O descarte de antimicrobianos e bactérias através de efluentes hospitalares tem contribuído para o aumento e disseminação da resistência antimicrobiana no ambiente natural. Esta resistência pode estar associada à aquisição de genes de resistência, presença de bombas de efluxo ou características intrínsecas das bactérias. O presente trabalho teve como objetivos: caracterizar o perfil de resistência em 60 isolados de Pseudomonas para os antimicrobianos Ceftazidima e Imipenem, detecção fenotípica das bombas de efluxo e detecção dos genes mex B, mex D, mex Y e mex F de bomba de efluxo da família Resistência-Nodulação- Divisão (RND), associados a fenótipos de resistência neste gênero bacteriano. O perfil de resistência dos isolados foi avaliado através da Concentração Inibitória Mínima (CIM) dos antimicrobianos Ceftazidima e Imipenem. Para a detecção fenotípica das bombas de efluxo foi comparada a CIM do Imipenem (IMP) e da Ceftazidima (CAZ) na presença e na ausência de carbonil cianida mclorofenilhidrazona (CCCP). A presença dos genes foi verificada através da Reação em Cadeia da Polimerase (PCR). Os isolados de Pseudomonas aeruginosa (n=32) mostraram-se mais resistentes aos antimicrobianos testados do que as outras espécies do gênero (n=28). Na avaliação fenotípica das bombas de efluxo 10 isolados reduziram a CIM do IMP e 6 isolados reduziram a CIM da CAZ na presença do CCCP, indicando que bombas de efluxo possivelmente são responsáveis por esta resistência nestes isolados. Dos 60 isolados utilizados no estudo, 95% amplificaram o gene mex Y, 88% o gene mex B, 88% mex F, 48% mex D, no entanto, quando os resultados do PCR foram comparados com a análise fenotípica, revelou que nem todos os isolados expressam os genes encontrados, portanto a resistência pode ser associada também a outros mecanismos. / The disposal of antimicrobial and bacteria through hospital effluents has contributed to the increase and spread of antimicrobial resistance in the natural environment. This resistance can be associated to gene acquisition, presence of efflux bombs or intrinsic characteristics of the bacteria. This study aimed to: characterize the resistance profile of 60 Pseudomonas isolates for Ceftazidime and Imipenem l, to detect phenotypically efflux pumps systems and detect the genes mex B mex D, mex Y and mex F responsible by the efflux pump family resistance-nodulation-division (RND). The isolates resistance profile was evaluated by Minimum Inhibitory Concentration (MIC) of ceftazidime and imipenem l. For the phenotypic detection of efflux pumps the MIC of imipenem (IMP) and ceftazidime (CAZ) in the presence and absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP was compared. The presence of the genes was verified by Polymerase Chain Reaction (PCR). Isolates of Pseudomonas aeruginosa (n=32) were more resistant to antimicrobials tested than other species of the genus (n=28). In the phenotypic evaluation of efflux pumps 10 isolates reduced the MIC of IMP and 6 isolates reduced the CAZ MIC in the presence of CCCP, indicating that an efflux system can be responsible for the resistance of the isolates. Of the 60 isolates used in the study, 95% amplified the mex Y, 88% mex B, 88% mex F and 48% mex D, however, when the PCR results were compared with the phenotypic analysis the results revealed that neither all isolates express genes found, therefore the resistance observed might be associated to another mechanism.

Submitted by MURILO RAPHAEL DE MELO null (engenhariasp@piraflora.com.br) on 2017-03-29T19:30:16Z No. of bitstreams: 1 Dissertação Modelo novo Final.pdf: 1070767 bytes, checksum: 9448e5eb4a8f905c67d161d2933a9827 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-30T18:20:46Z (GMT) No. of bitstreams: 1 melo_mr_me_bot.pdf: 1070767 bytes, checksum: 9448e5eb4a8f905c67d161d2933a9827 (MD5) / Made available in DSpace on 2017-03-30T18:20:46Z (GMT). No. of bitstreams: 1 melo_mr_me_bot.pdf: 1070767 bytes, checksum: 9448e5eb4a8f905c67d161d2933a9827 (MD5) Previous issue date: 2017-02-03 / Um dos fatores que mais contribuem para queda da produtividade em mudas de Eucalyptus spp. são as doenças. Nos últimos anos tem se observado o aumento gradativo de doenças de origem bacteriana, principalmente de ocorrência foliar. A mancha foliar bacteriana ocorrem em mudas de eucalipto em todas as fases de produção do viveiro, podendo facilmente ser levada a campo, prejudicando o desenvolvimento inicial da muda. No presente estudo foi avaliado o efeito da severidade de bacteriose e da desfolha no crescimento pós-plantio de mudas de Eucalipto. As plantas avaliadas apresentaram um retardo no desenvolvimento devido a doença e a desfolha, no entanto, mantendo a adubação, após a recuperação, as mudas tiveram seu crescimento normalizado. / Diseases are one of the factors that most contribute to decrease the Eucalyptus spp. productivity. Now a day observe the gradual increase of bacterial origin diseases, mainly of foliar occurrence. The bacterial leaf spot occurs in eucalyptus seedlings at all stages of nursery production, and it can easily take to the field, damaging the initial development of the seedlings. In this study was evaluated the effect of bacterial leaf spot severity and defoliation on post-planting growth of Eucalyptus seedlings. The evaluated plants presented a delay in the development due to disease and defoliation; however, maintaining the fertilization, after the recovery, the seedlings had their growth normalized.

