Identification and Characterization of Human Breast Milk and Infant Fecal Cultivable Lactobacilli Isolated in Bulgaria: A Pilot Study.

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.

Cuspidothrix Is the First Genetically Proved Anatoxin A Producer in Bulgarian Lakes and Reservoirs.

The paper presents the first proof of planktonic cyanoprokaryote genus Cuspidothrix as an anatoxin A (ATX) producer in Bulgarian wetlands. The results from polymerase chain reaction (PCR) obtained from two summer sampling campaigns in 26 selected lakes and reservoirs demonstrated presence of the anaC gene, responsible for ATX production in 21 strains of the genus. They were found in three waterbodies sampled in 2018 (coastal lake Vaya, coastal reservoir Poroy, inland reservoir Sinyata Reka) and in four waterbodies sampled in 2019 (inland reservoirs Duvanli, Koprinka, Plachidol 2, Sinyata Reka). The detected genetic diversity generally corresponds to the observations conducted by conventional light microscopy, by which we distinguished three species of Cuspidothrix (Cuspidothrix issatschenkoi, Cuspidothrix elenkinii and Cuspidothrix tropicalis, the latter considered alien in the country). Eleven strains showed high similarity to two sequences of C. issatschenkoi available from the National Centre for Biotechnology Information (NCBI). Ten other strains assembled in a group, which-in lack of available from NCBI genetic sequences-were presumed related to C. tropicalis and C. elenkinii after comparison with the results from light microscopy. Cuspidothrix strains found in Bulgarian waterbodies showed high genetic similarity to those isolated and sequenced from Asia (Japan, China) and Northern Europe (Norway, Finland).

First Report on Microcystis as a Potential Microviridin Producer in Bulgarian Waterbodies.

Bulgaria, situated on the Balkan Peninsula, is rich in small and shallow, natural and man-made non-lotic waterbodies, which are threatened by blooms of Cyanoprokaryota/Cyanobacteria. Although cyanotoxins in Bulgarian surface waters are receiving increased attention, there is no information on microviridins and their producers. This paper presents results from a phytoplankton study, conducted in August 2019 in three lakes (Durankulak, Vaya, Uzungeren) and five reservoirs (Duvanli, Mandra, Poroy, Sinyata Reka, Zhrebchevo) in which a molecular-genetic analysis (PCR based on the precursor mdnA gene and subsequent translation to amino acid alignments), combined with conventional light microscopy and an HPLC analysis of marker pigments, were applied for the identification of potential microviridin producers. The results provide evidence that ten strains of the genus Microcystis, and of its most widespread species M. aeruginosa in particular, are potentially toxigenic in respect to microviridins. The mdnA sequences were obtained from all studied waterbodies and their translation to amino-acid alignments revealed the presence of five microviridin variants (types B/C, Izancya, CBJ55500.1 (Microcystis 199), and MC19, as well as a variant, which was very close to type A). This study adds to the general understanding of the microviridin occurrence, producers, and sequence diversity.

Morphological and Molecular Identification of Microcystin-Producing Cyanobacteria in Nine Shallow Bulgarian Water Bodies.

The paper presents results from the first application of polyphasic approach in studies of field samples from Bulgaria. This approach, which combined the conventional light microscopy (LM) and molecular-genetic methods (based on PCR amplified fragments of microcystin synthetase gene mcyE), revealed that almost all microcystin-producers in the studied eutrophic waterbodies belong to the genus Microcystis. During the molecular identification of toxin-producing strains by use of HEPF × HEPR pair of primers, we obtained 57 sequences, 56 of which formed 28 strains of Microcystis, spread in six clusters of the phylogenetic tree. By LM, seven Microcystis morphospecies were identified (M. aeruginosa, M. botrys, M. flos-aquae, M. natans, M. novacekii, M. smithii, and M. wesenbergii). They showed significant morphological variability and contributed from <1% to 98% to the total biomass. All data support the earlier opinions that taxonomic revision of Microcystis is needed, proved the presence of toxigenic strains in M. aeruginosa and M. wesenbergii, and suppose their existence in M. natans. Our results demonstrated also that genetic sequencing, and the use of HEPF × HEPR pair in particular, can efficiently serve in water quality monitoring for identifying the potential risk from microcystins, even in cases of low amounts of Microcystis in the water.

Archaeal and bacterial diversity in two hot springs from geothermal regions in Bulgaria as demostrated by 16S rRNA and GH-57 genes.

Archaeal and bacterial diversity in two Bulgarian hot springs, geographically separated with different tectonic origin and different temperature of water was investigated exploring two genes, 16S rRNA and GH-57. Archaeal diversity was significantly higher in the hotter spring Levunovo (LV) (82°C); on the contrary, bacterial diversity was higher in the spring Vetren Dol (VD) (68°C). The analyzed clones from LV library were referred to twenty eight different sequence types belonging to five archaeal groups from Crenarchaeota and Euryarchaeota. A domination of two groups was observed, Candidate Thaumarchaeota and Methanosarcinales. The majority of the clones from VD were referred to HWCG (Hot Water Crenarchaeotic Group). The formation of a group of thermophiles in the order Methanosarcinales was suggested. Phylogenetic analysis revealed high numbers of novel sequences, more than one third of archaeal and half of the bacterial phylotypes displayed similarity lower than 97% with known ones. The retrieved GH-57 gene sequences showed a complex phylogenic distribution. The main part of the retrieved homologous GH-57 sequences affiliated with bacterial phyla Bacteroidetes, Deltaproteobacteria, Candidate Saccharibacteria and affiliation of almost half of the analyzed sequences is not fully resolved. GH-57 gene analysis allows an increased resolution of the biodiversity assessment and in depth analysis of specific taxonomic groups. [Int Microbiol 18(4):217-223 (2015)].

Phenotypic diversity of Erwinia amylovora in Bulgaria.

Fifty-one strains of Erwinia amylovora isolated from nine host plants in Bulgaria were characterized phenotypically and identified by the API 20E and BIOLOG system. The identification was confirmed by PCR amplification of a specific region of the plasmid pEA29 and the genome ams region. The phenotypic diversity of the strains was studied on the basis of their API 20E and BIOLOG metabolic profiles, as well as of their SDS-PAGE protein profile. Metabolic diversity among the strains was established, but no connection with the origin of the strains was revealed. The Bulgarian strains showed API 20E metabolic profiles not found in previous studies of E. amylovora. The strains formed a homogenous group on the basis of their protein profiles. All the strains were sensitive to the antibiotics streptomycin, tetracycline and oxytetracycline. This study was an initial step towards an investigation of the diversity and evolution in the Bulgarian population of E. amylovora, and it was the first characterization of E. amylovora strains isolated from different host plants in the period 1995-2005 in Bulgaria.