Detection of bacterial DNA in cardiac vegetations by PCR after the completion of antimicrobial treatment for endorcarditis

PCR with broad-range primers for prokaryotic 16S rRNA genes was used to identify bacterial DNA in tissue from patients undergoing valve replacements following a previous episode of infective endocarditis (IF). Of eight valves investigated, bacterial DNA was detected in three from patients for whom IE had been treated by antibiotic therapy 5, 12 and 18 months previously. The demonstration of bacterial DNA within resected heart valves suggests either recurrence of infection, treatment failure or the persistence of bacterial debris within the cardiac vegetation. There may also be implications for routine use of PCR in the diagnosis of infection. © 2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases.

Characterizations of the major coral diseases of the Philippines: Ulcerative white spot disease and novel growth anomalies of Porites

Coral reefs are in decline worldwide and coral disease is a significant contributing factor. However, etiologies of coral diseases are still not well understood. In contrast with the Caribbean, extremely little is known about coral diseases in the Philippines. In 2005, off Southeast Negros Island, Philippines, I investigated relationships between environmental parameters and prevalence of the two most common coral diseases, ulcerative white spot (UWS) and massive Porites growth anomalies (MPGAs). Samples were collected along a disease prevalence gradient 40.5 km long. Principal component analyses showed prevalence of MPGAs was positively correlated with water column nitrogen, organic carbon of surface sediments, and colony density. UWS was positively correlated with water column phosphorus. This is the first quantitative evidence linking anthropogenically-impacted water and sediment to a higher prevalence of these diseases. Histological and cytological alterations were investigated by comparing tissues from two distinct types of MPGA lesions (types 1 and 2) and healthy coral using light and electron microscopy. Skeletal abnormalities and sloughing, swelling, thinning, and loss of tissues in MPGAs resembled tissues exposed to bacterial or fungal toxins. Both lesion types had decreases in symbiotic zooxanthellae, which supply nutrients to corals. Notable alterations included migrations of chromophore cells (amoebocytes) (1) nocturnally to outer epithelia to perform wound-healing, including plugging gaps and secreting melanin in degraded tissues, and (2) diurnally to the interior of the tissue possibly to prevent shading zooxanthellae in order to maximize photosynthate production. Depletion of melanin (active in wound healing) in type 2 lesions suggested type 2 tissues were overtaxed and less stable. MPGAs contained an abundance of endolithic fungi and virus-like particles, which may result from higher nutrient levels and play roles in disease development. Swollen cells and mucus frequently blocked gastrovascular canals (GVCs) in MPGAs. Type 1 lesions appeared to compensate for impeded flow of wastes and nutrients through these canals with proliferation of new GVCs, which were responsible for the observed thickened tissues. In contrast, type 2 tissues were thin and more degraded. Dysplasia and putative neoplasia were also observed in MPGAs which may result from the tissue regeneration capacity being overwhelmed.

Characterizations of the Major Coral Diseases of the Philippines: Ulcerative White Spot Disease and Novel Growth Anomalies of Porites

Coral reefs are in decline worldwide and coral disease is a significant contributing factor. However, etiologies of coral diseases are still not well understood. In contrast with the Caribbean, extremely little is known about coral diseases in the Philippines. In 2005, off Southeast Negros Island, Philippines, I investigated relationships between environmental parameters and prevalence of the two most common coral diseases, ulcerative white spot (UWS) and massive Porites growth anomalies (MPGAs). Samples were collected along a disease prevalence gradient 40.5 km long. Principal component analyses showed prevalence of MPGAs was positively correlated with water column nitrogen, organic carbon of surface sediments, and colony density. UWS was positively correlated with water column phosphorus. This is the first quantitative evidence linking anthropogenically-impacted water and sediment to a higher prevalence of these diseases. Histological and cytological alterations were investigated by comparing tissues from two distinct types of MPGA lesions (types 1 and 2) and healthy coral using light and electron microscopy. Skeletal abnormalities and sloughing, swelling, thinning, and loss of tissues in MPGAs resembled tissues exposed to bacterial or fungal toxins. Both lesion types had decreases in symbiotic zooxanthellae, which supply nutrients to corals. Notable alterations included migrations of chromophore cells (amoebocytes) (1) nocturnally to outer epithelia to perform wound-healing, including plugging gaps and secreting melanin in degraded tissues, and (2) diurnally to the interior of the tissue possibly to prevent shading zooxanthellae in order to maximize photosynthate production. Depletion of melanin (active in wound healing) in type 2 lesions suggested type 2 tissues were overtaxed and less stable. MPGAs contained an abundance of endolithic fungi and virus-like particles, which may result from higher nutrient levels and play roles in disease development. Swollen cells and mucus frequently blocked gastrovascular canals (GVCs) in MPGAs. Type 1 lesions appeared to compensate for impeded flow of wastes and nutrients through these canals with proliferation of new GVCs, which were responsible for the observed thickened tissues. In contrast, type 2 tissues were thin and more degraded. Dysplasia and putative neoplasia were also observed in MPGAs which may result from the tissue regeneration capacity being overwhelmed.

