The development of Lactococcus lactis as an antimicrobial agent

Non-pathogenic lactic acid bacteria are economically important Gram-positive bacteria used extensively in the food industry. Due to their “generally regarded as safe” status, certain species from the genera Lactobacillus and Lactococcus are also considered desirable as candidates for the production and secretion of recombinant proteins, particular those with therapeutic applications. The hypothesis examined by this thesis is that Lactococcus lactis can be modified to be an effective antimicrobial agent. Therefore, the aims of this thesis were to investigate the optimisation of the expression, secretion and/or activities of potential heterologous antimicrobial proteins by the model lactic acid bacterium, Lactococcus lactis subsp. cremoris MG1363. L. lactis strains were engineered to express and secrete the recombinant CyuC surface protein from Lactobacillus reuteri BR11, and a fusion protein consisting of CyuC and lysostaphin using the Sep promoter and secretion signal. CyuC has been characterised as a cystine-binding protein, but has also been demonstrated to have fibronectin binding activity. Lysostaphin is a bacteriolytic enzyme with specific activity against the Gram-positive pathogen, Staphylococcus aureus. These modified L. lactis strains were then investigated to see if they had the ability to inhibit the adhesion of S. aureus to host extracellular matrix (ECM) proteins. It was observed that the cell extracts of the L. lactis strain with the vector only (pGhost9:ISS1) was able to inhibit the adhesion of S. aureus to fibronectin, whilst the cell extracts of the L. lactis strain expressing lysostaphin was able to inhibit adhesion to keratin. Finally, this thesis has identified specific lactococcal genes that affect the secretion of lysostaphin through the use of random transposon mutagenesis. Ten mutants with higher lysostaphin activity contained insertions in four different genes encoding: (i) an uncharacterised putative transmembrane protein (llmg_0609), (ii) an enzyme catalysing the first step in peptidoglycan biosynthesis (murA2), (iii) a homolog of the oxidative defence regulator (trmA), and (iv) an uncharacterised putative enzyme involved in ubiquinone biosynthesis (llmg_2148). The higher lysostaphin activity observed in these mutants was found to be due to higher amounts of lysostaphin being secreted. The findings of this thesis contribute to the development of this organism as an antimicrobial agent and also to our understanding of L. lactis genetics.

In vivo performance of melimine as an antimicrobial coating for contact lenses in models of CLARE and CLPU

Purpose: One strategy to minimize bacteria-associated adverse responses such as microbial keratitis, contact lens–induced acute red eye (CLARE), and contact lens induced peripheral ulcers (CLPUs) that occur with contact lens wear is the development of an antimicrobial or antiadhesive contact lens. Cationic peptides represent a novel approach for the development of antimicrobial lenses.---------- Methods: A novel cationic peptide, melimine, was covalently incorporated into silicone hydrogel lenses. Confirmation tests to determine the presence of peptide and anti-microbial activity were performed. Cationic lenses were then tested for their ability to prevent CLPU in the Staphylococcus aureus rabbit model and CLARE in the Pseudomonas aeruginosa guinea pig model. ---------- Results: In the rabbit model of CLPU, melimine-coated lenses resulted in significant reductions in ocular symptom scores and in the extent of corneal infiltration (P < 0.05). Evaluation of the performance of melimine lenses in the CLARE model showed significant improvement in all ocular response parameters measured, including the percentage of eyes with corneal infiltrates, compared with those observed in the eyes fitted with the control lens (P ≤ 0.05). ---------- Conclusions: Cationic coating of contact lenses with the peptide melimine may represent a novel method of prevention of bacterial growth on contact lenses and consequently result in reduction of the incidence and severity of adverse responses due to Gram-positive and -negative bacteria during lens wear.

Evaluating Two Mass Screening Methods for Tungro Disease Resistance

Tungro is one of the most destructive viral diseases of rice in South and Southeast Asia. It is associated with two viruses---rice tungro bacilliform virus (RTBV) ,and rice tungro spherical virus (RTSV) (Hibino et al 1978). Both viruses are transmitted by the green leafhopper (GLH) Nephotettix virescens (Ling 1979), However, prior acquisition of RTSV is required for Ihe transmission of RTBV alone (Hibino 1983). Plants infected with both viruses show severe stunting and yellowing. Those infected with RTBV alone show mild stunting but no leaf discoloration whereas those infected with RTSV alone do not show any apparent symptoms (Hibino el al 1978). Since the late 1960s, tungro has been mainly managed through varietal resistance (Khush 1989). The instability of resistant varieties in the field (Dahal et .a1 1990) led to a reexamination of the nature of the incorporated sources of resistance and to the adoption of more precise and more accurate screening methods.

Mechanism of Resistance to Rice Tungro Spherical Virus (RTSV)

RTSV is one of two viruses that cause tungro disease. RTSV is independently transmitted, whereas the other virus, rice tungro bacilliform virus (RTBV), is dependent on RTSV for its transmission by the green leafhopper (GLH), Nephotettix virescens. The occurrence and spread of tungro disease therefore depend on the presence of RTSV in the field. Resistance to RTSV infection would slow down the spread of the disease.

