Class of 2012 Abstract / Specific Aims: Determine the incidence of scheduled co-administration times in handwritten (paper) and electronic prepared medication administration records of oral ciprofloxacin and oral moxifloxacin with interacting substances that can affect fluoroquinolone gastrointestinal absorption. Also, determine the incidence of actual co-administration of oral ciprofloxacin and moxifloxacin with interacting substances that can affect fluoroquinolone gastrointestinal absorption with electronic and handwritten prepared medication administration records. Methods: Retrospective data was obtained by a chart review of patients from an academic medical center for a one month period before (May 2010) and after (August 2010) implementation of an electronic prepared medical administration record system. The scheduled time and actual time given for all fluoroquinolone antibiotics, as well as all possible interacting substances, were recorded. Main Results: A total of 99 subjects were included in this study (36 paper and 63 electronic). There was no statistical difference (p=0.47) between the percentage of scheduling errors for the electronic prepared medication administration records, 25.3%, compared to the paper medication administration records, 22.1%. However, there was a decrease in the percentage of actual co-administrations of fluoroquinolones with interacting substances for the electronic prepared MARs compared to paper prepared medication administration records; 22.3% and 32.1% respectfully (p=0.03). Conclusions: After implementing electronic prepared medication administration records at an academic institution, co-administration errors went down even though the amount of scheduling errors did not decrease.

Class of 2012 Abstract / Specific Aims: Determine the incidence of scheduled co-administration times in handwritten (paper) and electronic prepared medication administration records of oral ciprofloxacin and oral moxifloxacin with interacting substances that can affect fluoroquinolone gastrointestinal absorption. Also, determine the incidence of actual co-administration of oral ciprofloxacin and moxifloxacin with interacting substances that can affect fluoroquinolone gastrointestinal absorption with electronic and handwritten prepared medication administration records. Methods: Retrospective data was obtained by a chart review of patients from an academic medical center for a one month period before (May 2010) and after (August 2010) implementation of an electronic prepared medical administration record system. The scheduled time and actual time given for all fluoroquinolone antibiotics, as well as all possible interacting substances, were recorded. Main Results: A total of 99 subjects were included in this study (36 paper and 63 electronic). There was no statistical difference (p=0.47) between the percentage of scheduling errors for the electronic prepared medication administration records, 25.3%, compared to the paper medication administration records, 22.1%. However, there was a decrease in the percentage of actual co-administrations of fluoroquinolones with interacting substances for the electronic prepared MARs compared to paper prepared medication administration records; 22.3% and 32.1% respectfully (p=0.03). Conclusions: After implementing electronic prepared medication administration records at an academic institution, co-administration errors went down even though the amount of scheduling errors did not decrease.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / David G. Renter / Salmonella and Campylobacter are two leading causes of human foodborne disease. Cattle can asymptomatically shed these organisms in their feces. Fluoroquinolones are antimicrobials used to treat both humans and animals. With concerns over antimicrobial resistance, antimicrobial use in livestock has become scrutinized. Data on prevalence and susceptibility of Salmonella and Campylobacter in feedlot cattle, particularly those exposed to fluoroquinolones, are sparse. The purpose of the research described in this dissertation was to determine the prevalence and quinolone susceptibility of Salmonella and Campylobacter isolated from feedlot cattle and to determine whether these outcomes were associated with fluoroquinolone use. First, an observational study was performed at five commercial feedlots that used enrofloxacin (a fluoroquinolone) as first-line treatment for bovine respiratory disease (BRD). Fecal samples were collected from cattle pens with