Studies on physiologic specialisation of albugo candida causing white blister on broccoli in Victoria

White blister caused by oomycete Albugo candida (Pers. Ex. Lev.) Kuntze, (AC), is an important disease affecting many cruciferous hosts, including vegetable brassicas. The outbreaks of white blister in broccoli and cauliflower crops (Brassica oleracea var. italica and var. botrytis) in Southern Australia in the last three years led to restrictions on movement of fresh produce and seedlings from the disease-affected areas. Current classification of AC races is based on physiologic specialisation of this pathogen. Race 9 has been identified to cause white blister on B. oleracea in the USA. We report on specialisation of AC causing disease in Victorian broccoli crops and the use of molecular tools for the separation of AC races. In a glasshouse, 12 Brassicaceae species/varieties replicated 6 times, were inoculated twice at the fully developed cotyledon stage with a distilled water suspension of zoosporangia (1x104 per ml) collected from a single broccoli leaf. Two weeks after inoculation the incidence of white blister on cotyledons and seedling leaves of cauliflower, broccoli, black mustard and Indian mustard was 79.7, 78.4, 73.7 and 6.9% respectively. Cabbage plants were symptomless indicating that further specialisation of the pathogen may have occurred in Australia. High disease incidence among black mustard plants shows that the Australian isolate differs from overseas AC race 9. The interaction of a number of B. oleracea varieties to a range of AC isolates from various hosts will be investigated. Degenerate primers are now being used to amplify actin and β-tubulin genes to identify race specific polymorphisms in AC isolates from three different hosts (wild radish, Chinese cabbage, and broccoli). Differing PCR amplification efficiencies from broccoli and wild radish isolates using degenerate actin primers indicates sequence differences in the two isolates. The fragments are now being cloned and sequenced for race comparison.

Production of Omega-3 Triacylglycerol concentrates using a new food grade immobilized Candida antarctica Lipase B

Triacylglycerol concentrates of eicosapentaenoic and docosahexaenoic omega-3 fatty acids were synthesized either via transesterification or esterification of glycerol with the corresponding ethyl ester or free fatty acid concentrates, respectively. A newly developed food grade immobilized Candida antarctica lipase Β system using an Amberlite FPX-66 hydrophobic matrix, was compared with a commercially available non-food grade commercial system, for their ability to catalyze these reactions. For either system, the transesterification required higher temperature (90◦C) than esterification (70°C) to achieve maximum triacylglycerol yields. The newly developed immobilized system efficiently catalyzes the esterification of free fatty acids with glycerol and differs from the existing commercial system in that it is food grade and has a more uniform and larger particle distribution. The new system significantly improves flow in a packed bed reactor, enabling multiple reuse of the catalyst for up to 80 repeats.

Molecular phylogenetic relationships between Albugo candida collections on the Brassicaceae in Australia

Recent phylogenetic analyses of Albugo candida using the mitochondrial cytochrome c oxidase subunit II (cox2) gene, the nuclear ribosomal RNA large subunit (LSU) gene and the nuclear ribosomal RNA internal transcribed spacer (ITS) gene region have revealed significant genetic variation and led to the description of new species in the A. candida complex. This study examined the genetic diversity within Australian collections of A. candida from various Brassicaceae species in a range of geographic locations. Phylogenetic analysis of 31 Australian A. candida collections from 11 hosts using the rDNA ITS region, rDNA LSU region and cox2 mtDNA showed that the majority of Australian A. candida collections were the common form of A. candida. One collection from a common weed host, hairy bitter cress (Cardamine hirsuta), was found to belong to a previously reported but undescribed species, while three collections, also from C. hirsuta, were found to belong to a new undescribed species.

Races of Albugo candida causing white blister rust on brassica vegetables in Australia

Albugo Candida races of Australian isolates from B. oleracea var. italica (broccoli), B. rapa var. pekinensis (pak choi) and var. chinensis (Chinese cabbage), and Capsella bursa-pastoris (shepherd's purse) were identified on a set of Brassicaceae hosts. Isolates from broccoli were identified as A. candida race 9 (Ac 9), from pak choi and Chinese cabbage as the sub-race V of Ac 7 or as a mixed population of sub-races A and V of the same race. Isolates from Shepherd's purse were identified as Ac 4. Australian Ac 9 isolates caused white blister disease on broccoli, broccolini, cauliflower, Brussels sprout and other related Brassicaceae hosts including B. nigra (black mustard), B. napus (oilseed rape), and on B. rapa (turnip rape) 'Torch' (a differential host of Ac 7). All cultivars of cabbage (B. oleracea var. capitata) and one new broccoli 'Booster' inoculated with isolates from broccoli were immune to the isolate tested. This result indicates that the Australian A. candida varies from the European one that causes disease on cabbage as well as on other B. oleracea varieties and additionally on shepherd's purse and an American one that causes disease on cabbage. The genotype of B. nigra tested was susceptible to both Ac 9 and Ac 7. This result indicates that B. nigra can serve as a host for both races. This study provides the first record of white blister disease on B. napus ('Hobson' and 'Regent') in Australia.

