抗动物病原菌芽孢杆菌的筛选、初步鉴定和抗菌活性

从鸡肠道分离、挑选的18株芽孢杆菌经培养特征、形态观察、生理生化实验,初步被鉴定为枯草芽孢杆菌(Bacillus subtilus)、地衣芽孢杆菌(Bacillus licheniformis)、蜡状芽孢杆菌(Bacillus cereus)、巨大芽孢杆菌(Bacillus megaterium)和凝结芽孢杆菌(Bacillus coagulans)。同时测定了它们对5种常见动物病原菌大肠杆菌(E.coli)、金黄色葡萄球菌(Staphylococcus aureus)、肠炎沙门氏菌(Salmonella typhimurium)、猪链球菌(Streptococcus suis)、奇异变形杆菌(Proteus mirabilis)的抑菌活性,其中有4株芽孢杆菌对5种病原菌都有抑制作用。还分别测定了它们产木聚糖酶(从0.895 U1到3.239 U1)和纤维素酶活性(分别为0.391 U2和0.465 U2)。结果表明,芽孢杆菌分离株BC17、BC32、BC106、BC228、BC247和BC261具有作为益生菌的开发潜力。     

鸡肠源芽胞杆菌的分离、鉴定和抗菌活性

从人工饲养的健康三黄肉鸡的盲肠和大肠中分离出10株芽胞杆菌,对其进行菌落形态观察、革兰染色、芽胞染色和生理生化特性鉴定,确定为枯草芽胞杆菌、地衣芽胞杆菌、蜡状芽胞杆菌、巨大芽胞杆菌、球形芽胞杆菌、短小芽胞杆菌、凝结芽胞杆菌。同时测定了它们对动物病原性大肠埃希菌和链球菌的抑菌活性。其中有9株芽胞杆菌对大肠埃希菌有抑制作用,8株芽胞杆菌对链球菌有抑制作用。结果表明芽胞杆菌分离株Y2、Y3、Y4、Y5、Y6、Y7、Y8具有作为益生菌开发的潜力。     

Cocultivation of the amoeba Naegleria fowleri and the amoebicin- producing strain Bacillus licheniformis M-4.

Antagonism between Bacillus licheniformis M-4 and the pathogenic amoeba Naegleria fowleri HB-1 during cocultivation was influenced by the composition of the medium and the initial amoeba/bacterium ratio. While a ratio of 50 caused complete lysis of amoebae in soil extract with 0.3% glucose (SEG) before 72 h, this ratio had to be at least 12-fold lower in order to obtain similar results in Cline medium. Sporulation of B. licheniformis M-4 took place much earlier in SEG. Amoebicin production was stimulated by the presence of amoebae by either shortening the time of production (as in SEG) or increasing the amount of amoebicins released (as in Cline medium). Electron microscopy showed that amoebae cocultivated in the Cline medium contained bacteria enclosed in digestive vacuoles, while amoebae from SEG cocultures did not.     

Inhibitory effects of Bacillus probionts on growth and toxin production of Vibrio harveyi pathogens of shrimp

Aim:  To investigate the effects of Bacillus subtilis , Bacillus licheniformis and Bacillus megaterium in terms of toxin and growth of pathogenic Vibrio harveyi .
Methods and Results:  Three Bacillus probionts were isolated from probiotic BZT aquaculture and identified using a 16S rDNA sequence. Growth inhibition assay showed that supernatants from the 24-h culture of three Bacillus species were able to inhibit the growth of V. harveyi (LMG 4044); B. subtilis was the most effective based on the well diffusion method. Results of a liquid culture model showed that B. subtilis was also widely effective in inhibiting three strains of V. harveyi (isolated from Thailand, the Philippines and LMG 4044), and that both B. licheniformis and B. megaterium inhibit the growth of V. harveyi isolated from the Philippines. Moreover, a haemolytic activity assay demonstrated that V. harveyi (IFO 15634) was significantly decreased by the addition of B. licheniformis or B. megaterium supernatant.
Conclusions:  Bacillus subtilis inhibited Vibrio growth, and both B. licheniformis and B. megaterium suppressed haemolytic activity in Vibrio .
Significance and Impact of the Study:  The cell-free supernatants produced by Bacillus probionts inhibit Vibrio disease, and Bacillus probionts might have an influence on Vibrio cell-to-cell communications.     