various levels of BRD and exposure to enrofloxacin. Salmonella and Campylobacter prevalence and susceptibility to quinolones, nalidixic acid and ciprofloxacin, were evaluated. Prevalence of Salmonella and Campylobacter was highly variable among and within feedlots. All but one Salmonella isolate was susceptible to nalidixic acid and ciprofloxacin, whereas 49% (126/256) of the Campylobacter isolates were resistant to both antimicrobials. However, the number of enrofloxacin treatments was not associated with the prevalence or susceptibilities of either organism. A second, experimental study assessed prevalence and quinolone susceptibilities of Salmonella and Campylobacter in feces of feedlot cattle administered enrofloxacin for the control of BRD (metaphylaxis). Cattle with no history of fluoroquinolone exposure were randomly assigned to either an enrofloxacin treated pen or a non-treated, control pen. Cattle feces were repeatedly collected and cultured for Salmonella and Campylobacter, with isolates tested for susceptibilities to nalidixic acid and ciprofloxacin. Overall, Salmonella and Campylobacter prevalence estimates were relatively low and decreased over time. Resistance prevalence was negligible for Salmonella, but was high for Campylobacter. However, there was no evidence that enrofloxacin metaphylaxis impacted the prevalence of Salmonella or Campylobacter, nor did it significantly affect their susceptibility to human quinolones. In conclusion, enrofloxacin use in feedlot cattle does not appear to have a significant impact on the prevalence or resistance of Salmonella and Campylobacter.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Chronic infections are often attributed to bacterial biofilms. These biofilms are extremely tolerant to antimicrobial treatment due to the presence of dormant persister cells. Whilst a number of persister genes and pathways have been identified, it is likely that others remain. Investigating persistence of S. Typhimurium was therefore undertaken. A csp null mutant of Salmonella enterica sv. Typhimurium, lacking all six cold shock protein (CspA) paralogues was previously constructed (Hutchinson 2005). At 10°C, this strain is unable to divide, but remains viable for several weeks. However it remains capable of growth at 37°C and thus is conditionally dormant. Using this strain, the link between dormancy and persistence was investigated. Treatment of stationary phase planktonic cultures with fluoroquinolones revealed persister cells in SL1344. In contrast the csp null mutant was completely eliminated by treatment at 37°C; this could be prevented by cspC or cspE expression, implicating a role for cspA paralogues in persistence. Cold shock (10°C) substantially increased persister levels, although csp null cultures remained hypersensitive. Chloramphenicol pre-treatment also reduced elimination of the csp null mutant, linking translation with the persister phenotype. Mutations in 5 genes affecting chromosomal structure and function were investigated, 3 of which (hns, hfq, rpoS) were found to reduce persister levels, suggesting a possible role for DNA supercoiling in persistence. Plasmid topologies in the csp null mutant were highly supercoiled compared to SL1344, a phenotype prevented by cspC or cspE expression. Altered supercoiling is therefore proposed as a mechanism for fluoroquinolone sensitivity in the csp null mutant. Persister levels were also characterised in biofilms of SL1344 and the csp null mutant. In contrast to stationary phase planktonic cultures, the CspA paralogues did not appear to play a role in biofilm persistence under the experimental conditions tested. However, the study revealed a novel role for CspA paralogues in pellicle formation at the air-liquid interface. A plasmid library was used to identify chromosomal regions capable of rescuing the planktonic persister phenotype of the csp null mutant. One region which delayed fluoroquinolone elimination of the csp null mutant, contained components of the hpa gene cluster, replicated in 11 isolates. This locus is involved in hydroxyphenylacetate (HPA) catabolism, indicating a possible role of cellular metabolism in the persistence. Overall this study has revealed novel information about antibiotic persistence in S. Typhimurium and the involvement of the CspA paralogues. These results provide an important foundation for further investigations and contribute towards knowledge of the complex processes of dormancy, persistence and biofilm formation in bacteria.

