Piperazinyl-linked fluoroquinolone dimers possessing potent antibacterial activity against drug-resistant strains of Staphylococcus aureus

The synthesis of symmetric and asymmetric piperazinyl-linked dimers of the fluoroquinolone class of antibiotics is described. Specific dimers are shown to possess potent antibacterial activity against drug-resistant strains of Staphylococcus aureus, including strains possessing resistance due to the NorA multidrug efflux pump and a mutation in the quinolone resistance-determining region of topoisomerase IV.     

Antibacterial activity of flavonoids against methicillin-resistant Staphylococcus aureus strains

An experimental and theoretical study was performed on the anti-staphylococcal activity of 18 natural and synthetic flavonoids against methicillin-resistant Staphylococcus aureus strains. The analysed flavonoids belong to three well-differentiated structural patterns: chalcones, flavanones and flavones. The quantitative analysis of the anti-staphylococcal activity of the compounds was carried out by determining their percent inhibition degree. The hierarchical cluster analysis method was used to analyse the anti-MRSA activity of the compounds. With this methodology, the flavonoids were classified into four groups according to their anti-staphylococcal activity (high, sufficient, intermediate and low). The carbonylic region is of importance because it is part of the bioactive region inducing anti-MRSA activity in the flavonoid molecules. The introduction of OH groups in positions 2' of chalcones and 5 of flavanones (or flavones) increases flavonoid activity, while the OCH(3)groups produce the reverse effect. Using the experimental anti-MRSA activity data of flavonoids and six quantum chemical parameters calculated by means of the AM1 semiempirical molecular orbital method, a very good quantitative structure-activity relationship was obtained (confidence range: 95%; significance level for tests: 0.05; correlation coefficient=0.9842). The selected parameters explain 96.86% of the percent inhibition degree. The obtained relation is consistent with the conclusions formulated in this paper and serves as a theoretical support for some of them. Finally, it is concluded that the flavonoids chalcone, 2'(OH)-chalcone, 2',4'(OH)(2)-chalcone and 2',4(OH)(2)-chalcone might constitute promising therapeutic agents against infections with methicillin-resistant S. aureus strains.     

金黄色葡萄球菌拮抗菌株的筛选鉴定及其抗菌物质分析

【背景】植物内生菌的次生代谢产物是新型天然活性物质的重要来源。【目的】从芍药内生细菌中筛选对金黄色葡萄球菌有抑菌活性的菌株和次生代谢产物。【方法】采用平板对峙法筛选拮抗菌株,根据形态学特征和分子生物学的方法鉴定菌株,PCR扩增检测合成脂肽类物质的功能基因;运用牛津杯法依次测定内生细菌发酵液和脂肽类粗提物的抑菌活性,利用Sephadex LH-20凝胶层析分离脂肽类物质,利用基质辅助激光解吸电离飞行时间质谱分析具有抑菌作用的分离组分。【结果】共筛选出13株对金黄色葡萄球菌具有不同程度抑制作用的内生菌株,其中菌株SY11的抑菌作用最为显著,其发酵液和脂肽类粗提物均具有较强的抑制作用。结合形态学鉴定以及16S r RNA基因序列分析,鉴定其为解淀粉芽孢杆菌(Bacillusamyloliquefaciens)。PCR扩增检测表明菌株SY11含有3个合成脂肽类物质的功能基因fenA、ituD和srfkn,推测该菌株可能具有合成脂肽类物质的能力。根据具有抑菌活性分离组分的质谱分析结果,推测其有效物质的主要成分为Bacillomycin D。【结论】解淀粉芽孢杆菌SY11对金黄色葡萄球菌有良好抑制效果,其脂肽类粗提物也具有较强的体外抑菌活性。本研究为芍药内生细菌的开发应用奠定了基础。     

Phosphodiesterase activity of CvfA is required for virulence in Staphylococcus aureus

