Distinct Communities of Poplar Endophytes on an Unpolluted and a Risk Element-Polluted Site and Their Plant Growth-Promoting Potential In Vitro.

Numerous studies demonstrated that endophytic microbes can promote plant growth and increase plant stress resistance. We aimed at isolating poplar endophytes able to increase their hosts' fitness both in nutrient-limited and polluted environments. To achieve this goal, endophytic bacteria and fungi were isolated from roots and leaves of hybrid poplars (Populus nigra × P. maximowiczii clone Max-4) on an unpolluted and a risk element-polluted site in the Czech Republic and subsequently screened by a number of in vitro tests. Bacterial communities at the unpolluted site were dominated by Gammaproteobacteria with Pseudomonas sp. as the prominent member of the class, followed by Bacilli with prevailing Bacillus sp., whereas Alphaproteobacteria, mostly Rhizobium sp., prevailed at the polluted site. The fungal endophytic community was dominated by Ascomycetes and highly distinct on both sites. Dothideomycetes, mostly Cladosporium, prevailed at the non-polluted site while unclassified Sordariomycetous fungi dominated at the polluted site. Species diversity of endophytes was higher at the unpolluted site. Many tested endophytic strains solubilized phosphate and produced siderophores, phytohormones, and antioxidants. Some strains also exhibited ACC-deaminase activity. Selected bacteria showed high tolerance and the ability to accumulate risk elements, making them promising candidates for use in inocula promoting biomass production and phytoremediation. Graphical Abstract ᅟ.

Monitoring COVID-19 spread in selected Prague's schools based on the presence of SARS-CoV-2 RNA in wastewater.

The COVID-19 pandemic has demanded a broad range of techniques to better monitor its extent. Owing to its consistency, non-invasiveness, and cost effectiveness, wastewater-based epidemiology has emerged as a relevant approach to monitor the pandemic's course. In this work, we analyzed the extent of the COVID-19 pandemic in five primary schools in Prague, the Czech Republic, and how different preventive measures impact the presence of SARS-CoV-2 RNA copy numbers in wastewaters. Copy numbers were measured by reverse transcription-multiplex quantitative real-time PCR. These copy numbers were compared to the number of infected individuals in each school identified through regular clinical tests. Each school had a different monitoring regime and subsequent application of preventive measures to thwart the spread of COVID-19. The schools that constantly identified and swiftly quarantined infected individuals exhibited persistently low amounts of SARS-CoV-2 RNA copies in their wastewaters. In one school, a consistent monitoring of infected individuals, coupled with a delayed action to quarantine, allowed for the estimation of a linear model to predict the number of infected individuals based on the presence of SARS-CoV-2 RNA in the wastewater. The results show the importance of case detection and quarantining to stop the spread of the pandemic and its impact on the presence of SARS-CoV-2 RNA in wastewaters. This work also shows that wastewater-based epidemiological models can be reliably used even in small water catchments, but difficulties arise to fit models due to the nonconstant input of viral particles into the wastewater systems.

Occurrence of Cronobacter spp. in retail foods.

AIMS: To study the occurrence of Cronobacter spp. in foods and to investigate the phenotypic properties of the strains isolated. METHODS AND RESULTS: A total of 53 strains of Cronobacter spp. isolated from 399 food samples were identified using conventional biochemical methods and MALDI-TOF mass spectrometry. Foods of plant origin were the most frequently contaminated samples. No Cronobacter spp. were found in infant milk formula, wheat-based infant food, pasteurized and raw cow milk, mincemeat, chicken, chickpea and potato dumpling powder. The individual species were identified as Cronobacter sakazakii (54·7%), Cronobacter malonaticus (28·4%), Cronobacter dublinensis (7·5%), Cronobacter muytjensii (7·5%) and Cronobacter turicensis (1·9%). Cronobacter sakazakii and C. malonaticus belong to biotype 1, 2, 2a, 3, 4 and 5, 5a, respectively. Cronobacter dublinensis strains were subdivided into biotypes 6 and 12. All strains were resistant to erythromycin and two of them were resistant to both erythromycin and tetracycline. CONCLUSIONS: Cronobacter spp. were isolated from various food samples pre-eminently of plant origin and dried food ingredients. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will increase and detail our knowledge of the presence and diversity of Cronobacter spp. in foods.

