The influence of extraction medium and sample preparation on the total antioxidant capacity (TAC) of pea plants leaves is investigated. Sodium acetate buffer (pH 5), potassium phosphate buffer (pH 7.5), methanol or 0.1% w/v trichloroacetic acid (TCA) were used for homogenization. TAC was measured in the crude supernatants (one-step procedure). The pellets were extracted with acetone and their TAC values were added to those from the previous assay (two-step procedure). The values for TAC of the acetone extract, the low-molecular and protein fractions derived from a partial fractionation of the probes are used in the proposed by us new three-step method. The results indicate that TAC of the probes processed with the two or three-step procedure is significantly higher than that of the crude supernatants (one-step procedure). On the basis of this study we recommend two-step protocol as a minimum and buffer with slightly acid pH or TCA for extraction when plant material is investigated. The highest results were obtained when the new three-step procedure for sample preparation and sodium acetate buffer as an extracting agent were applied. Moreover, the separation of the cellular antioxidants into three different fractions is especially useful for studying the effects of aging, processing and storage on the antioxidant capacity.

The present study was focused on the pathosystem pepper plants (Capsicum annuum L.)-phytopathogenic bacterium X. euvesicatoria (wild strain 269p)-bacteriophage BsXeu269p/3 and the possibility of bacteriophage-mediated biocontrol of the disease. Two new model systems were designed for the monitoring of the effect of the phage treatment on the infectious process in vivo. The spread of the bacteriophage and the pathogen was monitored by qPCR. A new pair of primers for phage detection via qPCR was designed, as well as probes for TaqMan qPCR. The epiphytic bacterial population and the potential bacteriolytic effect of BsXeu269p/3 in vivo was observed by SEM. An aerosol-mediated transmission model system demonstrated that treatment with BsXeu269p/3 reduced the amount of X. euvesicatoria on the leaf surface five-fold. The needle-pricking model system showed a significant reduction of the amount of the pathogen in infectious lesions treated with BsXeu269p/3 (av. 59.7%), compared to the untreated control. We found that the phage titer is 10-fold higher in the infection lesions but it was still discoverable even in the absence of the specific host in the leaves. This is the first report of in vivo assessment of the biocontrol potential of locally isolated phages against BS pathogen X. euvesicatoria in Bulgaria.

Wastewater-based epidemiology (WBE) was applied to monitor human-pathogenic respiratory viruses, including SARS-CoV-2, in the four largest Bulgarian cities during the summer and autumn of 2024. Starting from 1 liter wastewater samples, concentra-tion of the virus fraction with PEG 8000/NaCl, extraction and purification of DNA/RNA were applied. Detection of SARS-CoV-2, FluA/B, RSV and HCoV229E was performed with TaqMan qPCR. A total of 42 samples were processed over a 7-month period. Two summer peaks of SARS-CoV-2 were detected in the capital Sofia, in July and September 2024. With some differences, a similar result was observed in Plovdiv and Burgas. The concentration of SARS-CoV-2 in wastewater decreased significantly in November and December. Other respiratory viruses started to be detectable only at the beginning of October. The results of this study show an unusual summer peak of SARS-CoV-2 in wastewater from large cities, followed by a significant decrease of vi-rus concentrations in the fall. The established trend correlates with the statistics of COVID-19 incidence in Bulgaria.

In response to the alarming rise in antimicrobial resistance, bacteriophages have re-emerged as a promising alternative to conventional antibiotic therapy. The main objective of this paper was to characterize a newly isolated phage (vB_SEC_3) in the context of its suitability for phage therapy against MDR E. coli, which is considered a priority pathogen. The phage was characterized at the morphological, genomic, and biological levels relevant to phage therapy. TEM analyses revealed a non-enveloped icosahedral capsid lacking tail structure. Phylogenetic and tANI analyses placed the phage within the α3 phages (genus Alphatrevirus) of the less-studied family Microviridae and revealed <95% similarity to its closest relatives, suggesting vB_SEC_3 is a putative novel species within this genus. The genome (6085 bp, GC content 45.3%) displayed the conserved organization typical for these phages, including overlapping genes. No known genes associated with lysogeny, antibiotic resistance, or virulence were detected. Briefly, vB_SEC_3 was able to effectively lyse two MDR strains of E. coli (S1 and B5, EOP 0.735 and 0.961, respectively). Tolerance to a wide pH range (4–10.5) and to temperatures up to 80 °C was established. Six-month storage of the crude lysate at 4 °C resulted in a slight decrease (<0.16 log₁₀ PFU/mL) in phage titer. This study provides additional insights into the biology and diversity of Microviridae phages and offers a basis for future investigations into their potential relevance in the context of combating MDR bacteria.

Influent wastewater, characterized by a diverse bacterial composition of human and animal origin, is a suitable source for studying the spread of antibiotic resistance and virulence genes, particularly in Enterococcus faecalis and Enterococcus faecium. This study aimed to evaluate the potential of these two species as reservoirs and indicators of antibiotic resistance and virulence genes. Wastewater treatment plants near the three largest cities of Bulgaria (Sofia, Varna and Burgas) were selected for sampling. Influent wastewater samples were collected for a period of 3 months (February, March and May) and used in the analyses. Overall, 34 bacterial isolates (E. faecalis, n = 13 and E. faecium, n = 21) were isolated and identified by MALDI-ToF. Phenotypic and genotypic methods were used to evaluate their antibiotic resistance profiles (focusing on clinically relevant antibiotics) and virulence potential. High dissemination of phenotypic ampicillin and quinupristin-dalfoprisin resistance (97% of all tested strains and 81% of all E. faecium strains, respectively) was established. In March, the highest number of antibiotic-resistant profiles was observed, including the emergence of MDR strains, primarily among E. faecium strains. Molecular analyses revealed a dissemination of genes encoding resistance to aminoglycosides and β-lactam antibiotics. The most prevalent virulence genes were gelE and ace, found only in E. faecalis strains. All gelE-positive strains also exhibited phenotypic gelatinase activity. In this study, E. faecium shows greater antibiotic resistance potential, whereas E. faecalis exhibits increased virulence capacity. These exploratory findings support the usefulness of influent wastewater as a matrix for monitoring antibiotic-resistant and potentially virulent enterococcal isolates, while larger-scale and seasonally representative studies are needed to confirm temporal and geographic patterns.