Evaluation of a selective medium for Brucella isolation using natamycin

AIMS: To select an anti-fungal agent to replace cycloheximide in the media used for isolation of Brucella. METHODS AND RESULTS: One potential agent, natamycin, was evaluated using 28 Brucella isolates, 18 yeasts and 14 fungi. The material for the evaluation included 37 bovine milk samples, six bovine vaginal swabs and 45 milk samples artificially infected with Brucella. The recovery of Brucella only from the artificially-inoculated milk samples increased with the use of the medium containing natamycin instead of cycloheximide, at the same time significantly inhibiting the growth of yeasts, fungi and other bacteria. The inclusion of either anti-fungal agent allowed growth of the 28 Brucella isolates and totally prevented the growth of all 18 yeasts and 13 of the 14 fungi. CONCLUSIONS: Based on the results it was concluded that natamycin would be a suitable alternative to cycloheximide. SIGNIFICANCE AND IMPACT OF THE STUDY: Cycloheximide has become unavailable worldwide and is currently an anti-fungal constituent of the medium often used for isolation of Brucella organisms. The use of natamycin as a replacement in the formulation did not inhibit growth of Brucella and was effective at eliminating most contaminants.     

Effect of natamycin on the enumeration, genetic structure and composition of bacterial community isolated from soils and soybean rhizosphere

Natamycin is commonly used to control fungal growth on agar media used for bacterial enumeration or strain isolation. However, there is no conclusive report on the possible effect of this antibiotic on bacterial growth or on the diversity of the recovered soil bacteria. Therefore, the possible effects of natamycin on the numbers of bacteria isolated at 12 degrees C from three different soils and soybean rhizosphere soil were investigated using natamycin concentrations ranging from 0 to 200 mg l(-1). Our results demonstrate that natamycin concentrations, which inhibit the growth of fungi on the media, have a small but significant inhibitory effect on the number of bacterial colony forming units. A natamycin concentration of 50-200 mg l(-1) is required for an efficient control of fungal growth on media in our experimental conditions depending on the soil type. Bacterial community structure was assessed on culturable cells (cells washed from enumeration plates: plate-wash approach) obtained at 12 degrees C from soybean rhizosphere soil by performing Ribosomal Intergenic Spacer Analysis (RISA) fingerprinting. We demonstrate that all natamycin concentrations used alter the structure of the recovered, culturable bacterial community, compared to control without natamycin. Using ARDRA (amplification of the 16S rDNA gene and restriction analysis) genotyping of individual isolates, some differences were observed between the bacterial isolates obtained in the presence or absence of natamycin. Bacterial isolates recovered in the presence of natamycin are more tolerant (maximal growth rate and lag phase) to this compound than those isolated without natamycin, indicating a possible selection of resistant strains. Therefore, high concentration of natamycin cannot be used for isolation of bacterial strains with the aim of studying biodiversity and could bias a selection of strains for practical applications.     

Cycloheximide Insensitive Amoebo-Flagellate Transformation in Starved Amoebae of Physarum polycephalum

The requirement of protein synthesis for amoebo-flagellate transformation of Physarum polycephalum was re-examined. When amoebae were grown on nutrient agar in association with live food bacteria and harvested in mid-exponential phase of growth, it took ca. 2 hours for half the cells to form flagella after suspension in phosphate buffer. The transformation was completely inhibited by 5 μg/ml cycloheximide. To the contrary, when the amoebae in mid-exponential phase were starved for 3 hr on non-nutrient agar and then suspended in phosphate buffer, the duration required for this process was shortened to ca. 8 min and it was not inhibited by up to 100 μg/ml cycloheximide. A similar result was obtained using bactobolin, another inhibitor of protein synthesis. When amoebae were starved on non-nutrient agar containing 5 μg/ml cycloheximide, however, the starvation effect described above was not observed. The results indicate that protein(s) necessary for the transformation might be synthesized during the starvation period, and that the amoebo-flagellate transformation may or may not require concomitant protein synthesis depending upon preculture conditions.     

Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37°C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H₂SO₄ both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5·5 resulted in lower mycobacterial counts than egg media at pH 6·5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green–cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6·5.     

