Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola

The aim of this study was to isolate bacteria with antimicrobial activities from the marine sponges Aplysina aerophoba and Aplysina cavernicola. The obtained 27 isolates could be subdivided into eight phylogenetically different clusters based on comparative sequence analysis of their 16S rDNA genes. The sponge isolates were affiliated with the low (Bacillus) and high G+C Gram-positive bacteria (Arthobacter, Micrococcus), as well as the alpha-Proteobacteria (unknown isolate) and gamma-Proteobacteria (Vibrio, Pseudoalteromonas). One novel Bacillus species was identified and two species were closely related to previously uncharacterized strains. Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the alpha-Proteobacteria. The sponge isolates show antimicrobial activities against Gram-positive and Gram-negative reference strains but not against the fungus Candida albicans. A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates. Antimicrobial activities were also found against clinical isolates, i.e. multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients. The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential.     

常见变质水性工业产品中的微生物种类和耐药性分析

为了科学治理常见变质水性工业产品中的腐败微生物,对其进行分离、鉴定和分类,同时以卡松、布罗波尔、甲基异噻唑啉酮和苯并异噻唑啉酮四种杀菌防腐剂对6种标准细菌菌株的最小抑菌浓度(MIC)作为耐药性标准,对腐败细菌进行耐药性评估分析.结果显示,日化用品变质样中革兰氏阴性细菌约占80.00％(克雷伯氏菌属、假单胞菌属、伯克霍尔...     

Comparative study of microbial communities from cultured and natural population of the mussel Mytilus trossulus in Peter the Great bay

The 525 strains of heterotrophic bacteria isolated from natural and cultured populations of the mussel Mytilus trossulus and the surrounding seawater were identified to a genus level on the basis of phenotypic analysis and the fatty acid composition of cell lipids. Gram-negative isolates were dominated by six genera of the family Enterobacteriaceae and by the genera Pseudoalteromonas, Vibrio, Photobacterium, Cytophaga/Flavobacterium/Bacteroides, Pseudomonas, and Moraxella, Gram-positive isolates were mainly represented by the genus Streptomyces. The taxonomic compositions of natural and cultured populations of the mussel M. trossulus in Peter the Great Bay were similar.     

Bladder cytopathic effects associated with Gram-negative bacterial genera

The interaction of bacteria belonging to several genera with the bladder epithelium was studied. An in vivo rat bladder model system was used to evaluate the cytopathic effects of members of eight representative genera of bacteria and six strains ofEscherichia coli on the bladder surface. Alterations were noted mainly with the Gram-negative bacteria (Escherichia, Klebsiella, Enterobacter, Proteus, Pseudomonas), although some strains were associated with more cytopathology than others. Foci of cytopathology consisted of swelling, exfoliated epithelial cells, strand formation, and ulceration. Aggregates of bacteria, epithelial cells, and debris were shed into the bladder lumen. In contrast, members of three genera of Gram-positive organisms (Lactobacillus, Streptococcus, Staphylococcus) caused little or no cytopathology. The study strongly suggests that the bladder responds in a relatively uniform manner to viable Gram-negative organisms, while the response to Gram-positive organisms is minimal.     

Multidrug resistance in hydrocarbon-tolerant Gram-positive and Gram-negative bacteria

New Gram-positive and Gram-negative bacteria were isolated from Poeni oily sludge, using enrichment procedures. The six Gram-positive strains belong to Bacillus, Lysinibacillus and Rhodococcus genera. The eight Gram-negative strains belong to Shewanella, Aeromonas, Pseudomonas and Klebsiella genera. Isolated bacterial strains were tolerant to saturated (i.e., n-hexane, n-heptane, n-decane, n-pentadecane, n-hexadecane, cyclohexane), monoaromatic (i.e., benzene, toluene, styrene, xylene isomers, ethylbenzene, propylbenzene) and polyaromatic (i.e., naphthalene, 2-methylnaphthalene, fluorene) hydrocarbons, and also resistant to different antimicrobial agents (i.e., ampicillin, kanamycin, rhodamine 6G, crystal violet, malachite green, sodium dodecyl sulfate). The presence of hydrophilic antibiotics like ampicillin or kanamycin in liquid LB-Mg medium has no effects on Gram-positive and Gram-negative bacteria resistance to toxic compounds. The results indicated that Gram-negative bacteria are less sensitive to toxic compounds than Gram-positive bacteria, except one bacteria belonging to Lysinibacillus genus. There were observed cellular and molecular modifications induced by ampicillin or kanamycin to isolated bacterial strains. Gram-negative bacteria possessed between two and four catabolic genes (alkB, alkM, alkB/alkB1, todC1, xylM, PAH dioxygenase, catechol 2,3-dioxygenase), compared with Gram-positive bacteria (except one bacteria belonging to Bacillus genus) which possessed one catabolic gene (alkB/alkB1). Transporter genes (HAE1, acrAB) were detected only in Gram-negative bacteria.     

