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.

The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

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.

Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

Background Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia. Conclusion We describe an Australian origin for B. pseudomallei, characterized by a single introduction event into Southeast Asia during a recent glacial period, and variable levels of lateral gene transfer within populations. These patterns provide insights into mechanisms of genetic diversification in B. pseudomallei and its closest relatives, and provide a framework for integrating the traditionally separate fields of population genetics and phylogenetics for other bacterial species with high levels of lateral gene transfer.

Occurrence of iron and associated bacterial populations in soils of a forested subtropical coastal catchment

Iron (Fe) biogeochemistry is potentially of environmental significance in plantation-forested, subtropical coastal ecosystems where soil disturbance and seasonal water logging may lead to elevation of Fe mobilization and associated water quality deterioration. Using wet-chemical extraction and laboratory cultivation, we examined the occurrence of Fe forms and associated bacterial populations in diverse soils of a representative subtropical Australian coastal catchment (Poona Creek). Total reactive Fe was abundant throughout 0e30 cm soil cores, consisting primarily of crystalline forms in well-drained sand soils and water-logged loam soils, whereas in water-logged, low clay soils, over half of total reactive Fe was present in poorly-crystalline forms due to organic and inorganic complexation, respectively. Forestry practices such as plantation clear-felling and replanting, seasonal water logging and mineral soil properties significantly impacted soil organic carbon (C), potentially-bioavailable Fe pools and densities of S-, but not Fe-, bacterial populations. Bacterial Fe(III) reduction and abiotic Fe(II) oxidation, as well as chemolithotrophic S oxidation and aerobic, heterotrophic respiration were integral to catchment terrestrial FeeC cycling. This work demonstrates bacterial involvement in terrestrial Fe cycling in a subtropical coastal circumneutral-pH ecosystem.

Apoptosis-related genes confer resistance to Fusarium wilt in transgenic ‘Lady Finger’ bananas

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, ‘Lady Finger’, were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3’ UTR, and independently transformed plant lines were regenerated for testing. Following a 12 week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 x Bcl-xL, 3 x Ced-9 and 2 x Bcl-2 3’ UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible ‘Lady Finger’ banana plants used as positive controls. Of these, one Bcl-2 3’ UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23 weeks post-inoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type ‘Lady Finger’ plants consistent with a necrotrophic phase in the lifecycle of this pathogen. This was further supported by the observed reduction of these effects in the roots of the resistant Bcl-2 3’ UTR transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.

Photocatalytic disinfection of bacterial pollutants using suspended and immobilized TiO(2) powders

The photocatalytic disinfection of Enterobacter cloacae and Enterobacter coli using microwave (MW), convection hydrothermal (HT) and Degussa P25 titania was investigated in suspension and immobilized reactors. In suspension reactors, MW-treated TiO(2) was the most efficient catalyst (per unit weight of catalyst) for the disinfection of E. cloacae. However, HT-treated TiO(2) was approximately 10 times more efficient than MW or P25 titania for the disinfection of E. coli suspensions in surface water using the immobilized reactor. In immobilized experiments, using surface water a significant amount of photolysis was observed using the MW- and HT-treated films; however, disinfection on P25 films was primarily attributed to photocatalysis. Competitive action of inorganic ions and humic substances for hydroxyl radicals during photocatalytic experiments, as well as humic substances physically screening the cells from UV and hydroxyl radical attack resulted in low rates of disinfection. A decrease in colony size (from 1.5 to 0.3 mm) was noted during photocatalytic experiments. The smaller than average colonies were thought to occur during sublethal (•) OH and O(2) (•-) attack. Catalyst fouling was observed following experiments in surface water and the ability to regenerate the surface was demonstrated using photocatalytic degradation of oxalic acid as a model test system

A cost-outcome description of the septic work-up for bacterial infection in neonates in a tertiary care hospital

Analysis of the septic work-up of 194 neonates at Women's College Hospital, Toronto, showed that the only antepartum condition predicting neonatal sepsis was the mother being on antibiotics. The only postnatal condition predicting sepsis was a maternal postpartum white blood cell count over 11,000. The average cost for tests for a septic work-up in these 194 mother-neonate pairs was $71.48 (Canadian dollars), and the average cost of tests to find a septic case was $1,066.77.