Bacterial and fungal endophyte communities in healthy and diseased oilseed rape and their potential for biocontrol of Sclerotinia and Phoma disease.

Phoma stem canker (caused by the ascomycetes Leptosphaeria maculans and Leptosphaeria biglobosa) is an important disease of oilseed rape. Its effect on endophyte communities in roots and shoots and the potential of endophytes to promote growth and control diseases of oilseed rape (OSR) was investigated. Phoma stem canker had a large effect especially on fungal but also on bacterial endophyte communities. Dominant bacterial genera were Pseudomonas, followed by Enterobacter, Serratia, Stenotrophomonas, Bacillus and Staphylococcus. Achromobacter, Pectobacter and Sphingobacterium were isolated only from diseased plants, though in very small numbers. The fungal genera Cladosporium, Botrytis and Torula were dominant in healthy plants whereas Alternaria, Fusarium and Basidiomycetes (Vishniacozyma, Holtermaniella, Bjerkandera/Thanatephorus) occurred exclusively in diseased plants. Remarkably, Leptosphaeria biglobosa could be isolated in large numbers from shoots of both healthy and diseased plants. Plant growth promoting properties (antioxidative activity, P-solubilisation, production of phytohormones and siderophores) were widespread in OSR endophytes. Although none of the tested bacterial endophytes (Achromobacter, Enterobacter, Pseudomonas, Serratia and Stenotrophomonas) promoted growth of oilseed rape under P-limiting conditions or controlled Phoma disease on oilseed rape cotyledons, they significantly reduced incidence of Sclerotinia disease. In the field, a combined inoculum consisting of Achromobacter piechaudii, two pseudomonads and Stenotrophomonas rhizophila tendencially increased OSR yield and reduced Phoma stem canker.

Hydrolysis of benzonitrile herbicides by soil actinobacteria and metabolite toxicity.

The soil actinobacteria Rhodococcus rhodochrous PA-34, Rhodococcus sp. NDB 1165 and Nocardia globerula NHB-2 grown in the presence of isobutyronitrile exhibited nitrilase activities towards benzonitrile (approx. 1.1-1.9 U mg(-1) dry cell weight). The resting cell suspensions eliminated benzonitrile and the benzonitrile analogues chloroxynil (3,5-dichloro-4-hydroxybenzonitrile), bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) (0.5 mM each) from reaction mixtures at 30 degrees C and pH 8.0. The products were isolated and identified as the corresponding substituted benzoic acids. The reaction rates decreased in the order benzonitrile >> chloroxynil > bromoxynil > ioxynil in all strains. Depending on the strain, 92-100, 70-90 and 30-51% of chloroxynil, bromoxynil and ioxynil, respectively, was hydrolyzed after 5 h. After a 20-h incubation, almost full conversion of chloroxynil and bromoxynil was observed in all strains, while only about 60% of the added ioxynil was converted into carboxylic acid. The product of ioxynil was not metabolized any further, and those of the other two herbicides very slowly. None of the nitrilase-producing strains hydrolyzed dichlobenil (2,6-dichlorobenzonitrile). 3,5-Dibromo-4-hydroxybenzoic acid exhibited less inhibitory effect than bromoxynil both on luminescent bacteria and germinating seeds of Lactuca sativa. 3,5-Diiodo-4-hydroxybenzoic acid only exhibited lower toxicity than ioxynil in the latter test.

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediation.

The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.

Biodegradation of methyl tert-butyl ether using bacterial strains.

