Inhibition of staphylothrombin by dabigatran reduces Staphylococcus aureus virulence

Summary. Background: Staphylocoagulase and von Willebrand binding protein (VWbp) bind to prothrombin to form the staphylothrombin complex that converts fibrinogen into fibrin. Objectives: To study the role of staphylothrombin and its inhibition by dabigatran on Staphylococcus aureus virulence. Methods: We studied the effect of staphylothrombin inhibition on bacterial attachment to polystyrene surfaces, leukocyte activation and bactericidal activity for S. aureus ATCC 25923, S. aureus Newman, and staphylocoagulase‐ and VWbp‐negative S. aureus Newman mutants in the presence or absence of prothrombin and fibrinogen. We measured the abscess size after subcutaneous (s.c.) injection of S. aureus ATCC 25923 and S. aureus Newman, as well as an S. aureus Newman mutant strain lacking staphylocoagulase and VWbp, in mice treated with either dabigatran or placebo. Results: Staphylothrombin‐mediated fibrin increased the association of S. aureus to polystyrene surfaces and reduced the bactericidal activity of leukocytes. The absence or inhibition of staphylothrombin decreased the bacterial association, enhanced leukocyte activation and reduced bacterial survival in vitro. Abscess size was smaller in mice treated with dabigatran or infected with a coagulase‐negative mutant. Conclusion: Inhibition or the absence of staphylothrombin reduced S. aureus virulence in in vitro and in vivo models.     

Subinhibitory quinupristin/dalfopristin attenuates virulence of Staphylococcus aureus

OBJECTIVES: The semi-synthetic streptogramin quinupristin/dalfopristin antibiotic exerts potent bactericidal activity against Staphylococcus aureus. We investigated whether, like other bactericidal antibiotics used at subinhibitory concentrations, quinupristin/dalfopristin enhances release of toxins by Gram-positive cocci. METHODS: The activity of quinupristin/dalfopristin on exotoxin release by S. aureus was investigated by 2D SDS-PAGE combined with MALDI-TOF/MS analysis and by western blotting. RESULTS: We show that quinupristin/dalfopristin at subinhibitory concentrations reduces the release of S. aureus factors that induce tumour necrosis factor secretion in macrophages. Furthermore, quinupristin/dalfopristin but not linezolid attenuated S. aureus-mediated killing of infected host cells. When added to S. aureus cultures at different stages of bacterial growth, quinupristin/dalfopristin reduced in a dose-dependent manner the release of specific virulence factors (e.g. autolysin, protein A, alpha- and beta-haemolysins, lipases). In contrast, other presumably non-toxic exoproteins remained unchanged. CONCLUSIONS: The results of the present study suggest that subinhibitory quinupristin/dalfopristin inhibits virulence factor release by S. aureus, which might be especially helpful for the treatment of S. aureus infections, where both bactericidal as well as anti-toxin activity may be advantageous.     

不同来源耐甲氧西林金黄色葡萄球菌毒力基因的研究

目的对临床分离的耐甲氧西林金黄色葡萄球菌(MRSA)感染状况进行分析,并通过对MRSA菌株、甲氧西林敏感金黄色葡萄球菌(MSSA)菌株毒力基因的检测,分析比较不同金黄色葡萄球菌(SA)的毒力基因分布。方法收集衢州市人民医院2009年1月至2011年6月临床标本分离的MRSA菌株60株,MSSA菌株54株。采用聚合酶链反应(PCR)检测不同标本分离的SA的杀白细胞素(pvl)、肠毒素C(sec)、肠毒素H(seh)、α-溶血素(hla)、β-溶血素(hlb)、黏附素凝聚因子(clfA、clfB)、纤连蛋白结合蛋白(fnbA、fnbB)毒力基因。结果60株MRSA菌株主要分布在神经外科、普外科、小儿科、骨科等病区,其中17株为社区获得性MRSA(CA-MRSA),43株为医院获得性MRSA(HA-MRSA)。MRSA菌株中pvl的检出率明显高于MSSA菌株,而clfA、fnbA、sec、she的检出率明显低于MSSA。而pvl、fnbA在CA-MRSA中的检出率要高于HA-MRSA。结论临床分离的SA中,MSSA菌株毒力相对MRSA可能更高,而CA-MRSA相对HA-MRSA毒力也较强,他们之间携带毒力基因的差别可能与之侵袭的感染部位相关。     

