Soňa Javoreková

Characteristics of microbial biomass carbon and respiration activities in arable soil and pasture grassland soil.

The aim of our work was to survey and state the representative values and range of the microbial biomass carbon (Cmic) and microbial respiration activities in arable soils of three types (Chernozem, Luvisol, Planosol) and in pasture grassland soil (Cambisol). In arable soils no significant differences between the soil types were found concerning the content of Cmic. Way of soil use significantly influenced Cmic with higher values on pasture grassland soil. In Cambisol, a relatively strong relationships between Cmic and Corg (r = 0.919) as well as between Cmic and Ntot (r = 0.922) were determined. The basal respiration (BR) was no affected neither by a soil type nor by a way of soil use. On all monitored sites microbes responded positively to the addition of glucose (potential respiration - PR), but the strongest response was observed on the soil types with good mineralisation ability, such as Chernozems and Luvisols. On pasture grassland soil, the substrate availability index (PR/BR) with values of 2.32 was observed, which is lower compared to 7.60 of arable soil. This fact to indicate that pasture grassland soil had more easy decomposable organic compounds than arable soils.

Antimicrobial and enzymatic activity of actinomycetes isolated from soils of coastal islands

Microbiological investigation of unexplored ecosystems is crucial for discovering of antibiotic producing actinomycetes. The present study was conducted to determine antimicrobial activity and identify the most active strains. Actinomycetes were isolated using the spread plate technique following by serial dilution of samples on starch casein agar. The screening method consists of primary and secondary testing. The most active isolates were identified based on molecular and cultural methods. 42 out of 66 isolates displayed antimicrobial potential. 63% exhibited antibacterial activity, 16% antifungal activity, and 16% displayed both activities. Identified isolates, Streptomyces scabrisporus, Streptomyces sparsogenes, Streptomyces misakiensis, Streptomyces cirratus, Streptomyces lincolnensis, Streptomyces endophyticus, Streptomyces chartreusis, and Streptomyces alboniger showed a broad spectrum of enzymatic activities. The results indicated that these isolates may serve as antibiotic and enzyme-producing microbes.

Keratinophilic Fungi Isolated from Soils of Long-Term Fold-Grazed, Degraded Pastures in National Parks of Slovakia

A total of 939 isolates of 11 genera representing 15 species of keratinophilic fungi were isolated and identified from the soils of three long-term fold-grazed pastures in national parks of Slovakia (Pod Ploskou, Strungový príslop, and Pod Kečkou) and one non-fold-grazed pasture in sierra Stolicke vrchy (Diel) using the hair-baiting technique. Keratinophilic fungi were present in all soil samples with a prevalence of Trichophyton ajelloi and Paecilomyces lilacinus. These fungi were more abundant in soil from fold-grazed pasture (Strungový príslop) compared to non-fold-grazed pasture (Diel). The occurrence of the other keratinophilic fungi was substantially lower, likely because of low pH in some soils.

Effect of Dursban 480 EC (chlorpyrifos) and Talstar 10 EC (bifenthrin) on the physiological and genetic diversity of microorganisms in soil

This investigation was undertaken to determine the impact of the insecticides Dursban 480 EC (with organophosphate compound chlorpyrifos as the active ingredient) and Talstar 10 EC (with pyrethroid bifenthrin as the active ingredient) on the respiration activity and microbial diversity in a sandy loam luvisol soil. The insecticides were applied in two doses: the maximum recommended dose for field application (15 mg kg−1 for Dursban 480 EC and 6 mg kg−1 for Talstar 10 EC) and a 100-fold higher dose for extrapolation of their effect. Bacterial and fungal genetic diversity was analysed in soil samples using PCR DGGE and the functional diversity (catabolic potential) was studied using BIOLOG EcoPlates at 1, 3, 7, 14, 28, 56 and 112 days after insecticide application. Five bacterial groups (α, β, γ proteobacteria, firmibacteria and actinomycetes) and five groups of fungi or fungus-like microorganisms (Ascomycota, Basidiomycota, Chytridiomycota, Oomycota and Zygomycota) were analysed using specific primer sets. This approach provides high resolution of the analysis covering majority of microorganisms in the soil. Only the high-dose Dursban 480 EC significantly changed the community of microorganisms. We observed its negative effect on α- and γ-proteobacteria, as the number of OTUs (operational taxonomic units) decreased until the end of incubation. In the β-proteobacteria group, initial increase of OTUs was followed by strong decrease. Diversity in the firmibacteria, actinomycetes and Zygomycota groups was minimally disturbed by the insecticide application. Dursban 480 EC, however, both positively and negatively affected certain species. Among negatively affected species Sphingomonas, Flavobacterium or Penicillium were detected, but Achromobacter, Luteibacter or Aspergillus were supported by applied insecticide. The analysis of BIOLOG plates using AWCD values indicated a significant increase in metabolic potential of microorganisms in the soil after the high-dose Dursban application. Analysis of respiration demonstrated high microbial activity after insecticide treatments; thus, microbial degradation was relatively fast. The half-life of the active insecticide compounds were estimated within the range of 25 to 27 days for Talstar and 6 to 11 days for Dursban and higher doses stimulated degradation. The recommended dose levels of both insecticides can be considered as safe for microbial community in the soil.

Soil myxobacteria as a potential source of polyketide-peptide substances.

