Psoriasis vulgaris ist eine chronisch-entzündliche Hauterkrankung, welche mit verdickten, erythematös-schuppigen Plaques und systemischer Inflammation einhergeht. Bisher veröffentlichte epidemiologische Studien, sowie Studien an Tiermodellen und Zellkulturen lassen vermuten, dass psoriatische Inflammation den Knochenstoffwechsel verändert. Um dieser Fragestellung nachzugehen, wurden in der vorliegenden Studie zwei Serummarker des Knochenstoffwechsels, das n-terminale Propeptid des Typ 1-Prokollagens (P1NP) sowie das c-terminale Telopeptid des Typ 1-Kollagens (CTX-I) anhand von 42 Patienten mit Psoriasis vulgaris im Vergleich zu 40 gesunden Kontroll-Probanden mittels ELISA, einem enzymgekoppelten Immunadsorptionstest, analysiert. Unsere Daten zeigen eine Verminderung des Knochenaufbaumarkers P1NP bei Psoriasis-Patienten unabhängig von Geschlecht, Alter und BMI im Vergleich zu gesunden Kontroll-Personen, wohingegen CTX-I als Knochenresorptionsmarker unbeeinflusst bleibt. Dies lässt einen Psoriasis vulgaris-assoziierten Netto-Verlust an Knochensubstanz bei vermindertem Knochenaufbau und gleichbleibendem Knochenabbau vermuten. Unsere Studie zeigt, dass sowohl bei männlichen als auch weiblichen Patienten mit Psoriasis vulgaris die Konzentrationen von P1NP signifikant verringert sind. Hinsichtlich der Beeinflussung dieser Marker durch das Körpergewicht zeigte die vorliegende Arbeit, dass sowohl bei übergewichtigen als auch normalgewichtigen Patienten mit Psoriasis vulgaris die P1NP-Konzentrationen erniedrigt sind. Nach Unterteilung der Patienten-Kohorte nach Geschlecht und BMI konnten bei den Patienten mit Psoriasis signifikant bzw. tendenziell verringerte P1NP Serumlevel festgestellt werden, sodass von einem geschlechter- sowie BMI-unabhängigen Effekt der Psoriasis vulgaris ausgegangen werden kann. Hierbei muss jedoch beachtet werden, dass durch die Aufteilung in Unter-Kohorten die Fallzahlen pro Gruppe zum Teil sehr klein werden und damit die statistische Aussagekraft eingeschränkt ist. Interessanterweise korrelieren die P1NP-Level weder mit der Erkrankungsschwere anhand des PASI (Psoriasis Severity Index), BSA (Body surface area) und PGA (Physican´s Global Assessment), noch mit der Erkrankungsdauer oder der Gesamtleukozytenzahl im Blut als Entzündungsparameter. Dies könnte bedeuten, dass bereits eine geringe psoriatische Entzündung beziehungsweise eine kurze Erkrankungsdauer in den Knochenstoffwechsel eingreift und den Knochenaufbau hemmt. Da Psoriasis-Patienten Risikofaktoren für reduzierte Knochendichte aufweisen, wie beispielsweise hohe Raten an Tabakkonsum sowie geringer sportlicher Aktivität, erfolgte eine Analyse der Knochenresorptionsmarker in entsprechenden Unter-Kohorten. Dennoch zeigten sich keine signifikanten Unterschiede bezüglich der P1NP- und CTX-I-Serumwerte zwischen Psoriasis vulgaris Patienten mit/ohne Tabakkonsum und aktivem/inaktivem Lebensstil. In Zusammenschau unserer hier vorgestellten Daten und anderen bisher veröffentlichten epidemiologischen Studien sowie Studien an Tiermodellen und Zellkulturen welche unsere Ergebnisse stützen, lässt sich vermuten, dass die psoriatische Entzündung den Knochenstoffwechsel verändert und damit möglicherweise das Osteoporose-Risiko erhöht. Folglich könnte eine konsequente anti-psoriatische Therapie sowie ein frühzeitiges Screening der Knochenqualität hilfreich sein, um psoriasis-assoziierten Knochenveränderungen vorzubeugen.:1 Einleitung ................................................................................................................ 1 1.1 Psoriasis .......................................................................................................... 1 1.1.1 Definition und klinisches Bild ................................................................. 1 1.1.2 Epidemiologie ........................................................................................ 1 1.1.3 Klassifikation .......................................................................................... 2 1.1.4 Histologie der Psoriasis vulgaris ............................................................ 7 1.1.5 Pathomechanismen ............................................................................... 9 1.1.6 Therapieformen ................................................................................... 12 1.1.7 Psoriasis-assoziierte Komorbiditäten .................................................. 18 1.2 Knochenstoffwechsel ................................................................................... 19 1.2.1 Definition und Physiologie ................................................................. 19 1.2.2 Knochenstoffwechselregulation ......................................................... 22 1.2.3 Zusammenhang zwischen Entzündung und Knochenstoffwechsel ... 27 2 Fragestellung ..................................................................................................... 30 3 Materialien und Methoden .................................................................................. 31 3.1 Klinische Methoden ..................................................................................... 31 3.1.1 Patientenselektion und Studiendesign ............................................... 31 3.1.2 Einschlusskriterien ............................................................................. 