Fluoroquinolones are broad spectrum antibiotics that have been in use for nearly 50 years. These agents are used to treat a variety of bacterial infections from simple urinary tract infections to tuberculosis. The protein targets of fluoroquinolones are bacterial type II topoisomerases. Fluoroquinolones inhibit the function of these topoisomerases by intercalating in the nick site of the DNA and forming an interaction with helix-4 of the enzyme through a magnesium-water bridge. The binding of a fluoroquinolone stabilizes the DNA-topoisomerase-fluoroquinolone ternary complex. Helix-4 is where some of the most important fluoroquinolone resistance mutations occur. While the fluoroquinolone class of antibiotics has been successful at treating a variety of infections over the past few decades, a number of problems exist. These problems include the inability of many fluoroquinolones to kill non-growing cells, the emergence of fluoroquinolone resistant mutants, and adverse side effects of this antibiotic class. Thus, various aspects of fluoroquinolone structure and activity are explored in this study. The first topic explored is the question of what structural features are necessary for a fluoroquinolone to be able to kill bacteria in the presence and absence of the protein synthesis inhibitor, chloramphenicol (to mimic a dormant, non-growing state of the bacteria). Previous studies have shown that steric bulk at the C-8 position (especially a methoxy group) is necessary to support the ability of a fluoroquinolone to kill non-growing cells. In this study, the N-1 position of a series of C-8 methoxy fluoroquinolones was explored to gain an understanding of what substituents at the N-1 position of C-8 methoxy fluoroquinolones support the ability to rapidly kill bacteria in the presence of a protein synthesis inhibitor. In a second study the N-1 position is further explored, but with different goals. A recent crystal structure of a fluoroquinolone bound in the ternary complex with topoisomerase IV and DNA has revealed that the N-1 position of the fluoroquinolone is near in space to the catalytic tyrosine residue. It was reasoned that new interactions can be made with active site tyrosine residue through the N-1 position of the fluoroquinolone core. A number of N-1 fluoroquinolone derivatives were designed, synthesized, and evaluated for their ability to inhibit the DNA supercoiling activity of DNA gyrase, as well as the poisoning ability of the fluoroquinolones. The advantages of targeting the catalytic tyrosine residue are that this amino acid cannot be mutated without loss of enzyme function, and that by forming a new binding contact to the enzyme, activity can be maintained against helix-4 mutants. Finally, in a step toward the goal of mitigating the tendon related side effects of fluoroquinolones (thought to be due to Ca2+ coordination), the metal binding domain of the fluoroquinolone was altered. These fluoroquinolones were tested for their ability to inhibit and poison DNA gyrase. From the studies described, we have learned that the N-1 position is very sensitive to modification, that novel binding contacts to bacterial topoisomerases can be made through the N-1 position, and that modifying the metal binding domain of fluoroquinolones can lead to retention of activity against DNA gyrase. These accomplishments all push the fluoroquinolone field ahead by introducing a novel binding interaction to optimize (with the goal of creating a fluoroquinolone that is active against current fluoroquinolone resistant mutants) and by showing that fluoroquinolone activity can be retained even when the metal binding domain is altered, thus moving us closer to the goal of reducing tendon-related side effects.

Fluoroquinolones are broad-spectrum antibacterial agents based on the structure of nalidixic acid. For nearly five decades it has been known that fluoroquinolones inhibit bacterial growth by blocking the enzymatic action of type II topoisomerases such as DNA gyrase and topoisomerase IV. Only recently has it been discovered that some fluoroquinolones are capable of a mechanism that results in fragmented DNA and leads to rapid bacterial cell death. This mechanism is not well understood. Presented here are studies towards understanding the structure activity relationship (SAR) of fluoroquinolones, specifically to determine what leads to the novel mechanism termed "rapid lethality." This work is based on the hypothesis that structurally unique fluoroquinolones interact with the DNA-topoisomerase complex in a unique manner that ultimately leads to rapid cell death. The first approach to understand SAR for killing was to evaluate the effect of a ring fusion between N-1 and C-8 of the fluoroquinolone core. Known lethal fluoroquinolones are substituted by N-1 cyclopropyl and C-8 methoxy, but some clinically important fluoroquinolones contain a 2-methylmopholino moiety between these two positions. Novel fluoroquinolones were synthesized and clinically available agents were obtained to create a panel of drug molecules with one of six C-7 substituents and either the morpholine ring system or N-1 cyclopropyl and C-8 methoxy. Bacteriostatic and bactericidal activities of these compounds were determined. Bactericidal studies were conducted both in the presence and absence of chloramphenicol, a protein synthesis inhibitor used to simulate non-growing bacteria. Lethality in the presence of chloramphenicol is also important when considering