The antimicrobial properties of thermophilic bacilli

Lytic activity and antagonistic properties of 11 thermophilic strains in different species of the genus Bacillus have been studied. It is shown that the spectra of lytic and antagonistic effect of the studied thermophiles do not coincide: filtrates of cultural liquids of these strains implement the degradation of living cells of Gram-negative bacteria and yeast but inhibit the growth of Gram-positive bacteria. Both the intensities of lytic and antagonistic actions of the extracellular products of the studied strains of bacilli do not correlate. An analysis of the obtained data shows that the lysis of test cultures takes place as a result of degrading action of extracellular hydrolases of thermophiles but not as a result of activation of autolytic systems of test cultures by the substances of antibiotic nature synthesized by the studied sporeforming bacteria.     

The antibacterial effect of river water onShigella Shigae in connection with the presence of corresponding antagonists and bacteriophages

Summary and conclusions It is emphasized that in the Tjiliwung river water no antagonists against the occurring typhoid bacteria are to be detected. Bacterial antagonists against dysentery bacteria can, however, easily be isolated, whereas the latter could not be isolated from the water by means of S.S. medium orLeifson plates. This elicited the question whether antibiotic substances in nature might cause a detrimental effect onShigella shigae.From 2054 colonies of water bacteria on broth agar 294 strains with an antagonistic effect on shigae bacteria were cultivated with methods based on the agar streak, the perforated Petri dish and that ofKelner. Among 3419 other colonies isolated from the same water samples no antagonists against typhoid bacteria could be detected. The method ofKelner proved 14% of the water bacteria growing on broth agar to be antagonistic against shigae bacteria. Some of these antagonists lost their activity soon after subinoculation on broth agar.In 14 out of 16 samples of Tjiliwung water a filtrable thermolabile agent could be detected, detrimental toSh. shigae when inoculated fairly profusely and in the presence of organic material (extract broth 1 : 10). The filtrated agent reduced the survival period ofSh. shigae to a maximum of 1/360 of the test period and inhibited the growth of these bacteria up to a dilution of 1 : 256. Heating at 100°C. destroyed the agent. In all cases this thermolabile anti-bacterial factor was associated with the presence of shigae phage and did not occur in two samples lacking the latter. The agent could not be separated from the phage particles by means of phage-tight glacial acid collodion filters through which human hemoglobine passes easily. The anti-shigae factor acted as a bacteriophage, not as an antibiotic. Propagation of the phage in three successive cultures could be demonstrated.No anti-typhoid agent was found in the filtrates lacking typhoid phages. It is concluded that the unheated river water filtrates owe their anti-shigae action mainly to the presence of corresponding phages. It is possible that in nature other factors may change the influence of the shigae-phage. Anti-bacterial substances produced by the numerous antagonists present are of no importance for the reduction in number of shigae bacteria in the river water.     

The ecological role of killer yeasts in natural communities of yeasts

The killer phenomenon of yeasts was investigated in naturally occurring yeast communities. Yeast species from communities associated with the decaying stems and fruits of cactus and the slime fluxes of trees were studied for production of killer toxins and sensitivity to killer toxins produced by other yeasts. Yeasts found in decaying fruits showed the highest incidence of killing activity (30/112), while yeasts isolated from cactus necroses and tree fluxes showed lower activity (70/699 and 11/140, respectively). Cross-reaction studies indicated that few killer-sensitive interactions occur within the same habitat at a particular time and locality, but that killer-sensitive reactions occur more frequently among yeasts from different localities and habitats. The conditions that should be optimal for killer activity were found in fruits and young rots of Opuntia cladodes where the pH is low. The fruit habitat appears to favor the establishment of killer species. Killer toxin may affect the natural distribution of the killer yeast Pichia kluyveri and the sensitive yeast Cryptococcus cereanus. Their distributions indicate that the toxin produced by P. kluyveri limits the occurrence of Cr. cereanus in fruit and Opuntia pads. In general most communities have only one killer species. Sensitive strains are more widespread than killer strains and few species appear to be immune to all toxins. Genetic study of the killer yeast P. kluyveri indicates that the mode of inheritance of killer toxin production is nuclear and not cytoplasmic as is found in Saccharomyces cerevisiae and Kluyveromyces lactis.     