31 3.1.3 Ausschlusskriterien ............................................................................ 32 3.1.4 Kohortencharakterisierung ................................................................. 33 3.1.5 Studienablauf ..................................................................................... 35 3.1.6 Fragebogen ........................................................................................ 36 3.2 Labormethoden ............................................................................................ 37 3.3 Statistische Analyse ..................................................................................... 37 4 Ergebnisse .......................................................................................................... 38 4.1 P1NP-Serumkonzentrationen ...................................................................... 38 4.1.1 P1NP in Zusammenhang mit dem Geschlecht ................................. 39 4.1.2 P1NP in Zusammenhang mit dem BMI .............................................. 40 4.1.3 P1NP in Zusammenhang mit dem Geschlecht und BMI..................... 41 4.1.4 P1NP in Zusammenhang mit systemischer Therapie......................... 43 4.1.5 P1NP in Zusammenhang mit Krankheitsschwere, Erkrankungsdauer und Entzündungsparametern.....................................................................45 4.1.6 P1NP in Zusammenhang mit den äußeren Einflussfaktoren Tabakkonsum und inaktiver Lebensstil ...................................................................... 46 4.2 CTX-I-Serumkonzentrationen ................................................................ 47 4.2.1 CTX-I in Zusammenhang mit dem Geschlecht .................................. 48 4.2.3 CTX-I-Serumkonzentrationen in Zusammenhang mit dem Geschlecht und BMI ............................................................................................. 50 4.2.4 CTX-I in Zusammenhang mit systemischer Therapie ....................... 52 4.2.5 CTX-I in Zusammenhang mit Krankheitsschwere Erkrankungsdauer und Entzündungsparametern ............................................................54 4.2.6 CTX-I in Zusammenhang mit den äußeren Einflussfaktoren Tabakkonsum und inaktiver Lebensstil ............................................. 55 5 Diskussion .......................................................................................................... 56 6 Zusammenfassung ............................................................................................. 71 7 Literaturverzeichnis ............................................................................................ 73 8 Abbildungsverzeichnis ........................................................................................ 81 9 Tabellenverzeichnis ............................................................................................ 82 10 Anlagen ............................................................................................................. 83 10.1 Fragebogen der Studie .............................................................................. 83 10.2 Selbstständigkeitserklärung ....................................................................... 85 10.3 Lebenslauf ................................................................................................. 86 10.4 Danksagung .............................................................................................. 87

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Enterobactérias produtoras de ESBLs são descritas tanto no ambiente hospitalar quanto na comunidade em todo o mundo. No Brasil, esses microrganismos também têm emergido como uma causa importante de infecções, sendo as enzimas CTX-M as prevalentes. O objetivo deste estudo foi analisar diferentes aspectos genotípicos relacionados à expressão da resistência aos antimicrobianos em cepas Escherichia coli e de Salmonella spp, tais como: a diversidade de ESBLs, os genes de resistência aos antimicrobianos e o conteúdo plasmidial. Os aspectos epidemiológicos das cepas produtoras de ESBLs também foram investigados. Foram estudadas 88 cepas de enterobactérias, sendo 43 E. coli e 45 cepas de Salmonella spp., de origem hospitalar e da comunidade (principalmente alimentos), isoladas na cidade do Rio de Janeiro. A expressão de ESBL foi observada em sete cepas de E. coli (7/43, 16,3%) e em uma cepa de Salmonella Typhimurium (1/45, 2,3%) e as enzimas foram identificadas como variantes de CTX-M e SHV-5, respectivamente. Entre as cepas de E. coli, a enzima CTX-M-2 foi a mais frequente (n = 4), sendo detectada em cepas isoladas de swab retal de pacientes hospitalizados, enquanto as enzimas CTX-M-59 (uma variante de CTX-M) (n = 1) e CTX-M-9 (n = 2) foram identificadas em cepas isoladas a partir de espécimes clínicos. Salmonella Typhimurium produtora de SHV-5 foi isolada do ambiente hospitalar (fórmula infantil). As cepas de E. coli produtoras das enzimas CTX-M pertenceram a grupos filogenéticos (A, B1, D) e STs (ST34, ST69, ST101) diferentes, sendo os genes blaCTX-M identificados em plasmídeos com tipo de replicon IncA/C de cerca de 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) ou 80 kb (blaCTX-M-2). A cepa de S. Typhimurium produtora de SHV-5 pertenceu a um único clone (A-ST19) e o gene blaSHV-5 foi identificado em plasmídeo com o replicon IncL/M com aproximadamente 55Kb. Foi identificado