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The use of bioenergy crops such as Miscanthus x giganteus in phytoremediation could have both environmental and economic benefits, such as biomass production and soil conservation for crops. In our previous work, we showed that rhizobacteria from the rhizosphere of M. x giganteus stimulated metal extraction and uptake and enhanced the phytoremediation ability of treated M. x giganteus. In the present study, we conducted transcriptome analysis and qPCR to elucidate the molecular mechanisms underlying these interactions in response to bacterial treatment by identifying the candidate genes involved in growth and development processes and metal uptake. Using high-throughput RNA sequencing of root samples, we found that 5134 and 4758 genes were up- and downregulated in plants treated with the rhizobacteria consortium. Gene ontology analysis showed that the upregulated DEGs were significantly enriched in 32 terms, while the downregulated genes were significantly enriched in 63 terms. Our results confirmed the increased expression of two genes: the multidrug and toxic compound extrusion, also known as multi-antimicrobial extrusion (MATE) 40, known for its role in plant response to biotic and abiotic stress, and COBRA-like protein 1 belonging to the COBRA-like (COBL) gene family, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein involved in cell wall thickening, cell elongation, and biomass increase when compared to untreated plants. We present the first insight into a mechanism whereby the interaction between the rhizobacterial consortium and M. x giganteus fosters plant growth and enhances its capacity for phytoremediation.

Background/Aim. Bacterial vaginosis (BV) is one of the most common microbial dysbiosis, characterized by a decrease of Lactobacillus spp. with an increase of other anaerobic bacteria species [Gardnerella (G.) vaginalis, Atopobium (A.) vaginae, Prevotella spp, Mobiluncus spp, etc.] causing serious gynecological and obstetric complications. There-fore, it is particularly important to have accurate and reliable diagnostic standards. The aim of this study was to compare the results of various diagnostic methods for detecting BV, such as Amsel, Nugent, and Ison and Hay criteria, as well as multiplex quantitative real-time polymerase chain reaction (mqRT-PCR) test. Methods. This study involved vaginal swabs from 235 patients of reproductive age. Nugent criteria were used as the ‘gold standard’ compared with Amsel and Ison/Hay criteria as well as mqRT-PCR test based on the detection and quantification of G. vaginalis, A. vaginae, Lactobacillus spp., and total concentration of bacterial DNA. The kappa coefficient was employed to measure agreement between tests. Results. Our analysis demonstrated excellent agreement between Ison/Hay criteria and Nugent scores (kappa = 0.95), good agreement between Amsel and Nugent criteria (kappa = 0.78), while between Nugent criteria and mqRT-PCR test agreement was moderate (kappa = 0.59). Total agreements of Ison/Hay, Amsel, and mqRT-PCR against Nugent scores were 94.9%, 90.2%, and 74%, respectively. Nugent methods classified the highest number of intermediate patients - 60 (25.2%). The largest number of BV patients was detected by the mqRT-PCR method, while the largest number of healthy patients was detected by Amsel criteria. Conclusion. The mqRT-PCR is the best choice for BV diagnosis because it is more efficient at differentiating patients with intermediate results. Compared to Amsel and Nugent methods that group patients into 2 or 3 categories, the mqRT-PCR method recognizes other conditions of vaginal flora important for correct diagnoses and application of better therapeutic approaches, as well as preventing possible clinical consequences of this dysbiosis.

