The potential role in pathogenicity of extracellular products (ECP) of the fish pathogen Aeromonas salmonicida was investigated. Evidence is presented that determinants of host damage are produced extracellularly by A. salmonicida. In vitro, the bacterium produced a number of extracellular enzymic and ctyolytic activities, including proteases and an heterogeneous haemolysin which was active against rainbow trout erythrocytes. Solubilization of trout erythrocytes in vitro resulted from the cooperative effects of the haemolysin and a caseinolytic protease. The caseinolytic protease was implicated in host damage; the in vivo effects of the caseinase may have been potentiated by haemolysin. The production of extracellular proteolytic and haemolytic activities by a number of isolates of A. salmonicida was studied; some potentially important differences between isolates were found. Evidence is presented that rainbow trout possess humoral mechanisms of resisting the toxic products of A. salmonicida. Proteolytic and haemolytic activities of ECP were inhibited, and the in vivo toxicity of ECP was neutralized, by normal trout serum. In vitro, factors in normal trout serum appearaed to form soluble complexes, as well as insoluble precipitates, with component(s) of ECP. In vitro, ECP caused a reduction in circulating levels of an alpha-2 macroglobulin analogue in rainbow trout. As putative determinants of pathogenicity, extracellular factors of A. salmonicida are potentially important in conferring protective immunity against furunculosis. The humoral immune response of rainbow trout and rabbits to components of ECP was investigated. At least 15 components of ECP, including the caseinase and haemolysin, were immunogenic in the rabbit. In the trout, antibodies to only 4 components of ECP were detected and no antibody to the caseinase or haemolysin was found. It is suggested that the effectiveness of furunculosis vaccines might be improved if the immunogenicity in trout of certain ECP components could be enhanced.

Call number: LD2668 .T4 1950 P67 / Master of Science

Candida albicans colonizes the gastrointestinal tract of up to 75 % healthy individuals. It usually cohabits the gut as an innocuous commensal. But in critically ill patients whose gut barrier, immune system and normal gut microbiota are compromised, C. albicans often transmigrates the gut barrier, transforms into an invasive pathogen and causes fatal systemic infections. The genetic transitions that drive this transformation in C. albicans have been a major focus of research and have led to the identification of key transcription factors that regulate this commensal-to-pathogen transition. However, the current challenge lies in identifying the downstream pathways and effectors that bring this transition into effect. This thesis addressed this challenge by developing 11 new bioinformatics tools, including 6 comprehensive databases, 4 novel software packages and 1 analysis framework. These databases included a comprehensive topological map of the mammalian gut biogeography, a C. albicans microarray database comprising of 3,091 publically available microarray transcript profiles, C. albicans RNA-seq gene expression and small variant databases extracted from 1,177 publically available RNA-seq samples, a C. albicans gene alias database comprising of 113,297 gene aliases representing the 6,735 open reading frames of C. albicans, and a C. albicans gene ontology slim comprising of 1,194 C. albicans-specific gene ontology terms. These databases were accompanied by a robust analysis framework which brought together these resources for quality control, batch correction and weighted gene co-expression network analysis. All these tools were finally employed in a pilot exploration of the C. albicans gut commensal-to-pathogen transition, which demonstrated the effectiveness of these bioinformatics resources. The analysis unveiled known regulators, uncharacterized gene networks, pathways and effectors potentially crucial for the C. albicans gut commensal-topathogen transition. These resources are a step towards a better understanding of this transition and can also be utilized for examining various other aspects of C. albicans biology.

