An integrated method for the enrichment and selective isolation of Actinokineospora spp. in soil and plant litter

AIMS: To devise and evaluate a method for isolating the rare, zoosporic actinomycetes, Actinokineospora spp. in soil and plant litter. METHODS AND RESULTS: The newly developed method consists of two enrichment stages followed by plating on a selective medium. The source material is initially incubated with calcium carbonate to multiply the population of Actinokineospora spp., and is then air-dried. The second stage consists of rehydration-centrifugation, in which the amended substrate is immersed in phosphate buffer-soil extract to liberate actinomycete zoospores, and nonmotile microbial associates are then eliminated by centrifugation. Portions of the supernatant enriched with zoospores are plated on humic-acid vitamin agar supplemented with fradiomycin, kanamycin, nalidixic acid and trimethoprim. We examined 39 soil and plant-litter samples taken from fields, forests and stream banks. The proposed method consistently enriched and selectively isolated Actinokineospora spp. in 17 samples. Evidence for antimicrobial activity was found in most of the isolates. CONCLUSION: A combination of enrichment and a medium containing selective antibiotics can be used successfully for efficient isolation of certain rare actinomycete taxa. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of new methodologies with which to isolate rare actinomycetes is of great importance to extend our understanding of their ecology, taxonomy and bioactivity.     

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。     

嗜酸丝状放线菌的选择性分离与多样性

摘要:【目的】针对酸性土壤中的嗜酸丝状放线菌,建立有效的选择性分离方法,并了解其多样性。【方法】用不同的样品预处理方式和分离培养基,并添加不同的抑制剂进行分离;根据放线菌的菌落数和出菌率确定最佳分离方法组合。采用最佳分离方法对从江西采集的17份酸性土壤样品进行分离;根据培养特征对分离菌株进行分群,进一步通过对各类群的显微形态观察和pH梯度生长实验确定代表菌株;对代表菌株进行16S rRNA基因序列分析研究其多样性。【结果】嗜酸丝状放线菌的最佳分离方法为：土壤样品经分散差速离心预处理后,涂布添加了放线菌酮、制霉菌素和萘啶酮酸（各50 mg/L）的GTV培养基。用此方法共分离到放线菌369株,归为10个不同的颜色类群,其中6.6%为严格嗜酸放线菌,72.4%为中度嗜酸放线菌,21.0%为耐酸放线菌。52株嗜酸放线菌代表菌株分布于放线菌目中的12个属：链霉菌属(Streptomyces)、小单孢菌属(Micromonospora) 、诺卡氏菌属(Nocardia)、野野村菌属(Nonomuraea) 、韩国生工属(Kribbella) 、小双孢菌属(Microbispora)、马杜拉菌属(Actinomadura)、拟无枝菌酸菌属(Amycolatopsis)、指孢囊菌属(Dactylosporangium)、伦茨氏菌属(Lentzea)、游动四孢菌属(Planotetraspora) 和链嗜酸菌属(Streptacidiphilus),其中链霉菌分离菌株在系统发育树上形成12个不同的进化类群。【结论】所建立的选择性分离方法可用于土壤嗜酸丝状放线菌的高效分离;江西酸性土壤含有丰富多样的嗜酸丝状放线菌种属。     

Application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter

The present paper describes a simple enrichment technique which enables rapid and selective isolation of diverse zoosporic actinomycete genera directly from soil and plant litter. This technique, designated the rehydration and centrifugation (RC) method, consists of immersing the air-dried source material in 10 mM phosphate buffer containing 10% soil extract, letting the preparation stand at 30 °C for 90 min, followed by centrifugation of the fluid at 1,500×g for 20 min. Portions of the supernatant containing actinomycete zoospores are plated on the humic acid-vitamin agar which is supplemented with nalidixic acid and trimethoprim as the selective inhibitors for Gram-negative bacteria and bacilli. The phosphate buffer-soil extract solution significantly promoted liberation of motile zoospores from the source material. The centrifugation stage greatly eliminated streptomycetes and other non-motile actinomycetes from the liquid phase, thereby facilitating selective growth of rare, motile actinomycetes on the isolation plates subsequent to inoculation. Ten different soil and leaf-litter samples, taken from fields, forests, and stream banks, were examined. The RC method consistently achieved preferential isolation of motile actinomycetes in all samples, which accounted for 37–86% of the total microbial population recovered. The most frequently isolated motile actinomycetes were Actinoplanes and Dactylosporangium. Strains of Actinokineospora, Catenuloplanes and Kineosporia were also recovered, depending on the nature of the samples examined. Other motile actinomycetes that were occasionally isolated in small numbers included Actinosynnema, Geodermatophilus and Sporichthya.     

