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.

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.

The ability of certain human pathogens to adapt to plants without losing their virulence toward people is a major concern today. Thus, the aim of the present work was the investigation of the presence of cross-over pathogenic bacteria in infected tomato and pepper plants. The objects of the study were 21 samples from seven different parts of the plants and three from tomato rhizosphere. In total, 26 strains were isolated, identified by MALDI-TOF, and phenotypically characterized. The PCR amplification of the rpoB gene was applied as an approach for the rapid detection of cross-over pathogens in plant samples. A great bacterial diversity was revealed from tomato samples as nine species were identified (Leclercia adecarboxylata, Pseudesherichia vulneris, Enterobacter cancerogenus, Enterobacter cloacae, Enterobacter bugandensis, Acinetobacter calcoaceticus, Pantoea agglomerans, Pantoea ananatis, and Pectobacterium carotovorum). Polymicrobial contaminations were observed in samples T2 (tomato flower) and T10 (tomato fruit). Five species were identified from pepper samples (P. agglomerans, L. adecarboxylata, Pseudomonas sp., Pseudomonas putida, and Enterococcus sp.). Antibiotic resistance patterns were assigned in accordance with EFSA recommendations. All isolates showed varying resistance to the tested antibiotics. The genetic basis for the phenotypic antibiotic resistance was not revealed. No genes for the virulence factors were found among the population. To our knowledge, this is the first overall investigation of tomato and pepper cross-over pathogenic bacterial populations in Bulgaria.

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.

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.

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.