Tesis presentada a la Universidad de Chile para optar al grado de Doctor en Farmacología / En abril del año 2015, la organización mundial de la salud (OMS), presentó un informe que reveló la situación actual en torno a la resistencia a antimicrobianos de 114 países, lo que dejó en evidencia un preocupante escenario: el fenómeno de la resistencia se encuentra diseminado en todo el orbe y está presente en una gran variedad de patógenos responsables de infecciones comunes, tales como: las del tracto urinario, la gonorrea, la diarrea, la neumonía y la sepsis. Dentro de estos agentes se encuentran diversos bacilos Gram negativos, Staphylococcus aureus resistente a meticilina y Enterococcus spp. resistente a vancomicina, siendo estos últimos responsables de enfermedades con altos índices de mortalidad, como son la sepsis y la endocarditis. A este escenario se suma el escaso avance e inversión por parte de las grandes industrias farmacéutica en el desarrollo de nuevos antimicrobianos. Frente a esto, el año 2010, la Sociedad Americana de Enfermedades Infecciosas (IDSA) en conjunto a otras entidades, lanzaron el programa “The 10x‘20 Initiative: Pursuing a Global Commitment to Develop 10 New Antibacterial Drugs by 2020”, una iniciativa que busca hacer un llamado a los diferentes actores de la salud pública mundial, a incentivar el desarrollo de nuevas entidades moleculares (NEMs) con actividad antimicrobiana para el año 2020. El Laboratorio de Desarrollo de Fármacos de la Universidad de Chile ha trabajado en la desarrollo de compuestos arilmercaptoquinonicos diseñados racionalmente a partir de la estructura de la ubiquinona, buscando intervenir la cadena de transportadora de electrones bacteriana (CTeB). Resultados previos han demostrado que los compuestos obtenidos poseen una interesante actividad sobre bacterias Gram positivas tales como S. aureus resistente a meticilina, S. aureus sensible a meticilina y E. faecalis. De la serie de compuestos sintetizados destacaron; DFUCh-O5 y DFUCh-P4. En la presente investigación se buscó determinar el potencial de aplicación terapéutica de los derivados sintetizados y con ello discernir cuál es el mejor candidato para poder avanzar a fases preclínicas que consideren la utilización de modelos animales. En primer término, se estudió la toxicidad de los compuestos sobre células humanas y murinas a través del método 15 de MTT. Además, se determinaron los valores Concentración Inhibitoria Mínima90 (CIM90) y Concentración Bactericida Minima90 (CBM90) para los derivados en estudio de acuerdo a las normas de CLSI, sobre población de aislamientos clínicos de S. aureus y E. faecium multirresistentes, obtenidos desde distintos sitios estériles (sangre, huesos, etc.) y diferentes centros hospitalarias de todo Chile durante el año 2014, provistos por el Instituto de Salud Pública de Chile. Se caracterizó la actividad antibacteriana de los derivados, a través de la cuantificación del efecto post-antibiótico, el estudios de las curvas de muerte de los derivados, el potencial de inducción de mutaciones de los derivados sobre cultivos expuesto en forma aguda y crónica a estos. Por otra parte, se evaluó la actividad de los compuestos en asociación con linezolid, vancomicina y daptomicina, por determinación del índice de CFI (Concentración Fraccionaria Inhibitoria). Adicionalmente, se determinó el porcentaje de unión a albumina de los derivados y se estudió cómo influye esta proteína en la actividad de los compuestos in vitro. Por último, se estudió la efectividad in vivo utilizando un modelo de infección en larvas de Galleria mellonella. Se evidenció que DFUCh-O5 y DFUCh-P4 no afectan la viabilidad de células mamíferas; HeLa, SH-SY5Y y H4-II-E-C3, a las concentraciones en que son activos como antibacterianos. Los compuestos poseen CIM90 similar a la exhibida por vancomicina sobre los aislamientos clínicos de S. aureus resistentes a meticilina. Frente a los aislamientos de E. faecium resistentes a vancomicina estudiados, DFUCh-P4 resultó 64 veces más activo que vancomicina y DFUCh-O5 128 veces más activo que esté fármaco. Los derivados son bactericidas en todos los aislamientos estudiados. En los estudios de cinética de muerte, los compuesto tardaron menos de 2 horas en reducir el 99,9% de la población bacteriana ensayada. Los derivados no tienen efecto post antibiótico significativo sobre las especies S. aureus y Enterococcus sp. prototipo. Ambos derivados tienen potencial de inducir mutaciones frente a una exposición aguda y cronica