Yoana Kizheva

In November 2025, an outbreak of Enterococcus cecorum in two commercial broiler flocks in southern Bulgaria were investigated. Clinical signs, including lameness and recumbency, appeared at approximately 20 days of age, and mortality reached 2.8% and 3.7%. Necropsy revealed systemic and osteoarticular lesions, including fibrinous pericarditis, hepatitis, splenomegaly, and osteomyelitis. E. cecorum was isolated in pure culture from 11 of 13 examined birds and confirmed by sodA-targeted PCR. The isolates carried the virulence-associated genes psaA, gelE, and ace, five tetracycline resistance genes (tetM, tetK, tetC, tetS, and tetT), and the macrolide resistance gene ermB. This study documents early-onset systemic E. cecorum disease in Bulgarian broilers and highlights the need for further phenotypic and genomic investigation of antimicrobial resistance.

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.

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 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 (10 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.

Enterococcus faecalis is an opportunistic pathogen associated with nosocomial infections, food spoilage, and reduced efficacy of orally administered medications in patients with Parkinson’s disease. Its genetic adaptability, particularly in acquiring virulence and antibiotic resistance genes, poses a significant challenge in treatment. Тhus, the development of new and effective approaches, such as phage therapy, is crucial in the fight against E. faecalis . The main goal of this study was to establish the biological characteristics of three bacteriophages (designated as vB_SEF_8, vB_SEF_13 and vB_SEF_15) isolated from wastewater in Bulgaria and their potential to eliminate E. faecalis . The host ranges of the phages were determined primarily using E. faecalis strains ( n = 29), although other species within the genus Enterococcus were also included. All three phages targeted only E. faecalis strains, including antibiotic-resistant or multidrug-resistant strains. The phages showed broad pH (4.0–10.5) and temperature (up to 80 °C) stability, formed clear plaques, with maximal titers reached at various MOIs. After 9 months at 4 °C, only a slight titer reduction was observed (up to 2 log ₁₀ PFU/mL). RFLP analysis revealed genetic diversity among the three phage isolates. The phage with the broadest host range (vB_SEF_8) was characterized in more details. TEM observation revealed elongated head and long noncontractile tail. vB_SEF_8 possessed linear dsDNA and lacked genes associated with lysogeny, antibiotic resistance, or virulence. Phylogenetic analysis and the calculated pairwise intergenomic distance showed that vB_SEF_8 is a novel species within the Saphexavirus genus, class Caudoviricetes . The phage also successfully inhibited E. faecalis in a milk-based matrix. The collected data demonstrate that vB_SEF_8 holds significant potential as an antibacterial and therapeutic agent against E. faecalis in settings where the presence of this bacterium is undesirable.

The objective of this study were fifty strains of seven lactic acid bacteria (LAB) species (L. paracasei, L. casei, L. rhamnosus, L. plantarum, L. fermentum, L. delbrueckii subsp. bulgaricus and L. koreensis), previously isolated from various biotopes (human breast milk, newborn feces, and fermented vegetables). As part of their probiotic potential, their ability for adhesion, biofilm / anti-biofilm formation, auto-aggregation and co-aggregation with different pathogenic species were evaluated.Based on their capacity to form biofilm, strains were grouped as poor (34 %), moderate (33 %), and strong (33 %) biofilm forming bacteria (BFB). L. fermentum 18V, showed the strongest biofilm formation ability and 70 % anti-biofilm activity against P. aeruginosa. The auto-aggregation capability of LAB ranged from 12 % to 68 %. The highest auto-aggregation (85 %) was established for L. fermentum 12V and L. casei 8V (78 %). The best co-aggregation ability with all tested pathogenic species was demonstrate by L. casei 8V. The highest percentage (85 %) of co-aggregation was determined for L. paracasei strain 29V with L. innocua. The housekeeping genes (ef-Tu, eno, gap, groEl, and srtA) involved in binding mechanisms shown 100 % presence in all of the tested strains. The great ability to adhere to mucin was determined for L. koreensis 35V (1. 9 x 106 CFU mL-1). The degree of adhesion varies both between individual species and between strains belonging to the same species. The obtained results showed that the LAB possess strain- and species-specifically probiotic properties with potential applications in the food industry.

Non-clinical enterococci are relatively poorly studied by means of acquired antibiotic resistance to tetracycline and by the distribution, functionality and role of their CRISPR systems. Background: In our study, 72 enterococcal strains, isolated from various non-clinical origins, were investigated for their phenotypic and genotypic (tet(M), tet(O), tet(S), tet(L), tet(K), tet(T) and tet(W)) tetracycline resistance. Methods: The genetic determinants for HGT (MGEs (Int-Tn and prgW), inducible pheromones (cpd, cop and cff), aggregation substances (agg, asa1, prgB and asa373) and CRISPR–Cas systems were characterized by PCR and whole-genome sequencing. Results: Four tet genes (tetM, tetO, tetS and tetT) were detected in 39% (n = 28) of our enterococcal population, with tetM (31%) being dominant. The gene location was linked to the Tn6009 transposon. All strains that contained tet genes also had genes for HGT. No tet genes were found in E. casseliflavus and E. gilvus. In our study, 79% of all tet-positive strains correlated with non-functional CRISPR systems. The strain E. faecalis BM15 was the only one containing a combination of a functional CRISPR system (cas1, cas2, csn2 and csn1/cas9) and tet genes. The CRISPR subtype repeats II-A, III-B, IV-A2 and VI-B1 were identified among E. faecalis strains (CM4-II-A, III-B and VI-B1; BM5-IV-A2, II-A and III-B; BM12 and BM15-II-A). The subtype II-A was the most present. These repeats enclosed a great number of spacers (1–10 spacers) with lengths of 31 to 36 bp. One CRISPR locus was identified in plasmid (p.Firmicutes1 in strain E. faecalis BM5). We described the presence of CRISPR loci in the species E. pseudoavium, E. pallens and E. devriesei and their lack in E. gilvus, E. malodoratus and E. mundtii. Conclusions: Our findings generally describe the acquisition of foreign DNA as a consequence of CRISPR inactivation, and self-targeting spacers as the main cause.

