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On souhaite reconstruire en ligne des signaux à échantillons manquants en utilisant une approche paramétrique. On propose alors des algorithmes adaptatifs d'identification et de reconstruction de processus AR à échantillons manquants. On s'intéresse premièrement à l'extension des algorithmes de gradient au cas des signaux à échantillons manquants. On propose alors deux alternatives à un algorithme existant fondées sur deux autres prédicteurs. Les algorithmes proposés convergent vers une estimation non biaisée des paramètres. Or les algorithmes de gradient souffrent d'une faible vitesse de convergence. Pour cela, on s'intéresse à l'extension de l'algorithme MCR au cas des signaux à échantillons manquants. On utilise alors l'algorithme MCR pseudo-linéaire pour l'identification conjointement avec un filtre de Kalman pour une prédiction optimale du signal au sens des moindres carrés. L'algorithme résultant permet une identification non biaisée des paramètres. De plus, il est rapide et bien adapté à l'identification de processus non stationnaires. Néanmoins, souhaitant contrôler la stabilité du filtre identifié, on s'intéresse ensuite à une identification fondée sur une structure en treillis du filtre. Ainsi, on propose une extension de l'algorithme de Burg adaptatif au cas des signaux à échantillons manquants, en utilisant pour la prédiction un filtre de Kalman. La stabilité du modèle ainsi identifié est garantie. De plus, l'algorithme s'adapte rapidement aux variations des paramètres. Finalement, on propose d'utiliser les algorithmes proposés dans un système à transmission non uniforme. On obtient ainsi l'amélioration simultanée du RSB et du débit de transmission moyen.

A pasteurelose é uma das doenças mais comuns do trato respiratório do coelho em criações comerciais e/ou em biotérios de animais destinados à pesquisa biomédica. A bactéria Pasteurella multocida é o patógeno responsável por uma série de manifestações clínicas em coelhos, incluindo rinite crônica, otite média, pneumonia, infecções no trato genital, formação de abscessos pulmonares e cutâneos, conjuntivite e septicemia hemorrágica. Porém, entre 50 e 70 % dos animais podem incubar o organismo de forma assintomática. Os fatores predisponentes para o desencadeamento dos sinais clínicos incluem acúmulo de amônia no ar (má ventilação), prenhez, aparecimento de doenças concomitantes, distúrbios no ambiente de criação ou na manipulação experimental. A doença está presente no Brasil, ocorrendo surtos com relativa freqüência, no entanto, o diagnóstico é feito com base nos sinais clínicos e necropsia. Dessa forma é difícil precisar a extensão dos prejuízos causados pela pasteurelose à cunicultura. Vacinas comerciais específicas contra a pasteurelose em coelhos não estão disponíveis no mercado. A prevenção, ainda que apresente resultados duvidosos, é realizada utilizando-se antibióticos dissolvidos na água, porém este tipo de tratamento normalmente não protege definitivamente os animais. Uma vez que não existem vacinas disponíveis e o tratamento com antibióticos não estabelece proteção contra a pasteurelose, foram desenvolvidos neste trabalho sistemas carreadores das proteínas antigênicas da membrana da P. multocida. Estes sistemas carreadores são formados por lipossomos, já conhecidos pelo seu potencial como imunoadjuvante, e por microesferas lipídicas, responsáveis por apresentar os antígenos às células apresentadoras de antígenos (APC). Inicialmente, foram obtidas colônias puras da bactéria as quais foram cultivadas em meio de crescimento específico (BHI). Os microrganismos foram isolados, rompidos e as proteínas antigênicas foram detectadas por SDS-PAGE e Western Blotting. Estes resultados mostraram que a maioria das bandas protéicas foi reconhecida pelo anticorpo policlonal contra a P. multocida. Visto que tínhamos um pool de proteínas as quais apresentavam antigenicidade, foi realizada uma solubilização incubando frações de membrana da bactéria com SDS 1 %. Este procedimento resultou em um rendimento de solubilização de 85 %. A obtenção dos proteolipossomos foi realizada pelo método da co-solubilização de lipídio, proteína e detergente. Um bom rendimento de incorporação das proteínas em lipossomos parecer estar relacionada com a metodologia utilizada para a remoção do detergente da mistura lipídio:proteína:detergente durante o processo de co-solubilização, e também com a natureza do fosfolipídio utilizado. Os resultados indicaram que a resina Calbiosorb® foi a mais eficiente para a remoção do SDS e, dentre os diversos