Common fish diseases and parasites affecting wild and farmed tilapia and catfish in central and western Uganda

Intensification of aquaculture production in Uganda is likely to result into disease out-breaks leading to economic losses to commercial fish farms and associated natural aquatic ecosystems. This survey assessed health profiles of selected commercial fish farms and adjacent natural aquatic ecosystemsto identify fish diseases and parasites affecting Nile tilapia (Oreochromis niloticus) and African catfish (Clarias gariepinus) in aquaculture systems in Uganda. Fish farms encounter disease out-breaks that cause low survival rates (0 - 30%), especially catfish hatcheries. Health management issues are not well understood by fish farmers, with some unable to detect diseased fish. Current control strategies to control aquatic pathogens include use of chemotherapeutants and antibiotics. Bacterial pathogens isolated included Flavobacterium columnare, Aeromonas sp., Edwardsiella sp., Psuedomonus sp., Steptococcus sp., Staphylococcus sp., Proteus sp., and Vibrio sp. A high occurrence of Flavobacterium columnare exists in both asymptomatic and symptomatic fish was observed. Parasites included protozoans (Ichthyopthirius multiphilis, Trichodina sp. and Icthyobodo sp.) and trematodes (Cleidodiscus sp. and Gyrodactylus sp.). Diagnosis and control of diseases and parasites in aquaculture production systems requires adoption of a regional comprehensive biosecurity strategy: the East African (EAC) region unto which this study directly contributes.

Effect of female sex hormones on Chlamydia trachomatis growth and gene expression

Transmissible diseases are re-emerging as a global problem, with Sexually Transmitted Diseases (STDs) becoming endemic. Chlamydia trachomatis is the leading cause of bacterially-acquired STD worldwide, with the Australian cost of infection estimated at $90 - $160 million annually. Studies using animal models of genital tract Chlamydia infection suggested that the hormonal status of the genital tract epithelium at the time of exposure may influence the outcome of infection. Oral contraceptive use also increased the risk of contracting chlamydial infections compared to women not using contraception. Generally it was suggested that the outcome of chlamydial infection is determined in part by the hormonal status of the epithelium at the time of exposure. Using the human endolmetrial cell line ECC-1 this study investigated the effects of C. trachomatis serovar D infection, in conjunction with the female sex hormones, 17β-estradiol and progesterone, on chlamydial gene expression. While previous studies have examined the host response, this is the first study to examine C.trachomatis gene expression under different hormonal conditions. We have highlighted a basic model of C. trachomatis gene regulation in the presence of steroid hormones by identifying 60 genes that were regulated by addition of estradiol and/or progesterone. In addition, the third chapter of this thesis discussed and compared the significance of the current findings in the context of data from other research groups to improve our understanding of the molecular basis of chlamydial persistence under hormonal different conditions. In addition, this study analysed the effects of these female sex hormones and C. trachomatis Serovar D infection, on host susceptibility and bacterial growth. Our results clearly demonstrated that addition of steroid hormones not only had a great impact on the level of infectivity of epithelial cells with C.trachomatis serovar D, but also the morphology of chlamydial inclusions was affected by hormone supplementation.

Prevalence and occurrence of zoonotic bacterial pathogens in surface waters determined by quantitative PCR

The prevalence and concentrations of Campylobacter jejuni, Salmonella spp. and enterohaemorrhagic E. coli (EHEC) were investigated in surface waters in Brisbane, Australia using quantitative PCR (qPCR) based methodologies. Water samples were collected from Brisbane City Botanic Gardens (CBG) Pond, and two urban tidal creeks (i.e., Oxley Creek and Blunder Creek). Of the 32 water samples collected, 8 (25%), 1 (3%), 9 (28%), 14 (44%), and 15 (47%) were positive for C. jejuni mapA, Salmonella invA, EHEC O157 LPS, EHEC VT1, and EHEC VT2 genes, respectively. The presence/absence of the potential pathogens did not correlate with either E. coli or enterococci concentrations as determined by binary logistic regression. In conclusion, the high prevalence, and concentrations of potential zoonotic pathogens along with the concentrations of one or more fecal indicators in surface water samples indicate a poor level of microbial quality of surface water, and could represent a significant health risk to users. The results from the current study would provide valuable information to the water quality managers in terms of minimizing the risk from pathogens in surface waters.

When and how to treat pulmonary non-tuberculous mycobacterial diseases

Nontuberculous mycobacteria are ubiquitous environmental organisms that have been recognised as a cause of pulmonary infection for over 50 years. Traditionally patients have had underlying risk factors for development of disease; however the proportion of apparently immunocompetent patients involved appears to be rising. Not all patients culture-positive for mycobacteria will have progressive disease, making the diagnosis difficult, though criteria to aid in this process are available. The two main forms of disease are cavitary disease (usually involving the upper lobes) and fibronodular bronchiectasis (predominantly middle and lingular lobes). For patients with disease, combination antibiotic therapy for 12-24 months is generally required for successful treatment, and this may be accompanied by drug intolerances and side effects. Published success rates range from 30-82%. As the progression of disease is variable, for some patients, attention to pulmonary hygiene and underlying diseases without immediate antimycobacterial therapy may be more appropriate. Surgery can be a useful adjunct, though is associated with risks. Randomised controlled trials in well described patients would provide stronger evidence-based data to guide therapy of NTM lung diseases, and thus are much needed.

