The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth,biofilm formation and proteolytic activity in Trichosporon spp.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 μg ml^?1) inhibited Trichosporon growth. RIT (100 μg ml^?1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 μg ml^?1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These results highlight the importance of proteases as promising therapeutic targets and reinforce the antifungal potential of protease inhibitors.     

Recovery and purification of the lipopeptide biosurfactant of Bacillus subtilis by ultrafiltration

Surfactin, a lipopeptide biosurfactant produced as micelles by Bacillus subtilis, was recovered from the fermentation broth by ultrafiltration with a 30 kDa MWCO membrane. The retained surfactin micelles were then ruptured and collected in the permeate by adding methanol to 50% (v/v). The final yield of surfactin was 95%.     

Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis

Aims:  Antibiotics from Bacillus subtilis JA show strong pathogen inhibition ability, which has potential market application; yet, the composition of these antibiotics has not been elucidated. The aim of this paper is to isolate and identify these antibiotics.
Methods and Results:  The antagonistic activity of JA was tested in vitro ; it exhibited strong inhibition against some important phytopathogens and postharvest pathogens. Crude antibiotic production was extracted with methanol from the precipitate by adding 6 mol l⁻¹ HCl to the bacillus-free culture broth. The crude extract was run on Diamonsil C₁₈ column (5  μ m, 250 × 4·6 mm) in HPLC system to separate the antibiotics. Major antibiotics were classified into three lipopeptide families according to electrospray ionization–mass spectrometry analysis. Subsequently, the classification of antibiotics was confirmed with typical collision-induced dissociation fragments.
Conclusions:  Three kinds of antibiotics were isolated from B. subtilis JA and were identified to the lipopeptide families, surfactin, iturin and fengycin. These compounds could function as biocontrol agents against a large spectrum of pathogens.
Significance and Impact of the Study:  This study provided a reliable and rapid method for isolation and structural characterization of lipopeptide antibiotics from B. subtilis .     

Isolation, characterization, and investigation of surface and hemolytic activities of a lipopeptide biosurfactant produced by Bacillus subtilis ATCC 6633

Bacillus subtilis ATCC 6633 was grown in BHIB medium supplemented with Mn2+ for 96 h at 37 degrees C in a shaker incubator. After removing the microbial biomass, a lipopeptide biosurfactant was extracted from the supernatant. Its structure was established by chemical and spectroscopy methods. The structure was confirmed by physical properties, such as Hydrophile-Lipophile Balance (HLB), surface activity and erythrocyte hemolytic capacity. The critical micelle concentration (cmc) and erythrocyte hemolytic capacity of the biosurfactant were compared to those of surfactants such as SDS, BC (benzalkonium chloride), TTAB (tetradecyltrimethylammonium bromide) and HTAB (hexadecyltrimethylammonium bromide). The maximum hemolytic effect for all surfactants mentioned was observed at concentrations above cmc. The maximum hemolytic effect of synthetic surfactants was more than that of the biosurfactant produced by B. subtilis ATCC 6633. Therefore, biosurfactant would be considered a suitable surface-active agent due to low toxicity to the membrane.     

Cell-free biosynthesis of surfactin, a cyclic lipopeptide produced by Bacillus subtilis

