Staphylococcus aureus resistente à meticilina (MRSA) é um dos principais microrganismos envolvidos nas Infecções relacionadas à Assistência à Saúde (IrAS). Porém, um clone de MRSA, o CA-MRSA, emergiu na comunidade e atualmente vem sendo agente de IrAS. O objetivo desta dissertação é avaliar fenotípica e genotipicamente 111 amostras de Staphylococcus aureus resistentes à meticilina e sensíveis a antibióticos não ß-lactâmicos de pacientes atendidos em cinco hospitais no município do Rio de Janeiro. Utilizando os critérios padronizados pelo CLSI 2012, foram determinadas as susceptibilidades a 11 antimicrobianos pelo método de disco difusão em ágar e concentração inibitória mínima para vancomicina e oxacilina pelo método da microdiluição em caldo. A multirresistência (resistência a 3 ou mais antimicrobianos não ß-lactâmicos) foi observada em 31,5% das amostras, sendo que 53,2% apresentaram resistência ao antimicrobiano clindamicina, uma das opções para o tratamento empírico das infecções de pele/tecidos moles. 86,4% apresentaram concentração inibitória mínima (CIM) para vancomicina ≥ 1,0 g/mL ou seja, elevado percentual de amostras associadas ao fenômeno MIC creep, o qual está associado ao insucesso na terapia antimicrobiana anti-MRSA. Não foi observado até o momento nenhuma amostra com CIM ≥ 4cg/mL para vancomicina, entretanto, já há resistência à linezolida em quatro hospitais do estudo. A tipificação do SCCmec nos permitiu classificar 4,5% das amostras em HA-MRSA e 86,5% em CA-MRSA, nas quais a resistência heterogênea típica à oxacilina foi observada em 57,2%. A toxina de Panton-Valentine (PVL) foi identificada pela metodologia de PCR em 28% das amostras com genótipo CA-MRSA. Os fatores de riscos clássicos, da literatura, relacionados à infecção por HA-MRSA foram também observados nos pacientes com infecção por CA-MRSA portadoras de SCCmec IV e V. No intuito de verificar a existência de similaridades genéticas ou a presença de clone predominante entre as amostras dos cinco hospitais, foi realizada a técnica de eletroforese em gel sob campo pulsado (PFGE) e observou-se diversidade genética assim como a presença de amostras com padrões similares aos clones OSPC (18,5%) e USA400. Não foram encontradas amostras com padrões de eletroforese similares aos clones USA300, USA800 e CEB. É essencial a vigilância da resistência aos antimicrobianos não ß-lactâmicos no CA-MRSA, em especial à vancomicina. A mudança na epidemiologia deste microrganismo vem impactando os padrões característicos dos genótipos limitando os critérios de diferenciação entre eles. Neste contexto, as técnicas moleculares atuam como excelentes ferramentas de caracterização. O conhecimento do patógeno auxilia na elaboração e implementação de medidas preventivas, contribuindo para o controle da doença tanto no ambiente hospitalar quanto na comunidade. / Methicillin-resistant Staphylococcus aureus (MRSA) is a major microrganism involved in healthcare associated infections (HAIs). However, a clone of MRSA, CA-MRSA, has emerged in the community and has been considered agent of HAIs. The goal of this dissertation is to evaluate phenotypically and genotypically 111 samples of methicillin-resistant Staphylococcus aureus susceptible to non ß-lactam antibiotics from patients treated in five hospitals in the city of Rio de Janeiro. Using the Clinical and Laboratory Standards Institute criteria were determined susceptibility to 11 antimicrobials by the disk diffusion method and minimal inhibitory concentration for oxacillin and vancomycin by broth microdilution method. The multidrug resistance (resistance to three or more non ß-lactam antibiotics) was observed in 31.5% of isolates, and 53.2% were resistant to the antimicrobial clindamycin, one of the choices in the empirical treatment of infections of skin / soft tissue. 86.4% showed minimal inhibitory concentration (MIC) for vancomycin ≥ 1.0 mg / mL, representing high percentage of samples associated with the MIC creep phenomenon, which can imply therapeutic failure. The typification of SCCmec enabled us to classify 4,5% of the samples in HA-MRSA and 86.5% in CA-MRSA, among which the typical heterogeneous oxacillin resistance was observed in 57.2%. The Panton-Valentine Leukocidin (PVL) toxin, one of the virulence factors involved in the pathogeneses of MRSA, was present in 28% of samples with genotype CA-MRSA. We performed uptake of demographic and clinical information on patients medical records and verified the presence of classical risk factors for HA-MRSA infection in individuals infected by CA-MRSA carrying SCCmec IV and V. In order to verify the existence of genetic similarities or the presence of predominant clone among the samples of the five hospitals, we applied the technique of pulsed-field gel electrophoresis (PFGE) and observed genetic diversity and the presence of samples with standards similar to OSPC clones (18.5%) and USA400. There were no samples with electrophoresis patterns similar to clone USA300, USA800 and CEB. Surveillance of resistance to non ß-lactam antibiotics is essencial in CA-MRSA, especially vancomycin. The change in the epidemiology of this microrganism has been impacting the characteristic patterns of genotypes limiting criteria of differentiation between them. In this context, molecular techniques serve as excellent characterization tools. Knowledge of pathogen assists in the development and implementation of preventive measures, contributing to disease control both in hospitals and in the community.

