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<p>The 20^th century introduction of antibiotics made once fatal infectious diseases readily treatable. This taken-for-granted therapy is now threatened by rising antibiotic resistance. The ability of pathogens to acquire numerous simultaneous resistance mechanisms has given rise to an alarming number of increasingly difficult to treat multi-drug resistant infections. When coupled with a sharp decline in development of novel antibiotic therapies, health practitioners today are left with limited therapeutic options. Several alternative methodologies have been employed to find novel therapeutics, including new techniques in natural product isolation and the production of semi-synthetic and synthetic antibiotics; however, there has been limited focus on targeting antibiotic resistance mechanisms directly to create synergistic therapies. We demonstrate the potential in using small molecules to target antibiotic kinases, thereby rescuing the antibiotic action of aminoglycosides and macrolides when used in combination. We conducted a thorough examination of these enzymes including: kinetic analysis; an assessment of phosphate donor specificity; and in-depth structural comparison, including a case study on the structure-function relationship of APH(4)-Ia. This analysis culminated in an intensive screening initiative of fourteen antibiotic kinases against a set of well defined protein kinase inhibitors. From this work, we have identified several inhibitors that have the potential for use in future combination therapeutics. This study illustrates the benefit of a structure-activity based approach to drug discovery, an important tool at a time when novel therapeutic strategies are required.</p> / Doctor of Philosophy (PhD)

<p>Antibiotic use is generally regarded as the major driver for resistance. Many studies reporting an association between antibiotic use and the emergence of resistance have been published. However, most studies have significant limitations such as single center data with comparably low number of cases, using retrospective designs with limited data availability, ecological studies with lack of assessing the individual level and risk for ecological fallacy, and inappropriate selection of controls in case-control studies.</p> <p>A cohort study in adult patients hospitalized in 15 participating acute care hospital sites in Ontario, Canada, was conducted from April 1 2005 to June 30 2006. Antibiotic use on the unit level in defined daily doses (DDD) was only available for 3 sites. In order to assess antibiotic use on both the individual as well as on the unit level as a risk factor for resistance, days of therapy (DOT) could be calculated. However, it was unclear whether this approach would results in similar findings as when using DDD. Thus, the impact of using either DDD or DOT on the risk estimates for resistance was assessed for three antimicrobial-bacteria combinations, i.e. fluoroquinolone use and fluoroquinolone resistance in enterobacteriaceae an in <em>Pseudomonas aeruginosa</em>, and the use of betalactams and resistance to third generation cephalosporins in enterobacteriaceae.</p> <p>The risk estimates for resistance were very similar for all three antimicrobial-bacteria combinations on acute care units, there were some discrepancies on the unit level on intensive care units, and discrepancies on both levels for step down and rehabilitation units.</p> <p>In conclusion, the approach to use DOT instead of DDD to measure antibiotic utilization revealed similar results. However, the lack of comprehensive information on patient transfers when calculating DOT may bias the findings on units with frequent patient transfers such as intensive care units and step down and rehabilitation units.</p> / Master of Science (MSc)

Objectives: Systemic antibiotics are generally recognized as providing a beneficial impact in treatment of both aggressive and chronic periodontitis. Since strains of periodontal pathogens among periodontitis patients may vary in their antibiotic drug resistance, the American Academy of Periodontology recommends antimicrobial susceptibility testing of suspected periodontal pathogens prior to administration of systemic periodontal antibiotic therapy, to reduce the risk of a treatment failure due to pathogen antibiotic resistance. E-test and MIC Test Strip assays are two in vitro antimicrobial susceptibility testing systems employing plastic- and paper-based, respectively, carriers loaded with predefined antibiotic gradients covering 15 two-fold dilutions. To date, no performance evaluations have been carried out comparing the Etest and MIC Test Strip assays in their ability to assess the in vitro antimicrobial susceptibility of periodontal bacterial pathogens. As a result, the purpose of this study was to compare the in vitro performance of E-test and MIC Test Strip assays in assessing minimal inhibitory concentration (MIC) values of four antibiotics frequently utilized in systemic periodontal antibiotic therapy against 11 fresh clinical subgingival isolates of the putative periodontal pathogen, Prevotella intermedia/ nigrescens, and to compare the distribution of P. intermedia/ nigrescens strains identified with interpretative criteria as "susceptible" and "resistant" to each of the four antibiotics using MIC values determined by the two antimicrobial susceptibility testing methods. Methods: Standardized cell suspensions, equivalent to a 2.0 McFarland turbidity standard, were prepared with 11 fresh clinical isolates of P. intermedia/nigrescens, each recovered from the subgingival microbiota of United States chronic periodontitis subjects, and plated onto