en S. aureus resistente a meticilina, pero estas mutaciones no se mantienen en la generaciones sucesivas de la población bacteriana. DFUCh-P4 y DFUCh-O5 poseen la misma 16 efectividad que vancomicina sobre los modelos de infección en G. mellonela provocado por S. aureus sensible a meticilina o E. faecalis. En este trabajo se demostró, que los derivados poseen potencial terapéuticos como compuestos antibacterianos de uso humano, ya que poseen una alta actividad sobre bacterias Gram positivas multirresistentes y no afectan la viabilidad de células mamíferas / In April 2015, the World Health Organization (WHO) presented a report that revealed the current situation regarding antimicrobial resistance in 114 countries, which revealed a dangerous scenario: the phenomenon of resistance It is disseminated throughout the world and is present in a wide variety of pathogens responsible for common infections, such as those of the urinary tract, gonorrhea, diarrhea, pneumonia and sepsis. Among these agents are Gram-negative bacilli, methicillinresistant Staphylococcus aureus (MRSA) and Enterococcus sp. Resistant to vancomycin (ERV), are the last responsible for diseases with high mortality rates, such as child sepsis and endocarditis. Added to this scenario is the scarce advancement and investment by large pharmaceutical companies in the development of new antimicrobials. In response to this, in 2010, the American Society for Infectious Diseases (IDSA), together with other entities, launched the program "The 10x'20 Initiative: Looking for a global commitment to develop 10 new antibacterial drugs by 2020", an initiative that. The Drug Development Laboratory of the University of Chile has worked on the development of new resources for the development of new molecular entities (NEMs) with antimicrobial activity for the year 2020. Arilmercaptoquinonics compounds rationally designed from the structure of ubiquinone, looking to block the bacterial electron transport chain (CTeB). Previous results have shown that the compounds obtained have an interesting activity on Gram-positive bacteria such as S. aureus susceptible to methicillin, MRSA and Enterococcus faecalis. From the series of synthesized compounds, it highlighted; DFUCh-O5 and DFUCh-P4. In the present investigation, we sought to determine the potential of therapeutic application of the synthesized derivatives and thereby discern which is the best candidate to be able to advance to preclinical phases that consider the use of animal models. First, the toxicity of the compounds was studied on human and murine cells through the MTT method. The values MIC90 and MBC90 were determined for the derivatives under study according to CLSI standards, on population of clinical isolates of Staphylococcus aureus and Enterococcus faecium multiresistant, isolated from different sterile sites (blood, bones, etc.) and different 18 hospital centers of Chile during 2014, provided by the Institute of Public Health of Chile. The antibacterial activity of the derivatives was characterized, through the quantification of the post-antibiotic effect, the studies of the time-kill curves of the derivatives, the potential for the induction of mutations of the derivatives on cultures exposed in acute and chronic form to these. On the other hand, the activity of the compounds in association with linezolid, vancomycin and daptomycin was evaluated by determining the CFI index (Fractional Inhibitory Concentration) In addition, the albumin binding percentage of the derivatives was determined and this protein was studied. in the activity of the compounds in vitro Finally, effectiveness was studied in vivo using a model of infection in larvae of Galleria mellonella. Evidence of DFUCh-O5 and DFUCh-P4 does not affect the viability of mammalian cells; HeLa, SH-SY5Y and H4-II-E-C3, at concentrations that are active as antibacterial drugs. The compounds possess a vancomycin-like activity on the clinical isolates of S. aureus resistant to methicillin. Faced with the isolates of vancomycin-resistant E. faecium studied, DFUCh-P4 was 64 times more active than vancomycin and DFUCh-O5 was 128 times more active than the drug. The activity exhibited is bactericidal in all isolates studied. In studies of death kinetics, isolation took less than hours to reduce 99.9% of the bacterial population tested. The compounds have no significant post-antibiotic effect on S. aureus and Enterococcus spp. prototype. Both derivatives have the potential to induce mutations in the face of acute and chronic MRSA exposure, but these mutations are not maintained in successive generations of the bacterial population. DFUCh-P4 and DFUCh-O5 have the same effectiveness than vancomycin on G. mellonela model infection by S. aureus or E. faecalis. In this study it was demonstrated that the derivatives have therapeutic potential as antibacterial compounds for human use, since they have a high activity on multiresistant Gram positive bacteria and do not affect the viability of mammalian cells

