Foi realizado um estudo de farmacocinética da polimixina B em pacientes críticos com desenvolvimento de um modelo populacional. Os critérios de inclusão foram pacientes internados em Unidade de Terapia Intensiva, com idade igual ou superior a 18 anos e em uso de polimixina B intravenosa por um período mínimo de 48 horas. Amostras de sangue, urina e dialisato foram coletadas durante um intervalo de doses no estado de equilíbrio. A concentração de polimixina B no plasma foi medida por meio de cromatografia líquida de alta performance associada à espectrometria de massas acoplada à espectrometria de massas, sua ligação às proteínas plasmáticas foi determinada por meio de diálise de equilíbrio rápido e a fração livre foi calculada. Foram realizadas análise farmacocinética populacional e Simulações de Monte Carlo. Foram incluídos 24 pacientes, dos quais dois estavam em hemodiálise contínua; 54,2% eram do sexo masculino e as medianas da idade, do escore APACHE e do peso corporal total foram de 61,5 anos, 21,5 e 62,5kg, respectivamente. As doses de polimixina B, conforme prescrição do médico assistente, variaram entre 0,45-3,38mg/kg/dia. O clearance estimado da creatinina nos 22 pacientes sem hemodiálise variou entre 10-143mL/min. A mediana da fração livre plasmática da polimixina B foi de 0,42 e a média (± desvio padrão) da fração livre da área sob a curva ao longo de um dia (fAUC0-24h) da polimixina B foi de 29,2±12,0mg•h/L, incluindo os pacientes em hemodiálise. A polimixina B foi excretada predominantemente por vias não renais e as medianas de sua recuperação urinária de forma inalterada foi de 4,04% e do seu clearance renal foi de 0,061L/hora. Nos pacientes 1 e 2 em hemodiálise foram identificados, respectivamente, clearance corporal total de 0,043 e 0,027L/h/kg, clearance da hemodiálise de 0,0052 e 0,0015L/h/kg; no dialisato foram recuperados 12,2% e 5,62% da dose como polimixina B não modificada. O clearance corporal total da polimixina B não mostrou nenhuma relação com o clearance da creatinina, escore APACHE II ou idade. A disposição da polimixina B no tempo foi adequadamente descrita pelo modelo de dois compartimentos com eliminação linear. O modelo farmacocinético populacional proporcionou ajustes excelentes para os perfis observados de concentração-tempo para pacientes individuais e as concentrações individuais e populacionais ajustadas foram precisas. O ajuste dos clearances e dos volumes de distribuição para o peso corporal total reduziu a variabilidade intersujeitos em 3,4% para o clearance e 41,7% para o volume de distribuição central; nos pacientes em diálise, após esse ajuste, os parâmetros estimados se assemelharam aos dos demais pacientes. As Simulações de Monte Carlo foram feitas com seis diferentes regimes de doses clinicamente relevantes escalonados pelo peso corporal total. O regime de doses de 1,5mg/kg 12/12h forneceu uma AUC0-24h de polimixina B no dia 4 de 90.4mg•hora/L para 50% dos pacientes, adequada para erradicação bacteriana em infecções graves por Pseudomonas aeruginosa ou Acinetobacter baumannii com concentração inibitória mínima para a polimixina B ≤2mg/L. Nas Simulações de Monte Carlo também foi possível identificar que uma melhor área sob a curva só foi atingida no dia 4 de tratamento. Este estudo mostrou que a dose de polimixina B intravenosa deve ser ajustada ao peso corporal total, que o melhor regime de doses é o de 1,5mg/kg 12/12h precedido de dose de ataque de 2,5mg/kg e que não há indicação de ajuste para a função renal, mesmo em pacientes em hemodiálise contínua. / A polymyxin B pharmacokinetics study in critically ill patients was conducted with the development of a population modeling. The inclusion criteria were patients from Intensive Care Unit, aged ≥18 years who received intravenous polymyxin B for ≥ 48 hours. Blood, urine and dialysate samples were collected over a dosing interval at steady state. Polymyxin B concentrations was measured by liquid chromatography- tandem mass spectrometry, its plasma protein binding was determined by rapid equilibrium dialysis and unbound fraction was calculated. Population pharmacokinetic analysis and Monte Carlo Simulations were conducted. Twenty four patients were