co-administration of fluoroquinolones with other antibiotic classes. In a second study, fluoroquinolones were synthesized with a C-2 thioalkyl substitution. Substitutions at the C-2 position are severely lacking in clinical fluoroquinolones, with only prulifloxacin, a newly developed antibiotic, being substituted by an N-1 to C-2 thiazetidine ring structure. Analogs of ciprofloxacin and moxifloxacin were synthesized such that the N-1, C-2, and C-8 positions were substituted with cyclopropyl, thioethyl/thioisopropyl, and methoxy groups, respectively. The compounds were then evaluated for antibiotic activity against three different bacterial strains to evaluate the contribution of the C-2 thioalkyl substituent to antibacterial activity. In a third study, quinazoline-2,4-diones, a new antibiotic class structurally and mechanistically similar to fluoroquinolones, were modified at the C-4 position in an effort to understand the binding interaction between these compounds and the target enzyme. Importantly, the quinazoline-2,4-diones typically retain activity against bacterial cells known to be resistant to fluoroquinolones and are less likely to select for resistant mutants. In this study, the C-4 carbonyl was replaced with either a thiocarbonyl or a hydroxylimine and the new compounds, bearing C-7 substituents common to potent antibiotic fluoroquinolones and quinazolines, were evaluated for activity against bacterial cells. Despite the findings of recently published X-ray crystallography, it was determined that one of the greatest determinants in antibiotic activity of fluoroquinolones is the C-7 substituent. Additionally, there is increasing evidence that the C-2 carbonyl of quinazoline-2,4-diones affords the increase in activity against resistant mutants by creating a unique binding interaction. Collectively, the conclusions reached here add to our understanding of the structure activity relationship of the fluoroquinolone antibiotic class for rapidly killing bacterial cells and overcoming resistant mutants.

Fluoroquinolones, a class of type-II topoisomerase inhibitors, have successfully been used as antibiotics for the last several decades, beginning with the use of nalidixic acid in urinary tract infections. This led to the broad-spectrum activity of ciprofloxacin in the 1980s. Unfortunately, use of fluoroquinolones has led to the emergence of resistant bacteria. Recently, this has generated new bacteria such as multidrug-resistant and extensive-drug-resistant strains of M. tuberculosis that are also fluoroquinolone-resistant. Infections caused by these bacterial strains are widespread, with high mortality rate in immune-compromised populations such as the elderly, infants, and in AIDS or HIV-positive patients. Fluoroquinolone resistance is acquired through amino acid substitutions of key fluoroquinolone-binding residues of the type-II bacterial topoisomerases DNA Gyrase and Topoisomerase IV, the enzyme targets of fluoroquinolones. Amino acid substitutions that result in fluoroquinolone resistance are located on Helix-4 of these enzymes, which is the site of a magnesium (Mg)-water bridge that is a crucial binding interaction for fluoroquinolones. When certain substitutions to Helix-4 occur, the Mg-water bridge is compromised and no longer available to anchor fluoroquinolones into a ternary complex composed of topoisomerase, fluoroquinolone, and DNA. This results in drug resistance. Herein are described attempts to generate fluoroquinolones that are capable of overcoming this mechanism of resistance. In the first study, attempts were made to generate a series of novel tricyclic fluoroquinolones and diones designed to exploit intercalative or pi-stacking binding interactions with the bacterial DNA in the ternary complex in order to lessen the importance of the Mg-water bridge interaction. Despite numerous attempts, no complete synthetic pathway to these core structures was ever discovered. The second study investigated the utility of a C7-aminomethylpyrrolidine group on the fluoroquinolone structure. This was done in order to explore the mechanistic reasons why previously generated fluoroquinolones possessing this C7-aminomethylpyrrolidine group maintained activity against common Helix-4 mutants. A panel of fluoroquinolones with C7-aminomethylpyrrolidine groups and diverse core structures was synthesized and docking studies with the original C7-aminomethylpyrrolidine fluoroquinolone and other fluoroquinolones were performed. Target compounds were synthesized and evaluated for inhibition/poisoning purified enzyme and for the ability to inhibit growth with wild-type and fluoroquinolone-resistant cells. In a third study, fluoroquinolones possessing structural variations of the C7-aminomethylpyrrolidine were designed and synthesized to explore structural requirements of the aminomethylpyrrolidine group binding and overcoming fluoroquinolone-resistance caused by alterations of Helix-4. This led to further exploration of the binding space around the C7-position of the fluoroquinolones. In both the second and third studies, the new fluoroquinolones were evaluated for the ability to specifically target bacterial topoisomerases over human topoisomerase. The results of these studies have contributed new knowledge to the binding requirements of fluoroquinolones that maintain potency against fluoroquinolone-resistant type-II topoisomerases, and represent a step towards methodology to overcome bacteria resistant to fluoroquinolones.

