Volume 65 (2016): Edition 3 (August 2016)

The excessive use of mineral fertilizers causes many negative consequences for the environment as well as potentially dangerous effects of chemical residues in plant tissues on the health of human and animal consumers. Bio-fertilizers are formulations of beneficial microorganisms, which upon application can increase the availability of nutrients by their biological activity and help to improve soil health. Microbes involved in the formulation of bio-fertilizers not only mobilize N and P but mediate the process of producing crops and foods naturally. This method avoids the use of synthetic chemical fertilizers and genetically modified organisms to influence the growth of crops. In addition to their role in enhancing the growth of the plants, biofertilizers can act as biocontrol agents in the rhizosphere at the same time. Biofertilizers are very safe for human, animal and environment. The use of Azotobacter, Azospirillum, Pseudomonas, Acetobacter, Burkholderia, Bacillus, Paenibacillus and some members of the Enterobacteriaceae is gaining worldwide importance and acceptance and appears to be the trend for the future.

Staphylococcus aureus colonizes the mucous membrane of the nasal vestibule of a significant number of healthy people. These microorganisms are opportunistic pathogens, that in favorable conditions, may cause infections of various course, location or manifestation. Secondary infections emerge in cases when other risk factors contribute to such a change. One of the diseases during which S. aureus changes its saprophytic character to a pathogenic one is atopic dermatitis (AD), an allergic skin condition of a chronic and recurrent nature. Patients with AD are highly predisposed to secondary staphylococcal infections due to active S. aureus colonization of the stratum corneum, damage of the skin barrier or a defective immune response. Microorganisms present in skin lesions destroy the tissue by secreting enzymes and toxins, and additionally stimulate secondary allergic reactions. The toxins secreted by strains of S. aureus also act as superantigens and penetrate the skin barrier contributing to a chronic inflammation of the atopic skin lesions. The S. aureus species also releases proinflam- matory proteins, including enzymes that cause tissue damage. When initiating treatment it is particularly important to properly assess that the onset of the secondary bacterial infection is caused by S. aureus and thus justifying the inclusion of antibiotic therapy. Depending on the severity and extent of the staphylococcal infection, topical antibiotics are used, usually mupirocin or fusidic acid, or general antibiotic treatment is introduced. Another therapeutic strategy without antibiotics has given a positive effect in patients.

Shiga toxin-producing Escherichia coli (STEC) strains also called verotoxin-producing E. coli (VTEC) represent one of the most important groups of food-borne pathogens that can cause several human diseases such as hemorrhagic colitis (HC) and hemolytic – uremic syndrome (HUS) worldwide. The ability of STEC strains to cause disease is associated with the presence of wide range of identified and putative virulence factors including those encoding Shiga toxin. In this study, we examined the distribution of various virulence determinants among STEC strains isolated in Poland from different sources. A total of 71 Shiga toxin-producing E. coli strains isolated from human, cattle and food over the years 1996–2010 were characterized by microarray and PCR detection of virulence genes. As stx1a subtype was present in all of the tested Shiga toxin 1 producing E. coli strains, a greater diversity of subtypes was found in the gene stx2, which occurred in five subtypes: stx2a, stx2b, stx2c, stx2d, stx2g. Among STEC O157 strains we observed conserved core set of 14 virulence factors, stable in bacteria genome at long intervals of time. There was one cattle STEC isolate which possessed verotoxin gene as well as sta1 gene encoded heat-stable enterotoxin STIa characteristic for enterotoxigenic E. coli. To the best of our knowledge, this is the first comprehensive analysis of virulence gene profiles identified in STEC strains isolated from human, cattle and food in Poland. The results obtained using microarrays technology confirmed high effectiveness of this method in determining STEC virulotypes which provides data suitable for molecular risk assessment of the potential virulence of this bacteria.

