“Unknown” prostate cancer stage at diagnosis in a population-based cancer registry: impact on epidemiological studies and use of multiple imputation

Routinely collected population-based cancer registry stage data are crucial to inform health service planning and to monitor variations in cancer outcomes. However, incomplete stage information is a major concern due to potential biases this introduces. This thesis examined the reasons why a large proportion of prostate cancer cases are recorded as “unknown” stage in the New South Wales (NSW) Cancer Registry (NSWCR) and validated the multiple imputation (MI) method for dealing with “unknown” stage data. NSW is the most populous state in Australia, with almost one third of the total national population. The NSWCR is the only population-based cancer registry in Australia that has collected stage information since its inception in 1972. The usefulness of long-term historical cancer registry stage data when examining cancer outcomes is illustrated in Chapter 2, with an investigation of geographical variation in long-term survival over time. The research reported in Chapter 3 shows that prostate cancer cases with “unknown” stage differ from those with a known stage, as survival and risk of disease progression for cases with “unknown” stage was intermediate between those for cases with localised and regional disease. Several possible reasons that could contribute to why “unknown” stage is recorded in the NSWCR are identified in Chapter 4. The publication included in Chapter 5 shows that MI appears to be valid for “unknown” stage when the MI is implemented according to the practical guidelines recommended in the literature. The application of MI to the NSWCR “unknown” stage data reported in Chapter 6 shows that the imputed stage data appear to be reliable. These findings provide important insights into prostate cancer cases with “unknown” stage recorded in the NSWCR, and an understanding of the potential biases in epidemiological studies that use these data. The validated MI method to handle “unknown” stage will help to increase the utility of the cancer registry data

Genes required for sediment fitness in Desulfovibrio desulfuricans G20.

97 genes required for sediment fitness of G20 were identified and the identification of chemotaxis genes validated our STM screening method since it would be expected to enhance sediment fitness. The growth of these sediment fitness mutants was monitored in laboratory growth media to determine whether growth rate influenced sediment fitness. Homology with other delta- proteobacteria was determined and the putative sediment functions of many of these genes were described. Amino acid biosynthesis mutants, hydrogenase mutants, and DNA repair mutants were characterized to prove the importance of specific functions for sediment fitness.A modified version of the signature-tagged mutagenesis (STM) procedure was developed, which incorporated microarray technology to streamline the entire screening process. A mini-Tn10 transposon that is capable of randomly mutagenizing our model sulfate-reducing bacterium was also identified. This system can therefore be applied to other sulfate-reducing bacteria to answer questions of ecological or economical significance. This is the first study using STM to study bacterial environmental fitness.Preliminary experiments were conducted on Desulfovibrio desulfuricans G20 to obtain some basic information of this bacterium. Strain G20 has a single flagellum and a doubling time of 3.2 hours at 37° C in the lactate-sulfate medium, compared to the doubling time of 7.3 and 5.0 hours for strain Essex6 and strain ASR, respectively. G20 is resistant to several antibiotics including nalidixic acid, spectinomycin, and streptomycin.The proteomes of the regulatory mutants were analyzed and compared to the proteome of the parent strain. The reproducible and reliable data produced by the accurate mass and time tag approach allow us to compare protein production in the mutants. The results showed that these regulators regulated different sets of genes which may be required for sediment survival, although the regulatory pathways remain to be elucidated. It is evident that the laboratory-adapted strain is different from sediment-adapted strain at the protein level

Immunogenicity and protective efficacy against enterotoxigenic Escherichia coli colonization following intradermal, sublingual, or oral vaccination with EtpA adhesin

Enterotoxigenic Escherichia coli (ETEC) strains are a common cause of diarrhea. Extraordinary antigenic diversity has prompted a search for conserved antigens to complement canonical approaches to ETEC vaccine development. EtpA, an immunogenic extracellular ETEC adhesin relatively conserved in the ETEC pathovar, has previously been shown to be a protective antigen following intranasal immunization. These studies were undertaken to explore alternative routes of EtpA vaccination that would permit use of a double mutant (R192G L211A) heat-labile toxin (dmLT) adjuvant. Here, oral vaccination with EtpA adjuvanted with dmLT afforded significant protection against small intestinal colonization, and the degree of protection correlated with fecal IgG, IgA, or total fecal antibody responses to EtpA. Sublingual vaccination yielded compartmentalized mucosal immune responses with significant increases in anti-EtpA fecal IgG and IgA, and mice vaccinated via this route were also protected against colonization. In contrast, while intradermal (i.d.) vaccination achieved high levels of both serum and fecal antibodies against both EtpA and dmLT, mice vaccinated via the i.d. route were not protected against subsequent colonization and the avidity of serum IgG and IgA EtpA-specific antibodies was significantly lower after i.d. immunization compared to other routes. Finally, we demonstrate that antiserum from vaccinated mice significantly impairs binding of LT to cognate GM1 receptors and shows near complete neutralization of toxin delivery by ETEC in vitro. Collectively, these data provide further evidence that EtpA could complement future vaccine strategies but also suggest that additional effort will be required to optimize its use as a protective immunogen

