Jarmila Pazlarova

Biodegradation of polychlorinated biphenyls by plant cells

Abstract The PCB biodegradative ability of plant cells cultivated in vitro in media containing a mixture of PCB congeners, Delor 103, is demonstrated. For experiments we used submerged cultures of Armoracia rusticana, Solanum aviculare, Atropa bella-donna , transformed hairy root or embryogenic cultures of Solanum nigrum . Transformation of PCB was followed by gas chromatography after cultivations of the above-mentioned cultures with Delor 103 (10 mg 100 ml −1 ). The overall PCB metabolizing capability and also degradation of individual congeners greatly differed from strain to strain. The highest capability to metabolize PCB was assayed with differentiated cultures of Solanum nigrum. Beside the capability of PCB degradation, total peroxidase activity in the medium and the cell extract was also followed. Differentiated or hairy root cultures exhibiting higher degradation abilities of PCB also showed increase of peroxidase activities.

PCB metabolism by Pseudomonas sp. P2

Abstract The degradation of Delor 103, a mixture of polychlorinated biphenyl (PCB) congeners, by Pseudomonas sp. P2, an indigenous soil bacterium was studied. In mineral medium with biphenyl as sole carbon source the extent of PCB degradation monitored by GC exceeded 70%. The efficiency of Delor 103 degradation by strain P2 was compared with that of Ralstonia eutropha H850. The addition of saccharose or agar improved PCB degradation, whereas the addition of glycerol or pyruvate substantially reduced the degradation efficiency. The presence of an amino acid mixture enhanced PCB degradation. The following chlorobenzoic acids (CBA): 2,5 CBA, 2,4 CBA, 4 CBA, 2 CBA and 2,5-diCBA were detected as metabolites of Delor 103, with 2,5-diCBA as the major product. Although 2,5-diCBA inhibited the growth of Pseudomonas sp. P2 the degradation of 2,5-diCBA was unaffected for upto 14 days.

Biofilm spatial organization by the emerging pathogen Campylobacter jejuni: comparison between NCTC 11168 and 81-176 strains under microaerobic and oxygen-enriched conditions

During the last years, Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed countries. Described as an obligate microaerophile, Campylobacter has puzzled scientists by surviving a wide range of environmental oxidative stresses on foods farm to retail, and thereafter intestinal transit and oxidative damage from macrophages to cause human infection. In this study, confocal laser scanning microscopy (CLSM) was used to explore the biofilm development of two well-described Campylobacter jejuni strains (NCTC 11168 and 81-176) prior to or during cultivation under oxygen-enriched conditions. Quantitative and qualitative appraisal indicated that C. jejuni formed finger-like biofilm structures with an open ultrastructure for 81-176 and a multilayer-like structure for NCTC 11168 under microaerobic conditions (MAC). The presence of motile cells within the biofilm confirmed the maturation of the C. jejuni 81-176 biofilm. Acclimation of cells to oxygen-enriched conditions led to significant enhancement of biofilm formation during the early stages of the process. Exposure to these conditions during biofilm cultivation induced an even greater biofilm development for both strains, indicating that oxygen demand for biofilm formation is higher than for planktonic growth counterparts. Overexpression of cosR in the poorer biofilm-forming strain, NCTC 11168, enhanced biofilm development dramatically by promoting an open ultrastructure similar to that observed for 81-176. Consequently, the regulator CosR is likely to be a key protein in the maturation of C. jejuni biofilm, although it is not linked to oxygen stimulation. These unexpected data advocate challenging studies by reconsidering the paradigm of fastidious requirements for C. jejuni growth when various subpopulations (from quiescent to motile cells) coexist in biofilms. These findings constitute a clear example of a survival strategy used by this emerging human pathogen.

Biodegradation of polychlorinated biphenyls and volatile chlorinated hydrocarbons in contaminated soils and ground water in field condition

Three sites with a long history of contamination with polychlorinated biphenyls (PCBs) or chlorinated ethenes (CIUs) were investigated to evaluate the natural attenuation process. Data showed that both the rates and mechanisms of biodegradation differed and that the occurrence of aerobic and anaerobic microorganisms differed qualitatively and quantitatively. Criteria for feasibility of spontaneous degradation of CIUs and PCBs in ground water and soil are formulated. (a) Congener analysis was applied to evaluate the long-term exposure of individual congeners of PCBs in the environment. In the course of approximately 20 years the small changes in congener composition could be probably due to slow biodegradation of light congeners in the relatively good oxygenated superficial layers of soil. (b) Enhanced reductive dehalogenation of PCBs was accomplished by the solid-state fermentation in reactors of 15 cubic metres of volume. Efficiency of biodegradation of individual congeners was evaluated and limits of the method were determined. (c) Different reaction rates of CIU degradation in ground water have been ascribed to the presence and/or activity of anaerobic bacteria in field conditions.