We previously identified the cvfA gene (SA1129) as a novel virulence regulator in Staphylococcus aureus using the silkworm infection model. The cvfA gene, which is conserved among various pathogenic bacteria, contributes to the expression of the agr locus, a global virulence regulator that controls the expression of genes encoding various exoproteins, such as hemolysin. CvfA protein has a transmembrane domain, an RNA binding domain (KH domain), and a metal-dependent phosphohydrolase domain (HD domain). We report here the purification of recombinant CvfA protein from a membrane fraction of Escherichia coli by measuring its phosphodiesterase activity. Purified CvfA protein hydrolyzed the phosphodiester linkage of 2',3'-cyclic AMP, 2',3'-cyclic GMP, and 2',3'-cyclic phosphate at the 3'-terminal of RNA in the presence of Mn(2+). CvfA mutant proteins with amino acid substitutions in the HD domain had significantly decreased phosphodiesterase activity. Furthermore, mutated cvfA genes encoding proteins with low phosphodiesterase activity did not complement the decreased hemolysin production or the attenuated killing ability against silkworms in the cvfA deletion mutant. These results suggest that the phosphodiesterase activity of CvfA protein is required for virulence in S. aureus.     

金黄色葡萄球菌耐药基因及新型抗菌药物的研究进展

金黄色葡萄球菌(Staphylococcus aureus,S.aureus),简称金葡菌,是一种临床常见致病菌,可引起皮肤化脓性感染、肺炎、心包炎、败血症及脓毒血症等多种疾病。目前临床上金黄色葡萄球菌的多重耐药情况已十分严峻,特别是耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)的出现,已成为临床抗感染治疗的棘手难题,严重危害人类健康。本文主要介绍金黄色葡萄球菌基因水平的耐药性、新型抗菌药物的研发及临床治疗金黄色葡萄球菌感染三方面的进展,为金黄色葡萄球菌的耐药性研究及耐药菌株的临床有效治疗提供科学依据。     

Antibacterial activity of flavanostilbenes against methicillin-resistant Staphylococcus aureus

Three phytochemical compounds (alopecurone A-C), flavanostilbenes which are produced by condensation between a hydroxyflavanone and a hydroxystilbene, were isolated as major components from the root of Sophora alopecuroides . They uniformly inhibited the growth of 21 strains of methicillin-resistant Staphylococcus aureus with minimum inhibitory concentrations of 3·13–6·25 μg ml^-1.     

Antibacterial activity of hypocrellin A against Staphylococcus aureus

The antibacterial activity and acting mechanism of hypocrellin A (HA) were conducted regarding in vitro activity of HA on Staphylococcus aureus GZ86 by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with HA. The experimental results indicated 1.5?mg/l HA could completely inhibit the growth of 10^7?CFU/ml S. aureus cells in liquid beef extract-peptone medium under a halogen?Ctungsten lamp for 120?min. Meanwhile, HA resulted in the leakage of reducing sugars and proteins and induced the respiratory chain dehydrogenases into inactive state, suggesting that HA were able to destroy the permeability of the bacterial membranes. When the cells of S. aureus were exposed to 2.5?mg/l HA under a halogen?Ctungsten lamp for 120?min, many pits and gaps were observed in bacterial cells by scanning electron microscopy, and the cell wall was fragmentary, indicating the bacterial cells were damaged severely. The experiments strongly confirmed the contribution of multiform reactive oxygen species (ROS) to bactericidal effect. In conclusion, the combined results suggested that ROS may damage the structure of bacterial cell wall and depress the activity of some membranous enzymes, which cause S. aureus bacteria to die eventually.     

