Antibiotics are a foundation of modern medicine, helping to save millions of lives since their discovery in 1928. But the improper and excessive use of these drugs over the last few decades has led to an alarming increase in antimicrobial resistance; coupled with the recent decrease in antibiotic discovery, it is widely thought that we are approaching a post-antibiotic era. A less well-understood problem is that of drug tolerance. Even at high doses, antibiotics often cannot kill all the bacteria in an infection because of cells that are able to tolerate antibiotic treatment. Evidence points to drug-tolerant cells, also called persisters, to be a major cause of treatment failure and chronic and recurring infections It is imperative that we develop insight and methods to prevent the spread of antimicrobial resistance and combat antimicrobial tolerance. One key effort is characterizing bacterial responses to antibiotic drug treatment to generate a more comprehensive understanding of the factors that contribute to cell death and to elucidate potential targets for new therapies. Quinolones are an important class of antibiotics that target DNA replication. They bind to topoisomerase II and IV, leading to eventual DNA fragmentation and death. However, the precise mechanism by which they work is not well understood. Because they inhibit DNA replication, quinolones lead to up-regulation of the SOS response, which allows for increased mutagenesis and the potential for increased antimicrobial resistance, thus making quinolones an interesting class of antibiotics to study. Although quinolones are one of the most effective classes of antibiotics, there are many conditions in which they do not kill, such as in stationary-phase cultures. Understanding the mechanism behind quinolone killing, quinolone-induced mutagenesis and tolerance to quinolones is important to improve quinolone efficacy. Here I have presented my work on understanding quinolones: sensitivity, mutagenesis and tolerance. In understanding quinolone sensitivity, I focus on DNA repair and its involvement in quinolone-mediated death. I then probe the field of stress-induced mutagenesis by quinolones, uncovering phenotypes of dose-dependent mutagenesis that have previously been uncharacterized. Finally, I focus on drug tolerance and how density-dependent tolerance to quinolones can be reversed by up-regulating cellular respiration through the addition of a carbon source and electron acceptor. / 2018-11-02T00:00:00Z

Urinary tract infection (UTI) is a major burden for healthcare due to high prevalence and increasing multi-drug resistance, and advance to urosepsis which accounts up to 20-30% of sepsis cases. Therapy failures due to inappropriate antibiotic treatment are a serious issue. We are studying the transcriptomic response of the uropathogenic strain E. coli CFT073 to antibiotic treatment during bloodstream infection (BSI) models in order to understand and avoid antibiotic therapy failures in bloodstream infection/urosepsis treatments which are caused by in vivo and in vitro differences of bacterial response to antibiotic challenge which cannot be explained by antibiotic resistance mechanisms. Bloodstream infection models and controls were established by growing E. coli CFT073 in Iso-Sensitest medium, pooled human serum, and pooled human whole blood with and without ciprofloxacin. The minimum inhibitory concentration (MIC) values for E. coli CFT073 in these models were identified and used to set the antibiotic challenge in the serum/blood cultures. The antibiotic challenge was introduced at mid logarithmic phase of growth of the organism to depict a clinical scenario. Global gene expression profiling of these conditions was examined using E. coli Genechip 2.0 from Affymetrix. Data analysis was performed using the Partek Genomics Suite. The difference in the growth medium was well reflected in the growth pattern. In contrast to the growth in IST, the growth in whole blood was double the duration and growth in human serum was 6 fold longer. This difference was also reflected in the transcriptomic study where metabolic genes were regulated differently in each medium as expected. When comparing the responses to antibiotic challenge, bacteria grown in the respective medium displayed specific responses to the antibiotic challenge which were not seen in the other media. The common functions of genes that responded to the ciprofloxacin challenge were SOS response, DNA repair, DNA replication, fimbrial genes and bacteriophage initiation. A subset of the bacteriophage genes showed similar responses between the three models. From genes that were differentially regulated, responses observed in the serum model appeared to have the highest fold changes; this could be an inverse proportion to the nutrient availability in the medium. In this study we have established new models to investigate bloodstream infections based on human serum and whole blood. They have been used to identify previously unknown differences in the molecular response to antibiotic treatment by the uropathogenic E. coli CFT073 depending on the media. In ongoing and future studies we will exploit these differences in gene expression as biomarkers of bacterial response to antibiotics for the development of diagnostic tools for antibiotic therapy monitoring and as well as tools for diagnostics and stratification in drug development.