GROWTH RESPONSE OF SALMONELLA SPP. TO CYCLOHEXIMIDE AMENDMENT IN MEDIA

The present study was designed to evaluate cycloheximide as a potential media amendment to prevent fungal overgrowth on selective media for salmonellae enumeration. The objectives were to determine the effect of cycloheximide on Salmonella spp growth rates and to determine the effect of cycloheximide addition on Salmonella enumeration in selective media. The bacteria tested included two strains of Salmonella typhimurium (NO/NA and LT2) and one strain of Salmonella arizonae. All strains were grown in tryptic soy broth containing cycloheximide to determine the effect of cycloheximide on bacterial specific growth rates. The growth rate of all strains grown in tryptic soy broth were not significantly influenced by addition of cycloheximide at concentrations up to 1,000 mg/L. Growth rates of S. typhimurium NO/NA in minimal media were significantly decreased by addition of cycloheximide aerobically (300 mg/L) and anaerobically (600 mg/L). However, S. typhimurium NO/NA populations on brilliant green agar, MacConkey agar, and from selenite cysteine broth and tetrathionate broth were not affected by cycloheximide additions at concentrations up to 1,000 mg/L. Cycloheximide has potential as a fungistat additive for salmonellae selective media.     

ABC转运蛋白SgnA/B促进纳他霉素胞外转运与高产

纳他霉素是一种天然、广谱、高效的多烯大环内酯类还原性抗真菌剂,广泛应用于食品真菌污染的防治和临床真菌感染的治疗。纳他霉素胞外转运效率可能是限制褐黄孢链霉菌(Streptomyces gilvosporeus)发酵高产纳他霉素的重要因素。通过生物信息学及分子对接技术分析纳他霉素胞外转运蛋白SgnA/B,发现SgnA和SgnB两个异源二聚体组成的ABC转运蛋白是内向开口构象的转运蛋白,且2个结合位点与纳他霉素结合能力有强弱差异,更有利于纳他霉素的胞外转运。本研究以纳他霉素生产菌株——褐黄孢链霉菌F607为出发菌株,构建了sgnA/B基因超表达菌株F-EX,以分析sgn A/B基因超表达对纳他霉素合成及胞外转运的影响。研究发现,纳他霉素对数合成期的F-EX菌株不仅提高了纳他霉素胞外/胞内比,其120 h发酵总产量也提高了12.5%,达到7.38 g/L。最后,通过转录组测序发现,sgnA/B基因超表达除提高纳他霉素胞外转运效率外,还影响了与多种氨基酸、丙酸盐、糖、五碳化合物代谢和TCA循环相关基因的表达。研究表明,强化纳他霉素胞外转运有利于纳他霉素的合成,是提高褐黄孢链霉菌纳他霉素产量的有效...     

Biosynthesis regulation of natamycin production from Streptomyces natalensis HDMNTE-01 enhanced by response surface methodology

Natamycin has been widely applied in medical treatments and food protection widely due to its effective inhibition to the growth of yeast and mold. As polyene macrolide antibiotic, the biosynthesis pathway of natamycin is relatively clear. To regulate the biosynthesis of natamycin, additions of precursors affecting cell growth and natamycin production were investigated. The results showed that 0.003% (w/v) potassium ferrocyanide and sodium propionate: n-butanol at a ratio of 4:1 was added into the broth at 0 and 24?hr, respectively, and they contributed to yield natamycin, reaching 1.62?g?L^?1 (174.6% higher than control). Furthermore, response surface methodology was undertaken to enhance natamycin production by Streptomyces natalensis HDMNTE-01 (a wild strain). The optimum conditions determined were: glucose 3.97%; soya peptone 2%; yeast extract 0.5%; original pH 7.0; inoculum volume 6%; growth in a 250-mL flask containing 24.68?mL of medium; shaken (220?rpm) at 28°C for 4 days. Under the optimized conditions, the yield was 2.81?g?L^?1 natamycin in 5-L fermentor when the fermentation was processed.     

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.     

