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Pesquisa do Estado do Rio Grande do Sul (FAPERGS) / Acinetobacter calcoaceticus-baumannii (ACB) complex comprises opportunistic and emerging pathogens that are responsible for several diseases, mainly affecting immunocompromised patients and those hospitalized in intensive care units. Therapeutic options for ACB infections are restricted, since these microorganisms are often resistant to most antimicrobials. In addition, these bacteria may also form persister cells, which constitute a small population of susceptible cells able to survive lethal concentrations of bactericidal antimicrobials and other stressors. This phenotype is associated with failure in antimicrobial therapy, especially in chronic and recurrent infections. Therefore, the aim of this study was to evaluate the presence of persister cells from ACB complex isolates exposed to meropenem and/or polymyxin B, in biofilm and planktonic cells, as well as to analyze these cells regarding their morphology and identify expression patterns of proteins possibly involved in the formation and maintenance of persistence. For this, 20 clinical isolates were characterized for the ability to form biofilm on polystyrene surface, and for meropenem and polymyxin B susceptibility, by the assessment of Minimum Inhibitory Concentration (MIC). All isolates were exposed to meropenem at different concentrations above the MIC, while five isolates were exposed to polymyxin B for the assessment of the persisters presence. For all experiments, in order to estimate the fraction of remaining cells, aliquots were removed at determined time points, followed by serial decimal dilutions and drop plating technique on nutrient agar. All isolates presented persisters at different proportions, in both culture conditions when exposed to meropenem or polymyxin B after 48 h. The higher fractions were verified in biofilm for both antimicrobials in comparison with planktonic cultures. Meropenem concentrations did not influence persisters levels. However, after polymyxin B exposure, persister cells fractions were dependent on the concentration employed. After 24 h polymyxin B exposure, a growth resumption of surviving cells was observed. These cells were again evaluated for susceptibility to this antimicrobial, remaining susceptible with MIC of 1 ?g/ mL. Moreover, integrity of polymyxin B in the supernatant of the cultures was verified by chromatographic assay, demonstrating that polymyxin B is not significantly degraded after 48 h exposure. On the other hand, when meropenem and polymyxin B were associated at different concentrations, no resumption of cell growth was observed, as well as this combination was able to eradicate persister cells from A. baumannii (Acb-1) cultured in late exponential phase. Furthermore, Nano-Liquid Chromatography Coupled to Tandem Mass Spectrometry was employed for the identification and relative quantification of proteins possibly associated with persistence in A. baumannii, after exposure to meropenem. Different patterns of expression were identified between persister cells present in planktonic and biofilm cultures, suggesting that persistence may be regulated by different mechanisms. Proteins involved in the cell division and DNA replication were overexpressed in planktonic persisters, in agreement with the electron scanning microscopy images that presented dividing cells in this culture condition. Overexpression of glucose dehydrogenase (GDH), NADH dehydrogenase 1 (NDH-1), succinate dehydrogenase and ATP synthase indicates the electron transfer from the GDH-catalyzed reaction to the electron transport chain as a possible energy source for persisters, supporting the presence of cell division observed in planktonic culture. Conversely, proteins involved in amino acid metabolism, as well as major elongation factors were underexpressed in Acb-1 persister cells, suggesting that protein synthesis is reduced, even though many proteins were overproduced. Increased expression of several membrane-related proteins has also been observed, indicating possible changes in its composition and function, although proteins related to lipid metabolism were underexpressed. Overall, proteomic analysis of the persister cells showed that these cells could be physiologically distinct when cultured under different conditions, as well as overtime in the same condition. Therefore, considering the different behaviors of Acb-1 when exposed alone to meropenem or polymyxin B, as well as when exposed to these drugs in combination, it was concluded that each antimicrobial might act as a different stressor, possibly leading and/or selecting distinct tolerance mechanisms among persisters, which enabled their eradication when the drugs were combined. / Os pat?genos pertencentes ao complexo Acinetobacter calcoaceticus-baumannii (ACB) s?o considerados oportunistas e emergentes, respons?veis por ocasionar diversas enfermidades, acometendo principalmente pacientes imunocomprometidos e internados em unidades de tratamento intensivo. As op??es terap?uticas para o tratamento de infec??es