Molts bacteris del grup fluorescent del gènere Pseudomonas són capaços de controlar malalties de les plantes causades per fongs i bacteris fitopatògens (ACBs) o mostren activitat com a bacteris promotors del creixement de les plantes (BPCPs). S'han descrit diversos metabòlits que intervenen de manera important en la seva activitat com a ACBs i BPCPs entre els quals en destaquen el 2,4-diacetilfloroglucinol (Phl), àcid fenazin-1-carboxílic (PCA), Pirrolnitrina (Prn), àcid cianhídric (HCN), àcid 3-indolacètic (IAA), sideròfors i quitinases.L'objectiu principal del nostre treball ha estat la comparació de les característiques d'un grup de Pseudomonas del grup fluorescent utilitzant una aproximació polifàsica amb la finalitat d'establir possibles relacions entre algunes de les característiques i la capacitat d'actuar com a ACB o BPCP.Atesa la importància en el biocontrol de la producció de metabòlits com Phl, PCA i Prn, l'objectiu preliminar ha estat la recerca i obtenció de soques productores d'aquests metabòlits. Per assolir aquest objectiu s'ha emprat una aproximació molecular basada en la detecció dels gens biosintètics implicats en la seva producció en lloc de la detecció directa dels metabòlits per evitar els efectes que poden tenir les condicions de cultiu en la inducció o repressió de la seva síntesi. S'han realitzat diferents protocols basats (i) en la cerca assistida de productors mitjançant l'ús de marcadors fenotípics i posterior confirmació per PCR i, (ii) en l'ús de la PCR per a la detecció dels gens directament dels extractes bacterians, d'enriquiments d'aquests extractes i la realització de la hibridació en colònies per al posterior aïllament. La cerca assistida de productors de Phl mitjançant marcadors fenotípics i posteriorment la utilització de tècniques moleculars (amplificació per PCR del gen phlD), ha estat el millor mètode en el tipus de mostres processades en el nostre treball, on la proporció de productors és relativament baixa. En total s'han aïllat a partir de diversos ambients 4 soques portadores dels gens de la síntesi de PCA, 15 de Phl i 1 de Prn.S'ha constituït una col·lecció de 72 soques de Pseudomonas del grup fluorescent que inclou 18 aïllats propis portadors dels gens biosintètics necessaris per la producció de Phl PCA i Prn; 6 soques de referència procedents de col·leccions de cultius tipus, 14 soques productores dels diferents antibiòtics cedides per altres investigadors i una selecció de 34 soques procedents d'un treball previ realitzat en el nostre grup de recerca. A la col·lecció s'hi troben soques candidates a ACB i BPCP de diverses malalties i plantes.Les 72 soques s'han caracteritzat fenotípica i genotípicament. La caracterització fenotípica s'ha portat a terme mitjançant la identificació a nivell d'espècie amb galeries API 20NE i proves bioquímiques específiques; la producció de metabòlits com PCA, Phl, Prn, IAA, HCN, quitinases i sideròfors mitjançant l'ús de diferents tècniques; antagonisme in vitro en diversos medis enfront dos fongs (Stemphylium vesicarium i Penicillium expansum) i tres bacteris fitopatògens (Erwinia amylovora, Pseudomonas syringae pv. syringae i Xanthomonas arboricola pv. juglandis); l'eficàcia de la inhibició de la infecció en bioassaigs in vivo sobre material vegetal enfront els fongs P. expansum en poma i S. vesicarium en fulles de perera i enfront el bacteri E. amylovora en fruits immadurs de perera i, finalment, en assaigs de promoció de creixement en dos portaempelts comercials de Prunus. Cal destacar que P. expansum causa la podridura blava en pomes i peres en postcollita, S. vesicarium la taca bruna de la perera i E. amylovora el foc bacterià de les rosàcies.El nombre de soques de Pseudomonas, sobre el total de les 72 estudiades, productores d'IAA (4) i quitinases (6) és baix, mentre que és elevat en el cas del HCN (32), que a més està associat a la producció de Phl. Els resultats obtinguts en l'antagonisme in vitro han mostrat en el cas dels bacteris que és dependent del patogen indicador i del medi de cultiu. La presència o absència de ferro no sembla ser un factor que potencií l'antagonisme. En el cas dels fongs no s'ha observat però, influència del medi de cultiu emprat. En el total de 72 soques s'ha observat un percentatge baix de soques que manifesten antagonisme en tots els medis assajats vers 3 o 4 dels patògens (7). Solament 2 d'aquestes 7 soques han mostrat ser també efectives en bioassaigs d'inhibició de les infeccions causades per 2 dels 3 patògens assajats. Algunes de les soques efectives en els bioassaigs no són antagonistes in vitro en cap dels medis assajats enfront el mateix patogen. En el cas de la promoció del creixement, s'han observat més soques promotores del creixement del portaempelts de prunera Marianna 2624 que no en l'híbrid de presseguer-ametller GF677 i les eficàcies assolides són també majors en el cas de Marianna 2624, detectant una elevada especificitat soca/portaempeltsLa caracterització genotípica s'ha realitzat mitjançant l'anàlisi dels polimorfismes en la longitud dels fragments de restricció de DNA ribosomal (RFLP-rDNA) i l'anàlisi dels