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During the last few decades, the main focus of numerous studies has been on the human breast milk microbiota and its influence on the infant intestinal microbiota and overall health. The presence of lactic acid bacteria in breast milk affects both the quantitative and qualitative composition of the infant gut microbiota. The aim of this study was to assess the most frequently detected cultivable rod-shaped lactobacilli, specific for breast milk of healthy Bulgarian women and fecal samples of their infants over the first month of life, in 14 mother-infant tandem pairs. Additionally, we evaluated the strain diversity among the most common isolated species. A total of 68 Gram-positive and catalase-negative strains were subjected to identification using the MALDI-TOF technique. Predominant cultivable populations belonging to the rod-shaped lactic acid bacteria have been identified as Lacticaseibacillus rhamnosus , Limosilactobacillus fermentum , Lacticaseibacillus paracasei , and Limosilactobacillus reuteri . Also, we confirmed the presence of Lactiplantibacillus plantarum and Lactobacillus gasseri. Up to 26 isolates were selected as representatives and analyzed by 16S rRNA sequencing for strain identity confirmation and a phylogenetic tree based on 16S rRNA gene sequence was constructed. Comparative analysis by four RAPD primers revealed genetic differences between newly isolated predominant L. rhamnosus strains. This pilot study provides data for the current first report concerning the investigation of the characteristic cultivable lactobacilli isolated from human breast milk and infant feces in Bulgaria.

Xanthomonas euvesicatoria is a major cause of bacterial spot disease in various crops. The present study was focused on the pathosystem pepper ( Capsicum annuum L.) - X. euvesicatoria 269p (wild strain). The infectious process was studied using several different modes of in vivo inoculation under controlled conditions. The spread of the pathogen in different parts of the plants was monitored by a new qPCR procedure developed for the detection of X. euvesicatoria , as well as by re-isolation of viable bacterial cells. Photosynthesis, the number of viable pathogens, oxidative stress markers, activities of the main antioxidant enzymes, and levels of nonenzymatic antioxidants in the novel single-leaf model system were studied. The most important observation is that the invasion of the pathogen causes local infection and the dissemination of bacteria to the healthy parts of the host is blocked. The plants limit bacterial colonization around the entry points. Oxidative burst and alterations in antioxidant defenses are detected in infectious leaf lesions. Localized ROS overproduction resembles a hypersensitive response, but several differences can be observed. We assumed that pepper plants are more likely to manifest an intermediate phenotype, similar to lesions simulating disease or leaf flecking. By localizing the infection, possibly involving oxidative stress, the plant survives. However, the same applies to bacteria. The pathogen multiplies at the infection spots and is transmitted to other plants. Our conclusion is that the intermediate phenotype in the studied pathosystem is an example of long and successful co-evolution for both species.

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.

Yersiniosis is the third most commonly reported foodborne zoonosis in the European Union. Here, we evaluated the prevalence of pathogenic Yersinia enterocolitica among healthy pigs (as a major reservoir) in a slaughterhouse in Bulgaria. A total of 790 tonsils and feces from 601 pigs were examined. Isolation and pathogenicity characterization was carried out by the ISO 10273:2003 protocol and Polymerase Chain Reaction (PCR), detecting the 16S rRNA gene, attachment and invasion locus ( ail ), Yersinia heat-stable enterotoxin ( ystA ), and Yersinia adhesion ( yadA ) genes. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE), and antimicrobial resistance by the standard disk diffusion method. Of all the pigs tested, 6.7% were positive for Y. enterocolitica . All isolates belonged to Y. enterocolitica bioserotype 4/O:3. ail , and ystA genes were detected in all positive strains (n = 43), while the plasmid Yersinia virulence plasmid (pYV) was detected in 41. High homogeneity was observed among the strains, with all strains susceptible to ceftriaxone, amikacin and ciprofloxacin, and resistant to ampicillin. In conclusion, a low prevalence of Y. enterocolitica 4/O:3 was found in healthy pigs slaughtered in Bulgaria, not underestimating possible contamination of pork as a potential risk to consumer health.