fosfolipídios testados o que melhor incorporou as proteínas foi o DPPC, com rendimento de incorporação de 93 % e diâmetro médio de 180 nm. Além disso, o SDS-PAGE dos proteolipossomos mostrou que todas as espécies protéicas presentes no extrato bruto solubilizado foram incorporadas nos lipossomos de DPPC. O Western Blotting mostrou que as proteínas incorporadas nos lipossomos continuavam a ser reconhecidas pelo anticorpo policlonal contra a P. multocida. Para os ensaios de imunização foram separados 3 grupos de coelhos: (i) imunizados com lipossomos; (ii) imunizados com extrato bruto solubilizado (EBS); (iii) imunizados com os proteolipossomos. Após 21 dias de imunização com as preparações descritas, os animais foram infectados com 105 ufc de bactéria. Todos os animais vacinados previamente com lipossomos ou EBS foram a óbito enquanto que os animais vacinados com os sistemas de proteolipossomos apresentaram sobrevida de 95 %. Além disso, um grupo controle vacinado com a bactéria atenuada na presença de hidróxido de alumínio como imunoadjuvante apresentou uma sobrevida de apenas 30 %, indicando que a vacina convencional não apresenta uma proteção satisfatória contra a pasteurelose. O soro dos animais vacinados com lipossomo, EBS e proteolipossomos foram coletados semanalmente antes e após a infecção experimental para a detecção da produção de anticorpos IgG, IgM e IgA, utilizando-se a técnica de ELISA. Como esperado, os animais vacinados com lipossomos não apresentaram estimulação de nenhum dos anticorpos específicos para P. multocida analisados. Os animais imunizados com EBS apresentaram um significativo aumento dos níveis de IgG sérico 7 dias após a imunização os quais se mantiveram constantes durante todo o período experimental. Os níveis de IgG no soro de animais imunizados com os proteolipossomos apresentam um aumento 7 dias após a imunização, porém não se mantiveram até o momento da infecção experimental. Após a infecção experimental, os níveis séricos de IgG nos animais imunizados com proteolipossomos apresentam um aumento significativo, enquanto que para os imunizados com EBS houve manutenção dos níveis antes obtidos. A análise de anticorpos IgM específicos para a P. multocida mostram uma produção significativamente maior destes anticorpos para animais imunizados previamente com proteolipossomos que para os animais imunizados com EBS. Além disso, após a infecção experimental, a produção de IgM nos animais imunizados com proteolipossomos continuou sendo estimulada, o que não foi observado para os animais imunizados com EBS. O sistema de proteolipossomos não produz anticorpos IgA sistêmicos específicos para a bactéria, porém após a infecção experimental foi possível observar o aparecimento gradativo deste anticorpo no lavado nasal dos animais, durante as semanas de observação. Os animais previamente imunizados com proteolipossomos sobreviventes da primeira infecção experimental foram observados durante 140 dias e novamente infectados, com nova carga bacteriana. Após a reinfecção a sobrevida destes animais foi de 100 % indicando que o sistema de proteolipossomos foi capaz de gerar uma memória imunológica. A análise conjunta dos resultados obtidos na detecção de anticorpos indica que a proteção proporcionada pelos proteolipossomos contra a pasteurelose é devida a estimulação de anticorpos IgG e, principalmente, de IgM. O outro sistema de delivery de proteínas antigênicas desenvolvido foi o de microesferas lipídicas. Foram experimentados diferentes protocolos, porém o que mais se adequou as nossas condições foi obtido da união e adaptação de duas metodologias descritas na literatura. Estudos de microscopia eletrônica de varredura mostraram que as microesferas lipídicas são formadas quando é utilizado 3 % (p/v) de PVA na formulação. Além disso, marcamos as proteínas com isoticiocianato de fluoresceína e a microscopia revelou a presença de estruturas esféricas fluorescentes, indicando a encapsulação das proteínas na região lipofílica das microesferas. Estudos sistemáticos variando a concentração de óleo, fosfolipídio, proteínas e PVA na formação das microcapsulas permitiram um rendimento de encapsulação de cerca de 99 %. Portanto, no presente trabalho, estabelecemos metodologias de incorporação das proteínas antigênicas em lipossomos constituídos de DPPC e em microesferas lipídicas. Além disso, os sistemas de proteolipossomos apresentaram uma satisfatória propriedade de proteção dos coelhos contra a pasteurelose (frente à infecção experimental com P. multocida) indicando que o sistema aqui proposto pode ser utilizado como