Functional properties of sodium and calcium caseinate antimicrobial active films containing carvacrol

Active edible films were prepared by adding carvacrol into sodium caseinate (SC) and calcium caseinate (CC) matrices plasticized with two different glycerol concentrations (25 and 35 wt%) prepared by solvent casting. Functional characterisation of these bio-films was carried out by determination of some of their physico-chemical properties, such as colour, transparency, oxygen barrier, wettability, dye permeation properties and antibacterial effectiveness against Gram negative and Gram positive bacteria. All films exhibited good performance in terms of optical properties in the CIELab space showing high transparency. Carvacrol was able to reduce CC oxygen permeability and slightly increased the surface hydrophobicity. Dye diffusion experiments were performed to evaluate permeation properties. The diffusion of dye through films revealed that SC was more permeable than CC. The agar diffusion method was used for the evaluation of the films antimicrobial effectiveness against Escherichia coli and Staphylococcus aureus. Both SC and CC edible films with carvacrol showed inhibitory effects on both bacteria.

Use of herbs, spices and their bioactive compounds in active food packaging

The interest and societal demand on the use of natural, biodegradable and renewable resources has increased in the last few years. In addition, food producers and consumers have improved their requirements for the quality of processed food, particularly in the field of increasing shelf-life while preserving organoleptic and nutritional properties. Active packaging technologies have greatly developed in the last decade by trying to satisfy the need for long-life processed food in addition to antioxidant/antimicrobial components in the packaging material. These components are intended to be released in a controlled way to food. These rising trends have been reflected in the field of food packaging by the use of chemicals extracted and obtained from plants in active packaging formulations. Herbs and spices have shown great potential to be used as renewable, biodegradable and valuable sources of chemicals, such as polyphenols, with high antioxidant/antimicrobial performance. This review aims to present the latest published work in this area.

Cost effectiveness of antimicrobial catheters in the intensive care unit : Addressing uncertainty in the decision

Introduction: Some types of antimicrobial-coated central venous catheters (A-CVC) have been shown to be cost-effective in preventing catheter-related bloodstream infection (CR-BSI). However, not all types have been evaluated, and there are concerns over the quality and usefulness of these earlier studies. There is uncertainty amongst clinicians over which, if any, antimicrobial-coated central venous catheters to use. We re-evaluated the cost-effectiveness of all commercially available antimicrobialcoated central venous catheters for prevention of catheter-related bloodstream infection in adult intensive care unit (ICU) patients. Methods: We used a Markov decision model to compare the cost-effectiveness of antimicrobial-coated central venous catheters relative to uncoated catheters. Four catheter types were evaluated; minocycline and rifampicin (MR)-coated catheters; silver, platinum and carbon (SPC)-impregnated catheters; and two chlorhexidine and silver sulfadiazine-coated catheters, one coated on the external surface (CH/SSD (ext)) and the other coated on both surfaces (CH/SSD (int/ext)). The incremental cost per qualityadjusted life-year gained and the expected net monetary benefits were estimated for each. Uncertainty arising from data estimates, data quality and heterogeneity was explored in sensitivity analyses. Results: The baseline analysis, with no consideration of uncertainty, indicated all four types of antimicrobial-coated central venous catheters were cost-saving relative to uncoated catheters. Minocycline and rifampicin-coated catheters prevented 15 infections per 1,000 catheters and generated the greatest health benefits, 1.6 quality-adjusted life-years, and cost-savings, AUD $130,289. After considering uncertainty in the current evidence, the minocycline and rifampicin-coated catheters returned the highest incremental monetary net benefits of $948 per catheter; but there was a 62% probability of error in this conclusion. Although the minocycline and rifampicin-coated catheters had the highest monetary net benefits across multiple scenarios, the decision was always associated with high uncertainty. Conclusions: Current evidence suggests that the cost-effectiveness of using antimicrobial-coated central venous catheters within the ICU is highly uncertain. Policies to prevent catheter-related bloodstream infection amongst ICU patients should consider the cost-effectiveness of competing interventions in the light of this uncertainty. Decision makers would do well to consider the current gaps in knowledge and the complexity of producing good quality evidence in this area.

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.

Development of a high-speed sensing and detection system for automatic removal of packages with leaky seals from a high-speed food processing and packaging line

