Biofilm formation by ica-positive and ica-negative strains of Staphylococcus epidermidis in vitro

Staphylococcus epidermidis is a clinically important opportunistic pathogen that forms biofilm infections on nearly all types of indwelling medical devices. The biofilm forming capability of S. epidermidis has been linked to the presence of the ica operon in the genome, and the amount of biofilm formation measured by the crystal violet (CV) adherence assay. Six S. epidermidis strains were characterized for their ica status using PCR, and their biofilm forming ability over 6 days, using the CV assay and a flow cell system. Ica-negative strains characterized as ‘negative for biofilm formation’ based on the CV assay were demonstrated to form strongly attached biofilms after 6 days. However, the biofilms were not as extensive as the ica-positive strains. It was concluded that ica is not required for biofilm formation, nor is the 24-h CV assay generalizable for predicting the 6-day biofilm-forming ability for all S. epidermidis strains.     

金黄色葡萄球菌生物膜形成机制研究进展

金黄色葡萄球菌是医院和社区获得性感染最常见的病原菌之一,而且可形成生物膜,从而导致生物膜相关疾病的产生。患者一旦发生金黄色葡萄球菌生物膜感染,难以彻底治愈。深入研究金黄色葡萄球菌生物膜形成的分子机制和调控网络,对寻找有效的防治、治疗药物和手段具有重要意义。我们就金黄色葡萄球菌生物膜形成过程和调控机制的研究近况做一综述。     

Mini-review: Staphylococcus epidermidis as the most frequent cause of nosocomial infections: old and new fighting strategies

Staphylococcus epidermidis is nowadays regarded as the most frequent cause of nosocomial infections and indwelling medical device-associated infections. One of the features that contributes to the success of this microorganism and which is elemental to the onset of pathogenesis is its ability to form biofilms. Cells in this mode of growth are inherently more resistant to antimicrobials. Seeking to treat staphylococcal-related infections and to prevent their side effects, such as the significant morbidity and health care costs, many efforts are being made to develop of new and effective antistaphylococcal drugs. Indeed, due to its frequency and extreme resistance to treatment, staphylococcal-associated infections represent a serious burden for the public health system. This review will provide an overview of some conventional and emerging anti-biofilm approaches in the management of medical device-associated infections related to this important nosocomial pathogen.     

Lavage with Allicin in Combination with Vancomycin Inhibits Biofilm Formation by Staphylococcus epidermidis in a Rabbit Model of Prosthetic Joint Infection

Background and Aim

The present anti-infection strategy for prosthetic joint infections (PJI) includes the use of antibiotics and surgical treatments, but the bacterial eradication rates are still low. One of the major challenges is the formation of biofilm causing poor bacterial eradication. Recently it has been reported that allicin (diallyl thiosulphinate), an antibacterial principle of garlic, can inhibit bacteria adherence and prevent biofilm formation in vitro. However, whether allicin could inhibit biofilm formation in vivo is unknown. The aim of this study was to investigate the effects of allicin on biofilm formation, and whether allicin could potentiate the bactericidal effect of vancomycin in a rabbit PJI model.

Methods

A sterile stainless-steel screw with a sterile ultra-high molecular weight polyethylene washer was inserted into the lateral femoral condyle of the right hind knee joint of rabbit, and 1 mL inoculum containing 10⁴ colony-forming units of Staphylococcus epidermidis was inoculated into the knee joint (n = 32). Fourteen days later, rabbits randomly received one of the following 4 treatments using continuous lavages: normal saline, vancomycin (20 mcg/mL), allicin (4 mg/L), or allicin (4 mg/L) plus vancomycin (20 mcg/mL). Three days later, the washer surface biofilm formation was examined by scanning electron microscopy (SEM). The bacterial counts within the biofilm of implanted screws were determined by bacterial culture.

Results

The lowest number of viable bacterial counts of Staphylococcus epidermidis recovered from the biofilm was in the rabbits treated with allicin plus vancomycin (P<0.01 vs. all other groups). The biofilm formation was significantly reduced or undetectable by SEM in rabbits receiving allicin or allicin plus vancomycin.

Conclusion

Intra-articular allicincan inhibit biofilm formation and enhance the bactericidal effect of vancomycin on implant surface in vivo. Allicin in combination with vancomycin may be a useful anti-infection strategy for the treatment of PJI.     

