Chitinases have been shown to be potential agents for biological control of the plant diseases caused by various phytopathogenic fungi and insect pests, because fungal cell walls and insect exoskeletons contain chitin as a major structural component. Chitinase has also been found to increase the efficacy and potency of Bacillus thuringiensis crystal (Cry) proteins toxic to larvae of insect pests. The reason of this synergy is the presence of chitin in the structure of the outer membrane of larval midgut. In this study, the gene encoding chitinase A (chiA) from Serratia marcescens Bn10, a local isolate of Trabzon province was amplified by PCR and cloned into the E.coli/Bacillus shuttle vectors, pNW33N and pHT315. For the expression in B. thuringiensis, the promoter region of cry3Aa11 gene of B. thuringiensis Mm2 was placed at the upstream of chiA. The vectors carrying both chiA and promoter site of cry3Aa11 was first introduced into E. coli and then into Bacillus subtilis 168 which were used as intermediate hosts in this study. pHT315PC carying chiA was then introduced into Coleoptera-specific B. thuringiensis cells (strain 3023) and the specific chitinase activity of the recombinant B. thuringiensis was measured as 5056 U/min/mg which was 6.3 fold higher than that of the parental strain. The specific activity corresponded to about one third of that produced by S. marcescens Bn10. The chiA gene was next sequenced and characterized. The sequence was submitted to GeneBank (Accession No. DQ165083). Chitinase A of S. marcescens Bn10 was found to be a 563 residue protein with a calculated molecular mass of 60.9 kDa. The mean G+C content of the gene is 58.75%. The deduced amino acid sequence was 99.3&ndash / 91.5% identical to those of known chitinases from S. marcescens, Burkholderia cepacia and Enterobacter sp. It was found that the chitinase of S. marcescens Bn10 has six amino acids difference from the consensus sequence of aligned chitinases. The production of chitinase by the local isolate S. marcescens Bn10 in different cultural conditions was also investigated. Optimum temperature and pH for chitinase production was found to be 30 oC and 7.5, respectively. Varying the concentration of colloidal chitin and the inclusion of NAG into the medium had no effect on chitinase production. The effect of different parameters such as temperature, pH, substrate concentration and certain inhibitory elements on enzyme activity were next assayed. The highest activity was obtained at 45 oC and in a pH range of 4.0 to 9.0. Activity of chitinase increased with increasing substrate concentration up to 35 mg/mL. Ca2+, Co2+, Cu2+, EDTA, Fe2+, Mg2+, Mn2+ and Zn2+ were tested for their effects on the activity of enzyme. The enzyme was inhibited by only 4% in the presence of 10 mM EDTA, whereas 10 mM Co2+ included in the assay mixture increased the activity by 20%.

<p dir="ltr"><i>Serratia marcescens </i>is a bacterium with a ubiquitous environmental distribution and the ability to cause opportunistic infections. This research explores three different group behaviors in <i>S. marcescen</i>s. These are biofilm formation, microbial hitchhiking, and responses to cannabinoid-induced stress. To study biofilm development, we used a crystal violet assay to measure biofilm and compared that to the bacterial growth of those cultures. We looked at the role of nutrients and temperature in biofilm produced by <i>S. marcescens</i> and tested four <i>S. marcescens</i> strains. We found that there were differences in the ratio of biofilm to growth when <i>S. marcescens</i> was grown in different media. The exact relationship between temperature and media composition requires more information than could be attained from these studies. Next, we wanted to investigate whether <i>S. marcescens</i> could also utilize movement of other, more highly motile species of bacteria through a process called microbial hitchhiking. <i>S. marcescens</i> was grown with a highly motile <i>Paenibacillus</i> sp. isolate. <i>S. marcescens</i> was tracked by the red pigment that it produces. It was observed that <i>S. marcescens</i> consistently spread farther across a surface when it was co-cultured with <i>Paenibacillus</i> sp. than when grown alone. This was repeated with three other <i>S. marcescens</i> strains and three different species of <i>Paenibacillus.</i><i> </i>Hitchhiking behavior was observed in all cases. To understand the mechanism by which <i>S. marcescens</i> hitchhikes on <i>Paenibacillus </i>spp., a <i>S. marcescens </i>flagellar mutant was used. Even in the absence of a flagellum, <i>S. marcescens</i> was still able to hitchhike implying that another mechanism must be involved. Finally, we evaluated the response of <i>S. marcescens </i>to cannabidiol (CBD) a phytocannabinoid with antimicrobial and antibiofilm properties, though it has limited potency against Gram-negative bacteria like <i>S. marcescens</i>. We found that CBD did not kill <i>S. marcescens </i>nor did it affect its biofilm development. Interestingly, <i>S. marcescens </i>cultures showed enhanced pigment production in response to high-dose CBD exposure compared to vehicle controls. This response was also observed with exposure to three additional phytocannabinoids. Results from these studies add to our understanding of how <i>S. marcescens</i> can access new areas and persist in a broad range of environments.</p>