Oligotrophy is Helpful for the Isolation of Bioactive Actinomycetes

It is necessary to develop new methods for the isolation of unknown actinomycetes from soils. To evaluate the effects of oligotrophic medium on the isolation of soil actinomycetes and develop a new isolation method, the Gause’s synthetic medium was diluted to one tenth the recommended concentration in the present study. Soil dilution plate technique was used to isolate actinomycetes from the soil samples. Oligotrophy decreased actinomycete and streptomycete counts, as well as the number of antagonistic actinomycete species. Oligotrophy also decreased the number of actinomycete species in five samples. Some actinomycete species were cultured only on the oligotrophic medium, whereas other species could not be cultured. Oligotrophy decreased actinomycete counts more significantly for soils with organic matter content >40 g/kg. We used 16S rRNA sequence analysis to identify 22 actinomycete species that were only cultured on the oligotrophic medium. Oligotrophic medium was helpful for the isolation of Streptomyces spp., Micromonospora spp. and Streptosporangium spp. Slightly more than 80 % of the identified actinomycete species were biologically active. Therefore, we could draw a conclusion that oligotrophic medium could be helpful for the discovery of new antibiotic producers and the exploitation and utilization of new, biologically active compounds.     

First human case of nocardiosis caused by Nocardia pseudobrasiliensis in Japan

Nocardia sp. IFM 0896, an actinomycete with biochemical characteristics that differed from Nocardia brasiliensis, was isolated from a 71-year-old Japanese man with a history of tuberculosis and cancer. Although the isolate was tentatively identified as N. brasiliensis, the morphological and physiological characteristics of strain IFM 0896 were different from those of N. brasiliensis IFM 0236T. The results of 16S rRNA gene sequence phylogenies and PCR-RFLP analysis of a heat shock protein revealed that Nocardia sp. IFM 0896 belongs to the species N. pseudobrasiliensis. This is the first clinical isolation report of N. pseudobrasiliensis in Japan.     

Use of phage battery to investigate the actinofloral layers of termite gut microflora

AIMS: The termite gut microbiota can include a variety of micro-organisms from the three domains: Bacteria, Archaea and Eucarya. The bacterial groups from the gut systems are mainly affiliated to the proteobacteria, the Gram-positive groups Bacterioiodes/Flavobacterium branch and the spirochetes, Firmicutes and Actinobacteria. However, culture independent molecular studies have revealed that the majority of these microbial gut symbionts have not yet been cultured, including actinobacterial clusters associated with termite guts. Accordingly, the aim of this study was to selectively isolate the actinofloral layers of gut associated microflora of the Coptotermes lacteus (Froggatt) species located at the Sunshine Coast Region of Queensland, Australia to increase our knowledge on the diversity of actinobacterial taxa present in the termite guts. METHODS AND RESULTS: Actinofloral layers associated with the guts of the wood-eating subterranean termite C. lacteus were investigated by exploiting the phage susceptibility of different gut associated bacteria which impede the growth of actinomycetes on isolation plates. These unwanted microbial taxa were removed by exposing the gut contents to polyvalent bacteriophages specifically targeting different background bacterial taxa and after their removal from the isolation plates previously undetected and novel actinomycetes were successfully cultured from the gut samples. CONCLUSIONS: Use of bacteriophages as a means of selective pressure successfully revealed the presence of novel actinomycete species within the guts of C. lacteus. SIGNIFICANCE AND IMPACT OF THE STUDY: Molecular ecology has undoubtedly revealed the fascinating diversity of micro-organisms, which cannot be cultured. However, these advances in the field still have not provided the ability to detect and isolate micro-organisms effectively from their ecological niches. Accordingly, studies like the one described here have importance in increasing the chances of uncultured taxa to be isolated to complement molecular microbial ecological efforts towards the establishment of an understanding on the diversity of termite gut microflora.     

