An investigation of gram-positive pathogens in powdered foods

The purpose of the project was to obtain and test a range powdered food products that are marketed for consumption by individuals that may be immunocompromised. Hence seventeen infant formula milks (0-6months), twelve over-the-counter, elderly, build-up products and nineteen sports powdered protein shakes were examined. These products were tested for the presence of four different Gram-positive pathogens: Two spore formers; Bacillus cereus and Clostridium perfringens and two non-spore formers; Listeria monocytogenes and Staphylococcus aureus. Products were tested according to the ISO standardised methods of testing for described for each of the organisms and samples plated on different diagnostic agars. The presumptively positive organisms that formed characteristic colonies were then further identified. This identification was confirmed in two ways; biochemically, using a wide range of tests including API, and molecularly using PCR-based assays. The results from the project showed that from the 48 products tested; 16 contained B. cereus, nine S. aureus, three L. monocytogenes and one with C. perfringens. To further investigate whether the non-spore forming organisms that survived in these products were more resilient than expected, a heat inactivation experiment was carried out. A simulated high-temperature-short-time (HTST) pasteurisation was set up and the results gained suggested that the S. aureus isolate was able to survive pasteurisation but not subsequent cold and heat shock. In contrast the L. monocytogenes was sub-lethally damaged by the heat treatment but then recovered during cold storage. Thus postulations about how or where these organisms entered food could be made

An investigation of gram-positive pathogens in powdered foods

The purpose of the project was to obtain and test a range powdered food products that are marketed for consumption by individuals that may be immunocompromised. Hence seventeen infant formula milks (0-6months), twelve over-the-counter, elderly, build-up products and nineteen sports powdered protein shakes were examined. These products were tested for the presence of four different Gram-positive pathogens: Two spore formers; Bacillus cereus and Clostridium perfringens and two non-spore formers; Listeria monocytogenes and Staphylococcus aureus. Products were tested according to the ISO standardised methods of testing for described for each of the organisms and samples plated on different diagnostic agars. The presumptively positive organisms that formed characteristic colonies were then further identified. This identification was confirmed in two ways; biochemically, using a wide range of tests including API, and molecularly using PCR-based assays. The results from the project showed that from the 48 products tested; 16 contained B. cereus, nine S. aureus, three L. monocytogenes and one with C. perfringens. To further investigate whether the non-spore forming organisms that survived in these products were more resilient than expected, a heat inactivation experiment was carried out. A simulated high-temperature-short-time (HTST) pasteurisation was set up and the results gained suggested that the S. aureus isolate was able to survive pasteurisation but not subsequent cold and heat shock. In contrast the L. monocytogenes was sub-lethally damaged by the heat treatment but then recovered during cold storage. Thus postulations about how or where these organisms entered food could be made

Development of rapid phage based detection methods for mycobacteria

MAP is the causative agent of a wasting disease in ruminants and other animals called Johne’s disease. Culture of the organism can take months and in the case of some sheep strains of MAP, culture can take up to a year. It can take several years for an animal infected with MAP to show clinical symptoms of disease. During this subclinical stage of infection, MAP can be shed into the environment contaminating their surroundings and infecting other animals. As well as this Johne’s disease is particularly difficult to diagnose during the subclinical stage of infection. Culture is very difficult and takes too long to be a viable method to diagnose Johne’s disease. Microscopic methods can be used on histological samples to detect MAP, however common acid-fast stains used are not specific for MAP and other mycobacteria and acid-fast organisms can be detected. Molecular methods, such as PCR, exist to rapidly detect the signature DNA sequences of these organisms, however they have the disadvantage of not being able to distinguish between live and dead organisms. Other immunological methods, such as ELISA tests, exist and are routinely used to diagnose Johne’s disease, however their sensitivity is very poor especially during the subclinical stage of disease. The aim of these studies was to develop novel rapid methods of detecting MAP to act as an alternative to methods already available. Sample processing using magnetic separation was carried out to allow good capture of MAP cells and to allow efficient phage infection. Using the phage assay, a specific, sensitive phage based method was developed that could detect approximately 10 cells per ml of blood within 24 h in the laboratory with a sensitive, specific plaque-PCR. This optimised detection method was then used to determine whether MAP cells could be detected in clinical blood samples of cattle suffering from Johne’s disease. The results suggest that animals experimentally and naturally infected with MAP harboured cells in their blood during subclinical and clinical stages of infection. A novel high-throughput method of detecting mycobacteria was also developed. Using phage D29 as a novel mycobacterial DNA extraction tool, viable MAP cells were detected within 8 h and the format of the assay means that it can be adapted to be used in a high-throughput capacity. Factors affecting phage infection and phage-host interactions were investigated to make sure the phage based methods of detection were as efficient as possible. It was found that periods of recovery were often necessary to not only make sure the phage were not inhibited but to also allow the host cells to be metabolically active as it was found that phage D29 can only infect mycobacteria cells that are metabolically active. A fluorescent fusion-peptide capable of specifically labelling MAP cells was also developed to be used as an alternative to acid-fast staining. Peptides that were found to specifically bind to MAP cells were fused with green fluorescent protein and cells mounted on slides were specifically labelled with the fluorescent fusion protein. This resulted in a good alternative to the generic acid-fast staining methods. The blood phage assay has shown that viable MAP cells can be found in the blood of animals suffering from Johne’s disease within 24 h and this can be confirmed using a MAP specific plaque-PCR protocol. A novel faster method to detect MAP was also developed, to cut down the time to detection of viable MAP cells to 8 h, which can be formatted to be used in a high-throughput capacity. The phage assay was used as a tool to determine different metabolic states of mycobacteria, and helped investigate optimal detection conditions when using the phage assay. Finally a novel fluorescent label was developed to detect MAP as an alternative to insensitive acid-fast staining. The development of these novel methods to rapidly, specifically and sensitively detect MAP will push further the understanding of Johne’s disease and help control it