The lipopeptide antibiotic surfactin is a potent extracellular biosurfactant produced by various Bacillus subtilis strains. Biosynthesis of surfactin was studied in a cell-free system prepared from B. subtilis ATCC 21332 and OKB 105, which is a transformant producing surfactin in high yield [Nakano, M. M., Marahiel, M. A., & Zuber, P. (1988) J. Bacteriol. 170, 5662-5668]. Cell material was disintegrated by treatment with lysozyme and French press. A cell-free extract was prepared by ammonium sulfate fractionation, which catalyzed the formation of surfactin at the expense of ATP. Lipopeptide biosynthesis required the L-amino acid components of surfactin and D-3-hydroxytetradecanoyl-coenzyme A thioester. D-Leucine which is present in surfactin was not utilized but inhibited the biosynthetic process. The structure of surfactin, synthesized enzymatically in vitro, was confirmed by chromatographic comparison with the authentic compound and by amino acid analyses. An enzyme fraction was prepared by gel permeation chromatography which catalyzed ATP/pyrophosphate exchange reactions dependent on the component amino acids of surfactin. This enzyme fraction was capable of binding substrate amino acids covalently, probably via thioester linkages. The formation of these intermediates was inhibited by various thiol blocking reagents and phenylmethanesulfonyl fluoride. De novo synthesis of the lipopeptide was not observed with this partially purified enzyme fraction most likely due to the lack of an acyltransferase activity required for linking the beta-hydroxy fatty acid to the peptide moiety.     

Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes

Fengycin is a biologically active lipopeptide produced by several Bacillus subtilis strains. The lipopeptide is known to develop antifungal activity against filamentous fungi and to have hemolytic activity 40-fold lower than that of surfactin, another lipopeptide produced by B. subtilis. The aim of this work is to use complementary biophysical techniques to reveal the mechanism of membrane perturbation by fengycin. These include: 1), the Langmuir trough technique in combination with Brewster angle microscopy to study the lipopeptide penetration into monolayers; 2), ellipsometry to investigate the adsorption of fengycin onto supported lipid bilayers; 3), differential scanning calorimetry to determine the thermotropic properties of lipid bilayers in the presence of fengycin; and 4), cryogenic transmission electron microscopy, which provides information on the structural organization of the lipid/lipopeptide system. From these experiments, the mechanism of fengycin action appears to be based on a two-state transition controlled by the lipopeptide concentration. One state is the monomeric, not deeply anchored and nonperturbing lipopeptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity. The mechanism, thus, appears to be driven mainly by the physicochemical properties of the lipopeptide, i.e., its amphiphilic character and affinity for lipid bilayers.     

枯草芽孢杆菌HS-A38产抗菌脂肽Bacillomycin D的组分分析及鉴定

对一株枯草芽孢杆菌HS-A38产生的脂肽类物质进行分离鉴定及抑菌活性研究。通过酸沉淀分离和有机溶剂抽提的方法,从枯草芽孢杆菌HS-A38发酵液中得到脂肽粗提物LP,产率为1.956 g/L。利用薄层色谱和茚三酮染色法确定该脂肽粗提物中存在四个组分,分别为LP1、LP2、LP3和LP4;抑菌活性检测显示,组分LP3对两株海洋致病菌副溶血性弧菌和铜绿假单胞杆菌的活性较高。组分LP3经硅胶柱层析纯化分离后,应用反相高效液相色谱(RP-HPLC)和基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)对该组分做进一步纯化和鉴定。分析表明,LP3样品在保留时间20 min~28 min产生单峰团LP3-1,其纯度为85.24%;经MALDI-TOF-MS分析和数据比对,组分LP3-1中的主要成分为杆菌霉素Bacillomycin D。     

阿萨希毛孢子菌的溶血活性及生物膜形成能力与其基因型关系研究

目的 分析临床分离自尿路感染患者的阿萨希毛孢子菌的体外溶血活性及生物膜形成能力与其基因型的关系,为临床诊治提供依据.方法 玻璃珠法提取总DNA,并采用PCR技术利用IGS1区特异性引物确定其基因型别;同时,平板法和XTT还原比色法分别检测阿萨希毛孢子菌的溶血活性及生物膜形成能力,并分析其与基因型的关系.结果 10株分离自尿路感染的阿萨希毛孢子菌基因型分别属于Ⅰ、Ⅲ、Ⅳ,其中以Ⅳ型为主;所分离菌株均具有不同程度的溶血活性;除1例分离株外,其余各分离株均具有在聚苯乙烯表面形成生物膜的能力;基因型Ⅲ型菌株具有较强的溶血活性和生物膜形成能力.结论 分离自尿路感染患者的10株阿萨希毛孢子菌以Ⅳ型为主,而其中Ⅲ型菌株表现出较强的溶血活性和生物膜形成能力.     