Bacterial proteases from the intracellular vacuole niche ; protease conservation and adaptation for pathogenic advantage

Proteases with important roles for bacterial pathogens which specifically reside within intracellular vacuoles are frequently homologous to those which have important virulence functions for other bacteria. Research has identified that some of these conserved proteases have evolved specialised functions for intracellular vacuole residing bacteria. Unique proteases with pathogenic functions have also been described from Chlamydia, Mycobacteria, and Legionella. These findings suggest that there are further novel functions for proteases from these bacteria which remain to be described. This review summarises recent findings of novel protease functions from the intracellular human pathogenic bacteria which reside exclusively in vacuoles.

Culture independent analysis of greyback canegrub-associated microflora and microbial community comparison using subtractive hybridisation

Greyback canegrubs cost the Australian sugarcane industry around $13 million per annum in damage and control. A novel and cost effective biocontrol bacterium could play an important role in the integrated pest management program currently in place to reduce damage and control associated costs. During the course of this project, terminal restriction fragment length polymorphism (TRFLP), 16-S rDNA cloning, suppressive subtractive hybridisation (SSH) and entomopathogen-specific PCR screening were used to investigate the little studied canegrub-associated microflora in an attempt to discover novel pathogens from putatively-diseased specimens. Microflora associated with these soil-dwelling insects was found to be both highly diverse and divergent between individual specimens. Dominant members detected in live specimens were predominantly from taxa of known insect symbionts while dominant sequences amplified from dead grubs were homologous to putativelysaprophytic bacteria and bacteria able to grow during refrigeration. A number of entomopathogenic bacteria were identified such as Photorhabdus luminescens and Pseudomonas fluorescens. Dead canegrubs prior to decomposition need to be analysed if these bacteria are to be isolated. Novel strategies to enrich putative pathogen-associated sequences (SSH and PCR screening) were shown to be promising approaches for pathogen discovery and the investigation of canegrubsassociated microflora. However, due to inter- and intra-grub-associated community diversity, dead grub decomposition and PCR-specific methodological limitations (PCR bias, primer specificity, BLAST database restrictions, 16-S gene copy number and heterogeneity), recommendations have been made to improve the efficiency of such techniques. Improved specimen collection procedures and utilisation of emerging high-throughput sequencing technologies may be required to examine these complex communities in more detail. This is the first study to perform a whole-grub analysis and comparison of greyback canegrub-associated microbial communities. This work also describes the development of a novel V3-PCR based SSH technique. This was the first SSH technique to use V3-PCR products as a starting material and specifically compare bacterial species present in a complex community.

Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.

Health risk from the use of roof-harvested rainwater in Southeast Queensland, Australia, as potable or nonpotable water, determined using quantitative microbial risk assessment

A total of 214 rainwater samples from 82 tanks were collected in urban Southeast Queensland (SEQ) in Australia and analysed for the zoonotic bacterial and protozoan pathogen using real-time binary PCR and quantitative PCR (qPCR). Quantitative Microbial Risk Assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to potential pathogens from potable and non-potable uses of roof-harvested rainwater. Of the 214 samples tested, 10.7%, 9.8%, and 5.6%, and 0.4% samples were positive for Salmonella invA, Giardia lamblia β-giardin , Legionella pneumophila mip, and Campylobacter jejuni mapA genes. Cryptosporidium parvum could not be detected. The estimated numbers of viable Salmonella spp., G. lamblia β-giradin, and L. pneumophila genes ranged from 1.6 × 101 to 9.5 × 101 cells, 1.4 × 10-1 to 9.0 × 10-1 cysts, and 1.5 × 101 to 4.3 × 101 per 1000 ml of water, respectively. Six risk scenarios were considered from exposure to Salmonella spp., G. lamblia and L. pneumophila. For Salmonella spp., and G. lamblia, these scenarios were: (1) liquid ingestion due to drinking of rainwater on a daily basis (2) accidental liquid ingestion due to garden hosing twice a week (3) aerosol ingestion due to showering on a daily basis, and (4) aerosol ingestion due to hosing twice a week. For L. pneumophila, these scenarios were: (5) aerosol inhalation due to showering on a daily basis, and (6) aerosol inhalation due to hosing twice a week. The risk of infection from Salmonella spp., G. lamblia, and L. pneumophila associated with the use of rainwater for showering and garden hosing was calculated to be well below the threshold value of one extra infection per 10,000 persons per year in urban SEQ. However, the risk of infection from ingesting Salmonella spp. and G. lamblia via drinking exceeds this threshold value, and indicates that if undisinfected rainwater were ingested by drinking, then the gastrointestinal diseases of Salmonellosis and Giardiasis is expected to range from 5.0 × 100 to 2.8 × 101 (Salmonellosis) and 1.0 × 101 to 6.4 × 101 (Giardiasis) cases per 10,000 persons per year, respectively. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically examined. Nonetheless, it would seem prudent to disinfect rainwater for potable use.