to the surfaces of culture plates containing enriched Brucella blood agar. After drying, pairs of antibiotic-impregnated, quantitative, gradient diffusion strips from two manufacturers (E-test, bioMérieux, Durham, NC, USA, and MIC Test Strip, Liofilchem s.r.l., Roseto degli Abruzzi, Italy) for amoxicillin, clindamycin, metronidazole, and doxycycline were each placed apart from each other onto the inoculated enriched Brucella blood agar surfaces, so that an antibiotic test strip from each manufacturer was employed per plate against each P. intermedia/ nigrescens clinical isolate for antibiotic susceptibility testing. After 48-72 hours anaerobic jar incubation, individual MIC values for each antibiotic test strip against P. intermedia/nigrescens were read in &mu;g/ml at the point where the edge of the bacterial inhibition ellipse intersected with the antibiotic test strip. MIC50, MIC90, and MIC range were calculated and compared for each of the test antibiotics, with essential agreement (EA) values determined per test antibiotic for the level of outcome agreement between two antimicrobial susceptibility testing methods. In addition, the identification of antibiotic "susceptible" and "resistant" strains among the P. intermedia/nigrescens clinical isolates was determined for each test antibiotic using MIC interpretative criteria from the MIC interpretative standards developed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for gram-negative anaerobic bacteria for amoxicillin, clindamycin, and metronidazole findings, and from the French Society of Microbiology breakpoint values for anaerobic disk diffusion testing for doxycycline data. Results: For amoxicillin, higher MIC50 and MIC90 values against the P. intermedia/ nigrescens strains were found with the MIC Test Strip assay than with E-test strips, resulting in a relatively low EA value of 45.5% between the two susceptibility testing methods. A higher percentage of amoxicillin "resistant" P. intermedia/nigrescens strains (72.7%) were identified by MIC Test Strips as compared to E-test strips (54.5%), although both methods found the same proportion of amoxicillin "susceptible" strains (27.3%). For clindamycin, both susceptibility testing methods provided identical MIC values (EA value = 100%), and exactly the same distributions of "susceptible" and "resistant" strains of P. intermedia/nigrescens. For metronidazole, only very poor agreement (EA value = 9.1%) was found between the two susceptibility testing methods, with MIC Test Strips exhibiting markedly higher MIC50 and MIC90 values against P. intermedia/nigrescens as compared to E-test strips. However, the distribution of "susceptible" and "resistant" P. intermedia/ nigrescens were identical between the two susceptibility testing methods. For doxycycline, relatively good agreement (EA value = 72.7%) was found in MIC concentrations between the two susceptibility testing methods, although generally lower MIC values were associated with MIC Test Strips. In addition, identical distributions of "susceptible" and "resistant" P. intermedia/nigrescens were provided by both susceptibility testing methods. Conclusions: Relative to MIC values measured against periodontal strains of P. intermedia/nigrescens, MIC Test Strips gave higher MIC values with amoxicillin and metronidazole, equal MIC values with clindamycin, and lower MIC values with doxycycline, as compared to MIC values measured with the E-test assay. Relative to the identification of antibiotic "susceptible" periodontal P. intermedia/ nigrescens strains, both susceptibility testing methods provided identical findings, suggesting that both methods appear to be interchangeable for clinical decision making in regard to identification of antibiotic-sensitive strains of periodontal P. intermedia/nigrescens. However, for epidemiologic surveillance of drug susceptibility trends, where exact MIC values are important to track over time, the relatively higher proportion of non-exact MIC differences between the two susceptibility testing methods argues against using them interchangeably. Instead, one or the other method should be used consistently for such studies. Further comparative studies of the E-test and MIC Test Strip assays are indicated using other periodontopathic bacterial species besides P. intermedia/ nigrescens, and to assess the reproducibility of MIC values provided by both in vitro susceptibility testing methods over time. / Oral Biology

Enterococcus faecalis are Gram-positive bacteria that colonize the gastrointestinal tracts of mammals, birds and invertebrates and are also found in sewage, soil, food and water. In addition to being commensal organisms, Enterococci can also cause nosocomial infections in humans including urinary tract infections, septicemia and endocarditis. Hospital-acquired infections often present a challenge in treatment due to the emergence of multi-drug resistant strains. Enterococcal plasmids may act as extremely stable reservoirs for resistance genes and other virulence factors. Pheromone responsive plasmids such as pCF10 mediate efficient transfer of genetic material within the species E. faecalis but may also be capable of transferring resistance genes across species and genus boundaries. Polymicrobial environments often found in nosocomial infections may expose plasmid-harboring enterococci to pathogenic species, poising cells for this type of promiscuous horizontal gene transfer of resistance determinants. Previous studies showed that prgW, which encodes the pCF10 replication initiation protein PrgW, is the minimal origin of replication for this plasmid. The replicon, which is usually limited to Enterococcal spp., can replicate in Lactococcus