ocasionadas por estes pat?genos s?o restritas, uma vez que estes apresentam frequentemente resist?ncia ? maioria dos antimicrobianos. Al?m disso, essas bact?rias podem ainda formar c?lulas persisters, que constituem uma pequena popula??o de c?lulas suscet?veis capazes de tolerar concentra??es letais de antimicrobianos bactericidas e outros agentes estressores. Este fen?tipo est? associado a falhas na terapia antimicrobiana, especialmente nas infec??es cr?nicas e recorrentes. Desta forma, o objetivo deste trabalho foi avaliar a presen?a de c?lulas persisters formadas por isolados do complexo ACB frente ? exposi??o ao meropenem e/ou ? polimixina B, na condi??o de biofilme e em cultivo planct?nico, assim como analisar estas c?lulas morfologicamente e identificar padr?es de express?o de prote?nas que pudessem estar envolvidos na forma??o e manuten??o da persist?ncia. Para tanto, 20 isolados cl?nicos foram caracterizados quanto ? capacidade em formar biofilme em superf?cie de poliestireno, e ? suscetibilidade ao meropenem e ? polimixina B, que foi avaliada por meio da determina??o da Concentra??o Inibit?ria M?nima (CIM) a estes f?rmacos. Todos os isolados foram submetidos ? exposi??o ao meropenem em diferentes concentra??es acima da CIM, enquanto que cinco foram expostos ? polimixina B para a avalia??o da presen?a de c?lulas persisters. Para todos os experimentos, a fim de estimar a fra??o de c?lulas remanescentes, al?quotas foram removidas em tempos determinados, efetuando-se dilui??es decimais seriadas e semeadura pela t?cnica da gota em ?gar nutriente. C?lulas persisters, em diferentes fra??es, foram encontradas nos cultivos de todos os isolados, tanto em biofilmes como na condi??o planct?nica, ap?s a exposi??o por 48 h ao meropenem e ? polimixina B, sendo as fra??es mais elevadas encontradas na condi??o de biofilme para ambos os antimicrobianos. As diferentes concentra??es de meropenem avaliadas n?o influenciaram os n?veis de c?lulas persisters; entretanto, frente ? exposi??o ? polimixina B, a fra??o de c?lulas mostrou-se dependente da concentra??o empregada. Ap?s exposi??o ? polimixina B por 24 h, foi observada retomada de crescimento das c?lulas sobreviventes, que foram avaliadas novamente quanto ? suscetibilidade a este antimicrobiano, mantendo-se suscet?veis com CIM de 1 ?g/mL, bem como foi verificada a integridade do antimicrobiano no sobrenadante destes cultivos por ensaios cromatogr?ficos, demonstrando que o mesmo n?o sofre degrada??o ap?s 48 h de exposi??o. Entretanto, quando se associou meropenem ? polimixina B em diferentes concentra??es, al?m de n?o ter sido observada a retomada de crescimento das c?lulas remanescentes, ocorreu a erradica??o das c?lulas persisters de um isolado de A. baumannii (Acb-1) cultivado em fase exponencial tardia. Al?m disso, a t?cnica de Nano-Cromatografia L?quida acoplada ? Espectrometria de Massas em Tandem foi empregada para a identifica??o e quantifica??o relativa de prote?nas possivelmente associadas ? persist?ncia em A. baumannii, ap?s a exposi??o ao meropenem. Diferentes padr?es de express?o foram identificados entre as c?lulas persisters presentes em cultivo planct?nico e em biofilme, sugerindo que a regula??o da persist?ncia possa ser realizada por mecanismos diferentes. Observou-se express?o aumentada de prote?nas envolvidas nos processos de divis?o celular e replica??o de DNA, especialmente no cultivo planct?nico, em concord?ncia com a presen?a de divis?o celular observada nas imagens obtidas a partir da microscopia eletr?nica de varredura nesta condi??o de cultivo. O aumento de express?o da glicose desidrogenase (GDH), NADH desidrogenase (NDH-1), succinato desidrogenase e ATP sintase indicam a transfer?ncia de el?trons a partir da rea??o catalisada por GDH para a cadeia de transporte de el?trons como uma poss?vel fonte de energ?tica para as persisters, corroborando a observa??o da presen?a de divis?o celular observada no cultivo planct?nico. Em contraste, prote?nas envolvidas no metabolismo de amino?cidos, bem como os principais fatores de elonga??o apresentaram express?o diminu?da em c?lulas persisters de Acb-1, sugerindo que a s?ntese proteica esteja reduzida, mesmo que muitas prote?nas tenham sido identificadas com express?o aumentada. Muitas prote?nas relacionadas ? membrana apresentaram a sua express?o aumentada, indicando poss?veis altera??es em sua composi??o e fun??o, embora prote?nas relacionadas ao metabolismo de lip?deos tenham apresentado express?o diminu?da. A an?lise prote?mica das c?lulas persisters, sobretudo, mostrou que estas c?lulas podem ser fisiologicamente distintas quando cultivadas em condi??es diferentes, bem como ao longo do tempo em uma mesma condi??o. Desta forma, considerando os distintos comportamentos do Acb-1 quando exposto isoladamente ao meropenem ou ? polimixina B, bem como quando exposto a estes f?rmacos ao mesmo tempo, pode-se concluir que cada antimicrobiano pode ter atuado como um diferente estressor, possivelmente, levando a e/ou selecionando mecanismos de toler?ncia diferentes entre as persisters, o que possibilitou a sua erradica??o quando os f?rmacos foram combinados.