Development of rapid phage based detection methods for mycobacteria

MAP is the causative agent of a wasting disease in ruminants and other animals called Johne’s disease. Culture of the organism can take months and in the case of some sheep strains of MAP, culture can take up to a year. It can take several years for an animal infected with MAP to show clinical symptoms of disease. During this subclinical stage of infection, MAP can be shed into the environment contaminating their surroundings and infecting other animals. As well as this Johne’s disease is particularly difficult to diagnose during the subclinical stage of infection. Culture is very difficult and takes too long to be a viable method to diagnose Johne’s disease. Microscopic methods can be used on histological samples to detect MAP, however common acid-fast stains used are not specific for MAP and other mycobacteria and acid-fast organisms can be detected. Molecular methods, such as PCR, exist to rapidly detect the signature DNA sequences of these organisms, however they have the disadvantage of not being able to distinguish between live and dead organisms. Other immunological methods, such as ELISA tests, exist and are routinely used to diagnose Johne’s disease, however their sensitivity is very poor especially during the subclinical stage of disease.\ud The aim of these studies was to develop novel rapid methods of detecting MAP to act as an alternative to methods already available. Sample processing using magnetic separation was carried out to allow good capture of MAP cells and to allow efficient phage infection. Using the phage assay, a specific, sensitive phage based method was developed that could detect approximately 10 cells per ml of blood within 24 h in the laboratory with a sensitive, specific plaque-PCR. This optimised detection method was then used to determine whether MAP cells could be detected in clinical blood samples of cattle suffering from Johne’s disease. The results suggest that animals experimentally and naturally infected with MAP harboured cells in their blood during subclinical and clinical stages of infection. A novel high-throughput method of detecting mycobacteria was also developed. Using phage D29 as a novel mycobacterial DNA extraction tool, viable MAP cells were detected within 8 h and the format of the assay means that it can be adapted to be used in a high-throughput capacity. Factors affecting phage infection and phage-host interactions were investigated to make sure the phage based methods of detection were as efficient as possible. It was found that periods of recovery were often necessary to not only make sure the phage were not inhibited but to also allow the host cells to be metabolically active as it was found that phage D29 can only infect mycobacteria cells that are metabolically active. A fluorescent fusion-peptide capable of specifically labelling MAP cells was also developed to be used as an alternative to acid-fast staining. Peptides that were found to specifically bind to MAP cells were fused with green fluorescent protein and cells mounted on slides were specifically labelled with the fluorescent fusion protein. This resulted in a good alternative to the generic acid-fast staining methods. The blood phage assay has shown that viable MAP cells can be found in the blood of animals suffering from Johne’s disease within 24 h and this can be confirmed using a MAP specific plaque-PCR protocol. A novel faster method to detect MAP was also developed, to cut down the time to detection of viable MAP cells to 8 h, which can be formatted to be used in a high-throughput capacity. The phage assay was used as a tool to determine different metabolic states of mycobacteria, and helped investigate optimal detection conditions when using the phage assay. Finally a novel fluorescent label was developed to detect MAP as an alternative to insensitive acid-fast staining. The development of these novel methods to rapidly, specifically and sensitively detect MAP will push further the understanding of Johne’s disease and help control it

Prevalence and Antimicrobial Resistance Patterns of Campylobacter Species Isolated from Poultry in Mathira, Nyeri County. Report for Fleming Fund Fellowship Programme.