Selection of Antagonistic Fluorescent Pseudomonas spp. and their Root Colonization and Persistence following Treatment of Seed Potatoes

One hundred and twelve fluorescent Pseudomonas isolates from potato periderm were screened for antagonistic activity in vitro against 5 pathogenic and saprophytic fungi, 5 Gram-positive bacteria including Streptomyces scabies and 4 Gram-negative bacteria including Erwinia car . var. carotovora and Erwinia car . var. atroseptica . Fifty-seven percent of the isolates showed wide spectrum inhibitory activity, mostly due to the production of fluorescent siderophores. Only 4 % of the isolates were very strong antagonists. By adding iron (Fe³+) to the test medium, the antagonistic activity drastically diminished but revealed some strong antagonists of which the antagonism was not affected. Apparently growth-inhibiting substances other than siderophores were involved. Antibiotic-resistant mutants of the antagonists were used to establish that the bacteria on the seed tubers migrated to the roots under different conditions. Drying of the treated potatoes gradually diminished the number of viable cells, but subsequent planting in moist soil led to substantial increases in the first three days. Initially high numbers of the antibiotic-resistant antagonists on the roots tended to drop quickly in time, probably due to loss of resistance to the antibiotics in the selective medium.     

Diversity of antagonistic bacteria isolated from medicinal plant Peganum harmala L.

The antimicrobial activity of plant extract of Peganum harmala, a medicinal plant has been studied already. However, knowledge about bacterial diversity associated with different parts of host plant antagonistic to different human pathogenic bacteria is limited. In this study, bacteria were isolated from root, leaf and fruit of plant. Among 188 bacterial isolates isolated from different parts of the plant only 24 were found to be active against different pathogenic bacteria i.e. Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium, Enterococcus faecalis and Pseudomonas aeruginosa. These active bacterial isolates were identified on the basis of 16S rRNA gene analysis. Total population of bacteria isolated from plant was high in root, following leaf and fruit. Antagonistic bacteria were also more abundant in root as compared to leaf and fruit. Two isolates (EA5 and EA18) exhibited antagonistic activity against most of the targeted pathogenic bacteria mentioned above. Some isolates showed strong inhibition for one targeted pathogenic bacterium while weak or no inhibition for others. Most of the antagonistic isolates were active against MRSA, following E. faecium, P. aeruginosa, E. coli and E. faecalis. Taken together, our results show that medicinal plants are good source of antagonistic bacteria having inhibitory effect against clinical bacterial pathogens.     

Production,purification, and characterization of a novel killer toxin from <Emphasis Type="BoldItalic">Kluyveromyces siamensis</Emphasis> against a pathogenic yeast in crab

The yeast Kluyveromyces siamensis HN12-1 isolated from mangrove ecosystem was found to be able to produce killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab. When the killer yeast was grown in the medium with pH 4.0 and 0.5% NaCl and at 25 °C, it could produce the highest amount of killer toxin against the pathogenic yeast M. bicuspidata WCY. The killing activity of the purified killer toxin against the pathogenic yeast M. bicuspidata WCY was the highest when it was incubated at 25 °C in the assay medium without added NaCl and pH 4.0. The molecular weight of the purified killer toxin was 66.4 kDa. The killer toxin produced by the yeast strain HN12-1 could kill only the whole cells of M. bicuspidata WCY among all the yeast species tested in this study. This is the first time to report that the killer toxin produced by the yeast K. siamensis HN12-1 isolated from the mangrove ecosystem only killed pathogenic yeast M. bicuspidata WCY.     

Natural wine yeasts as biocontrol agents

A total of 586 natural wine yeasts, belonging to different genera, were tested for their antagonistic effect on fungal pathogens. A low percentage of yeast strains completely inhibited the pathogens and the biocontrol activity was found to be a strain characteristic and did not solely depend on species or genus. Among the antagonists, two strains of Saccharomyces cerevisiae and one of Zygosaccharomyces showed a broad spectrum of antagonistic activity against 10 fungal pathogens.     

地锦草内生细菌多样性、拮抗及促生特性测定

【背景】植物内生细菌既能抑制病原菌对植物的侵染,也具有促生作用。分离具有拮抗和促生活性的内生细菌可为开发微生物菌肥提供理论依据。【目的】筛选内生细菌中的优势拮抗、促生菌种资源。【方法】以地锦草为材料,采用4种分离培养基分离该植物内生细菌,通过形态特征以及16S r RNA基因序列分析,鉴定内生细菌的分类归属。采用平板对峙法,测定内生细菌对棉花立枯丝核菌(Rhizoctonia solani)、小麦赤霉病菌(Fusarium graminearum)、玉米小斑病菌(Bipolaris maydis)的拮抗活性。通过固氮、解磷、产吲哚乙酸(Indole Acetic Acid,IAA)、产铁载体能力等指标初步检测地锦草内生细菌的促生活性。【结果】共分离到133株内生细菌,分属于4门5纲8目13科25属,其中变形菌门(Proteobacteria)为优势门(52.63%),优势属为芽孢杆菌属(Bacillus),占15.79%。发现有8株菌相似性小于98.65%,可能为潜在新物种。拮抗活性结果表明,22株菌有不同程度的抑菌作用,其中菌株DHL56、DHN17、DHP3、DHP8对这3种病原菌...     