We report the genetic similarity changes between a mutant mushroom (Pleurotus florida, designated as PfCM4) having increased cellulolytic activity developed through radiation mutagenesis and its wild type by amplified fragment length polymorphism (AFLP). On average, 23 AFLP fragments were amplified per primer combination, and a total of 286 polymorphic fragments (78.57% polymorphism) with maximal fragment length of 1365 base pairs (bp) were obtained. The genetic similarity between wild type and PfCM4 was found to be 22.30%. In addition, mycelial and secreted protein profiling by 2D-PAGE showed at least three and five different protein spots in the range of 25 kD to 100 kD, respectively, in PfCM4. It seems that the variation in genetic similarity and different expression of both mycelial and secreted proteins in PfCM4 in comparison to the wild type could likely be correlated with its increased cellulolytic activity effected by the irradiation.

During a screening of lactic acid bacteria producing bacteriocin from Cotija cheese, the strain QC38 was isolated. Based on the 16S rRNA gene nucleotide sequencing (516 pb accession no KJ210322) and phylogenetic analysis, the isolate was identified as Pediococcus acidilactici. Neutralized cell-free supernatant was tested for antimicrobial activity against 17 Gram-negative and Gram-positive pathogens. Growth inhibition was achieved against Listeria monocytogenes (supplier or indication or source), Staphylococcus aureus, Vibrio vulnificus, Vibrio cholerae O1 Ogawa, Vibrio cholerae NO 01 and Salmonella enterica subsp. Enterica serovar Typhimurium. Bacteriocin-like substance, after heating at 121°C for 15 min it remained stable and its antimicrobial activity was observed at pH ranging from 1.0 to 10.0 but inactivated by α-chymotrypsin and proteinase K. Strain QC38 was able to grow in 1–9% NaCl concentration. The plate overlay assay showed an approximate size of bacteriocin-like substance between 3.4 and 6.5 kDa. P. acidilactici QC38 harboured a plasmid that contains a gene for a pediocin (PA-1).

The capability of the biosurfactant-producing strain Rhodococcus wratislawiensis BN38 to mineralize both aromatic and aliphatic xeno- biotics was proved. During semicontinuous cultivation 11 g/l phenol was completely degraded within 22 cycles by Rhodococcus free cells. Immobilization in a cryogel matrix was performed for the first time to enhance the biodegradation at multiple use. A stable simultaneous hydrocarbon biodegradation was achieved until the total depletion of 20 g/l phenol and 20 g/l n-hexadecane (40 cycles). The alkanotrophic strain R. wratislawiensis BN38 preferably degraded hexadecane rather than phenol. SEM revealed well preserved cells entrapped in the heterogeneous super-macroporous structure of the cryogel which allowed unhindered mass transfer of xenobiotics. The immobilized strain can be used in real conditions for the treatment of contaminated industrial waste water.

The present study examined Polish strains of Flamulina velutipes as a potential source of nutraceuticals and found that their nutritional value is dependent on the fruiting bodies gathering time. To prove the above hypothesis protein, carbohydrate and phenolic substances concentration were determined. Moreover, catalase, superoxide dismutase, cellobiose dehydrogenase activities were assayed. In order to prove the healing properties of Enoki fruiting bodies the obtained extracts were tested for antioxidant and bacteriostatic abilities. We have proved that Polish F. velutipes fruiting bodies may be a rich source of antioxidants and that they are capable of inhibiting Staphylococcus aureus growth.

In the current study, 65 Vibrio spp. were isolated from the Monastir lagoon water, were characterized phenotypically and genotypically. In addition, we looked for the presence of three Vibrio parahaemolyticus virulence genes (tlh, trh and tdh) and ten Vibrio cholerae virulence genes (ctxA, vpi, zot, ace, toxR, toxT, tosS, toxRS, tcpA and cpP). We also investigated the antibiotic susceptibilities and the adherence ability of the identified strains to abiotic material and to biotic surfaces. The cytotoxicity activity against HeLa and Vero cell lines were also carried out for all tested strains. All Vibrio isolates were identified to the species level and produced several hydrolytic exoenzymes. The results also revealed that all strains were expressing high rates of resistance to tested antibiotics. The minimum inhibitory concentration (MIC) values showed that tetracycline and chloramphenicol were the most effective antibiotics against the tested bacteria. Vibrio alginolyticus and V. cholerae species were the most adhesive strains to both biotic and abiotic surfaces. Besides, V. alginolyticus isolates has the high levels of recombination of genes encoding V. cholerae and V. parahaemolyticus virulence factors. In vitro cytotoxic activities of several Vibrio extracellular product were also observed among HeLa and Vero cells.