The presence of pharmacologically active and persistent compounds in drinking water sources is an environmental and public health concern. Sources of pharmaceuticals in the aquatic environment include wastewater treatment plant effluents and veterinary use. Antibiotics are of special concern because of their role in the spread of bacterial resistance. Conventional drinking water treatment processes are often ineffective for removing trace organic contaminants. Ozonation processes have demonstrated the ability to remove pharmaceutical compounds from drinking water supplies. During the ozonation of drinking water, the primary oxidants are ozone and hydroxyl radicals formed during the decomposition of ozone. Both oxidants contribute to the removal of pharmaceutical compounds; however, the relative rates of destruction by these two oxidants depends on the treatment operating conditions, the background water chemistry and the structure and reactivity of the target compound. This study investigated the relative impact of natural water characteristics, such as pH, the carbonate system, and natural organic matter, on the removal of the fluoroquinolone antibiotic ciprofloxacin by ozonation processes. Rate constants for k"O3, Cip obtained at pH 7 were approximately one order of magnitude higher than at pH 5 because ciprofloxacin changes from a positively charged cation to a neutral species over this pH range. The results showed that there was very little variation of the rate constants for ciprofloxacin oxidation by O₃ or hydroxyl radicals regardless of the carbonate concentration or the presence of the two organic matters studied in this research. Typical values for k"O3, Cip and k"HO°, Cip obtained at pH 7 ranged between 1.49x10⁴ and 1.64x10⁴ M⁻¹s⁻¹ and 1.29x10¹⁰ to 1.80x10¹⁰ M⁻¹s⁻¹, respectively. However, the presence of carbonate and other hydroxyl radical scavengers did have an impact on O₃ and hydroxyl radical exposure. The relative impact of these two oxidants changed depending on the pH of the system and the presence of carbonate and natural organic matter. / text

Emergent multidrug resistant Escherichia coli increase clinical challenges. This thesis describes the resistance patterns, molecular epidemiology and mechanisms, for 315 E. coli from patients in the Vancouver Coastal Health Region for 2006/2007. Automated susceptibility testing was confirmed via E-test® for AmpC and/or ESBL production. PFGE, RFLP and PCR were used to assess genotypic relationships, and plasmid character. AmpC production was facilitated mainly by promoter mutations (54.5%). The principal ESBL detected was CTX-M-15 (49.5%). An unidentified ESBL-producer, with a pI near 8.3, was detected. A plasmid displayed variant resistance phenotypes dependent on selective growth media. A positive correlation between ST131 with CTX-M-15 and CIPR indicated the dissemination of companion phenotypes. Ciprofloxacin resistance resulted mainly (98.0%) from a double gyrA mutation. Overall fluoroquinolone resistance was not assessable due to exclusive selection parameters in this evaluation. Fluoroquinolone resistance factors require further examination to understand what causes resistant phenotypes exclusive of chromosomal mutations. ii

Emergent multidrug resistant Escherichia coli increase clinical challenges. This thesis describes the resistance patterns, molecular epidemiology and mechanisms, for 315 E. coli from patients in the Vancouver Coastal Health Region for 2006/2007. Automated susceptibility testing was confirmed via E-test® for AmpC and/or ESBL production. PFGE, RFLP and PCR were used to assess genotypic relationships, and plasmid character. AmpC production was facilitated mainly by promoter mutations (54.5%). The principal ESBL detected was CTX-M-15 (49.5%). An unidentified ESBL-producer, with a pI near 8.3, was detected. A plasmid displayed variant resistance phenotypes dependent on selective growth media. A positive correlation between ST131 with CTX-M-15 and CIPR indicated the dissemination of companion phenotypes. Ciprofloxacin resistance resulted mainly (98.0%) from a double gyrA mutation. Overall fluoroquinolone resistance was not assessable due to exclusive selection parameters in this evaluation. Fluoroquinolone resistance factors require further examination to understand what causes resistant phenotypes exclusive of chromosomal mutations. ii

Thesis (M.S.)--Michigan State University. Dept. of Biochemistry and Molecular Biology, 2008. / Title from PDF t.p. (viewed on July 29, 2009) Includes bibliographical references (p. 78-81). Also issued in print.

Thesis (M.S.)--Montana State University--Bozeman, 2004. / Typescript. Chairperson, Graduate Committee: Aleksandra Vinogradov. Includes bibliographical references (leaves 83-90).