Comparative Study of Microbial Communities from Cultured and Natural Populations of the Mussel Mytilus trossulus in Peter the Great Bay

The 525 strains of heterotrophic bacteria isolated from natural and cultured populations of the mussel Mytilus trossulus and the surrounding seawater were identified to a genus level on the basis of phenotypic analysis and the fatty acid composition of cell lipids. Gram-negative isolates were dominated by six genera of the family Enterobacteriaceae and by the genera Pseudoalteromonas, Vibrio, Photobacterium, Cytophaga/Flavobacterium/Bacteroides, Pseudomonas, and Moraxella. Gram-positive isolates were mainly represented by the genus Streptomyces. The taxonomic compositions of natural and cultured populations of the mussel M. trossulus in Peter the Great Bay were similar.     

Diversity analysis of diazotrophic bacteria associated with the roots of tea (Camellia sinensis (L.) O. Kuntze)

The diversity elucidation by amplified ribosomal DNA restriction analysis and 16S rDNA sequencing of 96 associative diazotrophs, isolated from the feeder roots of tea on enriched nitrogen-free semisolid media, revealed the predominance of Gram-positive over Gram-negative bacteria within the Kangra valley in Himachal Pradesh, India. The Gram-positive bacteria observed belong to two taxonomic groupings; Firmicutes, including the genera Bacillus and Paenibacillus; and Actinobacteria, represented by the genus Microbacterium. The Gram-negative bacteria included alpha-Proteobacteria genera Brevundimonas, Rhizobium, and Mesorhizobium; gamma-Proteobacteria genera Pseudomonas and Stenotrophomonas; and beta-Proteobacteria genera Azospira, Burkholderia, Delftia, Herbaspirillum and Ralstonia. The low level of similarity of two isolates, with the type strains Paenibacillus xinjiangensis and Mesorhizobium albiziae, suggests the possibility of raising species novum. The bacterial strains of different phylogenetic groups exhibited distinct carbon-source utilization patterns and fatty acid methyl ester profiles. The strains differed in their nitrogenase activities with relatively high activity seen in the Gramnegative strains exhibiting the highest similarity to Azospira oryzae, Delftia lacustris and Herbaspirillum huttiense.     

Biolog细菌自动鉴定系统应用初探

利用BiloogMicrostation细菌自动鉴定系统（3.50版）对已知的9个属23株菌进行了鉴定。24hBiolog系统鉴定结果：12林革兰氏阴性菌中，9株可准确鉴定到种的水平（75％），3株未达到属的水平。11株革兰氏阳性菌均属于芽孢杆菌属，全部鉴定到属的水平，9株准确鉴定到种的水平（81.8％）。总计属的水平准确率86.9％（20／23），种的水平准确率78.2％（18／23）。     

Classification and identification of bacteria by Fourier-transform infrared spectroscopy.

This study describes a computer-based technique for classifying and identifying bacterial samples using Fourier-transform infrared spectroscopy (FT-IR) patterns. Classification schemes were tested for selected series of bacterial strains and species from a variety of different genera. Dissimilarities between bacterial IR spectra were calculated using modified correlation coefficients. Dissimilarity matrices were used for cluster analysis, which yielded dendrograms broadly equated with conventional taxonomic classification schemes. Analyses were performed with selected strains of the taxa Staphylococcus, Streptococcus, Clostridium, Legionella and Escherichia coli in particular, and with a database containing 139 bacterial reference spectra. The latter covered a wide range of Gram-negative and Gram-positive bacteria. Unknown specimens could be identified when included in an established cluster analysis. Thirty-six clinical isolates of Staphylococcus aureus and 24 of Streptococcus faecalis were tested and all were assigned to the correct species cluster. It is concluded that: (1) FT-IR patterns can be used to type bacteria; (2) FT-IR provides data which can be treated such that classifications are similar and/or complementary to conventional classification schemes; and (3) FT-IR can be used as an easy and safe method for the rapid identification of clinical isolates.     