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

L’anthropisation médicamenteuse des eaux usées favorise l’émergence et la diffusion dans l’environnement de microorganismes résistants aux antibiotiques. Les effluents hospitaliers pourraient être doublement impliqués en véhiculant antibiotiques et bactéries multirésistantes. L’objectif de ce travail est de caractériser les effluents hospitaliers d’un Centre Hospitalo-Universitaire en évaluant simultanément les concentrations d’antibiotiques (fluoroquinolones et imipénème) et la diversité des bactéries résistantes à ces antibiotiques au sein de biofilms constitués in situ. Les concentrations en antibiotiques mesurées par chromatographie en phase liquide - spectrométrie de masse après collecte via un échantillonnage passif pendant 15 jours sont égales à 2,08±0,88μg/L (ciprofloxacine), 101,06±18.47 μg/L (ofloxacine), 6,43±0.56 μg/L (norfloxacine) et indétectable pour l’imipénème. Comparées aux données de consommation à l’hôpital pendant cette même période, les concentrations estimées sont 5,84±1,78μg/L (ciprofloxacine), 11.22±1.09μg/L (ofloxacine), 7.68±3,7μg/L (norfloxacine) et 3,61±0,24ug/L (imipénème). La mesure du risque potentiel écotoxicologique s’est avérée positive pour la ciprofloxacine et la norfloxacine (hazard quotient >1). En parallèle, des bactéries résistantes aux fluoroquinolones (n=115) ou aux carbapénèmes (n=38) ont été isolées de biofilms formés dans les effluents hospitaliers. 60 % des isolats, constitués majoritairement de bacilles à Gram négatif, notamment Aeromonas spp et Klebsiella spp, sont résistants à plusieurs familles d’antibiotiques dont certains sont exclusivement utilisés à l’hôpital. La majorité des souches hébergent des éléments génétiques mobiles dont des plasmides conjugatifs porteurs de la résistance à l’imipénème ou aux fluoroquinolones. La présence combinée de bactéries résistantes aux antibiotiques hébergeant des éléments génétiques mobiles en lien avec ces résistances et de faibles concentrations en antibiotiques permet de qualifier l’interface hôpital-environnement comme un lieu propice au transfert des résistances. / The presence of pharmaceutical compounds in waste water favors the emergence and the spreading of antibiotic resistant microorganisms. The hospital effluents could be involved gathering antibiotics and multiresistant bacteria. The aim of this work is to characterize the hospital effluents of a teaching hospital measuring simultaneously the concentrations of antibiotics (fluoroquinolones and imipenem) and the diversity of the bacteria resistant to these antibiotics within hospital effluent biofilms.The antibiotics concentrations were measured by liquid-phase chromatography - mass spectrometry via a passive sampling during 15 days. The measured environmental concentrations were 2.08 ± 0.88μg/L (ciprofloxacin), 101.06 ± 18.47 μg/L (ofloxacine), 6.43 ± 0.56 μg/L (norfloxacine). Imipenem was not detected. Compared with the data of hospital consumption during the same period, the predicted estimated concentrations are 5.84±1.78µg/L(ciprofloxacin), 11.22 ± 1.09µg/L (ofloxacin), 7.68 ± 3.7µg/L, 7.68 ± 3.7μg/L (norfloxacin) and 3.61 ± 0.24ug/L (imipenem). The ecotoxicological risk was confirmed for the ciprofloxacin and the ofloxacin (hazard quotient > 1).In parallel, fluoroquinolones (n=115) and carbapenem (n=38) resistant bacteria were isolated from hospital effluent biofilm. Sixty % of isolates, mainly composed by Gram negative bacilli in particular Aeromona spp and Klebsiella spp, are resistant to several antibiotics among which some are exclusively used at the hospital. The majority of these strains have mobile genetic elements such as conjugative plasmids harboring imipenem or fluoroquinolones resistances.The presences of both antibiotics resistant bacteria harboring mobile genetic elements in connection with these resistances and low antibiotics concentrations make the hospital effluent a convenient place for the transfer of resistance between the hospital and the environment.