Memoria para optar al título de Químico Farmacéutico / En la actualidad, y en los últimos diez años, la nanotecnología ha entrado en auge, dada su capacidad para resolver problemáticas de distinta índole. En particular, la nanomedicina permite generar distintas plataformas de entrega de fármacos, los cuales pueden ser protegidos y guiados a distintos tejidos y órganos. El objetivo del presente trabajo es desarrollar nanopartículas de óxido de zinc (ZnO) dopadas con magnesio, que permitan la conjugación a drogas y puedan disolverse a pH tumoral (pH 4.0 – 5.5). Las nanopartículas de ZnO poseen la particularidad de disolverse en condiciones ácidas. De esta forma, al ingresar a la célula por medio de endocitosis, las nanopartículas pueden componer un sistema de entrega de fármaco antitumoral controlado, con disolución de las mismas, liberando Zn+2 al medio intracelular de la célula en conjunto con la droga transportada, y con un potencial efecto sinérgico. Para explorar este sistema de entrega de fármacos, la primera parte del trabajo correspondió a la síntesis de las nanopartículas. Para lo cual se empleó el método de precipitación, el que consiste en la reacción de sales de zinc y magnesio con hidróxido de potasio. En la segunda parte del trabajo se evaluó el perfil toxicológico de las nanopartículas de ZnO en embriones de Danio rerio para corroborar su potencial toxicidad. Luego, las nanopartículas fueron funcionalizadas con dos polímeros, polialilamina y poliestirensulfonato, y un aminosilano APTES, con el objeto de obtener nanopartículas que puedan conjugarse con distintas drogas. Finalmente, se utilizó curcumina, como droga modelo, conjugada con la nanopartícula desnuda y funcionalizada, con el objeto de evaluar la capacidad de asociación de la droga a la nanopartícula. Se lograron desarrollar nanopartículas de ZnO dopadas con magnesio, las cuales tienen gran capacidad de adhesión a drogas que tengan estructura de tipo carbonilo-hidroxilo (por ejemplo, curcumina, doxorubicina y daunorubicina), posiblemente por medio de la formación de compuestos coordinados. En embriones de Danio rerio, son tóxicas, sin embargo, el recubrimiento del conjunto nanopartícula-droga, podría disminuir la toxicidad de estas. Así, próximos estudios biológicos podrán explorar estos aspectos en mayor detalle y la mejora del nanosistema / Currently, and in the last ten years, nanotechnology has boomed, given its ability to solve problems of various kinds. In particular, nanomedicine allows generating different drug delivery platforms, which can be protected and guided to different tissues and organs. The objective of this work was to develop zinc oxide nanoparticles (ZnO) doped with magnesium, which allows conjugation to drugs and can dissolve at tumoral pH (pH 4.0 - 5.5). The ZnO nanoparticles have the particularity of dissolving in acidic conditions. In this way, upon entering the cell by means of endocytosis, the nanoparticles can compose a controlled antitumor drug delivery system, with their dissolution, releasing Zn+2 in the intracellular medium of the cell in conjunction with the transported drug, and with a potential synergistic effect. To explore this drug delivery system, the first part of the work corresponded to the synthesis of nanoparticles. For this, the precipitation method was used, which consists of the reaction of zinc and magnesium salts with potassium hydroxide. In the second part of the work, the toxicological profile of the ZnO nanoparticles in Danio rerio embryos was evaluated to corroborate its potential toxicity. Then, nanoparticles were functionalized with two polymers, polyalylamine and polystyrene sulfonate, and an aminosilane APTES, in order to obtain nanoparticles that could be conjugated with different drugs. Finally, curcumin was used, as a model drug, conjugated with the nude and functionalized nanoparticle, in order to evaluate the association of the drug to the nanoparticle. ZnO nanoparticles doped with magnesium were developed and have great capacity of adhesion to drugs that have carbonyl-hydroxyl groups in their structure (for example, curcumin, doxorubicin and daunorubicin), possibly through the formation of coordinated compounds. In Danio rerio embryos, they are toxic, however, the coating of the nanoparticle-drug set, could diminish the toxicity of these. Thus, next biological studies will be able to explore these aspects in greater detail and the improvement of the nanosystem

