Studies on antagonistic marine actinomycetes from the Bay of Bengal

Screening of 26 marine sediment samples near 9 islands of the Andaman Coast of the Bay of Bengal resulted in the isolation of 88 isolates of actinomycetes. On the basis of sporophore morphology and structure of the spore chain, 64 isolates were assigned to the genus Streptomyces, 8 isolates to the genus Micromonospora, 5 to the genus Nocardia, 7 to the genus Streptoverticilium and 4 to the genus Saccharopolyspora. Among 64 Streptomyces spp., 44 isolates showed antibacterial activity and 17 isolates showed antifungal activity. Three isolates showed very promising antagonistic activities against multi-drug resistant pathogens.     

Isolation of faecal coliform bacteria from the American alligator (Alligator mississippiensis)

Aims: To determine whether American alligators (Alligator mississippiensis) are an unrecognized poikilothermic source of faecal coliform and/or potential pathogenic bacteria in South Carolina’s coastal waters. Methods and Results: Bacteria from the cloaca of American alligators, as well as bacteria from surface water samples from their aquatic habitat, were isolated and identified. The predominant enteric bacteria identified from alligator samples using biochemical tests included Aeromonas hydrophila, Citrobacter braakii, Edwardsiella tarda, Escherichia coli, Enterobacter cloacae, Plesiomonas shigelloides and putative Salmonella, and these were similar to bacteria isolated from the surface waters in which the alligators inhabited. Based on most‐probable‐number enumeration estimates from captive alligator faeces, faecal coliform bacteria numbered 8·0 × 10⁹ g^?1 (wet weight) of alligator faecal material, a much higher concentration than many other documented endothermic animal sources. Conclusions: A prevalence of enteric bacteria, both faecal coliforms and potential pathogens, was observed in American alligators. The high faecal coliform bacterial density of alligator faeces may suggest that alligators are a potential source of bacterial contamination in South Carolina coastal waters. Significance and Impact of the Study: These findings help to increase our understanding of faecal coliform and potential pathogenic bacteria from poikilothermic reptilian sources, as there is the potential for these sources to raise bacterial water quality levels above regulatory thresholds.     

地黄内生放线菌的分离及地黄轮纹病拮抗菌Streptomyces folium leaf-16的新种鉴定

药用植物内生放线菌具有合成天然活性化合物的潜力,放线菌新种是寻找新型抗生素先导化合物的一个重要来源。【目的】挖掘药用植物地黄内生放线菌资源,并对地黄轮纹病拮抗菌株leaf-16进行新种鉴定。【方法】本研究采用五步消毒法分离河南道地药材地黄的内生放线菌,以地黄轮纹病原真菌草茎点霉(Phoma herbarum)为指示菌,采用平板对峙法筛选对该病菌有抑制作用的菌株,16S rRNA基因测序发现一株抗地黄轮纹病的放线菌新种leaf-16。通过形态、生理生化、细胞壁化学组分和分子生物学等特征对菌株leaf-16进行多相分类学鉴定。【结果】经平板对峙实验得到8株抗地黄轮纹病的放线菌,其中菌株leaf-16经16S rRNA基因测序、形态比较、生理生化、化学组分和分子生物学以及DNA-DNA杂交分析,确定菌株leaf-16为1株链霉菌新种,并命名为Streptomyces folium。【结论】菌株leaf-16为1株链霉菌新种,具有抑制地黄轮纹病原真菌的活性,为进一步分离新型抗地黄轮纹病的生物制剂奠定物质基础。     

Isolation of endophytic actinomycetes from selected plants and their antifungal activity