Solubility properties and diffusional extraction behavior of natamycin from streptomyces gilvosporeus biomass

Natamycin is a type of polyene macrolide antibiotic and has been produced in submerged microbial cultures of some natural Streptomyces strains. Natamycin extraction from cellular biomass is greatly affected by the molecular and solubilization characteristics of the extraction solvent, and this is a major reason for the routine attainment of low volumetric titers, resulting from sparing natamycin solubility. In this work, a series of experiments were conducted to investigate the solubility of natamycin in some selected organic solvents in order to assess the influence on natamycin extraction yield. Natamycin showed the highest solubility in 75% aqueous methanol under the conditions of pH 2, 30°C and 1 atm. Furthermore, the extraction of natamycin using 75% aqueous methanol was performed and the highest extraction yield of 45.7% was obtained under pH 2. A mathematical model derived from Fick's law of the biomolecular diffusion process was developed to fit the experimental kinetic data of natamycin extraction. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb)

Oxygen deficiency is a critical factor during the fermentation production of natamycin. In order to alleviate oxygen limitation and enhance the yield of natamycin, the vgb gene, encoding Vitreoscilla hemoglobin (VHb) was inserted into pSET152 with its native promoter and integrated into the chromosome of Streptomyces gilvosporeus (S. gilvosporeus). The expression of VHb was determined by Western blotting. The activity of expressed VHb was confirmed by the observation of VHb-specific CO-difference spectrum with a maximal absorption at 419 nm for the recombinant. Integration of the empty plasmid pSET152 did not affect natamycin production of S. gilvosporeus. While the vgb-harboring strain exhibited high natamycin productivity, reaching 3.31 g/L in shake flasks and 8.24 g/L in 1-L fermenters. Compared to the wild strain, expression of VHb, increased the natamycin yield of the strain bearing vgb by 131.3 % (jar fermenter scale) and 175 % (shake flask scale), respectively, under certain oxygen-limiting condition. Addition of an extra copy of the vgb gene in S. gilvosporeus-vgb2 did not enhance the natamycin production obviously. These results provided a superior natamycin-producing strain which can be directly used in industry and a useful strategy for increasing yields of other metabolites in industrial strains.     

纳他霉素高产菌株Streptomyces gilvosporeus F607基因组及其生物合成基因簇分析

【背景】纳他霉素(Natamycin)是一种天然、广谱、高效的多烯大环内酯类抗真菌剂,褐黄孢链霉菌(Streptomyces gilvosporeus)是一种重要的纳他霉素产生菌。目前S. gilvosporeus基因组序列分析还未有报道,限制了该菌中纳他霉素及其他次级代谢产物合成及调控的研究。【目的】解析纳他霉素高产菌株S. gilvosporeus F607的基因组序列信息,挖掘其次级代谢产物基因资源,为深入研究该菌株的纳他霉素高产机理及生物合成调控机制奠定基础。【方法】利用相关软件对F607菌株的基因组序列进行基因预测、功能注释、进化分析和共线性分析,并预测次级代谢产物合成基因簇;对纳他霉素生物合成基因簇进行注释分析,比较分析不同菌种中纳他霉素生物合成基因簇的差异;分析预测S.gilvosporeusF607中纳他霉素生物合成途径。【结果】F607菌株基因组总长度为8482298bp,(G+C)mol%为70.95%,分别在COG、GO、KEGG数据库提取到5 062、4 428、5063个基因的注释信息。同时,antiSMASH软件预测得到29个次级代谢产物合成基因簇,其中纳他霉素基因簇与S.natalensis、S. chattanoogensis等菌株的纳他霉素基因簇相似性分别为81%和77%。除2个参与调控的sngT和sgnH基因和9个未知功能的orf基因有差异外,S. gilvosporeus F607基因簇中其他纳他霉素生物合成基因及其排列顺序与已知的纳他霉素基因簇高度一致。【结论】分析了S. gilvosporeus全基因组信息,预测了S. gilvosporeus F607中纳他霉素生物合成的途径,为从基因组层面上解析S. gilvosporeus F607菌株高产纳他霉素的内在原因提供了基础数据,为揭示纳他霉素高产的机理及工业化生产和未来新药的发现奠定了良好的基础。     

Formulation and antifungal performance of natamycin-loaded liposomal suspensions: the benefits of sterol-enrichment