Staphylococcus aureus small colony variants: formation and clinical impact

S. aureus small-colony variants are a naturally occurring subpopulation which grow slowly and produce small colonies on routine media. They also demonstrate a number of other characteristics that are atypical for S. aureus including reduced alpha-toxin production and delayed coagulase activity. The connection of S. aureus SCVs with persistent and relapsing infections has been defined over the past decade, especially in patients with chronic osteomyelitis and in cystic fibrosis patients as demonstrated by prospective studies. While the studies with clinical isolates of SCVs suggested a link between electron transport-defective strains and persistent infections, a defined hemB mutant with the SCV phenotype provided strong additional evidence for these connections. The hemB mutant was phagocytosed by cultured endothelial cells, but did not lyse these cells, because the mutant produced very little alpha-toxin. The intracellular location may shield the SCVs from host defenses and antibiotics, thus providing one explanation for the difficulty in clearing S. aureus SCVs from host tissues.     

甲氧西林耐药／敏感金黄色葡萄球菌基因分型和毒力基因检测

目的：分析耐甲氧西林金黄色葡萄球菌（金葡菌）（MRSA）和甲氧西林敏感金葡菌（MSSA）临床分离株基因分型及毒力基因分布特征是否存在差异,了解金葡菌耐药性演变与毒力变迁之间的相关性。方法采用脉冲场凝胶电泳（PFGE）方法和多位点序列分型（MLST）方法对呼和浩特地区住院患者中分离的30株 MRSA 和30株 MSSA 进行分子分型,同步采用聚合酶链反应（PCR）方法检测菌株毒力基因。结果60株金葡菌 PFGE 分型共分19个型,MSSA 菌株分布在 I 和 H 型等16个基因型中,而 MRSA 株主要集中分布在 K 和 M 2个基因型中。20株不同 PFGE 型菌株 MLST 分型结果显示,MRSA 株主要为 ST-239型;MSSA 株呈多样性分布特征,主要以 ST-5型、ST-7型、ST-15型为主。毒力基因在 MRSA 和 MSSA 中分布差异显著。MSSA 毒力基因整体携带率明显高于 MRSA（53．9％对40．0％,χ2＝32．7,P ＜0．01）。MRSA 株 sea、cna 和 cap 8基因携带率明显高于 MSSA 携带率（P ＜0．01）,而 sec、seg 、sei、sem、sen、seo、fnbB、ebpS、cap5基因携带率明显低于 MSSA（P＜0．05）。结论呼和浩特地区金葡菌临床分离株基因型呈现多样化分布特点,MRSA 主要以 ST-239型为主。MSSA 毒力基因携带率高,特定毒力因子在 MRSA 和 MSSA 株中呈现一定聚集分布特征,金葡菌特定耐药性的获得可能伴随特定毒力特征的变化。     

Subinhibitory concentrations of linezolid reduce Staphylococcus aureus virulence factor expression

The influence of the antibiotic linezolid on the secretion of exotoxins by Staphylococcus aureus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry and Western blot analysis. S. aureus suspensions were treated with grading subinhibitory concentrations of linezolid (12.5, 25, 50, and 90% of MIC) at different stages of bacterial growth (i.e., an optical density at 540 nm [OD(540)] of 0.05 or 0.8). When added to S. aureus cultures at an OD(540) of 0.05, linezolid reduced in a dose-dependent manner the secretion of specific virulence factors, including staphylococcal enterotoxin A (SEA) and SEB, bifunctional autolysin, autolysin, protein A, and alpha- and beta-hemolysins. In contrast, other presumably nontoxic exoproteins remained unchanged or even accumulated in supernatants in the presence of linezolid at a 90% MIC. Similarly, when added at OD(540) of 0.8, that is, after quorum sensing, linezolid reduced the release of virulence factors, whereas the relative abundance of nontoxic exoproteins such as triacylglycerol lipase, glycerol ester hydrolase, DnaK, or translation elongation factor EF-Tu was found to be increased. Consistently, linezolid reduced in a dose-dependent manner the tumor necrosis factor-inducing activity secreted by S. aureus into the culture supernatants. The results of our study suggest that the expression of virulence factors in S. aureus is especially sensitive to the inhibition of protein synthesis by linezolid, which should be an advantage in the treatment of infections with toxin-producing S. aureus.     