Bacteriocin-like substance production by Bacillus licheniformis strain P40

AIMS: To investigate the production of bacteriocin-like compounds by Bacillus spp. isolated from the Amazon basin. METHODS AND RESULTS: An antimicrobial substance produced by Bacillus licheniformis strain P40 was inhibitory to a broad range of indicator strains, such as Listeria monocytogenes, Bacillus cereus and clinical isolates of Streptococcus spp. The compound was stable at 100 degrees C, but lost its activity when treated at 121 degrees C/103.5 kPa for 15 min. It was resistant to the proteolytic action of trypsin and papain but sensitive to pronase E and was stable within a wide range of pH (3-11). The substance was bactericidal and bacteriolytic to L. monocytogenes. CONCLUSIONS: An antibacterial peptide produced by Bacillus licheniformis was characterized, presenting a broad spectrum of activity against pathogenic and spoilage organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of a substance active against important pathogens addresses an important aspect of food safety.     

Concerning the Differentiation of Bacillus subtilis and Related Species

The results of an investigation involving 45 strains of Bacillus subtilis , 31 strains of B. licheniformis and 29 strains of B. pumilus are reported. The hitherto recognized varieties B. subtilis var. niger and B. subtilis var. aterrimus appear to be only variants of B. subtilis. For a rapid differentiation of B. licheniformis from B. subtilis two tests are recommended—reduction of nitrites and splitting of arginine. The present tests, reduction of nitrate and hydrolysis of starch, are the most suitable for distinguishing between B. pumilus and B. subtilis.     

Toxigenic strains of Bacillus licheniformis related to food poisoning.

Toxin-producing isolates of Bacillus licheniformis were obtained from foods involved in food poisoning incidents, from raw milk, and from industrially produced baby food. The toxin detection method, based on the inhibition of boar spermatozoan motility, has been shown previously to be a sensitive assay for the emetic toxin of Bacillus cereus, cereulide. Cell extracts of the toxigenic B. licheniformis isolates inhibited sperm motility, damaged cell membrane integrity, depleted cellular ATP, and swelled the acrosome, but no mitochondrial damage was observed. The responsible agent from the B. licheniformis isolates was partially purified. It showed physicochemical properties similar to those of cereulide, despite having very different biological activity. The toxic agent was nonproteinaceous; soluble in 50 and 100% methanol; and insensitive to heat, protease, and acid or alkali and of a molecular mass smaller than 10,000 g mol(-1). The toxic B. licheniformis isolates inhibited growth of Corynebacterium renale DSM 20688(T), but not all inhibitory isolates were sperm toxic. The food poisoning-related isolates were beta-hemolytic, grew anaerobically and at 55 degrees C but not at 10 degrees C, and were nondistinguishable from the type strain of B. licheniformis, DSM 13(T), by a broad spectrum of biochemical tests. Ribotyping revealed more diversity; the toxin producers were divided among four ribotypes when cut with PvuII and among six when cut with EcoRI, but many of the ribotypes also contained nontoxigenic isolates. When ribotyped with PvuII, most toxin-producing isolates shared bands at 2.8 +/- 0.2, 4.9 +/- 0.3, and 11.7 +/- 0.5 or 13.1 +/- 0.8 kb.     

Cross-Species Induction of Antimicrobial Compounds, Biosurfactants and Quorum-Sensing Inhibitors in Tropical Marine Epibiotic Bacteria by Pathogens and Biofouling Microorganisms

Enhancement or induction of antimicrobial, biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions was investigated. Four marine epibiotic bacteria (Bacillus sp. S3, B. pumilus S8, B. licheniformis D1, and Serratia marcescens V1) displaying antimicrobial activity against pathogenic or biofouling fungi (Candida albicans CA and Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA and Bacillus pumilus BP) were chosen for this study. The marine epibiotic bacteria when co-cultivated with the aforementioned fungi or bacteria showed induction or enhancement in antimicrobial activity, biosurfactant production, and quorum-sensing inhibition. Antifungal activity against Y. lipolytica YL was induced by co-cultivation of the pathogens or biofouling strains with the marine Bacillus sp. S3, B. pumilus S8, or B. licheniformis D1. Antibacterial activity against Ps. aeruginosa PA or B. pumilus BP was enhanced in most of the marine isolates after co-cultivation. Biosurfactant activity was significantly increased when cells of B. pumilus BP were co-cultivated with S. marcescens V1, B. pumilus S8, or B. licheniformis D1. Pigment reduction in the quorum-sensing inhibition indicator strain Chromobacterium violaceum 12472 was evident when the marine strain of Bacillus sp. S3 was grown in the presence of the inducer strain Ps. aeruginosa PA, suggesting quorum-sensing inhibition. The study has important ecological and biotechnological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite production.     