The isolation of endophytic actinomycetes from surface-sterilized tissues of 36 plant species was made using humic acid–vitamin (HV) agar as a selection medium. Of the 330 isolates recovered, 212 were from roots, 97 from leaves and 21 isolates from stems with a prevalence of 3.9, 1.7 and 0.3%, respectively. Identification of endophytic actinomycetes was based on their morphology and the amino acid composition of the whole-cell extract. Most isolates were classified as Streptomyces sp. (n = 277); with the remainder belonging to Microbispora sp. (n = 14), Nocardia sp. (n = 8) and Micromonospora sp. (n = 4). Four isolates were unclassified and 23 were lost during subculture. The most prevalent group of isolates were the Streptomyces sp. occurring in 6.4% of the tissue samples of Zingiber officinale. Scanning electron microscopy investigation of this plant revealed that 7.5% of the root and 5% of the leaf samples contained endophytes. Three of the Streptomyces sp. isolates strongly inhibited Colletotrichum musae, five were very active against Fusarium oxysporum and two strongly inhibited growth of both test fungi.     

Zooplankton seasonal dynamics in a recently filled mine pit lake: the effect of non-indigenous <Emphasis Type="Italic">Daphnia</Emphasis> establishment

We examined the temporal and vertical dynamics of zooplankton in Weavers Lake, New Zealand, between October 2004 and October 2005, at a time when it was colonised by a non-indigenous Daphnia species. Zooplankton community composition changed during the study from one of rotifer dominance (e.g. Asplanchna, Polyarthra, Brachionus and Keratella species) to cladoceran (Daphnia dentifera) dominance. Temporal changes in zooplankton community composition were strongly associated with a gradual increase in lake water clarity, and were attributable to the highly efficient filter feeding of D. dentifera. The corresponding reduction in rotifer densities may have resulted from the superior competitive abilities of the newly established Daphnia. As Daphnia were rare inhabitants of New Zealand lakes before 1990, the arrival and rapid spread of the non-indigenous D. dentifera has lead to widespread changes in both water clarity and zooplankton community composition. An apparent lack of mixing in the lake was facilitated by the lake’s extremely small surface area:depth ratio. However, we conclude that physical features of the lake had minimal influence on water clarity relative to the invasion of D. dentifera.     

Dynamics of phytoplankton pigment characteristics in the Biya head water

Concentrations of phytoplankton photosynthetic pigments were measured at the Biya head water (in the immediate vicinity of Lake Teletskoe) every 10 days from July 1998 through December 2001. Comparison of phytoplankton pigment characteristics at the river’s head waters and at the surface of the lake’s pelagic zone for the ice-free period shows that the monitoring data give a clear picture of the formation and functioning of the lake phytoplankton. Analysis of seasonal fluctuations of pigment characteristics and their proportions revealed characteristic stages in the development of lake algal cenoses. We have demonstrated similarities and differences in the seasonal dynamics of the phytoplankton between Lake Teletskoe, a deep oligotrophic lake in the south of West Siberia, as compared with the general pattern observed in temperate lakes. According to the concentration of chlorophyll a, the trophic status of the lake’s pelagic zone and the Biya head water is ultraoligotrophic-oligotrophic. Yellow:green pigment ratio ranks Lake Teletskoe among “carotenoid lakes“. Low coefficients of linear correlation between chlorophyll a, water level and biogenic matter concentration shows that the relationships analyzed are far more complex than has been inferred from the combined effect of abiotic and biotic factors.     

Isolation and characterization of actinomycetes from healthy goat faeces

Aims: To isolate and characterize actinomycetes with probiotic activities from healthy goat faeces. Methods and Results: Faecal actinomycetes were isolated by dilution methods and identified by 16S rRNA gene sequence analysis. The hydrolytic enzyme activities were analysed by clear zone formation. The antimicrobial activities and resistance to heavy metals were tested by growth inhibition methods. The isolates belong to a small group of actinobacterial genera, including Streptomyces, Nocardiopsis and Oerskovia. The Oerskovia was the most widely distributed genus among the cultures. The proportion of streptomycete‐like strains producing amylase or protease is significantly higher than those of other actinomycetes (P < 0·05). Compared with streptomycete‐like strains, a higher proportion of (α‐ or β‐) galactase‐producing other actinomycetes was found in goat faeces. More than 50% of streptomycete‐like strains showed activities against test fungi. Streptomycetes could tolerate 0·25 mmol l^?1 Cr₂O₇^2?, 2 mmol l^?1 Ni²⁺; however, other actinomycetes are liable to 40 mmol l^?1 Fe³⁺ and 0·25 mmol l^?1 Cr₂O₇^2? and resistant to 5 mmol l^?1 Ni²⁺ and 2 mmol l^?1 Cu²⁺. Conclusions: The different physiological characteristics of the actinomycetes suggested that the cooperation in the actinomycetes might be involved in their association with goat. Significance and Impact of the Study: Probiotic mixtures based on faecal actinomycetes showed potentials in animal production.     

