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

Acute myeloid leukemia (AML) is a group of heterogeneous diseases characterized by an abnormal increase in myeloblasts in circulation and/or bone marrow. Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene occurs in about 30% of AML and is associated with an inferior prognosis. Tyrosine kinase domain (TKD) mutations occur in about 5% with uncertain prognostic significance. Intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment. However these approaches have reached a deadlock with a cure rate of 30-40%. Targeting FLT3 in AML with multi-tyrosine-kinase inhibitors has been evaluated in Phase II/III clinical trials. Despite an initial clearance of myeloblasts, the leukemia invariably progresses despite continuous treatment. The mechanisms of drug resistance and leukemia progression, hence the effective therapeutic strategies are currently unknown, limiting its clinical application. These issues were addressed in the present study. In the first part, 13 patients with chemo-refractory or relapsed FLT3-ITD+ AML received sorafenib 200-400 mg twice daily of whom 12 patients achieved clearance or near clearance of bone marrow blasts after a median of 27 days (range 21-84 days). There was evidence of myeloid differentiation of the leukemia blasts at remission. Leukemia progression occurred in 9 patients after a median of 72 days (range 54-287 days) and in 4 out of 6 patients it was dominated by clones carrying double FLT3-ITD and -TKD mutations. Microarray studies comparing myeloblasts before sorafenib treatment (sorafenib naïve) and at subsequent progression (sorafenib resistant) demonstrated up-regulation of 64 genes including ALDH1A1, JAK3 and TESC whose functions were unknown in AML. Transplantation of sorafenib naïve and resistant myeloblasts into NOD/SCID mice recapitulated their clinical behavior when the animals were treated with sorafenib. Both ITD and TKD mutations at D835 were identified in leukemia initiating cells (LICs) from sorafenib naïve samples. These results suggested that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations. In the second part, the gene encoding tescalcin (TESC), that was up-regulated at sorafenib resistance and was known to activate a sodium/hydrogen exchange (NHE1), was evaluated to examine its link with TKI resistance. TESC was highly expressed in FLT3-ITD+ AML cell lines MOLM-13 and MV4-11 and its knock-down by siRNA lowered intracellular pH and induced apoptosis. The results were recapitulated by treatment with a NHE1 inhibitor, 5-(N,N-Hexamethylene)amiloride (HMA). Induction of sorafenib resistance in MOLM-13 cell line (MOLM-13-RE) significantly increased its sensitivity to HMA. HMA treatment of MOLM-13 and MV4-11 as well as primary FLT3-ITD+ AML cells significantly reduced leukemia initiation in NOD/SCID mouse xenotransplantation. Normal CD34+ cells engraftment was not affected. HMA treatment significantly enhanced suppression of FLT3 signaling by sorafenib even in sorafenib resistant cell lines. These observations provided novel information about the pathogenetic role of TESC-NHE1-pHi in sorafenib resistance in AML. In conclusion, the information derived from the present study has provided mechanistic insights to the emergence of drug resistance during sorafenib treatment and important guide for future therapeutic strategies targeting FLT3-ITD+ AML. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Tuberculosis (TB) is a highly infectious disease that causes the second highest mortality rate in human worldwide. The emergence of multi-drug resistance tuberculosis (MDR-TB) leads to a major public health problem in controlling TB-caused mortality. Pyrazinamide (PZA) is an important first-line drug in the treatment of MDR-TB. However, since the challenge in performing susceptibility test on PZA, World Health Organization has not published any data on the prevalence of PZA resistance in Mycobacterium tuberculosis (M. tuberculosis). Since the occurrence of PZA resistance makes MDR-TB more difficult to treat with poor prognosis, rapid detection method in PZA resistance is urgently needed. Since pncA mutation is highly associated with up to 98% PZA resistant M. tuberculosis strains, it is worthwhile to develop rapid molecular method for detecting PZA resistance. This study aims to identify the mutations in PZA resistant M. tuberculosis strains. The first part of this study aims to characterize the pattern of pncA mutation among PZA-resistant and PZA-susceptible M. tuberculosis using Sanger sequencing method. Among