1株抗须癣毛癣菌枯草芽孢菌的鉴定

为了探讨抗须癣毛癣菌感染新的治疗途径,从患须癣毛癣菌病兔自然恢复皮肤分离了1株细菌,经16S rDNA基因比对,并结合菌落形态进行了鉴定,通过体内外试验测定其对须癣毛癣菌的拮抗作用。结果显示,分离株能在营养琼脂培养基生长,菌落为皱褶圆形,菌体呈杆状,革兰染色阳性,基因序列分析与GenBank中的枯草芽孢杆菌相似率为99.8%,该分离株被鉴定为枯草芽孢杆菌。抗菌谱分析显示,分离株对须癣毛癣菌能形成抑菌圈,对白色念珠菌、平滑念珠菌、热带念珠菌、烟曲霉菌、黄曲霉菌没有抑制作用。该分离株与须癣毛癣菌混合培养,能明显抑制须癣毛癣菌生长。在皮肤涂抹试验中,该分离株能逆转由须癣毛癣菌引起的皮肤病变。本试验证实了分离的枯草芽孢杆菌对须癣毛癣菌有明显的拮抗作用。     

羊毛皮张和土壤中炭疽杆菌PCR检测方法的建立

建立了检测炭疽芽孢杆菌染色体特异性基因序列 (Ba813)的PCR技术 ,并用于模拟污染的羊毛、皮张和土壤中炭疽芽孢 (Sterne菌株、PasteurⅡ菌株 )的检测。结果表明 ,该方法可特异、敏感地检出 0 .5 g羊毛中Sterne菌株的 4 5 2个芽孢和PasteurⅡ菌株的 6 88个芽孢 ;0 .5g皮张中Sterne菌株的 4 5 2个芽孢和PasteurⅡ菌株的 10 32个芽孢 ;0 .5 g土壤中Sterne菌株的 4 5 2个芽孢和PasteurⅡ菌株 1376个芽孢。证实PCR可用于羊毛、皮张和土壤等外环境中炭疽杆菌芽孢的检测。     

Trace detection of meglumine and diatrizoate from Bacillus spore samples using liquid chromatography/mass spectrometry

Following the September 11, 2001 terrorist attacks, letters containing Bacillus anthracis were distributed through the United States postal system killing five people. A complex forensic investigation commenced to identify the perpetrator of these mailings. A novel liquid chromatography/mass spectrometry protocol for the qualitative detection of trace levels of meglumine and diatrizoate in dried spore preparations of B. anthracis was developed. Meglumine and diatrizoate are components of radiographic imaging products that have been used to purify bacterial spores. Two separate chromatographic assays using multiple mass spectrometric analyses were developed for the detection of meglumine and diatrizoate. The assays achieved limits of detection for meglumine and diatrizoate of 1.00 and 10.0 ng/mL, respectively. Bacillus cereus T strain spores were effectively used as a surrogate for B. anthracis spores during method development and validation. This protocol was successfully applied to limited evidentiary B. anthracis spore material, providing probative information to the investigators.     

Bacterial Cross‐contamination Potential Associated with Contaminated Currency

Previous studies have shown a significant amount of contaminants on paper currencies. It is important to study the transfer of microorganisms between paper currencies to determine whether it meets the level of a human health threat. This cross‐contamination potential was analyzed by seeding new US 1‐dollar bills with Bacillus thuringiensis, and pressing or rubbing them against clean currency to determine the amount of bacteria transfer to the unseeded currency. The transferred amount of bacteria was recovered, plated, incubated, and the colony‐forming units were quantified. Among the recovery methods tested, the most efficient method, vortexing for 10 min with a recovery efficiency of 40 ± 8.1%, was used in this analysis. The resulting transfer rates were 4.8%, 8.6%, and 14.3% when pressed for 24 h, 72 h, and rubbed together, respectively. These transferred amounts of bacteria are significant and have the potential to spread infectious diseases.     

