiency of antimicrobial agents. The present review aims to focus on the current knowledge of interactions between C. albicans and major Gram-positive bacteria such as Staphylococcus aureus, coagulase negative Staphylococcus, Streptococcus spp. and Clostridium spp. within biofilms. A better understanding of this complicated, fast-paced world of multi-kingdom biofilms will contribute to develop new effective ways to fight biofilm-related infections.     

An immunoproteomic approach for characterization of dormancy within Staphylococcus epidermidis biofilms

Virulence of Staphylococcus epidermidis is mainly attributed to surface colonization and biofilm formation in indwelling medical devices. Physiological heterogeneity of biofilms may influence host immune response and sensitivity to antibiotics. Dormant cells, among others, contribute to biofilm heterogeneity. The aim of this study was to identify immunogenic proteins of S. epidermidis biofilms associated with dormancy mechanism, by using two-dimensional electrophoresis (2-DE) immunoblotting and mass spectrometry (MS). A total of 19 bacterial proteins, recognized by human serum samples, were identified. These proteins were mainly involved in small molecule metabolic biological processes. Catalytic activity and ion binding were the most representative molecular functions. CodY and GpmA proteins were more reactive to sera when biofilm dormancy was induced, while FtnA and ClpP were more reactive when dormancy was prevented. This is the first work that identifies differences in immunoreactive proteins within bacterial biofilms with induced or prevented dormancy. Considering the importance of dormancy within biofilms, further evaluation of these proteins can provide insights into the mechanisms related to dormancy and help to improve current understanding on how dormancy affects the host immune response.     

Targeted delivery of vancomycin to Staphylococcus epidermidis biofilms using a fibrinogen-derived peptide

This study reports on the use of a fibrinogen-derived peptide for the specific targeting and delivery of vancomycin to Staphylococcus epidermidis biofilms. One method by which S. epidermidis initially adheres to biomaterials uses the plasma protein fibrinogen as an intermediary, where the S. epidermidis surface protein SdrG binds to a short amino acid sequence near the amino terminus of the Bβ chain of fibrinogen. We mimicked this binding interaction and demonstrated the use of a synthetic fibrinogen-based β6-20 peptide to target and deliver vancomycin to S. epidermidis in vitro. The β6-20 peptide was synthesized and labeled with a Nanogold probe, and its targeting capabilities were examined through the use of scanning electron microscopy. The Nanogold component was then replaced by vancomycin, utilizing a flexible, variable length poly(ethylene glycol) linker between the peptide and antibiotic to create the targeted vancomycin products, β6-20-PEG(x) -VAN. Initial binding to surface adherent S. epidermidis was increased in a concentration-dependent manner relative to vancomycin for all equivalent concentrations ≥4 μg/mL, with targeted vancomycin content up to 22.9 times that of vancomycin alone. Retention of the targeted antibiotics was measured after an additional 24-h incubation period, revealing levels 1.3 times that of vancomycin. The results demonstrate the improved targeting and retention of vancomycin within a biofilm due to the incorporation of a specific targeting motif.     

Role of dimorphism in the development of Candida albicans biofilms.

Two model biofilm systems, involving growth on disks of catheter material or on cylindrical cellulose filters, were used to investigate the structure of Candida albicans biofilms. To assess the importance of dimorphism in biofilm development, biofilms produced by two wild-type strains were compared with those formed by two morphological mutants, incapable of yeast and hyphal growth, respectively. Scanning electron microscopy and thin sections of biofilms examined by light microscopy revealed that biofilms of the wild-type strains formed on catheter disks consisted of two distinct layers: a thin, basal yeast layer and a thicker, but more open, hyphal layer. The hypha- mutant produced only the basal layer, whereas the yeast- mutant formed a thicker, hyphal biofilm equivalent to the outer zone of the wild-type structures. Biofilms of the yeast- mutant were more easily detached from the catheter surface than the others, suggesting that the basal yeast layer has an important role in anchoring the biofilm to the surface. Biofilms formed on cylindrical cellulose filters were quite different in appearance. The hypha- mutant and both wild types produced exclusively yeast-form biofilms whereas the yeast- mutant generated a dense hyphal mat on the top of the filter. All these biofilms, irrespective of morphological form, were resistant to the antifungal agent, amphotericin B. Overall, these results indicate that the structure of a C. albicans biofilm depends on the nature of the contact surface, but that some surfaces produce biofilms with a layered architecture resembling to that described for bacterial systems.     