The prospective of endophytic microorganisms allied with medicinal plants is disproportionally large compared to those in other biomes. The use of antagonistic microorganisms to control devastating fungal pathogens is an attractive and eco-friendly substitute for chemical pesticides. Many species of actinomycetes, especially the genus Streptomyces, are well known as biocontrol agents. We investigated the culturable community composition and biological control ability of endophytic Streptomyces sp. associated with an ethanobotanical plant Schima wallichi. A total of 22 actinobacterial strains were isolated from different organs of selected medicinal plants and screened for their biocontrol ability against seven fungal phytopathogens. Seven isolates showed significant inhibition activity against most of the selected pathogens. Their identification based on 16S rRNA gene sequence analysis, strongly indicated that all strains belonged to the genus Strepto myces. An endophytic strain BPSAC70 isolated from root tissues showed highest percentage of inhibition (98.3 %) against Fusarium culmorum with significant activity against other tested fungal pathogens. Phylogenetic analysis based on 16S rRNA gene sequences revealed that all seven strains shared 100 % similarity with the genus Streptomyces. In addition, the isolates were subjected to the amplification of antimicrobial genes encoding polyketide synthase type I (PKS-I) and nonribosomal peptide synthetase (NRPS) and found to be present in most of the potent strains. Our results identified some potential endophytic Streptomyces species having antagonistic activity against multiple fungal phytopathogens that could be used as an effective biocontrol agent against pathogenic fungi.

Fifty seven bacterial isolates from root nodules of two spontaneous legumes (Astragalus corrugatus and Hippocrepis areolata) growing in the arid areas of Tunisia were characterized by phenotypic features, 16S rDNA PCR-RFLP and 16S rRNA gene sequencing. Phenotypically, our results indicate that A. corrugatus and H. areolata isolates showed heterogenic responses to the different phenotypic features. All isolates were acid producers, fast growers and all of them used different compounds as sole carbon and nitrogen source. The majority of isolate grew at pHs between 6 and 9, at temperatures up to 40°C and tolerated 3% NaCl concentrations. Phylogenetically, the new isolates were affiliated to four genera Sinorhizobium, Rhizobium, Mesorhizobium and Agrobacterium. About 73% of the isolates were species within the genera Sinorhizobium and Rhizobium. The isolates which failed to nodulate their host plants of origin were associated to Agrobacterium genus (three isolates).

Anaerobic reductive dechlorination of 2,3-dichlorophenol (2,3DCP) and 2,4,6-trichlorophenol (2,4,6TCP) was investigated in microcosms from River Nile sediment. A stable sediment-free anaerobic microbial consortium reductively dechlorinating 2,3DCP and 2,4,6TCP was established. Defined sediment-free cultures showing stable dechlorination were restricted to ortho chlorine when enriched with hydrogen as the electron donor, acetate as the carbon source, and either 2,3-DCP or 2,4,6-TCP as electron acceptors. When acetate, formate, or pyruvate were used as electron donors, dechlorination activity was lost. Only lactate can replace dihydrogen as an electron donor. However, the dechlorination potential was decreased after successive transfers. To reveal chlororespiring species, the microbial community structure of chlorophenol-reductive dechlorinating enrichment cultures was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Eight dominant bacteria were detected in the dechlorinating microcosms including members of the genera Citrobac- ter, Geobacter, Pseudomonas, Desulfitobacterium, Desulfovibrio and Clostridium. Highly enriched dechlorinating cultures were dominated by four bacterial species belonging to the genera Pseudomonas, Desulfitobacterium, and Clostridium. Desulfitobacterium represented the major fraction in DGGE profiles indicating its importance in dechlorination activity, which was further confirmed by its absence resulting in complete loss of dechlorination. Reductive dechlorination was confirmed by the stoichiometric dechlorination of 2,3DCP and 2,4,6TCP to metabolites with less chloride groups and by the detection of chlorophenol RD cprA gene fragments in dechlorinating cultures. PCR amplified cprA gene fragments were cloned and sequenced and found to cluster with the cprA/pceA type genes of Dehalobacter restrictus.