产酱香地衣芽孢杆菌CGMCC 3963耐受特征及基于转录组学的耐受机制分析

【目的】地衣芽孢杆菌是茅台酒高温大曲中能产酱香风味物质的主要微生物,对酱香型白酒的酿造具有重要价值。而酱香型白酒的酿造环境具有高渗、高温、酸性、高乙醇胁迫等特征,研究产酱香地衣芽孢杆菌在环境胁迫下的耐受特征有利于认识酱香型白酒的酿造特征。【方法】以一株产酱香地衣芽孢杆菌(Bacillus licheniformis CGMCC 3963)为研究对象,测定其耐渗、耐酸、耐乙醇特征,并从比较转录组学角度系统分析B.licheniformis CGMCC 3963的耐受机制。【结果】B.licheniformis CGMCC 3963在15%的KCl、15%的Na Cl、p H 4.0的酸性环境或6%乙醇浓度下的生长情况明显优于不产酱香的模式菌株B.licheniformis ATCC 14580。转录组比较分析显示B.licheniformis CGMCC 3963中一系列与耐受相关的基因表达有差异。【结论】来源于酿造环境的B.licheniformis CGMCC 3963耐受能力强于B.licheniformis ATCC 14580,一系列与耐受相关的基因表达有差异。编码脯氨酸和甜菜碱等溶质转运、离子外排、钾离子通道蛋白等基因的差异表达,使得高渗胁迫下B.licheniformis CGMCC 3963生长明显优于B.licheniformis ATCC 14580;编码II类热休克蛋白、乙醇脱氢酶、氧化应激、p H动态平衡等相关基因的差异表达,在提高菌株耐受酸性环境能力上起了重要作用;II类及III类热休克基因的高表达对B.licheniformis CGMCC 3963耐乙醇能力起了重要作用。     

Extracellular enzyme synthesis in a sporulation-deficient strain of Bacillus licheniformis.

A deletion of the spoIIAC gene of Bacillus licheniformis was prepared in vitro by using the splicing-by-overlap-extension technique. This gene was introduced into B. licheniformis on a temperature-sensitive plasmid, and following integration and excision from the chromosome, a precisely located deletion on the chromosomal gene was prepared. The mutated bacterium was totally asporogenous and formed abortively disporic cells characterized by asymmetric septa at the poles of the cells. Qualitative plate tests revealed that the bacterium synthesized normal levels of DNase, polygalacturonate lyase, protease, RNase, and xylanase, but the hydrolysis zones due to beta-1,3-glucanase and carboxymethyl cellulase activity were smaller in the mutant than in the parent strain. The synthesis of alkaline protease was the same in batch cultures of the mutant and the parent during prolonged incubation for 72 h, but the alpha-amylase yields were reduced by about 30% by the mutation.     

地衣芽胞杆菌对白色念珠菌等的拮抗作用

目的了解地衣芽胞杆菌在试管内与阴道正常菌群共生关系的情况。方法将地衣芽胞杆菌菌液分别与葡萄球菌、大肠埃希菌、白色念珠菌、德氏乳杆菌混合培养,定量计数各菌在不同时间内单独培养和混合培养时各菌的活菌数。结果地衣芽胞杆菌生长不受金黄色葡萄球菌、白色念珠菌和大肠埃希菌的影响,金黄色葡萄球菌和白色念珠菌在有地衣芽胞杆菌存在的情况下,其生长受到明显的抑制（P〈0.05）;乳杆菌在12-48 h内,有显著的抑制地衣芽胞杆菌生长的作用,而乳杆菌的生长不受地衣芽胞杆菌的存在与否而正常生长。结论地衣芽胞杆菌对金黄色葡萄球菌及白色念珠菌在体外具有明显的拮抗作用,地衣芽胞杆菌对大肠埃希菌、乳杆菌无明显的体外拮抗作用。     

Heterogenous expression of poly-gamma-glutamic acid synthetase complex gene of Bacillus licheniformis WBL-3

