Doctor of Philosophy / Department of Entomology / Ludek Zurek / Enterococci are a group of commensal bacteria that are important nosocomial pathogens. They are abundant in human sewage and wastewater treatment facilities (WWTF). This study focused on the role of house flies, Musca domestica, in the ecology of enterococci at WWTF in both field and laboratory experiments. The first study objective focused on sampling and characterizing enterococci from house flies and wastewater sludge from four WWTF in northeastern Kansas. Enterococci were quantified, identified, and screened for antibiotic resistance and virulence traits, and genotyped. The profiles of enterococci (spp. diversity, antibiotic resistance and virulence) from WWTF sludge and the house flies were similar, indicating that the flies successfully acquired the bacteria from the WWTF substrate. Enterococci with the greatest amount of antibiotic resistant and virulence traits originated from the WWTF that processed meat waste from a commercial sausage plant. Genotyping of E. faecalis revealed clonal matches from sludge and house flies. The second study objective involved tracking the fate of E. faecalis in the digestive tract of house flies in laboratory assays. Colony forming unit (CFU) counts were highest in the midgut at 1 h and declined during the first 24 h. In the labellum, foregut and hindgut, E. faecalis concentrations were more variable but were overall higher after 24 h. Observations from CFU counts and visualizations under a dissecting microscope revealed that E. faecalis peaked in the crop after 48 h suggesting active proliferation in this region. The third objective of the study involved tracking the emergence of calyptrate muscoid flies from stockpiled biosolid cake at one of the four WWTF. Traps were employed at the site for a total of 47 weeks, totaling 386 trap-weeks. A total of 11,349 calyptrate muscoid flies were identified with the two most common species being stable flies (Stomoxys calcitrans) (9,016, 80.2%) and house flies (2022, 18.0%). Numbers of stable flies and house flies peaked around mid-July each year and a second, smaller peak was observed for stable flies 5-8 weeks later. Estimated annual emergence of stable flies was 551,404 and for house flies 109,188.

Doctor of Philosophy / Department of Grain Science and Industry / Subramanyam Bhadriraju / Ludek Zurek / Feed samples and live stored-product insects from feed mills and swine farms were collected and cultured for Enterococcus spp. The mean concentration of enterococci in insect and feed were 2.7 ± 0.5 × 101 cfu/insect and 6.3 ± 0.7 × 103 cfu/g respectively. A total of 362 isolates of enterococci collected from 89 feed samples and 228 stored-product insects were identified to the species level using PCR. These isolates were represented by Enterococcus casseliflavus (53.0%), E. gallinarum (20.4%), E. faecium (16.2%), E. hirae (5.2%), and E. faecalis (5.0%). Enterococci were phenotypically resistant to tetracycline (48.0%), erythromycin (14.3%), streptomycin (16.8%), kanamycin (12.1%), ciprofloxacin (11.0%), ampicillin (3.3%), and chloramphenicol (1.1%). All isolates were susceptible to vancomycin and gentamicin. Tetracycline resistance was encoded by tetM (50.0%), tetO (15.1%), tetK (0.5%), tetS (0.2%) and other unknown tetracycline determinants. Enterococci carried virulence genes including gelatinase (gelE; 21.5%), an enterococcus surface protein (esp; 1.9%), and cytolysin (cylA; 2.2%). An aggregation substance (asa1) gene was detected in 61.0% of E. faecalis isolates. Fifty perncet of E. faecalis isolates were phenotipically tested positive for aggregation substances. Enterococci with cylA genes were hemolytic (52.0%) and with gelE genes were gelatinolytic (18.5%). The ermB gene, encoding erythromycin resistance was detected in 8.8% of the total isolates. The Tn916/1545 family of conjugative transposons was detected in 10.7% of the isolates. Laboratory experiments showed that adults of the red flour beetle, Tribolium castaneum (Herbst), fed on poultry and cattle feeds inoculated with E. faecalis OG1RF:pCF10, were able to successfully acquire enterococci and contaminate sterile poultry and cattle feeds. To assess the potential of horizontal gene transfer, conjugation assays were carried out with E. faecalis using a donor (wild strains) and recipient (E. faecalis OG1SSP) in ratio of 1:10. Only one isolate (1 out of 18 E. faecalis) could transfer tetM to a recipient using broth mating. However, filter mating assay, followed by PCR confirmation revealed that 89.0% (16 out of 18 E. faecalis) of isolates could transfer tetM to E. faecalis. Transfer ratios of transconjugant per recipients ranged from 2.6 × 10-4 to 1 × 10-9. In summary, feed (52.0%) and stored-product insects (41.6%) collected from feed mills and swine farms carried antibiotic-resistant and potentially virulent enterococci. Our study showed that T. castaneum, a pest commonly associated with feed, served as a potential vector for enterococci in the feed environment. Conjugation assays followed by PCR confirmed presence of the tetM gene on a mobile genetic element(s) such as Tn916 and may be horizontally transferred to other Enterococcus species and to other bacteria of clinical significance.

