类芽孢杆菌BD3526抑菌活性物质的初步分离及性质测定北大核心CSCD

本实验以一株类芽孢杆菌BD3526为研究对象,采用丙酮浸提菌体得到抑菌活性粗品,通过Sephadex LH-20对样品进行初步纯化,结果表明粗品对藤黄微球菌CGMCC1.1848的最低抑菌浓度(MIC)为12.5 mg/m L,对金黄色葡萄球菌CGMCC1.879和单增李斯特菌CGMCC1.9136的MIC为25 mg/m L;Sephadex LH-20纯化样品对藤黄微球CGMCC1.1848的MIC为1 mg/m L,对金黄色葡萄球菌CGMCC1.879和单增李斯特菌CGMCC1.9136的MIC为2 mg/m L。样品经链霉蛋白酶处理后,活性基本消失。经加热、酸碱、胰蛋白酶、胃蛋白酶、脂肪酶、蛋白酶K处理后,活性没有明显变化。因此,类芽孢杆菌BD3526产生的是一种细菌素类的抑菌物质,该抑菌物质在食品和医疗行业有广阔的应用前景。     

类芽孢杆菌BD3526抑菌活性物质的初步分离及性质测定

本实验以一株类芽孢杆菌BD3526为研究对象,采用丙酮浸提菌体得到抑菌活性粗品,通过Sephadex LH-20对样品进行初步纯化,结果表明粗品对藤黄微球菌CGMCC1.1848的最低抑菌浓度(MIC)为12.5 mg/m L,对金黄色葡萄球菌CGMCC1.879和单增李斯特菌CGMCC1.9136的MIC为25 mg/m L;Sephadex LH-20纯化样品对藤黄微球CGMCC1.1848的MIC为1 mg/m L,对金黄色葡萄球菌CGMCC1.879和单增李斯特菌CGMCC1.9136的MIC为2 mg/m L。样品经链霉蛋白酶处理后,活性基本消失。经加热、酸碱、胰蛋白酶、胃蛋白酶、脂肪酶、蛋白酶K处理后,活性没有明显变化。因此,类芽孢杆菌BD3526产生的是一种细菌素类的抑菌物质,该抑菌物质在食品和医疗行业有广阔的应用前景。     

产细菌素的细菌筛选鉴定及其产物抑菌性能研究

从水体改善制剂样品中筛选出一株产细菌素的芽孢杆菌XDF-2,经生理生化分析和分子生物学鉴定,确定为短小芽孢杆菌。发现该菌产细菌素受发酵时间和培养的影响。对该粗提的细菌素进行特性研究,发现该细菌素只对金黄色葡萄球菌和藤黄微球菌具有抑菌活性。但是该细菌素在不同pH、100℃以下的温度、表面活性剂和有机溶剂的环境下稳定。经酶解试验所得,推测该细菌素具有多肽结构和羧基酯的结构。     

小菜蛾溶菌酶Pxlys的基因克隆及其 重组蛋白的抗菌活性分析

【目的】本研究旨在通过研究小菜蛾Plutella xylostella溶菌酶的功能,进一步认识小菜蛾的免疫防御机理,为小菜蛾的生物防治提供新的思路。【方法】利用RACE技术克隆小菜蛾溶菌酶基因。构建原核表达载体pET-29a-Pxlys,利用原核表达系统表达并用镍柱亲和层析纯化重组蛋白Pxlys。利用牛津杯法检测重组蛋白Pxlys对停滞棒杆菌Corynebacterium stationis、藤黄微球菌Micrococcus luteus、金黄色葡萄球菌Staphyloccocus aureus、大肠杆菌Escherichia coli、志贺氏菌Shigella sp.、沙门氏菌Salmonella sp.和苏云金芽胞杆菌Bacillus thuringiensis的抑菌活性,并利用扫描电子显微镜观察重组蛋白Pxlys对停滞棒杆菌和大肠杆菌的溶菌特征。【结果】克隆获得开放阅读框长423 bp的小菜蛾溶菌酶基因Pxlys(GenBank登录号： MN702780)序列,它编码140个氨基酸,相对分子质量为15.79 kD。抑菌试验表明,重组蛋白Pxlys不仅对革兰氏阳性细菌的停滞棒杆菌、藤黄微球菌和金黄色葡萄球菌有较强的抑菌活性(抑菌圈直径分别为20.0±1.1, 19.0±0.5和16.5±0.5 mm),而且对革兰氏阴性细菌大肠杆菌、志贺氏菌和沙门氏菌也有抑菌活性(抑菌圈直径分别为16.3±0.5, 15.0±0.5和14.0±1.1 mm),重组蛋白Pxlys对革兰氏阳性细菌比对革兰氏阴性细菌表现出更强的抑菌活性。另外,重组蛋白Pxlys还表现出对苏云金芽胞杆菌的抑菌活性。扫描电子显微镜下,经重组蛋白Pxlys处理过的停滞棒杆菌和大肠杆菌的溶菌特征不同。【结论】Pxlys具有广谱的抗微生物活性,其对革兰氏阳性细菌和革兰氏阴性细菌的抑菌机理可能存在不同。研究结果为深入研究小菜蛾免疫防御系统提供基础。     