Prospective methyl tert-butyl ether (MTBE) degrading bacterial strains and/or consortia were identified. The potential for aerobic degradation of MTBE was examined using bacterial isolates from contaminated soils and groundwater. Using the 16S rDNA protocol, two isolates capable of degrading MTBE (Rhodococcus pyridinivorans 4A and Achromobacter xylosoxidans 6A) were identified. The most efficient consortium of microorganisms was acquired from contaminated groundwater. The growth of both strains and the consortium on MTBE was supported by various organic substrates, and monitored using Bioscreen. The biochemical oxygen demand of the cultures was measured using OxiTop, and their MTBE concentrations were estimated by gas chromatography. After 3 weeks of aerobic cultivation using n-alkanes as cosubstrate, the concentration of MTBE in R. pyridinivorans 4A was reduced to 62.4 % of its initial amount (50 ppm).

Response of the bioluminescent bioreporter Pseudomonas fluorescens HK44 to analogs of naphthalene and salicylic acid.

Pseudomonasfluorescens HK44 is a lux-based bioluminescent bioreporter capable of selective luminescence in the presence of naphthalene and/or salicylic acid intermediate of its metabolism. We attempted to induce bioluminescence (BL) in this strain with 72 compounds, viz. substituted naphthalenes, naphthalene-like compounds (e.g., quinoline), substituted salicylic acids, salicylic acid-like compounds (e.g., 2-anthranilic acid), oligocyclic aromates, and intermediates of naphthalene metabolism to better discriminate response specificity. From them, 42 induced BL significantly lower as compared to naphthalene, three (viz. isoquinoline, o-cresol, and salicylamide) induced BL significantly greater than naphthalene, and 27 yielded no bioluminescent response whatsoever. Strain HK44 is therefore not prone to extensive false-positive signaling and can serve as a fairly specific indicator organism for naphthalene bioavailability. At elevated concentrations, 41 compounds inhibited BL. Thus, the inclusion of constitutive bioreporter controls as indicators of sample toxicity is vital to successful biosensing application.

Cellular fatty acids analysis in the identification of lactic acid bacteria.

The main factors affecting the cellular fatty acid composition such as cultivation temperature, pH and NaCl content of cultivation medium, growth stage and the method of fat isolation are summarised. The effects of these factors on the fatty acids patterns are compared with those found after the cultivation of several strains of Lactobacillus sake and L. pentosus.

Sample processing effect on polymerase chain reaction used for identification of Campylobacter jejuni.

Model samples of Campylobacter 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/mL C. jejuni in the physiological saline and 100 CFU/mL in the basic Park and Sanders broth.

Bacterial aerobic degradation of benzene, toluene, ethylbenzene and xylene.

Several aerobic metabolic pathways for the degradation of benzene, toluene, ethylbenzene and xylene (BTEX), which are provided by two enzymic systems (dioxygenases and monooxygenases), have been identified. The monooxygenase attacks methyl or ethyl substituents of the aromatic ring, which are subsequently transformed by several oxidations to corresponding substituted pyrocatechols or phenylglyoxal, respectively. Alternatively, one oxygen atom may be first incorporated into aromatic ring while the second atom of the oxygen molecule is used for oxidation of either aromatic ring or a methyl group to corresponding pyrocatechols or protocatechuic acid, respectively. The dioxygenase attacks aromatic ring with the formation of 2-hydroxy-substituted compounds. Intermediates of the "upper" pathway are then mineralized by either ortho- or meta-ring cleavage ("lower" pathway). BTEX are relatively water-soluble and therefore they are often mineralized by indigenous microflora. Therefore, natural attenuation may be considered as a suitable way for the clean-up of BTEX contaminants from gasoline-contaminated soil and groundwater.

Characterization of polychlorinated biphenyl-degrading bacteria isolated from contaminated sites in Czechia.

