Fluoroquinolones, broad-spectrum bactericidal antibiotics, exert their effects by inhibiting type II topoisomerases through the formation of a fluoroquinolone-DNA-topoisomerase ternary complex. Recently, newer, structurally unique fluoroquinolones have been shown to kill bacteria by promoting chromosomal fragmentation in the presence and absence of protein synthesis, thus allowing fluoroquinolones to potentially be used in the treatment of microorganisms that go into a dormant state. There is a need to further understand the structure activity relationships (SAR) of fluoroquinolones to develop new antibiotics that can kill dormant bacteria and are active against current resistant strains. The hypothesis that structurally unique fluoroquinolones interact with the DNA- fluoroquinolone-topoisomerase ternary complex in a unique way that leads to different killing pathways is the basis of this work. The first approach to understand SAR for fluoroquinolones to kill non-growing bacteria was to evaluate the effect of modifications at the C-8 and C-5 positions on lethality. Novel, synthetically-derived and commercially-available fluoroquinolones were evaluated for ability to kill Escherichia coli in the presence and absence of chloramphenicol, a known protein synthesis inhibitor used to simulate non-growing bacteria. The second study was to understand SAR of fluoroquinolone-class agents necessary to maintain antibacterial activity against common fluoroquinolone resistance-causing bacterial mutations on topoisomerase IV. A panel of novel fluoroquinolones, 2,4-quinazoline diones, and fluoroquinolone-like analogues with unique substitution combinations at C-8 and C-7 was synthesized and evaluated for ability to poison wild-type and mutant Bacillus anthracis topoisomerase IV. The third study to understand the contribution of SAR of fluoroquinolone-class agents to novel killing mechanisms was to evaluate the binding interaction of fluoroquinolones to double-stranded and nicked DNA. Binding affinities of fluoroquinolones to DNA were determined; fluoroquinolones were found to bind different DNA types with varied affinities. The ability of a series of C-8 and C-7 modified fluoroquinolones to stabilize or destabilize DNA was assessed. The results of these studies also add broadly to the understanding of SAR associated with fluoroquinolone-class antibiotics for killing in the presence and absence of protein synthesis, maintaining activity in the presence of resistance-causing mutations in the target enzymes, and increasing binding interactions with different types of DNA.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Introduction: Has been observed the global spread of different variants of P. aeruginosa that is often associated with increased virulence or the emergence of new antimicrobial resistance genotypes. There are few studies describing the association of the Type III Secretion System (TTSS) with antibiotic resistance and outcome of patients with pneumonia and bacteremia. Objectives: Determine the relation among resistance to carbapenems and fluoroquinolones and the Type III Secretion System (TTSS) effector genotype and its association with the poor prognostic in patients with ventilator-associated pneumonia (VAP) and bacteremia, identify mutations in the Quinolone Resistance Determining Regions (QRDRs), Metallo-β-Lactamase genes (MβL), virulence genes (algD, lasB e toxA) and clonal spread of isolates producing MβL. Material and Methods: A retrospective cohort was conducted to determine the risk factors for 30-day mortality in patients with the first episode of bacteremia (157 patients) and VAP (60 patients) caused by P. aeruginosa. Genes blaIMP, blaVIM, blaSIM, blaGIM and blaSPM, TTSS genes (exoT, exoS, exoY, exoU ) and virulence genes (lasB, algD, toxA) were detected by PCR; the sequencing was conducted for QRDR genes (gyrA e parC) on fluoroquinolone-resistant strains and the Pulsed-Field Gel Electrophoresis (PFGE) for molecular typing of positive strains for the MβL genes Results: The multivariate analysis showed that predictors independently associated with death in patients with bacteremia were inappropriate therapy and cancer. Carbapenem resistance was more frequent among strains of VAP (53.3%), however with the detection of MβL genes in one isolate (blaIMP), unlike blood, where the frequency of these genes was 16.1%, being 10.7% blaSPM genotype and 5.4% blaVIM genotype. The exoS gene was