Purification, characterization and gene cloning of the killer toxin produced by the marine-derived yeast Williopsis saturnus WC91-2

As the killer toxin produced by Williopsis saturnus WC91-2 could kill many sensitive yeast strains, including the pathogenic ones, the extracellular killer toxin in the supernatant of cell culture of the marine yeast strain was purified and characterized. The molecular mass of the purified killer toxin was estimated to be 11.0kDa according to the data from SDS-PAGE. The purified killer toxin had killing activity, but could not hydrolyze laminarin. The optimal conditions for action of the purified killer toxin against the pathogenic yeast Metschnikowia bicuspidate WCY were the assay medium with 10% NaCl, pH 3-3.5 and temperature 16°C. The gene encoding the killer toxin from the marine killer yeast WC91-2 was cloned and the ORF of the gene was 378bp. The deduced protein from the cloned gene encoding the killer toxin had 125 amino acids with calculated molecular weight of 11.6kDa. It was also found that the N-terminal amino acid sequence of the purified killer toxin had the same corresponding sequence deduced from the cloned killer toxin gene in this marine yeast, confirming that the purified killer toxin was indeed encoded by the cloned gene.     

Protective antifungal yeast killer toxin-like antibodies

After several years of controversy, antibodies (Abs) are now believed to play an important role in the protection against fungal infections. Among them, recent data are strongly supporting the relevance of protective yeast killer toxin-like Abs ("antibiobodies", KT-Abs), which are able to exert a direct microbicidal activity by mimicking a killer toxin (PaKT) and its interaction with cell wall receptors on susceptible cells essentially constituted by beta-glucans. This review will focus on the implications of the yeast killer phenomenon, and, particularly, the occurrence and antimicrobial activity of protective antifungal KT-Abs, such as those produced during the course of experimental and natural infections caused by PaKT-sensitive microorganisms or produced by idiotypic vaccination with a PaKT-neutralizing mAb. The strong therapeutic activity exerted against different experimental mucosal and systemic mycoses by monoclonal and recombinant microbicidal KT-Abs (either in their soluble forms or expressed on human commensal bacteria) as well as by a synthetic killer peptide (KP, an antibody fragment engineered from the sequence of a recombinant KT-Ab) will be discussed. The surprisingly wide antimicrobial spectrum of activity against eukaryotic and prokaryotic pathogenic agents, such as fungi, bacteria and protozoa, of these Abs and Ab-derived molecules suggests new potential strategies for transdisease anti-infective prevention and therapy.     

Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards VERTICILLIUM: The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.     

Marine yeasts as biocontrol agents and producers of bio-products

As some species of marine yeasts can colonize intestine of marine animals, they can be used as probiotics. It has been reported that β-glucans from marine yeast cells can be utilized as immuno-stimulants in marine animals. Some siderophores or killer toxins produced by marine yeasts have ability to inhibit growth of pathogenic bacteria or kill pathogenic yeasts in marine animals. The virulent factors from marine pathogens can be genetically displayed on marine yeast cells, and the yeast cells displaying the virulent factors can stimulate marine animals to produce specific antibody against the pathogens. Some marine yeast cells are rich in proteins and essential amino acids and can be used in nutrition for marine animals. The marine yeast cells rich in lipid can be used for biodiesel production. Recently, it has been reported that some strains of Yarrowia lipolytica isolated from marine environments can produce nanoparticles. Because many marine yeasts can remove organic pollutants and heavy metals, they can be applied to remediation of marine environments. It has been shown that the enzymes produced by some marine yeasts have many unique properties and many potential applications.     

Production of a novel and cold-active killer toxin by Mrakia frigida 2E00797 isolated from sea sediment in Antarctica

The psychrotolerant yeast Mrakia frigida 2E00797 isolated from sea sediment in Antarctica was found to be able to produce killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab. When the psychrotolerant yeast was grown in the medium with pH 4.5 and 3.0% (wt/vol) NaCl and at 15°C, it could produce the highest amount of killer toxin against the pathogenic yeast M. bicuspidata WCY. The crude killer toxin activity against the pathogenic yeast M. bicuspidata WCY was the highest when it grew at 15°C in the assay medium with 3.0% (wt/vol) NaCl and pH 4.5. At temperatures higher than 25°C, the killing activity produced by M. frigida 2E00797 was completely lost and after the crude killer toxin was pre-incubated at temperatures higher than 40°C for 4 h, the killing activity was also completely lost. The killer toxin produced by M. frigida 2E00797 could kill only M. bicuspidata WCY, Candida tropicalis and Candida albicans among all the fungal species and bacterial species tested in this study.     

