Selective isolation and distribution of Actinobispora strains in soil

A simplified enrichment method for selective isolation of Actinobispora strains from soil is described. Actinobispora spores were tolerant to dry-heat treatment at 110 degrees C for 15 min. Actinobispora was more resistant to 1 microgram/mL leucomycin, 1 microgram/mL novobiocin, and 0.5 microgram/mL tunicamycin than Streptomyces dominant in soil, which prevents selective isolation of Actinobispora. Percentages of Actinobispora colonies on the isolation plate were increased by addition of antibiotics and dry-heat treatment of the soil samples. By combining the techniques described above, this genus was isolated from 105 out of 574 soil samples (18% of the samples tested). It was recovered from the soil samples with pH values ranging 5.0 to 8.9, and 78% of strains were isolated from neutral soil (pH 6.0-8.0). A number of Actinobispora strains were isolated from various soils around the world. Actinobispora strains are widely distributed in the world at relatively high frequency.     

热带药用植物根际放线菌的分离、鉴定及生物活性分析

从海南热带植物园采集12种药用植物的根际土样,采用选择性分离方法,分离得到400株根际放线菌。使用5种活性筛选模型对分离菌株进行生物活性评价,154株放线菌在一个或多个活性筛选模型中显示为阳性,菌株初筛阳性率达38.5%;根据菌株形态特征并结合代谢产物的生物活性,从中挑选出28株菌进行16S rRNA基因序列分析,发现其分属于链霉菌属、诺卡氏菌属、小单孢菌属和野野村菌属。     

高温链霉菌24＃的初步研究

从土壤中筛选得到一株产广谱、高活性抗真菌物质的链霉菌 2 4 # ,经测定对 2 5种植物病原真菌有显著拮抗作用 ,其次生代谢产物对病菌菌丝有断裂、扭曲、缢缩等致畸效应。 16SrDNA序列分析显示本菌株与模式菌株链霉菌DSM4 4 2 93T的 16SrDNA同源性为 98 93% ,但菌株形态特征、培养特征、细胞壁化学组分、生理生化特性等均不同于模式菌株 ,且DNA杂交率只有 2 2 5 4 % ,建议为链霉菌属的一个新种 ,命名为山东链霉菌 (Streptomycesshandon gensissp .nov .)。     

Occurrence of macrolide-lincosamide-streptogramin B resistant isolates of Staphylococcus aureus in clinical specimens in 2003-2004

The aim of the study was the analysis of frequency of macrolide-lincosamide-streptogramin B (MLS(B)) resistance among MSSA (n=1682) and MRSA (n=272) strains which were isolated in 2002-2004 from various clinical materials from patients hospitalized in the University Hospital at the L. Rydygier Collegium Medicum in Bydgoszcz, University of Nicolaus Copernicus in Toruń. Susceptibility testing and examination of methicillin-resistant strains were performed by the disc diffusion techniques according to recommendation of NCCLS. Resistance to the MLS(B) antimicrobials agents was higher among MRSA compared to MSSA isolates. The MLS, constitutive phenotype was more prevalent than the MLS(B) inducible phenotype among investigated MRSA (65.4%) and MSSA (7.6%) isolates. Inducible resistance had only 2.5% of the MSSA and 2.6% of the MRSA strains. Moreover in 2004 there were found increasing frequency of inducible MLS(B) resistance from 1.1% to 5.7% and decreasing frequency of constitutive MLS(B) resistance from 9.2% to 4.7% among MSSA strains, in comparison to 2003. The investigated MSSA MLS(B)-, MLS(B)- and MRSAMLS(B)+, MLS(B)- strains were the most frequently isolated from pus (adequately 5.2%, 28.8% and 30.5%, 10.7%) and also from nosopharynx swabs (1.7% MSSA MLS(B)+ and 22.9% MSSA MLS(B)-) and biomaterials (15,1% MRSA MLS(B)+ and 9.6% MRSA MLS(B)-). They mainly came from patients of the outpatient clinic (2,4% MSSA MLS(B)+ and 19.9% MSSA MLS(B)-) and patients treated at the neurosurgical ward (20.6% MRSA MLS(B)+ and 12.1% MRSA MLS(B)-).     

