Comparative Genomics of Listeria Species Recovered from Meat and Food Processing Facilities.

Listeria species (spp.) are contaminants that can survive in food, on equipment, and on food processing premises if appropriate hygiene measures are not used. Homologous stress tolerance genes, virulence gene clusters such as the prfA cluster, and clusters of internalin genes that contribute to the pathogenic potential of the strains can be carried by both pathogenic and nonpathogenic Listeria spp. To enhance understanding of the genome evolution of virulence and virulence-associated properties, a comparative genome approach was used to analyze 41 genome sequences belonging to L. innocua and L. welshimeri isolated from food and food processing facilities. Genetic determinants responsible for disinfectant and stress tolerance were identified, including the efflux cassette bcrABC and Tn6188_qac_1 disinfectant resistance determinant, and stress survival islets. These disinfectant-resistant genes were more frequently found in L. innocua (12%) than in L. welshimeri (2%). Several isolates representing the presumed nonpathogenic L. innocua still carried virulence-associated genes, including LGI2, LGI3, LIPI-3, and LIPI-4 which were absent in all L. welshimeri isolates. The mobile genetic elements identified were plasmids (pLGUG1 and J1776) and prophages (PHAGE_Lister_vB_LmoS_188, PHAGE_Lister_LP_030_3, PHAGE_Lister_A118, PHAGE_Lister_B054, and PHAGE_Lister_vB_LmoS_293). The results suggest that the presumed nonpathogenic isolates especially L. innocua can carry genes relevant to the strain's virulence and stress tolerance in the food and food processing facilities. IMPORTANCE This study provides genomic insights into the recently expanded genus in order to gain valuable information about the evolution of the virulence and stress tolerance properties of the genus Listeria and the distribution of these genetic elements pertinent to the pathogenic potential across Listeria spp. and clonal lineages in South Africa (SA).

Kombucha Multimicrobial Community under Simulated Spaceflight and Martian Conditions.

Kombucha microbial community (KMC) produces a cellulose-based biopolymer of industrial importance and a probiotic beverage. KMC-derived cellulose-based pellicle film is known as a highly adaptive microbial macrocolony-a stratified community of prokaryotes and eukaryotes. In the framework of the multipurpose international astrobiological project "BIOlogy and Mars Experiment (BIOMEX)," which aims to study the vitality of prokaryotic and eukaryotic organisms and the stability of selected biomarkers in low Earth orbit and in a Mars-like environment, a cellulose polymer structural integrity will be assessed as a biomarker and biotechnological nanomaterial. In a preflight assessment program for BIOMEX, the mineralized bacterial cellulose did not exhibit significant changes in the structure under all types of tests. KMC members that inhabit the cellulose-based pellicle exhibited a high survival rate; however, the survival capacity depended on a variety of stressors such as the vacuum of space, a Mars-like atmosphere, UVC radiation, and temperature fluctuations. The critical limiting factor for microbial survival was high-dose UV irradiation. In the tests that simulated a 1-year mission of exposure outside the International Space Station, the core populations of bacteria and yeasts survived and provided protection against UV; however, the microbial density of the populations overall was reduced, which was revealed by implementation of culture-dependent and culture-independent methods. Reduction of microbial richness was also associated with a lower accumulation of chemical elements in the cellulose-based pellicle film, produced by microbiota that survived in the post-test experiments, as compared to untreated cultures that populated the film. Key Words: BIOlogy and Mars Experiment (BIOMEX)-Kombucha multimicrobial community-Biosignature-Biofilm-Bacterial cellulose. Astrobiology 17, 459-469.

The First Space-Related Study of a Kombucha Multimicrobial Cellulose-Forming Community: Preparatory Laboratory Experiments.

Biofilm-forming microbial communities are known as the most robust assemblages that can survive in harsh environments. Biofilm-associated microorganisms display greatly increased resistance to physical and chemical adverse conditions, and they are expected to be the first form of life on Earth or anywhere else. Biological molecules synthesized by biofilm -protected microbiomes may serve as markers of the nucleoprotein life. We offer a new experimental model, a kombucha multimicrobial culture (KMC), to assess a structural integrity of a widespread microbial polymer - cellulose - as a biosignature of bacteria-producers for the multipurpose international project "BIOlogical and Mars Experiment (BIOMEX)", which aims to study the vitality of pro- and eukaryotic organisms and the stability of organic biomolecules in contact with minerals to analyze the detectability of life markers in the context of a planetary background. In this study, we aimed to substantiate the detectability of mineralized cellulose with spectroscopy and to study the KMC macrocolony phenotype stability under adverse conditions (UV, excess of inorganics etc.). Cellulose matrix of the KMC macrocolony has been mineralized in the mineral-water interface under assistance of KMC-members. Effect of bioleached ions on the cellulose matrix has been visible, and the FT-IR spectrum proved changes in cellulose structure. However, the specific cellulose band vibration, confirming the presence of ß(1,4)-linkages between monomers, has not been quenched by secondary minerals formed on the surface of pellicle. The cellulose-based KMC macrocolony phenotype was in a dependence on extracellular matrix components (ionome, viriome, extracellular membrane vesicles), which provided its integrity and rigidness in a certain extent under impact of stressful factors.