Immunological relatedness among Candida albicans and other pathogenic Candida species.

Membrane-mitochondrial (butanol-hot phosphate-buffered saline) and cytosol (soluble cytoplasmic substances) extracts from seven pathogenic species of Candida were used in in vivo and in vitro immunological assays to study antigenic similarities among the strains with respect to C. albicans. Mice were sensitized with C. albicans serotype A for footpad testing or to provide cells for lymphocyte stimulation assays, and guinea pigs were immunized with whole cells or butanol-hot phosphate-buffered saline extracts of C. albicans to obtain antisera for immunodiffusion assays. When extracts from each of the seven species were used in the assays, they consistently segregated, as determined by statistical or subjective analyses, into three groups. Extracts of C. albicans serotype A or B and C. stellatoidea were the most immunologically reactive in all assays, indicating close similarities between those two species, whereas extracts of C. tropicalis and C. parapsilosis elicited only moderate responses. Extracts from C. krusei, C. guilliermondii, and C. pseudotropicalis were hypo- or nonreactive in the assays, indicating a low level of antigenic relatedness to C. albicans.     

Microplate Alamar blue assay for susceptibility testing of Candida albicans biofilms.

Although biofilm-based fungal infections are an important cause of morbidity and mortality in patients, there is no standardized method for the in vitro evaluation of the drug susceptibility of biofilms. We investigated a high-throughput method for determining the susceptibility of Candida albicans biofilms that uses the oxidation reduction indicator Alamar blue (AB). Biofilms from the tested Candida albicans strains were markedly resistant to amphotericin B (AMB), nystatin (NYT), fluconazole (FLC) and 5-fluorouracil (5FC), but susceptible to Conflikt disinfectant. The latter was used in comparative studies of AB reduction with two other methods for assessing in vitro drug susceptibility i.e., 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and enumeration of viable colony counts (CFU/ml). AB results correlated well with XTT (r=0.88-0.93) and CFU/ml (r=0.93-0.99) for all four C. albicans test strains. This simple, reproducible method for determining in vitro drug susceptibility should facilitate discovery of antifungals active against Candida biofilms.     

Adherence of Candida albicans and other Candida species to mucosal epithelial cells.

To study the possible involvement of candidal adherence in mucosal colonization, we examined the in vitro adherence capabilities of seven Candida species. Adherence was evaluated by direct microscopic examination and by a quantitative radiometric adherence test. The results indicate that C. albicans adheres to vaginal and buccal epithelial cells to a significantly greater degree (P less than 0.01) than the other species tested. C. tropicalis and C. stellatoidea demonstrated moderate adherence capabilities, while C. parapsilosis adhered only to a slight degree. Other species failed to interact with isolated mucosal cells. These findings suggest that there is a relationship between the adherence capabilities of the Candida species and their abilities to colonize mucosal surfaces, since those species which adhere are those which most frequently colonize mucosal surfaces. C. albicans was found to be adherent under a variety of environmental conditions. Stationary-phase blastospores of C. albicans were found to be more adherent than logarithmic-phase yeasts, and larger blastospore cell-to-epithelial cell ratios resulted in greater adherence values. The actual number of adherent yeasts varied considerably when epithelial cells were obtained from different donors.     

Synergistic effect of Candida albicans and Staphylococcus aureus on mouse mortality.

A synergistic effect on mouse mortality was demonstrated in combined infection of mice with Candida albicans and a Staphylococcus aureus strain isolated from a patient with toxic shock syndrome. Mice exhibited high resistance when inoculated intraperitoneally by either pathogen alone. Dual infection with the two organisms together, however, at doses which separately caused no animal deaths, resulted in 100% mortality. This synergistic effect could not be reproduced when either of the agents was heat inactivated.     

