The contribution of neutrophils to bacteriophage clearance and pharmacokinetics in vivo.

With the increasing prevalence of antimicrobial-resistant bacterial infections, there is great interest in using lytic bacteriophages (phages) to treat such infections. However, the factors that govern bacteriophage pharmacokinetics in vivo remain poorly understood. Here, we have examined the contribution of neutrophils, the most abundant phagocytes in the body, to the pharmacokinetics of intravenously administered bacteriophage in uninfected mice. A single dose of LPS-5, an antipseudomonal bacteriophage recently used in human clinical trials, was administered intravenously to both wild-type BALB/c and neutropenic ICR mice. Phage concentrations were assessed in peripheral blood and spleen at 0.5, 1, 2, 4, 8, 12, and 24 hours after administration by plaque assay and qPCR. We observed that the phage clearance is only minimally affected by neutropenia. Indeed, the half-life of phages in blood in BALB/c and ICR mice is 3.45 and 3.66 hours, respectively. These data suggest that neutrophil-mediated phagocytosis is not a major determinant of phage clearance. Conversely, we observed a substantial discrepancy in circulating phage levels over time when measured by qPCR versus plaque assay, suggesting that substantial functional inactivation of circulating phages occurs over time. These data indicate that circulating factors, but not neutrophils, inactivate intravenously administered phages.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. Here, we use Dynamic Light Scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-year-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web-application (Phage-ELF) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and non-destructive tool for quality control of phage preparations in academic and commercial settings.

Personalized aerosolised bacteriophage treatment of a chronic lung infection due to multidrug-resistant Pseudomonas aeruginosa.

Bacteriophage therapy has been suggested as an alternative or complementary strategy for the treatment of multidrug resistant (MDR) bacterial infections. Here, we report the favourable clinical evolution of a 41-year-old male patient with a Kartagener syndrome complicated by a life-threatening chronic MDR Pseudomonas aeruginosa infection, who is treated successfully with iterative aerosolized phage treatments specifically directed against the patient's isolate. We follow the longitudinal evolution of both phage and bacterial loads during and after phage administration in respiratory samples. Phage titres in consecutive sputum samples indicate in patient phage replication. Phenotypic analysis and whole genome sequencing of sequential bacterial isolates reveals a clonal, but phenotypically diverse population of hypermutator strains. The MDR phenotype in the collected isolates is multifactorial and mainly due to spontaneous chromosomal mutations. All isolates recovered after phage treatment remain phage susceptible. These results demonstrate that clinically significant improvement is achievable by personalised phage therapy even in the absence of complete eradication of P. aeruginosa lung colonization.

Associations between sweet taste function, oral complex carbohydrate sensitivity, liking and consumption of ad libitum sweet and non-sweet carbohydrate milkshakes among female adults.

Excess energy intake is recognised as a strong contributing factor to the global rise of being overweight and obese. The aim of this paper was to investigate if oral sensitivity to complex carbohydrate relates to ad libitum consumption of complex carbohydrate foods in a sample group of female adults. Participants' ((n 51 females): age 23·0 (sd 0·6) years (range 20·0-41·0 years); excluding restrained eaters) sensitivity towards maltodextrin (oral complex carbohydrate) and glucose (sweet taste) was assessed by measuring detection threshold (DT) and suprathreshold intensity perception (ST). A crossover design was used to assess consumption of two different iso-energetic preload milkshakes and ad libitum milkshakes - (1) glucose-based milkshake, (2) maltodextrin-based milkshake. Ad libitum intake (primary outcome) and eating rate, liking, hunger, fullness and prospective consumption ratings were measured. Participants who were more sensitive towards complex carbohydrate (maltodextrin DT) consumed significantly more maltodextrin-based milkshake in comparison with less-sensitive participants (P = 0·01) and this was independent of liking. Participants who had higher liking for glucose-based milkshake consumed significantly more glucose-based milkshake in comparison with participants with lower hedonic ratings (P = 0·049). The results provide support regarding the role of the oral system sensitivity (potentially taste) to complex carbohydrate and the prospective to overconsume complex carbohydrate-based milkshake in a single sitting.

The Associations Between Oral Complex Carbohydrate Sensitivity, BMI, Liking, and Consumption of Complex Carbohydrate Based Foods.

