No description available.

No description available.

Members of the Burkholderia cepacia complex (Bcc) are plant and human opportunistic pathogens. Essentially all Bcc isolates demonstrate in vitro broad-spectrum antibiotic resistance. In fact, many clinical isolates are resistant to all currently available antibiotics, rendering therapy ineffective. There is a substantial need to develop new antimicrobial therapies. The potential use of phage-tail-like high molecular weight bacteriocins, or "tailocins", as alternative anti-bacterial agents against Bcc was investigated. A tailocin, designated Bcep0425, produced by B.cenocepacia strain BC0425 was determined to have broad host range activity against members of the Bcc. Targeted mutagenesis of genes involved in the biosynthesis of the bacterial lipopolysaccharide (LPS) was conducted to determine the receptor site and it was determined that L-rhamnose and alpha-glucose associated with the LPS core were the receptors. Genetic analysis and targeted mutagenesis of the tailocin encoding genes was conducted in the host strain, B. cenocepacia BC0425, to determine the genetic organization of the tailocin Bcep0425 gene cluster and to confirm gene functions. We report for the first time genes involved in replication and integration that are associated with a pyocin/tailocin gene cluster. Additionally, a new class (IV) of holin was identified as part of the lysis cassette. Genetic analysis of the tailocin encoding genes revealed a high degree of similarity to defective phages identified in sequenced Burkholderia genomes. Two novel transcriptional regulators, bctN and bctR, along with recA were found to be involved in the induction of Bcep0425. Numerous studies have focused on the characterization of pyocins from Pseudomonas, but there have been no molecular investigations of tailocins from Burkholderia. This constitutes the first molecular characterization of a phage tail-like bacteriocin from Burkholderia.

No description available.

Drug-resistant bacteria, especially those belonging to the Enterobacteriaceae family, have become increasingly problematic in the nosocomial setting. However, a solution may be to exploit bacteria’s ability to produce inhibitory proteins, like bacteriocins, to suppress competitors and synergistically pair these proteins with antibiotics. Our lab has discovered a potentially novel plasmid-mediated antimicrobial protein produced by as specific strain of Klebsiella pneumoniae. To verify the genetic elements of this plasmid necessary to produce the antimicrobial, a gene interruption plasmid library was generated by transposon mutagenesis using the EZ-TN5TM system. These transposon plasmids were then electroporated into competent E. coli. The resulting E. coli were then plated and screened on agar containing kanamycin to ensure successful plasmid uptake and were now able to secrete the antimicrobial protein. The transposon’s unique sequence allowed primer- binding sites, which were used to sequence the plasmid. Four different sequences were analyzed by NCBI BLAST comparisons and matched with high similarity to: 1) a predicted colicin; 2) an uncharacterized Klebsiella protein, 3) a TraM recognition domain containing protein. The K. pneumoniae antimicrobial protein has been shown, when spotted on lawns of Citrobacter freundii, Enterobacter cloacae and Enterobacter aerogenes to inhibit their growth. It has been additionally shown to inhibit the growth of closely related strains including Klebsiella pnuemoniae strain 9997 when spotted on a lawn. When the protein was synergistically paired with subinhibitory levels of common antibiotics, there was an increase in the effectiveness of the antibiotic, it was paired with. The optical density, MTT, and CFUs demonstrate that when the K. pneumoniae protein is paired with Streptomycin or Kanamycin, growth is inhibited greater than the antibiotic alone. These results demonstrate the importance of studying polymicrobial interactions as a means to combat drug resistance and discover novel antimicrobial derived proteins for new therapeutics.