Staphylococcus aureus resistente à meticilina (MRSA) é um dos principais microrganismos envolvidos nas Infecções relacionadas à Assistência à Saúde (IrAS). Porém, um clone de MRSA, o CA-MRSA, emergiu na comunidade e atualmente vem sendo agente de IrAS. O objetivo desta dissertação é avaliar fenotípica e genotipicamente 111 amostras de Staphylococcus aureus resistentes à meticilina e sensíveis a antibióticos não ß-lactâmicos de pacientes atendidos em cinco hospitais no município do Rio de Janeiro. Utilizando os critérios padronizados pelo CLSI 2012, foram determinadas as susceptibilidades a 11 antimicrobianos pelo método de disco difusão em ágar e concentração inibitória mínima para vancomicina e oxacilina pelo método da microdiluição em caldo. A multirresistência (resistência a 3 ou mais antimicrobianos não ß-lactâmicos) foi observada em 31,5% das amostras, sendo que 53,2% apresentaram resistência ao antimicrobiano clindamicina, uma das opções para o tratamento empírico das infecções de pele/tecidos moles. 86,4% apresentaram concentração inibitória mínima (CIM) para vancomicina ≥ 1,0 g/mL ou seja, elevado percentual de amostras associadas ao fenômeno MIC creep, o qual está associado ao insucesso na terapia antimicrobiana anti-MRSA. Não foi observado até o momento nenhuma amostra com CIM ≥ 4cg/mL para vancomicina, entretanto, já há resistência à linezolida em quatro hospitais do estudo. A tipificação do SCCmec nos permitiu classificar 4,5% das amostras em HA-MRSA e 86,5% em CA-MRSA, nas quais a resistência heterogênea típica à oxacilina foi observada em 57,2%. A toxina de Panton-Valentine (PVL) foi identificada pela metodologia de PCR em 28% das amostras com genótipo CA-MRSA. Os fatores de riscos clássicos, da literatura, relacionados à infecção por HA-MRSA foram também observados nos pacientes com infecção por CA-MRSA portadoras de SCCmec IV e V. No intuito de verificar a existência de similaridades genéticas ou a presença de clone predominante entre as amostras dos cinco hospitais, foi realizada a técnica de eletroforese em gel sob campo pulsado (PFGE) e observou-se diversidade genética assim como a presença de amostras com padrões similares aos clones OSPC (18,5%) e USA400. Não foram encontradas amostras com padrões de eletroforese similares aos clones USA300, USA800 e CEB. É essencial a vigilância da resistência aos antimicrobianos não ß-lactâmicos no CA-MRSA, em especial à vancomicina. A mudança na epidemiologia deste microrganismo vem impactando os padrões característicos dos genótipos limitando os critérios de diferenciação entre eles. Neste contexto, as técnicas moleculares atuam como excelentes ferramentas de caracterização. O conhecimento do patógeno auxilia na elaboração e implementação de medidas preventivas, contribuindo para o controle da doença tanto no ambiente hospitalar quanto na comunidade. / Methicillin-resistant Staphylococcus aureus (MRSA) is a major microrganism involved in healthcare associated infections (HAIs). However, a clone of MRSA, CA-MRSA, has emerged in the community and has been considered agent of HAIs. The goal of this dissertation is to evaluate phenotypically and genotypically 111 samples of methicillin-resistant Staphylococcus aureus susceptible to non ß-lactam antibiotics from patients treated in five hospitals in the city of Rio de Janeiro. Using the Clinical and Laboratory Standards Institute criteria were determined susceptibility to 11 antimicrobials by the disk diffusion method and minimal inhibitory concentration for oxacillin and vancomycin by broth microdilution method. The multidrug resistance (resistance to three or more non ß-lactam antibiotics) was observed in 31.5% of isolates, and 53.2% were resistant to the antimicrobial clindamycin, one of the choices in the empirical treatment of infections of skin / soft tissue. 86.4% showed minimal inhibitory concentration (MIC) for vancomycin ≥ 1.0 mg / mL, representing high percentage of samples associated with the MIC creep phenomenon, which can imply therapeutic failure. The typification of SCCmec enabled us to classify 4,5% of the samples in HA-MRSA and 86.5% in CA-MRSA, among which the typical heterogeneous oxacillin resistance was observed in 57.2%. The Panton-Valentine Leukocidin (PVL) toxin, one of the virulence factors involved in the pathogeneses of MRSA, was present in 28% of samples with genotype CA-MRSA. We performed uptake of demographic and clinical information on patients medical records and verified the presence of classical risk factors for HA-MRSA infection in individuals infected by CA-MRSA carrying SCCmec IV and V. In order to verify the existence of genetic similarities or the presence of predominant clone among the samples of the five hospitals, we applied the technique of pulsed-field gel electrophoresis (PFGE) and observed genetic diversity and the presence of samples with standards similar to OSPC clones (18.5%) and USA400. There were no samples with electrophoresis patterns similar to clone USA300, USA800 and CEB. Surveillance of resistance to non ß-lactam antibiotics is essencial in CA-MRSA, especially vancomycin. The change in the epidemiology of this microrganism has been impacting the characteristic patterns of genotypes limiting criteria of differentiation between them. In this context, molecular techniques serve as excellent characterization tools. Knowledge of pathogen assists in the development and implementation of preventive measures, contributing to disease control both in hospitals and in the community.

INTRODUÇÃO: A combinação de sepse e insuficiência renal com necessidade de diálise é bastante comum nas Unidades de Terapia Intensiva e esta situação tem elevada mortalidade. Um desafio neste cenário é prescrever a dose correta dos antimicrobianos para o tratamento destas infecções. Em pacientes críticos e hemodinamicamente instáveis que necessitam de terapia renal substitutiva, um dos métodos mais utilizados é a diálise contínua, entretanto, recentemente, tem-se utilizado a diálise de baixa eficiência - conhecida como SLED, da abreviação do inglês \"sustained low-efficiency dialysis\". Esta modalidade de terapia renal substitutiva combina características da hemodiálise contínua com a intermitente, utilizando o equipamento da diálise intermitente, com menores fluxos sanguíneos e de dialisato, e com vantagem de menor custo. Apesar do fluxo mais baixo, por ser utilizado tempo mais prolongado, a SLED frequentemente resulta em maior clearance e especula-se que a remoção dos fármacos seria maior. Há escassez de estudos que avaliaram a farmacocinética e farmacodinâmica de antimicrobianos em pacientes submetidos à SLED.OBJETIVOS: Avaliar adequação farmacodinâmica de meropenem e vancomicina em pacientes submetidos a diálise estendida de baixa eficiência. Avaliar a depuração paramêtros farmacocinéticos durante a sessão de SLED. MÉTODOS: Foi realizado estudo prospectivo descritivo observacional com coleta de material biológico julho de 2012 a julho de 2014 HC-FMUSP. Foram incluídos pacientes submetidos à SLED em uso de vancomicina e/ou meropenem. Foram coletadas amostras de sangue seriadas (tempos: imediatamente antes do início da sessão de diálise, 0,5h, 1h, 2h, 4h após o início do tratamento e ao final da sessão). A quantificação dos antimicrobianos foi realizada através dos métodos analíticos de quantificação em Cromatografia Líquida de Alta Eficiência (CLAE). Os parâmetros