Vingt souches d'actinomycètes ont été isolées à partir d'échantillons de sol et de bois prélevés à partir d'une vielle maison dans l'ancienne médina de Fès et d'échantillons de sol prélevés de la région de Moulay Yacoub et des rives d'Ouèd Sebbou. L'activité antimicrobienne, réalisée par la technique des cylindres d'agar et celle des stries croisées, a été déterminée contre des bactéries à Gram positif (Bacillus subtilis 5262, Bacillus cereus cip 14579, Staphylococcus aureus 7625, Staphylococcus epidermidis 6821) ; des bactéries à Gram négatif (Pseudomanas aeroginosa 76110, E.coli cip 7624) et la levure Candida albicans. Parmi les vingt isolats, douze (60 %) ont montré une activité contre au moins une des souches tests. Par ailleurs, l'analyse moléculaire des vingt isolats par amplification et séquençage partiel du gène de l'ARNr 16S a permis d'attribuer dix huit isolats au genre Streptomyces et deux aux genres Saccharothrix et Lentzea. De plus, le séquençage de la quasi-totalité de l'ADNr 16S (1460 pb) de cinq isolats (Sj32, Sj33, Sj38, Sj68 et Sj69) nous a permis d'assigner les isolats Sj38 et Sj69 à S. parvus, et Saccharothrix sp., respectivement. Les substances bioactives produites par les cinq souches d'actinomycètes sont extraites par des solvants organiques et les molécules produites sont purifiées par HPLC. Une étude de la stabilité de l'activité antibactérienne en fonction de la température et de la protéinase K a montré que les substances bioactives seraient de nature non protéique pour Sj32, Sj33, Sj68 et Sj69 et protéique pour Sj38. Une étude de la cytotoxicité de la fraction active de la souche Sj32 sur les cellules MRC-5 a montré qu'elle présente une forte cytotoxicité contre ces cellules. Enfin, l'étude de l'effet de la fraction active de la souche Sj32 sur la transcription et la traduction bactérienne in vitro, a montré que cette substance purifiée n'inhibe pas ces deux processus biologiques. / Twenty strains of actinomycetes were isolated from soil and wood samples taken from an old house in the old medina of Fez and also soil samples from the region of Moulay Yacoub and banks of Sebbou River. The antimicrobial activity, carried out using the agar piece and cross striations methods, were tested against Gram-positive bacteria (Bacillus subtilis 5262, Bacillus cereus cip 14579, Staphylococcus aureus 7625, Staphylococcus epidermidis 6821); bacteria Gram-negative (Pseudomonas aeruginosa 76110, E. coli cIP 7624) and the yeast Candida albicans. Among the 20 isolates, 12 (60%) showed activity against at least one of the test strains.Molecular analysis of the twenty isolates by amplification and partial sequencing of the 16S rRNA gene allowed us to assign eighteen isolates to the genus Streptomyces and two to genera Saccharothrix and Lentzea.In addition, sequencing of nearly complete 16S rDNA (1460 bp) of five isolates (Sj32, Sj33, Sj38, Sj68 and Sj69) allowed us to assign isolates Sj38 and Sj69 to S. parvus and Saccharothrix sp., respectively.Bioactive substances produced by the five strains of actinomycetes were extracted by organic solvents and the molecules produced are purified by HPLC. A study of the stability of the antibacterial activity according to temperature and proteinase K showed that the bioactive substances are of non-protein nature for Sj32, Sj33, Sj68 and Sj69 and of protein nature for Sj38.A study of the cytotoxicity of the active fraction of the strain Sj32 on MRC-5 cells showed that it had a strong cytotoxcity against these cells.Finally, the in vitro study of the effect of the active fraction of the strain Sj32 on bacterial transcription and translation, showed that the purified substance didn't inhibit these two biological processes

