Combinations of plant-derived compounds against Campylobacter in vitro

Campylobacter occur in fresh retail poultry products as a result of their colonization of the gastro-intestinal tract of chickens during growth. Feed additives could be used for suppression of Campylobacter levels in the chickens prior to slaughter. To address this opportunity, feed manufacturers are targeting natural antimicrobials from plant material as new forms of consumer-accepted feed additives. However, to be practical, these natural antimicrobials must be effective at low concentrations. The current study has validated an improved laboratory method to study minimal inhibitory concentrations of plant compounds and their combinations against Campylobacter. The assay was shown to be valid for testing lipid-soluble and water-soluble plant extracts and byproducts from the food industry. The study screened 29 extracts or plant-derived compounds and their mixtures for anti-Campylobacter activity using a laboratory assay. Combinations of oregano, lactic acid, and sorghum byproduct showed effective synergy in anti-Campylobacter activity. The synergies allowed a large reduction in the concentration of the individual compounds needed to kill the bacteria with an 80% reduction in concentration being achieved for oregano essential oil. The assay gives rise to further opportunities for the testing of a greater range of combinations of plant-derived compounds and other natural antimicrobials. The method is robust, simple, and easily automated, and it could be used to adjust the cost of feed formulations by reducing costs associated with antimicrobial feed additives.

Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library

Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a “bioactive” library of 4,182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 µM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 µM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide and pyridazinone. Exploitation of analogues of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.

Volatile compounds from the flowers of spathiphyllum Cannaefolium

Headspace analysis and solvent extraction of the pollenbearing flower spike of Spathiphyllum cannaefolium have been conducted by GC-MS, to determine the basis of the flower spike’s attractancy to certain fruit-fly species. The major components were benzyl acetate, methyleugenol, methylchavicol, p-methoxybenzyl acetate and fatty acids. Benzyl acetate is known to be attractive to D. cueurbitae, D. dorsalis and C. capitata (representing the three different ‘male-lure categories’) and methyleugenol (one of these male-lures) attracts D. cacuminatus, D. dorsalis and D. occipitalis. Thus the odoriferous flowerspike exhibits wide ranging attractancy and hence Spathiphyllum cannaefolium may have some application as a fruit-fly control measure for small orchards where ‘methyleugenol-attracted’ species (e.g. D. cacuminatus, D. dorsalis, D. occipitalis) are the dominant pests.

Antimicrobial susceptibility testing of Brachyspira intermedia and Brachyspira pilosicoli isolates from Australian chickens

Susceptibilities of predominantly Australian isolates of the pathogenic intestinal spirochaetes Brachyspira intermedia (n=25) and Brachyspira pilosicoli (n=17) from chickens were tested in agar dilution against four concentrations each of the antimicrobials tiamulin, lincomycin, tylosin, metronidazole, tetracycline and ampicillin. Based on available minimum inhibitory concentration (MIC) breakpoint values for Brachyspira hyodysenteriae or other Gram-negative enteric veterinary pathogens, isolates of both species generally were susceptible to tiamulin, lincomycin, metronidazole and tetracycline. Although not classed as resistant, four isolates of B. intermedia had an elevated MIC range for tiamulin (1 to 4 mg/l), 11 isolates of B. intermedia and five of B. pilosicoli had an elevated MIC range for lincomycin (10 to 50 mg/l), one isolate of B. pilosicoli had an elevated MIC range for tetracycline (10 to 20 mg/l), and one isolate of B. intermedia and five of B. pilosicoli had an elevated MIC range for ampicillin (10 to 50 mg/l). A clear lack of susceptibility to tylosin (MIC >4 mg/l) was seen in 11 isolates each of B. intermedia and B. pilosicoli, and to ampicillin (MIC >32 mg/l) in two isolates of B. pilosicoli. These data suggest that some resistance to common antimicrobials exists among intestinal spirochetes obtained from laying hens and supports the need of MIC data for clinical isolates before any treatment is considered.