In vitro activity of daptomycin against multidrug-resistant Staphylococcus aureus and S. aureus with known virulence factors, including community-acquired methicillin-resistant isolates

The in vitro activity of daptomycin was evaluated against 360 multidrug-resistant Staphylococcus aureus isolates (including hospital-acquired isolates) and multidrug-susceptible community-acquired methicillin-resistant S. aureus with known virulence genes. All isolates were inhibited at 相似文献   

Thrombocytopenia in Staphylococcus aureus bacteremia: risk factors and prognostic importance

OBJECTIVE: To identify risk factors and outcomes associated with thrombocytopenia at sepsis onset in Staphylococcus aureus bacteremia.PATIENTS AND METHODS: This single-center, retrospective, cohort study consists of all adult patients with a first episode of clinical S aureus bacteremia between April 1, 1988, and September 30, 1994, and between January 1, 1999, and December 31, 2007. Thrombocytopenia was defined as a platelet count less than 150 × 10⁹/L. The primary outcome was 30-day all-cause mortality. Risk factors for 30-day all-cause mortality were identified using univariate and multivariable analyses. Multivariable analysis was conducted using forward step logistic regression analysis. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated for risk of death.RESULTS: A total of 1052 patients had clinical S aureus bacteremia. Thrombocytopenia at sepsis onset was present in 235 patients (22.3%). Thrombocytopenia was associated with community-acquired bacteremia, infections caused by methicillin-sensitive S aureus, high-magnitude bacteremia (defined as >4 positive blood cultures [≥3 separate positive blood culture sets]), and endocarditis. Patients with thrombocytopenia presented more commonly with severe sepsis reflected by septic shock and acute renal failure. Thirty-day mortality was significantly higher among patients with thrombocytopenia (132/235 [56.2%]) vs those without thrombocytopenia (281/817 [34.4%]; P<.001). Higher mortality was associated with the degree of thrombocytopenia. In multivariable analysis, thrombocytopenia at baseline remained an independent risk factor for 30-day mortality (OR, 2.82; 95% CI, 1.87-4.24). The adjusted association between thrombocytopenia and death remained similar among the 917 patients with monomicrobial bacteremia (OR, 2.88; 95% CI, 1.83-4.53) and the 945 patients who did not die within the first 48 hours (OR, 2.88; 95% CI, 1.87-4.45.).CONCLUSION: We observed a strong association between thrombocytopenia at sepsis onset and all-cause mortality in S aureus bacteremia, possibly related to mechanisms other than sepsis alone.AUC = area under the curve; CI = confidence interval; Clf A = clumping factor A; DIC = disseminated intravascular coagulation; ICU = intensive care unit; INR = international normalized ratio; MRSA = methicillin-resistant Staphylococcus aureus; MSSA = methicillin-susceptible S aureus; OR = odds ratioStaphylococcus aureus is a leading cause of both community-acquired and nosocomial bacteremia and is currently the most common cause of infective endocarditis.¹ S aureus has a unique interaction with platelets that has not been fully investigated in clinical studies.Infection with S aureus can be accompanied by thrombocytopenia as a result of local activation of platelets and disseminated intravascular coagulation (DIC) if the activation of platelets becomes systemic.² S aureus can activate platelets, and this process is mediated by several surface-expressed proteins, such as clumping factor A (Clf A) and fibronectin-binding protein A³ or cell wall peptidoglycan.⁴ Moreover, S aureus is able to bind to adhered platelets, and this may be an important mechanism that contributes to colonization of endovascular epithelium or damaged heart valves, thus promoting infective endocarditis and endovascular infections.⁵ In turn, endocarditis may cause thrombocytopenia through shearing forces over the damaged valve.⁶ The adherence of S aureus to platelets leads to platelet activation and aggregation.⁷ When this occurs in a localized manner, it may result in formation of a thrombus; if the interaction occurs diffusely in the bloodstream, it may result in consumption of platelets, leading to thrombocytopenia. If the coagulation cascade is activated, DIC ensues.³In clinical practice, we observed an association between thrombocytopenia and adverse outcomes more frequently in patients with S aureus bacteremia than in those with other bacteria. We sought to examine this formally.     

Methicillin-resistant Staphylococcus aureus among companion and food-chain animals: impact of human contacts

Methicillin-resistant Staphylococcus aureus (MRSA), one of the major pathogens in humans, is a cause of infection and colonization among animals. The increasing number of companion animals and antibiotic use have made this population a reservoir of MRSA. In parallel, the evolution of new MRSA clones and mec homologues among animals of the food chain has emphasized the need for infection control practices in animals and humans in close contact. On the basis of a review of the literature, epidemiological and evolutionary data for MRSA infections and carriage, risk factors and control strategies are presented.     