西藏地区土壤放线菌种群多样性及拮抗活性研究

从西藏不同海拔、不同气候条件的5个地区采集的10份土样中,使用选择分离培养基、ISP 2（Yeast extract_malt extract agar）和高氏一号培养基,通过分散差速离心法（Dispersion and differential centrifugation,DDC）分离得到放线菌156株。根据形态和培养特征将其归入32个类群,并从中选出65个代表菌株进行全细胞壁氨基酸组分分析,结果表明：９株菌为非链霉菌胞壁类型,其余为链霉菌胞壁类型。16S rDNA扩增片段长度多态性（ARDRA）分析得到不同带型,对其序列分析结果表明：分离菌株分属链霉菌属（Streptomyces）和5个稀有放线菌属。同时测试了代表菌株抗耐药细菌和真菌的活性,其中38.5%的菌株具有拮抗活性。     

Mycolic acid-containing bacteria induce natural-product biosynthesis in Streptomyces species

Natural products produced by microorganisms are important starting compounds for drug discovery. Secondary metabolites, including antibiotics, have been isolated from different Streptomyces species. The production of these metabolites depends on the culture conditions. Therefore, the development of a new culture method can facilitate the discovery of new natural products. Here, we show that mycolic acid-containing bacteria can influence the biosynthesis of cryptic natural products in Streptomyces species. The production of red pigment by Streptomyces lividans TK23 was induced by coculture with Tsukamurella pulmonis TP-B0596, which is a mycolic acid-containing bacterium. Only living cells induced this pigment production, which was not mediated by any substances. T. pulmonis could induce natural-product synthesis in other Streptomyces strains too: it altered natural-product biosynthesis in 88.4% of the Streptomyces strains isolated from soil. The other mycolic acid-containing bacteria, Rhodococcus erythropolis and Corynebacterium glutamicum, altered biosynthesis in 87.5 and 90.2% of the Streptomyces strains, respectively. The coculture broth of T. pulmonis and Streptomyces endus S-522 contained a novel antibiotic, which we named alchivemycin A. We concluded that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the direct interaction between the mycolic acid-containing bacteria and Streptomyces. We used these results to develop a new coculture method, called the combined-culture method, which facilitates the screening of natural products.     

聚酮类化合物生物合成途径基因阳性菌株生物多样性研究

从中国云南省采集土样,采用GPY培养基、淀粉_酪素琼脂培养基和甘油天门冬酰胺琼脂培养基分离得到了876株细菌放线菌菌株。经聚酮类化合物基因筛选得到75株Ⅰ型和Ⅱ型聚酮类化合物生物合成基因双阳性的菌株,经抗菌活性、形态、生理等结果分析比较,选取其中的10株进行了16S rDNA序列分析。在物种多样性方面,分离到的菌分布至少有7个科,8个属。其中有链霉菌科的链霉菌属,分枝杆菌科的分枝杆菌属,链孢囊菌科的链孢囊菌属和野野村菌属,诺卡氏菌科的诺卡氏菌属,微球菌亚目的一新属、产碱杆菌科的无色杆菌属和β_亚纲中与紫色杆菌属紧邻的一革兰氏阴性菌新属。综合其表型、基因型特征,初步确定其中8株为新物种。研究表明利用新的思路和程序从自然环境分离、鉴定未知菌是微生物资源开发利用的关键。     

<Emphasis Type="Italic">Streptomyces roseoalbus</Emphasis> sp. nov., an actinomycete isolated from soil in Yunnan,China

A strain YIM 33098T (= CCTCC AA001027T = DSM 41831T) was isolated from a forest soil sample collected from Nanning in Guangxi Province, China, in the course of screening for producers of new drug lead compounds. This strain was identified by using a polyphasic approach. The results showed that it should be assigned to the genus Streptomyces. An almost complete 16S rRNA gene sequence of the strain was determined and compared with those of representative Streptomyces species. Strain YIM 33098T was clustered in the same subclade with Streptomyces tendae ATCC19812T and Streptomyces eurythermus ATCC14975T. Similarities of strain YIM 33098T with the two strains were 97.35% and 97.42%, respectively. Based on the phenotypic and genotypic evidence, it is therefore proposed that strain YIM 33098T should be classified in the genus Streptomyces as a new species under the name of Streptomyces nanningensis sp. nov.     