Orientador: Edson Luiz Furtado / Banca: Willian Bucker Moraes / Banca: Irae Amaral Guerrini / Resumo: Um dos fatores que mais contribuem para queda da produtividade em mudas de Eucalyptus spp. são as doenças. Nos últimos anos tem se observado o aumento gradativo de doenças de origem bacteriana, principalmente de ocorrência foliar. A mancha foliar bacteriana ocorrem em mudas de eucalipto em todas as fases de produção do viveiro, podendo facilmente ser levada a campo, prejudicando o desenvolvimento inicial da muda. No presente estudo foi avaliado o efeito da severidade de bacteriose e da desfolha no crescimento pós-plantio de mudas de Eucalipto. As plantas avaliadas apresentaram um retardo no desenvolvimento devido a doença e a desfolha, no entanto, mantendo a adubação, após a recuperação, as mudas tiveram seu crescimento normalizado. / Abstract: Diseases are one of the factors that most contribute to decrease the Eucalyptus spp. productivity. Now a day observe the gradual increase of bacterial origin diseases, mainly of foliar occurrence. The bacterial leaf spot occurs in eucalyptus seedlings at all stages of nursery production, and it can easily take to the field, damaging the initial development of the seedlings. In this study was evaluated the effect of bacterial leaf spot severity and defoliation on post-planting growth of Eucalyptus seedlings. The evaluated plants presented a delay in the development due to disease and defoliation; however, maintaining the fertilization, after the recovery, the seedlings had their growthnormalized. / Mestre

Introdução. Pseudomonas aeruginosa é um patógeno clinicamente importante. Existem diversos mecanismos de resistência aos antimicrobianos em P. aeruginosa, dentre eles a produção de enzimas (β-lactamases) e sistemas de efluxo se destacam, uma vez que são capazes de conferir resistência aos carbapenêmicos. Objetivo. Avaliar os sistemas de efluxo MexAB-OprM e MexXY em isolados clínicos de P.aeruginosa de pacientes atendidos no Hospital de Clínicas de Porto Alegre-RS e relacionar a expressão destes sistemas com a CIM de meropenem em isolados produtores e não produtores de metalo-beta-lactamases (MBL). Metodologia. Um total de 86 isolados de P. aeruginosa com suscetibilidade reduzida aos carbapenêmicos foram avaliados. A hiperexpressão dos sistemas MexAB e MexXY foi determinada fenotipicamente utilizando inibidor seletivo da bomba (PAβN). MBLs foi determinada por PCR utilizando primers específicos. Resultados. O fenótipo de hiperexpressão dos sistemas de efluxo estudados foi observado em 34 (47,8%) dos 71 isolados negativos para a produção de MBL e em 14 (93.3%) dos 15 isolados MBL positivos. Na presença de PaβN, todos os isolados não produtores de MBL apresentaram uma redução da CIM para meropenem para valores na faixa de suscetibilidade. Entretanto, das 13 P. aeruginosa produtoras de MBL que diminuíram a CIM, essa redução não foi para valores dentro da faixa de sensibilidade. Conclusão. Os isolados de P. aeruginosa não produtores de MBL apresentam resistência ao meropenem devido a hiperexpressão de MexAB-OprM. Na presença de PaβN, independente de apresentarem produção de MBL, o CIM de meropenem reduziu para valores ≤ 8 mg/L. Contudo, quando isolados não apresentavam MBL, a CIM de meropenem reduziu para níveis de sensibilidade. / Introduction. Bacterial efflux pump systems are resistance mechanisms which may lead to therapeutic failure of antibiotic treatment since many antimicrobial agents are substrate for these mechanisms. The aim of the study was to evaluate the expression of the MexAB, MexXY pump efflux and to determine its influence on meropenem MIC in carbapenemase producing and non-producing P. aeruginosa. Methods. A total of 86 non-repetitive clinical isolates of P. aeruginosa with reduced susceptibility to carbapenems were evaluated. Overexperession of MexAB and MexXY efflux systems were evaluated phenotypically using the PAβN selective inhibitor. Metallo-β-lactamases (MBL) were detected by PCR using specific primers. Results. The efflux pump-overexpressed phenotype was observed in 34 (47.8%) MBL-negative and in 14 (93.3%) MBL-positive isolates. In the presence of the PaβN, all non-producers of MBL presented a reduction of meropenem MICs to the range of susceptibility. In contrast, the 13 P. aeruginosa MBL-producing isolates decreased meropenem MICs at least 16-fold but this reduction did not reach the range of susceptibility. Conclusion. P. aeruginosa non-MBL-producing showed resistance to meropenem due to overexpression of MexAB-OprM. In the presence of PaβN, the isolates harboring or not MBL genes, the meropenem MICs were reduced to values ≤8 mg/L. However, when the isolates harbor MBL genes, the meropenem MIC values were reduced to the susceptibility.