Biphenyl-utilizing polychlorinated biphenyls (PCB)-degrading bacteria were isolated from sites highly contaminated by PCBs, and their degradation abilities were determined using GC for typical commercial PCB mixtures (Delor 103 and Delor 106). Out of twelve strains which utilized biphenyl as a sole source of carbon and energy, strains Pseudomonas alcaligenes KP2 and P. fluorescens KP12, characterized by the BIOLOG identification system and the NEFERM test, were shown to significantly co-metabolize the PCB mixture Delor 103. DNA-DNA hybridization was used to compare both strains with well-known PCB-degraders Burkholderia cepacia strain LB400 and Ralstonia eutropha strain H850. The strain KP12 employs the same meta-fission route for degradation of chlorobenzoates as a chlorobiphenyl degrader Pseudomonas cepacia P166. Both isolates KP2 and KP12 belong to different phylogenetic groups, which indicates that the same geographical location does not ensure the same ancestor of degradative enzymes. We confirmed that also highly chlorinated and the most toxic congeners, which are contained in commercial PCB mixtures, can be biotransformed by members of indigenous bacterial-soil community under aerobic conditions.

Decontamination of wastewater contaminated by polychlorinated biphenyls (PCBs).

Wastewater contaminated by PCBs obtained from three different sources was treated at both laboratory and pilot plant scale conditions by ultraviolet oxidation of organics at the presence of hydrogen peroxide after partial adsorption of impurities and PCBs on activated carbon and/or activated bentonite. The procedure was conducted both with and without a Fe(II) catalyst and considerable reduction of PCB concentration was achieved in both cases. In pilot plant scale experiments, activated carbon polishing step followed UV oxidation. The following three types of contaminated waste water were examined: a) aqueous extracts originated in the course of clean-up of contaminated soil by extraction with aqueous solvents. Concentrations of PCBs in extracts were between 1 microg/L to 3,000 microg/L; b) wastewater condensates originated in the process of thermal desorption of PCB from soils. Concentrations of PCBs in condensates were between 300 microg/L and 5,000 microg/L. c) underground water contaminated by PCBs extracted from the sites of old contamination. The content of PCBs was up to 50,000 ng/L. Biodegradation of PCBs with a mixture of indigenous soil bacteria (selected strains of Pseudomonas and Acitenotobacter) was also tested. It was carried out in a reactor with volume of 1.5 m3 by application of the bacteria in a slurry of bentonite with adsorbed PCBs.

Effect of recombinant divercin RV41, structural variants and the activators of potassium channels on Listeria monocytogenes EGDe.

The effect of recombinant divercin RV41 (DvnRV41) and its structural variants on the K-channel formation was determined. The growth of Listeria monocytogenes EGDe (sensitive phenotype) and its isogenic strain (resistant phenotype) was assessed in the presence of DvnRV41 combined or not with pinacidil, NS1619, cromakalim (as K-channel activators), iberiotoxin and glipizide (as K-channel blockers). The combined action of DvnRV41 and K activators permitted formation of ATP-dependent pores. The combination of DvnRV41 and ATP-dependent pore activator cromakalim inhibited the growth of sensitive strain. The antilisterial activity of structural variants was less important than that of DvnRV41 but their mode of action remained overall similar.

Reversed-phase chromatographic study of the binding of polychlorinated biphenyls to cyclodextrins and sodium dodecylsulphate.

The binding of polychlorinated biphenyls (PCBs) to hydroxypropyl-beta-cyclodextrin (HP-beta-CD), tau-cyclodextrin (tau-CD) and sodium dodecylsulphate (SDS) was studied by reversed-phase thin-layer chromatography and the relative strength of interaction was calculated. HP-beta-CD and SDS did not bind to PCBs making it unlikely that these compounds modify the adsorption capacity or decomposition rate of PCBs. Each PCB formed inclusion complexes with tau-cyclodextrin. The inclusion-forming capacity of PCBs increased with an increase in the number of chloro substitutions in the molecule, indicating that tau-CD can be successfully used for the modification of the physicochemical and biochemical characteristics of PCBs. The linear correlation between the hydrophobicity and specific hydrophobic area of PCBs indicated that they can be considered as a homologous series of compounds.