Exposure to antibiotics is an important factor influencing the development of bacterial resistance.  In an era where very few new antibiotics are being developed, a strategy for the development of optimal dosing regimen and combination treatment that reduces the rate of resistance development and overcome existing resistance is of utmost importance. In addition, the optimal dosing in subpopulations is often not fully elucidated. The aim of this thesis was to develop pharmacokinetic (PK) and pharmacokinetic-pharmacodynamic (PKPD) models that characterize the interaction of antibiotics with bacterial growth, killing and resistance over time, and can be applied to guide optimization of dosing regimens that enhance the efficacy of mono- and combination antibiotic therapy. A mechanism-based PKPD model that incorporates the growth, killing kinetics and adaptive resistance development in Escherichia coli against gentamicin was developed based on  in vitro time-kill curve data. After some adaptations, the model was successfully applied for similar data on colistin and meropenem alone, and in combination, on one wild type and one meropenem-resistant strain of Pseudomonas aeruginosa. The developed population PK model for colistin and its prodrug colistin methanesulfonate (CMS) in combination with the PKPD model showed the benefits for applying a loading dose for this drug. Simulations predicted the variability in bacteria kill to be larger between dosing occasions than between patients. A flat-fixed loading dose followed by an 8 or 12 hourly maintenance dose with infusion duration of up to 2 hours was shown to result in satisfactory bacterial kill under these conditions. Pharmacometric models that characterize the time-course of drug concentrations, bacterial growth, antibacterial killing and resistance development were successfully developed. Predictions illustrated how PKPD models based on in vitro data can be utilized to guide development of antibiotic dosing, with examples advocating regimens that (i) promote bacterial killing and reduce risk for toxicity in preterm and term newborn infants receiving gentamicin, (ii) achieve a fast initial bacterial killing and reduced resistance development of colistin in critically ill patients by application of a loading dose, and (iii) overcome existing meropenem resistance by combining colistin and meropenem

Current excessive use and abuse of antibiotics has resulted in increasing bacterial resistance to common treatment options which is threatening to deprive us of a pillar of modern medicine. In this work methods to optimize the use of existing antibiotics and to help development of new antibiotics were developed and applied. Semi-mechanistic pharmacokinetic-pharmacodynamic (PKPD) models were developed to describe the time course of the dynamic effect and interaction of combinations of antibiotics. The models were applied to illustrate that colistin combined with a high dose of meropenem may overcome meropenem-resistant P. aeruginosa infections. The results from an in vivo dose finding study of meropenem was successfully predicted by the meropenem PKPD model in combination with a murine PK model, which supports model based dosage selection. However, the traditional PK/PD index based dose selection was predicted to have poor extrapolation properties from pre-clinical to clinical settings, and across patient populations. The precision of the model parameters, and hence the model predictions, is dependent on the experimental design. A limited study design is dictated by cost and, for in vivo studies, ethical reasons. In this work optimal design (OD) was demonstrated to be able to reduce the experimental effort in time-kill curve experiments and was utilized to suggest the experimental design for identification and estimation of an interaction between antibiotics. OD methods to handle inter occasion variability (IOV) in optimization of individual PK parameter estimates were proposed. The strategy was applied in the design of a sparse sampling schedule that aim to estimate individual exposures of colistin in a multi-centre clinical study. Plasma concentration samples from the first 100 patients have been analysed and indicate that the performance of the design is close to the predicted. The methods described in this thesis holds promise to facilitate the development of new antibiotics and to improve the use of existing antibiotics.