pela primeira vez no Brasil o ST313 em um clone de S. Typhimurium (D-ST313), comumente associado com doenças invasivas severas, particulamente no continente africano. Genes que codificam para a resistência aos antimicrobianos não-beta-lactâmicos e integrons classe 1 foram identificados entre as cepas de E. coli e de Salmonella spp. multirresistentes produtoras ou não de ESBLs. Em conclusão: i) nossos resultados referentes à E. coli confirmaram a disseminação de enzimas CTX-M (principalmente variantes do grupo CTX-M-2) desde, pelo menos, o ano de 2000, em hospitais no Rio de Janeiro; demonstraram a implicação dos plasmídeos IncA/C na disseminação de genes blaCTX-M; indicaram a possível evolução intra-plasmídeo de blaCTX-M-59 a partir de blaCTX-M-2; a observação da diversidade e multiplicidade de plasmídeos poderiam fornecer plataformas genéticas para a dispersão de diferentes genes e/ou elementos de resistência aos antimicrobianos; ii) em relação à Salmonella spp. este estudo descreveu, pela primeira vez, o isolamento, a partir de fórmula infantil, de uma cepa de S. Typhimurium produtora de ESBL; foi demonstrada a associação do gene blaSHV-5 com plasmídeo do tipo IncL/M, que é considerado epidêmico; foi identificado o clone D-ST313 de S. Typhimurium, que está associado a doenças invasivas severas no continente africano, que reuniu cepas isoladas exclusivamente do ambiente hospitalar. / ESBL-producing Enterobacteriaceae have been described in hospitals and in the community worldwide. In Brazil, ESBL-producing Enterobacteriaceae have also emerged as an important cause of infections, being CTX-M enzymes the most prevalent ESBLs. The objective of this study was to analyze different genotypic aspects related to expression of antimicrobial resistance in isolates of Escherichia coli and Salmonella spp., such as: diversity of ESBLs, antibiotic resistance genes and plasmid content. Epidemiological features of ESBL-producing isolates were also investigated. We studied 88 isolates of enterobacteria, 43 E. coli and 45 Salmonella serotypes of hospital and community (mainly food) origin, isolated in the city of Rio de Janeiro. ESBL expression was observed in seven E. coli isolates (7/43; 16,3%) and in one Salmonella Typhimurium (1/45; 2,3%) and the enzymes identified as CTX-M variants and SHV-5, respectively. Among the E. coli isolates, CTX-M-2 was the most frequent (n=4), being detected in isolates recovered from rectal swabs of hospitalized patients, whereas CTX-M-59 (a CTX-M-2-variant) (n=1) and CTX-M-9 (n=2) were identified in E. coli isolated from clinical specimens. SHV-5-producing S. Typhimurium was isolated from the hospital environment (infant formula). CTX-M-producing E. coli belonged to different phylogenetic groups (A, B1, D) and STs (ST34, ST69, ST101), being blaCTX-M genes were identified in IncA/C plasmids of approximately 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) or 80 kb (blaCTX-M-2). SHV-5-producing S. Typhimurium belonged to a single clone (A-ST19) and blaSHV-5 gene was identified in IncL/M plasmids of approximately 55Kb. This study first described in Brazil the isolation of S. Typhimurium belonging to ST313 commonly associated with severe invasive diseases, particularly in Africa. Genes encoding resistance to non-beta-lactams and class 1 integrons were found among ESBL-producers and non-ESBL-producing multidrug-resistant E. coli and Salmonella spp. In conclusion: i) our results related to E. coli confirmed the dissemination of CTX-M-enzymes (especially CTX-M-2-variants) since, at least, the beginning of the last decade in Rio de Janeiro clinical settings; demonstrated the implication of IncA/C plasmids in the spread of blaCTX-M genes; indicated the possible intra-plasmid evolution of blaCTX-M-59 from blaCTX-M-2; observation of the diversity and multiplicity of plasmids would provide genetic platforms for spread of different antibiotic resistance genes and/or elements; ii) in relation to Salmonella spp. this study described for the first time, the isolation, from infant formula, ESBL- producing S. Typhimurium; has been demonstrated the association of blaSHV-5 to plasmids belonging to IncL/M group, that can be considered epidemic plasmids; was identified D-ST313 clone in S. Typhimurium, commonly associated with severe invasive diseases, particularly in Africa, among isolates recovered exclusively from hospital.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Enterobactérias produtoras de ESBLs são descritas tanto no ambiente hospitalar quanto na comunidade em todo o mundo. No Brasil, esses microrganismos também têm emergido como uma causa importante de infecções, sendo as enzimas CTX-M as prevalentes. O objetivo deste estudo foi analisar diferentes aspectos genotípicos relacionados à expressão da resistência aos antimicrobianos em cepas Escherichia coli e de Salmonella spp, tais como: a diversidade de ESBLs, os genes de resistência aos antimicrobianos e o conteúdo plasmidial. Os aspectos epidemiológicos das cepas produtoras de ESBLs também foram investigados. Foram estudadas 88 cepas de enterobactérias, sendo 43 E. coli e 45 cepas de Salmonella spp., de origem hospitalar e da comunidade (principalmente alimentos), isoladas na cidade do Rio de Janeiro. A expressão de ESBL foi observada em sete cepas de E. coli (7/43, 16,3%) e em uma cepa de Salmonella Typhimurium (1/45, 