Bacillus licheniformis WBL-3, one of poly-gamma-glutamic acid (gamma-PGA) producers, depends on the existence of glutamate in the medium. In this paper, gamma-PGA synthetase complex gene (pgsBCA) was cloned from Bacillus licheniformis WBL-3. pgsBCA gene of B. licheniformis WBL-3 was highly homologous with pgsBCA gene of B. licheniformis 14580. The similarity was 97%, but the similarity of pgsBCA gene between B. licheniformis WBL-3 and Bacillus subtilis IF03336 was only 74%. However, when pgsBCA was expressed in Escherichia coli, the E. coli clone produced gamma-PGA extracellularly. The yield of gamma-PGA was 8.624 g/l. This result infers that B. licheniformis and B. subtilis has the similar gamma-PGA biosynthesis mechanism, namely, glutamic acid is catalyzed by an ATP-dependent amide ligase to synthesize gamma-PGA.     

Chemical warfare? Effects of uropygial oil on feather‐degrading bacteria

Anti-microbial activity is a commonly suggested but rarely tested property of avian uropygial oil. Birds may defend themselves against feather-degrading and other potentially harmful bacteria using this oil. We preliminarily identified 13 bacterial isolates taken from the plumage of wild house finches Carpodacus mexicanus , measured bacterial production of the enzyme keratinase as an index of feather-degrading activity, and used the disc-diffusion method to test bacterial response to uropygial oil of house finches. For comparison, we performed the same tests on a type strain of the known feather-degrading bacterium Bacillus licheniformis . Uropygial oil inhibited the growth of three strongly feather-degrading isolates (including Bacillus licheniformis ), as well as one weakly feather-degrading isolate and one non-feather-degrading isolate. Uropygial oil appeared to enhance the growth of one weakly feather-degrading isolate. Growth of the remaining isolates was unaffected by uropygial oil. These results suggest that birds may defend themselves against some feather-degrading bacteria using uropygial oil.     

Numerical taxonomy of Bacillus isolated from orally administered drugs

Numerical taxonomy procedures were used to study 118 strains of Bacillus isolated from non-sterile drugs prepared for oral administration. Similarities between pairs of strains were calculated by the simple matching coefficient of Sokal and Michener (S_SM). Each strain was tested for 60 unit characters and three clusters were defined. The strains in each cluster presented a similarity level of at least 60%. Cluster A comprised the strains identified as Bacillus cereus (S_SM= 93·13%), cluster B contained three subgroups corresponding to the species B. pumilus, B. subtilis and B. licheniformis (S_SM= 84·35%) and cluster C also included three subgroups that belonged to the species B. firmus, B. lentus and B. badius (S_SM= 80·14%). The most discriminating tests were selected to differentiate the clusters from the subgroups. The feature with the highest discriminating power between clusters A and B was the lack of acid production from arabinose and mannitol. The Voges-Proskauer, methyl red tests and sensitivity to polymyxin B clearly distinguished cluster A from C. The Voges-Proskauer test and acid production from arabinose were the best to differentiate between B and C. Bacillus pumilus and B. subtilis differed in starch hydrolysis and B. licheniformis in growing anaerobically. To discriminate B. firmus from B. lentus the most important tests were the acid production from glucose and sucrose; intermediate strains were found. Bacillus badius was differentiated from B. firmus by 10 tests, and from B. lentus by the production of urease.     

Numerical taxonomy of Bacillus isolated from orally administered drugs

Numerical taxonomy procedures were used to study 118 strains of Bacillus isolated from non-sterile drugs prepared for oral administration. Similarities between pairs of strains were calculated by the simple matching coefficient of Sokal and Michener (SSM). Each strain was tested for 60 unit characters and three clusters were defined. The strains in each cluster presented a similarity level of at least 60%. Cluster A comprised the strains identified as Bacillus cereus (SSM = 93.13%), cluster B contained three subgroups corresponding to the species B. pumilus, B. subtilis and B. licheniformis (SSM = 84.35%) and cluster C also included three subgroups that belonged to the species B. firmus, B. lentus and B. badius (SSM = 80.14%). The most discriminating tests were selected to differentiate the clusters from the subgroups. The feature with the highest discriminating power between clusters A and B was the lack of acid production from arabinose and mannitol. The Voges-Proskauer, methyl red tests and sensitivity to polymyxin B clearly distinguished cluster A from C. The Voges-Proskauer test and acid production from arabinose were the best to differentiate between B and C. Bacillus pumilus and B. subtilis differed in starch hydrolysis and B. licheniformis in growing anaerobically. To discriminate B. firmus from B. lentus the most important tests were the acid production from glucose and sucrose; intermediate strains were found. Bacillus badius was differentiated from B. firmus by 10 tests, and from B. lentus by the production of urease.     