Three Streptococcal strains: S. milleri P213, S. milleri P35 and S. milleri B200 and three enterococcal strains: E. faecalis 123, E. faecalis 126 and E. faecium were used to test for vancomycin resistance. Two strains were used as reference strains that were already characterized as vancomycin resistant. E. faecium BM4147 was used as a VanA control and E. faecalis ATCC was used as a VanB control. Susceptibility of each strain to this antibiotic was tested by disk-diffusion assay and the MIC values for the strains were found to be between 5 - 10 ug/ml and for the VanA control, the MIC was > 64 ug/ml and for the VanB control was 32 ug/ml. These MIC values indicate that S. milleri P213, S. milleri P35, S. milleri B200, E. faecalis 123, E. faecalis 126, and E. faecium are all of the VanC phenotype. All strains were tested for lysis by means of addition of vancomycin (10 ug/ml) to the bacterial cultures. Lytic curves were constructed and the VanB control was found to be most autolytic upon addition of vancomycin and E. faecalis 123 was the least autolytic. However, under normal conditions in phosphate buffer, lytic curves showed that S. milleri P213 was the most autolytic and the VanA control, the least autolytic. PCR assays were performed to detect specific antibiotic resistant genes. Primers were selected from Dukta-Malen et al., 1995. The VanA primer yielded amplification of 732 bp for only the VanA control DNA and the VanB primer set yielded products for the VanB control DNA. S. milleri P213, P35, B200 and E. faecalis 123 and 126, and E. faecium DNA were amplified with the VanC primers. This supports the results obtained in MIC that these strains are possibly VanC resistant strains. Amplified VanA control and that of E. faecalis 126 were thereafter sequenced. VanA control amplicon was correctly amplified since it showed homology to E. faecium BM4147 as well as the VanB amplicons which was found to be homologous to the transposon Tn1549 found on the well-characterized E. faecalis strain which is known to harbour the VanB vancomycin-resistant genes. Whilst E. faecalis 126 which represented the VanC phenotype showed 96% homology to E. gallinarum BM4147 which is a well-characterized glycopeptide-resistant enterococci belonging to the VanC phenotype. Southern blots were performed using specific primers as a probe to verify whether the gene sequences for the specific genotype were present in these strains and results confirmed those found in the PCR assays and in DNA sequencing. The peptidoglycan precursors of each strain were arrested in vancomycin (20 ug/ml) to block transpeptidation and transglycosylation steps of peptidoglycan synthesis and bacitracin (100 ug/ml) was used to amplify precursors at the transglycosylation step. Precursors were extracted and analysed by reverse-phase HPLC. UDP-MurNAc-tetrapeptides cell wall precursors, which are found abundantly in vancomycin-resistant strains, were found in large proportions in all strains, except in E. faecalis 123 when arrested with vancomycin. This precursor has a noticeably decreased affinity for vancomycin, hence contributing to its resistance. The precursor accumulated when arrested with bacitracin, was, UDPMurNAc-tetrapeptide in all strains except in E. faecalis 126. UDP-MurNAc-pentapeptides were also found in moderate amounts in most strains. The molecular masses of the peptidoglycan precursors obtained from mass spectrometry correctly identified them. This confirmed that the bacterial strains investigated were in fact resistant to the antibiotic vancomycin and this study shows that results obtained from conventional phenotypical screening methods reliably correlated with the genotypes classified using more advanced techniques such as PCR, southern blot/hybridisation and DNA sequencing. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Introdução: Enterococcus faecium resistente à vancomicina (EFRV) surgiu como um importante patógeno multirresistente e de etiologia potencialmente letal nas infecções associadas aos cuidados de saúde em todo o mundo. Objetivo: O objetivo deste estudo de coorte retrospectivo foi avaliar os fatores associados à mortalidade em pacientes com bacteremia causadas por EFRV em um grande hospital de referência terciária no sul do Brasil. Métodos: Foram avaliados todos os casos documentados de bacteremia identificados entre maio de 2010 e julho de 2012. Regressão de Cox foi realizada para determinar se as características relacionadas ao hospedeiro ou o tratamento antimicrobiano estavam associadas com a mortalidade por qualquer causa em 30 dias. No total, 35 pacientes documentados com bacteremia por EFRV foram identificados durante o período de estudo. Resultados: A mediana do escore APACHE-II da população do estudo foi 26 (IQR 10). A mortalidade global em 30 dias foi de 65,7%%. Todos isolados de EFRV eram sensíveis à linezolida, daptomicina e quinopristina - dalfopristina. A linezolida foi o único agente antimicrobiano com atividade in vitro contra EFRV que foi administrada à coorte. Após a análise multivariada, o tratamento com linezolida (HR 0,08, 95% CI, 0,02-0,27) e a presença de insuficiência renal aguda no início da bacteremia (HR 4,01, 95% CI, 1,62-9,94) foram associadas de forma independente com o desfecho principal. Conclusão: Apresentação com insuficiência renal aguda e ausência de tratamento com um antibiótico eficaz representa um risco de mortalidade em pacientes com bacteremia por EFRV. / Background: Vancomycin-resistant Enterococcus faecium (VREF) has emerged as a relevant multidrug-resistant pathogen and potentially lethal etiology of health care-associated infections worldwide. Objective: The objective of this retrospective cohort study was to assess factors associated with mortality in patients with VREF bacteremia in a major tertiary referral hospital in southern Brazil. Methods: All documented cases of bacteremia identified between May 2010 and July 2012 were evaluated. Cox regression was performed to determine whether the characteristics related to the host or antimicrobial treatment were associated with the all-cause 30-day mortality. In total, 35 patients with documented VREF bacteremia were identified during the study period. Results: The median APACHE-II score of the study population was 26 (IQR 10). The overall 30-day mortality was 65.7%. All VREF isolates were sensitive to linezolid, daptomycin and quinopristin-dalfopristin. Linezolid was the only antimicrobial agent with in vitro activity against VREF that was administered to the cohort. After multivariate analysis, linezolid treatment (HR, 0.08; 95%CI, 0.02 – 0.27) and presence of acute kidney injury at the onset of bacteremia (HR, 4.01; 95%CI, 1.62 – 9.94) were independently associated with the main outcome. Conclusion: Presentation with acute kidney injury and lack of treatment with an effective antibiotic poses risk for mortality in patients with VREF bacteremia.

Introdução: Enterococcus faecium resistente à vancomicina (EFRV) surgiu como um importante patógeno multirresistente e de etiologia potencialmente letal nas infecções associadas aos cuidados de saúde em todo o mundo. Objetivo: O objetivo deste estudo de coorte retrospectivo foi avaliar os fatores associados à mortalidade em pacientes com bacteremia causadas por EFRV em um grande hospital de referência terciária no sul do Brasil. Métodos: Foram avaliados todos os casos documentados de bacteremia identificados entre maio de 2010 e julho de 2012. Regressão de Cox foi realizada para determinar se as características relacionadas ao hospedeiro ou o tratamento antimicrobiano estavam associadas com a mortalidade por qualquer causa em 30 dias. No total, 35 pacientes documentados com bacteremia por EFRV foram identificados durante o período de estudo. Resultados: A mediana do escore APACHE-II da população do estudo foi 26 (IQR 10). A mortalidade global em 30 dias foi de 65,7%%. Todos isolados de EFRV eram sensíveis à linezolida, daptomicina e quinopristina - dalfopristina. A linezolida foi o único agente antimicrobiano com atividade in vitro contra EFRV que foi administrada à coorte. Após a análise multivariada, o tratamento com linezolida (HR 0,08, 95% CI, 0,02-0,27) e a presença de insuficiência renal aguda no início da bacteremia (HR 4,01, 95% CI, 1,62-9,94) foram associadas de forma independente com o desfecho principal. Conclusão: Apresentação com insuficiência renal aguda e ausência de tratamento com um antibiótico eficaz representa um risco de mortalidade em pacientes com bacteremia por EFRV. / Background: Vancomycin-resistant