Near infrared spectroscopy as a rapid tool to measure volatile aroma compounds in Riesling wine: Possibilities and limits

Volatile chemical compounds responsible for the aroma of wine are derived from a number of different biochemical and chemical pathways. These chemical compounds are formed during grape berry metabolism, crushing of the berries, fermentation processes (i.e. yeast and malolactic bacteria) and also from the ageing and storage of wine. Not surprisingly, there are a large number of chemical classes of compounds found in wine which are present at varying concentrations (ng L-1 to mg L-1), exhibit differing potencies, and have a broad range of volatilities and boiling points. The aim of this work was to investigate the potential use of near infrared (NIR) spectroscopy combined with chemometrics as a rapid and low-cost technique to measure volatile compounds in Riesling wines. Samples of commercial Riesling wine were analyzed using an NIR instrument and volatile compounds by gas chromatography (GC) coupled with selected ion monitoring mass spectrometry. Correlation between the NIR and GC data were developed using partial least-squares (PLS) regression with full cross validation (leave one out). Coefficients of determination in cross validation (R 2) and the standard error in cross validation (SECV) were 0.74 (SECV: 313.6 μg L−1) for esters, 0.90 (SECV: 20.9 μg L−1) for monoterpenes and 0.80 (SECV: 1658 ?g L-1) for short-chain fatty acids. This study has shown that volatile chemical compounds present in wine can be measured by NIR spectroscopy. Further development with larger data sets will be required to test the predictive ability of the NIR calibration models developed.

An innovative microplate assay to facilitate the detection of antimicrobial activity in plant extracts.

A microplate assay was modified for the detection of antimicrobial activity in plant extracts. The aim was to develop an in vitro assay that could rapidly screen plant extracts to provide quantitative data on inhibition of microbial growth. A spectrophotometric assay using a microplate with serial dilutions of the plant extract and the bacteria was developed. Two bacteria, Staphylococcus aureus and Escherichia coli, were used for this study. Essential oils, oregano (Origanum vulgare) and lemon myrtle (Backhousia citriodora), and three active components carvacrol, thymol and citral were evaluated. The reproducibility of the assay was high, with correlation coefficients (r aureus and E. coli between 0.9321 and 0.9816. Similarly, r and 0.9814. This assay could also be used to measure antimicrobial activity in plant extracts which vary in pH and color.

Effect of Packaging Materials and Storage on Major Volatile Compounds in Three Australian Native Herbs

Lemon myrtle, anise myrtle, and Tasmanian pepper leaf are commercial Australian native herbs with a high volatile or essential oil content. Packaging of the herbs in high- or low-density polyethylene (HDPE and LDPE) has proven to be ineffective in preventing a significant loss of volatile components on storage. This study investigates and compares the effectiveness of alternate high-barrier property packaging materials, namely, polyvinylidene chloride coated polyethylene terephthalate/casted polypropylene (PVDC coated PET/CPP) and polyethylene terephthalate/polyethylene terephthalate/aluminum foil/linear low-density polyethylene (PET/PET/Foil/LLDPE), in prevention of volatile compound loss from the three native herbs stored at ambient temperature for 6 months. Concentrations of major volatiles were monitored using gas chromatography?mass spectrometry (GC-MS) techniques. After 6 months of storage, the greatest loss of volatiles from lemon myrtle was observed in traditional LDPE packaging (87% loss) followed by storage in PVDC coated PET/CPP (58% loss) and PET/PET/Foil/LLDPE (loss of 23%). The volatile loss from anise myrtle and Tasmanian pepper leaf stored in PVDC coated PET/CPP and PET/PET/Foil/LLDPE packaging was <30%. This study clearly indicates the importance of selecting the correct packaging material to retain the quality of herbs with high volatile content.

Urinary excretion of antioxidants in healthy humans following queen garnet plum juice ingestion: a new plum variety rich in antioxidant compounds

In recent years, there has been intense interest in the potential health benefits of dietary derived plant polyphenols and antioxidants. A new variety of Prunus salicina, Queen Garnet plum (QGP), was developed as a high anthocyanin, high antioxidant plum, in a Queensland Government breeding program. Following consumption of 400 mL QGP juice (QGPJ; 1,117 mg anthocyanins) by two healthy male subjects, QGP anthocyanins (cyanidin-3-glucoside and cyanidin-3-rutinoside) were excreted mainly as methylated and glucuronidated metabolites in urine (0.5% of the ingested dose within 24 h). Furthermore, QGPJ intake resulted in a threefold increase in hippuric acid excretion (potential biomarker for total polyphenols intake and metabolite), an increased urinary antioxidant capacity and a decreased malondialdehyde excretion (biomarker for oxidative stress) within 24 h as compared with the polyphenol-/antioxidant-free control. Results from this pilot study suggest that metabolites, and not the native QGP anthocyanins/polyphenols, are most likely the bioactive compounds in vivo.