<p>&nbsp;Bakteriozno su&scaron;enje tre&scaron;nje (Prunus avium L.) poslednjih nekoliko godina u mladim zasadima i plantažama tre&scaron;nje predstavlja značajan problem u proizvodnji ove voćne vrste. Simptomi bolesti se ispoljavaju u vidu su&scaron;enja grana, grančica ili celih stabala, &scaron;to se uglavnom zapaža na mestima rezidbe i oko pupoljaka, sa uočljivim promena boje tkiva kore, koje puca i nastaju rak rane. U periodu od 2012 &ndash; 2015 godine izvr&scaron;en je monitoring zdravstvenog stanja tre&scaron;nje kojim je obuhvaćeno nekoliko plantaža i manjih zasada tre&scaron;nje iz vi&scaron;e lokaliteta na području AP Vojvodine i centralne Srbije (Ritopek). Mlade voćke su očigledno najugroženije, jer smo na osnovu praćenja zdravstvenog stanja u vi&scaron;e lokaliteta i zasadima različite starosti, pojavu bakterioznog su&scaron;enja u jačem ili slabijem intenzitetu, konstatovali samo u mladim zasadima (do 3 godine starosti &ndash; Selenča, Gornji Tavankut, Donji Tavankut, Ljutovo, Mikićevo i Kanjiža). Izolacijama na strandardne hranljive podloge, iz prikupljenih obolelih uzoraka tre&scaron;nje, kao i sa zdravih pupoljaka i listova tre&scaron;nje (epifitna populacija), dobijeni su brojni izolati bakterija P. syringae pvs. od kojih je za dalja ispitivanja odabrano 155 izolata. Identifikikacija dobijenih izolata je izvr&scaron;ena je na osnovu fenotipskih i genotipskih metoda. Na osnovu LOPAT testova izolati pripadaju Ia grupi fluorescentnih vrsta Pseudomonas syringae. Prema GATTa testovima utvrđene su dve grupe izolata u okviru vrste P. syringae: I grupa (G+A+T&ndash;Ta&ndash;) i II grupa (G&ndash;A&ndash;T+Ta+). Dodatni testovi su potvrdili GATTa testove, na osnovu kojih je zaključeno da su&scaron;enje mladih stabala tre&scaron;nje prouzrokuju dve grupe bakterije P. s. pv. syringae (I grupa) i P. s. pv. morsprunorum rasa 1 (II grupa). Među ispitivanim izolatima nije bilo odstupanja u pogledu fenotipskih karakteristika u okviru iste grupe, osim sposobnosti stvaranja siringomicina pojedinih izolata I grupe (pv. syringae). Proverom patogenosti na raznim test biljkama i biljci domaćinu utvrđene su razlike, ali i određene sličnosti između izolata I i II grupe. Jasne razlike između grupa izolata utvrđene su pri inokulaciji zelenih plodova tre&scaron;nje, vi&scaron;nje, ringlova i kru&scaron;ke, paradajza, paprike i mahuna boranije. Pri inokulaciji odvojenih listova jorgovana izolati I grupe (pv. syringae), kao i većina izolata II grupe (pv. morsprunorum rasa 1) su pozitivno reagovali, &scaron;to ukazuje na heterogenost populacije bakterije P. s. pv. morsprunorum rasa 1. Pri inokulaciji sejanaca voćnih podloga (divlja tre&scaron;nja, magriva, divlja &scaron;ljiva, divlja kru&scaron;ka) svi izolati pv. syringae su prouzrokovali karakteristične patolo&scaron;ke promene na podlogama svih voćnih vrsta, a izolati pv. morsprunorum rase 1 takođe na svim vrstama, osim na sejancima divlje &scaron;ljive. Ovi rezultati ukazuju da je &scaron;irenje bakterija moguće i putem podloga koje takođe mogu biti zaražene. Inokulacijama dvogodi&scaron;njih grančica tre&scaron;nje u periodu mirovanja zaključeno je da su svi izolati pv. syringae i morprunorum rasa 1 podjednako patogeni na svim sortama tre&scaron;nje (Burlat, Summit, Hedelfigenska i Germerzdorfska). Najveća dužina nekroze najče&scaron;će je zabeležena na sortama Burlat i Summit u kombinaciji sa izolatima I grupe (pv. syringae) u pojedinim slučajevima i sa izolatima II grupe (pv. morsprunorum rasa 1), a najmanja uglavnom kod sorti Germerzdorfska i Hedelfigenska sa izolatima II grupe (pv. morsprunorum rasa 1). Identifikacija izolata KBNS71 &ndash; 84 (Gornji Tavankut) i KBNS85 &ndash; 94 (Selenča) na bazi MLST kori&scaron;ćenjem gena gyrB, rpoD, gapA i gltA, jasno je pokazala prisustvo dva patovara P. s. pv. syringae i P. s. pv. morsprunorum rasa 1. Pri poređenju sa sojevima H &ndash; 1, V &ndash; 85, V &ndash; 88 (vi&scaron;nja) i V &ndash; 109 (tre&scaron;nja) utvrđene su značajne razlike i postojanje genetskog diverziteta populacije ovih patogena. Simultana detekcija gena syrB i syrD utvrđena je kod 70 izolata I grupe (pv. syringae), a samo SyrB kod 9 izolata iste grupe (pv. syringae). Gen za sintezu koronatina detektovan je kod svih 76 izolata II grupe (pv. morsprunorum rasa 1). Rep &ndash; PCR metodom ustanovljene su značajne razlike (58%) između I i II grupe izolata (pv. syringae i pv. morsprunorum rasa 1). Ispitivani izolati sa tre&scaron;nje u okviru pv. syringae nisu ispoljili međusobne razlike, ali se razlikuju od sojeva sa drugih lokaliteta i ranije izolovanih sa istog domaćina (V &ndash; 109 i T6), kao i od sojeva sa drugih domaćina &ndash; vi&scaron;nje (V &ndash; 85) i uljane tikve (Tk21) do 37%. Razlike među izolatima