Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

<p>The study evaluated the Mutant Prevention Concentration (MPC) of Pradofloxacin on three Escherichia coli (E.coli) strains, 2 wildtypes and one first-step gyrA resistant mutant. We also measured the value of AUC (Under the Concentration)/MPC that prevents growth of resistant mutants. It is of importance to reach a concentration above MPC that prevent E.coli from developing resistance against the antibiotic.</p><p>We used an in vitro kinetic model where we added bacteria? and antibiotic. The culture flask was attached to a pump with an adjustable pump-speed. This made it possible to dilute the antibiotics in a satisfying elimination half-life (t1/2= 7 hours) pace. Samples were removed with a syringe at different times in the study. The samples where then cultured on agar- plates to enable counting of the viable colonies after incubation.</p><p>The optimal concentration to completely eradicate both E.coli wildtypes Nu14 and MG1655 with Pradofloxacin was Cmax ≥8 times MPC and AUC/MPC then became73. Additional experiments needs to be done on the resistant mutant LM378 before we can determine the optimal concentration. But results so far indicate that the concentration of Cmax would be about 8-12 timesMPC to completely eradicate that mutant.</p>

As fluorquinolonas são antimicrobianos frequentemente prescritos no Brasil e em outros países e utilizados no tratamento de diversos tipos de infecções, principalmente naquelas do trato urinário e gastrintestinal. A ação desses compostos ocorre devido a interação dos mesmos com a DNA-girase e a topoisomerase IV bacterianas. A DNA girase é codificada pelos genes gyrA e gyrB e a topoisomerase IV, pelos genes parE e parC. Estudos evidenciaram existir nesses genes regiões nas quais as mutações ocorrem de modo mais frequente, sendo esses sítios chamados de Quinolone Resistance Determining Regions (QRDR). Mutações nas QRDR levam a substituição de aminoácidos nas topoisomerases, diminuindo a afinidade destas enzimas pelas quinolonas, acarretando, portanto, em um fenótipo de resistência para essa classe de antimicrobianos. Este mecanismo de resistência mostrou-se um dos principais responsáveis pela resistência antimicrobiana para essa classe de antimicrobiano em isolados clínicos. Entretanto, outros mecanismos de resistência podem estar presentes, entre eles a aquisição de determinantes plasmidiais (qnrA, qnrB, qnrS, qepA, oqxA e oqxB). A maioria dos estudos foi realizada utilizando isolados bacterianos clínicos, contudo, pouco tem sido investigado sobre os mecanismos de resistência com bactérias de origem ambiental. Diante disso, o presente trabalho objetivou identificar os principais mecanismos de resistência às quinolonas em isolados bacterianos ambientais e os principais gêneros dessa microbiota que os albergam. Para tanto, 69 isolados bacterianos ambientais foram selecionados. Os principais gêneros bacterianos selecionados foram Stenotrophomonas, Achromobacter, Ochrobactrum e Escherichia para os quais foram pesquisados além dos genes Plasmid Mediated Quinolone Resistance (PMQR), mutações nos genes gyrA, gyrB, parE e parC e a tipagem plasmidial. No conjunto os resultados permitem evidenciar um padrão de mutações nos genes codificadores das topoisomerases semelhante ao observado em isolados bacterianos clínicos, atuando os genes PMQR de modo sinérgico na contribuição da elevação da concentração inibitória mínima (CIM) frente às fluorquinolonas. No entando, mecanismos adicionais que possam contribuir com os níveis de resistência não podem ser descartados, sobretudo sistemas de efluxo. A tipagem plasmidial evidenciou que a maioria dos isolados apresenta plasmídeos da família ColE-like. / Fluoroquinolones are antimicrobials frequently prescribed in Brazil and in other countries and used in the treatment of various types of infections, especially those of the urinary tract and gastrointestinal tract. The action of these compounds occurs due to their interaction with bacterial DNA-gyrase and topoisomerase IV. The DNA gyrase is encoded by the gyrA and gyrB genes and the topoisomerase IV, by the parE and parC genes. Studies have shown that there are regions in which the mutations occur more frequently, these sites being called QRDR (Quinolone Resistance Determining Regions). Mutations in QRDR lead to amino acid substitution in topoisomerases, reducing the affinity of these enzymes for the quinolones, thus leading to a resistance phenotype for this class of antimicrobials. This mechanism of resistance was shown to be one of the main factors responsible for antimicrobial resistance for this class of chemotherapy in clinical isolates. However, other mechanisms of resistance may be present, among them the acquisition of plasmid determinants (qnrA, qnrB, qnrS, qepA, oqxA and oqxB). Most studies were carried out using clinical bacterial isolates, however, little research has been done on the mechanisms. The present work aimed to identify the main mechanisms of resistance to quinolones in environmental bacterial isolates and the main genera of this microbiota that harbor them. For this purpose, 69 bacterial isolates were selected from environmental samples. The main bacterial genera selected were Stenotrophomonas, Achromobacter, Ochrobactrum and Escherichia for which the genes PMQR, gyrA, gyrB, parE and parC and the plasmid typing were investigated. The results pattern of mutations in the genes coding for topoisomerases are similar to that observed in clinical bacterial isolates, acting PMQR genes synergistically in the contribution of MIC elevation to fluoroquinolones. However, additional mechanisms that may contribute to resistance levels cannot be discarded, especially efflux systems. Plasmid typing evidenced that most of the isolates present plasmids from the ColE-like family