Attenuated virulence and biofilm formation in Staphylococcus aureus following sublethal exposure to triclosan

Subeffective exposure of Staphylococcus aureus to the biocide triclosan can reportedly induce a small-colony variant (SCV) phenotype. S. aureus SCVs are characterized by low growth rates, reduced pigmentation, and lowered antimicrobial susceptibility. While they may exhibit enhanced intracellular survival, there are conflicting reports regarding their pathogenicity. The current study reports the characteristics of an SCV-like strain of S. aureus created by repeated passage on sublethal triclosan concentrations. S. aureus ATCC 6538 (the passage 0 [P0] strain) was serially exposed 10 times to concentration gradients of triclosan to generate strain P10. This strain was then further passaged 10 times on triclosan-free medium (designated strain ×10). The MICs and minimum bactericidal concentrations of triclosan for P0, P10, and ×10 were determined, and growth rates in biofilm and planktonic cultures were measured. Hemolysin, DNase, and coagulase activities were measured, and virulence was determined using a Galleria mellonella pathogenicity model. Strain P10 exhibited decreased susceptibility to triclosan and characteristics of an SCV phenotype, including a considerably reduced growth rate and the formation of pinpoint colonies. However, this strain also had delayed coagulase production, had impaired hemolysis (P < 0.01), was defective in biofilm formation and DNase activity, and displayed significantly attenuated virulence. Colony size, hemolysis, coagulase activity, and virulence were only partially restored in strain ×10, whereas the planktonic growth rate was fully restored. However, ×10 was at least as defective in biofilm formation and DNase production as P10. These data suggest that although repeated exposure to triclosan may result in an SCV-like phenotype, this is not necessarily associated with increased virulence and adapted bacteria may exhibit other functional deficiencies.     

金黄色葡萄球菌sasX、psm-mec、pvl三种毒力基因的检测与分析

摘要：目的：分析54株金黄色葡萄球菌的mecA基因与sasX、psm mec、pvl三种毒力基因分布及临床相关性。 方法：收集2013年1～5月南京医科大学第一附属医院临床分离的54株金黄色葡萄球菌,用PCR及测序的方法分析mecA、sasX、psm-mec、pvl基因的携带情况,并结合患者的临床资料,探讨其临床相关性。 结果：54株金黄色葡萄球菌中有23株mecA基因检测阳性,31株mecA基因阴性。mecA基因阳性菌株较mecA基因阴性株体外药敏试验呈多重耐药表型。sasX基因阳性1株,阳性率为1.9%;psm-mec基因阳性10株,阳性率为18.5%;pvl基因阳性38株,阳性率为70.4%。mecA基因阳性菌株psm-mec基因阳性率显著高于mecA基因阴性菌株（P<0.01）,pvl基因阳性率显著低于mecA基因阴性菌株（P<0.05）。mecA基因和psm-mec基因与临床感染严重程度显著相关（P均<0.01）。 结论：联合检测mecA、psm-mec和pvl基因有利于金黄色葡萄球菌感染的诊断、治疗和预后综合评估。     

Clindamycin-resistant methicillin-resistant Staphylococcus aureus: epidemiologic and molecular characteristics and associated clinical factors

In this prospective, observational study of 618 consecutive adult patients with skin and soft tissue infections (SSTI) caused by methicillin-resistant Staphylococcus aureus (MRSA), the clinical characteristics, molecular epidemiology, and outcome of patients with clindamycin-resistant MRSA (n = 64) and clindamycin-susceptible MRSA (n = 554) were compared (including factors predictive of clindamycin-resistant MRSA SSTI). Patients with clindamycin-resistant MRSA were more likely to have had antibiotic exposure within 3 months (37.5% versus 17%, P < 0.01), surgery (25% versus 8%, P < 0.01), MRSA infection/colonization within 12 months (23% versus 7%, P < 0.01), or intravascular catheters (5% versus 0.5%, P = 0.02). On multivariate analysis, previous surgery (adjusted odds ratio [AOR] 2.97; 95% confidence interval [CI] 1.5-6.0), history of MRSA (AOR 3.4; 95% CI 1.7-7.1), and exposure to clindamycin (AOR 8.5; 95% CI 2.3-32) and to macrolides (AOR 7.2, 95% CI 1.6-31.8) were independently associated with presence of clindamycin-resistant MRSA. Clinical resolution was similar between groups (77% versus 68%; P = 0.26). Clindamycin-resistant MRSA was less often USA-300 (82% versus 98%, P = 0.004). Clindamycin resistance did not affect MRSA-SSTI clinical outcomes.