farmacocinéticos foram calculados apenas durante a sessão de diálise utilizando-se o software WinNonlin. A área sob a curva foi determinada para a vancomicina. Para o meropenem, calculou-se o tempo acima da MIC. Resultados: Foram incluídos 24 pacientes tratados com vancomicina e 21 com meropenem eforam obtidas 170 amostras de plasma. As concentrações médias de vancomicina sérica e meropenem: antes da sessão de SLED foram 24,5 e 28,0 ?g / ml, respectivamente; e após SLED 14 e 6 ?g / ml, respectivamente. A depuração média foi de 41% para a vancomicina e 78% para o meropenem. Para vancomicina, 22 (96%), 19 (83%) e 16 (70%) pacientes teriam atingido o alvo (AUC0-24 > 400) considerando-se MIC 0,5; <= 1mg/l e <= 2 mg/l respectivamente. Para meropenem, 19 (95%), 18 (90%) e 11 (55%) pacientes teriam atingido a meta (70% de tempo acima da CIM) se infectados com isolados com MIC <= 1, <= 4 e <= 8 mg/l, respectivamente. Conclusões: Em pacientes críticos, meropenem evancomicina foram removidas durante o SLED. Entretando, a maioria dos pacientes alcançaria alvo PK-PD, excepto para CIMs mais altas. Sugerimos doses de manutenção de 1g a cada 12 ou 8 horaspara meropenem. Para a vancomicina, deve-se utilizar abordagem mais individualizada com monitorização sérica, uma vez que ensaios comerciais são disponíveis / Background: Antibiotic dosing is a challenge in critically ill patients undergoing renal replacement therapy. Our aim was to evaluate pharmacokinetics and pharmacodynamics of meropenem and vancomycin in patients undergoing SLED.Methods: ICU patients undergoing SLED, receiving meropenem and/or vancomycin, were prospectively evaluated. Blood samples were collected at the start of SLED and 0.5; 1; 2; 4 and 6 hours later. Antimicrobial levels were determined by HPLC. Noncompartimental pharmacokinetic analysis was performed. Area under the curve was determined for vancomycin. For meropenem, time above MIC was calculated. Results: 24 patients receiving vancomycin and 21 receiving meropenem were included; 170 plasma samples were obtained. Median serum vancomycin and meropenem concentrations: before SLED were 24.5 and 28.0 ?g/ml, respectively; and after SLED 14 and 6 ?g/ml, respectively. Mean removal was 41% for vancomycin and 78% for meropenem. For vancomycin, 22 (96%), 19(83%) and 16(70%) patients would have achieved the target (AUC0-24>400) considering MIC 0.5; <= 1mg/l and <= 2 mg/l, respectively. For meropenem, 19 (95%), 18 (90%) and 11(55%) patients would have achieved the target (70% of time above MIC) if infected with isolates with MIC <= 1, <= 4 and <= 8mg/l, respectively. Conclusions: In critically ill patients, meropenem and vancomycin were removed during SLED. Despite this, overall high PK/PD target attainment was obtained, except for higher MICs. We suggest maintenance doses of 1g tid or bid for meropenem. For vancomycin, more individualized approach using therapeutic drug monitoring should be used, as commercial assays are available

A sepse após a injúria térmica é a maior causa de morbidade e mortalidade em pacientes queimados, uma vez que profundas alterações ocorrem na farmacocinética de agentes antimicrobianos. Investigaram-se trinta e um pacientes, portadores de sepse documentada e apresentando lesões ativas; utilizou-se o tratamento empírico conforme seguem os regimes de dose: 1 g, 12/12 h para a vancomicina, 1 g, 6/6 h para o imipenem e 2 g, 8/8 h para o cefepime. Sete coletas seriadas de sangue foram realizadas através de cateter venoso (2 mL/cada); o plasma foi obtido pela centrifugação e armazenado no congelador (-80o C) até o ensaio. A concentração plasmática dos antimicrobianos foi determinada simultaneamente pela aplicação do método bioanalítico desenvolvido no estudo. O método de cromatografia líquida de alta eficiência demonstrou boa linearidade, precisão e exatidão para a determinação simultânea da vancomicina, cefepime e imipenem plasmáticos; a plicação desse método bioanalítico permitiu o monitoramento plasmático terapêutico e o estudo farmacocinético. Com base nos resultados obtidos de concentração plasmática versus tempo, aplicou-se a modelagem para investigar a farmacocinética desses agentes antimicrobianos nos pacientes queimados. Os parâmetros cinéticos foram estimados com base no modelo aberto de um compartimento pela aplicação do programa PK Solutions v. 2.0; a estatística foi realizada pela utilização do programa GraphPad Prism v. 4.0. Com base na farmacocinética alterada, as concentrações obtidas para a vancomicina e imipenem se mostraram abaixo dos valores recomendados para atingir eficácia; por outro lado, as concentrações obtidas para o cefepime se mostraram dentro da faixa recomendada para atingir eficácia, uma vez que não se registrou alteração da farmacocinética deste antimicrobiano nos pacientes queimados. Desta forma, o monitoramento plasmático terapeutico se mostrou importante, permitindo o ajuste de dose para a vancomicina e para o imipenem, uma vez que a concentração minima efetiva (CME) não foi atingida para ambos pela utilização do regime de dose empírica nos pacientes queimados. Por outro lado, o monitoramento do cefepime plasmático também se mostrou de relevância, uma vez que os pacientes queimados com longa permanência na terapia intensiva podem apresentar disfunção renal em alguma fase da internação; consequentemente, a individualização de dose será recomendada para esses pacientes. Adicionalmente, investigou-se a disposição cinética da vancomicina em nove pacientes queimados após duas diferentes intervenções cirúrgicas. Comparou-se a farmacocinética da vancomicina pós-desbridamento versus pos-enxerto com base no monitoramento plasmático após o regime de dose empírica (1 g, 12/12 h). Após multiplas infusões, o vale da vancomicina plasmática foi obtido pela coleta de sangue imediatamente antes da infusão subsequente e está relacionado ao acúmulo no estado de equilíbrio. Em conseqüência da depuração aumentada e meia-vida biológica reduzida pós-desbridamento comparado ao pós-enxerto, registrou-se para a vancomicina vale abaixo da concentração efetiva mínima nos pacientes queimados. Finalmente, os resultados obtidos no presente estudo permitem concluir que a farmacocinética da vancomicina e do imipenem está alterada nos pacientes queimados com sepse, e recomenda-se o monitoramento das concentrações plasmáticas para garantir a eficácia de forma a previnir a emergência bacteriana. / Sepsis after thermal injury is the major cause of morbidity and mortality in burn patients, once deep changes on the pharmacokinetics of antimicrobials agents are expected. Thirty one burn patients were investigated, all of them had documented sepsis and presented active lesions; they were treated with empirical dose regimen as follows: 1 g, 12/12 h for vancomycin, 1 g, 6/6 h for imipenem and 2 g, 8/8 h for cefepime. A serial of seven blood samples were collected from the venous catheter (2 mL/each); plasma was obtained by centrifugation and storaged in an ultra-low freezer (-80o C) until assay. Drug plasma concentration was determined simultaneously by application of a bioanalytical method described previously. High performance liquid chromatographic method showed good linearity, precision and accuracy for vancomycin, cefepime and imipenem plasma measurements; its application permitted therapeutic drug monitoring and pharmacokinetic studies. Pharmacokinetic modeling was applied