pv. morsprunorum rase 1 iznosile su manje od 5%, a 24% u odnosu na soj CFBP2119 istog patogenog varijeteta. Rep &ndash; PCR analiza ukazala je na nizak nivo heterogenosti ispitivanih izolata u okviru istog patogenog varijeteta. RAPD metoda, kori&scaron;ćenjem većeg broja prajmera, bila je uspe&scaron;nija za poređenje ispitivanih izolata od rep &ndash; PCR. Od testiranih 11 prajmera, 4 (SPH1, DJP17, DJ15, DJ16) su selektovana za dalji rad na osnovu razlika među izolatima unutar patogenih varijeteta. Kumulativna RAPD analiza pokazala je da između ispitivanih izolata pv. syringae postoje razlike do 24%, a 41% u poređenju sa sojem KFB0103, dok su kod izolata pv. morsprunorum rase 1 razlike iznosile do 15%, a 36% u odnosu na soj</p><p>4<br />CFBP2119. Dobijeni rezultati RAPD analize ukazuju da u okviru populacije obe grupe ispitivanih izolata postoji određena heterogenost, ali je genetski diverzitet izraženiji kod pv. syringae. Proučavanjem epidemilogije ovih patogena u poljskim uslovima inokulacijom jednogodi&scaron;njih grana / mladara sortama Burlat, Germerzdorfska, Hedelfigenska i Droganova žuta, zaključeno je da tre&scaron;nja u na&scaron;im agroekolo&scaron;kim uslovima ranije postaje osetljiva (oktobar) prema P. s. pv. morsprunorum rasa 1 u odnosu na pv. syringae. Prvi pozitivni rezultati pri inokulaciji sojevima pv. syringae utvrđeni su pri inokulaciji u novembru. U pogledu dužine nekroze najuspe&scaron;nije su bile novembarske inokulacije (najduže nekroze; 2,17 &ndash; 3,35 cm), uspe&scaron;ne su bile i januarske i martovske inokulacije, ali je dužina nekroze bila sve manja, respektivno. Generalno najduže nekroze su ostvarene kod sorte Burlat, a najkraće kod sorte Germerzdorfska. Sve inokulacije urađene u periodu vegetacije su bile negativne. Inokulacijama dvo &ndash; trogodi&scaron;njih grana na sorti Summit prve uspe&scaron;ne inokulacije (oba patovara) su ostvarene tek u novembru (oktobarske su bile negativne), kada je utvrđena i veća agresivnost patovara syringae. Pri inokulacijama u januaru dužina nekroze je bila manja, a martovska je bila negativna. Sve inokulacije vr&scaron;ene u periodu od bubrenja pupoljaka do opadanja li&scaron;ća takođe su bile negativne. Ispitivanjem osetljivosti sotrimenta tre&scaron;nje i pojedinih sorti vi&scaron;nje zaključeno je da su prema oba patovara (syringae i morsprunorum rasa 1) najosetljivije sorte tre&scaron;nje Katalin, Linda, Summit, New Star i Burlat, srednje osetljive su sorte vi&scaron;nje Erdi Botermo i sorte tre&scaron;nje Droganova žuta, CarmCarmen, Germerzdorfska i Rana od Noara, a slabo osetljive sorte vi&scaron;nje &Scaron;panska i Ujfeheti firto&scaron; i sorta tre&scaron;nje Rita.</p> / <p>Bacterial die back (canker) of sweet cherry (Prunus avium L.) in young orchards and sweet cherry plantations in the past few years has been a significant problem in the production of this fruit species. Symptoms of the disease were manifested in the form of drying branches, twigs or whole trees, which were mainly observed in places of pruning or around the buds, bark changes a color, cracks and cankers has formed. In the period 2012 - 2015 monitoring of the health status of sweet cherries was carried out covering several plantations and smaller orchards of sweet cherries in several localities in Vojvodina and central Serbia (Ritopek). Young fruit trees are obviously the most susceptible, based on monitoring of the health status in many localities and plantations of different ages, the occurrence of bacterial canker in a stronger or weaker intensity was found only in young plantations (up to 3 years old - Selenča, Gornji Tavankut, Donji Tavankut, Ljutovo, Mikićevo and Kanjiža). From collected diseased samples of sweet cherries, as well as healthy buds and leaves of sweet cherry (epiphytic population) isolations on standard nutrient medium, were obtained numerous isolates of P. syringae pvs. and for further investigations was selected 155 isolates. Identification of isolates was performed on the basis of phenotypic and genotypic methods. Based on LOPAT tests isolates belonging to Ia group fluorescent Pseudomonas syringae. According to GATTa tests two groups of P. syringae isolates were identified, I group (G+A+T-Ta-) and II group (G-A-T+Ta+). Additional tests confirmed the GATT tests, on the basis which it was concluded that the drying of young sweet cherry trees caused P. s. pv. syringae (I group) and P. s. pv. morsprunorum race 1 (II group). Among the tested isolates was not exceptions in phenotypic characteristics within the same group, except for the ability to produced syringomycine for some isolates of I groups (pv. syringae). In pathogenicity tests on various plants and host plant were observed differences, but also and some