Genetic and metabolic diversity of streptomycetes in pulp and paper mill effluent treated crop fields

Irrigation of farm field with water mixed with pulp and paper mill effluent from Century pulp and paper mill in Uttrakhand state of India for over last 25 years in succession increased streptomycetes population (120 × 10⁵) compared to the fresh water irrigated fields (48 × 10³ in WIF). Denaturing gradient gel electrophoresis, amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, BIOLOG™ substrate usage, production of extracellular enzymes (xylanase and cellulase) and plant growth promoting attributes were applied to monitor changes in genetic and metabolic diversity of streptomycetes. Significant variation was observed for production of extracellular enzymes, Indolic compounds, siderophore and P-solubilisation among isolates. Metabolic substrate usage of Streptomyces isolates was evaluated using the BIOLOG™ GP2 plates and unique carbon substrate usage profiles were observed. Based on 16S rRNA gene sequencing, the isolates were identified as Streptomyces variabilis, Streptomyces spp. S. glaucescens, S. viridochromogenes, S. cinnabarinus, S. aburaviensis, S. viridis, S. xylophagus, S. macrosporeus, S. thermocarboxydus, and S. albogriseolus. The diversity index parameters like Shannon index, reciprocal of Simpson’s index (1/D), and Pielou index of evenness based on ARDRA revealed that streptomycetes community in effluent irrigated field (EIF) was more diverse. DGGE profiles of Streptomyces specific 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar in both soils.     

Chromosome topology and genome size of selected actinomycetes species

Information about the genome organization of actinomycetes species is restricted to a few genera: Corynebacterium, Mycobacterium, Rhodococcus, Saccharopolyspora and Streptomyces. Streptomyces species and Saccharopolyspora erythraea were shown to contain a single linear 8 Mb chromosome. In contrast, the Corynebacterium, Mycobacterium and Rhodococcus species studied were demonstrated to possess a smaller (3 Mb–6.5 Mb) single circular chromosome. To investigate whether linear chromosome topology and genome sizes above 7 Mb are unique features of Streptomyces and S. erythraea we have started to investigate the chromosome topology, the genome size and the status of accessory elements of additional actinomycetes species: Actinoplanes philippinensis, Amycolatopsis orientalis, Micromonospora chalcea, Nocardia asteroides, Rhodococcus opacus and Streptoverticillium abikoense. Our data which are based on PFGE experiments clearly suggest that large genome sizes and chromosome linearity are seen in mycelium forming actinomycetes genera. In addition we have identified large linear plasmids in Nocardia asteroides, Streptoverticillium abikoense and Rhodococcus opacus.     

库木塔格沙漠土壤细菌多样性及具有群体感应抑制活性放线菌筛选

【背景】群体感应抑制剂(quorum sensing inhibitor,QSI)作为抗生素潜在替代品,可有效降低致病菌传染性和毒性。沙漠土壤蕴藏着丰富的放线菌资源,是挖掘群体感应抑制剂的重要来源。【目的】解析库木塔格沙漠土壤细菌群落多样性,筛选并挖掘群体感应抑制活性放线菌资源。【方法】采用Illumina Nova Seq高通量测序技术揭示库木塔格沙漠土壤细菌群落组成,利用可培养方法进行土壤放线菌分离和鉴定;选用紫色杆菌CV026模型筛选群体感应抑制活性放线菌,并对其功能特性进行初步评价。【结果】Illumina Nova Seq高通量测序结果显示,样品土壤细菌涉及23门96目150属,优势菌门为变形菌门(Proteobacteria,61%)、放线菌门(Actinobacteria,28%),其中分枝杆菌属(Mycobacterium)为放线菌门最优势菌属(87.3%),其次为红球菌属(Rhodococcus,6.8%)和丙酸杆菌属(Cutibacterium,0.9%)。可培养方法共分离到108株放线菌,归属9科10属,其中优势菌属为链霉菌属(Streptomyces),占65....     