to data obtained based on drug plasma concentrations versus time, to investigate those antimicrobial agents in burn patients. Estimated kinetic parameters were based on the one compartment open model by application the software PK Solutions v. 2.0; statistics was performed by using the software GraphPad Prism v. 4.0. Based on altered pharmacokinetics, obtained plasma concentrations to reach drug efficacy were below the recommended values for vancomycin and imipenem; on the other hand, cefepime plasma concentrations to reach drug efficacy were in the recommended range, once its pharmacokinetics didnt change in burn patients. Then, therapeutic plasma monitoring was cost-effective permitting dose adjustment for vancomycin and imipenem, once the minimum effective concentration (MEC) wasnt reached for both antimicrobial agents by using the empirical dose regimen for burn patients. On the other hand, cefepime plasma monitoring was also cost-effective, since burn patients long term therapy can present renal dysfunction at the minimum one period in the intensive care unit; consequently, dose adjustment could be required for them. Additionally, vancomycin kinetic disposition was investigated in nine burn patients after two different surgical interventions. Vancomycin pharmacokinetics post-debridement versus post-skin grafting procedure was compared based on drug plasma monitoring by using the empirical dose regimen (1 g, 12/12 h). Trough vancomycin plasma level after multiple infusions, obtained by blood collection before de next dose, is related to drug accumulation at the steady state level; then, trough below the minimum effective concentration (MEC) were obtained after both surgical procedures performed in burn patients. Meanwhile, increased plasma clearance and reduced biological half-life were obtained after debridement compared skin grafting procedure. Finally, data obtained in the present study permit to conclude that the pharmacokinetics is altered for vancomycin and imipenem in burn patients with sepsis, and drug plasma monitoring is recommended to guarantee drug efficacy and to prevent the bacterial emergency.

Chaque année les infections nosocomiales touchent plus de 4 millions de patients en Europe, avec un impact important en termes de mortalité, de morbidité et de coût. Parmi ces infections, celles causées par des bactéries multi-résistantes aux antibiotiques (BMR) jouent un rôle majeur. La modélisation mathématique des épidémies est un outil essentiel qui permet de mieux comprendre la dynamique de diffusion des BMR et d’évaluer l’efficacité des mesures de prévention.L'objectif principal de ce projet est d'étudier la dynamique de propagation de BMR au sein d'un réseau d'hôpitaux, en prenant en compte différentes échelles : intra-service, inter-services et inter-hôpitaux. Il s'agit de mettre en place une recherche méthodologique basée sur la modélisation mathématique et informatique et validée par des données recueillies au sein du réseau de soins Paris Île de France Ouest (PIFO), afin de mieux comprendre le rôle joué par chaque hôpital dans l'émergence et la sélection de BMR, de quantifier le risque de leur dissémination (y compris dans la population générale), et d'identifier des mesures de contrôle efficaces. Ce travail s'appuiera sur des méthodes d'inférence statistiques, d'analyse de sensibilité et d'analyses d'incertitude. / Each year nosocomial infections affect more than 4 million patients in Europe, with a significant impact in terms of mortality, morbidity and cost. Of these infections, those caused by multi-resistant bacteria (BMR) play a major role. Mathematical modeling of epidemics is an important tool to better understand the dynamics of dissemination of BMR and evaluate the effectiveness of prevention measures.The main objective of this project is to study the BMR propagation dynamics within a network of hospitals, taking into account different levels: intra-ward and inter-wards and inter-hospitals. This is to establish a research methodology based on mathematical and computer modeling and supported by data collected in the Paris Île de France Ouest (PIFO), to better understand the role played by each hospital in the emergence and selection of BMR, to quantify the risk of their dissemination (including in the general population), and to identify effective control measures. This work will be based on statistical inference methods, analytical sensitivity and uncertainty analysis.

En ökad utbredning av vankomycinresistenta enterokocker (VRE) har setts i Sverige sedan 2007. Bakteriemi orsakad av VRE är mycket svårbehandlad, varför snabbare tillförlitlig resistensdiagnostik är betydelsefullt för att minska dödlighet, vårdtider, vårdkostnader och belastning på sjukvårdssystemet. På mikrobiologilaboratoriet, Region Jönköpings län (RJL), tar idag identifiering av fenotypisk vankomycinresistens vid optimala förhållanden 6 timmar, räknat från att enterokocker konstaterats växa i blodet. Resistensgenerna vanA och vanB, som bland andra orsakar vankomycinresistens hos enterokocker, kan genetiskt verifieras med loop-mediated isothermal amplification men tar idag upp till ett dygn då bakteriekolonier används som analysmaterial i arbetsrutinen på molekylärbiologilaboratoriet, RJL. Syftet med studien var att utvärdera bakteriepellet som analysmaterial för genetisk identifiering av vanA och vanB, på Genie® II Mk2 med eazyplex® VRE basic, hos enterokocker från positiva blododlingar. För att utvärdera bakteriepellet som analysmaterial analyserades isolat av Enterococcus faecium (n=17) och Enterococcus faecalis (n=5) från bakteriepellets tillverkade från simulerade positiva blododlingar med eazyplex® VRE basic på Genie® II Mk2, varpå resultaten jämfördes mot isolatens faktiska närvaro/frånvaro av vanA/vanB. Samstämmigheten av de uppmätta- och de förväntade resultaten var fullständig, vilket indikerar att bakteriepellet med hög tillförlitlighet kan användas som analysmaterial till eazyplex® VRE basic för att påvisa vanA och vanB hos enterokocker i blododlingar. / An increased prevalence of vancomycin-resistant enterococci (VRE) has been observed in Sweden since 2007. Treating bacteremia caused by VRE is difficult, which is why faster, and reliable resistance diagnostics are important. At the Microbiology laboratory, Region Jönköping County, the identification of phenotypic vancomycin resistance under optimal conditions takes 6 hours from when growth of enterococci in blood is determined. The genes vanA and vanB, which among others cause vancomycin resistance, can be genetically verified by loop-mediated isothermal amplification, but takes up to one day since bacterial colonies are used as analysis material. The aim of the study was to evaluate bacterial pellet as an analytical material for genetic identification of vanA and vanB, on Genie® II Mk2 with eazyplex® VRE basic, in enterococci from positive blood cultures. To evaluate the bacterial pellet, isolates of Enterococcus faecium (n=17) and Enterococcus faecalis (n=5) from bacterial pellets made from simulated positive blood cultures were analyzed with eazyplex® VRE basic on the Genie® II Mk2, and the results were compared to the actual presence/absence of vanA/vanB in the isolates. The complete coherence between the expected and measured results indicates that the bacterial pellet can be used as an analytical material for eazyplex® VRE basic.