enrolled, two of whom on continuous hemodialysis; 54.2% were male; the median of age, APACHE II score and total body weight were 61.5years, 21.5 and 62.5kg, respectively. The physician-selected dose of polymyxin B was 0.45- 3.38mg/kg/day. The creatinine clearance of the 22 patients without hemodialysis ranged from 10 to 143mL/min. The median unbound fraction in plasma of polymyxin B was 0.42 and the mean (± standard deviation) of the area under the curve over a day for unbound (fAUC0-24h) polymyxin B was 29.2±12.0mg•hour/L, including hemodialysis patients. Polymyxin B was predominantly nonrenally cleared with median unchanged urinary recovered of 4.04%; the median renal clearance was 0.061L/hour. Patients 1 and 2 in hemodialysis presented, respectively, total body clearance of 0.043 and 0.027L/h/kg, hemodialysis clearance of 0.0052 and 0.0015L/h/kg; 12.2% and 5.62% of the polymyxin dose were recovered intact in the dialysate. Polymyxin B total body clearance did not show any relationship with creatinine clearance, APACHE II score, or age. The time course of polymyxin B concentrations was well described by a 2-compartment disposition model with linear elimination. The population pharmacokinetics model provided excellent fits to the observed concentration-time profiles for individual patients and the individual-fitted and population-fitted concentrations were adequately precise. Linear scaling of clearances and volumes of distribution by total body weight reduced the between subject variability in 3.4% for clearance and 41.7% for the central volume of distribution; after this scaling, the estimated parameters in hemodialysis patients were within the range of estimates from the other patients. The population mean of the total body clearance of polymyxin B when scaled by total body weight (0.0276L/hour/kg) showed remarkably low interindividual variability. The Monte Carlo Simulations were performed for six different clinically relevant dosage regimens scaled by total body weight. The regimen of 1.5mg/kg/12 hours provided an AUC0- 24h of polymyxin B of 90.4 mg•h/L in day 4 for 50% of patients which is appropriate considering severe infections by Pseudomonas aeruginosa or Acinetobacter baumannii with minimal inhibitory concentration for polymyxin B ≤2mg/L. In Monte Carlo Simulations we also identified that the best area under the curve was attained only in the day 4 of the treatment. This study showed that doses of intravenous polymyxin B are best scaled by total body weight, that the best regimen of doses is 3mg/kg/day with a loading dose of 2.5mg/kg and that its dosage selection should not be based on renal function, even in patients in continuous hemodialysis.

INTRODUÇÃO: O aumento da incidência de infecções causadas por bactérias resistentes a múltiplos antimicrobianos limita cada vez mais as opções terapêuticas, dificultando o tratamento e aumentando os índices de morbidade e mortalidade, além dos gastos em saúde. Ao longo dos últimos cinco anos, essa limitação tem levado ao reestabelecimento do uso de antimicrobianos consideradas ultrapassados, como as polimixinas. Este grupo passou a ser utilizado com cada vez mais frequência no tratamento de infecções causadas por microrganismos gram-negativos resistentes aos carbapenêmicos. As enterobactérias, em particular a espécie Klebsiella pneumoniae, tem apresentado frequentemente esse perfil, porém, a resistência à polimixinas têm sido relatada, eliminando essa importante alternativa terapêutica. Apesar da importância do tema, são escassas as publicações sobre frequência de resistência às polimixinas em K. pneumoniae e a relação clonal entre isolados resistentes à polimixina B no Brasil. OBJETIVOS: Avaliar a relação genética, perfil de sensibilidade antimicrobiana e mecanismos de resistência às polimixinas em K. pneumoniae. MATERIAIS E MÉTODOS: A execução deste trabalho dividiu-se em duas partes principais: (i) levantamento de dados de culturas positivas para K. pneumoniae da rotina de pacientes hospitalizados em instituições atendidas pelo serviço de análises clínicas do Fleury Medicina e Saúde; (ii) confirmação das concentrações inibitórias mínimas (CIM) para polimixina B, avaliação da relação