牛樟芝发酵液提取物抗菌活性研究

牛樟芝作为一种珍稀食用和药用菌,具有极大的开发潜力。该研究以麦芽浸粉肉汤液体培养基(BD,美国BD公司)对牛樟芝菌丝体进行摇床培养60 d后,收获发酵液并用乙酸乙酯对其进行萃取,浓缩至干获得提取物;同时,采用抑菌圈法评价培养物对13种致病细菌抗菌活性(蜡样芽孢杆菌、缓慢芽孢杆菌、无乳链球菌、短小芽孢杆菌、福氏志贺氏菌、枯草芽孢杆菌、金黄色葡萄球菌、藤黄微球菌、副溶血性弧菌、溶血性葡萄球菌、铜尿假单胞菌、乙型副伤寒沙门氏菌、大肠埃希菌),并检测相应致病细菌的最低抑制浓度(MIC)。结果表明:牛樟芝麦芽浸粉肉汤发酵液提取物对供试的13种致病菌均有抑菌活性;在供试的13种致病菌中,提取物对缓慢芽孢杆菌、短小芽孢杆菌、枯草芽孢杆菌、副溶血性弧菌、藤黄微球菌5种致病菌的最低抑制浓度值均小于80μg·m L~(-1),其中对藤黄微球菌的最低抑制浓度最低为66.5μg·m~(-1);随着培养时间的增加,提取物的抗菌活性也增加。这说明牛樟芝菌丝体在液体培养条件下,能够产生广谱高效抑菌活性的次生代谢产物。该研究结果为牛樟芝进一步的有效利用开发奠定了理论基础。     

产广谱细菌素乳酸菌的筛选和鉴定

以大肠杆菌、金黄色葡萄球菌、藤黄微球菌、铜绿假单胞杆菌和枯草芽孢杆菌为指示菌,从分离自四川传统发酵食品中的267株乳酸菌中,采用平板打孔法初筛、牛津杯法复筛(排除酸、过氧化氢干扰以及胰蛋白酶和木瓜蛋白酶处理),筛选出1株分离自醪糟的具有较强抑菌作用的产广谱细菌素的乳杆菌菌株P158,结合形态学、生理生化特性和16S rDNA序列同源性分析,该菌株被鉴定为植物乳杆菌(Lactobacillus plantarum)。     

产广谱细菌素乳酸菌的筛选和鉴定

以大肠杆菌、金黄色葡萄球菌、藤黄微球菌、铜绿假单胞杆菌和枯草芽孢杆菌为指示菌,从分离自四川传统发酵食品中的267株乳酸菌中,采用平板打孔法初筛、牛津杯法复筛（排除酸、过氧化氢干扰以及胰蛋白酶和木瓜蛋白酶处理）,筛选出1株分离自醪糟的具有较强抑菌作用的产广谱细菌素的乳杆菌菌株P158,结合形态学、生理生化特性和16S rDNA序列同源性分析,该菌株被鉴定为植物乳杆菌（Lactobacillus plantarum）。     

片球菌素PediocinPA-1抑菌活性检测

目的检测通过基因工程获得的片球菌素Pediocin PA-1抑菌活性。方法采用琼脂扩散法检测片球菌素Pediocin PA-1对单核细胞增生李斯特杆菌、金黄色葡萄球菌、铜绿假单胞菌、沙门菌和大肠埃希菌O157的抑菌活性。结果片球菌素Pediocin PA-1对单核细胞增生李斯特杆菌、金黄色葡萄球菌、沙门菌、铜绿假单胞菌和大肠埃希菌O157等均有抑制作用。其中对单核细胞增生李斯特杆菌、沙门菌、大肠埃希菌和金黄色葡萄球菌的抑制作用效果明显,对铜绿假单胞菌有微弱的抑制作用。结论通过基因工程获得的片球菌素Pediocin PA-1具有抑菌活性。     