all clinical isolates, 12 out of 29 cases of M. tuberculosis were resistant to PZA. All PZA-resistant M. tuberculosis strains harbored pncA mutation, whereas no known mutations were found among those PZA-susceptible strains, giving the positive predictive value to be 100%. Eight mutation patterns were found among 12 resistant isolates. Four of these pncA mutations have not been described previously by other studies. Study also characterizes the pattern of pncA mutation in 19 sputum specimens, with 2 mutation patterns found. Overall 10 mutation patterns were found in this study. Results show that the mutation of pncA gene is highly associated with PZA-resistant M. tuberculosis. Results also suggest the scattered and more extensive mutations in pncA gene that confer PZA resistance to M. tuberculosis. The second and the last part of this study aims to evaluate the possibility of using molecular method to detect PZA resistance in routine clinical laboratory. Results show that using molecular sequencing to detect PZA resistance can shorten the turnaround time to about 3-4 working days. Since mutation of pncA was scattered along the entire pncA gene, using DNA sequencing approach may be the best strategy for the rapid detection of PZA resistance in M. tuberculosis. / published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The diminishing choice of effective antibiotics against resistant pathogens has forced clinicians to revive the use of old antibiotics. Hence, fosfomycin has been frequently suggested for alternative therapies given its track record of low resistance rates despite extensive use. However, there have been recent reports of plasmid-mediated fosfomycin resistance among animals and healthy humans in Asia. Accordingly, comparison of shared fosfomycin resistance mechanisms between animals and humans will shed light on the spread of resistance and guide future use of antimicrobials. This study aimed to investigate plasmid-mediated fosfomycin resistance in E. coli isolates collected from patients and animals in Hong Kong. Non-duplicate E. coli isolates were cultured from 1711 urinary isolates and 167 blood clinical samples collected from multiple centres during 1996-2008 and from fecal samples of 210 cattle, 214 pigs, 460 chickens, 398 stray cats, 368 stray dogs and 456 wild rodents during 2008-2010. A total of 2106 animal samples yielded 1693 E. coli isolates of which 831 were ESBL-producers. Fosfomycin-resistant isolates were more likely than fosfomycin-susceptible isolates to be ESBL-producers and multidrug resistant (≥3 antimicrobial classes). Of the 101 fosfomycin-resistant isolates, 97 (96.0%) were fosA3 positive and 94 (93.1%) were blaCTX-M positive. Of the 1878 clinical isolates, 18 were fosfomycin-resistant of which six were fosA3-positive and two were positive for another fosA variant (designated fosKP96). All but one fos-carrying clinical isolate was ESBL-producing. The majority of the fos-carrying E. coli strains belonged to diverse clones under two main clonal complexes CC58 accounting for 38 (36.2%) strains and CC10 for 32 (30.5%) strains. PCR mapping showed that all fosA3-containing regions were followed by a downstream IS26. In all clinical isolates and 81 (83.5%) of animal isolates, an IS26 was also found upstream. In 14 (14.4%) of animal isolates, the fosA3 gene was preceded by an upstream blaCTX-M-14-containing transposon-like structure (ΔISEcp1-blaCTX-M-14-ΔIS903 or ISEcp1-IS10-blaCTX-M-14-ΔIS903). For the remaining two animal isolates, the upstream region could not be defined. In a random subset of 18 animal isolates, fosA3 was carried on transferable plasmids with sizes of 50–200 kb and the following replicons: F2:A-:B- (n = 3), F16:A1:B- (n = 2), F24:A-B- (n = 1), IncHI2 (n = 3), IncN (n = 2), IncI1 (n = 1), B/O (n = 1) and untypeable (n = 5). Among six fosA3-carrying clinical isolates, the distributions were: F2:A-:B- (n = 2), IncN (n = 1), multi-replicon F-:A-:B1/IncN (n = 1) and untypeable (n = 2). Both fosKP96-carrying plasmids belonged to IncN. Restriction fragment length polymorphism analysis showed that the four F2:A-:B- plasmids carrying fosA3 and blaCTX-M-3 genes from a pig, dog and two patients shared an almost identical pattern. The complete plasmid sequences further demonstrated their homology. This study demonstrated the emergence of fosA3-mediated fosfomycin resistance among E. coli isolates from multiple sources. Highly similar IncFII plasmids and IS26 transposon-like structures appear to be the main vehicles for dissemination. This study also highlighted occurrence of plasmids carrying fosKP96 that may have been overlooked by others as this variant could not be detected by established PCR markers. / published_or_final_version / Microbiology / Master / Master of Philosophy