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 190 pages. Includes Vita. Includes bibliographical references.

Diante da presença de microrganismos resistentes à terapêutica endodôntica, novas formulações e alternativas medicamentosas têm sido investigadas. Desse modo, esse estudo teve como objetivo avaliar a citotoxicidade de diferentes medicações indicadas como medicação intracanal. Culturas de células de fibroblastos foram estimuladas de acordo com os seguintes grupos experimentais: Grupo I: controle; Grupo II: cloridrato de ciprofloxacina; Grupo III: cloridrato de clindamicina e Grupo IV: metronidazol. Para tal, as concentrações utilizadas para cada antibiótico foram de 5, 50, 150 e 300 mg/L, nos tempos experimentais de 24, 48, 72 e 96 horas. A citotoxicidade dessas substâncias foi avaliada usando a técnica de análise do MTT e leitura em espectrofotômetro de ELISA. Os resultados obtidos foram analisados pelo software BioEstat 4.0, por meio do teste de Kruskal-Wallis, complementado pelo teste de Dunn, com nível de significância de 5%. A viabilidade celular foi analisada diante da relação entre as diferentes concentrações e uma mesma concentração nos diferentes tempos experimentais. Diante dos resultados obtidos, foi possível concluir que: todos os antibióticos testados apresentaram citotoxicidade dosedependente. As concentrações de 5 e 50 mg/L, de todos os antibióticos, permitiram a viabilidade dos fibroblastos em todos os tempos avaliados. Independente da medicação avaliada, a viabilidade celular em 24 horas foi mais elevada em comparação aos demais tempos experimentais. A comparação entre os antibióticos nas mesmas concentrações, em diferentes tempos experimentais demonstrou que o gel de metronidazol apresentou menor citotoxicidade em 72 e 96 horas no confronto com os dois outros antibióticos, enquanto que o cloridrato de clindamicina apresentou maior viabilidade celular em relação ao cloridrato de ciprofloxacina em 72 horas. / New medicaments have been assessed due to the presence of resistantmicroorganism to therapeutic procedures. Thus, this study aimed to evaluate the citotoxicity of differents drugs indicated as intracanal medicament. The human gingival fibroblasts were estimulated according to the following experimental groups: Group I: control; Group II: Ciprofloxacin Hydrochloride; Group III: Clindamicin Hydrochloride and Group IV: Metronidazole. The concentration used for each drugs were 5, 50, 150, and 300 mg/L, in the experimental time of 24, 48, 72, and 96 hours. The citotoxicicty were evaluated with MTT analysis and ELISA spectrophotometer reading. The results were evaluated by BioEstat 4.0 software, according to Kruskal- Wallis test supplemented by Dunn test with significance level of 5%. Cell viability analysis has been assessed in different concentrations in each experimental period. According to the results, it is possible to conclude that: all the tested drugs showed dose-dependent citotoxicity. The concentrations of 5 and 50mg/L of all groups allowed fibroblast viability in all evaluated periods. The cell viability in 24 hours was higher than the others experimental periods. The comparing between the antibiotics at the same concentrations, in different times showed the gel metronidazole obtained lowest citotoxicity at 72 and 96 hours when compared with the others antibiotics, meanwhile, the Clindamicin Hydrochloride showed the highest cell viability compared to Ciprofloxacin Hydrochloride, at 72 hours.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-27T18:04:04Z No. of bitstreams: 1 Capett, Muniqui Scharamm [Dissertação, 2014].pdf: 1658363 bytes, checksum: 4be51a47b3655224f5200be235524e5d (MD5) / Made available in DSpace on 2017-03-27T18:04:04Z (GMT). No. of bitstreams: 1 Capett, Muniqui Scharamm [Dissertação, 2014].pdf: 1658363 bytes, checksum: 4be51a47b3655224f5200be235524e5d (MD5) / Escherichia coli é o principal patógeno associado a infecções do trato urinário (ITU) em pacientes com câncer ginecológico. As taxas de resistência dessas bactérias às fluoroquinolonas parecem elevadas nessa população. O objetivo deste trabalho foi caracterizar geneticamente as cepas de E. coli obtidas de ITU de pacientes com câncer ginecológico, bem