Microbial colonisation of chronic venous ulcers and synergism between bacterial species slow down the healing process. The study aimed at performing qualitative analysis of microbial flora in venous leg ulcers treated with platelet rich plasma (PRP). Twenty two women and twelve men aged 47–90 years were treated with PRP at our department between 2012 and 2015. Ulcer cultures collected before and after PRP therapy yielded 83 and 110 microbial isolates, respectively, of Gram positive, Gram negative bacteria and candida. Pseudomonas aueruginosa and Staphylococcus aureus were the most common pre- and post-treatment isolates. PRP therapy and increased the variety of microbial flora.

A broad series of 4,5,6,7-tetrahalogenated benzimidazoles and 4-(1H-benzimidazol-2-yl)-benzene-1,3-diol derivatives was tested against selected bacteria and fungi. For this study three plant pathogens Colletotrichum sp., Fusarium sp., and Sclerotinia sp., as well as Staphylococcus sp., Enterococcus sp., Escherichia sp., Enterobacter sp., Klebsiella spp. , and Candida spp. as human pathogens were used. MIC values and/or area of growth reduction method were applied in order to compare the activity of the synthesized compounds. From the presented set of 22 compounds, only 8, 16, 18 and 19 showed moderate to good inhibition against bacterial strains. Against Candida strains only compound 19 with three hydroxyl substituted benzene moiety presented high inhibition at nystatin level or lower.

Penicillin G oversecretion by Penicillium chrysogenum PQ-96 is associated with a strictly adjusted cellular organization of the mature and senescent mycelial cells. Abundant vacuolar phagy and extended cellular vacuolization combined with vacuolar budding resulting in the formation of vacuolar vesicles that fuse with the cell membrane are the most important characteristic features of those cells. We suggest as follows: if the peroxisomes are integrated into vacuoles, the penicillin G formed in peroxisomes might be transferred to vacuoles and later secreted out of the cells by an exocytosis process. The peroxisomal cells of the mycelium are privileged in penicillin G secretion.

A total of 181 cultivable endophytic bacterial isolates were collected from stems of 13 species of herbs inhabiting Europe (Poland): Chelido- nium majus L., Elymus repens L., Erigeron annuus L., Euphrasia rostkoviana Hayne, Foeniculum vulgare L., Geranium pratense L., Humulus lupulus L., Matricaria chamomilla L., Mentha arvensis L., Papaver rhoeas L., Rosmarinus officinalis L., Solidago gigantea L. and Vinca minor L. The isolates were screened for their antifungal activity and fifty three were found to inhibit fungal growth. Of these, five had strong antifungal properties. These selected isolates were identified as: Pseudomonas azotoformans, P. cedrina, Bacillus subtilis group and Erwinia persicina.

A study was carried out to determine the effects of paraquat, pretilachlor and 2, 4-D on growth and nitrogen fixing activity of Stenotropho- monas maltophilia (Sb16) and pH of Jensen’s N-free medium. The growth of Sb16 and pH of medium were significantly reduced with full (X) and double (2X) doses of tested herbicides, but nitrogen fixing activity was decreased by 2X doses. The nitrogenase activity had the highest value in samples treated with 1/2X of 2, 4-D on fifth incubation day, but 2X of 2, 4-D had the most adverse effect. An inhibition in the growth and nitrogenase activity was recovered on the last days of incubation.

Ganoderma boninense, a phytopathogenic white rot fungus had sought minimal genetic characterizations despite huge biotechnological potentials. Thus, efficient collection of fruiting body, basidiospore and protoplast of G. boninense is described. Matured basidiocarp raised under the glasshouse conditions yielded a total of 8.3 × 10⁴basidiospores/ml using the low speed centrifugation technique. Mycelium aged 3-day-old treated under an incubation period of 3 h in lysing enzyme from Trichoderma harzianum (10 mg/ml) suspended in osmotic stabilizer (0.6 M potassium chloride and 20 mM dipotassium phosphate buffer) yielded the highest number of viable protoplasts (8.9 × 10⁶single colonies) among all possible combinations tested (regeneration media, age of mycelium, osmotic stabilizer, digestive enzyme and incubation period).