The role of micro-organisms (staphylococcus aureus and Candida Albicans) in the pathogenesis of breast pain and infection in lactation women : study protocol

Background: The CASTLE (Candida and Staphylococcus Transmission: Longitudinal Evaluation) study will investigate the micro-organisms involved in the development of mastitis and “breast thrush” among breastfeeding women. To date, the organism(s) associated with the development of breast thrush have not been identified. The CASTLE study will also investigate the impact of physical health problems and breastfeeding problems on maternal psychological health in the early postpartum period.

Methods/Design: The CASTLE study is a longitudinal descriptive study designed to investigate the role of Staphylococcus spp (species) and Candida spp in breast pain and infection among lactating women, and to describe the transmission dynamics of S. aureus and Candida spp between mother and infant. The relationship between breastfeeding and postpartum health problems as well as maternal psychological well-being is also being investigated. A prospective cohort of four hundred nulliparous women who are at least thirty six weeks gestation pregnant are being recruited from two hospitals in Melbourne, Australia (November 2009 to June 2011). At recruitment, nasal, nipple (both breasts) and vaginal swabs are taken and participants complete a questionnaire asking about previous known staphylococcal and candidal infections. Following the birth, participants are followed-up six times: in hospital and then at home weekly until four weeks postpartum. Participants complete a questionnaire at each time points to collect information about breastfeeding problems and postpartum health problems. Nasal and nipple swabs and breast milk samples are collected from the mother. Oral and nasal swabs are collected from the baby. A telephone interview is conducted at eight weeks postpartum to collect information about postpartum health problems and breastfeeding problems, such as mastitis and nipple and breast pain.

Discussion: This study is the first longitudinal study of the role of both staphylococcal and candidal colonisation in breast infections and will help to resolve the current controversy about which is the primary organism in the condition known as breast thrush. This study will also document transmission dynamics of S. aureus and Candida spp between mother and infant. In addition, CASTLE will investigate the impact of common maternal physical health symptoms and the effect of breastfeeding problems on maternal psychological well-being.

Does Candida and/or Staphylococcus play a role in nipple and breast pain in lactation? A cohort study in Melbourne, Australia

Objective: To investigate Candida species and Staphylococcus aureus and the development of 'nipple and breast thrush' among breastfeeding women. Design: Prospective longitudinal cohort study. Setting: Two hospitals in Melbourne, Australia (one public, one private) with follow-up in the community. Participants: 360 nulliparous women recruited at 36 weeks' gestation from November 2009 to June 2011. Participants were followed up six times: in hospital, at home weekly until 4 weeks postpartum and by telephone at 8 weeks. Main outcome measures: Case definition 'nipple and breast thrush': burning nipple pain and breast pain (not related to mastitis); detection of Candida spp (using culture and PCR) in the mother's vagina, nipple or breast milk or in the baby's mouth; detection of S aureus in the mother' nipple or breast milk. Results: Women with the case definition of nipple/ breast thrush were more likely to have Candida spp in nipple/breast milk/baby oral samples (54%) compared to other women (36%, p=0.014). S aureus was common in nipple/breast milk/baby samples of women with these symptoms as well as women without these symptoms (82% vs 79%) (p=0.597). Time-to-event analysis examined predictors of nipple/breast thrush up to and including the time of data collection. Candida in nipple/breast milk/baby predicted incidence of the case definition (rate ratio (RR) 1.87 (95% CI 1.10 to 3.16, p=0.018). We do not have evidence that S aureus colonisation was a predictor of these symptoms (RR 1.53, 95% CI 0.88 to 2.64, p=0.13). Nipple damage was also a predictor of these symptoms, RR 2.30 (95% CI 1.19 to 4.43, p=0.012). In the multivariate model, with all three predictors, the RRs were very similar to the univariate RRs. This indicates that Candida and nipple damage are independent predictors of our case definition.

Antifungal synergy of theaflavin and epicatechin combinations against Candida albicans

New antifungal agents are required to compensate for the increase in resistance to standard antifungal agents of Candida albicans, which is an important opportunistic fungal pathogen that causes minor infections in many individuals but very serious infections in those who are immune-compromised. In this study, combinations of theaflavin and epicatechin are investigated as potential antifungal agents and also to establish whether antifungal synergy exists between these two readily accessible and cost-effective polyphenols isolated from black and green tea. The results of disc diffusion assays showed stronger antibacterial activity of theaflavin:epicatechin combinations against C. albicans NCTC 3255 and NCTC 3179, than that of theaflavin alone. Minimum inhibitory concentrations (MICs) of 1,024 μg/ml with theaflavin and 128-256 μg/ml with theaflavin:epicatechin combinations were found. The fractional inhibitory concentration indexes were calculated, and the synergy between theaflavin and epicatechin against both isolates of C. albicans was confirmed. Theaflavin:epicatechin combinations show real potential for future use as a treatment for infections caused by C. albicans.