Recent work suggests that humans may perceive complex carbohydrates and that their sensitivity to simple carbohydrates (i.e., glucose and sucrose) is independent from tasting complex carbohydrates. The aim of this study was to confirm whether humans could sense complex carbohydrates from a range of concentration levels; and if their oral sensitivity to complex carbohydrate relates to their BMI, liking, and consumption of complex carbohydrate-based foods using a large sample group of adults. Participants' (n = 92 females, age 23.7 ± 0.5 yr [range, 19 to 47 yr]) oral sensitivity towards complex carbohydrate (maltodextrin) and sweet taste function (glucose) was assessed by measuring detection threshold and suprathreshold intensity perception (gLMS). Participants were asked to complete an online version of a Food Frequency Questionnaire and a Likes and Dislikes Questionnaire. Hedonic ratings for complex carbohydrate and sweet solutions, as well as for a range of complex carbohydrate and sweet prototypical foods, were also measured. Consistent with previous findings, there was large interindividual variation in detection and intensity measures for maltodextrin and glucose. No significant associations were found between oral complex carbohydrate sensitivity, Body Mass Index (BMI), and frequency of consumption of complex carbohydrate-based foods measured. Similarly, no differences were observed between liking of complex carbohydrates, BMI, and food intake. All in, these results from a large sample group further support the proposition that complex carbohydrates are not invisible to the human palate, and can be sensed in the oral cavity even at low concentration levels.

Evidence supporting oral sensitivity to complex carbohydrates independent of sweet taste sensitivity in humans.

Compared to simple sugars, complex carbohydrates have been assumed invisible to taste. However, two recent studies proposed that there may be a perceivable taste quality elicited by complex carbohydrates independent of sweet taste. There is precedent with behavioural studies demonstrating that rats are very attracted to complex carbohydrates, and that complex carbohydrates are preferred to simple sugars at low concentrations. This suggests that rats may have independent taste sensors for simple sugars and complex carbohydrates. The aim of this paper is to investigate oral sensitivities of two different classes of complex carbohydrates (a soluble digestible and a soluble non-digestible complex carbohydrate), and to compare these to other caloric and non-nutritive sweeteners in addition to the prototypical tastes using two commonly used psychophysical measures. There were strong correlations between the detection thresholds and mean intensity ratings for complex carbohydrates (maltodextrin, oligofructose) (r = 0.94, P < 0.001). There were no significant correlations between the detection thresholds of the complex carbohydrates (maltodextrin, oligofructose) and the sweeteners (glucose, fructose, sucralose, Rebaudioside A, erythritol) (all P > 0.05). However, moderate correlations were observed between perceived intensities of complex carbohydrates and sweeteners (r = 0.48-0.61, P < 0.05). These data provide evidence that complex carbohydrates can be sensed in the oral cavity over a range of concentrations independent of sweet taste sensitivity at low concentrations, but with partial overlap with sweet taste intensity at higher concentrations.

Carbohydrate Taste Sensitivity Is Associated with Starch Intake and Waist Circumference in Adults.

Background: Recent studies have proposed that humans may perceive complex carbohydrates and that sensitivity to simple carbohydrates is independent of sensitivity to complex carbohydrates. Variation in oral complex carbohydrate sensitivity may influence food consumption.Objective: This study aimed to investigate the associations between oral complex carbohydrate sensitivity, anthropometry, and dietary intake in adults.Methods: We assessed oral sensitivity to complex carbohydrates (maltodextrin and oligofructose) by measuring detection thresholds (DTs) and suprathreshold intensity perceptions (STs) for 34 participants, including 16 men (mean ± SEM age : 26.2 ± 0.4 y; range: 24-30 y) and 18 women (age: 29.4 ± 2.1 y; range: 24-55 y). We also measured height, weight, and waist circumference (WC) and participants completed a 4-d food diary and a food-frequency questionnaire.Results: Measurements of oral sensitivity to complex carbohydrates were significantly correlated with WC and dietary energy and starch intakes (DT: r = -0.38, P < 0.05; ST: r = 0.36-0.48, P < 0.05). When participants were grouped into tertiles, there were significant differences in WC and total energy or starch intakes for those who were more sensitive or experienced high intensity compared with those who were less sensitive or experienced low intensity. Being more sensitive or experiencing high intensity was associated with greater energy (7968-8954 kJ/d) and starch (29.1-29.8% of energy) intakes and a greater WC (88.2-91.4 cm) than was being less sensitive or experiencing low intensity (6693-7747 kJ/d, 20.9-22.2% of energy, and 75.5-80.5 cm, respectively).Conclusion: Complex carbohydrate sensing is associated with WC and consumption of complex carbohydrates and energy in adults. This trial was registered at anzctr.org.au as ACTRN12616001356459.