clonal por eletroforese em campos pulsados (PFGE),e sequenciamento de múltiplos loci (MLST), avaliação da integridade do gene mgrB e da presença do gene mcr-1 por PCR entre isolados resistentes à polimixina B e aos carbapenêmicos (CPRKp). RESULTADOS e CONCLUSÕES: Na análise de 3.085 isolados de K. pneumoniae obtidos de pacientes internados em 11 hospitais da Grande São Paulo entre os anos de 2011 e 2015, foi evidenciado um aumento estatisticamente significativo na resistência aos carbapenêmicos de 6,8% em 2011 para 35,5% em 2015. Em 2015, KPC foi detectada em 96,2% dos isolados resistentes aos carbapenêmicos. A distribuição das concentrações inibitórias mínimas de polimixina B entre todos os isolados de K. pneumoniae evidenciou uma distribuição bimodal com a CIM de 2 mg/L como o valor de ponto de corte para a susceptibilidade à polimixina B; assim, 3,6% do número total de isolados sensíveis aos carbapenêmicos foram interpretados como resistentes enquanto essa proporção foi de 22,5% entre as resistentes aos carbapenêmicos (CRKp). Entre esses últimos isolados também houve um aumento estatisticamente significativo na tendência anual de resistência à polimixina B, de 0% em 2011 para 27,1% em 2015. Estas taxas variaram de 0,7% em 2011 para 3,9% até junho de 2014 entre os sensíveis aos carbapenêmicos. Entre os antimicrobianos alternativos, a amicacina e a tigeciclina foram os compostos mais ativos. A análise por PFGE de 60 isolados de CPRKp obtidos de pacientes distintos nos anos de 2014 e 2015 evidenciou dois grandes grupos clonais: CPRKp1 e CPRKp2, os quais segundo a análise por MLST pertencem, respectivamente, aos grupos ST11 e ST437, ambos do complexo clonal 258. Foi observado o mesmo grupo ST entre isolados obtidos dentro de um mesmo hospital e também entre diferentes hospitais, públicos e privados. O mecanismo de resistência mais comum entre os isolados de CPRKp foi a presença de sequências de inserção interrompendo o gene mgrB. O gene mcr-1 não foi detectado em nenhum dos isolados. / INTRODUCTION: The increasing incidence of infections caused by bacteria resistant to multiple antimicrobials increasingly limits therapeutic options, making treatment difficult and increasing the morbidity and mortality and health spending. Over the past five years, this limitation has led to the reestablishment of the use of antimicrobials deemed outdated, such as polymyxins. This group is now used with increasing frequency to treat infections caused by carbapenem-resistant gram-negative microorganisms. Enterobacteria, especially Klebsiella pneumoniae, have often presented this profile, however, resistance to polymyxins have been also reported, eliminating this important therapeutic alternative. Despite the importance of this issue, the publications are scarce on the polymyxins resistance frequency in K. pneumoniae and clonal relationship among isolates resistant to polymyxin B in Brazil. OBJECTIVES: To evaluate the genetic relationship, antimicrobial susceptibility profile and polymyxin B resistance mechanisms in K. pneumoniae. MATERIALS AND METHODS: The execution of this work was divided into two main parts: (i) survey data on routine cultures positive for K. pneumoniae from patients hospitalized in institutions attended by the clinical analysis service of Fleury Health and Medicine; (ii) confirmation of to polymyxin B minimum inhibitory concentrations (MIC), evaluation of clonal relationship by electrophoresis pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), evaluation of the integrity of the mgrB gene and the presence of mcr-1 gene by PCR among isolates resistant to polymyxin B and carbapenems (CPRKp). RESULTS AND CONCLUSIONS: The analysis of 3,085 K. pneumoniae isolates obtained from inpatients from 11 hospitals in the São Paulo urban area between 2011 and 2015, has shown a statistically significant increase in carbapenem resistance from 6.8% in 2011 to 35.5% in 2015. In 2015, KPC was detected in 96.2% of isolates resistant to carbapenems. The polymyxin B MIC distribution of all Klebsiella pneumoniae showed a bimodal distribution with the MIC of 2 mg/L as the cutoff value for polymyxin B susceptibility; thus, 3.6% of the total number of isolates