certain similarity between isolates of I and II groups. Clear differences between the groups of isolates were determined in the inoculations of green fruit of sweet cherry, sour cherry, cherry plum and pears, tomatoes, peppers and green bean pods. In the case of inoculation of separate lilac leaves isolates of I group (pv. syringae) and most isolates of II group (pv. morsprunorum race 1) reactions were positive, what indicating the heterogeneity of the population of P. s. pv. morsprunorum race 1. In the inoculation of fruit rootstock seedlings (wild cherry, Magriva, wild plum, wild pear) all isolates pv. syringae caused the characteristic pathological changes on the all fruit species, isolates of pv. morsprunorum race 1 also except on the seedlings of wild plum. These results suggest that the spreading of bacteria is possibly through the rootstock that can also be infected. Inoculations of two &ndash; years old branches of sweet cherry during dormancy, was concluded that all isolates pv. syringae and morprunorum race 1 were equally pathogenic in all sweet cherry cultivars (Burlat, Summit, Hedelfigen and Germersdorf). The longest length of necrosis usually was observed on the cultivars Burlat and Summit in combination with isolates of I groups (pv. syringae), in some cases with isolates of II group (pv. morsprunorum race 1), and the lowest mainly in cultivars Germersdorf and Hedelfigen with isolates of II group (pv. morsprunorum race 1). Identification of isolates KBNS71 - 84 (GornjiTavankut) and KBNS85 - 94 (Selenča) based on MLST using genes gyrB, rpoD, gapA and gltA genes clearly showed the presence of two patovars P. s. pv. syringae and P. s. pv. morsprunorum race 1. Comparison with strains H - 1, V - 85 V - 88 (sour cherry) and V - 109 (sweet cherry) showed significant differences and the existence of genetic diversity in the population of these pathogens. Simultaneous detection of syrB and syrD gene was found in 70 isolates of I group (pv. syringae) and only syrB gene in 9 isolates of the same group (pv. syringae). The gene for coronatine synthesis was detected in all 76 isolates of II group (pv. morsprunorum race 1). Rep - PCR method detected significant differences (58%) between isolates of I and II groups (pv. syringae and pv. morsprunorum race 1). The tested isolates from sweet cherry within pv. syringae did not show differences between them, but they were different from the strains from other locations and previously isolated from the same host (V - 109 and T6), as well as strains from other hosts - cherry (V - 85) and pumpkin (Tk21) to 37 %. The differences between isolates pv. morsprunorum race 1 were less than 5% and 24% compared to the same pathovar strain CFBP2119. Rep - PCR analysis indicated a low level of heterogeneity of isolates within the same pathovar. RAPD method using a large number of primers were more successful to compare isolates than rep - PCR. Among 11 tested primers, 4 (SPH1, DJP17, DJ15, DJ16) were selected for further work on the basis of the difference between isolates within same pathovar. Cumulative RAPD analysis showed up to 24% differences among tested isolates of pv. syringae and 41% compared to the strain KFB0103, while among isolates pv. morsprunorum race 1 differences were 15% and 36% compared to the strain CFBP2119. The results of RAPD analysis indicate that a certain heterogeneity<br />7<br />exists in the population of both tested groups of isolates, but genetic diversity is more pronounced among isolates of pv. syringae. Studying the epidemiology of this pathogen in field conditions, by inoculating one &ndash; year old branches / or shoots sweet cherry cultivars Burlat, Germersdorf, Hedelfigen and Droganova žuta, it was concluded that the sweet cherry in our agroecological conditions becoming sensitive (October) to P. s. pv. morsprunorum race 1 before in relation to the pv. syringae. The first positive results of inoculations with strains pv. syringae were determined in November. Regarding the length of necrosis most successful were inoculation in the November (necrosis longest; 2.17 to 3.35 cm), inoculations also were successful in the January and the March, but the length of necrosis was smaller, respectively. Generally longest necrosis were observed in the cultivar Burlat, and the shortest in cultivar Germersdorf. All inoculations carried out in the period of vegetation were negative. Inoculations of two &ndash; three &ndash; years old branches of the cultivar Summit, first successful inoculations (for both pathovar) were observed only in November (October was negative), when a greater aggressiveness of pathovar syringae were determined. In inoculations in January length of necrosis was smaller, and in March was negative. All inoculations carried out in the period from buds swelling to leaf falling were also negative. Investigation susceptibility of sweet cherry and some sour cherry cultivars was concluded that against to both pathovars (syringae and morsprunorum race 1) the most susceptible were cultivars of sweet cherry Katalin, Linda, Summit, New Star and Burlat, medium susceptible were cultivar of sour cherry Erdi Botermo and sweet cherry cultivars Droganova žuta, Carmen, Germersdorf and Rana od Noara and low susceptible cultivars of sour cherry &Scaron;panska and Ujfeheti firto&scaron; and cultivar of sweet cherry Rita.</p>