[Detection of potential sites of recombination in the Tick-borne encephalitis virus by the methods of comparative genomics].

The results of the bioinformatic search for the potential sites of the recombination in the genome-wide structures of the tick-borne encephalitis virus (TBEV) through a series of software techniques were presented in this work. The genomes of the 55 TBEV strains were assayed, 21 of them showed the presence of the recombination sites. Recombinant strains belonged to the Far Eastern (19 strains) and European (2 strains) genotypes. 22 sites of the recombination attributed were identified to five types based on position, strain, and regional characteristics. The parental strains were identified based on the genotypic and geographical parameters, which do not contradict the possibility of the formation of the recombinants. Nearly two-thirds of the sites are located in the regions of NS4a and NS4b genes, which are the "hot spots" of the recombination, most of them being concentrated in the gene NS4. It was shown that the recombination processes did not occur at the level of the genotypes (European genotype) or certain groups within the genotype (Far East) and were typical of the peripheral populations.

[The taxonomic study of the strain Bacillus sp. UCM B-7404--phytopathogenic fungae antagonist].

The polyphasic taxonomic analysis of strain Bacillus sp. UCM B-7404 active against phytopathogenic fungi and producing extracellular phytohormones, lytic enzymes and lipopeptide antifungal compounds has been carried out. The basic cell wall fatty acids presented by branched iso- and anteiso- C15:0 and C17:0 acids, contained 65-77% of the average pool. Phylogenetic assay of 16S rRNA gene nucleotide sequence, phenotypic and chemotaxonomic characteristics proved the attachment of strain Bacillus sp. UCM B-7404 to Bacillus amyloliquefaciens subsp. plantarum.

[Geno- and phenotypic characteristic of Bacillus strains--components of endosporin].

Endosporin is used in veterinary for the prophylaxis and treatment of disbacteriosis, intestinal infections, festering wounds and postpartum pyoinflammatory complications in agricultural animals. The probiotic is based on two Bacillus strains which inhibit growth of a broad spectrum of pathogenic microorganisms and synthesise proteolytic enzymes and other biologically active secondary metabolites, particularly - polysaccharides. The activity of these two strains was supplementary. For the species identification of these strains, sequences of 16S rRNA genes and fatty acid content of cell walls were analysed. It was found that the both strains belong to B. velezensis. Limitations of application of 16S rRNA sequences for identification of closely related species are discussed in the paper. A method of 16S rRNA sequence profiling by polymorphic nucleotides was proposed. It was also shown that usefulness of Bacillus strains in probiotics is mostly based on their unique strain specific properties rather than on general species characteristics.

Does the applicability of Bacillus strains in probiotics rely upon their taxonomy?

The taxonomic position and biological activities of two Bacillus strains used in veterinary probiotics were studied in this work. These microorganisms inhibit growth of a broad spectrum of pathogenic cultures. They synthesize proteolytic enzymes and other biologically active metabolites, and to some extent, supplement each other with probiotic activities. It is not clear whether these versatile activities are properties of individual strains or bacterial taxa as whole. 16S rRNA comparisons were conducted and illustrated the relatedness of these strains to Bacillus amyloliquefaciens . Their cell wall fatty acid contents were consequently analysed and specified a relation to the " Bacillus velezensis " ecomorph. On account of the previous observations, a simple method of 16S rRNA profiling by polymorphic nucleotides was proposed to determine a group of organisms closely related to "B. velezensis" and B. amyloliquefaciens subsp. plantarum, for they are biologically active strains suitable for use in biotechnology. The extreme genetic plasticity of these bacteria endowed each strain with a unique spectrum of antagonistic activity.

[PCR based identification of strains of endophytic ecotype of Bacillus amyloliquefaciens].

A search for genetic markers of an endophytic ecotype of Bacillus amyloliquefaciens was performed by the subtractive hybridization technique. The isolated fragments specificity was checked by DNA-DNA hybridization. The sequence of a DNA locus exhibiting the highest taxon specificity has manifested a 76% identity to the gene mtlA from Bacillus subtilis 168 genome. A set of discriminative primers has been derived from this sequence which could be used for the fast and reliable identification of isolates of the endophytic ecotype of B. amyloliquefaciens placing them separate against their close relatives including the type cultures of B. amyloliquefaciens and B. subtilis. Identification of strains of this ecotype is quite important as far as their characteristic ability to colonize the roots and internal tissues of plants in association with high antagonistic activity in respect of many phytopathogenic fungi and bacteria makes them valuable agents for plant protection.