Antagonism of Bacillus spp. Isolated from Marine Biofilms Against Terrestrial Phytopathogenic Fungi

We aimed at determining the antagonistic behavior of bacteria derived from marine biofilms against terrestrial phytopathogenic fungi. Some bacteria closely related to Bacillus mojavensis (three isolates) and Bacillus firmus (one isolate) displayed antagonistic activity against Colletotrichum gloeosporioides ATCC 42374, selected as first screen organism. The four isolates were further quantitatively tested against C. gloeosporioides, Colletotrichum fragariae, and Fusarium oxysporum on two culture media, potato dextrose agar (PDA) and a marine medium-based agar [yeast extract agar (YEA)] at different times of growth of the antagonists (early, co-inoculation with the pathogen and late). Overall antagonistic assays showed differential susceptibility among the pathogens as a function of the type of culture media and time of colonization (P < 0.05). In general, higher suppressive activities were recorded for assays performed on YEA than on PDA; and also when the antagonists were allowed to grow 24 h earlier than the pathogen. F. oxysporum was the most resistant fungus while the most sensitive was C. gloeosporioides ATCC 42374. Significant differences in antagonistic activity (P < 0.05) were found between the different isolates. In general, Bacillus sp. MC3B-22 displayed a greater antagonistic effect than the commercial biocontrol strain Bacillus subtilis G03 (Kodiak). Further incubation studies and scanning electronic microscopy revealed that Bacillus sp. MC3B-22 was able to colonize, multiply, and inhibit C. gloeosporioides ATCC 42374 when tested in a mango leaf assay, showing its potential for fungal biocontrol. Additional studies are required to definitively identify the active isolates and to determine their mode of antifungal action, safety, and biocompatibility.     

Marine killer yeasts active against a yeast strain pathogenic to crab Portunus trituberculatus

Some marine yeasts have recently been recognised as pathogenic agents in crab mariculture, but may be inhibited or killed by 'killer' yeast strains. We screened multiple yeast strains from seawater, sediments, mud of salterns, guts of marine fish, and marine algae for killer activity against the yeast Metchnikowia bicuspidata WCY (pathogenic to crab Portunus trituberculatus), and found 17 strains which could secrete toxin onto the medium and kill the pathogenic yeast. Of these, 5 strains had significantly higher killing activity than the others; routine identification and molecular methods showed that these were Williopsis saturnus WC91-2, Pichia guilliermondii GZ1, Pichia anomala YF07b, Debaryomyces hansenii hcx-1 and Aureobasidium pullulans HN2.3. We found that the optimal conditions for killer toxin production and action of killer toxin produced by the marine killer yeasts were not all in agreement with those of marine environments and for crab cultivation. We found that the killer toxins produced by the killer yeast strains could kill other yeasts in addition to the pathogenic yeast, and NaCl concentration in the medium could change killing activity spectra. All the crude killer toxins produced could hydrolyze laminarin and the hydrolysis end products were monosaccharides.     

Application of Bacillus-antagonists for biocontrol of fungi degrading raw wood

Species composition of micromycete complexes colonizing aspen, birch, and pine wood was studied. Calculation of the Sorens-Tchekanovsky similarity coefficients showed that these complexes shared a high degree of similarity. They were dominated by the representatives of the genera Penicillium, Paecilomyces, Trichoderma, and Rhizopus. Some antagonistic bacilli inhibited growth and development of wood-decay fungi in vitro and led to the formation of spheroplasts on growing hyphae. A study of possible involvement of bacillary mycolytic enzymes in the inhibition of wood-decay fungi demonstrated selectivity of their lytic effect, which was determined by the genus and species of micromycetes and did not correlate with their relative resistance to antagonists.     

Occurrence of killer yeasts in leaf-cutting ant nests

Killer activity was screened in 99 yeast strains isolated from the nests of the leaf-cutting antAtta sexdens against 6 standard sensitive strains, as well as against each other. Among this yeast community killer activity was widespread since 77, strains (78%) were able to kill or inhibit the growth of at least one standard strain or nest strain. Toxin production was observed in representatives of all the studied genera includingAureobasidium, Rhodotorula, Tremella andTrichosporon, whose killer activity has not yet been described.     