vacina, prevenindo a pasteurelose em criações de coelhos comerciais ou destinados à pesquisa biomédica. / Pasteurellosis is a common disease in the respiratory tract of commercial and/or biomedical rearing of research rabbits. The bacterium Pasteurella multocida is the pathogen responsible for a range of clinical syntomes, including chronic rhinitis (snuffles), otitis media, pneumonia, genital infection, pulmonary and cutaneous abscesses, conjunctivitis and hemorrhagic septicemia. However, between 50 and 70 % of the animals can harbour the microorganism asymptomatically. The factors that cause the clinical syntomes include the ammonium accumulation in the air (foul ventilation), pregnancy, another concomitant disease, disorder in the rabbit production environment and experimental manipulation. Outbreaks of this disease occur in Brazil with relative frequency; however diagnosis is generally based on the clinical signals and necropsy. Therefore, it is difficult to estimate the extent of losses caused by pasteurellosis druing cuniculture. However, specific commercial vaccines against pasteurellosis in rabbits are not available and prevention is through the use of antibiotics in drinking water, even though this type of treatment generally does not protect the animals. Initially, pure bacteria colonies were obtained, which were cultivated in specific growing media (BHI). The microorganisms were isolated, lysed and the antigenic proteins were detected by SDS-PAGE and Western Blotting. These results show that most protein bands were recognized by the policlonal antibody against P. multocida. Since this protein pool presented antigenicity, the protein mixture was solubilized by incubating 0,5 mg/ml of the membrane fraction with SDS 1 % (w/v) under constant agitation for 2 hours. This procedure resulted in a 85 % solubilization yield. The proteoliposomes wew formed using a lipid, protein and detergent co-solubilization method. A good yield of protein incorporation in liposomes seems to be related to the methodology used for the removal of the detergent from the lipid:protein:detergent mixture during the co-solubilization process, as well as the nature of the phospholipid used. The results indicated that the Calbiosorb® resin was the most efficient for SDS removal and, among the various phospholipids tested, DPPC best incorporated the proteins, presenting an incorporation yield of 93% and average proteoliposome diameter of 180 nm. In addition, SDS-PAGE of the proteoliposomes has shown that all the proteic species present in the crude solubilized extract were incorporated in the DPPC liposomes. The Western Blotting has shown that the proteins incorporated in the liposomes continue to be recognized by the policlonal antibody against P. multocida. For the immunization assays, three animal groups were separated: (i) rabbits immunized with liposomes; (ii) rabbits immunized with crude solubilized extract (CSE) and (iii) rabbits immunized with the proteoliposomes. After twenty-one days of immunization with the described preparations, the animals were challenged with 105 ufc of bacteria. All animals previously vaccinated with the liposomes or CSE died while the animals vaccinated with the proteoliposomes systems had 95 % survival after the challenge. Moreover, a control group vaccinated with the attenuated bacteria in the presence of aluminum hydroxide as an immunoadjuvant had only 30% survival, indicating that the conventional vaccine does not protect against pasteurellosis. The serum of animals vaccinated with liposome, CSE and proteoliposomes were collected weekly before and after the experimental infection for the detection of IgG, IgM and IgA antibodies production using ELISA. Animals vaccinated with liposomes did not present stimulation of any of the specific antibodies for the P. multocida analyzed. The animals immunized with CSE presented a significant increase in the IgA serum level seven days after the immunization, but these levels were not maintained until the moment of the experimental infection. After the experimental infection, the serum levels of IgG in rabbits immunized with proteoliposomes showed a significant increase, while for those animals immunized with the CSE the levels were maintained. The analysis of IgM antibodies specific for the P. multocida showed a higher production to animals vaccinated with proteoliposomes than for the animals immunized with CSE. Furthermore, after experimental infection, the production of IgM in animals immunized with proteoliposomes continued to be stimulated, which was not observed for those