Acinetobacter baumannii is an important nosocomial pathogen worldwide because of its remarkable ability to acquire antibiotic resistance. The global emergences of multidrug-resistant A. baumannii (MDR-AB) clones are predominated by a number of widely disseminated clones, namely clonal complex (CC) 1, CC2, and CC3. In early 2010, we reported two major clones of MDR-AB, designated HKU1 and HKU2 belong to sequence types (ST) 96 and ST92, widely disseminating in our hospitals. ST92 is a predominant clone that is prevalent in more than 30 countries, whereas ST96 has been identified recently and is geographically confined to certain parts of China. Our previous study only investigated the isolates collected in the year 2005-2006. We therefore extended our investigation over a six-year period (2005-2010) to generate a more complete picture of the molecular epidemiology and resistance mechanisms in A. baumannii. Firstly, we performed the susceptibility test on various antimicrobial agents and employed molecular methods to characterize the epidemiology of the target A. baumannii isolates. For the entire study period, increased resistance rates were noted for the seven antimicrobial agents, namely imipenem, piperacillin-tazobactam, cefoperazone, ticarcillin-clavulanate, ciprofloxacin, gentamicin and amikacin (P <0.01). Worryingly, an increased trend was also observed for the pandrug-resistant rate, from 0.2% in the year 2005-2006, to 1.9-2.9% in the year 2007-2008 and up to 6.0-8.1% in the year 2009-2010 (chi square for trend, P <0.001). Pulsed-field gel electrophoresis and multilocus sequence typing (PFGE/MLST) categorized 100 out of 108 (92.6%) isolates into four clones (PFGE/MLST), namely HKU2/ST92 (n = 14), HKU3/ST254 (n = 73), HKU4/ST137 (n = 5), and HKU5/ST362 (n = 8), respectively. PCR showed that 88.9% (96/108) of the amikacin-resistant isolates were armA positive and all isolates were found to harbour at least one of the OXA-type carbapenemases with frequencies as follows: OXA-51-like (98/108, 90.7%), OXA-23-like (85/108, 78.7%), OXA-58-like (9/108, 8.3%) and OXA-24-like (8/108, 7.4%). Secondly, we compared the biological fitness of the circulating clones by performing the doubling time and adhesion experiment. The results demonstrated that HKU3/ST254 has a higher capability for replication and adherence to human bronchial epithelial cells. Together with the higher antibiotic resistance rate, the selective advantages in terms of biological fitness may facilitate the clonal expansion and wide dissemination of this lineage. Finally, whole genome sequence data showed a high amount of resistance genes intermixed with various insertion sequence (IS) elements, integrons and transponsons clustering inside the resistance islands. The presence of a second genomic resistance island conferring aminoglycoside and sulphonamide resistance, additional loci outside the resistance islands harbouring resistance genes and the high amount of antibiotic efflux pumps in various A. baumannii genomes demonstrated that resistance islands contribute a significant part to the multidrug-resistant phenotype in A. baumannii but are not the only factor. The correlation analysis further demonstrated the significance of IS elements in the dissemination of antibiotic resistance genes in the A. baumannii genomes. As a whole, whole genome sequence data may provide an informative and efficient approach to generating a more comprehensive picture to study the resistance mechanism of the epidemic strains. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Chemoresistance remains a major clinical obstacle to effective management of ovarian cancer. Cancer stem cells (or tumor-initiating cells, TICs) have been discovered recently, and have played a pivotal role in changing the view of cancer development; however, the molecular mechanisms by which these cells escape conventional therapies remain elusive. In this study, TICs were isolated from ovarian cancer cells as tumor spheres with specific stem properties under TIC-selective conditions. Unlike non-TICs, TICs strongly express stem cell factor (SCF) and c-Kit. Blocking SCF-c-Kit by SCF neutralizing antibodies, c-Kit small interfering RNA (siRNA) or imatinib (Gleevec), a clinical drug that inhibits c-Kit signaling, significantly inhibited TIC proliferation. Although cisplatin and paclitaxel