PARENTE, T. M. L. Perfil de resistência a antibióticos e a terapia fotodinâmica antimicrobiana exibida por isolados ambientais, orais e extra-orais de Serratia marcescens. 2010. 90 f. Dissertação (Mestrado em Biotecnologia) - Curso de Medicina - Campus de Sobral, Universidade Federal do Ceará, Sobral, 2010. / Submitted by Djeanne Costa (djeannecosta@gmail.com) on 2016-04-18T15:28:12Z No. of bitstreams: 1 2010_dis_tmlparente.pdf: 50723625 bytes, checksum: 536c8be1802258847a68d8c4bdad58aa (MD5) / Approved for entry into archive by Djeanne Costa (djeannecosta@gmail.com) on 2016-04-18T15:39:33Z (GMT) No. of bitstreams: 1 2010_dis_tmlparente.pdf: 50723625 bytes, checksum: 536c8be1802258847a68d8c4bdad58aa (MD5) / Made available in DSpace on 2016-04-18T15:39:33Z (GMT). No. of bitstreams: 1 2010_dis_tmlparente.pdf: 50723625 bytes, checksum: 536c8be1802258847a68d8c4bdad58aa (MD5) Previous issue date: 2010 / Serratia marcescens is widely distributed in nature, but has emerged in the last years as important nosocomial pathogen with resistance of many antimicrobial drugs. This study aimed to verify the susceptibility of Serratia marcescens isolates from environment, from oral infections and from extra-oral infections to different antibiotics and evaluate the antimicrobial effect of photodynamic antimicrobial therapy as biotechnology tools reducing bacterial growth in planktonic cells and biofilm. E-test® were performed for fifty-five strains and the PACT for the thirty strains more resistant to antimicrobials tested. The antimicrobial effect of toluidine blue O, associated with 4,72 J cm-2 of a light-emitting diode , was evaluated. Before and after the treatments, bacterial inocula were analysed with regard to the number of colony- forming units. For antimicrobials, we observed that the 55 strains analyzed, 13 (23.63%) were resistant to doxycycline, but only one (1.81%) isolate showed resistance to ciprofloxacin, another to tobramycin and another to cefotaxime, 24 ( 43.63%) strains had intermediate sensitivity to doxycycline, all were sensitive to imipenem and most were sensitive to ciprofloxacin, tobramycin and cefotaxime Statistical analysis showed no significant differences in resistance of samples of different origins for drugs DX, CT, and IP. Considering the resistance to CI, the environmental samples were significantly more resistant than samples oral and extra-oral. For the drug TM, the oral samples were significantly more sensitive than the other samples. The irradiation of planktonic and biofilm cultures in the absence of TBO (L+S-), incubation with TBO alone (L-S+) and untreated control group (L-S-) had no significant effect on the viability of strains of S. marcescens studied (p <0.05). Significant decreases in bacterial viability was observed only when planktonic and biofilm culture of environmental strains, oral and extra-oral S. marcescens were exposed to toluidine blue O and LED light at the same time (L+S+). Significant reductions in bacterial counts were observed by antimicrobial photodynamic therapy ranging from 10-11 to 10-7.The association of TBO and light, with energy density 4,72 J cm-2, was effective in reducing the viability of bacterial strains in environmental, oral and extra-oral S. marcescens and can be a useful biotechnological tool in the control of bacterial resistance / Serratia marcescens se encontra largamente distribuída na natureza, mas tem emergido nos últimos anos como um importante patógeno nosocomial resistente a diversos antimicrobianos. Este estudo teve como objetivo verificar a susceptibilidade de isolados ambientais, orais e extra-orais de Serratia marcescens a diferentes antibióticos e avaliar a terapia fotodinâmica antimicrobiana na redução do crescimento bacteriano em culturas de células planctônicas e biofilme. O teste de susceptibilidade antimicrobiano E-test® foi realizado para as 55 cepas e o TFA para as 30 cepas mais resistentes aos antimicrobianos testados. O efeito antimicrobiano do azul de o-toluidina associado com 4,72 J cm-2 de luz emitida por um diodo (LED) foi avaliado. Antes e após os tratamentos, os inóculos bacterianos foram analisados com consideração do número de unidades formadoras de colônias. Considerando o perfil antimicrobiano observamos que das 55 cepas