Bacterial wilt of ginger in Queensland: Reappraisal of a disease outbreak

In 1955 a severe wilt disease occurring on ginger in the Near North Coast district of Queensland was incorrectly attributed to infection by a Fusarium sp., and later shown to be caused by a strain of Ralstonia solanacearum, now reclassified as R. sequeirae. The disease was brought from China into Australia on latently infected rhizomes, and possibly also with associated soil. Several DNA-based diagnostic methods have shown that the pathogen causing bacterial wilt of ginger in parts of China is indistinguishable from the pathogen uniquely associated with the disease in Queensland. © 2012 Australasian Plant Pathology Society Inc.

Recent insights into microbial triggers of interleukin-10 production in the host and the impact on infectious disease pathogenesis

Since its initial description as a Th2-cytokine antagonistic to interferon-alpha and granulocyte-macrophage colony-stimulating factor, many studies have shown various anti-inflammatory actions of interleukin-10 (IL-10), and its role in infection as a key regulator of innate immunity. Studies have shown that IL-10 induced in response to microorganisms and their products plays a central role in shaping pathogenesis. IL-10 appears to function as both sword and shield in the response to varied groups of microorganisms in its capacity to mediate protective immunity against some organisms but increase susceptibility to other infections. The nature of IL-10 as a pleiotropic modulator of host responses to microorganisms is explained, in part, by its potent and varied effects on different immune effector cells which influence antimicrobial activity. A new understanding of how microorganisms trigger IL-10 responses is emerging, along with recent discoveries of how IL-10 produced during disease might be harnessed for better protective or therapeutic strategies. In this review, we summarize studies from the past 5 years that have reported the induction of IL-10 by different classes of pathogenic microorganisms, including protozoa, nematodes, fungi, viruses and bacteria and discuss the impact of this induction on the persistence and/or clearance of microorganisms in the host.

A convenient sample preparation protocol for scanning electron microscope examination of xylem-occluding bacterial biofilm on cut flowers and foliage

Microbes and their exopolysaccharides (EPS) can block xylem vessels, thereby increasing the hydraulic resistance and decreasing the vase life of cut flowers and foliage. Scanning electron microscopy (SEM) provides a powerful tool for investigation of bacteria-induced xylem occlusion. However, conventional preparation protocols for SEM involving chemicals can cause loss of hydrated EPS material, and thereby damage the bacterial biofilms during dehydration. A modified chemical fixation protocol involving pre-fixation with 75 mM lysine plus 2.5% glutaraldehyde followed by the normal fixation in 3% glutaraldehyde was, therefore, tested for improved preservation of bacterial biofilm at the stem-ends of cut Acacia holosericea foliage stems. Stem-end segments with different stages of bacterial growth were obtained from stems stood into water. The lysine-based protocol was compared with four other processing protocols of critical point drying (CPD) without fixation (control), freeze-drying (FD), conventional chemical fixation followed by drying with hexamethyldisilazane (HMDS), and conventional chemical fixation with CPD. The non-fixed control. FD and the glutaraldehyde fixation with HMDS drying gave poor preservation of hydrated material, including bacterial EPS. Conventional glutaraldehyde fixation followed by CPD was superior to these three methods in terms of better preserving the EPS. However, this fourth method gave condensation of biofilms during dehydration. In contrast, the modified lysine-based protocol resulted in superior preservation of EPS and biofilm structure. Thus, this fifth method was the most appropriate for examination of bacterial stem-end blockage in cut ornamentals. (C) 2012 Elsevier B.V. All rights reserved.

Weakening of the Gram-negative bacterial outer membrane - A tool for increasing microbiological safety

Gram-negative bacteria are harmful in various surroundings. In the food industy their metabolites are potential cause of spoilage and this group also includes many severe or potential pathogens, such as Salmonella. Due to their ability to produce biofilms Gram-negative bacteria also cause problems in many industrial processes as well as in clinical surroundings. Control of Gram-negative bacteria is hampered by the outer membrane (OM) in the outermost layer of the cells. This layer is an intrinsic barrier for many hydrophobic agents and macromolecules. Permeabilizers are compounds that weaken OM and can thus increase the activity of antimicrobials by facililating entry of hydrophobic compounds and macromolecules into the cell where they can reach their target sites and inhibit or destroy cellular functions. The work described in this thesis shows that lactic acid acts as a permeabilizer and destabilizes the OM of Gram-negative bacteria. In addition, organic acids present in berriers, i.e. malic, sorbic and benzoic acid, were shown to weaken the OM of Gram-negative bacteria. Organic acids can poteniate the antimicrobial activity of other compounds. Microbial colonic degradation products of plant-derived phenolic compounds (3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid, 3-phenylpropionic acid and 3-hydroxyphenylpropionic acid) efficiently destabilized OM of Salmonella. The studies increase our understanding of the mechanism of action of the classical chelator, ethylenediaminetetra-acetic acid (EDTA). In addition, the results indicate that the biocidic activity of benzalkonium chloride against Pseudomonas can be increased by combined use with polyethylenimine (PEI). In addition to PEI, several other potential permeabilizers, such as succimer, were shown to destabilize the OM of Gram-negative bacteria. Furthermore, combination of the results obtained from various permeability assays (e.g. uptake of a hydrophobic probe, sensitization to hydrophobic antibiotics and detergents, release of lipopolysaccharide (LPS) and LPS-specific fatty acids) with atomic force microscopy (AFM) image results increases our knowledge of the action of permeabilizers.