2,3%) e as enzimas foram identificadas como variantes de CTX-M e SHV-5, respectivamente. Entre as cepas de E. coli, a enzima CTX-M-2 foi a mais frequente (n = 4), sendo detectada em cepas isoladas de swab retal de pacientes hospitalizados, enquanto as enzimas CTX-M-59 (uma variante de CTX-M) (n = 1) e CTX-M-9 (n = 2) foram identificadas em cepas isoladas a partir de espécimes clínicos. Salmonella Typhimurium produtora de SHV-5 foi isolada do ambiente hospitalar (fórmula infantil). As cepas de E. coli produtoras das enzimas CTX-M pertenceram a grupos filogenéticos (A, B1, D) e STs (ST34, ST69, ST101) diferentes, sendo os genes blaCTX-M identificados em plasmídeos com tipo de replicon IncA/C de cerca de 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) ou 80 kb (blaCTX-M-2). A cepa de S. Typhimurium produtora de SHV-5 pertenceu a um único clone (A-ST19) e o gene blaSHV-5 foi identificado em plasmídeo com o replicon IncL/M com aproximadamente 55Kb. Foi identificado pela primeira vez no Brasil o ST313 em um clone de S. Typhimurium (D-ST313), comumente associado com doenças invasivas severas, particulamente no continente africano. Genes que codificam para a resistência aos antimicrobianos não-beta-lactâmicos e integrons classe 1 foram identificados entre as cepas de E. coli e de Salmonella spp. multirresistentes produtoras ou não de ESBLs. Em conclusão: i) nossos resultados referentes à E. coli confirmaram a disseminação de enzimas CTX-M (principalmente variantes do grupo CTX-M-2) desde, pelo menos, o ano de 2000, em hospitais no Rio de Janeiro; demonstraram a implicação dos plasmídeos IncA/C na disseminação de genes blaCTX-M; indicaram a possível evolução intra-plasmídeo de blaCTX-M-59 a partir de blaCTX-M-2; a observação da diversidade e multiplicidade de plasmídeos poderiam fornecer plataformas genéticas para a dispersão de diferentes genes e/ou elementos de resistência aos antimicrobianos; ii) em relação à Salmonella spp. este estudo descreveu, pela primeira vez, o isolamento, a partir de fórmula infantil, de uma cepa de S. Typhimurium produtora de ESBL; foi demonstrada a associação do gene blaSHV-5 com plasmídeo do tipo IncL/M, que é considerado epidêmico; foi identificado o clone D-ST313 de S. Typhimurium, que está associado a doenças invasivas severas no continente africano, que reuniu cepas isoladas exclusivamente do ambiente hospitalar. / ESBL-producing Enterobacteriaceae have been described in hospitals and in the community worldwide. In Brazil, ESBL-producing Enterobacteriaceae have also emerged as an important cause of infections, being CTX-M enzymes the most prevalent ESBLs. The objective of this study was to analyze different genotypic aspects related to expression of antimicrobial resistance in isolates of Escherichia coli and Salmonella spp., such as: diversity of ESBLs, antibiotic resistance genes and plasmid content. Epidemiological features of ESBL-producing isolates were also investigated. We studied 88 isolates of enterobacteria, 43 E. coli and 45 Salmonella serotypes of hospital and community (mainly food) origin, isolated in the city of Rio de Janeiro. ESBL expression was observed in seven E. coli isolates (7/43; 16,3%) and in one Salmonella Typhimurium (1/45; 2,3%) and the enzymes identified as CTX-M variants and SHV-5, respectively. Among the E. coli isolates, CTX-M-2 was the most frequent (n=4), being detected in isolates recovered from rectal swabs of hospitalized patients, whereas CTX-M-59 (a CTX-M-2-variant) (n=1) and CTX-M-9 (n=2) were identified in E. coli isolated from clinical specimens. SHV-5-producing S. Typhimurium was isolated from the hospital environment (infant formula). CTX-M-producing E. coli belonged to different phylogenetic groups (A, B1, D) and STs (ST34, ST69, ST101), being blaCTX-M genes were identified in IncA/C plasmids of approximately 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) or 80 kb (blaCTX-M-2). SHV-5-producing S. Typhimurium belonged to a single clone (A-ST19) and blaSHV-5 gene was identified in IncL/M plasmids of approximately 55Kb. This study first described in Brazil the isolation of S. Typhimurium belonging to ST313 commonly associated with severe invasive diseases, particularly in Africa. Genes encoding resistance to non-beta-lactams and class 1 integrons were found among ESBL-producers and non-ESBL-producing multidrug-resistant E. coli and Salmonella spp. In conclusion: i) our results related to E. coli confirmed the dissemination of CTX-M-enzymes (especially CTX-M-2-variants) since, at least, the beginning of the last decade in Rio de Janeiro clinical settings; demonstrated the implication of IncA/C plasmids in the spread of blaCTX-M genes; indicated the possible intra-plasmid evolution of blaCTX-M-59 from blaCTX-M-2; observation of the diversity and multiplicity of plasmids would provide genetic platforms for spread of different antibiotic resistance genes and/or elements; ii) in relation to Salmonella spp. this study described for the first time, the isolation, from infant formula, ESBL- producing S. Typhimurium; has been demonstrated the association of blaSHV-5 to plasmids belonging to IncL/M group, that can be considered epidemic plasmids; was identified D-ST313 clone in S. Typhimurium, commonly associated with severe invasive diseases, particularly in Africa, among isolates recovered exclusively from hospital.