In critically ill patients, the pathophysiology of sepsis can affect the interactions between the antibiotic, the bacteria and the patient, leading to potential therapeutic failure and the development of antibiotic resistance. It is well acknowledged that research that optimises antibiotic exposure will assist improvement of outcomes in this patient group. Although beta-lactam antibiotics, such as piperacillin and meropenem, are commonly selected for empiric therapy of sepsis, dosing is unlikely to be optimal. In patients without renal dysfunction, data suggests that disease-induced alterations to pharmacokinetic parameters result in low trough concentrations for significant periods within a dosing interval. Administration of these time-dependent antibiotics by continuous infusion has been suggested to improve the pharmacokinetic-pharmacodynamic profile. Knowledge of concentrations in the extracellular fluid of human tissue, which is the target site of most pathogens, is particularly instructive. Extracellular fluid concentrations can be determined using techniques such as microdialysis. Therefore, the principal aims of this Thesis were to determine the plasma and subcutaneous tissue pharmacokinetics of piperacillin and meropenem administered by bolus dosing and continuous infusion in critically ill patients with sepsis; and to use Monte Carlo simulations to compare the ability of different dosing strategies to achieve pharmacodynamic endpoints. This Thesis also sought to compare the clinical outcomes of bolus dosing and continuous infusion of a beta-lactam antibiotic, ceftriaxone, in a prospective randomised controlled trial and to perform a meta-analysis on clinical outcomes from other similar published studies. Finally, this Thesis aimed to systematically review the published literature to determine any correlation between antibiotic dosing and the development of antibiotic resistance. The results of the pharmacokinetic studies, using piperacillin and meropenem, indicate that beta-lactam distribution into subcutaneous tissue, in critically ill patients with sepsis, is less than that observed in previous studies in healthy volunteers yet superior to studies in patients with septic shock. This supports the notion that the peripheral concentration of drugs may be inversely related to the level of sickness severity. Administration by continuous infusion was found to maintain statistically significantly higher trough beta-lactam concentrations in both plasma and subcutaneous tissue. Further analysis of the plasma data using population pharmacokinetic modeling and Monte Carlo simulations described significant pharmacodynamic advantages for administering meropenem or piperacillin by continuous infusion to organisms with high minimum inhibitory concentrations. Given the documented pharmacodynamic advantages for administering beta-lactams by continuous infusion, a prospective randomized controlled clinical trial was conducted using the beta-lactam antibiotic ceftriaxone. In 57 critically ill patients, we found equivalence between continuous infusion and bolus dosing in the intention-to-treat analysis. However, our a priori analysis criteria, requiring patients receive at least 4-days antibiotic treatment, found significant clinical and bacteriological advantages for administration by continuous infusion. To further investigate any clinical differences between bolus dosing and continuous infusion of beta-lactam antibiotics, we performed a meta-analysis of all published studies. Our analysis of the 13 published prospective randomized controlled trials (846 hospitalised patients) showed equivalence of continuous infusion and bolus dosing. Possible confounders observed within, and between the studies, make interpretation of these results challenging. However, two large retrospective cohorts not included in the meta-analysis, found definitive clinical and bacteriological advantages suggesting further research may be appropriate. The possible relationship between antibiotic dosing, or exposures, on the development of resistance was investigated using a structured review of the published literature. Our analysis of relevant papers found a wealth of data describing increasing levels of resistance with sub-optimal antibiotic dosing, particularly for fluoroquinolone antibiotics, but also for other classes including beta-lactams. These results demonstrate the importance of optimizing antibiotic dosing to decrease the development of antibiotic susceptibility from sub-optimal dosing, particularly for critically ill patients who are likely to have low drug concentrations. The results of this Thesis, suggest that a large, prospective, multi-centre randomised controlled trial in critically ill patients with sepsis, is required to definitively determine the clinical utility of administration of beta-lactam antibiotics by continuous infusion.