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.

<p>Antibiotic-resistant bacteria are increasingly a major healthcare problem but very few new classes of antibiotics have been discovered or launched in recent decades. Approaches to dealing with the problem include learning how bacteria evolve to resistance and improving dosing regimens with current antibiotics so as to reduce the selection of resistant bacteria. </p><p>This thesis presents studies examining whether antibiotic dosing at high levels can prevent the selection of fluoroquinolone-resistant mutants in <i>Escherichia coli</i>. It also addresses the genetics of fluoroquinolone resistance in <i>E. coli</i> in relation to fitness costs for the resistant bacteria, and the evolution of <i>E. coli</i> to reduce the costs of resistance.</p><p>The mutant prevention concentration (MPC) of ciprofloxacin was measured for a set of clinical urinary tract infection <i>E. coli</i> strains showing that MPC could not be predicted from the minimum inhibitory concentration (MIC). Results from an <i>in vitro</i> kinetic model showed that an AUC/MPC >22 for ciprofloxacin was the single best pharmacodynamic index that predicted prevention of resistance emergence in the wild-type. Simulating currently approved dosing regimens for three different fluoroquinolones it was found that only a few were effective in preventing the selection of a small sub-population of pre-existing mutants. </p><p>Step-wise selection of fluoroquinolone resistance showed that the accumulation of mutations usually reduced bacterial fitness<i> in vitro</i> and <i>in vivo</i>. Systematic construction of isogenic resistant strains confirmed this result and revealed that some combinations of resistance mutations mutually compensate and increase both resistance and fitness. It was discovered that mutations altering RNA polymerase could ameliorate the fitness costs of fluoroquinolone resistance. Thus, the major fitness cost of fluoroquinolone resistance is due to defective transcription. </p><p>The finding that fluoroquinolone resistance mutations can increase resistance while mutually compensating their fitness costs, shows that resistance to fluoroquinolones can continue to evolve in the absence of antibiotic selection.</p>

<p>The emergence of multidrug resistant bacteria world wide is a serious problem, and very few new drugs are under development. The selection of resistant bacteria is affected by factors such as mutation rate, biological fitness cost and the rate of fitness compensation. This thesis is focused on how mutation rate affects resistance to fluoroquinolones and on exploring a dosing strategy that might slow resistance development. </p><p>In a set of urinary tract <i>Escherichia coli</i> isolates MIC values above the breakpoint for the fluoroquinolones norfloxacin and ciprofloxacin carried at least three resistance-associated mutations. In these isolates the number of resistance mutations correlated with the mutation rate. During step-wise selection for decreased susceptibility to fluoroquinolones, the accumulation of mutations in <i>E. coli</i> was associated with an increasing biological cost both <i>in vitro</i> and <i>in vivo</i>. However, in some lineages an additional selection step for resistance was associated with a partial restoration of fitness. During step-wise selections we found, as expected, that reduced ciprofloxacin susceptibility frequently hitchhiked with a strong mutator phenotype. More surprisingly, we also found that reduced susceptibility was frequently associated with the emergence of rifampicin-resistant populations. We hypothesise that this correlation reflects selection for fitness-compensating mutations in RNA polymerase.</p><p>Mutant prevention concentration (MPC) dosing has been proposed as a strategy to reduce the selection of resistant bacterial populations. Based on limited data it had been thought that MPC might be a simple multiple of MIC, which can easily be determined. However, we showed for a collection of susceptible urinary tract <i>E. coli </i>that MPC could not be predicted from MIC and must be measured directly for relevant populations. Using an <i>in vitro</i> kinetic model we also showed that the pharmacodynamic index that best predicted prevention of resistance development in wild type <i>E. coli</i> was an AUC/MPC of > 22 for ciprofloxacin.</p>