神农架林区和自然保护区链霉菌资源考察

首次从神农架林区和自然保护区的原始林、次生林、荒地、耕地采集土样 78份 ,用 5种培养基分离放线菌。经挑选留取 96株 ,其中 86株放线菌为白长链霉菌 (Streptomyces .albolongus)等 5 5个种或类群种。对神农架林区和自然保护区链霉菌的分布规律作了初步探讨 ,讨论了土壤类型、植被类型、海拔高度、气候条件、人类活动等对神农架林区和自然保护区链霉菌分布的影响     

ε-聚赖氨酸产生菌TUST-2的分离鉴定

【目的】ε-聚赖氨酸是一种天然氨基酸同聚物,本研究目的为分离筛选新的ε-聚赖氨酸产生菌。【方法】采用一种新的分离方法从土壤中分离ε-PL产生菌。分离方法含3步:(1)富集培养ε-PL耐受菌;(2)通过改进的Nishikawa方法筛选;(3)挑选高浓度ε-PL耐受菌株。【结果】从海南省土样中分离获得ε-聚赖氨酸产生菌TUST-2。分类和形态特征属链霉菌属。16S rDNA序列分析比对结果表明TUST-2属淀粉酶产色链霉菌(Streptomyces diastatochromogenes)。经特征反应分析、水解物分析、红外光谱、1H NMR、13C NMR和MALDI-TOF-MS分析表明TUST-2发酵产物为ε-聚赖氨酸。【结论】根据16S rRNA基因序列比对和形态及生理生化特征表明ε-聚赖氨酸产生菌TUST-2属于淀粉酶产色链霉菌,命名为淀粉酶产色链霉菌TUST-2。     

Evolutionary relationship of the Bacillus licheniformis macrolide-lincosamide-streptogramin B resistance elements

Summary Naturally occurring erythromycin (Em) resistance was found in 11 of the 18 Bacillus licheniformis isolates tested but was absent from a wide variety of other Bacillus strains. The Em resistance elements confer inducible macrolide-lincosamide-streptogramin B (MLS) resistance and are related to ermD an MLS resistance element previously cloned from the chromosome of B. licheniformis 749. The MLS sensitive B. licheniformis strains and the other sensitive Bacillus strains tested, lack sequences with detectable homology to ermD. The sensitive B. licheniformis strains do exhibit homology to sequences which flank ermD in B. licheniformis 749. The relative sizes of the homologous DNA fragments suggest that the sensitive strains are lacking a 3.6 kb segment which contains ermD. It is shown that ermD is homologous to chromosomal DNA from Streptomyces erythreus ATCC 11635, an Em producing organism. These observations suggest to us that MLS resistance may have arisen in the Streptomyces and spread to B. licheniformis another gram positive bacterium found in soil. It is further proposed that ermD is or was located on a transposon-like element and has spread and evolved further to yeild a variety of related Staphylococcal and Streptococcal MLS determinants.     

Detection of the nec1 virulence gene and its correlation with pathogenicity in Streptomyces species on potato tubers and in soil using conventional and real-time PCR