Involvement of [beta]-glucans in the wide-spectrum antimicrobial activity of Williopsis saturnus var. mrakii MUCL 41968 killer toxin

BACKGROUND: Williopsis saturnus var. mrakii MUCL 41968 secretes a 85-kDa glycoprotein killer toxin (WmKT) that displays a cytocidal activity against a wide range of microorganisms, making WmKT a promising candidate for the development of new antimicrobial molecules. Although the killing mechanism of WmKT is still unknown, the toxin was recently proposed to bind to the surface of sensitive microorganisms through the recognition of beta-glucans. Indeed, Saccharomyces cerevisiae strains sensitive to the toxin become resistant when mutated in their beta-glucan synthesis pathway. MATERIALS AND METHODS: To investigate the interaction of WmKT with beta-glucans, we examined in agar diffusion assays the WmKT activity in the presence of enzymes displaying beta-glucanase activity. The toxin activity was also investigated using spheroplasts derived from sensitive yeast cells. The hydrolytic activity of WmKT was studied using specific glucosidase inhibitors as well as various sugar molecules covalently linked to p-nitrophenyl as potential substrates. Finally, the ultrastructural modifications induced by WmKT activity on sensitive yeasts were assessed by scanning electron microscopy. RESULTS: The data reported here support the hypothesis that WmKT binds to sensitive cells using surface-exposed beta-glucans. Indeed beta-glucanase exerts an antagonistic effect on WmKT activity and spheroplasts derived from WmKT-sensitive yeast cells are shown to be resistant to WmKT, suggesting that cell wall beta-glucans are required for WmKT lethal effect. Because WmKT exhibits amino acid sequence similarities with proteins suspected to be glucanase, we also investigated the effect of castanospermine, a potent glucosidase inhibitor, on WmKT activity. Castanospermine completely abolished WmKT killer activity as well as its hydrolytic enzymatic activity against p-nitrophenyl beta-D-glucopyranoside. The scanning electron microscopy analysis of sensitive yeast cells treated with the toxin reveals that WmKT causes cell wall modifications similar to those observed with zymolyase. CONCLUSION: The results reported in this study show that WmKT activity requires an interaction between the mycocin and the cell wall beta-glucans. Moreover, they indicate that WmKT acts on sensitive yeast cells through a hydrolytic activity directed against cell wall beta-glucans that disrupts the yeast cell wall integrity leading to death.     

A marine killer yeast against the pathogenic yeast strain in crab (Portunus trituberculatus) and an optimization of the toxin production

A pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculatus was identified to be Metschnikowia bicuspidate according to the results of routine yeast identification and 18S rDNA and ITS sequences. After screening of more than 300 yeast strains from different sources in marine environments, it was found that strain YF07b had the highest ability to produce killer toxin against the pathogenic yeast. Strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The optimal conditions for killer toxin production by strain YF07b were the production medium with 2.0% NaCl, pH 4.5, cultivation temperature of 20 degrees C and the optimal conditions for action of the crude killer toxin against the pathogenic yeast were the assay medium with 6.0% NaCl, pH 4.5 and temperature 15 degrees C.     

α-溶血性链球菌在健康人群口咽部分布及生物拮抗作用

目的 通过采集健康人群口咽部分泌物,分析上呼吸道中α-溶血性链球菌的分布状况,并对革兰阳性化脓性球菌进行生物拮抗试验,为进一步研究上呼吸道益生菌提供理论基础。方法 随机自愿原则,用无菌咽拭子采集沈阳市年龄在3～75岁的300名健康人群咽后壁分泌物,对α-溶血性链球菌进行鉴定和定量分析。对致病菌的生物拮抗试验采用小缸杯法。结果 定量分析显示不同年龄人群咽后壁的α-溶血性链球菌检出率均较高。在咽后壁菌群中α-溶血性链球菌构成比最多的是幼儿组,达到60.3%。其中唾液链球菌群在幼儿组所占比重最大;老年组人群格氏链球菌占比较大;儿童、青年、成人以缓症链球菌和口腔链球菌为主。对革兰阳性化脓性球菌的生物拮抗试验显示,1株婴儿链球菌婴儿亚种能够拮抗8株致病菌;4株分离菌只能拮抗1株病原菌,提示不同的菌株拮抗病原菌的能力差异较大。结论 α-溶血性链球菌在人群中分布广,数量多,不同年龄人群的菌群构成存在差异。并且某些菌株显示出对致病菌较强的生物拮抗作用,推测这些菌株在呼吸道黏膜保护中起到重要作用,可作为上呼吸道益生菌的备选菌株。