polimorfismes en la longitud dels fragments de macrorestricció genòmica de DNA cromosòmic separats per electroforesi en camp polsant (MRFLP-PFGE). Ambdues anàlisis van mostrar una gran heterogeneïtat genètica entre les soques caracteritzades i no s'ha pogut relacionar les agrupacions obtingudes amb les característiques fenotípiques o capacitat d'actuar com a ACB o BPCP. Els patrons de macrorestricció genòmica (MRFLP-PFGE) del bacteri model P. fluorescens EPS288 són estables en el temps i independents de les condicions de cultiu assajades al laboratori o en mostres naturals, mostrant ser una tècnica eficaç en la identificació de reaïllats de mostres naturals inoculades prèviament amb el bacteri.Una selecció de soques que comparteixen el fet de produir floroglucinol s'han caracteritzat mitjançant RFLP i seqüenciació del gen phlD. S'ha establert una relació entre les agrupacions obtingudes en les anàlisis RFLP-rDNA, RFLP-phlD i les seqüències del gen. En l'anàlisi filogenètica de les seqüències del gen phlD s'ha observat un elevat grau de polimorfisme obtenint-se 3 agrupacions principals. Les agrupacions semblen relacionar-se amb els patrons de producció de metabòlits (Phl, HCN i Prn en una primera agrupació; Phl i HCN en la segona i solament Phl en la tercera), però aquestes no s'han pogut relacionar amb l'origen geogràfic de les soques o la seva activitat com a ACBs i/o BPCP.Amb les dades obtingudes de la caracterització fenotípica i genotípica s'ha realitzat una anàlisi multivariant (correspondències, correlacions d'Spearman i de freqüències amb variables categòriques). S'ha demostrat la importància de disposar d'una tècnica que permeti depurar una col·lecció de soques descartant les soques genèticament idèntiques, ja que influeixen en els resultats de les anàlisis. Pels tres patògens assajats com a indicadors i els dos portaempelts emprats, no s'ha observat cap correlació entre la inhibició de la infecció o la promoció del creixement amb les característiques fenotípiques i genotípiques de les soques que fos significatiu i consistent en les tres tècniques emprades. / Many bacteria pertaining to the fluorescent Pseudomonas group are able to control plant diseases caused by bacterial and fungal plant pathogens (BCAs). Also, some of them exhibit activity as plant growth promoting bacteria (PGPBs). Several metabolites like 2,4-diacetylphloroglucinol (PHL), phenazin-1-carboxilic acid (PCA), pyrrolnitrin (Prn), hydrogen cyanide (HCN), indol-3-acetic acid, siderophores and chitinases has been described accounting for their ability of being BCAs or PBPBs.The aim of the present work was to compare the phenotypic and genotypic characteristics of a selected group of fluorescent Pseudomonas by means of a polyphasic approach in order to establish relationships with the ability of being BCA and PGPB.Due to the importance of production of metabolites like Phl, PCA and Prn in biocontrol, the preliminary objective of this work was to search and isolate of metabolite producing strains. To achieve this objective a molecular approach was used based on the detection of biosynthetic genes, which are implicated in the metabolite production, instead of direct detection of the metabolites. The procedure was performed to avoid the effect of culture conditions on induction or repression of metabolite synthesis. Two procedures were used (i) assisted search of metabolite producing strains by means of phenotypic markers and subsequent confirmation by PCR analysis, (2) direct use of PCR for gene detection in direct extracts or enrichments from samples and subsequent colony-hybridization for assistance in strain isolation. The best method for isolation of Phl producers in the type of samples processed in the present work, where the frequency of Phl producers is very low, was the assisted search by means of phenotypic markers followed of a confirmation by PCR amplification of phlD gene. Four strains harboring the PCA genes, 15 strains with Phl genes, and one with Prn genes were isolated.A collection of 72 strains of Pseudomonas pertaining to the fluorescent group was made including 18 isolates derived from the present work which harbor the biosynthetic genes for PhL, PCA and Prn production; 6 strains from reference collections; 14 strains producing several antibiotics which were supplied by other colleagues; and a selection of 34 strains from a previous work performed by our research group. Within the collection there are many strains with promising potential as BCAs and PGPBs of several diseases and plant hosts.The collection was characterized phenotypically and genotypically. The phenotypic characterization included identification at the species level with the aid of API20NE kit and several specific biochemical tests; the production of metabolites such as PCA, Phl, Prn, IAA, HCN, chitinases and siderophores; in vitro antagonism in several culture media against two fungi (Stemphylium vesicarium and Penicillium expansum) and three bacterial pathogens (Erwinia amylovora, Pseudomonas syringae pv. syringae and Xanthomonas arboricola pv. juglandis); efficacy in biossays of inhibition of