Virulence modulation of Candida albicans biofilms by metal ions commonly released from orthodontic devices

The installation of metal devices leads to an increase in the salivary concentration of metal ions and in the growth of salivary Candida spp. However, the relationship between released metal ions and Candida virulence has not been previously examined. The objective of this study was to evaluate whether metal ions affect fungal virulence. We prepared culture media containing Ni²⁺, Fe³⁺, Cr³⁺, Co²⁺ or a mixture of these metal ions at concentrations similar to those released in saliva of orthodontic patients. Biofilms of Candida albicans SC5314 were grown for 72 h and their biomasses were determined. The supernatants were analyzed for secretory aspartyl protease (SAP) and hemolysin activities. To verify changes in virulence following treatment with metals, proteolytic and hemolytic activities were converted into specific activities. The results revealed that all ions, except Co²⁺, caused increases in biofilm biomass. In addition, Ni²⁺ caused an increase in SAP activity and Fe³⁺ reduced hemolytic activity. However, the SAP and hemolysin activities in the presence of the mixture of ions did not differ from those of control. These results indicate that metal ions released during the degradation of orthodontic appliances can modulate virulence factors in C. albicans biofilms.     

Environmental pH influences Candida albicans biofilms regarding its structure,virulence and susceptibility to fluconazole

Candida albicans colonizes sites with different environmental pH. However, it is unclear how these conditions can interfere on biofilms. This study aimed to evaluate the influence of environmental pH on behavior of C. albicans regarding its structure, virulence and susceptibility to fluconazole (FLZ). Minimal inhibitory concentration, minimal fungicidal concentration and time kill were used to evaluate the susceptibility to FLZ in planktonic cells under three pH values (4.0, 5.5, 7.0). These pH values were used for biofilms analysis. C. albicans ATCC 90028 was developed on poly(methlymethacrylate) resin for 48 h. Then, 2.56 μg/mL of FLZ was added to experimental groups for 24 h, and biofilms were analyzed by cell quantification, bioactivity, secretion of proteinases and phospholipases and structure. All data were analyzed by two-way ANOVA, followed by Tukey's test (α = 0.05). For planktonic cells, changes in environmental pH decreased the susceptibility to FLZ. C. albicans biofilms developed at pH 5.5 showed higher cell counts, bioactivity, bio-volume, average thickness and roughness coefficient (p < 0.05). In contrast, the presence of FLZ at pH 4.0 did not influence the structural parameters (p > 0.05), but increased secretion of proteinase and phospholipase (p < 0.05). Within the conditions studied, it was shown that environmental pH modulates the structure, virulence and susceptibility of C. albicans to FLZ.     

Pheromone cross-inhibition between Staphylococcus aureus and Staphylococcus epidermidis

Cross-inhibition by quorum-sensing pheromones between Staphylococcus aureus and Staphylococcus epidermidis was investigated using all known S. aureus agr pheromone subgroups. All S. aureus subgroups were sensitive towards the S. epidermidis pheromone, with the exception of the recently identified subgroup 4. The subgroup 4 pheromone was also the only S. aureus pheromone able to inhibit the S. epidermidis agr response. The close relation of subgroup 4 to subgroup 1 suggests that subgroup 4 might have evolved from subgroup 1 by mutation under the selective pressure of competition with S. epidermidis. The competition between S. aureus and S. epidermidis by means of quorum-sensing cross talk seems to be generally in favor of S. epidermidis, which might explain the predominance of S. epidermidis on the skin and in infections on indwelling medical devices.     

Involvement of amyloid proteins in the formation of biofilms in the pathogenic yeast Candida albicans

Candida species represent a major fungal threat for human health. Within the Candida genus, the yeast Candida albicans is the most frequently incriminated species during episodes of candidiasis or candidemia. Biofilm formation is used by C. albicans to produce a microbial community that is important in an infectious context. The cell wall, the most superficial cellular compartment, is of paramount importance regarding the establishment of biofilms. C. albicans cell wall contains proteins with amyloid properties that are necessary for biofilm formation due to their adhesion properties. This review focuses on these amyloid proteins during biofilm formation in the yeast C. albicans.