como estudar a resistência ao ciprofloxacino nestas cepas. Para tal, 77 cepas de E. coli resistentes e 38 sensíveis ao ciprofloxacino, foram avaliadas. A diversidade clonal das cepas foi determinada pela técnica de PFGE. A tipificação filogenética e a presença dos genes de virulência iucC, fyuA, hlyC e cnf1, foram avaliadas por PCR. O estudo da resistência ao ciprofloxacino foi realizado pela detecção dos genes qnrA, qnrB e qnrS, aac(6’)-Ib-cr, e qepA, utilizando PCR; e pelo sequenciamento dos genes da DNA Girase, gyrA e gyrB, e da Topoisomerase IV, parC e parE, em nove cepas resistentes, do grupo filogenético B2. Testes de Fisher ou Qui-quadrado foram utilizados para análise estatística. Os resultados demonstraram grande variação genética entre as cepas resistentes ao ciprofloxacino analisadas pelo PFGE, no entanto, o grupo filogenético B2 foi o prevalente, tanto na população resistente (46,8%) quanto na população de cepas sensíveis (65,8%) ao ciprofloxacino. As cepas sensíveis tiveram maior detecção dos genes hlyC, cnf1 e fyuA (p<0,0001; p<0,0001; p=0,0008, respectivamente). O grupo B2 apresentou maior número de genes de virulência e foi associado com a presença de fyuA (p= 0,0123) na população resistente, e cnf1 (p=0,0008) na população sensível. Dentre todas as cepas, hlyC (p= 0,0255), cnf1 (p=0,0003) e fyuA (p= 0,0004) foram mais presentes no grupo B2. Com relação à resistência ao ciprofloxacino, os genes qnrA e qepA não foram detectados e o gene qnrS foi encontrado em uma única cepa. Número maior de detecção (6/ 7,8%) deu-se com relação ao gene aac(6’)-Ib-cr; porém, a detecção do gene qnrB foi muito acima do esperado, sendo este gene encontrado em 26% das cepas resistentes e em 13,2% das cepas sensíveis. As mutações encontradas foram Ser-83-Leu e Asp-87-Asn em gyrA, Ser-80-Ile, Glu-84-Val e Glu-84-Lys em parC. O número de mutações não teve correlação direta com a concentração mínima inibitória (CMI) / Escherichia coli is the major pathogen associated with urinary tract infections (UTI) in patients with gynecological cancer. The resistant rates of these bacteria to fluoroquinolones seem to be high in this population. The aim of this study was to genetically characterize and study the resistance to ciprofloxacin in E. coli strains obtained from UTI patients with gynecological cancer. For this purpose, 77 ciprofloxacin-resistant and 38 ciprofloxacin-susceptible E. coli strains were evaluated. The clonal diversity of the strains was determined by PFGE. Phylogenetic typing and the presence of virulence genes iucC, fyuA, hlyC, cnf1, were analyzed by PCR. The study of resistance to ciprofloxacin was performed by detection of qnrA, qnrB, qnrS, aac(6’)-Ib-cr and qepA genes using PCR; and the sequencing of DNA Gyrase genes, gyrA and gyrB, and Topoisomerase IV, parC and parE in nine resistant strains of phylogenetic group B2. Fisher or Chi-square tests were used for statistical analysis. The results showed large genetic variation among the ciprofloxacin-resistant strains analyzed by PFGE, however, the phylogenetic group B2 was the most prevalent in both the ciprofloxacin-resistant (46.8%) and ciprofloxacin-susceptible (65.8%) population of strains. The ciprofloxacin-sensitive strains had a greater detection of hlyC, cnf1 and fyuA (p<0.0001; p<0.0001; p=0.0008, respectively). The phylogenetic group B2 had a greater number of virulence genes and it was associated with the presence of fyuA (p= 0.0123) in the resistant population, and cnf1 (p=0.0008) in the sensitive population. Among all strains, hlyC (p= 0.0255), cnf1 (p=0.0003) and fyuA (p= 0.0004) were more present in phylogenetic group B2. Regarding to the ciprofloxacin-resistance, the qnrA and qepA genes were not detected, the qnrS gene was found in just one (1.3%) strain. A higher number of detection (6/ 7,8%) was found with respect to the aac(6’)-Ib-cr gene; however, the detection of the qnrB gene gene was far higher than expected, with this gene being noticed in 26% of resistant strains and 13.2% of susceptible strains. The mutations found were Ser-83-Leu and Asp-87-Asn in gyrA, Ser-80-Ile, Glu-84-Val and Glu-84-Lys in ParC. The number of mutations had no direct correlation with the Minimum Inhibitory Concentration (MIC)