[Some properties of Bacillus subtilis strains active against rotting agents on strawberries and fruit].

This work was aimed at the study of biological and genetic peculiarities of the strains Bacillus subtilis 2/10 and 63z which were selected for protection of strawberries from Botrytis fructigena and for apples under storage. The obtained strains were shown to possess a broad spectrum of antagonistic activity against phytopathogenic fungi and bacteria and this activity was manifested at low temperatures specific for the agriculture production storage. These cultures were not toxigenic or pathogenic in respect of plants and hot-blooded. By RAPD-typing and sequencing of the cloned parts of DNA the genetic specificity of the selected cultures was shown compared with other Bacillus subtilis strains which had not been so active. A set of genetic primes was selected to allow one to reliably identify the given strains during production of biopreparations and to control their use in the field conditions.

[Polymorphism of nonribosomal polypeptide synthetase genes in representatives of the Bacillus genus].

Polypeptide antibiotics are synthesized by Bacillus under control of nonribosomal polypeptide synthetases (NRPS). NRPS genes comprise the sequences of functional modules, each being responsible for incorporation of a certain amino acid to the synthesized oligopeptide. Dinucleotide frequency profiles calculated for DNA loci, covering the functional subunits of NRPS genes, were often observed to be significantly distant from those determined for the complete genome of the strain-producer, that suggests a possibility of horizontal transferring of the NRPS modules between microorganisms. A comparative analysis of the amino acid sequences of NRPS modules and statistical analysis of oligonucleotide usage frequencies allows following the pathway of evolution of genes controlling biosynthesis of antibiotics in Bacillus. A set of primers has been developed for genotyping of Bacillus strains with the purpose to identify producers of new polypeptide antibiotics among environmental isolates.

[Biological properties of the phosphate-mobilizing Bacillus subtilis strain IMV V-7023]. / Biologicheskiie svoistva fosfatmobilizuiushchego shtamma Bacillus subtilis IMV V-7023.

Biological characteristics of a new phosphate-mobilizing bacillus strain are reported. Species-level identification of the strain was performed according to morphological, cultural, and biochemical characteristics and the sequence of the 16S rRNA gene. The strain was identified as Bacillus subtilis IMV V-7023 and displayed a very high ability to mobilize phosphorus from its sparingly soluble inorganic and organic compounds and the capability of synthesizing biologically active substances; in addition, the strain essentially suppressed the growth of phytopathogenic bacteria, micromycetes, and agents causing various diseases of vegetable, cereal, leguminous, and other plants. The strain Bacillus subtilis IMV V-7023 is promising for developing bacterial preparations for crop production.

[Taxonomic contribution and antagonistic properties of antarctic fluorescent bacteria of Pseudomonas genus]. / Taksonomichnyi sklad ta antahonistychni vlastyvosti antarktychnykh fluorestsiiuiuchykh bakterii rodu Pseudomonas.

Fifty (50) strains of Pseudomonas genus have been isolated from the samples of soil, moss and bottom sediments taken on the Galindes, Piterman and Deception Islands (Ukrainian Antarctic station Akademik Vernadsky). Characteristic of the above strains by 84 phenotype features and identification by means of the computer program permitted them to be referred to the species Pseudomonas fluorescens (biovar V.), P. putida (biovars A and B), P. veronii. The belonging of the strain 23 to the species P. veronii has been confirmed by the results of sequence of 16S rRNA. Being grown at low temperatures (4 degrees C) the strains of the above species are characterized by high antagonistic activity as to Gram-positive, Gram-negative bacteria, yeast and microscopic fungi. Cultivation of antagonists at 26 degrees C led to the sharp decrease or complete disappearance of the antagonistic effect.

Genetic diversity and involvement in bread spoilage of Bacillus strains isolated from flour and ropy bread.

AIMS: To study Bacillus contamination of wheat flour and ropy bread, to analyse genetic diversity of isolated strains and to evaluate the ability of these strains to produce ropy bread. METHODS AND RESULTS: Classical and molecular methods [16S rDNA sequencing and random amplified polymorphic DNA (RAPD)-PCR] were used to identify and type-isolated strains. The predominant species isolated were Bacillus subtilis and B. licheniformis. RAPD analysis demonstrated that the same sample may harbor different strains. Ten of 15 strains of B. subtilis and four of six strains of B. licheniformis were able to cause rope spoilage of the laboratory-baked bread. CONCLUSION: RAPD typing can be useful in the tracking of Bacillus strains during bakery processing and in the understanding of the role of different Bacillus strains in the rope spoilage of bread. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate the variability of Bacillus strains isolated from flour and responsible for rope spoilage of bread.