susceptible to carbapenems were interpreted as resistant while this proportion was 22.5% among carbapenem-resistant isolates (CRKp). Among these isolates there was also a statistically significant increase in the annual trend of polymyxin B resistance, from 0% in 2011 to 27.1% in 2015. These rates ranged from 0.7% in 2011 to 3.9% by June 2014 between carbapenem-susceptible isolates. Among alternative antimicrobials, amikacin and tigecycline were the most active compounds. The analysis by PFGE of 60 CPRKp isolates obtained from different patients in the years 2014 and 2015 showed two major clonal groups: CPRKp1 and CPRKp2, which according to the analysis by MLST belong respectively to ST11 and ST437 groups, both from clonal complex 258. We observed the same ST group of isolates obtained within a hospital and between different public and private hospitals. The most common mechanism of polymyxin B resistance among CPRKp isolates was the presence of insertion sequences interrupting the mgrB gene. The mcr-1 gene was not detected in any of the isolates.

La colistine est un vieil antibiotique, utilisé à la fois en médecine humaine et vétérinaire. Cependant, l'arsenal antibiotique étant de plus en plus limité, la colistine apparait comme un des derniers remparts dans la lutte contre les bactéries multi-résistantes chez l'Homme. Afin de préserver l'efficacité de la colistine, deux problématiques ont été abordées dans cette thèse : (i) les risques de sélection de résistance à la colistine en lien avec la découverte fin 2015 d'un gène porté par un plasmide (mcr-1). Ainsi, l'impact de l'usage de colistine par voie orale en production porcine a été évalué in vivo et une absence de sélection a été observée dans nos conditions expérimentales. De façon similaire, l'usage (minoritaire) de colistine en médecine humaine comme prophylaxie de décontamination digestive sélective (SDD) a été étudié chez des rats hébergeant un microbiote intestinal humain. Les résultats préliminaires ne montrent pas non plus d'effet de sélection. (ii) le développement d'un modèle pharmacocinétique basé sur la physiologie (PBPK) chez le porc pour l'usage par voie systémique de la colistine et de sa prodrogue, le colistine méthanesulfonate (CMS). Ce modèle a permis d'explorer la distribution tissulaire du CMS et de la colistine, notamment au niveau rénal où la toxicité est la plus fréquente. Comme application de ce modèle, l'estimation des temps d'attente avant abattage lors d'usage de CMS chez le porc a été effectué. Enfin, la capacité des modèles PBPK à réaliser des extrapolations intra et inter-espèces a été utilisé pour adapter ce modèle chez l'adulte et l'enfant, afin de pouvoir prédire les concentrations plasmatiques de colistine lors d'un traitement. / Colistin is an old antibiotic used in human and veterinary medicine. However, as less and less antibiotics are discovered, colistin is considered as a last-line antibiotic to fight against multi-drug resistant bacteria in human. In order to preserve the efficacy of colistin, two issues were investigated in this thesis:(i) Risks of selection of bacteria resistant to colistin, in conjunction with the discovery by the end of 2015 of a plasmid-mediated resistance gene (mcr-1). Thus, the impact of oral use of colistin in pigs was assessed in vivo and no selection was observed in our experimental conditions. Similarly, the use of colistin in human medicine for selective digestive decontamination was studied thanks to human flora‐associated rats. Preliminary results were also neither in favour of a selective effect of colistin.(ii) development of a physiologically-based pharmacokinetic model (PBPK) in pigs for the systemic use of colistin and its prodrug, the colistimethate sodium (CMS). This model provided a further insight into CMS and colistin tissue distribution, especially in kidneys where toxic effects are frequent. As a model application, the withdrawal period after use of CMS in pigs was estimated. Then, we used the ability of PBPK models to carry out intra and inter-species extrapolations in order to adapt this model in adults and children and eventually predict the plasmatic concentrations of colistin during a treatment with CMS.