Contamination of packaged foods due to micro-organisms entering through air leaks can cause serious public health issues and cost companies large amounts of money due to product recalls, consumer impact and subsequent loss of market share. The main source of contamination is leaks in packaging which allow air, moisture and microorganisms to enter the package. In the food processing and packaging industry worldwide, there is an increasing demand for cost effective state of the art inspection technologies that are capable of reliably detecting leaky seals and delivering products at six-sigma. The new technology will develop non-destructive testing technology using digital imaging and sensing combined with a differential vacuum technique to assess seal integrity of food packages on a high-speed production line. The cost of leaky packages in Australian food industries is estimated close to AUD $35 Million per year. Contamination of packaged foods due to micro-organisms entering through air leaks can cause serious public health issues and cost companies large sums of money due to product recalls, compensation claims and loss of market share. The main source of contamination is leaks in packaging which allow air, moisture and micro-organisms to enter the package. Flexible plastic packages are widely used, and are the least expensive form of retaining the quality of the product. These packets can be used to seal, and therefore maximise, the shelf life of both dry and moist products. The seals of food packages need to be airtight so that the food content is not contaminated due to contact with microorganisms that enter as a result of air leakage. Airtight seals also extend the shelf life of packaged foods, and manufacturers attempt to prevent food products with leaky seals being sold to consumers. There are many current NDT (non-destructive testing) methods of testing the seal of flexible packages best suited to random sampling, and for laboratory purposes. The three most commonly used methods are vacuum/pressure decay, bubble test, and helium leak detection. Although these methods can detect very fine leaks, they are limited by their high processing time and are not viable in a production line. Two nondestructive in-line packaging inspection machines are currently available and are discussed in the literature review. The detailed design and development of the High-Speed Sensing and Detection System (HSDS) is the fundamental requirement of this project and the future prototype and production unit. Successful laboratory testing was completed and a methodical design procedure was needed for a successful concept. The Mechanical tests confirmed the vacuum hypothesis and seal integrity with good consistent results. Electrically, the testing also provided solid results to enable the researcher to move the project forward with a certain amount of confidence. The laboratory design testing allowed the researcher to confirm theoretical assumptions before moving into the detailed design phase. Discussion on the development of the alternative concepts in both mechanical and electrical disciplines enables the researcher to make an informed decision. Each major mechanical and electrical component is detailed through the research and design process. The design procedure methodically works through the various major functions both from a mechanical and electrical perspective. It opens up alternative ideas for the major components that although are sometimes not practical in this application, show that the researcher has exhausted all engineering and functionality thoughts. Further concepts were then designed and developed for the entire HSDS unit based on previous practice and theory. In the future, it would be envisaged that both the Prototype and Production version of the HSDS would utilise standard industry available components, manufactured and distributed locally. Future research and testing of the prototype unit could result in a successful trial unit being incorporated in a working food processing production environment. Recommendations and future works are discussed, along with options in other food processing and packaging disciplines, and other areas in the non-food processing industry.

The Gold Coast marine precinct : pre-packaging entrepreneurship

We investigate the evolving quality of entrepreneurship in the Gold Coast Marine Precinct, a purpose-built industrial district in Southeast Queensland, Australia. Our findings are that the environment in the Precinct can be conducive to a better quality of entrepreneurship than may be feasible for firms in other settings; that a successful industrial district can be created artificially, with appropriate social relationships evolving afterwards; and that improvements in information and communications technology have undermined some aspects of traditional behaviour in the Precinct, but the essential nature of internal relationships remains intact.

Genetic variability and antimicrobial resistance of Ureaplasma parvum in response to maternal erythromycin treatment : a study in pregnant sheep

Background: Ureaplasmas are the most frequently isolated microorganisms from the amniotic fluid (AF) of pregnant women and can cause chronic infections that are difficult to eradicate with standard macrolide treatment. We tested the effects of erythromycin treatment on phenotypic and genotypic markers of ureaplasmal antimicrobial resistance in sheep. Method: At 50 days of gestation (d, term=145d) 12 pregnant ewes received intra-amniotic injections of U. parvum serovar 3 (erythromycin-sensitive, 2x104 colony-forming-units). At 100d ewes received: erythromycin treatment (500 mg, q3h for 4 days, IM, n=6) or no treatment (n=6). Fetuses were delivered surgically (125d) and AF and chorioamnion were collected for: culture, minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) testing; 23S rRNA sequencing; and detection of macrolide-lincosamide-streptogramin resistance (MLSr) genes. Results: MICs of erythromycin, azithromycin and roxithromycin against AF isolates were low (range = 0.06 mg/L to 1.0 mg/L); however, chorioamnion isolates demonstrated increased resistance to roxithromycin (0.13 – 5.33 mg/L). 62.5% of chorioamnion ureaplasmas formed biofilms in vitro and mutations (125 nucleotides, 29.6%) were found in the 23S rRNA gene (domain V) of chorioamnion (but not AF) ureaplasmas. MLSr genes (ermB, msrC and msrD) were detected in 100% of chorioamnion isolates and only msrD was detected in AF isolates (40%). Conclusions: 23S rRNA mutations and MLSr genes occurred independently of erythromycin treatment, suggesting that the anatomical site of infection and microenvironment may exert selective pressures on ureaplasmas that cause genetic changes and alter antimicrobial sensitivity profiles. These results have serious implications for treatment of in utero infections.