Stomatitas yra dažna roplių burnos infekcinė liga. Didėjant namie auginamų roplių skaičiui, daugės ir klinikinių atvejų veterinarijos gydyklose. Svarbu identifikuoti sukėlėjus ir paskirti tikslingą antimikrobinę terapiją. Tyrimui burnos gleivinės tepinėliai paimti iš: 8 kukurūzinių žalčių, 2 karališkųjų gyvačių ir 13 stepinių vėžlių, auginamų Lietuvos zoologijos sode. Išskirta burnos mikroflora: kukurūzinių žalčių-Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Bacillus spp.; karališkųjų gyvačių: Staphylococcus spp., Streptoccocus spp.; stepinių vėžlių: Staphylococcus spp., Pseudomonas spp., Bacillus spp., Klebsiella spp. padermės. Jų atsparumui antimikrobinėms medžiagoms nustatyti atliktas testas pagal modifikuotą Kirby-Bauer (indikatorinių diskų) metodą. Panaudotos antimikrobinės medžiagos buvo: penicilinų, cefalosporinų, aminoglikozidų, sulfonamidų grupių. Atlikus sveikų kukurūzinių žalčių burnos mikrofloros tyrimus, nustatytas atsparumas antimikrobinėms medžiagoms- atspariausia sulfonamidui. Sergančio kukurūzinio žalčio išskirta burnos mikroflora atspari cefaklorui, oksitetraciklinui, amoksicilinui. Atlikus sveikų stepinių vėžlių burnos mėginių mikrofloros tyrimą, nustatytas atsparumas antimikrobinėms medžiagoms, atspariausia amoksicilinui ir klavulano rūgščiai. / Stomatitis (mouth infection) is common disease in reptiles. The number of reptiles kept as house pets is increasing so it is likely that clinical cases in veterinary clinics will also increase. It is necessary to identify infective agents and start optimal antimicrobial therapy. In this study microflora was isolated from reptiles kept at Lithuanian zoo: 8 corn snakes, 2 royal snakes, 13 russian tortoises. Isolated microflora was: corn snakes- Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Bacillus spp.; royal snakes: Staphylococcus spp., Streptoccocus spp.; russian tortoises: Staphylococcus spp., Pseudomonas spp., Bacillus spp., Klebsiella spp. Resistance to antimicrobial substances was identified using modified Kirby-Bauer (indicatory discs) method. Antimicrobial substances were: penicillins, cephalosporins, aminoglycosides, sulphonamides. Resistance to antimicrobial substances of healthy corn snakes mouth microflora was: highest resistance to sulphonamides. Mouth microflora of corn snake with stomatitis was most resistive to cephaclor, oxitetracyclin, amoxicillin. Russian tortoise mouth microflora was most resistive to amoxicillin and clavulanic acid.

Ausies uždegimas ne retai pasitaikanti liga porakanopiniams gyvūnams. Dėl racionalaus antimikrobinių vaistų vartojimo, svarbu išskirti ir identifikuoti sukelėjus ir jų jautrumą antimikrobinėms medžiagoms. Mėginiai imti iš dvylikos sveikų kamerūninių ožių, keturių šiaurinių elnių ir keturių lamų ausų landų. Visi gyvūnai yra laikomi Lietuvos zoologijos sode. Išskirti mikroorganizmai (Staphylococcus spp., Streptococcus spp., Escherichia coli, Bacillus spp., Enterococcus spp., Micrococcus spp.) ir nustatytas jų jautrumas antimikrobinėms medžiagoms. Kamerūniniams ožiams iš ausų landų išskirtos mikroorganizmų padermės Bacillus spp. ir Staphylococcus spp. Mikroflora jautriausia antimikrobinėms medžiagoms – amoksicilinui, cefaleksinui ir gentamicinui. Šiauriniams elniams ir lamoms iš ausų landų išskirtos mikroorganizmų padermės Bacillus spp. ir Staphylococcus spp. Mikroflora jautriausia antimikrobinei medžiagai - amoksicilinui. / Otitis is a common disease in artiodactyla animals. Identification the microorganism and begin the antibacterial treatment is important for rational use of antimicrobials. Samples were collected from twelve cameroons goats, four reindeers and four llamas external ear canal. They all are kept in Lithuanian zoo. Staphylococcus spp., Streptococcus spp., Escherichia coli, Bacillus spp., Enterococcus spp., Micrococcus spp. were identified in the samples and set their sensitivity to antimicrobials. In all twelve samples from healthy cameroon goats were identified Bacillus spp. and Staphylococcus spp. Our data showed that healthy cameroon goats sample's microflora were most susceptible to amoxicillini, gentamicini and cephalexini. In all eight samples from healthy llamas anr reinderrs Bacillus spp. and Staphylococcus spp. were identified. Reindeer and llamas external ear canal microflora samples were most susceptible to amoxicillin.