Pseudomonas aeruginosa is a bacterial pathogen that can infect any human tissue. The lungs of cystic fibrosis patients become chronically infected with Pseudomonas aeruginosa. Virulence factor gene expression is under elaborate regulatory control that remains poorly characterized. Understanding the regulatory hierarchy involved during infection is essential for identifying novel drug targets. RsmA is a post-transcriptional regulatory protein that controls expression of several virulence factors. Previous studies demonstrated alginate regulatory components AlgU and AlgR as regulators of rsmA expression. The aim of this study was to determine how AlgR controls rsmA expression. Western blot analysis of HA-tagged RsmA confirmed lower RsmA levels in an algR mutant. An electrophoretic mobility shift assay using purified AlgR demonstrated direct binding of AlgR to the rsmA promoter. These results indicate AlgR directly controls rsmA expression. We propose a mechanism whereby AlgR and AlgU work together to regulate rsmA.

No description available.

Determina la frecuencia de los genes blaIMP, blaVIM y blaNDM productores de Metalo-ß-lactamasas en aislamientos de Pseudomonas aeruginosa no sensibles a carbapenemes. Recolecta 149 aislamientos de Pseudomonas aeruginosa procedentes de muestras clínicas de los siguientes nosocomios: Hospital Nacional Daniel A. Carrión, Hospital Nacional Edgardo Rebagliati Martins – ESSALUD, Hospital Alberto Sabogal Sologuren – ESSALUD, Hospital General Fuerza Aérea Peruana (FAP) y el Hospital Nacional Docente Madre Niño San Bartolomé. Los aislamientos se almacenaron en el cepario del NAMRU-6 desde julio del 2010 hasta julio del 2012. Realiza el perfil de susceptibilidad a antibióticos mediante el método de “Kirby – Bauer” de acuerdo a los lineamientos del CLSI (Clinical Laboratory Standars Institute). La detección fenotípica de MBL se realizó mediante el método de aproximación de discos de EDTA (Ácido etilendiaminotetraacetico) y la detección de los genes blaIMP, blaVIM y blaNDM mediante un PCR multiplex. Encuentra que en 28 aislamientos de Pseudomonas aeruginosa se detecta la presencia del gen blaIMP mediante PCR, siendo la frecuencia de este gen 18.8%. No se detectaron los genes blaVIM y blaNDM. Concluye que la detección de los genes productores de MBL por PCR permitió detectar la frecuencia real de los aislamientos de Pseudomonas aeruginosa productores de MBL. / Tesis

Bacterial biofilm infection is a common (~ 2 to 4%) complication for recipients of surgically implanted medical devices. Due to the tremendous increase in antibiotic resistance when these bacteria enter the biofilm phenotype, present treatment requires explantation and replacement of the device, often with multiple surgeries and always with much longer patient recovery time. The specific objective of this study was to quantify the degree of biofilm deactivation from exposure to thermal shock for varying temperature and time durations. While extreme temperature (>150˚C) is routinely used to sterilize (e.g. autoclaves), such temperatures have a severe cost within the body. Despite extensive studies on thermal deactivation of bacteria in the planktonic phenotype over a wide range of temperatures (e.g., pasteurization protocols), surprisingly little is known about the thermal deactivation of biofilms except under extreme conditions. Here, the deactivation of Pseudomonas aeruginosa biofilms is reported. These biofilms were cultured at 37°C for 24 hours in a drip-flow reactor and subjected to heat shocks on the range of 50°C to 80°C for durations of 1 to 30 minutes. Heat shocks were delivered by immersion in thermostatted media for the prescribed time and the resulting concentration of colony forming units (CFU/mL) were quantified using direct enumeration. Up to 6.6 orders of magnitude reduction in CFU concentration was observed, indicating that thermal deactivation is a reasonable approach to biofilm mitigation. Integrating this approach with a magnetic nanoparticle implant coating will result in an innovative treatment for implant infections in situ without explantation or device replacement.