犬源枯草芽胞杆菌的分离鉴定及生物学特性分析

从犬用微生态制剂研制角度出发,采集健康幼犬粪样进行热处理,使用营养琼脂培养基分离出革兰阳性,类似芽胞杆菌菌株1株。之后,从形态学、生理生化特性及分子生物学分析进行菌种鉴定,最后确定为枯草芽胞杆菌,编号为YW1。通过耐酸、抑菌及药敏试验证实菌株YW1具有较好的耐酸特性及较高的抑菌能力与安全性,说明枯草芽胞杆菌YW1具有临床开发的潜能。本研究为后续进一步研究其在犬微生态制剂中的应用奠定了基础,也为开发具有抑菌活性的菌株提供了参考。     

Environmental survey for four pathogenic bacteria and closely related species using phylogenetic and functional genes

Bacterial species with high DNA sequence similarity to pathogens could affect the specificity of assays designed to detect biological threat agents in environmental samples. The natural presence of four pathogenic bacteria, Bacillus anthracis, Clostridium perfringens, Francisella tularensis, and Yersinia pestis and their closely related species, was determined for a large collection of soil and aerosol samples. Polymerase chain reaction (PCR) and gene sequencing were used using group-specific 16S rRNA primers to identify pathogens and related species, and pathogen-specific virulence genes. Close relatives of B. anthracis (B. cereus group species) were detected in 37% of the soils and 25% of the aerosol samples. The B. anthracis protective antigen (pag) gene or a close homolog was detected in 16 of these samples. For the other three pathogen groups, the frequency of detection was much lower, and none of the samples were positive with both the phylogenetic and virulence gene primer sets.     

蕈状芽孢杆菌代谢产物活性成分对小鼠免疫功能的影响

以蕈状芽孢杆菌代谢产物的4种提取物给小鼠腹腔注射,通过测定血红蛋白值、红细胞数、超氧化物歧化酶(SOD)活性、血清凝集抗体效价、离体白细胞吞噬活性和吞噬细胞杀菌活性,研究了不同提取物对小鼠免疫功能的影响。结果显示,注射提取物各组小鼠的免疫功能均有不同程度增强,乙酸乙酯提取物组、三氯甲烷提取物组、石油醚提取物组的白细胞吞噬活性、吞噬细胞杀菌活性、血清凝集抗体效价、SOD活性与对照组差异极显著(P<0.01),剩余液组的吞噬活性和杀菌活性与对照组差异显著(P<0.05),其他指标与对照组差异不显著;石油醚提取物组的血红蛋白值(13.52 g/100 mL)和红细胞数(11.35×106/mL)最高。各项指标的峰值均出现在第7~21 d。     

Evaluation of the rapid analyte measurement platform (RAMP) for the detection of Bacillus anthracis at a crime scene

The aim of this study was to evaluate the accuracy and reliability of the rapid analyte measurement platform (RAMP) for presumptive identification of Bacillus anthracis spores. Test samples consisted of serial dilutions of spore preparations of several Bacillus species, including B. anthracis, which were tested, using the RAMP Anthrax test cartridge, according to the manufacturer's instructions. The fluorescence labelled antibody-antigen complexes were detected in the portable reader after 15 min following sample addition. Dilutions of common environmental and household powders were also tested to identify possible false positive results. B. anthracis spores were identified reliably in test samples containing more than 6000 spores. The test kits were highly specific, showing no cross reactivity with other Bacillus species or any environmental powders tested. The RAMP system for detection of B. anthracis spores, from environmental samples, showed consistent results under a variety of analytical conditions, enabling the trained user to provide a rapid, accurate preliminary risk assessment of a suspected bioterrorism incident.     

芽胞杆菌的分离鉴定

为了确定实验室筛选的6株芽胞杆菌(Bacillus spp.)的种名,利用形态学、DNA促旋酶A亚单位基因(DNA Gyrase Subunit A,gyr A)和DNA促旋酶B亚单位基因(DNA Gyrase Subunit B,gyr B)序列的单基因系统发育分析和PCR相结合的方法进行鉴定。结果表明,6株芽胞杆菌的单菌落生长特征相似,革兰染色呈阳性,具有芽胞;以gyr A和gyr B基因构建的单基因系统发育树显示6株菌均与贝莱斯芽胞杆菌(Bacillus velezensis)归于同一分支,自展值(Bootstrap)较高;特异性PCR结果也证实它们均为贝莱斯芽胞杆菌阳性。结论,实验室分离的6株芽胞杆菌均属于贝莱斯芽胞杆菌,这为它们的深入研究奠定了基础。