Myxobacteria, a group of antimicrobial producing bacteria, have been successfully cultured and characterized from ten soil samples collected from different parts of Slovakia. A total of 79 myxobacteria belonging to four genera (Myxococcus, Corallococcus, Sorangium, and Polyangium) were isolated based on aspects of their life cycle. Twenty-five of them were purified, fermented, and screened for antimicrobial activities against 11 test microorganisms. Results indicated that crude extracts showed more significant activities against Gram-positive than against Gram-negative bacteria or fungi. Based on a higher degree and broader range of antimicrobial production, the two most potential extracts (K9-5, V3-1) were selected for HPLC fractionation against Micrococcus luteus and Staphylococcus aureus and LC/MS analysis of potential antibiotic metabolites. The analysis resulted in the identification of polyketide-peptide antibiotics, namely corallopyronin A and B (K9-5) and myxalamid B and C (V3-1), which were responsible for important Gram-positive activity in the observed strains. A sequence similarity search through BLAST revealed that these strains showed the highest sequence similarity to Corallococcus coralloides (K9-5, NCBI accession number KX256198) and Myxococcus xanthus (V3-1, NCBI accession number KX256197). Although screening of myxobacteria is laborious, due to difficulties in isolating cultures, this research represented the first report covering the isolation and cultivation of this challenging bacterial group from Slovakian soils as well as the screening of their antimicrobial activity, cultural identification, and secondary metabolite identification.

Streptomyces globosus DK15 and Streptomyces ederensis ST13 as new producers of factumycin and tetrangomycin antibiotics

Fifty seven soil-borne actinomycete strains were assessed for the antibiotic production. Two of the most active isolates, designed as Streptomyces ST-13 and DK-15 exhibited a broad range of antimicrobial activity and therefore they were selected for HPLC fractionation against the most suppressed bacteria Staphylococcus aureus (ST-13) and Chromobacterium violaceum (DK-15). LC/MS analysis of extracts showed the presence of polyketides factumycin (DK15) and tetrangomycin (ST13). The taxonomic position of the antibiotic-producing actinomycetes was determined using a polyphasic approach. Phenotypic characterization and 16S rRNA gene sequence analysis of the isolates matched those described for members of the genus Streptomyces. DK-15 strain exhibited the highest 16S rRNA gene sequence similarity to Streptomyces globosus DSM-40815 (T) and Streptomyces toxytricini DSM-40178 (T) and ST-13 strain to Streptomyces ederensis DSM-40741 (T) and Streptomyces phaeochromogenes DSM-40073 (T). For the proper identification, MALDI-TOF/MS profile of whole-cell proteins led to the identification of S. globosus DK-15 (accession number: KX527570) and S. ederensis ST13 (accession number: KX527568). To our knowledge, there is no report about the production of these antibiotics by S.globosus and S. ederensis, thus isolates DK15 and ST13 identified as S. globosus DK-15 and S.ederensis ST-13 can be considered as new sources of these unique antibacterial metabolites.

Isolation, antimicrobial activity of myxobacterial crude extracts and identification of the most potent strains

Broad spectrum antimicrobial agents are urgently needed to fight frequently occurring multidrug-resistant pathogens. Myxobacteria have been regarded as “microbe factories” for active secondary metabolites, and therefore, this study was performed to isolate two bacteriolytic genera of myxobacteria, Myxococcus sp. and Corallococcus sp., from 10 soil/sand samples using two conventional methods followed by purification with the aim of determining the antimicrobial activity of methanol extracts against 11 test microorganisms (four Gram-positive, four Gram-negative, two yeasts and one fungus). Out of thirty-nine directly observed strains, 23 were purified and analyzed for antimicrobial activities. Based on the broth microdilution method, a total of 19 crude extracts showed antimicrobial activity. The range of inhibited wells was more important in the case of anti-Gram-positive-bacterial activity in comparison with the anti-Gram-negative-bacterial and antifungal activity. In light of the established degree and range of antimicrobial activity, two of the most active isolates (BNEM1 and SFEC2) were selected for further characterization. Morphological parameters and a sequence similarity search by BLAST revealed that they showed 99% sequence similarity to Myxococcus xanthus − BNEM1 (accession no. KX669224) and Corallococcus coralloides – SFEC2 (accession no. KX669225). As these isolates had antimicrobial activity, they could be considered for use in the development of antibiotics for pharmaceutical use. https://doi.org/10.2298/ABS161011132C Received: October 11, 2016; Revised: November 14, 2016; Accepted: November 18, 2016; Published online: December 14, 2016 How to cite: Charousova I, Medo J, Javorekova S. Isolation, antimicrobial activity of myxobacterial crude extracts and identification of the most potent strains. Arch Biol Sci. 2017;69(3):561-8.

Effect of vermicompost on changes in the bacterial community in maize rhizosphere

The aim of the study was to observe changes in the diversity of bacterial community in maize rhizosphere influenced by organic and mineral fertilization. Four variants of fertilization were tested - vermicompost (VC) at recommended annual dose 40t*ha-1, doubled annual dose of VC, recommended dose of ammonium saltpeter with dolomite (LAD 27) and combination of VC and LAD 27. Experiment was conducted with potted maize plants in controlled conditions of greenhouse during 74 days. Using PCR-DGGE method, we investigated differences in total bacteria community as well as in community of ammonia oxidizing bacteria. Based on occurrence of operative taxonomic units (OTU) we found differences in bacterial species spectra among fertilization variants. The highest Shannon´s biodiversity index was observed in variant with VC addition in dose 80 t*ha-1.The fertilizers effect on diversity of ammonia oxidizing bacteria was not significant however in each variant with vermicompost addition was the occurrence of new specific OTU observed. This OTU was identified as Nitrosospira sp. It was proven that some bacterial species introduced to soil with vermicompost addition can survive for at least 74 days and these bacteria can influence basic functions of soil microbiocenosis in nitrogen cycle.