Characterization of actinomycetes isolated from the indoor air of the church of Saint Katherine Monastery, Egypt

Actinomycetes were isolated from the indoor air of the church of Saint Katherin Monastery during different visiting hours. Fifty air samples collected over one year by using automated air sampler were plated on four different media. The low nutrient-content medium 1/10 SC was significantly effective in recovery of actinomycetes compared to the other formula of rich media. Average counts of bacteria, actinomycetes and fungi were 487, 65 and 90 cfu/m³, respectively. Fifty-six morphologically different actinomycetes isolates were recovered during this study assigned into five different genera, in addition to three unidentified isolates. Tentative identification of the isolates indicated predominance of genus Streptomyces, representing 59% of the isolates. Isolates were screened for resistance to 11 antibiotics, antimicrobial activities towards seven microbial strains, Growth on 12 different carbon source, acid production and pigmentation. About 77% of actinomycete isolates were resistant to the antibiotics with different resistance patterns. 12.5% of the airborne actinomycetes exhibited antinmicrobial activities. The isolates showed wide variation in carbon source usage. Forty percent of the isolates were able to utilize all tested carbon sources while 80% were acid producers. Melanin production was produced by 34% of the isolates. While 45% of the isolates were able to produce other diffusible pigments, the majority of the pigments were brown in color however; yellow, orange and green pigments were produced by a number of isolates. The impact of these activities on the historical objects of the church and the tourist’s health is discussed.     

Actinobacterial diversity from marine sediments collected in Mexico

Seventeen different media known to support the growth and isolation of members of the class Actinobacteria were evaluated as selective isolation media for the recovery of this microbial group from marine sediments samples collected in the Gulf of California and the Gulf of Mexico. A general selective isolation procedure was employed for six sediments and nearly 300 actinomycetes were recovered from the selective isolation plates. Full 16S rRNA gene sequencing revealed that the isolates belonged to several actinobacterial taxa, notably to the genera Actinomadura, Dietzia, Gordonia, Micromonospora, Nonomuraea, Rhodococcus, Saccharomonospora, Saccharopolyspora, Salinispora, Streptomyces, “Solwaraspora” and Verrucosispora. Previous works on marine sediments have been restricted to the isolation of members of the genera Micromonospora, Rhodococcus and Streptomyces. This study provides further evidence that Actinobacteria present in marine habitats are not restricted to the Micromonospora-Rhodococcus-Streptomyces grouping. Indeed, this first systematic study shows the extent of actinobacterial diversity that can be found in marine sediments collected in Mexico and probably, worldwide. The 16S rRNA gene sequences of marine isolates A1, AA2, AA6, AB1, AB2, AG1, AI2, AK1, AL2, AO1, AO3, AR1, AW1, B1, BB1, BC1, C5, R1, R2, R3, AV1, AE1, AI1, AN1 and AP1 determined in this study have been deposited under GenBank accession numbers EU714241–EU714258 and FJ462359–FJ462365, respectively.     