Boronia heterophylla vase life is influenced more by ethylene than by bacterial numbers or vase solution pH

Bacterial proliferation in both vase solutions and in cut flower stems has been implicated in reducing the vase life of numerous genera. Boronia heterophylla F. Muell. (Red Boronia) vase life was assessed at two stages of floral maturity for nine vase solution treatments covering a pH range of 2.5-5.7. Vase life for advanced harvest maturity stems ranged from 4.2 d in 10 mM citric acid + 50 mg L-1 chlorine (pH 2.5) to 12.9 d after STS pulsing (pH 5.7). For normal harvest maturity stems, the corresponding range was 5.8-19.0 d, respectively. Vase solutions containing 50 mg L-1 chlorine biocide resulted in decreased longevity. In contrast, pulsing with the ethylene-binding inhibitor, STS, significantly increased vase life. The number of bacteria in the vase solutions after 11 d was determined in stems of advanced maturity. The solution with the greatest number of bacteria, 4.0 x 10(10) cfu mL(-1), was water used after STS pulsing and in which the flowers lasted longest. Vase solution bacteria were enumerated on days 0,3, 6, 9 and 12 of the vase period with stems of normal harvest maturity. There was no relationship between vase life and vase solution bacterial numbers ((R) over bar (2) = 0.000). Moreover, there was a negative relationship between numbers of bacteria in basal 0-5 cm stem segments and vase life. As no correlations were evident between longevity and either the pH or vase solution bacterial numbers, B. heterophylla vase life was evidently limited principally by ethylene action. (C) 2013 Elsevier B.V. All rights reserved.

Programming rumen bacterial communities in newborn Merino lambs

Establishment of the rumen microbiome can be affected by both early-life dietary measures and rumen microbial inoculation. This study used a 2 × 3 factorial design to evaluate the effects of inclusion of dietary fat type and the effects of rumen inoculum from different sources on ruminal bacterial communities present in early stages of the lambs’ life. Two different diets were fed ad libitum to 36 pregnant ewes (and their lambs) from 1 month pre-lambing until weaning. Diets consisted of chaffed lucerne and cereal hay and 4% molasses, with either 4% distilled coconut oil (CO) provided as a source of rumen-active fat or 4% Megalac® provided as a source of rumen-protected fat (PF). One of three inoculums was introduced orally to all lambs, being either (1) rumen fluid from donor ewes fed the PF diet; (2) rumen fluid from donor ewes fed CO; or (3) a control treatment of MilliQ-water. After weaning at 3 months of age, each of the six lamb treatment groups were grazed in spatially separated paddocks. Rumen bacterial populations of ewes and lambs were characterised using 454 amplicon pyrosequencing of the V3/V4 regions of the 16S rRNA gene. Species richness and biodiversity of the bacterial communities were found to be affected by the diet in ewes and lambs and by inoculation treatment of the lambs. Principal coordinate analysis and analysis of similarity (ANOSIM) showed between diet differences in bacterial community groups existed in ewes and differential bacterial clusters occurred in lambs due to both diet and neonatal inoculation. Diet and rumen inoculation acted together to clearly differentiate the bacterial communities through to weaning, however the microbiome effects of these initial early life interventions diminished with time so that rumen bacterial communities showed greater similarity 2 months after weaning. These results demonstrate that ruminal bacterial communities of newborn lambs can be altered by modifying the diet of their mothers. Moreover, the rumen microbiome of lambs can be changed by diet while they are suckling or by inoculating their rumen, and resulting changes in the rumen bacterial microbiome can persist beyond weaning.

