Resumen de Detecció i quantificació de bacteriòfags en mostres transparents i tèrboles
Most of the phage detection and quantification techniques are developed for applications in public health and industry environments, and for many years conventional methods like Double Agar Layer and Electron Microscopy were the only available option to detect and quantify phages. These methods are either labour intensive and time consuming or require expensive equipment, respectively. However, this has changed with the introduction of novel technologies that brings new solutions and alternatives to conventional methods. In order to build an effective tool, phage detection methods must take into account several factors: 1) type of phage; 2) level of sensitivity required; 3) availability of cultivable hosts; 4) possible interference by the sample matrix; 5) availability of sophisticated equipment and/or highly skilled personnel and, conditioning most of the above, 6) type of application (phage therapy, bio-control studies, food fermentation industry or environmental monitoring).
The work developed in this thesis explores the possibility of carrying out sensitive detection and quantification of specific phages, by analyzing the dynamic behavior of the phage-host system. The dataset has been published and made available to the community as it constitutes probably the first detailed study on phage-host concentration dependence that has been published from the discovery of bacteriophages in the 1920’s. All of these elements can be used as the basis for the development of specific methods tailored to any of the areas mentioned above.
In the first phase we have monitored optical density (OD) changes during phage-induced culture lysis in clear media in order to evaluate phage-host kinetics over 90 different combinations of bacteria/phage concentrations. In a second phase the fluorescent properties of the redox dye resazurin have been employed to overcome the problems observed when attempting to perform optical density kinetic measurements in media of high turbidity. Thanks to the fluorescent properties of resazurin, the phage-host kinetics of 186 different combinations of bacteria/phage concentrations have been evaluated in synthetic turbid media as well as in a high complexity matrix such as milk. Both OD and fluorescence-based approaches are complementary and have similar LOD and Time of Detection (5x101 pfu/mL in 3.5 h). OD-based assay is quantitative, and the resazurin assay is semi-quantitative but faster, detecting high phage numbers in less than 60 min. The working principle of both assays is highly flexible and can be easily adjusted to any specific application by choosing the right phage-host combination whether it be in clean or turbid samples. Finally, the technology used is relatively simple and well suited for further miniaturization, automatization and high-throughput analysis.