analisadas, 13 (23,63%) apresentaram resistência à doxiciclina, mas apenas um (1,81%) isolado apresentou resistência ao ciprofloxacino, outro à tobramicina e outro à cefotaxima; 24 (43,63%) cepas apresentaram sensibilidade intermediária à doxiciclina, todas foram sensíveis ao imipenem e a maioria foi sensível ao ciprofloxacino, à tobramicina e à cefotaxima. A análise estatística demonstrou não haver diferenças significativas no perfil de resistência das amostras de diferentes origens em relação as drogas DX, CT e IP. Considerando a resistência a CI, as amostras ambientais foram significativamente mais resistentes do que as amostras orais e extra-orais. Para a droga TM, as amostras orais foram significantemente mais sensíveis do que as demais amostras. A irradiação das culturas planctônicas e biofilmes na ausência de TBO (L+C-), a incubação com TBO sozinho (L-C+) e o grupo controle não tratado (L-C-) não apresentou efeitos significativos na viabilidade das cepas de S. marcescens estudadas (p < 0,05). Decréscimos significativos na viabilidade bacteriana foram observados somente quando cultura planctônica e biofilme de cepas ambientais, orais e extra-orais de S. marcescens foram expostas ao azul de orto toluidina e luz LED ao mesmo tempo (L+C+). Reduções significativas nas contagens bacterianas foram observadas pela Terapia Fotodinâmica Antimicrobiana com variação de 10-11 a 10-7. A associação de TBO e LED, com densidade de energia de 4,72 J cm-2 , foi efetivo na redução da viabilidade bacteriana em cepas ambientais, orais e extra-orais de S. marcescens podendo ser uma ferramenta biotecnológica útil no controle da resistência bacteriana

Cannabis is the only plant which contains cannabinoids and thanks to these compounds it has enormous potential. This thesis deals with the analysis of technical hemp. Effects of cannabinoids and methods used for cannabis analysis are discussed in the theoretical part. The experimental part includes spectrophotometric characterization of cannabis, it´s antimicrobial effects and thin layer chromatography analysis. Three sorts of Cannabis sativa L. were analyzed, namely Finola, Fedora and Kompolti. Firstly, the content of polyphenols, flavonoids and antioxidant activity in prepared tinctures were determined. Moreover, antimicrobial test were performed using disk test and turbidity determination. Gram-positive and Gram-negative bacteria and yeast organism were tested. It was found that cannabis tinctures possess good antimicrobial effects. Some of them are comparable with synthetic antibiotics. Finally, thin layer chromatography enabled visualization of cannabinoids in prepared tinctures.

Projet I : Les endospores représentent un état de dormance des bactéries leur permettant de résister à des conditions extrêmes et de persister pendant des années. La formation d'endospores a façonné l'évolution puisqu’elle se produit exclusivement chez les Firmicutes. Plusieurs études ont rapporté la formation d'endospores chez des espèces en dehors des Firmicutes, en particulier chez deux espèces de Protéobactéries, Rhodobacter johrii et Serratia marscescens, et une espèce d'Actinobacteries, Mycobacterium marinum. Le fait d’identifier les endospores en dehors des Firmicutes pourrait affecter la forme de l'arbre de vie et aiderait dans notre lutte contre les agents pathogènes humains. Par conséquent, nous avons visé d’étudier l'endosporulation chez ces trois espèces en utilisant des approches avancées d'imagerie et d'analyse, y compris la microscopie corrélative alliant la microscopie optique et électronique (CLEM), la tomographie de cryo- électron (cryo-ET) et la lipidomique. Nous avons utilisé la bactérie sporulante bien caractérisée Bacillus subtilis comme contrôle positif de la sporulation. L'examen de R. johrii, S. marcescens et M. marinum en utilisant CLEM et cryo-ET a montré que les objets à phase brillante ne ressemblaient à aucun stade de l'endosporulation. Les cryo-tomogrammes ont montré que les objets à phase brillante chez S. marcescens étaient des débris cellulaires agrégés de cellules mortes, alors qu'ils présentaient des structures granulaires typiques des cellules bactériennes chez les R. johrii et M. marinum. L'analyse lipidomique chez R. johrii a identifié les structures granulaires comme des granules de stockage potentiels enrichis en triacylglycérides (TAG). Nous pensons que les TAG peuvent fournir une source d'énergie pour résister à l'épuisement des nutriments. Des approches biochimiques et bioinformatiques supplémentaires ont soutenu nos conclusions selon lesquelles R. johrii, S. marcescens et M. marinum sont des bactéries non sporulantes. Projet II : Les plasmides jouent un rôle vital dans la propagation des gènes de résistance au sein et entre les espèces bactériennes. Par conséquent, il est essentiel de comprendre les systèmes bactériens