La colistine, antibiotique appartenant à la famille des polymyxines, est un polypeptide cyclique, cationique, ciblant les membranes bactériennes. Elle est produite par Paenibacillus polymyxa via des complexes multi-enzymatiques appelés Non-Ribosomal Peptides Synthétases (NRPS). Dans le cas de la mucoviscidose, et malgré des effets secondaires importants, la colistine est utilisée comme ultime recours pour lutter contre les bactéries Gram-négatives multirésistantes responsables d’infections pulmonaires dont Pseudomonas aeruginosa. Jusqu’ici les systèmes génétiques à l’origine de la production de la colistine étaient peu connus. Au cours de cette étude, nous avons caractérisé par LC-MS haute résolution des molécules antimicrobiennes, dont des colistines, produites par un nouveau Paenibacillus. Afin d’identifier et de cloner le cluster de gène responsable de la production de ces antibiotiques, une banque d’ADN génomique a été construite et criblée par homologie de séquence avec des systèmes de production déjà connus. Ce criblage a permis de sélectionner quatre clones d’intérêt. L’étude in silico de leurs séquences a permis d’identifier les différents modules d’un nouveau cluster NRPS qui serait à l’origine de la synthèse de variants de la colistine. À terme, cette découverte pourrait permettre de mieux contrôler la production de la colistine et d’obtenir des composés plus actifs et/ou présentant des effets secondaires amoindris. En parallèle à ce premier travail, nous avons également recherché la présence de nouvelles NRPS chez une centaine de micro-organismes issus d’une station d’étude environnementale du laboratoire (vasière intertidale). Ce travail a permis de découvrir des nouvelles séquences et d’isoler un nouveau micro-organisme producteur d’antibiotique(s). / Colistin is a cationic cyclic polypeptide antibiotic belonging to the polymyxin family and targeting bacterial membranes. It is produced by Paenibacillus polymyxa through a Nonribosomal Peptide Synthetase (NRPS) mechanism. In the context of cystic fibrosis (CF), colistin is used for the treatment of lung infections caused by multiresistant Gram-negative bacteria including Pseudomonas aeruginosa. Unfortunately, this molecule is also known for its strong side effects. So far, genetic systems controlling the production of polymyxins were little known. In this study we characterized by High-resolution LC-MS the antimicrobial molecules, including colistins, of a new Paenibacillus. A genomic library of this strain was constructed and screened to identify genes involved in the production of these antibiotics. A degenerated PCR screening was performed and allowed to select four clones in the genomic library. In silico study allowed to identify a new NRPS gene cluster responsible for the biosynthesis of colistin variants. In the future, this work might allow the harnessing of the production of colistin derived structures, more active and/or showing fewer side effects. In parallel, a second investigation was performed in order to find new NRPS genes in a collection of one hundred intertidal mudflat bacterial isolates. This work has allowed the identification of new sequences and the characterization of a new antimicrobial producing strain.