Enterococcus faecium (VREF) has emerged as a relevant multidrug-resistant pathogen and potentially lethal etiology of health care-associated infections worldwide. Objective: The objective of this retrospective cohort study was to assess factors associated with mortality in patients with VREF bacteremia in a major tertiary referral hospital in southern Brazil. Methods: All documented cases of bacteremia identified between May 2010 and July 2012 were evaluated. Cox regression was performed to determine whether the characteristics related to the host or antimicrobial treatment were associated with the all-cause 30-day mortality. In total, 35 patients with documented VREF bacteremia were identified during the study period. Results: The median APACHE-II score of the study population was 26 (IQR 10). The overall 30-day mortality was 65.7%. All VREF isolates were sensitive to linezolid, daptomycin and quinopristin-dalfopristin. Linezolid was the only antimicrobial agent with in vitro activity against VREF that was administered to the cohort. After multivariate analysis, linezolid treatment (HR, 0.08; 95%CI, 0.02 – 0.27) and presence of acute kidney injury at the onset of bacteremia (HR, 4.01; 95%CI, 1.62 – 9.94) were independently associated with the main outcome. Conclusion: Presentation with acute kidney injury and lack of treatment with an effective antibiotic poses risk for mortality in patients with VREF bacteremia.

Introdução: Enterococcus faecium resistente à vancomicina (EFRV) surgiu como um importante patógeno multirresistente e de etiologia potencialmente letal nas infecções associadas aos cuidados de saúde em todo o mundo. Objetivo: O objetivo deste estudo de coorte retrospectivo foi avaliar os fatores associados à mortalidade em pacientes com bacteremia causadas por EFRV em um grande hospital de referência terciária no sul do Brasil. Métodos: Foram avaliados todos os casos documentados de bacteremia identificados entre maio de 2010 e julho de 2012. Regressão de Cox foi realizada para determinar se as características relacionadas ao hospedeiro ou o tratamento antimicrobiano estavam associadas com a mortalidade por qualquer causa em 30 dias. No total, 35 pacientes documentados com bacteremia por EFRV foram identificados durante o período de estudo. Resultados: A mediana do escore APACHE-II da população do estudo foi 26 (IQR 10). A mortalidade global em 30 dias foi de 65,7%%. Todos isolados de EFRV eram sensíveis à linezolida, daptomicina e quinopristina - dalfopristina. A linezolida foi o único agente antimicrobiano com atividade in vitro contra EFRV que foi administrada à coorte. Após a análise multivariada, o tratamento com linezolida (HR 0,08, 95% CI, 0,02-0,27) e a presença de insuficiência renal aguda no início da bacteremia (HR 4,01, 95% CI, 1,62-9,94) foram associadas de forma independente com o desfecho principal. Conclusão: Apresentação com insuficiência renal aguda e ausência de tratamento com um antibiótico eficaz representa um risco de mortalidade em pacientes com bacteremia por EFRV. / Background: Vancomycin-resistant Enterococcus faecium (VREF) has emerged as a relevant multidrug-resistant pathogen and potentially lethal etiology of health care-associated infections worldwide. Objective: The objective of this retrospective cohort study was to assess factors associated with mortality in patients with VREF bacteremia in a major tertiary referral hospital in southern Brazil. Methods: All documented cases of bacteremia identified between May 2010 and July 2012 were evaluated. Cox regression was performed to determine whether the characteristics related to the host or antimicrobial treatment were associated with the all-cause 30-day mortality. In total, 35 patients with documented VREF bacteremia were identified during the study period. Results: The median APACHE-II score of the study population was 26 (IQR 10). The overall 30-day mortality was 65.7%. All VREF isolates were sensitive to linezolid, daptomycin and quinopristin-dalfopristin. Linezolid was the only antimicrobial agent with in vitro activity against VREF that was administered to the cohort. After multivariate analysis, linezolid treatment (HR, 0.08; 95%CI, 0.02 – 0.27) and presence of acute kidney injury at the onset of bacteremia (HR, 4.01; 95%CI, 1.62 – 9.94) were independently associated with the main outcome. Conclusion: Presentation with acute kidney injury and lack of treatment with an effective antibiotic poses risk for mortality in patients with VREF bacteremia.