Farmer’s Lung Disease and Microbiological Composition of Hay: A Case–Control Study

Previous studies performed in France have suggested that handling hay contaminated with high amounts of moulds, and especially Absidia corymbifera and Eurotium amstelodami, may favour farmer’s lung disease. The circumstances favouring farmer’s lung disease and the distinctive microbiological composition of hay samples that provoke attacks need to be specified. We present a case–control study which investigates the agricultural practices and the microbiological composition of hay handled in patients with farmer’s lung disease as compared to those of a representative control population. Ten cases identified the hay they were handling at the onset of symptoms. The location, type of farm and working conditions were similar to those of the control farms. Conversely, the microbiological composition of hay differed, with significantly higher amounts of E. amstelodami (P < 0.01), A. corymbifera (P = 0.003), mesophilic Streptomyces (P < 0.01), thermophilic Streptomyces (P < 0.01) and Saccharomonospora viridis (P < 0.01) than in the control population. Our results demonstrate that hay identified by patients as having a harmful effect is characterized by a higher total amount of microorganisms, notably five microorganisms that seem discriminative. Mean concentrations are 2- to 115-fold higher in hay suspected to cause symptoms than in hay from a representative panel of farms. Handling hay with high amounts of these five microorganisms constitutes a risk factor for farmer’s lung disease that should be considered for the development of prophylactic measures.     

Identification of Actinomycetes Producing Phospholipase D with High Transphosphatidylation Activity

Previously we isolated six actinomycetes strains, 9-4, 10-1, 10-2, 10-3, 10-6, and 21-4, that produce phospholipase D (PLD) with high transphosphatidylation activity. In this study, we identified these strains, and the PLD activities were compared with those of reference strains. 16S rDNA sequences and DNA–DNA hybridization tests indicated taxonomic affiliations of strain 9-6 with Streptomyces senoensis, strains 10-1 and 10-6 with S. vinaceus, and strains 10-2 and 10-3 with S. racemochromogenes. Strain 21-4, though identified as a Streptomyces sp., could not be identified with any known species. Meanwhile, most of the culture supernatants of reference strains demonstrated no or very weak PLD activity, while those of our strains exhibited significantly higher activity. All of the strains in this study were identified as Streptomyces species. The PLD activity of our strains exceeded most of the reference Streptomyces strains. The findings in this study imply that the Streptomyces strains, although they are members of the same species, can produce different quantities of PLD enzyme.     

Diversity of actinomycetes isolated from Challenger Deep sediment (10,898 m) from the Mariana Trench

Thirty-eight actinomycetes were isolated from sediment collected from the Mariana Trench (10,898 m) using marine agar and media selective for actinomycetes, notably raffinose-histidine agar. The isolates were assigned to the class Actinobacteria using primers specific for members of this taxon. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to the genera Dermacoccus, Kocuria, Micromonospora, Streptomyces, Tsukamurella and Williamsia. All of the isolates were screened for genes encoding nonribosomal peptide and polyketide synthetases. Nonribosomal peptide synthetase sequences were detected in more than half of the isolates and polyketide synthases type I (PKS-I) were identified in five out of 38 strains. The Streptomyces isolates produced several unusual secondary metabolites, including a PKS-I associated product. In initial testing for piezotolerance, the Dermacoccus strain MT1.1 grew at elevated hydrostatic pressures.     

Characterization of <Emphasis Type="Italic">Edwardsiella tarda rpoS</Emphasis>: effect on serum resistance,chondroitinase activity,biofilm formation,and autoinducer synthetases expression

In this study, rpoS gene was identified from Edwardsiella tarda EIB202 and its functional role was analyzed by using an in-frame deletion mutant ∆rpoS and the complemental strain rpoS ⁺. Compared with the wild type and rpoS ⁺, ∆rpoS was impaired in terms of the ability to survive under oxidative stress and nutrient starvation, as well as the resistance to 50% serum of Scophthalmus maximus in 3 h, demonstrating essential roles of RpoS in stress adaptation. The rpoS mutant also displayed markedly increased chondroitinase activity and biofilm formation. Real-time polymerase chain reaction revealed that the expression level of quorum sensing autoinducer synthetase genes luxS and edwI was increased by 3.7- and 2.5-fold in the rpoS mutant strain. Those results suggested that rpoS might be involved in the negative or positive regulation of chondroitinase and biofilm formation, or quorum sensing networks in E. tarda, respectively. Although there were no obvious differences between the wild-type and the rpoS mutant in adherence of epithelioma papulosum cyprini (EPC) cell and in the lethality on fish model, rpoS deletion leads to the drastically reduced capacity for E. tarda to internalize in EPC cells, indicating that RpoS was, while not the main, the factor required for the virulence network of E. tarda.     