The characterization of oil-degrading microorganisms from lubricating oil contaminated (scale) soil

AIMS: To isolate and characterize oil-degrading microorganisms from contaminated (scale) soil. METHODS AND RESULTS: Oil-degrading microorganisms were isolated from enrichment cultures of scale soil. Each isolate was identified using 16S rDNA gene and oil degradability was determined on both unused and used lubricating oil. The weight of the extracted remaining oil revealed that most isolates degraded unused lubricating oil more than used lubricating oil. Chemical composition of oil analysed by TLC-FID and GC-MS demonstrated that Nocardia simplex W9 degraded used oil the best, and resulted in a decrease in saturates, aromatics and resins to 52.46, 38.13 and 18.81%, respectively. CONCLUSIONS:Nocardia simplex W9 is the best degrader, among all the isolates, on both used and unused lubricating oil. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Nocardia simplex W9 in scale soil enables iron to be recycled by biodegradation.     

A biochemical protocol for the isolation and identification of current species of Vibrio in seafood

AIMS: We report a biochemical method for the isolation and identification of the current species of vibrios using just one operative protocol. METHODS AND RESULTS: The method involves an enrichment phase with incubation at 30 degrees C for 8-24 h in alkaline peptone water and an isolation phase on thiosulphate-citrate-salt sucrose agar plates incubating at 30 degrees C for 24 h. Four biochemical tests and Alsina's scheme were performed for genus and species identification, respectively. All biochemical tests were optimized as regards conditions of temperature, time of incubation and media composition. The whole standardized protocol was always able to give a correct identification when applied to 25 reference strains of Vibrio and 134 field isolates. CONCLUSIONS: The data demonstrated that the assay method allows an efficient recovery, isolation and identification of current species of Vibrio in seafood obtaining results within 2-7 days. SIGNIFICANCE AND IMPACT OF THE STUDY: This method based on biochemical tests could be applicable even in basic microbiology laboratories, and can be used simultaneously to isolate and discriminate all clinically relevant species of Vibrio.     

Application of actinomycetes to soil to ameliorate water repellency

AIMS: The aim of this study was to develop a novel isolation technique using a mixture of Bacillus and Streptomyces phages to selectively isolate wax-utilizing non-streptomycete actinomycetes effective in ameliorating water repellency in a problem soil. METHODS AND RESULTS: Phages added to a soil suspension reduced the dominance of Bacillus and Streptomyces isolates and significantly increased the number of non-streptomycete actinomycetes on isolation plates. Promising isolates, grown on a medium containing beeswax as sole carbon source, were selected for application to water repellent soil. Their addition significantly reduced water repellency. CONCLUSIONS: Phage application significantly increased the isolation of non-streptomycete actinomycetes. Wax-utilizing isolates were found to significantly reduce water repellency in a problem soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The phage technique can be used for the routine isolation of non-streptomycete actinomycetes. Beeswax medium can be used to selectively isolate wax-utilizing micro-organisms with the potential to ameliorate water repellency in soil.     

Use of HP selective medium to detect Helicobacter pylori associated with other enteric bacteria in seawater and marine molluscs

AIMS: This project investigated the utility of HP selective medium to isolate H. pylori cells from seawater and from marine molluscs. METHODS AND RESULTS: Nested-PCR was performed to reveal the presence of Helicobacter genus. All samples were cultured in HP selective medium and 16 cultures were initially selected as putative Helicobacter. Helicobacter spp. DNA were detected in 9/16 cultures and three of them had 99-100% homology to H. pylori based on 16S RNA gene sequence. Helicobacter pylori isolation was unsuccessful. On the basis of 16S RNA gene sequences the contaminating organisms were shown to be Proteus mirabilis and Vibrio cholerae. CONCLUSIONS: These results indicate the coexistence of three predominant bacterial genera in the cultures and that HP selective medium can grow other enteric bacteria besides Helicobacter. Additional assays will improve the HP selective medium formulation for marine samples avoiding P. mirabilis and V. cholerae interferents. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows the effectiveness of the selective HP medium for the Helicobacter culture from marine samples.     