Dans ce travail de thèse, l'efficacité de la voie intrapulmonaire et les paramètres biopharmaceutiques influençant la diffusion pulmonaire après nébulisation d'antibiotiques ont été évalués. La présence et l'impact de certaines pompes d'efflux dans un modèle in vitro de cellules primaires épithéliales pulmonaires de rat ont été testés. Trois fluoroquinolones et la colistine ont été utilisées comme molécules de référence. La combinaison des molécules testées a permis d'obtenir une vue d'ensemble des caractéristiques de diffusion intrapulmonaire des antibiotiques. L'étude in vivo avec les fluoroquinolones a démontré que les concentrations pulmonaires de ces molécules sont plus importantes que dans le plasma, probablement dû à la présence des transporteurs comme la glycoprotéine-P. La présence de ces transporteurs a été confirmée dans le modèle de cellules pulmonaires de rats. L'étude in vivo avec la colistine a montré qu'une lente diffusion pourrait conférer un avantage à la nébulisation par rapport à l'administration intraveineuse. En conclusion, l'administration par nébulisation des molécules, qui traversent les tissus lentement (colistine), pourrait être avantageuse, alors que pour d'autres, qui traversent vite la barrière (fluoroquinolones), la voie nébulisée pourrait ne pas présenter des avantages par rapport à la voie intraveineuse. De plus, les résultats ont démontré qu'une faible perméabilité à travers le poumon (colistine) pourrait donner un avantage à la nébulisation des antibiotiques, tandis qu'une affinité pour des transporteurs (fluoroquinolones) semble présenter un intérêt aussi bien dans le cadre d'une nébulisation que d'une administration intraveineuse. / The aim of this study was to investigate the efficiency of intrapulmonary administration and the biopharmaceutical parameters regulating the pulmonary diffusion following nebulization. We examined whether certain efflux pumps were present in an in vitro model of rat lung cells and whether these efflux pumps could be beneficial by increasing lung concentrations in vivo. Fluoroquinolones and colistin were the molecules used as reference. These different molecules allowed an overview of the intrapulmonary diffusion characteristics of antibiotics. The in vivo study with fluoroquinolones showed that their lung concentrations are higher than in plasma, probably due to glycoprotein-P. The presence of this efflux pump was confirmed in the model with rat lung cells. The in vivo study with colistin showed that a slow diffusion may confer an advantage for nebulization over intravenous administration. In conclusion, the nebulization molecules passing slowly (colistin) across the tissues may be advantageous, whereas for others, with a fast passage across the barrier (fluoroquinolones), the pulmonary route may not provide an advantage over the intravenous administration. Moreover, the results showed that a slow permeability across the lung (colistin) may confer an advantage for the antibiotic nebulization, while affinity by transporters (fluoroquinolones) is beneficial for both nebulization and intravenous administration.

Parmi les plus grandes menaces de la santé publique dans le monde entier, la résistance aux antibiotiques est à la pointe. Ceci en partie est dû à l'augmentation des infections causées par des bactéries pathogènes résistantes aux antibiotiques ainsi que la diminution du nombre actuel de nouveaux antibiotiques. Dans le souci de remédier à cette situation malheureuse, il y a eu récemment la ré-surfaçage des antibiotiques anciens et abandonnés comme les polymyxines. Colistine, un membre des antibiotiques de polymyxine, est maintenant considéré comme un antibiotique de «dernier recours» pour le traitement des infections bactériennes à Gram-négatives graves en raison de son action puissante contre ces agents pathogènes. Cependant, la résistance à la colistine parmi ces agents pathogènes a émergé dans plusieurs pays et est actuellement en augmentation. En raison de la nouvelle réintroduction relative de cet antibiotique, il ya un manque d'information complètes sur ses propriétés pharmacologiques ainsi que des mécanismes par lequel les bactéries développent une résistance contre celle-ci.Afin de combler ce manque d'information en ce qui concerne le mécanisme de résistance, nous avons donc entrepris ce projet. Tout d'abord, pour procéder à une surveillance épidémiologique des bactéries résistantes à la colistine chez les humains et les animaux domestiques et d'autre part, de décrypter les mécanismes moléculaires de résistance à la colistine parmi les bactéries résistantes isolées. / Among one of the greatest threats facing public health worldwide, antibiotic resistance is at the forefront. This is partly due to increase in infections caused by antibiotic-resistant pathogenic bacterial as well as the current dwindling number of new antibiotics. In a way to address this unfortunate situation, there have been recent resuscitation of old and abandoned antibiotics such as polymyxins. Colistin, a member of polymyxin antibiotics, is now regarded as a 'last-resort' antibiotic for the treatment of severe Gram-negative bacterial infections owing to its potent action against these pathogens. However, resistance to colistin among these pathogens has emerged in several countries and is currently on increase. Due to the relatively new reintroduction of this antibiotic, there is a lack of comprehensive information on its pharmacological properties as well as mechanisms by which bacteria develop resistance against it.In order to bridge this information gap in relation to the mechanism of resistance, we therefore undertook this project. First, to carry out an epidemiological surveillance of colistin-resistant bacteria in humans and domesticated animals and secondly, to decipher the molecular mechanisms mediating colistin resistance among the isolated resistant bacteria.