The emergence of multidrug resistant bacteria world wide is a serious problem, and very few new drugs are under development. The selection of resistant bacteria is affected by factors such as mutation rate, biological fitness cost and the rate of fitness compensation. This thesis is focused on how mutation rate affects resistance to fluoroquinolones and on exploring a dosing strategy that might slow resistance development. In a set of urinary tract Escherichia coli isolates MIC values above the breakpoint for the fluoroquinolones norfloxacin and ciprofloxacin carried at least three resistance-associated mutations. In these isolates the number of resistance mutations correlated with the mutation rate. During step-wise selection for decreased susceptibility to fluoroquinolones, the accumulation of mutations in E. coli was associated with an increasing biological cost both in vitro and in vivo. However, in some lineages an additional selection step for resistance was associated with a partial restoration of fitness. During step-wise selections we found, as expected, that reduced ciprofloxacin susceptibility frequently hitchhiked with a strong mutator phenotype. More surprisingly, we also found that reduced susceptibility was frequently associated with the emergence of rifampicin-resistant populations. We hypothesise that this correlation reflects selection for fitness-compensating mutations in RNA polymerase. Mutant prevention concentration (MPC) dosing has been proposed as a strategy to reduce the selection of resistant bacterial populations. Based on limited data it had been thought that MPC might be a simple multiple of MIC, which can easily be determined. However, we showed for a collection of susceptible urinary tract E. coli that MPC could not be predicted from MIC and must be measured directly for relevant populations. Using an in vitro kinetic model we also showed that the pharmacodynamic index that best predicted prevention of resistance development in wild type E. coli was an AUC/MPC of &gt; 22 for ciprofloxacin.

The study evaluated the Mutant Prevention Concentration (MPC) of Pradofloxacin on three Escherichia coli (E.coli) strains, 2 wildtypes and one first-step gyrA resistant mutant. We also measured the value of AUC (Under the Concentration)/MPC that prevents growth of resistant mutants. It is of importance to reach a concentration above MPC that prevent E.coli from developing resistance against the antibiotic. We used an in vitro kinetic model where we added bacteria? and antibiotic. The culture flask was attached to a pump with an adjustable pump-speed. This made it possible to dilute the antibiotics in a satisfying elimination half-life (t1/2= 7 hours) pace. Samples were removed with a syringe at different times in the study. The samples where then cultured on agar- plates to enable counting of the viable colonies after incubation. The optimal concentration to completely eradicate both E.coli wildtypes Nu14 and MG1655 with Pradofloxacin was Cmax ≥8 times MPC and AUC/MPC then became73. Additional experiments needs to be done on the resistant mutant LM378 before we can determine the optimal concentration. But results so far indicate that the concentration of Cmax would be about 8-12 timesMPC to completely eradicate that mutant.

Antibiotic-resistant bacteria are increasingly a major healthcare problem but very few new classes of antibiotics have been discovered or launched in recent decades. Approaches to dealing with the problem include learning how bacteria evolve to resistance and improving dosing regimens with current antibiotics so as to reduce the selection of resistant bacteria. This thesis presents studies examining whether antibiotic dosing at high levels can prevent the selection of fluoroquinolone-resistant mutants in Escherichia coli. It also addresses the genetics of fluoroquinolone resistance in E. coli in relation to fitness costs for the resistant bacteria, and the evolution of E. coli to reduce the costs of resistance. The mutant prevention concentration (MPC) of ciprofloxacin was measured for a set of clinical urinary tract infection E. coli strains showing that MPC could not be predicted from the minimum inhibitory concentration (MIC). Results from an in vitro kinetic model showed that an AUC/MPC &gt;22 for ciprofloxacin was the single best pharmacodynamic index that predicted prevention of resistance emergence in the wild-type. Simulating currently approved dosing regimens for three different fluoroquinolones it was found that only a few were effective in preventing the selection of a small sub-population of pre-existing mutants. Step-wise selection of fluoroquinolone resistance showed that the accumulation of mutations usually reduced bacterial fitness in vitro and in vivo. Systematic construction of isogenic resistant strains confirmed this result and revealed that some combinations of resistance mutations mutually compensate and increase both resistance and fitness. It was discovered that mutations altering RNA polymerase could ameliorate the fitness costs of fluoroquinolone resistance. Thus, the major fitness cost of fluoroquinolone resistance is due to defective transcription. The finding that fluoroquinolone resistance mutations can increase resistance while mutually compensating their fitness costs, shows that resistance to fluoroquinolones can continue to evolve in the absence of antibiotic selection.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.