Characterization and antimicrobial properties of lipopeptide biosurfactants produced by Bacillus subtilis SJ301 and Bacillus vallismortis JB201

Biosurfactant producing bacteria, terrestrial Bacillus subtilis SJ301 and marine Bacillus vallismortis JB201 were isolated from sites contaminated with crude oil and its by-products. Cellular growth and biosurfactant production of the isolates were studied with different carbon sources (glucose, fructose, glycerol and petrol). Both bacterial isolates synthesized biosurfactants in the presence of glucose at late log phase and in the presence of petrol at stationary phase at 35°C. Biosurfactants obtained from both bacteria reduced the surface tension of the growth medium below 33 mN/m and exhibited this capacity in cell-free filtrates also. Raising the temperature from 25 to 35°C, accelerated onset of biosurfactant production in both the isolates, however, change in pH values from 6.5 to 7.5 had no effect. Functional and structural characterization of the crude biosurfactants was carried out by FTIR and ¹H and ¹³C NMR spectroscopy and the compounds were identified as surfactin lipopeptides. Biosurfactant produced by the terrestrial B. subtilis SJ301 showed antimicrobial activity against Escherichia coli and Shigella dysenteriae whereas the marine B. vallismortis JB201 revealed antimicrobial activity against Klebsiella pneumoniae, Salmonella typhi and Streptococcus pneumoniae.     

Potential application of cyclic lipopeptide biosurfactants produced by Bacillus subtilis strains in laundry detergent formulations

AIMS: Crude cyclic lipopeptide (CLP) biosurfactants from two Bacillus subtilis strains (DM-03 and DM-04) were studied for their compatibility and stability with some locally available commercial laundry detergents. METHODS AND RESULTS: CLP biosurfactants from both B. subtilis strains were stable over the pH range of 7.0-12.0, and heating them at 80 degrees C for 60 min did not result in any loss of their surface-active property. Crude CLP biosurfactants showed good emulsion formation capability with vegetable oils, and demonstrated excellent compatibility and stability with all the tested laundry detergents. CONCLUSION: CLP biosurfactants from B. subtilis strains act additively with other components of the detergents to further improve the wash quality of detergents. The thermal resistance and extreme alkaline pH stability of B. subtilis CLP biosurfactants favour their inclusion in laundry detergent formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has great significance because it is already known that microbial biosurfactants are considered safer alternative to chemical or synthetic surfactants owing to lower toxicity, ease of biodegradability and low ecological impact. The present study provides further evidence that CLP biosurfactants from B. subtilis strains can be employed in laundry detergents.     

Naphthalene and crude oil degradation by biosurfactant producing Streptomyces spp. isolated from Mitidja plain soil (North of Algeria)

Petroleum and naphthalene (example of PAHs) degrading Streptomyces spp. isolates AB1, AH4, and AM2 were recovered from surface soils at Mitidja plain (North of Algeria). The degradation efficiencies were examined by HPLC and GC–MS analysis and the results showed that the biosurfactant producing isolates AB1, AH4 and AM2 could remove 82.36%, 85.23% and 81.03% of naphthalene after 12 days of incubation, respectively. During naphthalene degradation, a slight decrease in pH values was recorded for the three studied strains. Degradation metabolites were identified using GC–MS analysis of ethyl acetate extracts of the cell free-culture. The metabolism of degradation proceeds via the phthalic acid pathway for the three strains. Moreover, the selected strains showed an important degradation of the aliphatic fraction present in crude oil after 30 days of incubation. The finding suggests that the selected strains are suitable candidates for practical field application for effective in situ bioremediation of hydrocarbon-contaminated sites.     

Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2.