killed the non-TICs, they did not eliminate TICs. Importantly, the combination of cisplatin/paclitaxel with c-Kit siRNA or imatinib inhibited the growth of both non-TICs and TICs. Similar results were obtained when patient-derived TICs were used. The findings also indicate that tumor-predisposing microenvironment, such as hypoxia, may promote ovarian TICs through upregulating c-Kit expression. Furthermore, I have showed that c-Kit expression induced activation of Phosphatidylinositol 3-kinases (PI3K)/Akt, -catenin, and ATP-binding cassette G2, which could be reversed by treatment with the PI3K/Akt inhibitor or -catenin siRNA. I further studied potential gene expression in TICs using cDNA and microRNA (miRNA) microarrays. The result from these microarrays provided a general profile in gene expression of TICs compared with the bulk tumor cells. In particular, let-7a, b, and c were shown to be downregulated in TICs compared to bulk tumor cells, suggesting that their loss may contribute to ovarian cancer development. Together, this study reveals a previously undescribed therapeutic effect of SCF-c-Kit signaling blockade to prevent ovarian cancer progression by eliminating TICs and the altered genes or miRNAs may represent possible molecular targets. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Background A pandemic clone, Escherichia coli sequence type 131 (ST131), has been disseminated worldwide and represents an important cause of antimicrobial resistant infections. The spread of this resistant clone has become a great public health concern. Objectives The aims of this study were to investigate the prevalence of ST131 in Escherichia coli isolates from human urinary cultures in Hong Kong and study the antimicrobial phenotypes of ST131. Methodology This study included 340 E. coli clinical urinary isolates obtained from patients in four district hospitals between May 2013 and July 2013 in Hong Kong. Antimicrobial susceptibilities were assessed by disk diffusion method with reference to CLSI. The isolates were investigated by phylogroup-specific and ST131-specific PCR assays. ST131 strains were further assessed for subclone distribution, antimicrobial resistance and extended-spectrum β-lactamase (ESBL) type. Results A total of 18.5% (63/340) of the E. coli population was identified as ST131. ST131 isolates were significantly more likely than non-ST131 isolates to be ciprofloxacin resistant (69.8%, 44/63 versus 31.0%, 86/277; P <0.001), gentamicin resistant (38.1%, 24/63 versus 24.9%, 69/277; P=0.03) and ESBL producers (41.3%, 26/63 versus 18.8%, 52/277; P <0.001). Among the ST131 E. coli isolates, 68.3% (43/63) belonged to the H30 subclone. Most H30 isolates were ST131-O25b (97.7%, 42/43). Also, the ST131-H30 E. coli subclone was statistically associated with ciprofloxacin resistance compared with the non-H30 ST131 isolates (P <0.001). Additionally, strains which were co-resistant to ciprofloxacin, co-trimoxazole and gentamicin were overwhelmingly associated with the H30 subclone than non-H30 (23.3%, 10/43 versus 0%, 0/20; P=0.02). Conclusion This study showed that ST131 isolates were widespread among human E. coli urinary isolates in Hong Kong. The increase in antimicrobial resistance phenotypes are highlighted with ST131, especially the H30 subclone isolates. The dissemination of the ST131 resistant clonal group has aroused clinical attention and limited the choice of empirical treatment. / published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

No description available.

Resistance to ivermectin (IVM) in Haemonchus contortus has developed in many countries and its mechanism is still under investigation. P-glycoproteins (P-gp) are transmembrane proteins that can transport drugs out of cells. Researchers have found that there is polymorphism in a P-gp gene from H. contortus between IVM-selected and unselected worms. Three main P-gp polymorphs were identified, polymorph A was found to be related to IVM selection, while polymorphs B and X were associated with susceptibility. The purpose of this research is to investigate the genetic variations in P-glycoprotein that are associated with IVM selection or susceptibility in H. contortus. Total RNA and genomic DNA were extracted from individual male adult worms of IVM-selected and unselected strains of H. contortus. A fragment of the P-gp gene was amplified from the genomic DNA of individual worms and RFLP analysis was performed on the PCR product to genotype the corresponding worms. The homozygous worms that possessed polymorph A, B or X were identified.