Microorganisms isolated from the water phase of tropospheric clouds at the Puy de Dôme: major groups and growth abilities at low temperatures

This work constitutes the first large report on aerobic cultivable microorganisms present in cloud water. Seven cloud-event samples were collected at the Puy de D?me summit, and cultivation was performed leading to the isolation of 71 bacterial, 42 fungal and 15 yeast strains. Most of the fungi isolated were of Cladosporium or Trametes affiliation, and yeasts were of Cryptococcus affiliation. Bacteria, identified on the basis of their 16S rRNA gene sequence, were found to belong to Actinobacteria, Firmicutes, Proteobacteria (Alpha, Beta and Gamma subclasses) and Bacteroidetes phyla, and mainly to the genera Pseudomonas, Sphingomonas, Staphylococcus, Streptomyces, and Arthrobacter. These strains appear to be closely related to some bacteria described from cold environments, water (sea and freshwater), soil or vegetation. Comparison of the distribution of Gram-negative vs. Gram-positive bacteria shows that the number of Gram-negative bacteria is greater in summer than in winter. Finally, a very important result of this study concerns the ability of half of the tested strains to grow at low temperatures (5 degrees C): most of these are Gram-negative bacteria, and a few are even shown to be psychrophiles. On the whole, these results give a good picture of the microbial content of cloud water in terms of classification, and suggest that a large proportion of bacteria present in clouds have the capacity to be metabolically active there. This is of special interest with respect to the potential role of these microorganisms in atmospheric chemistry.     

Biodiversity of moderately halophilic bacteria in hypersaline habitats in Egypt

Screening bacteria from different saline environments in Alexandria. Egypt, lead to the isolation of 76 Gram-negative and 14 Gram-positive moderately halophilic bacteria. The isolates were characterized taxonomically for a total of 155 features. These results were analyzed by numerical techniques using simple matching coefficient (SSM) and the clustering was achieved by the unweighed pair-group method of association (UPGMA). At 75% similarity level the Gram-negative bacteria were clustered in 7 phena in addition to one single isolate, whereas 4 phena represented the Gram-positive. Based on phenotypic characteristics, it is suggested that the Gram-negative bacteria belong to the genera Pseudoalteromonas, Flavobacterium, Chromohalobacter, Halomonas and Salegentibacter, in addition to the non-identified single isolate. The Gram-positive bacteria are proposed to belong to the genera Halobacillus, Salinicoccus, Staphylococcus and Tetragenococcus. This study provides the first publication on the biodiversity of moderately halophilic bacteria in saline environments in Alexandria, Egypt.     

Bacteriocin-like substance (BLS) production in Aeromonas hydrophila water isolates

30 Aeromonas hydrophila water isolates were tested for bacteriocin-like substance (BLS) production using a target panel of closely related microorganisms and other Gram-positive and Gram-negative bacteria, including food-borne pathogens. A. hydrophila showed antibacterial activity against one or more indicator microorganisms, but the activity emerged only with non-phylogenetically related genera or species. In particular all A. hydrophila showed antibacterial activity against one or more of the tested Staphylococcus strains, five against Listeria spp. (Listeria seeligeri, Listeria welshimeri and Listeria ivanovii), and eight presented a weak antagonistic activity towards Streptococcus agalactiae and Lactobacillus spp. Inhibitory activity was not observed against the other Gram-positive (Listeria monocytogenes, Listeria innocua and Enterococcus spp.) and Gram-negative tested strains, including Aeromonas sobria, Aeromonas caviae and the same A. hydrophila, when used as indicator. Anti-staphylococcal activity was observed with a gradual increase of the inhibition zone during incubation and seemed to be influenced by A. hydrophila hemolytic expression. Extrachromosomal analysis showed the presence, in 70% of the strains, of one to five plasmids with molecular masses ranging from 2.1 to 41.5 MDa, but it was not possible to relate this result with BLS production.     

Genetic diversity of cultivable plant growth-promoting rhizobacteria in Korea

To elucidate the biodiversity of plant growth-promoting rhizobacteria (PGPR) in Korea, 7,638 bacteria isolated from the rhizosphere of plant species growing in many different regions were screened. A large number of PGPR were identified by testing the ability of each isolate to promote the growth of cucumber seedlings. After redundant rhizobacteria were removed via amplified rDNA restriction analysis, 90 strains were finally selected as PGPR. On the basis of 16S ribosomal RNA sequences, 68 Gram-positive (76%) and 22 Gram-negative (24%) isolates were assigned to 21 genera and 47 species. Of these genera, Bacillus (32 species) made up the largest complement, followed by Paenibacillus (19) and Pseudomonas (11). Phylogenetic analysis showed that most of the Grampositive PGPR fell into two categories: low- and high- G+C (Actinobacteria) strains. The Gram-negative PGPR were distributed in three categories: alpha-proteobacteria, beta- proteobacteria, and gamma-proteobacteria. To our knowledge, this is the largest screening study designed to isolate diverse PGPR. The enlarged understanding of PGPR genetic diversity provided herein will expand the knowledge base regarding beneficial plant-microbe interactions. The outcome of this research may have a practical effect on crop production methodologies.     

