ENABLING RAPID PHENOTYPIC DETECTION OF CEPHALOSPORIN RESISTANCE BEYOND THE CENTRAL LABORATORY

The so-called bacterial “superbugs” are largely resistant to some of the most commonly prescribed antibiotics, including a drug class known as cephalosporins used to treat many hospital and community-acquired infections. This major public health threat has been acknowledged for decades by the Centers for Disease Control (CDC) as a major concern; yet, the detection of superbugs has not been made routine since standard testing practices have been limited to specialized “central” laboratories with sophisticated yet bulky and expensive equipment and highly trained personnel. As a result, the lack of simpler testing methods that can be used in everyday clinics and doctor’s offices can be viewed as a source of error contributing to incorrect antibiotic treatment and poorer patient outcomes, factors that drive even more advanced resistance, depleting our drugs or last resort. In this dissertation, we explore new strategies for simplified methods to test for cephalosporin resistance in order to give higher accessibility in the timely detection of superbugs to support the improvement of patient care. To do this, we take an organic chemistry and biochemical approach to develop new detection molecules that report resistance activity in bacteria expressing extended-spectrum β-lactamase (ESBL) enzymes, one of the most prolific resistance strategies used by superbugs. Next, we describe methods of integrating these detection molecules into practical testing methods, and detail the engineering of simpler assays that allow for rapid readout of ESBL phenotypes using commonplace laboratory plate readers, portable Raman devices, and even handheld personal glucose meters (used for diabetes monitoring) purchased from the drugstore

XIII International Congress in Animal Hygiene, June 17-21, 2007, Tartu, Estonia "Animal health, animal welfare and biosecurity" : proceedings. Volume 2

KonverentsikogumikOn behalf of both the Organising Committee and the Scientific Committee, I am pleased to welcome you in Tartu, Estonia, to participate at the XIII International Congress of the International Society for Animal Hygiene (ISAH). The ISAH (www.isah-soc.org) was founded in 1970 and has today members from 48 countries throughout the world. ISAH can be considered as a group of scientists contributing to efficient, sustainable animal farming with healthy animals, providing wholesome food in a sound environment. Veterinarians and non-veterinary academic scientists (animal science, agricultural economics, engineers, microbiologists, public health professionals, epidemiologists etc., etc) and respective professionals in animal husbandry, who work and/or do research and education in the field of animal hygiene, can apply for a membership of ISAH, and are most welcome to attend ISAH congresses. The first ISAH congress was held in Budapest in 1973. The last ISAH main congress took place in Warsaw, Poland in 2005 and the last in-between symposium in Saint-Malo, France in 2004. Starting from Warsaw congress in 2005, the ISAH, considering the need for a more flexible and frequent exchange of scientific and practical knowledge, organizes its congresses every second year. The present, XIII ISAH congress in Tartu, Estonia, in June 17–21, 2007 is organised under the device "Animal health, animal welfare and biosecurity”. The scientific programme, trying to follow the scope of the ISAH and receive the feedback from modern animal husbandry and food production, concentrates with more profoundness on the following subjects: interaction between the environment and health and welfare of individual animal and herds; managing animal health in large dairy units; ensuring animal welfare during transportation and slaughter; economical implications considering animals’ health; possibilities of precision livestock farming in maintaining good health and welfare of animals; measures for prevention the development and spread of diseases and pathogens in animals including those posing risk to human health (zoonoses); food safety relevant infections and contaminations such as residues in food derived from animals; influence of the animal production on the environment and public health. The Proceedings from the XIII ISAH Congress are herewith presented. The papers on lectures from invited speakers, oral and poster presentations from 11 parallel sessions are included in this excellent compilation. In general, the printed contribution to the ISAH-2007 congress illustrates clearly the broad scientific field of the ISAH and related to it activities. I hereby would like to express my most sincere gratitude in the address of ISAH-2007 organising and scientific committees. Special thanks go to Frens Conference Services for their excellent organizational and technical contribution and to AS Triip for their outstanding printing job of these proceedings. We also appreciate different companies and organisations for their considerable financial support which gave us the opportunity to keep the registration fees affordable. Finally, we thank all participants, contributors, chairpersons, organisational and technical assistants for your considerable efforts – you made the ISAH-2007 in Tartu real success. We wish you all interesting and pleasant congress and enjoyable stay in Tartu. A. Aland Editor Chairman of the ISAH-2007 Organising Committe

Imaging polyphenolic therapeutic compounds in a eukaryotic model microbe

Flavonoids are polyphenolic metabolites that have a range of physiological and developmental functions in plants. They are the focus of much work as potential therapeutics, although investigation of specific mode of action remains a notably under-researched area. Monitoring transport and location of flavonoids in cells is difficult because, despite a role in UV-absorption in plants, they emit only low levels of fluorescence. Visualising them in plants is possible using the Naturstoff reagent (NA), reported historically to be a polyphenol-fluorescence-enhancing stain. We explored therefore whether this agent was effective during preclinical assessment of polyphenolic therapeutics in a microbial-model. The eukaryote Dictyostelium discoideum has been shown to be a useful model when identifying novel drug targets for treating various diseases. For example, in the case of polycystic kidney disease, naringenin decreased Dictyostelium cell division whereas a polycystin-2-null Dictyostelium line was resistant to the flavonoid, and, subsequently, naringenin treatment proved to reduce cyst-formation in mammalian-kidney model cell lines1. To monitor transport and site of action of the drugs investigated in such studies, we developed a method using NA-staining in this model organism. A range of polyphenolics were assayed in cells, cell-extracts and the cell-washes, and NA-enhanced imaging was evaluated in parallel with LCMS-quantification. NA-enhanced fluorescence of compounds at therapeutically relevant concentrations proved an effective and qualitative measure of transport and localisation in Dictyostelium, and could be used in concert with localisation dyes. Fluorescence-enhancement is limited to a subset of flavonoids, however, and not more widely applicable in our studies to date

The cyanobacterial rhomboid protease is a regulator of the CCM

Cyanobacteria are aquatic photosynthetic bacteria and useful models for study of the chloroplast and photosynthesis. We are studying a ‘rhomboid’ membrane-located proteases in Synechocystis sp. PCC 6803, which appears to function as a previously undiscovered regulator of the carbon concentrating mechanism (CCM) of this phototroph. Rhomboids are almost ubiquitous across evolution, and are known to activate diverse cellular processes via proteolysis of their specific, membrane-sequestered substrates. Although this well-conserved family has solved crystal structures of bacterial enzymes such as Escherichia coli GlpG, ironically, most work has been carried out on eukaryotic representatives. Following our study of the Arabidopsis thaliana chloroplast RBL10 protease, we identified cyanobacterial orthologues with the aim of discovering if roles might be conserved between these and organellar rhomboids. Molecular biology and reverse-genetics studies were made on slr1461, a mutant in the single rhomboid protease of Synechocystis. When photosynthetic parameters were investigated, it could be seen that inactivation of slr1461 did not affect nonphotochemical quenching, unlike the chloroplast rbl10 mutant, but Slr1461 was required for reduction of photosynthetic activity in mixotrophic conditions. This reduction allows cyanobacteria to avoid expending energy on the uptake of CO2 when an organic carbon source can be utilised: as might be expected, therefore, Slr1461 transcription was linked with downregulation of genes encoding proteins facilitating high-affinity CO2 import under high CO2 and mixotrophic conditions. Quantitative RT-PCR of CCM network genes suggested that Slr1461 is located upstream of known regulators, including another membrane protease, the Slr0228 FtsH, and a central, controlling transcription factor NdhR