infection of plant material by P. expansum on apple fruit tissue and S. vesicarium on pear leaves, E. amylovora on immature pear fruit, and of growth promotion in two commercial Prunus rootstocks. P. expansum causes blue mold rot of apple and pear in postharvest, S. vesicarium cause brown spot of pear and E. amylovora is the causal agent of fire blight of rosaceous plants.The number of strains, over a total of 72 studied, producing IAA and chitinases was very low (4 producing IAA and 6 producing chitinases), whereas a high number of HCN producing strains (32) was detected which was associated to Phl production. In vitro antagonism against bacteria was highly dependent on indicator pathogen and growth medium, but the presence or absence of iron did not seem to affect. However, in the case of antagonism against fungi, no influence of the medium composition was observed. Among the collection a low frequency of strains exhibiting antagonism against 3 or 4 pathogens a total of 7 strains was observed in all tested media. Only two out of the seven strains were effective in infection inhibition bioassays caused by two of the three pathogens tested. Some of the effective strains in the in vivo assays were not antagonist for the indicator pathogen in any of the media tested. In the case of plant growth promoting strains, the growth in rootstock Marianna 2624 was more stimulated than in GF677, and there was a strain-host specificity. Genotypic characterization of strains was performed by means of restriction fragment length polymorphism analysis of the ribosomal DNA (RFLP-rDNA) and macrorestriction fragment length polymorphism analysis of chromosomal DNA separated by pulsed field gel electrophoresis (MRFLP-PFGE). Both techniques showed a high level of genotypic diversity within the collection of strains, and no relationships were observed between clusters and phenotypic characteristics or ability to be BCA or PGPB. Patterns of MRFLP-PFGE for the model bacterium P. fluorescens EPS288 were found stable within time and independent of cultivation conditions in the laboratory or under natural conditions. Therefore the method is highly suitable for identification of strains reisolated from natural samples previously inoculated with the bacterium.A further selection of strains which produce phloroglucinol were characterized by RFLP analysis and phlD sequencing. The groups observed were consistent among the RFLP-rDNA, RFLP-phlD and gene sequence data. Phylogenetic analysis obtained using phlD sequences showed a high level of polymorphism revealing three main clusters. These clusters appear to be related with metabolite production: a first cluster producing Phl, HCN and Prn, a second producing Phl and HCN, and a third producing only Phl. However, this groups were related neither to the geographic origin of strains nor to the activity as BCA or PGPB.A multivariate analysis (correspondence, pairwise Spearman rank correlation, and frequency analysis)was performed using data of phenotypic and genotypic characterization of strains. The results emphasize the importance of discarding from the database those strains being genetically identical because skew the final results. The three types of analysis did not revealed a significant and consistent relationship between the activity of infection inhibition or growth promotion of the strains and their characteristics.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-09-06T20:24:13Z No. of bitstreams: 1 ThiagoAndreCidral_DISSERT.pdf: 3578139 bytes, checksum: bcb8c85feab01e028e946691a0776a04 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-09-06T23:31:16Z (GMT) No. of bitstreams: 1 ThiagoAndreCidral_DISSERT.pdf: 3578139 bytes, checksum: bcb8c85feab01e028e946691a0776a04 (MD5) / Made available in DSpace on 2016-09-06T23:31:16Z (GMT). No. of bitstreams: 1 ThiagoAndreCidral_DISSERT.pdf: 3578139 bytes, checksum: bcb8c85feab01e028e946691a0776a04 (MD5) Previous issue date: 2016-01-15 / Os Estafilococos Coagulase Negativos (ECN) s?o microrganismos pertencentes ? microbiota normal da pele e de mucosas dos seres humanos e de animais. A maioria das infec??es causadas por ECN est?o relacionadas ao uso de dispositivos m?dicos invasivos que ao lesionar a integridade da pele servem de base para a forma??o de biofilmes, um importante fator de virul?ncia. Grande parte dos isolados de coagulase negativo s?o provenientes de hemoculturas e pontas de cateter e nos ?ltimos anos vem se tornando um grave problema no que diz respeito ? antibioticoterapia, em virtude do n?mero elevado de cepas multirresistentes descritas. O objetivo deste trabalho foi pesquisar resist?ncia ? linezolida em estafilococos coagulase negativos resistentes ? meticilina isolados de ponta de cateter e hemocultura de hospitais p?blicos e privados da cidade do Natal. Os isolados bacterianos foram coletados a partir de demanda espont?nea em Hospitais P?blicos e Privados. O g?nero Staphylococcus foi confirmado atrav?s dos testes de rotina como colora??o de Gram, prova da catalase da coagulase livre. A identifica??o a n?vel de esp?cie foi realizada atrav?s de testes bioqu?micos