This study aims at the identification and characterization of five actinobacterial strains with presumed belonging to the species Curtobacterium flaccumfaciens isolated from tomato and pepper plants, and establishing the potential role of both plants as natural reservoirs of this phytopathogen. Species identification was performed via MALDI-ToF MS, 16S rDNA sequencing and PCR. The strains were Gram-positive with a coryneform cell shape having yellow/orange-pigmented colonies; positive for catalase and esculin, and starch and casein hydrolysis; oxidase-, urease-, indole- and nitrate-reduction-negative and were strictly aerobic. All isolates produced antimicrobial substances against various phytopathogenic bacteria. Tomato and pepper plants were artificially infected with newly isolated strains in order to establish their role as natural reservoirs of the bacteria. Morphological alterations were observed only in the tomato plants, with defoliation of the first two to four leaves at the 28th day. Then, viable coryneform bacterial isolates (n = 73) were successfully re-isolated only from the stems of the infected plants. The similarity between the re-isolates and the respective initial isolates was confirmed phenotypically and genotypically by RAPD-PCR, confirming that solanaceous vegetables can act as reservoirs of C. flaccumfaciens. This is the first report of C. flaccumfaciens in Bulgaria.

Background: Traditional Bulgarian fermented foods are prominent for their uniqueness of local ingredients, production methods, and endemic microbial species. The present research investigated the diversity and beneficial biological potential of lactic acid bacteria (LAB) isolated from various types of unique Bulgarian fermented foods. Methods: Species identification was performed via 16S rDNA sequencing. Biological activity was evaluated by determining antibacterial activity (via agar well diffusion assay), H2O2 production, spectrophotometrically determined auto- and co-aggregation, microbial adhesion to hydrocarbon, and biofilm formation. The biosafety of the isolated lactic acid bacteria was established based on hemolytic activity and phenotypic and genotypic antibiotic susceptibility. Results: Forty-five strains were isolated from fermented foods (sauerkraut, fermented green tomatoes, fermented cucumbers, kefir, white cheese, and Izvara (curdled milk)). Five species were detected: Lactiplantibacillus plantarum, Levilactobacillus koreensis, Levilactobacillus brevis, Lactobacillus helveticus, and Levilactobacillus yonginensis. The most prominent species was L. plantarum, at 47%. For the first time, L. koreensis and L. yonginensis, isolated from unique Bulgarian fermented foods, are reported in this study. The antibacterial effect of the cell-free supernatants was evaluated. An antagonistic effect was observed against Escherichia coli (57%) and Salmonella enterica subsp. enterica serotype Enteritidis (19%) for several L. plantarum strains. Only one L. brevis (Sauerkraut, S15) strain showed activity against E. coli. The best autoaggregation ability at hour 4 was observed for L. koreensis (fermented cucumbers, FC4) (48%) and L. brevis S2 (44%). The highest percentage of co-aggregation with Candida albicans, at hou 4 in the experiments, was observed for strains L. koreensis (fermented green tomato, FGT1) (70%), L. plantarum strains S2 (54%), S13 (51%), and S6 (50%), while at hour 24 for strains L. koreensis FGT1 (95%), L. brevis (Kefir, K7) (89%), L. plantarum S2 (72%), and L. koreensis FC2 (70%). Seven of the isolated LAB strains showed hydrophobicity above 40%. Our results showed that the ability of biofilm formation is strain–dependent. No hemolytic activity was detected. The antibiotic resistance to 10 antibiotics from different groups was tested phenotypically and genotypically. No amplification products were observed in any strains, confirming that the isolates did not carry antibiotic-resistance genes. Conclusions: Traditional fermented Bulgarian foods can be considered functional foods and beneficial LAB sources.

The pathway and the lifestyle of known enterococcus species are too complicated. The aim of the present study is to trace the path of pathogenicity of enterococci isolated from seven habitats (Cornu aspersum intestine; Bulgarian yoghurt; goat and cow feta cheese—mature and young, respectively; Arabian street food—doner kebab; cow milk; and human breast milk) by comparing their pathogenic potential. In total, 72 enterococcal strains were isolated and identified by MALDI-TOF, sequencing, and PCR. Hemolytic and gelatinase activity were biochemically determined. PCR was carried out for detection of virulence factors (cylB, esp, gls24, nucl, psaA, agg, gelE, and ace) and antibiotic resistance (erm, ermB, blaZ, vanA, aphA, mefA, gyrA, cat_pIP501, and aac6′-aph2″). Phenotypic antibiotic resistance was assigned according to EUCAST. Eleven representatives of the genus Enterococcus were identified: E. mundtii, E. casseliflavus, E. gilvus, E. pseudoavium, E. pallens, E. malodoratus, E. devriesei, E. gallinarum, E. durans, E. faecium, and E. faecalis. Twenty-two strains expressed α-hemolysis. Thirteen strains had the cylB gene. Only two strains expressed α-hemolysis and possessed the cylB gene simultaneously. Positive amplification for gelE was found in 35% of the isolates, but phenotypic gelatinase activity was observed only in three strains. All isolates showed varying antibiotic resistance. Only E. faecalis BM15 showed multiple resistance (AMP-HLSR-RP). Correlation between genotypic and phenotypic macrolide resistance was revealed for two E. faecalis strains.

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.