Substrate induction of isomaltulose synthase in a newly isolated Klebsiella sp. LX3

AIMS: Production of isomaltulose by newly isolated Klebsiella sp. LX3. METHODS AND RESULTS: The bacterial isolate LX3, which transforms sucrose to isomaltulose and trehalulose, has been isolated from a soil sample in Singapore. Morphological and biochemical analysis, as well as 16s rRNA sequence demonstrated that the isolate could represent a new member of genus Klebsiella. The strain has several interesting features. The immobilized cells of Klebsiella sp. LX3 convert more than 99% of sucrose to products that consist of more than 87% of isomaltulose, 11.6% of trehalulose, and <1% of glucose. CONCLUSIONS: The production of isomaltulose synthase in isolate LX3 is inducible by its substrate sucrose and the sugars containing a fructofuranosyl group. SIGNIFICANCE AND IMPACT OF STUDY: It would be useful for future biotechnological applications to understand the structural features or motifs of the isomaltulose synthases that determine the sucrose conversion efficiency and the ratio of the conversion products.     

The efficiency of 1M NH4Cl and 2 M NaCl for the isolation of pathogenic Nocardia from soil

The present paper reports an improvement to the classical method of the paraffin bait, by the usage of 1 M NH₄Cl or 2 M NaCl to eliminate contaminant microflora of soil.The purpose is to introduce a change in the paraffin bait method in order to reduce time required to isolate pathogenic strains of Nocardia from their natural sources.For this study three main criteria were used: a) Determination of the inhibitory effect of different concentrations of salts on soil microflora; b) The tolerance of Nocardia brasiliensis, Nocardia asteroides and Nocardia caviae (Nocardia otitidis caviarum) strains to these chemical inhibitors; c) Determination of the efficiency of salts in the isolation of Nocardia from soil when strains are grown on paraffin baits.     

Enumeration, isolation and identification of diazotrophs from Korean wetland rice varieties grown with long-term application of N and compost and their short-term inoculation effect on rice plants

AIM: This study has been aimed (i) to isolate and identify diazotrophs from Korean rice varieties; (ii) to examine the long-term effect of N and compost on the population dynamics of diazotrophs and (iii) to realize the shot-term inoculation effect of these diazotrophs on rice seedlings. METHODS AND RESULTS: Diazotrophic and heterotrophic bacterial numbers were enumerated by most probable number method and the isolates were identified based on morphological, physiological, biochemical and 16s rDNA sequence analysis. Long-term application of fertilizer N with compost enhanced both these numbers in rice plants and its environment. Bacteria were high in numbers when malate and azelaic acids were used as carbon source, but less when sucrose was used as a carbon substrate. The combined application promoted the association of diazotrophic bacteria like Azospirillum spp., Herbaspirillum spp., Burkholderia spp., Gluconacetobacter diazotrophicus and Pseudomonas spp. in wetland rice plants. Detection of nifD genes from different diazotrophic isolates indicated their nitrogen fixing ability. Inoculation of a representative isolate from each group onto rice seedlings of the variety IR 36 grown in test tubes indicated the positive effect of these diazotrophs on the growth of rice seedlings though the percentage of N present in the plants did not differ much. CONCLUSIONS: Application of compost with fertilizer N promoted the diazotrophic and heterotrophic bacterial numbers and their association with wetland rice and its environment. Compost application in high N fertilized fields would avert the reduction of N(2)-fixing bacterial numbers and their association was beneficial to the growth of rice plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitory effect of high N fertilization on diazotrophic bacterial numbers could be reduced by the application of compost and this observation would encourage more usage of organic manure. This study has also thrown light on the wider geographic distribution of G. diazotrophicus with wetland rice in temperate region where sugarcane (from which this bacterium was first reported to be associating and thereon from other plant species) is not cultivated.     

武陵山放线菌多样性

[目的]为了探究武陵山放线菌多样性,以便从新放线菌菌株中发现新的潜在药物先导化合物.[方法]从武陵山采集280份土样,采用纯培养的方法,用4种培养基分离到1134株放线菌.选择其中30株代表菌进行了初步分类鉴定;以3株细菌和7株农作物致病真菌作为指示菌,检测其抑菌活性;利用特异性引物扩增的方法,检测是否具有聚酮合酶(PKS Ⅰ、PKSⅡ)基因、非核糖体多肽合成酶(NRPS)基因和多烯类化合物合成酶(CYP)基因.[结果]分离到的武陵山放线菌中,链霉菌占70%以上,还有小单孢菌等8个科13个属,其中有5个菌株是潜在的新种.选取的30株实验菌对细菌、真菌有不同程度的抗菌活性;其中含有4类化合物合成基因的菌株占23%～60%.[结论]武陵山原始森林土壤中,放线菌多样性很丰富,且存在很多未开发的稀有类群.有抑菌活性的菌株,可用于进一步的药物开发利用.     