O uso da nisina como conservante natural tem aumentado cada vez mais, assim como os estudos para aprimorar o uso desta. Este trabalho visa o aumento da eficiência da nisina na inibição de microorganismos, como a Listeria, através da encapsulação da mesma na forma de nanolipossomas com incorporação de polissacarídeos. Foram testados nanolipossomas de nisina e nanolipossomas de nisina com incorporação de quitosana e incorporação de sulfato de condroitina. As análises mostraram que estes lipossomas tiveram a mesma eficiência que a nisina livre na inibição da L. monocytogenes 4b* e da Listeria sp.* e uma maior eficiência na inibição da L. monocytogenes 4b ATCC 6477. As análises de potencial zeta indicaram que os lipossomas de nisina com e sem incorporação de polissacarídeos possuem carga em torno de -20mV e estes apresentaram estruturas esféricas e não esféricas quando observados através de TEM. Apesar dos nanolipossomas de nisina com incorporação de quitosana apresentarem uma eficiência de encapsulaçãos mais baixa, em torno de 10%, estes tiveram maior estabilidade e também maior eficiência na inibição da Listeria monocytogenes ATCC 7644, quando comparados aos nanolipossomas de nisina com e sem incorporação de sulfato de condroitina, no tempo final de 24h a redução obtida foi de 2 log comparando-se com os demais tratamentos e de 7 log comparando-se aos controles. / The use of nisin, as a natural preservative, has increased, as well as studies to enhance the use of that. The aim of this study is to increase the efficiency of nisin in inhibition of microorganisms such as Listeria, by encapsulating as nanolipossomes incorporated with polysaccharides. Nisin nanoliposomes and nisin nanoliposomes with incorporation of chitosan and chondroitin sulfate were analysed. The analyzes showed that these liposomes had the same efficiency as free nisin in inhibiting L. monocytogenes 4b and Listeria sp., both isolated from bovine carcass, and greater efficiency in inhibiting L. monocytogenes 4b ATCC 6477. The zeta potential analyzes indicated that nisin liposomes with and without incorporation of polysaccharides have a load around -20mV and these showed spherical and nonspherical structures when observed through TEM. Despite of the efficience of nisin nanoliposomes incorporating chitosan present lower values of encapsulation efficiency, around 10%, they were more stable and also more efficient in inhibiting Listeria monocytogenes ATCC 7644 compared to nisin nanoliposomes with and without incorporation of chondroitin sulfate. In the final time of 24 hours the reduction obtained was 2 log compared with the other treatments and 7 log comparing to the controls.

O uso da nisina como conservante natural tem aumentado cada vez mais, assim como os estudos para aprimorar o uso desta. Este trabalho visa o aumento da eficiência da nisina na inibição de microorganismos, como a Listeria, através da encapsulação da mesma na forma de nanolipossomas com incorporação de polissacarídeos. Foram testados nanolipossomas de nisina e nanolipossomas de nisina com incorporação de quitosana e incorporação de sulfato de condroitina. As análises mostraram que estes lipossomas tiveram a mesma eficiência que a nisina livre na inibição da L. monocytogenes 4b* e da Listeria sp.* e uma maior eficiência na inibição da L. monocytogenes 4b ATCC 6477. As análises de potencial zeta indicaram que os lipossomas de nisina com e sem incorporação de polissacarídeos possuem carga em torno de -20mV e estes apresentaram estruturas esféricas e não esféricas quando observados através de TEM. Apesar dos nanolipossomas de nisina com incorporação de quitosana apresentarem uma eficiência de encapsulaçãos mais baixa, em torno de 10%, estes tiveram maior estabilidade e também maior eficiência na inibição da Listeria monocytogenes ATCC 7644, quando comparados aos nanolipossomas de nisina com e sem incorporação de sulfato de condroitina, no tempo final de 24h a redução obtida foi de 2 log comparando-se com os demais tratamentos e de 7 log comparando-se aos controles. / The use of nisin, as a natural preservative, has increased, as well as studies to enhance the use of that. The aim of this study is to increase the efficiency of nisin in inhibition of microorganisms such as Listeria, by encapsulating as nanolipossomes incorporated with polysaccharides. Nisin nanoliposomes and nisin nanoliposomes with incorporation of chitosan and chondroitin sulfate were analysed. The analyzes showed that these liposomes had the same efficiency as free nisin in inhibiting L. monocytogenes 4b and Listeria sp., both isolated from bovine carcass, and greater efficiency in inhibiting L. monocytogenes 4b ATCC 6477. The zeta potential analyzes indicated that nisin liposomes with and without incorporation of polysaccharides have a load around -20mV and these showed spherical and nonspherical structures when observed through TEM. Despite of the efficience of nisin nanoliposomes incorporating chitosan present lower values of encapsulation efficiency, around 10%, they were more stable and also more efficient in inhibiting Listeria monocytogenes ATCC 7644 compared to nisin nanoliposomes with and without incorporation of chondroitin sulfate. In the final time of 24 hours the reduction obtained was 2 log compared with the other treatments and 7 log comparing to the controls.

