Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xiii, 196 p.; also includes graphics (some col). Includes abstract and vita. Advisor: Robert W. Bruegemeier, Dept. of Pharmacy. Includes bibliographical references (p. 176-196).

Abstract Visceral leishmaniasis is a devastating neglected parasitic disease caused by infection with Leishmania donovani. It life cycle has two stages with promastigote (insect stage) and amastigote (animal stage) morphologies. Miltefosine is currently the only commercially available oral drug available to treat leishmaniasis and recent evidence suggests clinical resistance has emerged. Due to the importance of this drug and the scarcity of new drugs in the pipeline, work has been done on understanding the mechanism(s) of miltefosine, yet the mechanism of action for resistance is still not known. In previous studies investigators generated miltefosine resistance on the insect vector stage (promastigotes), yet there is an important gap in understanding miltefosine resistance in the human infectious stage (amastigotes) of the disease. Before we could accomplish this goal a L. donovani cell line was converted into a stable, continuously cultured axenic amastigote cell line and characterized by disease burden generated in vitro and in vivo. The axenic line of L. donovani (MHOM/SD/75/1246/130) retains characteristics of amastigotes infecting macrophages and proliferated better than metacyclic promastigotes in macrophages in vitro and hamsters. The axenic amastigote line was used to induce miltefosine resistance by using discontinuous stepwise increasing drug pressure. Stable high-grade resistance was established (62-fold) and characterized in vitro and in vivo. Lastly, the fitness of miltefosine resistant versus susceptible parasite was established for the first time. This work adds the first efforts characterizing and understanding the complex problem of miltefosine resistance in amastigotes.

published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Microbiology / Master / Master of Medical Sciences