Introduction Antimicrobial resistance is a growing global threat that is increasing animal and human health concerns. Antimicrobial resistance arises when antimicrobial agents fail to effectively kill microorganisms that were previously susceptible to them (Ayukekbong, 2017). The emergence of AMR is attributed to imprudent antimicrobial use arising from inappropriate practices in prescription, misuse, and overuse in both human and animal health (Muloi, 2019). This leads to exposure of animal and human normal flora and pathogens to selection pressure leading to the emergence of antimicrobial resistant strains which can withstand and survive in the presence of antimicrobial agents which would initially kill them. These new strains can be spread from animals to humans and the environment. In low and middle-level countries (LMICs), antibiotics are used for the treatment and prevention (prophylaxis) of infectious diseases in animals and humans. Most of these antibiotics are accessed from pharmacies and agro-veterinary shops over the counter without prescriptions from clinicians ad veterinary professionals and the data on antibiotic use in these countries is scarce (Muloi, 2019). Campylobacter species are bacterial pathogens recognized as a cause of gastroenteritis in the human population and pose a major public health threat worldwide. There are several species of Campylobacter which include C. jejuni, C.coli, C. lari and C.uppsaliensis, capable of causing human illness. However, C. jejuni and C.coli are the most commonly isolated species from poultry and poultry products and cause diarrhoea in patients, mainly children under the age of 1-year, young adults, and immunosuppressed persons. Campylobacter is present in the gut of most animals as normal flora and is transferred to foods from the faeces during slaughter and is acquired through consumption of undercooked poultry and other meat as well as water and unpasteurized milk. Fruits and vegetables can be contaminated by water containing faeces from birds and other animals. Campylobacter can also be transmitted through contact with dog and cat faeces as well as drinking contaminated water. Campylobacter infections are common in low- and middle-income countries as well as developed countries. Methods A total of 380 cloacal swabs were collected randomly from 53 farmers in Mathira, Nyeri, Kenya. They were transported in sterile Amies charcoal swabs under a cold chain and stored at 4°C. They were enriched in Preston broth and incubated for 24 h at 42 °C, after which they were streaked onto mCCDA agar and incubated for 48 h at 42 °C under microaerobic conditions generated by Campy Gen™ packs. Typical grey moist, swarming and discrete colonies were identified using MALD-TOF. Antimicrobial susceptibility was carried out with Ampicillin, Tetracycline, Ciprofloxacin, Gentamycin, Erythromycin, and Nalidixic acid by disk diffusion method, and disk diameters were measured and interpreted using the CLSI guidelines. Results A total of 271 out of 380 (71.32%) bacteria isolated on culture were Campylobacter spp. 190 (50%) were C. jejuni while 81 (22%) were C. coli. Resistance to Ampicillin was 40% for C. coli and 30% for C. jejuni respectively. 54% of C. coli isolates were resistant to Tetracycline while 52% were C. jejuni. 68% of the C. coli were resistant to Ciprofloxacin as compared to C.jejuni at 38%. Resistance to Erythromycin for C. coli 10% and 17% for C. jejuni. 65% of C. coli was resistant to Nalidixic acid as compared to 38% of C. coli. Conclusions The study found a high prevalence of Campylobacters at 71.3% with marked multiple drug resistance to Tetracyclines, Ciprofloxacin, and Nalidixic acid. Resistance to Gentamycin and Erythromycin was markedly low and these antibiotics can be reserved for treatment of human campylobacteriosis. Strengthening and support of surveillance activities for AMR should be enhanced across human and food animal sectors to establish the extent of emergence and and spread of resistance in Campylobacter. National and regional laboratories capacity for testing of pathogens of public health importance should be strengthened increased

Characterizing the Cool KOIs. IV. Kepler-32 as a Prototype for the Formation of Compact Planetary Systems throughout the Galaxy