The Asian dung beetle (Catharsius molossus L.; Coleoptera: Scarabeidae) has been shown to positively affect soil bacterial diversity and the agronomic features of crop plants. In this study, we used bioinformatic tools to investigate the differences in bacterial functional phenotypes and ecological functions between control soil, cow dung-amended soil (CD), and cow dung-amended soil composted by dung beetles (DB). The soil bacterial metagenomes were sequenced and analyzed with the bioinformatic packages BugBase, PICRUSt2, Tax4Fun, and FAPROTAX to evaluate the effects of dung beetle-mediated composting on bacterial functions such as human and plant pathogenicity, trophic strategies, and soil nutrient transformation. BugBase proved useful for the determination of differences in major functional phenotypes, whereas FAPROTAX was effective at identifying differences in bacterial ecological functions between the treatments. Both tools suggested a relative decrease in human pathogens in the DB soil. This was corroborated by the pairwise comparison of abundances in bacterial species, which showed a significant reduction in the abundance of the broad-host-range pathogen Pseudomonas aeruginosa in the DB soil. In addition, FAPROTAX suggested a decrease in plant pathogens and an increase in chitinolytic bacteria, meaning that the DB treatment might be beneficial to the plant-growth-promoting bacteria involved in biological control. Finally, FAPROTAX revealed an array of ecological functions related to trophic strategies and macro- and micronutrient metabolism. According to these results, the activity of C. molossus beetles enhanced methanotrophy, ammonification, nitrification, sulfate reduction, and manganese oxidation, whereas iron respiration was decreased in the DB-treated soil. Our results represent a collection of general insights into the effects of C. molossus beetles on soil bacterial functions, which also reflect on the nutrient composition of dung beetle-composted soil. [ABSTRACT FROM AUTHOR]

Aims This study aimed to evaluate the potential of endophytic plant growth-promoting bacterium (PGPB), Pseudomonas putida A32, to mitigate drought stress in two bell pepper genotypes, Amfora 19 and Amfora 26, and to assess the genotype-specific responses to bacterial treatment. Methods and results The isolate P. putida A32 was selected for its remarkable beneficial properties, exhibiting 13 out of 14 traits tested. Under drought conditions, Amfora 26 showed increased relative water content and decreased H2O2 and malondialdehyde following bacterial treatment, while Amfora 19 exhibited enhanced growth parameters but responded less to bacterial treatment regarding drought parameters. However, Amfora 19 displayed inherent drought tolerance mechanisms, as indicated by lower stress parameters compared to Amfora 26. Conclusions The study emphasizes the importance of genotype-specific responses to PGPB treatment and the mechanisms of drought tolerance in peppers. Pseudomonas putida A32 effectively mitigated drought stress in both genotypes, with differential responses influenced by plant genotype. Our study confirmed our initial hypothesis that Amfora 19, as a genotype tolerant to biotic stress, is also more tolerant to abiotic stress. Understanding these interactions is crucial for the development of customized strategies to improve plant productivity and tolerance to drought. [ABSTRACT FROM AUTHOR]

In this study we screened twelve newly synthesised N-(substituted phenyl)-2-chloroacetamides for antimicrobial potential relying on quantitative structure-activity relationship (QSAR) analysis based on the available cheminformatics prediction models (Molinspiration, SwissADME, PreADMET, and PkcSM) and verified it through standard antimicrobial testing against Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and Candida albicans. Our compounds met all the screening criteria of Lipinski’s rule of five (Ro5) as well as Veber’s and Egan’s methods for predicting biological activity. In antimicrobial activity tests, all chloroacetamides were effective against Gram-positive S. aureus and MRSA, less effective against the Gram-negative E. coli, and moderately effective against the yeast C. albicans. Our study confirmed that the biological activity of chloroacetamides varied with the position of substituents bound to the phenyl ring, which explains why some molecules were more effective against Gram-negative than Gram-positive bacteria or C. albicans. Bearing the halogenated p-substituted phenyl ring, N-(4-chlorophenyl), N-(4-fluorophenyl), and N-(3-bromophenyl) chloroacetamides were among the most active thanks to high lipophilicity, which allows them to pass rapidly through the phospholipid bilayer of the cell membrane. They are the most promising compounds for further investigation, particularly against Gram-positive bacteria and pathogenic yeasts.