Enterococcus faecium is a leading cause of hospital-acquired infection, disproportionally affecting immunocompromised and critically ill patients. Despite infection control measures, rates of vancomycin-resistant E. faecium (VREfm) bacteraemias have failed to decline in the United Kingdom, and Cambridge University Hospitals (CUH) report the highest numbers nationally. The aims of my PhD were to use epidemiological and genomic surveillance data to establish risk factors for acquisition and infection with E. faecium in patients at CUH, and to use a One Health approach to consider possible sources for hospital patients by relating bloodstream-associated isolates with those cultured from livestock and the environment in the same geographic region. A retrospective matched nested case control study was performed to determine risk factors for VRE bacteraemia relating to antibiotic exposure. 235 cases were matched to 220 controls for length of admission, year, specialty and ward type. Multivariable analysis demonstrated that duration of exposure to parenteral vancomycin, fluoroquinolones and meropenem were independently associated with VRE bacteraemia. This provides evidence for the importance of antimicrobial stewardship targeting high-risk antibiotics in patients at risk of VRE bacteraemia. VREfm bacteraemia may be complicated by disease recurrence. Whole genome sequencing was used to distinguish between relapse and reinfection in 14 episodes of recurrent VREfm bacteraemia. This demonstrated that 10 (71%) episodes were due to reinfection with a new strain, with reinfection being more likely with increasing time between two positive cultures. This study also evaluated 9 patients with blood cultures positive for both VREfm and vancomycin-susceptible E. faecium (VSEfm), the majority (78%) of which were found to be unrelated strains. More than half of all study isolates from these two patient groups were closely related to another isolate causing bacteraemia at CUH, suggesting that hospital acquisition of VREfm is a driver for infection and recurrence. A cross-sectional study of E. faecium in raw and treated wastewater from 20 municipal water treatment plants across the East of England revealed widespread dissemination of healthcare-associated lineages of VREfm in all sampled locations including rural areas, and environmental release in treated wastewater in 17/20 locations. Wastewater isolates were genetically intermixed with isolates causing bacteraemia at CUH, including highly related isolates indicating recent transmission between the two reservoirs. These findings are consistent with widespread distribution of healthcare-associated VREfm in community populations. A One Health approach incorporating sampling from livestock (10 pork, 10 cattle, 9 poultry farms) detected no VREfm in animals whilst 2 independent meat surveys demonstrated VREfm in 1-2% of uncooked products. Genomic comparison of >1400 E. faecium isolates from livestock, meat, wastewater and almost 800 people with bloodstream infection demonstrated that livestock and human isolates were genetically distinct. Analysis of the accessory genome added further evidence for distinct gene content associated with niche adaptation. An analysis of mobile genes encoding antibiotic resistance revealed limited evidence of sharing between human and animal populations. A prospective longitudinal study in haematology patients at CUH over 6 months revealed high rates of VREfm carriage (63% of cases) and environmental contamination (49% of samples). Genomic analysis elucidated complex colonisation dynamics with frequent loss and acquisition of subtypes, including unsuspected acquisition of new VREfm subtypes in patients already colonised with VREfm, and multiple transmission chains involving patients and the environment, including some leading to bacteraemia. These findings highlight the shortcomings of infection control and environmental cleaning and provide the basis for revised interventions.