Oxidative stress induced by ciprofloxacin in Staphylococcus aureus

Staphylococcus aureus with multiple sensitivity to ciprofloxacin, was investigated to detect alterations in the production of superoxide anion (O(2)(-)), other reactive oxidant species (ROS), and superoxide dismutase (SOD), and to relate them with ciprofloxacin accumulation and sensitivity. Oxidative stress was studied by means of Nitroblue Tetrazolium reaction (NBT) and chemiluminescence (CL); lucigenin was employed to detect O(2)(-), and luminol was used to measure other ROS. Sensitive strains exhibited higher intracellular O(2)(-) increase than resistant ones when incubated with ciprofloxacin. SOD was determined in normal conditions and induction was investigated in the presence of ciprofloxacin. These assays demonstrated that resistant and sensitive strains exported a great amount of SOD and that the induction of SOD intracellular was insufficient to counteract the augment of O(2)(-) in the cytoplasm of sensitive strains. Accumulation of ciprofloxacin, researched by spectrofluorometry, showed high levels of antibiotic in sensitive strains which increased the O(2)(-) causing more oxidative stress than in resistant S. aureus.     

Detection of low-enterotoxin-producing Staphylococcus aureus strains

Culture supernatant fluids from 26 (23.6%) monkey feeding test-positive Staphylococcus aureus strains, negative for enterotoxins by gel diffusion, were positive by enzyme-linked immunosorbent assay for one or more of the identified enterotoxins. Staphylococcal enterotoxin D (SED) was produced by 23 (88.5%) strains, SED and SEA were produced in two strains, and SED and SEC were produced in one strain. One strain produced only SEA, and two strains produced only SEC.     

Potentiation of methicillin activity against methicillin-resistant Staphylococcus aureus by diterpenes

Totarol is a diterpene compound extracted from the totara tree. Totarol and eight other diterpenes were found to potentiate methicillin, one reducing the minimum inhibitory concentration of methicillin against resistant Staphylococcus aureus 256-fold. Totarol did not inhibit the synthesis of DNA or peptidoglycan in S. aureus, but reduced the respiration rate by 70%. Under potentiation conditions, diterpenes had only a slight effect on the respiration rate, but had a significant effect on expression of PBP 2a. We conclude that the primary staphylococcal target for totarol is the respiratory chain, but that potentiation of methicillin by diterpenes is by interference with PBP 2a expression.     

Alpha-toxin is required for biofilm formation by Staphylococcus aureus

Staphylococcus aureus is a common pathogen associated with nosocomial infections. It can persist in clinical settings and gain increased resistance to antimicrobial agents through biofilm formation. We have found that alpha-toxin, a secreted, multimeric, hemolytic toxin encoded by the hla gene, plays an integral role in biofilm formation. The hla mutant was unable to fully colonize plastic surfaces under both static and flow conditions. Based on microscopy studies, we propose that alpha-hemolysin is required for cell-to-cell interactions during biofilm formation.     

Inflammasome-mediated production of IL-1beta is required for neutrophil recruitment against Staphylococcus aureus in vivo

IL-1R activation is required for neutrophil recruitment in an effective innate immune response against Staphylococcus aureus infection. In this study, we investigated the mechanism of IL-1R activation in vivo in a model of S. aureus infection. In response to a S. aureus cutaneous challenge, mice deficient in IL-1beta, IL-1alpha/IL-1beta, but not IL-1alpha, developed larger lesions with higher bacterial counts and had decreased neutrophil recruitment compared with wild-type mice. Neutrophil recruitment and bacterial clearance required IL-1beta expression by bone marrow (BM)-derived cells and not by non-BM-derived resident cells. In addition, mice deficient in the inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) had the same defects in neutrophil recruitment and host defense as IL-1beta-deficient mice, demonstrating an essential role for the inflammasome in mediating the production of active IL-1beta to promote neutrophil recruitment in host defense against S. aureus. This finding was further supported by the ability of recombinant active IL-1beta to control the infection and promote bacterial clearance in IL-1beta-deficient mice. These studies define a key host defense circuit where inflammasome-mediated IL-1beta production by BM-derived cells signals IL-1R on non-BM-derived resident cells to activate neutrophil recruitment in the innate immune response against S. aureus in vivo.