Detection, identification and quantification of Campylobacter jejuni, coli and lari in food matrices all at once using multiplex qPCR

BackgroundThermotolerant Campylobacter jejuni, coli and lari are recognized as leading food-borne pathogens causing an acute bacterial enteritis worldwide. Due to narrow spectrum of their biochemical activity, it is very complicated to distinguish between individual species. For reliable risk assessment, proper incidence evaluation or swift sample analysis regarding individual species, a demand for simple and rapid method for their distinguishing is reasonable. In this study, we evaluated a reliable and simple approach for their simultaneous detection, species identification and quantification using multiplex qPCR.ResultsSpecies specific primers and hydrolysis probes are directed to hippuricase gene of C. jejuni, serine hydroxymethyltransferase gene of C. coli and peptidase T gene of C. lari. Efficiencies of reactions were 90.85% for C. jejuni, 96.97% for C. coli and 92.89% for C. lari. At 95.00% confidence level and when cut off is set to 38 cycles, limits of detection are in all cases under 10 genome copies per reaction which is very appreciated since it is known that infectious doses are very low.ConclusionsProposed assay was positively validated on different food matrices (chicken wing rinses, chicken juice and homogenized fried chicken strips). No inhibition of PCR reaction occurred. Assay was evaluated in accordance with MIQE handbook.

The survival of micromycetes and yeasts under the low-temperature plasma generated in electrical discharge

The fungicidal effect of low-temperature plasma generated by positive direct current discharge and its influence on the growth dynamics was evaluated on three micromycete species and yeast in water suspensions. The fungicidal effect was lower than analogous bactericidal effect and differs substantially among various fungal species. Together with the cidal effects, the slower growth of exposed fungal spores was observed.

Bacteria Degrading PCBs and CBs Isolated from Long-Term PCB Contaminated Soil

Bacteria able to degrade polychlorinated biphenyls (PCBs) and chlorobenzoic acids (CBs) were isolated from soil that had been contaminated with PCBs for 15–30 years. Contaminated soil in which PCB content ranged between 10–470 mg/kg was naturally vegetated with different plants including ash (Fraxinus excelsior), birch (Betula pendula), black locust (Robinia pseudoacacia), Austrian pine (Pinus nigra) and goat willow (Salix caprea) trees as well as a variety of grasses and forbs. Bacteria able to use biphenyl as a sole source of carbon and energy were found in the root zone of all plants, but occurred in the largest numbers beneath pine and black locust. Bacteria able to degrade chlorobenzoic acids were isolated from the same location contaminated with PCBs. Strains that were taxonomically identified by 16S rDNA as Pandoraea were able to use 2-CB, 3-CB, 2,3-CB, 2,5-CB as sole carbon sources, and the strain Arthrobacter utilised 4-CB.

The biofilm matrix of Campylobacter jejuni determined by fluorescence lectin-binding analysis

Abstract Campylobacter jejuni is responsible for the most common bacterial foodborne gastroenteritis. Despite its fastidious growth, it can survive harsh conditions through biofilm formation. In this work, fluorescence lectin-binding analysis was used to determine the glycoconjugates present in the biofilm matrix of two well-described strains. Screening of 72 lectins revealed strain-specific patterns with six lectins interacting with the biofilm matrix of both strains. The most common sugar moiety contained galactose and N-acetylgalactosamine. Several lectins interacted with N-acetylglucosamine and sialic acid, probably originated from the capsular polysaccharides, lipooligosaccharides and N-glycans of C. jejuni. In addition, glycoconjugates containing mannose and fucose were detected within the biofilm, which have not previously been found in the C. jejuni envelope. Detection of thioflavin T and curcumin highlighted the presence of amyloids in the cell envelope without association with specific cell appendages. The lectins ECA, GS-I, HMA and LEA constitute a reliable cocktail to detect the biofilm matrix of C. jejuni.

Sample processing effect on polymerase chain reaction used for identification ofCampylobacter jejuni

Model samples ofCampylobacter jejuni for polymerase chain reaction (PCR) were prepared by rapid and simple procedures consisting of centrifugation, proteinase K treatment, Chelex 100 treatment, and boiling lyses. A PCR based on specific amplification of the variable sequence of 16S rRNA gene was performed using Tth DNA polymerase and the PCR products were visualized by agarose gel electrophoresis. The assay allowed the detection of 10 CFU/mLC. jejuni in the physiological saline and 100 CFU/mL in the basic Park and Sanders broth.

Application of Mouse Antibodies to Somatic Antigen for Detection of Salmonella enteritidis by Competitive ELISA

Competitive ELISA estimation based on application of polyclonal mouse antibodies to somatic antigen O:9, 12 was developed. The optimization of the protocol is reported. The optimal concentration of immobilized somatic antigen O:9, 12 was found to be 4.9 2 104 cells ml−1; optimal concentration of mouse IgG was 6.25 w g ml−1; and the optimal concentration of peroxidase labelled antibody to mouse IgG was 8 w g ml−1. The tested antibody exhibited neither cross reactions with chosen strains (serotypes) of salmonellas group 04 (B), 07 (C1), 08 (C2-C3), nor with members of Enterobacteriaceae: Escherichia coli, Klebsiella pneumonia, Citrobacter freundii and non-fermenting bacterium Pseudomonas fluorescens. Application of chemiluminiscent substrates increased the sensitivity of S. enteritidis detection up to three times. Competitive ELISA tested on model samples produced results comparable with standard cultivation techniques for Salmonella spp.