No description available.

Le traitement antibiotique local de l'infection osseuse par le polyméthacrylate de méthyle (PMMA), chargé de gentamicine ou de tobramycine, montre actuellement des limites. Ses inconvénients sont liés à la non résorbabilité du PMMA et à la rétention d'une grande partie de l'antibiotique intégré au PMMA. L’association fréquente à l’infection de pertes de substance osseuse a favorisé la recherche de vecteurs d’antibiothérapie locale, alternative au PMMA, parmi les substituts de comblement osseux résorbables et ostéoconducteurs. Les ciments phosphocalciques (CPC) pourraient devenir parmi les plus performants dans cette utilisation. Ils sont biocompatibles et offrent avec le Dicalcium Phosphate-ß-Tricalcium Phosphate (DCPD-ß-TCP), un CPC, la possibilité d'obtention d'un mélange DCPD-ß-TCP-gentamicine à une température de 43°C n'altérant pas l'antibiotique, contrairement aux céramiques phosphocalciques qui sont fabriquées par frittage à très haute température. Le but de notre travail était de tester in vitro (élution d’antibiotique) et in vivo (essai de traitement d'ostéite expérimentale) le DCPD-ß-TCP-gentamicine comme alternative possible au PMMA-gentamicine. [...] / Local antibiotic treatment of osteomyelitis is based on the use of gentamicin- (or tobramycin-) loaded polymethylmethacrylate (PMMA). These two aminoglycosides are effective against most cultured orthopedic microorganisms, including Staphylococcus aureus, the most frequent cause of infection. The extensive use of PMMA as a Local Antibiotic Delivery System (LADS) has various disadvantages. Firstly, only a small proportion (about 5 to 17%) of the antibiotic is released by the cement (trapping effect). Secondly, the most significant problem is that PMMA is not resorbable and presents a physical obstacle to osteogenesis. A second surgical operation is therefore always required to remove the PMMA and to fill the cavity caused by bone loss with a bone graft or a synthetic substitute. Several absorbable synthetic substitutes, such as calcium phosphate ceramics, calcium sulfate, and polymers of polylactic-polyglycolic acids, have been investigated as antibiotic carriers. These synthetic substitutes are largely underused as LADS in clinical practice. Polymers are not perfectly biocompatible, and ceramics provide a burst release of antibiotics as a consequence of their manufacturing techniques (Antibiotic adsorption onto the carrier, after sintering of the carrier at high temperature, 1000-1200°C). We have developed a possible alternative to gentamicin loaded-PMMA for local treatment of osteomyelitis in the form of novel calcium phosphate cement (CPC): dicalcium phosphate dihydrate-β-tricalcium phosphate (DCPD-β-TCP). The biocompatibility of such a cement has been demonstrated experimentally and has been clinically confirmed for the treatment of burst fractures and for filling bone cavities in osteoporotic fractures. DCPD-ß-TCP is made in granules from 2 to 3 mm in diameter to avoid the superficial ―creeping substitution‖ observed when DCPD-β-TCP is used as a cement block. [...]

The growing prevalence of antibiotic-resistant bacteria is a global health crisis that threatens the effectiveness of antibiotics in medical treatment. Increases in the number of antibiotic-resistant bacteria and a drop in the pharmaceutical development of novel antibiotics have combined to form a situation that is rapidly increasing the likelihood of a post-antibiotic era. The development of antibiotics with novel enzymatic targets is critical to stall this growing crisis. In silico methods of molecular modeling and drug design were utilized in the development of novel tryptamine analogs as potential antibiotics through their inhibition of the bacterial enzyme tryptophan synthase. Following the creation of novel tryptamine analogs, the molecules were analyzed in silico to determine their binding affinity to human MAOB and the E. coli α-subunit, E. coli β2-dimer and the M. tuberculosis β2-dimer of tryptophan synthase. Ten tryptamine analogs displayed significant increases in tryptophan synthase binding affinity and show promise as potential antibiotics and antibiotic adjuvants. Further in silico modeling determined that the binding sites of the tryptamine analogs were similar to wild-type tryptamine in the E. coli β2-dimer, the M. tuberculosis β2-dimer and human MAOB, while the analogs’ binding sites to the E. coli α-subunit differed. Although no tryptamine analogs increased tryptophan synthase binding affinity while decreasing human MAOB binding affinity, related increases in MAOB binding affinity warrants further research into the analogs’ potentials as MAO inhibitors. Given the increases in tryptophan synthase binding affinity and similar β2-dimer binding sites, a provisional patent was filed on the ten identified tryptamine analogs. Moving forward, we recommend the synthesis of the ten identified tryptamine analogs. Following synthesis, further research should be conducted to determine the in vitro and in vivo antibiotic properties of the ten tryptamine analogs.