immunized with EBS. The proteoliposome system does not induce IgA systemic antibodies that were specific for the bacterium. However, after the experimental infections it was possible to observe the gradual appearance of IgA in the nasal lavage of the infected animals on the time course of the experiment. Animals previously immunized with the proteoliposomes which survived the first experimental infection were observed during 140 days and re-infected. After the re-infection, the survival of these animals was 100 %, indicating that the proteoliposome system was able to generate a possible immunological memory. The global analysis of the results obtained in the antibody detection indicates that the protection given by the proteoliposome against pasteurellosis is due to the stimulation of antibodies IgG and mainly of IgM. The other delivery system of antigenic proteins developed during this work is of lipidic microspheres. Different protocols were tried, but the one which was more adequate to our experimental conditions was elaborated from joining and adapting two methodologies described in literature. Scanning electron microscopy studies have shown that the lipidic microspheres are formed when 3 % (w/v) of PVA is used in the formulation. Furthermore, we have marked the proteins with fluorescein isothiocyanate and the microscopy revealed the presence of fluorescent spherical structures which indicated the encapsulation of the proteins in the lipophilic region of the microspheres. Systematic studies varying the concentration of oil, phospholipid, proteins and PVA in the microcapsules formulation has given a yield of encapsulation of 99%. We have established methodologies of incorporation of the antigenic proteins in liposomes constituted of DPPC and lipidic microspheres. Moreover, the proteolipossome systems have shown a satisfying property of protection of rabbits against pasteurellosis in face of the experimental challenge with P. multocida indicating that the system proposed here can be used as a vaccine to prevent the pasteurellosis either in commercial or biomedical research rearing of rabbit.

O fungo Rhizopus microsporus var. rhizopodiformis foi submetido a determinadas condições de cultivo, objetivando produzir substâncias com atividade antimicrobiana. O microorganismo foi transferido da sílica, na qual foi armazenado, e incubado por sete dias em meio aveia-ágar para o crescimento prévio. Em seguida, 4 x 106 esporos/mL de meio foram transferidos para o meio pré-fermentativo e incubado por 24 horas, seguido de transferência para meio fermentativo (meio Czapek, meio Jackson e meio Vogel) e incubado por períodos de tempo determinados. Além disso, foi incubado em meio fermentativo de arroz por 20 dias. Após a fermentação nos diferentes meios, o caldo da cultura foi obtido por filtração e submetido a partições utilizando-se solventes orgânicos: acetato de etila e n-butanol, os quais foram recuperados por evaporação a vácuo. Os extratos obtidos dessas partições foram submetidos aos testes de bioautografia para determinação das frações ostentando atividade antimicrobiana. Os extratos em acetato de etila dos quatro meios fermentativos apresentam tal atividade. As frações ativas foram submetidas a diferentes modalidades cromatográficas para isolamento das substâncias, como a cromatografia liquida em coluna de sílica e a Cromatografia Liquida de Alta Eficiência. Foram isolados quatro metabólitos secundários a partir das frações obtidas em acetato de etila dos meio liquido Jackson e do meio sólido de arroz. Foram identificadas duas dicetopiperazinas (ciclos Leu-Pro e Leu-4-OH-Pro) e um derivado aromático contendo nitrogênio na porção alifática da molécula. Foram determinadas as concentrações inibitórias mínimas (CIM) das substâncias isoladas contra os seguintes microorganismos: Kocuria rhizophila (ATCC 9341), Staphylococcus aureus (ATCC 25923) Candida albicans (ATCC 10231), Escherichia coli (ATCC 25922) e Pseudomonas aeruginosa (ATCC 27853). Todos os extratos obtidos dos meios de cultura apresentaram atividade contra S. aureus e K. rhizophila, bem como o extrato metanólico dos micélios. Apenas os extratos em n-butanol não foram ativos. Nos ensaios de microdiluição, as três ix substâncias identificadas apresentaram CIMs entre 400 e 350 μg/mL. Os resultados obtidos indicam que o fungo em questão é uma fonte interessante para obtenção de metabólitos secundários. / The fungus Rhizopus microsporus var. rhizopodiformis was cultivated in different fermentative media aiming to produce secondary metabolites bearing antimicrobial activity. The