The thesis is focused on the research of stability of controlled drug release systems based on a blend of plasticized starch/polycaprolactone (TPS/PCL) that served as a carrier. Antibiotic vancomycin was used as a model drug, and its release from TPS/PCL pellets into aqueous environment was followed by UV-spectroscopy and the obtained time dependences were treated by a simple kinetic model. Moreover, the simultaneous release of starch particles to the surrounding liquid phase was studied by static and dynamic light scattering as well as transmission electron microscopy (TEM) in order to obtain information on the stability of biodegradable matrix and on the structure of the products of the pellet decomposition on a nanoscale level. Key words: vancomycin, starch, drug delivery system, polycaprolactone (PCL), particle release, dynamic light scattering (DLS), static light scattering (SLS)

La vancomycine est l'un des antibiotiques les plus prescrits, principalement utilisé pour les infections suspectées et confirmées à Staphylococcus aureus résistant à la méthicilline (SARM). Les infections par des souches de SARM font peser une charge importante sur le système de santé, à laquelle s'ajoute l'incertitude qui demeure quant à la posologie optimale de la vancomycine. Les récentes lignes directrices révisées sur le suivi thérapeutique de la vancomycine, publiées en 2020, avalisent principalement l'estimation directe de l'aire sous la courbe de concentration en fonction du temps (AUC) par l'utilisation d'équations bayésiennes ou pharmacocinétiques (PK) de premier ordre pour le suivi thérapeutique. Pour mieux informer la posologie de la vancomycine, nous avons d'abord mis à jour une revue précédente des analyses pharmacocinétiques de population (PopPK) de la vancomycine publiées chez les adultes et les enfants. Pour ce faire, nous avons déterminé les caractéristiques des modèles pharmacocinétiques rapportés et identifié les diverses sources potentielles de variabilité observées dans différentes sous-populations particulières. Motivés par la controverse existante autour des nouvelles directives de surveillance thérapeutique de la vancomycine et par l'absence d'une étude approfondie des méthodes recommandées, nous avons recueilli des données hospitalières et construit un cadre de modélisation qui nous a permis d'évaluer les recommandations des directives sur les méthodes de surveillance, tout en considérant une variété de scénarios et d'hypothèses cliniques réalistes. Notre analyse a confirmé que la surveillance bayésienne est la méthode la plus rapide et la plus fiable, à condition qu'elle soit correctement mise en œuvre, la plus importante condition pour cela étant l'utilisation de modèles bayésiens a priori appropriés. De plus, nous avons montré que le suivi bayésien ne nécessite pas nécessairement des niveaux de concentration de types creux ou pic et peut en fait être réalisé en utilisant un niveau aléatoire. Aussi, nous avons démontré que l'utilisation correcte des équations pharmacocinétiques de premier ordre exigerait au moins deux mesures de concentration à l'état d'équilibre. L’utilisation de la méthode creux-seulement de la vancomycine à l'état d'équilibre peut être tout aussi efficace dans certaines situations que nous avons explorées ici. En considérant la larges étendue et la grande variabilité des populations traitées à la vancomycine en termes d'âge, de gravité de l'infection et de scénarios cliniques, cette thèse adopte un regard objectif pour évaluer quantitativement le gain potentiel de chaque méthode de surveillance de la vancomycine, en explorant leur adéquation en termes d'effort nécessaire, de disponibilité des ressources et de gain potentiel. Compte tenu des lignes directrices sur la vancomycine récemment publiées et de la controverse qui persiste, nous pensons que cette thèse a permis de démêler la variété et la complexité de l'utilisation de la vancomycine et a apporté un éclairage supplémentaire plus objectifvement informé vers un suivi thérapeutique optimal de la vancomycine. / Vancomycin is among the most prescribed antibiotics, mainly used for suspected and confirmed methicillin-resistant Staphylococcus aureus (MRSA) infections. Infections by MRSA strains carry a substantial burden on the health care system, supplemented by the uncertainty that remains regarding vancomycin optimal dosing. The recent revised vancomycin therapeutic monitoring guidelines published in 2020, endorsed primarily the direct estimation of area under the concentration-time curve (AUC) through the use of Bayesian or first-order pharmacokinetic (PK) equations monitoring. To better inform vancomycin dosing, we first updated a previous review of published vancomycin population pharmacokinetic (PopPK) analysis in both adults and children. This was accomplished by determining the characteristics of the reported pharmacokinetic models and identifying the potential various sources of variability observed in different special subpopulations. Motivated by the existing controversy around the new vancomycin therapeutic monitoring guidelines and the lack of a thorough investigation of the recommended methods, we collected hospital data and built a modeling framework that allowed us to assess the guideline recommendations of monitoring methods while considering a variety of realistic clinical scenarios and assumptions. Our analysis affirmed that Bayesian monitoring is the fastest and most reliable method, conditional on its proper implementation, the most important being the use of proper Bayesian priors. Moreover, we showed that Bayesian monitoring does not necessarily require trough or peak concentration levels and can in fact be performed using a random level. Proper use of first-order PK equations required at least two steady-state concentration measurements. Alternatively, simpler trough-only vancomycin monitoring near steady-state can be as effective in certain cases that we explored here. By considering the wide ranges and the high variability in populations treated with vancomycin in terms of age, the severity of infection, and clinical scenarios, this thesis takes an objective look to quantitatively assess the potential gain of each vancomycin drug monitoring method, by investigating their suitability in terms of the effort needed, the availability of resources and the resulting gain. Considering the recently released vancomycin guidelines and the ensuing controversies between well-established clinical teams, we believe that this dissertation helped untangle the variety and complexity of vancomycin use and brought additional insights towards a more objective and optimal vancomycin therapeutic monitoring.