The crucial role of antibiotics in modern medicine, in curing infections and enabling advanced medical procedures, is being threatened by the increasing frequency of resistant bacteria. Better understanding of the forces selecting resistance mutations could help develop strategies to optimize the use of antibiotics and slow the spread of resistance. Resistance to ciprofloxacin, a clinically important antibiotic, almost always involves target mutations in DNA gyrase and Topoisomerase IV. Because ciprofloxacin is a substrate of the AcrAB-TolC efflux pump, mutations causing pump up-regulation are also common. Studying the role of efflux pump-regulatory mutations in the development of ciprofloxacin resistance, we found a strong bias against gene-inactivating mutations in marR and acrR in clinical isolates. MIC and fitness measurements revealed that amino acid substitutions conferred smaller susceptibility reductions and smaller fitness costs than gene-inactivating mutations, suggesting that resistance mutations in clinical isolates are selected for high fitness rather than high resistance (Paper I and II). We asked whether the high fitness costs of marR-inactivating mutations could be ameliorated without affecting the resistance phenotype. Multiple independent lineages were experimentally evolved to select for improved growth fitness. Whole genome sequencing revealed mutations affecting marA, lon and arcA as potential compensatory pathways. For the marA and lon mutations the improved growth rate was associated with an increased susceptibility (arcA is being investigated). (Paper III). An evolution experiment selecting for ciprofloxacin resistance revealed upon whole genome sequencing the expected mutations in drug target and efflux-regulatory genes, but also in genes encoding aminoacyl-tRNA synthetases. We investigated two independently selected leuS mutations, and concluded that they contributed to ciprofloxacin resistance by activating the stringent response that in turn caused up-regulation of genes involved in efflux. However, these leuS mutations incur a high fitness cost (Paper IV). To summarize, the research findings in this thesis suggest that the potential ciprofloxacin resistome may include more genes than previously thought, but a strong selection for high fitness selectively purifies many resistance mutations from clinical isolates. In conclusion, selection for high relative fitness constrains the spectrum of mutations that survive and get fixed in clinical populations of bacteria.

UA Open Access Publishing Fund / Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Objective To describe the use of Lactobacillus by hospitalized patients and to examine its relationship with various Clostridium difficile infection (CDI) related outcomes. Methods The characteristics of Lactobacillus users and non-users and the initiation of Lactobacillus with respect to initiation of antibiotic therapy and CDI treatment were described using national hospital discharge database. The relationships between Lactobacillus use and post-CDI length of stay, mortality, switch of CDI therapy, and readmission were analyzed. Results Lactobacillus users and non-users were different in most characteristics. Metronidazole and fluoroquinolones were the most frequently used antibiotics by Lactobacillus users. They were mainly CDI cases, used multiple antibiotics, extremely ill, and started Lactobacillus five or more days after initiation of antibiotics or CDI treatment. Lactobacillus use was associated with increased length of stay and switching of CDI therapy. Conclusions The true association between Lactobacillus use and CDI remains unclear. This study provides foundation for future research.

Microbiological Project Using the Disc Diffusion Method for Lower Secondary School ABSTRACT This thesis deals with project-based education and his involvement in the functional structure of the czech educational theory and practice. It refers to the project as an innovative teaching process, which is qualitatively able to shield asked RVP requirements and the implementation of which contributes to the development of educational goals and core competencies. This thesis contains both theoretical aspects are developed through the study of literature and other sources in order to summarize the current state of knowledge of the subject, so practical. Theoretical bases emphasize not only on the characteristics of project-based education as such, but also on the appropriate application of educational strategies in the learning process. Specifically mentioned are possibilities of project-based teaching in the teaching of natural history, biology and ecology. The theoretical background followed by the practical part, which shifts the theory of project learning into practice. There is particular designed a project called "Strength of antibiotics", which is then implemented and reflected. Is described in detail during the planning, implementation and evaluation of the project. KEYWORDS: project-based education, school...