可抑制致病菌的益生菌筛选及抑菌物质的初步确定

为了得到可抑制致病菌的益生菌并初步确定其抑菌物质。从土壤、饲料添加剂和肥料添加剂3种样品中初筛能产生抑菌圈的菌株。用牛津杯法,对初筛得到的菌株和实验室保存的菌株进行复筛。对复筛的未知菌株进行形态学、16S r RNA基因序列和生理生化特征鉴定。对复筛菌株的发酵上清液进行酸碱作用验证、高温处理、蛋白酶处理、盐析处理和透析处理,再进行抑菌活力测定。结果显示复筛中有8株对大肠杆菌、金黄色葡萄球菌和藤黄微球菌都有较强抑制作用的菌株。8株菌中有4株已知菌,4株未知菌。已知菌株分别为保加利亚乳杆菌、干酪乳杆菌、鼠李糖乳杆菌和植物乳杆菌。4株未知菌中3株被鉴定为地衣芽孢杆菌,1株被鉴定为乳酸片球菌。地衣芽孢杆菌能产生多种耐高温的抑菌物质,其中包含能透过8-14 k D透析袋的小分子和不能透过8-14 k D透析袋的大分子,初步判定为多肽类物质。5株产酸菌株产生的抑菌物质主要是酸性物质。     

一株来自于伊犁贝母的内生尖孢镰孢菌Y1的抑菌活性初步研究

从新疆巩留县伊犁贝母的新鲜鳞茎中分离到一株具有分泌抑菌活性物质的内生尖孢镰孢菌Fusarium oxysporumY1,该菌在7种不同培养基上生长时显示出不同的菌落生长特征,而且只在沙氏培养基中生长时才具有分泌抑菌活性物质的能力。抑菌活性筛选结果表明:由该菌及其发酵液制备的发酵液浸膏、菌体裂解液浸膏以及经进一步纯化后获得的乙酸乙酯浸膏和正丁醇浸膏均具有明显的抑菌活性,其中以发酵液的乙酸乙酯浸膏和菌体裂解液的正丁醇浸膏活性最强,它们对金黄色葡萄球菌Staphylococcus aureus、表皮葡萄球菌Staphylococcus epidermidis、枯草芽孢杆菌Bacillus subtilis、藤黄八叠球菌Sarcina lutea和大肠杆菌Escherichia coli的最低抑菌浓度均小于25μg/mL。     

Identification of a new Bacillus licheniformis strain producing a bacteriocin-like substance

The emergence of antibiotic resistance has spurred a great number of studies for development of new antimicrobials in the past decade. The purpose of this study was to screen environmental samples for Bacillus strains producing potent antimicrobial agents. A new strain, which showed strong antimicrobial activity against Staphylococcus aureus and Salmonella enterica ser. Pullorum, was isolated from soil and designated as B116. This new isolate was identified as Bacillus licheniformis by morphological, biochemical and genetic analyses. The production of bacteriocin-like substance (BLS) started at early exponential phase and achieved highest level at early stationary phase. The BLS was precipitated by ammonium sulfate and its molecular mass was determined as ～4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Culture supernatant of the new isolate exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, including Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Micrococcus luteus, Escherichia coli, and Salmonella spp. The BLS was resistant to heat, acid and alkaline treatment. Activity of the BLS was totally lost after digestion by pronase and partially lost after digestion by papain and lipase. The new isolate and relevant BLS are potentially useful in food and feed applications.     

Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom

The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme.     

Modulation of antibiotic resistance in bacterial pathogens by oleanolic acid and ursolic acid

Antibiotic resistance among bacterial pathogens is a serious problem for human and veterinary medicine, which necessitates the development of novel therapeutics and antimicrobial strategies. Some plant-derived compounds, e.g. pentacyclic triterpenoids such as oleanolic acid (OA) and ursolic acid (UA), have potential as a new class of antibacterial agents as they are active against many bacterial species, both Gram-positive and Gram-negative, and specifically target the cell envelope. The aim of the present study was to investigate the influence of OA and UA on the susceptibility of four bacterial pathogens (Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus and Staphylococcus epidermidis) to the β-lactam antibiotics ampicillin (Ap) and oxacillin (Ox). Antimicrobial assays were conducted with bacteria growing in liquid suspension cultures (planktonic cells) or as biofilms. Using FICI value estimation and the time-kill method it was demonstrated that in some combinations, the tested compounds acted in synergy to lower the susceptibility of S. aureus, S. epidermidis and L. monocytogenes to ampicillin and oxacillin, but no synergy was observed for P. aeruginosa. These results indicate that OA and UA may be useful when administered in combination with β-lactam antibiotics to combat bacterial infections caused by some Gram-positive pathogens.     

Detection and characterization of a novel antibacterial substance produced by a Lactobacillus delbrueckii strain 1043

A novel antibacterial substance produced by a strain isolated from Bulgarian yellow cheese was characterized. The producer strain was identified by molecular typing to belong to the species Lactobacillus delbrueckii , which is a rare producer of bacteriocins. The inhibitory agent was heat stable and active against lactic acid bacteria species and several food-borne pathogens : Listeria monocytogenes , Staphylococcus aureus , Enterococcus faecalis , Escherichia coli , Yersinia enterocolitica and Y. pseudotuberculosis . Its sensitivity to amylolitic enzymes and lipase suggested that a lipid and carbohydrate moiety could be important for the activity. The amino acid content of the purified bacteriocin was estimated to 29 amino acids. The bacteriocin was shown to be small (3·6–6 kDa) by three different methods : HPLC gel-filtration, SDS-PAGE and amino acid contents.     