Comparative genomic analysis and molecular examination of the diversity of enterotoxigenic Escherichia coli isolates from Chile

Enterotoxigenic Escherichia coli (ETEC) is one of the most common diarrheal pathogens in the low- and middle-income regions of the world, however a systematic examination of the genomic content of isolates from Chile has not yet been undertaken. Whole genome sequencing and comparative analysis of a collection of 125 ETEC isolates from three geographic locations in Chile, allowed the interrogation of phylogenomic groups, sequence types and genes specific to isolates from the different geographic locations. A total of 80.8% (101/125) of the ETEC isolates were identified in E. coli phylogroup A, 15.2% (19/125) in phylogroup B, and 4.0% (5/125) in phylogroup E. The over-representation of genomes in phylogroup A was significantly different from other global ETEC genomic studies. The Chilean ETEC isolates could be further subdivided into sub-clades similar to previously defined global ETEC reference lineages that had conserved multi-locus sequence types and toxin profiles. Comparison of the gene content of the Chilean ETEC identified genes that were unique based on geographic location within Chile, phylogenomic classifications or sequence type. Completion of a limited number of genomes provided insight into the ETEC plasmid content, which is conserved in some phylogenomic groups and not conserved in others. These findings suggest that the Chilean ETEC isolates contain unique virulence factor combinations and genomic content compared to global reference ETEC isolates

Dynamic interactions of a conserved enterotoxigenic Escherichia coli adhesin with intestinal mucins govern epithelium engagement and toxin delivery

At present, there is no vaccine for enterotoxigenic Escherichia coli (ETEC), an important cause of diarrheal illness. Nevertheless, recent microbial pathogenesis studies have identified a number of molecules produced by ETEC that contribute to its virulence and are novel antigenic targets to complement canonical vaccine approaches. EtpA is a secreted two-partner adhesin that is conserved within the ETEC pathovar. EtpA interacts with the tips of ETEC flagella to promote bacterial adhesion, toxin delivery, and intestinal colonization by forming molecular bridges between the bacteria and the epithelial surface. However, the nature of EtpA interactions with the intestinal epithelium remains poorly defined. Here, we demonstrate that EtpA interacts with glycans presented by transmembrane and secreted intestinal mucins at epithelial surfaces to facilitate pathogen-host interactions that culminate in toxin delivery. Moreover, we found that a major effector molecule of ETEC, the heat-labile enterotoxin (LT), may enhance these interactions by stimulating the production of the gel-forming mucin MUC2. Our studies suggest, however, that EtpA participates in complex and dynamic interactions between ETEC and the gastrointestinal mucosae in which host glycoproteins promote bacterial attachment while simultaneously limiting the epithelial engagement required for effective toxin delivery. Collectively, these data provide additional insight into the intricate nature of ETEC interactions with the intestinal epithelium that have potential implications for rational approaches to vaccine design

Comparative genomics and transcriptomics of Escherichia coli isolates carrying virulence factors of both enteropathogenic and enterotoxigenic E. coli

AbstractEscherichia coli that are capable of causing human disease are often classified into pathogenic variants (pathovars) based on their virulence gene content. However, disease-associated hybrid E. coli, containing unique combinations of multiple canonical virulence factors have also been described. Such was the case of the E. coli O104:H4 outbreak in 2011, which caused significant morbidity and mortality. Among the pathovars of diarrheagenic E. coli that cause significant human disease are the enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). In the current study we use comparative genomics, transcriptomics, and functional studies to characterize isolates that contain virulence factors of both EPEC and ETEC. Based on phylogenomic analysis, these hybrid isolates are more genomically-related to EPEC, but appear to have acquired ETEC virulence genes. Global transcriptional analysis using RNA sequencing, demonstrated that the EPEC and ETEC virulence genes of these hybrid isolates were differentially-expressed under virulence-inducing laboratory conditions, similar to reference isolates. Immunoblot assays further verified that the virulence gene products were produced and that the T3SS effector EspB of EPEC, and heat-labile toxin of ETEC were secreted. These findings document the existence and virulence potential of an E. coli pathovar hybrid that blurs the distinction between E. coli pathovars.</jats:p