The application of the in vitro method, which is the basis of my PhD research work, is the first implementation in Hungary of the so-called Mega-plate, a large (1200×600 mm) culture dish (600×300 mm) developed by researchers at Harvard University in 2016. This will be used to model in vitro the evolution of resistance of E. coli to increasing antibiotic concentrations and to monitor and map the evolution of resistance genes during bacterial selection using molecular genetic studies.
The first milestone is to assemble a Mega-plate (600×300 mm) culture dish adapted to the size of the thermostat available, to develop an efficient sterilisation of the dish and to test its operation.
The second milestone is the testing of each antibiotic. Using the international E. coli strain ATCC 25922, we first determine its MIC against the antibiotic and then calculate the starting antibiotic dose of 1x based on this and the CLSI standard recommendation. Using the Mega-plate dish, we can model the time course of resistance to increasing concentrations of a given antibiotic agent in 1x, 10x, 100x, 1000x in vitro conditions in E. coli. The mutation and selection of the bacteria can be visualized by breaking the concentration limits of each antibiotic. The dish is divided into 9 equal lanes, with no antibiotics in the medium of the two extreme lanes. Starting from each side, the second lane is filled with a single concentration of antibiotics, the third lane with a tenfold concentration, the fourth lane with a hundredfold concentration and the middle lane with a thousandfold concentration. The sensitive strain of bacteria is then dispersed into the two outer bands. It takes 12 to 15 days to test an antibiotic, the time it takes for the bacteria to break through all the antibiotic concentration limits. We then sample bacteria from each band, determine their new MIC value, and finally map the antimicrobial resistance gene pool using next-generation sequencing, investigate the encoding of each gene on chromosome, plasmid or phage, its encoding as a mobile genetic element and the effects of mutation-selection pressure by detecting point mutations.