Bacillus licheniformis JF-2 produces a very active biosurfactant under both aerobic and anaerobic conditions. We purified the surface-active compound to homogeneity by reverse-phase C18 high-performance liquid chromatography and showed that it is a lipopeptide with a molecular weight of 1,035. Amino acid analysis, fast atom mass and infrared spectroscopy, and, finally, 1H, 13C, and two-dimensional nuclear magnetic resonance demonstrated that the biosurfactant consists of a heterogeneous C15 fatty acid tail linked to a peptide moiety very similar to that of surfactin, a lipopeptide produced by Bacillus subtilis. Polyclonal antibodies were raised against surfactin and shown to exhibit identical reactivity towards purified JF-2 lipopeptide in competition enzyme-linked immunosorbent assays, thus providing further evidence for the structural similarity of these two compounds. Under optimal conditions, the B. licheniformis JF-2 biosurfactant exhibits a critical micelle concentration of 10 mg/liter and reduces the interfacial tension against decane to 6 x 10(-3) dyne/cm, which is one of the lowest interfacial tensions ever reported for a microbial surfactant.     

Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26

AIMS: To isolate an antagonist for use in the biological control of phytopathogenic fungi including Colletotrichum gloeosporioides, then to purify and characterize the biocontrol agent produced by the antagonist. METHODS AND RESULTS: Bacteria that exhibited antifungal activity against the causative agent pepper anthracnose were isolated from soil, with Bacillus thuringiensis CMB26 showing the strongest activity. A lipopeptide produced by B. thuringiensis CMB26 was precipitated by adjusting the pH 2 with 3 n HCl and extracted using chloroform/methanol (2:1, v/v) and reversed-phase HPLC. The molecular weight was estimated as 1447 Da by MALDI-TOF mass spectrometry. Scanning electron and optical microscopies showed that the lipopeptide has activity against Escherichia coli O157:ac88, larvae of the cabbage white butterfly (Pieris rapae crucivora) and phytopathogenic fungi. The lipopeptide had cyclic structure and the amino acid composition was L-Glu, D-Orn, L-Tyr, D-allo-Thr, D-Ala, D-Val, L-Pro, and L-Ile in a molar ratio of 3:1:2:1:1:2:1:1. The purified lipopeptide showed the same amino acid composition as fengycin, but differed slightly in fatty acid composition, in which the double bond was at carbons 13-14 (m/z 303, 316) and there was no methyl group. CONCLUSION: A lipopeptide was purified and characterized from B. thuringiensis CMB26 and found to be similar to the lipopeptide fengycin. This lipopeptide can function as a biocontrol agent, and exhibits fungicidal, bactericidal, and insecticidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Compared with surfactin and iturin, the lipopeptide from B. thuringiensis CMB26 showed stronger antifungal activity against phytopathogenic fungi. This lipopeptide is a candidate for the biocontrol of pathogens in agriculture.     

Ochrosin,a new biosurfactant produced by halophilic Ochrobactrum sp. strain BS-206 (MTCC 5720): Purification,characterization and its biological evaluation

A new bioactive molecule characterized as 4-dimethylaminobenzaldehyde (designated as Ochrosin) was isolated from a halophilic Ochrobactrum sp. strain BS-206 (MTCC 5720). Ochrosin exhibited good antimicrobial activity against both Gram-positive and Gram-negative bacteria and different Candida strains with minimum inhibitory concentration (MIC) values ranging from 4.68 to 150 μg mL⁻¹. It also exhibited anti-adhesive activity against various bacterial pathogens. Ochrosin caused an increase in the reactive oxygen species (ROS) levels to 32.4% and 28% and increased the NAD⁺/NADH ratio by 2.61 and 2.26 in S. aureus and E. coli, respectively. Ochrosin appears to interfere with ergosterol biosynthetic pathway in different Candida strains as it reduced the ergosterol content. Ochrosin showed insecticidal activity against three major stored-product grain pests like Tribolium castaneum, Callosobruchus chinensis and Sitophilus oryzae with LC₅₀ values of 7.3, 8.9 and 7.6 μg cc⁻¹, respectively. The anti-feedant index (ED₅₀) values observed were 1.07 and 1.35 μg cm⁻² of leaf area against Achaea janata and Spodoptera litura, respectively. This is the first report on Ochrosin, a multifunctional biosurfactant, produced by Ochrobactrum sp. BS-206 exhibiting promising antimicrobial, anti-adhesive, anti-feedant and insecticidal activities.     