A terapia renal substitutiva contínua (TRSC) é amplamente utilizada em pacientes criticamente enfermos com insuficiência renal aguda (IRA). O meropenem é um carbapenêmico usado em pacientes críticos que tem uma atividade antibacteriana dependente do tempo. O objetivo do estudo foi avaliar a farmacocinética do meropenem infundido em três horas em pacientes submetidos à TRSC. Estudamos as concentrações plasmáticas e de efluente em cinco pacientes submetidos à TRSC. As amostras foram coletadas em momentos 0, 30 min, e 1, 2, 4, 6 e 8 horas após o início de uma infusão de 3 horas. As determinações de meropenem foram feitas por cromatografia líquida de alta eficiência. Quatro pacientes do sexo masculino e um feminino, idade de 53,0 ± 19,7 (23 a 80 anos), 62,1 ± 10,6 kg, foram estudados. Parâmetros farmacocinéticos apresentados em mediana (amplitude): concentrações plasmáticas, 34.86mg / L (10,08-139,27); tempo de meia vida (t ½), 1,8 h (1,4-3,0); volume de distribuição (Vd), 8,29 L (5,8-15,3); depuração total (Dept ) 3,98 L / h (2,51-4,35); concentração máxima (Cmax) 48,5 mg/L (37,0-105,8); concentração mínima (Cmin) 20,1 mg / L (14,0-16,6); constante de eliminação (Kel), 0,38 (0,34-0,43); área sob a curva de concentração versus tempo (AUC 0 a 8 h), 251,1 mg / Lh (229,7-398,4); (AUC de 0a∞), 275,1 mg /Lh (263,8-453,6).A depuração total pela TRSC variou de 8,46 a 18,33 ml/min. No efluente as concentrações máximas foram 24,35 e 74,81 mg /L. A eliminação de meropenem por TRSC é semelhante ao que é relatado pelo rim normal, quando é infundido por 3 horas a cada 8 h. Os níveis plasmáticos foram sempre acima do MIC necessário. Podemos concluir que não houve necessidade de ajuste de dose do meropenem com a dose de TRSC prescrita. / Continuous renal replacement therapy (CRRT) is widely used in critically ill patients with acute kidney injury (AKI). Meropenem is a carbapenem used in critically ill patients, which has a time dependent antibacterial activity. The aim of the study was to assess the pharmacokinetics of meropenem on a 3-hour infusion in patients undergoing CRRT due to AKI. We studied the plasmatic and effluent concentrations in five patients undergoing CRRT. The samples were collected at moments 0, 30 minutes, and 1, 2, 4, 6 and 8 hours after the beginning of the 3-hour infusion. The meropenem determinations were made through high performace efficiency liquid chromatography (HPLC). Four male patients and one female patient, with a mean age of 53,0 ± 19,7 (23 to 80 years), weighing 62,1 ± 10,6 kgs were studied. Pharmacokinetic parameters presented in medians (range): plasmatic concentrations, 34.86mg / L (10,08-139,27); half-life (t ½), 1,8 h (1,4-3,0); volume of distribution (Vd), 8,29 L (5,8-15,3); total clearance (CLT) 3,98 L / h (2,51-4,35); (Cmax) (maximum plasma concentration), 48,5 mg / L (37,0-105,8); Cmin (minimum plasma concentration)20,1 mg / L (14,0-16,6); elimination constant (Kel), 0,38 (0,34-0,43); area under the concentration versus time curve (AUC 0 a 8 h), 251,1 mg / Lh (229,7-398,4); (AUC 0 a ∞) 275,1 mg / Lh (263,8-453,6). In the effluent, the maximum concentrations varied from 24,35 to 74,81 mg/L, and the clearance from the therapy varied from 8,46 to 18,33 ml/min. The elimination of meropenem through CRRT is similar to that of a normal kidney, given a 3-hour infusion every 8 hours. Plasmatic levels were always above the necessary MICs. We can conclude there was no need for dose adjustment of meropenem with the prescribed CRRT dose.

A terapia renal substitutiva contínua (TRSC) é amplamente utilizada em pacientes criticamente enfermos com insuficiência renal aguda (IRA). O meropenem é um carbapenêmico usado em pacientes críticos que tem uma atividade antibacteriana dependente do tempo. O objetivo do estudo foi avaliar a farmacocinética do meropenem infundido em três horas em pacientes submetidos à TRSC. Estudamos as concentrações plasmáticas e de efluente em cinco pacientes submetidos à TRSC. As amostras foram coletadas em momentos 0, 30 min, e 1, 2, 4, 6 e 8 horas após o início de uma infusão de 3 horas. As determinações de meropenem foram feitas por cromatografia líquida de alta eficiência. Quatro pacientes do sexo masculino e um feminino, idade de 53,0 ± 19,7 (23 a 80 anos), 62,1 ± 10,6 kg, foram estudados. Parâmetros farmacocinéticos apresentados em mediana (amplitude): concentrações plasmáticas, 34.86mg / L (10,08-139,27); tempo de meia vida (t ½), 1,8 h (1,4-3,0); volume de distribuição (Vd), 8,29 L (5,8-15,3); depuração total (Dept ) 3,98 L / h (2,51-4,35); concentração máxima (Cmax) 48,5 mg/L (37,0-105,8); concentração mínima (Cmin) 20,1 mg / L (14,0-16,6); constante de eliminação (Kel), 0,38 (0,34-0,43); área sob a curva de concentração versus tempo (AUC 0 a 8 h), 251,1 mg / Lh (229,7-398,4); (AUC de 0a∞), 275,1 mg /Lh (263,8-453,6).A depuração total pela TRSC variou de 8,46 a 18,33 ml/min. No efluente as concentrações máximas foram 24,35 e 74,81 mg /L. A eliminação de meropenem por TRSC é semelhante ao que é relatado pelo rim normal, quando é infundido por 3 horas a cada 8 h. Os níveis plasmáticos foram sempre acima do MIC necessário. Podemos concluir que não houve necessidade de ajuste de dose do meropenem com a dose de TRSC prescrita. / Continuous renal replacement therapy (CRRT) is widely used in critically ill patients with acute kidney injury (AKI). Meropenem