The composition of microbiota affects different traits of Drosophila throughout its life cycle and represents an important part of the adaptive response to environmental changes, such as increased levels of accumulated heavy metals in their habitats. We investigated the effects of lead added to the feeding substrate on the microbiota diversity in the fruit flies, Drosophila melanogaster and Drosophila subobscura. We compared the bacterial diversity of wild-caught flies and their progeny reared under standard laboratory conditions. We analyzed the shifts in bacterial diversity in the experimental groups of flies reared for 13 generations on standard and lead(II) acetate (Pb(CH3COO)2), Pb acetate-saturated substrates. Identification of the main bacterial genera was performed by next-generation sequencing (NGS) of the V3-V4 variable regions of the 16S rRNA gene. Results indicate significant species-specific differences in bacterial composition between natural and laboratory populations, and between the substrates. Diversity increased in both species under prolonged exposure to lead-polluted substrate, suggesting a potential adaptive response to an environmental stress.

Drug resistance of Pseudomonas aeruginosa is a leading problem in hospital infections. The aim of this study was to determine the best molecular genetic discrimination method for Pseudomonas spp. isolates among 94 outpatients and inpatients and see their grouping by phenotype characteristics (biofilm formation, frequency of serotypes, pigmentation, production of different class of beta-lactamases, and susceptibility to different antibiotic classes) and genotype. The most common serotypes were P1, P6, and P11, while co-productions of pyoverdine and pyocyanin were observed in 70 % of isolates. A total of 77.66 % isolates were mostly weak and moderate biofilm producers. Isolates were susceptible to colistin (100 %), aztreonam (97.87 %), imipenem (91.49 %), doripenem (90.43 %), and meropenem (84.04 %). MICs values confirmed susceptibility to ceftazidime and cefepime and singled out meripenem as the most effective inhibitor. Most isolates were resistant to aminoglycosides and fluoroquinolones. Only two isolates produced ESBL, eight were carbapenemase producers, and five isolates produced MBLs. Twenty-nine isolates were multidrug-resistant; 82.8 % of which produced both pigments, 58.3 % were non-typeable, while the P6 and P11 serotypes were equally distributed (16.7 %). Thirteen MDR isolates were strong enzyme producers. RAPD PCR analysis using primer 272 proved the best at discriminatory fingerprinting for Pseudomonas isolates, as it allocated 12 clusters. A correlation between DNA patterns and antibiotic resistance, production of pigments, serotypes distribution, and biofilm formation was not observed, and only confirmed higher genetic heterogeneity among P. aeruginosa isolates, which suggests that other molecular methods are needed to reveal potential relations between genotypic patterns and phenotypic characteristics.

The “Pirot ‘ironed’ sausage” (Pis) is a traditional, dry, fermented product originating from the town of Pirot, situated in southeastern Serbia. This product is made from different types of meat (goat, sheep, beef and donkey) and spices, without additives or starter cultures. Pis is an organic, unprocessed product, without heat or smoke treatment. The aim of this study was to characterize 120 isolates of lactic acid bacteria (LAB) from Pis produced by six different brands during a two-year period using phenotypic and genetic identification. Preliminary characterization of the LAB was based on general morphology and biochemical tests. Repetitive elements such as REP, BOX and GTG5, found in the genome of these bacteria, and randomly amplified polymorphic DNA-polymerase chain reaction (RAPD PCR) sequences were used for determination of genetic polymorphism. Identification by 16S rRNA gene sequencing showed the presence of only two LAB species, Lactobacillus sakei (76%) and Leuconostoc mesenteroides (24%). However, genetic polymorphism was detected using fingerprinting methods. In comparison with other primers, the profiles obtained with GTG5 showed the highest heterogeneity for most of the tested isolates, with sequencing results additionally confirming its discriminatory power. In addition, M13 RAPD primer also produced satisfactory separation of the tested isolates. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173026]

The study aimed to isolate potential biocontrol agents from mushroom substrate that could serve as an alternative to toxic chemicals commonly used for disease control in mushroom production. The antagonistic potential of ten native Bacillus subtilis strains against the causal agents of green mould disease of oyster mushroom, Trichoderma pleuroti and Trichoderma pleuroticola, was evaluated. The antagonistic potential of Bacillus spp. strains was quantified in vitro based on dual cultivation with the pathogen. Growth inhibition of T. pleuroti ranged from 54.44% to 62.22% and no significant differences in antagonistic activity were found between the tested B. subtilis strains. Inhibition of T. pleuroticola was slightly higher, ranging from 55.56% to 69.62% and B. subtilis strain B-358 induced the highest growth inhibition. This research confirmed mushroom substrate to be a good source of antagonistic microorganisms with potentials for use in biological control of green mould in oyster mushroom production.