Psychophysical Evaluation of Sweetness Functions Across Multiple Sweeteners.

Sweetness is one of the 5 prototypical tastes and is activated by sugars and non-nutritive sweeteners (NNS). The aim of this study was to investigate measures of sweet taste function [detection threshold (DT), recognition threshold (RT), and suprathreshold intensity ratings] across multiple sweeteners. Sixty participants, 18-52 years of age (mean age in years = 26, SD = ±7.8), were recruited to participate in the study. DT and RT were collected for caloric sweeteners (glucose, fructose, sucrose, erythritol) and NNS (sucralose, rebaudioside A). Sweetness intensity for all sweeteners was measured using a general Labeled Magnitude Scale. There were strong correlations between DT and RT of all 4 caloric sweeteners across people (r = 0.62-0.90, P < 0.001), and moderate correlations between DT and RT for both of the NNS (r = 0.39-0.48, P < 0.05); however, weaker correlations were observed between the DT or RT of the caloric sweeteners and NNS (r = 0.26-0.48, P < 0.05). The DT and RT of glucose and fructose were not correlated with DT or RT of sucralose (P > 0.05). In contrast, there were strong correlations between the sweetness intensity ratings of all sweeteners (r = 0.70-0.96, P < 0.001). This suggests those caloric sweeteners and NNS access at least partially independent mechanisms with respect to DT and RT measures. At suprathreshold level, however, the strong correlation between caloric sweeteners and NNS through weak, moderate, and strong intensity indicates a commonality in sweet taste mechanism for the perceived intensity range.

The Association between Sweet Taste Function, Anthropometry, and Dietary Intake in Adults.

Variation in ability to detect, recognize, and perceive sweetness may influence food consumption, and eventually chronic nutrition-related conditions such as overweight and obesity. The aim of this study was to investigate the associations between sweet taste function, anthropometry, and dietary intake in adults. Participants' (n = 60; mean age in years = 26, SD = ±7.8) sweet taste function for a range of sweeteners (glucose, fructose, sucrose, sucralose, erythritol, and Rebaudioside A) was assessed by measuring detection and recognition thresholds and sweetness intensity. Height, weight, and waist circumference were also measured, and participants also completed a Food Frequency Questionnaire. There was large inter-individual variation in detection, recognition and sweetness intensity measures. Pearson's correlation coefficient revealed no robust correlations between measures of sweet taste function, anthropometry, and dietary intake, with the exception of suprathreshold intensity, which was moderately correlated with total energy intake (r = 0.23-0.40). One-way analysis of variance revealed no significant differences between the most and least sensitive participants in terms of BMI, waist circumference, and dietary intake for all measures of sweet taste function and sweeteners (all p > 0.01). When stratified into BMI categories, there were no significant differences in any measure of sweet taste function between the normal weight and overweight/obese participants (all p > 0.01). Results show that that sweet taste function is not associated with anthropometry and sweetness intensity measures are the most appropriate measure when assessing links between sweet taste and food consumption.

An ultrastructural study of Paraphysoderma sedebokerense (Blastocladiomycota), an epibiotic parasite of microalgae.

Successful algal cultivation for biofuel production is one path in the transition to a renewable energy economy. The green alga Scenedesmus dimorphus is a candidate for biofuel production, but is subject to parasitism and subsequent population crash when cultivated in open ponds. From an open pond cultivating S. dimorphus for biofuel production in New Mexico, USA, an amoeboid parasite was isolated, designated as isolate FD61, and its rDNA operon sequenced. A BLAST search for nuc 18S rDNA (18S) sequence similarity identified the parasite as Paraphysoderma sedebokerense (Blastocladiomycota). Here, we examine the ultrastructure of P. sedebokerense and compare it with that of a sister taxon, Physoderma maydis. The parasite has thin-walled vegetative sporangia and thick-walled resting sporangia. Our observations indicate that amoeboid swarmers are produced in the vegetative phase, while either amoeboid swarmers or zoospores are the product of meiosis in resting sporangia. Meiosis is confirmed by the presence of synaptonemal complexes in resting sporangia nuclei. Notably, P. sedebokerense has a Golgi apparatus with stacked cisternae, a feature reported for P. maydis, but which is absent in all other examined taxa in Blastocladiomycota. This report furthers our knowledge of the life cycle of P. sedebokerense.