O presente estudo visou determinar a prevalência de bactérias Gram-negativas produtoras de produzem b-lactamases de amplo espectro (ESBL) e resistência adquirida a quinolonas mediada por plasmídeos (PMQR) em animais de estimação, investigando o potencial papel destes hospedeiros como portadores assintomáticos. Em 2012, foram coletadas 216 amostras (fezes e saliva) de 108 animais de companhia (29 gatos e 79 cães) abrigados em casas de família, um centro de acolhimento de animais abandonados, e no Centro de Controle de Zoonoses da Cidade de São Paulo. Do total de cepas estudadas, 85% apresentaram fenótipo sugestivo de PMQR; enquanto que 62% dos isolados exibiram um fenótipo característico e sugestivo para produção de ESBL, sendo na sua maioria identificadas como E. coli. Dentre os isolados, 14 carregaram variantes do gene blaCTX-M, 9 foram positivos para o gene blaTEM, e 6 foram positivos para blaSHV. Em relação às cepas resistentes às Q/FQ, 56% (n= 43) foram positivas para a presença do gene qnr, o qual foi identificado em 11 espécies diferentes. Os resultados apresentados demostram que animais de companhia podem ser portadores assintomáticos de cepas produtoras de ESBL e PMQR. / The present study aimed to determine the prevalence of Gram-negative bacteria producing b-lactamases producing broad-spectrum (ESBL) and acquired resistance to quinolones mediated by plasmids (PMQR) in pets, investigating the potential role of these hosts as asymptomatic carriers. In 2012, 216 samples (feces and saliva) of 108 companion animals (29 cats and 79 dogs) housed in shelters or a Zoonosis Control Center were collected from São Paulo city. Of the total strains studied, 85% had a phenotype suggestive for PMQR; while 62 % of the isolates exhibited a characteristic phenotype and suggestive for ESBL-producing genes, with the most identified as E. coli. Among the isolates, 14 carried variants blaCTX -M gene 9 were positive for blaTEM gene, and 6 were positive for blaSHV. Regarding resistant Q/FQ isolates, 56% (n = 43) were positive for the presence of qnr gene, which was identified on 11 different species. The results presented demonstrate that pets can be asymptomatic carriers of ESBL producing strains and PMQR.

O presente estudo visou determinar a prevalência de bactérias Gram-negativas produtoras de produzem b-lactamases de amplo espectro (ESBL) e resistência adquirida a quinolonas mediada por plasmídeos (PMQR) em animais de estimação, investigando o potencial papel destes hospedeiros como portadores assintomáticos. Em 2012, foram coletadas 216 amostras (fezes e saliva) de 108 animais de companhia (29 gatos e 79 cães) abrigados em casas de família, um centro de acolhimento de animais abandonados, e no Centro de Controle de Zoonoses da Cidade de São Paulo. Do total de cepas estudadas, 85% apresentaram fenótipo sugestivo de PMQR; enquanto que 62% dos isolados exibiram um fenótipo característico e sugestivo para produção de ESBL, sendo na sua maioria identificadas como E. coli. Dentre os isolados, 14 carregaram variantes do gene blaCTX-M, 9 foram positivos para o gene blaTEM, e 6 foram positivos para blaSHV. Em relação às cepas resistentes às Q/FQ, 56% (n= 43) foram positivas para a presença do gene qnr, o qual foi identificado em 11 espécies diferentes. Os resultados apresentados demostram que animais de companhia podem ser portadores assintomáticos de cepas produtoras de ESBL e PMQR. / The present study aimed to determine the prevalence of Gram-negative bacteria producing b-lactamases producing broad-spectrum (ESBL) and acquired resistance to quinolones mediated by plasmids (PMQR) in pets, investigating the potential role of these hosts as asymptomatic carriers. In 2012, 216 samples (feces and saliva) of 108 companion animals (29 cats and 79 dogs) housed in shelters or a Zoonosis Control Center were collected from São Paulo city. Of the total strains studied, 85% had a phenotype suggestive for PMQR; while 62 % of the isolates exhibited a characteristic phenotype and suggestive for ESBL-producing genes, with the most identified as E. coli. Among the isolates, 14 carried variants blaCTX -M gene 9 were positive for blaTEM gene, and 6 were positive for blaSHV. Regarding resistant Q/FQ isolates, 56% (n = 43) were positive for the presence of qnr gene, which was identified on 11 different species. The results presented demonstrate that pets can be asymptomatic carriers of ESBL producing strains and PMQR.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-20T11:52:49Z No. of bitstreams: 1 vanessacordeirodias.pdf: 663329 bytes, checksum: a6ecc4c9fdb1483811c9adc9cb8fad2c (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:23:46Z (GMT) No. of bitstreams: 1 vanessacordeirodias.pdf: 663329 bytes, checksum: a6ecc4c9fdb1483811c9adc9cb8fad2c (MD5) / Made available in DSpace on 2016-09-26T20:23:46Z (GMT). No. of bitstreams: 1 vanessacordeirodias.pdf: 663329 bytes, checksum: a6ecc4c9fdb1483811c9adc9cb8fad2c (MD5) Previous issue date: 2010-12-10 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / As infecções do trato urinário (ITU) são manifestações freqüentes na população, geralmente causadas por bacilos Gram-negativos. As β-lactamases são enzimas bacterianas que conferem resistência aos antimicrobianos do tipo β-lactâmicos (penicilinas, cefalosporinas, aztreonam e carbapenêmicos). A produção de β-lactamases de Espectro Estendido (ESBL) tem sido descrita como um importante mecanismo de resistência aos β-lactâmicos. De maneira geral, Escherichia coli e Klebsiella pneumoniae são as espécies bacterianas mais comumente encontradas produzindo ESBL, embora a detecção dessas enzimas já tenha sido observada em diversos gêneros dentro da família Enterobacteriaceae. O objetivo deste estudo