Bayesian statistical analysis of bacterial diversity

Bacteria play an important role in many ecological systems. The molecular characterization of bacteria using either cultivation-dependent or cultivation-independent methods reveals the large scale of bacterial diversity in natural communities, and the vastness of subpopulations within a species or genus. Understanding how bacterial diversity varies across different environments and also within populations should provide insights into many important questions of bacterial evolution and population dynamics. This thesis presents novel statistical methods for analyzing bacterial diversity using widely employed molecular fingerprinting techniques. The first objective of this thesis was to develop Bayesian clustering models to identify bacterial population structures. Bacterial isolates were identified using multilous sequence typing (MLST), and Bayesian clustering models were used to explore the evolutionary relationships among isolates. Our method involves the inference of genetic population structures via an unsupervised clustering framework where the dependence between loci is represented using graphical models. The population dynamics that generate such a population stratification were investigated using a stochastic model, in which homologous recombination between subpopulations can be quantified within a gene flow network. The second part of the thesis focuses on cluster analysis of community compositional data produced by two different cultivation-independent analyses: terminal restriction fragment length polymorphism (T-RFLP) analysis, and fatty acid methyl ester (FAME) analysis. The cluster analysis aims to group bacterial communities that are similar in composition, which is an important step for understanding the overall influences of environmental and ecological perturbations on bacterial diversity. A common feature of T-RFLP and FAME data is zero-inflation, which indicates that the observation of a zero value is much more frequent than would be expected, for example, from a Poisson distribution in the discrete case, or a Gaussian distribution in the continuous case. We provided two strategies for modeling zero-inflation in the clustering framework, which were validated by both synthetic and empirical complex data sets. We show in the thesis that our model that takes into account dependencies between loci in MLST data can produce better clustering results than those methods which assume independent loci. Furthermore, computer algorithms that are efficient in analyzing large scale data were adopted for meeting the increasing computational need. Our method that detects homologous recombination in subpopulations may provide a theoretical criterion for defining bacterial species. The clustering of bacterial community data include T-RFLP and FAME provides an initial effort for discovering the evolutionary dynamics that structure and maintain bacterial diversity in the natural environment.

Investigation of an emerging bacterial disease in wild Queensland groper, marine fish and stingrays with production of diagnostic tools to reduce the spread of disease to other states of Australia : final report

This project describes how Streptococcus agalactiae can be transmitted experimentally in Queensland grouper. The implications of this research furthers the relatedness between Australian S. agalactiae strains from animals and humans. Additionally, this research has developed diagnostic tools for Australian State Veterinary Laboratories and Universities, which will assist in State and National aquatic animal disease detection, surveillance, disease monitoring and reporting

Innate immunity and its control in the pathogenesis of inflammatory rheumatic diseases

The pathogenesis of inflammatory rheumatic diseases, including rheumatoid arthritis (RA) and spondyloarthropathies (SpAs) such as reactive arthritis (ReA), is incompletely understood. ReA is a sterile joint inflammation, which may follow a distal infection caused by Gram-negative bacteria that have lipopolysaccharide (LPS) in their outer membrane. The functions of innate immunity that may affect the pathogenesis, prognosis and treatment of these diseases were studied in this thesis. When compared with healthy controls, whole blood monocytes of healthy subjects with previous ReA showed enhanced capacity to produce TNF, an essential proinflammatory cytokine, in response to adherent conditions (mimicking vascular endothelium made adherent by inflammatory signals) and non-specific protein kinase C stimulation. Also, blood neutrophils of these subjects showed high levels of CD11b, an important adhesion molecule, in response to adherence or LPS. Thus, high responsiveness of monocytes and neutrophils when encountering inflammatory stimuli may play a role in the pathogenesis of ReA. The results also suggested that the known risk allele for SpAs, HLA-B27, may be an additive contributor to the observed differences. The promoter polymorphisms TNF 308A and CD14 (gene for an LPS receptor component) 159T were found not to increase the risk of acute arthritis. However, all female patients who developed chronic SpA had 159T and none of them had 308A, possibly reflecting an interplay between hormonal and inflammatory signals in the development of chronic SpA. Among subjects with early RA, those having the polymorphic TLR4 +896G allele (causing the Asp299Gly change in TLR4, another component of LPS receptor) required a combination of disease-modifying antirheumatic drugs to achieve remission. It is known that rapid treatment response is essential in order to maintain the patients work ability. Hence, +896G might be a candidate marker for identifying the patients who need combination treatment. The production of vascular endothelial growth factor (VEGF), which strongly promotes vascular permeability and angiogenesis that takes place e.g. early in rheumatic joints, was induced by LPS and inhibited by interferon (IFN)-alpha in peripheral blood mononuclear cells. These long-living cells might provide a source of VEGF when stimulated by LPS and migrating to inflamed joints, and the effect of IFN-alpha may contribute to the clinical efficacy of this cytokine in inhibiting joint inflammation.