conidia storage in silica gel was incubated in oat medium for conidia production. After seven days the conidia (4 x 106 conidia/mL) was transferred to a pre-fermentative medium and incubated for 24 h for mycelium production. After that, it was transferred to three fermentative media: Czapek, Jackson and Vogel, and incubated for different periods of time. After fermentation, cultures were filtered and the broth was submitted to partition using ethyl acetate and n-buthanol in sequence, which were, afterwards, recovered under vacuum. The obtained crude extracts were evaluated under bioautography assay aiming to select the most adequate medium for secondary antimicrobial metabolite production. The ethyl acetate extracts obtained from both Jackson and rice media displayed the higher activities. Therefore, they were submitted to different chromatographic means, including silica gel column and HPLC, furnishing four isolated compounds, from which three were identified: two diketopiperazines (cyclos Leu-Pro and Leu-4-OH-Pro) and one aromatic compound containing a nitrogen in the aliphatic moiety. The pure isolated compounds were submitted to microdilution tests against the following bacteria: Kocuria rhizophila (ATCC 9341), Staphylococcus aureus (ATCC 25923) Candida albicans (ATCC 10231), Escherichia coli (ATCC 25922) e a Pseudomonas aeruginosa (ATCC 27853), aiming to determine their Minimum Inhibitory Concentrations (MIC). All the ethyl acetate extracts displayed antimicrobial activity in the bioautography assays against S. aureus and K. rhizophila, as well as the mycelia methanol extracts. Only the n-butanol extracts did not display activity. Regarding the microdilution assay, three of evaluated pure compounds displayed MIC values between 400 and 350 μg/mL. The obtained results indicated that the studied fungus is an interesting source for biologically active secondary metabolites production.

Submitted by Natalie Mendes (nataliermendes@gmail.com) on 2015-07-28T23:01:37Z No. of bitstreams: 1 COMPOSI??O QU?MICA DO ?LEO ESSENCIAL DE Lippia origanoides Kunth E ATIVIDADE ANTIMICROBIANA FRENTE A DIFERENTES SOROTIPOS~1.pdf: 1434340 bytes, checksum: 8072381e4bdaa1aac1583317e834ac0d (MD5) / Made available in DSpace on 2015-07-28T23:01:37Z (GMT). No. of bitstreams: 1 COMPOSI??O QU?MICA DO ?LEO ESSENCIAL DE Lippia origanoides Kunth E ATIVIDADE ANTIMICROBIANA FRENTE A DIFERENTES SOROTIPOS~1.pdf: 1434340 bytes, checksum: 8072381e4bdaa1aac1583317e834ac0d (MD5) Previous issue date: 2014-03-28 / Pig farming has become increasingly important in recent years in Brazil, because of this, studies for the treatment of diseases that cause the loss of mass of meat animals has increased significantly, such as the Glasser's disease caused by Haemophilus parasuis. Some initial studies have shown human resistance to antibiotics due to the consumption of meat produced with high levels of these substances, and alternatively treatments have been developed from natural products. Lippia origanoides Kunth is presented as a natural source of antimicrobial substances due to the composition of the essential oil obtained, mainly, from the leaves of this plant. In this study the antimicrobial activity of the essential oil from Lippia origanoides Kunth, by determining the minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration (MBC) against Haemophilus parasuis serotypes 1,2,4,5,9,10,12,13,14 and one untypable was studied. Essential oils were obtained by hydrodistillation of the dried leaves and the chemical composition analysis revealed the presence of carvacrol as the predominant component, which characterizes the chemotype B. The results of the antimicrobial activity demonstrated the inhibitory effect of essential oil samples for all tested bacteria. The best result was 0.005% against the sample MV12315 (serotype 10) while the least satisfactory was 0.078% against the sample MV12196 (serotype 12). Results demonstrate the bactericidal action of the oil against the different serotypes of Haemophilus parasuis. / A suinocultura vem se sobressaindo nos ?ltimos anos no Brasil, por isso aumentam os estudos para tratamento das doen?as que causam perdas de carca?a dos animais, como a doen?a de Gl?sser, provocada pelo Haemophilus parasuis. Alguns trabalhos incipientes demonstram a resist?ncia humana a antibi?ticos devido ao consumo de carnes produzidas com altos ?ndices destas subst?ncias, e tratamento alternativos com produtos naturais vem sendo desenvolvidos. Lippia origanoides Kunth se apresenta como uma fonte natural de subst?ncias antimicrobianas devido ? composi??o