Rhodococcus equi is a soil-dwelling actinomycete with the ability to cause pyogranulomatous infections in different animal species and people. Young foals are particularly susceptible and develop a severe pulmonary illness known as rhodococcal pneumonia. The infection is endemic in many horse-breeding farms worldwide and poses a major challenge to the equine industry, as there is no commercial vaccine available. R. equi is a facultative intracellular pathogen. Intracellular survival in macrophages and hence virulence depends on the presence of large plasmids that carry a set of genes encoding virulence-associated proteins (Vaps) of largely unknown functions. Virulence plasmids are of different types and appear to determine host-specific infectivity for horses, pigs and cattle. In this thesis, I explored bacterial chromosomal factors that contribute to the virulence of R. equi. Previous microarray transcription profiling work from the laboratory showed that housekeeping metabolic genes from the R. equi chromosome were co-opted to serve a virulence function via co-regulation with plasmid virulence genes. Here, I identified a further virulence plasmid-co-expressed metabolic chromosomal locus with a key role in R. equi pathogenesis. The identified locus, gltAB1, encodes an NADPH-dependent glutamate synthase required for ammonia assimilation under low nitrogen conditions. Reverse-transcription quantitative rea-ltime PCR confirmed that gltAB1 was co-expressed with the vap genes from the plasmid whereas a homologous chromosomal locus encoding a second NADPH-dependent glutamate synthase, gltAB2, was not. In-frame deletion mutants were constructed and their virulence analysed. gltAB1 but not gltAB2, was found to be involved in virulence and required for intracellular proliferation in J774A.1 macrophages. The ΔgltAB1 mutant showed significant attenuation in vivo in a mouse infection model, in contrast to the ΔgltAB2, which behaved like the wild type. The ability of the ΔgltAB1 mutant strain to act as a live attenuated vaccine was tested in experiments in BALB/c mice. The mutant conferred protection against subsequent challenge of the animals with wild-type virulent bacteria, thus identifying a novel candidate vaccine for the control of R. equi pneumonia in foals. Furthermore, this thesis describes studies of the bovine-type plasmid, previously sequenced in our laboratory. The purpose of this work was to determine if VapN, the bovine-type allelic variant of the VapA protein encoded in the equine-type plasmid, was also essential for R. equi virulence. A plasmid-less derivative strain and a deletion mutant in the vapN gene were examined for their ability to proliferate in two different cell lines and to persist in BALB/c mice. These strains showed the same strong virulence attenuation observed with plasmid-less and ΔvapA strains derived from the equine isolate R.equi 103S, demonstrating that the bovine-type VapN protein also plays a central role in R. equi virulence. Additionally, the thesis includes preliminary work on approaches to explore the role and mechanism of Vap proteins in R. equi virulence. It also describes the construction of GFP-tagged R. equi strains for use in cell biological experiments and live imaging of infected cells.

Obligate intracellular bacteria are significant causes of morbidity and mortality with over two hundred and fifty million infections worldwide annually. One such bacterium, Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), a tick-transmitted febrile illness. Previous studies have shown that A. phagocytophilum lacks genes for cholesterol biosynthesis and solely relies on Niemann Pick protein type C (NPC)1-mediated low density lipoprotein (LDL)-derived cholesterol to complete its infection cycle.Acid sphingomyelinase (ASMase) is a lysosomal enzyme that is essential for diverse cellular processes including liberation of LDL-derived cholesterol from the lysosome. By first studying A. phagocytophilum, we found that functional inhibitors of acid sphinogmyelinase (FIASMAs) arrest the bacterium’s infection cycle in a dose-dependent manner. FIASMAs inhibit vacuole maturation, conversion to the infectious form, and eliminate the production of infectious progeny. NPC1-mediated LDL-derived cholesterol traffic to the ApV is abrogated in the presence of FIASMAs. Similar to the in vitro model, A. phagocytophilum cannot establish a productive infection in both ASMase-/-and FIASMA treated mice. Furthermore, we extended our studies to Coxiella burnetti (Q fever), and Chlamydia spp. (STD, infectious blindness, pneumonia). FIASMA treatment has a rapid bacteriocidal effect on C. burnettiwithin host cells. Additionally, FIASMA treatment inhibits C. trachomatis and C. pneumoniae inclusion expansion and infectious progeny generation, with C. pneumoniae being more severely impacted. These data highlight the critical, yet distinct roles that ASMase plays in these pathogens’ infection cycles. Furthermore, these results signify the therapeutic potential of FIASMAs for treating diseases caused by these pathogens.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