Major Australian tropical fruits biodiversity: Bioactive compounds and their bioactivities

The plant kingdom harbours many diverse bioactive molecules of pharmacological relevance. Temperate fruits and vegetables have been highly studied in this regard, but there have been fewer studies of fruits and vegetables from the tropics. As global consumers demand and are prepared to pay for new appealing and exotic foods, tropical fruits are now being more intensively investigated. Polyphenols and major classes of compounds like flavonoids or carotenoids are ubiquitously present in these fruits, as they are in the temperate ones, but particular classes of compounds are unique to tropical fruits and other plant parts. Bioactivity studies of compounds specific to tropical fruit plants may lead to new drug discoveries, while the synergistic action of the wide range of diverse compounds contained in plant extracts underlies nutritional and health properties of tropical fruits and vegetables. The evidence for in vitro and animal bioactivities is a strong indicator of the pharmacological promise shown in tropical fruit plant biodiversity. In this review, we will discuss both the occurrence of potential bioactive compounds isolated and identified from a selection of tropical fruit plants of importance in Australia, as well as recent studies of bioactivity associated with such fruits and other fruit plant parts.

Integrated pest management in cotton: exploiting behaviour-modifying (semiochemical) compounds for managing cotton pests

We review here research on semiochemicals for cotton pest management carried out in successive Cotton Co-operative Research Centres from 1998 to 2012. Australian cotton is now dominated by transgenic (Bt) varieties, which provide a strong platform for integrated pest management of key pests such as Helicoverpa spp., but new technologies are required to manage the development of resistance in Helicoverpa spp. to transgenic cotton and the problems posed by emerging and secondary pests, especially sucking insects. A long-range attractant for Helicoverpa moths, based on plant volatiles, has been commercialised as Magnet®. The product has substantial area-wide impacts on moth populations, and only limited effects on beneficial insects. Potential roles are being investigated for this product in resistance management of Helicoverpa spp. on transgenic cotton. Short-range, non-volatile compounds on organ surfaces of plants that do not support development of Helicoverpa spp. have been identified; these compounds deter feeding or oviposition, or are toxic to insect pests. One such product, Sero X®, is effective on Helicoverpa spp. and sucking pests such as whiteflies (Bemisia tabaci), green mirids (Creontiades dilutus), and other hemipteran insects, and is in the advanced stages of commercialisation.

Eco-Taxonomic Insights into Actinomycete Symbionts of Termites for Discovery of Novel Bioactive Compounds

Termites play a major role in foraging and degradation of plant biomass as well as cultivating bioactive microorganisms for their defense. Current advances in “omics” sciences are revealing insights into function-related presence of these symbionts, and their related biosynthetic activities and genes identified in gut symbiotic bacteria might offer a significant potential for biotechnology and biodiscovery. Actinomycetes have been the major producers of bioactive compounds with an extraordinary range of biological activities. These metabolites have been in use as anticancer agents, immune suppressants, and most notably, as antibiotics. Insect-associated actinomycetes have also been reported to produce a range of antibiotics such as dentigerumycin and mycangimycin. Advances in genomics targeting a single species of the unculturable microbial members are currently aiding an improved understanding of the symbiotic interrelationships among the gut microorganisms as well as revealing the taxonomical identity and functions of the complex multilayered symbiotic actinofloral layers. If combined with target-directed approaches, these molecular advances can provide guidance towards the design of highly selective culturing methods to generate further information related to the physiology and growth requirements of these bioactive actinomycetes associated with the termite guts. This chapter provides an overview on the termite gut symbiotic actinoflora in the light of current advances in the “omics” science, with examples of their detection and selective isolation from the guts of the Sunshine Coast regional termite Coptotermes lacteus in Queensland, Australia

Use of a proposed antimicrobial susceptibility testing method for Haemophilus parasuis

The aim of this study was to examine the antimicrobial susceptibility of 97 Haemophilus parasuis cultured from Australian pigs. As there is no existing standard antimicrobial susceptibility technique available for H. parasuis, methods utilising the supplemented media, BA/SN for disc diffusion and test medium broth (TMB) for a microdilution technique, were initially evaluated with the reference strains recommended by the Clinical and Laboratory Standards Institute. The results of the media evaluation suggested that BA/SN and TMB can be used as suitable media for susceptibility testing of H. parasuis. The proposed microdilution technique was then used with 97 H. parasuis isolates and nine antimicrobial agents. The study found that Australian isolates showed elevated minimum inhibitory concentrations (MICs) for ampicillin (1%), penicillin (2%), erythromycin (7%), tulathromycin (9%), tilmicosin (22%), tetracycline (31%) and trimethoprim-sulfamethoxazole (40%). This study has described potential antimicrobial susceptibility methods for H. parasuis and has detected a low percentage of Australian H. parasuis isolates with elevated antimicrobial MICs.