convencionais. Algumas amostras tiveram sua identifica??o confirmada pelos sistemas VITEK 2 e MALDI-TOF. O perfil de resist?ncia aos antimicrobianos foi avaliado atrav?s da t?cnica de disco-difus?o (CLSI 2013). A Concentra??o Inibit?ria M?nima para vancomicina e linezolida foi determinada atrav?s do uso de E-test e a presen?a dos genes mecA e cfr foi confirmada pela t?cnica da Rea??o em Cadeia da Polimerase. Algumas amostras tiveram a regi?o V da subunidade 23S do gene do rRNA sequenciadas e analisadas. Posteriormente, as mesmas foram submetidas a t?cnica do PFGE para determina??o do seu pulsotipo. Dos 43 estafilococos coagulase negativos resistentes ? oxacilina inclu?dos neste estudo, 33 (77%) foram identificados como S. epidermidis, 6 (14%) como S. haemolyticus, 3 (7%) como S. homins e 1 (2%) como S. capitis. Os isolados de hemocultura representaram 86% (37) e os de ponta de cateter 14% (6). As amostras apresentaram um perfil de multirresist?ncia, uma vez que 42 dos 43 isolados apresentaram resist?ncia ? 4 ou mais classes de drogas. Todas apresentaram o gene mecA. Nenhuma amostra apresentou resist?ncia ? vancomicina. Tr?s cepas de S. hominis e duas de S. epidermidis, apresentaram resist?ncia ? linezolida com CIM variando entre 6 e 64 ?L/mL. Quando investigadas, apresentaram duas muta??es pontuais (C2190T e G2603T) na regi?o V do gene para rRNA 23S. Nenhuma destas apresentou o gene cfr. O PFGE dos S. hominis revelou a presen?a de um ?nico pulsotipo em 3 hospitais, enquanto n?o foi encontrado semelhan?a gen?tica entre os S. epidermidis. Estes achados destacam a import?ncia da vigil?ncia continuada em rela??o a resist?ncia a linezolida no g?nero Staphylococcus. / Coagulase Negative Staphylococci (CoNS) belong to the normal microbiota of the skin and mucous membranes of humans and animals. The most of the infections caused by CoNS are a serious problem, since an elevated number of multi-drug resistant strains. The objective of this study was to investigate resistance to linezolid in methicillin-resistant coagulase negative staphylococci isolates from hospitals in the city of Natal. Bacterial samples were collected from spontaneous demand from Public and Private Hospitals of the city of Natal-RN. The identification staphylococci of the species were conducted by conventional biochemical. Some Samples had the identification to the species level confirmed by automated methodologies VITEK 2? and VITEK MS?. The resistance profile was evaluated with use of the disk diffusion technique (CLSI, 2013). The MIC to vancomycin and linezolid were determined by using E-test method. The antimicrobial resistance profile was evaluated by disk diffusion technique (CLSI 2013). The Minimum Inhibitory Concentration linezolid and vancomycin were determined by using E-test and the presence of the mecA gene and cfr was confirmed by the technique of Polymerase Chain Reaction. Some samples had the V region of the subunit 23S rRNA gene sequenced and subjected to PFGE technique for determining its pulsotype. Of the 43 coagulase negative staphylococci resistant to methicillin included in this study, 33 (77%) were identified as S. epidermidis, 6 (14%) as S. haemolyticus, 3 (7%) as S. homins and 1 (2%) as S. capitis. The catheter tip isolates accounted for 14% (6) and the blood culture 86% (37). Samples showed an alarming resistance profile, since 98% of the isolates were resistant to four or more class drugs. All were positive for mecA gene. No samples were resistant to vancomycin. Three S. hominis and two S. epidermidis exhibited linezolid resistance with MIC ranging from 6 to 64 ?L/mL. None of the samples had the cfr gene. When investigated, they showed two point mutations each (C2190T and G2603T) in the V region of the 23s rRNA gene. None of them was the cfr gene. The S.hominis of PFGE showed the presence of a single pulsotype in three hospitals, suggesting a clonal spread, while it was not found genetic similarity among S. epidermidis. These findings highlight the importance of continued vigilance of linezolid resistance in the genus Staphylococcus.

The aim of this master’s thesis was focused on characterization of carotenogenic yeast using molecular techniques. For this usage, interspecific variables of strongly conserved sequences of genomic DNA, especially rDNA D1/D2 large ribosomal subunit and ITS1 and 5,8-ITS2 rDNA regions were amplified. These sequences were subjected analysed by DGGE method, which approved differences of S. roseus in all analyzed rDNA sequencies compared to the other analyzed carotenogenic yeasts. Parameters of PFGE and isolation procedure of the intact DNA were optimized for caryotypic yeast characterization. At all, nine of carotenogenic yeast strains Rhodotorula, Sporobolomy-ces, Cystofilobasidium a Phaffia were analyzed by this techniques. Further part of this thesis was focused to application of molecular methods to analysis of region D1/D2 of large ribosomal subunit in mutant carotenogenic yeast strains. Mutant strains were pre-viously adapted to waste substrates - pasta and glycerol, and stability of their production properties was verified.