表皮葡萄球菌PhoU同源物(SERP0316)的抗体制备及免疫鉴定

表皮葡萄球菌可在植入性医疗材料表面形成生物膜,对多种抗生素耐受,而持留菌的形成是导致细菌生物膜耐药的原因之一。研究发现,大肠埃希菌中PhoU可影响持留菌的形成,与细菌对多种抗生素及压力条件的耐受相关,但表皮葡萄球菌中PhoU的功能仍不清楚。生物信息学分析显示,在表皮葡萄球菌中存在2个phoU同源基因,其中serp0956位于pst操纵子中,命名为phoU1;另一个编码功能未知蛋白的phoU基因同源物(serp0316)命名为phoU2。前期研究显示,敲除phoU1并不影响表皮葡萄球菌生长、生物膜形成等生物学特性,为此本研究对PhoU2进行了初步探讨。利用实时荧光定量反转录-聚合酶链反应(RT-PCR)检测phoU2基因在表皮葡萄球菌SE1457不同生长时期的转录水平,发现其在细菌生长不同时间点持续表达,其中对数期(6h)表达量相对较高。为进一步研究表皮葡萄球菌中PhoU2的表达情况,对PhoU2蛋白进行原核表达和纯化,并用纯化的重组PhoU2蛋白免疫小鼠制备PhoU2多克隆抗体。在此基础上,利用蛋白免疫印迹法检测PhoU2在SE1457菌株不同生长时间点的表达水平,结果显示PhoU2在细菌生长对数期和平台期均有表达。另外,在不同的表皮葡萄球菌临床分离株中均检测到PhoU2蛋白表达,表明PhoU2具有一定的保守性。以上研究提示,PhoU2可能作为PhoU的功能同源物发挥一定的作用,为进一步研究表皮葡萄球菌PhoU2的生物学功能及其在生物膜和持留菌形成中的作用奠定了基础。     

Inhibitory effect of the human liver-derived antimicrobial peptide hepcidin 20 on biofilms of polysaccharide intercellular adhesin (PIA)-positive and PIA-negative strains of Staphylococcus epidermidis

Staphylococcus epidermidis plays a major role in biofilm-related medical device infections. Herein the anti-biofilm activity of the human liver-derived antimicrobial peptide hepcidin 20 (hep20) was evaluated against polysaccharide intercellular adhesin (PIA)-positive and PIA-negative clinical isolates of S. epidermidis. Hep20 markedly inhibited biofilm formation and bacterial cell metabolism of PIA-positive and PIA-negative strains, but the decrease in biofilm biomass only partially correlated with a decrease in viable bacteria. Confocal microscope images revealed that, in the presence of hep20, both PIA-positive and PIA-negative strains formed biofilms with altered architectures and reduced amounts of extracellular matrix. Co-incubation of hep20 with vancomycin produced no synergistic effect, evaluated as number of viable cells, both in preventing biofilm formation and in treating preformed biofilms. In contrast, biofilms obtained in the presence of hep20, and then exposed to vancomycin, displayed an increased susceptibility to vancomycin. These results suggest that hep20 may inhibit the production/accumulation of biofilm extracellular matrix.     

Anti-biofilm properties of the antimicrobial peptide temporin 1Tb and its ability,in combination with EDTA,to eradicate Staphylococcus epidermidis biofilms on silicone catheters

In search of new antimicrobials with anti-biofilm potential, in the present study activity of the frog-skin derived antimicrobial peptide temporin 1Tb (TB) against Staphylococcus epidermidis biofilms was investigated. A striking ability of TB to kill both forming and mature S. epidermidis biofilms was observed, especially when the peptide was combined with cysteine or EDTA, respectively. Kinetics studies demonstrated that the combination TB/EDTA was active against mature biofilms already after 2–4-h exposure. A double 4-h exposure of biofilms to TB/EDTA further increased the therapeutic potential of the same combination. Of note, TB/EDTA was able to eradicate S. epidermidis biofilms formed in vitro on silicone catheters. At eradicating concentrations, TB/EDTA did not cause hemolysis of human erythrocytes. The results shed light on the anti-biofilm properties of TB and suggest a possible application of the peptide in the lock therapy of catheters infected with S. epidermidis.     

Extracellular Fibrinogen-binding Protein (Efb) from Staphylococcus aureus Inhibits the Formation of Platelet-Leukocyte Complexes

Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes.