Bacterial communities of plum phyllosphere and characterization of indigenous antagonistic Bacillus thuringiensis R3/3 isolate.

AIMS: The characterization of bacterial communities diversity on four local plum cultivars in two phenological stages using culture-dependent and culture-independent methods and screening among culturable plum community for indigenous bacteria active against phytopathogens. METHODS AND RESULTS: The bacterial communities associated with leaves and fruits of four local Serbian plum cultivars (Pozegaca, Ranka, Cacanska Lepotica and Cacanska Rodna) were investigated in two phenological stages during early (May) and late (July) fruit maturation. Metagenomic approach revealed Methylobacterium, Sphingomonas and Hymenobacter as dominant genera. The most frequently isolated representatives with cultivable approach were pseudomonads with Pseudomonas syringae and Pseudomonas graminis, the most likely resident species of plum community. Antagonistic Bacillus thuringiensis R3/3 isolate from plum phyllosphere had ability to produce exoenzymes, reduce the growth of phytopathogenic bacteria in co-culture environment and show quorum quenching activity. CONCLUSIONS: Plum cultivar and growth season contribute to the structure of the bacterial community associated with plum. Plum phyllosphere is good source of antagonists effective against phytopathogens. SIGNIFICANCE AND IMPACT OF STUDY: Knowledge of bacterial communities on plum will have an impact on studies related to phyllosphere ecology and biocontrol. The indigenous antagonistic isolate, B. thuringiensis R3/3, from plum could be further investigated for its potential use in biological control of plum diseases.

CHANGES OF CYTOLYSIS INDICATORS IN RATS' BLOOD RESULTED FROM SIMULTANEOUS INTOXICATION WITH TOBACCO SMOKE AND SODIUM NITRITE AFTER USING MILDRONATE.

The article is devoted to an experimental study of the effect of tobacco smoke and the effect of sodium nitrite on the background of tobacco intoxication on the body of rats of different age groups and to identify violations caused by these toxicants. It has been established that under the conditions of nitrite-tobacco intoxication, a more pronounced cytolysis of the cells takes place, which leads to a change in the permeability of the plasma membranes than in case of poisoning with only tobacco smoke. Immature rats were the most sensitive to the action of toxicants, in which the activity of membrane-dependent enzymes was more statistically significant (ALT activity in the serum increased 7,7 times, AST - 5 times, GGTP - 4,1 times, LDH - 1,8 times; p ≤ 0.05) at the end of the study. In the same group of rats, the highest percentage of permeability of the erythrocyte membrane was revealed, as indicated by its index (55,6% higher than normal, in mature animals 38% EII was higher than in intact control animals, in old ones - 54%). When using an antihypoxant, it was noted that mildronate significantly (p≤0.05) reduced the activity of indicator enzymes in the blood serum of rats during the whole experiment, and also led to a decrease in the erythrocyte index of intoxication in the blood of rats of all age groups. To confirm the obtained results, morphological studies of the organs of rats of different age groups were carried out after sodium was damaged by nitrite against the background of tobacco intoxication and the effect of the metabolic action of mildronate on them. The membrane-protective properties of mildronate have been proven, which makes it possible to use it in various pathologies accompanied by the development of cytolytic syndrome.

Biological control of Pseudomonas syringae pv. aptata on sugar beet with Bacillus pumilus SS-10.7 and Bacillus amyloliquefaciens (SS-12.6 and SS-38.4) strains.

AIM: Assessment of biological control of Pseudomonas syringae pv. aptata using crude lipopeptide extracts (CLEs) of two Bacillus amyloliquefaciens strains (SS-12.6 and SS-38.4) and one Bacillus pumilus strain (SS-10.7). METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of CLEs and their combinations against the pathogen and potential interaction between the extracts were determined in vitro. The most effective antibacterial activity was achieved with the CLE from B. amyloliquefaciens SS-12.6, with an MIC value of 0·63 mg ml-1 . Interactions between CLE combinations were mostly indifferent. The biocontrol potential of CLEs, mixtures of CLEs, and cell culture of B. amyloliquefaciens SS-12.6 was tested on sugar beet plants inoculated with P. syringae pv. aptata P53. The best result in inhibiting the appearance of tissue necrosis (up to 92%) was achieved with B. amyloliquefaciens SS-12.6 cell culture. CONCLUSION: This work demonstrated significant biocontrol potential of the CLE and cell culture of B. amyloliquefaciens SS-12.6 which successfully suppress leaf spot disease severity on sugar beet plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of biocontrol of sugar beet emerging pathogen will contribute to growers in terms of alternative disease control management. This study represents first assessment of biological control of P. syringae pv. aptata.

[APPLICATION OF VACUUM-THERAPY ON STAGES OF SURGICAL TREATMENT OF THE DIABETIC FOOT SYNDROME].

Vacuum therapy of an acute and chronic wounds was used in a complex of surgical treatment of 228 patients, suffering diabetic foot syndrome. There was established a positive local and systemic action of this method for the treatment of the wound defect. Vacuum therapy of the wounds guarantees the wound process clinical course stabilization, improvement of microcirculation, reduction of their microbial soiling, stimulation of regenerative processes, elimination of endogenous intoxication.

Microbiota associated with pollen, bee bread, larvae and adults of solitary bee Osmia cornuta (Hymenoptera: Megachilidae).

Using cultivation-dependant method, we isolated 184 strains from fresh and old bee bread, pollen, larvae and adults of solitary bee Osmia cornuta. The 16S rDNA sequencing of 79 selected isolates gave the final species-specific identification of strains. Phylogenetic analysis indicated that microbiota isolated from five different sources were represented with 29 species within three different phyla, Firmicutes with 25 species, Actinobacteria with only one species and Proteobacteria with three species of Enterobacteriaceae. Bacterial biodiversity presented with Shannon-Wiener index (H') was highest in the alimentary tract of adults and old bee bread (H' = 2.43 and H' = 2.53, respectively) and in the same time no dominance of any species was scored. On the contrary, results obtained for Simpson index (D) showed that in pollen samples the dominant species was Pantoea agglomerans (D = 0.42) while in fresh bee bread that was Staphylococcus sp. (D = 0.27). We assume that microbial diversity detected in the tested samples of solitary bee O. cornuta probably come from environment.

Novel antilisterial bacteriocin licheniocin 50.2 from Bacillus licheniformis VPS50.2 isolated from soil sample.

AIM: To isolate and characterize bacteriocin, licheniocin 50.2, from soil bacteria identified as Bacillus licheniformis. METHODS AND RESULTS: The strain B. licheniformis VPS50.2 was identified as bacteriocin producer, effective against Gram-positive bacteria, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and ß-haemolytic streptococci. The start of bacteriocin production coincides with the beginning of sporulation. Ammonium sulfate precipitation, chloroform extraction and ultrafiltration were used for bacteriocin purification. MALDI TOF/TOF mass spectrometry of purified sample detected the protein with molecular mass of 3253·209 Da. N-terminal sequencing recognized first 15 amino acids with the sequence: W E E Y N I I X Q L G N K G Q. We named the newly characterized bacteriocin as subclass II.3 bacteriocin, licheniocin 50·2. The bacteriocin activity was insensitive to lysozyme and proteinase K, heat stable after incubation at 100°C for 30 min and over wide range of pH (2-12). MICs of crude bacteriocin extract were determined for L. monocytogenes and MRSA. Time-kill study showed that licheniocin had bactericidal effect to L. monocytogenes. CONCLUSION: A novel, thermostable, pH-tolerant bacteriocin active against Gram-positive bacteria was isolated. SIGNIFICANCE AND IMPACT OF THE STUDY: Attributes of new, stable licheniocin 50.2 make it a promising agent for application as biopreservative in food industry.

The presence of prtP proteinase gene in natural isolate Lactobacillus plantarum BGSJ3-18.

AIMS: The study of proteolytic activity and examination of proteinase gene region organization in proteolytically active Lactobacillus plantarum strains from different natural sources. METHODS AND RESULTS: A set of 37 lactobacilli was distinguished by using multiplex PCR assay. Results showed that 34 strains were Lact. plantarum and three of them were Lact. paraplantarum. The examination of proteolytic activity revealed that 28 Lact. plantarum and two Lact. paraplantarum hydrolyse beta-casein. Further analyses of all proteolytically active Lact. plantarum with primers specific for different types of CEPs demonstrated that strain BGSJ3-18 has prtP catalytic domain as well as prtP-prtM intergenic region showing more than 95% sequence identity with the same regions present in Lact. paracasei, Lact. casei and L. lactis. No presence of prtB, prtH or prtR proteinase genes was detected in any of tested Lact. plantarum strains. CONCLUSIONS: One out of 28 analysed Lact. plantarum strains harbours the prtP-like gene. The other proteolytically active Lact. plantarum probably possesses a different type of extracellular proteinase(s). SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first report about the presence of the prtP-like gene in Lact. plantarum, which illustrates the mobility of this gene and its presence in different species.

Variation in specificity of the PrtP extracellular proteinases in Lactococcus lactis and Lactobacillus paracasei subsp. paracasei.

Comparison of cell-wall-bound extracellular proteinases (CEPs) from Lactobacillus paracasei (LBP) ssp. paracasei natural isolates BGHN14, BGAR75 and BGAR76 with Lactococcus lactis (LCL) ssp. cremoris Wg2, in their action on alpha(S1)-, beta- and kappa-casein was done. The CEPs of LBP strains were able to degrade alpha(S1)- and beta-caseins and their caseinolytic specificity depended on the type of buffer used. These CEPs, compared with LCL Wg2, differ in four amino acid residues in small segments predicted to be involved in substrate binding. The most striking features of this comparison are the presence of Ala instead of Ser(329) and the presence of Thr instead of Asn(256) and Ala(299), in the subtilisin-like region of the CEP in LBP natural isolates. Additional conservative amino acid substitution Leu to Ile(364) was found.

Molecular characterization of plasmids pS7a and pS7b from Lactococcus lactis subsp. lactis bv. diacetylactis S50 as a base for the construction of mobilizable cloning vectors.

AIMS: Strain Lactococcus lactis subsp. lactis bv. diacetylactis S50 harbours five theta-replicating plasmids (pS6, pS7a, pS7b, pS80 and pS140). The aim of this study was to characterize domains involved in the replication and conjugative mobilization of the small plasmids pS7a and pS7b, which are structurally very similar. METHODS AND RESULTS: Complete nucleotide sequences of pS7a and pS7b were determined by cloning DNA fragments of different sizes into Escherichia coli vectors. Linearized plasmids and four EcoRI fragments of the pS7a and pS7b were cloned into an origin probe vector. Constructed plasmids (pSEV10, pSK10, pISE1a and pISE1b) were able to replicate in the strain L. lactis subsp. cremoris MG1363. In addition, experiments showed that plasmids pS7a and pS7b contained oriT sequences and their conjugative transfer directly depended on the presence of pS80 in donor cells. CONCLUSIONS: Plasmids pS7a and pS7b contained typical lactococcal theta replication origin and repB gene that enable them to replicate in the strain L. lactis subsp. cremoris MG1363. Plasmid pS80 plays a key role in the conjugative transfer of small plasmids. SIGNIFICANCE AND IMPACT OF THE STUDY: Plasmids pS7a and pS7b-based derivatives could be valuable tools for genetic manipulation, studying processes of plasmid maintenance and horizontal gene transfer in lactococci.

Analysis of the presence of prtR proteinase gene in natural isolates of Lactobacillus rhamnosus.

The region of the prtR gene coding for the active site of PrtR proteinase was detected in natural isolates of lactobacilli, previously determined as Lactobacillus rhamnosus. This region was present in all L. rhamnosus strains with proteolytic activity. The PCR primers used were constructed on the basis of the sequence of the catalytic domain of the prtR proteinase gene. These primers generated in colony-PCR procedure specific 611 1-bp product with DNA from natural isolates of L. rhamnosus. No PCR amplifications using these primers were obtained for closely related bacteria of genus Lactobacillus, regardless of their proteolytic activity. In addition, these primers could be used singly or in multiplex PCR together with the Lactobacillus genus-specific primers. Compared with the other proteinases within the genus Lactobacillus (PrtP, PrtB and PrtH) which retained the activity in cell-free proteinase extracts, PrtR proteinase showed proteolytic activity only under in vivo conditions (whole cells of the producing strains).

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli.

The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6.5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both alphaS1-casein and beta-casein, showing very low activity towards kappa-casein. The BGPF1 proteinase completely hydrolysed only beta-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA-DNA hybridization and PCR analysis showed that BGPF1 contains the prtB-like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.

Characterization of natural isolate Lactobacillus acidophilus BGRA43 useful for acidophilus milk production.

Lactobacillus acidophilus BGRA43 was selected from a set of human origin isolates of Lact. acidophilus strains for the highest growth rates and antagonistic effect against both Gram-positive and Gram-negative bacteria. The strain BGRA43 also exhibited an inhibitory effect on the growth of Clostridium sporogenes. Inhibition of this strain seems to be due to lactic acid production rather than hydrogen peroxide or bacteriocin. Growth of Lact. acidophilus BGRA43 in non-fat skim milk for 6 h at 37 degrees C resulted in a lowering of the pH value to 4.53. Besides the fast acidification, this strain generated a high viscosity of skim milk. These characteristics make the strain BGRA43 attractive for acidophilus milk production. Lactobacillus acidophilus BGRA43 produces an extracellular proteinase. Whole cells efficiently degraded casein for 3 h at 37 degrees C especially alpha- and beta-casein fractions. Total DNA isolated from the strain BGRA43 did not show any hybridization with lactococcal proteinase probes indicating that this strain produces a distinctive proteinase.

A genetic study of apramycin-resistant mutants of Escherichia coli.

The apramycin-resistant mutants of E. coli were isolated spontaneously. Mutations, aprA and aprB, conferring resistance to apramycin were located at 72 min on the E. coli genetic map where most of ribosomal genes were mapped. One of the mutants, carrying the aprB mutation, has an altered translational fidelity expressed as severe restriction of amber suppressor activity in vivo. The chromosome location and altered translational fidelity indicate that the apramycin-resistant phenotype could be a consequence of an alteration of the ribosomal structure.

Characterization of the Cell Wall-Bound Proteinase of Lactobacillus casei HN14.

Lactobacillus casei HN14, which was isolated from homemade cheese, produces an extracellular, cell wall-bound proteinase. The HN14 proteinase can be removed from the cell envelope by washing the cells in a Ca-free buffer. The activity of the crude proteinase extract is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the HN14 proteinase is similar to the lactococcal PI-type enzyme, since it hydrolyzes beta-casein only. Lactobacillus casei HN14 appeared to be plasmid free, which suggests that the proteinase gene is chromosomally located. Chromosomal DNA of this strain hybridizes with DNA probes Q1 (which contains a fragment of the prtM gene) and Q6 and Q92 (which contain fragments of the prtP gene); all three probes originated from the proteinase gene region of Lactococcus lactis subsp. cremoris Wg2. A restriction enzyme map of the proteinase region of Lactobacillus casei HN14 was constructed on the basis of hybridization experiments. Comparison of the restriction enzyme maps of the Lactobacillus casei HN14 proteinase gene region and those of lactococcal proteinase gene regions studied so far indicates that they are highly similar.

Altered translational fidelity of a Salmonella typhimurium LT2 mutant resistant to the aminoglycoside antibiotic neamine.

A neamine-resistant mutant of Salmonella typhimurium LT2 with altered translational fidelity was isolated. The phenomenon was expressed in severe restriction of amber suppressor activity in vivo as well as in decreased misreading of poly(U) RNA in vitro. The mutation conferring resistance to neamine was mapped at 72 min on the Salmonella genetic map, where some of the ribosomal genes have already been mapped. This location indicates that the neamine-resistant phenotype as well as an altered translational fidelity could be a consequence of an alteration of the ribosomal structure.