地衣芽胞杆菌dif序列的功能鉴定

地衣芽胞杆菌是重要的工业菌株,如何为其建立一种有效的基因删除技术是对该菌株进行遗传改良的基础。枯草芽胞杆菌difB8序列已经被成功用于枯草芽胞杆菌多基因的删除。在分析地衣芽胞杆菌基因组序列并获得与枯草芽胞杆菌difB8以。序列十分相似的一段序列difBLi的基础上,构建了在庆大霉素抗性基因两侧具有difBLi的重组质粒pMD19-difGm和pHY-XI’：：difGm,通过电击转化法将质粒pHY-XI’：：difGm导入B．1icheniformis ATCC14580中,筛选获得了具有庆大霉素抗性的转化子。在转化子的传代过程中,重组质粒的庆大霉素抗性基因在体内Xer／dif／f位点特异性重组系统的介导下通过其侧翼的dif位点进行同源重组而被准确删除。确证了地衣芽胞杆菌中dif序列的功能,为地衣芽胞杆菌基因组中多基因的删除提供了一种新的实验途径。     

一种基于单交换原理的地衣芽孢杆菌基因敲除方法及应用

目的：基于同源单交换原理构建地衣芽孢杆菌基因快速敲除方法,提高基因敲除效率。方法：以地衣芽孢杆菌（Bacillus licheniformis）20085内切纤维素酶基因celb为拟敲除对象,利用重叠PCR技术将celb基因内约500bp片段与氯霉素抗性基因（Cm^r）相连接,经末端单酶切后电转化至B.licheniformis 20085感受态细胞中,仅通过一次同源单交换,将抗性基因Cm^r插入至celb基因内部,实现目的基因的敲除。结果：经过氯霉素抗性筛选和基因组PCR鉴定,成功获得celb基因缺失菌株B.licheniformis 20085Δcelb;发酵验证结果显示,B.licheniformis 20085Δcelb较原始菌株滤纸崩解能力显著降低,其中发酵60h后内切纤维素酶（CMC酶）活力由1.86U/ml降低至0.50U/ml,表明celb基因在地衣芽孢杆菌降解纤维素的过程中起着重要作用。结论：通过重叠PCR技术结合同源单交换原理能够实现地衣芽孢杆菌目的基因的快速敲除,为该菌株甚至其它微生物提供了一种基因功能快速鉴定的手段。     

酱香型白酒发酵中地衣芽孢杆菌与酿酒酵母的相互作用

【目的】为解析酱香型白酒酿造群体微生物的发酵过程, 研究了酱香型白酒酿造中重要微生物地衣芽孢杆菌与酿酒酵母之间的相互作用, 并对它们之间的作用机制进行初步探讨。【方法】通过地衣芽孢杆菌与酿酒酵母共培养体系的构建, 认识了两者的相互作用, 初步分析了酿酒酵母产生抑制物的分子量, 耐热性及对蛋白酶敏感性等特性。【结果】研究表明, 酿酒酵母发酵造成的酸性环境以及某些代谢物质能够抑制地衣芽孢杆菌的生长, 这些物质分子量大于10 kD, 对热和蛋白酶敏感。【结论】白酒酿造中酿酒酵母通过产酸以及大分子的蛋白质类物质对地衣芽孢杆菌生长形成抑制, 该研究促进了对白酒酿造群体微生物发酵过程的解析。     

Microorganisms associated with production lots of the nucleopolyhedrosis virus of the gypsy moth,Lymantria dispar [Lep.: Lymantriidae]

Samples of a gypsy moth nucleopolyhedrosis virus product, Gypchek^®, were taken each day during a 100-day production run and monitored for the presence of pathogenic bacteria and fungi. The standard plate count/g of product was 5.97±1.51×10⁸ over the 100-day period, while the sporulating bacteria count was 3.81±1.21×10⁶/g. We did not detect obligate anaerobic or fecal coliform bacteria in any of the samples.Bacillus cereus, Staphylococcus epidermidis, B. licheniformis, Streptococcus faecalis, Serratia liquefaciens, andAspergillus niger were the most frequently isolated microorganisms. We did not detect primary pathogenic bacteria or fungi, but the presence of opportunistic pathogens indicated that assiduous monitoring of the virus production facility and rigorous quality control of production batches are necessary.