Algumas actinobactérias habitantes da rizosfera são produtoras de substâncias capazes de combater micro-organismos patogênicos às plantas, o que as torna potenciais agentes de controle biológico, passíveis de serem utilizadas como princípio ativo de inoculantes de sementes e mudas. O presente trabalho teve como objetivo isolar e avaliar o potencial de isolados de actinobactérias no controle de doenças causadas por fungos nas espécies arbóreas Araucaria angustifolia (Araucária) e Pinus elliottii (Pinus). Além disso, foi iniciado o estudo do processo de elaboração de inoculante a base de actinobactérias antagônicas. Foram isoladas 215 estirpes de actinobactérias do rizoplano da Araucária, das quais 13 apresentaram, em testes in vitro, potencial como antagonistas contra os fungos fitopatogênicos Fusarium oxysporum, Cylindrocladium candelabrum, C. pteridis e Armillaria sp., e apenas os isolados Ac136 e Ac202 apresentaram os maiores valores de inibição nos testes com os quatro patógenos. Na avaliação de produção de algumas substâncias antimicrobianas, apenas celulases, quitinases e sideróforos foram produzidas pelos isolados, sendo este último o mais frequente. Nos testes de interação com organismos foi verificado que, embora os isolados de actinobactéria tenham inibido a germinação de esporos do fungo micorrízico arbuscular (FMA) Gigaspora rosea em teste in vitro, no experimento in vivo, em que foi utilizado o milho como planta hospedeira para inoculação com o FMA e os isolados A43, Ac136 e Ac202, os isolados Ac136 e Ac202 estimularam a colonização das raízes pelo FMA. Estes mesmos isolados também estimularam a germinação de sementes e desenvolvimento inicial de plântulas de Pinus, porém prejudicaram a germinação e desenvolvimento inicial de Araucária. Estes mesmos isolados foram capazes de reduzir a mortalidade de plântulas de Pinus em cerca de 25%, e esta diminuição foi atribuída à inibição de Fusarium sp.. No teste de viabilidade dos isolados em diferentes veículos o isolado que mostrou maior sobrevivência foi o Ac202, mantendo um número de propágulos viáveis correspondente a 5,48 log UFC mL-1, enquanto que o veículo mais apropriado para a elaboração de inoculante com actinobactérias foi a glicerina. A análise molecular mostrou que os isolados mais promissores apresentaram maior similaridade com S. kasugaensis. Dentre todos isolados Ac202 (S. kasugaensis) foi o mais promissor obtido neste trabalho para o uso como agente de biocontrole de doenças causadas por fungos, apresentando forte antagonismo contra os quatro patógenos testados, promovendo a germinação e desenvolvimento inicial de Pinus e aumentando a sobrevivência de plântulas contaminadas com fungos fitopatogênicos. / Some actinobacteria that inhabits the rhizosphere are producers of substances that are capable of combating plant-pathogenic microorganisms, what makes them potential biological control agents, which can be used as the active ingredient of seeds and seedlings inoculants. This study aimed to isolate and evaluate the potential of actinobacteria isolates in controlling diseases caused by fungi in Araucaria angustifolia and Pinus elliottii. In addition, the study initiated the process of elaboration of an actinobacteria-based inoculant. 215 actinobacterial strains were isolated from the Araucarias rhizoplane, and 13 of them showed potential as antagonists against the phytopathogenic fungi Fusarium oxysporum, Cylindrocladium candelabrum, C. pteridis and Armillaria sp. in in vitro tests, and only the Ac136 and Ac202 strains showed the highest inhibition in the tests against the four pathogens. Among the antimicrobial substances tested, only cellulases, chitinases and siderophores were produced, with the latter being the most frequent. In the interaction tests with other organisms it was found that although the actinobacterial strains have inhibited the germination of the arbuscular mycorrhizal fungi (AMF) Gigaspora rosea spores on the in vitro test, the in vivo experiment, with maize as host plant, inoculated with the AMF and A43, Ac136 and Ac202 actinobacterial strains, Ac136 and Ac202 stimulated root colonization by AMF. These strains also stimulated Pinus seed germination and seedling early development, but hindered the germination and early development of Araucaria. In addition, the same strains were able to reduce the mortality of pine seedlings by about 25%, and this decrease was attributed to the inhibition of Fusarium sp.. In the viability test of the strains in different vehicles the strain that showed the greatest shelf-life was Ac202, with 5.48 log CFU mL-1, and the most appropriate vehicle for the actinobacteriabased inoculant development was glycerin. Molecular analysis showed that the most promising isolates showed the greatest similarity with S. kasugaensis. Among all strains Ac202 (S. kasugaensis) was the most promising one for the use as biocontrol agent of fungal diseases, exhibiting a strong antagonism against the four pathogens tested, promoting germination and early development of Pinus and increasing survival of seedlings infected with pathogenic fungi.