Colistin is a polypeptide antibiotic used as a "last resort" effective against Gram-negative bacteria. However, increase in its consumption in veterinary medicine in the last 30 years has led to the development of bacterial resistance even to colistin. The application of slurry to soil containing unmetabolized colistin and resistant bacteria poses a risk, because the resistance of bacteria to colistin can be further developed and spread to other components of the environment. Therefore, this work was focused on the creation and optimization of extraction and analytical methods for soil samples containing colistin. Colistin was detected by liquid chromatography in connection with mass spectrometry (HPLC/MS and UPLC/MS/MS). Many extraction solutions have been tested in connection with ultrasonic extraction and purification on various SPE columns, yet colistin has not been successfully extracted from the soil. Colistin was successfully detected only in the slurry extract. The reason why colistin was not detected in soil extracts could be the sorption of colistin on soil organic matter or the formation of complexes of colistin with humic substances.

The continuous emergence of drug-resistance pathogens is a global concern. As a result, substantial effort is being expended to develop new therapeutics and mechanisms for controlling microbial growth to avoid entering a "post-antibiotic" era in which commonly used antibiotics are no longer effective in treating infections. In this work, we investigate the efficacy and application of ceragenins as non-peptide mimics of antimicrobial peptides (AMPs). First, this work examines the susceptibility of drug-resistant Gram-negative bacteria. The susceptibility of colistin-resistant clinical isolates of Klebsiella pneumoniae to ceragenins and AMPs suggests that there is little to no cross-resistance between colistin and ceragenins/AMPs. Furthermore, Lipid A modifications are found in bacteria with modest changes in susceptibility to ceragenins and with high levels of resistance to colistin. Next, we investigated the potential for cross resistance between chlorhexidine, colistin, AMPs and ceragenins as repeated exposure of bacteria to chlorhexidine might result in cross resistance with colistin, AMPs or ceragenins. Furthermore, a proteomics study on the chlorhexidine-resistant strains showed that chlorhexidine resistance is associated with upregulation of proteins involved in the assembly of LPS for outer membrane biogenesis and virulence factors in Pseudomonas aeruginosa.Second, this dissertation describes the antifungal activity of ceragenins against an emerging multidrug-resistant fungus, Candida auris. We found that lead ceragenins displayed activities comparable to known antifungal agents against C. auris isolates. We also found that fungal cell morphology was altered in response to ceragenin treatment, that ceragenins exhibited activity against sessile organisms in biofilms, and that gel and cream formulations including CSA-44 and CSA-131 resulted in a significant log reduction against established fungal infections in ex vivo mucosal tissues. Finally, a hydrogel film containing CSA-131 was generated on endotracheal tubes (ETTs). ETTs provide an abiotic surface on which bacteria and fungi form biofilms that cause serious infections. In this study, the eluting ceragenin prevented fungal and bacterial colonization of coated ETTs for extended periods while uncoated tubes were colonized by bacteria and fungi. Coated tubes were well tolerated in intubated pigs. The ability of ceragenins to eradicate established biofilms make them attractive potential therapeutics for persistent infections in the lung, including those associated with cystic fibrosis. In ex vivo studies, we initially found that this ceragenin, at concentrations necessary to eradicate established biofilms, also causes loss of cilia function. However, by formulating CSA-131 in poloxamer micelles, cilia damage was eliminated and antimicrobial activity was unaffected. These findings suggest that CSA-131, formulated in micelles, may act as a potential therapeutic for polymicrobial and biofilm-related infections in the lung and trachea.

No description available.

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