Detection of Salmonella in food samples by the combination of immunomagnetic separation and PCR assay.

A combination of immunomagnetic separation and polymerase chain reaction (IMS-PCR) was used to detect Salmonella in food samples. Pre-enrichment of samples was combined with filtration through a membrane for the removal of food debris. The IMS-PCR assay combines selective extraction of bacteria by specific antibodies with primer specific PCR amplification that enables to detect Salmonella in non-fatty food samples in 24 h. In comparison with conventional cultural methods, the IMS-PCR is a rapid and specific method. Combined with filtration bags, it partially reduces the negative effects of the food matrix and allows the quick detection of Salmonella cells. The shortened protocols for Salmonella spp. detection described here can improve considerably current methodologies.

Isolation and characterization of alpha-glucosidase from Aspergillus niger.

alpha-Glucosidase is an enzyme widely used in biochemical analytical methods. Aspergillus niger was selected as a potential source for its production. Conditions for glucosidase production were optimized and the enzyme was isolated from the culture supernatant by dialysis and anion-exchange chromatography. The activity of the enzyme was determined by maltose hydrolysis to glucose, which was determined using a glucose-specific electrode or by high-performance liquid chromatography. The isolated enzyme was further characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, substrate specificity and fast protein liquid chromatography. The Michaelis constant, optimal temperature and stability of the enzyme preparation were determined.

Dipodascus magnusii (Saccharomycetes) contains multiple glucose-6-phosphate dehydrogenases with different NAD+/NADP+ dependencies.

A cell-free extract of a morphologically unstable strain of Dipodascus magnusii contained six proteins with activity of glucose-6-phosphate dehydrogenase (G6PDH). Two of these proteins displayed only NADP(+)-dependent activity, two could utilize both NAD+ and NADP+, but had higher activity with NAD+, and two possessed only NAD(+)-dependent activity. When the cultivation was carried out in the presence of monoiodoacetic acid, only two proteins with G6PDH activity were produced, one of them NAD(+)-dependent and the other NADP(+)-dependent. In all cases, NAD(+)-dependent activity was less stable in the presence of proteinases than was the NADP(+)-dependent activity.

Analysis of PCB-degrading bacteria: physiological aspects.

Several aerobic co-cultures capable of co-metabolising polychlorinated biphenyls (PCBs) were acquired by cultivation on biphenyls (BP). The source of micro-organisms was PCB-contaminated soil taken from various sites in the Czech Republic. Several bacterial strains (Gram-negative rods) were isolated, and their capacity to degrade Delor 103 (a PCB mixture containing di- to hexachlorobiphenyls) was analysed. This study was focused on co-culture 319 and isolate 2. The growth parameters of both those cultures were studied on BP; for isolate number 2 the specific growth rate mu = 0.122 (h-1) was calculated. The degradation of the individual congeners was estimated and resulted in more than 50% of the degradation of nearly all congeners during a 2-week experiment. Toxicity of Delor 103 on the vitality of the cells was followed by using viable plate count. The viability of the tested strain was preserved in the 100 times higher Delor 103 concentration compared with conditions in degradation experiments.

Properties and stability of glycerophosphate oxidase isolated from a mutant strain of Aerococcus viridans.

The properties of microbial L-alpha-glycerophosphate oxidase (GPO) isolated from a mutant strain of Aerococcus viridans DBM 1509 were estimated. The stability at different temperatures and pH were detected. At 4 degrees C the enzyme lost activity during 15 d, at 20 degrees C and 30 degrees C GPO activity decreased during 30 and 25 h, respectively. The highest stability was measured at - 20 degrees C and pH 9. At 4 degrees C the stability was enhanced by the addition of 0.1 M EDTA or by lyophilization in the presence of dextrin. These conditions allow the prolongation of the low stability of microbial GPO which limited its use, and give the opportunity to increase the stability of other enzymes