Hormone-dependent bacterial growth persistence and biofilm formation - A pilot study investigating human follicular fluid collected during IVF cycles

Human follicular fluid, considered sterile, is aspirated as part of an in vitro fertilization (IVF) cycle. However, it is easily contaminated by the trans-vaginal collection route and little information exists in its potential to support the growth of microorganisms. The objectives of this study were to determine whether human follicular fluid can support bacterial growth over time, whether the steroid hormones estradiol and progesterone (present at high levels within follicular fluid) contribute to the in vitro growth of bacterial species, and whether species isolated from follicular fluid form biofilms. We found that bacteria in follicular fluid could persist for at least 28 weeks in vitro and that the steroid hormones stimulated the growth of some bacterial species, specifically Lactobacillus spp., Bifidobacterium spp. Streptococcus spp. and E. coli. Several species, Lactobacillus spp., Propionibacterium spp., and Streptococcus spp., formed biofilms when incubated in native follicular fluids in vitro (18/24, 75%). We conclude that bacteria aspirated along with follicular fluid during IVF cycles demonstrate a persistent pattern of growth. This discovery is important since it can offer a new avenue for investigation in infertile couples.

The role of HtrA as a chaperone and protease in bacterial pathogenesis

HtrA (High Temperature Requirement A) is a critical stress response protease and chaperone for many bacteria. HtrA is a multitasking protein which can degrade unfolded proteins, conduct specific proteolysis of some substrates for correct assembly, interact with substrates to ensure correct folding, assembly or localisation, and chaperone unfolded proteins. These functions are critical for the virulence of a number of bacterial pathogens, in some cases not simply due to the broad activities of HtrA in protection against the protein stress conditions which occur during virulence. But also due to the role of HtrA in either specific proteolysis or assembly of key protein substrates which function directly in virulence. Remarkably, these activities are all conducted without any requirement for ATP. The biochemical mechanism of HtrA relies both on the chymotryptic serine protease active site as well as the presence of two PDZ (protein binding) domains. The mechanism is a unique combination of activation by substrate motifs to alter the confirmation of the active site, and assembly into a multimeric complex which has enhanced degradation and may also act as a protective cage for proteins which are not degraded. The role of this protease in the pathogenesis of a number of bacteria and the details of its distinctive biochemical activation and assembly mechanisms are discussed in this chapter.

The role of HtrA as a chaperone and protease in bacterial pathogenisis

HtrA (High Temperature Requirement A) is a critical stress response protease and chaperone for many bacteria. HtrA is a multitasking protein which can degrade unfolded proteins, conduct specific proteolysis of some substrates for correct assembly, interact with substrates to ensure correct folding, assembly or localisation, and chaperone unfolded proteins. These functions are critical for the virulence of a number of bacterial pathogens, in some cases not simply due to the broad activities of HtrA in protection against the protein stress conditions which occur during virulence. But also due to the role of HtrA in either specific proteolysis or assembly of key protein substrates which function directly in virulence. Remarkably, these activities are all conducted without any requirement for ATP. The biochemical mechanism of HtrA relies both on the chymotryptic serine protease active site as well as the presence of two PDZ (protein binding) domains. The mechanism is a unique combination of activation by substrate motifs to alter the confirmation of the active site, and assembly into a multimeric complex which has enhanced degradation and may also act as a protective cage for proteins which are not degraded. The role of this protease in the pathogenesis of a number of bacteria and the details of its distinctive biochemical activation and assembly mechanisms are discussed in this chapter.