published_or_final_version / Public Health / Master / Master of Public Health

Background There has been an increasing awareness of the role of the hospital environment as a reservoir of Acinetobacter baumannii. A. baumannii is an important nosocomial pathogen and is difficult to control due to the increasing cases of resistance to carbapenem. Objectives The objectives of this study areto examine carbapenem-resistant Acinetobacter baumannii (CRAB) positive patients according to their environmental sample to determine how frequently the environment surrounding the patient becomes contaminated and which environmental surfaces are most commonly contaminated. Methodology During June 2011 to December 2011, data regarding 30 hospitalized patients with at least one positive CRAB clinical sample were collected from hospital X in Kowloon of Hong Kong. For 30 case patients, one patient in the ICU ward had been isolated in a single room and the other 29 patients stayed in a multi-room. Fifteen surfaces in the patient cubicle and nine surfaces in health care worker stations were evaluated for the presence of CRAB. 29 control environmental samples were obtained from the surroundings of patients without CRAB in the same cubicle and one control environmental sample was obtained from the surroundings of patients without CRAB in the other room of ICU. Pulsed-field gel electrophoresis was performed on all environmental isolates and clinical samples. Results Of the 30casepatients, 26 patients (86.7%) were found to have CRAB contamination in their surrounding environment and 6negative control patients (20%) were found to have CRAB in their environmental samples. The percentage of positive CRAB cultures in the case environment, control and health care worker stations was 28.9% (117/405), 3.4% (14/406) and 1.9% (5/265)respectively. In the surrounding case patient area, pillows (60% 18/30) and bed sheets on which the patients sleep on (60% 18/30), bed sheets covering the patients (50% 15/30) and bedside table tops (40% 12/30) were the most commonly contaminated. For 26casepatientswere found to have CRAB contamination in their surrounding environment, 23 (88.5%) of these patients were found to have the clone of isolates in the case environment related to the patients. Conclusion For patients with CRAB, the surrounding environment is frequently contaminated. Surfaces often touched by the patients are commonly contaminated. CRAB was also found on surfaces that were not closely related to the patient which are frequently touched by healthcare workers during patient care. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Dissemination of Carbapenem-Resistant Enterobacteriaceae (CRE) has raised a new challenge for health organizations all over the world. Acquisition of carbapenemase genes is the most worrisome among these CRE isolates. This study was constructed to investigate the dissemination of CRE isolates in Hong Kong and also to characterize plasmids harboring carbapenemase genes. CRE isolates were collected from public hospitals in Hong Kong from August 2006 to June 2012. Antimicrobial susceptibility of all CRE isolates was tested using disc diffusion method. Screening of carbapenemase genes (blaNDM , blaKPC, blaIMP, blaVIM and blaOXA-48) and ESBL genes (blaCTX-M and blaSHV) were also performed. Clonal relatedness was studied by multi-locus sequence typing. Characterization of plasmids was carried out by conjugation, S1-PFGE, hybridization and plasmid replicon typing. A total of 69 CRE isolates were collected including 50 K. pneumoniae, 15 E. coli, 2 E. cloacae, 1 E. aerogenes and 1 C. freundii. Eighteen carbapenemase producing Enterobacteriaceae were isolated from different patients with travel histories among these 69 isolates. Four K. pneumoniae were detected to carry blaKPC genes on different transferable plasmids as follows: 50 kb, IncX3 plasmid (ST258); 70 kb, un-typeable plasmid (ST258); 130 kb, un-typeable plasmid (ST11) and 140 kb, un-typeable plasmid (ST11). blaIMP genes were also detected in four CRE isolates to be harbored by different plasmids or located on chromosome: ST11 K. pneumoniae (50 kb, IncN), ST1 K. pneumoniae (150 kb, IncA/C), E. cloacae (130 kb, IncN-L/M) and ST899 K. pneumoniae (chromosomal located). NDM-1 (New Delhi Metallo enzyme) producing E. coli (n = 5), K. pneumoniae (n = 2), E. aerogenes (n = 1), E. cloacae (n = 1) and C. freundii (n =1) were also found in this study. Eight of them were isolated from patients travelled to different provinces of China blaNDM-1 was found to be carried by transferable plasmids in all ten isolates: IncX3 (n = 7, 50 kb), IncL/M (n = 1, 88 kb), IncA/C2 (n = 1, 140 kb) and FIIY- FIBS (n = 1, 110kb). Six of the seven IncX3 plasmids showed identical digestion profile while the other one only had two bands different from others using Restriction fragment length polymorphism (RFLP) analysis. An IncX3 plasmid pNDM-HN380 from a K. pneumoniae strain CRE380 was completely sequenced using Genome Sequencer FLX (Roche, USA). pNDM-HN380 was a 54,035 bp circular plasmid with 52 open reading frames (ORFs). The backbone of pNDM-HN380 was identical to those previous described IncX plasmids pIncX-SHV (accession number JN247852) and pEC14_35 (accession number JN935899). The blaNDM-1 gene was carried on an ISAba125 and IS26 flanked transposon-like element. And this element except IS26 and an interrupted ISAba125 was found to be identical to pNDM-BJ01 (accession number JQ001791). In conclusion, this is the first we describe a blaNDM-1 carrying IncX3 plasmid. This IncX3 plasmid was found to be predominant in the dissemination of blaNDM-1 in China. Future study of the nationwide dissemination of carbapenemase genes and also the novel IncX3 plasmids is needed. / published_or_final_version / Microbiology / Master / Master of Philosophy

Objective: To find out the spatial autocorrelation of antibiotic resistance of S. pneumonia and test the significance of distance as a risk factor. Methods: Descriptions of penicillin and macrolide resistance in EARS-Net countries from 2006 to 2010 were given. Global moran’s I and Anselin moran’s I were used to assess the spatial autocorrelation and gravity model was used to test the significance of distance and other socio – economic factors. Results: The trend of resistance in Europe was stable. Positive spatial autocorrelation existed from 2006 to 2010 for penicillin (Z(I): 0.16-0.2) and 2009 to 2010 for macrolide (Z(I): 0.11 -0.13). Some clusters (hotspots) were identified; they were Cyprus (2006-2010 for penicillin and 2009 to 2010 for macrolide), Spain (2006 for penicillin), France (2006 for penicillin), Romania (2009 for penicillin and macrolide) and Bulgaria (2009 for penicillin and macrolide). The result of gravity model showed that only parameters of population in 2007 for penicillin (p<0.05) and parameter of distance in 2009 for penicillin (p<0.05) in Cyprus were statistically significant. Conclusion: Distance was not a risk factor of high prevalence of antibiotic resistance of S. pneumoniae although there was a positive spatial autocorrelation. Improvement in surveillance system and appropriate public action were recommended for controlling the spread of resistant strain of S. pneumoniae. / published_or_final_version / Public Health / Master / Master of Public Health