Die Biosynthese von fragmentierten Fettsäuren (kurzkettige Dicarbonsäuren und deren Oxocarbonsäure-Vorstufen) ist in den meisten Pflanzen noch unklar. Wichtige, bekannte Dicarbonsäuren sind Pimelinsäure (PIM) und Azelainsäure (AZA) mit den putativen Vorstufen 7-Oxo¬heptanonsäure (OHA) und 9-Oxononanonsäure (ONA). Es besteht großes Interesse die Biosynthese¬mechanismen und die Regulation der Synthese dieser Substanzen aufzuklären, da Fettsäure¬fragmente an wichtigen biologischen Prozessen beteiligt sind. PIM ist eine essentielle Vorstufe von Biotin in Mikroben, Pilzen und Pflanzen. Bisher konnte die Biosynthese von PIM nur in Bakterien (E. coli und B. subtilis) aufgeklärt werden. Es gibt keine Hinweise auf einen analogen Mechanismus in Pflanzen. Eine biologische Aktivität von AZA bei Pflanzen konnte erst vor kurzem beschrieben werden. Eine Forschergruppe identifizierte AZA als Metabolit, der nach Infektion mit dem Pathogen Pseudomonas syringae vermehrt im Phloemsaft von Arabidopsis vorhanden ist und der in Pflanzen eine lokale und systemische Resistenz gegenüber dem Pathogen induziert. In Tieren sind Fettsäurefragmente ebenfalls Gegenstand aktueller Forschung. Es ist bekannt, dass eine nichtenzymatische oxidative Fragmentierung von Fettsäurehydroperoxiden in komplexen Membranlipiden als Folge von oxidativem Stress abläuft. Phospholipide mit veresterter ONA / AZA spielen aufgrund ihrer Struktur eine Rolle als endogene Liganden bei Reaktionen des angeborenen Immunsystems. Ziel dieser Arbeit war es, die Mechanismen der Oxidation von Fettsäuren und deren Fragmentierung in Pflanzen aufzuklären. Weiterhin sollte die Rolle der oxidierten Fragmente in der Immunantwort der Modellpflanze Arabidopsis thaliana untersucht werden. In Pflanzen wurden fragmentierte Fettsäuren im Rahmen dieser Arbeit erstmals in komplexen Lipiden identifiziert und verschiedene Hypothesen zur Bildung von Fettsäurefragmenten experimentell überprüft. Es konnte gezeigt werden, dass die Biosynthese der Fettsäurefragmente in A. thaliana ausgehend von zwei- oder dreifach ungesättigten Fettsäuren stattfindet. 9- und 13-Lipoxygenasen (LOX1, LOX5 und LOX2) spielen dabei keine essentielle Rolle. Die Fettsäurefragmente konnten in Arabidopsis in freier Form und in komplexen Lipiden verestert (ausschließlich in Galactolipiden) detektiert werden. Applikationsexperimente zeigten, dass die Biosynthese der Fettsäurefragmente in den komplexen Lipiden auf nichtenzymatischem Wege in situ stattfindet. Dabei wird in Übereinstimmung mit den experimentellen in vitro und in vivo Daten als Reaktionsmechanismus die Dimer-Hypothese der Arbeitsgruppe um Alan Brash vorgeschlagen. In grünen Pflanzenteilen verläuft die Biosynthese demzufolge in drei Schritten ab: Im ersten Schritt entsteht ein „Pool“ von oxidierten Galactolipiden mit Hydroperoxid-Acylketten (mit konjugierten Dienen). Diese Hydroperoxide entstehen fortlaufend durch Oxidation der Fettsäureacyle mittels Singulett Sauerstoff in Plastiden. Nach Infektion mit dem Pathogen P. syringae (avirulenter Stamm) wird der „Pool“ von Galactolipidperoxiden durch die katalytische Einwirkung von freien Radikalen und der LOX2 erhöht. Im zweiten Schritt findet eine Radikal-katalysierte Addition von Peroxylradikalen an Fettsäurehydroperoxide statt, wobei Lipid-Peroxid-Dimere gebildet werden. Diese instabilen Zwischenprodukte zerfallen spontan in vier Produkte, darunter zwei Aldehyd-Fragmente, ein Alkoxyradikal und ein Hydroxylradikal. Bemerkenswert ist, dass durch die Fragmentierung des Dimers weitere Radikale de novo entstehen. Im dritten Schritt können die in Galactolipiden veresterten Oxocarbonsäuren zu Dicarbonsäuren oxidiert werden. Hydroperoxide, die Vorläufer der Fettsäurefragmente, wurden in freier Form und in komplexen Lipiden verestert analysiert. Unter basalen Bedingungen liegt sowohl bei den freien, als auch bei den veresterten Hydroxyfettsäuren ein fast komplett Singulett Sauerstoff abhängiger Oxidationsmechanismus vor. Drei Galactolipid Hauptspezies (Monogalactosyldiacylglycerol (MGDG)-18:3-16:3, Digalactosyldiacylglycerol (DGDG)-18:3-18:3 und DGDG-18:3-16:3) sind hoch oxidiert (5 bis 9 Mol-%, relativ zur jeweiligen Vorstufe). MGDG-18:3-18:3, ebenso wie Phosphatidylglycerol-, Phosphatidylinositol- und Triacylglycerol-Hydroxyfettsäurespezies liegen basal nur schwach oxidiert vor (< 2 Mol-%). Nach Infektion mit dem Pathogen P. syringae kommt es zu einer massiven Lipid Biosynthese und Oxidation durch die 13-Lipoxygenase LOX2, Singulett Sauerstoff und freie Radikale. Der Oxidationsgrad der Hydroxyfettsäuren in den Galactolipiden ändert sich kaum. Innerhalb der Triacylglycerole kommt es zu