Margarita Stoilova-Disheva

Antimicrobial activity and resistance to heavy metals and antibiotics of heterotrophic bacteria isolated from sediment and soil samples collected from two Antarctic islands

In this study, 24 Antarctic bacteria, isolated from sediment and soil samples from Deception and Galindez Islands, were characterized for their antimicrobial activity and response to 13 antibiotics and seven heavy metals. Multiple antibiotic resistance was observed for 67% of sediment isolates and 92% of soil isolates, suggesting medium anthropogenic impact in these Antarctic regions. The results revealed a varying response of the Antarctic bacteria to the tested heavy metals. All isolates showed multiple metal resistance towards two to six heavy metals, with minimum inhibitory concentrations ranging from 1.0 mM to 23.2 mM. The majority of the strains in both groups were resistant to lead, nickel, copper and zinc. Plasmids were detected in 21% of isolates. A consortium of highly metal-resistant bacteria could be developed with potential application for biological treatment of wastewaters. Strains that are highly sensitive to cadmium would be promising for developing biosensors to detect this highly toxic heavy metal in environmental samples. All Antarctic bacteria were found to inhibit the growth of one to all eight of the tested indicator bacteria, and 87% inhibited the growth of one to all four of the yeast indicator cultures. Promising psychrotolerant strains were detected as a valuable source of new antimicrobial compounds.

Myroides guanonis sp. nov., isolated from prehistoric paintings

A novel psychrotolerant, strictly aerobic, non-motile, rod-shaped bacterial strain, designated IM13(T), was isolated from a sample taken from prehistoric guano paintings in Magura Cave, northwest Bulgaria and subjected to a polyphasic taxonomic study. Strain IM13(T) formed yellow colonies on LB agar plates and was Gram-staining-negative, heterotrophic and alkalitolerant. It grew optimally at pH 7.5 and 30 °C in the absence of NaCl. Phylogenetic analysis of the whole 16S rRNA gene revealed that strain IM13(T) branched with representatives of the genus Myroides with sequence similarity of 93-94 % with other species of the genus. The novel isolate contained iso-C15 : 0 (49.1 %), iso-C17 : 1ω9c (18.2 %) and iso-C17 : 0 3-OH (14.0 %) as dominant fatty acids. The DNA G+C content of strain IM13(T) was 33.5 mol%. Based on phylogenetic inference and phenotypic characteristics, it was concluded that strain IM13(T) represents a novel species of the genus Myroides, for which the name Myroides guanonis sp. nov. is proposed. The type strain is IM13(T) ( = DSM 26542(T) = NBIMCC 8736(T)).

Archaeal Diversity at Two Hot Springs, Rupi Basin, Bulgaria

ABSTRACT Archaea is the newest and the least understood domain which diversity and physiology are predominantly suggested after investigation of natural ecosystems. Recently many new archaeal groups have been detected by molecular phylogenetic methods. Diversity of thermophilic archaea in two hot springs at Rupi basin was studied by a culture-independent approach and high degree of covering biological diversity was reached in both clone libraries (RB and RB2). The 16S rRNA gene analysis of 379 clones grouped in 33 restriction profiles for each library showed presence of eleven groups from the domain Archaea, five of them being common for both springs. The retrieved sequences grouped together predominantly with environmental sequences and a small number of already known culturable Archaea was detected. A strong domination of HWCG III (Hot Water Crenarcheotic Group) was observed in both springs revealing strong domination of ammonia oxidizing metabolism. Although the difference in temperature between the two springs was only seven degrees, presence of hyperthermophiles was registered in the warmer spring. Four of the RB and nine of the RB2 sequences showed less than 97% similarity to the closest neighbour and were referred as representing new phylogenetic units.

Isolation and Characterization of a Psychrotolerant Streptomyces Strain from Permafrost Soil in Spitsbergen, Producing Phthalic Acid Ester

ABSTRACT An actinomycete, strain SB9, was isolated from a soil sample collected from permafrost in Spitsbergen, Arctic Ocean. The strain is psychrotolerant with growth at temperatures between 4°C and 30°C and optimal growth temperature between 20°C and 28°C. Phylogenetic analysis of the 16S rRNA gene sequence of the strain SB9 indicated a high level of sequence similarity (99%) to Streptomyces avidinii. The 16S rRNA gene sequence data of strain SB9 have been deposited in the GenBank databases under the accession number EU878377. The strain produces di-(2-ethylhexyl) phthalate (DEHP). The identification of (DEHP) was settled on the basis of electrospray mass spectrometry (ESI-MS), 1D NMR spectroscopy, and comparison with standards. Isolated compound showed antimicrobial activity against various microorganisms.