foi avaliar os perfis de susceptibilidade a antimicrobianos e correlacionar os testes fenotípicos com a detecção de marcadores genéticos para produção de β-lactamases dos tipos ESBL, AmpC e KPC em enterobactérias associadas à etiologia de ITUs em pacientes atendidos em um Laboratório de Análises Clínicas da cidade de Juiz de Fora, MG. Para o estudo restrospectivo (20012008), 66.660 isolados de urina com suspeita de ITU foram analisadas, e após a identificação bioquímica, as linhagens de enterobactérias foram submetidas a testes de susceptibilidade aos antimicrobianos, pelo método de disco-difusão, de acordo com as normas do Clinical and Laboratory Standards Institute/CLSI. A detecção fenotípica da produção de ESBL foi feita através do teste de aproximação dos discos. Para o estudo prospectivo (2009), 12.304 amostras com suspeita de ITU foram avaliadas, e após a identificação bioquímica das linhagens, foram feitos os testes de susceptibilidade aos antimicrobianos e o teste de aproximação dos discos, para a detecção fenotípica da produção de ESBL. A identificação dos marcadores de β-lactamase (SHV, TEM, CTX-M, AMPc e KPC) foi feita por reação em cadeia da polimerase (PCR). De 416 linhagens produtoras de ESBL entre 2001- 2008, E. coli foi a mais freqüente (74,4%). Altos níveis de resistência foram obtidos para sulfazotrim, gentamicina e ciprofloxacina neste período. Todas as linhagens foram sensíveis ao imipenem. No estudo prospectivo, 105 linhagens produtoras de ESBL foram isoladas, sendo E. coli a mais freqüente (63%). Foi observado um alto índice de resistência a amoxacilina-clavulanato (80,8%), e aproximadamente 70% das amostras foram resistentes à associação trimetoprim/sulfametoxazol e as fluoroquionolonas (ciprofloxacina e ácido nalidíxico). Dentre as drogas utilizadas como substrato para detecção de ESBL, a cefotaxima foi o substrato com maior índice de resistência (86,6%), seguido de aztreonam (60,9%) e ceftazidima (55,2%). Entre as 105 linhagens recuperadas, todos os marcadores genéticos pesquisados para ESBL foram detectados. Considerando-se a freqüência de detecção dos marcadores genéticos, TEM foi o mais frequente (86,6%), seguido por SHV (59%), CTX-M (31,4%) e AMPc (27,6%). Em todas as linhagens bacterianas avaliadas, foi detectado pelo menos 1 dos marcadores genéticos associados à produção de β-lactamases (22,8%), 52,4% apresentaram 2 marcadores, 20% apresentaram 3 marcadores e 4,8% apresentaram 4 dos marcadores pesquisados. β-lactamases do tipo KPC não foram detectadas. O conhecimento da epidemiologia, dinâmica de disseminação e circulação destes marcadores genéticos de resistência a drogas constitui um dado clínico relevante, pois possibilita a instauração de uma terapia antimicrobiana mais adequada, bem como a construção de um banco de informações epidemiológicas, como ferramenta para contenção da expansão da disseminação dos genes de resistência aos antimicrobianos β-lactâmicos. / The urinary tract infections (UTI) are highly frequent within the population and usually caused by Gram-negative rods. The bacterial β-lactamases are enzymes which confer resistance against β-lactam antibiotics (penicillins, cephalosporins, aztreonam and carbapenems) amongst which Extended Spectrum β-Lactamases (ESBL) has been described as an important resistance mechanism to the β-lactam in Gram-negative bacteria. Generally, Escherichia coli and Klebsiella pneumoniae are the most frequent ESBL producing bacterial species, but its production by representatives of other bacterial genus within the Enterobacteriaceae family has already been documented. The aim of this study were to evaluate the antimicrobial susceptibility patterns and to correlate phenotypic tests with the detection of genetic markers for β-lactamases production such as ESBL, AmpC and KPC in enterobacteria associated to the UTI etiology in patients assisted at a Clinical Analyses Laboratory in Juiz de Fora, MG. From a retrospective study (2001-2008), 66.660 urine samples were analyzed, and the biochemically identified bacteria were submitted to antibiotic susceptibility testing by the disk-diffusion method, according to the Clinical Laboratory Standards Institute/CLSI guidelines. Further, phenotypic detection of the ESBL production was carried out through the disk approximation test. From a prospective study (2009), 12.304 samples were evaluated, and after the microbial identification, antibiotic susceptibility tests and disk approximation assays were performed, for ESBL phenotypic detection. Identification of β-lactamase genetic markers (SHV, TEM, CTX-M, AMPc and KPC) were performed through polymerase chain reaction (PCR). Out of 416 ESBL producing bacteria identified between 2001 and 2008, E. coli was the most frequent (74.4%). High resistance levels were obtained for trimethopim-sulfamethoxazole, gentamicin and ciprofloxacin in this period. All of the strains were sensitive to imipenem. Regarding the prospective study, 105 ESBL producing bacteria were isolated, being E. coli the most frequent (63%). A high resistance rate was observed against amoxacilin/clavulanic-acid (80.8%), and almost 70% of the samples were resistant to the association trimethopim-sulfamethoxazole and the fluoroquionolones (ciprofloxacin and nalidixic acid). Among the drugs for ESBL detection, the cefotaxime was the substrate with the highest resistance rate (86.6%), followed by aztreonam (60.9%) and ceftazidime (55.2%). Among these strains, all of the researched genetic markers for ESBL were detected. In regard to the frequency of detection of the genetic markers, TEM was the most frequent (86.6%), following by SHV (59%), CTX-M (31.4%) and AmpC (27.6%). In all of the bacterial strains it was detected at least 1 of the genetic markers associated to the production of β –lactamases. Two markers were detected in 52.4% and in 20% and 48%, at least 3 or 4 markers were detected, respectively. The KPC type β -lactamase was not detected. The knowledge about the epidemiology, spread dynamics and circulation of these resistance genetic markers to drugs among the different bacterial populations constitutes a relevant issue and makes possible the establishment of a more appropriated chemotherapy. Besides, the generation of basic epidemiological information may sustain for contention of the antimicrobial resistance and the spread of resistant genetic markers related to inactivation of β-lactam drugs.