Bacillary Necrosis of Pangasius (BNP) caused by Edwardsiella ictaluri is considered to be the most serious disease occurring in farmed striped catfish (Pangasianodon hypophthalmus) in Vietnam. This disease has had an increasing impact over the last ten years and has been reported to cause 50-90% mortality of stocks during a single outbreak. Data obtained from natural outbreaks of E. ictaluri in striped catfish showed the role of environmental factors in the establishment and progression of this disease. At present, factors affecting the virulence and transmission of E. ictaluri in striped catfish are poorly understood. The central hypothesis of this thesis focuses on the complex picture of the environmental factors and infectivity of E. ictaluri in striped catfish. In this study, 80 isolates of E. ictaluri recovered from natural clinical disease outbreaks occurring in striped catfish farms between 2002 and 2011 located in 4 distinct geographical areas within Vietnam were characterised using a variety of methods. The biochemical profiles showed that E. ictaluri isolates from striped catfish in Vietnam have similar phenotypic characteristics to other E. ictaluri isolates from other infected fish species. These data showed high levels of phenotypic homogeneity between the E. ictaluri isolates investigated. The status of isolates recovered from natural infections over time and from geographically distinct farms was evaluated using pulsed-field gel electrophoresis (PFGE), plasmid profile identification and antibiotic sensitivity tests. The PFGE results showed 6 main groups with a similarity of 82% and the corresponding genotypes of the prevalent isolates illustrated annual differences. Three plasmid groups were identified distributed among the isolates investigated, in which high molecular weight plasmids of approximately 35 and 140 kb were found in two of the groups. Plasmid profiles of the present study did not show any trend of geographical region or year of isolation. The 140 kb plasmid has been considered as a multi-antibiotic resistance plasmid which confers resistance to tetracycline, trimethoprim and sulphonamides. All Vietnamese isolates showed a high level of resistance to Oxolinic acid, Sulfadimethoxine/Ormetoprim (Romet), Oxytetracycline and Amoxicillin. A reproducible bacterial immersion challenge model was developed and the LD60 estimated prior to performing subsequent experimental challenge studies. Fish were exposed to 107 cfu ml-1 of E. ictaluri by immersion for up to 30 seconds, resulting in a cumulative percentage mortality of 63%. Edwardsiella ictaluri was recovered and identified from all the dead and moribund fish during these experiments and affected fish showed similar clinical signs and pathology to those reported from natural E. ictaluri infections. The present study resulted in a successful experimental immersion challenge model for E. ictaluri infection in healthy striped catfish. Cohabitation challenges were also developed and produced 15-40% mortality, typical clinical signs and pathology, and successful recovery of the challenge organism demonstrating horizontal transmission of E. ictaluri in striped catfish. Experimental studies were then conducted to investigate the association between pH or salinity of water and susceptibility to E. ictaluri infection in striped catfish. The first experiments were performed in in vitro conditions in which E. ictaluri isolates were cultured in a variety of pH and salt concentrations. In vivo experiments were then designed where striped catfish were exposed to 107 cfu ml-1 of E. ictaluri for 30 seconds and then held at 4 different water pHs (5.5, 6.5, 7.5 and 8.5) or NaCl concentrations (0, 0.5, 1 and 1.5%). The results of in vitro experiments showed that a pH value between 5.5 to 6.5 and salt concentration between 0-0.5% were optimal for the growth of E. ictaluri. The in vivo experiments demonstrated that the cumulative mortality of striped catfish in water at pH 5 and pH 6 was significantly higher than that of fish maintained in more alkaline water (p<0.05). By contrast, the cumulative mortality of the striped catfish maintained in 0.5% salt concentration was significantly lower than those kept in 0%, 1% and 1.5% salt concentration (p<0.05). Clinical signs, lesions and histopathological changes in the affected fish were consistent with those reported in natural infections. This study highlighted the use of pH 8.5 and salinity of 0.5% NaCl as a means of decreasing the susceptibility of striped catfish to E. ictaluri. In conclusion, this study used a variety of methods in order to enhance the understanding of the biochemical, biophysical characteristics, plasmid profile and antibiotic resistance as well as the relatedness of E. ictaluri isolates recovered from farmed striped catfish in Vietnam. This study provided two reliable and reproducible bacterial challenge models (immersion and cohabitation) and emphasised the link between pH and salinity with the infectivity and pathogenicity of E. ictaluri in striped catfish.