The aim of this study is to develop and evaluate food-grade liposomal delivery systems for the antifungal compound natamycin. Liposomes made of various soybean lecithins are prepared by solvent injection, leading to small unilamellar vesicles (<130?nm) with controlled polydispersity, able to encapsulate natamycin without significant modification of their size characteristics. Presence of charged phospholipids and reduced content of phosphatidylcholine in the lecithin mixture are found to be beneficial for natamycin encapsulation, indicating electrostatic interactions of the preservative with the polar head of the phospholipids. The chemical instability of natamycin upon storage in these formulations is however significant and proves that uncontrolled leakage out of the liposomes occurs. Efficient prevention of natamycin degradation is obtained by incorporation of sterols (cholesterol, ergosterol) in the lipid mixture and is linked to higher entrapment levels and reduced permeability of the phospholipid membrane provided by the ordering effect of sterols. Comparable action of ergosterol is observed at concentrations 2.5-fold lower than cholesterol and attributed to a preferential interaction of natamycin–ergosterol as well as a higher control of membrane permeability. Fine-tuning of sterol concentration allows preparation of liposomal suspensions presenting modulated in vitro release kinetics rates and enhanced antifungal activity against the model yeast Saccharomyces cerevisiae.     

Potential of natamycin in enhancing the antagonistic activity of Bacillus amyloliquefaciens BGP20 against post-harvest bacterial soft rot of green pepper

Bacillus amyloliquefaciens BGP20 is a promising antagonist in controlling post-harvest bacterial soft rot of vegetables caused by Erwinia carotovora subsp. carotovora (Ecc). The objective of this study was to screen a kind of natural and safe additive which could enhance the bio-control activity of BGP20 against post-harvest bacterial soft rot of green pepper. The results of this study indicated that the additive natamycin had stronger inhibition against the pathogen Ecc compared with bamboo vinegar and chitosan in the 2× Yeast extract and Tryptone (2YT) medium. However, natamycin had a slight negative effect on the growth of BGP20 in the 2YT medium. In preventative treatments, natamycin significantly improved the bio-efficacy of BGP20, and enhanced its competitive position against Ecc in the wounds of green pepper. Compared with the treatment with BGP20 alone, the viable count of BGP20 after 72?h of incubation increased by 115.8% in the wounds of green pepper treated with BGP20 and 0.1% natamycin, while that of Ecc decreased by 92.1%. In addition, natamycin remarkably promoted the flocculation of Ecc cells in the 2YT medium, while promoting the dispersion of BGP20. Natamycin had no negative effects on the spore germination of BGP20 and its shelf life. These results indicated that natamycin had perfect compatibility with the antagonist BGP20, and it had a great potential in enhancing the bio-control activity of BGP20 against post-harvest bacterial soft rot of green pepper in preventative treatments.     

Multiple transporters are involved in natamycin efflux in Streptomyces chattanoogensis L10

Antibiotic‐producing microorganisms have evolved several self‐resistance mechanisms to prevent auto‐toxicity. Overexpression of specific transporters to improve the efflux of toxic antibiotics has been found one of the most important and intrinsic resistance strategies used by many Streptomyces strains. In this work, two ATP‐binding cassette (ABC) transporter‐encoding genes located in the natamycin biosynthetic gene cluster, scnA and scnB, were identified as the primary exporter genes for natamycin efflux in Streptomyces chattanoogensis L10. Two other transporters located outside the cluster, a major facilitator superfamily transporter Mfs1 and an ABC transporter NepI/II were found to play a complementary role in natamycin efflux. ScnA/ScnB and Mfs1 also participate in exporting the immediate precursor of natamycin, 4,5‐de‐epoxynatamycin, which is more toxic to S. chattanoogensis L10 than natamycin. As the major complementary exporter for natamycin efflux, Mfs1 is up‐regulated in response to intracellular accumulation of natamycin and 4,5‐de‐epoxynatamycin, suggesting a key role in the stress response for self‐resistance. This article discusses a novel antibiotic‐related efflux and response system in Streptomyces, as well as a self‐resistance mechanism in antibiotic‐producing strains.     

Iteratively improving natamycin production in Streptomyces gilvosporeus by a large operon-reporter based strategy

Many high-value secondary metabolites are assembled by very large multifunctional polyketide synthases or non-ribosomal peptide synthetases encoded by giant genes, for instance, natamycin production in an industrial strain of Streptomyces gilvosporeus. In this study, a large operon reporter-based selection system has been developed using the selectable marker gene neo to report the expression both of the large polyketide synthase genes and of the entire gene cluster, thereby facilitating the selection of natamycin-overproducing mutants by iterative random mutagenesis breeding. In three successive rounds of mutagenesis and selection, the natamycin titer was increased by 110%, 230%, and 340%, respectively, and the expression of the whole biosynthetic gene cluster was correspondingly increased. An additional copy of the natamycin gene cluster was found in one overproducer. These findings support the large operon reporter-based selection system as a useful tool for the improvement of industrial strains utilized in the production of polyketides and non-ribosomal peptides.     