Algumas actinobactérias habitantes da rizosfera são produtoras de substâncias capazes de combater micro-organismos patogênicos às plantas, o que as torna potenciais agentes de controle biológico, passíveis de serem utilizadas como princípio ativo de inoculantes de sementes e mudas. O presente trabalho teve como objetivo isolar e avaliar o potencial de isolados de actinobactérias no controle de doenças causadas por fungos nas espécies arbóreas Araucaria angustifolia (Araucária) e Pinus elliottii (Pinus). Além disso, foi iniciado o estudo do processo de elaboração de inoculante a base de actinobactérias antagônicas. Foram isoladas 215 estirpes de actinobactérias do rizoplano da Araucária, das quais 13 apresentaram, em testes in vitro, potencial como antagonistas contra os fungos fitopatogênicos Fusarium oxysporum, Cylindrocladium candelabrum, C. pteridis e Armillaria sp., e apenas os isolados Ac136 e Ac202 apresentaram os maiores valores de inibição nos testes com os quatro patógenos. Na avaliação de produção de algumas substâncias antimicrobianas, apenas celulases, quitinases e sideróforos foram produzidas pelos isolados, sendo este último o mais frequente. Nos testes de interação com organismos foi verificado que, embora os isolados de actinobactéria tenham inibido a germinação de esporos do fungo micorrízico arbuscular (FMA) Gigaspora rosea em teste in vitro, no experimento in vivo, em que foi utilizado o milho como planta hospedeira para inoculação com o FMA e os isolados A43, Ac136 e Ac202, os isolados Ac136 e Ac202 estimularam a colonização das raízes pelo FMA. Estes mesmos isolados também estimularam a germinação de sementes e desenvolvimento inicial de plântulas de Pinus, porém prejudicaram a germinação e desenvolvimento inicial de Araucária. Estes mesmos isolados foram capazes de reduzir a mortalidade de plântulas de Pinus em cerca de 25%, e esta diminuição foi atribuída à inibição de Fusarium sp.. No teste de viabilidade dos isolados em diferentes veículos o isolado que mostrou maior sobrevivência foi o Ac202, mantendo um número de propágulos viáveis correspondente a 5,48 log UFC mL-1, enquanto que o veículo mais apropriado para a elaboração de inoculante com actinobactérias foi a glicerina. A análise molecular mostrou que os isolados mais promissores apresentaram maior similaridade com S. kasugaensis. Dentre todos isolados Ac202 (S. kasugaensis) foi o mais promissor obtido neste trabalho para o uso como agente de biocontrole de doenças causadas por fungos, apresentando forte antagonismo contra os quatro patógenos testados, promovendo a germinação e desenvolvimento inicial de Pinus e aumentando a sobrevivência de plântulas contaminadas com fungos fitopatogênicos. / Some actinobacteria that inhabits the rhizosphere are producers of substances that are capable of combating plant-pathogenic microorganisms, what makes them potential biological control agents, which can be used as the active ingredient of seeds and seedlings inoculants. This study aimed to isolate and evaluate the potential of actinobacteria isolates in controlling diseases caused by fungi in Araucaria angustifolia and Pinus elliottii. In addition, the study initiated the process of elaboration of an actinobacteria-based inoculant. 215 actinobacterial strains were isolated from the Araucarias rhizoplane, and 13 of them showed potential as antagonists against the phytopathogenic fungi Fusarium oxysporum, Cylindrocladium candelabrum, C. pteridis and Armillaria sp. in in vitro tests, and only the Ac136 and Ac202 strains showed the highest inhibition in the tests against the four pathogens. Among the antimicrobial substances tested, only cellulases, chitinases and siderophores were produced, with the latter being the most frequent. In the interaction tests with other organisms it was found that although the actinobacterial strains have inhibited the germination of the arbuscular mycorrhizal fungi (AMF) Gigaspora rosea spores on the in vitro test, the in vivo experiment, with maize as host plant, inoculated with the AMF and A43, Ac136 and Ac202 actinobacterial strains, Ac136 and Ac202 stimulated root colonization by AMF. These strains also stimulated Pinus seed germination and seedling early development, but hindered the germination and early development of Araucaria. In addition, the same strains were able to reduce the mortality of pine seedlings by about 25%, and this decrease was attributed to the inhibition of Fusarium sp.. In the viability test of the strains in different vehicles the strain that showed the greatest shelf-life was Ac202, with 5.48 log CFU mL-1, and the most appropriate vehicle for the actinobacteriabased inoculant development was glycerin. Molecular analysis showed that the most promising isolates showed the greatest similarity with S. kasugaensis. Among all strains Ac202 (S. kasugaensis) was the most promising one for the use as biocontrol agent of fungal diseases, exhibiting a strong antagonism against the four pathogens tested, promoting germination and early development of Pinus and increasing survival of seedlings infected with pathogenic fungi.