is a carbapenem used in critically ill patients, which has a time dependent antibacterial activity. The aim of the study was to assess the pharmacokinetics of meropenem on a 3-hour infusion in patients undergoing CRRT due to AKI. We studied the plasmatic and effluent concentrations in five patients undergoing CRRT. The samples were collected at moments 0, 30 minutes, and 1, 2, 4, 6 and 8 hours after the beginning of the 3-hour infusion. The meropenem determinations were made through high performace efficiency liquid chromatography (HPLC). Four male patients and one female patient, with a mean age of 53,0 ± 19,7 (23 to 80 years), weighing 62,1 ± 10,6 kgs were studied. Pharmacokinetic parameters presented in medians (range): plasmatic concentrations, 34.86mg / L (10,08-139,27); half-life (t ½), 1,8 h (1,4-3,0); volume of distribution (Vd), 8,29 L (5,8-15,3); total clearance (CLT) 3,98 L / h (2,51-4,35); (Cmax) (maximum plasma concentration), 48,5 mg / L (37,0-105,8); Cmin (minimum plasma concentration)20,1 mg / L (14,0-16,6); elimination constant (Kel), 0,38 (0,34-0,43); area under the concentration versus time curve (AUC 0 a 8 h), 251,1 mg / Lh (229,7-398,4); (AUC 0 a ∞) 275,1 mg / Lh (263,8-453,6). In the effluent, the maximum concentrations varied from 24,35 to 74,81 mg/L, and the clearance from the therapy varied from 8,46 to 18,33 ml/min. The elimination of meropenem through CRRT is similar to that of a normal kidney, given a 3-hour infusion every 8 hours. Plasmatic levels were always above the necessary MICs. We can conclude there was no need for dose adjustment of meropenem with the prescribed CRRT dose.

A terapia renal substitutiva contínua (TRSC) é amplamente utilizada em pacientes criticamente enfermos com insuficiência renal aguda (IRA). O meropenem é um carbapenêmico usado em pacientes críticos que tem uma atividade antibacteriana dependente do tempo. O objetivo do estudo foi avaliar a farmacocinética do meropenem infundido em três horas em pacientes submetidos à TRSC. Estudamos as concentrações plasmáticas e de efluente em cinco pacientes submetidos à TRSC. As amostras foram coletadas em momentos 0, 30 min, e 1, 2, 4, 6 e 8 horas após o início de uma infusão de 3 horas. As determinações de meropenem foram feitas por cromatografia líquida de alta eficiência. Quatro pacientes do sexo masculino e um feminino, idade de 53,0 ± 19,7 (23 a 80 anos), 62,1 ± 10,6 kg, foram estudados. Parâmetros farmacocinéticos apresentados em mediana (amplitude): concentrações plasmáticas, 34.86mg / L (10,08-139,27); tempo de meia vida (t ½), 1,8 h (1,4-3,0); volume de distribuição (Vd), 8,29 L (5,8-15,3); depuração total (Dept ) 3,98 L / h (2,51-4,35); concentração máxima (Cmax) 48,5 mg/L (37,0-105,8); concentração mínima (Cmin) 20,1 mg / L (14,0-16,6); constante de eliminação (Kel), 0,38 (0,34-0,43); área sob a curva de concentração versus tempo (AUC 0 a 8 h), 251,1 mg / Lh (229,7-398,4); (AUC de 0a∞), 275,1 mg /Lh (263,8-453,6).A depuração total pela TRSC variou de 8,46 a 18,33 ml/min. No efluente as concentrações máximas foram 24,35 e 74,81 mg /L. A eliminação de meropenem por TRSC é semelhante ao que é relatado pelo rim normal, quando é infundido por 3 horas a cada 8 h. Os níveis plasmáticos foram sempre acima do MIC necessário. Podemos concluir que não houve necessidade de ajuste de dose do meropenem com a dose de TRSC prescrita. / Continuous renal replacement therapy (CRRT) is widely used in critically ill patients with acute kidney injury (AKI). Meropenem is a carbapenem used in critically ill patients, which has a time dependent antibacterial activity. The aim of the study was to assess the pharmacokinetics of meropenem on a 3-hour infusion in patients undergoing CRRT due to AKI. We studied the plasmatic and effluent concentrations in five patients undergoing CRRT. The samples were collected at moments 0, 30 minutes, and 1, 2, 4, 6 and 8 hours after the beginning of the 3-hour infusion. The meropenem determinations were made through high performace efficiency liquid chromatography (HPLC). Four male patients and one female patient, with a mean age of 53,0 ± 19,7 (23 to 80 years), weighing 62,1 ± 10,6 kgs were studied. Pharmacokinetic parameters presented in medians (range): plasmatic concentrations, 34.86mg / L (10,08-139,27); half-life (t ½), 1,8 h (1,4-3,0); volume of distribution (Vd), 8,29 L (5,8-15,3); total clearance (CLT) 3,98 L / h (2,51-4,35); (Cmax) (maximum plasma concentration), 48,5 mg / L (37,0-105,8); Cmin (minimum plasma concentration)20,1 mg / L (14,0-16,6); elimination constant (Kel), 0,38 (0,34-0,43); area under the concentration versus time curve (AUC 0 a 8 h), 251,1 mg / Lh (229,7-398,4); (AUC 0 a ∞) 275,1 mg / Lh (263,8-453,6). In the effluent, the maximum concentrations varied from 24,35 to 74,81 mg/L, and the clearance from the therapy varied from 8,46 to 18,33 ml/min. The elimination of meropenem through CRRT is similar to that of a normal kidney, given a 3-hour infusion every 8 hours. Plasmatic levels were always above the necessary MICs. We can conclude there was no need for dose adjustment of meropenem with the prescribed CRRT dose.