impliqués dans le transfert et la maintenance des plasmides pour mieux aider dans notre lutte contre la propagation de la résistance aux antibiotiques. Dans cette thèse de doctorat, nous avons cherché à caractériser l'opéron alp7ARC, en utilisant l'homologue de l'actine bactérienne Alp7A pour séparer le plasmide pLS20 codant pour la résistance à la tétracycline dans B. subtilis. La stabilité du plasmide s'est avérée dépendante de l'opéron alp7ARC, indiquant un rôle essentiel dans la ségrégation plasmidique. Nos résultats préliminaires sur Alp7A ont montré qu'il s'assemble dans une nouvelle nanostructure tubulaire plutôt que des filaments, suggérant un nouveau mécanisme de ségrégation de l'ADN par Alp7A. Nous avons également étudié la structure d'Alp7A in vivo en utilisant une combinaison d'approches, notamment la biologie moléculaire, la Cryo-ET et la fLM. Nous avons également utilisé la CLEM pour localiser Alp7A dans des cellules entières à une résolution macromoléculaire. En outre, nous avons étudié la structure et la fonction d'Alp7A in vitro en transfectant B. subtilis et E. coli avec diverses constructions plasmidiques incorporant des mutations dans le gène d’Alp7A. Nous avons déployé différentes méthodes pour la purification de la protéine Alp7A, y compris la séparation par chromatographie, et le fractionnement au sulfate d'ammonium. J'ai discuté des divers défis que nous avons rencontrés dans ces expériences, tels que la contamination, l'instabilité de la protéine Alp7A et l'épaisseur bactérienne. Enfin, j'ai proposé des approches expérimentales alternatives qui aideraient à étudier le mécanisme de ségrégation des plasmides par Alp7ARC. / Project I: Endospores represent a dormant state of bacteria that allows them to withstand extreme conditions and persist for years. Endospore formation has shaped evolution, whereby it exclusively occurs in Firmicutes. Several studies have reported endospore formation in species outside of Firmicutes, particularly in two species of Proteobacteria, Rhodobacter johrii and Serratia marcescens, and one species of Actinobacteria, Mycobacterium marinum. Identifying endospores outside of Firmicutes would affect the shape of the tree of life and aid in our fight against human pathogens. Therefore, we aimed to investigate endosporulation in these three species using advanced imaging and analytical approaches, including correlative light and electron microscopy (CLEM), cryo-electron tomography (cryo-ET), and lipidomics. We used the well-characterized sporulating bacterium Bacillus subtilis as a positive control of sporulation. Examination of R. johrii, S. marcescens, and M. marinum using CLEM and cryo-ET showed that phase-bright objects did not resemble any stages of endosporulation. Cryo-tomograms revealed that the phase-bright objects in S. marcescens were aggregated cellular debris of dead cells, whereas they displayed granular structures typical of bacterial cells in R. johrii and M. marinum. Lipidomic analysis in R. johrii identified the granular structures as potential storage granules enriched with triacyl-glycerides (TAGs). We speculate that TAGs may provide an energy source to withstand the nutrient depletion. Additional biochemical and bioinformatics approaches supported our conclusions that R. johrii, S. marcescens, and M. marinum are non-sporulating bacteria. Project II: Plasmids play a vital role in the spread of resistance genes within and across bacterial species. Therefore, it is essential to understand the bacterial systems involved in the transfer and maintenance of plasmids to better aid in our fight against the spread of antibiotic resistance. In this doctorate, we aimed to characterize the alp7ARC operon, employing the bacterial actin homolog Alp7A to segregate the tetracycline resistance-encoding plasmid pLS20 in B. subtilis. The stability of the plasmid was shown to be dependent on the alp7ARC operon, indicating an essential role in plasmid segregation. Preliminary results on Alp7A showed that it assembles into a novel tubular nanostructure rather than filaments, suggesting a novel mechanism for DNA segregation by Alp7A. We further studied the structure of Alp7A in vivo using combination of approaches, including molecular biology, cryo-ET, and fLM. We also used CLEM to localize Alp7A in whole cells to a macromolecular resolution. Besides, we investigated the structure and function of Alp7A in vitro by transfecting E. coli with various plasmid constructs and purification by several methods, including affinity chromatography and ammonium sulfate precipitation. I discussed the diverse challenges we encountered in these experiments, such as bacterial thickness, contamination, and Alp7A protein instability. Finally, I proposed alternative experimental approaches for investigating the mechanism of plasmid segregation by Alp7ARC.