Bactérias comensais do intestino de frangos tornaram-se objeto frequente de estudos, pois a alta taxa de exportação desses produtos tem aumentado a preocupação com a qualidade e a sanidade de granjas no que se refere à nutrição animal, ganho de peso e doenças infecciosas. O objetivo do presente estudo foi verificar a influência de diferentes dietas para frangos de corte na colonização, assim como no fenótipo e genótipo de resistência antimicrobiana de isolados Enterococcus sp. Amostras de “swabs” cloacais de frangos de corte foram utilizadas para isolamento de Enterococcus sp. Os frangos foram submetidos a diferentes dietas contendo promotores de crescimento, probióticos, óleos essenciais e coccidiostáticos ionóforos, e divididos em grupos conforme o tratamento empregado. Foram obtidos 240 isolados de Enterococcus sp. confirmados para gênero através da técnica de Reação em Cadeia pela Polimerase (PCR), submetidos a testes bioquímicos e moleculares para identificação de espécie, determinados os perfis de susceptibilidade a diversos antimicrobianos e testados para a presença de genes de resistência tet(M), tet(L) e erm(B) por PCR. Observou-se uma alteração na composição ou no número de espécies de Enterococcus sp. de acordo com as dietas empregadas. Todas as dietas, independentes da presença ou não de coccidiostático ionóforo, apresentaram um aumento na freqüência de Enterococcus resistentes quando comparados com o grupo controle. No entanto, nos grupos que os frangos receberam coccidiostático ionóforo, a taxa de Enterococcus sp. isolados foi menor quando comparada aos isolados dos grupos que não receberam essa composição. Do total de isolados resistentes à tetraciclina, 94% e 30%, continham os genes tet(M) e tet(L), respectivamente. Dos isolados resistentes a eritromicina, 97,9% possuíam o gene de resistência erm(B). Não houve correlação das diferentes dietas para frangos de corte na colonização, fenótipo e genótipo de resistência antimicrobiana de isolados de Enterococcus sp. / Commensal bacteria from the gut of chickens have been frequently object of studies because the high rate of exportation of these products the concern with the quality and health of poultry, in relation to animal nutrition, weight gain and infectious diseases has been increasing. The principal aim of this study was to verify the influence of different diets treated to broilers, in the colonization of Enterococcus strains, and as well the phenotype and genotype of resistance strains. Samples from cloacal swabs of broiler chickens were used for the isolation of strains of Enterococcus sp. The broilers were subjected to diets with growth promoter, probiotics, essential oils and ionophore coccidiostat and divided into groups according to treatment used. Two hundred forty isolates of Enterococcus sp. were confirmed the genus using the polymerase chain reaction (PCR), subjected to biochemical and molecular species identification, antibiotic susceptibility profile and verification of antibiotic resistance genes tet(M), tet(L) e erm(B) by PCR. Accordingly to the diets employed to the chickens a changing the composition or the number of Enterococcus sp. was observed. All diets, regardless to the presence or absence of ionophore coccidiostat showed an increase in the frequency of Enterococcus resistant when compared with the control group. However, the groups that received ionophore coccidiostat, the rate of resistant Enterococcus sp. was lower than the groups that did not administered this composition. The isolates that presented resistant to tetracycline, 94% and 30%, contained the genes tet(M) and tet(L), respectively. In the isolates resistant to erythromycin, 97.9% had the gene erm(B). There was no correlation of different diets for broiler chickens on colonization, phenotype and genotype resistance in the Enterococcus sp strains.

El objetivo del presente estudio fue comparar la efectividad antibacteriana de la pasta de hidróxido de calcio con clorhexidina al 2% y la pasta de hidróxido de calcio con yodopovidona al 1%, frente al Enterococcus faecalis. El diseño de contrastación fue experimental. Se distribuyeron 10 placas Petri que contenían agar Müller Hinton a 40° C, sobre las cuales fue inoculada la bacteria Enterococcus faecalis. Además, estas fueron divididas de manera aleatoria en 3 segmentos cada una de acuerdo al tipo de pasta medicamentosa que se aplicó: grupo P1 (hidróxido de calcio + clorhexidina al 2%), grupo P2 (hidróxido de calcio + yodopovidona al 1%) y el grupo P3 o control (hidróxido de calcio + agua destilada). Finalmente, se procedió a la lectura de halos de inhibición a las 24 horas, 48 horas, 7 días, 14 días. Los datos fueron procesados a través del análisis de Tukey para determinar la diferencia de medias entre los grupos experimentales y el análisis de ANOVA con un nivel de significancia del 95%, utilizando el programa SPSS 20. Se concluyó que la pasta de hidróxido de calcio con clorhexidina al 2% fue más efectiva que la pasta de hidróxido de calcio con yodopovidona al 1% frente al crecimiento in vitro del Enterococcus faecalis. / Tesis

published_or_final_version / Endodontics / Master / Master of Dental Surgery