The treatment options for multidrug resistant bacteria are dwindling and it is an important issue of research in medicine to solve. One of the more problematic bacterial species is Klebsiella pneumoniae, it can cause infections with high morbidity that are difficult to treat. Common antibiotics for treatment of these infections are carbapenems but K. pneumoniae can produce enzymes called carbapenemases that can hydrolyze carbapenems and most other beta-lactam antibiotics. In this study carbapenemase genes were introduced chromosomally to a previously susceptible K. pneumoniae strain using λ-Red recombineering. Further constructs were made with non-functional porins to examine how they affect combination treatment with carbapenems. Antibiotic combination therapy was evaluated against constructed carbapenemase- (KPC-2, NDM-1 and OXA-48) producing K. pneumoniae strains. Screening was done using time-lapse microscopy (oCelloScope), and combinations with better effect than treatment with a single antibiotic were chosen for time-kill assays. The results shows that a triple combination of colistin, meropenem and the beta-lactamase inhibitor avibactam gives an improved effect, up to twice the effect compared to monotherapy and up to 1.8 times increased effect compared to double combination. The synergistic effect was greater when adding colistin to treat the strains with non-functional porins, indicating that colistin can increase the permeability for other antibiotics into the cell. This is an interesting finding that need to be researched further.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:34:28Z No. of bitstreams: 1 DIS_BRUNA_KERN_DONAMORE_PARCIAL.pdf: 256997 bytes, checksum: a87e4fb183ebf7c784b824c77b71092c (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:34:34Z (GMT) No. of bitstreams: 1 DIS_BRUNA_KERN_DONAMORE_PARCIAL.pdf: 256997 bytes, checksum: a87e4fb183ebf7c784b824c77b71092c (MD5) / Made available in DSpace on 2017-06-30T18:34:43Z (GMT). No. of bitstreams: 1 DIS_BRUNA_KERN_DONAMORE_PARCIAL.pdf: 256997 bytes, checksum: a87e4fb183ebf7c784b824c77b71092c (MD5) Previous issue date: 2016-03-31 / Acinetobacter calcoaceticus-baumannii complex opportunistic human pathogens are responsible for several diseases affecting specially intensive care units and immunocompromised patients. These bacteria may also present the persistence phenotype, where a small population of susceptible bacteria survives after a high antimicrobial concentration treatment ? which the fraction of surviving cells may be affected by environmental conditions present in the medium. Thus, the aim of this study was to evaluate the influence of oxygen availability and galactose, sodium citrate and sheep blood presence in the formation of these cells upon meropenem and tobramycin exposure. For this, 10 clinical isolates, sent by the Clinical Pathology Laboratory of S?o Lucas Hospital, had their susceptibility characterized regarding the minimum inhibitory concentration (MIC) to tobramycin and meropenem. The influence of different environmental conditions was analyzed by exposing late exponential-phase cultures to meropenem and tobramycin for 48 h in the presence of sodium citrate, galactose and sheep blood; or in aerated or static condition. For all evaluations, aliquots were removed at determined time points, followed by serial decimal dilutions and drop plating technique on nutrient agar, to estimate the fraction of surviving cells. Aerated condition promoted a reduction of persister cells fraction ? independent of antimicrobial used ?, and, in addition, concentrations of tobramycin higher than 10X MIC provided even lower levels of these cells after 48 h of treatment. Regarding the presence of different sources of nutrients, it was observed that sodium citrate presence in 48 h of meropenem treatment promoted lower fraction of surviving cells when compared to this same antibiotic associated with galactose; whereas tobramycin exposure in the presence of galactose provided a reduced formation of persister cells in the first 6 h of treatment. Sheep blood, however, did not affect the fraction of surviving cells after 48 h of treatment, independently of the antimicrobial used. A remarkable heterogeneity in the behavior of all 10 isolates used in the study was present, regardless the conditions that have been exposed. Thus, it is possible that the conditions imposed in the experiments have influenced in the formation of persister cells by enhanced production of reactive oxygen species, and through the availability of more targets for antibiotic action by the bacterial growth stimulation. / Os pat?genos oportunistas do complexo Acinetobacter