Diversity, Productivity, and Stability of an Industrial Microbial Ecosystem.

Managing ecosystems to maintain biodiversity may be one approach to ensuring their dynamic stability, productivity, and delivery of vital services. The applicability of this approach to industrial ecosystems that harness the metabolic activities of microbes has been proposed but has never been tested at relevant scales. We used a tag-sequencing approach with bacterial small subunit rRNA (16S) genes and eukaryotic internal transcribed spacer 2 (ITS2) to measuring the taxonomic composition and diversity of bacteria and eukaryotes in an open pond managed for bioenergy production by microalgae over a year. Periods of high eukaryotic diversity were associated with high and more-stable biomass productivity. In addition, bacterial diversity and eukaryotic diversity were inversely correlated over time, possibly due to their opposite responses to temperature. The results indicate that maintaining diverse communities may be essential to engineering stable and productive bioenergy ecosystems using microorganisms.

A new isolate of Amoeboaphelidium protococcarum, and Amoeboaphelidium occidentale, a new species in phylum Aphelida (Opisthosporidia).

Microalgae used in the production of biofuels represents an alternative to fossil fuels. One problem in the production of algae for biofuels is attacks by algal parasitoids that can cause population crashes when algae are cultivated in outdoor ponds (Greenwell et al. 2010). Integrated solutions are being sought to mitigate this problem, and an initial step is pest identification. We isolated an algal parasitoid from an open pond of Scenedesmus dimorphus used for biofuel production in New Mexico and examined its morphology, ultrastructure and molecular phylogeny. A phylogenetic analysis placed this organism in Aphelida as conspecific with Amoeboaphelidium protococcarum sensu Karpov et al. 2013. As a result we re-evaluated the taxonomy of Amoeboaphelidium protococcarum sensu Letcher et al. 2013 and here designate it as a new species, Amoeboaphelidium occidentale.

Spectroradiometric monitoring for open outdoor culturing of algae and cyanobacteria.

We assess the measurement of hyperspectral reflectance for outdoor monitoring of green algae and cyanobacteria cultures with a multichannel, fiber-coupled spectroradiometer. Reflectance data acquired over a 4-week period are interpreted via numerical inversion of a reflectance model, in which the above-water reflectance is expressed as a quadratic function of the single backscattering albedo, which is dependent on the absorption and backscatter coefficients. The absorption coefficient is treated as the sum of component spectra consisting of the cultured species (green algae or cyanobacteria), dissolved organic matter, and water (including the temperature dependence of the water absorption spectrum). The backscatter coefficient is approximated as the scaled Hilbert transform of the culture absorption spectrum with a wavelength-independent vertical offset. Additional terms in the reflectance model account for the pigment fluorescence features and the water-surface reflection of sunlight and skylight. For the green algae and cyanobacteria, the wavelength-independent vertical offset of the backscatter coefficient is found to scale linearly with daily dry weight measurements, providing the capability for a nonsampling measurement of biomass in outdoor ponds. Other fitting parameters in the reflectance model are compared with auxiliary measurements and physics-based calculations. The model-derived magnitudes of sunlight and skylight water-surface reflections compare favorably with Fresnel reflectance calculations, while the model-derived quantum efficiency of Chl-a fluorescence is found to be in agreement with literature values. Finally, the water temperatures derived from the reflectance model exhibit excellent agreement with thermocouple measurements during the morning hours but correspond to significantly elevated temperatures in the afternoon hours.

Host cell pigmentation in Scenedesmus dimorphus as a beacon for nascent parasite infection.

Biofuels derived from the mass cultivation of algae represent an emerging industry that aims to partially displace petroleum based fuels. Outdoor, open-pond, and raceway production facilities are attractive options for the mass culture of algae however, this mode of cultivation leaves the algae susceptible to epidemics from a variety of environmental challenges. Infestations can result in complete collapse of the algal populations and destruction of their valuable products making it paramount to understand the host-pathogen relationships of known algal pests in order to develop mitigation strategies. In the present work, we characterize the spatial-temporal response of photosynthetic pigments in Scenedesmus dimorphus to infection from Amoeboaphelidium protococcarum, a destructive endoparasite, with the goal of understanding the potential for early detection of infection via host pigment changes. We employed a hyperspectral confocal fluorescence microscope to quantify these changes in pigmentation with high spatial and spectral resolution during early parasite infection. Carotenoid abundance and autofluorescence increased within the first 24 h of infection while chlorophyll emission remained constant. Changes in host cell photosynthesis and bulk chlorophyll content were found to lag behind parasite replication. The results herein raise the possibility of using host-cell pigment changes as indicators of nascent parasite infection.

