Antimicrobial Efficiency of Some Essential Oils in Antibiotic-Resistant Pseudomonas aeruginosa Isolates.

Pseudomonas aeruginosa is a non-fermentative Gram-negative opportunistic pathogen, frequently encountered in difficult-to-treat hospital-acquired infections and also wastewaters. The natural resistance of this pathogen, together with the frequent occurrence of multidrug-resistant strains, make current antibiotic therapy inefficient in treating P. aeruginosa infections. Antibiotic therapy creates a huge pressure to select resistant strains in clinical settings but also in the environment, since high amounts of antibiotics are released in waters and soil. Essential oils (EOs) and plant-derived compounds are efficient, ecologic, and sustainable alternatives in the management of various diseases, including infections. In this study, we evaluated the antibacterial effects of four commercial essential oils, namely, tea tree, thyme, sage, and eucalyptus, on 36 P. aeruginosa strains isolated from hospital infections and wastewaters. Bacterial strains were characterized in terms of virulence and antimicrobial resistance. The results show that most strains expressed soluble pore toxin virulence factors such as lecithinase (89-100%) and lipase (72-86%). All P. aeruginosa strains were positive for alginate encoding gene and 94.44% for protease IV; most of the strains were exotoxin producers (i.e., 80.56% for the ExoS gene, 77.78% for the ExoT gene, while the ExoU gene was present in 38.98% of the strains). Phospholipase-encoding genes (plc) were identified in 91.67/86.11% of the cases (plcH/plcN genes). A high antibiotic resistance level was identified, most of the strains being resistant to cabapenems and cephalosporins. Cabapenem resistance was higher in hospital and hospital wastewater strains (55.56-100%) as compared to those in urban wastewater. The most frequently encountered encoding genes were for extended spectrum ß-lactamases (ESBLs), namely, blaCTX-M (83.33% of the strains), blaSHV (80.56%), blaGES (52.78%), and blaVEB (13.89%), followed by carbapenemase-encoding genes (blaVIM, 8.33%). Statistical comparison of the EOs' antimicrobial results showed that thyme gave the lowest minimum inhibitory concentrations (MIC) and minimum biofilm eradication concentrations (MBEC) in P. aeruginosa-resistant isolates, making this EO a competitive candidate for the development of efficient and ecologic antimicrobial alternatives.

Local-scale impact of wind energy farms on rare, endemic, and threatened plant species.

BACKGROUND: Wind energy farms have become a popular solution to produce green energy worldwide. Their development within protected areas has increased dramatically in the past decade, and the effects on the rare, endemic and threatened plant species (i.e., protected plant species), essential for habitat conservation and management, are little known. Only a few studies directly quantify the impacts of wind energy farms on them. Our study analyzes the impact of wind energy farms on rare, endemic, and threatened plant species in steppic habitats and their recovery potential over a ten-year period on a wind energy farm within the Dealurile Agighiolului Natura 2000 site (Dobrogea Region, SE Romania). METHODS: We surveyed the rare, endemic, and threatened plant species within a radius of approximately 50 m around each of the 17 wind towers during the wind farm operational phase. We selected 34 plots to allow the investigation of two types of areas: (1) a disturbed area overlapping the technological platform, where the vegetation was removed before construction, and (2) an adjacent undisturbed area. To understand the effects of the wind energy farm on the rare, endemic, and threatened plant species diversity and the differences between the disturbed and undisturbed areas, we calculated under both conditions: (1) plant species richness; (2) sample-size-based rarefaction and extrapolation with Hill numbers parameterized by species richness; (3) non-metric multidimensional scaling of Jaccard dissimilarity index; (4) functional diversity; (5) beta-diversity (including replacement and nestedness of species). RESULTS: As a result of the disturbances caused by the wind energy farm's development, we identified a sharp contrast between the diversity of rare, endemic, and threatened plants inhabiting disturbed and undisturbed areas near the wind towers. Our research showed that less than 40% of the total inventoried rare, endemic, and threatened species colonized the disturbed sites. Species turnover within undisturbed plots was higher than disturbed plots, implying that the plant community's heterogeneity was high. However, a higher richness in rare, endemic, and threatened plant species was found in the plots around the wind towers in grasslands of primary type. Sample-size-based rarefaction and extrapolation with Hill numbers by observed species richness indicated an accurate estimation of species richness in disturbed habitats, demonstrating that recovery after wind energy farm construction was incomplete after ten years of low-intensity plant restoration and conservation activities. Thus, we consider that operating activities must be reconfigured to allow the complete recovery of the communities with rare, endemic, and threatened plant species.

Effects of Foliar Treatment with a Trichoderma Plant Biostimulant Consortium on Passiflora caerulea L. Yield and Quality.

The influence of spore concentration on the ability of a Trichoderma consortium to colonize the Passiflora caerulea phyllosphere was evaluated by determining the effects of foliar treatments with two spore concentrations, in two repeated treatments, on the morphological, physiological, and ultrastructural characteristics, and on the yield and quality of P. caerulea. The studied crop quality features were related to its nutraceutical use: the accumulation of polyphenols and flavonoids, antioxidant activity, and effects on mouse fibroblast L929 cells. The Trichoderma consortium consisted of two strains, T. asperellum T36b and T. harzianum Td50b, and the concentrations used were 106 colony forming units (cfu)/mL and 108 cfu/mL. As a reference treatment, a commercial product that was based on herbs and algal extracts was used. As compared to the negative control, the treatment with the Trichoderma consortium at 108 cfu/mL concentration determines the accumulation of higher level of polyphenols and flavonoids and increased antioxidant activity. This enhancement of P. caerulea quality characteristics after treatment with the higher concentration of Trichoderma consortium was associated with larger leaves, increased number and size of chloroplasts, improved plant physiology characteristics, and an increased yield. The treatment with high concentration of Trichoderma consortium spores promotes phyllosphere colonization and benefits both crop yield and quality.