found in all blood and lung isolates and the exoU gene only in 9.4%. Substitution of threonine to isoleucine at position 83 in gyrA was the most frequent mutation among fluoroquinolone-resistant strains. It was detected a mutation at position 91 in parC gene (Glu91Lys) associated with mutation in gyrA (Thre83Ile) in a strain of extensively drug-resistant P. aeruginosa, exoT+exoS+exoU+ genotype, isolate from lung, not described in Brazil yet. Among the strains that harboring the TTSS virulence genes it was observed high resistance to gentamicin (93.7%) and low for amikacin (37.5%). The evaluation of the clonal relationship between isolates producing blaSPM, blaVIM and blaIMP genes showed similarity (more than 80%) among the blaSPM strains, which was not observed for those producing blaVIM gene. Conclusions: Our results confirm previous findings regarding the spread of blaSPM clone, with indirect evidence of its cross-spread in our hospital and polyclonal those containing the blaVIM gene. Inappropriate therapy is significant factor for poor prognosis among patients with bacteremia caused by multidrug-resistant P. aeruginosa , independent of the virulence TTSS genotype associated. / Introdução: Vem sendo observado a disseminação global de diferentes variantes de P. aeruginosa que está frequentemente associada a maior virulência ou a emergência de novos genótipos de resistência aos antimicrobianos. Há poucos estudos descrevendo a associação do Sistema de Secreção Tipo III (TTSS) com resistência aos antibióticos e evolução dos pacientes com pneumonia e bacteremia. Objetivos: Determinar a relação entre resistência a fluorquinolonas e carbapenêmicos e a virulência de Pseudomonas aeruginosa pelo Sistema de Secreção Tipo III e sua associação com pior prognóstico em pacientes com Pneumonia Associada a Ventilação Mecânica (PAV) e bacteremia, identificar mutações na Região Determinante de Resistência as Quinolonas (QRDR), genes para Metalo-β-Lactamase (MβL), genes de virulência (algD, lasB e toxA) e disseminação clonal das amostras produtoras de MβL. Material e Métodos: Foi realizada uma coorte retrospectiva para determinar os fatores de risco para mortalidade em 30 dias em pacientes com primeiro episódio de bacteremia (157 pacientes) e PAV (60 pacientes) por P. aeruginosa. Os genes blaIMP, blaVIM, blaSIM, blaGIM e blaSPM, os genes do TTSS (exoT, exoS, exoY, exoU ) e os genes de virulência (lasB, algD, toxA) foram detectados por PCR; o sequenciamento foi realizado para os genes do QRDR (gyrA e parC) nas cepas resistentes a fluorquinolonas e o Pulsed-Field Gel Electrophoresis (PFGE) para tipagem molecular das amostras positivas para o gene produtor de MβL. Resultados: A análise multivariada mostrou que os preditores independentemente associados com mortalidade nos pacientes com bacteremia foram terapia antimicrobiana inapropriada e câncer. A resistência aos carbapenêmicos foi maior entre as amostras de PAV (53,3%), porém com a detecção dos genes que codificam MβL em apenas um isolado (blaIMP), ao contrário do sangue, onde a frequência desses genes foi de 16,1%, sendo 10,7% blaSPM e 5,4% blaVIM. O gene exoS foi encontrado em todas as amostras avaliadas de sangue e pulmão e o gene exoU em apenas 9,4% das mesmas. A substituição de uma treonina por isoleucina na posição 83 no gene gyrA foi a mais frequente entre as cepas resistentes a fluorquinolonas. Foi detectada uma mutação na posição 91 no gene parC (Glu91Lys) associada com a mutação em gyrA (Thre83Ile) em uma amostra de P. aeruginosa ,isolada do pulmão, extensivamente resistente, do genótipo exoT+exoS+exoU+ , ainda não descrita no Brasil. Entre as amostras que carreavam os genes de virulência TTSS observou-se alta resistência a gentamicina (93,7%) e baixa para amicacina (37,5%). A avaliação da relação clonal entre as amostras contendo os genes blaSPM, blaVIM e blaIMP , apresentou alta similaridade (maior que 80%), naquelas contendo blaSPM, o que não foi observado para as amostras contendo o gene blaVIM. Conclusões: Nossos resultados confirmam achados prévios com relação a disseminação do clone blaSPM, com evidências indiretas da sua disseminação cruzada no nosso hospital e policlonal daquelas contendo o gene blaVIM. A terapia inapropriada é fator significativo para pior prognóstico entre os pacientes com bacteremia por P. aeruginosa multirresistente, independente do genótipo de virulência TTSS associado. / Mestre em Imunologia e Parasitologia Aplicadas