AIMS: To evaluate the virulence gene nec1 as a reliable marker for the detection of pathogenic Streptomyces species on potato tubers and in soil samples using conventional and real-time quantitative PCR assays. Methods AND RESULTS: Two pairs of conventional primers (outer and nested) and one set of primers/probe for use in real-time PCR were designed to detect the necrogenic protein encoding nec1 gene of Streptomyces scabiei strain ATCC 49173(T). The conventional PCR primers were also incorporated into a multiplex PCR assay to simultaneously detect the nec1 gene in conjunction with the potato pathogens Helminthosporium solani and Colletotrichum coccodes. The specificity of each PCR assay was confirmed by testing 32 pathogenic and nonpathogenic reference strains of Streptomyces representing 12 different species and 74 uncharacterized streptomycete strains isolated from diseased tubers. A clear correlation between pathogenicity and the detection of nec1 by PCR was demonstrated. The sensitivity and specificity of both the conventional and real-time PCR assays allowed the detection of nec1 on potato tubers in the absence of visible symptoms of common scab, and in seeded soil down to a level equivalent to three S. scabiei spores per gram soil. CONCLUSIONS: Reliable and quantitative PCR techniques were developed in this study for the specific detection of the virulence gene nec1 of pathogenic Streptomyces species on potato tubers and in soil samples, and the data demonstrated a clear correlation between pathogenicity in Streptomyces species and the presence of the nec1 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Together with the DNA extraction protocols, these diagnostic methods will allow a rapid and accurate assessment of tuber and soil contamination by pathogenic Streptomyces species.     

Isolation and identification of Streptomyces spp. from Venezuelan soils: morphological and biochemical studies. I

The genus Streptomyces is represented in nature by the largest number of species and varieties among the family Actinomycetaceae. They differ greatly in their morphology, physiology, and biochemical activities, producing the majority of known antibiotics. The morphological and biochemical characteristics of 71 Streptomyces spp. isolated from soil samples collected at different places of Venezuela, are presented. A comparative analysis using the statistical software Minitab shows that 67 of these isolates are presumably new strains, since they possess a very low percentage of similarity with other reported species. Only four isolates shared 100% identity with one, two or three reported Streptomyces spp.     

Characterization of nickel-resistant bacteria isolated from serpentine soil

In the present study, heterotrophic nickel-resistant bacteria were isolated and characterized from three different serpentine outcrops in central Italy populated by the nickel-hyperaccumulating plant Alyssum bertolonii . Bacteria were isolated from the rhizosphere of the plant and from soil portions at various distances from the plant. The proportion of nickel-resistant cfu was higher in proximity to the plant than in free soil. A total of 138 isolates was collected and grouped into 47 different operational taxonomic units (OTUs) by means of amplified ribosomal DNA restriction analysis (ARDRA) and into 25 heavy-metal resistant phenotypes. The phylogenetic position of strains belonging to 20 OTUs, representing more than the 70% of the total isolates, was determined by 16S rDNA sequencing. These analyses showed that the most represented genera in all three different outcrops were Pseudomonas and Streptomyces . Pseudomonas strains were found to be predominant in the plant rhizosphere, whereas Streptomyces strains were mainly present in the soil.     

<Emphasis Type="Italic">Streptomyces gulbargensis</Emphasis> sp. nov., isolated from soil in Karnataka,India

During the course of screening for industrially important microorganisms, an alkali-tolerant and thermotolerant actinomycete, strain DAS 131T, was isolated from a soil sample collected from the Gulbarga region, Karnataka province, India. The strain was characterized by a polyphasic approach that showed that it belonged to the genus Streptomyces. Growth was observed over a wide pH range (pH 6-12) and at 45 degrees C. The 16S rRNA gene sequence of strain DAS 131T was deposited in the GenBank database under the accession number DQ317411. 16S rRNA gene sequence analysis revealed that strain DAS 131T was most closely related to Streptomyces venezuelae ISP 5230T (AY999739) with a sequence similarity of 99.5% (8 nucleotide differences out of 1,477). Despite this very high sequence similarity, strain DAS 131T was phenetically distinct from S. venezuelae. The DNA relatedness between these strains was 54%, indicating that strain DAS 131T is a distinct genomic species. On the basis of phenetic and genetic analyses, strain DAS 131T is classified as a new species in the genus Streptomyces, for which we propose the name Streptomyces gulbargensis sp. nov.     