Antimicrobial susceptibility of Histophilus somni isolated from clinically affected cattle in Australia

This study investigated antimicrobial resistance traits, clonal relationships and epidemiology of Histophilus somni isolated from clinically affected cattle in Queensland and New South Wales, Australia. Isolates (n = 53) were subjected to antimicrobial susceptibility testing against six antimicrobial agents (ceftiofur, enrofloxacin, florfenicol, tetracycline, tilmicosin and tulathromycin) using disc diffusion and minimum inhibitory concentration (MIC) assays. Clonal relationships were assessed using repetitive sequence PCR and descriptive epidemiological analysis was performed. The H. somni isolates appeared to be geographically clonal, with 27/53 (47%) isolates grouping in one cluster from one Australian state. On the basis of disc diffusion, 34/53 (64%) isolates were susceptible to all antimicrobial agents tested; there was intermediate susceptibility to tulathromycin in 12 isolates, tilmicosin in seven isolates and resistance to tilmicosin in one isolate. Using MIC, all but one isolate was susceptible to all antimicrobial agents tested; the non-susceptible isolate was resistant to tetracycline, but this MIC result could not be compared to disc diffusion, since there are no interpretative guidelines for disc diffusion for H. somni against tetracycline. In this study, there was little evidence of antimicrobial resistance in H. somni isolates from Australian cattle. Disc diffusion susceptibility testing results were comparable to MIC results for most antimicrobial agents tested; however, results for isolates with intermediate susceptibility or resistance to tilmicosin and tulathromycin on disc diffusion should be interpreted with caution in the absence of MIC results.

Downstream processing of reverse osmosis brine: Characterisation of potential scaling compounds

Reverse osmosis (RO) brine produced at a full-scale coal seam gas (CSG) water treatment facility was characterized with spectroscopic and other analytical techniques. A number of potential scalants including silica, calcium, magnesium, sulphates and carbonates, all of which were present in dissolved and non-dissolved forms, were characterized. The presence of spherical particles with a size range of 10–1000 nm and aggregates of 1–10 microns was confirmed by transmission electron microscopy (TEM). Those particulates contained the following metals in decreasing order: K, Si, Sr, Ca, B, Ba, Mg, P, and S. Characterization showed that nearly one-third of the total silicon in the brine was present in the particulates. Further, analysis of the RO brine suggested supersaturation and precipitation of metal carbonates and sulphates during the RO process should take place and could be responsible for subsequently capturing silica in the solid phase. However, the precipitation of crystalline carbonates and sulphates are complex. X-ray diffraction analysis did not confirm the presence of common calcium carbonates or sulphates but instead showed the presence of a suite of complex minerals, to which amorphous silica and/or silica rich compounds could have adhered. A filtration study showed that majority of the siliceous particles were less than 220 nm in size, but could still be potentially captured using a low molecular weight ultrafiltration membrane.

Transfer of Dietary Plant Natural Flavor Compounds From Essential Oils to Maternal Fluids in Pigs

Fetal flavor conditioning during the perinatal stage could be essential at the time of the weaning to reduce the stress and improve the feed intake in pigs. The transfer of flavor compounds from maternal diet to amniotic fluid and milk has been shown in behavioral experiments, but not through analytical procedures such as gas chromatography–mass spectrometry (GC–MS). The aim of the experiment was to trace the principal essential oils compounds supplied in the diet in maternal fluids. Twenty Large White sows around their 104th gestational day were allocated to individual farrowing crates. Two groups of 10 sows were fed either a standard gestation diet or the same diet supplemented with a mix of 8 essential oils at a rate of 1kg/ton during the last 10 days of gestation. At approximately the 113th gestational day, animals were individually treated with 10mg of Lutalyse IM was to induce farrowing. Fresh amniotic fluid was collected during the farrowing in 100-mL glass bottles and immediately stored at −20 °C freezer. During the second lactation day, 10–20 IU of Oxytocin IM was administered to each sow to facilitate collection of milk samples in 20-mL glass bottles. The samples were stored at −20 °C until analyzed by GC–MS. The presence of significant amounts of principal components of all the essential oils except one were found in the milk and amniotic fluid samples of the treated sows relative to the control sows. Our data prove the transfer of selected dietary flavors to maternal fluids and sets the scenario for further trials to manipulate postweaning behavior in piglets.