Bacteriophages have greatly engaged the attention of scientists worldwide due to the continuously increasing resistance of phytopathogenic bacteria to commercially used chemical pesticides. However, the knowledge regarding phages is still very insufficient and must be continuously expanded. This paper presents the results of the isolation, characterization, and evaluation of the potential of 11 phage isolates as natural predators of a severe phytopathogenic bacterium- Xanthomonas euvesicatoria . Phages were isolated from the rhizosphere of tomato plants with symptoms of bacterial spot. The plaque morphology of all isolates was determined on a X. euvesicatoria lawn via a plaque assay. Three of the isolates were attributed to the family Myoviridae based on TEM micrographs. All phages showed good long-term viability when stored at 4 °C and -20 °C. Three of the phage isolates possessed high stability at very low pH values. Fifty-five-day persistence in a soil sample without the presence of the specific host and a lack of lytic activity on beneficial rhizosphere bacteria were found for the phage isolate BsXeu269p/3. The complete genome of the same isolate was sequenced and analyzed, and, for the first time in this paper, we report a circular representation of a linear but circularly permuted phage genome among known X. euvesicatoria phage genomes.

Introduction: The gram-negative bacterium Porphyromonas gingivalis is a major causative agent of periodontitis in adults. It is also associated with disorders of the cardiovascular and endocrine systems, rheumatoid arthritis, pancreatic cancer, and Alzheimer's disease. Lactic acid bacteria (LAB) present in the oral cavity or introduced as probiotic preparations can support successful treatment of periodontitis due to their antagonism with the pathogen., Aim: The aim of this study was in vitro assessment of the antimicrobial activity of Lactobacillus spp. and Streptococcus thermophilus against P. gingivalis., Materials and Methods: The antimicrobial effect of lactobacilli or S. thermophilus from the LBB Culture collection against P. gingivalis DSM 20709 was evaluated with the well diffusion assay on Wilkins Chalgren blood agar. Inhibition of the pathogen was evaluated by measuring the diameter of clear zones around the wells., Results: Application of milk fermented with selected LAB resulted in а bacteriostatic effect. The most active culture was S. thermophilus 187/4, followed by L. delbr. ssp. bulgaricus (LBB.B1054, C3/2 and LBB.B120), L. helveticus LBB.H48/1 and L. rhamnosus I-1/13. The respective reconstituted freeze-dried preparations had a stronger inhibitory effect on the pathogen with the formation of clear bactericidal zones. The effect of milk acidified with lactic acid was apparent with minimal bactericidal zone observed at concentration of 0.1%. The effectiveness of the assay was confirmed with Elgydium and Eludril., Conclusions: P. gingivalis DSM 20709 was sensitive to the metabolites produced in fermented milk by selected strains of L. delbr. ssp. bulgaricus, L. helveticus, L. rhamnosus, and S. thermophilus. Reconstituted freeze dried fermented milk had а stronger inhibitory effect compared to fresh samples. Lactic acid produced by lactic acid bacteria was the key component for inhibition of the pathogen., (This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 24 th hour of incubation. TEM micrographs of the same isolates reveals that it belongs to family Podoviridae., (© 2021 The Author(s).)

Pulsed-field gel electrophoresis (PFGE) is a highly discriminative molecular typing method that is used for epidemiological studies and investigation of outbreaks caused by different pathogens, including phytopathogenic Xanthomonas species. Bacterial spot (BS) is the most common and one of the most destructive diseases of tomato and pepper plants in Bulgaria. Several Xanthomonas species are known to cause BS, but the global distribution and genetic diversity of these species are not well understood. A collection of 100 BS-causing strains, isolated during the period of 1985-2012 from different tomato cultivars and weeds associated with tomato production areas from 11 geographic regions in Bulgaria, were screened for genetic diversity by genomic DNA restriction with rare-cutting endonucleases (XbaI and SpeI) subsequently resolved by PFGE. Two haplotypes for Xanthomonas vesicatoria and one haplotype for Xanthomonas gardneri strains were found.