A Study on actinobacterial diversity of Hampoeil cave and screening of their biological activities

Caves are oligotrophic, dark and less-explored environments and are considered as sources of promising microbial strains in biotechnology. Hampoeil Cave is located in massive dolomite with thin bedded limestone in northwestern of Iran. In an isolation and screening program, various samples from soil, water, floor, wall and ceiling of Hampoeil cave and its invertebrates were collected. Four various treatments and 10 different isolation media were used for the isolation of the actinobacteria. Screening of the isolates for antimicrobial activity against 10 bacteria and fungi, 5 hydrolytic enzymes production and resistance to 5 heavy metals have been performed. Among 33 various samples, 76 actinobacteria from various genera, including Streptomyces, Micromonospora, Micrococcus, Kocuria and Corynebacterium were isolated. Eighty percent of the strains had one of the studied hydrolytic enzyme activity. At least one type of antimicrobial activity was seen in 25.3% of the isolates. Resistance to one metal or more was seen in 26.32% of the isolates. The ratio of rare-actinobacteria in the oligotrophic samples to enriched samples is 20% more than Streptomyces. Percentage of strains with the highest activity in esterase, amylase, DNase, protease or lipase activity that were isolated from organic-rich environmental samples were 100, 100, 100, 82 and 82%, respectively. Also, 26.32% of the actinobacterial isolates resisted to heavy metals. It was concluded that Hampoeil cave is a good source in finding cave-living actinobacteria potent in producing hydrolytic enzymes and bioremediation.     

Survey of susceptibility of clinical Clostridium diffiicile strains isolated from patients hospitalised in different departments of paediatric hospital to antimicrobial agents

This study was performed to determine the susceptibility of 50 C. difficile strains isolated from faecal samples of children suspected to antibiotic associated diarrhea (AAD) to antimicrobial agents: metronidazole, vancomycin, erythromycin, clindamycin, ciprofloxacin, moxifloksacin, gatifloksacin and imipenem. The all C. difficile strains were sensitived to metronidazole and vancomycin. Twenty six per cent of strains were resistant to erythromycin and clindamycin (MLS(B) type resistance). Resitance to ciprofloxacin, moxifloxacin, gatifloxacin and imipenem was detected in 98%, 8%, 8% and 30% of C. difficile strains, respectively.     

几株具有除莠活性的放线菌的鉴定

14 strains of Actinomycetes were isolated from soil samples collected in Yunnan China Experiments showed that they had activity of herbicide. In this paper,the morphological and chemotaxonomic characters of these strains were studied and they were identified as three genera of Actinomycetales respecitively, such as Streptomyces, Micromonospora and Nocardia.     

Isolation of soil Streptomyces strains capable of degrading humic acids and analysis of their peroxidase activity

Abstract Fifteen Streptomyces strains capable of decolorizing humic acids in presence of glucose were isolated from soil samples using the dilute suspension technique and spread on agar plates. Six strains, displaying a significant and stable activity, were selected for further characterization. Some features of these isolates (carbon source utilization, enzyme production, antibiotic resistance) were compared with those of the reference strain Streptomyces viridosporus ATCC 39115. Degradation properties studied in batch cultures at pH 7.0 showed that the catabolic activity on humic acids was generally stimulated by incubation with 100% oxygen and was cell surface-associated. Peroxidase activity from cell-free extracts was analysed by using the oxidation of N,N,N′,N′-tetramethyl-phenylene-diamine. PAGE analysis revealed the existence of two major types of peroxidases (molecular mass: about 39.2 and 61.6 kDa), dividing the strains into two groups. The role of cell surface-associated peroxidase activity in the breakdown of humic acids is discussed.     

三株杀粘虫放线菌的分类鉴定

菌株YIM31331、YIM31333、YIM31355是从云南省丽江、中甸采集的土壤样品中分离得到的具有产杀粘虫活性物质菌株。根据其形态学特征、生理生化特性及16S rDNA序列分析结果,认为菌株YIM 31331属于链霉菌属的Streptomyces subrutilus,YIM31333属于指孢囊菌属Dactylosporangium aurantiacum,YIM31355属于链孢囊菌属Streptosporangium vulgare。