La vancomycine est un antibiotique fréquemment utilisé dans le contexte hospitalier pour les infections cutanées et nosocomiales. Son utilisation nécessite un suivi thérapeutique pharmacocinétique (TDM) de la part du clinicien, étant donné l’index thérapeutique étroit et la variabilité de son profil pharmacocinétique entre les individus. Alors que le risque de néphrotoxicité associée à la vancomycine s’accroît avec sa durée de traitement, sa clairance et son volume de distribution deviennent difficiles à prédire dans le contexte des traitements prolongés, ce qui est souvent requis chez les patients avec infections ostéoarticulaires. Avec l’approche de modélisation pharmacocinétique de population (popPK), ce projet de maîtrise a cherché à évaluer les changements longitudinaux des paramètres pharmacocinétique de la vancomycine dans une population de patients atteints d’infections ostéoarticulaires. Dans un premier temps, nous avons décrit la pratique de TDM chez les patients qui recevaient de la vancomycine intraveineuse (IV) pour les infections ostéoarticulaires à l’Hôpital Général de Montréal entre décembre 2020 et décembre 2022. Dans un deuxième temps, nous avons identifié deux modèles popPK longitudinaux dans la littérature et évalué leur performance prédictive dans cette population. Bien que ces modèles proposent des approches intéressantes pour décrire les changements longitudinaux de la vancomycine, ils se sont avérés inadéquats pour décrire les paramètres pharmacocinétiques de cet antibiotique dans notre population. D’autres travaux seront nécessaires pour développer et valider des modèles longitudinaux de la vancomycine qui devront tenir compte des variables qui décrivent l’état inflammatoire du patient et des méthodes alternatives pour intégrer une structure longitudinale dans le modèle popPK. / Vancomycin is commonly used in the hospital setting to treat skin and soft tissues infections as well as nosocomial infections. As vancomycin has a small therapeutic window and its pharmacokinetic properties vary significantly across individuals, clinicians must ensure close therapeutic drug monitoring (TDM). As the risk of vancomycin induced nephrotoxicity increases with duration of therapy, clearance and distribution of vancomycin become difficult to predict in the context of long term treatment which is often required for osteoarticular infections. With the use of population pharmacokinetic (popPK) modeling, we aimed to examine the longitudinal changes in the pharmacokinetic properties of vancomycin in patients with osteoarticular infections. In the first part of this master’s project, we described the local practices of TDM in patients receiving intravenous (IV) vancomycin for osteoarticular infections at the Montreal General Hospital between December 2020 et December 2022. In the second part, we identified two longitudinal popPK models in the literature and assessed their predictive performance in this population. Although these models offer an interesting approach to integrate a longitudinal component in their structure, they were ultimately not applicable to our population. Further efforts to address the time related changes of vancomycin’s pharmacokinetics should take into account clinical factors such as the degree of systemic inflammation and consider alternative methods to integrate the duration of treatment and longitudinal components in the model structure.

Σκοπός της παρούσας ερευνητικής εργασίας ήταν η επιδημιολογική επιτήρηση της φορείας και των λοιμώξεων από Klebsiella pneumoniae που παράγει καρβαπενεμάση KPC (KPC-Kp), ανθεκτικό σε βανκομυκίνη Enterococcus (VRE) και ανθεκτικό σε μεθικιλλίνη Staphylococccus aureus (MRSA) σε ασθενείς που νοσηλεύονται στις Μονάδες Εντατικής Θεραπείας (ΜΕΘ) του Πανεπιστημιακού Γενικού Νοσοκομείου Πατρών (ΜΕΘ Α) και του Νοσοκομείου «Άγιος Ανδρέας» (ΜΕΘ Β) τη χρονική περίοδο Οκτώβριος 2009 έως Φεβρουάριος 2012. H διασπορά της KPC-Kp αποτελεί το σημαντικότερο πρόβλημα στις Ελληνικές ΜΕΘ, με τα ποσοστά της να αυξάνονται στις παθολογικές και χειρουργικές κλινικές. Κατά τη διάρκεια της παρούσας μελέτης, 12.8% των ασθενών που εισήχθηκαν στη ΜΕΘ Α (52 από 405 ασθενείς) ήταν αποικισμένοι από KPC-Kp κατά την εισαγωγή τους με την προηγηθείσα νοσηλεία σε ΜΕΘ, την χρόνια αποφρακτική πνευμονοπάθεια, τη διάρκεια προηγηθείσας νοσηλείας και την προηγηθείσα χορήγηση καρβαπενέμης ή συνδυασμού β-λακτάμης/αναστολέα λακταμάσης να συμβάλλουν στον αποικισμό. Παρατηρήθηκε μία σταδιακή αύξηση των αποικισμένων ασθενών που εισάγονται στη ΜΕΘ με 3.9% (4 από 102 ασθενείς) τους πρώτους 6 μήνες σε σύγκριση με 15.8% (48 από 300 ασθενείς) τους επόμενους 16 μήνες που αντικατοπτρίζει τη σταδιακή διασπορά της KPC-Kp σε κλινικές εκτός ΜΕΘ. Από τους 226 μη αποικισμένους ασθενείς κατά την εισαγωγή στη ΜΕΘ Α, 164 (72.6%) αποικίστηκαν κατά τη διάρκεια της νοσηλείας τους με σημαντικότερους παράγοντες που επηρεάζουν τον αποικισμό να είναι η παρουσία αποικισμένων ασθενών σε διπλανές κλίνες και η νοσηλεία σε κλίνη προηγουμένως αποικισμένου ασθενή, ενώ δε βρέθηκε συσχέτιση ανάμεσα στον αποικισμό και τη θνησιμότητα. Το υψηλό ποσοστό αποικισμού σε συνδυασμό με τους προηγούμενους παράγοντες υποδεικνύει την σημασία της διασποράς της KPC-Kp από ασθενή σε ασθενή μέσω του ιατρονοσηλευτικού προσωπικού και υποδηλώνει τη σημασία πιο αυστηρής εφαρμογής της πολιτικής ελέγχου λοιμώξεων. Συνολικά 53 ασθενείς της ΜΕΘ Α ανέπτυξαν βακτηριαιμία από KPC-Kp με 43.4% θνησιμότητα. Οι σημαντικότεροι παράγοντες που επηρεάζουν τη θνησιμότητα είναι η