<p>The first hospital outbreak of vancomycin-resistant enterococci (VRE) and carriage rates of VRE and ampicillin-resistant enterococci (ARE) in Sweden were investigated. Clonal relationships and mutations in fluoroquinolone resistance determining regions among ARE collected nation-wide were studied. Risk factors for ARE infection, shedding of ARE and the presence of the virulence gene <i>esp</i> in ARE isolates and patients on a hematology unit and other units at Uppsala University Hospital were further investigated. </p><p>The first Swedish hospital VRE outbreak was due to clonal spread of <i>E. faecium, vanA</i>. The nation wide carriage rates of ARE and VRE were 21.5% / 1% and 6% / 0%, among hospitalized patients and non-hospitalized individuals respectively. All ARE and VRE were <i>E. faecium</i> and >90% resistant to ciprofloxacin. All VRE carried<i> vanB</i>. Carriage of ARE was independently associated with >5 days of antibiotic treatment. Phenotypic and genetic typing showed a significantly higher homogeneity among ARE compared to matched ASE <i>E. faecium</i> isolates. Mutations conferring high-level ciprofloxacin resistance were found only in ARE. Risk factors for ARE infection included long duration of hospital stay and exposure to antibiotics. Skin carriage was associated with ARE shedding. ARE bacteremia was independently associated with prior ARE colonization and hematopoietic stem cell transplantation. Death was more common in ARE septicemia cases compared to controls. <i>Esp</i> was significantly more common in ARE surveillance compared to ARE blood isolates from patients on the hematology ward.</p><p>In conclusion, VRE were rare but clonally related multi-resistant ARE <i>E. faecium</i> were highly prevalent in Swedish hospitals. Spread of ARE in hospitals during the 1990s is suggested to be the main explanation for the emergence of ARE in Sweden. Spread was facilitated by use of antibiotics and probably by the presence of virulence genes in<i> E. faecium</i> isolates.</p>

The first hospital outbreak of vancomycin-resistant enterococci (VRE) and carriage rates of VRE and ampicillin-resistant enterococci (ARE) in Sweden were investigated. Clonal relationships and mutations in fluoroquinolone resistance determining regions among ARE collected nation-wide were studied. Risk factors for ARE infection, shedding of ARE and the presence of the virulence gene esp in ARE isolates and patients on a hematology unit and other units at Uppsala University Hospital were further investigated. The first Swedish hospital VRE outbreak was due to clonal spread of E. faecium, vanA. The nation wide carriage rates of ARE and VRE were 21.5% / 1% and 6% / 0%, among hospitalized patients and non-hospitalized individuals respectively. All ARE and VRE were E. faecium and &gt;90% resistant to ciprofloxacin. All VRE carried vanB. Carriage of ARE was independently associated with &gt;5 days of antibiotic treatment. Phenotypic and genetic typing showed a significantly higher homogeneity among ARE compared to matched ASE E. faecium isolates. Mutations conferring high-level ciprofloxacin resistance were found only in ARE. Risk factors for ARE infection included long duration of hospital stay and exposure to antibiotics. Skin carriage was associated with ARE shedding. ARE bacteremia was independently associated with prior ARE colonization and hematopoietic stem cell transplantation. Death was more common in ARE septicemia cases compared to controls. Esp was significantly more common in ARE surveillance compared to ARE blood isolates from patients on the hematology ward. In conclusion, VRE were rare but clonally related multi-resistant ARE E. faecium were highly prevalent in Swedish hospitals. Spread of ARE in hospitals during the 1990s is suggested to be the main explanation for the emergence of ARE in Sweden. Spread was facilitated by use of antibiotics and probably by the presence of virulence genes in E. faecium isolates.

Στην παρούσα μελέτη η βιοτυπία και η ανάλυση του DNA με ηλεκτρο-φόρηση σε παλλόμενο ηλεκτρικό πεδίο (PFGE) εφαρμόσθηκαν σε ένα σύνο-λο εντε-ρο-κόκ-κων που απομονώθηκαν από νερά αναψυχής και πόσιμα νερά, προκει-μένου να προσδιορισθεί πιθανή γενετική συγγένεια. Τα 200 στελέχη εντε-ρο--κόκκου απομονώθηκαν από 246 δείγματα νερών αναψυχής και 903 δείγματα πόσιμου νερού, από θάλασσα, νερό δικτύου, ποταμούς και πηγές της Ν.Δ. Ελλάδας. Η ταυτοποίηση σε επίπεδο είδους έγινε με το σύστημα API 20strep. Ο έλεγχος της ευαισθησίας στα αντιβιοτικά έγινε με προσδιορισμό της ελάχιστης ανασταλτικής πυκνότητας (MIC) με την μέθοδο ταινιών E-test. Η ηλεκτροφό-ρηση σε παλλόμενο ηλεκτρικό πεδίο εφαρμόσθηκε για τις γονοτυπικές δοκιμασίες. Ο χαρακτηρισμός σε επίπεδο είδους ταξινόμησε τους εντερόκοκκους σε 22 βιότυπους. Στην πλειοψηφία τους τα στελέχη ήσαν E. faecium (142 ή 71%), ακολουθούσε ο E. faecalis (40 ή 20%), o E. durans (9 ή 4,5%), o E. gallinarum (5 ή 2,5%) και o E. avium (4 ή 2%). Μεταξύ των στελεχών του E. faecium υπήρ-χε σχέση των βιοτύπων και της πηγής του δείγματος. Αυτό δεν παρατηρή-θη-κε στα στελέχη του E. faecalis ούτε στα υπόλοιπα είδη. Κανένα στέλεχος δεν βρέθηκε να παράγει β-λακταμάση. Δεν παρατηρήθηκε καμία σχέση μεταξύ της αντοχής στα αντιβιοτικά και της προέλευσης των στελεχών. Κανένα είδος εντεροκόκκου δεν παρουσίασε αντοχή στα γλυκοπεπτίδια. Κλινικά στελέχη E. faecium περιελήφθησαν στα γονοτυπικά πειράματα ως μάρτυρες για τους ήδη ταυτοποιημένους κλώνους που ενδημούν