Siting algae cultivation facilities for biofuel production in the United States: trade-offs between growth rate, site constructability, water availability, and infrastructure.

Locating sites for new algae cultivation facilities is a complex task. The climate must support high growth rates, and cultivation ponds require appropriate land and water resources, as well as transportation and utility infrastructure. We employ our spatiotemporal Biomass Assessment Tool (BAT) to select promising locations based on the open-pond cultivation of Arthrospira sp. and strains of the order Sphaeropleales. A total of 64,000 sites across the southern United States were evaluated. We progressively applied screening criteria and tracked their impact on the number of potential sites, geographic location, and biomass productivity. Both strains demonstrated maximum productivity along the Gulf of Mexico coast, with the highest values on the Florida peninsula. In contrast, sites meeting all selection criteria for Arthrospira were located along the southern coast of Texas and for Sphaeropleales were located in Louisiana and southern Arkansas. Results were driven mainly by the lack of oil pipeline access in Florida and elevated groundwater salinity in southern Texas. The requirement for low-salinity freshwater (<400 mg L(-1)) constrained Sphaeropleales locations; siting flexibility is greater for salt-tolerant species like Arthrospira. Combined siting factors can result in significant departures from regions of maximum productivity but are within the expected range of site-specific process improvements.

Industrial-strength ecology: trade-offs and opportunities in algal biofuel production.

Microalgae represent one of the most promising groups of candidate organisms for replacing fossil fuels with contemporary primary production as a renewable source of energy. Algae can produce many times more biomass per unit area than terrestrial crop plants, easing the competing demands for land with food crops and native ecosystems. However, several aspects of algal biology present unique challenges to the industrial-scale aquaculture of photosynthetic microorganisms. These include high susceptibility to invading aquatic consumers and weeds, as well as prodigious requirements for nutrients that may compete with the fertiliser demands of other crops. Most research on algal biofuel technologies approaches these problems from a cellular or genetic perspective, attempting either to engineer or select algal strains with particular traits. However, inherent functional trade-offs may limit the capacity of genetic selection or synthetic biology to simultaneously optimise multiple functional traits for biofuel productivity and resilience. We argue that a community engineering approach that manages microalgal diversity, species composition and environmental conditions may lead to more robust and productive biofuel ecosystems. We review evidence for trade-offs, challenges and opportunities in algal biofuel cultivation with a goal of guiding research towards intensifying bioenergy production using established principles of community and ecosystem ecology.

Characterization of Amoeboaphelidium protococcarum, an algal parasite new to the cryptomycota isolated from an outdoor algal pond used for the production of biofuel.

Mass culture of algae for the production of biofuels is a developing technology designed to offset the depletion of fossil fuel reserves. However, large scale culture of algae in open ponds can be challenging because of incidences of infestation with algal parasites. Without knowledge of the identity of the specific parasite and how to control these pests, algal-based biofuel production will be limited. We have characterized a eukaryotic parasite of Scenedesmus dimorphus growing in outdoor ponds used for biofuel production. We demonstrated that as the genomic DNA of parasite FD01 increases, the concentration of S. dimorphus cells decreases; consequently, this is a highly destructive pathogen. Techniques for culture of the parasite and host were developed, and the endoparasite was identified as the Aphelidea, Amoeboaphelidium protococcarum. Phylogenetic analysis of ribosomal sequences revealed that parasite FD01 placed within the recently described Cryptomycota, a poorly known phylum based on two species of Rozella and environmental samples. Transmission electron microscopy demonstrated that aplanospores of the parasite produced filose pseudopodia, which contained fine fibers the diameter of actin microfilaments. Multiple lipid globules clustered and were associated with microbodies, mitochondria and a membrane cisternae, an arrangement characteristic of the microbody-lipid globule complex of chytrid zoospores. After encystment and attachment to the host cells, the parasite injected its protoplast into the host between the host cell wall and plasma membrane. At maturity the unwalled parasite occupied the entire host cell. After cleavage of the protoplast into aplanospores, a vacuole and lipids remained in the host cell. Amoeboaphelidium protococcarum isolate FD01 is characteristic of the original description of this species and is different from strain X-5 recently characterized. Our results help put a face on the Cryptomycota, revealing that the phylum is more diverse than previously understood and include some of the Aphelidea as well as Rozella species and potentially Microsporidia.