Design of endoperoxides with anti-Candida activity

Broad antifungal structure-activity relationships governing epoxy-endoperoxides 2 and 3 and their parent endoperoxides 1 are reported. Their inhibitory activity against Candida albicans in conjunction with hemolytic activity and/or growth inhibition of cultured mammalian cells are reported. This information provided guidance for the further development of endoperoxide and epoxy-endoperoxides as topical antifungal agents.

Growth inhibition of Candida species and Aspergillus fumigatus by statins

Statins are a class of drugs widely used for lowering high cholesterol levels through their action on 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in the synthesis of cholesterol. We studied the effects of two major statins, simvastatin and atorvastatin, on five Candida species and Aspergillus fumigatus. The statins strongly inhibited the growth of all species, except Candida krusei. Supplementation of Candida albicans and A. fumigatus with ergosterol or cholesterol in aerobic culture led to substantial recovery from the inhibition by statins, suggesting specificity of statins for the mevalonate synthesis pathway. Our findings suggest that the statins could have utility as antifungal agents and that fungal colonization could be affected in those on statin therapy.

Novel endoperoxides: synthesis and activity against Candida species

Fifteen new endoperoxides have been synthesised and tested for activity against pathogenic Candida species. These endoperoxides can be prepared in high yields, in one to three steps, from inexpensive starting materials. Despite chemical and structural similarities, their inhibitory activity against Candida growth varied greatly from one endoperoxide to another, and one species to another. This study of susceptibility to endoperoxide compounds presented here may lead to the development of potent new antifungal agents.

Detection of Candida spp. in the vagina of a cohort of nulliparous pregnant women by culture and molecular methods: is there an association between maternal vaginal and infant oral colonisation?

BACKGROUND: Most studies describing vaginal Candida spp. in pregnancy focus on symptomatic vaginitis, rather than asymptomatic colonisation, and solely utilise microbiological culture. The extent to which asymptomatic vaginal carriage may represent a reservoir for infant oral colonisation has been highly debated. MATERIALS AND METHODS: This study formed part of the Candida and Staphylococcus Transmission Longitudinal Evaluation (CASTLE) study, in Melbourne, Australia, from 2009 to 2011 and used culture and molecular methods to examine vaginal swabs collected late in the third trimester of pregnancy for Candida spp. Oral swabs from infants were also examined using culture methods. RESULTS: Overall, 80 of 356 (22%) women were positive for Candida spp; the majority being Candida albicans (83%). Candida glabrata and other Candida spp. were also identified, but in much lower numbers. Molecular analysis identified numerous positive samples not detected by culture, including 13 cases of C. albicans. In addition, some positive samples only recorded to genus level by culture were accurately identified as either C. albicans or C. glabrata following molecular analyses. Eighteen infants recorded positive Candida spp. cultures, predominantly C. albicans. However, there were only four (25%) mother/infant dyads where C. albicans was detected. CONCLUSIONS: This study provides valuable data on asymptomatic colonisation rates of Candida spp. within an asymptomatic population of women late in pregnancy. The utilisation of molecular methods improved the rate of detection and provided a more accurate means for identification of non-albicans Candida spp. The low mother/infant colonisation rate suggests that non-maternal sources are likely involved in determining infant oral colonisation status.

Design of 1,2-dioxines with anti-Candida activity: aromatic substituted 1,2-dioxines

In an ongoing effort to rationally design new antimicrobials, 47 new 1,2-dioxines have been synthesised. Broad antifungal structure-activity relationships governing aromatically substituted epoxy-1,2-dioxines 2 and 3 and their parent 1,2-dioxines 1 were assessed primarily against the pathogenic yeast, Candida albicans, with haemolytic activity of selected examples also reported.

Candida infective endocarditis: an observational cohort study with a focus on therapy

Candida infective endocarditis is a rare disease with a high mortality rate. Our understanding of this infection is derived from case series, case reports, and small prospective cohorts. The purpose of this study was to evaluate the clinical features and use of different antifungal treatment regimens for Candida infective endocarditis. This prospective cohort study was based on 70 cases of Candida infective endocarditis from the International Collaboration on Endocarditis (ICE)-Prospective Cohort Study and ICE-Plus databases collected between 2000 and 2010. The majority of infections were acquired nosocomially (67%). Congestive heart failure (24%), prosthetic heart valve (46%), and previous infective endocarditis (26%) were common comorbidities. Overall mortality was high, with 36% mortality in the hospital and 59% at 1 year. On univariate analysis, older age, heart failure at baseline, persistent candidemia, nosocomial acquisition, heart failure as a complication, and intracardiac abscess were associated with higher mortality. Mortality was not affected by use of surgical therapy or choice of antifungal agent. A subgroup analysis was performed on 33 patients for whom specific antifungal therapy information was available. In this subgroup, 11 patients received amphotericin B-based therapy and 14 received echinocandin-based therapy. Despite a higher percentage of older patients and nosocomial infection in the echinocandin group, mortality rates were similar between the two groups. In conclusion, Candida infective endocarditis is associated with a high mortality rate that was not impacted by choice of antifungal therapy or by adjunctive surgical intervention. Additionally, echinocandin therapy was as effective as amphotericin B-based therapy in the small subgroup analysis.