lactis if it is engineered to produce pre-cCF10. Three conserved cysteines (C78/C275/C307) are important for plasmid stability and allow for replication of the pCF10 replicon in L. lactis in the absence of pre-cCF10. PrgW has a predicted molecular weight of 38,635. Four polyclonal antibodies targeting PrgW at the N-terminus (aa 1-20), C-terminus (aa 314-333) and two internal regions (aa 64-80 and aa 250-271) were used in current experiments and retrospective studies. When PrgW was overexpressed in E. faecalis, four different apparent approximate molecular weights were detected by Western blotting (p40*, p36*, p24* and p18*), suggestive of processing. In Enterococci where the replicon is active, p36* was consistently detected by all four antisera; when PrgW was overexpressed in Streptococcus mutans where the replicon is non-functional, p49* and p40* were detected but p36* was not observed. PrgW p24* was detected by a mixture of the internally targeting antibodies as well as the C-terminal targeting antibody, but not the N-terminal targeting antibody, suggesting that the N-terminal domain of PrgW has been cleaved off in p24*. The p24* form may play a role in pCF10 stability. Mutations to three cysteines in PrgW (C78/C275/C307), which reduce the stability of pCF10, result in the loss of p24*. Enterococcal conjugative plasmids have been previously implicated in the transfer of antibiotic resistance genes. The pCF10 plasmid contains the conjugative transposon Tn925, which possesses the tetM tetracycline resistance gene. Proximity of donor and recipient cells is a key part of pheromone-responsive conjugation. Aggregation substance allows for formation of clumps of E. faecalis in liquid mating experiments. E. faecalis forms biofilms; in contrast to filter mating experiments, polymicrobial biofilms provide an in vitro model of a natural scenario during which horizontal gene transfer may occur. Rates of cross-genus genetic transfer of tetM between E. faecalis OG1RF(pCF10) donor cells and Staphylococcus aureus recipient cells growing on glass coverslips as mixed-species biofilm populations were determined to be 10-8 after pheromone induction of pCF10 conjugation. This biofilm transfer model also holds potential to test the efficacy of synthetic peptides in the reduction or even prevention of pCF10 transfer, and the consequential dissemination of antibiotic resistance determinants throughout the genus Enterococcus and beyond. / Microbiology and Immunology

Widespread antimicrobial resistance, particularly in Gram-negative pathogens, is a serious threat facing the global community. Aminoglycosides are inactivated by enzymes such as aminoglycoside N-acetyltransferase-3 (AAC(3)) and O-nucleotidyltransferase-2” (ANT(2”)), while the New Delhi metallo-b- lactamase-1 (NDM-1) degrades carbapenems. Inhibition of these enzymes should result in bacteria becoming once again susceptible to aminoglycosides and carbapenems. This thesis describes the development of inhibitors to these enzymes, in an effort to rescue the utility of aminoglycoside and carbapenem drug classes through adjuvant therapy. High-throughput screening of protein kinase libraries identified two AAC(3)-Ia inhibitors with a common 3-benzylidene-2-indolinone core. New methods for purification of AAC(3)-Ia and monitoring its activity were developed. A chemical library was built around this scaffold and assessed for SAR. It was found that the initial hit (Z)-methyl 3-(3,5-dibromo-4-hydroxybenzylidene)-2- oxoindoline-5-carboxylate was the most active against AAC(3)-Ia, and alterations to either the 3,5-dibromo-4-hydroxybenzyl warhead or methyl ester substituent resulted in a decrease in activity. Previous whole-cell screening had identified two protein kinase inhibitors with a biphenyl isonicotinamide scaffold as inhibitors of ANT(2”)-Ia. A convergent parallel synthesis was developed, involving Suzuki and amide couplings and protecting group strategies. This methodology was used to assemble a focused chemical library for SAR analysis. Stepwise removal of extraneous complexity from the initial hits yielded a selective ANT(2”)-Ia inhibitor which demonstrated in vivo synergy with gentamicin. Aspergillomarasmine A (AMA) is a natural product with activity against NDM-1. Several derivatives of AMA have been synthesized to assess SAR, but the specific contributions of individual carboxylic acids have yet to determined due to difficulties accessing position 6. A synthetic approach was developed via reductive amination using Garner’s aldehyde as a serine equivalent. This strategy was used to synthesize an AMA analog with a hydroxyl group in place of the carboxylic acid in position 6. Additionally, an imine-promoted isomeric resolution was discovered. / Thesis / Doctor of Philosophy (PhD) / Antibiotics, such as aminoglycosides and carbapenems, are losing their effectiveness against bacteria responsible for deadly diseases. This is often due to resistance enzymes such as aminoglycoside N-acetyltransferase-3 (AAC(3)) and O- nucleotidyltransferase-2” (ANT(2”)), which inactivate aminoglycosides, and the New Delhi metallo-b-lactamase-1 (NDM-1), which destroys carbapenems. If these enzymes are blocked, the antibiotics should work against bacteria again. In order to develop compounds that will inhibit these enzymes, sets of similar compounds are made and tested. Patterns of what chemical groups improve or worsen inhibitory activity are noted and used to make another set of compounds in an iterative process. This thesis describes the development of inhibitors of AAC(3)-Ia and ANT(2”)-Ia by this process. Additionally, a specific compound was made to test if a particular chemical group has a role in inhibiting NDM-1.