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-01-21T11:23:35Z No. of bitstreams: 1 ritosantopereira.pdf: 1611336 bytes, checksum: 8393e7d27377653e27c6bd1d9654130b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-01-25T18:43:36Z (GMT) No. of bitstreams: 1 ritosantopereira.pdf: 1611336 bytes, checksum: 8393e7d27377653e27c6bd1d9654130b (MD5) / Made available in DSpace on 2016-01-25T18:43:36Z (GMT). No. of bitstreams: 1 ritosantopereira.pdf: 1611336 bytes, checksum: 8393e7d27377653e27c6bd1d9654130b (MD5) Previous issue date: 2014-11-28 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A resistência aos agentes antimicrobianos é um problema crescente, restringindo as opções de tratamento e resultando em falhas clinicas mais frequente. Carbapenemases são enzimas produzidas por bactérias Gram-negativas que conferem resistência aos β-lactâmicos. Klebsiella pneumoniae e Enterobacter aerogenes destacam-se pelo oportunismo e capacidade de produção destas enzimas e seu envolvimento nas infecções hospitalares. Nosso trabalho teve como objetivo avaliar os aspectos fisiológicos e moleculares relacionados à virulência e resistência aos carbapenêmicos em K. pneumoniae e E. aerogenes, isolados em um serviço de microbiologia clínica em um hospital terciário durante o ano de 2012. De maneira geral, 3437 espécimes clínicos coletados foram processados para isolamento microbiano. Amostras identificadas foram avaliadas quanto à susceptibilidade aos antimicrobianos e 42 enterobactérias resistentes aos carbapenêmicos foram consideradas. Foram realizados testes para avaliação da produção de carbapenemases, atividade hemolítica, estresse oxidativo, tolerância a biocidas e formação de biofilmes. Os microrganismos foram recuperados de urina, cateter, sangue e trato respiratório. Entre os pacientes, 61,9% eram de UTI. Os isolados apresentaram altos níveis de sensibilidade à amicacina e tigeciclina (>90%). Considerando-se a frequência dos marcadores genéticos, KPC foi a mais frequente (97,6%), seguido por TEM (95,2%), SHV (69,0%) e CTX-M (38,1%). Em todos os isolados avaliados, 2,4% apresentaram 1 marcador genético associado a resistência aos carbapenêmicos, 23,8% apresentaram 2 marcadores, 45,2% apresentaram 3 e 28,6% apresentaram 4. As espécies resistentes aos carbapenêmicos apresentaram características peculiares em relação às características fisiológicas avaliadas, sugerindo que essas bactérias possam apresentar comportamentos diferentes de microrganismos de mesma espécie sensíveis às drogas, com implicações não apenas para antibioticoterapia, mas também para sua virulência e persistência no ambiente nosocomial. Estes resultados confirmam a dificuldade de manejo terapêutico de pacientes com infecções associadas a microrganismos multi-resistentes e oportunistas com impactos diretos na mortalidade e no controle epidemiológico destes microrganismos nos centros de saúde. / The antimicrobial resistance is a growing problem, restricting treatment options and resulting in more frequent clinical failures. Carbapenemases are enzymes produced by Gram-negative bacteria which confer resistance to β-lactams. Klebsiella pneumoniae and Enterobacter aerogenes stand out by opportunism and production capacity of these enzymes and their involvement in nosocomial infections. Our study aimed to evaluate the virulence and related carbapenem resistance in K. pneumoniae and E. aerogenes physiological and molecular aspects, isolated from the Clinical Laboratory, in a tertiary hospital in the city of Juiz de Fora - Minas Gerais, during 2012. During the study period, 3437 clinical specimens were processed for microbial isolation. Identified samples were evaluated for antimicrobial susceptibility and 42 resistant Enterobacteriaceae to carbapenems were considered. Underwent tests for evaluation of carbapenemase production, hemolytic activity, biofilm formation, oxidative stress and biocides tolerance. The results showed that K. pneumoniae and E. aerogenes were recovered in urine, catheter, blood and respiratory tract. Among the patients, 61.9% were ICU. The isolates showed high levels of sensitivity to amikacin and tigecycline (>90%). Considering the frequency of genetic markers, KPC was the most common (97.6%), followed by TEM (95.2%), SHV (69.0%) and CTX-M (38.1%). In all isolates, 2.4% had one genetic marker associated with resistance to carbapenems, 23.8% had two markers, 45.2% had three and 28.6% had four. Carbapenemase producers showed particular characteristics regarding the assessed physiological characteristics, suggesting that these bacteria may exhibit different behaviors of microorganisms of the same species susceptible to carbapenem with implications not only for antibiotic therapy, but also for their virulence and persistence in the nosocomial environment. These results confirm the difficulty of therapeutic management of patients with infections associated with multi- resistant and opportunistic microorganisms with direct impacts on mortality and epidemiological control of these microorganisms in health centers.