Inheritance of Resistance to Rice Tungro Spherical Virus in a Near-Isogenic Line Derived from Utri Merah and in Rice Cultivar TKM6

Resistance to rice virus diseases is an important requirement in many Southeast Asian rice breeding programs. Inheritance of resistance to rice tungro spherical virus (RTSV) in TW5, a near-isogenic line derived from Indonesian rice cultivar Utri Merah, was compared to that in TKM6, an Indian rice cultivar. Both TKM6 and Utri Merah are cultivars resistant to RTSV infections. Crosses were made between TKM6 and TN1, a susceptible cultivar, and between TW5 and TN1, and F3 lines were evaluated for their resistance to RTSV using two RTSV inoculum sources and a serological assay (ELISA). In TKM6, the resistance to the mixture of RTSV-V + RTBV inoculum source was controlled by a single recessive gene, whereas in TW5, the resistance was controlled by two recessive genes. A single recessive gene, however, controlled the resistance in TW5 when another RTSV variant, RTSV-VI, was used, suggesting that the resistance in TW5 depends on the nature of the RTSV inoculum used. RT-PCR, sequence, and phylogenetic analyses confirmed that RTSV-VI inoculum differs from RTSV-V inoculum and accurate phenotyping of the resistance to RTSV requires the use of a genetic marker.

Research project for managing skid resistance is on the road

In December 2006, the Engineering and Technology Group of Queensland’s Department of Main Roads entered into a three-year skid resistance management research project with QUT Faculty of Built Environment and Engineering researchers and the QUT-based CRC for Integrated Engineering Asset Management (CIEAM). CIEAM undertakes a broad range of asset management research in the areas of defence, utilities, transportation and industrial processes. “The research project is an important activity of Main Roads’ Skid Resistance Management Plan published in June 2006.” said Main Roads project leader Mr Justin Weligamage. “The intended project output is a decision-support model for use by Road Asset Managers throughout a road network. The research objective is to enable road asset managers to better manage the surfacing condition of the road asset with specific focus on skid resistance,” said QUT project leader Professor Arun Kumar. The research project will review existing skid resistance investigatory levels, develop a risk-based method to establish skid resistance investigatory levels and improve the decision support methodology in order to minimise crashes. The new risk-based approach will be used to identify locations on the Queensland state-controlled road network that may have inadequate skid resistance. Once a high risk site is identified, the appropriate remedial action will be decided on. This approach will allow road asset managers to target optimal remedial actions, reducing the incidence and severity of crashes where inadequate skid resistance is a contributing cause.

Agroinoculation Allows the Screening of Rice for Resistance to Rice Tungro Bacilliform Virus

A system for agroinoculating rice tungro bacilliform virus (RTBV), one of the two viruses of the rice tungro disease complex, has been optimised. A nontumour-inducing strain of Agrobacterium (pGV3850) was used in order to conform with biosafety regulations. Fourteen-day-old seedlings survived the mechanical damage of the technique and were still young enough to support virus replication. The level of the bacterial inoculum was important to obtain maximum infection, with a high inoculum level (0.5 × 1012 cells/ml) resulting in up to 100% infection of a susceptible variety that was comparable with infection by insect transmission. Agroinoculation with RTBV was successful for all three rice cultivarss tested; TN1 (tungro susceptible), Balimau Putih (tungro tolerant), and IR26 (RTSV and vector resistant). Agroinoculation enables resistance to RTBV to be distinguished from resistance to the leafhopper vector of the virus, and should prove useful in screening rice germplasm, breeding materials, and transgenic rice lines.

Identifying relationship between skid resistance, road characteristics and crashes using probability-based risk approach

Road accidents are of great concerns for road and transport departments around world, which cause tremendous loss and dangers for public. Reducing accident rates and crash severity are imperative goals that governments, road and transport authorities, and researchers are aimed to achieve. In Australia, road crash trauma costs the nation A$ 15 billion annually. Five people are killed, and 550 are injured every day. Each fatality costs the taxpayer A$1.7 million. Serious injury cases can cost the taxpayer many times the cost of a fatality. Crashes are in general uncontrolled events and are dependent on a number of interrelated factors such as driver behaviour, traffic conditions, travel speed, road geometry and condition, and vehicle characteristics (e.g. tyre type pressure and condition, and suspension type and condition). Skid resistance is considered one of the most important surface characteristics as it has a direct impact on traffic safety. Attempts have been made worldwide to study the relationship between skid resistance and road crashes. Most of these studies used the statistical regression and correlation methods in analysing the relationships between skid resistance and road crashes. The outcomes from these studies provided mix results and not conclusive. The objective of this paper is to present a probability-based method of an ongoing study in identifying the relationship between skid resistance and road crashes. Historical skid resistance and crash data of a road network located in the tropical east coast of Queensland were analysed using the probability-based method. Analysis methodology and results of the relationships between skid resistance, road characteristics and crashes are presented.

Using data mining to predict road crash count with a focus on skid resistance values

Road crashes cost world and Australian society a significant proportion of GDP, affecting productivity and causing significant suffering for communities and individuals. This paper presents a case study that generates data mining models that contribute to understanding of road crashes by allowing examination of the role of skid resistance (F60) and other road attributes in road crashes. Predictive data mining algorithms, primarily regression trees, were used to produce road segment crash count models from the road and traffic attributes of crash scenarios. The rules derived from the regression trees provide evidence of the significance of road attributes in contributing to crash, with a focus on the evaluation of skid resistance.

Resistance is hopeless : assimilating queer theory

This paper seeks to assimilate Queer Theory: that is, to bring it within the gambit of a ‘mainstream’ or ‘dominant’ space: the academy. It does so by historicising Queer Theory, and investigating, if not what it is, then at least what it has been. This makes it possible to engage critically with Queer Theory. Suggesting that Queer Theory has often employed tropes of assimilation, the paper turns to another cultural site at which such language is popular - science fiction - in order to investigate the assumption of these metaphors. It goes on to suggest some of the assumptions about cultures which underlie these metaphors. Finally, it points to other sites in Queer Theory which undermines these assumptions, and provide other ways - quite uninterested in assimilation - in which to think Queer.