In vitro antagonistic potential of eleven isolates of Bacillus spp. against two phytopathogenic bacteria and one fungus was tested in order to identify potential biocontrol agents in vegetable crops. The Bacillus spp. isolates demonstrated different levels of antagonistic effect against the tested pathogenic microorganisms. Data in the study proved Xanthomonas vesicatoria to be more sensitive to Bacillus spp. strains than Clavibacter michiganensis subsp. michiganensis. Ten B. subtilis strains induced growth inhibition of X. vesicatoria, while a strain of B. pumilus did not affect the growth of that bacterium. The largest inhibition zones against X. vesicatoria were induced by strains B-319, B-325 and B-358. The pathogenic strain C. michiganensis subsp. michiganensis was most inhibited by two B. subtilis strains (B-338 and B-348) with mean inhibition zone diameters of up to 20 mm. B. subtilis strain B-319 which was the best in inhibiting X. vesicatoria, showed the lowest inhibitory effect on C. michiganensis subsp. michiganensis. The largest growth inhibition percentage of Verticillium sp. (PGI approximately 70%) was induced by B. subtilis strains B-310 and B-322. The other B. subtilis strains showed PGI values ranging from 45% to 68%, while B. pumilus strain B-335 had the least antagonistic potential (PGI =34.43%) against the pathogen. This study identified at least one suitable biocontrol candidate, B. subtilis strain B-358, as effective in vitro against all three vegetable pathogens.

Identification of 33 Bacillus spp. isolates from different environmental samples collected from the territory of Serbia was performed by sequencing of the 5ʹ-hypervariable section of 16S rRNA gene. Eight species were identified within four phylogenetic groups: B. pumilus, B. megaterium, B. subtilis and B. cereus. Determination of their antibiotic resistance was performed using the minimum inhibitory concentration (MIC) assay. We found that just one isolate was resistant to gentamicin, 9 were resistant to clindamycin and all were resistant to vancomycin. Based on the profile of resistance, the isolates were categorized into 4 categories. In silico analysis of the erythromycin-resistance (erm) gene for clindamycin resistance showed their distribution between related and nonrelated soil and human isolates including different species of Bacillus genera. This finding indicates that Bacillus spp. from the environment could be a source of resistance to clindamycin. The potential for the presence and spread of resistance determinants in the soil and similar ecosystems exists so that monitoring of antibiotic resistance genes in nonpathogenic Bacillus strains from the environment is advised. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173026]

Brown rot caused by Monilinia spp. is one of the main causes of pre- and postharvest losses in stone and pome fruit production. The use of beneficial microorganisms is considered one of the most promising, safe and effective alternative methods for controlling these pathogens. This study aimed to investigate the antagonistic potential of 33 Bacillus spp. strains, in order to identify the best candidate for brown rot biocontrol. Strains identified as Bacillus amyloliquefaciens B-241 and Bacillus subtilis B-313 and B-358 were chosen for further ex situ studies on detached apple fruit. The efficacy of B-241 (87.1–93.7%) did not differ significantly from a commercially available synthetic fungicide (p > 0.05). The putative mode of action of B. amyloliquefaciens B-241 against Monilinia species is competition for nutrients and antibiosis. The ethyl acetate extract of the strain, applied at 5 and 12.5 mg/mL, was bioactive in vitro and ex situ. A HPLC analysis confirmed the presence of surfactin and bacillomycin D in the extract. However, before developing a shelf-stable product and commercial production, the spectrum and efficacy on a larger scale of the B-241 strain should be determined, and its efficacy in combination with commercial biofungicides and fungicides tested in vivo. [ABSTRACT FROM AUTHOR]

