Galina Stoyancheva

Bacteriocin production and gene sequencing analysis from vaginal Lactobacillus strains

The human vagina is a complex and dynamic ecosystem containing an abundance of microorganisms. In women of childbearing age, this system is dominated by Lactobacillus spp. In the present work, seventeen newly isolated vaginal strains were identified by 16S rDNA sequencing and were investigated for their antimicrobial properties. Twelve of the isolated Lactobacillus strains showed activity against one or more microorganisms. Six and five of them produced substances that inhibited the growth of two different Klebsiella strains and Staphylococcus aureus, respectively. Two lactobacilli strains were active against an Escherichia coli strain, one isolate was active against an Enterococus faecalis strain and another lactobacilli strain showed antimicrobial activity against a Candida parapsilosis strain. The nature of the active compounds was additionally studied, and the presence of bacteriocin-like substances was proved. The genes related to the bacteriocin production in three of the newly isolated strains were identified and sequenced. The presence of gassericin A operon in the genome of the species Lactobacillus crispatus was described for the first time. The presence of antimicrobial activity contributes to their possible use as potential probiotic strains after further research.

Molecular identification of vaginal lactobacilli isolated from Bulgarian women

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in␣comparison with a set of 21 reference strains based␣on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of␣them to be classified as belonging to one of␣the␣following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.

Molecular Study of the useful and the Contaminant Microflora in Fermented Dairy Products

ABSTRACT Lactic acid bacteria have been used as starter strains in the production of fermented dairy products for centuries. Most of the dairy products contain lactic acid bacteria, but also other bacteria involved as contaminant microflora. We explored the microbial content of home-made dairy products and those purchased from the market. In our study twenty-six pure cultures were isolated. The isolated strains were investigated by a set of physiological and molecular-genetic methods for their accurate species identification and genotyping. From the microorganisms, involved in fermentation and ripening of dairy products with proven health benefits to human, in studied foods predominated Lactobacillus delbrueckii, Lactobacillus helveticus and Lactobacillus plantarum. Another part of the isolated strains, representatives of the genus Kluyveromyces, Rhodotorula and Candida were contaminant microflora, as a result of poor hygiene in the manufacture and storage of the dairy products. Some of these strains were isolated from commercially available dairy products. The obtained results raise again the question about the efficacy of microbiological quality control and food safety.

GENETIC DIVERSITY OF BACTERIOPHAGES HIGHLY SPECIFIC FOR STREPTOCOCCUS THERMOPHILUS STRAIN LBB.A

ABSTRACT Eight virulent bacteriophages φA1, φA2, φA5, φA6, φDmA, φSfA, φPtA and φA7, that showed high specificity for Streptococcus thermophilus strain LBB.A, the streptococcal component of an industrial yogurt starter, were isolated. The comparison of the HindIII and EcoRV restriction patterns of the phage DNA revealed limited relatedness between the isolates, clustering together only φA5 with φA6 and indicating similarity between φSfA, φPtA and φA7. All eight bacteriophages were classified as costype phages. An internal region within a conserved 2.2 kbp EcoRI DNA fragment typical for the majority of S. thermophilus bacteriophages was present in six of the phage isolates and was absent in φDmA and φA1. PCR amplification of the VR2 region of the antireceptor gene yielded an 800 bp product for seven of the phages and a 700 bp product for φA2. The genome size of the eight phage isolates was in the range of 33.4–37.3 kbp. Of a total of 62 other S. thermophilus cultures φA1, φDmA and φPtA were able to infect only two strains with reduced effectiveness of plaquing. Despite the broad genetic diversity of phages of S. thermophilus strain LBB.A, a two-step selection procedure, involving successive challenge of the bacterial culture with φA1 and φA7, was sufficient to obtain mutants resistant to all eight phases.

Potential of ligninolytic enzymatic complex produced by white-rot fungi from genus Trametes isolated from Bulgarian forest soil

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta. These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn‐dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP.

Genomic Approaches to Yeast Taxonomy

ABSTRACT Yeasts have ecological, medical and biotechnoloqical importance. They are primary drivers of global carbon cycle and they belong to the most valuable microorganisms for industrial applications. Despite their importance, yeast remain poorly classified, particularly at the level of strains. Accurate identification of yeasts is essential in medicine, biotechnology and for understanding interactions in the ecosystem. Our review will focus on genomic tools currently used in taxonomy of yeasts.

Isolation and Characterization of Plasmids from Strains of Streptococcus Thermophilus Bulgarian Yogurt Starters

ABSTRACT Twenty Bulgarian Streptococcus thermophilus strains used in industrial milk fermentation were analyzed for their plasmid content. Six of them were found to contain one or two plasmids. The newly isolated plasmids were divided into three groups based on DNA homology. Six plasmids, sharing homology with pt38 and p2992, were classified within group I, two plasmids were classified into group II and one plasmid, p2991, possessing homology only with itself, was classified in Group III. Hybridization experiments revealed low homology between streptococcal and Lactobacillus plasmids. Using plus-origin- specific (dso) DIG-labeled probes we determined that two of the plasmids (pt38 and p2992) belong to the pC194 plasmid family, that uses the rolling circle mode of replication. One plasmid from group I, pt38 was further characterized. We constructed a restriction map and identified the restriction fragment bearing the dso of replication. pt38 appears to be an excellent candidate for creation of a shuttle vector for gene expression in Escherichia coli and the Streptococcus thermophilus because it is small in size, has five unique restriction sites and known location of the plus-origin of replication.