O uso da nisina como conservante natural tem aumentado cada vez mais, assim como os estudos para aprimorar o uso desta. Este trabalho visa o aumento da eficiência da nisina na inibição de microorganismos, como a Listeria, através da encapsulação da mesma na forma de nanolipossomas com incorporação de polissacarídeos. Foram testados nanolipossomas de nisina e nanolipossomas de nisina com incorporação de quitosana e incorporação de sulfato de condroitina. As análises mostraram que estes lipossomas tiveram a mesma eficiência que a nisina livre na inibição da L. monocytogenes 4b* e da Listeria sp.* e uma maior eficiência na inibição da L. monocytogenes 4b ATCC 6477. As análises de potencial zeta indicaram que os lipossomas de nisina com e sem incorporação de polissacarídeos possuem carga em torno de -20mV e estes apresentaram estruturas esféricas e não esféricas quando observados através de TEM. Apesar dos nanolipossomas de nisina com incorporação de quitosana apresentarem uma eficiência de encapsulaçãos mais baixa, em torno de 10%, estes tiveram maior estabilidade e também maior eficiência na inibição da Listeria monocytogenes ATCC 7644, quando comparados aos nanolipossomas de nisina com e sem incorporação de sulfato de condroitina, no tempo final de 24h a redução obtida foi de 2 log comparando-se com os demais tratamentos e de 7 log comparando-se aos controles. / The use of nisin, as a natural preservative, has increased, as well as studies to enhance the use of that. The aim of this study is to increase the efficiency of nisin in inhibition of microorganisms such as Listeria, by encapsulating as nanolipossomes incorporated with polysaccharides. Nisin nanoliposomes and nisin nanoliposomes with incorporation of chitosan and chondroitin sulfate were analysed. The analyzes showed that these liposomes had the same efficiency as free nisin in inhibiting L. monocytogenes 4b and Listeria sp., both isolated from bovine carcass, and greater efficiency in inhibiting L. monocytogenes 4b ATCC 6477. The zeta potential analyzes indicated that nisin liposomes with and without incorporation of polysaccharides have a load around -20mV and these showed spherical and nonspherical structures when observed through TEM. Despite of the efficience of nisin nanoliposomes incorporating chitosan present lower values of encapsulation efficiency, around 10%, they were more stable and also more efficient in inhibiting Listeria monocytogenes ATCC 7644 compared to nisin nanoliposomes with and without incorporation of chondroitin sulfate. In the final time of 24 hours the reduction obtained was 2 log compared with the other treatments and 7 log comparing to the controls.