Cytotoxicity of topical antimicrobial agents used in burn wounds in Australasia

BACKGROUND: Burn sepsis is a leading cause of mortality and morbidity in patients with major burns. The use of topical antimicrobial agents has helped improve the survival of these patients. Silvazine (Sigma Pharmaceuticals, Melbourne, Australia) (1% silver sulphadiazine and 0.2% chlorhexidine digluconate) is used exclusively in Australasia, and there is no published study on its cytotoxicity. This study compared the relative cytotoxicity of Silvazine with 1% silver sulphadiazine (Flamazine (Smith & Nephew Healthcare, Hull, UK)) and a silver-based dressing (Acticoat (Smith & Nephew Healthcare, Hull, UK)). METHODS: Dressings were applied to the centre of culture plates that were then seeded with keratinocytes at an estimated 25% confluence. The plates were incubated for 72 h and culture medium and dressings then removed. Toluidine blue was added to stain the remaining keratinocytes. Following removal of the dye, the plates were photographed under standard conditions and these digital images were analysed using image analysis software. Data was analysed using Student's t-test. RESULTS: In the present study, Silvazine is the most cytotoxic agent. Seventy-two hour exposure to Silvazine in the present study results in almost no keratinocyte survival at all and a highly statistically significant reduction in cell survival relative to control, Acticoat and Flamazine (P<0.001, P<0.01, P<0.01, respectively). Flamazine is associated with a statistically significant reduction in cell numbers relative to control (P<0.05), but is much less cytotoxic than Silvazine (P<0.005). CONCLUSION: In this in-vitro study comparing Acticoat, Silvazine and Flamazine, Silvazine shows an increased cytotoxic effect, relative to control, Flamazine and Acticoat. An in-vivo study is required to determine whether this effect is carried into the clinical setting.

Hydrogels containing silver nanoparticles for burn wounds show antimicrobial activity without cytotoxicity

This research introduces a novel dressing for burn wounds, containing silver nanoparticles in hydrogels for infected burn care. The 2-acrylamido-2-methylpropane sulfonic acid sodium salt hydrogels containing silver nanoparticles have been prepared via ultraviolet radiation. The formation of silver nanoparticles was monitored by surface plasmon bands and transmission electron microscopy. The concentration of silver nitrate loaded in the solutions slightly affected the physical properties and mechanical properties of the neat hydrogel. An indirect cytotoxicity study found that none of the hydrogels were toxic to tested cell lines. The measurement of cumulative release of silver indicated that 70%–82% of silver was released within 72 hr. The antibacterial activities of the hydrogels against common burn pathogens were studied and the results showed that 5 mM silver hydrogel had the greatest inhibitory activity. The results support its use as a potential burn wound dressing.

DrsG from Streptococcus dysgalactiae subsp equisimilis inhibits the antimicrobial peptide LL- 37

SIC and DRS are related proteins present in only four of the more than 200 Streptococcus pyogenes emm-types. These proteins inhibit complement mediated lysis and/or the activity of certain antimicrobial peptides. A gene encoding a homologue of these proteins, herein called DrsG, has been identified in the related bacterium Streptococcus dysgalactiae subsp equisimilis (SDSE). Here we show that geographically dispersed isolates representing 14 of 50 emm-types examined possess variants of drsG. However not all isolates within the drsG-positive emm-types possess the gene. Sequence comparisons also reveal a high degree of conservation in different SDSE emm-types. To examine the biological activity of DrsG, recombinant versions of two major DrsG variants, DrsGS and DrsGL, were expressed and purified. Western blot analysis using antisera raised to these proteins demonstrated both variants to be expressed and secreted into culture supernatant. Unlike SIC, but similar to DRS, DrsG does not inhibit complement mediated lysis. However, like both SIC and DRS, DrsG is a ligand of the cathelcidin LL-37 and is inhibitory to its bactericidal activity in in vitro assays. The greatest similarity between DrsG and DRS/SIC is found in the signal sequence at the amino terminus and proline rich domains in the C-terminal half of the protein. Conservation of prolines in this latter region also suggests these residues are important in the biology of this family of proteins. This is the first report demonstrating the activity of an AMP inhibitory protein in SDSE. These results also suggest that inhibition of AMP activity is the primary function of this family of proteins. The acquisition of complement inhibitory activity of SIC may reflect its continuing evolution.

Development and characterization of a novel, antimicrobial, sterile hydrogel dressing for burn wounds : single-step production with gamma irradiation creates silver nanoparticles and radical polymerization

Patients with burn wounds are susceptible to wound infection and sepsis. This research introduces a novel burn wound dressing that contains silver nanoparticles (SNPs) to treat infection in a 2-acrylamido-2-methylpropane sulfonic acid sodium salt (AMPS-Na(+) ) hydrogel. Silver nitrate was dissolved in AMPS-Na(+) solution and then exposed to gamma irradiation to form SNP-infused hydrogels. The gamma irradiation results in a cross-linked polymeric network of sterile hydrogel dressing and a reduction of silver ions to form SNPs infused in the hydrogel in a one-step process. About 80% of the total silver was released from the hydrogels after 72 h immersion in simulated body fluid solution; therefore, they could be used on wounds for up to 3 days. All the hydrogels were found to be nontoxic to normal human dermal fibroblast cells. The silver-loaded hydrogels had good inhibitory action against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Results from a pilot study on a porcine burn model showed that the 5-mM silver hydrogel was efficient at preventing bacterial colonization of wounds, and the results were comparable to the commercially available silver dressings (Acticoat(TM) , PolyMem Silver(®) ). These results support its use as a potential burn wound dressing.