Antimicrobial and anti-pathogenic activity of some thioureides derivatives against Erwinia amylovora phytopathogenic strains.

A series of N-(1-methyl-1 Hpyrazole-4-carbonyl)-thiourea derivatives were assessed for their in vitro antimicrobial and anti-pathogenic activity against twenty-two strains of Erwinia amylovora isolated from different regions in Romania. The compounds were solubilised in dimethylsulfoxide and screened for their in vitro antimicrobial activity. The qualitative screening of the susceptibility spectra of various strains to the compounds was performed by adapted diffusion techniques (distribution of the tested compound solution directly on the solid medium previously seeded with the bacterial inoculums). The quantitative assay of the minimal inhibitory concentration (MIC, microg/mL) was based on liquid medium two-fold microdilutions. The subinhibitory concentrations of the tested substances were investigated for their influence on biofilm development on inert substrata. The present study showed that six new thiourea compounds exhibited a low antibacterial activity (MIC values > 500 microg/ml), but the subinhibitory concentrations inhibited the biofilm development on inert substrata. Thus, these results could suggest the usefulness of the tested compounds as control agents for preventing the first stage (colonization) of the infection with the fire blight pathogen.

Genetic characterization of Erwinia amylovora strains by random amplified polymorphic DNA fragments (RAPD).

Erwinia amylovora is the causative agent of fire blight, a destructive disease of rosaceous plants subjected to strict quarantine regulations worldwide. Previous studies showed that the population of E. amylovora in Romania is homogenous in its biochemical and serological characteristics, despite the different strains' geographical and host origin. The aim of the present study was to establish and test a typing method to quantify genetic diversity among the Romanian strains of this plant pathogen. Fourteen strains isolated from different hosts and geographical locations in Romania were examined by random amplified polymorphism DNA (RAPD) fragment analysis with two ten-base primers. This molecular method has not revealed any polymorphism, producing the same amplification patterns for all tested strains. Clustering of strains in the resulting dendrogram was not correlated with host, or region of isolation. The RAPD technique did not allow the detection of genetic markers in E. amylovora strains isolated in Romania and proved not to be discriminating among strains of this pathogen. The results presented in this study suggest that the population of E. amylovora in Romania is homogenous.

Novel antipathogenic strategies against adherent enterobacterial strains isolated from the hospital environment.

The emergence of the bacterial antibiotic multi-resistance made more and more stringent the developing of new anti-microbial strategies. The purpose of the present study was to investigate the antimicrobial potential of six (6) newly synthesized chemical compounds (derivating from phenantroline and dimethylguanin-copper complex combinations) versus 97 enterobacterial strains isolated from the hospital environment. The qualitative screening of the antimicrobial activity of the chemical compounds was performed by an adapted diffusion method. The minimal inhibitory concentrations (MIC) of the active chemical compounds were established by Mueller Hinton broth microdillution method. The tested chemical compounds were also tested for their ability to inhibit microbial adherence and biofilm development on inert substrata by a simple microtiter method. All six chemical compounds proved to have antimicrobial activity versus the most of the tested strains, the phenantroline derivatives exhibiting higher antimicrobial activity than the dimethylguanidine-copper complex combinations. The subinhibitory concentrations of the tested chemical products slightly inhibited the adherence ability of the bacterial strains to the inert substratum. Our results demonstrated that phenantroline derivatives may represent a new strategy of antimicrobial treatment, simultaneously with the bactericidal effect, the subinhibitory concentrations of these newly synthesized chemical compounds decreasing the adherence ability of bacteria to the inert substratum.

Erwinia amylovora strains isolated in Romania from outbreaks of fire blight disease: phenotypic characterization.

The fire blight disease was described for the first time in Romania, in 1992. Since then by continuous spreading, this disease has caused severe damages of the fruit trees production, particularly of the pear and quince orchards in different regions of the country, being advantaged by certain weather conditions (high temperatures and humidity). An epidemiological surveillance of this disease that was spreading over different regions of the country, has been instituted since 2002. During the year 2005 a total number of 785 samples were collected from the affected areas. The isolation and identification of Erwinia (E.) amylovora were performed on NSA and King's media and by indirect immunofluorescence (IFA) assay using monoclonal antiserum. Four hundred and fifty strains proved levan-type colonies on sucrose nutrient-agar and were IF-positive. Biochemical characterization of 46 selected strains, by help of the API 20E system, revealed a great homogeneity, for 80% of the strains, belonging to one of the two major API 20E profiles described for E. amylovora, the 20% remaining strains showing minor differences. Hypersensitivity test performed on tobacco leaves was positive. Six of the selected strains were susceptible to streptomycin. The present study can be considered as the first attempt of phenotypic characterization of E. amylovora strains isolated from Romanian area.