Today people focus on healthy lifestyle. It is well known that probiotics have a number of beneficial health effects in humans. They play an important role in protecting the host against harmful microorganisms, reduce metabolic disorders and support immune functions. In the present study, we investigated 26 probiotic products: 16 commercially available ones and 10 from a local manufacturer. None of the commercial products contained all labelled LAB and some of them contained unacceptable microorganisms. Of 890 isolated strains only 420 met the criteria for LAB. Ninety-seven strains were investigated by phenotypic and genotypic methods. 16S–23S rDNA amplification was performed for all putative LAB isolates. Fifty-seven rod-shaped bacteria referred to genus Lactobacillus and 16 coccus bacteria, to genus Weissela. Two-step multiplex PCR identified the rod-shaped strains to belong to four species: Lactobacillus delbrueckii ssp. bulgaricus (three strains), Lactobacillus acidophilus (four strains), Lactobacillus casei (three strains), Lactobacillus rhamnosus (two strains), Lactobacillus reuteri (four strains) and Lactobacillus plantarum (one strain). The transit tolerance of the isolated bacteria to in vitro simulated gastric juice (pH 2) was examined. Only four strains survived after 90 min of incubation. The antimicrobial activity of the native supernatants of the isolates was tested and 10 of them showed slight activity against three pathogenic bacteria. Our results demonstrated that only freshly produced products possessed the expected number of viable cells. The data revealed that one of the most common problems is the lower concentration of viable cells, misidentification and the presence of undesired microflora.

The urgent need of research of new approaches to control bacterial disease on economical important crops, focuses our attention on bacteriophages as alternative biocontrol agents. Thus, the purpose of this paper is to present the isolation and initial characterization of three bacteriophages (SfXv124t/1, 2 and 3) isolated from rhizosphere soil of a healthy tomato plant in Bulgaria that are capable to lyse three phytopathogenic bacteria. The initial characterization includes determination of: their host range, plaque morphology, optimal storage temperature of pure phage lysates, their sensitivity to UV light, thermal inactivation, optimal multiplicity of infection (MOI) and virion morphology. The obtained results showed that one of the phage isolates was capable to lyse wild strains from three phytopathogenic bacterial species: Xanthomonas vesicatoria, Xanthomonas euvesicatoria and Xanthomonas gardneri, and the two remaining phages were active against X. vesicatoria and X. euvesicatoria. On X. vesicatoria lawn, the phages produced the same plaque types that differed only in their size. Storage at 4 °C for 26 days did not lead to decrease in phage titer as opposed to storage at 28 °C followed by decrease to varying degree for all three phages. The results obtained after exposure of the phage lysates to sunlight (UVA + B) and UVC light in separate experiments showed that UVC had a potent phagocidal effect as after 50 min of exposure there were no viable phages in the samples. UVA an UVB had lethal effect for two of the phage isolates and absolutely no lethal effect for the third one as after 50 min of exposure to sunlight there was no decrease in the initial phage titer. Phage isolates were tested for their thermal inactivation after incubation of pure phage lysates at three different temperatures: 55 °C, 75 °C and 95 °C for a period of 10 and 30 min. The most lethal temperature turned out to be 95 °C as after 10 min there were no viable phages in the samples. Phage isolate SfXv124t/1 was the most susceptible as its titer decreased by 1 lg after 10 min of incubation at 55 °C and by another 1 lg after 30 min. The most thermally resistant isolate was SfXv124t/3 as its titer remained stable after 30 min of incubation at 55 °C and decreased only by lg after incubation at 75 °C for 10 min. The optimal MOI for SfXv124t/3 was 0,01 (tested range 0,01–100) with maximal phage titer, reported at the 24th hour of incubation. TEM micrographs of the same isolates reveals that it belongs to family Podoviridae.

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.

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.