αντοχή του στελέχους σε κολιστίνη/τιγεκυκλίνη/γενταμικίνη και η σηπτική καταπληξία, ενώ η θεραπεία με συνδυασμό τουλάχιστον δύο δραστικών αντιβιοτικών σχετίζεται με καλύτερη πρόγνωση επιβεβαιώνοντας τα αποτελέσματα προηγούμενων μελετών υπέρ της συνδυαστικής θεραπείας στην καταπολέμηση των λοιμώξεων από KPC-Kp. Η ανάπτυξη αντοχής των στελεχών KPC-Kp έναντι της κολιστίνης ή της τιγεκυκλίνης, οι οποίες αποτελούν τις τελευταίες θεραπευτικές επιλογές για το συγκεκριμένο παθογόνο, είναι ένα ανησυχητικό φαινόμενο. Συνολικά, 24.4% και 17.9% των ασθενών της ΜΕΘ Α αποικίστηκαν από στέλεχος KPC-Kp ανθεκτικό στην κολιστίνη και τιγεκυκλίνη, αντίστοιχα. Όπως αναμενόταν η λήψη των συγκεκριμένων αντιβιοτικών συνέβαλε στον αποικισμό, όμως ο σημαντικότερος παράγοντας για αποικισμό ήταν η παρουσία αποικισμένου ασθενή στις διπλανές κλίνες υποδηλώνοντας τη σημασία της διασποράς των στελεχών και όχι της de novo ανάπτυξη αντοχής. Η σύγκριση των δύο ΜΕΘ, ανέδειξε ότι μεγαλύτερο ποσοστό των ασθενών της ΜΕΘ Α αποικίζονται κατά τη διάρκεια νοσηλείας σε σχέση με τη ΜΕΘ Β (61.8% vs 34.1%) και σε συντομότερο χρονικό διάστημα (10.6 vs 19.9 ημέρες). Τα στοιχεία αυτά μπορούν να ερμηνευτούν από το υψηλότερο ποσοστό εισαγωγών αποικισμένων ασθενών (11.4% vs 1.8%), τη μικρότερη αναλογία νοσηλευτών/ασθενών καθώς και την αυξημένη κατανάλωση καρβαπενεμών στη ΜΕΘ Α. Συνολικά, 305 και 100 στελέχη K. pneumoniae που απομονώθηκαν από τη ΜΕΘ Α και Β, αντίστοιχα, ήταν θετικά για την παρουσία του γονιδίου blaKPC ενώ πέντε στελέχη της ΜΕΘ Α ήταν θετικά και για το γονίδιο blaVIM. Και στις δύο ΜΕΘ τα στελέχη ήταν ανθεκτικά σε πενικιλλίνες, στις κεφαλοσπορίνες, στην αζτρεονάμη, στην τριμεθοπρίμη-σουλφαμεθοξαζόλη (30% των στελεχών της ΜΕΘ Β ήταν ευαίσθητα), στην αμικασίνη, στην τομπραμυκίνη και στις κινολόνες. Η αντοχή στις καρβαπενέμες (67.9% vs 60%), στην κολιστίνη (35.1% vs 18%), στη γενταμικίνη (50.8% vs 24%) και στην τιγεκυκλίνη (17% vs 18%) στα στελέχη των δύο ΜΕΘ κυμαινόταν στα ίδια επίπεδα. Πενήντα επτά και 20 στελέχη της ΜΕΘ Α και Β, αντίστοιχα, ταυτοποιήθηκαν με PFGE, η οποία ανέδειξε την παρουσία δύο τύπων στη ΜΕΘ Α, με τον τύπο Α να απαρτίζεται από το 65.5% των στελεχών, ενώ στη ΜΕΘ Β όλα τα στελέχη ανήκαν στον τύπο Α. Τα ποσοστά αποικισμού από VRE στις δύο ΜΕΘ είναι χαμηλότερα σε σχέση με αυτά της KPC-Kp. Αποικισμός κατά την εισαγωγή στη ΜΕΘ παρατηρήθηκε σε 14.3% (71 από 497 ασθενείς), ενώ κατά τη διάρκεια νοσηλείας ήταν 14.4% (36 από 250 ασθενείς). Ο σημαντικότερος παράγοντας για αποικισμό από VRE κατά τη διάρκεια νοσηλείας είναι η νοσηλεία αποικισμένων ασθενών σε διπλανές κλίνες υποδεικνύοντας ότι η μη τήρηση των μέτρων υγιεινής των χεριών ίσως διαδραματίζει το σημαντικότερο ρόλο στη διασπορά του VRE. Συνολικά 107 στελέχη VRE απομονώθηκαν (100 E. faecium και 7 E. faecalis). Ογδόντα τέσσερα στελέχη έφεραν το γονίδιο vanA και ήταν ανθεκτικά στη βανκομυκίνη και στην τεϊκοπλανίνη, ενώ τα υπόλοιπα 23 έφεραν το γονίδιο vanB και χαρακτηρίζονταν από χαμηλού επιπέδου αντοχή στη βανκομυκίνη (12 στελέχη ήταν ευαίσθητα) και ευαίσθητα στην τεϊκοπλανίνη. Όλα τα στελέχη ήταν ευαίσθητα στη λινεζολίδη, στη δαπτομυκίνη και στην τιγεκυκλίνη. Η MLST αποκάλυψε ότι τα στελέχη E. faecium ανήκουν σε έξι διαφορετικούς κλώνους (STs: ST117, ST17, ST203, ST226, ST786, ST125) με το 90% των E. faecium, ανήκουν στο Κλωνικό Σύμπλεγμα 17 (Clonal Complex CC17). Τα στελέχη E. faecalis ταξινομήθηκαν σε τέσσερις κλώνους (STs: ST6, ST41, ST19, ST28). Τα ποσοστά αποικισμού από MRSA κατά την εισαγωγή και κατά τη διάρκεια νοσηλείας είναι χαμηλά (5.3% και 3.7%, αντίστοιχα) με το σημαντικότερο παράγοντα που σχετίζεται με τον αποικισμό να είναι ο εντερικός αποικισμός με vanA-θετικό στέλεχος Enterococcus. Ο έλεγχος φορείας για MRSA ανέδειξε 28 mecA-θετικά στελέχη S. aureus, με την πλειονότητα (ν=19) να είναι PVL-θετικά, να ανήκουν στον κλώνο ST80 και να είναι ανθεκτικά σε καναμυκίνη, τετρακυκλίνη και φουσιδικό, ενώ τα υπόλοιπα ταξινομήθηκαν σε τέσσερις κλώνους με MLST (6 στον ST239 και από ένα σε ST225, ST72 και ST30). Το στέλεχος που ανήκε στον ST30 ήταν tst-θετικό. Η σύγκριση των στελεχών φορείας S. aureus που απομονώθηκαν από αθενείς (ν=67) και προσωπικό (ν=23) των ΜΕΘ (Ομάδα Α) με τα στελέχη φορείας (ν=53) και βακτηριαιμιών (ν=75) μη νοσηλευόμενων σε ΜΕΘ (Ομάδα Β), ανέδειξε υψηλότερο ποσοστό MRSA (46.9% vs 31.1%) και PVL-θετικών στελεχών (39.8% vs 25.6%) στην Ομάδα Β, ενώ η Ομάδα Α χαρακτηρίζεται από υψηλότερο ποσοστό tst-θετικών στελεχών (21.1% vs 2.3%) υποδεικνύοντας τη σιωπηρή τους διασπορά στους ασθενείς και στο προσωπικό των ΜΕΘ. Προϊόν της παρούσας ερευνητικής εργασίας ήταν η ανεύρεση των παραγόντων κινδύνου για αποικισμό ή λοίμωξη από KPC-Kp, VRE και MRSA με στόχο την καθοδήγηση των μελλοντικών προσπαθειών περιορισμού της διασποράς τους στις δύο ΜΕΘ καθώς και στα ελληνικά νοσοκομεία, τα οποία στο σύνολο τους μαστίζονται από τα συγκεκριμένα παθογόνα. / The purpose of this study was to investigate the colonization and infections caused by KPC-producing Klebsiella pneumoniae (KPC-Kp), vancomycin-resistant Enterococcus (VRE) and methicillin-resistant Staphylococcus aureus in patients hospitalized in the Intensive Care Units of the University Hospital of Patras (ICU A) and the General Hospital “Saint Andrew” during October 2009 and February 2012. The dissemination of KPC-Kp constitutes the most important issue in Greek ICUs, with its percentage rising in medical and surgical wards. During the duration of this study, 12.8% of patients admitted in the ICU A (52 from 405 patients) were colonized upon admission