The polyene antimycotics nystatin and filipin disrupt the plasma membrane, whereas natamycin inhibits endocytosis in germinating conidia of Penicillium discolor

Aims:  To investigate the differences in membrane permeability and the effect on endocytosis of the polyene antimycotics nystatin, filipin and natamycin on germinating fungal conidia.
Methods and Results:  The model system was Penicillium discolor , a food spoilage fungus. Filipin resulted in permeabilization of germinating conidia for the fluorescent probes TOTO-1 and FM4-64, but not for ferricyanide ions. Nystatin caused influx of all these compounds while natamycin did not. Untreated germinating conidia internalize the endocytic marker FM4-64. Pretreatment of germinating conidia with natamycin showed a dose and time dependent inhibition of endocytosis as judged by the lack of formation of early endosomal compartments.
Conclusions:  The results obtained from this study indicated that, unlike nystatin and filipin, natamycin is unable to permeabilize germinating conidia, but interferes with endocytosis in a dose and time dependent manner.
Significance and Impact of the Study:  Natamycin acts via a different mode of action than other polyene antimycotics. These results offer useful information for new strategies to prevent fungal spoilage on food products and infection on agricultural crops. For laboratory use, natamycin can be used as a specific inhibitor of early endocytosis in fungal cells.     

A comparison between Dactylaria gallopava and Scolecobasidium humicola: first report of an infection in a tortoise caused by S. humicola

Scolecobasidium humicola, a soil fungus and etiologic agent of phaeohyphomycosis in fish, is herein reported to cause cutaneous lesions in a tortoise, Terrapine carolina var. carolina. S. humicola was isolated from lesions on the foot and dematiaceous hyphae were observed in KOH preparations of the biopsy and in stained preparations. This isolate and others were compared morphologically and physiologically with isolates of Dactylaria gallopava which it resembles. As a result of this investigation, we concluded that D. gallopava may be differentiated from S. humicola macroscopically, by the production in D. gallopava of an extensive diffusible purplish-red to reddish-brown pigment when cultured on Sabouraud dextrose agar; microscopically, by the presence and usually predominance of conidia, whose apical cell is markedly wider than the basal cell, and usually constricted at the septum; and physiologically, by the ability to grow on media containing cycloheximide and by the ability to grow well at 36-45 degrees C. In contrast, S. humicola does not usually produce a diffusible pigment on Sabouraud's dextrose agar or if present, is not extensive; it lacks the wider upper cell; is less constricted or non-constricted at the central septum; grows on media containing cycloheximide, although some inhibition may occur and lastly, does not grow at 36 degrees C or higher. Both species were urease positive, hydrolysed tyrosine but not casein, xanthine, or gelatin.     

Selective Medium for Recovering Specific Populations of Rhizobia Introduced into Tropical Soils

Experiments were designed to evaluate the usefulness of antifungal agents and streptomycin for recovering low densities of rhizobia inoculated into tropical soils. The results showed that yeast-mannitol agar (pH 6.0) containing 500 μg of streptomycin, 400 μg of cycloheximide and 50 μg of benomyl or chlorothalonil per ml was the best selective medium.     

Induced tolerance of Sclerotium cepivorum to antibiotics in the presence of onion exudates

The onion white rot pathogen Sclerotium cepivorum was cultured on agar media containing 2% malt extract and one of the antifungal antibiotics, endomycin, griseofulvin, venturicidin and cycloheximide at concentrations that reduced but did not prevent growth of mycelium. When onion seeds or agar discs impregnated with diffusates from onion bulbs were placed on the antibiotic media, radial growth of the fungus was greatly increased, and there was a profuse development of aerial mycelium. Gaseous diffusates from onion tissue and from impregnated agar discs were also effective. On the antibiotic media, tomato, cabbage and radish seeds did not stimulate the growth of S. cepivorum and the onion exudates did not stimulate the growth of four other fungi. This and other evidence is considered to show that the stimulation of growth of S. cepivorum was not caused by any direct effect on the antibiotics but by a tolerance of the fungus to them, which was specifically induced by an exudate from its host plant. The phenomenon may be related to the reported reversal by onion extracts of the inhibitory effects of soil mycostasis on germination of sclerotia of the fungus.