Molecular Characterization of the Archaeal Diversity in Vlasa Hot Spring, Bulgaria, by using 16S rRNA and Glycoside Hydrolase Family 4 Genes

ABSTRACT Biodiversity in the archaeal community from Vlasa hot spring, Velingrad, Bulgaria was investigated by sequence analysis of PCR amplified fragments of 16S rDNA and a metabolic gene of glycoside hydrolase 4 family (GH4). The 16S rRNA gene analysis demonstrated that the spring was inhabited predominantly by Crenarchaeota affiliated to two orders, Desulfurococcales (families Pyrodictiaceae and Desulfurococcaceae) and Thermoproteales. Almost half of the 16S rDNA clones were affiliated with hyperthermophilic anaerobic sulfate reducer Thermosphaera aggregans. Five 16S rDNA sequences were under cut off value of 97% homology to those of Genbank database and suggested the existence of novel phylogenetic units in the community. The archaeal diversity of the studied hot spring was further analyzed through sequence analysis of metagenomicaly cloned GH4 gene fragments. The comparative 16SrDNA and GH4 phylogenetic analyses demonstrated good correlation of the phylogenetic tree topology from both approaches, corresponding to the affiliation of the identified 16S rDNA sequences predominantly to organotrophic metabolizing taxons. The opportunity for simultaneous application of two molecular approaches, 16S rDNA and metabolite genes analyses for in dept characterization of environmental samples and directed metagenomic identification and cloning of metabolite genes of industrial interest is discussed.

Increase in tylosin production by a commercial strain of Streptomyces fradiae

Conventional mutagenesis (UV irradiation and exposure to nitrosoguanidine) as well as protoplast formation and regeneration were used to improve the antibiotic activity of a Streptomyces fradiae strain producing tylosin. Variants exceeding the activity of the initial producer strain by 0.5–28.3% were obtained. The most active variants were produced by a combined exposure to UV and nitrosoguanidine, as well as upon regeneration of protoplasts formed from the cells of clones produced by UV irradiation. Unstable inheritance of the trait of increased tylosin production was demonstrated.

DNA barcoding revealed Nematodospora valgi gen. nov., sp. nov. and Candida cetoniae sp. nov. in the Lodderomyces clade

During a yeast biodiversity survey conducted in 2009-2011 in Bulgaria (South Eastern Europe) five strains of a novel ascomycetous yeast species were isolated from the beetle Valgus hemipterus (Cetoniinae) collected from two localities, namely Osogovska Planina Mountain and Nature Park Zlatni Pyasatsi. Phylogenetic analysis using combined sequences of the D1/D2 domains of the large subunit ribosomal DNA (LSU rDNA) and the internal transcribed spacers 1 + 2 regions (ITS1+2) placed the novel species on a separate branch near the basal part of the Lodderomyces clade. The novel species has a unique ascospore morphology distinct from those of the closely related teleomorphic genus Lodderomyces. Based on phylogenetic analysis and morphology of the ascospores we propose Nematodospora valgi gen. nov., sp. nov. to accommodate these isolates (MB811804 D37S(T), MB802458). Two strains of a novel anamorphic yeast species were isolated from the beetles Cetonia aurata and Oxythyrea funesta (Cetoniinae) collected in East Rhodopies and Sofia city, respectively. DNA barcoding analysis placed the new yeast species within the Candida parapsilosis subclade. Here, we present the description of a new yeast species, Candida cetoniae sp. nov. (IMB1R2(T), MB803501) to accommodate these two strains. The ecology and biogeography of the insect-associated yeasts of the Lodderomyces clade is discussed.

DNA Barcoding of Yeasts from Selected Bulgarian Food Products

ABSTRACT Studies on biodiversity of yeasts in Bulgarian food ecosystems are scarce. In present article 14 yeast strains were isolated from various commercial Bulgarian food products and characterized by DNA barcoding analysis. The analysis has shown occurrence in food products various yeasts from the following genera- Saccharomyces, Kazachstania, Kluyveromyces, Pichia, Isatchenkia, Candida and Rhodotorula.

STRUCTURAL ELUCIDATION OF A BIOACTIVE METABOLITES PRODUCED BY STREPTOMYCES AVIDINII SB9 STRAIN, ISOLATED FROM PERMAFROST SOIL IN SPITSBERGEN, ARCTIC

ABSTRACT Streptomyces avidinii strain SB9 was isolated from permafrost soil samples in Spitsbergen, Arctic Ocean. The strain is psychrotolerant with optimal growth temperature between 20°C and 28°C. During our continuous search for unique secondary metabolites, we have investigated an arctic isolate of Streptomyces avidinii SB9 and we found for the first time, that Sreptomyces avidinii strain SB9 produces 2-amino-3-dodecanol [1], norophthalmic acid (2S)-2-amino-4-[[(1S)-1-(carboxymethylcarbamoyl) ethyl]carbamoyl] butanoic acid [2] and phthalic acid ester [3]. The compounds were purified by solvent extraction, silica gel column chromatography and preparative TLC consecutively. The structures of the compounds were elucidated by NMR experiments and mass spectrometric investigations. These compounds showed antimicrobial activity against important Gram-positive bacteria and fungi. Our studies are motivated by an interest in the functional role played by natural products in the ecological interactions of the strain with other members of the microbial community.

Preliminary Characterization of Yeasts and Actinomycetes Isolated from Mammalian Feces

ABSTRACT Yeasts and actinomycetes isolated from fecal pellets of various mammals from a zoo in Sofia, Bulgaria were identified using DNA barcoding analysis. The analysis has shown that yeast strains belong to the ascomycete genera Kluyveromyces, Clavispora, Pichia, Wickerhamomyces, Candida, Zygowilliopsis, Galactomyces, Hanseniaspora, and Debaryomyces. Basidiomycete yeasts belong to the genera Trichosporon, Cryptococcus, Rhodotorula and Guehomyces. All actinomycete strains were identified as Thermoactinomyces saccharii. Additionally, extracellular enzymatic activity and ability to grow at various temperatures of isolated strains were analyzed.