Orientador: Ranulfo Monte Alegre / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T21:39:29Z (GMT). No. of bitstreams: 1 Santos_LucielenOliveirados_M.pdf: 943021 bytes, checksum: 7237d5c1a050d0bfae7083ba9026d36f (MD5) Previous issue date: 2004 / Resumo: As bacteriocinas são substâncias protéicas produzidas por bactérias, com atividade bactericida e bacteriostática contra bactérias sensíveis. Estes compostos são produzidos por microrganismos gram-positivos e gram-negativos, incluindo as bactérias láticas. Estas bactérias são extensamente usadas no processamento de alimentos devido a sua contribuição na vida de prateleira, textura e propriedades organolépticas. Algumas cepas de enterococos podem produzir bacteriocinas ativas contra bactérias patogênicas, tal como a Listeria monocytogenes. As bacteriocinas produzidas por Enterococcus faecium e Enterococcus faecalis são peptídeos pequenos, hidrofóbicos e termoestáveis. Dentro deste contexto este trabalho teve como objetivo estudar a produção de enterocina por E. faecium e E. faecalis. Inicialmente, as fermentações foram feitas em frascos Erlenmeyers, incubados a 37°C por 24h, para determinar as melhores condições de fonte de carbono (glicose, sacarose comercial ou açúcar mascavo), pHinicial (4,0; 6,0; 8,0 ou 10,0) e agitação (0 ou 200rpm). O meio basal modificado sem glicose foi usado como meio base sendo complementado com diferentes fontes de carbono. Em um segundo estágio, os experimentos foram conduzidos em fermentador de bancada por 24h. Durante as fermentações foram coletadas amostras em intervalos de 24h ou 1h, para a determinação da atividade de enterocina, massa celular seca, pH e conteúdo de açúcar. Os resultados obtidos a partir dos testes em frascos Erlenmeyers indicam que usando o E. faecium para produção de enterocina as melhores condições de cultivo foram pHinicial 10,0, sacarose e sem agitação. A máxima produção de enterocina foi 2,81UE. Os resultados indicaram que as melhores condições para produção de enterocina usando E. faecalis foram pHinicial 10,0, 200rpm e açúcar mascavo, com atividade de enterocina de 1,68UE. No fermentador, a maior atividade de enterocina foi 2,94UE após 21h de fermentação no experimento com pHinicial 10,0, 200rpm, sacarose e E. faecium. A atividade de enterocina foi de 1,30UE após 12h de incubação no experimento com pHinicial 10,0, 200rpm, açúcar mascavo e E. faecalis. A L. monocytogenes Scott A foi utilizada como microrganismo teste e foi inibida pelas enterocinas produzidas por E. faecium e por E. faecalis / Abstract: Bacteriocins are protein substances produced by bacteria that are bactericidal or bacteriostatic against sensitive bacterial species. Both gram-negative and gram-positive organisms, including the lactic acid bacteria, produce these compounds. These bacteria are extensively used in food processing, for their contribution to shelf life, texture and organoleptic properties. Some strains of entecococci can produce bacteriocins active against pathogenic bacteria such as Listeria monocytogenes. Bacteriocins produced by Enterococcus faecium and Enterococcus faecalis are small, hydrophobic and termostable peptides. The aim of this work was the study of enterocin production by E. faecium and E. faecalis. Initially, fermentations assays were carried out in Erlenmeyers flasks for determining the best conditions of carbon sources (glucose, sucrose and brown sugar), pHinitial (4,0; 6,0; 8,0 or 10,0) and agitation (0 or 200rpm). The modified basal medium without glucose was used as base medium and the different carbon employed. The flasks incubated at 37°C for 24h. In a second stage, the experiments were carried out in laboratory scale fermentor for 24h. During the fermentations samples were collected at intervals of 24 h or 1h, for determination of enterocin activity, biomass (cell dry mass), pH and sugar contents. The first phase in Erlenmeyers flasks indicated that using E. faecium for enterocin production the best culture conditions were pHinitial 10,0, sucrose and without agitation. The maximum enterocin production was 2,81UE. The results indicated that the best conditions for enterocin production using E. faecalis were pHinitial 10,0, 200rpm and brown sugar, with enterocin activity of 1,68UE. In fermentor the highest enterocin activity was 2,94UE after 21h of fermentation in experiment using pHinitial 10,0, 200rpm, sucrose and E. faecium. The enterocin activity was 1,30UE after 12h of incubation in experiment with pHinitial 10,0, 200rpm, brown sugar and E. faecalis. When it was used L. monocytogenes Scott A as microorganism test occurred its inhibition by enterocin of E. faecium and E. faecalis / Mestrado / Mestre em Engenharia de Alimentos