published_or_final_version / Microbiology / Master / Master of Philosophy

Eleven fungal strains were received from the clinical microbiology laboratory collection of Queen Mary Hospital and Pamela Youde Nethersole Eastern Hospital in Hong Kong from 2010-2011. The collection comprised of ten ascomycetes and one zygomycete. They were identified down to the genus level based on the morphological criteria. Internal transcribed spacer (ITS), beta-tubulin, actin and 28S gene sequencing were used for genotypic characterization. The ITS sequences of four of the strains demonstrate <3%-base difference to a single fungal species. They were species of the genus, Acremonium, Aspergillus, Cladophialophora and Ochroconis respectively. Five strains belonging to the genus, Trichophyton, Monascus, Mucor, Arthrinium and Acremonium could not be identified to the species level due to low interspecies heterogeneity. The tubulin gene was used for two of the strains. The tubulin sequence of a strain of Phaeoacremonium was identified to the species level with 0%-base difference. The ITS, partial beta-actin and 28S rDNA genes were sequenced for a strain of Exophiala. They showed a distinct cluster, mostly closely related to, but distinct from, Exophiala xenobiotica, Exophiala jeanselmei and Exophiala oligosperma. The genotypic characteristics suggest the strain to be a novel species of Exophiala. More genotypic and phenotypic characterization are required to described this strain of Exophiala. / published_or_final_version / Microbiology / Master / Master of Research in Medicine

In clinical microbiology laboratories, the conventional method for identification of pathogenic fungi is based on fungal culture and observation of fungal phenotypic characters. However, it is time-consuming, subjective and unreliable due to the long incubation period and variations in fungal colony morphology. Thus, there is a need for a rapid, objective and accurate identification of pathogenic fungal isolates. ITS regions are most commonly used targets for molecular identification of fungal pathogens because of the optimal inter- and intra-species variations and large copies in fungal genome. In this study, twenty-two clinical fungal isolates were identified using the phenotypic method and ITS sequencing. The results showed that there were only thirteen isolates identified to species level by phenotypic method, while others were only differentiated in genus level. Due to the poor differentiation based on the conventional phenotypic approach, misidentification of fungal pathogens occasionally occurred. However, ITS sequencing successfully achieved accurate species-level identification of all fungal isolates. The results were demonstrated in phylogenetic trees with high bootstrap support. In conclusion, ITS sequencing is a rapid and reliable for the identification of pathogenic fungal isolates. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Identification of pathogens is one of the important duties of clinical microbiology laboratory. Traditionally, phenotypic tests are used to identify the bacteria. However, due to some limitations of the phenotypic tests, the bacteria may not be identified sometimes and cannot be identified promptly. 16S rRNA gene sequencing is a rapid and accurate method to achieve this target. It is especially useful for identify rare or slow growing bacteria. However, the interpretation of the 16S rRNA gene sequencing result is one of the challenging duties to laboratory technicians and microbiologists. Apart from the well known 16S rRNA gene databases such as Genbank, The Ribosomal Database Project (RDP-II), MicroSeq databases, Ribosomal Differentiation of medical Microorganism database (RIDOM), SmartGene IDNS, 16SpathDB is an automated and comprehensive database for interpret the 16S rRNA gene result. The 16SpathDB first version was established in 2011. In this study, 16SpathDB was updated based on the all clinical important bacteria present in the 10th edition of the Manual of Clinical Microbiology (MCM)(Versalovic. et al., 2011) into this new version of database, 16SpathDB 2.0. The database was evaluated by using 689 16S rRNA gene sequences from 689 complete genomes of medically important bacteria. Among the 689 16S rRNA gene sequences, none was wrongly identified by 16SpathDB 2.0, with 247 (35.8%) 16S rRNA gene sequences reported in only one single bacterial species with more than 98% nucleotide identity with the query sequence (category 1), 440 (63.9%) reported as more than one bacterial species having more than 98% nucleotide identity with the query sequence (category 2), 2 (0.3%) reported to the genus level (category 3), and none reported as “no species in 16SpathDB 2.0 found to be sharing high nucleotide identity to your query sequence” (category 4). 16SpathDB 2.0 is an updated, automated, user-friendly, efficient and accurate database for 16S rRNA gene sequence interpretation in clinical microbiology laboratories. / published_or_final_version / Microbiology / Master / Master of Medical Sciences