Phylogenetic diversity of aerobic saprotrophic bacteria isolated from the Daqing oil field

A diverse and active microbial community in the stratal waters of the Daqing oil field (China), which is exploited with the use of water-flooding, was found to contain aerobic chemoheterotrophic bacteria (including hydrocarbon-oxidizing ones) and anaerobic fermentative, sulfate-reducing, and methanogenic bacteria. The aerobic bacteria were most abundant in the near-bottom zones of injection wells. Twenty pure cultures of aerobic saprotrophic bacteria were isolated from the stratal waters. Under laboratory conditions, they grew at temperatures, pH, and salinity values typical of the stratal water from which they were isolated. These isolates were found to be able to utilize crude oil and a wide range of hydrocarbons, fatty acids, and alcohols. Phylogenetic analysis carried out with the use of complete 16S rRNA sequences showed that the isolates could be divided into three major groups: gram-positive bacteria with a high and a low G + C content of DNA and gram-negative bacteria of the gamma-subclass of the Proteobacteria. Gram-positive isolates belonged to the genera Bacillus, Brevibacillus, Rhodococcus, Dietzia, Kocuria, Gordonia, Cellulomonas, and Clavibacter. Gram-negative isolates belonged to the genera Pseudomonas and Acinetobacter. In their 16S rRNA sequences, many isolates were similar to the known microbial species and some probably represented new species.     

Isolation of Bacillus strains from the rhizosphere of cereals and in vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage micro-organisms

Bacillus strains were isolated from the rhizosphere of cereals in order to be used as natural biocontrol agents (BCAs). They were screened for antagonism in vitro against various test micro-organisms. The isolates showing antagonism were identified to species level. A combination of techniques was employed for the isolation of Bacillus species. Using the direct method, only one of the 25 isolates screened showed antagonistic properties. This strain (IFS-01) was identified by means of API test strips and the ATB Plus computer programme. It proved to be Bacillus subtilis and consequently has been designated as Bacillus subtilis IFS-01. This strain produced either a broad spectrum antimicrobial compound or several compounds with different activities. The fungi and Gram-positive bacteria were more sensitive to the antagonistic isolate than the Gram-negative bacteria. A Bacillus strain producing BCAs which can be used as biopesticides or organic preservatives has been isolated and identified.     

Compatible solutes in new moderately halophilic isolates

Abstract Using high performance liquid chromatography and nuclear magnetic resonance techniques, the compatible solutes of some moderately halophilic bacteria were studied. The following accepted species of moderately halophilic bacteria were included: Volcaniella eurihalina and Deleya salina among Gram-negative rods, and Salinicoccus roseus and Salinicoccus hispanicus among Gram-positive cocci. Besides these strains we have also screened other new isolates, including Marinomonas species and Gram-positive cocci and rods. The tetrahydropyrimidine carboxylic acid 'ectoine' was found to be the main compatible solute in the Gram-negative strains tested when these were grown in glucose-mineral medium. In addition, betaine was accumulated from complex media containing yeast extract. Among the Gram-positive strains investigated, the solutes proline (bacillus 30, Salinicoccus ) and hydroxyectoine (coccus 28) also played an important role, while alanine, glucose, glutamate, glutamine and trehalose occurred as minor components. We also detected two recently described compatible solutes: Nδ -acetylornithine and a homologous compound, Nε -acetyllysine. Representatives of distinct phenotypic groups of Gram-positive cocci and rods were clearly distinguished by their solute pattern.     

Distribution of glutathione transferases in Gram-positive bacteria and Archaea

Glutathione transferases (GSTs) have been widely studied in Gram-negative bacteria and the structure and function of several representatives have been elucidated. Conversely, limited information is available about the occurrence, classification and functional features of GSTs both in Gram-positive bacteria and in Archaea. An analysis of 305 fully-sequenced Gram-positive genomes highlights the presence of 49 putative GST genes in the genera of both Firmicutes and Actinobacteria phyla. We also performed an analysis on 81 complete genomes of the Archaea domain. Eleven hits were found in the Halobacteriaceae family of the Euryarchaeota phylum and only one in the Crenarchaeota phylum. A comparison of the identified sequences with well-characterized GSTs belonging to both Gram-negative and eukaryotic GSTs sheds light on their putative function and the evolutionary relationships within the large GST superfamily. This analysis suggests that the identified sequences mainly cluster in the new Xi class, while Beta class GSTs, widely distributed in Gram-negative bacteria, are under-represented in Gram-positive bacteria and absent in Archaea.     