Chitinolytic Activity of Cold Tolerant Antagonistic Species of <Emphasis Type="Italic">Streptomyces</Emphasis> Isolated from Glacial Sites of Indian Himalaya

Seventy-eight isolates of actinomycetes were isolated from the soil samples collected from alpine zones of Pindari glacier region in Indian Himalaya. Following a plate based rapid screening using two test fungi, five efficient isolates (nos. HA1, HA2, HA6, HA40, and HA142) were selected for further characterization with special reference to their antagonistic properties. Based on phenotypic and genotypic characters, the isolates were identified up to species level. All the isolates belonged to the genus Streptomyces. The isolate nos. HA1 and HA2 were S. sampsonii and HA6, HA40 and HA142 were S. griseobrunneus, S. aurantiacus, and S. griseoluteus, respectively. The isolates showed strong antifungal properties against phytopathogenic test fungi in plate assays. All the isolates hydrolyzed glycol–chitin as a substrate in denaturing conditions showing variable amount of different isoforms.     

A high‐throughput and quantitative hierarchical oligonucleotide primer extension (HOPE)‐based approach to identify sources of faecal contamination in water bodies

As faecal contamination of recreational and drinking water impairs the water quality and threatens public health, water bodies are routinely monitored for faecal coliforms to detect contamination. However, faecal coliforms are facultative anaerobes that survive and reproduce in ambient waters, and their presence does not depict the origin of contamination. Therefore, the use of Bacteroides‐Prevotella 16S rRNA gene to perform faecal source tracking has been proposed and applied. Here, we demonstrate the use of a new molecular method termed hierarchical oligonucleotide primer extension (HOPE) to simultaneously detect human‐associated Bacteroides spp. and three clusters of cow‐, pig‐ and dog‐specific uncultivated Bacteroidales. The method correctly identifies the origin of faecal contamination when tested against human, cow, pig and dog faeces (n = 17, 17, 16 and 13 respectively), and in waters contaminated with faeces of known origins. Subsequent tests with a total of 21 blind samples show that HOPE is able to accurately indicate single or multiple sources of faecal contamination originating from pigs, cows and humans in 81% of the blind samples. HOPE can further correctly detect and identify faecal contamination in five sampling sites located along a canal in southern Taiwan, and the results are validated against conventional faecal coliform tests and quantitative PCR. Overall, this study demonstrates HOPE as a quantitative and high‐throughput method that can identify sources of faecal contamination.     

Antimicrobial, antitumor and in vivo cytotoxicity of actinomycetes inhabiting marine shellfish

Sixty-three actinomycete strains isolated from the marine shellfish Donax trunculus anatinus were phenotypically identified as ten genera, in addition to two unidentified strains. Their metabolic extracts exhibited wide antimicrobial activities towards 11 reference and clinical cultures; and 17.5% showed antitumor activities with solid tumor selectivity of four Nocardioides, Kitasatosporia and Streptomyces strains. Streptomyces 23-2B was particularly noted for its high antitumor activity against Ehrlich’s ascites carcinoma with plateau inhibitory effect at 500, 250 and 50 μg/ml concentrations, promising solid tumor selectivity and high cytotoxicity to human carcinoma of liver (HEPG2), cervix (HELA) and breast (MCF7) (IC₅₀: 3.89, 9.4 and 10 μg/ml, respectively). In vivo cytotoxicity of S.23-2B metabolites showed common sign of unimpaired kidney and liver functions, as indicated from non-significant elevation in serum enzymatic activities, urea, creatinine, total protein and albumin levels in response to 0.5 and 5 μg/g doses after alternate-day injection for 2 weeks. Microorganisms associated with the marine shellfish are suggested to be potential source of bioactive metabolites.