einem großen Anstieg der oxidierten Spezies (auf 12 bis 38 Mol-%). Die Oxidation und Fragmentierung der Fettsäuren in den Galactolipiden unter basalen Bedingungen und induziert durch die Pathogenbehandlung, stellen einen wichtigen biochemischen Prozess dar, auf dem PIM und AZA entstehen. / The biosynthesis of fragmented fatty acids (short chain bicarboxylic acids and their precursor oxoacids) has not been established in plants. Important and common bicarboxylic fatty acids are pimelic acid and azelaic acid (PIM and AZA) and their putative precursors 7-oxoheptanoic acid (OHA) and 9-oxononanoic acid (ONA). Interest in the biogenetic origin of these unusual fatty acids stems from the fact that these fragmented fatty acids are involved in important biological processes. PIM is an established precursor of biotin in bacteria, fungi and plants. PIM biosynthesis has only recently been clarified in bacteria (E. coli and B. subtilis) and there is yet no evidence that an analogous pathway is operative in plants. Moreover, AZA has been identified as a pathogen-induced metabolite in Arabidopsis vascular sap that has been reported to prime local and systemic resistance against the pathogen Pseudomonas syringae. In animals, non-enzymatic fragmentation of fatty acid hydroperoxides is known to occur in complex membrane lipids during oxidative stress. ONA and AZA comprising phospholipids serve as endogenous pattern recognition ligands in the innate immune system of animals. The aim of this work was to clarify the mechanisms of fatty acid oxidation and fragmentation as well as the function of the oxidized fragments in the immune response of the model plant Arabidopsis thaliana. Within this work, fragmented fatty acids have been identified in complex lipids of plants for the first time and different hypotheses for the biosynthesis of fragmented fatty acids were examined. It could be shown that dienoic or trienoic fatty acids are precursors of fatty acid fragments in Arabidopsis thaliana. 9- and 13-lipoxygenases (LOX1, LOX5 and LOX2) are not essential for biosynthesis. In Arabidopsis fragmented fatty acids could be identified in free form and esterified in complex lipids, exclusively galactolipids. Application experiments revealed that biosynthesis of fatty acid fragments takes place in galactolipids in situ in a non-enzymatic way. Interpretation of in vitro and in vivo results lets suggest a reaction mechanism due to dimer hypothesis from Alan Brashs working group. The fragmentation process in green organelles was found to proceed through three steps. An initial and essential event is the formation of a pool of galactolipids comprising acyl hydroperoxides with conjugated dienes. These hydroperoxides are generated continuously in plastids through oxidation of fatty acyls by singlet oxygen. After infection with the avirulent strain of Pseudomonas syringae pathovar tomato the pool of galactolipid-peroxides is increased by catalytic impact of free radicals and lipoxygenases. In a second step, peroxy radical addition to fatty acid hydroperoxides results in dimerization of peroxides thereby forming an unstable intermediate that spontaneously decomposes to yield two aldehyde fragments, an alkoxy radical and a hydroxy radical. Notably, decomposition of the dimer leads to a de novo production of radicals. In a third step oxoacids esterified in galactolipids could be oxidized into bicarboxylic acids in a radical catalyzed process. Hydroperoxides, precursors of fatty acid fragments, were analyzed reduced to hydroxy fatty acids, in free form and esterified in complex lipids. Under basal conditions, some complex lipids were found to be highly and almost exclusively oxidized by singlet oxygen. Three galactolipid species (monogalactosyldiacylglycerole (MGDG)-18:3-16:3, digalactosyldiacylglycerole (DGDG)-18:3-18:3 and DGDG-18:3-16:3) showed highest level of oxidation with 5 to 9 mol-% (relative to their precursors). MGDG-18:3-18:3, as well as phosphatidylglycerol, phosphatidylinositol and triacylglycerol species are only slightly oxidized (< 2 mol-%). After P. syringae infection, massive lipid biosynthesis and oxidation by 13-lipoxygenase LOX2, singlet oxygen and free radicals was observed. Degree of oxidation in galactolipids was almost the same but hydroxylated triacylglycerol species showed a strong increase (up to 12 until 38 mol-%). Basal and pathogen-induced fatty acyl oxidation and fragmentation in galactolipids might be an important and general biochemical process yielding the essential biotin precursor PIM and AZA, which has previously been shown to prime the local and systemic immune response of A. thaliana.