一个金黄色葡萄球菌蛋白磷酸酯酶基因的克隆表达及酶活性测定

金黄色葡萄球菌是一种引起人畜多种感染的主要病原菌,开发治疗金黄色葡萄球菌感染的新药途径之一是药物寻靶。研究克隆了一个金黄色葡萄球菌基因,并进行了蛋白诱导表达和酶活性测定。结果表明,该基因编码的蛋白具有丝氨酸/苏氨酸蛋白磷酸酯酶活性。实验结果为进一步挖掘该蛋白作为药物靶标的潜在功能奠定了基础。     

Tilapia Piscidin 4 (TP4) Stimulates Cell Proliferation and Wound Closure in MRSA-Infected Wounds in Mice

Antimicrobial peptides (AMPs) are endogenous antibiotics that directly affect microorganisms, and also have a variety of receptor-mediated functions. One such AMP, Tilapia piscidin 4 (TP4), was isolated from Nile tilapia (Oreochromis niloticus); TP4 has antibacterial effects and regulates the innate immune system. The aim of the present study was to characterize the role of TP4 in the regulation of wound closure in mice and proliferation of a keratinocyte cell line (HaCaT) and fibroblast cell line (Hs-68). In vitro, TP4 stimulated cell proliferation and activated collagen I, collagen III, and keratinocyte growth factor (KGF) gene expression in Hs-68 cells, which induces keratin production by HaCaT cells. This effect was detectable at TP4 concentrations of 6.25 µg/mL in both cell lines. In vivo, TP4 was found to be highly effective at combating peritonitis and wound infection caused by MRSA in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. Taken together, our results indicate that TP4 enhances the survival rate of mice infected with the bacterial pathogen MRSA through both antimicrobial and wound closure activities mediated by epidermal growth factor (EGF), transforming growth factor (TGF), and vascular endothelial growth factor (VEGF). The peptide is likely involved in antibacterial processes and regulation of tissue homeostasis in infected wounds in mice. Overall, these results suggest that TP4 may be suitable for development as a novel topical agent for wound dressing.     

Assessment of the Antimicrobial Activity of Algae Extracts on Bacteria Responsible of External Otitis

External otitis is a diffuse inflammation around the external auditory canal and auricle, which is often occurred by microbial infection. This disease is generally treated using antibiotics, but the frequent occurrence of antibiotic resistance requires the development of new antibiotic agents. In this context, unexplored bioactive natural candidates could be a chance for the production of targeted drugs provided with antimicrobial activity. In this paper, microbial pathogens were isolated from patients with external otitis using ear swabs for over one year, and the antimicrobial activity of the two methanol extracts from selected marine (Dunaliella salina) and freshwater (Pseudokirchneriella subcapitata) microalgae was tested on the isolated pathogens. Totally, 114 bacterial and 11 fungal strains were isolated, of which Staphylococcus spp. (28.8%) and Pseudomonas aeruginosa (P. aeruginosa) (24.8%) were the major pathogens. Only three Staphylococcus aureus (S. aureus) strains and 11 coagulase-negative Staphylococci showed resistance to methicillin. The two algal extracts showed interesting antimicrobial properties, which mostly inhibited the growth of isolated S. aureus, P. aeruginosa, Escherichia coli, and Klebsiella spp. with MICs range of 1.4 × 10⁹ to 2.2 × 10¹⁰ cells/mL. These results suggest that the two algae have potential as resources for the development of antimicrobial agents.     

胸腺嘧啶依赖型金黄色葡萄球菌小菌落突变株的分离鉴定

本试验对由患慢性奶牛乳房炎奶样中分离出的1株疑似金黄色葡萄球菌小菌落突变株(SCVs)进行形态观察、金黄色葡萄球菌相关保守基因片段(nuc、nucA、16S rDNA) 多重PCR扩增鉴定、药敏试验、生理生化特性研究及补偿试验。结果显示分离出1株金黄色葡萄球菌SCVs,该菌含有金黄色葡萄球菌菌种特异性基因nuc和nucA;与金黄色葡萄球菌质控菌株ATCC 25923的抑菌圈大小明显不同;菌落形态主要表现为菌落细小、生长缓慢、溶血能力下降;凝固酶活性下降;耐盐能力降低;革兰氏染色为革兰氏阳性球菌,呈葡萄状排列;补偿试验鉴定该金黄色葡萄球菌SCVs为胸腺嘧啶依赖型。结果表明成功分离鉴定出1株胸腺嘧啶依赖型金黄色葡萄球菌SCVs,为由金黄色葡萄球菌SCVs引起的奶牛慢性乳房炎的预防和控制及其致病机制的研究奠定前期基础。     