Viren haben als Ursache lebensmittelassoziierter Infektionen eine große Bedeutung. Sie können vor allem über rohe oder unzureichend erhitzte Lebensmittel übertragen werden. In diesem Zusammenhang werden grüner Salat, Erdbeeren, Himbeeren, Frühlingszwiebeln, Muscheln, halbgetrocknete Tomaten, fäkal verunreinigtes Trinkwasser, Backwaren und Rohwürste als häufige Infektionsquellen genannt. Vor allem kurzgereifte Rohwürste gehören aus mikrobiologischer Sicht zu Risikoprodukten. Um eine gleichbleibende Qualität der Produkte zu gewährleisten, ist die Verwendung von Starterkulturen unerlässlich. Als sogenannte Schutzkulturen sollen sie gleichzeitig die Vermehrung unerwünschter bakterieller Pathogene unterbinden. Bisher ist allerdings nicht bekannt, inwieweit diese zur Virusinaktivierung in kurzgereiften Rohwürsten führen bzw. beitragen. Aus diesem Grund war es das Ziel dieser Arbeit, den Einfluss von rohwurstrelevanten Starter- und Schutzkulturen sowie deren Metabolite (Bacteriocine, Milchsäure) auf die Tenazität und Inaktivierungskinetik von Viren zu prüfen. Die Untersuchungen erfolgten mit dem murinen Norovirus (MNV) S 99 sowie dem humanen Influenzavirus H1N1 (A/WSN/33). Antivirale Effekte wurden zum einen anhand von in-vitro-Studien, zum anderen anhand von experimentell mit Viren kontaminierten kurzgereiften Rohwürsten (Mettwurst/Teewurst) geprüft. Die Bacteriocine Sakacin A und Nisin zeigten in phosphatgepufferter Salzlösung (PBS) keine viruzide Wirkung gegenüber MNV S 99 und H1N1 (pH 6,2; 24 °C; Exposition: 3 Tage). Weiterhin wurden anhand von in-vitro-Untersuchungen 29 verschiedene zellfreie Kulturüberstände [Milchsäurebakterien, Staphylococcus spp. (S.), Kocuria (K.) varians] hinsichtlich ihrer antiviralen Wirkung geprüft. Dabei konnte eine signifikante Titerreduktion von MNV S 99 bei Exposition mit dem Kulturüberstand eines Lactobacillus (Lb.) curvatus-Isolates festgestellt werden (p < 0,05). In mit dieser Kultur fermentiertem Tee- und Mettwurstbrät zeigte sich jedoch kein Effekt. Die Virustenazität von H1N1 und MNV S 99 konnte mit D,L-Milchsäure unter rohwurstrelevanten Bedingungen (pH 5,0 bis 6,2) sowohl in-vitro als auch im frischen Mettwurstbrät beeinflusst werden. In-vitro erzielte Titerreduktionen lagen bei 2,5 (H1N1) bzw. 3,25 log-Stufen (MNV S 99) nach drei Tagen (24 °C) Lagerung. Im Gegensatz dazu war MNV S 99 im Vergleich zu H1N1 im Mettwurstbrät stabiler. H1N1 konnte unterhalb von pH 5,5 bereits direkt nach dem Einmischen der Influenzaviren in das Wurstbrät nicht mehr nachgewiesen werden. MNV S 99 wurde hingegen erst nach einem Tag Lagerung (22 °C) maximal um 0,7 log-Stufen reduziert (pH 5,2). Die verwendeten Starter- und Schutzkulturen (Lb. sakei, Lb. curvatus, Lb. paracasei, Lb. plantarum, S. carnosus, S. xylosus, K. varians) zeigten im Mett- und Teewurstbrät im Vergleich zur Kontrolle (ohne Starterkultur) keinen zusätzlichen viruziden Effekt auf MNV S 99. Zunehmende Virustiterreduktionen konnten mit pH-Wert-Erniedrigung beobachtet werden. Nach der Reifung (1 Tag, 22 °C, pH 4,9) von Mettwurst mit Starterkulturen wurde das Virus um maximal 1,65 log-Stufen reduziert. In mit Einzel- beziehungsweise Mehrstamm-Mischkulturen fermentierter Teewurst (7 Tage, 22 °C, pH 4,9) betrug die Titerreduktion maximal 1,10 log-Stufen. Das Influenzavirus H1N1 konnte im Rohwurstbrät mit Starterkulturen auch nach Verwendung hoher Ausgangstiter bereits zu Beginn der Untersuchungen nicht mehr nachgewiesen werden. Aus den erzielten Daten kann geschlussfolgert werden, dass die Bacteriocine Sakacin A und Nisin nicht als antivirale Zusatzstoffe in Lebensmitteln (z. B. Rohwürste) geeignet sind. Das antivirale Potential von zellfreien Kulturüberständen war Bakterienstamm-spezifisch und nur in-vitro ersichtlich. Daher muss die Nutzung des Lb. curvatus 1-Stammes nicht anderen rohwurstrelevanten Starterkulturen vorgezogen werden. Die Verwendung von Milchsäure als Zusatzstoff im Rohwurstbrät eignet sich nur zum Ausschluss einer viralen Exposition im Zusammenhang mit H1N1. Frische Mettwurst muss allerdings hierzu adäquat gesäuert (pH < 5,5) werden. Neben dem antiviralen Effekt durch gebildete Säure, konnte keine weitere spezies-spezifische antivirale Wirkung verwendeter Starter- und Schutzkulturen auf MNV S 99 festgestellt werden. Die Säureleistung einzelner Kulturen ist demzufolge für eine Virusinaktivierung entscheidend. Das antivirale Potential verwendeter Starter- und Schutzkulturen in Rohwürsten ist im Zusammenhang mit MNV S 99 als gering einzuschätzen. Unter der Annahme, dass murine und humane Noroviren eine ähnliche Tenazität in kurzgereiften Rohwürsten aufweisen, sollten diese Produkte im Zusammenhang mit Noroviren als Risikoprodukte eingestuft werden.