and previous ICU stay, chronic obstructive pulmonary disease, duration of previous hospitalization and previous usage of carbapenem or combination of beta-lactamic/lactamase were found to influence colonization. A gradual increase of the percentage of colonized patients admitted at the ICU from 3.9% (4 from 102 patients) during the first 6 months to 15.8% (48 from 300 patients) the next 16 months that reflects the dissemination of KPC-Kp in non-ICU wards. Among the 226 non-colonized upon ICU A admission patients, 164 (72.6%) became colonized during their stay with the presence of colonized patients in nearby beds and the previous colonized occupant in the same bed were associated with colonization, which did not influence mortality. The high percentage of colonization in combination with the aforementioned factors indicates the importance of the dissemination of KPC-Kp among patients via the personnel and signifies the value of a strict implementation of infection control protocols. In total, 53 patients developed KPC-Kp bloodstream infection during ICU A stay with 43.4% mortality. The most important factors that influence mortality were the resistance of the strain to gentamicin/colistin/tigecycline and septic shock, while the treatment with two active antibiotics was associated with better survival confirming the results of previous studies favoring combination therapy for the treatment of KPC-Kp infection. The development of resistance against colistin or tigecycline, which are considered the last frontier in the treatment of KPC-Kp infections, is an alarming phenomenon. In total, 24.4% and 17.9% of ICU A patients became colonized by KPC-Kp resistant to colictin or tigecycline, respectively. As expected, the administration of colistin or tigecycline influenced colonization, while the most important factor favoring colonization was the presence of colonized patients in nearby patients, indicating the importance of dissemination of these strains against de novo resistance development. The comparison of the two ICUs, found a higher percentage of patients colonized during ICU A stay (61.8% vs 34.1%) and in a shorter period (10.6 vs 19.9 days). These results may be explained by the higher percentage of patients colonized upon admission (11.4% vs 1.8%), the lower nurse/patient ration and the higher carbapenem administration. In total, 305 and 100 strains of K. pneumoniae isolated from patients hospitalized in ICU A and B, respectively, were positive for the presence of blaKPC gene while five strains in ICU A were positive for the blaVIM gene also. All strains were resistant to penicillins, cephalosporins, aztreonam, trimethoprim sulfamethoxazole (30% of ICU B strains were sensitive), amikacin, tombramycin and quinolones. The resistance rates to carbapenems (67.9% vs 60%), colisitn (35.1% vs 18%), gentamicin (50.8% vs 24%) and tigecycline (17% vs 18%) among the ICUs strains were comparable. PFGE of 57 and 20 isolates from ICU A and B, respectively, revealed that ICU A strains belonged in two types, with type A comprising 65.5% of the isolates, while all ICU B isolates belonged in type A. The percentage of VRE colonization in both ICUs were lower in comparison with those of KPC-Kp. During ICU admission 14.3% (71 from 497 patients) was already colonized, while 14.4% (36 from 250 patients) became colonized during stay. The most important factor influencing colonization was the presence of colonized patients in nearby beds, indicating that non adherence with hand hygiene may play a predominate role in VRE dissemination. In total 107 VRE strains were isolated (100 E. faecium and 7 E. faecalis). Eighty four were positive for the vanA gene and resistant to vancomycin and teicoplanin, while the rest were vanB positive and were characterized by low level resistance to vancomycin (12 were in susceptibility range) and susceptible to teicoplanin. All strains were susceptible to linezolid, daptomycin and tigecycline. As MLST revealed, E. faecium strains belonged in six different Sequencing Types (ST117, ST17, ST203, ST226, ST786, ST125) with 90% among them belonging to the Clonal Complex CC17. E. faecalis strains were categorized in four STs (ST6, ST41, ST19, ST28). The proportion of colonized patients by MRSA upon admission and during ICU stay was very low (5.3% and 3.7%, respectively). The most important factor associated with colonization was enteric carriage of vanA-positive Enterococcus. Surveillance cultures revealed 28 mecA-positive S. aureus strains, with the majority (n=19) being PVL-positive, belonging to ST80 and resistant only to kanamycin, tetracycline and fucidic acid, while the remaining were categorized in four STs (6 strains in ST239 and one at ST225, ST72 and ST30). The ST30 strain was tst-positive. The comparison of colonization strains from patients (n=67) and personnel (n=23) of the ICUs (Group A) with the strains of colonization (n=53) and bloodstream infections (n=75) isolated from non-ICU patients (Group B), revealed a higher percentage of MRSA and PVL-positive strains in Group B, while Group A was characterized by higher percentage of tst-positive strains indicating their silent dissemination between ICU patients and personnel. The present study has identified the risk factors for colonization of infection by KPC-Kp, VRE and MRSA, in order to guide the future efforts towards containing their dissemination in the two ICUs, as well as, to the Greek hospitals, which in total are plagued by the aforementioned pathogens.