do seu ?leo essencial obtido principalmente das folhas desta planta. Este trabalho avaliou a atividade antimicrobiana do ?leo essencial de Lippia origanoides atrav?s da determina??o da Concentra??o Inibit?ria M?nima (CIM) e Concentra??o Bactericida M?nima (CBM) frente a amostras de campo do Haemophilus parasuis com sorotipos 1,2,4,5,9,10,12,13,14 e um n?o sorotip?vel. Os ?leos essenciais foram obtidos por hidrodestila??o das folhas secas ap?s tr?s horas, e na an?lise da composi??o qu?mica, o carvacrol foi identificado como componente predominante, caracterizando-o como quimiotipo B. Os resultados de atividade antimicrobiana demonstram o efeito inibit?rio do ?leo essencial para todas as amostras de bact?rias testadas. O melhor resultado encontrado foi de 0,005% frente a amostra MV12315 (sorotipo 10) enquanto o menos satisfat?rio foi de 0,078% contra a amostra MV12196 (sorotipo 12). Os resultados obtidos demonstram a a??o bactericida do ?leo para os diferentes sorotipos do Haemophilus parasuis.

Instead of planktonic growth in nature, many species of bacteria form biofilm to survive in harsh conditions. Although many chronic bacterial infections are caused by bacterial species in a biofilm lifestyle, previous research has focused on studying antibiotic resistance in planktonic growth. Here we used a modified MBEC assay, i.e. biofilm growth on pegs, to determine Escherichia coli biofilm inhibitory concentrations (BIC) of ciprofloxacin, streptomycin and rifampicin and to study the minimal selective concentration (MSC) for ciprofloxacin in E. coli biofilm. We could observe high inhibitory concentrations for all antibiotics in the biofilm pre-formed in media without antibiotics compared to the biofilm formed in antibiotics. We also show preliminary result indicating that sublethal concentrations of ciprofloxacin lead to the selection of ciprofloxacin resistant mutants in biofilm and that the selection level is lower than what was observed in planktonic growing E. coli. With more knowledge in how the biofilm formation precedes in different antibiotic settings, the treatment for chronic biofilm infections used today could be evaluated and changed so that the infections could be eradicated.

<i>Staphylococcus aureus</i> is a ubiquitous organism causing world-wide morbidity and mortality. This species readily develops resistance to antimicrobial agents. Current dosing strategies are based, in part, on minimum inhibitory concentrations (MICs). This susceptibility test fails to detect the presence of first-step resistant mutants often present in large heterogeneous populations of infecting bacteria. Dosing strategies based on MIC results may, in fact, allow for the selective proliferation of resistant subpopulations. The mutant-prevention concentration (MPC) is the drug concentration at which all first-step resistant mutants will be eradicated along with the susceptible cells. Determination of the mutant-selection window (MSW) is possible using MIC and MPC data. When considered together with achievable drug concentrations in human bodily sites, the MSW helps determine which antimicrobials are likely to select for resistance. MIC and MPC testing on clinical isolates of methicillin-susceptible (MSSA) and -resistant (MRSA) S. aureus was performed. Characterization via the polymerase chain reaction, sequencing, and electron microscopy (EM) was done on selected organisms recovered from MPC studies (MPC-recovered). MIC and MPC testing was performed on organisms isolated sequentially from patients with recurring S. aureus infections. Pulsed field gel electrophoresis was performed on these sequential isolates. Based on the MIC and the MPC values, the most potent agents for systemic MSSA and MRSA infections are gemifloxacin and vancomycin, respectively. Re-testing MPC-recovered populations by the MIC showed increased MIC results compared to the parent populations. Macrolide-resistance genes were discovered in S. aureus MPC-recovered populations; in contrast, parental isolates lacked these resistance determinants. EM revealed an increase in cell wall thickness of a vancomycin MPC-recovered population compared to its parental population. Moxifloxacin and vancomycin had the lowest and narrowest MSWs for systemic MSSA and MRSA infections, respectively, compared to the other agents tested. Sequential isolates showed no change in MIC and MPC values. The data presented provides evidence for the application of the MPC test to S. aureus organisms. The MPC data is significant when determining appropriate dosing strategies aimed at preventing resistance.