The isolation of bacteria was carried out from samples of straw and chicken manure, compost at various stages of the composting process and casing soil used for growing button mushrooms. A preliminary screening of 108 bacterial isolates for antagonistic activity against Trichoderma aggressivum f. europaeum showed that 23 tested isolates inhibited mycelial growth of the pathogenic fungus. Further screening with four indicator isolates of fungi revealed that all 23 bacterial isolates inhibited the growth of T. aggressivum f. europaeum, T. harzianum and T. koningii, while only 13 isolates inhibited the growth of T. atroviride. T. aggressivum f. europaeum proved to be the most sensitive, with many bacterial isolates generating a high percentage of growth inhibition. Only two bacterial isolates (B-129 and B-268) were successful in inhibiting the growth of all 4 tested pathogens. All 23 bacterial isolates were characterized as Gram-positive and catalase-positive and were subjected to molecular identification based on the partial sequence, the hypervariant region of the 16S rDNA. It was shown that the obtained bacterial strains belong to Bacillus subtilis, B. amyloliquefaciens, B. licheniformis and B. pumilus species. [Projekat Ministarstva nauke Republike Srbije, br. 31043 i br. 173026]

Pseudomonas syringae pv. aptata is a member of the sugar beet pathobiome and the causative agent of leaf spot disease. Like many pathogenic bacteria, P. syringae relies on the secretion of toxins, which manipulate host-pathogen interactions, to establish and maintain an infection. This study analyzes the secretome of six pathogenic P. syringae pv. aptata strains with different defined virulence capacities in order to identify common and strain-specific features, and correlate the secretome with disease outcome. All strains show a high type III secretion system (T3SS) and type VI secretion system (T6SS) activity under apoplast-like conditions mimicking the infection. Surprisingly, we found that low pathogenic strains show a higher secretion of most T3SS substrates, whereas a distinct subgroup of four effectors was exclusively secreted in medium and high pathogenic strains. Similarly, we detected two T6SS secretion patterns: while one set of proteins was highly secreted in all strains, another subset consisting of known T6SS substrates and previously uncharacterized proteins was exclusively secreted in medium and high virulence strains. Taken together, our data show that P. syringae pathogenicity is correlated with the repertoire and fine-tuning of effector secretion and indicate distinct strategies for establishing virulence of P. syringae pv. aptata in plants. [ABSTRACT FROM AUTHOR]

A diagnostic method based on nested-PCR, followed by ELISA and conventional bacteriology tests, for the rapid and reliable detection of halo blight pathogen Pseudomonas savastanoi pv. phaseolicola (Psp) collected from infected bean leaves and seeds is described. Psp formed white, small and flat colonies on nutrient agar medium, creamy white, flat and circular on Milk-Tween agar medium and light yellow, convex and shiny on modified sucrose peptone agar medium. Eighteen Gram-negative, catalase-positive and oxidase-negative strains were subjected to nested PCR with primers P 5.1/P 3.1 and P 5.2/P 3.2, which directed the amplification of the 450 bp target DNA fragment in all tested strains. According to the results of DAS- and PTA-ELISA with respect to reactivity to specific antibodies, all analyzed strains belonged to Psp bacterium. Pathogenicity was tested on bean pods and cotyledon leaves, on which greasy spots were formed. Psp did not cause hypersensitive reaction on the leaves of tobacco and geranium. Strains produced levan, fluorescent pigment, oxidative metabolism of glucose, did not reduce nitrate, did not produce indole and H2S, did not hydrolyze starch, gelatin and esculin; they produced acid from glucose, mannose, sucrose and glycerol, and did not produce acid from maltose, starch, esculin, dulcite, sorbitol, inositol and erythritol.