στο Πανεπιστημιακό Νοσοκομείο της Πάτρας. Η ανάλυση των τύπων PFGE μεταξύ των 104 στελεχών αποκάλυψε την παρουσία 18 κλώνων μεταξύ του E. faecium, 14 του E. faecalis, 2 του E. durans, 3 του E. gallinarum και ενός του E. avium. Αν και μεταξύ των εξετασθέ-ντων στελεχών παρατηρήθηκε γενετική ποικιλία, προσδιορίσθηκαν κοινοί κλώ-νοι μεταξύ διαφορετικών δειγμάτων νερών. Τα δενδρογράμματα αποκά-λυ-ψαν γενετική σχέση μεταξύ συγκεκριμέ-νων περιβαλλοντικών στελεχών του E. faecium και αυτών που ήσαν νοσοκομειακής προέλευσης. Για τη μελέτη της παρουσίας κλώνων μεταξύ εντεροκόκκων που απομονώνονται από το περιβάλλον πρέπει να εφαρμόζονται μέθοδοι ανάλυσης χρωμοσωμικού DNA. / In this study we have identified the enterococcal species isolated from different environmental sources and we have characterized their biotypes, antibiotic resistance patterns and PFGE types. Biotyping and DNA fingerprinting by pulsed-field gel electrophoresis was applied to a collection of enterococci recovered form recreational and drinking water, in order to identify possible genetic relationships. Clinical strains of hospital origin were compared to the environmental isolates. A total of 200 enterococci were isolated from 246 recreational water, and 900 drinking water. One hundred forty two isolates were characterized as Enterococcus faecium recovered from all sources, 40 E. faecalis, 9 E. durans, 5. E. gallinarum and E. avium. Biotypes, determined with API 20 strep, among E. faecium were correlated with certain environmental sources, while antibiotypes, determined with Etest, did not reveal any relationship with the sample origin. Even though genetic diversity was observed among the studied strains, common clonal types were also identified in different sources, suggesting a possible common origin of the enterococci. Cluster analysis revealed a genetic relationship between certain environmental E. faecium and clinical strains.

Listeria monocytogenes (L. monocytogenes) est un pathogène majeur en santé publique comme les épisodes de 2008 dans les fromages et les charcuteries l’ont démontré. Au Canada, il n’y a pas de surveillance règlementaire de ce microorganisme dans les étapes précédant la transformation de produits prêts-à-manger. Ainsi, la présence et la circulation de ce microorganisme dans ces environnements est peu documentée. Pour décrire ces phénomènes, nous avons effectué un échantillonnage dans une usine d’abattage et de découpe de porcs au Québec, principalement dans les parcs d’attente, et dans l’environnement de l’abattage et de découpe : les échantillonages ont été effectués après lavage et désinfection sur une période de 2 ans. Un nombre de 874 échantillons a été récoltés. Le protocole de détection utilisé était inspiré de la méthode MFHPB-30 de Santé Canada. Les sérotypes ont été obtenus par PCR et les isolats caractérisés par un génotypage RFLP-PFGE en utilisant les enzymes de restriction Apa1 et Asc1. Nous avons détecté la présence de Listeria monocytogemes dans toutes ces étapes de la production. De ces échantillons positifs, 4 sérotypes (principalement 1/2b) ont émergé. Les patrons PFGE ont démontré la présence d’une variété de génotypes dans les zones d’attente et d’abattage de l’usine et la présence d’un type majeur dans l’environnement de la zone de découpe (le type 1 représentant 96.1% des souches à cette étape). De plus, nous avons démontré des liens entre les souches retrouvés au début de la production, en attente, et les souches retrouvées dans la zone de découpe. Ces résultats suggèrent que Listeria monocytogenes entre dans l’usine avec les animaux, contamine les étapes suivantes de la production et que certaines souches peuvent être sélectionnées et leur croissance favorisé dans l’environnement, devenant majoritaires, persistantes et préoccupantes en regars de la santé publique. / Listeria monocytogenes (L. monocytogenes) is a major public health concern as it was illustrated by the 2008 episodes in cheese and ready-to-eat meat. In Canada, there is currently no surveillance policy of this microorganism in the production steps preceding transformation of ready-to-eat products. The presence and the circulation of this microorganism in these environments is not well documented either. To describe these parameters, we sampled a Quebec slaughtering and cutting plant in the lairage pens and on representative areas of the slaughter process and cutting rooms after washing and disinfection, during a two-year period. A total of 874 samples were collected. Listeria detection followed the MFHPB-30 Health Canada standard method, serotype confirmation was obtained by PCR and isolates were characterized by Apa1 and Asc1 RFLP-PFGE genotyping. We reported detection of Listeria monocytogenes in all stages of production. Among the positive samples, 4 different serovars (mainly 1/2b) emerged. PFGE patterns showed presence of a variety of different genotypes in the lairage and slaughtering areas of the plant and the presence of a major type in the environment of the cutting room (type 1 representing 96.1% of the strains at this step). Furthermore, strains found at the lairage pens were related to strains in the cutting room. These results suggest that Listeria monocytogenes can enter the plant with the animals, contaminate further production steps and that some strains can be selected and their growth promoted in the environment. Hence, becoming predominant, persistant and a food safety issue.