calcoaceticus-baumannii s?o respons?veis por diversas enfermidades, acometendo principalmente pacientes de unidades de tratamento intensivo e imunocomprometidos. Estas bact?rias podem ainda apresentar o fen?tipo de persist?ncia, onde uma pequena popula??o de bact?rias suscet?veis sobrevive ap?s o tratamento com elevadas concentra??es de antimicrobianos - podendo essa fra??o de c?lulas sobreviventes ser afetada pelas condi??es ambientais presentes no meio. Desta forma, o objetivo deste trabalho foi avaliar a influ?ncia da disponibilidade de oxig?nio e da presen?a de galactose, citrato de s?dio e sangue de carneiro na forma??o destas c?lulas frente ? exposi??o ao meropenem e ? tobramicina. Para isto, 10 isolados cl?nicos, cedidos pelo Laborat?rio de Patologia Cl?nica do Hospital S?o Lucas da PUCRS, tiveram sua suscetibilidade caracterizada quanto ? concentra??o inibit?ria m?nima (CIM) de tobramicina e de meropenem. A influ?ncia das diferentes condi??es ambientais foi analisada ao expor culturas de fase exponencial tardia ao meropenem e ? tobramicina por 48 h na presen?a de citrato de s?dio, galactose ou sangue de carneiro; ou em uma condi??o aerada ou est?tica. Para todas as observa??es, al?quotas foram removidas em tempos determinados, seguidas de dilui??es decimais seriadas e semeadura pela t?cnica da gota em ?gar nutriente, para estimar a fra??o de c?lulas sobreviventes. A condi??o aerada promoveu uma diminui??o na fra??o de c?lulas persisters - independente do antimicrobiano utilizado -, e tamb?m verificarmos que concentra??es superiores a 10X a CIM de tobramicina proporcionaram n?veis ainda menores destas c?lulas ap?s 48 h de tratamento. Quanto ? presen?a de diferentes fontes de nutrientes, foi observado que a presen?a de citrato de s?dio no tratamento de 48 h com meropenem proporcionou uma menor fra??o de c?lulas sobreviventes comparado a quando este mesmo antimicrobiano estava associado ? galactose; enquanto a exposi??o ? tobramicina na presen?a de galactose promoveu uma menor forma??o de c?lulas persisters nas primeiras 6 h de tratamento. O sangue de carneiro, entretanto, n?o afetou a fra??o de c?lulas sobreviventes ap?s 48 h de tratamento, independente do antimicrobiano utilizado. P?de-se ainda constatar uma not?vel heterogeneidade no comportamento de todos os 10 isolados utilizados no estudo, independente das condi??es as quais foram expostos. Desta forma, ? poss?vel que as condi??es avaliadas nos experimentos tenham influenciado a forma??o c?lulas persisters atrav?s da maior produ??o de esp?cies reativas de oxig?nio, e por disponibilizar mais alvos para a a??o dos antimicrobianos ao estimular o crescimento bacteriano.

Antibiotic resistance is a global and accelerating matter. Over time, the bacteria have evolved several defense mechanisms against the antibiotics. One of the defense mechanisms is that the bacteria can produce enzymes with the ability to hydrolyze the characteristic b-lactam ring of the antibiotics. These enzymes are called b-lactamases. There are three different generations of antibiotics clinically available, and b-lactamases have co-evolved with the antibiotics over the generations. The third generation of antibiotics are called the carbapenems and b-lactamases which hydrolyze carbapenems are called carbapenemases. Carbapenemases are promiscuous, which means that they hydrolyze a variety of antibiotics. The b-lactamase IMI-1 is an imipenem-hydrolyzing enzyme and imipenem is a carbapenem, hence IMI-1 is a carbapenemase. In this project, IMI-1 was investigated in complex with the carbapenems imipenem, meropenem and biapenem using computational methods. More specifically, a homology model of IMI-1 was generated and the carbapenems were docked into the model. The system was then used for MD simulations where the important molecular interactions were identified, and the binding free energies were calculated using the LIE method. The results indicate that IMI-1 has flexible loops that enables an open and a closed conformation of IMI- 1. All three carbapenems were docked and simulated in both conformations of IMI-1. The results indicate that open and closed conformations confirms the promiscuity of carbapenemases since the flexibility enables various initial binding mechanisms. in other words, the hydrolysis may occur so quickly that the binding does not have much bearing of the activity of the enzyme. Furthermore, the calculated binding free energies indicate that IMI-1 is optimized for the catalytic process rather than the binding affinity. In conclusion, IMI-1 and similar systems requires further research using computational methods to counteract antibiotic resistance based on knowledge.