<i>Staphylococcus aureus</i> is a ubiquitous organism causing world-wide morbidity and mortality. This species readily develops resistance to antimicrobial agents. Current dosing strategies are based, in part, on minimum inhibitory concentrations (MICs). This susceptibility test fails to detect the presence of first-step resistant mutants often present in large heterogeneous populations of infecting bacteria. Dosing strategies based on MIC results may, in fact, allow for the selective proliferation of resistant subpopulations. The mutant-prevention concentration (MPC) is the drug concentration at which all first-step resistant mutants will be eradicated along with the susceptible cells. Determination of the mutant-selection window (MSW) is possible using MIC and MPC data. When considered together with achievable drug concentrations in human bodily sites, the MSW helps determine which antimicrobials are likely to select for resistance. MIC and MPC testing on clinical isolates of methicillin-susceptible (MSSA) and -resistant (MRSA) S. aureus was performed. Characterization via the polymerase chain reaction, sequencing, and electron microscopy (EM) was done on selected organisms recovered from MPC studies (MPC-recovered). MIC and MPC testing was performed on organisms isolated sequentially from patients with recurring S. aureus infections. Pulsed field gel electrophoresis was performed on these sequential isolates. Based on the MIC and the MPC values, the most potent agents for systemic MSSA and MRSA infections are gemifloxacin and vancomycin, respectively. Re-testing MPC-recovered populations by the MIC showed increased MIC results compared to the parent populations. Macrolide-resistance genes were discovered in S. aureus MPC-recovered populations; in contrast, parental isolates lacked these resistance determinants. EM revealed an increase in cell wall thickness of a vancomycin MPC-recovered population compared to its parental population. Moxifloxacin and vancomycin had the lowest and narrowest MSWs for systemic MSSA and MRSA infections, respectively, compared to the other agents tested. Sequential isolates showed no change in MIC and MPC values. The data presented provides evidence for the application of the MPC test to S. aureus organisms. The MPC data is significant when determining appropriate dosing strategies aimed at preventing resistance.

Some yeast strains under certain conditions secrete polypeptide toxins which are inhibitory to sensitive fungal cells into the medium. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer proteins are classified into 11 typical types (K1-K11). These toxins have different killing mechanisms on sensitive cells. Some of them hydrolyze major cell wall component, beta-1,3- glucans. As mammalian cells lack cell walls research and development of novel highly selective antifungals are mostly focused on the agents which target the components of the fungal cell wall. K5 type killer protein was characterized in our labarotory previously. This protein is an exo beta-1,3-glucanase which is stable at pH&amp / #8217 / s and temperatures appropriate for its biocontrol usage. Beta-1,3-glucan hydrolyzing activity of the K5 type killer protein highlighted the potential use of this protein as a selective antifungal agent. According to CLSI methodology, antifungal activity of the K5 type yeast killer protein was tested against 6 fungal strains causing postharvest spoilage in citrus fruits and found to be effective on Botrytis cinerea, Penicillium digitatum, Penicillium italicum whereas non effective on Colletotrichum gloeosporoides, Phythophythora citrophthora, Alternaria citri. The MIC values of the toxin for B.cinerea, P.digitatum, P.italicum were found to be 16 mikrogram/ml while IC 50 values of the toxin were 2.12, 3.31, 2.57 mikrogram/ml respectively. The results showed that K5 type yeast killer protein would be used as a novel and selective agent against B.cinerea, P.digitatum and P.italicum.