Screening microalgae for some potentially useful agricultural and pharmaceutical secondary metabolites

Nearly two hundred microalgal strains (174 Chlorophyta and 23 Cyanobacteria) were screened against some bacteria, filamentous fungi and yeasts using a disc-diffusion type bioassay. From this initial screening, 10 Chlorophyta strains from three genera (Desmococcus, Chlorella and Scenedesmus) were selected because of their high antimicrobial activity. These 10 strains were partially purified and tested using MIC antimicrobial and microtiter IC₅₀ anticancer assays. These preselected algal strains showed a high incidence of antibacterial activity against both Gram-positive (9 out of 10 species) and Gram-negative (7 out of 10 species) bacteria. The extracts were also effective against some tumour cell lines.     

Impact of an urban effluent on the bacterial community structure in the Arga River (Spain), with special reference to culturable gram-negative rods

The Arga River is an interesting system in which to study the impact of urban effluent pollution because it receives a single effluent in the form of wastewater discharge from the city of Pamplona. To analyze the extent of this discharge, total bacteria, culturable heterotrophic bacteria, and Gram-negative heterotrophic bacteria were enumerated and 409 isolates of the latter were identified. One sampling station was located upstream from the inflow, while five were located up to 30 km downstream. Bacterial counts increased drastically downstream from the wastewater inflow. Total bacterial numbers decreased along the 30 km downstream, the last station attaining similar values to those recorded upstream from the discharge. However, culturable heterotrophic and Gram-negative heterotrophic bacteria levels generally remained significantly higher within the 30 km zone investigated. Among the 409 isolates identified, Aeromonas spp. were the most frequent both upstream and downstream from the discharge. In contrast, although strains belonging to different genera of Enterobacteriaceae were found in all stations, their occurrence was significantly higher just downstream from the polluted discharge. Acinetobacter spp., which were never found upstream, were detected in all stations below the discharge. Our results clearly show that the bacterial community structure changes definitively downstream from the discharge and that Aeromonas were common throughout the sampling zone. Thus they cannot be considered good indicators of pollution in this setting compared to some genera of Enterobacteriaceae or some species of Acinetobacter, the distribution of which correlated better with the distance from the wastewater discharge.     

Bacterial diversity in spent mushroom compost assessed by amplified rDNA restriction analysis and sequencing of cultivated isolates

Spent mushroom compost (SMC) is the residual by-product of commercial Agaricus spp. cultivation, and it is mainly composed of a thermally treated cereal straw/animal manure mixture colonized by the fungal biomass. Research on the valorization of this material is mainly focusing on its use as soil conditioner and plant fertilizer. An investigation of the bacterial diversity in SMC was performed using molecular techniques in order to reveal the origin of SMC microflora and its potential effect on soil microbial communities after incorporation into agricultural soils. The bacterial population was estimated by the plate count method to a mean of 2.7 10⁹ colony forming units (cfu) per g of dry weight, while the numbers of Gram-positive and Gram-negative bacteria were 1.9 10⁹ and 4.9 10⁸ cfu per g dw respectively as estimated by enumeration on semi-selective media. Fifty bacterial isolates were classified into 14 operational taxonomic units (OTUs) following ARDRA-PCR of the 16S rDNA gene. Sequencing of the 16S rDNA amplicon assigned 12 of the 14 OTUs to Gram-positive bacteria, associated with the genera Bacillus, Paenibacillus, Exiguobacterium, Staphylococcus, Desemzia, Carnobacterium, Brevibacterium, Arthrobacter and Microbacterium of the bacterial divisions Firmicutes and Actinobacteria. Two bacterial groups have phylogenetic links with the genera Comamonas and Sphingobacterium, which belong to β-Proteobacteria and Bacteroidetes respectively. Two potentially novel bacteria are reported, which are associated with the genera Bacillus and Microbacterium. Most of the bacteria identified are of environmental origin, while strains related to species usually isolated from insects, animal and clinical sources were also detected. It appears that bacterial diversity in SMC is greatly affected by the origin of the initial material, its thermal pasteurization treatment and the potential unintended colonization of the mushroom substrate during the cultivation process.