[Identification of the Pseudomonas genus bacteria by computer analysis]. / Identifikatsiia bakterii roda Pseudomonas metodami komp'iuternogo analiza.

A computer program for the simplified phenotypic identification of Pseudomonas has been developed. The information concerning 66 species included in up-to-date Pseudomonas genus characterized by 113 tests was accumulated in a database. The identification key is represented in interactive mode on a website http://www.imv.kiev.ua/PsmIK/default.htm. The program was used for the identification of 46 Pseudomonas strains isolated from rhizosphere. For 23 more strains unidentified by conventional technique, the level of similarity was 67-74%. This fact allows suggesting that they might be representatives of new Pseudomonas species.

[Endophytic bacilli--producers of type II restriction endonucleases]. / Endofitnye batsilly--produtsenty éndonukleaz restriktsii II tipa.

Two of thirteen bacillar strains isolated from the inner tissues of cotton plants were found to produce type II restriction endonucleases. The investigation of the site specificity of these enzymes showed that they are AsuI and Eco31I isoschizomers.

[Bsu50441--isoschizomer of endonuclease restriction AsuI]. / Bsu50441--izoshyzomer endonukleazy restryktsiï AsuI.

Endonuclease of restriction of type II has been found in Bacillus subtilis B-5044 during testing of endophytic strains of cotton plants bacilli. The restrictase Bsu5044I hydrolysed DNA of M13mp18 phage in 4 sites; pUC19 DNA in 5 sites; pBR322 in 15 sites. There were a lot of sites restriction in DNAs of phages T7 and lambda. A comparison of electrophoretical divisions of Bsu5044I and Cfr13I fragments of phages and plasmid DNAs showed their identity. Thus, the found restrictase Bsu5044I is an isoschisomer of AsuI.

Simplified technique for identification of the aerobic spore-forming bacteria by phenotype.

The use of modern research approaches of genetics, biochemistry and molecular biology has led to progress in bacterial taxonomy. Systematic study of the aerobic spore-forming bacteria has resulted in the realignment of the genus Bacillus into several new genera. In the meantime, the identification process has become more difficult for the non-specialist in Bacillus taxonomy. This paper presents a key for the simplified phenotypic identification of the mesophilic, aerobic, spore-forming bacteria belonging to the genera Bacillus, Paenibacillus, Brevibacillus, Aneurinibacillus, Geobacillus and Virgibacillus. A total of 81 species were included and 115 morphological and physiological tests were analysed for their discriminative efficiency. This key is practical for rough but quick identification of aerobic spore-forming bacteria isolated from nature. Such preliminary identification will be helpful for the selection of reference strains and methods for more precise identification using the newest techniques. The reliability of the proposed identification key was tested on 100 cultures from the Ukrainian Collection of Microorganisms. The developed identification key is represented in interactive mode on a website (http://www/imv.kiev.ua/key/).

[Type-II site-specific restriction endonucleases in endophytic strains of the genus Bacillus]. / Saitspetsyfichni endonukleazy restryktsiï II typu u endofitnykh shtamiv rodu Bacillus.

Using a modified Belavin's method the site-specific II type restriction endonucleases were determined for two endophytic strains of the Bacillus genus from the inner tissues of cotton plants.

[The identification of freshly isolated strains, the causative agents of human candidiasis and the search for effective antifungal probiotics]. / Identyfikatsiia svizhovydilenykh shtamiv--zbudnykiv kandydoziv liudyny i poshuk efektyvnykh protyhrybkovykh probiotykiv.

The species variety of yeasts of the genus Candida in children and patients with AIDS has been investigated. Composition of the studied species was found to be similar in newborns and in women with candidiasis. In order to create new probiotics with antifungal properties, the cluster analysis of antagonistic activity of some Bacillus strains against Enterobacteriaceae, Candida, normoflora and food-fermenting lactobacteria has been conducted. There were selected the combinations of some Bacillus strains, which effectively inhibited pathogenic and opportunistic microorganisms and at the same time had no effect on intestinal normoflora.

[Creation and practical use of a mathematical model of the antagonistic action of bacilli in designing probiotics]. / Sozdanie i prakticheskoe primenenie matematicheskoi model antagonisticheskogo deistviia batsill pri konstruirovanii probiotikov.

A major problem in constructing probiotics, preparations based on antagonistic action of live cultures of saprophytic bacteria, is finding antagonist strains with the required properties. A candidate strain must produce antagonistic action against a given set of pathogens, show high resistance to adverse environmental conditions, and be harmless for the macroorganism. Screening for such strains is a prolonged and laborious procedure.