Bactericidal activity of human peritoneal macrophages from patients undergoing peritoneal dialysis

Peritoneal macrophages (PM) play an essential role in the pathogenesis of bacterial peritonitis, the main complication of peritoneal dialysis (PD). We determined the antibacterial activity of PM from 31 PD patients using gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and gram-negative (Escherichia coli, Pseudomonas aeruginosa) test organisms. In an 8-hour test assay, PM revealed the highest antibacterial activity against E. coli [median bactericidal index (Bi) = 5.46 representing 0.74 log growth inhibition compared to controls] and the lowest against P. aeruginosa (Bi = 1.63, 0.21 log growth inhibition, p less than 0.05). The antibacterial activity against S. aureus (Bi = 1.99, 0.3 log growth inhibition) and S. epidermidis (Bi = 2.0, 0.31 log growth inhibition) was within this range. When compared to peripheral blood polymorphonuclear leukocytes, PM reached only 4% (S. aureus) and 8.1% (E. coli) of their antibacterial activity (p less than 0.05). Using E. coli as a test organism, PM isolated after a 4-hour dialysis period revealed the highest antibacterial activity when compared to PM isolated after longer dialysis periods (p less than 0.05). Increasing the duration of PD to 6 and 8 h subsequently decreased the antibacterial activity of PM, suggesting that unphysiologic concentrations of toxic metabolites in the peritoneal effluent might have a harmful influence on PM functions.     

新型乳杆菌素产生菌的筛选及菌株特性的研究

从酸菜汁中分离出 2 4株乳酸杆菌 ,采用牛津杯定量扩散法筛选出 1株抑菌活性较高的乳酸杆菌 ,经鉴定为戊糖乳杆菌。排除乳酸对指示菌作用的干扰 ,该菌株的离心发酵液不仅对革兰氏阳性细菌有抑制作用 ,而且对大肠杆菌 (E .coli.) ,沙门氏菌 (Salmonellatyphi)等革兰氏阴性细菌也有一定的抑制作用     

Antimicrobial activity of some Salvia species essential oils from Iran

The aerial parts of Salvia multicaulis, S. sclarea and S. verticillata were collected at full flowering stage. The essential oils were isolated by hydrodistillation and analyzed by combination of capillary GC and GC-MS. The in vitro antimicrobial activity of the essential oils were studied against eight Gram-positive and Gram-negative bacteria (Bacillus subtilis, Bacillus pumulis, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) and three fungi (Candida albicans, Saccharomyces cerevisiae and Aspergillus niger). The results of antibacterial activity tests of the essential oils according to the disc diffusion method and MIC values indicated that all the samples have moderate to high inhibitory activity against the tested bacteria except for P. aeruginosa which was totally resistant. In contrast to antibacterial activity, the oils exhibited no or slight antifungal property, in which only the oil of S. multicaulis showed weak activity against two tested yeasts, C. albicans and S. cerevisiae.     

双歧杆菌的粘附特性及其对肠道致病菌的体外拮抗作用

本文采用体外细胞培养法, 从实验室现有的20株不同生境来源的双歧杆菌中筛选具有较强粘附能力的菌株, 并通过混合培养和牛津杯方法, 研究了具有粘附特性的双歧杆菌对肠道致病菌的体外拮抗作用。结果显示, 长寿老人源菌株A03和I06的粘附能力最强, 其菌液对金黄色葡萄球菌及大肠杆菌均具有显著的抑制性, 但是菌体及中和后的发酵液均没有抑菌性, 说明受试菌主要通过其代谢产物中的有机酸来发挥其抑菌性能; 此外, 通过分析比较受试菌和肠道致病菌分别与Caco-2细胞粘附后释放的乳酸脱氢酶量, 证实双歧杆菌与致病菌对细胞的作用具有本质上的区别, 双歧杆菌的粘附能减缓致病菌对细胞所造成的损害。     

Antibiotic sensitivity of the main opportunistic pathogenic aerobic bacteria

Antibiotic sensitivity of 1421 strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa and Klebsiella spp. was studied. Gentamicin, levomycetin (chloramphenicol) and ristomycin proved to be the antibiotics of choice in treatment of purulent inflammatory diseases caused by S. epidermidis and S. aureus. For antibiotic therapy of infections caused by gram-negative organisms gentamicin and polymixin might be recommended.