In lactobacilli the aggregation phenotype is linked to their ability to colonize the intestinal and urogenital tracts and to counteract pathogenic bacteria. In all available complete genome sequences of Lactobacillus delbrueckii ssp. bulgaricus there are at least two genes putatively related to aggregation, one of which is annotated as aggregation-promoting factor (apf). Here we report the results from the in silico analysis of this gene and its product. The apf gene was present in the genome of all 70 tested L. delbr. ssp. bulgaricus strains. Its expression was confirmed for a selection of five strains with aggregation phenotype and two aggregation-negative strains. The mature Apf protein had a length of 257-284 amino acids with predicted molecular weight in the range of 28.64-30.36 kDa and isoelectric point of 10.6 ± 0.1, showing some similarity to Apf1 and Apf2 from L. johnsonii NCC533 and Apf1 and Apf2 from L. gasseri which are similar in size (28-35 kDa) and share a similar high isoelectric point (pI > 9). Predictive analyzes have indicated that Apf is a secretory protein. The 30 amino acid signal peptide and the predicted cleavage site in the pre-protein suggested that it was processed by Type I Signal protease. In the mature Apf protein a glutamine-rich N-terminal region was followed by an unusual lysine/alanine-rich region with variable length, supposed to be positively charged under physiological conditions, interacting with bacterial teichoic acids. The alignment of the C-termini of the Apf proteins showed similarity to conserved C-terminal domains in aggregation-related proteins in other lactobacilli such as Apf1 of Lactobacillus johnsonii ATCC 11506 and the secretory protein Sep of L. fermentum BR11, that may be involved in non-covalent binding to carbohydrates. The C-terminal anchor and the cationic domain in Apf may serve as mediators of physical cell-to-cell interaction in L. delbr. ssp. bulgaricus.

Lactobacillus delbrueckii subsp. bulgaricus LBB.B5 originates from homemade Bulgarian yogurt and was selected for its ability to form a strong association with Streptococcus thermophilus The genome sequence will facilitate elucidating the genetic background behind the contribution of LBB.B5 to the taste and aroma of yogurt and its exceptional protocooperation with S. thermophilus., (Copyright © 2016 Urshev et al.)

Among Streptococcus thermophilus cultures, the principle component of yoghurt and cheese starters, a minority of strains forms the group of 'H'-strains which show an unusually high acidification rate, grow faster and coagulate milk 3-5 hours earlier than the typical S. thermophilus cultures. A large-scale screening study was performed to select 'H'-strains of S. thermophilus from more than 100 samples of home-made yoghurt, industrial yoghurt starters and single cultures, maintained in the LBB culture collection. Only four strains - LBB.TN1, LBB.M23, LBB.M34 and LBB.M60 - were isolated/selected due to their ability to form large yellowish colonies on milk agar, supplemented with beta-glycerophosphate and bromocresol purple. While in general S. thermophilus is described as a species with limited proteolytic capacity and in contrast to all other tested S. thermophilus cultures, the four selected strains invariably gave positive amplification product with the polymerase chain reaction when primers, specific for the membrane proteinase-coding gene prt S were used. The macrorestriction profiles of the genomic DNA of the four strains confirmed that they are non-isogenic and not related to each other. When grown in milk and compared to the control industrial strain LBB.A, the four strains showed a dramatically faster acidification, coagulating milk within four hours. The application of strain TN1 or M23 as adjunct culture to industrial yoghurt starter LBB.BY5-12 resulted in shortening the fermentation time with more than 30 min.

The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-field gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profile. About 82% of the strains displayed a PFGE profile identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profiles, not reported so far, were established--one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profiles, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profile in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum.

A new Lactococcus lactis subsp. lactis B84, capable of utilizing starch as a sole carbon source and producing L(+)-lactate, was isolated from spontaneously fermented rye sourdough. Aiming at maximum lactic acid productivity, the components of the media and the cultivation conditions were varied. In MRS-starch medium (with absence of yeast and meat extracts), at 33 degrees C, agitation 200 rpm and pH 6.0 for 6 days complete starch hydrolysis occurred and 5.5 gl(-1) lactic acid were produced from 18 gl(-1) starch. The identification of strain B84 was based on genetic criteria. Amplified ribosomal DNA restriction analysis (ARDRA), PCR with species-specific primers and sequencing of the 16S rDNA proved its species affiliation. Four genes for enzymes, involved in starch degradation were detected in B84 genome: amyL, amyY, glgP and apu, coding cytoplasmic and extracellular alpha-amylases, glycogen phosphorylase and amylopullulanase, respectively. Reverse transcription PCR experiments showed that both genes, encoding alpha-amylases (amyL and amyY) were expressed into mRNAs, whereas apu and glgP were not. Amylase activity assay was performed at different pH and temperatures. The cell-bond amylase proved to be the key enzyme, involved in the starch hydrolysis with maximum activity at 45 degrees C and pH 5.4.