The aim of this study was to determine the prevalence of extended-spectrum b-lactamase (ESBL)- producing Klebsiella pneumoniae isolated from urinary tract and bloodstream infections in a rural hospital in Manhic¸a, Mozambique. ESBLs were investigated among ceftriaxone-non-susceptible K. pneumoniae clinical isolates recovered between 2004 and 2009. Characterisation of blaCTX-M, blaSHV, blaOXA and blaTEM genes was performed by PCR and sequencing. Epidemiological relationships were established by phylogenetic analysis, repetitive extragenic palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), whilst plasmid transferability was evaluated by conjugation. In addition,the presence of class 1 and 2 integrons was studied.A total of 19 K. pneumoniae were analysed. The blaCTX-M-15 gene was found in all strains. Other ESBL genes were found concomitantly, including blaSHV-5, blaSHV-2, blaSHV-2A, blaSHV-12 and blaSHV-38. In addition, other b-lactamases such as blaTEM-1 and blaOXA-30 were also detected. REP-PCR identified 15 different epidemiological profiles. MLST analysis also showed great variability of sequence types. The blaCTX-M-15 gene showed a high transfer capacity. The presence of class 1 integrons was high. High levels of multidrug resistance were also found. In conclusion, these data show the dominance of the CTX-M-type ESBL, particularly CTX-M-15, supporting its worldwide dissemination, including in areas with limited access to third-generation cephalosporins. This finding is a matter of concern for clinical management as third-generation cephalosporins are an alternative for treating severe cases of multidrug-resistant infections in this community. / Revisión por pares

The dissemination of members of the Enterobacteriaceae family with extended spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) has become a global concern. ESBLs and MBLs have been reported in humans, domestic animals, wildlife and the environment, and their isolation frequencies are increasing rapidly worldwide.  Most studies have been performed in developed countries and quite few in developing countries, where the antibiotic consumption is often poorly controlled. To explore the environmental contamination of antibiotic resistance in Bangladesh, and of ESBLs and MBLs in particular, fecal samples from poultry and wild birds were studied in this thesis. Samples were collected from both sick birds (poultry having Escherichia coli infections) and healthy birds (free-range poultry, seagulls and crows) residing in different environmental niches. Samples from patients and fresh/sea water were included, to follow the chain of antibiotic resistance in bacteria from humans to the environment. Information regarding the antibiotic usage in poultry production was also collected. The susceptibility of avian E. coli isolates cultured with and without selective pressure was tested against antibiotics commonly used in human and veterinary medicine in Bangladesh. Special attention was paid to ESBL-producing isolates, which were further characterized genetically. The results of the studies showed that E. coli isolates from commercial poultry, free-range poultry, gulls and crows were resistant to several classes of antibiotics, and that the level and spectrum of antibiotic resistance varied between different bird populations. There was no NDM-producer found among the birds, but ESBL-producing Enterobacteriaceae could be found in up to 59% of the crows, the birds with the highest carriage rate of multiresistant  Enterobacteriaceae of all bird species studied. The most common ESBL-type was CTX-M-15, which also is the most common in the human population in Bangladesh. Birds also shared clinically important sequence types with humans, including E. coli clone O25b-ST131. In conclusion, ESBL-producing bacteria with multiresistance are easily spread to wild birds. Their opportunistic feeding behavior at poorly managed hospital waste dumps and nearby water bodies makes them into both reservoirs and active spreaders. The high level of antibiotic resistant and ESBL-producing bacteria in the bird population of Bangladesh is worrying, and there is no easy solution in sight. Nationwide programs are necessary to both improve the management of hospital waste and sewage and the control of the antibiotic usage to prevent further environmental contamination. / <p>Time of defence has been changed to 09:00 am on 2013-05-27</p>

The objective of the present study was to investigate deformational behavior and functional adaptation of articular cartilage by comparing the changes of biochemical osteoarthritis markers&rsquo / concentrations due to 30-min exercise after 12-weeks of regular high impact, impact or non-impact exercise. Blood samples were drawn from 44 healthy sedentary males immediately before, immediately after and 0.5 h after a 30-min moderate walking exercise. Osteoarthritis biomarkers&rsquo / (Serum COMP and CTX-I) concentrations were determined with enzyme-linked immunosorbent assay. After the first measurements, participants were randomly assigned to running, cycling, swimming, and control groups. All groups except for control group trained for 12 weeks. After 12-weeks, post tests were applied. Multivariate tests indicated a significant fatigue and resting effect on serum COMP concentration in all groups at pre- and post-tests. Therefore, pair wise comparisons were conducted in order to assess the differences across all groups and conditions. Results indicated significant differences in post-test measurements among phases of groups except for running group. However, fatigue or resting did not change the concentration of serum CTX-I in any groups during the tests. According to results, moderate walking activity has an influence on the increase of serum COMP concentrations of young sedentary men. However, 12 weeks regular weight-bearing high impact physical exercise decreases the deformational effect of walking activity by functional adaptation of articular cartilage to specific environmental requirements.