Multi-Locus Next-Generation Sequence Typing of DNA Extracted From Pooled Colonies Detects Multiple Unrelated Candida albicans Strains in a Significant Proportion of Patient Samples

The yeast Candida albicans is an important opportunistic human pathogen. For C. albicans strain typing or drug susceptibility testing, a single colony recovered from a patient sample is normally used. This is insufficient when multiple strains are present at the site sampled. How often this is the case is unclear. Previous studies, confined to oral, vaginal and vulvar samples, have yielded conflicting results and have assessed too small a number of colonies per sample to reliably detect the presence of multiple strains. We developed a next-generation sequencing (NGS) modification of the highly discriminatory C. albicans MLST (multilocus sequence typing) method, 100+1 NGS-MLST, for detection and typing of multiple strains in clinical samples. In 100+1 NGS-MLST, DNA is extracted from a pool of colonies from a patient sample and also from one of the colonies. MLST amplicons from both DNA preparations are analyzed by high-throughput sequencing. Using base call frequencies, our bespoke DALMATIONS software determines the MLST type of the single colony. If base call frequency differences between pool and single colony indicate the presence of an additional strain, the differences are used to computationally infer the second MLST type without the need for MLST of additional individual colonies. In mixes of previously typed pairs of strains, 100+1 NGS-MLST reliably detected a second strain. Inferred MLST types of second strains were always more similar to their real MLST types than to those of any of 59 other isolates (22 of 31 inferred types were identical to the real type). Using 100+1 NGS-MLST we found that 7/60 human samples, including three superficial candidiasis samples, contained two unrelated strains. In addition, at least one sample contained two highly similar variants of the same strain. The probability of samples containing unrelated strains appears to differ considerably between body sites. Our findings indicate the need for wider surveys to determine if, for some types of samples, routine testing for the presence of multiple strains is warranted. 100+1 NGS-MLST is effective for this purpose

Immunoproteomics To Examine Cystic Fibrosis Host Interactions with Extracellular Pseudomonas aeruginosa Proteins ▿

The lungs of patients with cystic fibrosis (CF) are typically chronically infected with Pseudomonas aeruginosa. We used an immunoproteomics approach to analyze the responses of patients to secreted P. aeruginosa proteins. Extracellular proteins from P. aeruginosa strain PAO1 that had been grown to stationary phase were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by Western blotting using sera from four chronically infected patients. Sera from all four patients detected multiple extracellular proteins. The identities of selected proteins recognized by antisera were determined. Production of at least four of these proteins (azurin and three proteases: elastase, PrpL, and PasP) is governed by quorum sensing, consistent with active bacterial quorum sensing in the lungs of CF patients. The CF lung is generally thought to be an iron-deficient environment for infecting bacteria, and growing the bacteria in the presence of an iron-chelating agent, ethylene-diamine-di(o-hydroxyphenylacetic acid), enabled detection of additional proteins that were recognized by patient sera. The sera also detected multiple proteins from cells in the logarithmic growth phase, and protein identification suggested that most of these were the result of cell lysis or secretion in membrane vesicles. Comparison with extracellular proteins from a second P. aeruginosa strain, strain Pa4, showed that many proteins recognized by patient sera are common to both strains, although there are also some strain-specific extracellular proteins. Our data show that while there are some differences in the responses of different patients to P. aeruginosa, there are also many similarities, and that an immunoproteomics approach enables the identification of proteins that are made by P. aeruginosa during infection

Identifying individual possums using their oral bacteria

The New Zealand Brushtail possum, Trichosurus vulpecula, poses a threat to native forest and fauna. Furthermore, possums constitute both reservoir and vector for bovine tuberculosis. To better monitor possum ecology, new ways of population monitoring and management are required. Objectives: The aims of this study were (i) to determine whether possums can be individually identified by the bacteria they leave when biting Waxtags™; and (ii) to determine how long after biting can bacteria be recovered from Waxtags™. Methods: Eight possums from two locations were sampled by swabbing the central incisors and culturing the bacteria on Mitis-Salivarius agar (selective for streptococci). DNA was isolated from individual bacterial colonies and amplified by arbitrarily-primed polymerase chain reaction with OP A-2 as primer. The amplicons were separated by agarose electrophoresis and compared. Following biting by a human, plain and sugar-coated Waxtags™ were tied to trees (outside) and sampled at three, five and seven day intervals. Streptococci were cultured and the number of colonies compared. Results: Of the eight possums sampled, only two (one from each geographical location) had distinct dominant amplicon profiles. Three possums from each location shared dominant amplicon profiles. Possums from the same location possessed similar amplicon profiles but there were no similarities between possums from different locations. Streptococci were recovered from sugar-coated Waxtags™ after seven days, but from plain Waxtags™ streptococci could not be recovered after three days. Conclusions: Possums from the same geographical region harbor streptococci with indistinguishable DNA profiles and therefore cannot be identified by this method. However, possums from different locations have distinct streptococcal DNA profiles and this may aid in identifying animals from separate ranges. Sugar-covered Waxtags ™ offer an opportunity to monitor possum activities over extended periods and could provide a more cost effective method of population monitoring

LIPGENE food-exchange model for alteration of dietary fat quantity and quality in free-living participants from eight European countries

Controlled human intervention trials are required to confirm the hypothesis that dietary fat quality may influence insulin action. The aim was to develop a food-exchange model, suitable for use in free-living volunteers, to investigate the effects of four experimental diets distinct in fat quantity and quality: high SFA (HSFA); high MUFA (HMUFA) and two low-fat (LF) diets, one supplemented with 1.24g EPA and DHA/d (LFn-3). A theoretical food-exchange model was developed. The average quantity of exchangeable fat was calculated as the sum of fat provided by added fats (spreads and oils), milk, cheese, biscuits, cakes, buns and pastries using data from the National Diet and Nutrition Survey of UK adults. Most of the exchangeable fat was replaced by specifically designed study foods. Also critical to the model was the use of carbohydrate exchanges to ensure the diets were isoenergetic. Volunteers from eight centres across Europe completed the dietary intervention. Results indicated that compositional targets were largely achieved with significant differences in fat quantity between the high-fat diets (39.9 (SEM 0.6) and 38.9 (SEM 0.51) percentage energy (%E) from fat for the HSFA and HMUFA diets respectively) and the low-fat diets (29.6 (SEM 0.6) and 29.1 (SEM 0.5) %E from fat for the LF and LFn-3 diets respectively) and fat quality (17.5 (SEM 0.3) and 10.4 (SEM 0.2) %E front SFA and 12.7 (SEM 0.3) and 18.7 (SEM 0.4) %E MUFA for the HSFA and HMUFA diets respectively). In conclusion, a robust, flexible food-exchange model was developed and implemented successfully in the LIPGENE dietary intervention trial