Cytotoxicity of listeriolysin O produced by membrane-encapsulated Bacillus subtilis on leukemia cells

Encapsulation of biological material in the permiselective membrane allows to construct a system separating cells from their products, which may find biotechnological as well as biomedical applications in biological processes regulation. Application of a permiselective membrane allows avoiding an attack of the implanted microorganisms on the host. Our aim was to evaluate the performance of Bacillus subtilis encapsulated in an elaborate membrane system producing listeriolysin O, a cytolysin from Listeria monocytogenes, with chosen eukaryotic cells for future application in anticancer treatment. The system of encapsulating in membrane live Bacillus subtilis BR1-S secreting listeriolysin O was proven to exert the effective cytotoxic activity on eukaryotic cells. Interestingly, listeriolysin O showed selective cytotoxic activity on eukaryotic cells: more human leukemia Jurkat T cells were killed than human chronic lymphocytic B cells leukemia at similar conditions in vitro. This system of encapsulated B. subtilis, continuously releasing bacterial products, may affect selectively different types of cells and may have future application in local anticancer treatment.     

Production of biosurfactant at mesophilic and thermophilic conditions by a strain of Bacillus subtilis

The ability of a Bacillus subtilis strain to grow and produce biosurfactant on different carbon and nitrogen sources under thermophilic conditions (45°C) was studied. The strain was able to reduce surface tension to 34 dynes cm⁻¹ on 2% sucrose, and 32 dynes cm⁻¹ on starch after 96 h of growth. The biosurfactant was stable at 100°C and within a wide pH range (3.0–11.0). Biosurfactant formation at mesophilic conditions (30°C) was also studied. The organism was able to produce the maximum amount of biosurfactant when nitrate ions were supplied as the nitrogen source. The potential application of the biosurfactant in oil recovery from desert oil fields, acidic and alkaline environments is demonstrated. The biosurfactant was identical to surfactin as confirmed by TLC and IR analysis. Received 29 May 1997/ Accepted in revised form 03 October 1997     

Inhibitory activity of surfactin,produced by different Bacillus subtilis subsp. subtilis strains,against Listeria monocytogenes sensitive and bacteriocin-resistant strains

Three surfactin-producing Bacillus subtilis strains, C4, M1 and G2III, previously isolated from honey and intestines from the Apis mellifera L. bee, were phylogenetically characterized at sub-species level as B. subtilis subsp. subtilis using gyrA gene sequencing. The antagonistic effect of surfactin was studied against seven different Listeria monocytogenes strains, 6 of which were resistant to bacteriocins. Surfactin showed anti-Listeria activity against all 7 strains and a dose of 0.125 mg/mL of surfactin was enough to inhibit this pathogen. Surfactin sintetized by B. subtilis subsp. subtilis C4 inhibited the pathogen in lower concentrations, 0.125 mg/mL, followed by G2III and M1 with 0.25 and 1 mg/mL, respectively. In particular, a dose of 0.125 mg/mL reduced the viability of L. monocytogenes 99/287 RB6, a bacteriocin-resistant strain, to 5 log orders. Surfactin assayed maintained anti-Listeria activity within a pH range of between 2 and 10, after heat treatment (boiling for 10 min and autoclaving at 121 °C for 15 min) and after treatment with proteolytic enzymes. These results suggest that surfactin can be used as a new tool for prevention and the control of L. monocytogenes in different environments, for example, in the food industry.