The sixty-eight strains isolated from breastfed full-term infant feces and from another sources were identified using genus-specific polymerase chain reaction (PCR) for Lactobacillus, species-specific PCRs, multiplex PCR, pheS PCR, rep-PCR, RAPD-PCR and 16S rDNA sequencing into Lactobacillus species or group of species. Seven strains produced antimicrobial proteinaceous substances in the supernatants. Antimicrobial proteinaceous substances of three strains were tested on temperature, pH a detergent stability. All tested strains produced temperature-stable antimicrobial proteinaceous substances. Antimicrobial activity was not influenced by detergents with exception of SDS. Presence of genes for production of bacteriocins (acidocin B, gassericin A, gassericin T, gassericin K7A and gassericin K7B) were detected in DNA of fourteen strains using PCR and DNA/DNA hybridization. Selected PCR products were sequenced and analyzed using BLAST algorithm and CLUSTAL W2 programme. The sequences of specific PCR products in DNA of two strains had 100% similarity with the sequences from the database GeneBank. Selected strains of Lactobacillus acidophilus group were tested for the surveillance in gastrointestinal tract, for the production of antimicrobial substances, for the adhesion on Caco-2 cells and for the presence of genes of antibiotic resistance. DNA of strains was tested using specific primers on the presence of genes for histidine-decarboxylase, tyrosine-decyrboxylase and linoleate isomerase. The gene for histidine-decarboxylase production was detected in DNA of seven strains, for tyrosine-decarboxylase production in DNA of one strain and for linoleate isomerase in DNA of four strains. Imunomagnetic separation of the cells was optimized. Magnetic particles functionalized with streptavidin and the anti-Lactobacillus antidote was used for the separation of the cells of Lactobacillus rhamnosus LOCK 0900 from MRS medium, UHT milk and from the yogurt. The IMS-PCR was used for detection of imunomagnetic separated bacterial cells.

Present diploma thesis is focused on natural antimicrobial substances. The aim of the experimental part of thesis was testing of antimicrobial activity of selected natural substances in free and encapsulated forms, preparation of particles and monitoring of their long-term stability. Various teas extracts (aqueous, ethanoic and DMSO) and aqueous herbs extracts were used as plant antimicrobial substances. Liposomes and chitosan particles with encapsulated aqueous teas extracts were prepared by ultrasonication. Encapsulation efficiency, size, stability and antioxidant activity for all particles were specified. Consequently, within a two months period, long-term stability for these particles was monitored. Spectrophotometric method for determining total polyphenols was used for evaluating encapsulation efficiency as well as for monitoring of long-term stability. The DLS method was used for determination of size and stability of particles. Antioxidation activity was monitored by using spectrophotometric method using ABTS. For testing antimicrobial activity of various types of teas, herbs extracts and their mixtures and combianitons with lysozyme and antibiotics, broth dilution methods were used while using both Gram positive and Gram negative bacterial test strains. Antimicrobial activity of prepared particles was also monitored, the antimicrobial activity was especially significant with the chitosan particles. Suitable combinations of natural substances whatever with antimicrobial or antioxidation effect either in free or encapsulated form could be used for applications for example in cosmetics industry, food processing or in medicine.

Antimicrobial substances, or bacteriocins are substances produced by probiotic lactic acid bacteria. They have a positive effect on the gastrointestinal tract of humans and are especially suitable for the food, but also the pharmaceutical industry. The aim of the thesis was the molecular identification of lactic acid bacteria of the Lactobacillus genus, species and their subsequent inclusion PCR method. Using the PCR method were tested also genes responsible for the production of bacteriocins. To confirm the production of bacteriocins has been selected the microbiological method, agarose droplet spot-test. In the present study also included the bioinformatics part to assess the specificity and non-specificity of the primers using in Primer-BLAST program.