鸡舍空气致病菌分离鉴定及其对消毒剂的敏感性观察

本试验研究金黄色葡萄球菌与大肠杆菌对河北省某地区规模化鸡舍空气的污染情况及其对消毒剂的敏感性。根据细菌分离培养特性、形态学、生化试验、动物试验和玻板凝集试验分离出7株金黄色葡萄球菌、6株致病性大肠杆菌（血清型为O15、O26、O93、O78）。经统计,金黄色葡萄球菌、大肠杆菌在规模化鸡舍空气中的检出率分别为63.6%、27.27%。应用滤纸片法测定其对消毒剂的敏感性,2种致病菌对消毒剂的敏感性差异较大,这表明规模化鸡舍空气受致病菌的污染程度不同,并且致病菌对消毒剂的敏感性有差异。     

Effect of Staphylococcus aureus and Streptococcus uberis on apoptosis of bovine mammary gland lymphocytes

The aim of this study was to determine whether lymphocyte apoptosis is modulated by infections caused by Staphylococcus aureus and Streptococcus uberis. Samples of cell populations were obtained by lavage of the mammary glands at 4 intervals (24, 48, 72 and 168 h) following infection. The percentage of apoptotic lymphocytes peaked at 168 h after challenge with S. aureus or S. uberis. Subsequent experiments focused on in vitro cultivation of mammary gland lymphocytes with S. aureus and S. uberis. These experiments showed a lower percentage of apoptotic lymphocytes following 3 h of cultivating cells with bacteria than after cultivation without bacteria. The results demonstrate that during both experimental infection of bovine mammary glands with S. aureus or S. uberis and during in vitro cultivation of lymphocytes with S. aureus or S. uberis, apoptosis of lymphocytes is delayed.     

Differential responses of cells from human skin keratinocyte and bovine mammary epithelium to attack by pore-forming Staphylococcus aureus alpha-toxin

Human skin keratinocytes HaCat attacked by Staphylococcus aureus α-toxin showed a transient drop of cellular ATP levels whereas in toxin-perforated bovine mammary epithelial cells (BMEC), the ATP levels dropped more slowly. Morphologically, during the ATP level depletion, HaCat cell developed a spacious intracellular vacuole together with the transient influx of trypan blue. WST-1 signal, which tested the function of mitochondrial enzyme in viable cells, also decreased concomitantly. On the other hand, BMEC excluded trypan blue and vacuolation was not observed throughout the experiment. We conclude that mammary epithelial cells resist the toxin better than keratinocytes. This is the first report showing that α-toxin enhances transient membrane permeability to large molecules, temporary vacuole formation and the transient defect of mitochondrial enzyme in viable cells without cell lysis.     

金黄色葡萄球菌FnBA活性基因的克隆及其原核表达

根据GenBank中纤连蛋白结合蛋白A基因(FnBA)序列设计了1对特异性引物,以金黄色葡萄球菌基因组DNA为模板,进行PCR扩增;结果获得了1 735 bp的DNA片段.将PCR产物克隆至pMD18-T载体中,成功地构建了克隆质粒pMD18-T-FnBA.以HindⅢ和BamH Ⅰ双酶切pMD18-T-FnBA和pET28a(+),将纯化的基因FnBA亚克隆至pET28a(+)中,构建了原核表达质粒pET28a-FnBA,并将其转化至E.coli BL21感受态细胞中,经1 mmol/L IPTG诱导和SDS-PAGE分析,在约85 000处出现了与预期目的蛋白一致的外源蛋白带.又经Western-blotting分析表明,该蛋白具有金黄色葡萄球菌的抗原性.     

Activity of blue green microalgae extracts against in vitro generated Staphylococcus aureus with reduced susceptibility to vancomycin

Blue green microalgae have been identified as one of the promising groups of organism from which biochemically active natural products have been isolated. Aqueous and organic extracts of nine blue green microalgae strains were screened against in vitro generated vancomycin intermediate resistant Staphylococcus aureus (VISA) strains. Aqueous extracts of all the blue green microalgae cultures were found to be inactive, while all the organic (hexane, chloroform and methanolic) extracts of Anabaena virabilis and Anabaena sp. showed activity against VISA strains with MIC of 32-64 mug/ml.     

消毒威对几种菌株的杀灭效果研究

为了检测消毒威消毒剂对大肠杆菌、金黄色葡萄球菌、巴氏杆菌和沙门氏菌的杀灭效果．通过采用悬液定量杀菌法对几种标准菌株进行杀灭效果观察。发现20mg／L的消毒威作用5min．对大肠杆菌和金黄色葡萄球菌可达到全部杀灭作用,对沙门氏菌可达到消毒效能,对巴氏杆菌需适当加大浓度。以此笔者认为消毒威对大肠杆菌、金黄色葡萄球菌、沙门氏菌及巴氏杆菌均具有快速强效杀灭作用,适用于畜禽舍内外环境的消毒。