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and plasmid profile of soil and clinical isolates of Nocardia.

The aim of this study was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for generic and species-specific differentiation of Nocardia from other morphologically similar bacterial pathogens. To examine the utility of the PCR-RFLP approach in species identification, genomic DNA was prepared from 40 soil isolates, 10 clinical isolates and 8 reference strains of Nocardia. A set of oligonucleotide primers was designed from the consensus sequence of the highly conserved groEL gene that encodes the 65-kDa heat shock protein (hsp 65). The primers selectively amplified 422 bp DNA from the genomic DNA of all Nocardia species and isolates. The digestion of the amplicons with the restriction enzyme MspI produced DNA fragments that could differentiate between different Nocardia species regardless of their origin. Additionally, the RFLP patterns obtained with restriction enzymes MspI and BsaHI resulted in the differentiation of six Nocardia species which were earlier identified by biochemical tests. Apart from soil isolates of N. asteroides, which had shown some degree of genotypic polymorphism with BsaHI, the remaining taxa yielded more consistent results. Our results on the isolation of plasmids indicated that their occurrence is not a consistent feature in Nocardia species. It is neither related to the source of origin (clinical versus saprobic), nor to virulence, anti-microbial resistance or species specificity.     

繁茂膜海绵中可培养稀有放线菌的多样性

摘要：【目的】本文旨在尝试改进分离培养方法从大连海域繁茂膜海绵中筛选稀有放线菌,并对其多样性进行研究。【方法】根据繁茂膜海绵元素组成配制微量元素溶液,加入到放线菌分离培养基中,同时将部分培养基稀释成寡营养培养基,结合富集培养法,对繁茂膜海绵中放线菌进行分离培养。采用16S rDNA的限制性片断长度多态性(Restriction Fragment Length Polymorphism, RFLP)分析和序列分析,揭示其多样性。【结果】共获得可培养放线菌59株,通过形态、颜色观察,将其归为27个类群。RFLP分析表现为15种不同的图谱类型。16S rDNA序列分析表明：它们分别属于放线菌的10个属,其中布劳氏菌属（Prauseria）和糖单胞菌属（Saccharomonospora）是首次报道从海绵中分离培养。【结论】改进的分离培养基适合于繁茂膜海绵中稀有放线菌的分离培养,进一步揭示了该海绵中丰富的稀有放线菌,同样的方法有可能应用于其他海绵放线菌的分离培养。     

A new preferential medium for enumeration and isolation of desert actinomycetes

In order to facilitate the discovery of novel actinomycetes from the Egyptian deserts, which can be useful as new sources for bioactive metabolites, different media for enumeration and isolation of desert actinomycetes have been tested. For this purpose, 30 soil samples from different six sites representing the Western and Eastern deserts of Egypt were collected. The two deserts are considered hyper-arid and the soil characteristics were determined. The media used were glucose–yeast extract agar, soil extract agar and a new minimal medium (MM) containing glucose, yeast extract and mineral salts. The effects of the soil characteristics on the total viable actinomycete counts on the three media were evaluated. The results showed that the highest actinomycete count in samples from five out of six sites was obtained on MM. Also MM was more selective for actinomycetes and significantly decreased the number of fungal colonies and to a lower extent the number of bacterial colonies. Moreover, it supported the development of different and diverse groups of actinomycetes. From the results obtained in this study, MM is a new useful medium for enumeration and selective isolation of actinomycetes from the desert soils.     

First human case of nocardiosis caused by Nocardia pseudobrasiliensis in Japan

Nocardia sp. IFM 0896, an actinomycete with biochemical characteristics that differed from Nocardia brasiliensis, was isolated from a 71-year-old Japanese man with a history of tuberculosis and cancer. Although the isolate was tentatively identified as N. brasiliensis, the morphological and physiological characteristics of strain IFM 0896 were different from those of N. brasiliensis IFM 0236^T. The results of 16S rRNA gene sequence phylogenies and PCR-RFLP analysis of a heat shock protein revealed that Nocardia sp. IFM 0896 belongs to the species N. pseudobrasiliensis. This is the first clinical isolation report of N. pseudobrasiliensis in Japan.This revised version was published online in October 2005 with corrections to the Cover Date.