The Kepler space telescope has opened new vistas in exoplanet discovery space by revealing populations of Earth-sized planets that provide a new context for understanding planet formation. Approximately 70% of all stars in the Galaxy belong to the diminutive M dwarf class, several thousand of which lie within Kepler's field of view, and a large number of these targets show planet transit signals. The Kepler M dwarf sample has a characteristic mass of 0.5 M_☉ representing a stellar population twice as common as Sun-like stars. Kepler-32 is a typical star in this sample that presents us with a rare opportunity: five planets transit this star, giving us an expansive view of its architecture. All five planets of this compact system orbit their host star within a distance one-third the size of Mercury's orbit, with the innermost planet positioned a mere 4.3 stellar radii from the stellar photosphere. New observations limit possible false positive scenarios, allowing us to validate the entire Kepler-32 system making it the richest known system of transiting planets around an M dwarf. Based on considerations of the stellar dust sublimation radius, a minimum mass protoplanetary nebula, and the near period commensurability of three adjacent planets, we propose that the Kepler-32 planets formed at larger orbital radii and migrated inward to their present locations. The volatile content inferred for the Kepler-32 planets and order of magnitude estimates for the disk migration rates suggest that these planets may have formed beyond the snow line and migrated in the presence of a gaseous disk. If true, then this would place an upper limit on their formation time of ~10 Myr. The Kepler-32 planets are representative of the full ensemble of planet candidates orbiting the Kepler M dwarfs for which we calculate an occurrence rate of 1.0 ± 0.1 planet per star. The formation of the Kepler-32 planets therefore offers a plausible blueprint for the formation of one of the largest known populations of planets in our Galaxy

Small variations in reaction conditions tune carbon dot fluorescence

The development of robust and reproducible synthetic strategies for the production of carbon dots with improved fluorescence quantum yields and distinct emission profiles is of great relevance given the vast range of applications of CDs. The fundamental understanding at a molecular level of their formation mechanism, chemical structure and how these parameters are correlated to their photoluminescence (PL) properties is thus essential. In this study, we describe the synthesis and structural characterization of a range of CDs with distinct physico-chemical properties. The materials were prepared under three minutes of microwave irradiation using the same common starting materials (GlcNH2·HCl 1 and EDA 2) but modifying the stoichiometry of the reagents. We show that small changes in reaction conditions leads to the tailoring of the fluorescent behaviour of the CDs from apparent blue to green emission. Structural analysis of the different CD samples suggested different reaction pathways during the CD formation and surface passivation, with the latter step being key to the observed differences. Moreover, we demonstrate that the different materials also respond reversibly to changes in pH, which we can attribute to different behaviour towards protonation/deprotonation events of distinct emission domains present within each nanomaterial. Our results highlight the importance of understanding the reaction pathways that lead to the formation of this carbon-based nanomaterials and how this can be exploited to develop tailored materials towards specific applications

Using climate reanalysis data in conjunction with multi-temporal satellite thermal imagery to derive supraglacial debris thickness changes from energy balance modelling

Surface energy-balance models are commonly used in conjunction with satellite thermal imagery to estimate supraglacial debris thickness. Removing the need for local meteorological data in the debris thickness estimation workflow could improve the versatility and spatiotemporal application of debris thickness estimation. We evaluate the use of regional reanalysis data to derive debris thickness for two mountain glaciers using a surface energy-balance model. Results forced using ERA-5 agree with AWS-derived estimates to within 0.01 ± 0.05 m for Miage Glacier, Italy, and 0.01 ± 0.02 m for Khumbu Glacier, Nepal. ERA-5 data were then used to estimate spatiotemporal changes in debris thickness over a ~20-year period for Miage Glacier, Khumbu Glacier and Haut Glacier d'Arolla, Switzerland. We observe significant increases in debris thickness at the terminus for Haut Glacier d'Arolla and at the margins of the expanding debris cover at all glaciers. While simulated debris thickness was underestimated compared to point measurements in areas of thick debris, our approach can reconstruct glacier-scale debris thickness distribution and its temporal evolution over multiple decades. We find significant changes in debris thickness over areas of thin debris, areas susceptible to high ablation rates, where current knowledge of debris evolution is limited

The New Narrative: Applying narratology to the shaping of futures outputs

Both scenario development and design practices incorporate elements of storytelling, but this use remains undertheorised. This paper will draw upon literary theory, film theory and science fiction criticism to develop an analytical model of narrative structure and rhetorics which speaks to the concerns of scenario developers and designers when engaged in shaping the final outputs or deliverables of a futures project. After highlighting the differing role of telos in art and futures and defining the metacategory of “narratives of futurity”, this paper then defines the terms “story”, “narrative”, “narrator” and “world” in the literary context. It then shows how those concepts map onto futures practice, before going into detail regarding the variety of narrative strategies available across a range of different forms and media, and the qualitative effects that they can reproduce in audiences. There follows the construction of a 2 × 2 matrix based on the critical concepts of narrative mode and narrative logic, within which narratives of futurity might be usefully catalogued and compared, and from which certain broad conclusions may be reached as regards the relation between choice of medium and rhetorical effect. The implications of this analysis are explored in detail