El principal objetivo fue estimar la prevalencia y caracterizar las <i>E. coli</i> O157 de ganado vacuno faenado en Argentina. Se recolectaron un total de 1622 muestras de heces y carcasas en 9 frigoríficos de exportación. Todas las muestras se sometieron a separación inmunomagnética y las cepas fueron identificadas por PCR múltiple (rfb<SUB>O157</SUB>, stx<SUB>1</SUB>, stx<SUB>2</SUB>). Se aislaron 54 STEC O157 y 48 <i>E. coli</i> O157 toxina Shiga negativas (EC O157 TSN), de las que se establecieron sus características fenotípicas, genotípicas y las variantes stx. La prevalencia promedio de STEC O157 en materia fecal fue de 4,1% y 2,6% en carcasa; mientras que para EC O157 TSN la incidencia fue de 4,7 y 2,6%; respectivamente. No se observaron diferencias significativas por el género o raza de los animales. Los terneros y vaquillonas presentaron mayores porcentajes de prevalencia de STEC O157 en heces (10,5 y 8,5%, respectivamente). Todas las STEC O157 aisladas albergaban los genes stx<SUB>2</SUB>, eae, ehxA, y fliC<SUB>H7</SUB> y sólo el 16,7% presentó el gen stx<SUB>1</SUB>. El genotipo prevalente fue el stx<SUB>2</SUB>/stx<SUB>2c(vh-a)</SUB>, que también es frecuente en los casos de SUH. Mediante XbaI-PFGE se obtuvieron 29 patrones diferentes y 11 clusters. En cinco oportunidades, las cepas de STEC O157 aisladas de las carcasas fueron idénticas a las cepas de materia fecal. También 7 cepas idénticas se aislaron de carcasas muestreadas en dos visitas consecutivas a dos frigoríficos. Cinco perfiles de fago tipo-PFGE-stx fueron coincidentes con perfiles de cepas recuperadas de SUH. Las técnicas de subtipificación molecular mostraron que las cepas EC O157 TSN presentan un origen filogenético diferente a STEC O157. Se cuantificó la resistencia ácida (RA) por 3 mecanismos y se determinó que el sistema Glutamato-dependiente proporciona mejor protección en desafío ácido. Las EC O157 TSN fueron, en promedio, más resistentes que las cepas STEC O157 por los sistemas descarboxilasa dependientes.

Made available in DSpace on 2014-12-17T14:13:43Z (GMT). No. of bitstreams: 1 FranciscoCSJ.pdf: 1235219 bytes, checksum: 99fd9a9cd6382f5c2247c17ecd9cce0b (MD5) Previous issue date: 2009-10-09 / Staphylococcus aureus resistente ? meticilina (MRSA) ? um dos principais agentes de infec??es associadas a servi?os de sa?de em todo o mundo. No Brasil, h? a predomin?ncia de um clone de MRSA multirresistente denominando clone epid?mico brasileiro (CEB). Entretanto, novos clones n?omultirresistentes com alta virul?ncia t?m sido descritos em infec??es comunit?rias e hospitalares. O objetivo desse estudo foi realizar a caracteriza??o fenot?pica e genot?pica de cepas de MRSA isoladas na cidade do Natal/RN. Inicialmente avaliamos 60 amostras de S. aureus quanto a resist?ncia ? meticilina atrav?s de diferentes t?cnicas fenot?picas, utilizando a detec??o do gene mecA por PCR como padr?o. O antibiograma de todas as cepas foi realizado utilizando 12 antimicrobianos conforme descrito pelo CLSI. As cepas de MRSA foram caracterizadas geneticamente atrav?s da tipagem do cassete cromoss?mico estafiloc?cico mec (SCCmec) e da eletroforese em campo el?trico alternado (PFGE). Dos 60 S. aureus estudados, 45 foram resistentes ? meticilina. Observamos que para algumas cepas de MRSA os testes de triagem em ?gar com 6&#956;g/mL de oxacilina e difus?o em meio s?lido com oxacilina-1&#956;g apresentaram dificuldades na sua interpreta??o. No entanto, todas as 45 amostras de MRSA, foram facilmente detectadas pelos testes com o disco de cefoxitina-30&#956;g e pesquisa da PBP2a. A an?lise molecular das cepas de MRSA mostrou 8 padr?es distintos de PFGE (A-H), com predomin?ncia do padr?o A (73%), relacionado ao CEB. Estas carreavam o SCCmec tipo IIIA, e apresentaram uma consider?vel variedade de subtipos (A1-A16). Cinco cepas de MRSA portando SCCmec IV tamb?m foram xiv identificadas, tr?s delas relacionadas geneticamente ao clone USA800 (Padr?o B). Destas cinco, tr?s (2 padr?o F e 1 padr?o B) foram altamente suscept?veis as drogas testadas, entretanto, dois outros isolados, padr?o B, apresentaram multirresist?ncia. As amostras restantes pertenciam a padr?es de PFGE distintos dos clones internacionais predominantes em nosso continente. Para realiza??o deste projeto de pesquisa, a metodologia exigiu a intera??o com pesquisadores de ?reas como: infectologia, microbiologia e biologia molecular. Portanto, esta disserta??o apresentou um car?ter de multidisciplinaridade e transdiciplinaridade no seu desenvolvimento